Effect of temperature on the estimation of morphine in opium by the method of British Pharmacopoeia

Sections

Abstract
Review of literature
Experimental Preparation of raw good opium samples
Preparation of powdered opium samples
Air oven and steam (boiling water) oven
Infrared drying
Steam tables
Estimation of anhydrous morphine in opium
FIGURE 1
FIGURE 2
FIGURE 3
FIGURE 4
FIGURE 5
FIGURE 6
Preparation of Acid Alumina
Gravimetric estimation of morphine
Titration of morphine-dinitrophenylether
Gravimetric estimation of morphine in opium using 1-chloro-2,4-dinitrobenzene (B.D.H., U.K.)
TABLE 3 Percentage of anhydrous morphine in the dry opium powder samples by gravimetric estimation using 1-chloro-2,4-dinitrobenzene
Gravimetric estimation of morphine in morphine sulphate B.P. and morphine hydrochloride B.P.
Estimation of morphine in opium by the method of B.P. following the operational temperature at 20°C
Results and Discussions
Conclusion
Acknowledgement

Details

Author: V.S. RAMANATHAN ** and , C. RAMACHANDRAN
Pages: 69 to 82
Creation Date: 1974/01/01

Effect of temperature on the estimation of morphine in opium by the method of British Pharmacopoeia *

V.S. RAMANATHAN ** and
C. RAMACHANDRAN **
Government Opium and Alkaloid Works, Ghazipur, India.

Abstract

Effect of temperature on the estimation of morphine by the British Pharmacopoeia method in six representative samples of opium collected from the States of Uttar Pradesh, Madhya Pradesh and Rajasthan in India has been reported. The estimation of morphine on the samples was carried out once every month for a year, noting the atmospheric temperature and humidity at which the experiments were conducted. Some modifications were introduced in the method of estimation given in the British Pharmacopoeia. The precipitation of morphine was allowed for a period of 19 hours. The assay results of morphine in the opium samples were the highest at 20°-21°C in the month of January and lowest at 38°-39°C in the month of May indicating that the morphine assay results were affected by the temperature at which the experiments were carried out. Separate estimation carried out at 20°C gave the highest assay result of morphine in the same samples. The results of morphine estimations were also compared with those obtained by the gravimetric using 1-chloro-2,4-dinitrobenezne and 1-fluoro-2,4-dinitrobenzene. The gravimetric estimations gave higher values for morphine over the British Pharmacopoiea method. The values obtained by the perchloric acid titration of morphine-dinitrophenylether obtained by using 1-chloro-2,4-dinitrobenzene were comparable with the morphine assay results of the British Pharmacopoeia method carried at 20°C. The accuracy of morphine results obtained by the gravimetric method was also checked by estimating morphine in morphine sulphate B.P. and morphine hydrochloride B.P.

Review of literature

Morphine is the most important alkaloid of opium which exists as a salt in combination with the meconic acid, sulphuric acid and with other organic acids in opium. It is used in medicine as a pain killer. Besides it has the specific property to induce sleep along with the removal of pain. Derosne ( [ 1] )*** first obtained morphine as a crystalline substance. Serturner (2) described the isolation of morphine in a number of publications. The physical and chemical properties of morphine have been described in detail by Small ( [ 3] ), Henry ( [ 4] ), Bentley ( [ 5] ), Ginsburg ( [ 6] ) and by others ( [ 7] , [ 8] , [ 9] , [ 10] ).

