Instant Notes: Analytical Chemistry

Sample injection For classical electrophoresis, samples of 0.1–1 cm^3 are loaded into wells formed
in gel slabs or layered onto the tops of gel columns, often with the addition of a
sucrose solution to increase the density. For CEand CEC, much smaller samples
(1–50 nl) are drawn into one end of the capillary (usually the anodic end) from
a sample vial, either hydrodynamically using gravity, positive pressure or a
vacuum, or electrokinetically by applying a voltage for a short time when the
EOF causes the sample components to migrate into the capillary. The repro-
ducibility of sample injection into capillaries, typically 0.5–3%, is variable, and
electrokinetic methods may discriminate between components of a mixture
because of differences in electrophoretic mobilities. Time, temperature, pressure
drops and sample and running buffer viscosities are all sources of variability,
and automated sample injection is preferable to minimize these effects.

Solute detection For classical gel electrophoresis, solutes are detected on the gel after the separa-
tion is complete by treating it with a chromogenic reagent similar to those used
in TLC(Section D3), or a staining dye. The gels are immersed in a reagent or
dye solution, then the excess is removed by washing with a suitable solvent to
reveal the solute bands.
Detectors for CEand CECare similar to those used in HPLC (Topic D6), UV-
absorbancespectrometers, especially diode array detectors, and fluorimeters

180 Section D – Separation techniques

Capillary wall

Negatively charged

capillary surface due

to SiO– sites

Hydrated cations

accumulating at surface

Electro-osmotic

bulk flow profile

Cross-sectional flow profile

due to electro-osmotic flow

Cross-sectional flow profile

due to hydrodynamic flow

Fig. 3. EOF and comparisons of flow-profiles in a fused-quartz capillary and an HPLC

column.