Nondestructive measurement, assignment and replication of density gradients

Abstract

A system performs non-destructive measurements of density gradients to automatically replicate and distribute density gradients. The system obtains measurements at points along the length of the density gradient and generates a distribution of the density gradient based on the measurements. The system uses the distribution to replicate a density gradient of the component in the second container. The system inserts the distal end of the probe into a second container and pumps each individual component into a manifold and a mixing chamber connected to the proximal end of the probe to automatically dispense a density gradient in the second container.

Description

[0001] This application was filed as a PCT international patent application on July 21, 2023, and claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 369,299 filed on July 25, 2022, U.S. Provisional Patent Application No. 63 / 375,558 filed on September 14, 2022, U.S. Provisional Patent Application No. 63 / 369,306 filed on July 25, 2022, U.S. Provisional Patent Application No. 63 / 375,563 filed on September 14, 2022, U.S. Provisional Patent Application No. 63 / 369,318 filed on July 25, 2022, and U.S. Provisional Patent Application No. 63 / 375,564 filed on September 14, 2022, and the entire disclosures of the above U.S. Provisional Patent Applications are incorporated herein by reference in their entirety. Background Art

[0002] Proteins have a variety of cellular functions, structures, and mechanisms of action. Often, proteins bind to other biomolecules called ligands to accomplish their tasks. By purifying proteins bound to ligands (also called protein-ligand complexes), researchers can gain valuable knowledge about how proteins work in the cellular environment. For example, purified protein-ligand complexes are often used in downstream analyses such as high-resolution imaging, sequencing, or crystallography for the discovery of protein-based therapeutics.

[0003] Density gradients can be used to purify protein-ligand complexes, which are usually layered using an underlay or overlay method, wherein solutions are added in an order of gradually increasing or decreasing density. Step gradients can be used directly or allowed to diffuse in a controlled manner to produce a continuous or linear gradient. Step gradients have sharp interfaces between layers with different densities, while continuous or linear gradients have layers with increasing density from top to bottom. In some cases, the step gradient is rotated in a centrifuge, which diffuses the layers into a continuous or linear gradient.

[0004] The sinking technique for generating density gradients involves adding a first layer of low density to a tube, and then successively adding denser layers to the bottom of the tube via a long syringe so as not to disturb the previous layers. The overlaying technique for generating density gradients involves sequentially dispensing layers of decreasing density on top of the denser layers using a pipette. These layering techniques for generating density gradients can be tedious and time consuming, and difficult to replicate among researchers. Summary of the invention

[0005] In general, the present disclosure relates to a system for measuring and distributing a density gradient of a component. In one configuration, the system measures the density gradient without contacting or interfering with the density gradient. In another configuration, the system automatically distributes a density gradient of a component with multiple steps using a probe. In another configuration, the system measures the density gradient of a component distributed in a first container to create a distribution of the density gradient, and uses the distribution to replicate the density gradient of the component in a second container. Various aspects are described in the present disclosure, including but not limited to the following aspects.

[0006] One aspect relates to a system for non-destructively measuring a density gradient of a component used in centrifugation, the system comprising: a measuring device comprising: a sensor assembly; a motor coupled to the sensor assembly; and a processing circuit having a memory for storing instructions that, when executed by the processing circuit, cause the processing circuit to: move the sensor assembly along a length of the density gradient of the component using the motor; obtain measurements from the sensor assembly as the sensor assembly moves along the length of the density gradient of the component; and generate a distribution of the density gradient of the component based on the measurements.

[0007] Another aspect relates to a system for measuring the density gradient of a component dispensed in a container for use in centrifugation, the system comprising: a processing circuit having a memory for storing instructions which, when executed by the processing circuit, causes the processing circuit to: obtain measurements at points along the length of the density gradient of the component; generate a distribution of the density gradient of the component based on the measurements; and store the distribution of the density gradient of the component.

[0008] Another aspect relates to a method for non-destructively measuring a density gradient of a component, the method comprising: obtaining measurements at points along the length of the density gradient of the component; generating a distribution of the density gradient of the component based on the measurements; and storing the distribution of the density gradient of the component.

[0009] Another aspect relates to a system for automatically dispensing a density gradient of components for use in centrifugation, the system comprising: a processing circuit having a memory for storing instructions that, when executed by the processing circuit, cause the processing circuit to: insert a distal end of a probe into a container; pump each individual component into a mixing chamber connected to a proximal end of the probe, the mixing chamber generating a mixture of the individual components; dispense a plurality of steps into the container, each of the plurality of steps having a density based on a relative concentration of the individual components in the mixture generated by the mixing chamber, and each of the plurality of steps pushing a previously dispensed step away from the distal end of the probe; and remove the probe from the container without disturbing the plurality of steps.

[0010] Another aspect relates to a system for dispensing a density gradient of components for use in centrifugation, the system comprising: a processing circuit having a memory for storing instructions, which, when executed by the processing circuit, causes the processing circuit to: insert a distal end of a probe into a container; dispense a first step of a plurality of steps into the container, the first step being dispensed at a maximum dispensing speed; dispense additional steps of the plurality of steps into the container, each additional step initially being dispensed at a minimum dispensing speed and then increasing from the minimum dispensing speed to the maximum dispensing speed, each additional step of the plurality of steps having a density greater than a density of a previously dispensed step of the plurality of steps, such that the previously dispensed step of the plurality of steps moves away from the distal end of the probe; and remove the probe from the container without disturbing the plurality of steps.

[0011] Another aspect relates to a method for automatically dispensing a density gradient of components for use in centrifugation, the method comprising: inserting a distal end of a probe into a container; dispensing a first step of a plurality of steps into the container, the first step being dispensed at a maximum dispensing speed; dispensing additional steps of the plurality of steps into the container, each additional step initially being dispensed at a minimum dispensing speed and then increasing from the minimum dispensing speed to a maximum dispensing speed, each additional step of the plurality of steps having a density higher than a density of a previously dispensed step of the plurality of steps, such that the previously dispensed step moves away from the distal end of the probe; and removing the probe from the container without disturbing the plurality of steps.

[0012] Another aspect relates to a system for replicating a density gradient, the system comprising: at least one processing device; and a memory device storing instructions, which when executed by the at least one processing device causes the at least one processing device to: obtain measurements of a density gradient distributed in a first tube; store a distribution of the measurements; and replicate the density gradient in a second tube using the distribution.

[0013] On the other hand, it relates to a method for replicating a density gradient, the method comprising: obtaining measurements of a density gradient distributed in a first tube; replacing measurements at interfaces between steps of the density gradient; replacing measurements from a bottom portion of the first tube; removing measurements from a position of a meniscus of the density gradient; storing a distribution of the density gradient; and replicating the density gradient in a second tube based on the distribution.

[0014] Another aspect relates to a system for replicating a density gradient, the system comprising: at least one processing device; and a memory device storing instructions that, when executed by the at least one processing device, cause the at least one processing device to: obtain measurements of a density gradient distributed in a first tube; process the measurements by: replacing measurements at interfaces between steps of the density gradient; replacing measurements from locations in a bottom portion of the first tube; and removing measurements from locations of a meniscus of the density gradient distributed in the first tube; storing a distribution of the density gradient based on the processed measurements; and replicating the density gradient in a second tube using the distribution.

[0015] On the other hand, a method for replicating a density gradient of a component is provided, the method comprising: creating a first distribution by obtaining measured values ​​of a density gradient of a component distributed in a first container; creating a second distribution by replacing the measured values ​​of the first distribution; storing the second distribution; and replicating the density gradient of the component in a second container based on the second distribution.

[0016] On the other hand, it relates to a system for replicating a density gradient of components used in centrifugation, the system comprising: a first density gradient of components; a sensor assembly; a dispensing probe; a processing circuit having a memory for storing instructions, which, when executed by the processing circuit, causes the processing circuit to: obtain measured values ​​of a first density gradient of components contained in a first container using the sensor assembly; store a first distribution of the measured values ​​in the memory; create a second distribution based on the stored first distribution; and replicate the first density gradient of the components by dispensing a second density gradient of the components based on the second distribution into a second container using the dispensing probe.

[0017] Another aspect relates to a system for replicating a density gradient of a component used in centrifugation, the system comprising: a processing circuit having a memory for storing instructions which, when executed by the processing circuit, cause the processing circuit to: obtain measurements of a density gradient of a component dispensed in a first container, the density gradient of the component including a meniscus; process the measurements by: replacing measurements at interfaces between steps of the density gradient of the component; replacing measurements from a position in a bottom portion of the first container; and replacing measurements from a position based on a meniscus of the density gradient of the component dispensed in the first container; storing a distribution of the density gradient of the component based on the processed measurements; and using the distribution to replicate the density gradient of the component in a second container. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The following drawings, which form a part of this application, are illustrative of the described technology and are not meant to limit the scope of the present disclosure in any way.

[0019] Figure 1An example of a system for generating a density gradient for centrifugation is schematically shown.

[0020] Figure 2 Shown is housed in Figure 1 Example of a system with a manifold and a mixer within a mixing chamber.

[0021] Figure 3 yes Figure 1 An isometric view of a probe of the system having a distal end inserted into a container and a proximal end connected to a manifold and a mixing chamber.

[0022] Figure 4 Shown is the connection to the manifold and mixing chamber Figure 3 Example of the proximal end of the probe.

[0023] Figure 5 Schematically shows the use of Figure 1 An example of a method for generating a density gradient within a container by a system.

[0024] Figure 6 Schematically shows the completed Figure 5 Example of a density gradient formed inside a container following the method.

[0025] Figure 7 It is used to measure density gradient Figure 1 Isometric view of the measuring equipment in the system.

[0026] Figure 8 yes Figure 7 Detailed isometric view of measuring equipment.

[0027] Fig. 9 yes Figure 7 Front view of the measuring device.

[0028] Fig.10 Schematic diagram showing the installation Figure 7 Example of the electrical configuration of the sensor assembly on the measuring device.

[0029] Fig.11 Schematically shows the measurement distribution in Figure 6 An example of the method of density gradient in a container.

[0030] Fig.12 Schematically shows the measurement distribution in Figure 6 Another example of the method of a density gradient in a container.

[0031] Fig.13 The diagram shows Fig.11 or Fig.12 Example of a distribution of density gradients generated by the method.

[0032] Fig.14 Identification Figure 6 Example of a graph of voltage measurements of a vessel and density gradient characteristics.

[0033] Fig.15 Illustration of the method for identifying the distribution Figure 6 Example of a graph of voltage measurements of the position of the meniscus of the sample liquid in the container.

[0034] Fig.16 The diagram shows the distribution from Figure 6 A magnified view of a graph of the first derivative of the voltage measurement obtained for the sample liquid in the container.

[0035] Fig.17 Schematically shows the distribution of Figure 6 Example of a method to standardize measurement results by type of density gradient container.

[0036] Fig.18 Shown is shown in the completion Fig.17 Example of a graph of normalized measurement results following the method.

[0037] Fig.19 Shown is shown in the completion Fig.17 Another example of a graph of normalized measurement results following the method.

[0038] Fig. 20 Schematically shows the mitigation along Figure 6 Examples of the method of the effects of defects and wall thickness variations on the length of a container.

[0039] Fig.21 The diagram shows Fig. 20 The operation of the method Figure 6 An example of a graph of voltage measurements from a container when the container is empty.

[0040] Fig. 22 The diagram shows the completed Fig. 20 An example of a graph of mitigated container defects and / or container wall thickness variations based on DI water samples following the method.

[0041] Fig.23 shows the relative installation Figures 7 to 9 Example of a measuring device where the vertical axis of the sensor assembly is offset from the centerline of the container.

[0042] Fig.24 Schematically illustrates the mitigation from Fig.23 An example of a method for obtaining density gradient measurements using mechanical positioning errors on a vessel.

[0043] Fig.25 The diagram shows Fig.23 Example of a graph of voltage measurements taken across the outer diameter near the proximal end of a vessel.

[0044] Fig.26 The diagram shows Fig.23 Example of a graph of voltage measurements taken across the outer diameter near the distal end of a vessel.

[0045] Fig. 27 Shown by Figures 7 to 9 The measuring device holder holds the Fig.23 A cross-sectional view of a container from a perspective looking down into the container.

[0046] Fig.28 The diagram shows the Fig.24 Figure 2. Examples of methods for mitigation of mechanical positioning errors.

[0047] Fig.29 The diagram shows the Fig.24 FIG. 4 is another example of a method for mitigation of mechanical positioning errors.

[0048] Fig.30 The diagram shows the Fig.24 FIG. 4 is another example of a method for mitigation of mechanical positioning errors.

[0049] Fig.31 The diagram shows the Fig.23 A graph showing voltage measurements taken when the length of the container is rotated 45 degrees.

[0050] Fig.32 Another example embodiment of a measuring device is schematically shown, which can alleviate the Fig.23 The error occurs when the wall of a container does not have a uniform thickness around the circumference of the container.

[0051] Fig.33 Schematically shows the Fig.32 An example of a method in which a measuring device mitigates wall thickness variations around the perimeter of a container.

[0052] Fig.34 The diagram shows that by executing Fig.33 A diagram of the variation in wall thickness around the perimeter of a container is provided for the method of lightening.

[0053] Fig.35 The diagram shows Figure 1 Example of a system dispensing a density gradient into a container.

[0054] Fig.36 Schematically shows the generation of Fig.35 The density gradient method shown in .

[0055] Fig.37 Shown by Figure 1 Example of a system dispensing a density gradient into a container.

[0056] Fig.38 The generation of a density gradient (such as Fig.37 An example of the method of density gradient) is shown in FIG.

[0057] Fig.39 The diagram shows that Figure 1 System implementation Fig.38 An example of a diagram of the method.

[0058] Fig.40 The schematic diagram shows that Figure 1 An example of a method for replicating density gradients performed on a .

[0059] Fig.41 The diagram shows Figure 1 Example of a density gradient profile before system processing.

[0060] Fig.42 Schematically illustrates the process Fig.41 Density gradient distribution to remove possible interference Figure 1 Example of a method for systematically reproducing noise and imperfections in density gradient distributions.

[0061] Fig.43 The diagram shows Fig.42 An example of a modified density gradient distribution generated by the method for replacing a density gradient distribution.

[0062] Fig.44 The diagram shows the first density gradient and the Figure 1 Example of a graph comparing a second density gradient copied from a first density gradient.

[0063] Fig.45 Schematic diagram showing the processing density gradient distribution for Figure 1 Another example of a method of system replication.

[0064] Fig.46 The diagram shows Fig.45 Example of a density gradient distribution before processing with the .

[0065] Fig.47 The diagram shows Fig.46 An example of a differential plot after mathematical differentiation of a density gradient distribution.

[0066] Fig.48 yes Fig.47 A magnified view of the differential curve graph.

[0067] Fig.49 Picture shows Fig.46 The density gradient distribution is different from that in Fig.47 Example of comparison of adjusted density gradient profiles after replacing measured values ​​affected by optical effects (such as Gouy phase shift) with values ​​determined by differential curve graph of .

[0068] Fig.50 yes Fig.49 A magnified view of the adjusted density gradient profile.

[0069] Fig.51 It shows that Figure 1 An example of a processing device of a system executing to assign a density gradient to a text file in a container.

[0070] Fig.52 Shows Figure 1 Example computing hardware for a system. DETAILED DESCRIPTION

[0071] Figure 1 An example of a system 100 that can generate a density gradient for centrifugation is schematically shown. The system 100 is a computer controlled to accurately dispense any type, slope or shape of gradient. The system 100 can be used to generate linear density gradients and step density gradients, where the linear density gradient has a gradually increasing density from top to bottom and the step density gradient has at least two discrete steps of different density.

[0072] In addition, the system 100 can measure the density gradient dispensed inside the container 110 without contacting or disturbing the density gradient. The system 100 can use these measurements to replicate the density gradient in another container. In some examples, the container 110 is a tube in a centrifuge rotor for centrifugation.

[0073] The system 100 includes reservoirs 102, each of which holds a separate component for generating a density gradient within a container 110. Each reservoir 102 is connected to a pump 104 for pumping the component held in the reservoir 102 into a manifold and mixing chamber 106. Each pump 104 is programmed to pump the component from the reservoir 102 at a given volume and rate for mixing within the manifold and mixing chamber 106.

[0074] exist Figure 1In the example shown, the system 100 includes four reservoirs, such as a first reservoir 102a connected to a first pump 104a for pumping a first component into a manifold and a mixing chamber 106, a second reservoir 102b connected to a second pump 104b for pumping a second component into the manifold and the mixing chamber 106, a third reservoir 102c connected to a third pump 104c for pumping a third component into the manifold and the mixing chamber 106, and a fourth reservoir 102d connected to a fourth pump 104d for pumping a fourth component into the manifold and the mixing chamber 106. The system 100 may include more than four reservoirs for holding more than four separate components to generate a density gradient, or may include fewer than four reservoirs for holding fewer than four separate components to generate a density gradient in the container 110.

