DE4413121A1 - Method for separating enantiomers on chiral stationary phases by liquid chromatography in capillary columns - Google Patents

Abstract

The present invention relates to a method for separating enantiomers by liquid chromatography in open capillary columns using chiral stationary phases. The chiral component used is, for example, a polysiloxane to which alkylated cyclodextrin ( alpha , beta or gamma ) has been chemically bonded (e.g. CHIRALSIL-DEX) and which has been immobilised on the surface of the inner wall of the capillary. The method according to the invention is suitable for qualitative and quantitative analysis of enantiomers. In this respect there is no restriction to analytes which do not decompose on volatilisation as in capillary gas chromatography. A typical advantage of the method according to the invention is the smaller solvent usage, in the microlitre range, and the long lifetime of the arrangement used. The necessity, which is customary on use as addition to the mobile phase, of de novo preparation of the chiral separating component after each analysis run does not apply to the method according to the invention.

Description

Capillary gas chromatography is characterized by high efficiency (low floor height, HETP), separation performance (high resolution, R _1.2 ), short analysis times (small retention factors, k) and high detection sensitivity. Suitable coupling methods are also available. In contrast, liquid chromatography is carried out almost exclusively in packed columns using pressure. However, the universal Golay equation, which links the maximum efficiency of a capillary column (expressed by the height of a theoretical plate, HETP) with the mean flow rate (v), predicts that open tubular liquid chromatography (OTLC) is an important alternative for packed liquid chromatography (G. Guichon, H. Colin, in P. Kucera (Ed.), "Micro Column High Performance Liquid Chromatography", Elsevier, Amsterdam, (1984).)

However, the prerequisite for this method is the use of small columns Inner diameter (50 µm), covering with stationary phases (e.g. polysiloxanes), the preferably immobilized, low-volume or -free injection systems (split Technology) and on-column detection (UV, laser-induced fluorescence). Progress reports and original publications convincingly document the applicability of the OTLC (D. Ishi et al., J. High Resolut. Chromatogr., Chromatogr. Commun., 2 (1979) 371; T. Tsuda, G. Nakagawa, J. Chromatogr., 268 (1983) 369; H. Engelhardt, B. Lillig, J. High Res. Chromatogr., Chromatogr. Commun., 8 (1985) 531; O. van Berkel, H. Poppe, J.C. Kraak, Chromatographia, 24 (1987) 739. K. Jinno, Chromatographia, 25 (1988) 1004; S. Folestad, B. Josefsson, M. Larsson, J. Chromatogr., 391 (1987) 347.

Important advantages of OTLC are high efficiency, high permeability, simple equipment, Coupling options due to low flow rates and lowest consumption possibly environmentally harmful and toxicologically questionable solvents.

Enantiomer separation in liquid chromatography is carried out exclusively with packed columns operated. Over 500 chiral stationary phases are known and the published ones  Data can only be sensibly recorded through databases (e.g. chirabase). Although immobilized chiral polysiloxanes (CHIRASIL-DEX) in the capillary gas and supercritical fluid chromatography are routinely used according to ours Surprisingly, knowledge of no applications of this method in enantioselective OTLC before.

The present invention relates to the use of chirally modified surfaces of capillary columns for enantioselective liquid chromatography, as is shown using the example of immobilized CHIRASIL-DEX (peralkylated cyclodextrins which are bound to immobilizable polysiloxane via a polyalkylid chain, e.g. Fig. 1). The apparatus problems of OTLC are carried out particularly simply by using a capillary electrophoresis arrangement with the PRINCE injection system and on-column detection without voltage application. Since these devices are increasingly available in modern laboratories, the method according to the invention can be expected to spread rapidly.

The present invention differs from a previous one Disclosure (German patent application P 41 36 462.7) that the enantiomer separation with the same type of column by electromigration in an electrokinetic Process ("electrochromatography") describes, in that in the latter case the Migration of the analytes by electroosmosis and / or electrophoresis takes place during in the present example, the hike is due to an applied pressure difference.

