Identification of Codeine by Gas Chromatography – Mass Spectrometry (GC-MS)
Group Members:
Aim
The present practical exercise aims at examining a commercial Panadeine tablet for the presence of codeine and paracetamol using GC-MS.
Introduction
Methyl morphine, codeine, occurs in opium at low concentrations. Codeine is a pro-drug because it undergoes metabolism or demethylation, in vivo, to form the active analgesic morphine. The experiment 3 involves a pH dependent extraction technique. The process separates the active constituents of Panadeine. Before extracting Panadeine, however, Eosin B undergoes extraction in order to demonstrate the effect of pH on the solubility of the compound. The GC-MC allows for the separation of a mixture using the gas chromatograph. The mass spectrometer measures the mass spectra of the components of the mixture as they come off. The spectra provide information on the molecular mass and the fragmentation of the compounds.
Experimental Method
Results and Discussion
Part A:
The principle of solubility states neutral organic compounds are soluble in organic solvents while the charged or ionic compounds are soluble in water. The use of either acidic or basic aqueous phase is a refinement on extraction that utilizes the solubility principle. The technique is a pH dependent extraction. The extraction uses the principle that in acidic conditions, carboxylic acids and other acidic functional groups undergo protonation. A compound bearing such groups is, therefore, neutral and soluble in organic solvents. The same compound is, however, deprotonated in basic conditions. The compound becomes ionic and results in aqueous solubility. On the contrary, an organic compound bearing an amine or other basic groups is protonated under acidic conditions. Under basic conditions, the compound is deprotonated.
- In basic conditions, the carboxylic acid group of Eosin B forms a sodium salt as shown in Figure 1.
Figure 1. The carboxylic acid group of Eosin B in basic conditions.
- The extraction of Eosin B, before extracting Panadeine, allows the observation of the effect of pH on solubility. The chemical structure of Eosin B, shown in Figure 2, indicates that the compound has a phenol group and a carboxylic acid group (circled in Figure 3).
Figure 2. The chemical structure of Eosin B.
Figure 3. A chemical structure showing the carboxylic group (circled) of Eosin B.
- In acidic conditions, Eosin is organic soluble (Figure 4) but aqueous soluble in basic conditions (Figure 5).
Figure 4. Organic-soluble Eosin.
Figure 5. Aqueous-soluble Eosin.
- Eosin has a phenol group. In test tube reactions, therefore, Eosin is in the organic (top) layer in acidic conditions. In basic conditions, it is in an aqueous (bottom) layer.
Part B
- The functional groups of codeine include the amine, hydroxyl, and alkene groups (Figure 6) while the functional groups of paracetamol are hydroxyl and amine groups (Figure 7).
Figure 6. The functional groups of Codeine. (PubChem, n. d.).
Codeine is aqueous soluble in acidic conditions and organic soluble in basic conditions due to the presence of the amine group.
Figure 7. Chemical structure of paracetamol showing the functional groups (circled).
Paracetamol is organic soluble in acidic conditions and aqueous soluble in basic conditions due the presence of a phenol group.
- Paracetamol is a weak acid because it contains an OH functional group and an HN-CO-R (amide) functional group. Codeine is a weak organic base because there are fewer anions in its solution when compared to the strong organic bases. In addition, it has an amine functional group (Chemical Book, n. d). Amines neutralize acids to form salts and water.
- Paracetamol undergoes hydrolysis in acidic conditions to produce an amine and a carboxylic acid (Figure 8). The amine is aqueous soluble in acidic conditions and organic soluble in basic conditions. Figure 9 shows the paracetamol reaction in the basic conditions. In acidic conditions, codeine is organic soluble and aqueous soluble in basic conditions, due to the presence of the phenol group. The amine group undergoes hydrolysis in both acidic and basic conditions. The hydrolysis of amines under acidic conditions is acid induced because the process consumes acid. The hydrolysis of the amine group also occurs in basic conditions (Figure 11).
- The solubility of the compounds in different pH conditions is indicated in the Figures 8, 9, 10 and 11.
Figure 8. Paracetamol in acidic conditions. It is aqueous soluble.
Figure 9. Paracetamol in basic conditions. It is organic soluble.
Figure 10. The amine group of Codeine in acidic conditions. Aqueous soluble codeine.
Figure 11. Reaction of Codeine’s amine group in basic condition. The Codeine is organic soluble.
- The GC-MS results (Appendix A, B, and C) can be used to determine the compounds extracted in the basic and acidic layers. The chromatogram has three peaks. The peaks in the spectrum represent the components in the sample (ChemPages, n.d). The largest peak represents the solvent, dichloromethane. The Panadeine sample used in the experiment comprises of paracetamol (500 mg) and codeine phosphate (8 mg) (News Medical, n. d). The second largest peak represents paracetamol while the smallest is that of codeine. Codeine was extracted in the basic layer because it is unconjugated and organic soluble in basic conditions. Paracetamol was extracted in the acidic layer because it is organic soluble in acidic conditions.
GC-MS is better than GC in the identification of compounds because a gas chromatograph requires a mass spectrometer unless the reference sample undergoes analysis under the same conditions. Unlike the GC, the GC-MS allows the measurement of the mass spectra of the mixture’s components (Bitesize, n. d). A gas chromatography is useful in the assessment of the purity of the sample.
The separation of codeine and paracetamol was successful. GC-MS, however, has some limitations. A single peak, for example, may include several components that have not undergone separation. In addition, the MS part of the tool suffers from the inaccurate practice of interpreting mass spectra. The MS-MS analytical method has the advantage of increased sensitivity over the GC-MS. The process, however, is not widely used because the cost of the instrument is prohibitive (Douglas, n. d). The retention time reflects the substance’s affinity for the stationary phase. A long retention time indicates that the substance’s affinity for the stationary phase is high.
GC-MS technique is useful in the quantitative and qualitative analysis of organic compounds. It is quantitative because it involves measuring of the relative intensities of the mass spectra. The retention time and retention index are the important parameters in qualitative analysis.
Conclusion
The process of extracting a sample into an organic solvent is useful in the removal of organic compounds from a mixture in which the other components are soluble in water. Eosin B extraction allowed the study of the effect of pH on the compound’s solubility. In the GC-MS method, each substance has a specific mass spectrum under particular controlled conditions. The identification of a specimen, therefore, involves comparing the specimens’ mass spectrum with the known compounds. When used together, the GC and MS are useful tools for chemical analysis, despite their limitations.
References
Bitesize (n. d.) GC-MS. BBC. Retrieved from http://www.bbc.co.uk/schools/gcsebitesize/science/add_aqa/atomic_structure/analysing_substancerev2.shtml
ChemPages (n. d.) Gas Chromatography. Retrieved from http://chem.wisc.edu/deptfiles/genchem/lab/labdocs/modules/gaschrom/gaschrominterp.htm
Chemical Book (n. d.). Codeine. Retrieved from http://www.chemicalbook.com/ChemicalProductProperty_EN_CB3407233.htm
Douglas, F. (n. d). GC/MS Analysis. Scientific Testimony. Retrieved from http://www.scientific.org/tutorials/articles/gcms.html
News Medical (n. d). Panadeine. Retrieved from http://www.news-medical.net/drugs/Panadeine.aspx
PubChem (n. d.). Codeine. National Center for Biotechnology Information. Retrieved from http://pubchem.ncbi.nlm.nih.gov/rest/chemical/codeine
Appendix A
Appendix B
Appendix C
