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1) The document describes an experiment using gas chromatography to determine fatty acid content through fatty acid methyl ester (FAME) derivatives. Fatty acids are converted to volatile FAMEs via esterification to make them suitable for GC analysis. 2) Standard and sample FAME solutions are injected into a gas chromatograph with an FID detector and separated on a capillary column. Response factors are calculated from standard chromatograms and used to determine fatty acid amounts in samples. 3) Results are evaluated by comparing retention times and calculating fatty acid content in samples from standard response factors. The methodology, results, and conclusions are then discussed in the report.

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275 views12 pages

510 Exp 3

1) The document describes an experiment using gas chromatography to determine fatty acid content through fatty acid methyl ester (FAME) derivatives. Fatty acids are converted to volatile FAMEs via esterification to make them suitable for GC analysis. 2) Standard and sample FAME solutions are injected into a gas chromatograph with an FID detector and separated on a capillary column. Response factors are calculated from standard chromatograms and used to determine fatty acid amounts in samples. 3) Results are evaluated by comparing retention times and calculating fatty acid content in samples from standard response factors. The methodology, results, and conclusions are then discussed in the report.

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suraini
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CHM510: Experiment 3

FATTY ACID DETERMINATION USING GAS CHROMATOGRAPHY (GC)

The objectives of this experiment are:

1. To convert fatty acids to fatty acid methyl esters (FAME) using a derivatization
technique.

2. To calculate the amount of fatty acid methyl esters using the response factor method by
comparing their retention time to standard solution.

The first link contains a video that briefly explain how does esterification setup look like:
https://www.youtube.com/watch?v=ph-zGrS76fg

The second link shows how does esterification reaction happens:


https://www.youtube.com/watch?v=Wb4tSZhy-CU

Introduction

A flame ionization detector (FID) is commonly used for determination of fatty acid. The
advantages of FID are high sensitivity, large linear response range, robust and easy to use but
FID is a destructive detector as it can destroy the sample. Fatty acid is one of the components on
lipids that can be found in plants, animals and microorganisms. Fatty acid is a type of
hydrocarbon where it consists of number of carbon atoms and hydrogen along the chain. At the
end of the chain consist of functional group of –COOH (carboxyl group). There are two types of
fatty acid which are saturated and unsaturated fatty acid. Saturated fatty acid such as stearic acid,
lauric acid and myristic acid contain sigma bond only while unsaturated fatty acid such as oleic
acid, linoleic acid and palmitoleic acid where the carbon-carbon bond is double or triple bonds.

Any chemical that is volatile and chemically stable can be easily separate by gas
chromatography (GC). Fatty acids (non-volatile) are not suitable for GC analysis, therefore some
modification on the compounds so that they are suitable analysis. Unsuitable samples have high
risk of peak tailing thus affects the results. Though esterification reaction (derivatization
technique), fatty acids are converted to fatty acid methyl ester (FAME), which are volatile and
suitable for GC analysis.

• The amount of FAME is determined by using the response factor calculation:


Methodology

Sample and Reagent

 Oil or fat samples (margarine or butter)


 FAME standards (stearic acid methyl ester, lauric acid methyl ester, myristic acid methyl
ester, palmitoleic acid methyl ester and linoleic acid methyl ester)

Instrument

Gas chromatography (Agilent Technologies 6890N) equipped with flame ionization detector
(FID) and 30 m × 250µm × 0.25 µm HP5-MS capillary column.

Procedure

a) Preparation of standard solution

1. Prepare a FAME standard mixture solution containing of lauric acid methyl ester (0.10
mg mL-1), myristic acid methyl ester (0.10 mg mL-1), palmitoleic acid methyl ester (1.50
mg mL-1), stearic acid methyl ester (0.70 mg mL-1) and linoleic acid methyl ester (0.35
mg mL-1).

b) Preparation of fatty acid methyl ester samples from fat samples.

1. Weigh 2 g of oil or fat and record the actual weight.

2. Transfer the sample into 50 mL flask equipped with air condenser.

3. Add 5 mL of 0.5 M methanolic solution and reflux for 3 to 4 minutes

4. Add 15 mL of esterification reagent and reflux for 3 minutes.

5. Transfer the mixture into a separatory flask.

6. Add 50 mL of saturated sodium chloride and 25 mL of diethyl ether together.

7. Shake the mixture vigorously and vent for 2 minutes. Discard the aqueous layer.

8. Following with some addition, add 25 mL of saturated sodium chloride. Discard the
aqueous layer.

9. Transfer organic layer into a vial with a screw cap cover.

10. Seal the vial with parafilm for the quantitative analysis.
c) Instrument set-up

1. Injection port with split ratio (1:40)

2. Injection port Temperature: 250°C

3. Column temperature: 100°C to 290°C (increase at 40°C/min)

4. Carrier gas flow rate: 30 mL/s

5. Detector temperature: 250°C

d) Quantitative analysis of FAME

1. Inject 0.4 µL of FAME mix standard solution onto the injection port.

2. Repeat the injection to get reproducible peak areas.

3. Repeat step 1 and 2 for 0.4 µL of derivative samples.

4. Calculate the amount of each fatty acid from the sample using the data from the FAME
standard mixture solution.
Data – Standard Mixture FAME (Injection 1)
Data – Standard mixture FAME (Injection 2)
Data – Sample 1 (Injection 1)
Data – Sample 1 (Injection 2)
Data – Sample 2 (Injection 1)
Data – Sample 2 (Injection 2)
Data – Sample 3 (Injection 1)
Data – Sample 3 (Injection 2)
Data Evaluation

By referring to the chromatogram:

a) Calculate the Response Factor for each analyte (FAME) in the standard solution (from
the data/results of the standard mixture chromatograms).

b) Compare the retention time for each analyte (FAME) of standard and samples.

c) Calculate the amount of each analyte (FAME) in the samples.

Discussion

A. The discussion should include the justification of:

 Having temperature programming in the analysis.


 Derivatization/esterification step in the sample preparation
 Using gas chromatograph equipped with FID detector

B. The composition of the fatty acids obtained from analysis also should be discussed.

Conclusion

Should answer the objective of this experiment

References

List all references used to preparing the report

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