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Background: Sub/supercritical fluid chromatography is regarded as a greener separation technique due to the use of carbon dioxide as the main component of the mobile phase compared to conventional liquid chromatography techniques. Organic co-solvents are usually added to carbon dioxide to increase elution strength of the mobile phase. Therefore, it is of great importance to test applicability of green co-solvents in separation methods and to include them among commonly used mobile phase components., Results: A comprehensive study of the suitability of green solvent dimethyl carbonate as a co-solvent for enantioseparation in sub/supercritical fluid chromatography was conducted with a set of novel psychoactive substances from various groups. The experiments were performed on polysaccharide-based columns. For successful enantioseparation of these compounds, the presence of basic or mixed mobile phase additives was essential. The obtained results clearly show that dimethyl carbonate is a suitable co-solvent for enantioseparation on polysaccharide-based columns in sub/supercritical fluid chromatography and in some cases surpasses commonly used co-solvents as methanol and propan-2-ol., Significance: The use of more sustainable co-solvents, such as dimethyl carbonate, instead of conventional ones to carbon dioxide presents a greener approach to analytical applications and reduces the overall environmental impact of analytical processes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Ion-pairing reversed-phase liquid chromatography was utilized for the analysis of native and phosphorothioated oligonucleotides differing in the length (2-6mers and 21mer) and the number and position of phosphorothioate modifications. We investigated the influence of counterion (acetate vs. hexafluoroisopropanol) on the adsorption of eleven alkylamines on the stationary phases. A stronger adsorption of charged alkylamines on octadecyl- and phenyl-based stationary phases led to greater retention of oligonucleotides, and the adsorption of alkylamines was promoted with greater concentration of hexafluoroisopropanol in the mobile phase. Selected amines (triethylamine, dipropylamine, hexylamine) were used to study the resolution of n and n-x mers (main peak and its impurities shortened at 5´end), and diastereomeric separation of phosphorothioated oligonucleotides. The results confirmed a crucial role of alkylamine and counterion choice on the diastereomeric separation. The increasing hydrophobicity of alkylamine led to diminished diastereomeric selectivity which produced narrower phosphorothioated oligonucleotides peaks and led to improved n/n-x separation. Using hexafluoroisopropanol instead of acetate as counterion further enhances this effect (except for 100 mM concentration of hexafluoroisopropanol in combination with highly hydrophobic hexylamine). The elevated column temperature led to suppression of the diastereomeric resolution and improved resolution of n and n-x mers oligonucleotides. Baseline separation of oligonucleotides with different number of phosphorothioate linkages was achieved; this may be useful for therapeutic oligonucleotide analysis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

With the development of therapeutic oligonucleotides for antisense and gene therapies, the demand for analytical methods also increases. For the analysis of complex samples, for example plasma samples, where the use of mass detection is essential, hydrophilic interaction liquid chromatography is a suitable choice. The aim of the present work was to develop a method for separation and identification of the oligonucleotide impurities and metabolites by hydrophilic interaction liquid chromatography. First of all, the effects of different chromatographic conditions (e.g. pH of the aqueous part of the mobile phase, buffer concentration, column temperature) on the retention and separation of phosphorothioate oligonucleotides standards on the amide stationary phase were investigated. A set of model oligonucleotides containing a fully modified 21mer and its typical impurities (shortmers and oligonucleotides with different number of thiophosphate modifications) was used. The results showed that the concentration of the salt in the mobile phase as well as its pH, are the most influential parameters with regard to peak shape and separation. The knowledge gained was applied to the analysis of an unpurified 18mer oligonucleotides, analogues of the drug nusinersen used for the treatment of spinal muscular atrophy. The successful separation and identification of twenty-six and twenty-eight impurities was performed with the developed HILIC method. The method was applied to analysis of nusinersen metabolites of serum samples of patients treated with Spinraza., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Three sets of fluorinated chiral liquid crystals were used to explore the polar organic solvent chromatography mode for their enantioseparation. The materials include a set of newly synthesized compounds with chiral center derived from 2-hexanol and two sets of compounds with chiral center derived from 2-/3-octanol. Baseline enantioseparation of all materials was achieved using binary mobile phases without additives. For some of the compounds exceedingly high values of enantioresolution (> 20) and enantioselectivity (> 4) were found. The chromatographic behavior of the sample set was studied on three different polysaccharide-based chiral columns - Chiralpak IA-U, IG-U and IB-U. Comparison of results from Chiralpak IA-U and IB-U shows the effect of amylose vs. cellulose polysaccharide backbone while comparison of Chiralpak IA-U and IG-U reveals the effect of 3,5-dimethylphenylcarbamate vs. 3‑chloro-5-methylphenylcarbamate substituent. The mobile phases tested included whole range of acetonitrile/methanol mixtures to demonstrate that acetonitrile-rich and alcohol-rich mobile phases offer different enantiorecognition mechanisms and can provide complementarity to some extent. The effect of temperature on enantioseparation was investigated on Chiralpak IA-U by constructing van't Hoff plots for selected liquid crystals in pure acetonitrile and pure methanol as mobile phases., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The fungus Amanita muscaria is universally recognizable for its iconic appearance; it is also widely regarded as poisonous, inedible, and even deadly. In spite of that, there have been documented cases of use of A. muscaria -containing preparations against various diseases, including cancer, to no apparent ill effect. The search for compounds that can be used to treat cancer among various plants and fungi has been intensifying in recent years. In light of this, we describe an HPLC HILIC analytical method for the evaluation of the content of the anticancer compound ergosterol (ERG) and the neuroactive alkaloids ibotenic acid (IBO) and muscimol (MUS) that contribute significantly to the unpleasant physiological syndrome associated with A. muscaria consumption. A 'homemade' A. muscaria tincture made using 80-proof rye vodka as the solvent, an A. muscaria extract made with a standardized water-ethanol solution as the solvent, and fractions obtained from the second extract via liquid-liquid extraction with nonpolar solvents were analyzed. The study also presents the results of capillary zone electrophoresis with contactless conductivity detection and UHPLC-MS/MS analyses of the IBO and MUS content of the two native A. muscaria extracts and an evaluation of the standardized extract's cytotoxic effect against a small panel of lung cell cultures in vitro. Our results show that the standardized extract has a significant cytotoxic effect and does not contain the compounds of interest in any significant quantity.

