Apparatus for liquid-solid column centrifugation chromatography and method

Centrifugation chromatography columns including column members for containing a particle bed and a porous member underlying the bed. The column members have an upper receiving opening and a lower discharge opening. A receptacle is demountably secured to the column. Attachment is effected through cooperation between a lower portion of the column members and an upper portion of the receptacles. Support structures may be provided on the exterior of the column members to facilitate support thereof by a centrifuge. In another embodiment of the invention continuous column centrifugation chromatography is provided by means of a column rotor on which are mounted a plurality of columns. A collector rotor is provided with means for receiving material discharged from the columns. Relative synchronized movement between the column rotor and the collector rotor is provided. A distribution system for supplying liquid to be separated to the columns on a continuous basis is provided. Funnel means may be employed to facilitate transfer of effluent from the column to the collector rotor. A method for continuous column centrifugation chromatography employing a rotating array of columns which are subjected to a centrifugal force, effecting synchronized movement of an array of receptacles positioned to receive the liquid discharged from the columns and supplying on a continuous basis liquid to the columns.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
This invention relates to apparatus and methods for liquid-solid column 
centrifugation chromatography including automated, continuous systems 
therefor. 
2. Description of the Prior Art 
Chromatography is an analytical and preparative chemical process which 
separates molecules from each other according to their various physical 
and chemical properties, including their molecular size, electric charge 
and tendency to adsorb to various specific surfaces. This method is widely 
used throughout the chemical and medical sciences in research work, 
clinical laboratories and industry, both to analyze mixtures of chemicals 
and to purify specific chemcals. Liquid-solid chromatography involves the 
chromatography of molecules dissolved in liquids which are passed over 
beds of particles. When the particles are contained in a column, a porous 
plate is generally placed at the bottom and the particle bed is packed 
thereover. The porous plate serves to support the particle bed while 
allowing liquid to flow through the column and bed. 
Among the problems which exist with known types of liquid-solid 
chromatography, such as gel filtration or permeation chromatography is the 
fact that the technique is frequently unduly time consuming. Also, the 
process frequently leads to undesirable dilution of the molecules being 
chromatographed. 
It has previously been suggested to employ the centrifuge in 
chromatography. 
It has been suggested to employ centrifugal gel filtration methods in 
connection with basket centrifuges. See "A Procedure for Gel Filtration of 
Viscous Solutions" by N. I. Arne Emneus, 32 Journal of Chromatography, 
pages 243-257 (1968). Other disclosures of the use of a centrifuge in 
chromatography as well as tube construction and methods are found in "A 
Rapid Method for Desalting Small Volumns of Solution" by Neal and Florini, 
55 Analytical Biochemistry, pages 328-330 (1973) and "Micro-Step-Exclusion 
Chromatography" by Krieger and Tobler, 81 Analytical Biochemistry, pages 
450-453 (1977). 
U.S. Pat. No. 3,583,230 discloses sample injection methods and apparatus. 
The injector has an inlet opening which communicates with a central 
channel provided with chromatographic filter material and a capillary 
outlet. It is noted, however, the injection of the liquid samples into the 
column is for subsequent chromatographic development without use of 
centrifugation. 
U.S. Pat. No. 3,440,864 discloses liquid chromatographic columns with a 
threaded jacket. The system is not designed for centrifugal 
chromatography. 
U.S. Pat. No. 3,664,845 discloses the use of a bed of molecular 
fractionating gel under the influence of a centrifugal force such that the 
bed is expanded and contracted by means of pulsed centrifugation. 
It has been known to attempt various means for automating chromatography. 
U.S. Pat. No. 3,194,400 relates to a liquid chromatographic centrifuge 
wherein a rotor cooperates with radial channels which are provided with 
separate media. The disc 20 is provided with radial channels 40. After 
centrifugation, the various zones are examined to determine different 
zones of chemical fractions. These zones may then be separately eluted 
from portions of the sheet. The receptacles provided are intended to 
receive solvent liquid flowing from the sheet, but generally this liquid 
would not be collected in discrete fractions. The resolved chemicals stay 
on the sheet. 
