Interplay of chromatographic and electrophoretic processes in capillary electrochromatography

The separation mechanism in capillary electrochromatography (CEC) is a hybrid differential migration process, which entails the features of both high-performance liquid chromatography and capillary zone electrophoresis, i.e., chromatographic retention and electrophoretic migration. The adsorption of the different sample components on the stationary phase can be modified by the presence of the electric field across the column. Here, we use our previously published approach to decouple chromatographic retention from electrophoretic migration that allows us to investigate the "modification" of the retention process in CEC. This paper presents a methodology for characterization of changes in the retention of neutral and charged sample components, under identical conditions of stationary and mobile phase.     

Chromatographic and electrophoretic migration parameters in capillary electrochromatography

Chromatographic retention factor, k', as defined in high-performance liquid chromatography (HPLC) in terms of the migration times of the separand and the inert tracer, has limited applicability to capillary electrochromatography (CEC) when both chromatographic and electrophoretic processes determine the magnitude of the overall migration rates of the separands. This situation is unlike that in HPLC, where k' serves as a useful peak locator for the various sample components, as well as, provides thermodynamic insights into the interactions between the components and the stationary phase. Most publications have borrowed the definition of k' from HPLC and applied it on CEC. However, due to the dual separation mechanisms that are in action in CEC, the system is significantly complicated in comparison to that of HPLC. This paper discusses the impossibility of defining with a k' which would have all the attributes that it has in regular chromatography. The interplay of the two separation mechanisms in determining the overall migration process in CEC is discussed and the various definitions of the electrochromatographic retention factor are presented.     

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.     

Investigations on the behaviour of acidic, basic and neutral compounds in capillary electrochromatography on a mixed-mode stationary phase

This work describes the separation of acidic, basic and neutral organic compounds as well as inorganic anions in a single run by capillary electrochromatography employing a stationary phase which exhibits both strong anion-exchange and reversed-phase chromatographic characteristics. The positive surface charge of this stationary phase provided a substantial anodic electroosmotic flow. The analytes were separated by a mixed-mode mechanism which comprised chromatographic interactions (hydrophobic interactions, ion-exchange) as well as electrophoretic migration. The influence of ion-exchange and hydrophobic interactions on the retention/migration of the analytes could be manipulated by varying the concentration of a competing ion and/or the amount of organic modifier present in the background electrolyte. Additionally the effects of pH changes on both the chromatographic interactions as well as the electrophoretic migration of the analytes were investigated.     

Separation of acidic compounds by strong anion-exchange capillary electrochromatography

Separation of the acidic compounds in the ion-exchange capillary electrochromatography (IE-CEC) with strong anion-exchange packing as the stationary phase was studied. It was observed that the electroosmotic flow (EOF) in strong anion-exchange CEC moderately changed with increase of the eluent ionic strength and decrease of the eluent pH, but the acetonitrile concentration in the eluent had almost no effect on the EOF. The EOF in strong anion-exchange CEC with eluent of low pH value was much larger than that in RP-CEC with Spherisorb-ODS as the stationary phase. The retention of acidic compounds on the strong anion-exchange packing was relatively weak due to only partial ionization of them, and both chromatographic and electrophoretic processes contributed to separation. It was observed that the retention values of acidic compounds decreased with the increase of phosphate buffer and acetonitrile concentration in the eluent as well as the decrease of the applied voltage, and even the acidic compounds could elute before the void time. These factors also made an important contribution to the separation selectivity for tested acidic compounds, which could be separated rapidly with high column efficiency of more than 220000 plates/m under the optimized separation conditions.     

