GB2220867A

GB2220867A - Device and method for chromatographic separation

Info

Publication number: GB2220867A
Application number: GB8914243A
Authority: GB
Inventors: Rudolf Ehwald; Holger Woehlecke; Ulrich Klein; Ulrike Zimare
Original assignee: INDFORSCHZENTRUM BIOTECH VEB
Current assignee: INDFORSCHZENTRUM BIOTECH VEB
Priority date: 1988-06-22
Filing date: 1989-06-21
Publication date: 1990-01-24
Also published as: GB8914243D0; DD278482A3; FR2633397A1; SE8902254L; DE3916219A1; SE8902254D0

Description

1 M&C F0M0: 230P58703 Chromatocraphic Materials and Methods 22e2o S 67

WANGDOC: 0185D The present invention relates to materials and methods for chromatography in analytical and preparative use.

Devices for liquid chromatography with fixed beds made of tightly packed dry particles (filling materials) are known in numerous, common variants. Examples include thin-layer chromatography and the use of powders in dry columns (e.g. G.-J. Krauss and G. Krauss, 'Experimente zur Chromatographie', VBB Deutscher Verlag der Wissenschaften, Berlin. 1979). An advantage of devices with dry fixed beds, such as is described in DEPS 3043 608, is that the liquid solvent moves without external forces, using cohesion and adhesion, by capillary action through the intrinsically absorbent porous separation path. The chromatographic use of dry fixed beds does, however, cause difficulties in the case of separation materials with a high capacity for absorbing the liquid solvent, such as is the case with numerous xerogels, since the expansion of the chromatographic beds leads to destabilisation and detachment of the packing from the carrier, if the material swells too much. It is also known that chromatographic, swellable thin layers crack when drying.

Columns made of dry swellable material cannot be used because of the swelling pressure and the one-sided expansion of the column packing during swelling. For these reasons it has so far been impossible to combine, in chromatographic apparatus or chromatographic methods. the advantages of dry separation paths with those of swellable filling materials (large inner distribution space, exclusion-chromatographic efficacy. large inner surface).

2 In particular it is impossible, using hitherto known apparatus and methods, to separate completely, and without loss, a substance with high molecular weight, or a group of substances with high molecular weight, using the exclusion principle, from accompanying low molecular weight substances, without considerable thinning. Various "batch methods" are known in which the excluded high molecular weight components of a liquid mixture are selectively concentrated in the inter-particle volume by swelling of a xerogel (see, for example, Determann, H., 'Gel Chromatography', Springer-Verlag Berlin, Heidelberg, New York, 1967) or of incompletely swollen vesicular filling materials (DD-PS 247 570). Here, however, the low molecular weight components of the mixture are not completely separated, resulting either in losses because of the incompleteness with which the inter-particle liquid is separated, or in thinning effects when the packing is subsequently washed.

The object of the invention is the further development of chromatographic techniques for cleansing and separating substances with regard to economy and quality of the separation process.

Thus, in a first aspect, the present invention provides a chromatographic substrate at least a proportion of which comprises gas-filled chromatographic material.

By 'substrate, is meant a material or materials suitable to make a chromatographic bed, such as described above. 'Substrate' and 'packing' are used interchangeably herein.

The gas-filled chromatographic material is ideally a known chromatographic substrate which has been dried so as to preserve as much wet-volume as possible, with, for example, air as the filling gas, replacing the liquid. Such drying may be achieved by such methods as lyophilisation.

7 3 The chromatographic substrate preferably contains 50% or more of gasfilled material or particles, the remainder being made up from such suitable materials as may be desired.

In an alternative. the present invention provides apparatus characterised by a dry chromatographic packing, such as defined above, with gas-filled intra-particle and extra-particle capillary spaces. the volume of the gasfilled capillary spaces being over 80% of the total packing volume. the chromatographic packing consisting of known vesicular or cellular filling materials up to over 50% by volume, and the width of the intra-particle capillary spaces being predominantly between 0.5 and 100pm.

The present invention permits chromatography combining the advantages of filling materials which are highly capable of swelling and the advantages of a dry separation path. in particular enabling macromolecules to be cleaned by exclusion chromat.ography in a way almost or completely free from thinning.

