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Analytical Chemistry
Thin Layer Chromatography of Analgesic tablets


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Revised labscript :

Determination of analgesics by thin layer chromatography (TLC)

1. Introduction:

Because of its simplicity and speed, thin layer chromatography (TLC) has found many applications in medical, biological, chemical and pharmaceutical sciences (1,2). Some of the more common components found in over-the-counter analgesic products include aspirin, phenacetin (withdrawn), caffeine, paracetamol (acetaminophen), and codeine, (methylmorphine) (3). Find structures for these products in the Pharmacopedic.

Thin layer chromatography is a special application of adsorption chromatography, in which a thin layer of adsorbent coated onto a flat surface is utilized, instead of a column of adsorbent, as used in column chromatography. The most commonly used adsorbent in TLC is silica gel and the flat surface is a plain rectangular or square glass plate.

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 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, 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 (1,2).

Elution, or development of the chromatogram, is accomplished by capillary movement of the solvent up the thin layer of adsorbent. 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 visibly, but more often they are invisible and must be located by other means. Illumination with ultra-violet light will excite many compounds to fluoresce. Another possibility is to impregnate the plate in advance with a fluorescent dye; the presence of an ultra-violet-absorbing compound on the adsorbent will result in a dark spot on exposure to ultra-violet light, as the compound quenches the fluorescence of the dye. If these approaches still do not make the TLC components visible, 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:

Rf = Distance moved by compound Distance moved by solvent system

TLC can be used for small-scale preparative separations, employing thicker layers of adsorbent and applying the sample as a band instead of individual spots. The chromatogram can be developed, and the edges of the plate treated with visualizing agents to locate the bands of interest. The position of the bands on the developed TLC plate can alternately be located under 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. The dissolved components of the bands can then be analyzed by other techniques, including UV spectroscopy.

TLC may also be used as a means of identification of an analyte. For example, TLC analysis of an extract of a forensic sample, alongside standards of analytes suspected to be present in the sample, allows the analyte in the sample to be tentatively identified. Confirmatory tests can then be used to positively identify the analyte (4,5).

In this experiment, TLC plates will be prepared and used, , with UV spectroscopy, for the semi-quantitative determination of selected analgesics in pharmaceutical preparations.

2. Experimental procedure:

(A) Preparation of TLC plates:

(a) Obtain two 20cm x 20cm glass plates and two 5cm x 20cm plates, for coating with silica gel. Wash plates thoroughly with detergent and water, rinse with distilled water and allow to drain. 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 of the adsorbent.

(b) Mount plates on the plate spreader, with the 20 x 5cm plates on either end and clamp the plates to provide an even spreading surface.

(c) Mix the recommended weight of silica gel adsorbent with distilled water, to give a smooth slurry (25g in about 60-70mL water).

(d) Set the gap of the TLC applicator to 0.25mm, using the feeler gauge provided. Place applicator on an end-plate, with the gap away from the analytical 20 x 20cm plates.

(e) Pour the slurry and distribute evenly in the reservoir. With a single constant motion, draw the slurry along the plates, stopping only when the applicator is on the other end-plate.

(f) Tap the sides of the plate holder to smooth the surface of the slurry layer, then release the pressure on the plates. Separate the plates by means of a spatula, remove both end-plates and discard.

(g) Wash and drain the applicator, for use by the next group.

(h) Allow the analytical plates to air-dry, until the watery sheen has disappeared, then remove from plate holder.

(i) Place plates horizontally in a plate rack provided, and activate in an oven at 110 to 120C overnight and cool in a desiccator before use.

(B) Sample preparation:

(a) Weigh out accurately 1.5g of the finely powdered analgesic mixture provided and extract by swirling with 4mL analytical grade methanol/acetone (1:1 v/v), in a small beaker for about 5 minutes.

(b) Filter the mixture through filter paper (Whatman No.1) into a 10mL volumetric flask. Repeat the extraction procedure as described in (i) and make up to the mark with methanol.

(c) Sample application and development of chromatogram:

(i) On each of the activated TLC plates, gently mark the intended positions of samples and standards with a clean pointed glass rod, starting from one edge of the plate. Begin at least 1.5 cm from either vertical edge and keep samples at least 1.5 cm apart.

(ii) Apply the standards, starting from at least 2cm from one edge of the plate, using a separate graduated 10uL micropipette for each standard.

Take care not to make craters or holes on the adsorbent layer.

(iii) Apply the sample spots to the other marked positions on the origin line, using a clean 10uL micropipette

(iv) Score a horizontal line 15 cm from the origin line, to provide a stop mark for the developing solvent.

(v) Develop the plates in tanks pre-equilibriated for one hour with a toluene:acetic acid:diethyl ether:methanol mixture, in the volume ratio of 120:30:30:02.

N.B. Ensure that the level of the solvent is below that of the applied spots.

(vi) Remove the plates from the tank and allow the solvent to evaporate.

(C) Detection of sample spots:

(a) Some of the constituents in the mixture may not be very stable on the TLC plate. Thus, once the plates are dried, they must be examined immediately under short wavelength UV (254nm) and long wavelength UV (365nm) light.

(b) Using a clean pointed glass rod, mark the outline of the standard and sample spots, and note the color of each visualized spot.

(c) Identify the individual components in the mixture from their respective Rf values and colors, by comparison with those of the standards.

(D) UV Spectroscopy of sample extracts:

(a) Carefully scrape all the silica gel outlined for each sample component from the TLC plate, into the centre of a fluted 5cm diam. Whatman No. 1 filter paper. Take care not to contaminate a component with materials from other spots on the plate.

(b) Elute each sample component with 3 successive 3mL volumes of analytical grade methanol into a 10mL volumetric flask. Make up to the mark with methanol.

(c) Scan each analgesic standard solution provided and note their respective absorbances at the wavelength of maximal absorption. Scan each sample extract similarly and determine their absorbances.

(d) Hence calculate the concentration of each analgesic component identified in the sample, using tabulated absorption extinction coefficients of each standard.

3. Exercise:

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

4. References:

1. Stahl, E. (1969) Thin Layer Chromatography, 1st. Edition.
2. Freid, B., Sherma, J. Thin-Layer Chromatography (1986) 2nd. Edition. Marcel Dekker Inc.
3. The Merck Index, Merck and Co. Inc. (1983).
4. Lieu, V.T. (1971) Journal of Chem. Educ., 48(7) 479.
5. Ganshirt, H., Malzacher, A. (1960). Arch. Pharmz. 293(65) 925.


anal-chem resources
Chem. Dept. UWI. St. Augustine Campus




Original labscript :

TLC - Determination of Analgesics by Thin Layer Chromatography


Introduction

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 basic TLC equipment for identification and semi-quantitatave 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.

Theory

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.

Procedure

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. Pressurize 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 reservoir, 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 110° to 120° 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-equilibriated 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.
Detection:

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 Spactroscopy

  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.
Question

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.

References

  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).




anal-chem resources
Chem. Dept. UWI. St. Augustine Campus