Liquid chromatography coupled with mass spectrometry is widely used in the field of proteomic analysis after off-line protein digestion. On-line digestion with chromatographic column connected in a series with immobilized enzymatic reactor is not often used approach. In this work we investigated the impact of chromatographic conditions on the protein digestion efficiency. The investigation of trypsin reactor activity was performed by on-line digestion of N-α-benzoyl-L-arginine 4-nitroanilide hydrochloride (BAPNA), followed by separation of the digests on the mixed-mode column. Two trypsin column reactors with the different trypsin coverage on the bridged ethylene hybrid particles were evaluated. To ensure optimal trypsin activity, the separation temperature was set at 37.0 °C and the pH of the mobile phase buffer was maintained at 8.5. The on-line digestion itself ongoing during the initial state of gradient was carried out at a low flow rate using a mobile phase that was free of organic modifiers. Proteins such as cytochrome C, enolase, and myoglobin were successfully digested on-line without prior reduction or alkylation, and the resulting peptides were separated using a mixed-mode column. Additionally, proteins that contain multiple cysteines, such as α-lactalbumin, albumin, β-lactoglobulin A, and conalbumin, were also successfully digested on-line (after reduction and alkylation). Moreover, trypsin immobilized enzymatic reactors were utilized for over 300 injections without any noticeable loss of digestion activity., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Oxidative stress supposedly plays a role in the pathogenesis of Parkinson's disease (PD). Uric acid (UA), a powerful antioxidant, is lowered in PD while allantoin, the oxidation product of UA and known biomarker of oxidative stress, was not systematically studied in PD. We aim to compare serum and cerebrospinal fluid (CSF) levels of UA, allantoin, and allantoin/UA ratio in de novo PD patients and controls, and evaluate their associations with clinical severity and the degree of substantia nigra degeneration in PD. We measured serum and CSF levels of UA, allantoin, and allantoin/UA ratio in 86 PD patients (33 females, mean age 57.9 (SD 12.6) years; CSF levels were assessed in 51 patients) and in 40 controls (19 females, 56.7 (14.1) years). PD patients were examined using Movement Disorder Society-Unified Parkinson's Disease Rating Scale (MDS-UPDRS), Montreal Cognitive Assessment (MoCA), Scales for Outcomes in Parkinson Disease-Autonomic (SCOPA-AUT), the University of Pennsylvania Smell Identification Test (UPSIT), one-night video-polysomnography, and dopamine transporter single-photon emission computed tomography (DAT-SPECT). Serum allantoin and allantoin/UA ratio were significantly increased in the PD group compared to controls (p < 0.001 and p = 0.002, respectively). Allantoin/UA ratios in serum and CSF were positively associated with the SCOPA-AUT score (p = 0.005 and 0.031, respectively) and RBD presence (p = 0.044 and 0.028, respectively). In conclusion, serum allantoin and allantoin/UA ratio are elevated in patients with de novo PD. Allantoin/UA ratio in serum and CSF is associated with autonomic dysfunction and RBD presence, indicating that higher systemic oxidative stress occurs in PD patients with more diffuse neurodegenerative changes., (© 2023. The Author(s).)

The enantioselective potential of two macrocyclic glycopeptide-based chiral stationary phases for analysis of 28 structurally diverse biologically active compounds such as derivatives of pyrovalerone, ketamine, cathinone, and other representatives of psychostimulants and antidepressants was evaluated in sub/supercritical fluid chromatography. The chiral selectors immobilized on 2.7 μm superficially porous particles were teicoplanin (TeicoShell column) and modified macrocyclic glycopeptide (NicoShell column). The influence of the organic modifier and different mobile phase additives on the retention and enantioresolution were investigated. The obtained results confirmed that the mobile phase additives, especially water as a single additive or in combination with basic and acidic additives, improve peak shape and enhance enantioresolution. In addition, the effect of temperature was evaluated to optimize the enantioseparation process. Both columns exhibited comparable enantioselectivity, approximately 90% of the compounds tested were enantioseparated, and 30% out of them were baseline enantioresolved under the tested conditions. The complementary enantioselectivity of the macrocyclic glycopeptide-based chiral stationary phases was emphasized. This work can be useful for the method development for the enantioseparation of basic biologically active compounds of interest.

Analysis of diastereomers of phosphorothioate oligonucleotides in ion-pairing reversed-phase liquid chromatography is affected not only by the character and concentration of ion-pairing system, but also by the separation temperature. In this work, eight ion-pairing systems at two concentrations buffered with acetic acid were used with octadecyl column to investigate the effects of temperature (in the range from 20 °C to 90 °C) on retention, diastereomeric separation, resolution of mers of different length and resolution of oligonucleotides with different number of phosphorothioate linkages. It was observed that elevated temperature suppresses the diastereomeric separation and oligonucleotide peaks become narrower. This improves the resolution of n and n-1 mers at elevated temperature. Plots of ln k (k = retention factor) versus reciprocal absolute temperature show that for 100 mM ion-pairing systems the increase in temperature does not lead to simple decrease in oligonucleotides retention as generally observed in reversed-phase liquid chromatography. The aim of this work is to improve chromatographic method for analysis of phosphorothioate oligonucleotides., Competing Interests: Declaration of Competing Interest The authors declared no conflict of interest., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Ion-pairing reversed-phase liquid chromatography was utilized for the analysis of native and phosphorothioate oligonucleotides of the identical sequence but different amount and position of phosphorothioate modifications. The effects of ion-pairing amines nature (alkyl chain length, hydrophobicity) and concentration on retention, n/n-1 resolution, and diastereomeric separation of phosphorothioate oligonucleotides were investigated using octadecyl column. Eight different ion-pairing agents at two concentrations (10 mM and 100 mM) buffered with acetic acid were investigated. The resolution of n and n-1 mers oligonucleotides improved with hydrophobicity and concentration of ion-pairing amines. Ion-pairing amines with moderate hydrophobicity were most successful in suppressing diastereomeric resolution. However, a partial separation of phosphorothioate oligonucleotide diastereomers was observed with all ion-pairing systems, which resulted in wider peaks compared to phosphorodiester oligonucleotides of the same sequence. This phenomenon complicates the n and n-1 mers separation of oligonucleotides with high degree of phosphorothioate modifications. Separation of oligonucleotides with different number of phosphorothioate modifications was observed, which may be useful for therapeutic oligonucleotide analysis. The aim of the work was to identify the ion-pairing systems useful for chromatographic characterization of phosphorothioate oligonucleotides., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Liquid crystals can partake in enantioseparations in several different ways. Enantiomeric purity of chiral liquid crystals as analytes is often determined using enantioselective liquid or supercritical fluid chromatography. At the same time, chiral liquid crystalline materials can be applied as a chiral selector or they can function as an auxiliary material for a different chiral selector enabling enantioseparation of various solutes in miscellaneous separation techniques. They can also be used in fabrication of matrices or surfaces sensitive towards enantiomers or employed as chiral solvents for visualizing enantiomers in NMR. In this review, all of these aspects of relationship between liquid crystals and enantiodiscrimination are discussed in an effort to encompass their versatility and with emphasis on enantioseparations., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The coupling of columns in sub/supercritical fluid chromatography presents a great opportunity for influencing the separation efficiency and extending the selectivity of the separation system. Combinations of different types of chiral stationary phases could positively affect the enantioresolution if single ones are complementary to each other. In this work, two superficially porous particle (2.7 μm) macrocyclic glycopeptide-based columns, namely TeicoShell and NicoShell, were serially coupled and tested in sub/supercritical fluid chromatography for the first time. The influence of the column arrangement on the enantioseparation of structurally diverse biologically active compounds was examined. The obtained results showed how the column order crucially affected the enantioresolution of compounds tested, but the retention was negligibly affected in most cases. We also demonstrated that single TeicoShell and NicoShell columns are very promising towards the development of highly efficient and fast/ultrafast sub/supercritical fluid chromatography methods for structurally different chiral compounds. The optimized methods for sub-minute enantioselective separation of certain biologically important compounds were proposed., (© 2021 Wiley-VCH GmbH.)

