General Methods of Extraction and Isolation of Alkaloids

The general methods of extraction and isolation of the alkaloids from the plant sources one has to take into consideration the following steps in a sequential manner, namely:

(i) Separation of the alkaloid(s) from the main bulk of the non-alkaloidal substances,

(ii) Most of the alkaloid-containing plants, several alkaloids having closely related chemical structures are normally present, such as: the cinchona alkaloids consist of more than twentyfive alkaloids. There is hardly any known plant source that contains only one alkaloid exclusively,

(iii) Separation of each individual alkaloid from the mixture of alkaloids obtained from a particular plant source (e.g., cinchona bark) using latest separation techniques, for instance, preparative high-performances liquid chromatography, (HPLC) column chromatography, by the help of chromatotron, and high-performance thin-layer chromatography (HPTLC).

Nevertheless, the general methods of isolation of alkaloids largely depend upon several vital factors, for instance: (a) the alkaline nature of most alkaloids, (b) the ability and ease of formation of alkaloidal salts with acids, and (c) the relative solubilities of the resulting alkaloidal salts either in polar organic solvents e.g., ethanol, chloroform, isopropanol etc., or in aqueous medium.

The general methods of extraction of alkaloids from the plant sources solely depend upon the purpose and scale of the operation (e.g., pilot scale or commercial scale). It is also based on the quantum and bulk of the raw material to be employed in the operation. Of course, for research purposes column chromatography using ion-exchange resins have been used successfully and effectively to strip the plant materials of their alkaloidal contents. However, in the commercial scale large volumes of aqueous extracts of plant materials are normally pumped through huge metallic columns packed with cationic resins, which in turn pick up all basic components (cations). Subsequently, the alkaloids (i.e., the basic components are conveniently washed off by flushing the column with a moderately strong acid. The column having the cationic resins can be reused once again for the next drug substances.

By the advent of the latest separation techniques and the copious volume of informations accumulated through the intensive and extensive research carried out with regard to the conventional processes essentially associated with the separation as well as isolation of the hundreds of alkaloids from the natural plant sources, the following five steps are most important and vital, namely:

(i) Sample preparation

(ii) Liberation of free alkaloidal base

(iii) Extraction of alkaloidal base with organic solvent

(iv) Purification of crude alkaloidal extract

(v) Fractionation of crude alkaloids

All these five steps shall be discussed individually as under:

1. Sample Preparation

The first and foremost step is the sample preparation. The plant material is reduced to a moderately coarse powder by appropriate means using grinders and sieves, to facilitate maximum effective contact of the solvent with the ruptured alkaloid bearing tissues and cells. In the case of plant substances that are rich in oils and fats, such as: seeds, kernels, these non-alkaloidal chemical components need to be eliminated completely by extraction with a suitable non-polar solvent like nhexane, light petroleum ether, in a soxhlet apparatus, which would not extract the alkaloids in question.

However, it is always advisable to shake the light-petroleum ether or n-hexane fraction with a dilute mineral acid and subsequently test the acidic solution for the presence of alkaloids.
2. Liberation of Free Alkaloidal Base

It has been observed that the alkaloids invariably occur in the plant sources as the salt of acids, such as: oxalates, tannates etc. Therefore, when the plant substance is exposed to an alkaline medium, the alkaloidal salts are readily converted to the corresponding alkaloid bases.

Choice of Alkali Indeed, the choice of a suitable mineral base (alkali) for the ease of liberation of the alkaloid from the salts is not only very vital but also equally significant and largely depend on the following factors, namely:

(a) Natural state of the alkaloids: It has been observed that the salt of a strongly basic alkaloid with a mineral acid usually tends to undergo cleavage under the influences of a stronger base. Likewise, the corresponding salt of a weakly basic alkaloid and a relatively weak organic acid shall require a rather weaker base for its cleavage.

(b) Chemical characteristics of the alkaloidal base: The usage of strong alkali e.g., NaOH or KOH should be avoided as far as possible by virtue of the fact that certain alkaloids undergo hydrolysis on prolonged contact with a strong base.

(i) Hydrolysis of ester-alkaloids, e.g., cocaine, hyoscyamine;

(ii) Phenolic alkaloids e.g., cephaeline, morphine. These alkaloids normally get solubilized while in contact with a strong alkali and, therefore milder alkaline reagents e.g., dilute ammonia solution are necessary for their liberation.

