Paracetamol Isolation and Identification of codeine

The two potent types of over-the-counter painkillers available today include paracetamol and codeine. Codeine, also known as methyl morphine, is an alkaloid discovered in raw opium in 1832. It is also used for its analgesic, anti-diarrheal, and anti-tussive effects (Tremlett et al, 2010 p189). In the other hand, paracetamol, also known as acetaminophen, is a highly active nonsteroidal anti-inflammatory medication (NSAID) that is widely used to treat pain and fever.
The basic method for extracting paracetamol and codeine from their matrix has always been solvent extraction. The liquid- liquid extraction is composed of the application of two immiscible liquids, preferably water and chloroform, in the sample solution to result in two distinctive layers after a thorough mixture shake (BOGUSZ 2000 p 96). Thin layer chromatography follows to separate the extract components. The later method is the one commonly used in the identification of unknown mixture or drug analysis in most of the forensic laboratories. For the mixtures to be ideally separated, there is the need for the determination of its component’s polarity, pH, stationary layer phase and the solvent itself.

An ordinary supposed base chemical can have a pKa value since the terms bases and acids are denotations that connote whether a species can give up or uptake protons. Given a base Y with a 13 pKa, for example, will attract protons and result in YH. However, when it exceeds 13, the resultant will be deprotonated and form Y. That is to say, when either pKa or pH is given, the other value can be solved through the application of an approximation formula scientifically known as Henderson-Hasselbalch equation. The Henderson-Hasselbalch equation serves the purpose of relating the pH and the pKA.

pH = pKa+ log ({base conjugate}/{weak acid})

pH = pka + log ({A-}/{HA})

N/B the equation on most occasions is indicated for the value Ka as opposed to pKa making the relationship be pH= -log ka


The aims of the experiment are twofold. To isolate as well as identify Codeine and Paracetamol from their matrices using solvent extraction method.


The sample was dissolved in 20ml of distilled water. The resulting solution was then shaken gently and then basified with a NaOH solution till the pH rose to 11. The solution was then filtered. The aqueous solution was then extracted with 3X1 chloroform. The chloroform extract was then combined. The chloroform extract was then extracted using thin layer chromatography technique. In the analysis process, positive codeine controls, as well as paracetamol positive controls, were used with appropriate negative controls (Görög, 2015 p115).

Additionally, a diluted sample of chloroform extract was spotted to compare more accurately the resulting standard with the sample. As noted by Hewlett and Steiner (2011), separation of the mixture was carried out based on pH, component polarity and then thin layer stationary phase of the constituents. The solution was visualized using Dragendorff’s reagent under Ultra Violet (UV) light (Howlett & Steiner, 2011 p 1262).

Table 1: Observed pH table Results


Initial Ph

Final Ph

Basified sample



Table 2: Retention factor (Rf) table


Distance travelled by sample in centimeters (cm)

Distance travelled by solvent in centimeters (cm)

Value of retention factor (Rf)

Codeine positive control




Paracetamol positive control




Diluted sample




Chloroform (negative control)




N/B- Retention factor = distance traveled by sample/ distance traveled by solvent

3.1/4.0= 0.78

Figure 3: The TLC chromatogram

Figure 3: The TLC chromatogram

Adopted from


After running the chromatography extracts, diluted samples and the two positive and negative controls simultaneously on the single chromatography plates, the retention factors (Rfs) for the four different samples were determined. The obtained RF value of the sampled chloroform extract matched that of the positive codeine control (0.78) as well as that of the diluted sample control that matched that of the paracetamol positive control (1.00) (BOGUSZ, 2000 p95). This outcome indicates or confirms the presence of paracetamol and codeine in the sample.

The distance traveled by the mixed solvent otherwise referred to as solvent front was determined to be 4 centimeters. This approximate value is close to the individual solvent fronts for the constituent samples (3 to 3.5 cm). This finding disqualifies the use of retention factor as an accuracy of the actual values (Howlett & Steiner, 2011 p1027). Later on, the disparity in values was found to be a consequence of allowing the chromatographic plate (TLC) to develop for a much longer period. The quick drying of the solvent front upon removal from the solvent tank is also responsible for the wrong values obtained. The use of UV light and visualizing spray helped in solving the problem. The solvent –extraction method (liquid-liquid technique) used in this experiment involved the selective movement of a substance’s constituent components in minute quantities between two immiscible liquid phases. Its ability to invoke selective separation is based on pH control and solubility differences (Fifield & Kealey, 1995).

After adding chloroform to the basified filtrate, the solution was shaken and allowed to settle. Because of chloroform’s high density, it settles at the bottom most layer and the upper layer was made up of the aqueous phase. This layer chromatography which is used in the qualitative analysis of the non-volatile mixtures like pharmaceuticals was then used (Skoog et al., 2013; Sabino et al., 2015; Gorog, 2015). TLC is also useful in the process of identifying unknown samples. Since the codeine and paracetamol samples were dissolved without first weighing them also acts as a confirmation that TLC was not suitable for semi-quantitative analyses (Howlett & Steiner, 2011 p1028).


The experiment indicated that TLC could be used to accurately identify the individual components in a dissolved and non-volatile sample. The Rf values obtained in the experiment coupe with the visual indicator reaction applying UV lights shown that the extraction of codeine to a high degree is possible in a laboratory setting. Paracetamol was however extracted to a rather low level in the lab environment and was therefore positively identified. However, when it comes to the identification of compounds on the basis of their Rf values, care must be taken since several compounds share the same Rf values.

The spraying of TLC plates with identification reagents can, therefore, yield confusing outcomes that must further be investigated to ascertain the real identities of the constituent compounds. The work of Lewis and Evans (2011) indicated that developing a spot of an unknown compound on a TLC plate together with another spot for a known compound that has been suspected to be the one under investigation ultimately leads to obtaining similar Rf values if indeed the samples are from the sample substance or compound. This experiment concludes that TLC identification of Paracetamol, Codeine, and other substances is possible and the process scan is useful for the forensic investigation and determination of suspected substances (Sabino, Cardoso Jr & Romão, 2015 p280).


BOGUSZ, M. J. (2000). Forensic science. Amsterdam, Elsevier.

Fifield, F.W. and Kealey, D., 1995. Principles and practice of analytical chemistry. Blackie academic & professional.

Görög, S., 2015. Identification in drug quality control and drug research. TrAC Trends in Analytical Chemistry, 69, pp.114-122.

Howlett, S.E. and Steiner, R.R., 2011. Validation of Thin Layer Chromatography with AccuTOF‐DART™ Detection for Forensic Drug Analysis. Journal of forensic sciences, 56(5), pp.1261-1267.

Sabino, B.D., Cardoso Jr, A. and Romão, W., 2015. 16 Application of TLC–MS to Analysis of Drugs of Abuse. Planar Chromatography-Mass Spectrometry, p.281.

Skoog, D.A., West, D.M., Holler, F.J. and Crouch, S., 2013. Fundamentals of analytical chemistry. Nelson Education.

Tremlett, M., Anderson, B.J. and Wolf, A., 2010. Pro–con debate: is codeine a drug that still has a useful role in pediatric practice?. Pediatric Anesthesia, 20(2), pp.183-194.

Need help with your homework? Let our experts handle it.
Order form