A comprehensive study into the effects of mobile phase composition and column temperature on enantiomer elution order was conducted with a set of chiral rod-like liquid crystalline materials. The analytes were structurally similar and comprised variances such as length of terminal alkyl chain, presence of chlorine, number of phenyl rings, and type of chiral center. Experiments were carried out in polar organic and reversed-phase modes using amylose tris(3-chloro-5-methylphenylcarbamate) immobilized on silica gel as the chiral stationary phase. For all liquid crystals, reversal of elution order of enantiomers was observed based on type of used cosolvent and/or its content in the mobile phase; for some of the liquid crystals a temperature-induced reversal was also observed. Both linear and nonlinear dependencies of natural logarithm of enantioselectivity on temperature were found. Tested mobile phases comprised pure organic solvents and binary and tertiary mixtures of acetonitrile with organic solvents and/or water. Effect of acidic/basic mobile phase additives was also tested. Effect of structure of chiral selector is briefly discussed., (© 2021 Wiley-VCH GmbH.)

Introduction: Rapid eye movement (REM) sleep behavior disorder (RBD) is associated with an increased risk of developing Parkinson's disease (PD). Low uric acid (UA) levels are associated with the risk of development and progression of PD. Allantoin is the major oxidation product of UA and is considered as a biomarker of oxidative stress. We aimed to compare serum levels of UA, allantoin, and allantoin/UA ratio in RBD patients with those in healthy controls, and to examine their associations with clinical severity., Methods: We evaluated serum levels of UA, allantoin, and allantoin/UA ratio in 38 RBD patients (one female, mean age 66.8 (SD 6.3) years) and in 47 controls (four females, 66.8 (7.6) years). All RBD patients were assessed according to an examination protocol, which included structured interview, Montreal Cognitive Assessment (MoCA), Movement Disorder Society-Unified Parkinson's Disease Rating Scale (MDS-UPDRS), and dopamine transporter single-photon emission computed tomography (DAT-SPECT). The lower putaminal binding ratio from both hemispheres was used for analysis., Results: Mean serum allantoin concentration and allantoin/UA ratio were significantly increased in the RBD group compared to controls (2.6 (1.8) vs. 1.4 (0.7) μmol/l, p = 0.0004, and 0.008 (0.004) vs. 0.004 (0.002), p < 0.0001, respectively). There were no significant differences in UA levels between the two groups. No significant associations between any biochemical parameter and RBD duration, putaminal binding ratio on DAT-SPECT, MDS-UPDRS, or MoCA score were found., Conclusion: Serum allantoin and allantoin/UA ratio are increased in RBD patients in comparison to controls, which may reflect increased systemic oxidative stress in prodromal synucleinopathy., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The enantioselective synthesis of chiral [7]-helical dispirodihydro[2,1-c]indenofluorenes (DSF-IFs) was achieved for the first time in good yields with high er values (er up to 99 : 1). The crucial step of the whole reaction sequence was the enantioselective intramolecular [2+2+2] cycloaddition of tethered triynediols to indenofluorenediols, which was catalyzed by a Rh/SEGPHOS® complex. Further transformations led to the corresponding DSF-IFs. The prepared helically chiral DSF-IFs combine circularly polarized luminescence (CPL) activity (g lum =∼10 -3 ) with exceptionally high fluorescence quantum yields (up to Φ lum =0.97)., (© 2021 Wiley-VCH GmbH.)

In this work, two mixed-mode columns from a different manufacturers and one marketed as a reversed-phase column were characterized and compared in the terms of their interaction abilities, retentivity, peak symmetry, and applicability for peptide separation. All the tested columns contain octadecyl ligand and positively charged modifier, i.e. pyridyl group for the reversed-phase column XSelect CSH C18, quaternary alkylamine for mixed-mode column Atlantis PREMIER BEH C18 AX, and permanently charged moiety (details not available from the manufacturer) for mixed-mode column Luna Omega PS C18. For detailed characterization and comparison of their interaction potential, several approaches were used. First, a simple Walters test was performed to estimate hydrophobic and silanophilic interactions of the tested columns. The highest values of both parameters were observed for column Atlantis PREMIER BEH C18 AX. To investigate the effect of pH and buffer concentration on retention, mobile phases composed of acetonitrile and buffer (ammonium formate, pH 3.0; ammonium acetate pH 4.7 and pH 6.9) in various concentrations (5mM; 10mM; 15mM and 20mM) were used. The analysis of permanently charged compounds was used to describe the electrostatic interaction abilities of the stationary phases. The most significant contribution of electrostatic interactions to the retention was observed for Atlantis PREMIER BEH C18 AX column in the mobile phase with buffer of pH 3.0. A set of ten dipeptides, three pentapeptides and one octapeptide was used to investigate the effects of pH and buffer concentration on retention and peak symmetry. Each of the tested columns provides the optimal peak shape under different buffer pH and concentration. The gradient separation of the 14 tested peptides was used to verify the application potential of the tested columns for peptide separation. The best separation was achieved within 4 minutes on column Atlantis PREMIER BEH C18 AX., Competing Interests: Declaration of Competing Interest The authors declared no conflict of interest., (Copyright © 2021. Published by Elsevier B.V.)

