- Email: [email protected]
Comparison of retention of native cyclodextrins and its permethylated derivatives on porous graphite carbon and silica C18 stationary phases
Analytica Chimica Acta 537 (2005) 41–46
Comparison of retention of native cyclodextrins and its permethylated derivatives on porous graphite carbon and silica C18 stationary phases Arkadiusz Kwaterczak a , Anna Bielejewska a, b, ∗ a b
Institute of Physical Chemistry, Kasprzaka 44/52, 01-224 Warsaw, Poland Pharmaceutical Research Institute, Rydygiera 8, 02-187 Warsaw, Poland
Received 5 October 2004; received in revised form 21 January 2005; accepted 21 January 2005 Available online 19 February 2005
Abstract The retention of the solute in the stationary phase is the effect of interaction between the solute and the stationary phase and the solute and the mobile phase (solvatation phenomena). Additionally, the components of the mobile phase can modify the surface of the stationary phase, and thus the change the interaction between the solute and the stationary phase. The current paper presents a systematic comparison of retention of ␣-, - and ␥-cyclodextrins (CD) and its permethylated derivatives on the porous graphitic carbon and RP C18 columns. From temperature study, the contribution of H and S to G has been discussed. The physicochemical interactions of cyclodextrins seem to be similar only in the case of native cyclodextrins (except ␥-CD) with the RP C18 column when enthalpy–entropy compensation can be observed. In the case of permethylated cyclodextrins and the RP C18 column, interactions are only similar for individual alcohols. For the PGC column, the influence of individual interactions on individual cyclodextrins seems to be different. © 2005 Elsevier B.V. All rights reserved. Keywords: Porous graphitic carbon column; Cyclodextrins; Temperature effects; Liquid chromatography; Retention mechanism
1. Introduction Cyclodextrins (CDs) have a wide range of practical applications as chiral selectors in HPLC. They are used not only as chemically bonded stationary phases [1–4] but also as additives to mobile phase solutions [5–8]. Numerous chromatographic supports have been used for cyclodextrins separation processes [9,10]. Knowledge of cyclodextrins adsorption properties is important, especially in the case of preparing dynamically generated chiral stationary phases [11] as well as for investigation of stability constants of their complexes [12,7]. The porous graphitic carbon (PGC) stationary phase has an energetically homogeneous surface and is a very strong adsorbent [13,14]. This chromatographic support is often compared with C18 silicas and described as a stronger hydrophobic sorbent [15–17]. The retention mechanism for both sorbents appears to be different [18–20]. ∗
Corresponding author. Tel.: +48 22 6323221. E-mail address: [email protected] (A. Bielejewska).
0003-2670/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2005.01.046
The chromatographic behaviour of several cyclodextrins on RP C18 [21,22] as well as PGC columns [23] has already been investigated. Clarot et al. reported unusual behaviour of ␣-, - and ␥-CD retention on PGC with the methanolic aqueous mobile phase. They observed a dual retention mode depending on the mobile phase composition [24]. A careful comparison of retention data is needed for a better understanding of the difference in the retention mechanism for both phases. The current paper presents a systematic comparison of retention of ␣-, - and ␥-CD and its permethylated derivatives on the PGC and RP C18 columns. The thermodynamic parameters obtained for both phases are also discussed.
2. Experimental 2.1. Reagents All cyclodextrins were obtained from Chinoin (Budapest): ␣-cyclodextrin (␣-CD), -cyclodextrin (-CD),
42
A. Kwaterczak, A. Bielejewska / Analytica Chimica Acta 537 (2005) 41–46
analysis was used [25]. The first moment is the coordinate of the centre of gravity of the peak and this equals the retention time. The first moment µ1 is the sum of the products of the elution time (t) and the area (y) of each slice, sum divided by the peak area: ty µ1 = (1) y Fig. 1. Chromatogram of a mixture of ␣-, - and ␥-CD. Chromatographic conditions: PGC column, 150 mm × 4.6 mm; mobile phase, ethanol–water (15:85, v/v); flow rate, 0.9 ml/min.
␥-cyclodextrin (␥-CD), (2,3,6-tri-o-methyl)-␣-cyclodextrin (TM-␣-CD), (2,3,6-tri-o-methyl)--cyclodextrin (TM-CD) and (2,3,6-tri-o-methyl)-␥-cyclodextrin (TM-␥-CD). The solvents were of analytical reagent grade and were used without further purification. 2.2. Apparatus and procedure Chromatographic experiments were performed using a Waters (Vienna, Austria) pump model 590, a 5 l loop Rheodyne type injector and a polarimertic Knauer “Chiralyser 1.4” detector. The temperature was controlled using a Waters Temperature Control System. Two commercially available columns were used: a porous graphitic carbon Hypercarb column (150 mm × 4.6 mm i.d., particle size 5 m) from ThermoHypersil-Keystone and a Luna C18(2) column (250 mm × 4.6 mm i.d., particle size 5 m) from Phenomenex. Investigations were performed using different mobile phases: 1. Ethanol/methanol–water (different volume concentrations) for ␣-, - and ␥-CD on the RP C18 and PGC columns and methylated derivatives on the RP C18 column. Flow rate 0.9 ml/min. 2. Dioxane/2-propanol (IPA), (20:80, v/v) for methylatedCD on the PGC column. Flow rate 0.5 ml/min. As the shapes of the obtained peaks were not symmetric (see the chromatograms Figs. 1 and 2), the first moment
3. Results and discussion 3.1. The retention and elution order Aqueous ethanol or methanol solutions were found to be very suitable as the mobile phase for the separation of CDs on the RP C18 column [22] and for native CDs on the PGC column. The methyl derivatives of ␣-, - and ␥-CD are much more strongly adsorbed on the hydrophobic stationary phase than native CD themself. Their elution requires higher concentrations of organic additives in the aqueous mobile phase solutions for the RP C18 column. On the PGC column, it was impossible to investigate the retention behaviour of permethyleted CDs in an ethanol–water solution even using high concentration of ethanol. For the PGC column, the permethylated CDs were eluted in dioxane/2-propanol mixtures with 20:80 (v/v). Table 1a and b present the retention factors (k) for various CDs and their permethylated derivatives depending on the eluent composition. The retention of CDs is much stronger on the PGC than the RP C18 column. To obtain a similar retention time, a much higher concentration of organic modifier for the PGC column has been used. On the RP C18 column, the adsorption of -CD is much stronger than the adsorption of ␣-CD or ␥-CD. On the PGC, the order of elution is ␣-, - and ␥-CD, and is in accordance with their molecular weight, the retention increasing with the increasing size of cyclodextrin (see Fig. 1). It seems that for Table 1 Retention factors (k) of native CDs and permethylated CDs derivatives on different columns depending on the eluent composition at 25 ◦ C RP C18 5% EtOH (a) Native CDs ␣-CD 1.16 -CD 6.88 ␥-CD 2.32
PGC 7.5% MeOH
15% EtOH
50% MeOH
2.89 8.01 1.81
1.88 4.49 10.59
1.94 3.61 10.17
RP C18 50% EtOH
Fig. 2. Chromatogram of a mixture of TM-␣-CD, TM--CD and TM-␥CD. Chromatographic conditions: PGC column, 150 mm × 4.6 mm; mobile phase, dioxane–IPA (20:80, v/v); flow rate, 0.5 ml/min.
