Journal of Membrane Science 254 (2005) 1319

A molecular imprinted membrane for molecular discrimination of

tetracycline hydrochloride
F. Trotta
a,
, C. Baggiani
b
, M.P. Luda
a
, E. Drioli
c
, T. Massari
a
a
Dipartimento di Chimica Inorganica, Chimica Fisica e Chimica dei Materiali dellUniversit ` a, Via Pietro Giuria 7, 10125 Torino, Italy
b
Dipartimento di Chimica Analitica dellUniversit ` a, Via Pietro Giuria 5, 10125 Torino, Italy
c
Dipartimento di Ingegneria Chimica e dei Materiali dellUniversit ` a della Calabria, 87030 Arcavacata di Rende, Italy
Received 3 September 2004; received in revised form 25 October 2004; accepted 17 November 2004
Available online 2 April 2005
Abstract
Membranes of poly(acrylonitrile-co-acrylic acid) made by phase inversion technique in the presence of tetracycline hydrochloride showed
molecular recognition behavior towards the template molecule. The interactions of the carboxylic groups of the polymer chain played an
important role in the discrimination process. Prolonged washing with distilled water regenerated the membrane without any loss of binding
capacity. Chloramphenicol, of similar polarity and fair solubility in water, is recognized less efciently.
2004 Elsevier B.V. All rights reserved.
Keywords: Molecular imprinting; Molecular recognition; Tetracycline; Membrane; Antibiotic drugs
1. Introduction
Polymers made by means of the molecular imprinting
technique [1] are outstanding materials for discrimination
and separation of individual substances and they are more
frequently obtained by a high cross-linked co-polymerization
reaction in the presence of a selected template molecule. Sub-
sequent removal of the imprinted molecule leads to the for-
mation of complementary imprinted sites that often show
molecular discrimination properties. Two general procedures
are frequently used. In covalent imprinting, rst proposed
by Wulff and Sarhan [2], a template-monomer-complex is
formed by a covalent bond and polymerized; a successive
selective breakage of the covalent bond generates a poly-
mer network, which is able to recognize the imprinted tem-
plate. In non-covalent imprinting, proposed by Mosbach and
coworker [3], a highly cross-linked polymer interacts with the
template molecule simply by electrostatic interaction, hydro-
gen bonds or similar non-covalent bonds; after washing out

Corresponding author. Tel.: +39 011 6707550; fax: +39 011 6707855.
E-mail address: francesco.trotta@unito.it (F. Trotta).
of the retained template, the polymer shows the formation of
complementary sites with signicant molecular recognition
properties.
Recently, Kobayashi and coworkers [47] reported that
also membranes obtained by means of the phase inversion
technique in the presence of the imprinted molecule allow
molecular recognition. By using this technique, molecular
imprinted membranes were made for molecular recognition
of hardly water soluble theophylline [5] and naringin [8].
Common features to these molecules are the small solubility
in water (0.10.8 wt.%at rt) and the absence of a ionic charge
in their structure.
In this work we have considered the tetracycline
hydrochloride (TCH) [9]: ((4S-(48,1,4a,5a,612a))-
4-(dimethylamino)-1,4,4a,5,5a,6,11,12a-octahydro-3,6,10,
12,12a-pentahydroxy-6-methyl-1,11-dioxo-2-napthacenec-
arboxamide monohydrochloride). This is a water-soluble
compound that shows a neat positive charge on its molecular
structure. TCH is a well-known antibiotic molecule that has
been used as a drug for treatment of infections caused by
gram-positive microorganisms when bacteriological testing
indicated appropriate susceptibility to the drug. TCH is often
0376-7388/$ see front matter 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.memsci.2004.11.013
14 F. Trotta et al. / Journal of Membrane Science 254 (2005) 1319
an alternative drug for people allergic to penicillin. More
recently it seems that TCH could be useful in the treatment
of anthrax infections. The presence of traces of this drug
(and related ones) in meat, milk and eggs has been mostly
detected in developed countries. As a consequence, these
chemicals cause serious problems for human health and
represent an important target in analyses [10]. In the light
of this issue new methods for easy and selective extraction
and/or pre-concentration could be of interest.
