This chapter provides a deep dive into separation matrix and capillary column technology. First, gels and polymer network systems are discussed, paying close attention to the structures and properties of cross-linked (chemical) gels as well as transitionally cross-linked sieving matrices. Another important approach in capillary gel electrophoresis is the utilization of noncross-linked polymers (physical gels) including linear polyacrylamide and derivatized cellulose polymers. Alternative matrices such as composite gels, agarose, pluronics, sol–gel systems, and block polymers are also conferred in this chapter. Capillary coatings represent another important part of capillary gel electrophoresis, and accordingly the major techniques to generate covalent coatings and physical (noncovalent) surface coverage are introduced. Finally, examples are provided to the reader to help in choosing the appropriate sieving media and capillary coating for the separation of biologically important polymers including nucleic acids, proteins, and complex carbohydrates.

The introductory chapter gives a brief overview of the history of electrophoretic techniques with a special focus on the utilization of gel matrices as anticonvective and sieving media. As part of the early miniaturization efforts, narrow bore capillaries were utilized at the end of the last century resulting in the technique referred to as capillary electrophoresis (CE). CE represents a fully automated version of the electrophoresis techniques requiring unique injection, detection methods, and data acquisition systems, all similar to that is used in liquid chromatography. Then the basic separation modes of capillary gel electrophoresis are shortly introduced including capillary gel electrophoresis, capillary gel isoelectric focusing, capillary gel isotachophoresis, and capillary affinity gel electrophoresis. Finally, a brief comparison with slab gel and capillary gel electrophoresis is provided.

This chapter is devoted to the basic principles of capillary gel electrophoresis with the corresponding theoretical treatments on the fundamentals of the electromigration process of biopolymers. Efficiency, selectivity, and resolution terms are all thoroughly conferred. Band broadening in capillary gel electrophoresis is an important issue influencing separation performance and is discussed accordingly. Unlike in slab gel electrophoresis, the very high applied electric field strengths of dozens of kilovolts generate unwanted Joule heating that should be dissipated to maintain the high performance of the approach. Utilization of complexation-based equilibriums by various forms of affinity additives (free, chemically linked, or physically trapped) is also discussed as this phenomenon can significantly improve separation selectivity.

This chapter discusses the instrumentation details including sample introduction methods, detection systems, and the effects of operation variables. In addition to the regularly used electrokinetic and hydrodynamic injection methods, sample stacking techniques and injection-related artifacts are also conferred. The most often used capillary electrophoresis detection systems such as UV absorbance and diode array detection as well as laser-induced fluorescence detection are shown in detail. Other, not so frequently used methods including indirect, electrochemical, radioisotope detection as well as coupling to mass spectrometers are also discussed. The effect of operation variables on the electrophoresis separation including capillary dimensions, gel concentration (Ferguson plot), separation temperature (Arrhenius plots), as well as the utilization of aqueous and nonbuffer systems are all described.

The application chapter gives numerous examples of the use of capillary gel electrophoresis (CGE) of nucleic acids, proteins, and complex carbohydrates also having a capillary affinity gel electrophoresis section. Separation examples of single- and double-stranded DNA molecules include DNA sequencing applications and restriction fragment analysis, in special attention to mutation detection. Analysis of RNA and modified oligonucleotides is also included here. In the protein analysis section, the most frequently used sodium dodecyl sulfate CGE applications are described both for biopharma and biomedical applications. This is followed by discussing CGE options for carbohydrates, again with the main emphasis of biopharmaceutical applications, but also on food additive analysis. Finally, important subsets of capillary affinity gel electrophoresis applications are shown including the analysis of enantiomers.

Capillary electrophoresis-related microseparation techniques include ultrathin-layer gel electrophoresis and microchip gel electrophoresis. Since this latter can be a subject of another book, in this chapter we only discuss the former one. Analysis of DNA molecules was accomplished by ultrathin-layer gel electrophoresis in early sequencing applications and fragment analysis. Ultrathin gels were also utilized in multilane separation of sodium dodecyl sulfate–protein complexes, but showed their real potential in two-dimensional gel electrophoresis.