Experiment for Identification of Paracetamol

Experiment for Identification of Paracetamol PATRICK ANTWI MANU IDENTIFICATION OF PARACETAMOL THROUGH THE USE OF PHARMACOPIEA TEST ABSTRACT Paracetamol is widely used (NHS Choice, 2012) for the relief of minor pain, toothache, headaches, symptoms of cold and reduces of patient’s temperature (control fever symptoms). It is classified as a mild analgesic agent. The following procedures were used during the experiment of identification of paracetamol. TLC method is important for separation of mixture. This technique is commonly used to determine the presence of particular compound through comparison of Rf values of both unknown samples and the standard samples. Melting point analysis is also useful for identification of compound. The melting point range can be indicated and recorded with the help of a thermometer. For instance, the melting point of paracetamol ranges from168 to172 degree Celsius Infra-red spectroscopy is one of the most essentially useful methods for identification of compound. It is usually used to identify medicines that contain correct drug in most pharmaceutical industries. Structural information about the functional group of unknown compounds can be ascertained through the use of infra-red spectroscopy. Significantly, the results generated during this experiment attest the accuracy and importance of these methods for identification of compounds. For example, the Rf values of TLC method as well as infra-red spectroscopy analysis demonstrated the presence of paracetamol and cafeine in sample ‘B’ and ‘D’ respectively. INTRODUCTION Paracetamol is the best alternative choice for children and ulcer patients who have a minor pain such as headache and children. Consequently, too much it (NHS Choice 25/06/2012) can cause severe harm to the kidney and the liver. AIM OF THE EXPERIMENT Identify paracetamol from various unknown samples through pharmacopeia test. Hypothesis The range of melting point of the samples that contain paracetamol would be (168 to 172) degree Celsius. Again, the samples that contain paracetamol only would turn into violet without changing to red after the experiment D. The Rf values of unknown samples that contains paracetamol would be the same as the Rf value of the standard sample when compared. Apparatus used for the experiment Micro-spatula UV-light Indicator IR spectroscopy Melting apparatus Pencil Rule Chromatography tank TLC plate Thin capillary tube Conical flask Beaker Micro-pipette Materials used Ethyl acetate Four unknown samples Water Dichromate METHODS Melting point analysis Melting point analysis was conducted for all the four unknown samples labelled A, B, C, and D. Small amount of crystals of each unknown samples was fetched into the melting point capillary tube. The capillary tube contained the sample was placed into the melting point apparatus. The samples temperature was measured with digital thermometer. Hence, both initial and final melting point of the samples was observed and recorded. This experiment was repeated twice to obtain the ranges of the melting point. INFRA-RED TECHNIQUE During the experiment, the arm of IR spectroscopy was cleansed with (ethanol) alcohol. The background of IR spectroscopy was scanned. Hence, each sample was placed on the mouth of the IR spectroscopy and scanned. The wavelength detected the various bound in each compound. Copies of each compound scanned was printed out for observation and reading. EXPERIMENT D FOR IDENTIFICATION TEST ANALYSIS The experiment D was conducted for all the four unknown samples labelled A, B, C, and D. 0.1g of each unknown samples were measured with macro spatula and 1ml of concentrated hydrochloric acid was added to it. The solution was gently shake and heated to boil for about three minutes. 1ml of water was added to the boiling solution. After boiling, the solution was then placed in an ice bath to cool. Observation was made and there was no precipitation form. Therefore, 0.05m of 4.9/L solution of potassium dichromate was added to it and the colour developed for sample ‘B’ was violet without changing into red. However, the colour developed for sample ‘A’ was slightly red and changed to purpled whilst sample ‘C’ and ‘D’ developed yellow hint colour and diluted violet colour respectively. TLC METHOD A solvent system of about 20ml of ammonia: methanol: chloroform (1:19:80) was placed into the chromatography tank. A filter paper was placed against the wall inside the chromatography tank. The tank lid was placed on to prevent the evaporation of the solvent. The tank was left for about twenty minutes to allow saturated atmosphere to be formed. TLC method was conducted by using four unknown samples labelled A, B, C, D and ethyl acetate. During the experiment, a solution was made from each of the four unknown samples A, B, C, and D respectively. Thus, about 10mg of each sample was fetched with micro-spatula into the small beaker and dissolved with the small volume of ethyl acetate. Moreover, TLC