AU1474399A

AU1474399A - Annular chromatograph

Info

Publication number: AU1474399A
Application number: AU14743/99A
Authority: AU
Inventors: Adalbert Prior
Original assignee: Prior Separation Technology GmbH
Current assignee: Prior Separation Technology GmbH
Priority date: 1997-12-01
Filing date: 1998-11-30
Publication date: 1999-06-16
Anticipated expiration: 2018-11-30
Also published as: ATA203097A; CA2301570A1; EA199901022A1; AT405025B; WO1999028740A1; EA001640B1; EP1183531A1; AU754824B2

Description

1 Annular chromatograph The present invention relates to an annular chromatograph with charging in the form of a particulate bed. 5 Annular chromatography is a variant of preparative chromatographic separation which over the past few years has been recognised and used to an ever-increasing extent. Annular chromatography is used mainly when large quantities of mixed substances have to be separated, since this type of chromatography can be operated D continuously and with a relatively high degree of resolution. A typical P-CAC unit ("P-CAC" = preparative continuous annular chromatography) consists of an annular particulate bed which is packed into the space (annular gap) between two concentric cylinders. Whilst the particulate bed is being rotated around 5 its axis, feed solution and one or more eluents are introduced continuously at the top end. Such methods are known under prior art and are widespread (see for example EP-A-371.648). Apart from the advantage of the large throughputs in annular chromatography this 3 method is also characterised by high resolution and specificity. Nonetheless, for various specific separation problems it is necessary for further stages of annular chromatography to be added - for example, using different separating gels from those in the first stage - in order to achieve the required degree of separation. On the one hand this can be done relatively easily by comparison with conventional 5 column chromatography, since with annular chromatographic separation it is known at what point on the circumference the required product(s) will be discharged; on the other hand further annular chromatographs are necessary, which does of course mean a sharp increase in the amount of equipment, and thus the cost. The aim of the present invention is therefore to provide equipment for annular chromatography which can be used to efficiently increase the resolution and 2 specificity of this method of chromatographic separation without giving rise to an undue increase in the amount of equipment and thereby the cost of the method. The invention achieves this aim by means of an annular chromatograph with 5 charging in the form of a particulate bed, wherein said chromatograph is characterised by the fact that the particulate bed comprises at least two zones, arranged one on top of the other, that contain different bed material and that are preferably separated from each other by at least one separation layer. o In this way both the preliminary and the main separation stages, or several separations based on different methods-like for example exclusion chromatography or affinity chromatography and ion-exchange chromatography, etc.-can be carried out one after the other, continuously and with high resolution and specificity within the same separating equipment. In addition, the amount of time, money and 5 equipment needed to change columns, including for example concentration and conditioning of the mixture for the next stage of separation, regeneration or re equilibration of the columns, etc., as well as the danger of sample impairment during handling (e.g. in the case of substances which are sensitive to oxidation or moisture) are minimised or completely eliminated. 0 In a particularly preferred embodiment at least one of the zones can be adapted to create an electrophoresis zone by equipping it with electrical connections, preferably in the form of sliding contacts; electrophoretic separations can then also be carried out in the beneficial manner described above. This electrophoresis zone can 5 preferably be kept separate from the other zone(s) in each case by at least one electrically non-conducting separation layer in order to increase the efficiency of the electrophoresis. The bed material for the two or more zones can generally be selected from anion 0 exchange resins, cation-exchange resins, exclusion gels, gel permeation gels, affinity gels, hydrophobic interaction chromatography (HIC) gels, displacement resins, 3 reversed-phase gels, electrophoretic gels and other separating media used in practice, thereby offering a wide range of possibilities for combined separations, as described in more detail below. If one of the zones in the chromatograph according to the invention is an electrophoresis zone an electrophoretic gel will of course be 5 chosen as particulate bed for this zone. According to the invention the separation layer or separation layers are selected preferably from membranes, non-porous, inert particulate material, especially glass beads, and - specifically for any electrophoresis zones - electrically non-conducting ) material. In preferred embodiments of the invention the topmost zone of the particulate bed is covered with a covering layer and/or has a base layer beneath it; both the covering and base layer should preferably be of the same material as the separation layer(s). 