Experiment 6: Thin-Layer Chromatography

6.1 Background
Over the last few laboratories, we have learned about several techniques used by chemists to
purify organic compounds such as recrystallization, distillation, and extraction. In many cases, however,
the mixtures of products obtained from chemical reactions do not lend themselves to ready separation
by any of these techniques because the physical properties of the individual components are too
similar. In such cases, chemists resort to the use of chromatographic methods to purify mixtures of
organic compounds.
The word chromatography was first used to describe the colored bands observed when a
solution containing plant pigments is passed through a glass column containing an adsorbent packing
material. The term now encompasses a variety of separation techniques that are widely used for
analytical and preparative purposes.
All methods of chromatography operate on the principle that the components of a mixture will
distribute unequally between two immiscible phases. Two phases can be clearly distinguished in
chromatography

The mobile phase is generally a liquid or a gas and

Fixed stationary phase, which may be a solid or a liquid. The mobile phase flows over the
stationary phase.

The individual components of the mixture have different affinities for the mobile and stationary
phases. Since each component partitions between the two phases with a different equilibrium constant
or partition coefficient, the components divide into separate regions termed migratory bands (fig. 1)
The component that interacts with or binds more
strongly to the stationary phase moves more slowly in
the direction of the flow of the mobile phase. The
attractive forces that are involved in this selective
adsorption are the same forces that cause attractive
interactions between any two molecules: electrostatic
and dipole-dipole interactions, hydrogen bonding,
complexation, and van der Waals forces. The
chromatographic methods used by modern chemists
to identify and/or purify components of a mixture are characterized by the nature of the mobile and
stationary
phases.
In
thin-layer chromatography
(TLC), and high-performance
liquid
chromatography (HPLC) each involve liquid-solid phase interactions. Gas-liquid partition
chromatography (GLC), also known as gas chromatography (GC), involves distributions between a
mobile gas phase and a stationary liquid phase coated on a solid support. Although there are other
chromatographic techniques, such as ion exchange and paper chromatography, a review of those
methods is beyond the scope of this present lab experiment.

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6.2 THIN LAYER CHROMATOGRAPHY (TLC)

In this lab experiment, you will learn and perform Thin-layer chromatography (TLC). TLC is a
form of solid-liquid adsorption chromatography and is an important technique for rapid analysis of small
quantities of samples, sometimes as little as 10-5 g. Thus, TLC is frequently used to monitor the
progress of reactions and of column chromatographic separations as well as to determine the optimal
combinations of solvent and adsorbent for such separations.
To execute a TLC analysis, a small amount of the sample to be analyzed, or a solution of it, is
first applied to a solid adsorbent bound to a rectangular glass or plastic plate (fig. 2a).

Figure 2: Technique to apply a sample on the TLC plate.

The adsorbent serves as the stationary phase. Next, the plate, with its spotted end down, is
placed in a closed jar, called a developing chamber (fig. 3b).
The chamber contains a saturated atmosphere of an eluting
solvent, which is the mobile phase and may be comprised of either a single
solvent or mixture of two or more. A folded filter paper is often used to help
maintain solvent equilibration in the chamber. It is important that the level of
solvent in the chamber be below the location of the spot on the TLC plate.
Otherwise, the sample would be removed from the plate by dissolution in the
bulk solvent, thereby ruining the analysis.
Figure 3: TLC chamber. (a) Folded filter paper to be placed in developing
chamber for solvent equilibration. (b) Developing chamber with filter paper
and TLC plate.
When the plate is placed in the chamber, the solvent begins to ascend the plate. The individual
components of the sample, which initially were at the bottom, or origin, are carried up the plate along
with the eluting solvent at a rate that is dependent on their relative affinities for the solid adsorbent:
more weakly adsorbed compounds move up the plate faster than do those that are more strongly
adsorbed. Ideally, when the solvent front has nearly reached the top of the TLC plate, which normally
takes only a few minutes, each component of the original sample will appear as a separate spot on the
plate (fig. 2b). This completes the development of the chromatogram, and the plate is then removed
from the developing chamber for analysis.
The solid adsorbent in TLC is usually alumina or silica gel (silicic acid), both of which are polar.
Although TLC plates may be prepared in the laboratory, their ready commercial availability generally
makes this unnecessary.
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The strength of adsorption of organic compounds to the stationary phase of the TLC plate
depends on the polarity and nature of the adsorbent and the type of functional groups present in the
compounds. Substances containing carboxyl groups and other polar functional groups are more
strongly adsorbed than are those containing less polar moieties, such as those present in alkenes and
alkyl halides, as the elutropic series shown. In the schematic below, compound B travels faster than
compound A, because the OH group in A results in more attraction of A to the stationary phase (fig. 4)

Figure 4: Movement of compounds in a TLC.

