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Liquid Chromatography 1 and Solid-
Phase Extraction
Lecture Date: April 9
th
, 2008
Reading Material
Skoog, Holler and Crouch: Ch. 28
Cazes: Ch. 22, 26
For those using LC in their work, see:
L. R. Snyder, J . J . Kirkland, and J . L. Glajch, Practical HPLC
Method Development, 2
nd
Ed., Wiley, 1997.
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Basic LC Terminology
Adsorption chromatography
The stationary phase is an adsorbent (like silica gel or any other
silica-based packing)
The separation is based on repeated adsorption-desorption
steps.
Normal-phase chromatography
The stationary bed is strongly polar in nature (e.g., silica gel),
and the mobile phase is nonpolar (such as n-hexane or
tetrahydrofuran).
Polar samples are retained on the polar surface of the column
packing longer than less polar materials.
Reversed-phase chromatography
The stationary bed is nonpolar (hydrophobic) in nature,
The mobile phase is a polar liquid, such as mixtures of water
and methanol or acetonitrile.
The more nonpolar the material is, the longer it will be retained.
Size exclusion chromatography (SEC)
column filled with material having precisely controlled pore
sizes, and the sample is simply sieved or filtered according to
its solvated molecular size.
Larger molecules are rapidly washed through the column;
smaller molecules penetrate inside the pores of the packing
particles and elute later.
Also called gel permeation chromatography (GCP)
although the stationary phase is not restricted to a "gel"
Ion-exchange chromatography (IC)
the stationary bed has a charged surface of opposite charge
to the sample ions.
Used almost exclusively with ionic or ionizable samples.
The stronger the charge on the sample, the stronger it will be
attracted to the ionic surface and thus, the longer it will take
to elute
The mobile phase is an aqueous buffer, where both pH and
ionic strength are used to control elution time
Basic LC Terminology
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Analytical Applications of LC
- The branches of the LC family:
Note thismeansanalyte polarity
Basic Mechanisms used in LC Separations
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High Performance Liquid Chromatography (HPLC)
HPLC utilizes a high-pressure liquid mobile phase (ca.
100-300 bar) to separate the components of a mixture
These analytes are first dissolved in a solvent, and then
forced to flow through a packed small-particle
chromatographic column, where the mixture is resolved
into its components
HP =high pressure and high performance
Resolution depends upon the extent of interaction
between the solute components and the stationary
phase
Differences between HPLC and Classical LC
- Small ID (2-5 mm), reusable stainless steel columns
- Column packings with very small (3, 5 and 10 m)
particles and the continual development of new
substances to be used as stationary phases
- Relatively high inlet pressures and controlled flow of the
mobile phase
- Precise sample introduction without the need for large
samples
- Special continuous flow detectors capable of handling
small flow rates and detecting very small amounts
- Automated standardized instruments
- Rapid analysis
- High resolution
- From now on, LC refers to HPLC
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Advantages and Disadvantages of LC
Advantages:
Speed (minutes)
High resolution
Sensitivity
Reproducibility
Accuracy
Automation
Disadvantages:
Cost
Complexity
Low sensitivity for some compounds
Irreversibly adsorbed compounds not detected
Co-elution difficult to detect
More on Reversed-phase (RP) LC
- RP is the most widely used mode of HPLC (75%?)
- Separates molecules in solution on basis of their
hydrophobicity
Non-polar stationary phase
Polar mobile phase
- In practice: non polar functional group bonded to silica
Stationary phase
- functional group bonded to silica
- this corresponds to a volume (Van deemter)
- Alkyl groups ( C4, C8, C18)
- retention increases exp. with chain length
- Mobile Phases
Polar solvent (water) with addition of less polar solvent (acetonitrile
or methanol)
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The Packed Column and the Stationary Phase
- Packed LC columns, usually made of stainless steel and
carefully filled with material, are the heart of the LC
experiment
- The stationary phase fills the column its properties are
critical to the separation
Review of Molecular Interactions
- The basis of separations (and most of chemistry)
Name Energy (kcal/mol) Description
Covalent 100-300
Hold molecules together, orbital
overlap
Ionic 50-200 Electrostatic attraction
Polar
Hydrogen bonding
Dipole-dipole
t-stacking
3-10
Vary from electrostatic-type
interactions (e.g. hydrogen
bonds) to much weaker
Non-Polar
Van der Waals
(dispersion)
1-5 Weak, induced dipole
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Retention Mechanisms in LC
HPLC is a dynamic adsorption process. Analyte molecules, while
moving through the porous packing bead, tend to interact with the
surface adsorption sites. Depending on the HPLC mode, the different
types of the adsorption forces may be included in the retention process
Hydrophobic interactions are the main ones in reversed-phase
separations
Dipole-dipole (polar) interactions are dominant in normal phase mode.
