WO2004058378A1 - Dispositif pour separer des melanges de substances par chromatographie en phase liquide - Google Patents

Dispositif pour separer des melanges de substances par chromatographie en phase liquide Download PDF

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Publication number
WO2004058378A1
WO2004058378A1 PCT/EP2003/014908 EP0314908W WO2004058378A1 WO 2004058378 A1 WO2004058378 A1 WO 2004058378A1 EP 0314908 W EP0314908 W EP 0314908W WO 2004058378 A1 WO2004058378 A1 WO 2004058378A1
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WO
WIPO (PCT)
Prior art keywords
column
sample
head space
separation column
eluent
Prior art date
Application number
PCT/EP2003/014908
Other languages
German (de)
English (en)
Inventor
Bezhan Chankvetadze
Dieter Bergenthal
Heribert Wennemer
Original Assignee
Bezhan Chankvetadze
Dieter Bergenthal
Heribert Wennemer
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 Bezhan Chankvetadze, Dieter Bergenthal, Heribert Wennemer filed Critical Bezhan Chankvetadze
Priority to AU2003296737A priority Critical patent/AU2003296737A1/en
Publication of WO2004058378A1 publication Critical patent/WO2004058378A1/fr

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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
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/16—Injection
    • G01N30/20—Injection using a sampling valve
    • 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
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G01N30/10—Preparation using a splitter
    • 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
    • G01N30/60—Construction of the column
    • G01N30/6004—Construction of the column end pieces
    • 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
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/16—Injection
    • G01N30/20—Injection using a sampling valve
    • G01N2030/201—Injection using a sampling valve multiport valves, i.e. having more than two ports
    • 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
    • G01N30/38—Flow patterns
    • G01N2030/382—Flow patterns flow switching in a single column
    • G01N2030/383—Flow patterns flow switching in a single column by using auxiliary fluid
    • 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
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/16—Injection

