Wallace W. Yau

Broad-range linear calibration in high-performance size-exclusion chromatography using column packings with bimodal pores

Summary The linear size-separation range for size-exclusion chromatography (SEC) (gel permeation chromatography) is greatly extended by optimizing the pore-size distribution and the internal pore volume of particles in the columns. This approach permits easier, more accurate computation of molecular weights by the “single broad standard” method, which assumes a linear calibration graph. Linearities of 4–5 decades of molecular weight have been demonstrated, in comparison to the 2.5–3 decades of linearity obtained with conventional column arrangements. The new concept is to couple SEC columns containing only two discrete pore sizes, (bimodal), having about one decase difference in pore size and approximately equal pore volumes for the two pore sizes. This is in sharp contrast to the widely accepted approach of connecting several columns containing several packing materials of slightly different pore sizes and pore volumes. The desired pore sizes of this bimodal distribution are arranged so that the linear portion of the individual column molecular weight calibration graphs is substantially non-overlapping, and the pore volume of each mode is such that the linear portions of the calibration graphs are essentially parallel. The theoretical support and quantitative optimization guidelines for the bimodal approach of practical column selection are provided. The wide-linear molecular weight calibration also has been demonstrated with columns of new 10-μm single bimodal pore-size silica particles with narrow pore-size distributions. The use of a single packing material for SEC greatly simplifies column inventory and improves convenience, while maintaining the high chromatographic resolution and accurate molecular weight measurements associated with high-performance SEC.

Effect of column performance on the accuracy of molecular weights obtained from size exclusion chromatography (gel permeation chromatography)

This paper presents a functional molecular weight accuracy criterion for gel permeation chromatography column performance that connects the traditional concepts of column resolution with molecular weight accuracy in gel permeation chromatographic measurements. The criterion predicts accuracy of molecular weights directly from column parameters; conversely it can be used to specify needed column parameters to obtain a selected level of gel permeation chromatography-molecular weight accuracy. According to this accuracy criterion, the performance of the columns can be quantitatively rated in terms of the product of two fundamental column parameters: σ, the standard deviation of the peak (related to the column plate count for a monodispersed polymer), and D2, related to the slope of the linear portion of the chromatographic calibration curve. Use of this σD2 criterion is illustrated with data obtained on different types of column packings and various sets of columns. The effects of pore geometry and column operating variables on the column resolution are discussed in terms of separation and dispersion theories. A quantitative theory for interpreting the effect of packing pore size distribution on gel permeation chromatographic performance was developed. The theory indicates that the effect on D2 diminishes at narrow pore size distribution.

Sedimentation field flow fractionation at high force fields

New sedimentation field flow fractionation instrumentation with rotor speeds up to 32,000 rpm (about 100,000 g) permits separations of materials of somewhat less than 106 molecular weight, as well as larger particles. The apparatus has been constructed in an ultracentrifuge with a unique plastic channel rotor assembly. A key feature is that interchangeable plastic sedimentation field flow fractionation channels are mounted within a metal rotor bowl filled with liquid of a specified density. This unique construction allows liquid to surround the plastic rotor assembly, essentially equalizing stresses on the plastic parts at high force fields and permitting low-cost channels to be conveniently constructed. With this design, mechanical stress on the component plastic rotor parts is minimized and specified channel dimensions can be maintained over a wide range of force fields. The new apparatus has been utilized for characterizing a range of materials of biological and industrial interest.

Retention Characteristics of Time-Delayed Exponential Field-Programmed Sedimentation Field-Flow Fractionation

Abstract Sedimentation field-flow fractionation (SFFF) is a promising method for the high-resolution separation of a wide variety of suspended particulates and dissolved macromolecules. By using a new SFFF technique with a time-delayed exponential force-field (rotor speed) decay, quantitative particle-size distribution analyses in the 0.01–1 μm range can be performed in a few minutes. Relative to constant-field SFFF, programmed force-field operation can drastically decrease analysis time and improve detection sensitivity while maintaining adequate resolution. The linear relationship between particle retention and logarithm of particle diameter or mass for the new technique significantly simplifies data handling for convenient and accurate analyses. Standard graphs have been prepared to show how separation variables such as exponential decay time constant, τ, initial rotor speed, ωo, channel thickness, W, and flowrate, F, can affect particle retention. These simple and quantitative relationships are useful...

Asymmetric-channel flow field-flow fractionation with exponential force-field programming

Abstract Equipment and techniques have been developed to program the crossed-flow force fields with parallel-plate, asymmetric channels in flow field-flow fractionation (F1FFF). Force-field programming permits the rapid separation of samples with a wide range of molecular sizes; resolution is easily varied. Detectability of late-eluting components is enhanced as a result of band sharpening. Force-field programming probably can be performed with many functions. Exponential force-field decay method produces retention times vs. diffusion coefficient or particle size plots that are more linear than those from a constant force field. Resolution and measurement precision is more constant over the separation range. The exponential function also simplifies computer software measuring diffusion-coefficient and particle-size distributions. Optimum operating parameters for a desired F1FFF separation are predicted with a quantitative exponential force-field decay theory. Force-field programming significantly enhances the utility of the mild F1FFF method. Appropriate samples for this method include synthetic and natural polymers, organic and inorganic colloids and a variety of particulates.