[0075] The components held in the reservoirs 102 are liquids pumped into the manifold and mixing chamber 106 to distribute a uniform fluid stream into the container 110. As illustrative examples, the first reservoir 102a can contain deionized (DI) water, the second reservoir 102b can contain a density modifier, such as sucrose, glycerol, or iodixanol, the third reservoir 102c can contain a buffer solution, and the fourth reservoir 102d can contain additives, such as amino acids, proteins, chelating agents, stabilizers, detergents, salts, and biological sample materials. Illustrative examples of buffer solutions can include, but are not limited to, phosphate buffered saline (PBS), tris buffer solution concentrations (e.g., tris (hydroxymethyl) aminomethane, also known as tromethamine or THAM) and HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid).

[0076] All four component liquids are introduced into a single stream through the manifold and mixing chamber 106. The DI water and density modifier make up the majority of the volume in the stream, while the buffer solution and additives have smaller concentrations.

[0077] As an illustrative example, the first pump 104a and the second pump 104b are used to pump DI water and density modifier from the corresponding first reservoir 102a and the second reservoir 102b to the manifold and the mixing chamber 106. The first pump 104a and the second pump 104b may include a peristaltic pump to provide a smooth pumping flow for the DI water and density modifier components. The buffer solution and additives from the corresponding third reservoir 102c and the fourth reservoir 102d are pumped by the third pump 104c and the fourth pump 104d, respectively. In some examples, the third pump 104c and the fourth pump 104d include a peristaltic pump. In other examples, the third pump 104c and the fourth pump 104d may include a syringe pump, which can be used when the buffer solution and the additive require higher precision pumping.

[0078] Figure 2 An example of a mixer 200 housed within the manifold and mixing chamber 106 of the system 100 is shown. Figure 1 and Figure 2 , DI water, density modifier, buffer solution, and additives are introduced into a single stream through the manifold and mixing chamber 106. Within the manifold and mixing chamber 106, the mixer 200 includes mixing elements 202a to 202f that mix the components together as the components pass through the mixing elements. The mixer 200 mixes the components together to generate a uniform fluid flow for the probe 108 to dispense a step of a density gradient into the container 110, the step having a predetermined density based on the relative concentration of the components.

[0079] In some examples, the mixer 200 is a static mixer, and the mixing elements 202a to 202f include alternating spiral elements. In some examples, each spiral element is set to 90 ° with adjacent spiral elements to provide a thorough mixing of components on the length L of the mixer 200 in the manifold and mixing chamber 106. The mixing elements 202a to 202f mix the components together to eliminate the aggregation area of ​​low density and / or high density materials. The mixing elements 202a to 202f cut and rotate the DI water and density modifier together many times to produce a substantially uniform flow for the probe 108 to distribute the steps of the density gradient to the container 110. In an alternative example, the manifold and mixing chamber 106 can include an alternative type of mixer and mixing element.

[0080] Figure 3 1 is an isometric view of a probe 108 having a distal end 112 inserted into a container 110 and a proximal end 114 connected to a manifold and mixing chamber 106. Figure 3As shown, distal end 112 is positioned toward the bottom of interior volume 122 of container 110, such that probe 108 is ready to dispense a density gradient within the interior volume of the container. In some examples, probe 108 remains fixed in the same position while dispensing a density gradient within interior volume 122 of the container.

[0081] like Figure 3 As shown, during dispensing of the density gradient, the container 110 is fixedly positioned relative to the probe 108 by the holder 116. Figure 3 In the example of FIG. 1 , the retainer 116 includes a clamp for securely securing the container 110 to a frame 118 of the system 100 .

[0082] Figure 4 An example of the proximal end 114 of the probe 108 connected to the manifold and mixing chamber 106 is shown. The manifold and mixing chamber 106 includes a manifold portion 402 having inputs 404a to 404b, each of which receives a component pumped from a reservoir 102a to 102d by a pump 104a to 104d, respectively. The manifold and mixing chamber 106 also includes a mixing portion 406 that houses a mixer 200 for mixing the components pumped from the reservoirs together before they reach the proximal end 114 of the probe 108.

[0083] exist Figure 4 , the proximal end 114 of the probe 108 is shown as being secured by a set screw 408, which can be tightened or loosened about the proximal end 114 by using a rotatable handle 410. The manifold and mixing chamber 106 are attached to a motor drive mechanism that moves the probe 108 up and down to a desired position within the container 110. In other examples, the probe 108 can be manually lowered to a desired position within the container 110.

[0084] The probe 108 is coated with a non-stick material. In some examples, the probe 108 is coated with a non-stick material such as Teflon or a similar material. The non-stick material coating on the probe 108 minimizes the steps of the density gradient dispensed into the container 110 from adhering to the probe 108 or accumulating on the probe 108. Thus, the non-stick material coating enables the removal of the probe 108 while mitigating mixing between discrete steps of the density gradient.

[0085] Return to reference Figure 1 , the system 100 may include a control panel 130 for receiving input from a user to generate a desired density gradient. In some examples, the control panel 130 includes a display 132, such as a touch screen, which can be used by the user to create a desired density gradient, perform its measurement, and store the distribution of the density gradient. In other examples, the control panel 130 may include additional input devices, such as one or more physical buttons that can be selected to control the operation of the system 100.

[0086] Figure 5 An example of a method 500 for generating a density gradient within a container 110 using the system 100 is schematically shown. The method 500 includes an operation 502 of lowering the probe 108 to near the bottom of the container 110. Figure 1 and Figure 3 An example of a probe 108 positioned near the bottom of a container 110 is shown in FIG. In some examples, the distal end 112 of the probe 108 will remain positioned near the bottom of the container 110 as the probe 108 dispenses the density gradient.

[0087] The method includes an operation 504 of dispensing a first step (e.g., a first step consisting of DI water, a density modifier, a buffer solution, and an additive) made of components held in the reservoir 102 into the container 110. The first step has a first density based on the relative concentrations of the components. For example, increasing the amount of density modifier mixed by the manifold and the mixing chamber 106 will increase the density of the first step dispensed by the probe 108, while decreasing the amount of density modifier mixed by the manifold and the mixing chamber 106 will decrease the density of the first step dispensed by the probe 108.

[0088] Next, the method 500 includes an operation 506 of dispensing a second step made of components held in the reservoir 102 (e.g., a second step comprised of DI water, density modifier, buffer solution, and additives) into the container 110. The second step has a second density based on the relative concentrations of the components pumped from the reservoir 102. The second density is heavier than the first density, so that the second step pushes the first step upward, and the second step remains below the first step at the bottom of the container 110.

[0089] Next, method 500 includes an operation 508 of determining whether the density gradient includes an additional step. If the density gradient includes an additional step (i.e., "yes" in operation 508), method 500 repeats operation 506 to dispense an additional step made of the components held in reservoir 102 (e.g., an additional step made of DI water, density modifier, buffer solution, and additives). The additional step has a density based on the relative concentration of the components that is heavier than the density of the previously dispensed step, so that the additional step pushes up the previously dispensed step and remains below the previously dispensed step. Operation 506 can be repeated based on the desired number of steps of the density gradient. Each time operation 506 is performed, probe 108 remains in the same position (i.e., near the bottom of container 110).

[0090] If the density gradient does not include additional steps (i.e., "No" in operation 508), the method 500 proceeds to operation 510 of removing the probe 108 from the container 110. Operation 510 may include slowly removing the probe 108 so as not to disturb the steps of the density gradient. As described above, the probe 108 may be coated with a non-stick material to minimize the steps in the density gradient from adhering to the probe 108 during removal of the probe 108.

[0091] In method 500, each of pumps 104a to 104d is programmed to control the flow of each liquid component into manifold and mixing chamber 106 to have a given volume and / or velocity for generating each step of the density gradient. This enables system 100 to accurately control the concentration of each liquid component dispensed by probe 108 into container 110 for generating the density gradient in each step of the density gradient.

[0092] Figure 6 The example of density gradient 300 formed by system 100 in container 110 after method 500 is completed is schematically shown. Density gradient 300 is a medium (medium) generated for separating particles in ultracentrifugation. In this example, density gradient 300 is a step gradient (step gradient), so that density gradient 300 includes discrete steps with different densities. In this example, density gradient includes five discrete steps. In an alternative example, method 500 can be performed to form a continuous or linear gradient.

[0093] exist Figure 6 In the example of FIG. 1 , the density gradient 300 includes steps 302a to 302e. Each of the steps 302a to 302e has a unique density based on the relative composition of the components mixed in the manifold and mixing chamber 106. In this illustrative example, the density gradient 300 includes a first step 302a having a first density, a second step 302b having a second density, a third step 302c having a third density, a fourth step 302d having a fourth density, and a fifth step 302e having a fifth density. According to the above description, the fifth density of the fifth step 302e is the heaviest, and the first density of the first step 302a is the lightest, so that the density of the steps 302a to 302e increases from top to bottom inside the container 110. Each of the steps that form part of the density gradient 300 is shown as being separated by a boundary 310.

[0094] The system 100 and / or method 500 can form a density gradient having more than five individual steps, and / or form a density gradient having less than five individual steps. In addition, the system 100 can form a stepped gradient, such as Figure 6The density gradient 300 is shown as a stepped gradient, as well as a continuous or linear gradient having layers of increasing density from top to bottom. Therefore, the density gradient 300 is shown by way of illustrative example only.

[0095] like Figure 6 As further shown in FIG. 1 , the container 110 includes several features. For example, the container 110 includes a bottom portion 304, a cylindrical portion 312 extending from the bottom portion, and an opening 314 that allows liquid to be dispensed into the interior volume of the container 110. The container 110 includes a centerline CL extending along the center of the container 110. In some examples, the container 110 may include a seam between the bottom portion 304 of the container 110 and the cylindrical portion 312 of the container 110. In some examples, the boundary between the bottom portion 304 and the cylindrical portion 312 is used to establish an origin for positioning a sensor to begin measuring the density gradient 300. As will be described in more detail, the bottom portion 304 of the container 110 may include a curved surface that interferes with the measurement of the density gradient 300, so that the measurement results from this portion of the container 110 are filtered out and / or removed.

[0096] exist Figure 6 1 , the density gradient 300 exhibits features such as a meniscus 308 located at the top of the density gradient 300. The meniscus 308 is caused by surface tension between the density gradient 300 and the inner surface of the wall of the container 110. The meniscus 308 includes a bottom edge 309, where the centerline CL of the container 110 is located. The meniscus 308 also includes a top edge 311 near the wall of the container 110. The meniscus 308 can interfere with the measurement of the density gradient 300, making it impossible to obtain measurements from that portion of the density gradient 300 or filtering out measurements from that portion of the density gradient 300.

[0097] Container 110 includes a volume 306 located above meniscus 308 of density gradient 300 and below opening 314. Volume 306 can be filled with air or an inert gas. Measurement data can be obtained from volume 306 to determine the type of material from which container 110 is made. As an illustrative example, container 110 can be made of polypropylene, polycarbonate, copolyester resins such as polyethylene terephthalate glycol (PETG), and other materials. When light is transmitted through the empty portion of the container (e.g., volume 306), each type of material can exhibit unique characteristics. Measurement data from volume 306 can be used to standardize the measurement results of density gradient 300 for different types of containers made of different types of materials.

[0098] like Figure 6 As further shown in FIG. 1 , the container 110 has a length L extending from the bottom portion 304 to the opening 314 of the container. The density gradient 300 has a length L DIn this illustrative example, the length L of density gradient 300 is D is less than the length L of the container 110, such that the density gradient 300 occupies a portion of the length of the container. In some examples, the length L of the density gradient 300 D It can be about 80mm.

[0099] As will now be described in greater detail, the system 100 performs non-destructive density gradient measurements over the length L of the container 110. The density gradient measurements can be used to verify that the density gradient 300 conforms to a desired profile or meets desired quality controls. Additionally, the density gradient measurements can be stored in a memory for use in replicating the density gradient 300 within another container.

[0100] Figure 7 is an isometric view of an example of a measurement device 700 in a system 100 for performing nondestructive density gradient measurements over a length L of a vessel 110. For example, the measurement device 700 can measure the density gradient 300 without contacting or disturbing the density gradient. Figure 8 is a detailed isometric view of the measuring device 700 . Fig. 9 is a front view of the measuring device 700 .

[0101] Now refer to Figures 7 to 9 In this example, the measuring device 700 includes a platform 702 supporting a frame 704, and the frame 704 includes a rail 706. In some examples, the rail 706 includes a screw rail. The measuring device 700 also includes a sensor assembly 712, which is mounted on a bracket 708, and the bracket 708 is powered by a motor 710 to move up and down on the rail 706 while the container 110 is maintained in a fixed position. In some examples, the motor 710 is a stepper motor or other similar type of electric motor. The rail 706 and the motor 710 provide precise vertical movement of the sensor assembly 712 relative to the container 110. The container 110 can be held by a holder such as a clamp (see Fig. 27 ) is maintained relative to the measuring device 700.

[0102] For the length L along the container 110 and / or the length L of the density gradient D Alternative examples of moving the sensor assembly 712 are possible. For example, a gantry and pulley system may be used to move the sensor assembly 712 along the length L of the container 110 and / or the length L of the density gradient distributed within the container 110. D The mobile sensor assembly 712 is contemplated for use along the length L of the container 110 and / or the length L of the density gradient distributed within the container 110. D Additional structure of the mobile sensor assembly 712 is provided by way of illustrative example. Figures 7 to 9 The structure shown in .

[0103] The sensor assembly 712 is used to measure the length L of the density gradient 300 distributed in the container 110. D The density on Figure 8 and Fig. 9 As shown, the sensor assembly 712 includes an emitter 714 that emits a signal, such as light, and a detector 716 that measures the signal from the emitter 714 after it is transmitted through a density gradient distributed in the container 110. When mounted on the bracket 708, the emitter 714 and the detector 716 are in a fixed relationship relative to each other, which enables these components of the sensor assembly 712 to move together up and down on the guide rail 706. The bracket 708 enables the emitter 714 and the detector 716 to move in coordination up and down the length L of the container 110, and can be used to provide precise alignment of the emitter 714, the detector 716, and the centerline CL of the container 110.

[0104] exist Figures 7 to 9 In the example shown, the emitter 714 is mounted on one side of the carrier 708, and the detector 716 is mounted on the opposite side of the carrier 708. The container 110 having the density gradient dispensed therein is fixedly positioned relative to the central axis of the carrier 708 such that a signal (e.g., light) emitted by the emitter 714 passes through the container 110 and is received by the detector 716 on the opposite side of the container 110.

[0105] In an alternative example, the emitter 714 and the detector 716 may be mounted on the same side of the bracket 708. For example, the emitter 714 may transmit a signal from a first side of the bracket 708, the signal passing through the density gradient dispensed in the container 110, and the signal is reflected by a reflector mounted on a second side of the bracket 708 to reflect back toward the first side of the bracket 708, where the detector 716 is mounted on the first side. Other alternative arrangements for the sensor assembly 712 are contemplated.

[0106] In one example implementation, the emitter 714 emits light, and the detector 716 includes a photodiode that detects a current generated by the light being transmitted through the container 110. In some examples, the emitter 714 emits light within the infrared spectrum (e.g., light having a wavelength of about 700 nm to about 1000 nm). In some additional examples, the emitter 714 emits light having a wavelength of about 880 nm. In an alternative example, the emitter 714 emits light within the visible spectrum (e.g., from about 380 nm to about 750 nm).

[0107] When light from the emitter 714 that passes through the container 110 strikes the detector 716, a current is generated on the detector 716. The sensor assembly 712 may also include an amplifier circuit that converts the current into a voltage. Thus, the sensor assembly 712 measures and records the voltage at a plurality of points along the length L of the container 110 for measuring the density gradient distributed in the container 110. As an example, the sensor assembly 712 may measure and record the voltage at 320 points over a length of approximately 80 mm.

[0108] The voltage measurements recorded by the sensor assembly 712 are related to the refractive index along the length of the density gradient dispensed in the container 110 and can be used to calculate the density along the length of the density gradient. This is because the density affects the transmission of light from the emitter 714 through the density gradient. Therefore, the voltage measurements recorded by the sensor assembly 712 can be used to measure the density value at a given point along the length of the density gradient dispensed in the container 110.

