EP1342080A1 - Procede et systeme d'optimisation de processus en chromatographie - Google Patents

Procede et systeme d'optimisation de processus en chromatographie

Info

Publication number
EP1342080A1
EP1342080A1 EP01978338A EP01978338A EP1342080A1 EP 1342080 A1 EP1342080 A1 EP 1342080A1 EP 01978338 A EP01978338 A EP 01978338A EP 01978338 A EP01978338 A EP 01978338A EP 1342080 A1 EP1342080 A1 EP 1342080A1
Authority
EP
European Patent Office
Prior art keywords
column
optimized
parameters
solvent
parameter
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP01978338A
Other languages
German (de)
English (en)
Inventor
Sergey Golushko
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Merck Patent GmbH
Original Assignee
Merck Patent GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Merck Patent GmbH filed Critical Merck Patent GmbH
Publication of EP1342080A1 publication Critical patent/EP1342080A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/86—Signal analysis
    • G01N30/8658—Optimising operation parameters
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/86—Signal analysis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/86—Signal analysis
    • G01N30/8658—Optimising operation parameters
    • G01N30/8662—Expert systems; optimising a large number of parameters

Definitions

  • the present invention relates to a method and a system for method optimization of chromatography, in particular HPLC (high-performance liquid chromatography), with an analytical column (separation column) and a precolumn, which is provided for sample preparation, for example macromolecules of biological samples separate.
  • HPLC high-performance liquid chromatography
  • an analytical column separation column
  • precolumn precolumn
  • Chromatography is an analytical method frequently used in laboratory operations. It is a separation process in which different components of a sample to be examined move through a predetermined area at different speeds.
  • This predetermined range can be, for example, a so-called column of an adsorbent.
  • the adsorbent is a carrier of a thinly distributed phase, the so-called adsorption layer. If the sample is now passed through a column, some substances are adsorbed on the adsorbent, while other substances can flow through the column almost unhindered.
  • the moving liquid phase is generally referred to as the mobile phase, while the adsorbent layer is referred to as the stationary phase.
  • the solute is distributed between these phases in contact. This behavior, which is based on the law of mass action, ensures that the solutes flow very slowly through the column.
  • the different components of the sample will cross the column at different speeds. There is therefore a separation of the individual components of the sample. Details of various chromatographic separation processes, in particular reversed-phase chromatography, are known to the person skilled in the art.
  • Liquid chromatography is particularly suitable for the separation and analysis of high molecular weight compounds.
  • Liquid chromatography is generally divided into liquid-solid (adsorption) chromatography, liquid-liquid (distribution) chromatography, ion-exchange chromatography and gel filtration (gel permeation) according to the type of stationary phase chromatography).
  • the liquid is moved through the system with the aid of gravity or by pumps with a constant delivery rate.
  • gradient devices can provide gradual or continuous changes in the composition of the moving phase (gradient elution).
  • the individual components are detected using suitable detectors.
  • Detectors can, for example, differential refractometers, adsorption heat detectors, modified hydrogen flame ionization detectors, but also detectors based on photometry in the visible, infrared or ultraviolet range, or water-selective detectors. Sometimes the eluted compounds are also reacted with suitable substances, for example to form colored substances that can be easily detected.
  • method parameters are understood to mean all parameters that can characterize an analysis method.
  • the type of solvent to be used i.e. the mobile phase is a method parameter.
  • Other method parameters are e.g. B. the column temperature and the concentration of the solvent to be used.
  • the known chromatography methods and in particular also HPLC analysis fail to analyze biological samples that contain macromolecules, such as proteins.
  • macromolecules are namely precipitated by higher proportions of organic solvents in the mobile phase or irreversibly bound or denatured by hydrophobic groups on the surface of the chromatographic support.
  • the macromolecules are mainly adsorbed or precipitated on the outer surface of the porous stationary phase. The macromolecules thereby block access to the pores for the analytical substances actually to be investigated and thus reduce the number of chromatographic adsorption centers.
  • This sample preparation is preferably carried out in a precolumn in which the sample is separated into the sample matrix and the analyte or the analytes.
  • the protein matrix can be flushed directly into the waste while the analyte fraction is selectively extracted and enriched on the stationary phase of a guard column.
  • the analyte fraction can be transferred from the guard column to the separation column and separated and quantified there in the known manner.
  • This separation of the sample matrix takes place with the help of sorbents, which were developed as a special carrier material for the sample preparation of biological liquids. These sorbents have pore diameters that are significantly smaller than the diameter of the macromolecules. They have two chemically different surfaces.
  • hydrophilic, electroneutral groups are bound on the outer surface of the generally spherical particles.
  • This inert layer prevents any interaction with the protein matrix and any contamination during multiple use.
  • Only the inner surface of the porous particles has a hydrophobic distribution phase, essentially C, C 8 or C 18 alkyl chains.
  • the internal surfaces can also be modified by ion exchange groups.
