Forensic Examination of Drugs by Thin Layer Chromatography.
But Forensic Science
Skills for the Laboratory
Experiment Title: Forensic Examination of Drugs by Thin Layer Chromatography
Aims: To determine the constituents of four unknown drugs.
To establish good safe & organised working practice.
To encourage consistent & meticulous keeping of data & records.
To develop consistent & accurate laboratory procedures.
To establish team work & investigative approaches.
Background Info: The majority of evidence submitted to crime labs comes from drug-related crimes. Often, this evidence includes unidentified powders that may be illegal drugs. In order to prosecute individuals for possession of illegal substances, it is necessary for forensic scientists to positively identify any suspected drugs submitted to the laboratory. In addition, forensic toxicologists must determine the identity of drugs found in the bodies of drug-overdose victims. Although illegal substances can cause overdose, people also overdose on common over-the-counter (OTC) drugs, like aspirin, when attempting to take their own lives. Thin-layer chromatography (TLC) is one technique used to identify unknown drugs.
Thin-Layer Chromatography (TLC) is a simple & inexpensive technique that is often used to judge the purity of a synthesized compound or to indicate the extent of progress of a chemical reaction. In this technique, a small quantity of a solution of the mixture to be analyzed is deposited as a small spot on a TLC plate, which consists of a thin layer of silica gel (SiO2) or alumina (Al2O3) coated on an aluminium or plastic sheet. The plate constitutes the stationary phase. The sheet is then placed in a chamber containing a small amount of solvent, which is the mobile phase. The solvent gradually moves up the plate via capillary action, & it carries the deposited substances along with it at different rates due to the differential solubility of each of its components. The desired result is that each component of the deposited mixture is moved a different distance up the plate by the solvent. The components then appear as a series of spots at different locations up the plate. Substances can be identified from their so-called Rf values. The Rf value for a substance is the ratio of the distance that the substance travels to the distance that the solvent travels up the plate as you can see from the formula below:
Rf = Distance travelled by compound
Distance travelled by solvent
For example, an Rf value of 0.5 means that the spot corresponding to the substance travels exactly half as far as the solvent travels along the plate.
As a forensic application TLC can be found to be very useful to many aspects one of which being to determine the composition of unknown drugs. In this experiment we investigate this forensic application of thin-layer chromatography. Control samples must be used that provide Rf & colour reactions to compare the unknown samples to. It is essential that a control is used on every plate as Rf values are rarely identical between operators. Since in this experiment a team effort is employed each member should test each unknown with one of the three drugs supplied (Aspirin, Parecetemol & Caffeine). Since these compounds have different molecular structures they interact variably with the mobile & stationary phases in thin layer chromatography. The result is that each component travels at differing rates resulting in a separation of each component, hence each individual spot on the TLC plate represents a compound within the mixture. The unknown samples in this case consist of a mixture of the three compounds provided. Since this is the case by comparing each of the unknown samples to the tested known samples it is possible to determine the composition of each of the unknowns. In the forensic lab further examination of the samples would be carried out to determine the composition of the questioned powders, remember the TLC is only a screening test & would usually be carried out after a presumptive test has been used. Such tests would only be employed if there is sufficient sample & are used because they are cheep & quick. Presumptive tests are usually used to provide the analyst with an indication as to the class of drug involved. If the amount of sample is limited then only instrumental tests would be carried out. There are many presumptive tests available to the forensic toxicologist such as:
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- Mandelin test
- Marquis test
- Scott test
- Erlich test
- Liebermann test
- Dille-Koppani test
- Duquenois-Levine test
- Microcrystalline tests
Various separation techniques could be used, such as high performance liquid chromatography & gas chromatography. Also analytical techniques such as mass spectrometry, infra red spectrometry & 13C NMR spectroscopy could be used before or after separation has been achieved.
Materials: The apparatus used in this experiment are listed below.
- Filter Paper
- Aspirin, Paracetamol & Caffeine samples
- Unknown Samples A,B,C &D
- Small TLC Chamber
- Solvent 1= Ethyl acetate, Chloroform, 85% Formic acid (3:2:1)
- Solvent 1= Ethyl acetate, Chloroform, 85% Formic acid (3:2:1)
- Sample Solvent= 50% chloroform, 50% methanol
- Capillary Tubes
- TLC Plates
- U/V Lamp
Method: Student 1
Preparation of TLC solvent
The following solvents were provided:
Solvent 1 → Ethyl acetate, Chloroform, 85% Formic acid (3:2:1)
Solvent 2 → Ethyl acetate, Acetone, Butanol, 10% Ammonia (5:4:3:1)
Sample Solvent → 50% chloroform, 50% methanol
Solvent 1 was placed into the small TLC chamber to a depth of around 6mm & a watch glass was placed over the top to allow the vapour to fill the tank.
Preparation of samples
A micro spatula was used to put a small amount of each unknown sample (A,B,C & D) in to a cell in a test tray. Each of which was dissolved in a minimal volume of the sample solvent, this was also done with the aspirin sample to be used as a control.
Preparation of applicators
The applicators are prepared by holding capillary tubes at the tip of the blue flame provided by a Bunsen burner. When the capillary tube begins to glow red the two halves are quickly pulled apart. This produces a stretched & hence thinner end on the heated part of the tubes. The thinner circumference of the tubes allows for a more careful application of the samples & allows the samples to be collected using the tension.
Preparation of TLC plate
A pencil line is drawn 1 cm inside one edge of two TLC plates. This line must be drawn in pencil, if a pen was used the components of that pen would run as the samples do. On which three marks are made 1 cm apart labelled Aspirin A B on the first plate & Aspirin C D on the second. Great care is taken not to damage the delicate gel coating.
