EP1155315A1

EP1155315A1 - Utilization of supporting material in capillary electrochromatography

Info

Publication number: EP1155315A1
Application number: EP00909222A
Authority: EP
Inventors: Angelika Muscate-Magnussen
Original assignee: Evotec Biosystems GmbH
Current assignee: Evotec OAI AG
Priority date: 1999-02-22
Filing date: 2000-02-21
Publication date: 2001-11-21
Also published as: AU3157800A; WO2000050887A1; US20090032400A1; US20020046966A1

Abstract

The invention relates to the utilization of supporting material for capillary electrochromatography (CEC) which is characterized in that the supporting material is porous and the surface is composed of an outer surface and of a porous surface, whereby the outer surface has areas of varying derivatization and/or functionality which can be differentiated from the porous surface.

Description

Use of carrier material in capillary electrochromatography

The invention relates to the use of carrier materials in capillary electrochromatography (CEC).

In general, analytical methods are selective at best; however, few, if any, are really specific. Consequently, the separation of the analyte from interfering accompanying substances is essential in the context of an analysis.

In chromatographic separations, the sample is dissolved in a mobile phase, which is e.g. can be a gas, a liquid or a supercritical fluid. The mobile phase is moved by an immiscible stationary phase, which e.g. located in a column or fixed to a solid surface. The two phases are chosen so that the sample components are distributed to different degrees between the mobile and stationary phases. The components that are strongly retained by the stationary phase only move slowly with the mobile phase. In contrast, the components that are only slightly retained by the stationary phase move quickly. Because of these differences, the sample components separate into discrete bands.

A chromatography concept that combines the advantages of capillary liquid chromatography (eg HPLC) and capillary electrophoresis (CE) is the so-called capillary electrochromatography (CEC). Essentially, CEC can be viewed as a hybrid of HPLC and CE (Colon et al., Analytical Chemistry News & Features 1995; August 1, 461A-467A). As in HPLC, the components of a sample are separated by different distribution between a stationary and a mobile phase. In addition, however, as in the CE, an electroosmotic flow is generated by applying an electrical voltage. The separations can be carried out isocratically or with a gradient. The columns are preferably filled with silica gel particles, which typically have particle diameters in the range from 1 to 5 μm.

The advantage of this method is the possibility to separate anionic, cationic and neutral molecules. However, a major problem is the analysis of complex, especially biological, samples. These, e.g. Hemolyzed blood, plasma, serum, milk, saliva, spinal fluid, fermentation broth, urine, supernatants from cell culture, food and tissue homogenates or natural product extracts contain a large proportion of matrix components, such as proteins and salts, in addition to the analyte.

Proteins and other macromolecules are precipitated, for example, by high proportions of organic solvents in the mobile phase or unspecifically, irreversibly bound or denatured by residual silanol groups on the surface of a chromatographic support (JR Verart et al., J. Chromatography A 1999; 471-475). The proteins and other macromolecules block access to the pores when using a porous stationary phase and thus reduce the number of chromatographic adsorption centers. As a result of the reduced mass exchange between stationary and mobile phases, these processes lead to a loss of capacity and selectivity of the column. Non-specific adsorption also leads to fluctuations in the electroosmotic flow and to non-reproducible re retention times of the analytes. In all cases, the CEC column is severely damaged or rendered unusable. It is therefore necessary to remove these matrix components from the sample before CEC analysis.

This problem is all the more serious because it affects provisions that are carried out in large numbers: e.g. Metabolism studies, therapy control, determination of the body's own substances, quality control of food or also the high-throughput screening for potential pharmacological agents, especially using natural product extracts.

Common sample preparation methods include methods of dialysis, ultrafiltration, protein precipitation, liquid / liquid extraction, in particular methods of solid phase extraction e.g. Cartridge process or the use of guard columns, which are preferably filled with silica gel particles, the elution of the analyte preferably being carried out by liquid desorption (HPLC). The use of guard columns for the separation of analytes from protein or matrix-containing samples in HPLC is described, for example, by Rudolphi and Boos (LOGC 15 (9) 814-823, 1997).

However, the necessary sample pretreatment steps are often time, cost and labor intensive and lead to an increase in the volume of the sample due to the necessary transfer of the analyte to a separation column, which leads to a loss of selectivity and sensitivity of the separation process. In addition, sample volumes of at least 10 μl are required to carry out these sample pretreatment steps, which renders the use for sample preparation in high-throughput processes in which only nl to a few μl sample available, impossible. Pinkerton et al. (US Pat. No. 4,544,485) claim a carrier material for liquid chromatography based on silica or glass which enables proteins or macromolecules to be separated from a sample. The so-called Internal Surface Reverse Phase (ISRP) material is characterized by a hydrophilic outer surface and a hydrophobic inner or pore surface. In one modification, for example, glycine is bound to the outer particle surface. The pore surface is characterized by polypeptides bound via glycerol propyl, in particular tripeptides. These lead to limited access to the pores. Smaller target molecules (analytes) have access to the pores, while the large matrix molecules remain excluded.

Boos et al. (LC-GC 1997, 15, 602-611; LOGC 1996, 14, 554-560) describe a carrier material based on alkyl-diol-silica (ADS), which ensures quantitative separation of proteins and other macromolecular components. It is characterized by a surface that is inert to biomolecules and is coated with alkyl groups in the pores. The pore size allows small target molecules (analytes) access, while the large matrix molecules remain excluded. This material was specially developed for HPLC analysis.

LJ Glunz et al. (J. of Liquid Chromatography 1992, 15, 1361-1379) also developed a so-called restricted access material based on silica for HPLC, the functioning of which is based on a semipermeable membrane (SPS) on the particle surface. By covering the surface of the carrier material with polyethylene glycol or polyoxyethylene, a network is created that only small analytes can access the pores. Macromolecules are therefore unable to access the pores. The pore surface is hydrophobic Groups especially phenyl groups, C18, C8 and nitrile occupied.

ChromSper 5 Biomatrix (Chrompack), Hisep (Supleico) and Capcell Pak MF (Shiseido) are further restricted access carrier materials specially developed for HPLC based on porous materials, the outer surface of which has a different derivatization than the pore surface.

The methods of capillary electrochromatography and HPLC differ significantly from each other, particularly due to the electro-osmotic forces occurring in the CEC. Materials and conditions suitable for HPLC are therefore not easy to transfer to the CEC method (Colon et al., Analytical Chemistry & Features 1997; August 1).

It was therefore all the more surprising that the use of carrier material, which is characterized in that it is porous and whose surface is composed of an outer and a pore surface, the outer surface being distinguishable from the pore surface areas of different derivatization and / or functionality has, in capillary electrochromatography enables an essentially quantitative separation of the analyte from other sample components, in particular proteins and other macromolecular components (sample matrix) of the sample.

