WO2018156039A1

WO2018156039A1 - Centrifugal filtration device and method for concentrating liquid mixtures

Info

Publication number: WO2018156039A1
Application number: PCT/PT2018/050006
Authority: WO
Inventors: Vitor Manuel Geraldes FERNANDES; Viriato Sérgio DE ALMEIDA SEMIÃO; Miguel Ângelo Joaquim RODRIGUES; Carlos Duarte DA SILVA COMPLETO
Original assignee: Instituto Superior Técnico
Priority date: 2017-02-24
Filing date: 2018-02-23
Publication date: 2018-08-30
Also published as: PT109932A

Abstract

The present invention relates to a centrifugal filtration device and a method for concentrating, fractionating, purifying and/or desalinating small volumes of liquid samples, using reverse osmosis, nanofiltration or ultrafiltration membranes. The device comprises a shell, which comprises a sample chamber (102) with at least one ventilation orifice (123); a permeate chamber (106) with at least one ventilation orifice (145); a filtration chamber (104) with at least one semi-permeable membrane (105); a concentrate chamber (107) downstream of the filtration chamber (104); and at least one neck (103) connecting the sample chamber (102) to the filtration chamber (104) and the volume of which is less than the volume contained in the concentrate (107) and filtration (104) chambers. The device is particularly suited to performing nanofiltration or reverse osmosis on liquid samples with high osmotic pressures. Filtration takes place by means of the rotation of the centrifugal filtration device about a rotation shaft in a centrifuge. By using a long, narrow neck between the sample chamber, which is closer to the rotation shaft of the centrifuge, and the filtration chamber, which is further away from said rotation shaft, it is possible to maintain the pressure at high levels throughout the filtration process, which allows higher concentration factors to be achieved and the filtration time to be reduced.

Description

DESCRIPTION

CENTRIFUGAL FILTRATION DEVICE AND METHOD FOR CONCENTRATION OF LIQUID MIXTURES

FIELD OF INVENTION

The present invention relates to a centrifugal filtration device and a method for concentration, fractionation, purification, and / or desalination of small volume liquid samples using reverse osmosis, nanofiltration or ultrafiltration membranes. The centrifugal filtration device is particularly suitable for nanofiltration or reverse osmosis of liquid samples with high osmotic pressures.

TECHNICAL STATE

Liquid mixtures may be fractionated using pressure difference driven membrane separation processes, taking into account the molecular weight of the components of the liquid mixtures and the molecular exclusion limit of the membrane. Depending on the molecular weight range, microfiltration, ultrafiltration, nanofiltration and reverse osmosis membranes can be used, which operate at different pressure ranges. For volumes greater than hundreds of milliliters, tangential membrane filtration is generally the best alternative for concentrating such mixtures, as tangential flow minimizes concentration bias. For liquid mix volumes between a few milliliters and hundreds of milliliters, a common technique for fractionating and concentrating small volumes of liquid mixtures is centrifugal filtration. As its name implies, with this technique the difference in transmembrane pressure is created by centrifugal acceleration inside a centrifuge.

Compared to the front membrane filtration processes, centrifugal filtration has the advantage of propelling the denser fluid elements that accumulate on the membrane surface away from the axis of rotation under the effect of centrifugal force. With proper membrane orientation, the concentration boundary layer that forms near the membrane can be removed from the membrane by centrifugal force, providing a self-cleaning mechanism, reducing concentration bias and maintaining high filtration flow.

There are several centrifugal filtration devices described in the literature, but all of them have been developed for microfiltration and / or ultrafiltration. The apparatus, invented by PN Rigopulos, disclosed in U.S. Pat. US 3,488,768 was probably the first centrifugal filtration device for small volume liquid samples. This patent describes various embodiments of a centrifugal filtration device wherein the angle between the centrifugal force vector and the membrane surface is preferably less than 15 ° to ensure efficient removal of the concentrated layer in the vicinity of the membrane. The main drawback of this device is that it does not prevent filtration from continuing to complete concentrate dryness. Similarly, the same problem occurs in centrifugal filtration devices disclosed in U.S. Pat. No. 4,683,058 issued to GF Lyman and G. Mathus, and in U.S. Pat. EP 0298513, issued to A. Szabados, a Since these devices do not have locations where concentrate can accumulate without drying.

Filtration to dryness can be avoided by improving the design of centrifugal filtration devices. In Pat. US 4,632,761 issued to W.F. Bowers and P.N. Rigopulos, a centrifugal filtration device is described wherein the membrane is supported by a plate having permeate drainage channels. Filtration stops before dryness, when the liquid meniscus level reaches the radial level of the outermost edge of the outermost permeate drainage conduit, and consequently a certain amount of concentrate remains in the device at the end of the filtration process, regardless of of the elapsed filtration time. The main disadvantage of this device is that the concentrate is still in contact with the membrane, which can raise solute adsorption problems.

To minimize contact of highly concentrated solutions with the membrane, separate centrifugal filtration devices have been developed comprising a concentrate chamber receiving the concentrate. Typically, the concentrate chamber is located at the outermost radial level to maximize the dragging of the heavier and more concentrated fluid elements into that chamber. Examples of devices with this type of concentrate chamber are described in the following patents: Pat. US 4,722,792 issued to T. Miyagi et al. , US 5,647,990, issued to V. Vassarotti, US 6,357,601, issued to WF Bowers et al. , US 8,747,670 issued to L. Bonhomme et al. An interesting centrifugal filtration device has been disclosed in U.S. Pat. US 6,719,896 issued to P. Clark, in which the final volume of concentrate is adjusted by the user by clearance of permeate ducts. These permeate ducts are prepared in the device manufacturing process and the user only needs to uncover the ducts corresponding to the desired final concentrate volume.

