V O L U M E 26, N O . 3, M A R C H 1 9 5 4
609
ator is placed in the tube and the filter paper strip which has been spotted 2.5 cm. from the weighted end is dipped into the solvent to a depth of about 1.5 cm. The chromatographic process should be carried out a t a temperature between 15" and 25" C. in the dark or in subdued light-since the porphyrins are light-sensitive-and should he continued for 1 to 3 hours. The paper strip is then removed and air-dried for about 15 minutes, and the red fluorescent zones of porphyrins are observed and niarlced under ultraviolet light. The center of density of fluoreseerwe for each zone IS m:irked and the R/ value is calculated. RESULTS
In the arronipan! iny tnblc are given typical I?, values obtained on urine from patients with congenital or acute porphj-ria. Vsually four or five drtcct:zble zones nere observed. These consist of coproporphyrin, uroporphvrin, and unidmtified intcrmediates. i t 21' t h r follou-iiig I?, values w r e founcl:
R, 0 0 0 0 0
18 26 33 49
5s
.haunt Large Tiace Trace Imgr Sniall
Naturc Croporphyrin ? ? Coproporphyrin 1
Tlicse valups agrce i n grneral 1vit.h those obtained on :L mixture of pure samples of uropoiyliyrin I and coproporphyrin I. Pure protoporph,yrin showcd n i l K, value of 0.71 when cliromatographed in a mixture of ~ i u r cporphyrins. These values also agree well with those ohtnincd 11). Sicholas and Rimington ( 3antl by Kelil nnd Stich (Q.
violet light antl hence coritainetl 110 more than a negligible trace of porphyrins. Similarly, sufficient elutions of the lead precipitate removes the porphyrins quantitatively as evidenccd by the nonfluorescence of the third or fourth eluate and the practically white color of the lead residue. A number of modifications to the abovc proredure were studied. Barium chloridc was used as the precipitant and had the ativantage of being less solulile in 125; hydrochloric acid than the lead salts: however, the barium salts lacked the adsorbing capacity of the lend salts antl consequently not all of the porphyrins were removed from the urine. An attempt to reduce the solubility of the lead salts in acid by resorting to the common ion effect of dilute sulfuric and phosphoric acids was without success. Spotting with the acid eluate and drying in the presence of ammonia fumes gave poorer resolution, only two broad zones appearing rather than four or five. Thus, this time-saving procedure was not used. The cIiromatogrnphinI: must hc carried out a t a tcmperature between 15' and 25' C. Higher temperatures decreaw the solubility of water i n 2,&Iutitline and because the correct proportion of water in the developing mixture is necessary for separating the porphyrins, littlc or no resolution would be obtained. Of rourw, the use of 2,6-lutidine Tvould I m c n the restrictions iniposed by solvent immiscibility ( 2 ) . Chromatographing at tcmperatures Ion-er than 15" liken ise results in poorer resolution. The possible adaptahility of the above procedure to the atlsorption of the porphyrins from tissues, blood, and bone marrow extracts and the sensitivity of paper chromatography to small (1 to 107) quantities of porphyrins (4)makc further investigations of the method cx\trcmel!. desirablc.
1)ISCC'SSION
Because oi the relativc $iniplicity and efficienq. of tliip methotl it is well adapted for use in clinical as well as research laboratories. Following thc adsorption of the porphyrins on the lcati precipitate, the nicthotl may be shortened to afford a rapid and sensitive qualitativc, trst. for porphyrinuria. The red fluorescence i n ultraviolet light of thc hydrochloric acid eluate or thc lcad prceipitate is an extrenicly delicnte and specific trst for tht: ~ i o r p h ~ . iin~. The adsorption of porphyrins from urine appe:ti'a t o lie quantitative. This was demonstrated by adding 1.0- to 10-7 amounts or' protoporphyrin, cogroporphj-rin I, and uroporphyrin I per iiiillilitcr of urine and adsorliing on lead salts as described. X n estract of t,he supernatant'! using n 3 : l : l mist'ure of ethll acet:ite, ether, and glnc4:il ncvtir nritl, gave no fluorcxsccncr in ultra-
ACKNOWLEDGMENT
Appreciation is espressed to Earl G. Lersen of this laboratory for supplying the samples of pure porphyrin mcthj-1 esters used. LITER'iTURE CITED
(1) Chu, T. C., Green, A. A, and Chu, E. J.,,J. Biol. Chenz., 190, 1343 (1951). (2) Ericksen, I,.,Scand. J . C2in. Le: Lab. Znrest., 5, 155 (1953). (3) Falk, J. E., and Benson, A., Bi'ochem. ,J., 55, 101 (1953). (4) Kehl, R., and Stich, W., Z. physiol. Ciiern., 290, 151 (1952). (5) Sicholas, R. E. H., and Rimington, C., Scand. -1. Clin. R. Lab. Invest.,1, 12 (1949). RECEIVEDfor rei-iew .iugust 31, 1953. Accepted December 4 , l!j.j3. Work aided by a grant from the Sutrition Foiindation, Inc.
Use of Iodine Monochloride in the Kurt Meyer Titration ALEXANDER GERO H ah e m ann M e d i c a / College,
Ph i/ade/phia, Pa,
effort to im1irt)vr the technique of the I\urt !dryer I-utitration . [addition of bromine to the enol contairietl in AX
.
a
ketone (&$)I, iodine monochloride has been successfully substit'uted for the bromine. Recent 13-ork on simple olefins has shon-n ( 1 ) that iodine monochloride has t'he advantage of adding more rapidly and of causing no substitution. Methanol n-as used as n solvent because it dissolves most ketones e:iaily anti gives stable solutions with iodine monochloride. To eliminate the cnt:ilytic effect of the hydrochloric :icitl formed in the addition, sodium bicarbonate was added to the. miction misture in :miounts roughly equivalent to the amount, of enol present. Bring inso1ul)lc in methanol, t,he sodium biczrlmnnte does not interfere nit,h the equilibrium but it does neutrali7e the hydrochloric avid t'oi,nietl.
Sunieiuus titrations have shown the accurac~.of the method
to be of the order mole of enol. It is believed to be more reliable than the older versions of the Kurt Meyer titration. Details of the technique, as well as the enol content of various ketones determined by it, will be published elsewhere. LITERiTURE CITED
R. E.. J . 1 m . C'hem. .%e., 75,5119 (1953). (2) Lleyer, K. II., Ann., 380, 313 (1911). (3) Neyer, K. H., and Kappelmeier. P., Ber., 44, 2718 (1911). (4) Schwareenhnch, O., and Feldcr, E., H e h . Chim. Acta, 27, 1044 (1944). (1) Gero, A , Kershncr, ,J. .J.. and Perry,
R E C E I V E for D review J u n e 4, 1953. Accepted Soveniber 23. 1953.