The term "derivatization" refers to a covalent or, in particular, adsorptive binding of molecules to the surface of the carrier material. This can be, for example, synthetic or natural polymers which, as a chemical diffusion barrier, prevent macromolecules of the sample matrix on the carrier material adsorb or denature. The term "functionalization" denotes in particular the properties of a particular area. Certain areas of the carrier material can be made hydrophobic, while other areas have hydrophilic properties. Such functionalization can be achieved by different derivatization of the areas. Different molecules (eg fatty acids in one area, alcohols in another area) can be used for this. However, it is also possible to achieve a different functionalization by varying the occupancy density of areas with the same molecules.

The use of the support material according to the invention in the CEC makes it possible to separate the analyte from other components of the sample without diluting it. In connection with isotachophoresis, it is possible in one embodiment to transfer the analyte to a separation column, in particular a further CEC column or a μ-HPLC column, without significantly increasing the volume. Sample volumes <10 μl can even be processed.

When using the carrier material according to the invention, the reproducibility with regard to the number of plates, retention time and dissolution of the column is retained even after repeated injection of complex samples, in particular samples containing serum and cell culture media.

In a further embodiment, the use of the carrier material according to the invention even allows the combined sample preparation and separation of complex samples on a single CEC column. This is in relation to separation performance, sensitivity, signal-to-noise ratio, selectivity, column life and cost of sample preparation and separation carried out on separate columns equal or even superior. For the first time, this enables the use of such a system in high-throughput processes, such as high-throughput screening for potential pharmacologically active substances.

It may be preferred to carry out the analytes separated by means of CEC, preferably fully automatically, for further analysis, in particular using fluorescence correlation spectroscopy, in which the interaction of the analyte with other molecules is detected in particular. This can e.g. are receptor-ligand interactions.

The use of the carrier material according to the invention is therefore characterized overall by the following properties:

There is the possibility of repeated direct injection of untreated samples, in particular biological samples on a CEC column,

The protein matrix is removed quantitatively,

The analyte can be concentrated at the top of the column and quantitatively separated and separated independently of the matrix,

• high separation performance, sensitivity, accuracy, very good signal-to-noise ratio,

• high degree of reproducibility with regard to the number of trays, retention time and resolution of the separation in the column,

• automatic operation possible,

• high number of analysis runs, continuous operation of the column,

• low cost per analysis. It is particularly advantageous if the outer and / or pore surface of the carrier material is derivatized and / or functionalized with hydrophobic and / or hydrophilic groups and / or ion exchange groups and / or affinity ligands and / or chiral groups. The carrier material can thus be designed individually with regard to its chemical and / or physical separation properties.

The functional groups listed below are particularly suitable:

Chiral phases with the following functional ligands are particularly suitable for separating enantiomer mixtures: cyclodextrin, amylose tris (3,5-dimethylphenyl-carbamate), bovine serum albumin, ristocetin, 1,2-diphenylethyldiamine and vancomycin.

A number of suitable carrier materials are available with an outer diameter of 5 μm, in particular ChromSper 5 Biomatrix (Chrompack), ISRP GFF II and SPS (Regis Technologies), Hisep (Supleico) and Capcell Pak MF (Shiseido).

In addition to materials such as ChromSper 5 Biomatrix (Chromompack), ISRP GFF II and SPS (Regis Technologies), Hisep (Supleico) and Capcell Pak MF (Shiseido), the person skilled in the art can use different methods for producing the stationary phases for the use according to the invention .

Inorganic materials e.g. silicate-containing, in particular porous, silicate-containing, materials or glass are used. These can be modified, for example as described in US Pat. No. 4,544,485, on their outer and / or pore surface with glycerol propyl. A large number of starting materials are also commercially available, such as, for example, Hypersil (Separations Group), Spherisorb (Phase Separations), Nucleosil (Macherey-Nagel Co.), Zorbax Sil (DuPont), Micropack Si (Varian Associates) or Baker Silica gel (Baker Chem. Co.).

Organic polymers or copolymers containing hydroxyl groups can also be used as starting material. Hydrophilic organic copolymers made of, for example, oligoethylene glycol, glycidyl methacrylate or pentaerythrol dimethacrylate are also suitable as the starting material. These can be functionalized by acrylamide derivatives of the formula CH = CH-CO-NHR, where R is, for example, a linear and / or branched-chain aliphatic sulfonic acid group and / or carboxylic acid group. Copolymers of glycidyl methacrylate and ethylene dimethacrylate, dihydroxypropyl methacrylate and ethylene dimethacrylate or of glycerol monomethacrylate and glycerol dimethacrylate are also particularly suitable.

A modification of the Stöber method (Christian Kaiser, dissertation, Johannes Gutenberg University, 1996, W. Stöber et al., J. Colloid Interface Sci. 26, 62, 1968 ) for the production of highly ordered mesoporous materials based on silica gel. The person skilled in the art can also use the method described in DE 195 30 031, for example. Alternatively, polydisperse silica gels, as described under US 3,489,516, US 3,656,901 or US 2,385,217, can also be produced. Particles in a size range of in particular less than 5 μm are strongly enriched by a screening process, which particles can then serve as the starting material for the carrier material according to the invention.

Depending on the ligands with which the base material is to be derivatized, it may be advantageous that the surface of the starting material is already covered with epoxy groups. Crosslinked polymers consisting of so-called copolymers such as glycidyl methacrylate and ethylene dimethacrylate already contain epoxy groups. However, it is also possible to introduce epoxy groups into starting materials by a procedure known to the person skilled in the art. This can be done in particular by reacting a starting material with 3-glycidoxypropylsilane, or by reacting a copolymer of, for example, dihydroxypropyl methacrylate and ethylenedimethacrylate with epichlorohydrin.

Alternative regulations for the production of ion exchange materials based on the reaction of oxirane rings have been described in DE 43 33 674 (WO 95/09 964) and in DE 43 33 821 (WO / 95/09695): the person skilled in the art can these procedures are also applied to starting materials that are derivatized with oxirane groups only on the pore surface.

Further synthesis approaches have been described by Pinkerton (EP 0 173 233). Diol-containing bases are activated with 1,1-carboxydiimidazole (Bethell et al., J. Biol. Chem. 254, 2572, 1979; J. Cjrom., 219, 361, 1981) and then with a tripeptide, in particular glycine. L-Aspartic Acid-L-Aspartic Acid or Glycine-L-Serine-L-

Implemented glutamic acid. In a further step, the peptide unit on the outer surface of the carrier is hydrolyzed with the aid of a peptidase, for example carboxypeptidase A (exopeptidase, which is a peptide bond at the carboxy terminus) (Williams et al., FEBS Letters, 54, 353-357, 1975). When using a pore diameter of 4 nm, for example, the enzyme cannot penetrate into the pores because it has a molecular weight of 34 daltons. The selection of a suitable enzyme depends on the nature of the peptide and the size of the pore diameter. Alternatively, a modification of the method described under US 4,694,092 can also be used for the synthesis. Starting materials are carrier materials that are coated with ion exchange groups such as carboxypropyl on the outer and pore surface. The functional groups on the outer surface are converted into silanol groups by a plasma treatment, while the ion exchange groups on the inner surfaces of the pores are retained. In a further step, the silanol groups are reacted with 3-glycidoxy-trimethylsilanes and the epoxy group is then hydrolyzed with dilute sulfuric acid in order to obtain a hydrophilic outer surface.

