Instant Notes: Analytical Chemistry

separation of enantiomeric forms of drugs having different pharmacological

activities, chiral columns are expensive and most have very limited working

lives. Capillary electrophoresis(Topic D8) provides a cheaper alternative.

The optimum conditions for an HPLCseparation are those which give the

required resolutionin the minimum time. A stepwise approach based on the

characteristics of the solutes to be separated, and trial chromatograms with

different mobile phase compositions is usually adopted. The following is an

outline of the procedure for a reversed-phase separation where a hydrocarbon

stationary phase, usually C18(ODS), is the first choice.

● The mode of HPLCmost suited to the structures and properties of the

solutes to be separated is selected, having regard to their relative molecular

mass, polarity, ionic or ionizable character, and solubility in organic and

aqueous solvents.

● The stationary phase and columnare selected (Topic D6, Tables 2 and 3). The

shortest column and the smallest particle size of stationary phase consistent

with adequate resolution should be used.

● The detector, subject to availability, should match the solute characteristics.

UV-visible absorbancedetectors are suitable for many solutes except those

that are fully saturated. Fluorescence and electrochemical detectors should be

considered where high sensitivity is required.

● Mobile phase composition is optimized by obtaining and evaluating a

number of trial chromatograms, often with the aid of computer optimization

software packages. A typical series of chromatograms for a reversed-phase

separation on a hydrocarbon bonded phase column is shown in Figure 5.

Optimization of
separations

D7 – HPLC: modes, procedures and applications 171

Exclusion

Permeation

Separation

according to

molecular size

Vo Vi

Vo Vtot

107

106

105

104

103

102

Log relative molecular mass (RMM)

Retention volume (VR)

5 10152025303540

1. Thyroglobulin 670K


2. IgA 300K


3. IgG 150K


4. LDH 143K


5. Oralbumin 44K


6. Trypsin inhibitor 20.1K

1

2

3

4

5

6

Retention volume (cm^3 )

Fig. 4. A typical size exclusion calibration curve and chromatogram of the separation of a protein mixture. Column:
BIOSEP-SEC-S3000. Mobile phase: pH 6.8 phosphate buffer. Detector: UV abs. at 280 nm. Reproduced from W.J.
Lough & I.W. Wainer (eds), High Performance Liquid Chromatography, 1996, first published by Blackie Academic &
Professional.