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CH 4200, Fall 2004

Prof. Greenlief
FAMEs by GC-1
Fatty Acid Determination Using Gas Chromatography

Objectives
This experiment introduces a procedure that is used routinely for fat analysis in which
nonvolatile fatty acids are chemically converted to the corresponding volatile methyl esters. The
resulting volatile mixture can be analyzed by gas chromatography.

Background
Fatty Acids

Fats consist of glycerol esters and long chain aliphatic acids (fatty acids). The general
glycerol ester structure is shown below:

CH
2
-O-CO-R
1

|
CH
2
-O-CO-R
2

|
CH
2
-O-CO-R
3


The backbone of these compounds contains from 4 to more than 20 carbon atoms. Most natural
sources of these compounds have an even number of carbon atoms because the biosynthetic
pathway builds the backbone two carbons at a time. Fatty acid chains may contain one or more
double bonds at specific positions (unsaturated and polyunsaturated), or they may be fully
saturated. The physical and chemical properties of a fat depends on the composition of the fatty
acid mixture. Animal fats tend to have a larger proportion of long chain saturated acids and are
solids at room temperature. Fats from plant sources contain a higher proportion of unsaturated
acids and are often liquids at room temperature due to hydrogen bonding. Polyunsaturated fats
are usually of vegetable origin. Crisco is an example of a vegetable-derived, unsaturated fatty
acid that has been hydrogenated to form a solid material.

Fats are used in cooking because they are very high boiling compounds. Their high boiling
points therefore make this class of compounds ill suited for analysis by gas chromatography.
However, the glycerol esters can be chemically decomposed into methyl esters of each
individual fatty acid. In this lab, your samples will be inter-esterified in methanol using a BF
3

catalyst. The corresponding mixture of methyl esters will be extracted into hexane and
chromatographically separated. Methyl pentadecanoic acid (C
15
) will be added as an internal
standard. This compound is ideal as an internal standard because it should behave similarly to
the analytes of interest, but it is unlikely to be present in your natural fat sample since it has an
odd number of carbon atoms on the backbone.

CH 4200, Fall 2004
Prof. Greenlief
FAMEs by GC-2
Experimental
I. Preparation and analysis of fatty acid methyl ester (FAME) samples from fat samples

Weigh out about two grams of oil or fat in a small beaker and record the exact weight.
Dissolve the sample in 50 ml of chloroform and transferred to a 100 ml volumetric flask and
dilute to the mark. Transfer 1 ml of the unknown sample to a 10 ml screwtop culture tube with
Teflon liner. Now add exactly 1.00 ml of a standard solution of 0.814 mg/ml pentadecanoic
acid. When you esterify the glycerides in the fat sample, you will also esterify the pentadecanoic
acid standard. If we assume i.) the efficiency for esterification of the standard is the same as that
of the glycerides, and ii.) the response of the detector to each of the FAMES including the C
15

internal standard is the same, then we can quantify the amount of each ester in the fat by
comparison of the integrated areas with the known concentration of the standard.

Evaporate most of the chloroform under a stream of nitrogen until ~100 l of the solution
remains. If the solution is dried completely, it will be hard to re-dissolve with the esterification
reagent. Next, add 1 ml of interesterification reagent [25 vol% of a 12% BF
3
-methanol solution,
20 vol% benzene and 55 vol% methanol]. Flush the tube with nitrogen, seal it, and heat it in a
100C water bath for 30 minutes. After interesterification, extract the methyl esters with hexane
and H
2
O so that the final mixture of the reagent, hexane and water, is in a proportion of 1:1:1
(i.e., add 1 ml each of hexane and water to the reaction mixture). Shake the mixture vigorously
by hand for 2 min. If a stable emulsion is formed, break it by centrifugation. Transfer about half
of the top hexane phase to a small test tube for injection. Be careful to remove only the organic
layer. Do not inject directly from the reaction vial because of the risk of injecting water. Water
can ruin the GC column.

NOTE: You may want to run your calibration standards while the esterification reaction is in
progress.


II. Instrumental Set-Up

Refer to the Chromatographic Instrumentation: Gas Chromatography procedure for a
detailed description of the instrumental adjustments required for this analysis.

