Experiment 3: Analysis of Unknown Hydrocarbons by GC, NMR, and Chemical Tests for Unsaturation and Aromaticity In this experiment you will be determining the structures and checking the purity of the hydrocarbon distillates that you collected in Experiment 2. You will analyze the 1H NMR spectra and obtain gas chromatograms of your best fractions. You will also test for the presence of double bonds (unsaturation) in the compounds using a bromine test, and for aromaticity using an aluminum chloride test. In the bromine test, the presence of one or more alkene double bonds in a structure is evident from the disappearance of the reddish brown color of the bromine solution. The bromine adds to double bonds to form the dibromo substituted species as shown below: CH 2 Cl 2 C C + Br 2 C C Br Br In the aluminum chloride test, the presence of aromatic ring compounds is detected by formation of highly colored triarylmethane cation. The parent compound of these cation is a triarylmethane formed as shown in the example below with benzene. There are a large number of resonance structures that may be drawn to distribute a positive charge across the molecule. How many can you draw? AlCl 3 CHCl 3 3 H 3 HCl 34
Background: Gas Chromatography: Chromatography refers to a group of techniques for separating mixtures based on the different ways that they interact with stationary phase and a mobile phase. The molecules are divided or partitioned between the two phases. Molecules that interact more strongly with the stationary phase will move more slowly than those that interact more strongly with the mobile phase. Depending on the type of chromatography molecules may be separated based on a wide variety of differences in characteristics such as polarity, molecular weight, and ionic character, just to name a few. Chromatographic techniques are some of the most powerful tools in chemistry for both analysis and diagnostics. Drugs are purified by huge chromatography columns. Single proteins can be isolated from complex organisms. Drug testing often involves a combination of chromatography and mass determination. You already have some experience with thin layer chromatography from experiment 1. Gas chromatography (GC or GLC) is a common chromatographic technique in which the solutes are partitioned between a mobile gas phase (the carrier gas) and a stationary liquid phase present in the column. GC columns are very long, very thin capillaries that are coiled up inside the gas chromatograph. To get separation, your sample travels a very long distance. The gas-phase concentration of a solute depends on the vapor pressure of the solute, as does the boiling point of the solute. GC, then, is basically a separation method dependent on differences in boiling point of the components in a sample. You could think of it as a high efficiency distillation, with a very long fractionating column. This is, however, an overly simplified view. Gas chromatography is superior to an ordinary distillation in several ways. It is much more rapid than distillation, much more efficient (separation via GC is possible of solutes whose boiling points differ by no more than 2 ) and, most importantly, GC analysis requires only about 1-2 microliters (= 10-3 ml) of sample. GC can be an important tool for use in determining trace constituents in a sample mixture, and is especially useful where only tiny amounts of sample are available. It is not good, however, for separating or purifying large quantities of material. The five basic parts of a gas chromatograph are shown in the diagram below: 1. Carrier gas: usually He, sometimes N 2. 2. Injector: a T in the carrier gas line, one branch of which is closed with a rubber septum, allowing sample injection. 3. Column: usually metal, like a very long hollow needle, sometimes glass or teflon; packed with stationary phase (a high boiling liquid like stopcock grease) distributed over a material with high surface area (sand, firebrick, diatomaceous earth). The column is usually in an oven which keeps it at a specific temperature. Changing the temperature can change the quality of the separation. 4. Detector: most often is of a thermal conductivity type, shown in the diagram below. Two states are possible in the detector: either it sees only carrier gas (State I) or a mixture of carrier gas and an eluted component (State II). The detector itself is a filament which is heated 35
electrically; like a light bulb filament. When pure carrier gas flows through the detector the heat produced in the filament is carried away by the carrier gas, and the filament temperature is stable. In state II, however, some component is eluted, altering the thermal conductivity of the gas in contact with the filament. If the carrier gas is a light gas (H 2 or He) with high thermal conductivity, any solute present in the carrier will decrease its conductivity, so the filament heats up and its resistance changes. This change in resistance is converted to a change in voltage, which is plotted as a function of time, resulting in the chromatogram. It should be noted that the magnitude of the response from the detector is not the same for all compounds. To accurately quantify the components of a mixture using GC this difference in response factors must be accounted for. More on this when you need it. 5. Recorder: In this case, a laptop that plots and saves the data coming in from the detector. In the old days, there was a mechanical plotter that performed the same function. A schematic diagram of a gas chromatograph The GC has several temperature controls to maintain constant temperatures. Do not adjust these controls, they should be preset for the conditions that you need for the experiment. a. Injector: to vaporize the sample so that it is present in the gas phase and hence can enter the column. This means that the injection point is HOT and you can burn yourself if you touch it. b. Column: so that the separation can be performed at temperatures above room temperature. c. Detector: to maintain constancy of the detector s temperature to avoid signal drift, and so that solutes don t condense in the detector. The ability to operate at higher temperatures extends the boiling point range over which GC is a useful analysis technique. As long as the sample can be vaporized to be loaded onto the column, we can use GC to separate the mixture. However, heating some samples may cause them to decompose (sometimes explosively!), in these cases GC is not an appropriate analysis technique. 36
