Experiment 1: Thin Layer Chromatography

Experiment Description
In this experiment, you will experimentally determine which solvent is suitable for the separation of a mixture containing benzophenone, diphenylmethanol and biphenyl by thin layer chromatography (TLC) using silica gel adsorbent. Structures of these three compounds are shown below.

O

OH

Benzophenone

Diphenylme thanol

Biphenyl

Background: Thin Layer Chromatography
Chromatographic separations take advantage of possibility that substances will differently between two phases, a mobile phase and a stationary phase. You have already had some experience with gas chromatography where the mobile phase is an inert gas, usually helium, and the stationary phase is a high boiling liquid coating absorbed on the surface of a granular solid in a column. In thin layer chromatography, or TLC, the mobile phase is a liquid and the stationary phase is a solid absorbent.

Theory of Thin Layer Chromatography
In thin layer chromatography, a solid phase, the adsorbent (the stationary phase) is a powder which is coated onto a solid support, as a thin layer (about 0.25 mm thick). Thin plates of glass are the most common support, but plastic and aluminum can also be used. Like GC, TLC is usually used as a diagnostic tool it answers questions like “Is my reaction done yet?” You can watch the starting material go away and the product come in using TLC. “Is my product clean?” is another good question for TLC. It is also used to develop conditions for larger scale separations using a technique called column chromatography. In TLC, the mixture starts as a small spot near the bottom of the plate and the solvent (mobile phase) carries the compounds up the plate as it travels up from the bottom by capillary action. In most cases, the stationary phase (adsorbent) is very polar and the mobile phase (eluant) is fairly non-polar. Molecules that are more polar stick to the polar stationary phase more than fairly non-polar molecules which are carried along in the mobile phase. Keep in mind that this

Separation occurs because some things spend a higher percentage of the time standing still.is an equilibrium. more polar solvent is added. Drug purification frequently employs this technique. As the eluting power of the added solvent increases. “Medium polar” solvents like diethyl ether and ethyl acetate may be used 1%-50% combination with hexane making these mixtures very tunable and common. a drop or two is more common. but polar solvent molecules are also adsorbed by the stationary phase. Other common polar adsorbents include alumina. Stronger eluting solvents like methanol cannot be used as more than 10% of the solution or the silica gel will dissolve in them causing problems with the separation. Several factors determine the efficiency of a chromatographic separation. Thus. this is known as “reverse phase” chromatography because the nature of the stationary and mobile phases is inverted. More than 1% of pyridine or acetic acid isn’t often necessary. As you can see in the list provided below. Non-polar adsorbents may also be used with relatively polar solvent systems. charcoal and Florisil. Least Eluting Power Hexane or Pentane Cyclohexane Benzene Dichloromethane Chloroform Ether (anhydrous) Ethyl acetate (anhydrous) Acetone (anhydrous) Ethanol Methanol Water Pyridine Acetic Acid Greatest Eluting Power The non-polar solvents at the top of the list are often used as a base and a few percent of a stronger eluting. The “Eluting power” of a solvent is largely a measure of how well it is adsorbed onto the stationary phase. all molecules do a little of both. The adsorbent and solvent system chosen are the easiest factors to change. adsorbed on the stationary phase than others do. . there are many choices for solvents and solvent mixtures are quiet common. Silica gel (SiO2) is a very commonly used. but it does include the most commonly used ones. displacing other molecules. strongly polar adsorbent material that you will be using for these experiments. The most common factor that is adjusted to achieve good separation is the solvents used in the mobile phase. the amount that is generally added decreases. Molecules that are already adsorbed are displaced and “pushed along” by polar solvent molecules. The substances being separated are adsorbed onto the stationary phase. Eluting solvents for chromatography Note: This is not a complete list of possible solvents. everything moves up the plate faster in more polar solvent systems.

