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DEPARTAMENT DE QUIMICA ANALITICA

DESARROLLO DE MTODOS ANALTICOS PARA EL


CONTROL DE CALIDAD DE SURFACTANTES Y ADITIVOS
EN PRODUCTOS DE LIMPIEZA Y DE HIGIENE PERSONAL.

MIRIAM BENEITO CAMBRA

UNIVERSITAT DE VALNCIA
Servei de Publicacions
2011

Aquesta Tesi Doctoral va ser presentada a Valncia el dia 28


doctubre de 2011 davant un tribunal format per:
-

Dr. Michael Lmmerhofer


Dra. Coral Barbas Arribas
Dr. Alain Berthod
Dr. Jos Javier Laserna Vzquez
Dra. Mara Celia Garca lvarez-Coque

Va ser dirigida per:


Dr. Guillermo Ramis Ramos
Dr. Jos Manuel Herrero Martnez

Copyright: Servei de Publicacions


Miriam Beneito Cambra

I.S.B.N.: 978-84-370-8800-6
Edita: Universitat de Valncia
Servei de Publicacions
C/ Arts Grfiques, 13 baix
46010 Valncia
Spain
Telfon:(0034)963864115

FACULTAT DE QUMICA
DEPARTAMENT DE QUMICA ANALTICA

DESARROLLO DE MTODOS
ANALTICOS PARA EL CONTROL DE
CALIDAD DE SURFACTANTES Y
ADITIVOS EN PRODUCTOS DE
LIMPIEZA Y DE HIGIENE
PERSONAL
Memoria para alcanzar el grado de Doctor
(Doctorado Europeo) presentada por:
Miriam Beneito Cambra

Directores:
Dr. Guillermo Ramis Ramos
Dr. Jos Manuel Herrero Martnez
Valencia, 2011

II

D. Guillermo Ramis Ramos, catedrtico del Departamento de Qumica Analtica


de la Universidad de Valencia y D. Jos Manuel Herrero Martnez, profesor
titular del mismo departamento,

Certifican

Que la presente memoria, que lleva por ttulo Desarrollo de mtodos analticos
para el control de calidad de surfactantes y aditivos en productos de limpieza y
de higiene personal constituye la Tesis Doctoral de Da. Miriam Beneito
Cambra.

Asimismo, certifican haber dirigido y supervisado tanto los distintos aspectos del
trabajo, como su redaccin.

Burjassot, Junio de 2011

Guillermo Ramis Ramos

Jos Manuel Herrero Martnez

III

IV

Esta tesis se ha realizado gracias a una beca V-Segles-Empresa


entre la Universitat de Valncia y Qumicas Oro

VI

A mi familia

VII

VIII

AGRADECIMIENTOS
La realizacin de esta Tesis Doctoral no habr sido posible sin la ayuda y
el apoyo de un gran nmero de personas:
En primer lugar, quisiera agradecer a mis Directores, el Dr. Guillermo
Ramis Ramos y al Dr. Jos Manuel Herrero Martnez, por su ayuda y dedicacin.
Su confianza, apoyo y paciencia han sido fundamentales para poder llevar a cabo
este proyecto.
A si mismo, quisiera dar las gracias al Dr. Ernesto F. Sim Alfonso, por su
contribucin al desarrollo de mi trabajo y por haber estado dispuesto a apoyarme
cuando lo he necesitado.
Tambin quisiera agradecer a Da. Marisa Caellas y D. Miguel ngel
Chamorro de Qumicas Oro por esta oportunidad que se me ha brindado y la
paciencia durante estos aos.
Cmo no tambin agradecer a todos aquellos que, de un modo u otro
hicieron posible mi estancia en la Universidad de Viena, y principalmente al Dr.
Wolfgang Lindner y Dr. Michael Lmmerhofer, DANKE SCHN.
Por otro lado, agradecer a mis compaeros del Laboratorio 10, con los que
he compartido tantas cosas. cosas buenas, otras no tan buenas, agobios,
preocupaciones, trabajo y momentos de diversin (os acordis de Keops?):
Aarn, Alex, Amparo, Anna, Claudia, Cristina, Elisa, Enrique, Hugo, Isabel,
Ivn, Laura, Mara N., Mara V., Pascuali, Patty, Pietro, Roberto, Sandra,
Tamara, Victoria, Virginia y Yanelis. A la Dra. M Jess (MariJesu!!!!! Que ja
casi ho tinc, no masfixies!!! Moltes grcies per tot, pels consells, xarradetes i
espentes quan fan falta). Todos y cada uno, estis muy presentes, muchas gracias
por todo. A todos los dems compaeros y profesores del Departamento, con los

IX

que ha sido una verdadera satisfaccin relacionarme y trabajar. Tambin dar las
gracias a toda la gente con la que compart muchos momentos en Viena, los
compaeros de laboratorio y otra gente que estuvo ah, en especial al Dr. Xavier
Subirats, moltes grcies per tot, el teu suport, nim i consells son molt valuosos
per a mi.
Tambin agradecer a mis amigos, los de siempre: Amara, Amparo,
Enrique, John, Jose (Teuler), M Mar, Montse, Oscar, Rafa, Ramn, Rebeca,
Vicente y sin olvidar las ltimas incorporaciones (Aharn, Alexis y Nayara),
muchas gracias por estar siempre ah, apoyarme y motivarme para seguir hacia
delante. A todos los amigos de la facultad, que tantas horas hemos compartido en
los pasillos, clases, cafetera y por ah, gracias por entenderme, apoyarme y
aguantarme. Dar las gracias tambin a Pilar y Rosa, que tantos aos
compartiendo el mismo piso, al final une mucho, muchas gracias por todos
aquellos aos y por continuar manteniendo esa amistad.
Y como no, agradecer tambin a mi familia: mis abuelos, tos y primos,
por demostrar siempre tanto inters en mi trabajo y por estar ah, y en especial a
mis padres, quienes siempre han estado a mi lado y me han apoyado en los
momentos difciles, muchas gracias por vuestra ayuda incondicional en todos los
aspectos. Y como no, a mi to Batiste, tu no podies faltar, grcies per tot, ja veus,
tenim algo positiu.
A todos vosotros, y a los que aunque no haya nombrado han sido
partcipes de que todo haya llegado a buen fin, GRACIAS.

XI

NDICE

Captulo I Introduccin....

xx1

I.1. Detergentes....

xx3

I.1.1. Introduccin......

xx3

I.1.2. Componentes en las formulaciones de


los productos de limpieza.......

xx3

I.2. Surfactantes....

x12

I.2.1. Introduccin..

x12

I.2.2. Clasificacin de los surfactantes...

x14

I.2.3. APGs.

x20

I.2.4. Alcoholes no-etoxilados y etoxilados...

x25

I.2.5. AESs..

x29

I.3. Polmeros...

x31

I.3.1. Introduccin..

x31

I.3.2. Polmeros en detergentes..

x34

I.3.3. PVP-NO....

x36

I.3.4. PVP

x37

I.3.5. PVA...

x37

I.3.6. Mtodos de anlisis de polmeros.....

x38

I.4. Enzimas..

x41

I.4.1. Introduccin..

x41

I.4.2. Proteasas....

x43

I.4.3. Amilasas....

x45

I.4.4. Lipasas...

x46

XII

I.4.5. Celulasas........

x47

I.5. Tcnicas analticas.

x49

I.5.1. LC..

x49

I.5.1.1. Medida de parmetros cromatogrficos....

x52

I.5.2. CE..

x56

I.5.2.1. Mecanismo de separacin en CE...

x56

I.5.2.2. EOF

x57

I.5.2.3. Movilidad y tiempo de migracin..

x58

I.5.2.4. Tcnicas de electroseparacin capilar..

x59

I.5.3. CEC...

x60

I.5.3.1. Instrumentacin en CEC...

x60

I.5.3.2. Columnas empleadas en CEC...

x62

I.5.3.3. Polimerizacin de columnas monolticas


basadas en steres de metacrilato y acrilato..

x64

I.5.3.4. Caracterizacin de materiales monolticos...

x65

I.5.4. MS.

x66

I.5.4.1. Fuentes de iones.

x68

I.5.4.2. Analizadores de masas..

x70

I.5.4.3. Acoplamiento de una tcnica


de separacin a un MS...

x72

I.5.4.4. Resolucin.

x73

I.5.5. NMR..

x73

I.5.5.1. Espectrmetro de NMR..

x75

I.5.5.2. NMR de 1H.

x76

I.5.5.3. NMR de 13C....

x78

XIII

I.5.6. Algunas tcnicas de tratamiento estadstico de datos

x79

I.5.6.1. Anlisis clasificatorio supervisado.......

x79

I.5.6.2. LDA...

x80

Captulo II Objetivos y plan de trabajo.....

x83

II.1. Surfactantes..

x85

II.1.1. APGs...

x85

II.1.2. Alcoholes.

x86

II.1.3. AES..

x87

II.2. Polmeros..

x87

II.2.1. PVP-NO...

x87

II.2.2. PVP..

x88

II.2.3. PVA.

x88

II.3. Enzimas....

x89

II.3.1. CZE..

x89

II.3.2. Aminocidos por infusin directa en MS....

x89

II.3.3. Aminocidos derivatizados con OPA-NAC....

x90

II.3.4. Digestos con tripsina

x90

II.4. Columnas monolticas..

x91

II.4.1. Columnas monolticas de LM


fotopolimerizadas usando LPO como iniciador.....

x91

II.4.2. Comparacin de iniciadores para la preparacin


de columnas monolticas de metacrilato para CEC.....

x91

XIV

Captulo III Materiales y mtodos..................................................

x93

III.1. Surfactantes.

x95

III.1.1. APGs...

x95

III.1.1.1. Estndares, materias primas y otras muestras...

x95

III.1.1.2. Reactivos de uso general.

x95

III.1.1.3. Instrumentacin y condiciones de trabajo..

x95

III.1.1.4. Preparacin de estndares y muestras...

x96

III.1.2. Alcoholes....

x97

III.1.2.1. Estndares, materias primas y otras muestras...

x97

III.1.2.2. Reactivos de uso general.

x98

III.1.2.3. Instrumentacin y condiciones de trabajo..

x98

III.1.2.4. Preparacin de estndares y muestras...

x99

III.1.3. AESs...

101

III.1.3.1. Estndares, materias primas y otras muestras...

101

III.1.3.2. Reactivos de uso general.....

101

III.1.3.3. Instrumentacin y condiciones de trabajo..

102

III.1.3.4. Preparacin de estndares y muestras...

103

III.2. Polmeros sintticos

106

III.2.1. PVP-NO..

106

III.2.1.1. Estndares, materias primas y otras muestras...

106

III.2.1.2. Reactivos de uso general.

107

III.2.1.3. Instrumentacin y condiciones de trabajo..

107

III.2.1.4. Preparacin de estndares y muestras...

108

III.2.2. PVP.

108

III.2.2.1. Estndares, materias primas y otras muestras...

108

XV

III.2.2.2. Reactivos de uso general.

108

III.2.2.3. Instrumentacin y condiciones de trabajo..

109

III.2.2.4. Preparacin de estndares y muestras...

110

III.2.3. PVA....

111

III.2.3.1. Estndares, materias primas y otras muestras...

111

III.2.3.2. Reactivos de uso general.

111

III.2.3.3. Instrumentacin y condiciones de trabajo..

111

III.2.3.4. Preparacin de estndares y muestras...

114

III.3. Enzimas...

114

III.3.1. CZE.

114

III.3.1.1. Estndares, materias primas y otras muestras...

114

III.3.1.2. Reactivos de uso general.

116

III.3.1.3. Instrumentacin y condiciones de trabajo..

116

III.3.1.4. Preparacin de estndares y muestras...

117

III.3.2. Aminocidos por infusin directa en MS..

118

III.3.2.1. Estndares, materias primas y otras muestras...

118

III.3.2.2. Reactivos de uso general....

118

III.3.2.3. Instrumentacin y condiciones de trabajo..

119

III.3.2.4. Preparacin de estndares y muestras...

119

III.3.2.5. Construccin de las matrices de entrenamiento


y evaluacin......

121

III.3.2.6. Seleccin de variables predictoras para la


construccin de un modelo de LDA.....

122

III.3.3. Aminocidos derivatizados con OPA-NAC...

123

III.3.3.1. Estndares, materias primas y otras muestras...

123

III.3.3.2. Reactivos de uso general....

124

XVI

III.3.3.3. Instrumentacin y condiciones de trabajo..

124

III.3.3.4. Preparacin de estndares y muestras...

125

III.3.4. Digestos con tripsina..

126

III.3.4.1. Estndares, materias primas y otras muestras...

126

III.3.4.2. Reactivos de uso general....

126

III.3.4.3. Instrumentacin y condiciones de trabajo..

126

III.3.4.4. Preparacin de estndares y muestras...

127

III.4. Columnas monolticas.....

128

III.4.1. Monmeros, agentes entrelazantes, iniciadores,


estndares y reactivos de uso general....

128

III.4.2. Tratamiento de columnas...

129

III.4.2.1. Acondicionado de columnas...

129

III.4.2.2. Preparacin de columnas monolticas...

130

III.4.3. Instrumentacin y condiciones de trabajo..

132

Results..............

135

Chapter IV No ionic surfactants in cleaning products..

137

IV.1. APGs...

139

IV.1.1. APGs by HPLC-MS...

139

IV.1.1.1. Optimisation of working conditions using


continuous infusion MS....

141

IV.1.1.2. Separation of APGs by HPLCMS


in isocratic conditions......

141

IV.1.1.3. Separation of APGs by HPLCMS


with gradient elution.....

144

IV.1.1.4. Quantitation studies....

146

XVII

IV.1.2. Fragmentation of D-Glucose and AMGs...

150

IV.1.2.1. Optimization of the IT-MS detection conditions

152

IV.1.2.2. Fragmentation of D-Glucose and their isotopic


forms using MSn...

153

IV.1.2.3. Fragmentation of AMGp using MSn......

157

IV.1.2.4. Fragmentation of AMGf using MSn.......

159

IV.2. Non-ethoxylated and ethoxylated alcohols....

162

IV.2.1. Chromium(VI) oxide oxidation of non-ethoxylated


and ethoxylated alcohols for determination by ESI-MS.

162

IV.2.1.1. Optimization of the infusion medium..

164

IV.2.1.2. Relative sensitivity of carboxylic and


ethoxy-carboxylic acids as a function of n and m....

166

IV.2.1.3. Oxidation yields of primary non-ethoxylated alcohols


and extraction recoveries of the corresponding
carboxylic acids....

168

IV.2.1.4. Influence of water concentration


on the oxidation yield...

169

IV.2.1.5. Oxidation yields of ethoxylated alcohols....

171

IV.2.1.6. Application to industrial and


environmental samples..

173

Chapter V Anionic surfactants....

179

V.1. AESs.....

181

V.1.1. Determination of FAEs and AESs by SAX separation,


derivatization with a cyclic anhydride and HPLC...

181

V.1.1.1. Derivatization and HPLC separation


of the derivatives...

184

V.1.1.2. Optimization of the SAX separation of


the surfactant classes....

186

XVIII

V.1.1.3. Calibration studies...

190

V.1.1.4. Application to industrial raw materials


and commercial products.

193

V.1.1.5. Seawater analysis.....

194

Chapter VI Synthetic polymers...

197

IV.1. PVP-NO......

199

VI.1.1. Characterization of PVP-NO by FSCE and MEKC...

199

VI.1.1.1. FSCE studies in the absence and presence of PEA.

202

VI.1.1.2. Discussion on the results obtained by FSCE..

206

VI.1.1.3. MEKC studies in the absence and


presence of PEA...

208

VI.1.1.4. Discussion on the results obtained by MEKC.

210

VI.1.1.5. Quantitation studies and application to


real samples using FSCE..

215

VI.2. PVP.....

217

VI.2.1. Characterization and determination of PVP by


complexation with an anionic azo-dye and NECEEM..

217

VI.2.1.1. Electropherograms of PVP in


the presence of anionic azo-dyes......

219

VI.2.1.2. Formation rate of the PVPCR complexes.

222

VI.2.1.3. Application of NECEEM principles


to the interpretation of the electropherograms
of PVPdye mixtures..........................................

223

VI.2.1.4. Influence of MW on the location and shape


of the peak of the PVPdye complexes...

226

VI.2.1.5. Determination of the maximal stoichiometry


of the PVPdye complexes......

228

XIX

VI.2.1.6. Influence of MW on the mobility of


the PVPCR complexes at low q values.....

231

VI.2.1.7. Determination of PVPdye stability constants.......

233

VI.2.1.8. Analytical applications....

235

VI.3. PVA.....

239

VI.3.1. Evaluation of MW and tacticity of PVA by


NECEEM of a polymer and a dye...

239

VI.3.1.1. Effect of PVA on the UVVis absorption


spectrum of CR.....

241

VI.3.1.2. Selection of working conditions......

242

VI.3.1.3. Maximal stoichiometry of the PVACR complex


and its relationship with log MW and tacticity....

244

VI.3.1.4. Structure of the complex.....

247

VI.3.1.5. Influence of Mw and tacticity on


the electrophoretic mobility of the complex.

250

VI.3.1.6. Stability and dissociation rate


constants of the complex.....

254

Chapter VII Enzymes...

259

VII.1. Intact enzymes by CE...

261

VII.1.1. Identification of enzymes for the detergent


industry by CZE of the intact proteins...

261

VII.1.1.1. CZE with a basic BGE...

263

VII.1.1.2. CZE with an acid BGE..

264

VII.1.1.3. Relative sensitivities and application to


enzyme identification.

266

VII.1.1.4. Influence of surfactants commonly used in the


formulation of cleaning products...

267

XX

VII.2. Classification of enzymes by MS..........................

269

VII.2.1. Rapid classification of enzymes in


cleaning products by hydrolysis, MS and LDA.

269

VII.2.1.1. Mass spectra and normalization of the variables..

271

VII.2.1.2. Construction of LDA models..

272

VII.2.1.3. Evaluation of the prediction capability of


the optimal LDA model..

276

VII.3. Classification of enzymes by HPLC-UV-Vis....

276

VII.3.1. Enzyme class identification in cleaning products by


hydrolysis followed by derivatization with
o-phthaldialdehyde,HPLC and LDA.....

276

VII.3.1.1. Optimization of OPANAC derivatization and


chromatographic conditions..

278

VII.3.1.2. Data matrices and construction and


evaluation of the LDA models.

280

VII.4. Tryptic digests of enzymes, columns comparation...

284

VII.4.1. Comparison of microparticulate and monolithic


columns for RP-LC of tryptic digests of
industrial enzymes in cleaning products....

284

VII.4.1.1. Study of intact enzymes and tryptic digests using


a ProSwift polymeric monolithic column....

287

VII.4.1.2. Study of tryptic digests of enzymes using


microparticulate and silica monolithic columns....

290

VII.4.1.3. Influence of matrixes commonly used


in Cleaning product formulations...

295

Chapter VIII Monolithic columns...

297

VIII.1. Monolithic columns of LM.....

299

VIII.1.1. Photo-polymerized LMA monolithic columns


for CEC using LPO as initiator...

299

XXI

VIII.1.1.1. Preparation and characterization of


columns photo-initiated with LPO....

301

VIII.1.1.2. Comparison of LPO and AIBN as


initiators for photo-polymerization..

311

VIII.2. Comparison of photo-initiators for


methacrylate monolithic columns.

316

VIII.2.1. Comparison on photo-initiators for the preparation


of methacrylate monolithic columns for CEC.....

316

VIII.2.1.1. Preparation and characterization of LM


columns photo-initiated with AIBN or DMPA..

317

VIII.2.1.2. Preparation and characterization of LMA


columns photo-initiated with BPO or LPO...

324

Captulo IX Conclusiones generales...

329

IX.1. Surfactantes.

331

IX.1.1. APGs..

331

IX.1.1.1. Separacin y determinacin de APGs


por LC con deteccin ESI-MS....

331

IX.1.1.2. Estudio de fragmentacin de la D-Glucosa


y los AMG en presencia de iones de
sodio en IT-MS....

333

IX.1.2. Alcoholes....

335

IX.1.2.1. Oxidacin de alcoholes no etoxilados y


etoxilado con cromo(VI) y posterior
determinacin por ESI-MS.....

335

IX.1.3. AES....

341

IX.1.3.1. Determinacin de FAEs y AESs por separacin


mediante SAX, derivatizacin con un
anhdrido cclico y LC..

341

XXII

IX.2. Polmeros sintticos....

344

IX.2.1. PVP-NO.

344

IX.2.1.1. Caracterizacin de PVP-NO por CE y MECK...

344

IX.2.2. PVP.

347

IX.2.2.1. Caracterizacin y determinacin de PVP


por complejacin con un azo-colorante
aninico seguida de NEECEM....

347

IX.2.3. PVA....

350

IX.2.3.1. Evaluacin de la MW y tacticidad del PVA


mediante NEECEM de mezclas
polmero-colorante...

350

IX.3. Enzimas..

354

IX.3.1. CZE....

354

IX.3.1.1. Identificacin de enzimas de la industria de la


detergencia por CZE de enzimas intactas....

354

IX.3.2. Aminocidos por infusin directa en MS..

356

IX.3.2.1. Clasificacin rpida de enzimas en productos


de limpieza por hidlisis aminocidos, MS y LDA.

356

IX.3.3. Aminocidos derivatizados con OPA-NAC..

357

IX.3.3.1. Identificacin de la clase de las enzimas


presentes en los productos de limpieza
mediante hidrlisis seguida de
derivatizacin con OPA-NAC, HPLC y LDA.

357

IX.3.4. Digestos con tripsina..

359

IX.3.4.1. Comparacin de columnas microparticuladas


y monolticas para RP-LC de digestos trpticos de
enzimas industriales en productos de limpieza...

359

XXIII

IX.4. Columnas monolticas.

362

IX.4.1. Columnas monolticas de LMA para CEC....

362

IX.4.1.1. Columnas monolticas de LMA para CEC


utilizando LPO como iniciador.......

362

IX.4.1.2. Comparacin de fotoiniciadores para


la preparacin de columnas monolticas
de metacrilato para CE.......................................

364

Captulo X Referencias.

367

Abreviaturas.

399

Anexo I
Separation and determination of alkylglycosides by liquid
cromatography with electrospray mass spectrometric detection.

xA3

Anexo II
Study of the fragmentation of D-Glucose and
Alkylmonoglycosides in the presence of sodium
ions in an Ion-Trap Mass Spectrometer.......................................

A13

Anexo III
Chromium(VI) oxide oxidation of non-ethoxilated and
ethoxilated alcohols for determination by electrospray
ionization mass spectrometry..

A31

Anexo IV
Characterization of poly(4-vinylpyridine-1-oxide) by
free-solution capillary electrophoresis and
micellar electrokinetic chromatography..................................

XXIV

A41

Anexo V
Characterization and determination of poly(vinylpyrrolidone)
by complexation with an anionic azo-dye and
nonequilibrium capillary electrophoresis....

A51

Anexo VI
Evaluation of molecular mass and tacticity of polyvinyl alcohol
by non-equilibrium capillary electrophoresis of
polymer-dye equilibrium mixtures..

A61

Anexo VII
Rapid classification of enzymes in cleaning products by hydrolysis,
mass spectrometry and linear discriminant analysis.... A71

Anexo VIII
Enzyme class identification in cleaning products by hydrolysis
followed by derivatization with o-phthaldialdehyde,
HPLC and linear discriminant analysis...

A79

Anexo IX
Photo-polymerized lauryl methacrylate monolithic columns
for CEC using lauroyl peroxide as initiator....

A87

Anexo X
Comparison on photo-initiators for the preparation
of methacrylate monolithic columns
for capilary electrochromatography.....................................

XXV

A99

CAPTULO I

INTRODUCCIN

Captulo I. Introduccin

I.1. Detergentes

I.1.1. Introduccin
El jabn es conocido desde las culturas antiguas (egipcios y sumerios),
siendo usado tanto para lavar la ropa como para el aseo personal. Los jabones se
obtenan mediante saponificacin de grasas vegetales o animales con cenizas
vegetales o minerales como la sosa custica. Hasta el siglo XV, uno de los
principales ncleos de vida social en las ciudades europeas eran los baos
pblicos. En los siglos posteriores, los baos fueron considerados inmorales y el
jabn pas a ser algo a evitar. Se vesta la misma ropa durante semanas y los
malos olores se tapaban con perfumes. En Europa, el jabn no volvi a apreciarse
hasta bien entrado el siglo XVIII, cuando los mdicos se dieron cuenta de la
importancia de la higiene para la salud. Por otro lado, la industrializacin y las
importaciones de grasas baratas facilitaron la fabricacin de jabones a gran escala
[Ramrez-Corrales, 2006].

Los detergentes (en latn, detergere significa limpiar) son mezclas de


diferentes sustancias cuya finalidad es disolver la suciedad o las impurezas de un
objeto sin corroerlo. En 1907, una compaa alemana fabric el primer
detergente al aadir sales sdicas de perborato, silicato y carbonato al jabn
tradicional

[www.ambientum.com].

A partir de 1930 se empezaron a sintetizar

detergentes derivados de la industria del petrleo. Posteriormente se descubrieron


otros ingredientes que daban al conjunto una mayor capacidad limpiadora.

I.1.2. Componentes en las formulaciones de productos de limpieza


Para lograr su papel limpiador, un detergente debe producir numerosos
fenmenos, los cuales dependen del tipo de sustrato y de las condiciones de
lavado. As, se han diseado frmulas especficas capaces de actuar con

Miriam Beneito Cambra

eficiencia en casos particulares, y frmulas generales con resultados ms o


menos satisfactorios en la mayora de los casos. En la composicin qumica de
productos de limpieza, adems de los surfactantes, que constituyen la principal
materia activa de la formulacin, se encuentran aditivos de muy distintos tipos.
La composicin difiere en funcin del uso industrial o domstico del producto.
En las formulaciones existe un gran nmero de componentes cuyos papeles se
complementan uno a otro, a menudo con un efecto de sinergia. A continuacin se
comentan los diferentes tipos de componentes que se encuentran en
formulaciones de detergentes, as como el papel que stos desempean.

A) Surfactantes
Los surfactantes actan como agentes humectantes del sustrato, modifican
la tensin superficial del agua y emulsionan las partculas de suciedad. En
algunas aplicaciones se usan las propiedades bactericidas de ciertos surfactantes
en formulaciones desinfectantes [Showell, 2006].

B) Agentes secuestrantes (builders)


Estos agentes tienen como propsito mejorar la accin limpiadora del
surfactante mediante varios efectos. Su principal accin es secuestrar los cationes
divalentes del agua dura (calcio, magnesio) para evitar su interaccin con los
surfactantes. La eliminacin se hace en forma soluble (quelato), por
precipitacin, o por intercambio inico. Otra de las acciones de los builders es
mantener el pH de la disolucin del detergente a un valor alcalino, neutralizar los
cidos grasos libres y formar jabones in-situ en la interfase. Tambin aumentan el
potencial (negativo) de superficie de los textiles y de las manchas, y por tanto,
inhiben la redeposicin de la suciedad. Dentro de los agentes secuestrantes se
encuentran los compuestos inorgnicos solubles, como el tri(poli)fosfato

Captulo I. Introduccin

(Na5P3O10) y el pirofosfato (Na4P2O7) sdicos, as como los silicatos (xNa2O


ySiO2) y el carbonato sdico (Na2CO3). Por su parte, los compuestos inorgnicos
insolubles, como las zeolitas (alumino-silicatos naturales o sintticos), como el
aluminosilicato sdico (Na2O Al3O3 2SiO2 xH2O) eliminan los cationes
divalentes por intercambio inico [Showell, 2006].

C) Jabones como agentes dispersantes de calcio


Muchas formulaciones detergentes lquidas y en polvo para lavadoras
contienen sales alcalinas de cidos grasos, es decir jabones, cuyo papel es reducir
la formacin de espuma. Los jabones tienen excelentes propiedades limpiadoras,
son seguros y fcilmente biodegradables; sin embargo, son muy sensibles a los
cationes divalentes, especialmente al calcio, con el cual producen sales insolubles
en agua. Los agentes dispersantes de jabones de calcio son surfactantes aninicos
o anfteros que se co-micelizan con los jabones para formar disoluciones que no
precipitan en aguas duras [Salager, 1988].

D) Agentes antirredeposicin
Estas sustancias impiden que las suciedades separadas de los tejidos
durante el lavado vuelvan a depositarse sobre los mismos. Los agentes
antirredeposicin ms utilizados son la carboximetilcelulosa, y otros derivados
no inicos de la celulosa. Comercialmente tambin se utilizan polmeros
sintticos como PVP, PVA y algunos copolmeros de stos [Salager, 1988].

E) Agentes espumantes y no espumantes


Contrariamente a la creencia general, la produccin de espuma no tiene
nada que ver con el poder detergente. En ciertos usos, como champs o
detergentes para las manos, la generacin de grandes volmenes de espuma es un

Miriam Beneito Cambra

factor deseable. Son muchos los surfactantes capaces de generar y mantener la


espuma en ausencia de suciedad, sin embargo, sta fcilmente desaparece en
presencia de suciedad, especialmente en presencia de grasa. Para mantener la
espuma a lo largo del uso del detergente se aaden agentes espumantes como el
lauril sulfato sdico, y surfactantes no inicos nitrogenados
Showell, 2006].

[Salager, 1988;

Sin embargo, en otras aplicaciones, la generacin de espuma es un

inconveniente. Por ejemplo, en los lavavajillas, un elevado volumen de espuma


puede interferir en la rotacin del brazo, provocando una degradacin del
rendimiento. En estos casos se utilizan agentes no espumantes y antiespumantes,
que evitan la formacin o aceleran la desaparicin de la espuma. Entre los
agentes utilizados para el control de la espuma suelen emplearse surfactantes
etoxilados no inicos y jabones, y antiespumantes compuestos por partculas de
slice coloidal en suspensin, junto con aceites de silicona (polidimetil siloxano)
[Salager, 1988; Showell, 2006].

F) Agentes blanqueadores
La obtencin de blancura en textiles es quizs una de las propiedades ms
importantes para el consumidor. En el mercado existen dos tipos de agentes
blanqueadores para textiles, ambos con propiedades oxidantes: los hipohaluros,
como el hipoclorito de sodio, y las sales inorgnicas peroxigenadas, como el
perborato de sodio y el perxido de sodio estabilizado con carbonato sdico
(percarbonato). Los agentes blanqueadores oxidantes deben ser intrnsecamente
inestables para cumplir con su funcin (oxidar), paradjicamente, al mismo
tiempo deben ser estables cuando estn almacenados, solos o en la mezcla
detergente. El hipoclorito es un agente blanqueador ms activo y agresivo que el
perborato, siendo particularmente eficaz en la oxidacin de sustancias que
contienen nitrgeno. Adems de poseer una accin blanqueadora (an a baja

Captulo I. Introduccin

temperatura), es un efectivo bactericida. Por su accin sobre las sustancias


nitrogenadas no puede conservarse en forma de polvo o de lquido en
formulaciones detergentes que contengan sales de amonio, aminas, amidas, etc.
Por ello se suele emplear como un ingrediente aparte. Respecto a las sales
inorgnicas preoxigenadas, se usa el perborato de sodio tetrahidrato
(NaBO34H2O),

el

percarbonato

carbonato

de

sodio

peroxihidrato

(2Na2CO33H2O2), y el peroximonosulfato de potasio (KHSO5). El perborato y


otras sales semejantes exhiben una accin blanqueadora menos agresiva que el
hipoclorito, y son slidos compatibles con la gran mayora de los componentes
de los detergentes en polvo [Salager, 1988; Showell, 2006].

G) Blanqueantes pticos
El grado de blancura obtenido mediante agentes blanqueadores se puede
exaltar mediane el blanqueado ptico. Los blanqueadores pticos son colorantes
orgnicos poliaromticos que absorben luz ultravioleta y emiten una luz azulada
en el visible mediante fluorescencia. A la luz del sol, aaden un tono azulado que
compensa el tono amarillento de los tejidos, por lo que mejora la blancura o
profundidad de los colores. Segn la funcin a la que se destinen, poseen
grupos polares ms o menos importantes para adsorberse sobre fibras hidroflicas
como el algodn, o hidrfobas como el polister. Se han desarrollado agentes
fluorescentes solubles en agua y resistentes al hipoclorito u otros blanqueadores,
gracias a que los tomos de nitrgeno de dichas sustancias estn situados en
estructuras altamente aromticas de tipo benzotriazol o benzofurano
1988].

[Salager,

Miriam Beneito Cambra

H) Suavizantes
Despus de un lavado con detergentes sintticos formulados con agentes
secuestrantes, el textil seco presenta una superficie que no es agradable al
contacto con la piel. El residuo de los surfactantes sintticos adsorbidos aumenta
la carga esttica de las fibras, y la ausencia de sustancias con accin lubricante
vuelve al textil relativamente rgido. Los agentes suavizantes contrarrestan
ambos fenmenos, ya que por una parte reducen la carga esttica, y por otra
depositan una capa lubricante. Muchos surfactantes catinicos producen estos
efectos, pero son incompatibles con los surfactantes aninicos utilizados en la
mayora de las formulaciones, por lo que deben usarse por separado. Los mejores
suavizantes de este tipo son las sales de alquil amonio cuaternario y de
imidazolio. Existe una tendencia hacia la produccin de formulaciones que
contengan agentes suavizantes compatibles con los agentes limpiadores. Estos
suavizantes son surfactantes con cierto carcter catinico que se absorben sobre
las fibras textiles, siendo a la vez compatibles con surfactantes aninicos
comnmente empleados. Para este fin se usan surfactantes no inicos con un
grupo nitrogenado, o tambin algunos surfactantes anfteros, normalmente
conteniendo un grupo amido-amina (que tambin actan como dispersantes de
jabones de calcio) [Salager, 1988; Showell, 2006].

I) Enzimas
Se utilizan uso en las formulaciones de detergentes para eliminar las
manchas, gracias a la rotura cataltica de componentes especficos de las mismas.
Las proteasas, encargadas de la degradacin de las protenas, son las ms
comunes en la mayora de los detergentes. Sin embargo, tambin se emplean
otras enzimas como las amilasas (degradan el almidn), lipasas (degradan
lpidos) y celulasas (degradan la celulosa). Estas enzimas son capaces de

Captulo I. Introduccin

degradar rpidamente manchas en un medio de pH alcalino y a temperaturas de


hasta 60C. Estn diseadas para ser activas a temperatura ambiente, siendo
particularmente utilizadas en detergentes lquidos [Salager, 1988; Showell, 2006].

J) Polmeros
El uso de polmeros ha ido aumentando a medida que las formulaciones de
detergentes han evolucionado. Los polmeros han venido a sustituir a los fosfatos
que fueron cayendo en desuso a causa de su elevado impacto ambiental. Se han
usado principalmente polmeros de tipo poliacrilato, con el fin de remplazar a
agentes secuestrantes como el tripolifosfato de sodio. La carboximetilcelulosa
fue tambin uno de los primeros polmeros utilizados para este fin. Los xitos
obtenidos con los polmeros para otros propsitos ha impulsado la aparicin de
un gran nmero de stos

[Zini, 1999].

Entre otros muchos usos, se han

comercializado nuevos dispersantes y polmeros que inhiben la transferencia de


color entre los tejidos. Por cada polmero comercializado, existen otros muchos
patentados, lo que pone de manifiesto el inters dentro de este rea [Showell, 2006;
Watson, 2006].

K) Espesantes
A menudo es conveniente modificar la reologa, es decir, la consistencia
de la formulacin del detergente en condiciones dinmicas, lo que tiene
importantes consecuencias prcticas. As por ejemplo, los lavavajillas contienen
espesantes que ayudan a mantener en suspensin el fosfato y otras sustancias que
de otra forma quedaran separadas de la fase lquida. El espesado puede
conseguirse a partir de electrolitos inorgnicos, como por ejemplo, NaCl, arcillas
como la laponita o hectorita, o polmeros de un elevado peso molecular como la
carboximetilcelulosa y las gomas de guar o xantana. Existen polmeros

Miriam Beneito Cambra

particularmente efectivos como espesantes en las formulaciones de detergentes


para uso domstico. Por ejemplo, los modificadores reolgicos de la serie
Carbopol (Lubrizol) han demostrado ser excepcionales viscosantes, agentes de
suspensin y estabilizadores en una gran variedad de productos de cuidado
personal. La mayora de estos polmeros son homo- y copolmeros de cido
acrlico de alta masa molecular, entrecruzados con un polialquenil politer
[Showell, 2006].

L) Perfumes
En un sentido tcnico los perfumes no aaden un mayor poder detergente
a los formulados, sin embargo, desde el punto de vista del consumidor
representan un factor importante, ya que segn el aroma parece que el detergente
funcione mejor. El olor es una caracterstica que debe ser cuidadosamente tratada
en la formulacin de los detergentes para la aceptacin del producto por parte de
los consumidores. Los perfumes son complejas mezclas de compuestos
orgnicos, por ejemplo, el perfume de un detergente puede estar compuesto por
30, 50 o incluso ms de 100 compuestos. Esta compleja naturaleza puede dar
lugar a complejas interacciones con el resto de componentes del detergente, que
pueden afectar tanto al perfume como a las sustancias activas. En detergentes de
uso domstico, particularmente para lavavajillas y desinfectantes, se incorporan
perfumes, la mayora de los cuales son terpenos, cuyo esqueleto est compuesto
de 2, 3 ms unidades del isopreno (2-metil-butadieno)
2006].

10

[Salager, 1988; Watson,

Captulo I. Introduccin

M) Disolventes
La seleccin de los disolventes empleados en las formulaciones de
detergentes depende de la naturaleza de las sustancias activas presentes en los
formulados, de la aplicacin final del detergente y del factor econmico. El agua
es el disolvente ms ampliamente empleado en muchas de las formulaciones de
detergentes para uso domstico e industrial. Sin embargo, muchas de las
sustancias activas comnmente empleadas en formulacin de detergentes
presentan una limitada solubilidad en agua, por lo que se requiere la adicin de
un co-solvente (como el etanol) o de un hidrtopo (como el cumeno sulfonato)
[Showell, 2006].

N) Hidrtopos
En los detergentes lquidos, en ocasiones es necesario incluir hidrtopos
para garantizar la estabilidad del detergente en un amplio intervalo de
temperaturas. Los hidrtropos son sustancias hidroflicas con un grupo apolar,
cuya funcin es aumentar la solubilidad de los surfactantes en formulaciones
lquidas. Los hidrtropos no tienen propiedades surfactantes por ellos mismos,
pero actan como co-solubilizadores a alta concentracin. Los ms utilizados son
los sulfonatos de tolueno, etilbenceno y xileno [Salager, 1988; Showell, 2006].

O) Bactericidas
Ciertas frmulas desinfectantes contienen bactericidas, los cuales pueden
ser surfactantes anfteros que actan tambin como agentes dispersantes de
jabones de calcio. Tambin se usan compuestos catinicos que presentan un
efecto suavizante por sus propiedades antiestticas. Los desinfectantes pueden
tambin contener productos clorados bactericidas y sustancias con propiedades
desodorantes

[Salager, 1988].

En los detergentes lquidos, especialmente en los

11

Miriam Beneito Cambra

ms diluidos, donde el agua constituye una gran parte del volumen total del
producto, es normal que la formulacin incluya un agente antibacteriano
(normalmente un bacteriosttico) que alargue el tiempo de vida del producto
[Watson, 2006].

P) Agentes anticorrosin
En las formulaciones de detergentes tambin se pueden encontrar agentes
anticorrosin, con el objetivo de proteger las partes metlicas de las mquinas o
sistemas de lavado. Generalmente, se usa el silicato de sodio que adems posee
un papel secundario como builder [Salager, 1988].

I.2. Surfactantes

I.2.1. Introduccin
La palabra surfactante deriva de la contraccin de los trminos surfaceactive-agent (superficie-activo-agente) y engloba a una serie de especies
qumicas capaces de modificar las propiedades de las interfases de los lquidos
(acuoso o no acuoso) en los que estn presentes. Las propiedades caractersticas
de estas molculas residen en su carcter anfiflico, esto es, de la presencia de
una parte hidroflica y otra hidrofbica en la molcula. Los surfactantes se
concentran en la superficie libre del lquido y en las interfases entre fases
inmiscibles, disminuyendo la tensin superficial. El cambio en la tensin
superficial afecta a la capacidad para formar emulsiones, a la mojabilidad, al
poder dispersante, a la detergencia y a las propiedades solubilizantes.
Los surfactantes poseen una estructura molecular tpica, esencialmente
lineal y asimtrica, con dos zonas, una hidrofbica y la otra hidroflica. La parte
hidrfobica es una cadena aliftica, lineal o ramificada, conteniendo en general

12

Captulo I. Introduccin

entre 10 y 18 tomos de carbono. En los productos naturales y en los de


transformacin qumica predominan las cadenas no ramificadas, mientras que en
los derivados del petrleo y los obtenidos por sntesis (usualmente a partir del
carbn) existen multitud de cadenas ramificadas. Por su parte, el resto hidrfilo,
determinante de la solubilidad en agua, puede ser un grupo polar de carcter
cido tal como un grupo sulfato, sulfonato o carboxilato, o de carcter bsico
como una amina, una sal de amonio cuaternario o el ion piridinio, aunque
tambin puede ser un grupo polar no inico. Los surfactantes, ya sean naturales o
sintticos, organizan el medio formando micelas y otras microestructuras,
cambian la solubilidad de compuestos hidrofbicos y modifican el entorno de los
constituyentes del medio. Sobre las propiedades surfactantes de un compuesto
influyen, adems de la propia naturaleza del grupo hidrfilo, la situacin que ste
ocupa en la molcula. En principio se puede distinguir:
Posicin terminal: la estructura molecular es polar y totalmente asimtrica. El
grupo hidrfilo puede estar unido directamente al hidrfobo, o entre ambos
puede existir un resto de carcter aliftico o aromtico que posea cierto carcter
hidrfilo. Si la cadena hidrfoba tiene una longitud adecuada, estas estructuras
muestran bsicamente carcter detergente.
Posicin central: el grupo hidrfilo se intercala en cualquier punto de la cadena
hidrfoba, aunque si en ella existen puntos reactivos (enlaces dobles, grupos
hidroxilo, etc.) tiende a ocupar esos lugares. Conforme el grupo hidrfilo est
ms centrado en la cadena, ms mermada se encuentra la capacidad detergente
del compuesto.
Varios grupos hidrfilos: en una cadena pueden estar presentes varios grupos
hidrfilos, lo que exalta notablemente la solubilidad del surfactante en agua. Sin
embargo, esta estructura proporciona propiedades dispersantes al surfactante.

13

Miriam Beneito Cambra

I.2.2. Clasificacin de los surfactantes


Atendiendo a su carga, los surfactantes pueden dividirse en cuatro clases:
aninicos, catinicos, no inicos y anfotricos

[Oldenhove de Guertechin, 1999].

Aunque todos los surfactantes en s mismos presentan sus particularidades, hay


algunas caractersticas comunes a cada clase.

A) Aninicos
Se caracterizan por tener un grupo hidrfilo cargado negativamente, es
decir, el grupo hidrfilo tiene carcter cido y forma fcilmente un anin. Los
ms antiguos y conocidos son los jabones. Los surfactantes aninicos son los ms
utilizados y han sido considerados como el caballo de batalla en el mundo de
los detergentes. En consecuencia, su produccin es elevada, siendo adems muy
econmicos. Suelen distinguirse las siguientes familias: ABS, AS, AES, APES,
AOS, alquil sulfonatos, -sulfonatos de cidos grasos (inicos y steres de
alquilo), mono- y di-alquil sulfosuccinatos y sulfonatos derivados del petrleo
(Fig. I.1).
Los surfactantes aninicos son especialmente beneficiosos en cuanto a su
accin limpiadora, que se apoya en el hecho de que muchas superficies se
encuentran cargadas negativamente, por lo que no pueden quedarse adsorbidos
sobre las mismas, impidiendo as la redeposicin de sustancias indeseables. En
funcin de la naturaleza del grupo funcional que presenta la carga, muestran una
resistencia variable hacia la hidrlisis. As por ejemplo, los sulfatos se hidrolizan
con facilidad, mientras que los sulfonatos son muy estables. Algunos surfactantes
aninicos, presentan la propiedad de generar fases acuosas viscosas, pudiendo ser
empleados como espesantes. Una limitacin de los surfactantes aninicos es su
tendencia a precipitar en presencia de iones calcio y magnesio, que abundan en
las aguas duras, si bien, los AESs son mucho menos sensibles a los cationes

14

Captulo I. Introduccin

alcalinos que los ASs. Por otro lado, la baja solubilidad y las particulares
propiedades de las interfaces de los precipitados de sulfato de magnesio son
explotadas de forma positiva a la hora de optimizar el rendimiento de los
detergentes.
CH3(CH2)m

CH3(CH2)nCOO- Na+
n + 1 = 10 18

CH3(CH2)n

Jabones

n + m = 7 11
ABS

CH3(CH2)nOSO3 Na+
n + 1 = 12 18

CH3(CH2)n(OCH2CH2)mOSO3 Na+

AS

SO3 Na+

CH

AES
CH3(CH2)nCH

(OCH2CH2)mOSO3 Na+

CH(CH2)mSO3 Na+

m + n = 9 15
m = 1 8 R: cadena alqulica

AOS

APES
ROOCCH
-

CH3(CH2)nSO3 Na+

ROOCCH SO3 Na+

Alquil sulfonatos

Alquil sulfosuccinatos

Fig. I.1. Estructura y nomenclatura de las principales familias de


surfactantes aninicos.

B) Catinicos
Los surfactantes catinicos tienen el grupo hidrfilo de carcter bsico.
Suelen agruparse en derivados grasos de amida, amidoaminas, imidazolinas,
derivados del petrleo, nitrilos cclicos alifticos, aromticos, compuestos no
nitrogenados, polimricos catinicos y xidos de amina. En la Fig. I.2 se muestra
la estructura y nomenclatura de las principales familias de surfactantes
catinicos.

15

Miriam Beneito Cambra


CH3

CH3
CH2NH+

NH+

(CH2)nCH3

(CH2)nCH3

CH3

CH3
n + 1 = 7 18

n + 1 = 7 18

Sales de alquilbencildimetilamonio

Sales de alquilfenildimetilamonio

CH3
N+

(CH2)nCH3

R1

N+
CH3

n + 1 = 12 14

CH3
CH3

R1

N+

CH3

R2

R1, R2: cadenas alqulicas

Sales de alquil piridinio

Sales de alquil trimetil y dialquildimetil amonio

Fig. I.2. Estructura y nomenclatura de las principales familias de


surfactantes catinicos.

Los surfactantes catinicos de importancia industrial son compuestos


grasos nitrogenados y, especialmente, compuestos con nitrgeno cuaternario
[Wittcoff, 1987].

Son de poca utilidad en procesos de limpieza, ya que al presentar

la mayora de las superficies carga negativa, los cationes se retienen sobre ellas
en lugar de solubilizar la suciedad adherida. Sin embargo, y debido a estas
mismas propiedades, poseen numerosas aplicaciones especializadas. Por
ejemplo, las aminas y los compuestos cuaternarios inhiben el crecimiento de
microorganismos como bacterias y algas. Adems las aminas grasas primarias y
las aminopropilaminas grasas se utilizan como inhibidores de la corrosin, y en
la limpieza de metales, cuando se utiliza HCl para disolver el xido. La amina se
orienta en la interfase entre el metal y la disolucin cida, con las colas
hidrfobas comprimidas entre s, formando una capa protectora de una o dos
molculas de espesor. Esta capa es tan cerrada que evita el ataque del metal
limpio por parte del exceso de cido. Otra aplicacin ms de los compuestos
grasos nitrogenados, y que depende de la actividad de la superficie y orientacin

16

Captulo I. Introduccin

de los iones del surfactante, es el suavizado de textiles. El surfactante catinico


se adsorbe y orienta en la interfase formada entre el textil y el agua. Tambin,
tienen afinidad por la superficie del cabello, utilizndose como acondicionadores
y suavizantes en productos que se aplican despus del lavado, para contrarrestar
as el efecto apelmazante de los surfactantes aninicos.

C) No inicos
En esta clase de surfactantes, el grupo hidrfilo no es capaz de ionizarse y
formar sales. Los surfactantes no inicos son especialmente tiles por su baja
susceptibilidad a los iones Ca2+ y Mg2+ del agua dura. Aprovechando su
compatibilidad con especies inicas, se suelen mezclar con surfactantes
aninicos, dando lugar a asociaciones beneficiosas. Por ejemplo, los surfactantes
no inicos ayudan a solubilizar las sales de calcio o magnesio de los surfactantes
aninicos. El balance entre la parte hidroflica e hidrofbica en estos surfactantes
se obtiene teniendo en consideracin la cantidad y naturaleza de las unidades
polares y la parte hidrofbica de la molcula (longitud de la cadena
hidrocarbonada). La parte hidrfila de la molcula es casi siempre una cadena de
unidades de EO. Los grupos ter le proporcionan la polaridad necesaria para
garantizar su solubilidad en agua por aceptacin de puentes de hidrgeno. Los
FAEs son los surfactantes no inicos ms empleados en productos de limpieza,
cosmticos, herbicidas, etc. Los APEs, principalmente OPEs y NPEs, tambin
poseen una cadena de unidades de EO, pero a diferencia de los FAEs, absorben
en el UV. La aplicacin de los APEs en detergentes est sometida a restricciones
legales, debido a la difcil eliminacin biolgica (escasa biodegradabilidad) de
los metabolitos ms hidrfobos, concretamente, alquifenoles no etoxilados y
monoetoxilados. Los FAEs lineales se biodegradan ms rpidamente que los
APEs. Adems, tienen mejores propiedades de detergencia que los ABS sobre

17

Miriam Beneito Cambra

muchos tipos de suciedad y sobre la mayora de las telas, y son especialmente


eficaces para eliminar la grasa de las fibras sintticas. Tambin actan bien en
fro, por lo que actualmente aparecen en las formulaciones de los detergentes
domsticos como uno de sus principales y ms ferecuentes componentes. Los
derivados de aminas, amidas (FAA) y steres de cidos grasos, son tambin
bastante empleados en productos de aseo corporal. As por ejemplo, la
dietanolamida de coco posee buenas propiedades espumantes, estabilizando la
espuma de los surfactantes aninicos. Finalmente, los surfactantes no inicos a
base de azcares (APGs) tienen una biodegradabilidad sumamente rpida, baja
toxicidad y alta tolerancia desde el punto de vista dermatolgico. Adems,
pueden elaborarse a partir de materias primas naturales. En la Fig. I.3 se muestra
la estructura y nomenclatura de las principales familias de los surfactantes no
inicos.

CH3(CH2)nO(CH2CH2O)mH

FAEs

(CH2CH2O)mH

m = 2 100
APEs
O

(CH2CH2O)nH

RCHN
(CH2CH2O)mH
R: cadena alqulica
FAAs
HOH2C
HO
HO

O
OH

HOH2C
O

O
HO

(CH2)nCH3
OH

n = 8 18 m = 0 3
APGs
Fig. I.3. Estructura y nomenclatura de las principales familias de
surfactantes no inicos.

18

Captulo I. Introduccin

D) Anfotricos
Finalmente las molculas que tienen simultneamente grupos con carcter
cido y bsico son surfactantes anfteros o iones dobles. Son compuestos con
una estructura con una carga positiva y otra negativa simultneamente sobre la
misma molcula. Algunos de estos compuestos tienen grupos cidos o bsicos
dbiles, por lo que pueden comportarse como aninicos o catinicos en funcin
del pH. Usualmente se emplean junto con otros surfactantes (aninicos o
catinicos) para resaltar las propiedades deseadas, como puede ser la detergencia
o la formacin de espuma. Son especialmente utilizados en la formulacin de
productos de aseo personal (gel de bao, champs, etc.) por su suavidad y
compatibilidad con la piel, siendo menos irritantes que los surfactantes catinicos
y aninicos. La formulacin de estos productos es complicada por la posible
precipitacin del surfactante anftero cuando el pH est prximo a su punto
isoelctrico. Pueden utilizarse, junto con NaOH, en limpiadores alcalinos para
superficies grasas, y como limpiadores cidos junto con HCl para superficies
oxidadas, debido a que son estables y funcionales en un amplio intervalo de pH.
Un nmero importante de surfactantes anfteros son compuestos naturales
ampliamente conocidos, como por ejemplo la lecitina. Una familia adicional de
surfactantes anfteros que presentan un grupo amonio cuaternario son las
alquilbetanas (Fig. I.4).
C17H35COOCH2
C17H35COOCH2 O

CH3
CH3
R

CH2OPCH2CH2N+CH3
O-

N+CH2COOCH3

CH3

Lecitina

R: cadena alqulica
Alquil betanas

Fig. I.4. Estructura y nomenclatura de las principales familias de


surfactantes anfotricos.

19

Miriam Beneito Cambra

I.2.3. APGs
Los APGs son surfactantes no inicos obtenidos a partir de materiales
renovables (glucosa y cidos grasos)

[Koch, 1993; Balzer, 1996; Hill, 1997; Rybinski,

1998; Balzer, 2000; Biermann, 1993].

Se obtienen a partir de la alquilacin de

cadenas cortas de glucsidos procedentes de la alcoholisis cida de polisacridos


como el almidn. Para su produccin se han estudiado dos vas, la sntesis directa
o un proceso de transacetalizacin en dos pasos [Varvil, 2009]. Los oligmeros se
distinguen por la longitud de la cadena de alquilo, el nmero de unidades de
glucosa y la isomera

[Oldenhove de Guertechin, 1999].

En la Fig. I.5 se muestran

los efmeros de anillo de los alquilmonoglucsidos.


CH2OH

CH2OH

HOHC

HOHC

5
4

OH

H
3

HO

H
OH

HO

OCnH2n+1
3

H
H

OH

Alquil--D-glucofuransido

OH

OCnH2n+1
H

OCnH2n+1

CH2OH

H
4

OH

Alquil--D-glucofuransido

5
4

H
OH

OCnH2n+1
1

H
H

OH

CH2OH

OH

Alquil--D-glucopiransido

Alquil--D-glucopiransido

Fig. I.5. Estructura de los epmeros e ismeros del anillo de los


alquilmonoglucsidos.

Tres tipos de isomera estn presentes en los APGs: estereoisomera


(epmeros -y -), isomera del anillo (unidades de glucsido, que pueden ser
glucopiransidos o glucofuransidos), y con excepcin de los AMGs, ismeros
de posicin (con predominio de las uniones interglucosdicas 1,4 - y 1,6- [Beneito20

Captulo I. Introduccin
Cambra, 2007]).

Los diferentes oligmeros se pueden abreviar como CnGm,

donde n es el nmero de tomos de carbono en la cadena alqulica, y m el nmero


de unidades de glucosa. Cuando es necesario distinguir entre piransidos y
furansidos, en la presente memoria se emplea una "p" o una "f", al igual que se
aadir - - para distinguir entre los diferentes epmeros, as por ejemplo, C8G1p, hace referencia al oligmero -octilmonoglucopiransido.

A) Propiedades y aplicaciones
Los APGs se sintetizan controlando las condiciones para obtener
principalmente estructuras donde la cadena glucosdica presente un DP
comprendido entre 1,2 y 1,7 unidades, y cadenas alqulicas que se hallen en el
intervalo de 8 a 14 tomos de carbono

[Willing, 2006].

Se obtienen as mezclas

industriales muy complejas que contienen principalmente AMGs, con cantidades


importantes de alquildiglucsidos y menos importantes de otros APGs [Oldenhove
de Guertechin, 1999].

La presencia de numerosos grupos hidroxilo en las

molculas de glucosa asegura la completa solubilidad de la molcula en agua


[Partearroyo, 1991].

Los APGs con 16 o ms tomos de carbono en la cadena

alqulica son menos importantes, ya que son insolubles en agua cuando su DP es


inferior a 2, siendo solubles cuando est por encima de 5 [Willing, 2006].
Adems de la buena solubilidad en agua, presentan puntos de nube
(temperatura a la que los surfactantes se separan en dos fases) a temperaturas
generalmente superiores a 100C; son poco sensibles a la presencia de
electrolitos y raramente se ven influenciados por los cationes presentes en aguas
duras

[Oldenhove de Guertechin, 1999].

Este tipo de surfactantes son buenos

emulsificantes, especialmente para molculas como los aceites naturales y las


grasas. Los APGs proporcionan buenas propiedades de mojado y formacin de
espuma, siendo sus propiedades espumantes superiores a la de los alcoholes

21

Miriam Beneito Cambra

etoxilados

[Oldenhove de Guertechin, 1999].

Por estas propiedades favorables, as

como por la sinergia con otros surfactantes, y compatibilidad tanto


medioambiental como dermatolgica

[Willing, 2006],

en productos de limpieza e higiene personal


Biermann, 1993; Garca, 1997; Uppgard, 2000],

son ampliamente utilizados

[Balzer, 1996; Rybinski, 1998;

aunque tambin se han utilizado en

la industria del petrleo y del gas [McGregor, 2004].

B) Mtodos de anlisis
Dado que los APGs se estn convirtiendo en unos surfactantes cada vez
ms interesantes por los diversos aspectos comentados anteriormente, tiene
inters el desarrollo de mtodos para su caracterizacin y cuantificacin
2005].

[Thiele,

La cantidad total de APGs se puede determinar por hidrlisis seguida de

derivatizacin con antrona (9,10-dihidro-9-cetoantraceno) y colorimetra


[Buschmann, 1995; Buschmann, 1996-B],
1996-A],

valoracin potenciomtrica

espectrometra de infrarrojo cercano

[Kroh, 1999].

[Kim, 2001]

[Buchmann,

e hidrlisis enzimtica

Los APGs se han determinado en muestras ambientales mediante

hidrlisis, seguida de destilacin de los alcoholes y derivatizacin


1999].

[Meissner,

Para la concentracin de los APGs de muestras de agua se emplean

cartuchos de SPE C18, utilizando metanol para la elucin de los mismos


1995].

[Steber,

Se han descrito separaciones utilizando TLC con fases C8 y C18

[Buschmann, 1996-B; Buchmann, 1996-A; Buschmann, 1996-C; Spilker, 1996; Klaffke,


1998].

Los ismeros de los APGs pueden separarse sin derivatizar mediante GC a

alta temperatura, o bien, con una sililacin previa [Rybinski, 1998; Buchmann, 1996A; Spilker, 1996; Billian, 1998].

Las formas piransidas y furansidas pueden

distinguirse por los diferentes fragmentos obtenidos mediante GC-MS


1998].

[Billian,

Se ha empleado tambin MEKC con deteccin electroqumica para la

determinacin de APGs en champ

[Hbner, 2006].

Se han descrito distintos

procedimientos de HPLC con derivatizacin post-columna y deteccin


22

Captulo I. Introduccin

fotomtrica

[Kramer, 1992],

refractomtrica

[Klaffke, 1998],

ELSD

[Buschmann,

1996-B; Buchmann, 1996-A; Lafosse, 1992; Czichocki, 2002; Armari, 2003]


[Czichocki, 2002; Eichhorn, 1999; Klaffke, 1999; Khn, 2004].

o por MS

Se ha descrito el

comportamiento cromatogrfico de los alquilglucopiransidos en columnas C8,


C18, fenil y PVA

[Lafosse, 1992],

y se han realizado estudios comparativos

empleando columnas de slice, C18 y PVA [Czichocki, 2002]. Eichhorn y Knepper


[Eichhorn, 1999]

utilizaron HPLC-MS con ESI para determinar APGs en aguas

fluviales y residuales fortificadas. Utilizando una columna C18 y elucin en


gradiente con ACN/agua, es posible resolver los epmeros - y - y los ismeros
de

anillo.

Los

alquilglucopiransidos

pueden

distinguirse

de

los

alquilglucofuransidos por su distinta afinidad por el NH +4 . Kuhn y Neubert


[Khn, 2004]

usaron HPLC-ESI-QTOF-MS con una columna C18, fase mvil

MeOH/agua y elucin en gradiente para caracterizar mezclas industriales de


APGs; sin embargo, no llegaron a resolver los ismeros.

C) Estudios de fragmentacin de glucosa y oligosacridos


Las tcnicas de fragmentacin como el CID son poderosas herramientas
para la elucidacin estructural de oligosacridos. Con este fin se han empleado
iones alcalinos y iones amonio

[Harvey, 2000-A; Harvey, 2000B; Chiarelli, 1987;

Cancilla, 1999; Harvey, 1997; Harvey, 1994; Kovik, 1995; Penn, 1996; Asam, 1997],

cationes divalentes [Harvey, 2001; Madhusudanan, 2005] y sales de hierro [Carlesso,


2000; Carlesso, 2001],

sin embargo, las fragmentaciones con mayor sensibilidad y

las que ms informacin permiten obtener son aqullas que emplean iones sodio
y calcio

[Harvey, 2000-A; Harvey, 2000-B; Harvey, 2001].

La fragmentacin de los

oligosacridos tambin se ha estudiado utilizando ITMS

[Zaia, 2004],

y tambin

se han realizado anlisis de carbohidratos mediante MALDI-MS [Harvey, 2006].

23

Miriam Beneito Cambra

Al emplear bajas energas de fragmentacin, y de acuerdo con la


[Zaia, 2004; Domon, 1988],

nomenclatura de Domon-Costello

la rotura de los

enlaces glucosdicos produce principalmente fragmentos B e Y (Fig. I.6)


1997; Creaser, 2002].

Todas las uniones secuenciales de polisacridos lineales o

ramificados pueden analizarse a partir de los fragmentos


2001; Zaia, 2004].

[Asam,

[Asam, 1997; Harvey,

Por su parte, la utilizacin de altas energas de fragmentacin

produce roturas de anillo, lo cual proporciona informacin adicional de las


uniones

[Harvey, 2000-A; Harvey, 2000-B; Harvey, 2001; Cancilla, 1999; Asam, 1997;

Zaia, 2004; Creaser, 2002].

CH2OH
5

HO

Y0

5
4

OH

HO

CH2OH

0,2X

OH

Z0
2

OH

0,2A

B1

C1

CnH2n+1

O
1

Terminacin reductora

CnH2n+1

OH

Fig. I.6. Estructura de los -epmeros de AMGp (izquierda) y AMGf


(derecha). Se indican ejemplos de la nomenclatura de las fragmentaciones.
Los enlaces del anillo estn numerados en el sentido de las agujas del reloj
comenzando por cero tras el oxgeno. En los -epmeros, a diferencia de
los -epmeros, los grupos H y OR en el tomo de carbono 1 estn
invertidos.

En cuanto a los espectros CID de monosacridos y sus derivados, se han


demostrado diferencias estereoqumicas de monosacridos en presencia de Zn2+
[Gaucher, 1998]

y ion cloruro

[Carlesso, 2000].

Tambin se han distinguido

ismeros posicionales y diastereoismeros de monosacridos sulfatados


[Minamisawa, 2005]

fluorodesoxiglucosa

y se han comparado los espectros de la D-glucosa y 2[Macek, 2003].

Utilizando el espectro de CID, varias

pentosas y hexosas, incluyendo sus esteroismeros, pueden diferenciarse en

24

Captulo I. Introduccin

funcin de la competicin en los procesos de fragmentacin

[March, 2005].

Berman y colaboradores utilizaron el anlisis multivariante de los datos de TOFSIMS para distinguir entre varias furanosas, as como entre varias piranosas
[Berman, 2006].

Sin embargo, la fragmentacin CID ha sido poco utilizada para

distinguir ismeros de anillo. El tamao del anillo de la ltima unidad en el


extremo no reductor de los oligosacridos se ha relacionado con la abundancia de
los iones [M + Na-90]+ y [M + Na-104]+ en el espectro CID

[Kovik, 2001].

Usando TOF-SIMS, los monosacridos con anillos de cinco miembros fueron


ms estables que los correspondientes de seis miembros, dando diferentes
perfiles de pico en la fragmentacin [Berman, 2006].

I.2.4. Alcoholes no etoxilados y etoxilados


Los alcoholes primarios son componentes comunes en muchas muestras,
tanto biolgicas como industriales, adems de estar presentes a nivel de trazas en
el medio acutico. Los alcoholes grasos se obtienen mediante la hidrogenacin
cataltica de los cidos grasos correspondientes o de sus steres. La industria
actualmente se concentra en la hidrogenacin de steres de alcoholes grasos y
cidos grasos. Debido a que los aceites y grasas usadas como materia prima son
una mezcla de cidos grasos de distinta longitud de cadena, se obtienen como
productos alcoholes de nmero de carbonos variable

[Sad, 2007].

Los alcoholes

grasos a su vez se emplean tambin como materia prima en la produccin de


importantes clases de surfactantes, incluyendo AESs, FAEs y sulfonatos de
alcoholes grasos etoxilados [Farm, 2006.]. Tal como se ha indicado anteriormente,
los FAEs se obtienen en forma de complejas mezclas de oligmeros con la
estructura:
CH3(CH2)n-1(OCH2CH2)mOH
que puede abreviarse como CnEm, donde n adopta valores entre 8 y 18 y m, entre
0 y 30, el valor medio de EO se representa como m .
25

Miriam Beneito Cambra

A) Propiedades y aplicaciones
En los ltimos aos, los surfactantes basados en alcoholes grasos han
ganado importancia en el mercado de detergentes debido a sus excelentes
propiedades de lavado y su superior biodegrabilidad. Los alcoholes con una
cadena hidrocarbonada de entre 10 y 18 carbonos (alcoholes grasos) son potentes
feromonas, mientras que los de mayor masa molecular son componentes
esenciales en las ceras de las plantas

[Bianchi, 1995].

La importancia industrial de

los alcoholes deriva de su uso generalizado como disolventes y cargas


(espesantes) en productos industriales y del hogar. Actualmente, los alcoholes
grasos derivados de materias primas renovables son usados en la produccin de
surfactantes no inicos, como los ASs, AESs, FAEs y APGs
Fielder, 1989; Sparham, 2005].

[Marcomini, 1996;

Otro de los campos de aplicacin de los alcoholes

grasos es la industria de los cosmticos, donde se emplean en la formulacin de


jabones lquidos, champs, acondicionadores, cremas y lociones [Sad, 2007].
En cuanto a los FAEs, son los surfactantes no inicos ms empleados en
productos de limpieza, siendo tambin utilizados en la formacin de cosmticos,
estabilizantes o dispersantes de herbicidas, etc. Los grupos ter proporcionan la
polaridad necesaria para garantizar su solubilidad en agua. Si bien la cadena de
EO no es tan polar como un grupo ionizado, un conjunto de 5 a 10 unidades de
EO puede alcanzar una notable capacidad hidrfila. Los FAEs son excelentes
agentes humectantes, compatibles tanto con surfactantes aninicos como
catinicos, y su detergencia no se reduce en presencia de iones metlicos como
Ca2+ o Mg2+. Tienden a ser lquidos o ceras con bajo punto de fusin y, por
consiguiente, son difcilmente utilizables en la formulacin de detergentes en
polvo. Otro de sus inconvenientes es su tendencia a precipitar a temperaturas
elevadas o fuerzas inicas altas, debido a la menor solvatacin de la cadena de
EO en comparacin con los grupos inicos. Adems, a temperaturas ms

26

Captulo I. Introduccin

elevadas, se reduce el peso estadstico de las conformaciones polares de la


cadena de EO, de manera que la zona polar pierde buena parte de su carcter
hidroflico. En estas condiciones, el surfactante se separa del medio acuoso
formando otra fase (punto de nube). Los FAEs lineales se caracterizan, a grandes
rasgos, por el intervalo de valores de n (tambin llamado corte hidrofbico), y
por el nmero medio de unidades de EO; sin embargo, tanto para el control de
calidad industrial como medioambiental, se requiere informacin sobre la
distribucin de los oligmeros. La caracterizacin de los FAEs es importante
porque sus propiedades fisico-qumicas, as como el riesgo ambiental que
comportan, vienen fuertemente condicionadas por las distribuciones de sus
cadenas hidrofbica e hidroflica [Marcomini, 1996; Rudewicz, 1986; Eadsforth, 2006;
Belanger, 2006; Van Compernolle, 2006; Ribosa, 2007; Jurado, 2007].

Una clase especial de FAEs incluye compuestos de masa molecular baja


(tanto n como m < 4). Estos compuestos conocidos como cellosolves, se utilizan
por ejemplo, como aditivos anticongelantes en combustibles para aviacin y
como disolventes en algunas formulaciones para limpiadores

[Cheremisinoff,

2003].

B) Mtodos de anlisis
Los cellosolves y los alcoholes grasos con una cadena igual o menor a 26
tomos de carbono se han determinado con xito mediante GC [Nichols, 2006], sin
embargo, los FAEs con m > 4, no pueden determinarse por GC debido a su
limitada volatilidad

[Rudewicz, 1986; Crescenzi, 1995; Battersby, 2001].

El principal

inconveniente de los mtodos de HPLC para la determinacin de alcoholes


grasos y FAEs ha sido la falta de un detector adecuado
2001].

[Rudewicz, 1986; Petrovic,

Este problema se ha resuelto en parte con la introduccin de detectores

como el de ndice de refraccin

[Kudoh, 1984; Mengerink, 1991; Cho, 2003;

27

Miriam Beneito Cambra


Trathnigg, 2002]

y el ELSD

[Mengerink, 1991; Heinig, 1998; Bear, 1988; Miszkiewicz,

2000; Miszkiewicz, 1996-B; Kamiusuki, 2000],

sin embargo, los LODs en el detector

de ndice de refraccin son altos, mientras que la sensibilidad del ELSD se ve


mermada para compuestos voltiles como alcoholes no etoxilados y oligmeros
de FAEs con un bajo grado de etoxilacin (m < 3)
Zafn, 2006].

[Miszkiewicz, 1996-A; Bernab-

Los LOD ms bajos se consiguen mediante procesos de

derivatizacin pre-columna con agentes

cromognicos

y fluorognicos

[Marcomini, 1996; Schmitt, 1990; Kiewiet, 1995; Lemr, 1994; Zanette, 1996; Lemr, 1996;
Sun, 1997; Hoffman, 2004-A; Lemr, 2003; Okada, 1991; Okada, 1992; Hoffman, 2004-B;
Bachus, 2003; Desbne, 2005; Heinig, 1996-A; Mic-Tormos, 2008-A; Mic-Tormos,
2008-B; Mic-Tormos, 2009; Zu, 2010].

En la determinacin de FAEs mediante MS, la formacin de aductos con


iones positivos, ya sea utilizando la fuente ESI o APCI, resulta problemtica
debido a la disminucin de la sensibilidad conforme disminuye m lo que es
especialmente notable para m < 4. Finalmente, los alcoholes no etoxilados (m =
0) no pueden detectarse por MS [Rudewicz, 1986; Crescenzi, 1995; Petrovic, 2001; Zu,
2010; Chiron, 2000; Sherrard, 1994; Dunphy, 2001; Cassani, 2004; Sparham, 2005].

Para superar este inconveniente se han desarrollado procedimientos de


derivatizacin, en los que se adiciona un cromforo y/o una carga permanente a
los oligmeros de los FAEs

[Lemr, 1994; Lemr, 1996; Lemr, 2003; Desbne, 2005;

Heinig, 1996-A; Zu, 2010; Dunphy, 2001; Cassani, 2004; Sparham, 2005].

Sin

embargo, la derivatizacin de FAEs no es una tarea sencilla, ya que se requiere


un medio anhidro [Okada, 1992; Zu, 2010; Dunphy, 2001; Barry, 2003], existiendo un
mayor riesgo de interferencia tanto por el exceso de reactivo como por los
subproductos de la reaccin

[Sun, 1997; Hoffman, 2004-A; Mic-Tormos, 2008-A;

Mic-Tormos, 2008-B; Dunphy, 2001].

Por otra parte y debido al alto riesgo de

perder oligmeros con bajos valores de m por volatilizacin, el contenido de agua


de las muestras debe reducirse con precaucin, lo que aumenta el tiempo de

28

Captulo I. Introduccin

anlisis [Dunphy, 2001]. Sin embargo, en la derivatizacin con anhdridos cclicos


se tolera la presencia de pequeas cantidades de agua en las muestras

[Mic-

Tormos, 2008-A; Mic-Tormos, 2008-B; Mic-Tormos, 2009; Zu, 2010].

I.2.5. AESs
Los AESs son una clase de surfactantes aninicos ampliamente utilizados
en productos de limpieza e higiene corporal

[Fiedler, 1989].

Se obtienen por

esterificacin de FAEs, utilizando trixido de azufre o cido clorosulfnico. La


estructura molecular de los AESs est formada por una cadena de hidrocarburo
unida a una cadena de EO con un grupo sulfato en el extremo [Strain, 1959; Suter,
1944; Arthur D. Little, 1991].

Estos compuestos raramente son sustancias puras,

tratndose generalmente de mezclas, lo que es debido a la materia prima


(alcoholes) y a que su grado de etoxilacin es un valor medio. Los AESs se
obtienen como complejas mezclas de oligmeros con la estructura:

CH 3 (CH 2 ) n -1 (OCH 2 CH 2 ) m OSO3que se puede abreviar como CnEmS, donde n adopta valores entre 12 y 16,
mientras que m suele oscilar entre 0 y 3, el valor medio de EO se representa
como m .

