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WO2010057454A1 - Colonne monolithique comportant de l'avidine monomère immobilisée pour enrichir et identifier des espèces biotinylées - Google Patents

Colonne monolithique comportant de l'avidine monomère immobilisée pour enrichir et identifier des espèces biotinylées Download PDF

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Publication number
WO2010057454A1
WO2010057454A1 PCT/DE2009/001412 DE2009001412W WO2010057454A1 WO 2010057454 A1 WO2010057454 A1 WO 2010057454A1 DE 2009001412 W DE2009001412 W DE 2009001412W WO 2010057454 A1 WO2010057454 A1 WO 2010057454A1
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WO
WIPO (PCT)
Prior art keywords
avidin
monolithic
immobilized
monolithic column
groups
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/DE2009/001412
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German (de)
English (en)
Inventor
Jens Spross
Andrea Sinz
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.)
Martin Luther Universitaet Halle Wittenberg
Original Assignee
Martin Luther Universitaet Halle Wittenberg
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 Martin Luther Universitaet Halle Wittenberg filed Critical Martin Luther Universitaet Halle Wittenberg
Priority to EP09771267A priority Critical patent/EP2485836A1/fr
Priority to DE112009003262T priority patent/DE112009003262A5/de
Publication of WO2010057454A1 publication Critical patent/WO2010057454A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to polymers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
    • B01J20/26—Synthetic macromolecular compounds
    • B01J20/261—Synthetic macromolecular compounds obtained by reactions only involving carbon to carbon unsaturated bonds
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
    • B01J20/28014—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their form
    • B01J20/28042—Shaped bodies; Monolithic structures
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/305—Addition of material, later completely removed, e.g. as result of heat treatment, leaching or washing, e.g. for forming pores
    • B01J20/3064—Addition of pore forming agents, e.g. pore inducing or porogenic agents
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/3071—Washing or leaching
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3202—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating
    • B01J20/3206—Organic carriers, supports or substrates
    • B01J20/3208—Polymeric carriers, supports or substrates
    • B01J20/321—Polymeric carriers, supports or substrates consisting of a polymer obtained by reactions involving only carbon to carbon unsaturated bonds
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3244—Non-macromolecular compounds
    • B01J20/3246—Non-macromolecular compounds having a well defined chemical structure
    • B01J20/3248—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such
    • B01J20/3255—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such comprising a cyclic structure containing at least one of the heteroatoms nitrogen, oxygen or sulfur, e.g. heterocyclic or heteroaromatic structures
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3268—Macromolecular compounds
    • B01J20/3272—Polymers obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds
    • B01J20/3274—Proteins, nucleic acids, polysaccharides, antibodies or antigens
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2220/00—Aspects relating to sorbent materials
    • B01J2220/50—Aspects relating to the use of sorbent or filter aid materials
    • B01J2220/54—Sorbents specially adapted for analytical or investigative chromatography
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2220/00—Aspects relating to sorbent materials
    • B01J2220/80—Aspects related to sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J2220/82—Shaped bodies, e.g. monoliths, plugs, tubes, continuous beds
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2220/00—Aspects relating to sorbent materials
    • B01J2220/80—Aspects related to sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J2220/84—Capillaries
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2220/00—Aspects relating to sorbent materials
    • B01J2220/80—Aspects related to sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J2220/86—Sorbents applied to inner surfaces of columns or capillaries
    • 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
    • G01N30/50—Conditioning of the sorbent material or stationary liquid
    • G01N30/52—Physical parameters
    • G01N2030/524—Physical parameters structural properties
    • G01N2030/528—Monolithic sorbent material
    • 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