You may also want to label the positions where your spots will be if . Polar groups.while these two additives are next to each other on the list. 0. having different Rf values demonstrates that they are different. The mixture that you are separated is dissolved in a solvent. Table 3. aIkenyl halides Aromatic hydrocarbons. For a particular adsorbent/solvent/compound combination. First. carboxylic acids and amines. The functional groups of the molecules in your mixture effect how strongly they are adsorbed by the stationary phase. using a pencil gently draw a line across the bottom edge of the plate. Using a very thin glass tube. as shown in the figure. with oxygen and especially nitrogen are more strongly adsorbed. usually made entirely of carbon an hydrogen. This ratio is called the Rf value. they can have very different effects on a separation depending on the functional groups in the molecules being separated. Adsorbability of organic compounds by functional group Least Strongly Adsorbed Saturated hydrocarbons. The ability to hydrogen bond with the silica gel creates a strong adsorbing interaction in alcohols.5-1. Water is very strongly eluting and its presence as an impurity in your solvent can be problematic. aryl halides Polyhalogenated hydrocarbons Ethers Esters Aldehydes and ketones Alcohols Acids and bases (amines) Most Strongly Adsorbed TLC is useful because it is reproducible. the ratio of the distance the compound travels to the distance the solvent travels remains constant. Very “greasy” non-polar substructures. called a capillary you can put a tiny amount of solution in a specific spot on the plate. Rf value = distance traveled by substance distance traveled by solvent front) While having the same Rf value (under the same conditions) does not prove that two substances are the same. are hardly adsorbed by silica gel at all. Techniques: Thin Layer Chromatography Spotting Thin Layer Chromatography takes only a very small quantity of compound. alkyl halides Unsaturated hydrocarbons.0 cm up from the bottom.

The paper will soak up some of the solvent and help to saturate the atmosphere inside the jar with solvent vapor. below spots . just a few millimeters in diameter. you don’t want it to be TLC Plate Before it has Been Developed Spots of Compound Pencil Line Std Mix Developing a TLC Plate Cap the Jar Filter paper wet with solvent TLC Plate Leans against Wall Solvent level. If you need a heavier spot. rinse it with the solvent you’re going to put in it. (sometimes called a “TLC Spotter”) into the solution of your mixture and then briefly touch it to the plate. A simple number or letter code is probably best. As shown in the diagram above. Pour a small amount of solvent into the jar.you’re using more than one. Be careful not to scrape off too much adsorbent from the plate. Dip the tip of the capillary. place a clean dry piece of filter paper (cut if necessary) standing up against the wall of the jar. If it isn’t. This will make your plate run faster by slowing the evaporation of solvent from the plate. Keep the spot small. Developing Make sure that the jar you’re using is dry inside. Do not rinse it with water. Just a few milliliters. wait for the solvent to evaporate and spot again.

You should draw each plate in your notebook as part of your record of the experiment. Visualization One of the most common ways to visualize compounds on a TLC plate is to use a UV light. pull it out with your forceps and immediately mark the solvent front with a pencil line. Trace around any spots that you see so that you’ll know where they are later. Take your plate and a pencil to the UV lamp provided in the lab and look at it under the light. Compounds that are not UV active can be seen by staining the TLC plate.51. and spots appear. try again spotting less compound this time. The TLC plates that we are using have been treated so that they fluoresce a green color under UV light. if the plate runs too far. causing dark spots to appear where the compound is. If you can’t see anything.so deep that you’ll dunk your spots! Tip the jar to wet the paper with the solvent. Be careful not to let the solvent level get above the line at the bottom of the plate. you either accidentally washed the spots off in the solvent or didn’t spot enough to start with. The aromatic rings in the compounds that we are separating absorb the UV light before it reaches the fluorescent compound. This could take a while. The plate is sprayed with or dipped into a solution and then heated. or have long tadpole tails. Place a cap on top of the jar. especially if you have a less volatile solvent.0 cm from the top of the plate. you have to start over. If your compound streaked. When the solvent front has gotten 0. There is no need to screw it on. Place the TLC plate gently into the jar. you may want to use your forceps for this. so be patient and keep an eye on it. Remember. Reactions which cause a color change occur with the compound on the plate. TLC Plate After it has Been Developed Pencil Line to mark Solvent Front Separated Compounds (May not be visible) Std Mix Visualization of a TLC Plate with a Hand-held UV lamp . or your spots are huge. Wait and watch the solvent front travel up the plate.

