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8 Glutamate Dehydrogenase (Nash and Davies 1975)
Conduct the electrophoretic run as described. After electrophoresis incubate the
gels in a solution containing 8 mM monosodium glutamate, 0.2 mM methylthiazol
tetrazolium (MTT) or nitrobluetetrazolium (NBT), 0.1 mM phenazine methosulphate, 0.2 mM NAD, 1.2 mM sodium cyanide in 200 mM Tris-HCI buffer
pH 7.5. The staining solution for glutamate dehydrogenase (NADP-dependant)
contains 0.2 M NADP instead of NAD.
Indolacetic Acid Oxidase
Enzyme extract: As prepared for the assay of the enzyme.
Incubation of the gel: Incubate the gel in staining mixture of 1 mg potassium
indoleacetate, 0.08 mg 2,3,6-trichlorophenol and 2 mg Fast blue BB per ml in
60 mM phosphate buffer (pH 6.0) at 30 C for sufficient time (preferably overnight).
Malate Dehydrogenase (Honold et al. 1966)
Enzyme extract: Prepare the extract as described for the assay of the enzyme.
Staining of the gel after electrophoresis: First pre-incubate the gels for
15–20 min in 200 mM Tris-HCl (pH 7.5) buffer. Then transfer the gel to a solution
containing 16 mM L-malate, 0.2 mM NAD, 0.25 mM methylthiazol tetrazolium,
0.8 mM phenazine methosulfate, 4 mM MgCI2 and 1.2 mM sodium cyanide in
200 mM Tris-HCl (pH 7.5) buffer. To stain MDH (NADP-dependant), use NADP
instead of NAD.
Separation and Identification of Amino Acids
by Descending Paper Chromatography
Amino acids in a given mixture are separated on the basis of differences in their
solubilities and hence differential partitioning coefficients in a binary solvent
system. The amino acids with higher solubilities in stationary phase move slowly
as compared to those with higher solubilities in the mobile phase. The separated
amino acids are detected by spraying the air dried chromatogram with ninhydrin
reagent. All amino acids give purple or bluish purple colour on reaction
with ninhydrin except proline and hydroxyproline, which give a yellow coloured
product. The reactions leading to the formation of purple complexes are given
below (Fig. 12.1).
Whatman No. 1 filter paper sheet.
Oven (105 C)
Chromatographic chamber saturated with water vapours.
Developing solvent: Butanol, acetic acid and water in the ratio of 4:1:5 in
a separting funnel and mix it thoroughly. Allow the phases to separate out
completely. Use the lower aqueous phase for saturating the chamber. The upper
organic phase is used as mobile phase.
R. Katoch, Analytical Techniques in Biochemistry and Molecular Biology,
DOI 10.1007/978-1-4419-9785-2_12, # Springer Science+Business Media, LLC 2011
+ RCHO + NH3 + CH2
+ H 2N C H
Purple coloured product
Fig. 12.1 Reaction for ninhydrin test
8. Ninhydrin spray reagent: Prepare fresh by dissolving 0.2 g ninhydrin in 100 ml
9. Standard amino acids: Prepare solutions of authentic samples of amino acids
such as methionine, tryptophan, alanine, glycine, threonine, etc. (1 mg/ml of
10. A sample containing mixture of unknown amino acids.
1. Take Whatman No. 1 filter paper and lay it on a rough filter paper. Throughout
the experiment care should be taken not to handle the filter paper with naked
hands and for this purpose either gloves should be used or it should be handled
with the help of folded piece of rough filter paper.
2. Fold the Whatman No. 1 filter paper about 2.0–2 cm from one edge. Reverse
the paper and again fold it 2 cm further down from the first fold.
3. Draw a line across the filter paper with a lead pencil at a distance of about 2 cm
from the second fold. Put circular marks along this line at a distance of 2.5 cm
from each other.
4. With the help of a micropipette or micro-syringe apply 20 ml of solution of each
standard amino acid on a separate mark. Also apply spot of the sample or
mixture to be analyzed, preferably on the mark at centre of this base line. The
size of the spot should be as small as possible so that the developed spots are
compact and do not overlap.
5. Hang the filter paper in a chromatographic chamber which has previously been
saturated with aqueous phase of the solvent system. This is done by keeping
Petri plates containing the aqueous phase at the bottom of the chamber. The
paper is hung from the trough/tray and a glass rod is kept to hold it in place.
