DE3306127A1 - Fully automatic method of rapidly, specifically and selectively determining quantitatively low-concentration substances in extremely heterogeneous substance mixtures - Google Patents

Abstract

The system on which the method is based comprises a modular solvent purification and degassing system, a highly effective prepurification and a derivatisation system followed by a high-resolution chromotography system, and a final quantifying detector system. The entire system is synchronised by a process control system comprising two independent timing programmes, one of which controls the prepurification system and the start of the second programme, while the other controls the high-resolution chromatography system, including the detector devices. The solvent purification and degassing system is based on two liquid reservoirs which are connected via various filters and which are sealed from the atmosphere. The degassing takes place in the primary reservoir, while the secondary reservoirs contain the solvents in degassed and purified form. In the prepurification and derivatisation system, the substance mixture containing the analyte is applied to a column. Interfering substances are eliminated by "forward elution". The analyte fraction is then transferred largely selectively to a mixing section in which the medium of the analyte fraction or the analyte itself is altered in such a way that the analyte is retained on a subsequent column. After, if necessary, forward eluting interfering substances from this column, the analyte fraction is back-eluted and fed to a downstream high-resolution chromatography system. The analyte is quantified by a downstream detector system.

Description

FULLY AUTOMATIC PROCEDURE FOR FAST, SPECIFIC

FISHING AND SELECTIVE QUANTITATIVE DETERMINATION OF LOW CONCENTRATED SUBSTANCES FROM STRONGLY HETEROGENIC SUBSTANCE MIXTURES The invention relates to a fully automatic method for fast, specific and selective quantitative Determination of low-concentration substances from highly heterogeneous substance mixtures on a chromatographic basis.

The system on which the process is based consists in detail of a modular, expandable solvent degassing and cleaning system, at least a highly effective pre-cleaning and derivatization system based on liquid chromatography Basis, at least one high-resolution chromatography step and a final Quantification system. The entire process is carried out with an electronic Process control system synchronized.

In the field of clinical chemistry, fully automated Analytical devices introduced, which make it possible to selectively analyze any substances (analysands) to be quantified, regardless of the type and order of quantification Analysand. However, the selection of these analysands is largely limited to those that are accessible to photometric methods due to their concentration.

Procedures are currently being used for substances with a lower concentration applied and commercial offered based on immunological techniques and which thereby achieve a significant improvement in sensitivity. However, the specificity of these quantification techniques depends very much on the Specificity of the antisera used. As a result, the analytical quality is this quantitative methods limited by the fundamental problem of cross-reactivity, especially if powerful pre-extraction processes are not used.

Quantitative analyzes using high pressure liquid chromatography are far more specific (HPLC) or gas chromatography (GC).

For both methods, the quantitative analysis is less concentrated Substances made from highly heterogeneous mixtures of substances, especially those found in biological Liquids, e.g.

human serum, urine or tissue extracts are present, a pre-extraction an elementary necessity. A direct application of larger amounts of material is also not possible.

To work up the. Mixtures of substances have therefore been time-consuming, often lossy, non-reproducible and chemically decomposing manual operations required, often specifically designed for a particular analysand and therefore cannot be easily transferred to other analysands. So is a fast and selective quantitative determination of analy- .. ' sand not possible from heterogeneous substance mixtures.

M. Schöneshöfer applied for a patent in mid-1982, which makes it possible to largely select an analysand from a highly heterogeneous To extract the substance mixture and, if necessary, to derivatize it. This automatable Method and based on a solvent degassing and degassing process registered by A. Kage Cleaning process simplified processes form the basis of the patented Procedure for fully automatic, fast, specific and selective quantitative Determination of low-concentration substances from highly heterogeneous substance mixtures.

The obvious advantages of both methods have been summarized, Made more flexible by extensions and supplemented at the inadequate places. In addition, a process control system is used for synchronization.

