Fractionation methods carbohydrates

Glycoamine Synthesis. The covalent coupling of amino acid monomers and polypeptide fractions to carbohydrate backbones, previously described (30), was completed using a stationary pH modification (31) of a previously published method (32). 

Some of the substances that have been separated by this method are given in papers referred to by Morris and Morris (1964) amino acids, peptides (particularly those having molecular weights ranging from 500 to 5000), polypeptide antibiotics, proteins (including enzymes), carbohydrates (although for most compounds in this chemical class other fractionation methods are much more frequently applied), purines, pyrimidines, nucleic acid derivatives, tRNA s that are specific for various amino acids, organic acids, steroids, lipids, antibiotics that are not peptides, porphyrins, pterins, vitamin B,2 and other vitamins, lipoic acid, and alkaloids. The countercurrent-distribution procedure of Holley et al. (1965) is widely used, sometimes with modifications. Korte et al. (1965) have separated three isomers of tetrahydrocannabinol. 

FIGURE 2.7 SEC elution profiles of dextran in clinical samples, serum ( ) and urine ( ). The first peak represent dextran and the second peak inulin (used as a reference for clearance). The content of carbohydrates was determined in collected fractions with the anthrone method. [Reproduced from Hagel ef of. (1993), with permission.]... 

Chromatorapf c methods For gel filtration of polysaccharide fraction PI, a Sephacryl S-300 chromatographyc column (1,1 X 46,7 cm) was calibrated with standard dextrans (molecular mass range 266, 72, 40, and 17 KDa Sigma Chemicals), and the void volume determined with blue dextran. Polysaccharide sample (0.5 mL 2 mg/mL) was applied and eluted with 50 mM NaOH, fractions 1 mL being collected and carbohydrate absorbance (phenol-H2S04) being monitored. 

Sorensen and Haugaard147 showed that by a suitable fractionation of crystalline albumin there could be obtained a series of fractions which, although crystalline, varied widely in their properties, particularly in regard to their carbohydrate contents. The most soluble fraction contained about fifty times as much carbohydrate as the least soluble. In most of these studies the carbohydrates were estimated by modification of the orcinol colorimetric method of Tillmans and Philipi181 which could... 

Raunkjaer et al. (1994) have modified methods for the determination of carbohydrates, proteins and lipids to be applied for wastewater. Henze et al. (1995b) estimate the average composition of these three organic fractions in wastewater and propose a corresponding conversion to COD units (Table 3.3). The table also includes stoichiometric formulas for carbohydrate and protein, proposed by Kalyuzhnyi et al. (2000). 

Figure 3. Gel electrophoresis of cotton dust fraction 4 stained for protein (left) and carbohydrate (right). Conditions described in Material and Methods.

Chaplin used methanolysis for the analysis of carbohydrates in glycoproteins. His method was a variation of the foregoing procedures, with an improvement of using tert-hvAyX alcohol to remove hydrogen chloride by coevaporation, instead of prolonged trituration with silver carbonate. His method is useful for samples containing uronic acids and lipids. Mononen studied methanolysis, followed by deamination and reduction with borohydride, for determination of the monosaccharide constituents of glycoconjugates. This method was applied to a lipid-free, protein fraction of rat brain. 

Eor obtaining neutral fraction the column was eluted with water firstly. The acidic fractions were obtained by elution of linear NaCI gradient (0-1.4 M) in water. The carbohydrate elution profile was determined using the phenol-sulphiric acid method, finally two column volumes of a 2 M sodium chloride solution in water were eluted to obtain the most acidic polysaccharide fraction. The relevant fractions based on the carbohydrate profile were collected, dialysed and lyophilized. 

The work discussed in this article serves to illustrate the applicability of gel chromatography to a wide variety of carbohydrates. The technique can be used not only as a means of fractionation but also, provided that the relationship between molecular weight and such other molecular parameters as size and shape remains constant, as a method of estimating the molecular weights of polysaccharides. Often, information derived from gel chromatography has proved useful in structural elucidation. 

The principal constituents of milk, i.e. lipids, proteins and carbohydrates, can be readily fractionated and purified by relatively simple methods, for use as food ingredients. 

The method of Gaver147 for nonreducing carbohydrates (refluxing the carbohydrate in alcoholic alkali metal hydroxide solution without concomitant azeotropic distillation) might give results similar to those obtained by the Wolfrom method.8 -" However, the presence of water and hydroxide ion in the final reaction mixture would probably cause a. small fraction of the product to be the hydroxide adduct. 

The completion of a chromatographic experiment calls for a means to detect the presence of solutes in the collected fractions. The detection method used will depend on the nature of the solutes. Smaller molecules such as lipids, amino acids, and carbohydrates can be detected by spotting fractions... 

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