Thin-Layer Chromatography:
Over-the-Counter Analgesics
 2012, Sharmaine S. Cady
East Stroudsburg University
Skills to build:


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Spotting TLC plates
Developing TLC plates
Calculating Rf values
Identifying active ingredients in OTC drugs
Thin-Layer Chromatography
Chromatography, which means the graphing of colors, was a technique first used
in 1903 by the Russian botanist Michael Tswett to separate the pigments in green
leaves. Today chromatography is used to separate mixtures of colorless compounds as
well. In a forensics laboratory, drugs and dyes may be separated by thin-layer
chromatography. All forms of chromatography depend on the dynamic equilibrium that
exists between the solutes dissolved in the mobile phase and their adsorption onto the
stationary phase:
solute(mobile)
solute(stationary)
mobile phase
mobile phase
stationary phase
stationary phase
(a)
(b)
Figure 1. Solute equilibrium between mobile and stationary phases. (a) Solute prefers
the mobile phase. Few solute molecules remain adsorbed. (b) Solute prefers the
stationary phase. More solute molecules remain adsorbed than enter the eluent.
Thin-Layer Chromatography
Solute molecules that have a higher attraction for the stationary phase favor the
right side of the equilibrium; solute molecules that have a higher attraction for the mobile
phase favor the left side of the equilibrium (Figure 1). These differences in the
partitioning of dissolved solute molecules between the two phases permit the separation
of the components of a mixture. In turn, the partitioning depends on the polarities of the
mobile phase, the stationary phase, and the solute molecule. Table 1 shows the order
of polarity for various organic solvent types.
Table 1. Polarities of Organic Compounds
Carboxylic acids
Amines
Alcohols
Aldehydes
Ketones
Amides
Esters
Ethers
Aromatics
Alkenes
Alkanes
Most polar
Least polar
In thin-layer chromatography (TLC), the mobile phase is liquid, and the stationary
phase is a plate of silica gel usually supported by a backing of plastic, glass, or
aluminum. Sometimes the silica gel is impregnated with a fluorescent compound that
allows the detection of colorless solute molecules under UV light. When a TLC plate
spotted with solute molecules is partially submerged in a liquid solvent, the liquid mobile
phase moves by capillary action along the stationary silica gel on the TLC plate where it
encounters the solute molecules. When a nonpolar solvent is used as the mobile phase,
polar solute molecules have a strong affinity for the highly polar silica gel and do not
travel far along the stationary material. On the other hand, molecules of low polarity
have a weak affinity for the silica gel and migrate with the nonpolar mobile phase a
significant distance up the plate. Increasing the polarity of the solvent, however, lowers
the attraction of polar solutes for the silica gel and increases the distance they travel.
The "like-dissolves-like" rule of solubility can be used to predict these events. Figure 2
shows a finished TLC plate where a dye mixture has been separated into three
components. The component most soluble in the mobile phase is closest to the top of
the plate, while the component least soluble in the mobile phase is closest to the bottom
of the plate.
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Thin-Layer Chromatography
Figure 2. Thin-layer Chromatogram of a Dye
The choice of the developing solvent is critical to the successful separation of the
components in a mixture. A developing solvent that does not cause significant
differences between the solute molecules in their partitioning will produce incomplete
separation and overlapping of the solute molecules in the finished chromatogram.
Usually two or more liquids mixed in varying proportions are needed to prepare a
suitable developing solvent.
Differences in partitioning are mathematically determined by calculating an Rf
value from the equation found on the following page. The larger the Rf factor, the closer
the solute molecule is to the top of the plate and the higher its attraction for the mobile
phase. The numbers used in the equation are obtained by taking measurements once
the TLC plate is completely developed. Figure 3 shows the individual measurements
needed to calculate Rf values for the yellow component of the dye. Similarly,
measurements for the red and blue components can be obtained and their Rf values
computed. Example 1 shows the calculation of the Rf values for the TLC dye
separation.
Rf 
distance travelled by solute
distance travelled by solvent
distance
travelled
by solvent
distance
travelled
by solute
Figure 3. Measurements for Calculating Rf Values
3
Thin-Layer Chromatography
EXAMPLE 1
4.10 cm
3.35 cm
2.40 cm
1.05 cm
Calculation of Rf values for blue, yellow, and dye components:
Rf 
3.35 cm
 0.817
4.10 cm
Rf 
2.40 cm
 0.585
4.10 cm
Rf 
1.05 cm
 0.256
4.10 cm
Polarity of Molecules
The molecular composition and shape determine the polarity of a molecule. For
simple molecules, the directional moments of bond polarities may be drawn. If the bond
polarities cancel, the molecule is nonpolar; if not, the molecule has a net dipole moment
and is polar. Carbon tetrachloride is a tetrahedral molecule in which the bond moments
cancel; therefore, CCl4 is nonpolar. Water is bent molecule in which the bond moments
do not cancel; therefore, water is a polar molecule. The direction of a bond moment is
determined from the electronegativities of the two atoms that participate in the bond. A
bond moment points from the least electronegative to the most electronegative atom.
Figure 4 shows the direction of the bond moments in CCl4 and H2O and the net dipole
moment for water.
no net dipole
moment
net dipole
moment
Figure 4. Carbon tetrachloride's tetrahedral structure cancels its bond moments.
Water's bent structure produces a net dipole moment.
