Sample focusing device and method

Sample focusing method and device for field-flow fractionation techniques that lead to improved detection, improved separation resolution, and a compressed sample plug while permitting a more straightforward quantitation of peaks and reliable large volume injections. The method and technique can be implemented in separations that are performed by a variety of field-flow fractionation techniques, including thermal FFF, electrical FFF, sedimentation FFF, gravitational FFF, dielectric FFF, photophoretic FFF, flow FFF, asymmetric flow FFF, and symmetric flow FFF. The sample focusing device can be integrally built into a separation channel or it can be manufactured as an attachable independent piece.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates generally to analytical separation techniques 
such as field-flow fractionation. More specifically, the present invention 
relates to a method and a device for introducing samples in analytical 
separation apparatuses, and in particular to field-flow fractionation 
systems. 
2. Description of Related Art 
Field-flow fractionation is a separation and characterization technique 
that relies on the effects of an applied field on a sample that is carried 
by a fluid flow. This fluid flow moves down a channel that will 
hereinafter be referred to as the "channel". The stream flowing along the 
channel will be referred to by the term "channel flow". 
The character and strength of the interaction between the species in the 
sample and the field plays a decisive role in the separation. Species that 
more weakly interact with the field are more rapidly carried away by the 
fluid flow that moves perpendicular to the applied field. This leads to 
different retention times for different species in the sample. Field-flow 
fractionation was disclosed in U.S. Pat. No. 3,449,938, and it is an 
excellent technique to separate and characterize a great variety of 
species. Field-flow fractionation is also known as single phase 
chromatography, polarization chromatography and capillary hydrodynamic 
fractionation. These species include cells, subcellular particles, 
viruses, liposomes, protein aggregates, fly ash, colloids, industrial 
lattices and pigments, polymers, humic materials, proteins, and nucleic 
acid molecules such as DNA. Some of these species are dissolved in the 
fluid flow that carries the sample, whereas other species are better 
characterized as being suspended in the fluid flow. Consequently, the 
terms "carrier fluid" and "carrier" will hereinafter refer to the fluid 
flow that transports the sample species, regardless of the form in which 
such species are contained in the fluid medium (i.e., whether dissolved, 
dispersed, suspended or in any other form of aggregation in the fluid 
flow). Furthermore, terms such as "sample species", "particles" or 
"particle", and "component" or "components", will hereinafter characterize 
the entity or entities in the sample to be analyzed, or more particularly, 
in the sample that contains the entities to be separated. More 
particularly, these terms used in the specific context of field-flow 
fractionation refer to any sample species that can be retained and 
separated by any field-flow fractionation method, including rigid and 
deformable particles ranging in size from submicron to hundreds of 
microns, polymer molecules, aggregates and clusters, biological 
macromolecules, and particles including cells, DNA, proteins and any other 
molecules that are capable of analysis by field-flow fractionation. 
Consequently, these terms refer to the entities or components in the 
sample that is to be analyzed or separated, regardless of the nature, 
mass, size or any other specific characteristic of these entities, and the 
sample to be analyzed or separated is hereinafter referred to as the 
"sample". 
The great variety of sample species that can be separated and characterized 
by field-flow fractionation makes this technique an important tool for 
solving problems in a plurality of fundamental and applied research areas 
that include biology, medicine, and material and environmental sciences. 
More specifically, field-flow fractionation has been applied to sample 
species whose masses span a 10.sup.15 -fold range. These species encompass 
molecules with a mass of about 600 Dalton and increasingly bigger entities 
up to particles of about 100 micrometers in diameter. 
The choice of the applied field in field-flow fractionation depends on the 
particular property that controls the retention time of the sample species 
that is to be separated. The types of applied fields that can be used in 
implementing field-flow fractionation include thermal, gravitational, 
electric, and magnetic gradients. In addition, a cross flow with respect 
to the carrier is also used in flow field-flow fractionation, a very 
versatile and effective implementation of the field-flow fractionation 
principles. Other types of applied fields that have in fact been applied 
or that are of potential practical relevance as a driving force in 
field-flow fractionation include forces due to dielectrical, concentration 
gradient, photophoretic and shear effects. A short-hand notation that 
consists of the acronym FFF preceded by the name of the applied field is 
used hereinafter. Available commercial types of field-flow fractionation 
include flow FFF, thermal FFF, and sedimentation FFF. These types differ 
by the type of applied field. In flow FFF, the field that drives 
separation is a flow stream directed perpendicular to the channel flow 
longitudinal axis. A method and apparatus for flow FFF is described in 
U.S. Pat. No. 4,147,621. In thermal FFF, a thermal gradient is used as the 
field to drive separation. Acceleration is used to drive separation in 
sedimentation FFF. In particular, this acceleration is that of a 
centrifugal field in sedimentation FFF, and it is the gravitational field 
in gravitational FFF. Unless otherwise specified, the terms "field" or 
"applied field" will hereinafter refer to any applied field, to a cross 
flow, and to any appropriately generated potential gradient that creates a 
driving force that directs the sample species into a wall of the channel 
called the accumulation wall. Furthermore, the examples and illustrations 
offered herein refer in particular to flow FFF because this field-flow 
fractionation technique is currently established as a very versatile and 
effective technique. In addition, flow FFF has been characterized as the 
most universal of the field-flow fractionation methods. J. Calvin 
Giddings, Field-Flow Fractionation, Chemical and Engineering News, Vol. 66 
(1988), pp. 34-45; Particle Size Distribution II, ACS Symposium Series No. 
472, S. Kim Ratanathanawongs, Inho Lee, and J. Calvin Giddings, Separation 
and Characterization of 0.01-50-.mu.m Particles Using Flow Field-Flow 
Fractionation, 1991, chapter 15, pp. 229-46. 
For each applied field there are in turn a variety of operating modes. Each 
operating mode depends on the sample species separation mechanism. For 
example, sample species under the influence of an applied field may be 
subject to a diffusive, steric or hydrodynamic lift effects. Depending on 
which one of these effects is predominant, the field-flow fractionation 
operating mode is, respectively, a Brownian, steric or hyperlayer mode. 
Consequently, each appropriate choice of applied field and operating mode 
leads to a different field-flow fractionation subtechnique. 
Whereas sample species separation according to mass or size is often the 
goal of field-flow fractionation, this is not the only possible 
application of field-flow fractionation. With the appropriate choice of 
applied field, a field-flow fractionation apparatus can perform as a 
microbalance sensitive to forces of 10.sup.-16 N. Furthermore, field-flow 
fractionation permits the measurement of both particle size and density, 
from which a molar mass-can be calculated. Other properties that can be 
calculated include particle diameter and charge. The high sensitivity of 
sedimentation FFF to very small amounts of adsorbed material permits the 
measurement of the mass and thickness of adsorbed layers. When the sample 
species population is heterogeneous in any of these properties, the 
different components are separated by field-flow fractionation on the 
basis of the heterogeneous property, and a distribution curve relative to 
this property is obtained. These and other background materials pertaining 
to field-flow fractionation have been described by Ronald Beckett. John 
Ho, Yong Jiang, and J. Calvin Giddings, Measurement of Mass and Thickness 
of Adsorbed Films on Colloidal Particles by Sedimentation Field-Flow 
Fractionation, Langmuir, Vol. 7 (1991), pp. 2040-47; J. Calvin Giddings, 
Field-Flow Fractionation, Chemical and Engineering News, Vol. 66 (1988), 
pp. 34-45. 