Alternatively, a modification of the manufacturing method described under EP 0 537 461 can also be used. As the starting material, diols modified with diols are used, which are reacted with fatty acid derivatives to form an ester bond to give, for example, carboxylic acid-derivatized silica gels. In a second step, the ester bonds on the outer surface are hydrolyzed with particle-bound or free esterases and / or lipases.

For example, it is also possible to use epoxy-containing starting materials which, as described in DE 43 33 821, are converted into fatty acid-containing ion exchangers and hydrolyzed with esterases and / or lipases as described above.

According to the invention, it may be desirable to use carrier material which has regions on its outer and / or pore surface which contain a functional group in different densities. It is particularly preferred to use carrier material whose pores and / or outer surface have regions which are derivatized and / or functionalized with alkyl residues of the length C1 to C50, preferably C4 to C22, particularly preferably C4, C8 and C18.

It is also advantageous to use carrier material whose surface has areas which are derivatized and / or functionalized with diols.

It is also preferred to use carrier material which is modified on its outer surface by glycine and whose pore surface is modified by polypeptides, in particular tripeptides

Also suitable is carrier material which is modified on its outer surface with polyethylene glycol and / or polyoxyethylene and whose pore surface is modified with hydrophobic groups, in particular phenyl groups and / or C18 and / or C8 and / or nitrile.

Likewise preferred is the use of carrier material which is essentially spherical, with particularly good separation results being achieved when using carrier materials with an outside diameter D of 0.05 <D <20 μm, preferably 0.1 ≤ D ≤ 5 μm. For example, Carrier materials of the size 0.5 ≤ D <3 μm can be used.

The outside diameter of the particles can be determined in particular using a laser diffraction system, for example the Malvern Mastersizer from Malvern Instruments GmbH, Herrenberg. The principle of laser scattering according to Mie theory and Fraunhofer analysis is applied. The scattered light is measured. From these stray _« . _" -, _" -.- O 00/50887

The particle size distribution can be derived from light data. Another system for determining the particle size distribution uses the Sigraph 5100 Particle Sizer from Micrometrics. In this method, the particles to be determined are irradiated with X-ray radiation in a sedimentation solution and the radiation is detected after passing through the sample. The particle size distribution is then determined from the detected radiation.

If the support material is porous, it is advantageous for it to have a pore diameter d of 0.5 d d 100 100 nm, preferably 1 d d 50 50 nm, particularly preferably 2 d d <6 nm.

The pore diameter is preferably measured using the principle of gas adsorption, for example devices from Beckann Coulter (OMNISORB or SA3100) use this principle. For this purpose, any adsorbed gas is removed from the dry sample in a vacuum and the sample is cooled to 77K. At this temperature, inert gases such as. B. nitrogen, argon or krypton on the surface of the particles of the sample. An adsorption isotherm is recorded, i.e. the adsorbed volume of gas is plotted against the applied pressure. The pore size of the particles can then be determined from these isotherms using the BET (Brunauer, Emmet, Teller) equation. Devices for carrying out these measurements are offered in particular by Beckman Coulter

Comparable results for the pore diameter are provided by a method according to Walfort ("Chemically and enzymatically modified reverse-phase support materials for HPLC-integrated sample preparation", dissertation GH Paderborn, 1992), which uses the exclusion chromatographic properties of the support materials. Protein calibration Standards of gel permeation chromatography, such as lactate dehydrogenase, ovotransferrin, ovalbumin, caboanhydrase or cytochrome C with different molecular weights, are dissolved in a suitable buffer and injected onto columns filled with the carrier material and eluted with a suitable buffer or gradient. The composition of the eluate is determined by means of UV detection. The pore diameter results from the molecular weight of the retained molecules.

The methods described above, in particular that of Pinkerton (EP 0 173 233), Boss (0 537 461) and Takahata (US Pat. No. 4,694,092), can also be used to design a support material with affinity ligands on the pore surface and a hydrophilic outer surface. An example here is the reaction of oxirane groups with m-aminophenylboric acid, followed by plasma treatment, hydrophilization of the outer surface with 3-glycidoxy-trimethylsilane and final hydrolysis of the epoxy groups. Another example is the production of fatty acid-containing affinity sorbents according to DE 43 33 674, followed by hydrolysis of the ester bonds on the outer surface.

The method of 3. Haginaka et al. Is used to design a carrier material with a hydrophobic pore surface and a hydrophilic outer surface. (Anal. Chem. 61, 2445-2448, 1989), Pinkerton (US 4,544,485, EP 0 173 233), Boos (0 537 461), Kimata et al. (J. Chromatogr. 515, 73-84, 1990) or Takahata (US 4,694,092). An example here is the derivatization of the pore surface with glycine-L-phenylalanine-L-phenylalanine by the Pinkerton method or the covering of the pore surface with C-18 groups by the Takahata method. O 00/50887

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To produce carrier materials with a hydrophilic pore inner surface and a hydrophobic outer surface, the Pinkerton method can be modified in accordance with methods known to the person skilled in the art. The particle surfaces activated with 1,1-carboxydiimidazoles could, for example, be reacted with phenylalanine-L-glycine-L-glycine. After the addition of an enzyme such as carboxypeptidase A, the peptide unit on the outer surface is removed, so that a hydrophobic surface is formed by the derivatization with phenylalanine.

A modification of the Takahata method can be used to produce carrier material with a chiral inner surface and a hydrophilic outer surface. A support material coated with oxirane groups is reacted with 6-monodeoxy-6-monoamino-b-cyclodextrin and, in a second step, treated with plasma to remove the chiral ligands on the outer surface.

The use of the carrier material according to the invention in a CEC method for sample preparation is particularly advantageous, the sample consisting of analyte and other sample components (sample matrix)

Is applied to a CEC column system,

An electroosmotic flow is generated by applying an electrical voltage, through which the sample molecules are moved and / or the sample molecules migrate due to their charge-to-mass ratio,

The sample matrix is eluted by applying a washing buffer,

• the analyte is eluted by applying a transfer buffer. A CEC column in the sense of the method according to the invention is to be understood as a carrier material receiving device which can in particular be designed as a capillary column or part of a channel system on a chip.

