Analysis of polar peptides using a silica hydride column and high aqueous content mobile phases

The retention behavior of a set of polar peptides separated on a silica hydride stationary phase was examined with a capillary HPLC system coupled to ESI‐MS detection. The mobile phases consisted of formic acid or acetic acid/acetonitrile/water mixtures with the acetonitrile content ranging from 5 to 80% v/v. The effects on peptide retention of these two acidic buffer additives and their concentrations in the mobile phase were systematically investigated. Strong retention of the peptides on the silica hydride phase was observed with relatively high‐organic low‐aqueous mobile phases (i.e. under aqueous normal‐phase conditions). However, when low concentrations of acetic acid were employed as the buffer additive, strong retention of the peptides was also observed even when high aqueous content mobile phases were employed. This unique feature of the stationary phase therefore provides an opportunity for chromatographic analysis of polar peptides with water‐rich eluents, a feature usually not feasible with traditional RP sorbents, and thus under conditions more compatible with analytical green chemistry criteria. In addition, both isocratic and gradient elution procedures can be employed to optimize peptide separations with excellent reproducibility and resolution under these high aqueous mobile phase conditions with this silica hydride stationary phase.     

Synthesis and evaluation of silica hydride‐based fluorinated stationary phases

Two novel silica hydride‐based fluorinated bonded phases have been synthesized using a hydrosilation procedure to test combined fluorine and hydride selectivity. The bonded moieties were characterized by elemental and spectral analysis. Chromatographic testing was done using hydrophilic analytes in the aqueous normal phase mode. At higher amounts of the nonpolar solvent in the mobile phase, there should be increased retention for solutes such as acids, bases and other polar compounds, whereas nonpolar solutes can be retained when water is increased as in RP chromatography. The synergistic effects of the fluorinated phase selectivity and aqueous normal phase retention on a hydride surface have been explored for small polar molecules. The stability and repeatability of the hydride‐based fluorinated stationary phases were evaluated. The use of acetone as the organic component in the mobile phase was also tested.     

HPLC retention behavior on hydride-based stationary phases

Two stationary phases attached to a silica hydride surface, cholesterol and bidentate C18, are investigated with a number of pharmaceutically related compounds in order to illustrate the various retention mechanisms that are possible for these bonded materials. The test solutes range from hydrophilic to hydrophobic based on log P (octanol/water partition coefficient) and pKa values. The mobile phases consist of acidified (formic and perchloric acid) water/methanol or water/ACN mixtures. Of particular interest are the high organic content mobile phase compositions where the retention would increase if the bonded material was operating in the aqueous normal phase (ANP) mode. Plots of retention factor (k) versus mobile phase composition are used to elucidate the retention mechanism. A number of examples are presented where solutes are retained based on RP, ANP, or dual retention mechanisms. The silica hydride-based stationary phases can also retain compounds in the organic normal phase.     

Investigation of a novel mixed-mode stationary phase for capillary electrochromatography. Part III: Separation of nucleosides and nucleic acid bases on sulfonated naphthalimido-modified silyl silica gel

Capillary electrochromatography (CEC) with a novel stationary phase, 3-(4-sulfo-1,8-naphthalimido)propyl-modified silyl silica gel (SNAIP), proved useful for the separation of nucleosides and nucleic acid bases. The application scope of SNAIP, which is a relatively polar reversed-phase (RP)-type stationary phase, was successfully expanded to include the CEC separation of polar compounds although the combination of non-polar RP phase with highly aqueous mobile phase is often inadequate. Due to the permanently charged sulfonic acid groups and the naphthalimidopropyl moiety, the retention of charged and relatively polar nucleosides as well as bases on the SNAIP stationary phase was effected by electrostatic and hydrophobic interactions. This yielded a unique selectivity on SNAIP toward nucleosides and bases. The characteristic EOF on SNAIP, which was stronger at higher aqueous content in the mobile phase, proved suitable for the separation of polar compounds in reversed-phase mode with highly aqueous mobile phase. In addition, when a double stepwise gradient was employed to accelerate the latest peak (adenine), the elution time was shortened to less than half its original duration.     

