Thin Layer Chromatography (TLC) of Common Pain
Medication
Teacher’s Guide
by: Ian Kihara (Pocatello High School)
Lesson Overview: Laboratory experiment: thin layer chromatographic
separation and mixture composition analysis of common pain medication.
National Science Education Standards Addressed:
1. ?
Subject area(s): Analytical
Chemistry
Topic: Chemical analysis
Materials:
 Silica TLC plate(s) (pre-dyed)
 Stretched-out pasture pipette(s)
 UV lamp with short-wave setting
 2:2:1 acetone/methanol/hexanes solution
 TLC developing chamber(s)
 Crushed aspirin, acetaminophen, ibuprofen
Suggested time: 50 minutes
1. 10 min pre-lab discussion
2. 30 min lab work
3. 10 min post-lab discussion
Audience: AP chemistry
Resources:
Attached hand-out
Learning Objectives:
Students will:
1. Determine composition of pain medication mixture by comparison to standards using TLC
Background:
See attached handout
Assessment Strategy:
1. Are students able to correctly spot and run their TLCs?
2. Are students able to correctly identify the components of the medicine mixture?
Teaching Tips:
 Ensure that acetone is used to dissolve the medication for spotting.
 Make sure that students do not over saturate the plate when spotting.
Extension(s):
 Have students develop their own solvent mixture using observed correlations between solvent polarity
and rf.
References:
This material was developed with support from the National Science Foundation’s Graduate Teaching Fellows in K-12 Education (GK-12) program
(DGE-0338184) and Idaho State University. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the
author(s) and do not necessarily reflect the views of the NSF.
Idaho State University’s GK-12 Project
http://www.isu.edu/%7Esanghann/Index.html
1
Thin Layer Chromatographic (TLC) Separation and Mixture
Analysis of Common Pain Medication
Goals:
 Learn about chromatography
 Utilize an analytical method of chromatography
 Introduce students to more advanced laboratory techniques
References:
ADD REFERENCE!!!
Background:
Chromatography is used as a method of separation of a mixture into its
components using a variety of physical properties as means of separation including
molecular polarity, molecular weight, molecular geometry, and ionic charge. In the
laboratory, sometimes a quick and simple mixture composition analysis is needed;
thin layer chromatography (TLC) fulfils this need. Usually done with a silica (SiO2)
or alumina (Al2O3) support coated with an ultra-violet active dye. After an analyte
solution has been “spotted” onto the TLC plate an eluent (any solvent or
combination of solvents) is allowed to ascend the plate, in a closed chamber, toward
the top of the plate without reaching the top. This process is known as “developing”.
After being developed the plate
can be observed under a short-wave
solvent f ront
UV lamp were “spots” can be
ds
observed. If the eluent was chosen
correctly, spots representing
different compounds will appear at
different places. The distance
spotting
traveled by the spot is referred to as
line
the spot’s “rf”. This is calculated by
dx
dividing the distance the spot
traveled by the distance the solvent
traveled from the spotting line and
is written as a decimal:
cmpd 3
cmpd 1
rf 
cmpd 2
dx
ds
Each compound will have a
certain rf corresponding to the
respective support and eluent used
but may be coincident with other compounds. Using the proper eluent will help
ensure good separation between components.
mixture containing
cmpds 1,2
Idaho State University’s GK-12 Project
http://www.isu.edu/%7Esanghann/Index.html
2
Experimental:
1. With a dull pencil*, draw a line across the TLC plate approximately one cm from the
bottom and make equally spaced marks on the line and label them to mark where the
solutions will be spotted1.
2. Pour enough eluent solution into the TLC chamber to be ½ cm deep and rest the cap on
top of the chamber.
3. Slowly spot the desired solutions onto the plate at their labeled positions with the
stretched-out pipettes and allow to the plate to dry completely.
4. Using a pair of tweezers, gently place the TLC plate into the chamber with the spots on
the bottom**, replace the cap, and allow the eluent to climb the plate until it is
approximately ½ cm from the top.
5. Remove the plate, mark the solvent line, and allow it to dry completely before observing
it under the short-wave UV lamp.
6. While observing the plate under the UV lamp, trace the spots with a dull pencil and
compare each spotting lane.
7. If no spots appear, place the developed plate in a warm closed chamber with solid iodine
and allow it to stain for approximately 2 min.
8. If, still, no spots appear, then start over with a new plate and spot it with more solution
than before*** and repeat steps 3-6.
*
Do not pen because it will move with the solvent as well and may interfere with the
experiment.
**
Make sure that the eluent level is not higher than the bottom of the spots.
***
You may check the plate under the UV lamp prior to placing it into the chamber to
ensure that the spots show up.
1
2
3
Figure 1
This material was developed with support from the National Science Foundation’s Graduate Teaching Fellows in K-12 Education (GK-12) program
(DGE-0338184) and Idaho State University. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the
author(s) and do not necessarily reflect the views of the NSF.
Idaho State University’s GK-12 Project
http://www.isu.edu/%7Esanghann/Index.html
3