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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).
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
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.
Separation is the result of different partitions of molecules between the two phases. The high sensitivity. 2004. However. selectivity. In its initial stages of development it was applied to the analysis of gases and vapours from very volatile components.GAS CHROMATOGRAPHY-FID Optimization of flow rate and column temperature 1. in the case of preparative chromatography. Preparation chromatographic separations are generally not equilibrium processes. analytical chemists prefer to use chromatographic systems that are as near to the equilibrium state as possible. Modern Practice of Gas Chromatography. and reproducibility of chromatographic methods have been extensively exploited in food and nutrition science and technology.Barry. Fourth Edition. Factors that affect GC separations Efficient separation of compounds in GC is dependent on the compounds travelling through the column at different rates. 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 rate at which a compound travels through a particular GC system depends on the factors listed below: 1 Robert L.1 Instrument Background Gas chromatography is unique and versatile technique.Grob and Eugene F.0 Introduction: ______________________________________________________________________________ 1. Because the best separation of any solutes can be obtained under equilibrium conditions. (Page 37) 3 . 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. the situation is entirely different.
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. especially if the column is polarity. Length of the column: The longer the column. The flame ionization detector (FID) is a non-selective detector used in conjunction with gas chromatography. the FID or flame ionization detector detects analytes by measuring an electrical current generated by electrons from burning carbon particles in the sample. 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. Gas chromatography-FID (GC/FID). Column packing polarity: Usually. Longer columns are employed to obtain better separation. Differences in polarity of the compounds are only important if we are separating a mixture of compounds which have widely different polarities. but polar compounds will show a larger effect. the longer it will take all compounds to elute. Because it is non-selective. Column temperature: Raising the column temperature speeds up all the compounds in a mixture.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. all compounds will move slower on polar columns. 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 . Polarity of compounds: Polar compounds will move more slowly.
The current measured corresponds roughly to the proportion of reduced carbon atoms in the flame. P. When first designed. a graph is displayed that has time on the x-axis and total ion on the y-axis.98-1. two electrodes are used to provide a potential difference.) the FID detects all carbon containing compounds.0015%) the noise level is also very small (<10-13 amp) and with a well-optimized system. The ions thus are attracted to the collector plate and upon hitting the plate.GAS CHROMATOGRAPHY-FID Optimization of flow rate and column temperature hydrogen flame. negative electrode is positioned above the flame. Desty (London: Butterworths). Except for a very few organic compounds (e. Ed.02. Today. commonly referred to as a collector plate. This makes the detector sensitive to the mass rather than the concentration. the design has been modified into a tubular electrode. H. Vapour Phase Chromatography.(Page 131) 5 . 1957. Specifically how the ions are produced is not necessarily understood. the negative electrode was either tear-drop shaped or angular piece of platinum. at least five orders of magnitude with a response index between 0. The increased current due to electrons emitted by burning carbon particles is then measured. induce a current. etc. The positive electrode doubles as the nozzle head where the flame is produced. How the final data is displayed is based on the computer and software. A voltage of 100-200V is applied between the flame and an electrode located away from the flame.g. The detector also has an extremely wide linear dynamic range that extends over. This current is measured with a highimpedance picoammeter and fed into an integrator. Although the signal current is very small (the ionization efficiency is only 0. In general. carbon monoxide. 2 In order to detect these ions. which is useful 2 Scott. but the response of the detector is determined by the number of carbon atoms (ions) hitting the detector per unit time. W. R. The other.. sensitivities of 5 x 10-12 g/ml for n-heptane at a signal/noise ratio of 2 can be easily realized. D.
integrator.wikipedia. but the principles are the same. As the eluent travels up the FID. The eluent exits the GC column (A) and enters the FID detector’s oven (B). The effluent/fuel/oxidant mixture continues to travel up to the nozzle head where a positive bias voltage exists (E). This deposition would result in loss of effluent and errors in detection. it does not come out of the gaseous phase and deposit on the interface between the column and FID. FID Schematic The design of the flame ionization detector varies from manufacturer to manufacturer. 3(Shown in figure 4 at the Appendix A) 1. Most commonly.2 SAMPLES 3 http://en. The oven is needed to make sure that as soon as the eluent exits the column. which detects the ions hitting the plates.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. and then feed that signal (H) to an amplifier. the FID is attached to a gas chromatography system. This positive bias helps to repel the reduced carbon ions created by the flame (F) pyrolyzing the eluent.org/wiki/Flame_ionization_detector 6 . and display system. The products of the flame are finally vented out of the detector through the exhaust port (J). The ions are repelled up toward the collector plates (G) which are connected to a very sensitive ammeter. it is first mixed with the hydrogen fuel (C) and then with the oxidant (D).
