Lab 1 Chm 510 Complete 2011

GAS CHROMATOGRAPHY-FID Optimization of flow rate and column temperature

Title: ______________________________________________________________________________ Optimization of flow rate and column temperature

Objective: ______________________________________________________________________________ To optimize flow rate and column temperature to determine four type standard mixture of methyl;methyl laurate (0.20 mg/ml), methyl myristate (0.20 mg/ml) and methyl palmitate (1.0mg/ml).

Abstract: ______________________________________________________________________________

The factors which contribute to the efficient separation of mixture of methyl esters are examined. These factors included the affect of carrier gas flow rate on the isothermal and temperature programming GC separation of methyl esters. The elution rate of a compound depends on volatility of compound, column temperature, carrier gas flow rate and length of the column of the particular GC system. This experiment is examined gas chromatography, including the concepts of retention time and resolution using a mixture of methyl esters which were methyl 1

GAS CHROMATOGRAPHY-FID Optimization of flow rate and column temperature

laurate (0.20 mg/ml), methyl myristate (0.20 mg/ml) and methyl palmitate (1.0mg/ml).At the end of the experiment the resolution (RS) is measured to know of how well species are separated.

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GAS CHROMATOGRAPHY-FID Optimization of flow rate and column temperature

1.0

Introduction:

______________________________________________________________________________ 1.1

Instrument Background

Gas chromatography is unique and versatile technique. In its initial stages of development it was applied to the analysis of gases and vapours from very volatile components. 1 Chromatography is a common name for techniques based on the partition of the molecules to be analyzed between a mobile and a stationary phase. Separation is the result of different partitions of molecules between the two phases. Because the best separation of any solutes can be obtained under equilibrium conditions, analytical chemists prefer to use chromatographic systems that are as near to the equilibrium state as possible. However, in the case of preparative chromatography, where the main objective is not the optimal separation of solutes but the maximum yield of one or more solutes at a given purity, the situation is entirely different. Preparation chromatographic separations are generally not equilibrium processes. The high sensitivity, selectivity, and reproducibility of chromatographic methods have been extensively exploited in food and nutrition science and technology. Factors that affect GC separations 

Efficient separation of compounds in GC is dependent on the compounds travelling through the column at different rates. The rate at which a compound travels through a particular GC system depends on the factors listed below:

1

Robert L.Grob and Eugene F.Barry, Modern Practice of Gas Chromatography, Fourth Edition, 2004. (Page 37)

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GAS CHROMATOGRAPHY-FID Optimization of flow rate and column temperature



Volatility of compound: Low boiling (volatile) components will travel faster through the column than will high boiling components.



Polarity of compounds: Polar compounds will move more slowly, especially if the column is polarity.



Column temperature: Raising the column temperature speeds up all the compounds in a mixture.



Column packing polarity: Usually, all compounds will move slower on polar columns, but polar compounds will show a larger effect.



Flow rate of the gas through the column: Speeding up the carrier gas flow increases the speed with which all compounds move through the column.



Length of the column: The longer the column, the longer it will take all compounds to elute. Longer columns are employed to obtain better separation.

Generally the number one factor to consider in separation of compounds on the GCs in the teaching labs is the boiling points of the different components. Differences in polarity of the compounds are only important if we are separating a mixture of compounds which have widely different polarities. Gas chromatography-FID (GC/FID), the FID or flame ionization detector detects analytes by measuring an electrical current generated by electrons from burning carbon particles in the sample. The flame ionization detector (FID) is a non-selective detector used in conjunction with gas chromatography. Because it is non-selective, there is a potential for many non-target compounds present in samples to interfere with this analysis and for poor resolution especially in complex samples. The FID works by directing the gas phase output from the column into a 4

