GB2179564A

GB2179564A - Method of and apparatus for carrying out separation analysis investigations in a planar system

Info

Publication number: GB2179564A
Application number: GB08620632A
Authority: GB
Inventors: Erno Tyihak; Emil Mincsovics
Original assignee: Mta Novenyvedel Kuta Intezet; Mueszeripari Muevek Lab; Magyar Tudomenyos Akademia Novenyvedelmi Kutato Intezete
Current assignee: Mta Novenyvedel Kuta Intezet; Mueszeripari Muevek Lab; Magyar Tudomenyos Akademia Novenyvedelmi Kutato Intezete
Priority date: 1985-08-29
Filing date: 1986-08-26
Publication date: 1987-03-11
Anticipated expiration: 2006-08-26
Also published as: JPS6291859A; GB2179564B; HU196509B; GB8620632D0; HUT43964A; CH673779A5; FR2586812B1; FR2586812A1; DE3629394A1

Abstract

The method includes carrying out simultaneously or separately on-line chromatographic and/or electrophoretic measurements, of constituents of a complex mixture fed to a planar plate covered by a sorbent layer being the place of separation, continuing the on-line measurement up to reaching a given value limit following from the applied retention mechanism and then carrying out off-line measurements of constituents remaining on the separation plate. The apparatus includes at least one separation plate (18) covered by a sorbent layer (19), means for forcing flow of a carrier medium on the separation plate (18), means for feeding the carrier medium into the sorbent layer (19), means (22) for introducing a sample of a complex mixture into the sorbent layer (19), means (23) for conducting fluid medium from at least one edge of the separation plate (18), a detector unit (E) (suitably a flow chamber detector) including at least one element for real-time measurements of the fluid medium, and a sensing unit (F) (suitably a densitometer) arranged oppositely to the surface of the separation plate (18) covered by the sorbent layer (19). <IMAGE>

Description

SPECIFICATION Method Of and Apparatus For Carrying Out Separation Analysis Investigations in a Planar System Technical Field The application relates to chromatography and/or electrophoresis with possibility of multidimensional multiphase investigations employing real-time (on-line) and deferred (off-line) determination of characteristic parameters of constituents of a complex mixture. More particularly, it relates to a method and apparatus for carrying out separation analysis investigations employing a fluid medium for separating constituents of a complex mixture including a high number of constituents to be identified.The proposed method comprises the following steps known from the art: 1. introducing sample of a complex mixture to a sorbent layer covering a separation plate and forming a plurality of channels for transporting fluid medium along the channels to at least one edge of the separation plate; 2. feeding a fluid carrier medium into the channels of the separation plate; 3. arranging a separation plate carrying the sorbent layer in connection with means for forcing the carrier medium to flow in the channels of the sorbent layer; 4. forcing the carrier medium to flow and thereby to take away the components of the sample in a known order determined by retention of the components, the carrier medium with the sample forming the fluid medium; 5. conducting the fluid medium through the plurality of the channels to the edge of the separation plate, and therefrom to a sensing unit; and 6. determining at least one characteristic parameter of the components taken away in the fluid medium from the edge.

The apparatus includes a separation plate covered by a sorbent layer, means for forcing flow of a carrier medium on the separation plate, means for feeding the carrier medium into the sorbent layer, means for introducing a sample of a complex mixture into the sorbent layer, means for conducting fluid medium from at least one edge of the separation plate, and a detector unit including at least one element for real-time measurements of the fluid medium.

Background Art The separation of complex mixtures is a task of major importance for the analyst and there are several fields now where considerable efforts have been made for solving that kinds of problems in different organic chemistry fields especially.

The separation technique methods are mostly of electrophoretic and chromatographic character, the last being by nature the most efficient. The essence of these techniques is to force an appropriate carrier medium to flow in a sorbent layer. The carrier medium flows before or on entering the sorbent layer through a sample of a complex mixture taking away the constituents thereof in an order determined by the given retention mechanism. The flow of the carrier medium can involve e.g. by the capillary force. This generally results in slow separation. For accelerating the process it is known to apply forced flow generated by overpressure, electroosmosis, centrifugal forces, vacuum etc. The sample to be divided into constituents nature includes by nature ballast means and depending on the quantity of the last it is usual to carry out cleaning of the sample before separation.

