Mannose chromatography analysis

Analysis of glycosaminoglycan hydrolyzates is often complicated by the presence of basic and acidic glycoses, in addition to neutral monosaccharides. Adsorption chromatography on silica gel was used to classify such a hydrolyzate, and the neutral fraction was shown to contain D-glucose, D-galactose, and D-mannose.288 Separation on ion-exchange resins may also be used.51,149 The resolution of the acidic and basic fractions is discussed in Sections IX (p. 71) and X (p. 78). 

Lee, Acree, and Shallenberger have in some cases carried out actual isolations of the anomeric trimethylsilyl per-O-trimethylsilyl glycosides by preparative gas chromatography and have then characterized the separated derivatives by optical rotation, elementary analysis, and NMR spectroscopy (30). These characterizations, which have been done for glucose, mannose, and galactose (see below) confirm the usefulness of the GLC technique. 

To analyze potential interference of amino acids in monosaccharide analysis, each of the 20 amino acids (10 /xg each, each injected separately) was subjected to the chromatography conditions used for separating, detecting, and quantifying monosaccharides. In addition to PAD detection, we monitored UV detection at 215 nm after the electrochemical detector to verify amino acid electrochemical detection. Ten amino acids (R, K, Q, V, N, A, I, L, T and C) eluted between 2 and 25 min and were both PAD and UV active. Of these ten, two amino acids could potentially interfere with monosaccharide analysis. Glutamine was found to elute as a shoulder on mannose. However, acid hydrolysis conditions used to release monosaccharides from glycoproteins likely would oxidize glutamine. 

Figure 1. Monosaccharide analysis of MAb MY9-6. CarboPac PAl chromatography of 2M TFA hydrolysates (A B) and 6M HCl hydrolysates (C D). Chromatography of hydrolysates without prior OnGuard A sample pretreatment (A C) or with prior OnGuard A sample pretreatment (B D). Peaks are as follows 1. fiicose 2. rhartmose 3. mannosamine 4. glucosamme 5. galactose 6. glucose 7. mannose. Insets show UV absorbance monitored at 215 run...

The use of quantitative, paper chromatography is discussed in this Section, since one of the most important factors in its application to wood-cellulose analysis is that it permits a satisfactory determination of mannan. Reasonably reliable methods for the determination of xylan have been available for some time, but mannan determinations, for the reasons discussed above, have been less satisfactory. The fact that (on chromatograms) xylose, as well as other carbohydrates, can be determined simultaneously with mannose is an added attraction in the use of this technique. 

KoUer, A. et al. Analysis of high-mannose-type oligosaccharides by microliquid chromatography-mass spectrometry and capillary electrophoresis. Electrophoresis, 25, 2003, 2004. 

Trityl-D-mannose (10 mg), pyridine (70 fi ) and a solution of phosgene (2.4 mg) in toluene (20 fi ) are vigorously shaken together for 30 min in an ice bath at 0°C. The vial is taken out of the ice bath and, after 30 min at room temperature, the suspension in the vial is centrifuged and the supernatant is evaporated to a syrup. This is dissolved in chloroform and purified on a small silicic acid column, with chloroform elution. The clean-up may be monitored by gas chromatography, the appropriate fractions being combined and evaporated to a small volume for analysis [113],... 

Besides amino acids, traces of borate in the mobile phase can also affect monosaccharide analysis due to complexation. Borate ions are among the first to be detected in the slip of water purification systems when their mixed-bed ion exchangers approach the capacity limit (Borate can be determined with ion-exclusion chromatography and subsequent conductivity detection down to the lowest ng/L level utilizing pre-concentration see Section 5.5.) If de-ionized water used for preparing the mobile phase contains traces of borate, a significant loss of peak symmetry is observed this effect is most pronounced with mannose and other reduced saccharides. Even borate concentrations in the lowest mg/L levels are sufficient to cause this effect. With a specially-designed BorateTrap column, which is placed between the analytical pump and the injection valve, borate can be completely removed from the mobile phase. 

Gas chromatography techniques have been used for the quantitative analysis of sugars in invertebrates. Thus, Ueda and Sawada (1968) showed that the parasitic flatworm Paragonimus (lung fluke of man) contains about 70% glucose, 10% galactose, 8% mannose, and 6% ribose. 

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