The plant contains several constituents such as proteins, fats, waxes, pigments in addition to alkaloids. Therefore, isolation of alkaloids from alkaloid-bearing plants necessitates the following:

a) Separation of the alkaloids from the non-alkaloidal materials. b) Isolation of the pure alkaloids from the crude alkaloidal extract.

Preparation of the plant sample Liberation of the free alkaloidal bases

with a suitable alkali. Extraction of the alkaloids. Purification of the crude alkaloidal

extract. Fractionation of the total alkaloids.

The plant material is carefully dried and reduced to a suitable size

The plant material is then defatted with Petroleum Ether

Alkaloids usually occur in the plant as salts of organic acids e.g.: Tannates,Oxalates etc

The free bases can be liberated before extraction by treatment with a suitable alkali.

A salt of strong mineral acid and a strong basic alkaloid requires a strong alkali for their liberation

Alkaloidal esters like Atropine, Pilocarpine, require weaker bases like Sodium Carbonate in order to avoid hydrolysis of the ester group

Ammonium hydroxide is used in the case of Phenolic alkaloids like Morphine etc

Ammonium hydroxide is the most widely used base to extract alkali.

Extraction can be carried by using the following types of solvents

Water Immiscible Solvents Water Miscible Solvents Acidulated Water

EXTRACTION WITH WATER IMMISCIBLE SOLVENTS

Chloroform, ether, methylene chloride, and toluene may be used for extraction of all alkaloids.

The quaternary bases are more soluble in water than in organic solvents.

Extraction with immiscible organic solvents can be carried out by percolation or by continuous extraction (Soxhlet).

EXTRACTION WITH WATER MISCIBLE SOLVENTS

No pre-treatment with alkali is required, ethanol or methanol are, equally dissolve the alkaloidal salt and their free bases present in the plant.

Extraction with acidulated Water The plant material is extracted with acidulated

water. Acidulated extract is then made alkaline and the

alkaloids are extracted with an immiscible organic solvent.

This method is not selective because it dissolves large quantities of other materials present in the plant e.g. sugars, mucilage, colouring matters, proteins and tannins.

These impurities are usually extracted with an organic solvent before alkalization.

Dissolution in an Organic Solvent followed by an acid-base shake up.

Complex formation with a suitable precipitant followed by decomposition to recover the alkaloids.

E.g: Tannic acid complex is decomposed by treatment with Lead Carbonate

Mayer’s complex by treating with Hydrogen Sulphide

FRACTIONAL PRECIPITATION OR FRACTIONAL CRYSTALLIZATION

Usually performed after derivatization to Oxalates, picrates etc.

GRADIENT pH ExtractionThis method is suitable for separating

alkaloids with different basicity

For quantitative estimation of alkaloids they must be first extracted and purified by any of the previously described extraction methods.

When chloroform is used as a solvent, it should not be evaporated to dryness because some CHCI3 is converted to phosgene which is toxic and decomposition of alkaloids occurred.

CHCI3 should be evaporated to a small volume and then neutral ethanol is dded and evaporated to dryness.

The purified residue, which remains after evaporation of the solvent, consists usually of mixture of alkaloids.

For determination of each individual alkaloid, it must be first separated

Volumetric methods Gravimetric methods Colorimetric and spectrophotometric

methods Fluorirmetric methods Polarimetric methods Chromatographic methods

These methods are based on the basicity of alkaloids and their quantitative reaction with mineral acids to form salts. The purified residue is dissolved in a little amount of neutral alcohol and the solution is either titrated directly with a standard acid or a certain volume of standard acid is added and the excess acid is back titrated with standard alkali.

They depend on just weighing the purified residue which remains after evaporation of the solvent or a precipitating reagent is added and the dried complex formed is weighed and the percentage of the alkaloids is calculated.

This method is used for the determination of very weak basic alkaloids that can not be determined by volumetric methods, e.g. caffeine in coffee and tea, and mixtures of alkaloids that are obtained from the same plant but differ greatly in their molecular weight e.g. Cinchona and Rauwolfia alkaloids.

They are insensitive to micro-amounts of alkaloids.

Thermolabile alkaloids can not be determined

The presence of impurities such as fatty, waxy or colouring matters in the residue will be calculated as alkaloids.

Based on the colour reactions of alkaloids developed by a special reagent, which is measured in a suitable colorimeter at certain wavelength

1) Solanaceous alkaloids with Vitali's-Morin reagent give violet colour.

2) Ergot alkaloids with p-dimethylaminobenzaldehyde give blue colour

In UV spectrophotometric methods, the absorption of chromophores is measured under

UV light at certain wavelength, e.g. assay of vincristine and vinblastine.

It is based upon measuring of fluorescence of some alkaloids with certain reagents

e.g. Quinine and quinidine in oxygenated acids e.g. H2SO4.

It is based upon measuring the optical activity of certain alkaloids,

e.g. hyoscyamine,atropine.

These are carried out, using different techniques, e.g. CC, PC, TLC, GLC and HPLC.