EXPERIMENT 3 FATTY ACID DETERMINATION USING GAS CHROMATOGRAPHY
INTRODUCTION In chemistry chemistry,, and especially in biochemistry biochemistry,, a fatty acid is a carboxylic acid with acid with a long aliphatic aliphatic tail tail (chain (chain), ), which is either saturated saturated or or unsaturated unsaturated.. Fatty acids are merely carboxylic acids with long hydrocarbon chains. The hydrocarbon chain length may vary from 1!" carbons (most usual is 1#!1$). The non!polar hydrocarbon al%ane chain is an important counter balance to the polar acid functional group. In acids with only a few carbons, the acid functional group dominates and gives the whole molecule a polar character. character. &owever, in fatty acids, the non!polar hydrocarbon chain gives the molecule a non! polar character. character. The most common fatty acids are listed. 'ote that there are two groups of fatty acids!! saturated and unsaturated. ecall ecall that the term unsaturated refers to the presence of one or more double bonds between carbons as in al%enes al%enes.. saturated fatty acid has all bonding positions between carbons occupied by hydrogens. The melting points for the saturated fatty acids follow the boiling point principle observed principle observed previously. previously. *elting point principle+ as the molecular weight increases, the melting point increases. This observed in the series lauric (1#), palmitic (1-), stearic (1$). oom temperature temperature is #o, /auric acid which melts at 00o is still a solid, while arachidonic acid has long since melted at !o, so it is a liuid at room temperature. Fatty acids are important sources of fuel because, when metaboli2ed, they yield large uantities of T3 T3.. *any cell types can use either glucose glucose or or fatty acids for this purpose. In particular, heart and s%eletal s%eletal muscle prefer fatty acids. 4espite long!standing assertions to the contrary, brain can use fatty acids as a source of fuel in addition to glucose and %etone bodies. bodies.
OBJECTIVE • •
To To prepare the fatty acid acid sample To To modify compound through through derivation to procedure procedure a new compound which is suitable for gas chromatography
REAGENT AND SOLUTION • • • • • •
•
*ethyl laurate (.1 mg5ml) *ethyl myristate (.1 mg5ml) *ethyl palmitate (1. mg5ml) *ethyl strearate (.6 mg5ml) *ethanolic solution (.*) + 'a7& in methanol 8steri9cation reagent + added 6. ml &#:70 and g of 'al in a ml ;as% and re;ux for 1 minutes. :ample + oil or fat samples (margarine or butter) < 1g
ANALYTICAL PROCEDURE 1. 3reparation of fatty acid methyl ester samples from fat samples a. =eighed #g of fat and recorded the exact weight b. Transferred the sample into a ml ;as% euipped with air condenser c. dded ml of . * methanolic solution and re;uxed for " to 0 minutes d. dded 1 ml of esteri9cation reagent and re;uxed for " minutes e. Transferred the mixture into a separatory ;as%. dded ml of saturated 'al and # ml of diethyl ether. :ha%e the mixture vigorously for # minutes and discarded the aueous layer. f. dded another #ml of saturated 'al and again discarded the aueous layer. g. Transferred the oraganic layer into vial #. Instrument :et!up In>ection port + split(#+1) In>ection port temperature + #? 7ven temperature + 1? to #@? at 0?5min olumn ;ow rate + 0 ml5s 4etector temperature + #o
PROCEDURE 1. *ethyl strearate =eighed 16. mg of methyl strearate Then diluted in # ml diethyl ether in volumetric ;as% #. *ethyl laurate
Ased the micro pipette, pipette #.$60B/ of methyl laurate Then diluted in # ml diethyl ether in volumetric ;as% ". *ethyl myristate Ased the micro pipette, pipette #.@#0B/ of methyl myristate Then diluted in #ml diethyl ether in volumetric ;as% 0. *ethyl palmitate Ased the micro pipette, pipette 00.B/ of methyl palmitate Then diluted in #ml diethyl ether in volumetric ;as% . *ethanolic solution =eighed 0 mg and then diluted in ml water and ml methanol CALCULATION *ethyl /aurate 0.1 mg
ml
=
x 25 ml
2.5 mg 870 mg
/ cm 3
x 1000=2.874 μL
*ethyl myristate 0.1 mg
ml
=
x 25 ml
2.5 mg 855 mg
/ cm 3
x 1000=2.924 μL
*ethyl palmitate 1.5 mg
ml
=
x 25 ml
3.75 mg 852 mg
/ cm 3
x 1000 =44.0 μL
*ethyl strearate
0.7 mg
ml
=
x 25 ml
¿ 17.5 mg
8:7/ATI7' peak 1 ∧ peak 2 =
(
−3.303 ) 0.0331+ 0.0264
2 3.639
¿ 11.294
peak 2 ∧ peak 3 =
(
− 3.639) 0.0363 + 0.0264
2 4.543
¿ 28.836
peak 3 ∧ peak 4 =
(
− 4.543 ) 0.0363 + 0.0319
2 4.699
¿ 4.575
RESULT COMPOUND
RETENTION TIME (MIN)
3almitate 0.0" *yristate ".@" /aurate ".#6:tearate 0.61$ Table 1+ etention time for compound of 3almitate, *yristate, /aurate and :tearate in standard solution.
