USE of NON--INDIVIDUAL SORBENTIEN FOR ßSIMULATED MOVING BEDß SEPARATION PROCESS

The invention concerns the use of diaphragms as Sorbentien, in particular such, which contain Separationseffektoren, for “Simulated Moving Bed” separation process.

For economy from präparativen Stofftrennungen is the achievement of high Fluß guess, short Elutionszeiten and the adherence to moderate operating pressures substantial factors. With präparativen Stofftrennungen countercurrent processes at meaning won. Since it is technically only very with difficulty possible to realize an actual movement of a stationary phase the movement of the stationary phase is simulated. In addition the entire column bed is divided into cyclically connected in series single columns. The total number of the columns is typically a multiple of four, since such a system possesses four chromatographische zones. After a defined time the lines are switched, whereby a movement of the column bed in the opposite direction is simulated. For the continuous procedure “simulated moving bed” - chromatography (SMB chromatography) as separation materials individual Sorbentien is usually used. The column packings used thereby do not leave optimal Fluß advice, since the operating pressure is very high with individual carriers. Also the mechanical stability of the individual Sorbensbetten is not very good. Further it is necessary for the SMB chromatography to make a number available of chromatographischer columns (typically up to 24) with as same a characteristics as possible. This is with individual Sorbensbetten only with groß em expenditure with packing the columns and with the selection of the packed columns realizable.

Task of the invention is it to make chromatographische präparative separation processes available for the SMB procedures the Flu&szlig high with moderate operating pressure; guess/advise exhibit. Thus a higher throughput per time unit can be obtained.

It was found, daß non-individual Sorbentien, in particular monolithic Sorbentien or Sorbentien on the basis of derivatisierten diaphragms, for separation processes with high Fluß guess/advise to be used can; thus a higher throughput per time unit can be obtained with low operating pressure.

The subject of the invention are procedures for the präparativen chromatographischen separation of at least two substances in the SMB procedure, whereby as stationary phase a Sorbens on the basis of derivatisierten diaphragms gemäß Requirements 1 and 2 one uses.

Illustration 1 shows a schematic representation of the procedure of the counter current chromatography, which the basis “simulated moving bed” - chromatography (SMB chromatography) represents.

Illustrations 2a) to 2C) show a präparative separation of a test mixture from Tolulol, 2-Nitroacetanilid and 3-Nitroacetanilid with different Fließ speeds of the mobile phase (fig. 2a: 40 ml/min; Fig. 2b: 130 ml/min; Fig. 2C: 200 ml/min). Experimental details are descriptive in example 1.

Illustration 3 shows the separation from Dimethylphthalat/Dibutylphthalat at a C18-RP-Phase. Experimental details are descriptive in example 2.

Illustration 4 shows the Elutionsdiagramm of a separation from Chymotrypsinogen A and Lysozym in the SMB procedure at a parting agent from porous modified PP, the SO3^--Contains groups as Separationseffektoren, whereby the SO3^--Groups over a bridge grouping to the amino groups of the PP are bound. Experimental details are descriptive in example 3.

Common individual Sorbentien consists of discrete Sorbensteilchen, which are filled into a pipe abschlossenes by Filterelemente and which Sorbensbett result in. Individual Sorbentien consists of spherischen or unregelmäß industrial union formed particles, whose particle dimensions lie within the range of approx. 1 µm up to 1 mm, in particular from 5 µm to 500 µm. The attainable partition stage height depends on the Partikelgröß e and becomes with lower Partikelgröß e smaller, i.e. the attainable chromatographische dissolution becomes better. However rises with falling Partikelgröß e the decrease of pressure with same linear Fluß on, so daß with small particles the linear Fluß by the manageable operating pressure is limited. Auß erdem one observes a pronounced dependence of the separative power on the linear Flu&szlig with individual Sorbentien; (steep H/u curves). Altogether the parameter relevant for the separative power and economy exhibits a strong interdependence with individual Sorbentien, those the Prozeß optimization in particular for präparative applications makes more difficult. Contrary to common individual Sorbentien, with their use always a Kompromiß between decrease of pressure in the sorbensgefüllten column, attainable partition stage height and dependence of the separative power of the linear Fluß (H/u curve) to be received muß , it shows up, daß these parameters with non-individual Sorbentien are substantially less strictly coupled.

