Streaming potential in open and packed fused-silica capillaries

The surface properties of novel stationary phases in packed and open tubular columns for capillary electrochromatography (CEC) were examined by measuring the streaming potential in a home made apparatus. The surfaces investigated include materials such as porous styrenic sorbents and octadecyl-silica as well as fused-silica tubing, in both raw and surface modified forms. Functionalization of the surface was carried out, for instance, by reductive amination or organosilane grafting on to capillary inner wall. The dependence of the streaming potential on pH was examined with aqueous solutions in the pH range from 2.5 to 9.0. Electrokinetic properties of 50 microm I.D. fused-silica capillaries have been determined by both streaming potential and electrosmotic flow measurements. Both methods gave similar pH profiles of the zeta-potential and the isoelectric points. This confirms the viability of our approach to evaluate the specific charged groups of the packing which is one of the important factors influencing electrosmotic flow (EOF) velocity and protein adsorption during a chromatographic run. In addition to bare silica capillaries, styrenic monolithic columns with different surface functionalities, which have been extensively used in our laboratory for CEC separation of peptides and proteins, were employed for comparison of two methods. Plots of zeta potential as a function of percent ACN show a complex behavior, indicating that zeta potential cannot be predicted simply from binary mixture solvent properties. It is demonstrated that the evaluation of the zeta potential by the streaming potential method is nondestructive, relatively fast, without untoward effects introduced by Joule heating and yet another means for the characterization of the surfaces under conditions employed in CEC.     

Spacer-modified stationary phases for anion chromatography: Alkyl- and carbonylalkylspacers – A comparison

A comparison of two separation columns for high-capacity anion chromatography is presented. The distinctive feature of both packing materials is the structure of the alkyl-chain (spacer-group) between the polymer-backbone and the functional group. All other parameters, e.g. exchange capacity, functionality and length of the spacer-arm, are identical. The retention behavior of the so-called standard-anions is investigated on both columns under identical chromatographic conditions using an experimental design technique in the course of which the composition of the elution system applied is varied. The statistical treatment of the retention data offers the possibility to explain the different chromatographic behavior of both stationary phases, at least qualitatively.     

Improvement of the chromatographic separation performance of an imidazolium ionic liquid functionalized silica column by in situ anion‐exchange with dodecyl sulfonate and dodecylbenzene sulfonate anions

The anionic part of ionic liquids can provide additional interactions during chromatographic separations. In this work, the chromatographic separation performance of a silica column functionalized with 1‐propyl‐3‐methylimidazolium chloride ionic liquid was improved by in situ anion‐exchange from chloride anions to dodecyl sulfonate anions and dodecylbenzene sulfonate anions. The separation performances of these ionic liquid functionalized phases were investigated and compared with each other using polycyclic aromatic hydrocarbons, phthalates, parabens, and phenols as model compounds. Results indicated that the new columns presented a better chromatographic separation than the original one. This was ascribed retention mechanism from organic anions. The introduction of dodecyl sulfonate anions increased the hydrophobicity of stationary phase. Furthermore, the phenyl groups of dodecylbenzene sulfonate anions could provide an enhanced selectivity to aromatic compounds such as polycyclic aromatic hydrocarbons by π–π interactions. Analysis repeatability of the new columns was satisfactory (RSD of retention time, 0.10–0.40%; RSD of peak area, 0.66–0.84%).     

Mobile-phase pH influence on the retention of some benzoic acid derivatives in reversed-phase chromatography

Five end-capped octadecyl RP stationary phases, among which one was a polar embedded stationary phase, were tested for the analysis of benzoic acid derivatives using two mobile phases with or without addition of formic acid (water pH was measured by a common approach; pH of water with addition of formic acid was 3.0 and without formic acid 5.8). The influence of mobile-phase pH on the retention of benzoic acid derivatives was under study. Consequently, Purospher-STAR and Alltima columns provided symmetrical peaks for benzoic acid derivatives at pH 3.0 and also at pH 5.8. Reprosil and Symmetry stationary phases showed poor peak shapes at higher pH of the mobile phase. Differences between the tested columns may be caused by surface heterogeneity. Another reason may be the presence of some atoms creating additional adsorption sites on the surface of Reprosil and Symmetry stationary phases. This can lead to enhanced silanol activity resulting in peak tailing. The addition of formic acid into the mobile phase improved peak shapes. The polar embedded C18 stationary-phase Synergi-Fusion-RP appeared as not a suitable column for the analysis of benzoic acid derivatives. Synergi-Fusion-RP provided asymmetrical peaks even if formic acid was added into the mobile phase.     

