enable all workers throughout the United States engaged in the investigation of these products to arrive at just conclusions relative to their quality. For example, it is intended to fix an upper limit of the amount of foreign material that may be present in a leaf described by the United States Pharmacopoeia and to provide an ash limit for certain drugs. Considerable progress has also been made relative to testing existing analytical methods and formulating new methods for examining certain commodities.

In order to facilitate the investigation, and in harmony with the instructions of the association, the referee appointed several associates to take up specific features of the work, whose results will be given in separate papers.

A PRELIMINARY STUDY OF THE MICROCHEMICAL ANALYSIS AND IDENTIFICATION OF ALKALOIDS.

By B. J. HOWARD and C. H. STEPHENSON.

The large number of alkaloids used at present in drugs as well as the increasing number of synthetic products being placed on the market has made felt the want of additional means for their identification. Microchemical methods have been suggested and used to a limited extent by such workers as Wormley, Barth, and Behrens, but the principal application of the method has been rather for the purpose of localizing the alkaloid in the tissues, by such investigators as Errera, Maistriau, Clautriau, Bolling, and others.

At the suggestion of the Chief of the Division of Drugs this investigation was taken up by the Microchemical Laboratory and a study of several alkaloids begun. Only a preliminary report of progress can as yet be made, and the field has extended itself in many directions as the work progressed. The investigation as originally outlined anticipated several lines of work, among which the following might be mentioned:

(1) The normal reaction of each alkaloid with each of the various reagents which are known to be of service with one or more of them. This involves a study of dilution, the dilution of the alkaloid which will respond to the test and also the weight limit of alkaloid which will give positive tests with the reagent. It also involves a study of the forms of crystals produced at the various dilutions, the conditions for producing the reaction, or the manipulation, the determination of melting points, photographing the crystals as a matter of record, and in some cases the measurement of crystal angles.

(2) A study of the influence upon the reaction of another alkaloid present than the one sought.

(3) A study of the influence upon these reactions of such substances as glycerin, sugar, starch, oils, and fats, gums, waxes, and other compounds likely to be found in drugs, and from traces of which it is often difficult to remove, for testing, small traces of alkaloids in some medicinal preparations.

(4) The adaptation of alkaloidal purification methods for use microchemically so as to permit minute quantities of the alkaloids to be separated and prepared for testing. (5) The developing of an analytical scheme for systematically identifying microchemically the various alkaloids present in unknown mixtures. This last can only be accomplished after a considerable number have been studied and compared.

During the last year the work has been practically confined to the first two lines, which naturally constitute the foundation of the whole investigation. The alkaloids studied comprise a list of about forty, besides two or three salts of two of them, most of which were obtained through the Division of Drugs. They were commercial specimens and apparently of average purity. The list also embraces several synthetic compounds as well as the more common natural alkaloids. Among the natural alkaloids or their salts studied might be mentioned the following: Cocain, codein, atropin, cinchonin, morphin, papaverin, narcein, caffein, strychnin, tropacocain, hydrastin, coniin, berberin, solanin, etc., while among the synthetic bodies studied are anæs73673-Bull. 122-09

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thesin, beta-eucain, holocain, gujasanol, and acoin. The dilution of the alkaloid in the solvent in many cases has a most marked effect upon the form assumed by the precipitate. There is always a limit beyond which the dilution of the product is too great for crystallization to take place, while on the other hand the concentration may be so great as to cause too sudden precipitation and an unsatisfactory product results. In this work dilutions of 1:100 or 1:200 were most frequently tested. Other dilutions would possibly have given crystalline products where only noncrystalline products have thus far been obtained or where no reaction at all has been noted. The reagents used embraced a list of more than ninety compounds or mixtures and included the standard reagents and as far as known, the special alkaloidal reagents with the exception of two or three which have recently been brought to the authors' attention. Thus far crystalline precipitates have been obtained in about 400 combinations. Noncrystalline deposits resulted in nearly 600 other combinations, but their usefulness in identification is very limited and they can usually only be employed as corroborative tests.

Unfortunately some of the well known alkaloidal reagents, though giving reactions with most of the alkaloids, produce only noncrystalline precipitates. As ordinary analytical tests they may be satisfactory, but as microchemical reagents they leave much to be desired. To the analytical chemist they serve a good purpose as indicating alkaloidal presence, but rarely its identity. This is shown in the following examples: Mayer's reagent, 11 crystalline, 23 noncrystalline; Kraut's reagent 10 and 33 and Marme's reagent 11 and 25, respectively.

