A method for separating fine particulate matter with approximate sedimentation velocity using progressively accelerated centrifugation

Abstract

This invention proposes a method for progressively accelerated centrifugation to separate fine particulate matter with approximate sedimentation velocities. The method includes: adding a sample solution to a centrifuge assembly with an open, narrow tube; accelerating the centrifuge to N1, ensuring the sample solution at least submerges the opening of the narrow tube; then running the centrifuge at a constant speed of N1 for a time T1; further accelerating to N2, causing a small amount of sample solution to overflow from the separation chamber to the collection chamber; then running the centrifuge at a constant speed of N2 for a time T2. During this constant speed operation, the backflow distance of fine particles with lower sedimentation velocities is less than the pushing distance. This progressive acceleration and constant speed operation is repeated n times to repeatedly push and overflow fine particulate matter with approximate sedimentation velocities into the collection chamber. This method for progressively accelerated centrifugation to separate fine particulate matter with approximate sedimentation velocities is simple, low-cost, and economically solves the commercial problem of separating fine particulate matter with varying but not significantly different sedimentation velocities.

Description

Technical Field

[0001] This invention belongs to the field of fine particulate matter separation and purification technology, specifically a method for progressively accelerating centrifugal separation of fine particulate matter with approximate sedimentation velocity. Background Technology

[0002] The settling velocity of particulate matter suspended in a liquid in a centrifugal field is U = Jr 2 The formula is (ρ2-ρ1)α, where U represents the settling velocity of the particles, J represents the settling coefficient (the specific value of which is related to the viscosity of the liquid and the concentration of the particles), r represents the radius of the particles, ρ2 represents the density of the particles, ρ1 represents the density of the sample liquid, and α represents the centrifugal acceleration. Different particles of different volumes and densities have different settling velocities in the same liquid. When the ratio of the settling velocities of two particles is large, they can be separated by differential centrifugation. When the ratio of the settling velocities of the two particles is small, a large settling stroke is required for separation, resulting in a very large centrifuge and making it very difficult to maintain centrifuge balance. Current conventional centrifugation techniques cannot meet this requirement. To solve this problem, technicians have increased the liquid density to expand the ratio of the settling velocities of the two particles, thus separating them—this is density gradient centrifugation. However, it is not effective for particles with similar densities but different settling velocities mainly due to volume differences. Furthermore, due to the high cost of density liquids and the complexity of the operation, density gradient centrifugation is mainly used for separating small quantities of samples in scientific research. When extracting rectal epithelial cells from stool samples, although multi-stage filtration technology can remove impurities larger than 50 μm in diameter, the filtrate still contains a large number of impurities, including food residue, E. coli, and various crystals. Impurities account for more than 90% of the formed components by mass. These impurities vary in density (0.9 g / cm3-2.4 g / cm3) and diameter (1 μm-50 μm), covering the density range (1.05 g / cm3-1.1 g / cm3) and diameter range (10 μm-20 μm) of rectal epithelial cells. Separation using density methods is time-consuming, complex, costly, and ineffective. Washing rotor centrifuges utilize the principle that centrifugal force is in the opposite direction to liquid flow to separate two types of particles with similar settling velocities, regardless of whether the difference in settling velocity is due to density or volume differences. Theoretically, they can also separate rectal epithelial cells from stool. However, washing rotor centrifuges are complex in structure, cumbersome to operate, require specialized personnel, and are expensive, resulting in very limited use and making them difficult to implement in clinical laboratories. Therefore, a more economical, convenient, and large-scale separation of cellular components from impurities remains a challenge for clinical laboratories. Summary of the Invention

[0003] This invention aims to at least solve one of the technical problems existing in the prior art. To this end, this invention proposes a method for progressively accelerated centrifugal separation of fine particulate matter with approximate sedimentation velocity, comprising the following steps:

[0004] S1, add the specimen solution containing two or more fine particulate matter into a closed centrifuge bottle with an open slender tube, install the centrifuge bottle onto the separation chamber, and keep the separation chamber open to the atmosphere. The slender tube points to the bottom of the separation chamber. Then install the separation chamber into the collection chamber, and keep the collection chamber open to the atmosphere. The upper part of the separation chamber is provided with an overflow port connected to the collection chamber. The separation chamber includes a conical part and a container connected to the conical part.

