Electric resonance chromatography

A process and apparatus for enhancing the separation of molecules, such as cells or proteins, into distinct propulations based on their interactions with an externally imposed varying electric field during liquid gel chromatography (LGC). An electric field, at radio frequency (RF), is created between two plates of a capacitor structure and impressed through the gel media column situated between the plates. The varying field interacts with the molecules through polarization events that alter the molecular vibration and rotation motions. The induced changes cause the molecules to exhibit elution characteristics distinct from those normally obtained during LGC. Thereby, molecules can be further and more selectively partitioned according to their distinct polarization properties.

BRIEF SUMMARY 
The invention disclosed herein pertains to processes and apparatus for 
performing electric resonance chromatography (ERC). Mixtures of molecules 
passing through a liquid gel chromatography (LGC) column are 
simultaneously subjected to a varying frequency electric field. Resonance 
and dipole movement effects in the molecules shift, narrow and otherwise 
improve the distinctive character of the detected elution patterns 
attributable to the individual molecules in the sample population. 
The electric field to which the molecules are subjected is of a high 
intensity, and is impressed in a direction substantially transverse to the 
LGC column and molecular flowing therethrough. In one form, the varying 
frequency of this field is selected to oscillate near the resonant 
frequencies of the molecules and the dipoles within their structural 
chain. The net and individual dipoles of the molecules are stimulated into 
new vibration and rotation modes, causing the molecules undergoing ERC to 
exhibit new and unique elution patterns, such as ones in which the 
apparent molecular weight and strokes radius have increased. 
Partitioning by gel permeation type ERC generates separation patterns which 
are characterized by early elution and reduced zone broadening. 
Furthermore, since the observed effect on the partition coefficient 
appears to differ among various cell and protein molecules subjected to 
ERC, these distinguishing characteristics improve the identification of 
the various molecules comprising the population.

DETAILED DESCRIPTION 
Liquid gel chromatography (LGC) is an established laboratory technique for 
fractionation and separation of molecules according to weight. The 
underlying premise for the technique is that molecules elute from the gel 
bed in order of decreasing molecular weight. This may be shown by 
utilizing FIGS. 1a, 1b and 1c, in which the three stages of simple gel 
permeation partitioning in a descending column are schematically 
illustrated. Gel particles 1 fill column 2 to form a bed. When the 
molecules to be partitioned, 3, such as cells or proteins, are introduced 
and driven through the bed by a supplemental eluent, the molecules 
separate on the basis of molecular weight. The larger molecules, 4, 
separate and elute first. Thus, the elution order is one of descending 
size. Since LGC in general, and gel permeation type LGC in particular, are 
well known by those practicing in the related arts, and are adequately 
described in references such as U.S. Pat. No. 3,002,823 to P. G. M. Flodin 
et al, further elaboration is superfluous. 
Though a multitude of refinements have been developed to improve the 
distinctness of the partitioning and increase its rate, better resolution 
of molecular species on the basis of weight and shape continues to be 
sought. This is particularly true when complex cell or protein 
populations, such as biological fluids, are being separated. In such 
cases, the conventional approaches involve successive LGC filtration steps 
or the concurrent use of other separation methods in conjunction with LGC. 
Electric resonance chromatography (ERC) alters conventional LGC techniques 
to both accelerate separation and accentuate the ability to distinctly 
identify molecular groups within the population undergoing analysis. In 
gel filtration terms, the elution time is decreased while the zones 
representing particular molecular groups are narrowed. 
Consider the schematic block diagram of one embodying apparatus, as it 
appears in FIG. 2 of the drawings. The right side of the figure consists 
of the pieces forming a fairly conventional gel permeation type LGC 
structure, in this case using an ascending column orientation. The left 
side of the same figure depicts in conventional block diagram form the 
elements needed to generate and control the varying electric field. In the 
center of the figure the two interact to form the essential feature of the 
embodiment. As shown, the LGC column is centered between a set of parallel 
plates which create the electric field and impress it on the column and 
sample molecules passing through the column. A cross-section of the column 
itself is shown in FIG. 3, taken at a point about midway along its length. 
