JP2002503823A - Packed capillaries, especially for the separation of enantiomers - Google Patents

Abstract

  (57) [Summary] The present invention relates to a thin tube filled with a particulate adsorbent and having a constant inner diameter over its entire length, wherein the range of the adsorbent bed is defined by a solidified region generated by heat treatment. In particular, the invention relates to capillaries filled with a chiral adsorbent. The invention furthermore relates to a method for producing these capillaries and to the use of these capillaries for the separation of substances.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to improved capillaries filled with adsorbents and the use of these capillaries for high-performance liquid chromatography and capillary electrophoretic chromatography separations. The invention particularly relates to capillaries filled with chiral adsorbents and the use of these capillaries for high-performance liquid chromatography and capillary electrophoresis chromatography. Capillary tubes for separation by high performance liquid chromatography and capillary electrophoresis chromatography are known in the prior art. That is, EP-A-0 779 512 discloses that the adsorbent bed in the capillary is stabilized by using a capillary in which the terminal region of the adsorbent is different in inside diameter from another position of the capillary. Are disclosed. EP-A
-0 809 108 describes a capillary in which the adsorbent particles are fixed to the capillary by heat treatment, this heat treatment being carried out over the entire length of the adsorbent bed.

The method described in EP-A-0 779 512 requires a tubule having a stenosis or dilation at a predetermined location. This type of capillary must be specially manufactured for the adsorbent bed of the desired length, which is disadvantageous. The method of EP-A-0 809 108 requires heating the entire adsorbent bed, during which at least the heat-sensitive adsorbent is decomposed under certain circumstances. Therefore, there is a problem that the adsorbent in the capillary column is fixed without using a capillary having a narrowed portion or an expanded portion and without subjecting the entire adsorbent bed to heat treatment. A simplified method of making capillaries has been found. This method produces a stable adsorbent bed, even though only a very short portion of the adsorbent bed is heated. This method is particularly suitable for use with chiral separation materials.
Separation capillaries produced according to the invention are particularly suitable for HPLC. It is possible to provide a stable and miniaturized separation tubule filled with an adsorbent.

[0003] The present invention is directed to a capillary packed with a particulate adsorbent, having a constant inner diameter over its entire length, wherein the extent of the adsorbent bed is defined by a solidification zone generated by heat treatment. Regarding tubules. In particular, the invention relates to such capillaries filled with a chiral adsorbent. The present invention also relates to a process for the preparation of a capillary tube filled with a particulate adsorbent according to the invention, comprising the following steps: a) closing one end of a commercial quartz glass capillary tube with a frit; Attaching a storage vessel to the other end of the capillary; c) preparing a homogeneous suspension of the particulate adsorbent in a solvent; d) introducing this suspension into said storage vessel; Filling the tubing with adsorbent by pressure filtration from a storage container; f) exchanging the solvent for water; g) heating the narrow area of the tubing filled with adsorbent at both ends of the adsorbent bed; Forming a frit by heat treating the adsorbent.

The invention furthermore relates to the use of a capillary filled with a particulate adsorbent according to the invention for the separation of at least two substances, in particular by liquid chromatography. 1 to 6 show elution graphs for the separation of various racemates, and details of these experiments are given in Use Examples AF. The quartz glass capillaries used according to the invention are known;
μm to about 500 μm, and their length is 3 cm to about 1 m. Further details are known to those skilled in the art and are described, for example, in the cited prior publications. Capillary tubes having an inner diameter of 50 to 150 μm are mentioned as preferred.

[0005] A particulate adsorbent suitable for the purpose of the present invention preferably comprises silica gel (SiO ₂ ). Silica gel can be used as an adsorbent without further denaturation. However, to extend the range of types of separation, silica gel is usually
It is derivatized from a basic support (SiO ₂ ) and a separation effector is introduced. Such a separation effector causes separation. Depending on the separation principle (for example, ion exchange or reverse phase chromatography or enantiomeric separation), appropriate groups (for ion exchange chromatography, for example SO ₃ H or COOH)
Groups, for reverse phase chromatography, e.g. _C18 -alkyl or phenyl groups,
And for the enantiomer separation, a chiral group) is introduced by methods known to those skilled in the art. The particulate adsorbents used according to the invention can be porous or non-porous; their particle size is preferably between 2 and 10 μm (average particle size, medium); , Can be used in principle. For smaller capillaries (eg, 300 μm) with larger inner diameters, larger adsorbent particles can also be used (eg, 15 μm). Thus, the particle size can be adjusted in a manner that will be apparent to the skilled person, depending on the inner diameter of the capillary used.

