Experiment: Thin Layer Chromatography Chromatography is a technique widely used by organic chemists to separate and identify components in a mixture. There are many types of chromatography, but all involve the same basic principles. You should have received some instruction about chromatography in lab lecture, so what follows is a summary version of how chromatography works. Any chromatographic separation involves a stationary phase and a mobile phase. The stationary phase, as the name implies, does not move within the chromatographic system. It is either a solid or a viscous liquid held onto a solid support. The mobile phase, either a liquid or a gas, constantly flows through the particles that make up the stationary phase. The materials that undergo separation by chromatography have different attractions for the stationary and mobile phases, and an equilibrium is quickly established in which each substance distributes itself between the two phases. Consider substance X, a substance being chromatographed, and its distribution between the mobile and stationary phases. Xstationary phase Xmobile phase The direction in which this equilibrium lies depends on how strongly X is attracted to the two phases. These attractions are the typical attractions we see all the time in organic chemistry: London dispersion forces, dipole-dipole forces and hydrogen bonds. For molecules that are more strongly attracted to the stationary phase than to the mobile phase, the above equilibrium lies to the left. For molecules more strongly attracted to the mobile phase, the equilibrium lies to the right. Consider a solution having two solutes, A and B. Solute A is very polar, capable of hydrogen bonding while B is nonpolar, capable of forming only London forces with other molecules. Assume that the solid stationary phase of the chromatographic system is also very polar, while the mobile phase is nonpolar. [ote: Stationary phases do not have to be polar and mobile phases do not have to be nonpolar. They are described this way for this example to help you see how chromatography separation occurs.] Solute A when it comes into contact with both phases will be more attracted to the polar stationary phase. Its distribution equilibrium Astationary phase Amobile phase will lie to the left, on the stationary phase side. Solute B on the other hand will have a distribution equilibrium that lies to the right.
Bstationary phase Bmobile phase Because B spends a greater part of its time associated with the mobile phase, it will move more quickly through the chromatographic system. A, because it spends a greater part of its time associated with the stationary phase, does not move nearly as fast through the system. The separation of A and B in the above example shows how all chromatographic systems work. They separate components based on the differences between the components affinities towards the two phases. In this experiment, you will be working with a type of chromatography called thin layer chromatography, or TLC for short. The chromatographic separation takes place on thin sheets of plastic coated with a thin layer of stationary phase. The stationary phase can be many different solids, but the most common ones are silica gel (SiO2) and alumina (Al2O3). Alumina is more polar than silica gel, so it is used primarily for separating relatively nonpolar substances (hydrocarbons, ether, aldehydes, ketones, and alkyl halides) because better separation of components can be achieved. Compounds more polar than these would potentially bind too tightly to the alumina stationary phase, resulting in little or no migration up the TLC plate. To avoid this, silica gel is used to separate these more polar substances, ones that can hydrogen bond such as alcohols, carboxylic acids and amines. These substances will bind less tightly to the less polar silica gel stationary phase, so the compounds will move up the TLC plate with good separation, provided a good solvent system is selected. This solvent system, the mobile phase, is a liquid whose composition can vary widely depending on the substances being separated. In this experiment, you will be seeing the effect of different mobile phases on the effectiveness of the chromatographic separation. A solution is prepared of the mixture you are trying to separate dissolved in a low boiling solvent. Using micropipets, you will spot a tiny amount of this solution on the stationary phase so that the components to be separated are now adsorbed onto the stationary phase, the TLC plate. The TLC plate is then placed vertically in a container called the developing chamber. The bottom of this container is covered with liquid mobile phase to a depth of a few millimeters, but must be below the spot on the TLC plate. A filter paper is also placed in the chamber to ensure that the chamber is saturated with solvent vapor to prevent evaporation from the TLC plate during elution. The mobile phase rises up the solid stationary phase by capillary action and soon comes into contact with the spot of the sample. The components of the sample now partition themselves between the two phases and begin to move up the plate at