WO1992003726A1

WO1992003726A1 - Method of, arrangement and separation unit for carrying out separation analysis investigations in a planar system with forced flow of a fluid carrier medium, especially by the means of overpressurized layer chromatography

Info

Publication number: WO1992003726A1
Application number: PCT/HU1991/000041
Authority: WO
Inventors: Émil NINCSOVICS; Ern^'o TYIHÁK; Zoltánné BARANYI; Barnabás TAPA
Original assignee: Laboratóriumi Mu^'szergyár Rt.
Priority date: 1990-08-28
Filing date: 1991-08-28
Publication date: 1992-03-05
Also published as: HU905502D0; HUT60934A

Abstract

In a method of and an arrangement for carrying out separation analysis investigations in a planar system with forced flow of a fluid carrier medium, an overpressurizable chamber is created for receiving a sorbent layer (4) bearing a complex sample to be investigated and having channels where a fluid carrier medium (eluent) can flow. Within the overpressurizable chamber at least one barrier (7) is applied for interrupting the flow of the fluid carrier medium in the sorbent layer (4) is arranged. The separation unit comprises at least one separation plate having a sorbent layer for ensuring conditions of straight line front migration of a fluid carrier medium, and the at least one separation plate is connected with a closing frame having at least one barrier (7) for interrupting the flow of the fluid carrier medium in the sorbent layer (4) when the closing frame is pressed to the sorbent layer (4).

Description

METHOD OF, ARRANGEMENT AND SEPARATION UNIT FOR CARRYING OUT SEPARATION ANALYSIS INVESTIGATIONS IN A PLANAR SYSTEM WITH FORCED FLOW OF A FLUID CARRIER MEDIUM, ESPECIALLY BY THE MEANS OF OVERPRESSURIZED LAYER CHROMATOGRAPHY

FIELD OF INVENTION

The invention refers to a method of, an arrangement and a separation unit for carrying out separation analysis investigations in • planar system with forced flow of a fluid carrier medium, especially by the means of overpressurized layer chromatography. The proposed method is capable of carrying out investigations in one- or two-directional linear, planar and spatial systems, by the means of circular or anticircular (triangular) forced flow of the fluid carrier medium. The arrangement ensures by the means of mechanical forces applied in a pneumatical or hydraulical or other systems the conditions of carrying out overpressurized layer chromatography investigations and hinders the flow of the fluid carrier medium (eluent) in the directions where its presence is undesired. According to the method of the invention the known steps of introducing at least one sample of at least one complex mixture to a sorbent layer forming a part of a separation plate of a separation unit, the separation plate having at least one channel for transporting fluid carrier medium within the sorbent layer to at least one predetermined place on the separation plate, feeding a fluid carrier medium into the at least one channel of the separation plate, arranging at least one separation plate carrying a respective sorbent layer in connection with means for forcing the fluid carrier medium to flow in the at least one channel, forcing the fluid carrier medium to flow and thereby to take away the components of the sample in an order determined by known retention of the components, the fluid carrier medium with the samples forming a fluid medium, and conducting the fluid medium through the at least one channel to at least one predetermined place on the at least separation plate and therefrom to at least one sensing unit, than determining at least one characteristic parameter of the components taken away in the fluid medium from the pre¬ determined place in a continuous real-time process up to reaching a given value of the retention, and/or connecting the at least one separation plate with at least one sensing unit for carrying out measurements of at least one characteristic parameter of at least one component remaining on the surface of the separation plate are carried out. The novel arrangement comprises means for accomodating at least one separation plate made with at least one sorbent layer receiving a complex sample to be investigated, means for introducing the complex sample onto the sorbent layer, means for forcing a flow of a fluid carrier medium on the separation plate when held by the accomodating means, means for feeding the fluid carrier medium into the sorbent layer when the separation plate is held by the accomodating means, means for conducting the fluid carrier medium within the sorbent layer for taking away components of the complex sample when the separation plate is held by the accomodating means, a detector unit including at least one element for real-time measurements of the fluid carrier medium leaving the separation plate together with selected components of the complex sample and a sensing unit for measuring at least one characteristic parameter of the components remaining on the sorbent layer of the separation plate, the sensing unit being located opposite to the accomodating means or opposite a further means for accomodating the separation plate, wherein the accomodating means are constituted by a supporting plate bearing at least one separation plate made with the at least one sorbent layer and a covering plate, the supporting plate and the covering plate determining an overpressurizable chamber. The separation unit of the invention includes at least one separation plate having a sorbent layer for ensuring conditions of straight line front migration of a fluid carrier medium.

BACKGROUND OF THE INVENTION

The layer chromatography is a modified version of the traditional column chromatography wherein a fluid carrier medium called eluent is applied for making components of a sample migrate along a channel in an appropriate sorbent layer. The migration is caused by capillary or other forces and the components of the sample are separated from the liquid medium in a known order of their retention. On the basis of the retention the components can be determined and identified.

In the solid environment of the sorbent layer the flow of the fluid carrier medium comprising the components of the sample is very slow unless outer forces are applied. In linear and planar systems therefore the forced flow of the fluid carrier medium is ensured, e.g. by overpressurizing, creating electroos otic conditions, vacuum, electric field, centrifugal forces, etc. The overpressurized systems are shown among others in the GB-PS 1,570,760 wherein an apparatus with at least one sorbent layers is described. This apparatus can be applied to chro atcgraphic investigations and comprises the at least one sorbent layer arranged in an overpressurized chamber. The sorbent layer is covered within this chamber by a layer made of a relatively soft material, e.g. silica gel or alumina or other compositions showing absorbing features. In this way the vapour space over the sorbent layer can be excluded or its height diminished to a minimal extent. A micropump is applied for introducing the chromatographic eluent (fluid carrier medium) to a selected point of the carrier plate covered with the sorbent layer. At least one edge of the sorbent layer is closed on the carrier plate by impregnation or a thin plastic film. The eluent migrates on the plate in a linear front because before the place of introduction of the eluent a channel is prepared or a metallic wire or a plastic plate is arranged.

