DD247570A3 - VESICULAR DISTRIBUTION, FILLING AND SUPPLY MATERIAL - Google Patents

Abstract

The invention relates to a new type of particulate separation, filling and traeger material, which is particularly applicable in liquid chromatography, electrophoresis and filtration. The goal is the production and use of particulate separation, filling and carrier material, which is characterized by favorable mechanical properties and by a high intensity of Stoffaustauschvorgaenge. From this, the task is derived to develop a type of particulate separation, filling and Traegermaterial with new compared to the known porous solid particles new properties. According to the invention, the known porous solid particles, when used as separating, filling and carrier materials, are replaced by thin-walled hollow bodies (vesicles) with porous, potentially flexible and potentially hydrophilic solids hulls surrounding an interior space. Expediently, these hollow bodies are living, killed or additionally modified plant cells or microorganisms as single cells or in tissue combination.

Description

Field of application of the invention

The invention relates to a vesicular separating, filling and carrier material for substance separation, useful in liquid chromatography (distribution, adsorption, affinity, molecular sieve, ion exchange chromatography), centrifugation, ultrafiltration, electrophoresis and electrofocussing.

Characteristic of the known technical solutions j

Particulate porous separating, filling and carrier materials for the stated fields of application use solid particles which adsorb certain components of the liquid phase surrounding them in their pores and on their surface (Hrapia, H .: Introduction to chromatography, Akademie-Verlag, Berlin 1977th

Porath, J., Flodin, P .: Nature (London) 183, 1657 [1959]). The separation properties of these materials are largely determined by the particle size, since they depend on the specific surface or the rate of diffusion-controlled mass transfer with the pore space (inner phase) of the particles. The use of very small particles is opposed in chromatography by a high flow resistance of the column fillings (Hrapia, H .: Introduction to chromatography, Akademie-Verlag, Berlin 1977.

Kaiser, R.E., Oelrich, E .: Optimization in HPLC. Hüthig-Verlag, Heidelberg, New York, Basel 1975.

Eppert, G .: Introduction to fast liquid chromatography. Akademie-Verlag, Berlin 1979).

A principal disadvantage of the known porous solid particles is that the framework-forming solid is continuous and acts as a diffusion barrier in the entire particle volume. The manipulation of the inner phase is limited by the adsorption properties of the pores.

Object of the invention

The aim of the invention is to provide a new ^ universally applicable particulate release, fill and support material.

Explanation of the essence of the invention

The object is to develop a new type of porous particulate release, fill and support materials which have the following advantages, singly or in combination:

High speed of diffusion-related equilibration,

Favorable packing properties (continuity, homogeneity, low flow resistance of a column filling),

Good manipulability and interchangeability of the inner liquid phase,

- possibility to dry,

Low solids content with the possibility of setting a variable density,

Possibility of immobilizing colloids or macromolecules,

- Properties such as ion exchange, permselectivity and adsorption, which can be exploited for the separation of solution components.

The object is achieved by using thin-walled hollow bodies (vesicles) with a closed surface formed from porous solid instead of the previously used porous solid particles. The invention is based on the fact that for the spatial fixation of phase boundaries in liquid thin, porous and possibly flexible sheaths are suitable, which surround an inner cavity (interior space) can penetrate into the specific components of the outer liquid. Liquid-filled hollow bodies (vesicles) formed by such shells can be used as a separating, filling and carrier material in a manner similar to numerous known porous particles which have hitherto been used, but are distinguished by specific features:

- They consist in the liquid saturated state for the most part of a uniform liquid phase and only a small part of swollen solid substance, which is predominantly sheath-forming. This allows the benefits formulated in the task.

The diffusion inhibition by the solid affects only a thin, porous and potentially permselective envelope.

- The filling of the interior is highly exchangeable and manipulatable and can be done by a non-external phase miscible liquid. Density differences to the outer liquid can be varied.

- Since relatively large particles can be used with high activity of mass transfer, the flow resistance of a packing of such vesicles can be kept low with good separation efficiency and reactivity.

Depending on the deformability of the vesicle sheath, mass flows of the liquid can be generated mechanically.