(b) Permethylated CDs derivatives TM-␣-CD 5.28 TM--CD 4.64 TM-␥-CD 2.79
PGC 80% MeOH
20% Dioxane/IPA
7.17 4.45 2.81
11.74 4.29 5.45
A. Kwaterczak, A. Bielejewska / Analytica Chimica Acta 537 (2005) 41–46
43
Table 2 Retention factors (k) of native CDs and permethylated CDs derivatives on RP C18 and PGC columns depending on the temperature Temperature (◦ C)
RP C18
PGC
5% EtOH
7.5% MeOH
15% EtOH
50% MeOH
␣-CD
-CD
␥-CD
␣-CD
-CD
␥-CD
␣-CD
-CD
␥-CD
␣-CD
-CD
␥-CD
(a) Native CDs 25 30 35 40 45
1.16 0.72 0.41 0.30 0.22
6.88 4.38 2.79 2.03 1.45
2.32 1.42 0.88 0.66 0.47
2.89 1.99 1.23 0.92 –
8.01 5.56 3.56 2.69 –
1.81 1.30 0.85 0.65 –
1.88 1.42 1.36 1.19 1.04
4.49 3.40 2.91 2.41 2.01
10.59 9.50 6.18 5.36 4.31
1.94 1.73 1.69 1.55 1.45
3.61 3.32 3.10 2.90 2.69
10.17 8.83 7.83 7.21 6.53
Temperature (◦ C)
RP C18
PGC
50% EtOH TM-␣-CD (b) Permethylated CDs derivatives 20 – 25 5.28 30 5.15 35 5.04 40 4.91 45 –
80% MeOH
20% IPA/dioxane
TM--CD
TM-␥-CD
TM-␣-CD
TM--CD
TM-␥-CD
TM-␣-CD
TM--CD
TM-␥-CD
– 4.64 4.65 4.66 4.67 –
– 2.79 2.90 3.00 3.07 –
– 7.17 6.36 5.83 4.93 4.37
– 4.45 4.22 3.96 3.40 3.19
– 2.81 2.76 2.67 2.33 2.31
11.86 11.74 11.75 11.72 11.35 –
4.35 4.29 4.23 4.14 3.93 –
5.54 5.45 5.46 5.44 5.37 –
the RP C18 column, the solubility of CDs in the eluent plays greater role in the adsorption process, and consequently in separation. The solubility of -CD in water is much lower than ␣-CD and ␥-CD [26,27]. Although, for permethylated cyclodextrin, the TM-␣-CD is the strongest adsorbed cyclodextrin on both examined columns (see Fig. 2), methylated CDs also exhibit a different sequence of elution on both the examined adsorbents. On RP C18 column, TM-␥-CD is eluted before TM--CD and for PGC, TM--CD is eluted before TM-␥-CD. The elution order changes are proof that although the RP C18 and PGC columns act in reverse chromatographic mode, the mechanism of separation is different. 3.2. Temperature study The influence of temperature in the narrow range 25–45 ◦ C on retention for native and permethylated CDs was studied using a PGC and RP C18 columns with different mobile phase compositions. The temperature affect CDs retention on both stationary phases. The changes of temperature influence mass transfer between the mobile and stationary phase. Moreover, the study of the temperature influence can provide insight into the separation mechanisms and allows to design optimal conditions of analysis. Table 2a and b present some experimental data of temperature dependence. In a chromatographic system, the retention factor of the solute is dependent on the partitioning process between the mobile and stationary phases. The van’t Hoff expression for such a chromatographic system is given by the equation [28]:
◦
ln k =
◦
S −H + + ln ϕ RT R
(2)
where k is the retention factor, H◦ and S◦ represent standard enthalpy and entropy changes of transfer of the solute between the mobile and the stationary phase, R is the gas constant, T is the absolute temperature and φ is the volume phase ratio of the stationary to the mobile phase. The phase ratio for RP C18 column was calculated from the following equation [29]: Vs =
(%C)(M)(Wρ ) (100)(12.01)(nc )(ρ)
(3)
where Vs is the stationary phase volume, %C is grams of carbon per 100 grams of bonded silica, nc refers to the number of carbon atoms per mole of silane, M is the molecular weight of silane, Wρ is the weight (g) of the bonded packing material in the column. The density of the bonded octadecylsilyl (ρ) is 0.8607 g/cm3 [29]. The mobile phase volume in the column was assumed to be the same as the retention volume of a non-retained solute. The calculated phase ratio value was ϕ = 0.22. For the PGC column, Vs was calculated as the difference between the total volume of the column and its void volume. The obtained phase ratio was equal to 0.33. According to Eq. (2), the slope of the ln k plot against 1/T provides information about enthalpy differences, whereas the intercept is related to entropy changes. If H◦ , S◦ and ϕ are independent of temperature, the plots of ln k versus 1/T should be linear.
−8.87 −6.35 −6.97 7.53 ± 0.52 2.70 ± 0.72 5.99 ± 0.22 −1.34 ± 0.52 −3.65 ± 0.72 −0.98 ± 0.24 −8.66 −7.51 −6.34 −10.93 ± 1.14 −6.34 ± 1.56 −2.45 ± 1.78 −19.59 ± 1.18 −13.85 ± 1.61 −8.80 ± 1.85 T = 298 K.
