Andrea Weston

Factors affecting the separation of inorganic metal cations by capillary electrophoresis

Abstract Various alkali metals, alkaline earth metals, transition metals and lanthanides were separated by capillary electrophoresis, and factors influencing the separations were studied. The reproducible separation of fifteen metal cations was completed in 8 min. The detection system showed a linear relationship between peak area and analyte concentration. To permit the use of indirect photometric detection and to ensure symmetrical peak shapes, a highly UV-absorbing amine having an electrophoretic mobility similar to those of the analyte cations was chosen as the major component of the electrolyte. Complexing compounds were added to the electrolyte to maximize selectively the differences in the apparent mobilities of the cations and enhance the separations.

Purification of proteins on an epoxy-activated support by high-performance affinity chromatography

Abstract The use of a rigid silica-based packing material with large particle and pore size, 37–55 μm and 500 A pore, for affinity chromatography makes it possible to combine high selectivity with short analysis times. Both large and small molecules have been covalently bonded to the Protein-Pak TM Affinity Epoxy-Activated bulk packing for purification of glycoproteins, immunoglobulins, enzymes, lectins and other proteins. Recombinant protein A, GammaBind TM G, heparin, Cibacron Blue F3G-A, sulfanilamide, N-acetyl- D -glucosamine, concanavalin A and aminophenylboronic acid were covalently attached to the affinity packing for selective purification of proteins.

Instrumentation for High-Performance Liquid Chromatography

A high-performance liquid-chromatographic instrument consists of eluent containers, pump, injection device, column, detector, waste container, and data station. This chapter discusses the features of the pump, injector, column, and detector, which are connected together with narrow-inner-diameter tubing to minimize band broadening. The chapter also introduces approaches to sample preparation. The inner diameter of the tubing that is used between the injector and column, and also between the column and the detector, must be as narrow as possible to minimize band broadening. The choice of detector is based on the intrinsic properties of the solute. Often, more than one detector can be used to maximize sample information and confirm peak identities. An absorbance detector could be placed in series with a conductivity detector for the visualization of a charged, chromophoric solute. The length, diameter, and construction material of the column, which is the most essential part of a chromatograph, affect the lifetime, efficiency, and speed of separation.

Chapter 1 – High-Performance Liquid Chromatography

Publisher Summary This chapter introduces the basic theory and terminology governing chromatographic separations and the equations used to calculate the effectiveness of the analytical system. Liquid chromatography (LC), which is one of the forms of chromatography, is an analytical technique that is used to separate a mixture in a solution into its individual components. LC is used to describe any chromatographic procedure in which the mobile phase is a liquid. High-performance liquid chromatography (HPLC) is the term used to describe LC in which the liquid mobile phase is mechanically pumped through a column that contains the stationary phase. An HPLC instrument, therefore, consists of an injector, a pump, a column, and a detector. With this information, the best separation mechanism and column characteristics for a given problem can be chosen on the basis of the nature of the components in the mixture as well as the physical and chemical characteristics of the column.

Separations in Capillary electrophoresis

This chapter describes the capillary electrophoresis (CE) modes in terms of the separation mechanisms and the buffer systems. Examples of compounds separated by each mode, and the advantages and disadvantages of each mode, are provided in this chapter to enable investigators to choose the most appropriate mode for various applications, providing an understanding of the factors affecting the separation to be able to develop basic separations in each mode. Three distinct separation mechanisms have been developed for the separation of analytes by CE. The first and most often encountered separation mechanism in CE is based on the mobility differences of the analytes in an electric field; these differences are dependent on the size and charge-to-mass ratio of the analyte ion. The second separation mechanism is found in capillary isoelectric focusing, where analytes are separated on the basis of isoelectric points. The third mechanism is found in capillary isotachophoresis, where all of the solutes travel at the same velocity through the capillary, but are separated on the basis of differences in their mobilities.