Many methods have been reported for the quantitative estimation of morphine in opium, preparations containing opium and in morphine salts. Morphine being a narcotic, producing tolerance and dependence and also suppressing respiration on administering into the body, its correct estimation in opium, for prescribing as a medicine, for process control and for accounting in the manufacture of alkaloids in factories and for international control in the sale and consumption of opium is very essential. The evaluation of opium is also based on its morphine content. The earlier methods of estimation based on the quantitative precipitation of morphine by alkaloidal reagents like Mayer's reagent by Heikel ( [ 11] ) and Wagner's reagent by Prescott and Gordin ( [ 12] ) have been reported. Estimations were also based on the reducing property of morphine on ferricyanide or iodic acid ( [ 13] ). The nitroso colour reaction was also developed and used for the estimation of morphine ( [ 14] , [ 15] , [ 16] ). Pruner worked out the cerimetric estimation of morphine ( [ 17] , [ 18] ). Methods based on paper chromatography, spectrophotometry, electrophoresis, polarography and thin layer chromatography have been described in literature ( [ 19] , [ 20] , [ 21] , [ 22] , [ 23] , [ 24] , [ 25] , [ 26] , [ 27] ). Mannich developed a gravimetric method of precipitating morphine as its 2,4-dinitrophenylether and later improvements on this were also reported ( [ 28] , [ 29] , [ 30] , [ 31] , [ 32] , [ 33] , [ 34] , [ 35] , [ 36] , [ 37] ). The Pharmacopoeias of India, Britain, United States of America, Japan, Switzerland, USSR, International and of many other countries have prescribed the lime water method for the estimation of morphine. Several workers studied the pharmacopoeia procedure and have published their findings ( [ 38] , [ 39] , [ 40] , [ 41] , [ 42] ). To get comparative results, it is essential to carry out the determination precisely as laid down in the Pharmacopoeia. The British Pharmacopoeia method of assay of morphine in opium is adopted in the Government Opium & Alkaloid Works, Ghazipur. The British Pharmacopoeia however has not clearly stated the standard temperature at which morphine is to be precipitated and also the number of hours the flask is to be left over after the precipitation of morphine. Literature also does not show of any study made on the precipitation temperature and duration of time, and as to whether these factors affect the assay results of morphine. This investigation was therefore undertaken to see the effect of temperature on the assay results of morphine, in opium by the method of British Pharmacopoeia, 1963 ( [ 43] ). The British Pharmacopoeia states that the precipitation flask is to stand "over night". It was decided to keep this period to 19 hours. The assay results thus obtained were also compared with morphine results of the gravimetric method of estimation in the same samples of opium.

* This formed a part of the thesis submitted by V.S. Ramanathan for the award of his Ph.D. degree of the University of Madras.

** Present Address : V.S. Ramanathan, Chief Chemist, Central Revenues, CentralRevenues Control Laboratory, Hillside Road, Delhi-12; C. Ramachandran, Assistant Chemical Examiner, Central Revenues Control Laboratory, Hillside Road, Delhi-12.

*** Figures in parentheses refer to references, p. 81.

Experimental Preparation of raw good opium samples

Individual samples drawn from raw good opium and produced in the districts of Barabanki (Uttar Pradesh), Ratlam (Madhya Pradesh), Kota and Jhalawar (Rajasthan), received in bags from the District Opium Officers, were mixed and three separate composite samples were prepared. These three samples representing opium from Uttar Pradesh, Madhya Pradesh and Rajasthan were numbered 1, 2, 3 respectively. Good opium was stored in the opium factory in big vats ( [ 44] ). Representative samples were drawn from the opium of three selected vats and three composite samples were prepared and numbered as 4, 5 and 6.

Preparation of powdered opium samples

Experiments were carried out to study the most suitable and quickest way of drying the opium samples to a free flowing powder.

Air oven and steam (boiling water) oven

In the air oven and steam oven kept at 100°C and 98°C respectively, it took more than 24 hours (3 working days of 8 hours each) for a sample of moist raw opium (200 g) to dry to constant weight. Such prolonged heating of the opium near about 100°C was not desirable as it could have deleterious effect on the constituents of opium. Hence this method was not followed.

Infrared drying

Opium (50 g) was taken in a glazed porcelain plate. An infrared lamp (250 watts) clamped to a stand was kept over the opium on a plate. A thermometer was inserted into the opium to study the rise in temperature inside the opium. The opium was stirred by a glass rod. When the lamp was kept close to the opium, bubbles and fumes started coming out, indicating the decomposition of opium. The lamp was raised up in the stand, keeping it farther away from the opium. In this manner the optimum distance required to keep the temperature of opium below 70°C was studied and it was found to be 11 inches away from the opium. The opium could be dried safely and quickly in about 3 hours by this method.

Steam tables

Raw good opium (400 g) was taken in three glazed porcelain plates, placed on an open steam table, whose temperature was maintained between 65°-70°C by running steam (10 lbs p.s.i.). The surface of steam tables had a layer of one fourth of an inch thick cloth padding, with circular holes, into which the bottom of the porcelain plates fitted in for heating. The cloth padding prevented excessive heating. The opium on the plate was stirred with a stainless steel spatula to hasten the drying. In less than 2 hours, the opium was dried and powdered. The dry powder was sieved through 40 mesh to make it homogeneous. The sieved powder was dried in a steam oven for 1 hour at 98°C. The samples thus prepared were stored in amber coloured bottles filling the bottles full leaving practically no air space and keeping the mouths closed in an air-tight condition. The above described steam table drying procedure was adopted for the six samples of opium of Barabanki, Ratlam, Kota and Jhalawar and the three vat samples mentioned above were labelled as 1 to 6 respectively.

