JPH02504070A - Centrifugal high-speed chromatograph - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Centrifugal high-speed chromatograph division of powers The present invention relates to an improved centrifugal chromatograph.9 In particular, the present invention relates to an improved centrifugal chromatograph. It relates to centrifugal chromatography, which can be performed quickly and at relatively low cost.

l Rehabilitation! look High-pressure, high-resolution liquid chromatograph 2 is suitable for both industrial research laboratories and clinical trial stations. It is widely used for the separation of proteins, nucleic acids, metabolites, drugs, and a wide variety of compounds. came to be used. The systems currently in use are expensive and pulsating. A high-pressure pump without a pre-packed column and small uniform beads adapted to achieve high resolution; A spectrophotometer or colorimeter to monitor each column and a fraction monitor for each column. It includes a means for collecting information as an essential component. In addition, for chromatographic analysis a microprocessor system for integrating peaks, and a program for integrating peaks; A printer is used to print the quantitative results. The system uses columns Prevent irregular or clear flow and actively remove air bubbles to ensure ideal flow through the This prevents the column from drying out during use and directs the flow from the narrow-bore inlet tube to the separation column. in the headspace at the top of the column that extends to the entire diameter of the column, or The diameter narrows as it exits the system and is concentrated into the detection flow cell. Contains no active means to prevent irregular flow and mixing within the headspace. In addition, the rapid data processing capabilities of microprocessors are being fully utilized. i.e., today's microprocessors are only a dozen or so You must be able to process, manage, and display data from more than one column. Microprocessors are generally not used that much.

For systems currently in use, only one column is actuated, oscillated, The entire set of elements and monitors can only be analyzed once at a time, and It is expensive, has various operational problems that lead to anomalous results, and The separation power is lower than that which should be theoretically obtained. In addition.

Packing material into a new column or refilling a cleaned column is prohibited. , is difficult but not impossible in that field.

Recent developments have changed the instrumental requirements for achieving high resolution. This is very uniform It is a development of spherical beads or resin.

They reduced the bank pressure required to achieve high resolution. Previously it was 1 flat A pressure of several thousand pounds per square inch was required, but today it is several hundred pounds per inch. That's about it. This means that the pressure conditions found in centrifugal fields at very low speeds means. The mechanical strength of the beads improved their deformation resistance under pressure.

This high strength also creates resistance to deformation in the centrifugal field.

Traditional non-centrifugal chromatographs included many other problems. between different columns There was no automatic means of compensation for flow resistance. A non-centrifugal system is applied to many parallel columns. When pumped from one pump in the stem, the flow through different columns is Small differences occur due to differences in flow resistance within individual columns.

Until now, in column chromatography, the flow of a clear stream through a packing material has been There was no way to prevent such an irregular flow.

If the filler is non-uniform, i.e. the filler particles are of different sizes. If so, the flow resistance at different parts of the column will be different. liquid flows through that line, that is, a clear stream with low flow resistance, thereby It can be uneven. Flow distortion and clear flow reduce separation power.

Where there are many small anomalies, a broadening of the band width can be observed. strange Where the law is large, the band slopes. The sharp slanted band It is also observed as a broadened peak during elution. Minimize micro irregular flow Much effort has gone into producing column packings consisting of spherical particles of uniform diameter. was paid. Greatly improved resolution is achieved by using beads of homogeneous, uniform, particle size. However, microscopic flow control and minimization of clear flow are extremely important. Demonstrate. However, homogeneous particles do not actively prevent anomalous flow. homogeneity Even if the particles are packed, they may become uneven due to partial particle compression, or a sediment layer may form. Therefore, zero conditions that cause clogging in the column cause irregular flow.

'Separation power is determined by the flow of fluid from the tube into the column (thin layer mixing), Flow expands in the radial direction when flowing from a small diameter line to a wide diameter column. while the cross-sectional area is reduced at the bottom of the column, and the optical flow is increased by thin-layer mixing. - is lost during flow into the cells and intervening tubes.

It was also difficult to adjust the column path length consistently.

The fluid flowing through a chromatographic system is made up of many small fluid elements. It can be thought of as standing. In free fluid flow, these are density gradients It is held in order by the centrifugal force acting on it. However, these fluids The solute in the element is slowed down to a different degree by the flow through each column packing. If the path length from the packing material is different for different solute elements, the separation power will increase. It will be lost. If there is free space at the end of the column, there is no gradient. and combined with the centrifugal field, considerable mixing occurs.

If there is no free space at the column ends, the column simply becomes smaller in diameter at each end. If you are bound like that, then.

Fluid flowing along the edges of the column has a lower flow rate than fluid flowing along the central axis of the column. It flows through a long passage, resulting in a loss of separation power.

Finally, in traditional chromatography, attention is paid to removing air bubbles. We must also prevent their occurrence. in some cases , it is required to degas the tuim liquid.

These are just a few of the problems that traditional chromatographs have. this and other problems were addressed by the centrifugal high performance chromatograph of the present invention.

11F1” Elephant chromatography and any type of chromatographic separation quickly and simultaneously The objective is to provide a means to do many things efficiently and at relatively low cost.

