98 Artalyst, February, 1973, Vol. 98, pp. 98-102 Ionic Polymerisation as a Means of End-point Indication in Non-aqueous Thermometric Titrimetry Part III.* The Determination of Alkaloids and Alkaloidal Salts BY E. J. GREENHOW AND L. E. SPENCER (Defiartment of Chemistry, Chelsea College, University of London, Manresa Road, London, S. W.3) Strychnine, nicotine, atropine, quinine, papaverine, caffeine and theo- phylline have been determined in amounts down to 0.0001 mequiv, e.g., 33 pg of strychnine and 8.5 pg of nicotine, by catalytic thermometric titration. The hydrochlorides of quinine and ephedrine, ephedrine sulphate, codeine phosphate and atropine methonitrate have been determined by direct titra- tion by using the same technique. Addition of mercury(I1) acetate was not necessary in the titration of the hydrochlorides.Titrations were carried out in non-aqueous solution with 0.1, 0.01 and 0.001 M perchloric acid, with the ionic polymerisation of a-methylstyrene to indicate the end-point. Depending on sample size and the procedure adopted, each determination can be carried out in 2 to 6 minutes by using a manual method, with a thermometer for temperature measurement, or a simple automatic apparatus. The method would appear to be suitable for the determination of alkaloids and related basic compounds that have been extracted from crude drugs, formulations or natural materials with non-basic organic solvents. For most of the determinations it is not necessary to dry wet chloroform extracts before titration. IN Part I1 methods are described for the thermometric titration of organic bases by using ionic polymerisation to indicate the end-point.The organic bases examined were mainly simple derivatives of pyridine and aniline, although some polyfunctional heterocyclic com- pounds, such as benzimidazole, were titrated. It was shown that bases could be determined in amounts down to about 10 pg, depending on the equivalent mass of the base. Apart from the use of this technique in pollution studies, there is no widespread industrial analytical requirement for determinations of simple organic bases a t the 10-pg level. However, in the examination of many natural products and pharmaceutical preparations the determination of small amounts of organic bases is of considerable importance. The determination of alkaloids at the milligram level is a routine procedure in pharmaceutical analysis and forensic investigations.A standard procedure for the assay of alkaloids in crude drugs, galenicals, injections and tablets, which is prescribed in official publications of pharmaceutical organisations, involves solvent extraction of the alkaloid followed by its determination by a gravimetric, titrimetric or spectrophotometric method.2 Gravimetric methods are usually tedious and not necessarily accurate, while spectrophotometric methods, although more sensitive than titrimetric methods, require more careful calibration and more elaborate instrumentation than the latter. In some methods of assay use is made of direct non-aqueous titration with perchloric acid for the determination of the extracted alkaloid, although it is more usual to add an excess of standard acid to the extract and back-titrate in aqueous solution.Alkaloid hydro- chlorides dissolved in organic solvents can be titrated directly with perchloric acid but it is necessary to add mercury(I1) acetate to the sample to complex the chloride ions, which otherwise interfere with the titration. Recommended titrant concentrations are 0.1, 0.05 and 0.025 M for the perchloric acid and 0.1 and 0.02 M for the sodium hydroxide solution used in the back-titration, thus from 0.02 to 0.1 mequiv of sample can be treated with 1 ml of titrant, e.g., 33.4 to 6.7 mg of strychnine. Vajgand and co-workers3-6 have shown that tertiary amines, including caffeine, cinchonine and brucine, can be determined in amounts down to about 2 mg by thermometric titration by using the perchloric acid catalysed reaction between water and acetic acid to indicate the end-point , * For Parts I and I1 of this series, see pp.81 and 90, respectively. 