dilutions performed using RO water to prepare the reagents prior
to use. For the optimization of stage 1 of the protocol for your
specific biomass type, we would recommend a minimum series of
acid concentrations of 3, 6, 9, and 12 M (see note 1). However, a
more comprehensive optimization can be achieved using the experimental run conditions outlined in Table 1. Store reagents in a
hazardous chemical cabinet such as that designed for flammables.
1. RO water.
2. Algal biomass.
3. Sulfuric acid (H 2 SO 4 ).
4. A 2.5% (w/v) phenol solution is prepared by adding 2.5 g
phenol to 100 mL RO water (see note 2). Prepare in a fume
hood, and store in a hazardous chemical cabinet once prepared.
5. Acetic acid.
6. Methanol.
7. Analytical balance.
8. High-pressure liquid chromatography (HPLC).
9. C18 Techsphere ODS column (5 μm, 4.6 mm  250 mm;
HPLC Technologies, UK).
10. Water bath(s) (0–100
C).
11. Pyrex reaction tubes.
12. Test-tube rack.
13. Luer-Lok-type syringes.
14. Syringe filters (0.45 μm).
15. 15 mL plastic centrifuge tubes.
16. Suitable HPLC vials corresponding to the HPLC used.
3 Methods
Carry out all procedures at room temperature unless otherwise specified.
3.1 Total Acid
Hydrolysis Assay
1. Accurately weigh 30 mg of biomass into a 50 mL screw-capped
Pyrex reaction tube (Pyrex, UK) using an analytical balance
accurate to 4 decimal places.
2. Carefully add 1 mL of the required concentration of H 2 SO 4 (suggested acid concentration range is 3–12 M; see Sect. 2, item 2 for
guidelines) into each reaction vessel (see note 3) ensuring the
biomass is completely covered or submerged within the acid. Carefully ensure the lids are tight on all reaction vessels while ensuring
that the biomass stays submerged within the acid.
Complete Acid-Based Hydrolysis Assay for Carbohydrate Quantification in. . .
185
to use. For the optimization of stage 1 of the protocol for your
specific biomass type, we would recommend a minimum series of
acid concentrations of 3, 6, 9, and 12 M (see note 1). However, a
more comprehensive optimization can be achieved using the experimental run conditions outlined in Table 1. Store reagents in a
hazardous chemical cabinet such as that designed for flammables.
1. RO water.
2. Algal biomass.
3. Sulfuric acid (H 2 SO 4 ).
4. A 2.5% (w/v) phenol solution is prepared by adding 2.5 g
phenol to 100 mL RO water (see note 2). Prepare in a fume
hood, and store in a hazardous chemical cabinet once prepared.
5. Acetic acid.
6. Methanol.
7. Analytical balance.
8. High-pressure liquid chromatography (HPLC).
9. C18 Techsphere ODS column (5 μm, 4.6 mm  250 mm;
HPLC Technologies, UK).
10. Water bath(s) (0–100
C).
11. Pyrex reaction tubes.
12. Test-tube rack.
13. Luer-Lok-type syringes.
14. Syringe filters (0.45 μm).
15. 15 mL plastic centrifuge tubes.
16. Suitable HPLC vials corresponding to the HPLC used.
3 Methods
Carry out all procedures at room temperature unless otherwise specified.
3.1 Total Acid
Hydrolysis Assay
1. Accurately weigh 30 mg of biomass into a 50 mL screw-capped
Pyrex reaction tube (Pyrex, UK) using an analytical balance
accurate to 4 decimal places.
2. Carefully add 1 mL of the required concentration of H 2 SO 4 (suggested acid concentration range is 3–12 M; see Sect. 2, item 2 for
guidelines) into each reaction vessel (see note 3) ensuring the
biomass is completely covered or submerged within the acid. Carefully ensure the lids are tight on all reaction vessels while ensuring
that the biomass stays submerged within the acid.
Complete Acid-Based Hydrolysis Assay for Carbohydrate Quantification in. . .
185