Ultraviolet Spectrophotometric Study of Eugenol-Isoeugenol System

Jul 27, 2017 - Table I show a considerable difference between results obtained with and ... discrepancy mat" be explained by the difference in the war...
0 downloads 0 Views 342KB Size
ANALYTICAL CHEMISTRY

664 Roelter, Putiiani, and Lash ( 2 ) . The conclusion of these aut,hors with respect t o the neutral potassium iodide method is the same as that reached in the present, paper. However, there is consideral)le difference x i t h respect to the findings when boric acid is used as a buffer: they found no error, \Thereas the figures in Table I show it consideratile difference hetwcen results obtained with and without boric acid. Boelter ( 1 ) has suggested that this discrelmncv ma!. be explained l)?. the difference in the ~ i t ythP ozone was shsorhed by the iodide solution. IR the work of Boelter pi al. ( 2 ) the ozone w t s a h o r h e d directly from the gas phase into the iodide solution in a modified Hemprl hull) without much agitation of the solution. TVheii ozone rcarts with potassium indidc, potassium hydrositlc is a product of t,he reaction, and reaction of ozone with potaasium iodide is fast aiid the on of potassium hydrositle is slow, the liquid surface whew the ozone react,ed may ivcll havcx been a t a higher p H than the bulk of the solut,ioii. On the other hand, in the work summarized in Tahle I of this paper thc, ozonc was swept through a bubbler containiiig the boric acid-buffered iodide solution. The solution was strongly agitated by the buhbling and the pH a t n.hich t h r ozone was absorbed was therefore, more nearly that producwl tiy the boric acid-i.e., ahout 4.6. It is this lo\\ p H n h i c h hriiigs about thc crront:ourl!~ high iwults.

co~cLL~slox In thc concentration range investigated (1 to 25 mole ' C ) the use of a 2ci unhuffereti aqueous solution of potassium iodide for the analytical determination of ozone gives precise reiults which

ai'e accurate to 2 5 \\.hen compared with a physical method

The use of boric acid as a I)uffer is uniicccwar>- and can I P : ~to erronrous I~rsults.

LITER4TZ'RE CITED (1) Jhxltei., E. D., p r i n t e c o r n i i i u i i i ~ ~ : r t i o ~ ~ .

( 2 ) Hoelter, E. D . , Putnani. C;. I... ;and Lash. E . I.. ;lx.ir.. CHEXI.. 22, 1533 (1950). ( X Iirais. P.. mid 1Iarkei.t. H., d i i o e i c . C'herri,, 45, 309 (1932). (4) Ladenburg, -4.. and Quasig. I t . . B r r . . 34, 1183 (1901). (-16) Rieserifeld, E. H.. A I ~ Q W C'heu?., . 45, 304 ;1932). ((ii Riesenfeld, E. H . , a n d Henrker.. F., Z . nriorg. Clieiri.. 98, 16; (1916). 7 ) Riesenfeld. E. H.. a n d Schwab, G., H w . . 55, 2092 (1922). 8 ) Ruyssen, R . . .\-rrticicrci,. Tijdschr., 14, 24-16 1932). (9) Zhid.. 1 5 , 6-13 (1933). (10) Ti.eadwel1, F. P., a n d Ainiielei,, I.:.. X. ntLorg. Chei~i.,48, S6 (1906). (11) IVarburg, E.. . i / f r ( . P l t y a i k . . [I\-)9, 1286 (1902). ( I 2 ) \Vileoxon, Frank. .lmerican ('y;iiinniid Co., "Some Rapid .ipproximate Statistical Procedures," 1949. RP.( I . I V ~ . I I for w v i c w

, J ~ ~ I2I ;.

lrl.i1,

. i c ~ ~ e l ~ .iin~lars t ~ ~ L l 24 1952

.UIt raviolet Spectrophotometric Study of Eugenol-lsoeugenol System An ultraviolet spectrophotometric study of eugenol and isoeugenol was undertaken to ascertain the effect of position of the double bond in the side chain on the ultraviolet absorption spectra and to devise a spectrophotometric method for determining these compounds in synthetic mixtures. Eugenol and isoeugenol have characteristic ultraviolet absorption spectra amenable to the spectrophotometric analysis of a two-component system. The optimum ware lengths fbr absorbancy measure-

