High selectivity in new chiral separations of dansyl amino acids by cyclodextrin derivatives in electrokinetic chromatography

Journal of Chromatography A, 1155 (2007) 172–179

High selectivity in new chiral separations of dansyl amino acids by cyclodextrin derivatives in electrokinetic chromatography V. Cucinotta a,∗ , A. Giuffrida a , G. Grasso b , G. Maccarrone a , M. Messina a , G. Vecchio a a

Department Chemical Sciences, University of Catania, Viale A. Doria 6, 95125 Catania, Italy b Istituto di Biostrutture e Bioimmagini, CNR, Viale A. Doria 6, 95125 Catania, Italy Available online 8 February 2007

Abstract Enantiomeric pairs of 11 dansyl derivatives of ␣-amino acids were used as analytes in electrokinetic chromatography to test the ability as chiral selectors of two pure derivatives of ␤-cyclodextrin: the ethylendiamine derivative in primary position (CDen) and a member of a new class of receptors, the cysteamine-bridged hemispherodextrin THCMH. The selectivity obtained by the presence of the hemispherodextrin, appears particularly promising as shown by the large values of resolution obtained. The importance of a detailed analysis of these data is discussed in terms of suggestions for a rational approach to separation science. © 2007 Elsevier B.V. All rights reserved. Keywords: Chiral separation; Cyclodextrin derivatives; Electrokinetic chromatography; Amino acids derivatives

1. Introduction Enantiomers of dansyl derivatives of ␣-amino acids have been extensively used in literature as analytes to test the chiral selection ability of different kinds of selectors, as well as to test the influence of the experimental conditions for the same kind of receptor. Results of many investigations concerning such kinds of analytes are found, both in HPLC and in electrically driven techniques [1–6]. This extensive use is justified by their relatively low cost and easy availability, as well as their intrinsic fluorescence. Various alternative possibilities are available in the laboratory, since the reaction is a sulfonation of the amino group of the amino acid by the reactant 5-(dimethylamino)naphthalene-1-sulfonyl chloride, as reported in Fig. 1, and this reaction is also routinely used in the analysis of real samples [7,8]. In capillary electrophoresis, while laser-induced fluorescence (LIF) detection permits us to obtain the best values of sensitivity, together with the filtering of interferences from UV-adsorbing species in the background electrolyte (BGE), owing to the high value of their absorptivity coefficient (maximum value at about

∗

Corresponding author. Tel.: +39 0957385094; fax: +39 095580138. E-mail address: [email protected] (V. Cucinotta).

0021-9673/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2007.02.006

218 nm) induced by the naphthyl moiety, the detection of dansylated species by UV-Vis absorbance detectors is easy even at concentrations as low as 20 ␮M. In our laboratory, we synthesised and characterised several pure 3- [9,10] or 6- [11–14] derivatives of cyclodextrins. In addition, a new class of chiral selectors were synthesised, which we have called hemispherodextrins. They are capped derivatives of ␤-cyclodextrin, characterised by a molecule of ␣,␣ -d-trehalose in the capping moiety, bonded to the primary rim of cyclodextrin cavity through its primary positions. As shown in Fig. 2, the two saccharidic systems are bonded through moieties acting as a bridge. By varying such bridges, it is possible to obtain capping units of different types and lengths. By such derivatisation, owing to the trehalose moiety, it is possible to extend the saccharidic system with the capping unit, thus turning the toroidal cavity of the parent cyclodextrins into an approximately hemispheric shape, hence the use of the term “hemispherodextrin”. The use of a specific name for these compounds seems indeed justified by the peculiar receptor properties induced by this hemispherical shape, which gives rise to a better separation of the substrate from the aqueous solvent. The use of cyclodextrins in chiral separation, both in chromatography and in capillary electrophoresis, has had a long history, and in CE dates more than 20 years [15,16]. While in HPLC, the development of efficient chiral stationary phases has

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173

Fig. 1. Scheme of the reaction for the synthesis of dansyl derivatives.