[0005] S2, accelerate the centrifuge to N1, so that the sample liquid at least submerges the opening of the slender tube, but below the overflow port, and then the centrifuge runs at a constant speed of N1 for time T1.

[0006] S3, accelerate the centrifuge to N2, the sample liquid is thrown out through the thin tube, pushing the liquid level in the separation chamber to rise, so that a small amount of sample liquid overflows from the separation chamber to the collection chamber, but no large fine particles overflow from the separation chamber to the collection chamber with the liquid. Then run at a constant speed of N2 for time T2.

[0007] S4, the centrifuge is accelerated to N3, the sample liquid is thrown out through the thin tube, pushing the liquid level in the separation chamber to rise, so that an equal amount of sample liquid overflows from the separation chamber to the collection chamber, but no larger particles overflow from the separation chamber to the collection chamber with the liquid; then the centrifuge is run at a constant speed for T3 time; the constant speed operation for T3 time makes the back movement distance b3 of fine particles with small settling velocity less than the liquid column height a3 above the maximum cross section of the cone of the separation chamber, and the back movement distance c3 of particles with large settling velocity greater than or equal to the liquid column height a3;

[0008] S5, continue accelerating and running the centrifuge at a constant speed for n times (n≥4), until the centrifuge is accelerated to N. n Then with N n Running at T speed n Time, constant speed T n Time causes particles with smaller settling velocities to travel a distance b back. n The distance a of the liquid column in the specimen was less than the height of the column. n .

[0009] Furthermore, in step S2, the uniform running time T1 causes particles with higher settling velocity to move back to the bottom of the separation chamber by a distance greater than the distance L between the maximum cross-section of the cone-shaped part of the separation chamber and the overflow outlet.

[0010] Furthermore, the uniform running time T2 in step S3 causes particles with higher settling velocity to move back to the bottom of the separation chamber by a distance greater than the distance L between the maximum cross-section of the cone-shaped part of the separation chamber and the overflow outlet.

[0011] Furthermore, uniform speed T n Time causes particles with higher settling velocities to travel a greater distance c. n Greater than or equal to the column rise distance a of the specimen liquid n (where n≥4).

[0012] Furthermore, the centrifuge speed N1 is determined according to the following formula:

[0013]

[0014] V0 represents the volume of air inside the centrifuge bottle after the sample solution has been added and the bottle is sealed.

[0015] V f This indicates the volume of sample solution that flowed out of the centrifuge bottle before the outlet end of the slender tube was submerged in the sample solution, i.e., the sealing volume.

[0016] V1 represents the volume of the sample fluid ejected during the first acceleration.

[0017] K represents the eccentricity coefficient, calculated according to the following formula:

[0018]

[0019] Where R is the real-time centrifugation radius, H is the real-time liquid level in the centrifuge bottle, ρ1 is the sample liquid density, and P represents atmospheric pressure.

[0020] Centrifuge speed N2 is determined according to the following formula:

[0021]

[0022] Furthermore, the centrifuge speed N3 is determined according to the following formula:

[0023]

[0024] Furthermore, the centrifuge speed N n Determined according to the following formula,

[0025]

[0026] Furthermore, after the third acceleration, the volume of the ejected sample fluid was always equal to V3, and the propulsion distance was equal for each acceleration. n = a3, where n≥4, centrifuge speed N n Determined according to the following formula:

[0027]

[0028] In the formula, V2 represents the volume of the sample liquid ejected during the second acceleration, and V3 represents the volume of liquid required to generate a thrust distance a above the maximum cross-section, i.e., the volume of the liquid column segment of length a, where a can take any value in the range of 0.1-Lmm.

[0029] Furthermore, in order to ensure that the ratio of "push distance / return distance" of a specific particulate matter in the separation chamber is equal in each "push-return" stage, then T n =a n N3 2 T3 / N n 2 a3, where a n ≤L;

[0030] Furthermore, if the return distance of a specific particulate matter in the separation chamber is equal during uniform motion at each stage, then T n =N3 2 T3 / N n 2 .

