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WO1996012180A1 - Procede et dispositif pour effectuer des analyses par chromatographie liquide et des separations de substances - Google Patents

Procede et dispositif pour effectuer des analyses par chromatographie liquide et des separations de substances Download PDF

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Publication number
WO1996012180A1
WO1996012180A1 PCT/EP1995/003994 EP9503994W WO9612180A1 WO 1996012180 A1 WO1996012180 A1 WO 1996012180A1 EP 9503994 W EP9503994 W EP 9503994W WO 9612180 A1 WO9612180 A1 WO 9612180A1
Authority
WO
WIPO (PCT)
Prior art keywords
liquid
module
eluted
substances
mass
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.)
Ceased
Application number
PCT/EP1995/003994
Other languages
German (de)
English (en)
Inventor
Wolfgang Demmer
Thomas Ehlert
Khuong To Vinh
Jürgen Kaiser
Dietmar Nussbaumer
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.)
Sartorius AG
Original Assignee
Sartorius AG
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 Sartorius AG filed Critical Sartorius AG
Publication of WO1996012180A1 publication Critical patent/WO1996012180A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

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
    • G01N30/62—Detectors specially adapted therefor
    • 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
    • G01N30/80—Fraction collectors
    • G01N30/82—Automatic means therefor
    • 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
    • G01N2030/022—Column chromatography characterised by the kind of separation mechanism
    • G01N2030/027—Liquid chromatography
    • 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
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
    • G01N2030/324—Control of physical parameters of the fluid carrier of pressure or speed speed, flow rate
    • 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
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
    • G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
    • G01N2030/8831—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials involving peptides or proteins
    • 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
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/74—Optical detectors