a rate dictated by their affinities for the two phases. Those components more attracted to the stationary phase move more slowly and those more attracted to the mobile phase move more quickly. The mobile phase rises up the TLC plate fastest of all. When the solvent has almost reached the top of the plate, the plate is removed from the tank and a mark is made at the point near the top of the plate to which the solvent has moved. This point, marking the distance that the solvent traveled from the spot of sample, is called the solvent front. The question we are now faced with is, How far did the components in the sample move? If the components are colored, seeing them is easy. But if, as is more often the case, they are colorless, seeing them against the white stationary phase is impossible. There are many ways to help us see these invisible components. One way, the one that we ll be using, is to use a plate
whose stationary phase material is mixed with a fluorescent dye that glows in ultraviolet light. When UV light is shone onto a piece of such a treated TLC plate, it will glow green. During the chromatographic process, when one of the colorless components rises as a spot up the plate to a certain point, the spot will, when the plate is exposed to UV light, mask the fluorescent plate and appear as a dark spot against the green background. The ratio of the distance that a spot traveled compared to the distance that the solvent traveled is called the Rf value. distance traveled by the spot Rf = distance traveled by the solvent front The Rf value for a given component will be constant for a given chromatographic system of stationary and mobile phase. As a result, components being separated by TLC can be identified by their Rf values so long as the stationary and mobile phases of the chromatographic system are the same. In this experiment, you will be using thin layer chromatography to identify the components in an over the counter pain reliever. You will be identifying them by comparing their Rf values with the Rf values of four of the ingredients found in pain relievers: aspirin, acetaminophen, caffeine, and ibuprofen. The structures of these four compounds are given below.
COOH CHCOOH O C CHCH 2 aspirin O ibuprofen O H H 3 C O HO O acetaminophen caffeine Pre-lab Preparation Before coming to lab, you must do the following: 1. Explain how TLC separates compounds. Clearly indicate why compounds travel at different rates up the TLC plate. 2. A spot traveled 2.5 cm on a TLC sheet while the solvent front traveled 9.9 cm. What is the Rf value for the spot? Show your calculations. Experimental Procedure! Safety Considerations! Ultraviolet radiation is dangerous. The UV lamp has been placed in a viewing box with UV filters on the eyepieces. Looking at UV light through these eyepieces is safe. Do not remove the lamp from the viewing box and do not look directly at the UV radiation, the Blue light. It can permanently damage the eyes and lead to blindness. ever look directly into a source of ultraviolet light.
In this experiment, you will be developing three identical sets of spots using three different mobile phases. Consequently, you will prepare three identical TLC plates with the same set of spots. You will develop each plate in a different mobile phase. The most efficient way to do this is for you and your partner to spot the three plates at the same time and then develop them in the different mobile phases at the same time. 1. Use gloves at all times when handling the TLC plates to avoid contamination. Even with gloves, you should try to touch only the edges of the TLC plate. Take three pieces of the pre-cut silica gel TLC sheet and draw a light pencil line approximately one centimeter from the bottom on each of them. Draw these lines on the side of the plate which is coated with the silica; the silica gel will look like a white powder. Do not work on the shiny, plastic side of the plate. These lines, the origin lines, will be the lines on which you spot your reference compounds and your unknown pain reliever. You will be making five spots on each of these origin lines, evenly spaced. ( ote: ever use ink to draw your line because the ink, which is made of many pigments, may chromatograph and obscure your spots.) Prior to spotting your known and unknown samples, draw a picture of a TLC sheet in your notebook. Draw in the origin line and the location of each of the five spots you will be making in steps 4-6 below. The make certain that you have spotted your TLC plate exactly the same way that you drew it your notebook diagram. 2. Using a clean micropipet for each known standard, insert the small end of the pipet directly into the vial containing one of the known compounds. The standard solution will be drawn up into the micropipet by capillary action. Using a piece of scrap TLC sheet, practice your spotting technique. Since spots tend to get larger as they move up the plates, you want the spots that you make to be as small in diameter as possible. Take your filled micropipet and lightly touch the silica gel on a spot on the practice sheet. Practice until you become proficient at making small spots, approximately 0.5 mm in diameter. 