Fixed Settings: Generally the operator must adjust gas flows to the columns, the inlets, the
detectors, and the split ratio. In addition, the injector and detector temperatures must be set. The
detectors are generally held at the high end of the oven temperature range to minimize the risk of
analyte precipitation. All of these parameters should have been set to the correct values, but
double check all the parameters to ensure that the values are correct.

Detector Temperature

Detector A: 250C
Detector B: 300C

CH 4200, Fall 2004
Prof. Greenlief
FAMEs by GC-3
Injector temperature

Both injectors 220C

Integrator chart speed: 2 cm/min


III. Separation of Fatty Acid Methyl Esters (FAMES)

The following experiments will be run on column A using a standard sample containing the
following FAMES in approximately the following concentrations. (Get the exact
concentrations from your TA.)
Approximate
FAME Concentration
Methyl myristate C
14:0
0.10 mg/ml
Methyl pentadecanoate C
15:0
0.80 mg/ml
Methyl palmitate C
16:0
1.50 mg/ml
Methyl palmitoleate C
16:1*
0.15 mg/ml (* 16 carbon backbone, 1 double bond)
Methyl stearate C
18:0
0.70 mg/ml
Methyl linoleate C
18:2
0.35 mg/ml
Methyl oleate C
18:1
2.00 mg/ml
Methyl linolenate C
18:3
0.15 mg/ml

Note: You can often assign each peak of the chromatogram of each standard sample by
considering the melting point and the interactions of the analyte with the stationary phase
(column). The assigned peaks can be cross-checked by relating the concentration and the area of
the peak measured. The above list is given in the order of elution, but you will want to confirm
this by checking the concentrations against the relative areas of the peaks.

A. Isothermal Chromatogram of FAME Standard

Set the OVEN TEMP to 180C and allow the GC to warm up. While its warming, set:
SIG 1 A FINAL VALUE 181C
INIT VALUE 180C FINAL TIME 1 minutes.
INIT TIME 15 minute
RATE 0C/min

When the instrument is ready, the NOT READY light will turn off, and you can begin your
run. Inject a 1 microliter sample onto column A using proper injection technique as described in
the Chromatographic Instrumentation: Gas Chromatography document. Collect the
chromatogram and answer the following question.

Question 1: Do you get good separation of all peaks with the isothermal program? Why?

B. Temperature Program for FAME Standard
CH 4200, Fall 2004
Prof. Greenlief
FAMEs by GC-4

Next you will analyze the same sample with a temperature program. You will run the
temperature program listed below on column A of the instrument to compare the effect of
isothermal to temperature programmed methods. The effect should be noticeable by the change
in the retention times of the esterfied compound peaks. The program is as follows:

INIT VALUE 120C
INIT TIME 1 min
RATE 15C /min
FINAL VALUE 210C
FINAL TIME 7 min

(To get rid of tick marks on the chromatogram, press INTG, the number 8, and then ENTER on
the integrator keypad.)

Question 2: How do the retention times of the sample components differ in the two
chromatograms? Explain this result.

C. Temperature Program for FAME Sample
Using the same temperature program as in part III.B above, inject 1 l of the dry esterification
product for analysis. Assign the peaks according to the results of the standard chromatograms
collected in section III B.

IV. Quantitative Analysis of FAMES

From your chromatograms:

A. Identify the esters present in your sample qualitatively by retention times.
You will notice a relationship between carbon number and retention time, which can be
used to identify FAMES having chain lengths longer than C
18
.

B. Quantify the amount of each of the FAMES in your sample, but only do this for the
seven components (not including the C
15
internal standard) which were present in your
standard sample if they appear.
This requires several steps. You will need to compare the sample peak areas with the
standard peak areas. Since the concentrations of FAMEs responsible for the standard peaks are
known, this comparison will allow you to calculate a concentration from the sample peak area.
However, because the sample injection is different from the standard injection (e.g., small
differences in volume, split ratio, dilutions, etc.) you cannot make a direct comparison. This is
where the internal standard comes in. You can correct for variations in sample volume by taking
the ratio of all peak areas to the internal standard in both the sample and the standard. Since the
internal standard has a known concentration in both sample and standard, it can be used to
correct for sample variations. A
x
denotes the area of a peak due to compound x in the sample.
In order to relate this area to the area of compound x in the standard you must first correct for
sample variations to arrive at a corrected area denoted A
c,x
. You can find A
c,x
for each peak in
the chromatogram using the equation
CH 4200, Fall 2004
Prof. Greenlief
FAMEs by GC-5


