EA001640B1 - Annular chromatograph - Google Patents

Abstract

1. Annular chromatograph with charging in the form of a particulate bed, characterised in that the particulate bed comprises at least two zones (1, 2, 3, 4), arranged one on top of the other, which contain different bed material and are preferably separated from each other by at least one separation layer (5). 2. Annular chromatograph as claimed in claim 1, characterised in that at least one of the zones (4) is designed as an electrophoresis zone for carrying out electrophoretic separations, being equipped with electrical connections, preferably in the form of sliding contacts (6, 7). 3. Annular chromatograph as claimed in claim 2, characterised in that the electrophoresis zone (4) is separated off from the other zone(s) (1, 2, 3) in each case by at least one electrically non-conducting separation layer (5). 4. Annular chromatograph as claimed in any one of the claims 1 to 3, characterised in that the bed material for the at least two zones (1, 2, 3, 4) is selected from anion exchange resins, cation exchange resins, exclusion gels, gel permeation gels, affinity gels, hydrophobic interaction chromatography (HIC) gels, displacement resins, reversed-phase gels and electrophoretic gels. 5. Annular chromatograph as claimed in claim 4, characterised in that the bed material for the electrophoresis zone (4) is selected from electrophoretic gels. 6. Annular chromatograph as claimed in any one of the above claims, characterised in that the at least one separation layer (5) is selected from membranes, non-porous, inert particulate material, preferably glass beads, and electrically non-conducting material. 7. Annular chromatograph as claimed in any one of the above claims, characterised on that the particulate bed is covered with a covering layer (8) and/or has a base layer (9) beneath it, with both the covering and base layer (8, 9) preferably being of the same material as the separation layer(s) (5). 8. Annular chromatograph as claimed in any one of the above claims, characterised by the fact that the particulate bed contains at least one electrophoresis zone. 9. Annular chromatograph as claimed in any one of the above claims, characterised in that the particulate bed contains at least one exclusion gel zone and at least one adsorber resin zone. 10. Annular chromatograph as claimed in claim 9, characterised by the fact that at least one adsorber resin zone contains an ion-exchange resin. 11. Annular chromatograph as claimed in any one of the above claims, characterised in that a tempering (i.e. heating or cooling) jacket is provided for on the inner and/or outer circumference of the annular separating column (11).

Description

The present invention relates to a ring chromatograph loaded in the form of a layer of particles.

Ring chromatography is a proven over the years and increasingly practiced option preparative chromatographic separation. Ring chromatography is predominantly used if it is necessary to separate large amounts of mixtures of substances, since this type of chromatography can be operated continuously, and this at a relatively high degree of resolution.

Typical P-CAC equipment (P-CAC, Prégéréeu Soyuii, Applag Sygota 1 Edgar = Preparative Continuous Ring Chromatography) consists of an annular layer of particles packed in a gap (annular gap) between two concentric cylinders. When the layer of particles rotates around its axis, the feed solution, as well as one or more eluents, is continuously charged at the upper end. Such actions are known in the art and are widespread (see European Application No. 371648).

In addition to the advantages of high performance, ring chromatography is also characterized by high resolution and specificity. However, to solve various specific separation problems, it is necessary to introduce additional stages of ring chromatography, for example, using different separation gels than at the first stage, in order to achieve the desired degree of separation. On the one hand, in comparison with conventional column chromatography, this can be done relatively simply, since in the case of ring chromatographic separation it is known in which place of the circle the desired products will exit or exit. On the other hand, this requires additional ring chromatographs, which, of course, dramatically increases the hardware and, therefore, financial costs.

The aim of the present invention is therefore to provide a device for ring chromatography, with which you can effectively increase the resolution and specificity of this chromatographic separation method without excessive increase in hardware costs and, thus, technological costs.

The invention achieves this goal by means of a ring chromatograph loaded in the form of a layer of particles, which is characterized in that the particle layer has at least two zones located one above the other, containing different layer material and separated from each other by at least one separation layer each.

