FATTY ACID DETERMINATION USING GAS CHROMATOGRAPHY (GC)

(EXPERIMENT 3) FATTY ACID DETERMINATION USING GAS CHROMATOGRAPHY (GC) NAME:

MOHAMAD NOR AMIRUL AZHAR BIN KAMIS

STUDENT ID:

2014647344

PARTNERS’ NAMES: 1.

MOHAMAD HAMIZAN BIN MOHD ISA

2.

MOHAMAD SHAFIQ BIN PARMAN

3.

MOHAMAD AZMIZAM BIN MOHAMAD NOOR

DATE OF EXPERIMENT:

30/10/2014

DATE OF SUBMISSION:

8/12/2014

INTRODUCTION: 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 a new 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 non-volatile fatty acids are chemically converted to the corresponding volatile methyl ester (FAME) and to determine the amount of FAME in the derivatized samples. The amount of FAME is determined by using the response factor calculation. Response Factor (RF) = Amount of unknown compound = peak area of unknown compound × RF (std)

EXPERIMENTAL: a. Preparation of fatty acid methyl ester samples from fat samples: 1. 3 fat samples were prepared by weighing the mass of 2.0528g, 2.0894g and 2.0327g respectively. 2. The samples were transferred into a 50mL flask equipped with air condenser. 3. 5mL of 0.5M methanolic solution was added into the flask and was reflux for about 3 to 4 minutes until the mixture turned to golden brown colour. 4. 15mL of esterification reagent was added and continue refluxing for another 3 minutes. 5. The mixture then was transferred into a separatory flask and 50mL of saturated sodium chloride (NaCl) and 25mL of diethyl ether were added into the flask. The mixture was shaken vigorously and vented to release the pressure formed for about 2 minutes. The aqueous layer was discarded. 6. Step 5 was repeated with another 25mL of saturated NaCl and the aqueous layer was discarded too. 7. The organic layer was transferred into a screw cap vial for easies the analysis to be done with automated injector. 8. Note that, the preparation is the same for all 3 samples.

b. Instrument set up: Injector port: split (40:1) Injection port temperature: 250°C Column temperature: 100°C to 290°C at 40°C/min Carrier gas flow rate: 30mL/s Detector temperature: 250°C c. Quantitative analysis of FAME: 1. Each of the derivatized samples was injected into GC column by using automated injector. 2. FAME standard mixture was injected into the GC column. 3. The amount of fatty acid in each sample was calculated.

RESULT AND DISCUSSION: A. Response factor (RF) for analytes in standard FAME: Amount of Peak area (pA*s) FAME in standard (ppm) Peak 2 100 65.90409 Peak 3 100 435.19760 Peak 4 100 1244.67700 Peak 5 100 125.80314 Peak 6 100 116.25892 B. Comparison of retention time for standard and samples: Retention Retention Retention time for time for time for standard sample 1 sample 2 (min) (min) (min) Peak 2 1.269 1.275 1.276 Peak 3 1.671 1.684 1.684 Peak 4 2.366 2.390 2.390 Peak 5 3.537 3.613 3.605 Peak 6 3.739 7.822 -

Response Factor (RF) 1.5174 0.2298 0.0803 0.7949 0.8601

Retention time for sample 3 (min) 1.278 1.686 2.391 3.620 -

C. Amount of FAME in samples:

Sample 1

Sample 2

Sample 3

Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6

RF of corresponding peak 1.5174 0.2298 0.0803 0.7949 0.8601 1.5174 0.2298 0.0803 0.7949 0.8601 1.5174 0.2298 0.0803 0.7949 0.8601

Peak area (pA*s)

Amount of FAME (ppm)

62.79572 34.40598 888.78308 975.41412 81.3408 49.48989 1123.19543 65.98615 86.24370 36.68810 1133.67981 1315.67725 -

95.29 7.91 71.37 775.36 123.43 11.37 90.19 52.45 130.87 8.43 91.03 1045.83 -

D. Sample calculation: Response factor of peak 2 in standard = = 1.5174 Amount of FAME in peak 2 (sample 1) = 1.5174 × 62.79572 = 95.29ppm Amount of FAME in peak 2 (sample 2) = 1.5174 × 81.3408 = 123.43ppm Amount of FAME in peak 2 (sample 3) = 1.5174 × 86.24370 = 130.87ppm

The components in the samples are compared with the standard components 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 are too big. 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 un-complete 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 derivatize 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 derivatization technique used in this experiment is esterification to convert nonvolatile 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.

REFERENCES: 1. Nor’ashikin S., Ruziyati T., Mardiana S. (2012), Analytical Separation Methods Laboratory Guide (2nd edition), 3/10/2014. 2. Mardiana Saaid, Sample Preparation Lecture Notes, 15/10/2014.