Unusual retention mechanism and selectivity:

1. H-acceptor δ+ H-donors π-Select π-Select Eluent: 45% methanol Eluent: 18% methanol C: 4.7% k’=0.54 BCD Conventional phenyl-column Conventional phenyl-column k’=0.25 C: 7.0% 1. Benzothiophene 6. Benzo[a]pyrene 3 2 4 2 1 1 7. Benzo[g,h,i]perylene 3 2. Fluorene 7 5. Pyrene 5 6 α-CD 3. Fluoranthene 4. Dibenzothiophene π-Select 250x4 mm * Tyr-Pro-Phe-Atc-NH2 * racemic amino-acid Atc-NH2: 2-amino-decaline-2-carboxylicacid Plate number 16685 asymmetry: 1,189 CRISMEB DS=1.5 RAMEB DS=14 Z and E LiChrosphere Si 60 250x4 mm Plate number: 5649 asymmetry: 1,261 R and S * 1.: 4-OH-Phenylglycine 2.: Phenylglycine 3.: Phenylalanine 4.: Tryptophane 1.:N-methyl-ephedrine 2.: Athenolol 3.: Pindolol 4.: Sertraline 1.: Trasicor 2.: Propranolol Unusual retention mechanism and selectivity: CD-Screen column for analysis of cyclodextrin-derivatives and -Select column for general purpose applications Julianna Szemán1, Katalin Csabai1, Gábor Varga2 1CYCLOLAB Cyclodextrin R&D Laboratory Ltd., Budapest, Hungary, e-mail: szeman.j@cyclolab.hu 2CHIROQUEST Chiral Technologies Development Ltd., Budapest, Hungary, e-mail: chiroquest@chello.hu INTRODUCTION In our previous work novel stationary phase was prepared by bonding N-(4-nitrophenyl)-carbamide group to the silica gel matrix [1,2]. The new phase has primarily been developed for analysis of cyclodextrins (CDs) and their derivatives. Taking into account the structure of this selector it seemed to be a promising tool for the separation of various families of compounds. Based on theoretical considerationsseveral types of interactions can beexpected with different types of molecules. The main characteristic property of this stationary phase is the essential role of π-π interactions in the retention mechanism [3, 4]. Due to the electron-withdrawing nitro-group in para position, retention forces become stronger and shape selectivity can be significantly better compared to other commercially available phenyl-bonded silica phases [5]. In general, hydrogen bonding was regarded as one of the reasons of poor peak symmetry and low efficiency of chromatographic media. However, the rationally planned, well-defined, non silanol type hydrogen bonding capability can play an important role in the selectivity and retention.Due to the balanced ratio of several types of interactions, it can be supposed that this stationary phase would be suitable both for reversed and normal phase applications, as well. Although the numerous types of p-p active HPLC phases have been already designed and investigated [3, 4], columns having diverse and multiple interacting sites for selectivity tuning can still expect great interest to solve separation problems e.g. in orthogonal chromatographic systems. The aim of this work was to studythe separation potency of 4-nitrophenyl-carbamide stationary phases and to optimize its capability for different separation problems. STRUCTURE OF THE SELECTOR Electrostatic potential surface of 4-nitrophenyl and phenyl-carbamide in complex with phenol Illustration of hydrogen-bonding between 4-nitro-phenyl-carbamide and phenol PRINCIPLE OF SEPARATION Illustration of the interaction between the apolar cavity of cyclodextrin and the nitrophenyl-carbamide selector The steric possibilities of the interactions were examined by molecular modelling methods. Both selector and solute molecular models were geometry-optimized using HyperChem MM+ molecular mechanics computational method. The energy minimization of the system consisting of these molecules together was the next step using the same method. The resulted complexes show clearly the presence of one or more hydrogen-bridges, depending on the chemical structure of analyte. Examining the electrostatic potential surfaces of the molecules in these complexes the role of the electron- withdrawing nitro-group in the retention mechanism can be easily understood. RESULTS AND DISCUSSION p-SELECT CD-SCREEN • higher surface coverage compared to CD-Screen stationary phase to obtain strong p - p interaction • fully endcapped to eliminate the silanol interactions, to obtain well defined H-bonding • optimized surface coverage fitting to the size of cyclodextrin molecules • secondary interactions with free silanols to increase the selectivity Comparison to phenyl column Chromatographic profile of hydroxypropyl-b-cyclodextrin Polar analytes Apolar, aromatic analytes CD-Screen Phenyl column • Separation of phenol and caffeine • higher retention due to the - interactionsand well defined