Thus, the preliminary or main stages of separation can be carried out, or several separations can be carried out on various bases, for example, size exclusion or affinity or ion exchange chromatography, etc., sequentially within the same separation device, continuously and with high resolution and specificity, additionally minimized or completely eliminated the time, money and hardware costs for replacing columns, including, for example, narrowing and conditioning the mixture for the next step is divided I, regeneration or re-balancing the columns and the like, as well (e.g., susceptible to oxidation or substances humidity) the danger of deterioration of samples when handling them.

In a preferred embodiment, at least one of the zones can be adapted as an electrophoresis zone by providing electrical leads, mainly in the form of sliding contacts, whereby electrophoretic separation can also be carried out in the preferred manner described above. This electrophoresis zone can be advantageously limited by at least one electrically conductive separation layer from another zone (s) in order to increase the efficiency of electrophoresis.

The layer material for at least two zones can be selected as a whole from anion-exchange resins, cation-exchange resins, exclusion gels, gel-penetrating gels, affinity gels, hydrophobic chromatographic (H1C) gels, displacement (O18p1-network1) resins, reversed-phase (Кеега) gels, electrophoretic gels and other practical separation media, which provides many opportunities for combined separation, as described below in more detail. If one of the zones in the chromatograph according to the invention is an electrophoresis zone, then, of course, an electrophoretic gel is chosen as a layer of particles for this zone.

The separation layer or separation layers are selected according to the invention, preferably from membranes, a non-porous inert material of particles, in particular glass beads, and especially for possible electrophoresis zones, from an electrically non-conductive material.

In preferred embodiments of the invention, the uppermost zone of the bed of particles is coated with a coating layer and / or laid with a base layer, the coating layer and the base layer consisting mainly of the same material as the separation layer (s).

Particularly preferably, the chromatograph according to the invention includes at least one electrophoresis zone or at least one exclusion gel zone and at least one adsorption resin zone, in particular at least one adsorption resin zone contains ion-exchange resin, due to which it is possible to separate and purify, for example, proteins extremely selectively and with high resolution (sequentially in accordance with their size and charge), as well as numerous biotechnological products, for example ments, B-ECE (Bishai ep1betta1 dgo \\ 111 Gasyug human epidermal growth factor) and immunoglobulins.

According to one embodiment of the invention, a thermostatic (i.e. heating or cooling) jacket may also be provided on the inner and / or outer periphery of the annular separation column (11) so that the temperature can be set each time to be optimal.

The invention is described below by way of examples and with reference to the accompanying drawings, in which FIG. 1a) and 1B) schematically depict a ring chromatograph used according to the invention with 2 and 3 separation zones, FIG. 2a) schematically depicts a partial section view through another ring chromatograph according to the invention with an electrophoresis zone, and FIG. 2B) is a schematically enlarged fragment of a section indicated by a dashed line from FIG. 2a).

In FIG. 1a) and 1B) schematically depict two variants of a ring chromatograph according to the invention. In FIG. 1a) shows an embodiment with two, and in FIG. 1B) - with three separation zones of the particle layer. These zones 1, 2 and 1, 2, 3 consist mainly of separation resins selected, for example, from anion exchange resins, cation exchange resins, exclusion gels, affinity gels, hydrophobic chromatographic (H1C) gels, displacement (E | 5p1aetetep1) resins, electrophoretic gels, as well as, if necessary, from other stationary phases common in this field, for example silica gel, alumina, etc. This wide range of choices creates a wide variety of combinations of two or more of these release agents.

Thus, the invention includes the following combinations of two stationary phases:

- cation exchange resin - anion exchange resin;

- cation exchange resin - exclusion gel;

- anion exchange resin - exclusion gel;

- cation exchange resin - affinity gel;

- anion exchange resin - affinity gel;

- cation exchange resin - hydrophobic chromatographic gel;

- anion exchange resin - hydrophobic chromatographic gel;

- exclusion gel - affinity gel;

- exclusion gel - hydrophobic chromatographic gel;

- hydrophobic chromatographic gel an affinity gel, as well as similar combinations with an electrophoretic gel instead of one of the above gels or in addition to it (three-zone chromatography). The above enumeration of the possibilities of double combining does not describe how the gels are located in the column relative to each other, i.e. which is the upper gel and which is the lower. It depends solely on the task of separation.

