Relative peak broadening and pore size distribution of the stationary phase in exclusion chromatography

Summary It is shown experimentally that in size exclusion chromatography theh-values of a homologous series versus the relative molecular mass of the samples and the pore size distribution of the packing material determined by exclusion chromatography exhibit the same maximum.Part of the Ph.D. Thesis of W. Werner, University of Saarbrücken, Saarbrücken 1976.     

Direct determination of band broadening in size exclusion chromatography

A simple method to correct the measured extent of band broadening in size exclusion chromatography for the contribution of narrow (polydisperse) standards is presented. It is based on the assumptions that commercial polymer standards can be described by a Poisson distribution and the additivity of peak variances. Two sets of standards (polystyrene from two suppliers) were investigated under normal working conditions, i.e. a combination of four columns with different porosities and a flow rate of 1 ml/min. Furthermore, the polystyrene standards were used to determine the extent of band broadening for four additional combinations of columns (varying in their separation range and porosities) as a function of the elution volume. The assumption of a constant peak variance for band broadening turned out to be a (very) rough approximation for some combinations of columns, but all results taken together demonstrate that this assumption is not generally applicable. Qualitative agreement between theory and experiment was found with a rearranged van Deemter equation.     

Investigation and interpretation of band broadening in size exclusion chromatography

Study of Band Broadening occurring in Size Exclusion Chromatography (SEC) is reported using very narrow PS standards obtained and characterised by Temperature Gradient Interaction Chromatography (TGIC). Chromatograms are fitted by Exponentially Modified Gaussian functions (EMG) and mapping of band broadening is obtained for different column sets. Interpretation of the skewing of the chromatograms is proposed with a new model using Brownian motion properties inside the pores. That explains why band broadening and tailing become so important near total exclusion volume.     

Characterization of large water-soluble macromolecules by size exclusion chromatography

A method for characterizing very large, water-soluble polymers by size exclusion chromotography (SEC) has been developed. Sephacryl S1000 packing material, a precision syringe pump, and an eluent pressure detector have been utilized to produce highly accurate chromatograms of polymers having molecular hydrodynamic diameters up to 250 nm. Previous SEC analysis has been limited to polymers having hydrodynamic diameters of less than 120 nm.     

Alternative approaches for the estimation of the band broadening parameters in single-detection size exclusion chromatography

New approaches for the determination of the extent of symmetric and asymmetric band broadening (BB) in size exclusion chromatography (SEC) are presented. For this purpose raw data was simulated by starting with either a theoretical Poisson number chain length distribution (NCLD), or a log-normal weight chain length distribution (WCLD). Each distribution was first converted to a BB-free mass chromatogram, as typically obtained from a standard differential refractive index detector. Then, the broadened (or "measured") chromatograms were simulated by convoluting the BB-free chromatograms with a BB function, which was assumed to follow symmetrical (Gauss) as well as unsymmetrical (exponentially modified Gauss) function. A broad range of BB parameters (standard deviation, sigma(BB), and exponential decay, tau(BB)) was used for the simulations. The approaches are based on the determination of the points of inflection belonging to the peak of the broadened chromatogram, and closed as well as empirically derived equations connecting the peak width, its variance, and the parameters sigma(BB) and tau(BB). The developed methods are applicable for Poisson distributions well above a peak chain length of 100.     

Phenomena of insulin peak fronting in size exclusion chromatography and strategies to reduce fronting

Insulin peak fronting in size exclusion chromatography (SEC) results in more than 10% yield loss in the production of insulin. The goal of this study is to understand the mechanisms of peak fronting and to develop strategies to reduce fronting and increase insulin yield. Chromatography experiments ruled out pressure surge, viscous fingering, and adsorption as the cause for peak fronting. Theoretical analysis based on a general rate model indicated that reversible dimerization is the major cause for peak fronting and reducing the dimerization equilibrium constant is the most effective method for reducing fronting. Two strategies were developed and tested to reduce the degree of dimer formation. The first strategy was to use 0.1N acetic acid as the presaturant and eluent. The second strategy was to use 0.8 or 2.8N acetic acid in 20vol.% denatured ethanol as the mobile phase. The experimental results showed that both strategies can reduce insulin peak fronting in SEC, maintain desired product purity, and increase insulin yield to higher than 98%.     

The mechanism of peak broadening in paper chromatography

Summary The mechanism of peak broadening in paper chromatography was investigated by comparing the peak widths in paper chromatography with those caused only by diffusion in the paper for nine amino acids of widely differing R_f values.It appeared that eddy diffusion and resistance to mass transfer do not contribute to the peak broadening. By considering the peak widths as a function of R_f it could be shown that diffusion in the stationary phase cannot be neglected.

