Chm510 Experiment 3

EXPERIMENT 3 FATTY ACID DETERMINATION USING GAS CHROMATOGRAPHY INTRODUCTION In chemistry, and especially in biochemistry, a fatty acid is a carboxylic acid with a long aliphatic tail (chain), which is either saturated or unsaturated. Fatty acids are merely carboxylic acids with long hydrocarbon chains. The hydrocarbon chain length may vary from 10-30 carbons (most usual is 12-18). The non-polar hydrocarbon alkane chain is an important counter balance to the polar acid functional group. In acids with only a few carbons, the acid functional group dominates and gives the whole molecule a polar character. However, in fatty acids, the non-polar hydrocarbon chain gives the molecule a non- polar character. The most common fatty acids are listed. Note that there are two groups of fatty acids-saturated and unsaturated. Recall that the term unsaturated refers to the presence of one or more double bonds between carbons as in alkenes. A saturated fatty acid has all bonding positions between carbons occupied by hydrogens. The melting points for the saturated fatty acids follow the boiling point principle observed previously. Melting point principle: as the molecular weight increases, the melting point increases. This observed in the series lauric (C12), palmitic (C16), stearic (C18). Room temperature is 25oC, Lauric acid which melts at 44o is still a solid, while arachidonic acid has long since melted at -50o, so it is a liquid at room temperature. Fatty acids are important sources of fuel because, when metabolized, they yield large quantities of ATP. Many cell types can use either glucose or fatty acids for this purpose. In particular, heart and skeletal muscle prefer fatty acids. Despite long-standing assertions to the contrary, brain can use fatty acids as a source of fuel in addition to glucose and ketone bodies.

OBJECTIVE  

To prepare the fatty acid sample To modify compound through derivation to procedure a new compound which is suitable for gas chromatography

REAGENT AND SOLUTION       

Methyl laurate (0.1 mg/ml) Methyl myristate (0.1 mg/ml) Methyl palmitate (1.50 mg/ml) Methyl strearate (0.70 mg/ml) Methanolic solution (0.5M) : NaOH in methanol Esterification reagent : added 7.5 ml H2SO4 and 5g of NaCl in a 500ml flask and reflux for 15 minutes. Sample : oil or fat samples (margarine or butter) – 10g

ANALYTICAL PROCEDURE 1. Preparation of fatty acid methyl ester samples from fat samples a. Weighed 2g of fat and recorded the exact weight b. Transferred the sample into a 50ml flask equipped with air condenser c. Added 5 ml of 0.5 M methanolic solution and refluxed for 3 to 4 minutes d. Added 15 ml of esterification reagent and refluxed for 3 minutes e. Transferred the mixture into a separatory flask. Added 50ml of saturated NaCl and 25 ml of diethyl ether. Shake the mixture vigorously for 2 minutes and discarded the aqueous layer. f. Added another 25ml of saturated NaCl and again discarded the aqueous layer. g. Transferred the oraganic layer into vial 2. Instrument Set-up Injection port : split(20:1) Injection port temperature : 250˚C Oven temperature : 100˚C to 290˚C at 40˚C/min Column flow rate : 40 ml/s Detector temperature : 250oC

PROCEDURE 1. Methyl strearate Weighed 17.5 mg of methyl strearate Then diluted in 25 ml diethyl ether in volumetric flask 2. Methyl laurate

Used the micro pipette, pipette 2.874µL of methyl laurate Then diluted in 25 ml diethyl ether in volumetric flask 3. Methyl myristate Used the micro pipette, pipette 2.924µL of methyl myristate Then diluted in 25ml diethyl ether in volumetric flask 4. Methyl palmitate Used the micro pipette, pipette 44.0µL of methyl palmitate Then diluted in 25ml diethyl ether in volumetric flask 5. Methanolic solution Weighed 40 mg and then diluted in 50 ml water and 50 ml methanol

CALCULATION Methyl Laurate 0.1mg x = ml 25 ml 2.5 mg x 1000=2.874 μL 870 mg/cm3 Methyl myristate 0.1mg x = ml 25 ml 2.5 mg x 1000=2.924 μL 855 mg/cm3 Methyl palmitate 1.5 mg x = ml 25 ml 3.75 mg x 1000=44.0 μL 852mg/cm 3

Methyl strearate

0.7 mg x = ml 25 ml ¿ 17.5 mg

RESOLUTION peak 1∧peak 2=

2(3.639−3.303) 0.0331+ 0.0264

¿ 11.294

peak 2∧peak 3=

2(4.543−3.639) 0.0363+0.0264

¿ 28.836

peak 3∧ peak 4=

2( 4.699−4.543) 0.0363+ 0.0319

¿ 4.575

RESULT COMPOUND RETENTION TIME (MIN) Palmitate 4.543 Myristate 3.935 Laurate 3.276 Stearate 4.718 Table 1: Retention time for compound of Palmitate, Myristate, Laurate and Stearate in standard solution.