Peak shapes in open tubular ion-exchange capillary electrochromatography of inorganic anions

An experimental study of parameters influencing peak shapes in ion-exchange open tubular (OT) capillary electrochromatography (CEC) was conducted using adsorbed quaternary aminated latex particles as the stationary phase. The combination of separation mechanisms from both capillary electrophoresis and ion-exchange chromatography results in peak broadening in OT-CEC arising from both these techniques. The sources of peak broadening that were considered included the relative electrophoretic mobilities of the eluent co-ion and analyte, and resistance to mass transfer in both the mobile and stationary phases. The parameters investigated were the mobility of the eluent co-ion, column diameter, separation temperature and secondary interactions between the analyte and the stationary phase. The electromigration dispersion was found to influence peak shapes to a minor extent, indicating that chromatographic retention was the dominant source of dispersion. Improving the resistance to mass transfer in the mobile phase by decreasing the capillary diameter improved peak shapes, with symmetrical peaks being obtained in a 25 microm I.D. column. However, an increase in temperature from 25 degrees C to 55 degrees C failed to show any significant improvement. The addition of p-cyanophenol to the mobile phase to suppress secondary interactions with the stationary phase did not result in the expected improvement in efficiency.     

Why robust background electrolytes containing multivalent ionic species can fail in capillary zone electrophoresis

Previous models for the retention behaviour of carboxylic acids in ion-exclusion chromatography are applicable only when the degree of ionisation of the analyte is constant over the entire chromatographic peak. When solutions of sulfuric acid are used as eluents, this condition applies only when the eluent concentration is considerably higher than that of the analyte. Since it is common for dilute solutions of sulfuric acid to be used as eluents, a retention model which accounts for unbuffered eluents has been developed. This model also considers the effects on retention of hydrophobic adsorption of the undissociated and dissociated forms of the analyte onto the stationary phase substrate, as well as the effects of organic solvents added to the eluent. The derivation of this model is presented and it has been evaluated using a comprehensive set of retention data obtained using three different sulfonated stationary phases over a range of eluent conditions. The adsorption coefficients calculated from the model are in accordance with expected trends and showed that both the undissociated and dissociated forms of the analyte acids were retained by hydrophobic adsorption effects, although this adsorption was much stronger for the undissociated analytes.     

Deconvolution of electrokinetic and chromatographic contributions to solute migration in stereoselective ion-exchange capillary electrochromatography on monolithic silica capillary columns

A monolithic silica stationary phase functionalized with an enantioselective strong cation exchanger based on an aminosulfonic acid derivative was used for chiral separations of basic test solutes by nonaqueous CEC and capillary LC. The effects of the applied electric field as well as the ionic strength in the eluent on electrokinetic and chromatographic contributions to the overall separation performance in the electrically driven mode were investigated. Hence, under the utilized experimental conditions, i. e., at an electric field strength in the range of approximately 120-720 V/cm (applied voltages 4-24 kV) and an ionic strength of the counterion between 5 and 25 mM (at constant acid-to-base, i. e., co- to counterion ratio of 2:1), no deviations from the expected linearity of the EOF were observed. This led to the conclusion that an occurrence of the so-called electrokinetic effects of the second kind resulting from electric double layer overlap inside the mesopores of the monolithic stationary phase and concentration polarization phenomena were largely negligible. Additional support to this conclusion was inferred from the observed independence of CEC retention factors on the electric field strength across the investigated ionic strength range of the BGE. As a consequence, a simple framework allowing for calculation of the CEC mobilities from the individual separation contributions, viz. electroosmotic and electrophoretic mobilities as well as retention factors, could be applied to model CEC migration. There was a reasonable agreement between calculated and experimental CEC mobility data with deviations typically below 5%. The deconvolution of the individual contributions to CEC migration and separation is of particular value for the understanding of the separation processes in which electrophoretic migration of ionic sample constituents plays a significant role like in ion-exchange CEC and may aid the optimization procedure of the BGE and other experimental conditions such as the optimization of the surface chemistry of the stationary phase. In combination with the remarkable column performance evident from the low theoretical plate heights observed under CEC conditions for all test solutes (3.5-7.5 microm in the flow rate range of 0.4-1.2 mm/s, corresponding to (130,000-300,000 plates per meter), the presented framework provides an attractive tool as the basis for the assessment of chromatographic selectivities in a miniaturized CEC screening of new selectors and chiral stationary phases (CSPs), respectively, from experimental CEC data and known CE mobilities.     

