Identification of Alkaloids and Other Basic Drugs by Paper Partition Chromat ography LEO R. GOLDBAUM and LEO KAZYAK W a l t e r Reed A r m y M e d i c a l Center, Washington 72,
D. C.
A procedure is presented for the presumptive identification of microgram quantities of alkaloids and other basic drugs by means of the pattern of their R / values at four pH’s. The use of sensitive spotting agents makes possible the detection of microgram quantities. Recovery of the compounds from the paper is readily accomplished so that other confirmatory tests can be made. The Rj patterns of 44 commonly encountered basic drugs are reported and schematically arranged so that the identification of any one of these compounds is simplified. This procedure has been effectively used in the identification of microgram quantities of basic drugs in the toxicological examination of pharniaceu tical preparations and biological samples.
Sodium Sulfite Reagent. A,4% sodium sulfite solution in halfsaturated sodium borate solution. METHOD
Four strips of Whatman KO. 2 filter paper are Tyetted with buffers of pH 3.0, 5.0, 6.5, 7.5 (a different pH for each strip) and allowed to dry. These pH values are preferred because they produce the most characteristic differences with the largest number of compounds. An ethyl alcohol solution Qf the basic drug (50 to 100 y) is spotted near one end of the paper while the control compound (50 y of codine) is spotted alongside the unknown,
Table I. R / Values and Codeine Ratios of Basic Drugs and Related Compounds 0 1.01 1.51 2.01
0
S E of the most difficult analytical problems in toxicology is the identification of microgram quantities of basic drugs, which may be present in the isolated estracts of a sample or in a low concentration in a limited amount of sample. Paper partition chromatography offers a simple method for the identification of micro quantities of chemicals by means of sensitive spotting agents and characteristic R, values. Carless and Woodhead ( 1 ) introduced the concept of filter papers buffered at different pH’s in the development of chromatograms of alkaloids. These authors and others ( 2 , 3 ) showed that paper partition chromatography carried out at different pH levels produced marked changes in R / values. A method is presented here for the identification of alkaloids and other basic drugs based on the pattern produced by the Rt values a t four different pH’s. By the schematic arrangement of these Rf values, a tentative identification of an unknown basic drug can be made from chromatograms run simultaneously, each a t a different pH. Thus, an identification is not made from a Bingle value but from a pattern that is highly characteristic of a basic compound. To avoid the problem of reproducibilitj of Rf values, a control compound, codeine, is run on the chromatogram along with the unknown. The movement of codeine is compared with the movement of the basic drug to furniqh codeine ratios n-hich are more reproducible than R, values. In this n a y the effect of changes in temperature, humidity, and solvent preparation are minimized. APPARATUS AND REAGENTS
MacIlvaine’s Buffers. pH 3.0: 790 ml. of 0.1M citric acid diluted to 1.0 liter with 0.2M sodium dibasic phosphate. p H 5.0: 480 ml. of 0.1M citric acid diluted to 1.0 liter with 0.2M sodium dibasic phosphate. Sorenson’s Buffers. p H 6.5: 680 ml. of M / l 5 potassium monobasic phosphate diluted to 1.0 liter with iM/15 sodium dibasic phosphate. pH 7.5: 164 ml. of M/15 potassium monobasic phosphate diluted to 1.0 liter with M/15 sodium dibasic phosphate. Developing Solution. n-Butyl alcohol saturated with buffers. Paper. Whatman No. 2 filter paper, 55- to 60-em. lengths. Chromatocab. Model A, Research Equipment Corp., Oakland, Calif. Mineralight. A source of short-wave ultraviolet light; Model SL 2537, Ultra-violet Products, Inc., Pasadena, Calif. Iodoplatinic Acid Reagent. Mix 45 ml. of 10% potassium iodide, 5 ml. of 5y0 platinum chloride, and 100 ml. of distilled water.
