Cell wall polysaccharides are typically hydrolyzed to monosaccharides by concentrated acids, such as 1 M H2SO4 for 1 h at 100°C, 1 M HCL for 1 h at 100°C, or 2 M trifluoroacetic acid (TFA) for 1.5 h at 120°C. The former is called a ‘Saeman hydrolysis’ and actually, concentrated H2SO4 as added at ambient temperature for 15 min, and then the hydrolysate is diluted to 1 m and heated to 100°C for 1h. After hydrolysis, the sample may be alkalinized to 0.2 M NaOH (plus 1 M Na2SO4), and the monosaccharides separated by HPAEC and detected by PAD (see Section VII). However, one of the most routine assays for monosaccharides, which affords baseline separation of all common cell wall monosaccharides: rhamnose, fucose, arabinose, xylose, mannose, galactose, and glucose, is the separation of alditol acetates by GLC and their verification by EIMS. For this, the TFA hydrolysis is by far preferred because it is volatile and can be removed by evaporation under a stream of warm air or nitrogen.

A. Alditol Acetate

1. Use 1 mg cell wall material in a 1-dram screw-cap vial. In a fume hood, add 1 mL of 2 M TFA without myo-inositol and cap tightly with cap lined with Teflon.

2. Heat at 120°C for 90 min with vortex every 30 min. to break up any chunks. Make sure that the cap hasn't loosened during heating or you'll launch the tube across the fume hood.

3. After cooling, dry under stream of air at 40-45°C.

4. Add 0.6 mL solution containing 0.5 mL of fresh 20 mg/mL NaBH4 in DMSO and 0.1 mL of 1 M NH4OH; incubate at 40-45° C in water bath for at least 90 min, with vortexing every 30 min.

5. Neutralize mixture with 100 µL gl. acetic acid, mix thoroughly until fizzing stops.

6. Add 100 µL of 1-methylimidazole (kept at 4°C) [wear gloves when using imidazole].

7. In the fume hood, add 0.75 mL anhydrous acetic anhydride; cap tightly; mix thoroughly and incubate at 40-45° C in water bath for 30 min. Make sure that you work quickly with reagent acetic anhydride, leaving the cap off only as long as necessary.

8. Add 1.5 mL H2O, shake vigorously and wait until vial cools to room temp.

9. When cool, fill remaining volume with dichloromethane, cap tightly and shake igorously for 1-2 min. Spin for 5 min at 2000rpm.

10. Aspirate off the water fraction, being careful not to suck up the lower CH2Cl2 phase. Refill with H2O, cap tightly and shake vigorously for 1-2 min, spin at 2000 rpm. Aspirate off the water. Repeat water wash a total of five more times. Evaporate CH2Cl2 at 40-45°C.

11. Redissolve the alditol acetates in 500µL CH2Cl2 for chromatography.