E

UGEXOL, l-hydroxy-2-niet~hoxy-4-allylbenzene, aiid isoeugenol, l-hydroxy-2-met~hoxy-4-propenylbenzene, are two

constituents found in many essential oile and eynthetics. The purpose of t,his investigation was to compare the ultraviolet absorption spectra of these isomers and to explore the possibility of a simultaneous ultraviolet spectrophotometric determination of t,hese two constituents in sj-nthetic mixtures. h survey of the literature shoTved that, the spectrophotometric method had not been applied to this problem (S-O), although the ultraviolet absorption spectra of cis- and trans-isoeugenol and eugenol have been determined (9). G E N E R A L E X P E R I n l E N T A L WORK

Apparatus. The absorbancy measurements were made with a Beckman Model DUspectrophotometer and 1.000-cm. quartz cells. Reagents. Eugenol and isoeugenol, USP XI11 grade, Fritzsche Bros., Kere distilled a t reduced pressure until samples gave the

nients and molar absorbancy indexes were determined. The double bond in conjugation with the benzene ring in the case of isoeugenol resulted in the existence of an absorbancy maximum at a lower wave length than for eugenol and a much larger niolar absorbancy index. Simultaneous spectrophotometric determination of eugenol and isoeugenol was possible in solutions in which the eugenol-isoeugenol ratio varied from about 0.5 to 50, to IO-j molar. concentration of eugenol being

required refractive indcr va1ut.a. Ethyl alcohol and water were used as solvents. General Procedure. h definite amount of each liquid was weighed in a weighing bottle and transferred by washing with ethyl alcohol to a 100-ml. volumetric flask and the volume was adjusted to the mark TTith ethyl alcohol. Aliquots of this solution were then t,ransferred to a 1-liter volumetric flask and diluted t o the mark with distilled water. Ahsorhnncy measurements were taken from 220 to 350 nip at 2-nip intervals. Distilled water 11-as used in the reference cell. R E S U L T S A Y D DISCUSSIONS

Ultraviolet Absorption Spectrum of Eugenol. Curve 1 in Figure 1 shows the ultraviolet absorption spectrum of eugenol. The curve is characterizrd h y an absorbancy maximum at 270 nip. Beer's law was found to apply a t wave lengths 254 and 282 mp for concentrations ranging from 0 to 60 p.p.m. of eugenol. The reason for ?electing these n-ave lengths for testing for con-