Description of the invention

The invention relates to a process for the selective separation of enantiomers by Liquid chromatography in capillary columns with chiral surface-bound and / or cross-linked, immobilized enantioselective stationary phases, e.g. B. CHIRASIL-DEX (a polysiloxane to which cyclodextrin is chemically bound). In this process, the enantiomers to be separated are separated by pressure difference  the separation capillary moves. The cyclodextrin is applied to the surface through previous chemical bonding to a polysiloxane and its Immobilization. According to the invention, however, the connection does not have to be exclusive by prior chemical bonding to an insoluble or immobilizable polymer take place, but can in principle also by direct reaction on silanol groups Surface. The capillary inner wall can also be occupied by chiral selectors take place, which are dissolved in a solvent (e.g. a polysiloxane, Carbowax) become, as usual in gas chromatography. To wash out the unbound To complicate the stationary phase, the mobile phase can be saturated with the solvent become. However, the use of chiral polymers based on polysiloxane is preferred such as CHIRASIL-DEX (or other chiral polymers) (V. Schurig, D. Schmalzing, M. Schleimer, Angew. Chem. 103 (1991) 994). In these polymers the chiral selector is covalently attached to the polymer. Because of the often high enantioselectivity combined with the advantages of polysiloxanes, such as simple coverage of surfaces, easy immobilizability and high chemical stability chiral stationary phase CHIRASIL-DEX has proven itself as in other processes, e.g. B. Gas Chromatography and Supercritical Fluid Chromatography (V. Schurig et al., J. High Resolut. Chromatogr. 13 (1990) 713 and 14 (1991) 58) and electrochromatography (German patent application P 41 36 462.7).

According to the invention, the problem of avoiding peak broadening due to extra-column effects (injection, detection), which is critical for OTLC, is optimally solved by reference to the technology that is now available for electronic processes. For example, the capillary electrophoresis system from Bischoff (Leonberg) PRINCE was used to perform an enantiomer separation using capillary liquid chromatography ( Fig. 2-4).

PRINCE is an automatic, variable pressure injection device for capillary electrophoresis. By using controlled pressure ramps in a closed System are the physical laws of Boyle (pressure) and Poiseuille (flow) consistently realized. The device produces reliable and accurate injection results  with an error rate below 1%. So it was only natural to use this system for capillary liquid chromatography (OTLC). Problems with HPLC with capillary columns occur, such as B. dead volumes, due to injection technology and multiple Screw connections do not occur here. The PRINCE system also creates one compared to conventional HPLC pumps pulse-free pressure, which is the system for coupling methods (e.g. LC-MS etc.) is predestined. The overpressure built up for the injection is made usable for the migration of the enantiomers in the capillary column.

However, the method according to the invention can also be carried out in one for a greater effort the OTLC designed HPLC arrangement, as known from the literature, operated become.

Advantages of the invention described here over conventional packed HPLC In addition to simple handling, low consumption is required for enantiomer analysis mobile and stationary phase (cost factor), no support material, increase in Mass sensitivity for quantity-specific detectors, high precision, high efficiency and therefore better enantiomer resolution, simple coupling (hyphenated techniques) with spectroscopic (LC-MS, LC-FTIR) and secondary chromatographic Systems due to lower flow rates. In a multidimensional arrangement, Connect capillary columns more easily in series or in series. Because of the low Column volume, the heat capacity of the system is low and the capillary columns are suitable especially for temperature programming.

A variant of the invention is the addition of the chiral selector to the mobile phase and separation of the enantiomers by OTLC on an achiral stationary phase (e.g. polysiloxane) or the combination of both methods (chiral stationary phase and chiral mobile phase).

The following examples are intended to explain the process according to the invention, but without this restrict.

Examples Example 1: Instrumental parameters of capillary liquid chromatography (OTLC)

A capillary electrophoresis device from the company Bischoff (Leonberg) PRINCE was used, equipped with an on-column UV detector. The injection was done by dynamic pressure injection (PRINCE system). All samples were dissolved in methanol and adjusted to a concentration of 0.1 mg in 1 ml. The detection took place at 220 nm. Before the columns were installed in the device, 17 cm in front of the Column end by burning off about 3 mm of polyimide layer with a weak flame the optical window for on-column UV detection is attached. The column lengths were chosen so that the effective length 80 cm (from the injector to the optical window) scam. The pressure required for the measurement was generated using the "PRINCE system". The chromatograms were taken with an integrator CR-6A from Shimadzu recorded.

Example 2: Production of the capillary columns

1 m fused silica capillary columns from Ziemer (Mannheim) with a Inner diameter of 50 microns used. Before loading, the columns were weak Hydrogen stream dehydrated at 250 ° C for two hours. The connection of the Cyclodextrin derivatives of the polysiloxane are carried out by hydrosilylation (V. Schurig et al., J. High Resolut. Chromatogr. 13 (1990) 713 and 14 (1991) 58). The pillars were covered with carefully filtered 2.0% (w / v) solutions of Chirasil-Dex in diethyl ether at 30 ° C and 0.01 Torr statically occupied, so that a film thickness of approx. 0.25 µm resulted. Subsequently the capillaries were conditioned for 12 hours at 0.5 bar hydrogen and 100 ° C.  

