CHM 510ANALYTICAL SEPARATION METHODS

GAS CHROMATOGRAPHY (GC):

1. OPTIMIZATION OF FLOW RATE AND COLUMN TEMPERATURE 2. DETERMINATION OF FATTY ACID

NAME : NUR ADAWIYAH BINTI MANSOR (2012663606)LAB PARTNER : NUR ANITH BINTI MOHD SAHARUDIN (2012441998)GROUP : AS2253BLECTURER : MRS. HALIZA KASSIMDATE OF EXPERIMENT : 12th NOVEMBER 2013

DATE OF SUBMISSION: 26th DECEMBER 2013OBJECTIVE : 1. To study the effect of carrier gas flow rate and column temperature on isothermal GC separation of methyl esters.

2. To perform separation of methyl esters using temperature programming.

3. To identify each component in methyl ester mixture.

4. To determine concentration of fatty acid by using gas chromatography.

ABSTRACT :Gas chromatography(GC) is an analytical technique for separating

components/ solutes based primarily on their volatilities which means that the separation is based on differences in boiling points of the solutes. It may also based on the solutes interaction with the stationary phase.This experiment was carried out to optimize the flow rate and column temperature of methyl esters by using Gas chromatography(Agilent Technologies 6890N) equip with flame ionization detector(FID) ABD 30m x 250μm x 0.25μm HP5-MS capillary column. The effect of carrier gas flow rate and column temperature on isothermal GC separation of methyl esters can be observed by varying the flow rate and the temperature of column. The suitable condition for separation of methyl ester is at column temperature 2100C and flow rate 70cm3/sec.

For identification of each component in methyl ester mixture, we must compare the retention time of individual sample with standard mixture. The first eluted is methyl laurate, followed by methyl myristate and last eluted is methyl palmitate. For separation of methyl esters, standard mixture two using column temperature programming the resolution is smaller compare to resolution for standard mixture one, that using isothermal method.

For determination of fatty acid, this analysis is to determine type of fatty acid which contain in a majerin product but this analysis was done through the use of gas chromatography technique. This creates a problem

because fatty acid is not volatile enough, reactive and too polar for the column to separate them. To overcome this problem, that fatty acid was converted into their corresponding volatile methyl esters first by esterification process. . Fatty acids are converted to esters by reaction with excess alcohol, using acid catalyst or a lipase. In preparing methyl esters for gas chromatography analysis, boron trifluoride, sulfuric acid, or anhydrous hydrogen chloride in methanol are commonly used. The reaction is completed by refluxing. This involves the condensation of carboxyl group of an acid and hydroxyl group of alcohol, with water elimination. After we managed to prepare the methyl esters and we must keep it in refrigerator to keep it cold and inject it into gas chromatography machine under temperature programming condition. From the chromatogram GC of fatty acid, we can calculate the concentration of methyl ester and standard mixture two as our response factor (RF).

Table A : Type of metyhl esters that present in this experiment.

M.Myristate

Formula: C15H30O2 Melting point: 18°C Boiling point: 323°C Molar mass: 242.4 g/mol Density: 0.855 g/cm³ Classification : Ester

M. Laurate

Formula: C13H26O2 Melting point: 5 °C Boiling point: 261-262 °C Molar mass: 214.34 g/mol Density: 0.87 g/cm³ Classification : Ester

M.Palmitate

Formula: C17H34O2 Melting point: 28-34°C Boiling point: 135-137 °C Molar mass: 270.45g/mol Density: 0.853 g/cm³ Classification : Ester

M.Linoleate

Formula: C19H34O2 Melting point: −35  °C Boiling point: 192°C Molar mass: 294.47g/mol Density: 0.889g/cm³ Classification : Ester

M.Stearate

Formula: C19H38O2

Melting point: 37-41 °C

Boiling point: 215°C

Molar mass: 298.50 g/mol

Density: 0.84g/cm³

Classification : Ester

INTRODUCTION:Gas chromatography is a chromatographic technique of analytical

chemistry that is used in separation of volatile organic compounds. The technique is applied for testing the purity of a substance, separating the different components of a mixture, and identification of a compound. A gas chromatograph consists of a flowing mobile phase, an injection port, a separation column containing the stationary phase, a detector, and a data recording system.