GAS CHROMATOGRAPHY-FID Optimization of flow rate and column temperature

hydrogen flame. A voltage of 100-200V is applied between the flame and an electrode located away from the flame. The increased current due to electrons emitted by burning carbon particles is then measured. Although the signal current is very small (the ionization efficiency is only 0.0015%) the noise level is also very small (<10-13 amp) and with a well-optimized system, sensitivities of 5 x 10-12 g/ml for n-heptane at a signal/noise ratio of 2 can be easily realized. Except for a very few organic compounds (e.g. carbon monoxide, etc.) the FID detects all carbon containing compounds. The detector also has an extremely wide linear dynamic range that extends over, at least five orders of magnitude with a response index between 0.98-1.02. 2

In order to detect these ions, two electrodes are used to provide a potential difference.

The positive electrode doubles as the nozzle head where the flame is produced. The other, negative electrode is positioned above the flame. When first designed, the negative electrode was either tear-drop shaped or angular piece of platinum. Today, the design has been modified into a tubular electrode, commonly referred to as a collector plate. The ions thus are attracted to the collector plate and upon hitting the plate, induce a current. This current is measured with a highimpedance picoammeter and fed into an integrator. How the final data is displayed is based on the computer and software. In general, a graph is displayed that has time on the x-axis and total ion on the y-axis. The current measured corresponds roughly to the proportion of reduced carbon atoms in the flame. Specifically how the ions are produced is not necessarily understood, but the response of the detector is determined by the number of carbon atoms (ions) hitting the detector per unit time. This makes the detector sensitive to the mass rather than the concentration, which is useful 2

Scott, R. P. W., 1957, Vapour Phase Chromatography, Ed. D. H. Desty (London: Butterworths).(Page 131)

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GAS CHROMATOGRAPHY-FID Optimization of flow rate and column temperature

because the response of the detector is not greatly affected by changes in the carrier gas flow rate. FID Schematic The design of the flame ionization detector varies from manufacturer to manufacturer, but the principles are the same. Most commonly, the FID is attached to a gas chromatography system. The eluent exits the GC column (A) and enters the FID detector’s oven (B). The oven is needed to make sure that as soon as the eluent exits the column, it does not come out of the gaseous phase and deposit on the interface between the column and FID. This deposition would result in loss of effluent and errors in detection. As the eluent travels up the FID, it is first mixed with the hydrogen fuel (C) and then with the oxidant (D). The effluent/fuel/oxidant mixture continues to travel up to the nozzle head where a positive bias voltage exists (E). This positive bias helps to repel the reduced carbon ions created by the flame (F) pyrolyzing the eluent. The ions are repelled up toward the collector plates (G) which are connected to a very sensitive ammeter, which detects the ions hitting the plates, and then feed that signal (H) to an amplifier, integrator, and display system. The products of the flame are finally vented out of the detector through the exhaust port (J). 3(Shown in figure 4 at the Appendix A)

1.2

3

SAMPLES

http://en.wikipedia.org/wiki/Flame_ionization_detector

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GAS CHROMATOGRAPHY-FID Optimization of flow rate and column temperature

Fatty Acids are aliphatic carboxylic acid with varying hydrocarbon lengths at one end of the chain joined to terminal carboxyl (-COOH) group at the other end. The general formula is R(CH2)n-COOH. Fatty acids are predominantly unbranched and those with even numbers of carbon atoms between 12 and 22 carbons long react with glycerol to form lipids (fat-soluble components of living cells) in plants, animals, and microorganisms. Fatty acids all have common names respectively lilk lauric (C12), MyrIstic (C14), palmitic (C16), stearic (C18), oleic (C18, unsaturated), and linoleic (C18, polyunsaturated) acids. The saturated fatty acids have no solid bonds, while oleic acid is an unsaturated fatty acid has one solid bond (also described as olefinic) and polyunsaturated fatty acids like linolenic acid contain two or more solid bonds. Lauric acid (also called Dodecanoic acid) is the main acid in coconut oil (45 - 50 percent) and palm kernel oil (45 - 55 percent). Nutmeg butter is rich in myristic acid (also called Tetradecanoic acid) which constitutes 60-75 percent of the fatty-acid content. Palmitic acid (also called Hexadecylic acid) constitutes between 20 and 30 percent of most animal fats and is also an important constituent of most vegetable fats (35 - 45 percent of palm oil). Stearic acid (also called Octadecanoic Acid) is nature's most common long-chain fatty acids, derived from animal and vegetable fats. It is widely used as a lubricant and as an additive in industrial preparations. It is used in the manufacture of metallic stearates, pharmaceuticals, soaps, cosmetics, and food packaging. It is also used as a softener, accelerator activator and dispersing agent in rubbers. Oleic acid (systematic chemical name is cis-octadec-9-enoic acid) is the most abundant of the unsaturated fatty acids in nature.