The relationship between the distance traveled by the solvent front and the substance is usually expressed as the Rf value: Rf value = distance traveled by substance distance traveled by solvent front) Rf = B/A Distance the Solvent Traveled =A Distance the Compound Traveled = B Std Mix Using a Ruler measure both distances in millimeters and record the Rf values . is a constant.Calculating Rf Values The distance that a compound travels in a specific chromatography system. compared to how far the solvent has traveled.

From the solvents listed below.Experimental: O OH Benzophenone Diphenylme thanol Biphenyl Pre-lab Questions: 1. 2. Keep in mind that phenyl groups are fairly “greasy” and non-polar. Table: Eluting solvents for chromatography Least Eluting Power Hexanes (a mixture of isomers) Toluene Dichloromethane Diethyl Ether Ethyl acetate Acetone Ethanol Methanol Water Acetic acid Greatest Eluting Power Note: Bring a ruler and pencil to lab for this experiment . predict the order in which the compounds will move up the plate from highest Rf (fastest moving) to lowest Rf (slowest moving). diphenyl methanol and biphenyl (structures above) using TLC on silica gel. Assuming you separate the above compounds by TLC using your solvent of choice. select a single solvent that you think would give you the best chance of separating benzophenone. Explain the reasoning behind your choice.

measuring in millimeters to the center of the spots. The standards are separate. gently circle the spots that you see with your pencil and make a sketch in your lab notebook of the appearance of each plate showing the positions of all spots. Test the new solvents by preparing and developing two additional plates. as with the first. 2. After developing the plates. prepare one final TLC plate in which you spot the mixture of three compounds sideby-side with each of the three standards. consider how the polarity might be changed from the solvent that worked to improve the separation. indicate the position of the solvent front with a pencil mark. 3. Be sure to label the spots. If both of the solvents you tested in the first place provided separation of all three compounds. then. one in each of the different solvents. make sketches of each TLC plate showing the positions of the spots. diphenylmethanol. identified solutions of each compound. once with the mixture and once with each of the three standards. Be careful not to contaminate the standards. Then select two new solvents that you believe will change the polarity in the right direction to achieve separation. . Prepare a TLC chamber with each solvent. and biphenyl. In the second experiment. based on the appearance of each plate and the relative polarity of the two solvents. Test two new solvents to see if you can improve the separation. based on the appearance of each plate. There is more than one solvent which will achieve this separation. If neither of the first two solvents separated all three compounds on the TLC plate. but the other didn’t. choose two solvents to start with. and allow the plates to air dry for a few minutes. Depending on the results of this first TLC experiment.Based on your answers to pre-lab question 1. decide if the solvent used was too polar or not polar enough. Then develop this final plate using the best solvent from your earlier experiments and compare Rf values to confirm the identity of the three spots in the mixture. The plate you prepare will then be spotted four times. remove them from the chamber. Spot two TLC plates using the dichloromethane stock solution of the mixture containing benzophenone. as described in the Techniques section. and calculate Rf values. Calculate Rf values for each spot. design and conduct a second TLC experiment using two other solvents: 1. Develop the plates simultaneously. Examine them under UV light. conduct a second experiment to determine if the separation can be improved by increasing or decreasing the polarity of the solvent from the two solvents that worked. If one of the first two solvents separated the three compounds on the TLC plate. After conducting at least two pairs of TLC experiments (developing four plates) as described above and finding one or more solvents that can be used to separate the three compounds in the mixture. then.

In your lab report: Report the Rf values of the spots in each plate that you ran. conduct additional experiments (as time permits) until you achieve a good separation of all three compounds. Explain why they elute in the order that they do. Identify the components of the mixture. Discuss the decisions that you made to choose a solvent system which separated the mixture. You should have used your knowledge of the principles of chromatography to guide your choices. and make sketches of all plates in your lab notebook.If you were not able to achieve a very good separation of the compounds in the first or second experiment. Be sure to carefully consider the results of all previous experiments to make the best choice of solvents. ! .

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