Monosaccharides analysis

The anomeric configurations of the sugar residues were determined by chromium trioxide oxidation [14], Oxidation of the fully acetylated polysaccharide and subsequent monosaccharide analysis by GLC indicated that the D-Xyl units are P-linked (oxidized more rapidly) and that die D-GlcA are a-linked (Table II). 

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...

Figure 3. Five gum samples from species of major artistic importance. Note the similarity in shade of the kuteera, karaya, and pale gum arabic tears. All three tears of gum arabic are from the same tree yet one is as dark as the cherry gum. Chemically, the pale and dark samples are identical by monosaccharide analysis. Note the opacity of the sample from Anogeissus latifolia.

Townsend RR, Manzi A, MerUe RK, Rohde ME, Spellman M, Smith A, Carr SA (1997) Quantitative Monosaccharide Analysis A Multi-Center Study. ABRF News, epubUcation... 

In Table 3 a summary is compiled of the carbohydrate content and the monosaccharide analysis data of Hcs from typical species belonging to the four mollusc classes Gastropoda, Cephalopoda, Bivalvia, and Amphineura [10]. 

Linkage and monosaccharide analysis by a combination of derivitization and LC/MS continues to provide useful carbohydrate characterization, especially in development, but where more structural detail is needed, NMR still remains a powerful tool. 

These data in combination with monosaccharide analysis established the structure of the acidic trisaccharide as Glc/>A-( 1— 2)-Xylp-( l- 4)-Xylp. As with the disaccharide approximately 25% of the Glc A residues were endogenously methylated at position 4. These studies were consistent... 

Hardy, M. R., Townsend, R. R. and Lee, Y. C. Monosaccharide analysis of glycoconjugates by anion exchange chromatography with pulsed amperometric detection. Anal Biochem, 170,54,1988. 

Although NMR analysis applied to the general carbohydrate composition in LCCs presents some fundamental interest, from a practical point of view a routine wet chemistry monosaccharide analysis is much simpler and it provides more reliable and, more importantly, quantitative information compared to NMR methods (see, e.g.. Table 1). NMR analysis of carbohydrate units in LCCs should be focused on detailed structural characteristics, a piece of information that routine monosaccharide compositimial analysis methods cannot provide, similarly to the NMR investigatimis conducted on isolated hemicel-luloses (see, for instance, [83]). 

Some recent progress has been made. For example, a recent study employing both proton NMR and monosaccharide analysis has suggested that there are at least two major classes of UCM. The major component is acyl polysaccharide, a biopolymer rich in carbohydrates, acetate, and lipid and that this can account for between 50% and 80% of the total high molecular weight dissolved organic carbon in surface samples. 

Individual dissolved monosaccharides have been determined after desalting of the sample by ion-exchange membrane electrodialysis followed by evaporative concentration and ion-exchange LC using colorimetric detection with Cu -aspartic acid-disodium-2,2 bic-inchoninate. More recently a number of papers have described the use of anion-based LC with pulsed am-perometric detection for individual monosaccharide analysis though it has been noted that temperature control of the system is particularly important. 

Figure 3.221 Influence of the AminoTrap column on lysine as an interfering amino add in monosaccharide analysis. Separator column CarboPac PA10 eluent 18mmol/L NaOH flow...

Figure 3.223 Comparison between CarboPac PA1 and PA10 for monosaccharide analysis in a glycoprotein hydrolysate at high sensitivity. Chromatographic conditions see Figure 3.222 ...

Dionex Corporation (1998) Dionex Technical Note No. 40 Glycoprotein Monosaccharide Analysis Using HPAE-PAD. Dionex Corporation (now part of Thermo Fisher Scientific), Sunnyvale, USA. 

Fig. 3-164. Influence of the AminoTrap column on lysine as an interfering amino acid in monosaccharide analysis. -Separator column CarboPac PAIO eluant 18 mmol/L NaOH flow rate 1.5 mL/min detection see Fig. 3-160 solute concentrations 1 nmol each of fucose (1), galactosamine (2), glucosamine (3), galactose (4), glucose (5), mannose (6), and 100 nmol lysine (7).

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. 

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