Reduction of silanophilic interactions in liquid chromatography with the use of ionic liquids

A suppression of silanophilic interactions by the selected ionic liquids added to the mobile phase in thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) is reported. Acetonitrile was used as the eluent, alone or with various concentrations of water and phosphoric buffer pH 3. Selectivity of the normal (NP) and the reversed (RP) stationary phase material was examined using a series of proton-acceptor basic drugs analytes. The ionic liquids studied appeared to significantly affect analyte retention in NP-TLC, RP-TLC and RP-HPLC systems tested. Consequently, the increased separation selectivity was attained. Due to ionic liquid additives to eluent even analytes could be chromatographed, which were not eluted from the silica-based stationary phase materials with 100% of acetonitrile in the mobile phase. Addition of ionic liquid already in very small concentration (0.5%, v/v) could reduce the amount of acetonitrile used during the optimization of basic analytes separations in TLC and HPLC systems. Moreover, the influence of temperature on the separation of basic analytes was demonstrated and considered in practical HPLC method development.     

Separation of small peptides by electrochromatography on silica-based reversed phases and hydrophobic anion exchange phases

Peptide separations are regarded as a promising application of capillary electrochromatography (CEC) and, at the same time, a suitable model to elucidate its mixed separation mechanism when charged analytes are involved. In this paper, studies on the separation of small peptides (2-4 amino acids) on a Spherisorb octadecyl silane (ODS) phase at acidic pH and on a strong anion exchange (SAX)/C18 mixed mode phase at weakly basic pH are reported. For the ODS phase a comparison of CEC, capillary zone electrophoresis (CZE) and high-performance liquid chromatography (HPLC) under identical buffer/eluent conditions is presented. The predicted retention factors for CEC under the assumption of simple superposition of HPLC retention and CZE migration matched the measured results for the peptides that had small retention factors in HPLC. For both types of stationary phases, a variation of the acetonitrile content in the mobile phase led to a wide range of retention factors, including negative values when co-electroosmotic migration was dominant. Though both the ODS and the SAX/C18 phase offer unique advantages, the SCX/C18 phase at pH 9 provides more flexibility to alter separation selectivity for the selected peptides.     

Separation of peptides by multimode chromatography on a column packed with vinyl alcohol copolymer gel

Several mechanisms of peptide separation in high-performance liquid chromatography were observed to occur on the Asahipak GS-320 packed with vinyl alcohol copolymer. Neutral rather than acidic mobile phases were employed as they were found to result in higher retention of many peptides on the GS-320. For neutral peptides, the retention volume corresponded to the Rekker's hydrophobic fragmental constant, with a correlation coefficient of 0.71. Peptides with acidic residues eluted early, as an effect of ionic exclusion; those with basic residues were retained longer, owing to an ion-exchange effect. The ionic interactions were shown to involve the carboxylic group present on the gel polymer. The net result was found to be excellent separation of hydrophilic as well as hydrophobic peptides, related to differences in their isoelectric points. The combination of these complex mechanisms, together with the size-exclusion effect of the GS-320 gel for separation of proteins and other large molecules and for analysis with a mobile phase high in acetonitrile content, makes possible high-resolution isocratic analysis of peptides, which cannot be achieved on octadecylsilica or ion-exchange columns.     

CEC separation of peptides using a poly(hexyl acrylate-co-1,4-butanediol diacrylate-co-[2-(acryloyloxy)ethyl]trimethyl ammonium chloride) monolithic column

A polyacrylate-based monolithic column bearing cationic functionalities and designed for capillary electrochromatography (CEC) has been prepared via photopolymerization of a mixture of hexyl acrylate, butanediol diacrylate, 2-(acryloyloxy) ethyltrimethyl ammonium chloride (monomers), azobisisobutyronitrile (photoinitiator), acetonitrile, phosphate buffer, and ethanol (porogens). The polymerization process was initiated with UV light at 360 nm. The column performance was evaluated via the separations of alkylbenzenes, substituted anilines, basic drugs, peptides, and a protein digest. The separation of complex peptide mixtures was then studied since such separations constitute a promising application of capillary electrochromatography. In particular, the effects of mobile phase composition, including ionic strength of the buffer solution and the percentage of acetonitrile on the retention factor, the column efficiency, and the resolution were determined. The separations were affected by both interaction of the peptides with the stationary phase and their own electrophoretic mobility. Excellent separations with column efficiencies of up to 160 000 plates/m were achieved for both a mixture of ten well-defined peptides and a tryptic digest of cytochrome c. The fractions of eluent containing peptides of the digest separated in the monolithic column were collected and characterized using matrix-assisted laser desorption ionization mass spectrometry.     