Novel psychoactive substances (NPS) are synthetic compounds that have been designed to produce the physiological and psychological effects of known recreational drugs, while circumventing current drug control laws and scheduling guidelines. Such "designer drugs" pose problems in detection and prevention of use, and they are no less dangerous than known controlled substances. Among the various classes of NPS, many are chiral. As they are synthetic products, most are racemates. Not unexpectedly, there is limited information about different the pharmacological and toxicological properties of their pure enantiomers. Hence, fast and reliable enantioselective methods are of great interest. In this work, superficially porous particle (SPP) vancomycin-based chiral stationary phases were used for development of fast enantioselective separation methods for various classes of NPS in supercritical fluid chromatography and liquid chromatography. The NPS tested included pyrovalerones, benzofurans, phenidines and phenidates. The effect of mobile phase composition on the retention and resolution of NPS in supercritical fluid chromatography was examined. The amount as well as the ratios of additives used is crucial for enantioseparation efficiency. Results showed the high enantioselective potential of vancomycin-based columns in both chromatographic techniques; 88% of NPS tested were enantioseparated in supercritical fluid chromatography and 69% of NPS tested were enantioseparated in liquid chromatography. Moreover, under optimized conditions, simultaneous enantioseparations of some NPS were achieved, which indicates great suitability of vancomycin-based columns for this purpose. The proposed methods can serve as guides for method development and for enantioseparation of further upcoming NPS., Competing Interests: Declaration of Competing Interest The authors declared no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Increasing number of reported works dealing with macrocyclic glycopeptide-based columns in sub/supercritical fluid chromatography (SFC) points to the growing interest in this area. With the development and production of sub 2 µm fully porous particles and superficially porous particles with bonded macrocyclic glycopeptides, significant improvements have been made in ultrafast high efficiency chiral SFC. This review article gives an overview of macrocyclic glycopeptide-based chiral selectors that were used in theoretical studies and/or applications in SFC. The review covers the period from 1997 when macrocyclic glycopeptides were first used in SFC till the end of July 2020 according to Web of Science. This work can also be helpful to analysts searching for an appropriate method for the separation/determination of enantiomers of their interest., Competing Interests: The authors declare no conflict of interest., (© 2020 The Authors. Analytical Science Advances published by Wiley‐VCH GmbH.)

In this work we utilized basic and acidic analytes to investigate the ionic interaction participation in retention behavior of selected reversed-phase and polar columns. The test analytes included nitrate, benzenesulfonate and trimethylphenylammonium ions. The fully aqueous mobile phase comprising 10 mM dichloroacetic acid buffered with ammonia solution to desirable pH was used for retention experiments. Developed method was utilized to study the ionic interactions of stationary phases in pH range between 2.5 and 9.0. We demonstrate that selected sorbents used for reversed-phase and hydrophilic interaction chromatography separations exhibit cation- or anion-exchange interactions. We compare the results to novel Atlantis PREMIER BEH C18 AX mixed-mode column that combines reversed-phase and anion-exchange interaction modes. We evaluated the relative retention strength of selected columns for anionic and cationic analytes., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

Comprehensive study of enantioselective potential of eight different chiral stationary phases for chiral liquid crystal-forming molecules was conducted. The tested columns were: (i) polysaccharide-based Trefoil AMY1, CEL1 and CEL2 and (ii) superficially porous particles packed TeicoShell, VancoShell, TagShell, DMP-MaltoShell, and NicoShell. To test their enantioselective potential for these separations, twenty racemic mixtures of rod-like liquid crystalline materials comprising three different types of chiral centres and various other structural differences were used. Mobile phases consisting of supercritical carbon dioxide and alcohol as cosolvent were used; selected alcohols were methanol, ethanol and propan-2-ol. Effect of acidic and/or basic additives on enantioselectivity was also evaluated. Chiral stationary phases based on polysaccharides were found to have the greatest enantioselective potential for rod-like molecules that form liquid crystals, followed by TeicoShell, which proved suitable for enantioseparation of non-halogenated liquid crystals with lactic acid-based chiral centre., Competing Interests: Declaration of Competing Interest The authors declared no conflict of interest., (Copyright © 2020. Published by Elsevier B.V.)

Application of the superficially porous particles (SPPs) grafted with chiral selectors can substantially improve resolution in chromatographic techniques. In this work, we carried out a deeper study on supercritical fluid chromatography systems with 2.7 µm SPPs bonded with teicoplanin and vancomycin. Fast separations of the majority of enantiomers of phytoalexins, substituted tryptophans, and ketamine derivatives, as representatives of important biologically active and structurally diverse chiral compounds have been achieved. The chromatographic behavior of the structurally different analytes served to characterize these separation systems. The influence of separation conditions, namely mobile phase composition, i.e. type of co-solvent and additive on retention, enantioselective resolution and enantioselectivity was examined. The success rate of baseline and partial separations in individual groups of compounds differed with the chiral stationary phase and also with mobile phase composition. The best, baseline separations for the phytoalexins were achieved on the TeicoShell column using methanol as a co-solvent and trifluoroacetic acid as an additive if used. Mostly partial separations were achieved on the vancomycin-based column for all groups of analytes. Complementary separation behavior of these CSPs was confirmed for the majority of the chiral compounds examined in this work., Competing Interests: Declaration of Competing Interest None., (Copyright © 2019. Published by Elsevier B.V.)