G (kJ/mol) TS (kJ/mol) G (kJ/mol) TS (kJ/mol)
−7.86 −7.55 −6.29
H (kJ/mol) H (kJ/mol)
4.15 ± 0.10 7.93 ± 0.025 11.32 ± 0.35
TS (kJ/mol) 20% IPA/doixane 80% MeOH 50% EtOH
H (kJ/mol)
PGC RP C18
(b) Permethylated CDs TM-␣-CD −3.71 ± 0.12 TM--CD 0.38 ± 0.03 TM-␥-CD 5.03 ± 0.37
−4.35 −5.92 −8.45 −6.49 ± 1.02 −5.48 ± 0.20 −8.72 ± 0.84 −10.84 ± 1.06 −11.40 ± 0.21 −17.18 ± 0.86 −4.27 −6.53 −8.71 −19.95 ± 1.78 −28.02 ± 1.46 −28.69 ± 1.88 −24.22 ± 1.81 −34.55 ± 1.50 −37.40 ± 1.90 −6.37 −8.89 −5.23 −54.41 ± 3.39 −48.73 ± 2.95 −49.06 ± 3.10 −60.77 ± 3.48 −57.63 ± 3.01 −54.28 ± 3.16 −3.98 −8.42 −6.18
TS (kJ/mol) G (kJ/mol) TS (kJ/mol)
−61.89 ± 4.41 −52.87 ± 2.30 −56.36 ± 3.37
H (kJ/mol) H (kJ/mol) H (kJ/mol)
G (kJ/mol)
15% EtOH 7.5% MeOH
(a) Native CDs ␣-CD −65.87 ± 4.56 -CD −61.29 ± 2.37 ␥-CD −62.55 ± 3.49
TS (kJ/mol) Fig. 4. The van’t Hoff plots for permethylated-CDs in 50% EtOH on RP C18 column.
5% EtOH
For native CDs for both alcohols on the RP C18 column, both H and S are negative, thus the transfer between the mobile and stationary phases is enthalpically driven. A similar situation can be observed on the PGC column. Comparing, the ratio TS/H for both columns, lower values have been obtained for PGC than for RP C18 column
PGC
3.3. Native cyclodextrins
RP C18
The results from all the studied systems are presented in Table 3a and b. The van’t Hoff plots show linear behaviour in the examinated temperature range. As an example, Fig. 3 shows the van’t Hoff plots for native CDs in 5% EtOH on the RP C18 column. The retention time of CDs for all investigated eluents decreases with the increase of column temperature, except the retention for TM--CD and TM-␥-CD on RP C18 column in the ethanolic solution, where the retention was independent or increases (see Fig. 4). The values of free energy can be considered as a measure of the total efficiency of the transfer of the solute from the mobile phase to the stationary phase. The more negative H value indicates that the solute is more effectively transferred to the stationary phase from the point of view of energetic interaction. The negative values of entropy changes indicate that the solute is more organized in the stationary phase than in the mobile phase.
TS (kJ/mol)
(4)
Table 3 Thermodynamic parameters of native CDs and permethylated CDs determined with various eluents on RP C18 and PGC columns
G = H − TS
G (kJ/mol)
The H and S values obtained from the plot can be used to obtain G according to the equation:
H (kJ/mol)
50% MeOH
Fig. 3. The van’t Hoff plots for native CDs in 5% EtOH on RP C18 column.
G (kJ/mol)
A. Kwaterczak, A. Bielejewska / Analytica Chimica Acta 537 (2005) 41–46 G (kJ/mol)
44
A. Kwaterczak, A. Bielejewska / Analytica Chimica Acta 537 (2005) 41–46
(e.g. for ␣-CD on PGC column, it is 0.82 and for RP C18 column it is 0.94). This indicates that for the PGC column the unfavourable entropy influence plays lesser role in the adsorption process than for RP C18 column, thus it may be responsible for the general rule of increasing of CDs retention on PGC material. In the RP C18 column, the H value of -CD was less negative for the ethanolic solution compared to other native CDs, but the longest retention time was obtained. Thus, less unfavourable entropy changes for -CD induce its high retention. The most favourable interactions enthalpy with the RP C18 column occurred for ␣-CD, but unfavourable entropy interactions induced shorter retention. ␣-CD is eluted first in ethanol and as the second in methanol. On the PGC column for both eluents, the favourable H and G changes increased with the increasing size of the cyclodextrin. Thus, the ␥-CD was the most effectively transferred from the mobile to the stationary phase. 3.4. Permethylated cyclodextrins For the ethanolic solution on the RP C18 column, the slopes of the van’t Hoff plots change from negative to positive with the increasing of molecular weight of CDs. This is connected with the different behaviour of TM-␣-CD, TM--CD and TM-␥-CD. For TM-␣-CD, the retention time decreases with increasing temperature, for TM--CD the retention time is almost independent of temperature. The retention time of TM␥-CD increases with the increasing temperature. The positive value of enthalpy changes indicates that it is energetically more favourable for the solutes to be in the mobile phase. In such situation, the adsorption of the stationary phase occurs only due to entropic advantages (TM-␥-CD). On RP C18 column, for the ethanolic mobile phase, the entropy values are positive for all studied permethylated cyclodextrins, thus entropy facilitates the transfer of permethylated CDs to the stationary phase in these conditions. The favourable entropic interaction increases with the increasing size of cyclodextrin. As one can see for the results on the RP C18 column, the mobile phase composition can play an essential role in the adsorption process. Adsorption on the RP C18 column is an entropy-favourable process in the presence of ethanol (positive value of entropy) and entropy-unfavourable for the methanolic solution. In the latter case, the entropy changes receive negative values. The negative values of enthalpy and entropy changes for the methanolic solution indicate that the transfer between the mobile and stationary phases is enthalpically driven. On the PGC column, TM--CD has the most negative H values, but is eluated first because the other CDs have much more favourable entropy interactions with the stationary phase. On the PGC support, the process seems similar to that on RP C18 in the ethanolic solution. Entropy is greater than enthalpy (TM-␣-CD and TM-␥-CD) and facilitates the adsorption process.
45
Fig. 5. Correlation between ln k at 30 ◦ C and H for native cyclodextrins on RP C18 column. For chromatographic conditions see Section 2.
4. Enthalpy–entropy compensation To obtain more information about the interaction of the cyclodextrin with both stationary phases, the correlation between the logarithm of the retention factor measured at 30 ◦ C and the enthalpies for the particular cyclodextrin have been demonstrated in Figs. 5–7. The plots of the ln k of various cyclodextrins versus corresponding enthalpy are linear when enthalpy–entropy compensation occurs, meaning that the reversible binding of the cyclodextrin on the stationary phase is subject to an essentially identical mechanism [30]. Depend-
Fig. 6. Correlation between ln k at 30 ◦ C and H for permethylated cyclodextrins on RP C18 column. For chromatographic conditions see Section 2.