Examples of separation methods used in the invention are, including:

- ion exchange chromatography:

for example, mono 8 (Pharmacy) or toyopirl EEL 6508 (TosoHaas);

- reverse phase chromatography: for example, Amberchrome C-161eb (Rum und Haas) or Sephazyl C8 (Pharmacy);

- hydrophobic chromatography: phenylsepharose (Pharmacy) or T8K gelphenyl 5PA (TosoKhaas);

- exclusion (8 | / e Exc1i8yup) and gel permeation chromatography: for example, Sephadex C15 (Pharmacy) or toyopirl HA 40E (TosoKhaas);

- affinity chromatography: for example, toyopirl AE Tresml-650M (TosoKhaas) or EAN Sepharose-4B (Pharmacy);

- adsorption chromatography: for example, amberlite HAO (Rum und Haas) or purolite ΜΝ200 (Purolit).

Combinations of two or more gels of the same type, however, for example, from different manufacturers are also included in the scope of protection of the invention. For example, two cation exchange gels or two exclusion gels (for example, Sephadex C15 over TOopyr HA 40E and the like) can also be located one above the other.

According to the invention, ring chromatographs with a layer of particles from at least one zone of the affinity gel and at least one zone of the adsorption resin are particularly preferred, in particular at least one zone of the adsorption resin contains an anion exchange resin, and ring chromatographs with at least one electrophoretic gel in one of the zones.

Two or three gels are sequentially placed in the annular gap of the annular column 11 of an inert material, mainly glass, which is inert with respect to the components of the separation solutions, each of the gel zones being coated with a separation layer 5 before the next one is applied to it, so as to prevent mixing zone materials. In both cases, the particle layer is bounded above by a coating layer.

8. In FIG. 1a) a layer layer 9 is additionally shown, which, in addition to a porous bottom plate (not shown), for example, a porous glass filter, a membrane washer and the like, serves to prevent the exit of particle material at the bottom of the column. The material for the separation 5 covering 8 layers and the base layers 9 is selected from membranes, as well as a non-porous material of particles that is inert with respect to all components of the respective separation solutions, and it can be the same or different for all three layers, especially for electrophoretic separation It must be electrically conductive. According to the invention, glass beads are preferable since in almost all cases of application it is inert and easy to apply.

For the ring chromatographs of FIG. 1a) loading thus takes place in the sequence 9-2-5-1-8, and in FIG. 1b) - in the sequence 3-5-2-5-1-8.

The remaining structural elements of the ring chromatograph can be performed in the usual way. So, the column 11 (driven by a motor not shown) is rotatably mounted on the axis 12 and is fed through connecting pipes 13 for the feed solution and eluents, as well as through a distribution head 14 rigidly mounted on the axis, the feed channels 15 of the distribution head 14 being predominantly immersed in cover layer 8 so as to ensure uniform loading. Channels can take the usual form of execution, i.e. single, composite or slotted nozzles and the like, however, for the invention, curved slotted nozzles of different widths adapted to the periphery of the columns are preferred in order to match the flows of the feed solution and eluents as precisely as possible.

Outlets or tubes 16 for collecting eluates are provided at the lower end of the columns. These outlets 16 can either be connected to the column 11 (i.e., they rotate with it around the axis 12), or are fixed on the axis 12 and, for example, be in contact with the column rotating relative to it, the latter form runtime preferred.

