Capillary ion chromatography at high pressure and temperature

The application of high pressure and temperature in ion chromatography (IC) can significantly improve the efficiency and reduce the analysis time. In this work, the kinetic-performance limits of capillary IC columns with inner diameters of 400 μm packed with 4 and 7 μm macroporous anion-exchange particles were investigated employing a capillary ion-exchange instrument allowing column pressures up to 34 MPa and column temperatures up to 80 °C. Plate heights below 10 μm could be realized using capillary columns packed with 4 μm particles. Compared to conventional IC using 7 μm particles and pressures up to 21 MPa, a 40% improvement in plate number could be achieved when working at the kinetic performance limits at 34 MPa and using columns packed with 4 μm particles. Using coupled columns with a total length of 400 mm, a mixture of seven anions was separated within 7.5 min while yielding 20?000 plates. Increasing the temperature improved the performance limits when operating in the C-term region (for fast IC separation using columns <75 cm). Temperature also affected the retention properties and hence the selectivity. At higher temperature, retention for monovalent ions was mainly governed by ion diameter. An increase in retention with temperature was observed for small ions, and there was a decrease for ions having a larger diameter. The retention factor for divalent and trivalent anions increased with temperature.     

Prediction of analyte retention for ion chromatography separations performed using elution profiles comprising multiple isocratic and gradient steps

This study addresses the simulation of ion chromatographic (IC) separations performed under conditions where the elution profile consists of a sequence of isocratic and gradient elution steps (referred to as "complex elution profiles"). First, models for prediction of retention under gradient elution conditions in IC were evaluated using an extensive database of gradient elution retention data. It is shown that one such model is preferred on the basis that it can be used to predict gradient retention times on the basis of isocratic input data. A method is then proposed for using this model for complex elution profiles whereby each step of the elution profile is treated separately and analyte movement through the column is mapped. An empirically based algorithm for predicting peak width under complex elution conditions is also proposed. Evaluation of the suggested approaches was undertaken on a set of 24 analyte anions and 13 analyte cations on 5 different Dionex columns using a range of 5-step complex elution profiles that gave R2 values for correlations between predicted and observed retention times of 0.987 for anions and 0.997 for cations. The simulation of separations of anions and cations using a 3-step complex elution profile is demonstrated, with good correlation between observed and predicted chromatograms. The proposed approach is useful for the rapid development of separations when complex elution profiles are used in IC.     

A multicomponent mobile phase for ion chromatography applied to the separation of anions from the residue of low explosives

A multicomponent mobile phase utilizing ion-exchange, ion-exclusion, and ion-pairing principles for the rapid isocratic separation of anions in low explosives residue by ion chromatography (IC) has been developed. The notable feature of this system is that an ion-pairing reagent and an ion-exclusion reagent are combined in the same mobile phase. Contrary to expectation, these reagents act independently of each other in solution. The stock mobile-phase composition consisted of boric acid, D-gluconic acid, lithium hydroxide, and glycerol. Tetrapropylammonium hydroxide, an ion-interaction reagent was used to achieve pH 8.5. Acetonitrile (ACN) was added to enhance resolution and octanesulfonic acid, an ion-exclusion reagent, was added to adjust the retention time of perchlorate. Separation of a mixture of anions common to low explosives residue was achieved in less than 16 min using a Waters IC-Pak Anion HR column. Optimization studies were performed by changing the concentration of the ACN and by altering the pH or the type of ion-interaction or -exclusion agents. Simulated case studies were performed using postblast residues from pipe bombs. The results show this method to be a valid and reproducible procedure for forensic casework analysis. The practical significance of this system is that a reduction in the analysis time and an improvement in efficiency of late-eluting peaks can be achieved without resorting to gradient elution techniques. For the analysis of anions detected in explosives residue, the Waters IC-Pak Anion HR column has proven to be a suitable replacement for the Vydac 300IC405 column, which has been discontinued by the manufacturer.     