A) Propiedades y aplicaciones
Las disoluciones acuosas de sulfatos muestran un comportamiento
especial, debido a que su viscosidad, primero aumenta por adicin de electrolitos
como el cloruro de sodio en bajas concentraciones, disminuyendo con adiciones
posteriores. La mxima viscosidad, en cuanto a la concentracin de la sal a
aadir, depende de la estructura del ter sulfato. En comparacin con los ASs, los
correspondientes AESs son ms solubles en agua y muestran mejor la tolerancia
a la presencia de iones Ca2+ y Mg2+. La introduccin de una cadena de EO en el

29

Miriam Beneito Cambra

AS tiende a aumentar el poder espumante del surfactante, especialmente en agua


dura, ya que permite un aumento de la adsorcin interfacial. Tambin se mejoran
las propiedades humectantes y emulsionantes. Por ello se usan en las
formulaciones de jabn en barras, en diversos productos cosmticos y
farmacuticos y en el tratamiento de textiles [Salager, 2004; Spilker, 2005].

B) Mtodos de anlisis
Las

principales

caractersticas

de

estos

surfactantes

(viscosidad,

detergencia, formacin de espuma y compatibilidad con la piel), as como su


impacto ambiental, dependen de la distribucin de cadenas de alquilo y EO
[Arthur D. Little, 1991; Tadros, 2005; Marcomini, 1996; Rudewicz, 1986; Eadsforth,
2006; Belanger, 2006; Van Compernolle, 2006; Ribosa, 2007; Jurado, 2007].

Por lo

tanto, tiene inters el desarrollo de mtodos para su caracterizacin y


determinacin. Sin embargo, debido a la complejidad de las muestras, la falta de
cromforos, y los amplios intervalos de polaridad de los oligmeros, el anlisis
de AESs no es fcil. Adems, la ausencia de estndares comerciales de AESs
dificulta su determinacin.
La determinacin de AESs puede realizarse con el mtodo del azul de
metileno, en el que se determina el contenido total de surfactantes aninicos
[Llenado, 1983].

Para determinar ASs y AESs se ha utilizado la cromatografa de

pares inicos con deteccin UV indirecta

[Boiani, 1987; Shamsi, 1995],

cromatografa inica con deteccin conductimtrica

[Pan, 1995; Nair, 1998]

HPLC con derivatizacin post-columna y deteccin fluorimtrica


A].

la

y la

[Smedes, 1982-

Para la deteccin UV indirecta de AESs con separacin previa por HPLC se

han utilizando tampones acuosos con agentes reveladores UV como el veronal


[Nielen, 1991],

el sulfonato de naftaleno

[Romano, 1991; Shamsi, 1994],

el sulfonato

de tolueno [Shamsi, 1995] o los cidos benzoico y p-hidroxibenzoico [Heinig, 1996B].

Tambin se ha llevado a cabo la determinacin de AESs mediante GC tras


30

Captulo I. Introduccin

hidrlisis de los analitos con cidos diluidos para obtener sus correspondientes
alcoholes y alcoholes etoxilados, seguida de derivatizacin a trimetil sililteres
[Sones, 1979; Kirby, 1975].

En otro mtodo se utiliza GC-FID, con derivatizacin

previa de los AESs a sus correspondientes bromuros de alquilo [Neubecker, 1985].


La determinacin de AESs mediante HPLC-ESI-MS se ha aplicado a diversas
matrices ambientales como aguas, lodos de depuradora y sedimentos marinos
[Popenoe, 1994; Bruno, 2002; Lara-Martn, 2005].

I.3. Polmeros

I.3.1. Introduccin
Un polmero es una macromolcula constituida por la unin repetida de
pequeas unidades (monmeros) a travs de enlaces covalentes. Ejemplos de
polmeros de origen natural son las protenas (seda, enzimas, colgeno), los
polisacridos (almidn, celulosa) y los cidos nucleicos, los cuales cumplen
funciones especficas en los seres vivos. Dentro de los polmeros sintticos, el
ms simple es el polietileno, siendo el etileno el monmero a partir del cual se
forma. La unidad estructural que se repite a lo largo de la cadena polimrica se
denomina unidad repetitiva, y la reaccin en la cual los monmeros se unen entre
s para formar el polmero se denomina polimerizacin. Los polmeros consisten
en mezclas de molculas de distintas longitudes de cadena, y por ello se habla de
la MW promedia.

31

Miriam Beneito Cambra

A) Clasificacin de los polmeros


Existen diferentes formas de clasificar a los polmeros:
i) Segn su composicin:
- Homopolmeros: Formados por una nica unidad repetitiva. Por ejemplo, el
polimetacrilato de metilo, donde el nico monmero se repite.
- Copolmeros: formados por ms de una unidad repetitiva. Por ejemplo,
aqullos que estn formados por 2 monmeros, pudiendo las unidades
repetitivas distribuirse de distintas maneras a lo largo de la cadena del
polmero. Por ejemplo, siendo A una unidad repetitiva y B otra, pueden
disponerse:

al

azar

(ABBBBABABBAAABBA),

de

forma

alternada

(ABABABABABABABAB) o por bloques (AAAABBBAAAABBB). Los


copolmeros presentan propiedades intermedias entre las de los homopolmeros
que se formaran a partir de cada tipo de monmero por separado.

ii) Segn su estructura:


- Lineales: formados por monmeros difuncionales. Ejemplo: polietileno,
poliestireno.
- Ramificados: se requiere el agregado de monmeros trifuncionales, por
ejemplo, glicerol.
- Entrecruzados: se forma un material compuesto por una molcula
tridimensional continua, unida por enlaces covalentes. Por ejemplo, resinas
urea- formaldehdo y fenol-formaldehdo.

iii) Segn la reaccin de polimerizacin:


- Polimerizacin por reaccin en cadena (o adicin): se genera una partcula
reactiva (radical, anin o catin) a partir de una molcula de monmero, y sta
se adiciona a otro monmero de manera repetitiva.

32

Captulo I. Introduccin

- Polimerizacin por crecimiento en pasos (o condensacin): los monmeros que


reaccionan tienen un grupo funcional reactivo en cada extremo de la molcula y
la unin entre los monmeros requiere la prdida de una molcula pequea,
normalmente agua.

B) Estereoisomera en polmeros vinlicos


La tacticidad (del griego taktikos, orden) o estereoregularidad de los
polmeros es la organizacin estructural espacial de un polmero, esto es la
disposicin que adquieren los sustituyentes en el espacio. As cuando un par de
sustituyentes R adyacentes apuntan en la misma direccin en el espacio, la pareja
o dada se denomina m, mientras que si los sustituyentes apuntan en direcciones
contrarias del espacio se tiene una pareja o dada r (Fig. I.7).
R H

R H

Dada m
R H

R H

Dada r
R H

R H

n
Fig. I.7. Estructura de los polmeros isotcticos (A), sindiotcticos (B) y
atcticos (C).

Cuando se considera un mayor nmero de grupos R adyacentes, existen


tres ordenamientos posibles con respecto al plano del esqueleto carbonado del
polmero:

33

Miriam Beneito Cambra

i) Isotctico: con todos los grupos R hacia el mismo lado de la cadena


polimrica extendida (Fig. I.7-A).
ii) Sindiotctico: con los grupos R alternando a uno y otro lado (Fig. I.7-B).
iii) Atctico: con los grupos R distribudos al azar (Fig. I.7-C).
El tipo de estereoregularidad se establece en la reaccin de polimerizacin
y no es posible convertir un estereoismero en otro por simple rotacin de los
enlaces sigma a lo largo de la cadena polimrica.

I.3.2. Polmeros en detergentes


Se pueden enumerar varias caractersticas importantes que los polmeros
pueden ofrecer, y que en distinto grado, pueden ayudar a obtener como resultado
final un buen lavado. Existen polmeros que se emplean en el formulado de
detergentes que actan como agentes para la prevencin de la re-deposicin de
suciedad, secuestradores de los iones calcio y magnesio, agentes dispersantes y
otros que actan como DTI (inhibidores de la redeposicin de colorantes)

[Zini,

1999].

A) Agentes antirredeposicin
El efecto de los polmeros en las operaciones de lavado se conoce desde
los aos 60 del siglo XX. La eliminacin de las manchas de las superficies es una
compleja

combinacin

de

procesos

fsico-qumicos

como

disolucin,

desplazamiento y desorcin. Estos procesos son reversibles y las partculas de


suciedad pueden readsorberse sobre los tejidos tras permanecer en la disolucin
de lavado. Para evitar esto, se utilizan en las formulaciones componentes que
garanticen un rendimiento ptimo de la dispersin de la suciedad en la
disolucin. Sin embargo, las propiedades de la superficie pueden variar
considerablemente, existiendo diferentes afinidades entre las fibras y los

34

Captulo I. Introduccin

diferentes tipos de suciedad, dependiendo principalmente de la polaridad. As, las


partculas que se eliminan fcilmente de fibras de polaridad intermedia pueden
ser fuertemente adsorbidas por fibras tanto hidroflicas como hidrofbicas,
provocando un envejecimiento de los distintos tejidos tras numerosos lavados
[Waldhoff, 2005].

Para evitar estos iconvenientes, las formulaciones contienen

polmeros que interactan fuertemente tanto con las partculas de suciedad en la


disolucin de lavado, como con las fibras textiles, sobre las que se adsorben de
manera irreversible. En ambos casos, se impide la redeposicin de la suciedad.
Los polmeros derivados de la celulosa y del almidn son los ms utilizados
como agentes antirredeposicin, aunque tambin se pueden emplear otros como
el PVP o PVA.

B) Agentes secuestrantes
Los agentes secuestrantes enmascaran los cationes divalentes del agua
dura, evitando su interaccin con los surfactantes. Los polmeros tambin
utilizados con este fin suelen ser homopoliacrilatos o copolmeros de acrlico y
maleico [Zini, 1999].

C) Agentes inhibidores de la transferencia de color


Desde los aos 90 del siglo XX, la industria de los detergentes ha lanzado
al mercado varios polmeros solubles en agua con capacidad para inhibir
parcialmente la transferencia de color de unos tejidos a otros durante el proceso
de lavado

[Bertleff, 1998; Oakes, 2003-A].

Estos polmeros, denominados DTIs,

actan atrapando los colorantes desprendidos, mantenindolos en disolucin, y


por lo tanto, previniendo su redeposicin en los tejidos. Estos polmeros
muestran una fuerte afinidad con los grupos funcionales aromticos. Los DTI,
quedan adsorbidos sobre los colorantes en las aguas de lavado, mediante una

35

Miriam Beneito Cambra

combinacin de interacciones dipolo-dipolo inducido y -, previniendo


eficazmente la redeposicin del color

[Jger, 1991].

Los DTI estn constituidos

por monmeros con residuos ricos en electrones y fuertemente bipolares o


fcilmente polarizables, por lo general, heterociclos aromticos o alifticos con
sustituyentes vinlicos. Los polmeros que se utilizan con este fin suelen ser PVP
y PVP-NO y tambin el copolmero PVP-PVI.

I.3.3. PVP-NO
El PVP-NO es un polmero empleado como DTI en las formulaciones de
detergentes. Durante aos, se us principalmente PVP y sus copolmeros, sin
embargo, la eficacia de estos polmeros para atrapar colorantes se reduce en
presencia de surfactantes aninicos

[Oakes, 2003-A].

Por esta razn, se han

desarrollado nuevas generaciones de DTIs, con propiedades superiores de


complejacin, y con mayor tolerancia frente a los surfactantes aninicos. Un DTI
de nueva generacin relativamente comn es el PVP-NO, obtenido a partir de
una polimerizacin radicalaria de la 4-vinil-piridina utilizando AIBN como
iniciador, seguida de oxidacin con perxido de hidrgeno (Fig. I.8)

[Lee, 1996].

Los diferentes tipos de PVP-NO comercial para detergencia estn constituidos


por especies con masas moleculares entre 9 y 36 kDa, que corresponden a
cadenas de polmero que contienen desde 75 hasta 300 monmeros,
respectivamente [Oakes, 2003-A; www.ispcorp.com].
CH2 - CH

CH2 CH

N+
N

O-

4-vinilpiridina

PVP-NO

Fig. I.8. Estructura de la 4-vinilpiridina y del PVP-NO.

36

Captulo I. Introduccin

I.3.4. Poli(vinilpirrolidona)
De la primera polimerizacin de la N-vinilpirrolidona se obtuvo un
polmero soluble, el PVP (Fig. I.9), patentado en 1939. La polimerizacin se
realiza mediante radicales libres en agua o en 2-propanol, utilizando como
iniciador perxido de hidrgeno o un perxido orgnico, respectivamente [Bhler,
2005].

C H C H2
N
O

C H C H2
N
O

n
N-vinilpirrolidona

PVP

Fig. I.9. Estructura de la N-vinilpirrolidona y del PVP.

El PVP es un polmero no inico que puede aplicarse en gran variedad de


campos gracias a caractersticas como su solubilidad en agua, as como en
diversos disolventes orgnicos polares, buena afinidad con diferentes polmeros y
resinas, alta higroscopicidad, capacidad de formacin de pelculas, buena
adherencia a diversos sustratos y posibilidad de formar complejos/quelatos
[www.shokubai.co.jp].

El PVP es un polmero verstil utilizado en cosmticos,

textiles, tintes, productos farmacuticos y adhesivos


1985].

[Frauenfelder, 1974; Sheth,

En la formulacin de productos de limpieza, el PVP se emplea como DTI,

mantenimiento los colorantes desprendidos durante el lavado en disolucin, por


lo que inhibe su transferencia a otros tejidos presentes en el lavado [Bertleff, 1998;
Oakes, 2003-A; Oakes, 2003-B; Oakes, 2005].

I.3.5. Alcohol polivinilico


El PVA (Fig. I.10) es un polmero sinttico obtenido por primera vez en
1924 por Hermann Staudinger, mediante la hidrlisis del PVAcO con hidrxido

37

Miriam Beneito Cambra

de potasio en etanol

[Gallardo, 1999].

El PVA presenta un amplio abanico de

aplicaciones en medicina, cosmtica, alimentacin, farmacia, detergencia, etc.,


como

consecuencia

de

propiedades

tales

como

su

inocuidad,

alta

biocompatibilidad y solubilidad en agua [Park, 2010]. Segn la ruta de sntesis, se


obtienen PVAs con diferentes tacticidades, como corresponde a una estructura
que presenta centros quirales adyacentes repetidos a lo largo de la cadena
polimrica.
CH2

CH
O

CH2
O

C
CH3

CH
OH

PVAcO

PVA

Fig. I.10. Estructura del PVAcO y del PVA.

El PVA es un polmero capaz de complejar cationes metlicos como Cu2+,


y de formar steres con algunos aniones como borato, titanato, antimoniato y
vanadato. En disolucin acuosa, los complejos polmero-catin tienen
propiedades similares a las observadas en las disoluciones de polielectrolitos
(especies con cargas positivas o negativas). La carga de los grupos PVA-catin
puede provocar la ionizacin de la molcula de agua [Tsujimoto, 2002].

I.3.6. Mtodos de anlisis de polmeros


Durante los ltimos aos, la CE se ha aplicado a la caracterizacin y
determinacin

de

polmeros

sintticos

[Gallardo,

1999;

Cottet,

2005].

Principalmente, la FSCE y la CGE han sido empleadas para separar


polielectrolitos y polianfolitos (especies con cargas tanto positivas como
negativas), y la MEKC se ha usado para separar polmeros no cargados [Gallardo,
1999].

Tanto la FSCE como la MEKC proporcionan informacin sobre el

38

Captulo I. Introduccin

comportamiento

de

polmeros

en

disolucin,

permiten

cuantitativamente algunas de las disoluciones de polmeros

estimar

[Bohrisch, 2000].

Gallardo y col. [Gallardo, 1999] separaron por MEKC una fraccin mayoritaria en
metacrilato y otra rica en vinilpirrolidona a partir de copolmeros no inicos.
Tambin, el empleo de MEKC ha proporcionado informacin sobre el progreso
de la sntesis, naturaleza y composicin de copolmeros inicos

[Aguilar, 2002].

Por otro lado, en CGE es posible conseguir la discriminacin por masa molecular
de polmeros inicos mediante un proceso de tamizado molecular, a travs de un
BGE que contiene un polmero no inico, como el PEG
derivados de celulosa, como dextrano u otros polisacridos

[Grosche, 2000]

[Bohrisch, 2000; Clos,

1998; Starkweather, 2000; Welch, 2001].

En FSCE, la movilidad electrofortica para polielectrolitos de cadena


corta es funcin de la longitud de la cadena, de modo que la movilidad se
incrementa cuantos ms monmeros cargados existan en la misma. Sin embargo,
la movilidad alcanza un mximo y despus comienza a decrecer cuando el
polielectrolito cambia su conformacin extendida a la enrollada u ovillada. En el
caso de polielectrolitos ms largos, la relacin masa/carga permanece constante,
y el polmero migra con una movilidad que se denomina de drenaje libre
[Cottet, 2000],
2000].

proporcionando un pico nico en FSCE

[Bohrisch, 2000; Grosche,

No obstante, algunos polmeros pueden dar dos o ms seales a diferentes

tiempos de migracin en lugar de un pico nico. As por ejemplo, se ha


demostrado la formacin de micelas en disolucin acuosa de copolmeros
compuestos por cadenas largas polianinicas de polisulfonato de estireno y
cadenas hidrofbicas cortas de polietileno-propileno

[Cottet, 2001].

En estos

copolmeros, las cadenas libres (unmeros) presentan una movilidad elevada (en
trminos absolutos), mayor que la movilidad de los polmeros micelizados.

39

Miriam Beneito Cambra

Adems, el pico del polmero micelizado es ms ancho que el pico del polmero
libre, lo que se ha atribuido a cierta dispersin en el nmero de agregacin.
Gyorffy y col.

[Gyrffy, 1998]

analizaron un copolmero aninico de PVP-

NO y cido maleico mediante FSCE, encontrando un pico estrecho seguido de


uno ms ancho. Para explicar este comportamiento se propuso la existencia de
dos componentes en el polmero. Utilizando SEC y FSCE, tepnek y col.
[tepnek, 2001]

demostraron que algunos copolmeros forman micelas en

disolucin, y que stas se encuentran en equilibrio dinmico con los unmeros.


Adems, se demostr que el equilibrio micelas-unmeros est cinticamente
ralentizado en medio acuoso, donde las micelas se comportan como
nanopartculas independientes.
Para la caracterizacin de polmeros mediante CE puede emplearse la
tcnica descrita por S. N. Krylov y col.

[Berezovski, 2002; Krylov, 2003; Drabovich,

2006; Lin, 2008; Krylov, 2006; Krylov, 2007],

desarrollada inicialmente para estudiar

las interacciones entre una protena o un fragmento de DNA con un marcador


fluorescente. En dicha tcnica, denominada NECEEM, los enlaces no covalentes
de marcadores fluorescentes se equilibran con una protena diana o con DNA, y
la mezcla se inyecta en el capilar. Se obtiene una banda debida al complejo
seguida del pico producido por el exceso de marcador libre. Adems, entre
ambas seales se obtiene una regin intermedia, con el aspecto de una cada
exponencial, debida al marcador liberado por el complejo durante la separacin
electrofortica siguiendo una cintica de pseudo-primer oden. La formacin de
complejos entre polmeros solubles, como PVP, y azocolorantes, se conoce desde
hace dcadas [Frauenfelder, 1974].
Otra tcnica empleada para la caracterizacin de los polmeros es la NMR,
con la que puede determinarse la tacticidad o estereoregularidad de los polmeros
[Moritani, 1972.; Wu, 1977].

La espectrofotometra UV-Vis se ha utilizado para la

40

Captulo I. Introduccin

determinacin de copolmeros, siempre y cuando al menos uno de los


monmeros presente un grupo cromforo. Por ejemplo, el copolmero de
metacrilato de metilo y acenaftileno se pueden caracterizar por esta tcnica
[Snchez, 2000; Skoog, 1994].

Tambin se han caracterizado ciertos polmeros

naturales mediante la formacin de complejos polmero-ion coloreado [Berezovski,


2002; Krylov, 2003; Drabovich, 2006; Lin, 2008; Krylov, 2006; Krylov, 2007].

Una tcnica habitual para caracterizar especies de elevada masa molecular


es la SEC
capilar
2008],

[Snchez, 2000; Skoog, 1994].

[Snchez, 2000.; Skoog, 1994]

Otras tcnicas, como la viscosimetra

o la osmometra de presin de vapor

[Karimi,

tambin pueden emplearse para la caracterizacin de polmeros, ya que los

resultados obtenidos pueden correlacionarse con la masa molecular y otras


propiedades de los polmeros. Algunos polmeros empleados como DTI, en
particular la PVP, tambin pueden determinarse mediante pirlisis seguida de
GC-MS [Uchiyama, 1998].

I.4. Enzimas

I.4.1. Introduccin
Las enzimas aparecieron en el mercado de los productos de limpieza en la
dcada de 1960, para ayudar a eliminar las manchas proteicas. Hoy en da, la
presencia de diferentes enzimas en las formulacioens mejora la detergencia, ya
que facilitan la degradacin de grandes y complejas molculas tales como las
protenas, almidones y grasas. Los productos de reaccin obtenidos tras la
actuacin de las enzimas son ms solubles en las aguas de lavado, por lo que son
arrastrados ms fcilmente por los surfactantes. Por otro lado, las enzimas
tambin ayudan a mantener la blancura y brillo en los tejidos, as como a

41

Miriam Beneito Cambra

clarificar los colores, ya que pueden eliminar las fibras desprendidas de los
tejidos (pelusa) [Crutzen, 1999].
La incorporacin de las enzimas a los detergentes en polvo es muy
habitual, siendo menos frecuente en detergentes lquidos, por la mayor dificultad
para preservar su estabilidad. Tampoco suelen usarse en patillas de jabn ni en
lavamanos, debido a su inestabilidad durante los procesos de fabricacin, y por
posibles procesos de irritacin cutnea. Los detergentes enzimticos representan
un 30% del consumo mundial total de detergentes, el 80% del mercado en USA
[Krawczyk, 1996]

y un 85% en Europa [Whalley, 1994].

El amplio uso de enzimas en detergentes se justifica por sus favorables


caractersticas:
- Su alto rendimiento a bajas concentraciones favorece el desarrollo de
detergentes cada vez ms concentrados.
- Su ventajosa relacin precio/efectividad.
- Su excelente perfil medio-ambiental.
Las enzimas son protenas que se producen en todas las clulas vivas. Son
las responsables de la catlisis de muchas reacciones qumicas biolgicas, que
generalmente tienen lugar a bajas temperaturas y a pH neutro o cercano al neutro,
con una alta eficiencia y especificidad [Krawczyk, 1995]. Las enzimas presentes en
los detergentes no son tan especficas. Por ejemplo, las enzimas proteolticas
(proteasas) son menos discriminantes y aumentan la velocidad de rotura de
muchas protenas. Del mismo modo, las enzimas amiolticas (amilasas) ayudan a
fragmentar los almidones, las enzimas lipolticas (lipasas) catalizan la hidrlisis
de los triglicridos y las enzimas celulticas (celulasas) ayudan a romper la
celulosa. La mayor velocidad de rotura de molculas grandes, procedentes de
comida, secreciones corporales, etc, a molculas ms pequeas y solubles hace
que las enzimas faciliten la eliminacin de varias manchas que de otra manera

42

Captulo I. Introduccin

serian difciles de quitar slo con los detergentes. Tambin cabe mencionar que
al actuar como catalizadores, las enzimas suelen ser muy efectivas a bajas
concentraciones (menos de 0,1% en peso de enzima en el detergente)

[Crutzen,

1999].

Las enzimas utilizadas en los detergentes son producidas por cepas de


baterias resitentes a medios alcalinos, que producen, por lo tanto, enzimas que
tambin son resistentes a pHs altos. Adems se trata de enzimas exocelulares, es
decir, que son secretadas por las bacterias en el medio circundante. Por lo tanto,
estas enzimas pueden ser aisladas sin necesidad de romper las clulas
bacterianas, lo que hace que su extraccin y purificacin sea fcil y poco costosa.
Las enzimas son sensibles a algunos de los ingredientes presentes en los
detergentes, tanto durante el tiempo de almacenaje del producto como en el agua
de lavado. Dentro de ellos, los surfactantes catinicos son los ms perjudiciales,
aunque stos no son muy utilizados

[Baas, 1997].

Los surfactantes aninicos,

especialmente los ABS, tambin degradan las enzimas, mientras que los
surfactantes no inicos no las desestabilizan

[Bahn, 1987].

Las enzimas se

desactivan por la presencia de agentes oxidantes, sin embargo, son ms estables


en productos que contienen slo perborato sin ningn tipo de activador. An en
este caso, la oxidacin de las enzimas durante el tiempo de almacenaje puede
resultar un problema.

I.4.2. Proteasas
Las proteasas son las enzimas encontradas mayoritariamente en los
detergentes. Como se ha descrito anteriormente, las proteasas catalizan la rotura
de largas protenas en molculas ms pequeas, como pptidos o aminocidos,
que pueden ser ms fcilmente eliminadas por los surfactantes. Segn la posicin
activa, las proteasas pueden clasificarse en dos grupos:

43

Miriam Beneito Cambra

i) Endopeptidasas: rompen los enlaces peptdicos dentro de la cadena


polipeptdica, dando lugar a pptidos solubles en agua.
ii) Exopeptidasas: rompen los enlaces peptdicos terminales, obtenindos
aminocidos libres.
Tambin pueden ser clasificadas en funcin de las diferentes cepas de
Bacillus, las cuales exhiben diferentes sensibilidades en funcin del pH, y
distintas resistencias frente a los dems ingredientes de los detergentes. Las
enzimas procedentes del Bacillus licheniformis, como por ejemplo la Alcalase
(Novo Nordisk) o la Maxatase (Genencor), ofrecen una excelente relacin
coste/prestaciones, especialmente a bajas temperaturas y a un pH moderado (pH
7-10). Otras proteasas, procedentes del Bacillus lentus/alcalophilus, como la
Esperase, la Savinase (Novo Nordisk) y el Maxacal (Genencor), presentan una
mayor actividad a pH ms alto, y una mejor resistencia al pH, con un intervalo de
pH ptimo entre 8 y 12. Tambin difieren de otras proteasas en su masa
molecular y en el punto isoelctrico. Estas proteasas tambin presentan una
mayor estabilidad y actividad a altas temperaturas, as como una mejor eficacia
en presencia de perborato, pero tambin tienen una mayor sensibilidad a la
presencia de surfactantes aninicos [Crutzen, 1999; Maurer, 2005].
La actividad proteoltica se puede estimar mediante una gran variedad de
mtodos

[Sarath, 1989].

Numerosos mtodos utilizan la degradacin de la

hemoglobina o la casena como protenas estndar para medir dicha actividad


[Maurer, 1997].

Hasta ahora, todas las proteasas empleadas en detergentes

pertenecen a la familia de la subtilisina

[Egmond, 1997; Bott, 1997].

Las protenas

de esta familia tienen una estructura tridimensional y un tamao casi idnticos.


Las diferentes variantes de la enzima se distinguen slo por pequeas variaciones
en la secuencia de aminocidos producidos de forma natural, o bien, mediante
ingeniera gentica

[Ballinger, 1998].

Estas modificaciones pueden dar lugar a

44

Captulo I. Introduccin

pequeas diferencias en el punto isoelctrico, y a ligeros cambios en los


electroferogramas obtenidos mediante enfoque isoelctrico, electroforesis en gel
o capilar

[Vinther, 1992-A].

Cuando estas pequeas diferencias no son suficientes

para la identificacin de la enzima, es necesario recurrir a un mtodo de


asociacin o la secuenciacin completa de la protena para resolver el problema
[Christianson, 1994].

En algunas variantes de proteasas el residuo de metionina en

la posicin 222 se ha sustituido por un residuo de aminocido ms estable frente


a la oxidacin, lo que conduce a un aumento de la estabilidad en el periodo de
almacenaje. Estas proteasas se pueden identificar y determinar mediante
oxidacin con perxido de hidrgeno en un tampn de cido brico [Estell, 1985].

I.4.3. Amilasas
Las amilasas aumentan la eficacia de los detergentes ayudando a disolver
las manchas constituidas mayoritariamente por almidn, si bien, la mejora en la
eficacia limpiadora es menor que la obtenida con proteasas. Estas enzimas son amilasas de origen bacteriano (Bacillus licheniformis) que catalizan la hidrlisis
de los enlaces -1,4 glicosdicos de la amilosa y la amilopectina en dextrinas,
oligosacridos y azcares, dando lugar a molculas solubles. Las amilasas
presentan una mayor estabilidad frente a pHs alcalinos y a temperaturas altas que
las proteasas. Para mantener la actividad de las amilasas, el medio debe contener
suficiente concentracin de ion Ca2+, necesaria para estabilizarlas frente a la
desnaturalizacin y al ataque de las proteasas [Crutzen, 1999].
Las amilasas pueden detectarse mediante el uso de tabletas de Phadebas
o productos similares constituidos por almidn entrecruzado, insoluble y
coloreado; en presencia de amilasa se produce su degradacin, con formacin de
dextrinas y liberacin de colorantes solubles. Hasta ahora, las principales
amilasas usadas en detergentes derivan del Bacillus licheniformis, cuya enzima

45

Miriam Beneito Cambra

natural es termoestable. Mediante ingeniera gentica, se han desarrollado


variantes de dicha enzima que mejoran su estabilidad frente a la oxidacin
(Duramyl, Purastar OxAmTM), mostrando adems un mejor rendimiento que la
variante natural original, especialmente en los detergentes para lavavajillas. Estas
variantes de la amilasa pueden identificarse por su capacidad de resistir una
incubacin en tampones que contienen perxido de hidrgeno, perborato o
percarbonato [Estell, 1985; Maurer, 2005].

I.4.4. Lipasas
Los triglicridos son los principales constituyentes de las manchas
procedentes de grasas animales y aceites vegetales. Las lipasas empleadas en
detergencia catalizan la hidrlisis de los enlaces ster de los principales
componentes de las grasas, los TAGs. Por lo tanto, las lipasas ayudan a eliminar
las manchas grasas, las cuales se adhieren fuertemente a los tejidos, y adems
evitan su redeposicin durante el lavado. Las lipasas son una buena muestra de la
cooperacin enzima-surfactante. Estas enzimas actan a temperaturas a las cuales
la solubilizacin de las grasas por parte de los detergentes es baja. La reaccin
enzimtica conduce a una mezcla de diglicridos, monoglicridos y glicerol, los
cuales son menos hidrofbicos que los materiales originales, siendo por tanto
ms fcilmente arrastrados por los surfactantes [Crutzen, 1999].
La primera lipasa industrial, Lipolase, de la casa comercial Novo, y
aislada del hongo Humicola lanuginosa, apareci en el mercado en 1988. Gracias
a la ingeniera gentica, la Lipolasa puede ser producida con buenos
rendimientos a partir del inofensivo microorganismo Aspergillus oryzae. En
relacin con la accin limpiadora, la lipasa difiere de proteasas y amilasas, ya
que se necesitan varios lavados para observar sus beneficios
Gormsen, 1991].

[Aaslyng, 1991;

La diferencia es debida al efecto del agua en la actividad lipdica:

46

Captulo I. Introduccin

la enzima es poco activa cuando el contenido de agua en los tejidos est por
debajo del 5%, o por encima del 80%, siendo mxima con un contenido de agua
del 80%

[Gormsen, 1991].

Este nivel de agua se alcanza durante el proceso de

secado, por lo que la cantidad de material graso que permanece en el tejido


durante el primer lavado es alta, sin embargo, durante el segundo lavado los
triglicridos que han sido hidrolizados son eliminados mucho ms fcilmente
[Crutzen, 1999].

En cuanto al anlisis cualitativo, las lipasas pueden detectarse mediante


incubacin con laurato o palmitato de p-nitrofenilo, con la subsiguiente aparicin
del color amarillo del p-nitrofenol [Brockmann, 1981; Novo Nordisk, 1995; Martinelle,
1996; Pencreach, 2001; Eggert, 2000].

El ensayo enzimtico es extremadamente

sensible y debe ser controlado por la elevada probabilidad de falsos positivos


debidos a la actividad de fondo de la Humicola, o por la autolisis del sustrato. En
electroforesis en gel, la enzima puede teirse con steres de naftilo, como por
ejemplo el acetato de -naftilo

[Stander, 2000; Sims, 1965; Maurer, 2005].

Ya que

las lipasas hidrolizan especficamente a los TAGs en cidos grasos libres y


glicerol, se pueden determinar por valoracin de los cidos grasos que se han
liberado durante la incubacin de un estndar
1995; Martinelle, 1996; Maurer, 2005].

[Brockmann, 1981; Novo Nordisk,

Por otro lado, tambin pueden determinarse

por deteccin fluorimtrica los componentes alcohlicos (glicerol) previo


marcaje con resorufina [Junge, 1983]. En todos los casos, es necesario calibrar los
mtodos utilizando la correspondiente lipasa como estndar.

I.4.5. Celulasas
Todas las enzimas consideradas hasta el momento solubilizan las manchas
por la degradacin de los principales constituyentes. Las celulasas ejercen un rol
bastante diferente y proporcionan otros beneficios: suavidad de las fibras,

47

Miriam Beneito Cambra

luminosidad del color, propiedades antipelusa y antirredeposicin [Maurer, 1998].


Estos beneficios son resultado de la eliminacin de microfibras formadas en la
superficie de los tejidos de algodn

[Christensen, 1987].

Estos efectos son

acumulativos, incrementndose de forma considerable con el nmero de ciclos de


lavado realizados

[Whalley, 1994].

Las celulasas catalizan la hidrlisis de las

uniones 1,4--glucosdicas de la celulosa. Las celulasas empleadas en


detergencia son mezclas de endocelulasas, las cuales degradan aleatoriamente las
cadenas de celulosa, y exocelulasas, que atacan los extremos de las cadenas de
celulosa, liberando glucosa y celobiosa (un disacrido). Puesto que este ltimo
compuesto inhibe la actividad de la exocelulasa, las formulaciones tambin
contienen -glucosidasas, que transforman la celobiosa en glucosa
Crutzen, 1999].

[Boyce, 1986;

Las celulasas son las enzimas con mayor grado de variabilidad

entre las distintas clases de enzimas utilizadas en detergentes. As, las celulasas
pueden obtenerse a partir de Humicola insolens, diversas especies de los gneros
Trichoderma, Thielavia o Melanocarpus, o varias especies de Bacillus, as como
a partir de hongos.
Las celulasas se pueden detectar mediante el uso de mtodos de tincin
basados en el CR o en el azul de tripn. Estos mtodos, bien conocidos en el
mbito de la Bioqumica Analtica

[Cantwell, 1983; Bartley, 1984],

se basan en la

formacin de complejos entre el colorante y las cadenas de glucosa. Tambin se


han descrito mtodos de anlisis de celulasas basados en la derivatizacin
cromognica de la celulosa

[Fernley, 1963; McHale, 1981].

La identificacin de

celulasas separadas previamente mediante electroforesis est basada en la


utilizacin de 5-bromoindoxil--D-cellobiosido, que se hidroliza a 5bromoindoxilo, el cual reacciona con el colorante Rojo Rpido (Fast Red),
previamente acoplado a la celulosa mediante un enlace azo

[Chernoglazov, 1989].

La carboximetilcelulosa, derivado soluble de la celulosa, es tambin ampliamente

48

Captulo I. Introduccin

utilizada para el ensayo de la actividad de la celulasa. Este sustrato es especfico


para la endo-1,4--glucanasa. Slo la actividad de las endoglucanasas es
relevante para la funcin que desempean las celulasas dentro de los detergentes,
mientras que las exocelulasas (celobiohidrolasas) desempean una funcin
limitada o nula [Ghose, 1987]. Adems, tampoco se ha observado sinergismo entre
exocelulasas y endoglucanasas

[Eriksson,

1985].

Por tanto, la actividad

predominante de las endoglucanasas justifica la utilizacin de la carboxicelulasa


como el estndar ms adecuado para la determinacin de la actividad de las
celulasas. La carboxicelulasa se determina mediante la medicin del aumento de
azcares reducidos o la disminucin de la viscosidad de la disolucin. El mtodo
basado en la disminucin de la viscosidad tiene la ventaja de ser robusto, pero
presenta el problema de ser laborioso y difcil de automatizar [Maurer, 2005].

I.5. Tcnicas analticas

I.5.1. LC
La cromatografa lquida es un mtodo fsico de separacin en el cual los
componentes a separar se distribuyen entre dos fases: la fase estacionaria, que
permanece fija, y la fase mvil, que se mueve en una direccin definida. Un
sistema cromatogrfico (Fig. I.11) se compone al menos de un sistema de
bombeo, un dispositivo para la introduccin de la muestra o inyector, una
columna y un detector, y un sistema adecuado para la adquisicin de datos y
control.
Las bombas empleadas en LC pueden ser de dos tipos: bombas isocrticas
o bombas de gradiente. Los mdulos de bombas para gradiente generan,
mediante su control programado, mezclas de composicin variable. Segn el tipo

49

Miriam Beneito Cambra

de bomba, las mezclas se preparan a baja o a alta presin, siendo distintas las
caractersticas, ventajas y limitaciones de cada tipo.

Columna particulada o monoltica

Fase mvil
Detector

Inyector
Sistema de
reparto

Residuo
Registro
de datos

Inyeccin de la
muestra

Conc.

A B C

tR
Cromatograma

Fig. I.11. Diagrama de bloques de los componentes esenciales de un


cromatgrafo de lquidos.

Tanto en LC como en otras tcnicas analticas, la muestra se inserta en el


flujo de corriente de la fase mvil a travs de un bucle de inyeccin mediante una
vlvula de 6 vas y 2 posiciones, que puede ser manual o automtica. El
contenido del bucle se intercala entre el mdulo de bombas y la entrada de la
columna.
El tipo de columna a emplear depender del mecanismo de retencin con
el que se desee trabajar. Los tipos de LC ms habituales son:
i) Cromatografa de reparto: se utiliza una columna con una fase lquida
enlazada. Dependiendo de las polaridades relativas de las fases mvil y
estacionaria, se distinguen dos modalidades: RP, cuando la fase estacionaria es
apolar y la fase mvil es polar, o bien NP, cuando la fase estacionaria es polar y
la fase mvil es apolar. Un modo especial de cromatografa en fase normal es el
HILIC, en el que la fase mvil es menos polar que la fase estacionaria, si bien
mantiene su miscibilidad con agua.
50

Captulo I. Introduccin

ii) Cromatografa inica: la fase estacionaria es un intercambiador catinico o


aninico de tipo cido fuerte, base fuerte, cido dbil o base dbil.
iii) Cromatografa de exclusin molecular: como fase estacionaria se utilizan los
poros de un slido microporoso o de un gel.
Por otro lado, es importante desgasificar los disolventes para prevenir la
formacin de burbujas en el equipo. Para ello, los cromatgrafos lquidos suelen
incluir un desgasificador en lnea, insertado antes del paso de la fase mvil por el
mdulo de bombas.
Las tcnicas de deteccin ms empleadas en LC son la espectrofotometra
UV-Vis y la MS. Como detectores alternativos se utilizan los de ndice de
refraccin, as como los detectores evaporativos. Para aplicaciones especficas se
usan los detectores amperomtricos y fluorimtricos. Finalmente, la deteccin
conductimtrica es habitual en cromatografa inica. En espectrofotometra UVVis, la seal es proporcional a la concentracin molar del soluto, cuya
absortividad molar depende de la naturaleza del grupo o grupos absorbentes.
Cuando los LODs no son lo suficientemente bajos, se utilizan tcnicas de
preconcentracin, o se exalta la absortividad de los solutos aumentando su
conjugacin por derivatizacin. Los detectores espectrofotomtricos obedecen a
dos tipos de diseo: longitud de onda variable y DAD. En los primeros, se
selecciona una longitud de onda de medida fija, mientras que los segundos son
capaces de barrer todo el intervalo del espectro UV-Vis varias veces por
segundo. En este ltimo caso, el monocromador, est situado despus del paso de
la radiacin por la muestra, de modo que sobre sta incide radiacin de todas las
frecuencias. Despus del paso del haz por la muestra, se dispersa la radiacin
transmitida, de modo que cada fotodiodo mide la intensidad correspondiente a un
pequeo intervalo de longitudes de onda.

51

Miriam Beneito Cambra

En MS, el detector proporciona informacin de alto nivel sobre las


estructuras moleculares de los analitos, permitiendo distinguir grupos
funcionales, elementos qumicos e istopos, de acuerdo con los valores m/z de
los iones y sus fragmentos inicos. Un detector de MS acoplado a un
cromatgrafo puede diferenciar compuestos con caractersticas de retencin muy
similares, siendo posible identificarlos y/o cuantificarlos aunque slo estn
parcialmente resueltos, o incluso no resueltos en absoluto.
Ms recientemente, se han introducido modalidades cromatogrficas que
implican miniaturizacin, bien en el tamao de la columna o en el dimetro de
partcula. Entre las ventajas de estas tcnicas, cabe citar la reduccin de los
tiempos de anlisis, del consumo de reactivos, del volumen de residuos
generados y del tamao de muestra. A su vez, se pueden obtiener mejoras en la
sensibilidad (los LODs son menores), y mayores eficacias. Entre sus desventajas,
hay que indicar las mayores exigencias instrumentales, como el uso microbombas y micro/nano-nebulizadores.

I.5.1.1. Medida de parmetros cromatogrficos


Para llevar a cabo una separacin cromatogrfica, el analista debe
establecer si se puede separar adecuadamente al analito del resto de componentes
de la muestra, y si la cantidad en la que se halla es suficiente como para poderlo
detectar y/o determinar. El tiempo que transcurre desde la inyeccin hasta la
deteccin de un analito es su tiempo de retencin o tR. Por su parte, el factor de
capacidad o retencin relativa (k) expresa la retencin neta en unidades de tiempo
muerto, t0, o tiempo que tarda en eluir un compuesto que no presenta retencin:
ki =

t R ,i t0
t0

(E.I.1)

donde tR,i es el tiempo de retencin del analito i. El intervalo ptimo de valores


de k se sita entre 1 y 5, si bien valores entre 0,2 y 10 son aceptables. Valores de
52

Captulo I. Introduccin

k inferiores a 0,2 indican poca retencin, preferencia excesiva del soluto por la
fase mvil. Por el contrario, valores de k superiores a 20 indican una retencin
demasiado alta, producida por una preferencia excesiva del soluto por la fase
estacionaria, lo que implica tiempos de anlisis muy largos y en general picos
anchos y de baja altura, difciles de detectar y de medir con precisin, y por
tanto, con LODs mayores de lo deseable.
La capacidad de un sistema cromatogrfico para distinguir entre dos
solutos se expresa mediante el factor de selectividad i,j, que se calcula como el
cociente entre las retenciones relativas de ambos solutos:
i, j =

kj

(E.I.2)

ki

siendo i y j dos picos adyacentes, e i el soluto menos retenido.


El grado de separacin entre dos solutos se mide mediante la resolucin,
R:
R=

t R ,i t R , j

(E.I.3)

0,5( wi + w j )

siendo wi y wj las anchuras de las bases de los picos de los compuestos i y j.


Por su parte, la eficacia describe el grado de ensanchamiento de bandas en
relacin al volumen de retencin. Se obtiene una elevada eficacia cuando los
picos se mantienen estrechos a pesar de haber requerido un volumen elevado de
fase mvil para su elucin. La eficacia global de un sistema se describe mediante
el nmero de platos tericos (N), y la eficacia por unidad de longitud por la altura
equivalente a un plato terico (H), o por su inversa (1/H).
Para un soluto determinado, N puede calcularse a partir de las expresiones:
t
N = 16 R
w

t
N = 5,54 R
w1 / 2

(E.I.4)

donde tR es el tiempo de retencin del soluto y w y w1/2 son la anchura de la base

53

Miriam Beneito Cambra

del pico y su anchura a media altura, respectivamente. Por su parte, H se


relaciona con N a travs de la expresin:
N=

L
H

(E.I.5)

donde L es la longitud de la columna.


La ecuacin de Van Deemter describe las contribuciones a H, esto es,
indica como los diversos factores de construccin y funcionamiento de la
columna influyen sobre la eficacia. Para el caso de columnas microparticuladas
tanto en HPLC como en CEC, se tiene la expresin simplificada siguiente:
H = A+

B
+ C u
u

(E.I.6)

donde u es la velocidad lineal promedia de la fase mvil.


El trmino A de la ecuacin de van Deemter se conoce como difusin de
remolino o torbellino, y se debe a la distinta longitud de los caminos recorridos y
a las distintas velocidades de los solutos en su avance por el lecho
cromatogrfico (Fig. I.12, parte A). La contribucin al ensanchamiento de
bandas se debe a que las molculas se mueven a distinta velocidad segn la
anchura del camino seguido. Adems, la fase mvil que avanza por el centro de
los canales se mueve ms rpidamente que la que avanza pegada a las paredes.
Esta contribucin a H es funcin slo de la geometra del relleno, esto es, no
depende de u .
A
A

Trmino A

BB

Fig. I.12. A) Difusin de remolino y B) difusin molecular longitudinal.

54

Captulo I. Introduccin

El trmino B representa la difusin molecular longitudinal (en la direccin


axial), que es debida a la difusin de los solutos a nivel molecular (Fig. I.12,
parte B). Esta difusin es proporcional a la difusibilidad de los solutos y al
tiempo de residencia de la muestra en la columna. Conforme aumenta el tiempo
de permanencia, mayor es la difusin, y por tanto el trmino B slo cobra
importancia a velocidades de flujo bajas. Esta dependencia con el tiempo de
permanencia se refleja en la proporcionalidad inversa de esta contribucin
repecto a u .
El trmino C corresponde a la contribucin combinada de las velocidades
de transferencia de masa en la fase mvil y en la fase estacionaria (CM y CS). Este
trmino es proporcional a u ya que el avance de la fase mvil compite en
trminos de velocidad con las transferencias de masa del soluto entre ambas
fases, de modo que la importancia del trmino C aumenta con la velocidad de la
fase mvil al ser menor el tiempo de equilibrado entre ambas fases. La lentitud o
retraso con la que se efectan las transferencias de masa despus de cada etapa
de avance de la fase mvil origina un ensanchamiento de la zona ocupada por el
soluto.
Los trminos A y C de la ecuacin de van Deemter indican que la eficacia
de la columna puede mejorarse utilizando partculas de fase estacionaria ms
pequeas, o tambin rellenos ms uniformes. La forma de la curva de van
Deemter proporciona informacin acerca de la calidad del empaquetado o relleno
de la columna cromatogrfica. Cuanto menores sean las contribuciones de los
trminos A y C mayor ser el nmero de platos tericos a una determinada
velocidad de la fase mvil. En la Fig. I.13 se muestra una representacin tpica
de esta ecuacin.

55

Altura equivalente a un plato terico

Miriam Beneito Cambra

H = A+

B
+ C u
u
u

C u
B
u

A
Uptima
Velocidad lineal promedia

Fig. I.13. Representacin de H frente a (curva de van Deemter).

I.5.2. CE
La CE es una tcnica de separacin que se basa en la migracin diferencial
de especies cargadas en el seno de un tubo capilar y en presencia de un campo
elctrico. La fuerza que impulsa el flujo en CE se genera en la misma pared
interna del capilar mediante el fenmeno conocido como electromosis

[Landers,

1994; Li, 1992; Camilleri, 1993; Heiger, 1992; Brown, 1995; Rogan, 1993].

I.5.2.1. Mecanismo de separacin en CE


La velocidad que adquiere un soluto cargado en el seno de un campo
elctrico es proporcional al campo:
v = e E

(E.I.7)

donde v es la velocidad del ion, e la movilidad electrofortica del mismo y E el


campo elctrico aplicado. La movilidad electrofortica es una constante
caracterstica del ion en un determinado medio. Viene determinada por el balance
entre la fuerza electrosttica, Fe, y las fuerzas de rozamiento, Ff:
Fe = q E

(E.I.8)

Ff = 6rv

(E.I.9)

56

Captulo I. Introduccin

donde r es el radio efectivo del ion hidratado o solvatado y es la viscosidad de


la disolucin. Cuando se alcanza el estado estacionario, ambas fuerzas son de la
misma intensidad pero signo opuesto, por lo que:
e = q/ (6r)

(E.I.10)

De esta ecuacin se deduce que las partculas con mayor densidad de carga
elctrica tendrn movilidades mayores. La movilidad se considera positiva
cuando el ion se dirige al ctodo. La movilidad medida en funcin del tiempo de
migracin se conoce como movilidad aparente o efectiva (ap), y difiere de la
intrnseca o electrofortica cuando se observa en presencia de EOF no nulo. La
movilidad aparente corresponde a la suma de las movilidades electrofortica y
electroosmtica, siendo la velocidad total de las partculas cargadas la suma de
sus velocidades electrofortica y electroosmtica:
v = ve + vEOF = apE = (e +EOF)E

(E.I.11)

I.5.2.2. EOF
El EOF es uno de los principales fenmenos que afectan a las
separaciones electroforticas. En contacto con un medio acuoso, la superficie del
capilar de slice tiene generalmente un exceso de carga negativa que es debido a
la ionizacin de los grupos silanol. Para capilares de slice fundida, el EOF se
puede controlar reduciendo o aumentando el nmero de grupos silanol en forma
ionizada. As, el EOF es prcticamente nulo por debajo de pH 3, y aumenta con
el pH. Para la slice se tiene: log Kmedio 5,5.
Los iones que se encuentran en la disolucin tienden a neutralizar la carga
de la superficie del capilar, formando una doble capa elctrica, y creando una
diferencia de potencial residual conocida como potencial zeta. La primera capa, o
capa de adsorcin primaria, est fuertemente retenida, pero sobre ella se
establece una capa difusa en la que predominan los iones del signo contrario al

57

Miriam Beneito Cambra

potencial zeta de la superficie. Esta segunda capa est menos retenida, por lo que
puede moverse por aplicacin de una diferencia de potencial, y en su movimiento
arrastra a todo el lquido. En la Fig. I.14 se representa esquemticamente este
fenmeno.

+
+
-

Capa de
adsorcin
primaria

+
-

+
+

+
-

+ +

+ +
-

+
-

+
-

+ +

+
+

+
+

+
+

+
+
-

+
+
+

+
-

+
+

EOF
-

+
-

+
-

+
-

+ -

+
-

+
+
+ +

+
-

+
-

+
+

+
-

+
+

+
-

+ +
+
-

+ + + + +
+
+
+
+
+
+
-

+
+
-

+
-

Capa
difusa

+
+

+ + +

Fig. I.14. Esquema de la generacin de EOF por aplicacin de campo


elctrico.

La velocidad del EOF (vEOF) es proporcional al campo:


vEOF = EOFE

(E.I.12)

donde EOF es la movilidad electroosmtica que es proporcional al potencial zeta


sobre la superficie:
EOF = /

(E.I.13)

donde es el potencial zeta o densidad de carga residual sobre la superficie de la


pared interna del capilar, y es la permisividad elctrica o constante dielctrica
del medio. La EOF depende de la naturaleza de la pared del capilar y de la
composicin del medio, especialmente del pH y de la fuerza inica. El potencial
zeta es funcin de la carga por unidad de superficie, y depende tambin del pH, y
de la concentracin y la naturaleza de todos los iones presentes.

I.5.2.3. Movilidad y tiempo de migracin


El tiempo que un soluto necesita para migrar desde el punto de inyeccin
hasta el punto de deteccin se denomina tiempo de migracin. El tiempo de
migracin se relaciona con los siguientes parmetros experimentales:
58

Captulo I. Introduccin

ap =

l
lL
=
tE tV

(E.I.14)

donde la ap es la movilidad aparente, V es el voltaje aplicado, l es la longitud


efectiva del capilar o distancia desde la entrada hasta el detector, L es la longitud
total del capilar, t es el tiempo de migracin y E es el campo elctrico aplicado.

I.5.2.4. Tcnicas de electroseparacin capilar


La electroseparacin capilar abarca diversas tcnicas caracterizadas por su
gran versatilidad. Las tcnicas ms utilizadas son:

A) FSCE o CZE
En la FSCE o CZE, el capilar se llena slo con el BGE, y la separacin
tiene lugar gracias a las diferentes movilidades electroforticas de los solutos. La
seleccin del BGE es extremadamente importante, ya que la selectividad de la
separacin depende en gran medida de su naturaleza. Los reactivos utilizados
deben cumplir las siguientes condiciones:
i) Buena capacidad amortiguadora en el intervalo de pH requerido.
ii) Baja absorbancia a la longitud de onda de deteccin.
iii) Baja movilidad electrofortica de sus componentes para minimizar la
corriente.

B) MEKC
La MEKC fue introducida en 1984 por Terabe y col. [Terabe, 1984; Terabe,
1989; Terabe, 1992; Otsuka, 1989],

y se utiliza para separar especies sin carga

elctrica neta. La separacin se basa en las diferencias de las constantes de


asociacin entre solutos y micelas. El BGE contiene un surfactante inico a
concentracin superior a su CMC. Tanto las micelas como los iones libres del
surfactante experimentan interacciones hidrofbicas y electrostticas con los

59

Miriam Beneito Cambra

solutos. Las especies no cargadas que interaccionan con las micelas adquieren
movilidad, distinguindose del EOF, y las que no interaccionan se mueven con el
mismo. Se puede aplicar tambin a especies inicas, si bien, en este caso el
mecanismo de separacin es mixto, cromatogrfico y electrofortico a la vez.

I.5.3. CEC
La CEC es una tcnica analtica de separacin en fase lquida que combina
la elevada eficacia de la CZE con la alta selectividad y reproducibilidad
proporcionadas por la HPLC. En CEC, la separacin se lleva a cabo en columnas
capilares rellenas total o parcialmente con una fase estacionaria. Como en CZE,
el EOF es generado por el campo elctrico. Para que se genere EOF debe
asegurarse la presencia de cargas sobre la superficie del relleno de la columna.
Por otro lado, el EOF es el responsable del bombeo en CEC y en otras tcnicas
de electroseparacin. El bombeo mediante el EOF da lugar a un perfil plano de
velocidades en el seno de la fase mvil, a diferencia del perfil parablico que se
obtiene cuando el bombeo es por presin externa, como en HPLC. El perfil plano
del flujo es uno de los factores que permite obtener elevadas eficacias.
En CEC el mecanismo de separacin es doble [Rathore, 1996]. Por un lado,
hay un mecanismo cromatogrfico, ya que se produce un reparto de los solutos
entre una fase mvil y una estacionaria. Por otro lado, los solutos inicos tambin
se separan mediante un mecanismo electrofortico, esto es, en base a las
diferencias de movilidad electrofortica, por lo que la naturaleza del relleno de la
columna determina el EOF e influye sobre la selectividad de la separacin.

I.5.3.1. Instrumentacin en CEC


Un instrumento para CEC (ver el esquema de la Fig. I.15) est constituido
bsicamente por una fuente de alto voltaje, un sistema de suministro de

60

Captulo I. Introduccin

disolvente y/o muestras en los viales de entrada y de salida de la columna, una


columna capilar con una fase estacionaria en la cual se genera el EOF y tambin
tiene lugar la separacin electrocromatogrfica, un compartimento isotrmico
para la columna capilar, y un sistema de deteccin capaz de registrar los perfiles
de concentracin de los analitos en el eluyente.
Fuente de
campo elctrico
Detector

Capilar
relleno
Fuente de
presin

BGE

Fig. I.15. Esquema de un instrumento de CEC.

La misma instrumentacin utilizada en CE sirve para CEC, si bien, en este


caso se debe de disponer de un sistema de presurizacin de los viales de entrada
y salida. Esta presurizacin es necesaria para evitar la formacin de burbujas, que
podran interrumpir la corriente. Las burbujas pueden originarse por diversas
causas, ya sea por diferencias locales en la velocidad del EOF [Rathore, 1998], en
el campo elctrico, por prdida del gas atrapado en los poros de la fase
estacionaria, o por gas formado electroqumicamente
calentamiento

[Knox, 1988; Tsuda, 1987],

[Carney, 1999],

por

o en el caso de columnas empaquetadas,

por la presencia de las fritas que mantienen la integridad estructural del relleno
[Rebscher, 1994].

La presurizacin se puede aplicar sobre el vial de entrada o en el

de salida, aunque generalmente se aplica sobre ambos viales, ya que as se


asegura un flujo reproducible. Para presurizar los viales se usa un gas inerte,
normalmente N2, a presiones prximas a 10 bares. Para obtener resultados
reproducibles en CEC, es necesario el control de parmetros como la temperatura

61

Miriam Beneito Cambra

de la columna, el voltaje aplicado y la presin. En los equipos comerciales de


CE, estos parmetros se controlan automticamente, lo que da lugar a mejoras
significativas de la reproducibilidad y seguridad de las separaciones.
La espectrofotomtrica UV-Vis es la tcnica de deteccin ms utilizada en
CEC

[Choudhary, 2000; Rozing, 2001; Devowsky, 2002; Cahours, 2002],

siendo

posible trabajar tambin en el modo de deteccin indirecto. La deteccin se


realiza en la misma columna, utilizando como celda de deteccin una pequea
seccin de la misma, adyacente al relleno, y de la cual se ha retirado la capa
protectora de polmero. Otras tcnicas de deteccin tambin ampliamente
utilizadas son la fluorescencia inducida por lser [Wall, 2002; Horstktter, 2002; Liu,
2001],

y la MS [Shamsi, 2004; Klampfl, 2004; Barcel-Barrachina, 2004].

I.5.3.2. Columnas empleadas en CEC


Las columnas para CEC se preparan normalmente a partir de capilares de
slice fundida con dimetros internos comprendidos entre 100 y 200 m. En base
a las diferencias en los soportes cromatogrficos, se pueden distinguir tres tipos
de columnas: abiertas, empaquetadas y monolticas.
Las columnas monolticas, constituidas por un lecho continuo, poseen una
estructura porosa que permite trabajar en HPLC con caudales elevados, y por
tanto, obtener separaciones rpidas sin que ello conlleve un aumento excesivo de
la presin necesaria para mantener el caudal, a diferencia de lo que sucede con
las columnas particuladas. En CEC las columnas monolticas constituyen
igualmente una alternativa a las empaquetadas, con algunas interesantes ventajas.
As, dada su estructura continua, no es necesario el empleo de fritas en los
extremos del lecho monoltico, ya que ste est anclado directamente sobre la
pared del capilar mediante enlaces covalentes. Adems, pueden prepararse in
situ, por lo que la fabricacin de lechos monolticos es relativamente sencilla en

62

Captulo I. Introduccin

comparacin con las tcnicas de empaquetado de partculas.


Las columnas monolticas pueden clasificarse en dos categoras
principales, de slice y polimricas. Las columnas de slice se preparan usando la
tecnologa sol-gel. La estructura de un monolito de slice muestra esqueletos
interconexionados que crean una distribucin determinada de poros.
Por otro lado, la preparacin de las columnas monolticas polimricas se
lleva a cabo fcilmente mediante el relleno de las columnas capilares con una
mezcla de polimerizacin constituida por monmeros, un agente entrelazante
(cross-linker), una mezcla porognica de disolventes y un iniciador radicalario.
La hidrofobicidad del monolito resultante se puede controlar seleccionando la
naturaleza del monmero [Liao, 1996; Palm, 1997]. El EOF se asegura mediante la
presencia de monmeros derivados de los cidos acrlico o sulfnico, o mediante
sales de amonio cuaternario en la mezcla de polimerizacin

[Ericson, 1999].

La

polimerizacin se inicia por medios trmicos, qumicos o mediante radiacin


UV.
Para evitar cualquier desplazamiento del monolito a lo largo de la
columna, es necesario anclar el polmero a la pared interna del capilar. Para ello,
antes de introducir en el capilar la mezcla de polimerizacin, se silaniza la pared
interna del mismo. Con este fin, en la mayor parte de los casos, se utiliza 3(trimetoxisilil)propil metacrilato (silano binding).
Para construir monolitos se han utilizado diferentes tipos de polmeros,
pudiendo distinguirse principalmente entre los derivados de acrilamida,
poliestireno y steres de metacrilato o acrilato. En las primeras columnas
monolticas que fueron descritas se utiliz acrilamida y metacrilamida
1989;

Fujimoto,

1995;

Hoegger,

2001].

[Hjertn,

Estos polmeros se preparan por

polimerizacin de acrilamida, metacrilamida o sus derivados en presencia de


metilenbisacrilamida o piperazina diacrilamida como agentes entrelazantes. Las

63

Miriam Beneito Cambra

columnas monolticas basadas en poliestireno

[Gusev, 1999; Petro, 1996]

se

obtienen por polimerizacin de estireno o sus derivados con divinilbenceno como


agente entrelazante. Los monolitos basados en steres de metacrilato
2003;

Zhang,

2003;

Peters,

1998-A;

Peters,

1998-B]

[Merthar,

se preparan mediante

polimerizacin de butilmetacrilato u otros steres derivados del metacrilato,


empleando etilenglicol dimetacrilato como agente entrelazante.

I.5.3.3. Polimerizacin de columnas monolticas basadas en steres de


metacrilato y acrilato
Las columnas monolticas basadas en polimetacrilato son las ms
extendidas y mejor caracterizadas, habiendo sido ampliamente desarrolladas por
Svec y col. [Peters, 1998-A; Peters, 1998-B], quienes han descrito aplicaciones tanto
en HPLC como en CEC. Los polmeros de metacrilato y acrilato poseen
caractersticas mecnicas y qumicas que los hacen altamente apropiados como
fases estacionarias. Son estables en un amplio intervalo de valores de pH (212),
a diferencia de las fases estacionarias basadas en slice, que se degradan con gran
facilidad por encima de pH 9. Su sntesis es rpida y sencilla, siendo posible
partir de monmeros de polaridades muy diversas.
La polimerizacin de los monolitos basados en steres de metacrilato y/o
acrilato se realiza mediante una reaccin radicalaria, iniciada generalmente por
una temperatura elevada, por irradiacin UV, o por agentes qumicos a
temperatura ambiente. Para la iniciacin trmica de la polimerizacin se suele
adicionar a la mezcla de monmeros AIBN
2001],

[Peters, 1998-A; Peters, 1998-B; Chirica,

perxido de benzoilo [Xie, 1997], u otros perxidos [Cant-Mirapeix, 2008-D].


Svec y col.

[Peters, 1998-A; Peters, 1998-B]

han demostrado que las

propiedades cromatogrficas (eficacia, selectividad, permeabilidad, etc) de estos


materiales pueden alterarse variando la composicin de la mezcla de

64

Captulo I. Introduccin

polimerizacin, lo que constituye una va interesante, no slo para el desarrollo y


optimizacin de las separaciones cromatogrficas, sino para sus aplicaciones de
inters ambiental, bioqumico e industrial.

I.5.3.4. Caracterizacin de materiales monolticos


Los materiales monolticos se pueden caracterizar estudiando tanto sus
propiedades morfolgicas como electrocromatogrficas. Existen numerosas
tcnicas que proporcionan informacin acerca de la influencia de diversos
factores sobre las propiedades morfolgicas de los materiales monolticos
Para el estudio de las propiedades morfolgicas de los materiales
monolticos, existen numerosos mtodos y herramientas analticas. Entre ellas
cabe citar la SEM
[Doneanu, 2002],

[Baeuml, 2002],

la porosimetra de intrusin de mercurio

la adsorcin/desorcin de nitrgeno, evaluada mediante la

ecuacin de Brunauer-Emmet-Teller

[Brunauer,

1938]

y la permeabilidad

cromatogrfica. Las columnas monolticas utilizadas a lo largo de esta Tesis


Doctoral se han caracterizado principalmente mediante SEM, por lo que esta
tcnica se comenta ms extensamente a continuacin.
Mediante SEM se obtienen imgenes de la estructura de un material.
Sobre la superficie del material se enfoca un haz de electrones. Este haz barre la
superficie del material, produciendo principalmente la emisin de electrones
secundarios de baja energa y electrones retrodispersados de mayor energa,
recogindose ambos mediante adecuados sistemas de deteccin [Aballe, 1996]. La
informacin obtenida vara segn las caractersticas del detector empleado. Los
electrones secundarios se forman en una delgada capa superficial, del orden de 5
a 10 nm de espesor. La seal est constituida en parte por electrones que emergen
de la muestra con una energa inferior a 50 eV. El nmero de electrones de este
tipo es suficientemente elevado como para establecer un buen contraste entre las

65

Miriam Beneito Cambra

estructuras que se quieren describir. Por otra parte, al tratarse de electrones de


baja energa, pueden desviarse fcilmente de su trayectoria emergente inicial, y
se puede obtener informacin de zonas que no estn a la vista del detector. Esta
particularidad es fundamental para otorgar a la seal la posibilidad de aportar una
informacin tridimensional de la topografa de la muestra, siendo quizs la
caracterstica ms conocida de esta tcnica.
Por otro lado, la principal utilidad de la seal de electrones
retrodispersados, compuesta por aquellos electrones que emergen de la muestra
con una energa superior a 50 eV, reside en que su emisin depende fuertemente
del nmero atmico de los elementos de la muestra. Por esta razn, dos zonas
con distinta composicin qumica se revelarn con distinta intensidad, aunque no
exista ninguna diferencia de topografa entre ellas.
Para aplicar SEM la muestra a analizar debe estar seca. En caso contrario,
la baja presin existente en el microscopio causara la evaporacin de los
componentes voltiles que saldran despedidos violentamente, alterando la
estructura de la muestra. Adems, la superficie debe ser conductora, lo que se
consigue recubrindola con una pelcula de un material conductor. Para ello, se
utilizan tcnicas de pulverizacin catdica a alto vaco. Por otro lado, la reducida
estabilidad trmica de los polmeros limita el voltaje que puede aplicarse para
obtener las imgenes.

I.5.4. MS
La MS es una tcnica de aplicacin general, capaz de suministrar
informacin sobre la composicin cualitativa y cuantitativa, tanto de analitos
orgnicos como inorgnicos, en muestras complejas. Los espectros de masas se
obtienen por conversin de los componentes de una muestra en sus respectivos

66

Captulo I. Introduccin

iones en fase gas, que se separan en funcin de su relacin m/z, siendo la seal
analtica la abundancia o intensidad para cada valor m/z.
En la Fig. I.16 se muestra un esquema de los componentes principales de
un espectrmetro de masas.
Muestra

10-5 - 10-8 torr


Sistemade
de
Sistema
entrada
entrada

Analizador
Analizador
demasas
masas
de

Fuentede
deiones
iones
Fuente

Sistema
Sistema
devaco
vaco
de

Detector
Detector

Procesador
Procesador
dela
laseal
seal
de

Dispositivo
Dispositivo
delectura
lectura
de

Fig. I.16. Componentes de un espectrmetro de masas.

Como se ilustra en la figura, un espectrmetro de masas est formado en


primer lugar por un sistema de entrada, que permite la introduccin de una
pequea cantidad de muestra en el espectrmetro. El sistema de entrada elegido
ser diferente segn se quieran introducir muestras slidas, lquidas o gaseosas.
La muestra se puede introducir de manera discreta mediante una jeringa o una
sonda directa, o de forma continua mediante el acoplamiento con un sistema de
inyeccin en flujo o mediante un sistema cromatogrfico o electrofortico.
Junto al sistema de entrada, la fuente de iones es la encargada de convertir
los componentes de la muestra en iones por bombardeo con electrones,
molculas, fotones o por otros medios. En muchas ocasiones el sistema de
entrada y la fuente de iones estn combinados en un nico componente. Una vez
producidos los iones, stos son acelerados hacia el analizador por aplicacin de
campos elctricos. En el analizador de masas los iones se separan en base a su
relacin m/z, de forma que llegan al detector en diferentes momentos. Esta

67

Miriam Beneito Cambra

pequea corriente de iones se amplifica mediante el transductor, normalmente un


multiplicador de electrones que puede estar combinado con un multiplicador de
fotones

[Rubinson, 2001; Skoog, 1996].

Estos cuatro componentes se encuentran,

generalmente, dentro de un sistema de vaco, a unas presiones de 10-7 10-10 atm,


necesarias para evitar colisiones con el gas de fondo u otras molculas. Slo en
algunos casos, el vaco se aplica tan slo al analizador de masas y al detector. Por
ltimo, una vez registrada la seal analtica, se procede a su procesado y anlisis,
obtenindose el espectro de masas. En el espectro de masas se muestran las
cantidades de masa recogidas a valores crecientes de la relacin m/z. Cada pico
del espectro proporciona informacin sobre la masa de un determinado
fragmento de la molcula que lo origin. Para las fuentes habituales en HPLC y
CE, y para los espectros obtenidos en modo positivo, el fragmento de mayor
tamao suele ser la molcula sin fraccionar con prdida de un electrn, M+, o
con la adicin de un protn, [M+H]+, u otro catin.

I.5.4.1. Fuentes de iones


Las fuentes de iones ms habituales son las siguientes [Rubinson, 2001]: EI,
CI, ESI, APCI, APPI, ICP y FAB. Dentro de estas, las fuentes ms utilizadas en
el acoplamiento LC-MS, son la ESI, la APCI y la APPI. En esta seccin slo se
explica la fuente ESI, que es la que se ha empleado en alguno de los trabajos de
esta Tesis.
Dos caractersticas importantes de la ESI (Fig. I.17) son la generacin de
iones en fase gas en la misma forma en que se encuentran en disolucin, y la
relativa facilidad para impedir la entrada de grandes cantidades de disolvente en
el analizador de masas. Por esta ltima razn, la ESI es una fuente muy utilizada
en los acoplamientos HPLC-MS. En ESI la disolucin de la muestra fluye a
travs de una aguja hueca. La aguja se encuentra sometida a un campo elctrico

68

Captulo I. Introduccin

elevado respecto a las paredes de la cmara de nebulizacin, por lo que se forman


pequeas gotas con carga elctrica. La aguja tiene dos flujos concntricos, el
interior que lleva la muestra, y el exterior que lleva un gas que ayuda a la
formacin del aerosol. Las gotas que poseen carga neta son atradas hacia un
electrodo a travs del espacio abierto de la cmara de nebulizacin. En la cmara,
las gotas se mueven en contra del gas de secado, que evapora parte del
disolvente.
Iones
Gas nebulizador

Gas de secado
Aerosol

Entrada del capilar

Fig. I.17. Esquema de una fuente ESI.

Como consecuencia de este proceso, el volumen de las gotas disminuye y


los iones de la superficie se ven forzados a aproximarse unos a otros. En un
momento dado, la repulsin de los iones se hace mayor que la tensin superficial
que mantiene unidas a las gotas y stas se rompen (explosin de Coulomb). De
estas gotas se generan los iones en fase gas, que son atrados hacia el orificio de
entrada del analizador de masas por la accin de un segundo campo elctrico.
Dependiendo de la polaridad de este campo, en el analizador de masas entran
slo los aniones (operacin en modo negativo) o slo los cationes (modo
positivo). Los procesos de generacin de iones y evaporacin del disolvente
tienen lugar en la superficie de las gotas, y cualquier cambio en la misma puede
ser muy importante. La fuente de ionizacin por electronebulizacin puede

69

Miriam Beneito Cambra

producir iones con carga mltiple, lo que permite el anlisis de compuestos de


elevada masa.

I.5.4.2. Analizadores de masas


La funcin de los analizadores de masas es la de separar iones con
diferentes relaciones m/z

[Rubinson,

2001].

Existen diferentes tipos de

analizadores, y el poder de resolucin del espectrmetro de masas depender


principalmente de las prestaciones del analizador. Los analizadores de masas ms
ampliamente utilizados en HPLC y CE son el cuadrupolo (simple o triple), la IT
y el de tiempo de vuelo. En esta seccin slo se explica la trampa de iones, por
ser la empleada en algunos de los trabajos de esta Tesis.
Una IT permite analizar cationes y aniones en un amplio intervalo de
valores de m/z. Mediante la accin de campos elctricos y magnticos, los iones
se pueden confinar en un espacio reducido durante largos periodos de tiempo. Un
diseo sencillo de trampa de iones consiste en un electrodo en forma de anillo y
un par de electrodos colectores (electrodos de entrada y de salida). Al electrodo
anular se le aplica un potencial de radiofrecuencia variable, mientras que los
electrodos colectores estn a potenciales alternos adecuados para estabilizar las
rbitas de los iones en el plano transversal o plano del anillo. Los iones que
tienen el valor apropiado de m/z se mueven en rbitas estables, cuyo radio
depende de un valor de m/z, dentro de la cavidad formada por el anillo.
Los iones procedentes de la fuente de ionizacin se introducen a travs de
una abertura practicada en el electrodo colector superior, quedando atrapados en
rbitas estacionarias (Fig. I.18). A continuacin, se desestabilizan mediante una
rampa de potencial continuo superpuesta al potencial alterno del electrodo de
salida. Los iones dejan la cavidad del anillo a travs del electrodo de salida,

70

Captulo I. Introduccin

pasando al detector. En una IT, el proceso de carga y transmisin de iones al


detector es discontinuo.
Captura de iones

Eyeccin secuencial

Incremento de V
Fig. I.18. Esquema de funcionamiento de una IT.
Entrada
Gas
nebulizador

Nebulizador

Desecho

Vaporizacin
y ionizacin
(ESI)

Gas de
secado

Enfoque

Analizador
Deteccin
(IT)

Fig. I.19. Esquema de un espectrmetro de masas con fuente ESI y


analizador IT.

La capacidad de retener iones en la IT permite obtener conjuntos de iones


hijos, que se pueden aislar o fragmentar para barrer espectros de segunda
generacin o MS2. A su vez, los iones resultantes pueden ser de nuevo aislados y
fragmentados para barrer espectros de tercera generacin o MS3, y as
71

Miriam Beneito Cambra

sucesivamente. Las fragmentaciones en el interior de la IT se consiguen mediante


colisiones con molculas de He.
En la Fig. I.19 se muestra un esquema de las diferentes partes de un
espectrmetro de masas compuesto por una fuente ESI y un analizador IT.

