DE19801575A1 - Preparative chromatographic separation, e.g. of enantiomers - Google Patents

Abstract

Preparative chromatographic separation of at least two substances, by a continuous or batch process, is effected using a monolithic sorbent (I) based on a shaped article of silicon dioxide, having macropores of diameter 2-20 mu m and meso-pores having a diameter of 2-100 nm (both median values).

Description

The invention relates to the use of monolithic sorbents, especially those containing separation effectors for preparative chromatographic separation processes, as well as processes for preparative chromatographic separation of at least two substances below Use of monolithic sorbents.

The goal in preparative chromatographic separation processes is Isolation of the purified substance. In contrast, analy table chromatographic separation processes for high selectivity narrow band width aligned. Analytical methods are not used to isolate the substance, at most additional analyzes will follow procedure (e.g. mass spectrometry, UV / VIS spectrometry) the separation proceed on. For preparative chromatographic separation processes the optimal compromise between chromatographic resolution and Productivity essential.

For the economy of preparative separations are the Achieving high flow rates, short elution times and compliance moderate operating pressures essential factors. The separation performance a chromatographic column is determined by the floor height (or the number of trays per meter). The connections with Flow and diffusion processes are carried out by the van Deemter Equation described. If you increase the diameter of the Sorbent particles, the number of trays is reduced and it must be Column length can be increased in order not to reduce the separation performance reduce. If the diameter of the sorbent particles is increased, the number of trays becomes more dependent on the flow rate. In many cases this is the achievable chromatographic separation performance to a large extent depending on the selected flow rate (steep  H (u) curve). The relationships mentioned are known to the person skilled in the art generally known and in manuals such. B. "Handbook of HPLC "(K.K. Unger, ed .; GIT-Verlag, Darmstadt, DE). In contrast, it was found that the invention according to the use of monolithic sorbents in the pores diameter of the macropores can be varied without the Dimension of the skeleton located between the macropores phase must be varied. This can cause the pressure drop through Selection of a sorbent with larger macropores can be reduced while the increased flow rate hardly affects the separation performance.

Counter-current processes are used for preparative separations Gained meaning. Since it’s very difficult technically, to realize an actual movement of a stationary phase, the movement of the stationary phase is simulated. This will be Entire column bed in cyclically arranged single columns divided. The total number of columns is typically one Multiple of four, since such a system has four chromatographic Owns zones. After a defined time, the lines are switched switched, causing movement of the column bed in the opposite opposite direction is simulated. For continuous ver driving the "simulated moving bed" chromatography (SMB-Chroma topography) are usually particulate as separating materials Sorbents used. Leave the column packs used no optimal flow rates because the operating pressure at particulate Porters is very high. The mechanical stability is also particulate Sorbent beds are not very good. It is also for the SMB Chromato graphic necessary, a series of chromatographic columns (typical wise up to 24) with the same properties as possible. With particulate sorbent beds, this is very difficult Packing the pillars and realizing when choosing the packed pillars bar.  

The object of the invention is chromatographic preparative separation procedure, in particular for the SMB procedures, which are available at moderate operating pressure have high flow rates.

It has been found that monolithic sorbents are used for separation processes high flow rates can be used; thus a higher through rate per unit of time, d. H. improved productivity can be achieved. The after separation processes according to the invention using mono productivity achievable with lithic sorbents is typically around one Order of magnitude higher than when using separation processes particulate sorbents.

The invention relates to methods for preparative chromato graphic separation of at least two substances, especially after the SMB process, with a monolithic as the stationary phase Sorbent is used.

Fig. 1 and 2 show separations of toluene, 2- and 3-nitro acetanilide on two unmodified monolithic sorbents with different pore sizes of the macropores, produced according to PCT / EP97 / 06 980; Experimental details: see Example 1. Fig. 3 shows the separation of dimethyl phthalate and dibutyl phthalate on a porous molded body which has been end-capped by conventional RP 18 processes; Experimental details: see Example 2. Fig. 4 shows the separation of a mixture of xylose, fructose, glucose and sucrose on the particulate sorbent modified with aminopropyltrimethoxysilane; Experimental details: see Example 3. Fig. 5 shows the separation of the enantiomers of 2,2,2-trifluoro-1-anthrylethanol on the chiral monolithic sorbent (Pirkle type) prepared according to Example 4; Experimental details: see Example 4. Fig. 6 shows the separation of the enantiomers of chlorothalidone on the chiral monolithic sorbent prepared according to Example 5; Experimental details: see example 5.

