Chromatographic separation of carbohydrates

Unlike dissolved amino acids, carbohydrates have not seen the development of sensitive techniques that are capable of routine chromatographic analysis at natural concentrations. Although there are procedures that in principle should allow determinations of dissolved saccharides at natural concentrations, these have so far not been tested in routine analysis. Mopper et al. (1992) used pulsed amperometric detection after separation of underivatised monosaccharides with an anion-exchange column. Thus, techniques which require sample pre-concentration/desalting and post-column derivatisation are still the ones that have been shown to be reliable for determinations of dissolved free monosaccharides in seawater (Dawson and Liebezeit, 1983). 

Carbohydrate determination by HPLC has been treated by Ben-Bassat and Grushka (1990), while Lee (1990) reviewed applications of anion-exchange chromatography. Gas chromatographic techniques have been described particularly for the determination of particulate carbohydrates after hydrolysis (e.g., Leskovsek et al., 1994). 

Josefsson, B., Lindroth, R, Ostling, G. (1977), AnaL Chim. Acta, 89,21. 

Liebezeit, G., Dawson, R, (1981), J. High Res. Chrom. Chrom. Comm., 4,354. 

(1966), in Automation in Analytical Chemistry, Technicon Symposium 1966. New York Mediad, Vol. 1 pp. 440-444. 

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Niederhauser, T. L., Hailing, J., Poison, N. A., and Lamb, J. D. (1998) High-performance Anion-exchange Chromatographic Separations of Carbohydrates on a Macrocycle-based Stationary Phase with Eluents of Relatively Low pH and Concentration, J. Chromatogr. A 804, 69-77. 

The GC and HPLC methods were developed for the chromatographic separation of carbohydrates. Gas chromatographic methods are very time consuming, because carbohydrates, owing to their non-volatile character, have to be derivatized prior to the determination [97], On the other hand, the LC method using strong-acid cation exchangers in the calcium form and de-ionized water as the eluent [98] is widespread, but has several drawbacks ... 

Kdnig WA, Benecke I, Bretting H (1981) Gas Chromatographic Separation of Carbohydrate Enantiomers on a New Chiral Stationary Phase. Angew Chem Int Ed Engl 20 693... 

Chromatographic Separation of Carbohydrates, U. S. Patent 2,504,169 (April 18, 1950), Melville L. Wolfrom and Wilfred Wendell Binkley. 

The analytical application of particle-dispersed-modified electrodes to the selective detection of a single analyte is limited because of broad catalytic activities thus their use as electrochemical detectors following chromatographic separation of carbohydrates is often suggested. Similar nonspecific catalytic PMEs consisting of electrocatalytic RUO2 particles and Ru(OH2)6 in Nafion have been shown to catalyze the oxidation of catechol and of alcohols, respectively these could presumably be used in place of the carbon paste Ru02-modified electrode developed for postseparation detection of carbohydrates and alcohols. Other electrocatalytic particle electrodes were prepared from lead dioxide in polypyrrole and CoPc entrapped behind a permselective cellulose acetate film. ... 

Allen, D., and El Rassl, Z. (2004). Capillary electrochromatography with monolithic silica columns III. Preparation of hydrophilic silica monoliths having surface-bound cyano groups chromatographic characterization and application to the separation of carbohydrates, nucleosides, nucleic acid bases and other neutral polar species.. Chromatogr. A 1029, 239—247. 

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. 

A general essay on column chromatography of carbohydrates was written by Capek and Stanek [123], the same authors [224] reviewed polysaccharides, and Juficova and Deyl [225] summarized chromatographic separation of polysaccharide-protein complexes. Reviews specialized on ion exchange chromatography of oligosaccharides... 

Derenbach (1970) made a distinction between soluble and combined RNA of planktonic material from the different behaviour of these compounds after different extraction procedures. He and others (see Derenbach, 1970 for references), employed the orcinol/hydrochloric acid reagent according to Ceriotti (1955). Since a variety of other sugars may interfere (see section 3.1 orcinol/sulphuric acid has also been used as a reagent for total carbohydrate determinations) correction factors or preferably chromatographic separations of the extracts are required for quantitative results. 

Table 1 lists some examples for the electrophoretic separation of carbohydrates on the supports described above. Table 2 lists the electrophoretic mobilities of some common mono-, di-, oligosaccharides, sugar alcohols, and aldonic acids. Table 3 summarizes the electrophoretic mobilities of sugar acids, sugar phosphates, and amino sugars. In Table 4, the chromatographic conditions used in Tables 2 and 3 can be deduced by the use of the corresponding reference letters (A-G). 

Aminolysis of the intact rings with taurine leads to the formation of poly(2-sulfoethyl aspartamide) silica and the reaction with ethanolamine to the formation of poly(2-hydroxyethyl aspartamide) silica. Poly(succinimide)-based silica phases are manufactured by PolyLC (Columbia, MD, USA) under the trade names of PolyCAT A for poly(aspartic acid) silica, PolySulfoethyl A for poly(2-sulfoethyl aspartamide) silica, and PolyHydroxyethyl A for poly(2-hydroxyethyl aspartamide) silica. All three poly(succinimide)-based columns have a pore size of 200 A and a surface area of 188 m /g. Various poly(succinimide)-based columns have been used for the separation of carbohydrates, phosphorylated and nonphosphorylated amino acids, petides and glycopeptides, oligonucleotides, and various other polar analytes under HILIC conditions, but lately lost some of their momentum due to a lower chromatographic efficiency in comparison to more modern HILIC phases and column bleed [44]. 

Figure 10.265 Separation of carbohydrates in an instant coffee sample. Chromatographic conditions and peak identification see Figure 10.264.

The reaction of polyhydroxy compounds with boric acid or boronic acids has been used for derivatization and separation of carbohydrates and other compounds containing vicinal diols using different chromatographic and electrophoretic techniques (1,9,69). The mechanism of reaction is a complex between cis diol moieties and borate or boronate groups. It has been demonstrated that it is the borate ion, rather than boric acid, which is complexed by the polyol (70,71). The reaction is pH dependent and the optimum conditions are usually at pH > 8.0. In a pH ranging from 8 to 12, aqueous borate solutions contain tetrahydroxyborate ions and also more highly condensed polyanions such as triborate and tetraborate. Equilibrium between the different species depends on the pH and the total borate concentration. 

Chromatographic separations of cyclodextrins and glucosylcyclodextrins and various other carbohydrates on Hypercarb, a graphitized carbon column, have been reported and three positional isomers of dimaltosylcyclomaltoheptaose, prepared from maltose and 3-cyclodextrin, were separated and structually characterized. The same group has also reported on several diglucosyl p-cyclodextrins. ... 

Hyakutake, H. and Hanai, T. (1975), Further studies of practical high-speed liquid chromatographic separations of tricarboxylic cycle organic acids and carbohydrates. J. Chromatogr., 108,385. 

The above chemicals can be obtained by fermentation (qv) of other sugars. However, some compounds require sucrose as a unique feedstock. Examples are the polysaccharides dextran, alteman, andlevan, which are produced by specific strains of bacteria (48,54—56). Dextrans are used to make chromatographic separation media, and sulfated dextran derivatives are used as plasma extenders (41). Levans show promise as sweetness potentiators and, along with alteman, have potential as food thickeners and bulking agents in reduced-caloric foods (55,56) (see Carbohydrates). 

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