It showed up surprisingly, daß when using non-individual, in particular monolithic Sorbentien the H/u curves is flat. Further with non-individual Sorbentien, which cause a small decrease of pressure, low partition stage heights are found. Thus präparativ chromatographische separation processes, in particular such can be optimized in the SMB procedure, using non-individual Sorbentien substantially better than in use of individual Sorbentien; the economy of these processes can be improved importantly.

The term “non-individual” Sorbentien used here represents the contrast to well-known characterized above individual Sorbentien, with which the Sorbensbett consists of individual discrete particles. Both monolithic Sorbentien and with Separationseffektoren derivatisierte diaphragms becomes of the term non-individual Sorbentien umfaß t.

Monolithic Sorbentien is in principle well-known from the literature; in addition above all porous ceramic molded articles belong, how they are revealed in WHERE 94/19 687 and in WHERE 95/03 256. By the term monolithic Sorbentien becomes in the broader sense also molded articles from polymers umfaß t, like it in EP 0,407,560 and of F. Svec and J.M. Frechet (1992) Anal. Chem. 64, pages 820 - 822, and of S. Hjerten et al. (1989) J. Chromatogr. 473, pages 273 - 275, was described. Monolithic Sorbentien on the basis of porous molded articles, which exhibit among themselves connected macropores as well as Mesoporen in the walls of the macropores, is particularly preferential, whereby the diameter of the macropores a median größ it as 0.1 µm exhibits, and whereby the diameter of the Mesoporen exhibits a median of 2 and 100 Nm. Such monolithic Sorbentien and procedures for their production are for example in WHERE 95/03 256 revealed.

Monolithic Sorbentien consists thus of materials, as they are for individual Sorbentien the used. In many cases (e.g. SiO2) this Sorbentien can be used easily for chromatographische separations. More frequently however the basis carriers are derivatisiert, in order to improve the separation characteristics; additional groupings are introduced, those under the designation Separationseffektoren zusammengefaß t become, and which for the separation of the Analyte are substantial.

With Separationseffektoren adsorbate effective diaphragms derivatisierte is revealed in WHERE 91/03 506 and in WHERE 96/22 316. as well as in WHERE 97,149,754. Erfindungsgemäß the use in WHERE 96/22 of the 316 is and in WHERE 97,149,754 revealed derivatisierten PP diaphragms. Procedures for the production of these diaphragms are indicated in these block letters. Such diaphragms are usually used in devices, which can be essentially handled like chromatographische columns. The use of a device is particularly preferential, as it is revealed in DE 196 03 523,6 (AKZO Nobel).

Separationseffektoren and procedures for their introduction to the basis carrier are the specialist in principle well-known.

Examples of reactions, with which Separationseffektoren can be introduced into SiO2 or similar oxidic basis carriers, are:

1. a) The Derivatisierung with silane derivatives of the formula ISiXnR1_(3-n) R2 where

   X

   Methoxy, Ethoxy or halogen or Aminoderivate,

   G 1

   C1-C5-Alkyl,

   n

   1, 2 or 3

   mean and

   R2

   one in the following indicated meanings possesses:

   1. a1) unsubstituiertes or substituted alkyl or aryl, like e.g. n-Octadecyl, n-Octyl, Benzyloder Cyanopropyl;
   2. a2) anionische or sour remainders, like e.g. Carboxypropyl;
   3. a3) kationische or basic remainders, like e.g. Aminopropyl, Diethylaminopropyl or Triethylammoniumpropyl;
   4. a4) hydrophilic remainders, like e.g. (2,3-Dihydroxypropyl) - oxypropyl;
   5. a5) connectionable activated remainders, like e.g. (2,3-Epoxypropyl) - oxypropyl.