In FIG. 2a) shows a partial view of a ring chromatograph according to the invention with an electrophoretic separation step, namely, an electrophoresis separation zone 4 bounded from other above and lower zones (not shown) by separation layers 5 of an electrically conductive material. Both at the upper and lower ends of the electrophoresis zone 4, mainly directly adjacent to the respective separation layer (s), sliding contacts are provided, consisting of each of the conductive rings 6 mounted on the outer side of the axis 12, supplied with current through the network output 10, and from the ring-shaped current collector 7 provided on the inner side of the column 11, which fits snugly into the conductive ring 6 and is in electrical contact with it. The latter in the zone of sliding contact passes either pointwise or superficially through the column jacket to create the electrical voltage inside the column necessary for electrophoretic separation.

FIG. 2b) is an enlarged view of the dashed line portion of FIG. 2a) and in more detail a sliding contact.

The following are examples of the implementation of the present invention. The invention is in no way limited, however, to these examples. Preferred applications for ring chromatographs according to the invention are the separation of base and noble metals, as well as numerous separations and purification steps in biotechnology.

Example 1

Cation exchange resin - exclusion gel: ion-exchange exclusion chromatography.

Liquid chromatographic separation of platinum group metals with simultaneous removal of base metals.

Platinum group metals: rhodium, palladium, platinum and iridium can be continuously separated from each other by preparative ring chromatography. The base metals present at the same time, such as, for example, iron, copper, nickel or cobalt, can also be continuously separated from the noble metals.

Chromatographic separation of platinum group metals using exchange gels (for example, Sephadex, Toyopirl or biogel) has already been described in US Pat. No. 4,885,143. Concentrated noble metal solutions from leaching processes in separation plants typically contain additional base metals, such as iron, copper or nickel in different concentrations.

After the leaching process, a solution of noble metals is present mainly in concentrated hydrochloric acid. Due to the high concentration of chlorides, all noble metals are present as anionic chlorocomplexes. Base metals have the property of attaching to cation exchanger under certain conditions, while noble metals as anionic complexes pass through the cation exchanger without delay. This property can be used to separate at the first stage of the process the noble associated metals from noble metals, and at the second stage of the method, to separate the noble metals from each other. Using the P-CAC system according to the invention, this becomes possible with the help of simple equipment.

The column shown in FIG. 1a), they are filled to a certain height (the exact experimental conditions are given in Tables 1 and 2) with an exclusion gel 2 (for example, Sephadex, toyopirl or biogel) and glass beads 5 are poured on it (b = 150-240 microns); cation exchanger 1 is poured onto this layer 5 of glass beads (for example, dovex, amberlite, purolite), and again layer 8 of glass beads is placed on it (Fig. 3).

The feed solution is pumped in a radial position of 0 ° in an annular column. While the feed channel 15 is immersed in the upper layer 8 of glass beads. The main eluent (top eluent) is HC1 0.5-1 m high. Noble metals pass without interaction through cation exchanger layer 1 and are then separated from each other in exclusion gel layer 2. At the end of the column, individual fractions of rhodium (CB), palladium (Pb), platinum (P!) And iridium (1g) can be captured separately.

Base metals are adsorbed on cation exchanger and in the radial position in which the last noble metal is washed out (1 g), they are separated using a step eluent (HC1 with a height of 2 m or more). HC1 with a height of 2 m desorbes base metals from cation exchanger and drives them to the layer of exclusion gel. There, base metals do not interact with the stationary phase. Base metals can now be recovered as a co-fraction.

A solution (HC1, 6 m high) of the following concentration is continuously divided into individual noble metals and to a fraction containing base metals.

Table 1

The concentration of metals in the feed solution

P !: 30 g / l RB: 15 g / l KB: 3 g / l 1g: 1 g / l Be: 4 g / l Co: 4 g / l Νί: 4 g / l C: 4 g / l

Glass beads were poured onto a Sephadex layer 6-15 with a height of 26 cm and a layer of 5 cm. A 54 cm thick Dovex 50-X8 layer was poured onto this layer, which was again covered with a glass bead layer. The solution was washed with HC1 0.5 m high. The noble metals passed through layer 1 of cation exchange resin without interaction and were separated on layer 2 of Sephadex. In this case, 4 differently colored bands were formed — the KB band (red), the RB band (brown), and the P band! (yellow) and strip 1g (brown).

