Post on 07-Apr-2023
Accepted Manuscript
Title: Cationic permethylated6-monoamino-6-monodeoxy-�-cyclodextrin as chiral selectorof dansylated amino acids in capillary electrophoresis
Author: Krisztina Nemeth Celesztina Domonkos ViragSarnyai Julianna Szeman Laszlo Jicsinszky Lajos Szente JuliaVisy
PII: S0731-7085(14)00316-1DOI: http://dx.doi.org/doi:10.1016/j.jpba.2014.06.028Reference: PBA 9631
To appear in: Journal of Pharmaceutical and Biomedical Analysis
Received date: 18-4-2014Revised date: 17-6-2014Accepted date: 18-6-2014
Please cite this article as: K. Nemeth, C. Domonkos, V. Sarnyai, J. Szeman, L.Jicsinszky, L. Szente, J. Visy, Cationic permethylated 6-monoamino-6-monodeoxy-rmbeta-cyclodextrin as chiral selector of dansylated amino acids in capillaryelectrophoresis, Journal of Pharmaceutical and Biomedical Analysis (2014),http://dx.doi.org/10.1016/j.jpba.2014.06.028
This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.The manuscript will undergo copyediting, typesetting, and review of the resulting proofbefore it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers thatapply to the journal pertain.
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Cationic permethylated 6-monoamino-6-monodeoxy-β-cyclodextrin as chiral selector of 1
dansylated amino acids in capillary electrophoresis 2
3
Krisztina Németh1*, Celesztina Domonkos1, Virág Sarnyai1, Julianna Szemán2, László 4
Jicsinszky2, Lajos Szente2, Júlia Visy1 5
1Institute of Organic Chemistry, Research Centre for Natural Sciences, Hungarian Academy 6
of Sciences, H-1519 Budapest P.O.B.:286, Hungary 7
2CycloLab R&D Ltd., H-1097 Budapest, Illatos út 7, Hungary 8
9
*Corresponding author: K. Németh 10
Postal address: 1Institute of Organic Chemistry, Research Centre for Natural Sciences, 11
Hungarian Academy of Sciences, H-1519 Budapest P.O.B.:286, Hungary 12
Tel.: +36 1 382 6659; E-mail address: nemeth.krisztina@ttk.mta.hu 13
14
15
Highlights 16 17 • Permethylated 6-monoamino-6-monodeoxy-βCD resolves dansylated amino acids. 18 • Maximal chiral resolutions could be achieved at pH 6 or pH 4. 19 • The separations improved with increasing concentration of the selector. 20 • Low CD concentration was enough for the separation of the most apolar Dns-AAs. 21 • Resolution power of PMMABCD was proved by the separation of Dns-AAs’ mixture. 22 23
24
25
Abstract 26
27
The resolution power of permethylated 6-monoamino-6-monodeoxy-βCD 28
(PMMABCD) - a single isomer, cationic CD derivative - developed previously for chiral 29
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analyses in capillary electrophoresis was further studied here. Dansylated amino acids (Dns-30
AA) were chosen as amphoteric chiral model compounds. Changes in the resolutions of Dns-31
AAs by varying pH and selector concentrations were investigated and correlated with their 32
structures and chemical properties (isoelectric point and lipophilicity). Maximal resolutions 33
could be achieved at pH 6 or pH 4. The separations improved with increasing concentration of 34
the selector. Baseline or substantially better resolution for 8 pairs of these Dns-AAs could be 35
achieved. Low CD concentration was enough for the separation of the most apolar Dns-AAs. 36
Chiral discrimination ability of PMMABCD was demonstrated by the separation of an 37
artificial mixture of 8 Dns-AA pairs. 38
39
Keywords: cationic CD derivative, dansylated amino acid, enantioseparation, isoelectric 40
point, lipophilicity 41
42
Abbreviations: 43
Dns-AA: dansylated amino acid; Dns-Abu: dansylated α-amino butyric acid; Dns-Asp: 44
dansylated aspartic acid; Dns-Glu: dansylated glutamic acid; Dns-Leu: dansylated leucine; 45
Dns-Met: dansylated methionine; Dns-Nle: dansylated norleucine; Dns-Nva: dansylated 46
norvaline; Dns-Phe: dansylated phenylalanine; Dns-Thr: dansylated threonine; Dns-Trp: 47
dansylated tryptophan; Dns-Val: dansylated valine; PMMABCD: permethylated 6A-48
monoamino-6A-monodeoxy- β-cyclodextrin; 49
50
51
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1. Introduction 51
52
Analytical methods particularly capillary electrophoresis (CE) using chiral selectors 53
are appropriate for stereoselective separations [1-6]. Cyclodextrin (CD) derivatives are one of 54
the most favorable selectors to be applied in CE [7]. Development of new chiral selectors, 55
including charged cyclodextrins is important due to the increasing demand to analyze a great 56
number of pharmaceutical, environmental, and nutritional compounds. The advantage of the 57
charged selectors is that they provide relatively large separation window in the case of the 58
oppositely charged analytes [8]. Great numbers of anionic and even cationic CD derivatives 59
have been synthesized and released in the last decades [9-21]. Amino substituted CDs are 60
among the most widely investigated new selectors. Application of cationic –principally 61
amino- CD derivatives for chiral separation of several underivatized amino acids (AA) or 62
their fluorescent derivatives - for more sensitive detection –has been demonstrated [8,13,14-63
16,18-25]. Iványi et al. [26] reported that permethylated 6-monoamino-6-monodeoxy-βCD 64