Three chiral stationary phases were prepared by dynamic coating of sulfobutylether-β-cyclodextrin (SBE-β-CD) with different degrees of substitution, onto strong anion-exchange stationary phases. The enantioselective potential and stability of newly prepared chiral stationary phases were examined using a set of structurally different chiral analytes. Measurements were performed in RP-HPLC. Mobile phases consisted of methanol/formic acid, pH 2.10, and methanol/10 mM ammonium acetate buffer, pH 4.00, in various volume ratios. SBE-β-CDs with degrees of substitution (DS) 4, 6.3, and 10 proved suitable for the enantioseparation of 14, 11, and 8 analytes, respectively. The SBE-β-CD DS 4 based chiral stationary phase enabled the enantioseparation of the nearly all basic and neutral compounds. Chiral stationary phases with higher sulfobutylether-β-cyclodextrin substitution (especially DS 10) yielded higher enantioresolution values for acidic compounds., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A series of chiral indole phytoalexins with potential anticancer and antimicrobial activity were enantioseparated in supercritical fluid chromatography. Two polysaccharide-based chiral stationary phases composed of tris-(3,5-dimethylphenylcarbamate) derivatives of amylose or cellulose coated on 2.5 μm silica particles were successfully used. The influences of the polysaccharide backbone, co-solvent type and co-solvent content in the mobile phase on retention, enantioselectivity and enantioresolution of indole phytoalexins were investigated. Fast baseline separations were achieved for 26 from 27 tested compounds. Amylose-based chiral stationary phase provided higher number of baseline resolutions of the indole phytoalexins than the cellulose-based one. However, certain complementary enantioresolution results towards the studied compounds were observed between the investigated columns. The relationship between structure of the indole phytoalexins and their chromatographic behavior in supercritical fluid chromatography was discussed., (Copyright © 2019 Elsevier B.V. All rights reserved.)

A fast and simple supercritical fluid chromatography method for the enantioseparation of twenty newly synthesized orthoconic antiferroelectric liquid crystals is reported for the first time. The effects of alkoxy spacer length and fluorine atom presence and position in the phenyl ring on chromatographic behavior were investigated. Baseline enantioseparation of all compounds was achieved using simple mobile phases consisting of carbon dioxide and alcohol as cosolvent on (3,5-dimethylphenylcarbamate) derivative of amylose as chiral stationary phase. The analysis times ranged from 2 to 4 and from 4 to 7 min for most samples when using methanol and propane-2-ol, respectively. The significant effect of cosolvent type on the enantioseparation of these compounds was assessed and partial complementarity of methanol and propane-2-ol was observed., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The chromatographic behavior of new biogenic purine nucleosides in hydrophilic interaction liquid chromatography was examined on three different stationary phases, namely bare silica, and amide- and cyclofructan-based stationary phases. The effects of buffer concentration, pH and acetonitrile-to-aqueous-part ratio in the mobile phase on retention and peak shape were assessed. The retention coefficients and peak symmetry values substantially differed with respect to analytes´ structures, stationary phase properties and mobile phase composition. The bare silica column was unsuitable for these compounds under the chromatographic conditions tested due to very broad and asymmetrical peaks. Furthermore, the cyclofructan-based stationary phase provided almost Gaussian peak shapes of all deazapurine nucleosides under most conditions tested. Therefore, the cyclofructan-based stationary phase is the most suitable choice for the chromatographic analysis of nucleosides., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A cellulose tris-(3,5-dimethylphenylcarbamate)-based chiral stationary phase was studied as a tool for the enantioselective separation of 21 selected analytes with different pharmaceutical and physicochemical properties. The enantioseparations were performed using supercritical fluid chromatography. The effect of the mobile phase composition was studied. Four different additives (diethylamine, triethylamine, isopropylamine, and trifluoroacetic acid) and isopropylamine combined with trifluoroacetic acid were tested and their influence on enantioseparation was compared. The influence of two different mobile phase co-solvents (methanol and propan-2-ol) combined with all the additives was also evaluated. The best mobile phase compositions for the separation of the majority of enantiomers were CO 2 /methanol/isopropylamine 80:20:0.1 v/v/v or CO 2 /propan-2-ol/isopropylamine/trifluoroacetic acid 80:20:0.05:0.05 v/v/v/v. The best results were obtained from the group of basic β-blockers. A high-performance liquid chromatography separation system composed of the same stationary phase and mobile phase of similar properties prepared as a mixture of hexane/propan-2-ol/additive 80:20:0.1 v/v/v was considered for comparison. Supercritical fluid chromatography was found to yield better results, i.e. better enantioresolution for shorter analysis times than high-performance liquid chromatography. However, examples of enantiomers better resolved under the optimized conditions in high-performance liquid chromatography were also found., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A new ion-exchanger type chiral stationary phase for high-performance liquid chromatography was prepared. The synthetic protocol is based on derivatization of silica with (3-iodopropyl)trimethoxysilane in the first step followed by immobilization of strychnine via quaternization of nitrogen atom of the alkaloid strychnine. The synthesized chiral stationary phase was chromatographically characterized. The main effort was headed towards the evaluation of the enantioselectivity of the novel sorbent. For that purpose a set of suitable chiral probes, specifically, binaphthyl derivatives, was employed. The influence of methanol content, concentration of aqueous ammonium acetate buffer, and its pH on retention factors, separation selectivity, and resolution of the atropoisomers of the mentioned chiral solutes was studied in detail. It was demonstrated that the new chiral stationary phase was capable to separate atropoisomers of four out of seven testing compounds. Despite the strong influence of the above mentioned variables on retention, their impact on selectivity and resolution was rather moderate. Concerning retention mechanism, it seems that electrostatic interaction between the positively charged quaternary nitrogen of the chiral stationary phase and anionic solute participates significantly in the retention process., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Trypsin is the most widely used enzyme in proteomic research due to its high specificity. Although the in-solution digestion is predominantly used, it has several drawbacks, such as long digestion times, autolysis, and intolerance to high temperatures or organic solvents. To overcome these shortcomings trypsin was covalently immobilized on solid support and tested for its proteolytic activity. Trypsin was immobilized on bridge-ethyl hybrid silica sorbent with 300Å pores, packed in 2.1×30mm column and compared with Perfinity and Poroszyme trypsin columns. Catalytic efficiency of enzymatic reactors was tested using N α -Benzoyl-l-arginine 4-nitroanilide hydrochloride as a substrate. The impact of buffer pH, mobile phase flow rate, and temperature on enzymatic activity was investigated. Digestion speed generally increased with the temperature from 20 to 37°C. Digestion speed also increased with pH from 7.0 to 9.0; the activity of prototype enzyme reactor was highest at pH 9.0, when it activity exceeded both commercial reactors. Preliminary data for fast protein digestion are presented., (Copyright © 2017 Elsevier B.V. All rights reserved.)

