4. Add HPLC grade n-hexane to each of the new sample vials that
require a dilution (see Note 12).
5. Add the necessary amount of hexane upper phase from each
of the samples to the new sample vials (Fig. 1) (see Notes 12
and 13).
6. Seal the 1.5 mL vials containing the dilutions with PTFE/
silicone/PTFE crimp caps.
3.4 Preparation of
FAME Standards for GC
Analysis
1. Label a set of 300 μL insert vials for each level of FAME
standard, 1 through 5 and a calibration verification standard
(CVS) (see Note 14).
2. To each of the vials, add the following: 5 μL of the same
C13:0ME internal standard (10 mg/mL) used in the transesterification of the samples (see Note 15) and 200 μL of the
appropriate stock standard level or CVS (see Note 15).
3. Cap the vials immediately with PTFE/silicone/PTFE crimp caps.
4. Vortex the vials to thoroughly mix the contents.
3.5 FAME GC Setup
and Data Analysis
1. Suggested GC analysis temperature and flow program for a
DB-WAX GC column 30 m  0.25 mm inner diameter  0.25 μm film thickness is as follows: 1 μL injection at
10:1 split ratio; inlet temperature of 250
C; constant flow,
1 mL/min helium; oven temperature, 100
C for 1 min,
25
C/min up to 200
C, and hold for 1 min and 5
C/min
up to 250
C and hold for 7 min (23 min total); and FID,
280
C, 450 mL/min zero air, 40 mL/min H 2 , and 30 mL/
min helium.
2. Create a calibration table with the amount of each compound
in μg/mL based on the certificate of analysis (percent and
purity) and the weight of the 461C working solution
(or equivalent). See Fig. 1 for a typical fatty acid methyl ester
chromatogram.
3. Update the C13:0ME internal standard amount in μg/mL in
the software to represent the exact concentration of the
prepared standard
4. Quantification by ChemStation (Agilent) or respective GC
software (see Note 16).
3.6 FAME
Calculations
1. Export the FAME concentrations in μg/mL for each individual
FAME from the GC software, and normalize for the quantity
of the internal standard C13:0ME (this may also be done by
most software packages). The outcome is the total FAME
content normalized for the recovery of the internal standard
added at the start of the reaction:
208
S. Van Wychen and L.M.L Laurens
require a dilution (see Note 12).
5. Add the necessary amount of hexane upper phase from each
of the samples to the new sample vials (Fig. 1) (see Notes 12
and 13).
6. Seal the 1.5 mL vials containing the dilutions with PTFE/
silicone/PTFE crimp caps.
3.4 Preparation of
FAME Standards for GC
Analysis
1. Label a set of 300 μL insert vials for each level of FAME
standard, 1 through 5 and a calibration verification standard
(CVS) (see Note 14).
2. To each of the vials, add the following: 5 μL of the same
C13:0ME internal standard (10 mg/mL) used in the transesterification of the samples (see Note 15) and 200 μL of the
appropriate stock standard level or CVS (see Note 15).
3. Cap the vials immediately with PTFE/silicone/PTFE crimp caps.
4. Vortex the vials to thoroughly mix the contents.
3.5 FAME GC Setup
and Data Analysis
1. Suggested GC analysis temperature and flow program for a
DB-WAX GC column 30 m  0.25 mm inner diameter  0.25 μm film thickness is as follows: 1 μL injection at
10:1 split ratio; inlet temperature of 250
C; constant flow,
1 mL/min helium; oven temperature, 100
C for 1 min,
25
C/min up to 200
C, and hold for 1 min and 5
C/min
up to 250
C and hold for 7 min (23 min total); and FID,
280
C, 450 mL/min zero air, 40 mL/min H 2 , and 30 mL/
min helium.
2. Create a calibration table with the amount of each compound
in μg/mL based on the certificate of analysis (percent and
purity) and the weight of the 461C working solution
(or equivalent). See Fig. 1 for a typical fatty acid methyl ester
chromatogram.
3. Update the C13:0ME internal standard amount in μg/mL in
the software to represent the exact concentration of the
prepared standard
4. Quantification by ChemStation (Agilent) or respective GC
software (see Note 16).
3.6 FAME
Calculations
1. Export the FAME concentrations in μg/mL for each individual
FAME from the GC software, and normalize for the quantity
of the internal standard C13:0ME (this may also be done by
most software packages). The outcome is the total FAME
content normalized for the recovery of the internal standard
added at the start of the reaction:
208
S. Van Wychen and L.M.L Laurens