A N A L Y T I C A L CHEMISTRY
608
filling the tube with water before attaching the adapter. The tube is then turned around 180 degrees, the upper adapter I S removed, and the barium is eluted with 75 ml., twice with 25 ml., and again with 75 ml. of 12% hydrochloric acid. Thc eluate is concentrated to about 50 ml. on a steam bath, thc barium is precipitated with sulfuric acid and is filtered, heated, and weighed as before. Care must be taken that the level of the liquid in the tubc never drops below the top of the exchange resin. As that has occasionally happened, the apparatus has been modified as shoR 11 in Figure 1 (right-hand column). The Erlenmeyer flask has been replaced by a dropping funnel and the adapter with the stopcock has been replaced by an adapter with a syphon tubc,. The latter \vi11 keep the liquid level aln-ays above the top of the resin and will overflow a t the same rate a t which the solution iadded from the dropping funnel. After the sulfonic acid has been eluted, another adapter with a syphon tube filled with water is put on the upper ground of the adRorption tube and the latter is turned 180 degrees. The barium is then eluted nitli hydrochloric acid as described before.
fonic acid, a small ion exchange column (Figure 1, left-hand column) is used. It consists of a chromatographic tube, size B, with standard 19/22 ground-glass joints a t each end. The bottom ground-glass joint has an adapter with a standard ground-glass stopcock. The tube is filled with a column, 15 cm. in height, of Amberlite IR 120 cation exchange resin topped by a porcelain filter plate and some glass wool. Before use, the column is regenerated by passing 75 ml. of 15% hydrochloric acid through i t and then by washing until neutral with distilled water. The determination of the sulfur is carried out as follows: Dissolve about 0.1500 to 0.1800 gram of the barium sulfonate in 75 ml. of distilled water in a 150-ml. Erlenmeyer flask provided with a standard ground-glass outlet tube. Pass the solution through the freshly regenerated and well-washed cation exchange column a t a rate of 8 to 10 drops per minute without allowing the level of the liquor to drop beloly the surface of the exrhange resin laycr. Collect the filtrate directly in a Icjeldahl flask. Rinse the column three timrs (each time with 25 ml. of distilled water) by pouring the water into the Erlenmeyer flask from a wash bottle without removing the stopper. Then vash the column with 75 ml. of distilled water. Bfter adding a pinch of sodium nitrate, evaporate the filtrate, including the wash water in the Kjeldahl flask, to about 5 ml. Then add 25 ml. of fuming nitric acid and 5 ml. of perchloric acid and again evaporate the solution to about 5 ml. At this point the solution should be water clear-if it is not, the oxidation with nitric acid and perchloric acid must be repeated. Xeut add 25 ml. of concentrated hydrochloric acid and heat again until yellow or brown fumes are no longer formed. Concentrate the solution to about 5 ml., transfer the residue quantitatively into a 150-ml. beaker. and precipitate the sulfuric acid with barium chloride in the usual way. Filter the barium onto a Berlin filter crucible, heat it to a dull red color, cool, and weigh. For the elution of the barium a standard ground-glass adapter, equipped with a porous glass plate and a stopcock filled with water, is put into the upper ground-glass joint of the tube in such a way that the formation of an air bubble between the resin and the filter plate is avoidrd. This is readily achieved by
Thc method has been tested n ith barium toluenesulfonate(1) and barium tliphenylamine-p-sulfoiiatc(I1) with the following results: For I. Calculated: sulfur, 1 3 . 4 9 ; barium 28.GS. Found: sulfur 13.58, 13.50, 13.54; baiiuni 28.35, 26.51, 28.90 For 11. Calculated: sulfur, 10.12; barium 21 6 8 5 . Found: sulfur 1020, 10.12, 10.20, 10.14; baiiuni 21.45, 21.58, 21.58, 21.40, 21.08v0. For barium lignosulfonate. Found: ~ u l f u r :cExvE;D
for re\.ierv April 13, 1953.
h c c c p t e d Sovember 20, 1953.
