Protein Protocols Protein Protocols

Monosaccharide Analysis by GC 809
110
Monosaccharide Analysis by Gas Chromatography (GC)
Elizabeth F. Hounsell, Michael J. Davies, and Kevin D. Smith
1. Introduction
Although the methods given in Chapter 109 can give an approximate idea of oligosaccharide
amount or composition, they would not be able to distinguish the multiple
monosaccharides and substituents present in nature. For this, the high resolution
of gas chromatography (GC) is required (1,2). The most unambiguous results are provided
by analysis of trimethylsilyl ethers (TMS) of methyl glycosides with on line
mass selective detection (MS).
2. Materials
1. 0.5 M Methanolic HCl (Supelco, Bellefont, PA).
2. Screw-top PTFE septum vials.
3. Phosphorus pentoxide.
4. Silver carbonate.
5. Acetic anhydride.
6. Trimethylsilylating (TMS) reagent (Sylon HTP kit, Supelco: pyridine hexamethyldisilazane,
trimethylchlorosilane; use care; corrosive).
7. Toluene stored over 3-Å molecular sieve.
8. GC apparatus fitted with flame ionization or MS detector and column, e.g., for TMS ethers
25 m × 0.22 mm id BP10 (SGE, Austin, TX) 30 m × 0.2 mm id ultra-2 (Hewlett Packard,
Bracknell, Berkshire, UK).
3. Method
1. Concentrate glycoproteins or oligosaccharides containing 1–50 µg carbohydrate and
10 µg internal standard (e.g., inositol or perseitol) in screw-top septum vials. Dry under
vacuum in a desiccator over phosphorus pentoxide.
2. Place the sample under a gentle stream of nitrogen, and add 200 µL methanolic HCl
(see Note 1).
3. Cap immediately, and heat at 80°C for 18 h (see Note 2).
4. Cool the vial, open, and add approx 50 mg Silver carbonate.
5. Mix the contents, and test for neutrality (see Note 3).
6. Add 50 µL acetic anhydride, and stand at room temperature for 4 h in the dark (see Note 4).
7. Spin down the solid residue (see Note 5), and remove the supernatant to a clean vial.
8. Add 100 µL methanol, and repeat step 7, adding the supernatants together.
From: The Protein Protocols Handbook, 2nd Edition
Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ
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