Since the LGC apparatus consists of well known commercially available 
equipment, its operation need not be described with particularity. In 
general, though, sample material 1 and eluent 2 are selectively regulated 
by valve 3 and then driven through gel column 4 by pump 6. Valve 3 has 
three positions; a position in which both sample and eluent are off, a 
position in which only the sample passes, and a position in which only the 
eluent passes. The output from column 4 is directed through a detector, 
flow cell 7, and then enters fraction collector 8. UV monitor 9 responds 
to variations in the material passing through flow cell 7 and permanently 
records those signals on strip chart recorder 11. Column 4, and the 
materials passing through it, are maintained at a substantially constant 
temperature by the circulation of distilled water 12 at 25.degree. C. 
through its outer jacket, clearly appearing in FIG. 3, by means of water 
bath circulator and temperature control 13. 
More specifically as to the structure of this embodiment, sample material 1 
consists of three purified globular proteins mixed in equal amounts by 
weight, bovine serum albumin (BSA, m.w. 69,000d), ovalbumin (OVA, m.w. 
45,000d), and ribonuclease - A (R-A, m.w. 13,700d). All three were 
supplied by Calbiochem-Behring (San Diego, Calif.). Eluent 2 used in this 
embodiment is commonly known as phosphate buffered saline, with a pH of 
6.95 at 25.degree. C., formed from 0.9% W/V saline in glass distilled 
H.sub.2 O to which is added 0.05 M phosphate buffer. Column 4 is a model 
K-26 manufactured by Pharmacia Fine Chemicals (Piscataway, N.J.), while 
reference numerals 7, 8, 9 and 11 designate pieces of automated 
monitor-collector equipment from Instrument Specialties Company (Lincoln, 
Nebr.). Gel media 14, for purposes of this particular embodiment, was 
Sephadex G-200 (superfine), though similar results were later obtained 
using Sephadex G-100 and Sephacryl G-200. All three gel media materials 
are trademark products manufactured by Pharmacia Fine Chemicals. 
Since the control of the material admitted into pump 6 from valve 3 
conformed to the manufacturer's instructions when using gel media 14 and 
column 4, it need only be noted that the embodying example used 1-2 mg of 
the mixture in sample 1 to create the response described and plotted 
herein. Procedurally, valve 3 is first set to fill column 4 with eluent 2. 
Once the column is filled, valve 3 is set to allow sample 1 flow until 1-2 
mg are introduced. Thereafter, the valve is returned to its eluent flow 
position. Eluent flow is maintained until the column is cleared of sample 
material. 
The electrical aspects of this invention focus on the action of the varying 
electric field as it interacts with the material in column 4. As is 
depicted in FIG. 2, the varying electric field is created by a set of 
near-field synthesizer electric field capacitor plates 16 and 17. Column 4 
is situated substantially parallel to and midway between the capacitor 
plates. A faraday cage or shielded room, represented by dashed line 18, 
encloses the plates and column. The faraday cage is sufficiently large to 
prevent self-resonance at any frequency of interest. The field between 
plates 16 and 17 is substantially uniform, with a peak electric field 
intensity magnitude of 8500 V/m at the carrier frequency of 10 MHz. The 
carrier is further modulated at a rate of 16 Hz for the particular 
embodiment described herein. Inside gel media 14 the corresponding 
electric field intensity was calculated to be approximately 20.5 V/m. The 
detailed structure of the near-field synthesizer creating these electric 
fields is described with great particularity in the National Bureau of 
Standard (NBS) Technical Note 652, issued in May 1974 and entitled 
"Development and Construction of an Electromagnetic Near-Field 
Synthesizer". 
The excitation for field plates 16 and 17 is coupled through impedance 
matching device 21; this device is also described in the NBS Technical 
Note. Impedance matching device 21 is a tuneable network, for adjusting 
the resonant frequency of the two capacitor plates, and further includes a 
balun transformer for impedance matching the 75 ohm coaxial input cable to 
the 300 ohm input impedance of the capacitor plates and preceeding tuning 
network. 
The 10 MHz RF power entering impedance matching device 21 comes from linear 
amplifier 22. The RF signal controlling the amplifier is generated in RF 
generator 23 and modulated by signals from square wave pulse generator 24. 
Power meter 26 and dipole and electrometer 27 monitor the RF drive and 
electric field. 
