Alkaloids: Isolation and purification

1 downloads 0 Views 4MB Size Report
The Literature Cited (1-15) includes some articles select- ed from the alkaloids research field in which the svstematic working sequence described herein hasĀ ...
Alkaloids: Isolation and Purification Betty Maldoni Universidad Nacional del Sur. Bahfa Bianca, Argentina

It is the ohject 'of this article to give a detailed general method of isolation and purification of alkaloids in plants that can he useful for students that want to begin working in this field. The Literature Cited (1-15) includes some articles selected from the alkaloids research field in which the svstematic working sequence described herein has been followed. Alkaloids generally have vronounced ~hvsioloeicalaction on the animal organism; many are poisono&. ~ h kthey , are of considerahle importance in ~harmacolow, -.. medicine. and toxicology. The term "alkaloid" (alkali-like) is applied t o the large group of basic nitrogen-containing natural products of vegetable origin. The nitrogen content is usually present in a heterocyclic ring, such as in pyrrole, pyridine, pyrrolidine, quinoline, or isoquinoline, but occasionally is found in an aliphatic side chain. Alkaloids may he regarded as amines, derived theoretically from (NH& most are tertiary (NR3, but occasionally secondary amines (NHR*) occur. Less commonly, quaternary compounds (R4N+ OH-) are found. Alkaloids are basic com~ounds:the nitroeen atom accents protons from a n acid andform additive compounds kndwn as salts when treated with either ormnic acids such as acetic acid, or inorganic acids, such as hyhrochloric acid (see Fig. 1).

Most alkaloids are insoluble in water and more or less soluble in such organic solvent as ether and chloroform, whereas their salts have just the ovvosite soluhilitv characteristics. This combination of propekies is employed in separating alkaloids from other kinds of com~ounds:if a mixture of an alkaloid and another compounh is treated with dilute aqueous acid and shaken with a suitahle organic solvent, the other compound can he extracted into the solvent while the alkaloid remains in the acid layer. After the two lavers are sevarated from each other. theaddition of excess sodium hydioxide to the aqueous (aeid) layer will liberate the free tertiary alkaloid, which can then he extracted with organic solvent (chloroform, ether). Quaternarv alkaloid hvdrochloride with sodium hvdroxidem41 forman hydroxide that is soluble in water andinsoluble in chloroform. This sequence of operations will separate alkaloids from acidic and neutral compounds. This is the basis of isolation and purification of alkaloids in plants (see Fig. 1). General Classlflcatlon The classification scheme shown in the tahle is based on the type of nucleus or predominating chemical structure in the molecule.

Alkalold Detection If one considers the large amount of vegetable material that is available to a research lahoratorv and the time and cost required for studying each one, the isefulness of carrying out preliminary tests in order to select the most oromisCain Method Dried and pulverized vegetahle material (50 g) is extracted in Soxhlet with methanol, and the solvent is evaporated in vacuo t o dryness. The methanolic extract (50 mg) is placed in a small test tube, and 1 N HCl (1 mL) is added. The mixture is stirred with a glass rod for 10 min in order to achieve complete disolution of the alkaloids, which, due to their basic character, pass to the acid aqueous solution as salts. A clear extract usually is obtained after filtration, hut sometimes centrifugation is necessary. To the solid residue 1N HCl(0.5 mL) is added, and the operation is repeated. The combined filtrates (1.5 mL) are divided into three portions, and each is placed into a small test tube. Then, two or three drops of the orecioitationreagents-Maver. Bouchardat. and Draeendorff~ , . ~ ore added. A posi~iv~rest is indicated hy the formation o f n colored ~recipitiltr:while ~Mayrr), orange rDragenddfo, and brown Houchardat). Results are registered as abundant (+++), moderate (++I, scarce (+I, and negative (-).Precipitates can also be formed by proteins, purines, coumarins, and some polyphenols. Because a negative test is indicative of the absence of alkaloids, these reagents are used like presumptive tests for their presence. Mayer Reagent. Solution A: HgClz (1.36 g), H?O (60 mL). Solution B: KI (5 g), Hz0 (10 mL). The two solutions are mixed and diluted with water up to 100 mL. This reagent must be used in acid solution, but contamination by other protic solvents must he avoided; the precipitate is soluble in, for example, acetic acid or ethanol. Due to high solubility of some alkaloids in excess of reaeent, only a few drops must he added. DragendorffReagent.Solution A: Bi(NOJs.5HnO (8g), HNOs (20 mL). Solution B: K I (27.2g1, H20(50mL). Bothsolutions aremixed ~~

