Separation of oligosaccharides

Reversed phase HPLC is only rarely used for the chromatographic resolution of saccharides however, in instances where only occasional separations are required the wide availability of reversed phase columns in many laboratories saves the purchase of a specialised carbohydrate column. Reversed phase HPLC has been used primarily for the separation of oligosaccharides and separation is dependent on the degree of polymerisation (Cheetham et al., 1981). Organic phase modifiers such as -alkylamines can be used to provide increased capacity and selectivity for saccharides (Lochmuller and Hill, 1983). 

HPLC analysis of oligosaccharides from animal tissues or body fluids has been used to determine their molecular weight distribution. The majority of complex oligosaccharide separations has been carried out using chemically bonded amine columns which separate molecules on the basis of chain length, although reversed phase HPLC has been used to separate human milk oligosaccharides (Dua and Bush, 1983). 

Chemical modification of saccharides is an ideal tool for shifting their absorption maximum into a range which facilitates classical UV detection but suffers from inherent problems (Section 11.4.4) which precludes the popular use of this methodology. Detection of changes in refractive index is the preferred method of detection of the saccharides and detection limits are in the range 10-40 fig and depend upon the specific system employed. More recently mass detectors have been described which can be used with gradient elution and which achieve a 10 times greater sensitivity than refractive index detectors (Macrae and Dick, 1981). Less common techniques for the 

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The remarkable performance of P-6 in the separation of oligosaccharides, particularly of glucans and fructans, is well known (31,32) and is illustrated... 

Figure 1. Separation of oligosaccharides from PMII by HPAEC-PAD.

Plant material fractionation. The detailed steps of isolation and separation of oligosaccharide fractions were described earlier [10]. Pectin was separated by boiling the cell walls in 0.5 M ammonium-oxalate buffer, pH 5.2 at 100 C for Ih. The dialyzed solution of pectin was hydrolyzed with 0.15M HCl for 3h at 100 C. Neutralized and desalted hydrolysate was loaded to the column (lx90cm) filled with biogel TSK HW-40 (Toyo Soda, Japan) equilibrated and eluted with 50mM sodium acetate (pH 5.2) at a rate of 0.3ml/min. In all fractions (1ml) the sugars were determined by o-toluidine method (Resnikov et al., 1982) and fraction IP was collected as shown on Figure 1. 

Scobell, H. D. and Brobst, K. M., Rapid high-resolution separation of oligosaccharides on silver form cation-exchange resins, /. Chromatogr., 212, 51,1981. 

Guttman, A., Cooke, N., and Star, C. M., Capillary electrophoresis separation of oligosaccharides. I. Effect of operational variables, Electrophoresis, 15,1518, 1994. 

Although CE separations can be reasonable well described by the classical theoretical relationships for electrophoretic migration, slight deviations from the theory occur in the case of many classes of solutes. Thus, it has been reported that the CE separation of oligosaccharides follow the general rule [124], while the description of the separation of DNA in polymer solutions necessitated a new mathematical model. The drag forces were expressed by... 

B. Lagane, M. Treilhou and F. Couderc, Capillary electrophoresis theory, teaching approach and separation of oligosaccharides using indirect UV detection. Biochem. Mol. Biol. Educ. 28 (2000) 251-255. 

Figure 3-3 Separation of oligosaccharides by gel filtration. The sugars dissolved in distilled water were passed through a column of Sephadex G-25. The peaks contain (right to left) glucose, cellobiose, cellotriose, etc. From Flodin and Aspberg.64...

Figure 10.4 Separation of oligosaccharides by Dionex HPLC. Conditions column PA-100 5 p,m 250 X 4 mm solvent = 0.002 M sodium acetate in 0.1 M sodium hydroxide flow rate 1 ml/min solvent B1M sodium acetate in 0.1 M sodium hydroxide flow rate 1 ml/min using pulsed amperometric detection, time 0 = 100% A then a gradient to 46% B over 50 min using curve 9.

In the separation of oligosaccharides, sulfonic acids (mono-, di- or tri-) of aminonaphthalene have been used [29]. Polyuronic acids hke hyaluronic acid have also been characterized by CE [30]. 

ADSORPTIVE SEPARATION OF OLIGOSACCHARIDES INFLUENCE OF CROSSLINKING OF CATION EXCHANGE RESINS... 

Although results have been reported using RPLC, HPAEC is the method-of-choice for the efficient separation of oligosaccharides. 

The separation shown in Figure 1 illustrates the versatility of such a resin (12,13) packed in a 6 mm i.d. by 60 cm long jacketed column, maintained at 80°C. In this case, separation of oligosaccharides c7 to C2... 

Figure 1. Ion exchange separation of oligosaccharides, sugars, and alcohols, conditions as given in text.

Although HPLC may eventually become the method of choice for separating and quantifying neutral monosaccharides, it is at present unable to achieve the resolution of GLC. HPLC does offer considerable promise for the separation of oligosaccharides and their derivatives (see Section VI). 

Mass spectrometry provides a rapid, sensitive method for the structural analysis of oligosaccharides and, especially in combination with g.l.c., offers a valuable technique for the analysis of oligosaccharides from natural sources and from polysaccharide hydrolyzates. Methyl ethers " or trimethylsilyl ethers are suitable derivatives for the g.l.c. separation of oligosaccharides. 

Townsend, R., et al. (1989). Separation of Oligosaccharides Using High-performance Anion Exchange Chromatography with Pulsed Amperometric Detection, Methods Enzymol. 179 65-76. 

FIGURE 7.1 High-resolution CE separation of oligosaccharide structural isomers. Traces from the bottom (A)... 

Townsend, R. R., Hardy, M. R. and Lee, Y. C. Separation of oligosaccharides using high-performance anion-exchange chromatography with pulsed amperometric detection. Methods Enzymol, 179, 65, 1989. 

Microfluidic CE separation chips were also interfaced to MALDI-MS detection. The CE separation of oligosaccharides and peptides (0.5-5 mg/mL) was performed in open CE channels ( 250 ftm deep) that contained buffer and MALDI matrix. The chips were prepared in glass. After separation, the solvent was evaporated, the chips were placed into a specially designed MALDI source, and the CE channels was scanned with the laser beam. It is anticipated that the CE chip-MALDI-MS protocol could provide a fast and effective alternative for applications that utilize 2D-gel separations followed by MS detection. 

Flodin and Aspberg (1961) have reported a marked separation of oligosaccharides on a column of gel of small pore size. Ringertz (1960) has reported the fractionation of acid polysaccharides from mouse tumors on a Sephadex column. The fractions differed in several properties, including the type of amino sugar present. Tanaka (1966) has reported on the fractionation and isolation of acid mucopolysaccharides. 

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