The purity of commercial diphenylcarbazone

Arictl_vficcc Chirtrictr Ac’tcr. 68 ( 1974) 11 1-I 18 @:I Elscvicr Scientific Publishing Company. Amsterdam

THE

PURITY

OI= COMMERCIAL

G. J. WILLEMS Daptrrtrnent

111 -‘Printed

in The Ncthcrlonds

DIPHENYLCARBAZONE

and C. J. DE RANTER

oj’ Phurtncccy.

Ldmwtory.

(t/’ AtitrtjTicul

Chonistr~~.

Uuir?ersity

of

Lc~ftwtI, 3000

.!cuvct~

(Belgirm)

(Rcccived

29th

May

1973)

In a previous paper’ it has been shown that most commercial diphenylcarbazide (DPCI) samples are approximately equimolar mixtures of DPCI (610!,) when they are prepared by heating urea and phenylsemicarbazide (PSCI) (39%) with phenylhydrazine. Since diphenylcarbazone (DPCO) is prepared by partial oxidation of DPCI with hydrogen peroxide, it would be expected that commercially available DPCO would be a mixture of DPCI. DPCO and also of PSCI. Krumholz and Krumholz nevertheless stated’ that DPCO is an equimolar mixture of DPCI and D.PCO. They also described a method for separating the ,DPCO (K, = lo-“) from the DPCI, which shows no acidic character, by extracting *he latter with ether from an alcoholic solution of sodium hydroxide. After acidification and recrystallization, they obtained DPCO with a melting point of 127O, which is considerably lower than the 156-158” given for the commercial mixture. The purified product did not react with chromium(VI), in contrast to the reaction mechanism proposed by Bose3 : Cr(VI)+3

DPCO-+[Cr(II)(DPCO)]

+2 DPCDO+6

H+.

Da Silva et ala4 achieved separation by thin-layer chromatography on silica gel, by which they also concluded that commercial DPCO is a mixture of DPCI and DPCO. These authors also described a method for separating DPCO from DPCI, based on continuous liquid-liquid extraction. Earlier, Krumholz and Watzek5 reported that DPCO solutions undergo an oxidative decomposition to diphenylcarbadiazone (DPCDO) catalyzed by traces of copper(I1) ions. The possibility of further oxidation of DPCO to DPCDO had previously been noted by HelIe@ and by Bamberger et (il.‘, who defined it as diphenyltetrazolium betaine. Although various papers have been published about this problem, it has not been properly solved. A rigorous examination of the DPCO reagent was considered necessary, because a purification method was required on a preparative scale in order to obtain the pure DPCO reagent that can be used in complex formation studies with chromium. EXPERIMENTAL

The

chromatographic

experiments

and

the melting

point

determinations

G. J. WILLEMS.

112

The U.V. spectra were performed as dcscribcd previously’. Unicam spectrophotometer, and the mass spectra on a MS meter (Manchester, U.K.).

C. J. DE

RANTER

were recorded on an 12 AET mass-spectro-

Mcrtericlls

The following DPCO samples were investigated: (A) BDH. Poole, G.B.; (B) Fluka, Buchs, Switzerland;(C) Matheson. Coleman and Bell. ‘Norwood, U.S.A.; (D) Carlo Erba. Milano, Italy; (E) Schuchardt, Miinchen. W. Germany; (F) U.C.B., W. Germany: (H) Eastman Kodak, Brussels, Belgium; (G) Merck. Darmstadt. N.Y.. U.S.A.; (I) Aldrich. Milwaukee, U.S.A.; (J) DPCO prepared in this laboratory by reaction of hydrogen peroxide in alkaline solution on DPCI from Schuchardt; (K) DPCO prepared in this laboratory by reaction of hydrogen peroxide in alkaline solution on DPCI from B.D.H.. Analar. Pure DPCDO (melting point 160.8”) was prepared ‘from DPCO by the method described by Da Silva et ul. ‘. The following commercial t.1.c. sheets were used for the chromatographic experiments: t.1.c. aluminium sheets Silica Gel (without fluorescence indicator) and t.1.c. aluminium sheets Silica Gel Fzs4, both from Merck, Darmstadt. W. Germany; MN-polygram polyamid- 11, t.l.c.-grade and MN-polygram t.l.c.-grade, both from Macherey-Nagel & Co, Diiren, W. polyamid- I 1. UV,,, Germany. The separations on an analytical scale were achieved on a column SR 25/45 (Pharmucia Fine Chemicals. Uppsala, Sweden): the separations on a preparative scale were made with a column 3500-75 x 90. fitted with a plunger 3500-A-75, an eluent reservoir and a sample valve (Glenco, Houston, Texas, U.S.A.). Solvent system A (ethanol 42,. chloroform 96?:,) was used for thin-layer separations on silica gel sheets, and solvent system B (water+zthanol-acetic acid, 1: 3 : 0.04) for the thin-layer experiments on polyamide sheets. When non-fluorescing sheets were used, the spots were detected by irradiation with light at 366 nm during 1 11. With fluorescing sheets, instant detection was possible by irradiation with U.V. light of 254 nm. The column chromatographic experiments on an analytical scale were performed with a two-fold aqueous dilution of solvent B. The effluents from the column were collected at intervals of 160 s, at a rate of 1 drop per s, and monitored with the spectrophotometer at a wavelength of 234 nm. The preparative column was charged with about 1 g of commercial DPCO sample, and the separations were performed with a three-fold diluted aqueous solution of solvent B, except for the last fraction, which was finally eluted with solvent B as such. RESULTS