Selectivity differences between alkyl and polar‐modified alkyl phases in reversed phase high performance liquid chromatography

Compared to conventional C18 phases, polar‐modified phases have distinct differences with regards to chromatographic behavior. In the present study, ODS phases and polar‐modified phases were synthesized. The columns containing these new packings demonstrated satisfactory stability under both acidic (pH 1.5) and basic (pH 10) conditions. We evaluated the selectivity differences between alkyl and polar‐modified alkyl RP columns by using a range of neutral analytes. The polar‐modified alkyl phases showed excellent peak shapes for almost all compounds. We also compared the selectivity differences between them for separating nucleotides by using 100% aqueous mobile phase and tricyclic antidepressants in the intermediate pH mobile phases. The results demonstrated that polar‐modified phases display a significantly reduced hydrophobic nature and a significantly reduced silanol activity compared to the conventional C18 phases.     

The use of derivatized cyclodextrins as solubilizing agents in the preparation of macroporous polymers employed as amphiphilic continuous beds in capillary electrochromatography

Employing solubilization by complexation with CDs, new mixed-mode monolithic stationary phases for CEC and micro-LC were synthesized. Free radical copolymerization was performed in aqueous solution with a CD-solubilized hydrophobic monomer, a water-soluble crosslinker (piperazinediacrylamide), and a charged monomer (vinylsulfonic acid). Different hydrophobic methacrylate monomers (isobornyl, adamantyl, cyclohexyl, and phenyl methacrylate) were investigated. Chromatographic properties of the synthesized monoliths were studied with aqueous and nonaqueous mobile phases with hydrophobic and polar analytes. Due to the amphiphilic nature of the polymers synthesized, the elution orders obtained correspond to the RP mode and to the normal-phase mode dependent on the polarity of the mobile phase. However, observations made with polar solutes and polar mobile phase can only be explained by a mixed-mode retention mechanism. The influence of the total monomer concentration (%T) on the chromatographic properties and on the specific permeability was elucidated. Run-to-run, day-to-day, and capillary-to-capillary reproducibility of electroosmotic mobility and retention factors were determined. Comparison of retention data with those of a commercial octadecyl silica gel HPLC column reveals that the methylene selectivity of the monolithic capillaries prepared in this study is very similar to that of routinely used octadecyl silica gels.     

Comparison of retention on traditional alkyl, polar endcapped, and polar embedded group stationary phases

The development of new RP stationary phases containing polar groups has provided the chromatographer with a variety of stationary phase choices with differing selectivities. Polar endcapped and polar embedded group stationary phases have found use in solving a wide variety of separation problems, especially for the efficient separation of organic bases as well as separations necessitating the use of highly aqueous mobile phases. In this report, the retention thermodynamics of small, nonpolar solutes on traditional alkyl, polar endcapped, and polar embedded group stationary phases are compared. It is found that the nonpolar (methylene) transfer enthalpy is less favorable when polar embedded group phases are used, when compared to traditional or polar endcapped phases. In contrast, the transfer enthalpy of a phenyl group is found to be more favorable when a polar endcapped phase is used. In addition, the retention characteristics of these phases are compared using a set of solutes with differing solvatochromic parameters. Hydrogen-bond acids appear to have enhanced retention on polar embedded group phases, while hydrogen-bond bases have enhanced retention on polar endcapped phases.     

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.     

Selectivity of calixarene‐bonded silica phases in HPLC: Description of special characteristics with a multiple term linear equation at different methanol concentrations

Retention and selectivity characteristics of different calixarene‐, resorcinarene‐ and alkyl‐bonded stationary phases are examined by analyzing a set of test solutes covering the main interactions (hydrophobic, steric, ionic, polar) that apply in HPLC. Therefore Dolan and Snyder's multiple term linear equation has been adapted to fit the properties of calixarene‐bonded columns. The obtained parameters are used to describe retention and selectivity of the novel Caltrex^® phases and to elucidate underlying mechanisms of retention. Here, differences of stationary phase characteristics at different methanol concentrations in the mobile phases are examined. Both selectivity and retention were found to depend on the methanol content. Differences of these dependencies were found for different stationary phases and interactions. The differences between common alkyl‐bonded and novel calixarene‐bonded phases increase with increasing methanol content.     

