Chromatographic device, the porous self-sustaining structure and method of its manufacture

 

Usage: in liquid chromatography for separation and purification of compound substances. The inventive porous self-sustaining system containing at least two porous component a and b, and a porous component comprises a porous component And in which the surface of the pores of these, at least two components a and b, provided with the chemical components to interact with the substances passing through the pores, and the pores of the porous components have uniform mnegative size distribution throughout the polymer structure. The technical result of the invention is to provide a rigid porous polymeric medium of a large size with certain uniform characteristics of the pores and exhibiting low back pressure even at high flow velocities. 6 N. and 14 C.p. f-crystals, 12 ill.

The invention relates to a self-sustaining porous structure containing at least two porous component a and b, the product containing porous self-sustaining structure that contains at least two porous component and the housing for use in the product of this invention, the terminal fittings for use in this product is brilliant invention.

Liquid chromatography is one of the most important tools for discrete analysis, as well as for the separation or purification of compound substances. Chromatography operates mainly through the interaction of the molecules dissolved in the liquid phase (mobile phase) and solid phase (stationary phase). Almost perfect chromatographic process performs the efficient separation of a composite substance, sometimes a large sample volume, within a very short time. Conventional chromatographic process is carried out by passing the liquid phase containing the sample, which must be separated through a stationary phase (matrix).

Because different components interact differently with the stationary phase, the transition time is different, and the result is separation. The usual stationary phase is constructed in the form of porous spacers, providing a sufficiently large active surface for interaction. They are Packed in columns, usually a few centimetres in length and a few millimeters wide, and fixed porous Frits at both ends. Due to their porosity and structure of the washer has a relatively low mechanical strength. When the system chromatographies is to be used by large columns. The combination of large flow rates and greater height of installation (i.e. hydrostatic pressure) leads to a large reduction in pressure in the matrix, causing compression of the matrix material. This changes the characteristics of columns due to lower total porosity and irregularity. One attempt to solve this problem was to include short columns with large cross-section. However, the uneven distribution of samples in cross-section and a large amount of dead space creates additional problems. The design of the chromatographic column, using horizontal flow solves the problems associated with pressure, by using a column of cylindrical shape, as described by Saxena (U.S. patent numbers 4.627.918, 4.676.898 and 4.840.730). Separating matrix is placed between two porous Frits tubular shapes of different diameters. The mobile phase passes through the outer porous Frit through the matrix. Since the installation height of the matrix is small, the hydrostatic pressure does not play an important role. In addition, the thickness of the installation is small, which creates only low backpressure.

However, thanks to the structure of the particles remain two inherent disadvantage regarding the efficacy particles), and the time of the separation is not significantly reduced due to the diffusion limitations inside closed with one hand then the particles.

The first attempt to overcome both of the problems above was taken by Hartana and others, Journal of chromatography, 473 (1989) 273-275, WO 90/07965 by polymerization of a mixture of acrylic acid and methylenebisacrylamide for the production of stationary phase. The resulting polymer tube contains channels that are large enough to allow hydrodynamic flow. The polymer, however, is very soft and must be maximally compressed before use. At larger sizes, this leads to a disadvantage, because the compression creates an uneven channels inside the tube, leading to less than ideal efficiency speakers. Almost at the same time, was opened by SOCOM and other so-called “membrane chromatography” (U.S. patent N 4889632, 4923610 and 4952349). Used membranes have a rigid structure that contains ductive distribution of pores open channels and excellent hydrodynamic characteristics, leading to a short separation times. Although in principle the size of the membrane is not limited to, mechanical instability and irregular distribution of the sample restricts approx (U.S. patent numbers 5334310 and 5453185) by polymerization of monomers in a rigid porous tube in a hollow steel column chromatographic limited diameter. The porous tube has characteristics similar to the characteristics of the above-mentioned membranes. High pressure tube, however, specifies the upper limit of the flow velocity, which together with the small diameter column preclude the application by preparative level. Josic etc. describes a rigid porous pipe on the basis of methacrylates (WO-A-96/06158), where the mobile phase is installation in the radial direction, resulting in significantly less back pressure even at elevated flow rates. This design allows a very rapid separation of prepreparation level.

During the bulk polymerization tubes of large diameter pipes and a large thickness generated a significant amount of heat. Because the monomer mixture has a relatively low thermal conductivity, the temperature of the mixture increases dramatically during the polymerization (Peters, E. K., F. Svec, J. M.J.Frechet, Chemical materials, 9 (1997) 1898). Since the distribution of pore size depends on the temperature (Svec and Frechet, Chemical materials, 7 (1995) 707), the obtained polymer has a variable structure and cannot be used for good chromatographic separation. Peters and other Chemical materials, 9 (1997) 1898, poly offers the tion slower and that it has little effect on the distribution of pore sizes. However, there is no separation efficiency of such columns. In addition, this approach extends the time to complete the polymerization and requires very precise adding the monomer mixture, in order to avoid temperature increase.

Another way to control the amount of heat generated by or reduce this number is to add the polymer particles of the same structure in the monomer mixture. Since the diameter of the particles is typically in the range of microns, the monomer can diffuse in their pores, and the polymerization results in uneven distribution of pore size. To avoid changes in the polymer structure, the pores of the particles must be completed by the inhibitor. If the concentration of particles is too high, the inhibitor also inhibits polymerization of the monomer mixture around the particles.