AND

DISCUSSION

The t.l.c. results on silica gel sheets confirm the presence of DPCI and DPCO in all the DPCO samples together with a third component, that according to the preceding discussion could be PSCI, DPCDO or a mixture ofthem. Better separations were obtained on polyamide sheets, from which it was concluded that the commer-

PURITY

OF COMMERCIAL

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l.SU(

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1’”

0

0

113

DIPHENYLCARBAZONE

.

d’

O.S’M

2.6’“.

0

0

Q

A

DPCDC

34”

PSC

e

B

C

D

Fig. 1. Thin-luycr chrom:Itoprnms

E

F

of samples A-K

G

1

Ii

on polyaniidc

J

I

K

I I.

cial DPCO reagents are mixtures of up to six compounds, two of which-only visible after irradiation and present as traces--could not be identilied. In addition to the main components, DPCI and DPCO, the presence of PSCI and DPCDO was confirmed; the relative amounts of the components varied very considerably in the different commercial samples (Fig. 1). The trace components, with hRF- IO-20 are not indicated in this Figure and will be not further discussed. The DPCDO spots, when present, fluoresced on irradiation with U.V. light of 366 nm; the fluoresdue to the formation of 2,2’cence, according to Da Silva et al., is probably diphenylene-5-oxytetrazolium betaine”. The sequence of the AR,, values on silica gel sheets (Table I). with slightly acidic surface properties, can easily be understood from the pK, of the different components as the more acidic DPCO (K, = lo-“) will migrate much faster than the other more basic compounds. On the polyamide sheets, the sequence of the hRI: values is reversed, because of the basic properties of the polyamide surface (Table I). Since the identification of the PSCI and the DPCDO spot could not be done at this stage of the analysis, it was necessary to separate the fraction on a preparative TABLE

I

IrR,: VALUES

OBTAINED .----

Of1 silictr gel, cliffed wirh .w~/t:crtr .sy.~lcr~~A .PSCI 10 DPCI I5 DPCO 55

_ __._ _~_---_---

--_~-.-_--01, polytmidc Il. sohwt .~y.vren~B

clfrretl with --

__-DPCO 32 DPCI 50 PSCI 73 DPCDO 85 (llttorcscing)

---

-___-

--

G. J. WILLEMS.

114

C. J. DE RANTER

r

Fraction

Fig. 3. Elution

pattern

of sample

H on ;tn analytical

numbers

column

(Polyamide

6) monitored

at 234 nm.

scale. Liquid column chromatography (1.s.c.) and, as polyamide- 11 powder was not available in column quality, polyamide-6-CC was used for all the column investigations. Preliminary separations, however, on an analytical column showed that the results on 1s.~. were not identical with the t.1.c. data. For the different commercial products, only three elution peaks were obtained on separation by 1.s.c. monitored at 234 nm, even for products for which on t.1.c. sheets four spots were located (Fig. 2). The reasons for these differences between the elution patterns could be the different quality of polyamide powder, used in t.1.c. and I.s.c. separations, as well as the more fundamental differences in experimental conditions between the two techniques. Although DPCDO and PSCI could not be separated by 1.s.c. on polyamide-6 the composition of fraction 1 could be determined by comparison of the mass spectrum of this fraction residue with the mass spectra of the pure PSCI and DPCDO (Figs. 3 and 4). The identification was based on the most characteristic ion peaks for PSCI, with molecular ion peak 151 and fragment peaks 107 and 108. and for DPCDO. with molecular ion peak 238 and fragment peaks 210 and 105. In this way, it could be concluded that the first 1.s.c. fraction of the different DPCO

PURITY

OF COMMERCIAL

DIPHENYLCARBAZONE

7 ‘7

I I

210 ;_---------. ~N=N-C~H~ oc: ‘1 ?.fy+_y!o 705

77

IOB

M0

134

II Fig. 4. Mass spectrum of DPCDO.

230 210

I

I

m ‘;-

reagents, is either pure PSCI or pure DPCDO or a mixture of both. These results confirmed the results of the t.1.c. experiments, as illustrated in Fig. 1. Once the composition of fraction 1 was known, the relative quantitative determination of the elution chromatograms was possible, in as far as fraction 1 was pure. When fraction 1 was a mixture of PSCI and DPCDO, the relative amount of the total mixture against the two other fractions could be estimated, because of the small difference in the molar absorptivities of the two components at 234 nm (&?~r = 11,000; .&$nO= 11,600; Fig. 5). Evaluation was done by determining the peak area of the different fractions of the’elution chromatogram. These peak areas were referred to the PSCI peak and then corrected for their differences in molar absorptivities at 234 nm (Table II). The molar absorptivities of PSCI and DPCI had been formerly determined’;

G. J. WILLEMS.

116

3.0

aso

320

C. J. DE RANTER

I

3.0

300

Wavelength

Fig. 5. The uliraviolct (dotted line). TABLE

spectra (in methanol)

of diphcnylcarbazonc

(solid line) and diphcnylcurbadi~~zonc

II

THE RELATIVE AMOUNTS (IN ‘,‘;,),,OF THE REAGENTS ________~ .__-___-Sotrrw -.-.----I3 D /I E c --.

