WO1999028740A1

WO1999028740A1 - Annular chromatograph

Info

Publication number: WO1999028740A1
Application number: PCT/AT1998/000290
Authority: WO
Inventors: Adalbert Prior
Original assignee: Prior Separation Technology Gmbh
Priority date: 1997-12-01
Filing date: 1998-11-30
Publication date: 1999-06-10
Also published as: EA199901022A1; AU754824B2; CA2301570A1; EA001640B1; EP1183531A1; AT405025B; ATA203097A; AU1474399A

Abstract

The present invention relates to an annular chromatograph comprising a coating in the shape of a particulate bed, wherein said chromatograph is characterised in that the particulate bed comprises at least two superimposed areas which contain different materials and which are separated from each other by at least one separation layer.

Description

Annular chromatograph

The present invention relates to a circular chromatograph with a feed in the form of a particle bed.

Annular chromatography has been a variant of preparative chromatographic separations that has been recognized for a number of years and is increasingly being used. Annular chromatography is preferably used when large amounts of substance mixtures have to be separated, since this type of chromatography can be operated continuously, and with a relatively high degree of resolution.

A typical P-CAC apparatus ("P-CAC", preparative continuous annular chromatography = preparative continuous annular chromatography) consists of an annular particle bed which is packed in the space (annular gap) between two concentric cylinders. While rotating the particle bed around its axis, feed solution and one or more eluents are continuously added at the upper end. Such procedures are known and widely used in the prior art (see e.g. EP-A-371 .648).

In addition to the advantage of the high throughput of annular chromatography, this is also characterized by high resolution and specificity. Nevertheless, for various specific separation problems it is necessary to follow up further annular chromatography steps - for example using different separation gels than in the first step - in order to achieve the desired degree of separation. On the one hand, this is relatively easy to achieve in comparison to conventional column chromatography, since in the case of annular chromatographic separations it is known at which point on the circumference the desired product or products emerge; on the other hand, further annular chromatographs are required for this, which of course greatly increases the outlay in terms of equipment and therefore money. The aim of the present invention is therefore to provide a device for annular chromatography, by means of which the resolution and specificity of this chromatographic separation process can be increased efficiently without excessively increasing the outlay on equipment and thus the process costs.

The invention achieves this aim by means of an annular chromatograph with a feed in the form of a particle bed, which is characterized in that the particle bed has at least two zones arranged one above the other, which contain different bed material and are preferably separated from one another by at least one separating layer.

In this way, preliminary and main separation steps or several separations on a different basis, such as Carry out exclusion or affinity and ion exchange chromatography etc., successively within the same separating device, continuously and with high resolution and specificity, with the additional time, cost and equipment required for column changes, including e.g. Concentration and conditioning of the mixture for the next separation step, regeneration or re-equilibration of the columns, etc., as well as (e.g. in the case of substances sensitive to oxidation or moisture) the risk of impairment of the samples when handling them is minimized or completely eliminated.

In a particularly preferred embodiment, at least one of the zones can be adapted as an electrophoresis zone by providing electrical connections, preferably in the form of sliding contacts, as a result of which electrophoretic separations can also be carried out in the advantageous manner described above. This electrophoresis zone can preferably be delimited by at least one electrically non-conductive separating layer to the other zone (s) in order to increase the efficiency of the electrophoresis. The bed material for the at least two zones can generally consist of anion exchange resins, cation exchange resins, exclusion gels, gel permeation gels, affinity gels, hydrophobchromatography (HIC) gels, displacement (displacement) resins, reversed phase (reversed phase) gels, Electrophoresis gels and other separation media used in practice, which offers a variety 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 electrophoresis gel is of course chosen as the particle bed for this zone.

According to the invention, the separating layer or separating layers are preferably selected from membranes, non-porous, inert particle material, in particular glass beads, and - especially for any electrophoresis zones - from electrically non-conductive material.

In preferred embodiments of the invention, the uppermost zone of the particle bed is covered with a cover layer and / or underlaid with a base layer, the cover and base layers preferably being made of the same material as the separating layer (s).