Separation and indirect detection of small-chain peptides using chromophoric mobile phase additives

Ruthenium(II) 1,10-phenanthroline, Ru(phen)3(2+), and ruthenium(II) 2,2'-bipyridyl, Ru(bipy)3(2+), salts were evaluated as mobile phase additives for the liquid chromatographic separation of small-chain peptides on a polystyrene-divinylbenzene copolymeric (Hamilton PRP-1) stationary phase. In a basic mobile phase peptides are anions, and retention, resolution and detection occur because of the interactions between the stationary phase, the RuII complex and the peptide anion. Since the RuII complex concentration changes in the analyte band relative to the background eluent RuII complex concentration, the peptide can be detected by indirect photometric detection using the wavelength where the RuII complex absorbs. Peptide analyte peaks may be positive or negative depending on the counter-anion and its concentration. Small-chain peptides that do not contain chromophoric side-chains are detected without derivatization at about 0.1 nmol injected at a 3:1 signal-to-noise ratio. Factors that affect retention, resolution and indirect photometric detection are the RuII complex, its mobile phase concentration, mobile phase pH and solvent composition, and the type and concentration of the mobile phase counter-anion and/or buffer anion.     

Effects of ionic liquid as additive and the pH of the mobile phase on the retention factors of amino benzoic acids in RP-HPLC

As an organic salt, ionic liquids are widely used as new solvent media. In this paper, three positional isomers, such as o-amino benzoic acid, m-amino benzoic acid, and p-amino benzoic acid are separated with four different ionic liquids as additives to the mobile phase using reversed-phase (RP) high-performance liquid chromatography (HPLC). Amino benzoic acids are biologically active substances; the p-isomer is present in a group of water-soluble vitamins and is widely known as a sunscreen agent. The ionic liquids used are 1-butyl-3-methylimidazolium tetrafluoroborate, 1-ethyl-3-methylimidazolium tetrafluoroborate, 1-ethyl-3-methylimidazolium methylsulfate, and 1-octyl-3-methylimidazolium methylsulfate. The effects of the length of the alkyl group on the imidazolium ring and its counterion, the concentrations of the ionic liquid, and the effect of the pH of the mobile phase on the retention factor of the amino benzoic acid isomers are studied. Separation with the ionic liquid in the eluent was better than the separation without the ionic liquid. The pH mainly affected the retention and elution order of the solutes in RP-HPLC.     

Modelling retention in liquid chromatography as a function of solvent composition and pH of the mobile phase

The aim of this work was to develop a model that accurately describes retention in liquid chromatography (LC) as a function of pH and solvent composition throughout a large parameter space. The variation of retention as a function of the solvent composition, keeping other factors constants, has been extensively studied. The linear relationship established between retention factors of solutes and the polarity parameter of the mobile phase, E(N)T, has proved to predict accurately retention in LC as a function of the organic solvent content. Moreover, correlation between retention and the mobile phase pH, measured in the hydroorganic mixture, can be established allowing prediction of the chromatographic behavior as a function of the eluent pH. The combination of these relationships could be useful for modelling retention in LC as a function of solvent composition and pH. For that purpose, the retention behavior on an octadecyl silica column of a group of diuretic compounds covering a wide range of physico-chemical properties were studied using acetonitrile as organic modifier. The suggested model accurately describes retention of ionizable solutes as concomitant effects of variables included and is applicable to all solutes studied. We also aimed to establish an experimental design that allows to reproduce to a good approximation the real retention surface from a limited number of experiments, that is from a limited number of chromatograms. Ultimately, our intention is to use the model and experimental design for the simultaneous interpretive optimization of pH and proportion of organic solvent of the mobile phase to be used in the proposed separation.     

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.     