Modelling of migration behaviour of inorganic anions in ion-exchange capillary electrochromatography

A theoretical model to explain the observed mobility of inorganic anions in capillary electrochromatography (CEC) using ion-exchange (IE) stationary phases has been derived. The model divides contributions to the observed mobility of an analyte ion into capillary electrophoretic (CE) and IE components. The CE component includes the influence of varying the ionic strength of the background electrolyte on the electrophoretic mobility of the analyte, while the IE component accounts for the variation in retention of the analyte ion caused by changing the composition of the background electrolyte. The model was verified using a mixture of UV-absorbing inorganic ions in electrolytes of differing eluotropic strength in both packed and open-tubular CEC systems, with excellent agreement (r2 > 0.98) for both systems. Values of constants in the model equation determined by nonlinear regression were used to estimate the relative strengths of the interactions of different analytes with the stationary phase and these were found to agree well with elution orders observed in conventional IE chromatography.     

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.     

HPLC of synthetic polymers characterization of polystyrenes by high performance precipitation liquid chromatography (HPPLC)

Summary The advantages and disadvantages of high performance precipitation liquid chromatography have been demonstrated for polystyrene homopolymers. Depending on the mobile phase composition at the dissolution point of the polymeric sample and surface properties of the stationary phase, elution is governed either by a solution process or by adsorption. A contribution by adsorption was noticed on silica as well as on reversed phases based on silica with a normal phase gradient of increasing polarity (heptane to dichloromethane). Elution was solely governed by solubility of the polymers on both types of stationary phase for polystyrenes with a molecular weight above 35 000 and reversed phase gradient of decreasing polarity (methanol to dichloromethane). Under these conditions an identical dependence of elution solvent composition on sample size was found as for turbidity titrations. Due to differences in the velocity of the eluent front and the polymeric sample with porous stationary phases the polymers can be eluted as colloidal solutions Non-porous stationary phases are superior in this respect because the velocities of eluent and solutes are identical.     

Non-trivial temperature effects on the cation exchange chromatography and chelation ion chromatography of metal ions

The effect of column temperature upon the retention of metal ions on sulfonated and mono-, di-, and amino-carboxylated cation exchange columns has been investigated. The retention of alkali, alkaline earth and transition metal ions on each of the above types of cation exchanger was studied over the temperature range 19-65 degrees C. A major difference between the behaviour of mono- and divalent metal ions was shown on each of the above stationary phases, with the monovalent alkali metals exhibiting clearly exothermic behaviour (a decrease in retention with increased temperature) under acidic eluent conditions and an apparent relationship between retention factor and the magnitude of the temperature effect. The effect of temperature upon alkaline earth metal ions was less defined, although strongly endothermic behaviour (increase in retention with temperature) could be seen on all stationary phases through correct choice of eluent. The transition metal ions studied showed endothermic behaviour on all four stationary phases, with the sulfonated column unexpectedly showing the largest increases in retention. The above behaviour can be partially explained through the dominance of the type of solute-stationary phase interaction governing retention. In several of the above columns, both ion-exchange and surface complexation interactions can occur, with the effects of temperature indicating which process dominates under specific eluent conditions.     

Comparison of the Retention of Aliphatic Hydrocarbons with Polar Groups in RP-HPLC Systems with Different Modifiers of the Binary Eluent

The retention of aliphatic hydrocarbons with polar groups has been compared in respect to the separation selectivity changes in reversed-phase high-performance liquid chromatography with C18 stationary phase type and binary water eluent composed of methanol, acetonitrile, or tetrahydrofuran as modifiers. The changes in separation selectivity when one modifier is replaced by another in the eluent is explained, taking into consideration molecular interactions of the solutes with components of the stationary phase region, i.e., extracted modifier, and ordering of the stationary phase by the modifier.     