The following is a list of commonly used solvents in order of eluting power which correlates with
polarity.
Hexane or Cyclohexane <Cyclohexane <Benzene <Dichloromethane <Chloroform <Ether <Ethyl
acetate <Acetone <Ethanol <Methanol <Water <Pyridine <Acetic Acid (Greatest Eluting Power)
Selecting the eluant or the mobile liquid phase is an important decision that must be made when
planning a TLC analysis. The best solvent or combination of solvents is determined by trial and error;
however certain criteria guide the selection of the eluant.

An effective eluting solvent must readily dissolve the solute but not compete with it for binding sites
on the stationary phase.
The effectiveness of a separation is influenced by the selection of stationary phase. However, it is
the selection of the solvent (the mobile phase here), that is usually varied in order to obtain optimal
separation. In general, with increase in solvent polarity; compounds travel faster through the plate.
Solvents compete with the compounds for binding to the adsorbent, and therefore polar solvents
such as water adhere to the stationary phase more strongly, allowing the compounds being
analyzed to travel faster (fig. 5).

Figure 5: Typical TLC experiment

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Streaking in TLC: Sometimes a TLC analysis produces a streak on the plate rather than one or
more spots. This may result from the presence of so many compounds in the sample that the
individual spots are not separated, and they appear as a streak instead of a series of spots. Using a
different eluant may eliminate the streaking and produce individual spots. Another cause of
streaking may be using a solution of the sample to spot the plate that is too concentrated. In this
case, diluting the solution may minimize streaking.
Compounds such as carboxylic acids and amines that are strongly adsorbed to the alumina or silica
gel coating of the TLC plate tend to produce streaks rather than spots. The streaks observed on a
TLC plate by such compounds occur because of the equilibrium between neutral and ionized forms
of the respective functional groups. One easy way to address this problem is to deactivate the
adsorbent by adding some acetic acid to the eluant when analyzing carboxylic acids and some
ammonium hydroxide in the case of amines; only a drop or two of the acid or base is normally
required.
Both alumina and silica gel used as stationary phase are polar compounds. When a nonpolar
adsorbent is used, this is called reverse phase chromatography.

6.3 Visualizing the TLC plate:

There are several ways of visualizing the TLC plate. The easiest situation is that in which the
compounds being separated are colored, so visual detection is easy. However, many organic
compounds are colorless and a variety of methods have been developed to detect their presence on
the plate:
1. Compounds that fluoresce may be located by placing the plate under an ultraviolet light.
2. The chromatographic plate may be sprayed with a variety of reagents such as sulfuric acid,
potassium permanganate, phosphomolybdic acid, and ninhydrin; these reagents react with the
individual components to produce colored or dark spots.
3. The chromatographic plate may be exposed to iodine vapor by placing it in a closed chamber
containing several crystals of iodine. As the iodine forms complexes with the various organic
compounds, the brown spots appear on the plate. Since the process is reversible and the spots fade,
it is necessary to circle the spots with a pencil to have a permanent record of the chromatogram.
4. Once the separation of the components of the mixture is complete and the individual spots have
been detected, the retention factor of each compound is calculated. The Rf-value for a compound is
a physical constant for a given set of chromatographic conditions, so the adsorbent and the eluting
solvent should be recorded along with the experimentally determined Rf -values. Rf represents the
ratio of the distance traveled by the substance versus the distance traveled by the solvent (The
maximum, distance the substance could have traveled). The rate of elution of a particular substance
can be quantified in terms of its Rf value, Rf = distance traveled by substance/distance traveled by
the solvent (This value should be between 0 and 1). The Rf value for compound A for this particular
run is a/c. Likewise the Rf value for compound B is b/c.

