Electrophoresis

Electrophoretic separations

are based on differences in migration of charged analytes (ions) in an electric field in solution

Capillary Electrophoresis

electrophoretic separation carried out in free buffered solution

Capillary Gel Electrophoresis

electrophoretic separation carried out in buffered solution immobilised in a gel or a similar support

Capillary zone electrophoresis (CZE)

can be used to separate proteins, peptides and nucleic acids

Micellar electrokinetic chromatography (MEKC)

is based on solutes partitioning between micelles and the running buffer

Capillary gel electrophoresis (CGE)

can be used for size-based separation of biological macromolecules

Isoelectric focusing (IEF)

is separation based on isoelectric point difference; can be used for separation of amphoteric molecules such as proteins and peptides

CZE fused silica capillary has a length of

30-100 cm

Chip CZE

is CZE performed on a minuature device

Electrophoretic mobility/velocity

velocity per unit field strength

Electro-osmotic flow (EOF)

the motion of liquid induced by an applied potential across a capillary tube or microchannel with a surface charge on the wall

The EOF is caused by

an electrical double layer that forms at the stationary/solution interface

Inner Helmholtz layer

is formed due to build up of fixed layr of cations at the deprotonated silanol groups

Outer Helmholtz layer

is a layer of mobile cations from the carrier electrolyte

When voltage is applied outer Helmholtz layer moves to

cathode

Movement of outer Helmholtz layer to cathode causes

flow of the whole electrolyte to the cathode

Outer Helpholtz layer contains more … charges.

positive

EOF gives … peak broadening than hydrodynamic laminar flow.

less

EOF leads to … peak shape.

triangular

Increase of ionic strength of carrier electrolyte … EOF

decreases

Organic solvents in carrier electrolyte generally … EOF

decrease

Addition of EOF-modifiers can … or … EOF

reduce, reverse

Example of EOF-modifier

tetradecyltrimethylammonium

In electrophoresis sample can be injected

by pressure and electrokinetically

Injection of sample by pressure or electrokinetically leads to … sample sizes

small (10^-9 liters)

Electrokinetic injection

A voltage is applied for a certain time while placing the sample vial at the inlet end of the separation capillary.

Ionic compounds with the respective charge (+ or -) migrate into the capillary.

Additionally the induced EOF transports liquid from the sample vial into the capillary or in the case of reversed polarity liquid from the capillary is brought back into the vial.

Electrokinetic injection is highly

discriminating

Electrokinetic injection is highly discriminating. How is it useful?

It can be employed to inject a much larger number of analyte ions of interest into the capillary than in simple pressure injection.

Direct UV detection

detects UV-active sample cations/anions in UV-transparent background electrolyte (phosphate, borate)

Indirect UV detection

detects UV-active cations/anions of carrier electrolyte in imidazole (cations) or chromate napthalenedicarboxylic acid (anions)

CZE mechanism

Capillary is filled with (buffered) carrier electrolyte.

Separation according to electrophoretic mobilities (radius/charge ratio of analyte)

Optimization of CZE separation can be done by

changing pH

addition of complex-forming agents to the carrier electrolyte

Changing pH in CZE

changes the degree of dissociation of the analytes or even reverses the charge

Introducing complex-forming agents

changes size and/or size

Chiral separations by CZE

addition of cyclodextrines (chiral selectors) to the carrier electrolyte

in chiral CZE separations

diastereomeric complexes form between enantiomeric analyte and the chiral selectors

In isoelectric focusing

a capillary is filled with mixture of ampholytes (aminocarboxylic acids of different pH) leading to a pH gradient after applying voltage.

in IF ampholytic analytes are separated according to their

isoelectric point values

In IF analytes migrate

in the pH gradeint until they reach the pH zone corresponding to their pI value and become neutral

In gel electrophoresis

a layer if an anticonvective support (polyacrylamide, agarose, cellulose acetate) is coated on a glass plate.

SDS-PAGE

Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis

In SDS-PAGE samples are separated

in two dimentions (according to pI and then size)