Direct field evidence of autocatalytic iodine release from atmospheric aerosol.

Reactive iodine plays a key role in determining the oxidation capacity, or cleansing capacity, of the atmosphere in addition to being implicated in the formation of new particles in the marine boundary layer. The postulation that heterogeneous cycling of reactive iodine on aerosols may significantly influence the lifetime of ozone in the troposphere not only remains poorly understood but also heretofore has never been observed or quantified in the field. Here, we report direct ambient observations of hypoiodous acid (HOI) and heterogeneous recycling of interhalogen product species (i.e., iodine monochloride [ICl] and iodine monobromide [IBr]) in a midlatitude coastal environment. Significant levels of ICl and IBr with mean daily maxima of 4.3 and 3.0 parts per trillion by volume (1-min average), respectively, have been observed throughout the campaign. We show that the heterogeneous reaction of HOI on marine aerosol and subsequent production of iodine interhalogens are much faster than previously thought. These results indicate that the fast formation of iodine interhalogens, together with their rapid photolysis, results in more efficient recycling of atomic iodine than currently considered in models. Photolysis of the observed ICl and IBr leads to a 32% increase in the daytime average of atomic iodine production rate, thereby enhancing the average daytime iodine-catalyzed ozone loss rate by 10 to 20%. Our findings provide direct field evidence that the autocatalytic mechanism of iodine release from marine aerosol is important in the atmosphere and can have significant impacts on atmospheric oxidation capacity.

Tailor-made approach for selective isolation and elution of low-density lipoproteins by immunoaffinity sorbent on silica.

Immunoaffinity procedure was developed for isolation of low density lipoprotein (LDL) from biological samples by using silica-derived immunoaffinity sorbent. Sorbent was prepared by immobilization of monoclonal anti-apoB-100 antibody onto macroporous silica particles, using carefully optimized binding chemistry. Binding capacity of the sorbent towards LDL was determined by batch extraction experiments with solutions of isolated LDL in phosphate-buffered saline, and found to be 8 mg LDL/g. The bound LDL fraction was readily released from the sorbent by elution with ammonia at pH 11.2. The total time needed for isolation procedure was less than 1 h, with LDL recoveries being essentially quantitative for samples containing less than 0.3 mg LDL/mL. With higher concentrations, recoveries were less favorable, most probably due to irreversible adsorption caused by LDL aggreggation. However, reusability studies with isolated LDL at concentration 0.2 mg/mL indicate that the developed immunoaffinity material may be used for multiple binding-release cycles, with minor losses in binding capacity. Finally, the sorbent was successfully applied to isolation of LDL from diluted plasma. Apart from its practical implications for LDL isolation, this study provides crucial insights into issues associated with LDL-sorbent interactions, and may be useful in future efforts directed to development of lipoprotein isolation approaches.

Chromatographic Enantiomer Separation Using 9-Amino-9-(deoxy)-epiquinine-derived Chiral Selectors: Control of Chiral Recognition via Introduction of Additional Stereogenic Centers.

Three new cinchona-type chiral selectors have been prepared by attaching N-pivaloyl-glycine, N-pivaloyl-(S)-valine and N-pivaloyl-(R)-valine segments to the C9-amino function of 9-amino-9-(deoxy)-epiquinine (eAQN), and immobilized to silica to provide the corresponding chiral stationary phases (CSPs). Evaluation of the chromatographic enantioseparation characteristics of these CSPs with a broad assortment of N-carbamoyl protected amino acids under polar organic mobile phase conditions revealed modest chiral recognition capabilities for N-Fmoc-, N-Cbz- and N-Boc-derivatives. It was found that the enantioselective analyte binding to these CSPs is strictly controlled by the absolute stereo-chemistry of the amino acid functionalities attached to the C9-amino group of the eAQN framework. Specifically, the CSP derived from (S)-valine-based selector exhibits preferential binding of N-carbamoyl-(S)-amino acids, while the CSPs featuring (R)-valine- and the glycine-derived selectors show opposite enantioselective binding preference. The observed impact of analyte structure on enantioselectivity and the specific preferences in enantioselective binding point to chiral recognition mechanisms capitalizing on intermolecular ion pairing, hydrogen bonding and subtle steric interactions, with the latter making the crucial contributions to stereodiscrimination. The finding that the chiral recognition characteristics of epiquinine can be readily controlled via incorporation of additional stereogenic centers remote from the cinchona scaffold might be useful information for the design of new enantioselective receptors and organocatalysts.

