1519
V O L U M E 2 7 , NO. 1 0 , O C T O B E R 1 9 5 5 different solvent systems. Desoxycorticosterone and dehydrocorticosterone in 2 5 , 50-, and 75-r amounts were chromatographed in the methylcyclohexane-propylene glycol system until the steroids had migrated approximately two thirde of the distance down the strip. After drying in air, the strips were sprayed, washed, and dried according to the above procedure. The colored zones viere then cut out and eluted, and the color density vias determined. The same procedure was followed using cortisone and hydrocortisone in the toluene-propylene glycol system. I n all cases, the color density of the sprayed and eluted chromatograms gave values identical with that indicated by the standard curve prepared according to the method.
(3) (4) (5)
Heard, R. H. D., and Sobel, H., J . B i d . Chem., 165,697 ( 1 9 4 6 ) . Henly, A . A , , Sature, 169, 877 ( 1 9 5 2 ) . Hoffmann, H., and Standinger, H., Baochem. Z.,322, 2 3 0 (1952).
(6) Msder, W.J., and Buck, R. R., A s ~ L .CHEM.,24, 6 6 6 ( 1 9 5 2 ) . (7) RZeyer, A. S.,and Lindberg, 11.C., J A m . Chem. Soc., 76, 3034 (1954).
(8) Richardson, E. %I., Touchstone, J. C., and Dohan, F. C., J .
Clzn. Inzest., 34, 2 8 5 ( 1 9 5 5 ) .
(9)
Touchstone, J. C., Richardson, E. AI., Bulaschenko, H., Landolt, I., and Dohan, F. C., J . C l m . Endocrmol. &: Metab-
(10)
Wheeler, J., Fieeman, S.,and Chen, C., J . Lab. Clan. Med., 42,
(11)
Zaffaroni, A., “Recent Progress in Hormone Research,” vol. 8, Academic Press, New Tork, 1953.
olwm, 14, 676 ( 1 9 5 4 ) . 758 ( 1 9 5 3 ) .
LITERATURE CITED (1) (2)
Chen, C.. Tewell, H. E.. Federation Proc., 10, 377 ( 1 9 5 1 ) . Chen, C., Wheeler, J.. and Tewell, H. E., J . Lab. Clin. X e d . , 42, 7 5 4 ( 1 9 5 3 ) .
Received for review November 1 1 , 1951. -4ccepted June 20, 1955. Investigation supported in part by a research grant H-922 from the National Heart Institute, National Institute of Health, Public Health Service.
Identification of Imidazole Compounds by Paper Chromatography ROBERT W. COWGILL Department of Biochemistry, University of California, Berkeley, Calif.
The locations of 20 imidazole compounds are described following paper chromatography. Three solvent systems were used, singly or in combination. The identity of spots was further established by their appearance with ultraviolet light, a p-phenyldiazonium sulfonate spray, or an iodine spray. The iodine spray revealed the location of 1-substituted imidazole compounds which other sprays for imidazole compounds did not reveal.
