Investigation of the novel mixed-mode stationary phase for capillary electrochromatography. I. Preparation and characterization of sulfonated naphthalimido-modified silyl silica gel

A novel packing material, 3-(4-sulfo-1,8-naphthalimido)propyl-modified silyl silica gel (SNAIP), was prepared for the use as a stationary phase of capillary electrochromatography (CEC). The sulfonic acid groups on SNAIP stationary phase contributed to the generation of electroosmotic flow (EOF) at low pH and served as a strong cation-exchanger. In CEC with SNAIP, a mixed-mode separation was predicted, comprising hydrophobic and electrostatic interactions as well as electrophoretic migration process. In order to understand the retention mechanism on SNAIP, effects of buffer pH, concentration, and mobile phase composition on EOF mobility and the retention factors of barbiturates and benzodiazepines were systematically investigated. Moreover, the retention behavior of barbiturates on SNAIP was investigated and compared with those on octadecyl silica (ODS), phenyl-bonded silica, and 3-(1,8-naphthalimido)propyl-modified silyl silica gel to confirm the presence of pi-pi interaction on its retention mechanism. It was observed that a column efficiency was more than 85,000 N/m for retained compounds and the relative standard deviations for the retention times of EOF marker, thiourea, and five barbiturates were below 2.5% (n = 4). Under an applied voltage of 20 kV and a mobile phase consisted of 5 mM phosphate (pH 3.8) and 40% methanol, the baseline separation of five barbiturates was achieved within 3 min.     

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.     

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.     

Chiral anion exchangers applied to capillary electrochromatography enantioseparation of oppositely charged chiral analytes: investigation of stationary and mobile phase parameters

Weak anion-exchange (WAX) type chiral stationary phases (CSPs) based on tert.-butyl carbamoyl quinine as chiral selector (SO) and different types of silica particles (porous and non-porous) as chromatographic support are evaluated in packed capillary electrochromatography (CEC). Their ability to resolve the enantiomers of negatively charged chiral analytes, e.g., N-derivatized amino acids, in the anion-exchange mode and their electrochromatographic characteristics are described in dependence of several mobile phase parameters (pH, buffer type and concentration, organic modifier type and concentration) and other experimental variables (electric field strength, capillary temperature). The inherent "zwitterionic" surface character of such silica-based WAX type CSPs (positively charged SO and negatively charged residual silanols) allows the reversal of the electroosmotic flow (EOF) towards the anode at pH values below the isoelectric point (pI) of the modified surface, whereas a cathodic EOF results at pH values above the pI. Since for negatively charged analytes also an electrophoretic transport increment has to be considered, which can be either in or against the EOF direction, several distinct modes of elution have been observed under different stationary phase and mobile phase conditions: (i) co-electrophoretic elution of the negatively charged solutes with the anodic EOF in the negative polarity mode, (ii) counter-electrophoretic elution with the cathodic EOF in the positive polarity mode, and (iii) electrophoretically dominated elution in the negative polarity mode with a cathodic EOF directed to the injection end of the capillary. Useful enantioseparations of chiral acids have been obtained with all three modes. Enantioselectivity values as high as under pressure-driven conditions and theoretical plate numbers up to 120000 per meter could be achieved under electrically driven conditions. A repeatability study yielded RSD values below 2% for retention times and RSD values in the range of 5-10% for theoretical plate numbers and resolution, thus clearly establishing the reliability of the investigated anion-exchange type CEC enantioseparation methods.     

Investigation of the ion-exchange properties of methacrylate-based mixed-mode monolithic stationary phases employed as stationary phases in capillary electrochromatography