On the other hand, if the number of polymer particles added to the mixture of polymerization is too low, the particles can be deposited in the form during the polymerization. In this way, the local concentration of particles in the mixture of monomer in the upper part of the form is low, thus the amount of generated heat is again very high. Therefore in the highest degree difficult to manufacture large porous polymers with well-defined the second environment is large in size with well-defined uniform characteristics then.

An additional object of the present invention is to provide a rigid porous polymeric medium of a large size, exhibiting low back pressure even at high flow velocities.

Another object of the present invention is to provide a rigid porous polymeric medium of the large size of a large variety of monomers.

Another object of the present invention is to provide a rigid porous polymeric medium large size easy and inexpensive way.

These and additional objectives of the present invention will be apparent from the following description of the present invention, and examples.

In order to achieve the above and other objectives and in accordance with the present invention as embodied and described here, the present invention is related to the chromatographic unit, contains a porous polymeric tube having a greater thickness, and the case for porous polymeric tube. The resulting block can be used as a chromatographic column, for different bioconversion, adsorption and diagnostic processes, and as a matrix for peptide or oligonucleotide synthesis due to its ways is more sustained fashion than 0.45. The porosity is determined using the water return or mercury measurement of porosity.

The material contains small pores, i.e. pores with a diameter less than 200 nm, but also large pores with a diameter of at least about 700 nm. The porous polymeric tube is preferably a cylinder having an inner diameter of at least 1 mm, and outer diameter of at least 10 mm Porous polymeric tube may consist of a single monolith single monolithic porous polymer tube or from a set of tube-like monoliths, tightly inserted into one another porous polymeric tube from a variety of monoliths. Each tubular monolith can have different sorption properties, thus the sorption properties of porous polymer tubes can be tailored according to your specific requirements. Also a single monolithic porous polymer tube can have different sorption properties due to the two-step method of manufacture described herein. The porous polymeric tube of this invention is placed in the housing, adjusted for the size of the pipe. The distributor and the collector housing is designed to minimize dead volume of the entire unit. The case can be shotokai as stainless steel.

Porous polymer tube made from a mixture of moulinrouge monomer and polyvinyl monomer in the presence of porogen (pore-forming) and initiator. Can be used various mixtures of each tubular monolith to obtain desired characteristics. A given feature may be, for example, non-polar surface of the polymer tube. This can be achieved by introducing, for example, aliphatic groups With4or C18. It may also be desirable polar surface. In this case, there must be various groups, such as hydroxyl or amino group. The wall thickness of the tubular monolith must be installed so that during the polymerization the temperature increase (thermal reaction) in a mixture do not exceed that affects the hydrodynamic characteristics of the final product. The height of the porous polymeric tube, however, is not limited.

In the case of forming the porous polymeric tube from a variety of monoliths each tubular monolith polymerized separately so that the outer diameter of the inner tubular monolith tightly fit to the inner diameter of the outer tubular is s, affecting the porous structure. In the case of porous polymeric tube from one monolith preferably first polymerized tubular monoliths having a wall thickness less than critical. The outer diameter of the inner tubular monolith is slightly smaller than the inner diameter of the outer tubular monolith. Tubular cylindrical monoliths and placed one inside the other, the gaps between them are filled with the mixture of monomer. The monoliths can be connected in series together during the polymerization, producing the single monolith. After the porous polymeric tube prepared in one way or another, progeny washed with a suitable liquid.

Fig.1: schematic view of D in the collection of the porous polymeric tube of components a, b and component C.

Fig.2: technical drawing of the hull.

Fig.3: view of the chromatographic unit.

Fig.4: the second end of the fitting.

Fig.5: housing according to the invention.

Fig.6: component D, the porous polymeric tube of this invention.

Fig.7: the first end fitting of the invention.

Fig.8: collecting element to reduce dead volume.

Fig.9: comparison of the distribution of pore sizes for internal and external monasteries and cleanup the pipe from many monoliths, containing DEAE and srodstvenny active group.

Fig.12: the curve of performance measurement according to the invention in a 50 ml tube from a variety of monoliths.

According to this invention, created a self-sustaining porous structure containing at least two porous component a and b, and a porous component comprises a porous component And in which the surface of the pores of these, at least two components a and b are equipped with chemical components to interact with the substances passing through the pores, and the pores of the porous components include mnegative distribution of pore size throughout the polymer structure.

According to this invention, mnegative distribution of pore size (figure 9) indicates that at least three of the maximum pore size measured by the range from 5 nm to 10 μm, which are separated by areas where the volume of small pores or pores absent. It provides the following benefits:

- Pores with a pore diameter of more than 700 nm find low back pressure with increasing number of noise material.

- A large number of pores with a diameter less than 700 nm provides a large surface area required to obtain high binding capacity. monomers, having at least two polymerized constituents or two types of monomer, and the first type of monomer has one curable component, and another type of monomer capable of cross link the polymer chain derived by the polymerization of the first monomer.