plate was prepared by measuring 1.5cm distance from the edge of the plate with a rule. A horizontal line was drawn and marked the intervals with a pencil. The distance in between each interval was about 1.5cm apart. The line was demarcated into six intervals for the four unknown samples as well as the standard samples, which comprises of paracetamol and caffeine solution. Each solution was fetched with the help of a micro-pipette and spotted on the TLC plate at different intervals. In addition, the standard sample solutions of paracetamol and caffeine were spotted on the same plate at different position. Thus, spot A, B, C, D, Par and Caff in that order. The TLC plate was placed into the chromatography tank and covered with the lid. The solvent then moved up gradually through capillary action. Hence, the solute spotted on the TLC plate moved up along with the solvent (thus, mobile phase). TLC plate was removed from tank when the solvent reached about 2cm distance to the edge and marked with a pencil. Moreover, the plate was left to dry for about 20 minutes. After the evaporation of the solvent has taken place, TLC plate was then placed under the UV light for observation. A drawing was made with a pencil around the new spots formed on the plate. A measurement of the distance travelled by both solvent and substance were recorded. The Rf value was calculated for both unknown samples and the standard samples. Therefore, the Rf value was calculated base on the formula below. Rf value = distance travelled by substance divided by the distance travelled by solvent. Finally, the Rf values of unknown samples were compared with Rf values of the standard samples. The Rf value for paracetamol Rf = 3.90/6.1 Rf value = 0.639 = 0.64 RESULTS (b). Melting point analysis results SAMPLES A B C D MELTING POINT RANGE 171- 175 171 172 200 206 159 The sample ‘A’ is a bit higher than the normal range of the standard paracetamol sample. Equally, sample ‘B’ indicated the presence of paracetamol as the ranges 171-172 degree Celsius (c). (d). EXPERIMENT D FOR IDENTIFICATION ANALYSIS results SAMPLES A B C D Colour of solution Slightly red and turned to purple Violet Yellowish hint Dilute violet Sample ‘A’ developed purple colour which shows para-aminophenol The colour achieved for sample ‘B’ was violet which show positive result and it was therefore indicated the presence of paracetamol. The colour developed for sample ‘C’ was yellow hint which indicated the presence of caffeine. However, sample ‘D’ developed diluted violet colour which shows the presence of caffeine and paracetamol. Infra-red analysis results Sample AÂ   Sample B Bond Functional group Frequency / wave number absorption cm-1 Intensity v N-H amine 3319.12 stretch strong v C-H 2794.94 stretch strong/medium v O-H Hydrogen bond in alcohol, phenol 3109.51 stretch Strong v C=C 1650.71 Aromatic stretch s v C=O 1609.82 amide stretch N-H delta amide 1562.66 amide bend v C=C Aromatic stretch 1504.96 aromatic stretch The functional group obtained on sample ‘B’ indicated the presence of paracetamol. Sample C Sample D (g). Results for TLC analysis STANDARD SAMPLES Paracetamol Caffeine Rf values from TLC analysis 0.64 0.89 SAMPLES A B C D Rf values from TLC analysis 0.74 0.64 0.89 0.73 The Rf values calculated for TLC analysis indicated that sample B is paracetamol when compare with the standard samples. Thus, 0.64 Compound A, C, and D are less polar since they travelled faster and further in the mobile phase and they are more attracted to the mobile phase than compound B. However, compound B is more polar and travel slowly in the mobile phase. It is most attracted to the stationary phase. Discussion The experiment D of sample ‘B’ clearly showed positive outcome and indicated the presence of paracetamol as violet colour was achieved. In addition, TLC analysis also indicated that sample ‘B’ was paracetamol when Rf value of unknown samples ‘compared to the standard samples. Therefore, this shows the accuracy and precision of the positive outcomes of the experiment. Equally, the ranges obtained from melting analysis for sample ‘B’ also confirmed the presence of paracetamol. Experiment D of sample ‘A’ showed deep purple colour and the infra-red analysis confirmed that, it is para-aminophenol. Moreover, both experiment D, TLC method and infra-red analysis confirmed the presence of caffeine in sample ‘C’. However, sample ‘D’ developed dilute violet colour TLC analysis which shows a mixture of (two compounds) paracetamol and caffeine. The infra-red analysis also confirmed that, sample ‘D’ was a mixture of two compounds. Time SUMMARY REFERENCE Barber, J., Rostron, C.,(2013). Pharmaceutical Chemistry Hill, G., Holman, J,. (2011) Chemistry in Context. 6th edition NHS Choice, (25/06/2012). Available at: http://www.nhs.uk/Conditions/Painkillers-paracetamol/Pages/Introduction.aspx APPENDICES Mobile phase: Rf value: retention factor for thin layer chromatography. Stationary phase: TLC: thin layer chromatography UV light: ultraviolet light