Monolithic sorbents are generally known from the literature this includes, above all, porous ceramic moldings, as described in WO 94/19687 and WO 95/03256. Particularly preferred are monolithic sorbents based on porous form bodies, the interconnected macropores and mesopores in have the walls of the macropores, the diameter of the Macropores has a median value greater than 0.1 µm, and the Diameter of the mesopores have a median of 2 and 100 nm points. In particular, monolithic sorbents are based on porous shaped bodies whose macropores have a diameter between 0.3 and 20 µm, especially between 2 and 15 µm (each median values), and their mesopore diameter between 2 and 100 nm (Median values). Porous shaped bodies with such an enlarged Macropores are particularly accessible by methods such as those in the Patent application PCT / EP97 / 06 980 are disclosed.

Monolithic sorbents consist of inorganic materials as are used for particulate sorbents. In many cases (e.g. SiO ₂ ) these sorbents can easily be used for chromatographic separations. However, the base supports are derivatized more frequently in order to improve the separation properties; additional groups are introduced, which are summarized under the name Separationseffekto ren.

Separation effectors and methods for their introduction into the base support are known in principle to the person skilled in the art. Examples of reactions with which separation effectors can be introduced are:

• a) The derivatization with silane derivatives of the formula I.
  SiX _n R ¹ _(3-n) R ² (I)
  wherein
  X is a reactive group, such as. B. methoxy, ethoxy or halogen, R ¹ C ₁ -C ₅ alkyl,
  n 1, 2 or 3
  mean and
  R ^{2 has} one of the meanings given below:
  • a1) unsubstituted or substituted alkyl or aryl, such as. B. n-octadecyl, n-octyl, benzyl or cyanopropyl;
  • a2) anionic or acidic residues, such as. B. carboxypropyl;
  • a3) cationic or basic radicals, such as. B. aminopropyl, diethylaminopropyl or triethylammonium propyl;
  • a4) hydrophilic residues, such as. B. (2,3-dihydroxypropyl) oxypropyl;
  • a5) bindable activated residues, such as. B. (2,3-epoxypropyl) oxypropyl.
• b) The adsorption or chemical binding of polymers such as poly butadiene, siloxanes, polymers based on Styroll Divinyl benzene, (meth) acrylic acid derivatives or other vinyl compounds and peptides, proteins, polysaccharides and poly saccharide derivatives on the base support;
• c) The chemical bonding of the polymers mentioned under b) via the derivatives mentioned under a); these include graft polymers of Poly (meth) acrylic acid derivatives on diol-modified silica gel EP-B-0 337 144.  
• d) The adsorption or chemical binding of chiral phases, such as. B. of amino acid derivatives, peptides or proteins, or of cyclo dextrins, polysaccharides or polysaccharide derivatives.

Other common derivatization options and derivatization procedures are known to the person skilled in the art and are known in common manuals such as Unger, K.K. (ed) Porous Silica, Elsevier Scientific Publishing Company (1979) or Unger, K.K. Packings and Stationary Phases in Chromatographic Techniques, Marcel Dekker (1990).

Further examples of different separation effectors and of processes for introducing the separation effectors into monolithic sorbents are given in the following publications:

• a) DE 38 11 042, inter alia, discloses monomers which are suitable for the preparation of ion exchangers; These include, for example, acrylic acid, N- (sulfoethyl) acrylamide, 2-acrylamido-2-methyl propanesulfonic acid, N, N-dimethylaminoethyl-acrylamide, N, N-diethylaminoethyl-acrylamide, and trimethylammoniumethyl-acrylamide.
  Other monomers mentioned in this publication allow the binding of affinity ligands or enzymes, or are suitable for reversed phase chromatography: these include, for example, acrylic acid, acrylamide, allylamine or acrylonitrile.
• b) DE 43 10 964 discloses monomers which contain an oxirane ring, an azlactone ring or a group which can be converted into an azlactone ring. Polymers containing such monomers are particularly well suited for binding affinity ligands or enzymes. Affinity ligands are disclosed by way of example in DE 43 10 964.
  Furthermore, the epoxide groups can be reacted further in such polymers in an advantageous manner, whereby ion exchangers thiophilic sorbents or sorbents for metal chelate or hydrophobic chromatography are provided. For example, phosphoric acid, ammonia, diethylamine, trimethylamine, sulfurous acid or complexing agents such as iminodiacetic acid are added to the oxirane ring.
  The production of thiophilic sorbents and of sorbents for metal chelate chromatography is disclosed in DE 43 10 964.
  DE 43 33 674 and DE 43 33 821 disclose such reactions which can be used to provide ion exchangers.
  DE 43 23 913 describes sorbents for hydrophobic interaction chromatography.