[0031] The progressively accelerated centrifugation method of this invention for separating fine particulate matter with approximate sedimentation velocities allows the sample liquid in the centrifuge bottle to enter the separation chamber in an orderly manner, gradually pushing various particles in the separation chamber towards the outlet. Utilizing the centrifugal force exerted on the particles in the liquid during uniform rotation, pointing towards the bottom of the separation chamber, the particles settle towards the bottom. Particles with higher sedimentation velocities move a greater distance in the same time interval, while particles with lower sedimentation velocities move a smaller distance in the same time interval. After multiple pushes, the aggregation range of particles with different sedimentation velocities is expanded, and finally, as the liquid level rises, the particles overflow the separation chamber and reach the collection chamber. This progressively accelerated centrifugation method for separating fine particulate matter with approximate sedimentation velocities is simple to operate. Compared with the washing rotor centrifuge used in the washing rotor centrifugation method, the equipment used is extremely simple and low-cost. Compared with the gradient density method, it does not require a density solution as a cell suspension medium, thus maintaining good biological activity. Compared with ordinary centrifugation methods, it does not require an excessively long stroke, and its separation effect is particularly good, making it suitable for widespread application. Attached Figure Description

[0032] Figure 1 This is a schematic diagram of the apparatus used in the present invention for a method of progressively accelerating centrifugal separation of fine particulate matter with approximate sedimentation velocity;

[0033] Figure 2 V is the sealed volume of the specimen solution. f Schematic diagram;

[0034] Figure 3 The effective volume V of the separation chamber c Schematic diagram;

[0035] Figure 4 V1 represents the volume of the sample liquid that was ejected.

[0036] Figure 5 This is a schematic diagram of the volume V3 of the booster unit;

[0037] Figure 6 This is a schematic diagram showing the distance from the maximum cross-section of the conical part of the separation chamber to the overflow outlet;

[0038] Figure 7 This is a schematic diagram showing the state of a centrifuge bottle after sample solution has been added.

[0039] Among them, 1. Centrifuge shaft; 2. Centrifuge bottle; 21. Sealing cap; 22. Slender tube; 23. Bottom of centrifuge bottle; 3. Separation chamber; 31. Conical section; 32. Cylindrical section; 33. Overflow port; 34. Outer edge platform; 35. Bottom of separation chamber; 4. Collection chamber. Detailed Implementation

[0040] Embodiments of the present invention are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0041] In the description of this invention, it should be understood that the terms "center", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this invention.

[0042] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "installation," "connection," and "joining" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal communication between two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0043] This embodiment is an invention made for fine particulate matter, especially fine particulate matter at the cellular level. The terms "some difference", "relatively large" and "relatively small" used in this document are also for fine particles such as those in cellular science, and are not mechanical or everyday terms.

[0044] In the description of this invention, "settlement velocity ratio" refers to the ratio of faster settlement velocity to slower settlement velocity. A ratio much greater than "1" is called a larger ratio, and a ratio close to "1" is called a smaller ratio.