The process to be patented looks like this: It consists from a modularly expandable solvent degassing and cleaning system, a highly effective pre-cleaning and possibly

Derivatization system and at least one high-resolution chromatography step with a subsequent quantifying detector system, all with an electronic Process control unit synchronized, controlled and monitored will.

The basic unit of the solvent cleaning and degassing system consists of a primary reservoir that is sealed off from the atmosphere is and in addition to a derivation for the solvent contained in the primary reservoir a gas supply line from a gas pressure system (pump, pressurized gas cylinder, etc.), which a promotes gas that is poorly soluble and chemically largely inert in the solvent, and a supply line to a vacuum pump, and a secondary reservoir, the is also sealed off from the atmosphere. The derivation of the in The solvent contained in the primary reservoir carries with it through mechanical filters sufficiently small pores and columns with suitable chemical adsorption materials to purge the solvent to a secondary reservoir.

The procedure for degassing and cleaning the solvents looks like this: a.a. The vacuum pump is initially used to create a sub-atmospheric Pressure is generated in the primary reservoir, which, along with the heating of the contained Solvent leads to degassing.

A reflux of solvent is through a check valve or another valve prevents.

away. After sufficient degassing, the connection becomes the vacuum pump interrupted and there is a pressure equalization by supplying Gas from the gas pressure system.

This creates a pressure that is slightly above atmospheric pressure Pressure level generated in the primary reservoir, so that the respective solvent up to Pressure equalization between the primary and secondary reservoir slowly via the discharge the primary reservoir and the mechanical filters and chemical cleaning columns flow into the secondary reservoir.

a.c. The solvent that has been worked up in this way is now always out of the Secondary reservoir available.

The pressure prevailing in the secondary reservoir depends on the pressure resistance of the valve systems downstream of the secondary reservoirs and the properties of the pumps.

While the primary reservoirs are as large as possible, around a common one The dimensions of the secondary reservoirs are to avoid refilling the solvent kept as small as possible, so that on the one hand a longer retention of the solvent is avoided in the secondary reservoir, on the other hand the individual secondary reservoir is still large enough to be connected to the connected at maximum removal speed Pumps for a limited time the low flow capacity of the connection of primary bridge to the secondary reservoir due to the necessarily only slight overpressure to be able to. A negative pressure is at all times to avoid pall, thru the suction effect of the pumps otherwise through gases dissolved in the solvents or Low-boiling solvents create gas bubbles that interfere with the detector signals or a reduction or failure of the pumping capacity of the individual pumps cause.

Solvent-conducting pipes lead from the individual secondary reservoirs Connections directly to the inputs of various multi-way valves (= valves with several inputs and one output, each of which has a conductive connection can be varied between one of the different inputs and the output), of which the outputs of at least two valves to those in the system Run pumps (P11, P12, P13, P14, P21, P31 in sketch 2). For every single solvent all that is required is a primary and a secondary reservoir.

Another valve system (a lot, V12, V13, V21, V31 in sketch 2), each one output of two or more multi-way valves (MVll - MV18, MV21 and MV22 in sketch 2). via a mixing chamber with the individual suction valves of the Pumps connects, can be operated variably with a time control.

This is dependent on the opening times of this valve system to achieve a specified mixing ratio for the respective solvent. The duration of one of these cycles is based on the mixing capacity of the mixing chambers and is approx. 2 - 3 seconds. Depending on the Number of inputs of the Multi-way valves, the number of different solvents can be varied; only one primary reservoir and one secondary reservoir are required for each solvent including the filter system.

Sketch 1 shows schematically the structure of a solvent cleaning system used and degassing module. (DG) provides the pressurized gas system, (VP) the vacuum pump, (PR) the primary reservoir, (SR) the secondary reservoir, (R) a check valve and (F) represents the mechanical and chemical filter column system. (MV ..) is one of the Multi-way valves and (H) an adjustable heating plate.

A scheme of a possible circuit of the priority ..