Ionic interactions are responsible for the retention in ion-exchange
chromatography.
Retention in LC is competitive:
Analyte molecules compete with the eluent molecules for the
adsorption sites. So, the stronger analyte molecules interact with
the surface, and the weaker the eluent interaction, the longer
analyte will be retained on the surface.
Retention Mechanisms in LC
- Remember the elution order!
- Normal-phase vs. reversed-phase LC
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Physical Properties of Stationary Phase Particles
- HPLC separations are based on the surface interactions,
and depends on the types of the adsorption sites (surface
chemistry). Modern HPLC adsorbents are the small rigid
porous particles with high surface area.
- Key parameters:
Particle size: 3 to 10 m
Particle size distribution: as narrow as possible, usually within 10%
of the mean
Pore size: 70 to 300
Surface area: 50 to 250 m
2
/g
Bonding phase density (number of adsorption sites per surface
unit): 1 to 5 per 1 nm
2
Electron microphotograph of spherical and irregular silica particles. [W.R.Melander, C.Horvath,
Reversed-Phase Chromatography, in HPLC Advances and Perspectives, V2, Academic Press, 1980]
The Most Popular Particle: Silica
Macroporous spherical silica particle. [K.K.Unger,
Porous silica, Elsevier, 1979]
- Different morphology for different applications:
- Different chemistry:
Si OH Si OH O
H
H
Si
OH
OH
Free Silanol Adsorbed Water Geminal Silanol
Si
O
Si
O
Dehydrated
Oxide Siloxane
O
H
O
H
Si
Si
O
Bound and
Reactive
Silanols
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Chemical Modifications to Silica
- Silica (or zirconia, or alumina) by itself cannot do the job needed by
modern LC users it must be functionalized and modified to suit the
analytical problem
Residual
silanols
Si
O
Si
O
Si
O
Si
O
S
O
Si
Si
O
Si
O
Si
OH
S
OH
OH
OH
i
i
O
O
DiagramfromCrawford Scientific
Functionalized
groups
Chemical Modifications to Silica
- Groups are usually attached via reaction
of an organosilane (which can be pre-
polymerized in solution)
- Besides attaching groups, it is also
possible to polymerize the silica (or the
attached group)
- Purpose: stability at low pH, more
coverage
High-carbon load
- Monomeric phases are more
reproducible (easier reactions to control)
Monomeric phases are also known
as sterically-protected
- Endcapping: fully react the silica
surface, remove silanols and their
acidity, more coverage
DiagramfromK. A. Lippa et al., Anal. Chem.2005, 77,7852-7861
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Common LC Stationary Phases
Name Structure Description
Silica
Normal phase, for separating polar, non-ionic
organics
Propyl
Reversed-phase, for hydrophobic interaction
chromatography (proteins, peptides)
C8
Reversed-phase, like C18 but less retentive,
used for pharmaceuticals, steroids,
nucleotides
C18
Reversed-phase, retains non-polar solutes
strongly. When bonded to 300A silica can be
used for large proteins and macromolecules
Cyano
Reversed-phase and normal-phase, more
polar than C18, unique selectivity
Amino
Reversed-phase, normal-phase, and weak
anion exchange. RP used to separate
carbohydrates
Si C
3
H
7
Si C
8
H
17
Si C18H37
Si CH2CH2CH2CN
Si CH2CH2CH2NH2
Si OH
Common LC Stationary Phases
Name Structure Description
Phenyl
Reversed-phase, retains aromatic
molecules. Also used for HIC
(proteins)
Diol
Both reversed-phase and normal-
phase utility. Used for RP SEC,
also used for NP separations as a
more robust alternative to silica
(not ruined by trace water)
Nitro
Normal-phase, separates aromatic
and alkene-containing molecules
Si NO2
Si O
OH
OH
Si CH2CH2CH2
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Polar Stationary Phase Interactions
Sorbents Interactions
CN
NH2
2OH
Dipole/ Dipole
Hydrogen-
Bonding
Hydrogen-
Bonding
O
H
Si N
H
H
Si
N
O
H
C
O Si
O
H
O O
H
H
Source: Crawford Scientific.
