7. After the 15 min incubation time, turn off the block, and immediately add 1 mL of the ferric solution while the glass tubes are still
on the block (see Note 17).
8. Once the samples have reacted with the ferric solution, add
2.5 mL 18.2 MΩ water, and mix well by pipetting or vortexing.
9. Place an aliquot of the samples and standards into the appropriate cuvettes, and obtain an absorbance on the spectrophotometer at 620 nm (see Note 18).
10. Use the glucose calibration curve and linear regression to
quantify the total carbohydrate concentration in mg/mL
(see Note 19).
3.4 Liquid
Chromatography
Determination of
Monomeric Sugars
1. Hydrolyzed samples for spectrophotometric analysis should be
prepared by separating the solids from the hydrolysate (see
Note 12). See Fig. 1b for a typical hydrolysate.
2. Aliquot or dilute standards and samples into the labeled screwtop vials (see Note 20).
3. Prepare the HPAEC-PAD instrument for analysis (see Note 21).
4. Run standards and samples on HPAEC-PAD (see Note 22).
See Fig. 1a for a typical chromatogram of the monomeric
sugars.
5. Quantify the monomeric sugars by Chromeleon software
(Thermo Fisher Scientific) (see Note 23).
Fig. 1 (a) Representative HPAEC-PAD chromatogram of standards: (1) fucose, (2) rhamnose, (3) arabinose,
(4) galactose, (5) glucose, (6) mannose, (7) xylose, and (8) ribose. (b) Typical hydrolyzate of algal biomass in a
4% sulfuric acid solution in a total of 7.25 mL
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S. Van Wychen and L.M.L. Laurens
on the block (see Note 17).
8. Once the samples have reacted with the ferric solution, add
2.5 mL 18.2 MΩ water, and mix well by pipetting or vortexing.
9. Place an aliquot of the samples and standards into the appropriate cuvettes, and obtain an absorbance on the spectrophotometer at 620 nm (see Note 18).
10. Use the glucose calibration curve and linear regression to
quantify the total carbohydrate concentration in mg/mL
(see Note 19).
3.4 Liquid
Chromatography
Determination of
Monomeric Sugars
1. Hydrolyzed samples for spectrophotometric analysis should be
prepared by separating the solids from the hydrolysate (see
Note 12). See Fig. 1b for a typical hydrolysate.
2. Aliquot or dilute standards and samples into the labeled screwtop vials (see Note 20).
3. Prepare the HPAEC-PAD instrument for analysis (see Note 21).
4. Run standards and samples on HPAEC-PAD (see Note 22).
See Fig. 1a for a typical chromatogram of the monomeric
sugars.
5. Quantify the monomeric sugars by Chromeleon software
(Thermo Fisher Scientific) (see Note 23).
Fig. 1 (a) Representative HPAEC-PAD chromatogram of standards: (1) fucose, (2) rhamnose, (3) arabinose,
(4) galactose, (5) glucose, (6) mannose, (7) xylose, and (8) ribose. (b) Typical hydrolyzate of algal biomass in a
4% sulfuric acid solution in a total of 7.25 mL
196
S. Van Wychen and L.M.L. Laurens