In a preferred embodiment of the invention the porous self-sustaining patterns of this invention, the surface of the pore modified functional groups, such as ion exchange groups, hydrophobic components, a reactive group for covalent binding of ligands, such as srodstvenny ligands, preferably proteins, enzymes, antibodies, antigens, lectins, sugars, nucleic acids, cellular organelles or dyes, etc.,

Preferably porous, self-sustaining structure of this invention uses to build polyvinyl monomers and monovinyl monomers.

In particular, the group of polyvinyl monomers include divinylbenzene, Divinington, diphenylpyridine, alkylen dimethylacrylate, oxyalkylene dimethylacrylate, oxyalkylene diacrylate, oligoamine glycol diacrylate, vinyl polycarboxylic acid, divinyl ether, di-, tri - or tetramethylurea or dy and mixtures thereof.

According to this invention, the group monovinyl monomers include styrene substituted in the nucleus of styrene, in which the substitution includes chloromethyl, alkyl with up to 18 carbon atoms, hydroxyl, t-butyloxycarbonyl, halogen, nitro, amino groups, protected hydroxyl or amino group, vinylnaphthalene, acrylate, methacrylate vinyl acetate and pyrrolidone and mixtures thereof.

Polyvinyl monomer or polyvinyl monomer plus monomineralic monomer used in the polymerization mixture in an amount of from 20 to 60% for the production of porous self-sustaining patterns of this invention.

The first component In a porous self-sustaining patterns of this invention is a similar pipe structure having an inner cylindrical tube 10 with an inner diameter of 12 and an outer diameter of 11, which is capable of receiving the second component And having an inner cylindrical tube 20 with an outer diameter of 21 and the inner diameter 22, provided that the outer diameter 21 of the component coincides with the inner diameter 12 of the component B, and component a is inserted into the component Century.

Component a and component b can be of the same material or from different materials, for example, component a and the tion, the inner cylindrical tube 20 of the component And serves as a collection of samples.

The present invention relates also to a product containing a porous self-supporting structure and means for implementation of chromatographic processes. According to this invention, the product is preferably a chromatographic unit 30, a column or cartridge, or bioconversion reactor, or a matrix for the synthesis of peptide or oligonucleotide.

The preferred embodiment of the invention includes a housing 36, which includes the distributor 23 samples that hosts the component D, the housing 36 has at least one inlet opening 41 and at least one outlet port 40, the inner surface 42 and outer surface 43, and a pipe-like element or pipe-like elements 72 on the Central part of its inner surface 42 forming the dispenser of samples 23, while the remainder of the inner surface 42 smooth.

Especially preferred is a pipe-like element 72 made in the form of a helical or spiral grooves 25, beginning in the area of the inlet 41 of the chromatographic unit and in direct contact with him,the hole 40 of the chromatographic unit 30. The product of this invention, in particular, is a chromatographic unit 30, optionally containing a second end fitting 38 and the first end fitting 32, with 0-rings 33, 34, 35, 37 and sealing couplings 31, 39.

In a preferred embodiment, the chromatographic unit 30 of this invention, the first end fitting 32 has an upper portion 62, the lower portion 63 and the housing, the first end fitting 32 has an essentially cylindrical shape, the first end fitting 32 includes an annular flange 61 that divides the cylindrical end fitting 32 into two parts 62, 63, where part of the end fitting 32 nearest the annular rib 61, has a bottom portion 62 containing the connector 60, which is in connection with a Central hole 64 extending through the first end of the fitting 32, and the O-ring 35, located in an annular groove in the casing in the upper part 63 of the first end of the fitting 32, and O-rings in the upper part 63 of the first end of the fitting 32.

In a preferred embodiment, the chromatographic unit 30 of this invention, the second end fitting 38 has an upper portion 52, the lower portion 53 and the housing, and the second end fitting 38 is essentially a cylinder is ting 38 into two parts 62, 63, where part of the end fitting 38 nearest the annular flange 51, a lower portion 52 containing the connector 50, in connection with the stub center hole 54, associated with the hole 55, which is perpendicular to the stub Central hole 54, and the hole 55 begins in the annular groove 56 at the surface of the body of the second end of the fitting 38 and leads to the stub end of the Central hole 54.

The housing 36 for use in the product under item 13 of the claims dispenser contains samples 23, in which a pipe-like element 72 made in the form of a helical or spiral groove 25. As noted, the component And is inserted into the component C. In the case when the component And has a relatively large inner cylindrical tube 20, it will lead to a relatively large dead volume around the chromatographic unit. In order to reduce or eliminate this disadvantage, it is possible to insert in part a prefabricated element 80. Precast element corresponds to the inner cylindrical tube component either by fitting tightly into the inner hole of the component, or by leaving a gap. When formed, the gap between the precast element 80 and the inner cylindrical is significantly less. When prefabricated element is a tight fit in the component And, of course, must be made a tool that removes the liquid flowing through the porous components a and B. This can be done by performing on the outer surface of the precast element 80 channels or pipe-like elements. Preferably the channels or pipe-like elements 82 are formed in spiral or helical groove 81. Precast element with the upper portion 84 and the lower part 85, provides on its upper part 84 of the flat surface, preferably a smooth surface. However, the lower portion 85 includes a channel 83, which passes inward from the outer side, preferably to the center. A pipe-like element 82 continues in the channel 83 on the lower part 85 of precast element 80. Preferably the channel 83 is connected with the hole 64 of the first end of the fitting 32. The upper portion 84 is in contact with the bottom part 53 of the second end of the fitting 38.

