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SOXHLET EXTRACTION
AND
THIN LAYER CHROMATOGRAPHY
Introduction to Phytochemistry
Phytochemistry is the chemistry of plant constituents or chemicals produced
by plants. It should be remembered that not all chemical compounds found
in plants are important in pharmacy, only ‘’Bio-active compounds are
important in Pharmacy and Pharmacognosy”.
More work has been done on the chemistry of alkaloids and glycosides due
to their pharmacological activities. Most of the carbohydrates, fats and
proteins are of dietic value. Starches and gums are used in pharmacy.
Calcium oxalate, silica, lignin and colouring matters are used for
identification of drugs and adulterants.
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Extraction
For study of plant constituent chemistry, it is important to extract it from
plant material followed by:
• Characterization of isolated compounds.
• Investigation of its biogenetic pathway.
• Quantitative evaluation.
• Before extraction, the following points should be considered:
• All plant materials should be authenticated or identified.
• Choice of extraction procedure to be applied.
• Plant material is dried.
• It must be powdered.
• Fresh leaves may be macerated with solvent like alcohol.
• Alcohol is a general solvent for many plant constituents
• Light petroleum is used for fixed oils, volatile oils and steroids.
• Ether and chloroform may be used for alkaloids. 3-Jul-17 3
4
Ready to harvest
Harvest
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Primary processing Cleaning
The herbs must be washed immediately after harvesting.
6 Ready for grinding
Store
Drying
•Sun or oven dry, •temperature not exceeding • 45oC
SOXHLET EXTRACTOR
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Solvent strength 1. Hexane 2. Isooctane 3. Toluene 4. Chloroform 5. Dichloromethane 6. THF (Tetrahydrofuran) 7. Ethyl ether 8. Ethyl acetate 9. Acetone 10. Acetonitrile 11. Isopropyl alcohol 12. Methanol 13. Water
Assending
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Thin Layer Chromatography
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Stationary phase = a piece of glass, metal, or plastic
coated with a thin, uniform layer of a solid adsorbent.
› Usually silica gel (SiO2), alumina (Al2O3), or
cellulose
› A substance which fluoresces under UV light often
incorporated into the stationary phase.
Zinc sulfide
Mobile phase = suitable liquid solvent or mixture of
solvents.
TLC Procedure
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Step 1: Preparing the chamber
A) Choose a container
that is large enough
and can be sealed.
B) Add the a few cm
of the mobile phase
solvent to the chamber.
TLC Procedure
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Step 1: Preparing the chamber
C) Seal the chamber and allow it to sit overnight if possible.
The atmosphere of the chamber should be saturated with the solvent vapors before running samples.
You may line part of the inside of your chamber with filter paper to aid in this saturation process. Stops the solvent from evaporating as it
rises up the stationary phase plate.
Allows for better development of the chromatograms.
TLC Procedure
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Step 2: Preparing the stationary phase
A) Prepare the TLC plate:
Mix:
Adsorbent
Small amount of an inert binder
Water
Spread a thin layer (no more than a few mm) of the mixture on an non-reactive support.
After the plate is dried, it is activated by heating in an oven for approximately 30 minutes at 110˚C.
TLC Procedure
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Step 2: Preparing the stationary phase
-TLC plates are
also commercially
prepared and can be
purchased ready
for use.
B) Draw a line of origin approximately 0.5cm from the bottom
of the filter paper.
C) Indicate where each sample will be added.
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Step 3: Spotting the samples
A) If the sample isn’t in solution, dissolve it in an appropriate
solvent.
As a rule of thumb, a concentration of about 1%
(1g in 100ml) is good.
If the sample is too concentrated = smear
If the sample is too dilute = no results
TLC Procedure
The image shows a sample ran at three
different concentrations. The left plate was
ran too concentration and the spots are
running together. The other two plates
yielded good separation.
TLC Experimental Setup
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Step 4: Developing the chromatograms
Allow the solvent to rise until it almost reaches the
top of the plate.
Remove the plate from the chamber and mark the
position of the solvent and front before it can
evaporate.
If the sample spots are visible, mark their positions.
Step 5: Identify the spots and interpret the data.
TLC Procedure
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*What if the compounds being separated are
colorless? How are the spots visualized?
Two ways to get around
this problem:
A) Use fluorescence
B) Use chemical methods
Visualizing Colorless Compounds
Final Chromatogram
Using Fluorescence to Visualize Spots
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Substance which can fluoresce under
UV light is added to stationary phase.
› So, when the TLC plate is
exposed to UV light, the
entire plate will glow.
› On the final chromatogram,
the glow will be masked at
positions were spots are
located.
› Examples: zinc sulfide
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Interpreting the Results
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Relative mobility: Rf
The larger the Rf value, the farther the compound
traveled up the plate.
An Rf value is a physical property that can be used for
identification purposes.
But it does depended on the conditions under which it is
measured.
solventthebytraveledDistance
compoundthebytraveledceDistanR f
Sample Rf Calculation
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Visualizing Spots Chemically
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In some cases it may be possible to visualize the spots by reacting them with something that produces a colored product.
Iodine Crystals: › The dried chromatogram is placed into a closed container
containing iodine crystals.
› The iodine vapor either:
Reacts with the spots
Sticks more to the spots than it does to the rest of the chromatogram
› The sample spots will be brownish in color.
The spots are more intense for unsaturated compounds
Visualizing Spots Chemically
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Ninhydrin:
The dried chromatogram is sprayed with a ninhydrin solution.
Reacts with amino acids to produce a colored product.
Mainly brown or purple
Rhodamine B:
Visualization of lipids
Aniline phthalate:
Visualization of carbohydrates
Before After
Column Chromatography
Introduction
Column Chromatography is another common and useful separation technique in
organic chemistry. This separation method involves the same principles as TLC, but
can be applied to separate larger quantities than TLC. Column chromatography can
be used on both a large and small scale. The applications of this technique are
wide reaching and cross many disciplines including biology, biochemistry,
microbiology and medicine. Many common antibiotics are purified by column
chromatography.
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TLC and Column Chromatography
In the TLC experiment, we separated and analyzed the different components that
makeup over-the-counter painkillers. The technique of TLC was useful in determining
the type and number of ingredients in the mixture, but it was not helpful for collecting
the separated components. We could only separate and visualize the spots. If we
needed to collect the separated materials, column chromatography could be used. We
could load 100 mg of a crushed Anacin tablet on a column made up of a silica
stationary phase and separate the aspirin from the caffeine and collect each of these
compounds in separate beakers. Column chromatography allows us to separate and
collect the compounds individually. In this experiment, Column Chromatography
(abbreviated CC) will be used to separate the starting material from the product in the
oxidation of fluorene to flourenone and TLC will be used to monitor the effectiveness of
this separation.
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Examples
Stationary phase
Mobile phase
Column chromatography
How does Chromatography work?
Chromatography is a method for separating the components of a mixture by different
Absorption between a stationary phase and a mobile phase (Moving phase).
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UV
IR
1D NMR - 1H NMR, 13C NMR, DEPT 45, DEPT 90 and DEPT 135
2D NMR –COSY-45(1H-1H), XHCORR, NOESY, and HMQC
Elemental Analysis
GC-MS/MS (EI mode)
Structure elucidation of the isolated compounds