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Figure 6: Calculation of retention factor in TLC.
In the following example, a sample of benzophenone is spotted alongside a sample of an unknown
compound. Since the two compounds moved up the plate at the same rate (same Rf), the compounds
could indeed be the same. However, the unknown could be a different compound that happens to have
a similar Rf value under the same conditions. Compounds with different Rf values are more definitely
different.
As stated earlier, more polar compounds are attracted more strongly to the plate and thus will
have smaller Rf values. Compounds that can donate a hydrogen bond such as alcohols, amines,

Figure 7: Separation of unknown and

standard benzophenone on the TLC plate

amides and carboxylic acids move especially slow on the plate and thus require more polar solvents.
6.4 Spotting the TLC plate
1. When handling the TLC plate, avoid touching the silica gel. Grab the plate on its sides. You dont
want to be analyzing the compounds in your fingers!
2. Carefully and lightly draw a line with a pencil about cm from the bottom on the silica gel or
alumina (white grainy) side. (The exact distance is not critical). This is the line where you will spot
your sample or samples.
3. Using a spotter (prepared from heating the center of an open-ended capillary tube with a Bunsen
burner and pulling on it to create an even thinner tube), dip the skinnier end into your sample
(typically 1% of the compound in a volatile solvent such as dichloromethane) and lightly press on
the plate.
4. You should see a small wet dot appear then slowly disappear as the solvent evaporates.
5. The compound itself remains on the plate. If you wish to add more sample, lightly press again on
the same spot as before, but not until the first spot evaporates.

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6. You want to have the spot as small as possible for optimal separation. Ideally the diameter should
be no more than a few millimeters.
6.5 Running the TLC plate
1. You will need a beaker, which are provided in one of the common drawers. The beaker should
be dry. If it is not, wash it with the solvent you are going to use. Do not wash it with water
unless water is the solvent.
2. Cut a piece of filter paper in half and stand it up against the beaker wall (fig. 3b). The purpose
of the filter paper is to help saturate the chamber with solvent vapor. This solvent saturation will
help prevent solvent from evaporating from the plate and thus will make the solvent run faster
up the plate.
3. Pour a small layer of solvent in the jar.
4. Be sure the solvent level is low enough such that is below the spots on the TLC plate.

5. Once you place the plate in the jar, place the cap on top (does not need to be screwed on) and
watch the solvent level creep up the plate.
6. Remove the plate when the solvent level reaches near the top, but not at the top (about the 0.5
cm from the top). After removing the plate, immediately mark the solvent level with a pencil.
This needs to be done immediately since the solvent will quickly begin to evaporate.
7. After the solvent evaporates from the plate, view the spots under a UV lamp and trace the
spots with a pencil.
PROCEDURE
Safety: Hexane, diethyl ether, and ethyl acetate are flammable. Avoid using open flames.
There are six steps involved in the TLC experiment
Step 1: Preparation of the developing chamber
A developing chamber consists of a beaker, a g, a piece of filter paper and a rubber band
Place a piece of filter paper into the beaker, as demonstrated by the image
below. This will ensure that the TLC plate will remain saturated with the vapor
from the aqueous mobile phase, so that the plate will run correctly
Carefully pour hexane into the beaker, to a depth of approximately 1 cm,
making sure that the entire filter paper is saturated, swirling if necessary.
Step 2: Preparation of the TLC plate
Using a ruler and a pencil, draw a across the TLC plate 2.0 cm from the
bottom, as indicated in the picture below pressing the pencil lightly as to
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not damage the coating on the TLC plate. This line will serve as the origin line on which you or
use the capillary tubes to place the stock and unknown solutions. Repeat this step for 8 TLC plates
Step 3: Spotting the TLC plate
You will be testing 4 compounds (Biphenyl, vanillin, 2-napthol and benzophenone) and mixtures of
these compounds on a TLC plate. Stock solution A: Biphenyl (BP), Stock solution B: vanillin (VA), Stock
solution C: 2-napthol (NA), Stock solution D: benzophenone (BE), Stock solution E: unknown 1 (UN1)
and Stock solution F: unknown 2 (UN2)
You will be using 4 solvent systems

100% hexane (or simply hexane)

100% ethyl acetate
100% diethyl ether
50:50 hexane and ethyl acetate
Solvent
100% Hexane
100% Ethyl acetate
100% Diethyl ether
50:50 hexane and ethyl acetate
Composition of your choice based on results