5 It is particularly preferred that a chromatograph in accordance with the invention should have at least one electrophoresis zone or at least one exclusion gel zone and at least one adsorber resin zone, with, especially, at least one adsorber resin zone containing an ion-exchange resin, with which for example proteins (one after the other according to their size and their charge) and numerous products of biotechnology processes-like for example enzymes, h-EGF (human epidermal growth factor) and immunoglobulins-can be separated and purified with an extremely high degree of selectivity and high resolution. In one embodiment of the invention a tempering (i.e. heating or cooling) jacket can be provided for on the inner and/or outer circumference of the annular separating column (11) so that the optimum temperature can be set for the particular separation. The invention is described below with examples and with reference to the enclosed drawings, where Fig. 1a) and 1b) are schematic representations of an annular chromatograph with 2 and 3 separation zones, respectively, which can be used in accordance with the present invention, Fig 2a) is a schematic partial view of a section 4 through another annular chromatograph in accordance with the invention with an electrophoresis zone and Fig. 2b) is a schematic detail enlargement of the broken line box from Fig. 2a). 5 Two variants of the annular chromatograph of the present invention are shown in schematic form in Fig. 1a) and 1b). Fig. 1a) shows an embodiment with two separation zones in the particulate bed; Fig lb shows an embodiment with three separation zones. These zones 1 and 2, or 1, 2 and 3, consist preferably of separation resins, selected for example from anion-exchange resins, cation 0 exchange resins, exclusion gels, affinity gels, hydrophobic interaction chromatography (HIC) gels, displacement resins, electrophoretic gels, and possibly other stationary phases which are usual in the field, like for example silica gel, aluminium oxide, etc. This broad range of possibilities provides a large variety of combinations of two or more of these separating agents. 5 Thus the invention encompasses for example the following combinations of two stationary phases: " cation exchanger - anion exchanger 3 e cation exchanger - exclusion gel " anion exchanger - exclusion gel " cation exchanger - affinity gel e anion exchanger - affinity gel " cation exchanger - hydrophobic interaction chromatography gel 5 e anion exchanger - hydrophobic interaction chromatography gel e exclusion gel - affinity gel " exclusion gel - hydrophobic interaction chromatography gel * hydrophobic interaction chromatography gel - affinity gel as well as such combinations with an electrophoretic gel instead of one of the two gels referred to above or in addition to them (three-zone chromatography). The 5 above list of possible binary combinations does not describe how the gels are arranged in the column relative to one another, i.e. which is the higher gel and which is the lower one. This depends solely on the separating problem. 5 Examples of the separating techniques which can be used in the invention include: " ion-exchange chromatography: e.g. Mono S (Pharmacia) or Toyopearl DEAE 650S (TosoHaas); e reversed-phase chromatography: e.g. Amberchrom CG-161cd (Rohm & Haas) or Sephasil C8 (Pharmacia); e hydrophobic interaction chromatography: Phenyl Sepharose (Pharmacia) or TSK Gel Phenyl 5PW (TosoHaas); e size exclusion chromatography and gel permeation chromatography: e.g. Sephadex G15 (Pharmacia) or Toyopearl HW 40F (TosoHaas); e affinity chromatography: e.g. Toyopearl AF Tresyl-650M (TosoHaas) or EAH Sepharose-4B (Pharmacia); e adsorption chromatography: e.g. Amberlite XAD (Rohm & Haas) or Purolite MN200 (Purolite). The scope of protection of the invention also includes combinations of two or more gels of the same type, which may however come for example from different manufacturers. For example, two cation-exchange gels or two exclusion gels may also be arranged one over the other (e.g. Sephadex G15 above Toyopearl HW 40F or similar). According to the invention there is a particular preference for annular chromatographs with a particulate bed consisting of at least one exclusion gel zone and at least one adsorber resin zone, where in particular at least one adsorber resin zone contains an ion-exchange resin; and for annular chromatographs with at least one electrophoretic gel in one of the zones.