Picralonic acid gave 21 crystalline precipitates out of 37 positive reactions, but the forms unfortunately are in most cases too much alike to be of much sevice for identification. The alkaloids studied showed a great diversity in the character of the precipitate formed, as is seen from the following examples, which serve to illustrate the extremes, the first four giving a high number of crystalline forms, the last five giving a high number of noncrystalline.

With piperin, sanguinarin, emetin, and apocodein noncrystalline precipitates only have been obtained thus far, though it may be that by some change of manipulation crystals may yet be produced.

The melting point of the products is likely to be of service at times in establishing the identity of certain compounds though some of the precipitates apparently are too unstable for this test. For this purpose, however, an apparatus which had been devised in the Bureau of Chemistry for use on the stage of the microscope has been tested with promising results. It allows of the microscopic examination and determination of the melting point of an individual crystal in a mixture of various kinds either with plain or polarized light. In some crystals, especially some of the compact spherical forms, this last point is an important means of telling where melting begins, since as soon as a crystal melts it loses its polariscopic activity, and as all systems, except those belonging to the regular system, are active this feature can be used to advantage in determining the point where melting begins and where it ends even on small crystals.

The alkaloid thus far most thoroughly examined is cocain. Crystalline deposits have been obtained with each of the following eleven reagents, viz, palladous chlorid, platinum chlorid, gold chlorid, picric acid, chromic acid and hydrochloric acid, potassium dichromate and hydrochloric acid, potassium permanganate, potassium chromate, sodium carbonate, ferric chlorid, and potassium hydrate or sodium hydrate; noncrystalline deposits were obtained with chlorzinc iodid, picralonic acid, Mayer's reagent, phosphomolybdic acid, phosphotungstic acid, Kraut's reagent, Wagner's reagent, barium mercuric iodid, and potassium cyanid.

The following observations were noted concerning the various reactions with cocain in which crystals were produced:

Palladous chlorid.-This is one of the most characteristic tests for cocain, though not quite so sensitive as gold chlorid. The crystals vary in form greatly, according to the conditions of precipitation. There is at first formed, except in very dilute solutions (1:300 and up), an orange-colored amorphous-like or oily precipitate from which, on standing, crystalline forms of golden brown color are produced. One of the most common forms is that obtained with a 1:100 dilution, when feathery crystals are formed which have a strong tendency to twin. With a solution of 1:20 a dense precipitate is thrown down, out of which hexagonal plates are at first formed and frequently followed later by sheaf-like clusters of fine-pointed acicular crystals. A dilution greater than 1:500 gives crystals only with difficulty, crystallization being induced by rubbing the slide with the glass stirring rod. The limit of the test is

0.2 ugr.

Platinum chlorid.-With a 1:20 solution a dense white precipitate is formed and quickly followed by the production of very narrow feathery crystals—many times twinned so as to resemble a bird with outspread wings. Clusters of more than two are also abundant. If the reagents are mixed slowly the crystals are more like those of greater dilution. With a 1:100 dilution the feather type is much more prominent, the secondary branches being well developed into frost-like forms. With 1:1,000 solutions either short thick crystals are formed or else plate crystals twinning in a most characteristic manner are produced. The dilution limit is about 1:4,000, and the limit in 1:1,000 is 0.2 ugr.

Gold chlorid. This is the most sensitive reagent for cocain so far found. At 1:100 feathery frost-like crystals are produced, together with some nearly smooth star-like aggregates. At 1:1,000 the form is much the same, but the branches usually bear a rough outline. Diamond plates are also produced. At 1:4,000 a cross-like form predominates, the cross-bar being short. A few rosette crystals frequently are present. Crystals can be obtained in dilution up to 1:20,000 and the limit of the test for dilutions of about 1:3,000 is 0.033 μgr.

Picric acid. This is a good reagent for dilutions up to 1:800, though the crystals produced are not very characteristic for this alkaloid. They are produced in spherical rosettes (or sheafs) of fine lemon-yellow acicular forms. The reaction takes place quickly, and no difficulty is experienced in producing them nearly to the limit of dilution. At 1:300 the limit is 0.2 μgr.