[0045] like Figure 1 The diagram shows a device for progressively accelerating centrifugal separation of fine particulate matter with approximate sedimentation velocity, comprising a centrifuge, a centrifuge bottle 2, a separation chamber 3, and a collection chamber 4. The centrifuge is equipped with a centrifuge shaft 1; one end of the centrifuge bottle 2 is equipped with a sealing cap 21, and the other end is the bottom 23 of the centrifuge bottle, with a slender tube 22 communicating with the inner cavity of the centrifuge bottle on the bottom 23; the separation chamber 3 includes a connected upper section and a lower section, the upper section being a cylindrical section 32 and the lower section being a conical section 31, the cylindrical section 32 being connected to the large end of the conical section 31, the other end of the cylindrical section 32 being an open end, and the small end of the conical section 31 being a closed separation chamber bottom 35, which can be either pointed or flat; an outer edge platform 34 is provided at the opening of the cylindrical section 32. Several overflow ports 33 are provided near the opening of the separation chamber 3, connecting the inside and outside of the separation chamber 3. The overflow ports 33 can be through holes or pipes opened on the steps between the outer edge platform and the cylindrical section, or openings or pipes opened on the pipe wall near the opening of the cylindrical section. The collection chamber 4 is an open container. The outer edge platform 34 of the separation chamber 3 is installed at the open end of the collection chamber 4, and the bottom 35 of the separation chamber faces the collection chamber 4. The outer edge platform 34 of the separation chamber is connected to the bottom 23 of the centrifuge bottle. The slender tube 22 of the centrifuge bottle is inserted into the separation chamber and points to the bottom 35 of the separation chamber, but the end of the slender tube 22 of the centrifuge bottle does not contact the bottom 35 of the separation chamber. The centrifuge bottle 2, separation chamber 3, and collection chamber 4 are combined into a centrifuge kit and fixed in a basket on the centrifuge shaft 1, rotating with the centrifuge shaft 1. This device is mainly used for centrifugal separation and collection of biological cells, especially in commercial applications such as extracting rectal epithelial cells from feces, separating lymphocytes from blood, separating cells from pleural and peritoneal fluid, separating primary cells, and sperm selection.

[0046] This example uses the separation of rectal epithelial cells from feces as an example:

[0047] The steps for separating rectal epithelial cells from feces using the aforementioned progressively accelerated centrifugal separation device that separates fine particles at approximately sedimentation velocities are as follows:

[0048] Mix 3-5g of fecal sample with 20-25ml of preservation solution to obtain the stock solution. Filter the stock solution by shaking to obtain the sample solution. Seal the outlet of the slender tube 22 of centrifuge bottle 2, inject the sample solution into centrifuge bottle 2, and seal the mouth of the centrifuge bottle with the sealing cap 21. The centrifuge bottle used in this experiment has a capacity of 42ml, a slender tube length of 30mm, an inner diameter of 2.2mm at the open end, and 20ml of sample solution is added to the centrifuge bottle. Feces contain rectal epithelial cells and impurities. Relatively speaking, rectal epithelial cells settle at a slower rate, while larger and denser impurities settle at a faster rate. E. coli and smaller impurities settle at an even slower rate. It also contains impurities with a sedimentation rate very similar to that of rectal epithelial cells.

[0049] Install the separation chamber 3 onto the collection chamber 4, remove the seal from the opening of the centrifuge bottle's slender tube 22, and insert the centrifuge bottle's slender tube 22 into the separation chamber 3. The effective volume V of the separation chamber is then determined. C 2.6ml (e.g.) Figure 3 As shown), the bottom of the centrifuge bottle is placed on the outer edge platform 34 of the separation chamber 3, and then the centrifuge bottle 2, separation chamber 3, and collection chamber 4 are combined into a centrifuge kit and fixed on the centrifuge shaft 1.

[0050] Start the centrifuge and accelerate it to speed N1, causing the sample solution in the centrifuge bottle to be ejected into the separation chamber at a speed of V1 (e.g., Figure 4 As shown), V1 is slightly smaller than the effective volume V of the separation chamber. C In this experiment, V1 = 2.4 ml, V c =2.6ml, then run at a constant speed of N1 for time T1.

[0051] Centrifuge speed N1 is determined according to the following formula:

[0052]

[0053] N represents the centrifuge speed; correspondingly, N1 represents the centrifuge speed after the first acceleration, and N2 represents the centrifuge speed after the second acceleration. n This represents the rotational speed of the centrifuge after the nth acceleration.

[0054] V0 represents the volume of air inside the centrifuge bottle after the sample solution has been added and the bottle is sealed. Figure 7 As shown;

[0055] V represents the volume of the sample fluid ejected during the first acceleration, and V2 represents the volume of the sample fluid ejected during the second acceleration. n This represents the volume of the sample liquid ejected during the nth acceleration.

[0056] V fThis indicates the volume of sample solution that flowed out of the centrifuge bottle before the outlet end of the slender tube was submerged in the sample solution, i.e., the sealing volume (e.g. Figure 2 (As shown), K represents the eccentricity coefficient, calculated according to the following formula:

[0057]

[0058] Where R is the real-time centrifugation radius, H is the real-time liquid level in the centrifuge bottle, ρ1 is the sample liquid density, and P represents atmospheric pressure.