Diagram A: Component of Gas Chromatogram

Carrier gas acts as mobile phase for gas chromatography. It functions to transport the analyte through the column without interacting with the molecules of the analyte. The characteristic of carrier gas is that it must be chemically inert, dry, oxygen free and high purity. To achieve high purity level, molecular sieve acts to filter and remove any contaminants. The common type of gasses used include nitrogen, hydrogen, helium, argon, and carbon dioxide, depending on the type of detector used. The carrier gas system also contains a molecular sieve to remove water and other impurities. The flow rate of carrier gas are controlled the flow controller.

There are two types of column, the packed column and the open tubular (capillary) column.  Packed columns are typically a glass or stainless steel coil (typically 1-5 m total length and 2-6 mm inner diameter) that is filled with the stationary phase, or a packing coated with the stationary phase. Capillary columns have a very small inner diameter (0.1-0.5 mm) with length of 5-100m long and have 0.1-5µm thick stationary phase coated on inner walls. They provide much higher separation efficiency than packed columns (broader peak, longer retention time, less resolution) but are more easily overloaded by too much sample. There are three types of capillary column. Wall-coated open tubular (WCOT) having the inner wall directly coated with liquid stationary phase without support material. Support-coated open tubular (SCOT) have the liquid stationary phase coated on solid support (thin film of support, around 30µm) attached to the inside wall of column. As for porous-layered open tubular (PLOT) the stationary phase is a solid substance that is coated to the column wall. Column temperature is set slightly above the average boiling point of the sample. For various mixtures having different boiling point, temperature programing is needed.

Column selection is based on stationary phase, column diameter and length, and the thickness of stationary phase. Long and narrow column and also a suitable stationary phase can yield good resolution. The stationary phase of gas chromatography is a non-volatile liquid coated on inside of column or on a fine solid support. It mus have low volatility,

thermal stability, chemical inertness and low viscosity. The type are chosen by their polarity, thus it depends on whether analyte and mobile phase are polar or not. Injector port act as introducer of sample to column. The inlet is a piece of hardware attached to the column head. Sample is injected through the inlet using a microsyringe, then instantly evaporated to gas carried by carrier gas into the column. To ensure fast and complete vaporization, the injector port temperature is set 50oC higher than column temperature. Three types of injectors are the split injector, splitless injector, and on-column injector. Split injection used for high analyte concentrations. Splitting only allow only a small amount of sample into the column. Excessive part of sample will be vented out, then the split outlet is closed. The reason of splitting are to prevent overloading of the column and produces narrow and sharp peaks. While splitless injection is used for low concentration samples or trace analysis of high boiling compounds in low boiling solvents. Split vent are opened for 30 to 90 seconds after injection, removing the bulk of the solvent to avoid large solvent peak overlaping with the analyte peak, but leaves most of the sample condensed at the top of the column. This will give higher peak, making it suitable for trace samples. As for on-column injection, it is applied for samples that decompose above their boiling point or thermally labile compounds. This type of injection can handle dilute or concentrated solutions and relatively large or small volumes.There are four categories of detectors; specific detector that responds to a single chemical compound, selective detector responds to a range of compounds having a common physical or chemical property, a non-selective or universal detector responds to all compounds except the carrier gas and a non-destructive detector does not destroy the sample used. The most commonly used detectors are the flame ionization detector (FID) and the thermal conductivity detector (TCD). Recorder will then record the signal from the detector as the analyte elute from column. In this experiment, we are trying to determine the concept of gas chromatography retention time and resolution by using mixture of methyl

esters, methyl laurate, methyl myristate, methyl palmitate, methyl stearate and methyl linoleate. We are also trying to see the effects of column temperature and flow rate on the compounds’ separation

Fatty acid is a carboxylic acid having a long carbon chain. They exist as saturated or unsaturated form. Fatty acids bearing carbon-carbon double bonds are known as unsaturated fatty acid, while those that did not have double bond are saturated fatty acid. Most fatty acids contain an even number of carbon atoms in the hydrocarbon chain. The chain lengths may vary, but most natural fatty acids are have 4 to 22 carbons, with 18 carbons the most common one. Those with odd number of carbon mostly exist in bacteria and lower plants or animals. Fatty acids are the main constituents of oils and fats. Unsaturated fatty acids posseses one or more double bonds on the carbon chains. The double bonds can be broken by the addition of hydrogen atoms, converting it to saturated fatty acid, hence naming it “unsaturated”.