Potassium permanganate.-With cocain, solutions up to a dilution of 1:700 give purple-colored square plates, or aggregates of this form. Vigorous rubbing of the slide is often necessary to start the crystallization, which then proceeds readily. When they begin to crystallize spontaneously, the plates are sometimes deposited in spherical aggregates. The limit at 1:400 is 2 gr.

Chromic acid and hydrochloric acid. This test is made by adding a small drop of 5 per cent chromic acid solution to the test drop. A precipitate is formed which on stirring disappears (if too much has not been added). A small drop of strong hydrochloric acid is added and a yellowish deposit is produced, which after rubbing of the slide should in a few moments be transformed into loose spherical clusters of an acicular crystal. This test appears to be one of the most uncertain because of the difficulty with which the crystallization is sometimes induced to begin in dilutions greater than 1:60. A concentration of 1:1,000 has produced positive results on standing several minutes. The limit appears to be for 1:100 about 3 μgr.

Potassium bichromate and hydrochloric acid. This test gives the same form of crystals as the chromic acid and the test is conducted in a similar manner. The limit of dilution is about 1:1,000 while at 1:100 the limit is 3 gr.

Ferric chlorid.-The crystals are spherical aggregates of rather coarse blade-like crystals with chisel-shaped ends. The limit of dilution is about 1:1,000 and in a dilution of 1:100 the limit is 3 ugr.

Potassium hydrate, or sodium hydrate. This produces a white amorphous precipitate which changes into crystals on standing or by rubbing the slide with a glass rod.

The crystals are rod-like, frequently with more or less chisel-like ends and a V-shaped recess extending backward into the crystal. There is a strong tendency to form coarse clusters up to about 15 branches. In open drops tree-like forms are frequent. For each of these reagents dilutions up to 1:1,000 give the reaction and the limit at 1:100 is 3 ugr.

Sodium carbonate. This gives a precipitate with cocain like that produced by potassium hydrate, both in the amorphous and crystalline forms. Limit of dilution is 1:1,000. In 1:100 solution the limit is 3 gr.

In order to determine the usefulness of some of the above tests when other alkaloids are present the palladous chlorid test was made on test drops to which had been added solutions of one of the following alkaloids, codein sulphate, atropin sulphate, heroin, dionin, acoin powder, cinchonin sulphate, hydroxylamin hydrochlorid, apomorphin hydrochlorate, narcotin, papaverin, brucin, narcein, morphin, thebain, gujasanol, orthoform (new), cinchonidia sulphate, quinidia sulphate, beta-eucain, holocain, caffein, quinin sulphate, strychnin, and tropacocain. In each case the crystals of the cocain compound were obtained and in the case of brucin, gujasanol, caffein, strychnin, and tropacocain, with which the palladous chlorid regularly gives a crystalline precipitate, it was found that when cocain was also present the cocain product was given in addition to that for the other alkaloid, though occasionally with modified form.

The foregoing serves to give an idea of the scope of the work undertaken, which it is hoped will be carried much further during the coming year.

COOPERATIVE WORK ON HEADACHE MIXTURES.

By W. O. EMERY.

After making investigations of various suggested methods for determining the different constituents present in the many headache mixtures containing acetanilid and similar agents, a method was finally devised which proved quite satisfactory to the members of the Division of Drugs, and it was therefore decided to place this method in the hands of as many chemists interested in this line of work as could assist. A circular letter requesting cooperation was sent out, and a gratifying number responded, signifying their willingness to assist, eleven of whom sent in results. All who expressed a desire to cooperate were supplied with a sample of a mixture containing known amounts of acetanilid, sodium bicarbonate, and caffein, with the following instructions, the U. S. Pharmacopoeia, eighth revision, as amended and corrected May 1 and June 1, 1907, being used as a basis for all calculations and reagents unless otherwise specified:

SEPARATION OF CAFFEIN, ACETANILID, AND SODIUM BICARBONATE.

Caffein.