[0059] T represents the time the centrifuge operates at a constant speed. Correspondingly, T1 represents the time the centrifuge operates at a constant speed after its first acceleration, and T2 represents the time the centrifuge operates at a constant speed after its second acceleration. n This represents the time it takes for the centrifuge to maintain a constant speed after the nth acceleration. During the constant speed operation of the centrifuge, cells and impurities in the separation chamber will migrate back. The migration distance of the cells is b. The migration distance of the cells during the first constant speed operation is b1, the migration distance of the cells during the second constant speed operation is b2, and the migration distance of the cells during the nth constant speed operation is b... n The distance the impurities with larger settling velocities return is c. The distance the impurities return during the first uniform speed run of the centrifuge is c1. The distance the impurities return during the second uniform speed run of the centrifuge is c2. The distance the impurities return during the nth uniform speed run of the centrifuge is c... n The time T1 for uniform running after the first acceleration should ensure that particles with higher settling velocities settle below the maximum cross-section of the cone-shaped part of the separation chamber. This ensures that when the sample solution enters the separation chamber during the second acceleration, no particles with higher settling velocities will overflow from the separation chamber into the collection chamber. The time T1 can be determined by taking the maximum value based on past experience or by using a trial-and-error method. The uniform running time T1 ensures that impurities with higher settling velocities move back a distance greater than L to the bottom of the separation chamber, where L represents the distance between the maximum cross-section of the cone-shaped part of the separation chamber and the overflow outlet (e.g., ...). Figure 6 (As shown).

[0060] The second acceleration involves speeding the centrifuge to speed N2, causing the sample solution in the centrifuge bottle to be ejected at V2, with a small amount overflowing from the separation chamber into the collection chamber. The centrifuge is then run at a constant speed for time T2. Time T2 should ensure that when the sample solution enters the separation chamber for the third time, no impurities with high settling velocities overflow from the separation chamber into the collection chamber. The maximum value for T2 can be determined based on past experience, or it can be obtained through trial and error. The centrifuge speed N2 is determined using the following formula.

[0061]

[0062] The third acceleration involves accelerating the centrifuge to speed N3, causing the sample solution in the centrifuge bottle to be ejected from V3, and ensuring that an equal amount of liquid overflows from the separation chamber to the collection chamber from the separation chamber. Then, the centrifuge is run at a constant speed for time T3. The constant speed running for time T3 ensures that the back migration distance b3 of cells with lower sedimentation velocity (rectal epithelial cells) is less than the sample solution column rise distance a3 above the maximum cross-section of the cone of the separation chamber, and the back migration distance c3 of impurities with higher sedimentation velocity is greater than or equal to the sample solution column rise distance a3.

[0063] 'a' represents the thrust distance, which is the height of the liquid column above the maximum cross-section of the cone-shaped part of the separation chamber, corresponding to the volume of the ejected sample fluid. Figure 5 As shown, correspondingly, the propulsion distance during the second acceleration of the centrifuge is a2, and the propulsion distance during the nth acceleration of the centrifuge is a. n When the pushing distance is *a* mm, the liquid at the maximum cross-section of the cone in the separation chamber is propelled *a* mm towards the outlet. Cells and impurities originally present at this location are also propelled *a* mm towards the outlet. The centrifuge speed *N*3 is determined according to the following formula:

[0064]

[0065] Then the centrifuge is accelerated—at a constant speed—accelerated—at a constant speed…accelerated to N. n After undergoing n acceleration cycles (n≥4 in this embodiment), the centrifuge finally stops running and operates at a constant speed for T cycles. n Time causes cells with lower sedimentation rates (rectal epithelial cells) to migrate back a distance of b. n The distance a of the liquid column in the specimen was less than the height of the column. n The distance c that impurities with larger settling velocities travel back to their original location. n Greater than or equal to the column rise distance a of the specimen liquid n Ultimately, the fecal impurities with higher settling rates in the sample solution are retained in the separation chamber, while the rectal epithelial cells with lower settling rates are pushed up and separated into the collection chamber through the overflow outlet. Centrifuge speed N n Determined according to the following formula,