In this work we prepared a molecular imprinted mem-
brane from a viscous solution of a suitable co-polymer. This
membrane was able to recognize the TCH to a relevant
extent.
2. Experimental
Tetracycline hydrochloride (TCH), chloramphenicol (i.e.
d-(-)threo-2,2-dichloro-N-[-hydroxy--(hydroxymethyl)-
-(4-nitrophenyl)ethyl] acetamide reagents and solvents
were obtained from SigmaAldrichFluka (USA) Stabiliz-
ing agent, namely hydroquinone mono methyl ether, was
removed from commercial acrylonitrile and acrylic acid by
distillation under vacuum.
The poly(acrylonitrile-co-acrylic acid) co-polymer,
P(AA-co-AN), was 16.16 mol% of acrylic acid (based on
elemental analyses and on acid titration in DMSO) [11] and
was synthesized according to the reported method [4] with
minor modications; the feed was 6/1 AN/AA mol/mol.
A block AA/AN co-polymer, poly(acrylic acid)-block-
polyacrylonitrile (PAA-block-PAN), was synthesized by
living radical co-polymerization by using dithiocarbamate
iniferter method [12]. UVvis spectra were performed on a
Perkin-Elmer 15 spectrophotometer. FT-IR spectra were
run in transmission mode on a thin lm of the co-polymer
by using a Perkin-Elmer 2100 spectrophotometer.
The membranes were prepared by phase inversion tech-
nique [13] which permits the production of membranes with
asymmetric pore structure. In a typical experiment a solution
(8 wt.%) of P(AA-co-AN) was prepared by dissolving the
co-polymer in DMSO. The casting solution was centrifuged
for 15 min at 4000 rpm in order to remove unwanted pos-
sible macroscopic impurities. The co-polymer-DMSO solu-
tion was cast on a glass plate with a Gardner knife. The cast
polymer was then immersed at 24

C in a coagulation bath
of 5 l of distilled water, which is a non-solvent for the co-
polymer, and transferred to fresh distilled water after 15 min.
Membranes of about 200 mof thickness resulted fromthese
operation.
The membranes containing the TCH template were pre-
pared by the same methodology but adding the requested
amount of the template molecule (2 wt.%). Additional mem-
branes with known amounts of TCH (T1 =2%, T3 =6%,
T4 =8%) were prepared for IR analyses. The membranes
were stored in distilled water. TCH was then removed from
the membranes by prolonged washing with distilled water up
to the TCH absorption at 359 nm was no further detectable
in the UVvis.
The viscosity measurements were carried out on an Ubbe-
lohde type viscometer by using anhydrous DMSO or DMF
as a solvents at 30 and 25

C, respectively. Based on viscosity

coefcients calculated from data of Ref. [14], the molecular
weight (Mn) of the present P(AA-co-AN) co-polymer was
roughly estimated 71,000 amu.
The permeation experiments were carried out in a Mil-
lipore stainless steel lter holder into which the mem-
brane was placed (15.9 cm
2
area). A water solution of TCH
(5.2 mol/l =2.5 mg/l) was passed through the membrane by
means of a peristaltic pump (Masterex 7013-20) with a ow
rate of 0.45 ml/min.
An UVvis calibration curve of TCH was made previ-
ously; the content of TCH permeated through the membrane
was determined by the absorbance at 359 nm ( =10,536 at

max
359 nm).
The same procedure was also used for chloramphenicol
solution whose value at
max
278 nm is 9547. Permeation
curves were performed at least in triplicate with experimental
error less than 5%.
A SEM scanning electron Microscope Leica Stereoscan
420 was used for membrane micrographs.