Histidine‐modified organic‐silica hybrid monolithic column for mixed‐mode per aqueous and ion‐exchange capillary electrochromatography

A novel organic‐silica hybrid monolith was prepared through the binding of histidine onto the surface of monolithic matrix for mixed‐mode per aqueous and ion‐exchange capillary electrochromatography. The imidazolium and amino groups on the surface of the monolithic stationary phase were used to generate an anodic electro‐osmotic flow as well as to provide electrostatic interaction sites for the charged compounds at low pH. Typical per aqueous chromatographic behavior was observed in water‐rich mobile phases. Various polar and hydrophilic analytes were selected to evaluate the characteristics and chromatographic performance of the obtained monolith. Under per aqueous conditions, the mixed‐mode mechanism of hydrophobic and ion‐exchange interactions was observed and the resultant monolithic column proved to be very versatile for the efficient separations of these polar and hydrophilic compounds (including amides, nucleosides and nucleotide bases, benzoic acid derivatives, and amino acids) in highly aqueous mobile phases. The successful applications suggested that the histidine‐modified organic‐silica hybrid monolithic column could offer a wide range of retention behaviors and flexible selectivities toward polar and hydrophilic compounds.     

Separation behavior of basic compounds on unbonded silicon oxynitride and silica high‐performance liquid chromatography stationary phases with reversed‐phase eluents

Unbonded silicon oxynitride and silica high‐performance liquid chromatography stationary phases have been evaluated and compared for the separation of basic compounds of differing molecular weight, pK_a, and log D using aqueous/organic mobile phases. The influences of percentage of organic modifier, buffer pH, and concentration in the mobile phase on base retention were investigated on unbonded silicon oxynitride and silica phases. The results confirmed that unbonded silicon oxynitride and silica phases demonstrated excellent separation performance for model basic compounds and both the unbonded phases examined possessed a hydrophobic/adsorption and ion‐exchange character. The silicon oxynitride stationary phase exhibited high hydrophilicity compared with silica with a reversed‐phase mobile phase. An ion‐exclusion‐type mechanism becomes predominant for the separation of three aimed bases on the silicon oxynitride column at pH 2.8. Different from silicon oxynitride stationary phase, no obvious change for the retention time of three model bases on silica stationary phase at pH 2.8 can be observed.     

Dual Retention Mechanism in Two-Dimensional LC Separations of Barbiturates,Sulfonamides, Nucleic Bases and Nucleosides on Polymethacrylate Zwitterionic Monolithic Micro-Columns

In-house prepared zwitterionic polymethacrylate micro-columns using in situ polymerization of N,N-dimethyl-N-metacryloxyethyl-N-(3-sulfopropyl) ammonium betaine (MEDSA) functional monomer with bisphenol A glycerolate dimethacrylate (BIGDMA) cross-linker provided excellent stability and reproducibility of preparation and separation efficiency of 60,000–70,000 theoretical plates m⁻¹ for small molecules under isocratic conditions. The column showed a dual retention mechanism, reversed-phase (RP) in highly aqueous mobile phases and aqueous normal-phase (HILIC) in acetonitrile-rich mobile phases. This property can be used to obtain complementary separation and combined information on the sample from repeated injections of a sample on a single column, in different mobile phases characteristic for the HILIC and for the RP modes, which is in fact a form of offline two-dimensional chromatography on a single column. The dual retention mechanism has been observed with a variety of columns, however, often with impractically narrow retention range in one of the two modes. To take full advantage from the combined single-column RP–HILIC experiments, the column should provide a sufficiently broad mobile phase interval both in the RP and in the HILIC mode. The BIGDMA-MEDSA micro-columns proved suitable earlier for the combined RP–HILIC separations of some phenolic compounds and flavonoids. In the present work, we investigated the effects of the mobile phase composition on the retention of a variety of polar compounds over full retention range of buffered aqueous acetonitrile mobile phases, to find potentially useful HILIC and RP retention ranges for barbiturates, sulfonamides, nucleosides and nucleic bases. In the HILIC mode, proton donor–acceptor interactions show a major effect on retention and selectivity of separation, whereas the size of the non-polar hydrocarbon part of the sample molecule is the most important factor in the water-rich mobile phases. The sample structure strongly affects the composition of aqueous–organic mobile phases at which the transition between the two retention modes occurs. Of the investigated sample types, barbiturates show better separation under reversed-phase conditions, whereas nucleosides and nucleic bases in the HILIC mode. Aromatic carboxylic acids and sulfonamides can be separated either in the reversed phase or under HILIC conditions, the two separation modes showing complementary selectivity of separation.