强疏水性AS-15色谱柱测定茶叶中F-、Cl-、NO3-、PO4（3-）、SO4（2-）、BrO3-、Br-、NO2-的研究

建立了一种利用强疏水性分离柱,通过KOH梯度淋洗,同时测定茶叶中F-、Cl-、NO3-、PO43-、SO42-、BrO3-、Br-、NO2-的方法。采用沸水冲泡茶样,茶水比为1：50,时间为5 min,过滤后稳定30 min待测;样液经过活性炭净化,再用0.20μm滤膜过滤后待测。结果表明：8种无机阴离子色谱峰能达到完全的基线分离,且浓度与峰面积呈良好的线性关系,检出限均低于0.056mg/L,加标回收率为96.5%~105.1%,该法用于茶样中8种无机阴离子含量的测定,结果稳定且重复性较好。     

强疏水性AS-15色谱柱测定茶叶中F-、Cl-、NO3-、PO43-、SO42-、BrO3-、Br-、NO2-的研究

建立了一种利用强疏水性分离柱,通过KOH梯度淋洗,同时测定茶叶中F-、Cl-、NO3-、PO43-、SO42-、BrO3-、Br-、NO2-的方法。采用沸水冲泡茶样,茶水比为1:50,时间为5 min,过滤后稳定30 min待测;样液经过活性炭净化,再用0.20μm滤膜过滤后待测。结果表明:8种无机阴离子色谱峰能达到完全的基线分离,且浓度与峰面积呈良好的线性关系,检出限均低于0.056mg/L,加标回收率为96.5%~105.1%,该法用于茶样中8种无机阴离子含量的测定,结果稳定且重复性较好。     

A mechanism of separation in electrostatic ion chromatography

The retention mechanism of electrostatic ion chromatography (EIC) is currently under debate and is the focus of this paper. A comprehensive set of retention data has been obtained on a C18 column coated with the zwitterionic surfactant 3-(N,N-dimethylmyristylammonio)propanesulfonate used with a range of mobile phases in which both the mobile-phase anion and cation have been varied systematically. Electro-osmotic flow measurements were also obtained on fused-silica capillaries coated with the zwitterion (and also some monofunctional surfactants) and were used to evaluate the nature of the surface charge on the layer of adsorbed surfactant in the presence of various background electrolytes. A new retention mechanism for EIC was developed on the basis of these data. This mechanism proposes that equilibration of the bound zwitterions with a mobile phase containing a suitable electrolyte causes the establishment of a charged layer created by the terminal sulfonate groups of the zwitterion, which acts as a Donnan membrane. The magnitude and polarity of the charge on this membrane depends on the nature of the mobile-phase ions. The Donnan membrane exerts weak electrostatic repulsion or attraction effects on analyte anions. A second component of the retention mechanism is chaotropic interaction of the analyte anion with the quaternary ammonium functional group of the zwitterion. This interaction exerts the major effect on the separation selectivity of EIC, such that analyte anions are eluted in order of increasing chaotropic interactions in accordance with the Hofmeister series.     

