Instant Notes: Analytical Chemistry

Section D – Separation techniques

D6 HIGH-PERFORMANCE LIQUID

CHROMATOGRAPHY:

PRINCIPLES AND

INSTRUMENTATION

Key Notes

High-performance liquid chromatography (HPLC) is a technique for the

separation of components of mixtures by differential migration through a

column containing a microparticulate solid stationary phase. Solutes are

transported through the column by a pressurized flow of liquid mobile

phase, and are detected as they are eluted.

The mobile phase is either a single solvent or a blend of two or more

having the appropriate eluting power for the sample components. It

ranges from a nonpolar liquid to aqueous buffers mixed with an organic

solvent.

The solvent delivery system comprises a means of degassing, filtering

and blending up to four solvents which are then delivered to the top of

the column under pressure by a constant flow pump.

Liquid samples or solutions are introduced into the flowing mobile phase

at the top of the column through a constant or variable volume loop and

valve injector that is loaded with a syringe.

Columns are straight lengths of stainless steel tubing tightly packed with

a microparticulate stationary phase. The column packings are chemically-

modified silicas, unmodified silica or polymeric resins or gels.

Solutes are detected in the mobile phase as they are eluted from the end

of the column. The detector generates an electrical signal that can be

amplified and presented in the form of a chromatogram of solute

concentration as a function of time.

A dedicated microcomputer is an integral part of a modern high-

performance liquid chromatograph. Software packages facilitate the

control and monitoring of instrumental parameters, and the display and

processing of data.

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

chromatography: modes,

procedures and applications

(D7)

Principles

Mobile phase

Solvent delivery

system

Sample injection

Column and

stationary phase

Solute detection

Instrument control

and data processing