I.5.4.3. Acoplamiento de una tcnica de separacin a un MS


Los MS se pueden acoplar con GC, LC, SFC o con sistemas de CE o
CEC. Para mostrar todos los componentes eluidos en la separacin se representa
un TIC. La intensidad en el TIC es la suma de las respuestas del detector a todos
los iones presentes en cada momento. Tambin se pueden representar
cromatogramas a determinados valores de m/z, uno o varios. Estos se pueden
obtener de dos formas: como SIMs y, a partir de los datos almacenados en un
TIC, como EICs.
Cuando el cromatograma se registra a un valor m/z nico (o a unos pocos
valores de m/z), en lugar de hacer un barrido en un intervalo de m/z, la tcnica se
denomina SIM. Esta monitorizacin proporciona un lmite de deteccin
considerablemente menor que un EIC, especialmente si el EIC se ha obtenido a
partir de un barrido muy amplio de valores de m/z. Adems, un SIM permite
simplificar la seal en cromatogramas complejos que presenten numerosas
interferencias.
Para introducir en el espectrmetro el efluente procedente de un
cromatgrafo lquido o de una separacin electrofortica, es necesario utilizar
una interfaz adecuada. La misin de la interfaz es eliminar en lo posible la fase
mvil sin distorsionar el perfil de concentraciones de los analitos. Actualmente,
las fuentes ESI, APCI y APPI son las interfaces ms utilizadas en los
acoplamientos HPLC-MS, CE-MS y CEC-MS. Una limitacin de los
acoplamientos LC-MS o CE-MS es que los modificadores de la fuerza eluyente,

72

Captulo I. Introduccin

los tampones o electrolitos de fondo deben ser voltiles, para reducir en lo


posible los lmites de deteccin y minimizar las posibles interferencias en el
espectrmetro.

I.5.4.4. Resolucin
La capacidad de un espectrmetro de masas para distinguir entre masas
similares se expresa normalmente en trminos de resolucin, R, que se define
como:
R = m/m

(E.I.15)

Siendo m la masa nominal del primer pico, e m el poder resolutivo del


espectrofotmetro que se define como la anchura del primer pico a una fraccin
dada de la altura, con frecuencia a un 10% o a un 50%.
La resolucin que se necesita en un espectrmetro de masas depende en
gran parte de su aplicacin. Por ejemplo, para diferenciar entre iones de la misma
masa nominal pero con diferentes masas exactas, se necesitan equipos de elevada
resolucin.

I.5.5. NMR
La espectroscopa de NMR fue desarrollada a finales de los aos cuarenta
para estudiar los ncleos atmicos. En 1951, se descubri que la espectroscopa
de resonancia magntica nuclear poda ser utilizada para determinar las
estructuras de los compuestos orgnicos. Esta tcnica espectroscpica puede
utilizarse slo para estudiar ncleos atmicos con un nmero impar de protones o
neutrones (o de ambos). Esta situacin se da en ncleos de una docena de
elementos, entre ellos los de 1H,

13

C,

19

F y

31

P. Este tipo de ncleos son

magnticamente activos, es decir poseen espn no nulo. En el seno de un campo

73

Miriam Beneito Cambra

magntico estos ncleos se comportan como pequeos imanes, pudiendo adquirir


un movimiento de precesin, cuyos niveles estn cuantizados.
En ausencia de campo magntico, los espines nucleares se orientan al azar.
Sin embargo, cuando una muestra se coloca en un campo magntico, los ncleos
con espn positivo se orientan en la misma direccin del campo, en un estado de
mnima energa denominado estado de espn , mientras que los ncleos con
espn negativo se orientan en direccin opuesta a la del campo magntico, en un
estado de mayor energa denominado estado de espn . Existen ms ncleos en
el estado de espn que en el siendo esta diferencia suficiente para establecer
las bases de la espectroscopa de NMR.
La diferencia de energa entre los dos estados de espn y , depende de
la fuerza del campo magntico aplicado H0. Cuanto mayor sea el campo
magntico, mayor diferencia energtica habr entre los dos estados de espn.
Cuando una muestra es irradiada brevemente por un pulso intenso de radiacin,
los ncleos en el estado de espn son promovidos al estado de espn . Esta
radiacin se encuentra en la regin de las radiofrecuencias (rf) del espectro
electromagntico, por ello se le denomina radiacin rf. Cuando los ncleos
vuelven a su estado inicial, emiten seales cuya frecuencia depende de la
diferencia de energa (E) entre los estados de espn y . El espectrmetro de
NMR detecta estas seales y las registra como una grfica de frecuencias frente a
intensidad, que es el llamado espectro de NMR. El trmino NMR procede del
hecho de que los ncleos estn en resonancia con la radiofrecuencia de la
radiacin rf. Es decir, los ncleos pasan de un estado de espn a otro como
respuesta a la radiacin rf a la que son sometidos. La siguiente ecuacin muestra
la dependencia entre la frecuencia de la seal () y la fuerza del campo
magntico H0 (medida en Teslas, T).
E = h = h

H0
2
74

(E.I.16)

Captulo I. Introduccin

donde h es la constante de Planck y el radio giromagntico. El valor de este


ltimo depende del tipo de ncleo que se est irradiando.

I.5.5.1. Espectrmetro de NMR


En la Fig. I.20 se muestran de forma esquemtica los principales
componentes de un equipo para medidas de NMR.
Espectro
de NMR

Tubo con
muestra

Detector
y
Amplificador

Generador de
radiofrecuencia y
ordenador

Imn
superconductor

Fig. I.20. Esquema de un espectrmetro de NMR.

Como se aprecia, el espectrmetro de NMR consta de cuatro partes: un imn con


un controlador que produce un campo magntico preciso y estable, un transmisor
de radiofrecuencias, un detector para medir la absorcin de energa de rf de la
muestra, ordenador y un registrador para obtener el espectro de NMR.
Para obtener un espectro de NMR, se coloca una pequea cantidad del
analito en un disolvente en un tubo de vidrio largo que se sita dentro del campo
magntico. El campo magntico se mantiene constante mientras un breve pulso
de radiacin rf excita a todos los ncleos simultneamente. Dado que el corto
pulso de radiofrecuencia cubre un amplio intervalo de frecuencias, los protones
individualmente absorben la radiacin de frecuencia necesaria para entrar en
resonancia (cambiar de estado de espn). A medida que dichos ncleos vuelven a
75

Miriam Beneito Cambra

su posicin inicial emiten una radiacin de frecuencia igual a la diferencia de


energa entre estados de espn. La intensidad de esta frecuencia disminuye con el
tiempo a medida que todos los ncleos vuelven a su estado inicial. Un ordenador
recoge la intensidad respecto al tiempo y convierte dichos datos en intensidad
respecto a la frecuencia. La operacin matemtica conocida como transformada
de Fourier (FT) permite realizar dicha conversin entre los dominios del tiempo
y de la frecuencia.

I.5.5.2. NMR de 1H
Hasta ahora se ha descrito el concepto de resonancia de un ncleo aislado
dentro de un campo magntico, pero los ncleos se encuentran rodeados de
electrones que los protegen parcialmente del campo magntico externo al que se
ven sometidos. Los electrones se mueven generando un pequeo campo
magntico inducido que se opone al campo magntico externo. En cualquier
molcula, la nube electrnica que existe alrededor de cada ncleo acta como
una corriente elctrica en movimiento que, como respuesta al campo magntico
externo, genera una pequea corriente inducida que se opone a dicho campo. El
resultado es que el campo magntico que realmente llega al ncleo es ms dbil
que el campo externo, por tanto, se dice que el ncleo est apantallado. Este
apantallamiento es importante desde el punto de vista experimental, ya que el
campo magntico efectivo (Hef) que siente un protn dentro de una molcula es
siempre menor que el campo externo, y por lo tanto, para que el ncleo entre en
resonancia dicho campo externo debe ser mayor.
Si todos los protones (1H) de una molcula estuvieran apantallados de
igual forma, todos entraran en resonancia con la misma combinacin de
frecuencia y campo magntico. Sin embargo, los protones se hallan dentro de
entornos electrnicos diferentes y, por tanto, diferentemente protegidos o

76

Captulo I. Introduccin

apantallados. Por lo general, los efectos de apantallamiento de las nubes


electrnicas que rodean a cada protn son diferentes, lo que provoca diferentes
frecuencias de emisin. El resultado es un espectro de diversas frecuencias donde
cada conjunto de ncleos especficos da origen a una seal nica de NMR. Las
variaciones en las frecuencias de absorcin de NMR, que tienen lugar debido al
distinto apantallamiento de los ncleos, reciben el nombre de desplazamientos
qumicos (unidades ppm).
En la prctica, es difcil medir con suficiente exactitud y en trminos
absolutos el campo magntico al que un protn absorbe, lo que impide distinguir
tipos de protones individuales, ya que las absorciones entre uno y otro tipo slo
varan en unas pocas milsimas. Un mtodo ms exacto para expresar
desplazamientos qumicos es determinar el valor respecto a un compuesto de
referencia que se aade a la muestra. La diferencia en la intensidad del campo
magntico necesario para la resonancia de los protones de la muestra y de los
protones de referencia se puede medir con mucha exactitud. El compuesto de
referencia ms comn en NMR es el TMS ((CH3)4Si). Como el silicio es menos
electronegativo que el carbono, los grupos metilo del TMS son relativamente
ricos en electrones, estando sus protones fuertemente apantallados. Como
consecuencia, estos protones absorben a una intensidad de campo mayor que el
resto de protones enlazados al carbono o a otros elementos, de manera que casi
todas las seales de NMR aparecen a campos ms bajos. Adems, todos los
protones del TMS absorben con el mismo desplazamiento qumico dando una
nica absorcin intensa. Las escala ms comn de desplazamiento qumico es la
escala , en la que la absorcin del TMS se define como 0,00 . La mayor parte
de los protones absorben a campos menores que el TMS, de modo que la escala
aumenta hacia los campos menores. La mayora de las seales de protones (1H)
varan entre 0 y 12 , mientras que las seales del 13C varan de 0 a 250 .

77

Miriam Beneito Cambra

I.5.5.3. NMR de 13C


La resonancia magntica nuclear del

13

C es complementaria a la del 1H.

Esta ltima se utiliza para deducir la estructura del esqueleto carbonado,


observando para ello los entornos magnticos de los tomos de hidrgeno,
mientras que la espectroscopa de RMN del 13C determina el entorno magntico
de los tomos de carbono.
Aproximadamente el 99% de los tomos de carbono en una muestra
natural son del istopo

12

C. Este istopo posee un nmero par de protones y un

nmero par de neutrones, por tanto, no tiene espn magntico y no puede dar
lugar a seales de NMR. El istopo de

13

C menos abundante tiene un nmero

impar de neutrones, lo que le confiere un espn magntico de 172, igual al del


protn. La espectroscopia de NMR de 13C es menos sensible que la de 1H, debido
a que slo el 1% de los tomos de carbono presentes posee espn no nulo y a que,
adems, la frecuencia de resonancia del 13C, para un campo magntico dado, es la
cuarta parte de la que se da en la RMN del 1H.
Los desplazamientos qumicos del carbono son de 15 a 20 veces mayores
que los del hidrgeno, debido a que el carbono est directamente unido a los
tomos que resultan ser bien apantallantes o desapantallantes. Adems, las
seales en el espectro del

13

C son lneas verticales aisladas, es decir, no hay

desdoblamientos de espn-espn. Esto se debe a que slo el 1% de los tomos de


13

C entran en resonancia, ya que la probabilidad de que un ncleo de

adyacente a otro ncleo de 13C es muy pequea.

78

13

C sea

Captulo I. Introduccin

I.5.6. Algunas tcnicas de tratamiento estadstico de datos

I.5.6.1. Anlisis clasificatorio supervisado


En el anlisis clasificatorio supervisado, se construyen modelos capaces
de pronosticar la pertenencia de un objeto a una categora a partir de variables
cuantitativas o de escala. La matriz de datos contiene al menos una variable
categrica, que indica la categora a la que pertenece cada objeto y que constituye
la respuesta o variable que se quiere predecir, y una o ms variables de escala que
describen otras tantas caractersticas de los objetos y que se utilizan como
predictoras.
Para construir el modelo, es necesario disponer de una muestra de objetos
cuya categora sea conocida y para los que tambin se conozcan los valores de
las variables predictoras. La pertenencia de los objetos a las categoras puede ser
supuesta, esto es, puede tratarse de una hiptesis a comprobar. La asignacin de
los objetos a las categoras debe ser exhaustiva (todos los objetos pertenecen a
alguna categora) y mutuamente exclusiva (ningn objeto pertenece a ms de una
categora). Estos objetos forman el conjunto de entrenamiento (training set), con
el cual se construye el modelo de clasificacin. Una vez construido, el modelo se
utiliza para predecir la categora de nuevos objetos a partir de la medida de las
predictoras. La prediccin sobre un conjunto de evaluacin (evaluation set)
permite validar el modelo, que luego se aplicar a predecir la categora de las
muestras problema.
Se utilizan diversos tipos de tcnicas clasificatorias, tales como el anlisis
discriminante, que puede ser lineal (LDA) o cuadrtico (QDA) y la tcnica de las
ANN.

79

Miriam Beneito Cambra

I.5.6.2. LDA
En LDA se utiliza un algoritmo que busca funciones o vectores
discriminantes, esto es, combinaciones lineales de las variables manifiestas que
maximizan la varianza entre categoras, a la vez que minimizan las varianzas
intra-categoras. Para construir el modelo, es necesario asignar los objetos del
conjunto de entrenamiento a una categora dada. Para ello, se aade una variable
categrica a la matriz de datos conteniendo tantas categoras como sean
necesarias. El LDA estima los coeficientes a1, a2, , am de la funcin
discriminante lineal, f, que es capaz de predecir la pertenencia de los objetos a
una u otra categora:
f = a1 x1 + a2 x2 + ... + am xm

(E.I.17)

Las funciones discriminantes se contruyen de una en una, buscando las


direcciones del espacio que hacen mxima la expresin:
'=

SC D
SCI

(E.I.18)

donde SCD es la suma de cuadrados de las distancias eucldeas entre los objetos
que pertenecen a distintas categoras en la direccin que indica la funcin
discriminante buscada, y SCI es la suma de los cuadrados de las distancias
eucldeas entre los objetos que pertenecen a la misma categora, tambin en la
direccin de la funcin discriminante. A partir de q categoras se obtienen q-1
funciones discriminantes (aunque si el nmero de variables predictoras, N, es
menor que q, se obtendrn N-1 funciones discriminantes). Las funciones
discriminantes se obtienen en orden decreciente de su valor de , y manteniendo
la ortogonalidad entre ellas.
La funcin no est acotada, por lo que vara ampliamente con el
nmero de objetos y con la separacin entre ellos. Por ello, en lugar de
maximizar , se suele minimizar la lambda de Wilks, que se define como:

80

Captulo I. Introduccin

W =

1
SCI
=
1 + ' SCI + SCD

(E.I.19)

Esta funcin toma valores entre 0 y 1. Categoras bien resueltas proporcionan


valores de w prximos a 0, mientras que categoras solapadas dan valores de w
cercanos a la unidad.

81

CAPTULO II

OBJETIVOS Y PLAN DE TRABAJO

Captulo II. Objetivos y plan de trabajo

La presente Memoria se enmarca dentro de una de las lneas de


investigacin del grupo, financiada principalmente por el MICINN y fondos
FEDER (proyecto CTQ2007-61445, cuyo investigador principal es el Dr.
Guillermo Ramis), y cuyo objeto general es el desarrollo de mtodos analticos
para el control industrial y ambiental de componentes de los productos de
limpieza. En consecuencia, el objetivo principal de los trabajos presentados en
esta Memoria es la puesta a punto de mtodos de anlisis rpidos y fiables para
diferentes analitos de la industria de la detergencia. La presente Memoria puede
dividirse en cuatro grandes bloques. El primer bloque est dedicado a los
surfactantes, tanto no inicos como aninicos (captulos IV y V). El segundo y
tercer bloque estn dedicados a los polmeros sintticos (captulo VI) y a las
enzimas (captulo VII), utilizados en productos de limpieza. Por ltimo, y
entroncando con otra de las lneas de investigacin del grupo, uno de los bloques
de esta Memoria est dedicado a la preparacin de columnas monolticas para
CEC. En particular, en este bloque se muestra el trabajo realizado en el campo de
la optimizacin de columnas, con el objeto de aplicar dichas columnas a la
separacin de digestos de enzimas obtenidos con tripsina, trabajo actualmente en
desarrollo, y que por razones de tiempo no se incluye en esta Memoria.

II.1. Surfactantes

II.1.1. APGs
Se propone el desarrollo de mtodos para la separacin y determinacin de
APGs. El estudio de la separacin de mezclas de APGs se llev a cabo mediante
HPLC-MS usando columnas de alquilamida y cianopropilo, con mezclas
ACN/agua como fases mviles. Se optimizaron las condiciones de elucin para
conseguir la resolucin completa de epmeros (- y -) e ismeros de anillo

85

Miriam Beneito Cambra

(piransidos y furansidos) de los APGs, tanto en materias primas industriales


como en productos manufacturados. Asimismo, se estudi la influencia de
distintos parmetros en los factores de respuesta de los APGs. Los
procedimientos establecidos se aplicaron a la caracterizacin y determinacin de
APGs en productos de aseo personal.
Por otro lado, se propone el estudio de la fragmentacin secuencial de los
AMGs, que son los principales componentes de los APGs industriales. La
produccin industrial de APGs conduce a mezclas que, adems de contener
importantes cantidades de AMGp, (ismeros de anillo con 6 miembros)
presentan pequeas aunque significativas concentraciones de AMGf (ismeros
de anillo con 5 miembros). As pues, se obtendrn y describirn los espectros
CID tanto para los AMGp como para AMGf, utilizando diferentes formas
isotpicas de la glucosa para ayudar a su interpretacin. Para la obtencin de los
espectros, se emple infusin directa en MS-IT, o bien, HPLC-MS-IT.

II.1.2. Alcoholes
Se pretende el desarrollo de un procedimiento de derivatizacin que
permita aumentar la sensibilidad de los alcoholes no etoxilados y etoxilados por
ESI-MS-IT. Para ello, los alcoholes primarios sern previamente oxidados a sus
correspondientes cidos carboxlicos con el reactivo de Jones. Los extractos se
infundirn directamente en ESI-MS-IT. El estudio se extender a FAEs y a los
disolventes conocidos como Cellosolves, que al oxidarse proporcionan los
correspondientes cidos etoxi-carboxlicos. Los procedimientos establecidos se
aplicarn a la caracterizacin y determinacin de alcoholes grasos en muestras
complejas, tales como cosmticos y productos de aseo personal, as como a
muestras ambientales como agua de mar.

86

Captulo II. Objetivos y plan de trabajo

II.1.3. AES
En trabajos anteriores del grupo de investigacin en el seno del cual cual
se ha realizado la presente Tesis, y en el marco de una de sus lneas de
investigacin, se desarrollaron diferentes procedimientos para la determinacin
de FAEs mediante RP-HPLC-UV, previa derivatizacin de los alcoholes con un
anhdrido cclico

[Mic-Tormos, 2008-A; Mic-Tormos, 2008-B; Mic-Tormos, 2009;

Mic-Tormos, 2010].

Sin embargo, se ha observado que los anhdridos cclicos

causan la derivatizacin tanto de FAEs como AESs, dando lugar a exactamente


los mismos derivados, en concreto, los hemisteres del cido correspondiente al
anhdrido utilizado. Se pretende desarrollar un mtodo para la separacin,
caracterizacin y determinacin mezclas de FAEs y AESs en muestras tanto
industriales como ambientales. Para ello la separacin de las dos familias de
surfactantes se llevar a cabo mediante SPE utilizando un cartucho SAX. Tras la
optimizacin de los parmetros de extraccin se emplearn las tcnicas de
derivatizacin anteriormente publicadas con diferentes anhdridos cclicos.
Finalmente se separarn los oligmeros obtenidos mediante HPLC-UV. El
mtodo resultante se aplicar a la caracterizacin y determinacin de FAEs y
AESs en muestras de diferente naturaleza.

II.2. Polmeros

II.2.1. PVP-NO
Se estudiar la migracin caracterstica del PVP-NO, tanto en FSCE como
en MEKC. Estas dos tcnicas pueden proporcionar informacin sobre el
comportamiento cualitativo de polmeros en disolucin, as como informacin
cuantitativa sobre muestras que contienen estos aditivos. Se discutir la
naturaleza de las seales obtenidas en base a los estudios de la bibliografa, as

87

Miriam Beneito Cambra

como a los nuevos resultados. Para prevenir la adsorcin del polmero sobre las
paredes del capilar se utilizar PEA, que en disoluciones cidas existe como ion
doble de baja masa molecular (sin carga elctrica neta). Este tipo de aditivo
puede usarse en elevadas concentraciones sin causar un aumento significativo de
la conductividad del BGE. Finalmente, se demostrar la utilidad de la FSCE para
determinar este polmero en aditivos comerciales para el lavado de textiles.

II.2.2. PVP
Se pretende desarrollar de un mtodo para la caracterizacin y
determinacin de PVP en productos de limpieza mediante CZE. Dado que la
movilidad electrofortica del PVP es casi cero, se emplearn colorantes azoicos
que formen complejos con el polmero. Estos sistemas pueden interpretarse a la
luz de los estudios de Krylov y col., con aplicacin de los principios en que se
basa la tcnica conocida como NECEEM. A partir de los datos obtenidos y en
base a los estudios de la bibliografa, se buscarn diferentes parmetros
analticos. Posteriormente los procedimientos establecidos se aplicarn a la
caracterizacin y determinacin de PVP en productos de limpieza y formulados
farmacuticos.

II.2.3. PVA
Siguiendo con la determinacin de polmeros no inicos mediante tcnicas
de CE, se pretende el desarrollo de un mtodo para la caracterizacin y
determinacin de este polmero. Dado que la movilidad electrofortica del PVA
es casi cero, al igual que se ha indicado ms arriba para el PVP (apartado II.2.2),
se emplearn colorantes que formen complejos coloreados y cargados con el
PVA, seleccionando los colorantes que mejores resultados proporcionen;

88

Captulo II. Objetivos y plan de trabajo

finalmente, se interpretarn los resultados aplicando los principios de la


NECEEM.

II.3. Enzimas

II.3.1. CZE
Se pretende examinar enzimas intactas (protenas nativas) mediante CZE
con deteccin UV, como medio de clasificacin e identificacin de enzimas. Se
estudiarn las cuatro clases principales de enzimas utilizadas en productos de
limpieza: proteasas, amilasas, lipasas y celulasas. Para ello, las enzimas se
precipitarn con acetona, se redisolvern e inyectarn en el equipo de CE. Dado
que la carga y estructura de las protenas dependen en gran medida del pH, y con
el objetivo de reunir ms informacin para la identificacin de enzimas, se
emplearn dos BGEs diferentes, uno a pH cido y otro a bsico. Se utilizar el
ensayo de Bradford para medir las concentraciones de protena en las diferentes
enzimas industriales (seleccionando el BSA como protena de referencia).

II.3.2. Aminocidos por infusin directa en MS


Se pretende desarrollar un mtodo rpido y simple para la clasificacin de
enzimas presentes en productos de limpieza (proteasas, lipasas, amilasas y
celulasas). Para ello, se llevar a cabo su precipitacin con acetona, seguida de
hidrlisis cida, para obtener as los aminocidos constituyentes. Los
correspondientes digestos de aminocidos sern infundidos en ESI-MS-IT. Las
abundancias de los iones procedentes de los diferentes aminocidos se utilizarn
para construir modelos de LDA, capaces de distinguir entre las diferentes clases
de enzimas.

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Miriam Beneito Cambra

II.3.3. Aminocidos derivatizados con OPA-NAC


Se pretende desarrollar un mtodo para la identificacin de las clases de
enzimas en la industria de los detergentes. Para ello, las enzimas concentradas
industriales y las presentes en productos de limpieza se aislarn por precipitacin
con acetona y se hidrolizarn con HCl. Los aminocidos resultantes se
derivatizarn con OPA en presencia de NAC, y sus perfiles de concentracin
sern establecidos por RP-HPLC con deteccin UV-Vis. A partir de las reas de
los picos observados en los cromatogramas se construirn modelos LDA capaces
de predecir la clase de enzima: proteasa, lipasa, amilasa o celulasa. Los conjuntos
de entrenamiento se construirn a partir de los concentrados industriales de
enzimas, como tambin mediante bases de detergente aditivadas con enzimas.

II.3.4. Digestos con tripsina


Siguiendo en la lnea de intentar identificar las diferentes enzimas
utilizadas en las formulaciones de productos de limpieza, se pretende buscar un
mtodo alternativo basado en la digestin con tripsina, seguida de separacin de
los pptidos resultantes por RP-HPLC con deteccin UV-Vis. Se compararn los
perfiles peptdicos obtenidos en los digestos utilizando diferentes soportes
cromatogrficos,

incluyendo

columnas

monolticas

comerciales

(tanto

polimricas, como de slice) y columnas partculadas (tanto con relleno


convencional como con relleno de partculas con tecnologa corteza-ncleo
(shell-core). Bajo las condiciones ptimas, se llevar a cabo el anlisis de los
digestos con tripsina de enzimas de diferentes clases. Se utilizarn parmetros
como el nmero de picos, capacidad de pico y resolucin global para evaluar
cada lecho cromatogrfico. El mtodo propuesto se aplicar tanto a la
caracterizacin de las enzimas presentes en bases de detergentes aditivadas,
como en detergentes comerciales.

90

Captulo II. Objetivos y plan de trabajo

II.4. Columnas monolticas

II.4.1. Columnas monolticas de lauril metacrilato fotopolimerizadas usando


LPO como iniciador
Se pretende describir la preparacin de columnas monolticas para CEC
usando LMA como base para sintetizar el polmero. La polimerizacin se llevar
a cabo mediante radiacin UV utilizando LPO como iniciador. Para obtener
resultados satisfactorios se optimizar la composicin de la mezcla de
polimerizacin

(es

decir,

relaciones

de

monmeros/porgenos

monmeros/agente entrelazante, as como la composicin de los disolventes


porognicos). La caracterizacin morfolgica de las columnas se llevar a cabo
mediante fotografas SEM. Las prestaciones de estas columnas desde el punto de
vista de CEC se realizar mediante medida de los factores de retencin y
eficacias de una serie de analitos no cargados. Adems, se compararn las fases
estacionarias obtenidas mediante fotopolimerizacin con las obtenidas con
iniciacin trmica.

II.4.2. Comparacin de iniciadores para la preparacin de columnas


monolticas de metacrilato para CEC
Se describir la preparacin de columnas monolticas de LMA para CEC
utilizando diferentes fotoiniciadores radicalarios. Los iniciadores a estudiar sern
AIBN, DMPA, BPO y LPO. Se estudiar la influencia de cada iniciador y su
contenido

en

una

mezcla

1,4-butanodiol/1-propanol

como

disolventes

porognicos. La caracterizacin morfolgica de las columnas se realizar


mediante

fotografas

SEM.

Adems,

se

evaluarn

sus

prestaciones

cromatogrficas mediante la medida de la retencin y la eficacia de mezclas de


analitos no cargados.

91

CAPTULO III

MATERIALES Y MTODOS

Captulo III. Materiales y mtodos

III.1. Surfactantes

III.1.1. APGs

III.1.1.1. Estndares, materias primas y otras muestras


Se utilizaron como estndares metil-- (>99%), metil-- (>99%), hexil-(>98%), octil-- (>98%), octil-- (>99%), decil-- (>99%) y dodecil--Dglucopiransidos (>99%, Fluka, Buchs, Suiza). En los estudios de determinacin
de APGs se emple el nonil--D-glucopiransido (>99%, Glycon Bioch. GmbH.
Biotechnol., Luckenwalde, Alemania) como patrn interno. En los estudios de
fragmentacin de la glucosa y APGs tambin se emple D-glucosa anhidra
(Fluka, Buchs, Suiza), D-glucosa-13C6 (99% tomos de

13

C), D-glucosa-6,6-d2

(98% de tomos deuterados), D-glucosa-1-13C (99% tomos

13

C) (Sigma-

Aldrich, Steinheim, Alemania). Tambin se usaron las mezclas comerciales de


APGs Glucopone 215 CS UP y Plantacare 818 UP (Fluka). Los productos de
cuidado personal como crema de manos y champ para nios se adquirieron en
establecimientos locales.

III.1.1.2. Reactivos de uso general


Los diferentes disolventes empleados fueron de grado HPLC. Se utiliz
ACN, MeOH, HAcO, HCl, NaHCO3 (Scharlab, Barcelona, Espaa), NaAcO
(SigmaAldrich, Steinheim, Alemania) y agua desionizada (desionizador
Barnstead, Sybron, Boston, MA, USA).

III.1.1.3. Instrumentacin y condiciones de trabajo


Se emple un espectrmetro de masas 1100 Series VL IT-MS, provisto de
una fuente ESI (Agilent Technologies, Waldbronn, Alemania). Para infusin

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Miriam Beneito Cambra

directa en MS se emple una bomba de jeringa (kdScientific, Holliston, MA,


USA) con la cual se mantuvo un flujo constante de entrada de 0,3 mL h1 (5 L
min1). En HPLC, se emple una columna de alquilamida (100 mm 2,1 mm, 3
m ID, Supelco, Bellefonte, USA) y una de cianopropilo (75 mm 3 mm, 3 m
ID, Supelco). Como fases mviles se utilizaron mezclas de ACN/agua, que
contenan 40 mM de tampn HAcO/NaAcO (pH 4,7), a un flujo de 0,2 mL
min1. Para acelerar la elucin isocrtica con fases mviles que contenan un
20% de ACN tambin se emple un flujo de 0,4 mL min1. El intervalo de
barrido de masas fue de m/z 50800, siendo el voltaje del capilar de 4 kV. Se
utiliz el valor de m/z del ion ms abundante como masa objetivo cuando se
inyectaban mezclas de estndares, y el valor m/z correspondiente a [M+Na]+
cuando se inyectaban los estndares. En todos los casos, el mximo de carga de
la trampa de iones fue de 3104 cuentas, con un tiempo mximo de acumulacin
de 300 ms. En los experimentos de infusin directa, los espectros se obtuvieron
promediando la seal durante 2 min. Como gas de nebulizacin y de secado se
emple nitrgeno (generador Gaslab NGLC MS 20, Equcien, Madrid, Espaa).
La nebulizacin se efectu a 25 psi y 8 L min1, y la temperatura del gas de
secado fue de 300 C. Como gas de colisin se utiliz He (C-50, Carburos
Metlicos, Aranjuez, Espaa). Se emple el software Agilent LC/MSD ver. 4.2
para el tratamiento de datos. Para obtener los espectros de MS de alta resolucin
se emple un VG Autospec (VG Analytical, Micromass Instruments,
Manchester, UK) provisto de una fuente FAB.

III.1.1.4. Preparacin de estndares y muestras


Las disoluciones de estndares y de los APGs comerciales se prepararon a
una concentracin de 1000 g mL1 en MeOH/agua 50:50 (v/v), excepto para el
estndar C12G1, cuya concentracin fue de 500 g mL1 por razones de

96

Captulo III. Materiales y mtodos

solubilidad. En infusin directa, las disoluciones fueron diluidas en proporcin


50:50 (v/v) con una mezcla de MeOH/agua 50:50 (v/v) conteniendo NaAcO 20
mM. En HPLC, se emplearon concentraciones de 2000 g mL1 de Glucopone y
Plantacare en mezclas 50:50 (v/v) de MeOH/agua. Una crema de manos y un
champ para nios se diluyeron en proporcin 1:100 con una mezcla 50:50 (v/v)
de MeOH/agua. Las disoluciones inyectadas se pasaron a travs de filtros de
nylon de 0,45 m de tamao de poro (Albet, Barcelona). Los volmenes de
inyeccin en HPLC fueron de 20 L. Los espectros de alta resolucin se
obtuvieron con disoluciones diluidas de D-glucosa y hexil, octil- y decil--Dglucopiransido en 50:50 (v/v) MeOH/agua.

III.1.2. Alcoholes

III.1.2.1. Estndares, materias primas y otras muestras


Los alcoholes primarios no etoxilados y etoxilados se designarn como
CnEm como se indica en la seccin I.2.4. Los cidos carboxlicos y
etoxicarboxlicos provenientes de la oxidacin con xido de cromo(VI) de los
alcoholes primarios no etoxilados y etoxilados, respectivamente, se designarn
como CnEmA.
Como estndares se emplearon los alcoholes no etoxilados: C3E0
(Scharlab, Barcelona, Espaa), C8E0, C10E0, C12E0, C14E0, C16E0 y C18E0
(Fluka, Buchs, Suiza); los alcoholes etoxilados: C2E1, C4E2, C8E1, C12E1,
C12E2, C18E1 (Fluka), C12E3, C12E4, C12E5, C12E6 (suministrados por C.
Solans, CSIC, Barcelona); los cidos carboxlicos: C2E0A, C3E0A, C8E0A,
C10E0A, C12E0A (Fluka), C14E0A, C16E0A y C18E0A (Sigma-Aldrich,
Steinheim, Alemania) y los cidos etoxicarboxlicos: C1E1A, C1E2A, C1E3A y
C2E1A (Fluka). Tambin se emple la mezcla comercial FINDET 10/18 (una

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Miriam Beneito Cambra

mezcla de oligmeros C10Em con un EO promedio nominal de 6, y que contiene


aproximadamente un 20% de agua, Molins-Kao, Barcelona). Los cosmticos y
productos para el cuidado corporal fueron adquiridos en las tiendas locales. Las
muestras de agua de mar se recogieron en contenedores de polietileno, en una
playa cerca a la zona urbana de La Pobla de Farnals (Valencia, Espaa).

III.1.2.2. Reactivos de uso general


Los diferentes disolventes empleados fueron de grado HPLC. Se utiliz
HAcO, MeOH, acetona, acetato de etilo, ACN, HCl y H2SO4 (Scharlab),
butilamina (Fluka), TMBA (como patrn interno, Sigma-Aldrich), sulfito de
sodio anhidro, y el xido de cromo(VI) (Panreac, Barcelona), todos de grado
analtico. Tambin se emple agua desionizada (vase apartado III.1.1.2).

III.1.2.3. Instrumentacin y condiciones de trabajo


Se emplearon un espectrmetro de masas y una bomba de jeringa para la
infusin directa de las muestras (vase apartado III.1.1.3). El intervalo de barrido
de masas fue de m/z 50800, en los modos NI y PI. El voltaje del capilar fue de 4
kV, el voltaje de la segunda mscara se fij en 6 V, mientras que el voltaje de la
primera mscara se selecciona automticamente en funcin de la masa objetivo.
La nebulizacin se efectu a 15 psi y 3 L min1, y la temperatura del gas de
secado fue de 250C. En los estudios de cuantificacin, la intensidad mxima
(tanto de los analitos como del patrn interno) se determinaron utilizando la masa
de cada pico como masa objetivo para reducir al mnimo la influencia de ste
parmetro en la sensibilidad. Otros parmetros o mtodos de trabajo son los
indicados en el apartado III.1.1.3. Para estudiar el efecto de la temperatura sobre
el rendimiento de la reaccin, se utiliz un bao de agua equipado con una sonda
de temperatura (RTC+ETS-D4, IKA, Staufen, Alemania).

98

Captulo III. Materiales y mtodos

III.1.2.4. Preparacin de estndares y muestras


A menos que se indique lo contrario, el reactivo de Jones se prepar con
6,7 g de CrO3 y 6 mL de cido sulfrico, completando el volumen hasta 50 mL
con agua

[Burke, 1999].

Las disoluciones madre de los estndares se prepararon

disolviendo los alcoholes (50 mg de cada uno) en acetona (25 mL). Alcuotas de
250 L de estas disoluciones se introdujeron en tubos de centrfuga provistos de
tapn de rosca, aadindose 3 mL de acetona y 1 mL del reactivo de Jones, este
ltimo fue aadido gota a gota con agitacin constante a temperatura ambiente.
Tras 5 minutos, se adiciona 0,5 mL de HCl 2M, obtenindose una disolucin de
sales de cromo(III). El exceso de reactivo de Jones, indicado por el color amarillo
de la disolucin, se elimin aadiendo, con agitacin, unos 150 mg de Na2SO3.
Posteriormente, se aadi 4 mL de acetato de etilo. La mezcla se agit para
favorecer la extraccin y luego se centrifug para acelerar la separacin en dos
fases. La fase superior transparente e incolora (con un volumen aproximado de 7
mL) contiene los cidos carboxlicos y etoxicarboxilicos en un medio de acetato
de etilo/acetona (4:3; v/v). Las sales de cromo(III) permanecen en la fase acuosa
(capa inferior) con un volumen de aproximadamente 1 mL. A alcuotas de 2 mL
de la fase orgnica se les aadi 0,2 mL de una disolucin acuosa de butilamina
(0,33 M) que contena TMBA (5,5 mM), este ltimo incorporado como patrn
interno. Esta mezcla se introdujo directamente en el equipo MS.
Para la preparacin de la mezcla industrial FINDET 10/18, y las muestras
de productos cosmticos y de cuidado personal, se tom 1 y 2 g,
respectivamente, se aadi 10 mL de acetona, la mezcla se agit magnticamente
durante 10 min, se centrifug y se tomaron alcuotas del sobrenadante (3 mL).
Como se estudia en la seccin IV.2.1, la reduccin de la cantidad de agua antes
de la adicin del reactivo de Jones es un factor importante para obtener unos
altos rendimientos de la oxidacin. Por este motivo, para muestras con un alto

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Miriam Beneito Cambra

contenido de agua se tomaron volmenes ms pequeos del sobrenadante (0,25 1 mL); estas alcuotas fueron diluidas con acetona hasta un volumen final de 3
mL.
El anlisis de agua de mar se realiz tomando dos muestras de 5 L cada
una. Un volumen de 2 L, tomado de la parte superior de cada contenedor, se pas
a travs de cartuchos de SPE (C18, 500 mg/ 6 mL, 55 m, 140 , Phenomenex,
Torrance, CA, USA) a un flujo de 5 mL min-1. Estos cartuchos se eluyeron con
tres porciones de 2 mL de acetona. El volumen de los eluatos se redujo de 6 a
unos 3 mL por evaporacin en corriente de nitrgeno a temperatura ambiente.
Tras la evaporacin, se efectu la oxidacin y extraccin, tal como se ha descrito
antes. Para construir un blanco de referencia, se tom un tercer volumen de 2 L
de agua de mar, y se procedi a extraccin (SPE) y reduccin del eluato a 3 mL,
como anteriormente se ha indicado. En el procedimiento de oxidacin de este
eluato, el reactivo de Jones fue sustituido por cido sulfrico diluido, siendo el
resto del procedimiento de oxidacin y de extraccin el indicado previamente.
Los espectros MS de los extractos de acetato de etilo/acetona se
registraron en modo NI, y se midieron las abundancias de los iones [M-H]- de los
cidos carboxlicos y etoxicarboxlicos. En los estudios de rendimiento de
oxidacin-extraccin, los espectros tambin se obtuvieron en modo PI, y los
picos de los iones [M+H]+ correspondientes a los alcoholes etoxilados se
compararon con los espectros de los correspondientes cidos etoxicarboxlicos
obtenidos en modo NI. Para obtener espectros en modo PI, alcuotas de 1 mL de
los extractos de acetato de etilo/acetona se acidificaron con 0,1 mL de HCl 2 M
(en lugar de la disolucin de butilamina), esta disolucin se diluy con 1 mL de
ACN, y se infundi en la interfaz ESI del MS. Cuando fue necesario, se emple
el TMBA como patrn interno, ya que proporciona los picos [M-H]- y [M+H]+ en
los modos NI y PI, respectivamente.

100

Captulo III. Materiales y mtodos

III.1.3. AESs

III.1.3.1. Estndares, materias primas y otras muestras


Como estndares de FAEs se emplearon: C8E0, C12E0, C14E0 (SigmaAldrich, Steinheim, Alemania) y Dehydol LT-7 (mezcla comercial industrial de
FAEs, 12 n 18, m = 7, Cognis, Monheim, Alemania). Como estndares de
AESs se emplearon: C8E0S, C12E0S (SDS) (Fluka, Buchs, Suiza) y una mezcla
industrial comercial nominalmente lauril sulfato, si bien otros residuos de alcohol
graso estn tambin presentes en cantidades importantes (LES, 12 n 18,
m =3, suministrado por Qumicas Oro). Con el objetivo de estudiar posibles

interferencias con otras clases de surfactantes comnmente empleados en las


formulaciones, se utilizaron las mezclas comerciales de LAS (mezcla industrial
de oligmeros con 10 n 13) y SAS (mezcla industrial de oligmeros con 14
n 16) (suministrados por Qumicas Oro). Otras muestras industriales de AESs
fueron suministradas por Qumicas Oro e Industria Jabonera Lina (Torras de
Cotillas, Murcia, Espaa). Los detergentes comerciales fueron adquiridos en las
tiendas locales. Las muestras de agua de mar se recogieron en contenedores de
polietileno, en una playa cerca a la zona urbana de La Pobla de Farnals
(Valencia, Espaa).

III.1.3.2. Reactivos de uso general


Se utilizaron los siguientes disolventes (grado HPLC) y reactivos (grado
analtico): MeOH, HAcO, ACN, NH3, DMSO (Panreac, Barcelona, Espaa),
anhdrido ftlico ( 99%), urea (99,5%) (Fluka), HCl, anhdrido difnico (98%) y
1,4-dioxano (99,8%, Sigma-Aldrich). Tambin se emple agua desionizada
(vase apartado III.1.1.2).

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Miriam Beneito Cambra

III.1.3.3. Instrumentacin y condiciones de trabajo


Se emple un cromatgrafo de lquidos (HP 1100, Agilent, Waldbronn,
Alemania), constituido por una bomba cuaternaria, un desgasificador en lnea, un
compartimento termostatizado de columnas, un muestreador automtico y un
DAD. Cuando fue necesario, el cromatgrafo se acopl a un espectrmetro de
masas 1100 Series VL IT-MS, provisto de una fuente ESI (vase apartado
III.1.1.3). La columna empleada fue una C8 del tipo ncleo fundido (AscentisExpress, 2,7 m, 15 cm x 4,6 mm de ID, Supelco, Bellefonte, PA, USA). La
elucin se llev a cabo mediante la mezcla de dos disoluciones que contenan
ACN/agua, con un 50% (A) y un 100% de ACN (B), en presencia de un 0,1% de
HAcO en ambas mezclas. El flujo fue de 1 mL min-1, y a no ser que se indique lo
contrario, la fase mvil se vari de forma lineal de A a B en 50 minutos. Los
volmenes de inyeccin fueron de 20 L, siendo las alcuotas filtradas
previamente mediante un filtro de nylon (Albet, Barcelona) de 0,45 micras de
tamao de poro. La deteccin se realiz a 230 10 nm con una referencia fijada
a 360 60 nm. Las reas de los picos se midieron con el software ChemStation
para LC v.10.02 (Agilent). Cuando fue necesario, se utiliz HPLC-MS, con un
rango de barrido de masas de m/z 100900 en modo NI. El voltaje del capilar fue
de 4 kV, el voltaje de la segunda mscara se fij en 6 V, mientras que el voltaje
de la primera mscara se seleccion automticamente en funcin de la masa
objetivo. La masa objetivo se fij a un valor de m/z de 400. La nebulizacin se
efectu a 35 psi y 7 L min1, y la temperatura del gas de secado fue de 300 C.
Otros parmetros o condiciones de trabajo son las indicadas en el apartado
III.1.1.3. Tambin se emplearon los siguientes cartuchos de SPE (Phenomenex,
CA, EE.UU.): Strata C18-E (500 mg/ 6 mL, 55 m, 140 ) y Strata SAX (1000
mg/ 6 mL, 55 m, 70 ). Cuando fue necesario, parte de los disolventes fueron
evaporados en una centrfuga a vaco (miVac, Genevac, Ipswich, Reino Unido).

102

Captulo III. Materiales y mtodos

III.1.3.4. Preparacin de estndares y muestras


Para los estudios de calibracin, se prepararon disoluciones madre de
C8E0 y C12E0 (2 mg mL-1) en 1,4-dioxano. Tambin, se utilizaron disoluciones
madre de C8E0S y C12E0S (2 mg mL-1) en 1,4-dioxano con un 7% de DMSO
para facilitar su disolucin. Por otro lado, en los estudios de separacin con
cartuchos SAX, se prepararon disoluciones madre de C14E0 y C12E0S (5 mg
mL-1) en MeOH. Estas disoluciones madre se utilizaron para preparar las mezclas
de 0,5 mg mL-1 de cada estndar, en medios con diferentes proporciones de
MeOH/agua (20:80, 50:50 y 80:20, todos como v/v). Para los estudios de
separacin en SAX, tambin se prepararon disoluciones concentradas (40 mg
mL-1) de mezclas industriales de FAEs (Dehydol LT-7) y AES (LES), as como
mezclas de las mismas (aproximadamente 20 mg mL-1 de cada clase de
surfactante) en 50:50 MeOH/ agua. Alcuotas de 1 mL de estas disoluciones se
diluyeron a 5 mL con las cantidades adecuadas de MeOH y agua, para obtener
mezclas con diferentes proporciones MeOH/ agua (20:80, 50:50 y 80:20).
En el anlisis de productos comerciales (detergentes), se llev a cabo una
etapa de limpieza, previa a la separacin con los cartuchos SAX. Para ello, se
pesaron 0,1 g del detergente y se diluy hasta 25 mL con agua. Esta disolucin se
pas por un cartucho de SPE C18, previamente acondicionado con agua. La
elucin se efectu con dos alcuotas, primero una de 2,5 mL de MeOH/ agua
50:50, seguida de otra de 2,5 mL de MeOH/ agua 80:20. Tras la elucin, se
aadi la cantidad de agua necesaria para reducir la concentracin de MeOH al
50% antes de la separacin de las dos clases de surfactantes el cartucho SAX. El
anlisis del agua de mar se realiz tomando dos muestras de 5 L cada una. Un
volumen de 2 L, tomado de la parte superior de cada contenedor, se pas a travs
de cartucho de SPE C18, a un flujo de unos 5 mL min-1. La elucin del cartucho

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Miriam Beneito Cambra

SPE C18, se efectu como se ha indicado anteriormente en la elucin de los


detergentes.
El procedimiento optimizado de la separacin con el cartucho SAX para
SPE se indica en el esquema de la Figura III.1. En primer lugar, el cartucho
SAX se acondicion con una mezcla 50:50 MeOH/ agua. Luego, la muestra que
contiene FAEs y AESs en un medio MeOH/ agua 50:50, se pas a travs del
mismo. Con esta operacin los AESs se quedan retenidos en el cartucho,
mientras que la mayora de los FAEs son eluidos (fraccin 1). El cartucho se lav
con el mismo medio MeOH/ agua 50:50, para eliminar as los restos de cationes
(fraccin 2), quedando los AES retenidos fuertemente sobre los sitios activos del
cartucho catinico. Una pequea parte de los FAEs ms hidroflicos tambin
eluyen con la fraccin 2. Seguidamente, el cartucho se lav con una mezcla
MeOH/ agua (80:20); el mayor contenido de MeOH de esta mezcla facilita a la
elucin de los oligmeros ms hidrofbicos de los FAEs, constituyendo la
fraccin 3. Las fracciones de FAEs 1 + 2 + 3 se combinan en un nico tubo con
tapn de rosca de 15 mL. La elucin de los AES de los cartuchos SAX se realiz
con mezclas MeOH:HCl, en diferentes proporciones, utilizando HCl acuoso
concentrado. Para ello, la fraccin 4 formada por los AESs hidroflicos se eluye
con una mezcla MeOH:HCl 80:20 (concentracin de cido en la mezcla, 2,4 M).
Los AESs ms hidrofbicos (fraccin 5) se eluyen con una mezcla MeOH:HCl
95:5 (concentracin de cido en la mezcla, 0,6 M). Las fracciones de AESs 4 + 5
se combinan en un segundo tubo con tapn de rosca de 15 mL. Seguidamente
esta ltima fraccin combinada se neutraliza gota a gota con NH3 acuoso
concentrado en presencia de una gota de fenolftalena al 1% en MeOH, , hasta
viraje del indicador. Las disoluciones de FAEs y AESs recogidas fueron
evaporadas bajo corriente de nitrgeno. La eliminacin de los ltimos restos de
disolvente se realiz en una centrfuga evaporadora a vaco.

104

Captulo III. Materiales y mtodos


FAEs + AESs (50:50 MeOH:H2O)
Fraccin 1 (FAEs hidroflicos)
Fraccin 2
Fraccin 3

SAX

Fraccin 4
Fraccin 5

50:50 MeOH:H2O (eliminacin de cationes)


80:20 MeOH:H2O (FAEs hidroflicos)
80:20 MeOH:HCl (AESs hidroflicos)
95:5 MeOH:HCl (AESs hidrofbicos)
Eluatos de FAEs:
Fracciones 1 + 2 + 3
combinadas

Evaporacin de
disolventes

Neutralizacin
Eluatos de AESs:
Fracciones 4 + 5
combinadas

Evaporacin de
disolventes

Fig. III.1. Esquema de separacin de los FAEs y AESs, empleando un


cartucho de SPE tipo SAX. Fracciones eluidas: FAEs (1), disolucin de
lavado para eliminacin de cationes (2), FAEs hidrofbicos (3), AESs
hidroflicos (4) y AESs hidrofbicos (5).

Se utilizaron los procedimientos de la bibliografa para la derivatizacin de los


FAEs con anhdridos ftlico [Mic-Tormos, 2008-B] y difnico [Mic-Tormos, 2009].
En este trabajo, se evalu la aplicacin de estos mismos procedimientos a la
derivatizacin de AESs que tienen lugar, mediante una reaccin de
transesterificacin, de acuerdo con los esquemas de reaccin de la Figura III.2.
Tras la evaporacin de los disolventes, se llevaron a cabo las derivatizaciones de
FAEs y AESs en sus respectivos tubos. Para ello, a cada uno de los tubos se
aadi 0,25 g de urea finamente molida, 2 mL de 1,4-dioxano, y 1 g del
anhdrido ftlico o 0,5 g del difnico. El anhdrido ftlico se emple para las
muestras industriales o comerciales, mientras que el difnico se utiliz para
derivatizar las muestras ambientales. Los tubos con la mezcla de reaccin, se
agitaron e introdujeron en un bao termosttico de aceite de silicona a 105 C
durante 90 min. Tras enfriar a temperatura ambiente, los residuos fueron
disueltos por adicin de 10 mL de una mezcla MeOH/ agua 2:1, que contiene 0,1
M de NH3, sin embargo, slo 2 mL de dicha mezcla se aadieron a los extractos

105

Miriam Beneito Cambra

de agua de mar derivatizados. Las disoluciones se inyectaron inmediatamente o


se almacenaron a -20 C hasta su inyeccin.

O
O
R
R

OH
o

+
-

SO3

COO-

H2O
o
SO3

COO-

Fig. III.2. Esquemas de reaccin para la esterificacin de FAEs y


transesterificacin de AESs con los anhdridos ftlico y difnico.

III.2. Polmeros sintticos

III.2.1. PVP-NO

III.2.1.1. Estndares, materias primas y otras muestras


Se utilizaron muestras de PVP-NO (Chromabond S-403E, 9-17 kDa, con
polmeros constituidos por unos 74 - 140 monmeros, International Specialty
Products, Colonia, Alemania). Con el objetivo de estudiar posibles interferencias
con diferentes clases de surfactantes comnmente empleados en las
formulaciones, se utilizaron las mezclas comerciales Dehydol LT-7 (mezcla de
FAEs, Cognis, Dsseldorf, Alemania), LAS, LES y p-isopropil sulfonato de
sodio (cumeno sulfonato de sodio, sustancia incorporada a las formulaciones por
sus propiedades como hidrtopo). Estos tres ltimos componentes fueron
suministrados por Qumicas Oro.

106

Captulo III. Materiales y mtodos

III.2.1.2. Reactivos de uso general


Se emplearon los siguientes reactivos H3PO4 (Panreac, Barcelona),
NaH2PO4, Na2HPO4, NaOH, HAcO, NH4AcO (Probus, Badalona, Espaa),
acetona, MeOH (Scharlab, Barcelona), PEA, xido de mesitilo (Fluka), SDS
(Merck, Darmstadt, Alemania). Tambin se emple agua desionizada (vase
apartado III.1.1.2).

III.2.1.3. Instrumentacin y condiciones de trabajo


Se emple un sistema de electroforesis capilar HP3D (Agilent,
Waldbronn, Alemania) provisto de detector espectrofotomtrico de fila de
diodos, y capilares de slice fundida (Polymicro, Phoenix, AZ, USA) de 33,5 cm
(25 cm de longitud efectiva) 50 m ID (375 m OD). Los capilares nuevos
fueron acondicionados a 60 C con NaOH 1 M, NaOH 0,1 M y agua durante 10
minutos cada uno. Posteriormente, la temperatura del capilar se redujo a 25 C, y
se pas el BGE durante 10 min ms. Diariamente, antes de iniciar la sesin de
trabajo, el capilar se lav sucesivamente con disoluciones de cido fosfrico 1 M
y 20 mM durante 15 minutos cada vez, seguido del BGE durante 10 min ms.
Entre inyecciones, el capilar se lav con el BGE durante 10 min. La inyeccin
fue hidrodinmica, aplicando 50 mbar 3 s. Las separaciones se llevaron a cabo
a +20 y -20 kV en ausencia y presencia de PEA, respectivamente. La deteccin
se realiz a 214 y 276 nm. El BGE se prepar semanalmente, conservndolo a 4
C. Antes de las inyecciones, las disoluciones se pasaron a travs de un filtro de
nylon de 0,45 am de dimetro de poro (Albet).
Para las medidas de tensin superficial se emple un mtodo basado en la
masa de un nmero fijo de gotas

[Mukherjee, 1971].

Las gotas se generaron

mediante un capilar de vidrio de extremo plano y de 3 mm de dimetro externo,


sostenido en posicin vertical por un soporte colocado sobre la mesa

107

Miriam Beneito Cambra

antivibratoria de la balanza. Para cada medida se tom el peso de 40 gotas de


disolucin (por triplicado), a temperatura ambiente (22 C). La calibracin se
realiz con agua (tensin superficial, 72,4 mN m-1 a 22 C) [Lide, 2003].

III.2.1.4. Preparacin de estndares y muestras


Se prepar una disolucin madre de PVP-NO de 10 mg mL-1 en una
mezcla de 50:50 (v/v) MeOH/ agua. Los productos comerciales de limpieza se
diluyeron en proporcin 1:100 con una mezcla 50:50 (v/v) de MeOH/ agua y se
inyectaron en el sistema de CE.

III.2.2. PVP

III.2.2.1. Estndares, materias primas y otras muestras


Los estndares de PVP (Fig. I.9), con unas masas moleculares promedio
de MW = 10, 27,9, 40, 160 y 360 kDa se obtuvieron de Fluka (Buchs, Suiza),
mientras que aquellos con MW = 60 y 400 kDa fueron suministradas por
International Specialty Products (Colonia, Alemania). Los productos de limpieza
y farmacuticos, se obtuvieron en los comercios locales.

III.2.2.2. Reactivos de uso general


Se emplearon los colorantes azoicos RC (Panreac, Barcelona, Espaa) y
AB (Sigma-Aldrich, Steinheim, Alemania); cuya estructura se muestra en la Fig.
VI.9. Tambin se utilizaron tetraborato de sodio decahidratado, acetona (SigmaAldrich) como marcador del EOF, as como otros reactivos de grado analtico, y
agua desionizada (vase apartado III.1.1.2).

108

Captulo III. Materiales y mtodos

III.2.2.3. Instrumentacin y condiciones de trabajo


Para el sistema de electroforesis capilar HP3D, capilares y acondicionado
de los capilares nuevos, vase el apartado III.2.1.3. Diariamente, antes de iniciar
la sesin de trabajo, el capilar se lav sucesivamente con disoluciones de NaOH
0,1 M y agua durante 10 minutos en cada caso, seguido del BGE durante 10 min
ms. Entre inyecciones, el capilar se lav con NaOH 0,1 M y agua, durante 2 y 3
minutos, respectivamente, seguido por el BGE durante 5 minutos ms. El BGE
estaba compuesto por Na2B4O710H2O 25 mM, con el pH ajustado a 9,0 por
adicin de HCl 0,1 M. Todas las disoluciones se pasaron a travs de filtros de
nylon de 0,45 m de tamao de poro (Albet, Barcelona, Espaa). La inyeccin
fue hidrodinmica, aplicando 50 mbar 2 s. Las separaciones se llevaron a cabo
a 15 kV (polaridad positiva) a una temperatura de 25 C. La deteccin se realiz
a 215 nm, as como a una longitud de onda cercana al mximo de absorcin para
los colorantes, tanto en presencia como en ausencia de un exceso de PVP. Las
longitudes de onda seleccionadas para CR y AB fueron 500 y 565 nm,
respectivamente.
Los espectros de absorcin UV-Vis se registraron empleando un
espectrofotmetro UV-Vis modelo 8453 (Agilent Technologies), utilizando una
celda de cuarzo de 1 mm de camino ptico (Hellma, Mllheim, Alemania). Los
espectros de los colorantes se obtuvieron con disoluciones compuestas por 0,1
mM del colorante y 25 mM de tetraborato sdico en agua. Los espectros de los
complejos de PVP-colorante se obtuvieron en el mismo medio, despus de la
adicin de 100 g mL-1 (0,9 mM en monmeros) del PVP de 60 kDa. El espectro
de esta mezcla se registr cada 10 minutos durante 5 horas, con el fin de estudiar
el grado de formacin de los complejos PVP-CR con el tiempo. A menos que se
indique lo contrario, las mezclas de PVP-colorante se mantuvieron a temperatura
ambiente durante un mnimo de 5 horas antes de la inyeccin en el capilar de CE.

109

Miriam Beneito Cambra

III.2.2.4. Preparacin de estndares y muestras


Se prepararon disoluciones madre acuosas de los colorantes azoicos (5
mM) y PVP de distintas MW (40 mg mL-1), y a partir de ellas se prepararon
disoluciones ms diludas, tambin en agua. Se construyeron curvas de calibrado
externas para la determinacin de PVP mediante la representacin del rea de la
banda del complejo PVP-colorante (vase el mtodo de medida de la misma en la
seccin VI.2.1.1) frente a la concentracin de monmeros de PVP. A tal efecto,
las concentraciones de PVP utilizadas fueron 100, 250, 500, 1000 y 1500 g mL1

. Las curvas de calibrado interno se obtuvieron mediante la adicin de 250 y 500

g mL-1 de PVP a las disoluciones de las muestras. Dos bases de detergente, cuya
composicin viene dada en la Tabla III.1, se fortificaron con PVP 60 kDa,
obtenindose una concentracin final de 0,99 y 0,97% (en peso) para las bases I
y II, respectivamente.
Tabla III.1. Composicin de las bases de detergente empleadas en este trabajo
(porcentajes en peso).
Componente

Base I (%)

Base II (%)

AS

AESa

Oleina

3,5

FAEb

11

SDS

Cumeno sulfonato sdico

1,5

Agua y otrosc

75,5

80,5

Cadenas entre 12 y 18 tomos de carbono y nmero medio de EO 3.

Cadenas entre 12 y 18 tomos de carbono y nmero medio de EO 7.

Componentes minoritarios como KOH, trietanolamina, bactericidas y perfume.

A continuacin, se pesaron 0,5 g de cada mezcla y se aadi 4 mL de una


disolucin 5 mM de CR, completndose el volumen a 5 mL con agua. Alcuotas
de esta disolucin se inyectaron en el equipo de CE. Con los productos de
110

Captulo III. Materiales y mtodos

limpieza y farmacuticos se sigui el mismo procedimiento que el descrito con


las bases de detergente. La cuantificacin se realiz a partir de una curva de
calibracin externa obtenida con el CR y el PVP de 60 kDa. Todas las
inyecciones se realizaron por triplicado.

III.2.3. PVA

III.2.3.1. Estndares, materias primas y otras muestras


Los estndares de PVA (Fig. I.10) utilizados presentan las siguientes MW
y porcentajes de monmeros residuales acetilados (nac 100%, valores entre
parntesis): 15 (0,0%), 49 (0,1%) y 100 (12,5%) kDa (Fluka, Buchs, Suiza), y 31
(10,8%), 61 (1,5%), 130 (10,8%), 145 (0,6%) y 205 (10,8%) kDa (Sigma
Aldrich, Steinheim, Alemania).

III.2.3.2. Reactivos de uso general


Se emple el colorante azoico RC (Panreac, Barcelona, Espaa), cuya
estructura puede verse en la Fig. VI.9. Se utilizaron tetraborato de sodio
decahidratado, acetona (Sigma-Aldrich) como marcador del EOF, dimetil
sulfxido deuterado (DMSO-d6, SigmaAldrich) as como otros reactivos de
grado analtico. Tambin se emple agua desionizada (vase apartado III.1.1.2).

III.2.3.3. Instrumentacin y condiciones de trabajo


Para el sistema de electroforesis capilar HP3D, capilares y acondicionado
de los capilares nuevos, vase el apartado III.2.1.3. Diariamente, antes de iniciar
la sesin de trabajo, el capilar se lav sucesivamente con disoluciones NaOH 0,1
M y agua durante 5 minutos cada uno, seguido del BGE durante 10 min ms.
Entre inyecciones, el capilar se lav con NaOH 0,1 M y agua durante 2 y 3

111

Miriam Beneito Cambra

minutos, respectivamente, seguido por el BGE durante 5 minutos ms. Al


finalizar cada sesin de trabajo, el capilar se lav con agua y HCl 0,1 M durante
10 min en cada caso, con agua 5 minutos ms, y con NaOH 1 M seguido de ms
agua durante 20 min ms cada uno. El BGE estaba compuesto por
Na2B4O710H2O 25 mM, ajustndose a pH 9,0 con HCl 0,1 M. Todas las
disoluciones se pasaron a travs de filtros de nylon 0,45 m de tamao de poro
(Albet, Barcelona, Espaa). Las disoluciones con PVA no se filtraron para evitar
que el polmero se quedara retenido en el filtro. La inyeccin fue hidrodinmica,
aplicando 50 mbar 3 s. Las separaciones se llevaron a cabo a 15 kV (polaridad
positiva) y a 25 C. La deteccin se realiz a 500 nm, longitud de onda cercana al
mximo de absorcin de los colorantes en ausencia de PVA.
Los espectros UV-Vis se obtuvieron tanto con el equipo de CE, como con
un espectrofotmetro (indicado en la seccin III.2.2.3). Para obtener los
espectros en CE, el capilar se rellen con una disolucin de brax 20 mM que
contena CR 4 mM y concentraciones crecientes de PVA (muestra de MW = 49
kDa). En el espectrofotmetro, para no saturar la seal, se emplearon las
disoluciones anteriores diluidas en un factor 1:40 (0,1 mM CR). El tiempo de
pre-equilibrado de los complejos PVA-CR se estudi tanto por CZE como por
espectrofotometra.
La tacticidad (estereorregularidad) de las muestras de PVA se estableci
registrando los espectros de NMR 13C con un espectrmetro de NMR Ultrashield
DRX 400 (Bruker, Silberstreifen, Alemania). Para ello, las muestras de PVA se
deshidrataron en una centrfuga evaporadora a vaco durante 8 horas a 60 C
(miVac, Genevac, Ipswich, Reino Unido) y se prepararon disoluciones de PVA
al 1% con DMSO-d6. Se acumularon 2 104 espectros en todos los casos. Las
estructuras de las tres posibles tradas en una cadena de PVA (mm, rm y rr) se
muestran en la Figura. III.3.

112

Captulo III. Materiales y mtodos

HO H HO H HO H

HO H

H OH H OH

HO H

H OH HO H

Fig. III.3. Estructuras de las tres posibles tradas en una cadena de PVA:
(A) mm, (B) rm y (C) rr.

Los porcentajes de cada trada se estimaron a partir de las reas relativas de los
tres multipletes que se encuentran dentro del rango de 64-69 ppm [Moritani, 1972;
Wu, 1973; Wu, 1977; Fukae, 2000].

En la Figura III.4, se representan los

porcentajes de las triadas rr encontrados en las muestras, frente el porcentaje de


las tradas mm. En la misma figura, se representaron tambin datos obtenidos de
diversas publicaciones [Moritani, 1972; Wu, 1973; Wu, 1977; Fukae, 2000].

40
61 (1.72)

rr %

100 (1.09) M

30

31 (1.67)

145 (1.04)

15 (1.39)

130 (0.85)

20

49 (1.05)

205 (0.71)

6
2
15

20

25

70

mm %
Fig. III.4. Porcentajes de las triadas rr y mm, estimados por 13C NMR en
las muestras de PVA empleadas en este trabajo (). Los nmeros que se
encuentran junto a los smbolos (), son la MW (kDa) y la relacin rr/mm
(valores entre parntesis). Los otros smbolos corresponden a datos
bibliogrficos

[Moritani, 1972; Wu, 1973; Wu, 1977; Fukae, 2000]

para muestras

de PVA altamente sindiotctico (), atctico () y altamente isotctico ().

Como se observa en esta figura, las muestras de PVA utilizadas en este trabajo
resultaron agrupadas como sigue: un grupo de tres muestras con proporciones

113

Miriam Beneito Cambra

rr/mm dentro del rango 1,72 - 1,39, otro grupo de tres muestras dentro del rango
1,09 - 1,04 y finalmente, las dos muestras con mayor MW mostraron los menores
valores de la relacin rr/mm, en concreto 0,85 y 0,71. Para facilitar la discusin
en la seccin IV.3, estos grupos se denominaron en funcin de la relacin rr/mm
como H (alto), M (medio) y L (bajo).

III.2.3.4. Preparacin de estndares y muestras


Se prepararon disoluciones madre acuosas de RC (5 mM) y PVAs de
distinta MW (10 mg mL-1, 227 mM en monmeros), preparndose diluciones y
mezclas de los mismos con los tampones adecuados.

III.3. Enzimas

III.3.1. CZE

III.3.1.1. Estndares, materias primas y otras muestras


Los concentrados de enzimas industriales utilizados en esta seccin se
muestran en la Tabla III.2. Estos fueron suministrados en forma lquida o como
slidos granulares. Para el estudio de posibles interferencias y efectos de la
matriz se utilizaron las siguientes mezclas industriales comerciales: Dehydol LT7 (mezcla de FAEs, Cognis, Dsseldorf, Alemania), LAS y LES (donados por
Qumicas Oro).

114

Captulo III. Materiales y mtodos


Tabla III.2. Clase, nombre comercial, fabricante, seccin de esta memoria en la
que se utilizan las diferentes enzimas estudiadas, as como el conjunto asignado
para la construccin de modelos de LDA en las secciones indicadas.

Clase
Proteasa

Amilasa

Celulasa

Lipasa

a
b

Nombre comercial
Alcalase 2,5 L
Savinase 16L, Type EX
Everlase 16 L, Type EX
Esperase 8.0 L
Polarzyme 12Ta
Bioproteasa L 450
Bioproteasa L 800
Enziprot 450L
Deterzyme L 660
Deterzyme Apy L 560
Properase 1600L
Purafect Prime HA
Properase 4000Da
Excellase 2250Da
Purafect OX 8000Da
Termamyl Ultra 300L
Duramyl 300 L, Type DX
Stainzyme 12L
Enziamilasa
Purastar ST 15000L
Purastar ST 6000Da
Endolase 5000L
Carezyme 4500L
Celluzyme 0,7Ta
Deterzyme CL-5
Puradax HA 400Ea
Puradax EG 7000L
Lipolase 100L, Type EX
Lipex 100L
Lipolase 100Ta
Lipex 100Ta

Fabricantee
Novozymes
Novozymes
Novozymes
Novozymes
Novozymes
Biocon
Biocon
ChemWorld
Enmex
Enmex
Genencor
Genencor
Genencor
Genencor
Genencor
Novozymes
Novozymes
Novozymes
ChemWorld
Genencor
Genencor
Novozymes
Novozymes
Novozymes
Enmex
Genencor
Genencor
Novozymes
Novozymes
Novozymes
Novozymes

Seccin
VII.1; VII.2c; VII.3b,c; VII.4
VII.1; VII.2d; VII.3d
VII.1; VII.2d; VII.3d; VII.4
VII.1; VII.2d; VII.3d
VII.3d
VII.2d; VII.3c
VII.2d; VII.3c
VII.2c; VII.3d
VII.2c; VII.3d
VII.2d; VII.3d
VII.3d
VII.2d; VII.3d
VII.3d
VII.3d
VII.3d
VII.1; VII.2c; VII.3b,c
VII.1; VII.2c; VII.3d; VII.4
VII.1; VII.2d; VII.3c
VII.2c; VII.3c
VII.2d; VII.3d; VII.4
VII.3d
VII.1; VII.2c; VII.3d
VII.1; VII.2c; VII.3b,c
VII.1; VII.2d; VII.3d
VII.2c; VII.3c; VII.4
VII.3d; VII.4
VII.2d; VII.3c
VII.1; VII.2c; VII.3c; VII.4
VII.1; VII.2c; VII.3b,c; VII.4
VII.2c; VII.3c
VII.2d; VII.3d

Muestras suministradas como slidos granulados (el resto de muestras son lquidas).
Se emplean tanto como materia prima industrial como para aditivar las bases de
detergente (Tabla III.3).

Empleado en el conjunto de entrenamiento.

Empleado en el conjunto de evaluacin.

Donadas por las correspondientes casas comerciales: Novozymes (Bagsvaerd,


Dinamarca), Biocon (Bangalore, India), ChemWorld (Barcelona, Espaa), Enmex
(Tlalnepantla, Mxico), Genencor (Rochester, NY, USA)

115

Miriam Beneito Cambra

III.3.1.2. Reactivos de uso general


Se emple IDA, urea, PVA de MW media de 70 a 100 kDa (Sigma, St.
Louis, MO, EE.UU.), y HEC con una MW media de 27 kDa (Polysciences,
Warrington, PA, EE.UU.). Tambin se utilizaron NaOH y sodio tetraborato
decahidrato (Probus, Badalona, Espaa), acetona (Scharlau, Barcelona, Espaa) y
sdio dihidrgeno fosfato (Panreac, Barcelona, Espaa). El contenido de protena
de las muestras se calcul por el mtodo de Bradford

[Bradford, 1976]

utilizando

el Protein Quantification Kit Rapid (Fluka, Buchs, Suiza) y la BSA (Fluka)


como estndar. Tambin se emple agua desionizada (desionizador Barnstead,
Sybron, Boston, MA, USA)

III.3.1.3. Instrumentacin y condiciones de trabajo


Para el sistema de electroforesis capilar HP3D, capilares y acondicionado
de los capilares nuevos, vase apartado III.2.1.3. Para trabajar en medio bsico,
se utiliz un BGE compuesto por NaH2PO4 50 mM, Na2B4O7 25 mM y PVA al
0,1%, ajustado a pH 9,0 con NaOH 0,1 M. Al comienzo de cada sesin de
trabajo, el capilar se lav con NaOH 0,1 M y agua durante 5 minutos cada uno, y
10 minutos ms con el BGE. Entre inyecciones el capilar se lav con el BGE
durante 10 min. Las separaciones con el BGE bsico se realizaron a 25 C a 15
kV (polaridad positiva). Para trabajar en medio cido, se emple un BGE
compuesto por 50 mM de IDA (pH = 2,30 a 25 C), HEC al 0,5% y urea 6 M
(pHaparente = 3,1). Diariamente, antes de iniciar la sesin de trabajo, el capilar se
lav con el BGE cido durante 30 minutos y entre inyecciones el capilar se lav
durante 5 minutos con el mismo BGE. La temperatura del capilar se fij a 25 C
y separaciones se realizaron a 25 kV (polaridad positiva), lo que dio lugar a una
corriente tpica de 30 mA. Para ambos BGEs, las disoluciones de las posiciones
andica y catdica se renovaron tras cada serie de cinco inyecciones [Bossi, 1999].

116

Captulo III. Materiales y mtodos

Los BGEs se pasaron a travs de filtros de nylon de 0,45 micras de tamao de


poro (Albet, Barcelona, Espaa). Para las inyecciones hidrodinmicas se
aplicaron 50 mbar 2 s. La seal se monitoriz a 214 nm.
Para medir la absorbancia a 600 nm en el ensayo de Bradford, se emple
un espectrofotmetro UV-Vis modelo 8453 (Agilent Technologies), utilizando
una celda de cuarzo de 1 cm de camino ptico (Hellma, Mllheim, Alemania). El
contenido proteico en las distintas muestras se obtuvo siguiendo el protocolo
descrito en el Protein Quantification Kit Rapid (Fluka). Los resultados
obtenidos con las diferentes enzimas, expresados como porcentaje de BSA se
indican en la Tabla VII.1.

