Make your own free website on
C30F Analytical Chemistry Experiments
Thin Layer Chromatography - TLC

Separation of analgesic mixture and detection by uv-vis spectrophotometry

Back to Homepage

Thin Layer Chromatography

Back to List of Experiments

TLC separation and UV detection of an analgesic mixture


Because of its simplicity and speed,thin layer chromatography (TLC) has found many applications in medicine, biology, chemistry and pharmacy. The object of this experiment is to describe an inexpensive and practical thin layer chromatographic method to demonstrate the basics and TLC equipment required for identification and semi-quantitave analysis of the active components in an analgesic mixture. Further quantitation of the separated identified components will be carried out by VU-VIS spectroscopy.
Some of the more common components found in over-the-counter analgesic products include aspirin, phenacetin, caffeine, tylenol or 4-acetamido-2-methyl-1-naphthol (panadol), and codeine. Find the structures for these products in the Pharmacopedia.
In this experiment, a mixture of at least three (3) of the above in an inert matrix will be analyzed.


Thin layer chromatography is a special application of adsorption in which a thin layer of adsorbent supported on a flat surface is utilized instead of a column of adsorbent. The most commonly used adsorbent is silica gel and the flat surface is a plain glass plate (a cut piece of sheet glass).
The separation of the components of a mixture depends on adsorption-desorption equilibria between compounds adsorbed on the solid stationary phase and in the moving liquid phase. The extent of adsorption of a single component depends upon the polarity of the molecule, the activity of the adsorbent, and the polarity of the mobile liquid phase. The actual separation of the components in a mixture is dependent on the relative values of the adsorption-desorption equilibrium constants for each of the components in the mixture. In general, the more polar a functional group in the compound is, the more strongly it will be adsorbed on the surface of the solid phase. The activity of the adsorbent (adsorptive power) depends on the type of material and on the mode of its preparation. The choice of the proper adsorbent will depend on the types of compounds to be chromatographed.
Elution, or development of the chromatogram is accomplished by capillary movement of the solvent up the thin layer of adsorbent. Unfortunately, one is restricted to the use of a simple solvent system. However, after use of one solvent system, the chromatogram may be dried and developed further by use of a second solvent system, either in the same direction, or at right angles to the direction of the first development. The sample is applied in a small drop a short distance from one end of the plate, and the solvent evaporated off. The plate is then placed vertically into a closed jar with its lower edge dipping into a pool of eluting solvent. Separation is stopped by removal of the plate when the solvent front approaches the top edge.
If the components are colored, they can be located readily, but more often they are invisible and must be located by other means. Illumination with ultra-violet light will excite fluorescence in many compounds. Another possibility is to impregnate the plate in advance with a fluorescent dye; the presence of an ultra-violet absorbing compound will result in a dark spot on exposure to ultra-violet light, as the compound quenches the fluorescence of the dye. If this approach is not applicable, a color or fluorescence producing reagent can be sprayed onto the dried plate to render the spots visible. Once the spots are located, their Rf values can be calculated from the equation:

Distance moved by compound
Distance moved by solvent system

This is used for a number separation problems including the analysis of crude products or unknown mixtures to determine the number of components, and for checking the efficiency of purification processes. TLC can be used for relatively small preparative separations employing thicker layers of adsorbent and applying the sample as a band instead of spots. The chromatogram can be developed by use of indicators to locate the bands of interest. The bands can alternately be observed by ultra-violet light. Once the bands are located, they are scraped from the plates and the material leached from the adsorbent using an appropriate solvent. TLC may also be used as a tentative means of identification. If plate conditions are maintained constant, a compound in several initial spots will progress up the plate at the same rate relative to an added standard or to the solvent front. For identification purposes, a comparism of known and unknown should be carried out with a number of different solvent systems on several adsorbents. A final unambiguous identification with TLC is not possible in that many compounds have very similar adsorption properties.


Preparation of thin layer plates:

  1. Obtain two glass plates 20cm x 20cm and two smaller plates 5cm x 20cm. for coating with silica gel.
  2. Wash plates thoroughly with detergent and water, rinse with distilled water and allow to drain.
  3. Wipe plates free of grease and dirt with acetone-soaked tissue. It is important that the surface of the glass be kept free of grease and dirt to ensure complete spreading.
  4. Mount plates on spreader with the 20 x 5 cm plates on either end. Presurise the spreader to provide an even spreading surface.
  5. Mix the recommended weight of silica gel adsorbent with distilled water to give a smooth slurry (25g in 63ml water)
  6. Set the gap of the applicator to 0.25 mm with the feeler gauge provided and place on the end-plate with the gap away from the analytical 20 x 20 cm plates.
  7. Pour the slurry into the resevoir, and with a single constant motion, draw the slurry along the plates.
  8. Tap the sides of the plate holder to smooth the surface of the slurry layer, then release the pressure on the plates.
  9. Separate the plates by means of a spatula, remove both end-plates and the applicator and wash and dry for use by the next group.
  10. Allow the analytical plates to air-dry until the watery sheen has disappeared, then remove from plate holder.
  11. Place plates horizontally in a plate rack provided, and activate in an oven 120° to 130° overnight.
Sample preparation:

  1. Weigh out accurately 1.5g of the finely powdered analgesic mixture provided and extract with 4 ml methanol/acetone in a small beaker for several minutes.
  2. Filter the mixture through filter paper (Whatman No.1) into a 10 ml volumetric flask.
  3. Repeat the extraction procedure as described in (1) and (2) and finally make up to the mark with methanol.
Sample application and development of Chromatogram

  1. On two thin layer plates, gently mark the intended positions of samples and standards with a clean pointed glass rod at one horizontal edge of the plate.
  2. Begin at least 1.5 cm from either vertical edge and keep samples at least 1.5 cm apart.
  3. Apply the samples and standards at least 2.0 cm from the horizontal edge where the marks were made using a graduated micropipette, taking care not to make craters or holes on the adsorbent layer.
  4. Score a horizontal line 15 cm from the origin line to provide a stop mark for the developing solvent.
  5. Develop the plates in tanks pre-equilibrated for one hour with a Toluene:acetic acid:diethyl ether:methanol: in the ratio 120:30:30:02 solution.
    N.B. Ensure that the level of the solvent is below that of the spots.
  6. Remove the plates from the tank and allow the solvent to dry off.

Because some of the constituents in the mixture may not be very stable, the chromatoplate after development must be examined immediately under normal, short wavelength UV (254 nm) and long wavelength UV (366 nm) light, and all observations recorded.
Determine the individual components in the mixture from their Rf values.

UV-VIS Spectroscopy

  1. Excise each spot on the plate into a 5 cm Whatman No. 1 filter paper and elute it with three portions of methanol (3 ml) into a 10 ml volumetric flask.
  2. Make up to the mark with methanol.
  3. Run the UV spectra for components and standards.
  4. Calculate the concentration of the individual components in the mixture.

Explain how it might be possible to analyse a mixture such as APC by UV-VIS spectroscopy without separating the individual components from the mixture.


  1. Stahl, E. Thin Layer Chromatography, 1st. Ed. (1969).
  2. Lieu, V.T. Journal of Chem. Educ., 48(7) 479 (1971).
  3. Ganshirt, H., Malzacher, A., Arch. Pharmz. 293(65) 925 (1960).
  4. The Merck Index, Merck and Co. Inc. (1983) [available in the Chemistry Reference Library]
  5. Fried, B., sHERMA, j. Thin-Layer Chromatography 2nd. Ed. Marcel Dekker Inc. (1986).

delloyd infolab