Thermal field-flow fractionation of water-soluble macromolecules

Abstract While previous thermal field-flow fractionation (TFFF) separations have been carried out with mobile phases containing organic solvents, we have found Our studies suggest that the thermal diffusion coefficients for highly water-soluble macromolecules are often very small in pure water. Adjustment of t For useful separations in aqueous systems, the TFFF equipment should generate relatively large temperature differences between the channel faces. Fract

Computer simulation study of size-exclusion chromatography with simultaneous viscometry and light scattering measurements

Abstract The integration of an on-line viscosity or light scattering (LS) detector with size-exclusion chromatography (SEC) improves the accuracy with which polymer molecular mass distributions can be measured. The coupling of a viscometer and a light scattering detector in one SEC instrument potentially offers improved precision and dynamic range for SEC polymer conformation studies. However, the increased complexity of these experiments and the subsequent data handling introduce a number of problems not present in conventional SEC. A computer simulation of the multiple detector SEC experiment was developed in order to study these effects in detail. The computer model is described and preliminary data are presented to illustrate some of the additional features of SEC with multiple detectors.

Comparison of sedimentation field flow fractionation with chromatographic methods for particulate and high-molecular-weight macromolecular characterizations

Abstract Quantitative particle size and molecular weight determinations by time-delayed exponential force-field sedimentation field flow fractionation (TDE-SFFF) can currently be carried out in 15–20 min using automated apparatus with force fields of up to 50,000 gravities. New resolution parameters provide a common basis for comparing the ability of the commonly used separation methods for particle size analyses. These parameters show that TDE-SFFF has a 5–10 fold and 10–50 fold greater specific resolution than size-exclusion chromatography (SEC) and packed column or capillary hydrodynamic chromatography (HDC), respectively. Because of high resolving power and other characteristics, TDE-SFFF provides superior accuracy in particle size distribution analyses relative to these other separation methods, as confirmed by direct comparisons with typical literature data for a range of particulate samples. TDE-SFFF also has similar advantages over conventional non-chromatographic methods. For example, SFFF exhibits approximately the same resolving power as disc centrifugation but a much wider dynamic range of particle diameter separation in a single analysis. SFFF provides higher separation resolution than SEC and HDC because of intrinsic differences in retention mechanisms. These latter chromatographic methods separate species by size-exclusion effects —peaks elute prior to the mobile phase solvent —therefore, HDC and SEC are basically limited by available fractionation volume. On the other hand, SFFF exhibits true retention like the affinity liquid chromatography (LC) methods —peaks elute after the unretained mobile phase solvent. In contrast to SEC and HDC, but like LC, TDE-SFFF has the potential for very high peak capacity.

Polymer Characterization by SEC-Viscometry: Molecular Weight (MW), Size (Rg) and Intrinsic Viscosity (IV) Distribution

Abstract New polymer characterization capabilities have recently been added to our size exclusion chromatography (SEC) using an on-line viscosity detector. In addition to molecular weight distribution (MWD) capabilities, we now also can determine the intrinsic viscosity distribution (IVD), and the molecular size distribution, i.e., polymer radius-of-gyration, or Rg-distribution (RGD) of polymer samples. Polymer conformation and branching features can now be studied by the log(Rg) versus log(MW) results of a single SEC-viscometry experiment. Also added to our SEC-viscometry analyses is the absolute Mn method recently proposed by J. M. Goldwasser for handling the difficult problems of determining molecular weight (MW) of copolymers and polymer blends. In this new method, the number-average molecular weight (Mn) of a complex polymer sample can be determined by SEC using an on-line viscosity detector, without the need of an on-line concentration detector.

Separation Mechanisms in Gel Permeation Chromatography

Abstract This paper presents flow rate studies, vacancy chromatography, and a static mixing experiment. Data obtained on an unpacked column (a straight tube) and on a column packed with nonporous glass beads are also reported. The results reveal that peak dispersion in GPC arises mainly from lateral diffusion in the stationary phase (permeation in and out of the porous substrate) and from lateral diffusion in the mobile phase. GPC peak separation is mainly dominated by the process of steric exclusion. Pore size distribution data obtained on Bio-Rad porous glass are shown to illustrate the preference of random coil theories over theories of the equivalent sphere in the interpretation of steric exclusion of flexible polymers. The data are discussed in terms of Hermans diffusion theory and Cassasas exclusion theory.