Exercise in gas chromatography
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- Osborne Richardson
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1 AM0925 Exercise in gas chromatography Introduction The purpose of this exercise is to investigate the effect of different carrier gases (helium and nitrogen) on the Golay equation and the optimal carrier gas velocity (u opt ). In addition, you should identify two unknown compounds by using retention indices. For the data analysis you need access to a Windows computer with Matlab, and preferentially a memory stick with 500 MB free capacity. Some of the groups will be analysing the samples using helium as carrier gas, while other groups will use nitrogen. Pay attention to which carrier gas you use when the experiment is started. Working with the chromatograms The first thing you have to do after the samples have been analysed is to work with the chromatograms to get the retention times and the peak widths. The data and the Matlab code can be downloaded from [ as a zipfile named "CC_GR" followed by your group number, which you find at the same page. Download the zip file and move the folder "CC_GR{group number}" to a memory stick or the hard disk on the computer you are using. The program can not be run from server. The most practical solution is to use a memory stick because it allows you to move the work to a different computer if you need help. In the folder "CC_GR{group number}" there is a program file called "Chrombox C". Run this file. Matlab should then start (which can take up to a minute) and you should see a window similar to Figure 1, except that it has no chromatogram. Now you have to import the data. Press the [Import Box] button, which will take you to a new window. Select your data in the list to the left (there should only be one option in the list). Check that the files appear in the list to the right, press [Import All] followed by [Accept as new] in the list of buttons to the right. You should now be back in the main window and the first chromatogram should be displayed. You will see one large peak. This is the solvent peak and not one of the peaks we are interested in. If you click in the chromatogram and press arrow down on the keyboard a few times you should see five peaks in a pattern similar to the one in Figure 2. These are the C12 to C17 n-alkanes. If the baseline raises so the peaks disappear out of the window ensure that the lower value next to "Y:" on the scale line is set exactly to 0. 1
2 Chromatogram File name Scale Exit Report Integrate Import Load/Save Select Chrom/peak Info Box Figure 1, the main window of the program after import of data The next thing you have to do is to integrate the peaks. Press the [Integrate] button. In addition to the alkanes there will probably be other peaks that will be integrated. The areas of these should be deleted before the results are reported. You delete a peak by right-clicking on the area or the peak number and selecting "Delete". Note that the solvent peak will also be integrated, but you may not see the area or the peak number if you have scaled the chromatogram to show the smaller peaks. You can see the entire solvent peak by pressing the [Auto] button to the right on the scale line, or by increasing the scale by the arrow up button on the keyboard. 350 Solvent C12-C17 alkanes SAM140731_K230_T2 ALK_ C12 C13 C C15 C16 C Figure 2. n-alkane sample after proper scaling of the y-axis It is also possible that you may have to add an area if one of the peaks of interest is not integrated. You do this by right-clicking in the background of the chromatogram and selecting "Add peak". In the same menu you also find the options for copying and printing the chromatogram. 2
3 Before you can report the data you also need to check that the peaks are properly integrated. The area should start and end at the baseline. To correct the integration of a peak you click on the area and thereafter click on one of the two red points that appear. Move the axis cross to the horizontal position you want to be the start or end of the area and click again. Ensure that the axis cross is above the baseline when you set the position. The area will then be fitted to the baseline. An example of an incorrectly integrated peak is shown in Figure 3. You will not use the peak areas, but you will use the peak with (w b ). Peak width is measured at half peak height (w h ) by the program as shown in the figure, and thereafter converted to w b by multiplication with The reported peak widths are therefore peak widths at baseline. If the baseline is not set correctly the peak width will be measured at the incorrect position. To better see the areas you can zoom in and navigate on the x-axis (retention time in minutes), by the [+]/[-] buttons and the slider to the left on the scale line. [+] will zoom towards the selected peak if the [()] toggle button is selected, otherwise it zooms towards the centre of the visible area. If the [Auto] button is pressed the Y-scale will be automatically scaled to the height of the visible peaks. 300 (a) (b) w h 150 w h Figure 3. Incorrectly integrated peak (a) and corrected integration (b) When the right peaks are correctly integrated you generate the report by pressing [Report Sgl]. An Excel file will be created and it should open automatically. The generated Excel reports are also found in the folder "reports" under "CC_GR{group number}". The numbers you need are the retention times in the "Rt" column and the corresponding peak widths in the "Width column". The reported peak widths are w b. Both retention times and peak width are reported in minutes. Repeat the procedure for all the chromatograms and build tables of retention times and peak widths for the alkanes. The alkanes are found in samples with the name starting with "ALK". The number that follows is the estimated average carrier gas velocity in cm/s. If the file name ends with "PRG" it is acquired with a temperature program. Sample names starting with "UNK" contain two unknown compounds, but there may also be other minor peaks. In these samples you should only acquire the retention times of the two unknowns. If you need to save the work and resume it later (or if you need to ask for help), you save it by the [Save Box] button. To read saved works, select the file in the list next to the [Load Box] button and press [Load Box]. Alternatively, you can use the [Tempsave] and [Resume] buttons. 3