Chromatographic unit 30, presented here, contains all the details with figures 2, optional element 80 with figures 8 and the porous polymeric tube D, is shown in Fig.1-6. The casing is preferably made of an inert plastic material such as polypropylene is one and the same material. Figure 1 shows the Assembly D, the porous polymeric tube, of three different components: component A, component b and component C. These three components can be inserted into one another for the formation of porous polymeric tube from a variety of monoliths in which the component forms the inner part, component a is the middle part and component is In the outer portion concentric Assembly D. Component contains an inner hole 10, the diameter of which 12 is large enough to fit the outer diameter 21 of the component A. in Order to have a component inserted into the component And, of course, the diameter should correspond to the inner diameter 22 of the component A. the Component has a Central hole extending through the entire length of the component C. the Central hole acts as the collection of samples in the Assembly D with figure 1. If the Central hole has a larger diameter can be inserted element 80 from figure 8, in order to minimize the dead space of the collection, as well as to provide additional mechanical strength.

The figure 2 shows a detailed view of the chromatographic unit 30 with figure 3. Chromatographic unit comprises sealing couplings 31 and 39 containing cell 32, 33, 34, 35, polymeric tube from a variety of monoliths or a single monolith, such as Assembly D in the housing 36, as well as components 37 and 38. Component 32, the first end fitting and the second end fitting 38 are located at opposite ends. The second end fitting is inserted into the housing 36 and sealing O-ring 37 in the middle part of the fitting 38. The o-ring 37 fitted into the groove in the Central part of the body of the end fitting 38. The first end fitting 32 is inserted into the housing 36 and is sealed O-shaped rings 33, 34 and 35.

The figure 4 shows the second end fitting 38 having an upper portion 52 and lower portion 53 and the housing. End fitting 38 has a cylindrical shape and has an annular flange 51 which divides the end fitting 38 has a cylindrical shape asymmetrically into two parts. The longer part is inserted into the housing 36, and the annular flange 51 prevents complete the entry end of the fitting into the housing 36. The width of the annular flange 51 corresponds to the outside diameter of the body 36, so that the sealing sleeve 39 can be screwed on top of the annular flange 51 and the housing 36 to lock the end fitting 38. End fitting 38 has a Central opening that has a stub end in the lower portion of the end fitting Central hole 54. The thread that enters the chromatographic unit connector 50, passes through the Central hole and out the end hole 55. Perpendicular to the hole 55 is connected with the groove 56, which has a round shape.

The figure 5 shows the location in the body. Component D with figures 1 - 6 is located in the housing 36 so that the lower part is fitted to the end of the distributor 23, but the distributor 23 is above the top of the component D. the position of the component D inside the housing 36 is shown dash-dotted lines dl and d2. The Central part of the inner wall 42 of the housing 36 shows the grooves in the spiral or wit the device forming the dispenser of samples 23. End fitting 38 is inserted so as to touch the component D, and is connected with a spiral or twisted grooves 25, so that the liquid part of the housing through the opening 55 and the annular groove 56, is passed through the spiral or twisted groove 25 in the inner surface 42 of the housing 36 to the outer side of the component D. the O-ring 37 of the end fitting 38 is located over the grooves 25 of the distributor 23 and seals the inner surface 42. However twisted or spiral groove 25 is not associated with the Central hole of the end fitting 32, shown in the hole of the porous polymeric tube, acting as collector, and optionally containing element 80 from figure 8, the liquid is collected and conducted to the Central hole 64 of the end fitting 32.

The figure 6 shows schematically the component D, representing the porous polymer tube, which is depicted in figure 5.

The figure 7 shows the first end fitting 32, which has a shape similar to the second terminal fitting against the annular flange 61, the connector housing 60, and the upper part 62 and bottom 63. However, the Central hole 64 passes through the middle of the end fitting and through the fitting. In the lower part of the end fitting 32, O-ring 35, 34 and 33 condense the end fitting and the housing 36. The liquid coming from the center hole of the porous polymeric tube is routed through the Central opening 64 and can be collected.

The figure 8 shows the collecting element 80 having on its outer surface channels or pipe-like elements 82, forming a manifold with spiral or twisted groove 81. The upper portion 84 is smooth, while the lower part contains 85 channel 83 extending from the outside to the center. A pipe-like element 82 continues in the channel 83 on the lower part 85 collecting e is showing the end fitting 38.

Porous polymer tube is made from a mixture of moulinrouge monomer/monomers and polyvinyl monomer/monomers in the presence of proginov and on the choice of initiator. The polymer contains small pores, i.e. pores less than 200 nm in diameter, and large pores with a diameter of at least about 700 nm. The porosity of the polymer is greater than approximately 0.2, preferably greater than 0.45. Can be used in different mixtures of each tubular monolith to obtain desired characteristics. The wall thickness of the tubular monolith preferably set so that during the polymerization the temperature rise in the mixture did not exceed values that have negative effects on the hydrodynamic characteristics of the polymer. Typical thickness trooptube monolith obtained in a single step polymerization is in the range from a few millimeters to several centimeters.