(c) Presence of fatty substances: The usage of strong alkali is strictly prohibited in the case of fat containing plant materials because of the formation of saponified products causing troublesome emulsions. In such cases, it is always preferred to defat the plant substance before proceeding for the liberation of free alkaloids.

Ammonium Hydroxide Solution Dilute aqueous ammonium hydroxide solution is one of the choicest alkali most frequently used for the liberation of alkaloids from the plant sources. It enjoys a two-fold advantage. First, being its adequate alkalinity to liberate most of the common alkaloids, and second by, its volatile nature so that it may be removed by evaporation of the solvent. As it has a tendency to be extracted by solvent ether from the aqueous solution, therefore, it is almost necessary to get rid of it by evaporation and subsequent washing repeatedly. In normal practice, usually even the last traces of ammonia are removed when the combined ethereal extract is reduced to half of its original volume under vacuum.

NaOH or KOH Solution The alkaloids that occur naturally as their tannate salts specially require either NaOH or KOH solution for their subsequent liberation. In certain typical instance even the use of KOH or NaOH fails to cleave the tannate salts because of their intimately strong bondage with the alkaloid and extremely insoluble nature.

(i) Cinchona Bark: It has got to be treated first by heating with dilute HCl so as to decompose the salts and liberate the alkaloids in the form of water soluble hydrochlorides, and

(ii) Pomegranate Bark: It does not have the tannin so tenaciously bound to the alkaloids as in the case of cinchona bark. Hence, NaOH solution is strong enough to cause on effective split of the alkaloidal salts. It also acts to control the solubility of the water-soluble pomegranate alkaloids by preventing their dissociation.
3. Extraction of Alkaloidal Base

The extraction of alkaloidal base may be accomplished by three different types of solvents that are discussed below, namely:

[A] Extraction with Water-Miscible Solvents A plethora of alkaloids and their respective salts are soluble in alcohols, such as: methanol, ethanol, isopropanol; therefore, these very solvents may also be employed for the extraction of the plant substances. The usual pretreatment of the crude drug with alkali may be avoided completely, because alcohol appears to affect dissolution of not only the alkaloidal salts but also the free bases found in the plant substances. It is, however, believed that alcohol predominantly exerts a hydrolyzing effect upon the alkaloidal tannates and other salts. In actual practice, neither pretreatment of the crude drug with an alkali nor acidification of the alcohol with a small amount of a mineral acid or an organic acid is required.

1. The penetration and hence the subsequent extraction of the crude drug is almost complete with the help of four successive extractions with an alcohol. Further, the loss of solvent is comparatively less than the chlorinated solvents e.g., chloroform.

2. The extraction of total alkaloids with alcohol is highly recommended because of its maximum efficiency and economical viability.

[B] Extraction with Water-Immiscible Solvents In reality, the most widely used water-immiscible solvents for the extraction of alkaloids are: chloroform, diethyl ether (solvent ether) and isopropyl ether. However, a few other specific organic solvents, namely: ethylene chloride, carbon tetrachloride and benzene* may be employed with an evident advantage for certain specific alkaloids. Interestingly, chloroform is regarded as the choicest water-immiscible solvent for a broad-spectrum of alkaloids present in the plant kingdom and extracts them with varying degrees of ease.

Note: Chloroform is not suitable for the extraction of quaternary alkaloids e.g., tubocurarine.

[C] Extraction with Water The crude drug is subjected to extraction with water previously acidified with dilute solution of HCl, H2SO4 or CH3COOH, which is subsequently rendered alkaline, preferably with dilute NH4OH solution and finally extracted with a water-immiscible solvent as stated in [B] above.

Undoubtedly, water being an excellent and absolutely inexpensive polar solvent for the extraction of alkaloids, but if offers an enormous volume of disadvantages because it carries along with it a large number of other plant components, for instance: sugar, pigments (e.g., chlorophylls), starches, tannins, proteins etc., which ultimately puts across a collosal waste of time, energy and chemicals. Hence, its usage has been resulting to a bear minimum level.