The above and other objects are achieved by the centrifugal fast chromatograph (CFC) of the present invention. be done. The CFC automatically performs, monitors, and performs many sample elutions. Also, regenerate the chromatography column. In addition, the present invention allows multiple samples to be Including a method of simultaneous gradient elution. CFC is a chromatography rotor, Many chromatographic columns, including rotor drives, are mounted on a chromatographic rotor. Conformally mounted, each chromatographic column has an optical flow with a transparent window. It has a cell. The transport disk that holds the sample is inside the chromatography rotor. placed in the heart. The transport disks are arranged equiangularly around its circumference. It has a large number of sample holding holes. Each sample holding hole corresponds to each chromatocolor. It corresponds to The flow distribution blade is located in the distribution ring of the chromatography rotor. The solution from the sample holding hole is placed conformally on the internal edge of the chromatography column. Guided within. Its flow distribution blade also allows the elution gradient to be allocate within the group. A line carrying two fluids runs from the border of the conveying disc to each It extends to the chromatography column. Both have valves directing fluid to an outlet drain . One line is connected to the inner end of the chromatography column, and the other line is connected to the inner end of the chromatography column. connected to the outer end. The light source is located on one side of the chromatography rotor near its outer edge. mounted and directed to shine a beam of light through an optical flow cell.

A photodetector is placed on the opposite side of the light source to detect the light shining through the optical flow cell. It is mounted on the top or bottom of the chromatography rotor.

The gradient device passes the flow through the distribution ring containing the distribution blades to the chromatocolumn. are in fluid communication with each other, where flow distribution blades divide the elution gradient into individual Distribute into all chromatography columns simultaneously through the ram feed line. microphone A processor-type computer parallels the operation of a CFC. that mic The processor controls the gradient device and the rotor drive. That's cool Controls and monitors chromatograph processes and monitors all chromatography CRT display of RAM in real time. It also Controls the automatic pipetter used to fill the pull.

The CFC of the present invention includes a microprocessor that controls and monitors its separation. Includes a 118 cathode ray tube (CRT) that displays chromatographic separations in real time. On the other hand, a gradient device creates its gradient from a solution and also Controls playback At the start of analysis, the sample is transferred to the transport disk in the disk loader. loaded onto the screen. The transport disk is connected to the chromatography rotor at the start of driving. and the rotor is loaded into the rotor under microprocessor control. - Accelerated by a drive. The samples were each fitted with a matching flow distribution blade. It is transported by centrifugal force from the transport disk between Inflow. The transport disc and its distribution blades attached to the distribution ring are all Because the blades rotate in unison, each sample flows independently between a pair of matching blades, It reaches inside the column. The elution gradient then flows towards the same blade and The sample is then distributed into the column delivery line. The water flowing from the rotor is drained. or collected in a co-rotating fraction collection ring. that running water The absorbance of the stream is measured by a light source and photodetector, and the signal is sent to the microprocessor. Sent to processor. The position of the rotating rotor is determined using a synchronous signal pickup. It is determined by Two distribution rings, one with distribution blades, one with the other The one directly above is used. One distributes the elution gradient during analysis, the other dispenses regeneration and equilibration solutions. The lip that protrudes inward between the two rings is , preventing cross-flow between its rings.

The centrifugal high-speed chromatograph of the present invention works by using centrifugal force. required for column packing, chromatographic separation, column regeneration, and sample loading. Zero rotation provides a hydrostatic pressure that distributes liquid flow among multiple columns and traverses a single detection beam, allowing reproducible absorbance measurements. Provide the beam required for

Centrifugal high-speed chromatographs are designed to prevent columns from drying out during periods of rest or rotation. It also has a trap that controls the direction of flow through the column during analysis and column regeneration. It has a valve to control it. Two types of traps are essential in the design. first type prevents the column from drying out during rest. It goes in and out of the column through the valve. Includes upwardly slanted center and edge line registration that connects the ends to the center distribution ring The second type, which operates during rotation, involves the column and the valve inside it. Contains an edge line that connects to the If the liquid supply is stopped during rotation, The liquid simply drains into the open valve and the column does not dry out. If the flow is in one revolution When restarted, the air in the supply line is quickly replaced by the centrifuged liquid. The air moves centripetally.

The separated fractions are collected from time to time, and the hydrostatic pressure on the column is controlled by the rotational speed. or changing the length of the fluid column within the separation column. It can be changed by

The density gradient i@ separation in a centrifugal field actively directs the flow through the system. Stabilized, sample and separated regions are tilted and widened by anomalous flow. Prevent this from happening. Liquid gradient generation used in centrifugal high-speed chromatographs A microprocessor containing the machine controls the entire analysis procedure.

Centrifugal high-speed chromatography uses a larger chamber in the distribution ring. introduces the entire elution gradient into its rotor at once, and then It includes means for allowing flow through the separation column without the introduction of liquid.

Sample and elution gradient separately during rotation and within each column unit individually Includes additional options for introducing. Furthermore, within the rotor itself to drive its rotor by using an integrated printed motor. Selective measures are used. Printed motor is mandatory for rotor It's electrical lines "printed" onto plastic coated metal It has windings. The printed motor is made in a 1-strong centrifugal field. The direction of liquid flow during 8 rotations is centrifugal (system A and (referred to as system B) or centripetal (referred to as system B).

The preferred direction is centrifugal.

