2612
Anal. Chem. 1982, 64, 2652-2654
TECHNICAL NOTES
Purification of @-Cyclodextrin Alkis J. Sophianopoulost and Isiah M. Warner'J Department of Chemistry, Emory University, Atlanta, Georgia 30322
INTRODUCTION The interactions of cyclodextrins (CDs) with a variety of other molecules to form inclusion complexes have been studied extensively because of their importance in understanding the factors involved in complex formation as well as for their practical applications. Many studies involve spectral methods.'-3 As the approaches to the detail of such interactions become more refined, the requirement of eliminating the intrusion of even a small amount of contaminants has become essential. Commercial preparations of the three cyclodextrins which have been studied extensively, a-, 8-, and y C D , are nearly free from any significant contamination of one saccharide by any of the other two. However, some preparations, particularly 8-CD,may contain contaminants which produce turbid solutions. This problem has been recognized for some time. Methods for the preparation of the three crystalline CDs have been described.415 A refinement of one of these methods4 involves allowing a 2 5% aqueous solution of 8-CD to stand at room temperature a considerable time in order to promote aggregation of insoluble impurities and complexes; the solution is then filtered through a fine fritted-glass filter before recrystallization.6 Filtering reduces turbidity considerably; however, more turbidity may appear on standing. Another approach is to filter individual solutions of the mixtures of the reactants through membrane filters prior to usee7 Occasionally, 8-CD is recrystallized from water,3,g13but in our experience, some insoluble contaminants seem to coprecipitate with the 8-CD crystals. Preparations 8-CD have also been recrystallized from ethanol and treated with ~harc0al.l~
* Author to whom correspondence should be addressed. Department of Biochemistry, School of Medicine, Emory University. Department of Chemistry, Louisiana State University, Baton Rouge, LA 70803. (1)Iehizuka, Yasuko; Nagawa, Yoshinory; Nakanishi, Hiroshi; Kuboyama, Akira. J. Incl. Phenom. 1990,9,219-225. (2) Saenger, V. W. Angew. Chem., Ind. Ed. Engl. 1980,19,344-362. (3)Sbnchez, F. Garcia; Lopez, M. Hernandez; Gbmez, J. C. Marquez. Analyst 1987 112,1037-1040. (4)French, Dexter; Pazur, John; Levine, Melvin L.; Norberg, Ethelda. J . Am. Chem. SOC.1949,71,353-356. (5)Cramer, Friedrich; Henglein, Friedrich-Martin. Chem. Ber. 1958, 91,308-310. (6) French, Dexter. In Aduances in Carbohydrate Chemistry; Wolfrom, M. L., Ed.; Academic Press: New York, 1957;Vol. 12,p 216. (7)Mularz, Edward A.; Cline-Love, Linda J.; Petersheim, Mathew. Anal. Chem. 1988,60, 51-55. (8)Harata, Kazuaki; Uedaira, Hisashi. Bull. Chem. SOC.Jpn. 1975,48, 87.5-878. - . - - . -. (9)Shimizu, Hiroshi; Kaito, Akira; Hatano, Masahiro. Bull. Chem. Soc. Jpn. 1979,52,2678-2684. (10)Shimizu, Hiroshi; Kaito, Akira; Hatano, Masahiro. Bull. Chem. SOC.Jpn. 1981,54,513-519. (11)Uekama, Kaneto; Hirayama, Fumitoshi; Wakuda, Toku; Otarigi, Masaki. Chem. Pharm. Bull. 1981,29,213-219. (12)Kurono, Yukihisa; Stamoudis,Vassilios; Bender, Myron L. Bioorg. Chem. 1976,5,393-402. (13)Buvari, A.; Szejtli, J.; Barcza, L. J . Incl. Phenom. 1983,I, 151t
* Present address:
167
_I..
(14)Ricci, Robert W.Carbohydr. Res. 1984,129,278-286. 0003-2700/92/0384-2652$03.00/0
Another approach is to crystallize 8-CD from boiling water and then rinse it with ethanol or acetone.15 A communication on a reaction involving 8-CD suggested the presence of noncarbohydrate impurities.'6 We describe here a method for preparing 0-CD which is essentially free from light-absorbing contaminants. The starting material is a commercial preparation of &CD, and therefare the method described simply improves the purity of what is already available.
