Nanoengineered analytical immobilized metal affinity chromatography stationary phase by atom transfer radical polymerization: separation of synthetic prion peptides.

Atom transfer radical polymerization (ATRP) was employed to create isolated, metal-containing nanoparticles on the surface of nonporous polymeric beads with the goal of developing a new immobilized metal affinity chromatography (IMAC) stationary phase for separating prion peptides and proteins. Transmission electron microscopy was used to visualize nanoparticles on the substrate surface. Individual ferritin molecules were also visualized as ferritin-nanoparticle complexes. The column's resolving power was tested by synthesizing peptide analogs to the copper binding region of prion protein and injecting mixtures of these analogs onto the column. As expected, the column was capable of separating prion-related peptides differing in number of octapeptide repeat units (PHGGGWGQ), (PHGGGWGQ)(2), and (PHGGGWGQ)(4). Unexpectedly, the column could also resolve peptides containing the same number of repeats but differing only in the presence of a hydrophilic tail, Q-->A substitution, or amide nitrogen methylation.

Applications of ion chromatography with electrospray mass spectrometric detection to the determination of environmental contaminants in water.

Ion chromatography (IC) is widely used for the compliance monitoring of common inorganic anions in drinking water. However, there has recently been considerable interest in the development of IC methods to meet regulatory requirements for analytes other than common inorganic anions, including disinfection byproduct anions, perchlorate, and haloacetic acids. Many of these new methods require the use of large injection volumes, high capacity columns and analyte specific detection schemes, such as inductively coupled plasma mass spectrometry or postcolumn reaction with UV-Vis detection, in order to meet current regulatory objectives. Electrospray ionization mass spectrometry (ESI-MS) is a detection technique that is particularly suitable for the analysis of permanently ionized or polar, ionizable compounds. The combination of IC with MS detection is emerging as an important tool for the analysis of ionic compounds in drinking water, as it provides increased specificity and sensitivity compared to conductivity detection. This paper reports on the application of IC-ESI-MS for the confirmation and quantitation of environmentally significant contaminants, i.e. compounds with adverse health effects which are either regulated or being considered for regulation, such as bromate, perchlorate, haloacetic acids, and selenium species, in various water samples.

Initial study of using a laminar fluid diffusion interface for sample preparation in high-performance liquid chromatography.

This report describes a new microfluidic device called the H Filter for sample preparation prior to HPLC. The H Filters make possible a diffusional transfer of an analyte from a sample stream into a stream of a "receiver" fluid. Existing mathematical models can be used for optimizing experimental conditions. The authors have selected the extraction of the antibiotic cephradine from blood to demonstrate the utility of the new device. The extracts of blood samples spiked with cephradine levels between 0.2 and 100 microg/ml were analyzed using a C8 reversed-phase column and UV detection at 260 nm. The HPLC results were in good agreement with theory. The recovery of 32.2+/-2.8% was uniform over the entire range of cephradine concentrations. The new method completely avoids the use of centrifuges, that is otherwise typical for most current methodologies for the preparation of blood samples prior to HPLC analysis.

Protein variant separations using cation exchange chromatography on grafted, polymeric stationary phases.

We developed a set of cation exchange column packings (ProPac WCX-10 and ProPac SCX-10) that are based on a hydrophilic coated, pellicular polymeric support grafted with polymer chains bearing ion exchange functionalities. The supports are highly suited to resolving closely related protein variants. These column packings (1) afford minimal band spreading in conjunction with extremely high selectivity, (2) exhibit a very hydrophilic character, and (3) have moderate loading capacity. Cytochrome C variants (bovine, horse, rabbit) were baseline-separated, as was native ribonuclease A and its two deamidation products, the Asp67 and isoAsp67 forms. Humanized monoclonal antibody variants differing in the number of lysine residues at the C terminus of their heavy chains were baseline-resolved. Finally, the separation of hemoglobin variants found in a sample containing elevated levels of glycated hemoglobin was also demonstrated.

Simultaneous analysis of homocysteine and methionine in plasma.