A polyaniline coating was used to modify the surface of bare silica gel and octadecyl silica stationary phases to characterize the properties of altered materials. It was assumed that the mixed-mode retention was established on the basis of the polyaniline chemical structure and its combination with the original sorbents. Polyaniline was deposited onto the original surfaces during the chemical polymerization of aniline hydrochloride. The prepared materials were slurry packed into capillary columns and systematic chromatographic characterization was performed using the linear solvation energy relationship, also employing descriptors that allow inclusion of ionic interactions in the proposed retention mechanism. The retention times of 80 solutes with various chemical structures were measured in the hydrophilic interaction liquid chromatography mode. The obtained results demonstrated the significant contribution of the polyaniline coating to the retention mechanism under the given conditions; the assumed mixed-mode retention was confirmed. The dominant retention interaction for both modified stationary phases was based on the protonation of nitrogen atoms in the polyaniline structure, leading to suitable retention and selectivity for the hydrophilic analytes, especially anionic and zwitterionic species. Thus, especially, the polyaniline-coated bare silica gel sorbent seems to be promising for potential applications related to the separation of polar compounds., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In this short communication we report optimized procedures for the chiral separation of non-charged [6]helicene (1) and cationic derivative 1-butyl-3-(2-methyl[6]helicenyl)-imidazolium bromide (2) using high-performance liquid chromatography (HPLC) and supercritical fluid chromatography (SFC) methods. The possibility of using capillary electrophoresis (CE) was also tested. The satisfactory results were obtained with SFC, where the highly selective resolution of four enantiopure 1 and 2 helicenes was achieved in a single run within 5min. The semi-preparative procedure for the isolation of P and M enantiomers of compound 2, including circular dichroism data, is reported here for the first time. The results could be used in further separations and analytical applications targeting carbohelicenes vs. positively charged helicene derivatives., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Various amino acids, dipeptides and their isomers were (enantio)separated using sulfobutylether-β-cyclodextrin as a chiral selector. Two different approaches were employed: first, dynamic coating of sulfobutylether-β-cyclodextrin onto a strong anion-exchange stationary phase and, second, use of sulfobutylether-β-cyclodextrin as a mobile phase additive in a separation system with a C18 column. Measurements were carried out using RP-HPLC and hydrophilic interaction liquid chromatography. Mobile phases composed of organic modifier (methanol) and four different aqueous parts: (i) deionized water, (ii) an aqueous solution of formic acid (pH 2.1), (iii) ammonium acetate buffer (pH 4.7), and (iv) ammonium acetate buffer (pH 8.8) in various volume ratios. Under these separation conditions, out of 23 chiral analytes, 9 were baseline enantio-resolved and 7 were partially separated. Of 9 mixtures of dipeptide isomers, 8 were baseline-separated. Sulfobutylether-β-cyclodextrin proved to be suitable for the separation of chiral and also achiral analytes. The use of sulfobutylether-β-cyclodextrin as a dynamic coating agent or as a mobile phase additive depends on the particular chromatographic system and analytes of interest., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The enantioseparation of basic compounds represent a challenging task in modern SFC. Therefore this work is focused on development and optimization of fast SFC methods suitable for enantioseparation of 27 biologically active basic compounds of various structures. The influences of the co-solvent type as well as different mobile phase additives on retention, enantioselectivity and enantioresolution were investigated. Obtained results confirmed that the mobile phase additives, especially bases (or the mixture of base and acid), improve peak shape and enhance enantioresolution. The best results were achieved with isopropylamine or the mixture of isopropylamine and trifluoroacetic acid as additives. In addition, the effect of temperature and back pressure were evaluated to optimize the enantioseparation process. The immobilized amylose-based chiral stationary phase, i.e. tris(3,5-dimethylphenylcarbamate) derivative of amylose proved to be useful tool for the enantioseparation of a broad spectrum of chiral bases. The chromatographic conditions that yielded baseline enantioseparations of all tested compounds were discovered. The presented work can serve as a guide for simplifying the method development for enantioseparation of basic racemates in SFC., (Copyright © 2016 Elsevier B.V. All rights reserved.)

This is the first experimental evidence that both self-assembly and surface activity are common features of all water-soluble boron cluster compounds. The solution behavior of anionic polyhedral boranes (sodium decaborate, sodium dodecaborate, and sodium mercaptododecaborate), carboranes (potassium 1-carba-dodecaborate), and metallacarboranes {sodium [cobalt bis(1,2-dicarbollide)]} was extensively studied, and it is evident that all the anionic boron clusters form multimolecular aggregates in water. However, the mechanism of aggregation is dependent on size and polarity. The series of studied clusters spans from a small hydrophilic decaborate-resembling hydrotrope to a bulky hydrophobic cobalt bis(dicarbollide) behaving like a classical surfactant. Despite their pristine structure resembling Platonic solids, the nature of anionic boron cluster compounds is inherently amphiphilic-they are stealth amphiphiles.

Trypsin, a high fidelity protease, is the most widely used enzyme for protein digestion in proteomic research. Optimal digestion conditions are well-known and so are the expected cleavage products. However, missed cleavage sites are frequently observed when acidic amino acids, aspartic and glutamic acids, are present near the cleavage site. Also, the sequence motifs with successive lysine and/or arginine residues represent a source of missed cleaved sites. In spite of an adverse role of missed cleaved peptides on proteomic research, the digestion kinetics of these problematic sequences is not well-known. In this work, synthetic peptides with various sequence motifs were used as trypsin substrates. Cleavage products were analyzed with reversed-phase high performance liquid chromatography, and the kinetic constants for selected missed cleavage sites were calculated. Relative digestion speed for lysine and arginine sites is compared, including the digestion motifs flanked with aspartic and glutamic acid. Our findings show that DK and DTR motifs are cleaved by trypsin with 3 orders of magnitude lower speed than the arginine site. These motifs are likely to produce missed cleavage peptides in protein tryptic digests even at prolonged digestion times.