The most reactive sites in fatty acids are the carboxyl group and double bonds, as the carbon chain rarely show reactivity. Conversion of acids to esters and vice versa, also the exchange of ester groups are one of the most widely done in industry and chemistry. Fatty acids are converted to esters by reaction with excess alcohol, using acid catalyst or a lipase. In preparing methyl esters for gas chromatography analysis, boron trifluoride, sulfuric acid, or anhydrous hydrogen chloride in methanol are commonly used. The reaction is completed by refluxing. This involves the condensation of carboxyl group of an acid and hydroxyl group of alcohol, with water elimination.

The hydrocarbon chains of carboxylic acid plays important role in determining polarity. It is non-polar, thus counter balancing the polar acid functional group. In acids with only a few carbons, the acid functional group dominates, giving polar character to the molecule. However, in fatty acids, the non-polar hydrocarbon chain gives the molecule a non- polar character.

Margarine is a semi-solid emulsion mostly contains vegetable fats and water composition. The making of margarine involves emulsifying hydrogenated vegetable oils with skimmed milk, then chilling it to solidify the mixture. Vegetable and animal fats are similar compounds but different in melting points, due to the difference in carbon-carbon double bonds in the fatty acids components. The higher number of double bonds will result in lower melting

point. Margarines contain saturated and unsaturated fats. Vegetable fats contain around 7% to 86% saturated fatty acids margarines contain more saturated fat. In firmer margarines, the amount of saturated fats is higher. Unsaturated oils exist in two variants, mono- and poly-unsaturated fats. In margarine development, some of the unsaturated fats are converted into hydrogenated fats or trans fats, making them have higher melting point so that they are solid at room temperatures. In this experiment, we used margarine’s fatty acid to convert them to methyl esters, then test their separation in gas chromatography.

ANALYTICAL PROCEDURE :1. OPTIMIZATION OF FLOW RATE AND COLUMN TEMPERATURE

a) Instrument set-up

Injection port : Split (40:1)Injection port temperature : 250 oCColumn temperature : 210 oC

Carrier gas flowrate : 30 cm sec -1

Detector temperature : 250 oC

b) Effect of carier gas flow rate on isothermal GC separation of methyl esters.

0.4µL standard mixture injected isothermally at 210 oC at carrier gas flowrate of 30 cm sec-1 . then the flowrate increased to 50 cm sec -1 . the system was allowed to equilibratefor a few minutes and the standard mixture injected again. the same procedure repeated at flowrate of 70 cm sec -1. The most suitable flowrate was determined.

c) Effect of column temperature on the isothermal GC separation of methyl esters.

0.4µL standard mixture injected isothermally at 170 oC, followed by 190 oC , at the optimal carrier gas flowrate . the effect of column temperature on the separation, resolution, and the analysis time were evaluated.

d) Separation of methyl esters using column temperature programming.

The standard mixture injected at the optimal carrier gas flow rate using a linear temperature ramp from 100 oC to 290 oC at the optimal flow rate.

e) Identification of components in methyl esters mixture.

Each mehyl esters were injected individually to identify the various compounds in the standard mixture using the optimized GC conditions.

- Methyl laurate - Methyl myristate- Methyl palmitate- Methyl stearate- Methyl linolate

2. DETERMINATION OF FATTY ACID

a) Preparation of fatty acid methyl ester samples

i. 2g of margerine is weighed and the reading is recorded.

ii. Then, the sample is transferred into a 50 mL flask that equipped with air condenser

iii. 5 ml of 0.5 M methanolic solution is added into the flask and refluxed for 3-4 minutes.

iv. 15 ml of esterification reagent is added into the flask and refluxed again for another 3 minutes.

v. Next, the mixture is transferred into a separatory flask. 50 ml of saturated NaCl and 25ml of diethyl ether is added together in the separatory flask. The mixture is shaken vigorously for 2 minutes and the aqueous layer is discarded.

vi. Step (v) is repeated with another 25ml of saturated NaCl and the aqueous layer is discarded once again.

vii. The organic layer is transferred into a screw cap vial. Make sure that only the organic layer is injected into the GC as water can ruin the GC column.

b) Instrument set-up

Injection port : Split (40:1)Injection port temperature : 250oCColumn temperature : 100oC to 290oC at 40oC /minCarrier gas flow rate : 30 mL/sDetector temperature : 250oC

c) Quantitative analysis of FAME

i. 0.4µL of standard esters are injected to the column. The injection is repeated in order to get reproducible peak areas.

ii. 0.4µL of derivatized samples are injected to the column. The injection is repeated in order to get reproducible peak areas.

iii. The amount of each fatty acid in the sample were calculated by using the data from the standard esters.