Estimation of anhydrous morphine in opium

Dry powdered opium (8 g) was accurately weighed and placed in a glass mortar. Water (5 ml) was added and grounded with a glass pestle for 5 minutes to make the mass into a homogeneous paste. More water (10 ml twice) was added and triturated to make a uniform thinner paste. Calcium hydroxide B.P. free from carbonate (2 g) was added and mixed well. In the beginning the paste became thick which on continued grinding became thinner. Water (10 ml) was added, triturated and the whole slurry was then transferred into a tared conical flask (250 ml). The mortar and pestle was washed with small quantities of water and the entire material transferred into the flask. The net weight of the slurry in the flask was adjusted to 90 g. All the above operations were completed within 30 minutes. The conical flask was tightly stoppered with a rubber cork and shaken for half an hour in a mechanical shaker at 90 rounds per minute. After the shaking was over, the contents were transferred to a clean, dry, Buchner funnel fitted to a Buchner flask and filtered using Edwards vacuum pump. The filtration was over in less than 8 minutes. The filtrate was very clear. Instead of using a Buchner funnel and flask, the clear opium extract could be obtained using a laboratory centrifugal machine. The opium-calcium hydroxide slurry in the flask was transferred into a glass thimble (100 ml) and centrifuged for 3 to 4 minutes at 2000 r.p.m. The residue settled down at the bottom of the thimble and the supernatant liquid was filtered quickly in 3 minutes through a Whatman filter paper No. 1. The filtrate obtained by using a centrifugal machine or a Buchner funnel was of the same clarity. Hence in all the experiments the Buchner funnel was used in getting the clear alkaline extract of opium.

The filtrate (52 ml) representing the sample of opium (5 g) was pipetted out into a conical flask (200 ml), alcohol (90 per cent, 5 ml) and then ether B.P. (25 ml) were added and shaken for 5 minutes. Ammonium chloride B.P. (2 g) was then added, the flask was stoppered and shaken well for 5 minutes and allowed to stand for 5 minutes. Shaking and standing was continued, the total shaking being six times each for a duration of 5 minutes amounting to a total period of 30 minutes. The whole operation of shaking and standing took one hour. Morphine crystals separated and the flask was allowed to stand overnight (19 hours).

The next day the flask was gently shaken, once for the crystals of morphine to settle down at the bottom of the flask. A glass funnel (4 inches diameter) was tightly packed at the tail portion with surgical cotton wool B.P. through which ether B.P. (5 ml) was filtered to keep it wet and to see the rate of flow as drop by drop. The ether layer in the flask was then gently transferred over the cotton plug, avoiding the crystals as far as possible. The flask was rinsed with fresh ether B.P. (10 ml) and shaken. The ether layer was then decanted over the cotton plug. The ether washing was repeated twice more with 10 ml each time. When all the ether had been transferred, the aqueous mother liquor was decanted slowly, holding the crystals in the flask. When the aqueous layer had filtered, the crystals in the flask were rinsed with morphinated water (prepared according to B.P.) and the wash water slowly decanted. The crystals were washed till the wash water was free from chlorides as indicated by silver nitrate solution. Thereafter the cotton plug with the crystals was carefully transferred back into the original flask. The funnel was also washed down with distilled water into the original flask. O.l.N. sulphuric acid (25 ml) was added to the flask through the funnel and the funnel rinsed with distilled water into the flask. The flask was warmed over a steam bath to dissolve the crystals. The flask was cooled and titrated against 0.1 N sodium hydroxide using methyl red (2 drops) as indicator. 0.1 N sulphuric acid was equivalent to 0.02853 g of anhydrous morphine. As a correction factor for the solubility of morphine in 52 ml of opium solution taken, 0.052 g was added to the calculated value obtained by titration. The figure multiplied by 20 gave the percentage of anhydrous morphine in the sample of opium tested.