Another significant factor that may influence filtration performance is the angle between the membrane surface and the centrifugal force vector. For a given centrifugal filtration device, this angle varies with the centrifuge rotor type. In a tilting rotor centrifuge the device rotates horizontally, while in a fixed angle rotor centrifuge the device rotates at a given inclination relative to the axis of rotation. Many configurations with different angles between the membrane surface and the axis of the centrifugal filtration device have been disclosed in previous inventions. When the membrane surface is perpendicular to the axis of the centrifugal filtration device, there is no alignment between the membrane surface and the centrifugal force vector, which leads to poor membrane self-cleaning in the case of fixed angle centrifuges, or even without self-cleaning in the case of tilting rotor centrifuges, which may result in membrane clogging (see, for example, U.S. Pat. Nos. 3,583,627, OH Wilson, US 4,632,761, WF Bowers and PN Rigopulos , US 4,683,058, issued to GF Lyman and G. Mathus, US 5,601,711, issued to E. Sklar et al., US 5, 733, 449, issued to WF Bowers and B. Yankopoulos, US 7,658,982, issued to D. Schwarzwald and European Patents EP 0298513, issued to A. Szabados, EP 0709132, issued to G. Cianci and EP 2260943, issued to MM Klerks). The self-cleaning action of the membrane surface may be enhanced if the membrane surface is parallel to the axis of the centrifugal filtration device or if it is inclined at a slight angle to this axis (see, for example, US Patent 4,600,507 issued to A. Shimizu and

5. Otsubo, US 4,722,792, issued to T. Miyagi et al., US 4,769,145, issued to M. Nakajima, US 5,112,484, issued to P. Zuk Jr. , US 5,647,990, issued to V. Vassarotti, US 6, 344, 140, issued to P. Zuk Jr. , US 6,375,855 to V. Vassarotti, and US 8,747,670 to L. Bonhomme et al. ) · Another measure to keep the membrane clean is to reverse the orientation of the membrane such that the normal vector on the active side of the membrane (membrane face towards the permeate chamber) points, at least to some extent, to the same extent. direction of the centrifugal force vector. In this type of centrifugal filtration device, the membrane is usually placed at the base of a piston that presses against the liquid mixture to be filtered. The permeate passes through the membrane into an internal permeate chamber (inside the piston) and the concentrated fluid is drawn away from the membrane due to centrifugal force (see, for example, the devices disclosed in US Patent 3,661,265, to DJ Greenspan). , US 3,960,727, to HT Hochstrasser, US 4,522,713, to D. Nussbaumer et al., US 4,832,851, to WF Bowers and DB Tiffany, US 5,490,927, to AE Herczeg, and US 6,302,919 to B Chambers et al.

Although a larger membrane area per unit volume may result in a greater extent of solute adsorption, in recent years, some of the invented centrifugal filtration devices have maximized the membrane surface to increase filtration flow, as described in US Pat. . US

6,357,601 issued to WF Bowers et al. , and in Pat. US 8,980,107 issued to MJ Domanico et al. In many cases, filtration may also be used in conjunction with an adsorption process. In this case, some kind of physical or chemical modification of the membrane surface is first performed between it and a specific ligand that will bind, during filtration, to a target molecule present in the liquid sample. Depending on the purpose of filtration, the bound target molecule can then be recovered or discarded. Examples of such devices have been disclosed in U.S. Pat. US 5,552,325, issued to S. Nochumson and BS Goldberg, US 5,778,037, to DR Vlock et al. , US 5,833,860, issued to W. Kopaciewicz et al. , US 7,045,064 issued to TN Warner.

In centrifugal filtration devices, the permeate mass produced during the filtration process is conveyed to a permeate reservoir where it is easily collected at the end of the process. However, the concentrate mass remains in the chamber near the membrane and some manipulation is required to remove it. In the inventions disclosed in the literature, there are three main methods for collecting the concentrated liquid: 1) using a syringe-coupled tube, 2) using additional centrifugation after the filtration process, attaching a complementary concentrate removal chamber (see U.S. Patent 5,112,484 issued to P. Zuk Jr., and U.S. Pat. 5,490,927 issued to AE Herczeg) or 3) further centrifugation by inverting the centrifugal filtration device (see U.S. Patent 4,632,761). , issued to WF Bowers and PN Rigopulos, US Pat. 5,501,841, issued to YC Lee et al., and US Pat. 8,747,670, issued to L. Bonhomme et al.). There are several commercial centrifugal filtration devices available for sample volumes from about 0.5 mL to 100 mL, but all use only microfiltration or ultrafiltration membranes. Ultrafiltration membrane devices have a molecular exclusion limit typically between 1 kDa and 1000 kDa. This means that centrifugal filtration devices can now be applied to concentrate or purify viruses, bacteria or macromolecules such as proteins. For a molecular weight of less than 1 kDa in the range of nanofiltration and reverse osmosis there are no commercially available centrifugal filtration devices. This means that there are no centrifugal filtration devices capable of concentrating small peptides, drugs, toxins, biomarkers, etc. These small molecules can only be concentrated by nanofiltration or reverse osmosis membranes. However, with such membranes, to achieve reasonable filtration flows and solute rejections, as well as high concentration factors, it is necessary to operate at pressures typically between 5 and 80 bar.

Adapting centrifugal filtration devices to operate at high pressure is not simple. In fact, although centrifugal force can easily be increased in the centrifuge, the supply pressure begins to decrease rapidly as soon as the liquid level in the sample chamber decreases. This poses a serious problem as it is at the end of the concentration cycle that the supply pressure must be raised to compensate for the osmotic pressure of the concentrated solution, which is maximum at this final concentration phase. Increasing centrifugal acceleration too much does not adequately solve this problem as nanofiltration and reverse osmosis membranes can compact. irreversibly at high pressures. In fact, when the maximum pressure recommended by membrane manufacturers is far exceeded, severe irreversible membrane compaction is observed (McConnon, 2015), which causes the permeation flow to decrease dramatically throughout the concentration process.

An object of the invention is a centrifugal filtration device and a method for the fractionation, purification, concentration and / or desalination of liquid sample mixtures over a wide molecular weight range, including molecular weights below 1 kDa.

It is another object of the invention to provide a centrifugal filtration device that can use microfiltration, ultrafiltration, nanofiltration and reverse osmosis membranes.

It is another object of the invention to provide a centrifugal filtration device that is easy to handle, and has simple sample injection, and permeate and concentrate removal procedures.

The problems and limitations of state-of-the-art centrifugal filtration devices, related to the difficulty in concentrating and / or purifying liquid samples by nanofiltration and reverse osmosis, are overcome by providing a method and a device for performing said method. as defined in the appended claims. SUMMARY OF THE INVENTION

The device of the invention enables the fractionation, purification, concentration and / or desalination of liquid samples of substance mixtures over a wide range of molecular weights, including the particular case of high osmotic pressure liquid samples, also using nanofiltration and reverse osmosis to perform separation. By using a long, narrow neck between the sample chamber, which is closest to the spin axis of the centrifuge, and the filtration chamber, which is farthest from said axis of rotation, the pressure can be maintained at high levels throughout the filtration process, which allows to reach higher concentration factors and reduce the filtration time.