[0109] like Figure 8 As shown, the bracket 708 includes a slot 718 positioned in front of the detector 716. The slot 718 focuses the light emitted from the emitter 714 through a narrow slice of the density gradient dispensed in the container 110. The slot 718 enables the measurement device 700 to measure the narrow slice along the length of the density gradient dispensed in the container 110. In addition, multiple measurements can be made for each slice of the density gradient (e.g., 100 measurements per slice), and the measurements can be averaged to reduce the variation of the measurements made along the entire length of the density gradient.

[0110] To further reduce sensitivity due to positional errors between the container 110, the emitter 714, and the detector 716, the detector 716 is provided with a large surface area. In some examples, the detector 716 includes a surface area of ​​about 8.5 mm. 2 The surface area of ​​the photodiode.

[0111] Fig.10 An example of an electrical configuration of the sensor assembly 712 is schematically shown. Fig.10 As shown, resistor 720 sets the current of transmitter 714. As an illustrative example, resistor 720 can set the current of transmitter 714 in the range of about 19mA to about 33mA. In some examples, resistor 720 can have a resistance of about 90Ω. Transmitter 714 can be powered by a stable, high-precision DC power supply to maintain a stable level of infrared (IR) radiation. In an alternative example, the DC power supply for transmitter 714 can be replaced with a constant current source, which can reduce variations due to power supply drift. Additional examples for powering transmitter 714 are possible.

[0112] The transimpedance amplifier is designed to convert the detection current (I d ) is converted to a voltage across the feedback resistor 722. In some examples, the feedback resistor 722 has a resistance of about 47 kΩ. The operational amplifier 724 with a precise input current is used for its ability to operate at very low currents. The second DC power supply can provide + / -6 VDC for the operational amplifier 724.

[0113] When the density gradient is dispensed into the vessel 110, the vessel becomes a cylindrical lens such that the spacing between the emitter 714, the vessel 110, and the detector 716 can affect the voltage measurements obtained from the sensor assembly 712. For example, a spacing of about 1.10 inches (28 mm) between the emitter 714 and the detector 716 can be used for a vessel having a 9 / 16 inch diameter, and a spacing of about 2.44 inches (62 mm) between the emitter 714 and the detector 716 can be used for a vessel having a 1 inch diameter.

[0114] In addition, the voltage of the emitter 714 can be adjusted based on the distance between the emitter 714 and the detector 716 to optimize the level of infrared (IR) radiation transmitted through the container. Table 1 provides illustrative examples of optimal voltages for the emitter 714 and optimal distances between the emitter 714 and the detector 716 based on different container sizes and material types. Table 1 shows voltage measurements recorded by the detector 716 for a density gradient having a first step of 0% density modifier (e.g., sucrose) and a second step of 40% density modifier (e.g., sucrose) and the difference between these measurements.

[0115]

[0116] Table 1

[0117] As shown in Table 1, the sensor assembly 712 measures voltage to determine the concentration level of density modifiers such as sucrose, glycerol, and iodixanol in containers having different diameters (e.g., 9 / 16 inch or 1 inch) and made of different materials (e.g., Material Type 1 = polypropylene, Material Type 2 = polyethylene terephthalate glycol (PETG), Material Type 3 = polycarbonate). The concentration level of the density modifier is used to determine the density at a specific location along the length of the density gradient.

[0118] Fig.11 Schematic diagram of measuring a density gradient distributed in a container (such as distributed in Figure 6 100 ). The method 1100 may be performed by the measurement device 700 of the system 100 . The method 1100 may measure the density gradient non-destructively, such that the density gradient is not disturbed or changed by the method 1100 .

[0119] Method 1100 includes an operation 1102 of obtaining a measurement result from a density gradient, which will be referred to as Figures 7 to 9 and Fig.12 The method 1100 includes additional operations for improving density gradient measurements, such as an operation 1104 of filtering or removing measurements from the bottom portion 304 of the container 110, an operation 1106 of filtering or removing measurements at the location of the meniscus 308 of the density gradient 300, an operation 1108 of normalizing measurements based on container material and / or size, an operation 1110 of mitigating the effects of container defects and wall thickness variations along the length L of the container 110, an operation 1112 of mitigating the effects of wall thickness variations along the circumference of the container 110 on density gradient measurements, and an operation 1114 of mitigating mechanical positioning errors. Each of these additional operations will be described in more detail below.

[0120] Fig.12 An example of a method 1200 for obtaining a measurement result from a density gradient dispensed in a container 110 is schematically shown. In some examples, the method 1200 forms part of operation 1102 in the method 1100. The method 1200 may be used by the system 100 Figures 7 to 9 The measurement is performed using the measurement device 700 shown in FIG.

[0121] like Fig.12 As shown, method 1200 includes step 1202 of initiating measurement of a density gradient dispensed into container 110. Step 1202 may occur after receiving a user input / command on control panel 130 of system 100.

[0122] Next, method 1200 includes step 1204 of positioning sensor assembly 712 relative to container 110 for measurement. Sensor assembly 712 can be positioned by motor 710 while container 110 remains stationary. Motor 710 can move sensor assembly 712 up and down the entire length L of container 110 in precise steps.

[0123] In some examples, the sensor assembly 712 is initially positioned toward the bottom of the container 110 in step 1204. In alternative examples, the sensor assembly 712 is initially positioned toward the top of the container 110 in step 1204. In further examples, the sensor assembly 712 is positioned between the top and the bottom of the container 110 in step 1204.

[0124] Method 1200 includes step 1206 of measuring a voltage at the location of sensor assembly 712 located in step 1204. The voltage is measured by transmitting light from emitter 714 through container 110 and received by detector 716 on the opposite side of container 110. Step 1206 may include measuring the voltage multiple times and calculating an average voltage at the location of sensor assembly 712.

[0125] The method 1200 includes a step 1208 of determining whether additional locations along the length L of the container 110 require measurement. When it is determined that no additional locations require measurement (i.e., "no" in step 1208), the method 1200 may terminate at step 1210. When it is determined that additional locations require measurement (i.e., "yes" in step 1208), the method 1200 may repeat steps 1204 to 1208 to move the sensor assembly 712 to a new location along the length L of the container 110, take measurements at the new location, and determine whether there are additional locations that require measurement.

[0126] In some examples, when the density gradient measurement is obtained starting from the bottom of the container 110, the new position is upward relative to the previous position. In an alternative example, when the density gradient measurement is obtained starting from the top of the container 110, the new position is downward relative to the previous position. The length L of the container 110 can be divided into a different number of positions, and steps 1204 to 1208 are repeated for each position to generate a distribution of the density gradient. As an example, the method can obtain measurements at 320 points over a length of 80 mm.

[0127] Fig.13 An example of a profile 1300 of a density gradient measured according to method 1100 is illustrated. In this illustrative example, profile 1300 shows the voltage recorded by detector 716 for a 5% to 40% sucrose density gradient. The voltage represents the refractive index of the density gradient dispensed in container 110, which can be used to determine the concentration and density level of a density modifier (e.g., sucrose).

[0128] The bottom of the container 110 is on the left side of the distribution 1300, and the top of the container 110 is on the right side of the distribution 1300. The distribution 1300 excludes the bottom portion of the container 110 that may interfere with the optical path of the light from the emitter 714 (see operation 1104 of method 1100). The distribution 1300 also excludes the meniscus 308 that may be formed between the density gradient and the wall of the container 110 (see operation 1104 of method 1100). Figure 6 )'s top portion of the density gradient affected (see operation 1106 of method 1100).

[0129] like Fig.13As shown in the example of , the voltage is highest near the bottom of the container 110, where the densest step of the density gradient is located (i.e., it has about 40% sucrose), and the voltage gradually decreases as one moves up the container 110 to where the lowest density step of the density gradient is located (i.e., it has about 5% sucrose). By scanning along the length L of the container 110, the change in refractive index forms a profile 1300, which is related to the change in density modifier concentration and density level.

[0130] Fig.14 An example of a graph 1400 of voltage detected by the sensor assembly 712 identifying a characteristic of the container 110 and the density gradient dispensed therein is illustrated. In this example, the container 110 is a polypropylene container having a diameter of 9 / 16 inches, and the voltage is acquired along the entire length L of the container 110.

[0131] Graph 1400 may be used to identify the locations of various features of interest on container 110 and / or density gradient 300. For example, graph 1400 illustrates a location 1402 of bottom portion 304 of container 110; a location 1404 of cylindrical portion 312 of container 110; a location 1406 of meniscus 308 of density gradient 300; a location 1408 of volume 306 above density gradient 300; a location 1410 of opening 314 of container 110; and a location 1412 of air above container 110.

[0132] Identifying these features of interest can help improve the system 100's measurement of the density gradient 300. For example, according to operation 1104 in the method 1100, the location 1402 identifying the bottom portion 304 in the graph 1400 can be used to filter out and / or remove voltage measurements from that location of the container 110. Similarly, according to operation 1106 in the method 1100, the location 1406 identifying the meniscus 308 in the graph 1400 can be used to filter out and / or remove voltage measurements from that location of the density gradient 300.

[0133] As an example, location 1404 where cylindrical portion 312 begins can be selected as an origin for locating sensor assembly 712 in step 1204 in method 1200. As another example, location 1406 before meniscus 308 can be selected as an endpoint for terminating measurement of a density gradient by sensor assembly 712. As another example, location 1408 of volume 306 above density gradient 300 in container 110 can be selected to obtain a measurement to identify the material of container 110 because each type of container can be made of a material that has unique properties when an illumination signal, such as infrared light, is sent through an empty portion of the container.

[0134] The height and / or position of the meniscus 308 may be difficult to measure because the shape and / or size of the meniscus 308 may vary based on the amount of surface tension (i.e., adhesion) between the density gradient and the wall of the container 110. For example, liquids with different densities will have different surface tensions with the wall of the container 110.

[0135] Meniscus 308 may cause optical effects that may interfere with the accuracy of density measurements by sensor assembly 712. For example, bottom edge 309 of meniscus 308 may cause a higher voltage reading by sensor assembly 712. Additionally, top edge 311 of the meniscus may cause a lower voltage reading by sensor assembly 712. The following techniques are implemented in system 100 to identify the location of meniscus 308 regardless of the shape and / or size of meniscus 308. By identifying the location of meniscus 308, measurements obtained from sensor assembly 712 may be filtered based on the location of the meniscus to improve the accuracy of density gradient measurements by system 100.

[0136] Fig.15 An example of a graph 1500 of voltage for identifying the position of the meniscus of the sample liquid dispensed in the container 110 is illustrated. In this embodiment, five liquid samples are analyzed. The x-axis of the graph 1500 represents the height in millimeters, and the y-axis represents the voltage in millivolts detected by the sensor assembly 712. The left side 1502 of the graph 1500 shows the voltage measurement result of the liquid sample in the container. The right side 1504 of the graph 1500 shows the voltage measurement result of the volume 306 (e.g., air) above the sample. The transition 1506 between the left side 1502 and the right side 1504 in the middle of the graph 1500 shows the meniscus. The unexpected result from the graph 1500 is that, although the meniscus looks thin to the human eye, the optical effect of the meniscus is several millimeters wide.

[0137] The shape of the meniscus may vary due to different surface tensions between the sample liquid and the wall of the container 110. For example, the peak of the voltage waveform is located near a height of about 11 mm, and the valley of the voltage waveform is located near a height of about 15 mm. The locations of the peaks and valleys may vary due to different surface tensions exhibited by each of the sample liquids analyzed in the graph 1500.

[0138] Fig.16An enlarged view of a graph 1600 of the first derivative of a voltage obtained from a sample liquid dispensed in the container 110 is illustrated. A technique for accurately measuring the height of a density gradient dispensed in the container 110 includes identifying the location of a meniscus using a first derivative minimum of the voltage measured by the sensor assembly 712. While other features of the meniscus may also be identified by the sensor assembly 712, the first derivative minimum is a consistent and reliable source for identifying the meniscus.

[0139] The minimum of the first derivative is at the point where the voltage slope is most negative. This point occurs at the midpoint between the bottom edge 309 and the top edge 311 of the meniscus, regardless of the shape of the meniscus (see Figure 6 ). The use of the first derivative effectively eliminates the changes in meniscus shape that would normally occur due to surface tension. Fig.16 In the example of graph 1600 shown, the minimum of the first derivative is located at approximately 13.8 mm, with an uncertainty of approximately 0.25 mm.

[0140] Additional data shows that in a container with a total volume of 13 mL, the volume of a liquid sample dispensed into the container can be calculated from the height identified using the first derivative minimum with an error of approximately + / - 37 μL. This compares favorably to other more expensive methods for measuring the volume of a liquid sample dispensed into a container.

[0141] Fig.17 An example of a method 1700 for normalizing measurements based on the type of container in which the density gradient is dispensed is schematically illustrated. In some examples, the method 1700 forms part of operation 1108 in method 1100.

[0142] The container 110 can have different sizes (e.g., 9 / 16 inch diameter, 1 inch diameter, etc.), and the container 110 can be made of different materials, including but not limited to polypropylene, polycarbonate, and co-polyester resins, such as polyethylene terephthalate glycol (PETG). Each size and material can cause the container to exhibit unique characteristics when light is transmitted therethrough, thereby causing changes in the measurement results of the density gradient 300 when dispensed into different containers having different sizes and made of different materials.

[0143] Method 1700 includes an operation 1702 of measuring a voltage across a void volume of container 110. In some examples, operation 1702 is performed by system 100 prior to dispensing density gradient 300 into container 110.

[0144] In other examples, operation 1702 is performed by system 100 after density gradient 300 is dispensed into container 110. In such examples, operation 1702 includes measuring a voltage across volume 306 above meniscus 308 and below opening 314 of density gradient 300. The position of volume 306 may be determined based on the relative positions of meniscus 308 and opening 314, such as by identifying Fig.14 The ability to measure the voltage across the empty volume of the container 110 before or after dispensing the density gradient 300 can provide flexibility to the user of the system 100.

[0145] Method 1700 includes an operation 1704 comparing the voltage measured in operation 1702 to an expected voltage range known for different material types and container sizes. For example, each type of material produces a unique voltage distribution corresponding to the optical properties and quality of the material. In addition, the voltage measurement results can vary based on the size or diameter of the container. Table 2 is provided below to show the expected voltage range for the first type of material and the second type of material and different container sizes (e.g., 1 inch diameter and 9 / 16 inch diameter). As an illustrative example, the first type of material may include a polyester resin, such as polyethylene terephthalate glycol (PETG), and the second type of material may include polypropylene.

[0146]

[0147] Table 2

[0148] Next, the method 1700 includes an operation 1706 of determining a material and / or size of the container 110 based on the comparison in operation 1704. For example, when the voltage measured in operation 1702 falls within a voltage range expected for a particular material or a particular combination of material and container diameter, it is determined in operation 1706 that the container 110 has the particular material and / or container diameter.

[0149] Next, method 1700 includes normalizing the density gradient 300 over its entire length L D Operation 1708 of standardizing the voltage measurements obtained from the measuring device 700. The voltage measurements may be obtained according to the steps of the above method 1200. Operation 1708 enables the system 100 to standardize the voltage measurements obtained from the measuring device 700 for different types of containers made of different types of materials and / or having different sizes.

[0150] Fig.18An example of a graph 1800 showing normalized measurements after completing method 1700 is shown. In some cases, the process of accurately scanning density gradients in container 110 may be technically challenging due to the sensitivity of the measurements to variations in wall draft and / or thickness of the container. The wall draft of a container may be affected by the type of material and process used to manufacture the container. For example, injection molding may cause a container to exhibit wall draft along its length.

[0151] During injection molding, the container 110 is formed by forcing hot molten material into a mold cavity under high pressure and temperature. The molten material conforms to the shape of the mold and then cools. To aid in removing the container 110 from the mold, a small amount of draft is added to the mold so that the inner diameter of the container 110 is slightly larger at the top of the container 110 than at the bottom of the container 110. The draft causes an increase in the wall thickness near the bottom portion 304 of the container 110 because the outer diameter of the container 110 is the same along the length L of the container 110. Although the draft is invisible to the naked eye, it can be detected by the measurement device 700. For example, during a precise optical scan measuring the refractive index of a gradient sample dispensed in the container 110, the draft is revealed by a slight slope in the measurement results. As described above, the measurement results from the bottom portion 304 of the container 110 can be ignored or filtered out because the curved shape and increased wall thickness of the bottom portion interfere with the measurement results of the density gradient.

