Instrumental electrophoresis, general

Electrophoresis is the most powerful method available for separation and analysis of complex mixtures of charged biopolmers. This chapter provides an overview of modern electophoresis as a general introduction to the chapters which follow. The basic electrophoretic operating modes and formats for these modes are described. Means for detection of separated zones are reviewed. Finally, an approach to fully instrumental electrophoresis is discussed. 

Parallel to the development of mass spectrometric instrumentation and methodologies, the improvements of separation techniques, such as gas chromatography (GC), high performance liquid chromatography (HPLC) and capillary electrophoresis (CE), and of their coupling with MS allowed the study of complex mixtures, that are generally encountered in most studies. 

The first work on pKa determination by zone electrophoresis using paper strips was described by Waldron-Edward in 1965 (15). Also, Kiso et al. in 1968 showed the relationship between pH, mobility, and p/C, using a hyperbolic tangent function (16). Unfortunately, these methods had not been widely accepted because of the manual operation and lower reproducibility of the paper electrophoresis format. The automated capillary electrophoresis (CE) instrument allows rapid and accurate pKa determination. Beckers et al. showed that thermodynamic pATt, (pATf) and absolute ionic mobility values of several monovalent weak acids were determined accurately using effective mobility and activity at two pH points (17). Cai et al. reported pKa values of two monovalent weak bases and p-aminobenzoic acid (18). Cleveland et al. established the thermodynamic pKa determination method using nonlinear regression analysis for monovalent compounds (19). We derived the general equation and applied it to multivalent compounds (20). Until then, there were many reports on pKa determination by CE for cephalosporins (21), sulfonated azo-dyes (22), ropinirole and its impurities (23), cyto-kinins (24), and so on. 

Capillary electrophoresis (CE) is the most rapidly expanding separation technique in pharmaceutical analysis and is a rival to HPLC in its general applicability. The instrumentation is quite straightforward, apart from the high voltages required, but the parameters involved in optimising the technique to produce separation are more complex than those involved in HPLC. The technique is preferred to HPLC where highly. selective separation is required. 

In comparing separation techniques, we generally find a striking difference in methods based on continuous (c) chemical potential profiles and those involving discontinuous (d or cd) profiles. There is, for example, a glaring contrast in instrumentation, applications, experimental techniques, and the capability for multicomponent separations between the two basic static systems, Sc (e.g., electrophoresis) and Sd (e.g., extraction). Similarly, there... 

Ref [i] AltriaKD (1996) General guidelines to the operation of capillary electrophoresis methods and instrumentation. In Altria KD (ed) Capillary electrophoresis guidebook principles, operation, and applications. Methods in molecular biology, vol. 52. Humana Press, Totowa... 

Electrophoretic techniques, such as two-dimensional electrophoresis or isoelectric focusing, lend themselves under appropriate conditions to the separation with excellent resolution of small amounts of samples. They have mainly found use as powerful methods of analysis since their general applicability in the areas of peptide purification and isolation has, until recently, been severely restricted by limitations in sample capacity and instrumental design. 

Analytical instrumentation continues to increase in sophistication, and as a consequence, the range of materials that can now be almost routinely analysed has increased accordingly. Books in this series which are concerned with the techniques themselves will reflect such advances in analytical instrumentation, while at the same time providing full and detailed discussions of the fundamental concepts and theories of the particular analytical method being considered. Such books will cover a variety of techniques, including general instrumental analysis, spectroscopy, chromatography, electrophoresis, tandem techniques. 

Table 12.1 shows the variety of capillary electrophoresis detection methods that have been tested to date, as well as their reported detection limits. While detection limits for instrumental methods are usually reported in concentration units, those reported for CE methods are generally given in moles because of zone broadening (the peak concentration at the detector is always less than the concentration injected) and the variety of injection volumes that are possible between instruments with different sized capillaries and different injection and operating potentials. 

In this section, instrumentation, general electrophoretic operations, technical and practical considerations, and types of conventional electrophoresis are discussed. 

In assessing the yield and quality of DNA/RNA extracts, electrophoretic methods provide better precision and more information regarding quality, for instance the size distribution of the isolated DNA/RNA. Yet, these methods generally require significantly higher amounts of the extracted sample when compared with spectrophotometric assessment. However, automated analyzers that use prefabricated chips with microfluidic channels and are designed for the electrophoresis of microvolumes of DNA and RNA have become available. These instruments allow the sensitive and precise quantification and qualitative assessment of nucleic acids. 

The initial cost of equipment and the expense of maintenance for electrophoresis is generally significantly lower than for ion chromatography and atomic spectroscopy. Thus, commercial electrophoretic instruments are marketed in the price range of 10,000 to 65,000. ><>... 

Identification of components in environmental samples and in samples from laboratory studies of biodegradation and biotransformation is generally based on the application of MS coupled to either GC or LC systems. For environmental samples which may contain only small amounts of the relevant compounds, MS is particularly attractive in view of the extremely small amounts of samples — of the order of nanograms — which are required. An important additional advantage is that since the mass spectrometer can be interfaced with GC, LC, or capillary electrophoresis (CE) systems which incorporate separation procedures, pure samples are not required. Some salient issues in MS in the context of environmental application are summarized briefly as an introduction. Reference should be made to an exhaustive review (Burlingame et al. 1998) for instrumental details and aspects that are not covered here, such as MS of synthetic and natural polymers. 

---