U.S. patent number 3,856,669 [Application Number 05/375,882] was granted by the patent office on 1974-12-24 for elution centrifuge-apparatus and method.
This patent grant is currently assigned to The United States of America as represented by the Secretary of the. Invention is credited to Robert L. Bowman, Yoichiro Ito.
United States Patent |
3,856,669 |
Ito , et al. |
December 24, 1974 |
ELUTION CENTRIFUGE-APPARATUS AND METHOD
Abstract
An apparatus for elution centrifugation used in countercurrent
chromatography comprises a high speed centrifuge head which
revolves around a central vertical axis, a cylindrical column
holder which is horizontally and rotatably carried by the
centrifuge head, and a separation column fixedly disposed within
the holder. The separation column includes a fine tube which passes
externally introduced fluids to and from the centrifuge head and
the cylindrical column holder without the use of rotating seals.
The seals are eliminated by rotating the cylindrical column holder
about its own axis while, simultaneously, revolving it around the
central vertical axis of the apparatus at the same angular
velocity.
Inventors: |
Ito; Yoichiro (Bethesda,
MD), Bowman; Robert L. (Bethesda, MD) |
Assignee: |
The United States of America as
represented by the Secretary of the (Washington, DC)
|
Family
ID: |
23482752 |
Appl.
No.: |
05/375,882 |
Filed: |
July 2, 1973 |
Current U.S.
Class: |
210/635; 210/657;
210/198.2 |
Current CPC
Class: |
G01N
30/42 (20130101) |
Current International
Class: |
G01N
30/00 (20060101); G01N 30/42 (20060101); B01d
015/08 () |
Field of
Search: |
;210/31C,198
;55/67,197,386 |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Adee; John
Claims
What is claimed is:
1. An elution centrifuge comprising:
a separation column the axis of which is generally perpendicular to
a main axis of revolution;
a feed tube for introducing fluids to said separation column and
means for passing fluid to said feed tube;
a return tube for discharging fluids from said separation column
and means for receiving fluid therefrom;
centrifugation means for revolving said separation column about the
main axis of revolution and for simultaneously rotating said
separation column about its axis at the same angular velocity to
prevent twisting of said feed and return tubes; and
a stationary guide means, located outside said centrifugation means
coaxial with the main axis of revolution, for guiding said feed and
return tubes.
2. The device of claim 1 wherein said centrifugation means
includes:
a drive shaft located on said axis of rotation;
a motor for rotating said drive shaft; and
a centrifuge head perpendicularly attached to said drive shaft,
said centrifuge head comprising at one end a separation column
holder rotatable about the axis of rotation of said separation
column and an adjustable counterweight located at the opposite
end.
3. The device of claim 2 wherein said centrifugation means for
simultaneously rotating said separation column further
includes:
a first drive means coaxially located about said drive shaft;
a second drive means engaging said rotatable separation column
holder and disposed in a plane perpendicular to said first drive
means;
an endless belt connecting said first and second drive means;
and
means disposed on both sides of said first and second drive means
for changing the direction of said endless belt by 90.degree..
4. The device of claim 3 wherein said first and second drive means
are pulleys of equal diameter.
5. The device of claim 4 wherein said means for changing the
direction of the endless belt are idler pulleys, said idler pulleys
being rotatable about an axis defined by the intersection of the
planes of rotation of said pulleys and spaced apart a distance
approximately equal to the diameter of the pulleys.
6. The device of claim 1 wherein said stationary guide means
comprises:
a cylindrical hollow sleeve; said sleeve having first and second
ends, the first end lying approximately on the axis of rotation of
said separation column; and
a polytetrafluoroethylene collar disposed about and extending past
said first end.