Normal- and Reversed-Phase Behaviour of 16,17-Secoestrone Derivatives on Cyanopropyl-Bonded Silica Gel

The retention behaviour of a number of 16,17-secoestrone derivatives has been studied by LC and HPTLC on a polar cyanopropyl-bonded stationary phase using non-aqueous and aqueous-organic mobile phases. The retention behaviour has been discussed in terms of nature of the solute, eluent and stationary phase. The correlation between retention constants of 16,17-secoestrone derivatives obtained from reversed-phases and commercially available ACD log P software (Advanced Chemistry, Toronto, Canada) has also been examined.     

Excess Adsorption of Organic Eluent Components from Mobile Phases Containing Electrolytes

The excess adsorption isotherms of organic eluent components from solutions containing electrolytes on a C18-bonded stationary phase are investigated by frontal analysis in staircase mode. The excess adsorption of acetonitrile increases when NaHSO₄, NaH₂PO₄, NaCl, or NaOAc is added to the eluent, but decreases upon addition of NaBr or NaClO₄. The excess adsorption of acetonitrile increases in the order of NaCl, NaHSO₄, NaH₂PO₄?>?NaOAc?>?NaBr, NaClO₄. On the other hand, the effect of electrolyte addition on the excess adsorption of methanol is not significant. The effect of electrolytes on the retention of alkylbenzenes in reversed-phase liquid chromatography is discussed on the basis of the excess adsorption of organic eluent components. The retention of alkylbenzenes shows negative correlation with the excess adsorption of acetonitrile. This indicates that the acetonitrile layer on the stationary phase does not act as a part of the stationary phase. A developed acetonitrile layer reduces the retention of alkylbenzenes by the competitive adsorption at the interface between the organic layer and the stationary phase.     

A simple method for measuring excess adsorption isotherms of organic eluent components on reversed‐phase packing materials

A simple frontal analysis method has been developed for the reliable measurement of excess adsorption isotherms of an organic component on reversed‐phase adsorbents in a series of programmed concentration steps. In the present method, a peak, which is produced by refractive index change in column eluate, is detected at 589 nm; it represents the elution volume of the boundary. The method is applied to the measurement of the excess adsorption isotherms of organic eluent components from water on commercially available reversed‐phase stationary phases. The results are in good agreement with the previously reported isotherms. We also measure the excess adsorption isotherms of organic eluent components from solutions containing electrolytes. There are not any interference peaks on the elution traces. The method is thus reliably applicable to the evaluation of the excess adsorption of organic eluent components in practical systems.     

Vitamin U-bonded stationary phase in capillary ion chromatography

A vitamin U-bonded stationary phase was prepared and the retention behavior of inorganic anions was examined using ion chromatography. Inorganic anions were retained on the vitamin U-bonded stationary phase under acidic as well as neutral eluent conditions in the ion-exchange mode. The elution order of the examined anions under neutral eluent conditions was nearly the same as that observed in common ion exchange mode, while the elution order observed under acidic eluent conditions was completely different from that observed in common ion exchange mode. The retention of the analyte anions under the neutral eluent conditions was due to the sulfonium groups of the vitamin U, while protonated primary amino groups caused retention of the analyte anions with different selectivity under acidic conditions. The retention factor of the analyte anions increased with decreasing eluent concentration under both eluent conditions. The present system was applied to the determination of bromide and nitrate contained in seawater.     

Retention in analytical ion exchange chromatography-1. Strong electrolytes on strong ion exchangers

A retention equation of general validity is adapted to ion exchange chromatography providing a self-consistent interpretation of retention of solutes and system peaks. Discussion is limited to eluent mixtures containing ions of invariant charge and to stationary phases with pH-independent surface charge. An interpretation is proposed for the retention volume of system peaks in binary eluents (mixtures of two eluent counter-ions). Retention volumes of labelled counter-ions of the eluent appear to provide useful information about the chromatographic system with clear interpretation in any multicomponent eluent.     

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.