The laboratory atmospheric temperature and humidity were noted during the period the estimations were performed. The above procedure of estimating morphine was exactly followed in all the six samples of opium and the estimations were repeated once in the middle of the month on all the samples for twelve months. Each sample was tested in duplicate and the average of the two results was taken as the percentage of morphine in the sample. The results obtained are shown in table 1 and in figures 1 to 6.

TABLE 1

Percentage of anhydrous morphine in the six samples of dry opium powder by the method of British Pharmacopoeia, 1963

Winter and spring seasons

Month and date

October 17.10.66

November 15.11.66

December 15.12.66

January 16.1.67

February 15.2.67

March 15.3.67

Temperature°C
29-30
24-25
21-22
20-21
18-19
28-30
Relative humidity
47 60 57 65 35 49
Opium sample (Powdered, dry)
           
1.
10.51 10.58 10.90 10.93 10.81 10.34
2.
11.54 11.46 11.49 11.66 11.46 11.09
3.
10.71 10.69 10.84 10.95 10.93 10.48
4.
11.22 11.28 11.33 11.43 11.40 11.15
5.
11.38 11.44 11.49 11.58 11.56 11.31
6.
12.78 12.88 13.17 13.20 12.93 12.78

Summer, rainy, and autumn seasons

Month and date

April1 7.4.67

May 3.5.67

May 29.5.67

June 15.6.67

July 17.7.67

August 16.8 67

September 15.9.67

Temperature °C
31-33
34-36
38-39
36-37
33-34
31-32
31-32
Relative humidity
39 27 28 55 77 82 80
Opium sample (Powdered, dry)
             
1.
10.07 10.02 9.91 10.04 10.35 10.48 10.40
2.
10.90 10.83 10.79 10.83 10.90 11.02 10.94
3.
10.29 10.06 10.01 10.17 10.24 10.45 10.45
4 .
10.93 10.83 10.67 10.77 10.92 10.96 10.99
5.
11.00 10.98 10.82 10.91 10.99 10.99 11.10
6 .
12.51 12.41 12.24 12.39 12.58 12.55
12.62

FIGURE 1

Percentage of Anhydrous Morphine in Opium Sample No. I

Full size image: 7 kB, Percentage of Anhydrous Morphine in Opium Sample No. I

FIGURE 2

Percentage of Anhydrous Morphine in Opium Sample No. 2

Full size image: 7 kB, Percentage of Anhydrous Morphine in Opium Sample No. 2

FIGURE 3

Percentage of Anhydrous Morphine in Opium Sample No. 3

Full size image: 7 kB, Percentage of Anhydrous Morphine in Opium Sample No. 3

FIGURE 4

Percentage of Anhydrous Morphine in Opium Sample No. 4

Full size image: 7 kB, Percentage of Anhydrous Morphine in Opium Sample No. 4

FIGURE 5

Percentage of Anhydrous Morphine in Opium Sample No. 5

Full size image: 6 kB, Percentage of Anhydrous Morphine in Opium Sample No. 5

FIGURE 6

Percentage of Anhydrous Morphine in Opium Sample No. 6

Full size image: 7 kB, Percentage of Anhydrous Morphine in Opium Sample No. 6

Preparation of Acid Alumina

Aluminium oxide, anhydrous extra pure, E. Merck (100 g) was mixed with normal hydrochloric acid (400 ml) and the mixture was stirred well and allowed to settle.

The supernatant liquid and suspended particles were decanted. The alumina was washed with distilled water a number of times until the wash water was very slightly acidic to litmus. The alumina was then dried at 100°C.

Gravimetric estimation of morphine

Dry opium powder (1 g) was well triturated in a small glass mortar with distilled water (1 ml) to a homogeneous paste. Acid alumina (5 g) was added to this in small parts and grounded again. A pad of cotton wool was placed at the bottom of a chromatographic tube (300 mm long, 300 mm diameter) above the stop-cock. The tube was half filled with water. Acid alumina (5 g) was slowly poured into the tube and allowed to settle. This was stirred by a glass rod and again allowed to settle. The water was run out until a few cm remained above the surface of acid alumina. The alumina was washed with water 4 or 5 times, until the washings were no more turbid. In the final washing the water layer was drained out until the supernatant water was 2 cm above acid alumina. The opium-alumina triturate was then transferred into the chromatographic tube and mixed with supernatant water by stirring with a glass rod. The mortar and pestle were finally wiped with a little cotton wool and placed on top of the column The water from the column was drained out, drop by drop into the graduated measuring cylinder (100 ml) with the help of the glass stopper, until the water surface reached the cotton wool pad. The eluate was transferred back into the chromatographic column, and percolated dropwise. No suction was applied. The alumina column and the tube was washed with water (5 ml, 5 times) to obtain an eluate of 35 ml.