" o ~ c ~ ~ c ~ c ~ c ~ 2 c H "a 2 ~ ~ c H"= C3H M1O C H 3 P H~2 N I C H 3 1 ,

"1-

OC",

08-

OCH)

SINAPINE CHLORIDE

SINAPlNE r r P R O X I D E (water soluble1

Figure 1. Alkaloids: basicily arm formation of salts.

700

Journal of Chemical Education

Group

Example

Plant ol

Type of Nucleus

(structures)

Origin (anus)

Steroidal

&

Phenylelhylernine

INSOLUBLE I N

RCIO AQUEOUS SOLUTION a . Balificatlun La pHlo

Fyridine

Fyrrolidine

Figure 2. Alkaloid isolation method.

A Piperidine

Q I

I

snd all~,wedroatand for24 h; airerfilrrarion the volume i i made up u, 100 mL with water.'l'h~areagrnt is alway used on acidic samples. Bouehordar Reogeor. A rdution of KI r4 p.1 in water (lob ml.) is saturated with Is.

Coniine

Ouinoiine

Prepiyation of Vegetable Material Vegetable material (roots, leaves, seeds, or flowers), selected on the basis of results from precipitation reactions, is dried in an oven below 50 "C and in vacuo. Then, the material is pulverized and taken to complete dryness. Extraction and Isolation of Alkaloids (For Summary, See Fig.

2)

m

Morphine

~~

~

.~~ ~

Ciavicers plvpwea

Lysergic acid

Vegetable powder is extracted in Soxhlet with petroleum ether, 60-80 'C, in order to eliminate lipids and carotenoids. Afterwards, it is extracted with ethanol, and the solvent is removed in vacuo below 50 ' C to ohtain the ethanolie extract. This is extracted with 0.5 N HC1. which removes the bases as soluble salts. The acid solution is basified with 15% NaOH to pH 10, and free bases are extracted in chloroform (secondary and tertiary alkaloids). The aqueous solution may have water-soluble alkaloids (quaternary alkaloids), which are determined by precipitation reagentsReinecke or Mayer-in acid solution; the precipitate is dissolved in aeetone:methanol(1:1), and the solution is passed through aeolumn of anion exchange resin in the chloride form, giving after evaporation, a mixture of crude chlorides. This method is used especially for alkaloids with ouaternarv nitroeen. Different chromatoeranhic " - . terhninuea are med to reoarate from mixture of bases. ...-~ - ~ -. ~~-~ ~ ~. ~ . .comoonents -~~~ Extraction with Petroleum Ether. Plant powder (100 g) is extracted in Soxhlet with 500 mL of petroleum ether 6R-80 T. The solution is refluxed, and after 8 h 20 mL is taken out via the lateral branch and evaporated to dryness in vacuo. Extraction is considered complete if the residue is less than 5 mg. The solvent from the total extraction is removed in vacuo giving a residue principally formed for lipids and carotenoids. Vegetable powder is air dried and used fnr extraction. .~ethnnnlir ..~~~ .~~~~~ ~.. Ertracrioo "8th Ethonol. Plant powder is extracted in Soxhlet with 500 rnl.of ethanol during 8 h, and thrsolvent is removed until a 50-mL fraction is obtained. Recovered ethanol is used for another 8 h extraction from which 20 mL is taken out via lateral branch. After