partly superseded the use of cyclodextrins in the mobile phase, in capillary electrophoresis, CEC excluded, the absence of such competition has permitted the growing use of chiral selectors in BGE and the electrokinetic chromatography has developed also in the absence of micelles, at least for readily hydrosoluble analytes [17–21]. As concerns cyclodextrins specifically, the term CD-EKC (electrokinetic chromatography by cyclodextrins) has been introduced advantageously. The use of neutrally charged parent cyclodextrins, while being limited to charged analytes, relies exclusively on differences in electrophoretic mobilities of the diastereoisomeric complexes obtained, mainly with respect to the corresponding free analyte. There is an obvious advantage in introducing ionisable groups which, by electrically charging cyclodextrins, both widen and improve the chiral separation properties of these molecules. Results obtained with the derivatives synthesised in our laboratory appear more than encouraging. At this point, a more than significant amount of data show that, by opportunely optimising both the pH of the BGE and the analytical concentration of the selector, high chiral resolutions of most of the racemates can be obtained by the suitable selector. In order to direct the search for optimum conditions correctly, the availability of pure derivatives of cyclodextrins is required, though, unfortunately, generally only mixtures of derivatives are commercially available. In this series of investigations verifying the chiral selector ability of synthesised derivatives, an investigation concerning dansyl amino acids could not be missing. Here, we report the results obtained in CD-EKC in the separation of racemates of the dansylated derivatives of

the 11 ␣-amino acids listed in Fig. 3, by using one of the hemispherodextrins, the first synthesised, namely the THCMH (6A,6D-dideoxy-6A,6D-[6,6 -dideoxy-6,6 -di(S-cysteamine)␣,␣ -trehalose]-␤-cyclodextrin), schematically shown in Fig. 2. This compound, where the bridge is due to cysteamine moiety, has been synthesised and characterised [22], and was elsewhere used to separate phenoxy acids [23] and phenylpropionic (profen) racemates [24], while its protonation constants were determined by CE experiments by a computer program, developed by ourselves [25]. 6-Deoxy-6-[1-(2-amino)ethylamino]-␤-cyclodextrin (CDen), also shown in Fig. 2, is the ethylenediamine derivative of ␤-cyclodextrin in the primary position [26], whose receptor properties we already used in CD-EKC [27]. The same selector was used by exploiting its ligand properties towards copper(II) both in ligand exchange chromatography (LEC) [26] and ligand exchange capillary electrophoresis (LECE) [28]. In order to have a term of comparison that could underline the effect of the capping moiety in THCMH, the analogous systems formed by CDen with the same analytes reported in Fig. 2 were also investigated. A wide range of concentrations of selectors was explored (in some cases from 0.15 to 1.8 mM) in order to obtain information about the interaction between selector and analytes. 2. Experimental 2.1. Materials Dansyl amino acids were purchased from Fluka. THCMH and CDen were synthesised in our laboratory as described elsewhere [22,26]. 2.2. CE measurements

Fig. 2. Schematic formula of the cyclodextrin derivatives used as chiral selectors.

CE measurements were carried out on a Beckman P/ACE MDQ equipped with a diode array detector. An uncoated fusedsilica capillary (Beckman, 49.6 cm total length, 59.6 cm effective length, 75 ␮m I.D.) was held at a constant temperature of 20 ◦ C. The separation was carried out in the direct polarity mode at 25 kV. BGEs for the chiral separation experiments were prepared by dissolving each cyclodextrin derivative (0.15–1.8 mM) in 20 mM CH3 COONH4 (pH 6.8). Though ammonium acetate cannot be considered a real buffer, nonetheless it has been calculated and verified that during experiments, at the used ana-

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Fig. 3. Schematic formula of the enantiomeric pairs of dansyl amino acids used as analytes.

lytical concentrations, no appreciable variation of pH occurs. The sample solution (0.05 mM in racemate, 0.01 mM in lenantiomer excess) was obtained by dissolving the analyte in the same condition. The sample was hydrodinamically injected (0.6 psi, for 8 s). Before each experiment, the capillary was flushed (pressure of 2.0 × 106 Pa) with 20 mM CH3 COONH4 ,

0.10 mM NaOH and the BGE used in separation. The capillary was rinsed daily with 100 mM HCl, water, 100 mM NaOH and finally the used BGE. Values of current were found to increase from 26 to 37 ␮A with increase in the selector concentration. As values of EOF mobility are concerned, they significantly decrease with increase in the selector concentration. While in