Care should be taken to ensure that the base line should not get submerged
Separation and Identification of Amino Acids by Descending Paper . . .
when the mobile phase is added to the trough otherwise the spotted material
would get dissolved in the solvent.
Close the chamber firmly so that it is airtight. Allow sufficient time for
cellulose fibres of the paper to get fully hydrated.
Pour the mobile phase through the holes provided on the lid of the chamber into
the trough. Replace the rubber bungs in the hole and allow the mobile phase to run
down the paper till the solvent front reaches about 5 cm from the opposite edge.
Remove the paper and mark the solvent front with lead pencil and let it dry at
Spray the filter paper (chromatogram) with ninhydrin reagent and after drying it
at room temperature, transfer it to an oven at 105 C for 5–10 min.
Blue or purple coloured spots would appear on the paper. Mark the boundary of
each spot with lead pencil.
Measure the distance between the centre of the spots and also the distance of
the solvent front from the base line.
Calculate the Rf value of standard amino acids as well as those in the given
mixture or sample.
Distance traveled by unknown amino acid
Distance traveled by the solvent system
13. Identify the amino acids in the mixture or sample by comparing their Rf values
with those of the reference standards.
Observations and Calculations
Distance travelled by the solvent front from base line ¼ x cm
Distance travelled by glycine from base line ¼ a cm
Distance travelled by alanine from base line ¼ b cm
Distance travelled by threonine from base line ¼ c cm
Distance travelled by methionine from base line ¼ d cm
Distance travelled by spot no.1 in sample from base line ¼ a cm
The sample contains glycine since Rf value of spot no. 1 is identical to that of
• The chromatography should be carried out in temperature controlled room
because any fluctuation in the temperature would cause the uneven flow of the
solvent and may alter the Rf value.
• The paper should not be touched with naked hands because sweat on hands
contains significant amount of amino acids.
• The spots of the applied sample should be as compact as possible. Larger the
spot, poorer will be the resolution.
• At the time of fixing paper in chromatography chamber, it should be ensured that
the base line on which the sample has been applied does not dip into the solvent
otherwise the sample might get washed away in the solvent.
• Allow sufficient time for filter paper to absorb sufficient water (which will act as
a stationary phase) before pouring the solvent into the tank. Inadequate conditioning or equilibration will result in improper or poor quality resolution.
• Solvent front should advance in a straight line and should not be zig-zag or
sloping but should be parallel to the base line.
• Dry the paper thoroughly before spraying with the detection reagent. Wet paper
may interfere with the appearance of evenly shaped compact spots.
Separation and Identification of Amino Acids
by Ascending Paper Chromatography
Reagents and Materials
Same as given in above experiment except that cylindrical chromatography
chambers are needed for this experiment.
1. Take Whatman No. 1 filter paper sheet of appropriate size so that it can
be rolled into a cylinder and can be accommodated in the cylindrical
2. Draw a base line 2 cm from one of the breadthwise edge of the paper. Put small
circular marks along the base line in such a way that the distance from the edge
of the paper and the first spot and the distance between the adjacent spots is not
less than 2.5 cm.
3. Apply 20 ml aliquots of the standard amino acids and of the sample on different
spots. Diameter of the spotted material should be as small as possible and, if
required, the applied solution may be dried prior to loading additional volume.
4. Roll the paper into a cylinder; fasten its edges with a paper clip. Pour sufficient
volume of the mobile phase into the chromatographic jar which has been earlier
saturated with water vapours by lining the tank with filter paper saturated with
aqueous phase of the solvent system.
Separation and Identification of the Amino Acids by Two-Dimensional. . .
5. Gently place the rolled filter paper upright in the jar ensuring that it does not
touch the sides of the chamber and at the same time taking care that the base
line where the spots have been applied does not dip into the solvent.
6. Close the tank with n airtight lid or a glass plate to which sufficient amount of
silicon grease has been applied.
7. Leave the set up undisturbed and allow the solvent to move up fill it reaches
about 5 cm from the upper edge.
8. Remove the chromatogram from the chamber and air dry it.
9. Spray the paper with ninhydrin reagent and let it dry again at room temperature
prior to transferring it to an oven at 105 C for 5–10 min. Locate the position of
amino acids from the bluish or purple coloured spots on the chromatogram.