3. Results and discussion
Water was the coagulant in phase inversion preparation of
the imprinted membranes. Although TCHis freely soluble in
water a relevant part of the template molecule was retained
in the solidied membrane. TCH is quite a rigid molecule
(Fig. 1) with several hydroxyls and polar groups that allowthe
formation of strong hydrogen bonds with the carboxylic acid
groups of the co-polymer, as schematically shown in Fig. 2.
Probably, the immediate coagulation of the viscous DMSO
solution in water promotes the partial entrapment of the tem-
plate in the membrane. Complete removal of the template,
leading to the generation of the complementary imprinted
sites for TCH, could be only accomplished by prolonged
washing with distilled water until no detectable amount of
TCH could be revealed by UV analyses. No efforts were
made in order to determine the amount of the template re-
leased. The membranes were mainly characterized by FT-IR
analyses.
Fig. 1. Molecular structure of tetracycline.
F. Trotta et al. / Journal of Membrane Science 254 (2005) 1319 15
Fig. 2. Illustration of membrane imprinted site formation for tetracycline.
The FT-IR spectrum of the original P(AA-co-AN) is de-
picted in Fig. 3A together with that of a PAA-block-PAN
co-polymer (Fig. 3B). Assignments of the main absorption
are given in Table 1 according to [1416].
Fig. 3. IR spectra of P(AA-co-AN) obtained by radical co-polymerisation
(A) and of (PAA-co-PAN) by living radical co-polymerisation (B).
The IR spectrum of P(AA-co-AN) strictly resembles that
of a similar co-polymer reported in Refs. [7,17]. However,
it should be noted that the C O stretching shows absorption
somewhat displaced to higher frequencies (1730 cm
1
) than
that expectedfor a saturatedcarboxyl acid(17101705 cm
1
)
as shown in Ref. [15] for IR of poly(acrylic acid). The
dimer form of the carboxyl is highlighted by the broad ab-
sorption at 3223 and 2521 cm
1
[15,16]. In addition a shift
Table 1
Assignment of the main absorption of the P(AA-co-AN) co-polymer
Position Assignment Structure References
3520 Free OH AA [15,16]
3223 OH dimer AA [15,16]
2521 COOH dimmer AA [15,16]
2242 CN AN [15,16]
17301717 C O (monomer +dimer) AA [15,16]
1452 CH
2
AN [15,16]
1248 C O (dimer) AA [16]
1160 C O (monomer) AA [16]
16 F. Trotta et al. / Journal of Membrane Science 254 (2005) 1319
from11301180 to 12801315 cm
1
of the C Osingle bond
stretching is reported for the dimer form in Ref. [16]. All ab-
sorption of the dimer are more evident in the spectrum of the
PAA-block-PAN co-polymer than in that of P(AA-co-AN).
The blue shift of the C O bond is here observed at some-
what shorter wavelength (1245 cm
1
)
.
It is likely that COOH
dimers are formed to a major extent in the block co-polymer
rather than in P(AA-co-AN) because of the long sequences
of adjacent COOH in the block structure. On the other hand
P(AA-co-AN) is a statistic co-polymer with the structural
units of acrylic acid (AA) randomly distributed along the
chains making the dimer formation less probable. The statis-
tic structure of the P(AA-co-AN) co-polymer is supported
by the reactivity ratios r
AN
and r
AA
calculated both by Q
e scheme [18] (r
AN
=0.370.50; r
AA
=1.82.2) and by ele-
mental analysis [19] (r
AN
=0.97; r
AA
=3.79). Microstructure
determined by NMR [19] shows that a constant feed compo-
sition AN/AA6 mol/mol results in more than one-third of the
AA units in between two AN units in the co-polymer. Adja-
cent COOH should be suitable for dimer formation whereas
isolated COOH should be easier available to interaction with
the template.