cosmetics. accelerator activator and dispersing agent in rubbers. stearic (C18).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. and food packaging. Lauric acid (also called Dodecanoic acid) is the main acid in coconut oil (45 . polyunsaturated) acids.50 percent) and palm kernel oil (45 . oleic (C18.45 percent of palm oil). 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. 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 . Nutmeg butter is rich in myristic acid (also called Tetradecanoic acid) which constitutes 60-75 percent of the fatty-acid content. palmitic (C16). The general formula is R(CH2)n-COOH. Stearic acid (also called Octadecanoic Acid) is nature's most common long-chain fatty acids. and linoleic (C18. 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. MyrIstic (C14). pharmaceuticals. and microorganisms. unsaturated). Fatty acids all have common names respectively lilk lauric (C12). soaps. It is also used as a softener. The saturated fatty acids have no solid bonds. It is used in the manufacture of metallic stearates. It is widely used as a lubricant and as an additive in industrial preparations.55 percent). Oleic acid (systematic chemical name is cis-octadec-9-enoic acid) is the most abundant of the unsaturated fatty acids in nature. animals. derived from animal and vegetable fats. 7 .
2 Standard mixture of methyl laurate (0.0mg/ml) and methyl stearate (0. methyl myristate (0.7 mg/ml).methyl palmitate and methyl stearate.3 Instrument: Gas chromatography (Agilent Technologies 6890N) equipped with flame ionization detector (FID) and 30m×250 mm× 0. methyl palmitate (1.2. 2.2 Apparatus: Glass Vial.2.1 2. (Shown figure 2 at Appendix A) 2.20 mg/ml).25mm HP5-MS capillary column.0 Research Methodology: ______________________________________________________________________________ 2. 2.20 mg/ml).methyl myristate.4 Analytical Procedures: 1) Instrument Set-up: Injection port Injection port temperature Oven temperature Column temperature Detector temperature : Split (20:1) : 250 °C : 100 °C : 20cm3/s : 250 °C 8 .GAS CHROMATOGRAPHY-FID Optimization of flow rate and column temperature 2.1 Individual methyl esters compounds: Methyl laurate. Reagents and Solutions/Samples: 2.
190 °C and 210 °C at the optimal carrier gas flow rate.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. the flow rate was increased to 50cm3/sec. Each methyl esters was injected individually to identify the various compounds in the standard mixture using the optimized GC conditions. 9 . the system was allowed to equilibrate. 5) Identification of components in methyl esters mixture. 4) Separation of methyl esters using column temperature programming.4 µL standard mixtures were injected isothermally at 170 °C. 0. 3) Effect of column temperature on isothermal GC separation of methyl esters.4 µL standard mixtures were injected isothermally at 170 °C at carrier gas flow rate of 20 ml/s. Then. Before injected the standard mixture again. 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. resolution and analysis time was investigated. The effect of column temperature on the separation.
553 4.147 10 .661 17.041 17.4922 0.2319 0.383 14.200 7.773 21.1 Standard mixture 1 (GC1: Table 1: Flow Rate 30 cm3/sec and Column Temperature 170°C Rs2. tR Width 1&2 2&3 Average Peak Average 1 1 2 3 1 2 2 3 4. Rs1.2268 0.206 7.1264 0.386 14.504 18.1311 0.GAS CHROMATOGRAPHY-FID Optimization of flow rate and column temperature 3.peak Repeatability Peak Retention Time.4545 18.0 Results: ______________________________________________________________________________ Refer to Appendix B 3.599 0.174 19.
609 6.2087 0.1413 0.350 8.577 6. tR Width Rs1.3698 8.872 2.860 0.peak 1&2 Average Rs2.127 3.1268 0.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.841 2.3623 0.2199 0.295 11.159 3.668 11 .230 11.345 8.peak 2&3 Average 1 1 2 3 1 2 2 3 1 3 2 3 2.602 11.134 3.414 11.3510 0.572 6.1264 0.2083 0.416 8.
GAS CHROMATOGRAPHY-FID Optimization of flow rate and column temperature Table 3: Flow Rate 70 cm3/sec and Column Temperature 170°C Rs1.0964 0.1914 0.771 12 .1834 0.587 2.peak Average 1 1 2 3 1 2 2 3 1.3739 0.2856 0.1968 3. tR Width 1&2 2&3 Average Rs2.686 5.692 5.peak 1&2 Average Rs2.832 4.1412 0.154 Table 4: Flow Rate 70 cm3/sec and Column Temperature 190°C Repeatability Peak Retention Time.592 2.703 2.239 1.4205 4.948 3.672 1.354 4.1444 0.613 4. tR Width Rs1.669 0.911 0.886 1.2657 0.1750 0.peak Repeatability Peak Retention Time.471 6.231 1.598 4.729 3.peak 2&3 Average 1 1 2 3 1 2 2 3 1.708 2.82 5.588 7.509 6.1110 0.
773 Flow rate 30 cm /sec Temp 170°C 8.058 1.297 1. Rs1 Resolution.416 3 Resolution.086 Table 6: Comparison of Resolution in Different Flow Rate and Temperature Flow Rate and Temperature Temp 170 °C 17.471 Flow rate 70 cm3/sec Temp 190°C 3. tR Width Rs1.820 Flow rate70 cm3/sec Temp 210°C 2.104 2.927 3.0872 0.291 1.1218 0.071 1.1240 0.230 3.661 13 . Rs2 19.167 2.1726 2.727 1.1608 0.731 0.729 6.832 11.007 5.167 Flow rate 70 cm3/sec 3.peak 1&2 Average Rs2.peak 2&3 Average 1 1 2 3 1 2 2 3 1.007 2.414 Flow rate 50 cm3/sec Temp 170°C 4.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.0908 0.
