Experiment 1: Thin Layer Chromatography

Experiment Description

In this experiment, you will experimentally determine which solvent is suitable for theseparation of a mixture containing benzophenone, diphenylmethanol and biphenyl by thin layerchromatography (TLC) using silica gel adsorbent. Structures of these three compounds areshown below.

O OH

Benzophenone BiphenylDiphenylmethanol

Background: Thin Layer ChromatographyChromatographic separations take advantage of possibility that substances will differentlybetween two phases, a mobile phase and a stationary phase. You have already had someexperience 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 granularsolid in a column. In thin layer chromatography, or TLC, the mobile phase is a liquid and thestationary phase is a solid absorbent.

Theory of Thin Layer Chromatography

In thin layer chromatography, a solid phase, the adsorbent (the stationary phase) is a powderwhich is coated onto a solid support, as a thin layer (about 0.25 mm thick). Thin plates ofglass are the most common support, but plastic and aluminum can also be used. Like GC, TLCis usually used as a diagnostic tool it answers questions like “Is my reaction done yet?” You canwatch the starting material go away and the product come in using TLC. “Is my productclean?” is another good question for TLC. It is also used to develop conditions for larger scaleseparations using a technique called column chromatography.

In TLC, the mixture starts as a small spot near the bottom of the plate and the solvent (mobilephase) 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) isfairly non-polar. Molecules that are more polar stick to the polar stationary phase more thanfairly non-polar molecules which are carried along in the mobile phase. Keep in mind that this

is an equilibrium, all molecules do a little of both. Separation occurs because some things spenda higher percentage of the time standing still, adsorbed on the stationary phase than others do.

Several factors determine the efficiency of a chromatographic separation. The adsorbent andsolvent system chosen are the easiest factors to change. Silica gel (SiO

2) is a very commonly

used, strongly polar adsorbent material that you will be using for these experiments. Othercommon polar adsorbents include alumina, charcoal and Florisil. Non-polar adsorbents mayalso be used with relatively polar solvent systems, this is known as “reverse phase”chromatography because the nature of the stationary and mobile phases is inverted. Drugpurification frequently employs this technique.

The most common factor that is adjusted to achieve good separation is the solvents used in themobile phase. As you can see in the list provided below, there are many choices for solventsand solvent mixtures are quiet common. The substances being separated are adsorbed onto thestationary phase, but polar solvent molecules are also adsorbed by the stationary phase.Molecules that are already adsorbed are displaced and “pushed along” by polar solventmolecules. Thus, everything moves up the plate faster in more polar solvent systems. The“Eluting power” of a solvent is largely a measure of how well it is adsorbed onto the stationaryphase, displacing other molecules.

Eluting solvents for chromatographyNote: This is not a complete list of possible solvents, but it does include the most commonlyused ones.

Least Eluting Power Hexane or PentaneCyclohexaneBenzeneDichloromethaneChloroformEther (anhydrous)Ethyl acetate (anhydrous)Acetone (anhydrous)EthanolMethanolWaterPyridine

Greatest Eluting Power Acetic Acid

The non-polar solvents at the top of the list are often used as a base and a few percent of astronger eluting, more polar solvent is added. As the eluting power of the added solventincreases, the amount that is generally added decreases. “Medium polar” solvents like diethylether and ethyl acetate may be used 1%-50% combination with hexane making these mixturesvery tunable and common. Stronger eluting solvents like methanol cannot be used as more than10% of the solution or the silica gel will dissolve in them causing problems with the separation.More than 1% of pyridine or acetic acid isn’t often necessary, a drop or two is more common,

while these two additives are next to each other on the list, they can have very different effectson a separation depending on the functional groups in the molecules being separated. Water isvery strongly eluting and its presence as an impurity in your solvent can be problematic.

The functional groups of the molecules in your mixture effect how strongly they are adsorbedby the stationary phase. Very “greasy” non-polar substructures, usually made entirely ofcarbon an hydrogen, are hardly adsorbed by silica gel at all. Polar groups, with oxygen andespecially nitrogen are more strongly adsorbed. The ability to hydrogen bond with the silicagel creates a strong adsorbing interaction in alcohols, carboxylic acids and amines.