Definitions

  • the invention relates to a method and a device for carrying out liquid chromatographic analyzes and material separations. those with high reproducibility. Accuracy and speed are executable.
  • the invention can be used in the laboratory, on a technical and in production scale for the implementation of chromatography tasks in the fields of biotechnology. Medicine. Chemistry and for the extraction of valuable materials.
  • a sample of a substance mixture is placed on a chromatographic carrier and separated by means of elution liquids (eluents) which are pressed through the carrier under pressure.
  • elution liquids eluents
  • the individual constituents are held to different degrees by the carrier and emerge in fractions. They are indicated by a detection device and collected in a fraction collector.
  • the extinctions displayed on the detection device are usually plotted as a function of time (retention time). Using the graph, the separated substances can be determined qualitatively and quantitatively.
  • the sample can be placed on the support in such a way that it takes up only part or all of its capacity.
  • the so-called frontal chromatography the carrier is loaded with the mixture of substances to be separated to such an extent that the desired substance appears at the outlet of the module which contains the carrier. It can be eluted with suitable elution liquids and separated from the other substances attached to the support.
  • the frontal chromatography is also used to determine breakthrough curves, the knowledge of which is required to describe the effectiveness of the support and the entire module for the desired material separation process.
  • the carrier is completely loaded with the mixture of substances to be separated and the time Course of the extinction measured in the course of the wearer From the course of the extinction curve for this substance, conclusions can be drawn about the quality and usability of the separation system. A steep increase in the extinction is desirable
  • liquid chromatography is understood to mean chromatography processes such as HPLC, column chromatography and separation chromatography.
  • Modules are the modules which contain the chromatographic support between the liquid inlet and outlet.
  • the modules in HPLC are capillaries, in column chromatography as columns, and in separation chromatography as Dead-end modules. as they are used in separation technology (filtration technology), designed All suitable media are suitable as chromatographic supports, in particular resins.
  • porous nonparticulate polymeric adsorbers such as fleeces or membranes (membrane adsorbers) in the form of tubes, hollow fibers and flat materials
  • porous membrane adsorbers are membranes that adhere to their surface functional groups. Wear ligands or reactants that are capable of interacting with at least one component of a liquid phase in contact with it. The liquid phase is transported through the membrane in a convex manner.
  • membrane adsorber is a generic term for various types of membrane adsorbers such as membrane ion exchangers.
  • Ligand membranes and activated membranes Nonwovens and membranes are preferably used as a stack to increase the module capacity
  • ERS ⁇ TZBl- ⁇ T (RULE 26) flattened so that the modules appear to be unsuitable for the particular separation task.
  • the flow rate of elution liquid through the module decreases with increasing duration of the chromatography, that is to say with increasing elution time and number of batches
  • the time difference in absorbance and the associated disadvantages are primarily due to a decrease in the flow rate of elution liquid by the module.
  • the decrease in the flow rate can be caused by leaks on the pressure side of the device, poor quality in the pressurization, for example due to defective pumps and by Reduction of the permeability of the module, for example due to blocking of the pores of the carrier, such as the formation of a covering layer on the surface (membrane structure).
  • Inadmissible swelling behavior or compaction of the wearer To remedy this deficiency, measures have been proposed such as.
  • Use of high performance precision pumps regular checking of the device for leaks and their elimination, additional regeneration steps of the wearer. Calibrations with internal standard substances and recalculation of the data by computer programs
  • the invention is therefore based on the object of providing an improved method for carrying out liquid-chromatographic analyzes and material separations, which is characterized in particular by a high reproducibility of the results, an improved separation performance and, if appropriate, a higher speed, and to provide a device for carrying out the method
  • the object is achieved in that the extinction of the eluted substances is determined as a function of the amount of eluted liquid and / or the flow rate of eluted liquid is kept constant and that a device for carrying out liquid-chromatographic analyzes and material separations with a device for determining the amount of eluted liquid and / or combined with a device for controlling the flow rate of eluted liquid
  • separation tasks with a constant sharpness between the substances and high reproducibility can be carried out if the flow rate is kept constant.
  • the delivery rate of dosing pumps is regulated depending on the eluted amount measured in the fraction collector so that the flow rate after passage of the module.
  • the separation tasks can be carried out in routine operation with the same amount of time for each batch Carrying out the process with a constant flow rate is a dramatic shift in the elution peaks which occurs according to the prior art. which is caused by blocking the adsorber unit is prevented, a reliable identification of the individual substances is guaranteed even in routine operation
  • the determination of the amount of eluted liquid is preferably carried out g ⁇ av ⁇ met ⁇ sch or volumet ⁇ sch.
  • feedback is applied to the pressurization of the module with elution liquid in such a way that a constant flow rate within a chromatography batch and across all batches is guaranteed
  • the amount of eluted liquid is determined gravimetrically by means of a balance and the flow rate is kept constant by means of a device for pulsation-free continuous gravimetric metering, as is known, for example, from German Patent 39 38 898
  • FIG. 1 shows the schematic representation of an exemplary embodiment of a device according to the invention with a gravimetric device
  • FIG. 2 is a schematic representation of an exemplary embodiment of a device according to the invention with a device for keeping the
  • FIG. 4 A is a chromatogram with regulation of the flow rate according to the invention, in which the absorbance is shown as a function of time, and FIG. 4 B is a chromatogram. in which the extinctions are shown as a function of time, but the flow rate was not regulated
  • the exemplary embodiment according to FIG. 1 consists of a combination of an HPLC
  • Data acquisition -.- control and evaluation unit 6 is shown. with an electronic
  • Scale 7 for measuring the mass of eluted liquid
  • the detector 4 and the electronic scale 7 are with the PC-controlled data acquisition.
  • Control and identification unit 6 connected in terms of data processing technology The PC-controlled
  • Data acquisition, control and identification unit 6 provides extinction curves as a function of mass
  • Example 1 With an HPLC device (Millennium 2010 with refractometer detector from Waters
  • Table 1 shows those measured in a first and a 5 batch
  • the device consists of a combination of a device for separation chromatography with a module 8, vessels for receiving the sample solution 9, 9 'to be separated.
  • Eluent reservoirs 1. 1 '. a fraction collector 5 for eluted liquid, a detector 4 and a device for gravimetric metering of a liquid mass flow in the form of an electronic balance 7, an interface 10, a PC-controlled data acquisition.
  • the module 8 contains a chromatograph 1 see carrier 11 in the form of a membrane adsorber.
  • a membrane adsorber stack of at least two membrane systems which is arranged in a fluid-tight manner between a liquid inlet 12 and a liquid outlet 13 for permeate (eluted liquid) arranged before the liquid outlet 13 flows into a fraction collector 5 located on an electronic balance 7.
  • the solution of the substance mixture to be treated is in the sample vessels 9 or 9 '. It is made from 9', for example by hand using a piston tip, or from 9 automatically via the metering pump 2 or one optionally available (auxiliary) dosing pump 2 '. which are connected on the pressure side to the liquid inlet 12 of the module 8, placed on the carrier 11.
  • the device for gravimetric metering of the liquid mass flow requests the liquid with a preset value from the sample vessel 9 and
  • Table 2 shows the measured flow rates according to Example 2 at the beginning and end of the charee
  • a mixture of the three commercially available proteins is chymotrypsinogen.
  • Cytochrome C and lysozyme 0.5 mg / ml each. Total protein content 1.5 mg / ml
  • the proteins were adsorbed completely, as the absence of proteins in the course of the unit showed.
  • the proteins become chymotrypsinogen.
  • SPARE BLADES (RULE 26) The chromatogram obtained with the regulation of the flow rate according to the invention. in which the absorbance is shown as a function of mass. 4 A shows a comparison with a chromatogram. where the absorbance is shown as a function of time. and which was obtained under the same conditions as in Example 3, but with no regulation of the flow rate (FIG. 4B) shows that by blocking the adsorber unit, the elution peaks are shifted dramatically, so that reliable identification of the individual substances is not more is guaranteed