Ionic Stationary Phase Interactions
Sorbents Interactions
PRS
CBA
SAX
Electrostatic
Electrostatic
Electrostatic
H
3
+
N
SO
3
-
Si
Si
H
3
+
N
O
-
O
N
+
(CH
3
)
3
Si
-
O
3
S
Source: Crawford Scientific.
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Non-Polar Stationary Phase Interactions
Sorbents Interactions
C8
PH
C2
van der Waals
van der Waals
van der Waals
Si
Si
Si
Source: Crawford Scientific.
A Good Choice of Stationary Phase Depends on
the Anal yte
N
N
H
H
2
2
N
N
H
H
3
3
+
+
N
N
H
H
2
2
Functionality Analyte Mechanism
Hydrophobi c
H-Bonding
Ioni c
Non-Polar
Pol ar
Ion-Exchange
Source: Crawford Scientific.
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More Subtle Effects
- Shape selectivity (correlates with stationary phase order), temperature,
coverage (and the role of bonding chemistry):
DiagramfromK. A. Lippa et al., Anal. Chem.2005, 77,7852-7861
More Subtle Effects
- The effects of
temperature on the
order of the stationary
phase are often
surprising:
DiagramfromK. A. Lippa et al., Anal. Chem.2005, 77,7852-7861
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Chiral Stationary Phases
- Interactions between chiral analytes (enantiomers and
molecules with more than 1 chiral center) and chiral
stationary phases are also possible
- Normal-phase is most common because of binding modes
A. Berthod, Chiral Recognition Mechanisms, Anal. Chem. 78, 2093-2099 (2006).
Chiral Stationary Phases
- Interactions between chiral analytes and chiral stationary
phases are also possible.
- Common chiral stationary phases:
Adapted fromL. R. Snyder, J . J . Kirkland, and J . L. Glajch, Practical HPLC Method Development, 2nd Ed., Wiley, 1997. Pg 545.
Name Chiral Recognition Mechanism
Analyte and Mobile Phase
Requirements
Protein based
Hydrophobic and electrostatic
interactions
Analyte must ionize, helpful if it
contains an aromatic. RP only.
Cyclodextrin Inclusion complexation, H-bonding
Polar and aromatic groups, RP
and NP.
Polymer-
based
carbohydrates
Inclusion interactions, attractive
interactions
H-bonding donors/acceptors, steric
bulk at chiral center, RP and NP.
Pirkle
H-bonding, t interactions, dipole-
dipole interactions
H-bonding donor/acceptors, mostly
NP.
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A Chiral LC Separation
- Example: separation of
naproxen enantiomers
- Chiral AGP column
AGP =o
1
-acid glycoprotein
(orosomucoid), 181 amino
acid residues and 14 sialic
acid residues
- Isocratic (no change in mobile
phase composition during
separation)
Adapted fromL. R. Snyder, J . J . Kirkland, and J . L. Glajch, Practical HPLC Method Development, 2nd Ed., Wiley, 1997. Pg 545.
O
HO
(S)
O
(S)-naproxen
O
HO
(R)
O
(R)-naproxen
Ion Chromatography (IC)
- Form of LC, also known as ion-exchange chromatography
- Basic mechanism is electrostatic exchange:
Source: Rubinson and Rubinson, Contemporary Instrumental Analysis, Prentice Hall Publishing.