Therefore, centrifugal high-speed chromatographs, in a single instrument, The sample is aligned from the transport disc in the sample chamber of that transport disc. a means of transporting the gradient flow into each of several columns, and separation by means of equal distribution in all columns and by using centrifugal force. and means for creating the hydrostatic pressure required to achieve. Everything in rotation means for measuring the optical absorption of a flowing water stream from a column and from all columns. Means for collecting (or rejecting the flowing water flow) a fraction of is also formed. 8 The present invention also provides for regeneration of all columns and, if necessary, Includes means for monitoring playback. Anything essentially orthogonal to the direction of radial flow Actively maintain the area and boundary of means to actively prevent flow and whether the column is rotating or at rest. means to prevent drying and separation during flow during sample application and detection. All parts of the sample or separated area are separated by exactly the same distance within the medium. Also included is a means for controlling flow 2 to advance. Centrifugal high-speed chromatograph is also , a means to ensure that no air bubbles are retained in the analytical fluid, and a predetermined means for collecting samples at intervals, i.e. rejecting unnecessary fluid portions to waste; and also means for packing or refilling the column in place.

The flow during elution can be either centrifugal (quickly outward) or centripetal (quickly inward). , the flow is in the opposite direction during playback.

Most of the design descriptions discussed below are based on the rotor system that provides the elution flow to the system (System A) and central flow column regeneration.

The type of analysis performed on a centrifugal high-speed chromatograph is for the analysis of human plasma proteins. including, but not limited to, nucleotides and their test conductors, amino acids and their derivatives; body, urinary metabolites, therapeutic drug monitoring, drugs of abuse including monitoring for antimicrobials and analysis of proteins in wheat and other seeds.

Various additional advantages and novel features characterizing the invention are further described in the claims. has been identified. However, in order to better understand the invention and its advantages, Reference is made to the drawings and illustrative material illustrating and describing suitable embodiments of the invention. It should be done.

1. Smoking "Shu I" Figure 1 shows an overview of the centrifugal gradient elution chromatography system according to the present invention. It is a diagram.

Figure 2 shows a side view of the centrifugal-driven gradient elution chromatography system according to the present invention. It is a diagram.

Figure 3 is a chromatograph of the centrifugal oscillating gradient W1 separation chromatography system. FIG. 3 is a schematic view of the rotor from above.

FIG. 4 is a diagram illustrating the operating process of the chromatography rotor.

Figure 5 is a diagram explaining the inward elution flow in the chromatography rotor. Ru.

Figure 6 shows how gradients are used to control flow through a long path flow cell. It is a diagram explaining how it is used.

Figure 7 shows how the path length is controlled in a chromatographic column through the column packing material. FIG.

FIG. 8 shows the centrifugal percussion gradient elution chromatography system of the present invention. FIG. 2 is a diagram illustrating a piston-operated valve system.

A small day The central high-speed chromatograph of the present invention is a centrifugally driven gradient elution chromatograph. It is a tograph system. This system is shown in FIG. micro pro Sessa-1o.

The separation is controlled and further monitored. Cathode ray tube (CRT) 12 is a chromatograph The gradient device 14 displays the separation of the solutions 16 and 16 in real time. 2 analytical stars that create the gradient from 18 and control the regeneration of the column. During the test, the sample is transferred to a Discloro, an automatic quantifier known in the art. On the transport disk 20 (shown in FIG. 2) in the loader 21 is loaded into. The conveyance disk 20 is attached to the chromatography rotor 2 at the start of operation. 2, whose rotor is driven by a microprocessor 24 by a drive device 24. accelerated under the control of The water flowing from the rotor is directed to the drain 26 or The fractions are collected in a co-rotating fraction collection ring 28. Light from the flowing water The absorbance of light from the source 3° is measured using a photodetector 32, and the signal is The position of the rotor during 0 rotations, which is supplied to the processor 10, is determined by the synchronization signal pick-up. is determined using tap 34.

FIG. 2 shows a side view of the system. control center controls Panel, microprocessor-10° CRT display panel 12. Speed and temperature display and a gradient device 14. The centrifugal chromatography rotor 22 is The drive device 24 is driven by a control center drive device 24. Controlled by the control panel. Chromatography column 36 is centrifugal chromatography It is mounted to rotate on the graph rotor 22. Transport that holds the sample The disk 20 is mounted on the center of the rotor 22. optical flow cell 3 8 has a transparent window 39 and is mounted on the centrifugal chromatography rotor 22 . The light source 30 is mounted opposite the photodetector 32, and the photodetector 32 is a photoelectric detector. It is like a multiplier tube 4o, and measures the absorbance of flowing water. The upper part Tsujiline distribution ring 42 is a regenerating solution. ion) to the edge line, and is arranged at the end of the solution supply line 44. The solution supply line 44 is connected to the gradient on the control center. Extending from device 114. Edge line 46 is a supply line that carries fluid; It has an anti-backflow slope and is disposed between the upper distribution ring 42 and the line valve 48. It is placed. The lower or centerline distribution ring 50 is the centerline 5 It is located at one end of 2. The center line 52 carries fluid with an inclination to prevent backflow. It is a supply line that carries between the central end of the chromatographic column 36 and the distribution ring 5o. It is located.

The centerline valve 54 (FIG. 3) is located at the center end of the chromatography column 36 and is located at the center of the column 36. 12 are placed on the interface line 52.

Edgeline valve 48 is mounted on one end of edgeline train 56.

A drain and fraction collection ring 28 is located on the other end of the edge line drain 56. It is installed in.

The stationary drain collection ring 60 includes a collection tube and a collection port 58 leading to a stationary drain collection ring 60. The drain collection ring 6o is mounted on the side of the rotor 22, and is designed to collect fluid discharged from the The supply line 62 is connection between the center line distribution ring 50 and the center line distribution ring 50.

The distribution ring 50 is used to separate 36 kl of chromatographic columns.