7. A method of countercurrent chromatography comprising:
filling a separation column through a feed tube with a first
solvent, said separation column having an axis of rotation
perpendicular to a main axis of revolution, the feed tube being
located coaxial with the main axis of rotation;
centrifuging said filled separation column by revolving said filled
separation column in a plane perpendicular to the main axis of
revolution, the main axis of revolution lying outside said
separation column, at various predetermined angular velocities and
in a predetermined direction while simultaneously rotating said
separation column about its own axis of rotation at the same
angular velocity as the revolution of said separation column and in
a direction whereby the feed tube will not twist;
introducing a sample solute to be separated into the moving
separation column;
pumping a second solvent, immmiscible with said first solvent, into
the moving separation column; and
recovering the separating solute fractions leaving the separation
column.
8. The method of claim 7 wherein said first solvent is heavier than
said second solvent.
9. The method of claim 7 wherein said first solvent is lighter than
said second solvent.
Description
FIELD OF THE INVENTION
The present invention relates to the separation of samples and,
more pertinently, to an elution centrifuge method and device which
permits elution and elutriation on both analytical and preparative
scales.
BACKGROUND OF THE INVENTION
Various separation techniques are known in which two immiscible or
partially soluble liquid phases are brought into contact for the
transfer of one or more components. Among such liquid-liquid or
solvent extraction techniques are partition chromatography and
countercurrent chromatography. This latter technique can be carried
in various ways (see our copending application Ser. No. 275,777 now
U.S. Pat. No. 3,775,309) among which is helix countercurrent
chromatography, in which a horizontal helical tube is filled with
one phase of a two-phase liquid and the other phase is introduced
at one end of the helix and passes through the first phase. To
enable the countercurrent process to take place inside a very small
diameter tube having a maximum number of turns, it is desirable to
enhance the gravitational field by the use of a centrifuge.
Prior art helix countercurrent chromatography devices using a
centrifuge employ rotating syringes or rotating seals. A major
problem with these devices is that the rotating syringes or seals
make gradient or stepwise elution difficult, if at all possible,
and, therefore, decrease the efficiency of the devices.
The efficiency of prior art flow-through coil planet centrifuges
also suffers because they provide for a non-universal application
for solvent systems such as polymer phase systems used for the
separation of macromolecules and particulates.
SUMMARY OF THE INVENTION
It is, accordingly, an object of the present invention to overcome
the defects of the prior art, such as indicated above.
Another object is to provide for improved separation of one
material from another.
Another object of the present invention is to provide a centrifuge
capable of elution and elutriation on both analytical and
preparative scales.
Another object is to provide an elution centrifuge capable of
various applications including countercurrent chromatography.
Another object is to provide an elution centrifuge that yields a
resolving power at least equivalent to that in prior art helix
countercurrent chromatography devices.
A further object is to provide an elution centrifuge for
countercurrent chromatography which has a higher efficiency than
that of refined gas chromatography.
In furtherance of these and other objects, a principal feature of
the present invention is an elution centrifuge which eliminates
rotating seals, thereby permitting an accurate flow through the
feed tube under high feed pressure and preserving the narrow bands
of the separated samples in passing through the negligibly small
dead space of the return tube. Another feature is the particular
fluctuation of the centrifugal acceleration field in which
stability of the field is a function of radii of both revolution
and rotation. Thus, the shortcomings of prior art centrifuges used
for countercurrent chromatography are satisfactorily overcome by
the present invention.
The elution centrifuge of the present invention is characterized by
a flow-through separation column including fine lead tubes without
rotating seals. The device also includes a cylindrical column
holder, the holder being horizontally and rotatably disposed in a
centrifuge head which revolves around a central vertical axis. The
column holder revolves around the vertical axis while,
simultaneously, rotating around its own horizontal axis at the same
angular velocity and in a direction which avoids any twisting of
the lead tubes caused by revolution.
BRIEF DESCRIPTION OF THE DRAWING
FIG. 1 is a schematic diagram of the elution centrifuge.
FIG. 2 is a graph showing the motion of an arbitrary point on the
centrifuge column holder in the x-y-z planes.
