11. If the rest of the procedure cannot be completed on the same day,
vials must be recapped and then stored overnight (no longer than
24 h to avoid co-extraction of unwanted compounds) in a freezer
(À20
C). Always store vials upright. If longer storage is required,
transfer the hexane layer (after allowing for complete extraction of
at least 1 h) to a new 1.5 mL GC vial, cap the vial, and store in a
freezer (À20
C) for up to a week.
12. Use the following dilution guidelines when preparing to run
the hexane (upper) phase of the transesterified samples. Estimated sample lipid content of <10%, no dilution. Estimated
sample lipid content of 10–40%, 400 μL of HPLC grade hexane and 100 μL of the upper phase of the sample. Estimated
sample lipid content of 40–80%, 450 μL of HPLC grade hexane and 50 μL of the upper phase of the sample.
13. The upper phase of the sample can be withdrawn without
removing the caps. Use caution not to disturb the lower
phase when drawing the upper phase of the sample. Rinse the
syringe thoroughly with hexanes between samples. Work in
groups of ten samples or less to minimize evaporation of hexane, and cap immediately after adding the sample.
14. Prepare a series of calibration standard levels using the ~10 mg/
mL 461C working solution. Use the recommendations in
Table 1 to prepare levels 1–5 and a CVS (from an independent
working solution). Label 1.5 mL GC vials with the standard
level and date. Prepare each standard level before moving on to
the next: add the necessary amount of HPLC grade hexane to
the labeled 1.5 mL GC vial using a gas-tight syringe, and place a
PTFE/silicone/PTFE cap on the vial (you do not need to crimp
it yet). Add the necessary amount of FAME working solution
(~10 mg/mL) using a gas-tight syringe, and crimp the cap.
Then, move onto the next standard level. The prepared standard levels may be stored in PTFE/silicone/PTFE-capped (not
punctured) 1.5 mL GC vials in a freezer (À20
C). Always store
vials upright. Standards may be stored for up to 6 months.
Table 1
Standards prepared using the NuChek 461C ~10 mg/mL working solution
Standard level
~10 mg/mL 461C standard working solution (μL)
Hexane (μL)
5
500
500
4
250
750
3
100
900
2
30
970
1
10
990
CVS
90
910
Total Fatty Acid Content Determination of Whole Microalgal Biomass Using In. . .
211
vials must be recapped and then stored overnight (no longer than
24 h to avoid co-extraction of unwanted compounds) in a freezer
(À20
C). Always store vials upright. If longer storage is required,
transfer the hexane layer (after allowing for complete extraction of
at least 1 h) to a new 1.5 mL GC vial, cap the vial, and store in a
freezer (À20
C) for up to a week.
12. Use the following dilution guidelines when preparing to run
the hexane (upper) phase of the transesterified samples. Estimated sample lipid content of <10%, no dilution. Estimated
sample lipid content of 10–40%, 400 μL of HPLC grade hexane and 100 μL of the upper phase of the sample. Estimated
sample lipid content of 40–80%, 450 μL of HPLC grade hexane and 50 μL of the upper phase of the sample.
13. The upper phase of the sample can be withdrawn without
removing the caps. Use caution not to disturb the lower
phase when drawing the upper phase of the sample. Rinse the
syringe thoroughly with hexanes between samples. Work in
groups of ten samples or less to minimize evaporation of hexane, and cap immediately after adding the sample.
14. Prepare a series of calibration standard levels using the ~10 mg/
mL 461C working solution. Use the recommendations in
Table 1 to prepare levels 1–5 and a CVS (from an independent
working solution). Label 1.5 mL GC vials with the standard
level and date. Prepare each standard level before moving on to
the next: add the necessary amount of HPLC grade hexane to
the labeled 1.5 mL GC vial using a gas-tight syringe, and place a
PTFE/silicone/PTFE cap on the vial (you do not need to crimp
it yet). Add the necessary amount of FAME working solution
(~10 mg/mL) using a gas-tight syringe, and crimp the cap.
Then, move onto the next standard level. The prepared standard levels may be stored in PTFE/silicone/PTFE-capped (not
punctured) 1.5 mL GC vials in a freezer (À20
C). Always store
vials upright. Standards may be stored for up to 6 months.
Table 1
Standards prepared using the NuChek 461C ~10 mg/mL working solution
Standard level
~10 mg/mL 461C standard working solution (μL)
Hexane (μL)
5
500
500
4
250
750
3
100
900
2
30
970
1
10
990
CVS
90
910
Total Fatty Acid Content Determination of Whole Microalgal Biomass Using In. . .
211