Combination of chromatography

The combination of chromatography and mass spectrometry (MS) is a subject that has attracted much interest over the last forty years or so. The combination of gas chromatography (GC) with mass spectrometry (GC-MS) was first reported in 1958 and made available commercially in 1967. Since then, it has become increasingly utilized and is probably the most widely used hyphenated or tandem technique, as such combinations are often known. The acceptance of GC-MS as a routine technique has in no small part been due to the fact that interfaces have been available for both packed and capillary columns which allow the vast majority of compounds amenable to separation by gas chromatography to be transferred efficiently to the mass spectrometer. Compounds amenable to analysis by GC need to be both volatile, at the temperatures used to achieve separation, and thermally stable, i.e. the same requirements needed to produce mass spectra from an analyte using either electron (El) or chemical ionization (Cl) (see Chapter 3). In simple terms, therefore, virtually all compounds that pass through a GC column can be ionized and the full analytical capabilities of the mass spectrometer utilized. 

Other combinations of chromatography techniques with MS which may be useful in environmental studies are the coupling of high performance liquid chromatography (LC) with MS [84,384,504,506,530,585-593],LC with MS-MS [181, 594 - 599], LC with atmospheric pressure chemical ionization MS (LC-APCI-MS) [600], and Fourier transform infrared spectroscopy-fast atom bombardment coupled to LC-MS (FTIR-FAB-LC-MS) [514]. 

Sechenov, G. P., Bunina, N. N., Al tshuler, V. S. A method for analysing low molecular weight gases by combination of chromatography and volumetric analysis. Tr. Inst. Goryuch. Iskop., Akad. Nauk. SSSR. 18, 253 (1962). - Chem. Abstr. 58, 6624 (1963). 

Some good books are available on some of the subjects discussed in this article, for example, on combination of chromatography and FTIR (2), FTIR spectroscopy in general (3,4), and IR spectra of phosphorus-containing chemicals (5). 

Another drawback is the fact that there are over 60,000 chemical compounds in commerical use, and each one cannot have a distinctive retention volume on a given system. If a compound or a mixture is truly unknown, its chromatogram will not provide characteristic retention volumes on the basis of which identifications can be made. In short, chromatography can be used for qualitative analysis for a limited set of chemicals, but its main use is not for screening unknowns. In fact, one of the best methods of qualitative analysis is the combination of chromatography (an excellent separation method) with mass spectrometry (an excellent identification method). This topic is included in Chapter 11. 

The development of hyphenated techniques has depended on advances in at least three areas interfacing, scanning speed of the measuring technique, and adequate data systems. The different combinations of chromatography and on-line measuring instruments have different requirements, but a few generalizations can be made. Since the GC/MS combination is the most popular and successful, it will be described as a typical example of the types of considerations required for interfacing instruments. Problems associated with other interfaces will be mentioned briefly later. 

Weber, G., Jakubowski, N., Stuewer, D. Speciation of platinum in plant material. A combination of chromatography, elemental mass spectrometry and electrochemistry. In Zereini, F., Alt, F. (eds.) Anthropogenic platinum-group element emissions. Their impact on men and environment, pp. 183-190. Springer, Berlin (2000)... 

The analytical techniques that best meet these criteria are those based on the combination of chromatography with MS, either liquid chromatography/MS (LC/MS) or GC/MS. The most commonly used approaches for trace level organic impurity identification are therefore MS based. Note that an overview of MS and its application in the pharmaceutical industry was presented in the previous edition of this treatise. 

M. Kaspereit, Separation of Enantiomers by a Process Combination of Chromatography and Crystallisation, PhD thesis, Shaker Verlag, Aachen, Germany, 2006... 

The combination of chromatography/mass spectrometry with MS/MS methods can in fact markedly enhance the analytical performance of the identification of phenols. This was demonstrated in the case of hydroxy aromatic components in coal-derived liquids. The analytical performance can be further improved by using chemical derivatization, as also shown in an MS/MS study of some methylphenols and methylnaphthols. In the course of GC/MS/MS analytical studies on nonylphenol in biological tissues, derivatization proved to be favourable in an indirect way The El mass spectrum of nonylphenol... 

This methodology easily can be extended to the analysis of combinatorial chemistry samples. In combinatorial chemistry, the combination of chromatography and MS has proved a powerful method for substance identification based primarily on molecular weight. Analogously, HPLC-FTIR provides powerful identification possibilities, but based on molecular structure. As identifying a substance by MS requires knowledge about its possible molecular structure, the two techniques form a highly complementary system of identification. 

The progressive use of computational methods in chemistry laboratories leads to an amount of data that is barely manageable even by a team of scientists. Specialized methods in instrumental analysis, like the combination of chromatography with mass spectrometry, may produce several hundred megabytes of data in every run. Combinatorial approaches support this trend. 

Final Purification. A pharmaceutical product such as an AdV requires a high degree of purity. This level of purity is usually achieved with a combination of chromatography and filtration operations. The goal is to separate the viral particles from the most recalcitrant impurities that persist in the streams. The chromatographic operations described in the literature for virus purification explore properties such as size, charge, hydrophobicity, and metal affinity [41, 80,102-107]. 

An LC—MS chromatogram is essentially a three-dimensional data set with separate chromatographic, mass-to-charge (m/z) and ion count (intensity) dimensions. The combination of chromatography and mass spectral approaches can take on many incarnations and are dependent on the nature of the metabonomics investigation. Because of the complexity of most metabonomics samples, the final solution is often a compromise between good science and expediency since the ideal analytical conditions for 1000+ metabolome components is an um-ealistic goal. In this section, we will discuss each of these elements separately with an emphasis on biomedical metabonomic applications. 

Trypsin splits the protein at 11 different points, yielding 12 different polypeptides that can readily be separated by a combination of chromatography and electrophoresis. The further digestion of each polypeptide according to methods that have now become standard provides the exact amino acid composition and sequence of the polypeptide. By this procedure, Wittman and Wittman and Liebold were able to localize the amino acid changes in a number of mutants. 

In recovering each of the separated compounds of a chromatographic separation when they are solids, the various correct fractions are combined and evaporated. If the combined fractions contain sufficient material, they may be purified by recrystallization. If the compounds are liquids, the correct fractions are combined, and the solvent is evaporated. If sufficient material has been collected, liquid samples can be purified by distillation. The combination of chromatography-crystallization or... 

Dwyer [51] used a combination of chromatography and IR spectroscopy to provide a versatile tool for characterisation of polymers. HPLC-Fourier-transform IR spectroscopy interface systems deposit the output of a chromatograph on an IR optical medium, which is then scanned to provide data as a time-ordered set of spectra of the chromatogram. Polymer analysis applications described include the identification of polymer additives, the determination of composition/molecular weight distributions in copolymers, the mapping of components of polymer alloys and blends, molecular configuration changes in polymers, and component identification in complex systems. 

The elimination of interspecies reactive anti-IgG antibodies has been achieved by chromatography of anti-IgG sera on immobilized heterologous IgG.618 Using a combination of chromatography on immobilized concanavalin A followed by gel chromatography, IgG has been separated from serum albumin, ... 

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