High-performance liquid chromatographic method for the determination of prolyl peptides in urine

A rapid and accurate method is described for the determination of prolyl peptides in urine, with specific reference to the dipeptide prolylhydroxyproline, and free hydroxyproline and proline. Free amino acids and peptides were isolated from urine on cation-exchange minicolumns, and free imino acids and prolyl-N-terminal peptides were selectively derivatized with 4-chloro-7-nitrobenzofurazan, after reaction of amino acids and N-terminal aminoacyl peptides with o-phthalaldehyde. The highly fluorescent adducts of imino acids and prolyl peptides were separated on a Spherisorb ODS 2 column by isocratic elution for 12 min using as mobile phase 17.5 mM aqueous trifluoracetic acid solution containing 12.5% acetonitrile (eluent A), followed by gradient elution from eluent A to 40% of 17.5 mM aqueous trifluoroacetic acid solution containing 80% acetonitrile in 20 min. Analytes of interest, in particular the dipeptide prolylhydroxyproline, can be easily quantified by fluorimetric detection (epsilon ex = 470 nm, epsilon em = 530 nm) without interference from primary amino-containing compounds.     

Chromatofocusing of peptides on a sulfo-cation-exchange sorbent

Simple mobile phases containing no more than two active components were proposed for the formation of ascending pH gradients in a column filled with a sulfo-cation-exchange sorbent. The smoothest nearly linear pH gradients were obtained with the use of citric acid and Tris or NaH₂PO₄ and Tris as active components of the eluent and adjusting ionic strength (up to 0.1–0.3) in the starting solution or eluent; however, in the case of UV detection, the use of NaH₂PO₄ is preferable because of lower light absorption. Potentialities of the proposed approach in the chromatography of peptide mixtures on a sulfo-cation-exchange sorbent were demonstrated. Additions of acetonitrile to mobile phases improve the selectivity of the separation of peptides.     

Modeling of chromatographic retention of the selected antiarrhythmics and structurally related compounds in the hydrophilic interactions under the TLC and HPLC conditions

Abstract

High-performance liquid chromatography (HPLC) plays an important role in testing the pharmaceutically active compounds. In despite of the advantages of HPLC, thin-layer chromatography (TLC) retains its applicability to the different experimental tasks. The experimental conditions which allow hydrophilic interactions in the chromatographic system were tested in the HPLC and TLC systems for ivabradine, its related compounds, diltiazem and verapamil. Under the TLC conditions, retention behavior of the investigated compounds was tested on silica gel modified with cyanopropyl ligands as stationary phase and acetonitrile?+?methanol containing 25% v/v formic acid. Under the HPLC conditions, we used silica gel modified with cyanopropyl ligands as a column packing and the acetonitrile + 0.25% aqueous solution of formic acid as mobile phase. Retention behavior of the investigated analytes depending on the changing volume fractions of the mobile phase modifier was characterized both for TLC and HPLC data sets by the Soczewiński–Wachtmeister equation. Linear relationships were established between the retention coefficients characterizing the retention mechanism (R_M⁰/m, logk₀/m) and molecular properties of the investigated compounds. The Quantitative Structure Retention Relationship (QSRR) modeling was performed with the use of the stepwise multiple linear regression, in order to select molecular properties which influence retention.     

Overpressured layer chromatography in comparison with thin-layer and high-performance liquid chromatography for the determination of coumarins with reference to the composition of the mobile phase

The retention behaviour of fifteen closely related coumarins in normal-phase overpressured layer chromatography (OPLC) was studied with the aim of comparing the retentions with those in normal-phase thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) when optimization of the mobile phase was carried out according to the PRISMA system. The mobile phase optimization was carried out on TLC plates in unsaturated chambers. The resulting mobile phases were transposed to off-line, non-equilibrated OPLC and further to HPLC. The retention in TLC was measured at 37 selectivity points and in OPLC and HPLC at 13 points. Capacity factors (k′) and separation factors () were calculated in order to study the retention behaviour in the different systems. Two- and three-dimensional evaluations of k′ against selectivity points showed similar retention behaviours for the coumarins in TLC, OPLC and HPLC. The values for TLC, OPLC and HPLC showed similar patterns in the three-dimensional evaluations. The retention behaviour at different solvent strengths was also examined. According to quadratic regression, k′ showed a dependence on the change in solvent strength. OPLC, which can be considered as a “planar column” technique, and TLC are closely related methods, whereas HPLC shows a different behaviour in the elution process with regard to solvent strength.     