6 The two or three gels are introduced one after the other into the annular gap of an annular column 11 made of a material which is inert in relation to the components of the separating solutions, preferably of glass; each of the gel zones is covered with a separation layer 5 before the next is added to prevent mixing of the various materials. 5 The particulate bed is closed off at the top in both cases by a covering layer 8. Fig. 1a) also shows a base layer 9 which (in addition to a porous base (not shown), e.g. frit, membrane disc, etc.) serves to prevent particulate material from escaping at the bottom of the column. The material for the separation, covering and base layers 5, 8 and 9 is selected from membranes as well as non-porous particulate material which is inert in relation to all the components of the particular separating solutions and may be the same or different for all three layers, though specifically for electrophoretic separations it must not be electrically conductive. According to the invention there is a preference for glass beads since these are inert in practically all the usual applications and are easily introduced. Charging for the annular chromatograph of Fig. 1a) is thus in the order 9-2-5-1-8 and for that of Fig. 1b) in the order 3-5-2-5-1-8. The remaining components of the annular chromatograph can be designed in a conventional manner. Thus column 11 (driven by a motor which is not shown) is rotatable and mounted on a shaft 12, and it is fed via connections 13 for feed and eluent and a distributor head 14 fixed to the shaft; the feed ducts 15 of the distributor head 14 should preferably be immersed in the covering layer 8 to ensure an even feed. The ducts may be of the usual designs, i.e. single, multiple or slit-shaped nozzles or similar, though bent slit-shaped nozzles of different widths, adapted to the circumference of the column, are preferred for the invention, so that the flows of feed and eluent can be matched as precisely as possible to each other. Outlet ducts and tubes 16 are provided at the bottom end of the column to collect the eluates. These outlets 16 may either be connected to column 11 (i.e. they rotate with 7 the column around shaft 12) or be fixed to shaft 12 and be in contact - e.g. by way of a slip-ring - with the column which rotates relative to it; the latter design is preferred. Fig. 2a shows a partial view of an embodiment of an annular chromatograph in 5 accordance with the invention with an electrophoretic separating stage, namely the electrophoretic separation zone 4 which is separated off from a zone above it and a zone below it (neither of which is shown) by means of a separation layer 5 of electrically non-conducting material. Both at the top and the bottom end of the electrophoresis zone 4, preferably directly adjacent to the relevant separation D layer(s), there are sliding contacts consisting of a conductive ring 6, mounted on the outside of shaft 12 and supplied with electricity from a mains connection 10, and an annular current pick-up 7 which is provided for on the inside of column 11, lies close to the conductive ring 6 and is in electrical contact with this ring. The pick-up is passed through the column casing close to the sliding contact - either at a certain 5 point or over an area - to build up the electrical voltage necessary for electrophoretic separation inside the column. Fig. 2b) represents an enlarged view of the broken-line box from Fig. 2a and shows the sliding contact in a more detailed form. Specific examples of applications for the present invention follow. The invention is however by no means restricted to these possible uses. Preferred areas of use for annular chromatographs according to this invention include for example separations of base metals and noble metals as well as numerous types of separation and purification stages in biotechnology. Example 1: Cation exchan-ger - exclusion gel: lon-exchange / size-exclusion chromatography Separation of platinum group metals by liquid chromatography with simultaneous removal of base metals 8 The platinum-group metals rhodium, palladium, platinum and iridium can be separated from each other continuously by means of preparative annular chromatography. Base metals present at the same time, like for example iron, copper, nickel or cobalt, can also be separated continuously from the noble metals. 5 Chromatographic separation of the platinum-group metals using exchanger gels (e.g. Sephadex, Toyopearl or Biogel) has already been described in US-A-4.885.143. Concentrated noble-metal solutions from the leaching processes of separator units normally also contain base metals such as iron, copper or nickel at different 0 concentrations. After the leaching process the noble-metal solution is usually present in concentrated hydrochloric acid. Because of the high chloride concentration the noble metals are all present as anionic chlorocomplexes. Base metals have the characteristic of 5 binding themselves to a cation exchanger under certain conditions, whereas the noble metals run through the cation exchanger as anionic complexes without any retention. This characteristic can be used to separate the accompanying base metals from the noble metals in an initial process stage and then to separate out the various noble metals in a second process stage. With the P-CAC system of the ) present invention this can be done using a very simple set-up of equipment. A column, as shown in Fig. 1a), is filled up to a certain height (exact test conditions in Tables 1 and 2 below) with an exclusion gel 2 (e.g. Sephadex, Toyopearl or Biogel) and glass beads 5 (d = 150-240 <Dm) are arranged in a layer on top of it; a layer of 5 cation exchanger 1 (e.g. Dowex, Purolite, Amberlite) is placed over this layer of glass beads 5 and is in turn finally covered with a layer of glass beads 8. The feed solution is pumped into the annular column at the radial position 00. The feed duct 15 is immersed in the top layer of glass beads 8. 0.5-1 m HCI is used as main eluent. The noble metals run through the layer of cation exchanger 1 without any interaction and are then separated from one another in the layer of exclusion gel 9 2. The various fractions of rhodium (Rh), palladium (Pd), platinum (Pt) and iridium (Ir) can be collected separately at the end of the column. The base metals adsorb on the cation exchanger and are stripped with a step eluent 5 (2 m or higher HCI) at the radial position, at which the last noble metal (Ir) elutes. The 2 m HCI desorbs the base metals from the cation exchanger and drives them to the layer of exclusion gel. There the base metals do not have any interaction at all with the stationary phase. The base metals can now we recovered as a combined fraction. Specifically a solution (6 m HCI) with the following concentration is separated continuously into the various noble metals and into a fraction containing the base metals. 5 Table 1 Concentration of metals in the feed solution Pt: 30 g/l Pd: 15 g/l Rh: 3 g/l Ir: 1 g/l Fe: 4 g/l Co: 4 g/l Ni: 4 g/l Cu: 4 g/l A 26 cm high layer of Sephadex G-1 5 was covered with a 5 cm layer of glass beads. A 5 cm thick layer of Dowex 50-W X8 was added on top of this layer and was in turn covered with a layer of glass beads. The solution was eluted with 0.5 m HCI. The noble metals passed through the layer of cation exchanger 1 without any interaction and became separated on the layer of Sephadex 2. Four bands of different colours formed: Rh band (red), Pd band (brown), Pt band (yellow) and Ir band (brown). The step eluent (2 m HCI) for stripping the base metals was added at a radial position of 2700 relative to the feed inlet. The step eluent desorbed the base metals from the cation exchanger and a greenish-yellow band formed and ran through the column without any interaction. The base metals were recovered as a combined fraction after the iridium fraction.

