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Two-step separation of xanthine derivatives on dowex-50 utilizing an eluant effect
ANALYTICAL
BIOCHEMISTRY
Two-Step
53, 288-289 (1973)
Separation
on Dowex-50
of
Xanthine
Utilizing
Derivatives
an Eluant
Effect
An improved method of purifying xanthine derivatives in urine samples or in complex chemical reaction mixtures relies on an eluent effect which influences the elution pattern of several xanthine derivatives from Dowex-50. Normally, the stronger the eluting acid, the more readily a compound is eluted from a given column of Dowex-50. We have now, however, observed changes in the ratios of elution volumes and even reversal of elution sequences on elution with different concentrations of acids1 An example is given in Table 1, which shows the elution volumes of 1-hydroxyxanthine and several of its methyl derivatives (3). Upon preparative ion-exchange chromatography with 0.1 N HCl as eluent, 1-hydroxy-3-methylxanthine and 1-hydroxy-9-methylxanthine are well separated from I-methoxyxanthine and 1-hydroxyxanthine. The latter two compounds are not as well separated from one another, but subsequent chromatography on the same column with 1.1 N HCl separates this pair very well. One single chromatography will not separate a11 four compounds in one run. Also, when NaCl is present it is eluted together with the l-hydroxy-9-methylxanthine by 0.1 N HCl, but Elution
TABLE 1 Volumes of Xanthine Derivatives 0.1
I-Hydroxy-3-methylxanthine I-Methoxyxanthine 1-Hydroxyxanthine 1-Hydroxy-9-methylxanthine
N
55 77 120 208
from Dowex-BOO
HCI
1.1
(12) (18) (23) (29)
28 35 21 24
N
HCl
(5.5) (7.0) (4.2) (4.5)
Pk’ 0.27 0.6 0.85 1.23
* The chromatography column, 9 X 140 mm, contained BioRad AG 5OW-X8, minus 400 mesh, cation-exchange resin, eluted with 40 ml/hr at 23”. The elution volumes in milliliters are from the beginning of elution to the center of each peak. Peak widths at H/2, in milliliters, are given in parentheses. These are with samples of 0.2 mg. The pK’s of first protonation were determined spectroscopically according to Albert and Serjeant
(2). ‘Reversal of the elution sequence for xanthine and 8-methylmercaptoxanthine on Dowex-50 by 1 N as compared with 0.05 N HCI has been reported in a previous paper (1). 288 Copyright @ 1973 by Academic Press, Inc. All rights of reproduction in any form reserved.
289
SHORT COMMUNICATIONS
rechromatography with 1.1 N HCI separates 1-hydroxy-9-methylxanthine from the salt. In several instances we have utilized this effect to purify urinary trace metabolites in two steps in the presence of considerable amounts of impurities that could not be separated by a single step of chromatography (3). A similar effect of the change of acid concentration was described for anion-exchange chromatography by Cohn (4)) and for metal ions by Diamond (5). The results described here for xanthine derivatives’ may be more general. It. allows the advantages of Dowex-50 chromatography to be utilized in two subsequent chromatographical steps with different separation characteristics. We are unable to relate this effect to the pK values of the compounds studied (Table 1). Perhaps the hydration of the purine cations is involved, as noted by R. M. Diamond (5) for hydrated metal ions. ACKNOWLEDGMENT This Institute
investigation (Grant CA
was supported 08748) and from
in part by funds from the National the American Cancer Society (Grant
Cancer BC-32).
REFERENCES 1. BIRDSALL, N. J. M., W~LCKE, U., LEE, T.-C., AND BROWN, G. B. (1971) Tetrahedron 27, 5969. 2. ALBERT, A., AND SERJEANT, E. P. (1962) “Ionization Constants of Acids and Bases,” Wiley, New York. 3. ST~HFLER,G. (1972) Biochemistry 11, 4844. 4. COHN, W. E. (1955) in “The Nucleic Acids” (E. Chargti and J. N. Davidson, eds.), Vol. 1, p. 218, Academic Press, New York. 5. DIAMOND, R. M., AND WHITNEY, D. C. (1966) in “Ion Exchange” (J. A. Marinsky, ed.), Vol. 1, p. 329, Marcel Dekker, Inc., yew York.