Chiral separation materials for the separation of enantiomers are in a large number are known in the prior art. It is exclusive around particulate separation materials. The well-known chiral separation materials either consist of the chiral connection itself (for Example is cellulose triacetate) or a chiral separation effector mounted on a support or chemically bound to a support (e.g. chemically bound amino acid derivatives). Besides, it is possible to use chiral separation effectors with a stationary phase Interact, add in the eluent (dynamic Assignment with z. B. cyclodextrins).

Chiral separation effectors are known in large numbers; the most important groups of known chiral separation effectors are:

• a) amino acids and their derivatives, e.g. B. L-phenylalanine, or D-phenyl alanine, esters or amides of amino acids or acylated amino acids or oligopeptides;  
• b) natural and synthetic polymers with asymmetry or dis symmetry in the main chain; these include proteins (e.g. acidic α ₁ -glycoprotein, bovine serum albumin, cellulase; see J. Chrom. 264, pages 6368 (1983), J. Chrom. 269, pages 71-80 (1983), WO 91/12221) , Cellulose and cellulose derivatives, as well as other polysaccharides and their derivatives (e.g. cellulose tribenzoate, cellulose tribenzyl ether, cellulose trisphenyl carbamate, cellulose tris-3-chlorobenzoate, amylose tris- (3,5-dimethylphenyl carbamate), cellulose tris- (3, 5-dimethylbenzoate), cellulose tris (3,5-dimethylphenyl carbamate); see EP 0 147 804, EP 0 155 637, EP 0 718 625);
• c) Cyclodextrins and their derivatives (e.g. J. High Resol. Chrom. & Chromat. Comm. 3, pp. 147-148 (1984); EP 0 407 412; EP 0 445 604);
• d) polymers with asymmetry centers in the side chain (e.g. EP 0 249 078; EP 0 282 770; EP 0 448 823).
• e) polymers which have cavities, the cavities being an impression represent the analyte (imprinted polymers; WO 93/09 075).

The enantiomerically pure chiral separation effectors can to a suitable base carrier, if necessary after derivatization be adsorbed. It is also possible to have the enantiomerically pure the chiral separation effectors, if necessary after introduction suitable functional groups to bind to the base support. For bifunctional reagents can also be used for this purpose. Suitable implementations for these process variants are Known to those skilled in the art and are described in manuals.

In the examples, moldings suitable according to the invention are also included called chiral separation effectors.

Further details of the manufacture of the various sorbents and their use can be found in the above-mentioned documents  men will; the relevant disclosure of these documents is incorporated by reference into the present application.

The above monolithic sorbents can be used in devices be included for separation, which is essentially like chromato Have graphic columns handled. The known Separation processes are used: batch process, continuous Methods such as B. the simulated moving bed (SMB) method, or other countercurrent processes, as described, for example, in US Pat. No. 5,630,943 be disclosed.

It has been found that using these preferred sorbents the flow rate can be varied over a wide range, without the separation properties being impaired. Under Taking advantage of this property, it is possible to control the flow rate adjust the elution profile without reducing the separation efficiency This can greatly reduce the time required for the separation. Ins There are therefore great advantages especially for preparative separations. The low pressure drop is also important for the application of the SMB process relevant at high flow rates, since a Number of columns can be connected in series.  

Examples

The following examples are intended to illustrate the invention; they mean no limitation of the inventive concept.

example 1 Separation of toluene, 2-nitroacetanilide and 3-nitroacet anilide on monolithic sorbents with different Macropores diameter

Porous monolithic molded articles made of SiO ₂ (83.7.2 mm) with different diameters of the macropores (1.8 and 6 µm) were produced in accordance with PCT / EP97 / 06 980. A solution of toluene, 2- and 3-nitro acetanilide is applied and separated into eluent in heptane / dioxane 80:20 (flow rate: 8 ml / min; UV detection).

The elution diagrams are shown in Fig. 1 (1.8 µm pore size) and 2 (6 µm pore size).