While not involving centrifugation chromatography in columns, U.S. Pat. No. 
3,395,093 discloses separations which are carried out on a disc of paper 
or gel under the dual influence of centrifugal and electrical forces. 
Structure for handling the materials is disclosed. 
U.S. Pat. No. 3,666,105 discloses continuous liquid-solid chromatography 
employing a pair of cylinders which cooperate to define a column. It is 
noted that the fraction collector rotates with the column as a result of 
the design. 
A number of other disclosures suggest the use of discs. U.S. Pat. No. 
4,077,886 employs discs and makes no provision for collection of the 
resolved components into eluted fractions for analysis and subsequent use. 
U.S. Pat. No. 3,417,548 discloses the use of a pair of spaced discs. U.S. 
Pat. No. 3,527,350 discloses centrally introduced flow and resolution 
within a disc bed. U.S. Pat. No. 3,617,557 discloses continuous 
centrifugation chromatrography in a disc material. Under the influence of 
Coriolis forces, different separated components come to different regions 
of the rim of the disc. Cups are provided to receive eluate from discrete 
regions of the rim. U.S. Pat. No. 3,201,921 discloses selective treatment 
of a fluid by adsorption. Pumping action is the primary moving force for 
the liquid which is fed in at least two zones with a plurality of cells 
being provided adjacent the periphery. U.S. Pat. No. 3,113,103 discloses 
the use of centrifugal force in the disc. No provision is made for elution 
of separated components and, as a result, no fraction collector is 
provided. Means are provided for loading of samples. 
There remains, therefore, a significant need for effective apparatus for 
both batch and continuous liquid-solid column centrifugation 
chromatography which produces improved resolution in reduced time. 
SUMMARY OF THE INVENTION 
The present invention has solved the above-described problems by providing 
an effective means of improving liquid-solid column centrifugation 
chromatography. 
In one embodiment of the present invention, column means are provided with 
a particle bed and an underlying porous member. The column means have 
downwardly depending first attaching means. Receptacle means are 
positioned under the column means and by means of second attaching means 
are demountably secured to the column means. Various means for effecting 
such demountable securement are disclosed. Also, support means for 
faclitating securement of the column meansreceptable means assembly in a 
centrifuge are provided. 
In another embodiment of the invention, automated means for providing 
substantially continuous liquid-solid column centrifugation chromatography 
are provided. A column rotor means is rotarably mounted and has a 
plurality of centrifugation columns secured thereto. Coaxially mounted for 
synchronous rotation with the column rotor means is a collector rotor 
means having a plurality of receptacles adapted to receive fluid 
discharged from the columns. Power means established the synchronized 
movement of the column rotor means and collector rotor means. Distribution 
means provided to the columns a continuous supply of liquid to be 
separated. 
The method of the present invention provides for continuous liquid-solid 
column centrifugation chromatography. 
It is an object of this invention to provide a uniquely configurated 
column-collection receptacle assembly for use in liquid-solid 
centrifuation chromatography. 
It is a further object of this invention to provide such a 
column-receptacle combination wherein the receptacle is readily secured to 
and demounted from the overlying column member. 
It is a further object of this invention to provide such a 
column-receptacle assembly which has means to facilitate securement 
thereof to a conventional centrifuge. 
It is another object of the present invention to provide efficient systems 
for discontinuous and continuous liquid-solid column centrifugation 
chromatography. 
It is another object of this invention to provide such apparatus which may 
be economically manufactured and efficiently employed. 
It is another object of this invention to provide a method of automated 
liquid-solid column centrifugation chromatography. 
These and other objects of this invention will be more fully understood 
from the following description of the invention on reference to the 
illustrations appended hereto.

DESCRIPTION OF THE PREFERRED EMBODIMENTS 
In order to accomplish the objectives of the present invention, as 
conventional chromatography columns are not suited to use in a centrifuge, 
special column-receptacle assemblies are provided in the first embodiment 
of the present invention. 