EXPERIMENTAL SECTION Apparatus. Absorption spectra were recorded on a Varian Cary 3 UV-vis spectrophotometer, using 1-cm quartz cells. Circular dichroism measurements were performed on a JASCO 5-600 spectropolarimeter. Steady-state fluorescence measurements were performed on a SPEX-Fluorolog Model F2T21I spectrofluorometer. Materials. Although we used 8-CD from three sources initially,the 8-CDpreparation used for most of the work described here, as well as a-CD, were obtained from American Maize Products (Hammond, IN). The 2-propanol, electronic-grade, ACS-certified,and HPLC-grade water were obtained from Fisher Scientific. All other reagents were ACS certified grade. Membrane filters, Supor-200, 0.2 pm, were obtained from Gelman Sciences, Inc., Ann Arbor, MI. Methods. If possible, an all-glass apparatus should be used in the purification process. Since glass stoppers are inconvenient in many steps, polyethylene or polypropylene stoppers are safe if, after heating in 2-propanol-water mixtures, no light-absorbing impurities can be detected in the solvent. Rubber tubing and rubber stoppers, even neoprene or silicon ones, contain lightabsorbing impurities which may be extracted by the solvent. Also, the 2-propanol used was from freshly opened containers. The volumes stated are additive, so that a 35% v/v 2-propanol solution means that 35 mL of 2-propanol were mixed with 65 mL of water. The concentration expressed as percent of 6-CD means grams of 6-CD dissolved in 100 mL of the solvent mixture. The increase in volume produced by dissolution of 6-CD is ignored. To increase the concentration of 2-propanol from 35 % to 60 % , the volume of 2-propanol to be added is calculated by considering the known volumes of 2-propanol and water in the 35% 0-CD solution. Thus, to a solution of 0-CD which contains 35 mL of 2-propanol and 65 mL of water, 62.5 mL of 2-propanol are added. The volume of 0.1 M NaOH or HCl to be added is counted as part of the water present. The values of the pH of the mixture stated are pH meter readings, not necessarily true pH. The largest amount of starting material used has been 15 g. Purification Procedure. The steps of the preparation are as follows: 1. A solution of 7.5-8.5% 0-CDsolution in 35 % v/v 2-propanol is prepared by stirring and heating if necessary, and approximately 1.5 mL of 0.1 M NaOH per 100 mL of solution is added, to raise the pH to 10.5-11. The solution may be turbid at this point. It is stoppered and heated at 70 "C for approximately 20 h in a (15)Wei,Yansheng; Liu, Changsong;Zhang,Sushe.HuaxueShiji 1991, 13, 249-251. (16)French, Dexter; Pazur, John; Levine, MelvinL.; Norberg, Ethelda. J. Am. Chem. Soc. 1948,70,3145. 0 1992 Amerlcan Chemical Society
ANALYTICAL CHEMISTRY, VOL. 64, NO. 21, NOVEMBER 1, 1902 2853 0.14
o.80M 1 0.40
0.00 L 220
I
240
280
280
220
300
Flgure 1. Absorption spectra of a 12 mM aqueous solution of @-cyclodextrlnbefore purification. Curve 1: Spectra taken shortly after preparation of the solution. Curve 2: The same solution at pH 10, after standing at room temperature for approximately 1 h. Curve 3: The same solution after heating at 70 O C for 16-20 h.
t
Anal. Chem. 1982, 64, 2652-2654
TECHNICAL NOTES
Purification of @-Cyclodextrin Alkis J. Sophianopoulost and Isiah M. Warner'J Department of Chemistry, Emory University, Atlanta, Georgia 30322
INTRODUCTION The interactions of cyclodextrins (CDs) with a variety of other molecules to form inclusion complexes have been studied extensively because of their importance in understanding the factors involved in complex formation as well as for their practical applications. Many studies involve spectral methods.'-3 As the approaches to the detail of such interactions become more refined, the requirement of eliminating the intrusion of even a small amount of contaminants has become essential. Commercial preparations of the three cyclodextrins which have been studied extensively, a-, 8-, and y C D , are nearly free from any significant contamination of one saccharide by any of the other two. However, some preparations, particularly 8-CD,may contain contaminants which produce turbid solutions. This problem has been recognized for some time. Methods for the preparation of the three crystalline CDs have been described.415 A refinement of one of these methods4 involves allowing a 2 5% aqueous solution of 8-CD to stand at room temperature a considerable time in order to promote aggregation of insoluble impurities and complexes; the solution is then filtered through a fine fritted-glass filter before recrystallization.6 Filtering reduces turbidity considerably; however, more turbidity may appear on standing. Another approach is to filter individual solutions of the mixtures of the reactants through membrane filters prior to usee7 Occasionally, 8-CD is recrystallized from water,3,g13but in our experience, some insoluble contaminants seem to coprecipitate with the 8-CD crystals. Preparations 8-CD have also been recrystallized from ethanol and treated with ~harc0al.l~
* Author to whom correspondence should be addressed. Department of Biochemistry, School of Medicine, Emory University. Department of Chemistry, Louisiana State University, Baton Rouge, LA 70803. (1)Iehizuka, Yasuko; Nagawa, Yoshinory; Nakanishi, Hiroshi; Kuboyama, Akira. J. Incl. Phenom. 1990,9,219-225. (2) Saenger, V. W. Angew. Chem., Ind. Ed. Engl. 1980,19,344-362. (3)Sbnchez, F. Garcia; Lopez, M. Hernandez; Gbmez, J. C. Marquez. Analyst 1987 112,1037-1040. (4)French, Dexter; Pazur, John; Levine, Melvin L.; Norberg, Ethelda. J . Am. Chem. SOC.1949,71,353-356. (5)Cramer, Friedrich; Henglein, Friedrich-Martin. Chem. Ber. 1958, 91,308-310. (6) French, Dexter. In Aduances in Carbohydrate Chemistry; Wolfrom, M. L., Ed.; Academic Press: New York, 1957;Vol. 12,p 216. (7)Mularz, Edward A.; Cline-Love, Linda J.; Petersheim, Mathew. Anal. Chem. 1988,60, 51-55. (8)Harata, Kazuaki; Uedaira, Hisashi. Bull. Chem. SOC.Jpn. 1975,48, 87.5-878. - . - - . -. (9)Shimizu, Hiroshi; Kaito, Akira; Hatano, Masahiro. Bull. Chem. Soc. Jpn. 1979,52,2678-2684. (10)Shimizu, Hiroshi; Kaito, Akira; Hatano, Masahiro. Bull. Chem. SOC.Jpn. 1981,54,513-519. (11)Uekama, Kaneto; Hirayama, Fumitoshi; Wakuda, Toku; Otarigi, Masaki. Chem. Pharm. Bull. 1981,29,213-219. (12)Kurono, Yukihisa; Stamoudis,Vassilios; Bender, Myron L. Bioorg. Chem. 1976,5,393-402. (13)Buvari, A.; Szejtli, J.; Barcza, L. J . Incl. Phenom. 1983,I, 151t
* Present address:
167
_I..