0 SAC and the authors.GREENHOW AND SPENCER 99 In this paper the procedures described in Part I1 have been applied to the determination of some representative alkaloids, purine bases and alkaloidal salts. The aim has been to determine the lower limit of sample size at which reproducible results can be obtained, and to study the application of the method to alkaloidal salts and to extracts obtained by standard separation procedures that are used in assays for alkaloid content. EXPERIMENTAL REAGENTS- Toluene and 1,4-dioxan were AnalaR grade, and lJ2-dichloroethane and nitroethane were laboratory-reagent grade materials.All four solvents were dried over molecular sieve 4A before use. Chloroform (AnalaR grade) was extracted with distilled water so as to remove the ethanol and was used in the wet state or, when required, after drying over molecular sieve 4A. Other solvents, a-methylstyrene, alkaloids, purine bases and alkaloidal salts were labdratory-reagent grade materials and were used as received. Perchloric acid, 0.1 M solution in acetic acid-Prepare this solution and standardise it by the method described in Part 1.l Prepare 0.01 and 0.001 M solutions by diluting the 0.1 M titrant with lJ2-dichloroethane. APPARATUS- A. Manual method-Use a -5 to +50 “C thermometer, graduated in 0.1 “C, the “closed” flask (5 or 15 ml) described in Part II,s a 5-ml burette, graduated in 0.01 ml with the tip drawn out to a capillary about 1 inch long, and a magnetic stirrer.C.* Automatic method-The motor-driven micrometer syringe , 5-ml titration vessel and thermistor are shown diagrammatically in Fig. 1. The motor, a 12 r.p.m. Smith’s Clocks motor, drives at two speeds, 24 and 2.4 r.p.m., through a gear box, and the syringe mounting [Burkard Scientific (Sales) Ltd.] can accommodate 10, 5, 2 and l-ml glass syringes, which gives the choice of eight rates of titrant addition. To bridge circuit and 50-mV recorder \ D B A C Fig. 1. Apparatus for thermometric titrimetry : A, synchronous motor (12 r,p.m.); B, gear box; C, gear change (24 r.p.m.; disengaged; 2-4 r.p.m.); D, micrometer syringe; E, thermistor, 2 kfi; F, insulation; and G, titration flask PROCEDURE A.MANUAL METHOD- Prepare a solution of the base in a suitable solvent, e.g., acetic acid (dry), 1,4-dioxan nitroethane, lJ2-dichloroethane or chloroform. Suitable concentrations are 100, 10 and 1 mequiv 1-1 with 0.1, 0.01 and 0.001 M titrants, respectively. Add 0.1 to 1.0 ml of sample solution to 1 ml of a-methylstyrene in the 5-ml titration flask with a grade A l-ml pipette or an Agla micrometer syringe, add titrant at the rate of 0-4mlmin-1 to within 0.3 ml of the end-point, noting the temperature at 16-s intervals, and complete the titration with addition of titrant at a rate not exceeding 0.2 mlmin-l. Titrate larger sample volumes (up to 3 ml) in the 15-ml flask and use 2 ml of a-methylstyrene.* A semi-automatic method, B, is described in Part I, but was not used in the present investigations.100 GREENHOW AND SPENCER : IONIC POLYMERISATION FOR END-POINT [ArtdySt, VOl. 98 C. AUTOMATIC METHOD- Use the same volumes of sample solution and a-methylstyrene as in the manual procedure but add titrant at a constant rate (not greater than 0.2 ml min-l) and record the temperature on a 50-mV recorder at a chart speed of 600 mm h-1. End-points are measured as in Part I at the “upturn” temperature [method (i)] or at the intersection point of the tangents to the two component parts of the titration curve [method (ii)]. Use of method (i) is preferred when the upturn temperature can be located unambiguously as it gives a titration value corresponding closely to that obtained by potentiometric titration.RESULTS AND DISCUSSION The results obtained from a series of titrations in which standard solutions of the alkaloids and purine bases were titrated with 0.1, 0.01 and 0.001 M perchloric acid, the amount of sample taken being chosen so as to give titres in the range 0.1 to 1.0m1, are summarised in Table I. In all instances it was found that the sample size and titre were linearly related, except