As a vesicular separating, filling and carrier material according to the invention are living, killed and additionally purified or modified plant cells or microorganisms (single cells, cell aggregates or tissue parts) suitable. These objects allow the use of the cell wall matrix to stabilize boundary layers between multiple phases (eg, mobile and stationary) as well as the receptor and reaction site. Their cell wall cladding has hydrophilic and permselective character as well as ion exchange properties. The interior can be used as a manipulable volume and, depending on the treatment, can be loaded with molecules and colloids. The swollen vesicles are sufficiently pressure stable in the column packing, although they have a flexible shell. This may be due to capillary forces or osmotic phenomena. The solution of the interior can be exchanged, pressed off or removed by evaporation.

embodiments

The invention will be explained below by way of examples.

Example 1:

Beta vulgaris L suspension cells were used as vesicular solid particles after being killed in 2% toluene solution and washed with distilled water. The schematic structure of the vesicles is shown in FIG. 1. They consist of the sleeve 1 (cell wall), filled with a liquid phase interior 2 and coagulated residues of the cell interior. 3

 Characteristic sizes:

Y ^ diameter diameter: 50 ... 100 / um

Thickness of the shell 1: <1μπη * "

Volume fraction of the interior 2: ca.70 ... 95%

Shape of the vesicles: approximately ellipsoid

In the example described, vesicle complexes (FIG. 2) were used. The size of the complexes was 0.5mm, their shape was irregular. Examples of the kinetics of the mass transfer to the vesicles are shown in FIG. 3 shows the polarimetric measurement of the absorption kinetics of melibiose 4, inulin 5 and extran 6 (MW 200000) after addition of 0.5 ml of vesicle suspension (volume fraction of the vesicles about 40%). Dextran does not diffuse into the interior of the vesicles (volume of the effective interior AV). However, the pore size of the shell allowed the penetration of melibiose and inulin with a half-life of <20s. To demonstrate the separation of non-electrolytes by molecular size (molecular sieve effect) by the described vesicles, a conventional glass liquid chromatography column was filled with vesicle complexes by sedimentation

 Column data:

Length of the column: 500 mm

Diameter of the column: 15 mm

mobile phase: H ₂ O

Volume speed: 0.87 m / min

spec. Permeability: 8.57-10 ^-3 ms ~ ¹ MPa ~ ¹

(corresponds to applied pressure difference of 5.5 kPa

above the column a volume flow of 1 ml / min)

The packing of the column is fast and homogeneous, the filling process is straightforward. The column was operated vertically, descending. For the separation of substances, 0.5 ml of a disaccharide-polysaccharide mixture (sucrose, dextran) were injected, using as a detector a commercial polarimeter (Perkim bucket). 4, the chromatogram parameters can be taken:

Gross retention time of the dextranpeak: 7:78 min

Gross retentate volumetric dextran peaks: 68 ml

Gross retort time of the sucrose peak 8: 116 min

Gross Retention Volume of sucrose peaks: 101ml

Selection coefficient a = ^T 2 / t, = 1.49

Peak solution R = ^ z ₁ Z ₂ = 1.72

SvmmetrieDarametera / b = 1,1

The vesicles used offer the advantage of an easily accessible, large stationary volume (interior) with relatively large particle and aggregate size, good separation properties and low solids content. Accordingly, a high flow rate of the mobile phase can be achieved with a low pressure gradient. The washability of the interior is complete.

Example 2:

As in Example 1 used porous Vesilkomplexen of Beta vulgaris L. and Chenopodium album L were dried by hot air treatment. They shrank due to their flexible shell to about ¹ A of the initial volume. Renewed addition of an aqueous solution causes swelling to the initial volume while maintaining the permselective properties of the shell. According to the principle of ultrafiltration, substances that can not permeate through the envelope of the vesicles (eg Dextran 250000) are concentrated.

