Model based robustness analysis of an ion-exchange chromatography step

Process development, optimization and robustness analysis for chromatographic separation are often entirely based on experimental work and generic knowledge. This paper describes a model-based approach that can be used to gain process knowledge and assist in the robustness analysis of an ion-exchange chromatography step using a model-based approach. A kinetic dispersive model, where the steric mass action model accounts for the adsorption is used to describe column performance. Model calibration is based solely on gradient elution experiments at different gradients, flow rates, pH and column loads. The position and shape of the peaks provide enough information to calibrate the model and thus single-component experiments can be avoided. The model is calibrated to the experiments and the confidence intervals for the estimated parameters are used to account for the model error throughout the analysis. The model is used to predict the result of a robustness analysis conducted as a factorial experiment and to design a robust pooling approach. The confidence intervals are used in a "worst case" approach where the parameters for the components are set at the edge of their confidence intervals to create a worst case for the removal of impurities at each point in the factorial experiment. The pooling limit was changed to ensure product quality at every point in the factorial analysis. The predicted purities and yields were compared to the experimental results to ensure that the prediction intervals cover the experimental results.     

Optimization of gradient profiles in ion-exchange chromatography using computer simulation programs

Optimization procedure of gradient separations in ion-exchange chromatography using simplex optimization method in combination with the computer simulation program for ion-exchange chromatography is presented. The optimization of parameters describing gradient profile for the separation in ion chromatography is based on the optimization criterion obtained from calculated chromatograms. The optimization criterion depends on the parameters used for calculations and thus exhibits the quality of gradient conditions for the separation of the analytes. Simplex method is used to calculate new gradient profiles in order to reach optimum separations for the selected set of analytes. The Simplex algorithm works stepwise, for each new combination of parameters that describe the gradient profile a new calculation is performed and from the calculated chromatogram the optimization criterion is determined. The proposed method is efficient and may reduce the time and cost of analyses of complex samples with ion-exchange chromatography.     

Optimisation and robustness analysis of a hydrophobic interaction chromatography step

Process development, optimisation and robustness analysis for chromatography separations are often entirely based on experimental work and generic knowledge. The present study proposes a method of gaining process knowledge and assisting in the robustness analysis and optimisation of a hydrophobic interaction chromatography step using a model-based approach. Factorial experimental design is common practice in industry today for robustness analysis. The method presented in this study can be used to find the critical parameter variations and serve as a basis for reducing the experimental work. In addition, the calibrated model obtained with this approach is used to find the optimal operating conditions for the chromatography column. The methodology consists of three consecutive steps. Firstly, screening experiments are performed using a factorial design. Secondly, a kinetic-dispersive model is calibrated using gradient elution and column load experiments. Finally, the model is used to find optimal operating conditions and a robustness analysis is conducted at the optimal point. The process studied in this work is the separation of polyclonal IgG from BSA using hydrophobic interaction chromatography.     

Computer simulation of ion chromatography separation: an algorithm enabling continuous monitoring of anion distribution on an ion-exchange chromatography column

A computer algorithm for the calculation of ion chromatography separation is presented. It is based on the calculation of equilibrium concentrations of present analyte in discrete column segments. The continuous column is treated as a number of discrete cells or segments where the equilibration process between the stationary phase and the eluent is simulated. The ion-exchange equilibration process is supposed to be instantaneous and quantitative. The continuous flow of the eluent is rendered by discrete transfers. The size of each transfer of the eluent corresponds to a portion of the volume contained in one column segment. The equilibrium calculations in all column segments are repeated for each transfer of the eluent, through all the stages of the chromatographic process. The distribution of the analytes between the stationary phase and the eluent can be monitored at any step and in any column segment which means that the described algorithm provides the spatial and time concentration profiles. The simulated chromatogram is acquired as a time-concentration profile in the last column segment. The obtained chromatograms are in good agreement with the experimental ones. The distribution of ions between the stationary phase and the eluent in the early stages of the ion chromatographic process can thus be studied with confidence.     

