Reminder: These notes are meant to supplement, not replace, the laboratory manual.
Thin Layer and Column Chromatography
History and Application:
Chromatography is a very old and widely used analytical and preparative
technique which separates mixtures. Chromatographic separations are used in
forensics (CSI), research, chemical manufacturing plants, environmental testing
(pesticides, air, water), water purification in remote locations and drug testing of athletes
urine. It is one of the most common separation and identification methods used in the
world.
Thin Layer Chromatography (TLC) is a quick, simple and inexpensive way to
analyze small samples.
Column Chromatography is useful in isolating sizable amounts of pure solid
materials from mixtures. The sample size can vary from a few micrograms to tens of
grams.
1.
Here is some terminology related to this experiment:
Chromatography is a technique in which compounds in a mixture are separated
based on differing affinities between a mobile phase and a stationary
phase.
Mobile phase is a medium used in chromatography which moves through the
stationary phase. In TLC and column chromatography, the mobile phase
is an organic liquid. In Gas Chromatography (GC) the mobile phase is a
gas.
Stationary phase is a material used in chromatography which does not move.
The mobile phase passes through the stationary phase. The stationary
phase is either a pure solid powder such as alumina or silica or a thin
coating of liquid on a solid support or a gel.
In TLC the stationary phase is a thin layer of silica or alumina spread on an inert
support, usually a plastic sheet or a glass plate. The mobile phase moves
upward by capillary action.
In column chromatography a glass or plastic tube is filled with the powder
stationary phase. The mobile phase moves through the stationary phase
by gravity.
Partitioning is the separation of the compounds into distinct groups or
assemblages between two or more phases. In chromatography this is
commonly based on polarity and affinity between the mobile phase and
the stationary phases.
Affinity is a qualitative measurement of the attractive force between a compound
and a phase. In TLC and column chromatography this is based on
polarity. Materials with similar polarity will have a high affinity or
attractiveness toward each other. Materials with a dissimilar polarity will
have a low affinity for each other and will self-separate.
Elution is the removal or exit of a compound from the stationary phase. In column
chromatography this is when the material leaves the column.
Many compounds are not visible to the eye when dissolved in a solvent or
adsorbed on a TLC plate. Visualization processes make these
substances visible. Techniques used include UV lights that cause
fluorescence or phosphorescence and chemical reactions that give
colored compounds.
The Rf value in TLC is the ratio of the distance a compound moved from the
origin to the distance between the origin and the solvent front.
TLC is used primarily as an analytical technique. It can be used to identify
components of a mixture (by comparing Rf values), monitor the progress
of a reaction and check the purity of a sample (more than one spot
indicates an impure sample). Rarely is TLC used to isolate and purify
compounds into discrete samples.
Column chromatography is rarely used as an analytical technique, as is TLC.
Column chromatography is primary used to separate relative large
samples into pure components. Column chromatography is a preparative
technique.
2. The mobile phase moves through the stationary phase and may carry with it some of
the compounds. The ability of a solvent to carry and elute compounds
from a column or TLC plate is related to the solvents polarityi. In general
the stronger the eluting power the more polar the solvent. The stronger the
eluting power, the quicker the compounds will elute from the column or
plate.
A ranking of the eluting power of classesii of organic compounds can be made:
hydrocarbons<halogenated hydrocarbons<ethers<esters<ketones <alcohols<acids
The eluting power of specific organic solvents are as follows.
heptane<toluene<methylene chloride<MTBE
<ethyl acetate<acetone<ethanol<acetic acid
Heptane is much less polar than MTBE. Heptane has a lower eluting power than MTBE.
Sometimes the desired polarity or eluting power of a solvent is difficult to achieve with a
pure solvent and a mixture of solvents is used. The mobile phase for the
TLC is a 80:20 Heptane: Ethyl Acetate. The 1/5 part ethyl acetate in the
4/5 of heptane increases the eluting power of the solvent.
When separating two or more compounds via chromatography, it is very important to
choose the correct solvent as a mobile phase. If too weak of an eluting
power solvent is chosen, it will take a very long time and a very large
volume of solvent to elute the compounds. If too strong of an eluting
power solvent is chosen, all the compounds will elute immediately and no
separation will occur.
3. The two most common stationary phases used in TLC and Column are alumina and
silica gel. Both of these materials are fine powders which are exceedingly
polar. Alumina is aluminum oxide or Al2O3.The surface of alumina is
covered by oxygen atoms. Because of the small particle sizeiii, a cubic
centimeter of alumina may have a surface area of approximately 100 m2.
This means that there is just over half a volley ball court of surface area
per cubic centimeter of alumina! Silica is SiO2. Silica has similar
properties to Alumina.
