What Is Electrophoresis?

Electrophoresis is a separation process that sorts charged particles suspended in a fluid by driving them through that fluid with an applied electric field. A molecule carrying a net charge migrates toward the electrode of opposite polarity at a velocity set by its charge, size, and shape, together with the viscosity, ionic strength, and pH of the surrounding buffer. Because that velocity differs from one species to another, a mixture injected at a single point resolves into discrete zones as the run proceeds, and those zones can then be stained, imaged, or collected. Electrophoresis sits alongside chromatography and centrifugation in the family of analytical separations, distinguished by the fact that its driving force is electrical rather than a pressure gradient or a rotating reference frame.

The technique dates to Arne Tiselius, whose moving boundary apparatus of the 1930s resolved blood serum into albumin and the alpha, beta, and gamma globulin fractions, work recognized with the 1948 Nobel Prize in Chemistry. Free solution experiments of that era were limited by thermal convection and by Joule heating in the conducting buffer. Holding the buffer inside a porous gel or a narrow capillary suppressed both effects, and those two geometries define the two dominant formats in use today.

Gel Electrophoresis

In slab gel formats, the buffer is immobilized in a cross-linked matrix whose pore structure adds a sieving effect, so that separation depends on molecular size rather than charge density alone. Agarose gels resolve nucleic acid fragments across a broad range, and the accepted physical picture of how a long chain threads through the pores is biased reptation, in which the leading segment advances and drags the remainder behind it. A detailed treatment of DNA mobility in agarose, polyacrylamide, and free solution sets out how sieving and free-draining behavior trade off with fragment length. Polyacrylamide gels, with smaller and more uniform pores, handle proteins and short oligonucleotides. Denaturing the sample with sodium dodecyl sulfate confers an approximately constant charge-to-mass ratio, which is why SDS-PAGE reports molecular weight so directly.

Capillary Electrophoresis

Capillary electrophoresis replaces the slab with a fused silica tube of roughly 25 to 100 micrometers internal diameter. The high surface-to-volume ratio carries heat away efficiently, so field strengths of tens of kilovolts per meter become practical and separations finish in minutes with very sharp peaks. NIST defines capillary electrophoresis in terms of differential migration through a capillary filled with a liquid polymer, the configuration that multiplexed sequencing instruments used to read the human genome. Filling the capillary with a sieving polymer gives capillary gel electrophoresis, and a review of protein separation in that format covers the polymer chemistries and detection schemes that made it a purity assay for biologics. Electroosmotic flow, generated by the charged silica wall, adds a bulk transport term that must be controlled or deliberately exploited.

Isoelectric Focusing and Two-Dimensional Separations

Isoelectric focusing runs the sample through a stable pH gradient instead of a uniform buffer. Each protein migrates until it reaches the pH at which its net charge vanishes, its isoelectric point, and then stops, giving a steady-state rather than a time-dependent separation. Combining focusing in one dimension with SDS-PAGE in the second produces the two-dimensional gel maps long used in proteomics, where thousands of spots are resolved by charge and mass together. Imaged capillary versions of the same principle are now standard for measuring the charge heterogeneity of therapeutic monoclonal antibodies.

Applications

Electrophoresis has applications in a wide range of fields, including:

• Clinical chemistry, for serum protein and hemoglobin variant analysis
• Forensic science, for short tandem repeat DNA profiling
• Genomics and DNA sequencing instrumentation
• Biopharmaceutical quality control and lot release testing
• Proteomics and biomarker discovery
• Microfluidic lab-on-a-chip and point-of-care devices