The OHstretching region of the IRspectra of the template
containing membrane which contains the template (Fig. 4a)
Fig. 4. (a) IR spectra in the range 40002000 cm
1
of P(AA-co-AN)
membrane and of the imprinted membranes at different TCH/P(AA-co-
AN) weight ratio: T1 =2/8; T3 =6/8; T4 =8/8. (b) IR spectra in the range
22501100 cm
1
of P(AA-co-AN) membrane and of the imprinted mem-
branes at different TCH/P(AA-co-AN) weight ratio: T1 =2/8; T3 =6/8;
T4 =8/8.
is modied by the introduction of the template; in particu-
lar a strong reduction of the dimer absorption at 3223 and
2521 cm
1
only occurs for the membrane with the larger
amount of template. Thus, it appears that the rst molecules
of templates are blocked by the free COOH. Further intro-
duction of template molecules involves interaction with the
dimer form.
The 20001000 cm
1
region of the IRspectra of the mem-
branes that contains the template (Fig. 4b) also shows distinc-
tive features in comparison to that of simple P(AA-co-AN).
Indeed, the intensity of the carbonyl stretching at 1730 cm
1
of acrylic acid is progressively reduced as the amount of tem-
plate increases. In the meantime, newabsorptions at 1568 and
1412 cm
1
clearly rise in their IR that can be assigned to the
out-of-phase and in-phase stretching of the carboxylate ion
COO

[15b, p. 292] where two equivalent bond-and-a-half

replace the original C O double bond. IR spectra of sodium
salts of polyacrylate reported in Ref. [15a] (no. 2078 and
3299) both show these absorptions instead of the acid car-
bonyl absorption in the 1700 cm
1
region; on the contrary,
the CN stretching maintains the same position (2242 cm
1
)
in all spectra.
It is likely that the interaction co-polymer/template occurs
through an ionic interaction between the AA units and TCH.
Bearing in mind its structure, we speculate that a partial and
not frequently observed protonation of the oxygen atom of
the amide group of TCH could occur [20]. Apparently the
acrylonitrile units do not participate in this interaction.
Moreover, the viscosity also is greatly inuenced by the
presence of the template molecule. Fig. 5 reports the intrinsic
viscosity obtained by Hugging relation for the co-polymer
alone and by adding 24 wt.% of TCH. The co-polymer
shows a high value of intrinsic viscosity (1.7 dl/g), in agree-
ment with the literature data [5]. Besides, Hugging relation
is correctly followed meaning that carboxyl groups are not
Fig. 5. Intrinsic viscosity of the P(AA-co-AN) co-polymer alone () and
with 2 wt.% () and () 4 wt.% of tetracycline hydrochloride.
F. Trotta et al. / Journal of Membrane Science 254 (2005) 1319 17
ionized and the co-polymer, at least in DMSOsolution, do not
behave as a polyelectrolyte [21]. By adding 2 wt.% of TCH
intrinsic viscosity of the solution declines to 1.5 dl/g. This
effect is magnied by doubling the amount of the template
molecule (4 wt.%) where intrinsic viscosity is only 0.8 dl/g.
To explain this behavior we suggest that the presence of the
TCH molecule favors the curling up of the single polymer
chain around the template, thus generating the imprinted site.
The viscosity data tend to exclude interaction of the template
with several macromolecules.
Further characterization was obtained carrying out SEM
analyses. As reported in Fig. 6 the cross-section of the dry
membrane containing 2 wt.%of TCHhas a typical asymmet-
ric structure with a very thin top layer (less than 10 m) and a
porous layer (Fig. 6a). Asimilar structure was also noticed for
the blank membrane although the latter shows smaller porous
cavities (Fig. 6b). In any case, both membranes behave like
a micro-ltration membrane.
By owing a water solution of TCH through the molec-
ular imprinted membrane (Fig. 7), the membrane could re-
tain TCHbecause the solute molecules bond to the imprinted
sites. According to [5], the retained amount of TCH could be
expressed by the following equation:
[S] =

(C
0
C
i
) V
i
/W
where [S] (mol/g) is the concentration of the TCH retained
in the membrane, C
0
the initial concentration (mol/l) of
TCH, C
i
the concentration (mol/l) at the time i, V
i
the vol-
ume of the sample (l), and W the dry weight (g) of the im-
printed membrane used.