A cross-sectional view of the chromatographic rotor of System A is shown in FIG. chroma The tograph rotor 22 includes a transport disk 20 that holds the sample, and transports the sample. A plurality of sample holding holes 64 are disposed within the disk 20. sample retention Holes 64 are located around the outer edge of the transport disk 20 and in each chromatographic column 36. are placed in corresponding positions. The chromatography column 36 is connected to the center line 52. is connected around the transport disk 20. The blades 66 that distribute the flow are , within the centerline distribution ring 50 to transport the fluid sample to the disk 20. and the center line 52. The center line valve 54 is the center line 52 to prevent feedstock from being wasted during column regeneration in reverse flow. ing. Flow cell 38 has a transparent window 39 and is connected to edge line 46. It is located on the outer end of the talomad column 36 adjacent to the section where it is located. Etsujirai An intake valve 48 is located on the edge line 46 to direct the wastage of material during elution. It is now converted to . A device that splits two coaxial flows, that is, a distribution This is an upper distribution ring 42 (not shown in FIG. 3) that feeds the edge line. ) and the lower distribution ring 50 that supplies the center line. columns are used to split the flow into multiple columns.

The chromatographic system of the present invention has excellent flow properties resulting in high resolution. do. Although outward flow during elution (system A) is recommended, , the sample is located at the center end of the column, an optical flow cell or other sensing The column should be located at the outer end of the column, and the elution should be performed using a gradient that reduces the physical density. request that something be done at the event.

It also means that the liquid in the column is at the start of the sample i.e. the elution gradient. The start of the test (thicker than the sample) should be thicker than the end of the sample. . The interrelationship of these specific requirements is explained below.

When a central elution stream is used (system B), the fluid in the column is , at the end of the start of the sample or elution solution introduced through the edge line. The eluent fluid is not more concentrated than the center line above the center line. The elution gradient increases from the start to the end. increases in physical density. The sample is delivered to the bottom of the column through the edge line. is supplied through the centerline valve on the centerline, and exits through the centerline valve on the centerline, and the light The chemical flow cell is located at the top (center end) of the column as shown in FIG.

A system that works in any way and has a flow cell at both the top and bottom of the column. It is possible to build a system.

The sequence of steps during column packing, column regeneration and/or equilibration, and analysis is shown in Figure 4. is shown in Figure 2, where the same column is used for a series of tests including column packing, regeneration and analysis. are shown at each stage. The valve is set to change the direction of flow . In particular, during column packing, the suspension being packed passes through a bottom flow splitter; into the centerline, through the closed centerline valve, and into the column. flows into. The liquid that exits the column past the bottom frit is - flows through the cell to a drain that leads to an open Edgeline valve. column Once filling is complete, a small stopper is introduced into the centerline and held in place by centrifugal force. will be moved to This allows the filler to move and reset when the rotor is at rest. prevent. The slope of the center and edge lines is determined by the column and floor when the rotor is at rest. - Prevents fluid from escaping from the cell and outer ring.

In FIG. 4A, a suspension of inert particles 70 passes through center line 52 and crosses the It is introduced into the matocolumn 36. Edge line valve 48 and center line valve Both valves 54 are set for outward flow, ie, valve 48 is open. At this point, the valve 54 is closed. The inert particles 70 fit into the lower cone of the chromatography column. Ru. The active packing material 80 that creates the chromatographic separation is then introduced and As shown in Figure 4B, the chromatographic column 36 is partially filled by centrifugal force. There is. In Figure 4C, all the active packing material is in place and the chromatography The upper conical part of the ram 36 is partially filled with an inert material 7o. Fourth In figure D.

The chromatography column 36 is fully packed, and the upper perforated stopper 72 is connected to the upper part of the chromatography column. It is located within the center line 52 of. In Figure 4E, the edge liner Lube 48 and centerline valve 54 are set so that the flow is inward. .

During rotation, cleaning or regeneration or balancing liquid 74 is pumped through the upper distribution ring 42. is introduced into the tube line 46, thereby allowing the column to regenerate or equilibrate.

The density of the liquid is greater than the density at the rich end of the elution gradient. controlled so that it remains in the Sample 76 was sampled while the rotor was stationary. It is placed in the transport disk 20 adjacent to the center line as shown in Figure F. sump The edge line valve 48 and the The turbine line valve 54 once again allows flow outward through the chromatographic column. is set. At the 4th G[, the rotor is accelerated and the sample 76 is and is transported centrifugally into the center line 52. While the rotation continues.

The elution gradient 77 is introduced into the center of the rotor as shown in Figure 4H. Ru. The gradient elution of the chromatographic column was performed using the chromatographic column for the separation of sample 76. It does not occur in the inert particles in the upper cone of the mold, but it has started. In Figure 41 .

Sample components 1, 2, 3. and 4 were separated in chromatography column 36. Fourth In Figure 3, peaks and bands 1, 2, 3, and 4 represent each component of the sample, respectively. The solution continues to flow through the outer container of the chromatographic column 36, corresponding to Let go. The peak travels back through the container without mixing. Fourth Once the analysis shown is complete, backflow column regeneration is performed and the elution graph is A solution that is denser than the concentrated end of the agent remains inside. The analysis is shown in Figure 4F. Can be repeated by starting from the steps indicated.

The flow passes through the column during column regeneration and equilibration (referred to as system A). As it heads aft, the liquid enters through the upper flow divider and follows the edge line. through the closed edge line valve and into the centrifugal (outer) end of the column. and flows inward through the optical flow cell and column, and opens. flow out through the state's internal line valve and reach the drain (or collection).