(PMMABCD) developed previously as stereoselective agent for CE [27] was a good chiral 65
selector for dansyl-phenylalanine and dansyl-leucine as well as for many other – acidic - 66
chiral substances [26,28-32]. This cationic (pKa = 9.05 [28]), single-isomer βCD derivative is 67
completely O-methylated and substituted by an amino group in C6 position of one of the 68
glucopyranoside units (Degree of substitution = 20+1). The advantage of single isomer 69
selectors over randomly substituted ones is their outstanding batch-to-batch reproducibility. 70
Our study aimed to demonstrate the applicability of PMMABCD in chiral separation 71
of further dansylated amino acids (Dns-AAs) as amphoteric model compounds. Using Dns-72
AAs is explained by the increasing importance of the evaluation of the enantiomer ratio of 73
amino acids in biological samples and in food analysis [33-38] since the biological activity, 74
pharmacology and toxicology of D-amino acids are widely studied [39-42]. The pH and 75
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selector concentration dependencies of chiral resolutions were characterized and correlated 76
with the charged states of the analytes and selector and with the structure and lipophilicity of 77
the analytes. The chiral resolution power of PMMABCD was further examined with an 78
artificial mixture of Dns-AAs. 79
80
2. Materials and methods 81
82
2.1. Materials 83
84
Background electrolyte (BGE) components phosphoric acid, sodium hydroxide, 85
glacial acetic acid, boric acid and ethanol were purchased from Merck GmbH (Darmstadt, 86
Germany), eleven racemic (Table 1) and nine L-Dns-AA were obtained from Sigma-Aldrich 87
(St.Louis, USA). PMMABCD was the product of CycloLab Ltd. (Budapest, Hungary). Its 88
synthesis route has been described in details [27]. 89
90
2.2. Capillary electrophoresis 91
92
Capillary electrophoresis was performed with an Agilent Capillary Electrophoresis 93
3DCE system (Agilent Technologies, Waldbronn, Germany) applying bare fused silica 94
capillary having a 64.5 cm total and 56 cm effective length with 50 μm I.D. (Agilent 95
Technologies, Santa Clara, CA, USA). On-line absorption at 220 nm was monitored by DAD 96
UV-Vis detector. The capillary was thermostated at 25°C. Between measurements, the 97
capillary was rinsed subsequently with 0.1 M HCl, 1.0 M NaOH, 0.1 M NaOH and distilled 98
water for 3 min each and with BGE for 5 min. Various BGE buffers were prepared either 99
from 82 mM boric acid (pH 9); or from 16 mM phosphoric acid (pH 8); or from 22 mM 100
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phosphoric acid (pH 7); or from 35 mM phosphoric acid (pH 6); or from 65 mM acetic acid 101
(pH 5); or from 255 mM acetic acid (pH 4); or from 50 mM phosphoric acid (pH 3) with 102
sodium hydroxide to attain the desired pHs as indicated and the same ionic strength (I= 44 103
mM). The appropriate amounts of PMMABCD were dissolved in the running buffers. Dns-104
AA samples were dissolved in 50% ethanol and further diluted with distilled water, final 105
concentrations were 0.1 mg/ml. The racemic Dns-AAs were spiked by the pure L-enantiomers 106
(cL-Dns-AA/cD-Dns-AA= 7/3) – with the exception of Dns-Nle and Dns-Abu, their pure ‘L’ 107
enantiomers were lacking. The artificial mixture was prepared by mixing the stock solutions 108
of the Dns-AA samples and further diluted with distilled water. The ethanol content of the 109
mixture was higher, the concentrations of Dns-AAs were 0.03 mg/ml and enantiomer ratios 110
were similar with the individual samples. Samples were injected by 5×103 Pa pressure for 3 111
sec. Runs were performed in the positive-polarity mode with 30 kV. Enantiomer separations 112
were carried out with 2.5, 5, 10, 20 mM concentrations of PMMABCD. The resolution (Rs) of 113
the enantiomers is given by the following equation [43]: 114
( )( ) ( )25.015.0
12s ww
tt18.1R+
−×= Eq. 1 115
where t is the migration time of the enantiomers (1, 2 in lower index), w(0.5) is the peak width 116
at half height. 117
Selectivity values are given as αt,migr = t migr,2/tmigr,1 , where t migr,1 and t migr,2 are the migration 118
times of the Dns-AA enantiomers, and are also given as αµ,eff = µeff,2/µeff,1 , where µeff,1 and µeff,2 119
are the mobilities of the Dns-AA enantiomers, respectively. 120
121
2.3. Estimation of pI values of Dns-AAs 122
123
pIs of the Dns-AAs were determined from the effective mobilities (μeff) measured at 124
several pHs. The effective mobilities were plotted against the applied pHs and the intercept of 125
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the fitted curve on the abscissa gave the pI of the given analyte (Fig. S1, Table S1) [44,45]. 126
Predicted logP values were calculated by ChemAxon’s Marvin Calculator Plugin 127
software (version 5.9.2; URL: http://www.chemaxon.com/marvin) called as “Marvin” in the 128
followings. 129
130
3. Results and discussion 131
132
Chiral separations of 11 Dns-AA enantiomer pairs (Table 1) were studied by the 133
cationic PMMABCD. Separations were carried out in the pH 3-9 range and by applying the 134
selector in the 2.5 mM to 20 mM concentration range. Results are summarized in (Table 2, 135