A
PAPER chronxttographic method for the separation and identification of imidazole compounds is required in connection with biochemical studies in this laboratory. Current methods described in the literature (1, 4, 5 ) for the chromatographic separation of imidazole compounds either do not encompass the compounds of interest in these studies, or are not applicable to them. The range of imidazole compounds for which current paper chromatographic methods are applicable is limited by the nearly exclusive use of p-phenyldiazonium sulfonate or a similar diazonium compound as a spray to reveal the spot. Exceptions to this are sprays which react with certain substituents on the imidazole ring-for example, the ninhydrin spray for the a-amino group of histidine. The p-phenyldiazonium sulfonate spray (diazonium spray) is a sensitive test for many imidazoles. Unfortunately, it fails t o produce colored spota with all such compounds. An important group which does not react with the diazonium spray consists of the 1-substituted imidazoles. This group includes the biologically important 1-methyl histidine which occurs in muscle as the dipeptide anserine. Use of iodine t o reveal a variety of organic bases following paper chromatography has been described by several v,-orkers ( d 6). Such an iodine spray also reveals spots of these imidazoles refractory with the diazonium spray, as well as all other imidazole compounds tested. The combination of the iodine spray, diazonium spray, and the ultraviolet lamp is capable of revealing spots and in many cases of distinguishing between spots of a large variety of imidazole compounds (Table 11). Further, solvent mixtures described in the literature do not effect a distinct separation of several of the compounds listed in Table I. Two new solvent mixtures (solvent I and 11) that separate most of these compounds were devised. The solvent
mixture of Inoue ( 5 ) m-as found t o separate the compounds of Table I which are not separated by solvent mixtures I and 11; also the former solvent leads t o higher Rj values for imidazoles that bear carboxyl groups, Since the solvent mixture of Inoue complements solvent mixtures I and TI, the Rrm values are reported for all three (Table I). EXPERIMENTAL DETAILS
Conventional paper chromatographic techniques are used. Imidazole compounds are spotted on 11 4 X 40 cm. sheets of Whatman N o 5 2 paper. The sheets are hung lengthwise in jars for development by solvent flow in descent. Jars are equilibrated with the solvent mixture a t least 1 hour before the papers are introdured. Three single-phase solvent mixtures are used for development of the chromatogram. Ethyl alcohol, diethyl ether, water, and 28y0ammonia in a 4 t o 5 to 1 t o 0.1 ratio (solvent I) and acetone, chloroform, water, and 28% ammonia in a 30 to 5 to 4 to 0.2 ratio (solvent 11) were formulated in this laboratory. The third, acetic acid, n-butyl alcohol, ethyl acetate, water, in a 1 t o 1 t o 1 to 1 ratio (solvent 111) was described by Inoue (6). All solvents are distilled before use, except 28% ammonia and glacial acetic acid. The rates of solvent flow over a 30-em. path are 5 cm. per hour for solvent I, 9 em. per hour for solvent 11, and 3.5 em. per hour for solvent 111.
Table I.
hIovement of Compounds Relative to Movement of Imidazole (Whatman No. 52 paper with solvent flow in descent)
R I Values ~ Compound Imidazole 4(0r 5)-COOH Imidazole 4,5-(COOH)z Imidazole 4(or 5)-COOCHa Imidazole 4(or 5)-CHO Imidazole 4(or 5)-CH20H Imidazole 1-CHa Imidazole 2,4,5-(.Me)a Imidazole 4(or 5)-Br Imidazole 2,4,5-(Br)a Imidazole Benzimidazole L-Histidine L-Histidine anhydride Pht halyl-DL-histidine Methyl ester of L-histidine Methyl ester of phthalyl-L-hist,idine PilocarDine Histamhe XH4Cl
Solvent
I
1 .oo 0.10 0.02 0.98 0.81 0.72 1 05 1.26 1.16 1 .oo 1.15 0.08 0:39 0.68
1.14 1.03 0 53 0.25
Solvent I1 1.00 0.02 0.02 1.14 0.89 0.48 1.20 1.35 1 48 0.98 1.42 0.02 0.02 0.08 0.97 1.45
1.88
0.80
0.07
Solvent I11 1.00 0.63 0.60
1.37 1.06 0.85 1.03 1.34 1.56 1.83 1.37 0.37 0.41 1.26 0.81 1.50 1.17 0 28 0 38
1520
ANALYTICAL CHEMISTRY
