Properties and Applications of Mixed Packing Capillary Electrochromatography

Mixed packing capillary electrochromatography (MP CEC) with the stationary phase comprising a physical mixture of strong cation exchange (SCX) phase and octadecysilyl (ODS) phase was developed. With the existence of a sulfonic acid group on the surface of SCX, not only could the electroosmotic flow (EOF) remain high at low pH, but also the hydrophilicity of the stationary phase was increased greatly, leading to broad adaptable ranges of both pH and organic modifier concentration in the mobile phase. At the same time, with the coexistence of C₁₈ on the surface of ODS, both the retention and the resolution of samples were improved. Accordingly, MP CEC combined the advantages of both SCX and ODS columns. Effects of operation parameters on EOF and the capacity factors of solutes as well as the retention mechanism of such a column were studied systematically. In addition, MP CEC columns were used in the analysis of strong polar solutes as well as for the high speed separation of acidic, basic, and neutral compounds in a single run.     

Application of hydrophobic anion-exchange phases in capillary electrochromatography

Capillary electrochromatography (CEC) requires stationary phases that enable appropriate electroosmotic propel under various conditions. Analyte retention can be controlled through hydrophobic or electrostatic interaction with the packing material. The development and characterization of new strong anion-exchange materials with additional hydrophobic moieties (SAX/C18 mixed-mode phases) is described. The synthesis was based on polymer encapsulation of porous silica. The phases were systematically characterized by means of elemental analyses, HPLC frontal analyses and CEC experiments. The studies focused on the influence of various parameters (e.g., pH, kind of buffer, capillary wall) on the electroosmotic flow (EOF). Phases with high anion-exchange capacity generated a fast and constant EOF over a wide pH range. Long-time stability of EOF and hydrophobic retention under CEC conditions were demonstrated within the course of 100 consecutive injections. The applicability of the SAX/C18 phases in appropriate buffer systems is demonstrated for neutral, acidic and basic compounds.     

Capillary-electrochromatographic separations with copolymeric reversed-stationary phase and ion-exchanger-packed columns

A macroporous, spherical, 7 μm, polystyrene–divinylbenzene (PS–DVB), reversed-phase adsorbent (PRP-1) was evaluated as a stationary phase for the capillary electrochromatographic (CEC) separation of neutral, acidic, and basic analytes of pharmaceutical interest. Electroosmotic flow (EOF) for a PRP-1 packed capillary is nearly constant over the pH 2 to 10 range and is higher than for a silica-based C₁₈ packed capillary on the acidic side. EOF increases with an increase in buffer acetonitrile concentration or as applied potential increases. As analyte hydrophobicity increases, analyte retention and migration time increases. Increasing buffer acetonitrile concentration reduces analyte partitioning with the PS–DVB stationary phase and analyte retention and migration time decreases. When exchange sites are present on the PS–DVB copolymer, EOF (EOF is reversed for the anion-exchanger) increases as the exchange capacity increases. An increased exchange capacity also reduces partitioning of the analyte with the PS–DVB matrix and analyte retention and migration time decrease. Because of excellent stability in an acid environment, the PRP-1 packed capillary can be used in strong acid buffer solution and weak acid and base analytes depending on pK_a values can be separated as neutral species and cations, respectively. CEC separations on a PRP-1 capillary of neutral steroids, weak base pharmaceuticals (separation as cations), purines and pyrimidines (as cations), fatty acids (as undissociated species), and sulfa derivatives (as cations) are described. Efficiency for the PRP-1 packed capillary for acetone or thiourea as the analyte is about 6·10⁴ plates m⁻¹.     

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.     

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.     

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.     