Investigations of mobile phase contributions to enantioselective anion- and zwitterion-exchange modes on quinine-based zwitterionic chiral stationary phases.

Novel chiral stationary phases (CSPs) based on zwitterionic Cinchona alkaloid-type low-molecular mass chiral selectors (SOs), as they have been reported recently, were investigated in HPLC towards effects on their chromatographic behavior by mobile phase composition. Mobile phase characteristics like acid-to-base ratio and type of acidic and basic additives as well as effect of type of bulk solvents in nonaqueous polar organic and aqueous reversed-phase (RP) eluent systems were varied in order to illustrate the variability and applicability of zwitterionic CSPs with regard to mobile phase aspects. Chiral SOs of the five zwitterionic CSPs investigated herein contained weak and strong cation-exchange (WCX, SCX) sites at C9- and C6'-positions of the Cinchona alkaloid scaffold which itself accommodated the weak anion-exchange (WAX) site. The study focused on zwitterion-exchange (ZX) operational mode and chiral amino acids as target analytes. Besides, also the anion-exchange (AX) mode for chiral N-blocked amino acid analytes was considered, because of the intramolecular counterion (IMCI) property available in AX mode. Overall, most general and successful conditions in ZX mode were found to be weakly acidic methanolic mobile phases. In aqueous eluents RP contributions to retention came into play but only at low organic modifier content because of the highly polar character of zwitterionic analytes. At higher acetonitrile content, HILIC-related retention phenomena were observed. When using weakly basic eluent system in AX mode remarkably fast enantiomer separations involving exclusion phenomena were possible with one enantiomer eluting before and the other after void volume.

Stationary phase-related investigations of quinine-based zwitterionic chiral stationary phases operated in anion-, cation-, and zwitterion-exchange modes.

The concept of recently introduced Cinchona alkaloid-type zwitterionic chiral stationary phases (CSPs) is based on fusing key cation- and anion-exchange (CX, AX) moieties in one single low-molecular mass chiral selector (SO) with the resulting CSPs allowing enantiomer separations of a wide range of chiral ionizable analytes comprising acids, bases, and zwitterionic compounds. Herein, we report principal, systematic investigations of the ion-exchange-type retention mechanisms available with the novel zwitterionic CSPs in nonaqueous polar organic mode. Typical CX and AX processes, corresponding to the parent single ion exchangers, are confirmed also for zwitterionic CSPs. Also the mechanism leading to recognition and retention of zwitterions was found to be ion exchange mediated in a zwitterion-exchange (ZX) mode. In both AX and CX modes the additional ionizable group within the SO besides the site responsible for the respective ion-exchange process could be characterized as an intramolecular counterion (IMCI) that effectively participates in the ion-exchange equilibria and thus, contributes to solute elution. In the ZX mode both oppositely charged groups of the zwitterionic SO were found not only to be the sites for simultaneous ion pairing with the analyte but also functioned as IMCIs at the same time. The main practical consequences of the IMCI feature were significant reduction of the amounts and even elimination of acidic and basic additives required in the eluent systems to afford analyte elution while still providing faster analysis than the parent single ion-exchanger-type CSPs. The set of ten structurally different zwitterionic CSPs employed in this study facilitated the establishment of correlations between chromatographic behavior of the CSPs with particular SO elements, thereby supporting the understanding of the working principles of these novel packing materials on a molecular level.

Enantiomer separation of imidazo-quinazoline-dione derivatives on quinine carbamate-based chiral stationary phase in normal phase mode.