The new isocratic cation exchange method separates up to eight different amino thiols. The separated sample components are detected electrochemically using a gold electrode and the integrated pulsed amperometry. The eluent composition is, for example, 0.15 M sodium perchlorate, 0.02 M perchloric acid and 5% acetonitrile. The report describes the optimization of chromatographic parameters such as column diameter and eluent composition. Quantitative performance is discussed for eight different amino thiols using standards. Also presented is a long term quantitative study for homocysteine and methionine in plasma samples. The preparation of plasma samples is simpler than with the previously reported version of the method. Only a reduction step is required, and neither column switching nor derivatization are necessary.

Ion chromatography on-chip.

On-chip separation of inorganic anions by ion-exchange chromatography was realized. Micro separation channels were fabricated on a silicon wafer and sealed with a Pyrex cover plate using standard photolithography, wet and dry chemical etching, and anodic bonding techniques. Quaternary ammonium latex particles were employed for the first time to coat the separation channels on-chip. Owing to the narrow depths of the channels on the chip, 0.5-10 microm, there were more interactions of the analytes with the stationary phase on the chip than in a 50-microm I.D. capillary. With off-chip injection (20 nl) and UV detection, NO2-, NO3-, I-, and thiourea were separated using 1 mM KCl as the eluent. The linear ranges for NO2- and NO3- are from 5 to 1000 microM with the detection limits of 0.5 microM.

Simplified in-line sample preparation for amino acid analysis in carbohydrate containing samples.

This report describes a new, automated chromatographic procedure eliminating carbohydrates from amino acid samples prior to their analysis by anion-exchange chromatography and integrated amperometric detection. In the first step, a sample is brought onto a short cation-exchange column (trap column) in hydrogen form. Carbohydrates are passing through this column, while only amino acids are retained. Subsequently, the cation-exchange column, holding the amino acid fraction, is switched in-line with the gradient pump and separator column. The mobile phase used at the beginning of the separation (NaOH; pH 12.7) transfers amino acids from the trap column onto the anion-exchange column and the amino acid separation is completed without any interference by carbohydrates. All common amino acids are recovered following the carbohydrate removal step. The average value of their recovery is 88.1%. The calibration plots were tested between 12.5 and 500 pmol (amounts injected). The mean value of correlation coefficients of calibration plots was calculated as 0.99. The mean value of relative standard deviations from five replicates was 3.9%. The usefulness of the method is illustrated with two chromatograms of a carrot juice sample obtained before and after the in-line removal of carbohydrates.

Ion-exchange-based eluent-free preconcentration of some anions.

Preconcentration procedures based on ion-exchange methods are often used to enhance the sensitivities of analytical techniques where the eluent used for eluting the preconcentrated ions does not influence the subsequent analytical step. Until recently, only a limited use of ion-exchange-based sample preconcentration procedures has been found in those analytical techniques where the eluent components strongly influence the separation procedure [e.g., capillary electrophoresis (CE)]. In this paper, we present a preconcentration procedure based on (i) the preconcentration of anions on an ion-exchange resin, (ii) the subsequent elution of analytes, and (iii) on-line removal of eluent components by chemical suppression using an appropriate suppressor device (either packed-bed suppressor column or micromembrane suppressor). The adjustment of the system parameters, combined with a computer-controlled, sensing/switching system, resulted in a minimal additional dilution of the eluted preconcentrated anions. The efficiency of the proposed enrichment/matrix removal procedure was tested by using off-line CE analysis of collected preconcentrated samples, reaching a LOD of 1 microg/l for a selected anion.

Prediction of retention times for anions in linear gradient elution ion chromatography with hydroxide eluents using artificial neural networks.

The feasibility of using an artificial neural network (ANN) to predict the retention times of anions when eluted from a Dionex AS11 column with linear hydroxide gradients of varying slope was investigated. The purpose of this study was to determine whether an ANN could be used as the basis of a computer-assisted optimisation method for the selection of optimal gradient conditions for anion separations. Using an ANN with a (1, 10, 19) architecture and a training set comprising retention data obtained with three gradient slopes (1.67, 2.50 and 4.00 mM/min) between starting and finishing conditions of 0.5 and 40.0 mM hydroxide, respectively, retention times for 19 analyte anions were predicted for four different gradient slopes. Predicted and experimental retention times for 133 data points agreed to within 0.08 min and percentage normalised differences between the predicted and experimental data averaged 0.29% with a standard deviation of 0.29%. ANNs appear to be a rapid and accurate method for predicting retention times in ion chromatography using linear hydroxide gradients.

