5. Gas-tight syringes with PTFE plungers and 22 gauge, point
style 2 needles. Covering a volume range of 5–1000 μL.
6. Adjustable pipet covering 300 μL.
7. Clear, 11 mm crimp-top glass vials for GC analysis with 1.5 mL
fill volume.
8. Clear, 11 mm crimp-top glass vials with inserts for GC analysis
with 300 μL fill volume.
9. Aluminum crimp caps, 11 mm PTFE/silicone/PTFE septa for
the GC vials.
10. Gas chromatograph (GC) equipped with a variable split-flow
injector or equivalent device and a flame ionization detector
(FID).
11. Capillary column with polyethylene glycol stationary phase:
Agilent J&W GC Column DB-WAX length 30 m, internal
diameter 0.25 mm, film thickness 0.25 μm, or equivalent.
2.2 Transesterification Reagents
1. Prepare 2:1 (v/v) chloroform/methanol by adding 100 mL of
HPLC grade methanol to 200 mL of HPLC grade chloroform.
Use a glass graduated cylinder to measure out chloroform and
methanol. Store the final solution in a glass bottle with a lid
appropriate for organic solvent storage. This reagent may be
prepared ahead of time and stored for up to 2 months in the
dark. Chloroform is a trihalomethane and possible human
carcinogen. Methanol is a slight health hazard and moderately
flammable.
2. Prepare 0.6 M (2.1%, v/v) hydrochloric acid:methanol by
adding 5 mL of concentrated ACS grade HCl to HPLC
grade methanol. Use a graduated cylinder to measure out
methanol into a glass bottle. Use a pipet to add hydrochloric
acid directly to the methanol. Never add methanol to the acid,
as this may cause an explosion. This reagent may be prepared
ahead of time and stored for up to 2 months in the dark.
Hydrochloric acid is toxic and corrosive. Methanol is a slight
health hazard and moderately flammable.
3. For dilutions and extractions, use HPLC grade n-hexane. Hexane is a significant health hazard and extremely flammable.
4. Hexanes are used for gas-tight syringe rinses and do not need
to be HPLC grade quality.
2.3 Standards
1. Prepare a ~10 mg/mL tridecanoic acid methyl ester (C13:0ME)
internal standard in hexane. Use a certified tridecanoic acid methyl
ester (C13:0ME) standard (e.g., Sigma-Aldrich #91558-5ML).
To make up a 10 mg/mL solution, weigh out approximately
100 mg of the C13:0ME into a 10 mL, class A, volumetric flask.
Record the weight of the C13:0ME to the nearest 0.1 mg. Bring
Total Fatty Acid Content Determination of Whole Microalgal Biomass Using In. . .
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style 2 needles. Covering a volume range of 5–1000 μL.
6. Adjustable pipet covering 300 μL.
7. Clear, 11 mm crimp-top glass vials for GC analysis with 1.5 mL
fill volume.
8. Clear, 11 mm crimp-top glass vials with inserts for GC analysis
with 300 μL fill volume.
9. Aluminum crimp caps, 11 mm PTFE/silicone/PTFE septa for
the GC vials.
10. Gas chromatograph (GC) equipped with a variable split-flow
injector or equivalent device and a flame ionization detector
(FID).
11. Capillary column with polyethylene glycol stationary phase:
Agilent J&W GC Column DB-WAX length 30 m, internal
diameter 0.25 mm, film thickness 0.25 μm, or equivalent.
2.2 Transesterification Reagents
1. Prepare 2:1 (v/v) chloroform/methanol by adding 100 mL of
HPLC grade methanol to 200 mL of HPLC grade chloroform.
Use a glass graduated cylinder to measure out chloroform and
methanol. Store the final solution in a glass bottle with a lid
appropriate for organic solvent storage. This reagent may be
prepared ahead of time and stored for up to 2 months in the
dark. Chloroform is a trihalomethane and possible human
carcinogen. Methanol is a slight health hazard and moderately
flammable.
2. Prepare 0.6 M (2.1%, v/v) hydrochloric acid:methanol by
adding 5 mL of concentrated ACS grade HCl to HPLC
grade methanol. Use a graduated cylinder to measure out
methanol into a glass bottle. Use a pipet to add hydrochloric
acid directly to the methanol. Never add methanol to the acid,
as this may cause an explosion. This reagent may be prepared
ahead of time and stored for up to 2 months in the dark.
Hydrochloric acid is toxic and corrosive. Methanol is a slight
health hazard and moderately flammable.
3. For dilutions and extractions, use HPLC grade n-hexane. Hexane is a significant health hazard and extremely flammable.
4. Hexanes are used for gas-tight syringe rinses and do not need
to be HPLC grade quality.
2.3 Standards
1. Prepare a ~10 mg/mL tridecanoic acid methyl ester (C13:0ME)
internal standard in hexane. Use a certified tridecanoic acid methyl
ester (C13:0ME) standard (e.g., Sigma-Aldrich #91558-5ML).
To make up a 10 mg/mL solution, weigh out approximately
100 mg of the C13:0ME into a 10 mL, class A, volumetric flask.
Record the weight of the C13:0ME to the nearest 0.1 mg. Bring
Total Fatty Acid Content Determination of Whole Microalgal Biomass Using In. . .
205