The centrifugal filtration devices described above have a large part of their volume occupied by the sample chamber extending to the membrane region. However, as the sample is filtered, its volume decreases and the liquid pressure decreases to practically zero. Since the pressure generated by the centrifugal force varies quadratically with the distance from the air / liquid interface of the sample to the membrane, after some time has elapsed since the filtration process began, the low liquid level and the pressure and flow values of permeate are reduced to a small fraction of their initial values. Thus, to achieve a high concentration factor, the filtration time needs to be greatly increased. Low flow operation also results in reduced solute rejection, as rejection tends to decrease with decreasing permeate flow. To mitigate these adverse effects, ie increased filtration time and reduced rejection coefficient, the filtration pressure can of course be increased by gradually increasing the centrifuge's rotation speed throughout the test. However, controlling the spin speed of the centrifuge is difficult or even impossible to implement because the liquid level in the sample chamber is not known in real time. To overcome these important limitations of common centrifugal filtration devices, a centrifugal filtration device that maintains high pressure throughout most of the filtration process is described herein.

In this invention, in order to maintain the high pressure near the membrane for most of the filtration process, the liquid sample is placed as far as possible from the membrane in terms of radial distance. In practical terms, this means that the liquid sample is placed in a chamber, hereinafter referred to as the sample chamber, located as far as possible from the chamber from which the membrane is located, here designated as a filtration chamber, and both chambers are connected. by a channel as thin as possible, which is called a narrow neck. In addition, most of the initial liquid sample volume is confined to the sample chamber to ensure an always high liquid height during the filtration cycle. Using this innovative concept, pressure is maintained at a high level throughout most of the filtration process and pressure drop occurs only at the end of the filtration phase, with only a small portion of the liquid sample remaining in the sample chamber. initial To prevent filtration to dryness, the centrifugal filter device also has a chamber for collecting the sample elements. concentrates moving out of the membrane by centrifugal force. This chamber is called a concentrate chamber.

At the end of the filtration cycle, the supply pressure can be advantageously increased by increasing the centrifuge speed, provided this does not compromise the mechanical integrity of the centrifugal filtration device. With this procedure, the liquid sample may be further concentrated to the point where the osmotic pressure of the concentrated solution is reached or the permeate flow tends to zero due to membrane compaction.

BRIEF DESCRIPTION OF THE INVENTION

The centrifugal filtration device of the invention comprises, but is not limited to, a housing comprising a sample chamber, a filtration chamber, a concentrate chamber, a permeate chamber, a narrow neck connecting the sample chamber to the chamber. and a cap to prevent liquid from spilling out of the device. The body of the centrifugal filtration device described above may be made of one piece or several pieces depending on the embodiments of the invention. In addition, the system also includes chamber ventilation channels to prevent pressure imbalance between the permeate chamber and the filtration chamber after the centrifuge is stopped.

The operating mode of the device is briefly described below. When the centrifugal filtration device rotates about the axis of rotation in a In the centrifuge, the filtration process takes place in the filtration chamber, which is fed fresh liquid from the sample chamber through the narrow neck. In addition, during rotation of the centrifugal filtration device about the axis of rotation, the centrifugal force scans the heaviest elements of fluid that accumulate on the membrane surface located inside the filtration chamber. This self-cleaning mechanism minimizes concentration polarization in the membrane. At the same time, the liquid solution permeating through the membrane flows into the permeate chamber through at least one narrow permeate channel or porous support.

The method for fractionation, purification, concentration and / or desalination of liquid samples of substance mixtures consists of three main steps. The first step is the introduction of the liquid sample to be processed in the centrifugal filtration device. In the second step, filtration is performed by placing the centrifugal filtration device in a centrifuge at a rotational speed that creates the desired transmembrane pressure difference, typically between 0 and 10 bar for ultrafiltration and microfiltration, between 10 bar and 60 bar for nanofiltration and between 20 bar and 80 bar for reverse osmosis. The spin speed of the centrifuge can be increased at the end of the step to increase transmembrane pressure and thereby increase the final concentration of concentrated liquid. Finally, in the last step, the separated fluids are removed from the centrifugal filtration device. Removal of the permeate is easy because access to the permeate chamber below the membrane is sufficient. The concentrate can be removed by suctioning it through a thin tube that enters the narrow neck into the filtration chamber. In Alternatively, the concentrate may be returned to the sample chamber through the narrow neck, reversing the position of the centrifugal filtration device and turning the centrifuge at low speed.

The following table summarizes the method for fractionation, purification, concentration and / or desalination of liquid samples of substance mixtures.

Table 1 - Method for fractionation, purification, concentration and / or desalination of liquid samples of substance mixtures.

Step 1 Place the sample into the sample chamber of the centrifugal filtration device.

Step 2 Perform Centrifugal Filtration

• place the centrifugal filtration device and the counterweight in the centrifuge as recommended by the centrifuge manufacturer

• select the speed, or a rotational speed time profile, and the rotational time appropriate for the purpose of filtration

• perform centrifugation

• remove centrifugal filtration device from centrifuge

Step 3 Collection of Concentrated and Permeate Liquids from Centrifugal Filtration Device

• separate the permeate chamber from the centrifugal filtration device and collect the permeate

• remove concentrated liquid; Depending on the embodiment of the device, removal of the concentrate may be by suction, using a flexible thin tube through the narrow neck, or by centrifugation at low rotation speed of the inverted centrifugal filtration device. DESCRIPTION OF THE FIGURES

These and other objects, features and advantages of the invention will be more apparent from the following detailed description when read in conjunction with the accompanying drawings.

FIG. 1 is a schematic of a generic centrifugal filtration device according to the object of the invention.

FIG. 2 is the schematic of the centrifugal filtration device during the filtration process outlining the operating fundamentals.

FIG. 3 is the isometric view of the object of the invention according to certain embodiments (in particular with embodiments 1 and 2).

FIG. 4 is an exploded view of the object of the invention according to embodiment 1.

FIG. 5 is a top view of the object of the invention according to embodiment 1.

FIG. 6 is a sectional view of the object of the invention at level A-A of FIG. 5 according to embodiment 1.

