There are several methods reported for the extraction of alkaloids and the choice of method is dependent on the purpose of isolation and scale of operation. For small-scale isolations column chromatography will effectively separate the alkaloids from the initially prepared plant extracts. However, on a commercial scale, large volumes of aqueous extracts of plant materials are pumped through huge metallic columns packed with cationic resins, which in turn collect all basic components (cations). Subsequently, the basic alkaloids are conveniently washed off by flushing the column with a moderately strong acid. Such cationic resins may be reused.

It is rare to find alkaloids occurring single and they are found in plant parts as a mixture of closely related compounds. Hence, the total alkaloids are to be initially extracted prior to attempting separation of individual alkaloids.

Being basic, they occur either free or as salts and can be normally extracted from the plant material into a weakly acidic alcoholic solvent. Most alkaloids may be extracted into an organic solvent after basification. Volatile alkaloids may be isolated by steam distillation and all alkaloids have to be subsequently purified wither by chromatography or by recrystallization using a combination of solvents.

Following is one general method for the isolation of total alkaloids from plant material.

1. Plant material is dried at a temperature not exceeding 60°C and finely powdered.
2. Macerate the powdered material with sufficient quantity of ethanol and set aside overnight. Most alkaloids and their salts being alcohol soluble will get dissolved in the solution.
3. Filter and concentrate the extract to 1/4^th the initial volume. Complete the evaporation of the remaining solvent at a temperature not exceeding 50°C.
4. Treat the residue with dilute sulphuric acid and filter to remove resins, fatty matter, and other unwanted substances.
5. Basify the solution by the addition of alkali such as ammonia as it effectively precipitates most alkaloids and because of its volatility, it can be conveniently removed by heating in subsequent processing steps.
6. Extract this solution with successive portions of chloroform or till complete extraction of all the alkaloids is effected.
7. Concentrate the pooled organic layer to yield a crude mixture of total alkaloids of the plant material.
8. Dissolve the above residue in dilute sulphuric acid and filter if necessary.
9. Basify the solution with ammonia and successively extract with chloroform.
10. Run the pooled chloroform layers through a bed of anhydrous sodium sulphate and evaporate to dryness. Note the weight of the mixture of total alkaloids.

Fractionation of the mixture thus obtained into individual alkaloids may be done using a number of techniques such as column chromatography, ion exchange chromatography, counter current liquid–liquid partitioning, HPLC, and preparative TLC.

However, they may also be separated by fractional crystallization or by salt formation.

Alkaloids may be separated from one another based on their relative differential solubilities either in a single solvent or in a mixture of miscible solvents. Most alkaloids are more soluble in chloroform than in other solvents such as acetone, ethanol, methanol, ethyl acetate, ether, benzene, and hexane. Thus the mixture of alkaloids may be dissolved in minimum quantity of chloroform by warming and another solvent such as ethanol may be added in drops. The alkaloid which is less soluble in ethanol than chloroform will crystallize out of the solution. On cooling, crystals of this alkaloid, less soluble in ethanol, can be separated. In this way, the other alkaloids of the mixture could be fractionally crystallized using any of the other solvents in which they may be less soluble compared to chloroform.

For separation of alkaloid by salt formation, the mixture of alkaloids is dissolved in minimum quantity of a warm solution of 10% acetic acid or hydrochloric acid in methanol. Cool the solution and add ether drop by drop for the precipitation of the salt of the alkaloid. Separate the precipitated material by suction filtration and carefully transfer the precipitate into a very small quantity of hot acetone. Addition of drops of methanol will precipitate the salt of the alkaloid. Volatile alkaloids such as nicotine may be isolated by simple distillation of the plant