用强阴离子交换毛细管电色谱分离阴离子化合物

对阴离子性化合物在强阴离子交换毛细管电色谱中的保留行为进行了研究。发现样品中固定相上的吸附使样品的保留因子k^*变小,柱小的分离能力减小;而电压的增大,使酸性样品的k^*也增大,并且电压的改变也能改变分离的选择性;样品保留因子的对数值随着缓冲液离子强度的对数值的增大而线性减小;样品也强阴子交换毛细管电谱和毛细管区带电泳中有不同的保留行为。     

新型吡咯烷键合反相高效液相色谱固定相的制备与研究（英文）

正A new ionic liquid-based high-performance liquid chromatography stationary phase is reported.A derivative of N-methyl pyrrolidinium tetrafluoroborate was covalently immobilized on the surface of silica particles to prepare silica-based N-methyl pyrrolidinium tetrafluoroborate(SilprMP BF4)stationary phase.The obtained ionic liquid-modified silica was evaluated and confirmed by elemental analysis,infrared spectroscopy,and thermogravimetric analysis.A column was packed with the modified particles.The retention behavior of aromatic compounds,alkyl benzenes,and acidic and basic compounds on the SilprMP BF4 stationary phase was studied under reversed-phase liquid chromatography conditions.The effect of the eluent pH on the separation of the acidic and basic compounds was also studied.The new stationary phase involves multiple retention mechanisms,such as electrostatic,hydrophobic,ion-dipole,and anion-exchange interactions,which might lead to multipurpose separation media.     

High performance liquid chromatography separation of structurally related enkephalins on quaternary ammonium-embedded stationary phase in isocratic mode

Separation of twelve enkephalins was investigated on a quaternary ammonium-embedded stationary phase (Stability BS-C23). Variation of buffer pH of the mobile phase highlighted the complex relationship between repulsive/attractive electrostatic interactions and the reversed-phase partitioning mechanism. The effect of three different anions employed as additives (phosphate, chloride and perchlorate) was examined at various concentrations and two pH values (acidic and neutral). At pH 2.5, an increase in the anion eluent concentration resulted in a higher retention factors of positively charged enkephalins. This effect was more pronounced when perchlorate ions were added to the mobile phase rather than phosphate and chloride ions, due to chaotropic and ion-pairing effects. In contrast, at pH 7.5, retention factors of negatively charged enkephalins decreased when these salts were added, due to an anion-exchange mechanism. Perchlorate caused a sharper decrease than chloride and phosphate anions did. The results presented here provide insight into the possible adjustment of retention and separation of peptides on a mixed-mode stationary phase (BS-C23) by a careful control of the buffer pH, the nature and concentration of anions, added to the buffer, and organic modifier content.     

十二烷基键合氧化锆填充电色谱柱的研究

以十二烷基键合氧化锆(C12-ZrO2)作为固定相,制备了填充毛细管电色谱(CEC)柱,较为系统地研究了流动相条件对电渗流的影响、填充CEC柱的稳定性、碱性与中性化合物的保留与流动相pH值和有机溶剂含量的关系。C12-ZrO2固定相填充CEC柱在pH3～11．7范围内具有极好的稳定性;利用磷酸盐与氧化锆表面之间较强的相互作用,能够有效解决传统硅胶键合烷基固定相在有机溶剂含量低的流动相条件下不稳定的问题;同时吸附磷酸盐的固定相表面使得在更宽的流动相pH值范围内CEC柱有足够的电渗流,进一步拓宽CEC的应用领域。     

毛细管反相电色谱法分离行为的研究

对乙睛-水-磷酸二氢销体系毛细管反相电色谱分离行为进行了研究。采用柱上紫外检测,在75μmi.d.×30cm的毛细管ODS（3μm）填充柱上获得了小于2.0的折合培板高度。同时还研究了乙睛的比例、电解质的浓度和电场强度等因素对电渗流和往效的影响。     

Monolithic silica columns with mixed mode of hydrophilic interaction and weak anion-exchange stationary phase for pressurized capillary electrochromatography

A silica-based monolithic column as polar stationary phase is proposed for pressurized CEC (pCEC). The monolithic silica matrix from a sol-gel process was chemically modified by 3-aminopropyltrimethoxysilane to produce a column for hydrophilic interaction applications. The amino groups on the surface of the polar stationary phase generated anodic EOF under acidic conditions and served at the same time as a weak anion-exchanger. The anion solutes such as nucleotides were separated by the mixed mode mechanism, which comprised hydrophilic interaction, weak anion-exchange, and electrophoresis. The influences of buffer concentration and organic modifier content on the separation of nucleotides by pCEC have been investigated. In addition, the monolithic silica columns were also able to separate various polar compounds such as phenols, nucleic acid bases, and nucleosides in the hydrophilic interaction CEC mode.     

