measurement of absorption frequencies of the B-0-B bond which is present in the Boresters. Initial experiments, using the double beam technique, gave sharp absorptions at 1412 cm-1 and 1370 cm-', corresponding to the doublet associated with the B-0-B stretching frequency. For each sample a full spectrum was recorded, and then the portion of spectrum between 1600 cm-1 and 1200 cm-' was repeated four times, each time with a different aliquot of sample in the sample cell. The mean of the four values for absorbance was used. It seems unlikely that the chemical analysis of boron in these mixtures would be feasible without much further research. This view is supported by the notorious difficulty of precise boron determination in many materials. The results show the unreliability of the refractive index method. The infrared spectra measurement is the most suitable of all the presently tried methods. There still remains the need for finding relatively cheap
boron compounds of high boron to hydrogen ratios, and stable with a high boiling point, to mix with various polyphenyls in large quantities, and to develop a simple and reliable method of determining their boron content to an accuracy of better than 1%. This then will provide physicists with a tool to make careful and detailed studies of the neutronic properties of polyphenyls. FARHANG SEFIDVASH
Department of Technical Physics Technical University of Helsinki Otaniemi, Finland RECEIVED for review October 31, 1967. Accepted January 12, 1968. Work performed under the auspices of the Imperial College of Science and Technology (University of London).
Solvent and Temperature Effects in the Determination of Pyrrolizidine Alkaloids with 4-Dimethylaminobenzaldeh yde SIR: Recently Mattocks (1) described the application of the Polonovsky reaction (2) t o the development of a reliable colorimetric method for the determination of microgram quantities of unsaturated pyrrolizidine alkaloids. The preliminary steps to the final reaction with 4-dimethylaminobenzaldehyde (Ehrlich reagent) to form a colored product are conversion of the alkaloid base to the N-oxide with methanolic hydrogen peroxide and dehydrogenation of the formed N-oxide to a pyrrole by means of acetic anhydride. For these steps to be reproducible, all excess peroxide must be removed, alkaloid must not be decomposed in the process, and dehydrogenation with acetic anhydride must be moderated by a suitable solvent. Since isoamyl acetate appeared to meet the criteria of a suitable moderating solvent, namely nonreactivity with acetic anhydride and adequate polarity for solution of N-oxides, while lacking the disadvantages of peroxide formation, it has been investigated as an alternative t o diglyme. Reproducible results were obtained with all the alkaloids tested except lasiocarpine which invariably gave erratic results both within a series and from series to series. By omitting the oxidation step and studying lasiocarpine N-oxide alone, reproducible values were obtained. This finding showed that the moderating solvent was not involved and that the effect observed was specific to the oxidation stage. Therefore, this stage was examined to determine whether use of a temperature high enough to volatilize methanol but low enough t o restrain the vigor of oxidation might yield more consistent results. A temperature of 77 f 1 "C was selected after tests had shown that methanol was completely volatilized from the tube in 20 to 30 min at this level. During this study of the Mattocks procedure, it was observed that solutions prepared from heleurine and supinine formed strong colors in the cold following addition of Ehrlich reagent (1) A. R. Mattocks, ANAL.CHEM., 39, 443 (1967). (2) M. Polonovsky and M. Polonovsky, Bull. SOC.Chim. France, 41, 1190 (1927).
1166
ANALYTICAL CHEMISTRY
Table I. Molar Absorptivity (e) of Reaction Product between Treated Pyrrolizidine Alkaloids and 4-Dimethylaminobenzaldehyde in Two Different Solvents E
x
10-
Alkaloid Isoamyl acetate Diglymeb Heliotrine 73.8 =k 0.96(8). 75.2 Heliotrine N-oxide 80.4 i 1 . 9 (7) ... Lasiocarpined 80.0 =t 1 . 3 ( 5 ) 80.5 Lasiocarpine N-oxided 79.0 i 1 . 8 (6) ... Heliotridine 62.0 55.2 i 1 . 6 ( 5 ) Heliotridine trachelanthate 7 6 . 2 1 1.1 (5) ... 78.0 Supinine 74.2 12 . 6 (7) 63.1 12 . 6 (5) ... Heleurine a Average value with standard deviation. * Data of Mattocks ( I ) . The figure in parentheses is the number of determinations. Temperature at oxidation step, 77 "C.
in contrast to those from other pyrrolizidine alkaloids which required heating before appreciable color developed. Therefore, to determine whether this difference might be used to differentiate between alkaloids, the extent of color development at room temperature, compared with that at the elevated temperature, was studied. EXPERIMENTAL
Apparatus. A Beckman Model B spectrophotometer fitted with Corex cells of 1-cm path length was used for all absorbance measurements. The absorbance maximum was generally in the region of 557-560 mp for all alkaloids examined, tending to 557 mp for supinidine base alkaloids and 559 mp for heliotridine base alkaloids; 559 mp was routinely used. Reagents. All reagents with the exception of isoamyl acetate were prepared essentially by the details given by Mattocks (1).