Table S2). Using these conditions eight enantiomer pairs can be resolved at least at baseline 136
or better and additional two pairs can be separated partially. Separations can be carried out in 137
reasonable running times. Changes in resolution of Dns-AAs as a function of pH and 138
PMMABCD concentration is discussed in details as follows. 139
140
[Table 1] [Table 2] 141
142
3.1. pH dependence of resolution of Dns-AA enantiomers 143
144
Changes in the chiral separations of the enantiomers of Dns-AAs were investigated by 145
varying the pH between pH 3 and pH 9. In order to evaluate charge relationships between the 146
host and guest molecules in this pH range the pI values of the analytes were measured by CE. 147
The pI values of the Dns-AAs are between 3.46-3.79 (Table 1) being in good agreement with 148
literature data (Table S1) [46]. Consequently, the Dns-AAs are positively charged below and 149
negatively charged above these values while the PMMABCD is positively charged 150
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permanently below pH 9. The effects of the pH on chiral separations of Dns-AAs were 151
studied by investigating several combinations of different charge states of the selector and 152
selectands (at pH 3, 4, 6 and 9). 153
Migrations of the Dns-AAs were accelerated (μcompl>μfree) by PMMABCD at pHs 154
above their pI values (Table S2). The negatively charged Dns-AAs if complexed with 155
PMMABCD migrate faster than the free analytes alone due to the decrease in their negative 156
charges and increase in their mass (i.e. hydrodynamic radius). Using this cationic selector 157
below the pIs of the Dns-AAs the complexes gain positive charges not only from the analyte 158
but also from the selector, in addition their sizes are increased. Therefore their mobility 159
changes are not substantial and could not be predicted well due to those counter-effects. 160
Resolutions show maxima at pH 4 or at pH 6 (Table 2, Fig. 1) where PMMABCD is 161
cationic and the Dns-AAs are at least partially negatively charged. The highest selectivities 162
can be observed for Dns-Abu, Dns-Nva, Dns-Nle, Dns-Leu, Dns-Phe, Dns-Trp and Dns-Thr 163
at pH 4 and for Dns-Met, Dns-Val and Dns-Asp at pH 6. Resolutions are decreased at pH 9 164
where PMMABCD is partially in uncharged form and the Dns-AAs are negatively charged. 165
Resolutions are weaker too at pH 3 where the selector is fully positively charged and the 166
analytes are partially cationic. 167
In general, the amount of the D-amino acids is substantially lower than that of their L 168
pairs in nature. In most of our cases the migration order of the enantiomers (EMO) is D,L in 169
the entire pH range investigated. So that EMO is advantageous because the evaluation of 170
peaks is more accurate when the smaller component migrates before the major one. 171
172
[Fig. 1] 173
174
3.2. CD concentration dependence of resolution of Dns-AA enantiomers 175
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176
Selectivities increase with increasing selector concentrations in most cases of the Dns-177
AAs investigated. Our finding that the maximal selectivities are in the high concentration 178
range of the selector corresponds [2,47,48] to the weak interactions between PMMABCD and 179
Dns-AAs (Kassoc ≤ 10 M-1) estimated by the well known non-linear regression of the 180
electrophoretic mobility shifts [49-53]. 181
Whereas, increasing the selector concentration results in the improvement of 182
separations usage of PMMABCD in concentrations substantially higher than 20 mM is not 183
recommended because of longer analysis times and peak distortions. In addition, due to the 184
elevated current other counterproductive effects of Joule heating can also occur. Furthermore, 185
it is noteworthy that the pH and selector concentration dependencies for Dns-Nle and Dns-186
Phe deviate from the others (Fig. 1) because in these two cases the migration time of the 187
peaks of the enantiomers overlaps with the time of the electroosmotic flow at high (15-20 188
mM) concentrations of PMMABCD at pH 4. 189
Baseline or better separation can be achieved for some of the enantiomer pairs of these 190
Dns-AAs even with low concentrations of PMMABCD. In the presence of only 2.5 mM-10 191
mM concentrations of the selector Dns-Abu, Dns-Nva, Dns-Nle, Dns-Leu, Dns-Phe, Dns-Trp 192
and Dns-Met exhibited good resolution with further improvements with elevated CD 193
concentrations. In the group of Dns-AAs with nonpolar aliphatic sidechains the higher is the 194
lipophilicity the better is the resolution. 15 mM - 20 mM concentrations of PMMABCD are 195
needed for the resolution of the more polar (cf. the logP values in Table 1) Dns-AAs (namely: 196
Dns-Val, Dns-Thr and Dns-Asp). Unfortunately, enantiomers of the polar Dns-Glu can not be 197
separated at all with PMMABCD. 198
Limit of detection (LOD at signal to noise ratio S/N = 3) and the limit of quantitation 199
(LOQ at S/N = 10) were determined for several dansylated D- and L-amino acids (Table 3). 200
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The LOD and LOQ values were between 0.2 - 3 µg/ml and 6 - 10 µg/ml, respectively. Most 201
