Instant Notes: Analytical Chemistry

Section D – Separation techniques

D9 ELECTROPHORESIS AND

ELECTROCHROMATOGRAPHY:

MODES, PROCEDURES AND

APPLICATIONS

There are several modes of classical gel electrophoresis. They are defined by the

format and the nature of the gel, the running buffer and any incorporated

additives.

● Slab gel, column and disk electrophoresis(Topic D8) are the principal

formats. They allow multiple samples and standards to be run simultane-

ously for comparison purposes, which is comparable to separations by TLC

(Topic D3). Samples are placed in wells near the centre of horizontal slabs

when both cationic and anionic species are to be separated, or at one end if

all solutes are expected to carry the same charge. With vertical slabs, samples

are placed in wells at the top of the slabs so that only downwards migration

is possible.

● Sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE) is

used to determine the relative molecular masses (RMM values) of individual

proteins in a mixture. The proteins are first de-natured with mercapto-

ethanolbefore bonding to the SDS, which is an anionic surfactant. The

negatively-charged proteins subsequently migrate at rates inversely propor-

tional to the logarithm of their RMM, the system being calibrated with

proteins of known RMM.

● Isoelectric focusingis a mode used to separate ampholytes such as amino

Modes of
electrophoresis
and electro-
chromatography

Key Notes

Modes of electrophoresis are defined by the nature and form of the

supporting medium, the running buffer and any incorporated additives.

For capillary electrochromatography (CEC), the capillary is filled with a

stationary phase similar to those used in HPLC.

For classical gel electrophoresis, unknown solutes are identified by

comparisons of the distances migrated with those of standards run

simultaneously. For capillary electrophoresis (CE) and CEC, migration

times and spectrometric data are used.

Classical gel electrophoresis provides only semi-quantitative information

at best. For CE and CEC, quantitative information is readily obtained

from peak area measurements and calibration graphs.

Related topic Electrophoresis and electrochromatography: principles and

instrumentation (D8)

Qualitative analysis

Modes of

electrophoresis and

electrochromatography

Quantitative analysis