Acceptable temperature range, the polymerization reaction is determined by performing the same polymerization of mixtures of monomers in a thin sheet-like form. The thickness of the shape is preferably such that when measuring the temperature of the middle layer of the monomer during the polymerization was observed polymerizate preferably performed at a substantially constant temperature polymerization above the lowest temperature, defined as optimal with respect to sorption properties related to optimal hydrodynamic characteristics for a particular polymer. The hydrodynamic characteristics of the polymer is determined by measuring the distribution of pore size by mercury porometry, porosity also mercury parametria or return water, and by measuring the back pressure depending on the flow velocity. The highest temperature at which the characteristics remain unchanged, is considered the upper limit of the allowable temperature.

The upper value of the thickness of the tubular monolith can be set so that the tubular form of different thickness were prepared and filled with a mixture of monomer/monomers. During the polymerization is recorded temperature in the middle layer of the mixture of monomer. The temperature during the polymerization should not exceed the tolerable upper limit for the receipt of the monolith suitable for a good run chromatogaphy.

In addition to the above considerations, there is another approach to the determination of the thickness of the tube-like monolith. In this case, the thick tubular form is filled with a mixture of monomer/monomers, and writings is mperature inside the polymerization mixture. By solving equations for a specific geometry, based on heat balances can be calculated generated specific heat and thermal conductivity. Based on these data can be used to calculate the temperature rise in the tube-like shape with a certain thickness. Moreover, using the upper limit of the permissible temperature, can be calculated the maximum thickness of the form.

When one way or another specified thickness forms for a particular monomer mixture may be made in tubular form with different inner and outer diameter, but with a certain difference between them.

For the manufacture of porous polymer tube from a variety of monoliths tubular monoliths are preferably made so that the inner porous polymeric tubular monolith is a tight fit in the outer porous polymeric tube-like monolith. The height of all the tube-like monoliths is not limited, but preferably it is one and the same. The number of tube-like monoliths generally not limited and thus can be obtained a porous polymeric tube from a variety of monoliths of any desired diameter.

For the manufacture of a single porous polymannuronic the diameter of the inner porous polymeric tubular monolith was less than the inner diameter of the outer porous polymeric tube-like monolith, thus there is a free space between the porous polymeric tube-like monoliths. This space can be filled subsequently with a mixture of monomer, and polymerization is performed a second time, or it can be left untreated. The thickness of the free space is also limited to the maximum allowable thickness specified for the pipe-like monoliths. During the polymerization of various porous polymeric tube-like monoliths are connected together to form a single porous polymer tube of the desired diameter.

Polyvinyl monomers include divinylbenzene, Divinington, diphenylpyridine, alkylen dimethylacrylate, oxyalkylene dimethylacrylate, oxyalkylene diacrylate, oligoamine glycol diacrylate, vinyl polycarboxylic acid, divinyl ether, di-, tri - or Tetra-acrylate or acrylate of pentaerythritol, trimethylacetate or acrylate of trimethylpropane, alkylene bis acrylamide or methacrylamide, and mixtures of any such suitable polyvinyl monomers.

Monovinyl monomers include styrene substituted in the nucleus of styrene, in which the substitution includes chloromethyl, al is roxannie or amino groups, vinylnaphthalene, acrylates, methacrylates vinyl acetate and pyrrolidone and mixtures thereof. Polyvinyl monomer or polyvinyl monomer plus monomineralic monomer in total are present in the polymerization mixture in an amount of from 20 to 60%.

Progeny can be selected from various types of materials, such as aliphatic hydrocarbons, aromatic hydrocarbons, esters, alcohols, ketones, ethers, solutions of soluble polymers and mixtures thereof.

Soluble polymers may also be added to the monomers. They are dissolved from the polymer after its formation and serve to increase the porosity. If they are present preferably in an amount of from 10 to 40%.

To initiate polymerization can be used conventional initiators that generate free radicals, such as azo compounds, for example, azo bis nitrile somaclonal acid and 2,2’-azo bis (amide somaclonal acid) dihydrate, or peroxides, such as benzoyl peroxide and dipropylthiocarbamate. Can be used in a variety of initiators to obtain different porous structure, relative to the speed of degradation initiators. The amount of initiator is typically in the range from about 0.5 to 4% by weight of the monomers.

Polymerization, for example, is performed in the usual way known to experienced professionals, in General at temperatures of from about 40 to 90In the period up to 48 hours. Preferably the temperature is accurately controlled to obtain a certain distribution of pore size throughout the polymer in a controlled and repeatable manner.

After the porous polymeric tube is formed, it is washed to remove any parohinog solution, and dissolve any soluble polymers, if present. The type of solvent is not critical. Can be used in many different solvents, such as methanol, ethanol, benzene, toluene, acetone, or tetrahydrofuran. To completely remove porogen and dissolved polymer, the step of washing must be repeated several times.