4. Separation occurs due to the different affinities of the compounds within a mixture to
the stationary and the mobile phase. Polar compounds have a high affinity
to other polar solids and polar solvents. Low polarity or non-polar
compounds have a low affinity to polar solids (like alumina) and a high
affinity to non-polar solvents. Compounds have a high affinity for solvents
with a similar polarity to themselves. This can also be paraphrased as like
materials like other materials like themselves or “like like like”.
Partitioning takes place as compounds within mixtures self-separate
based on affinities.
In most column and TLC, the stationary phase has a very high polarity and
the mobile phase has a lower polarity. (There are some instances where
the polarities are reversed, but this will not be discussed in this class.) The
compounds will partition based on their relative affinity to the mobile phase
(lower polarity) and the stationary phase (high polarity). Alumina is a very
very polar substance. Heptane has a relatively low polarity. Ferrocene is
less polar then acetylferrocene. This means that ferrocene will like, or
have a higher affinity to the less polar mobile phase
(heptane:ethylacetate) than the more polar stationary phase (alumina). In
contrast acetylferrocene will have a larger affinity to the stationary phase
(highly polar alumina) than the heptane:ethyl acetate mobile phase. If a
compound has a high affinity to the stationary phase it will elute slower
than a compound which has a lower affinity to the stationary phase. Which
compound, ferrocene or acetylferrocene, do you expect to elute the
column first?
5. The Rf in TLC is the ratio of the distance the spot traveled to the distance the solvent
traveled on a TLC plate. To obtain a Rf, measure the distance from the solvent
start line (NOT the bottom of the plate) to the middle of the spot. Measure the
distance from the solvent start line (again NOT the bottom of the plate) to the
solvent front (where the solvent stopped moving). The distance between the
smallest two lines on the ruler should be approximated. Using the rulers in the
lab, this means that the distance will be measured to 2 decimal places or 1/100
of a centimeter. If more than one spot is present in a lane, calculate the Rf for
each spot within each lane.
D
Dist tan ce _ spot _ traveled
2.63cm
 spot 
 0.489
Dis tan ce _ solvent _ traveled Dsolvent 5.38cm
The units cancel. Rf is a unit less value and will always be between 0 and 1,
0≤Rf≤1.0. If Rf equals 0 the spot did not move at all, the compound is very polar
and has little to no affinity to the mobile phase. If Rf value equals 1.00 the
compound traveled right with the solvent, and the compound is very non-polar
with little to no affinity to the stationary phase.
Rf 
The higher polarity the compound, the larger affinity of the compound to the
stationary phase and the smaller the Rf. The lower the polarity the compound, the
higher the affinity to the solvent and the larger the Rf. If a solvent is changed from
a low polarity solvent (like heptane) to a higher polarity (like ethyl acetate) the
eluting power will increase and all the Rf values will increase.
6. The TLC plates being used today are made up of a thin film of alumina powder on a
plastic sheet. The alumina is approximatelyiv 250 m (0.25 mm) thick.
Handle TLC plates only by the edges. If a greasy finger is placed on the
matte surface of the plate, the plate is ruined. The solvent or mobile phase
being used in today’s experiment is a 4:1 by volume mixture of heptane
and ethyl acetate. The mobile phase will move up the TLC by capillary
action. This same force is responsible for water creeping up a paper towel
whose corner has been dipped in a puddle.
7. Here is the apparatus used for thin-layer and column chromatography. The TLC
apparatus is on the left, and the column chromatography apparatus as
used in this experiment is on the right.
When placing your TLC plate in the TLC chamber, be sure the origin line
(containing the spots) is above the surface of the solvent. If the solvent
covers the origin line (and spots), the compounds will dissolve in the pool of
solvent you will need to dump the contents of TLC chamber, throw away the
plate and begin again.
The filter paper is soaked with solvent and puts solvent vapors in the atmosphere
inside the beaker, preventing excess evaporation of the solvent from the plate
during the experiment. The watch glass or piece of aluminum foil keeps these
vapors inside the beaker. Keep TLC chambers covered except when placing
plates within or taking plates out.
In the column chromatography apparatus, the top layer of sand prevents the
solvent (mobile phase) from disturbing the adsorbent (which is the stationary
phase) when it’s poured in. The bottom layer of sand provides a level surface to
keep the adsorbent layer uniform. The wad of tissue paper allows the mobile
phase to flow through while preventing the sand from doing the same. The
mobile phase moves through the stationary phase due to gravity.
8.
Here are the structures of the organic chemicals used in this experiment.
Ferrocene and Acetylferrocene are organometallic compounds. This means that they
contain both a metal atom and carbon atoms. These two compounds contain an iron
atom bonded between two cyclopentadiene units.
9.
Here are some pitfalls of TLC: If you use too much solvent in the bottom of the
beaker, or if you carry the beaker around during the experiment allowing the
solvent to slosh part way up the plate, the sample you are studying may wash off
the plate into the solvent at the bottom. If you leave the watch glass off the top of
the beaker, the solvent may evaporate from the plate, giving R f values that are
artificially high.