Application of samples
A sample of each ink is spotted onto the appropriate margin cross ensuring that each spot is no bigger than 2-3 mm across. The TLC plate is then carefully placed into the chromatography tank leaning one edge against the wall of the tank & the lid is replaced to allow the plate to develop.
Marking & recording Data
When the solvent front has travelled a sufficient distance of the plate it is removed & the solvent front immediately marked using a pencil. The plate is then allowed to dry before each spot that is visible under normal light is marked in the centre using a pencil. This is then repeated under UV light & the distance that the solvent front & each spot has travelled from the margin line is measured & the Rf value of each spot is calculated. It is important to calculate the Rf value to the centre of each spot as this is the most concentrated region of the spot.
Student 2 also does the above process using paracetamol instead of aspirin & student 3 does the above process with caffeine.
The whole of the above process is repeated using solvent 2.
Table 1 Rf value of each compound in solvent 1
Table 2 Rf value of each compound in solvent 2
From the above information the following can be concluded:
Unknown A contains the following compounds:
Unknown B contains the following compounds:
Unknown C contains the following compounds:
Unknown D contains the following compounds:
The following graphs will help to confirm this:
Graph 1: Solvent 1, Student 1 (Aspirin)
Graph 2: Solvent 1, Student 2 (Paracetamol)
Graph 3: Solvent 1, Student 3 (Caffeine)
Graph 4: Solvent 2, Student 1(Aspirin)
Graph 5: Solvent 2, Student 2 (Paracetamol)
Graph 6: Solvent 2, Student 3 (Caffeine)
Conclusion: Unknown Sample A: To look at student two & student three’s TLC results it is clear to see that it consists of two compounds. Unfortunately this is not apparent in student one’s findings, reasons for such may be due to human error. Fortunately in this case it appears that caffeine is not present in the mixture anyway. By looking at graphs 2, 3, 5 & 6 a clear comparison can be made although it is not quite as obvious in graph 3. The clear comparison on graph five justifies the statement that this mixture consists of the two compounds paracetamol & caffeine.
Unknown Sample B: We can see this mixture consists of two compounds, by comparing the Rf values on table one & on graph one & graph two there is a good comparison shown towards the control. Unfortunately the results found from solvent two did not show a sufficient comparison, in forensic practice the class of drug used would have determined the solvent used. The results from this experiment have led me to believe that solvent two is not as appropriate as solvent one for these particular drugs. Fortunately the results from using solvent one give good justification to the statement that unknown sample B mixture consists of the two compounds Paracetemol & aspirin.
Unknown Sample C: Initial conclusions can be taken by looking at the tabulated results that this is a mixture of two compounds (also noticeable from the fact that the TLC resulted in two spots). From the comparison of the controls & the tests from graphs one, three, for & six the constituents of this mixture become more apparent. Both solvent one & two showed a good comparison for evaluation & backed up the statement that unknown sample C contains the compounds caffeine & aspirin.
Unknown Sample D: It becomes immediately apparent that this mixture contains three components. In our situation we have been informed that the mixtures contain some or all of the following compounds:
So from this information we can immediately conclude that this unknown contains all three of the possible compounds. However in a real laboratory situation this would not be the case. Where we can make an immediate conclusion the forensic team would not have such a narrow row of possibilities. Hence further examination would be required. For this mixture comparison of all the above graphs is required, & clear comparison can be seen on all graphs with each control. So confidence can be shown in the statement that unknown sample D contains all three compounds (caffeine, aspirin & papacetamol).
General analysis of the TLC plates shows the constituents of each mixture. The further calculations of Rf values gave greater graphical information for comparison once tabulated & the graphs were made. The first thing to be noted from the plate should be that the colouring of each spot matches, only when this is noticed may we justify any calculation. Overall the experiment proved to be generally successful showing only small differences between the controls & the tests. The only large problem appeared to be the lack of agreement to student ones result which showed unknown sample A to only consist of one compound.
Evaluation: When we look at the TLC plates the spots are quite large, this is due to the plates being over loaded on application. Although the spots were only approximately 2-3mm in diameter as suggested the samples were too concentrated & hence we can see the results are less that perfect although suitable to give an answer to the aim. As a forensic application TLC (as a screening test) allows toxicologists to get a quick insight into the possibility of a sample containing a drug substance. Yet another advantage of using such tests is that they allow the analyst to analyse a large number of samples in a relatively short amount of time. One disadvantage is that such screening, & presumptive tests are only possible when there is a sufficient amount of specimen. In the forensic lab a positive tests from TLC is considered provisional & should be confirmed by instrumental analysis. The toxicologist follows a particular scheme demonstrated below in figure one:
The instrumental methods include those mentioned earlier, for example:
Figure 2: HPLC Equipment
Figure 3: GC-MS equipment
Such equipment would be used to confirm the identity of the drugs after initial screening has been completed (providing there is sufficient sample).
References: Table 1 Rf value of each compound in solvent 1
Table 2 Rf value of each compound in solvent 2
Graph 1 Solvent 1, Student 1 (Aspirin)
Graph 2 Solvent 1, Student 2 (Paracetamol)
Graph 3 Solvent 1, Student 3 (Caffeine)
Graph 4 Solvent 2, Student 1(Aspirin)
Graph 5 Solvent 2, Student 2 (Paracetamol)
Graph 6 Solvent 2, Student 3 (Caffeine)
Figure 1 Routes taken by toxicologist
Figure 2 HPLC Equipment
Figure 3 GC-MS equipment