If the polymer pipe must be modified to specific functional groups, the polymer can be treated with certain chemical compounds. In the case of glycidyl methacrylate (as one monomial is using oleum 1,4-dioxane, to give sulfo group (SP), Chloroacetic acid to give carboxymethylamino group (CM), or various amines as diethylamine to give N,N-diethylamino-2-oxypropylene group (DEAHP), triethylamine-hydrochloride for the Quaternary trimethylamino groups (Q) or Ethylenediamine for the introduction of amino groups (EDA). The hydrophobic group can be introduced using alcoholate as ethanolic sodium, butanolate or octanol. The polymer can also react with affininty for specific binding. Affinity can be, but are not limited to, proteins, enzymes, antibodies, antigens, lectins, sugars, dyes or cellular organelles. Polymers based on other monomers can also be treated with similar methods known to experts in this field of technology.

For the manufacture of porous polymer tube from a variety of monoliths tubular monoliths are washed to remove progeny solution, dissolve any present soluble polymers. If you do not want further modification of the tube-like monoliths, or all of the tube-like monoliths should bear the same functional group, the pipe-like monoliths are inserted into one another, that shall be entered, using one of the above reagents. Finally, the porous polymeric tube from a variety of monoliths placed in the housing. If everyone, or at least two of the tubular monoliths should bear different functional groups, the modification must be performed on each tubular monolith separately, and only after that they should be inserted into one another for the formation of porous polymeric tube from a variety of monoliths. Another approach is to modify the first external tubular monolith. After the modification reaction is completed, unreacted reagent is washed, and is inserted into the lower tubular monolith. The Assembly can be placed in a new a different reagent. As the reactive group of the larger tubular monolith already transformed, the reaction occurs only in a smaller tube-like monolith. This procedure can be repeated several times, depending on the number of tube-like monoliths.

For the manufacture of a single monolithic porous polymer tube, when there is no additional modification of the pipe-like monoliths, or identical functional groups should be introduced in all the tube-like monoliths, they vstavlyayte gaps between the tube-like monoliths, in addition to the Central hole. The form is sealed and the polymerization is performed at the same temperature at which the produced tubular monoliths. After the polymerization was completed, a single monolithic porous polymer tube is washed to remove any parohinog solvent, and dissolution of any existing polymers. If required, a functional group can be introduced into the polymer using the above-described reagents. The tube is then placed in the housing and the unit is ready for use.

When should be made porous polymeric tube from one monolith with different functional groups, the following manufacturing methods. If the required functional groups are part of the monomers and different from that from which were made in tubular monoliths, a procedure similar to that used in the manufacture of porous polymer tube from one monolith without additional modifications. The polymerization temperature may, however, be different from the one used during the manufacture of the tubular monolith. A different approach is used when the functional group must be introduced into the polymer is solvent, as well as the dissolution of any present water soluble polymer. In each tubular monolith or a set of required functional groups are introduced as described above. The tube-like monoliths are dried and placed in parohinog mixture or inert substance, which fills the pores and can be easily removed later. Thus the pores are filled, and any additional polymerization them slowed down or even completely eliminated. The tube-like monoliths produced in this way are inserted in the form and processed according to the method described for the manufacture of porous polymer tube from one monolith with a uniform sorption characteristics. The tube is then washed, placed in a case and the unit is ready for use.

The present invention will be further explained by the following non-limiting examples:

Example 1. Manufacturer chromatographic block containing the porous polymer tube from a variety of monoliths

Produced two forms of stainless steel. The first form consists of two stainless steel pipes: outer with an inner diameter of 35 mm and an internal with an external diameter 17,6 mm stainless steel Tubes have a thickness , pipe. Stainless pipe with a smaller diameter longer than stainless pipe with a large diameter to allow the flow through it of the coolant to remove the heat. One clutch seals the area between the inner and outer tubes to form a gap of cylindrical shape, in which is placed a mixture of monomer. The second coupling seals the same area on the upper side. This clutch has a partition, through which the monomer mixture is introduced into the form, and a small hole to release air during loading of the mixture of monomer. After filling ends, the hole is sealed to prevent the flow of air. The second form is made in a similar way to the inner diameter of the outer tube 17.5 mm and outer diameter of the inner pipe is 1.2 mm, the monomer Mixture is prepared by mixing glycidyl methacrylate, ethylene dimethacrylate, cyclohexanol, lauric aldehyde and benzoyl peroxide. The mixture barbatiruem nitrogen for 20 min to eliminate any presence of oxygen. The mixture is injected in the form of until, until it is completely filled, and the polymerization begins by placing both forms in a thermostatic water bath. After 16 zwickau.de form and placed in pure methyl alcohol. Methyl alcohol is replaced several times to remove porogen. Small tubular monolith is then inserted into the hole larger monolith and placed in the appropriate case.

Example 2

The porous polymeric tube from a variety of monoliths composed of two tube-like monoliths, was produced in accordance with example 1. For each tubular monolith was measured distribution of pore size, using mercury porometry. Both measurements give very similar mnegative distribution of pore size is shown in Fig.9.