III.3.1.4. Preparacin de estndares y muestras


Los concentrados de enzimas industriales lquidas con contenidos entre 110% de protena, fueron diluidos con agua para obtener disoluciones de
aproximadamente un 0,01% de protena. A partir de estas disoluciones, se
tomaron alcuotas de 1 mL y se les aadi 4 mL de acetona. Tras agitacin, se
centrifug a 5500 rpm 4 min, se descart el sobrenadante, y los precipitados se
disolvieron en 1 mL de agua o de urea 3 M, en funcin de que la muestra se fuese
a inyectar en el BGE bsico o cido, respectivamente. Para las enzimas
comercializadas granuladas, se pes aproximadamente 1 g y se aadi 5 mL de
agua. La suspensin resultante se agit vigorosamente, se sonic durante 15
minutos, y se centrifug a 5500 rpm durante 4 minutos, tomndose una alcuota
del sobrenadante. A continuacin, se adopt el procedimiento anteriormente
descrito para concentrados lquidos de enzima.
Para establecer el contenido de protena segn el ensayo de Bradford
[Bradford, 1976],

se prepar una disolucin de BSA de 4000 g mL-1 (0,4%) en

117

Miriam Beneito Cambra

urea 3 M. A partir de esta disolucin se prepar una curva de calibrado de nueve


puntos en un intervalo de 10 a 2000 g mL-1 (0,001 a 0,2%).
Tambin se estudiaron las interferencias ocasionadas por los surfactantes
ms utilizados en la formulacin de productos de limpieza. Para ello, a
disoluciones que contenan un 5% de LAS, LES o Dehydol LT 7, se les aadi
un 0,01% de enzima. La separacin de las enzimas de estas disoluciones se
realiz tomando alcuotas de 5 mL y aadiendo 20 mL de acetona. Tras
agitacin, se centrifug a 5500 rpm durante 4 min, se descart el sobrenadante y
el precipitado resultante se diluy en 1 mL de agua o urea 3 M, segn se fuese a
inyectar en el BGE bsico o cido, respectivamente.

III.3.2. Aminocidos por infusin directa en MS

III.3.2.1. Estndares, materias primas y otras muestras


Los concentrados de enzimas industriales utilizados en este trabajo se
muestran en la Tabla III.2, siendo suministrados en forma lquida o como
slidos granulares. Tambin, se emple Dehydol LT-7 (mezcla de FAEs, Cognis,
Dsseldorf, Alemania), LES, oleina y cumeno sulfonato sdico (donados por
Qumicas Oro). Dos detergentes para lavado de textiles que contenan proteasa
(segn declaraba el fabricante) se obtuvieron en los comercios locales.

III.3.2.2. Reactivos de uso general


Se emple acetona, etanol (Scharlau, Barcelona, Espaa) y HCl (37%)
(Panreac, Barcelona, Espaa). Tambin se emple agua desionizada (vase
apartado III.1.1.2).

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Captulo III. Materiales y mtodos

III.3.2.3. Instrumentacin y condiciones de trabajo


Se emplearon un espectrmetro de masas (otros detalles en III.1.1.3) y una
bomba de jeringa para la infusin directa de las muestras (vase apartado
III.1.1.3). Las condiciones de trabajo en el espectrmetro de masas se adaptaron
de trabajos de la bibliografa [Peris-Vicente, 2005; Lerma-Garca, 2007]. El rango de
barrido de masas en el modo PI fue de m/z 50300. El voltaje del capilar fue de
3,5 kV, el voltaje de la primera y segunda mscara fue de 19.6 y 6 V,
respectivamente. La nebulizacin se efectu a 25 psi y 8 L min1, y la
temperatura del gas de secado fue de 250 C. La masa objetivo se fij a m/z 122
([M+H]+ para la cistena). Otros parmetros o mtodos de trabajo son los
indicados en el apartado III.1.1.3. El tratamiento estadstico de los datos
espectrales se realiz con el paquete estadstico SPSS (v. 12.0.1; Statistical
Package for the Social Sciences Inc., Chicago, IL, USA).

III.3.2.4. Preparacin de estndares y muestras


Para los concentrados de enzimas industriales, tanto lquidos como
slidos, se siguieron dos procedimientos adaptados de la bibliografa
Adelantado, 2002; Peris-Vicente, 2005].

[Gimeno-

Para los concentrados industriales lquidos

de enzimas se pes alrededor de 1 g en un tubo con tapn de rosca, se aadieron


4 mL de acetona, y se recogi el precipitado por centrifugacin. Se desech el
sobrenadante y se aadieron 200 l de HCl 12 M, mantenindose este tubo bien
cerrado a 110 C durante 24 h. Para las enzimas suministradas como slidos
granulares, se pes alrededor de 1 g, se aadieron 5 mL de agua, y la suspensin
se someti a sonicacin durante 15 min. Tras centrifugacin se tom 1 mL del
sobrenadante, llevndose a cabo la precipitacin e hidrlisis enzimtica como la
descrita para los concentrados lquidos de enzima.

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Miriam Beneito Cambra

Para el estudio de las posibles interferencias causadas por la matriz, se


prepararon dos bases de detergente cuya composicin viene dada en la Tabla
III.3. Unos 5 g de las bases de detergente fueron aditivadas con 50 L o 50 mg,
de concentrado lquido o granulado industrial de enzimas, respectivamente. Para
hacer las extracciones de enzimas a partir de las bases de detergente, o de los
detergentes comerciales, se tomaron porciones de unos 5 g, se aadieron 20 mL
de acetona y se agit, separndose el precipitado por centrifugacin. El residuo
se disolvi en 1 mL de agua, y se efectu una reprecipitacin con 4 mL de
acetona. El precipitado se hidroliz tal y como como se ha indicado
anteriormente para los concentrados lquidos de enzimas industriales.
Tabla III.3. Composicin de las bases de detergente empleadas en este trabajo (%
en peso).
Componente

Base I (%)

Base II (%)

AES

10

Olena

10

FAE

10

15

Sodio Cumeno sulfonato

Agua

70

65

Tras la hidrlisis cida de la muestra, el residuo se agita con 5 mL de una


mezcla EtOH/HCl (0,1 M) 1:1 (v:v). La suspensin se hace pasar por un filtro de
nylon de 0,45 m y se infunde directamente en el espectrmetro de masas, o se
conserva a -20 C hasta su uso. Se hidrolizaron tres alcuotas de todos los
concentrados industriales de enzimas, las bases de detergentes aditivadas y los
productos de limpieza, realizndose asimismo un mnimo de tres infusiones por
alcuota.

120

Captulo III. Materiales y mtodos

III.3.2.5. Construccin de las matrices de entrenamiento y evaluacin


Como se indica en la Tabla III.2, se utilizaron tres concentrados
industriales diferentes, dentro de cada una de las cuatro clases de enzimas, para
construir el conjunto de entrenamiento. Por lo tanto, cada categora contena el
mismo nmero de muestras (y los espectros correspondientes) que las dems
categoras, para evitar que un peso excesivo en una de las categoras conduzca a
un modelo incorrecto. Cada enzima se precipit e hidroliz tres veces de forma
independiente, y cada hidrolizado se infundi tambin por triplicado, pero en el
conjunto de entrenamiento slo se incluye la media de los datos obtenidos en las
tres infusiones. De esta forma, la variacin interna de las diferentes categoras se
redujo, lo que es importante para reducir el nmero de variables seleccionadas
por el algoritmo que utiliza el SPSS para la construccin del modelo. Por lo
tanto, el conjunto de entrenamiento I estaba constituido por los datos de 36
objetos (4 clases de enzimas 3 enzimas de cada clase 3 hidrolizados de cada
enzima un promedio de las tres infusiones de cada hidrolizado).
Para evaluar la posible influencia de las matrices de la muestra,
generalmente productos de limpieza, las dos bases de detergente de la Tabla
III.3, fueron aditivadas con tres concentrados industriales de enzimas de cada
categora, y se procesaron como se ha indicado anteriormente. As pues, el
conjunto de datos II contena 72 objetos (2 bases de detergente 4 clases de
enzimas 3 enzimas de cada clase 3 hidrolizados de cada enzima un
promedio de las tres infusiones de cada hidrolizado). Como se comentar
posteriormente en la seccin VII.2, el conjunto de datos II, fue utilizado para
evaluar los modelos construidos con el conjunto de datos I, sin embargo,
posteriormente los datos de los conjuntos I y II fueron utilizados conjuntamente
para mejorar la capacidad de prediccin de los modelos.

121

Miriam Beneito Cambra

Por ltimo, el conjunto de datos III fue construido a partir de todos los
datos espectrales de los concentrados industriales de enzimas y productos de
limpieza que no se incluyeron en la construccin de los conjuntos anteriores
(Tabla III.2). Este tercer conjunto contena 42 objetos (12 concentrados
industriales de enzimas ms 2 detergentes comerciales 3 hidrolizados de cada
enzima un promedio de las tres infusiones de cada hidrolizado).

III.3.2.6. Seleccin de variables predictoras para la construccin de un modelo


de LDA
El LDA, es una tcnica de clasificacin supervisada, siendo una excelente
herramienta para obtener los vectores que muestran la resolucin mxima entre
las diferentes clases o categoras. En un LDA se obtienen los vectores que hacen
mnima la lambda de Wilks, W

[Vandeginste, 1998].

La W es una funcin de las

distancias eucldeas entre los puntos medidos en la direccin de los vectores


(funciones discriminantes) que constituyen el modelo. Usando el SPSS, las
distancias eucldeas se miden en un espacio normalizado, donde todas las
variables de entrada tienen una media de cero y uno de desviacin estndar. Para
obtener W, la suma de los cuadrados de las distancias eucldeas entre todos los
puntos pertenecientes a la misma categora se divide por la suma total de
cuadrados. Los valores de W cercanos a cero se obtienen con categoras bien
separadas, mientras que la presencia de por lo menos un par de categoras
superpuestas conduce a valores de W prximos a uno. Mediante el uso de LDA
se construyen hasta N-1 funciones discriminantes, donde N es el valor ms bajo,
entre el nmero de variables predictoras o el nmero de categoras.
Para seleccionar las variables predictoras a incluir en los modelos, se
utiliz el algoritmo paso a paso del SPSS. El valor de W disminuye cada vez
que una variable predictora con un poder discriminante significativo (de acuerdo

122

Captulo III. Materiales y mtodos

a las categoras establecidas) se incluye en el modelo. De acuerdo con el


algoritmo paso a paso, una variable predictora se incorpora al modelo, cuando
tras su inclusin en el mismo, se supera un umbral, Fin. El criterio Fin es un
ensayo F que compara la reduccin de varianza residual producida por la entrada
de la variable con la varianza residual que queda. Sin embargo, la entrada de un
nuevo predictor modifica la reduccin de varianza residual debida a las dems
predictoras presentes en el modelo. Por esta razn, tras la inclusin de una nueva
predictora, se aplica un criterio de rechazo, Fout, para decidir si la predictora debe
ser eliminada del modelo. El proceso termina cuando no hay predictoras que
puedan entrar o ser eliminadas del modelo. Inicialmente se tomaron los valores
de probabilidad que emplea el SPSS por defecto, siendo 0,05 y 0,10 para los
criterios Fin y Fout, respectivamente.

III.3.3. Aminocidos derivatizados con OPA-NAC

III.3.3.1. Estndares, materias primas y otras muestras


La Gly y los siguientes levo-aminocidos fueron utilizados como
estndares: Ala, Arg, Asp, Glu, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Tyr, Val y
Cys (Sigma). Otros aminocidos como el Trp, Asn y Gln, no se incluyeron en
este estudio, ya que el primero se destruye completamente durante la hidrlisis
cida, y la Asn y la Gln, se convierten en Asp y Glu

[Hurst, 2002].

Los

concentrados de enzimas industriales utilizados en este trabajo se muestran en la


Tabla III.2. Dehydol LT-7 (mezcla de FAEs, Cognis, Dsseldorf, Alemania),
LES, olena y sodio cumeno sulfonato (donados por Qumicas Oro). Dos
detergentes para lavado de textiles que contenan proteasa (segn declaracin del
fabricante) se obtuvieron en los comercios locales.

123

Miriam Beneito Cambra

III.3.3.2. Reactivos de uso general


Se emple acetona, etanol (Scharlau, Barcelona, Espaa) y HCl (37%)
(Panreac, Barcelona, Espaa). Tambin se emplearon otros reactivos como OPA,
NAC (Fluka, Buchs, Switzerland), cido brico (Panreac, Barcelona, Espaa) y
cido ctrico anhdrido (Sigma, St. Louis, MO, USA). Tambin se emple agua
desionizada (desionizador Barnstead, Sybron, Boston, MA, USA)

III.3.3.3. Instrumentacin y condiciones de trabajo

Se emple un cromatgrafo de lquidos (HP 1100, Agilent, Waldbronn,


Alemania), constituido por una bomba cuaternaria, un desgasificador en lnea, un
termostato para la columna, un muestreador automtico y un detector UV-Vis de
longitud de onda variable. La columna empleada fue una Kromasil C18 (5 m,
25 cm x 4 mm de ID, Anlisis Vnicos, Tomelloso, Espaa). Los gradientes de
elucin, incluyendo el gradiente ptimo multi-segmentado (Tabla VII.3), se
obtuvieron mezclando dos disoluciones que contenan agua con ACN al 5% y
50% (mezclas A y B, respectivamente), ambas tamponadas con cido
ctrico/citrato sdico 5 mM a pH 6,5. El flujo de la fase mvil fue de 1 mL min-1,
y los volmenes de inyeccin fueron de 5 L. La deteccin se realiz a 335 nm.
Las reas de los picos se midieron con el software ChemStation para LC v.10.02
(Agilent). Para la normalizacin de las variables, as como para transferir los
datos al paquete estadstico SPSS (v. 12.0.1; Statistical Package for the Social
Sciences Inc., Chicago, IL, USA) se emple el programa Excel (Microsoft). El
SPSS fue empleado tambin para el tratamiento estadstico de los datos
espectrales. Para la seleccin de las variables predictoras que se incluyeron en los
modelos, se utiliz el algoritmo paso a paso (seccin III.3.2.6) del paquete

124

Captulo III. Materiales y mtodos

estadstico SPSS, donde se adoptaron inicialmente los valores de Fin y Fout, 3,84 y
2,71, respectivamente.

III.3.3.4. Preparacin de estndares y muestras


Se siguieron los mismos procedimientos de hidrlisis y extraccin de
enzimas comentados en la seccin III.3.2.4. En este caso, se hidrolizaron dos
alcuotas de cada concentrado de enzima industrial, base de detergente aditivada
o muestra comercial. Las dos alcuotas hidrolizadas se diluyeron y derivatizaron
como se indica a continuacin, realizndose una inyeccin de cada alcuota.
El reactivo de derivatizacin estaba compuesto por una mezcla que
contena OPA 1,25 10-2 M y NAC 2,5 10-2 M, tamponada con una disolucin
de cido brico/sodio borato 1 M a pH 9,5. El reactivo de derivatizacin se
protegi con papel de aluminio y fue conservado a 4 C, renovndose
semanalmente. La derivatizacin se realiz directamente por la adicin de 1 mL
de la disolucin de OPA-NAC a una alcuota de 100 L de los hidrolizados. Para
identificar los picos correspondientes a cada aminocido a lo largo del
cromatograma, se prepararon disoluciones de un aminocido o tambin mezclas
de dos o tres aminocidos (1000 g mL-1 de cada) que fueron derivatizados como
anteriormente se ha explicado. Tras la derivatizacin, estas disoluciones fueron
usadas tanto para inyectar directamente o para aditivar los hidrolizados cuando
fue necesario. En la Figura III.5 se muestra la reaccin de derivatizacin de los
aminocidos.
O

H2N

R1
COOH

+ HS

HN

H
H

Aminocido

OH
O

HO
NAC

Fig. III.5. Reaccin de formacin de isoindoles.

125

HN
HO
COOH
R1

O
OPA

OH

Isoindol

Miriam Beneito Cambra

III.3.4. Digestos con tripsina

III.3.4.1. Estndares, materias primas y otras muestras


Se utiliz tripsina de pncreas bovino y BSA (Fluka, Buchs, Suiza). Los
concentrados de enzimas industriales utilizados en esta seccin estn indicados
en la Tabla III.2. Tambin se emple Dehydol LT-7 (mezcla de FAEs, Cognis,
Dsseldorf, Alemania), LES, oleina y sodio cumeno sulfonato (donados por
Qumicas Oro), as como otros componentes empleados para preparar las bases
de detergentes empledas en este trabajo (Tabla III.1). Dos detergentes
comerciales que contenan proteasa (segn declaracin del fabricante) se
adquirieron en los comercios locales.

III.3.4.2. Reactivos de uso general


Se emple TFA ( 99,5%), DTT y (NH4)HCO3 (Sigma-Aldrich, Viena,
Austria), cido frmico (98-100%, Riedel de Haen, Seelze, Alemania), as como
ACN grado HPLC y acetona (VWR, Viena).

III.3.4.3. Instrumentacin y condiciones de trabajo


Se emple un cromatgrafo de lquidos (HP 1100, Agilent, Waldbronn,
Alemania), constituido por una bomba cuaternaria, un desgasificador en lnea, un
termostato para la columna, un muestreador automtico y DAD. El sistema
HPLC estaba equipado con un inyector automtico termostatizado, que permit
que las muestras se mantuviesen a 5 C hasta su inyeccin. Las principales
caractersticas de las columnas estudiadas se presentan en la Tabla III.4. La
elucin se llev a cabo usando mezclas de dos disoluciones que contenan agua y
ACN (fases A y B, respectivamente), ambas en presencia de un 0,1% de TFA. A
menos que se indique lo contrario, la elucin se efectu en dos pasos, un primer

126

Captulo III. Materiales y mtodos

paso isocrtico con 1% de B durante 5 minutos, seguido de un gradiente lineal


del 1 al 40% de B durante los siguientes 58 minutos. Los volmenes de inyeccin
fueron de 5 L y los flujos se modificaron para obtener la misma velocidad lineal
(1,3 mm/s) en todas las columnas. La deteccin se realiz a 214 y 280 nm.
Tabla III.4. Columnas empleadas en este trabajo.
Caractersticas
Tipo
Tamao de
partcula (m)
Dimensiones,
longitud ID
(mm)
Fabricante
a

ProSwift
Monoltica
polimricaa

Chromolith
Monoltica
slice (C18)

Gemini 5 m
Particulada
(C18)

Gemini 3 m
Particulada
(C18)

Kinetex
Particulada
(C18)

2,6

50 4,6

100 4,6

150 4,6

150 3

100 3

Dionex

Merck

Phenomenex

Phenomenex

Phenomenex

Columna monoltica de fenil-poliestireno.

III.3.4.4. Preparacin de estndares y muestras


Los concentrados de enzimas industriales lquidas fueron diluidos en un
factor 1:10 con una disolucin de (NH4)HCO3 40 mM (pH = 8,5). Para las
enzimas suministradas como slidos granulares, se pesaron 0,2 g, se aadi 1 mL
de agua, y se sonic la suspensin durante 15 minutos. Tras centrifugar, se tom
una alcuota del sobrenadante y se trat como se ha indicado para los
concentrados lquidos de enzima. Se prepar una disolucin de BSA de 3,5 mg
mL-1 en el tampn de (NH4)HCO3. La digestin se efectu de la siguiente
manera: se tomaron alcuotas de 100 L de cada enzima diluida y de la
disolucin de BSA, y se aadieron 5 L de una disolucin de DTT 200 mM. Esta
mezcla se dej reaccionar bajo agitacin a 100 C para reducir los grupos
disulfuro. Tras dejar enfriar a temperatura ambiente, se aadieron 25 L de una
disolucin de tripsina de 0,5 mg mL-1 preparada en el mismo tampn. La mezcla
se digiere durante toda la noche a 37 C, utilizando un mezclador termosttico
con agitacin. La digestin se detiene por adicin de 5 L de cido frmico al

127

Miriam Beneito Cambra

10%. Los digestos resultantes se filtraron a travs de un filtro de 0,2 m Phenex


RC (Phenomenex, Torrance, IL, USA) y se inyectaron en el cromatgrafo.
Tambin se prepar un blanco de muestra siguiendo el mismo protocolo, pero en
esta ocasin la alcuota de 100 L de enzima diluida fue sustituida por el mismo
volumen de tampn.
Se pesaron alrededor de 2 g de las bases de detergente y de los detergentes
comerciales. Las bases de detergente se aditivaron con 20 L de una enzima
concentrada industrial. Las enzimas fueron precipitadas por adicin de 10 mL de
acetona con agitacin. Tras eliminar el sobrenadante, el precipitado se aisl por
centrifugacin a 4000 rpm 10 minutos, y se disolvi en 200 L de agua. Esta
disolucin se re-precipit con 1 mL de acetona y el precipitado se aisl por
centrifugacin a 13400 rpm 10 min. El residuo se disolvi en 100 L del
tampn de (NH4)HCO3 y se llev a cabo la digestin como se ha indicado
anteriormente.

III.4. Columnas monolticas

III.4.1. Monmeros, agentes entrelazantes, iniciadores, estndares y


reactivos de uso general
Para la preparacin de columnas monolticas se emplearon los siguientes
reactivos: LMA, EDMA, META (al 75% en agua), 1,4-butanodiol, metacrilato
de 3-(trimetoxisilil)propilo (como reactivo enlazante o silano binding), LPO y
DMPA (Aldrich, Milwaukee, WI, USA), 1-propanol, ACN y MeOH (Scharlau,
Barcelona, Espaa); AIBN, BPO y Tris fueron suministrados por Fluka (Buchs,
Suiza). Se utiliz tiourea como marcador del EOF, una mezcla de PAHs
conteniendo pireno, antraceno, naftaleno, fluoreno, benzo[a]antraceno y
benzo[k]fluoranteno (Riedel de Han, Seelze, Alemania), y una mezcla de

128

Captulo III. Materiales y mtodos

alquilbencenos

conteniendo

tolueno,

etilbenceno,

n-propilbenceno,

n-

butilbenceno, n-pentilbenceno y n-hexilbenceno. Como estndares de prueba se


utilizaron

los

siguientes

solutos

bsicos: anilina,

4-nitroanilina,

N,N-

dimetilanilina, 2,4,6-trimetilanilina y piridina (Aldrich). Se utiliz agua


desionizada obtenida con un desionizador Barnstead (Sybron, Boston, MA,
USA).

III.4.2. Tratamiento de columnas

III.4.2.1. Acondicionado de columnas


Antes de rellenar las columnas con las disoluciones que contienen las
mezclas para la sntesis de monolitos, se procede a modificar la superficie interna
del capilar de slice. Esta operacin tiene como objeto favorecer el anclaje
covalente del monolito a dicha superficie interna. Para ello se sigui el
procedimiento descrito por Frchet y col. [Peters, 1997]:
Los capilares que se emplearon eran de slice fundida con dimensiones
375 m OD 100 m ID, con la capa externa transparente a la radiacin UV
(Polymicro Technologies, Phoenix, Arizona, USA). Por una seccin de capilar de
4 m de longitud se hicieron pasar sucesivamente, mediante la ayuda de una
jeringa Hamilton conectada a una bomba de jeringa (kd Scientific, Holliston,
MA, USA), las siguientes disoluciones a un caudal de 200 L min-1 (salvo que se
especifique lo contrario):
1. Acetona, hasta ver aparecer algunas gotas a la salida del capilar, para
asegurar la limpieza de la pared interna.
2. Agua nanopura, hasta la eliminacin completa de la acetona.
3. NaOH 0,2 M, hasta observar pH bsico a la salida del capilar.
4. Agua nanopura, hasta eliminar el NaOH, para evitar cambios bruscos del

129

Miriam Beneito Cambra

pH.
5. HCl 0,2 M, hasta observar pH cido a la salida del capilar.
6. Agua nanopura, hasta pH neutro a la salida del capilar, para eliminar los
restos de cido.
7. EtOH hasta olor persistente, con el fin de eliminar el agua y evitar la
hidrlisis del reactivo enlazante (silano-binding) que se adiciona en la
siguiente etapa.
8. Disolucin de reactivo enlazante (silano-binding) al 20% (m/v) en EtOH,
acidulado con cido actico hasta pH 5; el enlazante se pasa a un caudal de
0,25 L min-1 durante 60 min.
9. Acetona, para eliminar el exceso de reactivo enlazante.
Para finalizar, se aplic una corriente de nitrgeno para secar el capilar,
dejndose en estas condiciones durante 24 h hasta completar la reaccin de
condensacin de los grupos silanol con el reactivo enlazante (silano-binding).
Tras este periodo, se retir la fuente de nitrgeno y se sellaron los extremos del
capilar con sendos tapones, con el fin de evitar la hidrlisis de los enlaces
siloxano.

III.4.2.2. Preparacin de columnas monolticas


Los monolitos se preparan mediante polimerizacin de mezclas de un
monmero base (LMA), un agente entrelazante (EDMA), un monmero con
carga para la generacin del EOF (META), y disolventes porognicos, siendo los
empleados aqu el 1,4-butanodiol y el 1-propanol. Estas mezclas se preparan
mediante la pesada de cada uno de sus componentes en una balanza analtica.
Las mezclas se polimerizan mediante fotoionizacin. Los iniciadores
empleados fueron AIBN, DMPA, BPO y LPO. Antes de iniciar la
polimerizacin, y con el fin de eliminar el oxgeno disuelto de las disoluciones,

130

Captulo III. Materiales y mtodos

stas se sonican durante 10 minutos y se purgan con nitrgeno durante 10 min


ms.
Los capilares, previamente pre-acondicionados, fueron cortados en trozos
de 33,5 cm de longitud, rellenndose un segmento de 8,5 cm de longitud con las
mezclas de polimerizacin. Una vez rellenos los segmentos, se sellan los
extremos del capilar mediante tapones y se procede a la polimerizacin. En el
caso de la fotopolimerizacin los capilares se sometieron a una irradiacin de 0,9
J/cm2 durante 10 minutos dentro de una cmara UV equipada con cinco lmparas
UV (5 8 W, 254 o 365 nm). La lmpara UV de 254 nm fue empleada para
polimerizar con todos los iniciadores, a excepcin del AIBN para el que se
utiliz radiacin de 365 nm [Peters, 1998-A; Eeltink, 2005; Geiser, 2007; Ngola, 2001].
Una vez terminada la polimerizacin, se cortaron ligeramente los extremos de las
columnas resultantes para liberar restos adheridos a los tapones, y garantizar la
homogeneidad del relleno.
A continuacin, las columnas se desobstruyeron con MeOH y la ayuda de
una bomba de HPLC, eliminando tanto los disolventes porognicos como los
posibles monmeros sin reaccionar, y los oligmeros no incorporados a la
estructura del monolito. A continuacin, se cortaron las porciones de capilar
necesarias para ajustar la posicin de la ventana ptica a 8,5 cm de uno de los
extremos, y tambin para que la longitud total del capilar fuera de 33,5 cm.
Cortar un extremo tambin garantiza una seccin transversal del monolito
perpendicular al eje longitudinal del capilar en dicho extremo. Finalmente, antes
de la inyeccin de estndares o muestras, se hace pasar fase mvil a travs del
capilar durante 30 min.

131

Miriam Beneito Cambra

III.4.3. Instrumentacin y condiciones de trabajo


Los ensayos de CEC se realizaron con un equipo de electroforesis capilar
Agilent, modelo HP3D (Agilent Technologies, Waldbronn, Alemania), dotado de
un DAD, y de un sistema auxiliar capaz de suministrar hasta 10 bar de presin
externa de nitrgeno sobre ambos extremos del capilar simultneamente. La
presurizacin del capilar es importante para evitar la aparicin de burbujas que
cortaran el paso de corriente elctrica por el mismo. Para la adquisicin de los
datos se utiliz el software ChemStation (Rev. A.10.01, Agilent).
Con el fin de proceder a su acondicionado con la fase mvil, la columna se
coloc en el equipo de CEC y se equilibr con la fase mvil a 25 C,
incrementndose el voltaje progresivamente en el rango de 5 a 25 kV,
presurizando en todo momento los viales de entrada y salida a 10 bares, hasta
observar una seal analtica y una corriente estables. Esta etapa de equilibrado
tuvo una duracin de 45-60 min, dependiendo de las caractersticas de flujo de
cada columna. Para los ensayos de CEC se emple, cuando fue necesario, una
muestra de tiourea como marcador del EOF.
Las separaciones se realizaron a 25 C y a varios voltajes. En todos los
casos se utiliz nitrgeno para presurizar ambos viales a 1 MPa. Se prepar una
disolucin de Tris en agua a pH 8.0 ajustado con HCl 1 M. Las fases mviles
fueron preparadas mezclando el tampn de Tris con diferentes proporciones de
ACN y agua para obtener disoluciones con una concentracin constante de 5 mM
de Tris. Se prepar una mezcla de seis PAHs (pireno, antraceno, naftaleno,
fluoreno, benzo[a]antraceno, benzo[k]fluoranteno) y tiourea (100 200 g mL-1,
de cada sustancia) para evaluar el funcionamiento de las columnas. La inyeccin
de la mezcla de PAHs se hizo de forma electrocintica (5 kV 3 s), y la
deteccin se fij a 214 y 254 nm.

132

Captulo III. Materiales y mtodos

La morfologa de los materiales monolticos se estudi mediante el


empleo de un microscopio electrnico de barrido Hitachi modelo S-4100
(Ibaraki, Japn), provisto de un sistema de captacin de imgenes EMIP 3.0.
Previamente, se metaliz la superficie expuesta de la seccin de las columnas con
un depsito de oro y paladio. Para ello, se utiliz un recubridor por pulverizacin
BIORAD modelo SC-500 (Hemel, Hempstead, Reino Unido).

133

RESULTS

CHAPTER IV

NON IONIC SURFACTANTS


IN CLEANING PRODUCTS

Chapter IV. No ionic surfactants in cleaning products

IV.1. APGs

IV.1.1. APGs by HPLC-MS


The APGs are non-ionic surfactants produced from renewable materials
(glucose and fatty alcohols)

[Koch, 1993; Balzer, 1996; Balzer, 2000; Hill, 1997;

Rybinski, 1998; Biermann, 1993].

Owing to their favourable properties in terms of

foaming performance, synergism with other surfactants, and environmental and


skin compatibility, they are widely used in cleaning and personal care products
[Balzer, 1996; Rybinski, 1998; Biermann, 1993; Garca, 1997; Uppgard, 2000],

the oil and gas industry

[McGregor, 2004].

and in

APGs are industrially obtained as

complex mixtures of alkylpoliglucosides. The oligomers are distinguished by the


alkyl chain length, the number of glucose units and the isomerism (Fig. I.5).
Three types of isomerism are present: stereoisomerism with - and -epimers,
ring isomerism (pyranoside and furanoside forms), and positional isomers, with
predominant 1,4-and 1,6-interglucosidic linkages between glucose units.
The total amount of APGs can be determined by hydrolysis followed by
derivatisation with anthrone and colorimetry [Buschmann, 1995; Buschmann, 1996B],

potentiometric titration after sulfonation with the SO3DMF complex

[Buchmann, 1996-A],

hydrolysis

near-infrared spectrometry

[Kroh, 1999].

[Kim, 2001]

and enzymatic

APGs can be determined in environmental samples after

hydrolysis, followed by distillation of the alcohols and fluorogenic derivatisation


[Meissner, 1999].

The separation by thin layer chromatography on C8 and C18

phases has been described

[Buchmann, 1996-A; Buschmann, 1996-B; Buschmann,

1996-C; Spilker, 1996; Klaffke, 1998].

Underivatised alkylmonoglycosides can be

separated with isomer resolution using high-temperature GC


Spilker, 1996].

[Rybinski, 1998;

APGs have been also separated by GC after silylation

1996-A; Spilker, 1996; Billian, 1998].

[Buchmann,

The pyranoside and furanoside forms can be

139

Miriam Beneito Cambra

distinguished by the different fragments obtained by GCMS

[Billian, 1998].

Micellar electrokinetic chromatography with electrochemical detection has been


applied to the determination of APGs in shampoo

[Hbner, 2006].

To implement

the photometric detection of APGs after HPLC separation, several post-column


chromogenic derivatisation procedures have been described

[Kramer, 1992].

Alternatively, refractive index detection [Klaffke, 1998], ELSD [Buchmann, 1996-A;


Buschmann, 1996-B; Lafosse, 1992; Czichocki, 2002; Armari, 2003]
[Czichocki, 2002; Eichhorn, 1999; Klaffke, 1999; Khn, 2004],

or MS detection

can be used. The

chromatographic behaviour of alkylglucopyranoside standards using C8, C18,


phenyl and polyvinylalcohol columns has been described

[Lafosse, 1992],

and the

separation of APGs with silica, C18 and polyvinylalcohol columns have been
compared

[Czichocki, 2002].

Eichhorn and Knepper

[Eichhorn, 1999]

have used

HPLCMS with an ESI to determine APGs in spiked river and waste waters.
Using a C18 column and gradient elution with ACN/water, the - and -epimers
and the ring isomers were resolved. Alkylglucopyranosides can be further
distinguished by their higher affinity to form [M+NH4]+ adducts with respect to
alkylglucofuranosides. Kuhn and Neubert

[Khn, 2004]

have used HPLCESI-

QTOFMS with a C18 column, a MeOH/water mobile phase and gradient


elution, to characterize industrial mixtures of APGs; however, isomers were not
resolved.
In this section, the HPLC separation of APG mixtures using either an
alkylamide or a cyanopropyl column, with ACN/water mixtures as mobile
phases, was studied. The alkylamide column in isocratic conditions was adequate
to separate alkylmonoglucosides with full resolution between epimers and ring
isomers. Retention was lower on the cyanopropyl column, but equilibration time
was much shorter. Using the cyanopropyl column with gradient elution the ring
isomers were also resolved. The influence of the alkyl chain length on the

140

Chapter IV. No ionic surfactants in cleaning products

response factors was studied. The procedures were applied to the characterization
and determination of APGs in toiletries.

IV.1.1.1. Optimization of working conditions using continuous infusion MS


Using continuous infusion MS in the NI mode, peaks of the [MH] ions
of the standards and the components of Glucopone and Plantacare were obtained.
In the PI mode, the corresponding [M+H]+ and [M+Na]+ peaks were observed.
The [M+ Na]+ peaks largely predominated when NaAcO was added to the
infused solution. Maximal intensity was obtained with 20 mM NaAcO. The PI
mode, which gave peak intensities ca. 20 times larger than those obtained with
the NI mode, was selected. Glucopone and Plantacare showed several groups of
[M+Na]+ peaks, each of them corresponding to the oligomers with a given
number of glucose units, up to m = 6. Within each group, two large peaks
corresponding to the C8Gm and C10Gm oligomers, and a small one due to the
C12Gm oligomer, were observed. The intensity also decreased as m increased.
Using infusion of the Glucopone solution, the dry gas flow, spray temperature
and spray pressure were optimized.

IV.1.1.2. Separation of APGs by HPLCMS in isocratic conditions


The alkylamide column and a mixture containing 20:80 ACN/water in the
presence of the HAcO/NaAcO buffer were first used. The EICs at the m/z values
observed in the infusion spectra were examined. The AMGp standards gave
single chromatographic peaks, and complex chromatograms were obtained for
the technical grade mixtures. As shown in Fig. IV.1 for Glucopone, a complete
separation of the four C8G1 isomers (- and -epimers, and ring isomers, on the
EIC at m/z 315) was achieved.

141

Miriam Beneito Cambra

-C8G1p

Abundance x 107

1.5

-C8G1p
1.0

-C8G1f
0.5

C8G2

-C8G1f
m/z 315

m/z 477
0

10

20

30

40

50

Time (min)

Fig. IV.1. EIC of Glucopone. The alkylamide column was used. The
mobile phase contained water with 20% ACN in the presence of 40 mM
HAcO/NaAcO buffer of pH 4.7. The flow rate was 0.4 mL min1.

The epimers and ring isomers of C10G1 and C12G1 appeared also resolved at
longer retention times (not shown). Also, a partial separation of the isomers of
the alkyldiglucosides (C8G2) was evidenced (EIC at m/z 477). The identification
of - and -C8G1p was made by injecting solutions of the corresponding
standards. As far as we know, standards of APGf are not available; however, the
two peaks of low intensity which appeared at longer retention times than the and -C8G1p peaks on the m/z 315 trace, were attributed to the corresponding and -C8G1f isomers. Retention should be higher for these isomers as a
consequence

of

the

longer

CH(OH)CH2OH

chain

bound

to

the

alkylglucofuranoside ring in comparison with the CH2OH chain of the


alkylglucopyranosides.
Next, to reduce the analysis time of the C10Gn and C12Gn isomers, the
ACN concentration in the mobile phase was increased. With 30% ACN (Fig.

142

Chapter IV. No ionic surfactants in cleaning products

IV.2), the - and -epimer pairs of C8G1 were not resolved, but the four isomers
of C10G1 were resolved within 20 min. As also shown in Fig. IV.2, the presence
of alkyldiglycosides (C8G2 and C10G2) and alkyltriglycosides (C8G3) with
partial isomer resolution was evidenced.
1.5

C8G1p

Abundance x 106

C10G2
C8G3

1.0

-C10G1p
m/z 639

m/z 505
-C10G1p

C8G2
0.5
C8G1f

-C10G1f
-C10G1f
m/z 315

m/z 343

m/z 477
0

6
8
Time (min)

12

14

16
18
Time (min)

20

Fig. IV.2. EIC of Glucopone. The alkylamide column was used. The
mobile phase contained water with 30% ACN in the presence of 40 mM
HAcO/NaAcO buffer of pH 4.7. The flow rate was 0.2 mL min1.

The chromatograms of Plantacare were similar to those of Glucopone, but with


higher proportions of the C10Gm and C12Gm oligomers with respect to those of
C8Gm (not shown). A chromatogram of a baby shampoo, eluted with 50%
acetonitrile, is shown in Fig. IV.3. The ring isomers of the AMGs of C8G1,
C10G1, C12G1 and C14G1 were resolved. The - and -epimers of C12G1f,
C14G1p and C14G1f were also resolved. The isomers of the alkyldiglycosides
C8G2, C10G2 and C12G2 were partially resolved.

143

Miriam Beneito Cambra

C8G2
m/z 477

2.5
C10G2

m/z 505

Abundance x 107

C12G1p

C12G2

-C14G1f
m/z 533
1.5

-C14G1f

C8G1p

C10G1p
m/z 399

1
m/z 343

0.5
m/z 315

C10G1f

-C14G1p
-C12G1f
-C12G1f
m/z 371

18 20
Time (min)

16

-C14G1p

C8G1f

m/z 399
8

10

14

18
Time (min)

Fig. IV.3. EIC of a baby shampoo. The alkylamide column was used. The
mobile phase contained water with 50% ACN in the presence of 40 mM
HAcO/NaAcO buffer of pH 4.7. The flow rate was 0.2 mL min1. The inset
shows the m/z 399 trace with the intensity axis multiplied by 50.

Retention was much weaker with the cyanopropyl column; for instance,
using 20% ACN, the C8G1 isomers were eluted within 6 min (not shown). Using
95 and 100% water in the mobile phase, the C8G1 isomers appeared within 30
and 40 min, respectively. In all cases, the ring isomers were well resolved, but
the epimer pairs were poorly resolved with this column.

IV.1.1.3. Separation of APGs by HPLCMS with gradient elution


The alkylamide column and mobile phases containing 60:40 (A) or 90:10
(B) ACN/water (v/v) in the presence of the HAcO/NaAcO buffer were used.
Mobile phase A was first pumped until stabilization of the pressure, thus the
Plantacare solution was injected and the composition of the mobile phase was
144

Chapter IV. No ionic surfactants in cleaning products

linearly varied from A to B in 20 min. The AMGs were separated with partial
resolution between the epimers, and with excellent resolution between the ring
isomers, within 12 min (not shown). However, the re-equilibration of this column
was observed to be rather slow. Pumping of mobile phase A during several hours
was required to achieve reproducibility between successive injections.
C12G1p

C16G1p

C8G1p

4
Abundance x 107

m/z 427
C16G1f
C10G1p

C12G1f
C14G1p
C14G1f

m/z 399
m/z 371

m/z 343

m/z 315
0

m/z 427

C10G1f

C8G1f

13 14
Time (min)

Time (min)

12

Fig. IV.4. EIC of Plantacare. The cyanopropyl column was used. The
composition of the ACN/water mobile phase (also containing 40 mM of
the HAcO/NaAcO buffer of pH 4.7) was linearly varied from 25 to 90%
acetonitrile in 15 min. The flow rate was 0.2 mL min1. The inset shows
the m/z 427 trace with the intensity axis multiplied by 20.

Using the cyanopropyl column, mobile phases containing 25:75 (A) and
90:10 (B) ACN/water (v/v) in presence of the acetic acid/sodium acetate buffer,
were used. The mobile phase was linearly varied from A to B in 15 min. As
shown in Fig. IV.4 for Plantacare, all the AMGs, from C8G1 up to C16G1, were
separated within 14 min. The epimer pairs were not resolved, but a good
resolution between the ring isomers was achieved. Successive injections of the

145

Miriam Beneito Cambra

sample were performed after pressure stabilisation (15 min). Excellent


reproducibility of the chromatograms was obtained. The chromatogram of a hand
cream, which has a rather complex matrix, showed the resolved peaks of the ring
isomers of C12G1 and C14G1 (Fig. IV.5).

Abundance x 105

C12G1p

6
C14G1p
4

2
C14G1f
C12G1f
m/z 371

m/z 399

0
10

12

Time (min) 14

Fig. IV.5. EIC of a hand cream. The cyanopropyl column was used. The
composition of the ACN/water mobile phase (also containing 40 mM of
the HAcO/NaAcO buffer of pH 4.7) was linearly varied from 25 to 90%
ACN in 15 min. The flow rate was 0.2 mL min1.

IV.1.1.4. Quantitation studies


The calibration curves were obtained using the cyanopropyl column in the
optimized gradient elution conditions. Solutions containing a mixture of the
standards (-pyranosides C1G1, C6G1, C8G1, C10G1 and C12G1), in the
presence of 50 g mL1 -pyranoside C9G1 as internal standard, were injected.
Mixtures of the -pyranosides of C1G1 and C8G1 were also injected. The

146

Chapter IV. No ionic surfactants in cleaning products

concentration of the standards was varied from 10 to 120 g mL1 (from ca. 60
600 M for C1G1 to 30360 M for C12G1). In all cases, the calibration curves
gave a good linearity, with r2 > 0.99 (6 points per curve).
As shown in Fig. IV.6, the relative sensitivities of the -epimers (or
response factors, calculated by using -C1G1p as reference) increased almost
linearly from C1G1 to C8G1, reached a maximum at C10G1 and decreased for
C12G1. Relative rather than absolute sensitivities were plotted in this figure to
enhance the differences between the standards, independently from detector
sensitivity and some working conditions. A range of relative sensitivities for
C14G1 was also plotted on Fig. IV.6. This range was obtained by injecting
Plantacare, and using the sum of the areas of the C14G1p and C14G1f peaks, and
the composition given by the manufacturer (6.19.5% total C14G1 for a 5153%
pure product), to calculate the relative sensitivity range. As deduced from Fig.
IV.6, this range indicated a further decrease of the relative sensitivity for alkyl
chains longer than 12 carbon atoms. Since a range of concentrations was not
declared (Table IV.1), the relative sensitivity for C16G1 could not be calculated.
The influence of the mobile phase composition on the relative sensitivities
was also studied. For this purpose, a 20:80 (A) or a 75:25 (B) ACN/water (v/v)
mixtures, containing 20 mM NaAcO and 20 mM HAcO, were used. Dilutions of
the stock solutions of the standards were prepared using both A and B. The
dilution factor was 1:20, but 1:10 was used for C12G1. All the diluted solutions
also contained 50 g mL1 of the internal standard, -C9G1p. The autosampler
was directly connected with the ESI source with a PEEK tube in the absence of
the separation column, and the solutions were injected successively using A and
B as mobile phases. No significant differences between the peak areas obtained
with mobile phases A and B were observed. Thus, the differences among the
response factors observed in Fig. IV.6 should be attributed to the length of the

147

Miriam Beneito Cambra

alkyl chains rather than to the increase of the acetonitrile concentration along the
elution gradient.

16

90

Relative sensitivity

50
8

LOD (M)

70

12

30
4
10
0
0

12

Fig. IV.6. Relative sensitivities (left axis and dashed line) of - () and alkylglycopyranosides () with respect to -C1G1, and LODs (, right
axis and dotted line) vs. the number of carbon atoms in the alkyl chain, n
(-C9G1p used as internal standard). All values were obtained from pure
standards, but for C14G1 the two points delimit the range of relative
sensitivity, which was estimated from its concentrations in Plantacare (6.1
9.5% C14G1 for a 5153% pure product, as declared by the manufacturer).

Taking into account that Na+ is bound to the same oxygen as the alkyl
chain

[Klaffke, 1999],

the increase of the relative sensitivity from C1G1 up to

C10G1 can be due to stabilization of the adduct by the higher electronic density
provided by the longer alkyl chain. The smaller volatility, the formation of ionpairs or micellization are possible explanations for the sensitivity decrease when
the alkyl chain has 12 and 14 carbon atoms. Thus, the response factor can be
approximately predicted by linear interpolation within the C1G1C10G1 range,
but not when n > 10. Owing to the large differences, large systematic errors
148

Chapter IV. No ionic surfactants in cleaning products

should be expected if calibration is not performed by using all the appropriate


CnG1 standards.
The relative sensitivities of the -epimers of C1G1p and C8G1p with
respect to -C1G1p (used as a reference compound) were also plotted on Fig.
IV.6. The -epimer/-epimer sensitivity ratio was 0.995 for C8G1, but 0.43 for
C1G1. Thus, the response factor of the -epimer was less than half that of the epimer for C1G1, approaching to the sensitivity of the corresponding -epimer
for longer alkyl chains. This can be explained by a lower ability of the -epimer
of C1G1 to bind Na+ with respect to the -epimer. Further, the higher electronic
density on the oxygen atom bound to the chain could explain both the sensitivity
increase when the alkyl chain becomes longer, and the small sensitivity
differences between the C8G1 epimers. Small or negligible systematic errors
should be expected by using the calibration curve of a CnG1 epimer (n 6) to
predict concentrations of the other epimer, or the sum of the concentrations of the
two epimers.
The LODs were obtained as the concentrations giving a peak area equal to
three times the standard deviation of the calibration points with respect to the
regression straight-line (standard deviation of the residuals). As observed in Fig.
IV.6, the LODs were 85 M for C1G1 and of ca. 25 M from C6G1 up to
C12G1, which agrees with the higher response factors of the latter. Also, from
the standard deviation of the residuals, the limits of quantitation (as the
concentrations giving a 10% precision in the determination of the analytes) were
280 M for C1G1 and 80 M from C6G1 up to C12G1.
When the industrial products were analyzed, significant differences
between the peak area ratios of the respective furanoside/pyranoside forms were
observed. Thus, integration of the areas for the C10G1 and C12G1 isomer pairs
showed that this ratio was 0.12 0.13 for Glucopone and the baby shampoo, 0.26

149

Miriam Beneito Cambra

0.30 for Plantacare and ca. 0.04 for the hand cream. Thus, the
furanoside/pyranoside concentration ratio can be used to characterize industrial
samples. However, owing to the lack of standards, only the total concentration of
each CnG1 group of isomers was obtained. For this purpose, the sum of the peak
areas of the pyranoside and furanoside forms, and the calibration curve of the
corresponding -pyranoside standard, were used. For Plantacare, the calibration
curves predicted concentrations within the ranges declared by the manufacturer
(Table IV.1). The analysis of a baby shampoo gave 0.5% C8G1, 0.2% C10G1,
and 0.6% C12G1. The peaks of the C14G1 isomers were also observed in the
chromatograms of this sample.
Table IV.1. Declared and found concentrations of alkylmonoglucosides in
Plantacarea
Compound Declared (%) Found (%)

C6G1

Max. 0.26

0.06

C8G1

12.2 15.9

13.0

C10G1

7.6 11.7

9.7

C12G1

18.9 22.3

18.9

C14G1

6.1 9.5

C16G1

Max. 2.12

Range from minimal up to maximal declared concentrations multiplied by

minimal (51%) and maximal (53%) declared purity, respectively.

IV.1.2. Fragmentation of D-Glucose and AMGs


CID fragmentation constitutes a powerful tool for the structural
elucidation of oligosaccharides. Alkaline ions and ammonium

[Harvey, 2000-A;

Harvey, 2000-B; Chiarelli, 1987; Cancilla, 1999; Harvey, 1994; Harvey, 1997; Kovik,
1995; Penn, 1996; Asam, 1997],
2005],

and iron salts

divalent cations

[Carlesso, 2000; Carlesso, 2001]

150

[Harvey, 2001; Madhusudanan,

have been used; however, the

Chapter IV. No ionic surfactants in cleaning products

most informative fragmentation with maximal sensitivities has been obtained


with sodium and calcium ions

[Harvey, 2000-A; Harvey, 2000-B; Harvey, 2001].

The

fragmentation of oligosaccharides using IT-MS [Zaia, 2004] and the application of


MALDI-MS to the analysis of carbohydrates and glycoconjugates

[Harvey, 2006]

have been reviewed.


When using low fragmentation energies, and according to the Domon
Costello nomenclature [Zaia, 2004; Domon, 1988] (see Fig. I.6), the cleavage of the
glucosidic bonds produces mainly B and Y fragments [Asam, 1997; Creaser, 2002].
Entire linkage sequences of linear and branched polysaccharides can be
elucidated by analyzing the fragments

[Asam, 1997; Harvey, 2001; Zaia, 2004].

Using higher fragmentation energies, cross-ring cleavages, which provide


additional linkage information, are also produced [Harvey, 2000-A; Harvey, 2000-B;
Harvey, 2001; Cancilla, 1999; Asam, 1997; Zaia, 2004; Creaser, 2002].

Less attention has been paid to the CID spectra of monosaccharides and
their derivatives. The stereochemical differentiation of monosaccharides in the
presence of Zn2+
demonstrated.

[Gaucher, 1998]

The

positional

and iron chloride


isomers

and

[Carlesso, 2000]

diastereomers

of

has been
sulfated

monosaccharides have been distinguished [Minamisawa, 2005]. The CID spectra of


D-glucose and 2-fluorodeoxyglucose have been compared [Macek, 2003]. Using
CID spectra, several pentoses and hexoses, including stereoisomers, were
differentiated by the ratio of competing fragmentation processes

[March, 2005].

Multivariate analysis of TOF-SIMS has been used to distinguish among several


furanoses, as well as among several pyranoses

[Berman, 2006].

However, studies

addressed to distinguish five- from six-membered ring isomers using CID


fragmentation are rather scarce. The ring size of the last unit at the non-reducing
end of oligosaccharides has been related to the abundance ratio of the [M+Na90]+ and [M+Na-104]+ ions of the CID spectrum

151

[Kovik, 2001].

Using TOF-

Miriam Beneito Cambra

SIMS, five-membered ring monosaccharides were more stable than the


corresponding six-membered ring isomers, also giving different peak profiles
upon fragmentation [Berman, 2006].
APGs, which are industrially produced as complex mixtures from glucose
and fatty alcohols, constitute an important class of nonionic surfactants
1997; Rybinski, 1998; Balzer, 2000].

[Hill,

Because of their foaming performance,

synergism with other surfactants, and environmental and skin compatibilities,


APGs are widely used in cleaning and personal care products
Garca, 1997; Uppgard, 2000]

[Rybinski, 1998;

and in the oil and gas industry [McGregor, 2004]. The

aim of this work was to study the ion-trap stepwise fragmentation of AMGs,
which are the major components of industrial APGs. Also, the industrial
production of APGs leads to mixtures containing major amounts of AMGp, and
minor but significant concentrations of their five-membered ring isomers, AMGf.
Thus, in this section we describe the CID spectra of both AMGp and AMGf,
using several isotopic forms of D-glucose to assist interpretation. The successive
MS2, MS3 and pseudo-MS4 spectra of D-glucose, D-glucose-13C6, D-glucose-6,6d2, D-glucose-1-13C and AMGp having up to 12 carbon atoms in the alkyl chain
were obtained, at increasing fragmentation energies, with an ESI-IT-MS.
Standards of the corresponding AMGf were not available; however, previous
separation of a commercial mixture of APGs by HPLC, and their MS and MS2
spectra were obtained on-line.

IV.1.2.1. Optimization of the IT-MS detection conditions


Using infusion in the IT-MS, both [M+H]+ and [M+Na]+ ions were
observed to predominate in the PI spectra of D-glucose, the AMGp standards and
Glucopone. The spectrum of Glucopone showed the predominant peaks of the
octyl- and decyl-monoglucosides, plus the peaks of the corresponding

152

Chapter IV. No ionic surfactants in cleaning products

diglucosides and triglucosides with lower abundances. Concerning to the alkyl


chain length, the octyl- and decylglucopyranosides predominated, the
corresponding

octyl-

and

decylglucofuranosides

showing

much

lower

concentrations. Optimization of the IT-MS detection conditions was performed


using Glucopone solutions. The abundance of the [M+Na]+ ions largely increased
when sodium acetate was added to the MeOH/water solutions. The abundance of
the [M+Na]+ ions increased with the Na+ concentration, and a plateau was
reached within the 10 20 mM range. Then, the drying gas flow, spray
temperature and spray pressure were optimised in the presence of 20 mM
NaACO for a maximal intensity of the [M+Na]+ peaks of the AMGs of
Glucopone. The rules to nomenclature of fragmentation are given in section I.2.3.

IV.1.2.2. Fragmentation of D-Glucose and their isotopic forms using MSn


Using continuous infusion of the standard solutions in the optimized
detection conditions, the MS2 spectra of D-glucose, D-glucose-13C6, D-glucose6,6-d2 and D-glucose-1-13C were obtained at increasing fragmentation energies.
In Fig. IV.7, the MS2 spectra of the [M+Na]+ ions of D-glucose and D-glucose13

C6, both obtained at an energy of e = 0.45, are given. Using this fragmentation

energy, the peak of the parent ion was still observed together with the peaks of
next ion generation. By comparing the MS2 spectra of D-glucose and D-glucose13

C6, it is deduced that the m/z 185 peak of D-glucose corresponds to an ion with

six carbon atoms. This peak was interpreted as due to a loss of a water molecule
from the [M+Na]+ parent ion (m/z 203). At lower m/z values, the MS2 spectra of
D-glucose showed an abundant peak at m/z 143 and a weak one at m/z 113,
which according to MS2 spectrum of D-glucose-13C6 (Fig. IV.7, part B),
corresponded to ions with four and three carbon atoms, respectively.

153

Miriam Beneito Cambra

1.5
413

[M+Na]+

Intensity x 103

143

1
0.5

593

185 203

571
557

113
0

100

200

400

500

m/z

600

147

413

[M+Na]+

Intensity x 103

1.5

300

0.5

191 209

116
0

599

100

577
563

200

300

400

500

m/z 600

Fig. IV.7. MS2 spectra of the [M+Na]+ parent ion of (A) D-glucose (m/z
203) and (B) D-glucose-13C6 (m/z 209), both obtained at e = 0.45 (, parent
ions).

By comprehensively computing all the possible cross-ring cleavages leading to


an m/z 143 positive ion with four carbon atoms, the following possibilities were
found:

0,2

A,

1,3

X,

2,4

X and

0,4

X; however, by using the corresponding [M+Na]+

peak as parent ion, the MS2 spectrum of D-glucose-6,6-d2 (not shown) yielded a
peak at m/z 145. This indicated that carbon atom 6 was retained by the m/z 143
ion fragment of D-glucose. This excluded the

0,4

X fragmentation. Further, the

MS2 spectrum of D-glucose-1-13C (not shown) presented a peak at m/z 143,


which evidenced that carbon atom 1 was not present in this ion fragment. This
definitely excluded all the X fragmentations. Therefore, the m/z 143 ion of the
MS2 spectrum of D-glucose should be exclusively produced by a

0,2

fragmentation. The two possible structures of this fragment ion are shown in Fig.
IV.8.

154

Chapter IV. No ionic surfactants in cleaning products

Similarly, the

0,3

A,

1,4

A,

0,3

X and

1,4

X fragmentations, plus a

3,5

fragmentation followed by loss of a water molecule, are possible routes leading


to an m/z 113 ion. However, the MS2 spectrum of D-glucose-6,6-d2 showed a
peak at m/z 115, indicating that carbon atom 6 should be retained by the m/z 113
ion of D-glucose. Further, the spectrum of D-glucose-1-13C showed a peak at m/z
113, indicating that carbon atom 1 should be excluded from the composition of
this ion. Thus, according to the spectra, only the two fragments obtained by a 0,3A
fragmentation (Fig. IV.8) are possible to explain the formation of the m/z 113
ion.
Cross-ring
cleavage

Possible structures
OH

0,2A

Na+
5

m/z 143

OH

Na+

HO

OH

OH
OH
0,3A
m/z 113

O
Na+

HO

OH
6

Na+
OH

5
4

HO

Fig. IV.8. The two possible structures of the m/z 143 and m/z 113 ions of
the MS2 spectrum of the [M+Na]+ parent ion of D-glucose, produced by
0,2

A and 0,3A cross-ring cleavages, respectively.

Concerning to the abundant m/z 413 peak which was present in the MS2
spectrum of the [M+Na]+ parent ion of D-glucose, no shifts with respect to the
equivalent spectra obtained with either D-glucose-13C (Fig. IV.7, part B), Dglucose-6,6-d2 and D-glucose-1-13C (not shown) were observed. This indicated
the absence of fragments of glucose in the composition of the m/z 413 peak.
However, this abundant peak was also present in the MS2 spectra of all the
AMGs, and peaks showing the mass of the unfragmented molecules plus 413 m/z

155

Miriam Beneito Cambra

units also appeared in the spectra of both D-glucose and all the AMGs. For these
reasons, further efforts to interpret the m/z 413 peak were performed. First, MS2
spectra of the [M+Na]+ parent ion of D-glucose were obtained both in the
absence of MeOH, and also using 20 mM NaHCO3 instead of 20 mM NaAcO. In
both cases, an m/z 413 ion, with a similar abundance as that observed using 50%
MeOH and 20 mM NaAcO, was obtained. Therefore, the presence of MeOH,
AcO- or HAcO in the composition of the m/z 413 ion was also discarded. On the
other hand, the m/z 413 peak was not observed when a 20 mM NaAcO solution
in the absence of D-glucose was infused. Thus, the m/z 413 peak was formed
only in the presence of D-glucose or an AMG, and could contain Na+, OH- and
H2O, but not carbon atoms.
Thus, fragmentation of the m/z 413 ion yielded peaks at m/z 301 and 189,
which corresponded to two successive losses of 112 Da, in the MS3 spectrum.
These losses could correspond to the uncharged fragment NaOH4H2O. A peak at
m/z 171 which was attributed to a loss of water with respect to the m/z 189 ion
was also observed. Further fragmentation of the m/z 301 ion was achieved by
setting the compound stability parameter at 300%. Again, the m/z 189 and 171
ions were exclusively observed on this pseudo-MS4 spectrum. Then, all the
possible combinations of Na+, OH- and H2O (up to 20 units of each, respectively)
matching m/z 413, with the restriction of having a single positive charge, were
computed. The following single combination was found: 4 Na+ + 3 OH- + 15
H2O. However, the exact mass of this combination, 413.1257 Da, did not match
with the m/z values of the peaks observed in the high-resolution spectra of Dglucose (413.2341 Da) and the AMGp (413.2349 - 413.2406 Da). Thus, no more
efforts to interpret the m/z 413 peak were done.
As deduced from the comparison of the MS2 spectra of Fig. IV.7, parts A
and B, the ion of D-glucose at m/z 593 had six carbon atoms. A likely

156

Chapter IV. No ionic surfactants in cleaning products

explanation is the formation of an adduct containing a molecule of D-glucose


plus the m/z 413 ion (m/z = 413 + 180 = 593). The MS2 spectra obtained from the
[M+Na]+ parent ions of D-glucose-6,6-d2 and D-glucose-1-13C (not shown) gave
equivalent peaks at m/z 595 and 594, respectively, which agreed with this
composition. Similarly, the D-glucose peaks at m/z 571 and 557 (Fig. IV.7, part
A) were interpreted as coming from the m/z 593 adduct by gain of a molecule of
water and loss of NaOH, and by loss of two molecules of water, respectively.
These m/z 571 and 557 ions also showed shifts of +6, +2 and +1 Da when the
MS2 spectra were obtained with D-glucose-13C6, D-glucose-6,6-d2 and Dglucose-1-13C, respectively, which agrees with the proposed compositions.

IV.1.2.3. Fragmentation of AMGp using MSn


As summarized in Table IV.2, and as showed in Fig. IV.9 for octyl-GP, the
MS2 spectra of the [M+Na]+ parent ions of the AMGp exhibited peak patterns
similar to those of D-glucose, but with a few significant differences. Thus, the
m/z 185 ion, which was present in the MS2 spectra of D-glucose and all the
AMGp, and which was interpreted as a loss of water in D-glucose, should
correspond to the loss of the alkyl alcohol in the AMGp (B1 fragmentation).
Differently from D-glucose, which can undergo water losses at several locations,
in the AMGp the alkyl alcohol chain is exclusively bound to carbon atom 1. The
m/z 143 ion, which was present in the MS2 spectra of all the AMGp, can be
exclusively formed by a

0,2

A fragmentation. Owing to the presence of the alkyl

chain, this m/z value did not matched with the possible fragment ions obtained by
other cross-ring cleavages. This also agrees with the

0,2

A cross-ring cleavage

deduced above for D-glucose. The MS2 spectra of all the AMGp also showed an
m/z 129 ion which was not present in the MS2 spectra of D-glucose. This m/z 129
ion was weak for methyl-MGp and abundant for the other AMGp. This suggested

157

Miriam Beneito Cambra

that the presence of the hydrocarbon chain was necessary to stabilize the
resulting non-ionic fragment. By computing all the possible combinations of C,
H, O and Na giving rise to an m/z 129 ion with a positive charge, the two
following possibilities were found: C4H10O3Na+ and C3H6O4Na+; however, a 2,5A
fragmentation is the only way to produce the former ion in a single step, whereas
the less likely concurrence of at least two fragmentations,

3,5

X and C1, are

required to obtain the latter.


Table IV.2. Ions observed on the MS2 spectra of the AMGp (m/z values).
Ion \ n
2,5
0,2

A
A

B1
[M+Na]+
Unknown
[M+413-2H2O]+
[M+413+H2O-NaOH]+
[M+413]+
Others

10

12

129
143
185
217
413
571
585
607
235

129
143
185
287
413
641
655
677
209
249

129
143
185
315
413
669
683
705
209
235
275

129
143
185
343
413
697
711
733
235
309

129
143
185
371
413
725
739
761
291
315

Concerning to the peaks at large m/z values, and as also occurred with Dglucose,

they

matched

with

the

following

compositions:

[M+413]+,

[M+413+H2ONaOH]+ and [M+4132H2O]+, where M = 180 + n 14 (n = carbon


atoms in the alkyl chain). Thus, analogously to that proposed for D-glucose, they
were attributed to the formation of adducts of the m/z 413 ion with a molecule of
the corresponding alkyl-GP, and to related ions produced by further gains and
losses of water and NaOH.
As also indicated in Table IV.2, in comparison to the MS2 spectrum of Dglucose, a few additional ions which depended on the length of the alkyl chain
were also observed in the MS2 spectra of the AMGp. The m/z 235 ion of methylMGp can be attributed to the gain of water to yield [M+Na+H2O]+. For octyl-

158

Chapter IV. No ionic surfactants in cleaning products

MGp, the abundant m/z 275 ion (Fig. IV.9) could be due to a loss of NaOH to
yield [M-OH]+. Finally, significant differences between the MS2 spectra of the and -epimer pairs of methyl- and octyl-MGp were not observed.

275

Intensity x 104

129
315

413
235
143 185209
0
100

200

669 683
300

400

500

600

m/z

705

700

Fig. IV.9. MS2 spectrum of the [M+Na]+ parent ion () of octyl-MGp


obtained at e = 0.80.

IV.1.2.4. Fragmentation of AMGf using MSn


Because of the lack of standards, the MSn spectra of AMGf could not be
obtained by continuous infusion in the IT-MS. Instead of this, injections of
Glucopone using HPLC with MS and MS2 detection were performed. Isocratic
elution with mobile phases constituted by ACN/water mixtures, also containing
the sodium acetate buffer, was employed. Using 40 % ACN, the ring isomers of
the octyl- and decyl-MGp were well resolved in a short time. The extracted ion
chromatograms at the m/z values of the [M+Na]+ ions of the octyl- and decylMGs are shown in Fig. IV.10. Then, HPLC with MS2 detection, with an increase
of the fragmentation energy between injections, was used. The MS2 total ion
chromatograms (MS2-TIC), and the MS2 spectra at the retention times of the
159

Miriam Beneito Cambra

octyl-MGp and octyl-MGf peaks, are shown in Fig. IV.11. At low fragmentation
energies (e < 0.6), the peak area of octyl-MGp was much higher than that of
octyl-MGf (see the MS-TIC chromatograms of Fig. IV.10, which correspond to
MS2-TIC with e = 0); however, at increasing energies, the peak area of the octylMGp decreased at a higher rate than that of the octyl-MGf.
octyl-MGp

Intensity x 108

1.5

1.0
decyl-MGp

0.5
octyl-MGf
decyl-MGf
m/z 315
0

m/z 343
2

5
Time (min)

Fig. IV.10. Chromatogram of Glucopone obtained using MS detection. The


traces are the extracted ion chromatograms at the m/z values of the
[M+Na]+ ions of the octyl- and decyl-monoglucosides. Isocratic elution
with a 40:60 ACN/water mobile phase containing 20 mM NaAcO and 20
mM HAcO at 0.3 mL min-1 was performed.

As shown in Fig. IV.11, left part, the area of the octyl-MGp peak was even
smaller than that of the octyl-MGf peak when e = 0.80. Also, as observed in Fig.
IV.11, right part, the MS2 spectra of the alkyl-MGf differed ostensibly from
those of the corresponding alkyl-MGp. Thus, in comparison with the octyl-MGf,
the m/z 413 ion and its adducts with unfragmented molecules were more
abundantly formed by the octyl-MGp. The differences between the MS2-TICs
indicated an easier fragmentation of the alkyl-MGp compared to the alkyl-MGf.
160

Chapter IV. No ionic surfactants in cleaning products

150
100
50

octyl-MGf

octyl-MGp

e = 0.7

octyl-MGp
e = 0.7

315
80

octyl-MGf

octyl-MGp

Intensity x 104

413

octyl-MGp
e = 0.8
683

316
185 315

0.5

705

octyl-MGf
e = 0.8

185

669
octyl-MGp
e = 0.9

e = 0.9
octyl-MGf

octyl-MGp

143

413

315

413

e = 0.8

octyl-MGf
e = 0.7

143

20

315

413

705
octyl-MGf
e = 0.9

143

0.6
683 705
0.2

2
3
Time (min)

185
100

315
300

500

m/z

413

185

669

705

315
700

100

300

500

m/z

700

Fig. IV.11. Total ion HPLC-MS2 chromatograms of Glucopone recorded at


the indicated fragmentation energies (left), and corresponding MS2 spectra
at the retention times of the peaks (right). The [M+Na]+ ions of octyl-MGp
and octyl-MGf (m/z 315) were used as parent ions (). Other conditions as
in Fig. IV.10.

On the other hand, as also deduced from the MS2 spectra of Fig. IV.11,
the cross-ring cleavage at

0,2

A to give the m/z 143 ion was much more likely in

the alkyl-GFs than in the alkyl-GPs. This could be due to a lower stability of the
five-membered furanoside rings in comparison to the six-membered pyranoside
rings. Finally, as also observed in Fig. IV.11, the alkyl-GPs and alkyl-GFs
showed a similar trend to lose the alkyl chain giving rise to the m/z 185 ion.

161

Miriam Beneito Cambra

IV.2. Non-ethoxylated and ethoxylated alcohols

IV.2.1. Chromium(VI) oxide oxidation of non-ethoxylated and ethoxylated


alcohols for determination by ESI-MS
Primary alcohols are common components of many biological and
industrial samples, in addition to being present at trace levels in the aquatic
environment. Alcohols in the C10C18 range (fatty alcohols) are potent
pheromones while those with higher molecular masses are essential components
of plant waxes [Bianchi, 1995]. The industrial importance of alcohols derives from
their widespread use as solvents and fillers in industrial and household products,
while fatty alcohols are common ingredients of body-care and cosmetic products
[Farm, 2006].

Fatty alcohols also serve as the raw materials in the production of

important surfactant classes, including alkyl ether sulfates and sulfonates, and
FAEs

[Farm, 2006.].

FAEs are mostly obtained from renewable resources as

complex mixtures of oligomers with the structure: CH3(CH2)n1(OCH2CH2)mOH.


A special class of ethoxylated alcohols comprises those of low molecular
mass (both n and m <4). These compounds, known as cellosolves, are used, for
example, as anti-icing additives in aviation fuels and as solvents in cleaning
solutions

[Cheremisinoff, 2003].

GC has been successfully employed for the

determination of cellosolves and nonethoxylated alcohols with 26 carbon atoms


[Nichols, 2006],

but not FAEs with m >4, due to the limited volatility of these

compounds [Rudewicz, 1986; Crescenzi, 1995; Battersby, 2001]. The major drawback
of HPLC methods for FAEs has been the lack of an adequate detector [Rudewicz,
1986; Petrovic, 2001].

Although this problem was partially resolved through the

introduction of refractive index


Trathnigg, 2002]

[Kudoh, 1984; Mengerink, 1991; Cho, 2003;

and ELSD methods

[Mengerink, 1991; Heinig, 1998; Bear, 1988;

Miszkiewicz, 2000; Miszkiewicz, 1996-B; Kamiusuki, 2000],

162

the LODs are high for the

Chapter IV. No ionic surfactants in cleaning products

former, while the sensitivity of ELSD is poor for volatile compounds such as
non-ethoxylated alcohols (m = 0) and FAE oligomers with a low degree of
ethoxylation (m <3) [Miszkiewicz, 1996-A; Bernab-Zafn, 2006]. Finally, low LODs
are achieved with chromogenic and fluorogenic pre-column derivatization
procedures [Marcomini, 1996; Schmitt, 1990; Kiewiet, 1995; Lemr, 1994; Zanette, 1996;
Lemr, 1996; Sun, 1997; Hoffman, 2004-A; Lemr, 2003; Okada, 1991; Okada, 1992;
Hoffman, 2004-B; Bachus, 2003; Desbne, 2005; Heinig, 1996-A; Mic-Tormos, 2008-A;
Mic-Tormos, 2008-B; Mic-Tormos, 2009; Zu, 2010].

In the determination of FAEs by PI-MS with ESI or APCI interfaces,


problems arise because the sensitivity of these methods decreases with
decreasing m; thus, sensitivity is low when m <4, and non-ethoxylated alcohols
(m = 0) are not detected

[Rudewicz, 1986; Crescenzi, 1995; Petrovic, 2001; Zu, 2010;

Chiron, 2000; Sherrard, 1994; Dunphy, 2001; Cassani, 2004; Sparham, 2005].

To

overcome this problem, derivatization procedures, in which a chromophore or a


permanent charge is added to the oligomers, have been described

[Lemr, 1994;

Lemr, 1996; Lemr, 2003; Desbne, 2005; Heinig, 1996; Zu, 2010; Dunphy, 2001;
Cassani, 2004; Sparham, 2005].

However, the derivatization of FAEs is difficult because it requires an


anhydrous medium [Okada, 1992; Zu, 2010; Dunphy, 2001; Barry, 2003], and there is
an increased risk of interference from both the reagent excess and the byproducts of the reaction

[Sun, 1997; Hoffman, 2004-A; Mic-Tormos, 2008-A; Mic-

Tormos, 2008-B; Dunphy, 2001].

Furthermore, owing to the high risk of losing

oligomers with low m values by volatilization, the water content of the samples
must be reduced with particular care, which increases analysis time
2001].

[Dunphy,

Derivatization with cyclic anhydrides is, by contrast, tolerant to the

presence of small amounts of water in the samples


Tormos, 2008-B; Mic-Tormos, 2009; Zu, 2010].

163

[Mic-Tormos, 2008-A; Mic-

Miriam Beneito Cambra

Chromium(VI) oxide (CrO3) in aqueous media containing sulfuric or


acetic acid (Jones reagent) is a well-known reagent used in the oxidation of
primary alcohols to carboxylic acids [Hudlik, 1990; Burke, 1999]. The carboxylate
group has a low molar absorptivity and is therefore of little interest for UV-Vis
detection, but can be detected with high sensitivity by MS, particularly when the
alkyl chain contains a large number of carbon atoms

[Holapek, 2005].

In this

section, the determination of non-ethoxylated primary alcohols by MS, previous


oxidation to carboxylic acids with the Jones reagent, was studied. In the
proposed procedure, the reagent excess was removed with sodium sulfite, and the
oxidized alcohols were separated from the resulting chromium(III) salts by
extraction in ethyl acetate-acetone. After addition of an amine to increase pH,
and water to further enhance ionization of the carboxylate groups, the extracts
were directly infused in the ESI source of the MS. The study was also extended
to FAEs and cellosolves, which yielded the corresponding ethoxy-carboxylic
acids. Application to the characterization and determination of fatty alcohols in
complex samples, such as cosmetics and body care products, and in
environmental samples as seawater, was also demonstrated.

