CZ214295A3 - Separation means - Google Patents

Abstract

Porous ceramic shaped bodies are used as a substance separating medium, in particular as a stationary phase for chromatography. Also disclosed are surface-modified porous ceramic shaped bodies, as well as chromatography columns and cartridges containing the porous ceramic shaped bodies as stationary phase.

Description

The invention relates to the use of porous ceramic shaped as a means for separation of substances, in particular as a stationary phase in chromatography and ^'first The invention further relates to ceramic fittings whose pore surfaces are fashioned.

BACKGROUND OF THE INVENTION

The separation of the substances according to the invention comprises essentially chromatographic separations, such as column chromatography, thin layer chromatography or gas chromatography, liquid-liquid extraction, adsorption and desorption processes involving gas or liquid phase, and electrophoretic separation. Distillation and mechanical separation, such as filtration, are not included under this term.

---------------- P-i - method ec h - column CO ^r p a ^l l o l o l o ^r o ^r o gmatom rar using using chromatographic tubes which are closed at both ends by filter elements and which are provided with connection pieces for supplying and discharging the eluents. Powder sorbents are filled into these tubes. Instead of chromatographic tubes in which all components have to be replaced, insert systems are also used where only tubes with filter elements containing powdered sorbents have to be replaced. The fitting is still usable. Such liner systems are disclosed in EP-B-0 268185 and EP-B-0 305817. Such column packings, consisting of powdered sorbents, undergo changes, for example, under mechanical stress, thereby reducing the reproducibility between chromatographic courses.

SUMMARY OF THE INVENTION It is an object of the invention to provide a stationary phase with a stable structure for separation of substances, in particular for chromatography, for example for thin layer chromatography and for column chromatography.

According to the invention, this object is achieved by using porous ceramic fittings for separating fabrics.

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According to the invention, the use of porous ceramic fittings for separating fabrics consists in that these fittings with a three-dimensionally interconnected pore system are not produced by shaping plastically defective and then reinforcing masses, but are formed in layers by a repeated sequence of operations.

- forming a layer of matter, the layer corresponding to a figuratively structured pore system,

- strengthening the layer, the image structures of the individual layers being transferred from the corresponding templates.

The subject of the invention is a porous ceramic fitting whose pore surfaces are treated and used for separating substances. According to a particularly preferred embodiment, the skirt surfaces of the fitting are, according to the invention, according to the invention. The invention further relates to a chromatographic column equipped with connection pieces for supplying and discharging an eluent comprising a porous ceramic fitting as a stationary phase.

The invention further relates to a liquid chromatography cartridge comprising as a stationary phase a porous ceramic fitting.

Fig. 1 shows a column according to the invention.

DE 42 05 969 describes a special application

1 * 1, * method for producing porous ceramic fittings, as well as the use of such fittings for chromatography: porous fittings with a three-layer interconnected pore system are produced by shaping plastically defensible and then reinforcing masses, whereby the fitting is formed in layers by repeated sequence of operations.

- forming a layer of matter, the layer corresponding to a figuratively structured pore system,

- strengthening the layer, and wherein the image structures of the individual layers are transferred i

- 3 of the corresponding templates.

However, it has been shown that porous ceramic fittings manufactured in other ways can also be used as a stationary phase in chromatography or other separation processes.

According to the invention, materials known to those skilled in the art can be used to produce ceramics that sinter at temperatures of 600 to 2000 ° C. These include oxidic and non-oxidic inorganic materials such as oxides, carbides, borides, nitrides, and mixtures thereof. Examples of such materials include, but are not limited to, phosphorous-containing compounds such as hydroxycarbonate and palladium. oxide:. silica, alumina, silicon carbide, SiOC, zirconia, titanium dioxide and bornitride. . ... _p .

Current chromatographic columns may exhibit a substantially monomodal pore size distribution when the particles used are sorted within a narrow range and are not porous; exhibit a substantially bimodal pore size distribution when, for example, the column fill consists of porous particles sorted within a narrow range.

Other pore size distributions are also known in current chromatographic columns. The porous fittings used according to the invention can accordingly exhibit a substantially monomodal or bimodal distribution in the pore bone. pore size separation. This also includes through-channel pores. Those skilled in the art are familiar with suitable methods of making porous ceramic fittings from materials of different kinds. Suitable starting materials and preparation variants can be found in manuals such as Ullman's Encyclopedia of Industrial Chemistry (1986), Chemie and Kingery WD, Bowen, HK-A, Uhlmann DR (1976) Introduction to Ceramics, John Wiley. Depending on the purpose, the porous ceramic fittings used according to the invention may have various geometric shapes, such as cylinders, prisms, blocks, cones, discs or plates. For example, cylindrical porous fittings, thin-layer chromatography of disc or plate-shaped fittings are preferred for use in column chromatography. ^;

In current chromatographic separation methods, time4 is used by chemically modified separation materials to achieve the most diverse selectivity for the various principles of chromatographic separation of substances:

adsorption chromatography

I reverse phase chromatography (RP) partition chromatography ‘gel-permeation · chromatography- hydrophobic interaction chromatography affinity chromatography ion exchange chromatography chromatographic separation of racemates and chiral carriers of selective adsorption and desorption.