In a field-flow fractionation apparatus the carrier flows under laminar 
regime conditions along a narrow channel and a field is applied 
orthogonally to the carrier flow. One of the characteristics of a laminar 
flow is that the flow velocity profile is parabolic. Accordingly, the 
carrier moves slower near the walls and increasingly faster in regions 
closer to the channel center line of the channel along the longitudinal 
axis. As applied, the field drives sample species to different 
cross-sectional regions of the carrier flow, where they are transported 
with different momenta depending on the carrier flow region to which they 
are driven. The sample species are initially and ideally concentrated in a 
very small spot on one of the channel walls called the accumulation wall. 
In the course of flow displacement, particles that weakly interact with 
the field will move farther from the accumulation wall than the particles 
that strongly interact with the field, thus reaching sooner the regions of 
the carrier flow that move faster. These particles are carried downstream 
more rapidly than the particles that interact more strongly with the 
field. Therefore, rapidly swept particles are part of an outflow fraction 
that leaves the field-flow fractionation apparatus sooner than the 
fractions that contain the particles that more strongly interact with the 
field. More succinctly, the retention time of a particle depends on the 
interaction between the relevant property of the particle and the applied 
field. 
In the initial operation of the FFF and other analytical separation 
techniques, a plug of sample, also referred to as a sample pulse is 
injected into the carrier flow at or near the channel inlet. Typically, a 
small volume of sample is injected to avoid dispersion or band broadening 
of the sample plug. Band broadening is detrimental as it reduces the 
resolution of separation. In current practice, the volume of the sample 
plug is limited by band broadening effects. The injected volume is 
typically 1-20 microliters, or less than 10% of the total volume of the 
FFF channel. 
Field-flow fractionation is dissimilar to other analytical separation 
techniques because it utilizes an applied field for separation. Because of 
this feature, an additional sample introduction step is required for 
optimal resolution of separation. This process is the relaxation of the 
sample species with respect to the applied field. Equilibration is 
equivalently used in this context for relaxation. When the sample is first 
introduced into the FFF channel, it is generally distributed broadly over 
the channel cross section. Before the sample migration step is 
implemented, the sample species are subjected to a relaxation process in 
which they approach a steady-state distribution within the channel, 
usually by accumulating near one channel wall. The steady state 
distribution normally corresponds to a balance of the sample-field 
interaction which drives sample components towards the accumulation wall 
and Brownian diffusion which drives sample away from the accumulation 
wall. 
There are several methods for introducing sample into the field-flow 
fractionation channel. When referring to a sample, the terms 
"introducing", "injecting" or derivatives thereof are used as equivalent 
terms that encompass any procedure for incorporating into a carrier a 
sample that is to be separated or for introducing a flow into a conduit. 
Some methods provide a relaxed sample distribution. Other techniques 
merely position the sample components next to a wall without providing 
equilibration of the sample component with the field. The stop-flow method 
is the most commonly used method, and it provides a fully relaxed sample 
distribution. This method involves turning off the channel flow 
immediately following the sample injection and allowing the applied field 
to act upon the sample. This process both positions the sample at the wall 
and allows the sample components to equilibrate. The disadvantage of this 
method is that the carrier flow must be turned on and off; this typically 
requires a switching valve and extra time for equilibration. Furthermore, 
turning the flow on and off generates a pressure transient. The pressure 
transient generation is a most detrimental effect because the detectors 
used in FFF systems are sensitive to pressure transients. As a consequence 
of the pressure transient, the detector signal is distorted from its 
normal baseline value and a significant amount of time may be required for 
the detector to return to baseline. Whenever the detector response is 
disturbed, the separation cannot be accurately monitored, especially for 
species that elute at the beginning of the separation stage. Additionally, 
the pressure transient may broaden or otherwise disturb the sample zone 
which is precisely positioned in its equilibrium distribution during the 
previous stop-flow period. Either of these reasons will cause poor 
separation resolution. In addition to these undesired pressure pulses, a 
stop-flow process may also lead to another undesirable effect, which is 
adhesion of sample species at the accumulation wall. 
A desirable feature of this method, however, is that the sample does not 
travel down the channel as it relaxes on the accumulation wall. This tends 
to reduce band broadening effects and broadening of the initial sample. 
Terms such as "dispersion", "broadening", "spreading", or equivalents 
thereof, will be used herein for describing the extension of the area or 
volume occupied by the sample whose components are to be separated. 
Focusing the sample is preventing sample spreading and thus avoiding the 
enlargement of the region occupied by the sample whose components are to 
be separated. The stop-flow method is described in Particle Size 
Distribution II, ACS Symposium Series No. 472, S. Kim Ratanathanawongs, 
Inho Lee, and J. Calvin Giddings, Separation and Characterization of 
0.01-50-.mu.m Particles Using Flow Field-Flow Fractionation, 1991, chapter 
15, pp. 229-46. 
Some methods have been suggested for positioning the sample near the 
accumulation wall. U.S. Pat. No. 5,141,651 describes a pinched channel 
inlet system. In this method the thickness of the channel is reduced in 
the area of injection. Specifically, the structure of the channel is 
modified so that the position of the top or depletion channel wall is 
lowered. Consequently, the injected sample is, from the start, positioned 
closer to the accumulation wall. A pinched inlet channel system, however, 
has some shortcomings. First, since the flow through the channel is not 
discontinued in this method, the sample travels down the channel while 
also being relaxed towards the accumulation wall. This leads to increased 
band broadening effects and a broadened initial sample plug. Second, 
engineering the pinched inlet may present difficulties because high 
performance FFF channels are already very thin, typically 100-200 
micrometers. Because of this small dimension, reducing the channel 
thickness near the inlet is difficult. Third, the reduced channel 
thickness in the pinched inlet must be even if the same flow velocity in 
all areas of the pinched inlet is to be maintained. Manufacturing a 
channel with an even channel thickness of just a few micrometers in the 
pinched inlet area is difficult. This dimension is determined by the 
typical thickness of an equilibrated sample zone, which is of the order of 
1-10 micrometers. Fourth, at high channel flow rates, eddy currents may be 
generated at the interface between the pinched inlet area and the full 
channel thickness. Such eddy currents are undesirable because they may 
disturb the distribution of sample next to the accumulation wall. Finally, 
the reduced thickness of the channel at the inlet is susceptible to 
clogging. 
Another process and apparatus for positioning sample near the accumulation 
wall are described in U.S. Pat. No. 5,193,688, and by Min-Kuang Liu, 
Stephen Williams, Marcus N. Myers, and J. Calvin Giddings, Hydrodynamic 
Relaxation in Flow Field-Flow Fractionation Using Both Split and Frit 
Inlets, Analytical Chemistry, Vol. 63 (1991), pp. 2115-22. This process is 
known as hydrodynamic sample relaxation, and it involves a permeable will 
element, or frit inlet, positioned close to the sample inlet. This element 
is used to provide a separate flow stream that hydrodynamically forces 
sample to the accumulation wall. The permeable flow element is placed in 
the top channel wall, known as the depletion wall, and the flow from this 
element is distributed over the frit area immediately above the small 
inlet section of the channel where hydrodynamic relaxation is to be 
achieved. Flow is directed into this element using a separate pump and/or 
a flow control valve "tee-ed" into the carrier pump flow line. The amount 
of flow can be externally controlled to adjust the amount of viscous force 
that is applied to push the sample next to the accumulation wall. Thus, 
this relaxation process may be manipulated externally. In comparison to 
the pinched inlet, the channel structure required for hydrodynamic sample 
relaxation is easier to implement and is not subject to clogging. 
