CH 2270

Chromatography (Adapted from Laboratory Manual to Accompany Organic Chemistry:

A Short Course, H. Hart, L. E. Craine, D. J. Hart, and T.K. Vinod 13th ed. Brooks/Cole, Cengage
Learning, Belmont, CA 2012.)

General Principles
Chromatography is a technique used to separate the components of a mixture and to purify substances. It
was first developed in connection with the separation of colored compounds (hence the name, from the
Greek chroma, color), but chromatography is now used to separate colorless substances as well.

There are two kinds of chromatographic separations. The first is called adsorption chromatography.
This type depends on selective adsorption or adhering of the substance being purified on some highly
porous surface known as an adsorbent. Different types of intermolecular interactions including H-
bonding, dipole-dipole interactions, and Lewis acid-base interactions cause organic molecules to bind to
the porous surface of the adsorbent. Adsorption chromatography may be carried out in vertical columns
packed with an adsorbent (column chromatography). Alternatively, the adsorbent may be spread out in
a thin layer on an inert surface as illustrated in this experiment (thin-layer chromatography).

The second general type of chromatographic separation is called partition chromatography. Here,
separation depends on partitioning of the mixtures components between two solvents. One of these
solvents (called the stationary phase) is adsorbed on a solid support. The other solvent (called the
mobile phase) passes through this support.

One kind of partition chromatography, illustrated in this experiment, is called gas-liquid

chromatography or vapor-phase chromatography. In this technique, vapors of the mixture being
purified (the mobile phase) are passed through a heated tube that contains a finely divided solid support
on which a nonvolatile liquid (the stationary phase) is adsorbed. Separation depends on the different
affinities of the mixtures components for the nonvolatile liquid phase.

Thin-Layer Chromatography (TLC)

References:
Organic Laboratory Survival Manual, p. 219, 223, 249
SuperOrganicLab: http://chemed.ces.clemson.edu/SOL/SuperOrganicLab/
Then: click on Lab Techniques, then Purification Procedures, then double click on
Chromatography and Thin-Layer Chromatography
MIT TLC Video: MIT OpenCourseWare TLC Video for Organic Lab

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Analysis of Analgesics (Pain Killers) by Thin Layer Chromatography

Common ingredients in pain killers are:

O N
CH3 NH2
O
CH3 N N
N
OH
O N N CH3O
CH3 N
CH3 CH3

Caffeine Salicylamide Pyrilamine

Your task is to identify the ingredients in a common pain killer (analgesic). You will be given known
samples of these compounds and you will need to compare them with the commercial samples, of which
you will not know the composition. You will analyze the six known samples with the other members of
your group.

Unfortunately, commercial pain killers contain mixtures of compounds so you will not be able to use
melting point as a comparison technique. Instead you will use thin layer chromatography, a method that
allows you to visualize the number of ingredients in a mixture, and by comparison with known samples,
to identify those materials. Each group will analyze their own pain killer.

First, prepare your developing chambers. To simplify your lives, we will have two solvent systems made
up: 90% ethyl acetate/10% toluene; and 97% dichloromethane/3% methanol. They will be in bottles in the
hood. You must experiment and see which system works better. Into one 250 mL beaker, place a piece of
9 cm filter paper, with one edge slightly folded down as shown in Figure 1. Pour 15 mL 90% ethyl
acetate/10% toluene into the beaker, letting it wash over the filter paper. Cover the beaker with a watch
glass. Prepare the developing chamber for the other solvent system similarly, pouring 97%
dichloromethane/3% methanol into a separate 250 mL beaker with folded filter paper. Allow both
developing chambers to equilibrate, covered, for 5 minutes before running a TLC plate.

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 Figure 1. TLC Developing Chamber
with TLC Plate and Solvent Running Up

To determine which analgesics are in the pain killer, you will need to co-spot some of the pure
compounds beside your commercial pain killer on the same plate to compare Rf values. Prepare a
solution of your pain killer by dissolving 100 mg (0.1 g), in a test tube, in 2 mL DCM. For each
analgesic, make a solution by weighing 100 mg of compound and dissolving it, in a test tube, in 2 mL of
dichloromethane (DCM). If it does not dissolve, add 2 mL of ethanol. Make separate solutions for each
of the common pain killer ingredients.

Obtain a TLC plate from the side shelf and with pencil, lightly mark a horizontal line one centimeter
from the bottom of the plate. Do not chip away the adsorbent from the TLC plate. DO NOT WASTE
THE PLATES. We have to cut them up in the stockroom. It is labor intensive and time consuming.
Mark three equidistant cross-hairs along this line, one in the very center and the other two splitting the
distance from the center to the edge of the plate. Use a 15 L disposable applicator pipet to spot the
dichloromethane solution of pain killer onto the plate on the center crosshair you marked. Try to keep the
spots at 1-2 mm in diameter. Spot one known analgesic on the crosshair to the left of your pain killer and
another known analgesic on the crosshair to the right. Use only one applicator pipet for each compound;
do not cross-contaminate. Spot once per sample, running three samples per TLC plate. Label each spot
(with pencil) corresponding to its analgesic/pain killer. Look at the TLC plate under the UV lamp after
you spot it be sure that you see three dark circles where you spotted your samples. If not, consult your
TA. Place the plate in the 90% ethyl acetate/10% toluene developing chamber, with the sample spots on
the bottom but NOT submerged in the solvent system and such that the TLC plate does not touch the filter
paper, and cover with a watch glass. See Figure 1. Allow the solvent to wick up the TLC plate. When
the solvent reaches 1 cm from the top, pull the plate out and draw a line where the solvent stopped.

