Non-destructive measurement, dispensing, and replication of density gradients

Abstract

A system performs non-destructive measurements of a density gradient and automatically replicates and dispenses the density gradient. The system takes measurements at points along the length of the density gradient and generates a profile of the density gradient based on the measurements. The system uses the profile to replicate the density gradient of the components in a second container. The system inserts the distal end of a probe into the second container, pumps the separate components into a manifold and mixing chamber connected to the proximal end of the probe, and automatically dispenses the density gradient into the second container.

Description

[Background technology]

[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, the entire disclosures of which are incorporated herein by reference in their entireties.

[0002] Proteins have a variety of cellular functions, structures, and mechanisms of action. To perform their functions, proteins typically bind to other molecules called ligands. Purification of proteins bound to their ligands, also known as protein-ligand complexes, allows researchers to gain valuable knowledge about how proteins function within 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] Protein-ligand complexes can be purified using density gradients, which are typically layered stepwise using a top-down or top-down approach, with solutions added in order of increasing or decreasing density. Step gradients can be used directly or allowed to diffuse in a controlled manner to create continuous or linear gradients. Step gradients have abrupt interfaces between layers with different densities, while continuous or linear gradients have layers of gradually increasing density moving from top to bottom. In some cases, step gradients are rapidly spun in a centrifuge, which causes the layers to diffuse into a continuous or linear gradient.

[0004] The bottom-up technique for generating density gradients involves adding a first layer with a low density into a tube, then adding successively higher density layers to the bottom of the tube using a long syringe so as not to disturb the previous layers. The top-up technique for generating density gradients involves using a pipette to sequentially dispense layers of decreasing density onto higher density layers. These layering techniques for generating density gradients can be tedious and time-consuming, making them difficult to replicate between researchers. Summary of the Invention [Means for solving the problem]

[0005] Generally, the present disclosure relates to a system for measuring and dispensing density gradients of components. In one configuration, the system measures the density gradient without touching or disturbing the density gradient. In another configuration, the system uses a probe to automatically dispense a density gradient of a component having multiple steps. In another configuration, the system measures the density gradient of a component dispensed into a first container, creates a density gradient profile, and uses the profile to replicate the density gradient of a 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 the density gradient of a component for use in centrifugation, the system comprising: a measurement device including a sensor assembly and a motor coupled to the sensor assembly; and processing circuitry having memory for storing instructions that, when executed by the processing circuitry, cause the processing circuitry to use the motor to move the sensor assembly along the length of the density gradient of the component, obtain measurements from the sensor assembly while the sensor assembly is moved along the length of the density gradient of the component, and generate a profile 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 for use in centrifugation dispensed into a container, the system comprising processing circuitry having memory for storing instructions that, when executed by the processing circuitry, cause the processing circuitry to obtain measurements at points along the length of the component density gradient, generate a component density gradient profile based on the measurements, and store the component density gradient profile.

[0008] Another aspect relates to a method for non-destructively measuring a density gradient of a component, the method including obtaining measurements at points along a length of the density gradient of the component, generating a profile of the density gradient of the component based on the measurements, and storing the profile 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 processing circuitry having a memory for storing instructions that, when executed by the processing circuitry, cause the processing circuitry to insert a distal end of a probe into a container, pump separate components into a mixing chamber connected to a proximal end of the probe, the mixing chamber generating a mixture of the separate components, dispense a plurality of stages into the container, each stage of the plurality of stages having a density based on the relative concentrations of the separate components in the mixture generated by the mixing chamber, and push a previously dispensed stage away from the distal end of the probe, and remove the probe from the container without disturbing the plurality of stages.

[0010] Another aspect relates to a system for dispensing a density gradient of components for use in centrifugation, the system comprising processing circuitry having memory for storing instructions that, when executed by the processing circuitry, cause the processing circuitry to insert a distal end of a probe into a container, dispense a first stage of a plurality of stages into the container, the first stage being dispensed at a maximum dispense rate, dispense additional stages of the plurality of stages into the container, each additional stage starting at a minimum dispense rate and then dispensed at an increasing rate from the minimum dispense rate to the maximum dispense rate, each additional stage of the plurality of stages having a higher density than a previously dispensed stage of the plurality of stages, move the previously dispensed stage of the plurality of stages away from the distal end of the probe, and remove the probe from the container without disturbing the multiple stages.

[0011] Another aspect relates to a method for automatically dispensing a density gradient of components for use in centrifugation, the method including the steps of inserting a distal end of a probe into a container; dispensing a first stage of a plurality of stages into the container, the first stage being dispensed at a maximum dispense rate; dispensing additional stages of the plurality of stages into the container, each additional stage starting at a minimum dispense rate and then dispensed at a rate increasing from the minimum dispense rate to the maximum dispense rate, each additional stage of the plurality of stages having a higher density than a previously dispensed stage of the plurality of stages, and moving the previously dispensed stage away from the distal end of the probe; and removing the probe from the container without disturbing the multiple stages.

[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 that, when executed by the at least one processing device, cause the at least one processing device to obtain measurements of a density gradient dispensed into a first tube, store a profile of the measurements, use the profile, and replicate the density gradient in a second tube.

[0013] Another aspect relates to a method for replicating a density gradient, the method including the steps of obtaining measurements of a density gradient dispensed into a first tube, substituting measurements at an interface between stages of the density gradient, substituting measurements from a bottom portion of the first tube, removing measurements from a meniscus location of the density gradient, storing a profile of the density gradient, and replicating the density gradient into a second tube based on the profile.

[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 acquire measurements of a density gradient dispensed into a first tube, process the measurements by substituting measurements at an interface between stages of the density gradient, substituting measurements from a location at a bottom portion of the first tube, and removing measurements from a location at a meniscus of the density gradient dispensed into the first tube, store a profile of the density gradient based on the processed measurements, and use the profile to replicate the density gradient in a second tube.

[0015] Another aspect relates to a method for replicating a density gradient of a component, the method including creating a first profile by obtaining measurements of a density gradient of a component dispensed into a first container, creating a second profile by substituting measurements of the first profile, storing the second profile, and replicating the density gradient of the component in a second container based on the second profile.

[0016] Another aspect relates to a system for replicating a density gradient of a component for use in centrifugation, the system comprising: a first density gradient of the component; a sensor assembly; a dispensing probe; and processing circuitry having a memory for storing instructions that, when executed by the processing circuitry, cause the processing circuitry to acquire measurements of the first density gradient of the component contained in a first container using the sensor assembly, store a first profile of the measurements in memory, create a second profile based on the stored first profile, and replicate the first density gradient of the component by dispensing the second density gradient of the component into a second container using the dispensing probe based on the second profile.

[0017] Another aspect relates to a system for replicating a density gradient of a component for use in centrifugation, the system comprising processing circuitry having a memory for storing instructions that, when executed by the processing circuitry, cause the processing circuitry to acquire measurements of a density gradient of a component dispensed into a first container, the component density gradient including a meniscus, process the measurements by substituting measurements at an interface between stages of the component density gradient, substituting measurements from a location of a bottom portion of the first container, and substituting measurements based on the location of the meniscus of the component density gradient dispensed into the first container, store a profile of the component density gradient based on the processed measurements, and use the profile to replicate the density gradient of the component in a second container. [Brief explanation of the drawings]

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

[0019] [Figure 1] FIG. 1 illustrates diagrammatically an embodiment of a system for generating a density gradient for centrifugation.

[0020] [Figure 2]FIG. 2 illustrates an example of a mixer housed inside the manifold and mixing chamber of the system of FIG.

[0021] [Figure 3] FIG. 3 is an isometric view of a probe of the system of FIG. 1, with the probe having a distal end where it is inserted into a vessel and a proximal end where it is connected to a manifold and a mixing chamber.

[0022] [Figure 4] FIG. 4 illustrates an example of the proximal end of the probe of FIG. 3 connected to a manifold and mixing chamber.

[0023] [Figure 5] FIG. 5 illustrates diagrammatically an example of a method for generating a density gradient inside a vessel using the system of FIG.

[0024] [Figure 6] FIG. 6 illustrates diagrammatically an example of a density gradient formed inside a vessel after completion of the method of FIG.

[0025] [Figure 7] FIG. 7 is an isometric view of the measurement device in the system of FIG. 1 for measuring density gradients.

[0026] [Figure 8] FIG. 8 is a detailed isometric view of the measurement device of FIG.

[0027] [Figure 9] FIG. 9 is a front view of the measurement device of FIG.

[0028] [Figure 10] FIG. 10 illustrates diagrammatically an example of an electrical configuration for a sensor assembly mounted on the measurement device of FIG.

[0029] [Figure 11]FIG. 11 illustrates diagrammatically an embodiment of a method for measuring a density gradient dispensed into the vessel of FIG.

[0030] [Figure 12] FIG. 12 illustrates diagrammatically another embodiment of a method for measuring a density gradient dispensed into the vessel of FIG.

[0031] [Figure 13] FIG. 13 graphically illustrates an example of a profile for a density gradient generated according to the method of FIGS.

[0032] [Figure 14] FIG. 14 graphically illustrates an example of a plot of voltage measurements identifying the density gradient and vessel features of FIG.

[0033] [Figure 15] FIG. 15 graphically illustrates an example of a plot of voltage measurements to identify the location of the meniscus of a sample liquid dispensed into the vessel of FIG.

[0034] [Figure 16] FIG. 16 graphically illustrates an expanded view of a plot of the first derivative of voltage measurements obtained from sample liquid dispensed into the vessel of FIG.

[0035] [Figure 17] FIG. 17 diagrammatically illustrates an example of a method for normalizing measurements based on the type of container into which the density gradient of FIG. 6 is dispensed.

[0036] [Figure 18] FIG. 18 illustrates an example of a chart showing the normalization of measurements following completion of the method of FIG.

[0037] [Figure 19] FIG. 19 illustrates another example of a chart showing the normalization of measurements following completion of the method of FIG.

[0038] [Figure 20] FIG. 20 diagrammatically illustrates an example of a method for mitigating the effects of imperfections and wall thickness variations along the length of the vessel of FIG.

[0039] [Figure 21] FIG. 21 graphically illustrates an example plot of voltage measurements from the container of FIG. 6 when empty, according to the method operations of FIG.

[0040] [Figure 22] FIG. 22 graphically illustrates an example of a chart showing the mitigation of container defects and / or container wall thickness variations for a sample of DI water following completion of the method of FIG.

[0041] [Figure 23] FIG. 23 illustrates an embodiment of a container having a centerline that is offset relative to the vertical axis of the sensor assembly mounted on the measurement device of FIGS. 7-9.

[0042] [Figure 24] FIG. 24 diagrammatically illustrates an example of a method for mitigating mechanical positioning errors for density gradient measurements obtained from the vessel of FIG.

[0043] [Figure 25] FIG. 25 graphically illustrates an example of a plot of voltage measurements taken across the outer diameter near the proximal end of the vessel of FIG.

[0044] [Figure 26] FIG. 26 graphically illustrates an example of a plot of voltage measurements taken across the outer diameter near the distal end of the vessel of FIG.

[0045] [Figure 27] FIG. 27 shows a cross-sectional view of the container of FIG. 23 held by the holder of the measurement device of FIGS. 7-9 from a perspective looking down into the container.

[0046] [Figure 28] FIG. 28 graphically illustrates an example chart showing the reduction in mechanical positioning error according to the method of FIG.

[0047] [Figure 29] FIG. 29 graphically illustrates another example of a chart showing the reduction in mechanical positioning error according to the method of FIG.

[0048] [Figure 30] FIG. 30 graphically illustrates another example of a chart showing the reduction in mechanical positioning error according to the method of FIG.

[0049] [Figure 31] FIG. 31 graphically illustrates a chart in which voltage measurements are taken at 45 degree rotations along the length of the vessel of FIG.

[0050] [Figure 32] FIG. 32 diagrammatically illustrates another exemplary embodiment of a measurement device that can mitigate errors from when the walls of the container of FIG. 23 do not have a uniform thickness around the circumference of the container.

[0051] [Figure 33] FIG. 33 diagrammatically illustrates an embodiment of a method for mitigating wall thickness variations around the periphery of a container with the measurement device of FIG.

[0052] [Figure 34] FIG. 34 graphically illustrates a chart showing the reduction in wall thickness variation around the periphery of a container by implementing the method of FIG.

[0053] [Figure 35] FIG. 35 graphically illustrates an example of a density gradient dispensed into a vessel by the system of FIG.

[0054] [Figure 36] FIG. 36 illustrates diagrammatically how to generate the density gradient shown in FIG.

[0055] [Figure 37] FIG. 37 illustrates an example of a density gradient dispensed into a container by the system of FIG.

[0056] [Figure 38] FIG. 38 illustrates diagrammatically an embodiment of a method for generating a density gradient such as that shown in FIG.

[0057] [Figure 39] FIG. 39 graphically illustrates an example of a chart showing the implementation of the method of FIG. 38 by the system of FIG.

[0058] [Figure 40] FIG. 40 illustrates schematically an example of a method for replicating density gradients that can be implemented on the system of FIG.

[0059] [Figure 41] FIG. 41 graphically illustrates an example of a density gradient profile prior to being processed by the system of FIG.

[0060] [Figure 42] FIG. 42 schematically illustrates an example of a method for processing the density gradient profile of FIG. 41 to remove noise and imperfections that may interfere with replicating the density gradient profile with the system of FIG.

[0061] [Figure 43] FIG. 43 graphically illustrates an example of a modified density gradient profile generated according to the method of FIG. 42 for replacing a density gradient profile.

[0062] [Figure 44] FIG. 44 graphically illustrates an example of a chart showing a comparison of a first density gradient and a second density gradient replicated from the first density gradient by the system of FIG.

[0063] [Figure 45] FIG. 45 schematically illustrates another embodiment of a method for processing density gradient profiles for replication with the system of FIG.

[0064] [Figure 46] FIG. 46 graphically illustrates an example of a density gradient profile prior to being processed by the method of FIG.

[0065] [Figure 47] FIG. 47 graphically illustrates an example of a derivative plot after the density gradient profile of FIG. 46 has been mathematically differentiated.

[0066] [Figure 48] FIG. 48 is an expanded view of the derivative plot of FIG.

[0067] [Figure 49] FIG. 49 graphically illustrates an example of a comparison of the density gradient profile of FIG. 46 with an adjusted density gradient profile after measurements affected by optical effects such as Gouy phase shift have been replaced by values determined from the derivative plot of FIG. 47.

[0068] [Figure 50] FIG. 50 is a magnified view of the adjusted density gradient profile of FIG.

[0069] [Figure 51] FIG. 51 shows an example of a text file that can be executed by the processing device of the system of FIG. 1 to dispense a density gradient into a vessel.

[0070] [Figure 52] FIG. 52 illustrates exemplary computing hardware for the system of FIG. DETAILED DESCRIPTION OF THE INVENTION

[0071] Detailed Description 1 schematically illustrates an example of a system 100 capable of generating density gradients for centrifugation. System 100 is computer controlled to precisely dispense gradients of any type, slope, or shape. System 100 can be used to generate linear density gradients, which have a gradually increasing density from top to bottom, and step density gradients, which have at least two discrete steps of different density.