Among other things, adsorption chromatography is also used in the preparation of samples as a special embodiment. In this case, sorbents, which consist essentially of inorganic carriers, for example silica or alumina, are modified on the surface to influence the separation ability of the material. In principle, the methods used to modify the chromatography of the carrier can be applied. in-d-r'C'r - '' tv'a'rO'v'e'k''d'd'l 'n'n invention; suitable methods of modification are known to those skilled in the art. In this sense, it is to be understood that the term " surface modified " Examples of such known surface modifications for said purpose are:

(a) derivatization ‘with silane derivatives of the general formula I

SiX ¹¹ R ¹ (3-n) R ² (I) where is' ^? X is methoxy, ethoxy or halogen, i

; R ¹ is alkyl having 1 to 5 carbon atoms and n signifies 1,2 or 3,

R ² radical from the group consisting of:

a1) unsubstituted or substituted alkyl or aryl groups such as n-octadecyl, noctyl, benzyl or cyanopropyl groups; a2) anionic or acidic residues such as carboxypropyl; A4) hydrophilic residues, such as a (2,3-dihydroxypropyl) oxypropyl group, and 5) bonds capable of activated residues, such as a group (2). 3-epoxypropyl) oxypropyl;

(b) adsorption or chemical bonding of polymers such as polybutadiene, siloxanes, styrene / divinylbenzole-based polymers, acrylic acid (m'e t h) derivatives, and the fight against n-e-eh in i-nyl o-starting compounds such as peptides, proteins, polysaccharides and nasal polysaccharide derivatives; (C) chemical bonding of the polymers listed under (b) through the derivatives of (a). This includes the graft polymer derivates of (meth) acrylic acid derivatives of diol-modified diatomaceous earth according to EP-B 0 337144;

d) adsorption or chemical bonding of chiral phases such as amino acid derivatives, peptides or proteins, or cyclodextrins, polysaccharides or polysaccharide derivatives. ·

Other conventional ways and means for derivatization are pracovní'lt UM ^'7' V 'on it γϊΓ ζ nam'y ~ - and ~: jsOUvp ops-Y- in é-b-y-chloro-i -Pr, .ru waits ch., _.... like, _t U n -. ger, KK (ed.) Porous Silica, Elsevier Scientific Publishing Company (1979) or Unger, KK Packirigs and Statars; Phases in Chromatographic Techniques, Marcel Dekker (1990).

The fittings according to the invention have the dimensions usual in liquid chromatography: diameter 2 to 10 mm for analytical use and larger diameters (up to approximately 0 to 5 ml per ml). The length is a few millimeters to 50 cm, but in special cases columns up to 2 m in length are used. předsloupců that are often used only once, great savings. fittings of this invention are _in: as compared to current fillers sor.pčními fixed, unchanging sorbent bed. After use, * 'I can be used according to the invention fitting easily get rid of, as opposed nomical SES6 from columns and inserts essentially only of ceramic material.

When used as a carrier for thin-layer chromatography, the fittings are formed as thin layers which may have an additionally thick non-porous part or which may be deposited on a non-porous auxiliary carrier. It is not necessary to apply the sorption layer to the plate or film for the use of the fittings according to the invention in a chromastograph as well as in a thin layer.

Porosity is an essential property of the ceramic fittings used according to the invention. Porosity has significant effects on the flow rate on the surface effective for chromatographic separation and on the possibility of derivatizing the fitting surface. Porosity is given as the ratio of the volume pores to the total volume of the fitting. This ratio can be determined, for example, by determining the mean density of the fitting, if the density of the armament material is known. Other methods for determining porosity are based on mass determination and saturation of the fitting with water or porosometry. The extent of porosity depends on the cutting method used and the dimensions of the fitting. For column liquid chromatography, a porosity degree of 20-75%, in particular 50-65%, is preferred. The surface of the porous fittings used according to the invention is typically 1 to 1000 m ² / g.