Spot two more TLC plates with the remaining four analgesics, two per plate, with your pain killer in the
center, and run in the 90% ethyl acetate/10% toluene developing chamber similarly. Additionally, spot
three more TLC plates with the six analgesics (two per plate) and pain killer (in the center) and run them
in the 97% dichloromethane/3% methanol developing chamber.

Visualize your developed plates with the UV lamp on the side shelf. Trace the outline of each spot. For
example, see the figure below. Draw each developed TLC plate in your notebook. The plates are 1 inch
x 3 inches. This is a perfect size for your notebooks. The grid on the pages is 4 lines to an inch. This
makes it easy for you to draw pictures of your TLC plates without having to trace the plates. Just use a
straightedge.

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 ~ 1 cm

Distance Distances
Traveled Traveled
By By Compounds
Solvent
~ 1 cm
Original
Spots of
Samples

Figure 2. TLC Plate with Markings

Determine and record the Rf value of each spot. The distance traveled by each spot is measured from the
origin to the center of the spot. Use a ruler with mm graduations to measure your plates. Report your Rf
values to three decimal places.

Rf = distance compound has traveled from origin

distance developing solvent has traveled from origin

Determine which is your best solvent system. A good solvent system for TLC should:
1) Space all of the different analgesic compounds out, so that none have the same Rf.
2) Carry all compounds off of the baseline.
3) NOT carry any of the compounds all the way to the top of the solvent front.
If you are unsure, consult with your TA as to which is the better solvent system for your group.

Using your best solvent system, compare Rf values of your pain killer spot(s) with those of the analgesics
to determine the component(s) of your pain killer. An identical substance will have the same Rf value in
the same solvent system, so match your pain killer spot Rf values with those of the known analgesics.
Confirm your conclusions with your TA before you leave.

Waste Disposal

Since we are using DCM this week, we will have special bottles in the hood labeled "Halogenated
Organic Waste." We will always use these bottles when disposing of halogenated organics. Discard the
developing solvents containing DCM into a halogenated organic liquids waste bottle. The ethyl
acetate/toluene developing solvents should go into the nonhalogenated organic liquids waste bottle.

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Gas-Liquid Chromatography (GLC)
Gas-liquid chromatography (GLC)* is an accurate and rapid process for separating and analyzing
the components of a volatile mixture. The apparatus required for this type of chromatography
consists of the following essential parts:

1. Injection block. A heated chamber for introducing and vaporizing the sample.
2. Packed column. Usually a length of metal tubing packed with a porous solid that is thinly coated with
a high-boiling liquid. The tube is located in an oven that can be heated at a controlled temperature.
Most modern gas-liquid chromatographs use capillary columns (0.5 mm 1.0 mm in diameter) that
are considerably longer instead of the packed columns.
3. Carrier gas. The gas (usually helium or nitrogen) that carries the sample through the heated column.
4. Detector. A device used to detect each component of the sample as it appears at the exit of the packed
column. The intensity or area of the signal indicates the quantity of the eluted component.

*Gas-liquid chromatography is also called vapor-phase chromatography (VPC).

The instrument works as follows: The injection block, column oven, and detector are heated to the desired
temperature, and the inert carrier gas is passed through the apparatus. Actually, the carrier-gas stream is
split. Part of it passes through the entire apparatus, and part of it goes directly to the detector. One
common type of detector, called a thermal conductivity detector, consists of two heated wire filaments,
one exposed only to the reference carrier-gas stream and the other exposed to the effluent gas from the
column.

First, a small sample of the material to be analyzed is injected. The sample vaporizes in the injection
block and is carried through the column, through the detector, and eventually out of the apparatus. As the
sample is eluted from the column and passes through the detector, one of the detector wires is exposed to
a mixture of carrier gas plus sample, and the other wire is exposed only to the reference stream of carrier
gas. The difference between the two gas streams causes a difference in the electrical conductivity of the
wires, and this difference is automatically recorded. In this way a gas-liquid chromatogram is obtained.

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PROCEDURE
Separation of a Two-Component Mixture
Inject a small sample, approximately 1 L of the second, fifth, and last fractions from the simple and
fractional distillations of the hexane-toluene mixture (Experiment 3, Distillation). Assume that the area
under each curve is approximately proportional to the amount of material present, and calculate the ratio
of the two substances in each fraction. Record the conditions used for your chromatographic separation in
your notebook.

Pour any leftover hexane or toluene into the waste bottle provided.