biological samples contain D- and L-amino acid enantiomers approximately in a ratio of 202
1:100 [54]. Accordingly, the sensibility of our system for the detection of the minor (D-Dns-203
AA) component followed by the major ‘L’ enantiomer is demonstrated here with some 204
examples (Dns-Phe, Dns-Trp, Dns-Leu, Dns-Val, Dns-Nva and Dns-Met) where the 205
enantiomer ratios is also 1 to 100. In these setups (20 mM PMMABCD at pH 6.0) the 206
concentrations of D-Dns-AAs is around at their LOQ values (10 µg/ml) and those of their ‘L’ 207
pairs were 1 mg/ml (Fig. 2). 208
209
[Table 3] [Fig. 2] 210
211
3.4. Resolution of an artificial mixture of Dns-AAs 212
213
The resolution power of PMMABCD is further studied by mixture of Dns-AAs. From 214
our enantiomer pairs we chose those eight Dns-AAs which were separated at baseline at least. 215
Migration times of the enantiomers were taken into account too in selecting optimal running 216
parameters (pH and CD concentration) in order to obtain separated peaks for each constituent 217
of the mixture. Despite that in some cases pH 4 can provide more improved resolution for the 218
individual Dns-AAs than pH 6 the number of the enantiomer pairs resolved is greater at pH 6 219
than at pH 4. Consequently, we have chosen 20 mM PMMABCD at pH 6.0 and 7 enantiomer 220
pairs separated well and two further peaks partially of that mixture containing 8 pairs (Fig. 3). 221
[Fig. 3] 222
223
224
225
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4. Conclusions 226
227
The amino and methyl substituted βCD derivative PMMABCD is introduced as an 228
excellent cationic selector for Dns-AAs having nonpolar aliphatic and aromatic side chains 229
and a usable selector for some Dns-AAs with polar uncharged or charged groups. The 230
advantage of using PMMABCD is that it consumes a small amount of CD derivative, the 231
separations can be carried out in reasonable running times, and it provides the preferable ‘DL’ 232
migration order for the resolution of Dns-AAs at pHs above their pI values. Furthermore, the 233
chiral recognition ability of PMMABCD is proved by the separation of a mixture of Dns-234
AAs. 235
236
Acknowledgements 237
238
We gratefully acknowledge Dr. Róbert Iványi, Katalin Tuza (Cyclolab R&D Ltd.) as well as 239
Prof. Miklós Simonyi, Dr. Gábor Tárkányi, Mrs. Ilona Kawka (RCNS, HAS) and Dr. 240
Zsuzsanna Lakatos for their helpful contributions. The authors thank for the financial support 241
from the following grants: NKFP_A3-2008-0211 NATURSEP, TECH-09-AI-2009-0117 242
NanoSEN9, the Hungarian Scientific Research Fund (OTKA, grant numbers K-100134 and 243
NN-110214) and the “Lendület” Program of the Hungarian Academy of Sciences (LP2013-244
55/2013). 245
246
Appendix A. Supplementary data 247
Supplementary data associated with this article can be found, in the online version, at doi: 248
249
The authors have declared no conflict of interest. 250
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amperometric biosensor for the determination of D-amino acids, Bioelectrochemistry 71 346
(2007) 91-98. 347
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[35] X. He, X. Cui, M. Li, L. Lin, X. Liu, X. Feng, Highly enantioselective fluorescent sensor 348
for chiral recognition of amino acid derivatives, Tetrahedron Lett. 50 (2009) 5853-5856. 349
[36] H. Zahradníčková, P. Hušek, P. Šimek, P. Hartvich, B. Maršálek, I. Holoubek, 350
Determination of D- and L-amino acids produced by cyanobacteria using gas chromatography 351
on Chirasil-Val after derivatization with pentafluoropropyl chloroformate, Anal. Bioanal. 352
Chem. 388 (2007) 1815-1822. 353
[37] Y. Miyoshi, R. Koga, T. Oyama, H. Han, K. Ueno, K. Masuyama, Y. Itoh, K. Hamase, 354
HPLC analysis of naturally occurring free D-amino acids in mammals, J. Pharm. Biomed. 355
Anal. 69 (2012) 42-49. 356
[38] M. Herrero, C., Simo, V. Garcia-Canas, S. Fanali, A. Cifuentes, Electrophoresis 31 357
(2010) 2106-2114. 358
[39] K. Hamase, Sensitive two-dimensional determination of small amounts of D-amino acids 359
in mammals and the study on their functions, Chem. Pharm. Bull. 55 (2007) 503-510. 360
[40] M. Friedman, Origin, Microbiology, Nutrition, and Pharmacology of D-Amino Acids, 361
Chem. Biodiversity 7 (2010) 1491-1530. 362
[41] K. Hamase, Analysis and biological relevance of D-amino acids and relating compounds, 363
J. Chromatogr., B: Anal. Technol. Biomed. Life Sci. 879 (2011) 3077-3077. 364
[42] H. Ohide, Y. Miyoshi, R. Maruyama, K. Hamase, K. R. Konno, D-Amino acid 365
metabolism in mammals: Biosynthesis, degradation and analytical aspects of the metabolic 366
study, J. Chromatogr., B: Anal. Technol. Biomed. Life Sci. 879 (2011) 3162-3168. 367
[43] S. Fanali, Controlling enantioselectivity in chiral capillary electrophoresis with inclusion-368
complexation, J. Chromatogr. A 792 (1997) 227-267. 369
[44] D. Koval, V. Kasicka, J. Jiracek, M. Collinsová, Determination of pK(a) values of 370
diastereomers of phosphinic pseudopeptides by CZE, Electrophoresis 27 (2006) 4648-4657. 371
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[45] S.K. Poole, S. Patel, K. Dehring, H. Workman, C.F. Poole, Determination of acid 372
