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of bases, which would be present as cations, an alkyl sulphonate such as sodium

heptanesulphonate would be used:

RCOO þ R^04 Nþ нRCOONþR^04 Š

carboxylic counter cation ion-pair

acid anion

RNþH 3 þ R^0 SO 3 нRH 3 NþO 3 SR^0 Š

conjugate acid counter anion ion-pair

of weak base

The technique can also be applied to proteins. The addition of trifluoroacetic acid (TFA)

suppresses the ionisation of exposed groupson the protein surface giving it greater

hydrophobic character. In practice, the success of the ion-pairing approach is variable

and somewhat empirical. The size of the counter ion, its concentration and the pH of the

solution are all factors that may profoundly influence the outcome of the separation.

Octyl- and octadecylsilane-bonded phases are used most commonly in conjunction

with a water/methanol or water/acetonitrile mobile phase. One of the advantages of ion-

pair reversed-phase chromatography is that if the sample to be resolved contains a mixture

of non-ionic and ionic analytes, the two groups can be separated simultaneously because

the ion-pair reagent does not affect the chromatography of the non-ionic species.

11.5.5 Chiral chromatography

This form of chromatography allows mixtures of enantiomers (mirror image forms,

denoted either as D or L or as S or R) to be resolved. One of these techniques is based

on the fact that diastereoisomers, which are optical isomers that do not have an

object–image relationship, have different physical properties even though they con-

tain identical functional groups. They can therefore be separated by conventional

chromatographic techniques, most commonly reversed-phase chromatography. The

diastereoisomer approach requires that the enantiomers contain a function group that

can be derivatised by a chemically and optically purechiral derivatising agent(CDA)

that converts them to a mixture of diastereoisomers:

ðRþSÞþ R^0 Ð RR^0 þSR^0

mixture of chiral mixture of

enantiomers derivatising diastereoisomers

agent

Examples of CDAs include the R or S form of the following:

For amines N-trifluoroacetyl-1-prolylchloride,a-phenylbutyric anhydride

For alcohols 2-Phenylpropionyl chloride, 1-phenylethylisothiocyanate

For ketones 2,2,2-Trifluoro-1-pentylethylhydrazine

For aliphatic and

alicyclic acids

1-Menthol, desoxyephedrine

458 Chromatographic techniques