[0152] exist Fig.18 In the example shown, the graph 1800 includes a first graph 1802 and a second graph 1804 of the voltage measured by the detector 716 (y-axis) and the length of the container (x-axis). Each of the first graph 1802 and the second graph 1804 is measured for a container made of a polypropylene material having a diameter of 9 / 16 inches. In the first graph 1802, the container is filled with a single step having a 0% sucrose density modifier (e.g., the container is filled with 100% DI water). In the second graph 1804, the container is filled with a single step having a 40% sucrose density modifier (e.g., the container is filled with 60% DI water and 40% sucrose). The bottom portion of the container occupies a length of 0 to 14 mm (x-axis), so that these portions of the first graph 1802 and the second graph 1804 can be ignored.

[0153] In both the first graph 1802 and the second graph 1804, the container is filled with a uniform sample solution (i.e., 0% sucrose vs. 40% sucrose) so that the voltage measurement (y-axis) should be consistent (i.e., flat) over the length of the container (x-axis). Fig.18 As shown in the example of FIG. 1 , the first graph 1802 and the second graph 1804 include voltage measurements having a slight downward slope due to wall draft and / or thickness variations of the container.

[0154] According to operation 1708 of method 1700, first graph 1802 and second graph 1804 may be normalized based on the material (e.g., polypropylene) and size (e.g., 9 / 16 inch) of the container identified from operation 1706 of method 1700. As an example, a compensation value is added based on the location where each measurement is made above the bottom portion of the container (e.g., 14 mm above) to normalize first graph 1802 and second graph 1804 based on the polypropylene material and 9 / 16 inch diameter of the container. In this illustrative example, the maximum compensation value occurs toward the right side of graphs 1802, 1804, which is before the meniscus 308 of density gradient 300. In this illustrative example, the maximum compensation value is approximately 1.6 mV / mm.

[0155] exist Fig.18 In the embodiment, after adding the compensation values ​​to the first graph 1802 and the second graph 1804, such as after completing operation 1708 of method 1700, a first normalized graph 1802' and a second normalized graph 1804' are generated. The first normalized graph 1802' and the second normalized graph 1804' are linear (e.g., flat) along the length (x-axis) of the container, such that wall draft and / or thickness variations of the container are compensated by the compensation values.

[0156] Fig.19 Another example of a graph 1900 is shown showing normalized measurement results after completing method 1700. Graph 1900 includes a plot 1902 of voltage (y-axis) and container length (x-axis) for a container made of polyethylene terephthalate glycol (PETG) material having a diameter of 9 / 16 inches. In this example, the container is filled with 0% sucrose density adjuster (e.g., the container is filled with 100% DI water).

[0157] exist Fig.19 In the example shown, for a container made of polypropylene material and filled with 0% sucrose density modifier, the downward slope of the graph 1902 is less than Fig.18 The downward slope of the first graph 1802 in FIG. These examples show that containers made of different materials may have different wall drafts and / or wall thickness variations. For example, Fig.19 Containers made of PETG are Fig.18 In view of the foregoing, the compensation value used to normalize the voltage measurement result can vary based on the material and / or size of the container identified in operation 1706 of method 1700.

[0158] exist Fig.19, a compensation value is added based on the location where each measurement is made above the bottom portion of the container (e.g., 14 mm above) to normalize the graph 1902 based on the PETG material and 9 / 16 inch diameter of the container. In this illustrative example, the maximum compensation value is on the right side of the graph 1902, such as just before the meniscus 308 of the density gradient 300 (e.g., about 0.5 mV / mm).

[0159] After adding the compensation values, such as after completing operation 1708 of method 1700, a normalized curve graph 1902' is generated. Normalized curve 1902' is linear (e.g., flat) along the length (x-axis) of the container, such that wall draft and / or thickness variations of the container have been compensated by the compensation values.

[0160] Given that Fig.18 and Fig.19 , operation 1708 may include a compensation value added to the voltage measurement detected by detector 716. The compensation value is based on the length at the location where the measurement is made to remove the effects of container wall draft and / or wall thickness variation. The compensation value may be based on characterization data identifying the material of the container (see operations 1702 to 1706). Method 1700 may be applied to open top containers because these types of containers are often injection molded and appear to exhibit greater wall draft than containers made by other manufacturing methods.

[0161] In addition to wall draft, the accuracy of the density gradient measurement may depend on other physical properties of the container 110. Defects including blemishes, scratches, cracks, stains, dirt, etc., may interfere with the optical path of light emitted from the emitter 714 as the light passes through the container 110 to be detected by the detector 716. These types of defects are often present on containers, and even new tubes may have one or more types of defects due to the molding process used to make the container.

[0162] Fig. 20 An example of a method 2000 for mitigating the effects of defects and wall thickness variations along the length L of the container on measurements obtained from the container 110 according to the method 1200 is schematically shown. Examples of defects on the container 110 may include, but are not limited to, blemishes, scratches, cracks, stains, dirt, etc. These types of defects may change the optical path of light through the container 110 and, therefore, affect density measurements. In some examples, the method 2000 forms part of the operation 1110 in the method 1100. The method 2000 may be performed by the system 100.

[0163] The method 2000 includes an operation 2002 of measuring the container 110 when the container is empty (i.e., before the container 110 is filled with a density gradient). The operation 2002 is performed by the motor 710 moving the sensor assembly 712 along the length L of the container 110 while the container 110 remains stationary. In the operation 2002, the emitter 714 emits light for transmission through the container 110. The light is received by the detector 716 for measuring the optical properties of the container 110 when the container is empty. The measurements recorded by the detector 716 may include voltage measurements along the length L of the container.

[0164] Fig.21 An example of a graph 2100 of voltage measurements recorded by the sensor assembly 712 from the container 110 when the container is empty according to operation 2002 is illustrated. The x-axis of the graph 2100 represents the length L of the container 110 and the y-axis represents the voltage measurements in millivolts (mV). The length of the container 110 from the bottom to the top is represented by the voltage moving from left to right on the x-axis of the graph 2100. Fig.21 As shown, curve 2100 exhibits a slight downward slope in the voltage detected by sensor assembly 712, which may be caused by a variation in the wall thickness of container 110. Variations in wall thickness are generally due to the wall being thicker near bottom 304 of container 110 and gradually thinning as it moves toward opening 314 of container 110. The reduction in wall thickness from the bottom to the top of the container may facilitate removal of container 110 from a mold during manufacturing.

[0165] Return to reference Fig. 20 , method 2000 next includes an operation 2004 of calculating an average value based on the measurements recorded along the length L of container 110 in operation 2002. As an illustrative example, Fig.21 The average voltage measurement shown in graph 2100 is approximately 751 mV for the entire length L of the container.

[0166] The method 2000 has an operation 2006 of generating a difference between the average value calculated in operation 2004 and the measurements recorded in operation 2002 along the entire length L of the container 110. Larger differences may indicate the possibility of a defect (such as a scratch or flaw) at a particular location along the length L of the container 110 because they indicate a larger deviation from the average value. Also, larger differences may indicate that certain portions of the container 110 are particularly thick or thin relative to the thickness of other portions of the container 110. After completing operation 2006, a set of difference data values ​​along the entire length L of the container is created.

[0167] Next, method 2000 includes an operation 2008 of dispensing the density gradient into container 110. Figure 5 The operations of the described method 500 assign a density gradient in operation 2008 .

[0168] Method 2000 includes an operation 2010 of measuring a density gradient dispensed into container 110. Fig.12 The operations of method 1200 are described, and in operation 2010 a density gradient is measured.

[0169] The method 2000 includes an operation 2012 of point-by-point adding the difference values ​​generated in operation 2006 to the density gradient measurements measured in operation 2010. This adjustment can mitigate the effects of defects such as flaws, scratches, cracks, stains, dirt, etc., present along the length L of the container 110, and can also mitigate the effects of wall thickness variations on the density gradient measurements because the difference data accounts for these effects. In some examples, the difference values ​​are as described above with respect to Figures 17 to 19 Compensation value discussed.

[0170] Fig. 22 An example of a graph 2200 is illustrated, which shows how the method 2000 can mitigate and / or eliminate the effects caused by container defects and / or container wall thickness variations on a DI water sample dispensed in a container 110. The x-axis of the graph 2200 represents the length L of the container 110, and the y-axis represents the voltage measurement in millivolts (mV). Since the container 110 is filled with DI water, the voltage measurement should be constant along the length of the container 110. However, variations in the wall thickness of the container 110 (e.g., the wall thickness decreases from the bottom to the top) may cause the voltage measurement to slope downward, such as at Fig. 22 In addition, as described above, defects such as blemishes, scratches, cracks, stains, dirt, etc. can affect density measurements.

[0171] Graph 2200 includes a first graph 2202 of voltage measurements of DI water before a difference data value is added to the voltage measurements, and a second graph 2204 after the difference data value is added to the voltage measurements. Fig. 22 As shown, the second graph 2204 is flatter and has a smaller slope than the first graph 2202, indicating that the container wall thickness variation has been normalized from the data. In addition, in the second graph 2204, the effect of small scratches and flaws (such as small ridges in the range of 30 mm to 37 mm) is virtually eliminated or greatly reduced. This is in Fig. 22 This is illustrated by the second graph 2204 having a more linear or smoother distribution than the distribution of the first graph 2202, which exhibits a greater variance.

[0172] Fig.23An example of a container 2300 is shown having a centerline CL that is offset relative to a vertical alignment VA of a sensor assembly 712 mounted on a measurement device 700. The container 2300 includes a length L extending from a proximal end 2302 to a distal end 2304. The container 2300 has an outer diameter D that is constant over the length L of the container. The outer diameter D and length L of the container 2300 are perpendicular to each other.

[0173] The process of accurately measuring the density gradient of a dispense in a container 2300 is technically challenging due to the sensitivity of the density gradient measurement to the circular surface of the container 2300. For example, the container 2300, when filled with fluid, exhibits optical properties such that the amount of light received by the detector 716 after the light is emitted from the emitter 714 and passes through the container 2300 is affected by the alignment of the container 2300 relative to the emitter 714 and the detector 716 of the sensor assembly 712.

[0174] Fig.24 An example of a method 2400 for mitigating mechanical positioning errors of a container 2300 on density gradient measurements is schematically shown. For example, the method 2400 can mitigate and / or eliminate errors that may be caused by misalignment between a centerline CL of the container 2300 relative to a vertical alignment VA of an emitter 714 and a detector 716 of a sensor assembly 712. In some examples, the method 2400 forms part of operation 1114 in the method 1100. The method 2400 can be performed by the system 100 using the measurement device 700.

[0175] Method 2400 includes an operation 2402 of positioning sensor assembly 712 near proximal end 2302 of container 2300. Fig.23 As shown, proximal end 2302 includes opening 2306 into which a probe may be inserted to dispense a density gradient into container 2300. Operation 2402 may include positioning sensor assembly 712 below opening 2306 by moving bracket 708 on which sensor assembly 712 is mounted along rail 706 using motor 710 while container 2300 remains stationary.

[0176] The method 2400 includes an operation 2404 of scanning across an outer diameter D of the container 2300 near the proximal end 2302. The carriage 708 may include one or more additional motors that move the sensor assembly 712 in a radial direction perpendicular to the length L of the container 2300, thereby enabling the sensor assembly 712 to scan across at least a portion of the outer diameter D of the container 2300. In some examples, the operation 2404 includes scanning a central portion of the outer diameter D of the container 2300, such that the entire outer diameter D of the container 2300 is not scanned. In some additional examples, the motor causes the container 110 to be moved perpendicular to the length L of the container 2300, thereby enabling the sensor assembly 712 to scan across at least a portion of the outer diameter D of the container 2300.

[0177] The method 2400 also includes determining a position 2310 of the centerline CL of the container 2300 near the proximal end 2302 (see Fig.23 ) in operation 2406. The position 2310 of the centerline CL is determined in operation 2406 based on the measurement made across the outer diameter D of the container 2300 in operation 2404.

[0178] Fig.25 An example of a graph 2500 of voltage measurements taken across the outer diameter D near the proximal end 2302 of the container 2300 is shown. Figure 23 to Figure 25 , when the emitter 714 strikes the centerline CL of the container 2300, the detector 716 detects a maximum voltage at a given location along the length L of the container 2300. Any misalignment to the left or right of the centerline CL results in a lower voltage detected by the detector 716. The location 2310 of the centerline CL is determined in operation 2406 by identifying the location of the peak voltage measurement. Fig.25 In the example shown, a peak voltage of approximately 2.3 V occurs at a position of approximately +0.01 inches.

[0179] In some examples, the method 2400 includes measuring the voltage across at least a portion of the outer diameter D at each measurement location along the length L of the container 2300, and using the maximum voltage detected at each measurement location to determine the density at a given measurement location. This technique, while time consuming, can eliminate errors caused by misalignment of the container 2300 and imperfections on the container 2300.

[0180] Return to reference Fig.24 , method 2400 includes an operation 2408 of positioning sensor assembly 712 near distal end 2304 of container 2300. Fig.23As shown, distal end 2304 includes bottom portion 2308. Operation 2408 may include positioning sensor assembly 712 above bottom portion 2308. Operation 2408 may include moving carriage 708 along rail 706 using motor 710 while container 2300 remains stationary.

[0181] Next, method 2400 includes operation 2410 of scanning across the outer diameter D of container 2300 near distal end 2304. In some examples, operation 2410 includes scanning a central portion of outer diameter D of container 2300 such that the entire outer diameter D of container 2300 is not scanned in operation 2410.

[0182] Next, the method 2400 also includes determining the position 2312 of the centerline CL of the container 2300 near the distal end 2304 (see Fig.23 ) in operation 2412. In operation 2412, the position of the centerline CL is determined based on the measurement made across the outer diameter D of the container 2300 in operation 2410.

[0183] Fig.26 An example of a graph 2600 of voltage measurements taken across the outer diameter D near the distal end 2304 of the container 2300 is illustrated. Similar to operation 2406, in operation 2412, a location 2312 of the centerline CL near the distal end 2304 is determined by identifying the location of the peak voltage measurement. Fig.26 In the example shown, a peak voltage of approximately 2.3 V occurs at an offset position of approximately -0.01 inches.

[0184] Return to reference Fig.24 , method 2400 has an operation 2414 for generating a centerline CL of container 2300 by connecting the location 2310 of the centerline CL near the proximal end 2302 (determined in operation 2406) with the location 2312 of the centerline CL near the distal end 2304 (determined in operation 2412). The centerline CL generated in operation 2414 is Fig.23 712 as extending between the proximal end 2302 and the distal end 2304 and offset relative to the vertical axis VA of the sensor assembly 712.

[0185] The method 2400 includes an operation 2416 of measuring a density gradient dispensed in the container 2300 along the centerline CL generated in the operation 2414. The operation 2416 may include moving the sensor assembly 712 in two dimensions, such as in a vertical dimension along the length L of the container 2300 and in a horizontal dimension along the outer diameter D of the container 2300. In some examples, the operation 2416 includes moving the sensor assembly 712 starting from a location 2312 of the centerline CL near the distal end 2304 of the container 2300 to a location 2310 of the centerline CL near the proximal end 2302 of the container 2300.

[0186] An advantage of the method 2400 is that the execution of the method 2400 can eliminate the need for the bracket 708 supporting the sensor assembly 712 and the holder of the container 2300 (see Fig. 27 ) and the need to maintain alignment over the entire length L of the container 2300 for each density gradient measurement. For example, the use of the centerline CL generated in the method 2400 can offset the effects of any misalignment of the container 2300 with the sensor assembly 712, and can simplify the mechanical complexity and sensitivity of the measurement device 700. In addition, the method 2400 can simplify the manufacturing and field service requirements of the measurement device 700, such as by eliminating the need to periodically calibrate the measurement device 700, and improve the long-term reliability of the measurement device 700. In addition, during long-term use, wear on the bracket 708 of the sensor assembly 712 and / or the holder of the container 2300 may have less impact on the accuracy of the density gradient measurement results.

[0187] Other advantages of the method 2400 include mitigating the effects of defects on density gradient measurements. Defects typically reduce the amount of light focused by the container 2300, such that defects on the container 2300 result in lower voltage measurements. In some examples, the method 2400 may include scanning along the outer diameter D at each measurement point along the length L of the container 2300, such that lower voltage measurements that may be caused by defects are ignored because the width of the scan along the outer diameter D may be greater than the width of the defect. In some additional examples, a best fit polynomial may be performed to match the curvature of the horizontal scan across the outer diameter D to replace the erroneous data input, which may further reduce the effects of defects, scratches, cracks, stains, dirt, etc. on the container 2300.

[0188] In some further examples, the entire surface area of ​​the container 2300 is scanned with a solid-state array device to capture all points in two dimensions at once. Such a technique can reduce the amount of time used to measure the density gradient along the length L of the container 2300, and also mitigate the effects of misalignment of the container and imperfections on the container.