IV.2.1.1. Optimization of the infusion medium


In order to increase ionization of the carboxylate groups, and therefore to
enhance sensitivity in the NI mode, the influence of the addition of butylamine,
water and ACN to the ethyl acetate-acetone extracts, was studied. A stock
solution of four standards (C12E0A, C14E0A, C16E0A and C18E0A, 200 g
mL-1 each) in a 4:3 (v/v) ethyl acetate-acetone mixture was prepared. Aliquots of
this solution were diluted in a 1:1 ratio to obtain the following media: (a) ethyl
acetate and acetone in a 4:3 ratio; (b) as in a but in the presence of 30 mM
butylamine; (c) as in b but the medium contained also 10% water; and (d) as in b

164

Chapter IV. No ionic surfactants in cleaning products

but the medium contained also both 10% water and 40% ACN. These solutions
were infused in the MS, and the abundances of the [M-H]- ions were measured.
As shown in Fig. IV.12, sensitivity of the fatty acids increased as n
increased, and also varied largely with the nature of the infusion medium. Thus,
sensitivity increased ca. 5 times when butylamine was added to the extracts (Fig.
IV.12, from a to b). This was probably due to ionization of the acids in a
medium with a higher pH. Sensitivity further increased ca. 360 times when water
was also added (Fig. IV.12, from b to c).
1.2
C12E0A
C14E0A

Abundance x 107

C16E0A

0.8

C18E0A

100 x

0.4

0.0
a

Fig. IV.12. Negative-ion MS relative sensitivities for four fatty acids (n =


12, 14, 16 and 18, 100 g mL-1 each) in several media: (a) ethyl acetate and
acetone in a 4:3 ratio; (b) as in a but in the presence of 30 mM butylamine;
(c) as in b but containing also 10% water; and (d) as in b but containing
both 10% water and 40% acetonitrile. In a and b, the bar lengths were
multiplied by 100.

This should be mainly attributed to ionization enhancement upon increasing the


permittivity (dielectric constant) of the alkalinized medium. Remarkably, the
addition of water produced a much larger sensitivity increase than that observed
by solely increasing the pH of the extracts. This suggests that water was rather
scarce in the ethyl acetate-acetone extracts. Retention of most water into the
165

Miriam Beneito Cambra

water-rich layer during extraction could be due to the large concentrations of


both mineral acids and chromium(III) and sodium salts, which should made ionic
strength to be very large. In a different series of experiments, up to 13% water
was added to 4:3 ethyl acetate-acetone mixtures before observing two-phase
separation. Then, as indicated in the recommended procedures, an aqueous
butylamine solution up to a final concentration of 10% was added to the extracts
before obtaining NI mode spectra. Finally, sensitivities were only slightly higher
in the presence of 40% ACN.

IV.2.1.2. Relative sensitivity of carboxylic and ethoxy-carboxylic acids as a


function of n and m
Relative sensitivities are useful in the qualitative interpretation of MS
spectral profiles of unknown samples. They are also necessary to avoid bias in
predicting alcohol concentrations after calibration with a standard different from
the addressed alcohol. To estimate the relative sensitivities of carboxylic and
ethoxy-carboxylic acids at increasing values of both n and m, the [M-H]- peaks of
the standards in ethyl acetate-acetone containing butylamine and water, and at
several concentrations of each acid (0, 100, 200 and 400 g mL-1), were
measured in the NI mode. Relative sensitivities with reference to the internal
standard were calculated as follows:
fi =

Ii
I s [M ]i

(E.IV.1)

where Ii and Is are the abundances of the [M-H]- ion of the compound of interest
and the internal standard, respectively, and [M]i is the molar concentration of the
compound. As shown in Fig. IV.13, part A, relative sensitivities increased as the
alkyl chain increased. This agreed with the reported APCI-MS response factors
of fatty acids [Holapek, 2005]. Standards of ethoxy-carboxylic acids having large
values of both n and m are not commercially available; then, only the relative
166

Chapter IV. No ionic surfactants in cleaning products

sensitivities of a few ethoxy-carboxylic acids with low values of both n and m


(cellosolves) could be studied. As also shown in Fig. IV.13, parts A and B,
relative sensitivity of ethoxy-carboxylic acids was higher than that of the
corresponding non-ethoxylated carboxylic acids, and increased when either n or
m increased.

log fi

m=3
m=2
m=1

-1

m=0
-2

-3
0

12

16

log fi

n=2

-1

n=1

-2

B
-3
0

Fig. IV.13. NI MS relative sensitivities of carboxylic and ethoxycarboxylic acids versus the number of carbon atoms in the alkyl chain, n,
and at increasing values of the number of EO units, m (A), and vice versa
(B). Relative sensitivities in logarithmic scales, log fi, are given with
reference to the internal standard (TMBA).

167

Miriam Beneito Cambra

IV.2.1.3. Oxidation yields of primary non-ethoxylated alcohols and extraction


recoveries of the corresponding carboxylic acids
Recovery of carboxylic acids with short and large chain lengths during the
extraction step, independently from the yield of the previous oxidation step, was
first evaluated. For this purpose, the proposed procedure was applied to solutions
of C3E0A and C14E0A in acetone, and the NI mode spectra were measured. The
abundance of the [M-H]- ions was compared to that obtained by using standard
solutions of the acids in the ethyl acetate-acetone extraction medium. Recoveries
were 100% (duplicated extractions).
Then, in order to study oxidation yields, the proposed procedure was
applied to a series of standard solutions containing increasing amounts of C10E0
in acetone. The concentrations used corresponded to 0, 100, 200 and 400 g mL-1
in the infused solutions (duplicated points). Using the NI mode, the abundance of
the [M-H]- ions was measured. A regression straight-line with an excellent
linearity (r2 > 0.999, calibration curve A) was obtained. The standard deviation
of the residuals was used to calculate the standard deviation of the slope
2000].

[Miller,

Assuming a zero intercept, and in relative terms, this standard deviation

was 1.3% of the slope, which corresponded to a confidence limit of 3.0% (95%
confidence

level,

two-sided).

Using

increasing

concentrations

of

the

corresponding carboxylic acid, C10E0A, a linear relationship (r2 > 0.999,


calibration curve B) was also obtained. The ratio of the slopes of the two
calibration curves (as A/B) was 1.016. Thus, the oxidation yield of C10E0 was
not statistically different from 100%.
The influence of the addition rate of the Jones reagent was also studied.
For this purpose, aliquots of the C10E0 solution in acetone were oxidized
according to the proposed procedure, but the manual dropwise addition of the
Jones reagent was substituted by manual quick addition, and also by slow

168

Chapter IV. No ionic surfactants in cleaning products

addition with a syringe pump under stirring during 40 min. The abundance of the
[M-H]- ions, measured in the NI mode, was compared to that obtained using
calibration curve B (expected concentration in the infused solutions, ca. 200 g
mL-1). Either, dropwise or slow addition of the Jones reagent equally led to a
100% yield.
The influence of the reaction temperature during oxidation was also
investigated. Alcohols with a short (C3E0) and a large (C12E0) chain length
were used. A water bath was used to set the initial temperature of the reaction
mixture. Then, the Jones reagent was added, and after extraction the abundance
of the [M-H]- ions was measured. The ion abundances did not increase nor
decrease by regulating the initial temperature of the reaction mixture to 25, 35
and 55 C.

IV.2.1.4. Influence of water concentration on the oxidation yield


To study the influence of water concentration on the oxidation yield of
non-ethoxylated alcohols, two series of experiments were done. In all cases, a
C10E0 stock solution in acetone was used, and the proposed procedure was
applied, but with the modifications which are next indicated. First, the Jones
reagent was prepared in the presence of decreasing water concentrations; for this
purpose, water was substituted by increasing concentrations of acetic acid
[Hudlik, 1990; Burke, 1999].

Aliquots of the C10E0 stock solution were further

diluted using acetone, and the proposed procedure was applied; however,
oxidation was performed with the series of Jones reagent prepared with
increasing acetic acid concentrations (decreasing water concentrations). Second,
aliquots of the C10E0 stock solution were diluted with acetone-water mixtures
containing increasing amounts of water, instead of using pure acetone. Jones
reagent prepared in aqueous sulfuric acid, as indicated in the proposed procedure,

169

Miriam Beneito Cambra

was added to these solutions. In all cases, the NI spectra were obtained, and
internal standard correction using TMBA was applied.
As shown in Fig. IV.14, the yield was maximal when the reagent was
prepared as indicated in the proposed procedure, and decreased moderately when

Oxidation yield, %

water was partially substituted by acetic acid (Fig. IV.14, part A).
100
80

60

Oxidation yield, %

40
20

40
60
80
H2O % in the Jones reagent

100

80
60
40
0

25
50
75
100
Initial H2O % in the sample solution

Fig. IV.14. Reaction yield of C10E0 in the presence of different water


concentrations during oxidation. The Jones reagent was prepared by
substituting water by increasing concentrations of HAcO (A), and the
analyte was dissolved in acetone-water mixtures with increasing amounts
of water instead of using pure acetone (B).

Oxidation yield also decreased when the Jones reagent was added to C10E0
solutions containing increasing amounts of water (Fig. IV.14, part B).
Therefore, in the recommended procedure, industrial samples containing water
(i.e. cosmetics and body care products) were diluted with acetone. In addition,

170

Chapter IV. No ionic surfactants in cleaning products

after centrifugation, a low-volume aliquot of the supernatant was further diluted


with acetone before Jones reagent was added.

IV.2.1.5. Oxidation yields of ethoxylated alcohols


The oxidation yield of a short-chain ethoxylated alcohol, C2E1, was first
studied. For this purpose, the C2E1-C2E1A pair of standards was used. The
proposed procedure was first applied to a series of solutions containing
increasing C2E1 concentrations, and the abundance of the [M-H]- ions were
measured in the NI mode. As performed above for C10E0, the slope of this
calibration curve was compared with that obtained using increasing C2E1A
concentrations. These two straight-lines were highly linear (r2 > 0.999), but the
slopes differed, indicating an oxidation yield of ca. 65%. As also observed for
C10E0, this yield was not modified by increasing or decreasing the addition rate
of the Jones reagent (both sudden manual addition and slow addition with a
syringe pump under stirring during 40 min). The yield was not modified either by
increasing the reaction time to more than 2 hours after addition of the Jones
reagent (before removing the reagent excess with sodium sulfite). The influence
of the sample temperature before addition of the Jones reagent was also studied.
Using a C4E2 solution, a constant 65% yield was obtained at 25, 35 and 55 C.
The oxidation yield of fatty alcohols with an increasing degree of
ethoxylation was studied. For this purpose, standards of the C12Em series were
used. Standards of the corresponding ethoxylated fatty acids were not available,
which hindered the comparison of the abundances of the [M-H]- ions obtained in
the NI mode. Instead, abundances of the [M+H]+ ions obtained in the PI mode
were measured. The proposed procedure was applied, and the abundance of the
[M+H]+ ion of the remaining non-oxidized FAEs was compared to that of the
corresponding ethoxy-carboxylic acid. As shown in Fig. IV.15, two intense

171

Miriam Beneito Cambra

peaks corresponding to the C12E3 - C12E3A pair were observed in the spectrum
obtained after oxidation of C12E3. The peak of the remaining non-oxidized
C12E3 was higher than that of C12E3A; however, taking into account that the
C12E3/C12E3A sensitivity ratio in the PI mode was close to 1.8, an oxidation
yield of ca. 60% was calculated. Two other low intensity peaks, corresponding to
the [M+H]+ ions of the C12E2 - C12E2A pair, were also observed. These two
peaks were attributed to loss of an EO unit during oxidation. From the peak
abundances, this side-reaction amounted to less than 4% of the initial C12E3
concentration. Along the C12Em series, oxidation yield, which was 100% for
C12E0, decreased to ca. 65 and 60% when m = 2 and 4, respectively. The
oligomers with m = 5 and 6 also gave a ca. 60% yield.

[C12E3+H]+
[C12E3A+H]+

Abundance x 106

1
[C12E2+H]+
[C12E2A+H]+
0
300

m/z

Fig. IV.15. PI MS spectrum of C12E3 after oxidation with Jones reagent


showing the [M+H]+ peaks of the C12E3 C12E3A and C12E2 C12E2A
pairs. Target mass of the ion trap was set at m/z 275 to enhance the
abundances of the diethoxylated species.

172

350

Chapter IV. No ionic surfactants in cleaning products

Finally, ethoxylated alcohols of the CnE1 and CnE2 series were used to
study the influence of n from n = 2 to 18. In all cases, ca. 65% yields were
obtained. In addition to the expected peaks, two additional peaks due to the FAE
with loss of an EO unit, and its corresponding ethoxy-carboxylic acid, were
observed. However, yields for this side reaction were small (4-8% of the initial
amount of the FAE).

IV.2.1.6. Application to industrial and environmental samples

A) Industrial raw materials


The NI mode spectrum of industrial raw material FINDET 10/18 showed the
intense peaks of the oxidized oligomers of the C10Em series, including the nonethoxylated alcohol, C10E0 (Fig. IV.16). All these peaks were not observed on
the NI mode spectrum obtained without previous oxidation of the sample (not
shown). Also without oxidation, peaks of the [M+H]+ ions of ethoxylated
alcohols were observed on the PI mode spectrum, but sensitivity was small for m
= 1 and 2, and a peak for C10E0 was not observed (spectrum not shown). Thus,
the proposed procedure is particularly useful for the quick identification of nonethoxylated fatty alcohols at low concentrations. However, owing to ion
suppression effects, previous separation by HPLC or CE should be recommended
for quantitative purposes. Quantitative evaluation of mixtures of FAEs by the
proposed procedure is also burdened by bias due to the 4-8% breakdown of the
EO chain during oxidation (with loss of an EO unit), as well as by lack of
available standards of ethoxylated fatty acids, which hinders the determination of
response factors of the oligomers.

173

500

m/z

[C10E13A-H]-

[C10E12A-H]-

[C10E11A-H]-

[C10E9A-H]-

[C10E8A-H]-

[C10E7A-H]-

[C10E6A-H]-

[C10E5A-H]-

[C10E2A-H]-

[C10E1A-H]IS

300

[C10E10A-H]-

[C10E0A-H]-

Abundance x 106

[C10E4A-H]-

[C10E3A-H]-

Miriam Beneito Cambra

700

Fig. IV.16. NI MS spectrum of the industrial surfactant FINDET 10/18


after oxidation with Jones reagent. The peaks of the [M-H]- ions of the
oxidized oligomers of the C10Em series are labelled (IS = internal
standard).

B) Body care and cosmetic products


The proposed procedure was also applied to the body care and cosmetic
products of Table IV.3. In the NI mode, all these samples showed the intense
[M-H]- peaks of C16E0A and C18E0A (cetyl and stearic acids), as well as those
of other fatty acids. By applying again the proposed procedure but with addition
of diluted sulfuric acid instead of Jones reagent (CrO3 was not added), the peaks
of these fatty acids were absent, or present with much lower abundances. The
spectrum of a varicose vein cream is shown in Fig. IV.17. In this sample, the
abundances of the peaks of C16E0A and C18E0A increased ca. 30 and 10 times,
respectively, upon oxidation with the Jones reagent. The procedure was then
applied to the determination of C16E0 and C18E0 in the commercial products of

174

Chapter IV. No ionic surfactants in cleaning products

Table IV.3. Solutions of C16E0A and C18E0A were used to construct external
calibration curves (6 duplicated points). To check possible matrix effects,
internal calibration by the standard addition method was also applied; for this
purpose, two duplicated calibration points per sample were performed (with
addition of alcohol standards to the samples before the oxidation step).

[C18E0H-H]-

Abundance x 106

[C16E0H-H]4

IS

0
200

250

m/z

300

Fig. IV.17. NI MS spectrum of a commercial varicose vein cream after


oxidation with Jones reagent showing the peaks of the cetyl and stearic
acids resulting from the oxidation of the cetyl and stearic alcohols,
respectively.

As shown in Table IV.3, most samples showed satisfactory agreement between


the alcohol contents found by external and internal calibration. Limits of
detection (LODs) for C16E0 and C18E0 in the varicose vein cream, calculated as
the concentration corresponding to a net signal equal to three times the standard
deviation of the blank, were 0.1 and 0.03%, respectively. However, taken into
account that the extract of this sample was diluted in a 1:40 ratio before infusion

175

Miriam Beneito Cambra

in the MS, LODs of 25 and 7.5 g per gram of sample, respectively, were
calculated.
Table IV.3. Mass percentages of cetyl and stearic alcohols found in several
commercial samples by external and internal (standard addition) calibration (ND =
not detected).
C16E0 (%)

Sample
Shampoo
Hair conditioner
Lip protector stick
Deodorant stick
Body-care oil
Varicose-vein cream

External
0.71
0.55
0.66
ND
0.10
3.8

Internal
0.80
0.35
0.69
ND
0.16
3.3

C18E0 (%)

External
ND
0.25
0.34
5.0
0.15
2.0

Internal
ND
0.24
0.34
5.6
0.15
2.6

C) Seawater
The presence of saturated and unsaturated fatty alcohols at the g L-1 level
has been reported in seawater samples taken from coastal and high seas
1992; Tolosa, 2003; Belanger, 2009].

[Parrish,

As shown in Fig. IV.18, peaks which could

correspond to linear fatty alcohols with zero, one and two double bonds, and to
phytol (a common diterpenoid), were observed in the spectra of the oxidized
extracts obtained with seawater. These peaks were present with much lower
abundances in the spectrum of the seawater extract which was not oxidized with
the Jones reagent. In Fig. IV.18, ratios calculated by dividing the ion
abundances obtained after application of the proposed procedure to the seawater
extract, by those obtained without oxidation with the Jones reagent (reference
sample), are indicated between parentheses.

176

Chapter IV. No ionic surfactants in cleaning products

C26:1 (12)

C26:2 (30)

C24:2 (10)

C22:0 (2)

C22:2 (25)
C22:1 (4)

C20:2 (92)
C20:1 + phytol (13)
C20:0 (4)

C18:1 (10)
C18:0 (15)

C16:1 (16)
C16:0 (30)

0.5

C14:0 (8)

1.0

C12:1 (140)
C12:0 (13)

Abundance x 106

C12:2 (600)

1.5

0
200

300

m/z

Fig. IV.18. NI MS spectrum of a seawater extract after oxidation with


Jones reagent. Several peaks could be ascribed to common saturated and
unsaturated fatty acids and to phytol, is indicated (e.g., C18:1 is
octadecenoic acid). Numbers within parentheses are abundance ratios
between the addressed peak in this spectrum and in the spectrum of the
seawater extract not oxidized with the Jones reagent.

177

400

CHAPTER V

ANIONIC SURFACTANTS

Chapter V. Anionic surfactants

V.1. AESs

V.1.1. Determination of FAEs and AESs by anionic exchange separation,


derivatization with a cyclic anhydride and HPLC
FAE and AES are two important surfactant classes, widely used in
cleaners and body care products

[Fiedler, 1989].

FAE are industrially obtained as

complex mixtures of oligomers with the following structure (shortened below as


CnEm):
CH3(CH2)n1(OCH2CH2)mOH
where n is the number of carbon atoms in the alkyl moiety of the molecule, and
m is the number of EO groups. FAE mixtures obtained from vegetal oils contain
linear hydrocarbon chains with even values of n, whereas both linear and
branched chains, with even and odd values of n, can be found in FAE obtained
from mineral oils [Sparham, 2005; Marcomini, 1996].
On the other hand, AES are obtained by esterification of FAE with either
sulfur trioxide or chlorosulfonic acid. Then, FAE and AES have essentially the
same molecular structure, with a hydrocarbon chain attached to an EO chain, but
AES oligomers end with a sulfate group in substitution of the OH group of FAE
oligomers [Strain, 1959; Suter, 1944; Arthur, 1991]:
Na+CH3(CH2)n1(OCH2CH2)mOSO3Accordingly, AES oligomers are shortened below as CnEmS. Important
characteristics of both FAE and AES, including viscosity of their aqueous
solutions, detergency, foam formation and skin compatibility, as well as their
environmental impact, depend on the variable distributions of both the alkyl and
EO chains

[Marcomini, 1996; Arthur, 1991; Tadros, 2005; Rudewicz, 1986; Eadsforth,

2006; Belanger, 2006; Van Compernolle, 2006; Ribosa, 2007; Jurado, 2007].

Both the

hydrocarbon cut (range of n for the predominant hydrocarbon series) and m

181

Miriam Beneito Cambra

(average number of EO units) are important in industrial quality control. Thus,


methods for their characterization and determination are required; however,
owing to the complexity of the sample matrices, lack of chromophore, and wide
ranges of polarity and volatility of the oligomers, the analysis of FAE and AES,
which are usually found in complex mixtures with other surfactant classes, is not
an easy task. In addition, the unavailability of commercial standards constitutes
an added difficulty of AES analysis.
In determination of FAE, the low volatility and thermal instability of long
EO chains limit the use of GC to the oligomers with m < 4
Crescenzi, 1995],

[Rudewicz, 1986;

and owing to the high volatility of the oligomers with short EO

chains, the employ of HPLC with ELSD provides biased distributions


[Miszkiewicz, 1996-A; Bernab-Zafn, 2006].

In addition, the PI MS response factors

for underivatized FAE oligomers decrease ca. two orders of magnitude when m
decreases from 4 to 1
2001].

[Sparham, 2005; Rudewicz, 1986; Crescenzi, 1995; Dunphy,

Further, non-ethoxylated alcohols (m = 0) are not detected in a mass

spectrometer [Sparham, 2005; Rudewicz, 1986; Crescenzi, 1995; Bernab-Zafn, 2006;


Dunphy, 2001; Sherrard, 1994].

Underivatized FAE can be also characterized and

determined using isocratic elution and a refractive index detector


1993; Trathnigg, 1994; Trathnigg, 2004; Trathnigg, 2005],

[Trathnigg,

but selectivity is poor and

the limits of detection are large. Derivatization procedures designed to increase


volatility of FAE oligomers, followed by GC analysis have been proposed
[Marcomini, 1996; Kiewiet, 1996; Hbner, 2007].

Several derivatization procedures

for FAE, addressed to add a chromophore or a charge to the oligomers, followed


by HPLC

[Marcomini, 1996; Dunphy, 2001; Hoffman, 2004-B; Sun, 1997; Zanette,

1996; Lemr, 2003; Desbne, 2005]

or CE

[Sparham, 2005; Bernab-Zafn, 2006;

Dunphy, 2001; Lemr, 2003; Desbne, 2005; Wallingford, 1996; Heinig, 1998],

have

been also described. FAE derivatives have been separated by either NP- or RP-

182

Chapter V. Anionic surfactants

HPLC using UV-Vis

[Marcomini, 1996; Kiewiet, 1996; Zanette, 1996; Bachus, 2003;

Mic-Tormos, 2008-A; Mic-Tormos, 2008-B; Mic-Tormos, 2009]

or MS detection

[Crescenzi, 1995; Bernab-Zafn, 2006; Levine, 2005; Jandera, 1998; Krogh, 2002].

On the other hand, nonspecific determination of AES and other anionic


surfactants can be jointly performed by the methylene blue active substance
method

[Llenado, 1983].

Non-ethoxylated alkyl sulfates have been studied using

ion pair chromatography with indirect UV detection

[Boiani, 1987; Shamsi, 1995],

ionic chromatography with conductimetric detection

[Pan, 1995; Nair, 1998]

and

HPLC with post-column ion-pair formation followed by membrane phase


separation and fluorimetry

[Smedes, 1982-B].

Also, conversion of AES to the

corresponding alkyl bromides followed by GC-FID


HPLC coupled to ESI-MS

[Neubecker, 1985],

as well as

[Popenoe, 1994; Bruno, 2002; Lara-Martn, 2005],

have

been applied to the determination of AES in waters, sewage sludge and marine
sediments.
In former works of research group, we have developed procedures for the
RP-HPLC-UV determination of FAE previous derivatization with a cyclic
anhydride

[Mic-Tormos, 2008-A; Mic-Tormos, 2008-B; Mic-Tormos, 2009; Mic-

Tormos, 2010].

However, we have observed that cyclic anhydrides also derivatize

AES to yield exactly the same derivatives as FAE, i.e. the hemiesters of the
alkyl- or alkyl-ethoxy residues. Thus, the peaks corresponding to the sum of both
FAE and AES oligomers are obtained on the chromatograms if the two surfactant
classes are present in the samples. Therefore, in this work, a procedure for the
separation of these two surfactant classes, followed by the independent
derivatization of each class with a cyclic aromatic anhydride, and RP-HPLC-UV
determination of the derivatized oligomers, was developed. Separation of the two
surfactant classes was achieved by SPE on a SAX cartridge. Then, using either
phthalic or diphenic anhydride, FAE are esterified and AES are transesterified.

183

Miriam Beneito Cambra

Separation of the derivatized oligomers was achieved by RP-HPLC using


gradient elution with ACN/ water in the presence of 0.1% acetic acid. The
proposed method was applied to the analysis of FAE and AES in commercial
products (liquid cleaners) and environmental samples (seawater extracts).

V.1.1.1. Derivatization and HPLC separation of the derivatives


As shown in Fig. V.I, an industrial mixture of AES (LES) was
quantitatively derivatized at 105 C in about 70 and 50 min using phthalic and
diphenic anhydrides, respectively. Similar results were reported for the
esterification of FAE

[Mic-Tormos, 2008-B; Mic-Tormos, 2009].

Thus, to assure

derivatization of the two surfactant classes, 90 min was selected. Chromatograms


of Dehydol LT-7 and LES, obtained by previous derivatization with phthalic
anhydride, are shown in Fig. V.2.

Relative sum of peak areas

100

Formation of
diphenates

80
60

Formation of
phthalates

40
20
0
0

40

80
Reaction time (min)

Fig. V.1. Relative sum of the chromatographic peak areas of the derivatives
of all the oligomers given by an industrial AES (LES) sample versus
reaction time using phthalic (rhombus and dashed line) and diphenic
anhydrides (squares and dotted line). Each point represents an independent
derivatization performed at 105 C in 1,4-dioxane.

184

Chapter V. Anionic surfactants

Both chromatograms showed the successive hydrocarbon series at increasing


values of n, from n = 10 to 18. Hydrocarbon series having exclusively even
values of n were observed with Dehydol LT-7, but significant amounts of the n =
13 and 15 odd series were also present in LES. The large difference between the
peak profiles of the same hydrocarbon series for Dehydol LT-7 and LES is due to
the different average EO number, namely m = 7 for Dehydol LT-7 and m = 3
for LES. The elution order of the oligomers within the series, which is indicated
in the Fig. V.2 for the n = 12 series, was established with UV-Vis detection, by
injecting standards of derivatized oligomers, and was also confirmed by HPLCMS using extracted ion chromatograms (EICs, not shown).

Absorbance at 230 nm (mAU)

300
200

C12E0 C12E1
+
+
C12E5 C12E4
C12E6
C12E7
C12E8
C12E3
C12E9
C12E2

n = 12

n = 14
n = 16

n = 18

100
0
n = 12

300
200
100

C12E0S
+
C12E5S

C12E1S
+
C12E6S C12E4S
C12E3S
C12E7S
C12E8S
C12E2S
C12E9S

n = 14
n = 13
n = 15 n = 16

n = 18

0
10

20

30

Time (min)

Fig. V.2. Chromatograms obtained after derivatization of Dehydol LT-7


(A) and LES (B) with phthalic anhydride. In both cases, ca. 40 mg were
derivatized and final volume before injection was 12 mL. Elution with a
linear gradient from 50 to 100% ACN in 50 min at 25C. The insets show
peak identifications for the n = 12 series.

185

40

Miriam Beneito Cambra

Except for m = 0 and 1, the oligomers within the series eluted by following the
order of decreasing m. The consecutive pairs of oligomers were also fairly well
resolved; however, the peaks of the oligomers with m = 1 and 0 overlapped with
other oligomers within their respective hydrocarbon series. At 25 C and for the n
= 12 and 14 series, overlapping of the pairs m = 1 and 4, and m = 0 and 5, was
produced. Reversion of the elution order for m = 1 and 0 within the hydrocarbon
series has been explained as due to the rigidity of the short hydrophilic moiety of
these oligomers of the EO chain, which hinders intramolecular solvation, thus
making their hydrophobicity to decrease with respect to the oligomers with m 2
[Mic-Tormos, 2008-A; Mic-Tormos, 2008-B; Mic-Tormos, 2009; Mic-Tormos, 2010].

V.1.1.2. Optimization of the SAX separation of the surfactant classes


Since derivatization of both FAE and AES leads to the same derivatives, a
previous separation of both surfactant classes was implemented. For this purpose,
the method proposed by Fendinger et al. for alkylsulfate analysis in water was
modified

[Fendinger, 1992].

The SAX separation procedure was optimized to

achieve quantitative isolation of the two surfactant classes, including both


hydrophilic (low n and high m values) and hydrophobic (high n and low m
values) oligomers. Conditioning of both sample and SAX cartridge with 80:20
MeOH/H2O led to the coelution of part of the AES jointly with the FAE. Thus,
according to the optimized scheme of Fig. III.1 and section III.1.3.4,
conditioning was performed with 50:50 MeOH/H2O. In this medium, partial
elution of FAE, but without coelution of AES oligomers, was achieved.
Solubilization of the most hydrophobic oligomers, together with disruption of
FAE-AES mixed micelles, was also procured with 50% MeOH. At this point of
the procedure, an increase of the MeOH concentration to 80% to complete FAE
elution led to the partial coelution of AES. Coelution was avoided by washing

186

Chapter V. Anionic surfactants

the cartridge with additional portions of 50% MeOH. In this way, residual
cations from the sample (mostly, Na+) were washed away, thus strongly fixing
AES on the SAX cartridge before increasing the hydrophobicity of the medium.
Experiments performed with LES in 50% MeOH showed the absence of AES
oligomers in the chromatograms obtained by increasing MeOH concentration to
80% after washing the cartridge first with more 50% MeOH.
300

Absorbance at 230 nm (mAU)

200

100

40

20

0
10

20

30

Time (min)

40

Fig. V.3. Chromatograms of Dehydol LT-7 derivatized with phthalic


anhydride: (A) FAE oligomers eluted with 50:50 MeOH:H2O (combined
fractions 1 and 2 of Fig. 1); (B) FAE oligomers eluted with 80:20
MeOH:H2O (fraction 3 of Fig. III.1). Chromatographic conditions as in
Fig. V.2; other details as indicated in section III.1.3.3.

As shown in Fig. V.3, application of the optimized procedure to a Dehydol LT-7


solution led to the elution of an 82% and an 18% FAE with 50% (fractions 1 + 2
of Fig. III.1) and 80% MeOH (fraction 3 of Fig. III.1), respectively. Further, in
comparison to the expected oligomer distribution for Dehydol LT-7, the 80%
187

Miriam Beneito Cambra

MeOH fraction showed a higher proportion of the hydrophobic oligomers,


including both oligomers with large values of n, and oligomers with low values
of m within the series. A residual amount of 0.4% of the total FAE was obtained
after washing the cartridges with two additional 4-mL volumes of 80:20
MeOH/H2O.
Next, AES were eluted using HCl solutions in MeOH as a means to
achieve both high concentrations of Cl- and a highly hydrophobic medium.
According to the chromatograms shown in Fig. V.4, which were obtained using
LES, a 62% of the AES oligomers was eluted with 80:20 MeOH:HCl (Fig. V.4,
part A, fraction 4 of Fig. III.1). An additional 35%, containing a higher
proportion of hydrophobic oligomers than the former fraction, was eluted by
increasing MeOH to 95% (Fig. V.4, part B, fraction 5 of Fig. III.1). Total
elution of LES oligomers was checked by washing the cartridge with two
additional 4-mL volumes of both 80:20 and 95:5 MeOH:HCl; in this way, a
residual 3% AES was recovered (chromatogram not shown). As indicated in the
optimized procedure (Fig. III.1 and section III.1.3.4), the combined 4 + 5
fractions containing the AES were neutralized with concentrated ammonia to
prevent the release of acid fumes during solvent evaporation. Finally, the isolated
FAE and AES fractions were derivatized and injected. Application of this
procedure to mixtures of Dehydol LT-7 and LES (ca. 20 mg each) according to
the scheme of Fig. III.1, gave rise to chromatograms of the FAE and AES
fractions closely resembling the chromatograms given in Fig. V.2, which were
obtained by directly derivatizing and injecting Dehydol LT-7 and LES solutions.
In addition, the optimized procedure, including the SAX separation into
two fractions, was also independently applied to Dehydol LT-7 and LES
solutions. For Dehydol LT-7 (ca. 40 mg), the chromatogram obtained from the
combined fractions 1 + 2 + 3 (see Fig. III.1) was closely similar to that observed

188

Chapter V. Anionic surfactants

in Fig. V.2, part A, whereas the chromatogram obtained with the combined
fractions 4 + 5 showed no significant peaks. Therefore, FAE were quantitatively
retained in the combined fractions 1 + 2 + 3.

A
Absorbance at 230 nm (mAU)

100

50

B
20

0
20

30

Time (min)

40

Fig. V.4. Chromatograms of LES derivatized with phthalic anhydride: (A)


AES oligomers eluted with 80:20 MeOH:HCl (fraction 4 of Fig. III.1); (B)
AES oligomers eluted with 95:5 MeOH:HCl (fraction 5 of Fig. III.1).
Chromatographic conditions as in Fig. V.2; other details as indicated in
section III.1.3.3.

Similarly, using LES, the chromatogram obtained for the combined fractions 4 +
5 was closely similar to that observed in Fig. V.2, part B. On the other hand, the
chromatogram obtained with the combined fractions 1 + 2 + 3 showed a series of
small peaks which followed the same pattern as that observed in Fig. V.2, part
B, but with much smaller peak areas. This later chromatogram was attributed to
the FAE impurities which are always present in industrial AES, due to the nonquantitative sulfatation of FAE during AES manufacture. The total peak area

189

Miriam Beneito Cambra

obtained from fractions 4 + 5 of LES, divided by the total peak area obtained
from fractions 1 + 2 + 3 indicated a molar percentage of ca. 1.2 % FAE in the
LES sample.

V.1.1.3. Calibration studies


Standard solutions of C8E0, C12E0, C8E0S and C12E0S were
independently used to construct independent calibration curves for FAE and
AES. For each standard, the proposed derivatization procedure was applied to
series of solutions containing increasing concentrations, ranging from 0.06 up to
1.7 mM in the injected solutions (six solutions per standard). Plotting the areas,
an excellent linearity was obtained for all the standards (r2 > 0.999). Further, the
slopes of the calibration plots obtained with the four standards did not differ
significantly from each other, i.e. the maximal slope difference was 1%.
Therefore, sensitivity was the same for non-ethoxylated alcohols and
alkylsulphates, independently from the length of the alkyl chain. The similarity
of the slopes indicated both a high degree of derivatization, presumably close to
100%, for the two surfactant classes. Further, since AES are quantitatively
converted to the same derivatives as those obtained with FAE, then, the UV-Vis
response factors of the FAE oligomers, which were established in previous work
for the derivatives obtained with the phthalic
anhydrides

[Mic-Tormos, 2009],

[Mic-Tormos, 2008-B]

and diphenic

should be also valid for the corresponding

derivatized AES oligomers. This is of practical interest, because standards of


non-ethoxylated fatty alcohols are widely available, whereas non-ethoxylated
alkylsulfates are rare and expensive. Further, FAE oligomers with m > 0 are
commercially available, which does not occur with the corresponding AES
oligomers. Thus, in sections V.1.4 and V.1.5, the unexpensive and widely
available dodecyl alcohol (C12E0) was exclusively used as a standard to evaluate

190

Chapter V. Anionic surfactants

without bias total surfactant class concentrations, hydrocarbon series and


distribution and average EO numbers ( m ) of the complex mixtures of FAE and
AES oligomers found in industrial and environmental samples.
For this purpose, six duplicated calibration points, up to 1.7 mM C12E0
were obtained using the peak areas. Then, the proposed procedure was applied to
the samples of Table V.1. A pair of chromatograms per sample, corresponding to
the FAE and AES fractions, was obtained in duplicate, and all the peak areas
were measured.
Table V.1. Declared and found composition for industrial samples.

In mass percentages; the average EO number, m number found for each AES
sample is indicated between parentheses.

Overlapping of the m = 0 and 1 peaks with the m = 5 and 4 peaks, respectively,


within their respective hydrocarbon series, made necessary to establish an
indirect way of estimating the individual areas corresponding to these oligomers.
To estimate first the area of the m = 5 and m = 4 peaks by interpolation is
straightforward and sufficiently accurate for most applications. Interpolation is
possible at 25 C due to the perfect overlapping of the peaks by pairs of
oligomers (m = 0 with m = 5, and m = 1 with m = 4)

[Mic-Tormos, 2008-B].

As

shown in Fig. V.5, the peak areas of the m = 5 and 4 oligomers were estimated
by non-linear interpolation of the peak areas of the 2 m 3 and m 6
oligomers of their respective hydrocarbon series. Thus, the peak areas of the

191

Miriam Beneito Cambra

Dehydol LT-7 ( m = 7) and LES ( m = 3) fitted well to the cubic equation, and to
exponential equations of the form A=be-am, respectively.

FAE, m = 7
n = 12

Relative area

AES, m = 3
n = 12

n = 14

0
2

n = 14

10

12

Number of EOs
Fig. V.5. Estimation of the uncorrected peak areas of the m = 4 and 5
oligomers of the n = 12 and 14 series in: Dehydol LT-7 (empty symbols,
solid lines by cubic interpolation); LES (full symbols, dashed lines by
exponential interpolation). Experimental (rhombus) and interpolated peak
areas (squares).

For simplicity, when the peak areas of the m = 4 and 5 oligomers of the n = 12
and 14 series of Dehydol LT-7 were estimated, the contribution of the oligomers
with m > 13 of the n = 14 and 16 series (see Fig. V.2), respectively, was
neglected. Then, the peak areas of the m = 0 and 1 oligomers were obtained by
difference. All the peak areas were next divided by the tabulated response factors
of the respective oligomers

[Mic-Tormos, 2008-B; Mic-Tormos, 2009],

and the

molar concentrations of the oligomers were obtained by dividing the corrected


peak areas by the slope of the C12E0 calibration curve. Then, the average EO
number, m , was calculated from the molar distribution of the oligomers. Total
192

Chapter V. Anionic surfactants

mass concentrations of the surfactant classes, and the mass distribution of


hydrocarbon series, were finally calculated by taking into account the molar
masses of the oligomers and the dilution factor in the injected solutions.

V.1.1.4. Application to industrial raw materials and commercial products


As indicated in Table V.1, a 70% generic LES and liquid laundry
products containing mixtures of several surfactant classes were analyzed. The
common anionic surfactants SAS and LAS were studied as potential
interferences. These anionic surfactants should be retained together with the AES
in the SAX cartridge; however, application of the procedure to a SAS sample
gave a chromatogram with no additional peaks with respect to that of the reagent
blank. Also, a 1:1 mixture of LES and a SAS sample (ca. 20 mg each) gave rise
to a chromatogram which was undistinguishable from that of LES. Then, SAS
showed no reaction with the anhydrides and did not cause interference either. On
the other hand, LAS are aromatic surfactants which absorb in UV-Vis. The
HPLC separation of a LAS sample in the conditions used to separate FAE and
AES derivatives lead to a series of large and wide bands, close to the dead
volume, on the chromatogram of the AES fraction. Further, this pattern was not
modified by application of the derivatization procedure to a LAS sample; then,
LAS did not react either with phthalic anhydride. Application of the procedure to
a mixture of LAS and LES gave rise to a chromatogram showing the bands of
LAS followed by the expected peak pattern of the derivatives of the LES
oligomers; however, the absorbance due to the LAS bands decreased largely
before the elution time corresponding to the AES hydrocarbon series with n = 8
(chromatogram not shown). Therefore, LAS did not cause interference either in
the evaluation of the AES series of industrial interest (n 8). The determination
of residual FAE in industrial AES is important to control both the sulfation

193

Miriam Beneito Cambra

process and the quality of the final product. As indicated in Table V.1,
application of the proposed procedure to the 70% generic LES sample showed a
65% AES and a residual 1.2% FAE. According to the data given in Table V.1,
the proposed procedure is also useful for the characterization and determination
of both FAE and AES in commercial cleaners.

V.1.1.5. Seawater analysis


Application of the proposed procedure using diphenic anhydride to

Absorbance (mAU)

seawater extracts led to the chromatograms shown in Fig. V.6.


12

40

FAE
n = 12

AES
n = 12

20

20

0
0

Intensity 105

AES
n = 14

2
0

2
3

24

26

24

22

Time (min)

2
1

0
22

26

0
30

Time (min)

32

34

Time (min)

Fig. V.6. Chromatograms of a seawater extract obtained by derivatization


with diphenic anhydride and using UV-Vis (upper parts) and MS detection
(EICs, lower parts): (A) FAE, n = 12 series; (B) AES, n = 12 series; (C)
AES, n = 14 series. The numbers at the peaks are m values. Elution
conditions: linear gradient from 60 to 90% ACN in 50 min at 25C.

Using both UV-Vis and MS detection, the seawater samples showed measurable
amounts of several FAE oligomers of the n = 12 series, and several AES

194

Chapter V. Anionic surfactants

oligomers of the n = 12 and n = 14 series. According to the C12E0 calibration


curve, 0.25, 2.3 and 0.99 g L-1 were found in seawater for C12E0, C12E0S and
C14E0S, respectively (calculated as 1500 times less than in the injected solutions).
Then, the proposed method was capable of distinguishing the oligomers of the two
surfactant classes in the seawater, AES being present at higher concentrations than
FAE. This could be due to the faster biodegradation of FAE in comparison to
AES. This also indicates that previously reported data for FAE in environmental
samples, obtained by methods based on derivatization with anhydrides, actually
informed about the sum of FAE and AES. Due to the labitity of the ester bond of
AES, the same problem could bias the FAE concentrations reported by using other
derivatization reagents. Therefore, where appropriate, the reported data should be
revised.

195

CHAPTER VI

SYNTHETIC POLYMERS

Chapter VI. Synthetic polymers

VI.1. PVP-NO

VI.1.1. Characterization of PVP-NO by FSCE and MEKC


Several water-soluble polymers capable of partially inhibiting the
unwanted dye transfer from colored to white fabrics are used as additives in
laundry products

[Bertleff, 1998; Oakes, 2003].

These polymers, which are called

DTIs, act as dye scavengers, keeping the washed-off dyestuffs in solution, thus
helping in the prevention of redeposition on the fabrics. For years, PVP and their
copolymers have been mainly used; however, the dye scavenger efficiency of
these polymers is reduced in the presence of anionic surfactants [Oakes, 2003]. For
this reason, new generations of DTI, with superior complexing properties and a
higher tolerance to anionic surfactants, have begun to be employed. A relatively
common DTI of the new generation is PVP-NO (see structure in Fig.
VI.1).Molecular masses between 9 and 36 kDa, which correspond to polymer
chains constituted by ca. 75 and 300 monomers, respectively, are found in
different types of commercial PVP-NO for laundry

[Oakes, 2003; Household

Industrial and Institutional Cleaning, 2006].

In spite of the use of increasing amounts of polymers in laundry and other


applications, analytical methodologies for quality control of these compounds
and for the evaluation of their environmental impact have been scarcely reported.
In addition, the study of the interaction of polymers with surfactants in aqueous
solution is important for both fundamental research and industrial application. In
this work, the migration characteristics of PVP-NO in both FSCE and MEKC
have been studied. The determination of the polymer in commercial additives for
laundry is also demonstrated.
CE has been widely applied to the separation of biopolymers and to the
characterization and determination of synthetic polymers

199

[Gallardo, 1999; Cottet,

Miriam Beneito Cambra


2005].

Mainly, FSCE and CGE have been employed to separate polyelectrolytes

(polymers with either positive or negative charges) and polyampholytes


(polymers with both positive and negative charges), and MEKC has been used to
separate non-charged polymers

[Gallardo, 1999].

The application of FSCE, CGE

and MEKC to the analysis of synthetic polymers has been reviewed [Cottet, 2005].
Both FSCE and MEKC can provide useful information about the behavior of
polymers in solution, as well as quantitative information
MEKC, Gallardo et al.

[Gallardo, 1999]

[Bohrisch, 2000].Using

were able to separate two different

components of a non-ionic copolymer, i.e. a fraction rich in methacrylate and


another one rich in vinylpyrrolidone. MEKC has been also used to obtain
information about the synthesis progress, nature and composition of ionic
copolymers

[Aguilar, 2002].

On the other hand, in CGE, molecular mass

discrimination of ionic polymers is achieved by sieving through a BGE which


contains a non-ionic polymer, such as a PEG
derivative, dextran or other polysaccharides

[Grosche, 2000],
[Bohrisch,

2000;

a cellulose
Clos,

1998;

Starkweather, 2000; Welch, 2001].

In FSCE, and for short-chain polyelectrolytes, the electrophoretic mobility


is a function of the chain length. Thus, the mobility increases as more charged
monomers are added to the chain; however, the mobility reaches a maximum and
begins to decrease when the polyelectrolyte changes from the rod-like to the coil
conformation. Finally, for longer polyelectrolytes the charge-to-mass ratio
become constant, and the polymer migrates with the so-called free draining
mobility [Cottet, 2000]. Therefore, over a given chain length, and independently of
the chain length range, a polyelectrolyte gives a single peak in FSCE
2000; Grosche, 2000].

[Bohrisch,

However, due to additional effects, some polymers can

actually give two or more signals at different migration times rather than a single
peak.

200

Chapter VI. Synthetic polymers

Thus, copolymers composed by a long polyanionic chain bound to a short


hydrophobic chain have shown to form micelles in aqueous solutions
2001].

[Cottet,

Using FSCE, two peaks, which were attributed to the very slow rate of

exchange between the free and aggregated or micellized states of the polymer in
comparison to the time scale of electrophoresis, were observed. The free chains
of the polymer (the unimers) exhibited a larger mobility (in absolute values) than
the micellized polymer. Further, the peak of the micellized polymer was broader
than that of the free polymer, which was attributed to scatter in the aggregation
numbers.
Using FSCE and a PVP-NO/ maleic acid anionic copolymer, Gyrffy et
al.

[Gyrffy, 1998]

also found a narrow peak followed by a wider one. The

presence of two components in the polymer was proposed, but the possible
nature of the components was no further discussed. Using both SEC and FSCE,
tpnek et al.

[tpnek, 2001]

have shown that some copolymers form micelles

in solution, and that the micelles coexist in a mobile equilibrium with the
unimers. Further, the unimer-micelle equilibrium was shown to be kinetically
frozen in aqueous media, the micelles behaving as independent nanoparticles.
In this work, a narrow peak was obtained for PVP-NO solutions using
both FSCE and MEKC; however, at least an additional broad band was also
present in most electropherograms. The nature of the signals obtained is
discussed at the sight of both the studies reported in the literature and the new
results. In addition, low-molecular-mass zwitterions, including trimethylglycine
(betaine) and sarcosine, have been used for a long time to reduce adsorption in
the FSCE separation of proteins

[Bushey, 1989];

however, betaine and sarcosine

have carboxylate groups, which hinders their application below pH 56. Thus, to
separate proteins in more acidic media (pH 25), PEA has been used [Chen, 1992].

201

Miriam Beneito Cambra

In this work, PEA is used to reduce adsorption of PVP-NO in phosphate buffers


of acidic pH.

VI.1.1.1. FSCE studies in the absence and presence of PEA


Separation of PVP-NO was first tried in the absence of PEA. As shown in
Fig. VI.1, using positive polarity and an acid BGE (10 mM HAcO, pH 4), a large
peak located after the EOF marker peak was observed. Two narrow peaks plus a
broader band in the middle were observed at pH 7 (10 mM NH4AcO)
(electropherograms not shown). All the peaks and band at both pH 4 and 7

Abs. (mAU)

showed the spectrum of the polymer (see the insets in Fig. VI.1).

CH2 - CH

Absorbance at 214 nm (mAU)

40

N+
O-

40
20
0
8

EOF

20
0
200

300

(nm)

400

0
0

8
Time (min)

10

Fig. VI.1. Electropherograms of an EOF marker (mesityl oxide) and PVPNO (2000 g/mL) obtained using positive polarity and a BGE containing
10 mM HAcO (pH 4). The insets show the chemical structure of PVP-NO,
and the UV spectra at both the peak maximum and at the baseline location
indicated by an arrow.

Taking into account the large molecular masses of this polymer (917 kDa), and
its nominally nonionic nature, a single peak at the EOF migration time was
actually expected. However, as discussed below in Section VI.1.1.2, the

202

Chapter VI. Synthetic polymers

electropherograms and associated spectra indicated the presence of at least two


PVP-NO components or forms in the solutions, these forms also exhibiting nonzero net electrophoretic mobilitites.
The reproducibility of the migration time and area of the PVP-NO peak at
pH 4 was 1.8 and 7.2%, respectively, and similar values were obtained for the
peaks and band at pH 7 (n = 5). However, as revealed by recording the UV
spectra along the baseline after the PVP-NO peaks, a drawback was the presence
of severe peak tailing due to adsorption (see the insets in Fig. VI.1). Then, FSCE
in the presence of large concentrations of PEA was tried.
The program SPARC

[Hilal, 1995]

output the following pKa values for the

ionization of PEA: 1.07, 5.75 and 8.91; thus, the PEA species without a net
charge predominates, at least on a 9:1 basis, within the 2.074.75 pH range.
Therefore, a BGE containing 20 mM H3PO4 (pH 2.2) was used to investigate the
effect of the PEA concentration on the migration behavior of PVP-NO and the
EOF. As shown in Fig. VI.2, using this BGE in the presence of increasing PEA
concentrations, PVP-NO gave a large narrow peak (after an initial small peak)
within the anionic migration region. A broader band at the beginning of the
cationic migration region (immediately after the peak of the EOF marker) was
also observed. Both the large narrow peak and the band exhibited the spectrum of
the polymer. Except when otherwise indicated, adsorption along the baseline
after the peak locations was not detected by using 150 mM or higher PEA
concentrations.
Contrary to what occurred in Fig. VI.1, negative polarity was required in
Fig. VI.2 to observe the PVP-NO peaks. As revealed by using acetone as a
marker, the EOF was reversed in the presence of large PEA concentrations. The
negative EOF could be due to the formation of esters between the phosphate
groups of PEA and the silanols. This would lead to the predominance of the

203

Miriam Beneito Cambra

positively charged amino groups of PEA covering the capillary walls, thus

Absorbance at 214 nm (mAU)

reversing the EOF.

A
40

B
20

0
0

8
12
Time (min)

Fig. VI.2. Electropherograms of 1000 g/mL PVP-NO, obtained using


negative polarity and a BGE containing 20 mM H3PO4 (pH 2.2) and the
following PEA concentrations: 150 (A), 250 (B) and 350 mM (C). The
arrow shows the location of the peak of the EOF marker (acetone).

The electroosmotic mobility increased (in absolute values) rapidly up to 150 mM


PEA, reaching a plateau between 225350 mM PEA (triplicate points, not
shown). The net electrophoretic mobility of PVP-NO (according to the large
narrow peak) progressively increased (in absolute values) when the PEA
concentration was increased from 150 to 200 mM. At PEA concentrations higher
than 225 mM and up to 300 mM, PVP-NO showed an almost constant mobility.
Baseline disturbances were produced with PEA concentrations over 300 mM;
accordingly, further studies were made using 250 mM PEA. The contribution of
PEA to the capillary current was negligible.

204

Chapter VI. Synthetic polymers

Then, the influence of pH in the presence of 250 mM PEA was studied.


First, H3PO4 (pH 2.2) was substituted by NaH2PO4 (pH 3.0). The net mobility of
the large PVP-NO peak increased, which suggested an intensification of the
anionic character of the polymer. As shown by comparing the electropherograms
of Fig. VI.2, part B and Fig. VI.3, the intensity of the broad band near the EOF
migration time also increased at rising pHs, and it was partially resolved in
several bands. Some of these bands were clearly located within the cationic
migration region.
Both the migration time and area of the large narrow peak of PVP-NO
were highly reproducible (0.3 and 2.4%, respectively). On the contrary, as also
shown in Fig. VI.3, along successive replicated injections on the same capillary
the broad band showed a progressive shifting towards shorter migration times.
As discussed below in Section VI.1.1.2, the large narrow peak and the broader
band were attributed to two forms of PVP-NO in solution. The progressive
shifting of the broad band along successive injections could be due to residual
adsorption of the corresponding form of the polymer. In fact, adsorption was not
observed in Fig. VI.3, parts A and B, but the spectrum of the polymer was
observed at migration times longer than that of the band in Fig. VI.3, parts C to
E. Significant differences regarding pH 3.0 were not observed at pH 4.3
(obtained using Na2HPO4). The concentration of NaH2PO4 (pH 3.0) was also
varied within the 540 mM range; however, significant differences regarding 20
mM were not observed either.
Significant variations of the shapes of the peak and band were not
observed

at

increasing

capillary

temperatures,

from

25

to

45C

(electropherograms not shown); however, the large narrow peak appeared at


slightly shorter migration times. Thus, the net electrophoretic mobility of this

205

Miriam Beneito Cambra

peak varied from -17.6 to -18.2 and -19.7 (in 109 m2s1V1) when the capillary
temperature was increased from 25 to 35 and 45C, respectively.
200

Absorbance at 276 nm (mAU)

160

B
120

C
80

D
40

E
0
0

12

16
Time (min)

Fig. VI.3. Electropherograms of 1000 g/mL PVP-NO, obtained using


negative polarity and a BGE containing 250 mM PEA and 20 mM
NaH2PO4 (pH 3.0). From (A) to (E), successive replicated injections on the
same capillary. The arrow shows the location of the peak of the EOF
marker (acetone).

VI.1.1.2. Discussion on the results obtained by FSCE


PVP-NO has large molecular masses and does not contain ionizable
groups, thus, a single narrow peak at the EOF migration time should be expected
using FSCE. Instead of this, a peak with a well-defined anionic behavior was
observed in Figs. VI.1-VI.3, and at least an additional broad band with a weak
cationic behavior was observed in Figs. VI.2 and VI.3. As next discussed, and as

206

Chapter VI. Synthetic polymers

supported by other reports from literature, the anionic character of PVP-NO can
be explained by the separation of charges along the N-O bond, and the
subsequent formation of a ionizable complex with water. A possible explanation
for the additional broad band is the presence of a micellized form of the polymer.
The nitrogen and oxygen atoms of pyridinium oxides exhibit a remarkable
separation of the respective positive and negative charges

[Ochiai, 1953].

charge separation has been also demonstrated in PVP-NO

This

[Okamoto, 1998].

According to viscosity and light scattering measurements in solution, PVP-NO


has been shown to behave like a polyelectrolyte, rather than as a noncharged
polymer

[Okamoto, 1998; Lee, 1996].

This behavior has been attributed to the

presence of strong interparticle forces, which result from the charge separation
across the N-O bond

[Lee, 1996].

In addition, PVP-NO has been observed to

exhibit a remarkable electrodonating character, which has been explained by


means of resonant structures

[Yamamoto, 1996].

Further, PVP-NO solutions in

water have a non-zero electrical conductivity (2.81073.4106 S/cm), which


contrasts with the electrically insulating properties of the corresponding nonoxidated poly(4-vinylpyridine) (<1014 S/cm)

[Yamamoto, 1996].

Finally, purified

PVP-NO fractions gave solutions with pHs as low as 3.44.5 when solved in
water, which was attributed to the formation and subsequent ionization of a PVPNO/water complex [Okamoto, 1998].
The formation and subsequent ionization of a PVP-NO/water complex
fully agrees with the net anionic electrophoretic mobility of the large narrow
PVP-NO peak observed in this work. Further, the acidity of some N-O bonds
along the polymer chain can be enhanced by the presence of neighboring N-O
bonds, as occurs with polycarboxylic acids [Gyrffy, 1998], which would lead to a
range of pKa values along the polymer. This would also explain the increase of

207

Miriam Beneito Cambra

the net anionic mobility showed by this PVP-NO peak (in absolute terms) at
rising pH values from 2.2 to 3.0 (Figs. VI.2, part B and VI.3).
Another point to be explained is the presence of the broad bands near the
EOF migration time. At the sight of the previous studies
1998; tpnek, 2001],

[Cottet, 2001; Gyrffy,

a possible explanation is the presence of associated forms

of the polymer. These associated or aggregated forms could coexist with the free
form (the unimers) in a very slow or frozen equilibrium between them, as
reported for some copolymers

[tpnek, 2001].

Since PVP-NO has not well-

defined hydrophobic and hydrophilic regions, association cannot be produced by


the action of the hydrophobic forces. However, in the case of PVP-NO, the likely
alternative is association through strong dipole-dipole interactions.

VI.1.1.3. MEKC studies in the absence and presence of PEA


The MEKC behavior of PVP-NO using 60 mM SDS was first studied in
the absence of PEA. As shown in Fig. VI.4, a small peak, followed by a large
peak with a shoulder and by a second large peak at longer migration times, was
observed in an acid medium (20 mM H3PO4, pH 2.2). These peaks were observed
using negative polarity. As discussed above for FSCE, the presence of two peaks
can be explained by the coexistence of a free and an aggregated form of PVPNO. The EOF should be small at this low pH, thus the PVP-NO peaks along the
anionic migration time region can be explained as due to association of the two
PVP-NO forms with the SDS micelles. The SDS concentration was varied within
the 20100 mM range. Electropherograms similar to that shown in Fig. VI.4
were obtained; however, a major drawback was the poor reproducibility of the
migration time of the second large peak. Along replicated injections of the same
solution, this peak appeared at progressively longer migration times. This

208

Chapter VI. Synthetic polymers

problem was not overcome by conditioning the capillary between injections with

Abs. at 214 nm ( mAU)

hot NaOH solutions, as indicated in section III.2.1.3 for new capillaries.

100
50
0
0

Time (min)

Fig. VI.4. Electropherogram of 2000 g/mL PVP-NO, obtained using


negative polarity and a BGE containing 20 mM H3PO4 and 60 mM SDS.
The two large peaks and the shoulder showed the spectrum of the polymer.

Lack of reproducibility was attributed to adsorption, then, the behavior of PVPNO at increasing SDS concentrations was studied in the presence of 250 mM
PEA. Under these conditions, and using pH 3.0 (20 mM NaH2PO4), PVP-NO
showed the reproducible but complex behavior which is outlined in Fig. VI.5.
Between 1 and 3 mM SDS (Figs. VI.5, parts A and B), a single peak at
increasingly longer migration times was observed. With 3 mM SDS, a new broad
band at a short migration time, and a new narrow peak, began to appear. The
band increased with 5 mM SDS, and reached a constant intensity within the 20
60 mM SDS range (Figs. VI.5, parts C-F). The narrow peak was large at 520
mM SDS (Figs. VI.5, parts C and D), and appeared at longer migration times
and became broader at higher SDS concentrations (Figs. VI.5, parts E and F).
Using 20 mM SDS and 35C (instead of 25C), an electropherogram similar to
that shown in Fig. VI.5, part D was obtained, but with the large narrow peak
located at a longer migration time (ca. 10 min). A possible explanation of the
results summarized in Fig. VI.5 is next given and discussed.

209

Miriam Beneito Cambra

Absorbance at 276 nm (mAU)

200

C
100

D
E
F
0
0

10

Time (min)

20

30

Fig. VI.5. Electropherograms of 1000 g/mL PVP-NO, obtained using


negative polarity and a BGE containing 250 mM PEA, 20 mM NaH2PO4
(pH 3.0) and increasing SDS concentrations. From A to F: 1, 3, 5, 20, 30,
60 mM SDS, respectively.

VI.1.1.4. Discussion on the results obtained by MEKC


The association between SDS micelles and non-ionic polymers, such as
PEG and PVP, has been studied by measuring the decrease of the surface tension
as the concentration of the SDS increases, and by other techniques [Cabane, 1977;
Arai, 1971].

According to Cabane [Cabane, 1977], PEG forms mixed micelles with

SDS. Mixed PEG-SDS micelles have a definite composition, with the excess
material, whether polymer or SDS, being present as free polymer molecules or
regular SDS micelles, respectively. When SDS is added to a solution of the
polymer, two concentrations, x1 and x2, mark the beginning and the completion of
the association of SDS with PEG, respectively. The association process starts
abruptly above a certain surfactant concentration, x1, which is lower than the
CMC, and saturates abruptly above another surfactant concentration, x2, where

210

Chapter VI. Synthetic polymers

the coverage of the polymer by adsorption of surfactant molecules along its chain
is completed up. Beyond x2 the surface tension of the solution is close to that of a
solution containing regular surfactant micelles. The difference, x2 - x1, measures
the amount of surfactant bound to the polymer.
According to NMR studies

[Cabane, 1977],

PEG is attached to the

hydrocarbon/water interface of the mixed PEG-SDS micelles, with some


monomers of the polymer replacing water molecules in the vicinity of the head of
the SDS molecules. About 10% of the monomers of the polymer are thought to
be directly bound to the micelles, while the others form loops in the surrounding
water. At the saturation point, x2, depending on the polymer concentration, and
independently from the polymer molecular mass, the composition corresponded
to 3.34.1 PEG monomers for one SDS molecule.
Arai et al.

[Arai, 1971]

also found two transition points on the surface

tension vs. SDS concentration curves when recorded in the presence of PVP.
Association of SDS with PVP to form mixed micelles began at an SDS
concentration 40% lower than the CMC. The PVP/SDS weight ratio at the point
where adsorption was completed, x2, was 1:2.3, regardless of the PVP
concentration. This corresponded to 1.1 PVP monomers for one SDS molecule.
According to literature, PVP-NO also interacts strongly with the SDS
micelles. Thus, the inhibition of the dye scavenger capability of PVP-NO in the
presence of SDS and other anionic surfactants has been attributed to
displacement of the dye from the polymer/dye complex to form a
polymer/surfactant complex
[Gyrffy, 1998]

[Oakes, 2003-A].

Using MEKC, Gyorffy et al.

also found a strong interaction between SDS micelles and an

anionic PVP-NO/maleic acid copolymer. In a UV-Vis spectroscopic study,


Oakes et al. [Oakes, 2003-C] have shown that PVP-NO binds SDS micelles. Since
the hydrophobic region of PVP-NO (the hydrocarbon skeleton) is not well

211

Miriam Beneito Cambra

separated from the hydrophilic regions, the interaction was explained by the
formation of mixed polymer/SDS micelles, in which short segments of the
polymer occupied substantial parts of the micelles. As illustrated in Fig. VI.6, in
these parts of the micelles, a segment of the hydrocarbon skeleton was proposed
to be located inside the micelle, and the polar pyridine N-oxide groups were
assumed to be positioned among the SDS sulfate groups. Since a single polymer
molecule can bind several micelles, mixed aggregates containing a number of
SDS micelles was proposed to be formed

[Cabane, 1977; Arai, 1971; Oakes, 2003-

C].
OO-

N+
SO2

Fig. VI.6. Scheme adapted from ref.

[Oakes, 2003-C],

showing the likely

structure of a free PVP-NO/SDS mixed micelle.

Curves of the surface tension against the SDS concentration, obtained both
in the absence and in presence of 1000 g mL-1 PVP-NO, are shown in Fig. VI.7.
These measurements were made in the conditions of Fig. VI.5, that is, in the
presence of both 20 mM NaH2PO4 and 250 mM PEA. In the absence of the
polymer, the CMC of SDS was 1.8 mM. This parameter, which is 8.3 mM in
pure water, decreases in the presence of salts

[Phillips, 1955; Thvenot, 2005],

which is consistent with the value obtained. Two transition points, indicating the

212

Chapter VI. Synthetic polymers

beginning and end of the association process of PVP-NO with the SDS micelles,
were observed in the presence of 1000 g mL PVP-NO.

Surface tension (mN m-1)

70
60
50
40
30
0

0.5

1.5
log ([SDS]+1)

Fig. VI.7. Surface tension plotted against log([SDS]+1), where [SDS] is


the SDS concentration in mM (the 1 was added to plot the [SDS] = 0
point). Data obtained both in the absence (, dotted lines) and in the
presence of 1000 g mL-1 PVP-NO (, continuous lines).

The first point, x1, was located at 0.48 mM SDS, which corresponds to a
concentration a ca. 70% lower than the CMC. This indicates a strong interaction
between the polymer and SDS. The second point, x2, was placed over the CMC,
at 9.7 mM SDS. The difference (x2-x1) corresponded to 0.9 PVP-NO monomers
for one SDS molecule taking part in the mixed micelles. This value is close to
that reported for the formation of PVP/SDS mixed micelles, 1.1

[Arai, 1971].

Assuming that PVP-NO would behave in a similar way as PEG, a mixed micelle
would have about 70 SDS molecules with only a 10% of the polymer taking part
of the micelles

[Cabane, 1977].

If this were true, 0.9 PVP-NO monomers for one

SDS molecule would correspond to 6.3 PVP-NO monomers per mixed micelle,
which is quite realistic. This value was used to draw the scheme of Fig. VI.6.

213

Miriam Beneito Cambra

On the other hand, according to the FSCE discussion of above, and in


agreement with the results of tpnek et al.

[tpnek, 2001],

two forms of the

polymer, namely, the free polymer and the pure polymer aggregates, probably in
a very slow-rate equilibrium between them, could be present in the aqueous
solutions of PVP-NO, in the absence of SDS. Further, in agreement with the
study of Oakes et al.

[Oakes, 2003-C],

both the free and the micellized polymer

forms could bind SDS micelles. Thus, it seems reasonably to assign the narrower
peaks of the electropherograms of Figs. VI.4, part A, VI.5, part C and D to the
association between the free polymer and SDS micelles, and the broader band at
a shorter migration time to the association between the aggregated polymer and
SDS micelles. In this latter, band broadening could be explained by scatter of the
aggregation number. The presence of two types of PVP-NO/SDS aggregates
could explain the persistent presence of a peak and a band (or group of bands) in
most electropherograms, being also compatible with the presence of the two
transition points observed in Figs. VI.7.
The EOF should be almost zero at the low pH used; however, as indicated
above, an anionic (reversed) EOF was observed in the presence of large PEA
concentrations. Thus, the reduction of this cathodic EOF produced by the
presence of increasing SDS concentrations could be enough to explain the
broadening and shifting effects on the large narrow peak observed in Fig. VI.5,
parts A and B. The first transition point of Fig. VI.7, x1, approximately
coincided with the appearing of the new peak and band between Fig. VI.5, parts
A and B. Thus, the sudden formation of two new peaks between 3 and 5 mM
SDS could be attributed to the simultaneous formation of both free polymer/SDS
and aggregated polymer/SDS mixed micelles.
Since the EOF was very low in the conditions of Fig. VI.5, parts B to F,
the short migration times of the band and peak indicated that the polymer species

214

Chapter VI. Synthetic polymers

had high anionic mobilities. These can be explained by both the negative charge
of the free polymer, and the association of the free and aggregated polymer forms
with the SDS micelles. As shown in Fig. VI.5, parts E to F, the band assigned to
the associated PVP-NO/SDS mixed micelles showed no further changes of shape
and location at higher SDS concentrations. On the contrary, the peak assigned to
the free polymer/SDS mixed micelles was progressively broader, and appeared at
longer migration times as the SDS concentration increased. An explanation for
this behavior was not found.

VI.1.1.5. Quantitation studies and application to real samples using FSCE


Intra- and inter-day repeatabilities of the large and narrow polymer peak in
the presence of 20 mM NaH2PO4 and 250 mM PEA were measured by
performing successive injections of a 1000 g mL-1 PVP-NO solution. The
results are given in Table VI.1. A calibration curve was constructed by injecting
seven standard solutions within the 502000 g mL-1 range, and by measuring
the peak area. Excellent linearity (r > 0.993) and an LOD of 23 g mL-1 (S/N =
3) were obtained (Table VI.1).
The FSCE method was applied to the determination of PVP-NO in two
different

commercial

DTI

concentrates

for

laundry.

representative

electropherogram is shown in Fig. VI.8. This electropherogram was closely


similar to those given in Figs. VI.2, part B and VI.3, which were obtained in the
same conditions, but it showed an additional intense peak (marked with an
asterisk in the figure). This peak increased upon spiking the sample with cumene
sulfonate, which is a common hydrotropic additive of cleaning products. The
other narrow peak and the broader bands at longer migration times showed the
spectrum of the polymer, and increased upon spiking the sample with PVP-NO.

215

Miriam Beneito Cambra

According to the calibration curve, the area of the narrow PVP-NO peak
corresponded to 161 g mL-1 in the injected solution, and to 1.53% in the sample.
Table VI.1. Migration time and peak area repeatabilities, efficiency and LOD for
the large narrow peak of PVP-NO.

Absorbance at 214 nm (mAU)

Parameter

Value

Migration time repeatabilities

0.30a), 1.0b)

Peak area repeatabilities at 1000 g/mL

2.4a), 5.5b)

N, m1

80 000

LOD, g/mL (S/N = 3)

23

a)

Intraday as RSD % (n = 5).

b)

Interday as RSD % (n = 3 5).

20

12

16

20
Time (min)

Fig. VI.8. Electropherogram of a commercial additive for laundry obtained


using negative polarity and a BGE containing 20 mM NaH2PO4 and 250
mM PEA. The sample was 1:100 diluted with 50:50 v/v MeOH/water. The
arrow shows the location of the peak of the EOF marker (acetone), and the
asterisk identifies the peak of cumene sulfonate.

Attempts of detecting 500 g mL-1 PVP-NO in the presence of non-ionic


and anionic surfactants were also made. Thus, the PVP-NO narrow peak was not
disturbed by the presence of a 5% of FAEs (Dehydol LT-7); however, it was not

216

Chapter VI. Synthetic polymers

detected when the sample solution contained a 5% of an anionic surfactant (both


LAS and LES were tried).

VI.2. PVP

VI.2.1. Characterization and determination of PVP by complexation with an


anionic azo-dye and NECEEM
A variety of nonaqueous and aqueous CE methods for the characterization
and determination of synthetic polyelectrolytes, including CZE, CGE and CIEF
methods, have been described
2004; Cottet, 2005].

[Clos, 1998; Grosche, 2000; Borisch, 2000; Engelhardt,

In CGE, solutions of nonionic polymers are used as sieving

media to separate polyelectrolytes [Welch, 2001]. However, as far as we know, CE


methods for the characterization and determination of nonionic polymers have
not been reported. To analyze PEGs by CZE, previous derivatization with an
anhydride, which provides both a chromophore and charge, has been used
[Wallingford, 1996; Barry, 1998];

however, most nonionic synthetic polymers

cannot be easily derivatized.


PVP is a versatile hydrophilic polymer which is widely used for a variety
of

purposes,

including

cosmetics

pharmaceuticals and adhesives

and

toiletries,

[Frauenfelder,

1974;

textiles
Sheth,

and

1985].

dyes,
In the

formulation of cleaning products, PVP is used as a dye scavenger, keeping the


washed-off dyestuffs in solution, thus inhibiting the unwanted dye transfer from
coloured to white fabrics [Bertleff, 1998; Oakes, 2003-A; Oakes, 2003-B; Oakes, 2005].
The determination of PVP has been carried out in urine and blood serum by
precipitation with iodine and titration with thiosulfate
infrared absorptiometry

[Behen, 1964].

[Dwyer, 1964],

and by

PVP has been determined in foods,

cosmetics and laundry products by preconcentration on silicagel followed by

217

Miriam Beneito Cambra

complex formation with an anionic azo-dye and colorimetry [Frauenfelder, 1974].


A spectrophotometric procedure for the determination of PVP in waste water of
pharmaceutical plants, based on formation of a coloured complex with a dye, has
been also reported

[Chmilenko, 2001-A].

PVP can be also determined in

pharmaceutical preparations by direct injection of the sample in a RP-HPLC


column

[Jones, 2004];

however, this entails a high risk of getting PVP

permanently retained by the stationary phase.


Over half a century, studies concerning to the complexation of anionic
organic dyes by PVP, with special reference to total number of binding sites
available for the interaction, free energy and heat of interaction, and changes in
the shape of polymer molecule, have been carried out

[Sheth, 1953; Hansen, 1954;

Barkin, 1955; Frank, 1957; Luck, 1958; Molineux, 1961; Runge, 1996; Chmilenko, 2001B; Oakes, 2003-C].

The aim of this work was to develop a CE method for the

determination of PVP in cleaning products. Since the electrophoretic mobility of


nonionic PVP is almost zero, mixtures containing PVP and an anionic azo-dye
were prepared and injected in the capillary. A band due to the PVPdye
complexes followed by a peak due to free dye was observed. The
electropherograms were interpreted in the light of the theory developed by
Krylov and co-workers

[Berezovski, 2002; Krylov, 2003; Drabovich, 2006; Lin, 2008;

Krylov, 2006; Krylov, 2007],

which described the method of nonequilibrium CE of

equilibrium mixtures (NECEEM) for the study of proteinprobe and DNA


protein interactions. In NECEEM, non-covalently bound fluorescent probes are
equilibrated with a target protein or DNA, and the mixture is injected in the
capillary. In this work, application of NECEEM to the study of the interaction
between PVP and an azo-dye is demonstrated. Information about the average
molecular mass of the polymer, and the maximal stoichiometry, average stability
constant and dissociation rate of the polymerdye complexes, can be obtained. In
addition, the method was applied to the characterization and determination of
218

Chapter VI. Synthetic polymers

PVP in commercial cleaners and pharmaceutical preparations. The proposed


method should also be useful to characterize and determine other synthetic and
natural nonionic polymers by using the appropriate probes.

VI.2.1.1. Electropherograms of PVP in the presence of anionic azo-dyes


Two anionic bisazo-dyes (Fig. VI.9), which form with PVP stronger
complexes than single azo-dyes, were selected. The electropherogram of a CR
solution is shown in Fig. VI.10, part A. In the same figure, parts BF, a series
of electropherograms showing the effect of the addition of increasing CR
concentrations to a PVP solution, are given. In the absence of CR, PVP absorbed
only at low wavelengths, and its electrophoretic mobility was close to zero (Fig.
VI.10, part B, trace obtained at 215 nm).

PVP

C H C H2
N
O

n
NH2
O
N
O

S
O
Na+ O-

O- Na+
S

Congo Red (CR)

O S
O- Na+
O

H2N

NH

Acid Blue 113 (AB)

O
Na+ O-

Fig. VI.9. Molecular structures of PVP and the azo-dyes used in this work.

