23 Gel and Capillary Electrophoresis*

Electrophoresis was recognized with the Nobel Prize in Chemistry in 1948. The first experiments were done with aqueous salt solutions in a narrow glass U-tube apparatus with electrodes at each end; however, the format would soon change to using starch or agar gels.

Slab Electrophoresis

Today, the two most common formats for gel electrophoresis are slab agarose gels (0.5–2.0% w/v) and slab polyacrylamide gels (7–20% w/v). The gels are formed with embedded sample wells and positioned in buffer-filled tanks with an electrode at each end. Samples are pipetted into the sample wells, often with an additive that increases the solution density and causes the sample to sink to the bottom of the well (rather than dispersing). The voltage is switched on to start the experiment. Applied field strengths of 4­–15 V/cm are typical, translating into applied voltages of 75–150 V for most apparatuses. EOF can be generated by the gel material, but is typically small.

In slab electrophoresis, the gel acts as a sieving medium such that molecules are separated based on their charge and size. Agarose and polyacrylamide gel electrophoresis are thus popular in biomolecular sciences for the separation of nucleic acids and proteins. These methods have a favourable balance of capability, low cost, and ease of use. Many gels are prepared to be homogenous along their length; however, improved resolution over a wider range of molecular weights is achieved with gradient gels, which gradually transition from a low percentage of gel near the sample wells to a high percentage at the other end. After running for 30–90 min, the applied voltage is switched off and bands of molecules are visualized by staining methods that impart visible colour or fluorescence.

Capillary Electrophoresis

Capillary electrophoresis (CE) is an instrumental method that offers high-resolution separation. A fused silica capillary (i.e. a tube with small inner diameter) spans between two buffer reservoirs, each with an immersed electrode. Typical capillaries have lengths of 25–100 cm with inner diameters of 25–100 µm. Field strengths tend to be on the order of 10³ V/cm, translating to applied voltages of tens of kilovolts (kV) for most experiments. Unlike slab electrophoresis, which does not have a substantial contribution from EOF, the mobilities of chemical species in conventional CE is determined by both their electrophoretic mobilities and the system electroosmotic mobility.

For a CE experiment, sample is injected from the end of the capillary near the anode. The injection can be hydrodynamic, using applied pressure to introduce a plug of sample solution, or electrokinetic, using applied voltage. The hydrodynamic method introduces all chemical species in a sample without bias, whereas the electrokinetic method is biased toward species that have higher mobilities toward the cathode. Typical hydrodynamic injection volumes are 25­–100 nL, depending on the length and inner diameter of the capillary. Electrokinetic injections typically apply the desired voltage for several seconds.

Injected samples are separated under the influence of the applied potential and must be detected as they approach the end of the capillary near the cathode. Common modes of detection include UV-visible absorbance, fluorescence, and mass spectrometry. The data obtained from a CE instrument is an electropherogram that, analogous to a chromatogram, plots a detector signal versus time with different peaks for different chemical species. Migration times and peak areas are used for qualitative and quantitative analysis.

Connections

• Slab and capillary gel electrophoresis are based on the fundamental principles of electrophoresis and electroosmosis (Ch. 22).
• A gradient gel serves a similar purpose to gradient elution in LC (Ch. 19).
• The core designs of LC instruments (Ch. 20) and CE instruments are mostly the same: injector, column/capillary, detector. Unlike LC instruments, CE instruments do not need a pump.
• Detectors for CE are based on the same technology as UV-visible spectrophotometers (Ch. 7), fluorescence instruments (Ch. 9),  and mass-spectrometers (Ch. 25).
• Migration times in CE are conceptually analogous to retention times in elution chromatography (Ch. 19).

Post-Reading Questions

1. What are the two main factors that determine the migration rate in slab gel electrophoresis?
2. A scientist applies a potential of 100 V to their electrophoresis experiment. Are they most likely doing slab gel electrophoresis or capillary electrophoresis?
3. Do anions and cations travel in opposite directions in slab gel electrophoresis? What about in capillary electrophoresis?
4. Does slab gel electrophoresis or capillary electrophoresis provide higher resolution?

Topic Learning Objectives

The chapter is a primer for the following learning objectives, which will be covered in lecture and/or with additional assigned reading:

• Draw block diagrams for slab gel electrophoresis and CE instruments.
• Compare and contrast the practical differences between slab gel electrophoresis and CE.
• Recognize whether a scenario is best suited for agarose or polyacrylamide gel electrophoresis.
• Understand the advantages and disadvantages of electrokinetic and pressure injection in CE.
• Be able to match the possible types of CE detectors to different analyte systems.