Fig. 7. Correlation between ln k at 30 ◦ C and H for native and permethylated cyclodextrins on PGC column. For chromatographic conditions see Section 2.
46
A. Kwaterczak, A. Bielejewska / Analytica Chimica Acta 537 (2005) 41–46
ing on the cyclodextrin and the chromatographic conditions, three situations have been observed. In Fig. 5, for native cyclodextrins and the RP C18 column, we can see that for ␣-, - and ␥-CD in the ethanolic mobile phase and for ␣- and -CD in the methanolic solution, compensation can be observed. This could mean that physicochemical interaction between these cyclodextrins and RP C18 column has the same nature for both alcohols (only for ␥-CD in the methanolic solution, the interactions seems to have a different physico-chemical basis). It is interesting to see that for these processes, more negative values of enthalpy do not mean longer retention. High values of favourable enthalpy interaction are followed by higher unfavourable entropy interactions with the stationary phase, which causes shorter retention. In Fig. 6, for permethylated cyclodextrin and the silica RP C18 column, two linear plots for methanolic and ethanolic solutions have been obtained. From these results one can deduce that the physico-chemical interactions of permethylated cyclodextrin with the mobile and stationary phases are similar for a given alcohol. For these cyclodextrins in these chromatographic conditions, shorter retention has been obtained for lower negative values of enthalpy. In Fig. 7, for native and permethylated cyclodextrin on the PGC column, there is no linear relationship between ln k and H. One can see that for very similar retention of ␣-, - and ␥-CD obtained with various alcohols, much higher values of negative enthalpy are obtained for ethanol than for methanol solutions.
5. Conclusion The methyl derivatives of ␣-, - and ␥-CD are much more strongly adsorbed on the hydrophobic stationary phase than native CD themself. The retention of CDs is much stronger on the PGC than on the RP C18 column. Depending on the column, elution order of native and permethylated CDs was different. The elution order changes are proof that although the RP C18 and PGC columns act in reverse chromatographic mode, the mechanism of separation is different. From temperature study, we can see that the transfer of the CDs between the mobile and stationary phase may be enthalpy or entropy driven. As the physico-chemical interaction of the given CD with various stationary and mobile phase seems to be different, the chiral discrimination obtained for the same CD in various environment may be different. The physico-chemical interactions of cyclodextrins seem to be similar only in the case of native cyclodextrins (except ␥-CD) with the RP C18 column when enthalpy–entropy compensation can be observed. In the case of permethylated cyclodextrins and the RP C18 column, interactions are only similar for individual alcohols. For the PGC column, the in-
fluence of individual interactions on individual cyclodextrins seems to be different. Acknowledgment ˙ We would like to thank Dr. Janusz Zukowski and Dr. Bronisław Gł´od for providing the PGC columns. References [1] H. Riering, M. Sieber, J. Chromatogr. A 728 (1996) 171. [2] D. Schumacher, C. Mitchell, T. Xiao, R. Rozhkov, R. Larock, et al., J. Chromatogr. A 1011 (2003) 37. [3] Y. Kawaguchi, M. Tanaka, M. Nakae, K. Funazo, T. Shono, Anal. Chem. 55 (1983) 1852. [4] Y. Liu, A.W. Lantz, D.W. Armstrong, J. Liq. Chromatogr. Related Technol. 27 (7–9) (2004) 1121. [5] R. Herr´aez-Hern´andez, P. Campins-Falco, J. Chromatogr. B: Biomed. Sci. Appl. 740 (2000) 169. [6] A. Bielejewska, R. Nowakowski, K. Duszczyk, D. Sybilska, J. Chromatogr. A 840 (1999) 159. [7] A. Bielejewska, K. Duszczyk, D. Sybilska, J. Chromatogr. A 931 (2001) 81. [8] M. Asztemborska, A. Bielejewska, K. Duszczyk, D. Sybilska, J. Chromatogr. A 847 (2000) 73. [9] I. Clarot, D. Cledat, Y.C. Guillaume, P.J.P. Cardot, Chromatographia 54 (2001) 447. [10] L. Chen, L. Zhang, C. Ching, C. Ng Siu, J. Chromatogr. A 950 (2002) 65. [11] R. Nowakowski, A. Bielejewska, K. Duszczyk, D. Sybilska, J. Chromatogr. A 782 (1997) 1. [12] K. Flood, E. Reynolds, N. Snow, J. Chromatogr. A 903 (2000) 49. [13] J. Knox, B. Kaur, J. Chromatogr. 352 (1986) 3. [14] E. Forgacs, J. Chromatogr. A 975 (2002) 229. [15] J. Oxelbark, S. Claeson, Tetrahedron, Asymmetry 13 (2002) 2235. [16] Q.H. Wan, P.N. Show, M.C. Davies, D.A. Barrett, J. Chromatogr. A 697 (1995) 219. [17] P.T. Jackson, M.R. Schure, T.P. Weber, P.W. Carr, Anal. Chem. 69 (1997) 416. [18] M.C. Hennion, V. Coquart, S. Guenu, C. Sella, J. Chromatogr. A 712 (1995) 287. [19] K.K. Unger, Anal. Chem. 55 (1983) 361. [20] V. N´emeth-Kiss, E. Forg´as, T. Cserh´ati, J. Chromatogr. A 776 (1997) 147. [21] R. Nowakowski, P.J.P. Cardot, A.W. Coleman, E. Villard, G. Guiochon, Anal. Chem. 67 (1995) 259. [22] A. Bielejewska, M. Ko´zbiał, R. Nowakowski, K. Duszczyk, D. Sybilska, Anal. Chim. Acta 300 (1995) 201. [23] A.K. Chatjigakis, Ph.J.P. Cardot, A.W. Coleman, H. Parrot-Lopez, Chromatographia 36 (1993) 174. [24] I. Clarot, D. Cl´edat, L. Boulkanz, E. Assidjo, T. Chian´ea, P. Cardot, J. Chromatogr. Sci. 38 (2000) 38. [25] E. Grushka, M.N. Myers, P.D. Schettler, J.C. Giddings, Anal. Chem. 41 (7) (1969) 889. [26] K. Fromming, J. Szejtli, Cyclodextrins in Pharmacy, Kluwer Academic Publisher, 1994. [27] J. Szejtli, Cyclodextrins and their inclusion complexes, Akademiai Kiado Budapest, 1982. [28] J. Chmielowiec, H. Sawitzky, J. Chromatogr. Sci. 17 (1979) 245. [29] K.B. Sentell, J.G. Dorsey, J. Liq. Chromatogr. 11 (1988) 1875. [30] W. Melander, D.E. Campbell, C. Horvath, J. Chromatogr. 158 (1978) 215.
