Light-scattering and turbidimetric detection of silica colloids in size-exclusion chromatography

Silica colloids were separated by size-exclusion chromatography and monitored by fluorimetric and UV detection. In the former means of detection, silica colloids were visualized by light-scattering. The signal intensity based on the light scattering increased with increasing size of the silica colloids. The maximum intensity was observed at excitation wavelengths around 270–290nm. In UV detection, silica colloids were visualized based on turbidimetry, and the signal intensity also increased with increasing size of the silica colloids and with decreasing detection wavelength. The signal intensities for both light-scattering and turbidimetric detection were a linear function of the concentration of the silica colloids. The detection limit at S/N = 3 for 78-nm colloids was 0.06 ppm for light-scattering detection whereas the LOD was 2.3 ppm for UV detection. Effects of mobile phase conditions and flow rate on resolution and peak shape were examined. Use of phosphate buffer allowed the separation of silica colloids of different sizes in size-exclusion chromatography.     

Polymer characterization by size-exclusion chromatography with multiple detection.

Size-exclusion chromatography with coupled multiangle light scattering and differential refractometry detectors has been used to obtain molecular mass and radius of gyration distributions of polydisperse polymer samples. From these data the scaling law between dimensions and the absolute molecular mass is obtained with just one sample of each polymer. Three different kinds of polymers are presented: polystyrene which serves as reference polymer, polyphosphazenes which behave abnormally in solution and poly(ethylene oxide) which is soluble in water. Since the relationship between dimensions and molecular mass depends on the extent of interactions between chain segments and solvent molecules, the scaling law provides information about the solution properties of the polymer.     

Longitudinal diffusion in size-exclusion chromatography: a stop-flow size-exclusion chromatography study.

Band broadening in size-exclusion chromatography (SEC) has an adverse effect upon calculated molecular mass averages, distributions, and dilute solution data generated using single- and multi-detector systems. In the past, the longitudinal diffusion contribution to band broadening in SEC has been considered negligible. This assumption has been investigated by using a stop-flow methodology (SF-SEC) that maximizes the potential for longitudinal diffusion while minimizing that for mass transfer. Under the given experimental conditions, the effects of B-term band broadening were manifest only below 30 KDa, irrespective of chemical functionality or molecular mass polydispersity. This type of broadening was found to be flow rate-independent for a representative high molecular mass polymer.     

Contactless conductivity detection of synthetic polymers in non-aqueous size-exclusion electrokinetic chromatography

A capacitively coupled contactless conductivity detection (CCD) system has been applied for the detection of neutral synthetic polymers in capillary size-exclusion electrokinetic chromatography (SEEC). Polystyrene standards, that were used as a model compounds, were separated on a capillary column packed with porous 10 microm silica particles with an electrokinetically driven mobile phase, and detected by CCD and UV detection simultaneously. Mass-calibration curves for polystyrene were constructed. Satisfactory results were obtained for the linearity, the run-to-run repeatability (<0.2% for the relative retention and <4% for the peak area) and the robustness of the detector. One of the major issues in this preliminary study was to investigate the origin of the peaks observed for the polystyrene standards. The effect of the molar mass of the polystyrenes on the sensitivity was small. Therefore, the signals obtained could not be explained as the result of an increased viscosity and a decreased solution conductivity of the solute zone. An alternative hypothesis is suggested, and recommendations for further research are given.     

Evaluation of size-exclusion chromatography and size-exclusion electrochromatography calibration curves

Size-exclusion chromatography (SEC) and size-exclusion electrochromatography (SEEC) are chromatographic techniques used to determine molecular mass (weight) distributions (MWD) of polymers. One important step in the data treatment to derive MWD parameters is the modelling of the calibration curves. The calibration curves applied in SEC and SEEC are generally not linear. In this study the modelling of calibration curves is being examined. Different polynomial models have been evaluated and compared, not only for model fit but also for their predictive properties. It was found that sometimes a straight line and sometimes a third-order polynomial model were best. The best model across the effective range (also called linear range) is not always found to be a straight line. The SEEC curves were found to have considerably higher prediction errors than the SEC ones. Reduction of the number of calibration standards to five or six did not greatly affect the predictive properties of the calibration curves, neither in SEC nor in SEEC.     

Thin-layer size-exclusion chromatography of polymers

Modern concepts of thin-layer chromatography of macromolecular compounds are reviewed, including the features and potentialities of thin-layer size-exclusion chromatography.     

Poppe plots for size-exclusion chromatography

Poppe plots provide a clear and unambiguous way to discuss the performance limits of separation systems. The effects of particle size, pressure drop and column permeability can be illustrated using such plots. The performance limits of size-exclusion chromatography are of interest, due to developments in combinatorial chemistry and high-throughput experimentation. In these fields, fast separations of high-molecular-weight analytes are required. In this paper, Poppe plots will be presented for size-exclusion chromatography. Because of the very high-reduced velocities encountered, the Poppe plots are found to be significantly different from those commonly observed in HPLC. Fast separations in size-exclusion chromatography are not as unfavourable as suggested by conventional theory. The results are based on experimental data obtained for a wide range of polystyrenes (1.7-3.25 kDa) using THF as mobile phase, but may be equally valid in other cases.     