Definitions

  • chromatography denotes a group of separation processes in which the separation depends on the redistribution of molecules of the mixture between a thinly distributed phase and one or more phases with larger, coherent areas.
  • a plurality of chromatographic processes are known for the chromatographic separation, of which the adsorption chromatography, the column chromatography, the gas chromatography and the liquid chromatography are mentioned as examples.
  • the present invention relates to an apparatus for performing liquid chromatography.
  • Liquid chromatography or liquid chromatography is the collective name for all methods of chromatography in which liquids are used as the mobile phase and is abbreviated as LC (Liquid Chromatography).
  • LC Liquid Chromatography
  • HPLC High Performance Liquid Chromatography
  • Liquid chromatography in particular also HPLC, is a method of column-liquid chromatography. It represents a separation process represents, in which the sample liquid is transported by means of a liquid phase - also called eluent - under high pressure over the stationary phase - the separation column.
  • the sample molecules are reversibly bound to the stationary phase through dipole-dipole interactions. Since the dwell time of the sample substances in the stationary phase is different due to the different interactions with the surface of the stationary phases, the sample substances are separated from each other. Distribution chromatography, on the other hand, takes advantage of the different solubilities of the sample substances in the liquid and the stationary phase.
  • LC device for performing liquid chromatography - hereinafter referred to as "LC device” for short - basically consists of four main components: high pressure eluent reservoir,
  • the separation column is usually between 5 and 100 cm long and has an inner diameter of 1 to 25 mm. It is filled with silica gel or similar porous material with a grain size between usually 3 and 10 ⁇ m. It achieves a high number of separation numbers (up to 65000 / m), but at the same time requires the overcoming of a relatively high back pressure when transporting the mobile phase through the separation column. All parts of the LC device must therefore be connected to each other with as little dead volume as possible and be pressure-stable.
  • the known LC devices In the known LC devices, electromechanical pumps are used to generate the flow of the eluent through the separation column. Since these have a high weight, the known LC devices are particularly suitable for stationary operation.
  • the LC device usually comprises a sample loop into which the sample is injected.
  • the two ends of the sample loop are connected to a z. B. four-way valve connected.
  • a supply line for the eluent flow, a waste container for used eluent and the separation column are connected to the other connections of the four-way valve.
  • the sample loop is depressurized so that the sample can be injected.
  • the eluent flow is led through the sample loop, whereby the sample gets into the separation column.
  • Optical and / or electronic detectors or spectrometers are used to detect the separated phases.
  • the conventional sample application devices are disadvantageous because, depending on the structure, they have an unfavorable dead volume and do not allow the targeted addition of defined sample amounts.
  • the amount of sample injected depends directly on the time for the control or switching thereof.
  • the usual injectors cause unsharp sample peaks (sample distributions in the column over the time or length of the column), i. H. the peaks have artifacts deviating from an ideal Gaussian distribution, e.g. B. tails or several maxima.
  • the invention has for its object to provide an LC device or a sample application device which does not have the disadvantages of the prior art and in particular allows the addition of defined sample quantities and generation of sharp sample peaks.
  • the head of the separation column is arranged in a column head space surrounding it and the column head space comprises at least one outlet line and a changeover valve with which the inlet of the column head space can optionally be connected to the device for adding the sample or to a supply line for the eluent, it is possible to add or apply defined sample amounts to the column and thereby generate sharp sample peaks.
  • the head of the separation column is arranged in the column head space in such a way that it projects into the column head space in the direction of the device for adding the sample such that it protrudes past the outlet line.
  • column head space has an approximately "T" shape in longitudinal section
  • conventional components can be used for the production.
  • the head of the separation column and the outlet line are preferably spaced apart in the column head space such that the eluent can flow past the head of the separation column into the outlet line.
  • outlet line includes a valve
  • it can be opened or blocked. It is also possible, for. B. to add further eluents to generate a binary or ternary gradient.
  • the usual corrosion and solvent resistant materials such as glass, stainless steel, rubber, Teflon etc. can be used to manufacture or assemble the LC device or the sample application device.
  • the inventive design of the LC device or the sample application device enables an almost dead volume-free sample input and the amount of sample solution required for the analysis is reduced to 1 to 10 ⁇ l volume. If the device for supplying the eluent under pressure comprises a compressed gas reservoir, no heavy and complex pumps are required to operate the device according to the invention. As a result, both the outlay associated with the manufacture of the device according to the invention is reduced, so that it can be manufactured more cheaply.
  • the elimination of the electromechanical pumps allows the device according to the invention to be used as a portable, autonomous analysis device, since on the one hand the weight of the device is reduced, and on the other hand it is not necessary to connect to an external energy source to generate the pressure required for operating the device the compressed gas reservoir has been filled before the device is used, for which purpose a suitably equipped, stationary filling device can be used.
  • the invention can also be carried out with mechanical pumps.
  • the compressed gas reservoir preferably comprises a compressed gas container which can be filled with a compressed gas before the device is operated.
  • Compressed gas containers of this type are commercially available, so that their use reduces the outlay associated with the manufacture of the device according to the invention.
  • a stationary filling station can be used to fill the pressurized gas container, which can include a larger reservoir of the pressurized gas and optionally a compressor.
  • the pressurized gas container can preferably be filled with an inert gas, for example helium or nitrogen, preferably under a pressure between 100 and 120 bar.
  • an inert gas for example helium or nitrogen
  • the compressed gas container is as small as possible is trained.
  • the pressurized gas container therefore preferably has a volume between 100 and 500 ml, since the amount of pressurized gas stored therein is sufficient for carrying out analyzes.
  • an adjustable reducing valve is preferably arranged between the pressure medium resi / oir and the separation column.
  • the regulation of the devices and their valves etc. is preferably controlled or operated by a data processing system (computer). This can then also be used to control the detector, record the measured values and evaluate them.
  • a data processing system computer
  • a conventional capillary column is preferably used as the separation column, i.e. formed with a small cross-sectional area, since this measure can reduce the flow of eluent through the separation column and thus the gas consumption.
  • the analysis speed increases and the amount of sample substance required decreases.
  • the device for adding the sample comprises a dispensing device which comprises a metering device for the sample. It is therefore possible to add the exact amount of the sample to the test procedure without special measures being required.
  • FIG. 1 shows the embodiment of the device according to the invention during the addition of the sample solution
  • Fig. 2 shows the same embodiment while performing the analysis
  • Fig. 3 shows schematically the application of the sample to the separation column.
  • the device designated 100 in the drawing as a whole comprises an injection block 1, designed as a multi-way valve, of an injector 50, which has six connections A, B, C, D, E, F for connecting lines for fluid media.
  • a connection 2 for a liquid eluent 3 is connected to the connection F, which is located in an eluent container 4 and stands in this up to the mirror 5.
  • a pressure line 6 also opens into the eluent container 4, via which an inert gas is supplied to the eluent container 4 under a pressure of approximately 100 to 120 bar.
  • the compressed gas container 7 is equipped with a pressure measuring device 9 measuring the internal pressure. It also has a feed line 11 which can be closed by means of a shut-off valve 10 and via which the compressed gas container 7 can be filled with the inert gas from a refill station which is not shown in the drawing.
  • An adjustable reducing valve 12 is switched on in the pressure line 6, so that the pressure with which the eluent 3 is applied can be set.
  • connection F is connected to the connection E, which is provided with a closure 13.
  • the latter can for example, for emptying the eluent container 4 and the feed line 15.
  • the device further comprises a device for adding the sample 14, which comprises a piston-cylinder unit 15 in the manner of an injection syringe. It is used to hold a sample solution 16 and can be removed from the injection block for this purpose. In the state shown in FIG. 1, the device 14 is connected to the connection B of the injection block 1.
  • a separating column 17 is connected via a column head space 18, which is packed with a carrier material that is not recognizable in the drawing, for example silica gel or the like.
  • the separation column 17 is a capillary column, i.e. formed with a small cross-sectional area.
  • the column head space 18 surrounds the head 27 of the separation column 18.
  • head is meant the entrance area of the column.
  • the head 27 of the separation column is arranged in the column head space 18 in such a way that it projects into the column head space in the direction of the device 14 for adding the sample 16 such that it projects past an outlet line 19.
  • the column head space 18 itself has an approximately "T" shape in longitudinal section.
  • connection A of the injector block At one end it is connected to connection A of the injector block, at the other end the separation column 17 projects into it and the "T" is formed by the outlet line 19.
  • the head 27 of the separating column 17 and the outlet line 19 are spaced apart in the column head space 18 such that the eluent 3 can flow past the head 27 of the separating column into the outlet line if it is not shut off.
  • the separation column 10 has a line 20 connected to the outlet line 19. It ends with her the other end into a connection D of the injection block, which is connected to a connection C of the injection block 1 in the operating state of the device shown in FIG. 1.
  • a line 21 is connected to the latter, the open end 22 of which ends in a collecting container 23 for receiving excess sample and eluent solution.
  • a line 25 leads from the separation column 17 to a further collecting container 24, above which a conventional, opto-electronically operating detection system 26 for analyzing the separation phases is arranged.
  • FIG. 2 The operating state of the exemplary embodiment of a device according to the invention shown in the drawing during the analysis process is shown in FIG. 2.
  • Connections F and A, B and C as well as D and E are now connected. This has the effect that the device for adding the sample 14 is no longer connected to the column head space 18 and the separation column 17, but rather via the line 21, so that excess sample solution 16 can be fed directly to the collecting container 23 by actuating the device 14.
  • Due to the connection of the connections A and F, eluent 3 is now supplied to the separation column 17 with the pressure provided by the pressure vessel 7 and set via the reducing valve 12, whereby the chromatography process is started.
  • the separated phases are - as is known from the prior art - registered and analyzed with the opto-electronic detection means system 26.
  • the line 20 and thus also the output line 19 are connected to the closure 13 via the connections D and E, so that no eluent can escape via the output line during the analysis process.
  • the operation of the device according to the invention by means of the pressurized gas container 7 ensures a pulsation-free flow of the eluent, the production of which always means difficulties or a considerable design effort when operating liquid chromatographs with pressure pumps.
  • the configuration of the injection block 1 such that the connection for the device for adding the sample can be connected directly to the separation column allows the sample solution 16 to be added virtually free of dead volume and metered with an accuracy in the nanoliter range.
  • the device according to the invention is particularly suitable for use as a portable and autonomous system, since electrical energy is only required for the system control and the detection system when the compressed gas reservoir is filled.
  • FIGS. 3a-3e show an example of the application of a sample using the device according to the invention. This is an enlarged section of the column head space 18 in the area of the outlet line 19 and the head 27 of the separation column 17.
  • the working pressure is removed from the column head space and sample liquid 30 is introduced.
  • the position of the injector block or the valves corresponds to that from FIG. 1.
  • the sample liquid can be introduced via a sample loop (not shown).
  • the open outlet line 19 offers less resistance than the column.
  • the outlet line 19 is closed and the column head space 18 is briefly pressurized with the eluent 3 under pressure by means of targeted valve control. The sample liquid then penetrates into the column head 27, that is to say reaches the column.
  • the sample volume is dependent on the time t while the pressure is applied, on the pressure drop ⁇ P, on the diameter of the capillary R, the length of the capillary column and the viscosity ⁇ in accordance with Hagen-Poiseuille's law.
  • the outlet line 19 is opened and the column head space 18 is acted upon by the eluent 3 by means of targeted valve control.
  • the column head space is therefore flushed, since the resistance via the output line 19 is again lower.
  • the sample liquid remains in its place in the top of the column. This "rinsing" is carried out until the sample liquid 30 has been removed from the column head space.
  • the outlet line is closed and the column head space 18 is pressurized with the eluent 3, ie. H. the sample or sample liquid is pressed through the column and separated for analysis (cf. FIG. 2).
  • 3f shows another, alternative operating mode in which the output line 19 becomes the feed line.
  • eluent can be supplied, which makes it possible in a simple manner to generate a binary gradient in the eluent by adding a suitable second eluent 31 (bold dots).
  • ternary mixtures or gradients can also be generated if there is more than one outlet line 19 or if a binary eluent mixture is already used. LIST OF REFERENCE NUMBERS