Nevertheless, this method has some disadvantages. First, a sample that 
relaxes according to this method is equilibrated relative to the field 
generated by the viscous force of flow through the permeable wall element. 
The magnitude of this hydrodynamically provided field is much larger than 
the field applied in the remainder of the channel. This increased 
magnitude is required by the necessity of positioning the sample at the 
accumulation wall very quickly to minimize band broadening effects. 
Because the hydrodynamic relaxation field typically does not match the 
field applied in the remainder of the channel, the sample must 
re-equilibrate when it is transported beyond the inlet region. Another 
disadvantage, common to the pinched channel inlet system, is that the 
sample has an increased opportunity for band broadening relative to the 
stop-flow method. This is because the carrier flow is not stopped during 
the relaxation process. 
Hydrodynamic relaxation can also be pursued with a split inlet system. This 
system requires a splitter in the inlet region of the channel. The 
concepts underlying hydrodynamic relaxation, whether pursued with a frit 
inlet or with a split inlet system, are the same. A splitting plane is 
created in the region where two streams collide. The first stream is the 
carrier flow with the sample. The second stream is another flow that 
contains no sample, that is typically identical to the carrier flow, and 
that is introduced from above the carrier flow. The region where these two 
streams meet can be visualized as a plane, called the splitting plane. The 
flow rate of the second stream must exceed that of the sample stream for 
displacing the splitting plane--and with it all incoming particles--below 
the midplane of the channel. The greater the flow rate margin by which the 
second stream exceeds the stream that carries the sample, the closer the 
compression of the particles toward the accumulation wall, and the more 
complete the hydrodynamic relaxation. Equivalently, as this flow rate 
margin increases, the elevation of the splitting plane with respect to the 
accumulation wall decreases. The expected similarity in the results 
produced by the split inlet and frit inlet systems has been substantiated 
by Min-Kuang Liu, Stephen Williams, Marcus N. Myers, and J. Calvin 
Giddings, Hydrodynamic Relaxation in Flow Field-Flow Fractionation Using 
Both Split and Frit Inlets, Analytical Chemistry, Vol. 63 (1991), pp. 2115 
et seq. 
The advantages common to both the pinched inlet and hydrodynamic relaxation 
techniques stem from the fact that the carrier flow need not be stopped. 
Thus, no pressure transient is generated, and the detector is not exposed 
to a pressure transient. In this context, the terms "detector" and 
"detector cell" are used interchangeably. The sample is also continually 
moving tangentially to the surface of the accumulation wall. This feature 
reduces the opportunity for sample adsorption on the accumulation wall. 
Furthermore, the pinched inlet and the hydrodynamic relaxation methods 
require a sufficiently short sample injection time to avoid sample 
diffusion during injection. Sample diffusion would otherwise form an 
undesirable and effectively larger sample plug. 
Neither the pinched inlet nor the hydrodynamic relaxation method, however, 
provide complete sample relaxation. More specifically, neither one of 
these two methods controls the width of the sample plug, even though a 
compressed sample plug is desirable because it improves the resolution of 
the separation. A more compressed sample plug is provided by the stop flow 
method, which produces peaks that are sharper than those obtained with 
hydrodynamic relaxation. Equivalently, hydrodynamic relaxation results in 
somewhat broader elution bands than those produced by the stop flow 
technique. Furthermore, the sample in the stop flow technique is carried 
onto the channel by a carrier that occupies the full thickness of the 
channel, and the band does not undergo the spreading that is associated 
with the merging of the two streams in the frit inlet and split inlet 
techniques. Unfortunately, the stop flow method is more time consuming 
than stopless flow injection, it is more conducive to particle adhesion to 
the accumulation wall, and it typically produces a false signal due to the 
pressure pulses that are induced by abrupt flow changes in the channel. 
For best results in field-flow fractionation, a minimum volume of sample 
should be introduced. Band broadening in hydrodynamic relaxation is 
increased by sample spreading as the sample flows into a split or a 
divided channel. 
A method for providing a narrow initial sample plug is the outlet flow 
sample focusing method. This method has been practiced in tubular and 
rectangular cross section channels. The practice in tubular channels is 
described by H. L. Lee, J. F. G. Reis, J. Dohner, and E. N. Lightfoot, 
AIChE Journal, Vol. 20 (1974), pp.776-84. The practice in rectangular 
cross section channels is described by K. G. Wahlund, and J. C. Giddings, 
Properties of an Asymmetrical Flow Field-Flow Fractionation Channel having 
One Permeable Wall, Analytical Chemistry, Vol. 59 (1987), pp. 1332-39 and 
by H. Lee S. K. R. Williams, and J. C. Giddings, Particle Size Analysis of 
Dilute Environmental Colloids by Flow Field-Flow Fractionation Using an 
Opposed Flow Sample Concentration Technique, Analytical Chemistry Vol. 70 
(1998), pp. 2495-2503. Whether tubular or rectangular cross section 
channels are used, the channels according to this method are constructed 
with one or more walls that are permeable to solvent flow. The tubular 
channel is a hollow fiber permeable to solvent flow. The rectangular 
channel used by Wahlund had a permeable bottom wall. The rectangular 
channel used by Williams had permeable top and bottom walls. According to 
the outlet flow sample focusing method, a flow additional and opposed to 
inlet flow is introduced from the outlet of the channel. Sample is 
introduced at or near the channel inlet, and the introduced sample is held 
stationary or is focused at the interface of the two opposing flows. The 
position of the interface is termed the sample focus plane and is related 
to the ratio of the two flow rates. Sample may be pumped in over a long 
period of time without causing a broad sample plug since the opposed flows 
continuously focus the sample. For tubular channels (for example, hollow 
fibers), sample is distributed radially to the outside perimeter of the 
tubular channel. For rectangular channels, sample is distributed towards 
the bottom channel wall. This process is capable of providing both a 
narrow and a fully equilibrated sample plug. 
The disadvantage of the outlet flow sample focusing method lies in the 
transition that must be made between focusing and separation. During the 
focusing stage there is a forward directed flow from the channel and/or 
sample inlet(s) and backward directed flow coming in through the channel 
outlet. During the sample migration or separation stage, only forward 
directed channel inlet flow is implemented. This inlet flow carries the 
focused sample through the channel and out the outlet to a detector where 
the separated components are monitored. During the transition between 
focusing and separation, the outlet flow focusing procedure used by 
Lightfoot, Wahlund, and Williams requires that the direction of flow 
through the channel outlet be reversed. This requires a complex 
arrangement of pumps, tubing, and valves. At the transition between 
focusing and separation, which is the period during which the outlet flow 
is in the process of being reversed, a pressure transient is created in 
the channel. Additionally, the flow lines from the focusing point to the 
outlet of the channel must be established. During the transition between 
sample focusing and separation, the direction from the sample focus plane 
to the outlet must reverse completely. Thus, there is a period of unstable 
flow during the transition period. A consequence of this flow instability 
is that the focused sample is disturbed during the period of unstable 
flow; furthermore, the focused sample is also disturbed by the pressure 
transient. The detector is also disturbed by the pressure transient and 
the flow reversal. The effects on the detector may be slightly alleviated 
by placing a valve between the channel outlet and the detector so the 
backward directed focusing flow bypasses the detector cell. However, this 
requires extra valves and a pressure transient is generated due to the 
action of the valve. 
Finally, an injector with minimal flow-interrupt transient is described in 
U.S. Pat. No. 4,506,558. This injector is a mechanical device that 
includes rotor and stator elements that can rotate relative to one another 
between load and injection positions. These elements have an interface and 
a series of conduits that run therethrough. The purpose of this mechanical 
device is to inject a sample at a high pressure into a chromatographic 
column, but it does not focus the injected sample. 