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Zusammenfassung Der Vorgang der Peak-Verbreiterung in der Papierchromatographie wurde untersucht. Dazu wurden die Peakbreiten nach Chromatographie mit den nur durch Diffusion erhaltenen verglichen. Neun Aminosäuren mit sehr unterschiedlichen R_f-Werten wurden verwendet. Wirbeldiffusion und Massentransport spielen bei der Peak-Verbreiterung keine Rolle, dagegen kann der Einfluß der Diffusion in der stationären Phase nicht vernachlässigt werden.

     

An analytical solution of a newly proposed peak broadening equation and extension of polydispersities to higher molecular weight averages in size exclusion chromatography (SEC)

Summary Herein is reported an analytical solution to the peak broadening or peak dispersion/flattening equation based on the recently proposed Instrumental Spreading Shape Function and its application to correction for imperfect resolution (inadequate peak separation and/or excessive peak broadening) for higher molecular weight averages. The relationship of these higher MW averages with the familiar Weight Average and number average molecular weights is also discussed. Criteria for perfect resolution are specified and a true molecular weight calibration curve is accordingly defined.     

Recent progress in carbohydrate separation by high-performance liquid chromatography based on size exclusion

Since the introduction of stationary phases based on microparticulate porous silica and polymeric sorbents, rigid and semi-rigid, size-exclusion chromatography (SEC) has become established as a form of high-performance liquid chromatography. In recent years, there have beeen revolutionary developments in detection systems for high-performance SEC, which have placed the use of the method for the determination of molecular-size and molecular-weight distributions of polymers on a sound theoretical basis andincreased the range of information on molecular characteristics that can be retrieved from SEC data. This review surveys these changes in SEC systems and their application to the separation and molecular-weight distribution analysis of carbohydrates.     

Bioaffinity chromatography: synergy between interactive chromatography and molecular recognition for the separation and analysis of macromolecules

Affinity chromatography, commonly regarded as an integral tool in macromolecular separation sciences, also provides an analytical method to study structure-function relationships of macromolecular interaction processes and to design recognition molecules. The latter, as found recently for the case of antisense peptides, may be useful as affinity agents in immobilized forms to effect new types of biomolecular separation.     

High-performance size exclusion chromatography as a rapid method for the separation of steroid hormone receptors

Triazine dyes, bound to polyethylene glycol, have been used to influence the partition of some enzymes within a dextran-polyethylene glycol-water two-phase system. The enzymes, present in a protein extract from baker's yeast, included glucose 6-phosphate dehydrogenase, glyceraldehyde phosphate dehydrogenase, 3-phospho-glycerate kinase and alcohol dehydrogenase. The partition coefficients of the enzymes could be changed by a factor of 10-500 in favour of the polyethylene glycol-rich phase, while the partition of bulk protein was much less affected. The influence of the concentration of polymer-bound dye and phase-forming polymers, temperature, pH, kind and concentration of salt and the presence of nucleotides on this affinity partitioning effect was studied. The extraction was effective even at high concentrations of dye and protein (40 g/l). A partial purification (32-fold) of glucose 6-phosphate dehydrogenase was carried out by an extraction in five steps.     

分子排阻色谱硅质填料的制备及其对蛋白质的分离机理

利用自制的四种不同粒径的硅溶胶,通过堆积法来制备分子排阻色谱多孔硅质填料,该填料在进行化学键合改性后,形成二醇固定相。利用二醇固定相对蛋白质进行分离分析方面的研究。此填料粒径小,有利于蛋白质生物大分子的高效快速分离分析。     

New peak broadening parameter for the characterization of separation capability in capillary electrophoresis

The influence of separation conditions on peak broadening is usually estimated by the number of theoretical plates. Using the data available in literature and experimental data, it is shown that in pressure‐assisted capillary electrophoresis the plate number is not directly related to the separation capability of conditions used. The experiments at different electrolyte flow velocities demonstrate that a higher plate number (the best separation efficiency) can be obtained with a lower peak resolution. Since ions are separated by electrophoresis due to the difference in electrophoretic mobilities, the peak width in terms of electrophoretic mobility is suggested as a new peak broadening parameter describing the separation capability of the conditions used. The parameter can be calculated using the tailing factor and the temporal peak width at 5% of the peak height. A simple equation for the resolution calculation is derived using the parameter. The advantage of the peak width in terms of mobility over other parameters is shown. The new parameter is recommended to be used not only in pressure‐assisted capillary electrophoresis but also in general capillary electrophoresis when in a number of runs the virtual separative migration distance and separation capability of the conditions used change widely.     