1-fluoro-2,4-dinitrobenzene (E. Merck), (0.25 g in 20 ml of acetone pure) and ammonia (4 ml, 25 per cent) were added to the eluate. The measuring cylinder was rinsed with further acetone (10 ml), stoppered and shaken for a minute. The cylinder was allowed to stand at room temperature for 30 minutes. The precipitate of morphine dinitrophenylether was filtered through tared sintered glass funnel (Fine G.4) under suction by a vacuum pump and rinsing with the filtrate. The precipitate was washed finally with cold acetone (2 ml, 4 times). The sintered glass funnel with the precipitate was dried at 80°C for half an hour, cooled in a dessicator and weighed. The percentage of morphine in the sample of opium was calculated as follows.

The percentage of anhydrous morphine = W x 63.2 where W is the weight of morphine-dinitrophenylether.

TABLE 2

Percentage of anhydrous morphine in the dry opium powder samples by gravimetric estimation using 1-fluoro-2,4-dinitrobenzene

 

Anhydrous morphine percent

Opium sample powdered, dry

By weighment

By titration

1.
11.47 11.17
2.
12.39 12.03
3.
11.63 11.17
4.
11.83 11.61
5.
12.03 11.77
6.
13.72
13.21

Effect of temperature on the estimation of morphine in opium 77

Titration of morphine-dinitrophenylether

The precipitate in the sintered glass funnel was dissolved in glacial acetic acid (20 ml) and sucked into a Buchner flask (100 ml). The sintered funnel was washed with glacial acetic acid (5 ml, 3 times). Acetic anhydride (5 ml) was added and the solution was titrated with 0.l N perchloric acid using crystal violet as indicator (2 drops of 0.5 per cent W/V in glacial acetic acid). 1 ml of 0.1 N perchloric acid is equivalent to 0.02853 g of anhydrous morphine. The results obtained are reported in table 2.

Gravimetric estimation of morphine in opium using 1-chloro-2,4-dinitrobenzene (B.D.H., U.K.)

The procedure followed was the same as that of l-fluoro-2,4-dinitrobenzene except that the precipitate of morphine-dinitrophenylether was allowed to stand overnight (19 hours) before filtration. The results obtained on the six opium samples are given in table 3.

TABLE 3 Percentage of anhydrous morphine in the dry opium powder samples by gravimetric estimation using 1-chloro-2,4-dinitrobenzene

 

Anhydrous morphine percent

Opium sample powdered, dry

By weighment

By titration

1.
11.27 11.04
2.
12.29 11.91
3.
11.25 11.04
4.
11.69 11.54
5.
11.98
11.70
6.
13.59 13.17

Gravimetric estimation of morphine in morphine sulphate B.P. and morphine hydrochloride B.P.

The percentage of morphine in morphine sulphate B.P. and morphine hydrochloride B.P. was estimated by precipitating morphine as its dinitrophenylether using 1-chloro-2,4-dinitrobenzene and l-fluoro-2,4-dinitrobenzene. The percentage of morphine in morphine sulphate B.P. and morphine hydrochloride B.P. was also estimated by the procedure laid down in the British Pharmacopoeia, by solvent extraction and titration. The assay of morphine in the morphine hydrochloride B.P. and morphine sulphate B.P. by gravimetric method was done to examine how far the gravimetric method and the titration of the morphine-dinitrophenylether gave correct values. These values could also be compared with the assay results by the B.P. procedure for morphine salts.

Morphine hydrochloride B.P. or morphine sulphate B.P. (0.2 g) was accurately weighed into a conical flask (100 ml) with a glass stopper. Water (20 ml) was added and the morphine salt dissolved in it. 1-fluoro-2,4-dinitrobenzene or 1-chloro-2,4- dinitrobenzene (0.3 g in 20 ml of acetone) and ammonia (4ml, 25 percent) were added to the morphine solution. The flask was stoppered and shaken to precipitate the morphine-dinitrophenylether. The rest of the procedure as described above for opium was followed. The results obtained are shown in table 4.