Spacer-modified stationary phases for anion chromatography: alkyl- and carbonylalkylspacers--a comparison

A comparison of two separation columns for high-capacity anion chromatography is presented. The distinctive feature of both packing materials is the structure of the alkyl-chain (spacer-group) between the polymer-backbone and the functional group. All other parameters, e.g. exchange capacity, functionality and length of the spacer-arm, are identical. The retention behavior of the so-called standard-anions is investigated on both columns under identical chromatographic conditions using an experimental design technique in the course of which the composition of the elution system applied is varied. The statistical treatment of the retention data offers the possibility to explain the different chromatographic behavior of both stationary phases, at least qualitatively.     

Controllable preparation of a hydrophilic/ion‐exchange mixed‐mode stationary phase by surface‐initiated atom transfer radical polymerization using a mixture of two functional monomers

Mixed‐mode chromatographic stationary phases require functionalization with at least two functional groups to yield multiple interactions with analytes. Departing from reported methods, a mixture of two different monomers, glycidyl methacrylate and 2‐dimethylaminoethylmethacrylate, was grafted onto the surface of silica by a one‐step surface‐initiated atom transfer radical polymerization to prepare a novel hydrophilic interaction/anion‐exchange mixed‐mode chromatographic stationary phase. The grafted amounts of functional groups were controlled via varying the ratio of monomers in the polymerization system. The influences of water content, salt concentration and pH in the mobile phase were investigated to illustrate the mixed interaction between the stationary phase and analytes. The retention of various solutes on three columns, especially acidic and basic solutes, showed an obvious dependence on the ratio of the two monomers in the polymerization system. The results indicated that the strategy proposed in this work was beneficial to develop various types of mixed‐mode chromatographic stationary phases with adjustable selectivity to meet the needs of complex samples. Finally, the column was successfully employed in the isolation of melamine in liquid milk.     

Solid-phase extraction (SPE) assays to ascertain the mechanisms of retention of antimony species in several stationary phases

Liquid chromatography is the most suitable technique for antimony speciation in several types of samples. However, efficiency can be poor for some of these peaks, especially Sb(III) and Me₃SbCl₂ (TMSb). Weak and strong anion exchange stationary phases are mainly used for antimony speciation in several chromatographic conditions. The present study examines the possible contribution of the interaction between antimony species (Sb(III), Sb(V) and TMSb) and stationary phase support to the overall retention mechanism in their chromatographic separation. Several SPE cartridges, selected from those mainly used as support in anion exchange columns, were assayed. Sb (V) was quantitatively eluted from the PSDVB (polystyrene divinylbenzene) and SiO₂ phases, showing the absence of interaction. Sb (III) showed some interaction with the PSDVB phase; TMSb showed strong retention with all the cartridges studied and it was only eluted from the PSDVB phase.     

Investigation of polar stationary phases for the separation of sympathomimetic drugs with nano-liquid chromatography in hydrophilic interaction liquid chromatography mode

In this study, the retention and selectivity of a mixture of basic polar drugs were investigated in hydrophilic interaction chromatographic conditions (HILIC) using nano-liquid chromatography (nano-LC). Six sympathomimetic drugs including ephedrine, norephedrine, synephrine, epinephrine, norepinephrine and norphenylephrine were separated by changing experimental parameters such as stationary phase, acetonitrile (ACN) content, buffer pH and concentration, column temperature. Four polar stationary phases (i.e. cyano-, diol-, aminopropyl-silica and Luna HILIC, a cross-linked diol phase) were selected and packed into fused silica capillary columns of 100 μm internal diameter (i.d.). Among the four stationary phases investigated a complete separation of the all studied compounds was achieved with aminopropyl silica and Luna HILIC stationary phases only. Best chromatographic results were obtained employing a mobile phase composed by ACN/water (92/8, v/v) containing 10 mM ammonium formate buffer pH 3. The influence of the capillary temperature on the resolution of the polar basic drugs was investigated in the range between 10 and 50 °C. Linear correlation of ln k vs. 1/T was observed for all the columns; ΔH° values were negative with Luna HILIC and positive with aminopropyl- and diol-silica stationary phases, demonstrating that different mechanisms were involved in the separation.To compare the chromatographic performance of the different columns, Van Deemter curves were also investigated.     