In a radial position of 270 ° relative to the loading point of the feed solution, a step eluent (HC1 2 m high) was loaded to separate base metals. A step-eluting agent desorbed base metals from cation exchanger, and a green-yellow strip formed, passing through the column without interaction. The base metals were recovered as a joint fraction after the iridium fraction.

table 2

Experimental conditions

Top flow rate Step flow rate Feed rate Rotational speed The cross-sectional area of the annular gap eighteen ml / min 4 ml / min 0.4 ml / min 125 ° / h 24.4 cm ²

The column height is 41 cm, of which 26 cm of Sephadex are 6-15, 5 cm of glass beads (b = 150-240 microns), 5 cm of Dovex 50 \ U-X8 in the form of H ⁺ , 5 cm of glass beads (b = 150- 240 μm).

If, in addition, Ki and Ok are also contained in the mixture to be separated, then Ki is washed between KB, RB and Ok after 1 g (see Example 2).

Example 2

Adsorption resin - exclusion gel: adsorption-exclusion chromatography.

Selective separation of gold from a solution of noble metals and the simultaneous separation of platinum group metals

The exclusion gel is filled in P-CAC (at least up to half the height), a separation layer (glass beads or membrane) is applied to the exclusion gel, an adsorption resin (XAE7 amberlite or purolite ΜΝ 200) is applied to this layer.

A solution of noble metals containing all the metals of the platinum group (however, at least two of them) and gold are used as a nutrient solution for RSAC. Gold is selectively adsorbed by an adsorption resin, while other metals of the platinum group pass without delay through the adsorber bed and are separated on an exclusion gel. After washing out the last component, the gold is separated using a step eluent from the adsorption resin (Fig. 4).

Example 3

Cation exchanger - anion exchanger: ion exchange chromatography.

The selection of base metals from a solution of Kb / 1g and the subsequent separation of rhodium and iridium.

Anion exchange resin is filled at P-CAC to at least half the height. A separation layer is applied to the anion exchange resin (a membrane or glass beads of the appropriate size), and cation exchange resin is applied to the separation layer to a maximum filling height. A metal solution containing rhodium and iridium, as well as base metals such as iron, nickel, cobalt or copper, is pumped as a feed solution into the ring column. Base metals adsorb to cation exchange resin, while rhodium and iridium pass to anion exchange resin. Adsorb 1g (IV) on the anion exchange resin, and rhodium passes. After all the rhodium is washed, base metals are washed in this angular position using the step eluent 1. After this washing out, 1g (IV) is restored to 1g (III) using step eluent 2 (Fig. 5).

Example 4

Unionite - exclusion gel: ion-exchange exclusion chromatography.

The separation of gold cyanide from a solution of noble metals with the simultaneous separation of noble metals.

P-CAC is filled at least up to half the height with an exclusion gel, then a separation layer is applied, and anion exchange resin is applied to it. A solution of noble metals, containing in addition to gold as a cyanide complex also platinum group metals, is placed as a feed solution in the annular gap. Gold cyanide binds as an anionic complex with anion exchange resin, and the remaining metals of the platinum group pass through the anion exchange resin without interaction and are separated on an exclusion gel. After the angle at which the last metal of the platinum group (iridium) leaves the column, a strip solution for gold desorption is placed in the annular gap (Fig. 6).

A combination of anion exchangers and exclusion gels in biotechnology is also possible, for example, for the preparation and purification of monoclonal antibodies. Moreover, by means of anion exchange resin, pyrogens, nucleic acids and proteases can be removed; in an exclusion gel, the salts of the buffer solution can then be removed.

Example 5

Affinity gel - exclusion gel: affinity exclusion chromatography.

The selection of base metals from a solution of noble metals, followed by the separation of noble metals.

The P-CAC column is filled at least 50% of the height with an exclusion gel, coated with a separation layer, and a super ligel is applied to the final filling height (super lig gels are gels with a crowfire as a functional group; trademark of the company IBC Advance

Technology). A solution of precious metals with a high content of base metals is placed in the annular gap P-CAC. Base metals do not have an affinity for Super League Gel and are therefore washed through the entire column and can be trapped in the form of a joint fraction. Noble metals are separated by superleagel gel and separated on an exclusion gel in the sequence HB-RN-R (- [g (Fig. 7).