Zwitterionic stationary phase with covalently bonded phosphorylcholine type polymer grafts and its applicability to separation of peptides in the hydrophilic interaction liquid chromatography mode

A novel phosphorylcholine type zwitterionic stationary phase was synthesized by graft polymerization of 2-methacryloyloxyethyl phosphorylcholine onto the surface of porous silica particles. The resulting material possesses both negatively charged phosphoric acid and positively charged quaternary ammonium groups, which renders it a low net charge over a wide pH range. The composition of the surface grafts were determined by elemental analysis and solid state NMR, and the surface charge (zeta-potential) in different buffer solutions were measured using photon correlation spectroscopy. Separation of several peptides was investigated on packed columns in the hydrophilic interaction liquid chromatography (HILIC) separation mode. It was shown that small peptides can be separated based on hydrophilic interaction and ionic interaction between the stationary phase and analyte. The organic solvent composition, the pH and the salt concentration of the eluent have strong effects on the retention time. Compared to native silica before grafting, the newly synthesized zwitterionic material gave more stable retention times for basic peptides over pH range 3-7 due to elimination of the dissociation of silanol groups.     

Hydrophilic interaction liquid chromatography with alcohol as a weak eluent

There has been a significant increase of interest in polar compound separation by hydrophilic interaction liquid chromatography (HILIC), in which acetonitrile is mostly used as a weak eluent. Although replacing acetonitrile with alcohols as organic modifiers has been previously reported, the separation mechanism was poorly understood. In this paper we explored the separation mechanism through the method development for the analysis of the trace amounts of polar and basic hydrazines, which were genotoxic in nature. Separation parameters such as the type and concentration of alcohol, acid modifier, and buffer in mobile phase as well as the choice of stationary phase and column temperature were studied. The data indicated that both electrostatic and hydrophilic interactions contributed to the retention and separation of the hydrazines. The results presented here provide insight into the adjustment of the retention and separation of analytes in HILIC mode with alcohol as a weak eluent. The optimized HILIC method coupled with chemiluminescent nitrogen detection (CLND) is simple and sensitive (reporting limit at 0.02%) and was applied to simultaneous analysis of hydrazine and 1,1-dimethylhydrazine in a pharmaceutical intermediate.     

反相硅胶整体柱离子对色谱法快速测定吡啶离子液体阳离子

建立了整体柱离子对色谱-紫外检测法梯度淋洗快速分离测定4种吡啶离子液体阳离子的方法。分离采用C18反相硅胶整体柱,以离子对试剂(用柠檬酸调节pH值)-乙腈为淋洗液,并采用多级梯度洗脱程序。实验考察了色谱柱、离子对试剂、乙腈浓度、色谱柱温度及流速对吡啶阳离子保留的影响,并讨论了其保留规律。咪唑阳离子的保留符合碳数规律。最佳色谱条件是:在流速3.0 mL/min,柱温30℃下,以1.0 mmol/L庚烷磺酸钠(pH 4.0)(A)+乙腈(B)为淋洗液进行梯度洗脱。淋洗梯度为0～2.0 min,10%B;2.0～2.5 min,10%～15%B;2.5～4.0 min,15%B;4.0～4.5 min,15%～20%B;4.5～10.0 min,20%B。在此条件下,4种吡啶阳离子可在7 min内基线分离。所测阳离子的检出限(S/N=3)为0.05～0.17 mg/L;峰面积的相对标准偏差(n=5)小于0.6%。将本方法用于实验室合成的离子液体样品和污水样品的分析,加标回收率在95.7%～99.0%之间。本方法准确、快速,具有较好的实用性。     

CEC separation of insect oostatic peptides using a strong-cation-exchange stationary phase

The separation of several insect oostatic peptides (IOPs) was achieved by using CEC with a strong-cation-exchange (SCX) stationary phase in the fused-silica capillary column of 75 microm id. The effect of organic modifier, ionic strength, buffer pH, applied voltage, and temperature on peptides' resolution was evaluated. Baseline separation of the studied IOPs was achieved using a mobile phase containing 100 mM pH 2.3 sodium phosphate buffer/water/ACN (10:20:70 v/v/v). In order to reduce the analysis time, experiments were performed in the short side mode where the stationary phase was packed for 7 cm only. The selection of the experimental parameters strongly influenced the retention time, resolution, and retention factor. An acidic pH was selected in order to positively charge the analyzed peptides, the pI's of which are about 3 in water buffer solutions. A good selectivity and resolution was achieved at pH <2.8; at higher pH the three parameters decreased due to reduced or even zero charge of peptides. The increase in the ionic strength of the buffer present in the mobile phase caused a decrease in retention factor for all the studied compounds due to the decreased interaction between analytes and stationary phase. Raising the ACN concentration in the mobile phase in the range 40-80% v/v caused an increase in both retention factor, retention time, and resolution due to the hydrophilic interactions of IOPs with free silanols and sulfonic groups of the stationary phase.     

