456
ANALYTICAL CHEMISTRY
amounts of each substance were taken, toget,her with a known amount of cerium(III), for analysis according to the above procedure with ammonium persulfate (20 mg.) as reagent. \Yith many metals it n'm evident from the absorption curves that the int,erference could he greatly reduced, a t comparatively slight cost in sensitivity of cerium determinat.ion, by ivorking at 340 rather than 320 nip. The results are given in Table IY :inti Figures 6 and 7. D a h for potassium nitrate are also incluc!cvi.
-
,
!
I
260
240
Figure 7 .
,
240
Figure 6.
280
\
320 340 WAVELENGTH ( m p )
300
360
380
400
,Absorption Spectra o f Varioiis Compounds iii Ultraviolet
\ V ~ Rhigher than expected on the b:tsis of its extinction coefficient, wit,h manganese, on the othrr hand, less interference was foiinii than expcrted.
,
,
260
280
--- --___ ..., 300 320
-___
\..
-*
340
WAVELENGTH ( m p l
,~j
___----- --.-&\
360
aeo
400
Absorption Spectra of Various Compounds i r i Ultraviolet
(1)
L I T E H T I I H E ClTED
Ireedman, A. J.. anti Hump, D. N.. AXIL. CHEJI.. 22,
932
(1950).
(2) Kasline. c. T., and >Ie1loii, 11. (;., Isn. EN;. CHEA!... $ X A I Eo.. 8, 4(i3 (l9.'36i. V.,J . . i f f /( ,' h t u z . .So,., 49, 2659 (1927); SO, 2080
( 3 ) King, C.
(11928). T h i . molar extinction coefficients a t 320 nip of the majority of t,hc compounds of Table IV arr of the order of 10% of that of oerium(1V). For this reason separations must be made from ii larger number of elements than if t.he color of thc ceriuni(1V) is mc:nsured in the violet, portion of the visible spectmm. On thc ot,her hand, the interference due to copper, particularly at 340 mp, is so slight that it is possihlr to determine cerium accuratel). i n a strong blue solution Ttw interfcrrnee found with iroii
('. V., and Giiawold. I . I,., Ibid., 32, 14Y3 (1:)3?). ( 6 ) Ilortiirn. G., Z . phusik. Chf,n?.,B43, 415 (1930). (ti) .\IarshiLll, H., Proc. Roy. ,Sot. Edinburgh, 23, 163 (19OUi. ( 7 ) 3Iarshall. H.. and Inglis, .I. li. H.. Ibid., 24, 58 (1902). is) &indell, E. R.,"CoIorinieti4c. Determination of Traces of .\letals," New York, I n Publishers, 1944. (91 ~chribe.G.. Ber., 59, '6). (10) Yost, D. &I., J . A V L .C'het~/.SOC..48, 374 (1026).
(4) Kiig.
RI.:CEI\-ED July 21. 1950. R e m a r r h carried oil: )under the aiisgicea of thr Atornic Energy Commission
Spectrophotometric Determination of 8-Quinolinol And Some of Its Halogenated Derivatives W. T. HASKINS AND GEORGE W . LUTTERMOSEH National Institutes of Health, Public Health Service, Bethesda 14, .\Id.