to to to to
Class I Dihydromorphinone (Dilaudid) Morphine Brucine Chloroquine Codeine Homatropine Atropine Class 11, Adenine0 Emetine hlethyldihydromorphinone (Metopon) Strychnine Etliylmor hine (Dionin? Quinacrine Procaine Class 111 Pilocarpine Scopolamine Diacetylmorphine (Heroin) Kicotine n-rillylnormorphine Gelsemine Cocaine Class I V Doxylamine Trimeton Alethapyrilene Pyribenzamine Chlof-Trimeton Quinine Cinchonidine Quinidine Cinchonine Class V Meperidine Tetracaine Thonzylamine Yohimbine
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Key for Codeine Ratio 1.00 = A 2.51 to 3 . 0 0 E 1.50 = B 3 . 0 1 t o 3.50 = F 2.00 = C 3 . 5 1 to 4.00 = G 2.50 = D 4.00 to= H pH3.0 pH 5 . 0 pH 6 . 5 pH7.5 R / Ratio Rf Ratio R / Ratio Rf Ratio
ABC 0.12 0.15 0.15 0.15 0.19 0.25 0.29
0.75 0.76 0.80 0.80 1.00 1.30 1.65
ABC
0.60 0.60 0.85 0.80 1.00 1.40 1.70
0.16 0.26 0.21 0.26 0.34 0.22 0.34
AB
AB 0.55 0.70 0.70 0.70 1.00 0.70 0.94
B
0.80 0.90 0.75 1.10 1.00 0.65 0.75
0.61 0.73 0.57 0.89 0.78 0.50 0.62
AB
1.05 0 . 4 1 0.50 1 . 5 0 0.89 1 . 2 0
0.19 0.21
1.00 0.39 1.10 0 . 2 7
1 . 8 5 0.39 1 . 5 0 0.45
0.24 0.25
1.25 0.22 1.50 0.23
1 . 1 5 0.37 1.40 0 . 3 1
1.05 0 . 8 2 1.00 0 . 7 4
1.00 1.00
0.29 0.31 0.30
1 . 5 5 0.29 1.55 0.35 1.55 0.36
1.45 0.55 1.55 0 . 4 8 1.64 0.53
1.50 0.86 1.20 0 . 9 5 1.50 0.90
1.10
ABCD
0.15 0.22
BCD
0.75 0.22 1.15 0.26
CD
1.05 0.67 1.30 0.58
2.15 1.65
1.15 1.10
B 0.81 1 . 0 5 0.86
1.10
0.31 1.70 0.33 0.19 0.90 0.32
1.75 0.56 1.85 0.86 1.70 0.69 2.20 0.92
1.15 1.15
0 . 3 2 1 . 7 0 0.39 0.41 2.00 0.41 0 . 4 0 2 . 0 0 0.52
1.95 0.73 1.95 0.49 2 . 4 0 0.09
BCD
EFG 0.56 3.00 0.59 3 . 1 5 0.60 3 . 2 0 0 . 6 8 3.40 0.68 3.35 0.74 3.50 0.72 3.45 0.79 3 . 7 0 0.72 3.60 EF EF 0 . 5 0 2 . 6 5 0.61 3.05 0.53 2.90 0 . 5 0 2.80 0 . 5 0 2 . 8 5 0.56 3.00 0 . 5 5 3.35 0.52 3 . 2 0 0.26 0.35 0.35 0.37 0.41 0.47 0.47 0.46 0.50
1.50 2.00 2.00 1.95 2.10 2.25 2.25 2.30 2.50
Class VI Q 3-Hvdroxv-nmkthylmorphinan (Dromoran) 0.60 3 . 6 5 Diphenhydramine 0 . 6 5 3 . 5 0 Methadone 0.71 3.70 dmbodryl 0.67 3.80 0 . 6 6 3.86 Antihistine Class VI1 EH Nicotine 0.45 2.75 Chlorcyclizine 0.71 4.15 Cyolaine 0.79 4.50 Pu-uperqaine 0.83 4.75 Coramine 0 . 8 5 4.40 a Compound frequently isolated
1289
A
ABC 0.12 0.13 0.15 0.16 0.20 0.25 0.37
FQ 0.56 0.63 0.74 0.75 0.71
3.40 3.55 3.55 3.90 3.90
2 . 1 0 0.88 1.10 1 . 6 0 0.87 1.10 1 . 9 3 0.96 1 . 2 0
CD
0.61 0.55 0.65 0.70 0.62 0.72 0.71 0.75 0.68
1.80 1.70 2.00 2.00 1.70 2.30 2.30 2.25 1.95
CD
0 . 7 2 2.05 0.68 2 . 2 5 0.62 1.90 0 . 7 4 2.45
CD
0.54 1.85 0.67 2.20 0.68 2.20 0 . 7 8 2.30 0 . 6 0 1.85 H EF 0.83 5 . 0 0 0 . 9 3 3 . 1 0 0 . 7 7 4.15 0 . 8 7 2.70 0 . 8 3 4.40 0.84 2.55 0.80 L O 0 0 . 8 3 2.80 0 . 9 2 4 . 3 5 0 . 9 0 2.90 from biological samples.
0.90 0.89 0.92 0.96 0.91 0.92 0.92 0.95 0.91 0.90 0.91 0.92 0.90
B
1.15 1.15 1.15 1.20 1.15 1.20 1.20 1.20 1.15
B
1.20 1.20 1.20 1.20
B 0 . 8 2 1.10 0.91 1.20 0 . 9 3 1.20 0 . 9 5 1.20 0 . 7 8 1.05 B 0.94 1.25 0.96 1.25 0 . 9 4 1.20 0.95 1.25 0.91 1.15
1290
ANALYTICAL CHEMISTRY