Polysaccharide-based chiral stationary phases can be used for the enantioselective separation of a wide range of structurally different compounds. These phases are available with chiral selectors coated or immobilized on silica gel support. The means of attachment of the chiral selector to the carrier can influence the separation performance of these stationary phases. This paper deals with evaluation of differences in the separation abilities of coated Chiralpak AD-RH versus immobilized Chiralpak IA amylose-based stationary phases in the reversed-phase mode of high-performance liquid chromatography. A set of chiral analytes was separated under acidic and basic conditions. Differences were observed in the enantioseparation potential of the tested phases. The linear-free energy relationship and additional evaluation of ionic interactions were used to ascertain whether the interactions that participate in retention and enantioseparation are affected by the means of preparation of these phases. All the interactions covered by the linear-free energy relationship were significant for the studied phases and their absolute values were almost always higher for the coated phase. Ionic interactions were found to be more important on the immobilized stationary phase but did not contribute to any improvement in the enantioselective separation performance., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Two novel metallacarborane-based stationary phases for high performance liquid chromatography were synthesized and characterized utilizing linear solvation energy relationships concept. Interactions taking place between selected probes (45 analytes) and the stationary phases and the mobile phases consisting of a mixture of acetonitrile with buffer were described by the linear solvation energy relationship model in a broad range of acetonitrile/buffer ratios. The interaction properties of the metallacarborane phases were compared with plain silica, the starting material for the preparation of the metallacarborane phases, and with a typical silica-based reversed-phase sorbent. It was clearly demonstrated that the metallacarborane sorbents provide qualitatively and quantitatively different interactions with respect to both the plain silica and reversed-phase stationary phase. It was shown that these novel phases might have also a separation potential in the area of hydrophilic interaction liquid chromatography., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Derivatized polysaccharide-based columns have high enantiodiscrimination potential mainly in normal phase separation mode. In this work chiral recognition ability of four immobilized polysaccharide-derived chiral stationary phases was evaluated under reversed phase conditions. A set of 30 chiral compounds, particularly drugs possessing various functional groups was used for testing. Baseline enantioseparation was achieved for 17 of them. In general, amylose-based chiral stationary phases showed higher enantioselectivity than the cellulose-based ones, mainly for acidic and bifunctional compounds. The influence of the type and pH of the aqueous mobile phase constituents as well as the role of the organic modifier on the enantioselective separation ability of the stationary phases were also investigated and compared. Complementary separations were obtained on the amylose- and cellulose-based columns. The immobilized polysaccharide-based chiral stationary phases were shown to be useful tool for the enantioseparation of a broad spectrum of chiral analytes in reversed phase separation mode., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Supercritical fluid chromatography (SFC) has become popular in the field of enantioselective separations. Many works have been reported during the last years. This review covers the period from 2000 till August 2013. The article is divided into three main chapters. The first one comprises a basic introduction to SFC. The authors provide a brief explanation of general principles and possibilities of this method. The advantages and drawbacks are also listed. Next part deals with chiral separation systems available in SFC, namely with the commonly used chiral stationary phases. Properties and interaction possibilities of the chiral separation systems are described. Recent theoretical papers are emphasized in this chapter. The last part of the paper gives an overview of applications of enantioselective SFC in analytical chemistry, in both analytical and preparative scales. Separation systems and conditions are summed up in tables so that they provide a helpful tool for analysts who search for a particular method of analysis., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Cyclofructans and preferentially their derivatives can serve as chiral selectors for the separation of different enantiomers/atropisomers. Moreover, the strong ionophoric nature of the 18-crown-6 ether core of cyclofructan 6 for barium cations may be exploited to enhance or modify enantioselectivity. In this work isopropyl-cyclofructan-6 was used as a chiral selector for the separation of binaphthyl atropisomers in HPLC and CE. The data from both separation systems were compared with each other. While in HPLC the chiral selector was bonded to silica gel to afford a chiral stationary phase, in capillary electrophoresis it was freely mobile in the background electrolytes (BGE). This significant difference is reflected in the separation potential of the two separation systems. All five analytes could be baseline separated in HPLC (reversed phase mode) while only one derivative was baseline resolved in CE. This result was attributed to the more rigid nature of the immobilized chiral selector. Addition of Ba(2+) to the mobile phase or BGE improved chiral separations in both systems. The results may help to elucidate the interaction mechanism in these systems with cyclofructan derivatives and to gain some general knowledge of their separation potential., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Hydrophilic interaction liquid chromatography is a separation technique suitable for the separation of moderately and highly polar compounds. Various stationary phases (SPs) for hydrophilic interaction liquid chromatography are commercially available. While the SPs based on the same type of ligand are available from different providers, they can display a distinct retention characteristics and separation selectivity. The current work is focused on characterization and comparison of the separation systems of two amide-based HPLC columns from two producers, i.e. XBridge Amide column and TSK gel Amide-80 column. Several characterization procedures (tests) were used to investigate the differences between these columns. The chromatographic behavior of selected analytes indicates that multimodal interactions are responsible for retention and separation on these columns. Multiple testing approaches were used in order to reveal subtle differences between the SPs. Both amide-based columns showed certain differences in retention, selectivity, and plate counts. Based on the tests used in this study, we conclude that the investigated columns provide a different degree of H-bonding interactions., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A new separation method involving hydrophilic interaction chromatography with tandem mass spectrometric detection has been developed for the analysis of pteridines, namely biopterin, isoxanthopterin, leucopterin, neopterin, xanthopterin and erythropterin in the cuticle of heteropteran insect species. Two columns, Atlantis HILIC Silica and ZIC(®)-HILIC were tested for the separation of these pteridines. The effect of organic modifier content, buffer type, concentration and pH in mobile phase on retention and separation behavior of the selected pteridines was studied and the separation mechanism was also investigated. The optimized conditions for the separation of pteridines consisted of ZIC(®)-HILIC column, mobile phase composed of acetonitrile/5mM ammonium acetate, pH 6.80, 85/15 (v/v), flow rate 0.5mL/min and column temperature 30°C. Detection was performed by tandem mass spectrometry operating in electrospray ionization with Agilent Jet Stream technology using the selected reaction monitoring mode. The optimized method provided a linearity range from 0.3 to 5000ng/mL (r>0.9975) and repeatability with relative standard deviation<8.09% for all the studied pteridines. The method was applied to the analysis of pteridines in the cuticle of larvae and three adult color forms of Graphosoma lineatum and one form of Graphosoma semipunctatum (Insecta: Hemiptera: Heteroptera: Pentatomidae). The analysis shows that different forms of Graphosoma species can be characterized by different distribution of individual pteridines, which affects the coloration of various forms. Only isoxanthopterin was found in all the five forms tested., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Cyclofructan-based chiral stationary phases were previously shown as a promising possibility for separation of chiral compounds in high performance liquid chromatography. In this work retention and enantiodiscrimination properties of the 3,5-dimethylphenyl carbamate cyclofructan 7 chiral stationary phase are described in supercritical fluid chromatography. The results obtained in both of the separation methods were compared. A set of compounds with axial or central chirality was used as analytes. The effect of mobile phase composition, that is, addition of different alcohol modifiers and/or trifluoroacetic acid to carbon dioxide, was examined in the supercritical system. Similarly, mobile phases composed of hexane modified with propan-2-ol and/or trifluoracetic acid were used in liquid chromatography. A linear free energy relationship model was utilized for characterization of interactions that are decisive for retention and separation in both techniques. Dispersion interactions showed similar negative values using both methods. The main contribution of hydrogen bond acidity was also comparable for both methods. The propensity to interact with n- and/or π-electron pairs of solutes was significant only in the supercritical system., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Hydrophilic interaction liquid chromatography (HILIC) offers very good possibilities for separation of polar compounds as an alternative to reversed phase HPLC where polar compounds are not sufficiently retained. HILIC is becoming more popular for the analysis of biologically interesting (active) analytes. Various stationary phases are commercially available however, development of new materials (sorbents) suitable for HILIC systems still continues. Silica gel columns can be used directly but their modification can improve separation ability of the stationary phases. Cyclofructan-based stationary phases are demonstrated as possible HILIC columns in this work. The effect of silica gel modification by cyclofructan and a derivatized cyclofructan was studied in detail. HILIC separation systems with silica gel, cyclofructan and isopropyl cyclofructan modified silica stationary phases were compared. The detailed study of chromatographic behavior of peptides revealed that multimodal retention mechanism is present in systems with these stationary phases. Mobile phase composition changes the types of interactions and their strengths. It appears that ability to donate protons and dispersion forces are the main interactions that affect retention in HILIC with cyclofructan-based columns while they are less important in separation systems with bare silica stationary phase. Suitability of cyclofructan-based stationary phases in HILIC for separation of pentapeptides and nonapeptides was demonstrated., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Cyclofructans (CFs), a new class of chiral selectors, have been recently introduced for application in liquid chromatography and capillary electrophoresis. So far, derivatized CFs have performed interesting separation possibilities for a variety of compounds. The current work is focused on characterization of three different CF-based chiral stationary phases (CF-based CSPs), i.e. isopropyl carbamate cyclofructan 6 (IP-CF6), R-naphthylethyl carbamate cyclofructan 6 (RN-CF6) and dimethylphenyl carbamate cyclofructan 7 (DMP-CF7). The linear free energy relationship (LFER) model was used to reveal the dominant interactions participating in the complex retention mechanism. A set of 44 different test solutes, with known solvation parameters, was used to determine the regression coefficients of the LFER equation under two mobile-phase compositions in normal separation mode. The LFER results showed that hydrogen bond acidity, hydrophobicity and dipolarity/polarizibility mostly affect the retention and separation process on the CF-based columns in the studied separation systems., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Derivatized cyclofructans have been recently introduced as a new class of chiral selectors with great application potential. In this study, a R-naphthylethyl-functionalized cyclofructan 6 based chiral stationary phase (RN CF6 CSP) was used for separation of substituted binaphthyl catalysts in the normal phase HPLC mode. Dominant interaction types that play a role in the separation mechanism were revealed by a linear free energy relationship (LFER) method. In order to evaluate the contribution of the substituent on the cyclofructan structure to retention, the R-naphthylethyl-functionalized β-cyclodextrin (RN CD) CSP was chosen for comparison. Retention factors of 46 widely different solutes, with known solvation parameters, were determined on each of the columns under the same mobile phase compositions used for the enantiomeric separations. The LFER results showed that hydrogen bond acidity and polarity/polarizibility have the greatest impact on retention and enantioresolution on the RN CF6 CSP. The equal influence of the naphthylethyl substituent on the both CSPs was also confirmed while the effects of the basic cyclofructan versus cyclodextrin structures were different. The addition of trifluoroacetic acid to the hexane/propane-2-ol mobile phase was negligible on the RN CF6 CSP for the majority of atropoisomers except for one with ionizable functional groups. The RN CF6 column was shown to be more suitable for enantioseparation of the binaphthyl catalysts than the RN CD column. Higher retention offered by the latter CSP had no positive effect on the enantioresolution., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Separation systems with a zirconia-based polystyrene HPLC column were characterized by different approaches, which allowed the recognition of interactions participating in the separation environments. Zirconia-based HPLC columns as an alternative to silica-based ones offer unique interaction mechanism based on Lewis acid-base theory. Besides hydrophobic interactions with the modified surface of the zirconia carrier it includes ion-exchange and ligand-exchange interactions that are helpful in the separation of many bioactive compounds. Three distinct approaches were applied for description of the complex separation mechanism. General chromatographic tests by Walters, Engelhardt and Galushko were applied to evaluate the fundamental properties of the systems - hydrophobicity and polarity. The complex model of linear free energy relationship described the interactions from the qualitative and quantitative points of view more in detail. Application of a set of basic compounds revealed the contribution of ion-exchange interactions participating in the separation systems.