A chromatograph according to the invention particularly preferably comprises at least one electrophoresis zone or at least one exclusion gel zone and at least one adsorber resin zone, in particular at least one adsorber resin zone containing an ion exchange resin, which, for example, proteins (one after the other according to their size and their charge), as well as numerous biotechnological process products such as eg enzymes, h-EGF ("human epidermal growth factor") and immunoglobulins, separated and cleaned extremely selectively and with high resolution. In one embodiment of the invention, a tempering (ie heating or cooling) jacket can also be provided on the inner and / or outer circumference of the annular separating column (11) in order to be able to set the optimum temperature for each separation.

In the following, the invention will be described by way of examples and with reference to the accompanying drawings, in which FIGS. 1 a) and 1 b) are schematic representations of an annular chromatograph with 2 or 3 separation zones that can be used according to the present invention, FIG. 2a) is a schematic FIG. 2b) is a schematic enlargement of a detail of the dashed area from FIG. 2a). FIG. 2b) is a partial view of a section through a further annular chromatograph according to the invention with an electrophoresis zone.

1 a) and 1 b) two variants of the annular chromatograph of the present invention are shown schematically. 1 a) shows an embodiment with two, in FIG. 1 b) one with three separation zones of the particle bed. These zones 1 and 2, or 1, 2 and 3, preferably consist of release resins selected from e.g. Anion exchange resins, cation exchange resins, exclusion gels, affinity gels, hydrophobchromatography (HIC) gels, displacement (displacement) resins, electrophoresis gels, and optionally from other stationary phases customary in the field, such as e.g. Silica gel, aluminum oxide, etc. This wide range of options results in a large variety of combinations of two or more of these release agents.

For example, the invention comprises the following combinations of two stationary phases:

• Cation exchanger - anion exchanger

• Cation exchanger - exclusion gel

• Anion exchanger - exclusion gel

• Cation exchanger - affinity gel • Anion exchanger - affinity gel

• Cation exchanger - hydrophobic chromatography gel

• Anion exchanger - hydrophobic chromatography gel

• Exclusion gel - affinity gel

• Exclusion gel - hydrophobic chromatography gel

• Hydrophobchromatography gel - affinity gel

and such combinations with an electrophoresis gel instead of or in addition to one of the two gels above (three-zone chromatography). The above list of binary combinations does not describe how the gels are arranged in the column relative to each other, i.e. which is the top and which is the bottom gel. This depends solely on the separation problem.

Examples of separation techniques that can be used in the invention include:

Ion exchange chromatography: e.g. Mono S (Pharmacia) or Toyopearl DEAE 650S (TosoHaas);

Reversed phase chromatography: e.g. Amberchrom CG-161 cd (Rohm & Haas) or Sephasil C8 (Pharmacia);

• Hydrophobchromatography: Phenyl Sepharose (Pharmacia) or TSK Gel Phenyl 5PW (TosoHaas);

Size exclusion and gel permeation chromatography: e.g. Sephadex G1 5 (Pharmacia) or Toyopearl HW 40F (TosoHaas);

Affinity chromatography: e.g. Toyopearl AF Tresyl-650M (TosoHaas) or EAH Sepharose-4B (Pharmacia);

Adsorption chromatography: e.g. Amberlite XAD (Rohm & Haas) or Purolite MN200 (Purolite).

Combinations of two or more gels of the same type, but for example from different manufacturers, are also included in the scope of the invention. - for example, two cation exchange gels or two exclusion gels can also be arranged one above the other (eg Sephadex G 15 via Toyopearl HW 40F or the like).

According to the invention, particular preference is given to annular chromatographs with a particle bed comprising at least one exclusion gel zone and at least one adsorber resin zone, in particular at least one adsorber resin zone containing an ion exchange resin, and annular chromatographs with at least one electrophoresis gel in one of the zones.

The two or three gels are introduced one after the other into the annular gap of an annular column 11 made of material which is inert to the components of the separation solutions, preferably glass, each of the gel zones being covered with a separation layer 5 before the next is applied thereon to prevent mixing of the different zone materials. In both cases, the top of the particle bed forms a cover layer 8. In FIG. 1 a), a base layer 9 is additionally shown, which serves (in addition to a porous base plate, not shown), for example frit, membrane disc, etc. to prevent particulate matter from escaping at the bottom of the column. The material for the separating, covering and base layers 5, 8, 9 is selected from membranes and non-porous particle material which is inert to all components of the respective separating solutions and can be the same or different for all three layers, but it is special must not be electrically conductive for electrophoretic separations. Glass beads are preferred according to the invention, since they are inert and easy to apply in practically all common applications.