Similar applications are possible with stationary phases bound by complexing agents such as dithizones, dehydrodithizones, hydroxyquinolinates, pyridylazone naphtholates, etc.) as reactive groups. Using these stationary phases, however, only in difficult conditions can noble metals be qualitatively separated from each other (one strip eluent must be used for each metal). Therefore, it is impractical to impose another additional phase on these stationary phases, since this would additionally complicate the leaching conditions and make the process uneconomical.

Example 6

Cation exchanger - anion exchanger: ion exchange chromatography.

Two-stage cellulase purification.

Anion exchange resin as a top layer (mono O. Pharmacy) serves for the preliminary purification of cellulase; Wash with Tris. HCl buffer. The recovered fraction containing cellulase is further fractionated in cation exchange resin as the bottom layer (mono 8, Pharmacy) using an acetate buffer as eluent. With cation exchange resin, a very high resolution can be achieved so that cellulase can be recovered in its pure form.

Example 7

Cation exchange resin - anion exchange exchange ion exchange chromatography.

Extraction of monoclonal DSS; from a fermentation solution.

Preliminary purification and antibody concentration is achieved using cation exchanger as the upper phase (8-Sepharose High Performance, Pharmacy). In this case, ME8 with NaC1 is used as a buffer. The pool (the fraction also containing the antibody) is then liberated by means of an exclusion gel (Superdex 200) as the lower phase using sterile sodium chloride buffer from dimers and oligomers, as well as from transferrins.

Example 8

Anion exchange resin - exclusion gel: ion-exchange exclusion chromatography.

Purification of the human epidermal growth factor (Y-E6E, Nitap Ep16etta 1 Cg \\ 111 Eac! From), which was expressed as an extracellular product by means of an 8 cell network 5.

Anion exchange resin (O-Sepharose. Pharmacy) as the upper phase serves for preliminary purification (charge-based purification) of the growth factor, and the lower phase, the exclusion gel (Superdex 75, Pharmacy), serves for the final purification of growth factor.

Example 9

Hydrophobic chromatographic (H1C) gel-exclusion gel: affinity-ion exchange chromatography.

Extraction of human pituitary prolactin (Nitap ryiagu rgo1asyi).

The upper stationary phase (H1C-gel, phenylsepharose C1., Pharmacy) serves for primary purification and prolactin concentration (concentration by a factor of 7). In the second stage, prolactin is finally purified on an exclusion gel 21 (for example, Sephadex 6100, Pharmacy).

Example 10

Cation exchange resin - hydrophobic chromatographic gel: ion-exchange affinity chromatography.

Extraction of α-2-macroglobulin.

Using cation exchanger (upper stationary phase, for example, mono 8, Pharmacy), it is possible to extract a protein pool containing proteins with the same charge, including macroglobulin. Macroglobulin is purified to homogeneity in the second layer, H1C-gel (for example, phenylsepharose, Pharmacy).

Example 11

Anionite - hydrophobic chromatographic gel: ion-exchange affinity chromatography.

Purification of Recombinant Reverse Transcriptase Ηΐν.

In the upper phase, in anion exchange resin (for example, ΌΕΑΕ Sepharose, Pharmacy), the fermentation solution is separated by charge. The pool in which the transcriptase is located passes through the second layer, the H1C gel (for example, phenylsepharose high performance, Pharmacy). After this layer, due to different hydrophobicity, the transcriptase is recovered in its pure form.

Example 12

Cation exchange resin - anion exchange resin, with one of the stationary phases in the displacement (E | 5p1asstsi1) mode: ion-exchange chromatography.