Preparation and evaluation of n-octadecylphosphonic acid-modified magnesia-zirconia stationary phases for reversed-phase liquid chromatography

Three n-octadecylphosphonic acid-modified magnesia-zirconia reversed stationary phases (C₁₈PZM) are prepared via the strong Lewis base interactions between organophosphonate and magnesia-zirconia composite. And two of them are end-capped by using trimethylchlorosilane as end-capping agent in different procedures. Stability studies at extreme high pH conditions (pH 9-12) show that both the non-endcapped and endcapped columns are quite stable at pH 12 mobile phase. The reversed-phase liquid chromatographic behavior of three C₁₈PZM stationary phases are comparatively investigated in detail using a variety of basic compounds as probes. The retention of basic compounds on the three phases is studied over a wide range of pHs. And the possible retention mechanisms of basic compounds on the three stationary phases are discussed. The results show that the basic solutes retain by a hydrophobic and cation-exchange interaction mixed mechanism on three stationary phases when they are operated in eluents at pH values near to the pK_a of the Brönsted conjugate acid form of the analyte, suggesting that inherent zirconol groups on ZM are not expected to interact with bases via cation-exchange interaction at lower pH. Nonetheless, the non-endcapped phase differs markedly from the edncapped ones in retention and selectivity of basic solutes using eluents at pH 4.1, implying a complex retention mechanism at this pH. The cation-exchange sites under such conditions are more likely due to the adsorbed Lewis base anionic buffer constituents (acetate) on accessible ZM surface sites than the chemisorbed phosphonate. Although the three phases exhibit very similar chromatographic behavior with eluents at pH 10.1, and show in general satisfactory separation of basic compounds and alkaloids studied, the performance for a specific analyte, however, differs largely from column to column.     

Retention characteristics of a new butylimidazolium-based stationary phase. Part II: anion exchange and partitioning

A surface-confined ionic liquid (SCIL) and a commercial quaternary amine silica-based stationary phase were characterized employing the linear solvation energy relationship (LSER) method in binary methanol/water mobile phases. The retention properties of the stationary phases were evaluated in terms of intermolecular interactions between 28 test solutes and the stationary phases. The comparison reveals a difference in the hydrophobic and hydrogen bond acceptance interaction properties between the two phases. The anion exchange retention mechanism of the SCIL phase was demonstrated using nucleotides. The utility of the SCIL phase in predicting logk _IL/water values by chromatographic methods is also discussed.     

Comparison of Reversed Stationary Phases for the Chromatographic Separation of Inorganic Analytes Using Hydrophobic Ion Mobile Phase Additives

Abstract

Alkyl-modified silica (RSi) and polystyrenedivinylbenzene (PRP-1) stationary phases are compared for the chromatographic separation of inorganic analyte anions and cations using hydro-phobic ions of opposite charge as mobile phase additives. Tetra-alkylammonium salts were used for anion separations and alkyl sulfonate salts for cation separations. Two major equilibria influence the retention of analyte ions on PRP-1. These are: retention of the hydrophobic ion on PRP-1 and an ion exchange selectivity between the hydrophobic counterion and the analyte ion. When using RSi retention is also influenced by ion exchange at residual silanol groups, which act as weak cation exchange sites. Mobile and stationary phase variables that influence analyte retention are identified. Optimization of these provides favorable eluting conditions for the separation of inorganic ionic analytes. Of particular interest is the potential use of PRP-1 and RSi columns for the separation of inorganic cations; conditions for the separation of alkali metals and alkaline earths are discussed.     

Relationships between LC retention, octanol-water partition coefficient, and fungistatic properties of 2-(2,4-dihydroxyphenyl)benzothiazoles

The retention behavior of newly synthesized compounds with antimycotic activity from the 2-(2,4-dihydroxyphenyl)benzothiazole group by high-performance liquid chromatography has been investigated. RP-18 stationary phase and methanol-acetate buffer aqueous mobile phases at pH 4 and 7.4 have been used. In the case of the mobile phase at pH 7.4, higher concentrations of water can be applied than at pH 4. The studied compounds showed regular retention behavior, their log k values decreasing linearly with an increasing concentration of methanol in the mobile phase. On the basis of these relationships, the lipophilicity (log kw), specific hydrophobic surface area (S), and isocratic chromatographic hydrophobicity index (psi0) were determined. Similar log kw values and sensitivity to changes in the structure of compounds studied for both mobile phases have been found. Moderate correlations between the chromatographic parameters and the calculated octanol-water log P values were found. Finally, the lipophilicity parameters were compared with the fungistatic properties of compounds expressed by log MIC (minimum inhibitory concentration) values to find quantitative structure activity relationship equations.     