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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 dispositif pour séparer des mélanges de substances par chromatographie en phase liquide, comportant une colonne de séparation (17), une unité servant à acheminer un éluant (3) sous pression à la colonne de séparation (17), et une unité (14) servant à introduire l'échantillon (16). La tête (27) de la colonne de séparation (17) est placée dans une chambre de tête de colonne (18) qui l'entoure et qui comprend une conduite de sortie (19). L'invention concerne également un dispositif pour appliquer un échantillon sur une colonne de séparation (17) notamment pour réaliser une chromatographie en phase liquide, comportant une unité servant à acheminer un éluant (3) sous pression à la colonne de séparation (17), et une unité (14) servant à introduire l'échantillon (16) dans une chambre de tête de colonne (18). La tête (27) de la colonne de séparation (17) est placée dans la chambre de tête de colonne (18) qui l'entoure et qui comprend une conduite de sortie (19). Ledit dispositif présente une soupape d'inversion qui permet de raccorder l'entrée de la chambre de tête de colonne (18) soit avec l'unité (14) servant à introduire l'échantillon (16) soit avec une conduite d'amenée (2) pour l'éluant (3).
PCT/EP2003/014908 2002-12-24 2003-12-24 Dispositif pour separer des melanges de substances par chromatographie en phase liquide WO2004058378A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2003296737A AU2003296737A1 (en) 2002-12-24 2003-12-24 Device for separating substance mixtures by means of liquid chromatography

