text: the central fast chromatograph of the present invention is a centrifugally driven gradient elution chromatographic system . this system is shown diagramatically in fig1 . a microprocessor 10 both controls and monitors the separations . cathode ray tube ( crt ) 12 displays the chromatographic separations in real time , while gradient maker 14 produces the gradient from solutions 16 and 18 and controls column regeneration . at the start of an analysis the samples are loaded on a transfer disk 20 ( shown in fig2 ) in disk loader 21 which is an automatic pipetter known in the art . transfer disk 20 is loaded into chromatograph rotor 22 at the start of a run , and the rotor is accelerated by drive 24 under control of microprocessor 10 . effluent from the rotor 22 may be either led to drain 26 or collected in corotating fraction collection ring 28 . absorbance of light by the effluent stream from light source 30 is measured using photodetector 32 which provides the signal to microprocessor 10 . the rotor position during rotation is determined using synchronization signal pickup 34 . fig2 illustrates a side view of the system . a control center includes control panel 35 , microprocessor 10 , crt display panel 12 , keyboard 37 , speed and temperature indicators 39 , and gradient maker 14 . centrifugal chromatograph rotor 22 is driven by rotor drive 24 which is controlled by the control panel of the control center . chromatographic columns 36 are mounted for rotation on centrifugal chromatograph rotor 22 . the sample - holding transfer disc 20 is mounted in the center of rotor 22 . optical flow cells 38 having transparent windows 39 are mounted on centrifugal chromatograph rotor 22 . light source 30 is mounted opposite photodetector 32 , which may be photomultiplier 40 , to measure absorbance of the effluent stream . upper or edge line distributor ring 42 for edge line introduction of the regenerating solution is disposed at the end of solution feed line 44 , which runs from gradient maker 14 on the control center 35 . edge lines 46 are fluid carrying feed lines having an antiregurgitation slope disposed between upper distributor rings 42 and edge line values 48 . lower or center line distributor ring 50 for center line introduction of the regenerating solution is disposed at the end of solution feed line 62 , which runs from gradient maker 14 on the control panel 35 . center lines 52 are fluid carrying feed lines having an antiregurgitation slope and are mounted between the center end of chromatographic columns 36 and distributor rings 50 . a center line valve 54 ( fig3 ) is disposed on each center line 52 at the center end of chromatographic columns 36 . an edge line valve 48 is mounted on one end of edge line drain 56 . drain and fraction collecting ring 28 is mounted on the other end of edge line drains 56 and includes collecting tubes and collecting ports 58 leading to stationary drain collecting ring 60 . stationary drain collecting ring 60 is mounted on the outside of rotor 22 to collect fluid drained from the rotor 22 . feed line 62 runs from gradient maker 14 to center line distributor ring 50 which is used to elute chromatographic column 36 . a horizontal section looking down through the system a centrifugally driven gradient elution chromatograph rotor is shown in fig3 . chromatograph rotor 22 includes a sample holding transfer disk 20 in which a plurality of sample wells 64 are placed . sample wells 64 are placed around the outer edge of transfer disk 20 and correspond to respective chromatographic columns 36 . chromatographic columns 36 are connected to the periphery of transfer disk 20 by center lines 52 . stream apportioning blades 66 in center line distributor ring 50 guide the fluid sample between transfer disk 20 and center lines 52 . center line valves 54 are disposed on center lines 52 to divert material to waste during reverse flow during column regeneration . optical flow cells 38 having transparent windows 39 are disposed on the outer end of chromatographic columns 36 adjacent the connection to edge lines 46 . edge line valves 48 are disposed on edge lines 46 to divert material to waste during elution . two coaxial stream segmentation devices or distributor rings , upper distributor ring 42 ( not shown in fig3 ) which feeds the edge lines 46 and lower distributor ring 50 which feeds the center lines 52 , are used to apportion the stream into multiple columns the chromatographic system of the present invention has superior flow characteristics which result in higher resolution . outward flow during elution ( system a ), which is preferred , requires that the sample be placed at the center end of the chromatographic columns 36 , that the optical flow cells 38 or other detector be at the outboard end of the columns 36 , and that elution be done with a gradient of decreasing physical density . it also requires that the liquid in the column 36 at the start be denser than the sample or the starting ( heavy ) end of the elution gradient . the implications of these special requirements are discussed below . when center elution flow is used ( system b ), the fluid in the column at the start is