FIG. 3 is a graph showing the fluctuation of the centrifugal
acceleration of the arbitrary point parallel to the axis of the
holder of FIG. 2 during one revolution.
FIG. 4 is a graph showing the fluctuation of the centrifugal
acceleration of the arbitrary point in a plane perpendicular to the
axis of the holder of FIG. 2 during one revolution.
FIG. 5 is a graph showing the undulation of the direction of the
centrifugal acceleration of FIG. 4 during half a revolution.
FIG. 6 is a perspective view of a preferred embodiment of the
elution centrifuge.
FIG. 7 is a cross-sectional view of the separation column and
column holder shown in FIG. 6.
FIG. 8 is a perspective view of a separation column and a stretched
helix tube arrangement.
FIG. 9 is a perspective view of a separation column and a coiled
helix tube arrangement. column and a parallel helix tube
arrangement.
FIG. 11 is a perspective view of a separation column and a
loop-type tube arrangement.
FIG. 12 is a graph of the results of dinitrophenyl (DNP) amino acid
separation achieved through the use of the device of FIG. 6.
For a better understanding of the invention a possible embodiment
thereof will now be described with reference to the attached
drawing, it being understood that this is to be intended as merely
exemplary and in no way limitative.
DETAILED DESCRIPTION
The principle of the system is illustrated in FIG. 1. A cylindrical
holder 10 containing a separation column (not visible) is held
horizontal. Both feed and return tubes 11 and 12, respectively,
pass through an axial hole 17 of the holder 10 and are wrapped
around the separation column. The tubes 11 and 12 are supported
tightly at the center of the apparatus by a guide tube 13 fixed to
the top frame 14 of the centrifuge. The holder 10 revolves around
the central vertical axis of the apparatus in the horizontal plane
and simultaneously rotates about its own horizontal axis at the
same angular velocity. The relative directions of the revolution
and rotation are shown by the arrows in FIG. 1, it being understood
that both revolution and rotation may be in the direction opposite
to that shown so that twisting of the tubes 11 and 12 is avoided.
This simultaneous rotation and revolution at the same angular
velocity is achieved by coupling a pulley fixed to the holder by
means of a toothed belt to a stationary pulley of equal diameter on
the axis of the centrifuge drive (e.g. see description below of
FIG. 6). This synchronous rotation unwinds the twist of the lead
tubes caused by the revolution of the holder 10.
A pump 15 supplies fluid through the feed tube 11 to the holder 10.
After centrifuging the fluid travels through return tube 12 to a
receptor 16. A polytetrafluoroethylene collar 18 located at the
lower end of the guide sleeve 13 protects the feed tube 11 and
return tube 12 throughout their freely moving portion, thereby,
extending their life expectancy. A piece of silicon rubber tubing
(not shown) is inserted in the tapered hole of the
polytetrafluoroethylene collar 18 to prevent the feed and return
tubes from directly contacting the polytetrafluoroethylene
collar.
The synchronous rotation also adds a peculiar effect to the
centrifugal force field at the column holder. A simple mathematical
analysis, introduced below, shows that any arbitrary points in the
column holder, except for those located on the axis of rotation,
are subjected to a periodic fluctuation of the centrifugal
force.
Referring now to FIG. 2, an arbitrary point, P (x,y,z), is located
at r.sub.1 from the axis of revolution and at r.sub. 2 from the
axis of rotation at the starting point P.sub.o. The point then
revolves around the z axis at angular velocity .omega. and
simultaneously rotates around the column axis in the x-y plane at
the same angular velocity. Consequently, point P travels always on
a spherical surface centered at point O with a radius of
(r.sub.1.sup.2 + r.sub.2.sup.2).sup.1/2, and at time t, (since
.theta. = wt.)
x = r.sub.1 cos .theta. + r.sub.2 sin.sup.2 .theta.
y = r.sub.1 sin .theta. - r.sub.2 sin .theta. cos .theta.
z = r.sub.2 cos .theta.