Also preferred is the use according to the invention in a CEC method for combined sample preparation and separation, the sample consisting of analyte and other sample components

Is applied to a CEC column system,

The sample matrix is eluted by applying a washing buffer,

• the analyte is separated and eluted by applying an elution buffer.

Stationary phases in CEC columns made of carrier materials with a hydrophilic outer surface, preferably by derivatization with alcohols and a pore surface modified with ion exchange groups, are particularly suitable for the purification of small charged organic molecules from complex aqueous solutions in the CEC. Examples are the purification of charged drugs such as antisense molecules from biological body fluids, for example serum, plasma or urine. Other applications are the purification of crop protection agents from extracts from soil samples or parts of plants or the monitoring of syntheses such as labeling of proteins with fluorescent dyes. An alternative application is the use of ion exchange materials for the targeted increase in the rate of separation in the CEC, especially at low pH.

For the purification of anionic antisense oligonucleotides from serum using methods of the CEC, it is advantageous to use in the CEC carrier materials as stationary phase, which are characterized by a hydrophilic outer surface, preferably by derivatization with alcohols, and have a pore surface, which is preferred by anion exchangers -NR ₃ ^{+ "} OH, with R = -Ethyl, -propyl, is derivatized.

A carrier material with affinity ligands such as borate groups on the pore surface and a hydrophilic outer surface is particularly suitable for the separation of carbohydrate-containing compounds, such as for example the control of the enzymatic reaction of phosphorylases with poly sugars such as (glucose) _n .

Further applications of the carrier materials are the separation of drugs such as hydrocortisone from serum or plasma. Carrier materials which are functionalized on the pore surface by hydrocortisone-specific Fab fragments and whose outer surface e.g. is made hydrophilic by diol groups. The hydrophilic outer surface prevents the sample components from being absorbed in aqueous solutions. The method is particularly suitable for complex samples with a very large number of ingredients such as serum and plasma.

For the purification of small hydrophilic molecules using the CEC method according to the invention from, for example, organic extracts of creams or the isolation of water-soluble vitamins from mar- O 00/50887

- 19 - garine, carrier material is used as the stationary phase, which is coated with alcohol groups on the inner surface of the pores and phenyl groups on the outer surface of the pores.

The reaction product can be separated from synthesis mixtures which contain hydrophilic polymers, in particular polyethylene glycol and hydrophilic reactants, such as oligonucleotides, for example, by using carrier materials as the stationary phase, which have the following properties: the pore surface is derivatized with diols or sugars. Its outer surface has hydrophobic properties due to derivatization with C8 alkyl residues.

A carrier material that has a hydrophilic outer surface and a chiral inner pore surface is suitable for the separation of enantiomer mixtures such as temazepam or warfarin in biological liquids. For example, the support can be coated with alcohols on the outer surface and with cyclodextrins on the inner surface of the pores. The chiral ligand 1,2-diphenylethyldiamine is particularly suitable for the separation of aryl carbinols.

For the purification of small cationic molecules from Hengst Buffered Saline Solution (HBSS) buffer such as Atenolol, a carrier material is used as the stationary phase, which is coated with carboxylic acid groups on the inner surface of the pore and diol groups on the outer surface of the pore. The column is preferably equilibrated here with an aqueous buffer at a pH greater than 6, the sample is then applied to the column and the HBSS constituents are removed by applying an electrical field. For efficient separation and detection of atenolol, change to a buffer with a pH less than 3 and carry out the separation by applying an electrical field. Particularly good separation results are obtained if the composition of the mobile phase is individually matched to the carrier material. In particular, it may be desirable to generate many charged ion exchange molecules by means of a pH value or to minimize the charge by means of a pH value, which in particular favors the protonation of the ion exchange molecules. An aqueous buffer can be used, but it can also be preferred to equilibrate the CEC column with an organic buffer, for example 95% acetonitrile in water, for example in the presence of ammonium acetate, pH 4.7. In addition, it may be preferred to use a different buffer to apply the sample to the CEC column than to separate and detect the analyte.

For the purification of vitamin C from liquid cream, for example, a carrier material is suitable that is coated with C-8 on the outer surface and diol on the pore surface. The column is equilibrated with a buffer containing, for example, more than 90% acetonitrile. The sample is applied and the hydrophobic components are removed by applying an electric field. For efficient separation and detection, a change is made to a buffer which contains more than 60% aqueous phase, and the separation is carried out by applying an electrical field. The separation can be optimized by adjusting the hydrophibicity of the mobile phase.

The separation of hydrocortisone from serum is particularly suitable with carrier material that hydrocortisone-specific Fab fragments are coated on the inner surface of the pores and alcohols on the outer surface of the pores. For example, the Fab fragment is selected so that the binding and elution of the antigen can be carried out at two different pH's. The binding takes place, for example, at a pH O 00/50887

- 21 - under 6 in aqueous buffer, so that the serum components are removed by applying an electric field. Elution and detection is carried out after the introduction of a buffer greater than pH 7.5 and the application of an electric field.

The mobile phase is preferably composed of buffer salts in the presence of anionic and / or cationic and / or zwitterionic ion pair reagents, whereby their composition can be directly adapted to the nature and properties of the analyte to obtain good separation performance, as is e.g. the examples demonstrate. However, it is also possible to use so-called universal buffers for unknown compositions of the analyte, which have been optimized for the separation and elution of analytes of unknown composition.

In order to achieve particularly good separation results, it is also desirable that wash buffer and elution buffer contain organic solvents, wherein wash buffer should contain at least 1% and elution buffer at least 20% organic solvent.

Overall, in a particularly preferred form of the method, the stationary and mobile phase should be selected so that the electro-osmotic flow remains constant or changes reproducibly during the binding and elution of the analyte, an electro-osmotic flow in the range from 0.5 to 10 mm / s is preferred. A DC high voltage is preferably used to generate the electroosmotic flow.

The application of the sample to the column and the quantitative removal of the matrix is preferably carried out hydrodynamically and / or electroosmotically and / or electrophoretically, which leads to a concentration tion of the analyte by a factor of 10 to 1000.

However, it can also be preferred to apply the sample to the column and wash it using the flash-back method. This reduces possible contamination of the column with matrix components and also leads to a concentration of the sample at the top of the column.

It is also advantageous to carry out the elution and separation of the analytes hydrodynamically and / or electroosmotically and / or electrophoretically.

Electroosmotic elution and separation of the analytes is particularly preferred in order to obtain a sufficient number of trays even when using very short columns, preferably <10 cm.

In a further embodiment, a further concentration of the analyte by a factor of 10 to 1000 is achieved during the elution by isotachophoresis. For example, the analyte can be transferred from a separate sample preparation column to a separation column without a large change in volume.

In order to increase the selectivity of the process and / or to increase the electroosmotic flow, mixtures of different carrier materials are preferably also used as the stationary phase. The particular advantage of these mixtures is that they allow an optimal adaptation to the properties of the mobile phase and the sample and thus ensure a sensitive and selective separation.