Fig. 7 shows that saturation of the membrane occurs in less
than20 min. About 140 g(0.29 mol) of TCHwere retained
per gram of imprinted membrane. The theoretically avail-
able sites in 1 g of membrane, calculated from the amount of
template used in the fabrication of the imprinted membrane
Fig. 6. SEM microphotographs of non-imprinted membrane 1060 (a) and molecular imprinted membrane 829 (b).
18 F. Trotta et al. / Journal of Membrane Science 254 (2005) 1319
Fig. 7. Uptaken amount of tetracycline hydrochloride and chloramphenicol
in the imprinted membrane for tetracycline hydrochloride and non-imprinted
membrane. () tetracycline hydrochloride in to PAANnon-imprinted mem-
brane () tetracycline hydrochloride in the PAAN molecular imprinted
membrane () chloramphenicol in the PAANimprinted membrane () chlo-
ramphenicol in the non-imprinted membrane.
(80 wt.% of P(AA-co-AN) and 20% of TCH) are 416 mol.
On this basis we speculate that under the reported conditions
only about 1/1500 of the TCH imprinted sites are able to
show effective molecular recognition properties. Rather sur-
prisingly, this value is of the same order of magnitude as those
the reported for the imprinted membrane made for naringin
template molecule [8].
Thus, it seems that the number of the imprinted sites in
the membrane does not signicantly depend on the template
used, but could be a characteristic of the process at least for
the co-polymer employed. Probably, most of the binding sites
are not correctly formed or are not easy reachable in the bulk
of the membrane.
To test the selectivity of the imprinted membrane, we also
carried out some experiments by using another well-known
antibiotic molecule namely chloramphenicol (Fig. 8). It is
well known that chloramphenicol shows appreciable solu-
bility in water, like TCH; in addition, despite of its differ-
ent structure, the presence of hydroxyls and amide groups in
chloramphenicol, leads to a polarity a similar to that of TCH.
Bypassinga water solutionof chloramphenicol (5.34 mol/l)
through a molecular imprinted membrane made for TCH,
detected uptake of chloramphenicol (Fig. 7) is considerably
smaller thanthat for TCH(nomore than0.16 mol/gof mem-
brane), thus conrming the molecular recognition behavior
of the process.
Fig. 8. Molecular structure of chloramphenicol.
The same experiment carried out on a blank membrane
leads to a similar uptake amount of chloramphenicol. This
behavior could be ascribed to non-specic interaction of the
chloramphenicol with the membrane. Only the imprinting
procedure can generate the suitable sites for molecular dis-
crimination.
Because we detected comparable uptake of chlorampheni-
col both for the TCH imprinted membrane and for the blank
one, we speculate that the imprinted sites formed for TCH
are not suitable for a smaller molecule like chloramphenicol.
We point out that in previous works about molecular im-
printed membranes the phase inversion technique was effec-
tive in the molecular recognition of scarcely water-soluble
molecules such as theophylline and naringin. In this paper
we had fully proved that also the well soluble TCH could be
retained by a suitable imprinted membrane. Only prolonged
washing with distilled water is able to remove the template
molecule fromthe imprinted sites in the membrane. This sim-
ply means that rather strong global interactions between the
functional groups of the polymeric chain and the template
molecule are formed.
4. Conclusions
A molecular imprinted membrane for tetracycline
hydrochloride was made by using the precipitation phase in-
version technique. The resultant membrane shows molecu-
lar recognition properties towards the highly water-soluble
tetracycline hydrochloride. Another molecule that has simi-
lar solubility was less recognized. Simply prolonged washing
with distilled water easily restores the membrane.
Acknowledgement
This work was partially supported by MIURRome
(grant COFIN 2002).
References
[1] R.A. Bartsch, M. Maeda, Molecular and Ionic Recognition with Im-
printed Polymers, ACS Symposium Series 703, American Chemical
Society, 1998.