dissociation constants by capillary electrophoresis, J. Chromatogr. A 1037 (2004) 445-454. 373
[46] A. Bianchi-Bosisio, P.G. Righetti, N.B. Egen, M. Bier, Isoelectric focusing of 374
dansylated amino-acids in immobilized pH gradients, Electrophoresis 7 (1986) 128-133. 375
[47] A. Rizzi, Fundamental aspects of chiral separations by capillary electrophoresis, 376
Electrophoresis 22 (2001) 3079-3106. 377
[48] S. Wren, The separation of enantiomers by capillary electrophoresis, Chromatographia 378
54 (2001) S1-S95. 379
[49] S. G. Penn, D. M. Goodall, J. S. Loran, Differential Binding of tioconazole enantiomers 380
to hydroxypropyl-beta cyclodextrin studied by capillary, J. Chromatogr. 636 (1993) 149-152. 381
[50] K. L. Rundlett, D. W. Armstrong, Examination of the origin, variation, and proper use of 382
expressions for the estimation of association constants by capillary electrophoresis, J. 383
Chromatogr. A 721 (1996) 173-186. 384
[51] S. A.C. Wren, Mobility measurements on dansylated amino acids, J. Chromatogr. A 768 385
(1997) 153-159. 386
[52] A. Salvador, E. Varesio, M. Dreux, J-L. Veuthey, Binding constant dependency of 387
amphetamines with various commercial methylated beta-cyclodextrins, Electrophoresis 20 388
(1999) 2670-2679. 389
[53] Y. Tanaka, S. Terabe, Estimation of binding constants by capillary electrophoresis, J. 390
Chromatogr. A 768 (2002) 81-92. 391
[54] Zs. Wagner, T. Tábi, T. Jakó, G. Zachar, A. Csillag, É. Szökő, Chiral separation and 392
determination of excitatory amino acids in brain samples by CE-LIF using dual 393
cyclodextrin system, Anal. Bioanal. Chem. 404 (2012) 2363-2368. 394
395
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Figure captions 395
396
Fig. 1. Changes in selectivities (αt,migr) of several Dns-AAs as a function of pH and selector 397
concentration 398
Fig. 2. Separations of D- and L-Dns-AA enantiomers in a concentration ratio of 1:100 (10 399
µg/ml and 1 mg/ml, respectively) in the presence of 20 mM PMMABCD at pH 6 400
Fig. 3. Separation of Dns-AA mixture by 20 mM PMMABCD at pH 6 401
402
403
404
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Table 1: Structure, isoelectric point (pI) and lipophilicity (logP) of dansyl-amino acids 404 405
406 407
408
R group pI logP
Nonpolar, aliphatic R group, linear sidechain:
Dns-DL-α-aminobutyric acid (Dns-Abu) -CH2CH3 3.68 -2.54
Dns-DL-norvalin (Dns-Nva) -(CH2)2CH3 3.67 -2.11
Dns-DL-norleucine (Dns-Nle) -(CH2)3CH3 3.67 -1.52
Nonpolar, aliphatic R group, branched sidechain:
Dns-DL-valine (Dns-Val) -CH(CH3)2 3.70 -2.26
Dns-DL-leucine (Dns-Leu) -CH2CH(CH3)2 3.63 -1.52
Nonpolar, aromatic R group:
Dns-DL-phenylalanine (Dns-Phe) -CH2C6H5 3.67 -1.38
Dns-DL-tryptophan (Dns-Trp) -CH2C8NH6 3.79 -1.05 Nonpolar, uncharged R group:
Dns-DL-methionine (Dns-Met) -(CH2)2SCH3 3.55 -1.87
Polar, uncharged R group:
Dns-DL-threonine (Dns-Thr) -CHCH3OH 3.66 -2.94
Polar, negatively charged R group:
Dns-DL-aspartic acid (Dns-Asp) -CH2COOH 3.46 -3.89
Dns-DL-glutamic acid (Dns-Glu) -(CH2)2COOH 3.50 -3.69
N
CH3
CH3
S
O
O
NHR
COOH
*
N
CH3
CH3
S
O
O
NHR
COOH
*
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Table 2: Migration times (t1), selectivities (α) and resolutions (Rs) of Dns-AAs at pH 9-3 in 408 the presence of 2.5-20 mM PMMABCD as chiral selector 409 410 pH t1 (min) αe Rs
2.5d 5 10 15 20 2.5 5 10 15 20 2.5 5 10 15 20
9 n.a. n.a. 4.63 4.79 5.23 n.a. n.a. 1.000 1.004 1.010 n.a. n.a. nr 0.5 1.56 5.90 6.54 6.83 7.33 7.79 1.000 1.012 1.025 1.042 1.073 nr 1.8 3.9 6.5 11.14 12.19 14.19 17.21 19.23 20.30 1.000 1.009 1.039 1.085 1.131 nr 1.0 4.0 8.1 11.8
Dns-Abu
3 n.a. n.a. 14.67 15.49 19.16 n.a. n.a. 1.000 1.008 1.000 n.a. n.a. nr nr nr
9 4.22 4.30 4.47 4.64 5.07 1.000 1.000 1.000 1.000 1.012 nr nr nr nr 1.76 5.59 5.94 6.32 6.51 7.58 1.000 1.013 1.029 1.042 1.092 nr 2.1 4.6 8.0 12.94 12.21 14.02 16.14 17.22 17.59 1.000 1.016 1.068 1.133 1.179 nr 1.6 6.8 13.2 17.3
Dns-Nva
3 12.56 12.65 20.52 22.62 26.37 1.000 1.000 1.000 1.000 1.016 nr nr nr nr 0.8
9 n.a. n.a. 4.45 4.55 4.89 n.a. n.a. 1.005 1.018 1.032 n.a. n.a. 0.6 2.5 4.96 5.49 5.68 5.92 6.05 6.40 1.006 1.032 1.068 1.088 1.160 0.9 5.2 10.1 14.0 19.54 12.14 13.20 13.42 14.84 17.52 1.015 1.065 1.197 1.149 n.a. 1.9 5.5 20.4 20.3 n.a.
Dns-Nle
3 n.a. n.a. 15.91 17.24 21.66 n.a. n.a. 1.022 1.067 1.060 n.a. n.a. 1.8 2.8 3.6
9 n.a. n.a. 4.56 4.73 5.19 n.a. n.a. 1.000 1.000 1.000 n.a. n.a. nr nr nr6 5.62 6.07 6.62 7.01 8.45 1.000 1.000 1.000 1.000 1.016 nr nr nr nr 2.34 11.53 13.43 16.16 18.85 20.59 1.000 n.a. 1.000 1.000 1.000 nr n.a. nr nr nr
Dns-Val
3 n.a. n.a. 15.64 16.60 n.a. n.a. n.a. 1.000 1.000 n.a. n.a. n.a. nr nr n.a.
9 4.17 4.24 4.41 4.58 5.08 1.003 1.005 1.007 1.006 1.004 0.5 0.9 1.2 1.5 0.66 5.85 6.66 6.91 7.27 7.91 1.004 1.000 1.000 1.013 1.035 0.6 nr nr 2.0 4.44 11.99 13.96 16.89 18.46 19.17 1.000 1.010 1.059 1.137 1.220 nr 1.2 6.0 12.8 19.5
Dns-Leu
3 12.91 13.19 24.46 27.14 34.52 1.000 1.000 1.012 1.023 1.036 nr nr 1.0 1.7 6.9
9 4.15 4.21 4.23 4.25 4.58 1.142 1.000 1.019 1.039 1.059 0.9 nr 3.4 5.9 6.76 5.63 5.95 5.66 5.77 6.07 1.016 1.071 1.115 1.148 1.168 2.5 10.5 16.3 21.2 21.24 11.43 12.26 12.70 16.23 17.24 1.021 1.085 1.216 n.a. n.a. nr 7.4 27.1 n.a. n.a.