Referring now more specifically to FIGS. 1 and 2 there is shown a 
liquid-solid chromatography column assembly. The assembly consists of a 
column 2 under which is demountably secured a receptacle 4. The column 2 
is provided with an upper liquid receiving opening 6 and a lower discharge 
opening 8. Within the column 2 is a bed 12 of chromatography bed material 
such as gel beads, for example, which is supported on a disc 14 which is 
adapted to resist passage of the bed material 12 therethrough, but permit 
the flow of liquid therethrough. The bed material may conveniently be any 
type suitable for use in liquid-solid chromatography. Examples of specific 
suitable materials are those sold under the trade designations Sephadex 
G-25 and G-50 (Pharmacia) or Bio-Gel P-4 and P-2 (Bio-Rad Laboratories). 
The discs 14 may conveniently be porous polyethylene discs. 
In the form shown, the lower portion of column 2 has a pour spout 18 which 
defines discharge opening 8 which is in communication with the interior of 
receptacle 4. In order to facilitate flow into receptacle 4, at least one 
vent opening 20, which may conveniently be a hole of about 1/16 to 1/32 
inch in diameter, is provided within the upper portion of the receptacle 4 
so as to facilitate exhaust of air from the receptacle 4 while resisting 
discharge of fluid therefrom. If desired, in addition or in lieu of vent 
opening 20 in receptacle 4, a vent opening may be placed in the wall of 
column 2 in a portion in communication with the interior of receptacle 4. 
It could be placed in the region underlying the connection between pour 
spout 18 and the wall of column 2, for example. Also, venting could be 
effected by maintaining a column2--receptacle 4 connection, which is not 
air tight. 
In effecting securement of the receptacle 4 in demountable underlying 
engagement with respect to the column, mechanical retention is provided in 
the embodiment shown in FIGS. 1 and 2. The lower portion of the column 2 
has first attaching means 24 in the form of an annular tubular downward 
projection. The first attaching means 24 cooperate with second attaching 
means 26 on receptacle 4 which in the form shown consists of an upwardly 
projecting annular mouth portion. Cooperating treads 28 are provided on 
first attaching means 24 and second attaching means 26 in order to improve 
intimacy of securement. 
The embodiment shown in FIGS. 1 and 2 provides means for securing the 
column-receptacle assembly rotatably within the rotor of a centrifuge. The 
upper portion of column 2 (within the upper one-half and preferably within 
the upper one-third of the axial length of the column) is provided with a 
substantially continuous exteriorly disposed rib 32. This rib 32 is 
preferably the portion of the column-receptacle assembly of greatest 
transverse dimension. 
In the form shown in FIG. 1, a yoke member 34 which is adapted to be 
rotatably secured on the rotor of a centrifuge defines an opening 36 which 
will receive the column-receptacle assembly in such fashion that the 
annular rib 32 will not pass through opening 36, but preferably will rest 
on upper surface 38. The contact between annular rib 32 and surface 38 
serves to resist undesired excessive passage of the column-receptacle 
assembly through opening 36. 
The following will provide an example of how the apparatus of FIGS. 1 and 2 
may be used in liquid-solid centrifugation chromatography in order to 
effect molecular separation. A column 2 of the type shown in FIGS. 1 and 2 
without the receptacle 4 attached is provided with a slurry of 
chromatographic bed material in order to establish bed 12. Either prior to 
or after introduction of the slurry into the column 2, the column 2 is 
introduced into yoke 34 through opening 36 until annular rib 32 contacts 
upper surface 38. The column 2 is then subjected to centrifugal force in 
order to remove excess liquid from the slurry. The receptacle 4 is then 
secured in underlying position with respect to the column 2. A liquid 
sample containing molecules which will bind to the chromatographic bed 
material 12 and others which will not is introduced into the column 2 
through opening 6. The column-receptacle assembly is then centrifuged with 
the nonbinding molecules being recovered in receptacle 4 in eluted liquid. 
