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Infrared identification of paper chromatographed compounds. II. Procedures for some acidic compounds
MICROCHEMICAL
VOL.
JOURNAL
Infrared Chromatographed
Identification
553358
(1961)
of Paper
Compounds.
for Some Acidic ALAK
V, PAGES
II.
Procedures
Compounds
1’. KRIVIS* and ARLINGTON A. FORIST, Company, Kalamazoo, Michigan
Z’he CJpjohn
The isolation and identification of individual organic compounds, such as drugs and their metabolites, in biological systems is usually complicated by the presence of small quantities of the desired material and overwhelming amounts of many extraneous substances. Yermentation beers and complex organic reaction mixtures may present a similar problem. Paper chromatography has proved to be an excellent tool for the separation of minute amounts of material in crude mixtures,’ and techniques for the resolution of a wide variety of mixtures containing difficultly separable, similar compounds have been developed. Biological samples, in particular, have been very effectively handled by the separatory paper chromatographic methods. Unfortunately, powers of paper chromatography have not been matched by the ability to positively identify or characterize the compounds isolated. Infrared spectroscopy, however, affords the most generally useful and exact, identification that any single measurement can provide. Recently the separation of compounds by paper chromatography, followed by infrared characterization of the isolated material has been studied and found to be a useful procedure.2-4 To date, m&hods involving elution of a chromatographed spot and infrared examination of the eluate have been limited to situations where the compound was reasonably soluble in an acceptable solvent,. It has been found t]hat a great many types of compounds cannot bc * Present address: Central Analytical Corporation, New Haven, Connecticut.
Laboratories, 553
Olin Mathieson
Chemical
554
A. F. KRIVIS
AND A. A. FORIST
eluted from the developed chromatographic spot with nonpolar organic solvents. In general, polar solvents must be avoided because of the extraneous contaminants which they elute from the paper. Therefore, ionic species, such as salts, or compounds strongly adsorbed on the paper, are not amenable to the treatment previously used. Accordingly, alternative methods have been developed to circumvent these difficulties.
EXPERIMENTAL Reagents Potassium bromide, powdered, infrared quality (Harshaw Chemical Co.) was used to prepare all potassium bromide pellets. The organic eluents were purified prior to use, as described previously. 2 All other organic compounds and solvents were USP or reagent grade and were used without further purification. All inorganic chemicals used were reagent grade.
Apparatus A Perkin-Elmer Model 21 infrared spectrophotometer was used to obtain all of the infrared spectra. A Perkin-Elmer potassium bromide die was used in the manner previously described2 to produce all of the micropellets.
Procedure Tolbutamide A portion of human serum containing 75-125 Mg. of tolbutamide from a subject having received this drug was acidified with 5 ml. of 1.2M hydrochloric acid and extracted with 25 ml. of chloroform. The chloroform layer was then separated, extracted with 5 ml. of 1% aqueous sodium carbonate solution, and the separated aqueous layer acidified with 1 ml. of 1M hydrochloric acid. The acidified aqueous solution was extracted 5 times with 20 ml. of chloroform and the combined extracts evaporated to dryness. The extract residue was dissolved in methylene chloride and applied, in a streak, to a pre-washed sheet of Whatman No. 1 paper MICROCHEMICAL
JOURNAL,
VOL.
V, ISSUE
4
IDENTIFICATION
OF PAPER COMPOUNDS.
