Physical Methods
Biological Methods
Chemical Methods
Sensitivities
PRACTICAL PROCEDURE
Discussion
Summary
Acknowledgements
APPENDIX A
Author: E. G. C. Clarke, Margaret Williams
Pages: 33 to 42
Creation Date: 1955/01/01
It is probable that more tests have been described for the opium alkaloids than for any other similar group or organic substances. Their widespread use in medicine, their toxicity, and, above all, their habit-forming properties make their detection in minute quantities a matter of more than academic interest, while the legal significance of the presence of one of these substances makes their absolute identification more important than their exact quantitative estimation. It is the purpose of this paper to review briefly the methods available for the detection of the opium alkaloids when present in microgramme quantities, and to describe the application to these bases of a microtechnique recently elaborated by the authors (Clarke and Williams[1] ).
The methods available for the micro-identification of alkaloids may be classified as physical, biological and chemical.
Physical methods, which have recently been reviewed by Farmilo and Levi [2] , although often requiring expensive apparatus, have the distinct advantage that in most cases they do not destroy the material used; this leaves it available for further examination. Consequently, one can employ the entire available sample in a single test, without the subdivision necessary when chemical or biological methods are used. This is not necessarily true, however, for melting-point determination, as some alkaloids are readily decomposed by heat. In any case this method is not really delicate enough for use on the micro-scale, although Fischer and Karawia [3] and Kofler and Muller [4] describe methods using some 50 &mug of material.
Numerous methods for the identification and estimation of alkaloids have been based on the optical properties of solutions. Thus, Cahen and Feuer [5] found that they were able to estimate quantities of morphine as small as 20 &mug by electrophotometric estimation of the colour produced in a modified Deniges' reaction, while Gettler and Sunshine [6] have described a method for the colorimetric determination of alkaloids in microgramme quantities by methyl orange.
The use of spectrophotometric methods in the ultra-violet region has recently been reviewed by Farmilo [7] . There is some difference of opinion as to the sensitivity of the method. Biggs, for example, was able to make rough quantitative estimations of 1 mg quantities of morphine recovered from a Stas-Otto extract, while, replacing the Stas-Otto process by a special extraction technique, Berman and Wright [9] Were able determine as little as 1 &mug of certain alkaloids. Their method unfortunately fails altogether with some substances, notably morphine and heroin. Identification of alkaloids by means of their infra-red spectra does not appear to have been applied on the microgramme scale (Marion, Ramsay and Jones [10] ; Pleas, Harley and Wibberley [11] .
Little use has been made of fluorescence as a means of identification, although Chase and Pratt [12] have applied it to powdered vegetable drugs, while Hansen [13] states that1 &mug of morphine on filter paper may be recognized by the blue zone it gives in ultra-violet light.
The properties of crystals also provide useful means of identification. Keenan [14] , for example, has recorded the optical properties of the crystals of certain derivatives of a number of important alkaloids. Powdered crystals may also be used for the production of X-ray diffraction patterns. This method has high sensitivity; Barnes [15] used 10-&mug samples while Gross and Oberst [16] obtained positive results with morphine using less than 1 &mug of material.
Paper chromatography affords a further means for the isolation and identification of alkaloids, based on the variation in R f value. By this method 20 &mug or less may be detected (Munier and Macheb&oeliguf [17] ; Curry and Powell [18] ). Borke and Kirch [19] record a similar sensitivity for surface chromatography.
The biological detection of morphine depends on the "Mauseschwanzphänomen" first recorded by Straub [20] .
He noticed that mice which had received a subcutaneous injection of morphine exhibited certain definite signs, the most characteristic being the catatonic rigidity of the tail, which is carried in an S-shaped curve. This phenomenon was investigated by Herrmann [21] , who showed that the effect was roughly quantitative, the smallest quantity of morphine capable of detection being 5 &mug. Other opium alkaloids produced similar effects, although to a widely varying extent.
