4 Notes
1. Suggested tubes: VWR#89079-404—Do not use the caps that
come with the tubes as they do not vent properly. Use a stopper
and tear-away cap system. Stopper—VWR#15507-144. Tearaway aluminum cap—VWR#16171-851. The tubes may be
washed and reused.
2. Use plastic (HDPE) pipets and LabWare when working with
basic solutions.
3. HPAEC system equipped with pulsed amperometric detection
(AgCl reference electrode, gold electrode), sodium hydroxide
eluent generator (may also use manually prepared eluent if
eluent generator is unavailable—follow Thermo Fisher Scientific/Dionex TN 71), gradient pump and mixer, and the following column: PA-20 and PA-20 guard column (Dionex/
Thermo Fisher Scientific).
4. Run singlets or duplicates of each sample with a triplicate QC
sample. Keep batches to a manageable size.
5. All microalgal biomass samples must be freeze-dried before
analysis, and final moisture content should be <10%. Care
should be taken to ensure a representative, and ground/homogenized (particle size <1 mm) sample is taken for analysis.
Storage of samples should ideally be in an inert (nitrogen)
atmosphere and at least at À20
C. Ensure sufficient quantities
of the QC sample are available for QC charting over multiple
analyses
6. Dry samples in vials overnight in a 40
C oven under vacuum.
After drying, take a final weight to calculate the moisture-free
sample amount. Allow samples to cool in a desiccator under
vacuum before recording a final weight. If no vacuum oven is
available, samples can be stored overnight in a vacuum desiccator prior to analysis, given that a corresponding moisture measurement is available for that material. Refer to Determination
of Total Solids and Ash in Algal Biomass for obtaining a moisture measurement [11]. Because moisture content can be variable in samples, it is important to correct for moisture and
report carbohydrate content on a dry weight basis.
7. After all the samples have been weighed out, add 250 μL of 72%
(w/w) sulfuric acid to each tube, and vortex to thoroughly mix
the acid and biomass. Vortex samples carefully to ensure all
solids remain at the bottom of the tube and immersed in the
acid. If samples have a tendency to clump, add sulfuric acid to
one tube at a time, and vortex the tube immediately after the
addition of acid. If the sulfuric acid is tinted yellow or brown, it
needs to be replaced. Sulfuric acid is corrosive and is a moderate
health hazard.
Total Carbohydrate Content Determination of Microalgal Biomass by Acid. . .
197
1. Suggested tubes: VWR#89079-404—Do not use the caps that
come with the tubes as they do not vent properly. Use a stopper
and tear-away cap system. Stopper—VWR#15507-144. Tearaway aluminum cap—VWR#16171-851. The tubes may be
washed and reused.
2. Use plastic (HDPE) pipets and LabWare when working with
basic solutions.
3. HPAEC system equipped with pulsed amperometric detection
(AgCl reference electrode, gold electrode), sodium hydroxide
eluent generator (may also use manually prepared eluent if
eluent generator is unavailable—follow Thermo Fisher Scientific/Dionex TN 71), gradient pump and mixer, and the following column: PA-20 and PA-20 guard column (Dionex/
Thermo Fisher Scientific).
4. Run singlets or duplicates of each sample with a triplicate QC
sample. Keep batches to a manageable size.
5. All microalgal biomass samples must be freeze-dried before
analysis, and final moisture content should be <10%. Care
should be taken to ensure a representative, and ground/homogenized (particle size <1 mm) sample is taken for analysis.
Storage of samples should ideally be in an inert (nitrogen)
atmosphere and at least at À20
C. Ensure sufficient quantities
of the QC sample are available for QC charting over multiple
analyses
6. Dry samples in vials overnight in a 40
C oven under vacuum.
After drying, take a final weight to calculate the moisture-free
sample amount. Allow samples to cool in a desiccator under
vacuum before recording a final weight. If no vacuum oven is
available, samples can be stored overnight in a vacuum desiccator prior to analysis, given that a corresponding moisture measurement is available for that material. Refer to Determination
of Total Solids and Ash in Algal Biomass for obtaining a moisture measurement [11]. Because moisture content can be variable in samples, it is important to correct for moisture and
report carbohydrate content on a dry weight basis.
7. After all the samples have been weighed out, add 250 μL of 72%
(w/w) sulfuric acid to each tube, and vortex to thoroughly mix
the acid and biomass. Vortex samples carefully to ensure all
solids remain at the bottom of the tube and immersed in the
acid. If samples have a tendency to clump, add sulfuric acid to
one tube at a time, and vortex the tube immediately after the
addition of acid. If the sulfuric acid is tinted yellow or brown, it
needs to be replaced. Sulfuric acid is corrosive and is a moderate
health hazard.
Total Carbohydrate Content Determination of Microalgal Biomass by Acid. . .
197