The receptacle 4 containing the eluted liquid and non-binding molecules is 
then emptied and the receptacle 4 is then reattached to the column 2 or a 
different receptacle may be attached. A new solution which elutes the 
bound molecules is then introduced into the column through opening 6 and 
the column is centrifuged once again. This time the bound molecule is 
recovered in the elution solution. This final step may be repeated several 
times, if desired. 
The prior art liquid-solid chromatography practice carried out in a bed of 
solid particles immersed in liquid has two principal problems which limit 
the speed at which the process can be performed. First of all, the 
liquid-filled spaces between the particles are usually very large in 
dimension compared to the size of the molecules being chromatographed. As 
a result, the liquid must be passed through the bed slowly if the 
molecules are to have an adequate chance to diffuse to the particle 
surfaces and interact with them. In addition, the flow of liquid through 
the bed must not be so rapid that uneven liquid flow across the bed 
developes due to hydrodynamic effects. Such uneven flow results in a 
decrease in the efficiency of separation of zones containing different 
types of molecules. 
In the present system, by contrast, the liquid-filled particle bed is 
centrifuged prior to chromatography. As a result the liquid between those 
particles is largely removed leaving a thin layer of liquid on the surface 
of the particles in the bed in addition to the liquid which may permeate 
them. As the liquid layer on the centrifuge column particles is very small 
in thickness, it permits a very rapid equilibrium of molecules dissolved 
in it with the particles. In essence, the liquid layer is so thin that 
random diffusion becomes effective at promoting particle-molecule 
interaction. It, therefore, becomes advantageous to load a sample of 
molecules to be chromatographed in a column which has previously been 
centrifuged to remove free liquid. If this column is then centrifuged 
again after loading, the liqud sample moves down the particle bed in a 
thin layer itself, under conditions where interactions between the 
particles and the molecules being separated are maximized. As these 
interactions are so efficient in a thin layer of liquid, the flow rate 
down the column can be much higher than in conventional liquid-solid 
chromatography in a liquid-filled column. At the same time, there is very 
little problem with uneven liquid flow across the column bed because the 
bed is not filled with liquid. Instead, the liquid is flowing as a sheet 
over the bed particles. The result is that liquid-solid chromatography in 
a centrifuge column can accomplish the same type of separation as in 
normal, liquid-filled columns but much more rapidly, without sacrificing 
the ability to resolve different molecules efficiently. As very little 
free liquid is added to the column, the amount of dilution of the 
chromatographed molecules is very little. 
Referring now to FIG. 3, another embodiment of the container-receptacle 
assembly adapted to cooperate with yoke 34 is illustrated. In this 
embodiment, the securement means consist of a series of segmented ribs 44, 
46, 48 positioned within the upper third of the axial height of the column 
42. The segmented ribs 44, 46, 48 serve to permit yoke 34 to support the 
column 42 and receptacle 4 against excessive penetration therethrough 
during centrifugation. 
Referring to FIG. 4, there is shown a tube of the type shown in FIGS. 1 and 
2 wherein the column-receptacle assembly may be employed without the need 
to use a yoke 34. In this embodiment of the flange 32 or an embodiment 
such as column 42 may be introduced directly into the rotor 52 by 
positioning the same within a hole 54 sufficiently small as to preclude 
the passage of annular rib 32 or rib segments 44, 46, 48 therethrough. 
Alternatively, the column-receptacle assemblies may be introduced into a 
slot 56 within a rotor. 
Referring now to FIG. 5, the embodiment of column 60 and receptacle 62 
assembly is provided with a pair of generally diametrically opposed 
outwardly projecting pins or lugs 64, 66. The pins or lugs 64, 66, may be 
used without a yoke so as to permit the column-receptacle assembly to 
pivot about a slot or other retaining means within the centrifuge rotor 
which rotatably secures pins or lugs 64, 66. Under the influence of 
centrifugal force, the column 60 will pivot about the pins 64, 66 to 
assume a position generally parallel to the centrifugal field. 