[0189] Fig. 27A cross-sectional view of a container 2300 held by a holder 2320 of the system 100 is shown from a perspective looking down into the container 2300. Fig. 27 As shown, the emitter 714 emits light LR toward the container 2300, and the light LR after passing through the container 2300 is received by the detector 716 at the opposite side of the container 2300. In this example, the container 2300 has a diameter of 9 / 16 inches. In some examples, the light LR is infrared. The container 2300 can be tilted in the +y and -y directions along the y-axis relative to the emitter 714 and the detector 716 of the sensor assembly 712, and the container 2300 can be tilted in the +x and -x directions along the x-axis relative to the emitter 714 and the detector 716.

[0190] Table 3 shows the voltage measurements detected by the detector 716 when the container 2300 is tilted in the +y and -y directions along the y-axis and when the container 2300 is tilted in the +x and -x directions along the x-axis. As shown in Table 3, the variance of the voltage measurements along the y-axis is greater than the variance along the x-axis, making the voltage measurements (i.e., density gradient measurements) much more sensitive to container position errors on the y-axis (perpendicular to the ray LR) than to container position errors on the x-axis (parallel to the ray LR).

[0191]

[0192] Table 3

[0193] Fig.28 An example of a graph 2800 showing the mitigation of mechanical positioning errors is illustrated. In this illustrative example, graph 2800 includes a first graph 2802, a second graph 2804, and a third graph 2804' of voltage (y-axis) and container length (x-axis). Each of first graph 2802, second graph 2804, and third graph 2804' is measured for a container made of a polypropylene material having a diameter of 9 / 16 inches and filled with 100% DI water.

[0194] In the first graph 2802, the container is properly positioned so that the centerline CL of the container is aligned with the vertical axis VA of the sensor assembly 712. In the second graph 2804, the container is intentionally tilted along the y-axis so that the centerline CL of the container is not aligned with the vertical axis VA of the sensor assembly 712 (see Fig.23 ). In the third graph 2804', the container is intentionally tilted along the y-axis as in the second graph 2804, however, the density gradient measurement is made along the centerline CL of the container according to the operation of the method 2400. Fig.28, the bottom portion of the container occupies a portion of about 0 to 14 mm, so that this portion of the first graph 2802, the second graph 2804, and the third graph 2804' can be ignored.

[0195] like Fig.28 As shown, the second graph 2804 has a greater downward slope than the slope of the first graph 2802. This may be due to the misalignment between the centerline CL of the container and the vertical axis VA of the sensor assembly 712 along the y-axis. As an illustrative example, the difference in slope between the first graph 2802 and the second graph 2804 is greatest near the top of the container (i.e., to the right of the x-axis) due to the larger displacement between the centerline CL of the container and the vertical axis VA of the sensor assembly 712 at the top of the container for the second graph 2804. The third graph 2804' substantially corresponds to the first graph 2802, showing that the method 2400 of making the sensor assembly 712 measure along the centerline CL of the container by moving the sensor assembly 712 in two dimensions (such as in the vertical dimension along the length L of the container and in the horizontal dimension along the outer diameter D of the container) to mitigate the mechanical positioning error present in the second graph 2804.

[0196] Fig.29 Another example of a graph 2900 illustrating the mitigation of mechanical positioning errors according to method 2400 is illustrated. In this example, graph 2900 includes a first graph 2902, a second graph 2904, and a third graph 2904' of voltage (y-axis) and container length (x-axis). Each of the first graph 2902, the second graph 2904, and the third graph 2904' is measured for a container made of PETG material having a diameter of 9 / 16 inches and filled with 100% DI water. In the first graph 2902, the container is properly positioned so that the centerline CL of the container is aligned with the vertical axis VA of the sensor assembly 712. In the second graph 2904, the container is intentionally tilted along the y-axis so that the centerline CL of the container is not aligned with the vertical axis VA of the sensor assembly 712 (see Fig.23 ). In the third graph 2904', the container is intentionally tilted along the y-axis as in the second graph 2904, however, the density gradient measurement is made along the centerline CL of the container according to the operation of the method 2400. Fig.29 , the bottom portion of the container occupies a length of about 0 to 14 mm, so that this portion of the first graph 2902, the second graph 2904, and the third graph 2904' can be ignored.

[0197] Similar to the above-described graph 2800, the second graph 2904 has a greater downward slope than the slope of the first graph 2902. This may be due to the misalignment between the centerline CL of the container and the vertical axis VA of the sensor assembly 712. In this example, the difference in slope between the first graph 2902 and the second graph 2904 is greatest near the top of the container (i.e., to the right of the x-axis) due to the larger displacement between the centerline CL of the container and the vertical axis VA of the sensor assembly 712 at the top of the container for the second graph 2904. The third graph 2904' substantially corresponds to the first graph 2902, illustrating the method 2400 of mitigating the mechanical positioning error present in the second graph 2904 by moving the sensor assembly 712 in two dimensions (e.g., in the vertical dimension along the length L of the container and in the horizontal dimension along the outer diameter D of the container) to make the sensor assembly 712 measure along the centerline CL of the container.

[0198] Fig.30 Another example of a graph 3000 illustrating the mitigation of mechanical positioning errors according to method 2400 is illustrated. In this illustrative example, a first graph 3002 and a second graph 3004 of voltage (y-axis) and container length (x-axis) are shown. Each of the first graph 3002 and the second graph 3004 are measured in a container made of PETG material having a diameter of 9 / 16 inches for a 5%, 15%, 25%, and 35% sucrose step gradient. In the first graph 3002, the container is tilted on the y-axis so that each of the steps in the first graph 3002 has a significant downward slope along the container length (x-axis). In the second graph 3004, the container is tilted on the y-axis (similar to in the first graph 3002). However, the second graph 3004 is generated by method 2400, which causes the sensor assembly 712 to make voltage measurements along the centerline CL of the container. As shown in FIG. Fig.30 As shown, the slope of each of the steps in the second graph 3004 is much more linear (e.g., flatter) than the steps in the first graph 3002. Fig.30 Further shown is a method 2400 of mitigating mechanical positioning errors.

[0199] At any given location along the length L of the container in which the density gradient is dispensed, the measurements recorded by the sensor assembly 712 should be the same regardless of the rotation of the container. However, in some cases, the container deforms such that the wall of the container does not have a uniform thickness around the circumference of the container.

[0200] Fig.31 Graph 3100 illustrates voltage measurements (y-axis) taken while rotating 45 degrees along the length of the container (x-axis). Fig.31For the different rotations shown (i.e., 0, 45, 90, 135, 180, 225, 270, and 315 degrees), changes in the voltage measurements along the length of the container may produce density gradient measurement errors, especially when it is desired to measure the container only in one rotational orientation. In addition, rotating the container during the density gradient measurement may increase the complexity of the measurement device 700.

[0201] Fig.32 Schematically illustrated is another example of a measurement device 3200 that can mitigate errors from when the wall of a container 3210 does not have a uniform thickness around the circumference of the container. In this example embodiment, the measurement device 3200 includes a first pair 3202 of an emitter 3206 and a detector 3208 and a second pair 3204 of an emitter 3206' and a detector 3208'. The emitters 3206, 3206' and the detectors 3208, 3208' are positioned at 90 degree intervals relative to each other. The emitters 3206, 3206' are similar to the emitters 714, and the detectors 3208, 3208' are similar to the detectors 716 of the measurement device 700. For example, the emitters 3206 , 3206 ′ may emit light (eg, infrared) and the detectors 3208 , 3208 ′ may include photodiodes that measure a voltage of light transmitted from the emitters 3206 , 3206 ′ through the container 110 for measuring a density gradient dispensed in the container 3210 .

[0202] In some examples, the first pair 3202 and the second pair 3204 of emitters and detectors are mounted on a bracket, such as Figures 7 to 9 706 . When mounted on the carriage, the emitters 3206, 3206 'and the detectors 3208, 3208 'are fixed relative to each other, which allows these components to move up and down together on the rails 706. The motor 710 can be used to move the first pair 3202 and the second pair 3204 up and down the length of the container 3210 by moving the carriage along the rails 706 of the measurement device 700.

[0203] Fig.33 An example of a method 3300 for mitigating wall thickness variations around a perimeter of a container 3210 when measuring a density gradient dispensed in the container is schematically illustrated. In some examples, the perimeter includes a diameter or circumference of the container 3210. In some examples, the method 3300 forms part of operation 1112 in method 1100. The method 3300 may be performed by the system 100.

[0204] The method 3300 includes an operation 3302 of positioning a first pair 3202 and a second pair 3204 at a predetermined position along a length of a container 3210. In some examples, the predetermined position is at a distal end of the container 3210. The operation 3302 may include moving the first pair 3202 and the second pair 3204 using a motor while the container 3210 remains stationary to position the first pair 3202 and the second pair 3204 relative to the container 3210.

[0205] Next, method 3300 includes operation 3304 of measuring a first voltage from the transmission of light emitted by emitter 3206 and received by detector 3208 using first pair 3202 and measuring a second voltage from the transmission of light emitted by emitter 3206′ and received by detector 3208′ using second pair 3204. In some examples, the first voltage and the second voltage are measured simultaneously in operation 3304. In other examples, the first voltage and the second voltage are not measured simultaneously in operation 3304.

[0206] The method 3300 includes an operation 3306 of calculating an average value based on the first voltage and the second voltage measured in operation 3304. The wall thickness of the container 3210 may be thicker at one location, but is unlikely to be thicker at a location 90 degrees apart, so that the average value of the first voltage and the second voltage effectively mitigates errors in density gradient measurements that may be caused by variations in wall thickness around the perimeter of the container 3210. In addition, the container 3210 may have a blemish or scratch at one location, but is unlikely to have a blemish or scratch at a location 90 degrees apart, so that the average value of the first voltage and the second voltage effectively mitigates errors in density gradient measurements from blemishes, scratches, cracks, stains, dirt, etc.

[0207] Next, the method 3300 includes an operation 3308 of determining whether additional density gradient measurements are required along the length of the vessel 3210. When an additional density gradient is required (i.e., "yes" in operation 3308), the method 3300 may repeat operations 3302 to 3306 to measure the density gradient at another location along the length of the vessel 3210. Otherwise, when an additional density gradient is not required (i.e., "no" in operation 3308), the method 3300 terminates at operation 3310.

[0208] Fig.34A graph 3400 is illustrated showing the mitigation of wall thickness variations around a perimeter of a container 3210 by the method 3300. As described above, in some examples, the perimeter includes a diameter or circumference of the container 3210. The graph 3400 includes a graph of average voltage measurements (y-axis) taken while rotating 45 degrees along the length (x-axis) of the container 3210. For example, the average voltage between the emitters 3206 and detectors 3208 of the first pair 3202 and the second pair 3204 spaced 90 degrees apart is taken at positions 0 to 90 degrees, 45 to 135 degrees, 90 to 180 degrees, 135 to 225 degrees, 180 to 270 degrees, 225 to 315 degrees, 270 to 360 degrees, and 315 to 45 degrees. Fig.34 In, with Fig.31 The variation in voltage measurements along the length of container 3210 is shown as being significantly reduced in graph 3400 as compared to graph 3100 of FIG. 3400. In some examples, the variation in voltage measurements is reduced by approximately 56.6%.

[0209] Fig.35 An example of a density gradient 3500 dispensed by system 100 into container 110 is illustrated. Density gradient 3500 includes dispense rates (y-axis) of DI water, density modifier, concentrated buffer solution, and additives for each step along the length (x-axis) of container 110. In this illustrative example, density gradient 3500 is a continuous gradient including 41 steps along its length, forming a 5% to 40% continuous gradient with 10 mM buffer solution and four steps (e.g., steps 3, 7, 21, and 30 to 33) including additives.

[0210] The concentration of the density modifier is greater than the maximum concentration of the density gradient 3500. In this illustrative example, a 50% concentration of density modifier is used to support a maximum concentration of 40%. As the dispensing rate of the density modifier increases, the density of a given step in the density gradient increases. As the dispensing rate of the density modifier remains constant, the density of the steps dispensed into the container remains constant along the length of the container. As the dispensing rate of the density modifier decreases, the density of a given step in the density gradient decreases.

[0211] Fig.36 Schematically shows the generation of Fig.35 Method 3600 of density gradient 3500 shown in FIG. Method 3600 includes operation 3602 of dividing density gradient 3500 into a plurality of steps. Fig.35 In the illustrative example shown, the density gradient 3500 is a continuous gradient divided into 41 individual steps. The number of steps can be increased or decreased depending on the desired shape and size of the density gradient.

[0212] Next, method 3600 includes an operation 3604 of calculating the distribution rate of the component in each step of density gradient 3500. Fig.35 As shown, DI water and density modifier constitute the majority of the volume in each step of density gradient 3500. The dispensing rate of DI water is proportionally reduced relative to the increase in the dispensing rate of density modifier to maintain a constant volume for each step of density gradient 3500 and simultaneously maintain a continuous gradient profile.

[0213] exist Fig.35 In the illustrative example shown, the rate of dispensing of the concentrated buffer solution is low and remains constant along the length of the density gradient 3500. Whenever an additive is dispensed (e.g., steps 3, 7, 21, and 30 to 33), the volume of the additive is subtracted from the DI water to maintain a constant volume of the steps in the density gradient 3500 while maintaining a continuous gradient profile.

[0214] The method 3600 includes an operation 3606 for dispensing each step based on the volume and dispensing rate calculated for the component in each step in operation 3604. Operation 3606 can follow the method 500 described above, such that the distal end 112 of the probe 108 is lowered toward the bottom of the internal volume of the container 110, and the probe 108 dispenses each step of successively increasing density to form the density gradient 3500.

[0215] exist Fig.35 , the steps of density gradient 3500 are dispensed from left to right along the x-axis, starting with the first step having the lightest density (e.g., the highest concentration of DI water and the lowest concentration of density modifier). As subsequent steps having heavier densities are dispensed into the container, the first step is pushed upward in container 110. The last step is the heaviest step (e.g., the lowest concentration of DI water and the highest concentration of density modifier). Density gradient 3500 is a continuous gradient from 5% to 40%, such that the first step has 5% density modifier and the last step has 40% density modifier.

[0216] In addition to the illustrative examples provided Fig.35In addition to the density gradient 3500 shown in , method 3600 can be performed to generate various types of density gradients with various volumes, gradients and / or shapes. For example, method 3600 can be performed to generate a 10% to 25% density gradient, such as by dividing the gradient (operation 3602) into a smaller number of steps than the 41 steps shown for density gradient 3500, calculating the volume and distribution rate of the components in each step (operation 3604), and distributing the steps to form a 10% to 25% density gradient (operation 3606). As in the above example, the lightest step (i.e., 10% density modifier) ​​is distributed first, and then the steps of successively increasing density are distributed until the heaviest step (i.e., 25% density modifier) ​​is distributed. In other examples, method 3600 can be performed to generate more complex density gradients, such as exponential gradients, s-shaped gradients, and other desired shapes.

[0217] Fig.37 An example of a density gradient 3700 dispensed into a container 110 by the system 100 is shown. In this illustrative example, the density gradient 3700 is a stepped gradient comprising five different steps 3702a to 3702e separated by an interface 3704. The density gradient 3700 may be formed after the operation of the method 3600. The interface 3704 between the steps 3702a to 3702e is visible as a slightly darker line. It may be desirable that each step in the steps 3702a to 3702e has a uniform density, and that the interface 3704 between the steps 3702a to 3702e is as narrow as possible. It may also be desirable to dispense the density gradient 3700 as quickly as possible to maximize the throughput of the system 100 while mitigating mixing between the steps 3702a to 3702e in the container.

[0218] Fig.38 An example of a method 3800 for generating a density gradient is schematically shown. System 100 may perform method 3800 to generate a stepped gradient, such as Fig.37 3700. Method 3800 reduces the time system 100 takes to generate density gradient 3700 while minimizing mixing at interfaces 3704 between steps 3702 of the gradient. In another example, system 100 may perform method 3800 to generate a continuous gradient, such as Fig.35 The density gradient 3500 is shown in FIG.

[0219] Fig.39 An example of a diagram 3900 showing method 3800 implemented by system 100 is illustrated. Fig.39 As shown, graph 3900 includes a first graph 3902 of voltage measurements (left y-axis) versus time (x-axis) for density gradient 3700, and a second graph 3904 of dispense velocity (right y-axis) versus time (x-axis).

[0220] Now refer to Figure 37 to Figure 39 , method 3800 includes an operation 3802 of dispensing a first rung 3702a for density gradient 3700. First rung 3702a has the lightest density (e.g., the highest concentration of DI water and the lowest concentration of density modifier) ​​such that when subsequent runs with heavier densities are dispensed into container 110, first rung 3702a will be pushed upward in container 110. Fig.39 As shown, the first rung 3702a is dispensed at maximum speed until it reaches the interface 3704 with the second rung 3702b (e.g., at a time of about 27 seconds). In this illustrative example, the maximum speed is about 20 mL / minute. The first rung 3702a is dispensed at maximum speed because there is no risk of mixing with other runs.