The normal phase mode liquid chromatographic enantiomer separation capability of a quinine tert-butyl-carbamate-type chiral stationary phase (CSP) has been investigated for a set of polar [1,5-b]-quinazoline-1,5-dione derivatives. This class of chiral heterocycles is currently under development as potential alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and/or N-methyl-D-aspartic acid (NMDA) receptor antagonists. The effect of the nature and concentration of polar modifier, i.e., ethanol and isopropanol, in n-hexane-based mobile phases, as well as the substituent pattern of the phenyl ring attached to the quinazolone framework on retention factor, enantioselectivity, and resolution was investigated. The Soczewinski competitive adsorption model was used to describe the relationship between the retention and the binary mobile phase compositions. According to this model, linear plots of the logarithms of retention factor versus molar fractions of the polar modifiers were obtained over a wide concentration range (X(B) between 0.15 and 0.35). Addition of equimolar ethanol yields higher resolution than isopropanol, R(S) values ranging between 1.54 and 2.75, whereas the latter allows to achieve moderately increased enatioselectivity. The resolution was further improved by using a ternary mixture of n-hexane:methanol:isopropanol/85:5:10 (v/v). The most pronounced selectivity factor alpha and resolution R(S) values were obtained for the para-hydroxy substituted compound, indicating that chiral recognition is sensitive to steric and stereoelectronic factors. In the course of optimization, the temperature-dependence on the chiral separation was also investigated. It turned out that the enantiomer separation is predominantly enthalpically driven in normal phase mode.

Investigation of monovalent and bivalent enantioselective molecular recognition by electrospray ionization-mass spectrometry and tandem mass spectrometry.

In this work is described the investigation of bivalent versus monovalent enantioselective molecular recognition in the context of enantioselective separations. Electrospray ionization-mass spectrometry (ESI-MS) and tandem mass spectrometry (MS/MS) are used for evaluating enantioselective systems through the measurement of (1) relative solution-phase binding constants via titration and (2) relative gas-phase binding via collision threshold dissociation. In HPLC, a cinchonane-type chiral stationary phase (CSP) based on tert.-butylcarbamoylquinine provides vastly increased retention and enantioselectivity for separation of bivalent versus monovalent alkoxy-benzoyl-N-blocked leucine enantiomers. The bivalent enantiomers are able to span and simultaneously interact with multiple interaction sites on the CSP surface, leading to enhanced separation. ESI-MS titration measurements also show an increased avidity for binding between bivalent selector and bivalent selectand, compared with the monovalent system. However, enhanced enantioselectivities measured in HPLC for the bivalent system cannot be reproduced by MS due to inherent mechanistic differences. Assumed discrepancies in relative response factors also give rise to systematic errors which are discussed. The results of MS/MS gas-phase experiments show that enantioselectivity is essentially lost in the absence of solvation, but that dissociation thresholds can provide a measure of relative dissociation energy in the bivalent interaction system compared to the monovalent counterpart. Such measurements may prove useful and efficient in better understanding multivalent interactions, in line with current theoretical considerations of effective concentrations and ion trap effects. This is the first application of mass spectrometric methods for assessing increased avidity of binding in multivalent enantioselective molecular recognition.

Complementary enantioselectivity profiles of chiral cinchonan carbamate selectors with distinct carbamate residues and their implementation in enantioselective two-dimensional high-performance liquid chromatography of amino acids.