[0066]

[0067] Although we understand the settling velocity patterns of particulate matter, calculating the actual settling velocity of various particulate matter is very complex. In experiments, we often use a trial-and-error method to assess the required settling time for particulate matter. After liquid overflows from the separation chamber, i.e., after the third acceleration, regardless of whether the thrust distances in each stage are equal, as long as T... n =a n N3 2 T3 / N n 2By setting T3, the "push-back" rates of various particles can be made equal. In other words, the separation effect achieved in each "acceleration-uniformity" cycle is consistent. Thus, it is only necessary to continuously adjust T3 by observing the experimental results and calculate T using T3. n This will allow us to achieve a more ideal separation effect.

[0068] In this embodiment, for ease of control, the volume of the sample liquid ejected after the third acceleration is always equal to V3, and the thrust distance is equal for each acceleration. n = a3, where n≥4, centrifuge speed N n Determined according to the following formula:

[0069]

[0070] In the formula, N n V3 represents the rotational speed after the nth acceleration (where n≥4), and V3 represents the amount of fluid required to generate a thrust distance a above the maximum cross-section (e.g., ...). Figure 5 As shown in the figure, this is the volume of the liquid column segment of length a, where a can take any value in the range of 0.1-L mm.

[0071] To ensure that the return distance of a specific particle in the separation chamber is equal during each stage of uniform motion, then T n =N3 2 T3 / N n 2 .

[0072] Its working principle is as follows: After the centrifuge starts running, the sample solution is quickly thrown into the separation chamber through a thin tube, causing the liquid level in the separation chamber to gradually rise and first submerge the end of the thin tube. The sample solution in the centrifuge bottle gradually decreases, and the air volume in the centrifuge bottle continuously increases, gradually forming a negative pressure. When the centrifuge gradually accelerates to N1, the sample solution flowing out of the centrifuge bottle reaches the vicinity of the separation chamber outlet. Then, it runs at a constant speed of N1 for T1 seconds. The centrifugal force no longer increases, and the force of the negative pressure in the centrifuge bottle acting on the sample solution in the bottle is balanced with the centrifugal force on the sample solution. The sample solution in the centrifuge bottle no longer flows out. The cells suspended in the sample solution in the separation chamber move back to the bottom of the separation chamber under the action of centrifugal force. T1 should be appropriately increased compared to T2 and T3 so that all the impurities in the liquid in the upper part of the separation chamber move back to below the maximum cross section of the cone part of the separation chamber.

[0073] The centrifuge is accelerated to N2, and V2 sample liquid enters the separation chamber, causing a small amount of liquid to overflow from the separation chamber. Then, it is run at a constant speed of N2 for time T2. T2 should be appropriately increased so that all impurities in the liquid in the upper part of the separation chamber are moved back below the maximum cross section.

[0074] The centrifuge accelerates to N3, and V3 of the sample solution enters the separation chamber, pushing the liquid level at the maximum cross-section up by a3. Then, it runs at a constant speed of N3 for time T3. The rectal epithelial cells, with lower density and volume, migrate back a distance b3 less than a3, while impurities with higher density and volume migrate back a distance c3 greater than a3. The centrifuge continues to accelerate. Since the separation chamber is now completely filled with liquid, an equal amount of sample solution entering will result in an equal amount of liquid being expelled. In the subsequent n accelerations, the pushing distance is a. n The specimen fluid near the overflow port of the separation chamber is continuously pushed into the collection chamber by the ejected specimen fluid. This part of the specimen fluid flowing into the collection chamber consists of the uppermost preservation fluid, cells, and impurities with a diameter smaller than the cells. The impurities with a higher settling velocity have a return distance c greater than the pushing distance a and are thus permanently confined below the maximum cross-section of the cone of the separation chamber, and will never be pushed out of the separation chamber. The rectal epithelial cells with a lower settling velocity have a return distance less than the pushing distance. During each accelerated and uniform cycle, the rectal epithelial cells rise a certain distance in the separation chamber and are eventually pushed out of the separation chamber.