DATA AND CALCULATIONS :Table 1: Analysis for temperature: 170 oC and flow rate: 70 cm/s

Injections

Peaks Retention time, (tR)

Width, (min)

Resolution time , RS,

peak 2 & 3 Peak 3&41 2 2.272 0.1111

2(4.540-2.272)0.2181+0.1111

= 13.78

2(10.072-4.540)0.6920+0.2181

= 12.16

3 4.540 0.21814 10.072 0.6920

2 2 2.270 0.11272(4.530-2.270)0.2227+0.1127

= 13.48

2(10.069-4.530)0.7117+0.2227

= 11.86

3 4.530 0.22274 10.069 0.7117

3 2 2.275 0.11242(4.545-2.275)0.2187+0.1123

= 13.72

2(10.051-4.545)0.7068+0.2187

= 11.90

3 4.545 0.21874 10.051 0.7068

Average Resolution time , RS, 13.78+13.48+13.723

= 13.66

12.16+11.86+11.903

= 11.97

Table 2: Analysis for temperature: 190 oC and flow rate: 70 cm/s

Injections

Peaks Retention time, (tR)

Width, (min)

Resolution time , RS,

peak 2 & 3 Peak 3&4

1 2 1.540 0.06572(2.561-1.540)0.0657+0.1366

= 10.09

2(4.853-2.561)0.2594+0.1366

= 11.58

3 2.561 0.13664 4.853 0.2594

2 2 1.542 0.06582(2.565-1.542)0.0658+0.1397

= 9.96

2(4.849-2.565)0.2530+0.1397

= 11.63

3 2.565 0.13974 4.849 0.2530

3 2 1.542 0.06652(2.565-1.542)0.0665+0.1387

= 9.97

2(4.863-2.565)0.2607+0.1387

= 11.51

3 2.565 0.13874 4.863 0.2607

Average Resolution time , RS, 10.09+9.96+9.973

= 10.00

11.58+11.63+11.513

= 11.57

Table 3: Analysis for temperature: 210 oC and flow rate: 30 cm/s

Injections

Peaks

Retention time, (tR)

Width, (min)

Resolution time , RS,peak 2 & 3 Peak 3&4

1 2 2.770 0.07022(3.915-2.770)0.0702+0.1336

= 11.24

2(6.306-3.915)0.2209-0.1336

= 13.49

3 3.915 0.13364 6.309 0.2209

2 2 2.766 0.07312(3.911-2.766)0.0731+0.1345

= 11.03

2(6.284-3.911)0.2142+0.1345

= 13.61

3 3.911 0.13454 6.248 0.2142

3 2 2.767 0.07222(3.911-2.767)0.0722+0.1293

= 11.35

2(6.291-3.911)0.2472+0.1293

= 12.64

3 3.911 0.12934 6.291 0.2472

Average Resolution time , RS, 11.24+11.03+11.35

3 = 11.21

13.49+13.61+1

2.643

= 13.25

Table 4 : Analysis for temperature: 210 oC and flow rate: 50 cm/s

Injections

Peaks

Retention time, (tR)

Width, (min)

Resolution time , RS,peak 2 & 3 Peak 3&4

1 2 1.663 0.05402(2.356-1.663)0.0965+0.0540

= 9.21

2(3.803-2.356)0.1782+0.0965

= 10.54

3 2.356 0.09654 3.803 0.1782

2 2 1.664 0.04922(2.359-1.664)0.0970+0.0492

= 9.51

2(3.806-2.359)0.1769+0.0970

= 10.57

3 2.359 0.09704 3.806 0.1769

3 2 1.666 0.04852(2.361-1.666)0.0485+0.0949

= 9.69

2(3.804-2.361)0.1869+0.0949

= 10.27

3 2.361 0.09494 3.804 0.1869

Average Resolution time , RS, 9.21+9.51+9.693

= 9.47

10.54+10.57+10.27

3 = 10.46

Table 5 : Analysis for temperature: 210 oC and flow rate: 70 cm/s

Injections

Peaks

Retention time, (tR)

Width, (min)

