OVERVIEW OF PLANAR CHROMATOGRAPHY

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OVERVIEW OF PLANAR CHROMATOGRAPHY
--Thin Layer chromatography (aka. planar
chromatography) is a classical liquid
chromatographic method of separating
mixtures A)--Stationary phase (S.P) in TLC is
solid, which is spread over a glass sheet
(support material), --In general, it is a porous
solid powder of small particles (5-40 mm) in
diameter coated on open bed.
B)--The mobile phase (M.P) is a volatile liquid,
which is drawn up the plate by capillary
action. ---Commonly used M.P includes -Chloroform
, Toluene, acetic acid, CH3OH and small amount
of H20
C) Component/s are eluted through the planar
chromatographic bed.
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Commonly used S.P s are silica gel, alumina,
polyamides and cellulose
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What are the main features, which distinguishes
gas chromatography (GC )and liquid
chromatography (HPLC) from planar chromatography?
GC and HPLC are both carried in columns while
planar chromatography (TLC) is performed on
glass, metal, or plastic sheet Coated with
silica, alumina, cellulose etc
What advantages might planar chromato- graphy
i.e., TLC have over GC and HPLC?
Cost--? This may be right, but In GC and HPLC
we inject samples into columns but we never are
absolutely Certain that all components are eluted
from the column into the detector
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TLC Plates
--TLC plates are available at costs range from
1-10/plate --Common sizes 5x20, 10x20,
20x20 --Commercial plates comes into two
categories
A) Thick Layer silica gel (200-250 mm), dp 20
mm N2000 --------------------? with development
time of 25-30 min in a 1cm plate
B) Thin Layer (HPTLC plates, 100 mm), dp 20 mm
N4000 ------------------? with development time
of 12 min in a 3cm plate Main Difference of
HPTLC and TLC - Particle and Pore size of
Sorbents.
HPTLC stands for High Performance Thin Layer
Chromatography. It is different from HPTLC as it
does not involve the use of column and pump.
Herein there is a silica-coated plate analogous
to the HPLC column where the analytes are
separated and the mobile phase is a developing
solvent, which moves along the length of the
plate through capillary action
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Procedures for doing TLC separations
http//orgchem.colorado.edu/hndbksupport/TLC/TLCpr
ocedure.html
A Prepare the developing container Generally,
one can simply use a beaker with watch glass
on top. A1. Pour solvent into the beaker to a
depth of just about than 0.5 cm. A2. To aid
in the saturation of the TLC chamber with
solvent vapors, line part of the inside of the
beaker with filter paper.
B. Prepare the TLC plate. TLC plates used are
purchased as 5 cm x 20 cm sheets. Each large
sheet is cut horizontally into plates which are 5
cm tall by various widths the more samples you
plan to run on a plate, the wider it needs to
be. B1. Using a pencil, draw a line across the
plate at the 0.5 cm mark. This is the origin the
line on which you will "spot" the plate. B2. Use
a pencil and do not press down so hard that you
disturb the surface of the plate. A close-up of
a plate labeled "1 2 3" is shown to the
right.
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• Spot the TLC Plate The sample to be analyzed is
  added to the plate in a process called
• "spotting".If the sample is not already in
  solution, dissolve about 1 mg in a few drops of a
• volatile solvent such as hexanes, ethyl
  acetate, or methylene chloride.
• -?As a rule of thumb, a concentration of "1" or
  "1 gram in 100 mL" usually works
• well for TLC analysis. If the sample is too
  concentrated, it will run as a smear or streak
• If the sample is not concentrated enough, you
  will see nothing on the plate.
• -?The "rule of thumb" above is usually a good
  estimate, however, sometimes only a
• Process trial and error (as in, do it over) will
  result in well-sized, easy to read spots.

The solution is applied to the TLC plate with a
1µL microcap.
touch the filled microcap to TLC plate to spot it
make sure you watch to see that all the liquid
has drained from the microcap
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D. Develop the Plate Place the prepared TLC
plate in the developing beaker, cover the beaker
with the watch glass, and leave it undisturbed
on your bench top. Run until the solvent is
about half a centimeter below the top of the
plate (see photos below
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E. Visualize the Spot
If your samples are colored, mark them before
they fade by circling them lightly with a
pencil. Most samples are not colored and need to
be visualized with UV lamp. Hold a UV lamp over
the plate and mark any spots which you see
lightly with pencil.
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Qualitative TLC For qualitative analysis, spots
on TLC plates are characterized by Retardation
Factor Rf
Calculating Rf values
Rf value importance Rf 0 -? means solute
interact strongly with the S.P (spot does not
move) Rf 1 -? means solute is not retention and
no separation from the solvent front
Rf between 0-0.99 Indicate solute is separated
from the dolvent front and move
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Efficiency and Resolution in Thin Layer
Chromatography
Efficiency in TLC
If Rs 1-? Two spots are reasonably
separated If Rs gt1 ---? Much better
separation If Rs lt1 ---? Poor separation
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Factors that Affect TLC Separations
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Prediction of Elution Order In TLC
Example Separate a mixture of butyl amine and
cyclohexane using TLC
Things to consider Polarity of each compound in
the mixture Butyl amine is polar
cyclohexane is non-polar Polarity of stationary
phase Silica gel (or alumina) is polar
Polarity of the mobile phase the
solvent you determine what solvent to use
Mobile Phase If the mobile phase is non-polar
(e.g, heptane) than cyclohexane will travel along
with it, but butyl amine will not as readily. If
the mobile phase is relatively polar
(e.g.,heptane ethyl ether) as a mixed solvent
system both cyclohexane and butyl amine will
travel with it, but butyl amine will be slower
because itll be interacting with silica gel as
its traveling.

• Prediction
• Cyclohexane will elute first/faster through the
  stationary phase.
• Butyl amine will elute last/slower.

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Two Dimensional Thin Layer Chromatography (2D-TLC)
Glut and Ser overlapped in solvent 2 but are
fully separated in solvent 1 Val and Ph-ALA are
overlapped in solvent 1, but are separated in
solvent 2
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Advantages of TLC
--? Simultaneous Analysis Number of
samples/standards can be analyzed simultaneously.
However, with column chromatography samples are
analyzed sequentially
-? Simultaneous Separation of Unresolved
Components Samples which are difficult to resolve
in one dimensions can be developed in two
different solvent system, which runs in
perpendicular direction For e.g., in 2D-TLC
-? Harsh separation conditions can be used for
separation Since TLC plates are used only once,
harsh separation conditions that would degrade
the S.P can be used. In contrast, in HPLC harsh
conditions or dirty samples can destroy the column
-? Solutes movement is always observable If no
components are lost to vapor surronding the
plate, all components must be somewhere on the
plate In contrast to HPLC or GC in which sample
components may never elute and are lost