Regeneration of tetrabutylammonium ion-pairing reagent distribution in a gradient elution of reversed phase ion-pair chromatography

The regeneration of ion-pairing reagent distribution on liquid chromatography columns after gradient elution has been well recognized as the cause for long column equilibration time, a major drawback associated with gradient elution reverse phase ion-pair chromatography. To date, the majority of studies have focused on optimizing the separation conditions to shorten the equilibration time. There is limited understanding of the ion-pairing reagent distribution process between the mobile phase and stationary phase in the course of gradient elution, and subsequent column re-equilibration. The focus of this work is to gain a better understanding of this process. An ion-pair chromatographic system, equipped with a YMC ODS C(18) column and a mobile phase containing tetrabutylammonium (TBA) hydroxide as the ion-pairing reagent, was used in the study. The TBA distribution profile was established by measuring its concentration in the eluent fractions collected during the gradient cycle using different column equilibration times with an ion chromatographic method. Furthermore, the analyte retention time was evaluated as the function of the column equilibration time and TBA concentration in the mobile phase. The column equilibration and its impact on the method robustness will also be discussed.     

Solvent-generated liquid-liquid chromatography with aqueous ternary systems

Summary It is shown that solvent-generated liquid-liquid chromatography can also be realized with aqueous ternary systems. One phase of such a liquid-liquid system is used as eluent and the column is packed with a porous solid support whose surface is better wetted by the other phase which is then generated dynamically on it by the mobile phase. The partition mode of retention was verified by linear regression of the chromatographic retention data and the partition coefficients measured at equilibrium. A further confirmation was obtained by the sharp change observed in the retention characteristics when approaching a composition of the mobile phase corresponding to the liquid-liquid equilibrium. The stationary phase is generated rather quickly needing between 10 and 100 times the column volume of mobile phase for equilibration depending on the magnitude of the change of composition of the mobile phase and the specific surface area of the solid support. The stability of solvent-generated LLC columns is very high allowing long term reproducibility of the selectivity coefficient of about 1 %. It is demonstrated that solvent-generated LLC offers a powerful means to optimize the separation of a mixture by chromatography. Presented at the 15th International Symposium on Chromatography, Nürnberg, October 1–5, 1984; including results already presented at the 5th International Symposium on Column Liquid Chromatography, Avignon, May 11–15, 1981.     

Hard modeling of ion chromatography separations on hydroxide-selective stationary phase

In the present paper the development and testing of the computer software for the simulation of the on-column ion chromatography separation processes are presented. The computer algorithm based exclusively on the selectivity coefficients of the tested analytes has been upgraded to cope with the 2:1 and 1:1 ratios of the analyte-to-eluent ion charge. The analytical solution of the cubic equation, which is needed for calculation of chromatograms for doubly charged analytes in the presence of singly charged eluent, is presented. The developed modeling approach was tested on the data sets produced by the system composed of hydroxide-selective stationary phase in combination with on-line electrolytically generated OH(-)-based eluents. Retention behavior of the selected anions on the AS15 (DIONEX, USA) stationary phase was investigated. The study of the dependence of the peak widths on the number of theoretical column segments considered in the calculated chromatograms enabled us to choose the optimal number of column segments. The average error in the retention time of the calculated chromatograms for the data set used in the study, i.e. seven different ions at eight different eluent concentrations was found to be 1.4%. A good match with the experimental chromatograms allows us to use the information of the intermediate states of calculations to get a detailed insight into the time-dependent on-column analyte distribution.     

Ligand-exchange chromatography of amino sugars and amino acids on copper-loaded silylated controlled-pore glass

The application of silylated controlled-pore glass (CPG) particles as a stationary phase for the liquid chromatographic separation of selected amino acids and amino hexoses is reported. Copper-loaded columns prepared from CPG particles whose surface was silylated to immobilize an ethylenediamine functional group were employed. Copper bleeding from the column occurred but was compensated by 10^-4M copper in the ammoniacal eluent. Hydrolysis of the siloxane bonded to the surface limited the practical lifetime of a column to 50 h of continuous operation.     

Retention profiles and mechanism of anion separation on latex-based pellicular ion exchanger in ion chromatography

A retention model based on stoichiometric approach has been developed in order to describe analyte retention of anions on latex-based pellicular ion exchanger. The chromatographic process entails two stepwise and complex equilibria, first is ion-pair forming of analyte or eluent ion with ion-exchange sites under the effect of electrostatic forces due to the sulfonic layer behind the aminated functional groups of stationary phase. Second component is the ion-exchange between the analyte and eluent ions. As a new parameter of the fractional electrostatic coefficient of the ion exchange capacity was introduced to develop retention profiles of anions. Analysis of the dependence of the capacity factors on the eluent concentrations at different values of fractional coefficient shed light on the possible complex mechanism. Extensive experimental retention data were obtained for 14 anions (formate, acetate, propionate, pyruvate, lactate, chloride, nitrate, oxalate, malonate, succinate, tartarate, fumarate, maleate, sulphate) using hydroxide eluents of varying concentration. The ion-pair formation and ion-exchange selectivity constants for analyte and eluent species are determined using derived retention equation from experimental data by nonlinear iterative calculation. The model was utilized to predict retention data under elution conditions of practical importance. The predicted and obtained retention factors are in good agreement, which confirms the predictive power of the model.     