II
555
and developed by descending chromatography for 18 hr. in a butanolwater-piperidine (81:17:2) system.5 The tolbutamide zone on the dried, developed sheet was located by its ultraviolet absorption and was cut out and mounted in a drip eluter. Elution was carried out with fifteen l-ml. portions of distilled water. The aqueous eluate was acidified to pH 1 with hydrochloric acid and extracted twice with 15 ml. of methylene chloride. The methylene chloride solution was evaporated to about 1 ml., potassium bromide added, the mixture evaporated to dryness, and a micropellet prepared. An infrared spectrum of the material suspended on the potassium bromide was then obtained. p-Toluenesuljonamide
Tolbutamide from a sample of serum was extracted, chromatographed, and eluted as described above. The isolated tolbutamide was then hydrolyzed by heating under reflux with 5 ml. of 2.4N hydrochloric acid for 2 hr. The cooled solution was extracted 5 times with 25 ml. of ethyl ether, the organic phase evaporated to a small volume and chromatographed as described previously. The sulfonamide zone on the developed chromatogram was eluted with 12 ml. of methylene chloride and an infrared spectrum of the eluate obtained. p-Carboxybenzenesul$onamide
A sample of urine from a subject having received tolbutamide was extracted, the metabolite, l-butyl-3-p-carboxyphenylsulfonylurea, isolated as described for tolbutamide, hydrolyzed, and the resulting sulfonamide fraction carried through paper chromatography. The chromatographic zone of the hydrolyzed metabolite was treated with hydrochloric acid gas in a closed vessel for 2 hr. The paper strip was stored overnight under vacuum in a desiccator containing Ascarite and then eluted with 15 ml. of ethyl ether. An infrared spectrum of the eluate residue in potassium bromide was then ohtained. DISCUSSION Compounds eluted from chromatographic zones by certain organic solvents are sufficiently pure so that meaningful infrared spectra can be obtained.2 However, a great many compounds lack the
55ti
A. F. HRIVIS
AND A. A. FORIST
nccessury solubility in acceptable solvents to permit this direct approach. I’olar and ionic species, e.g., many drugs and their metabolit’es, fall in this category. In addition, some of the most useful chromatographic systems contain bases or acids which yield salt,s of t,hc chromatographed compounds on the paper. Therefore, m&hods have been developed for the elution and infrared identification of such polar or ionic materials following isolation by paper chromat,ography. Tolbutamide, 1-butyl-3-p-tolylsulfonylurea, has been chosen as a weakly acidic substance typical of many which might be encountered in studies of drug metabolism. In addition to tolbutamide, the sulfonamides obtained on acid degradation of this drug and its metab&e, I-butyl-3-p-carboxyphenylsulfonylurea,6 have been studied in order to demonstrate the powerful potential of this general approach to the solution of structure problems on small amounts of material. The three compounds discussed have quite different solubilities in wat)rr and in organic solvents and cover a wide range of acid strengths. In addition, since the most useful paper chromatographic system for these compounds contains piperidine, the problem of salt formation on the paper is encountered. Tolbutamide, pK, 5.43,7 can be extracted from acidified biological samples with chloroform, and a paper chromatographic separation of the sulfonylurea can eliminate the remaining contaminants from the serum. Direct elution of tolbutamide from such a papergram wit’h methylene chloride is impossible, whereas elution is readily accomplished with water; this behavior is indicative of a piperidinium salt on the paper. In addition to the sulfonylurea salt and excess piperidine, the aqueous eluate contains hemicelluloscs from the paper. However, acidification of the eluate regenerates the tolbutamide and permits its successful extraction with mcthylene chloride. An identification of the extracted tolbutamidc can then be made from its infrared spectrum. The technique described is general for weakly acidic mat,erials which in the non-ionic form can be extracted into a suitable organic solvent. The solvent used must, of course, not simultaneously extract the various contaminants present in the aqueous eluate. Since the fundamental principal involved is the enhancement of the solubility in an organic solvent over an aqueous one by adjustment of t,he pH, it is to be expected that the same general approach might be MICROCHEMICAL
JOURNAL,
VOL.
V, ISSUE
4
IDl3NTIFICATION
OF PAPER
COMPOUNDS.
II
557
used with basic compounds; the latter would necessitate raising the pH of the aqueous eluate before extraction with an organic solvent. Acid degradation of tolbutamide and identification of the resulting p-toluenesulfonamide offers confirmatory proof of st’ructurr. p-Tohlenesulfonamide does not form a salt on the paper and simple dripelution with methylene chloride is satisfactory. The spectnlm of the chromatographed compound is essentially identical t,o a refcrcl1ce spectrum of pure p-toluenesulfonamide. Isolation and infrared characterization of p-carboxybenzencsl~lfonamide, produced by acid degradation of the urinary metabolite of tolbutamide, proved the most difficult problem in this study. Since this sulfonamide is a relatively strong acid and exists as a salt on the chromstogram, a simple elution cannot be used. Aqueous ehltion followed by acidification and extraction as employed for tolbutamide is unat,tractive because p-carboxybenzenesulfonamide is extracted only with considerable dif5culty.R To overcome these difliculties, a procedure has been developed based on regeneration of the sulfonamide on the chromatogram by exposure to gaseous hydrochloric acid. Following removal of the acid vapors, the p-carboxybenzenesulfonamide is eluted with ethyl ether. Care must be taken to remove all traces of hydrochloric acid vapors from the paper before elution is attempted since residual chlorinated vapors appear to cause decomposition of the sample. The infrared spectrum of the eluted material is essentially the same as the spectrum of authentic p-carboxybenzenesulfonamide. This technique should be generally useful for compounds possessing chemical and physical properties similar to p-carboxybenzenesulfonamide. Furthermore, a variation of the same technique should be applicable to basic compounds. Exposure of the chromatographic zone to a basic vapor, such as ammonia, should regenerate an alkaline compound and permit a subsequent elution to be carried out. CONCLUSIONS Procedures have been developed for the isolation and infrared characterization of some acidic materials separated by paper chromatography. Alkaline materials might also be handled by variations of the basic principles involved. For those compounds existing on the paper chromatogram as
558
A. F. KRIVIS
AND A. A. FORIST
salts, two approaches are possible. If the non-ionic speciesis readily extracted from water by an acceptable organic solvent, elution of the salt with water, acidification of the eluate, and extraction into the organic solvent constitute the method of choice. If, however, the non-ionic speciesis poorly extractable, an alternative procedure based on conversion of the salt to the corresponding acid directly on the paper by exposure to gaseous hydrochloric acid followed by dripelution with an acceptable organic solvent may be employed. Residues from the extract or the eluate yield useful infrared spectra. The authors are indebted to W. A. Struck and R. W. Rinehart for helpful
suggestionsand to Mrs. S. T. Madden and R. W. Judy for excellent technical assistance.