TABLE 4

Percentage of anhydrous morphine in morphine hydrochloride B.P. and morphine sulphate B.P.

 

Percentage of anhydrous morphine using 1-fluoro-2,4-dinitrobenzene

Percentage of anhydrous morphine 1-chloro-2,4-dinitrobenzene using

 

Name of the sample

By weighment

By titration

By weighment

By titration

Percentage of morphine by B.P. procedure

Morphine Hydrochloride B.P.
99.24 99.57 98.88 99.02 98.79
Morphine Sulphate B.P.
99.17 99.00 99.04 98.79 98.54

NOTE. - 1. The percentage of anhydrous morphine in morphine hydrochloride B.P. was calculated with reference to the sample dried at 130oC.

  1. Percentage of anhydrous morphine in morphine sulphate B.P. was calculated on sample dried at 145o C.

Estimation of morphine in opium by the method of B.P. following the operational temperature at 20°C

The procedure described above for the estimation of morphine in opium using calcium hydroxide was followed with the following modifications:

  1. The opium powder triturated with calcium hydroxide and water and transferred to the conical flask (250 ml) as a slurry was cooled to 20°C in ice water bath before the net weight was adjusted to 90 g.

  2. The clear alkaline filtrate of the opium extract, was cooled to 20°C before 52 ml was pipetted out for the precipitation of morphine.

TABLE 5

Percentage of anhydrous morphine in the dry opium powder samples by the method of B.P. following the operational temperature at 20°C

Opium sample powdered and dry

Percentage of anhydrous morphine

1.
10.99
2.
11.72
3.
11.10
4.
11.49
5.
11.61
6.
13.18
  1. After the morphine has been precipitated by adding ammonium chloride and shaking, the conical flask (250 ml) with the crystals of morphine was kept inside a widemouth thermos flask containing water (100 ml, at 20°C) and 3 to 4 small pieces of ice also put inside the thermos flask to maintain the temperature of 20°C for 19 hours. The conical flask was tightly closed with a rubber cork with the opium extract (52 ml) and cooled in ice water at 20°C before it was placed inside the thermos flask. The thermos flask was also tightly closed with a cork and metallic screw cap.

  2. The ether and morphinated water used for washing the morphine crystals were cooled to 20°C before usage.

The results obtained are shown in table 5.

These estimations were carried out on the 8th June, 1967, when the laboratory temperature was 40°C.

Results and Discussions

Opium collected in India from the three States of Uttar Pradesh, Madhya Pradesh and Rajasthan is received in the Opium Factory in polythene bags. The opium is emptied for storage in big vats. Hence the samples of opium representing the good opium as received from the above three states and what was stored in the factory were drawn for conducting the estimations of morphine.

The opium had to be dried to a powder to make the samples homogeneous. Drying of the moist opium was an important step. It had to be dried at a low temperature as quickly as possible to avoid destruction of morphine. Dott ( [ 45] ) and Annett ( [ 46] , [ 47] ) reported that opium dried at 60°C or 98°C showed practically no loss of morphine. Dunnicliff et al ( [ 41] ) reported that opium dried at 60°C and stored in contact with air showed loss of morphine. They had also reported that opium dried at 95°-100°C showed no loss of morphine on storage. It was, therefore, decided that in this investigation opium be dried quickly on steam table at about 65°C first and finally at 98°C in a steam oven for an hour. Infra-red lamp drying was also helpful below a temperature of 70°C and the opium dried in this way should also be finally dried at 98°C for an hour to remove the entire moisture in it for long storage purposes.

Certain modifications had to be made in the B.P. method to follow a uniform procedure as well as to get correct results. The morphine was extracted as calcium morphinate. Sulphuric and meconic acids were eliminated as calcium salts in the insoluble marc. All no-phenolic alkaloids in the opium like Narcotine, Codeine, Thebaine, Papaverine, Cryptopine, Neopine, etc., were precipitated and removed in the marc. This needed vigorous shaking in the mechanical shaker for 30 minutes. Opium contains ammonium salts and so ammonia is liberated when triturated with calcium hydroxide. This ammonia is held in the aqueous solution. Ammonia is also liberated when ammonium chloride is added to precipitate the morphine. A portion of the codeine dissolved in this ammoniacal lime water is removed during filtration. The addition of alcohol before the precipitation of morphine also removes codeine and other alkaloids. The alcohol also helps in keeping resinous matter in solution and allows better crystallization of the morphine. The washing of morphine crystals with ether (3 times) make it pure. The filtration of the lime water-opium extract was done in a Buchner funnel or centrifuged in a laboratory centrifugal machine to prevent the concentration of the filtrate which would occur particularly in summer months, if filtered through ordinary filter taking a longer period. The laboratory centrifugal machine would help in getting a clear extract even quicker than the Buchner funnel. The flask containing the morphine crystals was allowed to stand for a definite period of 19 hours (over night) for complete precipitation and for repeating the experiments to the same length of time.