The application of overpressure for forcing the flow of the carrier medium is known e.g. from the U.K. patent specification of the U.K. Letters Patent No. 1 570 760 granted to the Labor Muszeripari Muvek of Esztergom, Hungary. The chromatographic apparatus disclosed therein comprises a sorbent layer arranged in an overpressured chamber wherein a water-including cover member is applied over the surface of the sorbent layer. The water in the cover member is overpressured. The sorbent layer formed by silicagel, alumina or other organic or inorganic material known from the practice is thereby placed in a space without water vapour. The chromatographic eluent is fed to a separation plate carrying the sorbent layer. At least one edge of the separation plate is closed by impregnation which can be made e.g. of plastic thin film.In order to allow the eluentto migrate in the sorbent layer a channel or a plastic plate element is arranged before the inlet opening for the eluent.

The U.S. Letters Patent 4 469 601 granted to Beaver et al. shows the possibility of separating in two, or even more, three dimensions. The system proposed by Beaver et al. includes the step of drying the separation plate after carrying out investigations in a first dimension and removing thereby the eluent from its surface, then carrying out separation in a second dimension and the constituents taken away from the separation plate are determined quantitatively.

The known techniques, systems and apparatuses have brought a very intensive development in the planar version of the column chromatography methods (layer chromatography). They offer, however, no solution for the problem that the constituents remaining on the separation plate are not really subject of determination. This follows from the retention mechanism and the constituents remaining on the plate mean loss of information and degradation of the reliability of the measurements. Another problem is that they contaminate the sorbent layer.

The US-PS 4 469 601 mentioned above refers also to the possibility of continuously taking away the carrier medium from the separation plate. In layer chromatography this renders possible to elaborate methods of automatically carrying out the measurements because the material leaving the separation plate can be collected and measured in measuring units with flow detectors. The disadvantage of this method is that many constituents can leave only very slowly the separation plate and as time lapses the danger increases that the constituents remaining on the plate are bound there by different chemical and physical processes, they can not be detected and contaminate the sorbent layer. The constituents after binding can often not be removed by washing.

However, the spot capacity can be substantially increased by two- or more dimensional development, this means always two or more beginnings of the development which results in increasing quantity of the constituents bound on the separation plate in an irreversible process. The two or more beginnings of the developmentfollowfrom the fact that in each dimension the eluent should be added separately after drying the plate for removing the eluent applied in the separation of the former dimension. The known planar systems allow investigations of a single sample in case of applying two or more dimensional developments. The US PS-4 469 601 shows the investigation of a higher number of samplers fed into a carrier medium.

Another problem of the developments carried out more times on the same sample is that for accelerating the drying process the temperature is increased which can cause denaturation or other damage to the more sensitive organic constituents.

For separating ionic constituents, and especially different enzymes, peptides, etc. or inorganic ions'it is known to apply electrophoresis causing the constituents to migrate on the separation plate to one of the electric poles according to their polarity.

The electrophoresis can be carried out also in a system with forced flow of the solvent making the migration possible and then the problems are the same as analysed above. The reference to this technique is also made in the US--PSS-4 469 601 analysed above.

Disclosure of the Invention The object of the present invention is to offer a solution to the problems of the separation analysis discussed above. Methods and apparatuses based on the planar systems suffer from a relatively low efficiency. The present invention proposes a method and an apparatus (system) for increasing efficiency. The invention is based on the recognition that the on-line detection shown in US-PS- 4469 601 can be completed by the off-line detection disclosed in US--PSS-4 346 001. This is the main novel feature of the invention which results in increasing efficiency of the chromatographic investigations, in decreasing time demand thereof.

Another novel object should be seen in the simultaneous application of the electrophoresis with the chromatographic development.

The invention proposes a method of carrying out separation analysis investigations in a planar system with forced, partially overpressured flow of a carrier medium comprising the steps of introducing at least one sample of at least one complex mixture to a sorbent layer covering a separation plate and forming a plurality of channels for transporting fluid medium along the channels to at least one edge of the separation plate; feeding a fluid carrier medium into the channels of the separation plate; arranging at least one separation plate carrying the sorbent layer in connection with means for forcing the carrier medium to flow in the channels of the sorbent layer; forcing the carrier medium to flow and thereby to take away the components of the sample in a known order determined by retention of the components, the carrier medium with the samples forming the fluid medium;; conducting the fluid medium through the channels to the edge of the separation plate, and therefrom to at least one sensing unit; determining at least one characteristic parameter of the components taken away in the fluid medium from the edge; wherein for increasing the efficiency of the separation, improving the conditions of the developments it is important to continue the determination in a continuous real-time process up to reaching a given value of the retention; to connect the separation plate with at least one sensing unit for carrying out measurements on the surface of the sorbent layer and to measure at least one characteristic parameter of the component remaining on the separation plate.