Each of the afore-mentioned patents and references is hereby incorporated 
by reference in its entirety for the material disclosed therein. 
SUMMARY OF THE INVENTION 
It is desirable to achieve high resolution in separation processes. It is 
further desirable to inject a narrow pulse sample into a field-flow 
fractionation or other chromatographic separation apparatus to prevent an 
increase in sample spread or dilution that reduces the separation 
resolution. Sample spreading depends in turn on the flow dynamics in the 
medium where the separation takes place. Consequently, it is also 
desirable to control the relevant flow dynamics so that sample spreading 
is avoided. 
Because sample spreading can occur in the longitudinal and transverse 
directions relative to the carrier flow motion, it is desirable in 
particular to avoid both the longitudinal and transverse spread of the 
sample prior to separation. When sample relaxation with respect to an 
applied field is required such as in field-flow fractionation, it is also 
desirable to achieve this sample relaxation while avoiding sample spread, 
including longitudinal and transverse spread relative to the carrier 
motion. 
Because the detector to which most field-flow fractionation apparatuses are 
typically coupled is sensitive to transient perturbations, it is desirable 
to achieve sample focusing while avoiding the creation and propagation of 
any transient perturbation that would diminish detector performance or 
cause the detector to produce false readings. More specifically, it is 
desirable to achieve sample focusing by avoiding the generation and 
propagation of any pressure transient that would distort or in any other 
way negatively influence detector performance. 
It is also desirable to achieve sample focusing by minimally perturbing the 
current lines of the laminar flow when such a flow condition is required 
by the separation technique. This is particularly desirable in field-flow 
fractionation. 
It is also desirable to keep the focusing region close to the inlet end, 
because this proximity will contribute to a narrow starting zone. In this 
setting, a minimum duration of time for focusing the flow will be needed 
to adequately sharpen the starting zone. 
The objectives of this invention are achieved by a specially designed 
sample injection device which facilitates the implementation of a method 
including the steps of focusing a sample and separating the focused 
sample. At the focusing stage, a sample is introduced downstream with 
respect to the injection point of a first sample-free carrier. The sample 
is consequently pushed downstream until it is stopped by an opposing flow. 
Subjecting the sample to opposing flows focuses the sample. The opposing 
flow is generated by a second flow of sample-free carrier that is 
introduced through an inlet system at a point farther down the channel 
with respect to both the sample injection point and the first sample-free 
carrier injection point. When injected through the inlet system, part of 
the second sample-free carrier stream naturally flows in opposition to the 
flow that carries the sample, and part of it forms the channel flow that 
moves towards the other end of the channel. 
Once the sample is focused the operation evolves continually into the 
separation stage. Continuous evolution means that while going from the 
focusing stage to the separation stage the channel flow is not reversed 
and no significant channel flow perturbation or pressure transient is 
introduced. To achieve this continuous evolution, the second sample-free 
carrier flow is decreased while the first sample-free carrier flow is 
increased and thus the focused sample is pushed down with the channel flow 
for sample separation and subsequent detection. 
This method is implemented by means of the sample focusing device described 
below. This sample focusing device can be embodied by a channel that 
includes a first injection point for injecting a first sample-free 
carrier, a second injection point for introducing the sample to be 
analyzed, and an inlet system that provides a third injection point for 
injecting a second sample-free carrier. This second sample-free carrier 
generates the channel flow. In addition, the second sample-free carrier 
simultaneously provides a flow that opposes both the first sample-free 
carrier and sample-carrying flows. 
Another preferred embodiment of the sample focusing device would comprise a 
first injection point for simultaneously introducing a sample carrying 
flow and an inlet system for injecting sample-free carrier. Nevertheless, 
this is also a possible embodiment of the sample focusing device. 
The exemplary embodiments of the sample focusing device are shown herein as 
being integrally attached to the separation apparatus. Nevertheless, the 
sample focusing device could be designed and manufactured as a separate 
unit to be attached to the separation apparatus by appropriate fastening 
means. These fastening means will be obvious to anyone with ordinary skill 
in the art. This is an important difference with some conventional sample 
focusing methods and devices that rely on the entire design and operation 
of a field-flow fractionation apparatus for achieving sample focusing. 
Necessarily, this fastening means must be of a type such that the 
attachment of the sample focusing device to the channel is rendered fluid 
tight and no protuberances or discontinuities at the attachment joint 
which would induce unacceptable eddy currents or other perturbations in 
the flow along the channel or in the sample focusing device. 
Although the examples herein discussed refer to flow FFF, the problems and 
solutions that this invention addresses are common to other FFF 
techniques. The choice of the flow FFF technique is herein made only for 
visualizing and offering concrete examples of embodiments of the invented 
device and method. 
The objectives of this invention include the following. The general 
objective of this invention is to provide a sample focusing device and 
method for field-flow fractionation. It is an additional objective of this 
invention to provide a method and a device for focusing a sample plug in a 
channel, and in particular in a field-flow fractionation channel. 
"Channel" in this context refers to a conduit. This conduit's cross 
section's perimeter can be circular, ellipsoidal, ovoid, curved in any 
way, polygonal, regular or irregular, or it can have a combination of 
straight and curved sides. Furthermore, this conduit can have a constant 
cross section, or it can be tapered. 
It is a further objective of this invention to provide a method and device 
for sample focusing that operates while maintaining continuous flow 
through the channel outlet and the detector cell. 
It is a further objective of this invention to provide a sample focusing 
method and device that do not require reversing the flow through the 
channel outlet or the detector cell at any stage between sample loading 
and detection. 
It is a further objective of this invention to provide a method and device 
for sample focusing while also equilibrating the sample with respect to 
any field used in field-flow fractionation. 
It is a further objective of one preferred embodiment of this invention to 
provide a sample focusing method and device that do not require stopping 
the carrier flow down the channel at any stage between sample loading and 
detection. 
It is a further objective of this invention to provide a sample focusing 
method and device whose implementation minimizes pressure transients. 
It is a further objective of this invention to provide a method and device 
that permit external, easy and effective sample focusing control. 
It is a further objective of this invention to provide a sample focusing 
method and device applicable to both large and small sample injection 
volumes. 
It is a further objective of this invention to provide a sample focusing 
method and device applicable to both particulate and macromolecular 
samples. 
It is a further objective of this invention to provide a sample focusing 
method and device that produce a sufficiently stable flow in the 
transition between the focusing and separation stages. Equivalently, it is 
an objective of this invention to provide a focusing method and device so 
implemented that the flow lines established from the sample focus point to 
the channel outlet do not significantly differ in the focusing and 
separation stages. 
It is a further objective of this invention to provide a sample focusing 
method and device that maintain the focusing point close to the inlet end. 
It is a further objective of this invention to provide a sample focusing 
method and device that can be implemented in operational settings within 
broad temperature ranges, or that is temperature-independent. 
It is a further objective of this invention to provide a method and device 
for sample focusing, whether the sample is carried by an aqueous or a 
nonaqueous carrier. 
It is a further objective of this invention to provide a sample focusing 
method and device that permit the injection of the sample directly into an 
area that has the same cross section as the rest of the channel. 
It is a further objective of this invention to provide a sample focusing 
method and device that can easily be implemented as an integral part of 
the apparatus where a separation takes place or that can be manufactured 
as an independent unit to be attached to the apparatus in which a 
separation takes place. 