Elution of propranolol as an ion-pair complex by buffer solutions on C18-silica

Propranolol (pK(a) = 9.4) was eluted on C(18)-bonded Kromasil, equilibrated with buffer solutions of methanol and water (40/60, v/v) containing a constant concentration of a counteranion (12 mM). Nine different counteranions were studied: Cl(-), I(-), NO(3)(-), SO(4)(2-), CH(3)COO(-), HOOCC(2)H(4)COO(-), (-)OOCC(2)H(4)COO(-), HOOCCOHCOOHCOO(-), HOOCCOHCOO(-)COO(-), and (-)OOCCOHCOO(-)COO(-). The co-cation was K(+) or Na(+). Vacancy perturbations were measured on three concentration plateaus of propranolol hydrochloride, at 1.2, 12, and 24 mM, by injecting 100 microL of a pure mixture of methanol and water (40/60, v/v). Indirect detection of the solvent, the counteranion, the co-cation, and the chloride ion was carried out at 325 nm, a wavelength at which only propranolol responds. In a 1.2 mM propranolol hydrochloride solution, there is a 10-fold excess of counteranions and only a positive perturbation peak, due to the excluded co-cation and eluting before the column hold-up time, and a large vacancy peak, associated with propranolol, were recorded. Association between propranolol and the counteranion in excess determines the retention time of this second perturbation. The hydrophobicity of the complexes increases in the order Cl(-) < CH(3)COO(-) approximately HOOCC(2)H(4)COO(-) < NO(3)(-) < I(-) < HOOCCOHCOOHCOO(-) < (-)OOCC(2)H(4)COO(-) < SO(4)(2-) approximately HOOCCOHCOO(-)COO(-) < (-)OOCCOHCOO(-)COO(-). Propranolol retention is larger in the presence of the trivalent citrate anion than in that of the bivalent citrate, succinate, or sulfate anions. It is larger with these bivalent anions than with any monovalent anion. Equal concentration of propranolol hydrochloride and buffer in the mobile phase reveals five system peaks associated with the five components (solvent, counteranion, co-cation, chloride, propranolol molecules). In contrast with monovalent anions, bivalent anions (sulfate, succinate, citrate) or trivalent anions (citrate) cause a reversal of the elution order of the perturbation peaks of chloride anions and buffer molecules. This confirms a competition between chloride and buffer anions to form ion pairs with propranolol. The retention of the perturbation signal of the buffer increases with increasing anion charge because multivalent anions can bind to several molecules of propranolol. The perturbation measurements demonstrate the influence of the valence and hydrophobicity of the buffer on the retention of ionizable compounds. The inverse method allowed the derivation of the isotherm parameters from the overloaded band profiles of propranolol. These values confirm that adsorbate-adsorbate interactions increase with increasing valence of the anions.     

An automated on-line multidimensional HPLC system for protein and peptide mapping with integrated sample preparation

A comprehensive on-line two-dimensional 2D-HPLC system with integrated sample preparation was developed for the analysis of proteins and peptides with a molecular weight below 20 kDa. The system setup provided fast separations and high resolving power and is considered to be a complementary technique to 2D gel electrophoresis in proteomics. The on-line system reproducibly resolved approximately 1000 peaks within the total analysis time of 96 min and avoided sample losses by off-line sample handling. The low-molecular-weight target analytes were separated from the matrix using novel silica-based restricted access materials (RAM) with ion exchange functionalities. The size-selective sample fractionation step was followed by anion or cation exchange chromatography as the first dimension. The separation mechanism in the subsequent second dimension employed hydrophobic interactions using short reversed-phase (RP) columns. A new column-switching technique, including four parallel reversed-phase columns, was employed in the second dimension for on-line fractionation and separation. Gradient elution and UV detection of two columns were performed simultaneously while loading the third and regenerating the fourth column. The total integrated workstation was operated in an unattended mode. Selected peaks were collected and analyzed off-line by MALDI-TOF mass spectrometry. The system was applied to protein mapping of biological samples of human hemofiltrate as well as of cell lysates originating from a human fetal fibroblast cell line, demonstrating it to be a viable alternative to 2D gel electrophoresis for mapping peptides and small proteins.     

Enantiomer separation of amino acids by complexation with chiral reference compounds and high-field asymmetric waveform ion mobility spectrometry: preliminary results and possible limitations

We present a new method for separation of enantiomers with high-field asymmetric waveform ion mobility spectrometry (FAIMS), coupled to mass spectrometric detection. Upon addition of an appropriate chiral reference compound to the analyte solution and subsequent ionization of the solution by electrospray ionization, analyte enantiomers formed diastereomeric complexes, which were potentially separable by FAIMS. The methodology being developed is intended to be general, but here amino acid analytes are specifically considered. In the examples presented herein, six pairs of amino acid enantiomers were successfully separated as metal-bound trimeric complexes of the form [MII(L-Ref)2(D/L-A)-H]+, where MII is a divalent metal ion, L-Ref is an amino acid in its L form acting as chiral reference compound, and A is the amino acid analyte. For example, D- and L-tryptophan were separated in FAIMS as [NiII(L-Asn)2(D-Trp)-H]+ and [NiII(L-Asn)2(L-Trp)-H]+. As FAIMS separation typically takes place over a time scale of only a few hundred milliseconds, the presented separation method opens new possibilities for rapid analysis of one analyte enantiomer in the presence of the other enantiomer. Preliminary quantification results are presented, which suggest that fast and sensitive quantitative chiral analyses can be performed with FAIMS. Method limitations are discussed in terms of diverse phenomena, which are not yet understood.     