Maier [22] found that the duration of the " tail reaction " gave an approximate measure of the dose of morphine administered, but that a more accurate estimation could be made by finding the threshold of reaction. By this means, 20 &mug of morphine could be estimated quantitatively. Keil and Kluge [23] , interpreting their results graphically, found that they were able to estimate quantities of morphine as small as 12 &mug with an accuracy of 5%.
The detection of narcotics by bio-assay has been developed by Munch [24] [25] , who has used this method for the investigation of " doping " in racehorses. Using a standard technique, he found the threshold for a 20-g. mouse to be 1 &mug for heroin, 12 &mug for dilaudid, and 60 &mug for codeine and morphine. The method is, however, lacking in specificity. Although Munch [24] states that there is a qualitative difference between the effect of morphine and that due to heroin, it is not usually possible to distinguish between closely related substances. The great advantage of the method is the speed with which a result can be obtained. Body fluids such as saliva and urine can be injected directly without the tedious extraction and purification necessary for chemical and physical methods, and even with minimal doses effects become apparent in half an hour.
It is worth noting that Morgan and Gellhorn [26] , in a series of experiments designed to compare the sensitivities of biolo gical and chemical methods, found that, while the absolute threshold for certain substances (e.g., heroin and dilaudid) is lower for biological methods, in actual application the chemical method was not only more sensitive, but considerably more reliable.
The chemical identification of alkaloids may be carried out by means of colour or crystal tests. Both have been in use for over a century; in 1821 Orfila [27] mentioned the shape of morphine crystals, and the red colour produced with nitric acid, while in 1845 Christison [28] gave the ferric chloride and iodic acid tests for morphine, and described the shape of the crystals of the base and of various salts for morphine, codeine and narcotine. Wormley [29] in 1885 gave both colour and crystal tests in some detail. Since then, however, the colour test has become much the more popular. This may be seen by reference to various standard works on toxicology. Autenreith [30] , Glaister [31] , Kobert [32] , Nicholson [33] , Sydney Smith and Fiddes [34] , Van Oettinger [35] , and Witthaus [36] make no mention of crystal tests for the opium alkaloids. Bamford [37] , Lucas [38] , McNalley [39] , Peterson, Haines and Webster [40] and Thienes and Haley [41] give both crystal and colour tests, while the "Methods of Analysis" of the A.O.A.C.*[42] gives crystal tests only.
Both types of test have been described in almost endless variety, Bentley [43] referring to some 50 colour tests for morphine alone, while an even greater number of crystal tests for the various alkaloids of this group have been described by such authors as Vadam [44] , Stephenson [45] , Fulton [46] and Whitmore and Wood [47] . There is little to choose between colour and crystal tests from the point of sensitivity. In the case of the former, with morphine as an example, the limit of the Marquis test is variously put at 1 µg (Hansen [13] ), 2 µg (Fulton [48] ), or 5 µg (Farmilo, Levi, Oestreicher and Ross [49] ). For Froehde's test, Hansen [13] gives 0.1 µg, Wormley [29] 0.6 µg, Farmilo et al. [49] 1 µg, and Fulton [50] 0.3 µg, the last-named being for Buckingham's modification of the test. For the crystal test, Fulton [51] puts the limit at 0.1 µg for morphine with iodine reagent M-2, while Lucas [52] gives it as 1 µg or less with Marm?'s reagent.
In spite of the popularity of the colour test, and the fact that it is of equal sensitivity, in our opinion the crystal test is by far the more satisfactory. A pure substance, taken in sufficient quantity, may certainly give a series of definite colours with a certain reagent. The same substance, however, when recovered by means of a Stas-Otto extraction, is usually contaminated with material readily charred by sulphuric acid, which masks to a greater or less extent the colours due to the alkaloid. And although purification presents few difficulties when milligrammes are available, on the microgramme scale repeated manipulation tends to reduce the material to vanishing-point. Furthermore, the colour test is subjective, as the transient colour changes may be seen differently by different observers. The crystal test, on the other hand, is objective. The microcrystals are a definite entity and may be photographed for permanent record.
*Association of Official Agricultural Chemists (Ed. note).