Weigh out about 0.3 gram of headache powder on a small (5.5 cm) tared filter,a wash with successive small portions of chloroform to the amount of about 30 cc, collecting the solvent in a 100 cc Erlenmeyer. Distil off chloroform by means of a small flame until only a few cubic centimeters remain. Add 10 cc of dilute sulphuric acid, then continue the distillation till all the chloroform has gone over, disconnect from condenser, heat gently, first on wire gauze to complete solution, finally on a steam or hot

b

a In cases of powder mixtures or tablets containing ground celery seed, much coloring matter, cinchona alkaloids, laxative or extractive principles other than acetanilid or phenacetin, it is our practice to shake out the latter by means of chloroform from dilute sulphuric acid solution.

In case the preparation contains ground celery seed or certain oily principles, it sometimes happens that the acid solution does not become entirely clear at this

water bath until the contents of the flask have evaporated to about 3 to 4 cc. Cool, transfer by washing with water to a separatory funnel, so that the final volume does not greatly exceed 20 cc. Add four times the volume, or about 80 ce of chloroform, shake for some time vigorously, allow to stand until the chloroform clears perfectly, pass through a small dry filter into a dry 100 cc Erlenmeyer, distil off the solvent and use distillate for a second extraction, observing the same method of shaking, clearing, and filtering as above noted. Distil off chloroform to a small volume, transfer residue to a small tared beaker, or crystallizing dish, by means of a few cubic centimeters of chloroform. Allow to evaporate spontaneously, or if desired on a steam or hot-water bath to dryness, in the latter case partially covering the dish toward the end of operation with a watch glass in order to avoid possible loss from "popping." Cool in desiccator and weigh as caffein, dry alkaloid.a

Acetanilid.

First method. The acid solution remaining in the separator and containing anilin sulphate is run into a 100 cc Erlenmeyer, the filter through which the chloroform passed is washed once with a little water, allowing the latter to run into the separator. Rinse the latter thoroughly, adding the aqueous rinsings to the acid solution. Now, run in slowly and with constant agitation a standard solution of potassium bromidbromate to a faint but distinct yellow coloration. The number of cubic centimeters employed, multiplied by the value of 1 ce in terms of acetanilid, will give the amount of acetanilid present.

Second method.-The acid solution aforesaid is treated with successive small portions of sodium bicarbonate until an excess of this reagent is observed in the bottom of the separator. Add 50 cc of chloroform and 15 to 20 drops of acetic anhydrid, shake for some time vigorously, allow the chloroform to clear, then pass through the same filter used for the caffein into a 100 cc Erlenmeyer, and distil off most of the chloroform. Use this distillate for a second shake out, clear, filter, and distil down to a small volume, transferring the residue and the subsequent chloroform washings to a tared beaker or dish precisely as in the case of caffein. Allow the solvent to evaporate spontaneously or by means of a blast or fan, avoiding, however, undue heat. Dry in desiccator over quicklime to constant weight.

Verify the final weight by means of titration with standard potassium bromidbromate solution as in the first method. Heat the residue with 10 cc dilute sulphuric acid a half hour on the steam or vapor bath, cool, add 5 cc of water and titrate as directed above.

Sodium bicarbonate.

The residue left after the first treatment with chloroform is weighed when dry and represents very nearly the amount of sodium bicarbonate present. It may be more accurately estimated by titrating with tenth-normal sulphuric acid, using congo red as indicator, or it may be ignited with dilute sulphuric acid and weighed as sodium sulphate.

Calculate results in parts per 100.

a Should the caffein not be colorless or nearly so, the residue is dissolved in about 10 cc of water, filtered, if necessary (in case oily matters are present), through a wet filter, the filtrate acidified with dilute hydrochloric acid, the caffein precipitated with 15 to 20 cc of Wagner's reagent, allowed to stand a half hour, filtered, and the precipitate washed with a few cubic centimeters of same reagent, the filter, together with precipitate, transferred to separator, decolorized by means of sodium sulphite, and the caffein finally extracted with chloroform.

For this purpose the solution is prepared by adding bromin in slight excess to a concentrated aqueous solution of 50 grams caustic potash, the liquid diluted till the separated salts redissolve, boiled, to expel any excess of bromin, and finally made up to 1 liter. This solution is standardized with weighed amounts of acetanilid, or it may be so adjusted by further dilution that 1 cc is exactly equivalent to 1 centigram of acetanilid. For purposes of titration 1 to 2 decigrams are heated a half hour on the steam or water bath with 10 ce of dilute sulphuric acid.

cAcetanilid suffers appreciable loss when heated above 40°,