Analysis of Analgesics using Thin Layer Chromatography

Protocol written by Robert Booth

Montclair State University

Spring 2015 Semester

Introduction
Thin layer chromatography (TLC) is a quick and inexpensive method used to
qualitatively check the purity of an experimentally-obtained sample against a standard pure
sample of known identity. Like other chemical separation methods, TLC works by manipulating
differences in polarity of the samples to be analyzed, as well as the solvent being used. As you
may recall from the caffeine extraction lab, polarity is an electronic characteristic of a molecule
that is defined as a net difference in electronegativity within the molecule. Electronegativity is an
atomic characteristic, so highly electronegative atoms within the molecule will have increased
electron density in the space surrounding that atom. Some examples of highly electronegative
atoms are nitrogen (N) and oxygen (O); many polar molecules contain these atoms.

The TLC plate you will be using in this experiment is made of fused silica. Silica is very
polar (it is entirely made up of silicon and oxygen), so polar samples will not migrate as far as
nonpolar samples since the polar sample will bind strongly to the polar TLC plate. However,
polarity is relative, so molecules can exhibit a wide range of polarities; it is incorrect to say that a
molecule is either extremely polar or not polar at all. In reality, molecules may have structural
regions that are nonpolar and other regions on the same molecule that are considered polar. That
being said, molecules that exhibit a lesser permanent dipole moment (less polar) will migrate
further up the TLC plate than molecules that exhibit a larger permanent dipole moment (more
polar). This is the basis on which you determine your unknown sample; your unknown sample
will travel the same distance as one of the three standards (aspirin, caffeine, and acetaminophen).
The standard that travels the same distance up the TLC plate as your unknown sample is the
identity of your unknown.

Rf =distance traveled by sample ¿ origin ¿ origin ¿

distance traveled by mobile phase ¿

Equation 1

When using TLC, it is important to define a “stationary phase” and a “mobile phase.” As
mentioned before, the TLC plate is comprised of silica, so your stationary phase is the polar
silica. The solvent you will be using to determine your unknown is a mixture of 95% ethyl
acetate and 5% acetic acid. Both components of the solvent are considered polar, but they are a
lot less polar than silica. Since the solvent will move up the TLC plate by capillary action, the
solvent is considered to be the mobile phase. By measuring the distance traveled by your sample
and the distance traveled by the mobile phase (sometimes called the “solvent front”), an Rf value
can be calculated (Equation 1), which helps distinguish between each of the samples being
analyzed.

Procedure

Figure 1: Spotted TLC Plate

1) Obtain a TLC plate, ruler, and pencil. When obtaining the TLC plate, only touch the sides

of the plate (do not touch the surface). Use the ruler to measure 2.5 cm from the bottom

of the TLC plate, and then use the pencil to lightly draw a line across the TLC plate (do

not press hard with the pencil).

2) Measure the width of the TLC plate (the length of the line you just drew). Make four

equally spaced dots on the line using your pencil.

3) Label the top of your TLC plate in the order as shown above in Figure 1 (from right to

left, “Tyl” “Caf” “Asp” “A/B/C” [you will only have unknown A, B, or C, not all three]).

Make sure your labels are directly above each dot. Also label the top of the TLC plate

with your group’s initials.

4) Spot your TLC plate using the provided capillary tubes in each of the samples. Take the

capillary tube out of the sample vial and use it to spot the corresponding dot on your TLC

plate. After you finish spotting your TLC plate, put the capillary tube back in to the

correct sample vial (do not cross-contaminate!).

5) Place your TLC plate in to a development chamber, being careful not to submerge your

spots in solvent (the solvent in the chamber should be below the line you drew

previously).

6) Wait. (Chemistry is 50% waiting, 50% bad jokes, and 1% math errors).

7) When the mobile phase is about halfway up the TLC plate and stops migrating, remove

the TLC plate from the development chamber (remember to only handle the sides of the

TLC plate near the dry top part). If the solvent does not stop migrating, do not allow the

mobile phase to reach the top of the TLC plate, or you will have to start over.

8) Before the solvent evaporates, mark the solvent front with a pencil.

9) Allow the TLC plate to dry.

10) Bring your dry TLC plate to the UV lamp, and turn the lamp on (I will do this; do not

operate the lamp yourself). Observe where each of the migrated spots are located on the

TLC plate and outline each new spot with a pencil. Using this information, identify your

unknown compound.
Discussion Questions

1) What is the identity of your unknown compound? Make sure to state which unknown

compound you were assigned (A, B, or C).

2) Calculate the Rf values for each of the standard compounds using Equation 1.

3) Is it possible to have an Rf value greater than 1.0? Why or why not?