During analysis, when the eluate flow is in an inward or centripetal radial direction (system B and The location of the optical flow cell (referred to as "optical flow cell") is shown in FIG. That is, optical 2 The 0-cell is located centripetally to the column (above) on the centerline. system In column B, the eluent passes through edge line 46 toward chromatography column 36. flows. Its pressure head during elution is proportional to the distance to centerline valve 54. Give an example.

System B has the inherent problem of density reversal within the edge line. analysis At the start, the dilute solution used fills the system, the sample is adjusted to be slightly thicker and, when introduced, forms a fluid with the edge line. Mix quickly. When that sample passes through a hairpin loop at its end, the inward direction , and the stabilization of the density gradient effectively begins. That glaze As components flow into the rotor during analysis, constant backmixing occurs in the edge line. A steady flow continues within the column, flow cell, and internal lines. sample The solution to the problem of backward mixing during installation is to Inject the samples individually through the self-sealing rubber section 81 at the distal end of the system. Is Rukoto. This solves the problem but is undesirable from an automation standpoint It becomes a system.

During sample loading following gradient introduction, flow and valve settings are as shown in Figure 5. It is as shown. Regeneration of the column is achieved by the flow of solution entering through line 52. and discharge through the outlet of valve 48, i.e., its flow and valve settings. is no different from that for column packing.

The CFC automatically adjusts the flow resistance in different columns to maintain constant flow. to correct. In that CFC, the actual pressure head on each column is at the outlet line. is a function of the length of the liquid column center. If one column flows more than the other If the resistance is a little smaller, the fluid will flow quickly and the length of its liquid column will decrease, The driving pressure is reduced and the liquid gradient is introduced less abruptly. automatically adjusts the flow through that column to match the others, provided that . The top of the column center in the exit line contains a large amount of fluid, i.e. the entire gradient. can be expanded to include In the centrifugal field, many density gradients are stable. ing. The bubbles are forced inward by the centrifugal field, so they do not block the flow.

During elution with either system A or system B, the gradient used is bifunctional. The first phase is a gradient that controls the flow and maintains the fluid segments in the proper order. The second is the density or specific gravity of the components of the mixture being analyzed from that column. This is to change the elution power to achieve continuous elution.

In system A, the liquid in the rotor, the sample and the elution gradient are Many proteins and reversed-phase chromatographs must be organized to reduce For other materials containing Ratsufi, the gradient changes with increasing acetonitrile concentration. This is one of the things that increases Acetonitrile is 0.785g/m, l Having density or specific gravity. Therefore, the gradient from water or a normal slow rg solution is , the density is approximately 1. ○The range is from OOg/ml to 0.785g/ml. The ratio The weight is reduced as required for system A. If sucrose or other inert a gradient material is added to the equilibrium solution, optionally to the sample, and to the gradient material. When added to a concentrated solution used to make a gradient, it creates a steep density gradient. can be obtained.

The commonly used gradients increase the salt concentration and thereby When the density is increased, the inert density increasing material is added to the sample in small amounts. amount that must be added to its equilibrium solution and also reduces its physical density. The concentration in the gradient increases to increase the elution power. must be reduced. this is.

When used in column and elution procedures requiring very concentrated salt solutions, the system This is A's disadvantage. In system B.

Part 9 requires a gradient that increases the density, but 1 originally increases the density. An increasing gradient may also be used directly.

The disadvantage of system B is that it contaminates the sample area while flowing through the edge line. It is. This can be minimized by having very small diameter edge lines. Can be done. A small amount of column packing material at the start of the sample's flow through the column. If it is strongly absorbed by the material, i.e., it will be absorbed in a small amount from the beginning. When reconcentrating a sample, the contamination may have a small effect initially. Injecting the sample at one end of its rotor also solves this problem, but operationally resulting in disadvantages.

The advantage of system B is that the flow is opposite to the centrifugal force. Therefore, the flow is The column packing tends to become resuspended, whereas centrifugal force causes it to pack. Tend. This limits the flow rate without aggressive positioning of the top frit. Define the boundaries. In system A, the flow and centrifugal force are in the same direction, so the flow Centrifugal forces combine to pack the column packing material more tightly. this is a column packing that is deformable and can be overcompressed to reduce flow. This is a disadvantage for the.

In the system described here, it uses a density gradient, Special flow control is achieved by centrifugal force. This is for a field of 5.000g Density is 0. This is explained by considering the efficiency of increasing by Ool.

This produces an effect equivalent to a density difference of 5 g/ml.

Its gradient stabilizing effect is also seen in optical flow cells. the A long optical path is desired to increase detection sensitivity. This is often shown in Figure 6A. As shown in FIG. Achieved by using a cylindrical micro-aperture flow cell that allows flow. will be accomplished. The liquid is illuminated by light from light source 30 through window 39. static system In systems, this often creates cleaning problems and is hampered by the presence of small air bubbles. I can't stand up anymore. An additional 1 resolution is lost due to N mixing. These problems are It is not an element that centrifugal high-speed chromatography has, but if it makes sense A long channel flow cell 38 having an optical cross section is prepared as shown in FIG. 6B. If the gradient does not appear, layer mixing will greatly increase the peak width; and reduce resolution. However, centrifugal force stabilizes the gradient. As shown in FIG. 6C, the band or peak 100 where no mixing occurs is the flow It is stabilized as it passes through the cell. the peak without any appreciable change in quantity flows through the flow cell and the resolution obtained with that column is not degraded during detection; Figure 6D is an end view of the flow cell of Figures 6B and 6C, with a very narrow one-dimensional cross section. It shows the area.