FIG. 7 is a sectional view of the object of the invention at section level AA of FIG. 5 according to embodiment 1 when placed in a centrifuge at a certain angle with the axis of rotation. FIG. 8 is a sectional view of the object of the invention at section AA level of FIG. 5 according to embodiment 1 when placed in a centrifuge at a given angle with the axis of rotation prior to filtration with a liquid sample in the feed reservoir.

FIG. 9 is a cross-sectional view of the object of the invention at section A-A of FIG. 5 according to embodiment 1 when placed in a centrifuge at a certain angle with the axis of rotation at the end of filtration with the permeated liquid already in the permeate chamber.

FIG. 10 is an exploded view of the two parts of embodiment 2 differing from embodiment 1 of the object of the invention.

FIG. 11 is a top view of the object of the invention according to embodiment 2.

FIG. 12 is a sectional view of the object of the invention at section level B-B of FIG. 11 according to embodiment 2.

FIG. 13 is the enlarged view of the object of the invention in section C defined in FIG. 12 according to embodiment 2.

FIG. 14 is the isometric view of the object of the invention according to embodiment 3.

FIG. 15 is an exploded view of the object of the invention according to embodiment 3. FIG. 16 is a top view of the object of the invention according to embodiment 3.

FIG. 17 is a cross-sectional view of the object of the invention at section D-D of FIG. 16 according to embodiment 3.

FIG. 18 is a cross-sectional view of the object of the invention at section level E-E of FIG. 16 according to embodiment 3.

FIG. 19 is a cross-sectional view of the object of the invention at section D-D of FIG. 16 when an extra reservoir is used to collect the concentrate by centrifuging with the inverted centrifugal filtration device according to embodiment 3.

FIG. 20 is a graph of a sucrose solution filtration result set obtained with a centrifugal filtration device according to embodiment 1.

DESCRIPTION OF INVENTION

The present invention relates to a centrifugal filtration device (1) for concentrating liquid mixtures to be inserted into the rotor of a centrifuge comprising: a housing which comprises a sample chamber (2, 102, 302) with at least one vent (123, 312); a permeate chamber (6, 106, 306) with at least one vent (145, 351); at least one filtration chamber (4, 104, 304) with at least a semipermeable membrane (5, 105, 205, 305); a concentrate chamber (7,107,307) downstream of the filtration chamber (4,104,304); and at least one neck (3, 103, 303) connecting the sample chamber (2, 102, 302) to the filtration chamber (4, 104, 304) and whose volume is less than the volume contained in the concentrate chamber (7). , 107, 307) and the filtration chamber (4,104,304).

In a preferred embodiment of the invention, the volume of the neck (33,103,303) is less than 1/5 of the volume contained in the concentrate and filtration chambers.

In a preferred embodiment of the invention, the cross-sectional area of the neck (3,103,303) is less than 1 mm ² .

Using a long narrow neck between the sample chamber, which is closest to the spin axis of the centrifuge, and the filtration chamber, which is farthest from said axis of rotation, preferably a neck with a volume of less than 1/5 of the volume contained in the concentrate and filtration chambers, and a cross-sectional area preferably less than 1 mm ² , can maintain the pressure at high levels throughout the filtration process, allowing for greater factors to be achieved. concentration and reduce the filtration time.

In a preferred embodiment of the invention, the housing further comprises a concentrate removal channel (133, 333) that connects the sample chamber (102, 302) to the concentrate chamber (107, 307), and through which it can be insert a flexible tube to remove the liquid mixture concentrated. This channel also allows to easily eliminate air pockets in the filtration and concentrate chambers at the beginning of centrifugation. In a preferred embodiment of the invention, the device further comprises at least one permeate channel (144, 237, 346, 347) which connects the permeate side of the filtration membrane to the permeate chamber and permits the permeate flow to be conducted. to the permeate chamber.

In an even more preferred embodiment of the invention, the normal vector on the active side of the membrane is at an angle of approximately 90 ° with the centrifugal acceleration vector to maximize the self-cleaning action of the concentration boundary layer.

In another even more preferred embodiment of the invention, the centrifugal filtration device comprises two filtration chambers (304a, 304b) with two semipermeable membranes (305a, 305b) facing each other on opposite sides of the filtration chamber. 304, and a neck 303 leading into two independent conduits 303a, 303b, which in turn flow into the filtration chambers 304a and 304b.

In another preferred embodiment of the invention the semipermeable membranes (5, 105, 205, 305) have a molecular exclusion limit in the range of reverse osmosis and nanofiltration, i.e. between 100 Da and 1000 Da. Using membranes nanofiltration or reverse osmosis, to achieve reasonable filtration flows and solute rejections, as well as high concentration factors, it is necessary to operate at pressures typically between 5 and 80 bar. THE The device of the invention avoids the rapid decrease of the supply pressure as soon as the liquid level in the sample chamber decreases, keeping the pressure at high levels throughout the filtration process, which allows to reach higher concentration factors and reduce the filtration time. .

In another preferred embodiment of the invention, the semipermeable membranes (5, 105, 205, 305) have a molecular exclusion limit in the range of ultrafiltration or microfiltration, i.e. between 1 kDa and 1000 kDa.

The invention further relates to a method for concentrating a liquid mixture comprising the following steps: a) providing a centrifugal filtration device (100, 200 or 300) according to claims 1 to 8;

b) providing a rotor centrifuge capable of receiving said centrifugal filtration device;

c) introducing the sample of liquid mixture to concentrate into the sample chamber (2, 102, 302);

d) inserting the centrifugal filtration device into the centrifuge rotor;

e) turning on the centrifuge and setting the rotation speed to a value sufficient to reach a pressure sufficient for the sample to permeate through the membrane;

f) waiting long enough for the sample to permeate through the membrane and achieve the desired concentration factor;

g) removing the centrifugal filter device from the centrifuge rotor;

h) removing the permeate and concentrate from the centrifugal filtration device. In a preferred embodiment of the invention, in step e) of said method, the spin speed of the centrifuge is set to gradually increase from a given starting value to a given ending value throughout the filtration process.

This section describes the fundamentals of the operation of the object of the invention and of three proposed alternative embodiments.