[2] G. Wulff, A. Sarhan, Use of polymers with enzyme-analogous struc-
ture for the resolution of racemates, Angew. Chem. Int. Ed. Engl.
11 (4) (1972) 341.
[3] R. Arshady, K. Mosbach, Synthesis of substrate-selective polymers
by hostguest polimerization, Macromol. Chem. 182 (1981) 687.
[4] T. Kobayashi, T. Nagai, et al., Molecular imprinted membranes pre-
pared by phase inversion of polyacrylonitrile copolymers containing
carboxylic acid groups, J. Membr. Sci. 86 (1994) 47.
[5] T. Kobayashi, H.Y. Wang, N. Fujii, Molecular imprintig of theo-
phylline in acrylonitrile-acrylic acid co-polymer membrane, Chem.
Lett. 10 (1995) 927.
[6] H.Y. Wang, T. Kobayashi, N. Fujii, Molecular imprinted membranes
prepared by the phase inversion precipitation technique, Langmuir
12 (1996) 4850.
F. Trotta et al. / Journal of Membrane Science 254 (2005) 1319 19
[7] T. Kobayashi, H.Y. Wang, N. Fujii, Molecular imprinted membranes
of polyacrylonitrile copolymers with different acrylic acid segments,
Anal. Chem. Acta 365 (1998) 81.
[8] F. Trotta, E. Drioli, C. Baggiani, D. Lacopo, Molecular imprinted
polymeric membrane for naringin recognition, J. Membr. Sci. 201
(2002) 77.
[9] H.P. Rang, M.M. Dale, Pharmacology, Churchill Livingstone, New
York, 1987 (Chapter 30).
[10] W.G. Huber, Veterinary Pharmacology and Therapeutics, 6th ed.,
Iowa State University Press, 1988.
[11] (a) M. Kolthoff, et al., Treatise on Analytical Chemistry, vol. 1A,
The Interscience Encyclopedia, 1959;
(b) T. Kurcharsky, L. Safarik, Tritations in Non-aqueous Solvents,
Elsevier, Amsterdam, 1965.
[12] B. Rueckert, A.J. Hall, B. Sellergren, Molecularly imprinted com-
posite materials via iniferter-modied supports, J. Mater. Chem. 12
(2002) 2275.
[13] R. Kesting, Synthetic Polymeric Membranes, vol. 7, 2nd ed., Wiley,
New York, 1995.
[14] P.F. Onyon, Molecular weights and intrinsic viscosities of solution
polymerized polyacrylonitrile, J. Polym. Sci. 37 (1959) 315.
[15] D.O. Hummel, F. Scholl (Eds.), Atlas of Polymer and Plastic Anal-
ysis, vol. 2, Carl Hanser Verlag, Munich, 1988 ([a] part b/I; [b] part
a1/a2 spectra 2078 and 3299).
[16] D. Lin-Vien, N.B. Colthup, W.G. Fately, J.G. Grasselli, The Hand-
book of Infrared and Raman Characteristic Frequencies of Organic
Molecules, Academic Press, San Diego, USA, 1991.
[17] H.Y. Wang, T. Kobayashi, T. Fukaya, N. Fujii, Molecular im-
print membranes prepared by the phase inversion precipitation tech-
nique. 2. Inuence of coagulation temperature in the phase inver-
sion process on the encoding in polymeric membranes, Langmuir 13
(1997).
[18] J. Brandup, E.H. immergut, E.A. Grulle (Eds.), Polymer Handbook,
Wiley, 1999 (section II).
[19] T. Kelen, F. Tudos, Analysis of the linear methods for determining
copolymerisation reactivity ratios. I. New improved linear graphic
method, J. Macromol. Sci. 9 (1975) 1.
[20] A. Bagno, G. Scorrano, Selectivity in proton transfer, hydrogen bond-
ing and solvation, Acc. Chem. Res. 33 (2000) 609.
[21] P.J. Flory, Principles of Polymer Chemistry, Cornell University Press,
1953, pp. 629637.