(mushroom)

~

~

~

~~~

~~

Volume 68

Number 8

August 1991

70 1

evaporation, if the residue is less than 5 mg, the extraction is considered complete. The solvent is removed, and the residue is added to the 50-mL fraction obtained in the first extraction; then a further concentration up to 30 mL is cartied out. Treatment of Ethonolic Extract. 0.5 N HCI (60 mL) is added dropwise to the magnetically stirred extract (30 mL), cooled in an ice bath (0 "C), and the stirring is continued for 3 h. After being kept overnight in the refrigerator, the solution is filteredto remove gummv material that is washed with water (2 x 5 mL). Washine fracTworrnmr #,/A y u w u s Acid Solulion.The acid solution rooled in an ice bath is basifi~dwith 15%. XaOH to pH 10, and the liberated bases are extracted with CHCL; the Latter, after heing washed with water and dried, is evaporated in vacua giving the ehloroformie extract talkaloidir fractih A,. l).eatment of Aqueous Solution p H 10. The residual aqueous solution. free uf CHCl and cooled in an ire bath, is acidified wirh 2 N HC1 pH 2-2.5. ~kineckereagent is added to a 2 mL sample of the acid solution; if there is a precipitate, the Reinecke reagent is added to the total aqueous solution, and control of the complete precipitation is performed on a 2-mL sample of the corresponding eentrifugated liquid. After heing kept overnight in the refrigerator, the precipitate is filtered by Buchner and washed with cold water (3 X 5 mL) until a negative reaction to Congo Red is obtained. The product is dried to constant weight at room temperature and in the dark. Conversion to Alkaloid Chloride. The Reineckate salt is dissolved in acetone, followed by dilution with methanol (used acetone and methanol volumes corresponding to product weight X 50). The solution is passed through a column of anion exchange resin in the chloride form (corresponding to the Reineckate weight X 25), previnuslv washed with aeetone:methanol (1:l). . . After oassaee -~ , . .. is complete, the column is aavhed with acerone:methanol \ l : l , ; evaporation of eluentsgives the quaternarychlorider (nlkaloidic fraction B). The determination of alkaloids by precipitation reagents is earried out in both A and B fractions.

tb

~

~

~

~

Preparation of Ammonium Reineckate Solutlon AmoniumReineckateNH, [Cr(NHMSCN)J ( I g) isdissolvedin water (1M) mL) at room temperature; it must he magnetically stirred in the dark for 12 h. We mav assume that a veeatahle ethanolic extract contains the ~, followingalkaloids: ainapine and hygrine. Thus, the isolation should be as shown in Figwe 3. ~~

Chromatographic Methods Alkaloidic fractions A and B can be purified using differenc chromatographic methods. Column chromatography can be performed by using either silicagel or neutral alumina (activity 11or \'I: chloroform &d chloroform with increasing amounts of methanol are the most common eluents, and the progress of the columns is monitored by thin-layer chromatography. Preparative-layer chromatography can also be used for isolation and purification of alkaloids; detection is achieved by UV examination and chromogenic reactions, i.e., Munier reagent or potassium iodoplatinate. On the other hand, the use of gas chromatography has extended and increased the types of alkaloids under study not only because of improvements in the equipment (more efficient columns and packing, operations at higher temperatures) hut also due to working techniques, such as sililation, that increase stability and volatility of the alkaloids. Goad separations and quantitative determinations of alkaloids were carried out using high-performance liquid chromatography (HPLC), e.g., Solanurn, Papauer, Cinchona, Datura, and Catharanthus alkaloids. As a great many alkaloids have relatively high molecular