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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)

Abstract

L'invention concerne un procédé et un dispositif permettant d'effectuer des analyses par chromatographie liquide et des séparations de substances en laboratoire, à l'échelle technique et industrielle, et s'utilise dans les domaines de la biotechnologie, de la médecine, de la chimie et de la préparation des produits recyclables. L'invention vise à mettre au point un procédé amélioré qui permette d'effectuer des analyses par chromatographie liquide et des séparations de substances et se caractérise notamment par une grande reproductibilité des résultats, un meilleur pouvoir de séparation et éventuellement une plus grande rapidité. L'invention vise également à mettre au point un dispositif de mise en ÷uvre dudit procédé. A cet effet, l'extinction des substances éluées est déterminée en fonction de la quantité de liquide élué et/ou le débit de liquide élué est maintenu constant, et un dispositif destiné à effectuer des analyses par chromatographie liquide et des séparations de substances est combiné avec un dispositif servant à déterminer la quantité liquide élué et/ou avec un dispositif de régulation du débit de liquide élué. La quantité est de préférence déterminée sous forme de masse à l'aide d'une balance électronique.
PCT/EP1995/003994 1994-10-18 1995-10-11 Procede et dispositif pour effectuer des analyses par chromatographie liquide et des separations de substances Ceased WO1996012180A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE19944437163 DE4437163C1 (de) 1994-10-18 1994-10-18 Verfahren und Vorrichtung zur Durchführung flüssigkeitschromatographischer Analysen und Stofftrennungen
DEP4437163.2 1994-10-18

Publications (1)

Publication Number Publication Date
WO1996012180A1 true WO1996012180A1 (fr) 1996-04-25

Family

ID=6531035

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP1995/003994 Ceased WO1996012180A1 (fr) 1994-10-18 1995-10-11 Procede et dispositif pour effectuer des analyses par chromatographie liquide et des separations de substances

Country Status (2)

Country Link
DE (1) DE4437163C1 (fr)
WO (1) WO1996012180A1 (fr)

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3204448A (en) * 1961-11-24 1965-09-07 Nat Res Dev Gravimetric detector for gas chromatography
US4083690A (en) * 1975-10-02 1978-04-11 Kirin Beer Kabushiki Kaisha Automatic preparation of sample for analysis
EP0041793A1 (fr) * 1980-06-05 1981-12-16 Beckman Instruments, Inc. Procédé et appareil de contrôle du débit
SU1048328A1 (ru) * 1982-05-20 1983-10-15 Институт Белка Ан Ссср Устройство дл сбора фракций по массе
DE3226398A1 (de) * 1982-07-15 1984-01-19 ABC-Analytische Biochemie GmbH, 8039 Puchheim Fluessigkeitschromatograph
DE3608227A1 (de) * 1986-03-12 1987-09-17 Helmut Apfel Anordnung zur fluessigkeitschromatographie

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3204448A (en) * 1961-11-24 1965-09-07 Nat Res Dev Gravimetric detector for gas chromatography
US4083690A (en) * 1975-10-02 1978-04-11 Kirin Beer Kabushiki Kaisha Automatic preparation of sample for analysis
EP0041793A1 (fr) * 1980-06-05 1981-12-16 Beckman Instruments, Inc. Procédé et appareil de contrôle du débit
SU1048328A1 (ru) * 1982-05-20 1983-10-15 Институт Белка Ан Ссср Устройство дл сбора фракций по массе
DE3226398A1 (de) * 1982-07-15 1984-01-19 ABC-Analytische Biochemie GmbH, 8039 Puchheim Fluessigkeitschromatograph
DE3608227A1 (de) * 1986-03-12 1987-09-17 Helmut Apfel Anordnung zur fluessigkeitschromatographie

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DATABASE WPI Week 8426, Derwent World Patents Index; AN 163992, Y.U. ALAKHOV ET AL.: "Chemical liquids fractions-by-weight collector - has scales to pass continual weight signal to recording device and differentiator to calculate rate of change of flow" *

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