3.3 Quantification of
Sugars by HPAEC-PAD
1. This protocol utilizes the method described in [9].
2. Dilute samples 1000Â prior to analysis (see Sect. 3.1, step 11).
3. The HPAEC system uses pulsed amperometric electrochemical
detection (PAD).
4. Separation is achieved using a pa20 column (150 mm  3.0 mm;
Dionex, USA).
5. The system is operated using isocratic elution with 10 mM NaOH
at 0.5 mL/min flow rate with a column regeneration step using
200 mM NaOH at 0.5 mL/min after each injection.
6. Quantification is performed by comparison of peak areas of authentic standards of mannitol, fucose, galactose, arabinose, galactose,
glucose, and xylose (0.0625–1 g/L concentration range, dissolved
directly in RO water; see note 8). Dilute standards 1000Â (with RO
water) prior to analysis.
3.4 Quantification of
Total Reducing Sugars
Using a Colorimetric
Assay
1. This protocol utilizes the phenol-sulfuric acid method described
in [5].
2. Transfer a 100 μL aliquot of each sample into a 5 mL test tube.
3. Carefully add 1 mL phenol solution (2.5% v/v) and 2.5 mL
concentrated (72% v/v) H 2 SO 4 (see note 7).
4. Transfer 2 mL aliquots of this reaction mixture into quartz cuvettes.
5. Read absorbances at 490 nm using a spectrophotometer (after
zeroing the instrument using water as a blank).
6. Quantification is then achieved by comparison to the absorbance
(at 490 nm) of authentic glucose standards (0, 200, 400, 600,
800, and 1000 μg/mL).
4 Notes
1. Concentrated H 2 SO 4 is highly hazardous, and we advocate all
handling to be conducted in a fume hood (cabinet). In addition,
the dilution process for the acid to achieve the desired acid
molarities is highly exothermic, and care should be taken to
allow solutions to cool before use.
2. Phenol is also a highly hazardous compound and should always
be handled in a fume hood (cabinet).
3. The use of automatic Dispensette pipettes (BrandTech Scientific, USA) is highly recommended for the rapid, reproducible,
and safe dispensing of reagents.
Complete Acid-Based Hydrolysis Assay for Carbohydrate Quantification in. . .
189
Sugars by HPAEC-PAD
1. This protocol utilizes the method described in [9].
2. Dilute samples 1000Â prior to analysis (see Sect. 3.1, step 11).
3. The HPAEC system uses pulsed amperometric electrochemical
detection (PAD).
4. Separation is achieved using a pa20 column (150 mm  3.0 mm;
Dionex, USA).
5. The system is operated using isocratic elution with 10 mM NaOH
at 0.5 mL/min flow rate with a column regeneration step using
200 mM NaOH at 0.5 mL/min after each injection.
6. Quantification is performed by comparison of peak areas of authentic standards of mannitol, fucose, galactose, arabinose, galactose,
glucose, and xylose (0.0625–1 g/L concentration range, dissolved
directly in RO water; see note 8). Dilute standards 1000Â (with RO
water) prior to analysis.
3.4 Quantification of
Total Reducing Sugars
Using a Colorimetric
Assay
1. This protocol utilizes the phenol-sulfuric acid method described
in [5].
2. Transfer a 100 μL aliquot of each sample into a 5 mL test tube.
3. Carefully add 1 mL phenol solution (2.5% v/v) and 2.5 mL
concentrated (72% v/v) H 2 SO 4 (see note 7).
4. Transfer 2 mL aliquots of this reaction mixture into quartz cuvettes.
5. Read absorbances at 490 nm using a spectrophotometer (after
zeroing the instrument using water as a blank).
6. Quantification is then achieved by comparison to the absorbance
(at 490 nm) of authentic glucose standards (0, 200, 400, 600,
800, and 1000 μg/mL).
4 Notes
1. Concentrated H 2 SO 4 is highly hazardous, and we advocate all
handling to be conducted in a fume hood (cabinet). In addition,
the dilution process for the acid to achieve the desired acid
molarities is highly exothermic, and care should be taken to
allow solutions to cool before use.
2. Phenol is also a highly hazardous compound and should always
be handled in a fume hood (cabinet).
3. The use of automatic Dispensette pipettes (BrandTech Scientific, USA) is highly recommended for the rapid, reproducible,
and safe dispensing of reagents.
Complete Acid-Based Hydrolysis Assay for Carbohydrate Quantification in. . .
189