Spectrophotometric Determination of Cinchona AIkaloids HELEN S. GRANT AND J . H. JONES'
Armour Research Foundation of Illinois I n s t i t u t e of Technology, Chicago 16,111. of quinine-type and cinchonine-type alkaloids in such mixtures can be determined by the same procedure. A simple, rapid method for analysis of small samples of cinchona bark based on this fact has been developed. The method may be used to determine the total alkaloid content and the amount of quinine-type alkaloids. The latter value is a reasonably reliable estimate of the quinine content.
The composition of a mixture of quinine (or quinidine) and cinchonine (or cinchonidine) can be accurately calculated from the optical density at 316 and 348 mp of a solution of the alkaloids in 0.1 N hydrochloric acid by the use of simultaneous equations. Mixtures of cinchona alkaloids, such as the alkaloids from cinchona bark, behave spectrophotometrically as a simple two-component system and the amount
1
-A ery stock of cinchona trees is of value in the selection of KNOWLEDGE of the alkaloid content of the barkof nurs-
0.8
seedlings which will produce high-quinine-yielding trees. T o permit an intelligent selection of the planting stock with the least loss, analyses of a considerable number of very small samples of bark are needed. Although a complete analysis of the component alkaloids of the young bark would be preferable, a knowledge of the quinine and total alkaloid content is sufficient to permit intelligent selection of seedlings The work reported here was undertaken with these objectives in mind. Gravimetric methods for the analysis of cinchona bark are reasonably accurate, but are lengthy and tedious, and require large samples of bark; consequently, they are not suitable for the purposes being considered. A review of previous work on the spectrophotometry of rinchona alkaloids suggested that a spectrophotometric method of analysis would meet the objpctives of this work. The ultraviolet absorption spectra of the cinrhona alkaloids have been determined by a number of workers. In dilute acid solution, quinine and quinidine have absorption peaks of about 250, 318, and 348 mp. The peaks for rinchonine and cinchonidine are a t about 235 and 315 mp. Carol ( 1 ) has shown that quinine dissolved in 0.1 A' hydrochloric acid can be determined spectrophotometrically a t 280.5, 318, and 347.5 mp if interfering substances are absent. Recently, Loustalot and Pagan ( 3 ) have shown that the quinine plus quinidine content of cinchona bark can be determined from the absorption of an acidified alcoholic extract of the bark a t 380 mp (using a 30 mp slit width). They determined the total alkaloid content of the bark by a volumetric procedure. Stimson and Renter ( 4 ) have suggested that the quinine content of totaquine may be determined from tile ab-
:OO
310
320
340
330
350
WAVE LENGTH
Figure 1.
- rnp
360
Si0
380
390
Absorption Spectra of Principal Cinchona Alkaloids 1. 2.
Cinchonine and cinchonidine Quinine and quinidine Concn., 30 mg. per liter Solvent, 0.1 N HCI Cells, 1 em.
sorption a t 331 mp in neutral alcohol. By using tho total weight of the alkaloids and the ultraviolet absorption of the mixture, Fuchs and Kampitsch ( 8 ) have determined the quinine and cinchonine contents of mixtures containing these two alkaloids. This paper reports the results of a study of the spectrophotometric analysis of mixtures of cinchona alkaloids.
I Preeent address, Food and D r u g 4dministration, Federal Securlty Agency, Washington 25, D. C.
EXPERIMENTAL
Table I. Analyses of Known XIixtures of Cinchona Alkaloids XIlX-
tiire No.
4 5 6 7 8 9 10 11
12 13
Composition of Mixturea Qd C Cd 1.50 1.50 .. o:i5 0:?5 0.60 0.60 2 25 0 :7 3 0.75 2.25 3.00 0:60 ,. 0 30 3 00 .. 3 00 .. 0 30
Q
3 00
0.15 0.90 0.90 0.60
0 30
0 15 3 00
..
