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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5466_Библиотеки_им_академика_М_И_Перельмана.pdf
267
Chromatography
Some of the common solvent systems are as follows:
Cyclohexane: Ethyl acetate (95:5)
Chloroform: Acetone (9:1)
Ethyl acetate: Methanol (99:1)
3. Development: Almost similar to the development phase of paper
chromatography:
i. preparation of TLC plate,
ii. putting the spot of sample to be separated.
iii. selection of development chamber,
iv. addition of mobile phase to the chamber so that it is about 2 cm deep,
v. saturation of development chamber with mobile phase,
vi. development of chromatogram,
vii. drying of the chromatogram,
viii. marking of solvent front.
4. Visualization: Similar to the visualization phase of paper chromatography.
5. Calculation: Calculations are carried out in a simalar way as in paper
chromatography.
Experiment 1 To perform identification of given amino acid using TLC.
Also calculate its R
f
value.
Requirement
1. Solution of amino acid: 0.5 or 1% solution of standard amino
acid like glycine, aspartic acid, glutamic acid, etc., also prepare a solution
of unknown amino acid.
2. Mobile phase: Glacial acetic acid : Water : Butan-1-ol (1 : 1 : 3).
M
Mi t re t e eparated M
dr phii
ip phi i
na ti e
Atie
tiita r entdA
tati nar pha e
ent ent
M i e pha e
ar
np ar
Fig. 10.3
Stahl’s triangle
268
Practical Pharmaceutical Analytical Chemistry
3. Visualizing reagent: 0.2% ninhydrin solution in butan-1-ol and
acetic acid mixture (95:5).
Procedure
1. Wash and clean the glass plate.
2. Prepare a slurry of silica gel in water.
3. Spread the slurry on a glass plate to prepare a uniform plate.
4. Dry the plate at room temperature for 30 min.
5. Further activate the plate by heating it in an oven at 110
o
C for
another 30 min.
6. Meanwhile, prepare the mobile phase.
7. Pour the mobile phase into development chamber. Put the lid
of chamber so that the atmosphere of chamber gets saturated with the
mobile phase.
8. Make a line with the help of pencil on glass plate indicating
origin line.
9. On that origin line make the spotting of solution of unknown
amino acid along with standard amino acid with the help of capillary.
10. Insert the plate in the developing chamber containing mobile
phase but make sure the origin spots are above the solvent level in the
chamber.
11. After development take out the plate, i.e. when the solvent has
run more than 3/4th length of the plate.
12. Mark the solvent front with pencil and dry the plate at room
temperature or in oven.
13. Spray the plate with visualizing agent (ninhydrin reagent), dry
the plate and mark the spot.
14 Calculate the R
f
value of the unknown and compare it with the
standard.
Observation
Mobile phase: Glacial acetic acid : Water : Butan-1-ol (1 : 1 : 3)
S. no. Amino acid Distance from the origin
to the centre of the spot
Distance of solvent front
from the origin line
R
f
value
1 Glycine
2
Aspartic acid
3 Glutamic acid
4 Unknown
269
Chromatography
Calculation
R
f
value=
Distance from the origin to the centre of spot
Distance of th
ee solvent front from the origin line
Experiment 2: To perform identification of given sugar using TLC. Also
calculate its R
f
value.
Requirement
1. Solution of sugar: 0.5 or 1% solution of standard sugar like
glucose, fructose, sucrose, etc. Also prepare a solution of unknown sugar.
2. Mobile phase: n-Butanol : Pyridine : Water (6 : 4 : 3).
3. Visualizing reagent: Dissolve 1.66 g phthalic acid and 0.9 mL of
aniline in 48 mL of n-butanol. To this add 48 mL ether and 4 mL water
to get the spraying reagent for sugar solution.
Procedure
1. Cut a strip of Whatmann no. 1 chromatographic paper.
2. Draw a line about 2" from the top of the paper.
3. Mark four spots on the paper for different sugar and unknown
sample.
4. Meanwhile, prepare the mobile phase.
5. Pour the mobile phase into development chamber. Put the lid of
chamber so that the atmosphere of chamber gets saturated with mobile
phase.
6. On the origin line make the spotting of solution of unknown
sugar along with standard sugar with the help of capillary.
7. Suspend the chromatogram in the developing chamber
containing mobile phase but make sure the origin spots are not below
the solvent level in the chamber.
8. After development take out the paper.
9. Mark the solvent front with pencil and dry the paper in room.
10. Spray the paper with visualizing agent, dry the paper and mark
the brown spot.
11. Calculate the R
f
value of the unknown and compare it with the
standard.
270
Practical Pharmaceutical Analytical Chemistry
Observation
Mobile phase: n-Butanol : Pyridine : Water (6 : 4 : 3)
S. no. Amino acid Distance from the origin
to the centre of the spot
Distance of solvent front
from the origin line
R
f
value
1 Glucose
2 Fructose
3 Sucrose
4 Unknown
Calculation
R
f
value=
Distance from the origin to the centre of spot
Distance of th
ee solvent front from the origin line