EXPERIMENT 5 Gas Curomatocrapny (GC) THEORY/ BACKGROUND Chromatographic analysis is used to separate complex mixtures of compounds. First used in the early 1900s chromatography got its name because it was used to separate different mixtures of colored compounds. By the 1930s the popularity of chromatography had increased as chemists realized that this experimental technique could also be used to separate mixtures of colorless compounds. All chromatographic systems have two phases, a mobile phase and a stationary phase. The mobile phase is a liquid or a gas that carries a sample through a solid stationary phase. As the sample in the mobile phase passes through the stationary phase the compounds in the mixture will separate because of differences in their affinities for the stationary phase, and differences in their solubilities in the mobile phase. In chromatographic analysis, the eluted compounds are characterized by retention times, Qualitative analysis involves determination of t, of analytes and comparing them with t, of standards. Quantitative analysis is accomplished by comparing the areas of the analyte peaks with those of standards. Instrumentation Mobile phases are generally inert gases such as helium, argon, or nitrogen. The injection port consists of a rubber septum through which a syringe needle is inserted to inject the sample. The injection port is maintained at a higher temperature than the boiling point of the least volatile component in the sample mixture. Since the partitioning behavior is dependant on temperature, the separation column is usually contained in a thermostat-controlled oven. Separating components with a wide range of boiling points is accomplished by starting at a low oven temperature and increasing the temperature over time to elute the high-boiling point components. Most columns contain a liquid stationary phase on a solid support, Separation of low-molecular weight gases is accomplished with solid adsorbents. The sample is swept through an open tubular column by a carrier gas, and the separated elnents (the compounds exiting the column) flow through a detector, whose response is displayed on a computer screen. The column must be hot enough to produce sufficient vapor pressure for each solute to be eluted in a reasonable time, The detector is maintained at a higher temperature than the column so that all the solutes are gaseous at the point of detection. Figure 5.1 below shows the main components of a gas chromatograph.Baste nsraucenraL AWALSIS Figure 5.1: Schematic Diagram of a Gas Chromatograph Some GC detectors are as follows: 1. Flame-ionization detector (FID). FID stands for Flame Ionization Detector. What that means is that as the effluent (carrier gas and any organic compounds) comes out of the column they are ignited in a flame made of hydrogen and air. The compounds produce ions as they burn. These ions conduct electricity. Changes in current within the flame are measured and sent to the computer to be seen as peaks on the chromatogram. FID is a good general detector for organic compounds, and is able to detect at the nanogram level. The FID is extremely sensitive with a large dynamic range and its only disadvantage is that it destroys the sample. 2. Electron-capture detector (ECD). ECD stands for Electron Capture Detector. This detector is very sensitive to halogenated compounds, as well as compounds with very electronegative functional groups such as nitro groups and peroxides. The detection limit of this detector for halogenatd compounds can be as low as the picogram level. This detector cannot detect compounds such as hydrocarbons, amines, and alcohols, making it very useful in quantifying herbicides and insecticides. The ECD is as sensitive as the FID but has a limited dynamic range and finds its greatest application in analysis of organic molecules that contain electronegative functional groups, such as halogens, phosphorous, and nitro groups. 3. Thermal conductivity detector (TCD). The TCD is not as sensitive as other detectors but it is non-specific due to its response to both organic and inorganic species and is non-destructive. 46Bxrenuener 5: Gas Oronaroanseny (GO) OBJECTIVES 1. To determine the retention times t, of n-butanol and 2-propanol. 2. To identify the components present in a standard mixture based on the ty. 3. To identify the component(s) present in an unknown sample. 4. — To determine the effect of temperature on t, and R,. APPARATUS Syringe, Beaker. Dropper. CHEMICALS 2-propanol. n-butanol. Standard mixture of 2-propanol and n-butanol (1:1 ratio). Unknown sample. PROCEDURE A. Sample Handling 1, Rinse the syringe before filling it with the sample. The volume of the sample may be more than the required volume. Make sure there are no air bubbles in the syringe. To remove any air bubbles, you may tap the syringe gently. 