[0221] The method 3800 includes an operation 3804 of reducing a dispensing velocity at an interface 3704 between a first step 3702a and a second step 3702b. Fig.39 The maximum speed is shown decreasing to a minimum speed. In this illustrative example, the minimum speed is about 2 mL / min. Fig.39 As shown, the dispensing speed shows a significant decrease at the interface 3704 between the first step 3702a and the second step 3702b. For example, the maximum speed decreases to the minimum speed in a short time of about 1 second.

[0222] Next, method 3800 includes an operation 3806 of increasing the dispensing speed of the second step 3702b from a minimum speed until a maximum speed is reached. Fig.39 In the illustrative example shown, the dispensing speed increases exponentially until a maximum speed is reached at approximately 30 seconds. When the second rung 3702b is dispensed, this is caused by Fig.39 The exponential curve in the second graph 3904 is shown. Other shapes for increasing the dispensing speed from the minimum speed to the maximum speed are possible, so that by way of illustrative example, Fig.39 The exponential curve shown in .

[0223] The adjustments to the dispensing speed in operations 3804 and 3806 (e.g., decreasing the dispensing speed from a maximum speed to a minimum speed, increasing the dispensing speed from a minimum speed to a maximum speed) are performed by the pump 104, which adjusts the flow rate for pumping the components from the reservoir 102 into the manifold and mixing chamber 106. Alternative examples for adjusting the dispensing speed are possible.

[0224] Next, method 3800 includes an operation 3808 of determining whether the density gradient includes another step. When the density gradient includes another step (i.e., "yes" in operation 3808), method 3800 repeats operations 3804, 3806 to assign additional steps. Fig.37 and Fig.39 In the illustrative example shown, density gradient 3700 includes five separate steps, with interfaces 3704 occurring at approximately 27, 39, 51, and 63 seconds. Operations 3804, 3806 may be repeated to generate each step 3702 in density gradient 3700.

[0225] If the density gradient does not include additional steps (i.e., "No" in operation 3808), method 3800 stops dispensing at operation 3810. Method 3800 reduces the time used to generate the density gradient while also mitigating mixing between steps of the density gradient by adjusting the dispensing speed when creating the interface. For example, method 3800 mitigates mixing by slowly dispensing (e.g., at a minimum speed) when starting a new step at the interface. Then, by increasing the dispensing speed until a maximum speed is reached, method 3800 can reduce the total time to dispense the density gradient. Method 3800 enables system 100 to generate a sharp interface between steps of the density gradient while dispensing the density gradient in a minimum amount of time.

[0226] As will now be described, the process of manually making a collection of identical density gradients can be difficult because there is often a large amount of inherent variation between manually made density gradients. For example, sources of variation can include the concentration of the mixing components and the rate at which the mixing components are dispensed, which can result in day-to-day and user-to-user variation in the manually made density gradients. As will now be described in greater detail, the following methods and techniques can be implemented on system 100 to replicate a density gradient that closely matches a previous density gradient.

[0227] Fig.40 Schematically illustrated is an example of a method 4000 of replicating a density gradient that may be performed on the system 100. The method 4000 may be repeated to replicate as many density gradients as desired.

[0228] like Fig.40 As shown, method 4000 includes an operation 4002 of measuring a density gradient selected for replication. In some examples, the density gradient is generated by system 100, such as according to the operation of method 500 described above. In other examples, the density gradient is generated by another system. In addition, the density gradient can be generated by the same user of system 100 or by a different user.

[0229] Operation 4002 may include scanning the container 110 using the sensor assembly 712 to detect light transmission through the container for measuring a voltage corresponding to the density. The measurement results may be used to create a distribution of the density gradient. The distribution may include a correlation between the voltage measurement results and the position along the length L of the container 110. As an example, the system 100 may measure the voltage at about 320 points along a length of about 80 mm of the container 110 to create a distribution of the density gradient.

[0230] Next, method 4000 includes an operation 4004 of storing the distribution of density gradients on a non-volatile memory device. The distribution of density gradients can be stored on the non-volatile memory of system 100. In other examples, the distribution of density gradients can be stored on the non-volatile memory of an external storage device. In some examples, the external storage device can include a portable device, such as a USB flash drive and similar data storage devices that can be inserted into or otherwise connected to system 100. In other examples, the external storage device can be included on a remote server, which can be accessed via a communication network 5220 (such as Fig.52 5220) shown in the figure to connect to the system 100.

[0231] The profile stored in operation 4004 may be used by the system 100 at any time to replicate a density gradient. The profile of the density gradient may be stored as a "favorite" density gradient profile, which may be selected for replication using the display 132 of the system 100. The system 100 may store multiple favorite density gradient profiles.

[0232] Next, method 4000 includes duplicating the density gradient in operation 4006. Operation 4006 includes retrieving a profile stored for the density gradient and then controlling pump 104 to pump the duplicate density gradient into container 110 based on the profile. The duplication of the density gradient in operation 4006 may be performed according to the operations of method 500 described above.

[0233] Method 4000 may also include an operation 4008 of verifying the quality of the replicated density gradient from operation 4006. Operation 4008 may include measuring the replicated density gradient in a manner similar to measuring the original density gradient in operation 4002. For example, operation 4008 may include measuring the replicated density gradient at the same point measured for the original density gradient. The measurements and / or distribution of the replicated density gradient are compared with the measurements and / or distribution of the original density gradient to determine whether they are within a predetermined tolerance.

[0234] When the measurement and / or distribution is within the predetermined tolerance, the replicated density gradient is approved. Otherwise, when the measurement and / or distribution is outside the predetermined tolerance, the replicated density gradient is rejected.

[0235] In some examples, operation 4008 may also include displaying the density gradient and the distribution of the copied density gradient side-by-side on display 132 of system 100. This enables a user of system 100 to view and / or confirm the similarity between the copied density gradient and the original density gradient.

[0236] When replicating a density gradient based on a distribution of a previous density gradient, the measurements used to generate the distribution should be processed to remove noise that may interfere with the accuracy and fidelity of the replicated density gradient. For example, measurements obtained from scanning a container in which a density gradient is dispensed may include noise from optical effects that may interfere with density calculations. This is particularly true for graded density gradients that have larger steps in density, which result in larger differences in refractive index between steps, thereby causing noise in density measurements near the interfaces between steps. Noise should be removed from the distribution of the density gradient because otherwise the noise will cause errors when replicating a new density gradient based on the distribution.

[0237] Fig.41 An example of a density gradient distribution 4100 is illustrated prior to being processed by the system 100. The density gradient distribution 4100 includes noise that could potentially interfere with the reproduction of the density gradient distribution 4100 by the system 100. Fig.41 , density gradient profile 4100 includes voltage measurements (y-axis) indicating the density along the length of the container (x-axis) for a graded density gradient having 5%, 15%, 25%, and 35% steps dispensed in a 9 / 16 inch diameter container.

[0238] Flat portions 4102 in density gradient profile 4100 represent steps in a graded density gradient. Density gradient profile 4100 also includes measurement swings 4104 near lengths of approximately 30 mm, 52 mm, and 73 mm, which are noise caused by optical effects at the interfaces between steps of the density gradient. Measurement swings 4104 are caused by large differences in refractive index between steps of the density gradient.

[0239] It may be desirable to remove and / or replace the measurement swing 4104 prior to replicating a density gradient using the density gradient profile 4100. Otherwise, the measurement swing 4104 may result in errors and loss of fidelity when the density gradient is replicated by the system 100 (such as according to operation of the method 4000).

[0240] like Fig.41As shown, density gradient profile 4100 includes measurements 4106 from the bottom of the container (e.g., below 10 mm). The curvature of the bottom portion 304 of the container can optically interfere with the measurement of the density gradient, causing significant measurement swings. When replicating the density gradient, it is also desirable to remove and / or replace the measurements 4106 from the bottom portion 304.

[0241] Fig.42 An example of a method 4200 for processing a density gradient distribution 4100 to remove noise and defects that may interfere with the system 100 reproducing the density gradient distribution is schematically shown. In some examples, the method 4200 forms part of operation 4002 in method 4000, such that the distribution of the density gradient is processed according to the method 4200 before being stored in operation 4004.

[0242] Method 4200 includes an operation 4202 of identifying the locations of interfaces between steps in a density gradient. Operation 4202 may include using a mathematical differentiation technique on density gradient profile 4100 to produce Fig.41 The second graph 4100' shown in . The negative peaks 4104' in the second graph 4100' identify the locations of the interfaces between the steps of the density gradient. In this illustrative example, the interfaces are located at distances of approximately 30 mm, 52 mm, and 73 mm along the length of the container.

[0243] Next, method 4200 includes operation 4204 of replacing the measurement value at the location of the interface identified in operation 4202. Operation 4204 includes calculating a first average measurement value from a set of measurement values ​​before the location of the interface, calculating a second average measurement value from a set of measurement values ​​after the location of the interface, and replacing the measurement value at the location of the interface with the first average measurement value and the second average measurement value.

[0244] Fig.43 An example of a modified density gradient profile 4300 generated according to the operations of the method 4200 for replacing the density gradient profile 4100 is illustrated. Fig.43 Calculation of the first average measurement and the second average measurement in operation 4204 is shown. In this illustrative example, the interfaces between the steps of the density gradient each have a total length of about 8 mm, of which about 4 mm belongs to the previous step and about 4 mm belongs to the next step. The first average measurement is calculated from a set of 10 measurements before each interface, and the second average measurement is calculated from a set of 10 measurements after each interface. In other examples, the first average measurement and the second average measurement can be calculated from more or less than 10 measurements before and after each interface.

[0245] like Fig.43As shown, the measurement swings 4104 are each replaced by a first region 4302 based on a first average measurement value and a second region 4304 based on a second average measurement value. By replacing the measurement swings 4104 with the first region 4302 and the second region 4304, the modified density gradient profile 4300 is smoother than the density gradient profile 4100 because the measurement swings 4104 detected at the interfaces between the steps of the graded density gradient are eliminated. This can improve the accuracy and fidelity with which the system 100 replicates the graded density gradient.

[0246] Return to reference Fig.42 , the method 4200 may also include an operation 4206 of adjusting the measurement 4106 at the bottom of the container (e.g., below 10 mm), which is where the bottom portion of the container produces significant measurement swings in the density gradient profile 4100. The modified density gradient profile 4300 replaces the measurement 4106 with a linear ramp 4306 of increasing density dispensed below 10 mm. This can provide a buffer for very heavy density materials dispensed at the bottom of the container. The linear ramp 4306 prevents the creation of a sharp interface at the bottom of the container, which can improve the replication of the graded density gradient by the system 100.

[0247] Fig.44 An example of a graph 4400 showing a comparison of a first density gradient 4402 and a second density gradient 4404 copied from the first density gradient by system 100 is illustrated. In this illustrative example, first density gradient 4402 and second density gradient 4404 are closely aligned with each other such that second density gradient 4404 has good fidelity relative to first density gradient 4402.

[0248] Fig.45 Another example of a method 4500 for processing a density gradient profile for replication by the system 100 is schematically shown. The method 4500 includes an operation 4502 of obtaining a density gradient profile. The density gradient profile includes voltage measurements that can be detected by a sensor of the system 100 scanning a container in which the density gradient is dispensed. The sensor can scan the container from bottom to top or from top to bottom in one scan. The voltage measurements are related to density.

[0249] As an example, the density gradient profile may include voltage measurements detected for every 0.25 mm of the length of the container. In some examples, the density gradient profile includes about 320 voltage measurements along the length of the container.

[0250] Method 4500 includes an operation 4504 of determining whether the density gradient profile is from a stepped density gradient or a linear density gradient. The determination can be based on characteristics of the voltage measurements in the density gradient profile obtained in operation 4502. For example, when the voltage measurements decrease incrementally by a small amount along the length of the container, the voltage measurements can indicate a linear density gradient. As another example, when the voltage measurements remain constant and then decrease by a large amount, the voltage measurements can indicate a stepped density gradient.

[0251] When the density gradient is determined to be a linear density gradient (i.e., "linear" in operation 4504), method 4500 may skip certain processing operations and proceed to operation 4508, which is described in more detail below. When the density gradient is determined to be a graded density gradient (i.e., "graded" in operation 4504), method 4500 proceeds to operation 4506 to remove optical anomalies that may occur due to large changes in refractive index caused by different densities at the interfaces between steps of the graded density gradient. In some examples, the optical anomalies removed in operation 4506 include Gouy phase shifts.

[0252] Fig.46 An example of a density gradient profile 4600 prior to being processed by method 4500 is illustrated. Density gradient profile 4600 includes voltage measurements (y-axis) measured over the length of a container (x-axis) in which the density gradient is dispensed. The bottom of the container is on the left side of density gradient profile 4600 (e.g., 0 mm), and the top of the container is on the right side of density gradient profile 4600 (e.g., 80 mm). In this illustrative example, density gradient profile 4600 is for a graded density gradient having 0%, 15%, 30%, and 40% sucrose density modifier.

[0253] exist Fig.46 , the density gradient profile 4600 begins at a length of about 14 mm of the container and ends at a length of about 76 mm. The density gradient profile 4600 includes a shorter first step 4602a (e.g., 40% sucrose) because most of the first step 4602a is located at the bottom of the container, which is removed from the profile. In this example, the first step 4602a has a constant voltage measurement of about 2000 mV, representing a level of 40% sucrose.

[0254] The density gradient profile 4600 exhibits a large measurement swing at the interface 4604a that begins at a length of about 16 mm and ends at a length of about 25 mm. The large measurement swing is a Gouy phase shift at the interface 4604a between the first step 4602a and the second step 4602b due to the steps having different densities, which produces a fringe effect that affects the transmission of light through the graded density gradient.

[0255] The second step 4602b (e.g., 30% sucrose) of the density gradient profile 4600 begins at a length of about 25 mm and ends at a length of about 34 mm. In this illustrative example, the second step 4602b has a constant voltage measurement of about 1890 mV, representing a level of 30% sucrose. Steps 4602 and interfaces 4604 alternate in the density gradient profile 4600 along the length of the container until the meniscus 4606 of the density gradient is reached.

[0256] In operation 4506, optical anomalies such as Gouy phase shifts are removed. In some examples, Gouy phase shifts are removed using operations similar to those described above in method 4200. For example, Gouy phase shifts can be removed by determining the location of interface 4604 on density gradient profile 4600. This can be accomplished by using mathematical differentiation techniques on density gradient profile 4600.

[0257] Fig.47 An example of a differential graph 4700 is illustrated after mathematically differentiating the density gradient profile 4600. In this illustrative example, negative peaks 4704a-4704c at distances of approximately 21 mm, 38 mm, and 56 mm identify the locations of interfaces 4604 between steps 4602 of the density gradient, while negative peak 4704d identifies the location of the meniscus 4606 of the density gradient.

[0258] Fig.48 is an enlarged view of differential graph 4700. In operation 4506, the measured value affected by the Gouy phase shift at interface 4604 is replaced by value 4702 determined from differential graph 4700. For example, a difference having a zero or positive minimum value such that it is the measured value of the flattest point on a particular step may be selected for the entire step, including replacing the measured value affected by the Gouy phase shift at interface 4604. Negative slope values ​​should not be used. In another example, the first average measured value and the second average measured value calculated in method 4200 (see Fig.43 The modified density gradient profile 4300) can be used to replace measurements affected by the Gouy phase shift at the interface 4604.

[0259] Fig.49 4700 after the measured values ​​affected by optical effects such as Gouy phase shift are replaced by the values ​​determined from the differential curve graph 4700. Fig.46 An example of a comparison of the density gradient distribution 4600 and the adjusted density gradient distribution 4900. Fig.50 is a magnified view of the adjusted density gradient profile 4900. Fig.49 and Fig.50As shown, the adjusted density gradient profile 4900 has sharp interfaces 4904 between steps 4902 so that the optical effects from the Gouy phase shift are removed from the adjusted density gradient profile 4900.

[0260] Return to reference Fig.45 , method 4500 includes an operation 4508 of removing and / or replacing measurements obtained from the bottom of the container. As described above, the curvature of the bottom of the container may interfere with the transmission of light used to measure the density gradient. Measurements from the bottom are useless because they may include large measurement swings in the density gradient distribution. As an example, measurements from the length of the container below 14 mm are removed in operation 4508. This is shown on the left side of the adjusted density gradient distribution 4900.