[0072] Additionally, system 100 can measure a density gradient dispensed inside container 110 without touching or disturbing the density gradient. These measurements can be used by system 100 to replicate the density gradient inside another container. In some embodiments, container 110 is a tube for use in a centrifuge rotor for centrifugation.

[0073] System 100 includes reservoirs 102, each holding a separate component for generating a density gradient inside a container 110. Each reservoir 102 is connected to a pump 104 for pumping the component held in reservoir 102 into a manifold and mixing chamber 106. Pumps 104 are each programmed to pump the component from reservoir 102 at a given volume and rate for mixing inside manifold and mixing chamber 106.

[0074] 1 , system 100 includes four reservoirs: a first reservoir 102a connected to a first pump 104a for pumping a first component into manifold and mixing chamber 106, a second reservoir 102b connected to a second pump 104b for pumping a second component into manifold and mixing chamber 106, a third reservoir 102c connected to a third pump 104c for pumping a third component into manifold and mixing chamber 106, and a fourth reservoir 102d connected to a fourth pump 104d for pumping a fourth component into manifold and mixing chamber 106. System 100 can include more than four reservoirs for holding more than four separate components for generating a density gradient, or fewer than four reservoirs for holding fewer separate components for generating a density gradient in container 110.

[0075] The components held in the reservoirs 102 are liquids that are pumped into the manifold and mixing chamber 106 to dispense a homogenous stream of fluid into the container 110. As illustrative examples, the first reservoir 102a can hold deionized (DI) water, the second reservoir 102b can hold a density modifier such as sucrose, glycerol, or iodixanol, the third reservoir 102c can hold a buffer solution, and the fourth reservoir 102d can hold additives such as amino acids, proteins, chelating agents, stabilizers, detergents, salts, and biological sample materials. Illustrative examples of buffer solutions include, but are not limited to, phosphate-buffered saline (PBS), Tris buffer concentrate (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 that proceeds through a manifold and mixing chamber 106. The DI water and density modifier make up the majority of the volume in the stream, while the buffer and additives have lower concentrations.

[0077] As an illustrative example, DI water and density modifier are pumped from respective first and second reservoirs 102a and 102b into the manifold and mixing chamber 106 using first and second pumps 104a and 104b, respectively. The first and second pumps 104a and 104b may include peristaltic pumps to provide smooth pumping flow for the DI water and density modifier components. Buffers and additives from respective third and fourth reservoirs 102c and 102d are pumped by third and fourth pumps 104c and 104d, respectively. In some embodiments, the third and fourth pumps 104c and 104d include peristaltic pumps. In other embodiments, the third and fourth pumps 104c and 104d may include syringe pumps, which may be used when more precise pumping is desired for the buffers and additives.

[0078] 2 illustrates an example of a mixer 200 housed inside the manifold and mixing chamber 106 of the system 100. Referring now to FIGS. 1 and 2, DI water, density modifier, buffer, and additives are introduced into a single stream that travels through the manifold and mixing chamber 106. Inside the manifold and mixing chamber 106, the mixer 200 includes mixing elements 202a-202f that mix the components together as they pass through the mixing elements. The mixer 200 mixes the components together to generate a homogenous flow of fluid for the probe 108 to dispense stages of a density gradient into the container 110, the stages having predetermined densities based on the relative concentrations of the components.

[0079] In some embodiments, mixer 200 is a static mixer, and mixing elements 202a-202f include alternating helical elements. In some embodiments, each helical element is set at 90° to adjacent helical elements to provide thorough blending of the components throughout the length L of mixer 200 inside manifold and mixing chamber 106. Mixing elements 202a-202f mix the components together and eliminate pockets of low and / or high density material. Mixing elements 202a-202f slice and rotate the DI water and density modifier together multiple times to produce a substantially homogenous flow for probe 108 to dispense the stages of the density gradient into vessel 110. In alternative embodiments, manifold and mixing chamber 106 can include alternative types of mixers and mixing elements.

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

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

[0082] 4 illustrates an example of the proximal end 114 of the probe 108 connected to the manifold and mixing chamber 106. The manifold and mixing chamber 106 each include a manifold portion 402 having inputs 404a-404b that receive components pumped from the reservoirs 102a-102d by the pumps 104a-104d, respectively. The manifold and mixing chamber 106 further includes a mixing portion 406 that houses a mixer 200 for mixing together the components pumped from the reservoirs before they reach the proximal end 114 of the probe 108.

[0083] 4, the proximal end 114 of the probe 108 is shown secured by a set screw 408, which can be tightened or loosened around the proximal end 114 by using a rotatable handle 410. The manifold and mixing chamber 106 are attached to a motor-driven mechanism that moves the probe 108 up or down to a desired position inside the vessel 110. In other embodiments, the probe 108 can be manually lowered to a desired position inside the vessel 110.

[0084] The probe 108 is coated with a non-stick material. In some embodiments, 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 density gradient dispensed into the container 110 from sticking to or accumulating on the probe 108. Thus, the non-stick material coating allows the probe 108 to be removed while mitigating mixing between discrete stages of the density gradient.

[0085] 1 , system 100 can include a control panel 130 for receiving input from a user to generate a desired density gradient. In some embodiments, control panel 130 includes a display 132, such as a touchscreen, that can be used by a user to create the desired density gradient, perform its measurements, and store the density gradient profile. In further embodiments, control panel 130 can include additional input devices, such as one or more physical buttons, that can be selected to control the operation of system 100.

[0086] 5 schematically illustrates an example of a method 500 for generating a density gradient inside a vessel 110 using the system 100. The method 500 includes an operation 502 of lowering a probe 108 proximate the bottom of the vessel 110. An example of the probe 108 positioned proximate the bottom of the vessel 110 is shown in FIGS. 1 and 3. In some examples, the distal end 112 of the probe 108 will remain positioned proximate the bottom of the vessel 110 while the probe 108 dispenses the density gradient.

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

[0088] Next, method 500 includes operation 506 of dispensing into container 110 a second stage (e.g., a second stage of DI water, density modifier, buffer, and additive) made from components held in reservoir 102. The second stage has a second density based on the relative concentrations of the components pumped from reservoir 102. The second density is heavier than the first density, and therefore, the second stage pushes up against the first stage, and the second stage rests below the first stage at the bottom of container 110.

[0089] Next, method 500 includes operation 508, which determines whether the density gradient includes additional stages. When the density gradient includes additional stages (i.e., "yes" in operation 508), method 500 repeats operation 506 to dispense additional stages (e.g., additional stages of DI water, density modifier, buffer, and additive) made from components held in reservoir 102. The additional stages have densities based on the relative concentrations of the components that are heavier than the densities of the previously dispensed stages; therefore, the additional stages push up the previously dispensed stages and remain below them. Operation 506 can be repeated based on the desired number of stages for the density gradient. Each time operation 506 is performed, probe 108 remains in the same position (i.e., proximate the bottom of container 110).

[0090] When the density gradient does not include additional stages (i.e., "not applicable" in operation 508), method 500 proceeds to operation 510, which involves removing probe 108 from container 110. Operation 510 may include slowly removing probe 108 so as not to disturb the stages of the density gradient. As discussed above, probe 108 may be coated with a non-stick material to minimize stages in the density gradient from sticking to probe 108 during its removal.

[0091] In method 500, pumps 104a-104d are each programmed to control the flow of each liquid component into manifold and mixing chamber 106 at a given volume and / or speed to generate each stage of the density gradient. This allows system 100 to precisely control the concentration of each liquid component in each stage of the density gradient dispensed by probe 108 into container 110 to generate the density gradient.

[0092] 6 schematically illustrates an example of a density gradient 300 formed by system 100 in container 110 after completion of method 500. Density gradient 300 is a medium created for particle separation in ultracentrifugation. In this example, density gradient 300 is a step gradient, and thus, density gradient 300 includes discrete steps having different densities. In this example, density gradient 300 includes five discrete steps. In alternative embodiments, method 500 can be implemented to form a continuous or linear gradient.

[0093] 6, density gradient 300 includes stages 302a-302e. Each stage 302a-302e has a unique density based on the relative concentrations of the components mixed in manifold and mixing chamber 106. In this illustrative example, density gradient 300 includes first stage 302a having a first density, second stage 302b having a second density, third stage 302c having a third density, fourth stage 302d having a fourth density, and fifth stage 302e having a fifth density. According to the above description, the fifth density of fifth stage 302e is the heaviest and the first density of first stage 302a is the lightest, and thus the densities of stages 302a-302e increase from top to bottom inside vessel 110. Each stage forming part of density gradient 300 is shown separated by boundary 310.

[0094] System 100 and / or method 500 can form density gradients having more than five distinct steps and / or fewer than five distinct steps. System 100 can also form step gradients, such as those shown in Figure 6, as well as continuous or linear gradients having layers of gradually increasing density from top to bottom. Thus, density gradient 300 is shown only as an illustrative example.

[0095] As further shown in FIG. 6 , 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 that runs through the middle of the container 110. In some embodiments, 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 embodiments, the boundary between the bottom portion 304 and the cylindrical portion 312 is used to establish a starting point 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 measurements of the density gradient 300, thereby filtering and / or removing the measurements from this portion of the container 110.

[0096] 6 , density gradient 300 exhibits features such as a meniscus 308 located at the top of density gradient 300. Meniscus 308 is caused by surface tension between density gradient 300 and the interior surface of the wall of container 110. Meniscus 308 includes a bottom edge 309 where the centerline CL of container 110 is located. Meniscus 308 also includes a top edge 311 near the wall of container 110. Meniscus 308 may interfere with measurements of density gradient 300, such that measurements are not taken or are filtered from this portion of density gradient 300.

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

[0098] 6, the vessel 110 has a length L that extends from the bottom portion 304 of the vessel to the opening 314. The density gradient 300 has a length L D In this illustrative example, the density gradient 300 has a length L D is less than the length L of the vessel 110, such that the density gradient 300 occupies a portion of the length of the vessel. D may be about 80 mm.

[0099] As will be described in more detail herein, system 100 performs non-destructive density gradient measurements over the length L of vessel 110. The density gradient measurements can be used to verify that density gradient 300 matches a desired profile or meets desired quality controls. Additionally, the density gradient measurements can be stored in memory to replicate density gradient 300 inside another vessel.

[0100] FIG. 7 is an isometric view of an embodiment of a measurement device 700 in a system 100 for performing non-destructive density gradient measurements over a length L of a vessel 110. For example, the measurement device 700 can measure a density gradient 300 without touching or disturbing the density gradient. FIG. 8 is a detailed isometric view of the measurement device 700. FIG. 9 is a front view of the measurement device 700.

[0101] 7-9, in this embodiment, measurement apparatus 700 includes a platform 702 that supports a frame 704 that includes a rail 706. In some embodiments, rail 706 includes a threaded rail. Measurement apparatus 700 further includes a sensor assembly 712 mounted on a carriage 708 powered by a motor 710 for movement up and down rail 706 while container 110 remains in a fixed position. In some embodiments, motor 710 is a stepper motor or other similar type of electric motor. Rail 706 and motor 710 provide precise vertical movement of sensor assembly 712 relative to container 110. Container 110 can be held relative to measurement apparatus 700 by a holder, such as a clamp (see FIG. 27).

[0102] The length L of the container 110 and / or the length L of the density gradient D Alternative embodiments for moving the sensor assembly 712 along the length L of the container 110 and / or the length L of the density gradient dispensed into the container 110 are possible. D The sensor assembly 712 may be moved along the length L of the container 110 and / or the length L of the density gradient dispensed into the container 110. D Additional structures for moving the sensor assembly 712 along are envisioned, and therefore the structures shown in FIGS. 7-9 are provided as illustrative examples.

[0103] The sensor assembly 712 measures the length L of the density gradient 300 dispensed into the container 110. D8 and 9 , 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 transmission through a density gradient dispensed into the container 110. The emitter 714 and the detector 716 are in a fixed relationship to each other when mounted on the carriage 708, which allows these components of the sensor assembly 712 to be moved together up and down the rail 706. The carriage 708 allows for coordinated movement of the emitter 714 and the detector 716 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] 7-9, emitter 714 is mounted on one side of carriage 708 and detector 716 is mounted on the opposite side of carriage 708. A vessel 110 with a density gradient dispensed therein is fixedly positioned relative to the central axis of carriage 708 such that a signal (e.g., light) emitted by emitter 714 passes through vessel 110 and is received by detector 716 on the opposite side of vessel 110.

[0105] In an alternative embodiment, emitter 714 and detector 716 can be mounted on the same side of carriage 708. For example, emitter 714 can emit a signal that passes from a first side of carriage 708, through a density gradient dispensed into vessel 110, and, together with detector 716, is reflected by a mirror mounted on a second side of carriage 708 for reflection back toward the first side of carriage 708 on which detector 716 is mounted. Further alternative arrangements for sensor assembly 712 are envisioned.

[0106] In one exemplary embodiment, emitter 714 emits light and detector 716 includes a photodiode that detects a current resulting from the transmission of light through container 110. In some examples, emitter 714 emits light in the infrared spectrum (e.g., light having a wavelength between about 700 nm and about 1,000 nm). In some further examples, emitter 714 emits light having a wavelength of about 880 nm. In alternative examples, emitter 714 emits light in the visible spectrum (e.g., between about 380 nm and about 750 nm).

[0107] When light from emitter 714 passing through vessel 110 strikes detector 716, a current is generated on detector 716. Sensor assembly 712 may further include amplifier circuitry that converts the current into a voltage. Thus, sensor assembly 712 measures and records voltage at multiple points along the length L of vessel 110 to measure the density gradient dispensed into vessel 110. As an example, sensor assembly 712 may measure and record voltage at 320 points over a length of approximately 80 mm.

[0108] The voltage measurements recorded by the sensor assembly 712 correlate to the refractive index along the length of the density gradient dispensed into the vessel 110 and can be used to calculate the density along the length of the density gradient, since density affects the transmission of light from the emitter 714 through the density gradient. Thus, 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 into the vessel 110.

[0109] 8 , carriage 708 includes a slot 718 positioned in front of detector 716. Slot 718 focuses light emitted from emitter 714 and passing through a narrow slice of the density gradient dispensed into vessel 110. Slot 718 allows measurement device 700 to measure a narrow slice along the length of the density gradient dispensed into vessel 110. Additionally, multiple measurements can be obtained per slice of the density gradient (e.g., 100 measurements per slice), and the measurements can be averaged to reduce variability in measurements taken along the entire length of the density gradient.