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GAS CHROMATOGRAPHY-FID Optimization of flow rate and column temperature

2.0

Research Methodology:

______________________________________________________________________________ 2.1

Apparatus: Glass Vial.

2.2

Reagents and Solutions/Samples: 2.2.1

Individual methyl esters compounds: Methyl laurate,methyl myristate,methyl

palmitate and methyl stearate. 2.2.2

Standard mixture of methyl laurate (0.20 mg/ml), methyl myristate (0.20 mg/ml),

methyl palmitate (1.0mg/ml) and methyl stearate (0.7 mg/ml). 2.3

Instrument: Gas chromatography (Agilent Technologies 6890N) equipped with flame ionization detector (FID) and 30m×250 mm× 0.25mm HP5-MS capillary column. (Shown figure 2 at Appendix A)

2.4

Analytical Procedures:

1) Instrument Set-up: Injection port

: Split (20:1)

Injection port temperature

: 250 °C

Oven temperature

: 100 °C

Column temperature

: 20cm3/s

Detector temperature

: 250 °C 8

GAS CHROMATOGRAPHY-FID Optimization of flow rate and column temperature

2) Effect of carrier gas flow rate on isothermal GC separation of methyl esters. 0.4 µL standard mixtures were injected isothermally at 170 °C at carrier gas flow rate of 20 ml/s. Then, the flow rate was increased to 50cm3/sec. Before injected the standard mixture again, the system was allowed to equilibrate.

3) Effect of column temperature on isothermal GC separation of methyl esters. 0.4 µL standard mixtures were injected isothermally at 170 °C, 190 °C and 210 °C at the optimal carrier gas flow rate. The effect of column temperature on the separation, resolution and analysis time was investigated.

4) Separation of methyl esters using column temperature programming. Standard mixture at the optimal carrier gas flow rate was injected using a linear temperature ramp from 100 °C to 290 °C at 40 °C/min.

5) Identification of components in methyl esters mixture. Each methyl esters was injected individually to identify the various compounds in the standard mixture using the optimized GC conditions.

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GAS CHROMATOGRAPHY-FID Optimization of flow rate and column temperature

3.0

Results:

______________________________________________________________________________ Refer to Appendix B 3.1

Standard mixture 1 (GC1:

Table 1: Flow Rate 30 cm3/sec and Column Temperature 170°C Rs2, Rs1,peak Repeatability

Peak

Retention Time, tR

Width

Average

Peak

Average

1&2 2&3

1

2

1

4.206

0.1311

2

7.383

0.2319

3

14.553

0.4922

1

4.200

0.1264

2

7.386

0.2268

3

14.599

0.4545

17.504

18.147

17.773 18.041

19.661 21.174

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GAS CHROMATOGRAPHY-FID Optimization of flow rate and column temperature

