CZ286859B6 - Stationary phase for chromatography - Google Patents

Abstract

In the present invention there is disclosed a porous ceramic shaped piece consisting of compounds containing calcium phosphate, such as hydroxyapatite, further silicon dioxide, aluminium oxide, silicon carbide, silicon carbonyl, zirconium oxide, titanium dioxide and boron nitride, whose pore areas are modified by silane derivative of the general formula I or by siloxane {SiXinRe1 i(3-a)Re2}, wherein X represents a methoxy group, an ethoxy group or a halogen atom, Re1 represents an alkyl group containing 1 to 5 carbon atoms, a is the number 1, 2 or 3, Re2 represents the group being selected from a group comprising (a) a non-substituted or substituted alkyl or aryl group, such as a-octadecyl, a-octyl, benzyl or cyanopropyl group, (b) anionic or acid radicals such as carboxypropyl group, (c) cationic or basic radicals such as aminopropyl, diethylaminopropyl or triethylammoniumpropyl group, (d) hydrophilic radicals such as (2,3-dihydroxypropyl)oxypropyl group, (e) activated radicals capable to form bonds such as (2,3-epoxypropyl)-oxypropyl group.

Description

Stationary phase for chromatography

Technical field

The invention relates to a stationary phase in chromatography.

BACKGROUND OF THE INVENTION

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

Column liquid chromatography methods employ chromatographic tubes which are closed at both ends by filter elements and which are provided with connection pieces for supplying and withdrawing 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 described in EP-B-0 268185 and EP-B-0 305817. Such column packings consisting of powdered sorbents, for example, undergo changes under the influence of mechanical stress, thereby reducing the reproducibility between chromatographic courses.

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

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

SUMMARY OF THE INVENTION

According to the invention, the use of porous ceramic fittings for separating substances consists in the fact that these fittings with a three-dimensionally interconnected pore system are not produced by shaping plastically deformable and then reinforcing masses, but instead the layers 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 skin surfaces of the fitting according to the invention are non-porous and are sealed.

The invention furthermore relates to a chromatographic column equipped with connection pieces for supplying and withdrawing the eluent, which comprises a porous ceramic fitting as a stationary phase.

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

-1 CZ 286859 B6

Fig. 1 shows a chromatographic column according to the invention.

DE 42 05 969 describes a special process for the production of porous ceramic fittings as well as the use of such fittings for chromatography: porous fittings with a three-dimensional interconnected pore system are produced by shaping plastically deformable and then reinforcing materials, forming the fitting in layers repetitive 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 from corresponding templates.

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

According to the invention, materials known to those skilled in the art can be used to produce ceramics which 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, in particular, calcium phosphate containing compounds such as hydroxylapatite, silica, alumina, silicon carbide, SiOC, zirconia, titanium dioxide and bomitride.

Current chromatography 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 chromatography columns. The porous fittings used according to the invention may accordingly have a substantially monomodal or bimodal pore size distribution. Other pore size distributions may also be used according to the invention. This also includes passage 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 the Ullman Encyclopedia of Industral Chemistry (1986), Chemie, and Kingey W. D., Bowen, H.K. and Uhlmann D.R. (1976) Introduction to Ceramics, John Wiley. Depending on the purpose of use, 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 fittings are preferred for use in column chromatography.

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

adsorption chromatography 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.

-2GB 286859 B6

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 chromatographic support materials can also be applied to the fittings of the invention; suitable methods of modification are known to those skilled in the art. In this sense, it should be understood whether the term 'surface modified' is used. Examples of such known surface modifications for said purpose are:

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

SiXnR -njR ^ ² (I) wherein

X is methoxy, ethoxy or halogen,

R ^{1 is} C 1 -C 5 alkyl, n is 1, 2 or 3,

R ² radical from the group consisting of:

a1) unsubstituted or substituted alkyl or aryl groups such as n-octadecyl, n-octyl, benzyl or cyanopropyl; a2) anionic or acidic residues such as carboxypropyl; and3) cationic or basic residues such as aminopropyl; diethylaminopropyl or triethylammoniumpropyl, a4) hydrophilic residues such as the (2,3-dighydroxypropyl) oxypropyl group, and 5) activated-capable linkages such as the (2,3-epoxypropyl) oxypropyl group;