Application of ion-exchange chromatography with an ion-selective electrode detector to iodine determination in natural waters

A system is described which uses a selective electrode for potentiometric detection of anions in natural waters after ion-exchange separation. With an iodide-selective electrode in the flow cell, a linear relationship exists between iodide concentration and potential in the sub-Nernstian range (0–100 μM). The limit of detection is 0.015 μM for an injection of 125 μl of sample. For optimum electrode performance, a constant iodide background of about 0.5 μM is required in the column effluent passing through the detector. This background is added uniformly through a short section of hollow-fibre dialysis tubing in a concentric-flow diffusion cell. The detector also responds to other anions which affect the solubility of silver ion. This can be used to advantage if chromatographic separation is sufficient. Preconcentration of iodide in fresh waters on an anion guard cartridge allows determination down to 1 nM concentrations. For sea-water samples, simple chemical pretreatment permits quantitation of different iodine species.     

Simultaneous analysis of six inorganic anions in urine by double-suppress ion-exchange chromatography

This study is aimed to establish a simple, rapid, and accurate ion chromatography approach for the simultaneous detection of six inorganic anions in urine. Various performance parameters affecting the determination of anions were optimized, including the selection of sample protein precipitation agent, eluent, and flow rate. The final eluent was 3.6 mmol/L sodium carbonate and 12% isopropanol with a flow rate of 0.6 mL/min. Acetonitrile was used for pretreatment to precipitate proteins, and the volume ratio of urine to acetonitrile was 1:4. The correlation coefficient of the target anion calibration curve ranged from 0.9973 to 0.9999. The limit of detection ranged from 1.50 to 12.0 μg/L, and the method detection limit ranged from 15.0 to 120 μg/L. The standard recovery rate for low, medium, and high concentrations ranged from 90 to 110%. The inter-day and intra-day relative standard deviations were <5%. The method has high accuracy and good reproducibility and is suitable for the separation and determination of anions in urine.     

Automatic analysis of serum lactate dehydrogenase isoenzymes by high-performance ion-exchange chromatography

The repetitive analysis of serum lactate dehydrogenase (LDH) isoenzymes has been performed on a weak anion exchanger (TSKgel DEAE-5PW), which was developed by introducing diethylaminoethyl groups into TSKgel G5000PW (10 microns particle diameter)--a hydrophilic polymer-based material of large pore size--for high-performance gel chromatography. By use of this anion exchanger, a high-pH (greater than 8.0) solvent could be used and the albumin peak was completely separate from the LDH isoenzyme peaks. After 10 successive analyses with an autosampler, the coefficient of variation of the LDH isoenzyme elution times was less than or equal to 0.90%, and the coefficient of variation for peak areas was less than or equal to 3.85%. After 40 successive analyses, resolution between isoenzymes was generally greater than 1.25. This column can be used for more than 300 intermittent injections of human serum.     

Sorption processes in ion-exchange chromatography of viruses

Purified viruses are used in gene therapy and vaccine production. Ion-exchange chromatography (IEC) is the most common method for large-scale downstream purification of viruses and proteins. Published IEC protocols provide details for specific separations but not general methods for selecting operating parameters. To make the selection more systematic, we study adenovirus type 5 (Ad5) as a model virus and develop batch uptake and light scattering methods for optimizing the ionic strength and pH of adsorption, as well as providing heuristics for resin geometry. The static capacity for Ad5 was found to go through a maximum with increasing ionic strength. Comparison to a protein-resin system shows that resin capacity for the virus is at least an order of magnitude lower, even on a wide-pore resin. Virus penetration into the wide-pore resin is only partial and the uptake rate is an order of magnitude slower than the uptake onto a narrow-pore resin.     

General problems associated with the analysis of amino acids by automated ion-exchange chromatography

Some of the general problems commonly encountered with the analysis of amino acids have been described. These include problems associated with the preparation and storage of samples prior to analysis and those associated with the analytical stage itself. The effects on the accuracy of the determination of amino acids have been discussed, together with possible solutions to some of the problems.     