The apparatus disclosed in the citation analyzed above can be improved further according to the proposal shown in the US-PS 4,469,601 (Beaver et al) . The system of this specification can be applied to planar systems capable of .carrying out in some cases spatial chromatographic develop¬ ments. According to Beaver et al the carrier plate and the sorbent layer are dried after development in one direction for expelling the eluent, thereafter the sorbent layer undergoes development in another direction whereby at least one component of the sample to be investigated is taken away from the sorbent layer and its quantity is determined. By making developments on more sorbent layers this system offers very high productivity, wherein carrier plates are applied which fit one another with high precision and the place of introduction of the eluent is at the same place on all of the plates applied.

The solutions depicted above have disadvantages and especially they do not offer good conditions for separation in large samples. Hence, the proposal shown in the GB-PS -2,179,564 and in the corresponding DE-OS 36,29,394A1 was elaborated, according to which the fluid carrier medium is forced to flow up to the edge of the sorbent layer where it is collected and in separate streams different components can be measured. The streams are analyzed up to reaching a given retention assigned to the components migrating with the fluid carrier medium, thereafter the sorbent layer is forwarded in relation to chromatographic and/or electrophoretic detecting units for determining the components remaining on the sorbent layer.

The methods and apparatuses disclosed and proposed in the publications cited above constitute a very high level of the overpressurized layer chromatography but the solutions which have become known show some disadvantages. The most important problem follows from the fact that in the case of linear and planar developments the edges of the sorbent layers should be closed, for example by impregnation in order to avoid escape of the eluent which is fed to the layer under overpressure. The edge of the sorbent layer can be closed only by sophisticated arrangements or by manual methods and this is a complicated step, resulting in a remarkable in- crease of the costs of the sorbent layers and carrier plates bearing the last.

The closure of the sorbent layers results also in another problem which can be recapitulated as follows: it is really very difficult to find a composition for impregnating the edge of the sorbent layer which is capable of with¬ standing the very wide variety of the polar and apolar solvents used as eluents. A further problem is to be seen in the fact that the polymer compositions or other solutions applied to impregnating the edge of the sorbent layer may contaminate the surface of the sorbent layers, causing thereby dicrease of the effectivity of chromatographic separation and disturbance in the structural investigations because the material used for the impregnation of the edge(s) can be eluated together with the components of the sample to be investigated. Hence, in a mass—spectrometry analysis components can be present which are not intended to be identified at all.

The contamination caused by the components of the solutions applied to impregnating the edges of the sorbent layers is amplified by the components of adhesives used for fixing the sorbent layer on a carrier plate. These adhesives are generally organic compounds or mixtures of such compounds of middle or low molecular weight. The components of the adhesives can disturb both the separation and the structural analysis investigations, the last following the separating operations.

During the linear development it is sometimes difficult to separate all the components of a sample. In different places of the sorbent layer mixtures of compounds and components can accumulate. The accumulated mixtures cannot be separated by the planar development (i.e. carried out in two dimensions) .

The increase of the effectivity of separation carried out by the overpressurized layer chromatography is sometimes impossible without preliminary cleaning of the samples. The advantage of the known traditional chromatographic technics is that the separation is rather effective also without preliminary cleaning or it is sufficient to carry out a very rough cleaning before separation. It is a long felt need that this is not sufficient, a system for cleaning is necessary which makes use of the advantages of the basic technics and creates special connection between the liquid chromatography technics based on column and layers whereby the effectiveness of both can be exploited.

SUMMARY OF THE INVENTION

The invention has the object to give a solution to the problems of the overpressurized layer chromatography depicted above and to improve the effectiveness of the chromatographic and similar separation analysis investigations.

The invention is based on the recognition that the impregnation of the edges of the sorbent layers is not necessary if the elements forming the chamber wherein the sorbent layer is arranged are prepared in a special manner and are capable of ensuring closure of the sorbent layer by the means of hydraulic, mechanic or pneumatic forces. The recognition results in the fact that no impregnation is necessary at the edges of the sorbent layers and in given case the carrier plate bearing the sorbent layer is also not necessary. This recognition is completed by the statement that the chromatographic and similar development can be carried out on the same sorbent layer more times and that carrying out this development in an environment containing reduced quantity of oxygen (coming from the air) helps in hindering the decay processes of the components. A yet fur- ther recognition is that it is possible to carry out spatial development if the impregnation is not necessary.

A further important element of the invention is that the preliminary cleaning of the samples can be solved by the means of the overpressurized layer chromatography, when clear samples can be produced which are the basis of chromato¬ graphic investigations in both overpressurized layer systems and liquid column systems.

Hence, the present invention refers to a method of, an arrangement for and a separation unit for carrying out separation analysis investigations in a planar system with forced flow of a fluid carrier medium, especially by the means of overpressurized layer chromatography realized in different known systems.

The method of the invention comprises the steps of introducing at least one sample of at least one complex mixture to a sorbent layer forming a part of a separation plate of a separation unit, the separation plate having at least one channel for transporting fluid carrier medium within the sorbent layer to at least one predetermined place on the separation plate, feeding a fluid carrier medium into the at least one channel of the separation plate, arranging at least one separation plate carrying a respective sorbent layer in connection with means for forcing the fluid carrier medium to flow in the at least one channel, forcing the fluid carrier medium to flow and thereby to take away the components of the sample in an order determined by known retention of the components, the fluid carrier medium with the samples forming a fluid medium, and conducting the fluid medium through the at least one channel to at least one predetermined place on the at least separation plate and therefrom to at least one sensing unit than determining at least one characteristic parameter of the components taken away in the fluid medium from the predetermined place in a continuous real-time process up to reaching a given value of the retention, and/or connecting the at least one separation plate with at least one sensing unit for carrying out measurements of at least one characteristic parameter of at least one component remaining on the surface of the separation plate, wherein according to the invention the further step of connecting the at least one separation plate with at least one mechanical closing means for stopping flow of the fluid carrier medium in the at least one channel at the predetermined place is carried out.

