- Email: [email protected]
Use of cyclodextrins in isotachophoresis
Journal of Chromatography, 405 (1987) 379-384 Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands CHROM. 19 755
Note
Use of cyclodextrins I. Effect of cyclodextrin penicillins
in isotachophoresis on the isotachophoretic
separation
of related
I. JELiNEK* Research Institute for Pharmacy and Biochemistry, KouFimskb 17, I36 00 Prague 3 (Czechoslovakia) and J. SNOPEK and E, $MOLKOVti-KEULEMANSOVA Department of Analytical Chemistry, Charles University. Albertov 2030, 128 40 Prague 2 (Czechoslovakia) (First received May 7th, 1987; revised manuscript received June lst, 1987)
Separations by isotachophoresis (ITP) are usually performed in non-packed capillary columns. The separability of the injected components is determined by the differences in their effective mobilities which are in turn mainly influenced by their degrees of dissociation and absolute ionic mobilities. The degree of dissociation can easily be varied experimentally by changing the pH of the leading electrolyte. It is much more complicated to affect the ionic mobility. This problem can be overcome by using a complex-forming counter ion. The theory of eomplex formation equilibria and practical applications mainly to inorganic ions were described by Gebauer and BoCeki. Complex formation involving organic ions has been much less investigated. The use of Ca2 ’ and 1,3-bis[tris(hydroxymethyl)methylamino]propane for selective retardation of organic anions was described2s3. Some neutral complex-forming agents, such as l&crown-4 ether4 and a-cyclodextrin5, were used for the separation of inorganic ions. Qnly little attention has been devoted to the use of cyclodextrins (CDs) in isotachophoretic separation of organic ions. CDs may form inclusion complexes with many different compounds both in the solid state and in aqueous solution. The selective formation of inclusion complexes, preferentially with certain types of compounds, based on structural differences is widely utilized for separations and analytical purposes using various chromatographic procedureF*. The CDs can be used either as an aqueous mobile phase component in thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) or more effectively as a stationary phase in TLC, HPLC and gas chromatography. The aim of this paper is to present some experiments which may contribute to understanding the role of CDs in ITP separations. We tried to separate mixtures of sulbactam synthesis intermediates and of sulbactam (drug of penicillin type) and its alkaline degradation product with an open fi-lactam ring, which causes an allergic reaction All these compounds are structurally similar and their ITP separation in 0021-9673/%7/$03.50
@) 1987 Elsevier Science Publishers B.V.
NC.YI‘ES
380
classical electrolyte systems is very difficult. The effects of the various adjustable parameters, especially the concentration of CDs in the leading electrolyte (LE), the pH of the LE and the dimensions of the CD ring, were studied experimentally and used to optimize the ITP separation process. EXPERIMENTAL
Chemicals
Redistilled water was used in the preparation of the electrolyte solutions and of the solutions of model mixtures. All chemicals were of the highest quality commercially available and were used without any purification: 37% hydrochloric acid, sodium hydroxide, 4-morpholineethanesulphonic acid (MES), fl-alanine (Merck, Darmstadt, F.R.G.); 6-aminocaproic acid (EACA) (Sigma, St. Louis, MO, U.S.A.); hydroxypropylmethylcellulose (HPMC) (Dow Chemical, Midland, MI, U.S.A.); caproic acid (Lachema, Brno, Czechoslovakia); a- and y-cyclodextrins (CX-and y-CDs) (ASTEC, Whippany, U.S.A.); /I-cyclodextrin (/?-CD) (Chinoin, Budapes, Hungary). The solutes investigated were obtained from VUFB (Prague, Czechoslovakia). Their formulae are given in Table I.
ITP experiments were performed with a Tachophor 2127 (LKB, Bromma, Sweden) equipped with a conductivity detector and a polytetrafluorethylene (PTFE) capillary. Injections were made with lo-p1 Hamilton syringe. For conditions see Table II.