Resolution time , RS,peak 2 & 3 Peak 3&4

1 2 1.186 0.04432(1.687-1.186)0.0443+0.0780

= 8.19

2(2.729-1.687)0.0780+0.1629

= 8.65

3 1.687 0.07804 2.729 0.1629

2 2 1.188 0.04612(1.686-1.188)0.0461+0.0788

= 7.97

2(2.726-1.686) 0.1530+0.0788 = 8.97

3 1.686 0.07884 2.726 0.1530

3 2 1.189 0.04782(1.688-1.189)0.0804+0.0478

= 7.78

2(2.730-1.688) 0.0804+0.1610 = 8.63

3 1.688 0.08044 2.730 0.1610

Average Resolution time , RS, 8.19+7.97+7.78 8.65+8.97+8.6

3= 7.98

33

= 8.75

RESULTS:1. OPTIMIZATION OF FLOW RATE AND COLUMN TEMPERATURE Table 6 : The resolution for optimization of flow rate and temperature

TEMPERATURE(OC)

FLOWRATE(cm/s)

170 190 210

30 Rs 2,3 =11.21

Rs 3,4=13.25

50 Rs 2,3=9.47

Rs

3,4=10.46

70 Rs2,3=13.66

Rs 3,4=11.97

Rs 2,3=10.00

Rs 3,4=11.57

Rs 2,3= 7.98

Rs 3,4=8.75

Table 7 : The average retention time, tR for optimization of flow rate and temperature.

TFR

170 oC 190 oC 210 oC

30cm/s

tR,1=4.152

tR,2=3.912

tR,3=6.283

50cm/s

tR,1=1.664

tR,2=2.419

tR,3=3.804

70cm/s

tR,1=2.272

tR,2=4.538

tR,3=10.064

tR,1=1.541

tR,2=2.563

tR,3=4.855

tR,1=1.188

tR,2=1.687

tR,3=2.728

T = temperature RF = Flow rate

Table 8 : Analysis for temperature programming of standard mixture 2.

Injections

Peak

Retention

time, (tR)

Width,

(min)

Resolution time , RS,

Peak 2&3

Peak 3&4

Peak 4&5

Peak 5&6

1 2 1.988 0.0802

9.89 12.4 11.53 1.66

3 3.099 0.1444

4 5.393 0.2256

5 7.744 0.1822

6 7.983 0.1050

2 2 1.988 0.0784

10.10 12.31 10.26 3.0983 3.103 0.142

34 5.407 0.232

15 7.537 0.183

36 7.979 0.102

0Average Resolution time , RS, 10.00 12.36 10.90 2.37

Table 9 : Analysis for isothermal of standard mixture 2.

Injections

Peak

Retention

time, (tR)

Width,

(min)

Resolution time , RS,

Peak 2&3

Peak 3&4

Peak 4&5

Peak 5&6

1 2 1.188 0.0399

8.61 9.47 10.60 9.96

3 1.685 0.0756

4 2.713 0.1416

5 4.469 0.1897

6 4.825 0.2612

Table 10 : The Retention time, tR of standard mixture 2(temperature programming).

Peak Retention time, tR (min)1st Injection 2nd Injection Average

2 1.988 1.988 1.988+1.9882

= 1.9883 3.099 3.103 3.099+3.103

2 = 3.101

4 5.393 5.407 5.393+5.4072

= 5.4005 7.544 7.537 7.537+7.544

2 = 7.5405

6 7.983 7.979 7.983+7.9792

= 7.981

Table 11 : Comparison of Resolution,RS, between temperature programming and isothermal for standard mixture 2.

Peaks Resolution, RS,

Temperature programming

Isothermal

Peak 2&3 10.00 8.61Peak 3&4 12.36 9.47Peak 4&5 10.90 10.60Peak 5&6 10.90 9.96

Table 12 : Comparison of retention time between temperature programming and isothermal for standard mixture 2.

Peaks Average Retention time, (tR)

Temperature programming

Isothermal

Peak 2 1.988 1.188Peak 3 3.101 1.685Peak 4 5.400 2.713Peak 5 7.540 4.469Peak 6 7.981 4.825

Table 13: The Retention time, tR of standard individual sample.

Methyl Esters

Retention time, tR (min)1st Injection 2nd Injection Average

M.Laurate 1.190 1.185 1.1875M.Myristate 1.685 1.690 1.6875M.Palmitate 2.722 2.715 2.7185M.Linolate 4.458 4.435 4.4465M.Stearate 4.838 4.837 4.8375

Table 14 : Comparison of retention time for individual samples with standard mixture 1.