FIG. 1 shows a generic sketch of a centrifugal filtration device. This figure is used herein to describe and explain the fundamentals of operation of the object of the invention. Centrifugal filtration device 1 comprises a sample chamber (2), at least one narrow neck (3) connecting the sample chamber (2) to a filtration chamber (4), a semipermeable membrane (5) which allows permeate passage from the filtration chamber (4) to a permeate chamber (6), and a concentrate chamber (7). All components are placed inside a housing and in this case it is assumed that centrifugal acceleration acts horizontally from left to right.

FIG. 2 shows the general sketch of the centrifugal filtration device 1 shown in FIG. 1 during operation. At a given point in the filtration cycle, the centrifugal filtration device (1) rotates at an angular velocity ω about the axis of rotation of the centrifuge. At the beginning of filtration, the liquid sample (2a) placed in the sample chamber (2) flows into the narrow neck (3). From this neck it then flows into the filtration chamber (4) where permeation occurs. Part of the concentrated sample then accumulates in the concentrate chamber (7). As the process A portion of the liquid sample permeates through the membrane (5) and is collected as permeate (6a) in the permeate chamber (6). Another portion of the sample is retained and concentrated by the membrane (5), being partly collected as the concentrate (7a) in the concentrate chamber (7). Filtration occurs due to the pressure increase caused by centrifugal acceleration in the high-speed centrifuge interior. When the centrifugal filtration device (1) is rotated, the pressure difference, Ap, between levels r ... 8 and? ¾. 9 is given, in good approximation, by Ap = £ ω {τ -? †}, where p is the average specific mass of the liquid between radial positions? _j and ·? .

If the angle is greater than or equal to 0 and less than ^the 90 °, the centrifugal force creates a self-cleaning mechanism that causes the fluid elements concentrates the membrane deviate from this in the radial direction. For α greater or equal to 0 and less than ^the 90 °, the more concentrated fluid elements are projected toward the concentrate chamber (7), which ends up being largely concentrated in the liquid sample (7b).

For all fluid from the sample chamber (2) to pass through the narrow neck (3), the angle β between the wall surface (2c) and the centrifugal force vector must be between 90 ° and 180 °. The narrow neck (3) allows the high transmembrane pressure difference to be maintained throughout most of the filtration cycle. The centrifugal filtration device is designed so that the level of the sample feed reaches the narrow neck inlet when the desired concentration factor has already been reached. That way, transmembrane pressure never decays much throughout the filtration cycle.

To prevent membrane damage, vent holes must be provided in the sample chamber (2) and permeate chamber (6). Otherwise, when the centrifuge stops, the pressure in the permeate chamber may be greater than the pressure in the feed chamber and the membrane may rupture. The location of the ventilation holes should take into account the orientation of the liquid levels in the sample (2) and permeate (6) chamber when the centrifuge is at rest and in operation so that no liquid sample is lost through these holes. FIG. 3 shows an isometric sketch of embodiment 1, or embodiment 2, of the centrifugal filtration device of the invention. The housing of centrifugal filter device 100 (or 200 for embodiment 2) is comprised of several parts. The cap (110) fits into a hole in the upper surface (121) of the upper part (120). The bottom edge surface of the upper part 120 connects to the membrane support part 140 (240) for embodiment 2 which in turn connects to the bottom part 150 which preferably has , a hemispherical shape, suitable for insertion into typical centrifuge rotors. The support portion (140) has a porous plate or a micro channel surface beneath the membrane to allow the permeate to flow into the permeate chamber (106) within the bottom portion (150) (see FIG. 4).

All parts of embodiment 1 of the invention can be seen in FIG. 4 in the form of a view of the centrifugal filtration device 100 shown in FIG. 3. Embodiment 1 uses a single membrane (105) glued by its edges to the upper surface (141) of the membrane support portion (140). The adhesive for bonding the membrane edges to the upper surface (141) must be compatible with the liquid sample solvent and the upper surface material (141). For metal contact porous polyester backing polyamide membranes, epoxy or polyurethane based adhesives may be used.

The membrane support portion 140 is secured between the upper portion 120 and the bottom portion 150. Two o-rings (142 and 143) ensure fluid tightness in the filtration (104), concentrate (107) and permeate (106) chambers. At least one narrow permeate channel (144) interconnects the surface (141) of the membrane support (140) to the permeate chamber (106) at the bottom (150) of the centrifugal filtration device. The upper surface (141) may have a porous plate or a set of grooves to facilitate permeate flow towards the narrow permeate channel (144). A cavity, delimited by the membrane support portion (140) and the upper portion (120), defines the concentrate chamber (107). The filtration chamber (104) is defined as the void space between the membrane (105) and the lower surface of the inner block (130). The height of the filtration chamber (104) is determined by the height of the edge (132) of the lower surface of the inner block (130). For this reason, the effective membrane area is smaller than the upper surface area (141) of the membrane support (140) and depends on the edge dimensions (132). A clearer description of edge 132 can be seen in FIG. 10. The camera The sample (102) is bounded between the upper part of the inner block (130) and the inner surface of the upper part (120).

Preferably, the sample chamber 102 should be located as far away from the filtration chamber as possible and as close as possible to the axis of rotation. In the inner block (130), there are grooved narrow channels connecting the sample chamber (102) to the filtration (104) and concentrate (107) chambers. One of the grooved channels is the narrow neck (103), which corresponds to the narrow neck (3) outlined schematically in FIG. 1. Narrow neck 103, which is not visible in FIG. 3, but is visible in FIG. 6 connects the sample chamber (102) to the filtration chamber (104), allowing the liquid sample to be fed to the filtration chamber. In embodiment 1, there is a second narrow channel (133) (concentrate removal channel) that connects the sample chamber (102) to the concentrate chamber (107). Through the bore (122), a flexible capillary tube can be inserted into the narrow channel (133) (concentrate removal channel) and thereby sucked out the concentrate from the concentrate chamber (107). The narrow channel (133) (concentrate removal channel) also facilitates the passage of the liquid sample into the filtration chamber at the initial stage of centrifugation, allowing any air pockets to be eliminated.

At the top surface (121), there is a first vent hole (123) connected to the sample chamber (102). This prevents the occurrence of vacuum at the end of the filtering cycle, which could damage the membrane. A second vent (145) (see FIG. 6) is located in the membrane support portion (140) and connects to the permeate chamber (106). Finally, the upper part 120 may be closed with the lid (110), which has a sealing o-ring (111).

