Chm 510 Experiment 3

CHM 510 ANALYTICAL SEPARATION METHODS EXPERIMENT 3 FATTY ACID DETERMINATION USING GAS CHROMATOGRAPHY

NAME: NABILAH BINTI ABD RAHMAN STUDENT ID: 2015484718 LAB PARTNERS: 1. ANIZA BINTI ABDULLAH (2015827038) 2. NIK NURFARAHAIN RIFHAN BINTI NIK AZMAN (2015896328) GROUP: AS2453D1 LECTERUR’S NAME: DR. MARDIANA BINTI SAAID DATE PERFORMED: 6TH APRIL 2016

DATE OF SUBMISSION: 27TH MAY 2016 TITLE Experiment

3

–

Fatty

Acid

Determination

Using

Gas

Chromatography 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 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 vegetablederived, 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 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.

EXPERIMENTAL PROCEDURE a.

Preparation of fatty acid methyl ester samples from fat

samples 1.

3 samples of approximately 2 g of fat (butter) were weighed out and the exact weight were recorded.

2.

The samples were then transferred into a 50mL flask equipped with air condenser.

3.

5mL of 0.5 M methanolic solution was added into the flask and was refluxed for about 3 to 4 minutes until the mixture turned to golden brown colour.

4.

15mL of esterification reagent was added and continued to reflux again for another 3 minutes.

5.

The mixture was then 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 again.

7.

The organic layer was transferred into a screw cap vial and be made sure that only the organic layer was transferred.

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.

EXPERIMENTAL RESULTS, DATA AND CALCULATIONS A.

Response Factor (RF) for Analytes in Standard FAME

Peaks

Retention Time of Peaks (min)

Base Peak Width of Peaks (min)

Area of Peaks (pA*s)

Peak 2 Peak 3

2.084 2.765

0.0160 0.0217

107.40754 117.88110

Resolutio n of 2 peaks (peak 3 & peak 4)

Response Factor (Rf) of Peaks 0.93103 0.8483

Peak 4 Peak 5 Peak 6

3.921 5.899 6.865

0.0299 0.0496 0.0432

1022.17914 698.57605 276.47214

45

0.09783 0.1431 0.3617

Amount of Standard Sample (ppm) = 100 ppm

Calculation of resolution of 2 peaks: Rs(2,3) = 2( Rt,3- Rt,2 ) (Wb,2 + Wb,3) Rs(2,3) = 2( 3.921 – 2.765 ) (0.0299 + 0.0217)

= 45 #

Response Factor (Rf) of each peak (FAME) 1.

Peak 2 Response Factor (RF) = amount peak area = 100 (ppm) 107.40754

2.

= 0.93103 #

Peak 3 Response Factor (RF) = amount peak area = 100 (ppm) 117.88110

3.

Peak 4

= 0.8483 #

Response Factor (RF) = amount peak area = 100 (ppm) 1022.17914 4.

= 0.09783 #

Peak 5 Response Factor (RF) = 100 (ppm) 698.57605

1.

= 0.1431 #

Peak 6 Response Factor (RF) = amount peak area = 100 (ppm) 276.47214

B.

Peak 2

Peak 3

Peak 4

= 0.3617 #

Comparison of Retention Time in Standard and Samples Retention

Retention

Retention

Retention

Time, Rt for

Time, Rt for

Time, Rt for

Time, Rt for

Standard

Sample 1

Sample 2

Sample 3

(min) 2.084

(min) = 2.105 +

(min) = 2.106 +

(min) = 2.106 +

2.106

2.106

2.106

2

2

2

= 2.106 = 2.811 +

= 2.106 = 2.814 +

= 2.106 = 2.812 +

2.812

2.812

2.813

2

2

2

= 2.812 = 4.004 +

= 2.813 = 4.003 +

= 2.813 = 4.006 +

4.004

4.004

4.005

2

2

2

2.765

3.921

Peak 5

Peak 6

C.

5.899

6.865

= 4.004 = 6.360 +

= 4.004 = 6.363 +

= 4.006 = 6.371 +

6.369

6.365

6.365

2

2

2

= 6.365 = 7.005 +

= 6.364

= 6.368 = 7.023 =

7.004

-

7.015

2

2

= 7.005

= 7.019

Resolution of Each Sample of the FAME Retention Time

Base Width of

Resolution of

(Rt) of Peak 3 &

Peak 3 & 4

Peak 3 & 4

Sample 1

5 (min) 2.812, 4.004

(min) 0.0494,

18

Sample 2 Sample 3

2.813, 4.004 2.813. 4.006

0.08495 0.0493, 0.0857 0.0493, 0.0838

18 18

Calculation of resolution of 2 peaks: 1.

Sample 1 Rs(2,3) = 2( Rt,3- Rt,2 ) (Wb,2 + Wb,3) Rs(2,3) = 2( 4.004 – 2.812 ) (0.0494 + 0.08495) = 18 #

2.

Sample 2 Rs(2,3) = 2( Rt,3- Rt,2 ) (Wb,2 + Wb,3) Rs(2,3) = 2( 4.004 – 2.813) (0.0493 + 0.0857) = 18 #

3.

Sample 3 Rs(2,3) = 2( Rt,3- Rt,2 ) (Wb,2 + Wb,3) Rs(2,3) = 2( 4.006 – 2.813 ) (0.0493 + 0.0838) = 18 #

D.