Methyl Esters Retention time, tR (min)Average tR for individual

sampleAverage tR for std

mixture 1M.Laurate 1.1875 1.1877

M.Myristate 1.6875 1.687

M.Palmitate 2.7185 2.7283

Table 15 : Comparison of standard mixture 2 with individual sample in terms of retention time.

Methyl Esters Retention time, tR (min)

Average tR for individual sample

Average tR for std mixture 2

M.Laurate 1.1875 1.188M.Myristate 1.6875 1.685M.Palmitate 2.7185 2.713M.Linolate 4.4465 4.469M.Stearate 4.8375 4.825

2. DETERMINATION OF FATTY ACIDStandard mixture 2 Response factor,RF = peak area

sample amount

Table 16 : Response factor,RF of fatty acid.

Methyl Esters Response factor,RF

1st Injection 2nd Injection Average

M.Laurate1141.59460

250= 4.5664

1067.89673250

= 4.27164.4190

M.Myristate769.81403

220= 3.4992

711.45239220

= 3.23393.3666

M.Palmitate96.87212

1010= 0.0959

91.980031010

= 0.09110.0935

M.Linolate35.14044

781= 0.04499

33.44002781

= 0.04280.0439

M.Stearate20.71200

350= 0.0592

17.95727350

= 0.05130.0553

Sample 1 : FATTY ACID

Concentration of Methyl Esters = peak area response factor

Table 17 : Average concentration of fatty acid for sample 1Methyl Esters Concentration (ppm)

1st Injection 2nd Injection 3rd Injection AverageM.Laurate 27.75001

4.4190= 6.2797

29.625234.4190

= 6.7041

27.183564.4190

= 6.15156.3784

M.Myristate 16.415733.3666

= 4.8761

13.979773.3666

= 4.1525

11.448843.3666

= 3.40074.1431

M.Palmitate 224.153850.0935

= 2397.3674

228.100720.0935

= 2439.5799

211.086750.0935

= 2257.6123 2364.8532

M.Linolate 14.890660.0439

= 339.1950

8.471540.0439

= 192.9736

1.11003e-10.0439

= 2.5285266.0843

M.Stearate 377.139710.0553

= 6819.8863

239.021150.0553

= 4322.2631

221.572010.0553

= 4006.72714164.4951

Sample 2 : FATTY ACID

Table 18 : Average concentration of fatty acid for sample 2.

Methyl Esters Concentration (ppm)1st Injection 2nd Injection 3rd Injection Average

M.Laurate 55.561294.4190

= 12.5733

53.828134.4190

= 12.1811

53.692344.4190

= 12.1503

12.3016

M.Myristate 22.254843.3666

= 6.6105

28.235563.3666

= 8.3870

23.026733.3666

= 6.8398

6.7251

M.Palmitate 265.552340.0935

= 2840.1320

301.042790.0935

= 3219.7090

304.281590.0935

= 3254.3486

3237.0288

M.Linolate 2.27972e-10.0439

= 5.1930

8.377830.0439

= 190.8390

22.297790.0439

= 507.9223

234.6514

M.Stearate 285.074830.0553

= 5155.0602

293.880650.0553

= 5314.2975

314.115690.0553

= 5680.2114

5383.1897

Sample 3 : FATTY ACID

Table 19 : Average concentration of fatty acid for sample 3.

Methyl Esters Concentration (ppm)1st Injection 2nd Injection 3rd Injection Average

M.Laurate 11.734614.4190

= 2.6555

9.032264.4190

= 2.0440

11.953074.4190

= 2.7049

2.6802

M.Myristate 14.365073.3666

= 4.2669

3.996463.3666

= 1.1871

14.176613.3666

= 4.2110

4.2389

M.Palmitate 60.380260.0935

= 645.7782

63.395730.0935

= 678.0292

76.865930.0935

= 822.0955

661.9037

M.Linolate 30.024460.0439

= 683.9285

28.723280.0439

= 654.2889

1.748570.0439

= 39.8308

664.6087

M.Stearate 54.994150.0553

= 1037.6255

59.571370.0553

= 1123.9881

3.217980.0553

= 60.7166

1080.8068

Table 20 : Average concentration for Sample 1, Sample 2 and Sample 3.