V O L U M E 24, NO. 4, A P R I L 1 9 5 2

665 of Eugenol and Iso-

eugenol. The spectrophotometric determ i n a t i o n of t w o components by making absorbancy measu r e m e n t s a t tn-0 different xavelengths requires that crrtain conditions be met if niasinium accuracy and precision are t,o be obtained. 1. .\ b s o r l i a n c y measurements should be made a t tivo \cave lengths a t wliich there is only a slight overlapping of absorption spectra. It has been shown (8) that for niasinium accuracy in thr detrrmination of each coniponent the ratio of ahsorhaiic.? indeses at the two wave lengths Figtire 1. Cltraviolet Absorption Spectra of Eugenol Figure 2. Determination of Optimuni should give a maxiand Jsoeugenol Wave Lengths for Eugenol-Isnerrgenol mum and a niininium Analysis 1. 48.6 p.p.m. eugenol 2. 10 p . p . m . i-oeugenol value. Thus, the ir:i t io 3 ( a >u)? ' j 4/ (a.ll)F4 and ( U . ~ ~ ) ~ ~ should ~ / ( ULoth ~ ~ ~ ) niasinpni ~ ~ ~ or niiiiiformity to Beer's Ian- will be cvident 011 examination of Figure 2 . mum values. Vnder such conditions, o ~ i rromporient absorbs 1Iolar a1)sorhancy index values were calculated using the followstrongly at the wave length at which the otlirr component ahing equ:ttion ( 7 ) : sorbs weakly. Figure 2 shoLvs a plot of the ratio of the molar absorbancy indexes of eugenol to that of kJinUgeflol versus wave air = d,/h X c (1) length. A maximuni occurs at 282 nip and a minimum at 254 where a.,< is thcl molar a1)sorI)ancy index, b is the thickness of the nip. Thrse tn-o n-ave lengths \vexre selerted as the optimum \r-ave absorption cell, in centimeters, and c is the molar concentration lengths. of thc desired constituent. The niolar absorbancy indexes for 2 . It is not always a valid uxsumption that because two (.omponents show conformity t o I3eer's Ian- mixtures of the two conieugenol at 254 and 282 m p \yere 480 and 2650, respectively. Ultraviolet Absorption Spectrum of Isoeugenol. Curvc 2 in ponents \vi11 also show conformity. Thus, the ahPorbancies must Figure 1 sho~vsthe ultntviolet aliaorptioii rpectruni of isoeugenol. be additive. In order to test for the additivity of absorbancies The alisorbanr!. ni:tximum occurred a t 256 nip. Conformity to t.he folloiving test vias devised: Beer's la\\- was found at 254 and 282 nip for 0 to 15 p.p.m. of isoMixtures of knon-n concentrations of eugenol and isoeugenol eugmol. The molar absorli:inc.y indexes at 234 and 282 nip were were prepared and the atisorhancies measured :it 284' and 282 nip. 13,200 ntid 4640, i,espectivel\-. These experimental ahsorhancy values n-ere compared with calculated values obtained by using the molar alisorhancy index values Uyeo, lIiIva, and Sakaiiishi (9) found that the ultraviolet abplot of observed aband the caoncentrations of the mirtur sorption spwtra of cjs- and trans-isoeugenol are similar and also sorbanry versus calculated absorban at 254 and 282 1nM gave that trans-iioeugenoi is the iii:iin constituent of crude isorugenol. a straight line, indicating that the atisorharick $7-ereadditive. r2lthough slightlJ- larger molar ahsorl)ancy indexes \vould lie ob.Znumber of additional synthetic mirtures were then prepared. and the absorbancj- \vas measured a t 254 and 282 nip. The followtained for thr purr trans stcwoisonier, the experimental molar ahing equations xere used t o calculate the conc.cntratior1 of eiiprnol soi,l)anry indexes should g i w more relial~leresults in analyzing and isoeugenol. coninierci:il isoeuKcnol-(,ugr.iiol mixtures. Comparison of Ultraviolet Absorption Spectra. A comparison ,) = ai5' X bcs n?' X hci ( 21 of thr ultraviolct atisorption spectra of eugenol and isoeugenol = /$> x bcs n y x hcr sho\vs that having the double bond in conjugation with the ben(3) zrne ring results in L: pronounced effect upon the position of the where absorbancy maximum and the magnitude of t,he absorhancy index A = absorbanc5; of mixture value. The highcr iiiolar absorbancy index in the case of isoa E = molar absorbancy index of eugenol a~ = molar absorbancj- of isoeugenol eugenol indicates that the absorbancy maximum at 254 mp may b = cell length in centimeters (1.000 cm). actually he due t o the displacemelit of the primary (200 mp) band r E = molar concentration of eugenol of bcnzeiic, toward longer wave lengths, a bathochroniic effect. C I = molar concentration of isoeugenol The nature of surh displacements has been studied by Doub and The solution of these equat,ions gives the follon ing expwssions Tandelibelt ( 1 , 2 ) . The most significant observation from an used to calculate the concentration of the two roriiponcnts in a analytical viexpoint is that the absorbancy maxima for eugenol mixture. and isoeugenol occur at differeqt wave lengths and thus indicate the plausibility of a simult,aneous spectrophotometric method. Simultaneous Ultraviolet Spectrophotometric Determination 1 x 3

+ +

ANALYTICAL CHEMISTRY

666 Table I. Sample NO.

1

2

3 4 5

6 7 8

photometric analysis of synthetic eugenol-isoeugenol samples are presented in Tablc I.

Comparison of Known and Calculated Concentrations for Eugenol and Isoeugenol in Synthetic Samples

Eugenol, 3Iole,’Liter Known Calcd Error. 7” -0 8 306 X 10-4 3.02 X lo-‘ -0 3 2.41x 10-4 2 . 4 0 x I O - & -0 3 2 14 X 10-4 2.13 X IO-‘ -1.8 1.73 X 10-4 1.70 X 10-6 1.51 X 1.42 X -6 1 9.14 X 10-8 9.37 X 10-6 +2 0 2.55 X 10-a 2.56 X 10-5 +O j 2 26 X 10-5 2 . 0 3 X 10-5 -9 5

cI

=

a;5+a42sa ap54a:s2

The analytical results

Of

LITERATURE CITED

Isoeugenol, hlole/Liter Known Calcd. Error, % 7.44 X 10-6 8 4 5 X 10-6 4-7 8 1 . 2 6 x io-j 1 . 3 1 x 10-5 +3 i,i5 x 10-5 i 20 x 10-5 +5.; 1.83 X 10-5