In general, the interaction of the varying electric field with the gel and 
sample materials, as embodied, accelerates the elution rate while reducing 
the zone broadening effects of the elution response. The early elution 
aspect, as a response, is similar to that which would appear if the 
molecular weights of the sample were increased. The narrowing of zones in 
the response is a beneficial attribute of ERC, but has yet to be fully 
understood as to its mechanism. 
The presence of the RF electric field induces changes in the sample mixture 
of proteins, which respond as if they experienced an increase in molecular 
weight and stokes radius. These apparent increases are attributable to 
perturbations in the shape of the sample molecules; the degree of the 
distortion being dependent on net dipole moment and the orientations of 
the dipoles within the molecular chain. As the varying electric field 
induces new modes of rotation and vibration in the net and individual 
dipoles, the effective radii of the sample molecules increases. When 
compared to conventional gel permeation type LGC, the apparent rise in 
molecular weight and strokes radius inhibits the more polarizable 
macromolecules from penetrating the gel matrix, leading to earlier and 
narrower elution responses. 
Consider, in further explanation of this principle, a schematic protein 
molecule chain, 28, as depicted in FIG. 4. A multitude of individual 
dipoles 29 are dispursed along the chain at near-random orientations and 
locations. The application of an electric field acts on each dipole to 
alter its alignment. The final orientation of any single dipole is, 
nevertheless, still related to the redistributed alignments of all the 
dipoles along the chain. Since the magnitude of the electric field created 
aligning force 31 acting on any dipole, such as dipole 32 in FIG. 5, is 
influenced by both the magnitude of electric field 33 and the angle .phi., 
the complexity of the total interaction prevents direct theoretical 
analysis. 
The sample molecules undergoing ERC may or may not have a net dipole 
moment. In either case, the molecules will still have multiple individual 
dipoles distributed along the molecular chain. When the molecules do 
exhibit a net dipole moment, the interaction with the electric field will 
respond with the effects of both the net and individual dipoles. On the 
other hand, the absence of a net dipole moment does not preclude 
interaction with the field, but rather lessens the degree of molecular 
distortion. 
A full recognition of all the mechanisms and their degree of contribution 
toward altering the elution patterns during ERC is not readily 
discernible. To a degree, this is a result of concurrent interactions. One 
such interaction involves the presence of smaller molecules in the varying 
electric field for a longer period of time by nature of convention LGC 
action. The effects of the varying field are superimposed. Though 
recognized herein as contributing to the shape of the overall elution 
pattern, the effects of this interaction are well beyond the scope of the 
invention as disclosed and claimed. 
As the varying electric field is applied to the sample molecules, their 
shape is distorted. Reversing the direction of the field changes the 
distortion. And if the field is completely removed, the molecular shape 
reverts to its relaxed state. In this fashion, new rotation and vibration 
modes enlarge the apparent molecular weight and stokes radius, and 
thereby, alter its elution characteristics to create unique separation 
patterns related to the dipole distribution of the molecule. 
Another aspect of the dipole activity induced by the electric field relates 
to the degree of force 31 needed to overcome the rotary friction 
attributable to solvent visocity, and that necessary to compensate for the 
rotary diffusion force caused by the kinetic thermal energy of the 
molecules. 
In both general types of influences created by the varying electric field, 
a relaxation time is associated with each new equilibrium position for the 
molecule. For the particular three proteins being considered in the 
embodiment, one microsecond is equivalent to infinite time for both 
influences. Therefore, a field varying at 10 MHz is sufficiently slow to 
permit complete dipole reorientation between cycle peaks. 
To further elaborate on the process and apparatus concepts disclosed above, 
consider the specific embodiment in FIG. 2. Sample 1 is a mixture in equal 
proportions by weight of three purified globular proteins, BSA, OVA and 
R-A. The partitioning output response, in terms of absorbancy measured by 
UV monitor 9, verses effluent volume entering fractional collector 8, is 
graphically displayed in FIG. 6. Control samples of the protein mix were 
eluted before and after the run in which the RF electric field was imposed 
to verify calibration. As shown, the pre and post electric field elution 
curves, solid line 34, are identical. The presence of the RF electric 
field produced the elution profile of dashed line 36. 