The top view of embodiment 1 of the invention is in FIG. 5 showing the location of the cross-section A-A to be used in FIGS. 6, 7, 8 and 9.

FIG. 6 shows the sectional view of embodiment 1 of the invention according to section A-A defined in FIG. 5. The connections between the various chambers and the two ventilation holes are visible in FIG. 6. As mentioned above, the narrow neck (103) connects the sample chamber (102) to the filtration chamber (104). The narrow neck (103) is formed by a groove in the inner block (130). The sample chamber (102) is defined as the void space between the inner block (130) and the top part (120). The surface (131) of the inner block (130), which defines the sample chamber, makes an angle γ to the vertical axis of the centrifugal filtration device.

The connection between the sample chamber (102) and the concentrate chamber (107) is through the second narrow channel (133) (concentrate removal channel), which consists of two sections (134 and 135). Section 134 is a perforated channel in inner block 130 from surface 131. Section 134 is connected to section 135, which is a groove engraved in the inner block 130. To minimize the dead volumes of narrow neck 103 and second narrow channel 133 (concentrate removal channel), these two channels are as thin as possible. FIG. 7 shows a cross-sectional view of embodiment 1 of the invention according to section AA defined in FIG. 5, when the centrifugal filtration device is placed within the fixed angle rotor of a centrifuge with angle ε. As already mentioned, angle β should preferably be between 90 ° and 180 °, while angle a should preferably be close to angle ε of the rotor so that the membrane is aligned with centrifugal acceleration.

FIGs. 8 and FIG. 9 show that of embodiment 1 of the centrifugal filter device positioned in the same configuration shown in FIG. 7, but in two different filtration phases. FIG. 8 shows the initial phase, prior to filtration, where the liquid sample (102a) fills the entire space defined by the sample chamber (102), the narrow neck (103), the second narrow channel (133) (concentrate removal channel). ), the filtration chamber (104) and the concentrate chamber (107). FIG. 9 illustrates the final phase of the filtration process, where permeate chamber 106 already contains most of the permeate liquid 106a that has permeated through the membrane, while concentrated liquid 107a is trapped in the concentrate chamber (10). 107). The permeate chamber (106) must have a volume equal to or greater than the sample volume to be processed and the orifice (145) must be positioned such that, even with permeation of the entire sample, no permeate leakage occurs through the orifice ( 145). Embodiment 2 of the invention is similar to embodiment 1 already described, but includes an additional membrane in the filtration chamber. This doubles the membrane area and halves the time required to concentrate the fluid sample. This embodiment 2 of the centrifugal filtration device is illustrated in FIG. 10. The additional membrane (205b) is disposed on the lower surface of the inner block (230) and the second permeate channel (237) connects the permeate side of the membrane (205b) to the permeate chamber (106). The second permeated channel (237) comprises two sections: a channel section (238) within the inner part of the block (230) and another section (246) within the membrane support part (240). The perforated rod (236) connects the channel section (238) in the inner block (230) to the channel section (246) in the membrane holder (240). The second membrane 205b is glued to the lower surface of the inner block 230 using the same type of bonding as previously described for gluing the first membrane.

The top view of embodiment 2 of the invention is shown in FIG. 11 which shows the location of the cross section BB to be used in FIG. 12. Cut section BB changes direction in position aligned with the center of the permeate channel (144) to pass through the middle of the perforated rod (236). The region defined by the dashed line C is enlarged in FIG. 13. The permeated channel 237 is shown in FIG. 13 as a tube with bends. However, sections 238 and 246 of permeate channel 237 may be straight channels made by perforation. Another embodiment of the invention is shown in FIGS. 14, 15, 16 and 17. FIG. 14 shows an isometric perspective sketch of embodiment 3 of the centrifugal filtration device of the invention. The wrapper The external portion of the centrifugal filtration device (300) comprises an outer part (350) and a lid (310) at the top thereof. There are also two holes to prevent pressure imbalance at the end of filtration and to prevent membranes from rupturing. On the side surface of the outer part 350 is the hole 351 connected to the permeate chamber. On the top surface of the cap (310) is the hole (312) connected to the sample chamber. The shape of the centrifugal filtration device (300) should preferably be designed so that this device can be inserted into standard centrifuge rotors.

FIG. 15 shows the exploded isometric view of embodiment 3 of the invention. All internal parts of centrifugal filtration device (300) are supported on edge (352) of outer part (350). The novelty of embodiment 3 of the invention is the use of an inner block (330) separating the sample chamber (302) from the filtration chamber (304). In this embodiment 3 of the invention there are two membranes, namely membrane (305a) and membrane (305b). The membrane 305a is supported in the slot 342, while the membrane 305b is supported in a similar slot on the opposite side. The surfaces of these grooves should preferably have a set of protrusions or porous surfaces to facilitate passage of permeate flow to permeate reservoir (306). Each of the membrane support pieces (340 and 341) has at least one permeate channel (346 and 347), respectively, to conduct permeate flow into the permeate chamber (306). The permeate chamber (306) is defined by the inner walls of the outer shell (350). The membrane support pieces (340 and 341) are attached to the edges (361a and 361b) of the inner part (360). The assembly comprising the parts (340, 341 and 360) is capped by the lid (310) and fits within the outer part (350), the edge (362) of the inner part (360) being supported at the edge (352) of the outside (350). In this embodiment 3 of the invention, the inner block (330) fits into the inner part (360), creating the filtration chambers (304a and 304b) that lie between the inner block (330) and the membranes (305a and 305b). . The thickness of the filtration channels is determined by the dimensions of the rod (339) of the inner block (330).

Inner block (330) is supported by lip engagement (338) at end (363) of inner part (360). The sample chamber (302) is created by a cavity in the top of the inner block (330). The neck 303 connects the deepest part of the sample chamber 302 to the filtration chambers 304a and 304b through the auxiliary narrow channels 303a and 303b. Optionally, a narrow channel (333) (concentrate removal channel) may be used to remove the concentrate at the end of filtration with the aid of a flexible capillary tube. This channel also allows to easily eliminate air pockets in the filtration chambers at the beginning of centrifugation.

