2. Chromatography
Definition: Chromatography is a procedure by which components of a
mixture are separated by their carrying through a stationary phase by the
flow of a mobile phase.
Mobile Phase: The Phase that travels through the column (gas or liquid);
transport sample through the column.
Stationary Phase: Solid or liquid phase that fixed in place in the column or
on a solid support ; retain analytes within the column.
Band or Zone: Area across which analyte is distributed as bands progress
down column.
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4. Classification of chromatographic methods:
• Classification according to the following:
• A- According to the mechanism of separation
1- Adsorption chromatography
2- Partition chromatography:
3- Ion exchange chromatography (IEC)
4- Size exclusion chromatography (SEC)
5- Affinity chromatography
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5. B- According to the mobile phase used
1- Liquid chromatography
2- Gas chromatography
C- According to the mode of operation:
1- Normal phase
2- Reversed phase
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6. D- According to apparatus:
1- Column chromatography:
in which the stationary phase is held in a narrow tube and the
mobile phase is forced through the tube under pressure or by
gravity. (e.g. HPLC & GC)
2- Planner chromatography:
in which the stationary phase is supported on a flat plate (TLC) or in
the pores of a paper (PC).
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7. Thin Layer Chromatography
1- Stationary phase
Is a special finely ground matrix (silica gel,
alumina) coated on a glass plate, a metal
or a plastic film as a thin layer.
2- Preparing the plate
- Do not touch the TLC plate on the side with
the white surface.
- Draw a thin line with pencil.
- Do not use pen. Why?
- The start line should be 1 cm from the
bottom of the plate
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8. 3- Spotting the plate
- The thin end of the spotter is
placed in the dilute solution; the
solution will rise up in the capillary
(capillary forces).
- Allow the solvent to evaporate
and spot at the same place
again. This way you will get a
concentrated and small spot.
- Try to avoid spotting too much
material, because this will
deteriorate the quality of the
separation considerably
(‘tailing’).
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Figure 2
9. The spots should be far enough away from the edges
and from each other as well.
-You should spot the compound or mixture together
with the starting materials on the plate. They will
serve as internal reference since every TLC plate is
slightly different.
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10. 4- Developing a Plate
- A TLC plate can be developed in a beaker or closed jar.
- Place a small amount of solvent ( mobile phase) in the
container.
- The solvent level has to be below the starting line of the TLC,
otherwise the spots will dissolve away.
- The lower edge of the plate is then dipped in a solvent. The
solvent travels moving the components of the samples at
various rates because of their different degrees of interaction
with the matrix (stationary phase) and solubility in the
developing solvent.
- Non-polar solvents will force non-polar compounds to the top of
the plate, because the compounds dissolve well and do not
interact with the polar stationary phase.
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11. - Allow the solvent to travel up the plate until ~1 cm from the
top.
- Take the plate out and mark the solvent front immediately.
- Do not allow the solvent to run over the edge of the plate.
- Next, let the solvent evaporate completely.
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Figure 3
12. 5- Analysis
- The components, visible as separated spots, are
identified by comparing the distances they have
traveled with those of the known reference
materials.
- Measure the distance of the start line to the solvent
front.
- Then measure the distance of center of the spot to
the start line.
- Divide the distance the individual spot moved by
the distance the solvent moved. The resulting ratio
is called Rf value.
- The value should be between 0.0 (spot did not
moved from starting line) and 1.0 (spot moved
with solvent front) and it is unitless.
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Figure 4
14. Quantitative TLC determination of
phenol red – bromophenol blue mixture
Apparatus:
TLC aluminum sheets, silica gel F254 (10 x 3 cm),
100-ml capacity jar covered with aluminum
foil to assist vapour pressure equilibrium with
TLC surface, microsyringe, 10-ml volumetric
flasks and UV-Vis spectrophotometer.
Samples:
(1) Phenol red (2) Bromophenol blue (3) Mixture
from 1 and 2.
Mobile system:
Butanol - ethanol - 2N ammonia (3:1:1)
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15. Procedure:
1- Prepare 15 ml mobile system in a jar and cover to
equilibrate.
2- Mark the solvent start onto TLC plate with a pencil.
3- Carefully and with microsyringe, spot 5 μl of each sample
(three spots, in one line) onto TLC plate.
4- Develop until solvent front reach near the plate end and
mark the solvent front with a pencil.
5- Remove the plate from the jar and dry with a heating
fan.
6- Locate the separated spots with a fine pencil and
calculate the Rf values.
7- Scratch the spot of phenol red and that of bromophenol
blue with a weighing spatula and collect the silica in a
test tube containing 5 ml of ethanol for each spot.
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16. 8- Shake well each tube and filter into a 10-ml
volumetric flask, wash the residue two times each with
2 ml ethanol then complete to the mark with ethanol.
9- Measure the absorbance of the resulting solution
against ethanol blank at 430 nm for phenol red and
600 nm for bromophenol blue.
10- Calculate the amount of phenol red and
bromophenol blue in the mixture taking in your
consideration the following values:
for phenol red = 590
for bromophenol blue = 970
Conc. of each dye = A x 2000/ A 1% = % w/v
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