PEAK

COMPOUND

RETENTION RESOLUTION TIME (MIN) 1 Laurate 3.303 Peak 1 & 2 Peak 2 & Peak 3 2 Myristate 3.639 11.294 3 &4 3 Palmitate 4.543 28.836 4.575 4 Stearate 4.699 Table 2: Retention time of compound of Palmitate, Myristate,Laurate and Stearate in sample of fatty acid.

DISCUSSION Based on the result obtained, it shows that the fatty acid consist all the four esters. This can be seen by comparing both compounds retention time in standard and sample solutions. From the chromatogram, the four components peak is determined to be the highest compare to other components. This shows that they are the major compounds in the fatty acid. Palmitate shows it peak at 4.543 min as same as the retention time in standard solution. For Laurate and Stearate, the retention time in sample is not the same as standard yet almost nearest. However, for Myristate the retention time is a bit earlier in sample solution compare to standard. This might be due to the condition applied in the sample is more suitable as it elute faster. Based on the standard chromatogram for Stearate, the peak is too small to recognize. This might be because of the sample’s temperature is too broad compare to the setting temperature. Thus, the compound remained in the column as it fully unable to vaporize completely. From the table, the resolution for peak 1 & 2 and peak 3 & 4 shows a good value, which is 11.294 and 4.575 respectively. However, for peak 2 & 3, the resolution is above 20, which is 28.836, and this indicates a poor separation between this two peaks. From the chromatogram, it can be seen that the peak of Palmitate is not separated well. This might be due to the presence of another compound in the sample that have the closest retention time with the Palmitate. A new condition, for instance increase the temperature of the column, need to be conducted to separate the overlapping peaks. The peaks appear to be tailing. This is may be because the sample is too soluble with column. Thus, it been retained in the column and elute lately as

a band to the detector. A large amount of compound arriving at the detector causing the peak to occur as tailing. In this experiment, derivization method was used. This is a method of converting non-volatile method into volatile so that can be analyse using gas chromatography. Derivatization will render highly polar materials to be sufficiently volatile so that they can be eluted at reasonable temperatures without thermal decomposition. For GC analysis, compounds containing functional groups with active hydrogens such as -SH, -OH, -NH and -COOH are of primary concern because of the tendency of these functional groups to form intermolecular hydrogen bonds which affect the inherent volatility of compounds containing them. Since GC is used to separate volatile organic compounds, modification of the functional group of a molecule by derivatization enables the analysis to be conducted.

CONCLUSION Derivization is a effective method in converting non-volatile samples into volatile. The sample prepared using this method was successfully run using gas chromatography. The result shows all the four esters found to be in the sample fatty acid and they are the major components identified in this analysis.

REFERENCES 1. Skoog, West, Holler, Crouch, Fundamentals of Analytical Chemistry, Thomson Brooks/Cole Publishers,8th ed.,2004 2. Derivization Reaction and Reagents for Gas Chromatography Analysis, https://www.google.com.my/url? sa=t&rct=j&q=&esrc=s&source=web&cd=1&cad=rja&ved=0CDYQFjA A&url=http%3A%2F%2Fwww.intechopen.com%2Fdownload%2Fpdf %2F32817&ei=P0qGUYWLO8f7rAfvoYDYCw&usg=AFQjCNFHrAor4ES26 _dUGvBemmxgs8EPnA&sig2=naK2oglw3g06bth-JRYeQ&bvm=bv.45960087,d.bmk, retrieved on 4th May 2013.

3. Fatty acid, http://en.wikipedia.org/wiki/Fatty_acid, retrieved on 5 May 2013 4. Fatty acids, http://www.elmhurst.edu/~chm/vchembook/551fattyacids.html, retrieved on 5 May 2013