CHEM Experiment Five Gas Chromatographic Analysis Of A Phenolic Extract. Objectives:

Transcription

1 CHEM 3281 Experiment Five Gas Chromatographic Analysis Of A Phenolic Extract Objectives: The objectives of this experiment are: 1. to evaluate the fundamental parameters of gas chromatography; 2. to identify the components of a phenolic mixture and their order of elution by comparison of the retention times of standards to those of mixture components; 3. to determine detector selectivity and sensitivity 4. to measure the limit of detection for a specific component Text Reference: Introduction: Handbook of Instrumental Techniques for Analytical Chemistry, Frank Settle, editor. Prentice Hall, Upper Saddle River, NJ 1997, Ch 8. Willard et. al. Instrumental Methods of Analysis, 7th edition, Wadsworth Publishing Co., Belmont, CA 1988, Ch 17 & 18. Skoog, Holler and Nieman, Principles of Instrumental Analysis, 5th edition, Saunders College Publishing, Fort Worth, TX 1998, Ch 26 & 27. The separation of mixtures by gas chromatography is accomplished by moving a gas over a liquid phase. The components of the mixture are separated according to their differential solubility in the two phases. In this experiment, the stationary phase has been coated onto the walls of a widebore fused silica capillary. The capillary provides a very compact column with a high number of theoretical plates. Thus, very efficient separations are possible in relatively short periods of time. At the end of the column, the effluent is divided into two streams which flow in parallel into a flame ionization and an electron capture detector. The data acquisition system will display the responses from both detectors in real time. This display mode will permit the rapid assessment of detector selectivity for each of the components in the mixture. The samples to be analyzed are mixtures of substituted phenols dissolved in methanol. This sample simulates samples one encounters following the extraction of industrial plant effluents (wastewater) which contains phenolic residues. Substituted phenols are on the Environmental Protection Agency's Priority Pollutants list. The procedure which follows is a modification of a

2 standard method for the determination of these priority pollutants in water. NOTE: Phenols are toxic substances. Use extreme caution in handling these mixtures. Eye protection and disposable gloves are to be worn at all times. Handle the syringes with care. Notify the TA immediately if you come in contact with the phenolic solutions. Experimental: 1. Obtain from the TA samples of six of the standards listed below, an unknown sample and five serum bottles containing methanol. Identify which six of the following substituted phenols you have received: Compound Molecular weight Boiling Point 2-chlorophenol o C phenol bromophenol ,6-dimethylphenol bromo-4-methylphenol ethylphenol chlorophenol chloro-3-methylphenol bromophenol bromophenol In addition, obtain a 100 l syringe and a 10 l gas chromatographic syringe for use in this experiment. Ask the TA to show you the proper use of both syringes. 2. The chromatographic system to be used with this experiment consists of a Varian Model 3300 Gas Chromatograph which has been interfaced to a computer system. The data acquisition and analysis package is the STAR program written by Varian. The gas chromatograph is controlled by a microcomputer which is built-in to the Model One can set the desired instrument parameters using the soft keyboard located on the front panel of the chromatograph. To set or change the column parameters, press the BUILD/MODIFY key located in the OPERATIONS segment of the panel. The prompt on the display should read Select Section or Table. Select COLUMN in the SECTIONS panel. A series of prompts will be displayed. Respond to each prompt by depressing the appropriate key(s) in the ENTRY panel followed by ENTER. In this manner, set the chromatographic parameters to the following values: - Initial Column Temperature: 115 o C - Initial Column Hold Time: 10.0 min - Temperature Program Column: NO (isothermal elution)

3 - Injector Temperature: 200 o C - Detector Temp: 250 o C - Detector A or B? A - FID Initial Attenuation: 8 - FID Initial Range: 10 - FID Autozero ON? Y - Time Program FID A? NO - ECD B Initial Attenuation: 8 - ECD B Initial Range: 10 - ECD B Autozero ON? Y - Time Program ECD B? NO - Method Complete-End Time: Return to the OPERATIONS panel and press STATUS. Your detector parameters "program" will be stored in the onboard memory. NOTE: Find out what type of column you are using, length. bore size, what stationary phase, it s temperature limits before starting!! 3. ISOTHERMAL GC RUN: You have now setup the gc for isothermal operation. When the READY diode is illuminated in the GC CONTROL panel, rinse the 10 L GC syringe with the unknown sample a few times to minimize sample contamination by residue left in the syringe. Draw 1.0 L of the unknown sample into the syringe and inject this sample onto the column. The RUN diode should be illuminated once you have activated the sensing mechanism on the injector. The color monitor should also indicate that the run is in progress. In the lower two windows, the response from the Flame Ionization Detector (detector A) and the Electron Capture Detector (detector B) should be displayed. Note: your unknown should contain between 3 and 5 components plus a solvent peak. If you find a greater number of peaks, it may either be due to improper injection technique or a contaminated sample. Consult your TA if any problems arise at this point. When the program time has been exceeded, the run will automatically be halted. The printer will begin to output a hardcopy of the chromatograms from detectors A (FID) and B (ECD) as well as a table of peaks and their integrated areas. 4. TEMPERATURE PROGRAMMING RUN: Modify the chromatographic parameters to the following settings: - Initial Column Temperature: 85 o C - Initial Column Hold Time: 0.5 min - Temperature Program Column? YES - Program 1 Final Column Temp: 180 o C (temperature gradient elution) - Program 1 Col Temp Rate: o C/min - Program 1 Col Hold Time: 0 min

