Acetates alditol

With the advent of column packings capable of separating alditol acetates (see Part I, Section VII,2 p. 59), these derivatives were first utilized by Lindberg and coworkers, who showed that the same liquid phase (ECNSS-M) is suitable for the separation of these compounds having all degrees of methylation. The simplicity of the chromatogram, coupled with the fact that the pattern of methylation in alditol acetates may readily be determined by mass spectrometry (see Section XXV  

Lindberg and coworkers recommended that retention times be related to those of two standards, one of which has a short and the other a long retention-time. Suitable compounds that are readily available are l,4-di-0-acetyl-2,3,5-tri-0-methylarabinitol and l,3,5,6-tetra-0-acetyl-2,4-di-O-methylgalactitol, with retention times of 0.48 and 6.35 with respect to l,5-di-0-acetyl-2,3,4,6-tetra-0-methylglucitol (Tu = 1.00). By their use, the relative retention-times of the components of an appropriate mixtmre can be found by interpolation. 

Where the components of a mixture differ greatly in degree of methyl-ation, temperature programming may be used with advantage, and may be started at as low as 50°. Even with the use of this technique, there are some compounds not readily separated as alditol acetates, and for which other derivatives are preferable thus, 2- and 3-O-methylxyloses are not separable as the alditol acetates, but are resolved as the tri-methylsilyl derivatives. With mixtures of unknown composition, it is thus prudent to investigate the separation by trying different derivatives. 

Satisfactory separations of alditol acetates have been obtained for O-methyl-O-propylglucitols, where propylation was used because of the natural occurrence of an 0-methylglucose, and for O-ethyl-0-methylglucitols prepared during an investigation into the lengths of the side chains in dextrans.  

Perry and Webb ° studied the separation of the acetates of partially methylated 2-amino-2-deoxyglucitols, and such derivatives have been used in the examination of Serratia marcescens. The related N-methyl compounds were encountered during work on Salmonella hybrids, and Gorin and Magus made a detailed study of the acetates of 2-deoxy-O-methyl-2-(methylamino) derivatives of n-glucitol and D-galactitol. 

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The mass spectrum of alditol acetates are easy to interpret as they fragment at each C—C bond as shown. 

Glycosyl-linkages were determined by GC-EIMS of the partially methylated alditol acetates. RG-II samples (2 mg) were methylated using sodium methyl sulfmyl carbanion and methyl iodide in dimethyl sulfoxide [24] followed by reduction of the uronosyl groups with lithium triethylborodeuteride (Superdeuteride , Aldrich) [23,25]. Methylated and carboxyl-reduced samples were then submitted to acid hydrolysis, NaBIlt reduction and acetylation, partially methylated alditol acetates being analysed by EIMS on two fused-silica capillary columns (DB-1 and DB-225) [20]. 

Qualitative analysis of selected fractions was based on GLC separation of alditol acetates (5). Alditol acetates from uronosyl residues were prepared by methanolysis followed by reduction (4). 

Smith degradation of PI. A sample of FI (50 mg) was dissolved in 0.5 M NaOH (5 mL) and acetic acid added to pH 7, followed by NaI04 to a final concentration of 50 mM. At the conclusion of the oxidation (7 days) the product was reduced with NaBH4 and dialyzed. An aliquot was removed and the remainder immediately treated with NaI04 and then reduced with NaBH4. Samples of material subjected to one and two cycles of oxidation-reduction were hydrolyzed and reduced, and the products analyzed by GLC as alditol acetates. 

Methylation analysis of PI and FIbp. The fractions were methylated according to Ciucanu and Kerek [8]. The procedure was repeated until no absorbance was detected by I.r. at 2500-3500 cm-i and the per-O-methylated polysaccharides hydrolyzed and analyzed by GLC and GLC-MS of the derived partly O-methylated alditol acetates. 

Oxidation of PI with chromium trioxide. Fraction PI was twice acetylated as described above. The peracetylated polysaccharide (75 mg), together with 20 mg of mannitol hexacetate as internal standard was dissolved in 1.5 mL of HCCI3, and treated with 1.89 mL of glacial acetic acid and 189 mg of chromium trioxide, at 50°C. Aliquots were removed at zero, 30, 60 and 120 min, water then added, and the material recovered by extraction with chloroform, hydrolyzed and analysed by GLC of derived alditol acetates. 