Comparative study of capillary electroendosmotic chromatography and electrically assisted gradient nano-liquid chromatography for the separation of peptides

Capillary electroendoendosmotic chromatography (CEC), being a hybrid of high-performance liquid chromatography (HPLC) and capillary electrophoresis, offers considerable changes to enhance column efficiency, speed of analysis and additional selectivity as compared to the parent methods. The analytes are driven by the electroendosmotic flow (EOF) and separated by surface-solute interactions as well as by differences in electromigration. In this paper on the separation of peptides on C18 reversed-phase and mixed-mode (sulphonic acid-n-alkyl) packings in CEC and electrically assisted reversed-phase gradient nano-LC are investigated. It is shown that mixed mode packings generate a higher EOF than reversed-phase packings that is scarcely dependent on the pH of the eluent. Applying a potential in gradient elution reversed-phase nano-LC of peptides shortens the analysis time as compared to separations without a potential. Electrically assisted reversed-phase gradient elution nano-LC is a powerful separation tool for analysis of tryptic digests. Peptides can be successfully resolved in acidic organic mobile phase at pH 2-3 with and without trifluoroacid as ion pairing reagent under isocratic conditions. It is demonstrated that CEC with mixed mode packing and an eluent of pH 2.3 with varying acetonitrile content can be applied to monitor impurities in a synthetic peptide.     

An approach to the prediction of retention times in liquid chromatography

The utility of Rekker's hydrophobic fragmental constant has been examined for optimization of reversed-phase mode liquid chromatographic separations. The chromatographic behavior of about 60 non-ionic compounds was measured in different acetonitrile/water mixtures and the logarithm of their capacity factors (log k) was correlated with their calculated hydrophobicities (log P). Linear relations were found in each case between log k and log P. The slope of the various lines was related to the percentage concentration of acetonitrile in the mobile phase. It was shown that, by using nine stand ard compounds and measuring their capacity factors in five eluents with different acetonitrile concentrations, the retention time could be predicted for 60 compounds. Calculation of the concentration of the organic modifier was also possible in a system of well coated octadecyl bonded packings with acetonitrile/water mixtures as eluent. Prediction of the capacity factor was accomplished to within 5% error.     

Peptide separation in hydrophilic interaction capillary electrochromatography

Separation of small peptides by hydrophilic interaction capillary electrochromatography (HI-CEC) has been investigated. The negative surface charge of a hydrophilic, strong-cation-exchange stationary phase (PolySULFOETHYL A) provided a substantial cathodic electroosmotic flow (EOF). The influence of acetonitrile content, ionic strength, mobile phase pH as well as applied voltage on the migration of the peptides was studied. Possible retention mechanisms of the peptides in HI-CEC were discussed. It was found that hydrophilic interaction between the solutes and the stationary phase played a major role in this system, especially when mobile phases with high acetonitrile content were used. However, an ion-exchange mechanism and electrophoretic mobility also affect the migration of the peptides in HI-CEC. Elution order and selectivity was proved to be different in HI-CEC and capillary zone electrophoresis (CZE), thus revealing the potential of HI-CEC as a complementary technique to CZE for the separation of peptides. Efficiency and selectivity of HI-CEC for the separation of peptides were demonstrated by baseline separating nine peptides in 6 min.     

Optimisation of chiral separation of omeprazole and one of its metabolites on immobilized α1-acid glycoprotein using chemometricsglycoprotein using chemometrics

Summary A strategy for the optimisation of direct chiral separation of omeprazole and a metabolite, hydroxi-omeprazole, in reversed phase liquid chromatography is described. A factorial design was used, where mobile phase pH, concentration of a mobile phase modifier, ionic strength and column temperature were tested as the variables and enantioselective retention, column efficiency and asymmetry factor as the responses. The experimental results were evaluated with multivariate analyses, which demonstrated that the column temperature and content of mobile phase acetonitrile were by far the most important variables. The enantiomers of omeprazole and one of its metabolites were baseline resolved within 15 minutes. The optimised chromatographic system was used for a separation of the enantiomers of omeprazole and its main metabolite in a patient plasma sample.