Instant Notes: Analytical Chemistry

Section D – Separation techniques

D7 HIGH-PERFORMANCE LIQUID

CHROMATOGRAPHY: MODES,

PROCEDURES AND

APPLICATIONS

Modes of HPLC Almost any type of solute mixture can be separated by HPLCbecause of the
wide range of stationary phases available, and the additional selectivity
provided by varying the mobile phase composition. Both normal-and reversed-
phaseseparations are possible, depending on the relative polarities of the two
phases. Although these are sometimes referred to as modes of HPLC, the nature
of the stationary phase and/or the solute sorption mechanism provide a more
specific means of classification, and modes based on these and the types of
solutes to which they are best suited are summarized below.

● Adsorption chromatography. Separations are usually normal-phasewith a

silica gel stationary phase and a mobile phase of a nonpolar solvent blended

with additions of a more polar solvent to adjust the overall polarity or

eluting power, e.g. n-hexane + dichloromethane or di-ethyl ether. The choice

of solvent is limited if a UV absorbancedetector is to be used. Traces of

water in the solvents must be controlled, otherwise solute retention will not

be reproducible. Solutes are retained by surface adsorption; they compete

with solvent molecules for active silanol sites (Si-OH), and are eluted in

Key Notes

Modes of HPLC are defined by the nature of the stationary phase, the

mechanism of interaction with solutes, and the relative polarities of the

stationary and mobile phases.

After selection of an appropriate mode, column and detector for the

solutes to be separated, the composition of the mobile phase must be

optimized to achieve the required separation. A trial and error approach

or a computer aided investigation can be adopted.

Unknown solutes can be identified by comparisons of retention factors or

times, spiking samples with known substances or through spectrometric

data.

Quantitative information is obtained from peak area or peak height

measurements and calibration graphs using internal or external

standards, or by standard addition or internal normalization.

Related topics Principles of chromatography (D2) High-performance liquid

chromatography: principles

and instrumentation (D6)

Modes of HPLC

Optimization of

separations

Qualitative analysis

Quantitative analysis