The concentration increase of the dextran can be registered on the registering polarimeter (Perkim-Elmer polarimeter 141 M). The relative increase in concentration

corresponds to the volume fraction of the liquid absorbed in the vesicle interior

Example 3

The cell wall envelopes of the killed and extracted plant cells used (suspension cultures of Beta vulgaris, Chenopdium album and Lycopersicon esculentum) are permeable to low molecular weight protein molecules such as myoglybine (17,000 D), trypsin (21,000 D) and chymotrypsin (23,000 D). These proteins permeating into the interior are sharply separated from substances that can not diffuse into the vesicle interior, such as human serum album and dextran with molecular weights of 69,000 D and 250,000 D. Cell complexes of Chenopodium album z. For example, trypsin on a 12cm length column is completely separated from Dextran 250000, as is myoglobin from human serum albumin. The permeating proteins are eluted simultaneously with sugars and salts, without a peak broadening is observed. At the used elution rate (0.5 ml / min), therefore, the equilibration by diffusion of the proteins into the interior is not yet limiting for the quality of the chromatographic separation. In contrast to the gel permeation on Sephadex, two alternative possibilities for the diffusion-related distribution occur on the examined vesicular material, as already shown in Example 1. The porous cell wall has such a small proportion of the total volume of the packing that the amount of substance dissolved in its pores can be neglected relative to the quantities distributed in the interiors and the outer mobile phase. Due to the small thickness of the Versikelhüllen (1-2 / tm), the equilibrium is done quickly even with relatively large vesicle complexes, whereby good separation results can be achieved with low hydrodynamic resistance and high flow rate. The carrier material produced from plant cells is therefore suitable for separating proteins of two molecular size classes from one another. The distribution spaces of various substances are not gradually dependent on the molecular size as in porous gel particles of the Sephadex type. Gradual separation by molecular size can be achieved by preparing and mixing vesicles with gradually different molecular sieve properties of the shells.

Example 4

The material prepared from plant suspension cultures can be subjected to the following treatments without changing the ultrafiltration properties of the cell walls and the separation efficiency (cf. Examples 1 and 3): 5% acetic acid at 100 ^° C. (1 h), extraction in soxhlet -Apparate with acetone or chloroform / ethanol in the ratio 4/1, treatment with a solution of trypsin (0.5%), MgCl ₂ (5 mM), Na ₂ HPO ₄ (2%, pH 8.4) and NaN ₃ (2OmM) dissolved in water at room temperature or 37 ° C, washing with heated solutions of detergents in distilled water (about 50 ⁰ C) distilled water and ethanol, dry basis, of 96% ethanol, acetone or chloroform / ethanol mixtures. If the water was sufficiently extracted from the material with organic solvents, the cell complexes do not agglomerate on drying and form a fine-grained powder which swells rapidly in aqueous solutions or ethanol and provides a usable suspension for column chromatography. By the cleaning methods described above visible Protoplasmareste visible by light microscopy can be almost completely removed. The trypsin treatment greatly reduces the light scattering of the vesicle pack, and then the material is glassy. Already after extraction with hot acetic acid and Na ₂ HPO ₄ solution, the material is solvent-resistant in the pH range of 2-8 and gives no more detectable amounts of UV-absorbing or optically active substances. The repeated use was tested on the example of the separation of human serum albumin and fructose on a column packing of purified cell complexes of Beta vulgaris (2.5 × 8 cm). In 10 repeated separations, selectivity and peak width did not change significantly. After use, the material can be dried from 96% alcohol without degrading its performance characteristics. The material is autoclavable after swelling in distilled water.

Example 5

Since molecular disperse solutes penetrate into vesicles made from plant cells up to a molecular weight of about 30000 D, but human serum albumin (69000D) is already excluded from diffusion through the versicle wall, there are numerous possibilities for the immobilization of macromolecules and colloids. Particles which due to their size are excluded from diffusion through the vesicle wall can be formed in the vesicle interior from permeable building blocks and at the same time prevented from leaving the vesicles. Liposomes arise in the interior z. B. in that the prepared cell complexes are saturated with a solution of lecithin in ethanol and chloroform, then transferred to acetone and then into an aqueous buffer solution.