The potential of methacrylate-based mixed-mode monolithic stationary phases bearing sulfonic acid groups for the separation of positively charged analytes (alkylanilines, amino acids, and peptides) by capillary electrochromatography (CEC) is investigated. The retention mechanism of protonated alkylanilines as positively charged model solutes on these negatively charged mixed-mode stationary phases is investigated by studying the influence of mobile phase and stationary phase parameters on the corrected retention factor which was calculated by taking the electrophoretic mobility of the solutes into consideration. It is shown that both solvophobic and ion-exchange interactions contribute to the retention of these analytes. The dependence of the corrected retention factor on (1) the concentration of the counter ion ammonium and (2) the number of methylene groups in the alkyl chain of the model analytes investigated shows clearly that a one-site model (solvophobic and ion-exchange interactions take place simultaneously at a single type of site) has to be taken to describe the retention behaviour observed. Comparison of the CEC separation of these charged analytes with electrophoretic mobilities determined by open-tubular capillary electrophoresis shows that mainly chromatographic interactions (solvophobic and ion-exchange interactions) are responsible for the selectivity observed in CEC, while the electrophoretic migration of these analytes plays only a minor role.     

Enantiomer separation by nonaqueous and aqueous capillary electrochromatography on cyclodextrin stationary phases

Native beta- and gamma-cyclodextrin bound to silica (ChiraDex-beta and ChiraDex-gamma) were packed into capillaries and used for enantiomer separation by capillary electrochromatography (CEC) under aqueous and nonaqueous conditions. Negatively charged analytes (dansyl-amino acids) were resolved into their enantiomers by nonaqueous CEC (NA-CEC). The addition of a small amount of water to the nonaqueous mobile phase enhanced the enantioselectivity but increased the elution time. The choice of the background electrolyte (BGE) determined the direction of the electroosmotic flow (EOF). With 2-(N-morpholino) ethanesulfonic acid (MES) or triethylammonium acetate (TEAA) as BGE an inverse EOF (anodic EOF) was observed while with phosphate a cathodic EOF was found. The apparent pH (pH*), the concentration of the BGE, and the nature of the mobile phase strongly influenced the elution time, the theoretical plate number and the chiral separation factor of racemic analytes.     

Stepwise gradient of buffer concentration for capillary electrochromatography of peptides on sulfonated naphthalimido-modified silyl silica gel

The advantage of using a stepwise gradient of buffer concentration in CEC was demonstrated with the mixed-mode stationary phase, 3-(4-sulfo-1,8-naphthalimido)propyl-modified silyl silica gel (SNAIP). Before the application of a stepwise gradient, the effect of buffer concentration on the separations of six peptides and tryptic digests was investigated. Bubble formation caused by Joule heating at currents up to 95 microA was successfully suppressed by using SNAIP column even without pressurization, which contributed to a stepwise gradient of buffer concentration. Utilizing the stepwise gradient improved and shortened the separation of six peptides as compared to the separation under an isocratic elution.     

Capillary electrochromatography of peptides on a column packed with tentacular weak cation-exchanger particles

Silica-based, tentacular weak cation-exchanger particles were prepared for use as the stationary phase in the separation of positively charged sample components by capillary electrochromatography (CEC). Silica beads were first silanized with 3-(trimethoxysilyl) propyl methacrylate that served as a heterobifunctional linker, which reacted with 2-acrylarmidoglycolic acid in a second step by radical polymerization in aqueous solution. Baseline separation of basic peptides with good column efficiency was obtained on packed capillary columns by isocratic elution CEC with NaCl as the mobile phase modulator. The retention mechanism in the electrochromatographic process was studied by examining the effect of salt concentration on the migration behavior of the peptides. The chromatographic retention factor k'(lc) for charged sample components in the electrochromatographic process was estimated on the assumption that the overall migration rate of a charged migrant can be taken as the sum of the rate of chromatographic elution and the rate of electrophoretic migration. The estimated k(lc) values from experimental results were plotted against the molal salt concentration on a double logarithmic scale. The linear correlation is in good agreement with the prediction by the theory on the basis of traditional ion-exchange chromatography. The comparison of CEC results, obtained with open tubular and packed capillary columns having the same retentive functions as the stationary phase, supports the notion that variation of the phase ratio in the column offers an additional means to modulate the electrochromatographic migration behavior.     

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.     

Chromatography of substituted benzoic acids with methanol-water-carbon dioxide mixtures.

