liquid chromatography 1
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Liquid Chromatography 1 and Solid-Phase Extraction
Lecture Date: April 9th, 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 HPLCMethod Development, 2nd Ed., Wiley, 1997.
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Basic LC Terminology
Adsorption chromatography
The stationary phase is an adsorbent (like silica gel or any othersilica-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 ortetrahydrofuran).
Polar samples are retained on the polar surface of the columnpacking 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 waterand 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 f iltered according toits solvated molecular size.
Larger molecules are rapidly washed through the column;smaller molecules penetrate inside the pores of the packingparticles 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 taketo elute
The mobile phase is an aqueous buffer, where both pH andionic strength are used to control elution time
Basic LC Terminology
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Analytical Appl ications of LC
The branches of the LC family:Note this means analyte 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 thenforced to flow through a packed small-particlechromatographic column, where the mixture is resolvedinto its components
HP = high pressure and high performance
Resolution depends upon the extent of interaction
between the solute components and the stationaryphase
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 newsubstances to be used as stationary phases
Relatively high inlet pressures and controlled flow of themobile phase
Precise sample introduction without the need for largesamples
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 theirhydrophobicity 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 andcarefully filled with material, are the heart of the LCexperiment
The stationary phase fills the column its properties arecritical to the separation
Review of Molecular Interactions
The basis of separations (and most of chemistry)
Name Energy (kcal/mol) Description
Covalent 100-300Hold molecules together, orbital
overlap
Ionic 50-200 Electrostatic attraction
Polar Hydrogen bonding Dipole-dipole
-stacking
3-10Vary from electrostatic-typeinteractions (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, whilemoving through the porous packing bead, tend to interact with thesurface adsorption sites. Depending on the HPLC mode, the differenttypes of the adsorption forces may be included in the retention process
Hydrophobic interactions are the main ones in reversed-phaseseparations
Dipole-dipole (polar) interactions are dominant in normal phase mode.
Ionic interactions are responsible for the retention in ion-exchangechromatography.
Retention in LC is competitive:
Analyte molecules compete with the eluent molecules for theadsorption sites. So, the stronger analyte molecules interact withthe surface, and the weaker the eluent interaction, the longeranalyte 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 (surfacechemistry). Modern HPLC adsorbents are the small rigidporous 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 m2/g
Bonding phase density (number of adsorption sites per surfaceunit): 1 to 5 per 1 nm2
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, 1 979]
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
DehydratedOxide Siloxane
O
H
O
H
Si
Si
O
Bound andReactiveSilanols
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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 theanalytical problem
Residualsilanols
SiO
SiO
SiO
SiO
SO
Si
Si OSi
OSiOH
S
OHOH
OH ii
O
O
Diagram from Crawford Scientific
Functionalizedgroups
Chemical Modifications to Silica
Groups are usually attached via reactionof an organosilane (which can be pre-polymerized in solution)
Besides attaching groups, it is alsopossible to polymerize the silica (or theattached group)
Purpose: stability at low pH, morecoverage
High-carbon load
Monomeric phases are morereproducible (easier reactions to control)
Monomeric phases are also knownas sterically-protected
Endcapping: fully react the silicasurface, remove silanols and theiracidity, more coverage
Diagram from K. A. Lippa et al., Anal. Chem.2005, 77,7852-7861
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Common LC Stationary Phases
Name Structure Description
SilicaNormal phase, for separating polar, non-ionic
organics
PropylReversed-phase, for hydrophobic interaction
chromatography (proteins, peptides)
C8Reversed-phase, like C18 but less retentive,
used for pharmaceuticals, steroids,nucleotides
C18Reversed-phase, retains non-polar solutes
strongly. When bonded to 300A silica can beused for large proteins and macromolecules
CyanoReversed-phase and normal-phase, more
polar than C18, unique selectivity
AminoReversed-phase, normal-phase, and weak
anion exchange. RP used to separatecarbohydrates
Si C3H7
Si C8H17
Si C18H37
Si CH2CH2CH2CN
Si CH2CH2CH2NH2
Si OH
Common LC Stationary Phases
Name Structure Description
PhenylReversed-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 amore robust alternative to silica
(not ruined by trace water)
NitroNormal-phase, separates aromaticand 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
OH
Si NH
H
SiN
OH
C
OSi
OH
OOH
H
Source: Crawford Scientific.
Ionic Stationary Phase Interactions
Sorbents Interactions
PRS
CBA
SAX
Electrostatic
Electrostatic
Electrostatic
H3+N
SO3-Si
Si
H3+N
O-
O
N+(CH3)3Si
-O3S
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 Analyte
NNHH22
NNHH33++
NNHH22
Functionality Analyte Mechanism
Hydrophobic
H-Bonding
Ionic
Non-Polar
Polar
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):
Diagram from K. A. Lippa et al., Anal. Chem.2005, 77,7852-7861
More Subtle Effects
The effects oftemperature on theorder of the stationaryphase are oftensurprising:
Diagram from K. 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 chiralstationary 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 stationaryphases are also possible.
Common chiral stationary phases:
Adapted from L. R. Snyder, J. J. Kirkland, and J. L. Glajch, Practical HPLC Method Development, 2nd Ed., Wiley, 1997. Pg 545.