If the analytes are to be detected after separation and elution, it is desirable, in particular in the case of mass spectrometric detection, that the buffer salts and / or anionic and / or cationic and / or zwitterionic ion pair reagents are volatile at room temperature.

In order to characterize the composition of the analyte precisely both qualitatively and quantitatively, it is possible in a preferred embodiment to carry out various spectrometric and spectroscopic analysis methods after separation and / or elution. The methods of mass spectrometry and / or optical detection are particularly advantageous here, e.g. by light scattering, in particular condensation nucleation light scattering detection (Szostek et al., 1997, Analytical Chemistry, 69, 2955-2962) and / or fluorescence detection and / or electrochemical detection and / or conductivity detection and / or refractive index detection, in particular laser-based Refractive index detection coupled with absorption detection (Anal. Chem. 59, 1632-1636, 1987) and / or laser-based refractive index detection using backscatter (US 5,325,170) and / or chemiluminescence nitrogen specific detection (LC-GC 12, 5 , 287-293, 1999) and / or thermo-optical detection, in particular thermo-optical absorption detection (Anal. Chem. 61, 37-40, 1989) and / or laser-induced capillary vibration (Anal. Chem. 63, 2216-2218, 1991 ). For example, UV detection used in embodiment 1.

However, it can also be preferred to feed the analyte fractions to a fraction collector after the separation, thus to collect them individually and to use them for further use.

In a further embodiment, the transfer to a further column system for further separation may also be desirable. The possibility of carrying out the method in parallel in a multiplicity of CEC column systems which are connected to one another is particularly advantageous.

According to the invention, a CEC device preferably contains the following components:

Carrier material receiving unit with at least one inlet and at least one outlet, packed with carrier material, which is porous and whose surface is composed of an outer and a pore surface, the outer surface having areas of different derivatization and / or functionality that can be distinguished from the pore surface,

at least two vessels for receiving the mobile phase, and

at least one voltage source.

A CEC device is particularly preferred, in which a pressure generating device is additionally provided for pressurizing the carrier material receiving unit.

It is also preferred that a system for automated changing of the vessels is provided for taking up the mobile phase.

In addition, it is advantageous to couple the carrier material receiving unit to at least one detector, the detector in particular as a mass spectrometer and / or optical detector, in particular UV detector, light scattering detector, particularly preferably condensation nucleation light scattering detector and / or fluorescence detector and / or electrochemical detector and / or conductivity detector and / or refractive index detector, in particular laser-based refractive index detector coupled with absorption detection and / or laser-based refractive index detector using backscatter and / or chemiluminescence nitrogen specific detector and / or thermo-optical detector Detector, in particular thermo-optical absorption detector and / or laser-induced capillary vibration detector.

In a further embodiment of the device according to the invention, the carrier material receiving unit is designed as a capillary column.

It is also preferred that the carrier material receiving unit of the device according to the invention is designed as part of a channel system on a chip.

It is furthermore advantageous that the capillary column and the chip consist of plastic and / or glass and / or fused silica and / or ceramic and / or elastomer and / or polymers.

In a further embodiment of the device according to the invention, at least two carrier material receiving units are provided, which are connected to one another via a capillary system and / or a channel system, the channel system and / or capillary system preferably having at least one outlet.

In addition, in a preferred embodiment, it is advantageous for the outlet of the carrier material receiving unit to have an inside and / or outside diameter that deviates from the inlet.

It is particularly advantageous if the outlet is designed as an electrospray device. In a further embodiment, a multiplicity, in particular 2 to 50, particularly preferably 2 to 16 carrier material receiving units are arranged in parallel and / or in two dimensions.

It is also preferred that the at least one carrier material receiving unit of the device according to the invention contains a mixture of different types of carrier materials, wherein each type of carrier material is porous and its surface is composed of an outer and a pore surface, the outer surface being distinguishable from the pore surface areas of different derivatization and / or has functionality.

In a further preferred embodiment of the device, the sample receiving device is designed for receiving samples with a volume V of 0.5 nl V V 100 100 μl. The use of CEC columns with a length of 0.1 to 100 cm and a diameter of <500 μm is particularly advantageous.

A CEC column in the sense of the device according to the invention is to be understood as a carrier material take-up unit, which can in particular be designed as a capillary column or part of a channel system on a chip.

Embodiments of the device are explained below with reference to the attached figures.

Figure 1 shows a CEC column system consisting of a single column for sample preparation and / or separation.

Figure 2 shows the coupling of a CEC column system to a de- tector, here a mass spectrometer.

Figure 3 shows the coupling of a CEC column system to another column.

Figure 4 shows the coupling of a CEC column system to a fraction collector.

Figure 5 shows a CEC chip system.

Figure 6 shows a possible embodiment of a CEC chip system that is coupled to a capillary system.

Figure 7 shows an example of a possible embodiment of a μ-total analysis system.

Figure 8 shows an example of a particle of a porous carrier material.

Figure 9 shows the electropherogram of an analyte mixture, separated on a CEC column packed with ISPR GFFII-S5-80.

Figure 10 shows the electropherogram of an analyte mixture, separated on a CEC column packed with SPS 5 PM-S5-100-phenyl.

Figure 11 shows the electropherogram of an analyte mixture, separated on a CEC column packed with SPS 5 PM-S5-100-CN.

Figure 12 shows the electropherogram of benzocaine from pure rat serum, separated on a CEC column packed with SPS 5 PM-S5-100-CN. Figure 13 shows the electropherogram of benzocaine from pure dog plasma, separated on a CEC column packed with SPS 5 PM-S5-100-CN.

A particularly advantageous embodiment of the device is shown in Figure 1. A CEC column (30), which is packed with carrier material (60) according to the invention, dips with both ends into containers (90) with a mobile phase (120). The voltage source (10) is used to apply a voltage between both ends of the columns. The voltage enables an electroosmotic flow to form in the column. In addition, a device for pressurizing the containers can be attached. The application of pressure evenly at both ends of the column counteracts a degassing of the buffer solutions and thus the formation of air bubbles in the column. One end of the column is designed to hold the sample. A changing device enables the containers (90) to be changed and thus the exchange or adaptation of the buffer solutions (120) to the process step. For example, by attaching a detector (150) directly to the column, as outlined in Figure 1, the analyte can be directly detected and analyzed.

In a further embodiment of the device, it is preferred that the column system consists of at least one CEC column for sample preparation and at least one CEC column for separating the analyte, which are connected to one another via a capillary system, this capillary system at least in a particularly preferred embodiment has an outlet through which the sample matrix can be removed. In addition, it is also possible to use a CEC column (30) only for sample preparation. A possible embodiment is shown in Figure 3. This example shows the combination of a CEC column (30) with a further column (170), which are arranged on a chip. It is also possible to transfer the analyte to other analysis or separation systems after the sample matrix has been separated.