For the annular chromatograph from FIG. 1 a), the loading takes place in the order 9-2-5-1 -8, in FIG. 1 b) in the order 3-5-2-5-1-8.

The other components of the annular chromatograph can be designed in a conventional manner. So the column 1 1 (driven by a motor, not shown) rotatably mounted on an axis 12 and is fed via connecting lines 1 3 for feed and eluents and a distributor head 14 fixedly mounted on the axis, the feed channels 15 of the distributor head 14 preferably immersing in the cover layer 8 in order to ensure an even task. The channels can have the usual designs, ie single, multiple or slot nozzles or the like, but curved slot nozzles of different widths which are adapted to the column circumference are preferred for the invention, in order to be able to match the feed and eluent flows as closely as possible.

At the lower end of the columns, outlet channels 16 are provided for collecting the eluates. These outlets 16 can either be connected to the column 11 (i.e. they rotate with it about axis 12) or they can be fixed to the axis 12 and e.g. are in contact with the column rotating relative to it via a slip ring, the latter embodiment being preferred.

2a) shows a partial view of an embodiment of an annular chromatograph according to the invention with an electrophoresis separation step, namely the electrophoresis separation zone 4, which leads to another zone above and below (not shown) by means of a separation layer 5 made of electrically non-conductive material is limited. Slip contacts are provided both at the upper and at the lower end of the electrophoresis zone 4, preferably directly adjacent to the respective separation layer (s), each consisting of a conductor ring 6 mounted on the outside of the axis 12, each of which has a network connection 10 is supplied with current, as well as from an annular current collector 7 provided on the inside of the column 1 1, which lies closely against the conductor ring 6 and is in electrical contact therewith. The latter is guided through the column jacket in the area of the sliding contact - either selectively or flatly - in order to build up the electrical voltage required for the electrophoretic separation in the interior of the column. 2b) shows an enlarged view of the dashed area from FIG. 2a) and shows the sliding contact in somewhat more detailed form.

Specific application examples for the present invention follow. However, the invention is by no means restricted to these possible uses. Preferred fields of application for annular chromatographs according to the invention are e.g. Separations of base metals and precious metals as well as numerous separations and cleaning steps in biotechnology.

Example 1: Cation exchanger - exclusion gel: ion exchange-size exclusion chromatography

Liquid chromatographic separation of the platinum group metals with simultaneous

Removal of base metal

The platinum group metals rhodium, palladium, platinum and iridium can be continuously separated from one another by means of preparative annular chromatography. Common metals present at the same time, such as iron, copper, nickel or cobalt, can also be continuously separated from the precious metals.

The chromatographic separation of the platinum group metals using exchange gels (e.g. Sephadex, Toyopearl or Biogel) has already been described in US-A-4,885,143. Concentrated precious metal solutions from the leaching processes of the tailor shops usually also contain base metals such as iron, copper or nickel in different concentrations.

After the leaching process, the precious metal solution is usually in concentrated hydrochloric acid. Due to the high chloride concentration, the noble metals are all present as anionic chloro complexes. Base metals have the property of binding to a cation exchanger under certain conditions, while the noble metals as anionic complexes pass through the cation exchanger without retention. This property can be used to make the base in the first step Separate accompanying metals from the precious metals and separate the precious metals from one another in a second process step. With the P-CAC system of the present invention, this is possible in the simplest apparatus design.

A column, as shown in Fig. 1 a), is filled 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 layered over it (d = 1 50-240 µm); A cation exchanger 1 (e.g. Dowex, Purolite, Amberlite) and finally another glass bead layer 8 are layered over this glass bead layer 5.

The feed solution is pumped into the annular column at the radial position 0 °. The feed channel 15 is immersed in the upper glass bead layer 8. The main eluent (top eluent) is 0.5-1 m Hcl. The noble metals run through the cation exchange layer 1 without interaction and are then separated from one another in the exclusion gel layer 2. At the end of the column, the individual fractions of rhodium (Rh), palladium (Pd), platinum (Pt) and iridium (Ir) can be collected separately.