Simplified Procedure for Separating Porphyrins from Urine By Paper Chromatography LAURENCE
M. CORWIN
and
JAMES M. ORTEN
D e p a r t m e n t of Physiological Chemistry, W a y n e University College of M e d i c i n e , Detroit, M i c h . r ~ H L is R a~gron ing
need for a simpler, more direct procedure f o r
1identifying porphyrins in biological materials for both research
nnd clinical purposes. ,1number of methods are to be found in the literature, but these are too lengthy and involved, requirr 1 athcr specialized equipment, or lack sufficient sensitivity for thc tletection of the small amounts of porphyrins usually prescnt in tissues and body fluids. Chromatography on filter paper \voultl appear to offer a convenient means of porphyrin separation and has been satisfactorily emploj ed by several investigators for determining the prcsence of porphyrins in urine either :is the free porphyrins ( 2 , 4,5 ) or as the methyl esters (1, 3 ) . In thc formcr studies ( d ) , however, coproporphyrin was first extracted ivith ether from urine and the uroporphj rin fraction was thrn ‘idsorbed and scparated using lead acetate. In the latter studies ( I , 3 ) employing the meth) 1 esters, the added time-consuming step of esterification is required. Thc present investigation \vas designed to study the possibility of adsorbing the total porphyrins from urine on a lead precipitate, eluting, and then chromatographing the free porphyrins on filter paper in a simplc test tube apparatus devised in this laboratory. Excellent results havr been obtained by thr following procedure. JIETHOD
salts are eluted with 1 ml. of 12 to 15% hydrochloric acid (for quantitative elution this process is repe:Lted until the remaining precipitate appears almost white; usunliy txvo or three elutionq are sufficient). -40.2-ml. aliquot of the eluate is immediately evaporated i n :I stream of air or, better, under reduced pressure over sodium hydroxide pellets. The evaporation should be completed within 2 hours; otherwise considerable destruction of the porphyrins may occur. Tlir No. 4 rubber stopper 1)orphyrin-containing recidue is taken up in 0.2 mi. of 1 0 s ammonium hydroxide. .$ small drop of this Test tube solution is then spotted on n ( 2 5 x 200 mm.) filter paper strip for chroniatograjihy. In this laboratory “test Filter paper strip ( 1 8 x 1 7 5 mm.) tube” chromatography is used x i t h success. The allparatus consists of a large t’est tube (25 X 200 nini.), a filter paper strip (What-. man No. 1, 18 X 175 mm.), prepared with vertical slits a t one end to hold a small glass rod as a weight, anti a No. 4 rubber stopper with 0 - ------Storting point a slit to hold the filter paper . >’........_.. as shon-n in Figure 1. Five ..:..:.:.:,.: ., ----- Glass rod weight milliliters of the developing .. ::....>;:,.... :. , ....:... .,.*”. _ _ _ _ Developing solvent solution [ l to 1 mixture of 2,4-Iutidine, or better 2,6Figure 1. Test Tube Chrolutidine ( 2 )in water] which m a t o g r a p h i n g .4pparatus has been stored in a refriger-.
J ,
To 10 nil. of urine in a 15-mI. centrifuge tube is added 2 ml. of 10% lead acetate solution. The solutions are immediately mixed by inversion and are centrifuged for 3 to 5 minutes a t 2400 r.p.m. The precipitate is then mixed with 5 ml. of distilled water and recentrifuged. The supernatant is discarded. The pigments which are adEorbed on the precipitated lead
i.....: (
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 ni: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
ACKNOWLEDGMENT Because oi the relativc $iniplicity and efficienq. of tliip methotl it is well adapted for use in clinical as well as research laboraAppreciation is espressed to Earl G. Lersen of this laboratory tories. Following thc adsorption of the porphyrins on the lcati for supplying the samples of pure porphyrin mcthj-1 esters used. precipitate, the nicthotl may be shortened to afford a rapid and sensitive qualitativc, trst. for porphyrinuria. The red fluorescence LITER'iTURE CITED i n ultraviolet light of thc hydrochloric acid eluate or thc lcad prc(1) Chu, T. C., Green, A. A, and Chu, E. J.,,J. Biol. Chenz., 190, 1343 eipitate is an extrenicly delicnte and specific trst for tht: ~ i o r p h ~ . (1951). iin~. (2) Ericksen, I,.,Scand. J . C2in. Le: Lab. Znrest., 5, 155 (1953). The adsorption of porphyrins from urine appe:ti'a t o lie quan(3) Falk, J. E., and Benson, A., Bi'ochem. ,J., 55, 101 (1953). titative. This was demonstrated by adding 1.0- to 10-7 amounts (4) Kehl, R., and Stich, W., Z. physiol. Ciiern., 290, 151 (1952). (5) Sicholas, R. E. H., and Rimington, C., Scand. -1. Clin. R. Lab. or' protoporphyrin, cogroporphj-rin I, and uroporphyrin I per Invest.,1, 12 (1949). iiiillilitcr of urine and adsorliing on lead salts as described. X n estract of t,he supernatant'! using n 3 : l : l mist'ure of ethll aceRECEIVEDfor rei-iew .iugust 31, 1953. Accepted December 4 , l!j.j3. t:ite, ether, and glnc4:il ncvtir nritl, gave no fluorcxsccncr in ultraWork aided by a grant from the Sutrition Foiindation, Inc.
Use of Iodine Monochloride in the Kurt Meyer Titration ALEXANDER GERO H ah e m ann M e d i c a / College,
Ph i / a d e / p h i a , Pa,
effort to im1irt)vr the technique of the I\urt !dryer I-utitration . [addition of bromine to the enol contairietl in AX
.
a
ketone (&$)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
Gero, A , Kershncr, ,J. .J.. and Perry, 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)
R E C E I V E for D review J u n e 4, 1953. Accepted Soveniber 23. 1953.