Table 3. Adsorbability of organic compounds by functional group

Least Strongly Adsorbed Saturated hydrocarbons; alkyl halidesUnsaturated hydrocarbons; aIkenyl halidesAromatic hydrocarbons; aryl halidesPolyhalogenated hydrocarbonsEthersEstersAldehydes and ketonesAlcohols

Most Strongly Adsorbed Acids and bases (amines)

TLC is useful because it is reproducible. For a particular adsorbent/solvent/compoundcombination, the ratio of the distance the compound travels to the distance the solvent travelsremains constant. This ratio is called the Rf value.

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 twosubstances are the same, having different Rf values demonstrates that they are different.

Techniques: Thin Layer Chromatography

SpottingThin Layer Chromatography takes only a very small quantity of compound. The

mixture that you are separated is dissolved in a solvent. Using a very thin glass tube, called acapillary you can put a tiny amount of solution in a specific spot on the plate. First, using apencil gently draw a line across the bottom edge of the plate, 0.5-1.0 cm up from the bottom,as shown in the figure. You may also want to label the positions where your spots will be if

you’re using more than one. A simple number orletter code is probably best. Be careful not toscrape off too much adsorbent from the plate.

Dip the tip of the capillary, (sometimescalled a “TLC Spotter”) into the solution of yourmixture and then briefly touch it to the plate.Keep the spot small, just a few millimeters indiameter. If you need a heavier spot, wait for thesolvent to evaporate and spot again.

DevelopingMake sure that the jar you’re using is dry

inside. If it isn’t, rinse it with the solvent you’regoing to put in it. Do not rinse it with water.

As shown in the diagram above, place aclean dry piece of filter paper (cut if necessary)standing up against the wall of the jar. The paperwill soak up some of the solvent and help tosaturate the atmosphere inside the jar with solventvapor. This will make your plate run faster byslowing the evaporation of solvent from the plate.

Pour a small amount of solvent into thejar. Just a few milliliters, you don’t want it to be

TLC Plate Before it has BeenDeveloped

Std MixPencil Line

Spots of Compound

Developing a TLC Plate

Solvent level, below spots

Filter paperwet with solvent

Cap the Jar

TLC PlateLeans against Wall

so deep that you’ll dunk your spots! Tip the jar to wetthe paper with the solvent.

Place the TLC plate gently into the jar, youmay want to use your forceps for this. Be careful notto let the solvent level get above the line at the bottomof the plate. Place a cap on top of the jar. There is noneed to screw it on.

Wait and watch the solvent front travel up theplate. This could take a while, especially if you havea less volatile solvent. Remember, if the plate runstoo far, you have to start over, so be patient and keepan eye on it. When the solvent front has gotten 0.5-1.0 cm from the top of the plate, pull it out with yourforceps and immediately mark the solvent front with apencil line.

VisualizationOne of the most common ways to visualize

compounds on a TLC plate is to use a UV light. TheTLC plates that we are using have been treated so thatthey fluoresce a green color under UV light. Thearomatic rings in the compounds that we areseparating absorb the UV light before it reaches thefluorescent compound, causing dark spots to appearwhere the compound is. Take your plate and a pencilto the UV lamp provided in the lab and look at itunder the light. Trace around any spots that you seeso that you’ll know where they are later.

Compounds that are not UV active can be seenby staining the TLC plate. The plate is sprayed withor dipped into a solution and then heated. Reactionswhich cause a color change occur with the compoundon the plate, and spots appear.

If you can’t see anything, you eitheraccidentally washed the spots off in the solvent ordidn’t spot enough to start with. If your compoundstreaked, or have long tadpole tails, or your spots arehuge, try again spotting less compound this time.

You should draw each plate in your notebook aspart of your record of the experiment.