[0261] In some examples, the measurement from the bottom of the container is replaced by a value of the heaviest density below 14 mm greater than or equal to the length of the container. In some examples, the measurement from the bottom of the container is replaced by a linear ramp 4306 of increasing density, such as Fig.43 shown.

[0262] Then, method 4500 includes an operation 4510 of removing and / or replacing the measurement at which meniscus 4606 is located. As described above, the location of meniscus 4606 can be determined using Fig.47 4604d). Similar to the interface 4604 between the steps 4602 of the density gradient profile 4600, the meniscus 4606 may cause optical effects due to the large change in refractive index caused by the density difference between the last step of the density gradient (i.e., step 4602d) and the volume of air above the density gradient in the container. In some examples, the optical properties of the meniscus 4606 may also cause Gouy phase shifts. Fig.49 and Fig.50 The left side of the adjusted density gradient profile 4900 shows the removal and / or replacement of measurements at the location where the meniscus 4606 is located.

[0263] Still refer to Fig.45 , method 4500 may also include Fig.49 and Fig.50 Operation 4512 converts the adjusted density gradient profile 4900 shown in FIG. 4 into computer-readable instructions for distributing a density gradient that can be used by system 100 to replicate the density gradient. Operation 4512 is performed regardless of whether the density gradient is a linear gradient or a step gradient. In some examples, the computer-readable instructions for distributing the adjusted density gradient profile 4900 are stored in a text file.

[0264] Fig.51 An example of a text file 5100 that can be executed by a processing device of the system 100 to assign a density gradient to a container 110 is shown. As an example, the text file 5100 is Fig.49 and Fig.50 4900. In this illustrative example, the text file 5100 includes a text conversion of the adjusted density gradient distribution 4900 shown in FIG. 4900. In this illustrative example, the text file 5100 includes a text conversion of the adjusted density gradient distribution 4900 along the length L of the container 110 (e.g., Fig.49 and Fig.50 In this example, the position along the length L of the container is in the range of 80 mm to 0 mm. The text file 5100 includes a first column 5102 of positions of the voltage values ​​(e.g., Fig.49 and Fig.50 y-axis in the second column 5104.

[0265] The voltage values ​​in the second column 5104 represent the refractive index, which corresponds to the density of the mixture of components from the reservoirs 102a to 102d. The system 100 can use the text file 5100 to calculate the dispensing rate of DI water and density modifier (e.g., 40% sucrose) for each position in the first column 5102 along the length L of the container 110 based on the voltage values ​​in the second column 5104. The system 100 independently controls the pumps 104a to 104d to control the dispensing rate of the components into the container 110, wherein the least dense step is dispensed first, followed by successively denser steps. This is shown in Fig.49 and Fig.50 5100, where the least dense step is shown. When system 100 executes text file 5100, system 100 generates a density gradient as a clone of the original density gradient.

[0266] Fig.52 An example of computing hardware for implementing aspects of the present disclosure of system 100 is schematically shown. The computing hardware includes a processing circuit having a memory for storing instructions that, when executed by the processing circuit, cause the processing circuit to perform the functions described herein.

[0267] like Fig.52 As shown in the example provided in , system 100 includes one or more processing devices 5202, memory storage devices 5204, and a system bus 5206 that couples the memory storage devices 5204 to the one or more processing devices 5202. The one or more processing devices 5202 may include a central processing unit (CPU).

[0268] like Fig.52As further shown in FIG. 5 , memory storage device 5204 may include random access memory (“RAM”) 5208 and read only memory (“ROM”) 5210. Basic input and output logic having basic routines to help transfer information between elements within system 100, such as during startup, may be stored in ROM 5210.

[0269] The system 100 may also include a mass storage device 5212, which may include an operating system 5214 and store software instructions 5216 and data. The mass storage device 5212 is connected to the processing device 5202 via the system bus 5206. The mass storage device 5212 and associated computer-readable data storage media provide non-volatile, non-transitory storage for the system 100.

[0270] Although the description of computer-readable data storage media contained herein refers to mass storage device 5212, it should be understood by those skilled in the art that computer-readable data storage media can be any available non-transitory physical device or article from which system 100 can read data and / or instructions. Computer-readable storage media can consist entirely of non-transitory media. Mass storage device 5212 is an example of a computer-readable storage device.

[0271] Computer-readable data storage media include volatile and nonvolatile, removable and non-removable media implemented in any method or technology for storage of information such as computer-readable software instructions, data structures, program modules or other data. Example types of computer-readable data storage media include, but are not limited to, RAM, ROM, EPROM, EEPROM, flash memory or other solid-state memory technology, or any other medium that can be used to store information and can be accessed by a device.

[0272] The system 100 can operate in a networked environment using logical connections to other devices through a communication network 5220. The system 100 is connected to the communication network 5220 via a network interface unit 5218 connected to the system bus 5206. The network interface unit 5218 can be connected to additional types of communication networks and devices, including through Bluetooth, Wi-Fi, and cellular telecommunication networks (including 4G and 5G networks). The network interface unit 5218 can connect the system 100 to additional networks, systems, and devices. The system 100 also includes an input / output unit 5222 for receiving and processing input and output from peripheral devices.

[0273] The mass storage device 5212 and the RAM 5208 may store software instructions and data. The software instructions may include an operating system 5214 for operating the system 100. The mass storage device 5212 and / or the RAM 5208 may also store software instructions 5216 that, when executed by the processing device 5202, provide the functionality of the system 100 discussed herein. As described above, the mass storage device 5212 and / or the RAM 5208 may store the distribution of the density gradient measured by the measurement device 700.

[0274] The various embodiments described above are provided by way of illustration only and should not be construed as limiting in any way. Various modifications may be made to the above embodiments without departing from the true spirit and scope of the present disclosure.

[0275] Embodiments of the present disclosure may be described with reference to the following numbered clauses, with preferred features listed in the dependent clauses:

[0276] 1. A system for non-destructively measuring the density gradient of components used in centrifugation, the system comprising:

[0277] Measuring equipment comprising:

[0278] Sensor components;

[0279] a motor coupled to the sensor assembly; and

[0280] A processing circuit having a memory for storing instructions which, when executed by the processing circuit, cause the processing circuit to:

[0281] Using a motor to move the sensor assembly along the length of the density gradient of the component;

[0282] obtaining measurements from the sensor assembly as the sensor assembly moves along the length of the density gradient of the component; and

[0283] A distribution of the density gradient of the components is generated based on the measurement results.

[0284] 2. A system according to clause 1, wherein the instructions, when executed by the processing circuit, further cause the processing circuit to:

[0285] The distribution is adjusted by removing measurements from the bottom portion of the vessel in which the density gradient of the components is contained.

[0286] 3. A system according to clause 1, wherein the instructions, when executed by the processing circuit, further cause the processing circuit to:

[0287] identifying the location of the meniscus of the density gradient of the components; and

[0288] The distribution is adjusted to remove measurements from the location of the meniscus.

[0289] 4. A system according to clause 3, wherein the position of the meniscus is identified by identifying a first derivative minimum of the measurement result.

[0290] 5. A system according to clause 1, wherein the instructions, when executed by the processing circuit, further cause the processing circuit to:

[0291] The measurements are normalized based on at least one of a material and a size of a container in which the density gradient of components is contained.

[0292] 6. A system according to clause 5, wherein the instructions, when executed by the processing circuit, further cause the processing circuit to:

[0293] obtaining measurements from the sensor assembly along the length of the container prior to dispensing the density gradient of the components into the container;

[0294] comparing measurements from the container before the density gradient of components is dispensed into the container to an expected measurement range for a container of predetermined material and size; and

[0295] At least one of a material and a size of the container is determined based on the comparison to an expected measurement range.

[0296] 7. A system according to clause 1, wherein the instructions, when executed by the processing circuit, further cause the processing circuit to:

[0297] Mitigating the effects on the distribution of density gradients caused by defects and wall thickness variations on containers containing density gradients of components therein.

[0298] 8. A system according to clause 7, wherein the instructions, when executed by the processing circuit, further cause the processing circuit to:

[0299] obtaining measurements from the sensor assembly along the length of the container when the container is empty;

[0300] Calculating an average of the measurements along the length of the container when the container is empty;

[0301] generating a difference between an average of the measurements and the measurements along the length of the container when the container is empty; and

[0302] Adjust the distribution of the density gradient of the components by adding differences.

[0303] 9. The system of clause 1, wherein the instructions, when executed by the processing circuit, further cause the processing circuit to:

[0304] Positioning errors in the distribution of density gradients of components caused by misalignment of containers into which the density gradients of components are dispensed are mitigated.

[0305] 10. The system of clause 9, wherein the instructions, when executed by the processing circuit, further cause the processing circuit to:

[0306] generating a centerline of the vessel; and

[0307] The sensor assembly is moved in at least two dimensions along the centerline of the container to obtain measurements of the distribution of the density gradient used to generate the component.

[0308] 11. A system according to clause 10, wherein the instructions, when executed by the processing circuit, further cause the processing circuit to:

[0309] positioning the sensor assembly near the proximal end of the vessel;

[0310] Scanning a cross section of the vessel near the proximal end;

[0311] determining a first position of the centerline near a proximal end of the container;

[0312] positioning the sensor assembly near the distal end of the container;

[0313] Scan the diameter of the vessel near the distal end;

[0314] determining a second position of the centerline near a distal end of the container; and

[0315] The centerline of the container is generated by linearly connecting a first location near the proximal end of the centerline to a second location near the distal end of the centerline.

[0316] 12. The system of clause 1, wherein the sensor assembly comprises a first pair of emitters and detectors mounted on a bracket, and a second pair of emitters and detectors mounted on the bracket, the first pair and the second pair being spaced 90 degrees apart on the bracket.

[0317] 13. A system according to clause 12, wherein the instructions, when executed by the processing circuit, further cause the processing circuit to:

[0318] moving the carriage using a motor to position the first pair of emitters and detectors and the second pair of emitters and detectors at locations along the length of the container in which the density gradient of the components is dispensed;

[0319] obtaining a first measurement using a first pair of emitters and detectors;

[0320] obtaining a second measurement using a second pair of emitters and detectors;

[0321] calculating an average measurement result based on the first measurement result and the second measurement result; and

[0322] A distribution of the density gradient of the components is generated using averaged measurements to mitigate errors from wall thickness variations around the perimeter of the container.

[0323] 14. The system of clause 1, wherein the sensor assembly comprises:

[0324] a transmitter that transmits an illumination signal, and

[0325] A detector measures the intensity of the illumination signal after it has been transmitted through the density gradient of the components.

[0326] 15. The system of clause 1, wherein the instructions, when executed by the processing circuit, further cause the processing circuit to:

[0327] Multiple measurements are obtained at each point along the length of the density gradient of the component, and a distribution of the density gradient of the component is generated using an average of the multiple measurements calculated for each point along the length of the density gradient of the component.

[0328] 16. A system according to clause 1, wherein the distribution is generated for a graded density gradient of components or a continuous density gradient of components.

[0329] 17. A system for measuring the density gradient of components dispensed in a container for use in centrifugation, the system comprising:

[0330] A processing circuit having a memory for storing instructions which, when executed by the processing circuit, cause the processing circuit to:

[0331] measurements are obtained at points along the length of the density gradient of the components;

[0332] generating a distribution of density gradients of the components based on the measurement results; and

[0333] The distribution of the density gradient of the storage components.

[0334] 18. A system according to clause 17, wherein the instructions, when executed by the processing circuit, further cause the processing circuit to:

[0335] The distribution is adjusted by removing measurements from the bottom portion of the container.

[0336] 19. The system of clause 17, wherein the instructions, when executed by the processing circuit, further cause the processing circuit to:

[0337] identifying the location of the meniscus of the density gradient of the components; and

[0338] The distribution is adjusted to remove measurements from the location of the meniscus.

[0339] 20. A system according to clause 19, wherein the position of the meniscus is identified by identifying a first derivative minimum of the measurement results.

[0340] 21. The system of clause 17, wherein the instructions, when executed by the processing circuit, further cause the processing circuit to:

[0341] obtaining measurements from the container prior to dispensing the density gradient of components into the container;

[0342] comparing the measurement results from the container to an expected measurement range for a container of predetermined material and size;

[0343] determining at least one of a material and a size of the container based on the comparison to an expected measurement range; and

[0344] The measurements are normalized for at least one of a material and a size of the container.

[0345] 22. The system of clause 17, wherein the instructions, when executed by the processing circuit, further cause the processing circuit to:

[0346] obtaining measurements from the sensor assembly along the length of the container when the container is empty;

[0347] Calculating an average of the measurements along the length of the container when the container is empty;

[0348] generating a difference between an average of the measurements and the measurements along the length of the container when the container is empty; and

[0349] The density gradient distribution of the components is adjusted by adding differences to mitigate the effects of defects and wall thickness variations on the container.

[0350] 23. The system of clause 17, wherein the instructions, when executed by the processing circuit, further cause the processing circuit to:

[0351] generating a centerline of the vessel; and

[0352] Measurements are obtained in at least two dimensions along the centerline of the container to mitigate positional errors of the container on the distribution generated by the density gradient for the components.

[0353] 24. A system according to clause 17, wherein multiple measurements are obtained at each point along the length of the density gradient of the component, and the distribution is generated using an average of the multiple measurements calculated for each point along the length of the density gradient of the component.

[0354] 25. The system of clause 17, wherein the distribution is generated for a graded density gradient of components or a continuous density gradient of components.

[0355] 26. A method for non-destructively measuring the density gradient of a component, the method comprising:

[0356] measurements are obtained at points along the length of the density gradient of the components;

[0357] generating a distribution of density gradients of the components based on the measurement results; and

[0358] The distribution of the density gradient of the storage components.

[0359] 27. The method according to clause 26, further comprising:

[0360] The distribution is adjusted by removing measurements from the bottom portion of the vessel in which the density gradient of the components is contained.

[0361] 28. The method according to clause 26, further comprising:

[0362] identifying the location of the meniscus of the density gradient of the components; and

[0363] The distribution is adjusted to remove measurements from the location of the meniscus.

[0364] 29. A method according to clause 28, wherein the position of the meniscus is identified by identifying a first derivative minimum of the measurement results.

[0365] 30. The method according to clause 26, further comprising:

[0366] obtaining measurements from the container prior to dispensing the density gradient of components into the container;

[0367] comparing the measurement results from the container to an expected measurement range for a container of predetermined material and size;

[0368] determining at least one of a material and a size of the container based on the comparison to an expected measurement range; and

[0369] The measurements are normalized for at least one of a material and a size of the container.

[0370] 31. The method according to clause 26, further comprising:

[0371] Obtaining measurements along the length of the container when the container is empty;

[0372] Calculating an average of the measurements along the length of the container when the container is empty;

[0373] generating a difference between an average of the measurements and the measurements along the length of the container when the container is empty; and

[0374] The density gradient distribution of the components is adjusted by adding differences to mitigate the effects of defects and wall thickness variations on the container.

[0375] 32. The method according to clause 26, further comprising:

[0376] generating a centerline of a container in which a density gradient of components is distributed; and

[0377] Measurements are obtained in at least two dimensions along the centerline of the container to mitigate positional errors of the container on the distribution generated by the density gradient for the components.

[0378] 33. The method according to clause 26, further comprising:

[0379] obtaining a plurality of measurements for each point along the length of the density gradient of the component; and

[0380] A distribution of the density gradient of the component is generated using an average of multiple measurements at each of the points along the length of the density gradient of the component.

[0381] 34. The method of clause 26, wherein the distribution is generated for a graded density gradient of components or a continuous density gradient of components.

[0382] 35. A system for automatically dispensing a density gradient of components for use in centrifugation, the system comprising:

[0383] A processing circuit having a memory for storing instructions which, when executed by the processing circuit, cause the processing circuit to:

[0384] inserting the distal end of the probe into the vessel;

[0385] pumping each individual component into a mixing chamber connected to the proximal end of the probe, the mixing chamber generating a mixture of the individual components;

[0386] dispensing a plurality of steps into the container, each step of the plurality of steps having a density based on the relative concentration of each individual component in the mixture generated by the mixing chamber, and each step of the plurality of steps pushing a previously dispensed step away from the distal end of the probe; and

[0387] The probe is removed from the container without disturbing the multiple steps.

[0388] 36. The system of clause 35, wherein the individual components include deionized water, a density modifier, a buffer solution, and an additive.

[0389] 37. A system according to clause 36, wherein the instructions, when executed by the processing circuit, further cause the processing circuit to:

[0390] A dispensing rate is calculated for each of the individual components in each of the plurality of steps, the dispensing rate determining the relative concentration of the individual components in the mixture generated by the mixing chamber.