Enantiomer separation by strong anion-exchange capillary electrochromatography with dynamically modified sulfated beta-cyclodextrin

A novel mode of capillary electrochromatography (CEC) based on a dynamically modified stationary phase was presented for chiral separation. The capillary column was packed with strong anion-exchange (SAX) stationary phase packing; the sulfated beta-cyclodextrin (S-CD), which was added to the mobile phase, was dynamically adsorbed to the packing surface. Separation of enantiomers was achieved by their different abilities to form an inclusion complex with the adsorbed S-CD. The enantiomers of tryptophan, praziquantel, atropine, metoprolol, and verapamil were successfully separated in this system with a column efficiency of 36000-412000 plates/m. The resolution value obtained for atropine was as high as 11.23. The superiority of CEC with a dynamically modified stationary phase over that with a physically adsorbed stationary phase was demonstrated. The influence of ionic strength, S-CD concentration, and methanol content on separation was also studied.     

Preparation of a neutral porous monolith and its evaluation in pressurized capillary electrochromatography with neutral and charged solutes

A neutral, nonpolar monolithic capillary column was evaluated as a hydrophobic stationary phase in pressurized CEC system for neutral, acidic and basic solutes. The monolith was prepared by in situ copolymerization of octadecyl methacrylate and ethylene dimethacrylate in a binary porogenic solvent consisting of cyclohexanol/1,4‐butanediol. EOF in this hydrophobic monolithic column was poor; even the pH value of the mobile phase was high. Because of the absence of fixed charges, the monolithic capillary column was free of electrostatic interactions with charged solutes. Separations of neutral solutes were based on the hydrophobic mechanism with the pressure as the driving force. The acidic and basic solutes were separated under pressurized CEC mode with the pressure and electrophoretic mobility as the driving force. The separation selectivity of charged solutes were based on their differences in electrophoretic mobility and hydrophobic interaction with the stationary phase, and no obvious peak tailing for basic analytes was observed. Effects of the mobile phase compositions on the retention of acidic compounds were also investigated. Under optimized conditions, high plate counts reaching 82 000 plates/m for neutral compounds, 134 000 plates/m for acid compounds and 150 000 plates/m for basic compounds were readily obtained.     

Separation of basic and acidic compounds by capillary electrochromatography using monolithic silica capillary columns with zwitterionic stationary phase

A novel monolithic silica column with zwitterionic stationary phase was prepared by in-situ covalent attachment of phenylalanine to a 3-glycidoxypropyltriethoxysilane-modified silica monolith. Due to the zwitterionic nature of the resulting stationary phase, the density and sign of the net surface charge, and accordingly the direction and magnitude of electroosmotic flow in this column during capillary electrochromatography could be manipulated by adjusting the pH values of the mobile phase. CEC separations of various acidic and basic compounds were performed on the prepared column in anodic and weakly cathodic EOF modes, respectively. The peak tailing of basic compounds in CEC on a silica column could be alleviated at optimized buffer compositions. Besides the electrophoretic mechanism and weak hydrophobic interaction, weak cation- and anion-exchange interactions are also involved in the separations of acids and bases, respectively, on the zwitterionic column.     

Capillary electrochromatography with novel stationary phases. IV. Retention behavior of glycosphingolipids on porous and non-porous octadecyl sulfonated silica

In this investigation, capillary electrochromatography (CEC) with a novel stationary phase proved useful for the separation of neutral and acidic glycosphingolipids (GSLs). Four different gangliosides, namely G(M1a), G(D1a), G(D1b) and G(T1b), served as the acidic GSLs model solutes. The following four GSLs: galactosylceramide (GalCer), lactosylceramide (LacCer), globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer) served as the typical neutral GSLs. The stationary phase, octadecyl sulfonated silica (ODSS), consisted of octadecyl functions bonded to a negatively charged layer containing sulfonic acid groups. Porous and non-porous ODSS stationary phases were examined. The retention behavior of the acidic and neutral GSLs was examined over a wide range of elution conditions, including the nature of the electrolyte and organic modifier and the pH of the mobile phase. The porous ODSS stationary phase yielded the separation of the four different gangliosides using a hydro-organic eluent of moderate eluent strength whereas the non-porous ODSS stationary phase permitted the separation of the four neutral GSLs with a mobile phase of relatively high eluent strength.     

