With an increasing interest in glycobiology, especially in the area of carbohydrate analysis, characterization and composition of these glycosyl residues has become important. One useful method for determining the sugar composition of these residues is by converting the individual sugars to TMS derivatives by using Tri-Sil Reagent from Pierce. These derivatives can then be subjected to gas chromatography and determined by their mass spectra. In this way organic hydroxyl and polyhydroxyl compounds are converted into ethers. The Tri-Sil Reagent from Pierce offers the fastest and most accurate conversion of sugars, alcohols, phenols, steroids, sterols, bile acids, and some amines to TMS derivatives. Tri-Sil Reagent was designed based on the method by Sweeley, et al. as previously described (Sweely, C.C., et al. (1963). JACS 85: 2497).
The reagent is available in 10-1ml vials and a 50ml Hypo-Vial Sample storage vial. This versatility is convenient for any type of application in the lab. It comes ready to use when needed and does not have an expiration date when properly stored. The 1ml vials are especially useful for cutting down on contamination and destruction of the reagent from moisture and other factors. The reagent should be used immediately upon opening.
For glycosyl composition analysis a sample is subjected in its dry form to methanolysis, acetylation, and then converted to TMS derivatives with the Tri-Sil Reagent. The process is not at all tedious and can be done by any level of experience. Sample prep time prior to GC analysis takes approximately 18-20 hours, most of which is methanolysis. The methanolysis is done with 1M methanolic HCl heated at 80C for 16 hours, or overnight. The sample is then dried several times with methanol and then acetylated using 1:1 pyridine: acetic anhydride for 30 min. During the Tri-Sil step, the reagent is simply added, the sample tube sealed, and then heated at 80C for only 20 minutes. The sample can then be dried quickly under a steady stream of clean air or nitrogen, just to dryness, and brought up in hexane to be filtered through glass wool. The sample is then ready for GC-MS which takes approximately 32 minutes. These methods were carried out as previously described (Methods Enzymol. 230:1-15; York W. S., Darvill, A. G., McNeil, M., Stevenson, T. T., and Albersheim, P. (1985) Methods Enzymol. 118:3-40).
One box of 10-1ml vials is enough for analysis of 50 samples. The price of the vials is $69.00, which is $1.38 per sample. This reagent can also be used in analyzing alcohols, phenols, steroids, sterols, bile acids, and some amines. It helps to silylate any hydroxyl group that is not affected by methanolysis. For the price it is a very good reagent for analyzing sugars. The only drawback to using Tri-Sil is that one must be careful not to evaporate excessively because the sample is extremely volatile upon derivatization. The Tri-Sil Reagent from Pierce seems to work effectively each time I use it. I would certainly recommend this product for anyone doing carbohydrate analysis.
Matt Hilley
Research Technician III
CCRC, University of Georgia