CFC provides constant column path length. As shown in Figure 7, this is due to the role of separation power. Eliminate stress. Figure 7 shows the elution gradient as it moves through the column packing. How to control the path length.

and how mixing in free space at the ends of the column is prevented by centrifugal force. , Figure 7A shows a fully packed column that has been stopped and eluted. has been done. Different fluid elements pass different distances. Pee from aisles 1 and 2 It is shown that this involves a loss of resolution because all the passages are summed.

In Figure 7B, the column has been stopped and elution has taken place. Top and bottom fringe Mixing occurs in the free space above and below the cut 78. Therefore, as shown in the figure, The separation distance of all passages is the same, but the separation power is not lost in the free space at their ends. be exposed.

If the cone at the end of the column is filled with an inert material 70 that does not contribute to the separation. Then, in the centrifugal force field, the gradient and centrifugal force are spread evenly.

The band within the cone is stabilized so that only a small loss of resolution occurs. same All fluid elements having a density are separated by a separating filler 8゛o as shown in Figure 7C. pass equal distance within. A band of delayed solutes, as shown by the dark band. is therefore kept sharp and not tilted.

If one strong frit is available to resist centrifugal force, then in Figure 7D As shown, an open conical structure can be used at the bottom of the column. So Its frit, shown at the top of the spigot, is held in place by centrifugal force. It's something like. The flow is within the centrifugal field within the cone and column body, The separated bands are perpendicular to the direction of centrifugal force as shown in the figure. high resolution is achieved.

In CFC, the “wall effect” that occurs when particles settle in a centrifugal field Therefore, one separation chamber (i.e. column) can be a cube, one cylinder, a sector, or an inverted It can have almost any shape, including layered shapes and the like.

In CFC, there is an excess of gradients that causes mixing and progression. Very little occurs except in the original sample area or early in the elution. For this reason, the most A large density gradient immediately occurs between the initial sample and most of the samples in the first order. must occur within those parts of the elution gradient that contain the population of pulls; ``Turnover effect'' is well known in density gradient centrifugation. This is due to the differential diffusion of the sample solution into the gradient, and This is due to the diffusion of the gradient solution supplemented during the pull, but it This is not a problem in a tograph rotor, since the sample is designed to minimize diffusion. This is because the time between sample introduction and the initial stage of separation is short, and the gradient solution and sample This is because the difference in molecular weight between pulls is not greater than the difference in regional centrifugation of subcellular particles. be.

The air bubbles must be removed sufficiently such that the centrifugal field completely drives them out of the column and flow lines. This is not a problem in large chromatographs, so the air-filled Even in very small diameter lines, the sample flows quickly and overcomes the air. It replaces it centripetally. Also, the solubility of gas is a function of pressure; (or other bubbles).

They tend to re-melt rapidly due to the pressure that develops within the rotor during one revolution. be.

The design of that system prevents the column from drying out; all exit lines are connected to the column. centered and tilted at several points along their length. outside the column Providing a mouth line prevents the column from drying out during rotation. A trap.

Therefore, it is provided in the line as a necessary part of the design.

A gradient device is a device that mixes two or more liquids to create a liquid gradient. of active type by using dynamically driven pumps, or with supplementary forms; Two containers are passed through a mixer and ejected by gravity to create a gradient. There is a passive type. These are liquids that contain one or more liquids. 9. For ram regeneration: Required, these can be supplied either manually or automatically. After adjusting the valves on the effluent and discharge lines, it is introduced back-flow through the column. that grazi The agent or regeneration solution flows into the centrifugal chromatograph during rotation.

Signal acquisition, data processing, signal averaging, data display and separation of chromatographic data The analysis method is well known.

In the described design, the driving force is the radius of the central (centripetal) liquid column (r l) and the radius of the discharge port (r2). Centrifugal force is directly proportional to radius. Ru. Therefore, the pressure caused by this difference in radius is the average value r=(rl+r2)/ 2 times the centrifugal force times the liquid density times the column length, also However, when the pressurized column is expanded from 4 ■ to 20 ■ (rl and r2), the centrifugal force becomes 1.2C11 and the pressure column will be 16 cm long. 3000 At rpm.

The centrifugal force at 12a1 is 19,440g/aJ or 276. It becomes 5psi. The centrifugal force at 4000 rpm and 12011 is the pressure force at the bottom of the ram. The pressure at this point is 33,400 gm/cJ or 489.28 psi.

Various methods are possible for operating the two valves attached to each column. So Some of them are explained below. In the explanation so far, the two valves is operated independently and manually. The valves are always operated at the same time, so each Pairs may be interconnected, or all valves may be connected in one groove. It is clear that something that can be easily manipulated is also fine. the valve is operated by pressure centrifugally operated, electrically operated, or mechanically operated. It is something that will be done.

A pressure operated valve is shown in FIG. The light attached to the column When one of the tubes is filled with liquid.

Others are empty up to the outlet valve. Therefore, there is considerable hydrostatic pressure within one line. exist. If liquid suddenly flows through a series of lines during rotation, the line Sufficient pressure is created to close the valve attached to the. have a closed valve The continued flow of liquid through the line keeps the valve closed.