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175

Table 1 Values of selectivity S and resolution R for the CDen/dns-AA systems investigated C (CDen) (mM)

aba

0.15 0.25 0.40 0.55 0.80 1.10 1.80

asp

glu

leu

phe

ser

S

R

S

R

S

R

S

R

S

R

S

R

– – 0 0 0.005 0.008 0.012

– – – –

0.020 0.030 0.034 0.035 0.055 0.067 0.028

0.71 1.14 1.12 1.39 1.75 2.04 0.92

– 0 0.008 0.021 0.038 0.124 0.144

– –

– – 0 0.011 0.015 0.025 0.025

– – – 0.51 0.76 1.09 1.14

– 0.018 0.023 0.027 0.023 0.020 0.020

– 1.09 1.28 1.41 1.10 1.10 0.99

– – 0 0.006 0.013 0.025 0.028

– – – 0.30 0.54 0.87 0.86

a a a

a

1.26 1.65 2.35 4.09

The systems with threo, val, nvl, nle and met derivatives were omitted since no separation was obtained at any concentration of the selector. a Low value of R.

the case of CDen, this decrease is smaller (from 4.0 × 10−8 to 2.8 × 10−8 m2 V−1 s−1 ), in the case of THCMH this effect is more pronounced (from 4.0 × 10−8 to 1.9 × 10−8 m2 V−1 s−1 ). On increasing the selector concentration, either an increase of viscosity of the BGE or an increase of the interaction of the cationic selector with the silica wall can be invoked. It cannot be excluded that both of these effects influence the EOF values. Values of efficiency up to N = 1.6 × 105 were obtained.

3. Results and discussion Results concerning the dns-AA racemates separations in the presence of CDen are briefly summarised in Table 1, while the values of the mobilities, corrected for the electroosmotic flow, concerning the same experiments, are reported in Table 2. As can be seen, initially the minimum value of analytical concentration of the selector tried was 0.4 mM, but when separation was attained at this value, even lower values of concentrations were

Table 2 Mobilities (×10−8 m2 V−1 s−1 ) corrected for the electroosmotic flow of dns-AA enantiomers in the presence of CDen C (CDen) (mM) 0 aba μDcorr μLcorr asp μLcorr μDcorr glu μLcorr μDcorr leu μDcorr μLcorr met μ1corr μ2corr nle μ1corr μ2corr nvl μ1corr μ2corr phe μDcorr μLcorr ser μLcorr μDcorr thr μ1corr μ2corr val μ1corr μ2corr