(b) adsorption or chemical bonding of polymers such as polybutadiene, siloxanes, styrene / divinylbenzole-based polymers, (meth) acrylic acid derivatives, or other vinyl compounds, as well as peptides, proteins, polysaccharides and carrier polysaccharide derivatives;

(c) chemical bonding of the polymers referred to in paragraph (b) through the derivatives referred to in paragraph (a). This includes graft polymerizates of poly (meth) acrylic acid derivatives on 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 possibilities and methods of derivatization are known to those skilled in the art and are described in conventional manuals, such as Unger, K.K. (ed.) Porous Silica, Elsevier Scientific Publishint Company (1979) or Unger, K.K. (Packing and Stationary Phases in Chromatographic Techniques, Marcel Dekker (1990)).

Fittings according to the invention have the dimensions usual in column chromatography: diameter 2 to 10 mm for analytical use and larger diameters (up to about 0.5 m) for preparative

-3GB 286859 B6 use; the length is several millimeters to 50 cm, but in special cases columns up to 2 m in length are used. The fittings according to the invention are suitable both as separating columns and as pre-columns. In this case, the filling of the columns or inserts is eliminated, which results in a large saving, in particular in the case of pre-columns. The fittings according to the invention represent a solid, stable sorbent bed compared to the current sorption fillers. After use, the shaped pieces used according to the invention can be easily disposed of since they consist essentially of ceramic material, unlike columns and inserts.

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. For the use of the fittings according to the invention in thin-layer chromastography, the application of a sorption layer to a plate or film is eliminated.

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 surface of the fittings. Porosity is given as the ratio of the pore volume 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 type of separation 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 chromatography column according to the invention shown in FIG. 1 consists of a porous ceramic fitting 1 serving as a sorption bed, a liquid impermeable Teflon casing 2, a pressure casing 3 with end cap nuts 4 with connecting pieces 5. Between the fitting and the connecting piece a filter 6, for example a Teflon screen, is inserted. 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 3, the pressure transfer fluid being generally higher or same as mean elution pressure. Hydraulic oil, aqueous solution or eluent may serve as the pressure transfer fluid.

The invention is illustrated in more detail by the following examples and the accompanying drawings.

Overview of the drawings

Fig. 1 shows an exemplary chromatographic column according to the invention. 2, 3 and 4 show the chromatograms of the separation of the same mixture, FIG. 2 corresponds to the use of fittings according to the invention and FIGS. 3 and 4 to fittings according to EP 0 160 267. The x-axis always shows the retention time in minutes; intensity in mV.

-4GB 286859 B6

DETAILED DESCRIPTION OF THE INVENTION

A Processing and derivatization of fittings

Example A1

Processing of aluminum oxide fittings

A commercially available 4 mm diameter and 125 mm length alumina fitting (20-30% porosity degree) is placed in a mixture of 125 ml acetonitrile and 125 ml sodium hydroxide solution (25 mM) and is sonicated in a laboratory ultrasonic bath for 45 minutes. It was then sonicated again 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) was placed in a measuring cylinder filled with 25% hydrochloric acid and left there for 48 hours. It is then washed several times with methanol / water.

Example A3

Modification of porous ceramic fitting

A commercially available silica fitting of 4 mm diameter and 125 mm length (50% porosity degree) is chemically derivatized with methyloctadecyldichlorosilane in situ as described by Gilpin et al. (Anal. Chem. 46, p. 1314, 1974). To this end, a solution (10% w / w) of silane in toluene is pumped through the fitting. Finally, it is washed in pure toluene and conditioned with acetonitrile and acetonitrile-water (50:50 by volume) until a constant baseline is obtained.