TABLE I THE COMPOUNDS
INVESTIGATED
o=H&2 v
Rb3 as/p
0
0
$32 5
n
C”3
CH
R
CH3 coo-
1.2
m+
0’
N 3.4
CH 3
3
=t43
N
cooM+
A
A,
C”3
cooM+
Compound
R
M+
Name
I
H
Na+
2
Br
K+
3
Br
K+
4
H
Na+
5
H
Na+
Penicillanic acid sodium (3,3-dimethyl-7-oxo-4-thia-lazabicyclo[3.2.0]heptane-2-carboxylate sodium salt) 6,6-Dibromopenicilanic acid potassium salt (6,6-dibromo-3,3-dimethyl7-oxo-4-thia-l-azabicyclo[3.2.0]heptane-2-carboxylate potassium salt) 6,6-dibromopenicillanic acid sulphone potassium salt (6,6-dibromo-3,3dimethyl-4,4,7-trioxo-4-thia-l-azabicyclo[3.2.0]heptane-2-carboxylate potassium salt) Sulbactam sodium salt (penicillanic acid sulphone sodium salt; 3,3dimethyl-4,4,7-trioxo-4-thia-l-azabicyclo[3.2,O]heptane-2-carboxylate sodium salt) Alkaline degradation product of Sulbactam (2-carboxymethyl-5,5dimethyl-l.l-dioxothiazaohdine-4-carboxylate sodium salt)
NOTES
381
TABLE II ELECTROLYTE
SYSTEMS AND CONDITIONS
Leading electrolyte (LE)
FOR ITP
I, 5 mM Hydrochloric acid including 0.2% HPMC with EACA to pH 4.30, 4.55, 4.65 and 4.80 II, as I but including 0.2% HPMC with B-alanine to pH 3.50 and 4.00 I, 5 mM MES II, 5 mA4 caproic acid 400 mm x 0.5 mm I.D., 520 mm x 0.5 mm I.D. 18°C Conductivity 100 pA (9 min); for detection 50 PA 2 pl of mixture I (compounds l-4, each 5 mM); 4 ~1 of mixture II (compounds 4 and 5, each 2.5 mM)
Terminating electrolyte (TE) Capillaries Temperature Detection Current Injected volumes
RESULTS AND DISCUSSION
The choice of electrolyte system for ITP separation of related penicillins is influenced by several factors. The use of alkaline electrolyte systems’ may be partially
I
1lME
I
Fig. 1. Effect of b-CD on the ITP separation of mixture I in LE I (pH 4.80) with TE I and capillary of length 520 mm. Concentrations, of J-CD in LE: (A) without b-CD; (B) 0.5 mM; (C) 1 mM; (D) 5 mM; (E) 10 m&f.
382
NOTES
successful, but the stability of penicillins in alkaline solution is poor and degradation may occur during ITP. Therefore the stability of sulbactam and its intermediates in aqueous solution was studied. It is known that complex formation with CDs may change the reactivity of a guest molecule. This property, connected with the ability to catalyze some reactions, was recognized by Cramerg. The P-CD catalyzed hydrolysis of penicillins was studied by Tutt and Schwarz**. The rate of penicillin hydrolysis, in the presence of P-CD, is increased 20&X0fold relative to the non-catalyzed reaction. Our stability experiments showed, however, that the sulbactam degradation during ITP is negligible. This type of penicillin proved to be stable even in a slightly acidic aqueous solution, pH 3.548, saturated with /?-CD. Fig. 1 shows the ITP separation of mixture I (see Table II) in a slightly acidic electrolyte system pH 4.8 where all the solutes are stable. The resolution of four components without B-CD is very poor. Only partially separated mixed zones could be seen on isotachopherogram A. Qualitative and quantitative evaluation is impossible. With increasing amount of P-CD in the LE the separation is improved. A distinct separation of components 3 and 2 occurs in the presence of 0.5 mM #?-CD. Components 4 and 1 are still not separated (Fig. 1B). Optimum separation of all the solutes is achieved with 5 and 10 mM B-CD (Fig. 1D and E). A comparison between Fig. IA and E clearly shows great differences in separation quality. When /3-CD is used in electrolyte systems the shape and spatial arrangement of the molecules to be separated become important factors. Even more critical is the separation of Sulbac-
Fig. 2. ITP separation of mixture I1 in LE I (PH 4.80) with TE I and capillary of length 520 mm: (A) LE without P-CD: (B) 10 mM /?-CD in LE. Fig. 3. Effect of (A, C) starch (567.4 mg per 100 ml LE) and (B, D) D-ghcOSe (1134.8 mg per 100 ml LE) on ITP separation of mixtures I and II in LE 1 (pH 4.8) with TE I and a capillary lengths of 520 mm.