weights, their gas chromatographic analysis is impossible. That inconvenience is not find using HPLC. Also, this method is used to examine the purity of the alkaloids isolated by thin-layer and column chromatography (e.g., potato glycoalkaloids). Studies of Cactaceae alkaloids have successfully employed thinlayer gas chromatographic procedures for the separation and identification of alkaloids. Difficulties occur, however, in attempting total separation of some isomeric pairs of the major alkaloid groups: tetrahydroisoquinolines and phenethylamines. A high-performance liquid chromatographic (HPLC) method was developed to overcome these difficulties. Finally, mass spectrometry-gas chromatography has revolutionized the study of some alkaloids groups, i.e., arylalkylamines and quinolines of the usual type in the Cactaceae. 702

Journal of Chemical Education

Figure 3. Isolation of hygrine (tertiary alkaloid)and sinaplne (quaternary alkalold) horn ethsnollc exhact.

Chromogenic Reagents Munier Rengent. Solution A: Bismuth subnitrate (0.850 g), CH:COOH (10 mL), Hz0 (40 mL) are heated and filtered. Solution B: KI (8 g), H 2 0 (20 mL). Solution B is mixed with an equalvolume of solution A; CHsCOOH (20 mL) is added to 10 mL of the resulting solution and further up to 100 mL. The appearance of an orange or red color indicates a positive test, but the reaction is considered positive only if the color persists at least for 24 h. Potassium lodoplatinate. 5% chloroplatinic acid solutions (5 mL) and 10%KI solution (45 mL) are brought up to 100 mL with water. In use, this reagent requires aqueous dilution (reagent 1:2 HzO). Alkaloids can give different colors: blue, violet, white, and brown. Purlficatlon via Derlvatlves Alkaloids form insoluble precipitates with potassium thiocyanate, picric acid, sodium nitrite, chloroplatinic acid, oxalic acid, sodium tetraphenylboronate, and potassium perchlorate solutions. An individual alkaloid is identified by physical and spectroscopic properties and also by the preparation and characterization of derivatives. The alkaloid and as many of its salts as convenient are analyzed for C, H, 0 , and N to establish the empirical formula. The molecular weight necessary to establish the molecular formula is usually determined by mass spectrometry. Alkaloid samples resulting from column chromatography of fractions A and B and detected by thin-layer chromatography with Munier reagent, can be purified bv conversion into derivatives. Alkaloid derivatives are nurified bv recrystallization until they show constant melting points. The free base is recovered using different methods which will be described. Preparation and Puriflcatlon of Picrata The most versatile derivative from which the free base can be readily recovered is the picrate. The alkaloid (10 mg), dissolved in water, alcohol, or benzene (0.3 mL), is treated with an excess of a saturated solution of picric acid (20 mg) in water, alcohol, or henzene (0.3 mL), respectively, until separation of the picrate is complete. If separation does not occur, the solution is stirred vigorously and warmed for a few minutes, or diluted with aaolvent in which the pierate is insoluble. Thus, a solution of the alkaloid and picric acid in