Gas in metal block filament exit Thermal Conductivity Detector Chromatographic Analysis In terms of identifying a particular substance, GC is semiqualitative since the information it yields is supporting rather than conclusive. This information is the time that it takes the compound to travel through the column, called the retention time. The retention time is measured from the point of injection to the maximum of a peak eluted from the column (see illustration below). The retention time is characteristic of a substance on a particular column, at a given temperature and flow rate of carrier gas. Retention times decrease as the temperature or flow rate is increased. Since these variables are not easy to reproduce precisely, the retention time may vary slightly from one injection to the next. For that reason, it is sometimes convenient to measure the retention time relative to a known standard that is deliberately added to the mixture. The ratio of retention times will be independent of experimental variables, and this ratio can be useful in identifying an unknown substance. Modern analytical laboratories use more expensive instruments that allow precise control over GC temperature and flow rate. The retention times from such instruments are more reliable, and can often be used to identify unknowns. Two properties from a given chromatogram may be measured: (l) the distance on the recorder chart that a given component requires to be eluted, (i.e., the retention time) and (2) the relative size (area) of the peak produced by a component. 37
A Sample Chromatogram The first property, (the retention time) yields supporting qualitative information, while the second property (the magnitude of the elution peak) is proportional to the amount of the component of the mixture present in the peak. Although different components have differing thermal conductivities, in this experiment the differences are small enough that we will assume that peak area ratios correspond to molar ratios. This assumption does not work for more sophisticated GC instruments that use other types of detectors. For these highly sensitive instruments, a correction for peak areas has to be made using the equation: peak area = R x (moles of component) where R is the response factor. In principle, R is different for each component. Hence a chromatographic process must be calibrated using standard mixtures of known concentration (i.e., R values must be determined for each component) before exact quantitative data are feasible. The values of response factors are usually between 0.8 and 1.2, so a reasonable estimate can be made simple by measuring the peak area. Most peaks are approximately the shape of an isosceles or a right triangle, whose areas are simply: A = 1/2 x base x height 38
The measurement of the base of most GC peaks is difficult because more abnormalities in the shape occur in this region than in any other. A more accurate estimate of peak area is: A = height x width-at-half-height height of alcohol peak = 167 mm height of ester peak = 172 mm isoamyl alcohol isoamyl acetate width at half of the height of alcohol peak = 3.0 mm width at half of the height of the peak = 3.5 mm area of alcohol peak = 3 x 167 = 501 area of ester peak = 3.5 x 172 = 602 inject air where instead of measuring the width of the base and multiplying it by 1/2, you simply measure the width of the peak at half of its height (see illustration above for isoamyl alcohol and isoamyl acetate). The following equation is often useful: Areaof peak B %B AREA = allpeak areas 39
A calculation of %B yields the fraction of the total area that is due to B; this value is often close (to within 5-10%) of the weight % of B in the original sample, if a thermal conductivity detector is used. Therefore a chromatogram of a mixture allows: (1) rough calculation of component amounts and (2) exact calculation of the same amounts if the system has been calibrated Note that only those components which are volatile at the operating conditions of the chromatograph will be eluted and hence determined. Nonvolatile components will remain at the head of the column and their existence may not be detected without other information. Techniques: Making a GC Injection: The needle of the GC syringe is easily bent and damaged, handle it with care. Rinse the syringe once with your compound by pulling compound in and then squirting it out into the provided waste container, a small flask or beaker by the GC. Draw 2 microliters of sample followed by 2 microliters of air into the syringe. Push the syringe needle all the way into the septum at the injection point. The GC has two injection points, make sure you re using the right one! Caution: The injection point is HOT and may cause burns if you touch it. Inject the sample in one rapid smooth motion and immediately hit the green Go button in the GC software. You ll probably have to hit the no, I don t want to save the previous data button as well before it will start collecting data. You may want to have a colleague hit the computer buttons when you ve injected. While you wait for the sample to run, rinse the syringe two or three times in the provided solvent, probably acetone. Let the GC run, recording data until both product peaks have eluted. This shouldn t take much more than about 5 minutes. If after 5 minutes you still haven t seen a signal, talk to your TA for help trouble shooting the system. When you re done, hit the Stop button. You may need to re-scale the window so that the tops of the peaks are visible or they re a reasonable height to measure. Use the Rescale menu option. To Print, choose Print Current Window. Using the balance: If you are weighing a chemical, place a weighing boat or a piece of weighing paper with a crease down the middle on the balance pan and tare the balance. Gradually add the reagent to the weighing boat using a clean spatula until you have the desired quantity. Close the reagent bottle and put it back where it belongs. Clean up any spilled reagent. Take your weighed reagent back to your bench to use. Balances are fragile and precise instruments which work best if they re treated gently and kept clean. Please be patient with them, they can take a long time to tare, especially the one in the hood. To have the balance in the hood work faster, make sure that all of the glass doors are closed so that the pan is protected from the wind. 40