The device can also couple the column system to at least one detector, in particular mass spectrometer and / or light scattering detector or other optical detector and / or electrochemical detector and / or fluorescence detector and / or conductivity detector and / or refractive index detector, in particular laser-based refractive index detector coupled with absorption detection and / or laser-based refractive index detector using backscatter and / or chemiluminescence nitrogen-specific detector and / or thermo-optical detector, in particular thermo-optical absorption detector and / or laser-induced capillary vibration detector (150). This preferred embodiment is outlined in Figure 2 using the coupling to a mass spectrometer which has an electrospay device (230). For coupling to a detector, it can be preferred that the outlet of the column system has an inside and / or outside diameter that deviates from the inlet.

Furthermore, in a further preferred embodiment of the device, it is provided that the column system (30) can be coupled to a fraction collector and / or a further column system. This can be done, for example, by direct coupling. In a preferred embodiment (Figure 4) the fraction Collection applied a voltage between the inlet of the column and, for example, a gold-coated Maldi plate (200) (Meeting Abstract, Advances in Mass Spectrometry, Jan 7-8, 1999, Orlando, Florida, USA). The eluate is atomized and the analyte is collected specifically in individual wells of the plate.

In a further advantageous embodiment, the CEC column system is arranged on a chip (300) (Figure 5). Shown here is a column system consisting of a carrier material receiving unit (30) with a carrier material (60) according to the invention, a sample reservoir (290) and three buffer reservoirs (260). The system is designed so that each reservoir can hold one electrode. In this way, a voltage can optionally be applied between different reservoirs. In a preferred embodiment, the control and switching of the electrodes takes place automatically, and the exact circuit diagrams can be managed by computer programs.

In a particularly advantageous embodiment, the chip system (300) is combined with glass capillaries or CEC columns (30) made of fused silica. An example of this embodiment of the device according to the invention is outlined in Figure 6.

For the production of the capillaries and chip systems, it is preferred to use materials such as plastic, glass, fused silica, ceramic, elastomers or polymers.

Suitable chips can be produced, for example, by using photolithography in conjunction with etching techniques. This was described, for example, by JP Landers (Handbook of Capillary Electrophoresis, 1997, CRC Press, p. 828) for the production of chips for use in capillary electrophoresis. Materials such as glass or fused silica are coated with a photosensitive substance. The desired channel system is transferred to the substrate by exposure with the aid of a mask and is etched into the substrate, for example in a bath of dilute HF / NH ₄ F. For fused silica substrates, it is necessary to apply a thin gold / chrome film to the substrate as an etching mask.

Depending on the material that is used to manufacture the capillaries or support material receiving units for the CEC columns and chip systems, it may be desirable to prevent unspecific reactions of the sample with, for example, free silanol groups on the inside of the capillary, to coat them. This is advantageously done with PVA or polyacrylamide.

In a further special embodiment of the device, the column system is a component of a total analysis system.

A possible total analysis system is shown in Figure 7. Such a system represents a whole of an analysis system and can equally include sample preparation and analysis and possibly upstream and / or subsequent steps. The μTAS shown in Figure 7 comprises the labeling of a protein with a dye, the separation of the dye and the unlabeled protein via a CEC column (30) packed with the carrier material (60) according to the invention and the detection of the labeled protein. The system outlined here can e.g. be integrated on a chip, wherein the round recesses can each hold electrodes for applying voltage.

Another embodiment provides for the parallelization of a large number of CEC column systems in which the sample preparation and - separation is carried out in parallel. These column systems are chip systems or capillary systems or combinations of both. This coupling of several systems is particularly advantageous in high-throughput screening because it allows the parallel preparation and separation of a large number of samples, which can then be further examined.

Figure 8 shows an example of a particle of a porous carrier material. The surface of this carrier material can be divided into an outer surface (510) and a pore surface (540).

Figure 9 shows the electropherogram of an analyte mixture in a model matrix. The procedure was carried out using a CEC column filled with ISRP GFFII-S5-80 (pore size 8 nm). The length of the packed capillary: 8.3 cm. Inside diameter 100 μm. Detection wavelength: 210 nm. For further conditions see embodiment 1.

Figure 10 shows the electropherogram of an analyte mixture in a model matrix. The procedure was carried out using a CEC column filled with SPS 5 PM-S5-100-phenyl (pore size 10 nm). Length of the packed capillary: 8.3 cm. Inside diameter 100 μm. Detection wavelength: 210 nm. For further conditions see embodiment 2.

Figure 11 shows the electropherogram of an analyte mixture in a model matrix. The procedure was carried out with a CEC column, filled with SPS 5 PM-S5-100-CN pore size 10 nm). Length of the packed capillary: 8.3 cm. Inner diameter: 100 μm. Detection wavelength: 210 nm. For further conditions see embodiment 3. Figure 12 shows the electropherogram of benzocaine from pure rat serum. The procedure was a CEC column PLC 5 PM-S5-100-CN Company Regis ^® (pore size 10 nm). Length of the packed capillary: 8.3 cm. Inner diameter: 100 μm. Detection wavelength: 210 nm. For further conditions see embodiment 4.

Figure 13 shows the electropherogram of benzocaine from pure dog plasma. The procedure was carried out using a CEC column, SPS 5 PM-S5-100-CN from Regis ^® (pore size 10 nm). Length of the packed capillary: 8.3 cm. Inner diameter: 100 μm. Detection wavelength: 210 nm. For further conditions see embodiment 5.

Embodiment 1

Separation of an analyte mixture in a model matrix on a CEC column packed with ISRP GFFII-S5-80

Used material :

The CEC column having a length of 8.3 cm and an inner diameter of 100 microns was with Pinkerton ISRP GFFII-S5-80 Company ^® Regis Technologies, Inc., Austin, USA, packed. The particles used had a diameter of 5 μm and a pore size of 8 nm.

The wash buffer consisted of 5% acetonitrile, 95% water, 5 mM ammonium acetate (pH 8.5). The elution buffer consisted of 40% acetonitrile, 60% water, 5 mM ammonium acetate (pH 8.5).

The solution contained FBS (Fetal Bovine Serum) in a concentration of 10 mg / ml, thiourea, acetaminophen, benzocaine, propranolol and quinine each 1 mg / ml. This sample solution is referred to in the text below as "analyte mixture in model matrix".

contraption

The device shown in Figure 1 was used to carry out the separation of the analyte mixture in a model matrix. The column packed with ISPR GFFII-S5-80 was immersed at one end in a container for the buffer solution. A voltage was applied between both ends of the column using a voltage source (10).