Table 2: Flow Rate 50 cm3/sec and Column Temperature 170°C

Repeatability

Peak

Retention Time, tR

Width

Rs1,peak

Average

1&2

1

2

3

1

2.159

0.1413

2

3.609

0.2083

3

6.872

0.3510

1

2.127

0.1264

2

3.572

0.2199

3

6.841

0.3623

1

2.134

0.1268

2

3.577

0.2087

3

6.860

0.3698

8.602

Average

2&3

8.295

8.345

Rs2,peak

11.668

8.414

11.230

11.416

11.350

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GAS CHROMATOGRAPHY-FID Optimization of flow rate and column temperature

Table 3: Flow Rate 70 cm3/sec and Column Temperature 170°C

Rs1,peak Repeatability

Peak

Retention Time, tR

Average

Width

Rs2,peak

Average

1&2 2&3

1

2

1

1.587

0.1750

2

2.598

0.2657

3

4.886

0.3739

1

1.592

0.1834

2

2.613

0.2856

3

4.911

0.4205

7.154

4.588

4.471

6.832 6.509

4.354

Table 4: Flow Rate 70 cm3/sec and Column Temperature 190°C

Repeatability

Peak

Retention Time, tR

Width

Rs1,peak

Average

1&2

1

2

1

1.231

0.1110

2

1.703

0.1444

3

2.672

0.1914

1

1.239

0.0964

2

1.708

0.1412

3

2.669

0.1968

Rs2,peak 2&3

3.692

5.771

3.82 3.948

Average

5.729 5.686

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GAS CHROMATOGRAPHY-FID Optimization of flow rate and column temperature

Table 5: Flow Rate 70 cm3/sec and Column Temperature 210°C

Repeatability

Peak

Retention Time, tR

Width

Rs1,peak

Average

Rs2,peak

1&2

1

2

1

1.058

0.0872

2

1.291

0.1218

3

1.727

0.1608

1

1.071

0.0908

2

1.297

0.1240

3

1.731

0.1726

Average

2&3

2.230

3.086

2.167

3.007 2.927

2.104

Table 6: Comparison of Resolution in Different Flow Rate and Temperature Flow Rate and Temperature

Resolution, Rs1

Resolution, Rs2

17.773

19.661

8.414

11.416

4.471

6.832

3.820

5.729

2.167

3.007

Temp 170 °C 3

Flow rate 30 cm /sec Temp 170°C Flow rate 50 cm3/sec Temp 170°C Flow rate 70 cm3/sec Temp 190°C Flow rate70 cm3/sec Temp 210°C Flow rate 70 cm3/sec

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GAS CHROMATOGRAPHY-FID Optimization of flow rate and column temperature

3.2

Individual methyl ester

Table 7: Retention time for individual methyl ester at temperature 210°C, flow rate 70 cm3/sec Methyl ester

tR First injection

tR Second Injection

Average tR

Laurate

1.039

1.007

1.023

Palmitate

1.596

1.612

1.604

Myristate

1.186

1.204

1.195

Table 8: Flow Rate 70 cm3/sec and Column Temperature 210°C

Repeatability

1

2

Peak

Retention Time, tR

Average Retention Time, tR

1

1.058

1.065

2

1.291

1.291

3

1.727

1.729

1

1.071

2

1.291

3

1.731

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GAS CHROMATOGRAPHY-FID Optimization of flow rate and column temperature

Table 9: Comparison of Average Retention Time of Unknown Peak and Individual Standards at temperature 210°C, flow rate 70 cm3/sec Peak Number

Standard Mixture,tR

2

1.065

3

1.291

4

1.729

Methyl Laurate,tR

Methyl Myristate,tR

Methyl Palmitate,tR

1.023

1.195

1.604

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GAS CHROMATOGRAPHY-FID Optimization of flow rate and column temperature

4.0

Discussion:

______________________________________________________________________________

In this experiment, gas chromatography was used to identify the various components in the standard mixture of methyl ester using the optimized GC conditions. The standard methyl ester contains three individual components; methyl laurate; methyl myristate and methyl palmitate. The instrument set to use split injection because only small amount of sample introduced into the column. This type of injection will produced more sharp and narrow peak compared to splitless injection. For optimum column efficiency, the sample for the injection should not too large and introduced onto the column as a plug of vapor because slow injection of large sample will cause band broadening and loss resolution.

