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.
20. Thaw a vial of the ~2 mg/mL master sugar mix. Dilute the master
mix 1:10 to reach a final concentration of ~0.2 mg/mL. Create a
six-point calibration with the ~0.2 mg/mL sugar mix using the
dilutions outlined in Table 2. Prepare dilutions directly in the
screw-top vials. Typical sample dilution for most microalgal samples (10–40% carbohydrates) is 1:20 or 50 μL filtered or centrifuged hydrolysate in 950 μL water.
21. Equilibrate the HPAEC-PAD at the following conditions: 4
C
sample compartment temperature, 0.45 or 0.5 mL/min flow
rate, 30–35 mM NaOH (either from the eluent generator or
prepared from 100 mM NaOH eluent), isocratic, column temperature 35
C, and compartment/detector temperature
35
C. Equilibrate the HPAEC-PAD for at least 2 h before
running standards and samples. Choose the isocratic condition
that best separates the monomeric sugars eluting in the first
10 min; this may change based on column variability and
column age.
22. Run standards and samples at the following conditions: sample
compartment temperature of 4
C. Use a 0.45 or 0.5 mL/min
flow rate, 30–35 mM NaOH isocratic eluent concentration for
10 min, followed by a ramping of the 1 M NaOAc/100 mM
NaOH eluent from 2 to 19% (the remainder as water, 98–81%)
with a linear (5) increase from 10 min until 30 min; turn off
ramp; run 100 mM NaOH for 5 min; and then re-equilibrate
Table 2
Suggested standard concentrations for the HPAEC-PAD method
Concentration of stock/working solution
(mg/mL)
Dilution stock + water
(μL)
Final concentration
(mg/mL)
0.2
10 + 990
0.002
0.2
20 + 980
0.004
0.2
50 + 950
0.01
0.2
100 + 900
0.02
0.2
200 + 800
0.04
0.2
300 + 700
0.06
200
S. Van Wychen and L.M.L. Laurens
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.
20. Thaw a vial of the ~2 mg/mL master sugar mix. Dilute the master
mix 1:10 to reach a final concentration of ~0.2 mg/mL. Create a
six-point calibration with the ~0.2 mg/mL sugar mix using the
dilutions outlined in Table 2. Prepare dilutions directly in the
screw-top vials. Typical sample dilution for most microalgal samples (10–40% carbohydrates) is 1:20 or 50 μL filtered or centrifuged hydrolysate in 950 μL water.
21. Equilibrate the HPAEC-PAD at the following conditions: 4
C
sample compartment temperature, 0.45 or 0.5 mL/min flow
rate, 30–35 mM NaOH (either from the eluent generator or
prepared from 100 mM NaOH eluent), isocratic, column temperature 35
C, and compartment/detector temperature
35
C. Equilibrate the HPAEC-PAD for at least 2 h before
running standards and samples. Choose the isocratic condition
that best separates the monomeric sugars eluting in the first
10 min; this may change based on column variability and
column age.
22. Run standards and samples at the following conditions: sample
compartment temperature of 4
C. Use a 0.45 or 0.5 mL/min
flow rate, 30–35 mM NaOH isocratic eluent concentration for
10 min, followed by a ramping of the 1 M NaOAc/100 mM
NaOH eluent from 2 to 19% (the remainder as water, 98–81%)
with a linear (5) increase from 10 min until 30 min; turn off
ramp; run 100 mM NaOH for 5 min; and then re-equilibrate
Table 2
Suggested standard concentrations for the HPAEC-PAD method
Concentration of stock/working solution
(mg/mL)
Dilution stock + water
(μL)
Final concentration
(mg/mL)
0.2
10 + 990
0.002
0.2
20 + 980
0.004
0.2
50 + 950
0.01
0.2
100 + 900
0.02
0.2
200 + 800
0.04
0.2
300 + 700
0.06
200
S. Van Wychen and L.M.L. Laurens