Codeine or methyl morphine, an alkaloid, was first isolated in 1832 from raw opium. It concentration ranges from 0.2% to 0.8%. Mostly used for its analgesic, anti-tussive and anti-diarrheal capabilities (Tremlett, Anderson and Wolf, 2010). Paracetamol also known as acetaminophen (n-acetyl-p-aminophenol, APAP) on the other hand, is a useful non- steroidal anti- inflammatory drug (NSAID). It is commonly used in the management of pain and fever in a variety of patients (Kamberi, et al., 2004).
Fig 1: Codeine[NCBI, 2009] Fig 2: Acetaminophen[NCBI, 2009] One of the technique involved in the extraction of codeine and paracetamol from its matrix, is the solvent extraction otherwise known as liquid – liquid extraction. This process entails the use of two immiscible liquids usually chloroform and water; in dissolving the sample for two distinctive layers to form after the mixture had been thoroughly shaken together (Rubinson and Rubinson, 1998). Separating the components of the extract, is done through the use of Thin Layer Chromatography. It is one of the standard procedures used in many forensic laboratory when analysizing unknown drugs or mixtures (Howlett and Steiner, 2011). Separation of the mixtures occur based on the pH, polarity of its components, solvent and the thin layer stationary phase (Howlett and Steiner, 2011).
The finely divided sample was dissolved in 20ml of distilled water. This was then basified with NaOH solution to pH 12 using litmus paper. The resulting solution was later filtered. 1.0ml of chloroform was pipetted into the filtrate. After shaken and combined, two distinctive layers was observed. The bottom layer was extracted thrice using a micro- pipette. On a thin chromatography plate, five spots were placed ( as shown in table 2) and the plate was developed using chloroform/methanol. This was later visualized with dragendorff’s reagent under the UV light. All separated components were observed, identified and recorded. RESULTS:
Table of observed pH
SOLUTIONInitial pHFinal pH
Table of Retention factor (RF value)
Rf = Distance travelled by the substance (cm) Distance travelled by the solvent (cm) SUBSTANCEDistance travelled by substance (cm)Distance travelled by Solvent (cm)Retention factor value (Rf) Chloroform extract3.04.00.75
Codeine positive control3.04.00.75
Paracetamol positive control4.04.01.00
Chloroform (negative control) 3.54.00.86
Fig 3: The Developed Chromatographic Plate.
Running the chloroform extracts and diluted sample together with two positive controls and a negative control on a single chromatographic plate simultaneously, the retention factor(Rf) of five different samples were determined. The RF value of the chloroform extract(0.75) tallied with that of the codeine positive control and that of diluted sample(1.00) with the paracetamol positive control. This tentatively shows that, codeine and paracetamol were present in the sample. The solvent front(i.e distance travelled by the mixed solvents) is 4cm, this is quite close to the distances covered by all separated components(between 3 – 3.5cm), which makes the retention factors, not a true representative of their actual values. It was later discovered that, this is due to not allowing the chromatographic plate to develop for a longer period of time in the solvent tank. The solvent front also dried up quickly when the plate is taken out., making drawing a line at that point quite difficult. Fortunately, this was overcome by the use of visualizing spray and UV lamp. Solvent extraction(liquid-liquid), involved selective movement of components of a substance in microgram to gram quantities between two immiscible liquid phase; its separation and selectivity is based on solubility differences and pH control respectively (Fifield and Kealey, 1995).
This was observed when chloroform was added to the basified filtrate. After vigorous shaking and settling down, chloroform being more dense, composed the bottom layer, with the aqueous phase up. Liquid-liquid extraction often involved high volume of organic solvents and poor resolution of mixtures of organic materials (Fifield and Kealey, 1995). Thin Layer Chromatography is usually employed in the qualitative analysis of mixtures of non-volatile compounds like pharmaceuticals (Skoog, et al., 2000). TLC can also be used to confirm the identity of an unknown sample ( Lewis and Evans, 2011). Dissolution of the codeine and paracetamol tablet in distilled water without weighing, shows that, TLC was never designed for semi- quantitative analysis. This is due to difficulties in reproducibly applying aliquots of the mixture to the plate and then recovering all of the separated components from the plate (Skoog, et al., 2000). CONCLUSION: Using the Rf values obtained in the table 2 above and the visual indicator reaction with the substances under the UV light, codeine was extracted to a high degree during the solvent extraction, tentatively identified by TLC (due to its positive control having the same Rf values with the chloroform extract(0.75) and both were the only one that were seen under the UV light) while paracetamol was extracted to a low degree (due to its positive control having the same Rf with the diluted sample). Multiple compounds can share the same retention factor(Rf) or produce similar chromophores when sprayed with detection reagents (Howlett and Steiner, 2011). The study by Lewis and Evans( 2011) shows that if a spot from an unknown substance is developed on a TLC plate together with a spot from a substance that is suspected to be the unknown, and the two substance are found to have the same Rf value, they are probably the same substance.
FUTURE SUGGESTIONS AND RECOMMENDATIONS: Due to the limitation that is associated with using TLC to exactly identify a given sample, minimum standards for drug testing and reporting in the forensic community are recommended by the Scientific Working Group for the Analysis of seized drugs (SWGDRUG) (Howlett and Steiner, 2011). In order for a drug identification to be confirmed to SWGDRUG specification, additional tests must includes, Infrared spectroscopy and GC-MS (Howlett and Steiner, 2011).
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