[0006] Chiral separation materials for the separation of enantiomers are largely of the prior art.
Are known. These chiral separation materials use the chiral compound itself (eg,
, Cellulose triacetate) or, alternatively, chiral separation
The effector is adsorbed on or bound to the base support
. In this case, both the silica gel and the organic polymer are used as the base polymer.
In the present invention, silica gel (SiO 2) can be used as a base support. _Two ) Are preferred. Chiral separations that interact with the stationary phase
Effectors can also be added in the eluent [dynamic loading
]. Numerous chiral separation effectors are known; known chiral separation effectors
The most important groups of ters are listed below:

A) Amino acids and their derivatives, such as L-phenylalanine or D-
Phenylalanine, an ester or amide of an amino acid or an acylated amino acid,
Or oligopeptides; b) natural or synthetic polymers having an asymmetric or asymmetric backbone; these include proteins (eg, acidic α ₁ -glycoprotein, bovine serum albumin, cellulase; J. Chrom., 264, 63). Pp. 68 (1983), J. Chrom. , 269 , 71-80 (1983), WO 9
1/12221), cellulose and cellulose derivatives, and other polysaccharides and their derivatives (eg, cellulose tribenzoate, cellulose tribenzyl ether, cellulose trisphenylcarbamate, cellulose tris-
3-chlorobenzoate, amylose tris (3,5-dimethylphenylcarbamate), cellulose tris- (3,5-dimethylbenzoate), cellulose tris- (3,5-dimethylphenylcarbamate); EP 0 147 804, EP 0 155
637 and EP 0 718 625).

C) cyclodextrins and cyclodextrin derivatives (eg J. High R
esol.Chrom. & Chromat.Comm., 3, pp. 147-148 (1984); EP 0 407 412; EP 0 455 6
D) a polymer having an asymmetric center in the side chain (eg, EP 0 249 078; EP 0 282 770)
E) a polymer polymerized around a chiral structure [“imprint” (inpri
nt) Polymers, for example, J. Chromat. , 707 , pp. 199-203 (1995); J. Chromat. , 694 , 3
1313 (1995)]. The separating capillaries are preferably filled by a known suspension method. For this purpose, the adsorbent is suspended in a solvent. If necessary, the suspension can be homogenized, for example by treatment with ultrasound, and can be introduced into a storage container that is connected to the capillaries to be filled. The other end of the tubule is sealed off with a frit. The solvent is then pumped into the capillary via a storage vessel, thus forming an adsorbent bed within the capillary.

[0009] The storage vessels used are, for example, precolumns which are customary in chromatography.
-column). Suitable solvents and solvent mixtures for packing this column, as well as additional parameters such as frit pore size, pressure and pressure during column packing, are known to those skilled in the art and are described in the references. Generally, an organic solvent is used for capillary filling; the heat treatment to form the frit is preferably performed in an aqueous environment. To do this, the solvent used to fill the column is replaced by water, if necessary, by pumping the water through the packed column, optionally with an organic solvent that is readily miscible with water. This method
It is a method familiar to those skilled in the art.

The heat treatment conditions which lead to the formation of frits are known in principle; Ni-Cr wire loops are particularly suitable; the current forms a filled capillary frit (depending on the capillary diameter). The point to be heated is set to be heated for about 30 seconds to about 2 minutes. Typically, the temperature inside the capillary is raised to 100-400 ° C. The method for producing silica glass tubules packed with an adsorbent for chiral chromatography and having an inner diameter of 75-100 μm is described in detail in the examples. These capillaries allow for "nano-scale" enantiomeric separation and capillary electrophoresis chromatography in normal and reverse phase HPLC. Without further elaboration, it is believed that one skilled in the art can use the above description in the broadest scope. The preferred embodiments and examples are illustrative only and are not intended to be limiting in any way. All applications, patents and publications cited above and below, and 1998
The entire description of the corresponding patent application DE 198 07 063.2 filed on February 20, 2008 is incorporated herein by reference.