References 1. Cassidy, H. G., Fundamentals of Chromatography, Interscience, New YorkLondon, 1957. 2. Krivis, A, F., G. E. Bronson, W. A. Struck, and J. L. Johnson, Submicrogram Experimentation, N. D. Cheronis, Ed., Interscience, New York-London, 1961, p. 277. 3. Rosenkrantz, H., Ann. N. Y. Acud. Sci., 69,5 (1957). 4. Toribara, T. Y., and V. Di Stefano, Anal. Chem., 26, 1519 (1954). 5. Miller, W. L., Jr., J. J. Krake, M. J. Vander Brook, and L. M. Reineke, Ann. N. Y. Acad. Sci., 71,118 (1957). 6. Louis, L. H., S. S. Fajans, J. W. Corm,W. A. Struck, J. B. Wright, and J. L. Johnson, J. Am. Chem. Sot., 78, 5701 (1956). 7. Forist, A. A., and T. Chulski, Metabolism, 5,807 (1956). 8. Tillson, E. K., N. W. Pusey, and K. H. Beyer, J. Pharmacol. Exptl. Therap., 112,252 (1954).
Received January 12, 1961
MICROCHEMICAL
JOURNAL,
VOL.
V. ISSUE
4
VOL.
JOURNAL
Infrared Chromatographed
Identification
553358
(1961)
of Paper
Compounds.
for Some Acidic ALAK
V, PAGES
II.
Procedures
Compounds
1’. KRIVIS* and ARLINGTON A. FORIST, Company, Kalamazoo, Michigan
Z’he CJpjohn
The isolation and identification of individual organic compounds, such as drugs and their metabolites, in biological systems is usually complicated by the presence of small quantities of the desired material and overwhelming amounts of many extraneous substances. Yermentation beers and complex organic reaction mixtures may present a similar problem. Paper chromatography has proved to be an excellent tool for the separation of minute amounts of material in crude mixtures,’ and techniques for the resolution of a wide variety of mixtures containing difficultly separable, similar compounds have been developed. Biological samples, in particular, have been very effectively handled by the separatory paper chromatographic methods. Unfortunately, powers of paper chromatography have not been matched by the ability to positively identify or characterize the compounds isolated. Infrared spectroscopy, however, affords the most generally useful and exact, identification that any single measurement can provide. Recently the separation of compounds by paper chromatography, followed by infrared characterization of the isolated material has been studied and found to be a useful procedure.2-4 To date, m&hods involving elution of a chromatographed spot and infrared examination of the eluate have been limited to situations where the compound was reasonably soluble in an acceptable solvent,. It has been found t]hat a great many types of compounds cannot bc * Present address: Central Analytical Corporation, New Haven, Connecticut.
Laboratories, 553
Olin Mathieson
Chemical
554
A. F. KRIVIS
AND A. A. FORIST
eluted from the developed chromatographic spot with nonpolar organic solvents. In general, polar solvents must be avoided because of the extraneous contaminants which they elute from the paper. Therefore, ionic species, such as salts, or compounds strongly adsorbed on the paper, are not amenable to the treatment previously used. Accordingly, alternative methods have been developed to circumvent these difficulties.
EXPERIMENTAL Reagents Potassium bromide, powdered, infrared quality (Harshaw Chemical Co.) was used to prepare all potassium bromide pellets. The organic eluents were purified prior to use, as described previously. 2 All other organic compounds and solvents were USP or reagent grade and were used without further purification. All inorganic chemicals used were reagent grade.