10 Table 2 Test conditions Flow-rate: Flow-rate: Flow-rate: Rate of Cross-sectional top eluent step eluent feed rotation area of annular gap 18 ml/min 4 ml/min 0.4 ml/min 125 0 /h 24.4 cm 2 Height of column: 41 cm of which 26 cm Sephadex G-15 5 cm glass beads (d = 150-240 Dm) 5 cm Dowex 50 W-XB in the H' form 5 cm glass beads (d = 150-240 Dm) Top - Eluent Feed 'S GUl.ss beo.ed 0 360 Rh Pd Pt Ir bo.se metaLs If there are also Ru and Os in the mixture, Ru elutes between Rh and Pd and Os after Ir (see Example 2).

11 Example 2: Adsorption resin - Exclusion gel Adsorption / size-exclusion chromatography Selective separation of gold from a noble metal solution and simultaneous separation of platinum-group metals 5 The exclusion gel is filled into the P-CAC (at least up to half the height); a separation layer (glass beads or membrane) is placed over the exclusion gel, and the adsorption resin (Amberlite XAD7 or Purolite MN 200) is added on top of this layer. A noble metal solution containing all platinum-group metals (or at least two of them) 0 and gold is used as feed for the P-CAC. Gold is adsorbed selectively by the adsorber resin, whilst the other platinum-group metals flow through the layer of adsorber without being retarded and are separated out on the exclusion gel. Following elution of the last component the gold is stripped from the adsorber resin with a step eluent. Feed Eluent Step-Eluent dLscorloer resgn. exclusott. ge-L Rh RuPd Pt Ir Os Au Example 3: Cation exchanger - Anion exchanger Ion-exchange chromatography Separation of base metals from a RhIlr solution and subsequent separation of rhodium and iridium 12 The anion exchanger is filled into the P-CAC up to at least half the height. A separation layer (membrane or glass beads of appropriate size) is added on top of the anion exchanger; the cation exchanger is added on top of the separation layer up to the maximum filling height. A metal solution containing rhodium and iridium as 5 well as base metals such as iron, nickel, cobalt or copper is pumped into the annular column as feed. The base metals adsorb on the cation exchanger, whereas rhodium and iridium pass through as far as the anion exchanger. Ir (IV) adsorbs on the anion exchanger and rhodium passes through. After all the rhodium has been eluted the base metals are eluted at this angle position with step eluent 1. Following this elution ) Ir (IV) is reduced to Ir (ll) with step eluent 2. Feed Eluent Step 1 Step2 catiorn archkexm 9 er ca~on~ eX~Cheaaer Example 4: Anion exchanger - Exclusion qel lon-exchange / size-exclusion chromatography Separation of gold cyanide from a noble metal solution with simultaneous separation of the noble metals 13 The P-CAC is filled with an exclusion gel up to at least half the filling height, then covered with a separation layer, which is in turn covered with a layer of anion exchanger. A noble metal solution containing both gold as a cyanide complex and platinum-group metals is fed onto the annular gap as feed solution. The gold cyanide 5 binds itself to the anion exchanger as an anionic complex; the remaining platinum group metals pass through the anion exchanger without interaction and are separated on the exclusion gel. After the angle at which the last platinum-group metal (iridium) leaves the column a stripping solution is introduced into the annular gap to desorb the gold. ) A combination of anion exchangers and exclusion gels is also conceivable in biotechnology, e.g. to obtain and purify monoclonal antibodies. Here pyrogenes, nucleic acids and proteases can be removed by the anion exchanger; the salts of the buffer solution can then be removed in the exclusion gel. Feed Eluent Strip PxPusLo r.AeL Rh Pd Pt IrAu 14 Example 5: Affinity gel - Exclusion gel Affinity / size-exclusion chromatography Separation of base metals from a noble metal solution with subsequent separation of the noble metals 5 A P-CAC column is filled with an exclusion gel up to at least 50% of the filling height, covered with a separation layer and charged with Superlig gels up to the final filling height (Superlig gels are