Example 2 Separation on a monolithic sorbent modified with a C ₁₈ reversed phase

From a porous molded body (83.7.2 mm; 1.5 µm pore size) consisting of SiO ₂ produced according to EP 0 710 219, an RP-18 modified monolithic separating material is produced according to the standard procedure by reaction with a silane derivative and subsequent endcapping reaction. A solution of dimethyl phthalate and dibutyl phthalate is applied and separated in methanol / water 90:10 as eluent (flow rate 4 ml / min; UV detection).

The elution diagram is shown in Fig. 3.

Example 3 Separation on a modified with amino groups monolithic sorbent

• 1) Production: A porous molded body (83.7.2 mm, 1.8 µm pore size), manufactured according to EP 0710219, is manufactured using standard processes Pumping through a solution of aminopropyltrimethoxysilane in Toluene converted to a sorbent modified with amino groups.
• 2) Separation example: A solution of xylose, fructose, glucose and Sucrose are based on that with aminopropyltrimethoxysilane modified sorbent applied and with acetonitrile / water 80:20 as Flux separated (flow rate 4 ml / min; RI detection).

The elution diagram is shown in Fig. 4.

Example 4 Separation on a with (R) - (-) - N- (2,4-dinitrobenzoyl) - phenylglycine modified monolithic sorbent (Pirkle-type modified chiral sorbent)

• 1) Production: The one prepared according to Example 3 with amino groups derivatized porous moldings are further implemented by a Lö solution of (R) - (-) - N- (2,4-dinitrobenzoyl) phenylglycine and N-ethoxycarbonyl- 2-ethoxy-1,2-dihydroquinoline (EEDQ) is pumped through in toluene. It will received a chiral sorbent.
• 2) Separation example: The enantiomers of 2,2,2-trifluoro-1-anthrylethanol are made on the chiral pirkle-type produced as described above  Sorbent separated in heptane / i-propanol 99.5: 0.5 (flow rate: 8 ml / min; UV Detection).

The elution diagram is shown in Fig. 5.

Example 5 Separation on one with N-acryloyl-L-phenylalanine modified ethyl ester as monomer units monolithic sorbent

• 1) Production: A porous molded body (83.7.2 mm; 1.7 µm pore size), produced according to EP 0 710 219, is pumped through by a) a solution of 3-glycidyloxypropyltrimethoxysilane in toluene and b) a solution of N-acryloyl -L-phenylalanine ethyl ester in toluene with the addition of azoisobutyronitrile (AlBN) converted to a chirally modified sorbent.
  A modified monolithic shaped body is obtained, on which covalently bound L-phenylalanine ethyl ester groups are present as separation effectors.
• 2) Separation example: Fig. 6 shows the separation of the enantiomers of chlorothalidone on the chiral sorbent prepared as described above (conditions: heptane / dioxane 50:50; 4 ml / min; UV detection).

Comparative example Comparison of the separation on a particulate Sorbent with the separation on a monolithic Sorbent

The productivity of a LiChrospher® Si 100 15 µm sorbent is increased with the of a monolithic sorbent manufactured according to PCT / EP97 / 06 980 compared. The monolithic sorbent shows just like the trade  usual particulate sorbent LiChrospher® mesopores with 10 nm pores widen. The particle size of the LiChrospher® Si100 is 15 µm. The particulate sorbent has a skeleton size of 1.5-2 µm and macropores from 6 µm.

The following characteristic data were found for the particulate sorbent:

ΔP / L (bar / m) = 26540.u (m / sec)
H (m) = 0.03657.u (m / sec) + 5.5.10 ^-5 for u <2.10 ^-3 n / sec.

The following characteristic data were found for the monolithic sorbent:

ΔP / L (bar / m) = 8500.u (m / sec)
H (m) = 0.00403. (M / sec) + 1.8.10 ^-5 for u <2.10 ^-3 m / sec.

The best productivity is achieved when you are working at the desired number of trays with maximum pressure drop. The optimal flow rate and column length are then:

The speed is then 5.7 times greater on the particulate sorbent. The productivity (g / day) is then for the same column diameter increased fourfold. Since the required column length is shorter, the relative one is Productivity (g / day / L stationary phase) increased by a factor of 10.5.

Claims (3)

1. A process for the preparative chromatographic separation of at least two substances, characterized in that the separation is carried out using monolithic sorbents. 2. The method according to claim 1, further characterized in that the chromatographic separation is carried out in a batch process becomes. 3. The method according to claim 1, further characterized in that the chromatographic separation in a continuous Procedure is executed.