Thin
Layer Chromatography (TLC)
Purpose:
This laboratory exercise will introduce the student
to the technique known as Thin Layer Chromatography (TLC). After practicing the technique in Part I of
the experiment, the student will identify a mixture of unknown analgesics by
comparing the TLC of the mixture with the TLC of the known samples.
Introduction:
Chromatography is a technique which is widely used to
separate a mixture of substances into its component parts. There are several
techniques for separating milligram to gram quantities in a mixture, including
column chromatography and ion exchange chromatography. Both column chromatography and thin layer
chromatography (TLC) are forms of adsorption chromatography in which the sample
is absorbed on a solid surface and eluted with a solvent. In thin layer chromatography, the solid
adsorbent, generally silica (silicon dioxide), is spread out in a thin layer
over a glass plate or plastic sheet, and the substance under examination is
placed on this layer in the form of a spot.
The sheet is then placed on end in a covered beaker or jar containing a
shallow pool of solvent. The solvent
will rise by capillary action until it reaches the upper part of the sheet,
carrying along the components of the mixture.
The components are carried along at different rates, depending on the
characteristics of the component. On the
surface of the silica are many Si-OH groups.
These groups are quite polar and the intermolecular forces (IF) between
these groups and the substrates to be separated have different strengths. Substrates that have strong IF’s with the
silica are retained on the surface to a greater extent and move more slowly
with the eluting (or ‘developing’) solvent.
This exchange between the surface of the silica and solvent is known as
‘partitioning’.
The polarity of the solvent also influences the partioning;
the more polar the solvent, the further up the plate an individual component is
likely to travel. Therefore, the choice
of solvents used to move the various components of the mixture up the TLC plate
will depend upon the components in the mixture. For a very weakly adsorbed
component a very nonpolar solvent such as petroleum ether (a mixture of low boiling hydrocarbons-ironically there are no ethers
in petroleum ether) or benzene would be used. For more strongly adsorbed
components, a more polar solvent such as ether might be used. For very strongly
adsorbed components, a very polar solvent such as ethanol, water or even acetic
acid might be required to move the component up the plate. A list of common
solvents in order of increasing eluting power follows.
A typical TLC is shown
below. In a typical experiment, a thin
line is drawn with a pencil (no ink!) and the sample(s) of interest spotted on
the line. Note that more than one sample
may be analyzed on an individual sheet.
The sheet is then placed ‘end on’ in the eluting solvent (the
‘developing tank’). In preparing a TLC plate, it is critical that the pencil
line containing the spotted components is above the surface of the solvent when
placed in the eluting solvent; otherwise the solvent will wash the spots off
the plate. The plate is allowed to
‘develop’ over ~5 -10 minutes until the eluting solvent is near the top of the
plate (do not let the solvent reach the top).
A line is drawn at the solvent boundary, the solvent is allowed to
evaporate, and the spots visualized.
Visualizing the
Components
After the eluting solvent
reaches near the top of the plate, the plate is removed from the developing
tank and a line drawn in pencil to mark the point at which the solvent
migrated. The solvent is then allowed to
evaporate until the plate appears dry.
There are several techniques for visualizing the spots; if the components are ‘UV active’, the sheet
is placed under a UV lamp to reveal the location of the components. Most commercially available TLC sheets contain
a fluorescent indicator. When observed
under a UV lamp, components that absorb UV prevent the indicator from
fluorescing, resulting in a dark area on the plate that can be seen.
Another technique is to place
the sheet in a jar containing I2.
The I2 vapors often will concentrate organic material,
producing a brown spot indicating the location of the components.
Still another technique is to
spray the developed sheet with oxidizing agents such as phosphomolybdic
acid. This material oxidizes organic
compounds and turns a blue to green color to indicate the location of the components.
Retention
Factors (Rf values)
During the elution with the solvent, the sample will
partition itself between the stationary phase (the adsorbent layer) and the
moving phase (the solvent) so that the distance which the sample moves up the
plate is characteristic of that substance and will differ from one substance to
the next. The distance moved by the spot of sample divided by the distance
moved by the solvent is known as the Rf value and is characteristic
of that compound for the solvent system used. A mixture of substances will thus
give rise to a series of spots, one corresponding to each component. This
technique is extremely useful for analysis on a micro scale.