Name Chiral Recognition MechanismAnalyte and Mobile Phase
Requirements
Protein basedHydrophobic and electrostatic
interactionsAnalyte must ionize, helpful if itcontains an aromatic. RP only.
Cyclodextrin Inclusion complexation, H-bondingPolar and aromatic groups, RP
and NP.
Polymer-basedcarbohydrates
Inclusion interactions, attractiveinteractions
H-bonding donors/acceptors, stericbulk at chiral center, RP and NP.
PirkleH-bonding, interactions, dipole-
dipole interactionsH-bonding donor/acceptors, mostly
NP.
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A Chiral LC Separation
Example: separation ofnaproxen enantiomers
Chiral AGP column AGP = 1-acid glycoprotein
(orosomucoid), 181 aminoacid residues and 14 sialicacid residues
Isocratic (no change in mobilephase composition duringseparation)
Adapted from L. 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 formonovalent cations in ammonium
nitrate based explosives Detection limits 50-100 ppb, max
working range 40 ppm Method:
Sample Loop Volume: 50 L Columns: IonPac CS3 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 fullscale Injection Volume: 50 L
From Dionex Application Note 121 R
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 somepolar 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 mobilephase (eluent) is one of the variablesinfluencing LC separations
Desirable properties: Purity Detector compatibility Solubility of the sample Low viscosity Chemical inertness Reasonable price
Figure from P henomenex technical literature
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Isocratic elution: the eluent composition remains constantas it is pumped through the column during the wholeanalysis.
Gradient elution: the eluent composition (and strength) issteadily changed during the run.
Control of Eluent Polarity
time
%m
obilephase
k
kNR
s
11
4
*
*
11
4 k
kNRs
where k* is the k at the midpoint of the column
LC Instrumentation
Pumps, Mixers
and InjectorsColumn Detector(s) Computer
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LC Instrumentation
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
detectorsignal transductionamplification/scalingfiltering
A/Ddata acquisitiondigitization
tubing to detector flow cell
analog output
digital output
chromatogramdigital processingdata analysis
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The LC Pump(s)
Modern pumps have the following parameters:
Flow rate range: 0.01 to 10 ml/minPressure range from 1-5,000 psiPressure pulsations : less than1 %
Types of PumpsConstant pressure pumpsConstant flow pumps
Reciprocating Piston Pump (90% of HPLCs)small internal volumepulsed flow
Syringe type pumps (Displacement Pumps)limited solvent capacity
Pneumnatic Pumps (pressure)
Temperature Control in LC
Thermoelectric heating/cooling
the ability of a surface toproduce or absorb heatwhen current is appliedacross the junction of twodissimilar conductors orsemicondeucted
The effect can be reversed (i.e.heating turned to cooling) byreversing the DC currentthrough the junction
Also known as the Peltier effectafter its 1834 discoverer, aFrench 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 level2. High sensitivity (ability to discriminate between
small differences in analyte concentration)3. Fast response4. Wide linear dynamic range5. Low dead volume6. Cell design that eliminates remixing of separated
bands7. Insensitivity to changes in types of solvent, flow
rate, temp8. Operational simplicity and reliability9. 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 heightF = flow rate
M = mass of solute
s = chart speed
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Fixed / Variable Wavelength Detectors
mercury vapor lamp emit very intense light at 253.7nm. By filtering out all other emitted wavelengths,manufacturers have been able to utilize this 254 nmline to provide stable, highly sensitive detectorscapable of measuring subnanogram quantities ofany components which contains aromatic ring. The
254 nm was chosen since the most intense line ofmercury lamp is 254 nm, and most of UV absorbingcompounds have some absorbance at 254 nm.
Diode Array Detectors
Diode array detectors can acquire all UV-Visiblewavelengths at once.
Advantages:
Sensitivity(multiplex)
Speed
Disadvantages:
Resolution
Figure from Skoog, et al., Chapter 13
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Other Detectors
Fluorescence Detector
Electrochemical Detector
Evaporative Light Scattering
Putting i t 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 (Rs) > 1.5
Short separation time (5-30 minutes)
Good quantitative precision/accuracy
Acceptable backpressure
Narrow peaks
Minimal solvent use
Overall Strategy
First select anappropriate method
If LC is best, thendetermine nature ofthe sample
Exploratory RPruns, i.e. fast simplegradients with C18phases, are usuallyhelpful in assessingretention 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 compoundsbetween 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 Typical SPE Process
Conditioning: solvates the sorbent
Equilibration: removes excess conditioning solvent,matches with analytical conditions (prevents shock)
SampleApplication
InterferenceElution
AnalyteElution
ColumnConditioning
ColumnEquilibration
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Solid-phase Extraction
Conditioning the cartridge:
Not conditioned Conditioned
SPE cartridges have a range of chemistries that are oftensimilar to those of LC stationary phases, but are optimizedfor adsorption/desorption
Solid-phase Extraction
Automated SPE systems for sample cleanup the SparkSymbiosisTM
Images from www.sparkholland.com
Can be hyphenated
with LC, MS, NMR,etc or used as astand-alone samplepretreatment
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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, PracticalHPLC 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 ofreversed-phase HPLC gradient elution)
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