DPCI DPCO DPCDO PSCI DPCDO+ PSCI

DIFFERENT -

1.5

51 48 0.5

63 36 1

51 48 1

47 53 0.5

1N

DPCO

-_-.----_-

-_ ------__---.

F

-_---.__-----

43 55

COMPONENTS

6X 29 2.5

G

----

70 24

Ii

37 58

6

I

J

K

44 55 I

40 I6

75 25

34

0

-.---

_.

4.5

the value for DPCDO was obtained by U.V. spectrometry of the same sample as that used for the determination of the mass spectrum and the value for DPCO by U.V. spectrometry of a thrice chromatographed sample (Fig. 5). CONCLUSIONS

The DPCO reagents which are commercially available, are not only mixtures of DPCO and DPCI, as described by Krumholz and Krumholz’ in 1937, but also contain PSCI and DPCDO in varying concentrations. This was clearly proved by chromatography and mass spectra data. The quantitative estimations of DPCO and DPCI do not confirm the findings of Da Silva et ~1.. who reported in 1964 that under conditions of mild oxidation the

PURITY

OF COMMERCIAL

117

DIPHENYLCARBAZONE

reaction stops at a 1: 1 intermolecular compound. leaving exactly one half of the DPCI unoxidized, although the possibility of the existence of hydrogen bonds between the two compounds has not been refuted by the present work. The authors thank Prof. H. Vanderhaeghe the Laboratory of Pharmaceutical Chemistry recording and interpreting the mass spectra.

for mass-spectrometer facilities in at Leuven and Dr. G. Janssen for

SUMMARY

Nine different, commercially available, samples of diphenylcarbazone were examined by thin-layer and liquid column chromatography. From all of them, 34 components were isolated by elution chromatography and identified by mass spectrometry. The relative amounts of diphenylcarbazide. diphenylcarbazone, phenylsemicarbazide and diphenylcarbadiazone were evaluated from the peak areas of the elution pattern at 234 nm. The amount of diphenylcarbazone varied very considerably from sample to sample; there was no stoichiometric relationship between the carbazone and carbazide.

Des essais chromatographiques sur couche mince et sur colonne ont CtC mis au point pour controler et analyser le reactif diphenylcarbazone. Neuf tchantillons commerciaux de diffbrentes origines ont CtC examines, contenant tous de trois 5 quatre composants, isoles par chromatographie sur colonne et identifies par spectrometrie de masse. Les quantites relatives de diphenylcarbazide, diphenylcarbazone, phenylsemicarbazide et diphenylcarbadiazone ont et& Cvaluees par determination de la surface des pits d’elution 6 234 nm. Ces determinations permettent de constater que la quantite de diphenylcarbazone varie considerablement d’un reactif a i’autre; on a pu conclure qu’il n’existe pas de relation stoechiometrique entre diphenylcarbazone et diphenylcarbazide, comme d’autres auteurs le signalent. ZUSAMMENFASSUNG

Mittels Diinnschichtund Siiulcnchromatographie wurden neun verschiedene Handelsproben von Diphenylcarbazon untersucht. Diese enthielten drie bis vier Bestandteile, die durch SZulenchromatographie isoliert und massenspektrometrisch identifiziert werden konnten. Die relativen Mengen von Diphenylcarbazid, Diphenylcarbazon, Phenylsemicarbazid und Diphenylcarbadiazon wurden aus den Fliichen des Elutionschromatogramms berechnet. Der Behalt von Diphenylcarbazon variierte stark von Probe zu Probe; ein stiichiometrisches Verhaltnis zwischen dem Carbazon und dem Carbazid bestand nicht. REFERENCES I G. Willcms. R. Lontic and W. Seth-Paul. ,4ntrl. C/rim Acftr. 2 P. Krumholz and E. Krumholz, Mor~ctrsl~.. 70 ( 1937) 43 I.

51 (1970)

544.

118 3 4 5 G 7

G. J. WILLEMS.

M. Bose. n,tct/. Chirtl. /Ic.ftc. 10 ( 1954) 201. J. J. Da Silva. J. C. Calado and M. L. DC Moura. Trrltcrlro. I I (1964) 983. P. Krumholz and H. Wutzck. Morrcttsl~.. 70 (1937) 437. Hcllcr, Lic+ig/s ,lrrrt. Clww.. 363 ( 1891) 274. E. Bamberpcr. R. Podova and E. Ormcrod. Livhiys Arru. Clw~~.. 446 ( 1926) 260. 8 J. J. DC Silva. J. C. Calado and M. L. DC Mourn. kc*. Port. Qtrint.. VI (1964) 22.

C. J. DE RANTER