  • the adsorption centers which are predominantly bound to the surface inside the pore, are freely accessible for the low-molecular sample components, ie the analytes. Different sorbents with different hydrophobicity are available for different biological samples.
  • biological samples such as e.g. hemolyzed blood, plasma, serum, milk, fermentation broth, supernatants from cell culture, food and tissue homogenates allowed.
  • this object is achieved by a system and a method for method optimization in chromatography, in particular HPLC (high-performance liquid chromatography) with an analytical column and a precolumn, which is provided for sample preparation, for example macromolecules of biological samples to separate,
  • the system comprising: a device for inputting data for the acquisition of experimental data and / or chemical structural formulas, a computing unit which has access to a database, the computing unit being intended as a function of the acquired data To optimize at least one method parameter based on the database entries and / or physico-chemical models, an output unit which is intended to output the at least one optimized method parameter, a device for inputting method parameters being provided and at least one optimized method pair rameter is a parameter of the guard column.
  • HPLC high-performance liquid chromatography
  • optimization is to be understood here in the broadest sense.
  • the optimization can also only be carried out by an estimate based on chemical-physical models and / or on database entries.
  • the optimized parameter does not have to be an optimum, but merely an improved parameter.
  • the optimization can also take place in several (iteration) steps, from which it becomes clear that an optimal method parameter is not necessarily present after the first optimization.
  • An optimized method parameter of the guard column is preferably a parameter from the group comprising guard column type, type of solvent for the guard column, concentration of solvent for the guard column, separation time, type of solvent for transfer to the analytical column, concentration of solvent for transfer to the analytical column and transfer duration.
  • This optimization can take place, for example, by accessing database entries on the basis of the recorded data, which represent method parameters based on empirical values. It is therefore preferable to enter chemical structure formulas provided an editor for chemical formulas.
  • the loading of a file which contains the corresponding chemical structural formula in a conventional format can also be provided.
  • the device for entering method parameters can be used, for example, to tell the system which different types of guard columns are currently available, so that the method parameters are optimized on the basis of the experimental options actually available.
  • the database contains parameters from different guard columns, preferably for several concentrations of a mobile phase, preferably of methanol water and / or acetonitrile water.
  • the database contains optimized method parameters that are based on empirical values.
  • the optimization then takes place on the basis of a physically mathematical model and / or on the basis of the database entries.
  • experimental data can be acquired and at least one method parameter can be optimized on the basis of the experimental data. For example, it is possible to detect the separation process in a test run at the exit of the guard column in order to determine the required separation time. At least one method parameter is then optimized on the basis of the separation time determined in this way.
  • the computing unit is preferably provided for optimizing at least one further method parameter in the event that no experimental data were recorded, but rather only structural formulas and possibly method parameters were recorded.
  • the chemical structural formula and the type of guard column and the type of solvent can be communicated to the system, for example, whereupon the computing unit then determines, for example, an optimized solvent concentration and outputs it via the output unit. This is particularly advantageous if only one type of guard column and only one specific solvent is used Available.
  • the system can thus be easily adapted to the local conditions and optimizes the method parameters within the scope of the locally given degrees of freedom.
  • the system is able to actuate a switching valve which connects the guard column to the analytical column.
  • the guard column is connected to the analytical column so that the analyte retained in the guard column can be transferred to the analytical column.
  • the guard column is then separated again from the analytical column, so that the HPLC analysis can be carried out in the analytical column.
  • the switching times of the switching valve are optimized depending on the experimental data and / or on the basis of the database entries.
  • the sample matrix is separated from the actual analyte in the guard column.
  • the outlet of the guard column is connected to a corresponding waste or disposal container.
  • the analyte is retained in the guard column while the sample matrix passes quickly through the guard column.
  • the movement of the analyte in the guard column is so severely restricted that it can only leave the guard column after the sample matrix has left the guard column for a long time. It is therefore necessary to determine the point in time at which the sample matrix has been almost completely removed from the guard column.
  • the first switching time of the switching valve i.e.
  • the point in time at which the guard column is connected to the analytical column must therefore lie between the point in time when the sample matrix is completely separated and the point in time at which the analyte also leaves the guard column.
  • the analyte is transferred from the guard column to the analytical column. Only after this transfer is almost complete can the valve be switched on again, so that the guard column is again separated from the analytical column and the 'normal' analysis process can be started with the analytical column.