The base metals adsorb to the cation exchanger and are stripped at the radial position where the last precious metal elutes (Ir) with a step eluent (2 m or higher HCI). The 2 m HCI desorbs the base metals from the cation exchanger and drives them to the exclusion gel layer. There the base metals have no interaction with the stationary phase. The base metals can now be obtained as a common fraction.

In particular, a solution (6 m HCl) of the following concentration is continuously separated into the individual noble metals and into a fraction which contains the base metals. Table 1 Concentration of the metals in the feed solution

A 26 cm high layer of Sephadex G-15 was covered with 5 cm glass beads. A 5 cm thick layer of Dowex 50-W X8 was applied to this layer, which in turn was overlaid with a layer of glass beads. The solution was eluted with 0.5 m HCl. The noble metals passed through the cation exchange layer 1 without interaction and separated on the Sephadex layer 2. Four differently colored bands formed - Rh band (red), Pd band (brown), Pt band (yellow) and Ir band (brown).

In a radial position of 270 ° relative to the feed point, the step eluent (2 m HCI) was stripped for the base metals. The step eluent desorbed the base metals from the cation exchanger, and a greenish-yellow band formed that runs through the column without interaction. The base metals could be obtained as a common fraction after the iridium fraction.

Table 2 Test conditions

Height of the column: 41 cm, thereof

26 cm Sephadex G-15 5 cm glass beads (d = 150-240 μm) 5 cm Dowex 50 W-X8 in the H ⁺ shape 5 cm glass beads (d = 150-240 μm). Top eluent

If Ru and Os are also contained in the separation mixture, Ru elutes between Rh and Pd and Os according to Ir (see Example 2).

Example 2: Adsorption resin - exclusion gel adsorption Z size exclusion chromatography

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

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, the adsorption resin (Amberlite XAD7 or Purolite MN 200) is filled over this layer.

A precious metal solution containing all the platinum group metals (but at least two of them) and gold is used as the feed for the P-CAC. Gold is from the adsorber resin selectively adsorbed, while the other platinum group metals run unretarded through the adsorbent layer and are separated on the exclusion gel. After elution of the last component, the gold is stripped from the adsorber resin using a step eluent.

Adsorber resin

Exclusion gel

Example 3: Cation Exchanger - Anion Exchanger Ion Exchange Chromatography

Separation of the base metal from a Rh / Ir solution and subsequent separation of rhodium and iridium

The anion exchanger is filled at least halfway up the P-CAC. A separation layer is filled over the anion exchanger (membrane or glass beads of the appropriate size), and over the separation layer the cation exchanger up to the maximum filling level. A metal solution containing rhodium and iridium as well as non-precious metals such as iron, nickel, cobalt or copper is pumped into the annular column as a feed. The base metals adsorb on the cation exchanger, while rhodium and iridium pass through to the anion exchanger. Ir (IV) adsorbs on the anion exchanger and rhodium runs through. After all rhodium is eluted, turn on this angular position with the step eluent 1 elutes the base metals. After this elution, the reduction from Ir (IV) to Ir (III) takes place with the step eluent 2.

Feed Eluent Stepl Step2

Cation exchangers

Anion exchanger

Example 4: Anion exchanger - exclusion gel ion exchange-size exclusion chromatography

Separation of gold cyanide from a precious metal solution with simultaneous

Separation of the precious metals

The P-CAC is filled with an exclusion gel at least up to half the fill level, then overlaid with a separating layer, which is in turn overlaid with an anion exchanger. A precious metal solution, which in addition to gold as a cyanide complex also contains the platinum group metals, is applied to the ring gap as a feed solution. The gold cyanide binds as an anionic complex to the anion exchanger, the remaining platinum group metals pass through the anion exchanger without interaction and are separated on the exclusion gel. At the angle at which the last platinum group metal (iridium) leaves the column, a strip solution for the desorption of the gold is introduced into the annular gap. A combination of anion exchangers and exclusion gels in biotechnology is also conceivable; eg for the production and purification of monoclonal antibodies. The anion exchanger can be used to remove pyrogens, nucleic acids and proteases; the salts of the buffer solution can then be removed in the exclusion gel.