10.
The most common error which occurs while running a column chromatography is
if any part of the stationary phase is allowed to run dry. This will lead to the
column support cracking and will result in poor separation or incomplete elution
of the components.
TLC INTERPRETATION
11.
TLC interpretation can be a little confusing at first, but with a little practice it will
become second nature. Each unique compound has a distinct Rf value in a given
solvent system. This means that the Rf can be used to identify compounds.
Two samples were spotted on a TLC plate and run using the same mobile phase.
Note that the distance the spots moved are different and the distance the solvent
fronts moved are different. Calculate the Rf for each. What number did you get
for each? Do you believe these two samples contain the same compound? Why?
Remember the Rf is used to assign identity.
When discussing identity of these two spots one cannot say S1 is the same as
S2 because they moved the same distance, because they did not. Instead a
2.30 𝑐𝑚
reasonable way to discuss the identity would be to say “S1 has a Rf of (3.45 𝑐𝑚 =)
1.58𝑐𝑚
0.67 and S2 has a Rf of 0.66 (2.40𝑐𝑚), therefore S1 and S2 are probably the same
compound because 0.67 is approximately equal to 0.66.” Note that the units
cancel, and Rf has no units. Rf is used to assign identity.
Different compounds within a mixture usually have different Rf values and hence will be
seen as different spots. The number of spots in a given channel or lane are used
to determine the number of different compounds in that mixture. If a lane has
only one spot, that means the sample in that lane is composed of only one
compound which in term means the sample is pure. The number of spots
indicates purity.
More than one material may be run on a single TLC plate. Each initial compound
mixture should run parallel up the TLC plate and hence each initial sample will
have its own lane. This is similar to multiple cars moving in the same direction on
a multilane highway.
HOW TLC CAN MONITOR A COMPOUNDS BEING SEPARTED USING COLUMN
CHROMATOGRAPHY.
12.TLC is routinely used to monitor a column separation. The solvent which elutes from
a column is captured in different fractions. In today’s lab only two fractions are
collected. It is much more common to collect many fractions; 5 to 100 or more.
Below is a TLC plate and results from authentic A, authentic B, and four
fractions.
Lane
A
B
F1
F2
F3
F4
Rf of each spot
0.20
0.80
0.78
0.15, 0.78
0.18
Try to clearly and scientifically describe the results in fractions 1 through 4 (F1-F4). First
describe each spot in each lane, then clearly explain what it means in terms of the
column separation. Remember to discuss identity in terms of Rf and not distances.
Here are some examples.
F1. Fraction one does not contain either A or B because no spots are present. This
indicates that the compounds have not eluted from the column yet.
F2. Fraction two is a pure material. This is known because there is only one spot. The
Rf of the spot is 0.78. The identity of this material is probably B, because the Rf of
authentic B was found to be 0.80, and 0.78 is almost identical to 0.80.
This is a good pure fraction of compound B and shows the initial separation was
successful.
F3. Fraction three does not contain a pure material. This is known because there are
two spots present. The Rf of the first spot is 0.15. The identity of this spot is likely A
because authentic A has a Rf of 0.20, and 0.15 is very close to 0.20. The second spot s
0.78. The identity of this material is probably B, because the Rf of authentic B was
found to be 0.80, and 0.78 is almost identical to 0.80.
This shows the separation was not successful because the fraction contains a mixture
of A and B.
F4. Fraction four is a pure material. This is known because there is only one spot. The
Rf of the spot is 0.18. The identity of this material is probably A, because the Rf of
authentic A was found to be 0.20, and 0.18 is almost identical to 0.20.
This is a good pure fraction of compound B and shows the separation was successful.
Overall the separation was successful. Pure A was isolated in fraction 4 and pure B was
isolated in fraction 2. Fraction 3 contains a mixture. This material could be combined
with other A/B mixture and again run through a column to separate.
HOW TLC CAN MONITOR THE PROGRESS OF A REACTION
13. TLC can be used to easily, quickly and inexpensively monitor the progress of a
reaction. To illustrate this point a reaction is run in which compound “A” is
reacted to compound “B” to form a new compound “C”. A + B -> C. The reaction
is run five different times (R1-R5). Pure compound A and pure compound B is
spotted on a TLC plate along with each of the five reaction mixtures. The
developed TLC results are depicted below.
Lane
A
B
R1
R2
R3
R4
R5
Rf of each spot
0.07
0.77
0.07, 0.75
0.51, 0.78
0.08, 0.52
0.06, 0.49, 0.76
0.52
1. Which of these reactions did not proceed? It produced no new products, and both the
starting materials are still present.