Dns-Phe
3 13.05 13.16 22.41 24.90 31.77 1.000 1.000 1.015 1.017 1.000 nr nr 1.0 1.4 nr
9 4.16 4.26 4.48 4.64 5.11 1.000 1.000 1.000 1.000 1.000 nr nr nr nr nr6 5.47 5.89 6.37 6.64 7.33 1.000 1.000 1.014 1.019 1.045 nr nr 2.1 1.4 7.44 n.a. 12.21 14.33 n.a. 17.53 n.a. 1.006 1.026 n.a. 1.092 n.a. 0.8 2.8 n.a. 8.7
Dns-Trp
3 12.49 12.69 17.33 20.05 23.72 1.000 1.000 1.000 1.000 1.006 nr nr nr nr nr
9 n.a. n.a. 4.59 4.70 5.14 n.a. n.a. 1.000 1.011 1.021 n.a. n.a. nr 1.3 3.26 5.96 6.78 6.92 7.20 7.94 1.000 1.021 1.044 1.071 1.134 nr 3.3 6.8 10.9 17.04 n.a. n.a. n.a. n.a. n.a. n.a. 1.000 1.000 1.000 1.000 n.a. nr nr nr nr
Dns-Met
3 n.a. n.a. 18.25 20.60 24.35 n.a. n.a. 1.000 1.000 1.010 n.a. n.a. nr nr 0.5
Dns-Thr 4 12.74 14.96 19.15 24.44 29.21 1.000 1.000 1.000 1.000 1.015 nr nr nr nr 1.0
Dns-Asp 6 9.88 11.43 13.31 16.10 19.79 1.000 1.000 1.000 1.011 1.016 nr nr nr 1.0 1.4
411 a) n.a. not available 412 b) nr not resolved; Rs < 0.5 413 c) t1 migration time of the faster enantiomer 414 d) Selector concentrations applied is given in mM in the second row of the header 415 e) Selectivity is given as follows: α = t migr,2/tmigr,1 416 f) The enantiomer migration order is D,L in the majority of the cases. The selectivity values 417 are underlined where the enantiomer migration order is reversed (L,D). 418 419
420
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Table 3: Limit of detection (LOD) and quantitation (LOQ) of D- and L-Dns AAs 420 421
422 423
D-Dns-AA L-Dns-AA
LOD (µg/ml) LOQ (µg/ml)
LOD (µg/ml) LOQ (µg/ml)
Dns-Abu 0.4 5.8 0.4 6.4
Dns-Nva 0.2 5.7 0.2 6.3
Dns-Nle 0.1 5.2 0.1 6.5
Dns-Val 1.7 7.9 1.7 7.7
Dns-Leu 1.5 7.4 1.5 7.5
Dns-Phe 1.3 6.2 1.3 7.5
Dns-Trp 1. 8 6.1 1.8 6.1
Dns-Met 3.0 10.0 2.9 10.2
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6.5 7.0 7.5 8.0 8.5 9.0 9.5
Migration time (min)
Ab
so
rba
nce
(2
20
nm
)
Dns-A
bu
Dns-D
-Leu
Dns-L
-Nva
Dns-L
-Leu
Dns-D
-Val
Dns-A
bu
Dns-L
-Val
Dns-L
-Met
Dns-N
le
Dns-L
-Phe
Dns-D
-Phe
Dns-D
-Nva
Dns-N
le
Dns-D
-Met
EO
F
Dns-D
-Trp
Dns-L
-Trp
(OCH3)6 and (NH2)1
C(3) C(2)
(CH3O)7 (OCH3)7
C(6) *Graphical Abstract
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3 4 5 6 7 8 9
5
10
15
20
pH
CD
co
nce
ntr
atio
n (
mM
)
1.000
1.025
1.050
1.075
1.100
1.125
1.150
1.175
1.200
1.225
1.250
3 4 5 6 7 8 9
5
10
15
20
pH
CD
co
nce
ntr
atio
n (
mM
)
1.000
1.025
1.050
1.075
1.100
1.125
1.150
1.175
1.200
1.225
1.2503 4 5 6 7 8 9
5
10
15
20
pH
CD
co
nce
ntr
atio
n (
mM
)
1.000
1.025
1.050
1.075
1.100
1.125
1.150
1.175
1.200
1.225
1.2503 4 5 6 7 8 9
5
10
15
20
pH
CD
co
nce
ntr
atio
n (
mM
)
1.000
1.025
1.050
1.075
1.100
1.125
1.150
1.175
1.200
1.225
1.250
3 4 5 6 7 8 9
5
10
15
20
pH
CD
co
nce
ntr
atio
n (
mM
)
1.000
1.025
1.050
1.075
1.100
1.125
1.150
1.175
1.200
1.225
1.2503 4 5 6 7 8 9
5
10
15
20
pH
CD
co
nce
ntr
atio
n (
mM
)
1.000
1.025
1.050
1.075
1.100
1.125
1.150
1.175
1.200
1.225
1.2503 4 5 6 7 8 9
5
10
15
20
pH
CD
co
nce
ntr
atio
n (
mM
)
1.000
1.025
1.050
1.075
1.100
1.125
1.150
1.175
1.200
1.225
1.250
Dns-Abu
Dns-Leu Dns-Trp Dns-Phe
Dns-Nle Dns-Nva
Dns-Met Fig. 1
Figure(s)
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3 4 5 6 7 8 9
5
10
15
20
pH
CD
co
nce
ntr
atio
n (
mM
)
1.000
1.025
1.050
1.075
1.100
1.125
1.150
1.175
1.200
1.225
1.250
3 4 5 6 7 8 9
5
10
15
20
pH
CD
co
nce
ntr
atio
n (
mM
)
1.000
1.025
1.050
1.075
1.100
1.125
1.150
1.175
1.200
1.225
1.2503 4 5 6 7 8 9
5
10
15
20
pH
CD
co
nce
ntr
atio
n (
mM
)
1.000
1.025
1.050
1.075
1.100
1.125
1.150
1.175
1.200
1.225
1.2503 4 5 6 7 8 9
5
10
15
20
pH
CD
co
nce
ntr
atio
n (
mM
)
1.000
1.025
1.050
1.075
1.100
1.125
1.150
1.175
1.200
1.225
1.250
3 4 5 6 7 8 9
5
10
15
20
pH
CD
co
nce
ntr
atio
n (
mM
)
1.000
1.025
1.050
1.075
1.100
1.125
1.150
1.175
1.200
1.225
1.2503 4 5 6 7 8 9
5
10
15
20
pH
CD
co
nce
ntr
atio
n (
mM
)
1.000
1.025
1.050
1.075
1.100
1.125
1.150
1.175
1.200
1.225
1.2503 4 5 6 7 8 9
5
10
15
20
pH
CD
co
nce
ntr
atio
n (
mM
)
1.000
1.025
1.050
1.075
1.100
1.125
1.150
1.175
1.200
1.225
1.250
Dns-Abu
Dns-Leu Dns-Trp Dns-Phe
Dns-Nle Dns-Nva
Dns-Met Fig. 1 in grey
Figure(s)
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7.0 7.5 8.0 8.5 9.0
Absorb
ance (
220 n
m)
Migration time (min)
7.5 8.0 8.5 9.0
Absorb
ance (
220 n
m)