Anion exchanger

Exclusion gel

Example 5: Affinity Gel - Exclusion Gel

Affinity Z size exclusion chromatography

Separation of base metals from a precious metal solution under the following

Separation of the precious metals

A P-CAC column is filled with an exclusion gel to at least 50% of the fill level, overlaid with a separating layer and loaded with superlig gels up to the final fill level (superlig gels are gels with crown ether as a functional group; trademark of IBC Advanced Technology) . A precious metal solution containing a high proportion of base metals is applied as a feed to the ring gap of the P-CAC. The base metals have no affinity for the Superlig gel and therefore elute through the entire pillar and can be collected as a common fraction. The precious metals are stripped from the Superlig gel and separate on the exclusion gel in the order Rh - Pd - Pt - Ir.

Feed eluent strip

Similar applications are conceivable with stationary phases that have complexing agents (such as dithizones, dehydrodithizones, hydroxyquinolinates, pyridylazo-naphtholates, etc.) coupled as reactive groups. With these stationary phases, however, it is only possible under difficult conditions to qualitatively separate the noble metals from one another (a strip eluent must be used for each metal). It is therefore not sensible to overlay these stationary phases with another phase, as this would make the elution conditions even more difficult and would make the process uneconomical. Example 6: Cation Exchanger - Anion Exchanger Ion Exchange Chromatography Two-step purification of a cellulase

The anion exchanger as the upper layer (Pharmacia Mono Q) serves to pre-clean the cellulase; is eluted with a Tris.HCI buffer. The fraction obtained, in which the cellulase is located, is then further fractionated in a cation exchanger as under layer (Pharmacia Mono S) with an acetate buffer as the eluent. A very high resolution can be achieved with the cation exchanger, so that the cellulase can be obtained in a pure state.

Example 7: Cation exchanger - exclusion gel ion exchange-size exclusion chromatography

Obtaining monoclonal IgG _2B from a fermentation broth

A preliminary cleaning and concentration of the antibody is achieved with the cation exchanger as the upper phase (Pharmacia S Sepharose High Performance). MES with NaCI is used as a buffer. The pool (fraction which also contains the antibody) is then freed of dimeric oligomers and of transferrin via an exclusion gel (Superdex 200) as the lower 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, Human Epidermal Growth

Factor), expressed as an extracellular product by Saccharomyces cerevisiae

The anion exchanger (Pharmacia Q - Sepharose) as the upper phase serves for the pre-cleaning (cleaning based on the charge) of the growth factor, the lower phase, the exclusion gel (Pharmacia Superdex 75) serves for the final cleaning of the growth factor. Example 9: Hydrophobchromatography (HIC) gel exclusion gel

Affinity Z Size Exclusion Chromatography

Obtaining human pituitary prolactin

The upper stationary phase (HIC gel, Pharmacia Phenyl Sepharose CL) is used for the initial cleaning and concentration of the prolactin (concentration by a factor of 7). In the second step, the prolactin is finally purified on an exclusion gel (e.g. Pharmacia Sephadex G 100).

Example 10: Cation Exchanger - Hydrophob Chromatography Gel Ion Exchange ZAffinity Chromatography

Obtaining α-2 macroglobulin

With the cation exchanger (upper stationary phase, e.g. Pharmacia Mono S) one can

Protein pool can be obtained that contains proteins of the same charge, including the macroglobulin. The macroglobulin is in the second layer, the HIC gel

(e.g. Pharmacia Phenyl Sepharose) cleaned to homogeneity.

Example 11: Anion Exchanger - Hydrophob Chromatography Gel Ion Exchange ZAffinity Chromatography

Purification of a recombinant reverse transcriptase from HIV

In the upper phase, the anion exchanger (e.g. Pharmacia DEAE Sepharose), the fermentation broth is separated after loading. The pool in which the transcriptase is located passes through the second layer, the HIC gel (e.g. Pharmacia Phenyl Sepharose High Performance). Due to the different hydrophobicity after this layer, the transcriptase is obtained in pure form.

