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.     

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.     

Fast size-exclusion chromatography--theoretical and practical considerations

Fast SEC is a very interesting modification of conventional SEC. The need for it emerges from combinatorial chemistry and high-throughput experimentation, where high-speed analyses are required. The different approaches to change the speed of analysis are extensively described in this paper. Special attention is paid to the trade-off between analysis time and resolution and to the selection of optimal column lengths and flow rates. Simulations are used to design and to understand experiments. Integrity plots are constructed to judge the quality of various SEC systems. Fast separations in size-exclusion chromatography are found to be more favorable than suggested by conventional theory. The results are based on experimental data obtained for polystyrene using THF as mobile phase.     

Fast, comprehensive online two-dimensional high performance liquid chromatography through the use of high temperature ultra-fast gradient elution reversed-phase liquid chromatography

A new approach to high speed, comprehensive online dual gradient elution 2DLC (LCxLC) based on the use of ultra-fast, high temperature gradient elution reversed phase chromatography is described. Entirely conventional gradient elution instrumentation and columns are assembled in a system which develops a total peak capacity of about 900 in 25 min; this is equivalent to roughly one peak/2 s. Each second dimension gradient is done in a cycle time of 21 s and the peak retention times measured for a set of twenty six indole-3-acetic acid (IAA) derivatives are reproducible to 0.2 s. Each peak eluting from the first dimension column is sampled at least twice across its width, as the corresponding peaks on the second dimension column appear in two or three consecutive second dimension chromatograms, clearly indicating that there is little loss in the resolution gained in the first dimension separation. Application to the separation of the low molecular weight components of wild-type and mutant maize seedlings indicates the presence of about 100 peaks on a timescale of 25 min. Compelling illustrations of the analytical potential of fast, high temperature 2DLC are evident in the clear presence of nine distinct peaks in a single second dimension chromatogram from a single quite narrow first dimension peak, and the great power of 2DLC to solve the "analytic dynamic range" problem inherent in the measurement of small peaks that are neighbors to a gigantic peak.     

Molecular size fractionation of soil humic acids using preparative high performance size-exclusion chromatography

High performance size-exclusion chromatography (HPSEC) is useful for the molecular size separation of soil humic acids (HAs), but there is no method available for various HAs with different chemical properties. In this paper the authors propose a new preparative HPSEC method for various soil HAs. Three soil HAs with different chemical properties were fractionated by a Shodex OHpak SB-2004 HQ column with 10mM sodium phosphate buffer (pH 7.0)/acetonitrile (3:1, v/v) as an eluent. The HAs eluted within a reasonable column range time (12-25 min) without peak tailing. Preparative HPSEC chromatograms of these HAs indicated that non-size-exclusion effects were suppressed. The separated fractions were analyzed by HPSEC to determine their apparent molecular weights. These decreased sequentially from fraction 1 to fraction 10, suggesting that the HAs had been separated by their molecular size. The size-separated fractions of the soil HA were mixed to compare them with unfractionated HA. The analytical HPSEC chromatogram of the mixed HA was almost identical to that of the unfractionated HA. It appears that the HAs do not adsorb specifically to the column during preparative HPSEC. Our preparative HPSEC method allows for rapid and reproducible separation of various soil HAs by molecular size.     

Quantitative FTIR detection in size-exclusion chromatography

With the increasing popularity of evaporative interfaces, detection using Fourier Transform Infrared (FTIR) spectrometry in the mid-infrared region is becoming more important in size-exclusion chromatography (SEC). FTIR spectrometry is a powerful, and potentially very widely applicable, method for obtaining chemical functional group information for each molecular size fraction. Quantitative evaluation of polymer composition across the SEC chromatogram can provide more accurate characterization of heterogeneous polymer samples for problem solving and for material specification. The evaporative interface removes the SEC mobile phase at the exit of the column and deposits the eluting polymer as a continuous film stripe or as a series of discrete films on infrared transparent substrates. Initially this detection approach was used only for qualitative analysis. More recently, it is being used quantitatively. Previously we demonstrated that the quality of the film generated by the evaporative interface was critical to determining the suitability of the resulting FTIR spectra for quantitative analysis. In a continuation of this work, the objective of this paper is to develop a procedure for obtaining valid quantitative results for polymer blends with the interface. Experimental topics include improving the quality of polymer films by post-SEC treatments, off-line FTIR calibrating using other means to obtain high quality polymer films, and utilizing in-line SEC detectors in calibration. Interpretation aspects focus upon peak fitting of FTIR spectra, linear regression, partial least squares, and data pre-processing. PLS prediction with internal calibration using the second derivative of solvent-annealed film spectra was found to provide the best compromise between processing time, accuracy and precision.     

