CRIME SOLVING CHROMATOGRAPHY rev 8/12

Transcription

1 EXPERIMENT 10 CRIME SOLVING CHROMATOGRAPHY rev 8/12 GOAL Three different types of chromatography (thin layer chromatography, gas chromatography, and liquid chromatography) will be used to separate mixture samples, and aid in the investigation of a crime. INTRODUCTION Do you watch TV shows like CSI where science is used to help solve crimes? A large portion of the techniques they use are from analytical chemistry. When called to court, those technicians must be able to explain their methods and how the instruments work. It s the job of an analytical chemist to separate, identify, and quantify the mixture components. Real life samples are messy. How do we analyze these messy mixtures? In general, the approach is to separate the mixtures into pure individual components, and then identify the components individually. Chromatography is the science of separating mixtures into individual compounds. The name originally came from the separation of different naturally colored pigments (thus the name "chrom" for color) into the dyes that make them up. Chromatography is used extensively by almost all chemists, biochemists, and biologists in the normal course of their work. The fundamental idea behind chromatography is solubility. Separations of mixtures are accomplished through the use of two different solvents at the same time. One solvent moves and is called the mobile phase. The other solvent stays put and is called the stationary phase. Each component of the mixture will have its own distinctive solubility in each solvent. Let's see how that separation works. Consider Figure 1. Here we have a mixture of two compounds, A and B, being placed onto the top of a cylindrical tube (called the column) and being flushed through that column by a flow of solvent, the mobile phase. The stationary phase in this case is a coating of liquid on the solid particles that are packed into the column. Now, what happens as we push the A+B mixture through the column? The chemicals will tend to dissolve in whichever solvent they are more soluble. In the case shown here, B is more soluble in the stationary phase than A is. Conversely, A is more soluble in the mobile phase than B is. As the mixture proceeds down the column, A spends most of its time in the moving phase, and B spends most of its time in the stationary phase. Thus A moves more Figure 1: Column Chromatography quickly down the column, and B lags behind. By the time they reach the end of the column, they no longer overlap at all. A comes off the column first, as a pure compound dissolved in the mobile phase solvent. Then later, B comes off the column, also as a pure 1

2 compound dissolved in the mobile phase solvent. The mixture has been separated into two pure compounds. The separation is observed at the detector as two separate "peaks" in time, as first A, then B comes off the end of the column. Chromatography comes in many different variants. The nature of the stationary and mobile phases can vary significantly, but the basic idea of differential solubility in these two phases remains constant. In this experiment we will deal with three different types of chromatography, as shown in the table below. In general, thin layer chromatography (TLC) and liquid chromatography are used for nonvolatile samples (ones that don't turn into gases easily), while gas chromatography is used for volatile sample components. Table 1: Different Types of Chromatography Type of Chromatography Stationary phase Mobile phase Thin layer chromatography Silica gel on a plastic plate Liquid solvent Gas chromatography Liquid layer on inside walls of hollow tube. Gas Liquid chromatography Liquid layer coating solid particles packed in tube Liquid solvent In TLC, the components of our mixture are separated based upon their relative solubility in the mobile liquid solvent and the stationary solid silica gel. The more soluble a component is in the liquid mobile phase solvent, the further and faster it moves up the TLC plate. We may be able to see colored spots caused by each compound or use a UV light to make these spots visible. In GC, we use a syringe to inject our mixture at one end of a long tube called the column. Our mixture is immediately evaporated to form a gas. A continuous flow of helium gas acts as the mobile phase, moving the components to the other end of the column. A viscous liquid coating the inside walls of the column acts as the stationary phase. The more soluble a component is in this stationary phase, the longer it takes for that component to come out of the other end of the column. We can t see the components come out of the end of the GC column with our eyes so we detect their presence instrumentally. The detector in our GC is a Mass Spectrometer. The gas leaving the end of the GC column is analyzed. Basically, any compounds in that gas are turned into ions, and the number of ions present is measured at many different masses. Each compound gives a characteristic pattern of fragments with different masses, and the pattern is used to identify compounds. The number of ions detected is proportional to concentration. In HPLC, we also use a syringe to inject our mixture at one end of a column. This time the column is packed with solid particles that hold the stationary phase. Pumps move a liquid solvent mobile phase down the column. The more soluble a component is in the stationary phase, the longer it takes for that component to come out of the other end of the column. The detector in our HPLC shines UV light through the liquid exiting the column. When some of the UV light is absorbed, a signal is sent to the computer indicating that a compound has come off the column. TEAMWORK The in-class portion of this experiment will be done in teams, but the lab report will be done individually. Each student should read the experiment and make the preliminary entries into his/her laboratory notebook before coming to lab. Although you will work with a partner in lab, both students should record their work in their own lab notebooks. Each team member must have a full set of observations and notes to use while writing the report. Although your report should be an individual effort, feel free to consult your in-lab partner if your notes are unclear. 2