gels with crown ether as functional group; trademark of IBC Advanced Technology). A noble metal solution containing a high proportion of base metals is introduced as feed onto the annular gap of the P-CAC. The base metals do not have any affinity to the Superlig gel and therefore elute throughout the entire column and can be collected as a combined fraction. The noble metals are stripped by the Superlig gel and become separated on the exclusion gel in the sequence Rh Pd - Pt - Ir. Feed Eluent Strip Similar applications are conceivable with stationary phases which complexing agents (such as dithizones, dehydrodithizones, hydroxyquinolinates, pyridylazo-naphtholates etc.) have united as reactive groups. With these stationary phases, however, it is only possible to separate the noble metals qualitatively from one another under difficult conditions (a strip eluent must be used for each metal). It is therefore not 15 practicable to cover these stationary phases with yet another phase, since this would make the elution conditions more difficult and the process uneconomical. Example 6: Cation exchanger - Anion exchanger 5 Ion-exchange chromatography Two-stage purification of a cellulase The anion exchanger as top layer (Pharmacia Mono Q) is used for preliminary purification of the cellulase; a tris HCI buffer is used for elution. The fraction obtained with the cellulase in it is then fractionated further in a cation exchanger as bottom 0 layer (Pharmacia Mono S) with an acetate buffer as eluent. A very high resolution can be achieved with the cation exchanger, so the cellulase can be obtained in a pure condition. 5 Example 7: Cation exchanger - Exclusion gel Ion-exchange / size-exclusion chromatography Obtaining of monoclonal IgG2B from a fermentation broth Preliminary purification and concentration of the antibody is achieved with the cation exchanger as top phase (Pharmacia S Sepharose High Performance). For this MES is used with NaCl as buffer. The pool (fraction which also contains the antibody) is then separated from dimers/oligomers and from transferrin by means of an exclusion gel (Superdex 200) as bottom phase with a sterile sodium chloride buffer. Example 8: Anion exchanger - Exclusion gel Ion-exchange / size-exclusion chromatography Purification of Human Epidermal Growth Factor (h-EGF), expressed as extracellular product through Saccharomyces cerevisiae The anion exchanger (Pharmacia Q - Sepharose) as top phase is used for preliminary purification (purification based on charge) of the growth factor; the bottom 16 phase, the exclusion gel (Pharmacia Superdex 75), is used for final purification of the growth factor. 5 Example 9: Hydrophobic interaction chromatography (HIC) qel - Exclusion gel Affinity / size-exclusion chromatography Obtaining of human pituitary prolactin The upper stationary phase (HIC gel, Pharmacia Phenyl Sepharose CL) is used for initial purification and concentration of the prolactin (concentration by a factor of 7). ) In the second stage the prolactin undergoes final purification on an exclusion gel (e.g. Pharmacia Sephadex G100). Example 10: Cation exchanger - Hydrophobic interaction chromatography (HIC) gel Ion-exchange / affinity chromatography Obtaining of a 2 -macroglobulin With the cation exchanger (upper stationary phase, e.g. Pharmacia Mono S) it is possible to obtain a protein pool containing proteins with the same charge, including the macroglobulin. The macroglobulin is purified in the second layer, the HIC gel (e.g. Pharmacia Phenyl Sepharose), until there is homogeneity. Example 11: Anion exchanger - Hydrophobic interaction chromatography (HIC) gel Ion-exchange / affinity chromatography Purification of a recombinant reverse transcriptase of HIV The fermentation broth is separated according to charge in the upper phase, the anion exchanger (e.g. Pharmacia DEAE Sepharose). The pool containing the transcriptase passes through the second layer, the HIC gel (e.g. Pharmacia Phenyl Sepharose High Performance). The transcriptase is obtained in pure form as a result of the differences in hydrophobicity after this layer.