Preparation and evaluation of diblock copolymer‐grafted silica by sequential surface initiated‐atom transfer radical polymerization for reverse‐phase/ion‐exchange mixed‐mode chromatography

A novel approach that involved the grafting of diblock copolymer with two types of monomer onto substrate by sequential surface initiated‐atom transfer radical polymerization was proposed to prepare a mixed‐mode chromatographic stationary phase. The distinguishing feature of this method is that it can be applied in the preparation of various mixed‐mode stationary phases. In this study, a new reverse‐phase/ion‐exchange stationary phase was prepared by grafting hydrophobic styrene and cationic sodium 4‐styrenesulfonate by the proposed approach onto silica surface. The chromatographic properties of the prepared stationary phase were evaluated by the separation of benzene derivatives, anilines, and β‐agonists, and by the effect of pH values and acetonitrile content on the retention. Compared with typical RP columns, the prepared stationary phase achieved the better resolution and higher selectivity at a shorter separation time and lower organic content. Moreover, the application of the prepared column was proved by separating widely distributed polar and charged compounds simultaneously.     

Chromatographic properties of zirconia-based stationary phases for ion exchange chromatography having surface bound cationic functions

A series of non-porous, microspherical zirconia-based stationary phases with surface bound cationic functions have been introduced and evaluated in ion exchange chromatography of proteins and small acidic solutes. Different surface modification procedures were evaluated in the covalent attachment of weak, strong or hybrid anion exchange moieties on the surface of non-porous zirconia micropar-ticles. N,N-Diethylaminoethanol (DEAE) was used as the weak anion exchange ligand while glycidyltrimethylammonium chloride, which was covalently attached to poly(vinyl alcohol) layer (PVAN) on the zirconia surface, constituted the strong anion exchange moiety. Partially quaternarized poly(ethyleneimine) hydroxyethylated (PEI) was used as the hybrid type of anion exchange coating. DEAE-zir-conia microparticles acted as purely cation exchange stationary phases toward basic proteins indicating the predominance of electron donor-electron acceptor interaction (EDA) with surface exposed zirconium sites as well as cation exchange mechanism via electrostatic interaction with unreacted and unshielded hydroxyl groups. PVAN-zirconia stationary phase exhibited anion exchange chromatographic properties toward acidic proteins, but EDA interaction has stayed as an important contributor to solute retention despite the presence of a relatively thick layer of poly(vinyl alcohol) on the surface of the zirconia particles. The modification of zirconia surface with partially quaternarized PEI proved to be the most effective approach to minimize Lewis acidic metallic properties of the support. In fact, PEI-zirconia stationary phase operated as an anion exchanger toward acidic proteins and other small acidic solutes.     

Optimization of the mobile phase conditions for the purification ofTaql endonuclease

Summary Taql endonuclease was purified by high performance ion exchange liquid chromatography with linear gradient elution. Of the chromatographic media tested as mobile phases for the HPLC purification ofTaql endonuclease, sodium acetate (pH 5.0) and phosphate (pH 7.0) buffers were appropriate for use with cation exchange columns, and L-histidine (pH 6.0) and Tris (pH 8.0) buffers with anion exchange columns.     

Lipophilicity assessment of basic drugs (log P(o/w) determination) by a chromatographic method

A previously reported chromatographic method to determine the 1-octanol/water partition coefficient (log P(o/w)) of organic compounds is used to estimate the hydrophobicity of bases, mainly commercial drugs with diverse chemical nature and pK(a) values higher than 9. For that reason, mobile phases buffered at high pH to avoid the ionization of the solutes and three different columns (Phenomenex Gemini NX, Waters XTerra RP-18 and Waters XTerra MS C(18)) with appropriate alkaline-resistant stationary phases have been used. Non-ionizable substances studied in previous works were also included in the set of compounds to evaluate the consistency of the method. The results showed that all the columns provide good estimations of the log P(o/w) for most of the compounds included in this study. The Gemini NX column has been selected to calculate log P(o/w) values of the set of studied drugs, and really good correlations between the determined log P(o/w) values and those considered as reference were obtained, proving the ability of the procedure for the lipophilicity assessment of bioactive compounds with very different structures and functionalities.     

Reversed Phase Chromatography – the Mystery of Surface Silanols

The nature of surface silanols is reviewed and their influence on retention of basic solutes in reversed phase chromatography is demonstrated and evaluated. The influence of the type of organic modifier and/or pH of buffer on retention of basic analytes with silica based RP columns is evaluated, and means for characterization of RP columns are discussed. Attempts are described to determine the surface silanol concentration of RP columns by chromatographic methods. Surprisingly the experimentally measured silanol concentrations with bare silica and with RP are by one order of magnitude lower than discussed in literature. At pH 7.6 values of 0.12 µmol m^?2 have been measured for benzylamine with RP columns, corresponding to about 43% of the measurable silanols of plain silica. The break-through curves of amines let suggest that even at low pH values unprotonated species are present within the pores of the RP stationary phases and their interaction with the bonded alkyl groups contribute to retention.