Instrumentation for Capillary Electrophoresis

The main components of a capillary electrophoresis (CE) instrument are power supply, injection system, capillary, detector, and buffer vials. The final electropherogram looks similar to a chromatogram obtained from high-performance liquid chromatography (HPLC). The majority of the literature using CE describes the application of ultraviolet (UV) or fluorescence detection because these were the only detectors offered by the early commercial instrument manufacturers. Next-generation instruments offer conductivity and mass spectrometry detection in addition to fluorescence and UV detection; therefore, applications using these detectors are no longer limited to university research departments. One of the major differences among commercially available instruments is the approach to cooling the capillary; the options include liquid cooling, forced air-nitrogen convection, and no mechanical circulation. Liquid cooling provides the most control, but forced air-nitrogen is sufficient for the purposes of reproducibility. Capillary cooling is of the most importance when using high ionic strength buffers and high voltages.

Chapter 4 – Capillary Electrophoresis

Publisher Summary The electrophoretic mobility of a solute is a characteristic property of the solute that describes its movement under the influence of an applied field. Electrophoretic mobility is dependent both on the charge density of the solute and on the physical properties of the buffer system. The apparent mobility of a species, that is, the mobility that the species appears to possess, is the sum of the electrophoretic mobility and the mobility of the electroosmotic flow. Capillary electrophoresis (CE) has emerged as an alternative form of electrophoresis, where the capillary wall provides the mechanical stability for the carrier electrolyte. CE incorporates all of the electrophoretic modes that are performed within a capillary. CE methods may be classified according to the nature of the electrolyte system or according to the contribution of the electroosmotic flow. The former classification is the most popular, and two types of systems are identified: continuous systems, which include kinetic and steady-state processes, and discontinuous systems.

Chapter 8 – Miniaturization

Publisher Summary Interest in miniaturization is increasing with the growing environmental concerns over waste disposal and the high cost of many biological samples. Because of high resolution, selectivity, sensitivity, and speed, high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) have become the methods of choice for a vast array of analytical separations. Miniaturization of these techniques offers many advantages. This chapter presents an overview of the advantages and disadvantages of various types of miniaturization, and the requirements they impose on instrument design. One advantage is that HPLC, which uses high pressure and high flow rates, can be more readily interfaced with spectroscopic identification techniques such as mass spectrometry, which require much lower flow rates. Interest in the miniaturization of CE has progressed in the direction of CE on a silicon chip.

Chapter 7 – Data Manipulation

Publisher Summary Data manipulation can be divided into qualitative analysis and quantitative analysis. Qualitative analysis is performed to determine the nature of the analytes in the sample. Quantitative analysis is performed to determine the amount of each analyte in the sample. The sample passes through the instrument and generates a signal that is recorded by the data station or strip-chart recorder. The signal must then be converted into qualitative or quantitative information. Data manipulation is the final step of the analysis. There are a variety of techniques available to aid in the identification of sample components: matching retention times, standard addition, internal standard, isotopic labeling, enzyme peak shift, and ultraviolet (UV) and mass spectral libraries. After integration, the analytical response is converted into an analyte concentration with the aid of a calibration curve. There are four principal techniques for determining relative composition information about a sample: normalization, internal standard, external standard, and standard addition.

Separations in High-Performance Liquid Chromatography

A variety of chromatographic modes have been developed on the basis of the mechanisms of retention and operation. The key chromatographic modes are normal-phase, reversed-phase, ion exchange, size-exclusion, and affinity chromatography. This chapter introduces the chromatographic modes and explains how they work. In addition to the major modes, there are a number of techniques that could be viewed as submodes. Examples of compounds separated by each mode, and the advantages and disadvantages, are provided to help chromatographers choose the most appropriate mode for a given group of analytes. There are a number of applications of high-performance liquid chromatography (HPLC). Normal-phase chromatography is used for the separation of neutral species on the basis of polarity, reversed-phase chromatography is used for the separation of neutral species on the basis of hydrophobicity, ion-exchange chromatography is used for the separation of ionic solutes on the basis of charge, size-exclusion chromatography is used for the separation of molecules on the basis of differences in molecular size, and affinity chromatography is used for the separation of biomolecules on the basis of the lock-and-key mechanism prevalent in biological systems.