less dense than either the sample or the starting end of the elution solution introduced through the edge line . the elution fluid exits through center line valves 54 on center lines 52 , and the elution gradient increases in physical density from start to finish . the sample is applied to the bottom of the columns 36 , through the edge lines 46 , exits through center line valves 54 on center lines 50 , and the optical flow cells are at the top ( center end ) of the column 36 as shown in fig5 . it is feasible to construct a system that will work either way , and has flow cells at both the tops and bottoms of the columns 36 . the sequence of events during columns packing , column regeneration and / or equilibration , and analysis are shown diagramatically in fig4 where the same column is presented in a series of steps involving column packing , regeneration , and analysis . the valves 48 and 54 may be set to allow change in direction of flow . specifically , during column packing , the suspension being packed flows through the lower stream - segmentation device ( distributor ring 50 ) and into the center lines 52 , past the closed center line valves 54 , and into the columns 36 . liquid flowing out of the columns 36 past the lower frit flows through the optical flow cells 38 , and to the open edge line valves 48 to drain . when column packing is complete , a small plug may be introduced into the center lines 52 , and moved into position by centrifugal force . this prevents packing material from moving and resettling when the rotor 22 is at rest . sloping of the center and edge , 52 and 46 , respectively , lines is used to prevent fluid from flowing out of the columns 36 , flow cells 38 , and outer loop when the rotor 22 is at rest . in fig4 a a suspension of inert particles 70 is introduced into chromatographic column 36 through center line 52 . both edge line valve 48 and center line valve 54 are set for outward flow , i . e ., valve 48 is open and valve 54 is closed . the inert particles 70 pack the lower cone of chromatographic column 36 . the active packing 80 which produces the chromatographic separation is then introduced and , in fig4 b , chromatographic column 36 has been partially packed due to centrifugal force . in fig4 c , all of the active packing 80 is in place , and the upper cone of chromatographic column 36 has been partially packed with inert material 70 . in fig4 d , chromatographic column 36 has been fully packed and an upper porous plug 72 has been set in place in center line 52 above the chromatographic column 36 . in fig4 e , edge line valve 48 and center line valve 54 have been set for inward flow i . e ., valve 48 is closed and valve 54 is open . during rotation , a washing or regeneration or equilibration liquid 74 is introduced into edge line 46 through upper distributor ring 42 thereby causing column regeneration or equilibration . liquid density is controlled so that the a liquid whose density is greater than that of the dense end of the elution gradient is left in place in the column 36 . a sample 76 is placed in transfer disk 20 adjacent the center line 52 in fig4 f while the rotor 22 is stationary . the sample is less dense than the liquid in chromatographic column 36 . the edge line valve 48 and center line valve 54 are , once again , set for outward flow i . e ., valve 48 is open and valve 54 is closed through chromatographic column 36 . in fig4 g , the rotor 22 is accelerated and the sample 75 is transferred centrifugally into the rotor 22 and then into center line 52 . during continued rotation , an eluting gradient 77 is introduced to the rotor center line 52 as shown in fig4 h . gradient elution of chromatographic column 36 has begun although no separation of the sample 76 occurs in the inert particles 70 in the upper cone of chromatographic column 36 . in fig4 i , constituents 1 , 2 , 3 , and 4 of the sample have separated in chromatographic column 36 . in fig4 j , peaks or bands 1 , 2 , 3 , and 4 representing each respective constituent of the sample are eluted sequentially through the cuvet 79 outboard of chromatographic column 36 . the peaks move through the cuvet 79 without backmixing . on completion of the analysis illustrated in fig4 column regeneration by back flow occurs and a solution having a density greater than the dense end of the elution gradient is left in place . the analysis may be repeated by beginning with the step illustrated in fig4 f . when the flow is backward through the column 36 during column regeneration and equilibration ( hereinafter referred to as system a ), liquid flows in through the upper stream - segmentation device ( distribution ring 42 ), out through the edge line 46 , past the closed edge line valves 48 , to the centrifugal ( outer ) end of the column 36 , and inwardly through the optical flow cells 38 , and column 36 , and out through the open inner line valves 54 to drain ( or collection ) 60 . the location of the optical flow cells 38 when the elution flow is in an inward or centripetal radial direction during analysis ( hereinafter referred to as system b ) is shown in fig5 with the optical flow cells 38 located centripetal to ( above ) the column 36 on center lines 52 . in system b , the eluting solution flows through edge line 46 to chromatographic column 36 . the pressure head during elution is proportional to the distance d to center line valve 54 . system b has an inherent problem with density reversal in the edge lines 46 . at the start of an analysis the least - dense fluid used fills the system . the sample is adjusted to be slightly denser , and will mix rapidly with fluid in the edge lines 46 as it is introduced . once the sample has passed the hairpin loop at the edge , and begins inward flow , density - gradient stabilization begins to be effective . as the gradient flows into the rotor 22 during analysis , constant backmixing occurs in the edge lines 46 , followed by stable flow in the columns 36 , flow cells 38 , and center lines 52 . a solution to the problem of backmixing during sample introduction is to inject the samples individually through a self - sealing rubber port 81 at the centrifugal end of the columns 36 with the rotor 22 at rest . this solves the problem , but makes the system less desirable from the automation viewpoint . during sample loading followed by introduction of the gradient , flow and valve settings are as shown in fig5 . column regeneration involves flow of solution in through center line 52 , and drainage out through the exit port of valve 48 , i . e ., the flow and valve settings are the same as for column packing . the cfc has automatic compensation for flow resistance in different columns that maintains constant flow . in the cfc , the actual pressure head on each column is a function of the length of the liquid column center of the exit line . if one column has slightly less resistance to flow than the other columns , fluid will flow more rapidly , the length of the liquid column will decrease , there will be less driving pressure , and flow through that column will automatically adjust itself to match the others , provided that the liquid gradient is not introduced too rapidly . the upper portion of the column center of the exit line can be expanded to include a large volume of fluid , up to and including the entire gradient . in a centrifugal field large density gradient are stable . air bubbles will not impede flow as air bubbles are forced inwardly by the centrifugal field . during elution with either system a and system b , the gradient used is a bifunctional gradient , varying first in density or specific gravity to control flow and maintain fluid segments in their proper sequence , and second in eluting power , to sequentially elute components of the mixture being analyzed from the column . with system a , the regeneration or equilibrium liquid 74 in the rotor , the sample 76 and the elution gradient 77 must be arranged to be in order of decreasing density . for much protein and other work involving reverse phase chromatography , the gradient 77 is one of increasing acetonitrile concentration . acetonitrile has a density or specific gravity of 0 . 785 g / ml ; hence a gradient from water or a customary buffer solution will grade from a density of approximately 1 , 000 g / ml to 0 . 785 g / ml . the specific gravity will decrease as required for system a . if sucrose or other inert gradient material is added to the equilibration solution 74 , optionally to the sample 76 , and to the denser of the solutions used to make the gradient 77 , a steeper density gradient may be obtained . when the gradient 77 normally used is one of increasing salt concentration and is therefore of increasing density , an inert density - increasing material must be added to the equilibration solution , in lesser amount to the sample 76 , and must be in decreasing concentration in the gradient 77 to yield a gradient of decreasing physical density , but of increasing elution power . this is one disadvantage of system a when used with columns and elution procedures requiring very concentrated salt solutions . with system b , which requires a gradient 77 of increasing density , gradients which are natively of increasing density may be used directly . the disadvantage of system b is sample - zone smearing during flow through the edge lines 46 . this may be minimized by having very small bore edge lines 46 . the smearing may be of little effect in instances where the sample 76 is strongly absorbed in a small volume of column packing material 80 at the start of flow through the columns 36 , thereby reconcentrating the sample 76 at the outset into a small volume . injection of the sample 76 at the rotor edge initially also solves this problem but introduces operational disadvantages . an advantage to system b is that flow is counter to centrifugal force . therefore flow is tending to resuspend the column packing , while centrifugal force is tending to pack it down . this sets a limit to the flow rate without positive positioning of an upper frit . with system a , flow and centrifugal force are in the same direction , hence flow and centrifugal force will combine to pack down the column material 80 more . this is a disadvantage with column packings 80 which are deformable , and can be overcompressed causing flow reduction . with the systems described here , which use density gradients , extraordinary control of flow is achieved by centrifugal force . this is illustrated by considering the effect of 0 . 