In an advantageous embodiment of the proposed method for carrying out investigations by the means of the over- pressurized layer chromatography the steps of forcing the fluid carrier medium to flow by the means of overpressure created in a closed chamber determined by a supporting plate and a closing plate, the supporting plate and/or the closing plate connecting at least one sorbent layer of the separation unit, and stopping the flow of the fluid carrier medium with a barrier forming a protrusion on the surface of the supporting plate and/or closing plate for making the sorbent layer impervious by interrupting and/or packing its material are carried out. It is also advantageous to stop the flow of the fluid carrier medium in the sorbent layer by the means of a barrier having a sharp edge, the barrier arranged on a carrier plate of the sorbent layer.

Another preferred possibility of realizing the proposed method lies in stopping the flow of the fluid carrier medium by the means of a barrier including at least one frame interrupting and/or packing the material of the sorbent layer along at least one line.

It is also advantageous when the flow of the fluid carrier medium is stopped by the means of a barrier interrupting the material of the sorbent layer and causing deformation of the carrier plate. A further preferred possibility is to stop the flow of the fluid carrier medium by the means of a barrier pressed to at least one surface of the sorbent layer in a controlled manner. The flow of the fluid carrier medium can advantageously be stopped by the means of a barrier exerting pressure depending on the force pressing the supporting plate against the covering plate.

A further object of the invention is, as mentioned, the arrangement for carrying out separation analysis investiga-. tions in a planar system with forced flow of a fluid carrier medium, especially by the means of overpressurized layer chromatography, comprising means for accomodating at least one separation plate made with at least one sorbent layer receiving a complex sample to be investigated, means for introducing the complex sample onto the sorbent layer, means for forcing a flow of a fluid carrier medium on the separation plate when held by the accomodating means, means for feeding the fluid carrier medium into the sorbent layer when the separating means is held by the accomodating means, means for conducting the fluid carrier medium within the sorbent layer for taking away components of the complex sample when the separation plate is held by the accomodating means, a detector unit including at least one element for real-time measurements of the fluid carrier medium leaving the separation plate together with selected components of the complex sample and a sensing unit for measuring at least one characteristic parameter of the components remaining on the sorbent layer of the separation plate, the sensing unit being located opposite to the accomodating means or opposite a further means for accomodating the separation plate, wherein the accomodating means are constituted by a supporting plate bearing at least one separation plate made with the at least one sorbent layer and a covering plate, the supporting plate and the covering plate determining an overpressurizable chamber, and according to the invention within the over¬ pressurizable chamber at least one barrier for interrupting the flow of the fluid carrier medium in the sorbent layer is arranged and advantageously the overpressurizable chamber is connected with means for helding in its interior isothermal or programmed temperature conditions.

In a preferred embodiment of the arrangement of the invention within the overpressurizable chamber at least one sealing element is arranged for hindering any flow of the fluid carrier medium in the direction closed by the sealing element. In a further preferred embodiment of the proposed arrangement at least one rigid closing plate and/or flexible closing plate is arranged within the overpressurizable cham¬ ber in connection with the supporting plate and/or the covering plate. It is a very advantageous embodiment of the arrangement of the present invention wherein the overpressurizable chamber includes straight and/or circular and/or segmented channels for directing, collecting and taking away the fluid carrier medium, the channels having advantageously at least one filtering element and preferably a point shaped inlet element or inlet elements arranged along a circular line for introduction of the fluid carrier medium.

The automatization of the measurements is facilitated by the advantageous embodiment of the arrangement of the invention, wherein the overpressurizable chamber is arranged in a loop of introducing at least one sample and preferably within the overpressurizable chamber means for preliminary- cleaning of the sample is arranged.

The proposed arrangement is capable of carrying out electroosmotic investigations when within the overpressuriz¬ able chamber at least one sorbent layer is equipped with electrodes for generating electric field.

Generally it is advantageous under aspect of placing the arrangement of the invention, if the overpressurizable chamber is made with elements resulting in its cylindrical shape wherein the sorbent layers are made with nonlinear surface.

It is also the object of the present invention a separation unit for carrying out separation analysis inves¬ tigations in a planar system with forced flow of a fluid carrier medium, especially by the means of overpressurized layer chromatography, comprising at least one separation plate having a sorbent layer for ensuring conditions of straight line front migration of a fluid carrier medium, wherein according to the invention the at least one separation plate is connected with a closing frame having a at least one barrier for interrupting the flow of the fluid carrier medium in the sorbent layer when the closing frame is pressed to the sorbent layer.

It is sometimes very advantageous when the separation unit of the invention the separation plate consists of a carrier plate and at least one sorbent layer covering one or two surfaces of the carrier plate which increases the strength of the sorbent layer(s) .

Under aspect of the rigidity of the separation unit proposed by the invention it is advantageous, when the separation plate is connected with at least, one rigid closing plate covering it from at least one side, and if necessary the rigid closing plate is covered from outer siede with a flexible closing plate. The different kinds of the chromatographic investiga¬ tions can be carried out when the separation unit is equipped with a barrier forming a wall-type extrusion arranged along a closed line, especially in a rectangular or circular shape or a wall-type extrusion arranged along an opened line. In the separation unit as of the invention it is preferred to apply carrier plate of the sorbent layer made of glass, appropriate plastics or metal and sorbent layers is made of silica gel, silica gel modified in polar and/or apolar manner, alumina, talc, magnesium silicate, hydroxy- apatite, cellulose, modified cellulose, ion exchanger of organic or inorganic basis, sorbent composition based on plastics, an optically active composition.