2. Which reaction produced a single pure new product?
3. The reaction proceeded, a new product was formed and the compound A was the
limiting reagent. Remember, the limiting reagent is the material that is consumed
first and defines how much product can be produced.
4. The reaction proceeded, a new product was formed and the compound B was the
limiting reagent. Remember, the limiting reagent is the material that is consumed
first and defines how much product can be produced.
5. One reaction produced a new product but did not finish? (When a reaction is
finished, or runs to completion, that means that the limiting reagent has been
consumed.)
Look this over and try to figure out which reaction is which. Discuss what you believe
with friends or colleagues. If having problems, discuss with instructor.
[Answer 1. R1, 2. R5, 3. R2, 4. R3, 5. R4]
HOW TO CLEALY EXPLAIN TLC RESULTS
14. Try to clearly and scientifically describe the results in R1-R5. First describe each
spot in each lane, then clearly explain what it means in terms of reaction progression.
Remember to discuss identity in terms of Rf and not distances.
Here are some examples.
R1. Reaction mixture 1 contains two compounds and hence it is not pure. This is
known because there are two spots in the R1 lane. The lower spot in R1 has an Rf of
0.07. The identity of this material is likely unreacted A, because authentic A sample was
shown to have an Rf of 0.07. The second spot in R1 lane has an Rf of 0.75. The identity
of this spot is likely unreacted B because authentic B has an Rf of 0.77 and 0.75 is
almost equal to 0.77.
This shows that the reaction has not worked because only unreacted starting
materials (A and B) are present and no new product spot is present.
R2. Reaction mixture 2 contains two compounds and hence it is not pure. This is
known because there are two spots in the R1 lane. The lower spot in R1 has an Rf of
0.51. The identity of this material is a new material, likely C, because this Rf is
substantially different than the Rf values of either of the starting materials (0.07 and
0.75). The second spot in R1 lane has an Rf of 0.78. The identity of this spot is likely
unreacted B because authentic B has an Rf of 0.77 and 0.78 is almost equal to 0.77.
This shows that the reaction has gone to completion, a new product has been
formed, and the limiting reagent was A. The reaction is complete because it cannot
proceed any more due to the fact that there is no more A present. Since A has been
depleted before B, that means B was in excess and A was the limiting reagent.
R4. Reaction mixture 4 contains three compounds and hence it is not pure. This
is known because there are three spots in the R1 lane. The lower spot in R1 has an Rf
of 0.06. The identity of this material is likely unreacted A, because authentic A sample
was shown to have an Rf of 0.07 and 0.06 is almost identical to 0.07. The middle spot
in R1 has an Rf of 0.49. The identity of this material is a new material, likely C, because
this Rf is substantially different than the Rf values of either of the starting materials (0.07
and 0.75). The third spot in R1 lane has an Rf of 0.76. The identity of this spot is likely
unreacted B because authentic B has an Rf of 0.77 and 0.76 is almost equal to 0.77.
This shows that the reaction has progressed, and a new product has formed, but
it has not gone to completion. The reaction is not complete because in the reaction
mixture both starting materials are present (A &B) and hence more C could form.
What is shown in R3 and R5?
15. Safety Precautionsv,vi
Ferrocene causes irritation to the eyes and skin. Prolonged ingestion or inhalation may
cause liver damage. Acetylferrocene is toxic if ingested or absorbed through skin.
Do not eat or breathe these materials. Keep away from skin. Wear gloves. Wash
hands at end of lab.
Dichloromethane (Methylene chloride) is a chlorinated organic material. Most
chlorinated organics possess some negative health effects. High level exposure
to dichloromethane can cause central nervous system depression; long term
exposure may cause tumors. Handle in small quantities, minimize contact with
liquid and avoid breathing vapors.
Heptane, ethyl acetate and methyl t-butyl ether (MTBE) are flammable organic liquids.
Have no flames in lab. Avoid breathing vapors. Keep TLC chambers covered.
References
i
Experimental Organic chemistry, 2nd Ed, J. C Gilbert & S. F. Martin, Sanders New York 1994, P157
ii
Quantitative Chemical Analysis, D. C. Harris W.H Freeman 1982 p584
Sigma –Aldrich Chemical Company Product specifications
http://www.sigmaaldrich.com/etc/medialib/docs/SigmaAldrich/Product_Information_Sheet/a1772pis.Par.0001.File.tmp/a1772pis.pdf (September 14, 2011)
iv
Fisher Scientific Catalogue Product Specifications http\\www.fisher.sci.com (September 14, 2011)
v
Fisher Scientific MSDS sheet Ferrocene http://fscimage.fishersci.com/msds/03388.htm and Acetylferrocene
https://fscimage.fishersci.com/msds/69220.htm (September 14, 2011)
vi
Fisher Scientific MSDS for Dichloromethane methylene chloride MSDS (September 14, 2011)
iii
Revised: December 4, 2015 S.L. Weaver