[0110] To further reduce sensitivity due to positional errors between the vessel 110, the emitter 714, and the detector 716, the detector 716 has a large surface area. In some embodiments, the detector 716 has a surface area of approximately 8.5 mm 2 The photodiode includes a surface area of

[0111] FIG. 10 schematically illustrates an example electrical configuration for sensor assembly 712. As shown in FIG. 10, resistor 720 sets the current for emitter 714. As an illustrative example, resistor 720 can set the current for emitter 714 to be in the range of approximately 19 mA to approximately 33 mA. In some embodiments, resistor 720 can have an electrical resistance of approximately 90 Ω. Emitter 714 can be powered by a stable, high-precision DC power supply to maintain a constant level of infrared (IR) radiance. In an alternative embodiment, the DC power supply for emitter 714 can be replaced with a constant current source, which can reduce fluctuations due to power supply drift. Additional embodiments for powering emitter 714 are possible.

[0112] The transimpedance amplifier design reduces the detected current (I d) is used to convert it into a voltage across a feedback resistor 722. In some embodiments, the feedback resistor 722 has an electrical resistance of approximately 47 kΩ. An operational amplifier 724 with a precision input current is used for its ability to operate at very low currents. A second DC power supply can provide + / - 6 VDC for the operational amplifier 724.

[0113] When a density gradient is dispensed into the vessel 110, the vessel becomes a cylindrical lens, and therefore, 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] Additionally, 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) radiance for transmission 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. [Table 1]

[0115] As shown in Table 1, the sensor assembly 712 measures voltage to determine the concentration levels of density modifiers such as sucrose, glycerol, and iodixanol in containers having different diameters (e.g., 9 / 16 inch or 1 inch) and made from different materials (e.g., material type 1 = polypropylene, material type 2 = polyethylene terephthalate glycol (PETG), and material type 3 = polycarbonate). The concentration levels of the density modifiers are used to determine the density at specific locations along the length of the density gradient.

[0116] 11 schematically illustrates an example embodiment of a method 1100 for measuring a density gradient dispensed into a container, such as density gradient 300 dispensed into container 110 shown in FIG. 6. Method 1100 can be performed by measurement device 700 of system 100. Method 1100 can non-destructively measure the density gradient, such that the density gradient is not disturbed or altered by method 1100.

[0117] Method 1100 includes operation 1102 of obtaining measurements from the density gradient, which will be described in more detail with reference to FIGS. 7-9 and 12. Method 1100 includes additional operations to refine the density gradient measurements, such as operation 1104 of filtering or removing measurements from the bottom portion 304 of container 110, operation 1106 of filtering or removing measurements where meniscus 308 of density gradient 300 is located, operation 1108 of normalizing the measurements based on container material and / or size, operation 1110 of mitigating the effect of container imperfections and wall thickness variations along the length L of container 110, operation 1112 of mitigating the effect of wall thickness variations around the circumference of container 110 on the density gradient measurements, and operation 1114 of mitigating mechanical positioning errors. Each of these additional operations will be described in more detail below.

[0118] 12 schematically illustrates an example embodiment of a method 1200 for obtaining measurements from a density gradient dispensed into a container 110. In some embodiments, the method 1200 forms part of operation 1102 in the method 1100. The method 1200 can be performed by the system 100 using the measurement device 700 shown in FIGS. 7-9.

[0119] 12, method 1200 includes step 1202 of initiating measurement of a density gradient dispensed into container 110. Step 1202 can occur following receipt of a user input / command on control panel 130 of system 100.

[0120] Next, the method 1200 includes a step 1204 of positioning the sensor assembly 712 relative to the container 110 and taking a measurement. The sensor assembly 712 can be positioned by the motor 710 while the container 110 remains fixed. The motor 710 can move the sensor assembly 712 in precise steps up and down the entire length L of the container 110.

[0121] In some embodiments, the sensor assembly 712 is initially positioned toward the bottom of the container 110 in step 1204. In alternative embodiments, the sensor assembly 712 is initially positioned toward the top of the container 110 in step 1204. In further embodiments, the sensor assembly 712 is positioned between the top and bottom of the container 110 in step 1204.

[0122] Method 1200 includes a step 1206 of measuring a voltage at the location where sensor assembly 712 was positioned in step 1204. The voltage is measured by emitting light from emitter 714, which passes through container 110 and is received by detector 716 on the opposite side of container 110. Step 1206 can include measuring the voltage multiple times and calculating an average voltage at the location of sensor assembly 712.

[0123] Method 1200 includes step 1208 of determining whether additional locations along the length L of container 110 require measurement. When it is determined that no additional locations require measurement (i.e., "not applicable" in step 1208), method 1200 may end in step 1210. When it is determined that additional locations do require measurement (i.e., "yes" in step 1208), method 1200 may repeat steps 1204-1208, moving sensor assembly 712 to a new location along the length L of container 110 and taking a measurement at the new location to determine whether there are additional locations requiring measurement.

[0124] In some embodiments, the new location is upward relative to the previous location when density gradient measurements are taken starting at the bottom of the vessel 110. In alternative embodiments, the new location is downward relative to the previous location when density gradient measurements are taken starting at the top of the vessel 110. The length L of the vessel 110 can be divided into a number of distinct locations, and steps 1204-1208 are repeated for each location to generate a profile for the density gradient. As an example, the method can take measurements across 320 points over a length of 80 mm.

[0125] 13 graphically illustrates an example of a profile 1300 for a density gradient measured according to method 1100. In this illustrative example, profile 1300 shows the voltage recorded by detector 716 for a 5-40% sucrose density gradient. The voltage represents the refractive index of the density gradient dispensed into container 110, which can be used to determine the concentration and density level of a density modifier (e.g., sucrose).

[0126] The bottom of container 110 is to the left of profile 1300, and the top of container 110 is to the right. Profile 1300 excludes the bottom portion of container 110, which may interfere with the optical path of light from emitter 714 (see operation 1104 of method 1100). Profile 1300 also excludes the top portion of the density gradient, which may be affected by meniscus 308 (see FIG. 6 ) formed between the density gradient and the wall of container 110 (see operation 1106 of method 1100).

[0127] 13, the voltage is highest near the bottom of the container 110, where the densest step of the density gradient is located (i.e., having about 40% sucrose), and the voltage gradually decreases as it moves up the container 110, where the least dense step of the density gradient is located (i.e., having about 5% sucrose). By scanning along the length L of the container 110, the change in refractive index forms a profile 1300, which correlates to changes in density modifier concentration and density level.

[0128] 14 graphically illustrates an example plot 1400 of voltage detected by sensor assembly 712, identifying characteristics of container 110 and the density gradient dispensed therein. In this example, container 110 is a 9 / 16 inch diameter polypropylene container, and voltage is obtained along the entire length L of container 110.

[0129] Plot 1400 can be used to identify the locations of various features of interest on vessel 110 and / or density gradient 300. For example, plot 1400 shows location 1402 of bottom portion 304 of vessel 110, location 1404 of cylindrical portion 312 of vessel 110, location 1406 of meniscus 308 of density gradient 300, location 1408 of volume 306 above density gradient 300, location 1410 of opening 314 of vessel 110, and location 1412 of air above vessel 110.

[0130] Identification of these features of interest can help improve measurement of density gradient 300 by system 100. For example, identification in plot 1400 of location 1402 of bottom portion 304 can be used to filter and / or remove voltage measurements from this location of vessel 110, according to operation 1104 in method 1100. Similarly, identification in plot 1400 of location 1406 of meniscus 308 can be used to filter and / or remove voltage measurements from this location of density gradient 300, according to operation 1106 in method 1100.

[0131] As an example, location 1404 where cylindrical portion 312 begins can be selected as a starting point for positioning sensor assembly 712 in step 1204 of method 1200. As a further example, location 1406 before meniscus 308 can be selected as an ending location for ending measurement of the density gradient by sensor assembly 712. As another example, location 1408 of volume 306 above density gradient 300 in container 110 can be selected for taking measurements to identify the material of container 110, because each type of container can be made from a material that has unique properties when an illumination signal, such as infrared light, is transmitted through an empty portion of the container.

[0132] The height and / or location of the meniscus 308 can be difficult to measure because the shape and / or size of the meniscus 308 can vary based on the amount of surface tension (i.e., adhesion) between the density gradient and the walls of the container 110. For example, liquids with different densities will have different surface tensions with the walls of the container 110.

[0133] The meniscus 308 may cause optical effects that may interfere with the accuracy of density measurements by the sensor assembly 712. For example, the bottom edge 309 of the meniscus 308 may cause a higher voltage reading by the sensor assembly 712, and the top edge 311 of the meniscus may cause a lower voltage reading by the sensor assembly 712. The following technique is implemented in the system 100 to identify the location of the meniscus 308, regardless of the shape and / or size of the meniscus 308. By identifying the location of the meniscus 308, measurements obtained from the sensor assembly 712 can be filtered from the location of the meniscus, improving the accuracy of density gradient measurements by the system 100.

[0134] FIG. 15 graphically illustrates an example of a voltage plot 1500 for identifying the location of the meniscus of a sample liquid dispensed into the container 110. In this example, five liquid samples are analyzed. The x-axis of the plot 1500 represents height in millimeters, and the y-axis represents voltage in millivolts detected by the sensor assembly 712. The left side 1502 of the plot 1500 shows voltage measurements of the liquid sample in the container. The right side 1504 of the plot 1500 shows voltage measurements 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 plot 1500 indicates the meniscus. An unexpected result from the plot 1500 is that the optical effect of the meniscus is several millimeters wide, even though the meniscus appears thin to the human eye.

[0135] The shape of the meniscus may vary due to different surface tensions between the sample liquids and the walls 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 plot 1500.

[0136] 16 graphically illustrates a close-up plot 1600 of the first derivative of the voltage obtained from a sample liquid dispensed into the vessel 110. A technique for accurately measuring the height of the density gradient dispensed into the vessel 110 involves using the minimum of the first derivative of the voltage measured by the sensor assembly 712 to identify the location of the meniscus. While other features of the meniscus can also be identified by the sensor assembly 712, the minimum of the first derivative is a consistent and reliable source for identifying the meniscus.

[0137] The minimum of the first derivative is at the point where the slope of the voltage 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 meniscus shape (see FIG. 6). The use of the first derivative is effective for eliminating meniscus shape variations that normally occur due to surface tension. In the example of plot 1600 shown in FIG. 16, the minimum of the first derivative is located at approximately 13.8 millimeters with an uncertainty of approximately 0.25 millimeters.

[0138] Additional data shows that for a container having a total volume of 13 mL, the volume of a liquid sample dispensed into the container can be calculated from the identified height using the minimum value of the first derivative, with an error of approximately + / - 37 μL, which compares favorably to other, more expensive methods for measuring the volume of a liquid sample dispensed into a container.

[0139] 17 schematically illustrates an example of a method 1700 for normalizing measurements based on the type of container the density gradient is dispensed into. In some examples, method 1700 forms part of operation 1108 in method 1100.

[0140] The containers 110 can have different sizes (e.g., 9 / 16 inch diameter, 1 inch diameter, etc.) and can be made from different materials, including, but not limited to, polypropylene, polycarbonate, and copolyester resins such as polyethylene terephthalate glycol (PETG). Each size and material causes the container to exhibit unique properties when light is transmitted through it, which can cause variations in measurements of the density gradient 300 when dispensed into different containers having different sizes and made from different materials.

[0141] Method 1700 includes operation 1702 of measuring a voltage across the empty volume of vessel 110. In some embodiments, operation 1702 is performed by system 100 before density gradient 300 is dispensed into vessel 110.

[0142] In other embodiments, operation 1702 is performed by system 100 after density gradient 300 is dispensed into container 110. In such embodiments, operation 1702 includes measuring a voltage across volume 306 above meniscus 308 and below opening 314 of density gradient 300. The location of volume 306 can be determined based on the relative locations of meniscus 308 and opening 314, such as by identifying characteristics of these features shown in plot 1400 of FIG. 14 . The ability to measure voltage across the empty volume of container 110 before or after density gradient 300 is dispensed can provide flexibility for users of system 100.

[0143] Method 1700 includes operation 1704, which compares the voltage measured in operation 1702 to known expected voltage ranges 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 measurements may vary based on the size or diameter of the container. Table 2 is provided below to show expected voltage ranges for different container sizes, such as 1-inch diameter and 9 / 16-inch diameter, for a first type of material and a second type of material. As an illustrative example, the first type of material can include a polyester resin, such as polyethylene terephthalate glycol (PETG), and the second type of material can include polypropylene. [Table 2]

[0144] Next, method 1700 includes operation 1706 of determining the material and / or size of container 110 based on the comparison in operation 1704. For example, when the voltage measured in operation 1702 falls within an expected voltage range for a particular material or combination of materials and container diameter, container 110 is determined in operation 1706 to have that particular material and / or container diameter.

[0145] Next, the method 1700 calculates the total length L of the density gradient 300. D The method includes an operation 1708 of normalizing voltage measurements obtained across the measuring device 700. The voltage measurements may be obtained according to the steps of method 1200 described above. Operation 1708 enables system 100 to normalize voltage measurements obtained from measuring device 700 for different types of containers made from different types of materials and / or having different sizes.

[0146] 18 illustrates an example of a chart 1800 showing the normalization of measurements following completion of method 1700. In some instances, the process of precisely scanning the density gradient within container 110 can be technically challenging due to the sensitivity of the measurements to variations in the container's wall draft and / or thickness. The container's wall draft can be affected by the type of material and process used to manufacture the container. For example, injection molding can cause containers to exhibit wall drafts along their length.

[0147] During injection molding, the container 110 is formed by forcing hot molten material into a die cavity under high pressure and temperature. The molten material conforms to the shape of the die and then cools. To aid in the removal of the container 110 from the die, a small amount of draft is added to the die, so the inner diameter of the container 110 is slightly larger at the top of the container 110 compared to the bottom of the container 110. The draft causes an increased wall thickness near the bottom portion 304 of the container 110, since the outer diameter of the container 110 is the same along the length L of the container 110. While draft is not visible to the naked eye, it is detectable by the measurement device 700. For example, during a precision optical scan measuring the refractive index of a gradient sample dispensed into the container 110, draft is revealed by a slight tilt in the measurement. As explained above, measurements from the bottom portion 304 of the vessel 110 can be ignored or filtered out because the curved shape and increased wall thickness of the bottom portion interfere with measurements of the density gradient.

[0148] In the example shown in FIG. 18 , chart 1800 includes first and second plots 1802, 1804 of the voltage measured by detector 716 (y-axis) and the container length (x-axis). The first and second plots 1802, 1804 are each measured for a container made from a polypropylene material having a diameter of 9 / 16 inches. In the first plot 1802, the container is filled in a single stage with 0% sucrose density modifier (e.g., the container is filled with 100% DI water). In the second plot 1804, the container is filled in a single stage with 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 0-14 mm (x-axis); therefore, these portions of the first and second plots 1802, 1804 can be ignored.

[0149] In both the first and second plots 1802, 1804, the vessel is filled with a uniform sample solution (i.e., 0% sucrose vs. 40% sucrose), and therefore the voltage measurements (y-axis) should be consistent (i.e., flat) across the length of the vessel (x-axis). However, as shown in the example of Figure 18, the first and second plots 1802, 1804 contain voltage measurements with a slight downward slope due to wall draft and / or thickness variations of the vessel.