  • the two switching times of the switching valve are optimized on the basis of experimental data and / or on the basis of chemical-physical models and / or on the basis of empirical values entered in the database, in order to accelerate the analysis time and at the same time ensure that the sample matrix has been completely separated.
  • the empirical values cannot necessarily be obtained by "trial and error” tests, but preferably were originally obtained by the optimization according to the invention.
  • the optimized method parameters can therefore preferably be stored in the database, so that future analysis methods may refer to them can be used and the optimization may be based on these optimized parameters.
  • the above-mentioned object is also achieved by a chromatography device, in particular an HPLC chromatography device (high-performance liquid chromatography), with an analytical column and a guard column and a detector assigned to the guard column, the separation column being provided for this purpose Sample preparation, for example, to separate macromolecules from biological samples, the chromatography device having a system which was described at the beginning.
  • the chromatography device preferably has a number of different guard columns, which can optionally be used for the separation, which have a porous support material with a pore diameter, preferably of about 4-8 nm.
  • a preferably controllable switching valve is provided, which optionally connects the guard column with a pump for the guard column solvent or with several analytical columns.
  • a method for method optimization in chromatography which comprises the following steps: at least partially acquiring a characteristic data set consisting of one or more structural formulas of one or more analytes, the type of biological matrix, method parameters and experimental data and determining at least one optimized parameter of the precolumn and possibly further optimized method parameters as a function of the at least partially recorded characteristic data set.
  • a characteristic data record is understood to be a data record in which all the characteristic parameters are entered for an analysis process, including the separation of the sample matrix.
  • access is preferably made to a database in which inherent column parameters for a plurality of different precolumn types are stored, depending on different solvent types and their concentration.
  • the determination of the at least one optimized parameter on the basis of empirical values stored in the database and / or depending on the molecular volume of the or the analyte and / or depending on the interaction energy of the analyte or analytes with H 2 O and / or depending on the inherent column parameters stored in the database.
  • the theoretical retention time can be calculated, for example, as a function of the solvent concentration for each component of the analyte.
  • a compromise can be found between the best possible separation of the different components of the analyte and a minimal analysis time.
  • the optimized parameters are preferably the precolumn type, the type of solvent for the precolumn, the concentration of the solvent for the precolumn, optionally the type and the concentration of the transfer solvent for the transfer of the analyte or analytes from the precolumn to the analytical column and optionally the type the analytical column.
  • the optimized parameters are the concentration of the solvent for the precolumn and, if appropriate, the type of solvent for the precolumn.
  • the at least partial acquisition of a characteristic data set and the determination of the optimized parameter are repeated at least once, wherein when the step of at least partial acquisition of the characteristic data set is repeated, experimental data is obtained on the basis of the optimized method parameters determined in the previous step were recorded. In other words, (test) tests are carried out on the basis of the optimized parameters.
  • the experimental data determined in this way are used to further optimize the method parameters. In principle, the method can be continued as often as desired, although in general a few optimization steps are sufficient to obtain very suitable method parameters.
  • the method particularly preferably provides that the optimized method parameters, preferably in the form of an optimized characteristic data set, are stored in the database.
  • the present invention allows HPLC analysis to be used as a standard analysis method also for biological samples. Because the otherwise very expensive separation procedure can be significantly shortened with the aid of the method, an HPLC analysis on biological samples can be carried out very inexpensively.
  • Figure 1 A and 1 B is a schematic diagram of the analysis method.
  • FIGS. 1A and 1B A chromatography device in which the system according to the invention and the method according to the invention are used is shown schematically in FIGS. 1A and 1B.
  • a first mobile phase A is pumped into the precolumn VS via the openings 6 and 1 of the switching valve SV by means of a first pump P1.
  • the downstream part of the guard column is connected to a waste collecting container 7 via the openings 4, 5 of the switching valve.
  • the injector IN through which the biological sample is injected for analysis, is provided between the pump P1 and the precolumn VS.
  • the biological sample is advantageously centrifuged before injection.
  • the biological sample is injected via the injector IN and the fractionation, ie the separation of the analyte from the sample matrix, is carried out.
  • the injection can be done manually or via an automatic sampler.
  • the mobile phase conveyed by the first pump P1 rinses the sample into the guard column via valve positions 1-6.
  • the guard column is equipped with a special carrier material for the sample preparation of biological liquids as a sorbent.
  • This special carrier material is porous with a pore diameter of about 6 nm. This has the consequence that macromolecules (for example proteins) with a large molecular weight cannot penetrate into the pores and therefore elute in the dead volume of the column.