Methyl Esters Average Concentration (ppm)Sample 1 Sample 2 Sample 3

M.Laurate 6.3784 12.3016 2.6802M.Myristate 4.1431 6.7251 4.2389M.Palmitate 2364.8532 3237.0288 661.9037M.Linolate 266.0843 234.6514 664.6087M.Stearate 4164.4951 5383.1897 1080.8068

DISCUSSION :Gas chromatography (GC), is a common type

of chromatography used in analytical chemistry for separating and analyzing compounds that can be vaporized without decomposition. Typical uses of GC include testing the purity of a particular substance, or separating the different components of a mixture. In some situations, GC may help in identifying a compound. In preparative chromatography, GC can be used to prepare pure compounds from a mixture. In gas

chromatography, the mobile phase is a carrier gas, usually an inert gas such as helium or an unreactive gas such as nitrogen. The stationary phase is a microscopic layer of liquid or polymer on an inert solid support, inside a piece of glass or metal tubing called a column. The instrument used to perform gas chromatography is called a gas chromatograph. The gaseous compounds being analyzed interact with the walls of the column, which is coated with a stationary phase. This causes each compound to elute at a different time, known as the retention time of the compound. The comparison of retention times is what gives GC its analytical usefulness.

In this experiment, type of gas chromatogram used is Gas chromatography(Agilent Technologies 6890N) equip with flame ionization detector(FID) ABD 30m x 250μm x 0.25μm HP5-MS capillary column. Flame ionization detector(FID) consist of a hydrogen / air flame and a collector plate. The effluent from the GC column passes through the flame, which breaks down organic molecules and produces ions. The ions are collected on a biased electrode and produce an electric signal. The column used is capillary column.

The two major factors that control elution time in gas chromatography is flow rate and column temperature. Although solute elution rate increases linearly with flow rate, elution rate increases approximately exponentially with column temperature and, thus, is far more effective in eluting strongly retained solutes. Firstly we will, analyse the effect of carrier gas flow rate on isothermal GC separation of methyl esters. Three difference flow rate which is 30 cm/s , 50 cm/s and 70cm/s been analyse with temperature 210oC. Based on resolution flow rate of 30 cm/s , 50 cm/s and 70cm/s , the best resolution is resolution flow rate at 70 cm/s because for peak2,3 the resolution is only 7.98and for peak3,4 is only 8.75 it is the lowest resolution compare to flow rate at 30 cm/s and 50 cm/s which is 9.47 and 10.46 for flow rate at 30 cm/s and, 11.21 and13.25 for flow rate at 50 cm/s. Based on retention time, the most shortest analysis time also for flow rate at 70cm/s with retention time for

first peak 1.188min, second peak with 1.687min and 2.728min for the last peak. Compare to other retention time, flow rate at 30 cm/s the retention time is 4.152,3.912 and 6.283 for first, second and third peak respectively and flow rate at 50 cm/s the retention time is 1.664, 2.419 and 3.804 for first, second and third peak respectively. We can make a conclusion here that the shortest retention time is when at flow rate 70cm/s and the best flow rate for gradient elution of chromatography.

The second factors that control elution time in gas chromatography are temperature. We will analyse the effect of carrier gas on isothermal GC separation of methyl esters. Three difference temperature which is 170 oC, 190 oC and 210oC are been analyse at flow rate 70cm/s. The best temperature based on resolution, is 210oC because the resolution for temperature 210oC is only 7.98 and 8.75 which is the shortest resolution compare to resolution for temperature 170 oC which is 13.66 and 11.97 and resolution for temperature 190 oC is 10.00 and 11.57. Based on retention time, the retention time for temperature 210 oC is the shortest compare to other. The retention time for temperature at 210 oC are 1.188, 1.687 and 2.728 for first, second and last peak respectively. For temperature 170 oC the retention time is 2.272, 4.538 and 10.064, meanwhile for temperature at 109 oC the retention time is 1.541, 2.563 and 4.855 for first, second and last peak respectively. We can conclude that the best temperature for elution gradient is 210 oC by analysis of resolution and retention time at difference temperature. Based on the chromatograms of standard mixture, the optimum condition of this experiment achieved at temperature 210°C at flow rate of 70 cm3/sec. The injection of sample at temperature 210°C and flow rate of 70 cm3/sec gives the lowest resolution value compared to other temperature and flow rate.

Before this we used method isothermal in order to know the effect of carrier gas flow rate and column temperature for separation of methyl esters. Now we going to used column temperature programming for separation of methyl esters. Temperature programming is a

chromatography development technique used largely in gas chromatography to accelerate the elution rate of late peaks that, otherwise, would take a very long time to elute. It is achieved by continuously raising the column temperature, usually as a linear function of time, during the elution process. Column temperature is increased either continuously or in steps as the analysis proceeds. But practically, we did not get the result as we expected, based on Table 11 and Table 12 we can see that the best retention time is when using isothermal technique not temperature programming. The analysis time for temperature programming is longer than isothermal technique, to elute all samples, isothermal technique only take 4.469min but for temperature programming it take 7.981min. In term of resolution, the resolution for isothermal technique is better than resolution for temperature programming .