Example A4

Production of alumina fittings, suitable for columns

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

-5GB 286859 B6

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 fixed on the ends 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 such that 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 B3

Partition column made of chemically modified porous ceramic fitting

The fittings of Example A5 are inserted into the 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 against the solvent. This column is connected to a conventional HPLC apparatus.

Example B4

Dividing column with porous ceramic fitting

A commercially available porous zirconia fitting having a diameter of 4 mm and a length of 125 mm (porosity degree 20 to 30%) 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; the cylinder housing is closed tightly against the solvent. This column is connected to a conventional HPLC apparatus.

C Application examples

Example C1

Isomeric nitroanilide separator

A 5 μΐ mixture of 2-nitroacetanilide (88 pg / ml) and 3-nitroacetanilide (545 pg / ml) in n-heptane / diosane eluent (80:20 v / v) was added to the separation column of Example B1. Elute at 0.05 ml / min and detect by ultraviolet absorption at 254 nm. Both substances were separated, eluting after 2 minutes (2-nitroacetanilide) and after 51 minutes (3-nitroacetanilide).

-6GB 286859 B6

Example C2

Classification of aromatics

A 10 μΐ mixture of naphthaline (17 pg / ml), anthracene (3 pg / ml) and benzatracene (600 pg / ml) was applied to the separating column of Example B2 in acetonitrile / water (50:50 by volume). 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 benzantracen 6.8

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) is 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 were separated.

Incorporated in pg / ml

Retention time in minutes aniline

N-methylaniline N, N-dimethylaniline

13.2

8.6

12.9

1.05

1.74

4.90

Example C4

Classification of alkylated anilines

A 10 μΐ mixture of Ν, dim-dimethylaniline and N, N-diethylaniline was added to the separation column of Example B2 and eluted under the conditions of Example C3. Both substances eluted separately.

Retention time in minutes

Incorporated in pg / ml

N, N-dimethylaniline 5.8 1,22 N, N-diethylaniline 21.3 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.

Incorporated in pg / ml Retention time in minutes Benzylbutylphthalate 130 1.03 Dinonyl phthalate 410 5.66

Example C6

Protein division

A 10 μΐ protein mixture consisting of trypsin, ribonuclease A, cytochrome C, BSA, and ovalbumin dissolved in 0.1% trifluoroacetic acid was added to the separation column of Example B2. Elution under the following conditions: flow rate 0.8 ml / min, detection of UV absorption at 280 nm, eluent: A: water + 0.2% trifluoroacetic acid and B: acetonitrile + 0.2% trifluoroacetic acid. Gradient A: 80% + B: 20% to A: 0% + B: 100% in 10 minutes. The five proteins are separated.

Retention time in minutes

Concentration in mg / ml

Trypsin 5.5 0.70 Ribonuclease A 2.3 3.52 Cytochrome C 2.1 4.41 BSA 3.2 5.08 Ovalbumin 6.9 5,52

Example C7

Classification of isomeric nitroacetanilides

10 μΐ of a mixture composed of 2-nitroacetanilide (88 pg / ml) and 3-nitroacetanilide (545 pg / ml) in n-heptane / dioxane (99: 1 by volume) was applied to the separation column of Example B4. It is eluted at a flow rate of 0.2 ml / min and is detected by measuring UV absorption at 254 nm. Both substances separated after 4.4 minutes (2-nitroacetanilide) and after 9.7 minutes (3-nitroacetanilide).

D Comparative tests

Example D1

Cutting on fittings according to the invention

Sorbent

The commercial fitting of silicon dioxide was modified with C? Silane according to Example A3 and inserted into the holder.