The applied carried medium is a chromatographic eluent and/or a solvent for electrophoretic investigations.

The samples can be arranged either on predetermined points of the sorbent layer(s) or into the fluid carrier medium before feeding it into the channels of the separation plate(s).

The real-time process is advantageously carried out by a flow chamber detector means and the measurements of the remaining components buy a densitometer.

As it is shown in our co-pending application U.S.

Ser. No. 724,524 it can be advisable to control the temperature of at least one separation plate in time and/or in different surface regions thereof.

The forced flow of the carrier medium can be realised by overpressure to be released before the measurements, by centrifugal force action on the separation plate(s), by electroosmosis of vacuum.

The efficiency of the separation can be further improved by generating electric field around the separation plate(s) during forced flow of the carrier medium.

For checking the selected measurement conditions it is advantageous to connect at least one separation plate with the sensing unit before beginning the real-time process.

In the case of comprising high content of ballast material it is proposed also to clean the sample before mixing it with the carrier medium.

The invention proposes also an apparatus for carrying out separation analysis investigations, comprising at least one separation plate covered by a sorbent layer, means for forcing flow of a carrier medium on the separation plate, means for feeding the carrier medium into the sorbent layer, means for introducing a sample of a complex mixture into the sorbent layer, means for conducting fluid medium from at least one edge of the separation plate, and a detector unit including at least one element for real-time measurements of the fluid medium, including for increasing the efficiency of the investigations a sensing unit arranged oppositely to the surface of the separation plate covered by the sorbent layer.

The apparatus according to the invention can advantageously comprise electrodes arranged for generating electric field for ensuring difference of electric potential on the surface of the separation plate(s).

The apparatus according to the invention can advantageously be arranged with one separation plate based on a basic body and under a covering body in a closed chamber connected to the conducting means, wherein the separation plate is connected with means for transporting it from the closed chamber into the measurement region of the sensing unit.

The apparatus according to the invention can advantageously comprise a basic body with straight plane surface for bearing the separation plate, wherein the flowforcing means form an upper body pressed to the surface of the separation plate for forming channels of forced flow of the carrier medium in the sorbent layer.

Another possibility is to apply a cylindric shaped basic body for bearing a curved separation plate, wherein the flow forcing means form a cylindric upper body covering the separation plate and pressing it to the basic body; the upper body is made of quartz-glass and connected with the sensing unit.

The sorbent layer can be closed by at least one impregnated edge, it can consist of segments arranged in cross form or of radial segments forming a circular layer on the separation plate. The segments can be made of different materials.

Brief Description of the Drawings For a better understanding of the nature and objects of the present invention reference is made to the accompanying drawings and the following detailed description. In the drawings Figure 1 shows diagrams obtained by off-line, on-line methods and by the method of the invention, Figure 2 is the cross-section of a proposed over pressured layer chromatography apparatus, Figure 3 is a cross-section of a multi-channel overpressured layer chromatography apparatus, Figure 4 is a cross-section of the apparatus shown in Figure 3, Figure 5 is the scheme of a guiding unit applied in the apparatus shown in Fig. 3, Figure 6 is a schematic view of the two dimensional overpressu red layer chromatographic separation, Figure 7 is a scheme top view of a separation plate in simultaneous application of the layer chromatography and electrophoresis separation by use of overpressure, Figure 8 is the top view of a separation plate applied for linearized circular developments, Figure 9 is the top view of a separation plate applied for circular developments, Figure 10 is the top view of a cleaning system applied in the apparatus according to the application and Figure 11 is the schematic diagram of carrying out the method of the invention.

Best Modes of Carrying Out the Invention Before carrying out the method of the invention it is necessary to prepare separation plates covered by a sorbent layer creating conditions for migration a fluid carrier medium in one or more directions of transporting constituents, components of a complex mixture present in a sample to be investigated. The fluid carrier medium is an eluent (for chromatographic developments) and a solvent (for electrophoretic developments) selected according to the constituents to be separated. The invention proposes the simultaneous application of the electrophoretic and chromatographic developments, and in this case the eluent should be a solvent capable of transporting one or more constituents under influence of an outer electric field.