Comparison of retention of native cyclodextrins and its permethylated derivatives on porous graphite carbon and silica C18 stationary phases Arkadiusz Kwaterczak a , Anna Bielejewska a, b, ∗ a b
Institute of Physical Chemistry, Kasprzaka 44/52, 01-224 Warsaw, Poland Pharmaceutical Research Institute, Rydygiera 8, 02-187 Warsaw, Poland
Received 5 October 2004; received in revised form 21 January 2005; accepted 21 January 2005 Available online 19 February 2005
Abstract The retention of the solute in the stationary phase is the effect of interaction between the solute and the stationary phase and the solute and the mobile phase (solvatation phenomena). Additionally, the components of the mobile phase can modify the surface of the stationary phase, and thus the change the interaction between the solute and the stationary phase. The current paper presents a systematic comparison of retention of ␣-, - and ␥-cyclodextrins (CD) and its permethylated derivatives on the porous graphitic carbon and RP C18 columns. From temperature study, the contribution of H and S to G has been discussed. The physicochemical interactions of cyclodextrins seem to be similar only in the case of native cyclodextrins (except ␥-CD) with the RP C18 column when enthalpy–entropy compensation can be observed. In the case of permethylated cyclodextrins and the RP C18 column, interactions are only similar for individual alcohols. For the PGC column, the influence of individual interactions on individual cyclodextrins seems to be different. © 2005 Elsevier B.V. All rights reserved. Keywords: Porous graphitic carbon column; Cyclodextrins; Temperature effects; Liquid chromatography; Retention mechanism
1. Introduction Cyclodextrins (CDs) have a wide range of practical applications as chiral selectors in HPLC. They are used not only as chemically bonded stationary phases [1–4] but also as additives to mobile phase solutions [5–8]. Numerous chromatographic supports have been used for cyclodextrins separation processes [9,10]. Knowledge of cyclodextrins adsorption properties is important, especially in the case of preparing dynamically generated chiral stationary phases [11] as well as for investigation of stability constants of their complexes [12,7]. The porous graphitic carbon (PGC) stationary phase has an energetically homogeneous surface and is a very strong adsorbent [13,14]. This chromatographic support is often compared with C18 silicas and described as a stronger hydrophobic sorbent [15–17]. The retention mechanism for both sorbents appears to be different [18–20]. ∗
Corresponding author. Tel.: +48 22 6323221. E-mail address: [email protected] (A. Bielejewska).
0003-2670/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2005.01.046
The chromatographic behaviour of several cyclodextrins on RP C18 [21,22] as well as PGC columns [23] has already been investigated. Clarot et al. reported unusual behaviour of ␣-, - and ␥-CD retention on PGC with the methanolic aqueous mobile phase. They observed a dual retention mode depending on the mobile phase composition [24]. A careful comparison of retention data is needed for a better understanding of the difference in the retention mechanism for both phases. The current paper presents a systematic comparison of retention of ␣-, - and ␥-CD and its permethylated derivatives on the PGC and RP C18 columns. The thermodynamic parameters obtained for both phases are also discussed.
2. Experimental 2.1. Reagents All cyclodextrins were obtained from Chinoin (Budapest): ␣-cyclodextrin (␣-CD), -cyclodextrin (-CD),
42
A. Kwaterczak, A. Bielejewska / Analytica Chimica Acta 537 (2005) 41–46
analysis was used [25]. The first moment is the coordinate of the centre of gravity of the peak and this equals the retention time. The first moment µ1 is the sum of the products of the elution time (t) and the area (y) of each slice, sum divided by the peak area: ty µ1 = (1) y Fig. 1. Chromatogram of a mixture of ␣-, - and ␥-CD. Chromatographic conditions: PGC column, 150 mm × 4.6 mm; mobile phase, ethanol–water (15:85, v/v); flow rate, 0.9 ml/min.
␥-cyclodextrin (␥-CD), (2,3,6-tri-o-methyl)-␣-cyclodextrin (TM-␣-CD), (2,3,6-tri-o-methyl)--cyclodextrin (TM-CD) and (2,3,6-tri-o-methyl)-␥-cyclodextrin (TM-␥-CD). The solvents were of analytical reagent grade and were used without further purification. 2.2. Apparatus and procedure Chromatographic experiments were performed using a Waters (Vienna, Austria) pump model 590, a 5 l loop Rheodyne type injector and a polarimertic Knauer “Chiralyser 1.4” detector. The temperature was controlled using a Waters Temperature Control System. Two commercially available columns were used: a porous graphitic carbon Hypercarb column (150 mm × 4.6 mm i.d., particle size 5 m) from ThermoHypersil-Keystone and a Luna C18(2) column (250 mm × 4.6 mm i.d., particle size 5 m) from Phenomenex. Investigations were performed using different mobile phases: 1. Ethanol/methanol–water (different volume concentrations) for ␣-, - and ␥-CD on the RP C18 and PGC columns and methylated derivatives on the RP C18 column. Flow rate 0.9 ml/min. 2. Dioxane/2-propanol (IPA), (20:80, v/v) for methylatedCD on the PGC column. Flow rate 0.5 ml/min. As the shapes of the obtained peaks were not symmetric (see the chromatograms Figs. 1 and 2), the first moment
3. Results and discussion 3.1. The retention and elution order Aqueous ethanol or methanol solutions were found to be very suitable as the mobile phase for the separation of CDs on the RP C18 column [22] and for native CDs on the PGC column. The methyl derivatives of ␣-, - and ␥-CD are much more strongly adsorbed on the hydrophobic stationary phase than native CD themself. Their elution requires higher concentrations of organic additives in the aqueous mobile phase solutions for the RP C18 column. On the PGC column, it was impossible to investigate the retention behaviour of permethyleted CDs in an ethanol–water solution even using high concentration of ethanol. For the PGC column, the permethylated CDs were eluted in dioxane/2-propanol mixtures with 20:80 (v/v). Table 1a and b present the retention factors (k) for various CDs and their permethylated derivatives depending on the eluent composition. The retention of CDs is much stronger on the PGC than the RP C18 column. To obtain a similar retention time, a much higher concentration of organic modifier for the PGC column has been used. On the RP C18 column, the adsorption of -CD is much stronger than the adsorption of ␣-CD or ␥-CD. On the PGC, the order of elution is ␣-, - and ␥-CD, and is in accordance with their molecular weight, the retention increasing with the increasing size of cyclodextrin (see Fig. 1). It seems that for Table 1 Retention factors (k) of native CDs and permethylated CDs derivatives on different columns depending on the eluent composition at 25 ◦ C RP C18 5% EtOH (a) Native CDs ␣-CD 1.16 -CD 6.88 ␥-CD 2.32
PGC 7.5% MeOH
15% EtOH
50% MeOH
2.89 8.01 1.81
1.88 4.49 10.59
1.94 3.61 10.17
RP C18 50% EtOH
Fig. 2. Chromatogram of a mixture of TM-␣-CD, TM--CD and TM-␥CD. Chromatographic conditions: PGC column, 150 mm × 4.6 mm; mobile phase, dioxane–IPA (20:80, v/v); flow rate, 0.5 ml/min.