NOTES
383
tam and its degradation product. The good resolution with 10 mM /?-CD is compared to that without P-CD in Fig. 2. The results suggest that the geometrical arrangements of the molecules again play a significant role in the separation process. Comparative measurements with D-glucose and starch were carried out to show the role of the cyclic structure of CD in ITP separation. D-Glucose was used as a model of the building units of CD and starch as a compound with helical structure but without rings. Fig. 3 shows that neither of these additives improves the separation and only the cyclic structure of CD provides favourable resolution. The CD ring diameter is an important factor in the separation. Experiments in which P-CD was replaced by its a- and y-analogues did not provide statisfactory results. Only a slight improvement in separation of both dibromoderivatives, in comparison with classical electrolytes, was observed. The separation of mixture I (see Table II) depends significantly on the pH value of the LE (Fig. 4). Two electrolyte systems were used to cover the range from pH 3.50 to 4.80. Penicillanic acid(l) and its dibromosulphone derivative behave as a reversed ion pair, where the more mobile constituent has the lower equilibrium constant. The isotachopherogram shows that these constituents are not separated at pH 4.30. Both lower and higher pH values give a resolution. Tt can be concluded that the lower pH value (Fig. A) gives a better resolution and sharper zone boundaries than the higher one (Fig. 4F).
Fig. 4. Effect of pH on the ITP separation of mixture I in LE II with TE 11 at pH 3.50, (A) and 4.00 (B). and in LE I with TE I at pH 4.30 (C), 4.55 (D), 4.65 (E) and 4.80 (F). Capillary lengths: 400 mm Concentration of j-CD in LE is 10 mM.
384
NOTES
The results indicate that complex-formation equilibria with CDs are undoubtedly a significant factor which influence effective mobilities and thus the possibility of ITP separation. It can be assumed that CDs do not migrate in the column during ITP due to their high pK, values (12.2-12.6) l*. CD, as a complex-forming agent added to the LE, acts as a quasi-stationary phase. The nature of the separation effect of CDs in TTP and HPLC seems to be similar. The results of all our experiments are in accord with inclusion complex theory. The addition of a CD to the LE can be compared with the use of CD-bonded columns. The consumption of CD in ITP, compared with that in an HPLC mobile phase, is negligible. The ITP capillary column need not be equilibrated with large volumes of CD-containing mobile phase. Moreover there is no hydrodynamic flow during ITP. The composition of the two model mixtures, was not accidental. The possibility of analytical evaluation of the compounds present in these mixtures is important for estimation of the purity of sulbactam. Optimization of their ITP analysis in classical electrolyte systems was only partially successful. The effect of #I-CD in a slightly acidic electrolyte is very advantageous in ITP. The experiments with c+ and y-CD did not provide satisfactory results. The use of CDs in ITP will certainly not be restricted to penicillins. Further research other possible applications is being carried out, and on the detailed interaction mechanism, based on the investigation of ortha-, meta- and para-substituted benzoic acids. REFERENCES 1 P. Gebauer and P. Bocek, in P. BoEek (Editor), Isotachophoresis-Basic Course, Advanced Course, ITP-84, Hradec Krcilovd, September 2-6, 1984, Institute of Radioecology and Applied Nuclear Techniques, Plant for Development and Production of Nuclear Instruments, SpiSskl Nov9 Ves, 1984, p. 78. 2 D. Kaniansky and F. M. Everaerts, J. Chromatogr., 148 (1978) 441. 3 D. Kaniansky, V. Madajovb, I. Zelensky and S. Stankoviansky. J. Chromatogr., 194 (1980) Il. 4 F. S. Stover, J. Chromatogr., 298 (1984) 203. 5 M. TxZdki, M. Takagi and K. Ueno, Chem. Lett., (1982) 639. 6 W. L. Hinze, Sep. furif. Methods, 10 (1981) 159. 7 E. Smolkova-Keulemansova. J. Chromatogr., 251 (1982) 17. 8 T. J. Word and D. W. Armstrong, J. Liy. Chromazogr., 9 (1986) 407. 9 F. Cramer, Chem. Ber., 86 (1953) 1576. 10 D. E. Tutt and M. A. Schwarz, J. Am. Chem. Sot., 93 (1971) 767. 11 M. L. Bender and M. Komiyama, Cyclodextrin Chemistry, Springer, Berlin, Heidelberg, New York, 1978.