[0391] 38. The system of clause 37, wherein the successively higher densities result from an increase in the dispensing rate of the density modifier.

[0392] 39. The system of clause 38, wherein the dispensing rate of the deionized water decreases proportionally with respect to an increase in the dispensing rate of the density modifier.

[0393] 40. The system of clause 37, wherein the dispensing rate of the additive is subtracted from the dispensing rate of the deionized water.

[0394] 41. The system of clause 40, wherein the additive is dispensed in a smaller number of steps than a plurality of steps in the density gradient of the components.

[0395] 42. The system of clause 37, wherein the dispensing rate of the buffer solution is maintained constant.

[0396] 43. A system according to clause 37, wherein the instructions, when executed by the processing circuit, further cause the processing circuit to:

[0397] One or more pumps are independently controlled for adjusting the dispensing rate of each of the individual components pumped into the mixing chamber.

[0398] 44. The system of clause 35, wherein the instructions, when executed by the processing circuit, further cause the processing circuit to:

[0399] dispensing a first step of the plurality of steps at a maximum dispensing rate; and

[0400] A dispensing speed of each of a plurality of steps following the first step is adjusted.

[0401] 45. The system of clause 44, wherein adjusting the dispensing speed comprises decreasing the dispensing speed from a maximum dispensing speed to a minimum dispensing speed and then increasing the dispensing speed from the minimum dispensing speed to the maximum dispensing speed.

[0402] 46. ​​The system of clause 45, wherein the dispensing rate increases exponentially from a minimum dispensing rate until a maximum dispensing rate is reached.

[0403] 47. The system of clause 35, wherein the mixing chamber comprises a static mixer.

[0404] 48. A system for partitioning a density gradient of components for use in centrifugation, the system comprising:

[0405] A processing circuit having a memory for storing instructions which, when executed by the processing circuit, cause the processing circuit to:

[0406] inserting the distal end of the probe into the vessel;

[0407] dispensing a first step of the plurality of steps into a container, the first step being dispensed at a maximum dispensing rate;

[0408] dispensing additional steps of the plurality of steps into the container, each additional step initially dispensing at a minimum dispensing speed and then increasing from the minimum dispensing speed to a maximum dispensing speed, each additional step of the plurality of steps having a density greater than a density of a previously dispensed step of the plurality of steps such that the previously dispensed step of the plurality of steps moves away from the distal end of the probe; and

[0409] The probe is removed from the container without disturbing the multiple steps.

[0410] 49. The system of clause 48, wherein the dispensing rate for each additional step increases exponentially from a minimum dispensing rate until a maximum dispensing rate is reached.

[0411] 50. The system of clause 48, wherein each of the plurality of steps comprises a mixture of components including deionized water, a density modifier, a buffer solution, and an additive.

[0412] 51. A system according to clause 50, wherein the instructions, when executed by the processing circuit, further cause the processing circuit to:

[0413] A distribution rate of each of the components in each of the plurality of steps is calculated, the distribution rate determining a concentration of each of the components in each step.

[0414] 52. The system of clause 50, wherein the density of each of the plurality of steps is based on a dispensing rate of the density modifier.

[0415] 53. The system of clause 50, wherein the dispensing rate of the deionized water decreases proportionally with respect to an increase in the dispensing rate of the density modifier.

[0416] 54. The system of clause 50, wherein the dispensing rate of the additive is subtracted from the dispensing rate of the deionized water.

[0417] 55. A system according to clause 54, wherein the additive is distributed in a smaller number of steps than the plurality of steps in the density gradient of the components.

[0418] 56. The system of clause 50, wherein the dispensing rate of the buffer solution is maintained constant.

[0419] 57. A system according to clause 50, wherein the instructions, when executed by the processing circuit, further cause the processing circuit to:

[0420] One or more pumps are independently controlled for adjusting the dispensing rate of each of the components pumped into the mixing chamber to be mixed together.

[0421] 58. A method for automatically dispensing a density gradient of components for use in centrifugation, the method comprising:

[0422] inserting the distal end of the probe into the vessel;

[0423] dispensing a first step of the plurality of steps into a container, the first step being dispensed at a maximum dispensing rate;

[0424] dispensing additional steps of the plurality of steps into the container, each additional step initially dispensing at a minimum dispensing speed and then increasing from the minimum dispensing speed to a maximum dispensing speed, each additional step of the plurality of steps having a density greater than a density of a previously dispensed step of the plurality of steps such that the previously dispensed step moves away from the distal end of the probe; and

[0425] The probe is removed from the container without disturbing the multiple steps.

[0426] 59. The method according to clause 58, further comprising:

[0427] The dispense rate for each additional step increases exponentially from the minimum dispense rate until the maximum dispense rate is reached.

[0428] 60. The method according to clause 58, further comprising:

[0429] Components including deionized water, a density modifier, a buffer solution, and additives are mixed for use in generating each of the plurality of steps.

[0430] 61. The method according to clause 60, further comprising:

[0431] A dispensing rate is calculated for each of the mixed components, the dispensing rate determining a concentration of each of the components in each of the plurality of steps.

[0432] 62. The method of clause 61, wherein the density of each of the plurality of steps is based on a dispensing rate of the density modifier.

[0433] 63. The method according to clause 61, further comprising:

[0434] The dispensing rate of deionized water is decreased in proportion to the increase in the dispensing rate of the density modifier.

[0435] 64. The method according to clause 61, further comprising:

[0436] Subtract the additive dispensing rate from the deionized water dispensing rate.

[0437] 65. The method according to clause 64, further comprising:

[0438] The additive is dispensed in a smaller number of steps than the plurality of steps.

[0439] 66. A method according to clause 61, wherein the dispensing rate of the buffer solution is kept constant.

[0440] 67. The method according to clause 61, further comprising:

[0441] One or more pumps are independently controlled for adjusting the dispensing rate of each of the components pumped into the mixing chamber to be mixed together.

[0442] 68. A method of replicating a density gradient of a component, the method comprising:

[0443] creating a first profile by obtaining measurements of a density gradient of components dispensed in a first container;

[0444] creating a second distribution by replacing measurements of the first distribution;

[0445] storing the second distribution; and

[0446] The density gradient of the components is replicated in a second container based on the second profile.

[0447] 69. The method according to clause 68, further comprising:

[0448] The measured value at the interface between the first step and the second step is replaced with a first average value from the first step before the interface between the first step and the second step, and with a second average value from the second step after the interface between the first step and the second step.

[0449] 70. The method according to clause 68, further comprising:

[0450] The measurement value at the interface between the first step and the second step is replaced by a first measurement value having zero or minimum positive difference with the first step before the interface between the first step and the second step, and by a second measurement value having zero or minimum positive difference with the second step after the interface between the first step and the second step.

[0451] 71. The method according to clause 68, further comprising:

[0452] The measured values ​​from the bottom part of the first container are replaced by a linear ramp of measured values.

[0453] 72. The method according to clause 68, further comprising:

[0454] Verify the quality of the density gradient of the replicated components in the second vessel by following these steps:

[0455] measuring the density gradient of the replicated components in the second container; and

[0456] A determination is made as to whether a difference between a density gradient of the component replicated in the second container and a density gradient of the component contained in the first container is within a predetermined tolerance.

[0457] 73. The method according to clause 68, further comprising:

[0458] A first distribution of the density gradient of the components is processed by converting the first distribution into a text file including positions along the length and corresponding measured values.

[0459] 74. A system for replicating a density gradient of components used in centrifugation, the system comprising:

[0460] The first density gradient of the components;

[0461] Sensor components;

[0462] Assign probes;

[0463] A processing circuit having a memory for storing instructions which, when executed by the processing circuit, cause the processing circuit to:

[0464] obtaining, using a sensor assembly, a measurement of a first density gradient of a component contained in a first container;

[0465] storing a first distribution of measurement values ​​in a memory;

[0466] creating a second distribution based on the stored first distribution; and

[0467] The first density gradient of the components is replicated by dispensing a second density gradient of the components based on the second distribution into a second container using a dispensing probe.

[0468] 75. A system according to clause 74, wherein the instructions, when executed by the processing circuit, further cause the processing circuit to:

[0469] The quality of the second density gradient of the component is verified by determining whether a difference between the second density gradient of the component and the first density gradient of the component is within a predetermined tolerance.

[0470] 76. A system according to clause 74, wherein the instructions, when executed by the processing circuit, further cause the processing circuit to:

[0471] The second distribution is created by replacing the measurements at the interfaces between the steps of the first distribution.

[0472] 77. A system according to claim 76, wherein the measurement value at the interface between the first step and the second step is replaced by a first average value from the first step before the interface between the first step and the second step, and by a second average value from the second step after the interface between the first step and the second step.

[0473] 78. A system according to clause 76, wherein the measurement value at the interface between the first step and the second step is replaced by a measurement value having zero or a minimum positive difference with the first step before the interface between the first step and the second step, and the measurement value having zero or a minimum positive difference with the second step after the interface between the first step and the second step is replaced.

[0474] 79. The system of clause 74, wherein the instructions, when executed by the processing circuit, further cause the processing circuit to:

[0475] A second distribution is created by removing measurements from the bottom portion of the first container.

[0476] 80. The system of clause 74, wherein the instructions, when executed by the processing circuit, further cause the processing circuit to:

[0477] A second distribution is created by replacing the measurements from the bottom portion of the first container.

[0478] 81. A system according to clause 74, wherein the measurement values ​​from the bottom portion of the first container are replaced by a linear ramp of measurement values.

[0479] 82. The system of clause 74, wherein the instructions, when executed by the processing circuit, further cause the processing circuit to:

[0480] A second distribution is created by removing the measurements of the position of the meniscus from the first density gradient of the components.

[0481] 83. The system of clause 74, wherein the instructions, when executed by the processing circuit, further cause the processing circuit to:

[0482] The second distribution is created by converting the first distribution into a text file that includes locations along the length and corresponding measurements.

[0483] 84. A system for replicating a density gradient of components used in centrifugation, the system comprising:

[0484] A processing circuit having a memory for storing instructions which, when executed by the processing circuit, cause the processing circuit to:

[0485] obtaining a measurement of a density gradient of a component dispensed in a first container, the density gradient of the component including a meniscus;

[0486] Process the measurement results in the following steps:

[0487] replacing measurements at interfaces between steps of a density gradient of components;

[0488] replacing the measurement from the position of the bottom portion of the first container; and

[0489] replacing a measured value of a position of a meniscus based on a density gradient of a component dispensed in the first container;

[0490] storing a distribution of density gradients of the components based on the processed measurements; and

[0491] The profile is used to replicate the density gradient of the components in a second container.

[0492] 85. A system according to clause 84, wherein the instructions, when executed by the processing circuit, further cause the processing circuit to:

[0493] The measured value at the interface between the first step and the second step is replaced with a first average value from the first step before the interface between the first step and the second step, and with a second average value from the second step after the interface between the first step and the second step.

[0494] 86. A system according to clause 84, wherein the instructions, when executed by the processing circuit, further cause the processing circuit to:

[0495] The measurement at the interface between the first step and the second step is replaced by a measurement before the interface between the first step and the second step having zero or minimum positive difference with the first step, and by a measurement after the interface between the first step and the second step having zero or minimum positive difference with the second step.

[0496] 87. A system according to clause 84, wherein the instructions, when executed by the processing circuit, further cause the processing circuit to:

[0497] The measured values ​​from the bottom part of the first container are replaced by a linear ramp of measured values.

[0498] 88. A system according to clause 84, wherein the instructions, when executed by the processing circuit, further cause the processing circuit to:

[0499] Verify the quality of the density gradient of the replicated components in the second vessel by following these steps:

[0500] measuring the density gradient of the replicated components in the second container; and

[0501] A determination is made as to whether a difference between a density gradient of the component replicated in the second container and a density gradient of the component dispensed in the first container is within a predetermined tolerance.

[0502] 89. The system of clause 84, wherein the instructions, when executed by the processing circuit, further cause the processing circuit to:

[0503] The distribution of the density gradient of the components is processed by converting the distribution into a text file including the positions along the length and the corresponding measured values.

Claims

1. A system for automatically dispensing a density gradient of components used in centrifugation, the system comprising: A processing circuit having a memory for storing instructions which, when executed by the processing circuit, cause the processing circuit to: inserting the distal end of the probe into the vessel; pumping each individual component into a mixing chamber connected to the proximal end of the probe, the mixing chamber generating a mixture of the each individual component; dispensing a plurality of steps into the container, each step of the plurality of steps having a density based on the relative concentration of the respective individual components in the mixture generated by the mixing chamber, and each step of the plurality of steps pushing a previously dispensed step away from the distal end of the probe; as well as The probe is removed from the container without disturbing the plurality of steps.

2. The system according to claim 1, wherein: The individual components include deionized water, density modifiers, buffer solutions and additives.

3. The system according to claim 2, wherein: The instructions, when executed by the processing circuit, further cause the processing circuit to: A dispensing rate is calculated for each of the individual components in each of the plurality of steps, the dispensing rate determining a relative concentration of the individual components in a mixture generated by the mixing chamber.

4. The system according to claim 3, wherein: Successively higher densities result from increasing dispensing rate of the density modifier.

5. The system according to claim 4, wherein: The dispensing rate of the deionized water decreases in proportion to the increase in the dispensing rate of the density modifier.

6. The system according to claim 3, wherein: The dispensing rate of the additive is subtracted from the dispensing rate of the deionized water.

7. The system according to claim 6, wherein: The additive is dispensed in a smaller number of steps than the plurality of steps in the density gradient of the components.

8. The system according to claim 3, wherein: The instructions, when executed by the processing circuit, further cause the processing circuit to: One or more pumps are independently controlled for adjusting the dispensing rate of each of the individual components pumped into the mixing chamber.

9. The system according to claim 1, wherein: The instructions, when executed by the processing circuit, further cause the processing circuit to: dispensing a first step of the plurality of steps at a maximum dispensing rate; and A dispensing speed of each of the plurality of steps subsequent to the first step is adjusted.

10. The system according to claim 9, wherein: Adjusting the dispensing speed includes decreasing the dispensing speed from the maximum dispensing speed to a minimum dispensing speed and then increasing the dispensing speed from the minimum dispensing speed to the maximum dispensing speed.

11. The system according to claim 1, wherein: The mixing chamber includes a static mixer.

12. The system of claim 1, further comprising: Measuring equipment, comprising: Sensor components; a motor coupled to the sensor assembly; and Wherein, when the instruction is executed by the processing circuit, the processing circuit further causes the processing circuit to: moving the sensor assembly along the length of the density gradient of the component using the motor; obtaining measurements from the sensor assembly as the sensor assembly moves along the length of the density gradient of the component; and A distribution of the density gradient of the components is generated based on the measurement results.

13. The system of claim 1, wherein: The instructions, when executed by the processing circuit, further cause the processing circuit to: creating a first profile by obtaining measurements of a density gradient of the components dispensed in the container; creating a second distribution by replacing measurements of the first distribution; storing the second distribution; and The density gradient of the components is replicated in a second container based on the second profile.

14. A method for automatically dispensing a density gradient of components for use in centrifugation, the method comprising: inserting the distal end of the probe into the vessel; dispensing a first step of a plurality of steps into the container, the first step being dispensed at a maximum dispensing rate; dispensing additional steps of the plurality of steps into the container, each additional step being dispensed initially at a minimum dispensing speed and then increasing from the minimum dispensing speed to a maximum dispensing speed, each additional step of the plurality of steps having a density greater than a density of a previously dispensed step of the plurality of steps such that the previously dispensed step moves away from a distal end of the probe; and The probe is removed from the container without disturbing the plurality of steps.

15. The method according to claim 14, further comprising: The dispensing speed of each additional step is increased exponentially from the minimum dispensing speed until the maximum dispensing speed is reached.

16. The method according to claim 14, further comprising: A dispensing rate for mixing each of the components is calculated, the dispensing rate determining a concentration of each of the components in each of the plurality of steps.

17. The method according to claim 16, wherein: The density of each step in the plurality of steps is based on a dispensing rate of the density modifier.

18. The method according to claim 16, further comprising: The dispensing rate of deionized water is decreased in proportion to the increase in the dispensing rate of the density modifier.

19. The method according to claim 16, further comprising: Subtract the additive dispensing rate from the deionized water dispensing rate.

20. The method according to claim 19, further comprising: The additive is dispensed in a smaller number of steps than the plurality of steps.

21. The method according to claim 16, wherein: The dispensing rate of the buffer solution was kept constant.

22. The method of claim 16, further comprising: One or more pumps are independently controlled for adjusting the dispensing rate of each of the components being pumped into the mixing chamber to be mixed together.

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