(b) Permethylated CDs derivatives TM-␣-CD 5.28 TM--CD 4.64 TM-␥-CD 2.79
PGC 80% MeOH
20% Dioxane/IPA
7.17 4.45 2.81
11.74 4.29 5.45
A. Kwaterczak, A. Bielejewska / Analytica Chimica Acta 537 (2005) 41–46
43
Table 2 Retention factors (k) of native CDs and permethylated CDs derivatives on RP C18 and PGC columns depending on the temperature Temperature (◦ C)
RP C18
PGC
5% EtOH
7.5% MeOH
15% EtOH
50% MeOH
␣-CD
-CD
␥-CD
␣-CD
-CD
␥-CD
␣-CD
-CD
␥-CD
␣-CD
-CD
␥-CD
(a) Native CDs 25 30 35 40 45
1.16 0.72 0.41 0.30 0.22
6.88 4.38 2.79 2.03 1.45
2.32 1.42 0.88 0.66 0.47
2.89 1.99 1.23 0.92 –
8.01 5.56 3.56 2.69 –
1.81 1.30 0.85 0.65 –
1.88 1.42 1.36 1.19 1.04
4.49 3.40 2.91 2.41 2.01
10.59 9.50 6.18 5.36 4.31
1.94 1.73 1.69 1.55 1.45
3.61 3.32 3.10 2.90 2.69
10.17 8.83 7.83 7.21 6.53
Temperature (◦ C)
RP C18
PGC
50% EtOH TM-␣-CD (b) Permethylated CDs derivatives 20 – 25 5.28 30 5.15 35 5.04 40 4.91 45 –
80% MeOH
20% IPA/dioxane
TM--CD
TM-␥-CD
TM-␣-CD
TM--CD
TM-␥-CD
TM-␣-CD
TM--CD
TM-␥-CD
– 4.64 4.65 4.66 4.67 –
– 2.79 2.90 3.00 3.07 –
– 7.17 6.36 5.83 4.93 4.37
– 4.45 4.22 3.96 3.40 3.19
– 2.81 2.76 2.67 2.33 2.31
11.86 11.74 11.75 11.72 11.35 –
4.35 4.29 4.23 4.14 3.93 –
5.54 5.45 5.46 5.44 5.37 –
the RP C18 column, the solubility of CDs in the eluent plays greater role in the adsorption process, and consequently in separation. The solubility of -CD in water is much lower than ␣-CD and ␥-CD [26,27]. Although, for permethylated cyclodextrin, the TM-␣-CD is the strongest adsorbed cyclodextrin on both examined columns (see Fig. 2), methylated CDs also exhibit a different sequence of elution on both the examined adsorbents. On RP C18 column, TM-␥-CD is eluted before TM--CD and for PGC, TM--CD is eluted before TM-␥-CD. The elution order changes are proof that although the RP C18 and PGC columns act in reverse chromatographic mode, the mechanism of separation is different. 3.2. Temperature study The influence of temperature in the narrow range 25–45 ◦ C on retention for native and permethylated CDs was studied using a PGC and RP C18 columns with different mobile phase compositions. The temperature affect CDs retention on both stationary phases. The changes of temperature influence mass transfer between the mobile and stationary phase. Moreover, the study of the temperature influence can provide insight into the separation mechanisms and allows to design optimal conditions of analysis. Table 2a and b present some experimental data of temperature dependence. In a chromatographic system, the retention factor of the solute is dependent on the partitioning process between the mobile and stationary phases. The van’t Hoff expression for such a chromatographic system is given by the equation [28]:
◦
ln k =
◦
S −H + + ln ϕ RT R
(2)
where k is the retention factor, H◦ and S◦ represent standard enthalpy and entropy changes of transfer of the solute between the mobile and the stationary phase, R is the gas constant, T is the absolute temperature and φ is the volume phase ratio of the stationary to the mobile phase. The phase ratio for RP C18 column was calculated from the following equation [29]: Vs =
(%C)(M)(Wρ ) (100)(12.01)(nc )(ρ)
(3)
where Vs is the stationary phase volume, %C is grams of carbon per 100 grams of bonded silica, nc refers to the number of carbon atoms per mole of silane, M is the molecular weight of silane, Wρ is the weight (g) of the bonded packing material in the column. The density of the bonded octadecylsilyl (ρ) is 0.8607 g/cm3 [29]. The mobile phase volume in the column was assumed to be the same as the retention volume of a non-retained solute. The calculated phase ratio value was ϕ = 0.22. For the PGC column, Vs was calculated as the difference between the total volume of the column and its void volume. The obtained phase ratio was equal to 0.33. According to Eq. (2), the slope of the ln k plot against 1/T provides information about enthalpy differences, whereas the intercept is related to entropy changes. If H◦ , S◦ and ϕ are independent of temperature, the plots of ln k versus 1/T should be linear.
−8.87 −6.35 −6.97 7.53 ± 0.52 2.70 ± 0.72 5.99 ± 0.22 −1.34 ± 0.52 −3.65 ± 0.72 −0.98 ± 0.24 −8.66 −7.51 −6.34 −10.93 ± 1.14 −6.34 ± 1.56 −2.45 ± 1.78 −19.59 ± 1.18 −13.85 ± 1.61 −8.80 ± 1.85 T = 298 K.