Since the net centrifugal acceleration field,
.alpha. = [(d.sup.2 x/dt.sup.2) + (d.sup.2 y/dt.sup.2) + (d.sup.2
z/dt.sup.2 ].sup.1/2
gives a rather complicated picture of the three-dimensional change
of its direction with .theta., it is convenient to express it in
terms of two components, .alpha..sub.1 (centrifugal acceleration
parallel to the axis of the holder), and .alpha..sub.2 (centrifugal
acceleration in a plane perpendicular to the axis of the
holder).
The first component is given by the equation,
.alpha..sub.1 = -(d.sup.2 x/dt.sup.2) cos .theta. - (d.sup.2
y/dt.sup.2) sin .theta.
which reduces to
.alpha..sub.1 = r.sub.1 .omega..sup.2 (1 - 2.beta. cos .theta.)
where .beta. = r.sub.2 /r.sub.1 and r.sub.1 .noteq. 0.
Referring now to FIG. 3, the values of .alpha..sub.1 expressed in
terms of r.sub.1 .omega..sup.2 is plotted against angle .theta.
during one revolution. Four lines are drawn according to the .beta.
values of 1, 1/2, 1/4 and 0 as indicated. It illustrates that
.alpha..sub.1 oscillates around the .beta. = 0 line once in each
revolution with amplitude dependent upon the .beta. value. When
.beta. exceeds 1/2, .alpha..sub.1 crosses below the dotted 0 line,
indicating that the acceleration acts momentarily in the opposite
direction to cause the liquids to move toward the center of
revolution. As .beta. decreases, the amplitude of the oscillation
becomes smaller and finally reduces to zero at .beta. = 0 providing
a stable acceleration field similar to that in the conventional
centrifuge system.
The second component which acts in the plane perpendicular to the
axis of the holder is given by the equation,
.alpha..sup.2.sub.2 = [(d.sup.2 x/dt.sup.2) sin .theta. - (d.sup.2
y/dt.sup.2) cos .theta.].sup.2 + (d.sup.2 z/dt.sup.2).sup.2
or
.alpha..sub.2 = r.sub.2 .omega..sup.2 (1 + 3 sin.sup.2
.theta.).sup.1/2 = r.sub.2 .omega..sup.2 [(5 - 3 cos
2.theta.)/2].sup.1/2
Referring now to FIGS. 4 and 5, FIG. 4 shows the relationship
between .alpha..sub.2 (in terms of r.sub.2 .omega..sup.2) and
.theta. during one rotation. Note that .alpha..sub.2 oscillates
twice in each revolution between the values of r.sub.2
.omega..sup.2 and 2r.sub.2 .omega..sup.2, and that the wave form is
not accurately sinusoidal. The value of .alpha..sub.2 is
independent of r.sub.1 and reduces to 0 at r.sub.2 = 0. The acting
direction (.gamma.) of .alpha..sub.2 in the plane perpendicular to
the column axis is calculated by the equation.
tan .gamma. = [(d.sup.2 x/dt.sup.2) sin .theta. - (d.sup.2
y/dt.sup.2) cos .theta.]/(-d.sup.2 z/dt.sup.2)
which becomes
tan .gamma. = 2 tan .theta. indicating that the direction of
.alpha..sub.2 undulates about the 0 line twice in one rotation (see
FIG. 5).
The results of the above analysis show that the synchronous
rotation causes an oscillating centrifugal acceleration. The
relative amplitude of the oscillation becomes greater as the
distance (r.sub.1) from the axis of revolution decreases or the
distance (r.sub.2) from the axis of the rotation increases. Thus, a
stable centrifugal acceleration field can be obtained at or near
the axis of the holder with a great radius (r.sub.1) of revolution.
On the other hand, the mixing or vibration, if desired, can be
attained at the location remote from the axis of the holder with a
small radius (r.sub.1) of revolution.
