High resolution separation of biomolecules
Capillary electrophoresis is a method that utilizes narrow capillaries (typically made of silica) under high voltage for the separation of ions, molecules or molecular complexes. In many applications, it can replace traditional slab-gel electrophoresis of nucleic acids and proteins. It can be performed with or without a gel, or with a special polymer solution inside the capillaries, either under native or denaturing conditions. Modern instruments allow full control of all electrophoretic parameters and thus provide high reproducibility, which often is associated with high resolution; thus, in DNA analyses it is often possible to achieve 1 bp resolution. The diameter of the capillaries is generally between 20 and 100 μm and the length 17–100 cm, so that the capillary volume typically is 10–100 μl. The inner wall of the capillaries may be coated to control both the interaction of the analytes with the silica surface and the electroendoosmotic flow. The outer capillary surface is polymer-coated to improve its mechanical stability.
Samples can be loaded into the capillaries either by electrokinetic injection (applying a high voltage for a short, defined period of time) or by hydrostatic pressure. Another difference, in comparison to gel electrophoresis, is that in capillary electrophoresis a detector remains fixed, so that all of the analytes must travel the same distance. The migration time is then used to identify each analyte in every sample. In addition, many samples may be run simultaneously, each sample in its own capillary. The results are usually displayed as electropherograms. The analytes can be detected using a number of detection methods, including UV absorbance, fluorescence, refractive index, etc. Using appropriate standards, the peak area in each electropherogram can be correlated with the amount of each analyte, and thereby its concentration can be determined.
Capillary electrophoresis has many advantages compared to slab gel electrophoresis and HPLC; it gives high efficiency separations and it’s automated, affordable and environment-friendly. In addition, it only requires small sample volumes and it’s fast and easy to perform. Since the sensitivity that can be achieved by capillary electrophoresis is extremely high, the method also allows analysis of samples containing very low analyte concentrations. Different modes of CE, as for example capillary zone electrophoresis, capillary gel electrophoresis, capillary isoelectric focusing and affinity CE, microfluidic CE, provide a large choice of operating modes from which one can select the best suited for a particular analytical application.
Being a versatile, fast and accurate separation method, capillary electrophoresis can ideally be used for protein and nucleic acid analysis in a number of different applications, involving sequencing, size analyses and quality control. Apart from using it for direct analysis of PCR/RT-PCR amplicons, restriction fragments, cDNA and NGS libraries, it can also be advantageous to include it in workflows involving genotyping, CRISPR, gene expression and regulation, and next-generation sequencing.
For information about a product based on this technology, see QIAxcel Advanced.