An advantageous variant of the said device consists in a closed chromatography column which is filled with dry air-containing powder made of lyophilised vesicular filling materials according to DD-WP 247 570. A second advantageous variant of the said device consists in a plate or foil on which lies a packing of lyophilised vesicular filling materials for chromatography as an open separation path. A further advantageous variant of the said device is characterised in that the dry chromatographic packing. as well as the vesicular carrier material. contains a further powdery medium with certain adsorption properties.

4 A method according to the invention for cleaning macromolecules in a way almost or completely free from thinning. using the said device. is characterised in that, first. part of the dry fixed bed is saturated with the liquid used as a solvent. thus moistening one side of the fixed bed. and then the liquid mixture with the macromolecules to be cleaned is introduced into the fixed bed from the damp side. then. from the same side, the chromatographic solvent is again introduced into the fixed bed until the moistening front has passed through the whole fixed bed, and in that at least one liquid sample is eluted from the last-moistened part of the fixed bed.

surprisingly. it has been shown that the chromatographic fixed beds consisting of dry vesicular carrier material of the invention with a high gas or air content are. regardless of their high absorption capacity for liquids. volume-stable and intrinsically absorbent in the moistening process at high rates of flow. At the same time - contrary to previous experience with material with high liquid absorption capacity - the microscopic structure change. which is determined by swelling. is completed at the moistening front, without flow-inhibiting compression or any macroscopic swelling process which would destroy the integrity of the fixed bed.

The method described makes it possible for the first time to separate high molecular weight substances from low molecular weight substances substantially without loss and without concomitant thinning. For completeness of separation of low molecular weight substances, the length of the separation patht already damp before the mixed sample is applied, should be sufficient. For concentration of the eluted macromolecular components, the decisive factor tends to be. however. the length of the parts of separation path which are still dry when the sample is applied. If the volume ratio between the dry and the damp part of the fixed bed after the samplehas been applied is more than about 0.8, it is generally possible to concentrate a high molecular weight substance or a group of macromolecules in combination with the cleaning of low molecular weight substances by the described method. At the same time it is possible to elute the concentrated macromolecules without substantial loss. In a preferred variant. favourable to the concentration of the eluted macromolecules. before the sample mixture is introduced. the fixed bed is saturated up to at least about 20% of its volume with the solvent. leaving dry a volume portion of the fixed bed of at least about 30% after the liquid mixture with the macromolecules to be cleaned has been introduced.

The invention will be further illustrated with reference to the following Examples.

Example 1. Dry Column A sample of a vesicular carrier material made of vegetable cell units according to DD-PS 247 570 was lyophilised. Of the dry powder obtained, 330M9 were packed tightly into a chromatography column (V = 7 mI, 1 = 7 cm). The process is illustrated by the accompanying Figure. Two ml of a 0. 1 M Soerensen phosphate buffer (pH 7.0) were applied to the dry substrate. When the buffer solution had been absorbed. 0.4m1 of a liquid mixture of Dextran Blue 2000 (Pharmacia) and phenol red, both in a 0.025% aqueous solution. were applied to the already damp part of the intrinsically absorbent fixed bed (1 in the Figure). When the sample had penetrated. a layer of the elution buffer was applied to the column. While the liquids were being absorbed, no gas bubbles were observed in the moistened part of the fixed bed.

6 During absorption of the sample mixture and subsequent penetration of the elution buffer, there was a visible separation between the phenol red band (2 in the Figure, small dots) which moved as quickly as the moistening front, and the band of the macromolecular Dextran Blue (larger dots) which passed more quickly through the damp part of the fixed bed and reached the moistening front before the latter had reached the lower end of the packing (3 in the Figure). The macromolecular dye was concentrated at the moistening front and it was collected quantitatively just 0.2 ml of the first elution traction. The concentration of the 0.2 ml fraction of dextran was 0.05% (photometrically determined). Separation of phenol red was complete. In the same way, complete separation between proteins (cL-amylases from human saliva, ferritin) and ammonium sulphate was achieved with definite concentration of the proteins. The total quantity of protein applied in 0.4 ml was obtained free from sulphate in the first 0.2 ml of the eluate.