PEAK
COMPOUND
RETENTION TIME (MIN)
RESOLUTION
1 /aurate "."" 3ea% 1 C # 3ea% # C 3ea% " # *yristate ".-"@ 11.#@0 " C0 " 3almitate 0.0" #$.$"0.6 0 :tearate 0.-@@ Table #+ etention time of compound of 3almitate, *yristate,/aurate and :tearate in sample of fatty acid.
DISCUSSION Dased 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 pea% is determined to be the highest compare to other components. This shows that they are the ma>or compounds in the fatty acid. 3almitate shows it pea% at 0.0" min as same as the retention time in standard solution. For /aurate and :tearate, the retention time in sample is not the same as standard yet almost nearest. &owever, for *yristate 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. Dased on the standard chromatogram for :tearate, the pea% is too small to recogni2e. This might be because of the sampleEs temperature is too broad compare to the setting temperature. Thus, the compound remained in the column as it fully unable to vapori2e completely. From the table, the resolution for pea% 1 C # and pea% " C 0 shows a good value, which is 11.#@0 and 0.6 respectively. &owever, for pea% # C ", the resolution is above #, which is #$.$"-, and this indicates a poor separation between this two pea%s. From the chromatogram, it can be seen that the pea% of 3almitate 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 3almitate. new condition, for instance increase the temperature of the column, need to be conducted to separate the overlapping pea%s. The pea%s 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. large amount of compound arriving at the detector causing the pea% to occur as tailing. In this experiment, derivi2ation method was used. This is a method of converting non!volatile method into volatile so that can be analyse using gas chromatography. 4erivati2ation will render highly polar materials to be suciently volatile so that they can be eluted at reasonable temperatures without thermal decomposition. For G analysis, compounds containing functional groups with active hydrogens such as !:&, !7&, !'& and !77& are of primary concern because of the tendency of these functional groups to form intermolecular hydrogen bonds which aHect the inherent volatility of compounds containing them. :ince G is used to separate volatile organic compounds, modi9cation of the functional group of a molecule by derivati2ation enables the analysis to be conducted.
CONCLUSION 4erivi2ation is a eHective 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 ma>or components identi9ed in this analysis.
REFERENCES 1. :%oog, =est, &oller, rouch, Fundamentals of Analytical Chemistry , Thomson Droo%s5ole 3ublishers,$th ed.,#0 #. Derivization Reaction and Reagents for Gas Chromatography Analysis , https+55www.google.com.my5url saJtCrctJ>CJCesrcJsCsourceJwebCcdJ1CcadJr>aCvedJ4KLF> CurlJhttpM"M#FM#Fwww.intechopen.comM#FdownloadM#Fpdf M#F"#$16CeiJ3GAK=/7$f6rfvoK4KwCusgJFL>'F&ror08:# NdAGvDemmxgs$83nCsig#JnaO#oglw"g-bth!P! KeLCbvmJbv.0@-$6,d.bm%, retrieved on 0th *ay #1".
". Fatty acid, http+55en.wi%ipedia.org5wi%i5FattyNacid, retrieved on *ay #1" 0. Fatty acids, http+55www.elmhurst.edu5Qchm5vchemboo%51fattyacids.html, retrieved on *ay #1"