I
N CONR'ECTIOS Lvith a study of the mode of action of t,litx
iodine-cont,aining 8-quinolinol compounds uwd for the trentmerit, of amebiasis, it was desired to cstimate quantities of the ortiel, of it fraction of a milligram of the drugs arid their partially o r romplt~tc~ly dehalogcnatcd derivatives. Other investigators ( I , 9,4 , 7, IO, 1 2 ) and t,he authors (9) have studied the abporption, diet,rihtion, i t r i d excretion of these iodinated compounds heretofore by tiekrmining the amount of iodine prrwnt in the biological matrrials by chemical or tracer twhniqutw. I-Iowevcr, it, may not al\vays he assumed that the iodine measured in this way is still bound t>othe 8-quinolinol riuclcus, for it, may have beer reniovcd hy some met,abolic process and subsequently transferred t o some othrr moiety. Thus it t)ecomc:s desirable to determiiic. riot only the iodin(: associated with thcb drug but also the quaxitit>. o f 8-qUinOliIKJl prcsent. Published mc)thods for the determination of 8-quinolinol depend 011 t.itration or gravimetric precipitation with bromine (6, Y, 8, 11) and hence are not applicable to dcrivittives which are alrriidy s u b s t i t u t ~ ~ind thcb 5 and 7 positions. Fresenius (6) has
dcxribed a spectrophotometric method for the determinatioii of 5-chloro-7-iodo-8-quinolinol by measuring the color developed ill glacial acetic acid in the prtscnce of ferric iron. However, this method suffers because of its cstn:mcL sensitivity to the water content of thc solvent. Grabbe ( ? ) stated that a colorimetric mctthod employed for the estimation of small amounts of 8-quinolinol using ferric chloride in aqueous solution, but gave no details. It was recognized that the intense grwn color of the complrs formed by 8-quinolinols with ferric iron offered a wnsitivr and n~lativclyspecific met,hod for their determination by spcmcatrophotometry, provided a suit,ahlr solvent n-ere available. It \ V ~ R found that methyl Cellosolve (2-mcthosyethanol) was satisfnctory for the purpose, as it is a good eolvcnt, for &quinolinol, 5rhloro-8-quinolinol, S-chloro-7-iodo-8-quinolinol(vioform), 5,Tdiiodo-8-quinolinol (diiodoquin), and their iron complexes. The color produced by the iron complexes in this solvent, which is not hygroscopic, is insensitive to small changes in water content. RIet,hyl Cellosolve is not suitable for the determination of 8quinolinol-5-~ulfonicacid or T-iodo-8-quinolino1-5-sulfonic aciil.
V O L U M E 23, NO. 3, M A R C H 1 9 5 1
457
In connection with a study of the mode of action of iodine-containing 8-quinolinol compounds (5,7diiodo-8-quinolinol and 5-chloro-7-iodo-quinolinol) used for the treatment of amebiasis, it was desired to determine quantities of the order of a fraction of a milligram of the drugs and their partially or completely dehalogenated derivatives occurring in the urine of experimental animals. By employing the intense green color of the complex formed by 8quinolinols with ferric iron and the selection of methyl Cellosolve (2-methoxyethanol) as a solvent, a spectrophotometric method was developed which .
I n the procedure a considerable of iron over the theoretical ratio of 1 mole of iron t o 3 moles of 8-quinolinol compound was used in order to ensure the maximum depth of color. It was found t h a t acid concentrations below pII l destroyed or diminished the intensity of the color. When developed as described, the color was stable for several hour8 and showed little change over a 24-hour period when titored at room temperature in a stoppered container. The curves relating optical density, D , and wave length of incident light for the 8-quinolinol-iron complexes are given in Figure 1.
is applicaable to the determination of 8-quinolino1, 5-chloro-8-quinolino1, 5-chloro-7-iodo-8-quinolinol, and 5,7-diiodo-8-quinolinol in amounts of 0.05 to 0.50 mg. A procedure for the separation and determination of the components of mixtures of 8quinolinol with any one of its halogenated derivatives and application to the analysis of urine w n taining these compounds in both the free and conjugated states is given. This method offers a simple rapid method for the estimation of 8-quinolinol compounds in urine and, with modifications, could he applied to many other types of materials. __ __ ~
tiecituhc of thcir insolubility.