Maximizing peak capacity and separation speed in liquid chromatography

The practical effects of gradient time and flow rate on the peak capacities of a range of analytes of differing molecular weights (MWs) and physico-chemical properties have been evaluated using ultra high pressure LC instrumentation with sub-2 mum and superficially porous particle phases. Optimum peak capacity, in RP gradient LC, for small molecules, including typical pharmaceutical drugs and peptides with MWs up to 1300, was demonstrated at a maximum flow rate for a given gradient time (i.e. up to 40 min). Flow rates significantly higher than the optimum in the van Deemter plots and also higher than those typically employed by the majority of the chromatographers today are recommended for gradient LC (i.e. up to 1.0 mL/min on 50-150x2.1 mm 1.7 mum columns). This recommendation is applicable for temperatures above 40 degrees C, i.e. temperatures typically utilized for separations employing sub-2 mum particles to reduce column back pressure. Van Deemter and pseudo van Deemter plots were determined and combined with chromatographic gradient elution theory to explain our unexpected observations. The derived models exhibited good agreement between experimental and predicted peak capacities (absolute average error 4%, max. error 12%).     

Chemometric approach to the treatment of benzodiazepine separation and peak broadening in capillary electrophoresis.

A chemometric methodology was used to study capillary efficiency and the separation of ten benzodiazepines in capillary electrophoresis. The resolution between two adjacent peaks on the electropherogram was estimated and the overall quality of the separation was assessed by means of a new response function. The nature (methanol or acetonitrile) and proportion of the organic modifier both in the background electrolyte and the sample buffer and the injection time were considered. The results predicted that if the sample had a lower dielectric constant than the background electrolyte buffer then a much larger injection volume could be used. The computer optimization routine was experimentally validated and the result demonstrated that the fastest electrophoretic reparation was obtained with acetonitrile (7 min instead of 9 min with methanol).     

Separation technique based on electrophoresis,chromatography and magnetism phenomena: the migration time and peak broadening

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Adsorption and desorption of macromolecules on solid surfaces studied by on-line size exclusion chromatography 2. Preferential adsorption and exchange processes

Preferential and exchange adsorption of polymers differing in molar mass and/or chemical nature under dynamic conditions were investigated using on-line size-exclusion chromatography (SEC). The sample investigated dissolved in an appropriate solvent was injected into a small adsorption–desorption column packed with nonporous silica. A nonadsorbed or desorbed fraction of the polymer was directed into an SEC column for determination of both the amount and the molecular characteristics. This approach is in many aspects superior to other techniques for studies of polymer adsorption onto solid surfaces due to its low sample and time consumption. At a low degree of surface coverage, adsorption and desorption of macromolecules were rapid and were affected by the rate of supply of macromolecules to the adsorbent surface. The exchange between macromolecules at the stage of surface saturation was found to depend on the mean molar masses of preadsorbed and displacing polymer species and possibly also on the chain flexibility of the macromolecules. It was shown that the preferential adsorption driven by the chain-length difference upon saturation of the adsorbent surface was more noticeable if the preadsorbed macromolecules were smaller. Received: 7 April 1999 Accepted in revised form: 21 July 1999     

Interconversion of cyclosporin molecular form inducing peak broadening,tailing and splitting during reversed-phase liquid chromatography

Summary Reversed-phase liquid chromatography of cyclosporin A, a peptide immunosuppressant, at various temperatures produced remarkably different chromatograms. At 60°C one sharp peak was obtained, at 23°C this became a single broad peak and between 15° and 0°C this became one high sharp peak followed by a tailing or low plateau. Remarkably different chromatograms were produced also by varying the mobile phase flow-rate. The effects of both temperature and flow-rate on the chromatogram could be accounted for by interconversion between two forms of the cyclosporin molecule. Kinetic analysis showed that one form was converted by first-order kinetics with a half-life of 2.0 min at 20°C and that the apparent activation energy for the conversion was about 18 kcal/mol. The other two immunosuppressants, cyclosporin C and D, were also shown to undergo interconversion. This kinetic analysis of the interconversion should be helpful in clarifying the relationship between molecular structure and activity of the immunosuppressants.     

Information theory of optimization in chromatography: Optimum peak separation and dependency on peak area

Summary If a peak (j=2) moves along the time axis of a chromatogram with no change in the position ₁ of the other peak (j=1) or in areas A₁ and A₂, the optimum separation, _s, is defined as the resolution R_s which provides the maximum information, called FUMI, among all possible positions ₂ of the peak (₂₁). This paper demonstrates that optimum separation, _s, of chromatographic peaks critically depends on the peak areas. As the area ratio A₂/A₁ of overlapped peaks increases, greater separation (higher resolution R_s) is needed to obtain maximum information. The quantitative relationship between _s and A₂/A₁ is derived by computer simulation.