FIG. 6 shows the preferred embodiment of a device in accordance
with the instant invention. A centrifuge head 20, which is a
substantially rectangular box defined by four wall plates including
short side plates 23 and 24, may be made of aluminum or any other
suitable material. Vertical septums 21 and 22 are located inside
the head 20 between and parallel to the short side plates 23 and
24. The spacing between side plate 23 and septum 21, between septum
21 and septum 22, and between septum 22 and side plate 24 is
substantially equal. The centrifuge head 20 which is shown in FIG.
6 with an open top may also be closed if desired and may be
constructed by modifying a conventional centrifuge, such as the
Model II manufactured by the International Equipment Company.
On the column side (right side in FIG. 6) a thrust bearing (not
shown) and an ordinary ball bearing 25 are located within the
septum 22 and short side plate 24, respectively, to support a
cylindrical separation column holder 26 which extends horizontally
through the ball bearing encircled opening in the side plate
24.
The septum 21 and short side plate 23 include U-shaped openings for
horizontally supporting a counterweight 27 made of an aluminum
cylinder or other suitable material. The counterweight 27 includes
a threaded rod 28 which extends, substantially perpendicular, from
the flat outer surface thereof. An adjustable bolt 29 and an
adjustable weight 30 are mounted on the threaded rod 28.
The synchronous rotation of the separation column holder 26 is
accomplished by a system of toothed pulleys of the same diameter
including the pulleys 31 and 32. One collar-like pulley 31 is
fixedly attached about the outer surface of the holder 26,
approximately midway between the septum 22 and the side plate 24. A
shaft 33 for revolving the centrifuge passes from the electric
driving motor (not shown), protected within a housing 34; and the
other stationary pulley 32 is fixedly attached to the top surface
of the motor housing 34 coaxially about the rotating central shaft
33, thereby allowing the shaft 33 to rotate freely from the
stationary pulley 32. The shaft 33 passes through the approximate
center of the substantially rectangular bottom plate of the
centrifuge head 20 which is securely fastened to the shaft 33 by a
nut 35 or any other suitable fastening means.
The pulleys 31 and 32 are coupled by a toothed endless belt 36
which passes through a hole 37 in the bottom plate of the
centrifuge head 20 and over a pair of toothed idler pulleys 38
(only one being visible in FIG. 6) mounted on both ends of a block
39 which is fixedly attached to the bottom plate of the centrifuge
head 20. These idler pulleys 38 function to change the direction of
the endless belt by 90 degrees.
Feed and return tubes 40 and 41, respectively, which may be made of
polytetrafluoroethylene or other suitable material pass through a
vertical guide sleeve 42 and are led through a center hole in the
end of a separation column (not shown in FIG. 6) around which they
are wound. The guide sleeve 42 passes through and is fixedly
attached to the top of the centrifuge frame 43. A
polytetrafluoroethylene collar 44 located at the lower end of the
guide sleeve 42 protects the feed tube 40 and return tube 41
throughout their freely moving portion thereby preserving their
life expectancy for many hours at the maximal speed of
approximately 2,000 rpm. A piece of silicon rubber tubing (not
shown) is inserted in the tapered hole of the
polytetrafluoroethylene collar 44 to prevent the feed and return
tubes from directly contacting the polytetrafluoroethylene
collar.
FIG. 7 shows a cross section of the separation column holder 26 and
a separation column 45 contained therein. The separation column 45
includes two circular T-shaped end plates or plugs 46 and 47. The
smaller diameter ends of these plugs 46 and 47 are press fitted
into the open ends of a cylindrical pipe 48. Located on the pipe 49
approximately midway between the end plates 46 and 47 is an
O-shaped collar 50 which helps to support the center of the pipe
48. The collar 50 and end plates 46 and 47 are secured to the pipe
48 by set screws 49.