219

Miriam Beneito Cambra

A
60

Absorbance (mAU)

6
40

4.5

3.0

F 2.25

20

G 1.12

0
0

10

Time (min)
Fig. VI.10. Electropherogram of 0.5 mM CR (A) and electropherograms of
500 g mL1 PVP of 60 kDa (4.5 mM in monomers) without an azo-dye
(B), and in the presence of the following CR concentrations: 0.75 (C), 1
(D), 1.5 (E) and 2 mM (F). Trace G was obtained with 4 mM AB. The
numbers on the traces are the values of q (monomer/dye molar ratio). The
traces were recorded at 215 (B), 500 (A, CF) and 565 nm (G). On each
trace, the arrow indicates the location of the EOF marker peak. The area at
the left half of the peak of the PVPdye complexes, a, was used to estimate
the total area of this band (a = APVPD/2).

A significant parameter in all the experiments along this work was the
monomer/dye molar ratio, which will be indicated by q throughout the article. At
increasing CR concentrations (decreasing values of q), the band appeared at
increasing migration time values after the EOF time. The band was also
220

Chapter VI. Synthetic polymers

progressively divided into two peaks which showed a large absorptivity in the
visible region, with their respective maxima at ca. 505 and 485 nm, respectively.
At q < 4, the peak with a lower migration time was Gaussian shaped, whereas the
other peak was asymmetric (Fig. VI.10, parts E to F). Resolution between the
two peaks, and their areas at 500 nm, increased as q decreased; however, the area
of the Gaussian shaped peak remained constant when q < 4. The asymmetric
peak continued increasing, approaching the migration time of CR at q <4 (Fig.
VI.10, parts E and F). Interpretation of the electropherograms was made in the
light of both the UVVis spectra of the free dye and the PVPCR complexes,
which were obtained with a conventional spectrophotometer, and the NECEEM
theory (see Section VI.2.1.3).
Using a conventional spectrophotometer, the absorption spectrum of CR
showed two maxima located at 340 and 487nm ( = 16,845 and 21,400 M1 cm1,
respectively). Upon addition of an excess PVP (0.91 mM in monomer giving q =
9.1), the band at 340 nm was only slightly modified, but the maximum of the
other band shifted from 487 to 505 nm. The molar absorptivity of this band also
increased from 21,400 to 24,000 M1 cm1. Batochromic shifts between 7 and 19
nm have been reported for the 500650 nm band of azo-dyes upon complexation
with PVP

[Scholtan, 1953; Runge, 1996],

which agrees with the 18 nm shift found

in this work for the formation of the PVPCR complexes. Accordingly, the
Gaussian shaped peak of the electropherograms of PVPdye mixtures (Fig.
VI.10, parts B to F), was attributed to the corresponding PVPdye complexes,
and the asymmetric peak at a longer migration time to the free dye. As also
shown in Fig. VI.10, when q < 4, the asymmetric peak was close to the location
of the free dye peak obtained in the absence of the polymer (trace A). There are a
large number of potential binding sites along a PVP molecule. Thus, the increase
in the peak area of the complexes at rising dye concentrations was attributed to

221

Miriam Beneito Cambra

an increased number of bound sites along the PVP molecules. The increasing
mobility of the complexes at increasing dye concentrations (at decreasing q
values up to q = 4) was also attributed to the same cause.
On the other hand, at q > 4 (Fig. VI.10, parts C and D), the peak of the
free dye ions was located at a migration time which was only slightly higher than
that of the PVPCR complexes. Further, a similar behaviour was observed by
injecting PVPAB mixtures. The peak due to the free dye was expected at longer
migration times, namely, at 6.4 and 9.5 min for CR and AB, respectively;
however, a peak close to these locations was obtained only when q < 4. As
further discussed in Section VI.2.1.3, this, as well as the presence of an
intermediate exponential region when q < 4, were explained by applying
NECEEM concepts.

VI.2.1.2. Formation rate of the PVPCR complexes


The formation rate of the PVPCR complexes was first studied
spectrophotometrically. For this purpose, a mixture containing 0.1 mg mL1 60
kDa PVP (0.91 mM in monomers) and 0.1 mM CR (q = 9.1) was prepared, and a
spectrum every 10 min was obtained. The band at 487 nm shifted progressively
to 505 nm, and the molar absorptivity also increased in a process which lasted ca.
40 min. No further modifications of the spectrum were observed during the
following 5 h. Then, another mixture containing 1 mg mL1 60 kDa PVP (9.1
mM in monomers) and 4 mM CR (q = 2.25) was prepared, and aliquots were
injected in the capillary at increasing times after preparation. In these
experiments the peak area of the PVPCR complexes reached some stability 3 h
after preparation of the mixtures. At the same time, the mobility of the complexes
increased, also reaching a plateau between 3 and 5 h after preparation. The
slower formation of the complexes when monitored using CE instead of the

222

Chapter VI. Synthetic polymers

spectrophotometer could be due to the different values of q (9.1 vs. 2.25).


However, conformational changes of the complexes, leading to an increase in
their stability, could take place during several hours after preparation of the
mixture. As deduced from application of NECEEM principles, conformational
changes which could be blind to a spectrophotometer, could be revealed by the
electropherograms. Thus, on the electropherograms, an increase of the peak area
of the PVPCR complexes at increasing time values after preparation could be
due not to an increase of the concentration of the complexes, but to a slower
dissociation rate. This point is further commented upon in the next section.
Except when otherwise indicated, in all the experiments presented in this work,
the vials containing all the solutions were maintained in the carrousel of the CE
instrument at ca. 25 C during a minimum of 5 h before injection.

VI.2.1.3. Application of NECEEM principles to the interpretation of the


electropherograms of PVPdye mixtures
According to Krylov and co-workers

[Berezovski, 2002; Krylov, 2003;

Drabovich, 2006; Lin, 2008; Krylov, 2006; Krylov, 2007],

when a targetprobe

complex is injected in the capillary, due to the violation of the equilibrium


conditions during migration, at least two peaks and an intermediate exponential
region should be obtained. The two peaks are due to the remaining targetprobe
complex at the time of detection, and to the equilibrium concentration of the free
probe. The exponential region is due to the probe which is liberated from the
complex during migration. A third peak and its corresponding exponential
region, due to the equilibrium concentration of the free target and to the target
liberated during migration, respectively, will also appear if detection conditions
sensitive to the target are used. The peaks of the targetprobe complex, free
probe and free target, should appear at the migration time values corresponding

223

Miriam Beneito Cambra

to their respective electrophoretic mobilities. Since both target and probe are
carried away from the complex during migration, the complex dissociation rate
depends exclusively on its concentration. For this reason, a first-order kinetics,
which gives rise to an exponential liberation of both target and probe, is
followed. The stability constant of the targetprobe complex can be established
by measuring the peak area of the free probe, and the sum of the peak areas of
the remaining complex and the exponential region. Correction of the areas is
necessary if the complex and the probe have different sensitivities (e.g. different
fluorescence quantum yields or different molar absortivities). Finally, kinetic
information related to the complex dissociation rate can be obtained from the
exponential profile of the intermediate region.
The PVPdye mixtures used in this work gave rise to electropherograms
which closely resembled to those described for proteinprobe and DNAprotein
complexes when injected in NECEEM conditions. As shown in Fig. VI.10, parts
C to G, the Gaussian peak attributed to the PVPdye complexes was followed by
an exponential region which ended abruptly at the long migration time side;
however, a peak due to the equilibrium concentration of free dye could not be
distinguished in the electropherograms of Fig. VI.10. This was attributed to the
use of q values not sufficiently close to the maximal stoichiometry of the
complexes, which as shown later in Section VI.2.1.5, was q = 4. In fact, in Fig.
VI.10, this ratio was 4.5 and 3.0 for traces D and E, respectively. At large q
values, the excess PVP should reduce the equilibrium concentration of the free
dye below its detection limit. On the other hand, at too low values of q, the large
equilibrium dye concentration could not be distinguished from the much lower
amount of dye liberated by the complexes. However, as shown in Fig. VI.11, a
peak of the free dye, separated from the beginning of the exponential region, was

224

Chapter VI. Synthetic polymers

clearly observed in all the electropherograms when mixtures with q values


ranging from 3.1 to 4.4 were injected.

Absorbance (mAU)

A
6
20

3.1

Absorbance (mAU)

Ae

4.0

6
7
Time (min)

3
40

AD

3.1
2

Ae

4.0

5
6
Time (min)

Absorbance (mAU)

AD

C
80

3.1

10
AD
Ae

4.0
2

7.0
7.5
Time (min)

Time (min)
Fig. VI.11. Electropherograms of PVP of 10 (A), 60 (B) and 360 kDa (C)
with 4 mM CR at the values of q (monomer/dye molar ratios) indicated on
the traces. The insets show how the areas corresponding to AD and Ae were
established. The electropherograms were recorded at 500 nm. Other details
as in Fig. VI.10.

In order to characterize the PVPdye complexes, three areas should be


measured: (i) the area of the remaining PVPdye complexes, APVPD; (ii) the area
225

Miriam Beneito Cambra

due to the equilibrium concentration of the free dye, AD; and (iii) the area due to
the dye liberated from the complexes during separation, Ae. The values of these
three areas were measured as next explained. As indicated in Fig. VI.10, the
maximum of the PVPdye peak was first located, and the area of the left half of
this peak was taken as APVPD/2. Then, as indicated in Fig. VI.11 for the
experiments performed at 3.1 q 4.4, the area at the right of the small dip of
the asymmetric peak was assigned to the equilibrium concentration of the free
dye, AD. Finally, the area of the exponential region, Ae, was calculated as the total
area minus (APVPD + AD). These areas were used below in sections VI.2.1.5 and
VI.2.1.7 to estimate the maximal stoichiometry and average stability constant of
the PVPdye complexes, respectively.

VI.2.1.4. Influence of MW on the location and shape of the peak of the PVP
dye complexes
As deduced from the electropherograms of Figs. VI.10 and VI.11, when q
< 4 (the dye was in excess in relation to the saturation point), the mobility of the
PVPdye complexes was independent of the molecular mass of the polymer.
This agreed with the model of repeating units of polymer binding individual ions.
A single peak, at a migration time independent from the polymer molecular mass
should be obtained for sufficiently long polyelectrolytes; further, differences in
the mobility of large ionic polymers due to different MW values should be
obtained only upon addition of a sieving medium to the BGE [Cottet, 2005].
As shown below in section VI.2.1.8, the peak areas of the complexes
increased linearly with the monomer concentration, but were essentially
independent of MW. However, as observed in Figs. VI.11, the shape of the peak
of the PVPdye complexes varied with MW. In fact, the height/width ratio of the
peaks increased with both MW and the PVP concentration. Thus, a method to

226

Chapter VI. Synthetic polymers

estimate MW was immediately derived. In Fig. VI.12, part B, the logarithm of


the height/width ratio (in mAU min1, and estimated as indicated in Fig. VI.12,
part A) divided by the PVP concentration (CPVP in g mL1) was plotted against
log MW.
40
Absorbance (mAU)

w1/2

2
-0.5

Time (min)

log (h/w) - log CPVP

-1.5

1.2
2.2
3.1
3.6
4

-2.5
4

4.5

log MW

5.5

Fig. VI.12. Logarithmic plot of (h/w)/CPVP against MW; h and w are the
height and base width of the peak of the PVPCR complexes, which were
measured as indicated in part A (w =2w1/2). The units used were mAU, min
and mg mL1 for h, w and CPVP, respectively. The dashed lines indicate
confidence limits for a significance of 0.05. The legend in part B indicates
the q values of the series. Other details as in Fig. VI.11.

This plot is useful to predict MW using electropherograms obtained at q < 4


(excess dye in relation to the saturation point). These predictions should be
particularly accurate at low MW values, where both a high slope of the curve and

227

Miriam Beneito Cambra

a reduced dispersion of the points are observed. This relationship between peak
shape and MW could be due to the increase in the stability of the complexes as
MW increased. However, another possible reason could be an increase in the
polydispersity of the PVPdye complexes as MW decreased.

VI.2.1.5. Determination of the maximal stoichiometry of the PVPdye


complexes
To estimate the maximal stoichiometry of the PVPdye complexes, the
dye concentration was increased while the PVP concentration was maintained
constant. Then, the APVPD values and the remaining area, (Ae + AD), were plotted
against the dye/monomer molar ratio (1/q). The plot corresponding to CR and 60
kDa PVP is shown in Fig. VI.13, part A. The peak area of the PVPCR
complexes increased until reaching an approximately constant value at which
saturation of the polymer with the dye was produced. On the other hand, the
remaining area, (Ae + AD), increased only slightly at low values of 1/q, and
increased steeply after saturation. This agreed with the complexation of all the
available dye by the polymer at large q values, whereas the dye added to the
solution over the saturation point remained free. As shown in Fig. VI.13, part B,
the absolute mobility of the PVPCR complexes increased as the dye
concentration in the complexes increased, reaching a constant value immediately
after the saturation point. A higher absolute mobility of the complexes should be
attributed to the increase of their charge density as a result of the increasing dye
polymer ratios. Mixtures of CR with PVP, and AB with PVP, at all the available
values of MW, gave plots which were closely similar to those shown in Fig.
VI.13, parts A and B. In all cases, the increase in both the peak area and
mobility of the PVPCR and PVPAB complexes pointed out to q 4.0 0.5 at
the saturation point (1/q 0.25 0.04 in Fig. VI.13).

228

Chapter VI. Synthetic polymers


1000

A
200
500
100
60 kDa PVP
0
3

Relative Ae + AD values

Relative A PVP-CR values

300

(-108) (m2 s-1 V-1)

B
2
27.9 kDa PVP
60 kDa PVP
400 kDa PVP

0
0

0.2
0.4
0.6
1/q (reverse of the monomer/ dye ratio)

Fig. VI.13. Area (APVPD, rhombus and continuous line) (A) and
electrophoretic mobility (B) of the peak of the PVPCR complexes at
increasing CR concentrations, keeping a constant monomer concentration
of 4.5 mM. In A, the squares and dashed lines correspond to Ae + AD (right
scale). The MW values are indicated on the plots. The electropherograms
were recorded at 500 nm.

Next, the PVP concentration was increased, while the dye concentration
was maintained constant. As shown in Fig. VI.14, part A for the CR complexes
with 60 kDa PVP, the peak area of the complexes increased at increasing q
values. This plot, and similar plots obtained with PVP having other MW values, as
well as by using AB, also indicated the formation of a complex with a maximal
monomer: dye molar ratio of q 4. A difference with respect to Fig. VI.13, part
A, was the linearity of the relationship between APVPD and the PVP
concentration, at least up to q = 3.5. Linearity was attributed to saturation of the

229

Miriam Beneito Cambra

polymer in the presence of an excess dye in this region of the curves. As shown
later in this work, this was of interest for the CE determination of the polymer.
Another feature of Fig. VI.14, part A, which was not observed in Fig. VI.13,
part A, was an abrupt increase of the peak area of the PVPCR complexes
immediately before reaching the saturation point.

800
1000

A
400
60 kDa PVP

(-108) (m2 s-1 V-1)

0
3.0

Relative Ae + AD values

Relative APVP-CR values

1200

2.5

27.9 kDa PVP


60 kDa PVP
400 kDa PVP

2.0
0

q (monomer/ dye molar ratio)


Fig. VI.14. Area (APVPD, rhombus and continuous lines) (A) and
electrophoretic mobility (B) of the peak of the PVPCR complexes at
increasing PVP concentrations, and at a constant CR concentration of 4
mM. Other details as in Fig. VI.13.

This could be due to a higher stability of the complexes in the vicinity of the
saturation point, where a more favourable conformation could exist; however, a
slower dissociation rate of the complexes could also contribute. As also observed
in Fig. VI.14, part A, the remaining area, (Ae + AD), decreased at increasing PVP

230

Chapter VI. Synthetic polymers

concentrations, approaching zero when q > 4, as expected from the increasing


amount of complexed dye.
In Fig. VI.14, part B, the variation in the electrophoretic mobility of the
PVPCR complexes at increasing PVP concentrations while maintaining a
constant CR concentration (increasing q values), was plotted. At q values well
below the saturation point, the mobility of the PVPCR complexes did not vary,
showing a perturbation (a local decrease of the absolute mobility) when the
saturation point was approached. Finally, the absolute mobility of the complexes
decreased steadily in the presence of an excess PVP. Therefore, Fig. VI.14, part
B also suggested a conformation change of the PVPCR complexes in the
vicinity of the saturation point. Extrapolation of the approximately linear regions
at low and high values of q pointed out to values of the saturation point close to q
= 4. As also shown in Fig. VI.14, part B, the absolute mobility of the PVPCR
complexes when q > 4 decreased with a higher absolute slope as greater was MW.
This agreed with the curves of Fig. VI.13, part B, where at 1/q values
approaching zero, lower absolute slopes as higher was MW were observed. Series
of experiments performed with either CR or AB and PVP samples at all the
available values of MW, led to plots closely similar to those shown in Fig. VI.14,
parts A and B. Both the peak areas and the mobility of the PVPAB complexes
also indicated a saturation point close to q = 4 for this dye.

VI.2.1.6. Influence of MW on the mobility of the PVPCR complexes at low q


values
The relationship between the initial slopes of the curves in Fig. VI.13,
part B (variation of the absolute mobility of the complexes vs. increasing values
of 1/q) and MW was studied. To obtain accurate values of the initial slopes of the
curves, series of experiments constituted by five triplicated points per series at

231

Miriam Beneito Cambra

5002000 g mL1 PVP, and at 1/q values equal to zero, 0.05 and 0.1 were
performed. These series were monitored at 215 nm. The curves were fitted to the
cubic equation, and for each curve, the initial slope was obtained as the
regression coefficient of the first grade term. A plot of the initial slopes against
log MW is shown in Fig. VI.15. Although with some dispersion, this plot showed
a decrease in the initial slopes when log MW increased. The possible reasons of

/ (1/q) at (1/q) 0

this behaviour are discussed next.

200

100

0
4

log MW

Fig. VI.15. Initial slopes of the curves in Fig. VI.13, part B (variation in
the mobility of the complexes vs. 1/q) plotted against log MW. Data
obtained as indicated in the text.

First, at a constant q value, and at increasing values of MW, a lower absolute


mobility of the complexes could be produced by a lower amount of dye
complexed by the polymer. However, attending to the peak area of the PVPdye
complexes,

the

opposite

behaviour

was

actually

observed

when

electropherograms at constant 1/q values were compared. In fact, the APVPD


values indicated an increase in the degree of complex formation as MW increased,
which agreed with the increased stability of the complexes at increasing MW
values (section VI.2.1.7). Second, the surface area: volume ratio of the PVPCR

232

Chapter VI. Synthetic polymers

complexes should decrease as MW increases; however, this should lead to an


increase in the absolute mobility of the complexes at increasing values of MW.
Again, the opposite behaviour was actually observed in Figs. VI.13, part B and
VI.15, thus suggesting that the absolute mobility of the complexes at low values
of 1/q should be influenced by another factor. This factor could be the location of
the bound dye ions closer to the surface of the complexes, which would increase
their zeta potential. A higher zeta potential should be more important at low
molecular masses, since the surface area:volume ratio of the complexes probably
decreases as MW increases.

VI.2.1.7. Determination of PVPdye stability constants


Although the NECEEM theory was developed to be applied to protein and
DNA complexes with fluorescent probes, to adapt it to the CE of polymerdye
mixtures with spectrophotometric detection is straightforward. Thus, the
equilibrium concentration of free dye is proportional to AD:

[ D ] eq =

AD
b D

(E.VI.1)

where bD is the optical path multiplied by the molar absorptivity of the dye. The
equilibrium molar concentration of the complexes is dependent on APVPD and Ae
as follows:

[ PVP D ] eq =

APVP D
A
+ e
b PVP D b D

(E.VI.2)

where PVPD is the average molar absorptivity of the complexes, which should be
calculated with reference to the molar concentration of the complexed dye. This
is equivalent to considering that a dye ion is complexed on a 1:1 basis by a group
formed by a fixed number of monomers, complexing polymer units, or binding
sites. In this way, the possible influence of a stoichiometry different from 1:1 is
ruled out. The ratio of the two equilibrium molar concentrations is:

233

Miriam Beneito Cambra

R=

[ PVP D ] eq
[ D ] eq

APVP D ( D / PVP D ) + Ae
AD

(E.VI.3)

The stability constant, Ka, is given by:


Ka =

1+ R
[C ] 0 (1 + 1 R) [ D ] 0

(E.VI.4)

where [C]0 and [D]0 are the analytical molar concentrations of the complexing
polymer units and the dye, respectively. Since the maximal stoichiometry of the
PVP complexes with CR and AB is 4:1, we have:
N
4

[C ] 0 = [ PVP ] 0

(E.VI.5)

where N is the average number of monomers and [PVP]0 is the analytical


concentration of the polymer in mol L1. To estimate Ka values, series of
electropherograms obtained at q values ranging from 3.1 to 4.5, and at increasing
MW values, were used. The values of APVPD, AD and Ae were measured as
indicated in sections III.2.2.3 and VI.2.1.2. The D/PVPD ratio was measured at
500 nm; for this purpose, a conventional spectrophotometer was used (Section
III.2.2.3).
The values of log Ka are given in Table VI.2. From top to bottom in
Table VI.2, it is deduced that log Ka varied with q, showing a maximum value at
the saturation point (q = 4) and decreasing slightly at higher q values. It should
be indicated that this variation could be partially due to systematic errors
associated to the difficulty in estimating AD independently from Ae; however, the
presence of the perturbations observed in Fig. VI.14, parts A and B, suggests
that more stable complexes were actually formed in the vicinity of the saturation
point than at other values of q.

234

Chapter VI. Synthetic polymers


Table VI.2. Stability constants, log Ka, for the PVPCR complexes using PVP
with different molecular masses and at increasing q values.
MW (kDa)
q

10

27.9

40

60

360

3.1

4.350.05

4.290.01

4.330.01

4.300.01

4.300.01

4.450.02

3.6

5.010.05

5.040.05

5.220.03

4.170.71

5.460.07

4.0

5.040.03

4.940.12

5.23*

5.430.12

4.730.14

5.660.06

4.5

4.670.08

4.350.01

5.09*

4.990.11

4.820.15

5.230.11

400

n = 2; the other values are means with n = 3.

In addition, the log Ka values given in Table VI.2 also showed that the stability
of the complexes when q = 4 increased with MW. Finally, it should be noted that
owing to the Joule heating, the values in Table VI.2 were obtained at a capillary
temperature which was actually higher than 25C [Evenhuis, 2009].

VI.2.1.8. Analytical applications


The increase in the peak area of the PVPCR complexes at increasing PVP
concentrations, when an excess dye is present (q < 4), was used to quantify the
polymer. Calibration curves were constructed using either CR or AB and PVP
standards at all the available MW values. As indicated in section III.2.2.4, the
maximal PVP concentrations used were 1500 g mL1, which corresponded to a
maximal value of q = 3.4 (at a safe distance from the saturation ratio). Linear
calibrations were obtained in all cases (r2 > 0.98). As shown in Table VI.3, PVP
samples with different MW values gave similar sensitivities, although for
unknown reasons the 160 and 400 kDa PVP standards gave sensitivities higher
than the other standards. Therefore, systematic errors can be occasionally
produced by applying the proposed procedure when a PVP standard different
from the PVP contained in the sample is used for calibration. In addition, the
possible interference of an anionic surfactant was studied. For this purpose,

235

Miriam Beneito Cambra

mixtures containing 1000 g mL1 60 kDa PVP and 4 mM of either CR or AB


were injected both in the absence and presence of a 5% SDS. When SDS was
present, the peak shape and area of the PVPCR complexes were not modified,
but the peak area of the PVPAB complexes was reduced largely. In addition to
electrostatic repulsion between the PVPdye complexes and SDS, the polar
amino groups of CR could also contribute to the lack of matrix effect observed
when SDS was present in the sample solution.
Table VI.3. Relative slopes of the external calibration curves obtained with PVP
solutions of different MW values.

a)

MW (kDa)

CR

AB

10

0.94

0.87

27.9

0.95

0.96

40

1.07

1.01

60a

1.00

1.00

160

1.54

1.57

360

0.92

0.93

400

1.28

2.00

Taken as reference.

PVP of 60 kDa is commonly used in the formulation of colour care


cleaning products, thus the calibration curve constructed with this PVP and CR
(r2 = 0.997) was used in the quantitation studies that followed. First, two
detergent bases with the composition given in Table III.1 were spiked with 60
kDa PVP, mixed with CR as indicated in section III.2.2.4, and injected. An
electropherogram of spiked detergent base II, recorded at 215 and 500 nm, is
shown in Fig. VI.16. The interference of the peaks due to several sample
components was removed by using 500 nm. Using external calibration, the
found/expected PVP concentrations were 1.67/0.99% and 1.19/0.97% for
detergent bases I and II (Table III.1), respectively. Using internal calibration

236

Chapter VI. Synthetic polymers

with two standard additions, the found/expected concentrations were 1.18/1.17%


and 0.62/0.97%, respectively. Thus, the matrix effect due to the detergent
components was partially reduced using internal calibration. Then, the curve of
Fig. VI.12, part B was used to predict MW in the spiked samples. The values MW
= 51 and 60 kDa were obtained for detergent bases I and II, respectively.

Abs. 500 nm (mAU)

Absorbance (mAU)

150

100

20

10

0
3.0

Time (min)

4.0

A
50

215 nm

B
500 nm
0
0

Time (min)

Fig. VI.16. Electropherograms of detergent base II spiked with ca. 1% 60


kDa PVP. Data obtained at 215 (A) and 500 nm (B). The inset shows the
peak of the PVPCR complexes for a series of calibration points.

Several commercial cleaning products and pharmaceutical preparations


were also analyzed. To reduce iodine, drops of an ascorbic acid aqueous solution
were added to the topical antiseptic until decolouration. Then, aliquots of the
samples were mixed with 4 mM CR and injected. The PVP concentrations,
predicted by using external calibration with 60 kDa PVP as standard, are given in
Table VI.4. The found values agree with the expected concentrations for colour
care liquid cleaners, which may contain up to 1% PVP

237

[Prudhomme de Lodder,

Miriam Beneito Cambra


2006].

The concentrations found for the cough syrup and the antibacterial tablet

were well within the usual ranges, namely 0.55% as binders in tablets and <5%
for dispersing agents in syrups [Rowe, 2005]. Finally, the declared amount of PVP
in the topical antiseptic was 10%. Then, the plot of Fig. VI.12, part B was used
to predict the MW values of Table VI.4. Values below 35 kDa were predicted for
the laundry cleaners. Owing to the interference of the surfactants, these values
were probably lower than the actual values. A large value (MW = 400 kDa) was
obtained for the cough syrup, which was attributed to the presence of the anionic
azo-dye amaranth (E123). This dye could form also complexes with PVP with a
mobility similar to that of the PVPCR complexes, thus contributing to the peak
area. Finally, MW = 22 and 16 kDa were predicted for the antibacterial tablet and
topical antiseptic, respectively. The actual MW values in these products are to be
expected along wide ranges [Rowe, 2005].
Table VI.4. PVP concentrations predicted using external calibration with 60 kDa
PVP and MW values estimated by applying the plot of Fig. VI.12, part B.
MW (kDa)

Concentration (%wt)

MW (kDa)

Laundry cleaner I

0.89

35

Laundry cleaner II

0.31

10

Laundry cleaner III

0.45

<10

Cough syrup

0.95

400

Antibacterial tablet

2.42

22

Topical antiseptic

12.4

16

238

Chapter VI. Synthetic polymers

VI.3. PVA

VI.3.1. Evaluation of MW and tacticity of PVA by NECEEM of a polymer


and a dye
A variety of nonaqueous and aqueous CE methods for the characterization
of synthetic polyelectrolytes, including CZE, CGE and CIEF, have been
described

[Clos, 1998; Grosche, 2000; Borisch, 2000; Engelhardt, 2004; Cottet, 2005].

Using CE, information about size, shape, surface charge and formation of
intramolecular

associates

can

be

gained

[Engelhardt,

2004].

Further,

polyelectrolytes have been separated by CGE using solutions of nonionic


polymers as sieving media

[Borisch, 2000; Engelhardt, 2004; Cottet, 2005; Welch,

2001; Starkweather, 2000; Cottet, 1997].

Models describing the electrophoretic

mobility of polyampholytes in free solution CZE have been developed


1998],

[Long,

and CZE has been also used to study both the polymerization degree and

the sulfonation rate of polyestyrenesulfonates [Cottet, 2000]. MECK has been used
to characterize highly charged polysaccharides (heparins)
polyacrylic acids

[Collet, 1996],

[Stefansson, 1994],

and

as well as to study the synthesis progress and

composition of ionic copolymers [Aguilar, 2002]. However, the characterization of


non-charged polymers using CE has been scarcely investigated. In this
connection, polyethylene glycols have been analyzed by CZE previous
derivatization with an anhydride, which provides chromophore groups and
electrical charges at both polymer ends
previous work

[Beneito-Cambra, 2009-A],

[Wallingford, 1996; Barry, 1998].

In a

we described a CZE method to

characterize and evaluate PVP; for this purpose, we used the azo-dye CR which
forms a charged and colored PVPCR complex. The electropherograms of PVP
CR mixtures were interpreted at the light of the NECEEM theory, which was
developed by Krylov et al.

[Berezovski, 2002; Krylov, 2003; Drabovich, 2006; Lin,

239

Miriam Beneito Cambra


2008; Krylov, 2006; Krylov, 2007]

to obtain information about proteins and DNA

fragments using fluorescent markers.


PVA is a synthetic polymer which is widely used for a variety of purposes
within the fields of cosmetic, pharmaceutical and food technologies.
Dependingonthe route of synthesis, PVA with a different tacticity is obtained.
Tacticity or stereoregularity is a characteristic feature of those polymers which
have repeating adjacent chiral centers along the main chain. Tacticity depends on
the class percentages of pairs of adjacent monomers or diads. The two possible
classes of diads are: m or meso (with the same orientation) and r or racemic (with
opposite orientation). Accordingly, three classes of units constituted by three
adjacent monomers or triads can exist: mm, mr and rr. The relative percentages
of mm, mr and rr triads, established by 1H NMR, or preferably by 13C NMR, are
normally used to evaluate PVA tacticity

[Snchez, 2000; Moritani, 1972; Wu, 1977;

Fukae, 2000; Wu, 1973].

As far as we know, CE methods for PVA characterization have not been


reported, and the possibility of evaluating the tacticity of polymers using CE
methods has not been investigated either. In this work, we have applied the
NECEEM principles to the study of the electropherograms obtained by injecting
PVACR mixtures. The formation of complexes between PVA and azo-dyes has
been known for decades
Tsujimoto, 2002].

[Fraunenfelder, 1974; Ikkai, 1996; Atkin, 2001; Ikkai, 1994;

When excess borate was added to both the injected PVACR

mixtures and the BGE, the expected NECEEM pattern for a mixture of a noncharged macromolecule and a charged marker was obtained. Commercial PVA
samples with different molecular masses, also differing in tacticity, were studied.
The electropherograms of PVACR mixtures provided information about the
electrophoretic mobility, maximal stoichiometry, thermodynamic stability
constant and pseudo first-order dissociation rate constant of the PVACR

240

Chapter VI. Synthetic polymers

complex. These parameters were observed to depend on both molecular mass and
tacticity of PVA.

VI.3.1.1. Effect of PVA on the UVVis absorption spectrum of CR


The formation of PVACR complexes has been described
1974; Ikkai, 1996; Atkin, 2001; Ikkai, 1994; Tsujimoto, 2002].

[Fraunenfelder,

As indicated in section

III.2.3.3, the formation of the complexes was first studied by filling the capillary
with PVACR mixtures containing 20 mM borax. As illustrated in Fig. VI.17 for
the 49 kDa PVA sample, the UVVis spectrum of a 4 mM CR solution was
largely

modified

when

the

mixture

also

contained

increasing

PVA

concentrations.
q=6
q=9
Absorbance (mAU)

600

400
q = 12
q=3

200

q=0

0
300

400

500

600

Wavelength (nm)
Fig. VI.17. UVVis absorption spectra obtained by filling the capillary
with solutions containing 20 mM borax, 4 mM CR and the following PVA
concentrations (49 kDa): 0, 12, 24, 36 and 48 mM (the resulting q values
are indicated on the traces).

Thus, from q = [monomer]/[dye] = 0 to 6, the molar absorptivity at the maximum


of the main absorption band increased in a ca. 30%, and a large bathochromic
241

Miriam Beneito Cambra

shift of about 40 nm was also observed (from 479 to 519 nm). Similarly, an
intensity increase of ca. 55% and a bathochromic shift of ca. 50 nm (from 489 to
539 nm) were observed with a conventional spectrophotometer when 0.4 mM
PVA was added to a 0.1 mM CR solution (spectra not shown). This implies a
major change in the physico-chemical environment of CR [Olsen, 1975]. The large
modification of the CR spectrum could be partially due to replacing of water
molecules attached to the polar locations of CR by the polar groups of the
polymer; however, as discussed below in section VI.3.1.4, another possible
reason is the stacking of dye ions in proximity to each other within the structure
of the PVACR complex. An absorptivity decrease at all wavelengths was also
observed when q > 6. This could be due to an increase of the refraction index of
the solution or to the optical screening of the dye in the presence of a large
polymer excess.

VI.3.1.2. Selection of working conditions


In Fig. VI.18, traces A to D, electropherograms of mixtures of CR (4mM)
and PVA (15 kDa, increasing concentrations) obtained in a BGE containing 25
mM borax are given. The injected mixtures were also equilibrated with borax;
however, 20 mM borax was used to preserve sample stacking. Pre-equilibration
with borax improved peak repeatability. In Fig. VI.18, traces E and F,
electropherograms obtained by injecting 2.5 and 4 mM CR solutions,
respectively, in the absence of PVA, are also shown. Truncation of the peak for
the 4 mM CR solution was attributed to the local concentration increase
produced by stacking, followed by precipitation of the dye. Truncation of this
peak was not observed when PVA was present in the injected solution with q 1.
According to the NECEEM theory, when a solution containing an uncolored
macromolecule and a colored charged marker is injected into the capillary, two

242

Chapter VI. Synthetic polymers

peaks with a superimposed exponential decay region in the middle should be


observed

[Berezovski, 2002; Krylov, 2003; Drabovich, 2006; Lin, 2008; Krylov, 2006;

Krylov, 2007].

Thus, in Fig. VI.18, traces A to D, the first band was attributed to

the PVACR complex, and the peak that followed was assumed to be contributed
by both the initial CR excess in free form and the dye released by the complex
during migration (contributing mainly to the left half of the peak). As q increased
(from traces A to D), the area of the band due to the PVACR complex
increased, and the area of the peak due to the free dye decreased. Above q 4 the
electropherograms showed the single band of the complex, which progressively
widened when q further increased (trace D). In addition, a BGE containing 20
mM Na2HPO4 (pH 10) instead of borax was tried. Mixtures containing 4 mM CR
and 16 mM PVA (q = 4) were injected; however, a low intensity wide band due
to the PVACR complex, and a large peak and exponential decay due to the free
dye, were observed (electropherograms not shown). Thus, owing to the better
shape and higher intensity of the complex band, the BGE containing a borax
excess was preferred.
The time required to equilibrate the mixtures before injection was studied.
For this purpose, aliquots of a solution containing 20 mM borax, 4 mM CR and
16 mM PVA monomers (49 kDa, q = 4) were injected at regular time intervals
after mixing the reagents. The area due to the remaining complex, APVA-D, was
estimated as indicated in Fig. VI.18, traces A to D, by doubling the area of the
left half of the complex band. The rest of the area, due to both the initial free dye
and the dye released by the complex during migration, was measured as
ATotalAPVA-D. A slow increase of APVA-D (ca. 8%), a decrease of the rest of the
area (ca. 20%), and a small increase of the electrophoretic mobility of the
complex (ca. 4%), were observed during the first 2 h after mixing the reagents
(sequence of electropherograms with time, not shown). This suggested a slow

243

Miriam Beneito Cambra

reorganization of the complex involving an increase of complex stability and


compactness, or a decrease in its dissociation rate, or both processes at a time.
Thus, to obtain reproducible electropherograms in the experiments that followed,
sample injection was performed with a delay of ca. 23 h after mixing the
reagents.

Absorbance at 500 nm (mAU)

300
APVA-D / 2

EOF

q=1

200

q=2

q=3

D
E
F

q=9

100

0
0

4
Time (min)

Fig. VI.18. Electropherograms of mixtures of CR (4 mM) and PVA (15


kDa, increasing concentrations) (A to D). The q values are monomer/dye
molar ratios. The procedure used to calculate APVA-D (used to construct Fig.
VI.19) is indicated on the traces. Traces E and F are electropherograms of
2.5 and 4 mM CR, respectively. The BGE contained 25 mM borax (pH 9),
and all the injected solutions contained 20 mM borax.

VI.3.1.3. Maximal stoichiometry of the PVACR complex and its relationship


with log MW and tacticity
Series of electropherograms also obtained with 4 mM CR and increasing
PVA concentrations (increasing q values) were used to estimate the saturation
point or maximal stoichiometry of the PVACR complexes, qsat, for all the PVA
244

Chapter VI. Synthetic polymers

samples. As shown in Fig. VI.19 for the 15 kDa PVA sample, when q was
increased the area of the PVACR complex, APVA-D, increased linearly up to q
4, and the rest of the total area decreased proportionally. When q 5, that is,
above the saturation point, the peak due to the excess dye was not observed any
longer, and APVA-D decreased steadily. This agreed with the decrease in the
absorptivity of the complex which was observed by recording spectra at large q
values (Fig. VI.17).

100
Atotal APVA-D

APVA-D

0
0

4
6
q = [monomer] / [CR]

10

Fig. VI.19. Relative areas obtained from electropherograms of a series of


solutions containing 4 mM CR and increasing PVA (15 kDa)
concentrations in the presence of 20 mM borax. Other conditions as in Fig.
VI.18. The continuous and dashed lines join points corresponding to the
area assigned to the PVACR complex (APVA-D, estimated as indicated in
Fig. VI.18) and the rest of the area under the peaks, respectively.

The other PVA samples behaved similarly, although with differences in the
location of the saturation point indicating the maximal stoichiometry of the
complex. In order to establish the saturation point of PVA complexes, qsat, as
accurately as possible, mixtures with q values close to the expected qsat values

245

Miriam Beneito Cambra

were injected; however, repeatability of the electropherograms was poor when q


qsat. Therefore, the qsat values used in the discussion that follows were
established for the different PVA samples as the point where the linear
extrapolation of the decrease of ATotalAPVA-D crossed zero (Fig. VI.19, dashed
line).
In Fig. VI.20 (full symbols), the qsat values obtained in this way for the
PVA samples were plotted against log MW. As observed, H samples formed a
clearly resolved group with respect to the M+L samples (see section III.2.3.3).
Also, within each tacticity group, qsat decreased as log MW increased. Further, the
upper and lower limits of the qsat range were similar for the H and M+L sample
groups, starting at qsat 4.9 at a low molecular mass, and decreasing down to qsat
3.5 at a large molecular mass. The two L samples, which had very large
molecular masses, gave both qsat values close to 3.5. Thus, qsat decreased from
ca. 4.9 to ca. 3.5 at increasing molecular masses, but this variation took place at
different log MW values depending on tacticity.
5.0

qsat and qsat,c

4.5

4.0

3.5
1.5

2.0
Log MW

Fig. VI.20. Plots of the maximal stoichiometry of the complex against log
MW without (qsat, full symbols) and with correction for the acetyl
percentage (qsat,c, empty symbols). Symbols indicating tacticity groups: H
(diamonds), M (circles) and L (triangles).

246

Chapter VI. Synthetic polymers

Next, a correction of the qsat values, thus to take into account the different
proportions of residual acetyl groups in the PVA samples was tried. The molar
proportions of residual acetyl groups per mol of monomers, nac, for the PVA
samples used in this work, were given in section III.2.3.1. In a PVA molecule,
acetyl groups could reduce the number of OH groups which are actually
available to link the dye ions. Thus, in nac 100% acetylated PVA, only
(1nac)100% of the monomers would be available for bonding; however, this
should not reduce the complexing capacity of PVA in an equivalent
(1nac)100% factor, since acetylated monomers can also perform as
nonbonding bridges between bonded monomers. Nevertheless, we studied next
the effect which would have on qsat the extreme situation in which the
complexing capacity of PVA would be reduced in a full (1nac)100% factor.
Thus, the correction applied was: qsat,c = qsat/(1nac). As shown in Fig. VI.20
(empty symbols), full correction for the acetyl percentage essentially confirmed
the conclusions obtained above using uncorrected qsat values concerning both the
decrease of qsat at increasing molecular masses and the noticeable difference
between the H and M+L tacticity groups.

VI.3.1.4. Structure of the complex


When qsat 4.9, each dye ion could be bonded by a maximum of four and
probably a minimum of two monomers (since CR is a symmetrical structure),
leaving an average of 0.92.9 non-bonded monomers per dye ion, respectively.
Similarly, when qsat 3.5, each dye ion could be bonded by a maximum of three
and a minimum of two monomers, leaving an average of 0.51.5 non-bonded
monomers per dye ion, respectively. In both cases, a small number of nonbonded monomers are available to perform as bridges between adjacent dye ions.
Thus, in all the possible scenarios, the number of non-bonded monomers per dye

247

Miriam Beneito Cambra

ion is very low, which suggests a proximity between the dye ions. As depicted in
the temptative structure of Fig. VI.21, stacks of dye ions by pairs, or by groups
constituted by a higher number of dye ions, could be formed. As proposed in this
figure, it seems reasonably to stack the dye ions by alternating the sulfonate and
amino groups, rather than putting together all the sulfonate groups at one side
and all the amino groups at the other side of the stack. Also, from a geometrical
point of view, it seems reasonably to link the PVA monomers with the alternated
sulfonate and amino groups though hydrogen bonds. As indicated in the
literature, the azo groups are also possible sites for hydrogen bonding
2001; Olsen, 1975.];

[Atkin,

however, below the saturation point, and owing to the low

stoichiometry, the number of available monomers per dye ion is rather short.
Then, hydrogen bonding of the PVA monomers with both amino and azo groups,
which are located in outer and inner sites of the dye ion, respectively, seems to be
less likely than the structure proposed in Fig. VI.21 However, the probability of
bonding the OH groups with azo groups should increase after the saturation
point, when an excess of monomers are available.
Dye stacking, resulting in a large absorptivity increase and blue shifting of
the absorption maximum, has been described in solutions of plant pigments when
certain ligands and Mg2+ are present [Ellestad, 2006]. Thus, dye stacking could
explain both the low qsat values of Fig. VI.20 and the very large modification of
the absorption spectrum when PVA is added to CR solutions (Fig. VI.17).
Further, the distance between donoracceptor atoms in adjacent stacked dye ions
should be constricted by the distances between pairs of adjacent OH groups
along the PVA chain, and these later are longer for r than for m diads. Thus, in
PVA samples having a larger proportion of r diads, adjacent dye ions could be
stacked at longer distances from each other than in samples with larger
proportions of m diads. As discussed below, this could also explain the

248

Chapter VI. Synthetic polymers

correlations found between other electrophoretic parameters and PVA tacticity as


established by 13C NMR.

O
H

O
H

H
N

H O

N
H

N
N

O
N
H

H
H

OH

O-

O H

O-

O
S N
O
N
N

O-

H
H

O-

O
H

H
O

O-

H
H

H
O

Fig. VI.21. Temptative structure for two adjacent PVACR complex units
(with two stacked dye ions).

The complex is probably compelled to adopt a given structure when an


excess of either dye ions or unbound monomers is present. This could explain the
excellent reproducibility of the electropherograms when q < qsat or q > qsat,
respectively. Therefore, poor reproducibility of the electropherograms when q
qsat could be due to the different ways the dye ions could be arranged within the
complex when the available monomers are either in a small defect or a small
excess with respect to qsat.

249

Miriam Beneito Cambra

VI.3.1.5. Influence of molecular mass and tacticity on the electrophoretic


mobility of the complex
Electropherograms obtained with PVA samples of increasing molecular mass,
also corresponding to different tacticities, are shown in Fig. VI.22. These
electropherograms were obtained with a small CR excess with respect to the
saturation point. Thus, traces A and C, which correspond to complexes with qsat
= 4.51 were obtained at q = 4, and the other traces, which correspond to
complexes with qsat = 3.51 were obtained at q = 3. As further commented in
section VI.3.1.6, this was important to distinguish the AD area from that of the
exponential decay contribution, Ae. As observed in Fig. VI.22, the
electropherograms of M+L samples (traces CE) showed a sharper complex band
than that of the H samples (traces A and B). In addition, within each tacticity
group, the electrophoretic mobility of the complex was reduced at increasing
molecular mass (AB and CD pairs). The relationships among electrophoretic
mobility of the complex formed in the presence of an excess dye, molecular mass
and tacticity are better recognized in Fig. VI.23 (full symbols). Within each
tacticity group, the absolute electrophoretic mobility decreased slightly at
increasing log MW values. Therefore, electrophoretic mobilities indicated that
charge density of the complexes decreased at increasing molecular masses. In
addition, absolute mobility increased when the rr/mm ratio decreased between
the H and M sample groups. Therefore, absolute mobility increased when the
average distances between the OH groups of adjacent monomers decreased as a
result of the reduction of the proportion of r diads, probably giving rise to an
increase of complex compactness.
In free solution, the electrophoretic mobility of polyelectrolytes with the
same linear structure but with different molecular masses is very similar [Grosche,
2000; Long, 1998; Cottet, 2000].

This is due to the almost identical charge-to-

250

Chapter VI. Synthetic polymers

volume ratio which is achieved when units with a given charge-to-volume ratio
are increasingly added to the polyelectrolyte. This electrophoretic behavior of
polyelectrolytes has been called the free draining regime [Cottet, 2000].

APVA-D / 2

Absorbance at 500 (mAU)

200

AD

EOF
4; 4.5
AD

150

3; 3.5
A
D

4; 4.5

100
A
D

3; 3.5

50
AD

3; 3.5

0
0

4
6
Time (min)

10

Fig. VI.22. Electropherograms of PVACR mixtures containing 20 mM


borax, 4 mM CR and the following PVA monomer concentrations: (A, C)
16 mM and (B, D, E) 12 mM. The molecular masses were: (A) 15, (B) 31,
(C) 49, (D) 100 and (E) 205 kDa. Sample tacticities: H (A, B), M (C, D)
and L (E). The numbers on the traces are q and qsat values (in italics). The
expanded parts show how the AD areas were estimated; APVA-D values were
estimated as indicated in trace A. Other conditions as in Fig. VI.18, traces
AD.

251

- 105 (cm 2 V -1 s -1)

60
30

50

40
25
1.0

1.5
Log MW

- [qsat + (VD / Vm)] 104 (cm 2 V -1 s -1)

Miriam Beneito Cambra

2.0

Fig. VI.23. Electrophoretic mobility of the PVACR complex against log


MW, without (full symbols, dotted lines and axis at the left) and with
correction for the complex stoichiometry (empty symbols, dashed lines and
axis at the right); correction was performed with both VD/Vm = 15 and 10.
Symbols indicating tacticity groups: H (diamonds), M (circles) and L
(triangles). Other conditions as in Fig. VI.22.

As occurs with polyelectrolytes, the mobility of the PVACR complexes could


also reach a constant value at increasing molecular masses, not further dependent
on the molecular mass of PVA. However, in the case of the PVACR complexes
formed in the presence of an excess CR, to increase the molecular mass of the
polymer is not the only factor to be taken into account for a free draining regime
to be reached. At increasing molecular masses, both the stoichiometry and the
apparent charge density of the saturated complex, qsat, should be also maintained
constant. As discussed above in section VI.3.1.3, stoichiometry of the complex
formed in an excess dye varied with both molecular mass and tacticity; however,
to show if a free draining regime is approached at high molecular masses, a
model taking into account the stoichiometry variations can be constructed. For
this purpose, the concept of average electrophoretic mobility per complex unit,
unit, can be used. This parameter was defined as the mobility due to a unit
constituted by a single dye ion and the average number of monomers directly

252

Chapter VI. Synthetic polymers

attached to that ion, plus the average share of monomers which are necessary to
link the complexed dye ion to the neighboring complex units. The mobility per
complex unit, uni, should be directly proportional to the charge of a dye ion, 2,
and inversely proportional to the volume of the complex unit:
unit =

2f
VD + qsatVm

(E.VI.6)

where f is a coefficient of proportionality, VD is the volume of a dye ion bound to


the polymer chain, and Vm is the average volume of a monomer in the complex.
If both sides of equation E.VI.6 are multiplied by the electrophoretic mobility of
the complex, , and the equation is reorganized, we have:
2f
= [ qsat + (VD / Vm) ]
Vm unit

(E.VI.7)

Therefore, if a free draining regime would be reached at large molecular masses,


the product [qsat+ (VD/Vm)] would also reach a constant value. Since the VD/Vm
ratio was unknown, two approximations were used: first, VD/Vm was taken as the
ratio of the molecular masses of the dye ion and the monomers, 651/44 15, and
second, VD/Vm was taken as the ratio of the total number of C, H, O and N atoms
involved, 68/7 10. These two values of VD/Vm are rough approximations;
however, as shown below, the use of any of them led to essentially the same
conclusions. In Fig. VI.23 (empty symbols), product [qsat+ (VD/Vm)] was plotted
against log MW. As observed in this figure, the differences among tacticity groups
were enhanced upon correction of the electrophoretic mobility of the complex by
the effects of complex stoichiometry. This correction confirmed the independent
reduction of the absolute mobility at increasing log MW values for the H and
M+L tacticity groups. Therefore, both a higher molecular mass and a higher
rr/mm ratio implied a larger volume of the monomerdye complex units, that is,
a smaller packing density of the complex. This agreed with the longer distances
between the OH groups in r diads compared to m diads. On the other hand, the

253

Miriam Beneito Cambra

corrected mobilities of Fig. VI.23 also indicated that the complex did not reach a
free draining regime at increasing molecular masses. This meant that coefficient f
in equation E.VI.7 was not constant within any molecular mass range, that is,
packing density of the complex decreased at increasing molecular masses for all
the PVA samples used in this work.

VI.3.1.6. Stability and dissociation rate constants of the complex


In Fig. VI.22, it can be also observed that the area of the peak due the free
dye was larger for the H (A and B traces) than for the M+L (CE traces) tacticity
groups. This indicated either a higher complex stability or a slower dissociation
rate, or both features at a time, for the H group in comparison to the M+L groups.
Thus, next the stability and dissociation rate constants of PVACR complexes
were estimated. For this purpose, three areas are needed: (i) APVA-D, due to the
PVACR complex; (ii) AD, due to the free dye present in the initial equilibrium
conditions; and (iii) Ae, generated by the dye released by the complex during
migration. In the electropherograms of Fig. VI.18, AD could not be distinguished
from Ae. This was attributed to the use of q values far from the maximal
stoichiometry of the complexes, qsat. Thus, the free dye concentration should be
very low at large values of q, and conversely, at low q values, the large initial
concentration of the free dye made also difficult to distinguish its contribution
from that due to the dye released during complex migration. However, as shown
in the expanded regions of the electropherograms of Fig. VI.22, at q values
slightly lower than qsat, it was possible to distinguish between these two
contributions. Therefore, the maximum of the PVACR complex peak was
located, and as indicated in the same figure (trace A), the area of the left half of
the band was taken as APVA-D/2. In this band half, the contribution to the area due
to the dye released during migration was assumed to be negligible (since the free

254

Chapter VI. Synthetic polymers

dye migrates towards increasing migration times). Then, the area at the right of
the first depression of the free dye peak (see the expanded parts in Fig. VI.22)
was assigned to AD, and Ae was calculated as the total area minus the
contributions of the complex and initially free dye: Ae = ATotal (APVA-D + AD).
These assignments probably implied a systematic error in the estimation of AD,
and therefore also in the calculation of the stability constants; however, this made
possible to compare the constants obtained with the different PVA samples. To
estimate stability constants, the equilibrium molar concentration of free dye,
[D]eq, was obtained as:

[ D ] eq =

AD
b D

(E.VI.8)

where bD is the optical path multiplied by the molar absorptivity of the dye. The
equilibrium molar concentration of the complex is given by:

[ PVA D ] eq =

APVA D
A
+ e
b PVA D b D

(E.VI.9)

where PVA-D is the molar absorptivity of the complex. Instead of reasoning about
complex formation in terms of qsat monomers per dye ion, calculations are much
simpler if the formation of a 1:1 complex is assumed, being the ligand the
complexing units formed by groups of qsat monomers. The stability constant, Ks,
is then given by:
Ks =

1+ R
[C ] 0 (1 + (1/ R)) [ D ] 0

(E.VI.10)

where [C]0 and [D]0 are the total analytical molar concentrations of the
complexing polymer units and the dye, respectively, and where R is given by:
R=

[ PVA D ] eq
[ D ] eq

255

APVA D ( D / PVA D ) + A e
AD

(E.VI.11)

Miriam Beneito Cambra

The value of [C]0 can be calculated as:

[C ] 0 =

n
[ PVA] 0
q sat

(E.VI.12)

where n is the average number of monomers along the polymer chains, and
where [PVA]0 is the total analytical molar concentration of the polymer in mol
L1. The molar absorptivity ratio D/PVA-D = 0.77 was obtained with a
spectrophotometer at 500 nm by measuring several PVACR mixtures with q
values sligthly lower than the respective qsat values; in these conditions, the
formation of a predominant complex with a 1:1 stoichiometry, in which the
ligand was constituted by qsat monomers, was assumed. In Fig. VI.24, part A,
the resulting values of log Ks for q = 2 and 3 (full and empty symbols,
respectively) were plotted against log MW. Contrary to that observed for other
CZE parameters, log Ks values did not show any significant trend concerning to
either molecular mass or tacticity.
The electropherograms with q = 2 used to obtain log Ks were further
processed to estimate pseudo-first-order rate constants for the dissociation of the
PVACR complexes. For this purpose, half-life measurements were made, and
rate constants were estimated as k = ln2/t1/2, where t1/2 is the half-life. The
exponential decay curve within the region close to the peak of the excess dye,
where the contribution of the remaining PVACR complex was small, was used.
Five measurements of the half-life were made by selecting 5 different starting
points on the decay curve of each electropherogram of series of triplicated
injections. The starting points were evenly spaced from each other in about 5 s.
Estimations of k were obtained with low uncertainties (ca. 4.3%). In Fig. VI.24,
part B, the average values of k were plotted against log MW. Within each tacticity
group, the dissociation rate constant of the complex was reduced at increasing
molecular masses; in addition, H samples showed differences with respect to the
M+L samples.
256

Chapter VI. Synthetic polymers

5.0

4.0
3.0
2.0
1.0

5.0
4.0
3.0
2.0
1.0
1.0

1.5

2.0
Log MW

Fig. VI.24. Plots of (A) log Ks and (B) k vs. log MW. Data were calculated
from electropherograms obtained with (A) q = 3 and 2 (full and empty
symbols, respectively) and (B) q = 2. Symbols indicating tacticity groups:
H (diamonds), M (circles) and L (triangles). Other conditions as in Fig.
VI.22.

257

CHAPTER VII

ENZYMES

Chapter VII. Enzymes

VII.1. Intact enzymes by CE

VII.1.1. Identification of enzymes for the detergent industry by CZE of the


intact proteins
Today enzymes constitute important components of laundry cleaners,
dishwashers and other cleaning products

[Novozymes,

2002].

Since the

introduction of Alcalase, an alkali-tolerant bacterial protease in 1963 [Novozymes,


2002],

the role of enzymes in cleaning products has changed from that of a minor
[Olsen, 1998].

additive to becoming a key ingredient

Enzymes for cleaning

products constitute the largest segment of the world market for industrial
enzymes

[Novozymes, 2002].

Cleaning power enhancement by enzymes leads to a

reduction of washing times and temperatures, with the subsequent savings of


water and energy. Further environmental advantages arise from the lower
consumption of surfactants, hypochlorite and alkalis, with the additional benefit
of a better care of fabrics during washing [Olsen, 1998].
In spite of their interest in the detergent industry, and in environmental
and toxicological studies

[www.heraproject.com.],

identification and quantification

methods for enzymes in cleaning products have been scarcely investigated.


Enzymes are commonly detected and quantified by monitoring the hydrolysis of
a substrate, or by precipitation with an antiserum
Dunn, 1971; Kulkarni, 1999; Lorentz, 2000].

[Novozymes, 2002; Olsen, 1998;

Polyacrylamide gel electrophoresis has

been used to separate proteases in cleaners

[Deschreider,

1972].

Protein

characterization and quantification is frequently carried out by hydrolysis


followed by chromatographic determination of the resulting amino acids [Phillips,
1983].

Amino acid profiles can be established by a variety of separation and

detection techniques, including GC previous derivatization with ethyl


chloroformate

[Gimeno-Adelantado, 2002; De la Cruz-Caizares, 2004],

261

or with a

Miriam Beneito Cambra

silylating reagent [Rampazzi, 2002], and a wide variety of HPLC methods [MolnrPerl, 2000; Molnr-Perl, 2001; Tcherkas, 2001-A; Tcherkas, 2001-B; Concha-Herrera,
2007; Hanczk, 2007; Chernobrovkin, 2007; Pereira, 2008].

The resulting amino acid

profiles have been used to predict the enzyme class


Beneito-Cambra, 2009-B].

[Beneito-Cambra, 2008;

However, amino acid analysis using hydrolysis followed

by either GC or HPLC requires long analysis times.


Capillary electroseparation techniques have the advantages of being
simple, fast and highly efficient. The application of these techniques to the
analysis of peptides and proteins has been periodically reviewed
Dolnk, 2006; Dolnk, 2008; Cifuentes, 1997].

[Jmeian, 2009;

MECK has been used to analyze

Savinase (a protease) and various analogues in cultivation broth [Vinther, 1992-B],


and both CZE and MEKC have been applied to the identification of serine
protease analogues [Eriksen, 1996].
The aim of this work was to examine CZE of intact proteins as a means of
classifying and identifying enzymes in industrial raw materials of the detergent
industry. The four main enzyme classes used in cleaners were included in this
study, i.e. proteases, amylases, lipases and cellulases. Charge and structural
changes of proteins are largely dependent on pH; thus, in order to gather more
information for enzyme identification, both a basic and an acid BGEs were used.
An alkaline borate buffer of pH 9, also containing PVA as dynamic coating to
hinder protein adsorption

[Ruiz-ngel, 2002],

was selected. On the other hand, an

acidic BGE containing urea, iminodiacetic acid as low conductive isoelectric


buffer

[Righetti, 1997; Bossi, 1997; Righetti, 1998; Capelli, 1998; Stoyanov, 1997]

and

HEC to inhibit adsorption, was also used. Due to their much reduced
conductivities, large concentrations of isoelectric buffers are compatible with
high voltage gradients, thus further reducing adsorption and favouring high
resolution within short migration times
Capelli, 1998].

[Righetti, 1997; Bossi, 1997; Righetti, 1998;

Further, the total amount of protein in the samples was established


262

Chapter VII. Enzymes

by the Bradfords method. This made possible to use total peak areas of the
electropherograms obtained with both BGEs to establish relative sensitivities,
which were also useful for enzyme identification. The enzymes used in this work
are indicated in Table III.2.

VII.1.1.1. CZE with a basic BGE


With the basic BGE, most enzymes gave rise to a single predominant peak
or a group of partially resolved intense peaks, which was frequently followed by
several small peaks (Fig. VII.1, parts A to D).

A
Absorbance at 214 nm (mAU)

Esp

Sta

EOF

800

EOF

400
120

EOF

80

Cel

40

Car

Lx
Eve

80
200

400
Dur

40

Sav

EOF
Ls
End

Ter

Alc

0
2

0
2

Time (min)

Fig. VII.1. Electropherograms of enzyme brands in the basic BGE (pH


9.0): (A) proteases, (B) amylases, (C) lipases and (D) cellulases.
Experimental conditions in text (sections III.3.1.3 and III.3.1.4).

For all proteases (Fig.VII.1, part A), the main peak was located within the
cationic migration region in the vicinity of the electroosmotic flow (EOF) time
(ca. 2.14 min). At pH = 9.0 charge density should be low for proteases (8.4 < pI
< 11), which agrees with the cationic behaviour of the main peak. The other

263

Miriam Beneito Cambra

enzymes (Fig.VII.1, parts B to D), with the exception of Sta, gave also an
intense peak or a group of peaks within the anionic migration region. Sta gave
two partially resolved peaks close to the EOF time, but within the cationic
region. An amylase, Ter, and a lipase, Lx, showed two peaks of similar intensity,
with baseline resolution, instead of a single predominant peak (Fig.VII.1, parts
B and C). The presence of a second intense peak close to the EOF time made Ter
to be easily distinguishable from its engineered variant Dur (Fig.VII.1, part B).
Similarly, the sharp intense peak within the cationic region made Lx to be easily
distinguishable from the other lipase, Ls (Fig.VII.1, part C). Also, some
cellulases as Cel and End (Fig.VII.1, part D) showed a division of the main
band into several partially resolved intense peaks. In addition to the main peaks,
the electrophoretic profiles of most enzymes also provided several small peaks
which can be useful as fingerprints for identification.
Intra- and inter-day repeatabilities in this BGE were obtained by injecting
a 0.01% Alc aqueous solution. Five injections per day during three consecutive
days were performed. Intra- and inter-day migration times showed RSDs lower
than 3.5 and 7.5%, respectively. More than 100 injections were performed
without the need of replacing the capillary.

VII.1.1.2. CZE with an acid BGE


In agreement with literature, an excellent current stability was observed in
the acid BGE [Piergiovanni, 2005]. The EOF was also very low in this medium. As
shown in Fig. VII.2, parts A to D, the electropherograms also showed several
distinctive features according to both enzyme class and the nature of the
particular enzyme. Thus, proteases gave a predominant peak together with
several small peaks within the 5.5 6.5 migration time region (Fig. VII.2, part
A). Further, Eve and Sav also gave an additional and quite characteristic intense

264

Chapter VII. Enzymes

sharp peak at ca. 3.5 min. The presence of two large peaks in these two enzymes
could be due to the occurrence of two predominant protein fractions in the raw
material. It should be noted that Esp and Sav are two closely related proteases
with well known three-dimensional structures, and a high degree of structural
similarity

[Georgieva, 2001].

However, in spite of this structural similarity, these

two enzymes can be clearly distinguished by CZE in the acid BGE.

Sta

60

120

Absorbance at 214 nm (mAU)

200

Cel

Esp

40
Dur

40

80

Eve

100

Lx

Car

20

Sav

20

40
Ls

End

Ter
Alc
0

0
4

0
4

Time (min)

Fig. VII.2. Electropherograms of enzyme brands in the acid BGE (apparent


pH 3.1): (A) proteases, (B) amylases, (C) lipases and (D) cellulases.
Experimental conditions in text (sections III.3.1.3 and III.3.1.4).

Concerning to amylases (Fig. VII.2, part B), Ter showed a single


predominant peak, rather than the two large peaks which were observed in the
acid BGE (Fig. VII.1, part B). As observed in the basic BGE (Fig. VII.1, part
C), lipases also gave rather characteristic patterns in the acid BGE, with several
partially resolved sharp peaks (Fig. VII.2, part C). Thus, both Lx and Ls were
clearly distinguishable by CZE using either the basic or acid BGEs. Concerning
to cellulases (Fig. VII.2, part D), Car gave two peaks, whereas Cel and End

265

Miriam Beneito Cambra

gave a single asymmetric peak. Using Alc, migration time intra- and inter-day
repeatabilities were below 2.7 and 5.3%, respectively. At least 120 injections
were performed without the need of replacing the capillary. Therefore, both in
the basic and acid BGEs, most enzymes gave characteristic patterns which
allowed class identification, and in many cases the electropherograms also
showed enough distinctive details to allow the safe identification of the
individual enzyme within its class.

VII.1.1.3. Relative sensitivities and application to enzyme identification


In order to establish relative sensitivities, the protein contents in the raw
enzymes were previously measured by the Bradfords method. The protein
concentrations found using BSA as reference are given in Table VII.1.
Table VII.1. Class, commercial name, protein content and relative sensitivities of the enzyme
industrial concentrates used in this work.

Class

Protease

Amylase

Lipase

Cellulase

Relative
sensitivityc)
Basic
Acid
BGE
BGE
1.0
1.0

Alcalase 2.5 L

Alc

Protein content
(% BSA)b)
4.03 0.03

Savinase 16L, Type EX

Sav

1.81 0.07

1.8

1.7

Everlase 16L, Type EX

Eve

1.24 0.03

2.8

1.0

Esperase 8.0L

Esp

2.45 0.05

1.5

1.4

Termamyl Ultra 300L

Ter

2.95 0.02

1.9

1.1

Duramyl 300L, Type DX

Dur

4.87 0.03

0.8

0.3

Stainzyme 12L

Sta

2.69 0.01

1.0

0.9

Lipolase 100L, Type EX

Ls

1.79 0.01

1.2

1.2

Lipex 100L

Lx

2.90 0.01

0.8

0.7

Endolase 5000L

End

2.56 0.01

1.1

1.0

Carezyme 4500L

Car

0.74 0.08

1.5

1.4

Celluzyme 0,7Ta)

Cel

1.31 0.06

4.9

0.4

Commercial name

Abbreviation

a)

Sample supplied as granular solid (the other samples were liquid concentrates)
Estimated using the Bradfords assay (calibration curve with 9 points)
c)
Relative sensitivities for the total area of the electrophoretic peaks (respect to Alcalase 2.5L)
b)

266

Chapter VII. Enzymes

Then, for each industrial enzyme a calibration curve was constructed. For this
purpose, industrial enzymes were diluted with water at several concentration
levels within the 0.2-20% v/v range, and aliquots were taken. Precipitation with
acetone, re-dissolution and injection in the capillary using both the basic and acid
BGEs were then performed. The plots of total peak area versus protein
concentration were linear (r > 0.992). From these plots, relative sensitivities were
established as follows:
S r = S x S Alc

(E.VII.1)

where Sx and SAlc are the slopes for the assayed enzyme and Alc which was used
as reference, respectively. As shown in Table VII.1, the ranges were 0.8-4.9 and
0.3-1.7 for the basic and acid BGEs, respectively. The differences between
relative sensitivities were small in a number of cases, but several enzymes gave
rather characteristic values. Thus, Eve and Sav gave high sensitivities in the basic
and acid media, respectively, Dur gave low sensitivities in both media, and Cel
exhibited a very high sensitivity in the basic BGE but a very low one in the acid
BGE. As also deduced from Table VII.1, the differences among relative
sensitivities were predominantly due to the nature of the individual enzymes
rather than to their respective classes.

VII.1.1.4. Influence of surfactants commonly used in the formulation of


cleaning products
The influence of some common surfactants, widely used in the
formulation of cleaning products, on the migration times and areas of the
electrophoretic peaks was examined. For this purpose, solutions of surfactants
spiked with Alc were treated as indicated in section III.3.1.4. Using the basic
BGE, the electrophoretic profile of the enzyme (Fig. VII.3, part A) was slightly
modified in the presence of fatty alcohol ethoxylates (Dehydol LT-7) (Fig. VII.3,

267

Miriam Beneito Cambra

part B); however, rather different profiles were obtained when the sample
solution contained anionic surfactants (Fig. VII.3, parts C and D). Similar
experiments were performed in the acid BGE. The influence of Dehydol LT-7
gave electropherograms similar to that of Fig. VII.2, part A for Alc; however, in
the presence of anionic surfactants, a significant increase in the retention time of
the enzyme peaks was observed (data not shown). Similar results were obtained
for selected enzymes of the other three classes. Consequently, the proposed
method for enzyme identification can be safely applied in the presence of nonionic surfactants, but will fail probably when anionic surfactants would be
present at large concentrations. Work addressed to reduce the interference of
anionic surfactants is in progress.

Absorbance at 214 nm (mAU)

80

80

40

40

0
20

0
20

C
10

10

0
0

Time (min)
Fig. VII.3. Influence of surfactants on the electrophoretic profile of Alc
(0.01%) in the basic BGE: (A) absence of surfactant, (B) 5% Dehydol LT7, (C) 5% LAS and (D) 5% LES. Other conditions as in Fig. VII.1.

268

Chapter VII. Enzymes

VII.2. Classification of enzymes by MS

VII.2.1. Rapid classification of enzymes in cleaning products by hydrolysis,


MS and LDA
The first enzyme-containing detergent was commercialized in 1913;
however, enzymes were little used for cleaning purposes until the introduction of
Alcalase, an alkali-tolerant bacterial protease, in 1963

[Novozymes, 2002].

Since

then, the role of enzymes in cleaning products has changed from one of a minor
additive to becoming a key ingredient [Olsen, 1998]. Cleaning power enhancement
by enzymes leads to a reduction of washing times and temperatures, with the
subsequent savings of water and energy. Further environmental advantages arise
from the lower consumption of surfactants, hypochlorite and alkalis, with the
additional benefit of a better care of fabrics during washing [Olsen, 1998].
Mainly proteases, amylases, lypases and cellulases are used in the
formulation of modern cleaning products. Proteases are used to remove proteinrich soil stains, including blood and grass. Amylases remove starch-containing
stains, and prevent starch from adhering to fabrics and dishes. Lypases help in
removing fat and edible oil stains. Finally, cellulases are used to remove fuzz and
pills (small balls of fabric) from cotton. In spite of the importance of these
enzymes in the detergent industry, and in environmental and toxicological studies
[www.heraproject. com],

identification and quantification methods for enzymes in

cleaning products have scarcely been investigated. Enzymes are commonly


detected and quantified by monitoring the hydrolysis of a substrate, or by
precipitation with an antiserum

[Novozymes, 2002; Olsen, 1998; Dunn, 1971;

Kulkarni, 1999; Lorentz, 2000].

Enzymes of different classes differ in molecular structure, and they also


have rather dissimilar amino acid profiles, which can be useful for enzyme

269

Miriam Beneito Cambra

classification. After hydrolysis, the amino acid profile of the enzyme can be
established by a variety of separation and detection techniques, including GC
following previous derivatization with ethyl chloroformate
2002; De la Cruz-Caizares, 2004],

and a variety of HPLC methods

[Gimeno-Adelantado,

or with a silylating reagent

[Rampazzi, 2002],

[Molnr-Perl, 2000; Molnr-Perl, 2001; Tcherkas,

2001-A; Tcherkas, 2001-B; Garca Alvarez-Coque, 1989; Concha-Herrera, 2007;


Hanczk, 2007; Chernobrovkin, 2007; Pereira, 2008].

Useful sample classification methods without previous chromatographic


separation have been described by using MS. For this purpose, the samples have
been directly infused into the mass spectrometer using an ESI

[Goodacre, 2002;

Gama-Melo, 2006; Ng, 2004; Peris-Vicente, 2005; Catharino, 2005]


[Gmez-Ariza, 2006]

or an APPI

ion source. Amino acid profiles obtained by protein

hydrolysis have been used as fingerprints to classify protein-binding media used


in works of art according to their biological sources
2003],

vegetable oils in accordance to their botanical origin

rice cultivars

[Wang, 1998],

and tea varieties

authenticate high quality beer


principal component analysis
analysis
2007],

[Peris-Vicente, 2005; Llet,

[Poulli, 2005],

LDA

partial least-squares

[Erbe, 2000].

[Lerma-Garca, 2007],

[Alczar, 2007],

as well as to

Multivariate algorithms including

[Goodacre, 2002; Poulli, 2005],

hierarchical cluster

[Peris-Vicente, 2005; Gmez-Ariza, 2006; Lerma-Garca,


[Yang, 2002]

and ANN

[Garca-Gonzlez, 2004],

have

been used to treat the spectral data.


The aim of this work was to develop a quick and straightforward method
for enzyme identification and classification in cleaning products. For this
purpose, the enzymes were isolated by precipitation with acetone, hydrolyzed,
and the hydrolysates were directly infused in the ESI ion source of a mass
spectrometer. After normalization, the ion abundances of the amino acids were
used as predictors to construct LDA models for enzyme classification. The

270

Chapter VII. Enzymes

procedure was validated by analyzing enzyme industrial concentrates and


laundry products. The enzymes used in this work are indicated in Table III.2.

VII.2.1.1. Mass spectra and normalization of the variables


After protein hydrolysis, the mass spectra of the enzyme industrial
concentrates showed the [M+H]+ ions of the following amino acids: Gly (m/z
76.1), Ala (m/z 90.1), Ser (m/z 106.1), Pro (m/z 116.1), Val (m/z 118.1), Thr (m/z
120.1), Cys (m/z 122.2), Ile/Leu (single common ion at m/z 132.2), Asn (m/z
133.1), Asp (m/z 134.1), Lys (m/z 147.2), Glu (m/z 148.2), Met (m/z 150.2), His
(m/z 156.2), Phe (m/z 166.2), Arg (m/z 175.2), Tyr (m/z 182.2) and Trp (m/z
205.5). A typical mass spectrum obtained from the hydrolysate of a protease is
shown in Fig. VII.4.

1.2

Trp

Tyr

Arg

Phe

Asn
Asp
Lys
Glu
Met
His

Thr
Cys

Ala
Ala

Ser

Gly

0.4

Gly

Cys

Met

Ser

Relative abundance x 107

0.8

Val

Pro

Leu+Ile

0.05

0
80

160

120

m/z

Fig. VII.4. Typical ESI mass spectrum of the hydrolysate of a protease.