4 Return to the OPERATION panel and press STATUS. When the READY diode is illuminated in the GC CONTROL panel, inject 1.0 L of the unknown sample onto the column. Again, when the program time has been exceeded, the run will automatically be halted and the chromatograms and table of peaks for the detectors will be generated. 5. STANDARDS RUN. Look at your isothermal and temperature program runs for your unknown. You decide what temperature (program or not) to run your standards and unknown at. You are looking for good separation in a miminum amount of time. Check your parameter decisions with the TA and then input on the front of the GC. Inject 1.0 L aliquots of each standard provided. Appropriately label each chromatogram and results table. Use the information obtained in this part to identify and determine the FID's sensitivity to each component in your mixture. Note: to save time, terminate each run after the elution of the standard by clicking the mouse on the RESET circle and depressing the RESET key in the GC CONTROL panel of the Model 3300 chromatograph. Run your unknown at the same exact conditions if you have not already done this. 6. Select one of the components of your mixture and determine its limit of detection in the following manner [this component must give an ECD response and have a retention time greater than 4 minutes]. Draw a L aliquot from the standard solution provided by the TA and inject this volume into one of the serum bottles containing 900 l of methanol. The resultant mixture is a 1:10 dilution of the original standard. Prepare a serial dilution of the standard by withdrawing l of the diluted standard and injecting that volume into another serum bottle containing methanol. Continue this procedure until you have five serial dilutions of the original standard Obtain chromatograms on each of the diluted samples, printing out the hardcopy of the chromatogram and results for the FID and ECD responses for each diluted sample. With this data, you will determine the limit of detection of your selected component for each detector and standard deviation of retention times. Calculations 1. For your standards make a table with the following column headings: compound, ng injected, retention time, area counts (FID & ECD), and relative response factor (FID & ECD). Remember mass sensitive detectors do not give equal area counts per unit concentration. Relative response factor is calculated by mass solute injected/area.

5 Use ng/area count. 2. For your unknowns, calculate the concentration of the unknown in ppm (i.e. mg/l or µg/ml or ng/µl). Multiply response factor by area to obtain mass and then divide by volume injected. Make a table for the unknown mixture including retention times, area counts, Response factor x area, concentration (ppm) 3. Make a data table for your serial dilution study including concentration, area and retention time. Calculate standard deviation for the retention times. Plot the FID and ECD response (area counts) versus the concentration of your selected standard (ppm) using the data obtained in the serial dilution study in part 6. Reporting the Results: The report should consist of a brief description of gas chromatography, labeled diagram of your instrument, instrumental parameters used, raw data-labeled, calculated data (in tables, please), sample calculations for anything calculated, all plots and a short discussion of the results. In addition, the discussion section should contain answers to the following questions: 1. Compare and contrast the chromatograms obtained in part 3 and part 4. What effect did the programmed temperature gradient have on the speed of the analysis? Was the resolution better when the chromatogram was obtained under isothermal or temperature gradient elution? 2. Use the uncertainty in retention time calculated from the serial dilution study as a good estimate of average retention time variation. Look at the two WORST resolved peaks in your unknown mixture. Calculate the alpha value for this pair and then answer this question: Can all components be reliably assigned based on retention time comparisons alone? Why or why not? 3. Compare and contrast the response factors from the FID and ECD. Is there a relationship between FID response and component structure? How do the response factors obtained for the FID compare to those obtained for the ECD? 4. What is the linear dynamic range based on your results for each detector? What is the limit of detection of your selected component for each detector. Estimate these parameters from your calibration curves for ECD and FID (part 6.) Estimate the detection limit as 3b/m; where b is y-intercept and m is the slope of the line. Why not use the correct definition of detection limit?