Makalah KLT
Makalah KLT
Makalah KLT
Thin layer chromatography (TLC) is among the most useful tools for
following the progress of organic chemical reactions and for assaying the purity of
organic compounds. TLC requires only a fewing (yes that's right nano grams!) of
sample for a successful analysis and can be accomplished in a matter of minutes.
Like all chromatographic methods, TLC takes advantage of the different affinity
of the analyte with the mobile and stationary phases to achieve separation of
complex mixtures of organic molecules.
Stationary Phase
Mobile Phase
For silica gel chromatography, the mobile phase is an organic solvent or
mixture of organic solvents. As the mobile phase moves past the surface of the
silica gel it transports the analyte past the particles of the stationary phase.
However, the analyte molecules are only free to move with the solvent if they are
not bound to the surface of the silica gel. Thus, the fraction of the time that the
analyte is bound to the surface of the silica gel relative to the time it spends in
solution determines the retention factor of the analyte. The ability of an analyte to
bind to the surface of the silica gel in the presence of a particular solvent or
mixture of solvents can be viewed as a the sum of two competitive interactions.
First, polar groups in the solvent can compete with the analyte for binding sites on
the surface of the silica gel. Therefore, if a highly polar solvent is used, it will
interact strongly with the surface of the silica gel and will leave few sites on the
stationary phase free to bind with the analyte. The analyte will, therefore, move
quickly past the stationary phase. Similarly, polar groups in the solvent can
interact strongly with polar functionality in the analyte and prevent interaction of
the analyte with the surface of the silica gel. This effect also leads to rapid
movement of the analyte past the stationary phase. The polarity of a solvent to be
used for chromatography can be evaluated by examining the dielectric constant ()
and dipole moment () of the solvent. The larger these two numbers, the more
polar is the solvent. In addition, the hydrogen bonding ability of the solvent must
also be considered. For example methanol is a strong hydrogen bond donor and
will severely inhibit the ability of all but the most polar analytes to bind the
surface of the silica gel.
Table 2. Eluting solvents for chromatography
Since the amount of adsorbent involved is relatively small, and the ratio of
adsorbent to sample must be high, the amount of sample must be very small,
usually much less than a milligram. For this reason, thin-layer chromatography
(TLC) is usually used as an analytical technique rather than a preparative method.
With thicker layers (about 2 mm) and large plates with a number of spots or a
stripe of sample, it can be used as a preparative method. The separated substances
are recovered by scraping the adsorbent off the plate (or cutting out the spots if the
supporting material can be cut) and extracting the substance from
the adsorbent.
Figure 25. TLC plate showing distances traveled by the spot and the solvent after
solvent front nearly reached the top of the adsorbent.
Because the distance traveled by a substance relative to the distance
traveled by the solvent front depends upon the molecular structure of the
substance, TLC can be used to identify substances as well as to separate them. The
relationship between the distance traveled by the solvent front and the substance is
usually expressed as the Rf value:
The Rf values are strongly dependent upon the nature of the adsorbent and
solvent. Therefore, experimental Rf values and literature values do not often agree
very well. In order to determine whether an unknown substance is the same as a
substance of known structure, it is necessary to run the two substances side by
side in the same chromatogram, preferably at the same concentration.
Application of the Sample
The sample to be separated is generally applied as a small spot (1 to 2 mm
diameters) of solution about 1 cm from the end of the plate opposite the handle.
The addition may be made with a micropipet prepared by heating and drawing out
a melting point capillary. As small a sample as possible should be used, since this
will minimize tailing and overlap of spots; the lower limit is the ability to
visualize the spots in the developed chromatogram. If the sample solution is very
dilute, make several small applications in the same place, allowing the solvent to
evaporate between additions. Do not disturb the adsorbent when you make the
spots, since this will result in an uneven flow of the solvent. The starting position
can be indicated by making a small mark near the edge of the plate.
Development of thin layer plates
The chamber used for development of the chromatogram (Figure 26) can be as
simple as a beaker covered with a watch glass, or a cork-stoppered bottle. The
developing solvent (an acceptable solvent or mixture of solvents must be
determined by trial) is poured into the container to a depth of a few millimeters.
The spotted plate is then placed in the container, spotted end down; the solvent
level must be below the spots (see figure below). The solvent will then slowly rise
in the adsorbent by capillary action.
Thin layer chromatography (TLC) is among the most useful tools for
following the progress of organic chemical reactions and for assaying the
purity of organic compounds.
In thin layer chromatography, the mobile phase is liquid and stationary
phase is solid.
The Rf values are strongly dependent upon the nature of the adsorbent and
solvent.
The chamber used for development of the chromatogram can be as simple
as a beaker covered with a watch glass, or a cork-stoppered bottle.
If the components of the sample are colored, they can be observed directly.
If not, they can sometimes be visualized by shining ultraviolet light on the
plate or by allowing the plate to stand for a few minutes in a closed
container in which the atmosphere is saturated with iodine vapor.
Reference:
Wall,Peter E. 2005. Thin Layer Chromatography- A Modern Practical Approach.
Chambridge: the Royal Society of Chemistry.
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