Embodiment 3 of the invention can be further understood using the sectional sections DD and EE outlined in FIG. 16 illustrating the top view of embodiment 3 of the invention. Section DD is seen from the front in FIG. 17. During filtration, fresh sample liquid flows from the sample chamber (302) into the narrow neck (303). The narrow neck then flows into the ducts (303a and 303b) which in turn flow into the filtration chambers (304a and 304b), where are the membranes (305a and 305b). The concentrated sample is collected in the concentrate chamber of (307). The permeate passes through the channels 346 and 347 and is collected in the permeate chamber 306.

FIG. 18 shows the cross-sectional view of embodiment 3 of the invention along section E-E defined in FIG. 16. The narrow channel (333) (concentrate removal channel) in the center of the device may be used to withdraw concentrated sample at the end of filtration or to facilitate the elimination of air pockets that may initially exist in the filtration chambers.

Another option for removing the concentrate from embodiment 3 of the invention is to detach the lid (310) and the outer part (350) from the assembly, insert a reservoir (370) through the opening of the inner block part (330) and perform a low speed spin with the inverted device. This procedure is best understood with the aid of FIG. 19. After removing the cover (310) and the outside of the assembly (350), the additional reservoir (370) is inserted into the top of the inner block (330) and the new assembly is inverted and centrifuged at a low rotational speed. . As a result, the concentrated liquid that was in the concentrate chamber 307 flows into the chamber 371, where it can be collected as the concentrated sample 307a, after separating the reservoir 370 from the device.

The centrifugal filtration device may utilize reverse osmosis membranes, nanofiltration membranes with a molecular exclusion limit that is typically between 100 Da and 1 kDa, or ultrafiltration membranes with a molecular exclusion limit between 1 kDa and 1000 kDa. EXAMPLES

To evaluate the performance of the centrifugal filtration device disclosed in this invention, various centrifugal filtration experiments were performed with aqueous sucrose solutions using embodiment 1 of the object of the invention, with an angle γ of 30 ° and an angle δ of 34 °. . The prototype used has a height of 103 mm and a width of 28.7 mm and was manufactured from an aluminum light alloy (with the exception of the Teflon inner block 130 and the polyinitrile o-rings 111, 142 and 143). , sample chamber 102 has a volume of about 3.2 mL.

Sucrose was selected as the reference solute because it has a molecular weight of 342.3 g / mol which is in the range of nanofiltration molecular exclusion limit. Quantitative evaluation of centrifugal filtration device performance was based on concentration factor and apparent rejection. The concentration factor, CF, is the ratio of the final concentrate concentration, C _c ., To the initial sample concentration, C _a , ie CF = C / C _t7 . Apparent rejection, R _s , is calculated as the difference between the initial and permeate sample concentrations, C ^, and Ç _B , respectively, divided by the initial sample concentration, ie R _a = {C _a - £ _ρ .) / € _α . An apparent rejection close to 1 indicates that the membrane prevents the passage of solute to the permeate.

In all examples the NFX nanofiltration membrane manufactured by Synder Filtration (Vacaville, USA) was used. The membrane, which according to the manufacturer has a limit of Nominal molecular exclusion between 150 and 300 Da was bonded to the backing using two epoxy resins: Ceys Araldite Standard (Spain) and Omega Omegabond OB-101 (USA). The membrane was glued to the surface (141) of the membrane support portion as described below. A surface-shaped membrane piece (141) was cut from a new membrane sheet. A thin wire (about 1 mm) of Omegabond OB-101 blend was scattered about 2 mm from the edges of either the surface (141) of the centrifugal filter device or the surface of the membrane polyester support (105). The surface of the membrane polyester support (105) was brought into contact with the surface (141) of the membrane support part (140) and the time required for curing the adhesives was waited according to the manufacturers' instructions. glues. After the adhesives were cured, a thin layer of Araldite Standard mixture was spread to about 3 mm from the membrane edge (105) (including the lip) to ensure their sealing. After curing of the glue, the filtration device according to embodiment 1 was fit for use.

The permeate and concentrate sucrose concentrations at the end of filtration were determined using the Atago DD-5 differential refractometer (Japan). In all examples a Sorvall RC6 centrifuge with a fixed angle F10-6x500y rotor was used. An adapter was attached to this rotor such that the angle ε was 34 °. In the examples below, the sample chamber volume was about 3.2 mL, the narrow neck length was 1.9 cm, the volume contained in the narrow neck was 0.018 mL, and the volume contained in the sample chambers. Concentration and filtration was 0.1 mL in Examples 1 and 2 and 0.4 mL in Example 3. The ratio between the volume of the neck and the volume contained in the concentrate and filtration chambers was then always less than 1/5 (this ratio is 0.18 in examples 1 and 2 and 0.04 in example 3). The cross-sectional area of the narrow neck was 1 mm ² .

Example 1

A 3.2 mL volume of aqueous sucrose solution with a concentration of 7.7 g / l was filtered according to the present method disclosed in the invention (see Table 1). The height of the filtration chamber, ie the distance from the membrane surface 105 to the lower surface of the inner part of the block 130, was 0.2 mm.

Prior to filtration of the sample itself, the membrane was washed with water to remove the preservative and protective substances from the membrane placed by the membrane manufacturer. To this end, 3.2 mL of deionized water was filtered into the centrifugal filtration device at a rotation speed of 6000 rpm for 30 minutes and the process repeated once. Under these conditions, the average pressure in the filtration chamber 104 at the beginning of the filtration cycle is 16 bar. After washing the membrane, 3.2 ml of sucrose solution was filtered over 45 minutes at a rotation speed of 6000 rpm. This filtration time was sufficient for all the solution contained in the feed chamber to be filtered.

At the end of the filtration process, the permeate was removed from the permeate reservoir and the concentrated liquid sample was suctioned from the device using a flexible capillary tube and syringe according to the method described in the present invention. Under these conditions, it was possible to extract from the centrifugal filtration device about 105 μΐ concentrate, which had an average concentration of 117 g / l sucrose. The apparent rejection obtained in this assay was 97%.

In summary, this example proves that through embodiment 1 of the object of the invention it is possible to concentrate 3.2 ml of an aqueous sucrose solution with a concentration of 7.7 g / l to obtain 105 μΐ of concentrated solution to give a concentration factor of 15, with an apparent rejection of 97%.

Example 2

An aqueous sucrose solution with a concentration of 30.7 g / l was filtered for 60 minutes at a rotation speed of 9000 rpm. The membrane in this example was previously washed with water using the same procedure as example 1, but only for 15 minutes.