291 1. tR Average Retention Time.727 1.186 tR Second Injection 1. tR 1 1 2 3 1 2 2 3 1.604 1.612 1.023 1.596 1. flow rate 70 cm3/sec Methyl ester Laurate Palmitate Myristate tR First injection 1.291 1.GAS CHROMATOGRAPHY-FID Optimization of flow rate and column temperature 3.058 1.2 Individual methyl ester Table 7: Retention time for individual methyl ester at temperature 210°C.729 14 .007 1.039 1.291 1.065 1.195 Table 8: Flow Rate 70 cm3/sec and Column Temperature 210°C Repeatability Peak Retention Time.204 Average tR 1.071 1.731 1.
291 1.195 1.023 1.065 1.tR Methyl Palmitate.tR Methyl Laurate.tR Methyl Myristate.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.729 1. flow rate 70 cm3/sec Peak Number Standard Mixture.604 15 .tR 2 3 4 1.
The standard methyl ester contains three individual components. 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. methyl myristate and methyl palmitate. 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.0 Discussion: ______________________________________________________________________________ In this experiment.GAS CHROMATOGRAPHY-FID Optimization of flow rate and column temperature 4. The instrument set to use split injection because only small amount of sample introduced into the column. gas chromatography was used to identify the various components in the standard mixture of methyl ester using the optimized GC conditions. The ideal resolution value for chromatography separation is 16 . Based on the chromatograms of standard mixture. methyl laurate. the optimum condition of this experiment achieved at temperature 210°C at flow rate of 70cm3/sec. The standard mixture injected at flow rate of 30. The optimum condition for this experiment is determined by injection of sample at different temperature and flow rates. This type of injection will produced more sharp and narrow peak compared to splitless injection. 50 and 70 cm3/sec and temperature of 170. For optimum column efficiency. The resolution value at different temperature and flow rate is compared in order to determine the best separation. 190 and 210°C in order to determine the suitable flow rate and temperature for the separation. The effects of carrier gas flow rate and column temperature on gas chromatography separation of compounds mixture were investigated in this experiment.
Then the individual methyl esters are identifying by comparing the chromatograms of the individual compounds and the standard mixture. 1. 5.604. The three compounds are separated better at high temperature and flow rate based on the value of resolution have been calculated. methyl myristate and methyl palmitate at optimum GC condition is 1. methyl laurate eluted first followed by methyl myristate and methyl palmitate.GAS CHROMATOGRAPHY-FID Optimization of flow rate and column temperature around 1 to 20.195and 1. Therefore.291 and 1. 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. The average retention times of individual peaks of methyl laurate. So.729. the separation takes longer times to complete. 1.0 Conclusion: ______________________________________________________________________________ This experiment we can conclude that.023. 17 . The average retention times of the standard mixture at the same condition is 1. the objective in the experiment is achieved. If the resolution value between two peaks calculated is greater than 20. 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.065.
(Page131) 18 .2. Desty (London: Butterworths).0 References: ______________________________________________________________________________ 6.wikipedia.GAS CHROMATOGRAPHY-FID Optimization of flow rate and column temperature 6. Vapour Phase Chromatography. W.2. Laboratory Guide.wikipedia. Analytical Chemistry: An Introduction. West.. 2004.2 Robert L.org/wiki/Fatty Acid (Retrieved on 19 Jan 2011) 6. 7th Edition.1 Nor’Ashikin Saim and Ruziyati Tajudin.1.1 Internet References: 6.2. 2000.2 Book References: 6. (Page 974) 6. (Page 1-3) 6.org/wiki/Methyl Ester (Retrieved on 19 Jan 2011) 6. Brook/Cole Thomson Learning.1. H.wikipedia.Barry. Analytical Separation Method.3 http://en. (Page 37) 6.2 Wikipedia Methyl Ester-en.1 Wikipedia Fatty Acid -en. Ed. 2000.org/wiki/Flame_ionization_detector (Retrieved on 20 Jan 2011) 6. 1957. Holler and Crough. Modern Practice of Gas Chromatography.4 Scott. R. P. Fourth Edition.3 Skoog.Grob and Eugene F.2.1. D.
0 Appendix: ______________________________________________________________________________ Appendices A 7.GAS CHROMATOGRAPHY-FID Optimization of flow rate and column temperature 7.1 Instruments and Apparatus Figure Name Figure Figure 1: Schematic Diagram Of Chromatography FID Figure 2:Gas Chromatography FID 19 .1.
1.GAS CHROMATOGRAPHY-FID Optimization of flow rate and column temperature Figure 3: Flame Ionization Detector 7.2 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) 20 .
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