Chemistry 3200 Gas chromatography (GC) is an instrumental method for separating volatile compounds in a mixture. A small sample of the mixture is injected onto one end of a column housed in an oven. The column in this experiment is a six feet long, ¼ inch diameter stainless steel tube. The column is packed with a sand-like substance (small SiO 2 or Al 2 O 3 particles) and coiled. The particles have been coated with a viscous, high boiling liquid such as dodecane or substituted dodecane. This coated solid is referred to as the STATIONARY PHASE. The mixture of volatile components is transported through the column by an inert gas such as argon or helium. The gas is referred to as the MOBILE PHASE and is also sometimes called the CARRIER GAS. Consider a component of the mixture that does NOT interact with the stationary phase. It is always in the gas phase, and it moves down the column with the velocity of the carrier gas. If you divide the length of the column by the velocity of the carrier gas you have calculated the time it takes for a NON-RETAINED component to pass through the column. If a component adsorbs onto the stationary phase during its journey down the column, it is RETAINED by its interaction with the stationary phase, and it takes longer to pass through the column. In general, different components of a mixture will interact differently with the column, and they will be retained differentially. Thus the components of a mixture will exit the column at different RETENTION TIMES. This is the fundamental principle of chromatography; components of a mixture can be separated by differential retention on a column. The retention times are characteristic of the interaction of the stationary phase with the components. A detector is placed at the end of the column. In this experiment a thermal conductivity detector will be used. The detector has been designed to generate an electrical signal when a component other that the mobile phase is present. Experimentally you will observe a signal that increases and then decreases as the components of the mixture enter and then exit the detector. This gives a peak that is approximately Gaussian in shape. The retention time is found as the time between the injection and the center of the signal peak. If the same stationary phase and temperature is used, the retention time is characteristic of the component. Therefore the retention times can be used for qualitative analysis of the components of a mixture. This is normally done by comparing retention times in an unknown mixture with retention times of known compounds. The area under the peak is characteristic of the amount of the component and therefore is used for quantitative analysis. A hypothetical chromatogram obtained for the separation of a sample containing organic components A and B and a certain amount of air is shown in Figure 1. In this chromatogram, component A is eluted first and component B is eluted second. Therefore, B is retained more than A. In order to quantify the retention of each component you must account for the transport time of an unretained compound. In many cases (including our experiment) air is not retained by the column, so if you make sure to include some air in the injected sample, the air peak provides a marker indicating the transport time of an
unretained compound. The time elapsed from sample injection until the appearance of the air peak, t air, is a measure of the interstitial volume of the column including the dead space of the injector and detector. This volume, V air, is calculated from t air and the carrier gas flow rate,, as shown in equation 1. V air t air (1) The retention times for components A and B, t A and t B, respectively, are the times required for elution of these substances to their maximum concentrations. The corrected retention volumes for these two components, V A and V B, are calculated as shown in equations 2 and 3. V A V B t A t air (2) t B t air (3) V A and V B are related to the weight distribution coefficients, K A and K B, for components A and B between the stationary and mobile phases as shown in equations 4 and 5. V A K A V L (4) V B K B V L (5) In these equations, V L is the volume of the stationary liquid phase. A common method of reporting retention information is in the form of relative retention ratios, some solute being chosen as the standard and the retention volumes of other solutes being given as a ratio to the standard. For our example, selecting component A as the standard, the relative retention ratio for B, a, is given in equation 6. K B V B t B (6) K A V A t A
Relative retention ratios are unaffected by carrier gas flow rate, pressure gradient in the column, or weight of the solvent phase. Another measure of the time separation of the components peaks is called the relative peak separation, S. S is defined in equation 7 for our example. t S B t A (7) t A The relative peak separation serves as a convenient parameter to characterize the separation of two solute peaks and also the behavior of the particular column packing. The ability of a particular column to achieve good separation of component peaks is a measure of the effective number of theoretical plates in the column, N. The larger the value of N, the greater is the separation. The value of N also affects the shape of the elution peak observed for a particular solute species. The value of N can be estimated from the shape of an elution peak, e.g. peak A, using equation 8. 