sistent in this laboratory than movement relative to the solvent front (R,). If desired, Appearance with Light Appearance after UltraAfter Iz spray Diazonium Spray Rt values may be calculated violet, UltraRelative from the Rrm values in Table Compound initial Ordinary violet Color intensity I and from the R, values for None Brown Brown Yellow Medium Imidazole Blue Brown Dark brown Yellow High 4(or 5)-COOH Imidazole imidazole. The R/ values for Blue Dark brown Dark brown Yellow Medium 4 5-(COOH)z Imidazole imidazole are 0.73 in solvent Blue None or ext. Blue Yellow Low 4ior 5)-COOCHt Imidazole weak I, 0.57 in solvent 11, and 0.51 Faint brown Blue Blue Brown Low 4(or 5)-CHO Imidazole Brown Grayish brown None Orange High 4(or 5)-CHzOH Imidazole in solvent 111. .. None Brown Reddish brown S o color 1-CHn Imidazole The compounds histidine, 1Faint blueu Dark brown N o color Reddish bmwn 2,4,5-(CHsja Imidazole Hish Brown Faint brown None Red-orange 4(or 5)-Br Imidazole methyl histidine and 1,3-diBlue High Yellow Dark blue Grayish b m w n 2,4,5-(Br)i Imidazole Light blue KOcolor ... Light blue Brown Benzimidazole methyl histidine, are of special High Brown Red-orange None Dark b r o u n L-Histidine interest in connection with No color None Reddish brown Brown 1-Ma-L-histidine b ... Brown N o color None Reddish brown 1 3 4 hle)r-~-histidineb studies in this laboratory. The Brown Reddish brown Kone Red-orange High LlHistidine anhydride Sone Brown Brown Red-orange Medium Phthalyl-DL-histidine brief time of solvent flow used Rf edi u m None Yellow Brown Brown Methyl ester of L-histidine for the chromatograms deiMedi u m Blue Orange Brown Brown Methyl ester of phthalyl L-histidineb Brown High None Red-orange Brown Histamine scribed in Table I mas not Brown N o color ... None Reddish brown Philocarpine Low Yellow Blue None Yellowa h-HdCI sufficient to differentiate the above three compounds from No color with solvent 111. b Syntheses of these compounds described in a subsequent paper. each other. The three could be distinguished readily by longer periods of solvent flow. The solvent was allowed to The iodine spray is a $elution of 1.0 gram of iodine in 100 ml. flow down the sheet and drip off the bottom for periods of 24 to of 95% ethyl alcohol. The spray is stable for several days, but 48 hours. Results are shown in Table 111; spot location is excan not be kept for more than 1 week for best results with all pressed relative to movement of histidine. imidazoles. Papers to be sprayed are dried overnight in the Samples of imidazoles applied to the paper as their hydroopen, or 30 minutes in an oven a t 100' C. t o expel1 all solvent vapors. Both ammonia and acetic acid interfere with this spray; chloride salts, yielded a spot which apparently was due to amammonia by production of highly colored background and acetic monium ion retention a t the position of migration of the chloride acid by inhibition of color formation of the spot. The iodine ion. This effect was due to the ammonium cation rather than solution is sprayed lightly over the paper. As the iodine vaporthe chloride or other anion. In solvent I (which contains amizes from the surface of the paper, the imidazole spots appear. For most compounds these spots rapidly fade and must be monia) potassium chloride, magnesium chloride, sodium chloride, marked within a few minutes. However, the paper may be and ammonium chloride gave spots with the diazonium spray a t sprayed again, and the spots revived repeatedly if necessary. identical positions; sodium sulfate and ammonium sulfate gave Spots may be visualized by their absorption of ultraviolet spots a t identical positions, but distinct from those for chloride light. Certain compounds in Table I1 can be detected on the developed chromatogram without further treatment; others salts; and silver nitrate and ammonium nitrate gave a third set require the iodine spray for appearance. Whereas the spots of identical spots, but distinct from those for chloride or sulfate brought out by the iodine spray rapidly fade when viewed in salts. Conversely, in solvent I11 (which does not contain amordinary light, the spots remain visible permanently with the monia) ammonium sulfate, ammonium nitrate, and ammonium ultraviolet lamp. Spots are best seen by passage of rays from the lamp through the paper. The lamp used in this laboratory chloride, all gave yellow spots with the diazonium spray, but a t is equipped with a General Electric germicidal bulb and a 4 X different positions on the paper, whereas sodium sulfate, silver 15 em. window. The window serves as a filter to absorb most nitrate and sodium, potassium, or magnesium chlorides did not rays except those of longer wave lengths of the ultraviolet region give spots. These facts should be remembered :In the study of (black light). Although less convenient, a Mineralight (UltraViolet Products, Inc., South Pasadena, Calif.) with similar charchromatograms of mixtures which contain anions, because the acteristics may be used. ammonium spot reacts with all three spot tests of Table 11. The diazonium spray was prepared and applied as directed by Ames and Mitchell ( 1 ) . The diazonium spray, iodine spray, and ultraviolet light all may be applied to the same chromatogram. The best sequence Table 111. Movement of 3Iethyl Histidine Compounds is as follows: initial view of spots with ultraviolet light, applicaRelative to Moyement of Histidine ( R H ) tion of the iodine spray, and location of transient spots; a second (Whatman S o . 