1.50 1.50 3.00
Q:90 0.90 0.60
Q .4lkaloids Added Found 1.50 1.48 0.75 0.74 0.60 0.59 2.25 2.26 0.75 0.75 3.00 3.00 3 .OO 2.98 0.31 0.30 3.00 2.99 0 .13 0.15 1.20 1.18 0.90 0.00 0.60 0.61
C Alkaloids Added Found 1.50 0.75 0.60 0.73 2.25 0.60 0.30 3.00 0.15 3.00 2.40 2.40 3.60
In mg. per 100 ml. of solution taken f o r rprrtrophotometric analysis. chonine. Cd, cinchonidine.
1 53 0 73 0.60 0 74 2.30 0.59 0.26 2 99 0.14 2 97 2 43 2.43 3 60
Q3 quinine.
619
Total Alkaloids Added Found 3.00 1.50 1.20 3.00 3.00 3.60 3.30 3.30 3.15 3 15 3.60 3.30 4.20
Qd. quinidine.
3.01 1 48 1.19 3.00 3 05 3.59 3.25 3.30 3.12 3.12 3.62 3.32 4.21 C. cin-
The s p e c t r o p h o t o m e t r i c data given in this report were o b t a i n e d with a Beckman Model DU spectrophotometer. A slit width of approximately 2 mp was used in all measurements. M a t c h e d 1 - c m . Corex cells were used. The alkaloids used as standards were recr,vstalliaed several times from alcohol or benzene and dried t o constant weight The melting a t 100" C. points of the purified materials agreed with the literature values. The spectral a b s o r p t i o n curves of equal weights of
ANALYTICAL CHEMISTRY
680
, quinidine, cinchonine, and cinchonidine in 0.1 ,I' hyPine rochloric acid, measured against control of 0.1 hydrochloric acid, are shown in Figure 1. T h e curves for quinine and quinia
A'
dine appear t o be identical and are shown as a simple curve. Each compound hss absorption maxima at 318 and 348 mp. The curves for cinchonine and cinchonidine also appear t o be identical and are shown in Figure 1 as a single curve. In the region selected for study, these compounds have a single absorption peak a t 316 mp. The curves in Figure 1 are in good agreement with the results of other workers. Concentration ais. optical density data show that quinine, quinidine, cinchonine, and cinchonidine dissolved in 0.1 N hydrochloric acid obey Beer's law t o within *l.oy0a t the absorption peaks for concentrations hetween 10 and 60 mg. per liter. Each of the alkaloids may be determined spectrophotometrically, therefore, if interfering substances are absent. Examination of Figure 1 shows that in 0.1 N hydrochloric aeid the optical density per unit weight of quinine (and quinidine) is about one half that of cinchonine (and cinchonidine) a t 316 mp but is nearly seven times as great a t 348 mp. Because this is true, i t should be possible to calculate accurately the composition of a mixture of quinine (or quinidine) and cinchonine (or cinchonidine) dissolved in 0.1 A' hydrochloric acid from the optical density of the solution a t 316 and 348 mp by the use of simultaneous equations. A mixture of all four of the alkaloids should behave as a simple two-component system and the spectrophotometric analysis should give the quinine plus quinidine and the cinchonine plus cinchonidine content of the mixture. Obviously, the data cannot be used to resolve mixtures of quinine and quinidine, or cinchonine and cinchonidine. Results of the spectrophotometric analysis of mixtures of known amounts of the four principal cinchona alkaloids are shown in Table I. (In these analyses the amounts of quinine-type and cinchonine-type alkaloids were calculated from the densities of the standards and the mixtures a t 316 and 348 mp by the method of simultaneous equations.) The average error in the determination of the total alkaloids is Tess than 1%and the largest error is 2y0. In most cases equally accurate results are obtained for the amount of each type of alkaloid. The mixtures shown in Table I cont,ain only the four principal cinchona alkaloids. The alkaloids from cinchona bark contain a considerable proportion (10 t o 50%) of the "so-called" amorphous alkaloids. Because most of the alkaloids in the bark are closely related chemically and the available data indicate t h a t the absorption spectra of the closely related cinchona alkaloids are very similar, it appears probable that the amorphous alkaloids have absorption