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.

On-capillary ion-exchange preconcentration of inorganic anions in open-tubular capillary electrochromatogrpahy with elution using transient-isotachophoretic gradient. 2. Characterization of the isotachophoretic gradient.

Diffuse transient-isotachophoretic boundaries can be used as an elution gradient of increasing eluotropic strength to elute inorganic anions that have been preconcentrated on an open-tubular ion-exchange stationary phase prior to electrophoretic separation. The generation and characteristics of these gradients for elution after preconcentration have been investigated. The gradients are generated by placing a low-mobility, weak ion-exchange competing anion in the capillary (weak electrolyte, WE), and a high-mobility, strong ion-exchange competing anion in the electrolyte vials (strong electrolyte, SE). Application of voltage establishes a diffuse boundary with the composition changing from the weak anion to the strong anion. Comparison of elution gradients generated with different electrolyte systems was accomplished by comparing the eluotropic strength (a function of eluent concentration, ion-exchange selectivity coefficient, and charge) and the shape of the profile as it changes from WE to SE. The ion-exchange selectivity coefficient of the SE competing anion is important in establishing a sharp change in elution strength. A large difference in mobility between the WE and SE competing anions gives an SE with a higher final eluotropic strength, but the slope of the gradient is shallower. This results in a reduction in the efficiency of analyte focusing. To ensure maximum focusing efficiency, the WE and SE electrolytes should be selected such that the SE has the highest possible eluotropic strength for a given concentration of WE. The SE competing anion should also have a sufficiently low electrophoretic mobility to ensure focusing for the maximum number of analytes, and the mobility difference between the WE and SE competing anions should be as small as possible.

New technique for increasing retention of arginine on an anion-exchange column.

A method is described for enhancing retention of arginine on a pellicular anion-exchange column. Arginine exhibits adjustable increases of retention time that are dependent on the acidity of standard or sample matrix. This effect is based on interactions of the protonated form of arginine with the residual cation-exchange groups on the core beads of pellicular particles. The relative magnitude of retention time shift of arginine is evaluated for identical concentrations of hydrochloric, sulfuric, and perchloric acids. Although the direct addition of acid is very effective in influencing the retention of arginine, it affects peak shapes and retention of other peaks in the chromatographic separation. The new technique-acid coinjection-achieves a similar retention enhancement for arginine with only a minimal effect on the rest of the separation. Detection limits, reproducibility results, and calibration data are presented for the chromatography of amino acids with acid coinjection. Improved resolution of arginine is demonstrated with chromatograms of soybean hydrolysate and cell culture samples.

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.

Analyzing mixtures of amino acids and carbohydrates using bi-modal integrated amperometric detection.

Described in this work is a new detection methodology - bi-modal integrated amperometric detection - for identifying peaks and as a tool for solving difficult separation problems. Bi-modal integrated amperometry makes it possible to selectively detect amino acids, amino sugars, and carbohydrates following their separation by anion-exchange. Selectivity is gained by two different methods of integrating anodic current on an otherwise identical waveform. As with the single-mode integrated amperometry reported previously, the limits of detection are in the femtomole range and linear calibration plots are possible over three orders of magnitude. This new detection method does not require analyte derivatization. The practical utility of this new technique is demonstrated in the analysis of amino acids and sugars in a recombinant mammalian cell culture medium.

Analysis of the N-acetylneuraminic acid and N-glycolylneuraminic acid contents of glycoproteins by high-pH anion-exchange chromatography with pulsed amperometric detection.