Thermodynamic description of retention mechanism in liquid chromatography

The chemical and physical properties of the stationary and the mobile phase determine the retention and selectivity of the separated molecules in chromatographic process. It should explain the specific and non-specific interactions in the chromatographic system. Special emphasis should be placed on column selection through the adequate choice of column dimensions (diameter and length) and the type of stationary phase with required parameters, which contributes the system effectiveness. However, column effectiveness is unable to guarantee a satisfactory level of substance separation. For this reason, the interactions between the analyte, the stationary and the mobile phase need to be investigated to determine the retention factors of the separated substances and the optimal parameters of the chromatographic system. According to the principles of thermodynamics, in particular molecular interactions, the chromatographic process has to be carried out with a high level of control. This work can be treated a brief tutorial devoted to thermodynamics of liquid chromatography process with a special emphasis on molecular interactions between analyte species and the components of the mobile and stationary phases.     

磷酸根离子在阴离子交换树脂上的保留行为及其机理探讨

首次发现磷酸根离子在阴离子交换柱上以两个色谱峰流出。在研究磷酸根离子的保留行为的基础上,提出了Ｈ２ＰＯ－４在固定相中进一步离解的保留机理,即Ｈ２ＰＯ－４在与阴离子交换树脂交换基进行离子交换的过程中,由于树脂交换基和淋洗离子的电荷相互作用促使一部分Ｈ２ＰＯ－４进行第２级离解。由于Ｈ２ＰＯ－４和ＨＰＯ２－４在阴离子交换树脂上的保留值不同,导致磷酸根离子出现“双峰”。     

色谱柱分离过程的驰豫理论基本模型的建立

根据样品分子在色谱柱分离过程中的输运特征,认为样品在系统中以跃迁的方式完成迁移,建立了色谱柱分离过程驰豫理论的基本模型。假设溶质在两相间平衡,用母函数方法研究非连续递推形式的驰豫理论输运动力学微分方程组,得到了描述组分在柱后浓度分布规律的基本流出曲线方程,并通过数值解方法模拟谱图对各输运参量与流出曲线特征的关系加以讨论。     

A novel silica based click lysine anion exchanger for ion exchange chromatography

Ion chromatography (IC) is one of the most powerful analysis technologies for the determination of charged compounds. A novel click lysine stationary phase was prepared via Cu(I) catalyzed alkyne-azide 1,3-dipolar cycloaddition (CuAAC) and applied to the analysis of inorganic ions. The chromatographic evaluation demonstrated good performance (e.g. the plate number of thiocyanate is ～50,000 plates m(-1)) and effective separation ability for the common inorganic anions with aqueous Na(2)SO(4) eluent. The separation mechanism was observed to be mainly dominated by ion exchange interaction. The retention of these analytes is highly dependent on the pH value of eluent. Compared with the lysine stationary phase prepared via the conventional manner, the click lysine exchanger demonstrated shorter retention time and better ion separation characteristics under the same chromatographic conditions, which is a great advantage for rapid separation and analysis of inorganic ions.     

A theoretical plate model accounting for slow kinetics in chromatographic elution

The chromatographic elution has been studied from different perspectives. However, in spite of the simplicity and evident deficiencies of the plate model proposed by Martin and Synge, it has served as a basis for the characterization of columns up-to-date. This approach envisions the chromatographic column as an arbitrary number of theoretical plates, each of them consisting of identical repeating portions of mobile phase and stationary phase. Solutes partition between both phases, reaching the equilibrium. Mobile phase transference between the theoretical plates is assumed to be infinitesimally stepwise (or continuous), giving rise to the mixing of the solutions in adjacent plates. This yields an additional peak broadening, which is added to the dispersion associated to the equilibrium conditions. It is commonly assumed that when the solute concentration is sufficiently small, chromatographic elution is carried out under linear conditions, which is the case in almost all analytical applications. When the solute concentration increases above a value where the stationary phase approximates saturation (i.e. becomes overloaded), non-linear elution is obtained. In addition to overloading, another source of non-linearity can be a slow mass transfer. An extended Martin and Synge model is here proposed to include slow mass-transfer kinetics (with respect to flow rate) between the mobile phase and stationary phase. We show that there is a linear relationship between the variance and the ratio of the kinetic constants for the mass transfer in the flow direction (τ) and the mass transfer between the mobile phase and stationary phase (ν), which has been called the kinetic ratio (κ=τ/ν). The proposed model was validated with data obtained according to an approach that simulates the solute migration through the theoretical plates. An experimental approach to measure the deviation from the equilibrium conditions using the experimental peak variances and retention times at several flow rates is also proposed.     