The impact of the proportion of CO2 concentration in methanol-water-CO2 mobile phases on the separation of several substituted benzoic acids was explored by studying the variation of retention with mobile phase pH in these mixtures. As the amount of CO2 in methanol-aqueous buffer-CO2 mixtures increased, a more basic buffer was needed to control the dissociation of these acids. Differences in terms of retention, separation efficiency and peak asymmetry were shown for substituted benzoic acids with methanol-water-CO2 and methanol-aqueous buffer-CO2 mixtures. Variations of these chromatographic parameters with mobile phase pH were related to the dissociation of these acids and their interaction with methanol-aqueous buffer-CO2 mobile phases and the stationary phase. The addition of a buffer into methanol-aqueous solution-CO2 was an effective means to optimize separations of acidic analytes with high fluidity liquid mobile phases. The substituted benzoic acids had baseline separation in the least amount of time using the high fluidity liquid mobile phases.     

Separation of selected peptides by capillary electroendoosmotic chromatography using 3 microns reversed-phase bonded silica and mixed-mode phases.

The retention behaviour and selectivity of selected basic, neutral and acidic peptides have been studied by capillary electroendoosmotic chromatography (CEC) with Hypersil C8, C18, Hypersil mixed-mode, and Spherisorb C18/SCX columns, 250 (335) mm x 100 microns, packed with 3 microns particles, and eluted with mobile phases composed of acetonitrile-triethylamine-phosphoric acid (TEAP) at pH 3.0 using a Hewlett-Packard Model HP3DCE capillary electrophoresis system. The selected peptides were desmopressin (D), two analogues (A and B) of desmopressin, oxytocin (O) and carbetocin (C). The peptides eluted either before or after the electroendoosmotic flow (EOF) marker, depending on the concentration of acetonitrile used and the buffer ionic strength. The retention and selectivity of these peptides under CEC conditions were compared to their behaviour in free zone capillary electrophoresis (CZE), where the separation mode was based on the electrophoretic migration of the analytes due to their charge and Stokes radius properties. In addition, their retention behaviour in RP-HPLC was also examined. As a result, it can be concluded that the elution process of this group of synthetic peptides in CEC with a TEAP buffer at pH 3.0 is mediated by a combination of both electrophoretic migration processes and retention mechanisms involving hydrophobic as well as silanophilic interactions. This CEC method when operated with these 3 microns reversed-phase and mixed-mode sorbents with peptides is thus a hybrid of two well-known analytical methods, namely CZE and RP-HPLC. However, the retention behaviour and selectivity of the selected peptides differs significantly in the CEC mode compared to the RP-HPLC or CZE modes. Therefore this CEC method with these peptides represents an orthogonal analytical separation procedure that is complimentary to both of these alternative techniques.     

Rapid separation of peptides and proteins by isocratic capillary electrochromatography at elevated temperature

The use of capillary electrochromatography (CEC) for the separation by isocratic elution of synthetic peptides, proteins as well as the tryptic digest of cytochrome c has been demonstrated. The monolithic porous stationary phase was prepared from silanized fused-silica capillaries of 75 microm I.D. by in situ copolymerization of vinylbenzyl chloride and ethylene glycol dimethacrylate in the presence of propanol and formamide as the porogens. The chloromethyl groups at the surface of the porous monolith were reacted with N,N-dimethylbutylamine to form a positively charged chromatographic surface with fixed n-butyl chains. Results of studies on the influence of temperature and mobile phase composition on the retention and selectivity of separation by CEC demonstrated the feasibility of rapid polypeptide analysis and tryptic mapping at elevated temperature with high resolution and efficiency. Typically the chromatography of a tryptic digest of cytochrome c took about 5 min at 55 degrees C and 75 kV/m with hydro-organic mobile phases containing acetonitrile in 50 mM phosphate buffer, pH 2.5. For peptides and proteins plots of logarithmic k'cec against acetonitrile concentration were nonlinear, whereas Arrhenius plots for the mobilities were nearly linear. Comparison of the separation of such samples under conditions of CEC and capillary zone electrophoresis (CZE) indicates that the mechanism of separation in CEC is unique and leads to a chromatographic profile different from that obtained by CZE.     

Separation and control of the elution order of N-t-butyloxycarbonyl amino acids D/L isomers by reversed-phase HPLC using cyclodextrins as chiral selectors for the mobile phase.