It is a further objective of this invention to provide a sample focusing 
method and device whose implementation is simple. In particular, it is a 
further objective of this invention to provide a sample focusing method 
and device that do not rely for focusing the sample on any mechanical 
device that involves components such as moving elements. 
Additional objects, features and advantages of this invention will become 
apparent to persons of ordinary skill in the art upon reading the 
remainder of the specification and upon referring to the accompanying 
figures.

DETAILED DESCRIPTION OF THE EMBODIMENTS OF THE INVENTION 
The illustrative embodiments of the invention exemplify the application of 
the useful characteristics discussed below, and further reference to these 
and other useful and novel features is made in the following discussion of 
each illustrative embodiment. The exemplary embodiments discussed below 
are intended to limit neither the scope of the process nor apparatuses or 
materials that are needed for performing the process. 
FIG. 1a displays basic elements of a preferred embodiment of the 
intermediate sample focusing device 10 with respect to channel 12. Channel 
12 is the flow cell in which the separation, and in particular the 
flow-field fractionation, takes place. In its operational mode, a carrier 
is introduced into channel 12 through channel inlet 20 near closed 
terminal end 21, and the sample may be introduced into channel 12 through 
a separate inlet 22. Referring to FIG. 1c, in another embodiment of the 
invention, channel inlet 20 serves as both the channel inlet and the 
sample inlet. 
In the exemplary embodiments herein described and in other embodiments of 
the sample focusing device and method, the position of sample inlet 22 can 
be shifted to locations other than that indicated in FIG. 1a. For example, 
sample inlet 22 can be placed as an insertion point in the bottom channel 
wall. Analogously, and independently, the position of channel inlet 20 can 
also be shifted to locations other than that indicated in FIG. 1a. For 
example, channel inlet 20 can be placed further downstream, or as an 
insertion point into the bottom channel wall. In all embodiments of the 
invention, the sample inlet is located downstream of the channel inlet 
when the sample inlet is provided by all inlet that is diligent from the 
channel inlet. The sample inlet, however, is in some embodiments of this 
invention part of the channel inlet as shown in FIG. 1c. 
The shape of channel 12 shown in FIG. 1 a is merely exemplary. For example, 
channel 12 may also be tapered so that the breadth is larger at one end of 
the channel, as will be appreciated by those familiar with field-flow 
fractionation. Furthermore, a tubular channel or a channel with 
rectangular or differently shaped cross section could be used, and the 
breadth or thickness of the channel need not be constant over the length 
of the channel. 
As noted previously, "channel flow" is the main flow stream that travels 
down channel 12 and away from focusing device 10. A channel outlet 24 is 
located at the opposite end of channel 12. Channel 12 has a bottom or 
accumulation wall 28 that is typically constructed of a permeable material 
and a top or depletion wall 30 that is impermeable. Sample focusing is 
achieved when a fluid, typically the carrier with no sample, is introduced 
through injection system 26. In FIG. 1a, injection system 26 is located 
between channel inlet 20 and channel outlet 24. More specifically, 
injection system 26 is located between sample inlet 22 and channel outlet 
24. Preferably, injection system 26 is generally located within the first 
third of the length of channel 12. In a more preferred embodiment of this 
invention, injection system 26 includes a permeable wall element 26c that 
in operating conditions is flush with depletion wall 30, and an inlet 
opening 26a which can extend outwards from depletion wall 30 forming, for 
example, a tubular structure or any other means for introducing fluid 
through injection system 26 into channel 12. In this embodiment, permeable 
wall element 26c is a porous frit. Typical dimensions of this porous frit 
are 2 cm in length, 2 cm in breadth, and 0.635 cm in thickness. 
Permeable wall elements herein described can be constructed of materials 
other than a porous ceramic frit, such as stainless steel frits and 
polymeric membranes. Furthermore, the dimensions of permeable wall element 
26c herein described could be increased or decreased in size depending on 
specific applications, as will be appreciated by those of ordinary skill 
in the art. 
In the exemplary embodiments herein described and in other embodiments of 
the sample focusing device and method, the position of injection system 26 
can be shifted to locations other than that indicated in FIG. 1a and 
subsequent figures that show exemplary embodiments of this invention. For 
example, injection system 26 can be placed in the bottom wall. This latter 
choice is a particularly well suited when the sample focusing device is 
attached to a field-flow fractionation channel that operates according to 
the principles of thermal FFF, gravitational FFF, electrical FFF, or 
sedimentation FFF. 
Dotted line 32 indicates the perimeter of the plane or region that is 
totally or partially occupied by the focused sample. Discontinuity 14 in 
FIG. 1a stresses the fact that although preferred embodiments of this 
invention for flow FFF apparatuses are an integral part of the separation 
channel, sample focusing device 10 can also be manufactured as a separate 
device with attachment means at open end 16 of device 10. An embodiment of 
focusing device 10 as a separate device may be useful when device 10 is to 
be used in a plurality of field-flow fractionation separation channels or 
columns. 
The sample focusing device in the embodiment shown in FIG. 1b is an 
endpiece 10 that is one of the ends of a flow FFF apparatus 18. The 
exemplary embodiment in FIG. 1b has an asymmetrical flow FFF channel with 
rectangular cross section. Asymmetrical flow FFF is a type of flow FFF 
technique in which only the bottom channel wall is constructed of a 
carrier permeable material in the form of permeable wall element 34. In an 
asymmetrical flow FFF apparatus, the flow stream directed into the channel 
inlet provides both the cross flow and the axial channel flow. Cross flow 
is the flow stream used in flow FFF techniques that provides the driving 
force for the separation that takes place in channel 12. FIG. 1b shows 
that a reservoir 26b may be placed in between inlet opening 26a and 
permeable wall element 26c. FIG. 1b also shows that permeable wall element 
26c is preferably placed so that its bottom surface is flush with the 
surface of depletion wall 30. In the embodiment shown in FIG. 1b, the top 
surface of permeable wall element 34 serves as accumulation wall 28 of the 
asymmetrical flow FFF channel 12. A cross flow reservoir 36 is machined or 
formed into channel bottom 40, and fluid is evacuated from reservoir 36 
through cross flow outlet 38. Dotted line 32 in this and subsequent 
figures is a side view of the plane that represents the position of the 
focused sample. 
The embodiments of this invention exemplified in FIGS. 1a-1b, and in 
subsequent figures that show other exemplary embodiments of this 
invention, illustrate one of the features of the sample focusing method 
and device. Sample focusing according to this invention is achieved by 
using the entire cross section of channel 12, rather than a reduced 
portion of it. Furthermore, the absence of any pinched, constrained or 
otherwise narrowed part of the channel of the invented sample focusing 
device avoids the problems associated with the clogging of such narrower 
passage, which affect some conventional sample focusing techniques. 
Additionally, the absence of such narrower passages in embodiments of the 
sample focusing device leads to the avoidance of design and manufacture 
problems inherent to the engineering of conventional sample focusing 
devices that rely on narrower conduits. 
Permeable wall element 26c can be a frit as shown in FIGS. 2a and 2b, a 
hole, as shown in FIG. 2c, or a series of holes as shown in FIG. 2d. 
Furthermore, the surface of permeable wall element 26c can be circular, 
ellipsoidal, ovoidal, polygonal, or have any irregular shape, as shown in 
FIGS. 2a-2d. These figures exemplify, but they do not limit, geometric 
characteristics of injection system 26, and more particularly of permeable 
wall element 26c. Other geometric characteristics that are also within the 
scope of this invention can be obtained by obvious combinations or 
modifications of the examples shown in FIGS. 2a-2d, as will be appreciated 
by those of ordinary skill in the art. 
