Jonans Tusiimire Expt T2: Impurity profiling of paracetamol using TLC Page 1 of 3
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
To detetermine the presence of p-chloroacetanilide and p-aminophenol impurities in paracetamol testsample 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
Paracetamol [N-(4-hydroxyphenyl)acetamide] is a commonly used potent antipyretic and analgesic agent. During itssynthesis (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 paracetamoland specifies their maximum tolerated ranges in the finished product. Some of these impurities include: 4-aminophenol, 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 andthe classes of impurities commonly present in it.
Figure 1: Chemical structure of paracetamol
Figure 2: Substituted paracetamol e.g.
Figure 3: e.g. 1-(2-hydroxyphenyl)ethanone (X = O,R
= OH, R
= H)Figure 4: Para-substituted phenol e.g. 4-nitrophenol(R=NO
) and 4-aminophenol (R=NH
Thin layer chromatography was used in this exercise to examine a test sample of paracetamol for the presence of thetwo 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 theless it will migrate on the TLC plate and therefore the smaller its Rf value. Solvent systems are composed of amixture of a low and high polarity solvents. The higher the proportion of polar solvent the greater the distanceanalytes 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 organiccompounds 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.
MethodsApparatus and Chemicals
Commercial Kieselgel 60 F254 thin-layer sheets
UV light box
10 μl syringe
Standard paracetamol powder
Paracetamol test powder
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