In the embodiment illustrated in FIG. 6, the column 70 has a receptacle 72 
demountably secured thereto with the column-receptacle assembly received 
within a trunion 74 which pivots on the rotor (not shown) of a centrifuge 
under the influence of centrifugal forces to bring the column to a 
position generally parallel to the centrifugal field. 
While a vent opening 20 in order to facilitate efficiency of liquid 
transfer from the column 2 to receptacle 4 has been specifically shown in 
FIG. 2, for simplicity of disclosure, it will be understood that 
appropriate venting means will be provided in the columns, receptacles, or 
both in all embodiments of the present invention, but specific 
illustration and discussion of the vent opening will not be provided with 
respect to each embodiment as the form and positioning will be readily 
apparent to one skilled in the art. 
In the embodiment shown in FIG. 7, a generally cylindrical column 76 is 
provided with a disc 78 which supports a chromatography bed (not shown). A 
trunion member 80 is provided with a vent passageway 82 disposed within 
the upper half of the trunion 80 and defines a liquid receiver portion 84 
therewithin. The trunion 80 is provided with an annular undercut upper 
surface 86 which is in firm engagement with the lower portion of column 76 
so as to establish the column-receptacle assembly. 
The embodiment of FIG. 8 is substantially identical with that of FIG. 7 
except that the trunion 80' is provided with a step 90 positioned below 
the undercut upper surface 86 and a receptacle 88, which is removably 
secured within the trunion liquid receiver 84, is provided. 
This invention also contemplates a single trunion member providing multiple 
columns or receptacles. Referring now to FIG. 9 there is shown in upper 
trunion member 92 and a lower trunion member 94. In the form shown, the 
upper trunion member 92 is provided with two columns 96, 98 which are 
integrally formed within the upper trunion member 92 and are provided 
respectively with discs 100, 102 and overlying chromatographic bed 
material (not shown). The columns 96, 98 are provided, respectively, with 
discharge outlets 104, 106 which are in overlying communicating 
relationship with receivers 108, 110 respectively. The receiver 110 is 
provided with a removable tube 112. 
FIGS. 10 through 14 show examples of certain additional preferred 
embodiments of first and second attaching means for demountably securing 
an underlying receptacle to an overlying column. 
In FIG. 10, column 116 has an upper generally cylindrical portion and first 
attaching means which in the form shown is a lower generally cylindrical 
portion 118 of reduced diameter having internal threads (not shown). 
Receptacle 120 has second attaching means in the form of a generally 
cylindrical mouth portion 124 on which are formed external threads 124 
which are adapted to be threadedly engaged with threads contained in 
section 118. 
In the form shown in FIG. 11, the column 128 is generally cylindrical and 
has a lower portion 130 which has externally positioned threads 132. The 
underlying receptacle 134 has an upper generally cylindrical neck portion 
which has internal threads adapted to mate with threads 132. 
FIG. 12 illustrates a column 140 which has a generally cylindrical lower 
section 142 provided with a slot which has a generally vertically oriented 
portion 144 connected to a generally horizontally oriented portion 146. A 
similar second slot (not shown) is positioned generally diametrically 
opposite to slot 144, 146. Receptacle 150 has a mouth provided with a pair 
of generally diametrically opposed generally radially outwardly projecting 
lugs or pins 152, 154 which are adapted to be received within the two 
slots of lower portion 142 in order to demountably secure the receptacle 
150 to the column 140. 
In the embodiment shown in FIG. 13, the column 158 has a pair of generally 
downwardly depending hook-like members 160, 162. The receptacle 164 has a 
pair of upwardly directed tabs 166, 170 which respectively, have openings 
168, 172 within which hook-like members 160, 162, respectively, are 
received. The hook-like members 160, 162 are preferably resiliently 
deformable and have their free ends pointing generally radially outwardly. 
