Introduction to Thin-Layer Chromatography
Background Material:
Chromatography is one of the most useful methods of separating organic compounds for
identification or purification. Measure the migration distance of five common dyes and
calculate their Rf values to identify three unknown dyes.
Dye molecules are generally large molecules which vary greatly in structure and
composition. A typical dye molecule contains at least three functional chemical groups,
each responsible for a particular property of the dye. These three groups include the
chrotnophore, which is the color-producing portion of the dye, the auxochrome, which
influences the intensity of the dye and provides a site for bonding (as to fabric), and the
solubilizing group', which allows a dye to be water-soluble. Typical examples of each of
these groups can be found in Figure 1.
It is the variation in number and arrangement of these chemical groups that determines
the polarity of the dye molecule. In general, the chromophores tend to be non-polar,
while the auxochromes and solubilizing groups tend to increase the polarity of the dye
molecule, although, again, this is subject to the location and arrangement of the groups.
The structures of the dye molecules used in this activity can be seen in Figure 3 on the
following page.
Many different types of Chromatography are used but most work on the concept of
adsorbance. The two important components of Chromatography are the adsorbent and the
eluent. A good adsorbent is usually a solid material that will attract and bind the
components in a mixture. Paper, silica gel, or alumina are all very good adsorbents. The
eluent is the solvent that carries the materials to be separated through the adsorbent.
Chromatography works on the concept that the compounds to be separated are slightly
soluble in the eluent and will spend some of the time in the eluent (or solvent) and some
of the time on the adsorbent. When the components of a mixture have varying solubilities
in the eluent, they can then be separated from one another. The polarity of the molecules
to be separated and the polarity of the eluent are very important. Changing the polarity of
the eluent will only slightly change the solubility of the molecules but will greatly change
the degree to which they are held by the adsorbent. This affinity for the eluent versus the
adsorbent is what separates the molecules.
To separate complex organic molecules, thin-layer chromatography (TLC) is frequently
used. In TLC, the adsorbent is usually silica gel (SiO2) or alumina (A12O3) coated on a
glass plate or plastic sheet and the eluent is an organic solvent. The polarity of the eluent
is very important in TLC since a small change in polarity can dramatically increase or
decrease the solubility of some organic molecules. Many times, a mixture of a nonpolar
solvent (petroleum ether) and a polar solvent (acetone) is used to achieve an optimum
polarity. When placed in a chromatography chamber as shown in Figure 2, the eluent
(chromatography solvent, which is petroleum ether and acetone) moves up the plate,
being drawn by both capillary action and by the silica gel itself. The molecules, which
were "spotted" onto the TLC plate, separate as they are carried with the eluent up the
plate at different rates. Those molecules that have a polarity closest to the polarity of the
eluent will be the most soluble, and will move up
the plate the fastest.
The choice of the eluent or solvent is the most
difficult task. Choosing the right polarity is critical
because this determines the level of separation that
will be achieved. Common solvents used in TLC, in
order of increasing polarity, are: petroleum ether or
hexanes, cyclohexane, toluene, chloroform, ethyl ether, acetone, ethanol, and methanol.
Sometimes mixtures of solvents are used to achieve the desired degree of polarity. A
general rule of thumb is if the substances to be separated are polar, the developing solvent
should be slightly less polar. Likewise, non-polar substances would require slightly polar
solvents.
Materials:
Acetone chromatography solvent
Dye samples: Eosin Y , Fast green FCF solution, Fluorescein solution, Methylene blue
solution, Safranin solution, Unknown mixture
Capillary tubes, 6
Chromatography chamber (250-mL beaker with watch glass or Parafilm® cover)
Pencil, Ruler
TLC plates, 2
Watch glass or glass plate
Safety Precautions:
The chromatography solvent is flammable and a dangerous fire risk; toxic by ingestion
and inhalation. This lab should be performed only in an operating chemical fume hood or
well-ventilated area. Wear chemical splash goggles, chemical-resistant gloves, and a
chemical-resistant apron. Wash hands thoroughly with soap and water before leaving the
laboratory.
Procedure:
1. Obtain two TLC plates. Using a pencil, draw a horizontal line 1 cm from the bottom
edge of each TLC plate.
2. Again using a pencil, label the top of the first plate with the numbers "1," "2," and
"3." Label the top of the second plate with the numbers "4" and "5" and the letter "M"
(for "mixture") (see Figure 4). The numbers on the plates correspond to the dye
samples according to the following key:
1= Methylene Blue
2= Safranin
3= EosinY
4 = Fluorescein
5 = Fast Green FCF
3. Place one drop of Sample 1 (methylene blue) on a watch glass or glass plate.
4. You are now ready to "spot" the TLC plates. The sample spot will be placed on the
pencil line under its corresponding number. Touch the narrow end of a spotter to the
drop of sample. The sample will be drawn up the tube due to capillary action. Gently
and very briefly touch the tip of the spotter to the line on the TLC plate (under the
corresponding number), keeping your index finger over the end of the tube, so that
only a small amount of solution is transferred. It is extremely important to keep the
spot as^small as possible, as the dyes are very concentrated. It is also important not to
disrupt the silica gel, so a gentle touch is required.
5. Repeat steps 3 and 4 for the other dye samples (safranin, eosin Y, fluorescein, and
fast green FCF) according to the key provided in step 2.
6. Repeat steps 3 and 4 for one of the unknown dye mixtures (the spot will be placed on
the pencil line under "M").
7. Remove the watch glass or Parafilm cover of the chromatography chamber. Carefully
place the first TLC plate in the chromatography chamber with the sample end down
(as shown in Figure 2). Important: (1) Do not get any solvent on the upper portion
TLC plate, and (2) the sample spots must remain above the level of the solvent. If the
solvent level is too high, the sample will dilute into the solvent! Carefully place the
second TLC plate in the chamber, making sure the two plates do not touch. Replace
the watch glass or Parafilm cover.
8. The solvent will be drawn up the TLC plates. As it is drawn up, it will carry the dyes
in each sample up the plates at different rates depending on the characteristics of the
individual compounds.
9. When the solvent front is within 1-2 cm of the top of the TLC plate, the run is
stopped by removing the plate from the chamber.
10. Mark the location of each of the separated bands on the TLC plates and the final
solvent front, again using a pencil. This is done because some of the color and
brightness of each of the spots may be lost over time.
11. Measure the distance from the pencil line where the dyes were spotted to the solvent
front near the top of the TLC plate. Record in the data table
12. Repeat step 11 for each of the dyes, measuring from the pencil line to the center of
each colored band.
13. Optional: If an ultraviolet light is available, shine it on each of the TLC plates in a
darkened room. Note any differences in color. Can you make any additional
inferences about your unknown sample, or are your conclusions further confirmed?
Disposal:
The TLC plates may be placed in the trash. The chromatography solution should be
returned to the instructor.
Analysis
1. Since the samples are dyes they are relatively easy to see. Draw representations of
each of the TLC plates, including the dye colors and locations as well as the starting
spots and final solvent front locations. To compare and identify compounds
separated by TLC, calculate the Rf (rate of flow) values for each dye.
2. What can be inferred about the relative polarities of the various dyes knowing that the
solvent is quite polar?
3. Which dyes appear to be in your mixture?