The carrier plate is not necessary in the separation unit proposed by the invention when the sorbent layer is consisted of grains fixed in laminae by the means of a glue of organic or inorganic basis or by sintering applying a grained plastic or plastic fibers.

The samples to be investigated can be developed in three dimensions, i.e. in a spatial system, when the separa¬ tion unit of the invention consists of more sorbent layers arranged one over another without respective carrier plates in a column which is covered by a directing block for for¬ warding the fluid carrier medium into the column.

The method, the arrangement and the separation unit of the invention offer the possibility of widening the inves- tigation capacity of different methods of overpressurized and high-pressure layer chromatography and especially it creates the conditions of effective development in three dimensional sorbent layer systems.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be further described in more detail with reference to the accompanying drawings showing by way of example only some preferred realizations and embodiments of the method, arrangement and separation unit proposed by the invention. In the drawings Fig. la shows a first embodiment of an overpressurized chamber for chromatographic investigations in cross- section which offers the possibility of closing an edge of a sorbent layer by the means of a sharp barrier, Fig. lb shows a second embodiment of an overpressurized chamber in cross-section which offers the possibility of closing an edge of a sorbent layer by the means of a simple packing barrier and a sealing element, Fig. lc illustrates a third embodiment of an overpressurized chamber wherein the sorbent layer is connected with a rigid closing plate covered by a flexible closing plate, the chamber in cross-section which offers the possibility of closing an edge of a sorbent layer by the means of a sharp barrier crossing the rigid and flexible closing plates. Fig. 2a is the cross-section of a fourth embodiment of an overpressurized chamber offering the possibility of closing of two edges of a sorbent layer by the means of packed regions of the material of the sorbent layer. Fig. 2b shows the cross-section of a fifth embodiment of an overpressurized chamber offering the possibility of closing an edge of a sorbent layer by the means of a body pressed from outside to the sorbent layer, the sorbent layer being, if necessary connected with a rigid closing plate. Fig. 3 illustrates the cross-section of a sixth embodiment of an overpressurized chamber including a thick sorbent layer of indented surface and having support¬ ing plate and covering plate prepared for receiving a such layer, Fig. 4a is a side view of a carrier plate with a sorbent layer from one side, Fig. 4b shows a side view of a carrier plate with a sorbent layer from two sides. Fig. 4c illustrates a side view of a sorbent layer realized without carrier plate. Fig. 5a shows the top view of an embodiment of a sorbent layer prepared for on-line circular forced flow investigations, Fig. 5b illustrates the top view of an embodiment of a sorbent layer prepared for on—line anticircular forced flow investigations.

Fig. 6 is a side view of a box receiving a sorbent layer and having means for directing the flow of the eluent in an overpressurized measuring arrangement.

Fig. 7a is the top view of a sorbent layer for a more layer chromatography system, Fig. 7b is the top view of a covering plate receiving a sorbent layer shown in Fig. 7a,

Fig. 8 illustrates an elevational view of a two carrier plate sorbent layer system with sorbent layers facing one another and arranged in a closing frame, Fig. 9 illustrates an elevational view of a four carrier plate sorbent layer system with sorbent layers lying on the same sides of the carrier plates and arranged in closing frames, Fig. 10 is an elevational view of a three carrier plate sorbent layer system forming a box, Fig. 11a is the top view of a sorbent layer with separated introduction of the fluid carrier medium and the sample, Fig. lib is the top view of a sorbent layer with common introduction of the fluid carrier medium and the sample,

Fig. 12a illustrates the top view of a more channel over¬ pressurized layer chromatography arrangement of on- —line system based on an isokratic or gradient sorbent layer with common introduction of the fluid carrier medium (eluent) ,

Fig. 12b illustrates the top view of a more channel over¬ pressurized layer chromatographic arrangement of on- -line system based on an isokratic or gradient sorbent layer with more point introduction of the fluid carrier medium (eluent) ,

Fig. 13a illustrates the the top view of a first embodiment of a sorbent layer equipped with different elements causing the forced flow of the fluid carrier medium in combined on-line and off-line chromatographic systems, Fig. 13b illustrates the the top view of a second embodiment of a sorbent layer equipped with different elements causing the forced flow of the fluid carrier medium in combined on-line and off-line chromatographic systems, Fig. 13c illustrates the the top view of a third embodiment of a sorbent layer equipped with different elements causing the forced flow of the fluid carrier medium in combined on-line and off-line chromatographic systems, Fig. 14a is a schematic representation of the first step of a spatial development, i.e. the introduction of the sample, Fig. 14b is a schematic top view of a sorbent layer after linear development forming the first step, Fig. 14c is a schematic representation of the second step of a spatial development, i.e. the development of the sample in a second direction which is preferably per¬ pendicular to the direction of the linear development carried out in the first step, Fig. 14d is a schematic top view of a sorbent layer after linear development forming the second step, Fig. 14e shows the elevational view of a column of sorbent layers for spatial development and Fig. I4f illustrates the elevational view of a system for carrying out the spatial development with the column shown in Fig. 14e, and Fig. 15 shows a schematic representation of a system for carrying out on-line investigations by the means of overpressurized layer chromatography and traditional column chromatography. DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODI¬ MENTS