HPLC Chromatography Conditions:

eluent: acetonitrile / water (60/40 by volume) detection: 254 nm flow: 3.0 ml / min

-8EN 286859 B6

temperature: rooms injection volume: 5μ1 Sample: benzene 0.456 mg / ml toluene 0.338 mg / ml ethylbenzene 0.34 mg / ml propylbenzene 0.40 mg / ml butylbenzene 0.628 mg / ml pentylbenzene 0.256 mg / ml Results: the analyte elutes separately, i.e. the individual components are separated

Example D2

Cutting on fittings according to EP 0160267 (Comparative Example 1)

Sorbent

The commercial silica fitting is modified with polyethylene glycol to achieve a lipophilic character:

A 20% solution of PEG 1000 in methanol is pumped at a rate of 0.2 mL / min for 75 minutes (15 mL) via an HPLC pump through TR 2237 fittings (83-7.2 mm). The impregnated fittings are dried for six hours at 60 ° C and placed in a holder. The same alkylbenzene mixture was chromatographed as in Example D1.

HPLC Chromatography Conditions:

eluent: acetonitrile / water (60/40 by volume) detection: 254 nm flow: 1.0 ml / min temperature: rooms injection volume: 5 μΐ Sample: benzene 0.466 mg / ml toluene 0.338 mg / ml ethylenene 0.34 mg / ml propylbenzene 0.40 mg / ml butylbenzene 0.628 mg / ml pentylbenzene 0.256 mg / ml Results: all components elute together, that is, they do not separate.

Example D3

Cutting on fittings according to EP 0160267 (Comparative Example 2)

Sorbent

The cordierite according to EP 0160267 is modified with a C ₁₈ silane to obtain a lipophilic character according to Example A3 and placed in a holder.

-9EN 286859 B6

HPLC Chromatography Conditions:

eluent: acetonitrile / water (60/40 by volume) detection. 254 nm flow: 1.0ml / min temperature: room volume: 5 μΐ

Sample: benzene 0.456 mg / ml toluene 0.338 mg / ml ethylbenzene 0.34 mg / ml propylbenzene 0.40 mg / ml butylbenzene 0.628 mg / ml pentylbenzene 0.256 mg / ml

Results: all components eluted together, that is, they do not separate.

When using the porous ceramic fittings according to the invention in liner systems, no additional column tubes of glass or stainless steel are required, only reusable holders are required. Since the fittings consist of a uniform material, the disposal of spent inserts is greatly simplified. The solid, fixed incorporation of sorbents guarantees good reproducibility between different chromatographic columns.

Without further elaboration, it will be appreciated by those skilled in the art that the present disclosure can be utilized to a large extent.

Industrial applicability

The use of porous ceramic fittings with a modified pore surface as a substance separating agent, in particular as a stationary phase in chromatography.

Claims (4)

Stationary phase for chromatography, characterized in that it consists of a porous ceramic fitting comprising calcium phosphate containing compounds such as hydroxylapatite, silica, alumina, silicon carbide, silicon carbonyl, zirconium dioxide, titanium dioxide and a boronitride impregnated with a silane derivative of the formula I or a siloxane SiX _n R ' _{(3- n)} R ² (I) wherein is X is methoxy, ethoxy or halogen, R ^{1 is} C 1 -C 5 alkyl, n is 1, 2 or 3, -10GB 286859 B6 R ² radical from the group consisting of: (a) unsubstituted or substituted alkyl or aryl such as n-octadecyl, n-octyl, benzyl or cyanopropyl; b) anionic or acidic residues such as carboxypropyl, c) cationic or basic residues such as aminopropyl, diethylaminopropyl or triethylammoniumpropyl; d) hydrophilic residues such as (2,3-dihydroxypropyl) oxypropyl; e) bonds capable of activated residues such as (2,3-epoxypropyl) oxypropyl. Stationary phase for chromatography according to claim 1, characterized in that it is in the form of a cylinder with a circular or elliptical cross-section or a prism shape. Stationary phase for chromatography according to claim 2, characterized in that the surface or surfaces of the shells are non-porous. The stationary phase for chromatography according to claim 1, characterized in that it is in the form of a flat disc or plate.