Chelation ion chromatography as a method for trace elemental analysis in complex environmental and biological samples

The development and evaluation of a new method for the determination of trace transition and rare-earth elements based on the combination of chelation and ion chromatography are described. The new method, chelation ion chromatography (Chelation IC), uses a chelating column to concentrate and separate transition and rare-earth elements from the common alkali and alkaline-earth metals, as well as other matrix components, prior to analysis by ion chromatography. The sample fraction from the chelating column contains only the concentrated analyte ions, thus eliminating interfering matrix components from complex matrices such as seawater and digested biological, botanical, and geological materials. This combination of chelation and ion chromatography provides a technique that makes possible the determination of trace elements in complex matrices that have proven to be difficult or impossible to analyze by ion chromatography or conventional atomic spectroscopy techniques.     

Analysis of inorganic species by capillary electrophoresis-mass spectrometry and ion exchange chromatography-mass spectrometry using an ion spray source

Mixtures of inorganic ions separated by capillary electrophoresis (CE) and ion exchange chromatography (IC) are detected by mass spectrometry (MS) using an ion spray atmospheric pressure ionization source. The selectable degree of ion-adduct declustering and molecular fragmentation in the MS interface region allows the system to be operated as an elemental analyzer or as a molecular detector suitable for oxidation state determinations. Both inorganic anions and cations (including alkalis, alkaline earths, transition metals, and lanthanides) are analyzed by CE-MS. A variety of CE separation buffers are evaluated for the cation analyses (e.g., creatinine, ammonium acetate, and tris[hydroxymethyl]aminomethane). Only one of the buffers (i.e., creatinine) can be used for CE-indirect UV detection. A CE capillary permanently coated with strong anion exchange sites and a pyromellitic acid buffer (suitable for indirect UV detection) is used for the inorganic anion separations. The coated column eliminates the need for buffer modifiers to reverse the flow in the capillary, which then reduces background noise and mass spectral complexity. The separation and detection of 13 inorganic anions are also accomplished by IC using an anion exchange column with a carbonate-bicarbonate mobile phase, on-line suppressed conductivity detection, and mass spectrometric detection.     

Design and Optimization of a Corona Discharge Ion Source for Supercritical Fluid Chromatography Time-of-Flight Mass Spectrometry

The interfacing of capillary column supercritical fluid chromatography (SFC) to time-of-flight mass spectrometry (TOFMS) through atmospheric pressure chemical ionization (APCI) was investigated. An ion source chamber and a new, flexible, and efficient transfer line from the SFC to the TOFMS system were designed to accommodate the requirements of this study. Ionization of analytes was performed using a corona discharge needle. The interface was equipped with two multiple-axis translation stages for positioning of the transfer line tip and the discharge needle inside the ion chamber. The investigations were oriented toward the optimization of parameters which have a strong effect on the intensity and stability of the analyte signal, including background stability, corona discharge needle positioning in the ion source, transfer line tip and discharge needle relative positioning, curtain gas and makeup gas flow interactions, ion chamber temperature, and elution pressure of analytes from the SFC system.     

强疏水性AS-15色谱柱测定茶叶中F~-、Cl~-、NO_3~-、PO_4~(3-)、SO_4~(2-)、BrO_3~-、Br~-、NO_2~-的研究

建立了一种利用强疏水性分离柱,通过KOH梯度淋洗,同时测定茶叶中F-、Cl-、NO3-、PO43-、SO42-、BrO3-、Br-、NO2-的方法。采用沸水冲泡茶样,茶水比为1:50,时间为5 min,过滤后稳定30 min待测;样液经过活性炭净化,再用0.20μm滤膜过滤后待测。结果表明:8种无机阴离子色谱峰能达到完全的基线分离,且浓度与峰面积呈良好的线性关系,检出限均低于0.056mg/L,加标回收率为96.5%~105.1%,该法用于茶样中8种无机阴离子含量的测定,结果稳定且重复性较好。     