A cardinal requirement for effective 2D-HPLC separations is sufficient complementarity in the retention profiles of first and second dimension separations. It is shown that retention and enantioselectivity of chiral selectors derived from cinchona alkaloids can be conveniently modulated by structural variation of the carbamate residue of the quinine/quinidine carbamate ligand of such chiral stationary phases (CSP). A variety of aliphatic and aromatic residues have been tested in comparison to non-carbamoylated quinine CSP. Various measures of orthogonality have been utilized to derive the CSP that is most complementary to the tert-butylcarbamoylated quinine CSP (tBuCQN CSP), which is commercially available as Chiralpak QN-AX column. It turned out that O-9-(2,6-diisopropylphenylcarbamoyl)-modified quinine is most promising in this respect. Its implementation as a complementary CSP for the separation of amino acids derivatized with Sanger's reagent (2,4-dinitrophenylated amino acids) in the first dimension combined with a tBuCQN CSP in the second dimension revealed successful enantiomer separations in a comprehensive chiral×chiral 2D-HPLC setup. However, the degree of complementarity could be greatly enhanced when simultaneously the absolute configurations were exchanged from quinine to quinidine in the chiral selector of the first dimension separation resulting in opposite elution orders of the enantiomers in the two dimensions. The advantage of such a chiral×chiral over achiral×chiral 2D-HPLC setup, amongst others, is the perfect compatibility of the mobile phase because in both dimensions the identical eluent can be used.

Deuterium isotope effects observed during competitive binding chiral recognition electrospray ionization--mass spectrometry of cinchona alkaloid-based systems.

Deuterium isotope effects are reported for binding between tert-butylcarbamoyl-quinine/quinidine chiral selectors and isotopomeric quasienantiomers of N-(3,5-dinitrobenzoyl)leucine measured using electrospray ionization-mass spectrometry (ESI-MS) and competitive binding. Evaluation of mixtures of each selector with one labeled and one unlabeled enantiomeric selectand of identical configuration showed a significant difference in measured ion abundances of diastereomeric complexes between the selector and each selectand. It was found that in some cases, the complex containing the nondeuterated selectand was 15% more abundant than its deuterated counterpart. On the basis of an assessment of solution- and gas-phase isotope effects reported in the literature, a series of control experiments were performed to study the origin of the effects. On the basis of these measurements, our preliminary conclusion is that the differing gas-phase physicochemical nature of the deuterated versus nondeuterated selectand represents the strongest contribution to the observed effect in this chiral molecular recognition system.

Chiral recognition mass spectrometry: remarkable effects observed from the relative ion abundances of ternary diastereomeric complexes using electrospray ionization.

The relative abundances of ternary diastereomeric complexes (composed each from a cinchonane-type chiral selector, a model chiral acid, and an alkali cation) are shown to change remarkably and fortuitously with variation in concentration and type of alkali metal using electrospray ionization-mass spectrometry and competitive binding analysis.

Quantitative profiling of O-glycans by electrospray ionization- and matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry after in-gel derivatization with isotope-coded 1-phenyl-3-methyl-5-pyrazolone.

The potential and benefits of isotope-coded labeling in the context of MS-based glycan profiling are evaluated focusing on the analysis of O-glycans. For this purpose, a derivatization strategy using d0/d5-1-phenyl-3-methyl-5-pyrazolone (PMP) is employed, allowing O-glycan release and derivatization to be achieved in one single step. The paper demonstrates that this release and derivatization reaction can be carried out also in-gel with only marginal loss in sensitivity compared to in-solution derivatization. Such an effective in-gel reaction allows one to extend this release/labeling method also to glycoprotein/glycoform samples pre-separated by gel-electrophoresis without the need of extracting the proteins/digested peptides from the gel. With highly O-glycosylated proteins (e.g. mucins) LODs in the range of 0.4 µg glycoprotein (100 fmol) loaded onto the electrophoresis gel can be attained, with minor glycosylated proteins (like IgAs, FVII, FIX) the LODs were in the range of 80-100 µg (250 pmol-1.5 nmol) glycoprotein loaded onto the gel. As second aspect, the potential of isotope coded labeling as internal standardization strategy for the reliable determination of quantitative glycan profiles via MALDI-MS is investigated. Towards this goal, a number of established and emerging MALDI matrices were tested for PMP-glycan quantitation, and their performance is compared with that of ESI-based measurements. The crystalline matrix 2,6-dihydroxyacetophenone (DHAP) and the ionic liquid matrix N,N-diisopropyl-ethyl-ammonium 2,4,6-trihydroxyacetophenone (DIEA-THAP) showed potential for MALDI-based quantitation of PMP-labeled O-glycans. We also provide a comprehensive overview on the performance of MS-based glycan quantitation approaches by comparing sensitivity, LOD, accuracy and repeatability data obtained with RP-HPLC-ESI-MS, stand-alone nano-ESI-MS with a spray-nozzle chip, and MALDI-MS. Finally, the suitability of the isotope-coded PMP labeling strategy for O-glycan profiling of biological important proteins is demonstrated by comparative analysis of IgA immunoglobulins and two coagulation factors.