2. b) The adsorption or chemical connection of polymers such as polybutadiene, Siloxanen, polymers on the basis of styrene Divinylbenzol, of (Meth) acrylic acid derivatives or of other Vinylverbindungen, as well as of Peptiden, proteins, Polysacchariden and Polysaccharidderivaten at the basis carrier;
3. c) The chemical connection of polymers specified under b) over the derivatives specified under a); in addition grafting polymers of Poly (meth) belong to acrylic acid derivatives on diolmodifiziertem silicagel after EP-B-0 337,144.
4. d) The adsorption or chemical connection of chiralen phases, as e.g. of amino acid derivatives, Peptiden or proteins, or of Cyclodextrinen, Polysacchariden or Polysaccharidderivaten.

Further common Derivatisierungsmöglichkeiten and Derivatisierungsverfahren are the specialist admit and in usual manuals such as Unger, K.K. (OD) Porous Silica, Elsevier Scientific Publishing company (1979) or Unger, K.K. Packings and Stationary far in Chromatographic Techniques, Marcel Dekker (1990) described.

Other monomers specified in this block letters permit the connection of Affinitatsliganden, or are suitable for reversed phase to chromatography: in addition belong for example acrylic acid, acrylamide, allyl amine or acrylonitrile.

Further the epoxy groups in such polymers can be converted in favourable way further, whereby ion exchangers, thiophile Sorbentien or Sorbentien for the Metallchelatoder the hydrophobe chromatography are made available. For example phosphoric acid, Diethylamin, tri methyl amine, sulfurous acid or also complexing agents such as Iminodiessigsäure at the Oxiranring are added.

The production of thiophilen Sorbentien and of Sorbentien for the Metallchelatchromatographie is revealed in DE 43 10 964.

In DE 43 33 674 and in DE 43 33 821 is revealed such conversions, with those assistance ion exchanger to be made available can.

In DE 43 23 913 is described Sorbentien for the hydrophobe interaction chromatography.

Chirale separation materials for the separation from Enantiomeren are in groß it number while stationary the technology admits. It acts ausschließ lich around individual Trenrimaterialien. The well-known chiralen separation materials consist either of the chiralen connection (for example cellulose triac budget) or however a chiraler Separationseffektor is drawn up or chemically bound to a carrier on a carrier. Auß it is possible for erdem to add chirale Separationseffektoren, which step with a stationary phase into reciprocal effect, in the Elutionsmittel (dynamic allocation).

Chirale Separationseffektoren are in groß it number admits; the most important groups well-known chiraler Separationseffektoren are:

1. a) Amino acids and its derivatives, e.g. L-Phenylalanin. or D-Phenylalanin, ester or amides of amino acids or acylierte amino acids or Oligopeptide;
2. b) natural and synthetic polymers with an asymmetry or Dissymmetrie in the main chain; in addition proteins (e.g. sour belong α_{1-Glycoprotein}, cattle serum albumin, Cellulase; see J. Chrom. 264, pages 63,-68 (1983), J. Chrom. 269, pages 71 - 80 (1983), WHERE 91/12 221), cellulose and cellulose derivatives, as well as other Polysaccharide and their derivatives (e.g. Cellulosetribenzoat, Cellulosetribenzylether, cellulose trisphenylcarbamat, Cellulose-tris-3-chlorobenzoat, Amylosetris (3,5-dlmethylphenylcarbamat), cellulose trichloroethylene (3,5-dimethylbenzoat), cellulose trichloroethylene (3,5-dimethylphenylcarbamat); see to EP 0147 804, EP 0,155,637, EP 0,718,625);
3. c) Cyclodextrine and its derivatives (e.g. J. High Resol.Chrom. & chromate. COMM. 3, pages 147 - 148 (1984); EP 0,407,412; EP 0,445,604);
4. d) Polymere one with assymmetric centers in the Seitenkette (e.g. EP 0,249,078; EP 0,282,770; EP 0,448,823).