  • the sorbent used has two chemically different surfaces.
  • hydrophilic, electroneutral diol groups are bound on the outer surface of the generally spherical parts.
  • modification of the inner surfaces can also be carried out by ion exchange groups.
  • This layer serves to prevent any interaction between the sorbent and the protein matrix and thereby minimize contamination of the guard column when used multiple times.
  • the inner surfaces of the pores of the porous particles also have a hydrophobic distribution phase (C, C 8 or C 8 alkyl chains). These adsorption centers bound on the surface inside the pore are generally freely accessible for the low molecular weight sample components, ie the actual analyte.
  • the switching valve SV must also not be switched too late, since the precolumn VS only delays the analyte in the biological sample, so that if the position shown in FIG. 1A is maintained too long, the analyte also moves via the switching valve position 4-5 is flushed into the waste container 7.
  • the elution profile of the sample matrix is therefore first determined.
  • the guard column is connected directly to a detector, for example a UV detector.
  • the elution profile of the protein matrix is then determined.
  • the separation process ie the complete elution of the protein matrix, is complete when the protein peak reaches the baseline.
  • t M The time until complete fractionation is referred to as t M below.
  • the analyte elution profile is determined.
  • the analyte is eluted with the same mobile phase and flow rate as was used for the elution of the sample matrix.
  • Let t A be the time after which the analyte begins to elute.
  • t A > t M. This can be done by a suitable choice of the sorbent and the mobile phase.
  • the switching valve SV is switched, as already mentioned, so that the arrangement shown in FIG. 1B is produced.
  • a second mobile phase B is transferred to the precolumn via the switching valve position 3-4, which in turn is connected to the analytical column AS via the switching valve SV position 1-2.
  • the switching valve position 3-4 which in turn is connected to the analytical column AS via the switching valve SV position 1-2.
  • Waste container 7 arranged. There is a detector between the analytical column AS and the waste container 7 D provided. In this position, the analyte is rinsed out of the guard column and placed on the analytical column. After the analyte has been completely transmitted, the switching valve SV is switched again, so that the arrangement shown in FIG. 1A results again. The analyte is now on the analytical column and the actual analysis can be carried out in the known manner.
  • the mobile phase B is transferred to the analytical column by means of the pump P2 via the valve position 2-3, which in turn is connected to the waste container 7 via the detector D.
  • the second valve switching point is also of crucial importance.
  • the elution profile of the analyte transfer is measured.
  • the analyte is preferably applied directly to the precolumn without a sample matrix in a preliminary test.
  • the elution profile of the analyte from the precolumn is then measured on a detector, which is preferably arranged directly in front of the analytical column, in the arrangement of FIG. 1B.
  • the point in time at which the elution profile of the analyte transfer reaches the baseline is denoted by t ⁇ . This is the earliest point in time at which the switching valve SV can be switched back to the position shown in FIG. 1A, since only then has the analyte been completely transferred to the analytical column.
  • the optimum valve switching times can be calculated from the measurement results, so that for analytes which have a value of ⁇ 10 min for t A , a first valve switching time results which switches from the arrangement shown in FIG. 1A to the arrangement shown in FIG. 1B
  • t v1 y 2 (t M + t A ).
  • t ⁇ should be at least t M + 5 min. It should be noted that t M ⁇ t A. If this is not the case, the sorbent material of the guard column or the mobile phase of the guard column must be changed.
  • the second valve switching time t V2 at which the guard column is again separated from the analytical column, is preferably at least t ⁇ + 1min.
  • one embodiment of the system according to the invention provides that the user who wants to detect a certain analyte in a certain sample matrix informs the system of the chemical structural formula of the analyte and / or the sample matrix. The system then suggests a type of guard column and an optimized concentration of the mobile phase for the guard column to the user. Based on the proposal, the user can now experimentally determine the elution profile of the analyte and the sample matrix. After entering the experimental data into the system, the system calculates the valve switching time for the matrix separation step t v1 .
  • the system tration of the organic solvent for the transfer of the analyte determined.
  • the user now determines the elution profile for the transfer of the analyte.
  • the system uses the experimental data to determine the valve switching time t V2 for the transfer step of the analyte. Based on the results, the system performs the optimized method parameters.
  • the system is able to enter the optimized parameters in a database, so that in the event that the user later wants to analyze the analysis of an identical or chemically similar analyte in an identical or chemically similar protein matrix, Preliminary tests can be completely dispensed with and the system only suggests the method parameters that have already been optimized. For example, according to the invention it is possible to completely do without the preliminary tests, provided that there are entries in the database which already include method parameters which have been optimized in preliminary tests.
  • the present invention therefore makes it possible for the first time to use chromatography in general, in particular HPLC chromatography, in a simple and inexpensive manner also for analytes contained in a biological sample.
  • HPLC chromatography is also available as the standard analysis method for biological samples.