System peaks are important but often also disturbing phenomena occurring in separation systems. Behavior of system peaks was studied in reversed phase high performance liquid chromatography (RP HPLC) systems consisting of an RP Amide C16 column and aqueous solutions of organic acids with alkaline metal hydroxides as mobile phases. Binary mobile phases, composed of benzoic acid and lithium hydroxide (LiOH) or cesium hydroxide (CsOH), yielded two system peaks. The first peak was stationary and the second one moved with dilution of the mobile phase or with changes of the alkaline metal hydroxide concentration. The latter changes affected dissociation of the benzoic acid present in the mobile phase and thereby its retention. The presumption that the first system peak is not influenced by the type of alkaline metal cation and that it is related to the non-adsorbed component of the mobile phase was confirmed by a cyclic procedure. Three-component mobile phases composed of benzoic acid, tropic acid, and a hydroxide gave rise to three system peaks as expected. The first peak was again stationary and the two others shifted depending on the concentration variation of both acids. Resonance causing a zigzag peak, well described in capillary zone electrophoresis (CZE), was observed if 1-pentanol was injected into a chromatographic system with one-component mobile phase.

A new HPLC method for the separation and quantification of cloprostenol enantiomers was developed. The optimized separation system consisted of Chiralcel OD-RH column and acetonitrile-sodium dihydrogenphosphate (pH 3.0; 20mM) (33:67, v/v) as the mobile phase. Baseline resolution of (+/-)-cloprostenol (R=2.16) was achieved and the analysis time did not exceed 10 min. Limits of detection and quantification were units of micromol/l at 274 nm. The respective values decreased an order of magnitude at 210 nm. The R.S.D. values obtained for the retention factor, peak area and peak height of each enantiomer were less than 2%. Conditions for semipreparative separation of the enantiomers can be achieved easily just by a small adaptation of the mobile phase composition.