Separation of complex branched polymers by size-exclusion chromatography probed with multiple detection

Size-exclusion chromatography (SEC) separates polymers by hydrodynamic volume (the universal calibration principle). Molecular weights can be determined using viscometry (relying on universal calibration) and light scattering (independent of universal calibration). In the case of complex branched polyacrylates with tetrahydrofuran as eluent, universal calibration is valid, although the separation in term of molecular weight is incomplete: a given elution slice contains a range of molecular weights, described in terms of a 'local polydispersity'. The local polydispersity index decreases when the number of branches per chain increases and complete separation is reached for highly branched chains.     

Indirect fluorimetric detection of proteins via postcolumn mixing with fluorescence probe in size-exclusion chromatography

Summary Proteins were visualized by postcolumn mixing with 2-p-toluidinyl-6-naphthalene sulfonate or 1-anilino-8-naphthalene sulfonate in size-exclusion chromatography. The indirect detection is based on fluorescence enhancement of the fluorescence probe owing to hydrophobic interaction with proteins. Bovine serum albumin gave the highest signal intensity among the proteins examined.     

Analysis of heparins by size-exclusion and reversed-phase high-performance liquid chromatography with photodiode-array detection

High-performance liquid chromatography (HPLC) of heparins was carried out with on-line photodiode-array detection. Average molecular weights and molecular weight distribution were calculated using computer data acquisition and handling; size-exclusion chromatography was performed using different selective microparticulate columns and narrow molecular weight distribution heparin standards for calibration; heparin peak purity was investigated using several methods for evaluation. Additional UV-absorbing compounds present in heparin preparations were characterized and quantitated by reversed-phase HPLC. These methods are useful for the analysis of the molecular weight distribution and peak purity of heparins and for the determination of additional drugs, additives or impurities.     

体积排阻色谱法定量检测9种型别人乳头瘤病毒原液中病毒样颗粒

据统计,5％以上的人类癌症由人乳头瘤病毒(HPV)导致.HPV疫苗的使用,尤其是多价HPV疫苗的使用,可有效预防HPV感染和肿瘤的发生.例如,9价HPV疫苗可有效预防90％以上HPV相关癌前病变.人乳头瘤病毒样颗粒(VLP)是HPV疫苗的唯一抗原.VLP由360份衣壳蛋白L1组成.VLP的含量测定对HPV原液和HPV疫...     

Fast size-exclusion chromatography at high temperature

We report on the size-exclusion chromatography (SEC) operated at high column temperature to reduce the analysis time. The column temperature was raised beyond the normal boiling point of the eluent and a sufficient column backpressure was applied to prevent the mobile phase from boiling by inserting a narrow bore tubing between the separation column and the detector. The narrow bore tubing also functions to cool the effluent down to the room temperature before it reaches the detector. Therefore, normal SEC detectors can be used without any modification. It was confirmed that the SEC analysis time could be shortened significantly by the high-temperature operation without serious deterioration in the resolution.     

Transfer-volume effects in two-dimensional chromatography: adsorption-phenomena in second-dimension size-exclusion chromatography

Gradient-elution LC × LC is a valuable technique for the characterization of complex biological samples as well as for synthetic polymers. Breakthrough and viscous fingering may yield misleading information on the sample characteristics or deteriorate separation. In LC × SEC another phenomenon may jeopardize the separation. If the analytes adsorb on the SEC column under the injection-plug conditions, peak splitting may occur. In LC × LC the effluent from the first column is the sample solvent for the analytes injected into the second dimension. If a gradient-elution LC × SEC setup is used (i.e. if reversed-phase gradient-elution LC is coupled to organic SEC and if normal-phase gradient-elution LC is coupled to SEC with a polar solvent), the percentage of weak solvent may be significant, especially at short analysis times. In this case adsorption in the first-dimension-effluent zone on the second-dimension SEC column can become an issue and two peaks--the first eluting in size-exclusion mode and the second undergoing adsorption--can be obtained. The work presented in this paper documents peak splitting in LC × SEC of polymers. The adsorption of the polymer on the size-exclusion column was proven in one-dimensional isocratic runs. The observed effects were modeled and visualized through simulation. Studies on the influence of the transfer volume were carried out. Keeping the transfer volume as small as possible helped to minimize peak splitting due to adsorption.     

