7.2.3 Obtaining a Linear Calibration Line
After the temperature program has been optimized, the next task is to calibrate the instrument. As discussed in Chapter 1, instruments easily generate numbers but the analyst must always question the validity of numbers until they are sufficiently scrutinized. Chromatographic analysis has a special feature over most other analyses since the very nature of chromatography allows the analysis of several compounds at one time. If quantitative work is being performed, the instrument must be calibrated with respect to each analyte. This experiment will illustrate proper calibration of a GC-MS. We will use several components of gasoline as our analytes and service station samples of gasoline as our sample.
In this experiment, the analyst will (1) obtain reference standards of several components of gasoline, (2) make dilutions of the reference standards (in pentane) ranging from 1.00 ppm (parts per million) to 100 ppm, (3) inject these standards into the instrument, (4) analyze the samples (at an appropriate dilution), (5) use the software to calibrate the instrument, and (6) analyze the results (perform a linear least squares on the calibration line and calculate the concentration of each component in the gasoline sample).
Experimental Procedures
Chemicals and Supplies:
-25-mL, 50-mL, and 100-mL glass microsyringes
-1.00-mL and 2.00-mL Class A pipets
-Eight 10-mL volumetric flasks
-Two 25-mL volumetric flasks
-One 250-mL volumetric flask
-Neat (pure) samples of benzene, decane, ethyl benzene, n-heptane, isooctane, toluene, m-xylene, and o-xylene.
Instrumental Settings:
-GC-FID Settings (Flame Ionization Detector)
-Capillary Column: DB-5 : Poly(phenylmethyldimethyl) siloxane (5 % phenyl)
30 m x 0.53 mm; 1.5 mm phase coating
-
Injection Volume: 1.00 mL
-Splitless Injection for: 1.00 min.
-Split Flow Rate: 50 mL/min.
-Column Flow: 1.2 mL/min.
-Linear Velocity: 14 cm/s
-Injector Temperature: 230oC
-Detector Temperature: 250oC
-Oven Program: 40oC for five minutes, 4oC to 200oC, hold for 10 minutes
GC-MS Settings:
-Capillary Column: DB-5: Poly(phenylmethyldimethyl) siloxane (5 % phenyl)
30 m x 0.25 mm; 0.25 mm phase coating
-Injection Volume: 1.00 mL
-Splitless Injection for: 0.50 min.
-Split Flow Rate: 50 mL/min.
-Column Flow: 1.2 mL/min.
-Linear Velocity: 40 cm/s
-Injector Temperature: 230oC
-Detector Temperature: 250oC
-Oven Program: 40oC for five minutes, 4oC to 200oC, hold for 10 minutes
Calibration and Sample Preparation:
Calibration standards containing the major components of unleaded gasoline are required. An external calibration procedure will be used, with an internal standard to correct for injector errors and detector drift. The dilution solvent will be pentane because it has a very low boiling point and most other dilution solvents would co-elute with one or more analytes. Prepare a stock calibration standard, as described below, and use this standard to perform serial dilutions (using pentane containing decane as an internal standard) to obtain a range of calibration standards.
NOTES: (1) To minimize the volume (and expense) of GC grade solvents used, dilutions will be made with micro-syringes. This method is less accurate then when using Class A pipets, but will be sufficient for our demonstrations here. (2) All compounds used in this lab are very volatile and flammable. Work in a fume hood away form hot plates, flames, and combustion sources. To minimize volatilization of analytes during solution preparation, place approximately 10 to 15 mL of pentane in the volumetric flask. Since pentane has the lowest boiling point, it will be the first to volatilize, leaving the other analytes in solution.
Procedures:
(1) To add each analyte to the flask, fill a microsyringe to the desired volume (in Table 3.1 below), place the syringe needle on the inside neck of the flask (not in the solution), empty the syringe, withdraw it, and immediately rinse the walls of the flask with 1-3 mL of pentane. Rinse the syringe thoroughly with clean pentane and repeat the process. After all of the analytes have been added to the flask, fill it to the mark with pentane. This solution is the stock solution of each analyte.
Table 7.1 Preparation Guide for the Stock Calibration Solution.
Analyte (> 99% neat) |
Boiling Point oC |
Density of liquid |
mL of pure analyte to be added to a 25 mL volumetric flask |
Resulting ppm concentration in flask |
Benzene |
80 |
0.874 |
29.0 |
1010 |
Ethyl Benzene |
136 |
0.867 |
29.0 |
1010 |
n-Heptane |
98 |
0.684 |
37.0 |
1010 |
Isooctane |
99 |
0.692 |
36.0 |
996 |
Toluene |
111 |
0.865 |
29.0 |
1000 |
m-Xylene |
138 |
0.868 |
29.0 |
1010 |
o-Xylene |
144 |
0.870 |
29.0 |
1010 |
Decane (Internal Standard) |
174 |
0.73 |
14.0 |
101 |
(2) All solutions injected into the GC must contain internal standard (decane). Make 250 mL of pentane-internal standard solution for dilutions by adding 35 mL of pure decane with a microsyringe to a 250-mL volumetric flask and then filling the flask to the mark with pentane. Cap, mix, and use to make the following solutions.
(3) Make dilutions of the ~1000 mg/L solution made in step 1, according to the table below. Fill each flask with the internal standard-pentane solution made in step 2.
Table 7.2 Preparation of GC-MS Calibration Standards.
| 1 | 2 |
3 |
4 |
Approx. Conc. of each Analyte (ppm) |
Solution to be Used in Dilution |
mL of Solution from Column 2 to be added to Volumetric Flask |
Volumetric Flask Size to Use |
100. |
Stock 1000 ppm |
1000 (1.00 mL) |
10.00 |
80.0 |
Stock 1000 ppm |
2000 (2.00 mL) |
25.00 |
40.0 |
Stock 1000 ppm |
1000 (1.00 mL) |
25.00 |
20.0 |
100. ppm |
2000 (2.00 mL) |
10.00 |
10.0 |
100.0 ppm |
1000 (1.00 mL) |
10.00 |
4.00 |
40.0 ppm |
1000 (1.00 mL) |
10.00 |
2.00 |
20.0 ppm |
1000 (1.00 mL) |
10.00 |
1.00 |
10.0 ppm |
1000 (1.00 mL) |
10.00 |
0.400 |
4.00 ppm |
1000 (1.00 mL) |
10.00 |
0.200 |
2.00 ppm |
1000 (1.00 mL) |
10.00 |
(4) The compounds in pure gasoline are at too high of a concentration to be analyzed directly on the GC-MS. Most of the major constituents in gasoline are present between 5 and 20 percent on a mass basis. To dilute the gasoline to an acceptable level, add 40.0 mL to 100 mL of pentane-internal standard solution. Several samples of unleaded gasoline should be analyzed. Suggestions for selecting samples include brand, octane rating, and the presence of methanol and MTBE. Note: if methanol or MTBE are present in your sample, the calibration standards must also include these compounds.
(5) Analyze the standards and diluted samples by GC-MS using the instrumental conditions given earlier. Use the MS to identify each peak in the spectra and then calibrate your instrument. Calculate the % composition of each analyte. Finally, analyze the spectrum of each compound and review the fragmentation rules from Chapter 2.
Results:
Each compound should produce a linear calibration line over the concentration range of your external standards. Most modern instruments will do this relatively automatically. After you calculate the concentration of each analyte in your gasoline sample, convert the ppm concentrations to percent by mass. Compare this to published composition available on the Internet. NOTE: the power of chromatography is the separation of complex mixtures which we have accomplished in this experiment.
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