In comparing the sensitivities of these methods several points must be borne in mind. The usefulness of a test depends not only on its sensitivity, but also on its specificity and its range-i.e., the number of alkaloids for which individual results can be obtained. Some tests are more fully diagnostic than others. Thus, with the physical tests the X-ray diffraction pattern may afford complete identification, whereas the melting-point serves only to confirm a diagnosis already made. Biological tests, as already stated, are almost completely lacking in specificity and the sensitivity claimed for them is apparent rather than real, as no deductions can be drawn from the results of an experiment carried out with a single mouse. In the case of chemical tests the questions of specificity and range depend largely on the choice of reagent which is discussed in detail below.
1. Crystal Test
Reagents
With crystal tests, identification depends on recognition of the characteristic shape and arrangement of the crystals formed when a suitable reagent is added to a solution of the alkaloid. The success of the test depends to a great extent on the correct choice of reagents. As stated previously, several hundred reagents have been described. In addition to those mentioned by the authors cited above, others are given by Bachmann [53] , Denoel and Soulet [54] , Duquenois and Faller [55] , Fulton and Dalton [56] , Janot and Chaigneou [57] , Levi and Farmilo [58] , Oliverio [59] , Putt [60] , Rosenthaler [61] , Uffelie [62] , Wachsmuth [63] , Wagenaar [64] and White [65] . Use of all these reagents as a general routine is obviously impracticable, as it would entail excessive subdivision of the material available. In selecting the reagents most likely to prove useful, their specificity and range must be considered. Some reagents, such as zinc chloride or sodium nitroprusside, give crystalline derivatives with but few alkaloids. That is to say, their specificity is high but their range limited. Reagents of this type are most useful for confirmatory purposes. Other reagents, such as Reinecke's salt, react with most alkaloids but give crystals so similar in appearance as to be almost useless for purposes of differentiation. Others again, such as platinum chloride and potassium bismuth iodide, react with a reasonable number of alkaloids to give crystals varying so widely in form that they provide a useful basis for identification. This class of reagent is obviously the most useful for routine testing. In practice, we find that the use of some 20 of these reagents is sufficient to identify provisionally all the more common alkaloids. Details of those most useful in the case of the alkaloids of opium are given in Appendix A.
It will be noted that we have abandoned the use of such terms as " Wagner's reagent " and " Marm?'s reagent " as, in our opinion, the indiscriminate use of these can lead to considerable confusion.
Technique
The micro-technique recently introduced by the authors [1] is carried out as follows :* The test material is dissolved in 1% acetic acid or hydrochloric acid. A microdrop (volume approximately 0.1 cu. mm.) of this solution is transferred to a cover slip by means of a glass rod 1 mm. in diameter. A similar drop of reagent is added and the two mixed. The cover slip is inverted and placed on a cavity slide where it rests on thin glass distance pieces cemented on either side of the depression as shown in the diagram. The hanging drop is now examined under the microscope. As soon as a precipitate can be seen, the cover slip is ringed with 25% gum-arabic solution to prevent further evaporation. The drop is then examined every few hours and its appearance noted when crystallization is complete. This may take 24 hours or more. Once tentative identification has been made, similar crystals should be prepared from a known sample of the alkaloid and compared with those from the test solution. It is essential that all glassware should be well cleaned and thoroughly rinsed, as traces of synthetic detergents may interfere with precipitation (Casidio and Gallo [66] , Janot, Goutarel and Decay [67] ).
*See Fig.1.