*

The column according to the invention shown in FIG. 1, for example, consists of a porous ceramic fitting 1 serving as a sorption bed, a liquid-impermeable Teflon casing 2, a pressure casing 3 with terminal union nuts 4 with connecting pieces 5. A filter 6, for example a Teflon strainer, may be inserted between the fitting and the connecting piece. In order to relieve the liquid-impermeable Teflon casing 2 from the mean elution pressure, the connection sleeve 7 in the pressure casing 3 can be pushed into the gap 8 between the liquid impermeable casing 2 and the pressure casing 2, the liquid transferring using the pressure is generally higher than or equal to the mean elution pressure. Hydraulic oil, aqueous solution or eluent may serve as the pressure transfer fluid.

DETAILED DESCRIPTION OF THE INVENTION

A Processing and derivatization of fittings

Example Al i *

Processing of alumina fittings

A commercially available 4 mm diameter and 125 mm length alumina (20-30% porosity degree) fitting was placed in a mixture of 125 ml of acetonitrile and 125 ml of sodium carbonate. 25mM and sonicated for 45 minutes in a laboratory ultrasonic bath.

It was then sonicated once more in pure acetonitrile (15 minutes).

Example A2

Processing of fitting made of silica

A commercially available fitting of 4 mm diameter and 125 mm length (50% porosity degree) is placed in a graduated cylinder filled with 25% hydrochloric acid and left there for 4 ' It is washed with methanol / water mixtures.

Example A3 ....... '...... -

Modification of porous ceramic fitting

Commercially available fitting of 4 mm diameter and length. 125 mm (s-degree-por.oz.i. even those by volume _5_0%) is chemically deri-?

vatovat methyloctadecyldichlorsi by rope in situ in a way that: described by Gilpin et al. (Anal.Chem. 46, 1314, 1974). To this end, a solution (10% w / w) of silane in toluene is pumped through the fitting. Finally, it is washed in purified toluene and conditioned with acetonitrile and acetone / tri-water (50:50 by volume) until a constant baseline is obtained. .

Example A4

Production of alumina fittings, suitable for pre-columns

The shaped pieces produced according to Example A1 are divided into short (4 mm) pieces that fit into the pre-column holder.

Example · A5

Production of porous, chemically modified ceramic fittings

The fitting manufactured according to Example A2 is chemically modified according to Example A3.

B Chromatographic columns and inserts

Example B1

Dividing column with porous ceramic fitting

The fitting of Example A1 is inserted into the holder of FIG. 1 so that the inlet and outlet lines for the eluent can be attached to the ends of the cylinder and the cylinder housing is closed tightly against the solvent. This column is connected to a conventional HPLC apparatus.

Example B2

Dividing column made of derivatized porous ceramic fitting

The fitting of Example A3 is inserted into the holder of FIG. 1 so that the inlet and outlet lines for the eluent can be attached to the ends of the cylinder and the cylinder housing is closed tightly against the solvent. This column is attached to a conventional HPLC apparatus.

Example B3

Partition column made of chemically modified porous ceramic fitting

The fitting of Example A5 is placed in a holder such that the inlet and outlet lines for the eluent can be attached to the ends of the cylinder and the cylinder housing is closed tightly to the solvent. This column is connected to a conventional HPLC apparatus.

Example B4

Dividing column with porous ceramic fitting

Commercially available porous zirconia fitting with a diameter of 4 mm and a length of 1-2-5 ··-m® ((s.tu.pe.fí_ .. porosity_2-0 to 30%)) 1, so that the inlet and outlet lines for the eluent can be fixed on the ends of the cylinder and the cylinder housing is closed tightly against the solvent. This column is connected to a conventional HPLC apparatus.

C Application examples

Example C1

Separation of isomeric nitroanilides 5 Add 5 μΐ of the mixture to the separation column according to example B1

2-nitroacetanilide (88 μδ / πιΐ) and 3-nitroacetanilide (545 (xg / ml) in n-Heptane / dioxane (80:20 v / v) as eluent. 0.5 ml / min and detected by ultraviolet absorption at 254 nm. Both substances were separated, eluting after 22 minutes (2-nitroacetanilide) and after 51 minutes (3-nitroacetanilide).

Example C2

Classification of aromatics

10 μΐ of a mixture of naphthaline (17 μ§ / πι1), anthracene (3 µg / ml) and benzatracene (600 μξ / ml) in acetonitrile / water (50:50 v / v) was applied to the separation column of Example B2. Elute at 1 ml / min and detect by ultraviolet absorption at 254 nm. All three substances are separated:

Retention times in minutes:

naphthalene 1 _; , 6 anthracene 3, , 1 bezantracen θ, , 8

Example C3

Classification of methylated anilines

A 10 μΐ mixture of aniline, N-methylaniline and N, N-dimethylaniline in acetonitrile / water (75:25 by volume) was applied to the separation column of Example B2. It is eluted at a flow rate of 0.8 ml / min and is detected by measuring ultraviolet absorption at 254 nm. All three substances are separated:

Retention time in minutes: aniline 13.2 1.05

N-methylaniline 8.6 1.74

N, N-dime thylaniline 12.9 4.90

Example C4

Classification of alkylated anilines

The separating column according to Example B2 is applied

N, N-dimethylaniline and N, N-diethylaniline and eluting according to Example C3. Both substances eluted separately.