Amount of FAME individuals in Samples Response Factor, Rf of

Peak Area (pA*s)

Correspond

Sample 1

Sample 2

Sample 3

Amount of FAME (Fatty Acid),

Peak Peak Peak Peak Peak

2 3 4 5 6

ing peak 0.93103 0.8483 0.09783 0.1431 0.3617

35.89295 16.54252 234.95041 18.673855 6.16273

(ppm) 33.417 14.033 22.958 2.6729 2.2291

Peak Peak Peak Peak Peak

2 3 4 5 6

0.93103 0.8483 0.09783 0.1431 -

11.16741 3.72386 44.85535 33.72593 -

10.397 3.159 4.388 4.8261 -

Peak Peak Peak Peak Peak

2 3 4 5 6

0.93103 0.8483 0.09783 0.1431 0.3617

14.295845 5.26132 68.67184 44.42255 10.71028

13.310 4.4632 6.718 6.357 3.874

Calculation of Amount of Sample (ppm)

= Response Factor of Standard X Area (pA*s)

DISCUSSION Fatty acid is an important component of lipids (fat-soluble components of living cells) in plants, animals, and microorganisms. Generally, a fatty acid consists of a straight chain of an even number of carbon atoms, with hydrogen atoms along the length of the chain and at one end of the chain and a carboxyl group (−COOH) at the other end. It is that carboxyl group that makes it an acid (carboxylic acid). If the carbon-to-carbon bonds are all single, the acid is saturated; if any of the bonds is double or triple, the acid is unsaturated and is more reactive. GC can be used to analyze fatty acids either as free fatty acids or as fatty acid methyl esters. The primary reasons to analyze fatty acids as fatty acid methyl esters include; in their free, underivatized form, fatty acids may be difficult to analyze because these highly polar compounds tend to form hydrogen bonds, leading to adsorption issues. Reducing their polarity may make them more amenable for analysis and to distinguish between the very slight differences exhibited by unsaturated fatty acids, the polar carboxyl functional groups must first be neutralized. This then allows column chemistry to perform separations by boiling point elution, and also by degree of unsaturation, position of unsaturation, and even the cis vs. trans configuration of unsaturation. The esterification of fatty acids to fatty acid methyl esters is performed using an alkylation derivatization reagent. Methyl esters

offer excellent stability, and provide quick and quantitative samples for

GC

analysis.

The

esterification

reaction

involves

the

condensation of the carboxyl group of an acid and the hydroxyl group of an alcohol. Esterification is best done in the presence of a catalyst (such as boron trichloride). The catalyst protonates an oxygen atom of the carboxyl group, making the acid much more reactive. An alcohol then combines with the protonated acid to yield an ester with the loss of water. The catalyst is removed with the water. The alcohol that is used determines the alkyl chain length of the resulting esters (the use of methanol will result in the formation of methyl esters whereas the use of ethanol will result in ethyl esters). In this experiment, a gas chromatography technique is used to determine the amount of fatty acid present in the butter or margarine. Butter commonly contains a lot of fatty acid types, and fatty acids are derivatized from the butter using the techniques mentioned in the experimental procedure. Generlly, 5 types of fatty acids are contained and that is methyl laurate, methyl myristate, methyl palmitate, methyl stearate and methyl linoleate and they are commonly known as Fatty Acid Methyl Esters (FAME). Fatty Acid Methyl Esters (FAME) are esters of fatty acids. The physical characteristics of fatty acid esters are closer to those of fossil diesel fuels than pure vegetable oils, but properties depend on the type of vegetable oil. A mixture of different fatty acid methyl esters is commonly referred to as biodiesel, which is a renewable alternative fuel. FAME has physical properties similar to those of conventional diesel. It is also non-toxic and biodegradable. Since methyl laurate is least retained by the stationary phase, it eluted out first, at the 2.0th minute followed by methyl myristate at the 2.8th minute, methyl palmitate at 4.0th minute, methyl stearate at 6th minute and methyl linoleate at 7 th minute. By comparing the retention time of standard and the 3 samples, it can be told that the

retention time are almost the same, which means that the 5 compounds of samples elute out at almost the same time as the standard. Methyl laurate is represented by peak 2, and at the standard it eluted out at 2.084th minute and the samples it eluted out at 2.1 minute. Peak 3 is methyl myristate, and in the standard the compound elute out at 2.765th minute and the samples at 2.8th minute. The 4th peak is methyl palmitate, and in the standard it elute out at 3.921 minute and the in the samples methyl palmitate elute out at the 4th minute. Methyl stearate is represented by peak 5 and in the standard it elute out at the 5.899 th minute but in the samples peak 5 elute out at 6.3 rd minute, which differs slightly. And lastly at peak 6, is methyl linoleate and it elute out at 6.865 th minute and in the sample it elute out at 7th minute which also differs slightly. By the response factor calculated in the standard, we can tell the amount of each methyl esters in the sample. And the amount of fatty acids in each sample are calculated and stated in the results and data section. CONCLUSION The derivatization 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 that is the methyl esters. The concentration of each component is calculated by using the response factor of the standard. REFERENCES 1.

http://global.britannica.com/science/fatty-acid

2.

http://www.sigmaaldrich.com/analyticalchromatography/analytical-products.html? TablePage=105120181