10. Calculate the Rf values of the standard amino acids and those is the sample or
mixture as described in above experiment.
11. Identify the amino acids in the mixture or sample by comparing Rf values with
those of applied standard amino acids.
Separation and Identification of the Amino Acids
by Two-Dimensional Paper Chromatography
Amino acids having very close Rf values in a particular solvent system may appear
as a single or overlapping spots in a single dimensional chromatography and may be
mistaken as one component. They can be separated into individual components by
developing the chromatogram again in a direction perpendicular to the first run in a
second solvent system in which they have different Rf values. The main limitation
of this method is that only one spot either of the sample or of a standard amino acid
can be applied on each filter paper sheet necessitating running of a large number of
chromatograms for the standard amino acids.
1. As in previous experiment with an additional chromatographic chamber for the
second solvent system.
2. Solvent system No.2: Phenol: water (80:20, w/v) is used as second solvent
system. Add 125 ml of water to 500 g to phenol. Add a few drops of ammonia
(0.88%) to this mixture just before use (CAUTION-Phenol is corrosive and can
cause burns on the skin).
3. Standard amino acids: Prepare 1% solution of standard amino acids such as
aspartate, glycine, serine, arginine in 10% iso-propanol (u/v).
1. Lay the chromatographic paper sheet flat on the rough filter paper using gloves.
2. Draw a base line 5 cm from one of the edges of the paper.
3. Draw another line perpendicular to the first line again 5 cm away from the
4. Apply 60 ml of the sample solution or given mixture containing unknown amino
acids at the point of intersection of these two lines. The sample should be
applied in small volumes at a time with the help of a micropipette with
intermittent drying to ensure that the zone of applied solution is as small as
5. Repeat the same procedure for a mixture of three standard amino acids using a
separate chromatographic sheet for each mixture. The composition of mixture
of standard amino acids should be such that each amino acid is present in at
least two different mixtures so that its identity can be established.
6. Hang the paper in the chromatographic tank whose interior has previously been
saturated with aqueous phase of solvent system No.1 (Butanol: Acetic acid:
Water mixture is 4:1:5).
7. After allowing an equilibration period of half an hour, pour the solvent No.1
into the trough of the chamber and let it run till t\it is about 10 cm from the
opposite edge of the paper.
8. Take the paper out, air dry it, turn it at 90 C angle and now develop the paper in
the second chromatographic chamber using the solvent system No.2 (Phenol:
9. Remove it when the solvent has travelled upto about 10 cm from the
10. Dry it at room temperature and spray it with ninhydrin reagent. After air drying
it, keep the chromatogram in an oven at 105 C for 10 min. Mark the blue and/or
purple coloured zones which appear on the paper.
11. Calculate the Rf value of the standard amino acids and those in given mixture as
given in previous experiment in both the solvents. From these values identify
the amino acids in the given mixture.
Identification of Lipids by TLC
Lipids generally exists as lipoprotein complexes and these need to be isolated.
Lipids, being soluble in non-polar organic solvents and proteins being soluble in
polar aqueous solvents, the efficient lipid extraction can be achieved only when an
aqueous solvent like ethanol or methanol is included in the non-polar organic
solvent like chloroform and diethyl ether. This would help in breaking the
Identification of Lipids by TLC
lipoprotein complexes. Extracted lipid components can be separated on TLC base
on their differential mobility along the porous stationary phase such as silica gel and
these can be located by spraying the plates with either 2’,7’-dichlorofluorescein or
50% sulfuric acid.
Reagents and Materials
Oven set at 110 C
Glass plates (20 Â 20 cm) for TLC
Solvent system: Petroleum ether (b.p. 60–70 C) or hexane: diethyl ether: glacial
acetic acid (80:20:1, u/v)
6. Lipid standards: Various lipids such as cholesterol acetate, vitamin A palmitate,
triacyl glycerol (e.g., trioleate, tripalmitate, tristearate)
7. Sulfuric acid (50%, u/v)
8. 2’,7’-dichlorofluorescein: Prepare 0.2% solution of 2’,7’-dichlorofluorescein in
95% (u/v) ethanol
1. Extraction of lipids from sample: Grind 1 g of the tissue in the extraction solvent
[either ethyl ether: ethanol (3:1) or chloroform: methanol (2:1)] in pestle and
mortar. Transfer the homogenate to a separating funnel. Shake the contents
vigorously and allow it to stand till the two phases have completely separated.