In general, the alkaloids may be extracted by any of the following three well-defined and widely accepted processes, namely:

(a) Soxhlet Extraction Process

(b) Stas-Otto Process, and

(c) Kippenberger’s Process.

All these three processes shall now be discussed briefly in the sections that follows:

(a) Soxhlet Extraction Process: The soxhlet assembly is a continuous extractor which is generally suitable for the extraction of alkaloids from powdered plant materials with the help of organic solvents. In this instance, the powdered drug is usually moistened with dilute ammonia solution and then packed loosely in the thimble of the Soxhlet apparatus; and the organic solvent affords a deep penetration of the moist drug thereby allowing the greatest possible extraction of the alkaloids from the exposed surfaces of the cells and tissues of the crude drug. Once, the extraction is ascertained to have completed, the solvent is filtered and evaporated in a Rotary Thin-Film Evaporator and the residue is treated further for the isolation of individual alkaloids.

(b) Stas-Otto Process: The Stas-Otto process essentially consists of treating the powdered and sieved drug substance with 90–95% (v/v) ethanol, previously acidified with tartaric acid. The proportion of crude drug to solvent should be maintained as 1 Kg to 1 L. The alcohol is distilled off under vacuum and the resulting aqueous residue is treated with petroleum-ether (60-80°C) to remove the fatty components completely. If any alkaloid is removed by the petroleum ether, it must be recovered by treating it with dilute mineral acid. Thus, the resulting aqueous extract is mixed with the main bulk of aqueous extract. The combined aqueous extract is filtered and evaporated to dryness preferably in a Rotary Thin-Film Evaporator under vacuum. The residue is extracted with absolute ethanol thereby dissolving the total alkaloids.

(c) Kippenberger’s Process: In Kippenberger’s process the powdered and sieved plant substance is first and foremost digested with solution of tannin (100 g) in glycerol (500 g) at a constant temperature of 40°C for a duration of 48 hours. The resulting mixture is further heated to 50°C so as to help in the complete coagnlation of proteinous substances, cooled to ambient temperature and finally filtered. The resulting filtrate is thoroughly shaken with petroleum ether to get rid of faulty materials (oils, fats and waxes), and the last traces of petroleum ether is removed from the extract by heating either on a water-bath (electric) or exposure to Infra-Red Lamp. The fat-free crude plant extract is subsequently acidified and shaken with chloroform, successively to remove the bulk of the alkaloids, namely, atropine, codeine, colchicine, narcotine, nicotine, papaverine, spartenine and thebaine.

The resulting residual extract may still contain narceine, curarine and morphine. However, narceine and morphine may be isolated by passing freshly generated CO2 directly into extract so as to convert the alkali hydroxide into their corresponding carbonate, which is then ultimately subjected to solvent extraction using a mixture of alcohol and chloroform. Finally, the third alkaloid, curarine, may be extracted by agitation with a mixture of equal volumes of ether and chloroform.

However, a combination of Kippenberger’s process and Stas-Otto process by its application to the final alcoholic extract obtained by the latter process is found to give better separation of alkaloids.
4. Purification of Alkaloidal Extract

The main bulk of the crude alkaloidal extract is invariably subjected to further purification by means of either anyone or combination of the following methods:

(a) Extraction with Acid Solution The extraction of the alkaloid from the bulk of the crude alkaloid solution in immiscible organic solvent is invariably carried out by shaking with an acid solution. In usual practice, the use of HCl is restricted when chloroform remains as the solvent because of the fact that quite a few alkaloidal hydrochlorides are distinctly soluble in the latter. However, dilute H2SO4 is always preferred over HCl for general use in the extraction of alkaloids. Subsequently, the acid solution is rendered alkaline with dilute NH4OH solution to liberate the alkaloids which is then extracted with an organic solvent. The solvent is removed under reduced pressure and the traces of moisture is removed with anhydrous sodium sulphate.
Note: The following two precautions may be observed, namely

(i) To avoid the formation of stubborn and troublesome emulsions a solution of gumtragacanth is often added to the aqueous-phase. In case, it still persists the two phases may be got separated by centrifugation, and

(ii) To discard the presence of foreign interfering extractive components present in plant substances, such as: pigments, resins, waxes, oils and fats, the use of a 2.5-5% (w/v) solution of lead acetate is made to the alkaloidal extract which precipitates them effectively. The excess of lead present in the filtrate is removed by either passing H2S gas through the Kipp’s Apparatus or by adding sodium phosphate.