In the embodiment shown in FIG. 14, the column 176 has a pair of downwardly 
depending tabs 178, 180 which have, respectively, openings 182, 184. The 
receptacle 186 has a pair of generally upwardly and outwardly directed 
hook-like members 188, 190 which are adapted to be received within 
openings 182, 184. 
While in the foregoing embodiments, the columns and receptacles may be made 
of any suitable inert material possessing adequate strength, among the 
preferred materials are glass, plastics such as polyethylene, 
polypropylene, and polyvinychloride, for example. The economics of these 
materials are such that a disposable product may be produced. The trunions 
may be made of stainless steel, for example. 
In the practice of this invention virtually all liquid chromagography 
support materials and equivalents thereof may be employed as the 
chromatography bed material. Examples of suitable materials are beads for 
gel filtration (permeation), beads and particles of plastic resin or 
cellulose for ion exchange chromatography and silica gel particles. 
It will be appreciated that in the foregoing embodiments the 
column-receptacle assemblies are so designed as to fit into a centrifuge 
rotor in such fashion that the receptacle "hangs down" both under the 
influence of gravity when the rotor is at rest and under the influence of 
a centrifugal force field. 
In the second group of embodiments of the present invention, apparatus and 
method adapted for continuous column centrifugal chromatography is 
provided. 
As is shown in FIG. 15, a column supporting rotor 202 is fixedly secured to 
a drive shaft 208 and a collector rotor 204 is secured to a lower 
extension of the column rotor with an interposed bearing 206. This 
arrangment results in rotation of the drive shaft 208 about its 
longitudinal axis A providing synchronized relative rotation of the column 
rotor 202 and collector rotor 204. This embodiment provides direct 
securement of the column rotor 202 to the drive shaft 208. 
In the embodiment shown in FIG. 16, the collector rotor 212 is secured 
directly to drive shaft 216 which rotates about its longitudinal axis B. 
The column rotor 210 is secured to an upward extension of hub 213 of the 
collector rotor 212 with an interposed bearing 214. As a result, 
synchronized rotation of the collector rotor 212 and column rotor 210 
about axis B are provided. 
Referring now to FIG. 17 there is shown one preferred embodiment of the 
present invention. In this embodiment the column rotor 202 is fixedly 
secured to the drive shaft 208 which rotates about axis A and the 
collector rotor 204 is directly secured to the hub 220 of column rotor 202 
through bearing 207. The drive shaft 208 is rotated by a suitable motor 
with or without interposed speed reducing transmission means (not shown). 
In a preferred embodiment the drive shaft will rotate at about 100 rpm to 
about 2000 rpm. 
A post member 222 is preferably substantially coaxial with axis A and 
rotates thereabout. The post member 222 is through speed reducer 224 which 
is secured to column rotor 202 and contains suitable gearing (not shown) 
connected to stub shaft 226 which is secured to collector rotor 204. This 
serves to provide coordinated relative movement of rotors 202, 204 and 
post member 222. At least one chromatographic column 225 is provided with 
a chromatographic bed 226, a porous disc 228, a stopper 230 at one end and 
a discharge outlet 232 at the other. 
Referring still to FIG. 17, a reservoir 236 which is annular in shape and 
upwardly open and rigidly secured to post 222 for rotation therewith is 
adapted to receive liquid from nonrotating tube 240. Liquid delivered from 
nonrotating tube 240 will enter reservoir 236 and by means of tube 238 
under the influence of centrifugal force and gravity deliver liquid into 
stopper 230 and from there into the bed 226. It will be appreciated, 
therefore, that as drive shaft 208 rotates, the liquid will be processed 
through the column 225 and the elution solution will be discharged through 
opening 232. 
Considering still FIG. 17, a preferred form of collector rotor 204 will now 
be discussed. The outer portion of the collector rotor 204 has a generally 
upwardly and angularly outwardly directed ring 242 secured thereto. 