-4- Z47 & 7Ü

Example 6

The vesicular material can be filled with hydrophobic organic liquids. Killed and purified cells of Chenopodium album and Beta vulgaris are initially saturated with ethanol and then with various hydrophobic liquids (previously used: n-butanol, isobutanol, chloroform, carbon tetrachloride, mixtures of n-butanol or isobutanol with chloroform). If they are then transferred into the saturated with the corresponding organic liquid aqueous buffer solution, the cells swell significantly, leaving in the interior of an organic phase in the form of a spherical liquid droplet. If the buffer solution used is not saturated with the organic phase, the droplets dissolve or reduce until the saturation of the aqueous phase is reached. Complete dissolution of the organic droplets outside the vesicles is achieved by vigorous stirring of the vesicle suspension. In the clear solution, saturated with the organic phase, the Vesil complexes can be conveniently filled in columns. The hydrophobic droplets formed in the prepared plant cells remain stable because their union is prevented by the water-swollen fine-pored vesicle sheath. In this way, based on the vesicle wall, a stationary liquid hydrophobic phase is formed in the form of small droplets in a mobile aqueous phase. The vesicular material in this form enables a new type of liquid-liquid chromatography with numerous degrees of freedom for the targeted adaptation to the separation task. The following interactions can be used individually or in combination: distribution between the hydrophobic liquid droplets and the mobile aqueous phase, adsorption on the droplet surface, adsorption on the vesicle wall, exclusion of macromolecules from the diffusion into the vesicle interior and the interaction with the there hydrophobic phase. All these interactions could be made effective in model experiments for the chromatographic separation. The organic phases used were: chloroform / n-butanol and chloroform / isobutanol in the ratio 1/1. Extracted cell complexes from the suspension culture of Beta vulgaris and Chenopodium album were used as support phase. Well proven have column dimensions of 3.5 x 8cm and elution rates of 1-3 ml / min, which are achieved with low pressure differences (0.1-0.2 kPa). The sulfophthaleins bromothymol blue, bromophenol blue, and phenol red served to demonstrate the chromatographic interaction of acidic organic compounds with the entrapped hydrophobic droplets. At pH4, the indicators used are largely in the acid form and are expected to be concentrated in the droplets at the point of application and eluted very slowly. At pH 7, the distribution balance of the indicators used is on the aqueous phase side when shaken out in the chloroform / butanol / water system. Nevertheless, especially bromothymol blue and bromophenol blue are eluted at the separation columns from vesil complexes filled with chloroform / butanol with great delay, since the anions occurring at pH 7 are amphiphilic and are adsorbed on the surface of the immobilized droplets. At pH7, the dyes are eluted one after the other in a well reproducible separation in the order phenol red - bromophenol blue - bromothymol blue and separated completely in sharp bands over a distance of 4 cm. The good packing properties of the material prepared from plant suspension cultures are evidenced by the dye zones. The chloroform / butanol-filled cells were also shown to separate saccarose and dextran, adsorb denatured human serum albumin to the vesicle wall (see Examples 1 and 10).

Example 7

Commercially available baker's yeast (Saccharamyces cerevisiae) was subjected to the following treatment steps:

1. washing with distilled water,

2. Extraction mit3N NaOH (100 ⁰ C, 5 h),

3. Extraction with 0.5N acetic acid (100 ⁰ C, 1 h),

4. washing with ethanol,

5. Extraction with ethanol / chloroform in the ratio 4/1 in the Soxhlet apparatus,

6. Drying in the air. The result is vesicular material as a white powder with the following properties:

- Size of single vesicles in the swollen state (in H ₂ O): 2-6cm.

- Nitrogen content less than 0.3%.

- Closed, solvent-resistant cell wall shells of extensively purified yeast glucan (predominantly / 3-1,3-glucan), which are impermeable to polymeric materials with molecular weights above 6000D.

In contrast to sugars that diffuse into the interior without any measurable delay, inulin (6000D), dextrans and human serum albumin are excluded from the interior and concentrate when swelling the dried vesicles in the exterior solution. In addition to the vesicles purified in the manner described, a strongly adsorbing material can be prepared from baker's yeast if the yeast protein is extracted only incompletely (shorter extraction time in NaOH or extraction with hot 1 NH ₂ SO ₄ (100 ^° C., 3 h). The material is also present as a white powder after drying from ethanol or chloroform / ethanol, but contains larger amounts (up to 30%) of denatured protein in the interior, it buffers strongly in a wide pH range and adsorbs solutes according to the affinity properties However, human serum albumin is not adsorbed, as well as inulin or dextran, as these substances do not permeate through the vesicle wall, therefore a possible use is the purification of polymeric substances from adsorptive low molecular weight admixtures Normal pressure operation However, it is good as a polar support material for column chromatography with hydrophobic eluents (see. also example 8). With purified yeast vesicles as the stationary phase and a mobile phase of n-butanol / acetic acid / water in a ratio of 7/1/1, D-fructose and raffinose were separated in the model experiment. Column parameters: 6 χ 1.6 cm, running speed: 0.5 ml / min, applied amount of substance: 6 mg of each sugar in 0.3 ml. Elution volume of fructose peak: 38 ml, elution volume of raffinose peak: 164 ml, peak solution R 2 ; 3. Detector: Perkin Elmer Polarimeter141M.