(b) Precipitation of Alkaloid with Precipitating Reagent The usual precipitation of the alkaloid as a complex compound is accomplished by the addition of a suitable precipitating reagent. The resulting alkaloidal complex is further purified by filtration, recrystallization and ultimately decomposed to obtain the desired free alkaloid(s).

(i) Tannic-acid Complex: It is normally decomposed by treatment with freshly prepared Pb(OH)2 or Pb(CO3)2.

(ii) Precipitates obtained with HgCl2, AuCl3, PtCl4, Mayer’s Reagent: These precipitates are decomposed by passing a stream of H2S gas through its suspension.

(iii) Precipitates with Double Salts: The double salt obtained with Dragendorff’s Regent is quickly boiled with 5% (w/v) BaCO3 solution.

(iv) Precipitates with Nitrogenous Acids: The precipitates obtained with nitrogenous acids like picric acid and picrolonic acid are normally decomposed by treatment with either NH4OH or NaOH.


(v) Reineckate Complex: The complex obtained from alkaloid with Reinecke Salt, NH4 [Cr(NH3)2 (SCN)4], is normally decomposed by treating its solution in a mixture of acetone and water (1:1) with a silver sulphate solution. It is pertinent to mention here that the free liberated alkaloid from the complexes stated above, (i) through (v), may be further extracted for its final recovery with an appropriate organic solvent, such as: chloroform.

(c) The purification of alkaloids may also be accomplished by the formation of its crystallised alkaloidal salt by the addition of an appropriate mineral or organic acid, such as: hydrochloric, hydrobromic, perchloric, sulphuric, oxalic and tartaric acids.

(d) Various known separation techniques, namely: partition, ion-exchange and column chromatography are invariably used for the purification of a host of alkaloids.

Besides, various physical parameters like: specific rotation, melting point, solubility are frequently used as a definite criteria of ascertaining the purity of alkaloids.

5. Fractionation of Crude Alkaloids

It has been observed largely that most of the alkaloid-bearing plant materials usually contain a mixture of closely-related alkaloids. Therefore, it has become almost necessary to carry out an effective fractionation of crude alkaloids from the extract or solution of total crude alkaloids.

However, the traditional and orthodox methods of separation are not only difficult but also tedious and cumbersome. The commonly employed techniques of separation that were found to the reliable and dependable may be short-listed as follows:

(i) Fractional crystallization,

(ii) Fractional distillation, and

(iii) Derivatization with low solubility products.

The latest methods employed for the separation of alkaloids are the preparative high performance liquid chromatography (HPLC), high performance thin-layer chromatography (HPTLC), chromatotron, counter-current distribution and other chromatographic techniques including column chromatography, ion-exchange chromatography.

Following are some of the typical situations whereby the mixture of alkaloids may be separated effectively, such as:

(a) A larger section of the alkaloids are easily soluble in chloroform and relatively less soluble in other organic solvents. In general, the order of solubility is as stated below chloroform > acetone > ethanol > methanol > ethyl acetate > ether > n-hexane. Keeping in view the above solubility profile of alkaloids in organic solvents, if one of the alkaloids is much less soluble in ethanol than chloroform, the fractional crystallization of this alkaloid is possible. In this particular instance the chloroform-fraction is concentrated to an appropriate level, and hot ethanol added in small proportions at intervals. Thus, upon cooling the alkaloid, which is less soluble in ethanol, separates out conveniently.

(b) In case, the fractional crystallization of the mixture of closely related alkaloids become tedious and ineffective, one may try to form their respective salts,** and then carry out the separation indicated above.

(c) The various acids, namely: HCl, HBr, HI, HClO4, HNO3, C2H2O4, and C6H3N3O7, may either be employed in aqueous or methanolic solution. Thus, from the resulting methanolic solution, the salts of the respective alkaloids may be precipitated by the addition of ether. The precipitated crude alkaloidal salts may be further recrystallized from hot acetone containing a small proportion of methanol.

(d) In certain other specific instances, the salts of the respective oxalates, picrates and perchlorates may be precipitated from their solutions in acetone, by the addition of ethyl acetate.

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