Pillar preparation

The column preparation was carried out in two stages at 15 ° C: The column was first equilibrated with separation buffer. During this process, the voltage was increased in steps of 5 kV from -5 kV to -20 kV every 5 minutes. The inlet buffer tank (buffer tank into which the column inlet is immersed) was pressurized to 5 bar. Then both buffer vessels were pressurized with 10 bar and a voltage of -15 kV was applied. The stability of the column was checked in the meantime by measuring the current and the UV adsorption (210 nm).

The 2nd equilibration phase was carried out in washing buffer and lasted 12 min. A voltage of -15 kV was applied and a pressure of 10 bar was applied to both buffer tanks. The current and voltage were also checked.

After completion of the 2nd phase, the current and the UV adsorption were stable.

Separations

During the entire operation, the buffers, the samples and the separation capillary were kept at 15 ° C.

The sample (analyte mixture in model matrix) was electrokinetically charged to the column by applying a voltage of -5 kV for 3 seconds. Subsequently, a small amount of washing buffer, a so-called buffer plug, was loaded onto the column under the same conditions to prevent a possible diffusion of the sample into the buffer vessel.

The sample was then applied by applying the washing buffer at a voltage of -15 kV and applying pressure Both ends of the column were washed at 10 bar and the proteins and salts from the model matrix were removed from the CEC column. After 3 min there was a change from the wash buffer to the elution buffer. The conditions -15 kV and 10 bar were maintained. The thiourea eluted at 1.48 min, acetaminophen at 2.27 min, benzocaine at 4.88 min, propranolol at 4.99 min and quinine at 5.67 min. The electropherogram of this separation is shown in Figure 9.

Design example 2

Separation of an analyte mixture in a model matrix on a CEC column packed with SPS 5 PM-S5-100-Phenyl

Used material:

The CEC column having a length of 8.3 cm and an inner diameter of 100 microns was packed with SPS 5 PM-S5-100-Phenyl Company ^® Regis Technologies, Inc., Austin, USA. The particles used had a diameter of 5 μm and a pore size of 10 nm.

The wash buffer consisted of 5% acetonitrile, 95% water, 5 mM ammonium acetate (pH 4.7). The elution buffer consisted of 15% acetonitrile, 85% water, 5 mM ammonium acetate (pH 4.7).

Device tuner

The device shown in Figure 1 was used to carry out the separation of the analyte mixture in a model matrix. The The column packed with SPS 5 PM-S5-100-Phenyl was immersed at one end in a container for the buffer solution. A voltage was applied between both ends of the column using a voltage source (10).

Column preparation

The column preparation was carried out according to embodiment 1.

Separations

The sample was then washed by applying the washing buffer at a voltage of -15 kV and applying pressure to both ends of the column at 10 bar. The proteins and salts were removed from the model matrix. After 6 minutes, the wash buffer was replaced by an elution buffer. The conditions -15 kV and 10 bar were maintained. The thiourea eluted at 1.94 min, acetaminophen at 4.23 min, propranolol and quinine at 11.34 min and benzocaine at 18.25 min. The electropherogram of this separation is shown in Figure 10. Embodiment 3

Separation of an analyte mixture in a model matrix on a CEC column packed with SPS 5 PM-S5-100-CN

used material

The CEC column having a length of 8.3 cm and an inner diameter of 100 microns was the firm ^® Regis Technologies, Inc., Austin, USA packed with SPS 5 PM-S5-100-CN. The particles used had a diameter of 5 μm and a pore size of 10 nm.

The wash buffer consisted of 5% acetonitrile, 95% water, 5 mM ammonium acetate pH 4.7. The elution buffer consisted of 15% acetonitrile, 85% water, 5 mM, pH 4.7.

The solution contained FBS (Fetal Bovine Serum) in a concentration of 10 mg / ml, thiourea, acetaminophen, benzocaine, propranolol and quinine each 1 mg / ml. This sample solution is referred to in the following text as an analyte mixture in a model matrix.

contraption

The device shown in Figure 1 was used to carry out the separation of the analyte mixture in a model matrix. The column packed with SPS 5 PM-S5-100-CN immersed with one end each in containers for receiving buffer solution. A voltage source (10) was used to apply a voltage between the two ends of the column.

Column preparation

The column preparation was carried out according to embodiment 1 O 00/50887

- 39 -

leads.

Separations

The sample was then washed by applying the washing buffer at a voltage of -15 kV and applying pressure to both ends of the column at 10 bar. The proteins and salts were removed from the model matrix. After 5 minutes, the wash buffer was replaced by an elution buffer. The conditions -15 kV and 10 bar were maintained. The thiourea eluted at 1.87 min, acetaminophen at 3.43 min, benzocaine, propranolol and hydrocortisone at 10.39 min and quinine at 11.87 min. The electropherogram of this separation is shown in Figure 11.

Embodiment 4

Separation of benzocaine in rat serum

used material

The CEC column having a length of 8.3 cm and an inner diameter of 100 microns was the firm ^® Regis Technologies, Inc., Austin, USA packed with SPS 5 PM-S5-100-CN. The particles used had O 00/50887

- 40 -

a diameter of 5 μm and a pore size of 10 nm.

The sample consisted of rat serum doped with benzocaine in a concentration of 0.5 mg / ml serum.

contraption

The device corresponded to that from exemplary embodiment 3.

Column preparation

The column preparation was carried out according to embodiment 3.

Separations

The sample was electrokinetically charged to the column by applying a voltage of -5 kV for 3 sec. A small amount of washing buffer, a so-called buffer plug, was then loaded onto the column under the same conditions to prevent the sample from diffusing into the buffer vessel.

The sample was then washed by applying the washing buffer at a voltage of -15 kV and applying pressure to both ends of the column at 10 bar. The proteins and salts were removed from the model matrix. After 5 minutes the Wash buffer replaced by an elution buffer. The conditions -15 kV and 10 bar were maintained. The benzocaine eluted at 12.75 min. The electropherogram of this separation is shown in Figure 12.

Embodiment 5

Separation of benzocaine in dog plasma

used material

The sample consisted of dog plasma doped with benzocaine in a concentration of 0.5 mg / ml plasma

contraption

The device corresponded to that from exemplary embodiment 3.

Column preparation

The column preparation was carried out according to embodiment 3.

Separations

During the entire operation, the buffers, the samples and the Separate capillary at 15 ° C.

The sample was then washed by applying the washing buffer at a voltage of -15 kV and applying pressure to both ends of the column at 10 bar. The proteins and salts were removed from the model matrix. After 5 minutes, the wash buffer was replaced by an elution buffer. The conditions -15 kV and 10 bar were maintained. The benzocaine eluted at 11.38 min. The electropherogram of this separation is shown in Figure 13.

Claims

Expectations

1. Use of carrier material for capillary electrochromatography (CEC), characterized in that the carrier material is porous and the surface is composed of an outer and a pore surface, the outer surface being distinguishable from the pore surface by different areas of derivatization and / or functionality having.