     

Zirconia—A stationary phase capable of the separation of polar markers of myocardial metabolism in hydrophilic interaction chromatography

Creatine, phosphocreatine, and adenine nucleotides are highly polar markers of myocardial metabolism that are poorly retained on RP silica sorbents. Zirconia represents an alternative material to silica with high promise to be used in hydrophilic interaction chromatography (HILIC). This study describes a first systematic investigation of the ability of ZrO₂ to separate creatine, phosphocreatine, adenosine 5′‐monophosphate, adenosine 5′‐diphosphate, and adenosine 5′‐triphosphate and compares the results with those obtained on TiO₂. All analytes showed a HILIC‐like retention pattern when mobile phases of different strengths were tested. Stronger retention and better column performance were achieved in organic‐rich mobile phases as compared to aqueous conditions, where poor retention and insufficient column performance were observed. The effect of mobile phase pH and ionic strength was evaluated as well. The analysis of myocardial tissue demonstrated that all compounds were separated in a relevant biological material and thus proved ZrO₂ as a promising phase for HILIC of biological samples that deserves further investigation.     

HPLC separation of dihydropyridine derivatives enantiomers with emphasis on elution order using polysaccharide‐based chiral columns

The separation of enantiomers of five chiral dihydropyridine derivatives was studied on five different polysaccharide‐based chiral HPLC columns with various normal‐phase (NP), polar organic, and reversed‐phase eluents. Along with the successful separation of analyte enantiomers, the emphasis of this study was on enantiomer elution order (EEO) with various columns and mobile phase composition. The interesting phenomenon of reversal of EEO, recently reported in the case of amlodipine (AML) depending on the concentration of formic acid in acetonitrile, was also confirmed with NP eluents. Under RP conditions at relatively low water content, the EEO of AML could also be reverted by varying the concentration of formic acid in the mobile phase. However, at higher water content the same parameter did not affect the EEO, but only induced gradual decrease in resolution up to complete co‐elution of enantiomers. Additionally, in organic‐aqueous mobile phases retention factors decreased with increasing water content but only up to 20% (v/v), while above this concentration the expected typical RP behavior was observed. The presence of the commonly used additive diethylamine in the mobile phase seems important for observing a reversal in EEO with increasing concentration of formic acid. The reversal of the EEO was characteristic of AML only and was not observed for any of other dihydropyridines included in this study.     

Studies on the chromatographic behavior of nucleosides and bases on p-tert-butyl-calix[8]arene-bonded silica gel stationary phase by HPLC

The chromatographic behavior of some nucleosides, pyrimidines and purines on a new p-tert-butyl-calix[8]arene-bonded silica gel stationary phase (CABS) were studied by high performance liquid chromatography. Their retention behavior on CABS were compared with those on ODS. The influence of mobile phase variables, such as methanol content, pH and ionic strength on the retention behavior were studied. Some nucleosides, pyrimidines and purines on CABS were successfully separated. The results show that the calix[8]arene-bonded phase exhibits high selectivities for the above analytes in high aqueous mobile phases. According to the chromatographic data, it is indicated that hydrophobic interaction, hydrogen-bonding interaction, and dipole-dipole interaction are mainly responsible for the retention behavior. In addition, in some extent, the vertical stacking action of the analytes on CABS can also change the retention behavior. CABS was superior to ODS in the routine fast separation of nucleosides and bases.     

