CSI-GAC
January, 2005
Identification of Drugs & Poisons
CRIME SCENE
A law enforcement agency is alerted to the apparent sale of drugs by three persons living in a
house located in a middle-class residential area. The information states that an excessively
large number of persons have been visiting this house & that in two instances there have been
sounds of violent arguments coming from inside the house.
After obtaining a search warrant, the law enforcement officials enter the premises and find only
some unlabeled bottles containing white tablets. The tablets cannot be identified by
appearance. Two of the occupants of the house profess ignorance of the identity of the bottle’s
contents, while the third claims that they are “just aspirin”.
The tablets have been transferred to the state forensic laboratory for analysis. You now have
samples of the different tablets which have been ground into powders for ease of analysis.
Color tests (presumptive screening spot tests) have ruled out most of the typical narcotics and
other controlled substances. You will now use a presumptive test employing thin-layer
chromatography (TLC) to try to determine if these tablets contain certain simple over-thecounter analgesic medications. If the TLC results suggest that that is the case, then you will
need to positively confirm the identity of the ingredients by GC-MS and infrared spectroscopy.
CHE-101
January, 2005
Todd A. Swanson
1
CSI-GAC
January, 2005
TLC of Analgesics
PURPOSE
Learn how to perform TLC analysis of samples. Use the technique to identify unknown
analgesic compounds and components of a mixture, by comparison to known reference
standards.
INTRODUCTION
Thin-Layer Chromatography (TLC) is a simple, inexpensive analytical technique that is
widely used in the organic chemistry laboratory for a number of different purposes.
Some of these include:
• Checking sample purity
• Identifying components of a mixture
• Following the progress of a reaction
• Developing solvent conditions for purification of reaction products by column
chromatography.
TLC involves spotting a solution of a sample near one end of a plastic or glass plate that
has been coated with a thin layer of an adsorbent material. There are a variety of
adsorbent materials that can be used but the most common is silica gel (SiO2). The
plate is placed on end in a covered jar or beaker containing a shallow layer of solvent.
As the solvent rises up the adsorbent by capillary action, the components present in the
sample will move up the plate. The rate at which the various components migrate up
the plate depends on the differential partitioning of each component between the silica
gel surface (stationary phase) and the solvent (mobile phase). Since the silica gel is a
very polar material, a more polar component of a sample will spend more time on the
plate surface, and less time dissolved in the solvent. Thus, a more polar component of
a sample will move up the plate more slowly than a less polar component.
When the solvent “front” has nearly reached the top of the plate, the plate is removed
and the components of the sample are “visualized” as spots on the plate. By choosing
an appropriate solvent, the different components (spots) will often appear at distinct
locations on the plate. Of course a pure organic compound should produce only a
single spot on the TLC plate. Using a specific type of TLC plate and a specific solvent,
a particular compound will move at a certain rate, so-as to appear at a particular
location on the TLC plate. Each spot on the plate is assigned an “Rf” value. The Rf is
the ratio of the distance the spot travels from the origin, to the distance the solvent
travels (see Figure 1 below).
CHE-101
January, 2005
Todd A. Swanson
2
CSI-GAC
January, 2005
Rf = 40/80 = 0.5
Figure 1 Determination of Rf
ANALGESICS
Analgesics are substances that relieve pain. In this lab you will run TLC on solutions of
the most common components of analgesics. These components are Aspirin, Caffeine,
Acetaminophen, and Ibuprofen. These will be your reference standards.
O
O
O
OH
H3 C
O
O
O
Aspirin
N
CH3
N
N
N
CH3
Caffeine
HN
OH
OH
O
Acetaminophen
Ibuprofen
Using these reference standards you will use TLC to determine the identity of four
unknown powders or tablets. To identify an unknown by TLC, you must run a TLC
chromatogram spotting the unknown alongside a reference standard (or standards), on
the same plate. If the unknown has one or more spots that correspond to spots with the
same Rf values as the standards, then those substances are probably present.
CHE-101
January, 2005
Todd A. Swanson
3
CSI-GAC
January, 2005
PROCEDURE
Draw a light pencil line about 1 cm from the bottom of a TLC plate. On this line spot the
reference standard solutions. The spots should be no bigger than 0.5 - 1 mm in
diameter. Be sure to use a different spotter for each reference solution. Examine the
plate under a UV lamp to visualize the spots and verify that enough of each compound
has been applied. On other plates, run each unknown alongside one or more of the
standards.
An unknown solution is prepared by crushing a tablet or using one of the unknown
powders provided. About 100 mg of the powder is placed in a test tube and about 5 mL
of ethanol is added. The suspension is mixed well, and the insoluble material allowed
to settle. (This insoluble material is a binder used to hold the tablet together and may
be silica or starch.) The solution is used to spot onto the TLC plate.
The TLC plate is placed into a developing chamber (such as a beaker) containing 1%
acetic acid in ethyl acetate as the developing solvent. The level of the solvent must
be below the line where you have spotted the samples! There should also be a
piece of filter paper lining the inside of the beaker that is wet with the solvent. This
saturates the atmosphere inside the chamber. Cover the chamber with a cap or a piece
of aluminum foil and allow the solvent front to travel to within about 2 cm of the top of
the plate. Remove the plate, immediately (before the solvent evaporates) mark the
solvent front with a pencil, and allow the plate to dry. Place the TLC plate under the UV
lamp to visualize the spots. Carefully circle the spots with your pencil. Now you can
calculate the Rf’s and determine which analgesics are present in each unknown.
CLEANUP
The ethanolic extract of the powders may be rinsed down the sink. The insoluble
material may be placed in the trash. The 1% acetic acid in ethyl acetate used as the
developing solvent should be put in the waste container provided.
QUESTIONS
1.
When developing a TLC plate, why should the plate contact the solvent layer
horizontally and not at an angle?
2.
What is one method for "visualizing" spots on TLC plates that we used in this lab?
3.
If two samples have very similar Rf’s on TLC, such that it is difficult to tell if the two are
different compounds or the same compound, a technique called “over-spotting” may be
helpful. What do we mean by over-spotting and how does it work?
CHE-101
January, 2005
Todd A. Swanson
4
CSI-GAC
January, 2005
During lab and post-lab
•
Data that should be a part of the report includes:
o Structures of the various analgesics
o Traces (drawings) of the TLC results for the reference standards and
unknowns
o Calculated Rf’s for the reference standards and the unknowns
•
Part of your conclusion section should include your identification of the unknowns.
This includes the Name of the medication and the analgesics present in them.
CHE-101
January, 2005
Todd A. Swanson
5