3. Once you have become proficient at making small spots with your micropipet, refill your micropipet with the same known standard solution and lightly touch the silica gel on the origin line at the place you have designated for this compound. Gently blow on the spot to evaporate the solvent. Repeat this with the same standard solution several times on exactly the same spot. After 4-5 spottings, there should be enough standard on the spot to be visible under ultraviolet light. Repeat this with this same standard solution on the other two TLC sheets. 4. Repeat step three for the other standards. You should now have three identical TLC sheets, each with four spots evenly spaced along the pencil line, one for each of the four standards. There should be one space remaining on each sheet for your unknown analgesic. 5. There are several samples of ground analgesic tablets that have been prepared for you. Your instructor will assign each pair of students one of them. In your notebook, record
the number of the sample which has been assigned to you. Take approximately 0.1 gram of the powder and mix it thoroughly with 1 ml of ethanol. The ethanol will dissolve the active ingredients of the analgesic powder, leaving the inactive filler material undissolved. Decant the ethanol solution and use it to spot the three TLC plates next to the four standard spots. 6. After you have made all five spots, take the three TLC sheets to the ultraviolet lamp. Turn the lamp on and slide the sheets under the rubber flap. Look through the eyepieces to observe the spots. The spots should appear as dark spots on a fluorescent green background. If the spots are faint, continue to spot them with the standards or with your unknown analgesic solution at the same positions until all spots are sufficiently intense. 7. Into each of the chromatographic containers, place a piece of the cut filter paper into the beaker so that the side of the beaker is partially lined by the paper. This will insure that the air is saturated with solvent vapor. Pour enough (~15 ml) of one of the three mobile phases or developing solvents into the container so that the paper is completely saturated and the liquid level is approximately 0.5 cm deep. Cover the beaker with foil. Repeat this procedure by pouring one of the other two developing solvents into each of the other two chromatographic containers. In your notebook, write the composition of each of the solvent mixtures as noted on the bottles. 8. Uncover the chromatographic container and place the TLC sheet in it so that the origin line is just above the liquid level. The stationary phase side of the TLC sheet should be facing you. Do not allow the side edges of the sheet to touch the beaker walls or filter paper. This will cause the solvent to travel unevenly up the sheet. Cover the chromatographic container. The liquid will rise up to the spots and the chromatographic process will begin. 9. Because the analgesic compounds are all colorless, you will not see the spots move, but you will be able to see the solvent advancing up the sheet. When the solvent front gets to approximately 1 cm from the top of the sheet, remove the sheet from the chromatographic container and immediately mark the solvent line before it begins to disappear due to evaporation. The easiest way to do this is with a pencil mark on each side of the solvent front. After the sheet has dried, draw a pencil line across the top of the sheet to connecting these marks. 10. Take the sheet to the UV lamp and observe the spots. With a pencil, trace the outlines of the spots. Measure the distance from the origin to the center of each spot and record this distance neatly in your notebook. Also, measure the distance that the solvent front traveled from the origin and record this distance in your notebook. 11. When you have completed the experiment to your satisfaction, dispose of the developing solution in the designated waste container.
Post-Lab and Report Requirements On a sheet of white paper, turn in the following material at the end of the period. Your instructor will tell you how many points it will be worth. 1. Write down the distances traveled by all spots and the solvent front on each of your three chromatograms. 2. Calculate the Rf values for all of the standard spots on each of the three chromatograms. 3. Identify, based on the Rf values of the standards and the Rf values of the spots on your unknown, the active components of your unknown analgesic powder. 4. In this experiment you used three different mobile phases or developing solvents. Which one produced a chromatogram in which the four standards Rf values differed the most and which provided the best separation of the components in your unknown analgesic powder? Describe the chromatograms developed in the other two mobile phases. Give possible reasons why the other two solvents yielded less satisfactory chromatograms. 5. Aniline and toluene were analyzed using silica gel TLC plates and hexane as the mobile phase. a. Write structural formulas for these two solids. b. Which of the two solids will have the larger Rf value? Explain why in detail.