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

5

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

8

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).

32

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