0.15

0.25

0.40

0.55

0.80

1.10

1.80

−1.78 −1.78

– –

– –

−1.24 −1.24

−1.23 −1.23

−1.19 −1.20

−1.03 −1.05

−0.93 −0.95

−3.23 −3.23

−2.60 −2.62

−2.45 −2.51

−2.28 −2.34

−1.87 −1.90

−1.69 −1.80

−1.59 −1.72

−1.46 −1.52

−3.14 −3.14

– –

−2.01 −2.01

−1.67 −1.68

−1.62 −1.65

−1.56 −1.61

−1.44 −1.56

−1.15 −1.33

−1.75 −1.75

– –

– –

−1.10 −1.10

−1.06 −1.09

−1.02 −1.06

−0.93 −0.98

−0.83 −0.89

−1.81 −1.81

– –

– –

−1.21 −1.21

−1.20 −1.20

−1.17 −1.17

−1.16 −1.16

−1.03 −1.03

−1.80 −1.80

– –

– –

−0.98 −0.98

−0.98 −0.98

−0.94 −0.94

−0.92 −0.92

−0.82 −0.82

−1.82 −1.82

– –

– –

−0.81 −0.81

−0.80 −0.80

−0.80 −0.80

−0.79 −0.79

−0.71 −0.71

−1.82 −1.82

– –

−1.30 −1.35

−1.15 −1.23

−0.93 −1.01

−0.65 −0.72

−0.64 −0.72

−0.60 −0.68

−1.94 −1.94

– –

– –

−1.56 −1.56

−1.55 −1.56

−1.52 −1.55

−1.50 −1.53

−1.24 −1.28

−1.87 −1.87

– –

– –

−1.46 −1.46

−1.44 −1.44

−1.38 −1.38

−1.30 −1.30

−1.23 −1.23

−1.85 −1.85

– –

– –

−1.31 −1.31

−1.27 −1.27

−1.23 −1.23

−1.15 −1.15

−1.08 −1.08

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investigated. As concerns pH, a neutral value (6.8) was chosen. At this pH, the dns-AAs are present as anionic species (partly divalent in the case of glutamic and aspartic acids). The protonation constants of CDen (log K1 = 8.92, log K2 = 5.56) [26] show that this selector at pH value of the BGE will be almost completely present as monoanionic species. Thus, the mutual interaction between selector and each of the analytes can be assisted by their opposite electrical charge, and their complex is neutral: the higher the formation degree of the complex, the shorter its apparent migration time. Electrophoretic runs carried out using the selector used as analytes, showed that at the BGE pH CDen has a corrected mobility ␮ = 1.75 × 10−8 m2 V−1 s−1 , as expected by its electrical charge. It is interesting to observe the order of migration of the two enantiomers. In this respect, the two selectors behave in the same way, and the order is a function of the specific racemate involved. By addition of an excess of the l-enantiomer, it is possible to identify the peak of this enantiomer, which shows a shorter migration time in the case of the aspartate, glutamate, serine, threonine and norvaline derivatives. On the contrary, in the case of the derivatives of norleucine, and phenylalanine, the l-enantiomer shows a longer migration time with respect to the d-enantiomer. Lastly, in the cases of valine, leucine, methionine and ␣-amino butyrate derivatives, owing to the unavailability of either of the pure enantiomers, it has not been possible to deduce the identity of each peak. If we consider the first four racemates of the first group (l-enantiomer faster), we see that all of them show a polar side chain, ending either with a carboxylate or with an hydroxyl group, and thus they show two distinct arms both able to electrostatically interact with the protonated amino group of the selector. This, however, is not the case for the norvaline derivative. What really appears

Fig. 4. Electropherograms (␭ = 218 nm) of CDen/dns-phe systems at different concentrations of CDen.

common for all five of them is the absence of a large apolar side chain Thus, apparently, what makes the difference is the presence of a wide apolar system, as we find in leucine and phenylalanine systems. Tentatively, it can be hypothesised that in both leucine and methionine derivatives, due to their quite wide apolar side chain, it is the d-enantiomer having a shorter migration time, while the reverse behaviour should be observed for dans-val. In Fig. 4, the electropherograms concerning the dns-phe/CDen systems at the different concentrations explored are reported as an example. It can be seen in the figure that at certain values of selector concentrations, baseline separation is achieved. While the resolution is the quantity that defines the usefulness and feasibility of a separation, when appreciating the selector ability, the selectivity indicators should be considered. Capillary electrophoresis has borrowed most of its “language” from chromatography for fully justified historical reasons. Nonetheless, in our opinion, time has come to start to reconsider the models used in CE, in order to verify if some concepts need a more specific and autonomous development. Here, we would start from the selectivity indicator. Presently, Table 3 Values of selectivity S and resolution R for the THCMH/dns-AA systems investigated C (THCMH) (mM)

aba S R asp S R glu S R leu S R met S R nle S R nvl S R phe S R ser S R thr S R val S R a

0.15

0.25

0.40

0.55

0.80

1.1

1.8

0 –

0.005 a

0.013 0.50

0.025 1.38

0.042 4.31

0.051 3.06

0.051 3.03

0.078 2.11

0.113 2.69

0.120 3.50

0.150 3.44

0.160 3.94

0.161 5.72

0.164 6.00

0.038 1.46

0.071 1.89

0.082 2.68

0.127 3.53

0.191 5.49

0.290 4.35

0.332 7.64

0 –

0 –

0.015 0.50

0.023 0.99

0.037 0.82

0.039 0.92

0.050 1.15

– –

– –

– –

– –

– –

– –

0.006

0 –

0 –

0 –

0.006

0.007

a

a

0.014 0.61

0.014 0.63

0 –

0 –

0 –

0.003 a

0.010 0.57

0.010 0.60

0.012 0.44

0.034 1.46

0.056 1.69

0.057 2.25

0.085 2.65

0.096 2.57

0.110 2.88

0.065 1.69

0 –

0.009 0.43

0.016 0.92

0.032 1.55

0.053 1.52

0.063 2.86

0.121 4.55

0 –

0 –

0 –

0 –

0.004

0.011

0.008

a

a

a

0 –

0 –

0 –

0.005 0.50

0.017 0.83

0.024 0.95

0.032 1.20

Low value of R.