When carrying out the method proposed by the present invention the sorbent layers are of special importance. The arrangement thereto can be the same as disclosed in the GB-PS 2,173,125A with the difference that a barrier for inter¬ rupting the flow of a fluid carrier medium as described later is applied. The sorbent layers can be prepared in any known way or in the manner described hereinbelow. The invention offers the possibility of applying more than one sorbent layer in the chromatographic development of complex samples of different organic and inorganic compounds and the sorbent layers can be arranged in a complex system. The sorbent layers are prepared either according to the art on appropriate carrier plates, or according to the invention without such carrier plates on the basis of self supporting structures. The sorbent layers are realized with different compositions of specific surface (graininess, porosity) selected with regard to the given con¬ ditions, they are prepared on the basis of silica gel, alumina, magnesium silicate, talc as inorganic components, cellulose and cellulose derivatives, pulverized synthetic resin, polyamid as organic components. The practice has shown the best results when reverse phase sorbents bound chemically are applied. A very advantageous measure is to apply alcail chains of different length and bound by specific means, for example by Si-O-Si-C chains. The functional groups can be linked with the silica gel and among these groups the amino, aminocyano, nitro and diol groups are especially important. The sorbent layers based on these groups can be applied with very good results. The ion exchanger layers based on organic and inorganic compounds, the combination of such layers are also especially preferred and the same can be stated with reference to the different combination of ion exchanger compounds and inorganic sorbents. If the separation analysis investigations are carried out by the means of the size exclusion method the cross- —linked polymerized gels and the inorganic gels (e.g. the high porosity mixtures based on silica gel) can be applied. When separating optically active compounds the application of sorbent layers comprising at least one optically active com¬ ponent is also preferred.

The carrier plate of the sorbent layer can be made of appropriate plastics, metal or glass wherein the sorbent layer covering the carrier plate consists of a single substance, a homogeneous or heterogeneous composition of two or more different substances.

The thickness of the sorbent layer lies advantageously in the range from 0.05 mm to 5.0 mm, the specific value depends on the conditions and object of the separation analysis.

The introduction of the sample into the separation system is carried out according to the known principles by bringing it either in strips or in spots onto the dry or wet sorbent layer. The sample can be added to the stream of the fluid carrier medium or brought onto the sorbent layer independently on that stream.

The temperature conditions of the sorbent layer and its environment can be controlled according to a predetermined program both in space and time, whereby the effectiveness of the separation can be improved because the temperature can be selected according to the conditions of the process.

If the grains of the sorbent layer have to be arranged and fixed in layers, this can be ensured by adhesives of organic or inorganic basis, by pulverized plastics or synthetic fibers where the structure is built up and fixed by the means of sintering.

A very advantageous solution is when closing plates made of rigid and flexible materials are applied together with the sorbent layers and the closing plates are equipped with straight line or circular channels or channel systems having in given case segmented parts for collecting, taking away and directing the eluent; the channels can be completed also with filtering elements.

An especially effective and unique solution of the invention is when the development is carried out in a three- —dimensional, spatial system. The spatial development is a task which has been depicted many times in the literature but the present invention is the first offering a real solution to this problem in the following way: the sample is developed in a planar system in two directions and thereafter the sorbent layer including the spots of the components of the sample spread in two directions is arranged on the top of a "cube" consisting of sorbent layers realized without carrier plates. The cube is covered by a block for directing the flow of the eluent and the flow of the eluent forces the components of the sample to move into the interior of the cube. After terminating the development the sorbent layers of the cube are separated and the different components of the sample can be determined on them. The third step of the spatial development can be made also by traditional liquid chromatographic systems when the cube as a column is arranged in the eluent and the eluent migrates in the block of the sorbent layers under the influence of the capillary forces. The more step development offered by the invention increases the effectiveness of the separation, creates conditions where the oxygen of the air cannot deteriorate the components, the time of development is minimalized and there¬ by the danger of decay of the components can be remarkably limited. Hence, the advantages of the more step development can be used, the steps can be automatised.

The sorbent layers can be arranged in a box indepen¬ dently whether they are realized on a carrier plate or without it. In the covering element of the box a channel for directing the eluent can be created and frames are applied for closing the sorbent layers. A such system can be applied also in spatial developments.

An important element of the present invention is the combination of the process of cleaning the sample and its development in a forced flow system. This is a combination of high effectiveness, uniting the advantages of the layer and column chromatography methods. The overpressurized chamber can replace the injecting loop of a high pressure liquid chromatography system or the two systems are simultaneously applied. Another solution is when a preliminary cleaning of the sample is carried out in on-line system in the over¬ pressurized chamber and a fraction of given parameters is taken to the column. When separation is carried out by the traditional TLC chromatography system the main fractions can be eluated through the overpressurized chamber to the column of the high pressure liquid chromatography system. Another possibility is to collect and if necessary to take over to a layer or a column the components eluated on at least one outlet, or to spread the components onto the sorbent layer and after drying the last to carry out the development. The structural analysis of the fractions received by any of the methods depicted above, the determination of the physico-chemical parameters of the fractions can be carried out so that the composition and/or compound to be inves¬ tigated are taken away from the outlets and transported to a high sensitivity system for carrying out the prescribed measurements and analyses. The sensitive measurement can be done by infrared and ultraviolet spectrometers, mass spectro¬ meters, nuclear magnetic resonance (NMR) spectrometers, and ^"similar means. The arrangement realized according to the invention is schematically illustrated in Figures la, lb, lc, 2a, 2b and 3. In Figs la, lb, lc some selected possibilities of realiz¬ ing the closure of the edges of the sorbent layer by appropriate barriers are shown. In the arrangements of these Figures an eluent (fluid carrier medium) is introduced through an inlet 1 (not shown in more detail) onto an appro- priate surface section 6 of a sorbent layer 4 which may be arranged on a carrier plate 5 surrounded by a frame 8 for fixing its position. The carrier plate 5 and the sorbent layer 4 are arranged between a supporting plate 3 and a covering plate 3* pressed against one another in the direc¬ tion of arrows 2. The mentioned elements constitute an over¬ pressurizable chamber applied to the overpressurized layer chromatography investigations.