3 PRELIMINARY ACTIVITY The crime scene we will investigate is in room 470. You and your partner should visit the crime scene and make observations. As you do, consider the following questions. Only four to six people at a time will be allowed into the room, and your stay is limited to two minutes. You don t actually collect any samples just observe and think! Please don t touch the materials in the scene. Feel free to ask clarifying questions to the supervising faculty member or student assistant. 1. What crime has apparently been committed? 2. What kinds of chemical samples are available at the crime scene? 3. How are those chemical samples the same and different from a sample you typically might receive at the start of a chemistry lab experiment? 4. What questions might we ask about those samples? 5. How will the answers to those questions be used in the investigation of the crime, and in a trial of suspects found by that investigation? Each person should write their answers to these questions in his/her lab notebook, after discussing the questions with your partner. Each team should complete and submit one copy of the Crime Scene Visit Report form at the start of the group discussion. GROUP DISCUSSION Your instructor will lead a group discussion of the crime scene and the chemical samples that are available. The different types of chromatography will be discussed, with a particular focus on which techniques are best suited to different kinds of samples and different chemicals of interest. Specific information about the crime and the suspects will also be given during this discussion, so please pay careful attention! THE INVESTIGATION You and your lab partner are CSI technicians and must analyze samples collected from the suspects in the case. There is circumstantial evidence linking the suspects to the crime, but scientific evidence would greatly enhance the case (or perhaps it will clear one or more suspects). Your job is to see if physical evidence found on the suspect(s) matches up with physical evidence found at the crime scene, thus adding to the possibility that the suspect(s) had been at the crime scene. Your chemical analyses may also add to the understanding of what happened at the crime scene, based on the identity of the chemicals you find. Some preliminary analysis results related to this crime have already been generated by a state agency. The results of those tests will be provided at the group discussion following your inspection of the crime scene. You will analyze three samples taken from suspect A or suspect B. Details will be provided at the group discussion. HAZARDS Wear gloves when you handle any organic solvent (dichloromethane, methanol). Be sure to avoid skin contact with all chemicals as usual, and to wash your hands thoroughly at the end of lab, or in the event of contact with chemicals. Work in the fume hoods when using organic solvents, or when doing TLC. Used capillary tubes should be placed in the broken glass container for disposal. Wear goggles at all times, of course! When you use the UV lights to visualize TLC spots, do not look directly into those lamps. LABORATORY OBSERVATIONS AND DATA Record the names of your team members and the identification numbers for each of your unknown samples. Record initial observations on the appearance of each sample Record what you do in outline form. Record additional observations and data as directed in the procedures that follow. 3

4 PROCEDURE This experiment will be done in pairs, which your instructor will assign. Your instructor will also give you the order of the tests you should follow (to minimize congestion at the instruments). Everyone should still do his/her own preliminary laboratory notebook entries. The gas chromatograph mass spectrometer (GC/MS) is in room 472, and the high performance liquid chromatograph (HPLC) is located in room 572. The TLC test will be done in the hoods in our usual lab room. Get your team s assigned unknowns from the reagent bench. This set will contain several powder samples and possibly a solution. Record the ID numbers of your unknown set. You will do three tests: one by HPLC, one by GC/MS, and one by TLC. Sample Preparation One of the rotations for your team will be a sample preparation rotation. During that rotation, you should do some preliminary steps for your HPLC sample, and separately for your TLC sample. Save the samples you prepare for later use, when your HPLC or TLC rotation occurs. The sample preparation for GC/MS will be done as part of the GC/MS rotation, so you don t have to do those steps right now. HPLC Sample Preparation 1. Weigh out approximately 0.1 g +/ g (record mass) of your solid white sample. Transfer this solid to a small test tube and add 2.0 ml of methanol from the dispenser bottle. Use your stirring rod to crush the solid particles and to mix thoroughly with the solvent. Most of the sample will dissolve. Any small amount of undissolved solid can be allowed to sit on the bottom of the tube. This solution must be diluted, since it is too concentrated for our sensitive instrument. 2. Use a graduated cylinder to measure out 50 ml of distilled water and place this in a clean 125 ml Erlenmeyer flask. Use the special digital pipet and a fresh plastic tip (as demonstrated by your instructor) to measure out ml of your concentrated solution. Depress the pipet button to the first hesitation point. Insert the tip into your concentrated solution and SLOWLY release the button, sucking ml of your concentrated solution into the pipet tip. Then deliver that volume into the Erlenmeyer flask of water by pressing the pipet button all the way down (to the second stop point). Stir with a clean glass stirring rod. Discard the used pipet tip. 3 Use this one hundred fold diluted solution for the HPLC experiment. Save it until your HPLC rotation. TLC Sample Preparation. 1. Since your sample is a solid, you should prepare a solution version for use. Weigh out approximately 0.1 g +/ g (record mass) of your solid sample. Transfer this solid to a small test tube and add 1.0 ml of methanol from the dispenser bottle. Use your stirring rod to crush the solid particles and to mix thoroughly with the solvent. Most of the sample will dissolve. Any undissolved solid can be allowed to sit on the bottom of the tube. Use this solution when you get to your TLC rotation. Test 1: HPLC on the White Solid 1. Take the diluted liquid solution that you prepared earlier (in the Erlenmeyer flask) to room 572. Here, the instructor will assist you in obtaining a chromatographic tracing that will allow you to identify the solid. 2. The general procedure is to fill a syringe with 60 L (microliters) of your solution, fill a loop of tubing with that liquid, and then inject that loop of sample onto a chromatographic column. 4

5 Alternatively, you instructor may have you load a sample vial for use with the instrument s autosampler. A pump pushes mobile phase solvent along with the sample through the column. A UV detector measures the absorbance of the solution at the end of the column, recording a peak whenever a sample component comes off the column. Different components emerge from the column at different times (see Figure 1), and this retention time is measured and used to identify the components, by matching the retention time to the retention times of known compounds. 3. You will get a computer print-out from the instrument. One team member should keep the computer print-out and include it in the lab report. All team members should record the retention times of each major peak from your chromatogram in their lab notebooks. 4. Now compare these times as well as peak shapes to the chromatograms of known compounds displayed in room 572 or 473. Record your identification of your white solid unknown. Test 2: GC/MS (wear gloves) 1. Your sample is a solid, so measure 0.05 g (+/ g) of the sample into a test tube. Go to the hood and add 2.5 ml of CH 2 Cl 2 from the dispenser. While still working in the hood, mix the solutions in the test tube thoroughly. The solid may or may not dissolve. Prepare a test tube containing 5.0 ml CH 2 Cl 2. Use a special automatic pipet to transfer 500 μl (0.5mL) of the clear CH 2 Cl 2 layer from the test tube that contains your sample into the test tube containing 5.0 ml of CH 2 Cl 2, to do a dilution. Do not transfer any of the undissolved solid. Mix the diluted solution in the second test tube well. Use that diluted solution to almost fill a special GC/MS vial. Screw the special cap onto the vial. Take your vial to the GC/MS in room 472. The laboratory assistant will guide you as you place the vial into the autosampler tray, noting the sample position numbers. Then, enter your identifying information in the GC/MS software, adding one line of information to the sequence table for each sample. Have the assistant check your computer work and the placement of your sample in the tray. 2. The GC/MS instrument will automatically analyze your sample. The general procedure it uses is to fill a syringe with 1.0 μl of your sample, and inject that amount onto a chromatographic column. The injector, column, and detector are kept in an oven to maintain the samples in the gas state. The detector in this case (once again at the end of the column) changes the sample components into ions, and then measures the resulting masses of the ions. As before, the different components emerge from the column at different times, and the mass spectrum is used to identify the components by comparison to a library of known compounds. 3. You will get a computer print out the day AFTER your lab period. It should be available in room 465 by noon. Copies will be available for each member of the team. 5

6 Test 3: TLC 1. Locate the concentrated sample solution you prepared in the sample preparation rotation. 2. Obtain one TLC plate. Using a pencil, mark it with a horizontal line 3/4" from one end. Draw lightly. Do not scratch away the silica on the plate. This line represents where samples should be spotted onto the plate. Up to four samples can be spotted onto the plate, all on the pencil line, and spaced about 3/8 to 1/2" apart, as shown in the adjacent figure. To spot a sample, dip a capillary tube into the desired solution and then briefly touch the end of the tube to the desired spot on the TLC plate. Let the wet spot dry, then repeat several times. Holding the tube on the plate too long would transfer all of the solution at once, which would make the spot size too large. Put your unknown solution in a middle spot. For the other three spots, use solutions of known composition. For example, if you have a red solution, appropriate known solutions might be a red food dye in solution and a solution of red Kool-Aid. Your instructor will have appropriate comparison solutions available for use. Position Solution to spot 1 Kool-Aid standard 2 Your unknown sample 3 Red food dye standard 4 Caffeine standard Figure 2: Example of TLC plate loading 3. Prepare your TLC plate with samples as shown in the figure to the right, using a clean capillary tube for each of the four solutions. Place the used capillaries in the broken glass container for disposal. Be sure that you are recording what you are doing and what you are observing. 4. After the spots have dried, place your TLC plate in the appropriate TLC solvent tanks in the hood. If your sample was a red powder or solution, the appropriate solvent is methanol. The end closest to the line where your samples were spotted is the bottom end. The solvent should wet the TLC coating, but not come up to the pencil line. Allow the solvent to rise up the TLC plate until the top of the solvent is just below the top of the TLC plate. Do not move or open the tank during this process! Remove the plate from the tank and immediately mark with a pencil the top line of solvent. 5. Allow the plate to dry and then use your pencil to circle each spot you see and to label each sample. Also use the UV lamps provided to visualize spots that are not visible. Caution, do not look directly into the UV lamps! Use the rulers provided to measure the migration distances of different spots and the solvent itself, relative to the initial starting line. Use the center of the main part of the spot for this measurement. Also record the distance that the solvent traveled, from the starting pencil line to its maximum marked travel. For each of the four samples, record the distance that each spot traveled. If a sample contained more than one component, you will see more than one spot directly above its starting point. Record the distance traveled by each spot. 6. Sketch a picture of your TLC plate and its spots in your notebook, including the distances measured. Record observations on the spots colors and shapes. Identify your unknown as best you can, by comparing the spots of the unknown to the spot patterns of the known samples. Record this identification. Write your name on the back of your TLC plate. Your instructor will collect it. 6

7 FOR YOUR LAB REPORT Remember that you write up the lab report individually. Include the name of your in-lab partner on your cover sheet. RESULTS Begin the Results section of your lab report by giving the ID numbers of your unknowns. Summarize your results in your report, using the information below as a guide. Test 1, HPLC on the white solid - give the identity of the chemical or chemicals in your white powder sample. Test 2, GC/MS. You will get a computer print out the day AFTER your lab period. It should be available in room 465 by noon. Copies will be available for each member of the team. Look at the graph of the chromatogram. Each peak represents a different compound in your sample. How many compounds were found and identified in each of your samples? Next, look at the library search report, which identifies the chemical in each peak by comparing the mass spectrum to a reference library. The top 3 matches are shown for each chromatographic peak. For reliable compound identification, the match quality should be 80 or larger (shown on the report as Qual ). In your report, just report and discuss the one compound for each peak that matches best. Based on the chemical name, discuss the potential toxicity of the compounds found in this sample. Remember the information from the Group Discussion, about toxicity and about preliminary results from crime scene samples. Consult with your instructor if the list of compounds found in your sample is confusing, or if you are unsure about the toxicity of those chemicals. Test 3, TLC. First, calculate the R f values for each spot on the TLC plate using the equation. Prepare a four-column table, using Known A, Unknown Sample, Known B and Known C as the four columns (use the actual names for A, B, and C). In each column put the appropriate R f values for all the spots. One sample may have several spots, and thus several R f values. Below the table, include a sample calculation for R f. Different chemicals have different R f values, so the R f value is useful in identifying compounds once they are separated. Identify your unknown sample by comparing R f values to those of the different known samples. If your unknown doesn t match any of the knowns, we don t have a firm identification, but we know what it is not! Figure 3: Sample TLC plate DISCUSSION Summarize your findings for the three unknown samples in a ½ to one page essay. After stating the facts about what is present in these samples, discuss what your results mean about the guilt or innocence of the suspect. Try to explain how strong your evidence is, as you defend your conclusions. Be sure that you refer to information from the crime scene, and about what the suspects have stated the samples to be. Refer to your notes from the group discussion and the Forensic Science Lab Report. Your answer should be well organized. It will be graded for both correctness and clarity. 7

8 Be sure that either you or your lab partner includes the computer print-outs in the lab report. QUESTIONS 1. In an effort to identify an unknown, we run TLC and get the plate shown at right. a) How many compounds were in Sample A? In Sample B? In the unknown? Explain. b) A student concludes that the unknown is the same as Sample B because of the number of spots. Is this a valid conclusion? Explain. c) A second student concludes that the unknown is the same as Sample A because they both have spots with Rf values of 0.3. Is this a valid conclusion? Explain. d) Propose the most reasonable conclusion you can for the identity of the unknown. Explain your reasoning. A unknown B 2. Consider your three analysis results, focusing on what they mean about guilt or innocence. If you were able to analyze one more sample (with a single analysis method) from one of the suspects to clarify the guilt/innocence question, what sample would it be? State your choice, explain how it would be helpful to have that data, and why you think that piece of data is the most important one. 3. In several well-written paragraphs, summarize your understanding of chromatography: what it is good for and how it works. Compare TLC, GC, and HPLC, discussing several characteristics they share and several ways they differ. Support your general statements with examples you encountered in this experiment. (Re-read the Introduction for help.) 4. A block diagram for a GC-MS is shown below. Consult the Tro textbook, pp. 65, 568. a) Copy this diagram into your notebook. Label the following parts: Column, Detector, Injector/Injection port, Ion source, Mass analyzer, Oven. These are indicated by the numbers 3-5 and 7-9. b) Write a paragraph that explains how the instrument works, referencing the parts you labeled. 8

9 Crime Scene Visit Report Lab Partners: Provide an overview description of the scene. Do not exceed the space provided. What crime has apparently been committed? What kinds of chemical samples are available at the crime scene? How are those chemical samples the same and different from a sample you typically might receive at the start of a chemistry lab experiment? What questions might we ask about those samples? How will the answers to those questions be used in the investigation of the crime, and in a trial of suspects found by that investigation? What experimental problems might be expected for these samples compared to a pure sample of a chemical taken from a chemical reagent bottle? 9