001 increment in density in a field of 5 , 000 g . this produces an effect equivalent to a difference in density of 5 grams / ml . the stabilizing effect of the gradient will also be seen in the optical flow cells 38 . to increase the sensitivity of detection , a long optical path is desired . this is often attained by using a cylindrical micro - bore flow cell as shown in fig6 a in which liquid from chromatographic column 36 flows into optical micro flow cell 38 . the liquid is illuminated by light from light source 30 which passes through windows 39 . in static systems , this often presents cleaning problems , and becomes useless with the presence of small air bubbles . in addition , resolution is lost by laminar mixing . these problems are not a factor with the centrifugal fast chromatograph . if a long - path flow cell 38 with a reasonable optical cross - section is arranged as shown in fig6 b , then , when no gradient is present , laminar mixing will greatly increase peak width and decrease resolution . however , with centrifugal gradient stabilization , no mixing occurs as shown in fig6 c . the zone or peak 100 is stabilized as it passes through the flow cell 38 . the peak 100 flows through the flow cell 38 without an appreciable volume change , and the resolution obtained in the column 36 is not degraded during detection . fig6 d is an end view of the flow cell 38 of fig6 b and 6c , illustrating the very narrow one dimensional cross section . the cfc provides constant column path length . this overcomes resolution loss as illustrated in fig7 a - d . fig7 a - d shows how the path length is controlled as the eluting gradient 77 moves through the column packing 80 , and how mixing in a free space at the ends of the column 36 is overcome by the centrifugal field . in fig7 a , a fully packed column eluted at rest is shown . different fluid elements traverse different distances . peaks from paths 1 and 2 are graphically shown along with resolution loss as all paths are summed . in fig 7b , the column is also eluted at rest . mixing occurs in the free space above the upper frit and the free space below frit 78 . thus , as shown , the separation distance for all paths is identical , but resolution is lost in the free spaces at the ends . if the cones at the end of the column 36 are filled with inert material 70 which does not contribute to the separation , then in a centrifugal field the gradient 77 and centrifugal force will stabilize zones within the cones so that they widen evenly in the cones , and little resolution is lost . all fluid elements having the same density then pass through identical distances in separative packing 80 as shown in fig7 c . retarded solute zones , shown as dark bands , are therefore kept sharp and do not tilt . if strong frits are available which will withstand the centrifugal force , then an open - cone construction can be used at the bottom of the column as shown in fig7 d . the frit shown at the top may be a plug held in place by centrifugal force . flow is controlled by the gradient in the centrifugal force field in the cones and in the body of the column , and separated zones are kept perpendicular to the direction of centrifugal force as shown , and high resolution is achieved . with the cfc there are no &# 34 ; wall effects &# 34 ; such as occur when particles are sedimented in a centrifugal field . therefore , the separation chamber ( i . e ., the column 36 ) can have almost any configuration including that of a cube , a cylinder , a sector , or an inverted sector . in the cfc , gradient overloading which may cause mixing and inversions will rarely occur except at the original sample zone or early in the course of elution . hence the greatest density slope must occur immediately under the sample 76 initially , and later in those parts of the elution gradient 77 containing the most sample mass . the &# 34 ; turnover effect &# 34 ; which is well known in density gradient centrifugation and is due to differential diffusion of the sample solution 76 down into the gradient 77 , and the diffusion of the gradient solution 77 back up into the sample 76 will not be a problem in the chromatograph rotor because of the short time between sample introduction and initiation of separation which minimizes diffusion , and because the differences in molecular mass between the gradient 77 solution and the sample 76 are not as large as in the zonal centrifuge separation of subcellular particulates . air bubbles are not a problem in centrifugal chromatography where the centrifugal fields are sufficiently large to move any air bubbles completely out of the column 36 and flow lines 46 and 52 . thus , even in very small bore lines filled with air , samples will flow past the air and quickly displace it centripetally . also , because gas solubility is a function of pressure , should any air bubbles ( or other gas bubbles ) occur , they will tend to redissolve rapidly given the pressures existing in the