Working in the Hood Fume hoods are there for your protection. They work best if as many of the doors as possible are shut. If (as you are in this experiment) you are instructed to work with reagents in the hood, do not remove them from the hood. Procedure: Run the following tests on 3 samples: Your most pure low boiling fraction, most pure high boiling fraction (use the same fractions that you took NMRs of), and the sample of the original mixture that you saved. Note: you can do these three tests in any order there is no need to do the bromine test first. A. Bromine Test Safety Precaution: Bromine is volatile, toxic, irritating to the respiratory system and causes burns. Handle the Bromine Solution IN THE HOOD. Use GLOVES. Test each of the distillation fractions for the presence of alkenes using a test solution of 2% bromine in dichloromethane. Dissolve 2-3 drops of your sample in 0.5 ml of dichloromethane (CH 2 Cl 2 ). Carefully add the bromine test solution dropwise IN THE HOOD while gently shaking and swirling the test tube to mix the solutions and count the number of drops needed to produce a visible, orangebrown color. As a control experiment, repeat the test with pure dichloromethane in the test tube to decide whether your unknown behaved differently. What should happen if there are alkenes in the reaction mixture? Which (if any) distillation fractions contain alkenes? If you have doubts about the results of your test, run the test with a sample of one of the alkene reference compounds to compare results. As always, record all observations on your laboratory notebook. Place the entire reaction mixture in the halogenated waste container after you are done with the tests. B. Test for Aromaticity Safety Precaution: Aluminum chloride is highly reactive. It reacts rapidly with water to produce hydrogen chloride gas. The moisture in the air is sufficient to produce HCl fumes which are strongly acidic and irritating. Perform this test IN THE HOOD. Wear gloves. Place 1 ml of chloroform (CHCl 3 ) in a dry test tube; add 0.1 ml of the liquid sample and mix thoroughly. Weigh 0.25 g of anhydrous aluminum chloride in the hood using the aluminum 41
weighing boats provided. Tilt the test tube to moisten the wall of the tube. Then add the anhydrous aluminum chloride so that the powder strikes the wetted side of the test tube. Note any color change of the powder and the solution. Try the test with reference compounds available (possible components of your unknown mixture). Place the entire reaction mixture in the halogenated waste container after you are done with the tests. As always, record all observations on your laboratory notebook. C. Gas Chromatography Obtain a gas chromatogram of the original mixture from the unknown distillation experiment. The chromatography column conditions and instrument settings should be such that there is baseline separation between the two peaks (if this is not the case, talk to your TA). Determine the retention times of the two peaks and the relative amounts of the two compounds from the peak areas. Next, obtain a gas chromatogram of your two best distillation fractions to determine how successful you were at separating the two compounds. How different are the amounts of each component in the distillation fractions compared to the original mixture? Make sure that you wait long enough for the second peak to have come off the GC, that s why you ran the mixture first. Wait for the chromatogram to return to baseline for a bit before stopping the trace. Determine the retention times of the two peaks in each of your fractions. Compare the retention times of each component in the distillation fractions and the original mixture. Did they change? Explain why or why not. We will assume equal response of the detector to each compound therefore the relative amounts of the compounds are proportional to their peak areas. Record the estimated composition of your two best distillation fractions in your notebook. If time permits, obtain a gas chromatogram on the reference compounds that you believe to be the same as your high and low boiling compound to see if the retention times are the same. D. 1 H NMR Analysis Examine the 1 H NMR spectra of your two best distillation fractions. In each spectrum, look for signals in the alkane, alkene, and aromatic regions to try to determine which type of compound the major component is. Try to identify the major compound in each fraction from its NMR spectrum. Be sure to consider the chemical shift of the signals, the integrations, and any recognizable splitting patterns. Refer to the provided 1 H NMR spectra of the reference compounds to make a positive identification of the compounds you separated. 42
Compare the NMR spectra of your two fractions. How good was your separation? Can you detect any of the higher boiling compound in the NMR spectrum of the lower boiling fraction and vice versa? Do the relative amounts of the two compounds as indicated by NMR agree with your GC results? In your lab report: Identify the components of your unknown mixture. Support your conclusion with data from each of the tests and your NMR analysis. Your discussion should convey an understanding of how each of the tests works and what your results mean in terms of molecular structure. Don t forget to mention which unknown you had! Analyze your NMR by writing the structure of the compound on the spectrum and indicating which peaks correspond to the different hydrogens on the molecule. Letter the Hydrogens and then mark the peaks with the appropriate letter. Briefly discuss why the signals occur where they do and display the splitting pattern observed. Using the integration in the NMR, estimate the product ratio for each of the two purified samples. How successful were you at purifying your mixture? Assuming that the response factors are the same for each of the two components of the mixture, use the area of the peaks to determine the product ratios. Do these product ratios match those that you found using the NMR of the same samples? If they don t, which do you think is the better estimate of the purity of your fractions and why? 43