2 t N 16 A 5.55 t A In equation 8, Δt A is the width of the elution band in time units as measured at the baseline intercepts of the tangents to the peak (see Figure 1). N may vary with the solute as well as the column packing. t A w 1/2 2 (8) Note: In equations relating to peak width such as equation 8, the retention time DtA is calculated from time of injection not appearance of air peak. In this experiment, you will separate and quantitatively determine a mixture of organic compounds using the internal standard technique. The internal standard technique consists of accurately adding a known amount of arbitrary compound (internal standard) to the sample mixture. Calibration curves can then be established by one of two methods. The first is the by ratioing peak areas, by measuring width and height of the peaks, of various concentrations of components to the area of the internal standard. The second and preferred method for higher accuracy is to weigh the paper that corresponds to the peak. The mass of the paper is directly proportional to the area, and removes problematic measurements with drifting baselines. The internal standard is chosen to have a retention time about similar to that of other components in the mixture, and the amount added us chosen to approximate the concentration range of the components. In this manner, reproducibility of measurement will be quite satisfactory even if running conditions such as flow rate and temperature have varied slightly from run to run. The experimental parameters and instrument settings have been properly adjusted by your instructor due to the length of time needed for instrument warm-up to equilibrium. Use of Hamilton Syringe for Sample Injection Use a Hamilton Syringe for injecting all samples into the gas chromatograph. Be careful as the syringe is very expensive. When moving the wire piston, do not bend it from side-toside. You must completely clean the syringe when the composition of the next sample for
injection is different than that of the previous sample. Three or four good rinses with the next sample will be sufficient. Do not contaminate a sample by insertion of a syringe needle, which bears a small amount of the previous liquid. Wipe the needle clean with a clean Kimwipe. When inserting the syringe needle into the injection port, hold the syringe perpendicular to the side of the chromatograph and push straight toward the chromatograph. An insertion of about one half inch is sufficient although deeper insertion may be more convenient. After insertion, quickly push the wire piston to eject the entire sample and then remove the syringe from the injection port. Procedure (Students will work in groups for this experiment) NOTE: When finished with the experiments empty each flask and bottle into the used material container located in each hood. Rinse each with one small portion of diethyl ether (Please do not use water). Leave stoppers removed so organics remaining in the containers will evaporate. When preparing mixtures of organic liquids, work in the hood. 1. Collect about 5 ml of an unknown solution in a vial. Using a pipette, transfer 3.00 ml of the unknown solution to a 10 ml volumetric flask. Immediately add ml of toluene (internal standard) and dilute to 10.00 ml with diethyl ether. Mix, transfer to a 1 oz bottle and cap with rubber septum. 2. Prepare the following mixtures in 10.00 ml volumetric flasks. Mix, transfer to separate 1 oz bottles and cap with rubber septums. Standard number 1 2 3 4 5 6 benzene heptane n-propanol toluene diethyl ether 1.40 ml 1.20 0.80 0.60 0.40 0.40 ml 0.60 0.80 1.20 1.40 0.80 ml 1.20 1.40 0.40 0.60 ml dilute to 10.00 ml total volume with diethyl ether 3. The gas chromatograph will be set up for use for you to a temperature of 85-95 C and a mobile phase flow rate of 30 ml/min. 4. Obtain chromatograms for 5.0 µl portions of each solution prepared in Step 2. Be sure to include sufficient air with each injected portion to produce an air peak on the chromatogram. Adjust the attenuator so that the highest peak (excepting that for diethyl ether which will go off the chart) covers about 75 % of the chart width. Label each chromatogram. 5. Measure the retention time of all peaks, including air, of the chromatogram.
6. Identify the organic compound producing the peaks obtained in these chromatograms. This is easily accomplished by comparing relative changes in peak heights with changes in composition of the injected sample. 7. Determine the area under each peak (except that for diethyl ether) by approximating the peak as a triangle (area = ½w h) or weighing clippings of the chromatogram (preferred). Calculate the ratio, (area of component peak)/(area of internal standard peak). For each component, plot this ratio versus the volume percent of that component in the 10.0 ml mixture. These plots should be linear. 8. Obtain a chromatogram for a 5.0 µl portion of your unknown sample. Use the same instrumental operating conditions used in Step 4. Determine the identity of each component in your unknown by comparing the respective peak retention times with those observed in Step 4. Possible organic components are benzene, n-propanol, and heptane. 9. Determine the mass (or area) of each peak obtained for the unknown (except that for diethyl ether). Calculate the volume percent of each component in your unknown using the calibration curves produced in Step 7. Questions (answer these in your report) 1. From the ratios, (area of component peak)/(area of internal standard peak) decide whether the thermal conductivities are the same. 2. What are the advantages of using the internal standard method? 3. Why did you choose your particular method of area determination? 4. Could you have predicted the order of elution of components from the column without having determined their actual retention times? What factors determine retention time? 5. Do your peaks exhibit tailing? What causes tailing? What conditions could be changed to prevent tailing?
Student Name: Chemistry 3200 Date: Lab Instructor: Section: Unknown Number: Retention times (in min) of each component measured from the time of injection: air peak: solvent (diethyl ether) peak: Peak #1, component : Peak #2, component : Peak #3, component : Peak #4, component : Peak masses (in mg) of each component: Peak #1 Component: Standard #1 Standard #2 Standard #3 Standard #4 Standard #5 Standard #6 Unknown Peak #2 _ min _ min _ min _ min _ min _ min Peak #3 Peak #4 Calibration curves for benzene, heptane and n-propanol 1. Include a graph for each of the calibration curves and the regression data. 2. Give the mathematical formula (y = mx + b) for the best line describing the calibration curve. 3. The graph should plot ratio {(area of component peak)/(area of toluene peak)} versus volume percent of that component in 10.00 ml mixture. Formula for calibration curves: benzene: heptane: n-propanol:
Student Name: Calculation for volume percent of one component in your unknown: Quantity (volume percent) of each compound present in unknown: List the possible sources of errors in your volume percent calculation: