A Quantitative Method for the Isolation and Determination of Morphine in Opium and Medicinal Preparations


Selection of the adsorption column
Elution of the opium alkaloids
Removal of solvent
Recommended procedure
Accuracy of method


Author: I. Grosfeld-Nir, S. Gassner, E. Weissenberg
Pages: 23 to 26
Creation Date: 1954/01/01


A Quantitative Method for the Isolation and Determination of Morphine in Opium and Medicinal Preparations

I. Grosfeld-Nir
S. Gassner
E. Weissenberg1

An assay of morphine in crude opium and medicinal preparations is described, based upon chromatographic purification of the alkaloid, which is subsequently determined volumetrically.


In spite of the many methods published on the estimation of morphine in crude opium, there is at present no satisfactory method available which permits quantitative determination of the alkaloid with ease and reliable accuracy.

The methods of assay of morphine in opium given in the majority of pharmacopoeias are based upon lime [1] 2 treatment of sample with formation of calcium salt, its subsequent separation from accompanying alkaloids and other interfering substances and finally, volumetric determination of the isolated morphine. Other methods making use of polarographic [2] , colorimetric [3] , and ion-exchange [4] procedures, have also been described. Lately, methods advocating adsorption of opium alkaloids onto alumina [5] , and florisil [6] columns have been published.

The official pharmacopoeial methods have the disadvantage of being complicated and requiring many time-consuming and tedious manipulations, thus entailing loss of accuracy. (This latter fact is countered in the British Pharmacopoeia by the addition of a factor of ten per cent of the morphine content to the result obtained.)

The main problem in the assay of morphine in opium lies in the difficulty with which it can be separated from the other alkaloids occurring naturally in opium, owing to their similar physical and chemical properties. However, there exist some differences in their solubilities in organic solvents. Data on the different solubilities of opium alkaloids in various organic solvents were published by Klyachkina [7] .


Government Institute for the Standardization and Control of Pharmaceuticals, Ministry of Health, Govt. Central Laboratories, Jerusalem-Israel.


Figures in parentheses refer to figures in the Bibliography in this section.

From this work we know that morphine is practically insoluble in organic solvents like benzene, ether, etc., whereas the other opium alkaloids are fairly soluble in these solvents.

We therefore attempted to make use of these differences, under well defined and controlled conditions; i.e., by employing chromatography, when it was hoped that isolation of morphine could be achieved.

Our task consisted therefore in finding a simple and practical solution to each of the following problems:

  1. Complete extraction of morphine from crude opium.

  2. Choice of a suitable adsorbent for the extracted alkaloids.

  3. Removal of accompanying alkaloids other than morphine and interfering substances.

  4. Choice of suitable eluting agent for quantitative recovery of purified morphine.

In the following report a short and simple method is described in which the accompanying interfering substances are removed by use of an adsorption column, and the purified alkaloid in the eluate is determined volumetrically.


The first step in the determination of morphine consists in its complete extraction from the crude opium. After different procedures such as extraction with acids and alkalies in different concentrations and at various temperatures, and extraction with distilled water only, at various temperatures, were tried out, the technique described below was accepted as the most suitable.

The extraction with acids was found to be unsatisfactory, owing to the simultaneous release of darkly coloured material from the crude opium. The coloured substances could not be separated from the alkaloid in the subsequent chromatographic step.

The alkaline extraction on the other hand yielded a solution containing the corresponding morphinate, which proved exceedingly difficult to elute from the chromatographic column in the subsequent stage. Both procedures had thus to be discarded.

Distilled water ensured complete extraction of the morphine present provided extraction was carried out at 60 degrees C for at least two hours. A further rise in temperature or increase of extraction time merely added to the amount of interfering substances also going into solution. On the other hand, incubation at 37 degrees C for 24 hours gave us results similar to extraction at 60 degrees C. However the two hours period of extraction was finally adopted in the described assay.

The completeness of the extraction of total morphine from crude opium by the procedure described above was checked by means of the B.P. and U.S.P. methods for the determination of morphine in opium.

Selection of the adsorption column

A number of substances such as permutite, silicic acid, alumina, celite, mixtures of celite with alumina and celite with magnesia were tested as to their ability of adsorbing the different opium alkaloids and/or impurities.

These adsorbents were placed into a chromatographic column consisting of a glass tube 280mm. in height and having an internal diameter of 22mm. constricted at the lower end to fit into a holed rubber stopper. Various amounts of adsorbents giving columns from 2cm. to 10cm. in height were packed to different degrees of tightness by suitable application of a vacuum pump. Various amounts of the aqueous extract ranging from 2 to 10ml. were used in these preliminary experiments, which gave the following results:

Permutite adsorbed neither opium alkaloids, nor impurities. Silicic acid adsorbed morphine but did not permit its recovery without simultaneous removal of impurities. When using alumina, it was found that some eluting agents such as ether or benzene removed neither morphine, nor "other alkaloids", whereas solvents such as chloroform removed both morphine and accompanying alkaloids together. Celite was the most efficient and convenient substance to use. Mixtures of celite and alumina or magnesia did not improve the efficacy of the column, the alumina in fact causing undesirable colloidal solutions with the eluting agent.

Celite was therefore adopted as the most suitable adsorbent, six grammes of which were used in the above described chromatographic tube packed tightly to a column of about 80mm. in height. Under these conditions five ml. of the aqueous extract were found to be optimal.

To ascertain the optimum pH for the best separation of morphine from the other opium alkaloids and accompanying interfering substances, a number of experiments were run on columns adjusted to different pH values, by adding one of the following reagents in various concentrations to the aqueous extract before pouring it on the celite: hydrochloric acid, acetic acid, and mono-potassium-dihydrogenphosphate.

Acidic columns were rejected, as the partition of the solutes from the aqueous medium having a pH lower than 7, was found to give unsatisfactory results.

Columns having a pH higher than 7 were found to be much more suitable for our purpose. The addition of 0.3-0.5ml. of concentrated ammonia to five ml. of the opium extract before application to the column proved to permit the desired separation of the alkaloids without difficulty. Equally, by rendering the aqueous phase alkaline with a weak solution of sodium hydroxide, a good separation of the solutes was achieved, but the use of ammonia was found preferable because any traces of this base passing through the column into the eluate could easily be removed before titration by simple heating. Hence, the results of analyses thus obtained were found to be more reproducible.

Elution of the opium alkaloids

For the selective transport of the solutes from the aqueous phase the following organic solvents and mixtures were tried out: benzene, ethyl ether, ethanol, acetone, chloroform, iso-butanol, methanol, CCl 4, etherisobutanol 4:1, chloroform-isobutanol 4:1, phenol 1 per cent in chloroform, chloroform-methanol 3:1 and chloroform-ethanol 3:1 by volume. Of these benzene was found most suitable under the above conditions for the removal of alkaloids other than morphine, and chloroform-ethanol for the elution of the morphine itself, leaving the colouring matter, etc., adsorbed on the celite column. About 100ml. of the chloroform-ethanol mixture was found to be sufficient for the complete elution of the morphine from the adopted Celite column, prepared as described in the experimental procedure. (See figure 1.)

Removal of solvent

From the eluate obtained, containing the morphine, the chloroform-ethanol was distilled off on a boiling water bath. To ensure the complete removal of any possible traces of ammonia, without, however, exposing the residue to any oxidative alteration by excessive heating, we adopted the procedure as described below. Drying of the residue at 100oC. in an oven or under reduced pressure at lower temperatures did not give better results than simply boiling it on the water-bath. Incidently this latter procedure also caused the traces of colouring matter remaining with the morphine crystals in the residue to become baked together, so that they did not interfere in the subsequent titration of the isolated morphine.

Figure 1

Full size image: 8 kB, Figure 1


Celite No. 545 or 655 (Johns-Manville, diatomaceous earth)

Benzene, A.R.

Chloroform, U.S.P.

Ethanol absolute, U.S.P.

Ammonia solution, U.S.P.

Sodium hydroxide A.R. A N/100 solution is employed, suitably prepared by diluting a N/10 standard solution.

Sodium sulphate, anhydr.

Sulphuric acid, conc. A.R.

Chloroform-ethanol abs. mixture. Mix 3 parts of chloroform with 1 part absolute ethanol.

Shiro-Tashiro Indicator Solution. Mix 2 parts of 0.1 per cent solution of methyl red in 96 per cent ethanol with 5 parts of 0.1 per cent solution of methylene blue in 96 per cent ethanol.

pH 4.4
pH 5.4
pH 6.2
dirty blue

Recommended procedure

A. Extraction of the morphine.

Five grams of finely pulverised opium are triturated in a porcelain mortar with ca.10cc. of distilled water for 15 minutes. The resulting suspension is then transferred into a tared 250cc. Erlenmayer flask with the help of a spraying bottle containing warm distilled water, until the total weight increase is 50 grams. The flask is stoppered and shaken up vigorously for thirty minutes.

The flask is then immersed in a water-bath at 60 degrees C for two hours. The resulting extract is centrifuged at 2000 r.p.m. for ten minutes and the supernatant solution finally filtered through a paper filter (Whatman No. 1) The extract is now ready for the next step.

B. Separation of the alkaloids on a chromatographic column

Place five grams3 into a test tube, add 0.3-0.5ml. of concentrated ammonia, cover with a pad of cotton-wool and shake gently. Remove pad of cotton-wool with a pincette and pour contents of test-tube into the partition column, using the cotton-wool to transfer any particles of precipitated alkaloid adhering to the test-tube walls. Place cotton-wool on the column and press down with a glass-rod. Wash test-tube twice with five ml. portions of benzene each and pour into column.


For convenience sake take 5ml. and correct for specific gravity of 1.02.

Fill column with benzene and allow Celite to become soaked. As soon as the first drops of benzene start to drop from the bottom end of the tube, connect receiving flask to a vacuum pump, adjusting the rate of flow to approx. 2 drops per second. A total of about 150ml. of benzene is used for wetting the column and washing out the "other" opium alkaloids.

When practically all the benzene has passed through the Celite column, the vacuum is released slowly (in order to prevent displacement of the Celite column by the sudden formation of back-pressure).

The receiving flask is replaced by a clean vacuum flask and 150ml. of chloroform-ethanol abs.(3:l) is introduced into the column from a separating funnel suspended from the top of the column. The vacuum is again applied to produce the same rate of flow. Note that the Celite must at all times be covered with solvent.

When all but 1-2ml. of the solvent above the Celite level of the column has passed through, the vacuum is carefully released. The eluate is transferred quantitatively to a 400ml. Erlenmeyer flask.

C. Distilling off the solvent

The chloroform-alcohol mixture is now distilled off from a boiling water-bath. To the residue in the flask five ml. of absolute ethanol, neutral to Shiro-Tashiro indicator, are added and again distilled off. A further portion of five ml. ethanol abs. are added and the distillation repeated once more. The Erlenmeyer flask is left in the boiling water-bath for an additional 20 minutes to ensure complete removal of any remaining traces of ammonia.

D. Volumetric determination of the morphine

Fifty ml. of N/100 H 2SO 4 are added to the residue in the Erlenmeyer flask, and the solution is heated to boiling over an open flame for about 3 minutes. After cooling to room temperature by immersing the flask in ice water, the excess acid is back with N/100 NaOH, using 1ml. 4 Shiro-Tashiro as indicator.


Adsorption column

A small pad of cotton-wool is packed into the constricted stem of the partition tube 280mm. in height and 22mm. in inner diameter. Sodium sulphate anhydr. is added until a layer of about two centimetres thickness is formed. This is kept in place by cotton-wool packed down by a glass-rod. [4]

About 6 grms. of Celite is sucked down to a height of about 80mm. with the help of a glass-rod to ensure uniformity of the adsorbent in the column. Finally a small pad of glass-wool is placed on top of the Celite layer.