Example 8

When dried vesicles are suspended after their preparation from vegetable suspension cultures (Example 4) or baker's yeast (Example 7) in toluene, butanol or another hydrophobic liquid, they swell far less than when suspended in water. If one adds to such a suspension water, the proportion of aqueous

-- * *

Droplet takes place. First, the porous vesicle walls fill, then the interior spaces with the aqueous phase. Only when the vesicle interiors are filled to the maximum, it comes with further addition of the aqueous phase for segregation in the free liquid and at the same time to a greater aggregation of the vesicles. In this way, biogenic vesicles can be prepared in a variable manner for a partition chromatography in which a stationary hydrophilic and a mobile hydrophobic phase cooperate.

Example 9

Vesicular separating, filling and carrier material can be prepared from vegetable parenchyma tissues by suitable methods of comminution (enzymatic or chemical maceration, mechanical processes). Cell walls of beetroot (Beta vulgaris) parenchyma tissues extracted with hot water and ethanol are comparable in ultrafilter properties to those of cell complexes from suspension culture (see Examples 2 and 4). After complete extraction of the sugar, betanine and other soluble constituents, red-bellied parenchyma slices are dried from ethanol and swelled to their original size in aqueous solution. Here, human serum albumin and extrane (M = 250,000) are excluded from the cell interiors and concentrate in the outer fluid volume by the theoretically calculated from the amount of liquid absorbed amount. For example, a suitable material for chromatography is obtained by pressing extracted parenchyma slices through a fine sieve in hot water. With this material, a separation of human serum albumin and dextran from the sugars can be demonstrated, as described in Example 1. However, because of the still high proportion of destroyed cells and the necessary coarseness of the material used in the crushing process, a significantly longer separation distance (about 40 cm) is required for a complete separation of the high molecular weight from the low molecular weight fraction. The mechanical properties of the parenchymatic material are very stable in aqueous solution and allow comfortable working with low pressure gradients. The hydraulic conductivity of a column packing of prepared parenchymal particles of about 1 mm in diameter is about 4mm ² s ~ ¹ kPa ~ ¹ .

Example 10

Filter layers of biogenic vesicles are suitable for removing various substances from solutions. Biogenic vesicles from plant cells, for example, adsorb denatured proteins in weakly acidic medium. Turbidities arising from heating or acidification of human serum albumin solutions are completely removed by filtering through thin layers (about 2 cm) of the parenchymal material (see Example 9) or the purified and prepared Chenopodium cells (Example 4).

Claims (7)

Invention claim: 1. Vesicular separation, filling and support material for the absorptive, chromatographic and electrophoretical ^ separation, characterized in that it consists of hollow bodies (vesicles), which have a porous, thin shell and that it is in the solvent-saturated state for larger volume fraction from a liquid phase filling the interior and to a lesser extent from the swollen solid forming the sleeve. 2. Vesicular separating, filling and carrier material according to item 1, characterized in that the hollow bodies consist of living, killed or additionally modified plant cells or microorganisms. 3. Vesicular separating, filling and carrier material according to item 1 or 2, characterized in that they are individual separate vesicles or vesicle aggregates. 4. Vesicular separating, filling and carrier material according to item 1 to 3, characterized in that the interior space is filled with a liquid phase which does not mix with the outer phase. 5. Vesicular separating, filling and support material according to item 1 to 3, characterized in that the porous shell contains a liquid which can not be mixed with the liquid phases of the interior and the exterior space. 6. Vesicular separating, filling and carrier material according to item 2 to 5, characterized in that the interior of reactive molecules or colloids are included. 7. Vesicular separating, filling and carrier material according to one or more of the items 1 to 6, characterized in that the hollow bodies have a flexible shell.