Alkaloid |
Gold bromide |
Lead iodide |
Mercuric chloride |
Platinum chloride |
Platinum iodide |
Pot. bismuth iodide |
Pot. cadmium iodide |
Pot. chromate |
Pot. iodide |
Pot. mercury iodide |
Pot. tri-iodide ( 1 ) |
Pot. tri-iodide (2) |
Pot. tri-iodide (3) |
Sodium carbo-nate |
Sodium nitro-prusside |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Apomorphine |
X |
a |
a |
a |
a |
X |
a |
a |
a |
X |
o |
a |
o |
a |
a |
Benzyl-morphine |
a |
X |
a |
a |
a |
a |
a |
a |
X |
a |
a |
X |
a |
X |
a |
Codeine |
a |
X |
X |
a |
a |
a |
X |
- |
X |
X |
X |
- |
X |
- |
o |
Cotamine |
X |
X |
X |
X |
o |
a |
a |
- |
- |
X |
X |
o |
X |
- |
- |
Dihydro-codeine |
- |
- |
o |
- |
a |
X |
o/a |
- |
- |
a |
o |
- |
o |
- |
- |
Dihydro-codeinone |
a |
X |
X |
a |
a |
X |
a |
- |
- |
a |
a |
X |
o/a |
- |
o |
Dihydro-hydroxy-codeinone |
a |
- |
o |
X |
- |
a |
a |
- |
- |
a |
X |
- |
o/a |
X |
- |
Dihydro-morphine |
a |
- |
o |
a |
X |
a |
o |
- |
- |
X |
o |
- |
o |
- |
- |
Dihydro-morphinone |
a |
- |
o |
a |
a |
a(X) |
a |
- |
- |
a |
o |
- |
o |
- |
X |
Ethyl-morphine |
a |
- |
X |
a |
a |
a |
X |
- |
- |
X |
- |
- |
X |
- |
- |
Ethyl-narceine |
a |
a |
o |
a |
o |
a |
o/a |
X |
X |
a |
o |
X |
o |
o |
o |
Heroin |
a |
a |
X |
X |
a |
X |
a |
X |
- |
a |
o/a |
- |
o/a |
X |
o |
Morphhie |
a |
- |
X |
o |
a |
X |
X |
X |
- |
a(X) |
X |
- |
o |
- |
- |
Narceine |
a |
X |
X |
X |
o |
a |
o |
X |
X |
o/a |
o |
X |
o |
- |
- |
Narcotine |
a |
X |
o/a |
a |
a |
a |
a |
X |
o |
a |
o/a |
a |
o/a |
X |
o |
Neopine |
a |
- |
X |
- |
a |
a |
X |
- |
- |
X |
X |
- |
X |
- |
- |
Papaverine |
a |
o/a |
X |
X |
a |
a |
X |
o |
X |
X |
a |
X |
a |
o |
o |
Pseudo-morphine |
a |
X |
X |
X |
X |
X |
a |
o/a |
X |
a |
a |
X |
a |
- |
X |
Thebaine |
a |
o/a |
o/a |
X |
o/a |
a |
a |
a |
o |
a |
a |
- |
a |
X |
o/a |
X = Crystals; a = Amorphous; o = Oil droplets; o/a = Oily amorphous; a(X) = Crystals form from more dilute solutions; - = No precipitate.
Results
Table 1 shows the results obtained by treating some of the more important opium alkaloids with fifteen different reagents, under comparable conditions. In each case a microdrop of a 1% solution of the alkaloid (containing 1 µg) was mixed with a similar drop of reagent. The slide was sealed immediately to prevent evaporation and the appearance recorded after twenty-four hours.
Table 2 gives the appearance of the crystals and the sensitivity of the test, the latter quantity being the minimum amount of alkaloid required to give recognizable crystals. In order to obtain these figures serial dilutions of the alkaloidwere employed, sealing of the slide being deferred until the drop had been concentrated by evaporation.
Some of the crystals obtained are shown in Plates 1-12, the weight of material used in the test being indicated.
FIGURE 1
2. Colour Tests
As already stated, in our opinion colour tests are less reliable than crystal tests, although they have considerable value for confirmatory purposes. Details of these tests are given by such authors as Bamford [38] , Fulton [50] , Jackson [68] , Pesez [69] and Taylor [70] .
The most widely used colour reactions are those in which a solution of some substance in concentrated sulphuric acid is added to a minute portion of the solid alkaloid and the resulting colour changes noted. Many substances have been suggested for this purpose, the more important including formaldehyde (Marquis [71] ), ammonium molybdate (Froehde [72] ), selenious acid (Mecke [73] , Lafon [74] ), p-dimethyl-aminobenzaldehyde (Wasicky [75] ), sodium tungstate (Reichard [76] ), titanium dioxide (Flueckiger [77] ), and sucrose (Schneider [78] , Weppen [79] ).