Note
Use of cyclodextrins I. Effect of cyclodextrin penicillins
in isotachophoresis on the isotachophoretic
separation
of related
I. JELiNEK* Research Institute for Pharmacy and Biochemistry, KouFimskb 17, I36 00 Prague 3 (Czechoslovakia) and J. SNOPEK and E, $MOLKOVti-KEULEMANSOVA Department of Analytical Chemistry, Charles University. Albertov 2030, 128 40 Prague 2 (Czechoslovakia) (First received May 7th, 1987; revised manuscript received June lst, 1987)
Separations by isotachophoresis (ITP) are usually performed in non-packed capillary columns. The separability of the injected components is determined by the differences in their effective mobilities which are in turn mainly influenced by their degrees of dissociation and absolute ionic mobilities. The degree of dissociation can easily be varied experimentally by changing the pH of the leading electrolyte. It is much more complicated to affect the ionic mobility. This problem can be overcome by using a complex-forming counter ion. The theory of eomplex formation equilibria and practical applications mainly to inorganic ions were described by Gebauer and BoCeki. Complex formation involving organic ions has been much less investigated. The use of Ca2 ’ and 1,3-bis[tris(hydroxymethyl)methylamino]propane for selective retardation of organic anions was described2s3. Some neutral complex-forming agents, such as l&crown-4 ether4 and a-cyclodextrin5, were used for the separation of inorganic ions. Qnly little attention has been devoted to the use of cyclodextrins (CDs) in isotachophoretic separation of organic ions. CDs may form inclusion complexes with many different compounds both in the solid state and in aqueous solution. The selective formation of inclusion complexes, preferentially with certain types of compounds, based on structural differences is widely utilized for separations and analytical purposes using various chromatographic procedureF*. The CDs can be used either as an aqueous mobile phase component in thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) or more effectively as a stationary phase in TLC, HPLC and gas chromatography. The aim of this paper is to present some experiments which may contribute to understanding the role of CDs in ITP separations. We tried to separate mixtures of sulbactam synthesis intermediates and of sulbactam (drug of penicillin type) and its alkaline degradation product with an open fi-lactam ring, which causes an allergic reaction All these compounds are structurally similar and their ITP separation in 0021-9673/%7/$03.50
@) 1987 Elsevier Science Publishers B.V.
NC.YI‘ES
380
classical electrolyte systems is very difficult. The effects of the various adjustable parameters, especially the concentration of CDs in the leading electrolyte (LE), the pH of the LE and the dimensions of the CD ring, were studied experimentally and used to optimize the ITP separation process. EXPERIMENTAL
Chemicals
Redistilled water was used in the preparation of the electrolyte solutions and of the solutions of model mixtures. All chemicals were of the highest quality commercially available and were used without any purification: 37% hydrochloric acid, sodium hydroxide, 4-morpholineethanesulphonic acid (MES), fl-alanine (Merck, Darmstadt, F.R.G.); 6-aminocaproic acid (EACA) (Sigma, St. Louis, MO, U.S.A.); hydroxypropylmethylcellulose (HPMC) (Dow Chemical, Midland, MI, U.S.A.); caproic acid (Lachema, Brno, Czechoslovakia); a- and y-cyclodextrins (CX-and y-CDs) (ASTEC, Whippany, U.S.A.); /I-cyclodextrin (/?-CD) (Chinoin, Budapes, Hungary). The solutes investigated were obtained from VUFB (Prague, Czechoslovakia). Their formulae are given in Table I.
ITP experiments were performed with a Tachophor 2127 (LKB, Bromma, Sweden) equipped with a conductivity detector and a polytetrafluorethylene (PTFE) capillary. Injections were made with lo-p1 Hamilton syringe. For conditions see Table II.