-1
I.//
BXCPPS
06
,
1-
I
$;, 3
07
c
5-CHLORO-8-QUINOLINCL
5-CHLORO-7-IODO-8-OUlNOLlNOL
&
5.7-DIl000-8-QUINOLINOL
0-QUINOLINOL 5-CHLORO-8-QUINOLINOL 5-CHLORO-7-IODO-8-QUINOLINOL
MG
Figure 2. Optical Density TS. Concentration of 8-Quinolinol-Iron Complexes 1-om. cuvette
ured a t 650 mp using as a biitrik a solutiou of 0.5 nil. of ferric rhlorjde reagent diluted to 5 ml. with methyl Cellosolve. Calibration curves (Figure 2) were prepsred for each of the 80 , L , ._1 quinolinol compounds having a satisfactory solubility in methyl 500 550 600 650 700 750 Cellosolve. All were straight lines passing through the origin, X IN mfi with the exception of the curve for 8-quinolinol which deviates from a straight line at concentrations greater than 0.35 mg. In Figure 1. Optical Density us. Wave Length of 8practice, D values greater than 0.6: were avoided by reducing the Quinolinol-Iron Complexes volume of the aliquot or increasing the dilution of the sample. Concentration, 0.25 mg. of compound and 0.5 mg. of iron per 5 ml. This served to ensure a proper concentration of iron and to inof solution, 1-cm. cuvette crease the precision of the measurement by using the lower portion o f the logarithmic scale of the Beckman spectrophotometer. 'The maximum optical density was found t o occur at 650 mg fui .In iritcrcstiiig relationship was noted w h t a r i the calibration ibach of the compounds, indicating t h a t halogen substituents have curves were plotted with the concentration expreswd in nlicron o measurable effect on the shade of the color. moles instead of milligrams (Figure 3 ) . The lines lie very close toget,her, ahowing t h a t the amount of color produced is primarily METHOD a function of t h r amount of the 8-quinolinol moiety prewnt; the Tiit. reagents required are commercial grade methyl Cellokind or number of the halogcn substituents has only a minor effect. solve and a n aqueous solution of ferric chloride containing 1 I t \vas also found possible to analyzo a mixture of 8-quirlolinol gr:tm of ferric iron and 1 ml. of concentrated hydrochloric acid with any one of its halogm>itcd derivatives by nirapuring thr per liter. The S-quinolinol compound was dissolved in methyl Cellosolve optical d(1nsity of the iron coniplcs of the mixture, scparating the t i \ Ramling, if necessary, and diluted with the same solvent to halogenated compound, mcasuring its optical density, and suba'volumc such t h a t a 1-nil. aliquot contained 0.05 to 0.50 mg. of tracting the latter from the former to give the optical density the compound. The aliquot was pipetted into a 10-ml. graduated equivalent t o the amount of 8-cjuirrolinol prrwnt in thi. mistun,. cylinder, 0.5 ml. of the ferric chloride reagent was added, and the volume was adjusted to 5 ml. with methyl Cellosolve. The tube WLS stoppered and the contents were mixed by inverting the . The 8-quinolinol \v:w .vel)aruted from its halogelisted derivucylinder several times. T h e p H of this solution as measured with tives by pouring the methJd Cellosolve solution of the iron coina glass electrode was 1.6 to 1.8 for all the 8-quinolinol compounds plex of the mixture after the optical density had been determined measured Over the concentration range specified. T h e solution into 25 ml. of 1 -Y hydrochloric acid and extracting n-ith five 5\\its transferred to a 1-cm. cuvette and the optical density meas. 1111. pnrtions o f ethvlene dichloride. Lnder t,hcse conditions the ~
1
ANALYTICAL CHEMISTRY
458 halogenated compound was quantitatively extracted, leaving the 8-quinolinol in the aqueous phase. The ethylene dichloride was evaporated a t room temperature by a current of air and the residue was taken up in a small amount of methyl Cellosolve, 0.5 ml. of ferric chloride reagent was added, and the volume was adjusted to 5 ml. a s before. The optical density of this solution subtracted from the optical density of the mixture is the optical density of the 8-quinolinol present in the mixture. If desired, the 8-quinolinol may be recovered from the aqueous hydrochloric acid solution by making it alkaline with sodium bicarbonate and extracting r i t h several small portions of ethylene dichloride.
0 8-QUINOLINOL
a 5-CHLORO-8-OUINOLINOL a 5-CHLORO-7-IODO-8-QUlNOLlNOL @ 5,7-DllODO-8-QUINOLINOL 0.6
c
1
i
0.4
0.3
The butanol extract was centrifuged and filtered to break the emulsions and the solution was evaporated in vacuo, or by a current of air, a t room temperature. The residue was taken up in 5 ml. of methyl Cellosolve, transferred to a 10-ml. volumetric flask, and diluted to volume with the same solvent. An aliquot of appropriate volume, as determined by trial, was transferred to a 10-ml. graduated cylinder and diluted to 4 ml., 0.5 ml. of ferric chloride reagent was added, and the volume was adjusted to 5 ml. with methyl Cellosolve.
S o green color appeared on addition of the reagent, but Llithin a few minutes a color change was apparent, as the acidic reagent caused hydrolysis of the conjugate with concurrent formation of the 8-quinolinol-iron complex. The rate of hydrolysis was followed by observing the change of optical density with time a t 650 mp, A typical curve, given in Figure 4, shows that the hydrolysis is virtually complete in 8 hours. For routine estimations it was unnecessary to follow the course of the hydrolysis. The solutions were allowed to stand a t room temperature for 8 hours and then read; in fact, check determinations a t 8 and 18 hours showed that there was no significant difference in the reading when the solutions were made up in the late afternoon and read the next morning. The hydrolyzed solution may also be analyzed for the presence of a mixture, as previously described.