As we have reported previously [1] , we find that the sensitivity of most of these tests can be increased if the following procedure be adopted, Froehde's test being taken as an example. A microdrop of the test solution is placed on an opal-glass plate and a similar drop of a 0.5% aqueous solution of ammonium molybdate added. After evaporation, a microdrop of concentrated sulphuric acid is applied to the residue, and the colour changes noted. In the same way the tests ascribed to Mecke, Reichard and Schneider-Weppen may be carried out with aqueous solutions containing 0.5% of selenious acid, 1% sodium tungstate and 10% of sucrose respectively. For Wasicky's test a 10% solution of p-dimethylamino-benzaldehyde in glacial acetic acid is used.
Reagent |
Appearance of crystals |
Sensitivity (µg) |
---|---|---|
Apomorphine |
|
|
Gold bromide |
Serrated crosses |
0.0005 |
Potassium bismuth iodide |
Rosettes of dark needles |
0.005 |
Potassium mercury iodide |
Bunches of needles |
0.05 |
Benzylmorphine |
|
|
Lead iodide |
Hedgehogs (2 days) |
0.25 |
Sodium carbonate |
Rosettes of rods |
0.05 |
Potassium tri-iodide (2) |
Rosettes of plates (2 days) |
0.25 |
Potassium iodide |
Rosettes of needles or plates |
0.025 |
Codeine |
|
|
Potassium tri-iodide (1) |
Feathery rosettes |
0.05 |
Potassium cadmium iodide |
Small rosettes-small plates |
0.01 |
Potassium mercury iodide |
Irregular plates |
0.025 |
Lead iodide |
Rosettes of rods (2 days) |
0.5 |
Mereuric chloride |
Irregular needles in bunches |
0.25 |
Potassium iodide |
Rosettes of needles |
1.0 |
Potassium tri-iodide (3) |
Dense rosettes |
0.25 |
Cotarnine |
|
|
Platinum chloride |
Rods |
0.025 |
Mercuric chloride |
Bunches of needles |
0.05 |
Gold bromide |
Yellow rods |
0.1 |
Potassium tri-iodide (1) |
Very small plates |
1.0 |
Potassium tri-iodide (3) |
Small rods |
0.005 |
Potassium mercury iodide |
Irregular plates |
0.05 |
Lead iodide |
Needles or dendrites |
0.25 |
Dihydrocodeine |
|
|
Potassium bismuth iodide |
Bunches of hexagonal plates |
0.005 |
Dihydrocodeinone |
|
|
Mercuric chloride |
Dense rosettes |
0.05 |
Potassium tri-iodide (2) |
Very long rods (2 days) |
0.25 |
Lead iodide |
Dense rosettes (2 days) |
0.1 |
Potassium bismuth iodide |
Rhomboidal plates |
0.01 |
Dihydrohydroxycodeinone |
|
|
Potassium tri-iodide (1) |
Small dark plates |
0.0025 |
Sodium carbonate |
Long thin needles |
0.025 |
Platinum chloride |
Rosettes of rods |
0.25 |
Dihydromorphine |
|
|
Platinum iodide |
Smudge rosettes |
0.025 |
Potassium mercury iodide |
Sheaves of free needles |
0.1 |
Dihydromorphinone |
|
|
Sodium nitroprusside |
Rods |
0.5 |
Potassium bismuth iodide |
Grain-like crystals (2 days) |
0.05 |
Ethyl morphine |
|
|
Mereuric chloride |
Long blades |
1.0 |
Potassium mercury iodide |
Smudge rosettes |
0.05 |
Potassium cadmium iodide |
Smudge rosettes (2 days) |
0.1 |
Potassium tri-iodide (3) |
Bunches of feathery needles |
0.05 |
Ethyl narceine |
|
|
Potassium iodide |
Rosettes of rods |
0.025 |
Potassium chromate |
Bunches of rods |
0.1 |
Potassium tri-iodide (2) |
Rosettes of rods |