2. Use of carrier material according to claim 1, characterized in that the areas of different derivatization and / or functionality are homogeneously and / or heterogeneously distributed on the outer and / or pore surface.

3. Use of carrier material according to claim 1 and / or 2, characterized in that the pore and / or outer surface with hydrophobic and / or hydrophilic groups and / or ion exchange groups and / or affinity ligands and / or chiral groups derivatized and / or functionalized is.

4. Use of carrier material according to at least one of claims 1 to 3, characterized in that the pore and / or outer surface has areas which are with alkyl radicals of length C1 to C50, preferably C4 to C22, particularly preferably C4, C8 and C18 are derivatized and / or functionalized.

5. Use of carrier material according to at least one of claims 1 to 4, characterized in that the pore and / or outer surface has areas which are derivatized with diols and / or are functionalized.

6. Use of carrier material according to at least one of claims 1 to 5, characterized in that the carrier material is essentially spherical and has an outer diameter D of 0.05 <D <20 μm, preferably 1 <D <5 μm, particularly preferably 0.5 <D <3 μm.

7. Use of carrier material according to at least one of claims 1 to 6, characterized in that it has a pore diameter d of 0.5 <d <100 nm, preferably 1 <d <50 nm, particularly preferably 2 <d <6 nm.

8. Use of carrier material according to at least one of claims 1 to 7, characterized in that it consists of a hydroxyl-containing organic organic polymer or copolymer.

9. Use of carrier material according to at least one of claims 1 to 8, characterized in that it consists of silicate-containing material, modified on its outer surface with polyethylene glycol or polyoxyethylene and the pore surface with hydrophobic groups, in particular phenyl groups, C18, C8 and / or nitrile is modified.

10. Use of carrier material according to at least one of claims 1 to 9, characterized in that it consists of hydroxyl-containing material, modified on its outer surface by glycine and modified on the pore surface by polypeptides, in particular tripeptides.

11. Use of carrier material according to at least one of claims 1 to 10, characterized in that it consists of silica gel modified with glycerol propyl.

12. Use of carrier material according to at least one of claims 1 to 11, characterized in that it consists of glass modified with glycerol propyl.

13. CEC device with the following components

Carrier material receiving unit with at least one inlet and at least one outlet, packed with carrier material which is porous and the surface of which is composed of an outer and a pore surface, the outer surface having regions of different derivatization and / or functionality which are distinguishable from the pore surface,

• at least two vessels for receiving the mobile phase, and

• at least one voltage source.

14. CEC device according to claim 13, characterized in that a pressure generating device is provided for pressurizing the carrier material receiving unit.

15. CEC device according to claim 13 and / or 14, characterized in that a system for automatically changing the vessels is provided for receiving the mobile phase.

16. CEC device according to at least one of claims 13 to 15, characterized in that the carrier material receiving unit is coupled to at least one detector.

17. CEC device according to claim 16, characterized in that the detector as a mass spectrometer and / or optical detector in particular light scatter detector, UV detector and / or electrochemical detector and / or fluorescence detector and / or conductivity detector and / or refractive index detector, in particular laser based refractive index detector coupled with absorption detection and / or laser-based refractive index detector using backscatter and / or chemiluminescence nitrogen specific detector and / or thermo-optical detector, in particular thermo-optical absorption detector and / or laser-induced capillary vibration detector.

18. CEC device according to claim 16, characterized in that the detector is a condensation nucleation light scattering detector.

19. CEC device according to at least one of claims 13 to 18, characterized in that the carrier material receiving unit is a capillary column.

20. CEC device according to at least one of claims 13 to 19, characterized in that the carrier material receiving unit is formed as part of a channel system on a chip.

21. CEC device according to at least one of claims 13 to 20, characterized in that the capillary column and the chip Plastic and / or glass and / or fused silica and / or ceramic and / or elastomer and / or polymers.

22. CC device according to at least one of claims 13 to 21, characterized in that at least two carrier material receiving units are provided which are connected to one another via a capillary system and / or a channel system.

23. CEC device according to claim 22, characterized in that the channel system and / or capillary system has at least one outlet.

24. CEC device according to at least one of claims 13 to 23, characterized in that the outlet of the carrier material receiving unit has an inner and / or outer diameter which differs from the inlet.

25. CEC device according to at least one of claims 16 to 24, characterized in that the outlet is designed as an electrospray device.

26. CEC device according to at least one of claims 16 to 25, characterized in that a multiplicity, in particular 2 to 50, particularly preferably 2 to 16, of carrier material receiving units are arranged in parallel and / or in two dimensions.

27. CEC device according to at least one of claims 16 to 26, characterized in that the at least one carrier material receiving unit contains a mixture of different types of carrier materials, each type of carrier material being porous and its surface being composed of an outer and a porous material. Surface composed, wherein the outer surface of the pore surface distinguishable areas of different derivatization and / or functionality.

28. CEC column with at least one inlet and at least one outlet, packed with carrier material, which is porous and the surface of which is composed of an outer and a pore surface, the outer surface having areas of different derivatization and / or functionality that are distinguishable from the pore surface ,

29. CEC procedure for sample preparation with the following steps:

Applying a sample consisting of analyte and sample matrix to a CEC device according to at least one of claims 13 to 28,

Applying an electrical voltage to generate an electroosmotic flow,

Application of a washing buffer,

Elution of the sample matrix,

Application of a transfer buffer,

• Elution of the analyte.

30. CEC procedure for combined sample preparation and separation with the following steps:

• Applying an electrical voltage to generate an electrical troosmotic river,

Application of a washing buffer,

Elution of the sample matrix,

Application of an elution buffer,

• Separation and elution of the analyte.

31. CEC method according to at least one of claims 29 or 30, characterized in that after the separation of the analyte, its components and / or the concentration of the components are determined by a detector.

32. CEC method according to claim 31, characterized in that the detector is a mass spectrometer and / or optical detector, in particular light scatter detector, condensation nucleation light scattering detector and / or electrochemical detector and / or conductivity detector and / or refractive index detector, in particular laser-based Refractive index detector coupled with absorption detection and / or laser-based refractive index detector using backscatter and / or chemiluminescence nitrogen specific detector and / or thermo-optical detector, in particular thermo-optical absorption detector and / or laser-induced capillary vibration detector.

33. CEC method according to at least one of claims 29 to 32, characterized in that the application of the sample to the CEC device is carried out hydrodynamically and / or electroosmotically and / or electrophoretically.

34. CEC method according to at least one of claims 29 to 33, characterized in that the components of the analyte according to the fractionation collected.

35. CEC method according to claim 29, characterized in that the analyte after the elution is transferred to a separation device, in particular a high pressure liquid chromatography device, capillary electrophoresis device or liquid chromatography device.

36. CEC method according to at least one of claims 29 to 35, characterized in that the analyte is atomized after separation into its constituents when it emerges from the carrier material receiving unit by an electrospray device.