[0075] When the centrifuge uses N n When the rotation speed is constant, the velocity U of the particles in the centrifugal force field is directly proportional to the magnitude of the centrifugal force, and the velocity U = Jr 2 The sedimentation mobility coefficient J (ρ2-ρ1)α is related to the liquid viscosity and particle density. Since all particles are in the same liquid system, the J value is equal for all particles. The difference in velocity is determined by their volume and density. Particles with larger volume and density move faster, while those with smaller volume and density move slower. The relocation distance is equal to the product of the relocation velocity and the relocation time. n Within a given time, particles with different settling velocities travel different distances, thus separating particles with different settling velocities. This process can separate most impurities, resulting in a sample solution with fewer impurities.

[0076] In this embodiment, V0 = 22 ml, V1 = 2.4 ml, V f =0.1ml, R=11.6cm, H=6.4cm, ρ1=1.015, N1=340 rpm, T1=200 seconds. When the rotation speed reaches N1, the sample liquid ejected from the centrifuge bottle just reaches the vicinity below the overflow port.

[0077] N2 = 372 revolutions per minute, T2 = 100 seconds. When the rotation speed reaches N2, a small amount of liquid flows out from the overflow port.

[0078] N3 = 388 rpm, T3 = 52 seconds. When the rotation speed reaches N3, the volume of the push unit V3 = 0.25 ml, the push distance a = 1 mm, and the epithelial cell retraction distance is less than 0.9 mm.

[0079] In this embodiment, for ease of control, the volume of liquid ejected each time after N3 is equal, and the distance 'a' that the particles at the largest cross-section in the separation chamber are pushed up by each acceleration of the centrifuge is equal.

[0080] After 10-20 cycles of pushing and returning, rectal epithelial cells, E. coli, and particles with smaller diameters and densities are gradually pushed to the outlet of the separation chamber and eventually overflow into the collection chamber, while particles with larger diameters and densities are retained in the separation chamber.

[0081] After the above separation process is completed, the separation liquid in the collection chamber is subjected to a second separation experiment in the same way. This allows the rectal epithelial cells to remain in the separation chamber, while E. coli and smaller particles are separated into the collection chamber. Through two separations, we remove most of the impurities. Only those impurities with a sedimentation rate that is basically the same as that of the rectal epithelial cells are mixed in the separated specimen, which meets the requirements for observation and diagnosis.

[0082] Based on the same technical principles, we can also separate single-cell components from blood, separate target cells from primary cell culture media, separate specific cells from pleural and peritoneal fluid, remove leukocytes and dead cells from semen, and separate various particulate matter with different sedimentation rates.

[0083] Although embodiments of the invention have been shown and described, those skilled in the art will understand that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.

Claims

1. A method for progressively accelerating centrifugal separation of fine particulate matter with approximate sedimentation velocity, comprising the following steps: S1, a sample solution containing two or more fine particulate matter with similar settling velocities is added to a closed centrifuge bottle with an open, thin tube. The centrifuge bottle is then installed on the separation chamber, ensuring that the separation chamber is open to the atmosphere. The thin tube points to the bottom of the separation chamber. The separation chamber is then installed in the collection chamber, ensuring that the collection chamber is open to the atmosphere. The upper part of the separation chamber is provided with an overflow port that connects to the collection chamber. The separation chamber includes a conical part and a container connected to the conical part. S2, accelerate the centrifuge to N1, so that the sample liquid at least submerges the opening of the slender tube, but below the overflow port, and then the centrifuge runs at a constant speed of N1 for time T1. S3, the centrifuge is accelerated to N2, the sample liquid is thrown out through the thin tube, pushing the liquid level in the separation chamber to rise, so that a small amount of sample liquid overflows from the separation chamber to the collection chamber, but no large-diameter fine particles overflow from the separation chamber to the collection chamber with the liquid, and then runs at a constant speed of N2 for time T2. S4, the centrifuge is accelerated to N3, the sample liquid is thrown out through the thin tube, pushing the liquid level in the separation chamber to rise, so that an equal amount of sample liquid overflows from the separation chamber to the collection chamber, but no large-diameter particles overflow from the separation chamber to the collection chamber with the liquid; then run at a constant speed for T3 time; running at a constant speed for T3 time makes the back movement distance b3 of fine particles with small settling velocity less than the liquid column height a3 above the maximum cross section of the cone of the separation chamber, and the back movement distance c3 of particles with large settling velocity greater than or equal to the liquid column height a3; S5, continue to accelerate and run the centrifuge at a constant speed for a total of n times, n≥4, until the centrifuge is accelerated to Nn, and then run at a speed of Nn for Tn time. When running at a constant speed for Tn time, the distance bn of the particles with smaller sedimentation velocity is less than the distance an of the sample liquid column is pushed up.