[0150] According to operation 1708 of method 1700, the first and second plots 1802, 1804 can 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 where each measurement is taken above the bottom portion of the container (e.g., 14 mm above) to normalize the first and second plots 1802, 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 the plots 1802, 1804, which is in front of the meniscus 308 of the density gradient 300. In this illustrative example, the maximum compensation value is approximately 1.6 mV / mm.

[0151] 18, first and second normalized plots 1802′, 1804′ are generated after compensation values are added to the first and second plots 1802, 1804, such as following completion of operation 1708 of method 1700. The first and second normalized plots 1802′, 1804′ are linear (e.g., flat) along the length (x-axis) of the container, and thus wall draft and / or thickness variations of the container are compensated for by the compensation values.

[0152] 19 illustrates another example of a chart 1900 showing the normalization of measurements following completion of method 1700. Chart 1900 includes a plot 1902 of voltage (y-axis) and container length (x-axis) for a container made from polyethylene terephthalate glycol (PETG) material having a diameter of 9 / 16 inches. In this example, the container is filled with 0% sucrose density modifier (e.g., the container is filled with 100% DI water).

[0153] In the example shown in FIG. 19, the downward slope of plot 1902 is less than the downward slope of first plot 1802 in FIG. 18 for a container made from a polypropylene material and filled with 0% sucrose density modifier. These examples illustrate that containers made from different materials may have different wall draft and / or wall thickness variations. For example, the container made from the PETG material in FIG. 19 has less draft than the container made from the polypropylene material in FIG. 18. Given the foregoing, the compensation value used to normalize the voltage measurements can vary based on the material and / or size of the container identified in operation 1706 of method 1700.

[0154] 19, compensation values are added based on where each measurement is taken above the bottom portion of the container (e.g., 14 mm above) to normalize plot 1902 based on the container's PETG material and 9 / 16 inch diameter. In this illustrative example, the largest compensation value is to the right of plot 1902, such as just before meniscus 308 of density gradient 300 (e.g., approximately 0.5 mV / mm).

[0155] The normalized plot 1902′ is generated after the compensation values have been added, such as following completion of operation 1708 of method 1700. The normalized plot 1902′ is linear (e.g., flat) along the length (x-axis) of the container, and thus, wall draft and / or thickness variations of the container have been compensated for by the compensation values.

[0156] 18 and 19, operation 1708 can include a compensation value added to the voltage measurement detected by detector 716. The compensation value is based on the length over which the measurement is taken and removes the effects of wall draft and / or wall thickness variations of the container. The compensation value can be based on characterization data identifying the container's material (see operations 1702-1706). Method 1700 can be applied to open-top containers because these types of containers are typically injection molded and are believed to exhibit greater wall draft than containers made by other manufacturing methods.

[0157] In addition to wall draft, the accuracy of density gradient measurements may depend on other physical attributes of the container 110. Defects, including stains, scratches, cracks, dirt, dust, and the like, may interfere with the optical path of the light emitted from the emitter 714 as the light passes through the container 110 for detection by the detector 716. These types of defects are frequently present on containers, and even new tubes may have one or more types of defects due to the molding process used to manufacture the container.

[0158] 20 schematically illustrates an example embodiment of a method 2000 for mitigating the effects of defects and wall thickness variations along the length L of a container on measurements obtained from the container 110 according to method 1200. Examples of defects on the container 110 include, but are not limited to, blemishes, scratches, cracks, dirt, dust, and the like. These types of defects may alter the optical path of light through the container 110 and thus affect the density measurements. In some examples, method 2000 forms part of operation 1110 in method 1100. Method 2000 can be performed by system 100.

[0159] Method 2000 includes operation 2002 of measuring the container 110 when empty (i.e., before the container 110 is filled with a density gradient). Operation 2002 is performed by a motor 710 moving a sensor assembly 712 along the length L of the container 110 while the container 110 remains fixed. In operation 2002, an emitter 714 emits light for transmission through the container 110. The light is received by a detector 716 to measure the optical properties of the container 110 when empty. The measurements recorded by the detector 716 can include voltage measurements along the length L of the container.

[0160] FIG. 21 graphically illustrates an example plot 2100 of voltage measurements recorded by the sensor assembly 712 from the container 110 when empty, according to operation 2002. The x-axis of plot 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 bottom to top is represented by voltage moving from left to right on the x-axis of plot 2100. As shown in FIG. 21 , plot 2100 exhibits a slight downward slope of the voltage detected by the sensor assembly 712, which is likely caused by a change in wall thickness of the container 110. Wall thickness changes typically result from the walls being thicker near the bottom portion 304 of the container 110 and becoming progressively thinner as you move toward the opening 314 of the container 110. The decrease in wall thickness from the bottom to the top of the container can help aid in the removal of the container 110 from a mold during manufacturing.

[0161] 20, the method 2000 next includes an operation 2004 of calculating an average value from the measurements recorded along the length L of the vessel 110 in operation 2002. As an illustrative example, the average voltage measurement in plot 2100 shown in FIG. 21 is approximately 751 mV for the total length L of the vessel.

[0162] Method 2000 includes an operation 2006 of generating difference values between the average value calculated in operation 2004 and the measurements recorded in operation 2002 along the length L of the container 110. Larger difference values may indicate a possible defect, such as a scratch or stain, at a particular location along the length L of the container 110 because they indicate a greater deviation from the average value. Larger difference values may also indicate that a portion of the container 110 is particularly thick or thin relative to the thickness of other portions of the container 110. A set of difference data values along the length L of the container is created after completion of operation 2006.

[0163] Next, method 2000 includes an operation 2008 of dispensing a density gradient into container 110. The density gradient can be dispensed in operation 2008 according to the operations of method 500, described above with reference to FIG.

[0164] Method 2000 includes an operation 2010 of measuring a density gradient dispensed into container 110. The density gradient is measured in operation 2010 according to the operations of method 1200, described above with reference to FIG.

[0165] Method 2000 includes operation 2012 of adding the difference value generated in operation 2006 point by point to the density gradient measurement measured in operation 2010. This adjustment can mitigate the effects of imperfections such as stains, scratches, cracks, dirt, dust, and the like present along the length L of the container 110, and can also mitigate the effects of wall thickness variations on the density gradient measurement, because the difference data takes these effects into account. In some embodiments, the difference value is a compensation value as discussed above with respect to FIGS. 17-19.

[0166] FIG. 22 graphically illustrates an example of a chart 2200 showing how method 2000 can reduce and / or eliminate the effects caused by container defects and / or container wall thickness variations on a sample of DI water dispensed into container 110. The x-axis of chart 2200 represents the length L of container 110, and the y-axis represents voltage measurements in millivolts (mV). Because container 110 is filled with DI water, the voltage measurements should be constant along the length of container 110. However, variations in the wall thickness of container 110 (e.g., decreasing wall thickness from bottom to top) can cause voltage measurements to slope downward, such as in chart 2200 shown in FIG. 22. Additionally, defects such as stains, scratches, cracks, dirt, dust, and the like can affect density measurements, as explained above.

[0167] Chart 2200 includes a first plot 2202 of DI water voltage measurements before the differential data values are added and a second plot 2204 after the differential data values are added to the voltage measurements. As shown in FIG. 22, second plot 2204 is flatter and has less slope than first plot 2202, indicating that vessel wall thickness variations have been normalized from the data. Additionally, the effects of small scratches and blemishes, such as small bumps in the 30 mm to 37 mm range, are virtually eliminated or significantly reduced in second plot 2204. This is shown in FIG. 22 by second plot 2204 having a more linear or smoother profile than the profile of first plot 2202, which exhibits greater variance.

[0168] 23 illustrates an example of a container 2300 having a centerline CL that is offset relative to the vertical alignment VA of a sensor assembly 712 mounted on a measurement device 700. The container 2300 includes a length L that extends from a proximal end 2302 to a distal end 2304. The container 2300 has an outer diameter D that is constant across the length L of the container. The outer diameter D and the length L of the container 2300 are perpendicular to each other.

[0169] The process of precisely measuring a density gradient dispensed into container 2300 is technically challenging due to the sensitivity of density gradient measurements to the rounded surfaces of container 2300. For example, container 2300, when filled with fluid, exhibits optical properties such that the amount of light emitted from emitter 714 and received by detector 716 after passing through container 2300 is affected by the alignment of container 2300 with emitter 714 and detector 716 of sensor assembly 712.

[0170] 24 schematically illustrates an example of a method 2400 for mitigating mechanical positioning errors of a container 2300 on density gradient measurements. For example, method 2400 can mitigate and / or eliminate errors that may result from misalignment of a centerline CL of the container 2300 with respect to the vertical alignment VA of the emitter 714 and detector 716 of the sensor assembly 712. In some examples, method 2400 forms part of operation 1114 in method 1100. Method 2400 can be performed by system 100 using measurement device 700.

[0171] 23 , the proximal end 2302 includes an opening 2306 through which a probe can be inserted to dispense a density gradient into the vessel 2300. The operation 2402 can include positioning the sensor assembly 712 below the opening 2306 by using a motor 710 to move a carriage 708, on which the sensor assembly 712 is mounted, along a rail 706 while the vessel 2300 remains stationary.

[0172] 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 radially perpendicular to the length L of the container 2300, allowing the sensor assembly 712 to scan across at least a portion of the outer diameter D of the container 2300. In some embodiments, operation 2404 may include 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 further embodiments, the motors move the container 110 perpendicular to the length L of the container 2300, allowing the sensor assembly 712 to scan across at least a portion of the outer diameter D of the container 2300.

[0173] The method 2400 further includes an operation 2406 of determining a location 2310 of a centerline CL of the container 2300 near the proximal end 2302 (see FIG. 23 ). The location 2310 of the centerline CL is determined in operation 2406 based on measurements taken across the outer diameter D of the container 2300 in operation 2404.

[0174] FIG. 25 graphically illustrates an example of a plot 2500 of voltage measurements taken across the outer diameter D near the proximal end 2302 of the container 2300. Referring now to FIGS. 23-25, 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 of the centerline CL to the left or right will result in a lower voltage being 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. In the example illustrated in FIG. 25, a peak voltage of approximately 2.3 V occurs at a position of approximately +0.01 inches.

[0175] In some embodiments, method 2400 includes measuring voltage across at least a portion of outer diameter D at each measurement location along length L of container 2300 and using the maximum voltage detected at each measurement location to determine density at a given measurement location. This technique, while time consuming, can eliminate errors from inconsistencies and blemishes on container 2300.

[0176] 24, the method 2400 includes an operation 2408 of positioning the sensor assembly 712 near the distal end 2304 of the container 2300. As shown in FIG. 23, the distal end 2304 includes a bottom portion 2308. The operation 2408 can include positioning the sensor assembly 712 above the bottom portion 2308. The operation 2408 can include using the motor 710 to move the carriage 708 along the rails 706 while the container 2300 remains stationary.

[0177] Next, the method 2400 includes an operation 2410 of scanning across the outer diameter D of the container 2300 near the distal end 2304. In some embodiments, operation 2410 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 operation 2410.

[0178] Next, the method 2400 further includes an operation 2412 of determining a location 2312 of a centerline CL of the container 2300 near the distal end 2304 (see FIG. 23 ). The location 2312 of the centerline CL is determined in operation 2412 based on measurements taken across the outer diameter D of the container 2300 in operation 2410.

[0179] 26 graphically illustrates an example of a plot 2600 of voltage measurements taken across the outer diameter D near the distal end 2304 of the container 2300. As in operation 2406, the location 2312 of the centerline CL near the distal end 2304 is determined in operation 2412 by identifying the location of the peak voltage measurement. In the example illustrated in FIG. 26, a peak voltage of approximately 2.3 V occurs at an offset of approximately −0.01 inches.

[0180] 24 , the method 2400 includes an operation 2414 of generating a centerline CL of the container 2300 by connecting a location 2310 of the centerline CL near the proximal end 2302 (determined in operation 2406) with a location 2312 of the centerline CL near the distal end 2304 (determined in operation 2412). The centerline CL generated in operation 2414 is shown in FIG. 23 as extending between the proximal end 2302 and the distal end 2304, and as offset relative to the vertical axis VA of the sensor assembly 712.

[0181] The method 2400 includes an operation 2416 of measuring the density gradient dispensed into the container 2300 by following the centerline L generated in operation 2414. Operation 2416 can include moving the sensor assembly 712 in two dimensions, such as a vertical dimension along the length L of the container 2300 and a horizontal dimension along the outer diameter D of the container 2300. In some examples, operation 2416 includes moving the sensor assembly 712 starting from a location 2312 of the centerline CL near the distal end 2304 to a location 2310 of the centerline CL near the proximal end 2302 of the container 2300.

[0182] An advantage of method 2400 is that implementation of method 2400 can eliminate the need for precise alignment of the carriage 708 supporting the sensor assembly 712 with the container 2300's holder (see FIG. 27 ) and the need to maintain alignment throughout the length L of the container 2300 for each density gradient measurement. For example, the use of the centerline CL generated in method 2400 can counteract the effects of any misalignment between the container 2300 and the sensor assembly 712, simplifying the mechanical complexity and sensitivity of the measurement device 700. Method 2400 can also simplify manufacturing and field service requirements for the measurement device 700 and improve the long-term reliability of the measurement device 700, such as by eliminating the need to periodically calibrate the measurement device 700. Additionally, wear on the carriage 708 for the sensor assembly 712 and the container 2300's holder may have less of an effect on the accuracy of density gradient measurements over long-term use.

[0183] Additional advantages of method 2400 include mitigating the effect of stains on density gradient measurements. Staining typically reduces the amount of light focused by container 2300, and therefore stains on container 2300 result in lower voltage measurements. In some embodiments, method 2400 can include scanning along outer diameter D at each measurement point along length L of container 2300, such that the width of the scan along outer diameter D is likely to exceed the width of the stain, and therefore lower voltage measurements that may result from stains are ignored. In some further embodiments, a best-fit polynomial can be implemented to fit the curvature of the horizontal scan across outer diameter D and replace error data inputs, which can further reduce the effect of stains, scratches, cracks, dirt, dust, and the like on container 2300.

[0184] In some further embodiments, the entire surface area of the vessel 2300 is scanned using a solid state array device to capture all points in two dimensions at once. Such a technique can reduce the amount of time to measure the density gradient along the length L of the vessel 2300 and also mitigate the effects of inconsistencies and blemishes on the vessel.

[0185] FIG. 27 shows a cross-sectional view of a container 2300 held by a holder 2320 of the system 100 from a perspective looking down into the container 2300. As shown in FIG. 27 , the emitter 714 emits a light beam LR toward the container 2300, which passes through the container 2300 and is received by a detector 716 on the opposite side of the container 2300. In this example, the container 2300 has a 9 / 16 inch diameter. In some examples, the light beam 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 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 detector 716.