(14)Ricci, Robert W.Carbohydr. Res. 1984,129,278-286. 0003-2700/92/0384-2652$03.00/0
Another approach is to crystallize 8-CD from boiling water and then rinse it with ethanol or acetone.15 A communication on a reaction involving 8-CD suggested the presence of noncarbohydrate impurities.'6 We describe here a method for preparing 0-CD which is essentially free from light-absorbing contaminants. The starting material is a commercial preparation of &CD, and therefare the method described simply improves the purity of what is already available.
EXPERIMENTAL SECTION Apparatus. Absorption spectra were recorded on a Varian Cary 3 UV-vis spectrophotometer, using 1-cm quartz cells. Circular dichroism measurements were performed on a JASCO 5-600 spectropolarimeter. Steady-state fluorescence measurements were performed on a SPEX-Fluorolog Model F2T21I spectrofluorometer. Materials. Although we used 8-CD from three sources initially,the 8-CDpreparation used for most of the work described here, as well as a-CD, were obtained from American Maize Products (Hammond, IN). The 2-propanol, electronic-grade, ACS-certified,and HPLC-grade water were obtained from Fisher Scientific. All other reagents were ACS certified grade. Membrane filters, Supor-200, 0.2 pm, were obtained from Gelman Sciences, Inc., Ann Arbor, MI. Methods. If possible, an all-glass apparatus should be used in the purification process. Since glass stoppers are inconvenient in many steps, polyethylene or polypropylene stoppers are safe if, after heating in 2-propanol-water mixtures, no light-absorbing impurities can be detected in the solvent. Rubber tubing and rubber stoppers, even neoprene or silicon ones, contain lightabsorbing impurities which may be extracted by the solvent. Also, the 2-propanol used was from freshly opened containers. The volumes stated are additive, so that a 35% v/v 2-propanol solution means that 35 mL of 2-propanol were mixed with 65 mL of water. The concentration expressed as percent of 6-CD means grams of 6-CD dissolved in 100 mL of the solvent mixture. The increase in volume produced by dissolution of 6-CD is ignored. To increase the concentration of 2-propanol from 35 % to 60 % , the volume of 2-propanol to be added is calculated by considering the known volumes of 2-propanol and water in the 35% 0-CD solution. Thus, to a solution of 0-CD which contains 35 mL of 2-propanol and 65 mL of water, 62.5 mL of 2-propanol are added. The volume of 0.1 M NaOH or HCl to be added is counted as part of the water present. The values of the pH of the mixture stated are pH meter readings, not necessarily true pH. The largest amount of starting material used has been 15 g. Purification Procedure. The steps of the preparation are as follows: 1. A solution of 7.5-8.5% 0-CDsolution in 35 % v/v 2-propanol is prepared by stirring and heating if necessary, and approximately 1.5 mL of 0.1 M NaOH per 100 mL of solution is added, to raise the pH to 10.5-11. The solution may be turbid at this point. It is stoppered and heated at 70 "C for approximately 20 h in a (15)Wei,Yansheng; Liu, Changsong;Zhang,Sushe.HuaxueShiji 1991, 13, 249-251. (16)French, Dexter; Pazur, John; Levine, MelvinL.; Norberg, Ethelda. J. Am. Chem. Soc. 1948,70,3145. 0 1992 Amerlcan Chemical Society
ANALYTICAL CHEMISTRY, VOL. 64, NO. 21, NOVEMBER 1, 1902 2853 0.14
o.80M 1 0.40
0.00 L 220
I
240
280
280
220
300
Flgure 1. Absorption spectra of a 12 mM aqueous solution of @-cyclodextrlnbefore purification. Curve 1: Spectra taken shortly after preparation of the solution. Curve 2: The same solution at pH 10, after standing at room temperature for approximately 1 h. Curve 3: The same solution after heating at 70 O C for 16-20 h.
t
