Instant Notes: Analytical Chemistry

lamide or agarose, under the influence of an applied electric fieldthat creates a

potential gradient. Two platinum electrodes (cathode and anode) make contact

with the electrolyte which is contained in reservoirs at opposite ends of the

supporting medium, and these are connected to an external DC power supply.

Considerable amounts of heat may be generated during separations at higher

operating voltages (Joule heating), and many systems stabilize the operating

temperature by water-cooling. Buffer solutions undergo electrolysis, producing

hydrogen and oxygen at the cathode and anode, respectively, and the reservoirs

have to be replenished or the buffer renewed to maintain pH stability.

Cationic solute species(positively-charged) migrate towards the cathode,

anionic species(negatively-charged) migrate towards the anode, but neutral

speciesdo not migrate, remaining at or close to the point at which the sample is

applied. The rate of migration of each solute is determined by its electrophoretic

mobility, m, which is a function of its net charge, overall size and shape, and the

viscosity of the electrolyte. The latter slows the migration rate by viscous drag

(frictional forces) as the solute moves through the buffer solution and supporting

medium.

The distance travelled, d, after the application of a potential, E, between two

electrodes for time, t, is given by

dm¥t ¥ (1)

where Sis the distance between the two electrodes and E/Sis the potential

gradient. For two separating solutes with mobilities m 1 and m 2 , their separation,

Dd, after time, t, is given by

Dd(m 1 - m 2 ) · t ·  (2)

Ddis maximized by the application of a large potential gradient over a long

period. However, as in chromatographic separations, diffusion of the solute

species in the buffer solution causes band spreading which adversely affects

resolution, so excessive separation times should be avoided.

The role of buffers in electrophoresis is crucial because many solutes are

weakly acidic, basic or ampholytic, and even small changes in pH can affect

their mobility. Amino acids, peptides and proteins are particularly susceptible

as their direction of migration is a function of pH, e.g for glycine

Typical formats for classical electrophoresisare shown in Figure 1. In the slab

gel method, the supporting gel is pre-formed into thin rectangular slabs on

which a number of samples and standards can be separated simultaneously.

Alternatively, it can be polymerized in a set of short tubes. The whole assembly

is enclosed in a protective perspex chamber for safety reasons because of the

high voltages employed (500 V–2 kV DC, or up to 50 V cm-^1 ). A separation may

take from about 30 minutes to several hours, after which the gels are treated

with a suitable visualizing agent to reveal the separated solutes (vide infra). Slab

H 2 N-CH 2 - COO-

high pH

anion

OH-

̈Æ

H+

H 2 N-CH 2 - COOH

or

H 3 N+-CH 2 - COO-

neutral/zwitterion

OH-

̈Æ

H+

H 3 N+-CH 2 - COOH

low pH

cation

E

S

E

S

D8 – Electrophoresis and electrochromatography 175