TLC Plate After it has BeenDeveloped

Std Mix

Pencil Lineto mark Solvent Front

Separated Compounds(May not be visible)

Visualization of a TLC Platewith a Hand-held UV lamp

Calculating Rf Values

The distance that a compound travels in a specific chromatography system, compared to howfar the solvent has traveled, is a constant. The relationship between the distance traveled by thesolvent front and the substance is usually expressed as the Rf value:

Rf value = distance traveled by substance

distance traveled by solvent front)

Std Mix

Distance the Solvent Traveled= A Distance the

Compound Traveled = B

Rf = B/A

Using a Ruler measure both distances in millimeters and record the Rf values

Experimental:

O OH

Benzophenone BiphenylDiphenylmethanol

Pre-lab Questions:

1. From the solvents listed below, select a single solvent that you think would give youthe best chance of separating benzophenone, diphenyl methanol and biphenyl(structures above) using TLC on silica gel. Explain the reasoning behind yourchoice. Keep in mind that phenyl groups are fairly “greasy” and non-polar.

2. Assuming you separate the above compounds by TLC using your solvent of choice,predict the order in which the compounds will move up the plate from highest Rf(fastest moving) to lowest R

f (slowest moving).

Table: Eluting solvents for chromatography

Least Eluting Power Hexanes (a mixture of isomers)TolueneDichloromethaneDiethyl EtherEthyl acetateAcetoneEthanolMethanolWater

Greatest Eluting Power Acetic acid

Note: Bring a ruler and pencil to lab for this experiment

Based on your answers to pre-lab question 1, choose two solvents to start with.Prepare a TLC chamber with each solvent, as described in the Techniques section.

Spot two TLC plates using the dichloromethane stock solution of the mixturecontaining benzophenone, diphenylmethanol, and biphenyl. Develop the platessimultaneously, one in each of the different solvents. After developing the plates, remove themfrom the chamber, indicate the position of the solvent front with a pencil mark, and allow theplates to air dry for a few minutes. Examine them under UV light, gently circle the spots thatyou see with your pencil and make a sketch in your lab notebook of the appearance of eachplate showing the positions of all spots. Calculate R

f values for each spot, measuring in

millimeters to the center of the spots.

Depending on the results of this first TLC experiment, design and conduct a second TLCexperiment using two other solvents:

1. If neither of the first two solvents separated all three compounds on the TLC plate, then,based on the appearance of each plate, decide if the solvent used was too polar or notpolar enough. Then select two new solvents that you believe will change the polarity inthe right direction to achieve separation. Test the new solvents by preparing anddeveloping two additional plates.

2. If one of the first two solvents separated the three compounds on the TLC plate, but theother didn’t, then, based on the appearance of each plate and the relative polarity of thetwo solvents, consider how the polarity might be changed from the solvent that workedto improve the separation. Test two new solvents to see if you can improve theseparation. There is more than one solvent which will achieve this separation.

3. If both of the solvents you tested in the first place provided separation of all threecompounds, conduct a second experiment to determine if the separation can beimproved by increasing or decreasing the polarity of the solvent from the two solventsthat worked.

In the second experiment, as with the first, make sketches of each TLC plate showing thepositions of the spots, and calculate R

f values.

After conducting at least two pairs of TLC experiments (developing four plates) as describedabove and finding one or more solvents that can be used to separate the three compounds in themixture, prepare one final TLC plate in which you spot the mixture of three compounds side-by-side with each of the three standards. The standards are separate, identified solutions ofeach compound. Be careful not to contaminate the standards. The plate you prepare will thenbe spotted four times, once with the mixture and once with each of the three standards. Be sureto label the spots. Then develop this final plate using the best solvent from your earlierexperiments and compare R

f values to confirm the identity of the three spots in the mixture.

If you were not able to achieve a very good separation of the compounds in the first or secondexperiment, conduct additional experiments (as time permits) until you achieve a goodseparation of all three compounds. Be sure to carefully consider the results of all previousexperiments to make the best choice of solvents, and make sketches of all plates in your labnotebook.

In your lab report:

Report the Rf values of the spots in each plate that you ran.

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 yourchoices.

Identify the components of the mixture. Explain why they elute in the order that they do.

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