Definitions

  • analytes prior to separation and analysis within a complex sample or, ideally, separate from interfering (matrix) substances.
  • affinity chromatography has established itself as the method of choice. Since suitable interaction partners are available for a large number of analytes, their immobilization on a chromatographic medium allows specific analytes to be separated and / or accumulated in a targeted manner from complex mixtures.
  • biotin / avidin system which, due to its extremely high interaction (K D ⁇ 10 "14 M) and its high specificity, is used for a variety of analytical questions, indeed the interaction between biotin and avidin which is native in the tetrameric state, so strong that biotin-derivatized species (eg, ssDNA, RNA, proteins, peptides, etc.) are often attached to an avidin-loaded medium and used for further experiments can only be eluted from the avidin-loaded medium by means of denaturing reagents, such as guanidine hydrochloride or sodium dodecyl sulfate, these extreme conditions are naturally not suitable if further investigations are to be carried out on the previously bound analytes (eg proteins), which is a natural folding of the protein conditional to be meaningful An example of this is protein expression, in which a biotin label is used to isolate and purify the expressed protein.
  • analytes eg proteins
  • agarose monomeric avidin bound to particles
  • a major disadvantage of agarose materials is that they are broken down by bacteria and can only be poorly sterilized, making them poorly stored and allowing the analytes to be contaminated or degraded.
  • polystyrene or SiO 2 particles are used, which, however, have a lower avidin capacity than the agarose materials and are incompatible with some biologically important ions.
  • the particle format is unfavorable for LC / MS couplings, since it is only laborious to pack columns / Mirzaei, H., Proteomics, 2008, 8, 1516 /, which often have poor reproducibility due to the difficulty of the production process.
  • the monolithic column according to the invention consists of:
  • a monolithic separation medium prepared by: a) a radical polymerization, which is started by UV light or heat by means of appropriate reagents (eg ⁇ , ⁇ '-azobisisobutyronitrile, Benzoinmethylether), from acrylic acid derivatives (eg acrylamide), methacrylic acid derivatives (eg glycidyl methacrylate , Ethylene glycol dimethacrylate, alkyl methacrylates) and / or styrene derivatives (eg divinylbenzene, styrene, 4-chloro-styrene) in the presence of inert solvents (eg 1,4-butanediol, cyclohexanol, 1-dodecanol, 1-propanol) which are responsible for the pore properties of the Monoliths are in a pretreated with vinyl organosilicate (eg trimethoxysilyl-propyloxymethacrylate) quartz glass capillar
  • organosilicates e.g., tetramethoxysilane, trimethoxy- (glycidyloxy) -silane
  • inert solvents e.g., water, 1-propanol
  • the monolithic carrier material thus prepared is used by flushing the pore-forming solvent and remaining monomers with an organic solvent for attachment of the avidin.
  • various strategies are used for this: a) epoxy methacrylate monoliths:
  • Native avidin can be immobilized on the monolith directly at neutral to weakly basic pH (pH: 7-9) using the free, terminal amino groups of the amino acid side chains by utilizing the epoxide groups.
  • the epoxide groups of the monolith are reacted with amine derivatives (ammonia, 1,2-diaminoethane, etc.).
  • amine derivatives ammonia, 1,2-diaminoethane, etc.
  • the amine groups produced thereby react with a dialdehyde (glutaraldehyde, etc.), whereby aldehyde groups for an avidin bond are available on the monolith.
  • the terminal amino groups of the amino acid side chains of the native avidin react at neutral to weakly basic pH (pH 7 - 9) with the aldehyde groups.
  • the resulting Schiff base is reduced in a final step with a hydrogenation reagent (eg, sodium cyanoborohydride, sodium borohydride) to produce an amine bond that is stable over a wide pH range.
  • a hydrogenation reagent eg, sodium cyanoborohydride, sodium borohydride
  • monomeric avidin is generated from the immobilized, native (tetrameric) avidin.
  • Denaturing reagents (8 M urea, 6 M guanidine hydrochloride (pH 1.5), 10% SDS) are pumped through the monolithic column, denaturing the tetrameric avidin and rinsing out non-monolith bound subunits.
  • the monomeric avidin immobilized on the monolithic column is renatured by rinsing with aqueous buffer solutions having a neutral to weakly basic pH (pH 7-9) and stands for the reversible binding of biotin and biotinylated analytes (eg ssDNA, RNA, proteins, peptides, etc.).
  • the immobilized monomeric avidin monolithic columns can be shortened to a particular length as needed and used for offline or online HPLC / MS experiments, as well as other analytical techniques (e.g., capillary electrophoresis).
  • the process according to the invention produces a monolithic stationary phase with avidin for the first time since the process described in EP 0 423 938 Bl leads to a particle column which does not have the desired properties.
  • the solution according to the invention combines in an outstanding manner the advantages of previously used systems: the use of monomeric avidin enables elution under approximately native conditions, the monolithic carrier material causes improved contact between the biotinylated analytes and the interaction partner avidin due to the excellent mass transfer Carrier material for liquid chromatography and capillary electrophoresis is founded.
  • various detection methods such as UV spectroscopy, fluorescence spectroscopy and Especially the mass spectrometry results in an increase in the field of application, due to the high sensitivity of the individual methods.
  • Exemplary embodiment 1 is a diagrammatic representation of Exemplary embodiment 1:
  • a quartz glass capillary (internal diameter 100 .mu.m) is rinsed overnight with 1 M NaOH solution for activation and then washed with deionized water. • the activated capillary is now with a 50% solution (v / v) of [ ⁇ -
  • the resulting monolith is purged with acetonitrile for 8 hours to remove the pore-forming solvents. Subsequently, the monolith is flushed with conc. Ammonia + 50% acetonitrile (16h), 50% acetonitrile (8h), 50wt% glutardialdehyde solution + 50% acetonitrile (16h), 25% acetonitrile (4h) and 0.1M phosphate buffer (pH 7, 4h) for loading with avidin prepared. Immobilization of the avidin is accomplished by purging the monolith with a solution of avidin (2.5 mg / mL) in 0.1 M phosphate buffer (pH 7) for 16 h.
  • the monolithic immobilized monomer avidin column is equilibrated with 100% solvent A (10% ACN with 100 ⁇ M MES buffer pH 7.0) prior to the liquid chromatographic experiments, then the sample containing biotinylated analytes (eg ssDNA, RNA, proteins, peptides, etc .) are injected.
  • a typical gradient for an HPLC / MS experiment is described below: 100% solvent A (10% ACN with 100 ⁇ M MES buffer pH 7) for 18 min, change over 2 min to 100% solvent B (50% ACN + 0.4% formic acid).
  • FIGS. 1 and 2 are identical Legend to FIGS. 1 and 2:

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  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Health & Medical Sciences (AREA)
  • Thermal Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne une colonne monolithique comportant de l'avidine monomère immobilisée pour enrichir et identifier des espèces biotinylées, et consistant en une phase stationnaire de matériaux monolithiques qui contiennent un groupe réactif auquel l'avidine est reliée, monomérisée et renaturée. L'invention concerne également un procédé de production de cette colonne monolithique.
PCT/DE2009/001412 2008-10-09 2009-10-07 Colonne monolithique comportant de l'avidine monomère immobilisée pour enrichir et identifier des espèces biotinylées Ceased WO2010057454A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP09771267A EP2485836A1 (fr) 2008-10-09 2009-10-07 Colonne monolithique comportant de l'avidine monomère immobilisée pour enrichir et identifier des espèces biotinylées
DE112009003262T DE112009003262A5 (de) 2008-10-09 2009-10-07 Monolithische Säule mit immobilisiertem monomerem Avidin zur Anreicherung und Identifizierung biotinylierter Spezies

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE200810050588 DE102008050588A1 (de) 2008-10-09 2008-10-09 Monolithische Säule mit immobilisiertem monomeren Avidin zur Anreicherung und Identifizierung biotinylierter Spezies
DE102008050588.9 2008-10-09

Publications (1)

Publication Number Publication Date
WO2010057454A1 true WO2010057454A1 (fr) 2010-05-27

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PCT/DE2009/001412 Ceased WO2010057454A1 (fr) 2008-10-09 2009-10-07 Colonne monolithique comportant de l'avidine monomère immobilisée pour enrichir et identifier des espèces biotinylées

Country Status (3)

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EP (1) EP2485836A1 (fr)
DE (2) DE102008050588A1 (fr)
WO (1) WO2010057454A1 (fr)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108114706A (zh) * 2016-11-29 2018-06-05 中国科学院大连化学物理研究所 一种硅胶基质牛磺酸键合固定相及其制备和应用
FR3065376B1 (fr) * 2017-04-25 2022-02-04 Commissariat Energie Atomique Procede de preparation d'une phase stationnaire monolithique, procedes de fabrication d'une colonne chromatographique et d'extraction associes
CN108421542B (zh) * 2018-03-22 2020-08-07 河南科技学院 液态金属微球作为致孔剂在制备整体柱中的应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0423938A1 (fr) * 1989-09-29 1991-04-24 Rohm And Haas Company Milieu contenant un ligand pour la séparation chromatographique, son procédé de préparation et son utilisation pour isoler des molécules synthétiques ou naturelles de mélanges de fluides
WO2002066135A1 (fr) * 2001-02-20 2002-08-29 Advion Biosciences, Inc. Dispositif d'electronebulisation a micropuce et colonne comprenant des adsorbents d'affinite et utilisation

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0423938A1 (fr) * 1989-09-29 1991-04-24 Rohm And Haas Company Milieu contenant un ligand pour la séparation chromatographique, son procédé de préparation et son utilisation pour isoler des molécules synthétiques ou naturelles de mélanges de fluides
WO2002066135A1 (fr) * 2001-02-20 2002-08-29 Advion Biosciences, Inc. Dispositif d'electronebulisation a micropuce et colonne comprenant des adsorbents d'affinite et utilisation

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
H. ZOU: "Monolithic stationary phases for liquid chromatography and capillary electrochromatography", JOURNAL OF CHROMATOGRAPHY A, 19 April 2002 (2002-04-19), pages 5 - 32, XP002574792 *
QIANG ZHAO: "Aptamer-Modified Monolithic Capillary Chromatography for Protein Separation and Detection", ANALYTICAL CHEMISTRY, vol. 80, 26 March 2008 (2008-03-26), pages 3915 - 3920, XP002574911 *
R. MALLIK: "Affinity monolith chromatography", JOURNAL OF SEPARATION SCIENCE, vol. 29, 11 July 2006 (2006-07-11), pages 1686 - 1704, XP002574791 *
S.L. WILLIAMS: "Affinity capture of a biotinylated retrovirus on macroporous monolithic adsorbents: Towards a rapid single-step purification process", BIOTECHNOLOGY AND BIOENGINEERING , JOHN WILEY AND SONS INC. US, vol. 89, no. 7, 30 March 2005 (2005-03-30), pages 783 - 787, XP002574384 *
ZAIFA PAN: "Protein A immobilized monolithic capillary column for affinity chromatography", ANALYTICA CHIMICA ACTA, vol. 466, 21 August 2002 (2002-08-21), pages 141 - 150, XP002574920 *
ZHEN LIU: "Physically adsorbed chiral stationary phase of avidin on monolithic silica column for capillary electrochromatography and capillary liquid chromatography", ELECTROPHORESIS, vol. 23, no. 17, September 2002 (2002-09-01), pages 2973 - 2981, XP002574383 *

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Publication number Publication date
DE112009003262A5 (de) 2013-04-25
EP2485836A1 (fr) 2012-08-15
DE102008050588A1 (de) 2010-04-15

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