0.05 |
Heroin |
|
|
Platinum chloride |
Rosettes |
0.25 |
Potassium bismuth iodide |
Small dense rosettes (2 days) |
0.005 |
Mercuric chloride |
Fine dendrites |
0.1 |
Potassium chromate |
Smudge rosettes (hedgehogs) |
0.1 |
Sodium carbonate |
Rosettes of plates |
1.0 |
Morphine |
|
|
Potassium cadmium iodide |
Sheaves of fine needles |
0.01 |
Potassium tri-iodide (i) |
Plates |
0.1 |
Mereuric chloride |
Bunches of needles |
0.1 |
Potassium bismuth iodide |
Small rods and plates |
0.01 |
Potassium mercury iodide |
Tufts of thread-like crystals |
0.05 |
Potassium chromate |
Small plates |
0.5 |
Narceine |
|
|
Platinum chloride |
Rosettes of blades |
0.25 |
Mercuric chloride |
Small rosettes |
0.25 |
Lead iodide |
Thread-like crystals |
0.1 |
Potassium chromate |
Tufts of needles |
0.1 |
Potassium tri-iodide (2) |
Rosettes of needles |
0.025 |
Potassium iodide |
Fine needles |
0.025 |
Narcotine |
|
|
Sodium carbonate |
Rosettes and bunches of needles |
0.025 |
Potassium chromate |
Rosettes |
0.1 |
Lead iodide |
Rosettes of fine branching needles |
0.25 |
Neopine |
|
|
Potassium cadmium iodide |
Silvery rosettes - plates |
0.025 |
Potassium mercury iodide |
Oily rosettes |
0.025 |
Potassium tri-iodide (3) |
Small plates or rosettes |
0.1 |
Potassium tri-iodide (1) |
Rosettes of feathery needles |
0.05 |
Mercuric chloride |
Bunches of plates, rods and needles |
0.25 |
Papaverine |
|
|
Platinum chloride |
Yellow plates (2 days) |
0.05 |
Potassium iodide |
Irregular plates |
0.025 |
Potassium mercury iodide |
Plates and squares |
0.05 |
Potassium tri-iodide (2) |
Small rosettes |
0.1 |
Potassium cadmium iodide |
Rosettes of blades |
0.025 |
Mercuric chloride |
Very small needles |
0.25 |
Pseudomorpine |
|
|
Potassium iodide |
Petal-shaped crystals |
0.01 |
Platinum iodide |
Small dark rosettes (2 days) |
0.25 |
Platinum chloride |
Small rods |
0.025 |
Lead iodide |
Squares |
0.025 |
Potassium bismuth iodide |
Rhomboids and rods (2 days) |
0.05 |
Potassium tri-iodide (2) |
Plates |
0.05 |
Mercuric chloride |
Smudge rosettes - rods and plates |
0.1 |
Sodium nitroprusside |
Plates |
0.1 |
Thebaine |
|
|
Sodium carbonate |
Rosettes of small plates |
0.025 |
Platinum chloride |
Dense rosettes |
0.05 |
This modification is not practicable for Marquis' test or Flueckiger's test, which are carded out by treating the residue left by evaporating a microdrop of the test solution with a trace of the original reagent - namely, a mixture of one part of 40% formaldehyde solution with twenty parts of concentrated sulphuric acid for the former and a 0.5% solution of titanium dioxide in concentrated sulphuric acid for the latter.
Carried out in the above manner, these tests have sensitivities ranging from 1 µg to 0.025 µg, although the colour changes observed are not always the same as when larger quantities are employed. The colours obtained for 1 µg quantities of a number of alkaloids together with the absolute sensitivities are given in Table 3. It should be noted that with minimal quantities it is seldom that the whole range of colours is observed.