Example 3: Immobilization

The immobilization was carried out by installing the capillary columns in the gas chromatograph Carlo Erba (Fisons, Mainz) of the types Fractovap 2101, 2150, 2350 and Vega carried out. These were with split injector (1: 100) and flame ionization detector (FID). Hydrogen (purity 99.999%) and compressed air were used as carrier gas used.

For the thermal immobilization of capillary columns covered with Chirasil-Dex these are installed on the injector on the gas chromatograph and 24 hours in one extremely low hydrogen flow (about 30 bubbles per minute at the end of the column, at open split) heated to 190 ° C. To calculate the degree of immobilization Test chromatograms (n-decane, n-tridecane and 1-phenylethanol) after immobilization and after washing the capillaries with 5 ml of methanol, a mixture of Methanol / dichloromethane (1: 1) and 5 ml dichloromethane added. From the percentage Decrease in k values for the n-alkanes after immobilization and after Wash out the degree of immobilization (usually 80%) was calculated.

Example 4: Preparation of the buffers

All buffers were made with water for HPLC, stored in the refrigerator and before Use in the LC with a filter (0.45 µm pore size) (Machery Nagel, Düren) filtered. To prepare the borate / phosphate buffer, solutions of NaH₂P₄ were used  × 2 H₂O and Na₂B₄O₇ × 10 H₂O in the concentration of 20 mM prepared and to pH 7 adjusted by mixing the solutions of the same concentration. Depending on your needs added methanol to the buffer for HPLC.  

Example 5: Enantiomer separations ( Fig. 2-4)

The capillary column is inserted into the capillary electrophoresis system from Bischoff (Leonberg) PRINCE built in.

Care is taken to adjust the injection window carefully. The capillary column will slowly rinsed with buffer solution (e.g. borate-phosphate buffer pH 7). This flushing will carried out until there is a very sensitive setting of the detector and Integrators (e.g. Shimadzu CR-6A Chromatopac) to observe a stable baseline is.

According to Example 2, a capillary column was covered and the chiral stationary phase CHIRASIL-DEX according to Example 3 was immobilized. With this capillary column, the enantiomer separation of hexobarbital, methylphenobarbital and thiamylal was achieved ( Fig. 2-4).

The following parameters apply to all three separations:

Injection: 60 mbar, 0.12 min;
Column inner diameter: 50 µm;
effective column length up to the detector: 80 cm;
total length 97 cm;
a borate-phosphate buffer pH 7 is used, to which methanol has been added (buffer methanol 80/20 (v / v));
Detection 220 nm.

The enantiomer separation of hexobarbital is carried out at a pressure of 0.5 bar, that of Methylphenobarbital and thiamylal carried out at 1 bar. The pillar is after everyone Measurement 0.5 min at 2 bar with methanol and then 0.5 min at 2 bar with the appropriate buffer flushed.

Claims (6)

1. A process for the separation of enantiomers by liquid chromatography in capillary columns, characterized in that chiral stationary phases are used, which are applied as a film to the inner surfaces of the capillary. 2. Process for the separation of enantiomers by liquid chromatography in capillary columns, characterized in that chiral stationary phases are used, the applied as a film to the inner surface of the capillary and then immobilized become. 3. The method according to claim 1 and 2, characterized in that immobilized CHIRASIL- DEX is used for surface modification. 4. The method according to claim 1, 2 and 3, characterized in that the hydroxyl groups the cyclodextrin component in the CHIRASIL-DEX free or alkylated (unbranched, branched to C₁₀₀) or acylated or perfluoroacylated (unbranched, branched to C₁₀₀) are present and that α, β, γ or δ-cyclodextrin is used. 5. The method according to claim 1, 2 and 3, characterized in that the connection of the Cyclodextrin component to the polysiloxane in CHIRASIL-DEX via a branched or unbranched alkylidene chain of any length. 6. The method according to claim 1, 2 and 3, characterized in that the polysiloxane component in CHIRASIL-DEX, whose organic residues contain all radicals known to date (Alkyls, aryls, functionalized or unfunctionalized) can represent on capillary surfaces (preferably fused silica) is immobilized.