Laboratory temperature in Ghazipur varied from 18°C to 40°C in a year and in some years it was even higher than 40°C. The temperature in the months of April, August and September was 31° to 33°C. When the temperature was above 34°C ether would evaporate off from the conical flask in which morphine is precipitated. The conical flasks, therefore had to be kept immersed in water up to the ether level in a separate container to prevent its loss by evaporation.

From table 1 and figures 1 to 6 it is clear the assay results of morphine in all the samples of opium decreased as the atmospheric temperature increased. The assay results were highest in the month of January and lowest in May which correspond to winter and summer months. Figures 1 to 6 show that as the atmospheric temperature from January to March increased the assay results of morphine in the opium samples came down steeply. The morphine results in the months of April, August and September agreed fairly well. The difference between the highest and the lowest assay results in some samples was nearly one unit. The precipitation of morphine from the opium solution in summer months was incomplete. The assay results for morphine was the highest when the laboratory temperature was 20°-21°C. This would show that the precipitation of morphine was affected by the atmospheric temperature in which the estimation was carried. Buchi et al ( [ 37] ) claimed that by using l-fluoro-2,4-dinitrobenzene correct assay results for morphine in opium could be obtained. The estimations of morphine in opium samples both by gravimetric and titrimetric method using 1-fluoro- and l-chloro-2,4-dinitrobenzene were carried out and results reported in tables 2, 3 and 4. The assay results of morphine in the opium by titration of the morphine-dinitrophenyl ether was lower than gravimetric method by 0.30 to 0.50 units as shown in tables 2 and 3. The percentage of morphine obtained by gravimetric method using 1-fluoro-2,4-dinitrobenzene in all the six samples of opium was higher than by using 1-chloro-2,4-dinitrobenzene. The gravimetric assay results of morphine in opium determined using 1-chloro and l-fluoro-2,4-dinitrobenzene were much higher than those obtained by the method of B.P. at the operational temperature at 20°C as shown in table 5.

Results shown in table 4 for morphine sulphate B.P., and morphine hydrochloride B.P. indicate that the gravimetric method gives higher percentage of morphine in those samples also. However, there is a close agreement in the assay values in the samples of opium obtained by titration of the morphine dinitrophenyl ether using 1-chloro-2,4-dinitrobenzene with the assay results shown in table 5 and table 1 conducted by B.P. method at 20°C.

Conclusion

The percentage of anhydrous morphine in the opium samples obtained by the method of B.P. following an operational temperature at 20°C gave the highest results. To get at the correct and repeatable percentage of morphine in opium, the B.P. procedure is to be conducted at 20°C with the modifications followed in this investigation.

Acknowledgement

The authors are indebted to the Government of India, Ministry of Finance, Department of Revenue & Insurance, New Delhi, for permission granted to conduct this investigation and to publish the results.

References

001

Derosne, Ann. Chim., 1803, 45, 257.

002

Serturner, F.W., Trommsdorff's Journal der Pharmazie, 1805, 13(1), 234.

003

Small, L.F., Chemistry of the Opium Alkaloids, U.S. Treasury Dept., Publication Health Service, United States, Govt. Printing Office, Washington, Supplement No. 103, 1932, 139.

004

Henry, T. A., The Plant Alkaloids, IV Edition, J. and A. Churchill Ltd., London, 1949, 213.

005

Bentley, K. W., "The Chemistry of Morphine Alkaloids", Clarendon Press, Oxford, 1954, 16.

006

Ginsburg, D., "The Opium Alkaloids", Interscience Publishers, New York 1962, 3.

007

Norman Evers and Dennis Caldwell, "The Chemistry of Drugs", III Edition, Ernest, Benn Ltd., London 1959, 201.