spectra very similar to t h a t of either quinine or cinchonine. If this is true, the presence of the amorphous alkaloids should not introduce a n appreciable error in the spectrophotometric determination of total alkaloids. The results of the analysis of several samples of alkaloids t h a t contained amorphous alkaloids are shown in Table 11. It is apparent that the spectrophotometric method gives a good estimate of the total alkaloid content of such mixtures. It also appears reasonable to assume that the results for quinine-type and cinchonine-type alkaloids are valid indications of the composition of the mixture. The spectrophotometric method was applied t o the determination of the total alkaloids in cinchona bark as follows: A small sample of the powdered bark, usually 1 gram, was mixed with a few milliliters of 10% sodium h droxide solution in a 200-ml. flask, 100 ml. of benzene were ad&d, and the flask and contents were weighed. The flask was connected t o a reflux condenser and the benzene was boiled gently until the alkaloids were completely extracted (3 t o 6 hours). After cooling t o room temperature, any benzene lost was replaced. A 50ml. aliquot of the benzene was transferred t o a separatory funnel and the alkaloids were extracted with several small portions of 0.1 N hydrochloric acid. The combined acid extracts were boiled 2 to 3 minutes to expel any suspended benzene, cooled, and diluted t o exactly 1000 ml. The optical density of the acid solution of the alkaloids was determined at 316 and 348 mp. From these dat,a and density of
the standard solutions of quinine and cinchonine (30 mg. per liter in 0.1 ,V hydrochloric acid), the alkaloid content of the solution was calculated from the simultaneous equations,
Dais
=
XD:"
4- YD:"
Dad,
=
XD:'"
+ YD:4E
whLre X and Y are the amounts of quinine-type and cinchoninetype alkaloids; 0 3 1 6 and & are the optical densities of the sample solution at 316 and 348 mp; and D;le, D:48, D:'", and Dg4s are the optical densities per unit weight of the two standards a t the respective wave lengths.
T a b l e 11.
A n a l j s e s of Clixtures C o n t a i n i n g A m o r p h o u s 4lkaloids
Analysis Q C Total alkaloids alkaloids Alkaloids Oram Gram Gram Totaqiiine alkaloids 0.039 0.300 0.255 0.291 Totaqiiine alkaloids 0.300 0.040 0.253 0.293 Bark ( 1 ) alkaloids 0.300 0.096 0.207 0.303 Bark (2) alkaloids 0.300 0.099 0.206 0.305 0.600 Amorphous alkaloidsa 0.193 0.398 0.591 Bark alkaloids after removal of cinchonine, quinine, a n d cinchonidine hy U.S.P. XI1 method. Sample
Sample Weight Gram
Typical results obtained by this procedure are shown in Table
111, together with the results of gravimetric analysis of the same samples. In most cases the results by the spectrophotometric and gravimetric methods do not differ by more than 2% of the alkaloid content of the sample. The average deviation between replicates for the bark samples shown in Table I11 was approximately 2y0. DISCUSSION
The proposed spectrophotometric method separates the cinchona alkaloids into two groups-the quinine type and the cinchonine type. The amount of quinine-tj-pe alkaloids gives directly the maximum amount of quinine that could be present. Loustalot and Pagan (3) have shown that the absorption in dilute acid at 380 mp gives an accurate estimate of the quinine plus quinidine content of many cinchona barks. The quininetype alkaloids shown by the proposed method should give an equally accurate estimate of the quinine plus quinidine content. The quinine content of several samples analyzed in this work o the quinine-type alkaloids shown has varied from 65 to 1 0 0 ~of by spectrophotometric analysis. Even if the spectrophotometric method does not give a very accurate estimate of the quinine content in all cases, i t should still be valuable as a rapid preliminary test. The extraction procedure described has proved satisfactory. It would appear, however, t h a t spectrophotometric determination could be used with other