Enhancement of performance in sedimentation field-flow fractionation by temperature elevation.

Nonequilibrium theory, combined with the principles of time optimization, show that the time necessary to achieve a given separation in FFF is scaled to eta/T, where eta is viscosity and T is absolute temperature. The eta/T ratio for water (and other common liquids) decreases several-fold for modest temperature gains of approximately 20-60 degrees C, implying a significant advantage for FFF operation at elevated temperatures. This concept was tested by modifying a standard sedimentation FFF apparatus with a heating system. The separation of 0.220-0.742 microns polystyrene latex beads in aqueous carrier liquids was compared at room temperature and at elevated temperatures of 51 and 68 degrees C. Both separation power and speed were improved. In accordance with the predicted eta/T scaling, the separation time of five bead sizes at a given resolution level was reduced by a factor of approximately 2.4 (from 29 to 12 min) in elevating the temperature from 25 to 68 degrees C. Some other potential benefits of temperature elevation in FFF are discussed.

Hydrodynamic relaxation in flow field-flow fractionation using both split and frit inlets.

Two means are described for achieving hydrodynamic relaxation and thus avoiding the stopflow injection procedure in field-flow fractionation (FFF): split flow injection and frit inlet injection. The advantages, disadvantages, and the theoretical basis of these procedures are discussed. Incremental band broadening due to the final relaxation step is examined theoretically and shown to be negligible when the flow rate of the sample inlet substream is small compared to the total channel flow rate. The optimization of the sample inlet flow rate is discussed. Experimental results for both injection procedures are reported for flow/steric (or hyperlayer) FFF applied to latex standards, confirming the expected trends. However, closer examination shows that the observed incremental band broadening associated with hydrodynamic relaxation is somewhat larger than the value predicted.

Particle size distribution by sedimentation/steric field-flow fractionation: development of a calibration procedure based on density compensation.

Because of the important but mathematically complex role played by hydrodynamic lift forces in sedimentation/steric FFF, applied generally to particles greater than 1 micron in diameter, retention cannot readily be related to particle diameter on the basis of simple theory. Consequently, empirical calibration is needed. Unfortunately, retention is based on particle density as well as size so that a purely size-based calibration (e.g., with polystyrene latex standards) is not generally valid. By examining the balance between driving and lift forces, it is concluded that equal retention will be observed for equal size particles subject to equal driving forces irrespective of particle density. Therefore by adjusting the rotation rate to exactly compensate for density, retention can be brought in line with that of standards, a conclusion verified by microscopy. Linear calibration plots of log (retention time) versus log (diameter) can then be used. This approach is applied to two glass bead samples (5-30 and 5-50 microns) using both a conventional and a pinched inlet channel. The resulting size distribution curves are self consistent and in good agreement with results obtained independently.

Evaluation of pinched inlet channel for stopless flow injection in steric field-flow fractionation.

In this article the concept of utilizing a pinched inlet channel for field-flow fractionation (FFF), in which the channel thickness is reduced over a substantial inlet segment to reduce relaxation effects and avoid stopflow, is evaluated for steric FFF using one conventional channel and two pinched inlet channels. It is shown that with the proper adjustment of flow-rate, the stopflow process in FFF can be completely avoided, thus bypassing the flow interruption associated with stopflow and reducing separation time. The maximum flow-rate that can be used for stopless flow operation without incurring zone distortion is shown to agree reasonably well with simple theory; slight departures from theory are attributed to the existence of reduced transport rates of large particles through thin channel structures.

Hydrodynamic relaxation using stopless flow injection in split inlet sedimentation field-flow fractionation.