Fast separation of pyrimidine derivatives by capillary electrochromatography on ion-exchange/reversed-phase mixed-mode stationary phases

This work describes the use of mixed-mode stationary phases which exhibit both strong ion-exchange (either cation-exchange, SCX, or anion-exchange, SAX) and reversed-phase chromatographic characteristics in capillary electrochromatographic separations of pyrimidine derivatives. Different packing materials, namely C6, SCX/C6 and SAX/C6, were compared and the influence of the composition of the carrier electrolyte (concentration of acetonitrile and pH) on the retention behavior of the selected solutes was investigated. A separation of all eight pyrimidine derivatives could be obtained on a 6.5 cm column packed with the SAX/C6 stationary phase in less than 3 min, with good peak shapes and efficiencies in the range 39,000 to 81,000 plates per meter.     

Characterization of a microparticulate strong anion-exchanger in the HPLC of acidic drugs

The use of Waters Spherisorb S5SAX for the HPLC of acidic compounds, including a number of non-steroidal anti-inflammatory drugs (NSAIDs), has been investigated. Adequate retention, separation, and peak efficiency and symmetry were obtained for most analytes on a 250 x 4.6 mm i.d. column using methanol containing ammonium perchlorate (10 mmol L(-1), pH 6.7 or pH 8.3) as eluent. The results of changes in (i) eluent pH (constant ionic strength); (ii) eluent ionic strength (constant pH); and (iii) adding water to the eluent (constant pH) were consistent with a retention mechanism dominated by ion-exchange with the bonded strong anion-exchange (SAX) moieties. However, there were some unexpected observations, including (i) a general decrease in retention at eluent pH values above 7.7; (ii) a marked increase in retention on adding 1% (v/v) water to the eluent; (iii) a subsequent marked decrease in retention on adding 5% (v/v) or more water; and (iv) decreased column activity with time. These observations may be due to (i) interaction between the charged SAX moieties and ionised surface silanols (with ionization increasing at higher eluent pH values) and (ii) influence of the solvation of silanols, analytes, SAX moiety, and counter-ion varying with both pH and water content. Nevertheless, the factors influencing separation of individual NSAIDs remain unclear especially as no relation between log k and pKa exists for these compounds. Hydrophobic interactions are unlikely to be important since basic and neutral compounds were hardly retained. Ease of accessibility of the counter-ion to the SAX moiety for analyte displacement may be a factor.     

Capillary electrochromatography of peptides on a neutral porous monolith with annular electroosmotic flow generation

A new kind of monolithic capillary column was prepared for capillary electrochromatography (CEC) with a positively charged polymer layer on the inner wall of a fused-silica capillary and a neutral monolithic packing as the bulk stationary phase. The fused-silica capillary was first silanized with 3-glycidoxypropyltrimethoxysilane (GPTMS). Polyethyleneimine (PEI) was then covalently bonded to the GPTMS coating to form an annular positively charged polymer layer for the generation of electroosmotic flow (EOF). A neutral bulk monolithic stationary phase was then prepared by in situ copolymerization of vinylbenzyl chloride (VBC) and ethylene glycol dimethacrylate in the presence of 1-propanol and formamide as porogens. Benzyl chloride functionalities on the monolith were subsequently hydrolyzed to benzyl alcohol groups. Effects of pH on the EOF mobility of the column were measured to monitor the completion of reactions. Using a column with this design, we expected general problems in CEC such as irreversible adsorption and electrostatic interaction between stationary phase and analytes to be reduced. A peptide mixture was successfully separated in counter-directional mode CEC. Comparison of peptide separations in isocratic monolithic CEC, gradient HPLC and capillary zone electrophoresis (CZE) indicated that the separation in CEC is governed by a dual mechanism that involves a complex interplay between selective chromatographic retention and differential electrophoretic migration.     

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.