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.     

Determination of methylamphetamine and related compounds in human urine by high performance liquid chromatography/electrospray/mass spectrometry

The influence of tetrabutylammonium (TBA) as a hydrophobic charged additive of the mobile phase was investigated in a chromatographic system involving d,l-dansyl amino acids as the test solute enantiomers and immobilized human serum albumin as the chiral stationary phase. By varying the column temperature, van't Hoff plots for solute retention and enantioselectivity were performed and thermodynamic parameters were calculated. An enthalpy-entropy compensation study revealed that the type of interaction between the analyte and stationary phase was independent of TBA concentration in the eluent. The counterion dependence on retention indicated that the coulombic interactions between dansyl amino acid and the site II binding cavity were of crucial importance in this association process. Also, the increasing variations of chiral discrimination with a TBA addition were attributed, by an analysis of the thermodynamic parameter trends, to a great facilitation of enantioselective H-bonding between solute and polar residues at the cavity rim.     

Mobile-phase-viscosity dependence on DNA separation in slalom chromatography

Slalom chromatography (SC) is an alternative chromatographic procedure for the separation of relatively large double-stranded DNA molecules and is based on a new principle. The retardation of the DNA fragments from the cleavage of the Lambda DNA by the KpnI restriction enzyme was studied using an acetonitrile-phosphate buffer as a mobile phase with various concentrations of viscosity modifier (i.e. glycerol) and a C1 column as a stationary phase. The DNA molecule retention was accurately described over the glycerol concentration range using a model previously established. It was shown that the eluent viscosity increase enhanced the slalom chromatographic capacity to separate the DNA fragments. A connection between SC and 'hydrodynamic chromatography' processes was predicted to link the two processes in a global separation mechanism based on a non-equilibrium principle.     

Retention behaviour of metal complexes with 5-sulphoquinoline-8-ol in reversed-phase ion-pair liquid chromatography

Summary The reversed-phase ion-pair liquid chromatographic behaviour of several metal complexes with 5-sulphoquinoline-8-ol (HQS) on an octadecyl-modified silica column was investigated by using a fluorescence detector. The aluminum complex gave two peaks on the chromatograms when eluents containing phosphate buffer were used. The effects of the pH of the eluent, HQS concentration, the nature of anions in the eluent, and the column temperature on the retention behaviour of the aluminum complex are discussed on the basis of the equilibria and the kinetics of the complexation of the aluminum ion with HQS and anions in the eluent. The equilibrium constant for the interconversion reaction between the two Al(III)-HQS species which correspond to the two peaks observed and the second stepwise formation constant for the Zn(II)-HQS complex were evaluated from the HPLC data obtained.     

Dynamically modified silica — the use of bare silica in reversed-phase high-performance liquid chromatography

A new approach to reversed-phase high-performance liquid chromatography has been developed and investigated in some detail. Bare silica is used as the column packing material with an aqueous eluent containing a buffer, an organic solvent as the modifier and a long-chain quaternary ammonium ion. The eluent exhibits a high affinity to the silica surface at pH values above 5, thus, dynamically, forming a nonpolar stationary phase.     

Retention equilibrium and mass transfer characteristics in reversed-phase liquid chromatography using methanol-water mixtures

Pulse response experiments (i.e., elution chromatography) were made in reversed-phase liquid chromatography (RPLC) using a C18 silica gel column and methanol-water mixtures of different compositions (phi). The moment analysis of the elution peak profiles measured in the RPLC system provided some items of information about four parameters characterizing the retention equilibrium and the mass transfer kinetics in the column, i.e., adsorption equilibrium constant, isosteric heat of adsorption, surface diffusion coefficient and activation energy of surface diffusion. Characteristics of the chromatographic behavior were studied by analyzing the dependence of the four parameters on phi and the correlation between them. It was found that surface diffusion was one of the important processes of molecular migration having a significant contribution to the mass transfer kinetics in the column. Both the adsorption equilibrium constant and the surface diffusion coefficient varied depending on phi. The direction of their changes was approximately opposite, suggesting that the mass transfer in the manner of surface diffusion was restricted owing to the retention of the sample molecules on the stationary phase.     