TABLE I THE COMPOUNDS
INVESTIGATED
o=H&2 v
Rb3 as/p
0
0
$32 5
n
C”3
CH
R
CH3 coo-
1.2
m+
0’
N 3.4
CH 3
3
=t43
N
cooM+
A
A,
C”3
cooM+
Compound
R
M+
Name
I
H
Na+
2
Br
K+
3
Br
K+
4
H
Na+
5
H
Na+
Penicillanic acid sodium (3,3-dimethyl-7-oxo-4-thia-lazabicyclo[3.2.0]heptane-2-carboxylate sodium salt) 6,6-Dibromopenicilanic acid potassium salt (6,6-dibromo-3,3-dimethyl7-oxo-4-thia-l-azabicyclo[3.2.0]heptane-2-carboxylate potassium salt) 6,6-dibromopenicillanic acid sulphone potassium salt (6,6-dibromo-3,3dimethyl-4,4,7-trioxo-4-thia-l-azabicyclo[3.2.0]heptane-2-carboxylate potassium salt) Sulbactam sodium salt (penicillanic acid sulphone sodium salt; 3,3dimethyl-4,4,7-trioxo-4-thia-l-azabicyclo[3.2,O]heptane-2-carboxylate sodium salt) Alkaline degradation product of Sulbactam (2-carboxymethyl-5,5dimethyl-l.l-dioxothiazaohdine-4-carboxylate sodium salt)
NOTES
381
TABLE II ELECTROLYTE
SYSTEMS AND CONDITIONS
Leading electrolyte (LE)
FOR ITP
I, 5 mM Hydrochloric acid including 0.2% HPMC with EACA to pH 4.30, 4.55, 4.65 and 4.80 II, as I but including 0.2% HPMC with B-alanine to pH 3.50 and 4.00 I, 5 mM MES II, 5 mA4 caproic acid 400 mm x 0.5 mm I.D., 520 mm x 0.5 mm I.D. 18°C Conductivity 100 pA (9 min); for detection 50 PA 2 pl of mixture I (compounds l-4, each 5 mM); 4 ~1 of mixture II (compounds 4 and 5, each 2.5 mM)
Terminating electrolyte (TE) Capillaries Temperature Detection Current Injected volumes
RESULTS AND DISCUSSION
The choice of electrolyte system for ITP separation of related penicillins is influenced by several factors. The use of alkaline electrolyte systems’ may be partially
I
1lME
I
Fig. 1. Effect of b-CD on the ITP separation of mixture I in LE I (pH 4.80) with TE I and capillary of length 520 mm. Concentrations, of J-CD in LE: (A) without b-CD; (B) 0.5 mM; (C) 1 mM; (D) 5 mM; (E) 10 m&f.
382
NOTES
successful, but the stability of penicillins in alkaline solution is poor and degradation may occur during ITP. Therefore the stability of sulbactam and its intermediates in aqueous solution was studied. It is known that complex formation with CDs may change the reactivity of a guest molecule. This property, connected with the ability to catalyze some reactions, was recognized by Cramerg. The P-CD catalyzed hydrolysis of penicillins was studied by Tutt and Schwarz**. The rate of penicillin hydrolysis, in the presence of P-CD, is increased 20&X0fold relative to the non-catalyzed reaction. Our stability experiments showed, however, that the sulbactam degradation during ITP is negligible. This type of penicillin proved to be stable even in a slightly acidic aqueous solution, pH 3.548, saturated with /?-CD. Fig. 1 shows the ITP separation of mixture I (see Table II) in a slightly acidic electrolyte system pH 4.8 where all the solutes are stable. The resolution of four components without B-CD is very poor. Only partially separated mixed zones could be seen on isotachopherogram A. Qualitative and quantitative evaluation is impossible. With increasing amount of P-CD in the LE the separation is improved. A distinct separation of components 3 and 2 occurs in the presence of 0.5 mM #?-CD. Components 4 and 1 are still not separated (Fig. 1B). Optimum separation of all the solutes is achieved with 5 and 10 mM B-CD (Fig. 1D and E). A comparison between Fig. IA and E clearly shows great differences in separation quality. When /3-CD is used in electrolyte systems the shape and spatial arrangement of the molecules to be separated become important factors. Even more critical is the separation of Sulbac-
Fig. 2. ITP separation of mixture I1 in LE I (PH 4.80) with TE I and capillary of length 520 mm: (A) LE without P-CD: (B) 10 mM /?-CD in LE. Fig. 3. Effect of (A, C) starch (567.4 mg per 100 ml LE) and (B, D) D-ghcOSe (1134.8 mg per 100 ml LE) on ITP separation of mixtures I and II in LE 1 (pH 4.8) with TE I and a capillary lengths of 520 mm.