0.2
0.I
I O
2.0
i 3.0
o.4
MICROMOLES Figure 3.
t
i
/
Optical Density us. Concentration of 8-Quinolinol Complexes 1-cm. cuvette
.k mixture of 0.100 mg. of 8-quinolinol and 0.175 mg. of 5,:diiodo-8-quinolinol when analyzed as described gave D 0.372 for the mixture and D 0.167 for the diiodo compound. Subtracting, the optical density for 8-quinolinol was 0.205 or 0.102 mg. as read from the calibration curve, in good agreement with the theory. Application to Urine Samples. The urine from rabbits receiving 8-quinolinol compounds orally was analyzed as follow : The pH of the specimen v a s determined and, if it was below 7. sodium bicarbonate was added until a slightly alkaline reaction was obtained. A 50-ml. ali uot was extracted with five 10-ml. portions of ethylene dichlorile, and the combined extracts were centrifuged to break the emulsions which usually formed. The clarified ethylene dichloride was filtered and evaporated to dryness with a current of air. The residue was dissolved in methyl Cellosolve and the determination carried out as described in the method. In the caw of dosage with halogenated 8-quinolinols, it was also carried through the procedure for the determination of mixtures. These results gave the quantity of 8-quinolinol compounds present in the free state in the urine. I t has been shown by other investigators ( 3 , 7 , 12, IS) that 8quinolinols occur in the urine as conjugates with sulfuric and glucuronic acids. These conjugates were not extracted by ethylene dichloride and gave no color with iron because the hydroxyl group was blocked. Hydrolysis of the urine samples with various concentrations of hydrochloric acid, both at, room temperature and a t 100" C was unsatisfactory because of incomplete hydrolysis a t low temperature and partial dehalogenation accompanied by an apparent disappearance of varying amounts of the 8quinolinol nucleus a t high temperatures and acid concentrations. These difficulties were circumvented by extracting the same sample used for the estimation of the free 8-quinolinol compounds with butanol. ilt least 8 extractions with 25 ml. of butanol each , Mere required because of the relatively low solubility of the conjugates. I t was found advisable to mork up the last estract separately, in order to be sure that all the material had been extracted from the sample
0
0
1
2
3
4
5
6
7
8
TIME IN HOURS Figure 4. Hydrolysis of Conjugated 5Chloro-7-iodo-8-quinolinol from Urine of Rabbits Receiving Vioform p e r os
Blank runs were made on urine from rabbits receiving the same diet but no 8-quinolinol compounds, and in no case was a green color produced by the residues from the estractions, thus eliminating any need for control blanks. LlTERATURE CITED
(1) Albright, E. C., Tabern, D. L., and Gordon, E. S.,Am. J . T r o p . Med., 27, 533 (1947). (2) hnderson, H. H., David, N. A., and Koch, D. .4., Proc. Soc. E z p t l . Biol. Med., 28, 484 (1931). (3) Brahm, C., Hoppe-Sylers 2. physiol. Chem., 28, 439 (1899). (4) David, N. il., Phatak, N.M.,and Zener, F. B., -4m. J . T r o p . Med., 24, 29 (1944). (5) Fleck, H. R., Greenane, F. J., and Ward, A. hl.,Analgist, 59,325 (1934). (6) Fresenius, Suddeut. Apoth. Ztg., 79, 68 (1939). (7) Grabbe, C., Arch. Ezptl. P a t h . Pharmakol., 137,96 (1928). (8) Grant, E. H., J . Assoc. Ofic.Agr. Chemists, 29,280 (1947). (9) Haskins, W. T., Luttermoser, G. W., and Brady, F. J., Am. J . T r o p . M e d . , 30, 599 (1950). (10) Knight, A. A , , and Miller, J., A n n . Internal M e d , 30, 1180 (1949). (11) Luther, F., Deut. med. Wochschr., 57, 1739 (1931). (12) Palm, A,, Brch. Erptl. Path. Pharmakol., 166, 176 (1932). (13) Rost, E., Arb. kaiserl. Gesundh. 15, 288 (1899). RECEIVED October 28, 1950