μΐ mixtures under conditions

Introduced in μ§ / ιη1 Ν, Ν-dimethylanine 5.8

N, N-diethylaniline 21.3

Min retention time: 1.22 8.13

Example C5

Separation of phthalic acid esters

10 μΐ of a mixture of benzylbutyl phthalate and dinonyl phthalate is applied to the separation column of Example B2 and eluted under the conditions of Example C3. Both substances eluted separately.

Inserted in gg / ml Retention time in minutes: Benzylbutyl phthalate 130 1.03

Dinonyl phthalate 410 5.66

Example C6

Protein splitting The column was ee according to example B2.J5JS, Y, iese 1.0% protein mixtures consisting of trypsin, ribonuclease A, cytochrome C, BSA, and ovalbumin dissolved in 0.1X trifluoroacetic acid. Elute under the following conditions: flow rate 0.8 ml / min, detection

UV absorption at 280 nm, eluent: A: water + 0.2X trifluoroacetic acid and B: acetonitrile + 0.2% trifluoroacetic acid. Gradient A: 80 X + B: 20 X to A: 0 X + The five proteins are separated. B: 100 ALIGN! X in 10 minutes. Concentration in mg / ml Retention time in ‘minutes: Trypsin 5,5 0.70 R rddukreá ⁿ sa ~ A '---- 2-, 3 - - - ----- * - · -  .. 52 ^ _______ Cytochrome C 2.1 4,41 ' BSA 3.2 ''  .5.08 -. . Ovalbumin 6.9 5,52? Example C7 , ·. * Separation of isomeric nitoacetanides .....— .......... ‘ On the dividing, column according to example B4 Apply 10 μl of the mixture.

consisting of 2-nitroacetanilide (88 µg / ml) and 3-nitroacetanilide (545 µg / ml) in n-beptan / dioxane (99: 1 by volume). It is eluted at a flow rate of 0.2 ml / min and is detected by measuring UV absorption at

254 nm. Both substances were separated after 4.4 minutes (2-nitroacetanilide) and after 9.7 minutes (3-nitroacetanilide).

When using the porous ceramic fittings according to the invention in liner systems, no additional stainless steel column tubes are required, only reusable holders are required. Since the fittings consist of a uniform material, the disposal of spent inserts is greatly simplified. The rigid, fixed incorporation of the sorbent guarantees good reproducibility between the various chromatography steps. .........

Without further elaboration, it will be appreciated by those skilled in the art that the present disclosure can be utilized to a large extent. Preferred embodiments are given merely as illustrative, non-limiting examples.

The full disclosure of all of the provisional and subsequent patent applications, patents and publications is incorporated herein by reference.

Technical usability

Use of porous ceramic shaped pieces with modified pore surface as a means for separating substances, especially as a sufficient phase in chromatography. ¹ ο · C71

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PATENTS

Claims (8)

Claims The use of porous ceramic fittings for separating fabrics, wherein the fittings with a three-dimensionally interconnected pore system are not produced by molding a plastically defectable and then refillable mass, characterized in that the fitting is formed in layers by a repetitive sequence of operations. - forming a layer of matter, wherein the layer corresponds to an image-structured pore system, in the form of a layer, wherein the image structures of the individual layers are transferred from the corresponding of 'originals'. ‘-.............. ...... ...... 2. Use of porous ceramic fittings for separating fabrics, characterized in that fittings whose pore surfaces are modified are used. Use according to claim 1 or 2, characterized in that the substances are separated by chromatography. 4 '.' Pore-free kerosene-like-y-z-y-y-y-y-y-y-y-y-y-y-y-y-y-y-y-y-y-y-y-yy ί, ο ,,. _ί: .__ T s__e_ in that the pore surfaces are modified. 5. A porous ceramic fitting as claimed in claim 4, having the shape of a cylindrical or elliptical cross-section or a prism. J i c i réz. Porous ceramic fitting according to claim 5, characterized in that the surface or surfaces of the combs are a non-porous ceramic fitting according to claim 4, characterized in that it has the shape of a flat disc or plate. 8. Chromatographic column equipped with connection pieces for elution inlet and outlet. and characterized in that the stationary phase is a ceramic porous fitting. 4 · 9. A liquid chromatography cartridge, characterized in that the stationary phase is a ceramic porous fitting. 10 '. A method for separating the mixture by column chromatography, characterized in that a column according to claim 6 is used.