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.     

High throughput liquid chromatography with sub-2 microm particles at high pressure and high temperature

In this study, ultra performance liquid chromatography (UPLC) using pressures up to 1,000 bar and columns packed with sub-2 microm particles has been combined with high temperature mobile phase conditions (up to 90 degrees C). By using high temperature ultra performance liquid chromatography (HT-UPLC), it is possible to drastically decrease the analysis time without loss in efficiency. The stability and chromatographic behavior of sub-2 microm particles were evaluated at high temperature and high pressure. The chromatographic support remained stable after 500 injections (equivalent to 7,500 column volumes) and plate height curves demonstrated the capability of HT-UPLC to obtain fast separations. For example, a separation of nine doping agents was performed in less than 1 min with sub-2 microm particles at 90 degrees C. Furthermore, a shorter column (30 mm length) was used and allowed a separation of eight pharmaceutical compounds in only 40s.     

Two-dimensional liquid chromatography analysis of synthetic polymers using fast size exclusion chromatography at high column temperature

In recent years, two-dimensional liquid chromatography (2D-LC) has been used increasingly for the analysis of synthetic polymers. A 2D-LC analysis provides richer information than a single chromatography analysis at the cost of longer analysis time. The time required for a comprehensive 2D-LC analysis is essentially proportional to the analysis time of the second dimension separation. Many of 2D-LC analyses of synthetic polymers have employed size exclusion chromatography (SEC) for the second-dimension analysis due to the relatively short analysis time in addition to the wide use in the polymer analysis. Nonetheless, short SEC columns are often used for 2D-LC analyses to reduce the separation time, which inevitably deteriorates the resolution. In this study, we demonstrated that high temperature SEC can be employed as an efficient second-LC in the 2D-LC separation of synthetic polymers. By virtue of high temperature operation (low solvent viscosity and high diffusivity of the polymer molecules), a normal length SEC column can be used at high flow rate with little loss in 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.     

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.     

Observations on the rapid size-exclusion chromatography of proteins

Summary The retention behaviour of reference proteins on commercial siliceous size-exclusion supports was studied. Sorption was observed on both surface modified and unmodified supports. When sodium dodecylsulfate was added to the aqueous mobile phase, normal elution patterns were found. With this system, proteins, such as those isolate from different alfalfa genotypes, may be compared rapidly. Comparisons were facillitated by use of on-line central data processing capability.     

Fractionation of apple procyanidins by size-exclusion chromatography.

Oligomeric constituents of apple procyanidins were fractionated by size-exclusion chromatography using a TSKgel Toyopearl HW-40F column. The best separation was obtained using a mobile phase of acetone-8 M urea (6:4; adjusted to pH 2) at a flow-rate of 1.0 ml/min. In this chromatographic system, the use of 8 M urea in the mobile phase resulted in a molecular sieve effect without any surface affinity interaction between the gel beads and the procyanidin molecules. Each fraction obtained was examined by reversed-phase high-performance liquid chromatography and time-of-flight mass spectrometry. The order of elution of the procyanidins from the column was coincident with their degree of polymerization.     

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.     

Determination of naturally occurring hydroxynaphthoquinone polymers by size-exclusion chromatography

Summary Polymerization of alkannin, shikonin, and their derivatives, potent pharmaceutical substances, crucially affects their use in pharmaceuticals, cosmetics, and as food colorants, because it leads to loss of their antimicrobial activity, reduction of the lustre of their red coloration, and a decrease in their solubility. In this study size-exclusion chromatography (SEC) has been used for the first time for qualitative and quantitative analysis of monomeric and polymeric hydroxynaphthoquinone alkannin and shikonin derivatives. The purity and degree of polymerization has been determined to evaluate severalAlkanna tinctoria root samples from different geographical sources, and commercial samples of alkannin and shikonin, as pharmaceutical raw materials. Conditions for extraction of hydroxynaphthoquinones fromAlkanna tinctoria roots with olive oil were optimized in terms of polimerization, aiming to improve the biological activity of the final pharmaceutical product, Helixderm.