Mounted within the rings are a series of receptacles 244, 246 (only two of 
which are shown in FIG. 17). It is understood that a substantially 
continuous circumferential array of receptacles would be provided so as to 
permit substantially continuous receipt of eluted liquid from the 
discharge opening 232. In order to facilitate efficiency of fluid transfer 
from discharge opening 232 into the array of tubes 244, 246 and the 
remaining tubes (not shown) a series of funnels 248, 250 are interposed 
between the column opening 232 and the receptacles. 
Referring to FIGS. 18 through 24, certain preferred constructions of liquid 
transfer reservoirs will now be considered. In the embodiment shown in 
FIGS. 18 and 19 the reservoir 252 is generally frustoconical in shape, has 
a single discharge tube 254 and a liquid receiving opening 256. It has a 
single interior chamber 258 
In the embodiment shown in FIGS. 20 and 21, the reservoir 262 also has a 
frustoconical configuration and is provided with a series of interior 
liquid receiving or storage chambers 272, 274, 276, 278. Four outlet tubes 
264, 266, 268, 270 are each associated with an interior liquid receiving 
or storage chamber 272, 274, 276, 278 for supplying liquid to a column. 
In the embodiment shown in FIGS. 22 and 23, the reservoir 282 has a single 
outlet tube 284 and is provided with an annular storage chamber 286 
surrounding an interior opening 288. 
In the embodiment shown in FIGS. 24 and 25, the reservoir 294 has three 
chambers 302, 304, 306 and associated discharge tubes 294, 296, 298 
respectively. The chamber 306 is generally frustoconical in shape and the 
other chambers 302, 304 are annular. 
It will be appreciated from the foregoing that it is preferred that the 
number of chambers in the reservoir equal the number of supply tubes 
emerging therefrom to provide liquid to columns. 
FIG. 26 shows a preferred form of stopper which facilitates even 
disbursement of liquid within a column. A supply tube 310 delivers liquid 
to a column 312 which has a column bed 314 and a stopper 316. The stopper 
316 has a main passageway 318 which is in communication with the tube 310 
and a number of branch passageways 320, 322 preferably of reduced size 
with respect to the main passageway 318. In the form shown the stopper has 
a tubular extension 324 which is received within tube 310. 
Referring now to FIG. 27 there is shown a modified form of collector 
system. Collector rotor 328 has annular outer ring 332. A column 330 is 
adapted to discharge material into a receptacle 334. The receptacle at 
rest 334 is generally vertically oriented, but under the influence of 
centrifugal force is adapted to assume the dotted position shown at 336 by 
pivoting about trunion pins 338 (only one shown) which project outwardly 
from opposite sides of receptacle 334 and are rotatably received within 
ring 332, as by grooves therein. The pivot pins 338 may take the form of a 
pair of outwardly projecting lugs provided on a ring which supports a 
receptacle upper flange. In a preferred form, the trunion pins 338 or 
other pivot means will be positioned within the upper one-third of the 
longitudinal axial extent of receptacle 334 to facilitate efficient 
rotation. 
FIGS. 28 through 39 shown certain preferred means for distributing liquid 
from a column to a series of receptacle tubes. In FIG. 28, the rotor 340 
is provided with an angularly disposed annular ring 344 within which is 
mounted a receptacle 346 having its longitudinal axis directed generally 
angularly upwardly and inwardly. The column 342 has a discharge opening 
350 which is generally aligned with the opening in the receptacle 346. 
In the embodiment shown in FIG. 29, the collector rotor 352 is adapted to 
receive liquid from column 354. An outer annular ring 356 has a lower 
generally vertically oriented portion and an upper angularly outwardly 
directed portion which has a recess which supports receptacle 358. A 
funnel member 360 has its lower end mounted adjacent the annular ring 356, 
its receiving end aligned with the column 354 and its discharge end 
inserted into receptacle 358. 
In the embodiment shown in FIG. 30, the rotor 364 is adapted to receive 
liquid from column 366. The rotor 364 is provided with a series of 
receptacles 368 (one shown) which have an outwardly diverging mouth which 
serves as a funnel to improve efficiency of transfer of liquid into the 
receptacle 368. 