G (kJ/mol) TS (kJ/mol) G (kJ/mol) TS (kJ/mol)
−7.86 −7.55 −6.29
H (kJ/mol) H (kJ/mol)
4.15 ± 0.10 7.93 ± 0.025 11.32 ± 0.35
TS (kJ/mol) 20% IPA/doixane 80% MeOH 50% EtOH
H (kJ/mol)
PGC RP C18
(b) Permethylated CDs TM-␣-CD −3.71 ± 0.12 TM--CD 0.38 ± 0.03 TM-␥-CD 5.03 ± 0.37
−4.35 −5.92 −8.45 −6.49 ± 1.02 −5.48 ± 0.20 −8.72 ± 0.84 −10.84 ± 1.06 −11.40 ± 0.21 −17.18 ± 0.86 −4.27 −6.53 −8.71 −19.95 ± 1.78 −28.02 ± 1.46 −28.69 ± 1.88 −24.22 ± 1.81 −34.55 ± 1.50 −37.40 ± 1.90 −6.37 −8.89 −5.23 −54.41 ± 3.39 −48.73 ± 2.95 −49.06 ± 3.10 −60.77 ± 3.48 −57.63 ± 3.01 −54.28 ± 3.16 −3.98 −8.42 −6.18
TS (kJ/mol) G (kJ/mol) TS (kJ/mol)
−61.89 ± 4.41 −52.87 ± 2.30 −56.36 ± 3.37
H (kJ/mol) H (kJ/mol) H (kJ/mol)
G (kJ/mol)
15% EtOH 7.5% MeOH
(a) Native CDs ␣-CD −65.87 ± 4.56 -CD −61.29 ± 2.37 ␥-CD −62.55 ± 3.49
TS (kJ/mol) Fig. 4. The van’t Hoff plots for permethylated-CDs in 50% EtOH on RP C18 column.
5% EtOH
For native CDs for both alcohols on the RP C18 column, both H and S are negative, thus the transfer between the mobile and stationary phases is enthalpically driven. A similar situation can be observed on the PGC column. Comparing, the ratio TS/H for both columns, lower values have been obtained for PGC than for RP C18 column
PGC
3.3. Native cyclodextrins
RP C18
The results from all the studied systems are presented in Table 3a and b. The van’t Hoff plots show linear behaviour in the examinated temperature range. As an example, Fig. 3 shows the van’t Hoff plots for native CDs in 5% EtOH on the RP C18 column. The retention time of CDs for all investigated eluents decreases with the increase of column temperature, except the retention for TM--CD and TM-␥-CD on RP C18 column in the ethanolic solution, where the retention was independent or increases (see Fig. 4). The values of free energy can be considered as a measure of the total efficiency of the transfer of the solute from the mobile phase to the stationary phase. The more negative H value indicates that the solute is more effectively transferred to the stationary phase from the point of view of energetic interaction. The negative values of entropy changes indicate that the solute is more organized in the stationary phase than in the mobile phase.
TS (kJ/mol)
(4)
Table 3 Thermodynamic parameters of native CDs and permethylated CDs determined with various eluents on RP C18 and PGC columns
G = H − TS
G (kJ/mol)
The H and S values obtained from the plot can be used to obtain G according to the equation:
H (kJ/mol)
50% MeOH
Fig. 3. The van’t Hoff plots for native CDs in 5% EtOH on RP C18 column.
G (kJ/mol)
A. Kwaterczak, A. Bielejewska / Analytica Chimica Acta 537 (2005) 41–46 G (kJ/mol)
44
A. Kwaterczak, A. Bielejewska / Analytica Chimica Acta 537 (2005) 41–46
(e.g. for ␣-CD on PGC column, it is 0.82 and for RP C18 column it is 0.94). This indicates that for the PGC column the unfavourable entropy influence plays lesser role in the adsorption process than for RP C18 column, thus it may be responsible for the general rule of increasing of CDs retention on PGC material. In the RP C18 column, the H value of -CD was less negative for the ethanolic solution compared to other native CDs, but the longest retention time was obtained. Thus, less unfavourable entropy changes for -CD induce its high retention. The most favourable interactions enthalpy with the RP C18 column occurred for ␣-CD, but unfavourable entropy interactions induced shorter retention. ␣-CD is eluted first in ethanol and as the second in methanol. On the PGC column for both eluents, the favourable H and G changes increased with the increasing size of the cyclodextrin. Thus, the ␥-CD was the most effectively transferred from the mobile to the stationary phase. 3.4. Permethylated cyclodextrins For the ethanolic solution on the RP C18 column, the slopes of the van’t Hoff plots change from negative to positive with the increasing of molecular weight of CDs. This is connected with the different behaviour of TM-␣-CD, TM--CD and TM-␥-CD. For TM-␣-CD, the retention time decreases with increasing temperature, for TM--CD the retention time is almost independent of temperature. The retention time of TM␥-CD increases with the increasing temperature. The positive value of enthalpy changes indicates that it is energetically more favourable for the solutes to be in the mobile phase. In such situation, the adsorption of the stationary phase occurs only due to entropic advantages (TM-␥-CD). On RP C18 column, for the ethanolic mobile phase, the entropy values are positive for all studied permethylated cyclodextrins, thus entropy facilitates the transfer of permethylated CDs to the stationary phase in these conditions. The favourable entropic interaction increases with the increasing size of cyclodextrin. As one can see for the results on the RP C18 column, the mobile phase composition can play an essential role in the adsorption process. Adsorption on the RP C18 column is an entropy-favourable process in the presence of ethanol (positive value of entropy) and entropy-unfavourable for the methanolic solution. In the latter case, the entropy changes receive negative values. The negative values of enthalpy and entropy changes for the methanolic solution indicate that the transfer between the mobile and stationary phases is enthalpically driven. On the PGC column, TM--CD has the most negative H values, but is eluated first because the other CDs have much more favourable entropy interactions with the stationary phase. On the PGC support, the process seems similar to that on RP C18 in the ethanolic solution. Entropy is greater than enthalpy (TM-␣-CD and TM-␥-CD) and facilitates the adsorption process.
45
Fig. 5. Correlation between ln k at 30 ◦ C and H for native cyclodextrins on RP C18 column. For chromatographic conditions see Section 2.
4. Enthalpy–entropy compensation To obtain more information about the interaction of the cyclodextrin with both stationary phases, the correlation between the logarithm of the retention factor measured at 30 ◦ C and the enthalpies for the particular cyclodextrin have been demonstrated in Figs. 5–7. The plots of the ln k of various cyclodextrins versus corresponding enthalpy are linear when enthalpy–entropy compensation occurs, meaning that the reversible binding of the cyclodextrin on the stationary phase is subject to an essentially identical mechanism [30]. Depend-
Fig. 6. Correlation between ln k at 30 ◦ C and H for permethylated cyclodextrins on RP C18 column. For chromatographic conditions see Section 2.
Fig. 7. Correlation between ln k at 30 ◦ C and H for native and permethylated cyclodextrins on PGC column. For chromatographic conditions see Section 2.
