Instant Notes: Analytical Chemistry

Principles High-performance liquid chromatography(HPLC) is a separation technique
where solutes migrate through a column containing a microparticulate
stationary phase at rates dependent on their distribution ratios (Topic D2).
These are functions of the relative affinities of the solutes for the mobile and
stationary phases, the elution order depending on the chemical nature of the
solutes and the overall polarity of the two phases. Very small particles of
stationary phase are essential for satisfactory chromatographic efficiency and
resolution, and the mobile phase must consequently be pumped through the
column, resulting in the generation of a considerable back-pressure. The compo-
sition of the mobile phase is adjusted to elute all the sample components reason-
ably quickly. Solutes eluted from the end of the column pass through a detector
that responds to each one. There are a number of modes of HPLCenabling an
extremely wide range of solute mixtures to be separated. The modes (Topic D7)
are defined by the type of stationary phase and associated sorption mechanism.
A schematic diagram of a high-performance liquid chromatographis shown
in Figure 1. It consists of five major components:

● solvent delivery system;

● sample injection valve;

● column;

● detection and recording system;

● microcomputer with control and data-processing software.

These are described in the following sections.

Mobile phase The mobile phase, or eluent, is most frequently a blend of two miscible solvents
that together provide adequate eluting powerand resolution. These are deter-

156 Section D – Separation techniques

He He

Solvent

reservoirs

Syringe

application

of sample

Guard

column

Dual-port

valve

Scavenger

column

Mixing

chamber

Pump

Detector

Chromatography

control station

Analytical

column

Chromatogram

Integrator/

computer

Fig. 1. Schematic diagram of a high-performance liquid chromatograph. Reproduced from

A. Braithwaite & F.J. Smith, Chromatographic Methods, 5th edn, 1996, first published by

Blackie Academic & Professional.