Quantitative Determination of Ethanol Using GC and Headspace Sampling

Gas Chromatography I. Introduction Temperature programming is one of the most efficient means of effecting the separation of closely related compounds using gas chromatography. Temperature influences the retention time of a component by changing the vapor pressure of the component that is dissolved in the stationary phase. As the column temperature is increased, a component’s vapor pressure increases. Thus, the equilibrium between the component dissolved in the liquid stationary phase and in the mobile (gas) phase shifts toward the gas phase and the retention time of the component decreases. Consequently, a high initial column temperature is not very effective in separating components because each component strongly favors the mobile phase. Temperature programming is carried out by gradually increasing (ramping) the temperature during the run. In this experiment, you will utilize temperature programming to obtain an efficient elution of an alkane mixture. Generally, one of the principal limitations of gas chromatography is the requirement that the sample be easily vaporized. When an analysis is done on a volatile component in a less volatile matrix (for example, the analysis of hydrocarbons in an aqueous solution), headspace sampling removes this limitation. Instead of injecting the entire sample, only vapor in equilibrium with the liquid sample is injected. This not only eliminates the need to inject a less volatile sample matrix which might cause damage to the column, but the more volatile analytes are concentrated in the aliquot that is injected. In this experiment, you will also make a quantitative determination of the amount of ethanol in an unknown using an internal standard and headspace sampling. II. Equipment A. Hewlett Packard 5890 Series II Plus Gas Chromatograph with HP ChemStation Computer Interface, Split/Splitless Injection Port, Capillary Column and Flame Ionization Detector B. Hewlett Packard 6890 Gas Chromatograph with HP ChemStation Computer Interface, Split/Splitless Injection Port, Capillary Column, Flame Ionization Detector and HP 7694 Headspace Sampler C. 5.0-µL syringe (obtain from the instructor) D. 5-mL glass transfer pipet E. 1-mL glass transfer pipet F. 2-mL glass vials G. 20-mL headspace sampling vials with caps (obtain from the instructor) III. Reagents A. B. C. D. E. alkane standard mixture (octane, nonane, decane and dodecane in hexane) hexane absolute ethanol 0.3% propanol solution ethanol unknown IV. Procedure A. Gas Chromatograph Settings 1. Turn on the gas chromatograph and the ChemStation computer. Open the hydrogen, compressed air and nitrogen tanks and ensure that the line pressure for each is set to the marked level and that all valves in the gas line between the tanks and the chromatograph are open. 2. Your instructor will demonstrate the use of the ChemStation software. Ensure that the “chem422" method is loaded. Take careful note of the oven temperature, flow rate and split ratio used by this method. 3. A flame ionization detector will be used to monitor the separated components. To establish the hydrogen flame, press the Signal A button twice to monitor the detector signal, then open the air and hydrogen needle valves for Detector A to a full counterclockwise position and open the N2 valve one full turn. Press the Ignite button until the flame lights. You can detect the flame by the signal (should be about 10 for just the carrier and flame gases present) or by looking for fogging (water condensation) on a spatula held at an angle just above the detector outlet. 4. Allow the system to warm up for at least 10 minutes. B. Temperature Programming 1. Rinse a 5-µL syringe several times with the alkane standard sample and inject a 1.0-µL sample and start data collection. After completion of the run (about 8 min), associate the large peaks in the chromatograms with the major components of the standard mixture and note the retention time (tR) for each of these peaks. 2. Adjust the temperature program to improve the elution of the alkane standard mixture. This will involve changes in the initial temperature, initial time, ramp rate and final temperature. The final temperature should not exceed 250EC. Strive to elute the major peaks with baseline resolution in less than 4.0 minutes. Record all temperature programming parameters used during this part of the experiment. Print out the chromatogram for your most successful separation. C. Ethanol Unknown 1. Using the method of successive dilution, prepare a series of 4 aqueous ethanol solutions of precisely known concentration in the range 0.01% to 0.1% ethanol by volume. Make 10 mL of each solution. 2. Using glass transfer pipets, place 5 mL of a standard prepared in C.1 into a headspace sampling vial, add 1.0 mL of the 0.3% propanol solution and seal the vial cap as demonstrated by your instructor. Repeat this process for the other standards, your ethanol unknown and a blank solution. 3. Your instructor will demonstrate the use of the 6890 ChemStation software and the Headspace Sampler. Ensure that the “Chem422L” method is loaded and that “Method 4" for the Headspace Sampler is being used. Note the various analysis parameters on the two instruments that are being used for your measurements. 4. Run each standard and the unknown and collect the ethanol and propanol peak area data for your calculations. V. Treatment of Data A. Tabulate the retention times for octane, nonane, decane and dodecane obtained in IV.B.1 and IV.B.2. B. Turn in all chromatograms (make sure each is properly identified) and report the temperature program used to obtain the best elution results for the alkane mixture. C. To calculate the ethanol concentration in your unknown, first, normalize the area of the ethanol peak in each sample by dividing by the propanol peak area for that sample. Plot this ratio as a function of ethanol concentration for your standards and fit these data with a linear least-squares line. From the normalized area for your unknown and the equation of the least-squares line, determine the ethanol concentration in your unknown. Report this value. Lab Cautions for Gas Chromatography 1. THE SYRINGES USED FOR INJECTING SAMPLES ARE RATHER FRAGILE. Be especially careful not to bend the needle. When you are finished using a syringe, thoroughly rinse it with hexane and return it to your instructor. 2. Before you leave the lab, make sure the detector is turned off by closing the needle valves for all of the detector gases. 3. DO NOT EXIT THE DATA COLLECTION SOFTWARE OR CHANGE THE CHEMSTATION VIEWING WINDOW FOR YOUR INSTRUMENT. 4. Recover all sample vials used in this experiment. Dispose of samples in the Organic Waste container and put vials in the Used Vials bucket.