Migration time (min)
8.0 8.5 9.0 9.5 10.0
Absorb
ance (
220 n
m)
Migration time (min)
6.0 6.5 7.0 7.5 8.0
Migration time (min)
Absorb
ance (
220 n
m)
7.5 8.0 8.5 9.0 9.5
Absorb
ance (
220 n
m)
Migration time (min)
8.5 9.0 9.5 10.0
Absorb
ance (
220 n
m)
Migration time (min)
Dns-D
-Phe
Dn
s-L
-Ph
e
Dns-D
-Nva
Dn
s-L
-Nva
Dns-D
-Val
Dn
s-L
-Val
Dns-D
-Trp
Dns-L
-Trp
Dns-D
-Le
u
Dns-L
-Le
u
Dns-D
-Met
Dns-L
-Met
Fig. 2 Figure(s)
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6.5 7.0 7.5 8.0 8.5 9.0 9.5
Migration time (min)
Ab
so
rba
nce
(2
20
nm
)
Dns-A
bu
Dns-D
-Leu
Dns-L
-Nva
Dns-L
-Leu
Dns-D
-Val
Dns-A
bu
Dns-L
-Val
Dns-L
-Met
Dns-N
le
Dns-L
-Phe
Dns-D
-Phe
Dns-D
-Nva
Dns-N
le
Dns-D
-Met
EO
F
Dns-D
-Trp
Dns-L
-Trp
Fig. 3 (OCH3)6 and (NH2)1
C(3) C(2)
(CH3O)7 (OCH3)7
C(6) Figure(s)
Supplementary data
Cationic permethylated 6-monoamino-6-monodeoxy-β-cyclodextrin as chiral selector of
dansylated amino acids in capillary electrophoresis
Krisztina Németh1*, Celesztina Domonkos
1, Virág Sarnyai
1, Julianna Szemán
2, László
Jicsinszky2, Lajos Szente
2, Júlia Visy
1
1Institute of Organic Chemistry, Research Centre for Natural Sciences, Hungarian Academy
of Sciences, H-1519 Budapest P.O.B.:286, Hungary
2CycloLab R&D Ltd., H-1097 Budapest, Illatos út 7, Hungary
*Corresponding author: K. Németh
Postal address: 1Institute of Organic Chemistry, Research Centre for Natural Sciences,
Hungarian Academy of Sciences, H-1519 Budapest P.O.B.:286, Hungary
Tel.: +36 1 382 6659; E-mail address: nemeth.krisztina@ttk.mta.hu
Determination of the pI values of the dansylated amino acids (Dns-AAs)
According to Eq. S1 the effective mobilities (µeff) of the analytes are zero when their charges
(q) are also zero (Fig S1A). Therefore the intercept of the fitted straight line at the abscissa
gives the isoelectric points of the amphoteric Dns-AAs (Fig. S1B; Table S1). Our
experimental pI data are in a good agreement with data in the literature (Table S1).
r6
qeff Eq. S1
Where r is the Stokes radius of the analyte and is the viscosity of the electrolyte.
-3.5
-3.0
-2.5
-2.0
-1.5
-1.0
-0.5
0.0
0.5
1.0
1.5
3 5 7 9pH
μeff
(1
0-4
cm
2/V
s)
Dns-Asp Dns-Trp
Fig. S1A: Effective mobility of dansylated aspartic acid and tryptophan as a function of pH
-1.1
-0.6
-0.1
0.4
0.9
1.4
3.0 3.2 3.4 3.6 3.8 4.0
pH
μeff
(1
0-4
cm
2/V
s) Dns-Abu Dns-Nva
Dns-Nle Dns-Val
Dns-Leu Dns-PheDns-Trp Dns-Met
Dns-Thr Dns-Asp
Dns-Glu
Fig. S1B: Intercepts at the abscissa of the straight lines of the effective mobilities of
dansylated amino acids vs. pH give the isoelectric points.
Table S1: Comparison of calculated isoelectric points (pI) and data in the literature
a: A. M. Rizzi,L. Kremser, Electrophoresis 20 (1999) 2715-2722.
b: A. Bianchi-Bosisio, P.G. Righetti, N.B. Egen, M. Bier, Electrophoresis 7 (1986) 128-133.
pI
Litrerature 1a Litrerature 2b calculated
Nonpolar, aliphatic R group, linear sidechain:
Dns-DL-α-aminobutyric acid (Dns-Abu) 3.68
Dns-DL-norvalin (Dns-Nva) 3.67
Dns-DL-norleucine (Dns-Nle) 3.67
Nonpolar, aliphatic R group, branched sidechain:
Dns-DL-valine (Dns-Val) 3.68 3.76 3.70
Dns-DL-leucine (Dns-Leu) 3.69 3.63
Nonpolar, aromatic R group:
Dns-DL-phenylalanine (Dns-Phe) 3.65 3.72 3.67
Dns-DL-tryptophan (Dns-Trp) 3.64 3.81 3.79
Nonpolar, uncharged R group:
Dns-DL-methionine (Dns-Met) 3.56 3.55
Polar, uncharged R group:
Dns-DL-threonine (Dns-Thr) 3.67 3.66
Polar, negatively charged R group:
Dns-DL-aspartic acid (Dns-Asp) 3.43 3.46
Dns-DL-glutamic acid (Dns-Glu) 3.55 3.50
pH and CD concentration dependence of mobility and selectivity
Effective mobilities of the Dns-AA enantiomers have been calculated in the presence of
PMMABCD at several pHs (pH 9-3). Selectivity data given as a ratio of the migration times
(t,migr) of the Dns AA enantiomers (Table 2) are completed with selectivity data calculated
from the effective mobilities (µ,eff,) (Table S2). t,migr and µ,eff change similarly as a function
of pH and CD concentration, but in some cases values of µ,eff are extremely high because one
of the enantiomers migrates close to the EOF and the other one remains slower, so their
mobilities differ in an order of magnitude from each other and therefore their ratio gives so
extraordinary (e.g. µeff = 22) values.
