Alditol acetate peaks

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,...

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 ... 

Although many analyses are performed on alditol acetates (see Section VII, p. 56), in order to avoid the formation of multiple peaks, such a reduction is not practical when the mixture contains ketoses, notably fructose. Such analyses are mainly encountered with medical samples and in the examination of sugars occurring free in Nature. Furthermore, the peak-area ratios may be used as a means of identification, to check on the completeness of trimethylsilylation,67,89 and, despite the complex chromatograms obtained from trimethyl-silyl derivatives, they have the merit of being rapidly formed.89 For all of these reasons, improvements in the separation of monosaccharides as their trimethylsilyl derivatives continue to be of considerable importance. 

With the development of new liquid phases, interest in the separation of alditol acetates has revived, and this now may be considered the most widely used method for analyzing carbohydrate mixtures, provided that reduction does not introduce an ambiguity into the analysis also, it must be borne in mind that the multiplicity of peaks obtained by trimethylsilylation of a free sugar or of its methyl glycosides may serve a useful purpose in characterization. 

Adapted from Kariya et al. (1997). With permission from Elsevier Publisher. a Partially methylated alditol acetate with methoxy groups at the positions shown. b Retention time on a SP2330 capillary column relative to 2,3,4-tri-O-methyl-fudtol. c The molar ratios were based on the peak area. d Not detected. 

Determination of the monosaccharide composition by gas chromatography of alditol acetates is commonly used for cell wall analyses because the procedure gives a single peak for each sugar. An alternative is to prepare trimethylsilyl (TMS) derivatives. However, these are easily hydrolyzed by moisture in the atmosphere and therefore should be analyzed in the gas chromatograph immediately, whereas alditol acetates are relatively more stable and could be rerun on the gas chromatograph the following day if required. 

The amount of each neutral monosaccharide in the samples can be calculated relative to the internal standard, allose, using response factors. The relative response of the detector for the individual alditol acetates can be calculated from the areas under the peaks for each alditol acetate, relative to the area under the peak for allitol acetate. There should be no peaks in the chromatogram of the water control and only one peak, corresponding to allitol hexaacetate from the internal standard allose, in the chromatogram of the TFA control. 

Order of elution Alditol acetate Retention time (min) Peak area Response factor relative to allitol hexaacetate... 

Figure 9. Open tubular gas chromatogram of partially methylated alditol acetates obtained from blood-group A active tetraglycosylceramide (A) and hexaglycosyl-ceramide (B), respectively. Stationary phase was OV-1, and carrier gas was Ns. Column temperature was kept at 175°C for 14 min, then raised l°C/min. The designation above the peaks indicate actual binding positions.

GLC is a versatile and widely used method for the separation of volatile sugar derivatives. Although a number of derivatives have found application, the most commonly used derivative is the alditol acetate which gives a single peak for each sugar (Sawardeker et al., 1965). The monosaccharides commonly found in plant cell walls can, as their alditol acetates, be separated readily on a number of commercially available liquid phases such as OV-225 (Selvendran et al., 1979) and SP-2330 (Englyst et al.,... 

PEAK NUMBER R r ALDITOL ACETATE DIAGNOSTIC PRIMARY FRAGMENT IONS ... 

The base peak in the specti a of all alditol acetates is the acetylium ion. 

The component in peak A gave a mass spectrum identical with that of the alditol acetate of a 3,6-dideoxy-2,4-di-0-methyIhexose. As tyvelose is the only 3,6-dideoxy-hexose present, the component of the first peak must be the corresponding alditol acetate, methylated at 0-2 and 0-4. The component in peak B gave a mass spectrum characteristic of a mixture of the alditol acetates from a 6-deoxy-2,3-di-0-methylhexose (27) and a 2,3,4,6-tetra-O-methylhexose (28). 

Table V). Mizuno and coworkers quantitatively detected 33 peaks by g.Lc. of the alditol acetate derivatives of the sugars in formose [see Table IV (p. 188) and Fig. 14].

Methyl glycosides are of their greatest use when only a few methylated sugars are anticipated, as with polysaccharides of simple structure and, in particular, with oligosaccharides. They are useful where identification must be based on peak profile and retention times, but, when characterization is to be achieved by mass spectrometry, other derivatives, such as alditol acetates, are preferable (see Section XXI,2 p. 30). Glycosides have the advantage over alditols that they may be hydrolyzed to the free sugar for further characterization. 

Elimination of multiple peaks may also be achieved by conversion of a sugar into the dithioacetal or the nitrile. The suitability of the former type of derivative for g.l.c. appears to have been explored only in a model study on xylose methyl ethers. Lance and J. K. N. Jones also demonstrated that the acetylated nitriles derived from diese ethers give sharp, single peaks, and permit 2- and 3-0-methyl-i>-xylose to be distinguished from each other, a resolution not achieved by using alditol acetates. Nitriles have been used by Bouhours and Cheshire to demonstrate the occurrence of the same two xylose ethers in peat, and by Bacon and Cheshire to prove the natural existence of 3-O-methyl-D-galactose in leaves. 

Aldononitriles give sharp, single peaks on g.l.c., and their methylated derivatives give mass spectra characteristic of the pattern of substitution. Their use instead of alditol acetates has been recommended, as the possible ambiguity introduced on reduction of the sugar is avoided. 

Reduction of the saccharide gives alditols and only one peak per sugar. The same occurs in GC but, in addition, as sugars are not volatile, they must be transformed into alditol acetates by reduction and peracetylation to get a single peak per sugar in GC (57). 

Figure 8-4. Methylation analysis of in vitro P-D-glucans. (a) Gas chromatography of the permethyl-ated alditol acetate obtained from methylation analysis of the cellulose synthesized in vitro by the enzyme from blackberry. Peak 1, derivative characteristic of (1 4) linked glucosyl units. Peak 2, internal standard (mj o-inositol). The derivative characteristic of (1 3) linked glucosyl units usually elutes 1 min before the major derivative visible in the chromatogram (not shown see Bulone et al. 1995). (b) Structural characterization by electron impact mass spectrometry of the 1,4,5-tri-0-acetyl-2,3,6-tri-0-methyl-D-glucitol derivative corresponding to peak 1 in A and characteristic of (1 4) linked glucosyl units.

Per-O-acetyl aldononitriles and methylated aldononitrile acetates have been separated by g.I.c. on a mixed liquid phase. Chemical ionization m.s. showed the presence of peaks at M + 1 in each instance. The retention times of a number of partially methylated alditol acetates have been recorded on a variety of stationary phases. Using a combination of silica gel chromatography and g.I.c., fifteen methyl ethers of methyl a-D-galactopyranoside have been isolated from the partial methylation products of D-galactose. ... 

---