Preparative Uses
Larger size TLC plates are available that can be used to
isolate milligram quantities of purified components from a mixture. The technique is similar except that once the
components have been located on the plate, that portion of the adsorbent is
removed from the plate and extracted with an organic solvent to dissolve the
organic component. The resulting slurry
of adsorbent and dissolved component is then filtered to remove the insoluble
adsorbent and the solvent removed from the filtrate by evaporation to leave the
purified component behind.
Procedure:
Note:
All solvents should be used under the snorkel hood. At the conclusion of the lab, all organic
solvents must be placed in the organic waste.
Place all solvent containing methylene chloride (aka dichlormethane CH2Cl2)
in the container marked ‘halogenated waste’.
Part I:
Separating aromatic compounds of different polarities
Sample solutions: 5 wt/vol% benzaldehyde, benzoic acid, benzyl
alcohol, methyl benzoate, t-butylbenzene in acetone
1.
Obtain
a TLC plate and 5 capillary spotting tubes for spotting the samples on the
plate. It is recommended that you use
the disposable gloves when handling the plate, since touching the plate with
your fingers will contaminate the adsorbent with oils from your skin. Handle the plate by grasping the side edges,
not the surface.
2.
Using
a pencil (no ink-it will migrate with the eluting solvent) draw a light line ~
1cm from the bottom of the plate. Along
the line draw 5 ‘tic marks’ evenly spaced along the line. Do not place a tic mark at the very edges of
the plate.
3.
Dip
the end of the spotting tube into one of the sample solutions, making sure to
note which sample is spotted. Spot the
plate by lightly touching the end of the tube to the left most tic mark. As soon as liquid is seen at the tic mark,
remove the tube, allow the area to dry, and then re-spot using the same
technique. Repeat until the tube is
empty. It is important that the liquid
circle does not exceed ~2mm in diameter.
4.
Using
a separate spotting tube for each sample, repeat the process described in step
3. Be sure to note which tic mark
corresponds to each sample. For example,
you may label each tic mark A, B, C etc and record the identity of A, B, C etc.
5.
Use
an 8 oz jar with lid as the developing chamber.
Cut a 2 in strip of filter paper and place it on one side of the
jar. Add 10mL of the development solvent
to the developing chamber. The
developing solvent is prepared as a 50:50 mixture of hexane and 0.5% acetic
acid in ethyl acetate.
6.
Place
the TLC plate in the developing chamber for ~10 minutes or until the solvent
elutes to ~ 1cm from the top of the plate.
Remove the plate and mark the location of the solvent front with a
pencil line.
7.
Allow
the solvent to evaporate and place the plate under an ultraviolet light. Trace the outline of the spots with
pencil. Make a sketch of the plate and
pencil tracings in your notebook or photograph the plate for documentation in an
electronic report.
8.
Calculate
the Rf values of each component.
Part II: Identifying
analgesic components in an unknown mixture.
Sample solutions: 5 wt/vol%
caffeine, acetylsalicylic acid, acetaminophen, ibuprofen in acetone. Unknown solutions A,B,C,D
1.
Obtain
a TLC plate and 5 TLC spotting capillary tubes.
Handle with care as described in step 1, Part I.
2.
Using
a pencil (no ink-it will migrate with the eluting solvent) draw a light line ~
1cm from the bottom of the plate. Along
the line draw 5 ‘tic marks’ evenly spaced along the line. Do not place a tic mark at the very edges of
the plate.
3.
Spot
each solution of the known analgesic solutions and one of the unknowns on a tic
mark, noting which mark corresponds to each known solution. You will need to develop your own labeling
protocol to keep track of the samples. Leave the center tic mark for the
unknown solution.
4.
Develop
the plate as in Part I, but use only the 0.5% acetic acid in ethyl acetate as
the developing solvent.
5.
Allow
the solvent to evaporate and visualize the spots as done in Part I.
6.
Determine
the Rf of each known sample as well as the Rf of each
component of the unknown.
Conclusion
At minimum, your conclusion
should address the following concepts:
·
What
is the order of polarity of the samples analyzed (least polar to most polar)?
·
Explain
the observed order of polarity by analyzing the functional groups present in
each sample and its interaction with the polar surface of the TLC plate.
·
How
does the presence of the aromatic system (the benzene ring) aid in the
visualization of the spots
·
Identify
the components present in your unknown sample.
Developed by C. Snelling
Updated 5/21/2010 by J. Neilan