Introduction:

Chromatography is a technique which is widely used to separate a mixture of substances into its component parts. The term chromatography encompasses a number of different techniques which, although they will be discussed separately, are all based on common principles. Chromatography may be divided broadly into three kinds: adsorption, partition and ion exchange, and the simplest type of these is adsorption chromatography. In this technique, the substance under investigation is adsorbed onto a solid support (the stationary phase), such as alumina (aluminum oxide) or silica gel (oxides of silicon), and separation of a mixture into the component parts is achieved by elution with solvents of different polarity. Adsorption chromatography may be carried out in two ways, by column, or thin layer techniques, and these will be discussed separately. In this experiment you will use the thin layer technique but a brief discussion of the column technique is included for additional background. In column chromatography a finely divided adsorbent such as silica or alumina is placed in a glass column, supported at the bottom by a wad of glass wool, as shown below:

A layer of sand is placed over the top of the adsorbent, and the whole column is whetted with the solvent to be used. A solution of the substance to be purified in this solvent is then applied evenly to the top of the column, and this solution is allowed to pass down into the column so that the dissolved solid is adsorbed at the top of the column. The column is then eluted by passing down a number of solvents of increasing polarity. In this way, weakly adsorbed substances will pass rapidly through the column while the more strongly adsorbed substances will pass through at a slower rate. By eluting with a series of solvents of increasing polarity it is therefore possible to separate the components of a mixture and to elute them, one after the other, from the solid adsorbent.

The order in which the compounds are eluted will depend on how strongly they are adsorbed on the surface of the stationary phase. Alumina ­­ unless specially pretreated ­­ is slightly basic and hence strongly adsorbs acidic substances or materials capable of forming hydrogen bonds to the basic oxygen atoms of the alumina. Compounds without the ability to form hydrogen bonds but with substantial dipole moments will be somewhat less strongly adsorbed due to electronic interactions between their dipoles and those of the alumina. Compounds with neither acidic hydrogen nor dipole moments are only very weakly adsorbed due to dipole, ­induced dipole interactions.

The choice of solvents used to elute the various components of the mixture from the column will depend upon the components in the mixture. For a very weakly adsorbed component a very non­polar solvent such as petroleum ether or benzene would be used. For more strongly adsorbed components, a more polar solvent such as ether might be used. For very strongly adsorbed components, a very polar solvent such as ethanol, water or even acetic acid might be required to displace the material from the column. A list of common solvents in order of increasing solvent strength follows:

Thin layer chromatography (TLC) is another type of adsorption chromatography. Here, the solid adsorbent, again usually alumina or silica gel, is spread out in a thin layer over a glass or plastic sheet, and the substance under examination is placed on this layer in the form of a spot. A suitable solvent is then allowed to run up the sheet by capillary action, as shown below:

During the elution with the solvent, the sample will partition itself between the stationary phase (the adsorbent layer) and the moving phase (the solvent) so that the distance which the sample moves up the plate is characteristic of that substance and will differ from one substance to the next. The distance moved by the spot of sample divided by the distance moved by the solvent is known as the R_f value and is characteristic of that compound for the solvent system used. A mixture of substances will thus give rise to a series of spots, one corresponding to each component. This technique is extremely useful for analysis on a micro scale and for the purification of small quantities (usually less than 0.1 g) of material. In preparative work, a substance is recovered from the plate after development by removing the particular region of the adsorbent layer containing that substance from the plate, followed by the removal of the substance from the adsorbent layer by extraction with a suitable solvent.  Click her to see a short video on TLC.

The second general type of chromatography is partition chromatography, the three major types here being gas-liquid chromatography, liquid-liquid chromatography and paper chromatography, which is an application of liquid-­liquid chromatography. In the first two cases, the stationary phase commonly consists of a liquid which is bonded to an inert solid substance. For liquid chromatography, eluting the system with a moving liquid leads to separation of the components of a mixture by the partitioning of these components between the stationary and moving liquid phases so that the components will move at differing rates through the column. Separation is thus achieved by collecting the moving liquid phase in different fractions as it leaves the column. In gas chromatography (see Experiment 6), however, the moving phase is a stream of an inert gas, such as nitrogen or helium. The sample under investigation is volatilized and the vapor swept through a column of the stationary phase by the carrier gas. Partition of the sample between the stationary and mobile phases will occur. The sample may be recovered from the gas as it leaves the column by condensing it at a low temperature. Paper chromatography is analogous to thin layer chromatography except that in this case, the support material consists of a sheet of specially prepared paper, and the stationary phase is considered to be water adsorbed on the paper. Elution with a solvent, measurement of the R_f value and preparative work is carried out in the same way as for thin layer chromatography.

The third type of chromatography, ion ­exchange chromatography, is of somewhat more limited application. Here, the solid support consists of a resin which can have either basic or acidic properties, and mixtures of acidic or basic substances can be separated using these resins by eluting with buffers of different pHs.