GERHARD STSHRER ELAINE CORBIN GEORGE BOSWORTH Division of Biological Chemistry The Sloan-Kettering Institute for New York 10021 Received July 8’, 1972; accepted
Cancer November
Research
Z4, 1972
BROWN
BIOCHEMISTRY
Two-Step
53, 288-289 (1973)
Separation
on Dowex-50
of
Xanthine
Utilizing
Derivatives
an Eluant
Effect
An improved method of purifying xanthine derivatives in urine samples or in complex chemical reaction mixtures relies on an eluent effect which influences the elution pattern of several xanthine derivatives from Dowex-50. Normally, the stronger the eluting acid, the more readily a compound is eluted from a given column of Dowex-50. We have now, however, observed changes in the ratios of elution volumes and even reversal of elution sequences on elution with different concentrations of acids1 An example is given in Table 1, which shows the elution volumes of 1-hydroxyxanthine and several of its methyl derivatives (3). Upon preparative ion-exchange chromatography with 0.1 N HCl as eluent, 1-hydroxy-3-methylxanthine and 1-hydroxy-9-methylxanthine are well separated from I-methoxyxanthine and 1-hydroxyxanthine. The latter two compounds are not as well separated from one another, but subsequent chromatography on the same column with 1.1 N HCl separates this pair very well. One single chromatography will not separate a11 four compounds in one run. Also, when NaCl is present it is eluted together with the l-hydroxy-9-methylxanthine by 0.1 N HCl, but Elution
TABLE 1 Volumes of Xanthine Derivatives 0.1
I-Hydroxy-3-methylxanthine I-Methoxyxanthine 1-Hydroxyxanthine 1-Hydroxy-9-methylxanthine
N
55 77 120 208
from Dowex-BOO
HCI
1.1
(12) (18) (23) (29)
28 35 21 24
N
HCl
(5.5) (7.0) (4.2) (4.5)
Pk’ 0.27 0.6 0.85 1.23
* The chromatography column, 9 X 140 mm, contained BioRad AG 5OW-X8, minus 400 mesh, cation-exchange resin, eluted with 40 ml/hr at 23”. The elution volumes in milliliters are from the beginning of elution to the center of each peak. Peak widths at H/2, in milliliters, are given in parentheses. These are with samples of 0.2 mg. The pK’s of first protonation were determined spectroscopically according to Albert and Serjeant
(2). ‘Reversal of the elution sequence for xanthine and 8-methylmercaptoxanthine on Dowex-50 by 1 N as compared with 0.05 N HCI has been reported in a previous paper (1). 288 Copyright @ 1973 by Academic Press, Inc. All rights of reproduction in any form reserved.
289
SHORT COMMUNICATIONS
rechromatography with 1.1 N HCI separates 1-hydroxy-9-methylxanthine from the salt. In several instances we have utilized this effect to purify urinary trace metabolites in two steps in the presence of considerable amounts of impurities that could not be separated by a single step of chromatography (3). A similar effect of the change of acid concentration was described for anion-exchange chromatography by Cohn (4)) and for metal ions by Diamond (5). The results described here for xanthine derivatives’ may be more general. It. allows the advantages of Dowex-50 chromatography to be utilized in two subsequent chromatographical steps with different separation characteristics. We are unable to relate this effect to the pK values of the compounds studied (Table 1). Perhaps the hydration of the purine cations is involved, as noted by R. M. Diamond (5) for hydrated metal ions. ACKNOWLEDGMENT This Institute
investigation (Grant CA
was supported 08748) and from
in part by funds from the National the American Cancer Society (Grant
Cancer BC-32).
REFERENCES 1. BIRDSALL, N. J. M., W~LCKE, U., LEE, T.-C., AND BROWN, G. B. (1971) Tetrahedron 27, 5969. 2. ALBERT, A., AND SERJEANT, E. P. (1962) “Ionization Constants of Acids and Bases,” Wiley, New York. 3. ST~HFLER,G. (1972) Biochemistry 11, 4844. 4. COHN, W. E. (1955) in “The Nucleic Acids” (E. Chargti and J. N. Davidson, eds.), Vol. 1, p. 218, Academic Press, New York. 5. DIAMOND, R. M., AND WHITNEY, D. C. (1966) in “Ion Exchange” (J. A. Marinsky, ed.), Vol. 1, p. 329, Marcel Dekker, Inc., yew York.
GERHARD STSHRER ELAINE CORBIN GEORGE BOSWORTH Division of Biological Chemistry The Sloan-Kettering Institute for New York 10021 Received July 8’, 1972; accepted
Cancer November
Research
Z4, 1972
BROWN