Radiochemical analysis of the polar agrochemicals validamycin and the metabolite melamine by ion-exchange chromatography

Suitable analytical methods to determine the radiochemical purity of validamycin and melamine are not described in the literature. High performance ion-exchange chromatography for the detection of underivatized validamycin and melamine has been proposed. The compounds were separated on HPLC using a styrene-divinylbenzene copolymer based cation-exchanger. For the separation of validamycin a resin in the hydrogen-form has given a good separation and for melamine in the Na⁺-form. For the first time an ion-exchange separation of validamycin is described allowing the separation of at least four components of commercial validamycin: validamycin A, validamycin B or G, validoxylamine A and validoxylamine B or G without prior derivatization. The methods were applied to determine the radiochemical purity of ¹⁴C- or ³H labeled compounds.This revised version was published online in November 2005 with corrections to the Cover Date.     

Sequence-specific predictive chromatography to assist mass spectrometric analysis of asparagine deamidation and aspartate isomerization in peptides

Deamidation of asparagine and spontaneous isomerization of aspartic acid in proteins and peptides occur frequently. These modifications result in a mixture of peptide variants containing all three residues in the sequences. Identification and isomer quantification for these systems are challenging tasks for tandem mass spectrometry commonly utilized in protein analysis. Chromatographic data provide a set of sequence-specific information complementary to mass spectrometry. In order to compare measured retention times (RTs) with those calculated from the sequences derived from protein databases, it is necessary to develop chromatographic models and tools allowing the prediction of RT and elution order for peptides with modified residues. In this work we extended recently introduced critical liquid chromatography of biomacromolecule model for prediction of RTs for peptides containing asparagines, aspartic acid, and isoaspartic acid residues.     

Using computer modeling to predict and optimize separations for comprehensive two-dimensional gas chromatography

In order to fully realize the separation power of comprehensive two-dimensional gas chromatography (GC x GC), a means of predicting and optimizing separations based on operating variables was developed. This approach initially calculates the enthalpy (DeltaH) and entropy (DeltaS) for the target compounds from experimental input data, and then uses this information to simultaneously optimize all column and runtime variables, including stationary phase composition, by comparing the performance of large numbers of simulated separations. This use of computer simulation has been shown to be a useful aid in conventional separations. It becomes almost essential for GC x GC optimization because of the large number of variables involved and their very complex interaction. Agreement between experimental and predicted values of standard test samples (Grob mix) using GC x GC separation shows that this approach is accurate. We believe that this success can be extended to more challenging mixtures resulting in optimizations that are simpler and transferable between GC x GC instruments.     

Purification to homogeneity of bovine prolactin by high-performance ion-exchange chromatography

Homogeneous bovine prolactin (bPRL) has been obtained using a procedure based on high-performance anion-exchange chromatography. The procedure enables up to 6 mg of 99.4% pure bPRL to be obtained per hour, with a recovery of 32.4%. The purity of the protein was checked by N-terminal sequencing and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The highly purified bPRL obtained with this method is suitable for complete structural and immunochemical studies.     

Dynamic coating ion-exchange chromatography of cations on an octadecyl-bonded silica stationary phase

It was found that common cations (Na+, NH4+, K+, Mg2+ and Ca2+) could be strongly retained on an ODS stationary phase when aqueous solutions of carboxylic acids were used as eluents. The chromatographic conditions used in this work were the same as in common cation-exchange chromatography on a cation-exchange resin and the retention behavior of the above-mentioned cations on the ODS column was quite similar to that on a cation-exchange column. The retention behavior and mechanism have been investigated using a number of carboxylic acids as eluents. The retention mechanism of the cations in these experiments was considered to be a dynamic coating ion-exchange mechanism. The carboxylic acids in the mobile phase were coated onto the surface of the ODS stationary phase and formed a dynamic carboxylic acid functional layer which could act like the functional group layer of a carboxylic group cation exchanger.     

Use of sodium pyrophosphate in the ion-exchange chromatography of oligonucleotides

All-Union Scientific-Research Institute of Molecular Biology. Vektor Scientific-Production Association, Ministry of the Medical Industry, Koltsova, Novosibirsk. Novosibirsk Institute of Bioorganic Chemistry, Siberian Branch, Russian Academy of Sciences. Translated from Khimiya Prirodnykh Soedinenii, No. 1, pp. 148–149, January–February, 1992.