in the region close to zero sample size when the 0.001 M titrant was used. In the 0.1 to 1.0-ml range it is possible to determine 0-01 to 0.1 mequiv of alkaloid with 0.1 M titrant, 0.0001 to 0.001 mequiv of alkaloid with 0.001 M titrant and intermediate amounts with titrants of intermediate molarity.Calibration graphs for nicotine, which are similar to those for the other alkaloids, are shown in Fig. 2. TABLE I THERMOMETRIC TITRATION OF ALKALOIDS WITH PERCHLORIC ACID : RANGES OF SAMPLE SIZE SHOWING LINEAR CORRELATION WITH TITRANT VOLUME Samples sizes in milligrams Perchloric acid titrant Alkaloid 0:1 M 0.01 M 0.00 i M Atropine .. . . 29.0-2.9 2’9-0.29 ’ 0.29-0.029 Nicotine . . .. . . 8-6-0.86 0.86-0.0 85 0.085-0.0085 Papaverine . . 6 . 17.0-3.4 3.4-0.34 0.34-0-034 Quinine . . .. . . 384-1.9 1-9-0.19 0*19-0*0 19 Strychnine . . . . 33-3-3.3 3.3-0.33 0.3 3-0.0 3 3 Caffeine . . .. . . 19-4-1.9 1.9-0.19 0.19-0.019 Theophylline . . . . 19.7-2.0 2-0-0.2 0-2-0.02 The manual method (A) was used for the 0.1 M titrations and the automatic method (C) for the 0.01 and 0.001 M titrations.The precision of the thermometric titration method was assessed in Table I, Part I,1 and shown to be of the order of 1.5 per cent., and is marginally better with semi-automatic and automatic methods than when the manual procedure was used. Results obtained with the alkaloids indicate the same order of precision (Table 11). The lower limit of 0.1 ml of titrant was chosen as being a reasonably reproducible titrant volume when the manual procedure is used. Typical titration curves obtained with this procedure are shown in Fig. 2, Part I; titration curves obtained with the automatic method are shown in Fig. 3 in this paper. With the automatic method, total titrant volumes of 0.01 ml can be measured, with a slow rate of titrant addition to give a measurable chart length, but the precision is lower than that which can be obtained with volumes in the 0.1 to 1.0-ml range.With the exception of atropine, the alkaloids gave perchlorates that were insoluble in the titration mixture, and with strychnine and papaverine the precipitates were voluminous. With papaverine the precipitate gave rise to high titration values at the 0.1 mequiv level, owing to occlusion of titrant in the precipitate, and it is recommended that with 0.1 M titrant, the determination of papaverine should be restricted to the 0.05 to 0.01 mequiv range. No special precautions are necessary with 0.1 and 0.01 M titrants but 0-001 M perchloric acid is sensitive to moisture present in the atmosphere; it should be freshly prepared before use and the titration should be carried out in a dry atmosphere.February, 19731 INDICATION IN NON-AQUEOUS THERMOMETRIC TITRIMETRY.PART 111 101 a b c 1 .o - 0.5 - I 110.0 0.1 F .; \ C .- .I- 0.05 Perchloric acid/ml (1 division = 1 ml) Fig. 2. Calibration graphs for the thermometric titration of nicotine, Titvant- a b C Molarity . . .. . . 0.1 0.01 0.001 Rate of additionlml min-’ 0.2 0-2 0.2 Method . . .. . . A C C Titvation- End-point . . .. . . (2i) (f’ (22) a-hlethylstyrene/ml . . 1 Dry acetic acid is a convenient solvent for most of the alkaloids, alkaloid hydrochlorides and purine bases; quinine was soluble with difficulty in this solvent but could be dissolved in dioxan. Nicotine was titrated in solution in 1,2-dichloroethane. With the exception of nicotine and quinine, which were titrated as diacidic bases, all the compounds examined reacted with perchloric acid stoicheiometrically in a 1 : 1 ratio.The analysis of alkaloidal extracts was simulated by dissolving the alkaloid in chloroform that was saturated with water. With the 0.1 and 0.01 M titrants the solution could be titrated satisfactorily without drying it, but it is essential that water droplets are removed as these completely inhibit the’ cationic polymerisation. With 0.001 M titrants it is advisable to dry all sample solutions. It was found that solutions of the quinine and ephedrine hydrochlorides in dry acetic acid could be titrated directly with perchloric acid without adding mercury(I1) acetate so as TABLE I1 RESULTS FOR PRECISION FROM THE THERMOMETRIC TITRATION OF ALKALOIDS WITH 0.1 TO 0.001 M SOLUTIONS OF PERCHLORIC ACID Titration? * Amount Titrant End- Alkaloid taken/mg molarity* Method point Theophylline .. 