The separation plates can be made in different forms, and it is possible to use the chromatographic plate described by Beaver et al. in US-PS- 4469 901. The separation plate is covered partly by a sorbent layer consisting of e.g. silica-gel, alumina, magnesium-silicate, talc or cellulose, synthetic resin in pulverised form, poliamid etc. These are only non-limitative examples taken from organic and inorganic materials and the artisan can select the best material according to the well-known practical principles. As a specially advantageous basic material can be regarded the sorbent comprising chemically bound inverted phase. The alkyl chains bound by Si-O-Si-C binding having different length are also advantageous.The separation layers can be selected with different specific surface value, different magnitudes and distributions of the pores etc. In practice it can be proposed to apply sorbent layers with chemically bound normal phases capable of carrying of dial (vicinal hydroxyl), aminocyano, amino and nitro functional groups. The ion exchange layers consisting of organic and/or inorganic compounds can serve as sorbent layers, wherein appropriate function groups can be applied for giving the required cationic or anionic character.

When carrying out separation based on size exclusion the application of polymerized gels formed by cross-binding or of inorganic gels (as silica-gel of wide pore range in sylilized or chemically modified form) is proposed.

For separating optically active components chiralic (optically active) compounds are recommended.

The separation plate is made of eloxated aluminium or appropriate organic material, e.g. by sintering process. In some cases synthetic resins can be also very advantageous.

The sorbent layer can be homogeneous, of mixed material or consist of more segments. The last possibility includes applications of different basic materials for different segments, however, the same material in modified form can be applied also. The segments form e.g. a cross-shaped element (Fig. 8), a circular sorbent layer (Fig. 9) or a rectangular shape (Fig. 7).

The thickness of the sorbent layer generally is in the range from to 0.05 to 5.0 mm and can be selected according to the conditions of the separation.

In the method of the invention the first step of separation is the feeding of one or more samples with constituents to be measured to one or more predetermined points of one or more separation plates bearing appropriate sorbent layer. According to the conditions of the separation the sorbent layer should be dried or wet. The samples can be transported also by an appropriate fluid carrier medium (the chromatographic eluent and/or the solvent necessary for electrophoretic investigation) to the sorbent layer. The samples can be placed in spots or in strips on the sorbent layer.

The fluid carrier medium should be conducted onto the surface of the separation plate in a way ensuring its contact with the sample. This can be done independently on feeding the samples or by the way described above: the samples are introduced into the fluid carrier medium before connecting the last with the sorbent layer.

The fluid carrier medium should be forced to flow in the sorbent layer after contacting the samples (mixing it with the sample). This can be done by applying overpressure, electroosmotic conditions, by means of centrifugal forces (rotating the separation plate around an axis lying outside of the plane of the plate), by placing it into vacuum (sucking the medium) or by other appropriate means known from the practice.

The fluid carrier medium forced to flow in the sorbent layer transports the constituents of the sample in a predetermined time order following from the solubility, the retention mechanism.

As it can be seen from Figure 1, by measuring selected parameters and especially by measuring the optical density some optical characteristic data of the constituents taken away from the sample by the fluid carrier medium can be obtained. Taking into account the constituents remaining on the separation plate the diagram A of Figure 1 can be determined by measuring the optical density. The curves in bands 1 to 7 correspond to constituents giving (in an off-line measurement) valuable measurement results, and the measured values in bands 8to 14 reflect levels hardly acceptable.

Collecting the carrier medium leaving the edge of the separation plate in separate channels for each sample in an on-line (real-time) measurement the diagram B can be obtained. In the last the bands 1 to 7 give peaks hardly valuable because of the relatively high material quantity remaining on the separation plate, and peaks in band 8 to 14-being substantially higher than that in bands 1 to 7correspond to the constituents the dominant quantity of which is taken away in by the fluid carrier medium. This means the measurements give well acceptable values in parts a of the diagram A and b' of the diagram B wherein the parts b and a' in these diagrams include values hardly acceptable after a relatively short development.

The essence of the invention is to be seen therein that after creating the necessary conditions the material constituents corresponding to the bands 1 to 7 should be measured on the separation plate, the other constituents corresponding to the bands 8 to 14 in a measuring system collecting the medium leaving the separation plate. This means, it is proposed to use two measuring units in a system wherein the separation takes place by means of a chromatography and/or electrophoresis. The method of the invention gives a diagram C (Fig. 1 ) with well distincted peaks in all bands 1 to 14 (parts a and b').