In this experiment, gas chromatography was used to identify the various components in the standard mixture of methyl ester using the optimized GC conditions at temperature 210°C and at flow rate of 70 cm3/sec. For standard mixture 1, from the chromatogram we can see 4 peak appear first peak is solvent peak and to know the other three peaks, we must compare the retention time between individual sample with standard mixture 1. Based on Table 14 we can predict the component that will be eluted first is methyl laurate, follow by methyl myristate and lastly methyl palmitate. For standard mixture 2, we also compare the retention time between standard mixture and individual sample. Based on Table 15 we can see that the order of elution, component that will be eluted first is methyl laurate, follow by methyl myristate, then methyl palmitate, methyl linolate and lastly, methyl stearate.

For determination of fatty acid using gas chromatography(GC), the order of elution of compound same with order of elution for standard mixture 2 when using temperature programming. The experiment was done using margarine to obtain fatty acid. Since fatty acid was not volatile to be analyzed in gas chromatography, the sample was modified

into methyl ester. Short fatty acids, having fewer than six carbon chains tend to be volatile. The longer chained are much difficult to be tested with gas chromatography due to little volatility, plus the highly polar compounds tend to form hydrogen bonds, leading to adsorption problems. This leads to the esterification process of the acid, thus reducing their polarity. Esterification reaction involves the condensation of the carboxyl group of an acid and the hydroxyl group of an alcohol, resulting in a type of fatty acid ester named fatty acid methyl esters (FAME). Base-catalyzed methanolysis proceeds faster under mild temperature conditions compared to acid-catalyzed reactions. BF3 is a commonly used acid catalyst for methylation and methanolysis. However, it is hazardous and has a limited shelf life. Another effective acid catalyst for FAME synthesis is H2SO4, but another downfall is it has a very corrosive property and must be handled with care. 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 along with the water. Methyl esters offer excellent stability, and provide quick and quantitative samples for gas chromatography analysis Moisture must be removed prior to analysis, as to prevent any damage towards the GC. The sample was also neutralized from any form of acidity by adding the NaCl solution.

To calculate the concentration of the component we must calculate the response factor first. The result of response factor tabulated in Table 16. After that, we can calculate the concentration of each component for sample 1, sample 2 and sample3.Based on Table 20, we can see that methyl stearate has the highest concentration among the other, second highest in term of concentration is methyl palmitate, than methyl linolate, methyl myristate and lastly methyl laurate.

CONCLUSION :As a conclusion, the separation of analytes mixture using gas

chromatography affected by the column temperature and carrier gas flow rate. Compounds separated better at temperature, 210oC and flow rate,

70cm/s based on the value of resolution calculated. The elution order of standard mixture, first is methyl laurate, follow by methyl myristate, then methyl palmitate, methyl linolate and lastly, methyl stearate.

The derivatization procedure is routinely used for fat analysis in which nonvolatile fatty acids are chemically converted to the corresponding volatile methyl esters (FAME) so that it can be analyzed by gas chromatography.From the experiment, we can conclude that of methyl stearate was the largest compund present in margarine, and methyl laurate being the least.

REFERENCES :1. Holler, Skoog, and Crouch, Principles of Instrument bAnalysis 6th

Edition(2007)2. 124-10-7 CAS MSDS (METHYL MYRISTATE) Melting Point Boiling Point

Density CAS Chemical Properties.3. https://www.google.com.my/webhp?

hl=en&tab=ww#hl=en&q=ester+Methyl+Myristate+properties4. https://www.google.com.my/webhp?

hl=en&tab=ww#hl=en&q=ester+Myristate+wiki5. http://en.wikipedia.org/wiki/Prefix 6. http://www.telecomabc.com/p/prefix.html 7. https://www.google.com.my/webhp?hl=en&tab=ww#hl=en&q=C14H28O2

APPENDIX :

Conc. of Methyl Esters = peak area response factor

Response factor,RF = peak area sample

amount Resolution, Rs = 2[(tR)A – (tR)B] WA + WB

Formula of plate height, H = L

N