The end of the filter paper nearest the spotted section is immersed in the buffered butyl alcohol of corresponding pH. The system is allowed to develop, descending, for about 15 hours in a Chromatocab. The papers are removed and the solvent front 1s marked and air dried. To locate the compounds, the papers are first examined with a source of short-wave ultraviolet light for areas of fluorescence or absorption, then sprayed with iodoplatinate reagent to develop characteristic dark spots of the iodoplatinate complex of the basic drugs. The front of the spot is used to measure the movement and the Rj and codeine ratios distance traversed by the compound distance traversed by codeine
(
) are computed for each dr
To recover the compounds, the iodoplatinate complex is cut out of the pa er, placed in a, separatory funnel, and covered with 5 ml. of a 4 8 sodium sulfite solution in half-saturated sodium borate (pH 9.5) to destroy the complex and free the basic compound. When the paper is decolorized, a suitable solvent is added and the separatory funnel is shaken. The solvent containing the basic drug is evaporated to dryness. Chemical teste, characteristic absorption spectra, or other tests can be made to confirm the identity of the compound. RESULTS
Table I lists the Rj values and codeine ratios of 44 pure coinpounds commonly encountered by toxicologists and pharmacologists. These values represent an average of at least three determinations for a particular compound. The R , values and codeine ratios vary approximately 15% and 10%) respectively, a t the lower pH values, and about 5% and 2%, respectively, a t the higher pH values. An attempt was made to decrease the deviations by more careful control of such conditions as buffer saturation of the mobile phase, temperature, humidity, and the like but this was cumbersome, time-consuming, and not always successful. The compounds are grouped into seven classes according to the pattern produced by the codeine ratios at four pH's, so that the identification of any one of these compounds can be more easily made. Consideration must be given to members of the adjoining class in borderline instances. When an unknown is found to have the following Rj values and codeine ratios, it is located as follows. pH 3 . 0 R f Ratio
R/ Ratio
1.90
0.62 3.20
0.31
pH 5 . 0
pH 6 5
Rj
Ratio
0.72 2.10
pH 7 . 5
R,
I n this group, ppibenzamine most closely resembles the Rj values and codeine ratios of the unknown. Methapyrilene and Chlor-Trimeton are possibilities. Because Chlor-Trimeton shows a pattern that retrogresses at pH 6.5, the unknown is less likely to be this compound. The conclusive identification can be made by running another chromatogram m-ith the unknown along with pyribenzamine and methapyrilene or other suspected compounds. DISCUSSION
The above chromatographic procedure offers a relatively simple method for the identification of microgram quantities of an unknown drug even in the presence of other basic compounds. Along with the characteristic pattern of Rj values and codeine ratios, the procedure provides the following additional aids in the identification of an unknox-n. Examination of the paper with ultraviolet light (nave length, 255 mp) shous the presence of fluorescent compounds as ne11 as those compounds that strongly absorb