Details of the production of the different Sorbentien and their use can be inferred from the block letters specified above.

The non-individual Sorbentien specified above can be contained in devices for Stofftrennung, can which be essentially handled like chromatographische columns.

In illustration 1 the procedure of the counter current chromatography, which the basis “simulated moving bed” - chromatography (SMB chromatography) represents, is schematically represented. Therein means (1) the river of the Sorbens. In the SMB procedure that physically river of the Sorbens simulated by cyclisches switching of multiple way valves, which can be realized only with difficulty, which several to a cycle switched columns connect.

The experimental realization of the separation wurd on a SMB plant implemented, which works after that in the following described four-zone model. Erfindungsgemäß can also SMB plants be used, those according to other models, the three-zone model be e.g. worked. Suitable procedure variants are well-known the specialist from the literature.

By the countercurrent principle the SMB for isolating of Zweistoffgemischen (e.g. the two Enantiomere of a Racemates) is suitable in ideal way. In addition, for other chromatographische separation processes SMB procedures are well-known. Thus becomes in J. Chromatogr. 719, pages 267 - 274 (1996) the application of a SMB procedure to the cleaning of monoclonal antibodies described. Also for the separation from Mehrstoffgemischen SMB procedures are usable. Such a separation is for example in Biosci. Biotech. Biochem. 56, pages 801 - 802 (1992) described. Processing parameter for other separations can specify the specialist by optimization.

Separations in the SMB procedure are described in the following exemplarily for separations of two substances:

The continuous function of the SMB of procedure, as it is schematically represented exemplarily in illustration 1, permits the attitude of a temporally stationary condition with that continuously to Eluent (3), as well as a solution of the Zweistoffgemisches which can be separated (feed; (4)) the system supplied and just as continuously the two separate components (raffinate (6) and excerpt (5)) from the system to be led out can. The Zuund would effected drive out the material flow mentioned by 4 pumps (not represented). The main stream of the Eluenten (2) with a further pump in the cycle one leads (recycling pump; not represented). There therefore the system only a smaller quantity of fresh Eluenten to be supplied muß (Feed + Eluent_(new) = raffinate + excerpt), solvent consumption per Produkteinheit is clearly smaller with the SMB than in case of the batch chromatography. The column bed of a stationary phase is divided with the SMB into 4 zones (ever a Adsoprtionsund desorption zone for the two components which can be separated), which relative to Zufuhrund Auslaß score are defined:

Zone I

- between Eluentund excerpt line

Zone II

- between Extraktund feed line

Zone III

- between Feedund raffinate line

Zone IV

- between raffinate and Eluent line

In case of the separation from Zweistoffgemischen now conditions, i.e. Flu&szlig leave themselves; guess/advise in the zones I-IV, are, with those the more weakly retinierte component with the mobile phase and the more strongly retinierte component with the stationary phase moves. The separate components can be inferred then in pure form with the excerpt relations way the raffinate stream.

It is technically only very with difficulty possible, an actual movement of a stationary phase to realize (1). Therefore this movement of the stationary phase is simulated. In addition the entire column bed is divided into cyclically connected in series single columns. The total number of the columns is a multiple of the number of 4, since the system, as mentions above, possesses 4 chromatographische zones. Between the single columns are 4 two-way valves each, the one connection to 4 Zufuhrund Auslaß lines represent. Due to these valves, thus each point between the columns knows each function (Eluent, feed supply or Raffinatbez. Excerpt Auslaß) take. At a given time the situation 4 Zufuhrund Ausla&szlig defines; - Lines the 4 chromatographischen zones. Becomes now the position of the 4 lines after a defined time around a column unit toward the Fließ central movement advanced, then corresponds this to a movement of the column bed in the opposite direction. By Weiterschaltung of the Speisepunkte in defined time intervals thereby each single column goes through successively all 4 zones, until Zufuhrund Auslaß - Lines again their original position take and thus a cycle is final.