The processing and derivatization system is shown in sketch 2. It guarantees the following process flow: b.a. This is done using the pump (P1) heterogeneous substance mixture loaded onto column (C1) together with the analysand.

b.b. During this time, the high-resolution chromatography system (CS3) prepared for the chromatography of this analysand.

Depending on the type of chromatography (HPLC or GC or a combination of both) different procedures.

b.c. With the pump (P1) interfering substances are removed by suitable Eluent in 'forward flow' from column (C1) elu-'inert and discarded.

b.d. The analysand is then eluted largely selectively.

b.a. The elution medium for the analysand fraction is in the mixing section (M) or, if necessary, the analysand himself has changed in such a way that he or his derivative is based on the Column (C2) is retained.

The question of whether and how the analysand is derivatized depends on the conditions of the high resolution chromatography system (GC) and the one used Detector system.

b.f. With a suitable valve position further interfeirerende Substances (e.g. also substances or undesired reaction products) from the column (C2) eluted and discarded.

b.g. The column (C2) is then passed through with the analysand fraction Switching the valve (V2) backwards in the flow from the pump (P3) to the chromatography system (C3S) switched on.

The analysand fraction is thus largely purified and if necessary, transferred in derivatised form to the high-resolution chromatography system (C3S), this system consisting of an HPLC, a GC or a combination of both methods consists.

bra. The analysand fraction is displayed on the high-resolution chromatography system (C3S) chromatographed.

bi. Immediately after switching the valve (V2) (b, g,) it is activated by switching of the valve (V1), the column (C1) flows through the column (C1) in a reverse flow of solvents and thereby cleaned of still retained residues of the heterogeneous mixture of substances and for the following Chromatography run activated.

b.j. The analysand fraction that has meanwhile been chromatographed over the system (C3S) is fed to a quantifying detection system.

W photometers, fluorimeters, chemical ones are used as detection systems or electrochemical detectors, immunological techniques, mass spectrometry or similar

in question.

For derivatization of the analysand, temperature control of the Mixing part (M) ensures and a variability of the length of the reaction path intended. Pumps (P3) and (P4) can be used to stop or change the derivatization reaction possible. If further reaction steps are required, the mixing part (M) is appropriate to expand; Sketch 2 shows only one possible circuit of the pre-cleaning and Derivatization system.

In the event that the chromatographic properties of the columns (C1) or (C2) are not suitable for the respective analysand in sufficient To retain the dimensions, it is possible to use the valve systems (CV1) and (CV2) Replace the column with another, more retentive one.

The entire process flow is independent of each other by two working time programs controlled, whereby program 1 the process steps b.a., b.c. - b.f. and b.h. controls and program 2 the process steps b.b., b.g. and bi. including the setpoints for the appropriate nete detector device and the valve (DV), which the final chromatographed analysand fraction directed there. The procedural steps a.a. - a.c. the solvent degassing and cleaning system become independent of the two main control programs of controlled by a secondary process monitoring system; programs 1 and 2 only control the positions of the multi-way valves that control the supply line of the individual solvents to the pumps (Pl) - (P4).

Both programs are linked by program 1 in such a way that that program 1, which is specific to one or a group of analysands Program 2 starts so that for action b.g. the activation or selection of the high resolution Chromatography system is complete (step b.b.).

A secondary process monitoring system controls next to the Solvent degassing and purification system the actual values of a variety of Parameters1 whose target values are specified by programs 1 and 2, e.g.

Column interior and solvent temperature, pressure and flow as well as compliance with the specified mixing ratio of the solvents on Entrance of the individual pumps.

This monitoring system is shown in the circuit example shown in sketch 2 continue the actions of an automatic sampler whose start impulse is triggered the program is given and that of the entire system the individual Samples consisting of a heterogeneous mixture of substances and analysand are supplied. By a valve circuit (V3) puts the sample in the chromatographic system behind the pump (P1) is switched on and after the sample is out of the loop switched on washed out, the loop is removed from the chromatographic system taken out and loaded with a new sample after using a suitable solvent the contents of the sample loop have been removed. The length of the sample loop determines the amount of sample fed to the system.

