EXPERIMENT NO.

VII, VIII, IX

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 General precipitation and specific color tests for alkaloids

Background: Most alkaloids are precipitated from neutral or slightly acidic

solution by Mayer's reagent (potassium mercuric iodide solution), Dragendorff's
reagent (solution of potassium bismuth iodide), Wagner's reagent (aqueous
solution of iodine), Hagar's reagent (saturated solution of picric acid) and tannic
acid. These precipitates are of various colors; cream (Mayer's), reddish brown
(Wagner's), orange red (Dragendorffs's), yellow (Hagar's) and white (tannic acid).
Some alkaloids like caffeine do not give these precipitates with the above
reagents. Alkaloids show great variation in their botanical and biochemical
origin, in chemical structure and pharmacological action. Consequently, many
different systems of classification are possible. But, the usual method of used
classification is based on the basic chemical structures from which they are
derived. Accordingly the following groups of Alkaloids are recognized: pyridine-
piperidine, tropane, quinoline, isoquinoline, indole, imidazole, purine and
steroid derived. Because of these basic structures found in a group, chemical
reactions exist that help in the determination of the specific group to which an
individual alkaloid belongs to that specific group. In this experiment some of
these tests are carried out.

Materials and Equipment:

Test tubes, test tube holders, electronic balance, spatula, Dragendorff’s reagent,
Mayer’s reagent, ephedrine, strychnine, morphine, codeine, quinine, caffeine,
atropine, nicotine, pilocarpine, reserpine,

Method: General precipitation test for alkaloids:

1. Place about 2 ml of a solution of alkaloids in test tube.
2. Add five drops of an alkaloidal reagent and observe the formation of any
precipitate.
3. Try to compare the relative sensitivities of the different precipitating
reagents for the sample provided.

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 Extraction of alkaloids from the leaves of Calpurnea aurea and performing
spot test on the extract
Introduction
Majority of alkaloids are basic nitrogenous compounds with marked
physiologic activity. Most of them are tertiary amines and their nitrogen is in
heterocyclic ring. The general procedures for the extraction, isolation and
purification of alkaloids are largely based on the alkaline nature of most
alkaloids, their subsequent ability to form salts with acids and on the
difference between the relative solubility of the alkaloid bases and their salts in
aqueous and organic solvents. Generally alkaloidal salts are soluble in water but
insoluble in organic solvents such as chloroform. Alkaloidal bases are insoluble
in water but soluble in organic solvents such as chloroform. Defatting the plant
material before any extraction is advisable especially for leaves and seeds.

In this experiment alkaloids are extracted from the leaves of Calpurnia aurea
by different methods.

Apparatus and materials: Defatted powder of Calpurnia aurea leaves, 2%

sulfuric acid, 10% ammonium hydroxide solution, concentrated ammonium
hydroxide solution, Dragendorffs’s reagent, chloroform, diethyl ether,
anhydrous sodium sulfate, filter paper and capillary tubes.

Procedure:
A. Extraction with acidic solvent
1. Weigh 20g of the defatted dried plant material and grind using mortar and
pestle to a fine powder.
2. Transfer the powder to a suitable container (flask/beaker/maceration
chamber)
3. Add sufficient amount of dilute aqueous solution of acid (e.g. 2% sulfuric
acid), macerate for 30 minutes and filter.

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4. Transfer the filtrate to a separatory funnel
5. Wash the filtrate with diethyl ether until no color is further removed and
collect lower layer.
6. Transfer the aqueous layer to a beaker and adjust the pH of the acidic
extract to 9 by adding sufficient amount of concentrated ammonium
hydroxide
7. Transfer the basified mixture to a separatory funnel
8. Partition the aqueous layer with 20ml of chloroform (2X), collect the
chloroform layer and combine the portions.
9. Remove any moisture from the chloroform extract by adding anhydrous
sodium sulfate and filter.
10.Concentrate the chloroform extract on water bath and confirm the presence
of alkaloids init by spot test.

Spot test for alkaloids:

1. Using a capillary, make a spot of the solution of your extract obtained from
the Calpurnea leaves on a piece of filter paper and dry.
2. Apply a drop of Dragendorffs's reagent on the above spot and observe any
color change.
3. The production of orange to reddish brown color indicates the possible
presence of alkaloids.

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Isolation & purification of active constituents-Column Chromatography

Introduction

There are two general reasons for performing separations on mixtures. To

remove contaminants and/or pick the desired compound- the process of
isolating and thus removing substances considered to be contaminants is called
purification and to perform analysis. Heterogeneous groups of chemicals in
crude extracts are separated as single components by different methods. These
methods can be grouped as Classical- Precipitation, Fractional distillation,
Crystallization, Chemical derivatization and Sublimation and Modern
techniques-The various chromatographic techniques.

Column Chromatography-It is also known as classical (traditional)

chromatography. This is simply to differentiate it from other advanced
techniques. It utilizes a vertical glass column filled with packing material with
the sample on top it. The rest of the column is filled with a solvent which,
under the influence of gravity, moves the sample through the column.

Materials and Equipment

Electronic balance or mechanical balance, conical flask 500 ml, spatula, iron
stand, extension clamp, clamp holder, glass column, Silica gel for column
chromatography, methanol.

Method
Attach the column to a ring stand and make sure that the column is securely
fastened in a vertical position. Prepare slurry of silica gel with methanol in
conical flask. Pour the slurry in the glass Column. Put piece of sand on the
column followed by whatmann filter paper on the top. Condition the column
with methanol for 12 hours. Put 1 g of the sample to be analyzed dissolved in a
very small amount of solvent and transfer to the top of the column and run the
mobile phase. Collect the different effluents (based on colour bands) with 25 ml
conical flasks.
Keep the fractions for further work.
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