Apparatus A Perkin-Elmer Model 21 infrared spectrophotometer was used to obtain all of the infrared spectra. A Perkin-Elmer potassium bromide die was used in the manner previously described2 to produce all of the micropellets.
Procedure Tolbutamide A portion of human serum containing 75-125 Mg. of tolbutamide from a subject having received this drug was acidified with 5 ml. of 1.2M hydrochloric acid and extracted with 25 ml. of chloroform. The chloroform layer was then separated, extracted with 5 ml. of 1% aqueous sodium carbonate solution, and the separated aqueous layer acidified with 1 ml. of 1M hydrochloric acid. The acidified aqueous solution was extracted 5 times with 20 ml. of chloroform and the combined extracts evaporated to dryness. The extract residue was dissolved in methylene chloride and applied, in a streak, to a pre-washed sheet of Whatman No. 1 paper MICROCHEMICAL
JOURNAL,
VOL.
V, ISSUE
4
IDENTIFICATION
OF PAPER COMPOUNDS.
II
555
and developed by descending chromatography for 18 hr. in a butanolwater-piperidine (81:17:2) system.5 The tolbutamide zone on the dried, developed sheet was located by its ultraviolet absorption and was cut out and mounted in a drip eluter. Elution was carried out with fifteen l-ml. portions of distilled water. The aqueous eluate was acidified to pH 1 with hydrochloric acid and extracted twice with 15 ml. of methylene chloride. The methylene chloride solution was evaporated to about 1 ml., potassium bromide added, the mixture evaporated to dryness, and a micropellet prepared. An infrared spectrum of the material suspended on the potassium bromide was then obtained. p-Toluenesuljonamide
Tolbutamide from a sample of serum was extracted, chromatographed, and eluted as described above. The isolated tolbutamide was then hydrolyzed by heating under reflux with 5 ml. of 2.4N hydrochloric acid for 2 hr. The cooled solution was extracted 5 times with 25 ml. of ethyl ether, the organic phase evaporated to a small volume and chromatographed as described previously. The sulfonamide zone on the developed chromatogram was eluted with 12 ml. of methylene chloride and an infrared spectrum of the eluate obtained. p-Carboxybenzenesul$onamide
A sample of urine from a subject having received tolbutamide was extracted, the metabolite, l-butyl-3-p-carboxyphenylsulfonylurea, isolated as described for tolbutamide, hydrolyzed, and the resulting sulfonamide fraction carried through paper chromatography. The chromatographic zone of the hydrolyzed metabolite was treated with hydrochloric acid gas in a closed vessel for 2 hr. The paper strip was stored overnight under vacuum in a desiccator containing Ascarite and then eluted with 15 ml. of ethyl ether. An infrared spectrum of the eluate residue in potassium bromide was then ohtained. DISCUSSION Compounds eluted from chromatographic zones by certain organic solvents are sufficiently pure so that meaningful infrared spectra can be obtained.2 However, a great many compounds lack the
55ti
A. F. HRIVIS
AND A. A. FORIST
nccessury solubility in acceptable solvents to permit this direct approach. I’olar and ionic species, e.g., many drugs and their metabolit’es, fall in this category. In addition, some of the most useful chromatographic systems contain bases or acids which yield salt,s of t,hc chromatographed compounds on the paper. Therefore, m&hods have been developed for the elution and infrared identification of such polar or ionic materials following isolation by paper chromat,ography. Tolbutamide, 1-butyl-3-p-tolylsulfonylurea, has been chosen as a weakly acidic substance typical of many which might be encountered in studies of drug metabolism. In addition to tolbutamide, the sulfonamides obtained on acid degradation of this drug and its metab&e, I-butyl-3-p-carboxyphenylsulfonylurea,6 have been studied in order to demonstrate the powerful potential of this general approach to the solution of structure problems on small amounts of material. The three compounds discussed have quite different solubilities in wat)rr and in organic solvents and cover a wide range of acid strengths. In addition, since the most useful paper chromatographic system for these compounds contains piperidine, the problem of salt formation on the paper is encountered. Tolbutamide, pK, 5.43,7 can be extracted from acidified biological samples with chloroform, and a paper chromatographic separation of the sulfonylurea can eliminate the remaining contaminants from the serum. Direct elution of tolbutamide from such a papergram wit’h methylene chloride is impossible, whereas elution is readily accomplished with water; this behavior is indicative of a piperidinium salt on the paper. In addition to the sulfonylurea salt and excess piperidine, the aqueous eluate contains hemicelluloscs from the paper. However, acidification of the eluate regenerates the tolbutamide and permits its successful extraction with mcthylene chloride. An identification of the extracted tolbutamidc can then be made from its infrared spectrum. The technique described is general for weakly acidic mat,erials which in the non-ionic form can be extracted into a suitable organic solvent. The solvent used must, of course, not simultaneously extract the various contaminants present in the aqueous eluate. Since the fundamental principal involved is the enhancement of the solubility in an organic solvent over an aqueous one by adjustment of t,he pH, it is to be expected that the same general approach might be MICROCHEMICAL
JOURNAL,
VOL.
