Electrophoresis

Introduction

Electrophoresis is a separations technique that is based on the the mobility of ions in an

electric field. Positively charged ions migrate towards a negative electrode and
negatively-charged ions migrate toward a positive electrode.For safety reasons one
electrode is usually at ground and the other is biased positively or negatively. Ions have
different migration rates depending on their total charge, size, and shape, and can
therefore be separated.

Instrumentation

An electrode apparatus consists of a high-voltage supply, electrodes, buffer, and a

support for the buffer such as filter paper, cellulose acetate strips, polyacrylamide gel, or
a capillary tube. Open capillary tubes are used for many types of samples and the other
supports are usually used for biological samples such as protein mixtures or DNA
fragments. After a separation is completed the support is stained to visualize the
separated components.

Resolution can be greatly improved using isoelectric focusing. In this technique the
support gel maintains a pH gradient. As a protein migrates down the gel, it reaches a pH
that is equal to its isoelectric point. At this pH the protein is netural and no longer
migrates, i.e, it is focused into a sharp band on the gel.

Schematic of zone electrophoresis apparatus

Discontinous Electrophoresis
Introduction

Discontinous electrophoresis uses two gels that are buffered at different pHs. When
proteins migrate from one gel to the other they become concentrated into sharp bands,
which produce higher resolution than in conventional electrophoresis.

Schematic of discontinous electrophoresis technique

Capillary Electrophoresis (CE)

Introduction

Performing electrophoresis in small-diameter capillaries allows the use of very high

electric fields because the small capillaries efficientlydissipate the heat that is produced.
Increasing the electric fields produces very efficient separations and reduces separation
times.

Instrumentation

Capillaries are typically of 50 µm inner diameter and 0.5 to 1 m in length. The applied
potential is 20 to 30 kV. Due to electroosmotic flow, all sample components migrate
towards the negative electrode. A small volume of sample (10 nL) is injected at the
positive end of the capillary and the separated components are detected near the negative
end of the capillary. CE detection is similar to detectors in HPLC, and include
absorbance, fluorescence, electrochemical, and mass spectrometry.
The capillary can also be filled with a gel, which eliminates the electroosmotic flow.
Separation is accomplished as in conventional gel electrophoresis but the capillary allows
higher resolution, greater sensitivity, and on-line detection.

Schematic of capillary electrophoresis

Electroosmotic flow

The surface of the silicate glass capillary contains negatively-charged functional groups
that attract positively-charged counterions. The positively-charged ions migrate towards
the negative electrode and carry solvent molecules in the same direction. This overall
solvent movement is called electroosmotic flow. During a separation, uncharged
molecules move at the same velocity as the electroosmotic flow (with very little
separation). Positively-charged ions move faster and negatively-charged ions move
slower.

Schematic of the double layer on the capillary surface

SDS-PAGE
Introduction

SDS-PAGE stands for sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis
(PAGE) and is useful for molecular weight analysis of proteins. SDS is a detergent that
dissociates and unfolds oligomeric proteins into its subunits. The SDS binds to the
polypeptides to form complexes with fairly constant charge to mass ratios. The
electrophoretic migration rate through a gel is therefore determined only by the size of
the complexes. Molecular weights are determined by simultaneously running marker
proteins of known molecular weight.