The effects of carrier gas flow rate and column temperature on gas chromatography separation of compounds mixture were investigated in this experiment. The optimum condition for this experiment is determined by injection of sample at different temperature and flow rates. The standard mixture injected at flow rate of 30, 50 and 70 cm3/sec and temperature of 170, 190 and 210°C in order to determine the suitable flow rate and temperature for the separation. The resolution value at different temperature and flow rate is compared in order to determine the best separation. Based on the chromatograms of standard mixture, the optimum condition of this experiment achieved at temperature 210°C at flow rate of 70cm3/sec. The injection of sample at temperature 210°C and flow rate of 70cm3/sec gives the lowest resolution value compared to other temperature and flow rate. The ideal resolution value for chromatography separation is

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GAS CHROMATOGRAPHY-FID Optimization of flow rate and column temperature

around 1 to 20. If the resolution value between two peaks calculated is greater than 20, the separation takes longer times to complete.

Then the individual methyl esters are identifying by comparing the chromatograms of the individual compounds and the standard mixture. The average retention times of individual peaks of methyl laurate, methyl myristate and methyl palmitate at optimum GC condition is 1.023, 1.195and 1.604. The average retention times of the standard mixture at the same condition is 1.065, 1.291 and 1.729. So, the individual components of methyl ester can be identified. Based on the comparison of retention times of standard mixture and individual components of methyl esters, methyl laurate eluted first followed by methyl myristate and methyl palmitate.

5.0

Conclusion:

______________________________________________________________________________ This experiment we can conclude that, the efficient separation of the mixtures of methyl ester using gas chromatography-FID is affected by changing the column temperature and carrier gas flow rate. The three compounds are separated better at high temperature and flow rate based on the value of resolution have been calculated. Therefore, the objective in the experiment is achieved.

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GAS CHROMATOGRAPHY-FID Optimization of flow rate and column temperature

6.0 References: ______________________________________________________________________________ 6.1

Internet References:

6.1.1 Wikipedia Fatty Acid -en.wikipedia.org/wiki/Fatty Acid (Retrieved on 19 Jan 2011) 6.1.2 Wikipedia Methyl Ester-en.wikipedia.org/wiki/Methyl Ester (Retrieved on 19 Jan 2011) 6.1.3 http://en.wikipedia.org/wiki/Flame_ionization_detector (Retrieved on 20 Jan 2011)

6.2

Book References:

6.2.1

Nor’Ashikin Saim and Ruziyati Tajudin, Analytical Separation Method, Laboratory Guide. (Page 1-3)

6.2.2

Robert L.Grob and Eugene F.Barry, Modern Practice of Gas Chromatography, Fourth Edition, 2004. (Page 37)

6.2.3

Skoog, West, Holler and Crough, 2000, Analytical Chemistry: An Introduction, 7th Edition, 2000, Brook/Cole Thomson Learning. (Page 974)

6.2.4

Scott, R. P. W., 1957, Vapour Phase Chromatography, Ed. D. H. Desty (London: Butterworths).(Page131)

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GAS CHROMATOGRAPHY-FID Optimization of flow rate and column temperature

7.0

Appendix:

______________________________________________________________________________ Appendices A 7.1.1

Instruments and Apparatus

Figure Name

Figure

Figure 1: Schematic Diagram Of Chromatography FID

Figure 2:Gas Chromatography FID

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GAS CHROMATOGRAPHY-FID Optimization of flow rate and column temperature

Figure 3: Flame Ionization Detector

7.1.2

Samples

Figure Name

Figure

Figure 5: Methyl Laurate Structure

Figure 6:Methyl Myristate Structure

Figure 7:Methyl Palmitate Structure

Figure 7: Methyl Laurate Structure Appendices B (Experimental Results)

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