52 paper with solvent flow in descent) view with ultraviolet light for spots brought out by the iodine RH spray; and finally the application of the diazonium spray. Compound Solvent I Solvent 111 Paper chromatography in two dimensions may be carried out. c-Histidine 1.00 1.00 For this purpose the procedure described by Redfield (7') is suit1 . 2 6 1-Me-L-Histidine 0.87 able. The author used 20 X 20 em. squares of Whatman S o . 1,3-(hfej?-~-Histidine 1 05 0.63 52 paper rather than the smaller sheets of Schleicher and Schuell Xo. 507 specified by Redfield. Cylinders formed from these larger sheets can be accommodated in inexpensive, 1-gallon jars. The best sequence of solvent development is the use of solvent When possible, solvents I or I1 were used in preference to 111,then I or 11, or the use of solvent I, then solvent 11. solvent 111. Spots were more compact, and background with the iodine spray was less with solvents I and 11. With solvent DISCUSSION I11 the chromatogram was overloaded more readily with the result that spots were streaked. Spots of not more than about All compounds listed in Table I may be separated by one or 0.05 micromole should be developed with solvent 111, but tenfold more of the three solvent mixtures. [4(or 6)-Chloromethylor even fiftyfold more may be developed with solvents I and 11. imidazole decomposed in all three solvent mixtures. ] Further After application of spots, the formation of streaks with solvent distinction between compounds may be secured by the characI11 was reduced by exposure of the paper to ammonia vapors teristic response to the combination of sprays and ultraviolet for 15 minutes. The paper was aerated well to expel all ammonia light described in Table 11. Movement of spots is reported in before it was placed in the jar for development with solvent 111. terms of R I , in preference to R,. Movement of spots relative to A second exposure to ammonia vapors was of advantage after readily obtainable imidazole (RI,) has been found more con-
Table 11. Appearance of Spots of Imidazole Compounds after Paper Chromatography
V O L U M E 2 7 , NO. 10, O C T O B E R 1 9 5 5 development with solvent I11 in the event that spot formation with the iodine spray was poor. Sensitivity of the diazonium spray and iodine spray vary with the nature of the imidazole compound, but an order of magnitude may be indicated. The diazonium spray is capable of detecting micromoles of imidazole and the iodine spray is capable of detecting lo-* micromoles. Whether maximum sensitivity with the iodine spray occurs with the transient color in ordinary light, or with the ultraviolet light, depends on the nature of the conipound. For example, the transient spot in ordinary light was the most sensitive indicator of 1-methylimidazole whereas the corresponding spot with 4(or 5)-bromoimidazole was nTeak. The latter compound gave a strong spot when viewed with ultraviolet light. The nature of the reaction with the iodine spray is not known ( 2 ) . Substitution on the imidazole ring is unlikely since imidazoles which bear substituents on the ring nitrogen are reported not to undergo iodination (3). -4ddition of iodine to the unsaturated imidazole ring is unlikely also in view of the known resistance of the imidazole ring to this type of reaction. Possibly a reversible physical combination of iodine with the imidazole c~ompoundoccurq. This is indicated by the fact that the
1521
colored spots formed with most imidazole compounds rapidly faded, but could be revived repeatedly by further application of the iodine spray. Highly-colored unstable products were observed by Brunings ( 3 ) . He has proposed that these products are iodinated on the ring nitrogen at position Yo. 3. ACKNOWLEDGMENT
The author takes this opportunity to thank Reuben Jones for a sample of the methyl ester of 4(or 5)-carboxyimidazole and Eva Wormser for the preparation of several other compounds used in these studies. LITERATURE CITED
(1) Ames. Bruce S . , and Mitchell, H. K., J . Amer. Chem. Soc., 74, 252 (1952). ( 2 ) Brante, Gunnar. Suture, 163, 651 (1949). (3) Biunings, Karl J., J . Am. Chem. Soc., 69, 205 (1947). (4) Huebner, Charles F., Ibid., 73,4667 (1951). (5) Inoue, Mitsuji, h'ugasulci Igakkai Zassi, 28, 1283 (1953). (6) LIarini-Bettolo, G. B., Congr. intern. biochim., Resumes communs., 28 Congr., Paris, 1952, p. 490. (7) Redfield. R. R., Biochim. et Biophys. Acta, 10, 344 (1953).