I1 or V), which is washed with the same solvent used in the hefore-mentioned solution. Evaporation in vacuo of the total eluent affords the free alkaloid. The picric acid is adsorbed on the column. (2) The free base is recovered by adding excess of 2 M aqueous sodium hydroxide and warmed a little. Because of the limited solubility of sodium picrate, excess hot water must be added. The solution is cooled, the alkaloid is extracted with a suitable solvent such as chloroform or ethyl ether, washed with 5 M sodium hydroxide until the alkaline solution remains colorless, then with water, and the extract is dried with anhydrous sodium sulfate. The solvent is distilled off, and the alkaloid is recrystallized. (3) Picrate or another derivative is converted into the hydrochloride by passage of its solution inacetonemethanol (1:l) through a column of anion exchange resin in the chloride farm previously washed with acetone:methanol(l:l). After completing the operation, the column is washed again with acetone:methanol(l:l), and the total eluent is evaporated giving the alkaloid hydrochloride. This is dissolved in water, hasified, and extracted several times with CHClr. Chlorofarmic extract is washed with water, dried (MgSOJ, and evaporated. The free base can be purified either by chromatography or recrystallization until constant melting point. Application of chemical and physical methods leads to structure determination of the isolated and purified alkaloids.

Acknowledgment I wish toexpress my deepgratitude t o t h e IateO. 0. Orazil a n d R. A. Corral (Divisiim Quimica Orginica Superior, L'niversidad d e L a Plata, Argentina) for everything they taught me a b o u t t h i s subject. Figwe 4. Free alkaloid from alkaloid derivative.

LRerature Clted ethanol or benzene can be treated with henzene or light petroleum, respectively, to precipitate the picrate. Alternatively, the alkaloid can he dissolved in alcohol and aqueous picric acid added. The picrate is filtered off and recrystallized from boiling water, ethanol, methanol, aqueous ethanol or methanol, or chloroform or henzene. Another derivative can he prepared in an analogous manner using o d i c acid, potassium perchlorate, potassium thiocyanate, etc.

Free Alkalold from Alkaloid Derivative (See Fig. 4) Three procedures can be used: (1) A ehlorofomic (or another solvent) solution of an alkaloid derivative is passed through a column of neutral alumina (activity

1. Corral,R.A.;Orazi,O. 0. Tetrahedron 1965,21,909-916. 2. Corral, R. A.;Orazi, O.O.:Pizzorno, M. T. Anolss Asor. Quim. A ~ ~ e n l i n1972,60,37o *9

3. Orazi.0. O:Corrsl, R.A.;Bmages, I. A. Tetrahedron 1913,29,206211. 4. Dorninguu, Xarge A. MOlodnr de Invesligocibn Fitoquimica: Lirnusa: MLxico, 1973. 5 . Sangster. A. W. J. Chem Educ. 1960.37.154-459. 6. Maldnni, B. E. An&x Asor. Qufm A~genfino1984, 72. 265-267. 7. Msldoni. B.E.Rru.Lolinoarner. Quim. 1984,lS. 88-U. 8. Dhar, K. L.; Jain, M. P.:Koul,S. K.:Atal,C. K. Phytochemi8t~y1981,20,319-321. 9. Luanratena.0.: Grilfin, W. J.Phytorhemi~try1982.21.449-451. 10. Murakabhi.1.: Kidoguchi.E.;lkrarn. M.; 1srar.M;ShaB.N;Haginiwa. J.:Ohmiya.S.:

Otornesu, H. Phytoehrmirtry 1982,21,1313-1315. 11. Uluhel~n,A.Phytochemisfry 1985,24,372-374. 12. G0h.S. H.:Junsn.S.A. A.Phylorhrmirlry 1985.24.880-881. 13. Charls.A.S.:Redha,F.M. J.; Jacksan,A.H.Phyloehamirlry 1985,[email protected]. 14. Mahmood, U.: Shuk1a.Y. N.:Thakur, R. S.Phytoch~misfr),1985,24,161%1619. 15. Bouillard, L.: Homes J.: Vanhaelen. M. Phylorhamistry 1987,26.2265-2266.

Second Edition of Modern Experiments for Introductory College Chemistry The second ednion uf fllod~m Expxnmenls for inlrorlurtor~Chrmtulr), containing experiment.+ from the Journal of Chemical Erluration rhnt were jelrctcd hernure they rellect exciting innnvst~nnsin laboratory work, is nvailsble. Compiled by H. Anrhnny Netdigand WilmrrStratton. thccuperimmtsarr written for professors rouse, not a prestylrd, specific laboratory dwections, but ns msplration and guide for adoption and adaptation in rheir indlwdual wursts. Thr 144-page paperback is $1850 ($1950 forrign, and can be purchased hy ssndmg a prepaid order to Subscription and Book Order D c p a n m ~ n r.Jwrnal , of Chemwal Education, 1991 Snnhnmptm Street, Easton, I'A 18012 All orders must be prepaid, and the price includes the rh~pping.

Volume 66

Number 8

August 1991

703