Detailed characterization of cationic hydroxyethylcellulose derivatives using aqueous size-exclusion chromatography with on-line triple detection

A more complete understanding of polymeric, cationic cellulose derivatives, including polyquaterium-10 (Polymer JR), has become increasingly important in the eye care industry as thorough characterization of raw materials helps promote product quality and process control. Often such detailed information requires utilization of a combination of analytical techniques. In this work three Polymer JR samples with different viscosities were characterized using aqueous size exclusion chromatography (SEC) with a light scattering detector, a differential viscometer, and a differential refractometer (triple detection). Detailed molecular information such as absolute molecular weights, molecular weight distributions, intrinsic viscosities, and molecular conformations were obtained. One major challenge of analyzing cationic polymers is abnormal size exclusion separation, which could be caused by the ionic interaction between sample molecules and the column packing material. A selection of mobile phases varying in pH, buffer, organic solvent content, and molar concentration of salts was employed to evaluate the correlation of obtained molecular weight values and mobile phase composition. Universal calibration concept was used to examine the abnormal size exclusion separation phenomenon of Polymer JR samples when using different mobile phases. It was observed that the abnormal size exclusion was dependent on both the separation conditions and molecular weights of the samples. Despite the changes in separation parameters and uncharacteristic polymeric structure compared to conventional SEC samples, the use of aqueous SEC with triple detection provided reproducible and valuable molecular information of Polymer JR samples with low to medium molecular weights. By using a combination of high buffer content and adding organic solvent, the abnormal exclusion separation of high molecular weigh Polymer JR could be considerably reduced.     

Band broadening in size-exclusion chromatography of polydisperse samples

Understanding and controlling the band broadening is essential to obtain accurate molar-mass distributions by size-exclusion chromatography (SEC). In this paper, band broadening in SEC is reviewed from a contemporary perspective. The observed band broadening is due to dispersion inside and outside the chromatographic column (undesirable band broadening) and to the polydispersity of the sample (desirable SEC selectivity). The various contributors to band broadening are discussed. Integrity plots are introduced as a tool to evaluate the performance of specific SEC columns at given experimental conditions. For narrow polymer standards on single SEC columns the observed peak width is dominated by the chromatographic dispersion. MALDI-ToF-MS is demonstrated as an alternative to determine the PDI of narrowly distributed samples. The plate heights encountered at very high reduced velocities are found to be lower than expected. This is advantageous for fast separations by SEC.     

Micellar gradients in size-exclusion simulated moving bed chromatography

The selectivity of size-exclusion chromatography (SEC) can be modified by adding non-ionic micelles to the mobile phase. Surfactant-aided size-exclusion chromatography (SASEC) can therefore very well be performed in a gradient mode on an SMB, as is reported in this paper. A method has been developed for correctly positioning a micellar gradient over an SMB. The method is applied for size-exclusion chromatography with the non-ionic surfactant C12E23 as gradient forming solute, and demonstrated by applying it to a relevant chromatographic protein separation problem.     

Non-radioactive detection of MHC class II-peptide antigen complexes in the sub-picomole range by high-performance size-exclusion chromatography with fluorescence detection

In order to avoid chemical or structural modification of T-cell epitopes by labelling, a high-performance size-exclusion chromatographic fluorescence binding assay was developed, based on the intrinsic Trp fluorescence of major histocompatibility complex (MHC) proteins. The increase in Trp fluorescence intensity of the isolated human MHC product HLA-DR 1 on complex formation with unlabelled influenza matrix peptide[18-29] (IM[18-29]) was examined. Binding of IM[18-29] to the heterodimeric form of HLA-DR 1 (Kd = 4.8 mM) and to the disassembled alpha-and beta-subunits (Kd = 9.2 mM) could be demonstrated. In addition, the assay showed the peptide-induced formation of a dimeric conformer of HLA-DR 1, the nature of which is still undefined. Detection of HLA-DR 1 subunit-peptide complexes was possible in amounts of 25 ng in 10 microliter (80 fmol/microliter). The technique proved to be reproducible and less time consuming than common methods that need fluorescence or radioactive labelling.     

Observation of Ficoll charge using size-exclusion chromatography

The retention volumes of Ficoll samples of varying molecular weight were determined on porous glass and Superose columns, and compared with those of Pullulan. The retention of Ficoll is pH- and ionic strength-dependent, indicating that it bears a weak negative charge at moderate pH. Comparisons were made with similar charge repulsion effects for proteins on Superose to provide an estimate of the extent of charge on Ficoll, with the conclusion that only a few charges exist per molecule at neutral pH.     

Conductivity detection for molecular mass estimation of per-O-sulfonated glycosaminoglycans separated by high-performance size-exclusion chromatography

Chemically per-O-sulfonated polysaccharides, including glycosaminoglycans (GAGs) and hyaluronan oligosaccharides were analyzed using high-performance size-exclusion chromatography (HPSEC) with suppressed conductivity detection. The results were compared to those obtained by gel filtration HPLC using UV detection or fluorescence detection after the post-column reaction with 2-cyanoacetamide in strong alkaline solution. Analysis was performed on a TSKgel G3000SWXL HPSEC column in 5 mM boric acid (pH 7.0 adjusted by 10 mM NaOH). The use of conductivity detection, in the absence of any derivatization and under isocratic conditions gave a limit of detection in the picogram range. Preliminary studies suggest that this approach may be particularly useful in examining sulfonated polysaccharides and oligosaccharides having no UV chromophore, such as those prepared from O-sulfonated fucans and galactans isolated from algae.