Examples of the crystals obtained with various reagents
Ammonium molybdate (Froehde) |
Selenious acid (Mecke) |
Sodium tungstate (Reichard) | ||||
---|---|---|---|---|---|---|
Alkaloid |
in µg |
in µg |
in µg | |||
Apomorphine |
Deep green - blue green |
0.1 |
Blue black - green - brown |
0.1 |
Black violet |
0.025 |
Benzylmorphine |
Violet - green |
0.05 |
Green |
0.1 |
Violet |
0.25 |
Codeine |
Blue, slowly fading |
0.1 |
Blue green - yellow green - brown |
0.5 |
Pale violet |
0.5 |
Cotarnine |
Pale violet - green |
0.25 |
Yellow brown |
0.5 |
Yellow - brown |
0.5 |
Dihydrocodeine |
Green - blue |
0.1 |
Green - yellow brown |
0.1 |
______ |
|
Dihydrocodeinone |
Green - blue |
0.1 |
Yellow green |
0.25 |
______ |
|
Dihydrohydroxycodeinone |
Yellow - green - blue |
0.1 |
Orange - olive green |
0.25 |
______ |
|
Dihydromorphine |
Blue violet |
0.025 |
Brown - green |
0.25 |
Violet |
0.25 |
Dihydromorphinone |
Purple - blue - green |
0.05 |
Orange - brown |
0.25 |
Dark brown |
0.25 |
Ethylmorphine |
Green - blue |
0.1 |
Green |
0.5 |
Blue violet |
0.5 |
Ethylnarceine |
Green - blue |
0.05 |
Green - blue |
0.1 |
Orange - green |
0.1 |
Heroin |
Red violet - blue - light green |
0.05 |
Blue green - olive green - brown |
0.5 |
Deep violet |
0.5 |
Morphine |
Violet - blue - light green |
0.05 |
Blue green - grey green |
0.1 |
Violet |
0.25 |
Narceine |
Brown - grey - blue - green |
0.05 |
Bright green - grey - orange |
0.25 |
Orange - green |
0.05 |
Narcotine |
Brown - green - blue - pale green |
0.05 |
Green - orange |
0.25 |
Blue violet |
0.25 |
Neopine |
Blue - green |
0.05 |
Reddish purple - brown |
0.1 |
______ |
|
Papaverine |
Faint green - blue |
0.5 |
Grey - grey green fading |
0.25 |
______ |
|
Pseudomorphine |
Blue - violet - green |
0.1 |
Purple - brown |
0.1 |
Deep violet |
0.1 |
Thebaine |
Greenish brown - red brown |
0.1 |
Green - brown - orange |
0.25 |
Brownish green |
0.25 |
Sucrose (Schneider-Weppen) |
p-dimethylamino benzaldehyde (Wasicky ) |
Formaldehyde (Marquis) |
Titanium dioxide (Flueckiger) | |||||
---|---|---|---|---|---|---|---|---|
Alkaloid |
in µg |
in µg |
in µg |
in µg | ||||
Apomorphine |
Brown |
0.25 |
Red |
0.25 |
Purple - black |
0.05 |
Deep purple |
0.05 |
Benzylmorphine |
Pink |
0.5 |
Orange |
0.25 |
Red - purple |
0.025 |
Purple |
0.05 |
Codeine |
Pink |
0.5 |
Orange |
0.25 |
Violet |
0.05 |
_________ |
|
Cotarnine |
Orange |
0.5 |
_________ |
|
_________ |
|
Yellow - brown |
0.5 |
Dihydrocodeine |
Pink |
0.5 |
Orange |
0.25 |
Purple |
0.1 |
_________ |
|
Dihydrocodeinone |
Pink |
1.0 |
Orange |
0.5 |
Yellow - brown - purple |
0.25 |
_________ |
|
Dihydrohydroxy-codeinone |
Pink |
1.0 |
Faint orange |
0.1 |
Yellow - brown - purple |
0.5 |
_________ |
|
Dihydromorphine |
Pink |
0.25 |
Bright orange |
0.25 |
Red purple |
0.025 |
Deep purple |
0.025 |
Dihydromorphinone |
Pink |
1.0 |
Orange |
0.25 |
Yellow - red - purple |
0.25 |
Deep purple |
0.025 |
Ethylmorphine |
Pink |
1.0 |
Bright orange |
0.1 |
Yellow - purple - black |
0.1 |
_________ |
|
Ethylnarceine |
Yellow |
0.5 |
Yellow |
0.5 |
Brown - green - blue |
0.1 |
Orange - brown |
0.1 |
Heroin |
Pink |
0.5 |
Orange |
0.1 |
Violet |
0.05 |
Deep purple |
0.025 |
Morphine |
Pink |
0.25 |
Orange |
0.1 |
Violet |
0.05 |
Deep purple |
0.025 |
Narceine |
Yellow |
0.25 |
Yellow |
0.25 |
Brown - deep brown - green |
0.05 |
