Experiment T2: Chromatographic purity profiling of a sample of paracetamol pure substance

Jonans Tusiimire, MSc. Pharmaceutical Analysis, University of Strathclyde, Glasgow, UK

Aims and objectives (a) To detetermine the presence of p-chloroacetanilide and p-aminophenol impurities in paracetamol test sample by thin layer chromatography (TLC) (b) To determine the Rf values of the impurities present in the test sample and explain why they differ (c) To determine if any other related impurities were present in the paracetamol test sample Introduction Paracetamol [N-(4-hydroxyphenyl)acetamide] is a commonly used potent antipyretic and analgesic agent. During its synthesis (from p-nitrophenol) and/or storage, degradation may occur leading to the production of a number of impurities. The British Pharmacopoiea, 2011 names more than 10 impurities which can be present in paracetamol and specifies their maximum tolerated ranges in the finished product. Some of these impurities include: 4aminophenol, 4-nitrophenol, 4-chloroacetanilide, 1-(4-hydroxyphenyl)ethanone, N-(2-hydroxyphenyl)acetamide, 4(acetylamino)phenyl acetate among others. The figures below represent the chemical structure of paracetamol and the classes of impurities commonly present in it.

Figure 1: Chemical structure of paracetamol Figure 3: e.g. 1-(2-hydroxyphenyl)ethanone R2 = OH, R4 = H) (X = O,

Figure 2: Substituted paracetamol e.g. 4-chloroacetanilide (R1=R2=R4=H, R3=Cl)

Figure 4: Para-substituted phenol e.g. 4-nitrophenol (R=NO2) and 4-aminophenol (R=NH2)

Thin layer chromatography was used in this exercise to examine a test sample of paracetamol for the presence of the two impurities 4-aminophenol and chloroacetanilide. Principle of the Method TLC on Silica gel 60 (Keiselgel 60) separates compounds according to their polarity. The more polar the analyte the less it will migrate on the TLC plate and therefore the smaller its Rf value. Solvent systems are composed of a mixture of a low and high polarity solvents. The higher the proportion of polar solvent the greater the distance analytes will migrate as their bonding to the polar stationary phase is weakened by a polar solvent system. Water although being the most polar solvent is not generally used in high amounts in mobile phases because many organic compounds are not very soluble in it. In this experiment UV light was used to detect the compounds on the basis of their light absorption which quenches the fluorescence of the stationary phase containing a fluorescent indicator showing as a dark spot. Methods Apparatus and Chemicals Commercial Kieselgel 60 F254 thin-layer sheets UV light box 10 l syringe Standard paracetamol powder Paracetamol test powder
Jonans Tusiimire

4-aminophenol and chloroacetanilide Unlined TLC tank containing chloroform:butanol (75:25 by volume) solvent system 2x10 ml glass vials with stoppers 2x50 ml volumetric flasks
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Expt T2: Impurity profiling of paracetamol using TLC

Procedure A total of four solutions were prepared. Solution #1 was 10 ml of a 1 % w/v standard solution of paracetamol in methanol, solution #2 was a 10 ml of 1 % w/v solution of the paracetamol test sample in methanol, solution #3 was 50 ml of a 0.1% w/v solution of p-chloroacetanilide standard in methanol, and solution #4 was 50 ml of a 0.1% w/v solution of p-chloroacetanilide standard in methanol. They were each prepared by separately weighing ca. 0.1g, 0.1g, 0.05g, and 0.05g respectively of paracetamol standard, paracetamol test sample, p-chloroacetanilide and p-aminophenol into separate 10 ml glass vials (paracetamol standard and sample) or 50 ml volumetric flasks (impurity standards) and adding the required amounts of methanol to dissolve the samples and form solutions to the mark. Separate spots of 10 l of each solution were applied slowly to the TLC plate taking care to rinse the syringe between applications and not allowing the spots to spread too far. The plate was then developed in the solvent system provided which composed of chloroform:butanol solvent system (75:25 by volume). The development of the plate was done for ca. 40 minutes while it lay in a slightly slanting position in a tank with its spotted end dipping in the mobile phase to a level slightly lower than the spotting line. When the solvent front moved to within 90% of the total height of the plate, a horizontal line was drawn at the level of the solvent front and the development of the plate stopped by taking it out of the mobile phase. The developed plate was allowed to dry in the fume cupboard and then observed under UV light (254 nm wavelength) and small circles drawn around the spots to locate their positions. Results The location and Rf values of the observed spots were as shown in the figure below:

Figure 5: Developed TLC plate showing the relative positions and Rf values for paracetamol and its major impurities. Note that the colours shown are not the actual colours of the spots as observed under the UV.

Jonans Tusiimire

Expt T2: Impurity profiling of paracetamol using TLC

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Table summarizing the results of the TLC observations Sample Number of spots Distance moved/cm #1: Paracetamol standard 1 4.3 #2: Paracetamol test sample 3 Spot 1: 4.2 Spot 2: 4.3 Spot 3: 5.5 #3: P-Chloroacetanilide standard 1 5.5 #4: P-Aminophenol standard 1 4.2

Solvent front/cm 7.6 7.6

7.6 7.6

Rf 0.566 0.553 0.566 0.724 0.724 0.553

Appearance under UV light When the spots were observed under the UV light they appeared to fluoresce with a yellow/cream colour. However under visible (normal) light, none of the spots were visible. The p-aminophenol spot in paracetamol test sample was being overlapped by the paracetamol spot because their Rf values were very close. So the band appeared unreasonably broadened with no clear distinction between the two bands. When the plate was left standing in the laboratory for 24 hours, the p-aminophenol spots changed from colourless under visible light to reddish brown. So it was at his point that I could clearly see the spot corresponding to paminophenol in paracetamol. Discussion The experiment showed the presence of the two impurities in paracetamol test sample, that is: p-aminophenol and pchloroacetanilide. Since there were no other spots apart from those identified, it is not farfetched to state that there were certainly no other detectable impurities in the test sample provided. The Rf value of p-chloroacetanilide was higher than that of p-aminophenol implying that the former was less retained in the stationary phase compared to the latter. This was not surprising since p-aminophenol is more polar than pchloroacetanilide due to the presence of the free hydroxyl group which interacts with the free silanol groups in the silica gel (polar) stationary phase leading to its retention. Also the Rf value of paracetamol was slightly higher than that of p-aminophenol for the same reason that the latter is more strongly held to the polar stationary phase than the former leading to more retardation. P-aminophenol is more polar than paracetamol because the free ionisable amino group in p-aminophenol is acylated in paracetamol to form a neutral secondary amide. The phenomenon of florescence leads to the observation of the spots under the UV. Florescence occurs at higher wavelengths that those at which molecules absorb radiation. Thus whereas the chromophoric and auxochrome rich paracetamol and its impurities will absorb in the UV, they will emit in the visible region which is a lower energy (higher wavelength) region than UV. The reason for this shift is due to energy loss by collisions in solution of the fluorescing substance. The phenomenonal visual appearance of p-aminophenol after exposure to air for 24 hours on the developed TLC plate probably arises due to a chemical change which occurs when it reacts with oxygen in the air. This oxidation probably leads to the formation of a dye which absorbs at a longer wavelengths in the visible region (blue region) thus appearing reddish brown. Conclusion The presence of p-aminophenol and p-chloroacetanilide in paracetamol has been demonstrated. These two appeared to be the only impurities in the paracetamol sample provided since no other bands were seen on the developed TLC chromatogram. References British Pharmacopaoie (BP), Volume III, 2011 Laboratory Manual, Separation Techniques, SIPBS (2011/12)
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