Another embodiment of the sample focusing device 10 can be illustrated by 
an integral implementation into a symmetrical flow FFF apparatus. This 
implementation is shown in FIG. 3a. Symmetrical flow FFF differs from 
asymmetrical flow FFF in that both the top and bottom channel walls are 
constructed of carrier permeable materials. The bottom surface of 
permeable wall element 40 is depletion wall 30. A reservoir 42 is placed 
above permeable wall element 40 and fluid is introduced into reservoir 42 
through inlet 44. In a conventional symmetrical flow FFF channel, 
permeable wall element 40 extends along the entire length of channel 12, 
from channel inlet 20 to channel outlet 24. In the embodiment shown in 
FIG. 3a, however, the lengths of reservoir 42 and permeable wall element 
40 have been shortened and sample focusing device 10 is integrally 
attached to the symmetric flow FFF apparatus. 
Because other field-flow techniques do not commonly employ permeable wall 
elements. FIG. 3b illustrates an exemplary embodiment of sample focusing 
device 10 attached to a field-flow fractionation apparatus that can be 
operated under thermal FFF, electrical FFF, gravitational FFF and 
sedimentation FFF regimes. In the embodiment shown in FIG. 3b, applied 
field 46 is, respectively, a thermal gradient, an electric, a 
gravitational or a centrifugal field. Applied field 46 in this embodiment 
plays a role that is analogous to the role played by the cross flow in the 
embodiments shown in FIGS. 1b and 3a. Together, these embodiments 
illustrate that the performance of the claimed sample focusing device 10 
and the implementation of the claimed sample focusing method are not 
dependent on the driving force that is used in the separation, and it does 
not depend on the type of field-flow fractionation subtechnique. These 
embodiments also illustrate that neither the invented sample focusing 
device 10 nor the invented sample focusing method depends on how sample 
focusing device 10 is attached to the rest of the separation system. This 
is regardless of whether the separation system is a field-flow 
fractionation apparatus or any other separation apparatus that requires 
sample focusing like that provided by the invented device and method. The 
invented sample focusing method and device can be used to create a narrow 
sample plug in field-flow fractionation operating modes other than the 
Brownian (or normal) mode, such as the hyperlayer mode. The sample plug, 
however, will not relax in some of these operating modes that are not the 
Brownian mode. 
Referring to FIG. 3b, a permeable wall element 48 is placed at the head of 
channel 12 in accumulation wall 28. Permeable wall element 48 is 
positioned so that edge 53 is at or near closed terminal end 21; the other 
edge 55 is across channel 12 at or near a locus directly below permeable 
wall element 26c of injection system 26. Typically, the dimensions of 
permeable wall element 48 are 6 cm in length, 2 cm in breadth, and 0.635 
cm in thickness. A typical material for the construction of permeable wall 
element 48 is porous ceramic frit. Fluid from channel 12 that passes 
through permeable wall element 48 is evacuated through outlet 52. Outlet 
52 extends out and away from permeable element 48, and it may be connected 
directly to permeable wall element 48 or it may be connected to reservoir 
50 that is located between permeable wall element 48 and outlet 52. 
The exemplary embodiments of this invention herein shown and discussed 
illustrate the ease with which the sample focusing method and device can 
be implemented in various apparatuses and particularly in field-flow 
fractionation systems. The inlets of the sample focusing device 10 can be 
built while the field-flow fractionation apparatus is manufactured, with 
no requirement of additional materials, more complex designs, or 
significantly different manufacturing techniques and machinery. 
FIG. 4 illustrates how the components of an exemplary embodiment of the 
sample focusing device and method claimed herein can be integrated in a 
fully operational set-up. Other set-ups that in light of the diagram shown 
in FIG. 4 would be obvious to those of ordinary skill in the art are also 
possible. 
In the embodiment illustrated in FIG. 4. channel inlet 20 is connected 
through 1/16 teflon tubing to channel pump 54. Injection system 26 is 
similarly connected through 1/16 teflon tubing to sample focusing pump 56. 
These connections are achieved by means of optional four-way switching 
valve 58. Switching valve 58 can redirect the flow from channel pump 54 to 
injection system 26 and the flow from focusing pump 56 to channel inlet 
20. In an embodiment that has both channel inlet 20 and sample inlet 22, 
sample inlet 22 is connected through 1/16 tubing to pump 60. Typically, 
channel pump 54, sample focusing pump 56, and pump 60 are HPLC type pumps 
which can deliver flow rates in the range of 0.01 to 10 mL/min and which 
can pump against back pressures up to 3000 psi. Detection system 62 may 
comprise one or more detectors and it is attached through 1/16 teflon 
tubing to channel outlet 24. Reverse pump 64 is connected through 1/16 
teflon tubing to detection system 62. Reverse pump 64 is typically a 
syringe or single piston pump which has similar capabilities as HPLC 
pumps. However, these pumps can equally pump in a reverse direction so 
that flow is reliably controlled. All pumps, valves, and tubing in the set 
up represented by the diagram in FIG. 4 must be able to withstand the back 
pressure of the field-flow fractionation channel. Typically, this pressure 
is on the order of 100-1000 psi. 
Although HPLC pumps are used in the preferred embodiment described above, 
other pumps instead of HPLC and syringe or single piston pumps can be 
employed in other embodiments of this invention. For example peristaltic 
or dual piston pumps could be employed. Analogously, and independently, a 
refractive index detector is appropriate for most separations; a detector 
system that is not a refractive index detector can also be used when 
appropriate. For example, any detector used with HPLC applications could 
be employed. Furthermore, computer controlled pumps facilitate the 
operation of the set-up described above, but manual control of channel 
pump 54 and focusing pump 56 could alternatively be used instead of 
computer control. 
During the focusing stage, sample is introduced through sample inlet 22. 
Pump 60 is used to gradually pump the sample into channel 12. The 
flowstream provided by channel pump 54 through channel inlet 20 helps to 
sweep sample down channel 12. An additional flowstream which opposes these 
flows is introduced through injection system 26. The flowstreams provided 
by channel inlet 20 and by sample inlet 22 flow opposite to the flow 
provided by injection system 26. These opposing flows meet within channel 
12 in the region represented by sample focus plane 32. The particles that 
are entrained in the flowstream formed by the streams coming from channel 
inlet 20 and from sample inlet 22 are gradually pushed into a region 
symbolized by sample focus plane 32. The focusing stage culminates in a 
sample that is focused near the inlet end of channel 12, as shown by 
sample focus plane 32 in FIGS. 1a, 1b, 1c 3a, 3b, and 4. The position of 
the focus plane 32 can be located anywhere between channel inlet 20 and 
injection system 26. 
In a preferred embodiment of the sample focusing device invented, sample is 
injected through sample inlet 22. Alternatively, sample can be injected in 
a different embodiment of this invention through channel inlet 20, in 
which case sample inlet 22 can be removed, plugged, or simply not built 
into the embodiment. 
An idealized visualization of the flows in the invented sample focusing 
method and device may be described as follows. A down-stream flow is 
defined as a flow that generally moves from the inlet region (channel 
inlet 20 and sample inlet 22), to the outlet region (channel outlet 24). 
Conversely, an up-stream flow is defined as a flow that generally moves 
opposite to the down-stream flow. In this setting, both the fluid injected 
through channel inlet 20 and the sample injected through sample inlet 22 
flow substantially down-stream. In this context, "substantially" means 
that, but for minor immaterial flow disturbances that do not cause 
measurable or undesirable effects, the flow is as herein characterized. 