The enantiomeric resolution of N-t-butyloxycarbonyl (N-t-Boc) amino acids D/L isomers by reversed-phase HPLC was investigated using cyclodextrins (CD's) as chiral selectors for the mobile phase. The use of a low pH (pH<4) for the mobile phase enabled the enantioseparation of N-t-Boc amino acids. The opposite elution order of D/L isomers was observed when hydroxypropyl-derivatized beta-CD was used instead of native beta-CD. A computer simulation of the enantioseparation showed that the ratio of the retention factors of the chiral selector and the sample determined the elution order and the resolution. When the retention factor of the chiral selector is smaller than that of the sample, an isomer having larger complex formation constant eluted faster. However, when the chiral selector had a larger retention factor than the sample, an opposite elution order of the isomers was obtained. The large difference in the retention factors between the chiral selector and the sample led to good enantiomeric separation.     

Retention mechanisms and selectivity in internal-surface reversed-phase liquid chromatography. Studies with cyanobacterial peptide toxins

Summary Microcystins-LA,-LR,-RR,-YR and nodularin, cyanobacterial peptide toxins, were separated by internal-surface reversed-phase (ISRP), high-performance liquid chromatography. The capacity factors of the toxins were measured in the range pH 2–8 using acetonitrile, isopropanol or tetrahydrofuran in potassium dihydrogenphosphate mobile phase. The main retention mechanism of the ISRP column was reversed-phase interaction but cation-exchange offered additional selectivity at neutral and slightly acidic pH. At neutral pH (10% modifier, 0.1 M buffer) the elution order was microcystin-LA (two nonpolar residues leucine and alanine as the variable amino acids), nodularin, microcystin-LR,-YR and-RR (two basic arginines as the variable amino acids). The retention times of all toxins except microcystin-RR were substantially longer at acidic pH. At pH 2 (10% modifier, 0.1 M buffer) where the cation-exchange mechanism was inoperative the elution order was changed to microcystin-RR, nodularin, microcystin-LR,-YR and-LA. The best separation was achieved at pH 2 where even two desmethylated microcystin-RR analogs could be separated from microcystin-RR.     

Hydrophilic interaction chromatography separation mechanisms of tetracyclines on amino-bonded silica column

Effects of mobile-phase variations on the chromatographic separation on amino-bonded silica column in hydrophilic interaction chromatography (HILIC) were investigated for four zwitterionic tetracyclines (TCs): oxytetracycline, doxycycline, chlortetracycline, and tetracycline. A mixed-mode retention mechanism composed of partitioning, adsorption, and ion exchange interactions was proposed for the amino HILIC retention process. Buffer type and pH significantly influenced the retention of TCs, but showed similar separation selectivity for the tested analytes. Experiments varying buffer salt concentration and pH demonstrated the presence of ion exchange interactions in TCs retention. The type and concentration of organic modifier also affected the retention and selectivity of the analytes, providing direct evidence supporting the Alpert retention model for HILIC. The retention time of the analytes increased in the following order of organic modifiers: tetrahydrofuran < methanol < isopropanol < acetonitrile. The linear relationships of logk' versus %water (v/v) curve and logk' versus logarithm of %water (v/v) in the mobile phase indicated that TCs separation on the amino phase was controlled by partitioning and adsorption. The developed method was successfully utilized in the detection of TCs in both river water and wastewater samples using solid-phase extraction (SPE) for sample cleanup.     

硅胶电色谱分离机理的研究

对硅胶电色谱柱的性能进行了考察,发现在水／有机溶剂流动相条件下,几乎不存在气泡问题,流动相的组成在有机溶剂浓度、电解质浓度、ＰＨ值等方面可以在较大范围变化,选用５种典型样品,对硅胶电色谱的分离机理进行了系统研究,发现有反相分离机理、正相吸附机理、离子交换机理以及电泳机理参与作用。同时考察了有机溶剂浓度、电解质浓度、ＰＨ等对分离的影响。此外,还首次提出了一种全新的电色谱模式－动态改性硅胶电色谱。     