Plate 1
BP, VA, NA
BP, VA, NA
BP, VA, NA
BP, VA, NA
BP, VA, NA

Plate 2
BE, UN1, UN 2
BE, UN1, UN 2
BE, UN1, UN 2
BE, UN1, UN 2
BE, UN1, UN 2

1. Dissolve each of the compounds using 1% solutions (pre-prepared) in ether. You will be using the
capillary tubes to spot both the known and unknown solutions to your TLC plates. You will use
about 5l of each sample to spot on the plate. Each solution will require a separate capillary tube to
prevent cross-contamination.
2. Using a piece of tape wrapped around the top of the tube, carefully label each tube, using the same
symbols you used to label the compounds (shown above). Use caution with the capillary tubes, as
they are fragile and very sharp when broken.
3. Take the capillary tube named BP and place the pointed end into stock solution A. You should be
able to see the solution rise up into the capillary, through capillary action.
4. Next, using plate #1, touch the end of the capillary tube gently to on the origin line at the spot
indicated for that solution. Your goal is to make only a small spot. DO NOT let all of the contents of
the capillary tub run onto the paper. You will not use all of the solution inside the capillary tube.
Repeat this process for unknowns.
5. You will now repeat this process for the remaining solutions, putting one spot at the indicated spot
on each TLC plate, until each solution has been spotted on its appropriate plate.
6. After each spot is dry, you will repeat the entire process 2 more times, so that each solution has
been spotted 3 times on every plate.
7. It is VERY IMPORTANT that you allow each spot to fully dry before re-spotting the solution. Blowing
on the spot, or fanning the TLC plates may help to speed drying time.
8. Proceed to step four when all the spots on both plates are dry.
Step 4: Developing the TLC plate
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Before placing your TLC plates into its developing chamber, measure and compare the height of
the solvent in relation to the line you have drawn on your TLC plate. If it appears that the solvent may
cover the line, then remove some of the solvent from the beaker. This step is critical! If the solvent
covers the line when you place the TLC plate into the developing chamber, you will have to start
the lab over from the beginning. If you are unsure if the solvent will cover the line, then err on the
side of caution, and remove some of the solvent. You can always add solvent back with no negative
effects if there isnt enough solvent in the beaker to develop the plate.
1. Carefully place the prepared TLC plate in the developing beaker, so that it is sitting on the bottom of
the beaker, and leaning against the side of the beaker that is not covered by the filter paper. Be
very careful! If the plate falls into the water, you will have to start the lab over from the beginning!
Make sure you record what time you first placed the plate into the chamber.
2. Cover the beaker with the saran wrap, and carefully secure the saran wrap with a rubber band,
making sure not to dislodge the TLC plate within the chamber. DO NOT pick up the chamber!
3. Repeat this process for the remaining plate.
4. Watch as the mobile phase runs up the TLC plate. When the mobile phase is between 2- 4 cm from
the top of the TLC plate, remove it from the chamber using the tweezers, and place it on a paper
towel. Make sure you remove the plate before the mobile phase runs off the end of the plate. If this
happens, you will have to start the lab over from the beginning.
5. Using a pencil, trace the line of the mobile phase on your plate. This is a critical step for the
calculation of the Rf factor.
6. Leave the plate on the paper towel until it is completely dry.
7. Proceed to step 5 only when the plates are totally dry.
Step 5 Visualizing the spots
Warning: UV light is damaging both to your eyes and to your skin. Make sure you are wearing your
goggles and do not look directly into the lamp. Protect your skin by wearing gloves.
1. With a lab partner, hold the UV lamp over the plate, facing down, and mark any spots which you
see lightly with a pencil, tracing around their outline. Ideally, you should see individual dots for
each compound in the mixture.
2. Now that you can visualize the molecules that are on the plate, you can being begin collecting
the data which will allow you to determine the identity of the unknown solutions.
Step 6 Evaluating the data
Plate #1 - The purpose of plate one is to compare the distance travelled by the spot with known
standards. In addition, plate one allows you to calculate the Rf factor for the known solutions.
Plate # 2 - The purpose of plate two is to identify the unknown standards using a comparison of Rf
values. To do this, you will need to calculate the Rf value for each spot on both plates, using the
formula provided below, and record your information on the student work sheet.
Sample Calculation: This calculation is based upon a sample that is made up of a combination of
substances, and therefore has more than one spot. The procedure for determining the Rf value for
each spot within one sample is the exact same as the procedure for determining the Rf value for many
individual samples on one plate

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The solvent front is the distance the mobile phase traveled on the
plate, and is what you recorded with your pencil when you took your
plates out of the developing chambers.
#1

The formula for calculating the Rf values is
Rf = Distance moved by the molecule (location of the spot)/ Distance
moved by the mobile phase (solvent front)
The Rf value for the substance (marked by the yellow spot) would be
Rf = 5.5 cm/6.0 cm = 0.92 cm

For all TLC plates developed, sketch the results in your notebook
Identify unknowns and write basis for identification in your formal lab report

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