Carboxyl redution. A sample of pennethylated PI (5 mg) was carboxyl-reduced by a modification of the method described by Lindberg and Lbnngren [9], as follows. The methylated fraction was solubilized and added a mixture of LiAlH4 (25 mg) in THF (5 mL) at 20 °C for 4 h. and refluxed during 1 h. The excess of reagent was destroyed with ethyl acetate (5-6 drops) and water (10 drops) and the pH of the mixture adjusted to neutrality with acetic acid. The insoluble residue was removed by centrifugation. The reduced fi action was precipitated with EtOH. The reaction was monitored by l.r. specroscopy. Hydrolysis products were analysed by GC-MS as methyl alditol acetates... 

The removal of arabinose suggested the presence of peripheric a-L-arabinofuranosyl units. Flap was methylated by the method of Ciucanu and Kerek and products of derived O-methyl alditol acetates analysed by GLC-MS... 

Neutral sugars were quantified by trifluoroacetic acid hydrolysis (3) and gas chormatography of alditol acetates (4). Uronic acids were determined by Blumenkrantz method... 

Oligosaccharides were isolated from PMII by weak acid hydrolysis and separation by SEC and HPAEC-PAD. The isolated oligosaccharides were desalted, reduced and methylated. GC-MS analysis of the partially methylated alditol acetates has been used to reveal the structure of the oligosaccharides. 

The content of neutral monosaccharides was determined after an acid hydrolysis, performed as follows with 72 % sulphuric acid for 1 hr at 30°C and then, after dilution to IM sulphuric acid, for another 3 hr s at 100°C. The determination was performed by GC analysis of the prepared alditol acetates (13,14)... 

Sugar composition. Desalted fractions (IPN1-IPN14) were hydrolyzed using 2N TFA for 1.5h at 121 C. The released neutral sugars were converted to their alditol acetates and analysed by GC as described [12]. 

Fox, A. Krahmer, M. Harrelson, D. Monitoring muramic acid in air (after alditol acetate derivatization) using a gas chromatography-ion trap tandem mass spectrometer. J. Microbiol. Meth. 1996, 27,129-138. 

The chemical-ionization (c.i.), mass spectra of some methylated alditol acetates have been reported.51,52 The main intensities in the c.i. 

Multiple-ion monitoring is, however, of considerable value in structural studies, but only if model compounds of known structure are available for comparison. Such an approach has been used in the study of the carbohydrate structures of glycoproteins from different tissues.50 Separation of glycopeptides obtained from various tissues was performed on columns of concanavalin A-Sepharose. Structural analysis by multiple-ion monitoring of partially methylated, alditol acetates derived from the various fractions indicated that the glycopeptides were separated according to the linkage pattern of mannose (see Fig. 1). 

Fig. 1.—Mass Fragmentograms of Partially Methylated Alditol Acetates Obtained from Rat-brain Glycopeptides. [(A) Fraction A glycopeptides, (B) fraction B glycopep-tides, and (C) fraction C glycopeptides. Peak 1, 2,3,4-tri-O-methylfucitol peak 2,...

Chromatographic analysis ages G.l.c. separation of substituent, partially methylated alditol acetates On-line e.i.- or c.i.-m.s. analysis G.l.c. analysis after sequential hydrolysis, reduction, and acetylation... 

Figure 3a. GLC of alditol acetate derivatives of the hydrolyzed methylated fructan.

Take 0.25 ml of the mixed sugars standard solution, add 0.25 ml allose internal standard solution and 0.5 ml 2 M sulphuric acid, mix and substitute instead of the hydrolysate solution in the above procedure for preparation of alditol acetates. The concentration of sugars should give a linear relationship with peak area over the normal ranges. The GLC determination conditions will depend on the instrument, but the following are suggested ... 

Englyst, H.N. and Cummings, J.H. (1984) Simplified method for the measurement of total non-starch polysaccharides by gas-liquid chromatography of constituent sugars as alditol acetates. Analyst 109, 937-942. 

The difficulty of acetylating in pyridine in the presence of borate was observed by Oades,91 who used an acid-catalyzed acetylation that was insensitive to large proportions of borate. Unfortunately, this method produced artifacts that had retention times similar to those of the alditol acetates, but they could be eliminated by passage through a short column of silica gel. This treatment also removed com-... 

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