3.2 mL of sucrose solution was introduced into the centrifugal filtration device. The average pressure in the filtration chamber 104 at the beginning of the filtration cycle was 35 bar for the rotation speed of 9000 rpm. Under these conditions, at the end of centrifugal filtration, a volume of 325 μL concentrate was extracted with a concentration of 233 g / l sucrose. Apparent rejection of the membrane was 99.2%. In summary, this example proves that the centrifugal filtration device, operating at 9000 rpm for 60 minutes, is capable of concentrating a 3.2 ml sample of a solution. sucrose, increasing its concentration from 30.7 g / l to 233 g / l.

Example 3

In this example, the temporal evolution of filtration of an aqueous sucrose solution with a concentration of 30.7 g / l, operating at a rotation speed of 9000 rpm, was studied. For this, filtration tests were performed with increasing filtration times. In this example, the filtration chamber height of the centrifugal filtration device was 1.3 mm. FIG. 20 shows the evolution of the average permeate flow rate at each time interval and the sucrose concentration in the concentrate at the end of each interval. The average permeate flow rate is the average permeate volume that crosses the membrane per unit time and was obtained by determining the accumulated permeate volume in the permeate chamber 106 at the different time intervals shown in FIG. 20. Since the centrifugal filtration device disclosed in this invention achieves high repeatability between assays, and the membrane used in the different assays was always the same, it was possible to determine the average permeate volume between two generic t1 and t2, ie in the time interval [tl, t2], calculating the accumulated permeate volume difference in centrifugal filtration tests with different filtration times tl and t2. Thus, for each interval [tl, t2] a filtration time test t2 was performed always starting from

Claims

of the initial conditions. At the end of the assay, permeate and concentrate volumes and concentrations were determined, and the average permeate flow rate was obtained by difference with the accumulated permeate value for the immediately preceding filtration time assay (and dividing by the respective interval time ).

In FIG. 20, concentrate concentration values at each time t report to filtration test with filtration time t. In this example, the concentrate designation refers to the fraction of liquid present in the concentrate chamber (107) and / or the filtration chamber (104) after removal of the remaining liquid fraction in the sample chamber (102). This is because at the initial time intervals, although the permeate flow rate is high, the filtration time is insufficient for full filtration to occur. Therefore, at these initial time intervals there was always the presence of liquid in the sample chamber (102) after the centrifuge was stopped. However, analysis of the liquid in the sample chamber (102) revealed that there was no mixture between this liquid and the liquids in the filtration chamber (104) and / or the concentrate chamber (107), as their concentration remained unchanged. equal to the initial concentration of the sample.

This example shows that for the centrifugal filtration device used, the maximum concentration of about 170 g / l sucrose is reached after about 40 minutes of filtration. This type of test should always be done at an early stage to identify the minimum filtration time required to filter the entire sample and thus to achieve the desired maximum concentration factor. Centrifugal filtration device (1) for concentrating liquid mixtures for insertion into the rotor of a centrifuge comprising: a housing which comprises a sample chamber (2, 102, 302) with at least one vent hole (123,312); a permeate chamber (6, 106, 306) with at least one vent (145, 351); at least one filtration chamber (4, 104, 304) with at least one semipermeable membrane (5, 105, 205, 305); a concentrate chamber (7,107,307) downstream of the filtration chamber (4,104,304); and at least one neck (3, 103, 303) connecting the sample chamber (2, 102, 302) to the filtration chamber (4, 104, 304) and which volume is less than the volume contained in the concentrate chamber ( 7, 107, 307) and in the filtration chamber (4,104,304).

Centrifugal filtration device according to claim 1, characterized in that the volume of the neck (3,103,303) is less than 1/5 of the volume contained in the concentrate and filtration chambers.

Centrifugal filtration device according to the preceding claims, characterized in that the cross-sectional area of the neck (3, 103, 303) is less than 1 mm ² .

Centrifugal filtration device according to any one of the preceding claims, characterized in that said housing further comprises a concentrate removal channel (133, 333) connecting the sample chamber (102, 302) to the concentrate chamber (107, 307), and through which a flexible tube may be inserted to remove the concentrated liquid mixture.

Centrifugal filtration device according to any one of the preceding claims, further comprising at least one permeate channel (144, 237, 346, 347) connecting the permeate side of the filtration membrane to the permeate chamber.

Centrifugal filtration device according to any one of the preceding claims, characterized in that the normal vector on the active side of the membrane is at an angle of approximately 90 ° to the centrifugal acceleration vector so as to maximize the self-cleaning action of the concentration boundary layer.

Centrifugal filtration device according to any one of the preceding claims, characterized in that it comprises two filtration chambers (304a, 304b) with two semi-permeable membranes (305a, 305b) facing each other on opposite sides of the filtration chamber (304 ), and a neck (303) which flows into two independent ducts (303a, 303b), which in turn flow into the filtration chambers (304a and 304b).

Centrifugal filtration device according to any one of the preceding claims, characterized in that the semipermeable membranes (5, 105, 205, 305) have a molecular exclusion limit in the range of reverse osmosis and nanofiltration, preferably between 100 Da and 1000 µm. Gives.

Centrifugal filtration device according to any one of claims 1-7, characterized in that the semipermeable membranes (5, 105, 205, 305) have a molecular exclusion limit in the range of ultrafiltration or microfiltration, ie between 1 kDa and 1000 kDa.

A method for concentrating a liquid mixture comprising the following steps: a) providing a centrifugal filtration device (100, 200,300) according to any one of claims 1 to 9;

b) providing a rotor centrifuge capable of receiving said centrifugal filtration device; c) introducing the sample of liquid mixture to concentrate into the sample chamber (2, 102, 302) of said device (100, 200, 300);

d) inserting the centrifugal filtration device (100, 200, 300) into the centrifuge rotor;

e) turning on the centrifuge and setting the speed of rotation to a sufficient pressure to allow the sample to permeate through the at least one semipermeable membrane of said device (100, 200, 300);

f) waiting long enough for the sample to permeate through said at least one membrane and reach the predefined concentration factor;

g) removing the centrifugal filter device from the centrifuge rotor;

h) removing the permeate and concentrate from the centrifugal filtration device. Method of concentrating a liquid mixture sample according to claim 10, characterized in that, in step e), the spinning speed of the centrifuge is gradually increased from a given initial value to a given final value throughout the process. filtration.