Presence or absence of N-acetylneuraminic acid (Neu5Ac) can change a sialylated glycoprotein's serum half-life and possibly its function. We evaluated the linearity, sensitivity, reproducibility, and accuracy of a HPAEC/PAD method to determine its suitability for routine simultaneous analysis of Neu5Ac and N-glycolylneuraminic acid (Neu5Gc). An effective internal standard for this analysis is 3-deoxy-d-glycero-d-galacto-2-nonulosonic acid (KDN). We investigated the effect of the Au working electrode recession and determined that linear range and sensitivity were dependent on electrode recession. Using an electrode that was 350 microm recessed from the electrode block, the minimum detection limits of Neu5Ac, KDN, and Neu5Gc were 2, 5, and 2 pmol, respectively, and were reduced to 1, 2, and 0.5 pmol using a new electrode. The response of standards was linear from 10 to 500 pmol (r2>0.99) regardless of electrode recession. When Neu5Ac, KDN, and Neu5Gc (200 pmol each) were analyzed repetitively for 48 h, area RSDs were <3%. Reproducibility was unaffected when injections of glycoprotein neuraminidase and acid digestions were interspersed with standard injections. Area RSDs of Neu5Ac and Neu5Gc improved when the internal standard was used. We determined the precision and accuracy of this method for both a recessed and a new working electrode by analyzing Neu5Ac and Neu5Gc contents of bovine fetuin and bovine and human transferrins. Results were consistent with published values and independent of the working electrode. The sensitivity, reproducibility, and accuracy of this method make it suitable for direct routine analysis of glycoprotein Neu5Ac and Neu5Gc contents.

Improvements to in-line desalting of oligosaccharides separated by high-pH anion exchange chromatography with pulsed amperometric detection.

High-pH anion exchange chromatography with pulsed amperometric detection (HPAEC/PAD) (1) is routinely used to separate neutral and charged oligosaccharides differing by branch, linkage, and positional isomerism. Oligosaccharides are eluted in 0.1 M NaOH with gradients of sodium acetate (up to 0.25 M). Analyses of HPAEC/PAD-purified oligosaccharides generally require neutralization and removal of eluent salts. To facilitate the process, we designed and produced a cation-exchange system to remove sodium ions (Na+) from the eluent after oligosaccharide detection [the Carbohydrate Membrane Desalter (CMD), with a volatile regenerant]. Exchange of >99.5% of eluent Na+ for hydronium ions (H3O+) within the CMD generates dilute acetic acid (removable by vacuum evaporation). The exchange process desalts up to 0.35 M Na+ at 1.0 ml/min. Oligosaccharides collected after on-line desalting, evaporated and resuspended in their original volume of deionized water contained < or = 350 muM residual Na+ when the eluting sodium concentration was 300 mM. This represents a desalting efficiency of >99.8%. Recovery of neutral and sialylated oligosaccharides under these conditions ranged from 75 to 100%. With the CMD system and postcollection evaporation, HPAEC/PAD can purify oligosaccharides ready for further characterization. As a proof test, oligosaccharides from a human monoclonal antibody were separated by HPAEC/PAD, desalted with the CMD system, dried, and analyzed by matrix-assisted laser desorption-ionization, time-of-flight mass spectrometry.

Electrically polarized ion-exchange beds in ion chromatography: eluent generation and recycling.

This paper describes how electrically polarized ion-exchange beds pumped with water can produce electrolyte of steady and controllable concentration. Such devices make it possible to use water as the pumped phase in ion chromatography (IC), thus avoiding off-line eluent preparation. Control of the electrical current flowing through the devices allows precise control of the concentration of eluent that they deliver. This provides a new way of performing gradient and isocratic elutions. Using water as the carrier and two small beds of resin, one as a generator the other as a suppressor, and periodically reversing their roles through automatically switched valves, we have developed a form of continuous IC that involves little intervention by the user. The paper presents the principles of the new method and examples of its use in anion analysis.

Protein variant separations by cation-exchange chromatography on tentacle-type polymeric stationary phases.

We developed a set of prototype cation-exchange column packings that are based on a hydrophilic coated, pellicular polymeric support with a grafted tentacular surface chemistry that is highly suited to resolving closely related protein variants. These column packings (1) afford minimal band spreading in conjunction with extremely high selectivity, (2) exhibit a very hydrophilic character and (3) have moderate loading capacity. Cytochrome c variants (bovine, horse, rabbit) were baseline-separated, as was native ribonuclease A and its two deamidation products, the Asp67 and isoAsp67 forms. Humanized monoclonal antibody variants differing in the presence of lysine at the C terminus of the heavy chains were baseline-resolved. Finally, the separation of hemoglobin variants found in a sample containing elevated levels of glycated hemoglobin was also demonstrated.