End plate 46 which faces towards the center of the centrifuge
includes a central aperture 51 through which pass the feed and
return tubes 52 and 53 from outside the separation column 45
through the aperture 51 in end plate 46 to the interior of the pipe
48. A hole 54 in the wall of pipe 48 allows the feed and return
tubes to pass from the interior to the exterior thereof. The feed
and return tubes are wound about the exterior surface of pipe 48 in
one of many configurations which will be described hereinafter.
Collar 50 includes a longitudinal hole 55 through which the feed
and return tubes pass so that they may be wound about the entire
length of the pipe 48.
The larger diameter of the circular T-shaped end plates 46 and 47
and the outer diameter of collar 50 are approximately equal to the
inner diameter of the column holder 26 thereby allowing the
separation column assembly 45 to be securely press fitted into the
column holder 26. The feed and return tubes 52 and 53 are
accordingly protected within the annular space between the pipe 48
and the column holder 26.
Referring now to FIGS. 8 - 11, it is seen that the feed and return
tube arrangement may take various forms. FIG. 8 shows the tubing
which is folded in two to form the feed and return tubes 52 and 53,
respectively. The tubes are then twisted together to make between
10,000 and 20,000 turns and stretched in a helical pattern about
the cylindrical pipe 48. The feed and return tubes are anchored at
the end of pipe 48 fartherst from the central aperture 51 by a pin
54 which extends radially outwardly from the surface of the pipe
48.
FIG. 9 shows the feed and return tubes 52 and 53 wrapped about the
pipe 48 in a coiled helix pattern. FIG. 10 shows the coiled feed
and return tubes 52 and 53 wrapped about the pipe 48 in a parallel
helix configuration. FIG. 11 shows the feed and return tubes 52 and
53 in a parallel loop configuration.
FIG. 12 shows the result of dinitrophenyl (DNP) amino acid
separation acheived with the instant device and described more
fully below.
In order to more fully describe the operation of the apparatus and
the method as applied to countercurrent chromatography, separation
of nine dinitrophenyl (DNP) amino acids on a two phase system
composed of chloroform, glacial acetic acid and 0.1 N HCl (2:2:1)
will now be discussed. The separation column is filled with the
stationary lower (heavier) phase of the solvent and 3 .mu.l of
sample solution (solute), containing each component at about 1%
where solubility permits, is introduced through the feed tube. The
immiscible upper (lighter) phase of the solvent is pumped with any
suitable syringe drive, such as Model 933 manufactured by the
Harvard Apparatus Co. at a rate of 120 .mu.l per hour while the
apparatus is spun at 700 rpm at room temperature. It should be
noted that the order of introduction of the heavier and lighter
solvents may be reversed if desired.
The continued injection of the upper moving phase causes it to
percolate through the stationary lower phase which is trapped by
gravity and centrifugal force. Sample solution or solute which is
introduced into the device as described above is, thereby, exposed
to each stationary segment, attaining a degree of equilibration
dependent upon the degree of mixing that results from the
percolation, filming, and surface tension changes as the solute is
partitioned between the phases. Consequently, such a solute
introduced into the device is subjected to a partition process
between the oscillating alternate segments of the two phases and
finally eluted out through the return tube of the separation
column. The eluate may be monitored by an LKB Uvicord II or similar
device at 280 mm.
As shown in FIG. 12, nine DNP amino acids are eluted out with 54
hours. The efficiency of the present method ranges between 10,000
and 6,400 theoretical plates estimated according to the formula
used in gas chromatography and indicates that a resolving power at
least equivalent to that reported in helix countercurrent
chromatography may be achieved.
The foregoing description of the specific embodiment will so fully
reveal the general nature of the invention that others can, by
applying current knowledge, readily modify such specific embodiment
and/or adapt it for various applications without departing from the
generic concept, and, therefore, such adaptations and modifications
should and are intended to be comprehended within the meaning and
range of equivalents of the disclosed embodiment. It is to be
understood that the phraseology or terminology employed herein is
for the purposes of description and not of limitation.
* * * * *