ultraviolet light of this wave length. For example, quinine or quinacrine TI-ill be seen as fluorescent areas n hen present on the paper in concentrations of less than 1 y , n hile compounds like strychnine or adenine appear as dark areas n-hen present in concentrations of about 10 y . Khen the filter paper is sprayed n ith iodoplatinic acid reagent, all the compounds listed in Table I, ewept adenine, coramine, and pilocarpine, appear as blue or black spots when the concentration is approximately 50 y. As little as 10 y of many compounds listed in Table I can be detected with this reagent. Adenine and coramine produce yellow spots at all pH values, pilocarpine produces a black spot at pH 3.0, a brown spot at pH 5.0, and a yelloE spot a t pH 6.5 and 7 5 . The ability to recover the compounds from the paper in a relatively pure state is also a great advantage. Confirmatory tests can be made on the recovered extract to make a conclusive identification of the unknoL7.n drug. When sufficient amounts of the basic compounds listed in Table I are available, as in pharmaceuticals and in many toxicological samples, the use of the above procedure has proved eff ertive. LITERATURE CITED
Ratio
0.95 1.25
From the key in Table I, the codeine ratios are replaced by their letter equivalents: 1.90 = C, 3.20 = F, 210 = D, and 1.25 = B. The pattern CFDB is checked against the classes in Table I. Only Class I V compounds include this particular pattern (Class IV: pH 3.0, BCD; 5.0, EFG; 6.5, CD; 7 . 5 , B ) .
(1) Carless, J. E., Woodhead, H. B., A-ature 168, 203 (1951). (2) Moerloose, P. de, Mededel. Vlaam. Chem. Ver. 15, 13-18 (1953). (3) Munier, R., hlachehoeuf, AI., Cherrier, N.. Bull. soc. chim.bid. 34, 204 (1952). RECEIVED for review February 8, 1956. Accepted M a y 1, 1966. Preliminary report presented a t American Academy of Forensic Sciences, 5th Annual Meeting. Chicago, Ill., February 1953.
Quantitative Paper Chromatography of Methylated Aldose Sugars Improved Colorimetric Method Using Aniline Hydrogen Phthalate W. C. SCHAEFER and J. W. VAN C L E V E Northern Utilization Research Branch, Agricultural Research Service,
A n improved procedure is presented for the colorimetric determination of methyl ethers of D-glucose which have been separated by paper chromatography. Aniline hydrogen phthalate is the chromogenic agent in this procedure, and the optimum range of sugar is 90 to 200 y. Similar color development was observed with methyl ethers of D-mannose, D-galactose, and D-xylose, indicating the general value of this method for the determination of methyl ethers of aldose sugars.
0
U. S.
Department o f Agriculture, Peoria,
111.
F CONSIDER-\BLE importance in structural studies of polysaccharides by the methylation technique is a reliable method for the determination of methylated reducing sugars after their separation from mixtures by means of paper chromatography. Such a method has been developed by modification of a known colorimetric procedure using aniline hydrogen phthalate as chromogenic agent. The color-forming reaction of the simple aldose sugars with this reagent was first demonstrated by Partridge (20), v-ho used