Quantitative fingerprinting of O-linked glycans released from proteins using isotopic coded labeling with deuterated 1-phenyl-3-methyl-5-pyrazolone.

Investigation of oligosaccharides attached to proteins as post-translational modification remains an important research field in the area of glycoproteomics as well as in biotechnology. The development of new tools for qualitative and quantitative analysis of glycans has gained high importance in recent years. This is particularly true with O-glycans for which quantitative data are still underrepresented in literature. This fact is probably due to the absence of an enzyme for general release of O-linked saccharides from glycoproteins and due to their low ionization yield in mass spectrometry (MS). In this paper, a method is established aimed at improved qualitative and quantitative analysis of mucin-type O-glycans. A chemical reaction combining release and derivatization of O-glycans in one step is combined here with mass spectrometric quantification. For the purpose of improved quantitative analysis, stable-isotope coded labeling by d0/d5 1-phenyl-3-methyl-5-pyrazolidone (PMP) was performed. The "heavy"-version of this label, penta-deutero (d5)-PMP, was synthesized for this purpose. Beneath improving the reproducibility of quantitation, PMP derivatization contributed to an enhancement of ionization yields in MS. By introducing an internal standard (e.g. GlcNAc3) the reproducibility for quantification can be improved. For higher abundant O-glycans a mean coefficient of variation (CV) less than 6% could be attained, for very low abundant CV values between 15 and 20%. For the determination of O-glycan profiles in mixtures, a HPLC separation was combined with a high resolution Qq-oaTOF instrument. RP-type stationary phases were successful in separating glycan species including some of isomeric ones. This separation step was particularly useful for removing of salts avoiding so the presence of various sodium clusters in the MS spectrum.

Investigations on the chromatographic behavior of hybrid reversed-phase materials containing electron donor-acceptor systems. II. Contribution of pi-pi aromatic interactions.

The chromatographic behavior of three naphthalimide-type stationary phases were elucidated in terms of hydrophobic, silanophilic and pi-pi interaction properties, employing besides common chromatographic column test methods from Engelhardt and Tanaka, also new test mixtures of geometrical and functional aromatic isomers. It was found that the presence of electron donor/acceptor moieties within a reversed phase system did not only increase the overall retention times for aromatic solutes, but also lead to an enhanced shape selectivity of the hybrid stationary phase. In this context, shape discrimination is primarily based on the number of accessible pi-electrons for pi-pi interaction with the embedded electron deficient ligand moieties. The most outstanding results were obtained for the 1,4,5,8-naphthalenediimidic selector with its horizontal arrangement on the silica surface, which enables a direct face-to-face pi-pi interaction with aromatic solutes, with only little hydrophobic contribution.

Liquid chromatographic-mass spectrometric separation of oligoalanine peptide stereoisomers: influence of absolute configuration on enantioselectivity and two-dimensional separation of diastereomers and enantiomers.