Nonmolecular solvents in separation methods: dual nature of room temperature ionic liquids

Room temperature ionic liquids (RTIL) are molten salts starting to be used as nonmolecular solvents in separation methods mainly for their extremely low vapor pressure and thermal stability. RTILs are formed by an anion associated to a cation. This intrinsic structure gives them a dual nature. When used as additives in RPLC mobile phases to enhance basic compound separation, RTILs lose their particular physicochemical properties to become just salts. However, a given RTIL is not equivalent to another one made with the same cation. It is shown that both the anion and the cation contribute to solute retention and peak efficiency extending beyond simple "salting-out" or ion-pairing effects. Nine different alkyl-methyl-imidazolium ionic liquids with different alkyl chain length and chloride or BF(4-) or PF(6-) anions were used as additives (50 mM max. conc.) in the liquid chromatography separation of some cationic basic solutes on a Kromasil C18 column. It is shown with sodium salts and an acetonitrile-water 30/70 v/v mobile phase that anions can adsorb on the stationary phase surface according to their lyotropic character. They can also form ion pairs with the cationic basic solutes. Alkyl-imidazolium cations also adsorb on the C18 bonded stationary phase due to hydrophobic character depending on their alkyl chain length. Anion adsorption dramatically increases the cationic solute retention factors when cation adsorption decreases them. The cation adsorption is mainly responsible for peak shape and efficiency enhancements. RTILs are additives that enhance the basic cationic solute peak shape changing peak position. A wise choice of the appropriate combination of anion lyotropy with imidazolium cation hydrophobicity allows playing with solute selectivity and analysis duration.     

Application of atmospheric pressure ionization time-of-flight mass spectrometry coupled with liquid chromatography for the characterization of in vitro drug metabolites

Atmospheric pressure ionization time-of-flight mass spectrometry coupled with high-performance liquid chromatography was used to characterize the in vitro metabolites of glyburide. Metabolic products formed in vitro by human microsomes were separated using a C18 column with gradient elution at a flow rate of 200 microL/min without postcolumn splitting. In-source collision-induced dissociation (CID) by automated nozzle potential switching was employed to obtain both abundant protonated molecules and characteristic fragments whose accurate masses were measured simultaneously by internal mass calibration, performed by continuous postcolumn infusion of two reference standards. The mass errors were within 9 ppm for all ions measured, whose abundance was greater than 5%, relative to the most abundant isotopic "A" ion. Exact mass differences between the parent drug and metabolite(s) were determined and these values corresponded to a unique elemental composition. The elemental compositions of all metabolite fragment ions were generated based upon the known compositional elements of the protonated molecule. The structures of metabolites and their fragment ions were proposed based on the determined elemental composition and in-source CID spectra. The elemental composition and fragmentation pathways of four cyclohexyl hydroxylation metabolites and one ethylhydroxy metabolite are discussed.     

Preparation and application of methacrylate-based cation-exchange monolithic columns for capillary ion chromatography

Polymer-based strong cation-exchange monolithic capillary columns with different capacities were constructed for ion chromatography by radical polymerization of glycidyl methacrylate (GMA) and ethylene dimethacrylate in a 250-microm-i.d. fused-silica capillary and its subsequent sulfonation based on ring opening of epoxides with 1 M Na(2)SO(3). The cation-exchange capacities can easily and reproducibly be controlled in the range of up to 300 microequiv/mL by changing the immersion time of the epoxy-containing polymer in the Na(2)SO(3) solution. The chromatographic performance of the produced monolithic capillary columns was evaluated through the separation of a model mixture of common cations such as Na(+), NH(4)(+), K(+), Mg(2+), and Ca(2+). As an example, these cations could be well separated from one another on a 15-cm-long cation-exchange monolithic column (column volume, 7.4 microL) with a capacity of 150 microequiv/mL by elution with 10 mM CuSO(4). The pressure drop of this 15-cm column was approximately 1 MPa at a normal linear velocity of 1 mm/s (a flow rate of 3 microL/min), and the numbers of theoretical plates for the cations were above 3000 plates/15 cm. This GMA-based cation-exchange monolithic column could withstand high linear velocities of at least 10 mm/s. Over a period of at least two weeks of continuous use, no significant changes in the selectivity and resolution were observed. The applicability of a flow rate gradient elution and the feasibility of direct injection determination of major cations in human saliva sample were also presented.     