[0011] Examples Example 1: chiral adsorbent having a packed length 40cm and internal diameter 100μm manufacture of separation capillary (the chiral poly methacrylamide induction member on silica gel), a commercially available silica glass capillary, a commercially available metal HPLC frit To one side. The other side is screwed onto an HPLC precolumn (4.6 x 50 mm). This precolumn serves as a storage container for the suspension of the packing material. 50 mg of chiral adsorbent [this is chiral polymethacrylamide covalently bonded to silica gel; particle size 5 μm;
(ChiraSpher) (registered trade name), Merck KGeA; Darmstadt, DE]
Suspend in a 2-propanol mixture (9/1 (v / v)) and then treat in an ultrasonic bath for 15 minutes. This suspension is introduced into the precolumn and the pressure is then increased to 40
Gradually increase to 0-600 bar. When the desired pressure is reached, continue pumping for an additional 30 minutes, then stop the pump. Decrease pressure gradually. The tubule is then pumped with water for at least 60 minutes. Then 2
At this point, the smallest possible area of the chiral adsorbent is heated using a loop of Ni-Cr wires for 40 seconds. The capillary is then rinsed with the organic or aqueous eluent used for chiral separation. If a conductive eluent is used, electrophoretic chromatography can be performed.

Example 2 Preparation of Separation Capillary Filled with Chiral Adsorbent (Cellulose Derivative on Silica Gel) In a manner corresponding to that described in Example 1, silica gel as base support and cellulose tris (3 , 5-dimethylphenylcarbamate) is charged into a capillary. For this purpose, a commercial product, CHIRACEL OD (registered trade name) (Daicel, Japan) is used. Use example A : Separation of enantiomer of lopirazepam The separation capillary filled according to the above method (Example 1) was set in a commercially available capillary electrophoresis chromatography apparatus (HP 3D, Hewlett-Packard, Walobronn, Germany),
n-hexane / 1,4-dioxane / 2-propanol mixture (60/39/1
, V / v) for about 30 seconds. The chiral drug lopirazepam (racemic, 1 mg / ml) was then injected under a pressure of 12 bar for 6 seconds and the nanochromatographic enantiomer separation was performed at relatively low pressure (12 bar). A baseline separation of this enantiomer was obtained:

[0013]

[Table 1] The elution graph is shown in FIG. 1 (by UV absorption at λ = 254 nm). Use Example B : Separation of enantiomers of bendroflumethiazide Separation capillaries packed according to the above method (Example 1) were loaded onto a commercially available capillary electrophoresis chromatography apparatus (HP 3D, Hewlett-Packard, Walobronn, Germany). Install,
Flush with a methanol / water mixture (60/40, v / v) for about 30 seconds. Then, the chiral diuretic bendroflumethiazide (racemic, 3 mg / ml)
Was injected under a pressure of 12 bar for 6 seconds and the nanochromatographic enantiomer separation was performed at relatively low pressure (12 bar / 2.5 bar counter pressure). A baseline separation of this enantiomer was obtained:

[0014]

[Table 2] The elution graph is shown in FIG. 2 (by UV absorption at λ = 214 nm). Use Example C : Separation of enantiomers of bendroflumethiazide Separation capillaries packed according to the above method (Example 1) were loaded on a commercial capillary electrophoresis chromatography apparatus (HP 3D, Hewlett-Packard, Walobronn, Germany). Install,
About 3% with a mixture of methanol / 50 mM NaH ₂ PO ₄ (60/40, v / v)
Flash for 0 seconds. The chiral diuretic bendroflumethiazide (racemic, 3 mg / ml) is then injected for 6 seconds under a pressure of 12 bar and at a relatively low pressure (12 bar / 4 bar counter pressure) the nanochromatographic enantiomer separation. Was done. A baseline separation of this enantiomer was obtained:

[0015]

[Table 3] The elution graph is shown in FIG. 3 (by UV absorption at λ = 214 nm). Use Example D : Separation of enantiomers of bendroflumethiazide Separation capillaries packed according to the above method (Example 1) were transferred to a commercial capillary electrophoresis chromatography apparatus (HP 3D, Hewlett-Packard, Walobronn, Germany). Install,
About 3% with a mixture of methanol / 50 mM NaH ₂ PO ₄ (60/40, v / v)
Flash for 0 seconds. The chiral diuretic bendroflumethiazide (racemic, 3 mg / ml) is then infused under a pressure of 12 bar for 6 seconds, at a relatively low pressure (12 bar / 4 bar counter pressure) and an applied voltage of 10 kV, Nanochromatography / electrophoresis chromatography Enantiomer separation was performed. A baseline separation of this enantiomer was obtained:

[0016]

[Table 4] The elution graph is shown in FIG. 4 (by UV absorption at λ = 214 nm). Use Example E : Separation of Enantiomers of Stilbene Oxide According to the method of Example 2, the tubing is filled with OD material and then placed in a commercial capillary electrophoresis apparatus (HP 3D, Hewlett-Packard, Walobronn, Germany). Flush with a mixture of n-hexane / isopropanol for about 30 seconds. Then stilbene oxide (racemic, 1 mg / ml) was injected for 30 seconds under a pressure of 10 bar and the nanochromatographic separation was performed under a pressure of 10 bar. A baseline separation of the two enantiomers was obtained:

[0017]

[Table 5] The elution graph is shown in FIG. 5 (by UV absorption at λ = 214 nm). Use Example F : Separation of Enantiomers of Glutethimide According to the method of Example 2, the tubing is filled with OD material and then placed in a commercially available capillary electrophoresis apparatus (HP 3D, Hewlett-Packard, Walobronn, Germany). Flush with methanol for about 30 seconds. Then, glutethimide (racemic, 1 mg / m
l) was injected under a pressure of 10 bar for 30 seconds and the nanochromatographic separation was performed under a pressure of 10 bar. A baseline separation of the two enantiomers was obtained:

[0018]

[Table 6] The elution graph is shown in FIG. 6 (by UV absorption at λ = 214 nm). The special properties of these capillaries include the following properties: 1) Enantiomeric separation in one and the same capillary at least three different modes (normal phase HPLC, reverse phase HPLC and electrophoresis). Chromatography). The same capillary is also highly expected to be suitable for enantiomeric separation by SFC (critical fluid chromatography). 2) High speed HPLC can be performed at very low pressure. This allows performing high efficiency chiral HPLC separations using commercially available capillary electrophoresis equipment (eg, HP 3D, Hewlett-Packard, Walobronn, Germany, or MDQ, Beckmann Instruments, Fullerton, CA, USA). I do.

3) The formation of frit in the separation capillary is on-capillary.
Enable detection. This also eliminates the need for additional transfer from the separation tubing to the detector. At the same time, the special nano-injector and nano-detector cells required when using conventional narrow- and micro-pore HPLC capillaries.
ll) is no longer necessary. 4) The preparation of the capillaries is simple, in particular the chiral adsorbent can be filled under a pressure of 400-600 bar, then the two frits (inlet and outlet)
It can be formed by short-time local heating in the region filled with the chiral adsorbent. Thus, there is no need to repeatedly fill and then empty the capillaries.

[Brief description of the drawings]

FIG. 1 is a graph showing the elution of use example A (by UV absorption at λ = 254 nm).

FIG. 2 is a graph showing the elution of use example B (by UV absorption at λ = 214 nm).

FIG. 3 is a graph showing the elution of use example C (by UV absorption at λ = 214 nm).

FIG. 4 is a graph showing the elution of Use Example D (by UV absorption at λ = 214 nm).

FIG. 5 is a graph showing the elution of Use Example E (by UV absorption at λ = 214 nm).

FIG. 6 is a graph showing the elution of Use Example F (by UV absorption at λ = 214 nm).

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 30/48 G01N 30/48 W 30/88 30/88 PC (71) Applicant: Frankfighter Str. 250, D-64293 Darmstadt, Federal Republic of Germany (72) Inventor Shankvetaz, Bezhan Germany Day-48149 Münster, Huefferstraße 61, Apartment 19 (72) Inventor Schultu, Michael Germany Day-65438 Russell Rusheim, Im Groschen-Romsee 20 (72) Inventor Gilrod, Marko Germany Day-33790 Hull, Am Leibach 22 (72) Inventor Krause, Kerstin Germany-48147 Münster, Kettererstrasse 18 F term (reference) 4D017 AA03 BA03 CA14 CB01 DA03 DB02 EA05 EB03

Claims (6)

[Claims] 1. A thin tube filled with a particulate adsorbent and having a constant inner diameter over its entire length, wherein the range of the adsorbent bed is determined by a solidified region generated by heat treatment. . 2. The filled capillary according to claim 1, which contains a chiral adsorbent. 3. A process for producing a capillary filled with a particulate adsorbent, comprising the following steps: a) closing one end of a commercially available quartz glass capillary with a frit; b) the other end of the capillary. C) preparing a homogeneous suspension of the particulate adsorbent in a solvent; d) introducing said suspension into said storage container; e) pressurizing from said storage container. F) exchanging the solvent with water; g) heating the narrow area of the adsorbent-filled capillary at both ends of the adsorbent bed by heat treatment of the adsorbent by filtration; Forming a frit. 4. Use of a capillary packed with a particulate adsorbent according to claim 1 or 2 in the separation of at least two substances. 5. Use according to claim 4, wherein the separation is performed by liquid chromatography. 6. Use according to claim 4, wherein the separation is carried out by electrophoretic chromatography.