Experiment 3: Fatty Acid Determination Using Gas Chromatography (gc)

CHM 510 Experiment 3 FATTY ACID DETERMINATION USING GAS CHROMATOGRAPHY (GC) Name Group

: Nur Hafikah Bt Mustapha (2019602814) : RAS2453B

Lecturer’s name

: Dr Sharizal

EXPERIMENT 2: FATTY ACID DETERMINATION USING GAS CHROMATOGRAPHY (GC)

OBJECTIVE 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.

INTRODUCTION Fats consist of glycerol esters and long chain aliphatic acids (fatty acids). 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 depend on the composition of the fatty acid mixture. Animal fats tend to have a larger proportion of long chainsaturated 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. Gas chromatography separates the analytes that is volatile and chemically stable. Fatty acids are not sufficiently volatile for GC analysis, so that it needs to be modified chemically to produce anew compound, which has properties that are suitable for analysis. If the unsuitable sample is

introduced into GC analysis, it tends to cause peak tailing due to the adsorption and non-specific interaction with the column. In this experiment, the fatty acid is changed to fatty acid methyl ester (FAME) that is more volatile, suitable for GC analysis by using esterification reaction that used metholic solution with catalyst of esterification reagent. The objective for this experiment is to introduce a derivatization procedure routinely used for fat analysis in which nonvolatile fatty acids are chemically converted to the corresponding volatile methyl ester (FAME) and to determine the amount of FAME in the derivatized samples. .

CHEMICALS Oil or fat samples (margarine or butter)

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

PROCEDURE Approximately 2g of margarine was weighted and the exact weight was recorded. The sample was transferred into a 50 ml flask equipped with air condenser. 5 ml of 0.5 M methanolic solution was added and reflux for 3-4 minutes. 15 ml of esterification reagent was added and reflux for 3 minutes. The mixture was transferred into separatory flask. 50 ml of saturated NaCl and 25 ml were added. For 2 minutes, mixture was shaken vigorously and aqueous layer was discarded. Repeated the previous step with another 25 ml of saturated NaCl aqueous layer discarded again. The organic layer was transferred into a screw cap vial. Only organic layer was injected into the GC as water ruined the GC column.

RESULTS

FIGURE 1 STANDARD FAME FIGURE 2 SAMPLE 1 (INJECTION 1)

FIGURE 3 SAMPLE 1 (INJECTION 2)

FIGURE 4 SAMPLE 2 (INJECTION 1)

FIGURE 5 SAMPLE 2 (INJECTION 2)

FIGURE 6 SAMPLE 3 (INJECTION 1)

FIGURE 7 SAMPLE 3 (INJECTION 2)

DISCUSSION The component in the samples are compared with the standards component by the retention time. From the retention time of standard and samples, it is proven that component 5(peak 6) is not present in all 3 samples because of the difference of the retention time between standard and samples is too far. The amount of each component is different in each samples may due to the different mass of the fat initially. Peak 5 in each sample give very large different in the amount of FAME, this may be due to the incomplete separation process during shaking process or the discarding process, it is necessary to discard little organic layer to make sure that there is no aqueous layer anymore to be injected onto GC. That condition also affected by the contaminants in the flask that is not clean before using. The other peaks that correspond to specific component show small difference that assumed to be correct. There is another way to derivative or modified the low volatility fatty acid to more volatile compound called silylation that BSTFA to yield trimethylsilyl (TMS) ester that is more suitable to be analysed in GC.

CONCLUSION The derivation to technique used in this experiment is esterification to convert non-volatile fatty acids to more volatile fatty acid methyl ester (FAME). There are 5 components in the standard mixture while the 3 samples only indicate 4 components as shown in the standard mixture by comparison of the retention time. The concentration of each component is calculated by using the response factor of the standard.

REFRENCES 1.Retrieved from May 9 2020, https://www.ncbi.nlm.nih.gov/pubmed/8520689 2.Retrieved from May 9 2020 , http:www.doiserbia.nb.rs/img/doi/0352-5139/2015/035251391400073w.pdf