extraction procedures t h a t quantitatively extract the alkaloids and eliminate interfering substances. The presence of excessive coloring matter is recognized as a possible source of error, although no interference from this source could be detected in samples examined in the course of this work. Benzene extracts much less of the coloring matter fron cinchona bark than does alcohol. Although relatively few bark samples were analyzed, those examined were said to represent young, mature, and very old bark from several commercial species of cinchona. The U.S.P. X I 1 method for the analysis of totaquine consists of the following steps: Cinchonine is precipitated as the alkaloid and weighed. Quinine and cinchonidine are precipitated together as thc tartrates and the amount of each alkaloid is calculated from the
V O L U M E 2 2 , NO. 5, M A Y 1 9 5 0 weight of the precipitate and the optical rotation of a solution of the precipitate. Quinidine is precipitated as the iodide and the quinidine content is calculated frorn the w i g h t of the precipitate. Each of the above precipitates can be analyzed conveniently for its alkaloid content spectrophotometrir,ally. The precipitates are dissolved in a measured volume of 0.1 ,V hydrochloric arid and the optical density of the solution a t the appropriate wave length (or wave lengths) is determined. The spectrophotometric determination is particularly advantageous in the analysis of the tartrate precipitate, inasmuch as the accuracy of the results obtained hy the U.S.P., method, when applied to natural mixtures, is questionable. Tests of several tartrate precipitates indicate that the spectrophotometric method gives results that agree with those calculated from the niethosJ.1 and nitrogen content of the precipitate. These data together with the results on known mixtures indicate t h a t the spectrophotometric method gives accurate Irsults for the quinine :ind cinc*honidinccontent of the tartratr prwipitatc. SUMWARY
The composition of a mixture of quinine (or quinidine) and cirrrhonine (or cinchonidine) can be determined accurately from the optical density a t 316 and 348 mp of a solution of the mixture in 0.1 S hydrochloric acid. A mixture of all four alkaloids behaves spectrophotometrically as a simple two-component mixture :inti the quinine plus quinidine and the cinchonine plus cinchonidine content of the mixture can be determined in the same way. The spectrophotometric method is also applicable to the analysis of mixtures containing the so-called amorphous cinchona :ilkaloids. A simple, rapid method for the determination of the total alkaloids in cinchona bark, based on this fact, may be used to determine the total alkaloid content and the amount of quinine-type alkaloids in the bark. The.latter value should be a reasonably reliable estimate of the quinine content. IThch of the precipitates obtained in the U.S.P. XI1 method of
681 Table 111.
Analyses o f Cinchona Bark and
Saiiil,lca
_Spertropiioto,iietrii. ______ _ Cinriionine Qiiininc Total alkaloids alkaloid+ alkalmris %
Totaqiiine Bark 1 Bark 2 Bark 3 Bark 4a Bark 4b Bark 4c Bark 4 d Bark 4e Hark 4 (av.)
02.1 6.5 6 ,I ;7 2 2 4 2.0 2.7 2.2 2 2
?c
10.8 2 9 2 !I 3.0 1 6 2.8 2.0 2.1 3.2
..
% 72.9 9.5 9 ,'3 8 2 3.9 4.7 4.7 4.3 5.4 4.6
All rewlts are the averaxe of two or inore detrrininations
4 7 cxi.ci11
bark 4 . Barks 4a to 4e are Fajnples frorn individual y o u n g tree.. iiirtrir valiie for bark 4 \('ab obtained o n a coiniiositr satn1iIe of c(IiiaI of harks 4 s t o l e .
LITER.4TURE CITEI) (1) Carol, .J.. J . Assoc. O f l c . ..lor. Cho?iists, 26, 238 (1943).
( 2 ) Fuchs. V. L., and Kanrpitsch, A.. Scientin Phnrrn.. 6, 135 (1935). (3) Loustalot, -4. J.. a n d Pagan. C . , J . Assoc. Ofic.A g r . Chemists. 30, 154 (1947). (4) Stinison, XI. 11.. a n d Reriter. .\I. A . , .J. A m Cheni.Soc.. 6 8 , 1192 ( 1Y4(i). R E C E I I . E October D 27, 1!44Y, Prriented a t the All-Day Techiucal Co11fer. cnce, Chicago Sertinn. A \ I E R I C .
