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CHAPTER 28

LIQUID CHROMATOGRAPHY
- classified by type of stationary phase or separation mechanism: partition, adsorption, ion exchange, size
exclusion, affinity, chiral
- because smaller particle size increases efficiency, high pressures were used => almost all LC is done
using pressurized flow

Column efficiency
- increases with decreasing particle size
- extracolumn band broadening occurs because of differences in flow rate between the layer of liquid
adjacent to the wall and that in the center of the tube, which causes the center part to move more quickly;
largely offset by diffusion in GC
- better and more reproducible chromatograms are obtained by maintaining constant column temperature
Guard columns
- introduced before the analytical column to increase its lifetime by removing particulate matter,
contaminants and sample components that bind irreversibly to the stationary phase ; cheaper to replace
LC Detectors

- can be used with flame and thermal conductivity detectors (universal)


- the combination of HPLC and mass spectrometry gives high selectivity because unresolved peaks can be
isolated by monitoring only a selected mass
=> LC/MS can provide fingerprinting of a particular eluate instead of relying on retention time

- ion cyclotron resonance and ion traps MS can be operated in n stages => MS^n systems
Partition chromatography
- the stationary phase is a liquid that is immiscible with the liquid mobile phase
- in liquid/liquid chromatography, the liquid was held in place by physical adsorption
- in bonded phase chromatography, it is by chemical bonding, resulting in highly stable packings
insoluble in the mobile phase
-in normal phase chromatography, a polar stationary phase is used with a nonpolar mobile phase => elutes
least polar cpd first
- in reversed phase chromatography, the stationary phase in nonpolar while the mobile phase is polar =>
elutes most polar cpd first

- retention in most types of normal phase chromatography seems to be governed by adsorptiondisplacement processes
- the major advantage of reversed-phase separations is that water can be used as the mobile phase
- longer chains produce packings that are more retentive and that permit the use of larger samples
- when using silica, pH>7.5 needs to be avoided because formation of soluble silicate species can cause
the stationary phase to dissolve; likewise, pH<2.5 should also be avoided to prevent acidic hydrolysis of
the siloxane
- in LC, sample components interact with both the stationary and mobile phase while in GC, the mobile
phase simply carries the sample components through the stationary phase and does not contribute to the
separation process
=> GC is not affected by the identity of the carrier gas while LC is strongly dependent on the identity of
the mobile phase
- successful LC requires a proper balance of intermolecular forces among the solute, stationary phase, and
mobile phase => depends on their relative polarity
- usually, the polarity of the stationary phase is matched ~ with that of the analyte; a mobile phase is
considerably different polarity is then used for elution
- in LC, the retention factor is the easiest to manipulate b/c of its strong dependence on the mobile phase
composition
- the selectivity factor can also be changed by varying mobile phase composition or by choosing a
different column packing
- solvents that interact strongly with solutes are "strong" solvents; often but not always polar solvents
- the polarity index P'ab of a mixture of solvents A and B is:

Ion pair chromatography

- a subset of reversed phase chromatography in which easily ionizable species are separated on reversedphase columns
- possible mechanism 1: the counterion forms an uncharted ion pair with an oppositely charged solute ion
in the mobile phase; this ion pair then partitions into the nonpolar stationary phase, giving differential
retention of solutes based on the affinity of the ion pair for the two phases
- possible mechanism 2: the counterion is retained strongly by the normally neutral stationary phase and
imparts a charge to this phase; separation of organic solute ions of the opposite charge then occurs by
formation of reversible ion pair complexes with the more strongly retained solutes forming the strongest
complexes with the stationary phase
Chiral chromatography
- uses either chiral mobile phase additives or chiral stationary phases => separation occurs by preferential
complexation between the chiral resolving agent and one of the enantiomers
Ion exchange equilibria
- based on exchange equilibria between ions in solution and ions of like sign on a surface
- the active sites for cation-exchange resins uses -SO3H or -CO2H; those for anionic exchange use
NMe3OH or NH3OH

- polyvalent ions are much more strongly held than singly charged ones
- polymer based packings (functionalized with acidic or basic functional groups) have higher capacities
than silica based packings and can be used over a broader pH range although silica based ion exchangers
give higher efficiencies
Size exclusion chromatography
- particularly applicable to high molecular mass species
- contain pores into which solute and solvent molecules can diffuse => the average residence time in the
pores depends on the effective size of the analyte molecules, leading to fractionation according to
molecular size and shape
- does not involve chemical or physical interaction between analytes and stationary phases (avoided since
they lower column efficiencies)
- has an upper limit to retention time since no analyte species is retained longer than those that totally
permeate the stationary phase
Affinity chromatography
- an affinity ligand (antibody, enzyme inhibitor, etc) is covalently bonded to a solid support
=> only molecules that selectively bind to the affinity ligand are retained; after the undesired molecules
are removed, the retained ones can be eluted by changing the mobile phase conditions (pH, ionic strength)

- extraordinary specificity
- allows for rapid isolation of biomolecules
Planar chromatogaphy
- thin layer chromatography
- paper chromatography
- the mobile phase moves through the stationary phase by capillary action assisted by gravity or an
electrical potential
- Rf values can be used to identify compounds

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