Carbohydrate coupling chromatography

Pulsed amperometric detection (PAD), introduced by Johnson and LaCourse (64, 65) has greatly enhanced the scope of liquid chromatography/electrochemistry (66). This detection mode overcomes the problem of loss of activity of noble metal electrodes associated with the fixed-potential detection of compounds such as carbohydrates, alcohols, amino acids, or aldehydes. Pulsed amperometric detection couples tlie process of anodic detection with anodic cleaning and cathodic reactivation of a noble metal electrode, thus assuring a continuously cleaned and active... 

Conboy, J. J. Henion, J. High performance anion exchange chromatography coupled with mass spectrometry for the determination of carbohydrates. Biol. Mass Spectrom. 1992, 21, 397—407. 

Jeffrey, A.M., Zopf, D.A., and Ginsburg, V. (1975) Affinity chromatography of carbohydrate-specific immunoglobulins coupling of oligosaccharides to sepharose. Biocbem. Biopbys. Res. Comm. 62, 608-613. 

In principle, mass spectrometry is not suitable to differentiate enantiomers. However, mass spectrometry is able to distinguish between diastereomers and has been applied to stereochemical problems in different areas of chemistry. In the field of chiral cluster chemistry, mass spectrometry, sometimes in combination with chiral chromatography, has been extensively applied to studies of proton- and metal-bound clusters, self-recognition processes, cyclodextrin and crown ethers inclusion complexes, carbohydrate complexes, and others. Several excellent reviews on this topic are nowadays available. A survey of the most relevant examples will be given in this section. Most of the studies was based on ion abundance analysis, often coupled with MIKE and CID ion fragmentation on MS " and FT-ICR mass spectrometric instruments, using Cl, MALDI, FAB, and ESI, and atmospheric pressure ionization (API) methods. 

Z)-awh-4-Hydroxy-l-aIkenyl carbamates 363, when subjected to substrate-directed, vanadyl-catalysed epoxidation , lead to diastereomerically pure epoxides of type 364 (equation 99)247,252,269 qqjggg epoxides are highly reactive in the presence of Lewis or Brpnsted acids to form -hydroxylactol ethers 366 in some cases the intermediate lactol carbamates 365 could be isolated . However, most epoxides 364 survive purification by silica gel chromatography . The asymmetric homoaldol reaction, coupled with directed epoxidation, and solvolysis rapidly leads to high stereochemical complexity. Some examples are collected in equation 99. The furanosides 368 and 370, readily available from (/f)-0-benzyl lactaldehyde via the corresponding enol carbamates 367 and 369, respectively, have been employed in a short synthesis of the key intermediates of the Kinoshita rifamycin S synthesis . 1,5-Dienyl carbamates such as 371, obtained from 2-substituted enals, provide a facile access to branched carbohydrate analogues . 

This detection system is coupled with anion-exchange chromatography (48,49), which has the same alkaline pH requirements. The sensitivity of PAD is considerably higher than that yielded using the refractometric detector, with detection limit being of the order of 10 pmol (29). On the other hand, its response also depends heavily on the nature of the carbohydrate. Finally, other alternatives described in the literature include the use of nickel-based or copper-based electrodes and catalytic oxidation (50). 

All the anti-carbohydrate antibodies purified by affinity chromatography are isomers of a different number of isoforms. The forms can be separated by electrofocusing and all have been found to have combining activity for the immunodeterminant of the same antigen by the method of coupled analysis of electrofocusing and agar diffusion [88], The isoforms of antimonosaccharide antibodies are shown in Fig. (48). 

The majority of reports have used electrospray ionization mass spectroscopy (ESI-MS) as an analytical detection method because of its sensitivity and the soft namre of its ionization procedure, which generally only leads to the detection of the molecular ions of the positive library members. Many separation techniques have been coupled to ESI-MS, including affinity chromatography (49), size exclusion chromatography (50, 51), gel filtration (52), affinity capillary electrophoresis (53-58), capillary isoelectric focusing (59), immunoaffinity ultrafiltration (60), and immunoaffinity extraction (61). ESI-MS has also been used alone (62) to screen a small carbohydrate library. Other examples reported alternative analytical techniques such as MALDI MS, either alone (63, 64) or in conjunction with size exclusion methods (65), or HPLC coupled with immunoaffinity deletion (66). 

Bernal, J.L., Del Nozal, M.J., Toribio, L., Del Alamo, M. (1996). HPLC Analysis of carbohydrates in wines and instant coffees using anion exchange chromatography coupled to pulsed amperometric detection. J. Agric. Food Chem.,44, 507-511. 

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