This contribution describes the chromatographic separation of peptide stereoisomers. Thereby, one focus is laid on the influence of the absolute configurations of peptide enantiomer pairs on their enantioselective separation. Three different N-terminal protecting groups and three different chiral stationary phases (CSPs) based on cinchona alkaloid derivatives were employed and oligoalanine di-, tri- and tetra-peptides were used as model set. The absolute configurations of the individual enantiomeric pairs were found to profoundly influence both the elution order and the enantioselectivity. The stereoselective molecular recognition mechanism was observed to be dependent on the combination of configuration and the chosen protecting group and CSP. As the CSPs on their own exhibited insufficient diastereoselectivity, a two-dimensional liquid chromatography-mass spectrometry (LC-MS) system was developed for the separation of both diastereomers and enantiomers of peptides in the second part of this study. Diastereomers were separated by reversed phase (RP) and the resulting enantiomeric pair fractions were transferred to a CSP for enantioseparation. All eight stereoisomers of a tripeptide (Ala-Ala-Ala) and 9 out of 10 stereoisomers of a tetrapeptide (Ala-Ala-Ala-Ala) could be successfully resolved.

Towards ochratoxin A selective molecularly imprinted polymers for solid-phase extraction.

Molecularly imprinted polymers (MIPs) displaying selective binding properties for the mycotoxin ochratoxin A (OTA) in polar/protic media were prepared. Crucial to the success of these efforts was the implementation of rationally designed OTA mimics as templates and a set of novel basic and neutral functional monomers, allowing the maximization of the template-functional monomer association via ion-pairing, hydrophobic and steric interactions. MIPs prepared with a 20:1:1:3 molar ratio of cross-linking agent, template mimic, basic functional monomer and hydrophobic auxiliary monomer produced polymers with superior recognition properties compared to materials generated with other stoichiometries. Chromatographic evaluation using the OTA mimics, OTA and a set of structurally closely related compounds as analytes revealed pronounced substrate selectivity of these MIPs in polar/protic media, the templates and OTA being bound with significantly higher affinities. Complementary substrate selectivities/affinities were observed in aprotic and apolar solvents. The possibility of solvent-dependent tuning of substrate selectivity/affinity and the high binding capacity recommend the developed MIPs as promising solid-phase extraction adsorbents for clean-up and pre-concentration of OTA from various biologically relevant matrices.

Novel urea-linked cinchona-calixarene hybrid-type receptors for efficient chromatographic enantiomer separation of carbamate-protected cyclic amino acids.

Two novel diastereomeric cinchona-calixarene hybrid-type receptors (SOs) were synthesized by inter-linking 9-amino(9-deoxy)-quinine (AQN)/9-amino(9-deoxy)-epiquinine (eAQN) and a calix[4]arene scaffold via an urea functional unit. Silica-supported chiral stationary phases (CSPs) derived from these SOs revealed, for N-protected amino acids, complementary chiral recognition profiles in terms of elution order and substrate specificity. The AQN-derived CSP showed narrow-scoped enantioselectivity for open-chained amino acids bearing pi-acidic aromatic protecting groups, preferentially binding the (S)-enantiomers. In contrast, the eAQN congener exhibited broad chiral recognition capacity for open-chained as well as cyclic amino acids, and preferential binding of the (R)-enantiomers. Exceedingly strong retention due to nonenantioselective hydrophobic analyte-calixarene interactions observed with hydro-organic mobile phases could be largely suppressed with organic mobile phases containing small amounts of acetic acid as acidic modifier. With the eAQN-calixarene hybrid-type CSP particularly high levels of enantioselectivity could be achieved for tert-butoxycarbonyl (Boc)-, benzyloxycarbonyl (Z)- and fluorenylmethoxycarbonyl (Fmoc)-protected cyclic amino acids using chloroform as mobile phase, e.g. an enantioselectivty factor alpha >5.0 for Boc-proline. Increasing amounts of acetic acid compromised enantioselectivity, indicating the crucial contributions of hydrogen bonding to chiral recognition. Comparison of the performance characteristics of the urea-linked eAQN-calixarene hybrid-type CSP with those of structurally closely related mutants provided evidence for the active involvement of the urea and calixarene units in the chiral recognition process. The urea linker motif was shown to contribute to analyte binding via multiple hydrogen bonding interactions, while the calixarene module is believed to support stereodiscrimination by enhancing the shape complementarity of the SO binding site.