46
A. Kwaterczak, A. Bielejewska / Analytica Chimica Acta 537 (2005) 41–46
ing on the cyclodextrin and the chromatographic conditions, three situations have been observed. In Fig. 5, for native cyclodextrins and the RP C18 column, we can see that for ␣-, - and ␥-CD in the ethanolic mobile phase and for ␣- and -CD in the methanolic solution, compensation can be observed. This could mean that physicochemical interaction between these cyclodextrins and RP C18 column has the same nature for both alcohols (only for ␥-CD in the methanolic solution, the interactions seems to have a different physico-chemical basis). It is interesting to see that for these processes, more negative values of enthalpy do not mean longer retention. High values of favourable enthalpy interaction are followed by higher unfavourable entropy interactions with the stationary phase, which causes shorter retention. In Fig. 6, for permethylated cyclodextrin and the silica RP C18 column, two linear plots for methanolic and ethanolic solutions have been obtained. From these results one can deduce that the physico-chemical interactions of permethylated cyclodextrin with the mobile and stationary phases are similar for a given alcohol. For these cyclodextrins in these chromatographic conditions, shorter retention has been obtained for lower negative values of enthalpy. In Fig. 7, for native and permethylated cyclodextrin on the PGC column, there is no linear relationship between ln k and H. One can see that for very similar retention of ␣-, - and ␥-CD obtained with various alcohols, much higher values of negative enthalpy are obtained for ethanol than for methanol solutions.
5. Conclusion The methyl derivatives of ␣-, - and ␥-CD are much more strongly adsorbed on the hydrophobic stationary phase than native CD themself. The retention of CDs is much stronger on the PGC than on the RP C18 column. Depending on the column, elution order of native and permethylated CDs was different. The elution order changes are proof that although the RP C18 and PGC columns act in reverse chromatographic mode, the mechanism of separation is different. From temperature study, we can see that the transfer of the CDs between the mobile and stationary phase may be enthalpy or entropy driven. As the physico-chemical interaction of the given CD with various stationary and mobile phase seems to be different, the chiral discrimination obtained for the same CD in various environment may be different. The physico-chemical interactions of cyclodextrins seem to be similar only in the case of native cyclodextrins (except ␥-CD) with the RP C18 column when enthalpy–entropy compensation can be observed. In the case of permethylated cyclodextrins and the RP C18 column, interactions are only similar for individual alcohols. For the PGC column, the in-
fluence of individual interactions on individual cyclodextrins seems to be different. Acknowledgment ˙ We would like to thank Dr. Janusz Zukowski and Dr. Bronisław Gł´od for providing the PGC columns. References [1] H. Riering, M. Sieber, J. Chromatogr. A 728 (1996) 171. [2] D. Schumacher, C. Mitchell, T. Xiao, R. Rozhkov, R. Larock, et al., J. Chromatogr. A 1011 (2003) 37. [3] Y. Kawaguchi, M. Tanaka, M. Nakae, K. Funazo, T. Shono, Anal. Chem. 55 (1983) 1852. [4] Y. Liu, A.W. Lantz, D.W. Armstrong, J. Liq. Chromatogr. Related Technol. 27 (7–9) (2004) 1121. [5] R. Herr´aez-Hern´andez, P. Campins-Falco, J. Chromatogr. B: Biomed. Sci. Appl. 740 (2000) 169. [6] A. Bielejewska, R. Nowakowski, K. Duszczyk, D. Sybilska, J. Chromatogr. A 840 (1999) 159. [7] A. Bielejewska, K. Duszczyk, D. Sybilska, J. Chromatogr. A 931 (2001) 81. [8] M. Asztemborska, A. Bielejewska, K. Duszczyk, D. Sybilska, J. Chromatogr. A 847 (2000) 73. [9] I. Clarot, D. Cledat, Y.C. Guillaume, P.J.P. Cardot, Chromatographia 54 (2001) 447. [10] L. Chen, L. Zhang, C. Ching, C. Ng Siu, J. Chromatogr. A 950 (2002) 65. [11] R. Nowakowski, A. Bielejewska, K. Duszczyk, D. Sybilska, J. Chromatogr. A 782 (1997) 1. [12] K. Flood, E. Reynolds, N. Snow, J. Chromatogr. A 903 (2000) 49. [13] J. Knox, B. Kaur, J. Chromatogr. 352 (1986) 3. [14] E. Forgacs, J. Chromatogr. A 975 (2002) 229. [15] J. Oxelbark, S. Claeson, Tetrahedron, Asymmetry 13 (2002) 2235. [16] Q.H. Wan, P.N. Show, M.C. Davies, D.A. Barrett, J. Chromatogr. A 697 (1995) 219. [17] P.T. Jackson, M.R. Schure, T.P. Weber, P.W. Carr, Anal. Chem. 69 (1997) 416. [18] M.C. Hennion, V. Coquart, S. Guenu, C. Sella, J. Chromatogr. A 712 (1995) 287. [19] K.K. Unger, Anal. Chem. 55 (1983) 361. [20] V. N´emeth-Kiss, E. Forg´as, T. Cserh´ati, J. Chromatogr. A 776 (1997) 147. [21] R. Nowakowski, P.J.P. Cardot, A.W. Coleman, E. Villard, G. Guiochon, Anal. Chem. 67 (1995) 259. [22] A. Bielejewska, M. Ko´zbiał, R. Nowakowski, K. Duszczyk, D. Sybilska, Anal. Chim. Acta 300 (1995) 201. [23] A.K. Chatjigakis, Ph.J.P. Cardot, A.W. Coleman, H. Parrot-Lopez, Chromatographia 36 (1993) 174. [24] I. Clarot, D. Cl´edat, L. Boulkanz, E. Assidjo, T. Chian´ea, P. Cardot, J. Chromatogr. Sci. 38 (2000) 38. [25] E. Grushka, M.N. Myers, P.D. Schettler, J.C. Giddings, Anal. Chem. 41 (7) (1969) 889. [26] K. Fromming, J. Szejtli, Cyclodextrins in Pharmacy, Kluwer Academic Publisher, 1994. [27] J. Szejtli, Cyclodextrins and their inclusion complexes, Akademiai Kiado Budapest, 1982. [28] J. Chmielowiec, H. Sawitzky, J. Chromatogr. Sci. 17 (1979) 245. [29] K.B. Sentell, J.G. Dorsey, J. Liq. Chromatogr. 11 (1988) 1875. [30] W. Melander, D.E. Campbell, C. Horvath, J. Chromatogr. 158 (1978) 215.