Introduction
As part of investigation of substances as drugs, we need to use several analytical techniques.
The ones used for this report are as described below.
Different analytical techniques are available for the identification and characterisation of molecules. Chromatography techniques are amongst the most widely used. These techniques involve dissolving the substance in a liquid or gas and passing through a column made of small particles. Depending on the size of the solute, it will get separated out at a different rate from others.
High Performance Liquid Chromatography is the most commonly used chromatography technique for separating out mixtures into their individual components.
All chromatographic techniques involve the use of two phases: A stationary phase and a mobile phase and these two phases do not mix with each other. Unlike the traditional column chromatography, the solvent is forced through the column under pressures of ~ 400 atmospheres, which makes it faster. The smaller particular size of the column helps in better separation of the components. The mobile phase is a solvent mixture and the sample is injected into this. The stationary phase is made of porous silica gel beads. Because of the flexibility of HPLC, it can be used for the separation of a wide variety of substances. The components get eluted out from the column and are detected by the absorbance in the UV region.
For identification of components when small amount of the substance is there, Thin Layer Chromatography can be used. In this case, the solid phase made of silica gel particles, is coated onto a glass plate. The mixture is applied at the bottom end of the glass plate, which is immersed in a non polar liquid. As the liquid moves up, due to non polar attractions, the non polar molecules will be dragged up high.
LC-MS (Liquid Chromatography –Mass Spectrometry) this is a very popular technique w which combines the separation abilities of chromatographic techniques with the mass based identification of molecules.
The great advantages of this technique are its sensitivity and its selectivity and it can be used to separate and identify components of complex mixtures. In LC-MS, sample introduction is based on HPLC technique. The difference is with the choice of stationary phase/solvent combination. For LC-MS, Reverse Phase HPLC would be used for sample injection, where the stationary phase is an organic matrix while the mobile phase would be a water-organic m ix. The separation process is connected in tandem with MS. In Mass Spectrometry, the separated sample molecules are ionised to generate the charged molecules, which are separated on a charge/mass ratio, detected and quantitated to provide the abundances. The mass spectra is obtained as a result of this and shows the various species represented as peaks with their charge/mass ratio.
Infrared Spectroscopy is based on the fact that molecules absorb certain frequencies of light, which are typical of their structure. Based on the complexity of the molecule, specific bands called IR bands will be observed.
More complex the molecule, more complex will be the IR bands. Simple bands will be observed in the Raman Spectrum while more complex bands in the IR spectra region. Big molecules will have many peaks in their IR spectra. This technique can be used to assess the purity of the molecule. Simpler the molecule and the purer it is, simpler will be the IR spectra. Complex IR spectra will be obtained with more complex and impure molecules. Samples are prepared by different ways according to the nature of the sample, and then subjected to the IR radiation. Absorbance will occur when the frequency of the IR matches the resonating frequency of the bond. Measurement of the transmitted light would indicate the amount of energy absorbed at that frequency. Fourier Transform Infrared Spectroscopy can be used to measure the IR spectra.
How do we know which is the best technique to use? It is necessary to compare and contrast these techniques and understand the advantages and disadvantages of each of the techniques, before making a decision on selection. Let us look at the ones we have used here.
HPLC is very fast. Separation can be achieved in minutes. It is suitable for compounds which cannot be vaporised without breakdown, can be used for both identification as well as separation, and can be used with both polar and non polar solvents as mobile phases. It can detect ng to fg levels of molecules, is accurate and automatable. It is used for analysis of pollutants, eluents, pharmaceuticals etc. On the flip side, it is very expensive and has low sensitivity for some substances. Some other problems are irreversible adsorption of some substances which prevents their detection as well as some substances may be eluted out together, which again interferes in complete separation. The instrumentation has to be maintained very well as accumulation of dirt, improper mixing of sample in solvent, air bubbles etc may cause problems.
LC-MS is a far more superior technique to HPLC and IR spectroscopy, as it enables identification of components of far more complex mixtures. It can be used to analyse wide variety of organic compounds. Components, which would otherwise come together as a single peak in HPLC, can be separated out easily through this method. Mass Spectrometry is amongst the most sensitive techniques one can use as LC detectors. Each compound which is analysed through this has its own unique signature generated. This technique also provides molecular weight information, structural information, is much quicker than HPLC as it has no retention time. Also, MS can be performed many times on the sample, which would provide a complete picture of the sample. Hence, in terms of analysis, LC-MS would be a better approach as compared to HPLC and IR so far as range of purposes is concerned.
IR spectroscopy has the advantage that any substance in any physical state can be studied and can be analysed for its molecular components, both in terms of identity as well as amount. As an analytical technique, some limitations are its accuracy is affected by the limitations of the Beer Lambert’s Law. The molecular weight of the substance cannot be determined.
Observations and Results
Using the above mentioned techniques, we proceeded with the analysis of the given white powder. We used it to determine the type of cocaine, the amount of cocaine present and an estimation of its purity. LC-MS of 1µl of the sample (1 mg/ml concentration) along with other known samples of cocaine and its derivatives was carried out. LC chromatogram showed four peaks with the third peak being that of cocaine (elution time ~ 2 mins). LC followed by MS of these peaks. The Mass spectra of the cocaine peak showed the unionized molecular species as well as the ionised species and the weights of these correspond to the known ones. HPLC was carried out using two aliquots of the sample, both of which gave same results. The sample molecules got separated out based on their molecular weight. The HPLC chromatogram displayed the peak heights, widths and the retention time of the cocaine molecules and through this the amount of the molecule in the sample can be calculated. Infrared Spectroscopy of the samples was carried out. The IR spectra of the sample as well as that for a known concentration of cocaine were compared. By studying the peaks obtained in the IR spectrum of the unknown sample with that of a known sample of cocaine, one could determine the purity of the sample. It could be observed that the IR spectra of the sample were more discrete and sharper as compared to that for the known standards, which indicated that the sample cocaine was purer.
Discussion and Conclusion
HPLC, LC-MS and IR spectroscopy are very popular analytical techniques which are used for the separation and characterisation of molecules. In this case, LC-MS analysis was used to separate out the various components of the cocaine sample as indicated by the chromatogram and subsequent MS would indicate the different cocaine species, both the whole and the ions, as well as the derivatives present in the mixture. MS will indicate the correct cocaine species based on the charge/mass ratio which can be compared with the mass spectra of known cocaine sample.
One has to determine which technique one needs to use depending on the final purpose. HPLC is useful for quick separation of mixtures with reasonably high resolution and sensitivity. In conjunction with MS, in the form of LC-MS, it can be even more powerful by not only enabling high resolution but also characterisation and identification of the separate components. IR spectroscopy is another sensitive technique, which can provide information on the purity of the substance as well as its structure. Taken in conjunction, all these techniques can provide valuable information about a molecule.
References
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