After several cycles became to go through, a stationary condition adjusts itself, that it with suitable choice of the Fließ makes speeds possible in the system and suitable cycle time for the valve circuits to decrease the separate products in pure form as Extraktund of raffinate stream.

It was found, daß on use of these preferential Sorbentien the Fluß speed over a wide range to be varied can, without daß the separation characteristics thereby to be worsened. Under utilization of this characteristic it is possible, the Fluß to adapt speed to the Elutionsprofil, without daß the separative power is reduced. Thus the time requirement of the separation can be strongly reduced. In particular for präparative separations arise thus groß e of advantages. For the application of the SMB procedure also the small decrease of pressure is with high Fluß speed relevantly, since with this procedure a number of columns is switched one behind the other.

The usual Fließ speed of a column of the dimension 25 mm in diameter, which are packed with individual particles, amounts to 40 ml/min. like example 1 in detail shows, permits the not-individual carriers a substantially higher operating speed. Thus the task of separation can be solved substantially more economically.

By injection of rising concentrations of the components (for example 100, 300, 600 µl for each component) and evaluation by means of ECP method (Elution by characteristic POINTs) as in Guiochon, ski Razi, Katti, Fundamentals OF Preparative and Nonlinear Chromatography, Academic press, Boston, 1994, 83 f described page, can the adsorption isotherms of the components be determined.

For example (see example 2) became for the components A (Dimethylphthalat) and B (Dibutylphthalat) the following modified Langmuir isotherms found: C¯ = 1,1• C + 0,0735• C1+0,000735• CC¯ = 1,1• C + 1,75• C1+0,000735• C

From the determined isotherms F.Charton, J.Chrnmatogr can after in R.M.Nicoud. 702 (1995) 97 FF descriptive procedures, for example with the help of the simulation software HELP, which is intended separation conditions for a suitable SMB system:

Even without further remarks one assumes that, daß a specialist the above description to furthest extent to use can. The preferential execution forms are not therefore only as describing to understand by any means as into any way limiting revealing.

The examples 1 and 2, although not erfindungsgemäß , are just like the example 3 the invention to clarify.

A sample toluol, 2-Nitroacetanilid and 3-Nitroacetanilid becomes containing with different Fließ speeds of the mobile phase separately:

The results are zusammengefa&szlig in the illustrations 2a2 C; t. The usual Fließ speed of a column of the dimension 25 mm in diameter, which are packed with individual particles, amounts to 40 ml/min. the not-individual carriers permits thus a substantially higher operating speed, which leads too clearly improved economics of the tasks of separation.

Dimethylphthalat and Dibutylphthalat are laid on and separated into different quantities:

For the components A (Dimethylphthalat) and B (Dibutylphthalat) were found the following modified Langmuir to lsothermen: C¯ = 1,1• C + 0,0735• C1+0,000735• CC¯ = 1,1• C + 1,75• C1+0,000735• C

From the determined isotherms F.Charton became, J.Chromatogr after in R.M.Nicoud. 702 (1995) 97 descriptive procedures with the help of the simulation software HELP the separation conditions for the SMB separation intends:

Hollow fiber modules (FRACTOSEP® SO3, company Merck KGaA), as they are revealed in DE 195 01 726,9, are built in place of the usual columns into a SMB plant (company new fact SEPTEMBEr). 100 µl a solution, which Chymotrypsinogen A and Lysozym (in each case 10 g/l) in 0,3 M NaCl and 20 mm of sodium phosphate buffer (pH 7.0) contains, is laid on and developed isokratisch. The processing parameters according to determination of the adsorption parameters by the program “HELP”, the part of the plant control is determined. The Elutionsdiagramm is represented in illustration 4.