for 10 min at the initial NaOH concentration (30–35 mM),
with a total run time of 45 min. Column and compartment/
detector temperature is 35
C. Use the Gold Standard PAD
waveform (waveform A): E1, +0.1 V for 400 ms; E2, À2.0 V
for 1 ms; E3, +0.6 V for 1 ms; and E4, À0.1 V for 6 ms.
Run two to four water blanks before running standards and
samples. Run a water and one of the standard levels (typically
four) every ten samples to monitor system performance. Rerun
the full calibration every 40–60 samples. Monitor sample areas
during the run, and adjust dilutions as necessary. This is very
important as the calibration curves for the PAD are typically
quadratic.
23. Do not force calibration curves through the origin. Monomeric sugar curves on the PAD are typically the best fit with a
quadratic curve (second-order polynomial). Obtain the carbohydrate content as mg/mL for each sample. Remember to
correct the sample carbohydrate concentration for the amount
of hydrolysate that was used in the dilution (for a 1:20 in 1 mL,
use 50 μL), and then get a total carbohydrate content by
multiplying that concentration by the total volume from the
hydrolysis—7.25 mL. Divide the carbohydrate content by the
moisture-free weight of the sample to obtain the % monomeric
sugar content on an ovendry weight basis. The target relative
percent difference between duplicates or root mean square
between triplicates should be <10%. CVS recovery should be
between 95 and 105%. Track the results for the QC material
over time.
5 Acknowledgments
This work was supported by the US Department of Energy under
Contract No. DE-AC36-08GO28308 with the National Renewable Energy Laboratory. Funding provided by the US DOE Office
of Energy Efficiency and Renewable Energy Bioenergy Technologies Office. The US Government retains, and the publisher, by
accepting the article for publication, acknowledges that the US
Government retains a nonexclusive, paid-up, irrevocable, worldwide license to publish or reproduce the published form of this
work, or allow others to do so, for US Government purposes.
References
1. Templeton D, Quinn M, Van Wychen S et al
(2012) Separation and quantification of microalgal
carbohydrates. J Chromatogr A 1270:225–234
2. Williams PJLB, Laurens LML (2010) Microalgae
as biodiesel & biomass feedstocks: review &
analysis of the biochemistry, energetics & economics. Energy Environ Sci 3:554–590.
https://doi.org/10.1039/b924978h
3. Laurens LML, Nagle N, Davis R et al (2014)
Acid-catalyzed algal biomass pretreatment for
Total Carbohydrate Content Determination of Microalgal Biomass by Acid. . .
201
with a total run time of 45 min. Column and compartment/
detector temperature is 35
C. Use the Gold Standard PAD
waveform (waveform A): E1, +0.1 V for 400 ms; E2, À2.0 V
for 1 ms; E3, +0.6 V for 1 ms; and E4, À0.1 V for 6 ms.
Run two to four water blanks before running standards and
samples. Run a water and one of the standard levels (typically
four) every ten samples to monitor system performance. Rerun
the full calibration every 40–60 samples. Monitor sample areas
during the run, and adjust dilutions as necessary. This is very
important as the calibration curves for the PAD are typically
quadratic.
23. Do not force calibration curves through the origin. Monomeric sugar curves on the PAD are typically the best fit with a
quadratic curve (second-order polynomial). Obtain the carbohydrate content as mg/mL for each sample. Remember to
correct the sample carbohydrate concentration for the amount
of hydrolysate that was used in the dilution (for a 1:20 in 1 mL,
use 50 μL), and then get a total carbohydrate content by
multiplying that concentration by the total volume from the
hydrolysis—7.25 mL. Divide the carbohydrate content by the
moisture-free weight of the sample to obtain the % monomeric
sugar content on an ovendry weight basis. The target relative
percent difference between duplicates or root mean square
between triplicates should be <10%. CVS recovery should be
between 95 and 105%. Track the results for the QC material
over time.
5 Acknowledgments
This work was supported by the US Department of Energy under
Contract No. DE-AC36-08GO28308 with the National Renewable Energy Laboratory. Funding provided by the US DOE Office
of Energy Efficiency and Renewable Energy Bioenergy Technologies Office. The US Government retains, and the publisher, by
accepting the article for publication, acknowledges that the US
Government retains a nonexclusive, paid-up, irrevocable, worldwide license to publish or reproduce the published form of this
work, or allow others to do so, for US Government purposes.
References
1. Templeton D, Quinn M, Van Wychen S et al
(2012) Separation and quantification of microalgal
carbohydrates. J Chromatogr A 1270:225–234
2. Williams PJLB, Laurens LML (2010) Microalgae
as biodiesel & biomass feedstocks: review &
analysis of the biochemistry, energetics & economics. Energy Environ Sci 3:554–590.
https://doi.org/10.1039/b924978h
3. Laurens LML, Nagle N, Davis R et al (2014)
Acid-catalyzed algal biomass pretreatment for
Total Carbohydrate Content Determination of Microalgal Biomass by Acid. . .
201