Referring to FIG. 31, there is shown partially in section, as viewed from 
above an annular funnel plate 370 which is preferably mounted on the 
collection rotor and is provided with a series of openings 372 which 
converge generally radially outwardly and are aligned with a series of 
receptacles 374. In this fashion all liquid being discharged toward the 
funnel plate will be received within an opening 372 and distributed to an 
adjacent receptacle 374. It will be appreciated that while only three 
receptacles 374 have been shown in use, a receptacle would be preferably 
provided for each opening 372. 
In the embodiment shown in FIG. 32, the annular ring which is mounted on 
the collection rotor is provided with a series of receptacles integrally 
formed within the ring. It is noted that the receptacles have their widest 
opening adjacent the inner circumference of the ring. In order to minimize 
undesired spillage, the ring slopes downwardly and outwardly from its 
inner extremity. As a result, the receptacles would slope downwardly away 
from their mouths to the closed end so that they might retain fluid under 
the influence of both centrifugal and gravity forces. 
The ring of FIG. 32 is shown in FIG. 33 as well. In this embodiment the 
system has two columns 380 and 382 which have discharge outlets generally 
radially opposed from each other. Reservoir 384 is mounted on post 386 and 
supplies liquid to columns 380, 382, respectively, through tubes 388, 390. 
As the columns rotate they sequentally fill the openings 376. 
If desired, as in the FIG. 33 embodiment, for example, the columns may be 
subjected to rotation about their longitudinal axis by any means either 
coordinated with main drive shaft rotation as by appropriate reduction 
gears, for example, or any form of independent means. Axial rotation of 
the columns can result in even greater resolution as a result of the 
rotation which tends to conteract the Coriolis force. This resistance to 
the Coriolis force causes the liquid to flow down the column generally 
parallel to the tube's longitudinal axis rather than at an angle with 
respect thereto. This results in more uniform travel of the transverse 
zones or bands of molecules downwardly. Rotation of several revolutions 
per minute would be adequate for this purpose. 
In the method of this embodiment of the invention continuous column 
centrifugation chromatography is provided by applying a centrifugal force 
to one or an array of columns. Synchronized relative movement of an array 
of associated receptacles is effected in order that the receptacles may 
receive liquid discharged from the columns. A continuous supply of liquid 
is provided to the centrifuge columns through reservoir and supply means. 
It will be appreciated that it will generally be advantageous to employ 
with the automated version of the invention, reservoir means. The system 
may, however, if desired, be provided with a rotatable coupling to secure 
a stationary liquid supply tube to a rotating tube connected to the 
columns. 
It will be appreciated that the present invention is adapted for a wide 
range of uses including clinical chemical research and industrial uses. 
Examples of advantageous uses include: 
(a) removing salts and other small molecules from proteins in biological 
fluids such as blood serum and cerebrospinal fluid; 
(b) removing unbound small molecules, such as steroids, from larger 
molecules, such as antibody proteins, in diagnostic tests such as 
radio-immuno assay procedures; 
(c) separating large proteins, large polysaccharide, and large nucleic 
acids, from small molecules of these types of clinical samples, prior to 
or during diagnostic testing; 
(d) separate proteins such as serum proteins from salts; 
(e) separate proteins from steroids and other small hormones, including 
small peptide hormones; 
(f) separate proteins from drug molecules; 
(g) assay the binding of drugs to proteins; 
(h) assay the binding of hormones to proteins; 
(i) separate proteins of various sizes and/or shapes from each other; 
(j) separate nucleic acid molecules from salts; and 
(k) separate nucleic acid molecules of differing sizes from each other--for 
example, DNA fragments of different lengths. 
It will be appreciated that the present invention has provided an efficient 
means for obtaining the full benefits of liquid-solid column centrifuge 
chromatography in a rapid fashion without undesired dilution of the 
specimens. 
Whereas particular embodiments of the invention have been described above 
for purposes of illustration, it will be evident to those skilled in the 
art that numerous variations of the details may be made without departing 
from the invention as defined in the appended claims.