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE10260700.1 2002-12-24
DE2002160700 DE10260700A1 (de) 2002-12-24 2002-12-24 Vorrichtung zur Trennung von Substanzgemischen mittels Flüssigchromatographie

Publications (1)

Publication Number Publication Date
WO2004058378A1 true WO2004058378A1 (fr) 2004-07-15

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DE (1) DE10260700A1 (fr)
WO (1) WO2004058378A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8741149B2 (en) 2004-02-06 2014-06-03 Micromass Uk Limited Mass spectrometer

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2028732A1 (fr) * 1969-01-20 1970-10-16 Siemens Ag
FR2336673A1 (fr) * 1975-12-22 1977-07-22 Sandoz Sa Nouvelle tete d'introduction d'echantillons pour colonnes de chromatographie en phase liquide sous haute pression
DE2731652A1 (de) * 1977-07-13 1979-01-25 Siemens Ag Verfahren der fluessigkeits-chromatographie fuer eine vielzahl von proben
DE3145180A1 (de) * 1981-11-10 1983-05-26 Institut für angewandte Chromatographie, 1000 Berlin Trennsaeulen-einlass fuer die hplc
EP0355546A2 (fr) * 1988-08-22 1990-02-28 Fisons Instruments S.P.A. Appareil et procédé de contrôle des fonctions d'un chromatographe à gaz
EP0529201A2 (fr) * 1991-08-26 1993-03-03 Hewlett-Packard Company Procédé de chromatographie avec enrichissement micellaire sur colonne
DE19525856C1 (de) * 1995-07-15 1996-10-31 Merck Patent Gmbh Chromatographische Säule
US20020187557A1 (en) * 2001-06-07 2002-12-12 Hobbs Steven E. Systems and methods for introducing samples into microfluidic devices

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2028732A1 (fr) * 1969-01-20 1970-10-16 Siemens Ag
FR2336673A1 (fr) * 1975-12-22 1977-07-22 Sandoz Sa Nouvelle tete d'introduction d'echantillons pour colonnes de chromatographie en phase liquide sous haute pression
DE2731652A1 (de) * 1977-07-13 1979-01-25 Siemens Ag Verfahren der fluessigkeits-chromatographie fuer eine vielzahl von proben
DE3145180A1 (de) * 1981-11-10 1983-05-26 Institut für angewandte Chromatographie, 1000 Berlin Trennsaeulen-einlass fuer die hplc
EP0355546A2 (fr) * 1988-08-22 1990-02-28 Fisons Instruments S.P.A. Appareil et procédé de contrôle des fonctions d'un chromatographe à gaz
EP0529201A2 (fr) * 1991-08-26 1993-03-03 Hewlett-Packard Company Procédé de chromatographie avec enrichissement micellaire sur colonne
DE19525856C1 (de) * 1995-07-15 1996-10-31 Merck Patent Gmbh Chromatographische Säule
US20020187557A1 (en) * 2001-06-07 2002-12-12 Hobbs Steven E. Systems and methods for introducing samples into microfluidic devices

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8741149B2 (en) 2004-02-06 2014-06-03 Micromass Uk Limited Mass spectrometer

Also Published As

Publication number Publication date
AU2003296737A1 (en) 2004-07-22
DE10260700A1 (de) 2004-07-08

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