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Typical IC Results
- Example: an isocratic method for
monovalent cations in ammonium
nitrate based explosives
- Detection limits 50-100 ppb, max
working range 40 ppm
- Method:
Sample Loop Volume: 50 L
Columns: IonPacCS3 Analytical,
IonPac CG3 Guard
Eluent: 25 mM HCl, 0.1 mM DAPHCl,
4% Acetonitrile
Eluent Flow Rate: 1.0 mL/min
Suppressor: Cation MicroMembrane
Suppressor (CMMS)
Regenerant: 100 mM
Tetrabutylammonium Hydroxide
Detector: Conductivity, 30 S full
scale
Injection Volume: 50 L
FromDionex Application Note 121R
Mobile Phases in LC
- Mobile phases differ for each LC mode
Normal phase solvents are mainly nonpolar
Reversed-phase eluents are usually a mixture of water with some
polar organic solvent such as acetonitrile.
- Size-exclusionLC has special requirements for mobile phases
Must dissolve polymers
Must also suppress all possible interactions of the sample molecule
with the surface of the packing material
- The type and composition of the mobile
phase (eluent) is one of the variables
influencing LC separations
- Desirable properties:
Purity
Detector compatibility
Solubility of the sample
Low viscosity
Chemical inertness
Reasonable price
FigurefromPhenomenextechnical literature
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Isocratic elution: the eluent composition remains constant
as it is pumped through the column during the whole
analysis.
Gradient elution: the eluent composition (and strength) is
steadily changed during the run.
Control of Eluent Polarity
time
%

m
o
b
i
l
e

p
h
a
s
e
|
.
|

\
| +
|
.
|

\
|
=
k
k N
R
s
1 1
4

|
|
.
|

\
| +
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.
|

\
|
=
*
*
1 1
4 k
k N
R
s

where k
*
is the k at the midpoint of the column
LC Instrumentati on
Pumps, Mixers
and Injectors
Column Detector(s) Computer
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LC Instrumentati on
- The Agilent 1100, a typical modern LC system
Solvent reservoirs
Solvent degasser
Pump
Autosampler
Column oven
DAD
Review: The Purpose of Key LC Components
column separation chemistry
detector
signal transduction
amplification/scaling
filtering
A/D
data acquisition
digitization
tubing to detector flow cell
analog output
digital output
chromatogram
digital processing
data analysis
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The LC Pump(s)
Modern pumps have the following parameters:
Flow rate range: 0.01 to 10 ml/min
Pressure range from 1-5,000 psi
Pressure pulsations : less than1 %
Types of Pumps
Constant pressure pumps
Constant flow pumps
Reciprocating Piston Pump (90% of HPLCs)
small internal volume
pulsed flow
Syringe type pumps (Displacement Pumps)
limited solvent capacity
Pneumnatic Pumps (pressure)
Temperature Control in LC
- Thermoelectric heating/cooling
the ability of a surface to
produce or absorb heat
when current is applied
across the junction of two
dissimilar conductors or
semicondeucted
- The effect can be reversed (i.e.
heating turned to cooling) by
reversing the DC current
through the junction
- Also known as the Peltier effect
after its 1834 discoverer, a
French watch maker
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Overview of LC Detectors
- Common HPLC detectors
Refractive Index
UV/Vis
- Fixed Wavelength
- Variable Wavelength
- Diode Array
Fluorescence Detector
- Less common:
Conductivity
Mass-spectrometric (LC/MS)
Evaporative light scattering (ELSD)
Desirable Features of an LC Detector
1. Low drift and noise level
2. High sensitivity (ability to discriminate between
small differences in analyte concentration)
3. Fast response
4. Wide linear dynamic range
5. Low dead volume
6. Cell design that eliminates remixing of separated
bands
7. Insensitivity to changes in types of solvent, flow
rate, temp
8. Operational simplicity and reliability
9. Non-destructive
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Baseline Noise and Drift
Detector Response
The definition of detector response depends on whether
it is mass sensitive or concentration sensitive
Mass sensitive mV/mass/unit time
R =hw/sM
Concentration sensitive mV/mass/unit volume
R =hwF/sM
h = peak height mV
W =width at .607 of height
F =flow rate
M =mass of solute
s =chart speed
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Cell Efficiency
Example:
column 15,000 plates
15 cm long
2 min t
R
2 ml at 1 ml /min
peak width of 80uL
flow cell of 20 ul
only four measurements
Things to note:
parallel light beam
flow cell volume <1/10 of peak volume
optimization of cell geometry
Ultraviolet/Visi bl e Spectroscopi c Detectors
infrared (IR) 2,500 - 50,000 nm
near infrared 800 - 2,500 nm
visible 400 - 800 nm
ultraviolet (UV) 190 - 400 nm
Any chemical compound could interact with the electromagnetic field. Beamof the
electromagnetic radiation passed through the detector flow-cell will experience some change
in its intensity due to this interaction. Measurement of this changes is the basis of the most
optical HPLC detectors.
