Instant Notes: Analytical Chemistry

responsible for the movement of all species (cationic, anionic and neutral) along

the capillary towards the detector and is the basis of the technique. The strength

of the EOFin fused-quartz capillaries filled with a running buffer arises from

the ionization of the surface silanol groups (SiOH) on the inner wall above

about pH 4. Hydrated buffer cations accumulate close to the negatively charged

178 Section D – Separation techniques

Table 1. A comparison of classical gel and capillary electrophoresis

Classical gel Capillary

Gels

Polyacrylamide or agarose

slabs: length and width 5–25 cm

thickness 1–2 mm

columns: length 7–10 cm

internal diameter ~5mm

Applied field

100 V–2 kV (5–500 V cm-^1 )

Heat dissipation

Slow from columns, quicker from slabs,

aided by water cooling

Running buffer

0.05–0.5 M electrolyte providing

conductivity and pH stability

Buffer additives

Urea, surfactants, complexing agents,

organic solvents

Sample injection

0.1–1 cm^3 loaded into well in slab or

layered onto top of column

Solute detection

Chromogenic reagent or staining dye e.g.

ninhydrin for amino acids, coomassie

brilliant blue or ponceau S for proteins,

acridine orange for nucleotides including

DNA

Modes

Horizontal or vertical slab, disk or column

SDS –PAGE

Isoelectric focusing

Immunoelectrophoresis

Performance

Slow with limited resolution, but many

samples run simultaneously, may require

several hours

Tubing

Fused-quartz capillary

length 25–75 cm

internal diameter 20–100mm

Applied field

10–50 kV (up to 500 V cm-^1 )

Heat dissipation

Rapid due to high surface area to volume

ratio, aided by air cooling

Running buffer

As for classical gel

Buffer additives

Urea, surfactants, inorganic salts, organic

solvents, sulfonic acids, chiral

cyclodextrins, amines

Sample injection

1–50 nl by hydrodynamic or electrokinetic

method

Solute detection

Similar to HPLC detectors,

UV absorbance and fluorescence the most

common, see Table 3

Modes

Capillary zone electrophoresis (CZE)

Micellar electrokinetic chromatography

(MEKC)

Capillary gel electrophoresis (CGE)

Capillary isoelectric focusing (CIEF)

Performance

Very high efficiency, resolution and

sensitivity, moderately fast, 5 min to about

1h