Throughout this specification, the term “particle” refers to the constituents of liquid sample aliquots that may be molecules of varying types and sizes, nanoparticles, virus like particles, liposomes, emulsions, bacteria, colloids, etc. Their size range may lie between 1 nm and several thousand micrometers.
The separation of particles in a solution by means of field flow fractionation, FFF, was studied and developed extensively by J. C. Giddings beginning in the early 1960s. The basis of these techniques lies in the interaction of a channel-constrained sample and an impressed field applied perpendicular to the direction of flow. Among those techniques of current interest is cross flow FFF, often called symmetric flow or SF1FFF, whereby an impressed field is achieved by introducing a secondary flow perpendicular to the sample borne fluid within the channel. There are several variations of this technique including asymmetric flow FFF, or A4F, and hollow fiber, or H4F, flow separation.
Other FFF techniques include sedimentation FFF, SdFFF, wherein a gravitational/centrifugal cross force is applied perpendicular to the direction of the channel flow; electrical FFF, EFFF, wherein an electric field is applied perpendicular to the channel flow; and thermal FFF, ThFFF, wherein a temperature gradient is transversely applied. As the application of a particular FFF technique is achieved by means of a corresponding device, said application will be referred to herein as a type of “field flow fractionator.”
Common to all these field flow fractionators is a fluid, or mobile phase, into which is injected an aliquot of a sample whose separation into its constituent fractions is achieved by the application of the cross field. Many of the field flow fractionators allow for the control and variation, during the time the sample aliquot flows down the channel, of the channel flow velocity and the strength of the applied cross field. Common also to these field flow fractionators is the fact that only the cross field and channel flow rates may be varied and, only then, throughout the entire region in which the separation is occurring. Although such programming is capable of producing effective separations for a wide variety of particle classes, it has associated limitations.
An illustration of such limitations relates to the separation of particles by means of a symmetric flow cross flow fractionator. As the sample aliquot begins to undergo non-steric separation while it moves down the channel, the smaller particles lead the larger ones. By increasing the cross flow rate, the separation of all species continues yet the larger fractions begin to trail further behind their smaller sized companions. With sufficient cross flow, these larger fractions may be slowed down considerably while the smaller particles have already completed their traversal of the channel producing their associated fractionation. By this time, the smaller fractions, though separated, may have been diluted significantly so that their local peaks have broadened and their associated concentration diminished. Upon leaving the channel, subsequent analyses using various detection means may be unable to detect fractions of such correspondingly very low concentration. While the retained larger particles as yet may not have had sufficient time to complete their passage through the channel and to separate therein, the smaller particles may have long left the channel and no longer, therefore, be subject to the cross forces needed to continue their separation. Indeed, the separation of substantially larger particles, say, within the range of 500 to 1000 nm requires considerably different channel and cross flow rates than might be required to separate particles within the range of 5 to 10 nm. The separation of samples whose sizes extend over very large size ranges requires considerable flexibility in programming the relative channel and cross flow rates during the passage of such sample aliquots through the channel selected. Optimal fractionation of one group of sizes does not insure an equivalent or even comparable fractionation of another group of sizes.
A continuing problem for such FFF separations lies in the historic inability of these techniques to vary local flow conditions within the channel. Heretofore, control of the fractionation process, irrespective of the FFF method, has been directed to the entire channel. It is the major objective of the inventive methods and field flow fractionators described herein to permit localized control over the applied flow and forces. By these means, the flows and forces may be controlled at specific local regions throughout the length of the fractionating channel.
Although most of the illustrations of the new fractionation method presented will be in the context of cross flow based separations, as will be obvious to those skilled in the art, the methods disclosed will be applicable equally to other field flow fractionators. The most important class of such field flow fractionators, in terms of the sheer numbers of scientific papers referring thereto, is that referred to as asymmetric flow field flow fractionation, or A4F, and invented by Karl-Gustav Wahlund. A brief review of the technique is provided later.
The A4F fractionator is considered a variation of the earlier developed symmetrical flow field fractionator, SF1FFF. In this earlier device, a cross flow is provided to the channel by a separate pump. Thus each flow is produced by a separate pump providing, thereby, crossflow symmetry. For A4F, on the other hand, an effective cross-flow is established by restricting the channel out flow relative to the input flow. The difference between the two flows becomes the effective cross flow. Because the A4F fractionator produces the two basic flow fields of the traditional SF1FFF device with a single pump, many of the results characteristic of symmetrical cross-flow FFF have been assumed operative for A4F, as well. One of these, for example, is the so-called fractionating power F that is proportional to the product of cross flow fx times the square root of the ratio of cross flow to channel-flow, fc, i.e.
  F  ∝            f      x        ×                                        f            x                                f            c                              .      Since the source of the cross-flow for the symmetrical fractionator is independent of the channel flow, both may be varied and would be constant over the channel length. For the A4F device, on the other hand, the channel flow always varies along the length of the channel and reaches a minimum just before the sample leaves the channel.
In order to compensate for this decrease in channel flow and to provide an associated constancy of the cross-flow per unit area, a variety of techniques have been employed. These include programming the cross-flow by varying the mobile phase input flow rate and changing the impedance to the cross flow. A variety of channel shapes have been tried including trapezoidal and exponential with the hope of preserving a greater channel flow near the outlet. The tapered channel, decreasing its width along its length, allows the channel flow per unit area to be increased sufficiently to compensate for its diminution necessary to provide the corresponding cross flow.
There are other difficulties with the A4F fractionator despite its superficial simplicity. First, there is a common problem to both SF1FFF and A4F: the four surfaces that define the channel are of different materials and one of them may depart from the expected laminar flow patterns of the theory. Indeed, the frit-supported membrane, of the accumulation wall, is soft and porous which is quite distinct from the exemplar sedimentation field flow fractionator, or SdFFF, wherein all walls, generally the sides of stainless steel tubing, are smooth and of the same materials. Laminar flow confined by such surfaces does produce the expected parabolic flow profile with the tangential flow reaching zero at the walls. For both A4F and SF1FFF flow, however, conditions at the membrane boundary are not well understood.
It is a major objective of this invention to establish, by reference to the A4F fractionator and its associated methods of application, a new type of separation capability having greater versatility by means of a different type of frit support structure and an associated set of cross flow regulators. This inventive frit structure will permit the cross flow per unit membrane area to be variable at different regions of the channel. Because if this variability, another objective of the invention is achieved: the selective filtering of larger particle components of a sample undergoing fractionation. Another objective of the invention is to sharpen broadened species peaks that had been resolved/separated earlier within the channel.
All of the objectives of this new inventive A4F-based fractionator are applicable to most classes of FFF techniques that might be modified to allow for external control of the cross field at discrete regions along the associated channel. These include the symmetrical cross flow fractionator, hollow-fiber fractionator, and, to some extent, the electrical field flow fractionator.
A further objective of the inventive method is its enhanced ability to capture and identify outlier particle populations. This, in turn, may be used in appropriate circumstances to purify certain classes of solutions that may be contaminated by such undesirable and potentially dangerous particulates, returning them, thereby, to a requisite pristine condition.