2.59 X 4.86 X 5.45 X 3.84 X

- a~s2a~2s4 - a:s4a3s2

10-5 10-5 10-6 10-5

1 . 8 0 X 10-5 2 . 5 9 X 10-5 4 ( 9 X 10-5

5.37 x 10-5 3 87 X 10-6

(5)

the simukaneous ultraviolet sPectr0-

(1) Doub, L., and Vandenbelt J If, J . .4m C h e m Soc., 69, 2714 (1947). (2) Ibzd., 71, 2414 (1949). (3) Guenther, E., “The Essential Oils Vol I, p 291 -1 New York, D. Van Kostrand Co , 1949 00 (4) Guenther, E , and Langenau, E I: A s i ~CHEV -1 3 21, 202 (1949). -l.R t0.8 ( 5 ) Ibzd., 22, 210 (1950). (6) Ibid., 23, 217 (1951). (7) llellon, lf, G., “;inalytical Absorption Spectroscopy,” p. 191. S e w York, John %ley Br Sons. 1950. (8) Ihid., p. 370. (9) Uyeo, S.,Miwsa, T., and Sakaniahi, RI., J . Chem. Soc. J a p a n . 64, 659 (1943). I

RECEIVED f o r review

J U I ~23,

1051. Accepted January 24, 1952.

Colorimetric Determination of Cortisone and Related Keto1 Steroids W. J . 3I-iDER AND R . R . RUCK Chemical Control Division, Lklerck& Co., I n c . , Rahway, .\-.J . U ith the advent of the commercial production of cortisone rapid, accurate, and sensitive methods were needed for the quantitative and qualitative determination of this product in order to control plant production, dosage in pharmaceutical preparations, and body levels of this important ketol steroid. A colorimetric method based on the reducing properties of the ketol moiety in tetrazolium salts was investigated. A method is presented by which as little as 10 micrograms of cortisone or related ketol steroids can be determined and by obvious modifications much smaller quantities detected. This analytical procedure has been of help in production, control, and research on steroids.

0

Tu’ THE basis of the current knowledge of steroids, anti-

rheumatic activity is dependent upon specific chemical structures. The presenceof a ketone a t carbon 20 adjacent tc? the primary hydroxyl group a t carbon 21 and an unsaturated ketone group a t pdsitions 3, 4, and 5 in adrenal steroids is essential to cortical activity ( 3 ) . Antirheumatic activity requires in addition a hydroxyl group or a ketone group at, carbon 11 and a hydroxyl group a t carbon 17. Cortisone acet)at>e(11-dehydro-17-hydroxycortjicosterone-21-acetate) and 17-hydroxycorticosterone are the onh. known steroids that meet these requirements. The ketol group imparts to the steroid molecule reducing properties similar to those of fructose, which contains the same aketol group. Tetrazolium salts yield, upon reduction, deep red water-insoluble pigments knou-n as formazans ( I ) . The tetrazolium chlorides have a reduction poteiitinl of about -0.08 volt (2). Rutenburg et al. (4)have shom-n that a stable blue tiiforniazan is obtained from the reduction of 3,3’-dianisole-bis-4,4’-(3,5dipheny1)tetrazolium chloride. .Ilcoholic solutions of steroids which contain the primary a-keto1 group reduce tetsazolium salts in the presence of tetramethyl ammonium hydroside and form colored solutions. I n the case of 2,3,5-triphenyltetrazoliuni chloride and 3,3-dianisole-bis-4,4’-(3,~-diphenyl)tetrazoliiim chloride the color produced follows Lambert-Beer’s law over a suitable concentration range, as shown in Figure 1. The absorption curves for the formazan obtained from 2,3,5-triphenyltetrazolium chloride (TZ) and the diformazan from the dianisole bisdiphenyltetrazolium chloride (BT) and cortisone aceCate are given in Figure 2. As expected, the molecular absorption of the

diformazan from cortisone acetate and dianisole bisdiphenyltetrazolium chloride is twice that of the forniazan of cortisone acetat’e and 2,3,5-triphenyltetrazolium chloride. The laboratory procedure as reported can be used to determine cortisone acetate in a concentration range of 0.01 to 0.17 mg. per ml. By obvious modifications of the method, much snialler example, the dianisole bisdiamounts could he detectcd-for

0.9 0.8

‘ / . ’

0.7 .

BT

M G. c b RTI s ON E A C E T A T E

Figure 1. Absorbancy