Carbohydrate analysis applications

The earliest examples of analytical methods based on chemical kinetics, which date from the late nineteenth century, took advantage of the catalytic activity of enzymes. Typically, the enzyme was added to a solution containing a suitable substrate, and the reaction between the two was monitored for a fixed time. The enzyme s activity was determined by measuring the amount of substrate that had reacted. Enzymes also were used in procedures for the quantitative analysis of hydrogen peroxide and carbohydrates. The application of catalytic reactions continued in the first half of the twentieth century, and developments included the use of nonenzymatic catalysts, noncatalytic reactions, and differences in reaction rates when analyzing samples with several analytes. 

The purpose of the experiment is to illustrate the application of derivatisation in the analysis of sugar and related substances by gas-liquid chromatography. The silylation method described is an almost universal derivatisation procedure for carbohydrate analysis by GC.79... 

A typical application of the RI detector is in carbohydrate analysis. Carbohydrates do not adsorb in the UV, do not ionize and although fluorescent derivatives can be made, the procedure is tedious. Consequently, the RI detector can be ideal for detecting such materials and an example of such an application is shown in figure 18. 

Cotte, J. F., Casabianca, H., Chardon, S., Lheritier, J., and Grenier-Loustalot, M. F. (2003). Application of carbohydrate analysis to verify honey authenticity. /. Chromatogr. A 1021, 145-155. 

One of the applications of CE for carbohydrate analysis that has shown increase acceptance in the biotechnology industry is the relative distribution of the N-linked oligossacharides on the conserved asparagines residue on the Fc portion of rMAbs. The advantages of CE over traditional methods include enhanced separation efficiencies, shorter analysis time, and improved robustness. The oligosaccharides present on a monoclonal antibody are asialo-N-linked complex biantennary structures with a core fucose, typical of the structures produced in the Chinese hamster ovary (CHO) cells. The main forms share the fucosylated branched core structure but vary in their terminal galactose occupancy ... 

Several new phases applied to maltosaccharide analysis that seem to be promising have been described in the literature, for instance, a silica-phase covered with polymeric polyamine resin, and an entirely polymeric resin containing an amide function (35). In any case, partition chromatography is restricted mainly to the utilization of polar-bonded phases, as already described. Nevertheless, there have been a certain number of applications of reversed-phase chromatography that permit relatively simple separations to be achieved. Octadecyl-bonded silica phases are the most widely used, although few applications involve carbohydrate analysis. Their interest lies rather in the analysis of derivated sugars, where the selectivity increases (36). 

In spite of the fact that this chromatographic technique is not generally associated with carbohydrate analysis, some applications are found in the literature where silica gel has been employed either directly or indirectly (after modification of the phase or the analytes). The use of silica gel for this purpose involves a polar eluent such as ethylformate/EtOH/H20 (6 2 1, v/v/v) and has been applied to the chromatographic separation of some mono- and disaccharides. 

Table 6 Examples of Applications of HPLC to Carbohydrate Analysis...

In 1987, Anne Dell s now classic chapter entitled FAB-Mass Spectrometry of Carbohydrates appeared in this series.1 At the time, it provided a much needed introduction to and summary of the capabilities of fast atom bombardment (FAB)2 mass spectrometry (MS), its practicalities, and its potential applications for carbohydrate analysis. The chapter immediately became the authoritative work, and in the intervening almost 20 years it has been neither replaced nor updated. 

Any species that can acquire a charge can be separated by ion-exchange chromatography. Some of the more important applications include analyses of amino acids, nucleotides, carbohydrates, and proteins. Carbohydrate analysis is an interesting example, as most carbohydrates are uncharged... 

The aim of this chapter is to cover the theoretical and practical aspects of capillary gel electrophoresis. It also provides an overview of the key application areas of nucleic acid, protein, and complex carbohydrate analysis, affinity-based methodologies, as well as related microseparation methods such as ultra-thin-layer gel electrophoresis and electric field-mediated separations on microchips. It also gives the reader a better understanding of how to utilize this technology, and determine which actual method will provide appropriate technical solutions to problems that may have be perceived as more fundamental. Micropreparative aspects and applications are discussed in Chapter 12. 

NPC has also found some apphcations in the held of carbohydrate analysis. Typical stationary phases used for this application are alkyl amine-, diol-, or polyol-bonded silicas [50-53]. Alkyl amino-bonded silicas are commonly used for the separation of saccharides and oligosaccharides in various matrixs, such as food or biological huids. Although water is used as part of mobile phase, the retention behavior of carbohydrate follows the NPC retention behavior. 

It is impossible to conclude this discussion on FAB without mentioning the possibility of recording the spectrum of a substance directly from a thin-layer chromatography (TLC) plate (84AC109 84UP1), as this will have direct applications (on paper chromatography) in carbohydrate analysis. 

The basic approach of this edition is little changed from the first the emphasis is still on the review of methods and applications which are most useful for quantitative, analytical determination of ions in a wide variety of matrices. An ultimate practitioner of ion chromatography, the author has added a substantial amount of data from his own applications development work. The theoretical background description on various subjects of ion determination is short but informative, and is written so that a novice in the field will not only read and understand it, but also enjoy it. Experts in the field, on the other hand, will undoubtedly find Dr. Weiss s new text a useful reference for many applications and practical problems faced by an analytical chemist, ranging from the field of water purity analysis to the complex task of carbohydrate analysis of glycoproteins. 

In the procedure of Laine and Sweeley [405], methoximation and trimethylsilyla-tion are combined to avoid variation in the ratios of a- and j8-anomers of the pyranose and furanose forms. This approach was used to handle successfully 33 sugar derivatives, including aldoses, ketoses, polyols, acidic forms and A -acetylated amino sugars, on a fused silica capillary column [406]. Obviously, the problems of multiple peak formation can be overcome through the use of highly efficient capillary columns. The application of capillary columns in carbohydrate analysis, used probably for the first time by Tesarik [407] in 1972, has now become widespread. 

One promising approach to be exploited more in the future with respect to TCD analysis is microchip-based CE. It is expected to enable ultimate integrated and highly sensitive systems to be built [176-178], CE-MS coupling represents another area of fast development in carbohydrate analysis of TCD, as it has advanced in other applications in research, biopharmaceutical, and bioindustrial fields. 

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