hydrogen-bonding • residual silanols are eliminated, good peak shape • in pH 2-8 interval the retention time of phenol is relatively high and does not depend on the pH (on conventional phenyl column the retention time of phenol is near to t0at higher pH) • Separation of apolar, aromatic analytes • high retention on the -Select column, in spite of the fact that the carbon-content of this stationary phase is very low • the shape-selectivity and CH2-selectivity are higher, even diethyl-phthalate and biphenyl are well resolved. • The higher surface coverage of conventional phenyl columns causes steric hindrance, complex formation is restricted [5, 6]. • On CD-Screen column • The retention mechanism is based on the complex forming capability of CD rings with the functional groups of columns • Due to the strong retentionwider range of eluent composition can be used. • Higher efficiency - lower LOQ of unsubstituded cyclodextrins. • The separation of component groups gives the possibility to follow batch-to batch reproducibility. Examination of cyclodextrin derivatives on CD-Screen column Eluent: MeOH-water 60:40 1.: Dimethyl-phtalate; 2.: Diethyl-phtalate 3.: Biphenyl 4.: o-Terphenyl The commercially available cyclodextrin derivatives are statistically substitutedhaving different degree of substitution (DS) and substitution patterns. Detailed “fingerprint” chromatograms give the possibility to compare the identity or similarity of materials. Eluent: MeOH-water 30:70 1.: Phenol 2.: Caffeine Shape selectivity of p-Select columnPolyaromatic hydrocarbons Different batches of randomly methylated αCD, same production method and DS Determination of the remnant non-substituted αCD in the randomly methylated αCD Randomly methylated αCD prepared by different production methods, DS~12 Eluent: MeOH-water 85:15 Eluent: MeOH-water 75:25 Component distribution of randomly methylated βCD cyclodextrins having different DS Prostaglandine intermediate product Diastereomer peptides Decomposition of randomly methylated β-cyclodextrins Quality controll of single isomer6-monoamino β-cyclodextrin Degradation products Normal phase applicationEluent: n-hexane - t-buthyl-methyl-ether 97:3 Eluent: 0.1% TFA in water-0.1% TFA in MeOH 75:25 CONCLUSIONS Aromatic amino-acids Basic drugs Separation of various families of compounds have been investigated on 4-nitrophenyl-urea bonded stationary phase in both reversed and normal phase systems.The new chromatographic media kept its retentive capability even in extremely polar or apolar conditions and proved to be suitable for the separation of different type of substances: p-Select:  separation of polyaromatic hydrocarbons  separation of diastereomers in reversed and normal phase conditions, as well  analysis of aromatic amino-acids and basic drugs in acidic conditions CD-Screen:  characterisation of component distribution of statistically substituted CD derivatives, batch-to-batch reproducibility, control of degree of substitution or synthesis method  determination of parent CDs, and degradation products in substituted CD derivatives  quality control of single isomer 6-mono-amino-β-cyclodextrin Eluent: MeOH-0.1% H3PO4 30:70 0.8 ml/min pH: 2.6 Eluent: MeOH-0.1% H3PO4 60:40 0.8 ml/min pH: 2.6 Eluent: MeOH-0.1% H3PO4 30:70 0.8 ml/min pH: 2.6 EXPERIMENTAL REFERENCES Apparatus: Agilent 1050 HPLC system with UV-VISDetector at 205 or 254 nm. For detection of cyclodextrins Evaporative Light Scattering Detector PL-ELS 1000, (Polymer Laboratories) was used (Evaporation: 110°C, Nebulization: 90 °C, Gas flow: 1.2 l/min) Columns: The stationary phases (Hungarian Patent Application Pending 2004) were prepared by ChiroQuest Ltd. Column size: 250 mm x 4.0 mm I.D; Mobile phases: methanol – water or acetonitrile – water; Column temperature: 30 °C; Flow rate: 1.0 ml/min. Samples: CDs and CD derivatives of Cyclolab Ltd., Hungary and Wacker Chemie, Germany were analysed. [1] PCT Application Number PCT/HU 05/00043, May 30, 2005 [2] J. Szemán, K. Csabai, K. Kékesi, L. Szente, G. Varga; J. Chromatogr. A, submitted for publication [3]   J. Horak, W. Lindner ; J. Chromatogr. A, 1043, 177-194 (2004) [4]  J. Horak, N. M. Maier, W. Lindner; J. Chromatogr. A, 1045, 43-58 (2004) [5]  I. Caron, C. Elafkir, M. Dreux; Chromatographia 47, 383-390 (1998) [6] A. Salvador, B. Herbretau, M. Dreux; J. Chromatogr. A, 855, 645-656 (1999) ACKNOWLEDGEMENT The authors are grateful to Ms. Zs. Zachár and Ms. E. Erdei to their valuable technical assistance.The work was supplied by the National R&D programme (NKFP-1/A-04104).