008

Mitchell, A., Allen's Commercial Organic Analysis, V Edition, J. and A. Churchill and Sons Ltd., London, 1929, VII 655.

009

Merck Index, Chemicals and Drugs, VIII Edition, Rahway New Jersey, U.S.A., 1968, 702.

010

Manske, R.H.F. and Holmes, H.L., The Alkaloids, Chemistry and Physiology II, 1952, 2.

011

Heikel, Chem. Ztg. 1908, 32, 1149, 1162, 1212.

012

Prescott and Gordin, J. Am. Chem. Soc., 1898, 20, 706.

013

Keiffer, L., Ann., 1957, 103, 271.

014

Garratt, D.C., Quart. J. Pharm. Pharma., 1937, 10, 466.

015

Adamson, D. C. M. and Handisyde, F.P., Analyst, 1945, 70, 305, Quart. J. Pharm. Pharmacology 1946, 19, 350.

016

Ramanathan, V.S., Journal of Scientific and Industrial Research, 1962, 21D, No. 12, 399-460.

017

Pruner, G., U.N. Document, ST/SOA/SER.K/118, 15 March 1962.

018

Pruner, G., Rendi. Indst. Sup. Sanita, 1956, XIX, 492.

019

Fulton, C.C., U.N. Document ST/SOA/SER.K/34, 14 September 1954.

020

Pride, R. R. A. and Stern, E. S., Journal of Pharmacy and Pharmacology, 1954, 6, 590.

021

Clark, W. A. and McBay, A., J. Amer. Pharm. Association, 1954, XLIII, No. 1, 39.

022

Dyer, M.S. and McBay A., Amer. Pharm. Association, 1955, XLIV, No. 3, 156.

023

Farmilo, C.G. et al., U.N. Document ST/SOA/SER.K/58, 29 November 1957.

024

Van Der Pol, H. J., Journal de Pharmacie de Belgique, 1958, 9, 426.

025

Pfeifer, S., Bulletin on Narcotics, X, 3, p. 18.

026

Genest, K. and Farmilo, C. G., Journal of Amer. Pharm. Association, 1959, 48, 287.

027

Genest, K. and Farmilo C.G., Analytical Chemistry, 1962, 34 No. II, 1464.

028

Mannich, C., Arch. Pharmazie, 1935, 273, 97; 1942, 280, 386.

029

Van Pinxteren, J. A. C. and Smeets, M. A. G., Pharm. Weekblad, 1950, 85, 48.

030

Dann, O. and Wippern, F., Dtsch. Apoth. Ztg., 1951, 91, 905.

031

Fischer, R. and Folberth, K., Arzneimittel-Forschung, 1955, 5, 66.

032

Svendsen, A.B. and Aarnes, E.P., Scientia Pharmaceutica, 1955, 23(I}, 18.

033

Garratt, D. C. et al., Journal of Pharmacy and Pharmacology, 1957, 9, 914.

034

Toffoli, F., Rendi, Inst. Sup. Sanita., 1957, XX, 155.

035

Svendsen, A.B., U.N. Document ST/SOA/SER.K/92, 3 November 1959.

036

Pruner, G., U.N. Document ST/SOA/SER.K/101, 12 April 1960.

037

Buchi, J., Huber, R. and Schumacher, H., Bull. on Narcotics, XII, 2, p. 25.

038

Annett, H. E., Analyst, 1918, 43, 205.

039

Dott, D. B., Analyst, 1919, 44, 50.

040

Rakshit, J.N., Analyst, 1919, 44, 337; 1921, 46, 481; 1926, 51,491; 1931, 56, 711; Ann. Chem. Analytique, 1935, 11, 17.

041

Dunnicliff, H.B. and Ray, J. N., Proc. Nat. Inst. Sci. India, 1935, I No. 2, 107.

042

Wellingford, V.H. and Homeyer, A. H., Journal of Amer. Pharm. Association, 1936, XXV No. 5, 402.

043

British Pharmacopoeia, 1963, 544.

044

Vardhan, S. K., Bulletin on Narcotics, VIII, 2, p. 35.

045

Dott, D.B., Pharm. J., 1926, 116, 356.

046

Annett, H.E. and Singh H. D., J. Soc. Chem. Ind., 1918 37, 315.

047

Annett, H.E., Pharm. J., 1923, 57(4), 647.