Table 11. Effect of Temperatures of Oxidation on the Molar Absorptivity and Their N-Oxides and of Supinine E
x
(a)
of Heliotrine and Lasiocarpine
10-3a
Oxidation temperature 77 "C
a
Alkaloid Lasiocarpine Heliotrine Supinine Average value of triplicate
Not oxidized
100 "C
Base
N-oxide
Base
N-oxide
N-oxide
80.3 =t 1 . 5 73.5 =t 1 . 7 75.0 =I= 0.6
80.0 f 1 . 7 80.5 =t 2 . 3
55.0 f 11 72.8 + 2 . 2 74.3 f 1 . 8
69.1 i 1 . 7 79.7 =t1 . 4
77.9 jr 1 . 6 81.1 f 0 . 7
...
...
. ~ .
determinations with standard deviation.
Isoamyl Acetate. The reagent used was a commercial grade of isoamyl acetate. Acetone Diluent. Occasionally when stored samples of acetone were used as diluent, lilac-colored blanks developed in a short period and increased in intensity with time. When the acetone was redistilled, this interference was abolished. Fresh quantities of reagent were therefore tested, and used when no appreciable blank developed after 4 hr; if a n appreciable blank developed, the batch was redistilled. Standard Solutions of Alkaloids. Recrystallized samples of alkaloids were dissolved in reagent grade methanol to the required concentrations. Procedure. Aliquots of methanolic solutions containing microgram amounts of several alkaloids were transferred t o borosilicate glass test tubes and 0.5 ml of 0.12 methanolic hydrogen peroxide was added. After 30 min in a water bath at 100 "C (77 "C for lasiocarpine), the samples were cooled t o room temperature, 1 ml of isoamyl acetate was added to each, and they were mixed on a "Vortimix." Acetic anhydride (0.1 ml) was then added and the contents again mixed; the tubes were closed with loose fitting metal or glass caps and returned to the water bath at 100 "C for 1 min. They were then cooled in a water bath (10-15 "C), the caps were removed, and 1 ml of modified Ehrlich reagent was added. When mixed the tubes were transferred t o a water bath at 57.5 "C for 4.5 min, at the end of which time they were again placed in a water bath (10-15 "C),5.0 ml of acetone was then added, and after mixing the absorbance was determined at 559 mp in 1-cm cells within 1 hr. For the purpose of assessing the rate of color development of the different alkaloids at room temperature (17-18 "C), the same procedure as described above was followed with the difference that, after the addition of the modified Ehrlich reagent, a delay of 4.5 min elapsed before addition of the acetone diluent and a further 10 min before reading the absorbance.
supinine and heliotrine and its N-oxide manifest thermal stability at 100 "C, the same is not true for lasiocarpine and its N-oxide. At a temperature of 100 "C the latter two compounds appeared t o undergo a variable and significant amount of decomposition, but when the oxidation was carried out at 77 "C there was virtually n o degradation. Thermal instability is a known characteristic of pyrrolizidine alkaloids and in the light of the present findings, more moderate conditions of oxidation are called for when lasiocarpine is being assayed. Heliotrine, heliotridine, heliotridine trachelanthate, lasiocarpine, supinine, and heleurine were tested for color development at 17-18"C, and at 57.5 "C. It was found that while the first four named alkaloids after treatment developed color with Ehrlich reagent t o the extent of approximately 27 f 2% of that found at the higher temperature, supinine and heleurine developed 100 and 95% of maximal color, respectively. At 10 "C the color produced with heliotrine was about 5 % of the maximum but again supinine gave 100% color development. This effect was then used t o test the possibility of the determination of supinine in a mixture with heliotrine. The color developed at 10 "C in one of two aliquots of a mixture of supinine with heliotrine was compared with the color developed at 57.5 "C in a similar aliquot. The increment of color thus found was equal t o that of the same quantity of heliotrine determined in the absence of supinine. There is thus a clear difference in behavior toward Ehrlich reagent of supinidine base alkaloids which have hydrogens attached only to the carbon atom in ring position 7 and the heliotridine base alkaloids which possess an O H or a potential O H (hydrolyzed lasiocarpine) in this position (3). This configurational difference appears t o determine t o some extent the energy required for the development of the colored reaction product with Ehrlich's reagent.
RESULTS AND DISCUSSION
ACKNOWLEDGMENT
Table I compares the molar absorptivities obtained for a number of alkaloids when isoamyl acetate was used as moderating solvent with those obtained by Mattocks ( I ) using diglyme. The agreement is sufficiently close t o warrant the use of isoamyl acetate in future work. In addition new information is provided concerning the molar absorptivity of several alkaloids not previously studied and these values are high enough t o suggest that they t o o may be determined using the alternative solvent. Table I1 summarizes the effects of conducting the oxidation stage at two different temperatures on the recovery, as indicated by molar absorptivity, of heliotrine and lasiocarpine and their N-oxides and of supinine. It is evident that, while
The author thanks C. C. J. Culvenor for his interest in this work and for a specimen of heleurine.
J. B. BINGLEY Division of Animal Health Animal Health Research Laboratory C.S.I.R.O. Parkville, Victoria Australia
RECEIVED for review December 27,1967. Accepted February 19, 1968. (3) C. C. J. Culvenor, Australiatz J . Clzem., 3, 287 (1954).
VOL. 40, NO. 7,JUNE 1968
1167