When the liquid stops flowing, the level of that liquid gradually decreases to that of the other lines. (i.e., the outlet valve level) and one opposite line The sudden introduction of the body reverses the setting of that valve. The operation details are based on the piston-operated valve. It is shown in FIG. 8 using the lube. Figure 8A is a side section of the chromatography rotor. A top view is shown showing the position of the piston operated edge line valve 48. No. In Figure 8B, a piston-operated valve 82 (center line and edge line) is shown. (used in both columns) is in the stop position prior to column elution. fixie The piston 84 has a 2Il head and a spring 86 that biases the piston to the left. Ru.

The piston has 241 openings 88,90 through its center to restrict the passage of liquid. do. The first opening 88 creates a fluid path from the centerline 52 to the drain 92; A second opening 90 creates a fluid path from edge line 46 to drain 94 . piston When the edge is activated to the left, the connection between the edge line drains is open. , the connection between the center line drains is closed. In Figure 8C, the valve is in the column regeneration position. The edge line is filled during one revolution, Apply enough pressure to the left side of the piston to force the piston to move to the right. This closes the edge line leading to the drain, and the center line leading to the drain closes. The line is open. FIG. 8D is an end view of the piston showing the opening 88 in the center. .

In centrifugally operated valves, centrifugal force alone or in combination with water pressure Used to operate valves.

Spring biased valves are well known and remain in one position when stopped. centrifugal force (due to spring loading) and centrifugal force resists that spring. It can be adjusted to be in other positions in the field. The spring force is The position change can be adjusted to occur relatively quickly, regenerating the column at a first speed, and regenerating the column at a first speed. This makes it possible to elute at a speed of 2. Also, when that position transformation occurs, Immediately apply centrifugal force to the hydrostatically operated valve so that hydrostatic force holds its position. It is possible to combine operations according to

In electrically operated valves, an electroactive system is used to control the chromatographic flow. used to immediately change all valve positions within the controller.

In mechanically operated valves, the valve is operated by mechanical action. adjusted so that For example, the valve is on a shaft that extends above and below the rotor. , the pair of members are adjusted so that one is on top of the other. Vertical movement non-rotating ring , all the extended ones are pushed down from above at the same time, or the rotor In a similar ring located at the bottom, everything is pushed up.

Detection of valve operation errors is important when valves are operated over long periods of time. Ru. If the edge and center line drains are separated into separate drain collection rings, If it does, the fluid flow is from one ring and not the other. Deciding that is easy. If the flow is both collection rings If this occurs, it means that a valve defect has occurred.

The centerline can hold a significant amount of liquid, especially at its inner edge. In marginal cases, the entire elution gradient The system does not dry out during rotation and provides an elution buffer. There is no lower limit as a percentage. However, if the solution layer supply rate is too high, the liquid The body flows out of the centerline distribution ring. In one variation of that system This liquid is then guided to the centerline collection ring and detected. its elution The flow rate is increased until liquid is detected within the centerline collection ring and then Then, the proportion of elution buffer is reduced until the outflow and inflow match. Similarly, The overflow distributed in the Tsuji line is an essira which normally does not collect fluid during regeneration. Excess flow during regeneration is detected by the in-collection ring.

In most cases, no fractions are collected and the fraction collection described here The collector is omitted and the outlet line is led directly to the drain collection ring.

However, when fraction collection is interrupted, the fraction collection ring is provided, and this ring co-rotates with the CFC's chromatography rotor, but each The separated fractions from the ROMAT column can be directed to be collected. frac The initial configuration of the collection ring is to allow each exit line to pass through the collection ring. It is designed to be drained and wasted.

This allows regeneration of the column and the initial part of the elution gradient is discarded. It also makes it possible.

Fraction collection rings are designed to accept tubes, but more optimally Allows a large number of collection units to be compressed into the limited space of the collection ring It is designed to hold a flattened collection chamber that allows for tube or flat collection The chambers are curved so that they do not leak during rest or rotation.

The position of the rotor during rotation is determined by the electromagnetic position adjacent to the chromatography rotor. or sensed by an optical pickup, and the same pickup is positioned in the collection ring. determine the location. By using appropriate arithmetic methods, we can The location of the collection rings is determined, the collection containers used are the same, and the collection rings are To knit (one tube or collection container) various mechanical, electrical or hydraulic Use mechanisms. The preferred method is to apply a small force to the collection ring and The idea is to use a mechanical brake on the rough rotor to slow it down.

A gear locking mechanism with a ratchet allows the collection section to move only within the width of the collection container. You can do whatever you like. The ratchet goes through the ring at the top of the rotor. It is operated using a supplied downward force, electrical force, or deceleration force. here, The famous sword has a spring that resets its ratchet when the deceleration force is released. supply power.

Gear locking mechanisms suitable for this purpose are well known.

Isolate the center of the rotor from the rest of the rotor chamber and A large piece of Teflon is used to maintain atmospheric pressure at the center of the bar while it is being evacuated. using a solid seal or other self-lubricating material attached to a central bell-shaped chamber. Good. This creates an additional driving pressure of 1 atmosphere across the column in the rotor, The contents of the collection container may also be concentrated by evaporation or freeze-drying in a centrifugal field. and make it possible. This is an excellent feature when concentration is required. Also .

Centrifugal force makes the drying process very efficient and the material is not affected by bumping or boiling. Therefore, it does not scatter from the evaporation surface.

For column-equipped products, the column is pre-packed and attached to the chromatography rotor. Or it can be adjusted to be replaced if necessary.

Numerous features, advantages and embodiments of the invention have been described in the above description with reference to the accompanying drawings. It was explained in detail. However, the disclosure is illustrative only and the invention is not accurate. The invention is not limited to the specific examples described in . Various changes and modifications may be made within the scope of the invention. is done by experts in the technical field without departing from the spirit.