2. The method for progressively accelerating centrifugal separation of fine particulate matter with approximate sedimentation velocity according to claim 1, characterized in that, In step S2, the uniform running time T1 causes particles with higher settling velocity to move back to the bottom of the separation chamber by a distance greater than the distance L between the maximum cross-section of the cone-shaped part of the separation chamber and the overflow outlet.

3. The method for progressively accelerating centrifugal separation of fine particulate matter with approximate sedimentation velocity according to claim 1, characterized in that, In step S3, the uniform running time T2 ensures that particles with higher settling velocity move back to the bottom of the separation chamber by a distance greater than the distance L between the maximum cross-section of the cone-shaped part of the separation chamber and the overflow outlet.

4. The method for progressively accelerating centrifugal separation of fine particulate matter with approximate sedimentation velocity according to claim 1, characterized in that, When running at a constant speed for a time Tn, the distance cn that the particles with larger settling velocities move back is greater than or equal to the distance an that the liquid column of the specimen is pushed up, where n≥4.

5. The method for progressively accelerating centrifugal separation of fine particulate matter with approximate sedimentation velocity according to claim 1, characterized in that, Centrifuge speed N1 is determined according to the following formula: V0 represents the volume of air inside the centrifuge bottle after the sample solution has been added and the bottle is sealed. Vf represents the volume of sample solution that flowed out of the centrifuge bottle before the outlet end of the slender tube was submerged in the sample solution, i.e., the sealed volume; V1 represents the volume of sample solution ejected during the first acceleration. K represents the eccentricity coefficient, calculated according to the following formula: Where R is the real-time centrifugation radius, H is the real-time liquid level in the centrifuge bottle, ρ1 is the density of the sample liquid, and P represents atmospheric pressure.

6. The method for progressively accelerating centrifugal separation of fine particulate matter with approximate sedimentation velocity according to claim 1, characterized in that, Centrifuge speed N2 is determined according to the following formula: 。 7. The method for progressively accelerating centrifugal separation of fine particulate matter with approximate sedimentation velocity according to claim 1, characterized in that, Centrifuge speed N3 is determined according to the following formula: 。 8. The method for progressively accelerating centrifugal separation of fine particulate matter with approximate sedimentation velocity according to claim 1, characterized in that, The centrifuge rotational speed Nn is determined according to the following formula: 。 9. The method for progressively accelerating centrifugal separation of fine particulate matter with approximate sedimentation velocity according to claim 1, characterized in that, After the third acceleration, the volume of the ejected sample liquid is equal to V3, and the propulsion distance is equal in each acceleration, so an = a3. Where n≥4, the centrifuge speed Nn is determined according to the following formula: In the formula, V2 represents the volume of the sample liquid ejected during the second acceleration, and V3 represents the volume of liquid required to generate a thrust distance a above the maximum cross-section, i.e., the volume of the liquid column segment of length a, where a can take any value in the range of 0.1-Lmm.

10. The method for progressively accelerating centrifugal separation of fine particulate matter with approximate sedimentation velocity according to claim 1, characterized in that, To ensure that the ratio of "lift distance / return distance" is equal for a specific particulate matter in the separation chamber during each "lift-return" stage, then Tn = a n N3 2 T3 / N n 2 a3, where a n ≤L.

11. The method for progressively accelerating centrifugal separation of fine particulate matter with approximate sedimentation velocity according to claim 9, characterized in that, In the separation chamber, the return distance of a specific particulate matter is equal during uniform motion at each stage, Tn = N3. 2 T3 / N n 2 .

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