where

A
C
15
,standard
and

A
C
15
,sample
denote the internal standard areas in the standard solution
and the sample, respectively. Note that the C
15
signal area is proportional to the mass of analyte
passing through the detector. Since the mass is equal to the concentration of C
15
times the
volume, the ratio of Area/conc. is equal to a volume times a proportionality constant. The above
equation results in a cancellation of the proportionality constant, and so this equation can be
rewritten as

to emphasize the importance of the internal standard in correcting for sample volume. The
resulting A
c,x
is an area that has been corrected to give a volume equivalent to what would be
expected if the internal standard peaks has identical areas in sample and standard
chromatograms.

From the corrected areas, the concentration of each component of the mixture can be
calculated using the expression
This expression reflects the fact that the properly corrected area of compound x can be compared
with the area of compound x on the standard chromatogram, whose concentration is known.
Thus the ratio of the areas gives the ratio of concentrations, and the product of this ratio and the
known concentration of compound x in the standard gives the concentration of compound x in
the sample.

Use the results of this quantitative analysis to calculate the percentage composition of each
fatty acid in grams of compound per gram of oil. (Decide if C
15
should be included in this
calculation.) The development of an exact formula for this calculation is left to you.

Additional Questions

3. Some FAME sample chromatograms may contain additional peaks. What might these be due
to assuming they are not simply contamination of the sample?

4. Suppose we found out that the sensitivity scale used in the run of the standard was different
from that of the sample. Should we consider the change of the sensitivity? If not, why (Hint:
C
15
)?

standard
standard
, C
sample , C
sample , C
, C
x x , c
15
15
15
15
CONC
CONC
A
A
A A =
standard
standard
, x
, x
x , c x
A
CONC
A CONC =
sample , C
, C
x x , c
15
15
lume InjectedVo
lume InjectedVo
A A
standard
=
CH 4200, Fall 2004
Prof. Greenlief
FAMEs by GC-6
5. What is the resolution (R) for the C
16:0
and C
16:1
peaks in the standard chromatogram?



Lab Write-up Format for Gas Chromatography Lab
The Gas Chromatography Lab report will be a formal report. The report must be typed, and
must include the following sections

Title of Analysis, Name of Analyst, Date of Analysis
General methods (a page and a half maximum)
Raw Data, chromatograms
Data Analysis
Questions
Conclusions

For General Methods give a brief description of the techniques used in the analysis. This
should include, for example, the temperature program, flow rates, pressures, the column type,
detector type, esterification procedure, etc. It should include enough detail so that another
analyst can duplicate the experiment if necessary. This section must be written in the third
person (do not write I/We prepared a sample), and it must be written in the past tense. This is
standard writing practice. This section CANNOT be a list of steps; it must be in sentence form.
No credit will be given for procedural checklists.

For Raw Data, tables of retention times and integrations of relevant peaks must be included.
These tables should be labeled sequentially (Table 1, Table 2, Table 3, etc) and should be
referred to in the text of the Data Analysis. These should be Microsoft Excel Spreadsheet tables
(or a similar program like Sigmaplot, etc). This also MUST include copies of the
chromatograms. They can be reduced, if necessary.

For the Data Analysis section should include any graphs and tables of calculated numbers that
you might feel necessary. (This lab does require many graphs.) There is no need to show all of
the calculations performed (mostly the results of those calculations will be fine), but
representative calculations and/or formulas might provide some clarity to the section. You might
include sample calculations in an Appendix, or you may choose to include them in the Data
Analysis section. This is your choice, however, make it completely clear where each of the
numbers comes from for your data analysis.

Questions should be answered in this section of the report. The answers should be written in
essay style using complete sentences and coherent paragraphs.

Conclusions: This section will contain the final thoughts and recap of any important points
made in data analysis. This can be a paragraph, or a bulleted (or numbered) list would also be
acceptable. Any important results should be briefly noted in the conclusion.

CH 4200, Fall 2004
Prof. Greenlief
FAMEs by GC-7
The TAs will not ask to see any photocopies of the lab notebooks for grading at the time that the
labs reports are turned in, however, they may spot check lab notebooks to check for
completeness during some of the lab periods. Therefore, it would be to everyones advantage to
keep all notes and observations in the lab notebooks.