17 Example 12: Cation exchanger - Anion exchanger, with one of the stationary phases in displacement mode Ion-exchange / displacement chromatography 5 Protein separation An anion exchanger (e.g. Pharmacia Mono Q) is filled into an annular chromatograph. An inert layer, e.g. glass beads or a membrane, is placed on top of this anion exchanger; a cation exchanger (e.g. Pharmacia Mono S) is then added in turn on top of this. A mixture of different proteins is separated on the cation 0 exchanger because of the different charges; the separated fractions are concentrated on the anion exchanger using a displacement reagent (displacer) (e.g. Nalcolyte). In this way separation can be combined with simultaneous concentration. 5 Example 13: Exclusion qel - Electrophoretic gel Size-exclusion chromatography / electrophoresis Protein separation The electrophoretic gel forms the lower layer in the P-CAC and is covered with an exclusion gel. If proteins with different charges and different pi values are now 3 brought onto the exclusion gel they separate according to size. The electrophoretic layer then takes care of further separation of the proteins according to their charge. Example 14: Exclusion gel - Cation exchanger, with the cation exchanger in displacement mode 5 Size-exclusion / ion-exchange chromatography Working-up of an amino-acid mixture with simultaneous separating-off of proteins The upper stationary phase, an exclusion gel (e.g. Pharmacia Sephadex G25) is used to separate off proteins (e.g. albumins) from a feed solution containing the amino acids L-glutamic acid, L-valine and L-leucine. Separation in the exclusion gel yields a fraction containing the proteins and a fraction containing the amino acids. In 18 the second layer the amino acids are separated on a cation exchanger (e.g. Dowex 50 W-X8) with a displacer (e.g. 0.1 n NaOH) and simultaneously concentrated. The amino acids are obtained in the order glutamic acid - valine - leucine. A regenerating solution (e.g. dilute H 2

SO

4 ) has to follow-the displacer in order to bring the gel bed to 5 the initial state. The example of displacement elution is not limited to use in cation exchange. As can be seen from the above description, the present invention provides a new annular chromatograph with a particulate bed comprising several different zones 0 which can be used to carry out several types of chromatographic separation continuously in a single stage and thereby more quickly and more economically than has previously been possible according to the state of the art.

Claims

1. Annular chromatograph with charging in the form of a particulate bed, characterised by the fact that the particulate bed comprises at least two zones 5 (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 by the fact 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 by the fact 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 by the fact that the bed material fo'r 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 by the fact 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 by the fact 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 by the fact that the particulate bed is covered with a covering layer (8) and/or has 20 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. 5

9. Annular chromatograph as claimed in any one of the above claims, characterised by thefact 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. 0

11. Annular chromatograph as claimed in any one of the above claims, characterised by the fact 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).