Lab 1 Chm 510 Complete 2011

GAS CHROMATOGRAPHY-FIDOptimization 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

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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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1.0 Introduction:

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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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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

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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.

2In 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 high-

impedance 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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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 SAMPLES

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

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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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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

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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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3.0 Results:

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Refer to Appendix B

3.1 Standard mixture 1 (GC1:

Table 1: Flow Rate 30 cm3/sec and Column Temperature 170°C

Repeatability Peak Retention Time, tR WidthRs1,peak

1&2Average

Rs2,

Peak

2&3

Averag

e

1

1 4.206 0.1311

17.504

17.773

18.147

19.661

2 7.383 0.2319

3 14.553 0.4922

2

1 4.200 0.1264

18.041 21.1742 7.386 0.2268

3 14.599 0.4545

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Repeatabilit

y

Peak Retention Time, tR Width Rs1,peak

1&2

Average Rs2,peak

2&3

Average

1

1 2.159 0.1413

8.295

8.414

11.668

11.416

2 3.609 0.2083

3 6.872 0.3510

2

1 2.127 0.1264

8.345 11.2302 3.572 0.2199

3 6.841 0.3623

3

1 2.134 0.1268

8.602 11.3502 3.577 0.2087

3 6.860 0.3698

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Repeatability Peak Retention Time, tR WidthRs1,peak

1&2Average Rs2,peak

2&3

Average

1

1 1.587 0.1750

4.588

4.471

7.154

6.832

2 2.598 0.2657

3 4.886 0.3739

2

1 1.592 0.1834

4.354 6.5092 2.613 0.2856

3 4.911 0.4205


Repeatability Peak Retention Time, tR Width Rs1,peak

1&2

Average Rs2,peak

2&3

Average

1

1 1.231 0.1110

3.692

3.82

5.771

5.729

2 1.703 0.1444

3 2.672 0.1914

2

1 1.239 0.0964

3.948 5.6862 1.708 0.1412

3 2.669 0.1968


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Repeatabilit

y

Peak Retention Time, tR Width Rs1,peak

1&2

Average Rs2,peak

2&3

Average

1

1 1.058 0.0872

2.230

2.167

3.086

3.007

2 1.291 0.1218

3 1.727 0.1608

2

1 1.071 0.0908

2.104 2.9272 1.297 0.1240

3 1.731 0.1726

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

Flow Rate and Temperature Resolution, Rs1 Resolution, Rs2

Temp 170 °C

Flow rate 30 cm3/sec17.773 19.661

Temp 170°C

Flow rate 50 cm3/sec8.414 11.416

Temp 170°C


Temp 190°C

Flow rate70 cm3/sec3.820 5.729

Temp 210°C


3.2 Individual methyl ester

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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


Repeatability Peak Retention Time, tR Average Retention Time, tR

1

1 1.058 1.065

2 1.291 1.291

3 1.727 1.729

21 1.071

2 1.291

3 1.731

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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 Methyl Laurate,tR Methyl Myristate,tR Methyl Palmitate,tR

2 1.065

1.023 1.195 1.6043 1.291

4 1.729

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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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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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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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http://en.wikipedia.org/wiki/Flame_ionization_detector%20(Retrieved


7.0 Appendix:

______________________________________________________________________________

Appendices A

7.1.1 Instruments and Apparatus

7.1.2 Samples

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Figure Name Figure

Figure 1:

Schematic

Diagram Of

Chromatography

FID

Figure 2:Gas

Chromatography

FID

Figure 3:

Flame Ionization

Detector

Figure Name Figure

Figure 5: Methyl

Laurate Structure

Figure 6:Methyl

Myristate Structure

Figure 7:Methyl

Palmitate Structure

Figure 7: Methyl

Laurate Structure

http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=61689%7CFLUKA&N5=SEARCH_CONCAT_PNO%7CBRAND_KEY&F=SPECi

http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=61689%7CFLUKA&N5=SEARCH_CONCAT_PNO%7CBRAND_KEY&F=SPECi


Appendices B (Experimental Results)

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