Molecularly imprinted polymer-assisted sample clean-up of ochratoxin A from red wine: merits and limitations.

A new analytical method for the determination of the carcinogenic mycotoxin ochratoxin A (OTA) in red wines has been developed involving a two-dimensional solid-phase extraction (SPE) clean-up protocol on C18-silica and a target-selective molecularly imprinted polymer (MIP). Prior removal of the interfering acidic matrix compounds by C18 solid-phase extraction was crucial for a successful clean-up as direct sample loading onto the MIP led to poor recoveries. The combined solid-phase extraction protocol afforded extracts suitable for sensitive ochratoxin A quantification by HPLC-fluorescence detection. Preliminary validation of the method performance with spiked (0.033-1.0 ng OTA/ml) and commercial red wines provided recoveries >90% and < 10%, with limit of detection (LOD) and limit of quantification (LOQ) of 0.01 and 0.033 ng/ml. However, a similarly favorable performance characteristics was observed in control experiments in which the MIP was replaced by the corresponding non-imprinted polymer (NIP). These findings provide evidence that under the employed experimental conditions specific analyte binding to imprinted binding sites plays a minor role in selective OTA retention. In the framework of this study, other problems inherent to MIP-based solid-phase extraction have been addressed. These include the reproducible preparation of MIP materials with consistent molecular recognition characteristics, the potential for repeated use of MIP, unfavorable polymer swelling in application-relevant solvents, potential sample contamination by template bleeding, and slow analyte binding kinetics.

A practical method for the quantitative assessment of non-enantioselective versus enantioselective interactions encountered in liquid chromatography on brush-type chiral stationary phase.

A convenient experimental method for the quantitative assessment of enantioselective versus non-enantioselective interactions in liquid chromatography on brush-type chiral stationary phases (CSPs) is described. This procedure involves the systematic evaluation of the retention characteristics of resolved enantiomers as chiral selectands, SAs, on a set of CSPs containing chiral selectors, SOs, attached to an achiral support S with well-defined but different surface SO loading levels. The emerging body of retention data can be dissected into non-enantioselective and enantioselective increments by equations accounting separately for the two co-existing equilibrium processes, namely the "unproductive" adsorption of the enantiomers on the achiral domains of the CSP and their "productive" enantioselective association with the surface-anchored chiral SOs. The general applicability of this approach was demonstrated using a set of CSPs loaded with different densities of a quinine carbamate anion exchange-type SO, and three acidic model SAs, i.e., N-Fmoc-phenylalanine, N-[(3,5-dipropoxybenzyloxy)carbonyl]leucine and 2-(2,4-dichlorophenoxy)propanoic acid, employing nonpolar, polar organic and reversed phase mobile phase conditions. In all tested mobile phase environments, the global retention characteristics of the studied SO was found to be dominated by enantioselective SA/SO binding rather than by non-enantioselective SA/support interactions, providing evidence for chiral recognition processes primarily capitalizing on strong electrostatic intermolecular binding forces. For the investigated compounds, the polar organic mode was identified as the most favorable mobile phase scenario, producing the highest apparent enantioseparation factors, α(app), in combination with the lowest degree of non-enantioselective adsorption. It is anticipated that the described approach toward the deconvolution of non-enantioselective versus enantioselective interactions will also be applicable to other types of brush-type CSPs. Most important, the quantitative information on the inherent thermodynamics of SO-SA interactions emerging from these studies will permit an unambiguous interpretation of the changes of the chiral recognition characteristics occurring as a consequence of mobile phase modifications. We are confident that this knowledge will be helpful in the design and evaluation of new non-invasive surface and immobilization chemistries, and will also provide valuable guidance for the optimization of operation conditions for large scale preparative enantiomer separations. Finally, it should be stressed that the advanced strategy for distinguishing enantioselective from non-enantioselective contributions has general applicability to all liquid phase-based enantioseparation techniques, well beyond the realm of chromatography, such as liquid-liquid extraction and batch-binding processes.