R E C E I V Efor D review 1ZarcIi 12, 1955.
Accepted Xay 23, 1955.
Colorimetric Method for Analysis of Histidine and Certain Related Imidazole Compounds ROBERT W. COWGILL Department o f Biochemistry, University o f California, Berkeley, Calif.
A colorimetric method is described for the quantitative analysis of histidine and certain of its derivatives, histamine and imidazole. Phenols and aromatic bases which interfere with other colorimetric procedures for histidine did not interfere with this method unless present in relatively large amounts.
T
H E Pauly ( 6 ) diazo reaction has long been used for the colorimetric determination of imidazole compounds. This reaction between imidazoles and diazotized aromatic amines in alkaline solution leads to the formation of azo dyes. This is the basis of the classic Koessler-Hanke procedure ( 5 ) for histidine. A more specific method for histidine is the Kapeller-Adler method ( 3 ) . This method is based on the observation of Knoop ( h ) that a heated solution of histidine in bromine water yields a reddish-colored product. Both of these methods suffer from lack of specificity, and many compounds other than imidazoles contribute color. These include phenols, aromatic amines, pyrroles, and indoles. A more specific method was desired by the author in order to analyze for certain imidazole compounds in the presence of other imidazoles and other compounds known to interfere with the above methods. A number of reactions that might lead to colored products were investigated. Of these, the most promising reaction was based on the Bamberger degradation ( 1 ) of imidazoles with benzoyl chloride in alkali. I n order to obtain a colored product, p-nitrobenzoyl chloride was substituted for benzoyl chloride (Equation 1). Conditions were devised so that the amount of light absorbed a t the wave length of maximum absorption by ( I ) was proportional to the concentration of the imidazole compound. (See structural Reaction I.) The identity of the colored product of the reaction with imidazole was established to be 1,2-di(p-nitrobenzamid0)ethylene-
(I). Properties of the colored product from the procedure for the colorimetric analysis proved to be identical with those of a purified preparation of 1,2-di(p-nitrobenzamido)ethylene. The specificity of the colorimetric method was tested with 16 imidazole compounds. Of these, only imidazole, histamine, histidine, and derivatives of histidine were reactive. Other compounds such as tryptophane, tryosine, arginine, or phenol did not interfere with this procedure unless present in relatively large amounta. EXPERIMENTAL DETAILS
Procedure for Colorimetric Analysis. STOCK REAGESTS Aqueous sodium bicarbonate, l.OM, and 1 . 0 s aqueous sodium hydroxide. p-Nitrobenzoyl chloride, 0.06M1 in acetone. The p-nitrobenzoyl chloride from Eastman Kodak Co., was used without purification. The p-nitrobenzoyl chloride from Rlatheson Co. was crystallized from petroleum ether (boiling point 60" to 70" C.) before use. The acetone fiolution of p-nitrohenzoyl chloride
HC=CH N1 PU" 1
+SO2DCOCl+
\c/ I
H
H C - X H - C __ O ~
I/ H C - N H - C O ~
xo2
xo2+ HCOOH
(1)