Whether the term "substantially" is expressly used or not, it is 
understood that flow dynamics characterizations made herein are subject to 
minor immaterial flow disturbances that do not cause measurable or 
undesirable effects. 
For sample focusing, the fluid injected through injection system 26 
preferably has two major currents that flow simultaneously. One current 
flows substantially up-stream and the other current flows substantially 
down-stream. Less preferably, a third current that is comparatively minor 
can flow substantially across from permeable wall element 26c to 
accumulation wall 28. The presence of this third current, however, does 
not significantly affect the focusing of the sample. The fluid injected 
through injection system 26 that substantially forms the up-stream current 
retains and focuses the sample carried down-stream by the fluid injected 
through channel inlet 20. The fluid injected through injection system 26 
that substantially flows down-stream maintains fluid flow through channel 
12 and detector system 62. 
Focusing takes a few minutes, and more time may be required for more 
voluminous samples. This is because, as the sample is introduced through 
inlet 22, a finite time is required for the sample particles to travel 
from the point of introduction to the sample focus plane 32. While 
focusing, part of the flow introduced through injection system 26 flows 
down channel 12 towards outlet 24. Optionally, and to avoid changes in 
flow rate at the end of the focusing stage, reverse pump 64 controls the 
stream exiting channel 12 through outlet 24 by maintaining this stream's 
flow rate equal to the flow rate during the separation stage that follows 
the focusing stage. 
An exemplary set of flow rates while the set up shown in FIG. 4 operates in 
the focusing stage is given by the data in Table 1. 
TABLE 1 
______________________________________ 
Flow at Flow rate 
______________________________________ 
Channel inlet 20 0.25 mL/min 
Sample inlet 22 0.1 mL/min 
Injection system 26 4.5 mL/min 
Channel outlet 24 0.5 mL/min 
Cross flow outlet 38 4.35 mL/min 
______________________________________ 
Typically, the flow stream rates introduced through channel inlet 20 and 
sample inlet 22 are on the order of 0.01 to 0.5 mL/min; the flow stream 
rate through inlet 26a is typically on the order of 0.5 to 10 mL/min. 
Typical outward directed flow stream rates are: 0.5 to 5 mL/min at channel 
outlet 24 and 0.2 to 10 mL/min at the cross flow outlet 38. 
In contrast to conventional sample focusing methods, the sample focusing 
method and device permit the easy, external and effective tuning or 
control of the focusing process. As indicated in the preceding disclosure 
of actual focusing operations, rate regulation of the flows through 
injection system 26 on the one hand, and channel inlet 20 and sample inlet 
22, on the other hand, permit precise control and monitoring of the sample 
focusing process. This direct control and monitoring of sample focusing 
cannot be achieved by conventional methods that inject a sample that is 
subsequently confined to a region near the accumulation wall by the static 
or dynamic interaction with an element above the sample. In these 
conventional methods, the sample is still permitted to spread down-stream 
along the channel longitudinal axis. The invented sample focusing method 
and device further allow for precise control of the sample introduction 
process. In contrast, sample introduction is controlled by the channel 
dimensions in apparatuses that operate according to the pinched inlet 
principle. Another advantage of the sample focusing method and device 
operating as exemplified by the preceding description of the set-up shown 
in FIG. 4 is that sample focusing and sample equilibration are carried out 
simultaneously. 
A separation or sample migration stage follows the sample focusing stage. 
At the beginning of the separation stage, the flow through injection 
system 26 is ramped down and the flow through channel inlet 20 is ramped 
up so that in the most preferred embodiment the total amount of flow for 
these two flow streams is kept constant. Under these conditions, the flow 
rate in separation channel 12 during focusing is equal to the flow rate 
during separation. In another preferred embodiment, the flow through 
sample inlet 22 is discontinued at the beginning of the separation stage. 
In this preferred embodiment, the flow rate in separation channel 12 
during separation is less than the flow rate in the same channel during 
focusing. Optionally, by using computer controlled pumps, the flow rate 
through the injection system 26 is gradually decreased while the flow rate 
through channel inlet 20 and/or through sample inlet 22 is gradually 
increased. This is preferably accomplished by using computer controlled 
channel pump 54 and also a computer controlled focusing pump 56. In the 
most preferred embodiment, the total rate of the flows delivered by these 
two pumps is constant during the focusing and the separation stages. 
Alternatively, optional four-way valve 58 can be used to divert the flow 
from sample focusing pump 56 into channel inlet 20 and the flow from 
channel pump 54 into injection system 26. In any case, continuous flow 
through channel outlet 24 and detection system 62 is maintained throughout 
the focusing and separation stages. 
During the separation stage, the flow rates at channel outlet 24 and cross 
flow outlet 38 are approximately the same as those given in Table 1. The 
combined flow rates at channel inlet 20 and sample inlet 22, however, are 
approximately equal to the flow rate at the injection system 26 during 
focusing, and the injection system 26 flow rate is appropriately reduced 
to a rate on the order of 0.01 to 0.5 mL/min to maintain a flow rate 
through channel 12 that, in the most preferred embodiment, is constant. 
As illustrated by FIG. 4 and by the operational procedure described above, 
the invented sample focusing method and device rely on a simplified 
operation procedure because fewer switching valves are used for the 
focusing and separation processes. Furthermore, the flow paths used in the 
focusing stage are more similar to the flow paths used in the separation 
stage. The operational procedure described in relation to FIG. 4 also 
helps to explain the improved detection capability of the invented sample 
focusing method and device. This achievement is partly due to the absence 
of flow reversal or halting during the transition between the focusing and 
separation stages of the sample focusing method and device. 
This is because the invented sample focusing method and device are suitable 
to whichever conditions are imposed by the nature of the sample and 
operational parameters of the detector and the apparatus in which the 
separation takes. In particular, the temperature at which the separation 
is to be performed and the aqueous or nonaqueous character of the carrier 
fluid do not materially affect the performance of embodiments of the 
sample focusing method and device. 
EXAMPLES 
The sample focusing device and method of this invention were tested using a 
refractive index detector as the detection system. This type of detector 
is especially sensitive to pressure transients and so its response should 
indicate the presence and magnitude of a pressure transient. The specific 
refractive index detector used, the Optilab model DSP (Wyatt Technology 
Corp., Santa Barbara, Calif.), uses interferometry to detect refractive 
index changes. 
For comparison purposes, an asymmetrical flow FFF channel was set up under 
outlet focusing method conditions. FIGS. 5a and 5b show diagrams of this 
instrumental set up. The flow lines and positions of the switching valves 
are shown for the focusing stage according to the outlet focusing method 
in FIG. 5a; the valve positions and flow lines for the separation stage 
according to the same method are shown in FIG. 5b. The experimental 
conditions for the focusing stage were those given in Table 2. 
TABLE 2 
______________________________________ 
Flow Flow rate 
______________________________________ 
Channel inflow 0.25 mL/min 
Sample inflow 0.1 mL/min 
Outlet focusing 4.5 mL/min 
inflow 
Cross outflow 4.85 mL/min 
______________________________________ 
The flow rates during the separation phase were those given in table 3. 