Example 2. Surface Bed A 2 mm thick layer of the dry powder described in Example 1 was applied to a glass plate (2 x 7 em). The separation path was limited at the ends by a narrow strip of chromatography cardboard via which the elution agent and the sample solution were fed. After absorption of 1 ml of a Soerensen phosphate buffer (pH 7, 0.1 M), the same mixture as described in Example 1 of Dextran Blue and phenol. red was applied to the already damp part of the intrinsically absorbent fixed bed. The buffer was then absorbed into the fixed bed. The bed remained stable in shape at the moistening front and was saturated with the aqueous solution with a straight moistening front in 30 minutes without macroscopic swelling or formation of k 7 cracks. During this, the separation between Dextran Blue and phenol red became visible. Dextran Blue was concentrated at the moistening front as in the column packing described in Example 1.

Example 3. Mixed Bed Dry cellular or vesicular carrier material was mixed with diethylamino- cellulose in the ratio 1:1. As described in Example 1. the powder obtained was packed into a chromatography column and the elution agent and of the sample solution applied. The elution agent used was distilled water. The sample mixture consisted of 0.2 mg Dextran Blue 2000 and 0.2 mg Dextran T 250 in 0.4 ml distilled water. The neutral Dextran T 250 was concentrated. as described in Example 1, at the moistening front, while the Dextran Blue, derivatised by Cibachrom Blue F 3G-A and therefore anionic, was immobilised by adsorption on entry into the packing. The neutral Dextran T 250 was quantitatively separated from the anionic Dextran Blue molecules and in concentrated form.

8

Claims

1. A chromatographic substrate at least a proportion of which comprises gas-filled chromatographic material.

2. A substrate according to claim 1 wherein the gasfilled material comprises a wetted chromatographic substrate dried so as to preserve as much wet-volume as possible.

3. A substrate according to claim 2 wherein the wetted substrate has been dried by lyophilisation.

4. A substrate according to any preceding claim wherein the substrate contains 50% or more of the gas-filled material.

5. A substrate according to any preceding Claim, further comprising powdery media with a volume portion of the non-vesicular material in the dry mixture of below 50%.

6. A substrate according to Claim 5. wherein the non-vesicular powdery medium has selective adsorption properties.

7. Packing material for dry chromatographic fixed beds. comprising a known vesicular carrier material with gas-filled intra-particle and extraparticle cavities. the volume of the gas-filled cavities being over 80% of the total fixed bed volume and the width of the intra-particle cavities being predominantly between 0.5 and 100 lim.

8. A substrate as defined in any of claims 1 to 6 having any or all of the parameters of claim 7.

9 9. Apparatus for chromatographic separation. characterised in that it comprises a fixed bed of material according to any preceding claim.

10. Apparatus according to Claim 9, wherein the dry chromatographic fixed bed is in a closed chromatographic column. or lies as an open layer or packing on a foil or plate.

11. A method for chromatographic separation. comprising use of apparatus according to Claim 9 or 10. and first moistening a part of the fixed bed with a liquid solvent. resulting in a fixed bed moistened on one side, then the liquid mixture with macromolecules to be cleaned is introduced or sucked into the fixed bed from the same side. then the chromatographic solvent is again introduced or sucked into the packing from the same side, until the moistening front has passed through the whole fixed bed. at least one liquid sample being eluted from the last-moistened part of the fixed bed.

12. A method according to Claim 11, wherein, before the introduction of the liquid mixture with the macromolecules to be cleaned. the fixed bed is moistened on one side with the solvent up to at least 20% of the fixed bed volume. the dry volume portion of the fixed bed amounting to at least 30% after the liquid mixture with the macromolecules to be cleaned has been introduced.

13. A gas-filled chromatographic substrate, substantially as hereinbefore described.

14. Chromatographic apparatus, using gas-filled substrate, substantially as hereinbefore described.

15. A method of separating substances by chromatography using a gasfilled substrate, substantially as hereinbefore described.

Published 1990 a, The Patent Office. State House. 66 71 High Holborn. London WClR4TP_Futhercopies maybe obtained from The Patent Office Sales Branch. St Mary Cray. Orpington. Kent BR5 3RD Printed by Multiplex techniques ltd, St Mary Cray. Kent. Con. 1 87