4
Thin-Layer Chromatography
Predicting the polarity of more complicated molecules is not always foolproof. As
a guide, those structures that contain mostly carbon and hydrogen bonds (CH) are
considered nonpolar. Those structures that contain a large amount of oxygen and
nitrogen atoms bonded to hydrogen (OH and NH) are often considered polar. Many
molecules contain both nonpolar (hydrophobic) and polar (hydrophilic) groupings in their
structures. Hence, they may dissolve in both polar and nonpolar solvents.
For
example, the drug morphine contains both hydrophobic and hydrophilic groups as shown
in Figure 5. The solubility of morphine in water is 1 gram in 5000 mL, while its solubility
2
HO
3
1
11
4
H2
C
10
12
O
9
13
H3 C
17
14
N
5
CH3
H2
C
C
H2
OH
C
H2
15
HO
8
6
16
7
Figure 5. The structures of morphine (left) and 1-pentanol (right) with the nonpolar
areas in red.
in 1-pentanol is 1 gram in 114 mL. The larger amount of nonpolar character in pentanol
makes it a more suitable solvent for morphine's largely nonpolar structure than water.
The structures of several over-the-counter drugs are given in Figure 6. Note the
presence of polar and nonpolar areas in the structures. The drugs aspirin, ibuprofen,
ketoprofen, and naproxen are part of the non-steroidal anti-inflammatory drug family
commonly referred to as NSAIDS.
In this experiment, you will test the effects of developing solvent polarity and pH
on the separation of different drug components. You will select a solvent system and
determine the active ingredient(s) in an unknown drug tablet and identify it by its brand
name: Advil, Aleve, Bayer, Excedrin, or Tylenol.
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Thin-Layer Chromatography
O
C
O
OH
H3C
O
CH3
C
O
O
H2
C H CH3
C
CH3
N
N
N
H3C H
C
N
CH3
ASPIRIN
C
CAFFEINE
OH
IBUPROFEN
O
HO
O
C
O
CH3
C
HO
C
H
N
C
H CH3
O
CH
CH3
KETOPROFEN
CH3O
HO
NAPROXEN
C
CH3
O
ACETAMINOPHEN
Figure 6. Structures of Some Common Over-the-Counter Drugs
6
Thin-Layer Chromatography
Experimental Methods and Materials
Safety considerations
Wear suitable protective clothing, gloves, and eye/face protection!
You should read the online MSDS for:
Acetic Acid
1-Butanol
Ibuprofen
Acetaminophen
Caffeine
Methanol
Aspirin
Heptane
Naproxen Sodium
Preparation of developing chambers
Clean and dry a 600-mL beaker. In a 50-mL beaker, place 10 mL heptane, 5
mL 1-butanol, and 1 mL glacial acetic acid into the beaker and stir to mix. Place a
piece of filter paper along one side of the 600-mL beaker. Tilt the beaker at a 45
angle and pour the solvent mixture over the filter paper. Cover with plastic wrap and
let stand while the TLC plates are prepared. Similarly, place 5 mL heptane, 10 mL
1-butanol, and 1 mL glacial acetic acid into a second beaker and stir to mix.
Which solvent is more polar? Explain this in your report.
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Thin-Layer Chromatography
Preparation of drug sample
Record the number of your unknown in your notebook. Crush the tablet into a
fine powder with a mortar and pestle. Place 5 mL methanol in the mortar and stir
for 5 minutes to dissolve the active ingredients. Let the mixture stand for 5 minutes.
Preparation of TLC plates
Handle your TLC plate by the edges to avoid placing fingermarks in the area for
developing the chromatogram. Using a ruler, very lightly make a pencil line 1.5 cm
above the short edge of the TLC plate. Determine the number of standards plus
unknowns that you will be spotting. Make hash marks equal to this number on the
line. Using a different capillary pipette and hash mark for each known standard and
unknown drug solution, carefully spot the TLC plate at the intersections of the
horizontal lines and hash marks. Be careful to avoid pitting the surface of the silica
gel with the point of your pipette. Identify by code which hash marks correspond to
which standards and which unknowns. See Figure 7. Examine the TLC plate under
a UV lamp to determine if another application is needed to make the spots more
readily visible. Always wait until each spot has dried before reapplying more
solution. Check to see that the spots are not more than 1-2 mm in diameter to
prevent any overlapping of substances during the chromatographic process.
KNOWNS
1 cm
1 cm
A
B
C
D
E
F
G
1.5 cm
UNKNOWNS
Figure 7.
Prepared TLC plate.
8
Thin-Layer Chromatography
Developing the chromatogram
Once the TLC plate has been prepared, stand it vertically opposite the filter
paper with the silica gel side facing the filter paper. The developing solvent line
should be below the pencil line to prevent the removal of the solutes from the silica
gel surface. Reseal the beaker with plastic wrap. DO NOT DISTURB THE BEAKER
WHILE THE CHROMATOGRAM IS DEVELOPING. When the developing solvent is
within 2 cm of the top edge of the TLC plate (time varies with each solvent), remove
the plate from the beaker and draw a pencil line along the solvent front. Place the
TLC plate on a clean paper towel and allow to dry completely. Place the plate under
a UV light and circle the position of each spot with a pencil. Note if any of the spots
show fluoresce under UV light. Determine the Rf values for each spot. Determine
which mixture provides the better separation of components.
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