NOTES
383
tam and its degradation product. The good resolution with 10 mM /?-CD is compared to that without P-CD in Fig. 2. The results suggest that the geometrical arrangements of the molecules again play a significant role in the separation process. Comparative measurements with D-glucose and starch were carried out to show the role of the cyclic structure of CD in ITP separation. D-Glucose was used as a model of the building units of CD and starch as a compound with helical structure but without rings. Fig. 3 shows that neither of these additives improves the separation and only the cyclic structure of CD provides favourable resolution. The CD ring diameter is an important factor in the separation. Experiments in which P-CD was replaced by its a- and y-analogues did not provide statisfactory results. Only a slight improvement in separation of both dibromoderivatives, in comparison with classical electrolytes, was observed. The separation of mixture I (see Table II) depends significantly on the pH value of the LE (Fig. 4). Two electrolyte systems were used to cover the range from pH 3.50 to 4.80. Penicillanic acid(l) and its dibromosulphone derivative behave as a reversed ion pair, where the more mobile constituent has the lower equilibrium constant. The isotachopherogram shows that these constituents are not separated at pH 4.30. Both lower and higher pH values give a resolution. Tt can be concluded that the lower pH value (Fig. A) gives a better resolution and sharper zone boundaries than the higher one (Fig. 4F).
Fig. 4. Effect of pH on the ITP separation of mixture I in LE II with TE 11 at pH 3.50, (A) and 4.00 (B). and in LE I with TE I at pH 4.30 (C), 4.55 (D), 4.65 (E) and 4.80 (F). Capillary lengths: 400 mm Concentration of j-CD in LE is 10 mM.
384
NOTES
The results indicate that complex-formation equilibria with CDs are undoubtedly a significant factor which influence effective mobilities and thus the possibility of ITP separation. It can be assumed that CDs do not migrate in the column during ITP due to their high pK, values (12.2-12.6) l*. CD, as a complex-forming agent added to the LE, acts as a quasi-stationary phase. The nature of the separation effect of CDs in TTP and HPLC seems to be similar. The results of all our experiments are in accord with inclusion complex theory. The addition of a CD to the LE can be compared with the use of CD-bonded columns. The consumption of CD in ITP, compared with that in an HPLC mobile phase, is negligible. The ITP capillary column need not be equilibrated with large volumes of CD-containing mobile phase. Moreover there is no hydrodynamic flow during ITP. The composition of the two model mixtures, was not accidental. The possibility of analytical evaluation of the compounds present in these mixtures is important for estimation of the purity of sulbactam. Optimization of their ITP analysis in classical electrolyte systems was only partially successful. The effect of #I-CD in a slightly acidic electrolyte is very advantageous in ITP. The experiments with c+ and y-CD did not provide satisfactory results. The use of CDs in ITP will certainly not be restricted to penicillins. Further research other possible applications is being carried out, and on the detailed interaction mechanism, based on the investigation of ortha-, meta- and para-substituted benzoic acids. REFERENCES 1 P. Gebauer and P. Bocek, in P. BoEek (Editor), Isotachophoresis-Basic Course, Advanced Course, ITP-84, Hradec Krcilovd, September 2-6, 1984, Institute of Radioecology and Applied Nuclear Techniques, Plant for Development and Production of Nuclear Instruments, SpiSskl Nov9 Ves, 1984, p. 78. 2 D. Kaniansky and F. M. Everaerts, J. Chromatogr., 148 (1978) 441. 3 D. Kaniansky, V. Madajovb, I. Zelensky and S. Stankoviansky. J. Chromatogr., 194 (1980) Il. 4 F. S. Stover, J. Chromatogr., 298 (1984) 203. 5 M. TxZdki, M. Takagi and K. Ueno, Chem. Lett., (1982) 639. 6 W. L. Hinze, Sep. furif. Methods, 10 (1981) 159. 7 E. Smolkova-Keulemansova. J. Chromatogr., 251 (1982) 17. 8 T. J. Word and D. W. Armstrong, J. Liy. Chromazogr., 9 (1986) 407. 9 F. Cramer, Chem. Ber., 86 (1953) 1576. 10 D. E. Tutt and M. A. Schwarz, J. Am. Chem. Sot., 93 (1971) 767. 11 M. L. Bender and M. Komiyama, Cyclodextrin Chemistry, Springer, Berlin, Heidelberg, New York, 1978.