Introduction to Forensic Science - The Thin Layer Chromatography (TLC)

Introduction to Forensic Science
Chromatographic separations Online version
Instructions – Do not upload to Blackboard
Thin Layer Chromatography of a drug mixture
*You Tube link to two videos on TLC
https://www(dot)youtube(dot)com/watch?v=hKYhMxcweys
https://www(dot)youtube(dot)com/watch?v=HZzA9M0H40U
The video with the instructor is very similar to what we would have done in class .
The second video show the drugs separating while under U.V light
Make sure to read all the instructions and background material below. You will need it to answer the homework questions. Don’t relay on the videos alone!
Chromatography is an important tool for the forensic scientist. Many types of evidence present themselves in the form of mixtures. These can include hydrocarbons found as accelerants in fire debris, drugs mixed with diluents and poisons and other toxic substances in someone’s tissues taken at autopsy. There are several commonly used chromatographic techniques used in the forensic laboratory. They include Gas chromatography (GC), High performance liquid chromatography (HPLC) and thin layer chromatography (TLC).They all have in common separations conducted between a stationary phase and a mobile phase. Techniques such as TLC can provide the forensic scientist with a quick and inexpensive screening tool to separate mixtures and is a technique commonly used in the drug analysis laboratory.
In today’s lab, we will use TLC to identify the components in a mixture of analgesics .We will use a stationary phase of silica gel bound to a glass plate and a mobile phase of a solvent. The samples will move up the plate by capillary action and their speed will be dictated by there polarity and how much they are slowed done by adherence to the plates active sites
. Analgesics are pain relievers. We will be using over the counter analgesics such as aspirin, acetaminophen, ibuprofen. We will also be looking at caffeine which can be found in some analgesic preparations. In a forensic setting, drug chemists sometimes come across tablets that have controlled substance analgesics such as oxycodone mixed with over the counter analgesics such as aspirin or acetaminophen.
For your unknown you will be given either a tablet or a powder. You will run an extract of the unknown against 4 standards. It will be your job to compare the number of components separated in the unknown against the separated components in the standards and determine which drug(s) is (are) present in your unknown sample.
Safety requirements – Students must be wearing safety glasses/goggles during this exercise and are to work under the hood when handling chemicals. This also applies to the use of the ultraviolet light. Wear your safety glasses when using the light, only point the light at the TLC plate and do not point the light at yourself or anyone else. Ultra violet radiation when viewed directly can be dangerous to your eyes! Always follow the instructor’s safety directions when working in the laboratory.
At the end of the laboratory dispose of any chemicals used in this lab in the appropriate hazardous waste containers under the hood.
DO NOT DISPOSE OF CHEMICALS DOWN THE DRAIN!
Online version – Instead of conducting the TLC exercise in lab there will be a posted “ Developed TLC Plate “ for you to analyze. Read though what we would have done t and watch both videos to understand how to interpret the results
Materials required
Extraction solvent – Methanol
Developing solvent -0.5% Glacial acetic acid in ethyl acetate
Glass plates 250 um U.V 254 Silica gel
Micropipettes – 50 µl
Standards- aspirin, acetaminophen, caffeine
Filter paper
TLC tank with cover
Pencil
1. You will be given four tubes containing the standards for this lab as well as envelope containing your unknown drug sample (it may be in the form of a pill or powder)
2. If your unknown is in the form of a powder place it between two pieces of filter paper and crush it until it turns into a powder. Place the powder into a separate empty tube.
3. To each tube add 5 drops of the extraction solvent- methanol. Shake the tube and let sit for 5 minutes.
4. To the TLC plate draw a very faint line across the bottom of the plate at about 1cm from the end of the plate. This will be the application line for your extracted samples

5. Using one micropipette per tube draw up a sample of the liquid and spot the sample on the TLC plate. Repeat for each of the standards and your unknown (should be the last spot on the right - # 5)
6. Place a small amount of the developing solvent in the TLC tank such that the height of the liquid will be below the line drawn on the TLC plate. Place a small piece of filter paper upright in the TLC tank, swirl around the solvent to saturate the paper and cover with a watch glass. Wait 2-3 minutes,
7. Carefully place the TLC plate in the TLC tank and cover. Make sure the liquid level is below the application line when the plate is sitting in the tank.
8. Allow the solvent front to travel to a distance app. 1cm from the top of the plate.
9. With a pencil mark the leading edge of the solvent front.
10. Turn out the lights and with the U.V light set to 254 nm (short wave) illuminate your plate. Diagram the separation of the spots and measure the distance travelled by each from the application point. Measure from center of spot to application line.
Solvent front