The advantages of this method are in contrast to that of A. Kage patent pending process in that there is a loss of solvent caused by the necessary cleaning of the filter unit is avoided, as for each Solvent in addition to a primary reservoir and a secondary reservoir, a filter unit is available.

Compared to the proceedings registered by M. Schöneshöfer the process to be patented here represents a significant further development, since this System is not only used for simple sample preparation and derivatization, but a flexible and powerful method for the selective analysis of the most diverse Substances represents and the integration of high pressure liquid chromatographic and gas chromatographic techniques in routine analysis.

Claims (8)

PATENT CLAIMS: 1. Fully automatic process for fast, specific and selective quantitative determination of low-concentration substances strongly heterogeneous substance mixtures, - that from a modularly expandable solvent - degassing and cleaning system, a highly effective pre-cleaning and, if necessary, derivation system based on column chromatography and a high-resolution chromatography step with a subsequent quantifying detector system, and with a electronic process control unit is synchronized and controlled. 1 The basic unit of the solvent cleaning and degassing system consists of a primary reservoir, which is sealed off from the atmosphere and next to a discharge for the solvents contained in the primary reservoir a gas supply line from a gas pressure system, the one that is poorly soluble in solvents and largely chemically inert Promotes gas, and a feed line to a vacuum pump, and a secondary reservoir. The solvent contained in the primary reservoir is drained off mechanically Filters with outside rows and small pores and columns with chemical adsorption materials to purify the solvent to a secondary reservoir, which is also tight is shut off from the atmosphere. The procedure for degassing and cleaning the solvents looks like this: a.a. The vacuum pump initially becomes a sub-atmospheric Pressure is generated in the primary reservoir, which together with a heating of the contained Solvent leads to degassing. A backflow of solvent is prevented by suitable valves. away. After sufficient degassing, the connection becomes the vacuum pump interrupted and there is a pressure equalization by supplying gas from a Gas pressure system. This creates a pressure that is slightly above atmospheric pressure Pressure level generated in the primary reservoir, so that the respective solvent up to Pressure equalization between the primary and secondary reservoir slowly via the discharge the primary reservoir and the mechanical filters and chemical cleaning columns flow into the secondary reservoir. a.c. The solvent that has been worked up in this way is now always out of the Secondary reservoir available. Solvent-conducting pipes lead from the individual secondary reservoirs Connections directly to the inputs of various multi-way valves, one of which the outputs of at least two valves to the pumps in the system to lead. There is only one primary and one secondary reservoir for each individual solvent necessary. Another valve system, each having an output of two or more Multi-way valves via a mixing chamber with the individual suction valves of the Pumps connects, can be operated variably with a time control. This is in Depending on the opening times of this valve system for the respective solvent to achieve a given mixing ratio. Depending on the number of entrances the multi-way valves, the number of different solvents can be varied; only one primary and one secondary reservoir have to be included for each solvent a suitable filter system can be added. The basic sequence of the pre-cleaning and derivatization process takes place as follows: b.a. By means of a pump, the required solvent via the outputs of at least two multi-way valves from the secondary reservoir whose mixing ratio is obtained via the valve system upstream of the intake valves is regulated, a heterogeneous mixture of substances becomes together with the analysand loaded on a column. b.b. The high resolution chromatography system is used for chromatography this analysand prepared. This step requires depending on the type of chromatography (HPLC or GC or a combination of both) have a different approach. b.c. With the pump that loaded the analysand onto the column, Interfering substances are used by suitable eluents in the 'forward flow' from, the column eluted and discarded. b.d. The analysand is then eluted and largely selectively transferred to a mixing part. b.e. The elution medium for the analysand fraction is in this mixing section or possibly the analysand himself changed in such a way that he or his derivative on a second pillar is retained. b.f. With suitable valve switching, further interfering Substances are eluted from the second column and discarded. b.g. Then the second column with the analysand fraction is through Valve switching backwards in the stream leading to the high-resolution chromatography system leads, switched on. bra. The