V, ISSUE
4
IDl3NTIFICATION
OF PAPER
COMPOUNDS.
II
557
used with basic compounds; the latter would necessitate raising the pH of the aqueous eluate before extraction with an organic solvent. Acid degradation of tolbutamide and identification of the resulting p-toluenesulfonamide offers confirmatory proof of st’ructurr. p-Tohlenesulfonamide does not form a salt on the paper and simple dripelution with methylene chloride is satisfactory. The spectnlm of the chromatographed compound is essentially identical t,o a refcrcl1ce spectrum of pure p-toluenesulfonamide. Isolation and infrared characterization of p-carboxybenzencsl~lfonamide, produced by acid degradation of the urinary metabolite of tolbutamide, proved the most difficult problem in this study. Since this sulfonamide is a relatively strong acid and exists as a salt on the chromstogram, a simple elution cannot be used. Aqueous ehltion followed by acidification and extraction as employed for tolbutamide is unat,tractive because p-carboxybenzenesulfonamide is extracted only with considerable dif5culty.R To overcome these difliculties, a procedure has been developed based on regeneration of the sulfonamide on the chromatogram by exposure to gaseous hydrochloric acid. Following removal of the acid vapors, the p-carboxybenzenesulfonamide is eluted with ethyl ether. Care must be taken to remove all traces of hydrochloric acid vapors from the paper before elution is attempted since residual chlorinated vapors appear to cause decomposition of the sample. The infrared spectrum of the eluted material is essentially the same as the spectrum of authentic p-carboxybenzenesulfonamide. This technique should be generally useful for compounds possessing chemical and physical properties similar to p-carboxybenzenesulfonamide. Furthermore, a variation of the same technique should be applicable to basic compounds. Exposure of the chromatographic zone to a basic vapor, such as ammonia, should regenerate an alkaline compound and permit a subsequent elution to be carried out. CONCLUSIONS Procedures have been developed for the isolation and infrared characterization of some acidic materials separated by paper chromatography. Alkaline materials might also be handled by variations of the basic principles involved. For those compounds existing on the paper chromatogram as
558
A. F. KRIVIS
AND A. A. FORIST
salts, two approaches are possible. If the non-ionic speciesis readily extracted from water by an acceptable organic solvent, elution of the salt with water, acidification of the eluate, and extraction into the organic solvent constitute the method of choice. If, however, the non-ionic speciesis poorly extractable, an alternative procedure based on conversion of the salt to the corresponding acid directly on the paper by exposure to gaseous hydrochloric acid followed by dripelution with an acceptable organic solvent may be employed. Residues from the extract or the eluate yield useful infrared spectra. The authors are indebted to W. A. Struck and R. W. Rinehart for helpful
suggestionsand to Mrs. S. T. Madden and R. W. Judy for excellent technical assistance.
References 1. Cassidy, H. G., Fundamentals of Chromatography, Interscience, New YorkLondon, 1957. 2. Krivis, A, F., G. E. Bronson, W. A. Struck, and J. L. Johnson, Submicrogram Experimentation, N. D. Cheronis, Ed., Interscience, New York-London, 1961, p. 277. 3. Rosenkrantz, H., Ann. N. Y. Acud. Sci., 69,5 (1957). 4. Toribara, T. Y., and V. Di Stefano, Anal. Chem., 26, 1519 (1954). 5. Miller, W. L., Jr., J. J. Krake, M. J. Vander Brook, and L. M. Reineke, Ann. N. Y. Acad. Sci., 71,118 (1957). 6. Louis, L. H., S. S. Fajans, J. W. Corm,W. A. Struck, J. B. Wright, and J. L. Johnson, J. Am. Chem. Sot., 78, 5701 (1956). 7. Forist, A. A., and T. Chulski, Metabolism, 5,807 (1956). 8. Tillson, E. K., N. W. Pusey, and K. H. Beyer, J. Pharmacol. Exptl. Therap., 112,252 (1954).
Received January 12, 1961
MICROCHEMICAL
JOURNAL,
VOL.
V. ISSUE
4