Characterization of calixarene‐bonded stationary phases

Calixarene‐bonded stationary phases received growing interest in HPLC as stationary phases with special retention characteristics and selectivity. The commercially available unsubstituted and p‐tert‐butyl‐substituted Caltrex^® columns have been intensively studied and characterized in our workgroup. They can be used as reversed phases, yet they support additional interactions. Especially, their steric, polar and ionic properties differ from conventional alkyl‐bonded phases. However, also the hydrophobic interaction shows differences since adsorption and partition interactions on or in a bonded layer of calixarenes are not similar to those of alkyl‐bonded layers. The relative strength of the hydrophobic properties of the stationary phases has been found depending on the methanol concentration of the mobile phase. Generally, the dependencies of their interaction strengths on mobile‐phase conditions, e.g. the change of the intensity of the hydrogen‐bonding abilities with decreasing methanol content, are not similar from phase to phase either. This probably gives calixarene‐bonded stationary phases enhanced suitability for analyses at extreme compositions of the mobile phase. An overview about the synthesis, retention and selectivity properties of Caltrex^® columns is given here.     

Retention mechanism of the multifunctional solute on columns with different coverage densities using highly aqueous reversed‐phase conditions

A series of 11 homemade octadecyl bonded phases with different coverage densities were tested to determine the influence of the stationary phase on the retention in highly aqueous mobile phases. The concentrations of the organic modifiers (methanol and ACN) were in the range of 0–20%_v/v. The coverage density of bonded ligands and the presence of the end‐capping have strong influence on the solute retention. Amoxicillin (AMO) was chosen as the test compound. Dual properties of AMO, which contain hydrophobic skeleton and polar groups (amino, hydroxyl and carbonyl), cause irregular changes of the retention over the stationary phase hydrophobicity and silanol activity at given mobile phase composition. Presented data show that application of non‐standard low coverage density C18 phases allow to determine AMO in the RPLC condition with high retention.     

Dual‐mode hydrophilic interaction normal phase and reversed phase liquid chromatography of polar compounds on a single column

Adopting a stationary phase convention circumvents problematic definition of the boundary between the stationary and the mobile phase in the liquid chromatography, resulting in thermodynamically consistent and reproducible chromatographic data. Three stationary phase definition conventions provide different retention data, but equal selectivity: (i) the complete solid phase moiety; (ii) the solid porous part carrying the active interaction centers; (iii) the volume of the inner column pores. The selective uptake of water from the bulk aqueous‐organic mobile phase significantly affects the volume and the properties of polar stationary phases. Some polar stationary phases provide dual‐mode retention mechanism in aqueous‐organic mobile phases, reversed‐phase in the water‐rich range, and normal‐phase at high concentrations of the organic solvent in water. The linear solvation energy relationship model characterizes the structural contributions of the non‐selective and selective polar interactions both in the water‐rich and organic solvent‐rich mobile phases. The inner‐pore convention provides a single hold‐up volume value for the retention prediction on the dual‐mode columns over the full mobile phase range. Using the dual‐mode monolithic polymethacrylate zwitterionic micro‐columns alternatively in each mode in the first dimension of two‐dimensional liquid chromatography, in combination with a short reversed‐phase column in the second dimension, provides enhanced sample information.     

Evaluation of protein, peptide, and amino acid retention on C5 hydride-based stationary phases

A pentyl (C5) stationary phase is synthesized on a hydride surface using both 100 and 300 A silica particles. The aqueous normal phase properties of these new phases are evaluated using amino acids at high concentrations of ACN in the mobile phase containing 0.1% formic acid. The RP properties of the materials are tested by using peptides and proteins in order to evaluate the effect of pore size on retention. Comparisons of retention as well as efficiencies and peak symmetry with a C18 phase are used to determine the influence of the hydrophobic properties of the bonded material. An evaporative light scattering detector (ELSD) is used in all phases of the study and compared with UV detector for gradient elution of proteins.