The [M+H]+ ions of the amino acids used as variables in this study are
indicated.

271

200

Miriam Beneito Cambra

In order to reduce the variability associated with the total amount of


protein recovered from the samples, normalized rather than absolute ion
abundances were used. For this purpose, two normalization procedures were
tried. In procedure A, the ion abundance of each amino acid was divided by the
sum of the ion abundances of all the amino acids in the corresponding spectrum.
In procedure B, the ion abundance of each amino acid was divided by each of the
ion abundances of the other amino acids in the corresponding spectrum. In this
way, and taking into account that 18 peaks were measured on each spectrum and
that a pair of peaks should be considered only once, (1817)/2=153 nonredundant ion abundance ratios were obtained. The ion abundances of these 18
amino acids were either intermediate or low, but in all cases signal-to-noise ratios
adequate for data analysis were obtained. Significant differences between the
amino acid profiles given by different enzyme classes were observed. After
normalization according to procedures A and B, the significance of several
variables was checked using analysis of variance (ANOVA). Using 3 enzymes of
each class 3 infusions of each, all the variables checked showed significances
better than 0.05.

VII.2.1.2. Construction of LDA models


Using the normalized variables, LDA models capable of classifying the
samples according to the respective enzyme classes were constructed. Thus,
training data set 1 was used in combination with normalization procedures A and
B to constitute matrices M1A and M1B. Taking into account the 18 and 153
predictors generated by normalization procedures A and B, the dimensions of the
M1A and M1B matrices were 36 18 and 36 153, respectively. Similarly, data
set 2 was used to constitute matrices,M2A and M2B, whose dimensions were 72
18 and 72 153, respectively, and evaluation data set 3 was used to constitute

272

Chapter VII. Enzymes

matrices, M3A and M3B, whose dimensions were 42 18 and 42 153,


respectively.
Using training matrix M1A, an LDA model with 12 variables (selected out
of 18), which showed an excellent resolution between all the possible pairs of
categories, was obtained. Using training matrix M1B, an LDA model with 15
variables (selected out of 153) was obtained. This latter model showed a slightly
better W value (0.035) than the previous one (0.043). Then, matrices M2A and
M2B, which were obtained by using detergent bases spiked with enzymes, were
used to evaluate the models. With some exceptions, all the objects of matrices
M2A and M2B were erroneously assigned to the protease category. This revealed a
relevant sample matrix effect, which could be due to the modification of the ion
suppression effect in the mass spectrometer. Ion suppression may depend on both
the concentrations of the enzymes, which was much smaller in the spiked
detergent bases than in the enzyme industrial concentrates, and the saline
contents of the infused solutions, which could be higher in those obtained by
enzyme precipitation from spiked detergent bases.
Therefore, to take into account the sample matrix effect in the design of
the training set, matrices M1A and M2A were jointly used. Analogously, matrices
M1B and M2B were also jointly used to construct another LDA model. Both
models yielded excellent resolution between all the pairs of categories; however,
in comparison with the model obtained using normalization procedure A,
procedure B led to a smaller number of predictors (18 instead of 20), and to a
much lower value of W (0.019 instead of 0.047). Thus, model construction using
normalization by procedure B was selected for further studies.
The effect of requiring an entrance threshold of Fin=0.01 instead of 0.05 in
the stepwise algorithm for variable selection was studied. With Fin=0.01, the four
categories were also very well resolved, W increased only slightly (from 0.019 to

273

Miriam Beneito Cambra

0.022), and the number of predictors selected from the 153 variables initially
established decreased from 18 to 13. The predictors and their standardized
coefficients for the three discriminant functions obtained by using Fin=0.05 and
0.01 are shown in Table VII.2. Using normalization procedure B, the ion
abundance ratios of pairs of amino acids rather than the ion abundances of
individual amino acids are used as predictors.
Table VII.2. Predictors selected and standardized coefficients of the LDA models
constructed by jointly using training matrices M1B (enzyme industrial concentrates)
and M2B (detergent bases spiked with enzyme industrial concentrates) with two
different entrance thresholds.
Fin=0.05
f2
f3
6.34 -0.800

f1

Fin=0.01
f2

Selected variable
Val/Met

f1
3.44

f3

Val/Thr

-2.78

0.715

5.71

1.92

0.51

5.82

Leu+Ile/Met

-2.12

-0.744

-0.22

0.295

1.70

-0.326

Phe/His

3.24

0.805

0.92

-4.10

0.839

1.04

Phe/Lys

-6.78

12.1

3.74

3.02

2.57

3.75

Phe/Arg

-6.43

4.47

-2.02

6.41

0.172

-2.72

Phe /Ser

2.06

0.849

0.070

-1.67

-0.247

-0.237

Phe/Trp

3.08

-3.20

-0.261

-3.43

-2.16

-0.572

Phe/Cys

-1.63

-4.76

-0.424

Phe/Pro

1.78

-1.34

-2.07

-0.927

0.698

-1.22

Glu/His

-1.07

-1.70

3.69

1.85

0.275

4.21

His/Arg

0.95

-2.95

-0.831

-2.47

-0.545

-0.254

Lys/Arg

10.6

-17.0

-5.19

-5.17

-2.41

-3.99

Lys/Trp

-8.26

15.5

2.02

2.42

2.11

0.646

Gly/Trp

-1.21

0.846

0.426

Ser/Thr

0.513

-1.19

0.088

Thr/Trp

5.75

-4.06

-0.255

Tyr/Pro

-0.158

-3.63

0.022

Phe/Thr

2.14

0.290

-0.002

This prevented us from assigning any particular relevance to the discriminant


capability of any individual amino acid; however, the coefficients of Table VII.2

274

Chapter VII. Enzymes

suggested that Phe was important in distinguishing enzyme classes, and that the
pairs Lys/Arg, Lys/Trp, Phe/Lys and Phe/Arg, among others, were also
particularly important.
10

20

Third discriminant function

A
lipases

10
proteases

cellulases

amylases
-10
-60

proteases
lipases
0

cellulases

-10
amylases

-20
-10

-40
-20
0
20
First discriminant function

0
10
20
30
Second discriminant function

C
First discriminant function

Second discriminant function

30

proteases

lipases

20
0
-20

amylases
cellulases

-40

30

10

20
10

0
-10

Fig. VII.5. Score plots on the planes of the first and second (A), and
second and third discriminant functions (B), and on an oblique plane of the
3-D space defined by the three discriminant functions (C). Matrices M1B
(industrial concentrates of enzymes selected for training) and M2B (two
detergent bases spiked with the same industrial concentrates of enzymes)
were jointly used to construct the LDA model. The predictors were
selected using Fin=0.01.

275

Miriam Beneito Cambra

As observed in Fig. VII.5, part A, the variance gathered by discriminant


function 1 was mainly associated with the resolution between cellulases and the
other categories, whereas discriminant function 2 was associated with the
resolution between lipases and the rest of the categories. According to Fig. VII.5,
part B, amylases were resolved with respect to the other categories along
discriminant function 3. As illustrated in Fig. VII.5, part C by using a plane
oblique to the three discriminant functions, all the possible pair of categories
were very well resolved from each other.

VII.2.1.3. Evaluation of the prediction capability of the optimal LDA model


The LDA model obtained by jointly using matrices M1B and M2B for
training (with Fin=0.01) was used to predict the enzyme categories of the objects
of the evaluation set (M3B matrix). The enzymes of all the samples (42 objects),
including the two commercial laundry cleaners (6 objects), were correctly
classified, with assignment probabilities higher than 98%.

VII.3. Classification of enzymes by HPLC-UV-Vis

VII.3.1. Enzyme class identification in cleaning products by hydrolysis


followed by derivatization with o-phthaldialdehyde, HPLC and LDA
Today, enzymes are important components ofmost laundry and
dishwasher cleaners, spot removers and other household products [Olsen, 1998]. In
fact, enzymes for cleaning products constitute the largest division of the world
market for industrial enzymes

[Novozymes, 2002].

Using enzymes, substantial

reductions of washing times and temperatures, with the subsequent savings of


water and energy are achieved. Further, the concentrations of surfactants and
harsh chemicals as alkalis and strong oxidants are reduced

276

[Olsen, 1998],

with the

Chapter VII. Enzymes

additional benefit of a better care of fabrics duringwashing. Mainly proteases,


amylases, lypases and cellulases are used in the formulations. Proteases are used
to remove protein-rich soil stains (as blood and grass), amylases are addressed to
solubilise starch-containing stains, also preventing starch from adhering to
fabrics and dishes, lypases help in removing fat and edible oil stains, and
cellulases are used to remove fuzz and little balls of fibers from the surface of
cotton fabrics. In spite of their interest in the detergent industry, and in
environmental and toxicological studies

[www.heraproject.com],

identification and

quantificationmethods for enzymes in cleaning products have been scarcely


investigated. Enzymes are commonly detected and quantified bymonitoring the
hydrolysis of a substrate, or by precipitation with an antiserum

[Olsen, 1998;

Novozymes, 2002; Dunn, 1971; Kulkarni, 1999; Lorentz, 2000].

Enzymes of different classes largely differ in molecular structure, also


having rather dissimilar amino acid profiles, which can be useful for enzyme
class identification. After hydrolysis, the amino acid concentration profile of the
enzyme can be established by a variety of analytical techniques, including gas
chromatography previous derivatization with ethyl chloroformate
Adelantado, 2002; De la Cruz-Caizares, 2004],
[Rampazzi, 2002],
[Phillips, 1983],

[Gimeno-

or with a silylating reagent

automated ion exchange chromatography previous hydrolysis

and a variety of HPLC methods

[Molnr-Perl, 2000; Molnr-Perl,

2001; Tcherkas, 2001-A; Tcherkas, 2001-B; Garca-lvarez-Coque, 1989; ConchaHerrera, 2007; Hanczk, 2007; Chernobrovkin, 2007; Pereira, 2008].

Among these,

RP-HPLC with pre-column derivatization using either phenylisothiocyanate or


OPA, in the presence of a reagent containing an SH group, is most frequently
used [Molnr-Perl, 2000]. Using OPA, isoindoles, which can be detected by either
UVVis spectrophotometry

[Concha-Herrera, 2007],

2001; Tcherkas, 2001-B; Pereira, 2008]


2001-A],

fluorimetry

[Molnr-Perl,

or electrochemical techniques

[Tcherkas,

are quickly and easily obtained. Among the SH-group-containing


277

Miriam Beneito Cambra

additives, 3-mercaptopropionic acid, NAC


2004; Concha-Herrera, 2007]

[Molnr-Perl, 2001; Concha-Herrera,

and ethanethiol

[Hanczk, 2007],

which yield rather

stable isoindoles, have been recommended. Amino acid concentration profiles of


protein hydrolyzates have been used to classify protein-binding media used in
works of art

[Llet, 2003; Peris-Vicente, 2005],

and rice cultivars

[Wang, 1998].

used to classify tea varieties


[Erbe, 2000].

and LDA

vegetable oils

[Lerma-Garca, 2007],

The contents of free amino acids have been also

[Alczar, 2007]

and to authenticate high quality beer

Multivariate data treatment techniques, including ANN

[Peris-Vicente, 2005; Lerma-Garca, 2007],

[Llet, 2003]

have been used to construct

the models for class prediction.


In this work, we describe a method for the identification of the enzyme
class in raw materials of the cleaning industry and household cleaners by HPLCUV-Vis detection. The enzymes are first precipitated with acetone and
hydrolyzed with HCl, the resulting amino acids are derivatized with OPA in the
presence of NAC, and their concentration profiles are established by RP-HPLC
with UVVis detection. Then, either the normalized peak areas (divided by the
sum of the peak areas of the chromatogram), or ratios of pairs of peak areas, are
used as predictors in the construction of LDA models for enzyme class
prediction. The enzymes used in this work are indicated in Table III.2.

VII.3.1.1. Optimization of OPANAC derivatization and chromatographic


conditions
The conditions for OPANAC derivatization, initially taken from
literature

[Concha-Herrera, 2006],

were further optimized using hydrolyzates of

Alc. First, the OPA concentration was increased, while both an OPA/NAC molar
ratio of 1:2 and an OPANAC/hydrolyzate ratio (v/v) of 10:1 were maintained at
fixed values. The peak areas increased when the OPANAC concentration

278

Chapter VII. Enzymes

increased from 2.5104M

[Concha-Herrera, 2006]

1.25102 M, close to the OPA solubility in water

to 1.25102 M. Then,

[www.cas.org/SCIFINDER],

was

selected. Owing to the increase of the dilution factor, the peak areas were
reduced to ca. 50% when the OPANAC volume was increased from 1 to 2mL,
while maintaining an hydrolyzate volume of 100 L. The amino acid peak areas
also decreased when the hydrolyzate volume was increased to 0.5 mL, which
could be due to a reduction of the reaction yield at the decreasing pH of the
mixture. Thus, further studies were performed with an OPANAC/hydrolyzate
ratio of 10:1 (v/v). Under these conditions, the other enzymes of Table III.2,
section III.3.1.1 also provided satisfactory peak intensities.
To optimize the chromatographic separation of the amino acids, the multisegmented gradient of Concha-Herrera et al.

[Concha-Herrera, 2006]

was initially

used. This consisted of three linear steps where the ACN concentration was
increased as follows: 518.5% (30 min), 18.522% (40 min) and 2227.5% (10
min). However, with this gradient a few peaks overlapped at long retention times.
Then, careful trial-and-error optimization of the multi-segmented gradient was
carried out in order to improve resolution between the critical peak pairs. With
the gradient described in Table VII.3, and as illustrated in Fig. VII.6, all the
amino acid peak pairs, except the Phe/Leu pair, were baseline resolved.
Table VII.3. Optimal multi-segmented gradient accomplished by mixing 5% (A)
and 50% (B) ACN/water solutions, both buffered at pH 6.5 with 5mM sodium
citrate/citric acid.
Time (min)

A(%)

B(%)

100

28

72.5

27.5

36

70

30

42

44.4

55.6

50

42.4

57.6

55

100

279

Miriam Beneito Cambra

Phe + Leu

Thr

30
Ala
Asp

80

20

Val

ACN %

Absorbance at 335 nm (mAU)

120

Glu
40
His

10

Tyr

Ser

Gly

Arg

Ile

Lys

Met

0
0

10

20

30

40 Time (min)

50

Fig. VII.6. Chromatogram of a hydrolyzate of Alc showing the peaks of


the isoindoles of the amino acids and the optimal multi-segmented gradient
(dashed line and Table VII.3). Other conditions as indicated in section
VII.3.1.1.

Also, the Ser peak, which was well resolved in the chromatogram of Fig.
VII.6,was only partially resolved from an unidentified peak (see Fig. VII.6) in
the chromatograms of a few other enzymes. Thus, to construct LDA models, the
Ser peak was not used, and the Phe/Leu peak pair was jointly measured, 13 peak
areas corresponding to 14 amino acids being then selected.

VII.3.1.2. Data matrices and construction and evaluation of the LDA models
In order to reduce the variability associated to the total amount of protein
recovered from the samples and to their hydrolysis, normalized rather than
absolute values of the peak areas were used. For this purpose, two normalization
procedures were tried. In procedure A, the area of each amino acid peak was
divided by the sum of the areas of all the amino acid peaks of the chromatogram.
In procedure B, the area of each amino acid peak was divided by each one of the
280

Chapter VII. Enzymes

areas of the other 12 amino acid peaks; in this way, and taking into account that a
pair of peaks should be considered only once, (1312)/2=78 non-redundant ratios
of peak areas were obtained.
To construct LDA models for enzyme class prediction, the training set
was first constituted by the enzyme industrial concentrates indicated in Table
III.2, section III.3.1.1. Three enzymes from each one of the four enzyme classes
were selected. As indicated in section III.3.3.4, two aliquots of each enzyme were
hydrolyzed and injected; however, only the average of the peak areas of the two
injections was included in the training matrix. In this way, the internal variance
of the categories was reduced, which was important to also reduce the number of
variables selected by the stepwise algorithm during model construction.
Therefore, the training matrices were initially constituted by 12 objects each (3
enzymes 4 enzyme classes 1 average of two hydrolyzates) and either 13 or 78
variables obtained according to normalization procedures A and B, respectively.
The resulting LDA models were used to predict the enzyme class in the
two detergent bases spiked with the four enzymes indicated in Table III.2. Data
obtained from a total of 16 chromatograms were evaluated (4 enzyme industrial
concentrates 2 detergent bases 2 hydrolyzed aliquots of each mixture).
However, the two models showed a poor prediction capability (2530% of
correct assignments for a 95% probability level) which was attributed to the
matrix effect produced by the anionic surfactants and other components present
in large concentrations in the detergent bases. Thus, in order to increase the
prediction capability of the models, data obtained with the spiked detergent
baseswere also included in the training set. Thus, the expanded training matrices
had 20 objects (3 enzyme industrial concentrates plus 2 spiked detergent bases
4 enzyme classes 1 average of two hydrolyzates), and either 13 or 78 predictors
as indicated. Then, the duplicate hydrolyzates of the enzyme industrial

281

Miriam Beneito Cambra

concentrates of Table III.2 which were not included in the training set, plus the
duplicated hydrolyzates of the spiked detergent bases and the two commercial
cleaners, were used to construct the evaluation matrices. Thus, the two evaluation
matrices had 58 objects each (19 industrial concentrates of enzymes plus 8
spiked detergent bases and 2 cleaning products 2 hydrolyzates each).
Using normalization procedure A, an LDA model with 9 variables,
showing an excellent resolution between all the possible pairs of categories (W =
0.032), was obtained. However, only a 30% of the samples of the evaluation
setwere correctly classified by this model. On the other hand, using
normalization procedure B, an LDA model with 8 variables, showing a slightly
better value of W (0.019) than the previous model (0.032) was obtained. Further,
all the 58 samples of the evaluation set were correctly classified, with assignment
probabilities higher than 99%. The predictors and respective standardized
coefficients of the three discriminant functions of this model are shown in Table
VII.4. The use of ratios of areas of peak pairs as predictors, rather than individual
peak areas, prevented from reliably assigning any particular relevance to the
discriminant capability of individual amino acids.
Table VII.4. Predictors and their corresponding standardized coefficients of the
optimal LDA model.
f1

f2

Asp/Val

0.92

0.56

-0.55

Glu/His

0.63

-0.76

1.76

Glu/Ala

0.07

1.04

0.82

His/Thr

0.20

-3.77

2.29

Thr/(Leu+Phe)

1.09

0.25

1.46

Ala/Tyr

-0.45

2.35

-1.70

Ala/(Leu+Phe)

-1.26

0.15

0.15

Gly/Ile

0.97

1.47

0.81

Selected variables

282

f3

Chapter VII. Enzymes

As observed in Fig. VII.7, part A, the variance gathered by discriminant


function f1 was mainly associated to the resolution between the protease class and
the rest of the classes (amylase+lipase+cellulase), whereas discriminant function
f2 explained variance associated to the resolution of the amylase class with
respect to the rest of the classes (protease+cellulase+lipase).
10

cellulases

f2

cellulases

f3
0
lipases

amylases
proteases

-2

proteases
-4
-6

amylases
-10
-8 -6 -4 -2 0

lipases

-8
-10

8 10

10

f2

f1

C
10

f2

cellulases
lipases

0
proteases
amylases
4 2
0 -2
-4 -6
f
3

10
0

f1

Fig. VII.7. Score plots on the planes of the first and second (A), and
second and third discriminant functions (B), and on an oblique plane of the
3D space defined by the three discriminant functions (C) of the LDA
model of Table VII.4.

283

Miriam Beneito Cambra

Finally, according to Fig. VII.7, part B, the lipase class was resolved with
respect to the other classes (protease+cellulase+amylase) mainly along
discriminant function f3. As illustrated in Fig. VII.7, part C by using a plane
oblique to the three discriminant functions, all the possible pairs of classes were
very well resolved.
Then, the peak areas of the most stable amino acids (Ala, Arg, Asp, Glu,
Gly, His, Leu, Lys and Phe) were exclusively used as predictors to construct an
LDA model. In this case, the following four peak area ratios were selected by the
stepwise algorithm: Glu/His, Glu/Ala, His/Ala, Ala/(Leu+Phe). This model gave
W = 0.387, and the score plots along the discriminant functions showed well
resolved classes. The resolution between classes was only slightly lower than that
observed in Fig. VII.7 for the model obtained with the eight predictors of Table
VII.4.

VII.4. Tryptic digests of enzymes, columns comparation

VII.4.1. Comparison of microparticulate and monolithic columns for RP-LC


of tryptic digests of industrial enzymes in cleaning products
Today enzymes are commonly used in cleaning product formulations,
particularly in developed countries, with over half of all detergents presently
available containing enzymes

[Olsen, 1998].

In fact, the detergent industry is the

largest single market for enzymes, constituting 25 - 30% of total sales in the
enzyme market

[Novozymes, 2002].

Enzymes allow a reduction of washing times

and temperatures, jointly with lower consumptions of aggressive chemicals,


which translates into additional environmental benefits and better care of fabrics
during washing [Olsen, 1998; Novozymes, 2002]. An active research area within this
field is the development of enzymes capable of maintaining their activity in

284

Chapter VII. Enzymes

extreme temperatures and high pH values, and in the presence of chelate agents
for calcium ions. For this purpose, several techniques (i.e. DNA technology) are
used [Olsen, 1998; Novozymes, 2002].
Enzymes are used in small amounts (0.4 - 0.8% crude enzyme by weight)
in most formulations [Novozymes, 2002], being by far proteases, lipases, amylases
and cellulases the most commonly used enzyme classes. Proteases are used to
remove protein-rich stains (including blood and grass), amylases are addressed to
solubilize starch-containing stains, also preventing starch from adhering to
fabrics and dishes, lipases help in removing fat and edible oil stains, and
cellulases are used to remove fuzz and little balls of fibres from the surface of
cotton fabrics.
Analytical methods for enzyme identification and quantitation in raw
materials and manufactured products are required in industrial quality control.
Further, enzyme producers and their customers are also interested in
investigating the enzyme market trends, which also requires analytical support.
Finally, analytical methods for enzymes are also needed to assess their potential
impact on water treatment plants, as well as to optimize plant operation.
However, in spite of their interest in the detergent industry, and in environmental
and toxicological studies

[www.heraproject.com],

analytical methods for enzymes

in cleaning products have rarely been investigated. Enzymes are commonly


detected and quantified by monitoring the hydrolysis of a substrate, or by
precipitation with an antiserum
Kulkarni, 1999; Lorentz, 2000].

[Olsen, 1998; Novozymes, 2002; Dunn, 1971;

Other methods are based on complete hydrolysis of

the protein into its constitutive amino acids followed by derivatization and
separation/detection by GC [Gimeno-Adelantado, 2002; De la Cruz-Caizares, 2004;
Rampazzi, 2002]

or HPLC

[Molnr-Perl, 2000; Molnr-Perl, 2001; Tcherkas, 2001-A;

Tcherkas, 2001-B; Garca-lvarez-Coque, 1989; Concha-Herrera, 2007; Hanczk,


2007; Chernobrovkin, 2007; Pereira, 2008].

The resulting amino acid profiles have

285

Miriam Beneito Cambra

been also used to predict the enzyme class [Beneito-Cambra, 2008; Beneito-Cambra,
2009-B];

however, much information about the nature of the protein is lost during

total hydrolysis.
In proteomics, a common approach for protein analysis is the enzymatic
digestion with trypsin. In this way, the resulting peptides, which are much more
specific for protein identification than the amino acid profiles can be studied.
After digestion, profiles are studied by HPLC-MS
Opiteck, 1997-B; Opiteck, 1998].

[Nilsson, 2000; Opiteck, 1997-A;

Although this routine is well-established in

proteomics, it has been not applied to industrial enzymes in detergents.


Further, there is a need to develop highly efficient and/or fast analytical
methods to assess the quality of peptides produced by biotechnological
procedures in terms of identity, content and purity. Within this concern, several
analytical strategies related to column technology have been developed in LC,
including the use of monolithic supports

[Cabrera, 2004; Guiochon, 2007],

columns with fused-core (or core-shell) sub-3 m particles


Marchetti, 2007-A; Marchetti, 2007-B],

packed

[Cunliffe, 2007;

or with sub-2 m particles operating at ultra-

high pressure (UPLC) [Mazzeo, 2005; Guillarme, 2007].


In the present study, several commercial chromatographic supports
(monolithic and particulate columns) are comparatively applied to the LC-UV
analysis of enzymes of the different classes commonly used in the detergent
industry. Using industrial concentrates of the raw enzymes, both the intact
proteins and their tryptic digests were analyzed. A polymeric and a silica
monolithic, and particulate columns, including classical and core-shell particle
technology, were compared. For this purpose, and for each column, peak
capacity, resolution and number of peaks were evaluated. The best column was
also used to analyze tryptic digests of enzymes in spiked detergent bases and
commercial cleaners.

286

Chapter VII. Enzymes

VII.4.1.1. Study of intact enzymes and tryptic digests using a ProSwift


polymeric monolithic column
First, a commercial ProSwift RP polymeric monolithic column was
applied to the monitoring of raw industrial concentrates of enzymes using intact
proteins. This column has been employed for the analysis of proteins and
[Rao, 2006; Causon, 2010-B].

peptides

The gradient elution conditions for intact

proteins were as follows: an isocratic step with 1% ACN for 2 min followed by a
linear gradient up to 75 % ACN in 18 min.

Absorbance

Absorbance

120
80

100

40

50

40

20

0
10

40

20

150

15
20
Time (min)

10

15
20
Time (min)

Fig. VII.8. Chromatograms of intact enzymes using a ProSwift polymeric


monolithic column: (A) protease (Everlase), (B) amylase (Duramyl), (C)
lipase (Lipex) and (D) cellulase (Deterzyme). Elution conditions: isocratic
with 1% ACN for 2 min followed by a linear gradient up to 75 % ACN in
18 min at 1 mL min-1.

As shown in Fig. VII.8, most enzymes gave rise to a single predominant peak;
however, a comparison of enzymes of different classes showed a coelution of the
main peaks for the following enzyme class pairs: proteases and cellulases (main

287

Miriam Beneito Cambra

peak within the ca. 11-12 min range), and amylases and lipases (main peak
within the ca. 13-14 min range). No improvement in the resolution between
enzyme classes was achieved by modifying the elution conditions. Then, the
chromatograms of the intact enzymes are useful to assess the quality of the
protein for any enzyme class; however, chromatography of the intact enzymes
did not provide the necessary information to unequivocally identify unknown
enzymes. For this purpose, application of the tryptic digests of the enzymes was
investigated.

Absorbance

120
80
40
0
5

80

10

15

Absorbance

B
40

0
10

20

30

40

50
Time (min)

Fig. VII.9. Chromatograms of a tryptic digest of BSA using a ProSwift


polymeric monolithic column under different elution conditions: (A) as in
Fig. 1, and (B) isocratic step with 1% ACN for 5 min followed by a linear
gradient up to 40 % ACN in 58 min at 1 mL min-1.

In addition to the enzymes, BSA was also used as a reference protein to check
the performance of the tryptic digestion, as well as a probe to optimize the
288

Chapter VII. Enzymes

separation of the digests. Fig. VII.9, part A shows a chromatogram of a BSA


digest obtained with the same elution gradient employed in Fig. VII.8 for intact
proteins. A number of peptides, most of them partially resolved, were observed.
Optimization of elution conditions to improve resolution was performed. Fig.
VII.9, part B shows the best conditions achieved, selecting 50 min gradients as
compromise between analysis time and peak resolution.

Absorbance

10

20

30

40

50
Time (min)

Fig. VII.10. Chromatograms of a tryptic digest of (A) a protease (Alcalase)


and (B) a lipase (Lipolase) using the ProSwift polymeric monolithic
column under elution conditions as in Fig. VII.9, part B.

According to literature, an increase in the column temperature can lead to


an increase in efficiency of separations of tryptic digests
2010-A].

[Ruta, 2010; Causon,

Thus, a tryptic digest of BSA was chromatographed at several

temperatures (25, 40 and 60 C; chromatograms not shown). At 40 C, the

289

Miriam Beneito Cambra

resolution between the weakly retained peptides decreased, whereas at 60 C a


reduction of peak intensity, which could be due to peptide hydrolysis, was
observed. Then, 25 C was selected for further studies. Under these conditions,
tryptic digests of enzymes belonging to different classes were analyzed. The
chromatograms of a protease and a lipase, showing rather different fingerprints
are given in Fig. VII.10. A series of partially resolved peptides at retention times
below 20 min, were observed for the protease, whereas, the lipase provided a
wide range of satisfactorily resolved peptides. Also, acceptable separations were
achieved with amylases and cellulases (chromatograms not shown); however, as
evidenced, low efficiencies were obtained for the tryptic digests of the four
classes of enzymes. Changes in gradient elution conditions did not lead to
significant improvements in efficiency and resolution, then, other stationary
phases were investigated.

VII.4.1.2. Study of tryptic digests of enzymes using microparticulate and silica


monolithic columns
Chromatographic supports with C18 particulate packings and a silica monolithic
bed (see Table III.4) were investigated. Fig. VII.11 shows the chromatograms of
a tryptic digest of a protease obtained with different columns and using the
gradient elution conditions found in the previous section. Since the columns
differed in their cross section, the flow rate was adapted to maintain a constant
value of the average linear flow velocity. As evidenced, a satisfactory separation
of the tryptic digest of the protease was achieved for all these columns, in
contrast with that obtained for the same enzyme by using the ProSwift column
(Fig. VII.10, part A).

290

Chapter VII. Enzymes

Absorbance

20

*
C

Absorbance

20

0
0

20

40

Time (min)

20

40
Time (min)

Fig. VII.11. Chromatograms of a tryptic digest of a protease (Alcalase)


obtained using different columns: (A) Chromolith, (B) Gemini 5 m, (C)
Gemini 3 m and (D) Kinetex. The flow rate was 1.12, 1.5, 0.4 and 0.4 mL
min-1, respectively. Other elution conditions as Fig. VII.9, part B. The
asterisk indicates a blank peak i.e. a peak not originating from the enzyme
analyte.

In order to evaluate the chromatographic performance of the columns, the


number of resolved peaks, peak capacity (PC) and global resolution (RG) were
obtained by selecting as representative probes a protease (Fig. VII.11) and a
lipase. The PC was experimentally determined using the well-known equation
[Snyder, 1986.]:

PC = 1 + (t g / w)

(E.VII.1)

where w is the average peak width at 4 (13.4% of peak height) in time units
(experimentally measured) and tg is the gradient time. The global resolution, RG,
was also measured as the geometric mean of the resolution between the

291

Miriam Beneito Cambra

consecutive peak pairs. To perform this evaluation unresolved peak pairs or with
RS values 0.5 were excluded.
Fig. VII.12 shows the number of peaks, PC and RG found for a protease
and a lipase using the different columns. As it can be seen for the protease (Fig.
VII.12 part A), the PC values increased in the following order: ProSwift <
Gemini (5 m) < Chromolith ~ Gemini (3 m) < Kinetex, and a similar order
was obtained for the lipase, providing in this case the Chromolith column higher
PC values than the Gemini (3 m) column. A similar trend was observed for the
number of peaks and RG; in both cases, the Kinetex column showed the best
performance.
The differences in number of peaks, PC and RG values among the columns
are a consequence of differences in their morphological features. Comparing
monolithic beds, the results obtained for complex tryptic digests for the ProSwift
column suggest a larger globule/polymer skeleton diameter and/or bed
inhomogeneity than for its silica counterpart (Chromolith Performance RP18),
which translates into lower plate numbers and reduced peak capacities as well as,
along with absence of mesoporous structure, into lower surface area and less
retention of analytes. Silica monoliths show larger porosities than packed beds.
Hence, they exhibit a higher permeability than packed columns. The silica
skeleton in Chromolith is mesoporous and has mean thickness of around 1-3 m
leading to a mass-transfer kinetics faster than that of packed columns with 5-m
particles and comparable to those of columns packed with 34 m particles
[Guiochon, 2007],

in rough agreement with the results shown in Fig. VII.12.

The Kinetex column, packed with 2.6 m core-shell particles (porous shell
0.35 m, fused-core 1.9 m) showed a better performance than both Gemini
columns, packed with conventional 5- or 3-m particles and monolithic
supports.This can be explained by the particular characteristics of the Kinetex

292

Chapter VII. Enzymes

packing, where the particles are constituted by porous outer layer surrounding a
solid non-porous core [Snyder, 1979].

80
Number of peaks

A
60
40
20
0

Peak Capacity

400

300
200
100

Log (Global Resolution)

C
20

10

Fig. VII.12. Evaluation of separation performance of tryptic digests of a


protease (Alcalase, dotted bars) and a lipase (Lipolase, striped bars) using
different columns: (A) number of peaks, (B) peak capacity and (C) log
(global resolution). Elution conditions as indicated in Fig. VII.11.

293

Miriam Beneito Cambra

Absorbance

40

Absorbance

Absorbance

20

0
40

0
0

20

40

60
Time (min)

Fig. VII.13. Chromatograms of tryptic digests of (A) an amylase


(Purastar), (B) a lipase (Lipolase) and (C) a cellulase (Puradax) using the
Kinetex column. Other details as in Fig. VII. 11, part D.

In comparison to conventional particle technology, efficiency is gained due to the


faster mass transfer through the shorter diffusion distance provided by the porous
layer. In addition, the PC values obtained for the Kinetex column were similar to
those found in literature for other shell-core columns (Ascentis), and lower than
those reported for columns packed with sub-2 m particles (Acquity, BEH C18)
operating at much higher pressures (UHPLC technology)

[Ruta, 2010].

The

Kinetex column which showed the best chromatographic performance for tryptic
digests, was chosen for further studies. Chromatograms of a protease, an
amylase, a lipase and a cellulase are shown in Figs. VII.11, part D and Figs.
VII.13, parts A to C, respectively.

294

Chapter VII. Enzymes

VII.4.1.3. Influence of matrixes commonly used in cleaning product


formulations
In order to evaluate the feasibility of the proposed method to analyze
enzymes in cleaning products, two detergent bases containing surfactants and
other reagents commonly employed in the formulation of cleaning products (see
composition in section III.3.4.4) were used. These solutions, spiked with
different classes of enzymes, were treated as indicated in section III.3.4.4 and
injected into the HPLC system. Fig. VII.14, part A shows a representative
chromatogram of a tryptic digest resulting from detergent base II spiked with a
protease (Alcalase). This profile was closely similar to those obtained for
detergent base I spiked with the same enzyme (chromatogram not shown) and by
directly digesting the raw enzyme (Fig. VII.11, part D). Similarly, detergent
bases (I and II) spiked with other classes of enzymes also gave chromatograms
closely resembling those obtained with the corresponding raw enzymes.
Therefore, interference due to the matrix components used to prepare the
detergent bases was not observed. The method was also tested with several
commercial cleaning products containing mainly proteases, which constitute the
most common enzymes used today by the detergent industry [Watson, 2006.]. Fig.
VII.14, part B shows a chromatogram of a liquid detergent containing a protease
(Everlase, as declared by the manufacturer Qumicas Oro, S.A.). Again, a closely
similar profile was obtained for the tryptic digest of the industrial concentrate of
this enzyme (chromatogram not shown). Other commercial household cleaners
containing proteases of unknown origin were analyzed, and matrix interferences
were not evidenced in any case.

295

Absorbance

Miriam Beneito Cambra

40

Absorbance

40

0
0

20

40

60
Time (min)

Fig. VII.14. Chromatograms of tryptic digests of (A) detergent base II


spiked with a protease (Alcalase) and (B) liquid detergent containing
protease (Everlase). Other details as in Fig. VII. 11, part D.

296

CHAPTER VIII

MONOLITHIC COLUMNS

Chapter VIII. Monolithic Columns

VIII.1. Monolithic columns of lauryl methacrylate

VIII.1.1. Photo-polymerized LMA monolithic columns for CEC using LPO


as initiator
Polymer-based monolithic stationary phases have been developed over the
past two decades as an alternative to particle-packed phases for CEC and HPLC.
The advantages of these monolithic supports are very well documented
Lmmerhofer,

2005;

2003],

[Svec,

and include good efficiencies, simplicity of

manufacturing and low back-pressure in HPLC. These phases are usually


prepared by in situ free-radical polymerization of a mixture containing one or
more functional monomers, including a crosslinker, a porogenic solvent and an
initiator. Heat

[Peters, 1997; De Vries, 2008; Peters, 1998-A; Yu, 2002; Eeltink, 2005]

and UV irradiation

[Peters, 1998-A; Eeltink, 2005; Geiser, 2007; Ngola, 2001]

are the

most common ways of initiating polymerization, while other techniques, such as


microwaves

[Faure, 2007],

[Beiler, 2007]
Mirapeix,

-radiation

and chemical agents

2008-A;

Cant-Mirapeix,

[Zhang, 2008; Sfrny, 2005],

electron beam

[Bandari, 2007; Holdvendov, 2003; Cant2008-B]

have been scarcely employed.

Advantages of photo-initiation are speed and easy selection of polymerization


regions by using masks, which is particularly important in relation to the
manufacturing of microfluidic chips.
Acrylate and methacrylate-based materials have been proved to be
excellent as stationary phases, with outstanding chemical stability over a broad
pH-range

[Peters, 1997; De Vries, 2008; Peters, 1998-A; Yu, 2002; Eeltink, 2005;

Sfrny, 2005; Beiler, 2007; Cant-Mirapeix, 2008-C; Throckmorton, 2002; DelaunayBertoncini, 2004; Augustin, 2006].

Several authors have described the preparation of

monoliths using UV irradiation, mostly in the presence of either AIBN


1998-A;

Eeltink,

2005;

Geiser,

2007;

Ngola,

299

2001;

Cant-Mirapeix,

[Peters,
2008-C;

Miriam Beneito Cambra


Throckmorton, 2002; Delaunay-Bertoncini, 2004; Augustin, 2006]

or 2,2-dimethoxy-

2-phenylacetophenone [Augustin, 2008; Ro, 2004; Lee, 2004; Huo, 2007] as initiators.
LPO, as other diacyl peroxides, constitutes a source of free-radicals, when
decomposed by thermolysis, irradiation or activation by tertiary amines [Gu, 2007;
Redington, 1948; Sanchez, 1996; ODriscoll, 1960].

This compound has been

employed in the preparation of methyl methacrylate polymers by thermal


initiation

[Denisov, 2003; Bevington, 2004],

and recently, we have described its use

in the preparation of LMA-based monolithic columns for CEC also by thermal


polymerization

[Gao, 2005].

The monoliths obtained with this initiator provided

better permeability and a finer control of the pore size over the studied range of
1,4-butanediol/1-propanol ratio, than columns prepared with AIBN. This
suggested that the combination of LPO and UV irradiation could also be an
attractive way for the fast preparation of LMA-based monoliths.
In this work, the preparation of LMA-based monolithic columns for CEC
by UV irradiation using LPO as an initiator is described. In order to obtain
satisfactory column performances, the composition of polymerization mixture
(i.e. ratios of monomers/porogens and monomer/crosslinker, and the composition
of the porogenic solvent) was optimized. SEM photographs were used to
characterize the morphology of the resulting monoliths, and the CEC
performance of different columns was evaluated by measuring the retention
factors and efficiencies of test mixtures of non-charged solutes. Photopolymerized LMA stationary phases were compared with those prepared by
thermal initiation. These columns were also compared with those prepared using
the more common AIBN instead of LPO as an initiator.

300

Chapter VIII. Monolithic Columns

VIII.1.1.1. Preparation and characterization of columns photo-initiated with


LPO
The conditions to prepare photo-polymerized LMA-based monoliths were
adapted from our previous work, where CEC columns were thermally
polymerized using LPO as initiator [Gao, 2005]. Initially, the selected composition
of the polymerization mixture was 40 wt% monomers (59.8 wt% LMA, 39.9
wt% EDMA and 0.3 wt% META) and 60 wt% porogens (17 wt% 1,4-butanediol
and 83 wt% 1-propanol) in the presence of a 0.3 wt% LPO. However, when a
PAH test mixture was injected in this monolith (column C1), wide peaks of
components

with

several

overlappings

(naphthalene/fluorene

and

pyrene/benz[a]anthracene peak pairs) were evidenced (Fig. VIII.1). These peaks


showed much lower efficiencies than those reported for a thermally polymerized
LMA column [Gao, 2005].

Absorbance (mAU)

60

40
3
4

20

5+6
7

0
1

4
Time (min)

Fig. VIII.1. Electrochromatogram of a PAH test mixture obtained with an


LMA-based monolithic column photo-polymerized with LPO (column C1
of Table VIII.1) and SEM photograph of the monolith (inset). CEC
conditions: mobile phase, 80:20 v/v ACN/aqueous 5 mM Tris (pH = 8.0);
applied voltage, 10 kV; UV detection at 254 nm. Peak identification: (1)
thiourea, (2) naphthalene, (3) fluorene, (4) anthracene, (5) pyrene, (6)
benz[a]anthracene and (7) benzo[k]fluoranthene.

301

Miriam Beneito Cambra

It was attributed to that the photo-polymerized columns yielded monoliths with


larger pores and globules compared with those initiated thermally, which is
consistent with the literature

[Peters, 1998-A; Cant-Mirapeix, 2008-D].

This was

also corroborated by the SEM pictures, where the photo-polymerized column C1


showed larger flow-through pores and globule sizes (inset of Fig. VIII.1) than a
thermally initiated column obtained with the same polymerization mixture

[Gao,

2005].

In order to improve the CEC properties of the photopolymerized LMA


monoliths, the influence of the ratios of monomers/porogens and 1,4butanediol/1-propanol on the porosity and performance of the columns was first
studied (Table VIII.1). For this purpose, SEM pictures were obtained, and a
mixture of PAHs was used to measure retention and efficiency values (by giving
the minimum plate height, Hmin obtained from van Deemter plots). The ratio of
monomers/porogens was varied from 30:70 to 60:40 wt/wt at several 1,4butanediol percentages. For 17 wt% 1,4-butanediol and a 30:70 wt/wt ratio of
monomers/porogens, the polymerization inside the capillaries was not produced.
Stable monoliths were obtained with 40:60 and 50:50 ratios; however, for a
60:40 ratio and at all the studied 1,4-butanediol percentages, the bed permeability
was significantly reduced, thus, leading to column blockage. At 17 wt% 1,4butanediol, when ratio of monomers/porogens was increased from 40:60 (column
C1) to 50:50 (column C2), efficiency improved (see Table VIII.1); however the
pyrene/benz[a]anthracene pair was still not resolved by column C2. As shown in
Table VIII.1, the k-values increased from column C1 to column C2, while the
flow rate (u) remained almost the same. The increase in k-values can be
explained by the smaller globule structure of column C2 when compared with
column C1, and therefore, the column C2 gave higher surface/column volume
ratio.

302

303

Flow rates and retention measured at 5 kV. Mobile phase, 80:20 v/v ACN:5 mM Tris (pH = 8.0)

Weight percentages.

Solvents: pore-forming solvents.

columns with LPO as initiator and their CEC properties

Table VIII.1. Composition of the polymerization mixtures used for the preparation of photo-polymerized LMA-based monolithic

Chapter VIII. Monolithic Columns

Miriam Beneito Cambra

On the other hand, the similar u-values suggested similar flow-through pore sizes
for columns C1 and C2, which was consistent with their SEM pictures (data not
shown). The similarity of the pore sizes could be due to the excessively large 1,4butanediol content employed in the preparation of these two monoliths.

Fig. VIII.2. SEM photographs of LMA monoliths photo-polymerized with


LPO prepared with 10:90 wt/wt 1,4-butanediol/1-propanol, at several ratios
of monomers/porogens (wt/wt): 30:70 (column C3) (A), 40:60 (column
C4) (B) and 50:50 (column C5) (C). Part (D) shows a monolith prepared
with a 40:60 wt/wt ratio of monomers/porogens and a 6:94 wt/wt ratio of
1,4-butanediol/1-propanol

(column

C7).

Other

details

about

the

composition of the columns are given in Table VIII.1.

The influence of the ratio of monomers/porogens on the CEC properties


was also studied at 1,4-butanediol contents lower than 17 wt% (columns C3C8).
As shown in Table VIII.1, at 10 and 6 wt% 1,4-butanediol in the porogenic
mixture, and when the content of this mixture was reduced (from 30:70 to a
50:50 ratio of monomers/porogens), an increase in k- and a decrease in u-values

304

Chapter VIII. Monolithic Columns

were observed. This trend was consistent with the SEM pictures of these
monoliths. As a representative example, monoliths photo-polymerized with 10
wt% 1,4-butanediol showed a significant decrease in both flow-through pore and
globule sizes when the ratio of monomers/porogens was increased from 30:70 to
50:50 (columns C3C5) (Fig. VIII.2, parts A to C).

Hmin (m)

20

10

0
0

0.5

1.5

60

Hmin (m)

B
40

20

0
0

0.5

u (mm s-1)

1.5

Fig. VIII.3. Van Deemter plots of LMA monolithic columns


photopolymerized with LPO and using different polymerization mixtures
(wt/wt ratios): (A) 40:60 monomers/porogens and 10:90 1,4-butanediol/1propanol (column C4); (B) 50:50 monomers/porogens and 6:94 1,4butanediol/1-propanol (column C8); other details about the composition of
the columns are given in Table VIII.1. Compounds: () naphthalene, ()
anthracene, and () benzo[k]fluoranthene. CEC conditions: mobile phase,
80:20 v/v ACN: 5 mM Tris (pH=8.0); UV detection at 254 nm; injection, 5
kV for 3 s.

305

Miriam Beneito Cambra

Regarding to the efficiency, at 10 and 6 wt% 1,4-butanediol, a 30:70 wt/wt


ratio of monomers/porogens (columns C3 and C6) provided the worst Hmin
values. The best efficiencies for 10 and 6 wt% 1,4-butanediol were achieved at
ratios 40:60 (column C4) and 50:50 (column C8), respectively. Figure VIII.3
shows the van Deemter plots obtained for these columns for naphthalene,
anthracene and benzo[k]-fluoranthene. The column C4 gave Hmin values
comprised 8.911.1 m (at optimum u-values 0.650.67 mm/s), whereas for the
column C8, the Hmin values ranged between 18.622.7 m (u-values 0.300.33
mm/s) (see Table VIII.1). Additionally, this latter monolith showed higher mass
transfer contributions (C-term) (26.547 ms) than those obtained with the column
C4 (6.813 ms). The separation of the PAH test mixture on columns prepared
with these two monoliths was examined. In both cases, all the analytes were well
resolved, giving the monolith prepared with a 40:60 ratio (column C4) the
shortest analysis time (see Fig. VIII.4, parts A and B).
Next, the influence of the content of 1,4-butanediol in the porogenic
solvent was studied at a given ratio of monomers/porogens. At 40:60 wt/wt
monomers/porogens, by decreasing the 1,4-butanediol from 17 (column C1) to 6
wt% (column C7), the flow rates did not change significantly, but the k-values
progressively increased. This retention behavior was attributed to the reduction
of the globule size, which was corroborated by the SEM pictures (see inset of
Fig. VIII.1 and Fig. VIII.2, parts B and D for columns C1, C4 and C7,
respectively). A reduction of the globule size produced by a reduction of the
porogenic solvent content was also reported for LMA-based monoliths prepared
by thermal polymerization in the presence of LPO as initiator

[Gao, 2005].

The

increase in retention was also observed at 50:50 wt/wt monomers/porogens,


being more pronounced than 40:60 wt/wt monomers/porogens (see columns C2,
C5,

C8

compared

with

columns

306

C1,

C4,

C7).

At

50:50

wt/wt

Chapter VIII. Monolithic Columns

monomers/porogens, with decreasing 1,4-butanediol, u decreased from column


C2 to C5, for later did not change significantly for column C8. This behavior can
be explained as follows. The reduction of 1,4-butanediol in polymerization
mixtures prepared at 50:50 wt/wt monomers/porogens led to monoliths with
small macropore and globule sizes, which translated in a reduction in u and
increase in k-values. However, at 6 wt% 1,4-butanediol percentage (column C8),
no substantial changes in u and an increase in k-values was observed. It can be
explained by considering that this polymerization media produced a decrease in
globule size and non-significant variations in macropore size.
The influence of the 1,4-butanediol percentage in the mixture of porogenic
solvents on the CEC separation performance was also evaluated. When the PAH
test mixture was injected using columns prepared with a 40:60 wt/wt ratio of
monomers/porogens, the decrease of the 1,4-butanediol percentage from 17 wt%
(column C1, Fig. VIII.1) to 10 wt% (column C4, Fig. VIII.4, part A) led to an
improvement of the resolution between all the successive analyte pairs. The
column prepared with 6 wt% 1,4-butanediol (column C7, Fig. VIII.4, part C)
also provided baseline resolution of analytes; however, it showed lower
efficiency than the column C4. Thus, this monolithic bed, which represented the
best compromise between resolution and analysis time, was selected for further
studies.
Changes in the monomer to crosslinker ratio have been proved to have
significant effects on monolith porosity
Okay, 2000].

[Svec, 1995-A; Svec, 1995-B; Viklund, 1996;

Thus, the LMA:EDMA ratio was increased from 40:60 to 50:50,

60:40 and 70:30 wt/wt (columns C9, C4 and C10, respectively). When 40:60 was
used the columns exhibited a large flow resistance, which caused column
blockage.

307

Miriam Beneito Cambra


30
Absorbance (mAU)

4
20
6

10

0
0
Absorbance (mAU)

40

8
B

30
3

20

5 6

10

0
0

12

16

Absorbance (mAU)

60
4
40

1
3

20
5 6

0
0

8
Time (min)

Fig. VIII.4. Electrochromatograms of a PAH test mixture obtained using


LMA columns photo-polymerized with LPO and using different
polymerization mixtures (wt/wt ratios): (A) 40:60 monomers/porogens and
10:90

1,4-butanediol/1-propanol

(column

C4);

(B)

50:50

monomers/porogens and 6:94 1,4-butanediol/1-propanol (column C8); and


(C) 40:60 monomers/porogens and 6:94 1,4-butanediol/1-propanol
(column C7). Other details about the composition of the columns are given
in Table VIII.1. CEC conditions and peak identification are as in Fig.
VIII.1.

308

Chapter VIII. Monolithic Columns

When LMA:EDMA ratio was increased from 50:50 (column C9) to 60:40
(column C4), a decrease in retention without any separation improvement was
observed (see Table VIII.1). Finally, with 70:30 (column C10), overlapping of
the naphthalene/fluorene and pyrene/benz[a]anthracene peak pairs was produced.
This was consistent with the SEM pictures of these columns, which showed how
the decrease of the crosslinker concentration from 60 to 30 wt% led to monolithic
beds with larger globules (pictures not shown). This was in agreement with other
studies reported in the literature, where a decrease in the crosslinker percentage
was also accompanied by an increase in the average pore size [Svec, 1995-A; Svec,
1995-B; Okay, 2000].

This behavior can be explained as follows. When the

LMA/EDMA ratio was progressively increased from 50:50 to 70:30 wt/wt, the
hydrophobicity of the monomer mixture was increased, which resulted in an
earlier phase separation. The aggregates preferentially tended to swell with the
LMA monomers than with porogenic solvent. Overall, the globules and voids
formed in this system will be larger. It should translate in a reduction both in kvalues and efficiency along this series. However, similar Hmin values were
observed from column C9 to C4, whereas as expected an increase in Hmin was
observed for column C10. As shown in Table VIII.1, the LMA:EDMA ratio of
60:40 wt/wt (column C4) provided the best Hmin values for all the PAHs of the
test mixture, thus being this ratio selected for the studies that followed. These
efficiencies are comparable with the performance of microparticulate columns
packed with 5 m silica particles [Vlakh, 2007; Eeltink, 2004].
Using LPO, the photo-polymerized LMA-based monolithic columns were
also compared with those prepared by thermal polymerization. For this purpose,
the 1,4-butanediol and porogenic solvent contents, which were found to be
optimal for each polymerization processes were used. The monoliths of the two
types showed similar efficiencies for naphthalene, i.e. Hmin

309

naphthalene

= 11.1 and

Miriam Beneito Cambra

9.5 m for photo-polymerized and thermal initiated columns, respectively, but


the former showed much lower k-values than the latter (kpyrene = 1.76 and 4.58,
respectively).
A comparison in terms of efficiency with monolithic columns made with
either LMA or other similar bulk monomers (i.e. LA) was also performed.
Regarding to LMA columns initiated thermally with AIBN, the Hmin values found
were similar to the 915 m for alkyl benzenes (at optimum flow rates 0.51.0
mm/s) obtained by Buszewski et al.

[Buszewski, 2004].

Our efficiencies were also

comparable with the values of 513 m for PAH compounds (flow rates: 1.01.5
mm/s) obtained with photo-initiated LA monoliths with AIBN

[Geiser, 2007].

However, the plate heights achieved were slightly higher than those obtained for
LA columns chemically initiated with LPO

[Buszewski, 2004],

where the Hmin

values for PAH compounds ranged from 2.6 to 5.3 m (flow rates: 0.51.5
mm/s).
Table VIII.2. Reproducibility of CEC properties of LMA-based monolithic
columns photo-polymerized with LPOa
Column-to-column (n=3)
Parameter

Batch-to-Batch (n=3)

Mean

RSD (%)

Mean

RSD (%)

u (mm/s)

1.25

4.5

1.29

5.4

kanthracene

1.08

2.8

1.00

6.3

Hminb (m)

11.0

2.1

13.1

6.0

Polymerization mixtures prepared with 40 wt% monomers (59.8 wt%


LMA, 39.9 wt% EDMA and 0.3 wt% META) and 60 wt% porogens (10
wt% 1,4-butanediol and 90 wt% 1-propanol); LPO, 0.3 wt%. Applied
voltage, 15 kV; other conditions as in Fig. VIII.2.

Hmin values obtained for naphthalene.

To estimate the reproducibility of photo-polymerized columns initiated


with LPO, three independent batches of three columns each were prepared. As it

310

Chapter VIII. Monolithic Columns

can be observed in Table VIII.2, satisfactory reproducibilities were achieved for


all the tested parameters, with RSD values comprised between 2.1 and 4.5% for
column-to-column, and between 5.4 and 6.3% for batch-to-batch. These
reproducibilities were similar to those reported for thermally polymerized
monoliths using also LPO as initiator (<5.3%) [Gao, 2005].

VIII.1.1.2. Comparison of LPO and AIBN as initiators for photopolymerization


Since AIBN is used as initiator for UV polymerization in most of the
studies reported in the literature, the CEC performance of columns photopolymerized using either LPO or AIBN was next compared. The features of
columns prepared by using the same ratios of monomers/porogens (40:60 wt/wt)
and LMA/EDMA (60:40 wt/wt), and the same 1,4-butanediol range in the
porogenic solvent (176 wt%), are shown in Tables VIII.1 and VIII.3. Using
PAHs for testing, the columns photo-initiated with AIBN at 17 wt% 1,4butanediol in the porogenic solvent showed better efficiencies than the
corresponding columns obtained using LPO (column C1, Table VIII.1). At 6
wt% 1,4-butanediol, the columns polymerized with AIBN gave slightly better
efficiencies (see Table VIII.3 and column C7, Table VIII.1) and comparable
separations were achieved for both initiators (data not shown). As deduced from
Table VIII.3, the optimal efficiency for the monoliths photo-initiated with AIBN
was found at 10 wt% 1,4-butanediol in the porogenic mixture. Figure VIII.5
shows the electrochromatogram obtained with the column that provided the best
Hmin using AIBN. As it can be observed, the solutes of the PAH test mixture were
resolved at 10 kV with similar peak efficiencies as those obtained with the
optimal monolith initiated with LPO (see Fig. VIII.4, part A), but within a
longer analysis time.

311

Miriam Beneito Cambra


Table VIII.3. CEC properties of LMA-based monolithic columns prepared from
mixtures with different 1,4-butanediol/1-propanol ratios using AIBN as initiatora)

a)

The polymerization mixtures contained 40:60 wt/wt monomers/porogens and 60:40


LMA/EDMA.
b)
Weight percentages.
c)
Flow rates and retention measured at 5 kV. Mobile phase, 80:20 v/v ACN: 5 mM Tris
(pH = 8.0).

Absorbance (mAU)

80

40

3
6

2
0
2

10
Time (min)

Fig. VIII.5. Electrochromatogram of a PAH test mixture obtained with an


LMA-based monolithic column photo-polymerized with AIBN, and SEM
photograph of the monolith (inset). Polymerization mixture: 40 wt%
monomers (59.8 wt% LMA, 39.9 wt% EDMA and 0.3 wt% META) and 60
wt% porogens (10 wt% 1,4-butanediol and 90 wt% 1-propanol). CEC
conditions and peak identification are as in Fig. VIII.1.

As shown in Tables VIII.1 and VIII.3 for LPO and AIBN, respectively,
at the optimal ratios of monomers/porogens (40:60 wt/wt) and LMA/EDMA

312

Chapter VIII. Monolithic Columns

(60:40 wt/wt), when the 1,4-butanediol contents was decreased from 17 to 6


wt%, the u-values did not varied significantly. As also shown in these tables, the
LPO photo-initiated monoliths gave higher u-values than those initiated with
AIBN. Concerning to retention, for 10 and 17 wt% 1,4-butanediol, the monoliths
photo-polymerized with AIBN gave higher k-values than those obtained with
LPO. This could be explained by the presence of a higher number of micropores
in the globule structure of the AIBN columns, and consequently, by a higher
surface area. This was confirmed by the SEM pictures, which showed smaller
globules in beds made with AIBN than in those prepared with LPO (see for
instance inset of Figs. VIII.5 and VIII.2, part B for columns prepared with 10
wt% 1,4-butanediol).
The performance of these monolithic stationary phases as RP packings
was also evaluated using alkyl benzenes and a mixture of basic compounds (Figs.
VIII.6 and VIII.7, respectively). From Fig. VIII.6, it can be seen that the
column initiated with LPO showed a fast baseline separation of alkyl benzenes in
less than 4 min. Similar theoretical plates (7.118.3 m) were achieved with this
monolithic column than the monolithic column initiated with AIBN, which
yielded 8.216.4 m for a separation under 6 min.
The analysis of basic compounds in HPLC commonly undergoes of peak
broadening and tailing due to the interaction of these compounds with residual
silanol groups on RP columns

[Cant-Mirapeix, 2009-B].

Figure VIII.7, part A

shows the separation of five basic compounds in less than 12 min with column
efficiency of up to 20 m, whereas the column initiated with AIBN (Fig. VIII.7,
part B) as in Fig. VIII.6 showed longer analysis times with theoretical plates ca.
24 m. Similar efficiencies were achieved for RP columns, in particular XTerra
RP18 and XBridge C18, two columns with low-silanophilic activity
2003],

[McCalley,

with theoretical height plates ranged between 1625 m. In any case, it

313

Miriam Beneito Cambra

can be observed that symmetric peaks were obtained in CEC due to suppressed
electrostatic interaction between the neutral solutes and the positively charged
monolithic surface, then; these stationary phases were suitable for the analysis of
basic compounds.

3
Absorbance (mAU)

0
Absorbance (mAU)

3
2

7
5
1

1
0
0

5
6
Time (min)

Fig. VIII.6. Electrochromatograms of a mixture of alkylbenzenes obtained


by using LMA monoliths photo-polymerized with LPO (A) and AIBN (B)
as initiator. The composition of the polymerization mixtures is as in Figs.
VIII.4, part A and VIII.5, respectively. CEC conditions: mobile phase,
70:30 v/v ACN: 5mM Tris (pH = 8.0); UV detection at 214 nm; applied
voltage, 25 kV; injection, 5 kV for 3 s. Peak identification: (1) thiourea, (2)
toluene, (3) ethylbenzene, (4) n-propylbenzene, (5) n-butylbenzene, (6) npentylbenzene and (7) n-hexylbenzene.

314

Chapter VIII. Monolithic Columns

Absorbance (mAU)

1
4

3
2

Absorbance (mAU)

10

20

30

4
1
0
0

10

20

30
40
Time (min)

Fig. VIII.7. Electrochromatograms of a mixture of basic compounds


obtained by using LMA monoliths photo-polymerized with LPO (A) and
AIBN (B) as initiator. The composition of the polymerization mixtures is
as in Figs. VIII.4, part A and VIII.5, respectively. CEC conditions:
mobile phase, 40:60 v/v ACN: 5mM Tris (pH=8.0); UV detection at 254
nm; applied voltage, 10 kV; injection, 5 kV for 3 s. Peak identification: (1)
thiourea, (2) pyridine, (3) aniline, (4) 4-nitroaniline, (5) 2,4,6trimethylaniline, (6) N,N-dimethylaniline.

315

Miriam Beneito Cambra

VIII.2. Comparison of photo-initiators for methacrylate monolithic


columns

VIII.2.1. Comparison of photo-initiators for the preparation of methacrylate


monolithic columns for capillary electrochromatography
In the last years, monolithic columns have attracted considerable attention
due to their properties such as the ease of preparation, the good efficiencies, the
speed of chromatographic separations at low back-pressure and non-requirement
of frits, constituting a competitive technology to the conventional particlepacked
phases used in HPLC and CEC

[Svec, 2003; Legido-Quigley, 2003].

polymer-based monoliths are prepared in situ by thermal

Organic

[Peters, 1997; Peters,

1998-A; Eeltink, 2005; Geiser, 2007],

photoinduced

Delaunay-Bertoncini, 2004; Faure, 2007]

or chemical [Cant-Mirapeix, 2008-A; Cant-

Mirapeix, 2009-A]

[Geiser, 2007; Ngola, 2001;

polymerization of a mixture that contains one or more

functional monomers, a cross-linking agent, a porogenic solvent and an initiator.


Photo-initiation process is faster than other initiation ways and provides a
spatial and temporal control of the polymerization reaction, since the radiation
can be focused on a location of interest and stopped at a specified time. Thus, UV
irradiation constitutes the most common way of photo-polymerization

[Geiser,

2007; Ngola, 2001; Delaunay-Bertoncini, 2004; Faure, 2007; Yu, 2002; Augustin, 2006],

although other sources of electromagnetic radiation (e.g., electron beam, -rays


or microwaves [Bandari, 2007; Sfrny, 2005; Zhang, 2008]) have also been used to
induce the polymerization reaction.
Typically, the morphological properties of polymeric monoliths are
controlled by the type of porogenic solvent selected and the amounts of both
porogen and crosslinker used [Peters, 1997; Peters, 1998-A; Eeltink, 2005; Svec, 1995B; Eeltink, 2007].

In contrast, very less attention has been paid to the effects of

316

Chapter VIII. Monolithic Columns

other variables such as type of free-radical initiator and its concentration, which
both affect the kinetics of the free-radical polymerization. Consequently, these
will affect the morphology of the resulting polymer.
Most literature concerning UV-initiated polymeric-based monoliths has
reported the use of AIBN

[Geiser, 2007; Ngola, 2001; Delaunay-Bertoncini, 2004;

Faure, 2007; Yu, 2002; Augustin, 2006; Augustin, 2008; Bernab-Zafn, 2009]

or

DMPA [Ro, 2004; Lee, 2004; Huo, 2007; Gu, 2007] as initiators, whereas others, such
as BPO and LPO

[Chen, 2007; Du, 2007; Bevington, 2004; Cant-Mirapeix, 2008-D]

have been less employed.


In this work, the preparation of LMA-based monoliths for CEC by photopolymerization using several free-radical initiators is described. Fig. VIII.8
shows the decomposition schemes for the four initiators investigated (AIBN,
DMPA, BPO and LPO). Using a 1,4-butanediol/1-propanol mixture as porogenic
solvent, the influence of each type of initiator and its content in the
polymerization mixture was comparatively investigated. The morphology of the
resulting columns was characterized by SEM images, whereas their CEC
properties were evaluated by measuring retention factors, efficiencies and
resolution of a test mixture of non-charged solutes. The run-to-run and columnto-column reproducibilities of CEC columns were also studied.

VIII.2.1.1. Preparation and characterization of LM columns photo-initiated


with AIBN or DMPA
A series of LMA-based monolithic columns photo-initiated with AIBN
was prepared by modifying the percentage of this initiator in the polymerization
mixture. The composition of this mixture was taken from a previous work
[Bernab-Zafn, 2009].

It contained 40 wt% monomers (59.8 wt% LMA, 39.9

wt% EDMA and 0.3 wt% META) and 60 wt% porogens (10 wt% 1,4-butanediol

317

Miriam Beneito Cambra

and 90 wt% 1-propanol). The AIBN percentage was varied from 0.05 to 0.8 wt%
in the polymerization mixture. As it can be seen in Table VIII.4, minor changes
in efficiency (minimum theoretical plate, Hmin obtained from Van Deemter plots)
were observed for the range of AIBN tested. On the other hand, small variations
in u-values were observed with increasing AIBN content, while the k-values
increased from 0.05 to 0.30 wt% AIBN, followed by a decrease at contents
higher than 0.6 wt% (see Table VIII.4).

N
N

OCH3

N2

OCH3

OCH3

OCH3
OCH3

C OCH3

CH3

OCH3
O

O
O

CH3(CH2)9CH2

CH2(CH2)9CH3

CH3(CH2)9CH2

Fig. VIII.8. Photolytic cleavage schemes of four investigated photoinitiators: AIBN (A), DMPA (B), BPO (C) and LPO (D).

In order to understand this behaviour, SEM pictures of these monoliths


were taken. Thus, from 0.05 (Fig. VIII.9, part A) to 0.30 wt% AIBN (Fig.
VIII.9, part B), the characteristic dimensions of the globules are decreased,
which produce an increase in surface area, and consequently, larger retention.

318

Chapter VIII. Monolithic Columns

Fig. VIII.9. SEM photographs of LMA monoliths photo-polymerized with


AIBN at several contents: 0.05 wt% (A), 0.3 wt% (B) and 0.8 wt% (C).
Part (D) shows a monolith photo-polymerized with 0.05 wt% DMPA.
Polymerization conditions: 40 wt% monomers (59.8 wt% LMA, 39.9 wt%
EDMA and 0.3 wt% META) and 60 wt% porogens (10 wt% 1,4butanediol and 90 wt% 1-propanol); photo-polymerization at 0.90 J/cm2 for
10 min. The bar lengths stand for 3.0 m.

However, SEM photograph of monolith photo-initiated with 0.8 wt%


AIBN (Fig. VIII.9, part C) did not show significant changes in globule size
compared to Fig. VIII.9, part B. It would be reasonable to assume that lower
concentrations of initiator (i.e., 0.05 wt%) would lead to lower number of nuclei
and thus polymers with slightly large globules (Fig. VIII.9, part A). Increasing
the initiator concentration increases the polymerization rate, which would
produce the formation of a larger number of free-radicals and growing nuclei,
which would result in smaller globules.

319

320

Flow rate and retention measured at 5 kV. Mobile phase, 80:20% (v:v) ACN:water (5mM Tris buffer pH 8.0).
Mean and RSD values (%) obtained for run-to-run repeatability with n = 10 at optimal initiator content.
c
Mean and RSD values (%) obtained for column-to-column repeatability with n = 3 at optimal initiator content.
d
Global resolution as geometrical mean of resolution between the consecutive PAH pairs.

Tabla VIII.4. Electrochromatographic properties of LMA-based monolithic columns prepared with several photo-initiators.

Miriam Beneito Cambra

321

Flow rate and retention measured at 5 kV. Mobile phase, 80:20% (v:v) ACN:water (5mM Tris buffer pH 8.0).
Mean and RSD values (%) obtained for run-to-run repeatability with n = 10 at optimal initiator content.
c
Mean and RSD values (%) obtained for column-to-column repeatability with n = 3 at optimal initiator content.
d
Global resolution as geometrical mean of resolution between the consecutive PAH pairs.

Table VIII.4. Continue.

Chapter VIII. Monolithic Columns

Miriam Beneito Cambra

It was in agreement with Fig. VIII.9, part B and C, and with the increase in
retention observed from 0.05 to 0.3 wt% AIBN. However, a reduction in kvalues was observed at larger AIBN contents (Table VIII.4). Several possible
explanations could be given for this irregular retention behaviour. The effect of
initiator concentration on the final globule size is likely to be dependent on the
competition between the rates of nucleation and termination. With increasing
AIBN content, an increase in termination step could be favoured, which leads to
the formation of less chains able to grow a sufficient length either to become
nuclei or to stabilize the growing nuclei. It could reduce the available free sites
for interactions within the polymer, and would produce a decrease in retention of
analytes

[Paine, 1990; Lai, 1997].

Another potential explanation is taking into

account the screening effect of initiator, as has been reported [Lai, 1997; Gruber,
1992; Guthrie, 1986; Hutchison, 1973; Moad, 1995].

At large initiator concentrations,

the distribution of initiating species will become less uniform, being the majority
of these concentrated in a relatively narrow region close to the radiation source.
Thus, this surface layer will effectively screen the deeper layer of initiating
species against UV radiation. This non-uniformity will give rise to an overall
reduction in the rate of polymerization, and consequently, monoliths with less
binding capacity.
The possibility of employing DMPA as free-radicals source by photoinitiation instead of AIBN was also evaluated. Different amounts of DMPA
ranging between 0.02 and 0.2 wt% were tested (Table VIII.4). Concentrations
above 0.15 wt% led to a fast polymerization of mixture at room temperature,
making a proper filling of the capillaries unfeasible. Along the DMPA range,
similar flow rates and Hmin values were obtained, while the k-values decreased
from 0.02 to 0.05 wt% DMPA, remaining practically constant at 0.15 wt%.
However, SEM pictures of these monoliths did not show significant differences
in morphology. A representative example of porous structure of these monoliths
is given in Fig. VIII.9, part D.

322

Chapter VIII. Monolithic Columns

The retention behaviour could be explained by taking into account the


above considerations. Additionally, one should keep in mind that the initiation
efficiency of generated radicals depends on the type of photo-initiator. In fact,
each initiator will have its own specific decomposition rate, which would depend
on its concentration and the selected experimental conditions [Gruber, 1992].
A comparison of CEC performance of monoliths photo-initiated with
AIBN and DMPA was also accomplished. As it can be seen in Table VIII.4, at
0.05 wt% for each initiator, slightly better efficiency values were achieved for
columns photo-initiated with DMPA. The k-values were also higher in columns
obtained with DMPA. This fact suggests the presence of higher number of
micropores in the globule structure for monoliths photo-polymerized with
DMPA, and consequently, larger retention, which is confirmed by SEM pictures
(see Fig. VIII.9, parts A and D). Rohr et al.

[Rohr, 2001]

have reported the use

of DMPA as UV initiator for polymerization 2-hydroxyethyl methacrylate


monoliths providing a much faster polymerization rate than those photo-initiated
with AIBN. Faster polymerization rate results in a faster initiation and therefore
the formation of smaller globules.
Additionally, the RG, measured as the geometrical mean of the resolution
between the consecutive PAH pairs, was also evaluated. Monoliths prepared with
DMPA showed slightly better resolution values than those obtained for AIBN
taking into account the studied range for each initiator (Table VIII.4). Fig.
VIII.10, parts A and B shows the electrochromatograms of PAHs obtained with
monoliths photo-initiated at the optimal percentage of AIBN and DMPA,
respectively.

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Miriam Beneito Cambra

Absorbance (mAU)

80

40
6

20

5 6

0
1

160

Absorbance (mAU)

20

120

80
60

80
40

60

60
40

80

3
1
6

40
3

5 6

5
20

2
0

0
1

2
Time (min)

Time (min)

Fig. VIII.10. Electrochromatograms of a PAH test mixture obtained using


LMA-based monolithic column photo-polymerized with several initiators:
0.3 wt% AIBN (A), 0.05 wt% DMPA (B), 0.05 wt% BPO (C) and 0.3 wt%
LPO (D). Other details about the composition of the columns are given in
Fig. VIII.9. CEC conditions: mobile phase, 80:20% (v/v) ACN/aqueous
5mM Tris (pH 8.0); applied voltage, 25 kV. Peak identification: (1)
thiourea, (2) naphthalene, (3) fluorene, (4) anthracene, (5) pyrene, (6)
benz[a]anthracene and (7) benzo[k]fluoranthene.

VIII.2.1.2. Preparation and characterization of LMA columns photo-initiated


with BPO or LPO
LMA-based monolithic columns were prepared using BPO or LPO as
photo-initiators and the same polymerization mixture described in the previous
section was adopted. The BPO content was varied from 0.02 to 0.8 wt% in the
polymerization mixture, while the content of LPO ranged from 0.05 to 0.8 wt%
(see Table VIII.4). When BPO was increased from 0.02 to 0.30 wt%, a decrease
in the k-values was observed, followed by an increase at 0.6 wt%, and a later

324

Chapter VIII. Monolithic Columns

decrease at 0.8 wt%. The beds initiated with 0.05 (Fig. VIII.11, part A) and 0.6
wt% (Fig. VIII.11, part C) showed globule sizes smaller than those obtained
with 0.3 wt% (Fig. VIII.11, part B), which would explain its lower retention.
The monolith initiated at 0.8 wt% did not show significant changes in
morphology compared to 0.6 wt%.

Fig. VIII.11. SEM photographs of LMA monoliths photo-polymerized


with BPO at several contents: 0.05 wt% (A), 0.3 wt% (B) and 0.6 wt% (C).
Other details about the composition of the columns are given in Fig.
VIII.9.

As we commented in previous section, a possible explanation of this


retention behaviour could be related to the competition between the rates of
coagulation and nucleation along initiator concentration.
LMA-based monolithic columns using LPO as initiating system were also
prepared. At 0.05 wt% LPO (Fig. VIII.12, part A), large globule sizes were
observed, whereas beds initiated with higher LPO contents gave similar

325

Miriam Beneito Cambra

monolithic structures (see Fig. VIII.12, part B as representative example). Thus,


these latter monoliths showed small variations in u- and k-values.