2. Hold the syringe vertically, needle up, and push the plunger to the required volume at eye level. Remove the excess sample using a tissue. Note: Since the samples that you are working with are volatile, the preparation and injection of samples must be done as quickly as possible. 47Basie raueyras ANALY a Experimental 1. Switch on the instrument, 2. Set the GC using the following conditions: i, Initial oven temperature: 70°C ii, Final oven temperature: 70 °C iii, Injection temperature: 180°C iv. Detector temperature: 180 °C (always set the detector temperature higher than the initial and final oven temperature) 3. Inject the component sample and determine the retention time of each component individually. 4, Inject the standard mixture and identify each component present by comparing the retention time of each component with the retention time of each single component determined previously. 5. Inject the unknown sample and identify the component (s) present in the unknown. 6. Change the column temperature as follows: i. Initial column temperature: 100°C ii, Final column temperature: 100°C ii, Inject the standard mixture and comment on the effect of reducing the temperature on the retention time and R, of the components. 7. Change the oven temperature as follows: i, Initial oven temperature: 140°C ii. Final oven temperature: 140°C Inject the standard mixture and comment on the effect of increasing the temperature on the retention time and R, of the components. c. Operation of the GC Instrument: Agilent 6890. Operating instructions 1. For manual injection, inject the sample into the septa (liner). 2. Check the oven column. Make sure the instrument is in the off mode. 3. Switch on the instrument. Warning tone will ring if the gas is not enough. 4, — Click ‘Instrument | online’ in your window screen. The instrument is allowed to setup itself. 5. After setup is completed, click ‘method’—> ‘edit entire method’ (make sure all the items in this column are correct) -> ‘ok’. 48Exreruenr 5: Gas Chsousroenamy (GC) 6. Enter the title and click ok. 7. To select injection, click ‘manual’. To select injection location, click ‘back’. At instrument, edit inlet, choose ‘back’ and keep in information. 8. If sample is in high concentration, choose ‘split’ mode. If sample is in low concentration or in small quantities, choose ‘splitless’ in inlet. 9. Goto column section, choose ‘column no. 2’. At inlet column, choose ‘back’. At mode column, choose ‘constant pressure’. At outlet column, choose ‘ambient’. 10. Go to detector column. Use FID (‘front’). 11, Inject parameter (time: 5 min, 1 pL). 12. After doing all of the above, click ‘ok’. 13. Click ‘apply’ —> ‘ok’ — signal detail > ‘ok’ > ‘edit integration events’ > ‘ok’ > ‘specify report’ >’ ok’ — ‘run time checklist’ —> ‘ok’, 14. Go to ‘file’, click ‘save’, ‘method’. Keep in the command. 15. During injection, make sure no bubbles appear in the syringe. 16. Go to ‘run control’, click ‘sample info’. Set your name in ‘operator name’ column. 17. Set signal 2, fill in the sample name. 18. Click ‘run method’ and the instrument will wait for injection. 19. Inject the sample quickly (use both hands!) anc ‘hen press ‘start’ button quickly on the instrument. Then quickly pull out the syringe from thie septa. 20. Go to data analysis, click ‘file’ then ‘load signal’. 21. Enter ‘calibration’, click ‘new calibration table’. Enter your name and print the document. 22. For the next sample, click ‘data analysis’ and repeat steps 17-21. REPORT 1. + Include in your report the chromatograms you obtained and explain how you interpreted the peaks. 2. Explain the reasoning used to determine which peak in the ‘unknown’ chromatogram corresponded to each compound in the standard mixture. 49Basic Insrauuentas Auaysis 3. _ Discuss the effects of reducing and increasing the column temperatures on t, and R,. PRE-LABORATORY QUESTIONS 1. Define: volatile compound, retention time, resolution. 2. _ Find the boiling points of n-butanol and 2-propanol. 3. Predict which compound, n-butanol and 2-propanol will elute first in a GC analysis. Explain your answer. QUESTIONS 1, State the types of compounds which are suitable for analysis using GC. 2. Why is FID a suitable detector for this analysis? 3. _ List two factors which can increase the efficiency of a GC column, 50DATASHEET EXPERIMENT 5 GAS CHROMATOGRAPHY (GC) Name: Date: Student ID; Group: Name and model of instrument: Table 5.1: Retention Times of Standards and Unknown Sample ‘Temperature (°C) Compounds Retention time, t, (min) 70 Standard mixture 70 2-Propanol 70 n-Butanol 70 Unknown sample 100 ‘Standard mixture 140 Standard mixture Unknown number : Leéturer’s signature,