TABLE 3 
______________________________________ 
Flow Flow rate 
______________________________________ 
Channel inflow 4.5 mL/min 
Sample flow 0 mL/min 
Channel outflow 0.5 mL/min 
Cross outflow 4.0 mL/min 
______________________________________ 
A blank sample was injected through the sample inlet. FIG. 6 shows the 
detector's response when the outlet focusing method was used with the set 
up illustrated in FIG. 5a for the focusing stage and FIG. 5b for the 
separation stage. The focusing stage begins at reading 62. In the focusing 
stage the flow is directed from a focusing pump through the detection 
system into the channel. FIG. 6 shows that the detector signal swings up 
and down at this stage. This "ringing" 64 is a response peculiar to the 
Optilab detector. Other refractive index detectors would generate at this 
stage an off-scale saturated signal response. The separation stage begins 
at reading 66. The two four-way valves are switched at the beginning of 
this stage and the detector response to this action is a large peak 68 
that almost reaches 1750 counts. This peak reflects the pressure transient 
that occurs when the flow is reversed. Following initial peak 68, the 
detector response was saturated at negative reading 70 near -400 counts. 
Over the course of the separation phase the detector response 72 only 
slowly re-equilibrated to a zero baseline. Other features in the graph 
shown in FIG. 6 include deviations 73 recorded between 14 and 16 minutes, 
but this deviations should be ignored because they are due to syringe pump 
perturbations. 
FIG. 7 shows the improved detector performance found using the invented 
sample focusing device and method. The optional four-way valve was used in 
this test, and a blank sample was injected through the sample inlet. The 
experimental conditions were chosen to be similar to those under which the 
outlet focusing method experiment described above was performed. These 
conditions are given in Table 1. Referring to FIG. 7, a small deviation 74 
in the detector response is shown at the beginning of the focusing stage 
76. This is due to the operation of the four-way valve and the sample 
injector. The detector baseline 78 during the focusing stage is slightly 
negative, -100 counts, but it does not drift. When the valve is switched 
back to its separation stage position, separation phase begins with 
reading 80. Small deviation 82 occurs when the separation phase starts and 
then baseline 84 immediately returns to its zero position. 
A comparison of FIGS. 6-7 indicates that the pressure transient found with 
the operation of the invented sample focusing device is insignificant. The 
benefits of this method include the following. First, the sample zone is 
not disturbed by the change from focusing to separation stages and so the 
resolution of separation is improved. Second, the lack of the initial 
pressure transient leads to a more stable baseline so that detection is 
improved, especially for peaks eluting early in the separation stage. 
Finally, baseline drift found in the outlet focusing method is eliminated 
so that quantitation of the peaks is more straight-forward. That is, with 
a drifting baseline the user must make assumptions regarding the position 
of the baseline under a peak. These assumptions affect the volume of the 
peak which is related to the amount of the sample species measured by the 
user. 
The invented sample focusing device and method allows the injection of a 
large volume sample, and this is one way to demonstrate the ability to 
generate a narrow sample plug. With the conventional sample introduction 
procedures, such as the pinched inlet and frit inlet relaxation 
procedures, only small volume injections can be used. Otherwise, the 
excessive band broadening results in loss of resolution. In contrast, 
FIGS. 8a-8b show the results obtained with an embodiment of this invention 
with samples of a 5 microliter and a 1 milliliter injection volume, 
respectively, of bovine serum albumin. BSA. BSA comprises two species: BSA 
monomer and BSA dimer. The analysis shown in FIG. 8a was conducted by 
injecting a 5-microliter sample that contained BSA monomer and dimer at 
concentrations of 5 mg/mL and 2 mg/mL, respectively. The injection and 
focusing time was 10.7 min. The analysis that produced the results shown 
in FIG. 8b was conducted by injecting a 1-milliliter sample that contained 
BSA monomer and dimer at concentrations of 0.025 mg/mL and 0.01 mg/mL, 
respectively. The injection and focusing time for this run was also 10.7 
min. The total amount of solids, 0.035 mg, was the same for both 
injections, so that the results could be compared directly. 
FIGS. 8a-8b show that the resolution of the BSA monomer and dimer 
detection, peaks 86 and 88, respectively, did not experience any 
significant change as a consequence of the 200-fold increase in injection 
volume. FIGS. 8a-8b also show that there is no significant difference in 
the peak heights, shapes, or in the areas under the peaks when 
corresponding peaks in these figures are compared with each other. The 
features shown in FIGS. 8a-8b are not compared to the corresponding 
results that one would obtain according to the pinched inlet or frit inlet 
relaxation methods because these methods would produce such broad peaks 
that currently available detection systems would not detect them relative 
to background noise. 
The results shown in FIGS. 8a-8b also indicate that the invented sample 
focusing method and device generate a narrow sample plug. The benefits of 
generating a narrow sample plug include the following. First, large volume 
injections can be made so that dilute samples can be effectively analyzed. 
This allows analysis of samples in which the particles, macromolecules or 
other species in the sample are normally present at levels below detection 
limits. Second, relative to current relaxation procedures, resolution is 
improved because the sample plug is compressed. Finally, the flexibility 
in sample injection flow rates is increased. That is, since the focusing 
process compresses the sample plug, the user no longer is concerned with 
sample dispersion which occurs when sample is injected slowly over a long 
period of time. 
A comparison of results with those obtained according to conventional 
sample introduction techniques further demonstrates the advantages of the 
invented sample focusing device and method. Specifically, the resolution 
achieved with the invented sample focusing device and method is compared 
with the resolution achieved by the stop flow and the frit inlet 
relaxation techniques. 
Experimental conditions were chosen so that analysis times were similar in 
each case and identical samples were used. In particular, the frit inlet 
run was performed with a cross-flow rate of 6.0 mL/min, channel flow rate 
of 1.4 mL/min, and frit-inlet ratio of 10:1. The stop-flow run was 
performed with the same cross-flow and channel flow rates and a stop-flow 
time of 0.4 min. Furthermore, except for the band broadening effects of 
the sample introduction methods under comparison, experimental conditions 
that generate the same amount of resolution in each system were used. This 
comparison should further indicate the advantages gained by incorporating 
the invented sample focusing device and method of this invention into 
appropriate separation systems. In all cases the sample was a mixture of 
BSA monomer and dimer. 
FIG. 9a shows the results of a separation performed according to the 
invented focusing sample device and method of this invention, and FIGS. 
9b-9c show the results obtained according to the stop flow and the frit 
inlet injection techniques, respectively. Whereas peak 90 that corresponds 
to the BSA dimer in FIGS. 9b-9c appears as a shoulder of the near peak for 
the BSA monomer, peak 88 for the BSA dimer in FIG. 9a is fully resolved, 
and it appears as a peak that is completely independent of peak 86 for the 
BSA monomer in the same figure. 
The results herein discussed show that the invented sample focusing method 
and device have a significantly improved resolution of separation relative 
to the outlet flow sample focusing method because the sample focusing 
method and device do not disturb the sample zone during the transition 
from the focusing to the separation stage. This improved resolution is 
also obtained relative to current relaxation procedures because the sample 
focusing method and device provide a highly compressed sample plug. 
The continuity of flow maintained by the sample focusing method and 
technique during the separation and focusing stages and during the 
transition therebetween leads to improved detection. Other features of the 
sample focusing method and device also contribute to detection 
enhancement. These features include the absence of detector signal 
disturbance as a consequence of absence of pressure transient, and the 
absence of flow reversal or flow stopping during the transition between 
the focusing and the separation stages. 
The schematic diagrams shown in FIGS. 1a-b, 2a-d, 3a-b, and 4 are not meant 
to be mutually exclusive. On the contrary, features represented in these 
figures can be suitably combined to generate additional embodiments of the 
present invention. These additional combinations however, can be performed 
with the aid of the objectives and teachings herein contained and ordinary 
skills in the art; thus no other combinations are offered as additional 
explicit examples.