The choice where should be the limit between the bands, i.e. the width of the parts a and b' (the number of peaks to be analysed during the off-line and on-line measurements) depends on the conditions and in extreme cases it is possible to apply only on-line or off-line measurements. During off-line measurements only the diagram B is not taken, and during on-line measurements only the diagram A is not determined.

When carrying out the method of invention the following additional possibilities can be taken into account: 1. Temperature control. The temperature on the surface of the separation layer(s) is controlled according to the conditions of separation. The control can be realised in predetermined time and space sequences, i.e. the different surface regions of the separation layer can have different temperatures in different time moments. In this way the spot capacity and the peak capacity can be controlled according to some predetermined conditions. The increased temperature can be very important for sufficiently increasing the sensibility. The temperature is a factor of influencing the flow of the fluid carrier medium and thereby of decreasing the time of the investigations.

2. Carrying out a preliminary measurement on the separation plate before the fluid carrier medium reaches the edge of the separation plate. Thereby the preselected conditions of separation the efficiency and the reliability of the separation can be improved.

3. Application of separation plates usable only one or more times with a sorbent layer of homogeneous quality.

4. Preparation of a sorbent layer consisting of more segments made of different materials or of the same material with different modification ingredients.

5. Two dimensional and bidirectional separation, e.g. by simultaneous application of the electric field or temperature control.

6. The number of samples can be higher than 1 and in this case the controlled temperature renders higher efficiency and spot capacity possible than in the known systems. Some other means, as application of divided, segmented sorbent layer can be helpful in this case.

The apparatus according to the invention capable of carrying out the method of the invention will be described in more detail on the basis of the following figures. This apparatus realises a combined measuring system.

This system means that the dimensions of the separation plates used for chromatographic and/or electrophoretic developments must not be increased for improving the efficiency. The proposed planar system can be applied for analytic and preparative investigations and ensures separation of constituents characterized by low capacity factor.

The appropriate choice of the sorbent layers can be applied for influencing the order of separating the different constituents and thereby for increasing efficiency of the separation. It should be noted that the appropriate choice of the temperature conditions can intensify this effect.

The proposed apparatus in its version applying forced flow (Fig. 2) is based on a separation chamber D connected to a detector unit E with at least one flow chamber and to a transmission densitometer F working by reflection measurements in one or more channels.

As shown in Fig. 2 the separation chamber D comprise a closed chamber 15 wherein a space 16 is filled with a medium for transporting pressure, e.g.

water. The space, filled e.g. with water, is connected to a source of overpressure. The space 16 is closed from one side by a basic plate 17 bearing at least one separation plate 18. The upper surface of the separation plate 18 is covered with a sorbent layer 19 the material of which has been discussed above.

The sorbent layer 19 is covered by an appropriate upper body 20 for realising the overpressured conditions in the sorbent layer 19. The upper body 20 can be a second, third ... separation plate 18 covered by a respective sorbent layer 19 and in this case the overpressure realised in the space 16 is transmitted from one separation plate 18 to the other and they are pressed to the sorbent layer 19 lying underneath. In case of applying more separation plates 19 the upper body 20 will really be the upper closing element of the closed chamber 15.

The elements covering the sorbent layers 19, i.e. the upper body 20 and in case of applying more separation plates 18 the plates covering a sorbent layer 19 are equipped with means 21 for introducing an appropriate eluent and/or solvent, means 22 for introducing at least one sample of a complex mixture and means 23 for conducting the eluent or solvent to the detector unit E carrying out on-line measurements. The transmission densitometer F comprises a light source 25 and a sensor 24 receiving the light reflected from the surface of the separation plate 18 (sorbent layer 19) arranged in its measurement region.

With a dash line and array the arrangement and movement direction of a separation plate 18 is shown for off-line (deferred) measurement. In the measurement region the separation chamber D can be opened also by lifting up a cover element 27 signed by dash line.

Another possibility of realising the apparatus according to the invention is to prepare a cylindrical form upper body 20 covering a cylindric form separation plate 18. In this case the upper body 20 is made of transparent material as quartz-glass and an inner elastic tube member is applied for forcing the eluent to flow. Of course, the upper body 20 with the separation plate 18 can be rotated around the central axis of the upper body 20 and under action of the centrifugal force forced flow is initiated. The transparent upper body 20 has the advantage that after taking away the eluent and/or sorbent from the sorbent layer 19 and carrying out the on-line measurements up to reaching the required value of retention the separation plate 18 must not be removed from the cylindric body, it can undergo the off-line measurements without any movement.