The elution peaks corresponding to BSA, OVA and R-A are individually 
designated, with their molecular weights shown in parenthesis. The 
enhanced elution exhibited by dashed line 36 is quite evident not only as 
to its earlier occurrence, but also as to depth of troughs 37 between 
adjacent peaks. The first mentioned characteristic corresponds to an 
increased elution rate, typifying an increased molecular weight or stokes 
radius. The second is best described in terms of its effect, this being 
enhanced distinction of adjacent peaks for superior purification of mixed 
molecules. The interesting aspect of the latter characteristic is its 
presence in the elution pattern without an accompanying drop in the 
magnitudes of the peaks. 
Partitioning of mixtures, such as the three proteins described above, by 
permeation through a gel is characterized by a partition coefficient, 
K.sub.av. Partition coefficient K.sub.av is defined as a relationship of 
volumes; i.e. 
EQU K.sub.AV =(V.sub.e -V.sub.o)/(V.sub.t -V.sub.o), 
where 
V.sub.e =elution volume for the proteins, 
V.sub.o =void volume for the packed column, and 
V.sub.t =total volume for the packed column. 
This parameter is essentially independent of column dimensions and the 
compaction degree of the gel bed. 
FIG. 7 contains a plot of K.sub.av verses the log of molecular weight for 
the elution data plotted in FIG. 6. Lines 38 and 39 join data taken under 
comparable operating conditions during elution. A comparison of the pre 
and post RF electric field values for K.sub.av against the values during 
the RF electric field indicates again that the presence of the field 
causes an apparent rise in molecular weight. For instance, consider the 
case of R-A protein having a known molecular weight of 13,700 d; the log 
of this magnitude being 4.14. Without the RF electric field K.sub.av is 
calculated from the elution pattern to have a value of 
4.9.times.10.sup.-3, reference point 41 of FIG. 7. With the effects of the 
electric field, the elution response calculates to a K.sub.av of 
4.4.times.10.sup.-3, appearing as reference point 42. To determine what a 
K.sub.av of 4.4.times.10.sup.-3 represents in gel permeation type LGC, 
project across to point 43 on line 39. Following dotted line 44 down to 
the molecular weight axis of the plot, the corresponding weight is found 
to be 16,790 d, the antilong of 4.225. The elution response with an RF 
electric field present, thereby, can be said to increase the apparent 
molecular weight of R-A by 22.6%. Repeating this process for OVA and BSA 
reveals corresponding molecular weight rises of 7.6% and 0.27%, 
respectively. 
The above-exemplified three proteins are all globular in shape. ERA would 
be expected by those skilled in the art to alter the elution 
characteristics of asymmetric, fibrous proteins to an even further degree, 
since large frictional and rotary diffusion forces would act to prevent 
rapid relaxation during field variations. 
As another consideration, the invention as embodied and described in the 
foregoing recognized and fully contemplates other variations in the 
character of the electric field. One such is the absence of square wave 
pulse generator 22, so that the varying field, at RF or otherwise, is 
continuously present during elution. Preliminary experiments have shown 
this to be viable. In conjunction with this variant, selective tuning of 
the frequency or the electric field intensity to optimize the separation 
characteristics are similarly contemplated. 
The overall scope of the invention encompasses gel beds and sample 
materials beyond those in the embodiment. For instance, Sephadex G-100 
(fine) and Sephacryl G-200, trademark products of Pharmacia Fine 
Chemicals, have performed similarly. Because of their likeness to the 
above-name products insolubilized dextran, copolymerized acrylamide and 
agarose would be expected to respond substantially the same. The 
invention, therefore, broadly encompasses the use of all gel materials 
which selectively absorb or otherwise selectively interact with substances 
from a solution passing therethrough. 
The term liquid gel chromatography (LGC) when used herein implies the art 
in its broadest sense. Namely, the term includes, but is not limited to, 
species such as affinity liquid gel chromatography, ion exchange liquid 
gel chromatography, and fairly conventional gel permeation forms of such 
chromatography. Since the essential process and apparatus features of this 
invention are broad, yet situated within a highly fluid art, their 
equivalents should not be circumscribed by the structural or material 
limitations of the present art.