rotor during rotation . the design of the system prevents the columns 36 from ever running dry . the exit lines 46 and 52 are all center of the columns , and are tilted up at some point along their length . providing the exit lines 46 and 52 inboard of the columns 36 prevents their running dry during rotation . traps are thus provided in the lines as an integral part of their design . the gradient maker 14 may be of a positive type using differentially driven pumps to mix two or more liquids to produce a liquid gradient , or a passive type in which two containers having complementary geometries are drained by gravity through a mixer to form a gradient . these are well known . one or more liquids are also required for column regeneration , and these are introduced by reverse flow through the columns after adjusting either manually or automatically the feed line and drain line valves 48 and 54 . the gradient 77 or the regeneration solutions 74 flow into the centrifugal chromatograph during rotation . the methods for signal acquisition , data processing , signal averaging , data display , and the analysis of chromatographic data are well known . in the designs described , the driving forces are generated by the difference in radius of the center ( centripetal ) fluid column ( r1 ), and the radius of the drain exit ( r2 ), as shown in fig5 . centrifugal force is directly proportional to radius . hence , the pressure generated by this difference in radius is equal to the average r =( r1 + r2 )/ 2 multiplied by the centrifugal force , multiplied by the liquid density , and multiplied by the column length . if the pressurizing column extends from 4 cm to 20 cm ( r1 and r2 ), the centrifugal force is calculated at 12 cm , and the pressure column is 16 cm long . at 3000 rpm the centrifugal force at 12 cm results in a pressure head of 19 , 440 g / cm 2 , or 276 . 5 psi . at 4000 rpm the centrifugal force at 12 cm results in a pressure at the bottom of the pressure column of 33 , 400 gm / cm 2 , or 489 . 28 psi . a variety of methods for operating the two valves attached to each column are possible . several are discussed below . in the illustrations provided thus far , the two valves are operated separately and manually . it is evident that since the valves are always operated simultaneously that each pair may be interconnected , and that all valves may be operated by one mechanism . the valves may be pressure operated , centrifugally operated , electrically operated , or mechanically operated . pressure operated valves are illustrated in fig8 a - c . when one of the lines 46 or 52 attached to the column 36 is full of liquid , the other 46 or 52 is empty out to the exit valve 48 or 54 . hence there is considerable hydrostatic pressure in one line . if liquid suddenly runs through one set of lines during rotation , pressure sufficient to close the valve attached to that line is created . by continuing flow into the line with the closed valve , the valve is kept closed . when liquid flow in ceases , the liquid level will gradually drop to equal that of the other line ( i . e ., the exit valve level ), and sudden liquid introduction into the opposite line will reverse the valve setting . operating details are shown in fig8 using a piston operated valve . fig8 a is a side sectional view of the chromatograph rotor 22 showing the position of piston operated edge line valves 48 . in fig8 b , a piston operated valve 48 or 54 ( used for both center lines 52 and edge lines 46 , is its rest position preparatory to column elution . piston 84 has two head portions 85 and 87 and spring 86 biasing piston to the left . piston 84 has two openings 88 and a 90 through its center to permit passage of liquid . first opening 88 provides a fluid passageway from center line 52 to drain 92 and second opening 90 provides a fluid passageway from edge line 46 to drain 94 . when piston 84 is driven to the left , the edge line - drain connection 90 is open and the center line - drain connection 88 is closed . in fig8 c valve is in position for column regeneration . edge line 46 is filled during rotation and exerts a pressure on the left of piston 84 sufficient to drive piston 84 to the right , thereby closing edge line 46 to drain 94 and opening center line 54 to drain 90 . fig8 d is an end view of piston 84 showing the opening 88 in the center . in centrifugally operated valves , centrifugal force either alone or in combination with hydraulic pressure is used to operate the valves . spring loaded valves are well known , and can be arranged so that they are in one position at rest ( due to spring loading ), and in the alternate position in a centrifugal field where centrifugal force opposes the spring . spring tension can be set so that position change occurs at relatively high speed , making it possible to regenerate columns at one speed , and elute at a second . it is also feasible to combine centrifugal operation with hydrostatic pressure - operated valves so that once the positional change occurs , hydrostatic force maintains that position . in electrically operated valves an electric drive system is used in the chromatograph rotor to change