Duringthe chromatographic development the eluent leaving the sorbent layer 19 is measured in the flow chamber of the detector unit E comprising, if necessary a higher number of measuring elements. The arrangement shown in Fig. 2 is especially advantageous when before the measurements to be carried out by the one- or multi-channel transmission densitometer F the sorbent layer 18 should be treated in anyway-e.g.

sprayed by a reagent, dried, heated or cooled, etc.

The last operations generally require taking out the separation plate 18 out from the closed chamber. If such treatment is not required, the cylindric arrangement described above can be regarded as very advantageous.

The schematic arrangements of the Figures 3, 4, and 5, illustrate an overpressured layer chromatography apparatus carrying out multichannel off-line and on-line investigations. The apparatus is covered by a housing 28 the left part of which comprises an inlet 26 for forwarding drying gas and an openable covering element 27. In this left part it is possible to remove the rests of the eluent and/or the solvent from the sorbent layer 19 after carrying out the on-line measurements. The right part of this apparatus includes, similarly to the apparatus shown in Fig. 2, a space 16 filled with appropriate medium for transporting overpressure, a separation plate 18 based on wheels or balls 52 arranged in an appropriate guiding element and an upper body 20. An inlet 21 is applied for introducing eluent or solvent and inlets 30 for introducing one or more samples. An outlet 23 receives eluent and/or solvent together with the constituents of the sample(s) of the complex mixture taken away from the sorbent layer 19. An outlet 31 is foreseen for transporting drying gas away from the sorbent layer 19. In the upper body 20 there are connection means for sensing units 29 and detectors or quartz windows.

In this apparatus the samples can be conducted to the wet or dry sorbent layer 19 pressed thereafter (vertical array) to the upper body 20. The solvent (eluent) reaching the edge of the separation plate 18 flows before the detectors 24 and leaves the system through the output 23. This is the on-line detection.

The spots (strips) of the constituents remaining on the separation plate 18 can be evaluated in a different manner. A possibility is that the separation plate 18 is moved on the wheels or balls 52 in the sensibility region of the detectors 24 (horizontal array), or the separation plate 18 is moved to the left part of the apparatus, wherein gas is applied for drying the sorbent layer and the dry sorbent layer 19 moves before the detector 24 (off-line measurement). Of course, in this arrangement more separation plates 18 can be applied.

The overpressured layer chromatography separation can be realised in two-dimension on-line and off-line measurements by means of an apparatus with sorbent layer 19 shown in Fig. 6. The separation is carried out in an off-line and an on-line step: before development in the second dimension the separation plate with the sorbent layer 19 is taken out from the apparatus, dried and placed back-between the two developments the separation plate can be dried also in the apparatus itself. The development in the second dimension is carried out before detecting the constituents leaving the sorbent layer 18 in an on-line measurements, then spots 32 on the sorbent layer 19 are evaluated by moving the separation plate under appropriate detectors 24 shown in the previous figures.

The fluid carrier medium (eiuent or solvent) is led through the inlet 21 into the sorbent layer 19. Under action of the means for involving forced flow the fluid medium migrates toward a straight front in channels 33. The samples are introduced in a corner 34. The flow of the medium is stopped at impregnated edges 35 and thereby it is impossible that this medium escapes from the sorbent layer 19 under the action forcing it to flow. The detectors 24 give diagrams e.g. as shown under 36.

In Figure 7 a simultaneous two-dimensionl on-line and off-line separation is diagrammatically shown.

The sorbent layer 19 bears samples and is limited by electrodes 37. The eluent being a solvent enters the sorbent layer 19 through the inlet 21 and migrates in a straight front in the channels 33 limited by the impregnated edge 35. The electrodes 37 gives the possibility of simultaneous electrophoresis because of generating an electric field acting in a predetermined direction, e.g. perpendicularly to the direction of chromatography. As shown in Figure 3, the detector system 24 can be used for identifying the constituents remaining on the sorbent layer 18, and the eluent/solvent leaving the separation plate covered by this sorbent layer 18 is conducted away through an opening 39 connected to a collecting channel 38. After finishing the separation process the detectors 24 can be used for evaluating the spots of the constituents remaining on the sorbent layer 19.Arrays 40 and 41 represent the direction of forces acting during the separation.