Usually 15 drops were sufficient, but in some cases it was necessary to add additional drops of the indicator to obtain a sharp end point.

Accuracy of method

To determine the accuracy of our proposed method we made up a morphine solution containing 50mg. of the hydrochloride in 5ml. corresponding to 36mg. of the free base. The results of analyses obtained by our method were on the average 35.5mg. falling within the limits of 35.5 to 36.5mg.

Next, several mixtures of alkaloids were prepared, containing in addition to morphine, some of the other naturally occurring opium alkaloids in various proportions. Results of estimation of morphine in such mixtures, and of recovery of a weighed amount added to an opium extract, are given in table I below:


Table I


Alkaloid added

Recovery of Morphine titr. as free base


Sample No.

Weight of Mornphine HCI

Weight of Morphine calc. as free base




50 mg. 36.0
36.1 35.9
50 mg. 36.0
36.0 35.4
50 mg. 36.0
36.5 36.2
50 mg. 36.0
36.2 36.0
50 mg. 36.0
39.0 39.6
50 mg. 36.0
Mixture of 1-5
36.0 36.8
37.1 37.1
(B.) Recovery of Morphine Added to Opium Extract
55.6mg.       56.2 55.8

In the following table, results of morphine estimations carried out on various samples of opium of different origin by our proposed method are compared with results obtained on the same samples with the U.S.P. and B.P. methods.


Turkey I
16.6 16.2 15.5
Turkey II
13.6 13.1 12.3
Turkey III
17.2 18.0 17.6
Turkey IV
15.2 15.5 15.1
Turkey VI
13.0 12.7 12.0
Turkey VII
17.5 17.5 16.9
Turkey VIII
14.9 14.7 13.9
Turkey IX
16.4 16.8 16.3
Turkey X
13.5 12.0 11.2
Turkey XI
13.6 13.5 12.4
Turkey XII
14.8 14.5 13.8
Turkey XV
13.5 14.5 13.3
Yugoslavia I
17.0 17.8 17.6
Yugoslavia II
17.1 15.7 15.2
China I
12.9 10.2 9.5
China II
10.2 8.0 7.7


A simple and accurate method for the assay of morphine in crude opium and medicinal preparations is suggested, which is based upon its separation from accompanying alkaloids and other interfering materials by means of chromatography.

The results obtained by this method were found to be reproducible.

The proposed method is equally applicable to medicinal preparations, such as tinctures, extracts and mixtures of alkaloids containing morphine. Where ethanol is present in preparations it has firstly to be removed by distillation before application on to the column. This is easily achieved by driving off the ethanol by partial immersion in a boiling water-bath and then making up to the original volume with distilled water. Traces of ethanol do not interfere.

As to the "other" opium alkaloids which are removed by the benzene: although obtained in a purified state freed from accompanying colouring material and other impurities, they could not be determined volumetrically, as not all of them are stochiometrically titratable.

However, their separation and the isolation of individual alkaloids might now be possibly attempted with promising results.


  1. An assay of morphine in crude opium and medicinal preparations is proposed, based upon chromatographic separation of the alkaloid, which is then determined volumetrically.

  2. A simple chromatographic adsorption method has been developed for separating morphine from accompanying alkaloids and interfering substances present in crude opium. The separation system consists of a Celite adsorption column, wetted by an ammoniacal aqueous opium extract. The alkaloids other than morphine we washed out with the help of benzene, whilst a mixture of CHCl 3+ethyl alcohol was employed as eluting agent for the isolated, purified morphine.

The proposed method is short, simple, and gives reproducible results.


The authors wish to express their sincere thanks to the Chemist of the Secretariat of the United Nations for the kind interest he took in the course of this work and for the care in supplying the material used.



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De Prado, L., Pharm. Abstr. 1, 309 (1935)

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