[0186] Table 3 shows the voltage measurements detected by detector 716 when container 2300 is tilted along the y-axis in the +y and -y directions, and when container 2300 is tilted along the x-axis in the +x and -x directions. As shown in Table 3, the voltage measurements have a larger variance along the y-axis than along the x-axis, and therefore the voltage measurements (i.e., density gradient measurements) are much more sensitive to container position errors in the y-axis (perpendicular to light ray LR) than in the x-axis (parallel to light ray LR). [Table 3]

[0187] 28 graphically illustrates an example of a chart 2800 showing the mitigation of mechanical positioning error. In this illustrative example, the chart 2800 includes first, second, and third plots 2802, 2804, 2804' of voltage (y-axis) and container length (x-axis). The first, second, and third plots 2802, 2804, 2804' are each measured for a container made from polypropylene material having a 9 / 16 inch diameter and filled with 100% DI water.

[0188] In the first plot 2802, the container is properly positioned so that the container's centerline CL is aligned with the vertical axis VA of the sensor assembly 712. In the second plot 2804, the container is intentionally tilted along the y-axis so that the container's centerline CL is misaligned with the vertical axis VA of the sensor assembly 712 (see FIG. 23). In the third plot 2804', the container is intentionally tilted along the y-axis as in the second plot 2804; however, density gradient measurements are taken along the container's centerline CL in accordance with the operations of method 2400. In FIG. 28, the bottom portion of the container occupies approximately 0-14 mm; therefore, this portion of the first, second, and third plots 2802, 2804, 2804' can be ignored.

[0189] 28, the second plot 2804 has a greater downward slope than the slope of the first plot 2802. This may be due to a misalignment between the container centerline CL 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 plot 2802 and the second plot 2804 is greatest near the top of the container (i.e., to the right of the x-axis) due to a greater displacement between the container centerline CL and the vertical axis VA of the sensor assembly 712 at the top of the container relative to the second plot 2804. The third plot 2804′ substantially corresponds to the first plot 2802 and shows the mitigation of mechanical positioning error present in the second plot 2804 by the method 2400 of having the sensor assembly 712 take measurements along the container centerline CL by moving the sensor assembly 712 in two dimensions, such as the vertical dimension along the container length L and the horizontal dimension along the container outer diameter D.

[0190] FIG. 29 graphically illustrates another example of a chart 2900 showing the mitigation of mechanical positioning error by method 2400. In this example, chart 2900 includes first, second, and third plots 2902, 2904, 2904′ of voltage (y-axis) and container length (x-axis). The first, second, and third plots 2902, 2904, 2904′ are each measured for a container made from PETG material having a 9 / 16 inch diameter and filled with 100% DI water. In the first plot 2902, the container is properly positioned such that the centerline CL of the container is aligned with the vertical axis VA of sensor assembly 712. In the second plot 2904, the container is intentionally tilted along the y-axis such that the centerline CL of the container is misaligned with the vertical axis VA of sensor assembly 712 (see FIG. 23). In the third plot 2904', the container is intentionally tilted along the y-axis as in the second plot 2904; however, density gradient measurements are taken along the centerline CL of the container in accordance with the operations of method 2400. In FIG. 29, the bottom portion of the container occupies a length of approximately 0-14 mm, and therefore, this portion of the first, second, and third plots 2902, 2904, 2904' can be ignored.

[0191] As in chart 2800 described above, second plot 2904 has a greater downward slope than the slope of first plot 2902. This may be due to a misalignment between the container centerline CL and the vertical axis VA of sensor assembly 712. In this example, the difference in slope between first plot 2902 and second plot 2904 is greatest near the top of the container (i.e., to the right of the x-axis) due to the greater displacement between the container centerline CL and the vertical axis VA of sensor assembly 712 at the top of the container relative to second plot 2904. Third plot 2904′ substantially corresponds to first plot 2902 and illustrates the mitigation of mechanical positioning error present in second plot 2904 by method 2400, which causes sensor assembly 712 to take measurements along the container centerline CL by moving sensor assembly 712 in two dimensions (e.g., in the vertical dimension along the container length L and the horizontal dimension along the container outer diameter D).

[0192] FIG. 30 graphically illustrates another example of a chart 3000 showing the mitigation of mechanical positioning error by method 2400. This illustrative example includes first and second plots 3002, 3004 of voltage (y-axis) and container length (x-axis). The first and second plots 3002, 3004 are measured for 5%, 15%, 25%, and 35% sucrose step gradients, respectively, in a container made from PETG material having a 9 / 16-inch diameter. In the first plot 3002, the container is tilted in the y-axis; therefore, each step in the first plot 3002 has a significant downward slope along the container length (x-axis). In the second plot 3004, the container is tilted in the y-axis (as in the first plot 3002). However, second plot 3004 is generated by method 2400, which causes sensor assembly 712 to take voltage measurements along the centerline CL of the container. As shown in Figure 30, the slope of each of the steps in second plot 3004 is much more linear (e.g., flatter) than the steps in first plot 3002. Thus, Figure 30 further illustrates that method 2400 mitigates mechanical positioning errors.

[0193] At any given position along the length L of the vessel into which the density gradient is dispensed, the measurements recorded by the sensor assembly 712 should be the same regardless of rotation of the vessel. However, in some cases, the vessel is deformed and therefore the vessel walls do not have a uniform thickness around the circumference of the vessel.

[0194] Figure 31 graphically illustrates a chart 3100 in which voltage measurements (y-axis) are obtained at a 45-degree rotation along the length (x-axis) of the vessel. The variation in voltage measurements along the length of the vessel for the different degrees of rotation shown in Figure 31 (i.e., 0, 45, 90, 135, 180, 225, 270, and 315 degrees) can result in density gradient measurement errors, especially when it is desirable to measure the vessel in only one rotational orientation. Also, rotating the vessel during density gradient measurements can add complexity to the measurement apparatus 700.

[0195] 32 diagrammatically illustrates another example of a measurement device 3200 that can mitigate errors from when the walls of a container 3210 do not have a uniform thickness around the circumference of the container. In this exemplary 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 90 degrees apart relative to one another. The emitters 3206, 3206′ are similar to emitter 714, and the detectors 3208, 3208′ are similar to detector 716 of measurement device 700. For example, the emitters 3206, 3206′ can emit light (e.g., infrared light), and the detectors 3208, 3208′ can include photodiodes that measure the voltage of the light transmitted from the emitters 3206, 3206′ through the container 110 to measure the density gradient dispensed into the container 3210.

[0196] In some embodiments, the first and second pairs 3202, 3204 of emitters and detectors are mounted on a carriage, such as carriage 708 shown in FIGS. 7-9. The emitters 3206, 3206′ and detectors 3208, 3208′ are in a fixed relationship to one another when mounted on the carriage, which allows these components to be moved together up and down the rails 706. A motor 710 can be used to move the carriage along the rails 706 of the measurement device 700, thereby moving the first and second pairs 3202, 3204 up and down the length of the container 3210.

[0197] 33 schematically illustrates an example embodiment of a method 3300 for mitigating wall thickness variations around the periphery of a container 3210 when measuring a density gradient dispensed into the container. In some embodiments, the periphery comprises the diameter or circumference of the container 3210. In some embodiments, method 3300 forms part of operation 1112 in method 1100. Method 3300 can be performed by system 100.

[0198] The method 3300 includes an operation 3302 of positioning the first and second pairs 3202, 3204 at a predetermined location along the length of the container 3210. In some examples, the predetermined location is at a distal end of the container 3210. The operation 3302 can include using a motor to move the first and second pairs 3202, 3204 while the container 3210 remains fixed to position the first and second pairs 3202, 3204 relative to the container 3210.

[0199] 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 embodiments, the first and second voltages are measured simultaneously in operation 3304. In other embodiments, the first and second voltages are not measured simultaneously in operation 3304.

[0200] Method 3300 includes an operation 3306 of calculating an average from the first and second voltages measured in operation 3304. The wall thickness of the vessel 3210 may be thicker at one location but is unlikely to be thicker at a location 90 degrees away, and therefore, averaging the first and second voltages is effective to mitigate errors in the density gradient measurements that may result from wall thickness variations around the circumference of the vessel 3210. Also, the vessel 3210 may have a stain or scratch at one location but is unlikely to have a stain or scratch 90 degrees away, and therefore, averaging the first and second voltages is effective to mitigate errors in the density gradient measurements from stains, scratches, cracks, dirt, dust, and the like.

[0201] Next, method 3300 includes operation 3308 of determining whether additional density gradient measurements are desired along the length of vessel 3210. If additional density gradient measurements are desired (i.e., "yes" in operation 3308), method 3300 may repeat operations 3302-3306 to measure the density gradient at another location along the length of vessel 3210. Otherwise, if no additional density gradient measurements are desired (i.e., "no" in operation 3308), method 3300 ends at operation 3310.

[0202] 34 graphically illustrates a chart 3400 showing the mitigation of wall thickness variation around the periphery of the vessel 3210 by the method 3300. As discussed above, in some embodiments, the periphery includes the diameter or circumference of the vessel 3210. The chart 3400 includes a plot of average voltage measurements (y-axis) obtained at 45 degree rotations along the length (x-axis) of the vessel 3210. For example, the average voltage between a first pair 3202 and a second pair 3204 of emitters 3206 and detectors 3208 spaced 90 degrees apart is obtained at positions between 0 and 90 degrees, between 45 and 135 degrees, between 90 and 180 degrees, between 135 and 225 degrees, between 180 and 270 degrees, between 225 and 315 degrees, between 270 and 360 degrees, and between 315 and 45 degrees. In Figure 34, the variation in voltage measurements along the length of the vessel 3210 is shown as being significantly reduced in chart 3400 compared to chart 3100 of Figure 31. In some examples, the variation in voltage measurements is reduced by approximately 56.6%.

[0203] 35 graphically illustrates an example of a density gradient 3500 dispensed into container 110 by system 100. Density gradient 3500 includes dispense rates (y-axis) for DI water, density modifier, concentrated buffer, and additives per 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 5-40% continuous gradient with four steps (e.g., steps 3, 7, 21, and 30-33) containing 10 mM buffer and additives.

[0204] The density modifier has a concentration that exceeds the maximum concentration of the density gradient 3500. In this illustrative example, a 50% concentration of density modifier is used to support a 40% maximum concentration. When the dispensing rate of the density modifier increases, the density of a given step in the density gradient increases. When 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. When the dispensing rate of the density modifier decreases, the density of a given step in the density gradient decreases.

[0205] Figure 36 schematically illustrates a method 3600 of generating the density gradient 3500 shown in Figure 35. Method 3600 includes an operation 3602 of dividing density gradient 3500 into stages. In the illustrative example shown in Figure 35, density gradient 3500 is a continuous gradient divided into 41 separate stages. The number of stages can be increased or decreased depending on the desired shape and size of the density gradient.

[0206] Next, method 3600 includes an operation 3604 of calculating dispense rates for the components in each stage of density gradient 3500. As shown in Figure 35, DI water and density modifier make up the majority of the volume in each stage of density gradient 3500. The dispense rate of DI water decreases proportionally to the increase in the dispense rate of density modifier to maintain a constant volume per stage of density gradient 3500 while maintaining a continuous gradient profile.

[0207] In the illustrative example shown in Figure 35, the dispense rate of concentrated buffer is low and remains constant along the length of density gradient 3500. Whenever an additive is dispensed (e.g., stages 3, 7, 21, and 30-33), the volume of additive is subtracted from the DI water to maintain a constant volume for the stage in density gradient 3500, while maintaining a continuous gradient profile.

[0208] Method 3600 includes an operation 3606 of dispensing each stage based on the volume and dispense rate calculated for the components in each stage in operation 3604. Operation 3606 may follow method 500 described above, such that distal end 112 of probe 108 is lowered toward the bottom of the interior volume of container 110 and successively denser stages are dispensed by probe 108 to form density gradient 3500.

[0209] In FIG. 35, the stages of density gradient 3500 are dispensed from left to right along the x-axis, starting with the first stage having the lightest density (e.g., highest concentration of DI water and lowest concentration of density modifier). The first stage is pushed up the container 110 as subsequent stages having heavier densities are dispensed into the container. The last stage is the heaviest stage (e.g., lowest concentration of DI water and highest concentration of density modifier). Density gradient 3500 is a 5-40% continuous gradient, so the first stage has 5% density modifier and the last stage has 40% density modifier.

[0210] In addition to density gradient 3500 shown in FIG. 35 , which is provided as an illustrative example, method 3600 can be implemented to generate various types of density gradients having different volumes, slopes, and / or shapes. For example, method 3600 can be implemented to generate a 10-25% density gradient by dividing the gradient into fewer steps than the 41 steps shown for density gradient 3500 (operation 3602), calculating the volumes and dispense rates for the components in each step (operation 3604), dispensing the steps to form a 10-25% density gradient (operation 3606), etc. As in the example described above, the lightest step (i.e., 10% density modifier) is dispensed first, followed by successively denser steps until the heaviest step (i.e., 25% density modifier) is dispensed. In further examples, method 3600 can be implemented to generate more complex density gradients, such as exponential gradients, S-shaped gradients, and other desired shapes.

[0211] FIG. 37 illustrates an example of a density gradient 3700 dispensed into a container 110 by system 100. In this illustrative example, density gradient 3700 is a step gradient including five distinct stages 3702a-3702e separated by interfaces 3704. Density gradient 3700 can be formed according to the operations of method 3600. The interfaces 3704 between stages 3702a-3702e are visible as slightly darker lines. It may be desirable for each of stages 3702a-3702e to have a uniform density and for the interfaces 3704 between stages 3702a-3702e to be as narrow as possible. It may also be desirable to dispense density gradient 3700 as quickly as possible to maximize the throughput of system 100 while mitigating mixing between stages 3702a-3702e within the container.

[0212] Figure 38 schematically illustrates an example embodiment of a method 3800 for generating a density gradient. System 100 can perform method 3800 to generate a step gradient, such as density gradient 3700 shown in Figure 37. Method 3800 reduces the time for system 100 to generate density gradient 3700 while minimizing mixing at interface 3704 between gradient steps 3702. In a further example, system 100 can perform method 3800 to generate a continuous gradient, such as density gradient 3500 shown in Figure 35.

[0213] 39 graphically illustrates an example of a chart 3900 showing an implementation of method 3800 by system 100. As shown in FIG. 39, chart 3900 includes a first plot 3902 of voltage measurements (left y-axis) over time (x-axis) and a second plot 3904 of dispense rate (right y-axis) over time (x-axis) for density gradient 3700.

[0214] 37-39, a method 3800 includes an operation 3802 of dispensing a first stage 3702a of a density gradient 3700. The first stage 3702a has the lightest density (e.g., the highest concentration of DI water and the lowest concentration of density modifier), and therefore, the first stage 3702a will be pushed up the container 110 as subsequent stages having heavier densities are dispensed into the container 110. As shown in FIG. 39, the first stage 3702a is dispensed at a maximum rate until it reaches an interface 3704 with the second stage 3702b (e.g., at a time of about 27 seconds). In this illustrative example, the maximum rate is about 20 mL / min. The first stage 3702a is dispensed at a maximum rate because there is no risk of mixing with the other stages.