Protein Separation

Anion exchange resin (for example, mono O. Pharmacy) is filled into a ring chromatograph. An inert layer, such as glass beads or a membrane, is poured onto this anion exchange resin, and cation exchange resin (e.g., mono 8, Pharmacy) is placed on it. A mixture of different proteins is separated on a cation exchange resin based on a different charge, the separated fractions are concentrated on anion exchange resin using a displacer or displacer (for example, nalcolite). Thus, separation and simultaneous concentration can be combined.

Example 13

Exclusion gel - electrophoretic gel: size exclusion chromatography and electrophoresis.

Protein Separation

The electrophoretic gel forms a lower layer in P-CAC coated with an exclusion gel. When proteins of a different charge and different p1 values are applied to the exclusion gel, they are separated based on the value. The electrophoretic layer then carries out an additional separation of proteins based on their charge.

Example 14

Size exclusion gel - cation exchange resin, with cation exchange resin in displacement (E | 5p1asstsi1) mode: size exclusion-ion chromatography.

Separation of the amino acid mixture with the simultaneous release of proteins.

The upper stationary phase, an exclusion gel (e.g., Sephadex 625, Pharmacy), is used to isolate proteins (e.g., albumin) from a nutrient solution containing amino acids, L-glutamic acid, L-valine and L-leucine. Separation in an exclusion gel gives a fraction containing proteins and a fraction containing amino acids. In the second layer, the amino acids are separated on cation exchange resin (for example, Dovex 50 \ Y-X8) with a dispenser (for example, 0.1 N н αΝ) and simultaneously concentrated. Amino acids are isolated in the sequence: glutamic acid - valine - leucine. The dispenser should be followed by a regenerating solution (for example, dilute H ₂ 8 O ₄ ) in order to bring the gel layer back to its original state.

The displacement wash example is also not limited to use in cation exchange resin.

As can be seen from the above description, the present invention provides a new ring chromatograph with a layer of particles consisting of several different zones, with which several types of chromatographic separation can be carried out in one step and, thus, faster and more economical than it was still possible in accordance with the prior art.

Claims (11)

CLAIM 1. Ring chromatograph with loading in the form of a layer of particles, characterized in that the layer of particles has at least two zones located above each other (1, 2, 3, 4), consisting of various materials and mainly separated from each other, at least one separating layer (5) each. 2. Chromatograph according to claim 1, characterized in that at least one of the zones (4) due to the provision of electrical leads, mainly in the form of sliding contacts, is made as an electrophoresis zone for electrophoretic separation. 3. Chromatograph according to claim 2, characterized in that the electrophoresis zone (4) is limited by at least one electrically conductive separation layer (5) from another zone (other zones) (1, 2, 3). 4. Chromatograph according to one of claims 1 to 3, characterized in that the layer material for at least two zones (1, 2, 3, 4) is selected from anion-exchange resins, cation-exchange resins, exclusion gels, gel-penetrating gels , affinity gels, hydrophobic chromatographic (H1C) gels, displacement resins, reverse phase gels and electrophoretic gels. 5. Chromatograph according to claim 4, characterized in that the layer material for the electrophoresis zone (4) is selected from electrophoretic gels. 6. Chromatograph according to one of claims 1 to 5, characterized in that at least one separation layer (5) is selected from membranes of a non-porous inert material of particles, mainly glass beads, and from an electrically non-conductive material. FIG. 2a) 7. Chromatograph according to one of paragraphs. 1-6, characterized in that the particle layer is coated with a coating layer (8) and / or laid with a base layer (9), and the coating layer (8) and the base layer (9) consist mainly of the same material as the separation layer ( layers) (5). 8. Chromatograph according to one of claims 1 to 7, characterized in that the particle layer contains at least one electrophoresis zone. 9. Chromatograph according to one of claims 1 to 8, characterized in that the particle layer contains at least one zone of an exclusion gel and at least one zone of an adsorption resin. 10. The chromatograph according to claim 9, characterized in that at least one zone of the adsorption resin contains an ion exchange resin. 11. Chromatograph according to one of claims 1 to 10, characterized in that a thermostatic shirt is provided on the inner and / or outer periphery of the annular separation column (11).