Example 3

Chromatographic device was manufactured in accordance with example 1. The device is attached to preparatory HPLC system (liquid chromatography high pressure, ghvd) and tested at different flow rates up to 450 ml/min, the Device shows a low pressure even at high flow velocities. The relationship between pressure and flow rate was defined as linear (figure 10).

Example 4. The manufacture of pipes from a variety of monoliths reverse phase

Use the form described in example 1. A monomer mixture was prepared by mixing glycidyl methacrylate, stearyl methacrylate, ethyl is giving 20 min to eliminate any presence of oxygen. The mixture is injected in the form of until, until it is completely filled, and the polymerization begins by placing both forms in a thermostatic water bath. After 16 h of the form are removed from the bath, cooled to room temperature, and the coupling is removed. Polymer cylinders are extracted from the molds and placed in pure methyl alcohol. Methyl alcohol is replaced several times to remove porogen. Small tubular monolith is then inserted into the hole larger monolith and placed in the appropriate case. The porous polymeric tube from a variety of monoliths shows a strong hydrophobic character.

Example 5. Manufacturer chromatographic block containing the porous polymer tube from one monolith

For the polymerization of two tube-like monoliths were used two forms of stainless steel, such forms of example 1, but with different size diameters of the gaps. The internal diameter of the larger tubular monolith 2 mm larger than the external diameter of the smaller of the monolith. Less has been removed from the form. More tube-like monolith was left in a form from which it was removed the inner steel tube. The form was sealed on one side by the clutch. Less triratna on the other side of the bulkhead, through which was added deaerated mixture of monomer of example 1, to fill the gap between the two solid cylinders. The form was placed for 20 h in a thermostatic bath. After the polymerization was completed, the porous polymeric tube from one monolith was removed. The monomers were polymerized mixture between the two tube-like monoliths and inserted to the formation of porous polymeric tube from one monolith.

Example 6. The chromatographic separation of the block containing the porous polymer tube from a variety of monoliths

The porous polymeric tube from a variety of monoliths was manufactured according to example 1. A housing containing a porous polymeric tube from a variety of monoliths, was filled with pure diethylamine and left to react for 24 hours at a temperature of 30C. After the reaction was completed, excess diethylamine was removed by pumping through a porous polymeric tube from a variety of monoliths water with a flow rate of 5 ml/min. and Finally the tube was washed by buffer 20 mm Tris-HCl, pH 7.4.

The solution of myoglobin (5 mg/ml), conalbumin (10 mg/ml) and trypsin inhibitor (20 mg/ml) in buffer 20 mM Tris-HCl, pH 7.4 was injected check NaCl, pH 7.4. Was applied the following method of separation: 45 with 100% buffer a, gradient from 100% to 20% buffer And within 3 minutes the flow Rate was 42 ml/min For separation should check for ultraviolet (UV) spectrophotometer at 280 nm, and the corresponding chromatogram is shown in figure 11. Additionally, we measured the ability of the binding protein. A solution of whey protein man (11 mg/ml) was dissolved in a buffer of 20 mM Tris-HCl, pH 7.4 and pumped through the porous polymeric tube from a variety of monoliths with a flow rate of 10 ml/min. Capacity, estimated from the curve of the invention, presented in figure 12, was about 1 g of protein.

Example 7. Manufacturer chromatographic block containing porous polymer tube from a variety of monoliths containing different functional groups

Two tubular monolith were made using the forms and the procedure described in example 1. Both were placed in pure methyl alcohol. Methyl alcohol was replaced several times to remove porogen. Finally, the tubes were placed in a mixture of methyl alcohol and water 50:50, and in distilled water. After the washing step was completed, more tube-like monolith was placed in a clean diethylamin when temperatures several times, to remove the entire diethylamin from the pores.

A lower tubular monolith was placed in a solution of antibody (2 mg/ml IgG in buffer 0.5 phosphate, pH 8.0) at room temperature. After 24 h, the tube was placed in distilled water, which was replaced several times to remove any remaining protein. A lower tubular monolith was inserted into the hole of a large monolith for the formation of porous polymer tubes from multiple monoliths and placed in the case.

Sample myoglobulin (5 mg/ml), conalbumin (10 mg/ml), trypsin inhibitor (20 mg/ml) and protein A (10 mg/ml) in buffer 20 mM Tris-HCl, pH 7.4 was injected through the loop samples 500 ál.

Binding buffer was buffer 20 mM Tris-HCl, pH 7.4, vamiali buffer from the DEAE groups was buffer 20 mM Tris-HCl + 1 M NaCl, pH 7.4, and vamiali buffer of affinity groups was 0.5 M acetic acid, pH 2, 5.

All proteins were adsorbiroval on the outer part of the porous polymeric tube from a variety of monoliths on the DEAE groups in the regime of ion exchange. After applying the gradient salt described in example 6, all proteins were selectively washed. Since IgGs binds selectively only protein And other proteins were washed out of the porous polymeric tube with many monoliths. Protein A, but SV is Ohm salt, because the relationship is based on the interaction affinity. Protein And relieved by the change of the pH value. Using porous polymer tube from a variety of monoliths containing different active groups, it is possible to perform the separation and cleaning in one pass.