9-8 0.1 B (4 Nicotine . . . . 0.85 0.0 1 B (4 Nicotine . . . . 0.085 0.001 B (i) Strychnine . . 0.33 0.001 B (4 hydrochloride . . 18.0 0.1 A (i) Nicotine . . . . 0.086 0.001 B (ii) Quinine Mean titre/ nS ml 3 0.52 4 1.12 3 1.10 3 1-27 3 1.06 3 0.90 Standard deviation 0.010 0.008 0-0 17 0.008 0.006 0.01 1 Coefficient of variation, per cent. r S i n g l e n points value 1.93 1.12 0.71 0.36 1-57 0.91 0.64 0.37 0.54 0.31 1-21 0.70 * Nominal. t See Experimental. Number of titrations.102 GREENHOW AND SPENCER to complex chloride ions. Hydrogen chloride is not a catalyst for cationic polymerisation and does not, therefore, affect end-point sharpness.When mercury(I1) acetate was added to the titration solution a very high titre was obtained with perchloric acid and its value depended on the amount of mercury(I1) acetate added, suggesting that reaction occurs between the titrant and the mercury(I1) acetate either directly or, more probably, by way of an or-methylstyrene - mercury(I1) acetate adduct. I I I Perchloric acid/ml (1 division = 1 rnl) I Fig. 3. Thermometric titration curves obtained by the automatic method. Titvant- a b C Molarity . . .. .. 0.1 0.01 0.001 Rate of additionlml min-l 0.2 0.2 0.2 or-Methylstyrene/ml . . .. 2 2 1 Compound/mg . . . . Atropine, 29.0 Theophylline, 0.98 Nicotine, 0.086 Arrow indicates theoretical end-point (allowing for blank titration) The titration of other alkaloidal salts was investigated with 0.1 M perchloric acid.Ephedrine sulphate in dry acetic acid solution gave slightly low, although reproducible, titra- tion values, and it is possible that sulphuric acid liberated in the titration has a slow catalytic action on the a-methylstyrene. Very low titration values were obtained with atropine methonitrate dissolved in dry acetic acid, suggesting that the latter in some way causes de- composition of the alkaloid. A solution of the methonitrate in a mixture of acetonitrile, dioxan and chloroform (5 + 1 + 1) gave a satisfactory titration. Codeine phosphate is not easily soluble in the usual organic solvents but can be dissolved in a mixture of phenol and chloro- form (1 + l).’ However, when such a solution was titrated there was a large “background” rise in temperature, which made the end-point indistinct and, consequently, the precision poor.A dilute solution of codeine phosphate in dry acetic acid (0.01 mequiv ml-1) gave a satisfactory titration curve and reproducible results. It can be concluded that the thermometric method involving cationic polymerisation to indicate the end-point is suitable for the determination of alkaloids and alkaloid hydro- chlorides at a lower level than has previously been practicable by titrimetric methods. With the automatic apparatus about 10 pg of alkaloid can be determined with 0.001 M titrant. With this titrant, end-point sharpness is such as to suggest that even weaker titrants should be effective and, by taking extreme care to avoid contamination from the atmosphere and using well insulated automatic equipment, determinations at the sub-microgram level might well be possible. Messrs. G. P. Davis and S. F. George are thanked for construction of apparatus. REFERENCES 1. 2. 3. 4. 5. 6. 7. Greenhow, E. J . , and Spencer, L. E., Analyst, 1973, 98, 81. Beckett, A. H., and Stenlake, J. B., “Practical Pharmaceutical Chemistry,” University of London, Vajgand, V. J., and GaLl, F. F., Talanta. 1967, 14, 346. Vajgand, V. J., Kiss, T. A., Ga61, F. F., and Zsigrai, I. J., Ibid., 1968, 15, 699. Vajgand, V. J . , Ga&l, F. F., and Brunn, S. S., Ibid., 1970, 17, 416. Greenhow, E. J . , and Spencer, L. E., Analyst, 1973, 98, 90. Pernarowski, M., Chatten, L., and Levi, L., J. Amer. Pharm. Ass., Sci. Edn, 1964, 43, 746. NOTE-References 1 and 6 are to Parts I and I1 of this series, respectively. The Athlone Press, London, 1962. Received July 13th, 1972 Accepted September 26th, 1972