Purpose:

In this experiment, TLC will be used to examine the composition of various common over-the-counter medications.  These medications are classified into one or more basic categories:  analgesics (pain relieving), antipyretic (fever reduction), anti-inflammatory (reduces swelling), or uricosuric (relieves symptoms of arthritis and gout). The best known of these is aspirin, but several other chemically similar compounds are (or were) also used.  Among these are phenacetin, salicylamide and acetaminophen:

Procedure:

Developing Chamber

You will be using a small glass with a watch glass as your developing chamber.  To clean it, add approximately 5 mL of the developing solvent (1 vol% glacial acetic acid in ethyl acetate) to the glass.  Gently swirl the solvent around so it contacts the whole surface of the glass.  DISPOSE OF THE DEVELOPING SOLVENT IN THE LABELED CONTAINER IN THE HOOD.  Now add about 2-3 mLs of the developing solvent to your cleaned developing chamber, put the watch glass on and set it aside to equilibrate.    

Preparation of Samples

A series of unknown OTC medications have already been crushed for you.  Add approximately 125 mg of your unknown sample into a test tube and dissolve in 4 mL of ethanol (Be sure to note your unknown number!).  Use your stirring rod to mix vigorously.  Set the test tube aside for 10-15 minutes to make sure all of the active ingredients have dissolved  (NOTE: some insoluble material will not dissolve, this is normal).  If there are still materials that have not settled, use the centrifuge (Using a Centrifuge). The reference standards have a concentration of approximately 25 mg/mL. 

Your unknown will be one of those listed below:

You will be using commercially prepared fluorescent Silica Gel TLC plates, supported on plastic sheet, 2.5 x 7.5 cm. These sheets have been activated by heating at 120°C for 60 minutes (removes any water that was absorbed by the silica gel).

You will have five solutions (4 reference compounds and 1 unknown) to examine. All of these solutions need to be carefully 'spotted' on a single plate. They should be spotted on the coated side (NOT the shiny side) in a line about 1 cm from one end of the plate, equally spaced apart, with the outer two spots about 0.5 cm from the edge of the sheet. The unknown should be placed in the center, with two reference compounds on each side.  You should also draw a line about 1cm from the top of the plate to mark the solvent front.

To apply a sample, touch the end of your applicator to the solution, and then gently touch the Silica Gel plate at the proper spot. Use a fresh applicator for each sample. The sample spots should not be larger than 2 - 3 mm diameter.  Note:  you may find it useful to touch a paper towel to your applicator to remove most of the sample (leaving only 1/8" to 1/4" of liquid) before applying it to the TLC plate.

Development of the Chromatogram

After you have 'spotted' the standards and your unknown, carefully place it, spotted end down, into your developing chamber.  Make sure that the level of the solvent pool is below the line of your sample spots.  Cover the tank, do not disturb, and allow about 10 - 15 minutes for the solvent to rise until it touches the solvent front line you drew at the top of the plate - do not allow the solvent to run all the way to the top of the plate!  Remove the plate from the tank and allow the plate to dry completely (you will probably need to use a hair drier to evaporate all of the solvent).

Visualization

The colorless compounds are visualized by illumination of the plate with an ultraviolet lamp. Many substances, particularly aromatic compounds, will show a bright fluorescence, which may have a characteristic color. The thin layer plates that you are using contain a trace of fluorescent dye. Compounds which are fluorescent show up as bright spots on a light background; any others appear as a dark spot since they quench the fluorescence of the background dye. Circle the spots lightly in pencil, and note any distinctive colors.  Photograph the plate or draw a representation of your plate and any spots you detected and include this in your final report.

You can also use other methods to visualize your sample, such as iodine.  Place your TLC plate in the iodine bottle for 1-2 minutes.  The iodine will react with some compounds and not with others.  Remove your TLC plate and again note the color and position of any spots you see.  Photograph the plate or draw a representation of your plate and any spots you detected in your final report.

Warning:  These UV lamps produce intense UV radiation, do not look directly at them.  Also, if your are susceptible to sunburn, limit your exposure.

Results:
 

• Calculate the R_f values for the reference compounds and the components in the unknown.
• Photograph your TLC plate (chromatogram) under both UV and iodine conditions.  Identify and label the spots on the chromatogram, including as many of the spots in the unknown as possible.
  
• From the number, position and appearance of the spots in the unknown, and the composition of the possible unknowns (from the table above), identify your unknown analgesic (there may be several possibilities).
• Given that the stationary phase (TLC plate) is very polar and the solvent is non-polar, discuss why each compound moved the relative distance that it did.  Could you have predicted the relative order of elution knowing the relative polarity of each compound?
  

(Updated 9/30/12 by C.R. Snelling)