[0215] The method 3800 includes an operation 3804 of decreasing the dispense rate at an interface 3704 between a first stage 3702a and a second stage 3702b. FIG. 39 shows that the maximum rate is decreased to a minimum rate. In this illustrative example, the minimum rate is approximately 2 mL / min. As shown in FIG. 39, the dispense rate exhibits a dramatic decrease at the interface 3704 between the first stage 3702a and the second stage 3702b. For example, the maximum rate is reduced to the minimum rate within a short period of time, approximately 1 second.

[0216] Next, method 3800 includes operation 3806 of increasing the dispense rate of second stage 3702b from a minimum rate until a maximum rate is reached. In the illustrative example shown in FIG. 39, the dispense rate increases exponentially until the maximum rate is reached at approximately 30 seconds. This is shown by the exponential curve in second plot 3904 of FIG. 39 when dispensing second stage 3702b. Additional shapes for increasing the dispense rate from a minimum rate to a maximum rate are possible, and therefore the exponential curve shown in FIG. 39 is provided as an illustrative example.

[0217] The adjustment of the dispense rate in operations 3804 and 3806 (e.g., decreasing the dispense rate from a maximum rate to a minimum rate and increasing the dispense rate from a minimum rate to a maximum rate) is performed by pump 104, which adjusts the flow rate for pumping components from reservoirs 102 into manifold and mixing chamber 106. Alternative embodiments for adjusting the dispense rate are possible.

[0218] Next, method 3800 includes operation 3808, which determines whether the density gradient includes another stage. When the density gradient includes another stage (i.e., "yes" in operation 3808), method 3800 repeats operations 3804 and 3806 to dispense the additional stage. In the illustrative example shown in Figures 37 and 39, density gradient 3700 includes five distinct stages, with interfaces 3704 occurring at approximately 27, 39, 51, and 63 seconds. Operations 3804 and 3806 can be repeated to generate each of stages 3702 in density gradient 3700.

[0219] When the density gradient does not include additional stages (i.e., "not applicable" in operation 3808), method 3800 stops dispensing in operation 3810. Method 3800 reduces the time to generate the density gradient while also mitigating mixing between stages of the density gradient by adjusting the dispense rate when an interface is created. For example, method 3800 reduces mixing by dispensing slowly (e.g., at a minimum speed) when starting a new stage at the interface. Then, by increasing the dispense rate until a maximum speed is reached, method 3800 can reduce the overall time to dispense the density gradient. Method 3800 enables system 100 to generate sharp interfaces between stages of the density gradient while dispensing the density gradient in a minimal amount of time.

[0220] As will be described in more detail herein, the process of manually creating a set of identical density gradients can often be difficult due to the large inherent amount of variation that exists between manually created density gradients. For example, sources of variation can include the concentrations of the mixture components and the rate at which the mixture components are dispensed, which can cause day-to-day and user-to-user variations in manually created density gradients. As will be described in more detail herein, the following methods and techniques can be implemented on system 100 to replicate density gradients that closely match a prior density gradient.

[0221] 40 schematically illustrates an example of a method 4000 for replicating density gradients that may be implemented on system 100. Method 4000 can be repeated to replicate as many density gradients as desired.

[0222] 40, method 4000 includes an operation 4002 of measuring a density gradient selected for replication. In some embodiments, the density gradient is generated by system 100, such as in accordance with the operations of method 500 described above. In other embodiments, 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.

[0223] Operation 4002 may include scanning the container 110 using the sensor assembly 712 to detect light transmission through the container to measure a voltage corresponding to density. The measurements may be used to create a profile of the density gradient. The profile may include a correlation between voltage measurements and position along the length L of the container 110. As an example, the system 100 may measure voltage at approximately 320 points along an approximately 80 mm length of the container 110 to create a density gradient profile.

[0224] Next, method 4000 includes an operation 4004 of storing the density gradient profile on a non-volatile memory device. The density gradient profile can be stored on the non-volatile memory of system 100. In other examples, the density gradient profile can be stored on the non-volatile memory of an external storage device. In some examples, the external storage device can include portable devices such as USB flash drives and similar data storage devices that can plug into or otherwise connect to system 100. In further examples, the external storage device can be included on a remote server that can connect to system 100 via a connection through a communications network 5220, such as that shown in FIG. 52.

[0225] The profile stored in operation 4004 can be used at any time by system 100 to replicate a density gradient. The density gradient profile can be stored as a "favorite" density gradient profile that can be selected for replication using display 132 of system 100. System 100 can store multiple favorite density gradient profiles.

[0226] Next, method 4000 includes an operation 4006 of replicating the density gradient. Operation 4006 includes retrieving a stored profile for the density gradient and then controlling pump 104 to pump a replica of the density gradient into vessel 110 based on the profile. The replication of the density gradient in operation 4006 can be performed according to the operations of method 500 described above.

[0227] Method 4000 may further 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 the measurement of the original density gradient in operation 4002. For example, operation 4008 may include measuring the replicated density gradient at the same points that were measured for the original density gradient. The measurements and / or profile of the replicated density gradient are compared to the measurements and / or profile of the original density gradient to determine whether they are within a predetermined tolerance.

[0228] If the measurements and / or profile are within the predetermined tolerances, the replicated density gradient is approved; otherwise, if the measurements and / or profile are outside the predetermined tolerances, the replicated density gradient is rejected.

[0229] In some examples, operation 4008 may further include displaying the profiles of the density gradient and the replicated density gradient displayed side-by-side on the display 132 of the system 100. This allows a user of the system 100 to view and / or confirm the similarity between the replicated density gradient and the original density gradient.

[0230] When replicating a density gradient based on a prior density gradient profile, the measurements used to generate the profile 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 with a density gradient dispensed therein may contain noise from optical effects that may interfere with density calculations. This may be particularly true for stepped density gradients that have large steps in density, causing large differences in refractive index between the steps, resulting in noise in density measurements near the interfaces between the steps. Noise should be removed from the density gradient profile because otherwise the noise would cause errors when a new density gradient is replicated based on the profile.

[0231] 41 graphically illustrates an example of a density gradient profile 4100 prior to being processed by system 100. Density gradient profile 4100 includes noise that could potentially interfere with replication of density gradient profile 4100 by system 100. In FIG. 41, density gradient profile 4100 includes voltage measurements (y-axis) indicating density along the length of a vessel (x-axis) for a step density gradient having steps of 5%, 15%, 25%, and 35% dispensed into a vessel having a diameter of 9 / 16 inch.

[0232] The flat portions 4102 in density gradient profile 4100 represent steps in the step density gradient. Density gradient profile 4100 also contains measurement fluctuations 4104 near lengths of approximately 30 mm, 52 mm, and 73 mm, which are noise due to optical effects at the interfaces between the density gradient steps. Measurement fluctuations 4104 are caused by large differences in refractive index between the density gradient steps.

[0233] Before density gradient profile 4100 is used to replicate a density gradient, it may be desirable to remove and / or replace measurement runout 4104. Otherwise, measurement runout 4104 may cause errors and loss of fidelity when the density gradient is replicated by system 100, such as in accordance with the operations of method 4000.

[0234] 41, the density gradient profile 4100 includes measurements 4106 from the bottom of the vessel (e.g., below 10 mm). The curvature of the vessel's bottom portion 304 can optically interfere with the density gradient measurements, thus producing dramatic measurement deviations. When replicating a density gradient, it is further desirable to remove and / or replace the measurements 4106 from the bottom portion 304.

[0235] 42 schematically illustrates an example of a method 4200 for processing density gradient profile 4100 to remove noise and imperfections that may interfere with replicating the density gradient profile by system 100. In some examples, method 4200 forms part of operation 4002 in method 4000, and thus the density gradient profile is processed according to method 4200 before it is stored in operation 4004.

[0236] Method 4200 includes an operation 4202 of identifying locations of interfaces between stages in the density gradient. Operation 4202 may include using mathematical differentiation techniques on the density gradient profile 4100 to produce a second plot 4100' shown in Figure 41. Negative peaks 4104' in second plot 4100' identify locations of interfaces between stages 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 vessel.

[0237] Next, method 4200 includes an operation 4204 of replacing measurements at the interface location identified in operation 4202. Operation 4204 includes calculating a first average measurement from the set of measurements before the interface location, calculating a second average measurement from the set of measurements after the interface location, and replacing the measurements at the interface location with the first and second average measurements.

[0238] FIG. 43 graphically illustrates an example of a modified density gradient profile 4300 generated according to the operations of method 4200 for replacing density gradient profile 4100. FIG. 43 illustrates the calculation of first and second average measurements in operation 4204. In this illustrative example, the interfaces between the density gradient stages each have a total length of about 8 mm, with about 4 mm belonging to the previous stage and about 4 mm belonging to the next stage. The first average measurement is calculated from a set of 10 measurements before the start of each interface, and the second average measurement is calculated from a set of 10 measurements after each interface. In other examples, the first and second average measurements can be calculated from more or less than 10 measurements before and after each interface.

[0239] 43, measurement runout 4104 is replaced with a first area 4302 based on a first average measurement and a second area 4304 based on a second average measurement, respectively. By replacing measurement runout 4104 with first and second areas 4302, 4304, modified density gradient profile 4300 is smoother than density gradient profile 4100 because measurement runout 4104 detected at the interface between steps of the step density gradient is eliminated. This can improve the accuracy and fidelity of replicating step density gradients by system 100.

[0240] 42 , method 4200 may further include operation 4206 of adjusting measurement value 4106 at the bottom of the vessel (e.g., below 10 mm), where the bottom portion of the vessel produces dramatic measurement deviations in density gradient profile 4100. Modified density gradient profile 4300 replaces measurement value 4106 with a linear ramp 4306 of increasing density dispensed below 10 mm. This may provide relief for very heavy density material dispensed at the bottom of the vessel. Linear ramp 4306 prevents the creation of a sharp interface at the bottom of the vessel, which may improve replication of step density gradients by system 100.

[0241] 44 graphically illustrates an example of a chart 4400 showing a comparison of a first density gradient 4402 and a second density gradient 4404 replicated from the first density gradient by system 100. In this illustrative example, the first and second density gradients 4402, 4404 are closely matched to one another such that the second density gradient 4404 has good fidelity to the first density gradient 4402.

[0242] 45 schematically illustrates another example of a method 4500 of processing a density gradient profile for replication by system 100. Method 4500 includes an operation 4502 of acquiring a density gradient profile. The density gradient profile includes voltage measurements that can be detected by a sensor of system 100 that scans a container into which the density gradient is dispensed. The sensor can scan the container from bottom to top or from top to bottom in a single pass. The voltage measurements correlate to density.

[0243] By way of example, the density gradient profile may include voltage measurements taken every 0.25 mm along the length of the vessel, hi some embodiments, the density gradient profile includes approximately 320 voltage measurements along the length of the vessel.

[0244] Method 4500 includes operation 4504, which determines whether the density gradient profile is from a step density gradient or a linear density gradient. The determination can be based on characteristics of voltage measurements in the density gradient profile obtained in operation 4502. For example, the voltage measurements can indicate a linear density gradient when the voltage measurements incrementally decrease in small amounts along the length of the vessel. As another example, the voltage measurements can indicate a step density gradient when the voltage measurements remain constant and then decrease in large amounts.

[0245] When the density gradient is determined by a linear density gradient (i.e., "Linear" in operation 4504), method 4500 can omit certain processing operations and proceed to operation 4508, described in more detail below. When the density gradient is determined by a step density gradient (i.e., "Step" in operation 4504), method 4500 proceeds to operation 4506, which removes optical anomalies that may arise due to large changes in refractive index caused by different densities at interfaces between steps of a step density gradient. In some embodiments, the optical anomalies removed in operation 4506 include Gouy phase shifts.

[0246] 46 graphically illustrates an example of a density gradient profile 4600 prior to being processed by method 4500. Density gradient profile 4600 includes voltage measurements (y-axis) measured across the length (x-axis) of a vessel having a density gradient dispensed therein. The bottom of the vessel is to the left (e.g., 0 mm) of density gradient profile 4600, and the top of the vessel is to the right (e.g., 80 mm) of density gradient profile 4600. In this illustrative example, density gradient profile 4600 is for a step density gradient having steps of 0%, 15%, 30%, and 40% of sucrose density modifier.

[0247] In Figure 46, density gradient profile 4600 begins at approximately 14 mm of the vessel length and ends at approximately 76 mm of the vessel length. Density gradient profile 4600 includes a short first stage 4602a (e.g., 40% sucrose) because most of the first stage 4602a is at the bottom of the vessel, where it is removed from the profile. In this example, first stage 4602a has a constant voltage measurement of approximately 2,000 mV, representing a 40% sucrose level.

[0248] Density gradient profile 4600 exhibits a large measurement deviation at interface 4604a, starting at a length of approximately 16 mm and ending at a length of approximately 25 mm. The large measurement deviation is a Gouy phase shift at interface 4604a between first stage 4602a and second stage 4602b due to these stages having different densities, which creates a fringing effect that affects the transmission of light through the step density gradient.

[0249] The second stage 4602b (e.g., 30% sucrose) of density gradient profile 4600 begins at a length of approximately 25 mm and ends at a length of approximately 34 mm. In this illustrative example, second stage 4602b has a constant voltage measurement of approximately 1,890 mV, representing the 30% sucrose level. Stages 4602 and interface 4604 alternate in density gradient profile 4600 along the length of the vessel until meniscus 4606 of the density gradient is reached.

[0250] In operation 4506, optical anomalies, such as Gouy phase shifts, are removed. In some embodiments, the Gouy phase shifts are removed using operations similar to those in method 4200 described above. For example, the Gouy phase shifts can be removed by determining the location of the interface 4604 on the density gradient profile 4600. This can be accomplished by using mathematical differentiation techniques on the density gradient profile 4600.

[0251] 47 graphically illustrates an example of a derivative plot 4700 after density gradient profile 4600 has been mathematically differentiated. In this illustrative example, negative peaks 4704a-4704c at distances of approximately 21 mm, 38 mm, and 56 mm identify the location of interface 4604 between density gradient stages 4602, while negative peak 4704d identifies the location of the density gradient meniscus 4606.

[0252] Figure 48 is an expanded view of derivative plot 4700. In operation 4506, measurements affected by the Gouy phase shift at interface 4604 are replaced by values 4702 determined from derivative plot 4700. For example, a measurement with a derivative of zero or the smallest positive value can be selected for use across the entire stage, including replacing measurements affected by the Gouy phase shift at interface 4604, as this is the flattest point on the particular stage. Negative slope values should not be used. In a further example, the first and second average measurements calculated in method 4200 (see modified density gradient profile 4300 in Figure 43) can be used to replace measurements affected by the Gouy phase shift at interface 4604.