Comparison of C18 silica bonded phases selectivity in micellar liquid chromatography

The paper describes a new test designed in micellar LC (MLC) to compare the commercial C18 stationary phase properties. This test provides the total hydrophobicity, hydrophilicity, steric selectivity, hydrogen bonding, and ion‐exchange capacity properties calculation of the ODS stationary phases. Both the test compounds and chromatographic separation conditions choice for column characterization in MLC are detailed. The chromatographic performance of several stationary phases that are used in MLC was evaluated with specific chromatographic test comprising nine test compounds, possessing different physico‐chemical properties, which were injected on different supports with two micellar mobile phases: one at pH 7.0 (0.075 mol/L SDS and 1.5% v/v 1‐pentanol), and other at pH 2.7 (0.075 mol/L SDS and 1.5% v/v 1‐pentanol adjusted to pH by TFA). Fundamental column chromatographic properties were obtained under these conditions and were treated by hierarchical cluster analysis. From the results of cluster analysis, two closely related groups of columns are distinguished, and it was shown that the chosen column characteristic parameters allow characterizing both sorbent and micellar chromatographic system properties. Eleven columns were analyzed by this test, which allows a comparison of columns with the aim of the selection of suitable and analogous column for the analysis with MLC.     

Co-eluent effect in partition chromatography. Rhamnose-xylose separation with strong and weak cation-exchangers in aqueous ethanol

The effect of ethanol in aqueous eluent on the chromatographic separation was studied at 298 K. Two sugars, L-rhamnose and D-xylose, were separated by using strong and weak cation-exchangers as a stationary phase. The ionic form of the resins was Na+ or Ca2+. The separations were carried out with sugar feed concentrations up to 35 wt% and with both low (about 1%) and high (about 10%) feed volume to bed volume ratios. The separation of the sugars was improved by adding ethanol into the eluent. The separation was also significantly enhanced when the weak cation-exchangers with the greatest affinity for water were used instead of strong cation-exchangers as a separation medium for the sugars having different hydrophilicities. The experimental data were successfully explained with a rate-based column model, which accounted for the volume changes of the stationary phase. A thermodynamic sorption model was utilized in column calculations.     

High speed gradient elution reversed phase liquid chromatography of bases in buffered eluents. Part II. Full equilibrium

In this work we determined when the state of thermodynamic (full) equilibrium, i.e. time-invariate solute retention, was achieved in gradient elution reversed-phase chromatography. We investigated the effects of flow rate, temperature, organic modifier, buffer type/concentration, stationary phase type, n-butanol as eluent additive, and pore size. We also measured how selectivity varied with reequilibration time. Stationary phase wetting and the ability of the stationary phase to resist changes in pH strongly affect the time needed to reach full equilibrium. For example, full equilibrium is realized with many endcapped stationary phases after flushing with only two column volumes of acetonitrile-water containing 1% (v/v) n-butanol and 0.1% (v/v) trifluoroacetic acid. Trends in retention time (<0.010min) and selectivity become quite small after only five column volumes of reequilibration. We give practical guidelines that provide fast full equilibrium for basic compounds when chromatographed in buffered eluents.     

Iodide analysis by ion chromatography on a new stationary phase of polystyrene-divinylbenzene agglomerated with polymerized-epichlorohydrin-dimethylamine

A new stationary phase for iodide ion analysis has been developed. The cationic polymerepichlorohydrin-dimethylamine(PEPI-DMA) was served as modifier in synthesizing polyelectrolyte sorbents and the macroporous polystyrene-divinylbenzene(PS-DVB) resin was used as support. The positively charged polymer(PEPI-DMA) was electrostatically bonded to a negatively charged particle(PS-DVB sulfonated). The new stationary phase was characterized by scanning electron microscopy(SEM), Fourier transform infrared(FTIR), elemental analysis, chemical adsorption and desorption measurements. The chromatographic evaluation of the new stationary phase was performed using various anions with a conductivity detector. The new stationary phase was also applied to the determination of iodide directly with a DC amperometric detector using a platinum working electrode and an Ag/Ag Cl reference electrode. The chromatographic conditions were optimized and the eluent solution contained 5 mmol/L HNO3 and 15 mmol/L Na NO3 at a flow rate of 1.0 m L/min and column temperature of 30 8C. The applied voltage of the DC amperometric detector was 0.9 V. Under the optimum conditions, the linear range of the method was 0.2–50 mg/L for iodide ion with a correlation coefficient of 0.9990. The detection limit was 0.05 mg/L(calculated at S/N = 3) and the relative standard deviations(RSD, n = 6) were all less than 1% for retention time, peak area and peak height. This method was also utilized for the determination of iodide ions in samples of povidone iodine solution and kelp samples with satisfactory results.