In this paper relaxation effects in both the normal and steric operating modes of sedimentation field-flow fractionation are examined by using three different injection procedures: stop flow, stopless flow, and a new stopless flow procedure employing an inlet splitter. In the usual operation of field-flow fractionation (FFF), a stop procedure is used in which the channel flow is halted for an adequate period of time after injection for sample relaxation (in which the sample particles approach equillibrium near one wall) before the resumption of channel flow. If the channel flow is not stopped (stopless flow procedure), the elution profile is shifted and distorted due to the downstream migration of the particles during the relaxation process. To avoid peak distortion while retaining the advantages of the stopless flow procedure, a physical splitter at the channel inlet divides the entering flow stream into two substreams; sample is injected into only one of these. In this way a rapid (although not complete) hydrodynamic relaxation is realized. This stopless split flow injection procedure is compared to ordinary stop and stopless flow procedures using both submicrometer (normal FFF) and supramicrometer (steric FFF) polystyrene latex particles. It is found that much of the distortion normally accompanying stopless flow injection is eliminated by this new procedure. However, further optimization is needed to match the high resolution of the stop flow method.

Characterization of albumin microspheres by sedimentation field-flow fractionation.

Sedimentation field-flow fractionation (FFF) is a new technique that separates and characterizes submicron particles. In the present work, two independent sedimentation FFF methods are presented to characterize bovine serum albumin microspheres in terms of particle size, polydispersity, and diffusion coefficient. Particle diameters and polydispersities determined by the two sedimentation FFF methods were in excellent agreement with each other and in good agreement with values calculated from transmission electron microscopy (TEM) measurements. The diameters calculated from the two FFF methods and TEM were 0.349, 0.346, and 0.354 microgram, respectively.

Flow field-flow fractionation: new method for separating, purifying, and characterizing the diffusivity of viruses.

The nature and theory of flow field-flow fractionation is described, and its potential applicability to virus-like particles is discussed. Different virus types are shown to be retained at different levels. Retention can be controlled by variation of the experimental parameters, in good agreement with theory. However, a mild adsorption effect is indicated and requires the development of alternate strategies for measuring diffusion coefficients. For Qbeta, our value agrees well within 10% of literature values; the values obtained for other viruses, using Abeta as an internal standard, are untested. Finally, it is demonstrated that flow field-flow fractionation can cleanly fractionate two viruses from one another and from an albumin impurity, that samples as large as several milligrams in size can be analyzed, and that the method has potential utility in the quantitative and qualitative analysis of virus systems.

Flow-field-flow fractionation: a versatile new separation method.

Flow-field-fractionation is a new separation technique that seems likely to have broad applicability. Its theoretical scope includes any solute for which one can find a solvent and a semipermeable membrane. The principles on which the technique is based are presented. Some experiments with polystyrene beads, viruses, and proteins verify that retention depends solely on diffusion coefficients.

Electrical field-flow fractionation of proteins.

Protein separation has been achieved by electrical field-flow fractionation, a heretofore unrealized separation technique. Some advantages of this method relative to electrophoresis are the low voltage required, the lack of adverse heating and support effects, and the existence of the method as an elution technique. A comparison of theoretical and experimental retention shows good agreement.

Dense-gas chromatography of nonvolatile substances of high molecular weight.

Working at pressures of up to 2000 atmospheres, more than ten times higher than in previous gas chromatography, we used the solvent power of dense gases to enable migration of chromatographic substances of molecular weights as high as 400,000. Carotenoids, corticol steroids, sterols, nucleosides, amino acids, carbohydrates, and several polymers have been caused to migrate, separated, and detected in NH(3) and CO(2) carrier gases at temperatures of 140 degrees and 40 degrees C, just above the respective critical points. Previously such compounds either defied separation by gas chromatography or had to be chromatographed as their more volatile derivatives.

Turbulent-gas chromatography.

Turbulent flow in gas chromatography was achieved and its effect was studied with high-speed, high-pressure equipment. A gas-solid capillary and several packed columns were used. The onset of turbulence was associated with abrupt decrease in peak width in the capillary and gradual leveling off and decrease in the Packed width in the existence of separation under turbulent conditions was shown. The Potential of the method was demonstrated by its short elution times and generation of theoretical Plates.