Name Chromophore Wavelength [nm] Molar extinction, e
acetylide -C=C 175-180 6,000
Aldehyde -CHO 210 1,500
amine -NH
2
195 2,800
azo -N=N- 285-400 3-25
bromide -Br 208 300
carboxyl -COOH 200-210 50 - 70
disulphide -S-S- 194 5,500
ester -COOR 205 50
ether -O- 185 1,000
ketone >C=O 195 1,000
nitrate -ONO
2
270 12
nitrile -C=N 160 -
nitrite -ONO 220 - 230 1000-2000
nitro -NO
2
210 strong
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Fixed / Variable Wavelength Detectors
mercury vapor lamp emit very intense light at 253.7
nm. By filtering out all other emitted wavelengths,
manufacturers have been able to utilize this 254 nm
line to provide stable, highly sensitive detectors
capable of measuring subnanogram quantities of
any components which contains aromatic ring. The
254 nm was chosen since the most intense line of
mercury lamp is 254 nm, and most of UV absorbing
compounds have some absorbance at 254 nm.
Diode Array Detectors
- Diode array detectors can acquire all UV-Visible
wavelengths at once.
- Advantages:
Sensitivity
(multiplex)
Speed
- Disadvantages:
Resolution
FigurefromSkoog, et al., Chapter 13
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Other Detectors
Fluorescence Detector
Electrochemical Detector
Evaporative Light Scattering
Putting it All Together: LC Method Development
- The importance without a good method:
Co-elution can be missed
Unable to detect/assay key components
- Basic consequences of method changes:
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Choosing an LC Approach
- Goals of a separation:
Resolution (R
s
) >1.5
Short separation time (5-30 minutes)
Good quantitative precision/accuracy
Acceptable backpressure
Narrow peaks
Minimal solvent use
Overall Strategy
- First select an
appropriate method
- If LC is best, then
determine nature of
the sample
- Exploratory RP
runs, i.e. fast simple
gradients with C18
phases, are usually
helpful in assessing
retention and polarity
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Solid-phase Extraction (SPE)
- What is SPE?
The separation of an analyte or analytes from a mixture of
compounds by selective partitioning of the compounds
between a solid phase (sorbent) and a liquid phase (solvent)
- Comparison with conventional liquid-liquid extraction (e.g.
the organic sep funnel approach):
SPE: selective towards functional groups (better)
LLE: selective towards solubility
SPE: more choices because no miscibility (better)
LLE: must avoid miscible solvents
SPE: concentrates analytes (better)
LLE: can concentrate analyte after stripping
The Typi cal SPE Process
- Conditioning: solvates the sorbent
- Equilibration: removes excess conditioning solvent,
matches with analytical conditions (prevents shock)
Sample
Application
Interference
Elution
Analyte
Elution
Column
Conditioning
Column
Equilibration
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Solid-phase Extraction
- Conditioning the cartridge:
Not condi ti oned Condi ti oned
- SPE cartridges have a range of chemistries that are often
similar to those of LC stationary phases, but are optimized
for adsorption/desorption
Solid-phase Extraction
- Automated SPE systems for sample cleanup the Spark
Symbiosis
TM
Images fromwww.sparkholland.com
- Can be hyphenated
with LC, MS, NMR,
etc or used as a
stand-alone sample
pretreatment
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Homework and Further Reading
- Homework problems (for study only):
28-2, 28-3, 28-11, 28-14
- For a detailed discussion of method development in LC:
L. R. Snyder, J . J . Kirkland, and J . L. Glajch, Practical
HPLC Method Development, 2nd Ed., Wiley, 1997.
- For recent advances in understanding gradient elution,
see:
P. Nikitas and A. Pappa-Louisi, Anal. Chem., 2005, 77,
5670-5677 (a new derivation of the equation of
reversed-phase HPLC gradient elution)