Bright orange - brown |
0.1 |
Narcotine |
Brown |
0.1 |
Yellow |
0.25 |
Bluish violet fading |
0.1 |
Faint brown - purple |
0.5 |
Neopine |
Pink |
0.25 |
Orange |
0.1 |
Blue violet |
0.1 |
Faint purple |
0.5 |
Papaverine |
Brown |
0.1 |
_________ |
|
_________ |
|
Faint purple |
1.0 |
Pseudomorphine |
Green |
1.0 |
_________ |
|
Rose red |
0.1 |
Deep purple |
0.1 |
Thebaine |
Brown |
0.025 |
Brown |
0.25 |
Red - orange |
0.05 |
Green - brown - black |
0.1 |
The "smallest detectable quantity" of any substance depends on a number of factors including the efficiency of the extraction process employed, the number of different tests that must be carried out and the sensitivities of these tests. Leaving the first of these factors out of consideration and assuming the second to have been reduced to a minimum by correct choice of reagents, it is obvious that any technique that increases the sensitivity of the test causes a corresponding decrease in the amount that can be identified. As the results tabled above show at least one crystal test with a sensitivity of 0.05 µg for each alkaloid mentioned, it follows that, under favourable circumstances, one should be able to identify quantities of the order of a microgramme of any of these substances, provided that they are in a reasonable state of purity.
Although most of these alkaloids may be identified without difficulty, it is not easy to differentiate between codeine and neopine. This might be expected from their structures, which differ only in the position of a double bond. We find that potassium mercury iodide is the most satisfactory reagent to distinguish between these substances. In both cases gelatinous rosettes are formed, but whereas with codeine these quickly disintegrate into small plates, in the case of neopine no such change takes place.
The literature dealing with the subject is briefly reviewed.
Results are given of the application to a number of the opium alkaloids of new microtechniques for colour and crystal tests.
We wish to express our thanks to Professor E. C. Amoroso for his help and encouragement and to Mr. R. F. S. Creed and Mr. H. Burgess for taking the photographs. We acknowledge gratefully gifts of alkaloids from Messrs. T. and H. Smith Ltd., and Messrs. J. F. MacFarlan and Co. Ltd. We are also much indebted to Miss Ann Stanley for technical assistance.
Reagents for microcrystalline tests
(Unless otherwise indicated, the quantities shown are dissolved in 100 ml of water)
Gold bromide: 5 g gold chloride + 5 g sodium bromide.
Lead iodide: Dissolve 30 g lead acetate in 100 ml of water, adjust to pH 6 with acetic acid, and saturate with lead iodide.
Mercuric chloride: 5 g.
Platinum chloride: 5 g.
Platinum iodide: 5 g platinum chloride + 25 g sodium iodide.
Potassium bismuth iodide: 5 g bismuth subnitrate + 25 g
potassium iodide in 100 ml of 2% sulphuric acid.
Potassium cadmium iodide: 1 g cadmium iodide + 2 g potassium iodide.
Potassium chromate : 5 g.
Potassium iodide: 5 g.
Potassium mercury iodide: 1.5 g mercuric iodide + 5 g potas-sium iodide.
Potassium tri-iodide (1) : 2 g iodide + 4 g potassium iodide.
Potassium tri-iodide (2) : 0.1 g iodine + 0.2 g potassium iodide.
Potassium tri-iodide (3) : 1 g iodine + 50 g potassium iodide.
Sodium carbonate: 5 g.
Sodium nitroprusside: 1 g.
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