The covering plate 3' of the embodiment shown in Figs la, lb, lc is equipped with a barrier 7 protruding therefrom in the direction of the sorbent layer 4. The barrier 7 crosses the sorbent layer 4 and reaches the carrier plate 5 in the embodiment of Fig. la. Another possibility s (Fig. lb) when the barrier 7 is applied only for packing the material of the sorbent layer 4 causing thereby increase of the density of the sorbent layer 4 in predetermined places. The packed parts of the sorbent layer 4 are not capable of hindering fully the flow of the eluent, and therefor in order to close the way of flow a seal 10 is arranged behind the inlet l. A further possibility is shown in Fig. lc: the surface of the sorbent layer is connected with a rigid closing plate 9, the last is covered by a flexible closing plate 11 and in the covering plate 3• the barrier 7 is made to cross both the rigid and flexible closing plates 9 and 11 and the sorbent layer 4. According to Fig. 2a the supporting plate 3 is made with a protrusion where the material of the sorbent layer 4 is packed. This solution is advantageous when the eluent fed in by the inlet 1 should be taken away from the sorbent layer 4 through an outlet 12. The embodiments of Fig. 2b gives another solution: a pressing element 13 acting from below is applied under the sorbent layer 4 and it is arranged in the supporting plate 3. The pressing element 13 can be contacted with the carrier plate 5 by the means of a forcing unit 14 represented here only by arrows 14. This is a very effective solution for increasing locally the pressure within the overpressurized chamber, in predetermined places of the sorbent layer 4. The difference between the two arran¬ gements of Fig. 2b is that one of them is completed with a rigid closing plate 9 covering the sorbent layer 4.

The arrangement of Fig. 3. is the basis of a system intended to be used for preparative separation, wherein seals 10 are applied for closing the way of flow of the fluid car¬ rier medium and an inlet channel 15 equipped with filtering element(s) is coupled with the inlet 1 introducing the eluent (fluid carrier medium) . Thereby some preliminary cleaning can be done. The supporting plate 3 and the covering plate 3- have in this embodiment indented surface matching one another and leaving place for fixing a relatively thick sorbent layer 4 shaped according to the inner surface of the supporting and covering plates 3, 3 * . The separation analysis investigation can be carried out in the arrangements shown in Figs la, lb, lc, 2a, 2b and 3 according to the known principles of the overpressurized layer chromatography set forth e.g. in the specification of the British Patent No. 2,173,125A. The overpressurized cham- ber can be arranged also in a system for controlling and adjusting the temperature.

The separation unit is a very important element of the present invention. As known, the separation itself is ensured within the sorbent layer 4 which can be arranged on one side of a carrier plate 5 (Fig. 4a) or can cover two sides of the

^"carrier plate 5 (Fig. 4b) and an important novel feature of the invention is that the sorbent layer 4 can be made also without the carrier plate 5, wherein the material of the sorbent layer 4 is fixed by polymerization or divided in a ceramic body.

The sorbent layer applied in the methods of the on-line or off-line circular or anticircular overpressurized layer chromatographic investigations can be made according to Figs 5a or 5b wherein points 16 are selected for placing the complex sample before the development. The place of the barrier 7 pressed to the sorbent layer 4 according to Figs _la, lb, lc, 2a, 2b or 3 is shown by dashed circular line. The inlet 1 (Fig. 5a) or inlets 1 (Fig. 5b) and the outlets 12 (Fig. 5a) or outlet 12 (Fig. 5b) are in concentrical arrangement with the place of the barrier 7. The sorbent layers 4 of the invention can be positioned within a box 18 having an upper and a lower plates (not marked in Fig. 6) . The upper plate is equipped with a channel 17 for collecting the eluent and a closing frame 19 being simultaneously the barrier 7. When the box 18 is closed the barrier 7 surrounds that surface part of the sorbent layer 4 arranged in this embodiment on a carrier plate 5 with which the inlet 1 and outlet 12 is connected and where the points 16 for placing the samples to be investigated are selected. The barrier 7 may be open at a limited lenght, if necessary, as shown in Fig. 6.

One of the most attractive varieties of the over¬ pressurized layer chromatography is the method of carrying out investigations on more layers when a development is done on more than one sorbent layer 4 whereby a high number of components and samples can be separated during one develop¬ ment process. An example of a separation unit for carrying out such investigation is shown in Figs 7a and 7b. In this system a perforation 20 is made in the sorbent layers 4 and positioning elements 21, preferably holes are prepared in a frame forming a rigid closing plate 9. The barrier 7 pro¬ trudes from the surface of the rigid closing plate 7 behind the inlet 1 of the fluid carrier medium. The inlet 1 is aligned with the perforation 20 and two frames of Fig. 7b can be united through the positioning elements 21. in Figs 8. and 9. separation units consisting of two and four sorbent layers 4 with respective carrier plates 5 are illustrated, wherein the sorbent layers 4 of Fig. 8. are arranged facing one another and the sorbent layers 4 of Fig. 9 are divided by the carrier plates 5. Within the lower sor- bent layer 4 an inlet 1 with a surface section 6 for intro¬ duction of the eluent and a channel 17 for collecting the eluent is realized, the sorbent layers 4 are fixed by closing frames 19. In the systems of Figs 8. and 9. the place of the perforation 20 for forwarding the eluent (fluid carrier medium) is also shown and an outlet 12 for taking away the eluent can be seen, too.

A three layer separation unit is illustrated in Fig. 10. The sorbent layers 4 prepared, if necessary, on carrier plates 5 and fixed by closing frames 19 form together a box with framed carrier plates 22 bearing also the barrier 7 (not shown here) . Within the box the points 16 for introducing the samples, the perforation 20 of the sorbent layers 4 and the surface section 6 contacting the inlet 1 are also shown. The barrier 7 acts on the sorbent layers 4 when the bos of Fig. 10 is closed. The connection between the layer and the column system liquid chromatography arrangements is very clear when apply¬ ing forced flow layer system arrangements. In Figs 11a and lib sorbent layers 4 are shown which can be applied to inves¬ tigations in on-line system overpressurized liquid chro ato- graphy. In the sorbent layers 4 of Fig. 11a and lib the possibilities of common and separate introduction of the eluent and the sample are illustrated.