analysand fraction is displayed on the high-resolution chromatography system chromatographed. This high-resolution chromatography system can consist of either an HPLC or a GC. A combination of both is also in the way possible that the analysand fraction is first separated with the HPLC and then through a valve circuit that almost affects the analysand pure fraction contained is separated again by gas chromatography. bi. Immediately after switching the analysand fraction contained Column backwards into the flow to the high resolution chromatography system is through Switch a valve through the first column in the 'reverse flow' of solvents and thus cleaned of still retained residues of the heterogeneous substance mixture and activated for the subsequent chromatography run. Alternatively, another column can be switched into the solvent stream will. b.j. The chromatographed over the high-resolution chromatography system The analysand fraction is fed to a quantifying detector system. as Systems come from UV photometers, fluorimeters, chemical or electrochemical detectors, immunological techniques, mass spectrometry or the like. In the event that the chromatographic properties of the columns in the pre-cleaning and derivatization system are not suitable for the respective To retain the analysand sufficiently, it is possible to use a valve system to replace the column with another, more retentive one. The entire process flow is independent of each other by two working time programs controlled, program 1 the process steps b.a., b.c. - b.f. and b.h. and program 2 controls the process steps b.b., b.g. and bi. including the setpoints for the appropriate detector device in each case and the actions of the valve that controls the final chromatographed analysand fraction directed there. The procedural steps a.a. - a.c. of the solvent degassing and cleaning system are independent of the two main programs of one controlled secondary process monitoring system, which control programs 1 and 2 here only the positions of the multi-way valves and thus the type of solvent, which are conveyed by the pumps. Both programs are linked by program 1 in such a way that that program 1, which is specific to one or a group of analysands Program 2 starts so that for action b.g. the activation or selection of the high resolution Chromatography system is complete (step b.b.). A process monitoring system arranged next to it controls next to the Solvent degassing and purification system the actual values of a variety of Parameters whose target values are specified by programs 1 and 2, such as Column interior and solvent temperature, pressure and flow as well as compliance with the specified mixing ratio of the solvents on Entrance of the individual pumps. 2. A method according to claim 1, wherein the pre-purification and derivatization process is expanded as follows: b.a. - b.f. as in claim 1 Now will not die Analysand fraction fed backwards to the high-resolution chromatography system, but the analysand fraction by means of suitable valve switching forwards or backwards eluted and fed to a further mixing section. In this mixed part the analysand himself or his milieu can again changed in such a way that the analysand or its derivative is on a further column is retained. Interfering substances are eluted and discarded. This process can be repeated as often as you like. Complete the pre-purification and derivatization process as in claim 1, steps b.g. - b.j. Control of the inserted steps takes place via program 1, the other steps are controlled as in claim 1. 3. A method according to claim 1, wherein the supply of the analysand contained heterogeneous substance mixture to a different than that in claim 1 takes place, e.g. by being sucked in by pump 1 or with the help of an injector is injected into the system, as this is irrelevant for the entire process is. All that needs to be done is an exact volume control. 4. A method according to claim 1, wherein conveying the solvents from the primary reservoir to the secondary reservoir does not have an overpressure in the primary reservoir is done, but by some type of fluid pump. 5. A method according to claim 1, wherein the process control is not controlled by 2 largely independent programs, but by one program in which both programs are integrated, or through more than two programs in which the process control ---- tasks are divided. 6. A method according to claim 1, wherein the programs 1 and 2 on Data carriers of any kind are stored and retrievable. This can be done through a external control or by an implanted program that changes the order of the Programs 1 and, if applicable, 2 presets, take place. 7. A method according to claim 1, wherein one or more mixing parts, which are used in the pre-cleaning and derivatization system, from two or there are more feed lines, between each of which a reaction path is specified is. Thanks to a valve system, these mixing parts can be connected to one another as required be replaced. 8. A process that consists of any combination of the items listed under 1 - 7 described patent claims exists.