all valve positions at once . in mechanically operated valves , the valves are arranged so that they are operated by mechanical movement . for example , the valves may be arranged so that the members of a pair are one above the other , on shafts that extend above and below the rotor . with a vertically - moving non - rotating ring all of the extensions may be simultaneously pushed down from above , or with a similar ring placed below the rotor , all may be pushed up . detection of valve operation failure may be important in routine long term operation . if the edge and center line drains open into separate drain collection rings ( not shown ), then it is a simple matter to determine that fluid flow exists from one ring , and is absent from the other . if flow occurs from both collector rings , then valve failure has occurred . the center line can be enlarged , especially at its inboard end , to hold a considerable volume of liquid , in the limiting case , the entire elution gradient volume . because the system will not run dry during rotation , there is no lower limit for the rate of feed of elution buffer . however , if the elution feed rate is to high , liquid will flow out of the center line distributor ring . in one modification of the system , this liquid is guided to the center line collecting ring and is detected . the elution flow rate may then be increased until fluid is detected in the center line collecting ring , and then decreased until the rate of elution buffer outflow matches inflow . similarly , the edge line distributor overflow may be guided to the edge line collecting ring which normally collects no fluid during regeneration , and excess flow during regeneration detected . for most applications fractions are not collected , and the fraction collector described here is omitted , and the exit lines are led directly to the drain collecting ring . however , when fraction collection is desired , then a fraction collector ring 84 is provided which corotates with the chromatograph rotor of the cfc but can be indexed around to collect separate fractions from each chromatographic column 36 . the initial setting of the fraction collector ring 28 is designed so that each exit line drains through the collector ring 28 to the drain ring 60 and to waste . this allows column regeneration , and also allows the initial part of the elution gradient to be discarded . the fraction collector ring 28 may be designed to receive tubes 58 , but it is more optimally designed to hold flattened collection chambers which allow a very much larger number of collection units to be compressed into the limited space of the collector ring 28 . tubes or flattened collection chambers are angled so that they do not spill either at rest or during rotation . the position of the rotor 22 during rotation is sensed by a small electromagnetic or optical pickup positioned next to the chromatograph rotor 22 , and a similar pickup determines the position of the collector ring 28 . using a suitable algorithm the position of the ring 28 relative to the exit lines is determined , and the identity of the collection vessels being used is known . to advance the collecting ring 28 one unit ( one tube or collecting vessel ), a variety of mechanical , electrical , or hydraulic mechanisms may be used . the preferred method uses a mechanical brake to apply a small force to the collecting ring 28 , retarding it relative to the chromatograph rotor 22 . a ratcheted escapement mechanism allows the collector to move only the width of one collecting vessel . the rachet is then operated using either downward force applied through a ring above the rotor , electrical force , or a retarding force applying pressure to a spring which resets the ratchet when the retarding force is released . escapement mechanisms suitable for this purpose are well known . it is feasible to use a large seal of teflon or other self lubricating material attached to a central bell - shaped chamber to isolate the center of the rotor from the rest of the rotor chamber , and to keep the center at atmospheric pressure while a vacuum is produced in the rotor chamber . this produces an additional driving pressure of one atmosphere across the column in the rotor , and also allows the contents of the collecting vessels to be concentrated by evaporation of lyophilization in a centrifugal field . this is of great advantage when concentration is necessary . also , the centrifugal force makes the drying process much more efficient , and material does not fly off the evaporation surface due to bumping or boiling . the column fittings can be arranged so that columns may be prepacked and attached to the chromatograph rotor , or may be changed as necessary . numerous characteristics , advantages and embodiments of the invention have been described in detail in the foregoing description with reference to the accompanying drawings . however , the disclosure is illustrative only and the invention is not limited to the precise illustrated embodiments . various changes and modifications may be effected therein by one skilled in the art without departing from the scope or spirit of the invention .