As it is shown in Figures 8 and 9 the sorbent layer 19 can be constituted of more segments giving a cross-form (Figure 8) or a circular form (Figure 9).

The eluent or solvent is introduced at inlet 21 and inlet points 34 serve for receiving the samples. At the edge of the sorbent layer 19 outlets 23 are realised for conducting away the eluent or solvent leaving the separation surface.

According to Figure 8 the linearized circular layer chromatography can be realized, advantageously also by means of overpressure. The arrangements of this kind as the arrangement of Figure 9 make development of more samples possible. The last represents the circular layer chromatography which can be realised also in an on-line measuring system with appropriate detectors.

The samples contaminated or containing a high amount of ballast require cleaning which is realised e.g. by a system shown in Fig. 10, wherein the inlet 21 is connected through a valve member K with reservoirs I, 11,111 containing eluents. The separation plate 18 is covered in this case by a sorbent layer for retaining the ballast material.

The system of the invention is shown in Figure 11.

A sample 42 to be analysed enters means 22 for introducing the sample into a sorbent layer 19. The sorbent layer remains under action of a unit 43 whereby forced flow of a solvent and/or an eluent is ensured. This system includes means 49 for changing the separation plates 18, if more than one such plate 18 is applied in the system. The means 49 are of known design and ensure that the separation plate 18 to which the on-line measurement has been finished is forwarded to the off-line measurements.

This forced flow takes place under the action of overpressu re, centrifugal forces etc., wherein electric field can be applied, too. This is a combination of the electrophoretic and chromatographic developments giving, in a detector unit E and sensing unit F (they can be the same) chromatogram 44 which can be also an electrophoretogram. The detectors 24 supply signal to a data processing unit 45 of conventional programmable design, wherein a printed report 47 can be prepared. The main elements of the system are undertaken a control given buy a control unit 46 capable of regulating the temperature, forces acting on the sorbent layer etc.

The system of the invention includes the possibility of the temperature control whereby-in given conditions-the efficiency of the separation can be sufficiently improved. The related solutions are described in our co-pending U.S. Application Ser. No.724,524. The units of temperature control ensure the control of the temperature in given regions of the sorbent layer according to a predetermined function. It is obvious that the change of temperature influences the flow of the eluent or solvent.

The detectors applied in the system can be of ultraviolet, fluorescence type, based on measurements of the refraction and electrochemic detectors. Of course, this is a non-limitative listing.

The different ultraviolet detectors are e.g. the filter detectors, the detectors of variable wave length, the scanning detectors, the detector with arrays of photodiodes. The band or filter detectors based on fluorescent principles are also known. The electrochemical detectors sensitive to some definite constituents, the "bulk" type detectors, the reaction detectors are also applicable.

The electrophoretic separation requires application of contacting elements, electric inlet and outlet elements, electrodes leading the electric current to the separation plate. The electric current can be transmitted by appropriate element arranged in the covering elements of the chambers.

In the system of the invention the chromatographic and electrophoretic separation can be carried out separately or simultaneously in the same or different, e.g. orthogonal directions.

The practical investigations showed the most advantageous is to apply chromatographic development in one direction and electrophoretic development in the direction perpendicular to the first. In this case the mixed detection, i.e. on-line measurement of a part and off-line measurement of the other part of the constituents give especially well valuable results.

The data processing means in the invention form two subsystems, one of which ensures the interpretation of the chromatogram (electrophoretogram) and the other selects the data obtained by the measurements. The measurements give data from which the first subsystem determines the peaks and their characteristic.

The data processing unit ensure also storage of the measured data, preparation of printed reports and analysis of the measured data, if necessary.

The control units are generally of well-known design for regulating, coordinating and controlling the measurement conditions.

The method, and apparatus (system) ofthe invention ensures increased efficiency and improved conditions of measurements.

From the above description it should be understood that apparatuses, systems equivalent to those given above will be within the scope of the claimed invention and such apparatuses will depend on the field of application and the given circumstances, samples to be separated and analysed.