a

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177

Fig. 5. Electropherograms (␭ = 218 nm) of THCMH/dns-aba systems at different concentrations of THCMH.

most studies of capillary electrophoresis, either omit the use of a specific indicator of selectivity, reporting only mobilities, or use ␣-values, borrowed from chromatography. This parameter suffers from a paradox when used in CE: if a separation is carried out between a cationic and an anionic species, the opposite signs of the two mobilities give rise to a negative value of their ratio. More interestingly, even considering the absolute value only of mobilities, their separation is underestimated: if the two mobilities had the same absolute value, we could obtain |␣| = 1, as if they were not separated at all. This paradox is due to the fact that reference in chromatography is at one extreme of chromatogram, while in CE, the reference is at an intermediate value,

corresponding to the fact that the electrophoretic mobilities are algebraic (not arithmetic) quantities. S-values, as defined according to the following formula [29]: S=

2 × (μ2 − μ1 ) μ1 + μ 2

are strongly recommended. In this formula, the difference in apparent mobilities gives visual separations of the peaks in electropherograms, while their sum at the denominator takes into account how long the separation process has operated, thus acting as a normalising factor. This formula does not suffer from

Fig. 6. Electropherograms (␭ = 218 nm) of THCMH/dns-phe systems at different concentrations of THCMH.

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separation is obtained, the formation degree of the complex is higher than for well-separated systems. In Table 3, the results concerning the systems in the presence of THCMH are reported. Also for these systems, neutral BGE solutions were used (pH = 6.8), and THCMH is mainly present as mono-cationic species (log K1 = 8.8, log K2 = 7.47) [25]. Electrophoretic runs carried out using the selector used as analytes, showed that at the BGE pH THCMH has a corrected mobility ␮ = 2.20 ×10−8 m2 V−1 s−1 , as expected by its electrical charge. Firstly, it can be seen that all the 11 enantiomeric pairs investigated are resolved, at least partially, in the presence of this selector. Large values of S are obtained for some systems. Indicatively, a value of S ranging from 0.025 to 0.040, depending on the efficiency, is sufficient to obtain a baseline separation. Thus, values higher than 0.1 must be considered huge, particularly for a chiral separation. Indeed, for the system dns-glu in the presence of the highest explored concentration of THCMH (1.8 mM), a value much higher than 0.1 (0.332) is attained, and correspondingly, a value of 7.64 is obtained for the resolution: to our knowledge, these are among the highest values ever reported for chiral separations in CE. Differently from the systems with CDen, with the only exception of dns-phe, the highest selectivity

any paradox, and appears perfectly appropriate as an indicator of selectivity. The S-values in the function of the selector concentration show different trends in the different systems. Interestingly, for asp and phe, the S-values reach a maximum at an intermediate value of selector concentration. This behaviour would suggest that, even when successful separation is not attained in the presence of high concentrations of selector, it may be still worthwhile to explore lower concentrations, with a reasonable probability of obtaining the separation. The reason is readily seen when considering that the separation is obtained by a difference in the formation degree between the two enantiomers, independently as to how high the formation degree is for each enantiomer. The values of the mobilities, corrected for the electroosmotic flow, reported in Table 2, show this aspect very clearly: in all systems, even those where no separation is attained, the absolute values of mobility decrease with increase in the CDen concentration. This decrease is due to the increase in the formation degree of the neutral complex, at the expense of the anionic free analyte, as the migration time approaches the EOF time. Thus, we cannot even exclude that for some of the systems where no

Table 4 Mobilities (×10−8 m2 V−1 s−1 ) corrected for the electroosmotic flow of dns-AA enantiomers in the presence of THCMH C (THCMH) (mM)

aba μ1corr μ2corr asp μLcorr μDcorr glu μLcorr μDcorr leu μDcorr μLcorr met μ1corr μ2corr nle μ1corr μ2corr nvl μLcorr μDcorr phe μDcorr μLcorr ser μLcorr μDcorr thr μLcorr μDcorr val μ1corr μ2corr