[0253] Figure 49 graphically illustrates an example comparison of density gradient profile 4600 of Figure 46 with an adjusted density gradient profile 4900 after measurements affected by optical effects such as Gouy phase shift have been replaced with values determined from derivative plot 4700. Figure 50 is a close-up view of adjusted density gradient profile 4900. As shown in Figures 49 and 50, adjusted density gradient profile 4900 has an abrupt interface 4904 during stage 4902; therefore, optical effects from Gouy phase shift are removed from adjusted density gradient profile 4900.

[0254] 45, method 4500 includes operation 4508 of removing and / or replacing measurements taken from the bottom of the vessel. As explained above, curvature on the bottom portion of the vessel can interfere with the transmission of light for measuring the density gradient. Measurements from the bottom portion are not useful because they can include large measurement variations in the density gradient profile. As an example, measurements from lengths of the vessel below 14 mm are removed in operation 4508. This is shown on the left side of adjusted density gradient profile 4900.

[0255] In some embodiments, the measurement from the bottom of the container is replaced with a value greater than or equal to the heaviest density below 14 mm of the container length. In some embodiments, the measurement from the bottom of the container is replaced with a linear ramp 4306 of increasing density, as shown in FIG.

[0256] Next, method 4500 includes operation 4510 of removing and / or replacing measurements where meniscus 4606 is located. As explained above, the location of meniscus 4606 can be identified using derivative plot 4700 shown in FIG. 47. For example, the negative peak furthest to the right (i.e., negative peak 4704d) identifies the location of meniscus 4606 in the density gradient. Like interface 4604 between stages 4602 of density gradient profile 4600, meniscus 4606 can cause optical effects due to the large change in refractive index caused by the difference in density between the last stage of the density gradient (i.e., stage 4602d) and the volume of air above the density gradient in the container. In some embodiments, the optical properties of meniscus 4606 can also cause a Gouy phase shift. Removal and / or replacement of measurements where meniscus 4606 is located is shown on the left side of adjusted density gradient profile 4900 in FIGS. 49 and 50.

[0257] 45, method 4500 may further include operation 4512 of converting the adjusted density gradient profile 4900 shown in FIGS. 49 and 50 into computer-readable dispense instructions usable by system 100 to replicate the density gradient. Operation 4512 is performed regardless of whether the density gradient is a linear or step gradient. In some examples, the computer-readable dispense instructions from the adjusted density gradient profile 4900 are stored in a text file.

[0258] FIG. 51 shows an example of a text file 5100 that is executable by a processing device of system 100 to dispense a density gradient into a vessel 110. As an example, text file 5100 is a text translation of the adjusted density gradient profile 4900 shown in FIGS. 49 and 50. In this illustrative example, text file 5100 includes a first column 5102 of positions along the length L of vessel 110 (e.g., the x-axis in FIGS. 49 and 50). In this example, positions along the vessel length L range from 80 mm to 0 mm. Text file 5100 includes a second column 5104 of voltage values (e.g., the y-axis in FIGS. 49 and 50).

[0259] The voltage values in second column 5104 represent the refractive index, which corresponds to the density of the mixture of components from reservoirs 102a-102d. System 100 can use text file 5100 to calculate the dispense rate for DI water and a density modifier (e.g., 40% sucrose) based on the voltage values in second column 5104 for each position in first column 5102 along length L of container 110. System 100 independently controls pumps 104a-104d to control the dispense rate of components into container 110, with the lowest density stage dispensed first, followed by successively higher density stages. This is shown to the right of adjusted density gradient profile 4900 in FIGS. 49 and 50, where the lowest density stage is shown. When text file 5100 is executed by system 100, a density gradient is generated by system 100 as a clone of the original density gradient.

[0260] 52 schematically illustrates an example of computing hardware of system 100 for implementing aspects of the present disclosure. The computing hardware includes processing circuitry having memory for storing instructions that, when executed by the processing circuitry, cause the processing circuitry to perform functions described herein.

[0261] 52, the system 100 includes one or more processing devices 5202, a memory storage device 5204, and a system bus 5206 that couples the memory storage device 5204 to the one or more processing devices 5202. The one or more processing devices 5202 may include a central processing unit (CPU).

[0262] 52, memory storage device 5204 can include random access memory (“RAM”) 5208 and read only memory (“ROM”) 5210. Basic input and output logic with basic routines that help to transfer information between elements within system 100, such as during start-up, can be stored in ROM 5210.

[0263] The system 100 may also include a mass storage device 5212, which includes an operating system 5214 and may store software instructions 5216 and data. The mass storage device 5212 is connected to the processing device 5202 through 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.

[0264] Although the descriptions of computer-readable data storage media contained herein refer to mass storage device 5212, it should be understood by those skilled in the art that a computer-readable data storage medium can be any available non-transitory physical device or article of manufacture from which system 100 can read data and / or instructions. A computer-readable storage medium can consist entirely of non-transitory media. Mass storage device 5212 is an example of a computer-readable storage device.

[0265] 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. Exemplary types of computer-readable data storage media include, but are not limited to, RAM, ROM, EPROM, EEPROM, flash memory or other solid-state memory technologies, or any other media that can be used to store information and that can be accessed by the device.

[0266] System 100 can operate in a networked environment using logical connections to other devices through a communication network 5220. System 100 connects to communication network 5220 through a network interface unit 5218, which is connected to system bus 5206. Network interface unit 5218 can connect to additional types of communication networks and devices, including through Bluetooth, Wi-Fi, and cellular telecommunications networks, including 4G and 5G networks. Network interface unit 5218 can connect system 100 to additional networks, systems, and devices. System 100 also includes an input / output unit 5222 for receiving and processing input and output from peripheral devices.

[0267] The mass storage device 5212 and RAM 5208 can store software instructions and data. The software instructions can include an operating system 5214 for operating the system 100. The mass storage device 5212 and / or RAM 5208 can also store software instructions 5216 that, when executed by the processing device 5202, provide the functionality of the system 100 discussed herein. The mass storage device 5212 and / or RAM 5208 can store a profile of the density gradient measured by the measurement apparatus 700, as described above.

[0268] The various embodiments described above are provided by way of example only and should not be construed as limiting in any way. Various modifications can be made to the above-described embodiments without departing from the true spirit and scope of the present disclosure.