Example 12: Cation exchanger - anion exchanger, with one of the stationary phases in the displacement (displacement) mode ion exchange-Z-displacement chromatography protein separation An anion exchanger (eg Pharmacia Mono Q) is filled into an annular chromatograph. An inert layer, for example glass beads or a membrane, is layered on this anion exchanger and a cation exchanger is filled in (for example Pharmacia Mono S). A mixture of different proteins is separated on the cation exchanger due to different charge, the separated fractions are concentrated on the anion exchanger using a displacement reagent or displacer (eg Nalcolyte). Separation and simultaneous concentration can thus be combined.

Example 1 3: Exclusion gel - electrophoresis gel

Size exclusion chromatography electrophoresis

Protein separation

The electrophoresis gel forms the lower layer in the P-CAC, which is covered with an exclusion gel. If proteins of different charge and different pI values are now applied to the exclusion gel, they separate due to their size. The electrophoretic layer then takes over the additional separation of the proteins based on their charge.

Example 14: Exclusion gel - cation exchanger, with the cation exchanger in displacement mode, size exclusion zone exchange chromatography

Processing of an amino acid mixture with simultaneous separation of proteins

The upper stationary phase, an exclusion gel (eg Pharmacia Sephadex G25) is used to separate proteins (eg albumins) from a feed solution which contains the amino acids L-glutamic acid, L-valine and L-leucine. Separation in the exclusion gel gives a fraction containing the proteins and a fraction containing the amino acids. In the second layer, the amino acids are separated on a cation exchanger (eg Dowex 50 W-X8) with a displacer (eg 0.1 N NaOH) and concentrated at the same time. The amino acids are listed in the order glutamine Acid - valine - leucine obtained. The displacer must be followed by a regeneration solution (eg dilute H ₂ S0 ₄ ) in order to bring the gel bed into its original state.

The example of displacement elution is also 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 particle bed consisting of several different zones, with which several types of chromatographic separations can be carried out continuously, in one step and thus more quickly and cost-effectively than in the prior art was previously possible.

Claims

Claims:

1 . Annular chromatograph with a charge in the form of a particle bed, characterized in that the particle bed has at least two zones (1, 2, 3, 4) arranged one above the other, which contain different bed material and are preferably separated from one another by at least one separating layer (5).

2. Annular chromatograph according to claim 1, characterized in that at least one of the zones (4) is formed by providing electrical connections, preferably in the form of sliding contacts, (6, 7) as an electrophoresis zone for carrying out electrophoretic separations.

3. Annular chromatograph according to claim 2, characterized in that the electrophoresis zone (4) by at least one electrically non-conductive separation layer (5) to the or the other zone (s) (1, 2, 3) is limited.

4. Annular chromatograph according to one of claims 1 to 3, characterized in that the bed material for the at least two zones (1, 2, 3, 4) from anion exchange resins, cation exchange resins, exclusion gels, gel permeation gels, affinity gels, hydrophobic chromatography (HIC) Gels, displacement (displacement) resins, reversed phase (reversed phase) gels and electrophoresis gels is selected.

5. Annular chromatograph according to claim 4, characterized in that the bed material for the electrophoresis zone (4) is selected from electrophoresis gels.

6. Annular chromatograph according to one of the preceding claims, characterized in that the at least one separating layer (5) is selected from membranes, non-porous, inert particle material, preferably glass beads, and electrically non-conductive material.

7. Annular chromatograph according to one of the preceding claims, characterized in that the particle bed is covered with a cover layer (8) and Z or is underlaid with a base layer (9), the cover and base layers (8, 9) preferably made of the same material as that Separating layer (s) (5) exist.

8. Annular chromatograph according to one of the preceding claims, characterized in that the particle bed contains at least one electrophoresis zone.

9. Annular chromatograph according to one of the preceding claims, characterized in that the particle bed contains at least one exclusion gel zone and at least one adsorber resin zone.

10. Annular chromatograph according to claim 9, characterized in that at least one adsorber resin zone contains an ion exchange resin.

1 1. Annular chromatograph according to one of the preceding claims, characterized in that a tempering (i.e. heating or cooling) jacket is provided on the inner and outer circumference of the annular separating column (1 1).