0

0.15

0.25

0.40

0.55

0.80

1.1

1.8

−1.78

−1.10 −1.10

−0.99 −1.01

−0.96 −0.99

−0.82 −0.87

−0.61 −0.69

−0.56 −0.65

−0.20 −0.29

−3.23

−3.02 −3.19

−3.01 −2.80

−2.32 −2.61

−2.22 −2.59

−2.21 −2.54

−2.01 −2.44

−1.78 −2.22

−3.14

−2.62 −2.67

−2.49 −2.57

−2.17 −2.31

−1.97 −2.18

−1.55 −1.73

−1.16 −1.65

−1.08 −1.54

−1.75

−0.97 −0.97

−0.89 −0.91

−0.87 −0.91

−0.83 −0.88

−0.78 −0.85

−0.68 −0.74

−0.36 −0.44

−1.81

−1.09 −1.09

−1.08 −1.08

−1.07 −1.07

−1.06 −1.06

−1.03 −1.03

−0.94 −0.94

−0.64 −0.65

−1.80

−0.78 −0.78

−0.77 −0.77

−0.76 −0.76

−0.74 −0.75

−0.73 −0.74

−0.71 −0.73

−0.68 −0.70

−1.82

−0.88 −0.88

−0.88 −0.88

−0.87 −0.87

−0.85 −0.86

−0.84 −0.86

−0.76 −0.77

−0.45 −0.47

−1.82

−1.51 −1.58

−1.37 −1.47

−1.12 −1.29

−0.97 −1.17

−0.77 −0.95

−0.54 −0.76

−0.17 −0.30

−1.94

−1.59 −1.59

−1.51 −1.53

−1.48 −1.52

−1.43 −1.49

−1.38 −1.46

−1.33 −1.40

−0.97 −1.11

−1.87

−1.40 −1.40

−1.26 −1.26

−1.15 −1.15

−0.73 −0.73

−0.65 −0.69

−0.65 −0.66

−1.85

−1.19 −1.19

−1.18 −1.18

−1.12 −1.13

−1.12 −1.15

−1.09 −1.12

−0.84 −0.87

− − −1.15 −1.15

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is attained at the highest investigated concentration of THCMH. Examples of electropherograms are reported in Figs. 5 and 6 for, respectively, the system with dns-aba and dns-phe, where the exceptional resolution obtained can be appreciated. In the case of phenylalanine derivatives, a fronting effect is particularly noticeable, seen only slightly in other systems, which should be ascribed to electromigration dispersion [30]. In order to achieve a better understanding of the mechanism of separation, effective electrophoretic mobilities of the analytes in the different conditions have been reported in Table 4. Similarly to that described for the first selector, a qualitative analysis of these data once more clearly shows no direct correlation between selectivity and formation degrees of the complexes between the single enantiomer and the selector. Since selectivity depends on differences in formation degrees, a low stability of one enantiomer complex can help in separation as well as the high stability of the other enantiomer. The comparison between the serine systems and the corresponding systems with phenylalanine appears particularly interesting. If we consider the decrease of the absolute value of mobility of serine going from the lowest concentration (0.15 mM) to 1.8 mM, we obtain a poor decrease, about 38% as an average of the two enantiomers, compared with a corresponding value for phenylalanine of about 52%. This difference in decrease percentages, that reflects the formation degree of the complex, however, does not give rise to any significant gain in separation performance for dns-phe. On the contrary, it is the dns-ser that is better separated, since, as already mentioned, when going from 1.1 to 1.8 mM concentration of THCMH, the selectivity for dns-phe worsens. Lastly, the comparison between the systems of these two enantiomeric pairs with CDen compared to those with THCMH show how in this case an increase in the stability of the complexes generally improves separation, but, the small difference in stability of the complexes of dns-ser with THCMH compared to those with CDen is even more effective than the large difference in the analogous case of dns-phe. 4. Concluding remarks This study shows the usefulness of CD-EKC once more, especially when cyclodextrin derivatives bearing ionisable groups are used. The reproducibility and the useful comparison obtained when using pure compounds as selectors can easily be seen. In the field of cyclodextrin derivatives, a particularly promising role can be played by this new class of selectors, the hemispherodextrins, whose huge values of selectivity towards some enantiomeric pairs of dns-AAs are shown. The possibility of better isolating hydrophobic moieties of the substrate by using a saccharidic “cap” gives rise to more selective interactions with substrates, able to differentiate very efficiently between the two members of an enantiomeric pair.

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