[0269] Embodiments of the present disclosure can be described with reference to the following numbered appendices, where preferred features are expanded in the dependent appendices. 1. A system for non-destructively measuring density gradients of components for use in centrifugation, comprising: a sensor assembly; a motor coupled to the sensor assembly; a measuring device including: When executed by the processing circuitry, the processing circuitry using a motor to move the sensor assembly along the length of the density gradient of the component; obtaining measurements from the sensor assembly while the sensor assembly is moved along the length of the density gradient of the component; generating a density gradient profile of the component based on the measurements; processing circuitry having memory for storing instructions; A system comprising: 2. The instructions further, when executed by the processing circuitry, cause the processing circuitry to: 10. The system of claim 1, wherein the profile is adjusted by removing measurements from a bottom portion of a vessel containing a density gradient of components. 3. The instructions further, when executed by the processing circuitry, cause the processing circuitry to: Identifying the location of the meniscus of the density gradient of the components adjusting the profile to remove measurements from the meniscus location; 10. The system of claim 1. 4. The system of claim 3, wherein the location of the meniscus is identified by identifying a minimum in the first derivative of the measurement. 5. The instructions further, when executed by the processing circuitry, cause the processing circuitry to: 10. The system of claim 1, wherein the measurements are standardized based on at least one of the material and size of the container in which the density gradient of the components is contained. 6. The instructions further, when executed by the processing circuitry, cause the processing circuitry to: obtaining measurements from the sensor assembly along the length of the container before the density gradient of the component is dispensed therein; comparing measurements from the container before the density gradient of components is dispensed therein with an expected measurement range for a container of a given material and size; determining at least one of a material and a size of the container based on a comparison with an expected measurement range; 10. The system described in Appendix 5. 7. The instructions further, when executed by the processing circuitry, cause the processing circuitry to: 10. The system of claim 1, wherein the density gradient of the components is mitigated from the effects on the density gradient profile caused by imperfections and wall thickness variations on the vessel in which the density gradient is contained. 8. The instructions further, when executed by the processing circuitry, cause the processing circuitry to: obtaining measurements from the sensor assembly along the length of the container when empty; Calculate the average of the measurements along the length of the container when empty; generating a difference value between the average of the measurements along the length of the container when empty and the measurement; By adding the difference value, the density gradient profile of the component is adjusted. 7. The system of claim 7. 9. The instructions further, when executed by the processing circuitry, cause the processing circuitry to: 2. The system of claim 1, which reduces positioning errors on a component density gradient profile caused by misalignment of the container into which the component density gradient is dispensed. 10. The instructions further, when executed by the processing circuitry, cause the processing circuitry to: Generate a centerline for the container; moving the sensor assembly in at least two dimensions along the centerline of the vessel to obtain measurements used to generate a density gradient profile of the component; 10. The system of claim 9. 11. The instructions further, when executed by the processing circuitry, cause the processing circuitry to: positioning a sensor assembly adjacent the proximal end of the container; scanning a cross section of the vessel near the proximal end; determining a first location of the centerline near the proximal end of the container; positioning a sensor assembly adjacent a distal end of the container; scanning a diameter of the vessel near the distal end; determining a second location of the centerline near the distal end of the container; generating a centerline for the container by linearly connecting a first location of the centerline near the proximal end to a second location of the centerline near the distal end; 11. The system of claim 10. 12. The system of claim 1, wherein the sensor assembly includes a first pair of emitters and detectors mounted on the carriage and a second pair of emitters and detectors mounted on the carriage, the first and second pairs having a 90 degree separation on the carriage. 13. The instructions further, when executed by the processing circuitry, cause the processing circuitry to: using a motor to position the first and second pairs of emitters and detectors at locations along the length of the vessel where the density gradient of the components is to be dispensed, and moving the carriage; obtaining a first measurement using a first pair of emitter and detector; obtaining a second measurement using a second pair of emitter and detector; calculating an average measurement from the first and second measurements; Using the average measurements to generate a density gradient profile of the component, mitigating errors from wall thickness variations around the periphery of the vessel; 13. The system of claim 12. 14. The sensor assembly is an emitter that emits an illumination signal; a detector that measures the intensity of the illumination signal after transmission through the density gradient of the component; 2. The system of claim 1, comprising: 15. The instructions further, when executed by the processing circuitry, cause the processing circuitry to: 2. The system of claim 1, wherein multiple measurements are taken at each point along the length of the density gradient of the component, and the profile 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. 16. The system of claim 1, wherein the profile is generated for a step density gradient of the component or a continuous density gradient of the component. 17. A system for measuring density gradients of components dispensed into a container for use in centrifugation, comprising: When executed by the processing circuitry, the processing circuitry obtaining measurements at points along the length of the density gradient of the component; generating a density gradient profile of the component based on the measurements; storing a profile of the density gradient of the component; A system comprising processing circuitry having a memory for storing instructions. 18. The instructions further, when executed by the processing circuitry, cause the processing circuitry to: 18. The system of claim 17, wherein the profile is adjusted by removing measurements from a bottom portion of the container. 19. The instructions further, when executed by the processing circuitry, cause the processing circuitry to: Identifying the location of the meniscus of the density gradient of the components adjusting the profile to remove measurements from the meniscus location; 18. The system of claim 17. 20. The system of claim 19, wherein the location of the meniscus is identified by identifying a minimum in the first derivative of the measurement. 21. The instructions further, when executed by the processing circuitry, cause the processing circuitry to: obtaining measurements from the container before the density gradient of the component is dispensed therein; comparing the measurements from the container to an expected measurement range for a container of a given material and size; determining at least one of a material and a size of the container based on a comparison with an expected measurement range; standardizing the measurements with respect to at least one of the material and size of the container; 18. The system of claim 17. 22. The instructions further, when executed by the processing circuitry, cause the processing circuitry to: obtaining measurements from the sensor assembly along the length of the container when empty; Calculate the average of the measurements along the length of the container when empty; generating a difference value between the average of the measurements along the length of the container when empty and the measurement; Adding differential values allows adjusting the density gradient profile of the components and mitigating the effects caused by imperfections on the vessel and wall thickness variations; 18. The system of claim 17. 23. The instructions further, when executed by the processing circuitry, cause the processing circuitry to: Generate a centerline for the container; obtaining measurements in at least two dimensions along a centerline of the container to mitigate position errors of the container on a generated profile of the density gradient of the component; 18. The system of claim 17. 24. The system of claim 17, wherein multiple measurements are taken at each point along the length of the density gradient of the component, and an average of the multiple measurements calculated for each point along the length of the density gradient of the component is used to generate the profile. 25. The system of claim 17, wherein the profile is generated for a step density gradient of the component or a continuous density gradient of the component. 26. A method for non-destructively measuring density gradients of components, comprising: obtaining measurements at points along the length of a density gradient of the component; generating a density gradient profile of the component based on the measurements; storing a profile of the density gradient of the component; A method comprising: 27. The method of claim 26, further comprising adjusting the profile by removing measurements from a bottom portion of a vessel containing a density gradient of components. 28. Identifying the location of the meniscus of the density gradient of the components; adjusting the profile to remove measurements from the meniscus location; 27. The method of claim 26, further comprising: 29. The method of claim 28, wherein the location of the meniscus is identified by identifying a minimum in the first derivative of the measurement. 30. Obtaining measurements from a container before a density gradient of components is dispensed therein; comparing measurements from the container to an expected measurement range for a container of a given material and size; determining at least one of a material and a size of the container based on a comparison to an expected measurement range; standardizing the measurements with respect to at least one of the material and size of the container; 27. The method of claim 26, further comprising: 31. Taking measurements along the length of the container when empty; calculating an average of the measurements along the length of the container when empty; generating a difference value between the average of the measurements along the length of the container when empty and the measurement; adjusting the density gradient profile of the components by adding differential values to mitigate the effects caused by imperfections and wall thickness variations on the container; 27. The method of claim 26, further comprising: 32. Generating a centerline for a container into which the density gradient of the component is dispensed; obtaining measurements in at least two dimensions along a centerline of the container to mitigate positional errors of the container on a generated profile of the density gradient of the constituents; 27. The method of claim 26, further comprising: 33. Obtaining multiple measurements at each point along the length of the density gradient of the component; generating a profile of the density gradient of the component using an average of multiple measurements point-wise along the length of the density gradient of the component; 27. The method of claim 26, further comprising: 34. The method of claim 26, wherein the profile is generated with respect to a step density gradient of the component or a continuous density gradient of the component. 35. A system for automatically dispensing a density gradient of components for use in centrifugation, comprising: When executed by the processing circuitry, the processing circuitry Inserting the distal end of the probe into the container; pumping the separate components into a mixing chamber connected to the proximal end of the probe, the mixing chamber generating a mixture of the separate components; dispensing a plurality of stages into the container, each stage of the plurality of stages having a density based on the relative concentrations of the distinct components in the mixture generated by the mixing chamber, each stage of the plurality of stages pushing a previously dispensed stage away from the distal end of the probe; removing the probe from the vessel without disturbing the multiple steps; A system comprising processing circuitry having a memory for storing instructions. 36. The system of claim 35, wherein the separate components include deionized water, a density modifier, a buffer, and an additive. 37. The instructions further, when executed by the processing circuitry, cause the processing circuitry to: 37. The system of claim 36, wherein a dispense rate for each separate component in each of the plurality of stages is calculated, the dispense rate determining the relative concentration of the separate components in the mixture generated by the mixing chamber. 38. The system of claim 37, wherein successively higher densities result from increasing the dispensing rate of the density modifier. 39. The system of claim 38, wherein the dispensing rate of the deionized water is decreased proportionally to increasing the dispensing rate of the density modifier. 40. The system of claim 37, wherein the additive dispensing rate is subtracted from the deionized water dispensing rate. 41. The system of claim 40, wherein the additive is dispensed in fewer than multiple steps in a density gradient of the component. 42. The system of claim 37, wherein the buffer dispensing rate remains constant. 43. The instructions further, when executed by the processing circuitry, cause the processing circuitry to: 38. The system of claim 37, wherein one or more pumps are independently controlled to regulate the dispensing rate of each of the separate components pumped into the mixing chamber. 44. The instructions further, when executed by the processing circuitry, cause the processing circuitry to: dispensing a first stage of the plurality of stages at a maximum dispensing speed; adjusting the dispensing speed for each of the plurality of steps subsequent to the first step; 36. The system of claim 35. 45. The system of claim 44, wherein the step of adjusting the dispensing rate includes the steps of decreasing the dispensing rate from a maximum dispensing rate to a minimum dispensing rate, and then increasing the dispensing rate from the minimum dispensing rate to the maximum dispensing rate. 46. The system of claim 45, wherein the dispensing rate increases exponentially from a minimum dispensing rate until a maximum dispensing rate is reached. 47. The system of claim 35, wherein the mixing chamber includes a static mixer. 48. A system for dispensing a density gradient of components for use in centrifugation, comprising: When executed by the processing circuitry, the processing circuitry Inserting the distal end of the probe into the container; dispensing a first stage of the plurality of stages into the container, the first stage being dispensed at a maximum dispense rate; dispensing additional stages of the plurality of stages into the container, each additional stage being dispensed starting at a minimum dispense rate and then increasing from the minimum dispense rate to a maximum dispense rate, each additional stage of the plurality of stages having a density greater than the density of a previously dispensed stage of the plurality of stages; and moving the previously dispensed stage of the plurality of stages away from the distal end of the probe; removing the probe from the vessel without disturbing the multiple steps; A system comprising processing circuitry having a memory for storing instructions. 49. The system of claim 48, wherein the dispensing rate for each additional step increases exponentially from the minimum dispensing rate until the maximum dispensing rate is reached. 50. The system of claim 48, wherein each stage of the plurality of stages comprises a mixture of components including deionized water, a density modifier, a buffer, and an additive. 51. The instructions further, when executed by the processing circuitry, cause the processing circuitry to: 51. The system of claim 50, wherein a dispense rate is calculated for each component in each of the plurality of stages, the dispense rate determining a concentration for each component in each stage. 52. The system of claim 50, wherein the density of each of the multiple stages is based on the dispensing rate of the density modifier. 53. The system of claim 50, wherein the dispensing rate of the deionized water is decreased proportionally to increasing the dispensing rate of the density modifier. 54. The system of claim 50, wherein the additive dispensing rate is subtracted from the deionized water dispensing rate. 55. The system of claim 54, wherein the additive is dispensed in fewer than multiple steps in a density gradient of the component. 56. The system of claim 50, wherein the buffer dispensing rate remains constant. 57. The instructions further, when executed by the processing circuitry, cause the processing circuitry to: 51. The system of claim 50, wherein one or more pumps are independently controlled to adjust the dispensing rate of each of the components pumped into the mixing chamber to mix the components together. 58. A method for automatically dispensing a density gradient of components for use in centrifugation, comprising: inserting a distal end of a probe into a container; dispensing a first stage of a plurality of stages into a container, the first stage being dispensed at a maximum dispense rate; dispensing additional stages of the plurality of stages into the container, each additional stage being dispensed starting at a minimum dispense rate and then increasing from the minimum dispense rate to a maximum dispense rate, each additional stage of the plurality of stages having a density greater than the density of a previously dispensed stage of the plurality of stages, and moving the previously dispensed stage away from the distal end of the probe; removing the probe from the vessel without disturbing the multiple steps; A method comprising: 59. The method of claim 58, further comprising exponentially increasing the dispense rate for each additional step from the minimum dispense rate until the maximum dispense rate is reached. 60. The method of claim 58, further comprising mixing components including deionized water, a density modifier, a buffer, and an additive to generate each of the multiple stages. 61. The method of claim 60, further comprising the step of calculating a dispense rate for mixing each of the components, the dispense rate determining the concentration of each component in each of the multiple stages. 62. The method of claim 61, wherein the density of each of the multiple stages is based on the dispensing rate of the density modifier. 63. The method of claim 61, further comprising increasing the dispensing rate of deionized water in proportion to increasing the dispensing rate of the density modifier. 64. The method of claim 61, further comprising the step of subtracting the dispensing rate of the additive from the dispensing rate of the deionized water. 65. The method of claim 64, further comprising dispensing the additive in fewer than a plurality of stages. 66. The method of claim 61, wherein the buffer dispensing rate remains constant. 67. The method of claim 61, further comprising independently controlling one or more pumps to adjust the dispensing rate of each of the components pumped into the mixing chamber to mix the components together. 68. A method for replicating a density gradient of a component, comprising: generating a first profile by obtaining measurements of a density gradient of components dispensed into a first container; creating a second profile by substituting measurements from the first profile; storing a second profile; replicating a density gradient of the component in a second container based on the second profile; A method comprising: 69. The method of claim 68, further comprising replacing measurements at an interface between the first and second stages with a first average value from the first stage before the interface and a second average value from the second stage after the interface. 70. The method of claim 68, further comprising replacing measurements at an interface between the first stage and the second stage with a first measurement having zero or a minimum positive derivative from the first stage before the interface and a second measurement having zero or a minimum positive derivative from the second stage after the interface. 71. The method of claim 68, further comprising replacing measurements from a bottom portion of the first container with a linear ramp of measurements. 72. Measuring the density gradient of the replicate component in a second container; determining whether the difference between the density gradient of the replicated components in the second container and the density gradient of the components contained in the first container is within a predetermined tolerance; 69. The method of claim 68, further comprising the step of verifying the quality of the density gradient of the components replicated in the second container by: 73. The method of claim 68, further comprising processing the first profile of the density gradient of the component by converting the first profile into a text file including positions along the length and corresponding measurements. 74. A system for replicating density gradients of components for use in centrifugation, comprising: a first density gradient of the component; a sensor assembly; a dispensing probe; When executed by the processing circuitry, the processing circuitry obtaining a measurement of a first density gradient of components contained in a first container using a sensor assembly; storing a first profile of measurements in memory; creating a second profile based on the stored first profile; replicating the first density gradient of components by dispensing a second density gradient of components into a second container according to a second profile using a dispensing probe; processing circuitry having memory for storing instructions; A system comprising: 75. The instructions further, when executed by the processing circuitry, cause the processing circuitry to: 75. The system of claim 74, wherein the system verifies the quality of the second density gradient of the component 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. 76. The instructions further, when executed by the processing circuitry, cause the processing circuitry to: 75. The system of claim 74, wherein the second profile is created by replacing measurements at the interface between stages of the first profile. 77. The system of claim 76, wherein measurements at an interface between the first stage and the second stage are replaced with a first average value from the first stage before the interface and a second average value from the second stage after the interface. 78. The system of claim 76, wherein measurements at the interface between the first stage and the second stage are replaced with measurements from the first stage before the interface that have zero or a minimum positive derivative, and measurements from the second stage after the interface that have zero or a minimum positive derivative. 79. The instructions further, when executed by the processing circuitry, cause the processing circuitry to: 75. The system of claim 74, wherein the second profile is created by removing measurements from a bottom portion of the first container. 80. The instructions further, when executed by the processing circuitry, cause the processing circuitry to: 75. The system of claim 74, wherein the second profile is created by displacing measurements from a bottom portion of the first container. 81. The system of claim 74, wherein measurements from a bottom portion of the first container are replaced with a linear ramp of measurements. 82. The instructions, when executed by the processing circuitry, further cause the processing circuitry to: 75. The system of claim 74, wherein the second profile is created by removing measurements from a meniscus location of the first density gradient of the component. 83. The instructions further, when executed by the processing circuitry, cause the processing circuitry to: 75. The system of claim 74, wherein the second profile is created by converting the first profile into a text file, the text file including positions along the length and corresponding measurements. 84. A system for replicating density gradients of components for use in centrifugation, comprising: When executed by the processing circuitry, the processing circuitry obtaining a measurement of a density gradient of the component dispensed into the first container, the density gradient of the component including a meniscus; Substituting measurements at interfaces between stages of the density gradient of the components; Substituting measurements from a location at the bottom of the first container; replacing the measurement based on the location of the meniscus of the density gradient of the component dispensed in the first container; The measurements are processed by storing a profile of the density gradient of the component based on the processed measurements; Using the profile to replicate the density gradient of the component in a second container; A system comprising processing circuitry having a memory for storing instructions. 85. The instructions further, when executed by the processing circuitry, cause the processing circuitry to: 85. The system of claim 84, wherein measurements at an interface between the first and second stages are replaced with a first average value from the first stage before the interface and a second average value from the second stage after the interface. 86. The instructions may further, when executed by the processing circuitry, cause the processing circuitry to: 85. The system of claim 84, wherein measurements at an interface between the first and second stages are replaced with measurements from the first stage before the interface having zero or a minimum positive derivative and measurements from the second stage after the interface having zero or a minimum positive derivative. 87. The instructions may further, when executed by the processing circuitry, cause the processing circuitry to: 85. The system of claim 84, wherein measurements from a bottom portion of the first container are replaced with a linear ramp of measurements. 88. The instructions may further, when executed by the processing circuitry, cause the processing circuitry to: measuring the density gradient of the replicated components in a second container; determining whether the difference between the density gradient of the replicated component in the second container and the density gradient of the component dispensed in the first container is within a predetermined tolerance; 85. The system of claim 84, wherein the quality of the density gradient of the replicated components in the second container is verified by: 89. The instructions may further, when executed by the processing circuitry, cause the processing circuitry to: 85. The system of claim 84, wherein the component density gradient profile is processed by converting the profile into a text file containing positions along the length and corresponding measurements.

Claims

1. 1. A system for automatically dispensing a density gradient of components for use in centrifugation, said system comprising:

1. A processing circuitry having a memory for storing instructions that, when executed by the processing circuitry, cause the processing circuitry to: Inserting a distal end of a probe into a container; pumping the separate components into a mixing chamber connected to a proximal end of the probe, the mixing chamber generating a mixture of the separate components; dispensing a plurality of stages into the container, each stage of the plurality of stages having a density based on the relative concentrations of the separate components in the mixture generated by the mixing chamber, each stage of the plurality of stages pushing a previously dispensed stage away from the distal end of the probe; removing the probe from the vessel without disturbing the steps; A processing circuitry that performs A system comprising:

2. The system of claim 1 , wherein the separate components include deionized water, a density modifier, a buffer, and an additive.

3. The instructions further, when executed by the processing circuitry, cause the processing circuitry to:

3. The system of claim 2, further comprising: calculating a dispense rate for each of the separate components in each of the plurality of stages, the dispense rate determining the relative concentrations of the separate components in the mixture generated by the mixing chamber.

4. The system of claim 3 , wherein successively higher densities result from increasing the dispensing rate of the density modifier.

5. The system of claim 4 , wherein the dispensing rate of the deionized water decreases proportionally to increasing the dispensing rate of the density modifier.

6. The system of claim 3 , wherein the additive dispense rate is subtracted from the deionized water dispense rate.

7. The system of claim 6 , wherein the additive is dispensed in fewer stages than the plurality of stages in the density gradient of the component.

8. The instructions further, when executed by the processing circuitry, cause the processing circuitry to:

4. The system of claim 3, wherein one or more pumps are independently controlled to regulate the dispensing rate of each of the separate components pumped into the mixing chamber.

9. The instructions further, when executed by the processing circuitry, cause the processing circuitry to: dispensing a first stage of the plurality of stages at a maximum dispensing rate; adjusting the dispensing speed for each of the plurality of stages subsequent to the first stage; The system of claim 1 .

10. 10. The system of claim 9, wherein adjusting the dispense rate comprises decreasing the dispense rate from the maximum dispense rate to a minimum dispense rate and then increasing the dispense rate from the minimum dispense rate to the maximum dispense rate.

11. The system of claim 1 , wherein the mixing chamber comprises a static mixer.

12. A measuring device comprising: a sensor assembly; a motor coupled to the sensor assembly; a measuring device, Furthermore, The instructions further, when executed by the processing circuitry, cause the processing circuitry to: using the motor to move the sensor assembly along the length of the density gradient of the constituent; obtaining measurements from the sensor assembly while the sensor assembly is moved along the length of the density gradient of the constituent; generating a density gradient profile of the component based on the measurements; and The system of claim 1 .

13. The instructions further, when executed by the processing circuitry, cause the processing circuitry to: generating a first profile by obtaining measurements of a density gradient of the components dispensed into the container; creating a second profile by substituting measurements of the first profile; storing the second profile; replicating the density gradient of the component in a second container based on the second profile; and The system of claim 1 .

14. 1. 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 stage of a plurality of stages into the container, the first stage being dispensed at a maximum dispense rate; dispensing additional stages of the plurality of stages into the container, each additional stage beginning at a minimum dispense rate and then dispensed at a rate increasing from the minimum dispense rate to the maximum dispense rate, each additional stage of the plurality of stages having a density greater than that of a previously dispensed stage of the plurality of stages, and moving the previously dispensed stage away from the distal end of the probe; removing the probe from the vessel without disturbing the steps; A method comprising:

15. 15. The method of claim 14, further comprising exponentially increasing the dispense rate in additional steps from the minimum dispense rate until the maximum dispense rate is reached.

16. calculating a dispense rate for mixing each of the components, the dispense rate determining a concentration of each of the components in each of the plurality of stages; 15. The method of claim 14, further comprising:

17. The method of claim 16 , wherein the density of each of the plurality of stages is based on a dispensing rate of a density modifier.

18. 17. The method of claim 16, further comprising decreasing the dispensing rate of deionized water in proportion to increasing the dispensing rate of the density modifier.

19. 17. The method of claim 16, further comprising subtracting the dispense rate of the additive from the dispense rate of the deionized water.

20. 20. The method of claim 19, further comprising dispensing the additive in fewer than said plurality of stages.

21. 17. The method of claim 16, wherein the dispensing rate of the buffer remains constant.

22. 17. The method of claim 16, further comprising independently controlling one or more pumps to adjust the dispensing rate of each of the components pumped into the mixing chamber to mix the components together.

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