Table S2: Effective mobilities (µeff,1), selectivities () and resolutions (Rs) of Dns-AAs at pH
9-3 in the presence of 2.5-20 mM PMMABCD as chiral selector
pH µeff,1 (10
-4cm
2/Vs)
c e Rs
0d 2.5 5 10 15 20 2.5 5 10 15 20 2.5 5 10 15 20
Dns-
Abu
9 -1.84 n.a. n.a. -1.65 -1.55 -1.42 n.a. n.a. 1.000 1.010 1.027 n.a. n.a. nr 0.5 1.5
6 -1.80 -1.72 -1.53 -1.38 -1.20 -0.93 1.000 1.023 1.054 1.095 1.196 nr 1.8 3.9 6.5 11.1
4 -0.61 -0.59 -0.56 -0.45 -0.32 -0.19 1.000 1.022 1.098 1.258 1.620 nr 1.0 4.0 8.1 11.8
3 0.83 n.a. n.a. 1.11 1.11 0.96 n.a. n.a. 1.000 1.009 1.000 n.a. n.a. nr nr nr
Dns-
Nva
9 -1.76 -1.73 -1.66 -1.54 -1.47 -1.31 1.000 1.000 1.000 1.000 1.035 nr nr nr nr 1.7
6 -1.80 -1.67 -1.50 -1.32 -0.96 -0.81 1.000 1.030 1.071 1.132 1.288 nr 2.1 4.6 8.0 12.9
4 -0.63 -0.59 -0.53 -0.38 -0.20 -0.04 1.000 1.043 1.213 1.719 5.883 nr 1.6 6.8 13.2 17.3
3 0.76 n.a. n.a. 0.72 0.71 0.66 1.000 1.000 1.000 1.000 1.019 nr nr nr nr 0.8
Dns-
Nle
9 -1.72 n.a. n.a. -1.49 -1.33 -1.16 n.a. n.a. 1.016 1.058 1.111 n.a. n.a. 0.6 2.5 4.9
6 -1.78 -1.60 -1.34 -1.07 -0.77 -0.41 1.014 1.084 1.209 1.362 2.129 0.9 5.2 10.1 14.0 19.5
4 -0.65 -0.60 -0.44 -0.12 -0.01 0.00 1.043 1.212 3.118 21.181 n.a. 1.9 5.5 20.4 20.3 n.a.
3 0.75 n.a. n.a. 0.98 0.91 0.79 n.a. n.a. 1.027 1.081 1.068 n.a. n.a. 1.8 2.8 3.6
Dns-
Val
9 -1.77 n.a. n.a. -1.62 -1.52 -1.42 n.a. n.a. 1.000 1.000 1.000 n.a. n.a. nr nr nr
6 -1.79 -1.68 -1.57 -1.44 -1.21 -1.09 1.000 1.000 1.000 1.000 1.037 nr nr nr nr 2.3
4 -0.53 -0.50 n.a. -0.40 -0.30 -0.22 1.000 n.a. 1.000 1.000 1.000 nr n.a. nr nr nr
3 0.75 n.a. n.a. 1.02 1.03 n.a. n.a. n.a. 1.000 1.000 1.000 n.a. n.a. nr nr n.a.
Dns-
Leu
9 -1.72 -1.67 -1.60 -1.48 -1.41 -1.31 1.008 1.014 1.020 1.018 1.011 0.5 0.9 1.2 1.5 0.6
6 -1.74 -1.62 -1.46 -1.35 -1.20 -0.99 1.008 1.000 1.000 1.032 1.091 0.6 nr nr 2.0 4.4
4 -0.63 -0.60 -0.55 -0.43 -0.28 -0.13 1.000 1.026 1.157 1.487 2.548 nr 1.2 6.0 12.8 19.5
3 0.69 n.a. n.a. 0.56 0.54 0.47 1.000 1.000 1.018 1.030 1.044 nr nr 1.0 1.7 6.9
Dns-
Phe
9 -1.76 -1.66 -1.55 -1.30 -1.09 -0.89 1.350 1.000 1.067 1.163 1.279 0.9 nr 3.4 5.9 6.7
6 -1.75 -1.52 -1.10 -0.75 -0.45 -0.10 1.036 1.207 1.499 2.026 6.203 2.5 10.5 16.3 21.2 21.2
4 -0.57 -0.50 -0.34 -0.01 0.00 0.00 1.072 1.381 22.109 n.a. n.a. nr 7.4 27.1 n.a. n.a.
3 0.67 n.a. n.a. 0.63 0.61 0.54 1.000 1.000 1.021 1.022 1.000 nr nr 1.0 1.4 nr
Dns-
Trp
9 -1.67 -1.80 -1.61 -1.55 -1.46 -1.36 1.000 1.000 1.000 1.000 1.000 nr nr nr nr nr
6 -1.69 -1.61 -1.48 -1.33 -1.10 -0.98 1.000 1.000 1.032 1.053 1.128 nr nr 2.1 1.4 7.4
4 -0.40 n.a. -0.33 -0.24 n.a. -0.06 n.a. 1.032 1.149 n.a. 2.716 n.a. 0.8 2.8 n.a. 8.7
3 0.84 n.a. n.a. 0.90 0.82 0.75 1.000 1.000 1.000 1.000 1.007 nr nr nr nr nr
Dns-
Met
9 -1.76 n.a. n.a. -1.64 -1.51 -1.38 n.a. n.a. 1.000 1.032 1.059 n.a. n.a. nr 1.3 3.2
6 -1.77 -1.69 -1.50 -1.34 -1.17 -0.86 1.000 1.042 1.094 1.165 1.365 nr 3.3 6.8 10.9 17.0
4 -0.78 n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a.
3 0.48 n.a. n.a. 0.84 0.80 0.73 n.a. n.a. 1.000 1.000 1.011 n.a. n.a. nr nr 0.5
Dns-
Thr 4 -0.65 -0.62 -0.61 -0.60 -0.56 -0.52
1.000 1.000 1.000 1.000 1.020
nr nr nr nr 1.0
Dns-
Asp 6 -3.17 -3.12 -3.03 -2.95 -2.85 -2.75
1.000 1.000 1.000 1.005 1.006
nr nr nr 1.0 1.4
a) n.a. not available
b) nr not resolved; Rs < 0.5
c) effective mobility of the faster enantiomer
d) Selector concentrations applied is given in mM in the second row of the header
e) Selectivity is given as follows: = µeff,2/µeff,1
f) The enantiomer migration order is D,L in the majority of the cases. The selectivity values
are underlined where the enantiomer migration order is reversed (L,D).