Claims

1. Porous self-sustaining structure that contains at least two porous component a and b, and a porous component comprises a porous component And in which (i) the surface of the pores of these, at least, porous components a and b are equipped with chemical components to interact with the substances passing through the pores, and (ii) the pores of the porous components have a uniform mnegative distribution of pore sizes throughout the polymer structure.

2. Porous self-sustaining structure under item 1, in which the structure contains a polymer obtained by polymerization of monomers having at least two polymerized components, or two types of monomers, and the first type of monomer has one curable component, and the second type of monomer cross links the polymer chain derived by the polymerization of the first monomer.

3. Porous self-sustaining structure on p. 1,service components, reactive groups for covalent binding of ligands, such as affinity ligands, preferably proteins, enzymes, antibodies, antigens, lectins, sugars, nucleic acids, cell organelles, or dyes, and the like.

4. Porous self-sustaining structure on p. 2, in which the monomers are vinyl monomers and monolinolein monomers.

5. Porous self-sustaining structure on p. 4, in which the group of polyvinyl monomers include divinylbenzene, Divinington, diphenylpyridine, alkylen dimethylacrylate, oxyalkylene dimethylacrylate, oxyalkylene diacrylate, oligoamine glycol diacrylate, vinyl polycarboxylic acid, divinyl ether, di-, tri - or Tetra-acrylate or acrylate of pentaerythritol, trimethylacetate or acrylate trimethyl-propane, alkylene bis-acrylamide or methacrylamide, and mixtures thereof.

6. Porous self-sustaining structure on p. 4, in which the group monovinyl monomers include styrene substituted in the nucleus of styrene, in which the substitution includes chloro-methyl, alkyl with up to 18 carbon atoms, hydroxyl, t-butyloxycarbonyl, halogen, nitro, amino groups, protected hydroxyl or amino group, vinylnaphthalene, acrylic is/or 5, in which polyvinyl monomer or polyvinyl monomer plus monomineralic monomer present in the polymerization mixture in an amount of from 20 to 60%.

8. Porous self-sustaining structure under item 1, in which the first component contains a tubular structure having an inner cylindrical tube (10) with an inner diameter (12) and outer diameter (11), and this cylindrical tube (10) is configured to accept the second component And having an inner cylindrical tube (20) with an outer diameter (21) and inner diameter (22), provided that the outer diameter (21) of the component And corresponds to the inner diameter (12) of the component, and component And inserted into the component Century.

9. Porous self-sustaining structure on p. 8, in which the inner cylindrical tube (20) the component is a collection of samples.

10. The product containing porous self-sustaining structure according to any one of the preceding paragraphs and the means for implementation of chromatographic processes.

11. The product under item 10, in which the product is a chromatographic unit (30), column or cartridge, or bioconversion reactor, or a matrix for the synthesis of peptide or oligonucleosides.

12. Sleepus (36) has, at least one inlet (41) and at least one outlet opening (40), the inner surface (42) and outer surface (43), and a pipe-like element or pipe-like elements (72) forming the dispenser samples (23) on its inner surface (42).

13. The product under item 12, in which a pipe-like element (72) is made in the form of helical grooves (25), beginning in the area of the inlet (41) of the chromatographic unit (30) and being in direct contact with him, and ending after at least one full revolution, but not in direct connection with the outlet (40) of the chromatographic unit (30).

14. Product according to any one of paragraphs.11-13, in which the chromatographic unit (30) further comprises a first end fitting (32) and the second end fitting (38) with 0-rings (33, 34, 35, 37) and addictive clutch (31, 39).

15. The article on p. 14, in which the second end fitting (38) has an upper part (52), the lower part (53) and the housing and the second end fitting (38) has an essentially cylindrical shape and includes an annular flange (51), which divides the terminal fitting (38) has a cylindrical shape into two parts, where part of the end fitting (38), located closer to the ring Bur connected to the hole (55), which is perpendicular to the stub Central hole (54), and the hole (55) begins in the annular groove (56) at the surface of the body of the second terminal fitting (38) and leads to a dead-end Central hole (54).

16. The article on p. 14, in which the first end fitting (32) has an upper part (62), the lower part (63) and the housing and the first end fitting (32) has an essentially cylindrical shape and includes an annular flange (61), which divides the terminal fitting (32) has a cylindrical shape into two parts (62, 63), where part of the end fitting (32), located closer to the ring flange (61), has a bottom portion (62) containing the connector (60) in connection with the Central hole (64) extending through the first end fitting (32), and 0-ring (35) located in the annular groove in the casing at the upper part (63) of the first end fitting (32), and 0-rings in the circular grooves in the upper part (63) of the first end fitting (32).

17. The housing (36) for use in the product under item 12 that includes a dispenser samples (23), in which a pipe-like element (72) is made in the form of helical grooves (25).

18. The first end fitting (32) or the second end fitting (38), made with COI is LII under item 10.

20. A method of manufacturing a porous self-sustaining patterns under item 1, comprising the steps of mixing moulinrouge and polyvinyl monomers together with Porogaramu and not necessarily the initiators of polymerization, optional deaeration, infusion of the mixture in the mould trooptube patterns, temperature control in the range from 40 to 90With, with the subsequent formation of the polymer, removing proginov not reacted monomers and initiators or by-products.

 

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