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Claims (15)

[Claims] 1. Centrifuge height to automatically perform, monitor, and compare multiple sample elutions A rapid chromatograph, which includes: chromatography rotor; a rotor drive unit that rotates the chromatography rotor; a plurality of chromatography columns mounted conformally on the chromatography rotor; Each of the chromatography columns has a transparent window located at one end of the chromatography column. an optical flow cell having; A transport disk for holding a sample is placed in the center of the chromatography rotor. and where the transport disc is equiangularly disposed around the circumference of the transport disc. It has multiple sample holding holes, each with its own sample holding hole. corresponds to the chromatography column; coaxial with the chromatography rotor first and second distribution rings mounted on; from the transport disk to the inner end of the chromatography column via the first distribution ring. A fluid delivery center with a centerline valve that extends and directs fluid to an outlet drain. - line; extending from the outer end of the chromatography column to the second distribution ring; an edge line for fluid delivery with an edge line valve that directs fluid to the outlet drain; ; is sent to the chromatography column through both the center line and the edge line. to form a liquid gradient for column elution and regenerate for column regeneration. a gradient device for forming a solution; A solution from the sample holding hole and a liquid gradient from the gradient device. an equiangular part on the inner circumference of the chromatographic rotor to direct the flow to the chromatographic column. flow distribution blades arranged in such a manner; for backflowing through the column while maintaining the pressure and flow created by centrifugal force. a valve system; the optical flow cell on one side of the chromatography rotor; a light source arranged to project a beam of light through the chromatographic flow; the light source on a side of the tar, opposite the one side, and opposite the light source; a photodetector mounted to detect light incident through the optical flow cell; Beauty The chromatography flow is carried out through the gradient device and the rotor drive unit. control the chromatographic process, control the chromatographic process, Monitor the process and real-time CRT display of all chromatography columns A microprocessor type computer. 2. The centrifugal high-speed chromatograph according to claim 1, wherein the photodetector is configured to detect light. It includes a charge multiplier tube. 3. The centrifugal high-speed chromatograph according to claim 1, wherein the center and The edge line is sloped and further includes a trap, where the slope The slanted feed line and the trap prevent the chromatography column from drying out. It is something. 4. The centrifugal high-speed chromatograph according to claim 1, wherein the optical flow The cell has a long optical flow path that increases detection sensitivity and allows for fluid mixing and Loss of resolution is prevented by centrifugal force acting on the liquid density gradient. It is something that 5. The centrifugal high-speed chromatograph according to claim 1 further comprises a distribution ring. and the gradient device is connected to each gradient feed line and and the center line and the edge line through this distribution ring respectively. each distribution ring includes a distribution blade. 6. In the recirculating high-performance chromatograph according to claim 5, the above-mentioned Load the sample to be measured and confirmed into the sample holding hole of the transport disc It includes a system for measuring and transporting samples. 7. The centrifugal high-speed chromatograph according to claim 1, wherein the center line The edge line valve and each edge line valve are interconnected and operated simultaneously. It is something that moves. 8. The centrifugal high performance chromatograph according to claim 7, wherein the interconnected Center line and edge line valves are pressure actuated valves. a pair of valves, the pair of valves comprising: a double-headed piston having first and second head portions connected by an elongated member; , wherein each said head portion has an opening through its center to allow fluid to pass therethrough. wherein the first opening provides a fluid passage from the center line to a drain; The second opening provides a fluid path from the edge line to the drain. and biasing means for biasing said piston to a rest position prior to elution. 9. The centrifugal high-speed chromatograph according to claim 8, wherein the bias means includes a spring. 10. Gradient elution of many solutions simultaneously using centrifugal high-speed chromatography The method includes the following steps: positioning the sample within a transport disk of a chromatographic rotor; positioning a gradient solution in a gradient device; From the gradient device there is a gradient feed line, a distribution ring, and a feed line. conveying the gradient through a column into a chromatographic column; Elute the sample by rotating the gradient and the chromatographic column The process of; Check the state of the solution passing through the optical flow cell placed in each of the chromatographic columns. monitoring the separated solution by an emitting photodetector and a light source; Simultaneously control all aspects of the chromatographic process for the chromatographic column. monitoring, recording, and recording. 11. The method according to claim 10, further comprising: automatically distributes the sample into multiple streams using flow distribution blades on top of the This may include a step of transporting an equal amount of the sample into the chromatography column. It is something that 12. The method according to claim 10, further comprising the following steps: Ru: Center and edge line valves direct flow outward through the chromatography column. While the inert particle suspension is set up, the bottom cone of the chromatography column is a step of introducing to fill the portion; filling the chromatographic column with active particles by centrifugal force; filling the upper conical part of the chromatography column with insoluble particles; and arranging an upper perforated stopper in each of the chromatographic columns; 13. 13. The method of claim 12, wherein the rotor prevents back-mixing. While at rest, the sample is individually passed through a self-sealing rubber section at the centrifugal end. It is injected. 14. 11. The method according to claim 10, wherein hydrostatic pressure is provided by centrifugal force. Packing the chromatography column, putting the sample into the chromatography column separating the sample during elution, and regenerating the chromatographic column. It is intended to assist with this. 15. The method according to claim 10, wherein the density gradient in the centrifugal field Elution stabilizes the flow of sample through the chromatographic column and reduces the slope and spread of bands. This prevents irregular flow including width and groove flow.