A more channel separation unit for on-line over¬ pressurized liquid chromatography system with either divided or undivided sorbent field is illustrated in Figs 12a and 12b wherein the eluent is introduced either through one or more inlets l. The samples are arranged in points 16 before the inlets l. The outlet 12 is connected with channels 17 for taking away the eluent which is transported to the sorbent layer 4 by a gradient pump 24 from at least one container 25. The sorbent layers 4 are pressed together and to the barrier 7 by the means of positioning elements 21. The barrier 7 con¬ sists of more parallel and perpendicular protrusions which determine closed surface areas as shown in Fig. 12b by the continuous lines.

The effectiveness of the methods of the overpressurized liquid chromatography can be improved when they are combined with electrophoresis whereby an electrostatic field is applied. This solution gives the possibility of detecting and separating the electrically active components simultaneously. The components can be sensed by a series 26 of detecting elements and electrodes 27 are arranged before the inlet 1 and behind the outlet 12 of the eluent in the direction of the development (Fig. 13a) . According to another possibility the electrodes 27 create an electric field perpendicular to the direction of the development (Fig. 13b) . A further possibility follows from the Fig. 13c: the electrodes 27 are arranged at each edge of the sorbent layer 4 whereby electric fields of different directions can be generated.

A scheme of the spatial development is shown in Figs 14a to 14f. One complex sample is arranged in a selected point 16 of a sorbent layer 4 having no carrier plate 5 but closed from each side by a barrier 7 which forms a part of an outer closing element at the beginning of the development (Fig. 14a) . The sample is developed in one direction and this results in separated parts 29 (spots) comprising one or more components up to a front line 28 of the eluent (fluid carrier medium) . The planar development starts with the arrangement of Fig. 14b which is practically the same as that of Fig. 14a with the difference that the direction of forced flow of the eluent is perpendicular to that of applied in the arrangement of Fig. 14a. The development in this second direction results in a spot arrangement of the separated parts 29 as shown e.g. in Fig. 14d. The sorbent layer 4 received in the last step is applied as the covering element of a cube of sorbent layers 4 (Fig. 14e) which should be covered by a directing block 30 (Fig. 14f) for directing the eluent into the cube. According to this solution the sorbent layer 4 received in the second step of the development lies under the directing block 30 which can be capable of creating the conditions of forced flow of the eluent. As mentioned, the spatial development can be carried out also without overpressure. After development the sorbent layers 4 of the cube are separated from one another and the components of the sample collected in the different sorbent layers can be evaluated and determined with measurements based on the known principles. Basically the sorbent layers 4 of Fig. 14f after separating are characterized by a similar spot arrangement of the separated parts 29 as shown in Fig. 14d. This spot arrangement can be observed after steps carried out according to the art in a planar system. A complex overpressurized and high pressure liquid chromatography arrangement working in on-line system is shown schematically in Fig. 15. The eluent is fed by a dosing unit 31 into a loop 32 of introducing the samples and for the separation a high pressure liquid chromatography column 33 is applied. At the output of the last a system 34 of detecting means, e.g. flow detectors is applied. From the loop 32 an outlet 12 is realized to the sorbent layer 4 of an over¬ pressurized liquid chromatography system of the invention. The sample to be investigated are introduced by an inlet 23 to the loop 32. The system shown in Fig. 15. gives the possibility of full combination of the mentioned chromato¬ graphic investigations especially by the so-called column switching technics. This system very advantageous for preparing samples by solid phase extraction which is used to biologic samples.

Claims

1. Method of carrying out separation analysis investi¬ gations in a planar system with forced flow of a fluid carrier medium, especially by the means of overpressurized layer chromatography, comprising the steps of introducing at least one sample of at least one complex mixture to a sorbent layer forming a part of a separation plate of a separation unit, the separation plate having at least one channel for transporting fluid carrier medium within the sorbent layer to at least one predetermined place on the separation plate, feeding a fluid carrier medium into the at least one channel of the separation plate, arranging at least one separation plate carrying a respective sorbent layer in connection with means for forcing the fluid carrier medium to flow in the at least one channel, forcing the fluid carrier medium to flow and thereby to take away the components of the sample in an order determined by known retention of the components, the fluid carrier medium with the samples forming a fluid medium, and conducting the fluid medium through the at least one channel to at least one predetermined place on the at least separation plate and therefrom to at least one sensing unit than determining at least one characteristic parameter of the components taken away in the fluid medium from the predetermined place in a continuous real-time process up to reaching a given value of the retention, and/or connecting the at least one separation plate with at least one sensing unit for carrying out measurements of at least one characteristic parameter of at least one component remaining on the surface of the separation plate, characterized in the further step of connecting the at least one separation plate with at least one mechanical closing means for stopping flow of the fluid carrier medium in the at least one channel at the predetermined place.

2. The method as set forth in claim 1, characterized in forcing the fluid carrier medium to flow by the means of overpressure created in a closed chamber determined by a supporting plate and a closing plate, the supporting plate and/or the closing plate connecting at least one sorbent layer of the separation unit, and stopping the flow of the fluid carrier medium with a barrier forming a protrusion on the surface of the supporting plate and/or closing plate for making the sorbent layer impervious by interrupting and/or packing its material.

3. The method as set forth in claim 1 or 2, charac¬ terized in stopping the flow of the fluid carrier medium by the means of a barrier having a sharp edge, the barrier arranged on a carrier plate of the sorbent layer.

4. The method as set forth in any of claims 1 to 3, characterized in stopping the flow of the fluid carrier medium by the means of a barrier including at least one frame interrupting and/or packing the material of the sorbent layer along at least one line.