Fig. VIII.12. SEM photographs of LMA monoliths photo-polymerized


with LPO at several contents: 0.05 wt% (A) and 0.6 wt% (B). Other details
about the composition of the columns are given in Fig. VIII.9.

A comparison of CEC performance of both diacyl peroxides was also


performed. Taking into account the initiator content employed, for both types of
monoliths, some differences in the studied parameters could be observed. As it
can be seen in Table VIII.4, at low initiator contents (i.e., 0.05 wt%), the kvalues were slightly higher with BPO than those observed for LPO, which was
consistent with SEM pictures of these columns (see Figs. VIII.11, part A and

VIII.12, part A). On the other hand, for initiator contents comprising between
0.050.3 wt% BPO and 0.150.6 wt% LPO, the k-values obtained with BPO
initiated monoliths were lower than those observed for LPO ones. This result was
consistent with SEM pictures, which showed larger globules in beds made with
BPO than those prepared with LPO (see Figs. VIII.11, part B and VIII.12, part

B). However, for BPO contents above 0.6 wt%, the use of this initiator provided
again monoliths with larger retention. As we commented above, these variations
in retention could be probably ascribed to the yield of radical generation, which
depends on the type and concentration of photo-initiator [Gruber, 1992].

326

Chapter VIII. Monolithic Columns

As observed for LPO, similar k-values were obtained within the range
0.150.8 wt%, in contrast to their BPO counterparts. This fact would allow the
possibility of a fine control of morphological and electrochromatographic
properties of LMA-based monolithic columns prepared using LPO.
In terms of efficiency and resolution, similar values were achieved both
using BPO and LPO over the tested initiator range, except at 0.3 wt% BPO,
where the lowest efficiency and resolution values (Table VIII.4) were achieved,
probably due to its large globule size. Fig. VIII.10, parts C and D shows the
electrochromatograms of PAHs that provided the best chromatographic
performance obtained with the monoliths initiated with BPO and LPO,
respectively.
Finally, a comparison of four investigated photo-initiators was performed
taking into account the optimal initiator concentration found for each one. As
shown in Table VIII.4, the four initiating systems investigated showed in their
optimum conditions similar Hmin and RG values. Fig. VIII.13, parts A to D
shows the Van Deemter plots for several PAHs with the optimal monoliths found
for each photoinitiator. The AIBN and LPO monoliths showed low mass transfer
contributions (C-term): 5.612.1 and 6.812.5 ms, respectively, followed by
DMPA (8.214.3 ms) and BPO (7.816.6 ms). At the sight of these values and
Fig. VIII.13, the AIBN monoliths showed the flatter profile, which offered the
possibility of increasing the speed of analysis without a significant loss in
efficiency. Anyway, as shown in Fig. VIII.10, for all tested monoliths, the PAHs
were satisfactorily separated at 25 kV in less than 4 min. The monoliths initiated
with LPO gave the best compromise between separation performance and
analysis time, followed by AIBN, DMPA and BPO.
The repeatability of LMA-based monolithic columns prepared using the
optimal monolithic columns found for each photoinitiator was also examined

327

Miriam Beneito Cambra

(see Table VIII.4). Satisfactory run-to-run repeatabilities in the tested


chromatographic parameters were found for each initiator, whereas for columnto-column repeatability, the beds photo-polymerized with BPO showed the
largest RSD values (see Table VIII.4).

Hmin (m)

30

20

20

10

10

0
0

0.4

0.8

1.2

1.6

30

Hmin (m)

30

0.4

0.8

1.2

30

20

20

10

10

1.6

0
0

0.4

0.8

1.2
1.6
u (mm s-1)

0.5

1.5

Fig. VIII.13. Van Deemter plots of LMA monolithic columns photopolymerized with 0.3 wt% AIBN (A), 0.05 wt% DMPA (B), 0.05 wt%
BPO and 0.3 wt% LPO (D). Compounds: () naphthalene, () anthracene,
and () benzo[k]fluoranthene. Polymerization conditions as in Fig. VIII.9
and CEC conditions as in Fig. VIII.10.

328

2
u (mm s-1)

CAPTULO IX

CONCLUSIONES GENERALES /
GENERAL CONCLUSIONS

Captulo IX. Conclusiones generales

IX.1. Surfactantes

IX.1.1. APGs

IX.1.1.1. Separacin y determinacin de APGs por LC con deteccin ESI-MS


Los espectros de masas de los APGs en el modo PI estn constituidos por
picos de iones [M+Na]+. Utilizando una columna de alquilamida, elucin
isocrtica y deteccin MS, se consigui una buena separacin de los AMGs, con
resolucin entre los epmeros - y - y los ismeros de anillo. Los ismeros de
los APGs (alquildiglicsidos y alquiltriglicsidos) tambin se resolvieron
parcialmente. Alternativamente, los ismeros de anillo pueden resolverse en
menor tiempo utilizando una columna de cianopropilo y elucin en gradiente con
la ventaja de que esta columna requiere un tiempo de equilibrado mucho ms
corto que la columna de alquilamida. Ambos mtodos permiten la identificacin
y cuantificacin de APGs en mezclas industriales y productos de tocador.
Utilizando ESI-MS como sistema de deteccin, los factores de respuesta de los
AMG son independientes del contenido de ACN en la fase mvil. Los factores de
respuesta aumentan en ms de un orden de magnitud al pasar del C1G1 al
C10G1, y nuevamente disminuyen para cadenas de alquilo ms largas; por lo
tanto, para evitar un error sistemtico en la determinacin de los APGs de mayor
inters industrial (C10G1-C16G1) es necesario utilizar estndares de todos los
oligmeros.

The mass spectra of the APGs in the positive-ion mode were constituted by
the single peaks of the [M+Na] + ions. Using an alkylamide column, isocratic
elution and MS detection, the alkylmonoglycosides can be separated with
resolution between the - and -epimers and ring isomers. The isomers of the

331

Miriam Beneito Cambra

alkyldiglucosides and alkyltriglucosides are also partially resolved. The ring


isomers can be also resolved in a shorter time using a cyanopropyl column with
gradient elution. Also in comparison with the alkylamide column, the
cyanopropyl column requires a much shorter equilibration time. The APGs
present in industrial mixtures and products (toiletries) can be quickly identified
and quantified by any of these two methods. Using ESIMS, the response factors
of alkylmonoglycosides are independent from the acetonitrile contents of the
mobile phase, but increase more than one order of magnitude from C1G1 up to
C10G1, and decrease again for longer alkyl chains; thus, standards of all the
oligomers are required to avoid systematic error in the determination of the
alkylpolyglycosides of most industrial interest (C10G1C16G1).

Main points:
-

The ESI-MS spectra of the APGs in PI mode consist mainly of [M+Na] +


peaks.

A column of silica-alkylamide allows the separation of the AMGs, with


resolution between epimers and ring isomers, as well as the partial resolution
of the APGs (alkyldiglicosides and alkyltriglicosides). Its main drawback is
the long equilibration time, thus, isocratic elution should be used.

Using a cyanopropyl column and gradient elution, the AMG ring isomers are
resolved in shorter analysis times than using the alkylamide column.

The two methods can be applied to the identification and quantification of


APGs in industrial mixtures and toiletries.

In ESI-MS, the AMG response factors are independent of the content of ACN
in the mobile phase, increase from C1G1 to C10G1, and decrease for longer
alkyl chains.

332

Captulo IX. Conclusiones generales

IX.1.1.2. Estudio de fragmentacin de la D-glucosa y los AMG en presencia de


iones de sodio en IT-MS
En presencia de iones Na+, los espectros MS en modo PI correspondientes
a la D-glucosa y a los APGs con cadenas alqulicas de hasta 12 tomos de
carbono presentaron, predominantemente, iones [M+Na]+. Los espectros de MS2
obtenidos a partir de los iones progenitores [M+Na]+ mostraron un patrn de
fragmentacin comn con las siguientes caractersticas: (i) un ion a m/z 185, cuya
obtencin se debe a una deshidratacin en la D-glucosa, o a la prdida de la
cadena alqulica en forma de alcohol en los APGs; (ii) un ion a m/z 143
producido por una rotura 0,2A en el anillo; (iii) un ion a m/z 413, que no contiene
tomos de carbono, y que se debe probablemente a un cluster o grupo constituido
por Na+, OH- y H2O; (iv) aductos que contienen una molcula de D-glucosa o el
correspondiente APG (se representa como M), con las estructuras [M+413]+,
[M+413-2H2O]+ y [M+413+H2O-NaOH]+; y (v) unos iones adicionales, cuya
estructura depende de la longitud de la cadena de alquilo. Adems, la D-glucosa
tambin mostr un dbil in a m/z 113 producido por una rotura 0,3A en el anillo
(Fig. IV.8), mientras que todos los APGs mostraron un ion a m/z 129 producido
por una rotura transversal 2,5A del anillo.
Debido a la falta de estndares comerciales de AMGf, los patrones de
fragmentacin de estos ismeros de anillo se estudiaron mediante HPLC-MS y
HPLC-MS2 de mezclas industriales de APGs. Estas mezclas contienen pequeas
concentraciones de octil- y decil-MGf. En comparacin con los AMGf, el ion a
m/z 413 y los aductos formados a partir de ste con molculas no fragmentadas
se producen ms fcilmente con los AMGp. Por el contrario, el ion a m/z 143 se
produce ms fcilmente con los AMGf que con los AMGp, lo que puede deberse
a la menor estabilidad de los anillos de cinco miembros (furansidos) respecto a
los de seis miembros (piransidos).

333

Miriam Beneito Cambra

In the presence of Na+, the PI MS spectra of D-glucose and the APGs with
alkyl chains up to 12 carbon atoms gave predominantly [M+Na]+ ions. The MS2
spectra obtained from the isolated [M+Na]+ parent ions showed a common
fragmentation pattern with the following features: (i) an m/z 185 ion, which is
due to dehydration and to loss of the alkyl chain as an alcohol in the cases of Dglucose and the AGPs, respectively; (ii) an m/z 143 ion produced by a 0,2A crossring cleavage; (iii) an m/z 413 ion, not containing carbon atoms and probably
containing Na+, OH-, and H2O; (iv) adducts containing a molecule of either Dglucose or the corresponding AGP, M, with the structures [M+413]+, [M+413
2H2O] +, and [M+413+H2ONaOH] +; and (v) a few additional ions, whose
structures depended on the length of the alkyl chain. In addition, D-glucose also
showed a weak m/z 113 ion produced by a

0,3

A cross-ring cleavage (Fig. IV.8),

and all the AGPs exhibited an m/z 129 ion produced by a

2,5

A cross-ring

cleavage.
Standards of AMGf were not available; however, the fragmentation
patterns of these ring isomers were studied by using HPLC-MS and HPLC-MS2
of industrial mixtures of APGs. These mixtures contain minor concentrations of
octyl- and decyl-MGf. In comparison with the AMGf, the m/z 413 ion and its
adducts with unfragmented molecules were more easily formed by the AMGp. In
contrast, the m/z 143 ion occurred more easily in the AMGf than in the AMGp,
which can be due to the lower stability of the five-membered furanoside rings.

Main points:
-

In the presence of Na+, the [M+Na] + peak also predominates in the ESI-MS
spectrum of D-glucose in PI mode.

When obtained from [M+Na] + parent ions, the ESI-MS2 spectra of D-glucose
and the APGs show a common fragmentation pattern.

334

Captulo IX. Conclusiones generales

The spectra of AMGp y AMGf (ring isomers) can be separately studied by


using HPLC-MS y HPLC-MS2. The corresponding spectra show differences
that can be useful to identify the ring isomers.

IX.1.2. Alcoholes

IX.1.2.1. Oxidacin de alcoholes no etoxilados y etoxilados con cromo(VI) y


posterior determinacin por ESI-MS
Los alcoholes alifticos no pueden ser detectados por MS empleando ESI
u otras interfaces de ionizacin a presin atmosfrica. Sin embargo, en el
procedimiento propuesto, la oxidacin de alcoholes primarios alifticos con CrO3
(reactivo de Jones), para dar los correspondientes cidos carboxlicos de forma
rpida y cuantitativa, permite su deteccin mediante ESI-MS. Una vez oxidados
mediante el mtodo propuesto, los alcoholes primarios presentes en muestras
industriales y ambientales pueden ser identificados y cuantificados por infusin
directa en el espectrmetro de masas. Se infunde una disolucin transparente e
incolora constituida por extractos en acetato de etilo/acetona. La sensibilidad en
modo NI se exalta por la simple adicin de un 10% de agua alcalinizada a los
extractos. Por otra parte, la sensibilidad de este mtodo es mayor al aumentar la
masa molecular de los alcoholes, de manera que para alcoholes de cadena larga
se consiguen LODs particularmente bajos.
Los rendimientos combinados de las etapas de extraccin-oxidacin
fueron de prcticamente del 100% para alcoholes no etoxilados disueltos en
acetona, y disminuyeron progresivamente para muestras que presentaban
cantidades crecientes de agua. Por ejemplo, con un 50% de agua se obtuvo un
rendimiento del 75%. Por esta razn, las muestras industriales que contienen
agua en proporciones importantes se diluyeron con acetona antes de aadir el

335

Miriam Beneito Cambra

reactivo de Jones. Con ello se reduce considerablemente la concentracin final de


los analitos en las muestras, lo que no constituye ningn problema en control de
calidad industrial, donde las concentraciones de analito suelen ser grandes. Sin
embargo, en muestras ambietales, a causa de las bajas concentraciones de analito
normalmente presentes, se necesita una preconcentracin de la muestra. La
preconcentracin puede hacerse por SPE, lo que permite remplazar el agua por
otro disolvente (como acetona) durante la etapa de elucin.
La formacin de aductos con H+ permite el anlisis por MS de los
alcoholes etoxilados en modo PI, aunque la sensibilidad es baja para oligmeros
con un nmero pequeo de unidades de EO. Por otra parte, los espectros en
modo PI son ms complejos y con una peor relacin seal-ruido que los
espectros en modo NI. Por lo tanto, la oxidacin de los FAEs a los
correspondientes cidos etoxicarboxlicos, seguido por MS en el modo NI, tiene
mayor inters. En el procedimiento desarrollado, los FAEs se oxidan con unos
rendimientos del 65 y 60% para los oligmeros con m = 1-2 y m 2,
respectivamente. La oxidacin tambin puede dar lugar a la prdida de una
unidad de EO, sin embargo, el rendimiento de esta reaccin secundaria es bajo
(4-8%). Por otra parte, al variar el nmero de tomos de C y de EOs, los
rendimientos en la oxidacin de los FAEs fueron bastante constantes. Por otra
parte, las sensibilidades en MS fueron mayores para los cidos etoxicarboxlicos
que para los cidos carboxlicos con el mismo nmero de tomos de carbono
(vase Fig. IV.13).
Los alcoholes no etoxilados y alcoholes con un bajo grado de etoxilacin
se suelen determinar por GC-FID. Las ventajas del procedimiento propuesto en
este trabajo son la rapidez y las elevadas sensibilidades obtenidas para los
alcoholes de cadena larga, lo que tiene inters en el anlisis de muestras con
matrices complejas. En cosmticos, productos para el cuidado corporal y

336

Captulo IX. Conclusiones generales

extractos de muestras medioambientales, la presencia de muchos compuestos


neutros con volatilidades relativamente altas da lugar a cromatogramas muy
complejos en GC. En el procedimiento propuesto, esta complejidad se evita por
la oxidacin de los alcoholes a los correspondientes cidos carboxlicos, y por el
aislamiento de los mismos mediante extraccin lquido-lquido. Tras aumentar el
pH, los extractos se infunden directamente en un espectrmetro de masas
trabajando en modo NI. Adems, el procedimiento oxidacin-extraccin
propuesto permite tambin el anlisis de alcoholes por HPLC-MS, CE-MS, y
CEC-MS. Por el contrario, los alcoholes no etoxilados con altas masas
moleculares no se detectan (o bien se observan con una sensibilidad muy baja)
por HPLC-ELSD, mientras que la sensibilidad de alcoholes mono y dietoxilados
es tambin bajas con esta tcnica

[Bernab-Zafn, 2006].

Por esta razn, otra

ventaja del procedimiento propuesto es que permite ampliar el campo de


aplicaciones de los detectores evaporativos, haciendo posible la deteccin de
alcoholes no etoxilados, y mejorando la respuesta de alcoholes mono y
dietoxilados. Por ltimo, cuando los cidos carboxlicos correspondientes a la
oxidacin de los alcoholes de inters estn inicialmente presentes en la muestra,
las seales obtenidas por el mtodo propuesto corresponden a la suma de
alcoholes y cidos carboxlicos. En estos casos, se puede obtener un espectro o
cromatograma adicional a partir de la muestra sin la adicin del reactivo de
Jones, y utilizarlo para restar la contribucin de las seales derivadas de los
cidos carboxlicos inicialmente presentes en la muestra. De este modo, es
posible aislar las contribuciones de los alcoholes respecto a las debidas a los
cidos carboxlicos. Aunque la sustraccin de la seal aumenta el error aleatorio,
la prdida de precisin es pequea cuando la concentracin de alcohol en las
muestras es bastante mayor que la de los cidos carboxlicos correspondientes, lo
que es frecuente en muchos casos.

337

Miriam Beneito Cambra

Aliphatic alcohols cannot be detected by MS using ESI and other


atmospheric-pressure ionization interfaces. However, in the proposed procedure,
the oxidation of primary aliphatic alcohols with a CrO3 solution in aqueous
sulphuric acid (Jones reagent) resulted in their rapid and quantitative
conversion into the corresponding carboxylic acids. Primary alcohols present in
industrial and environmental samples can be identified and quantitated by
infusion of the transparent and uncoloured ethyl acetate-acetone extracts in a
mass spectrometer. Sensitivity in the NI mode is strongly enhanced by the
addition of 10% alkalinized water to the extracts. Moreover, the sensitivity of this
approach further increases as the molecular mass of the alcohols increases, such
that for long-chain alcohols the LODs obtained using the proposed procedure
were particularly low.
Oxidation-extraction yields, which were ca. 100% for nonethoxylated
alcohols dissolved in acetone, decreased moderately in samples containing
increasing amounts of water, e.g., a 75% yield was obtained in the presence of
50% water. For this reason, industrial samples containing large amounts of
water were diluted with acetone before Jones reagent was added. While this
greatly reduced the final concentration of the analytes in these samples, this
should not be a problem in industrial quality control, where analyte
concentrations are usually large. However, owing to the low analyte
concentrations typically present in environmental samples, preconcentration of
the sample, with replacement of water by another solvent, is usually required.
The formation of H+ adducts enables MS analysis of ethoxylated alcohols
in PI mode, although the sensitivity is low for oligomers with a small number of
EO units. Furthermore, PI mode spectra are potentially more complex and
noisier than NI mode spectra. Thus, oxidation of ethoxylated alcohols to the
corresponding ethoxycarboxylic acids, followed by NI mode MS, is also of

338

Captulo IX. Conclusiones generales

interest. In the procedure developed in this work, ethoxylated alcohols are


oxidized, with ca. 65 and 60% yields for m=12 and m 2 oligomers,
respectively. Oxidation may also result in the loss of an EO unit; however, the
extension of this side reaction is low (48%). Furthermore, oxidation yields of
ethoxylated alcohols were fairly constant, and MS sensitivities were larger for
ethoxycarboxylic acids than for carboxylic acids with the same number of carbon
atoms (see Fig. IV.13).Therefore, application of the proposed procedure not only
to nonethoxylated alcohols but also to ethoxylated alcohols is of much interest.
Non-ethoxylated alcohols and alcohols with a low degree of ethoxylation
are usually identified and determined by GC-FID. The advantages of the
procedure proposed in this work are its rapidity and the prominent signals
obtained from long-chain alcohols present within wide concentration ranges in
samples comprising complex matrices. In cosmetics, body-care products, and
extracts from environmental samples, the presence of many neutral compounds
with relatively large volatilities frequently gives rise to complex gas
chromatograms. In the proposed procedure, this is avoided by alcohol oxidation
to carboxylic acids and isolation of the acids by liquid-liquid extraction; after the
pH has been raised, the extracts can be directly infused in the mass spectrometer
working in the NI mode, without the need for further purification. Moreover, the
proposed oxidation-extraction procedure allows alcohol analysis by HPLC-MS,
CE-MS, and CEC-MS as well. By contrast, non-ethoxylated alcohols up to high
molecular masses are either not detected or are observed with low sensitivities
by HPLC-ELSD, while the sensitivities for mono- and diethoxylated alcohols are
likewise poor

[Bernab-Zafn, 2006].

Thus, a further advantage of the proposed

procedure is that it expands the range of applications of evaporative detectors to


include non-ethoxylated alcohols and improves the response of mono- and
diethoxylated alcohols. Finally, signals corresponding to the sum of alcohols and

339

Miriam Beneito Cambra

carboxylic acids are obtained when these acids are initially present in the
sample. In this case, an additional spectrum or chromatogram, obtained without
adding Jones reagent to the sample, can be used to subtract the signal
contributions arising from the carboxylic acids initially present in the sample
and thus to isolate the contributions of alcohols. Although signal subtraction
increases the random error, the loss of precision is small when the alcohol
concentration in the samples is larger than that of the corresponding carboxylic
acids.

Main points:
-

A procedure that combines the oxidation of aliphatic primary alcohols and


the extraction of the resulting carboxylic acids to obtain an extract free of
inorganic salts has been developed. The oxidation-extraction yields are close
to 100%.

Aliphatic primary alcohols are not detected in MS; however, using this
procedure, the identification and quantification of aliphatic primary alcohols
by ESI-MS is possible.

In NI mode, sensitivity increases with pH and the molecular mass of the


alcohol, being also higher when alcohol is ethoxylated.

The oxidation yield decreases with increasing water content in the sample.

After SPE preconcentration in SPE cartridges, followed by elution with


acetone, the method can be applied to samples of the aquatic environment.

Owing to the formation of adducts with H+, ethoxylated alcohol can be


analyzed by MS in the PI mode; however, the sensitivity is low for oligomers
with a low EO number. In addition, PI mode spectra are more complex tan Ni
mode spectra. Also, the signal to noise ratio in the PI mode is lower than that
usually found in NI mode spectra.

340

Captulo IX. Conclusiones generales

Oxidation of FAEs to ethoxycarboxylic acids results in loss of one EO unit,


but the yield of this side reaction is small (< 8%).

Ethoxylated alcohols are oxidized with lower yields (60-65%) than those
obtained with non-ethoxylated alcohols (~ 100%), however, oxidation yields
are independent from the number of carbon atoms and the number of EOs.

The proposed method allows the use of evaporative detectors and mass
spectrometry detection in the determination of aliphatic primary alcohols.
The method is direct, fast and results in lower LODs with complex samples of
industrial and environmental origin.

IX.1.3. AES

IX.1.3.1. Determinacin de FAEs y AESs por separacin mediante SAX,


derivatizacin con un anhdrido cclico y LC
En trabajos anteriores, se han desarrollado procedimientos para la
caracterizacin y la determinacin de FAEs basados en su derivatizacin con un
anhdrido cclico, seguida por separacin de los oligmeros mediante RP-HPLC
[Mic-Tormos, 2008-A; Mic-Tormos, 2008-B; Mic-Tormos, 2009; Mic-Tormos, 2010].

Sin embargo, cuando los FAEs se esterifican, los AESs se transesterifican, dando
lugar ambas clases de surfactantes a los mismos derivados. Por lo tanto, en el
caso en que las dos clases de surfactantes estn presentes en una muestra, los
cromatogramas resultantes muestran, en realidad, la suma de FAEs y AESs. Por
lo tanto, en este trabajo se ha desarrollado un procedimiento para la separacin
de ambas clases de surfactantes, empleando SPE con un cartucho de SAX. Tras
la separacin, los oligmeros se derivatizan independientemente y se determinan
por RP-HPLC-UV. La separacin casi cuantitativa de las dos clases de
surfactantes, incluyendo oligmeros hidroflicos e hidrofbicos, se logr

341

Miriam Beneito Cambra

mediante la elucin de las fracciones correspondientes a los FAEs y AESs en tres


y dos pasos, respectivamente. Como ya se demostr previamente para los FAEs,
la derivatizacin de los AESs tambin tiene lugar de forma cuantitativa. Para ello
se puede usar anhdrido ftlico o difnico en 1,4-dioxano a 105 C. La separacin
de los oligmeros derivatizados empleando RP-HPLC y elucin en gradiente con
fases de ACN/agua, mostr una buena resolucin entre las sucesivas series
hidrocarbonadas, y entre los oligmeros dentro de una misma serie. Tambin se
ha demostrado que los FAEs pueden utilizarse como patrones de calibracin para
AESs, lo que supone una ventaja, ya que los estndares de AESs son raros y
caros, mientras que los estndares de alcoholes grasos son ampliamente
disponibles; adems, los estndares de AES con m > 0 no se encuentran
comercialmente disponibles. Por otro lado, otras clases de surfactantes aninicos
de uso general en productos de limpieza, como son el SAS y el LAS, no
interfieren. El mtodo propuesto se aplic satisfactoriamente a la caracterizacin
y determinacin de FAEs y AESs en detergentes lquidos industriales y en
extractos de agua de mar. El procedimiento propuesto es tambin til para el
control de calidad industrial de los AESs, donde los FAEs suelen estar presentes
como impurezas, a causa de que en el proceso de obtencin de los AESs la
sulfatacin no es cuantitativa.

In previous work, procedures for FAE characterization and determination


based on derivatization with a cyclic anhydride, followed by RP-HPLC with UV
or MS detection, were developed [Mic-Tormos, 2008-A; Mic-Tormos, 2008-B; MicTormos, 2009; Mic-Tormos, 2010].

However, when FAE are esterified, AES are

also transesterified, leading to the same derivatives as FAE. Thus, if the two
surfactant classes are present in the samples, the resulting chromatograms show
the sum of both FAE and AES oligomers. In this work, a procedure for the SAX

342

Captulo IX. Conclusiones generales

separation of both surfactant classes, followed by their independent


derivatization and RP-HPLC-UV, was developed. Quantitative isolation of the
two surfactant classes, including both hydrophilic and hydrophobic oligomers,
was achieved by eluting the FAE and AES fractions in three and two steps,
respectively. As previously shown for FAE, quantitative derivatization of AES
with either phthalic or diphenic anhydrides has been demonstrated. Separation
of the derivatized oligomers, with good resolution between both the hydrocarbon
series and the successive oligomers within the series, was achieved by RP-HPLC
using gradient elution with ACN/ water. It has been also shown that FAE
oligomers can be used as calibration standards for AES, which is an advantage
because standards of alkylsulfates are rare and expensive, whereas standards of
fatty alcohols are widely available; further, standards for AES with m > 0 are
not commercially available. On the other hand, anionic surfactant classes
commonly used in cleaners, including SAS and LAS, did not interfere. The
proposed method was successfully applied to the characterization and
determination of FAE and AES in industrial liquid cleaners and seawater
extracts. The proposed procedure is also useful to control the quality of
industrial AES, where FAE are always present as an impurity, due to the nonquantitative sulfatation of FAE during AES manufacture.

Main points:
-

Reacting with a cyclic anhydride, FAEs are esterified, and AESs are
transesterified, quantitatively giving rise to the same derivatives (hemiesters
of the alcohol residues).

Using a SAX cartridge, a procedure for the quasi quantitative separation of


FAEs and AESs has been developed.

343

Miriam Beneito Cambra

A method for the characterization and determination of FAEs and AESs in


mixtures has been developed. The method is based on the separation of the
two surfactant classes with a SAX cartridge, followed by derivatization with a
cyclic anhydride and RP-HPLC-UV. A good resolution between consecutive
series (different hydrocarbon chain), and between the oligomers within a
series is obtained.

The method allows the use of FAEs as calibration standards for AESs.

The method is useful to characterize and determine FAEs and AESs in


industrial liquid detergents and in seawater. Other classes of anionic
surfactants such as SAS or LAS do not interfere.

IX.2. Polmeros sintticos

IX.2.1. PVP-NO

IX.2.1.1. Caracterizacin de PVP-NO por CE y MECK


Los estudios mediante FSCE realizados en este trabajo han mostrado que
el PVP-NO es un polmero aparentemente no inico, que se comporta como un
polielectrolito con un carcter aninico bien definido en disolucin acuosa. Los
resultados obtenidos por FSCE estn de acuerdo con los obtenidos previamente a
travs de estudios de viscosidad, conductividad, dispersin de luz y cido-base
[Okamoto, 1998; Lee, 1996; Yamamoto, 1996].

Los resultados tambin son

consistentes con la presencia de al menos dos formas del polmero en


disoluciones acuosas, probablemente una de ellas constituida por cadenas libres
(unmeros) y la otra por agregados. Este enfoque estara de acuerdo con los
estudios realizados por CE para otros polmeros y copolmeros
Gyrffy, 1998; tpnek, 2001].

[Cottet, 2001;

Por otro lado, los resultados obtenidos por MEKC

344

Captulo IX. Conclusiones generales

indican una fuerte interaccin del PVP-NO con las micelas de SDS, y son
coherentes con la formacin de micelas mixtas de dos tipos: las constituidas por
polmeros libres y SDS, y las que contienen agregados de polmero y SDS
1971;

Cabane,

1977].

[Arai,

Esta explicacin es consistente con los estudios

espectroscpicos por UV-Vis de Oakes y col.

[Oakes, 2003-C].

Por lo tanto, en el

presente estudio se ha demostrado la utilidad de la FSCE y la MEKC en la


investigacin del comportamiento de los polmeros sintticos en disolucin.
Tambin se ha demostrado la capacidad del PEA para reducir la adsorcin de
polmeros sintticos sobre la pared interna de los capilares en medio cido. Por
ltimo, la FSCE puede ser til en el control de calidad de materias primas y
productos comerciales que contienen PVP-NO, si bien, el procedimiento se
encuentra limitado a productos que no contienen surfactantes aninicos.

The FSCE studies performed in this work have shown that PVP-NO,
which is a nominally non-ionic polymer, behaves as a polylectrolyte, with a welldefined anionic character in aqueous solutions. In this concern, the results
obtained by FSCE agreed with those previously reported using viscosity, light
scattering, conductivity and acid-base studies
Yamamoto, 1996].

[Okamoto, 1998; Lee, 1996;

The results are also consistent with the presence of at least two

forms of the polymer in aqueous solutions, likely one of them constituted by free
chains (unimers) and the other one by aggregates. This would agree with the CE
studies performed with other polymers and copolymers
1998; tpnek, 2001].

[Cottet, 2001; Gyrffy,

On the other hand, the results obtained by MEKC indicated

a strong interaction of PVP-NO with the SDS micelles, and are consistent with
the formation of both free polymer/SDS and aggregated polymer/SDS mixed
micelles [Arai, 1971; Cabane, 1977]. This explanation is consistent with the UV-Vis
spectroscopic studies by Oakes et al.

[Oakes, 2003-C].

345

Thus, the present study

Miriam Beneito Cambra

further shows the usefulness of both FSCE and MEKC as complementary


techniques in the investigation of the behavior of synthetic polymers in solution.
The capability of PEA to reduce the adsorption of synthetic polymers in weak
acid media has been also demonstrated. Finally, FSCE can be useful in the
quality control of raw materials and commercial products containing PVP-NO,
although the procedure is limited to products not containing anionic surfactants.

Main points:
-

Using FSCE, it has been shown that PVP-NO (apparently a non-ionic


polymer) behaves as a polyelectrolyte with anionic character in aqueous
solution, which has been attributed to the deprotonation of water associated
with the NO- group.

The FSCE experiments are consistent with the presence of two forms of the
polymer in solution, one made up of free chains (unimers) and the other
constituted by aggregates.

PVP-NO interacts strongly with SDS micelles, giving rise to mixed micelles of
two types: free polymers bound to SDS, and polymer aggregates also bound
to SDS.

The usefulness of FSCE and MEKC in investigating the behavior of synthetic


polymers in solution has been demonstrated.

The ability of PEA to reduce adsorption of synthetic polymers on the inner


wall of the capillaries in acid medium has been demonstrated.

Although limited to samples having no anionic surfactants, FSCE is useful in


the quality control of industrial products containing PVP-NO.

346

Captulo IX. Conclusiones generales

IX.2.2. PVP

IX.2.2.1. Caracterizacin y determinacin de PVP por complejacin con un


azo-colorante aninico seguida de NEECEM
En disolucin acuosa, los colorantes aninicos estn enlazados al PVP
formando complejos que muestran movilidad aninica. Durante la separacin
electrofortica, estos complejos se disocian parcialmente siguiendo una cintica
de primer orden. En este trabajo, la teora NECEEM, que se desarroll para
estudiar las interacciones protena-marcador y protena-DNA, se aplica por
primera vez al estudio de polmeros sintticos. Mediante esta teora se puede
obtener informacin sobre la estabilidad de los complejos formados entre un
polmero y un colorante. A pesar de la mayor polidispersidad de los polmeros
sintticos en comparacin con las protenas

[Grosche, 2000; Borisch, 2000],

el

comportamiento electrofortico de las mezclas PVP-colorante ha podido ser muy


bien explicado por la teora NECEEM. As, a partir de la inyeccin de mezclas
con diferentes razones molares monmero/colorante, puede establecerse la
estequiometra mxima de los complejos, y trabajando con razones molares
monmero/colorante cercanas al punto de saturacin, puede estimarse tambin la
constante de estabilidad de los complejos. El mtodo NECEEM es, por lo tanto,
til para caracterizar polmeros no inicos, tanto sintticos como naturales, y
estudiar su interaccin con un marcador en disolucin. Adems, trabajando a
razones molares inferiores al punto de saturacin, puede obtenerse la
concentracin de monmeros mediante una curva de calibracin, y puede
estimarse un promedio de la masa molecular del polmero a partir de relaciones
sencillas basadas en la forma del pico o banda del complejo polmero-marcador.
En este trabajo se estim la estequiometra de los complejos formados por
el PVP con dos azo-colorantes aninicos, CR y AB. La saturacin de PVP con

347

Miriam Beneito Cambra

estos dos colorantes se produjo a una razn molar monmero/colorante prxima


a q = 4. Se estimaron tambin las constantes de estabilidad de los complejos
PVP-CR. Cuando se trabaja en presencia de un exceso de colorante (q < 4), la
banda de los complejos PVP-CR es til para predecir la concentracin del PVP,
as como su MW promedia, lo que se aplic a la caracterizacin de PVP en
productos de limpieza y productos farmacuticos.

Anionic dyes are bound to PVP in aqueous solutions, forming complexes


that show anionic mobility. During the electrophoretic run, the complexes
partially dissociate following a first-order kinetics. On the basis of the NECEEM
theory, which was formerly developed to study proteinprobe and DNAprotein
interactions, information about the stability of polymerdye complexes can be
obtained. In spite of the much larger polydispersity of synthetic polymers when
compared to proteins [Grosche, 2000; Borisch, 2000], the electrophoretic behaviour
of PVPdye mixtures is very well explained by the NECEEM theory. Thus, by
injecting mixtures at several monomer/dye molar ratios, the maximal
stoichiometry of the complexes can be established, and by working at
monomer/dye molar ratios close to saturation, the average stability constant of
the complexes can be estimated. The NECEEM method is thus useful to
characterize synthetic and natural nonionic polymers, and to study their
interaction with a probe in solution. In addition, at monomer/dye molar ratios
lower than saturation, the monomer concentration can be obtained by using a
calibration curve, and the average molecular mass of the polymer can be
estimated from a simple relationship provided by the shape of the peak of the
polymerprobe complexes.
In this work the stoichiometry of the complexes formed by PVP with two
anionic azo-dyes, CR and AB, was estimated. Saturation of PVP with these dye

348

Captulo IX. Conclusiones generales

ions was produced at a monomer/dye molar ratio close to q = 4. The stability


constants of the PVPCR complexes were also estimated. Upon addition of an
excess dye to the sample (q < 4), the band of the PVPCR complexes was useful
to predict both the concentration of PVP and the average molecular mass, Mw,
in cleaning products and pharmaceutical preparations.

Main points:
-

Anionic dyes are bound to PVP in aqueous solution, resulting in complexes


with anionic mobility.

During electrophoretic separation, the PVP-dye complex is partially


dissociated following a first-order kinetics.

It has been shown that the NECEEM theory, previously developed to


characterize proteins and DNA fragments, is also useful to characterize
synthetic nonionic polymers. Information on the stability of the complexes
and the nature of the polymer (average molecular mass) can be obtained.

Working at molar ratios below the saturation point, the stoichiometry of the
complexes (number of monomers per dye ion) can be estimated.

Saturation of the polymer (PVP) with CR or AB occurs at a monomer / dye


molar ratio close to 4.

Working in an excess dye (q < 4), the band of the PVP-CR complexes
contains information which is useful to predict the PVP concentration and
their average MW.

349

Miriam Beneito Cambra

IX.2.3. PVA

IX.2.3.1. Evaluacin de la MW y tacticidad del PVA mediante NEECEM de


mezclas polmero-colorante
Cuando una disolucin de PVA que contiene borato se mezcla con una
disolucin que contiene CR, se forma inmediatamente un complejo PVA-CR. La
formacin de este complejo se detecta por el aumento de la absortividad molar y
por el notable desplazamiento batocrmico de la banda principal del espectro de
absorcin UV-Vis del CR. Utilizando polaridad positiva, los electroferogramas
de las mezclas PVA-CR en presencia de un exceso de CR mostraron el patrn
predicho por la teora NECEEM para complejos formados por una
macromolcula sin carga y un marcador aninico. Cuando la concentracin de
PVA aument hasta el punto de saturacin, el rea de la banda del complejo
aument tambin linealmente, y las reas debidas al exceso de colorante liberado
durante la migracin disminuyeron igualmente de forma lineal. Se estim la
mxima estequiometra del complejo a partir de la variacin de reas a valores
crecientes de la razn molar monmero/colorante (valores de q). Esta razn
oscil entre qsat ~ 4,9 y 3,5 para PVA de MW bajo y alto, respectivamente. Por
otra parte, esta variacin se produjo a diferentes valores de log MW dependiendo
de la tacticidad del PVA. A la vista de los valores de la qsat, se discuti la posible
estructura del complejo de PVA-CR. Las correlaciones encontradas entre varios
parmetros electroforticos obtenidos en las condiciones NECEEM respecto a la
MW y la tacticidad del PVA, pueden explicarse suponiendo que los iones de
colorante se apilan muy prximos unos a otros dentro de la estructura de los
complejos PVA-CR. La correccin de la estequiometra para tener en cuenta el
porcentaje de monmeros acetilados no enlazados (qsat,c) confirm las diferencias
entre los grupos de muestras de distinta tacticidad. La forma de la banda de los

350

Captulo IX. Conclusiones generales

complejos PVA-CR, las reas relativas de la banda del complejo y del pico del
colorante libre, la movilidad electrofortica y la constante de disociacin de
pseudo-primer orden del complejo, tambin resultan estar correlacionadas con la
MW y la tacticidad del PVA. Las muestras ms sindiotcticas (tipo H) dieron
complejos con menor movilidad absoluta que las muestras ms atcticas (tipos M
y L). Este comportamiento podra deberse a las mayores distancias entre los
grupos OH adyacentes en las muestras tipo H, las cuales presentan una mayor
relacin rr/mm que las muestras M y L. El producto [qsat + (VD / Vm)]
disminuy para todas las MW, lo que indica que no llega a alcanzarse el rgimen
de drenaje libre (donde la movilidad es constante porque la relacin carga/masa
ya no aumenta al seguir creciendo la MW del polmero). La variacin de este
producto, tambin confirm la dependencia entre la movilidad del complejo
PVA-CR y la tacticidad. Por lo tanto, se puede obtener informacin til acerca de
la MW y la tacticidad del PVA a partir de la CZE de mezclas de PVA-CR. Sin
embargo, sera necesario recurrir a la calibracin multivariante, incluyendo la
variacin ortogonal de las variables MW y el cociente rr/mm, para construir el
correspondiente conjunto de estndares de calibracin. Sin una calibracin
independiente de estas variables no es posible construir modelos capaces de
hacer predicciones de la MW y la tacticidad con una precisin razonable. El
conjunto de muestras comerciales de PVA recopilado y empleado en este trabajo
fue suficiente para demostrar la dependencia de los parmetros de CZE obtenidos
en condiciones de NECEEM sobre la MW y tacticidad, pero no es lo
suficientemente bueno para hacer una calibracin multivariante. Por ltimo,
aunque esto tambin debe ser estudiado, la NECEEM podra ser til para evaluar
la MW y la tacticidad de otros polmeros solubles no cargados.

351

Miriam Beneito Cambra

A PVACR complex is immediately formed when a PVA solution


containing borate and a CR solution are mixed. Complex formation produced a
large increase of the molar absorptivity and a remarkable bathochromic shift of
the main band of the UVVis absorption spectrum of CR. Using positive polarity,
electropherograms of PVACR mixtures containing a CR excess showed the
pattern predicted by the NECEEM theory for a complex formed by an uncharged
macromolecule and an anionic marker. The area of the complex band increased
linearly, and the areas due to the excess dye and to the dye released by the
complex during migration decreased, when the PVA concentration increased up
to the saturation point. The variation of the areas at increasing monomer/dye
molar ratios (q values) was used to estimate the maximal stoichiometry of the
complex. This ranged from qsat ~ 4.9 to 3.5 for low and high MW PVA,
respectively, this variation being produced at different log Mw values depending
on PVA tacticity. At the sight of the values of qsat, the possible structure of the
PVACR complex was discussed. The correlations found along this work among
several electrophoretic parameters obtained in NECEEM conditions with respect
to both molecular mass and tacticity of PVA can be explained by assuming that
the dye ions are stacked in a proximity to each other within the structure of the
PVACR complex. Correction of the stoichiometry taking into account the
percentage of unbonding acetylated monomers (qsat,c) confirmed the differences
between tacticity groups. The shape of the PVACR complex band, the relative
areas of the complex band and free dye peak, and the electrophoretic mobility
and dissociation pseudo-first-order rate constant of the complex, were also
related to both MW and tacticity of PVA. Thus, the H samples gave complexes
with lower absolute mobilities than the M+ L samples. In comparison to M+ L
samples, these differences could be due to the larger distances between adjacent
OH groups in H samples, which have a higher rr/mm ratio than the M+ L

352

Captulo IX. Conclusiones generales

samples. Product [qsat + (VD / Vm)] decreased at all MW, thus indicating that a
free draining regime was not reached. The variation of this product also
confirmed the dependence of the mobility per complexed dye ion on tacticity.
Therefore, useful information about both MW and tacticity of PVA can be gained
by CZE of PVACR mixtures. However, multivariate calibration, including the
orthogonal variation of the molecular mass and rr/mm ratio values along the set
of standards, would be necessary to construct multivariate models capable of
making predictions of these two responses with reasonable precision. The set of
commercial PVA samples we collected and used in this work was adequate to
demonstrate the dependence of CZE parameters obtained in NECEEM
conditions on molecular mass and tacticity, but it was deficient to support
multivariate calibration. Finally, although this should be also investigated,
NECEEM could be useful to evaluate MW and tacticity of other soluble noncharged polymers.

Main points:
-

The PVA-CR complex is immediately formed upon mixing a solution of PVA


with a solution containing CR (in the presence of borate). Upon complex
formation, an increase in the molar absorptivity of the main UV-absorption
band of CR, and a large bathochromic shift, is produced.

In the presence of an excess of CR, the electropherograms of PVA show the


pattern predicted by the NECEEM theory.

Increasing the PVA concentration up to the saturation point, the band area of
the complex increases linearly, while the sum of the areas due to excess dye
and the dye released during migration, also decreases linearly.

353

Miriam Beneito Cambra

The maximum stoichiometry of the complex ranges from qsat ~ 4.9 to 3.5 for
samples of PVA of high and low MW, respectively. This variation occurs at
different values of log MW, depending on the tacticity of PVA.

The correlations between MW and tacticity of PVA with various


electrophoretic parameters obtained in the NECEEM conditions can be
explained by assuming that in these complexes, dye ions are stacked very
close together within the structure of the PVA-CR complex.

Correction of the stoichiometry of the complex to take into account the


percentage of unbound acetylated monomers, confirmed the differences
observed between groups of samples of different tacticity.

The shape of the band of PVA-CR complexes, the relative areas of the band of
the complex and free dye peak, the electrophoretic mobility and the
dissociation constant of the complex, are all correlated with MW and tacticity.

IX.3. Enzimas

IX.3.1. CZE

IX.3.1.1. Identificacin de enzimas de la industria de la detergencia por CZE


de enzimas intactas
Se ha demostrado que la separacin de protenas intactas por CZE,
utilizando dos BGEs, uno cido y otro bsico, es potencialmente til para
identificar las enzimas comnmente utilizadas en la formulacin de productos de
limpieza. En particular, el BGE bsico proporciona una lnea base habitualmente
plana, lo que permite distinguir una serie de pequeos picos que muestran pautas
caractersticas del tipo de enzima. Adems del tiempo de migracin del pico
principal, la presencia de un segundo pico de mediana intensidad tambin

354

Captulo IX. Conclusiones generales

constituyen un rasgo caracterstico de algunas enzimas utilizadas en la industria


de los detergentes. La cuantificacin del contenido proteico por el mtodo de
Bradford, permiti calcular las sensibilidades relativas a partir de las reas totales
de los picos obtenidos en los electroferogramas en los BGEs bsico y cido.
Estas sensibilidades tambin pueden ser tiles para clasificar o identificar las
enzimas. La aplicacin del procedimiento a muestras reales falla en presencia de
surfactantes aninicos, si bien, algunas muestras que contienen surfactantes no
inicos dan resultados aceptables.

It has been shown that CZE of the intact proteins using either basic or
acid BGEs is potentially useful to identify the enzyme brands commonly used in
the formulation of cleaning products. In particular, the basic BGE gives flat
baselines, which makes possible to distinguish a number of small peaks
constituting rather characteristic patterns. In addition to the migration time of
the main peak, the presence of a second large well resolved peak was also a
rather characteristic feature of some raw enzymes. Further, after quantitation by
the Bradfords method, relative sensitivities calculated from the total peak area
of the electropherograms obtained in the basic and acid BGEs can be also useful
to identify the enzymes. Application of the procedure to real samples is possible
in the presence of non-ionic surfactants, but not in samples containing large
concentrations of anionic surfactants, more research being needed to reduce this
interference.

Main points:
-

The separation of intact proteins by CZE is potentially useful to classify or


identify the enzymes used in the formulation of cleaning products.

355

Miriam Beneito Cambra

A basic BGE provides a flat baseline on which some small peaks are
distinguished; the peaks constitute characteristic patterns which can be useful
to classify or identify the enzymes.

Relative sensitivities in the acid and basic BGEs, calculated after protein
quantification by the Bradfords method, may also be useful for enzyme
identification.

The application of the procedure to real samples is limited to samples that do


not contain anionic surfactants, being tolerated the presence of nonionic
surfactants.

IX.3.2. Aminocidos por infusin directa en MS

IX.3.2.1. Clasificacin rpida de enzimas en productos de limpieza por


hidrlisis aminocidos, MS y LDA
Se ha desarrollado un procedimiento sencillo y rpido, capaz de clasificar
las enzimas presentes en los productos de limpieza de acuerdo con su clase
(proteasa, amilasa, celulasa y lipasa). Para ello, tras la precipitacin e hidrlisis
de las enzimas empleadas como materia prima, los hidrolizados fueron disueltos
en etanol y directamente infundidos en la interfaz ESI de un espectrmetro de
masas de trampa inica. Los datos espectrales fueron utilizados para construir
modelos LDA, obtenindose unas capacidades de prediccin excelentes. Los
efectos de matriz de la muestra, observados al aditivar las bases de detergente
con los concentrados de enzimas industriales, se redujeron en gran medida
mediante la inclusin de muestras aditivadas en el conjunto de entrenamiento. El
modelo de LDA resultante mostr una excelente capacidad de prediccin.

356

Captulo IX. Conclusiones generales

A simple and quick procedure capable of classifying enzymes in cleaning


products according to their protease, amylase, lypase and cellulase classes has
been developed. For this purpose, after precipitation and hydrolysis, the
hydrolysates were dissolved in ethanol and directly infused into the ESI ion
source of an ion trap mass spectrometer. The spectral data were used to
construct LDA models with excellent prediction capabilities. The sample matrix
effects, which were observed when detergent bases spiked with enzyme industrial
concentrates were used for model evaluation, were ruled out by also including
the spiked samples in the training set. The resulting model showed an excellent
prediction capability.

Main points:
-

A simple and rapid procedure for enzyme classification in cleaning products


has been developed; enzymes are classified according to the protease,
amylase, cellulase or lipase classes.

The spectral data of amino acids obtained by hydrolysis followed by ESI-MS


were used in the construction of LDA models which showed excellent
predictive capabilities.

Sample matrix effects were reduced by including spiked samples in the LDA
training set.

IX.3.3. Aminocidos derivatizados con OPA-NAC

IX.3.3.1. Clasificacin de enzimas presente en productos de limpieza mediante


hidrlisis, derivatizacin con OPA-NAC, HPLC y LDA
Se ha desarrollado un mtodo para la clasificacin de las enzimas
presentes en productos de limpieza, basado en la hidrlisis seguida de

357

Miriam Beneito Cambra

derivatizacin con OPA- NAC de los aminocidos resultantes, separacin de los


derivados (isoindoles) mediante HPLC-UV-Vis. La clasificacin se efecta en
funcin de las categoras ms comunes: proteasas, amilasas, lipasas y celulasas.
Para la separacin se utiliz una columna C18 y elucin de los isoindoles con un
gradiente multisegmentado. A partir del perfil de aminocidos se obtuvo un
modelo de LDA con una excelente capacidad predictora. Se utilizaron los
cocientes de las reas de pares de picos como variables predictoras para construir
el conjunto de entrenamiento, adems de los concentrados industriales de las
enzimas (materias primas), se incluyeron bases de detergentes reforzadas con
enzimas. Todos los objetos del conjunto de evaluacin, incluyendo concentrados
industriales de enzimas, bases de detergente aditivadas y productos de limpieza
comerciales, se asignaron correctamente, con una probabilidad del 99%.

An HPLCUVVis method for class identification of the enzymes found in


cleaning products according to the protease, amylase, lypase and cellulase
classes, has been developed. For this purpose, after precipitation and hydrolysis,
the amino acids were derivatized with OPANAC and aliquots were
chromatographed. A C18 column and multi-segmented gradient elution with
ACN/water were used to separate the isoindoles of the amino acids. An LDA
model with an excellent prediction capability was obtained by using ratios of the
areas of the peaks taken by pairs as predictors, and by including both enzyme
industrial concentrates and spiked detergent bases in the training set. All the
objects of the evaluation set, including enzyme industrial concentrates, spiked
detergent bases and commercial cleaners, were correctly assigned with a 99%
probability.

358

Captulo IX. Conclusiones generales

Main points:
-

A method for the classification of enzymes in cleaning products has been


developed. The method is based on the total hydrolysis of the enzymes to
amino acids, followed by derivatization with OPA-NAC, HPLC of the
resulting isoindoles and LDA, using the chromatographic peak areas as
predictors.

In addition to industrial enzyme concentrates, the inclusion of detergent bases


spiked with enzymes in the training set improves the prediction capability of
the model when applied to real samples.

IX.3.4. Digestos con tripsina

IX.3.4.1. Comparacin de columnas microparticuladas y monolticas para RPLC de digestos trpticos de enzimas industriales en productos de
limpieza
Se ha realizado un estudio comparativo de las prestaciones de diversos
soportes cromatogrficos comerciales, incluyendo tanto columnas monolticas
(una polimrica y otra de base slice) como columnas particuladas (dos basadas
en tecnologa convencional de partculas y una conteniendo partculas de ncleo
fundido), para el anlisis mediante HPLC-UV de digestos trpticos de las
enzimas ms comunes en la industria de los detergentes. Se desarroll tambin
un procedimento para el control de calidad de las enzimas intactas en
concentrados industriales, utilizando para ello la columna monoltica polimrica.
Sin embargo, esta columna proporcion unas bajas eficacias y resolucin global
en la separacin de los pptidos obtenidos por digestin con tripsina. Para este
fin, la columna Kinetex, empaquetada con partculas de 2,6 m, con un ncleo
fundido no poroso y una superficie porosa (tecnologa de partculas shell-core),

359

Miriam Beneito Cambra

fue la que mostr las mejores prestaciones. La columna monoltica de slice


Chromolith, y columnas convencionales empaquetadas con partculas de 5 y 3
m mostraron prestaciones intermedias. La columna Kinetex en las condiciones
ptimas de elucin se utiliz para el anlisis de los digestos trpticos de los
concentrados de materias primas industriales, bases de detergente aditivadas y
productos de limpieza comerciales. Los cromatogramas resultantes fueron muy
similares a los obtenidos directamente por la digestin de los concentrados
industriales de enzimas, por lo que no se observaron interferencias ni efecto
matriz.
El mtodo cromatogrfico propuesto es til tambin para la clasificacin
de las enzimas. Actualmente se est trabajando para mejorar este mtodo,
estableciendo los pptidos que llevan a una mejor identificacin y clasificacin
fiable de las enzimas a partir de datos de LC-MS.

The

chromatographic

performance

of

several

commercial

chromatographic supports, including monolithic (polymeric and silica-based)


and particulate columns (with conventional and core-shell particle technology),
for the HPLC-UV analysis of tryptic digests of enzymes commonly used in the
detergent industry was comparatively evaluated. In addition, quality control of
industrial enzymes concentrates was shown to be possible by chromatographing
the intact enzymes on the polymeric monolithic column (ProSwift). However, this
column gave a poor efficiency and a low global resolution for the separation of
the peptides obtained by trypsin digestion. The Kinetex column, packed with
superficially porous sub-3 m particles (shell-core particle technology), showed
the best performance when applied to the separation of tryptic digests of the
enzymes. The Chromolith silica monolithic column, and columns packed with 5
m or 3 m conventional particles showed intermediate performances. The

360

Captulo IX. Conclusiones generales

Kinetex column in the optimal elution conditions was applied to the analysis of
tryptic digests from both raw industrial concentrates of the enzymes as well as to
enzymes in spiked detergent bases and commercial cleaners. The resulting
chromatograms were quite similar to those obtained by directly digesting the
industrial enzyme concentrates; then matrix interferences were not observed.
Therefore, HPLC of intact enzymes is useful in the quality control of
industrial samples, as well as to discard enzyme classes; however, a more
selective approach is required for a reliable classification and identification of
the enzyme. For this purpose, HPLC of the tryptic digest of the enzyme using a
core-shell particulate column is an excellent option. Further work on this topic,
in order to establish characteristic peptides leading to the reliable identification
of the individual enzymes by coupling LC with MS is in progress.

Main points:
-

A polymeric monolithic column (ProSwift) is useful for the separation of


intact enzymes, giving rise to chromatograms which show a predominant
peak. These chromatograms are useful in quality control; however, they do
not provide enough information to reliably classify or identify the enzymes.

To separate the peptides obtained by enzyme digestion with trypsin, the


polymeric monolithic column provides low efficiencies and poor overall
resolution.

For the separation of tryptic digests of enzymes, a Kinetex column showed an


excellent performance. Columns packed with conventional 5 or 3 m
particles, and a silica monolithic column (Chromolith), presented
intermediate performances.

361

Miriam Beneito Cambra

No interference or matrix effects were observed in the separation of tryptic


digests of industrial enzyme concentrates, detergent bases spiked with
enzymes and commercial cleaning products, with the Kinetex column.

IX.4. Columnas monolticas

IX.4.1. Columnas monolticas de lauril metacrilato para CEC

IX.4.1.1. Columnas monolticas de LM para CEC utilizando LPO como


iniciador
Se ha optimizado la preparacin de columnas monolticas de LMA
mediante fotopolimerizacin UV en presencia de LPO como iniciador. Se ha
investigado la influencia de los cocientes monmeros/disolventes porognicos,
1,4-butanodiol/1-propanol y LMA/EDMA sobre las propiedades morfolgicas y
sobre las prestaciones de las columnas en condiciones de uso en modo CEC.
Utilizando LPO como iniciador, se compar la polimerizacin por va UV con la
polimerizacin trmica. Ambos mtodos proporcionaron columnas con una
eficacia similar, sin embargo, las columnas fotopolimerizadas mostraron menores
tiempos de anlisis que las iniciadas trmicamente. Otras ventajas de la
fotoiniciacin respecto a columnas obtenidas por polimerizacin trmica es el
menor tiempo de fabricacin de la columna, y la mayor permeabilidad de la
misma.
Por otra parte, se estudi la sustitucin del LPO por AIBN como iniciador
en la sntesis de columnas monolticas obtenidas por fotoionizacin. Bajo sus
respectivas condiciones ptimas de polimerizacin, los dos tipos de monolitos
proporcionaron eficacias semejantes. Las columnas obtenidas utilizando LPO
como iniciador dieron lugar a tiempos de anlisis eran menores. Asimismo, se

362

Captulo IX. Conclusiones generales

evalu el potencial de las columnas obtenidas con ambos iniciadores para separar
compuestos tanto neutros como bsicos. Teniendo en cuenta los resultados
obtenidos, el empleo de LPO y radiacin UV es la mejor opcin para la
preparacin

rpida

de

monolitos

de

LMA

con

buenas

prestaciones

cromatogrficas.

The preparation of photo-polymerized LMA monoliths in the presence of


LPO as initiator has been optimized, and the resulting columns have been
characterized. The influence of ratios of monomers/porogenic solvents, 1,4butanediol/1-propanol and LMA/EDMA on the morphological and CEC
properties was investigated. Under their respective optimal conditions, photoand thermal-polymerization using LPO gave rise to columns with similar
efficiencies; however, photo-polymerized columns showed shorter analysis times.
Other advantages of photo-initiation are shorter polymerization times and better
permeabilities.
Additionally, the LMA-based monolithic columns photoinitiated with
either LPO or AIBN were compared. Under their respective optimal
polymerization conditions, both types of monoliths provided similar efficiencies,
but those prepared in the presence of LPO gave faster separations than their
AIBN counterparts. The capability of columns obtained with both initiators to
separate neutral and basic compounds has been also demonstrated. Thus, the
employ of LPO and UV irradiation constitutes an attractive way for the fast
preparation of improved LMA-based monoliths.

Main points:
-

Using LPO as initiator, photopolymerization gives rise to better columns than


thermal polymerization. Efficiencies were similar, but analysis times were

363

Miriam Beneito Cambra

shorter with photoinitiated columns. Also, manufacturing time was shorter,


and the monoliths showed a higher permeability.
-

Using photoinitiation in the presence of either LPO or AIBN, similar


efficiencies were obtained; however, LPO led to shorter analysis times than
AIBN.

Excellent columns are quickly prepared using photoinitiation in the presence


of LPO.

IX.4.1.2. Comparacin de fotoiniciadores para la preparacin de columnas


monolticas de metacrilato para CE
Se ha estudiado la preparacin y caracterizacin de columnas monolticas
de LMA para CEC por fotopolimerizacin, utilizando varios iniciadores
radicalarios a distintas concentraciones. El tipo y la variacin del contenido del
iniciador produce cambios en la estructura monoltica. As, el tamao de los
glbulos puede variar, lo que influye en las propiedades electrocromatogrficas
de los monolitos. Por lo tanto, es importante encontrar una concentracin ptima
para cada uno de los fotoiniciadores investigados. En las respectivas condiciones
ptimas, se obtuvieron eficacias y resoluciones similares para los cuatro
fotoiniciadores estudiados. Los monolitos polimerizados con AIBN y DMPA
mostraron separaciones ligeramente mejores que aquellos polimerizados con
BPO y LPO; sin embargo, este ltimo iniciador proporcion menores tiempos de
anlisis y la posibilidad de controlar mejor las propiedades cromatogrficas del
monolito. Por otro lado, la peor repetibilidad columna-columna se encontr para
los lechos cromatogrficos fotoiniciados con BPO.

The preparation and characterization of LMA-based monolithic columns


for CEC by photo-polymerization using several free radical initiators have been

364

Captulo IX. Conclusiones generales

described. The influence of each type of initiator and its concentration in the
polymerization mixture was studied. As a result of this study, we have found that
the type and variation of initiator content can produce changes in the monolithic
structure, leading to an increase, a small influence, or a decrease on the final
globule size, and therefore, variations in the electrochromatographic properties
of monoliths. Consequently, it is important to find an optimum concentration for
each photo-initiator investigated. Thus, under their respective optimum
conditions, similar efficiencies and resolutions were obtained for the four
investigated photo-initiators. The monoliths initiated with AIBN and DMPA
showed slightly better separations than BPO and LPO, however, this latter
initiator provided shorter analysis time jointly with a fine control in the retention
properties. Additionally, the highest RSD values found for column-to-column
repeatabilities were obtained for beds photo-initiated with BPO.

Main points:
-

The type and concentration of the initiator may cause changes in the
structure of the monoliths obtained by photopolymerization, which implies
variations in the CEC properties.

Each photoinitiator has its own optimal concentration.

The four photoinitiators which have been studied show similar efficiencies
and resolutions. The monoliths polymerized with AIBN and DMPA showed
separations slightly better than those polymerized with BPO and LPO;
however, LPO provided the shortest analysis time for the test compounds. On
the contrary, BPO provided the worst column to column reproducibility.

365

CAPTULO X

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397

ABREVIATURAS
A
AB

Azul cido / Acid blue

ABS

Alquilbenceno sulfonatos / Alkylbenzenesulfonates

ACN

Acetonitrilo / Acetonitrile

AES

Alquil ter sulfatos / Alkyl ether sulfates

AIBN

,-azobisisobutironitrilo / ,-azobisisobutyronitrile

Ala

Alanina / Alanine

Alc

Alcalase 2.5 L

AMG

Alquilmonoglucsido / Alkylmonoglucoside

AMGf

Alquilmonoglucofuransido / Alkylmonoglucofuranoside

AMGp

Alquilmonoglucopiransido / Alkylmonoglycopyranoside

ANN

Red neuronal artificial / Artificial neuronal network

ANOVA

Anlisis de la varianza / Analysis of variance

AOS

-olefina sulfonatos / -olefin sulfonates

APCI

Ionizacin qumica a presin atmosfrica /


Atmospheric pressure chemical ionization

APE

Alquilfenol etoxilados / Alkylphenol ethoxylates

APES

Alquil fenol ter sulfatos / Alkyl phenol ether sulfates

APG

Alquilpoliglucsidos / Alkylpolyglucosides

APPI

Fotoionizacin a presin atmosfrica /


Atmospheric pressure photoionization

Arg

Arginina / Arginine

AS

Alquil sulfatos / Alkyl sulfates

Asn

Asparagina / Asparagine
399

Asp

cido asprtico / Aspartic acid

B
BGE

Electrolito de fondo / Background electrolyte

BPO

Perxido de dibenzoilo / Dibenzoyl peroxide

BSA

Albmina srica bovina / Bovine serum albumine

C
Car

Carezyme 4500L

CE

Electroforesis capilar / Capillary electrophoresis

CEC

Electrocromatografa capilar /
Capillary electrochromatography

Cel

Celluzyme 0,7T

CGE

Electroforesis capilar en gel / Capillary gel electrophoresis

CI

Ionizacin qumica / Chemical ionization

CID

Cationizacin seguida de colisin inducida /


Cationization followed by collision-induced

CIEF

Isoelectroenfoque capilar / Capillary isoelectric focusing

CMC

Concentracin micelar crtica /


Critical micelar concentration

CR

Rojo Congo / Congo red

Cys

Cistena / Cysteine

CZE

Electroforesis capilar zonal / Capillary zone electrophoresis

D
DAD

Detector de fila de diodos / Diode array detector

DMPA

2,2-dimetoxi-2-fenilacetofenona /
2,2-dimethoxy-2-phenylacetophenone

400

DMSO

Dimetilsulfxido / Dimethilsulfoxide

DNA

cido desoxiribonucleico / Deoxyribonucleic acid

DP

Longitud de la cadena de poliglucsido /


Degree of polymerization

DTI

Inhibidor de la transferencia de color / Dye-transfer inhibitor

Dur

Duramyl 300L, Type DX

E
EDMA

Dimetilacrilato de etileno / Ethylene dimethacrylate

EI

Impacto electrnico / Electron impact

EIC

Cromatograma de ion extrado /


Extracted ion chromatogram

ELSD

Detector evaporativo de luz dispersada /


Evaporative light scattering detection

End

Endolase 5000L

EO

xido de etileno / Ethylene oxide

EOF

Flujo electroosmtico / Electroosmotic flow

ESI

Ionizacin por electronebulizacin / Electrospray ionization

Esp

Esperase 8.0L

EtOH

Etanol / Ethanol

Eve

Everlase 16L, Type EX

F
FAA

Alcanolamidas / Fatty acid amide ethoxylates

FAB

Bombardeo con tomos rpidos / Fast atom bombardment

FAE

Alcoholes grasos etoxilados / Fatty alcohols ethoxylates

FID

Detector de ionizacin por llama / Flame ionization detector

401

FSCE

Electroforesis capilar en disolucin libre /


Free solution capillary electrophoresis

FT

Transformada de Fourier / Fourier transform

G
GC

Cromatografa de gases / Gas chromatography

Gf

Glucofuransido / Glucofuranoside

Gln

Glutamina / Glutamine

Glu

cido glutmico / Glutamic acid

Gly

Glicina / Glycine

Gp

Glucopiransido / Glucopiranoside

H
HAcO

cido actico / Acetic acid

HEC

Hidroxietilcelulosa / Hydroxyethylcellulose

HILIC

Cromatografa de interaccin hidroflica /


Hydrophilic interaction chromatography

His

Histidina / Histidine

HPLC

Cromatografa lquida de alta resolucin /


High performance liquid cromatography

I
ICP

Plasma acoplado por induccin / Inductively coupled plasma

ID

Dimetro interno / Internal diameter

IDA

cido iminodiactico / Iminodiacetic acid

Ile

Isoleucina / Isoleucine

IT

Trampa inica / Ion trap

402

L
LAS

Alquilbenceno sulfonatos lineales /


Linear alkylbenzenesulfonates

LC

Cromatografa lquida / Liquid cromatography

LDA

Anlisis discriminante lineal / Linear discriminant analysis

LES

Lauril ter sulfatos / Lauryl ether sulfates

Leu

Leucina / Leucine

LMA

Lauril metacrilato / Lauryl methacrylate

LOD

Lmite de deteccin / Limit of detection

LPO

Perxido de lauroilo / Lauroyl peroxide

Ls

Lipolase 100L, Type EX

Lx

Lipex 100L

Lys

Lisina / Lysine

M
MALDI

Desorcin-ionizacin lser asistida por matriz /


Matrix-assisted laser desorption/ionization

MEKC

Cromatografa micelar electrocintica capilar /


Micellar electrokinetic capillary chromatography

MeOH

Metanol / Methanol

Met

Metionina / Methionine

META

Cloruro de [2-(metacriloiloxi)etil] trimetilamonio /


[2-(methacryloyloxy)ethyl]trimethyl ammonium chloride

MGf

Monoglucofuransido / Monoglucofuranoside

MGp

Monoglucopiransido / Monoglucopyranoside

MS

Espectrometra de masas / Mass spectrometry

MW

Peso molecular / Molecular weight

403

N
NaAcO

Acetato sdico / Sodium acetate

NAC

N-acetil-cistena / N-acetyl-cysteine

NECEEM

Electroforesis capilar en condiciones de no equilibrio de


mezclas en equilibrio / Non-equilibrium capillary
electrophoresis of equilibrium mixtures

NH4AcO

Acetato amnico / Amonium acetate

NI

Ion negativo / Negative ion

NMR

Resonancia magntica nuclear / Nuclear magnetic resonance

NP

Fase normal / Normal phase

NPE

Nonilfenoles etoxilados / Nonylphenol ethoxylates

O
OD

Dimetro externo / Outside diameter

OPA

o-Ftaldialdehdo / o-Phthaldialdehyde

OPE

Octifenoles etoxilados / Octylphenol ethoxylates

P
PAH

Hidrocarburo poliaromtico / Polyaromatic hydrocarbon

PEA

o-fosfoetanolamina / o-phosphoethanolamina

PEEK

Poli-ter-ter-cetona / Polyether ether ketone

PEG

Polietilenglicol / Polyethylenglycol

Phe

Fenilalanina / Phenylalanine

PI

Ion positivo / Positive ion

Pro

Prolina / Proline

PVA

Polivinil alcohol / Polyvinyl alcohol

PVAcO

Acetato de polivinlo / Polyvinyl acetate


404

PVP

Polivinil pirrolidona / Polyvinyl pyrrolidone

PVP-NO

Poli(1-xido-4-vinilpiridina) / Poly(4-vinylpyridine-1-oxide)

PVP-PVI

Poli(1-vinilpirrolidona-co-1-vinilimidazol) /
Poli(1-vinylpyrrolidona-co-1-vinylimidazole)

Q
QDA

Anlisis discriminante cuadrtico /


Quadratic discriminant analysis

QTOF

Cuadrupolo-tiempo de vuelo / Quadrupole time-of-flight

R
RG

Resolucin global / Global resolution

RP

Fase reversa o inversa / Reverse phase

RSD

Desviacin estndar relativa / Relative standard deviation

S
Sav

Savinase 16L, Type EX

SAX

Intercambio aninico fuerte / Strong anionic exchange

SDS

Dodecil sulfato sdico / Sodium dodecil sulfate

SEC

Cromatografa de exclusin por tamao /


Size exclusion chromatography

SEM

Microscopa electrnica de barrido /


Scanning electron microscope

Ser

Serina / Serine

SFC

Cromatografa de fluidos supercrticos /


Supercritical fluid chromatography

SIM

Monitorizacin de iones seleccionados /


Selected ion monitoring

405

SIMS

Espectrometra de masas de iones secundarios /


Secondary Ion Mass Spectrometry

SPE

Extraccin en fase slida / Solid phase extraction

Sta

Stainzyme 12L

T
TAG

Triacilgliceroles / Triacilgliceride

Ter

Termamyl Ultra 300L

TFA

cido trifluoroactico / Trifluoroacetic acid

Thr

Treonina / Threonine

TIC

Cromatograma de iones totales / Total ion chromatogram

TLC

Cromatografa en capa fina / Thin layer chromatography

TMBA

cido 3,4,5-trimetoxibenzoico /
3,4,5-trimethoxibenzoic acid

TMS

Trimetilsilano / Trimethylsilane

Tris

Tris(hidroximetil)aminoetano /
Tris(hydroxymethyl)amino ethane

Trp

Triptfano / Tryptophan

Tyr

Tirosina / Tyrosine

U
UV

Ultravioleta / Ultraviolet

V
Val

Valina / Valine

Vis

Visible / Visible

406

ANEXO I

A nexo I

A3

M iriam B eneito Cam bra

A4

A nexo I

A5

M iriam B eneito Cam bra

A6

A nexo I

A7

M iriam B eneito Cam bra

A8

A nexo I

A9

ANEXO II

A nexo II

A13

M iriam B eneito Cam bra

A14

A nexo II

A15

M iriam B eneito Cam bra

A16

A nexo II

A17

M iriam B eneito Cam bra

A18

A nexo II

A19

M iriam B eneito Cam bra

A20

A nexo II

A21

M iriam B eneito Cam bra

A22

A nexo II

A23

M iriam B eneito Cam bra

A24

A nexo II

A25

M iriam B eneito Cam bra

A26

A nexo II

A27

ANEXO III

A nexo III

A31

M iriam B eneito Cam bra

A32

A nexo III

A33

M iriam B eneito Cam bra

A34

A nexo III

A35

M iriam B eneito Cam bra

A36

A nexo III

A37

M iriam B eneito Cam bra

A38

ANEXO IV

A nexo IV

A41

M iriam B eneito Cam bra

A42

A nexo IV

A43

M iriam B eneito Cam bra

A44

A nexo IV

A45

M iriam B eneito Cam bra

A46

A nexo IV

A47

M iriam B eneito Cam bra

A48

ANEXO V

A nexo V

A51

M iriam B eneito Cam bra

A52

A nexo V

A53

M iriam B eneito Cam bra

A54

A nexo V

A55

M iriam B eneito Cam bra

A56

A nexo V

A57

M iriam B eneito Cam bra

A58

ANEXO VI

A nexo V I

A61

M iriam B eneito Cam bra

A62

A nexo V I

A63

M iriam B eneito Cam bra

A64

A nexo V I

A65

M iriam B eneito Cam bra

A66

A nexo V I

A67

M iriam B eneito Cam bra

A68

ANEXO VII

A nexo V II

A71

M iriam B eneito Cam bra

A72

A nexo V II

A73

M iriam B eneito Cam bra

A74

A nexo V II

A75

M iriam B eneito Cam bra

A76

ANEXO VIII

A nexo V III

A79

M iriam B eneito Cam bra

A80

A nexo V III

A81

M iriam B eneito Cam bra

A82

A nexo V III

A83

ANEXO IX

A nexo IX

A87

M iriam B eneito Cam bra

A88

A nexo IX

A89

M iriam B eneito Cam bra

A90

A nexo IX

A91

M iriam B eneito Cam bra

A92

A nexo IX

A93

M iriam B eneito Cam bra

A94

A nexo IX

A95

ANEXO X

A nexo X

A99

M iriam B eneito Cam bra

A100

A nexo X

A101

M iriam B eneito Cam bra

A102

A nexo X

A103

M iriam B eneito Cam bra

A104

A nexo X

A105