4 The Golay equation From the tables of retention times and peak widths you should calculate the plate number (Eq 1) for the six n-alkanes for each of the carrier gas velocities. Calculate thereafter the mean plate number for each velocity by using average of the six n-alkanes. N=16( t R 2 w b) From the average plate number you calculate the plate height for each carrier gas velocity by Equation 2. The column length is 30 m. Calculate plate heights as mm. H=L/ N The plate heights should thereafter be fitted to the Golay equation. The normal Golay equation is given by Equation 3. (1) (2) H= B u +C u (3) However, because of the compressibility of the carrier gas the ordinary Golay equation will typically have a good fit to experimental data only in cases with a low pressure drop (short columns or large internal diameter). It has been claimed that Equation 4 should be used in cases with high pressure drop [L.M. Blumberg, Journal of Chromatography A 1218 (2011) 8722]. H= B u 2 +C u2 (4) The conditions used in our case will typically give intermediate pressure drop, so it can be expected that the exponent on u should be somewhere between 1 and 2. You should therefore use Equation 5 to fit the plate height to the velocities. H= B u x +C ux (5) When H is given by equation 5 the optimal velocity is found by Equation 6. u opt =( B 1/ x = C) ( B 1 C) 2 x (6) Templates for solving Equation 5 by the solver algorithm (CURVES_GC.xls and CURVES_GC.ods) can be found at [ Use these to find B and C, optimal velocity and predicted plate height at optimal velocity (minimum H). Include these values in the report together with the figure of the Golay plot and the calculated average H values. Note that there are two sheets in the template files, one for nitrogen and one for helium as carrier gas. Compare your results with the results for one of the other groups that used the other carrier gas. Discuss the differences and the cause of the different optimal carrier gas velocity. 4
5 Identification of unknown compounds The two peaks in the unknown sample should be tentatively identified by using Kováts retention indices. The principle for retention indices is that unknown compounds can be identified by their retention time relative to the retention times of a series of reference compounds, in this case n- alkanes. The principle is explained in Figure 4. n-alkanes are used to define a new retention scale where the values for the calibration compounds by definition is equal to the number of carbon atoms in the compounds. The unknown compounds are thereafter measured on the same scale. Contrary to retention time, which is very dependent on experimental conditions, the retention index for a compound will basically depend only on the type of stationary phase (and to some degree on temperature), and the compounds may therefore be tentatively identified by comparing calculated values with values from the literature. For isothermal conditions the retention indices are calculated by Equation 7. I=100( logt ' R (x) logt ' R (z) log t ' R(z+1) log t ' R (z)) +100 z (7) where t' R is adjusted retention time, x denotes the unknown compound, z denotes the number of C atoms in the nearest n-alkane eluting before x and z+1 denotes the number of C atoms in the nearest n-alkane eluting after x. To find t' R you need the holdup time, which can be estimated from the carrier gas velocity and the column length. Pay attention to the carrier gas velocity that was used when the unknowns were analysed, and ensure you use the chromatogram for the n-alkanes that was acquired with the same velocity. For temperature-programmed conditions, where the n-alkanes are more linearly spaced, the retention indices are calculated by Equation 8. I=100( t R (x ) t R (z) t R(z+1) t R (z)) +100z (8) Calculate the retention indices for the unknowns at both isothermal and temperature-programmed conditions. Show the calculations in the report. Compare your values to the list of retention indices given in Appendix 1 and guess which compounds you have been given. Report the deviations between your calculated values and the corresponding values in the table. Note that there are some deviations between the values for the different carrier gases because different temperatures were applied for Helium and Nitrogen. C16 I 1600 I = 1661 C17 I 1700 I = 1745 I = 1825 C18 I 1800 I = 1852 C19 I min 1600 Figure 4. Principle for retention indices (Kováts) I 5
6 Reporting In addition to what is described above you should give a brief description of the purpose of the exercise, and a brief description of the instrument and the experimental conditions (Appendix 2) that you consider important for the results. Include the following chromatograms: A) the n-alkanes at the conditions used to analyse the unknown at isothermal conditions. B) the unknown sample at isothermal conditions C) the n-alkanes at temperature-programmed conditions D) the unknown sample at temperature-programmed conditions Comment and on the difference between isothermal and temperature programmed conditions. Explain the cause of the differences. 6
7 Appendix 1 Retention indices at different conditions Compound CAS No. He He N 2 N 2 Isotherm 160 C Tmp. program Isotherm 180 C Tmp. Program 1,2,3,4-Tetramethylbenzene ,2,3,4-tetrahydro-1-naphtol Hexylbenzene Hexyl ether Methyl-naphthalene ,2-dihydroxybenzene Biphenyl Heptyl ether ,4-Dimethylnaphthalene Acenaphthylene Acenaphthene Fluorene Octyl ether ,6,10,14-tetramethylpentadecane
8 Appendix 2, Experimental conditions Common for all experiments: Equipment: Agilent 7890 gas chromatograph equipped with split-splitless injector, flame ionization detector and autosampler. Injector temperature: 250 C Detector temperature: 260 C Injection: 1 µl injected with a split ratio of 1:100 Column: DB5 (5% phenyl 95% methyl polysiloxane), L = 30 m, d c = 0.25 mm, d f = 0.25 µm. Helium, isothermal: Oven temperature: 260 C Estimated carrier gas velocity, cm/s in steps of 4 cm/s Nitrogen, isothermal: Oven temperature: 280 C Estimated carrier gas velocity, 7-23 cm/s in steps of 2 cm/s Helium, temperature program Injection at 100 C, 4 C/min to 220 C. Estimated carrier gas velocity, 26 cm/s Nitrogen, temperature program Injection at 100 C, 4 C/min to 220 C. Estimated carrier gas velocity, 14 cm/s 8
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