Crystal chemistry of stilbites: structure refinements of one normal and four chemically anomalous samples

Structural refinements were performed on four stilbites with ‘anomalous’ chemical compositions as regards the monovalent/divalent cation ratio, and a ‘normal’ stilbite. The four anomalous samples showed, moreover, a monoclinic angle lower than that expected from an empirical correlation between monovalent cation content and β angle. In all samples the two cationic sites characteristic of stilbite, were found; in only one sample was a new cationic site localized. One site is fully occupied by Ca alone, or, with some Na, whereas the other sites are fully or partially occupied by monovalent cations only. In the five stilbites, different numbers of water molecule sites were localized. The frameworks, however, seem to be unaffected by the number and composition of extraframework sites. The small β angle of the anomalous samples was only partially explained by structural data.     

Improved resolution of glycoproteins by chromatography with concanavalin A immobilized on microparticulate silica via temperature-programmed elution

The ability of the column temperature to control elution in the affinity chromatography of glycoproteins (e.g., ovalbumin and horseradish peroxidase) on silica immobilized concanavalin A has been studied. Column temperature programs can be achieved by placing a small HPLC column within a commercial mobile phase preheater assembly. It is shown that elution of adsorbed proteins can be initiated by changing the column temperature without altering the chemical composition of the mobile phase. Further, due to the enhancement in the rate of dissociation of the sample from the ligand, the peaks are narrowed. The resolution can be controlled by changing the initial temperature, dwell time at the initial temperature, and the rate of change of the temperature program. Addition of a competitive binding agent to the mobile phase decreases the temperature needed to elute strongly retained proteins. The effect of heating the column through many thermal cycles is assessed by periodically measuring the retention of a small monosaccharide that binds to the immobilized concanavalin A. The effect of two different immobilization procedures (glutaraldehyde and carbonyldiimidazole), as well as the effect of including a monosaccharide in the mobile phase, on the stability of the column is easily monitored by thermal elution chromatography. The effect of column temperature on the above glycoproteins has been assessed through studies of enzyme activities and anion exchange and isoelectric focusing patterns before and subsequent to temperature-programmed elution affinity chromatography.     

Concentration and purification of chromate from electroplating wastewater by two-stage electrodialysis processes

A designed two-stage electrodialysis system is proposed to concentrate and purify chromate from a low pH electroplating wastewater using monovalent selective electrodialysis membranes. With low pH of the raw water (pH 2.2) in the first stage, chromate was presented as HCrO(4)(-) and monovalent ions (HCrO(4)(-), NH(2)SO(3)(-), Na(+) and Cl(-)) were able to pass through the membrane thus chromate was concentrated up to 191%. Higher current density, flowrate and more membrane area all increased the chromium recovery. When pH was adjusted to 8.5 before entering the second stage, the chromate species was presented as divalent CrO(4)(2-) and retained in the concentrated stream, and the rest monovalent ions (NH(2)SO(3)(-), Na(+) and Cl(-)) were separated by passing through the membrane. For example, 45% of the chlorides were separated in this study. The separation efficiencies in the second stage were also increased when the current density, flowrate and membrane area were increased. Electron Spectroscopy for Chemical Analysis was used to examine the surface chromate species for stage 1, and anion exchange membrane showed more chromate fouling comparing to cation exchange membrane due to more adsorption and concentration polarization effects for the anion exchange membrane.     

水产品中氟化物的离子色谱法

本文提出用离子色谱检测水产品中的氟化物。样品经过600℃灰化,酸性下蒸馏后,用碱液吸收上机。采用阴离子柱分离,电导检测。流动相为Na2CO3/NaHCO3=6mM/1mM。50μL进样,检测限可达0.05mg/kg。线形范围为,0.05~5×103mg/kg。方法的回收率为86~93%。方法有选择性好,灵敏度高的优点。