Claims

1. Method of carrying out separation analysis investigations in a planar system with forced, particularly overpressured, flow of a carrier medium, comprising the steps of introducing at least one sample of at least one complex mixture to a sorbent layer covering a separation plate and forming one or more channels fortransporting fluid medium along the channels to at least one edge of the separation plate; feeding a fluid carrier medium into the channels of the separation plate; arranging at least one separation plate carrying the sorbent layer in connection with means for forcing the carrier medium to flow in the channels of the sorbent layer; forcing the carrier medium to flow and thereby to take away the components of the sample in a known order determined by retention of the components, the carrier medium with the samples forming the fluid medium;; conducting the fluid medium through the channels to at least one edge of the at least one separation plate, and therefrom to at least one sensing unit; determining at least one characteristic parameter of the components taken away in the fluid medium for the edge in a continuous realtime process up to reaching a given value of the retention; connecting the at least one separation plate with at least one sensing unit for carrying out measurements on the surface of the sorbent layer and measuring at least one characteristic parameter of the components remaining on the separation plate.

2. The method according to claim 1, wherein the carrier medium is a chromatographic eluent.

3. The method according to claim 1 or 2; wherein the carrier medium is a solvent for electrophoretic investigations.

4. The method according to any of claims 1 to 3, characterized in introducing the sample into the fluid carrier medium before feeding it into the channels of the separation plate.

5. The method according to any of claim 1 to 4, characterized in carrying out the reai-time process by a flow chamber detector means and the measurements of the remaining components by a densitometer.

6. The method according to any of claims 1 to 5, characterized in further comprising the step of controlling the temperature of the at least one separation plate.

7. The method according to claim 6, characterized in separately controlling the temperature in different surface regions of the separation plate.

8. The method according to any of claims 1 to 7, characterized in applying overpressure for forcing the carrier medium to flow.

9. The method according to claim 8, characterized in further step of releasing the overpressure before the measurements.

10. The method according to claim 8 or 9, characterized in applying centrifugal force action on the at least one separation plate for forcing overpressured flow of the carrier medium.

11. The method according to any of claims 1 to 7, characterized in applying electroosmosis for forcing the carrier medium to flow.

12. The method according to any of claims 1 to 11, characterized in further comprising the step of generating electric field around at least one separation plate during forced flow of the carrier medium.

13. The method according to any of claims 1 to 12, characterized in further comprising the step of connecting at least one separation plate with the sensing unit before beginning the real-time process.

14. The method according to any of claims 1 to 13, characterized in further comprising the step of cleaning the sample before mixing it with the carrier medium.

15. Apparatus for carrying out separation analysis investigations, comprising means for accommodating at least one separation plate covered by a sorbent layer, means for forcing flow of a carrier medium on the separation plate, means for feeding the carrier medium into the sorbent layer, means for introducing a sample of a complex mixture into the sorbent layer, means for conducting fluid medium from at least one edge of the separation plate, a detector unit including at least one element for real-time measurements of the fluid medium, a sensing unit arranged opposite the surface of the separation plate covered by the sorbent layer.

16. The apparatus according to claim 15, characterized in further comprising electrodes arranged for generating electric field for ensuring differences of electric potential on the surface of the separation plate.

17. The apparatus according to claim 15 or 16, characterized in that the separation plate is based on a basic body and arranged under a covering body in a closed chamber connected to the conducting means.

18. The apparatus according to any of claims 15 to 17, characterized in further comprising a basic body with straight plane surface for bearing the separation plate, and in flow forcing means forming an upper body pressed to the surface of the separation plate for forming channels of forced flow of the carrier medium in the sorbent layer.

19. The apparatus according to claim 18, characterized in further comprising means for transporting the separation plate from the closed chamber into the measurement region of the sensing unit.

20. The apparatus according to any of claims 15 to 17, characterized in further comprising a cylindric shaped basic body for bearing a curved separation plate and in flow forcing means forming a cylindric upper body covering the separation plate and pressing it to the basic body, wherein the upper body is made of quartz-glass and connected with the sensing unit.

21. The apparatus according to any of claims 15 to 20, characterized in further comprising at least one impregnated edge of the sorbent layer covering the separation plate.

22. The apparatus according to any of claims 15 to 21, characterized in that the sorbent layer consists of segments arranged in cross form on the separation plate.

23. The apparatus according to any of claims 15 to 21, characterized in that the sorbent layer consists of radial segments forming a circular layer on the separation plate.

24. The apparatus according to any of claims 15 to 23, characterized in that the sorbent layer consists of segments made of different materials on the separation plate.

25. Chromatographic system including means for real-time and deferred measurements of the components of a complex mixture according to any one of claims 15 to 24.

26. Chromatographic system including means for real-time and deferred measurements of the components of a complex mixture substantially as herein described with reference to and as shown in the drawings.