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  • Physics & Mathematics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Treatment Of Liquids With Adsorbents In General (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

L'invention concerne un système d'optimisation de processus en chromatographie, en particulier en chromatographie en phase liquide haute performance. Ledit système comporte une colonne analytique (colonne de séparation) et une colonne préliminaire destinée à séparer par exemple des macromolécules d'échantillons biologiques pour la préparation des échantillons. L'invention vise à mettre en oeuvre un système, un procédé, et un dispositif de chromatographie permettant de réaliser une analyse automatique et rapide par chromatographie en phase liquide haute performance d'une pluralité d'analytes différents dans divers échantillons biologiques tels que par ex. du sang hémolysé, du plasma, du sérum, du lait, des suspensions de fermentation, et des résidus d'homogénéisats de cultures cellulaires, d'aliments, et de tissus. A cet effet, Le système selon l'invention est composé d'une unité de saisie de données destinée à la saisie de données expérimentales et/ou de formules de structures chimiques de l'analyte et/ou de la matrice d'échantillon, d'une unité de calcul pouvant accéder à une base de données, ladite unité de calcul pouvant optimiser au moins un paramètre de processus en fonction des données relevées, et d'une unité d'émission destinée à émettre le ou les paramètres de processus optimisés, au moins un des paramètres de processus optimisés étant un paramètre de la colonne préliminaire.
EP01978338A 2000-10-02 2001-09-05 Procede et systeme d'optimisation de processus en chromatographie Withdrawn EP1342080A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE10049079 2000-10-02
DE2000149079 DE10049079A1 (de) 2000-10-02 2000-10-02 Verfahren und System zur Methodenoptimierung in der Chromatographie
PCT/EP2001/010219 WO2002029401A1 (fr) 2000-10-02 2001-09-05 Procede et systeme d'optimisation de processus en chromatographie

Publications (1)

Publication Number Publication Date
EP1342080A1 true EP1342080A1 (fr) 2003-09-10

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EP01978338A Withdrawn EP1342080A1 (fr) 2000-10-02 2001-09-05 Procede et systeme d'optimisation de processus en chromatographie

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EP (1) EP1342080A1 (fr)
AU (1) AU2002210485A1 (fr)
DE (1) DE10049079A1 (fr)
WO (1) WO2002029401A1 (fr)

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Publication number Priority date Publication date Assignee Title
DE10128546A1 (de) * 2001-06-13 2002-12-19 Merck Patent Gmbh Verfahren und Vorrichtung zur automatischen, optimierten Durchführung chromatographischer Analysen
DE10393819D2 (de) * 2002-09-19 2005-08-11 Charite Universitaetsmedizin Verfahren zum Auffinden geeigneter chromatographiebedingungen zur Trennung biologischer Moleküle
US20060186028A1 (en) 2004-02-06 2006-08-24 Micromass Uk Limited Mass spectrometer
CN103180727B (zh) * 2010-10-27 2015-12-16 通用电气健康护理生物科学股份公司 具有保护柱的色谱系统

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Publication number Priority date Publication date Assignee Title
DE4130475A1 (de) * 1991-09-13 1993-03-18 Merck Patent Gmbh Modifizierte chromatographische traegermaterialien
IT1277749B1 (it) * 1995-12-29 1997-11-12 Fisons Instr Spa Dispositivo e metodo per effettuare la separazione di un campione in singoli componenti in un condotto capillare di un apparecchio per la

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Title
See references of WO0229401A1 *

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AU2002210485A1 (en) 2002-04-15
DE10049079A1 (de) 2002-04-18
WO2002029401A1 (fr) 2002-04-11

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