Drain out the lower organic layer which contains the lipids. Evaporate the
solvent under organic layer which contains the lipids. Evaporate the solvent
under vacuum and keep the concentrated lipid extract protected from light under
2. Prepare the TLC plates using Silica Gel G as the adsorbent as described in
3. Activate the TLC plates at 110 C for 30 min, cool them in a desiccator and spot
the lipid samples, standards as well as unknown sample.
4. Develop the plates in the solvent system consisting of petroleum ether or hexane:
ethyl ether: glacial acetic acid (80:20:1) till the solvent has travelled upto 1 cm
from the opposite side of the plate.
5. Remove the plate and allow it to air dry.
6. Locate the lipid spots by either of the following methods:
(i) Spray the plate with 2’,7’-dichlorofluorescein and examine it under UV light.
Lipids show up as green fluorescent regions against the dark background.
(ii) Spray the plate carefully with 50% H2SO4 and heat it in an oven at 110 C
for 10 min. Areas containing lipids gel charred and appear as black spots.
7. Calculate the Rf value of the lipid components in the sample and identify them
by comparing their Rf values with lipid standards.
Separation of Pigments by Adsorption Column
Different pigments get adsorbed to alumina to different extents. They can be selectively desorbed by using mobile phase of increasing polarity in a stepwise manner.
Materials and Reagents
Pestle and mortar
Whatman No.1 filter paper
Benzene: methanol (2:1)
Sodium sulphate (anhydrous)
(a) Preparation of extract:
1. Homogenize 5 g leaves or flowers in a pestle and mortar, using sand as an
abrasive in 20 ml of benzene: methanol (2:1) adding a small amount of this
extractant at a time.
2. Filter the extract through Whatman No.1 filter paper and transfer the filtrate
to a separating funnel.
3. Add 10 ml of water to the filtrate and after shaking the contents and allowing
the phase to separate out, drain out the lower aqueous methanol layer. Repeat
this step. Avoid very vigorous shaking.
4. Collect the benzene layer in a beaker and add small amount of solid
anhydrous Na2SO4 to remove the traces of moisture.
5. Decant the clear benzene layer to another beaker and concentrate the extract
by evaporating the solvent on a boiling water bath.
Separation and Identification of Sugars by Adsorption TLC
(b) Column preparation:
1. Mount a burette or a glass column vertically on a burette stand with the help
2. Place lightly a plug of glass wool at the base of the burette and close the
stopcock or outlet at the bottom of the column.
3. Take 5 g of alumina (adsorbent) previously dried at 120 C for 8 h and prepare
its slurry in benzene. Pour the slurry carefully into the column or burette, by
gentle tapping of the column so that no air bubbles get trapped in the adsorbent.
4. Allow the adsorbent to settle by opening the outlet. After the adsorbent has
completely settled, add 20 ml of benzene and let it pass out of the column. Care
should be taken not to let the adsorbent to get dried. When about 1 cm layer of
the solvent remains at the top of the chromatographic bed, close the outlet.
1. Allow the solvent at the surface of the column to drain out slowly and
transfer the leaf or flower extract with the help of pipette without disturbing
the surface of the column adsorbent. Let it enter into the column and then
add a few drops of benzene to wash the traces of the extract sticking to the
wall of the column.
2. Add 20 ml more benzene to wash out the column of any unadsorbed material.
(c) Column development:
1. For desorption of the adsorbed substance change the polarity of the solvent in
a stepwise manner. After 20 ml of benzene has passed through the column
add 10 ml of 5% acetone (u/v) in benzene and let it percolate through the
column and collect 1–2 ml fractions of the effluent from the outlet. Continue
increasing the concentration of acetone in benzene at every succeeding step.
Finally pass pure acetone through the column.
• Note the change in the colour of the collected fractions. In case of the leaf
extract, the initial fractions are colourless followed by yellow coloured and
then by the green coloured ones. The colourless fractions do not contain
any pigments but it is quite possible that these fractions may contain some
UV absorbing materials.
Separation and Identification of Sugars
by Adsorption TLC
Sugars get separated on the basis of differential adsorption onto silica gel. The
sugars which have higher affinity for stationary phase are adsorbed more strongly