High-Performance Aqueous Size-Exclusion Chromatography Using Diol-Bonded Porous Glass Packing Materials. Retention Behavior of Some Proteins

Abstract

Retention volume of proteins increased or decreased with increasing phosphate buffer or neutral electrolyte concentrations in the mobile phase. This variation suppressed or accelerated by changing pH values in the mobile phase. The behavior of proteins can be interpreted by knowing isoelectric points (pI) of proteins and pKa value of the residual silanol groups on the surface of diol-bonded porous glasses. Positively charged surface of proteins below pH 8.0 (cytochrome c, lysozyme) retarded the elution by the ion-adsorption effects and negatively charged proteins around pH 7.0 (egg albumin, bovin serum albumin) eluted earlier than expected by the ion-exclusion effects. These effects suppressed by increasing phosphate buffer and neutral electrolyte concentrations in the mobile phase. Size-exclusion separation was attained in the mobile phase over 0.1 M phosphates and 0.1 M NaCl concentrations at pH 7.0. Mcllvaine buffer and Gomori buffer showed opposite action to proteins for retention comparing with Soerensen phosphate buffer. Potassium thiocyanate showed the different action for retention of proteins comparing with other neutral electrolytes and acted like sodium dodecyl sulphonate.     

Selectivity of dansyl amino acids in micellar liquid chromatography with an ion-exchange-induced stationary phase

Summary The retention behavior of dansyl amino acids in micellar liquid chromatography has been examined by using ionexchange-induced stationary phases. Several parameters affected the retention of the analytes, including the type and concentration of micellar agent and modifier ion and the concentration of acetonitrile in the mobile phase. The order of elution of dansyl amino acids obtained with the micellar mobile phase was very different from that observed in conventional reversed-phase liquid chromatography. Fluorescence intensities of some dansyl amino acids were enhanced by the micellar mobile phase.     

Separation of negatively charged nonsteroidal anti-inflammatory drugs by reversed-phase capillary electrochromatography

Reversed-phase capillary electrochromatography in a 5-microm C18 fully packed capillary was employed to optimize the separation of negatively charged nonsteroidal anti-inflammatory drugs. The effect of the physico-chemical parameters and different analysis modes on the separation of 2-arylpropionic acids was studied and evaluated. The mobile phase composition, buffer type, concentration and pH differently influenced the peak efficiency and resolution, selectively modulating the analytes interaction with the stationary phase. The use of zwitterionic MES or acetate mobile phases strongly modulated the analytes migration order and peak efficiency. The optimum experimental conditions were found in MES buffer, pH 5.0, containing the 75% acetonitrile-methanol (1:1). All the analytes were baseline separated in a mixture in less than 13 min with peak efficiencies in the range of 78,500-84,200 N/m. Under these conditions the analytes were negatively charged and their effective electrophoretic mobilities played a role in the separation. The analysis of different pharmaceutical preparations containing anti-inflammatory drugs, e.g. drops and tablets, is also presented after a very simple sample pretreatment.     

Selectivity differences of water‐soluble vitamins separated on hydrophilic interaction stationary phases

In this study, the retention behavior and selectivity differences of water‐soluble vitamins were evaluated with three types of polar stationary phases (i.e. an underivatized silica phase, an amide phase, and an amino phase) operated in the hydrophilic interaction chromatographic mode with ESI mass spectrometric detection. The effects of mobile phase composition, including buffer pH and concentration, on the retention and selectivity of the vitamins were investigated. In all stationary phases, the neutral or weakly charged vitamins exhibited very weak retention under each of the pH conditions, while the acidic and more basic vitamins showed diverse retention behaviors. With the underivatized silica phase, increasing the salt concentration of the mobile phase resulted in enhanced retention of the acidic vitamins, but decreased retention of the basic vitamins. These observations thus signify the involvement of secondary mechanisms, such as electrostatic interaction in the retention of these analytes. Under optimized conditions, a baseline separation of all vitamins was achieved with excellent peak efficiency. In addition, the effects of water content in the sample on retention and peak efficiency were examined, with sample stacking effects observed when the injected sample contained a high amount of water.