Lab Activity 7
IUG, Fall 2012
Dr. Tarek Zaida
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Chromatography
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Chromatography
• Definition
A physical method of separation in which the components to
be separated are distributed between two phases:
A stationary phase and a mobile phase that moves in a
definite direction.
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Types of Chromatography
Types of
chromatography
Based on
stationary phase
Based on mobile
phase
Column
Liquid
Planar
Gas
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Planar Chromatography
• A separation technique in which the stationary phase
serves as a plane.
• The plane can be either a paper (paper
chromatography) or a layer of solid particles spread
on a support such as a glass- or a plastic- plate (thin
layer chromatography).
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Thin Layer Chromatography (TLC)
• Principle
 Different compounds in sample mixture
travel different distances according to how
strongly they interact with the stationary
phase as compared to the mobile phase.
 The specific Retention factor (Rf) of each
chemical can be used to aid in the
identification of an unknown substance.
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Measuring Rf
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Determination of amino acids using thin layer
chromatography
Development of Ruhemann’s purple from ninhydrin and
amino acid.
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• Instruments, chemicals and glassware
• Eluent1. [Mix n-butanol, acetic acid (purity 98 – 100 %) and
distilled water in volume ratio 5:1:5. Stir for 10 minutes,
then let the layers separate. Use upper layer as eluent].
• Developing Solution: Dissolve 0.3 g of ninhydrin in 100 ml nbutanol. Add 3 ml of glacial acetic acid.
• 0.2% in 10% isopropyl alcohol solutions of diferrent amino
acids (e.g. leucine, methionine, alanine and serine) and
mixture of these amino acids.
• Chromatographic paper
• Elution chamber, Glass capillaries for spotting the samples.
• Drying oven at ~ 60° C.
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Procedure
1.
2.
3.
Rubber gloves must be used during this work to avoid contamination of
chromatographic paper with amino acids from skin, and for protecting
skin from solvents and ninhydrin while working with the sprayer or
sprayed paper.
While the paper is being prepared for chromatographic analysis it
should be kept on a piece of filter paper.
Mark the starting line to the paper - 8-9 mm from the edge of the plate
- with graphite pencil (very slight line!). Also mark the locations where
the samples will be spotted. The distance between neighboring spots
should be about 8 mm and the spots should be at least 5 mm away
from the paper’s edge. Usually the spot of unknown substance is
applied to the center of the starting line.
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4. Before applying samples to the paper and filling the
elution chamber fit the length of chromatographic paper
with the height of elution chamber.
5. The spots of individual amino acids and sample solutions
are applied to the chromatographic paper. Use separate
clean and dry glass capillary for each solution. Dip the
capillary into solution – some solution is drawn into the
capillary. With the filled capillary touch the prepared
location on chromatographic paper. The spot on the paper
should not be bigger than 2-3 mm. (You can exercise
spotting on a sheet of filter paper.)
6. After application of samples let the spots dry. Meanwhile
measure with a graduated test-tube 5 ml of eluent into
the elution chamber. Cover the chamber with lids and let
the chamber atmosphere saturate with eluent vapors for
at least 10 min.
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7. Elution is stopped when the solvent front has traveled
up the plate until 7-10 mm from the lid.
8. Remove the paper from elution chamber and place it
on a sheet of filter paper. After 2-3 minutes mark the
eluent front with pencil and dry the paper in oven.
9. When the paper is dry, take it into the fume hood and
spray it with solution of ninhydrin until the paper is
slightly damp. Chromatographic paper and the paper
supporting it should lie at 45° angle while spraying. The
chromatographic paper is again put in the drying oven
(60 C) for 15 min to speed up the reactions.
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10. Remove chromatographic paper from drying in the oven,
draw the contours and centers of the chromatographic
bands. Calculate RF values by the method described
above.
11. Compare retention of standard substances and
components in sample and determine which amino acids
were present in the sample.
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TLC For Sugars
• The procedure will be repeated with sugars,
with the difference that the spots at the end
will be developed by using Aniline hydrogen
phthalate instead of Ninhydrine.
• Chromatographs will be dried in oven at 100C.
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quiz
• 1. Write down the reactions on which the
determination of vitamin C is based.
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• 2. The principle of kjeldahl method?
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• 3. If you were given a food sample that weighs
10 g. How would be the percentage of protein
in this sample if you found that its nitrogen
content was 200 mg?
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