5. The method as set forth in any of claims 1 to 4, characterized in stopping the flow of the fluid carrier medium by the means of a barrier interrupting the material of the sorbent layer and causing deformation of the carrier plate.

6. The method as set forth in any of claims 1 to 5, characterized in stopping the flow of the fluid carrier medium by the means of a barrier pressed to at least one surface of the sorbent layer in a controlled manner.

7. The method as set forth in any of claims 2 to 6, characterized in stopping the flow of the fluid carrier medium by the means of a barrier exerting pressure depending on the force pressing the supporting plate against the cover- ing plate.

8. Arrangement for carrying out separation analysis investigations in a planar system with forced flow of a fluid carrier medium, especially by the means of overpressurized layer chromatography, comprising means for accomodating at least one separation plate made with at least one sorbent layer (4) receiving a complex sample to be investigated, means for introducing the complex sample onto the sorbent layer, means for forcing a flow of a fluid carrier medium on the separation plate when held by the accomodating means, means for feeding the fluid carrier medium into the sorbent layer (4) when the separation plate is held by the accomodat¬ ing means, means for conducting the fluid carrier medium within the sorbent layer (4) for taking away components of the complex sample when the separation plate is held by the accomodating means, a detector unit including at least one element for real-time measurements of the fluid carrier medium leaving the separation plate together with selected components of the complex sample and a sensing unit for measuring at least one characteristic parameter of the components remaining on the sorbent layer of the separation plate, the sensing unit being located opposite to the accomodating means or opposite a further means for accomodat¬ ing the separation plate, wherein the accomodating means are constituted by a supporting plate (3) bearing at least one separation plate made with the at least one sorbent layer (4) and a covering plate (3¹), the supporting plate (3) and the covering plate (3') determining an overpressurizable chamber, characterized in that within the overpressurizable chamber at least one barrier (7) for interrupting the flow of the fluid carrier medium in the sorbent layer (4) is arranged and advantageously the overpressurizable chamber is connected with means for helding in its interior isothermal or programmed temperature conditions.

9. The arrangement as set forth in claim 8, charac¬ terized in that within the overpressurizable chamber at least one sealing element (10) for hindering any flow of the fluid carrier medium in the direction closed by the sealing element (10) is arranged.

10. The arrangement as set forth in claim 8 or 9, characterized in that at least one rigid closing plate (9) and/or flexible closing plate (11) is arranged within the overpressurizable chamber in connection with the supporting plate (3) and/or the covering plate (3¹).

11. The arrangement as set forth in any of claims 8 to 10, characterized in that the overpressurizable chamber includes straight and/or circular and/or segmented channels for directing, collecting and taking away the fluid carrier medium, the channels having advantageously at least one filtering element.

12. The arrangement as set forth in any of claims 8 to 10, characterized in that the overpressurizable chamber includes a point shaped inlet element (1) or inlet elements (1) arranged along a circular line for introduction of the fluid carrier medium.

13. The arrangement as set forth in any of claims 8 to 10, characterized in that the overpressurizable chamber is arranged in a loop (32) of introducing at least one sample.

14. The arrangement as set forth in any of claims 8 to

13, characterized in that within the overpressurizable chamber means for preliminary cleaning of the sample is arranged.

15. The arrangement as set forth in any of claims 8 to

14, characterized in that within the overpressurizable chamber at least one sorbent layer (4) is equipped with electrodes (27) for generating electric field.

16. The arrangement as set forth in any of claims 8 to

15, characterized in that the overpressurizabl chamber is made with elements resulting in its cylindrical shape wherein the sorbent layers (4) are made with nonlinear surface.

17. Separation unit for carrying out separation analy¬ sis investigations in a planar system with forced flow of a fluid carrier medium, especially by the means of over¬ pressurized layer chromatography, comprising at least one separation plate having a sorbent layer for ensuring condi¬ tions of straight line front migration of a fluid carrier medium, characterized in that the at least one separation plate is connected with a closing frame (19) having at least one barrier (7) for interrupting the flow of the fluid carrier medium in the sorbent layer (4) when the closing frame (19) is pressed to the sorbent layer (4) .

18. The separation unit as set forth in claim 17, characterized in that the separation plate consists of a carrier plate (5) and at least one sorbent layer (4) covering a respective surface of the carrier plate (5) .

19. The separation unit as set forth in claim 17 or 18, characterized in that the separation plate is connected with at least one rigid closing plate (9) covering it from at least one side.

20. The separation unit as set forth in claim 19, characterized in that the rigid closing plate (9) is covered from outer siede with a flexible closing plate (11) .

21. The separation unit of any as set forth in claims 17 to 20, characterized in that the barrier (7) is a wall- type extrusion arranged along a closed line, especially in a rectangular or circular shape.

22. The separation unit as set forth in any of claims

17 to 20, characterized in that the barrier (7) is a wall- type extrusion arranged along an opened line.

23. The separation unit as set forth in any of claims

18 to 22, characterized in that the carrier plate of the sorbent layer (4) is made of glass, appropriate plastics or metal.

24. The separation unit as set forth in any of claims 17 to 23, characterized in that the sorbent layer (4) is made of silica gel, silica gel modified in polar and/or apolar manner, alumina, talc, magnesium silicate, hydroxyapatite, cellulose, modified cellulose, ion exchanger of organic or inorganic basis, sorbent composition based on plastics, an optically active composition.

25. The separation unit as set forth in any of claims 17 to 24, characterized in that the sorbent layer (4) is consisted of grains fixed in laminae by the means of a glue of organic or inorganic basis or by sintering applying a grained plastic or plastic fibers.

26. The separation unit as set forth in claim 17, characterized in that it consists of more sorbent layers (4) arranged one over another in a column covered by a directing block (30) for forwarding the fluid carrier medium into the column.