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Chromatographic profiling of paracetamol pure substance

Chromatographic profiling of paracetamol pure substance

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Published by: jontusiire on Jan 21, 2012
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01/27/2014

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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
(a)
 
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
Introduction
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.
4-chloroacetanilide(R1=R2=R4=H, R3=Cl)
 Figure 3: e.g. 1-(2-hydroxyphenyl)ethanone (X = O,R
2
= OH, R
4
= H)Figure 4: Para-substituted phenol e.g. 4-nitrophenol(R=NO
2
) and 4-aminophenol (R=NH
2
)
 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
 
Jonans Tusiimire Expt T2: Impurity profiling of paracetamol using TLC Page 2 of 3
Procedure
 A total of four solutions were prepared. Solution #1 was 10 ml of a 1 % w/v standard solution of paracetamol inmethanol, solution #2 was a 10 ml of 1 % w/v solution of the paracetamol test sample in methanol, solution #3 was50 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/vsolution 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 paracetamolstandard, 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 amountsof methanol to dissolve the samples and form solutions to the mark.
Separate spots of 10 μl of each solution were a
pplied slowly to the TLC plate taking care to rinse the syringe betweenapplications and not allowing the spots to spread too far. The plate was then developed in the solvent systemprovided which composed of chloroform:butanol solvent system (75:25 by volume). The development of the platewas done for 
ca.
40 minutes while it lay in a slightly slanting position
in a tank with its “spotted” end dipping in themobile phase to a level slightly lower than the “spotting” line
. When the solvent front moved to within 90% of the totalheight of the plate, a horizontal line was drawn at the level of the solvent front and the development of the platestopped 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 nmwavelength) 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.
 

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