WO1997024610A1 - Procede de separation electrophoretique capillaire a l'aide d'une solution polymere chargeable utilisee comme agent de separation - Google Patents

Procede de separation electrophoretique capillaire a l'aide d'une solution polymere chargeable utilisee comme agent de separation Download PDF

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Publication number
WO1997024610A1
WO1997024610A1 PCT/EP1996/005704 EP9605704W WO9724610A1 WO 1997024610 A1 WO1997024610 A1 WO 1997024610A1 EP 9605704 W EP9605704 W EP 9605704W WO 9724610 A1 WO9724610 A1 WO 9724610A1
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WO
WIPO (PCT)
Prior art keywords
weight
composition
capillary
water
composition according
Prior art date
Application number
PCT/EP1996/005704
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English (en)
Inventor
Gerardus Jacobus Maria Bruin
Aran Paulus
Iris Barme
Original Assignee
Novartis 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 Novartis Ag filed Critical Novartis Ag
Priority to JP09524006A priority Critical patent/JP2000502734A/ja
Priority to AU13750/97A priority patent/AU1375097A/en
Priority to EP96944010A priority patent/EP0870194A1/fr
Publication of WO1997024610A1 publication Critical patent/WO1997024610A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44704—Details; Accessories
    • G01N27/44747—Composition of gel or of carrier mixture

Definitions

  • the present invention relates to a substantially urea-free composition consisting of (a) 10 to 30 % by weight of a substantially non-crosslinked water-soluble polymer having an average molecular weight of less than 100000, (b) up to 50 % by weight of at least one organic solvent which is miscible with water, and (c) at least 20 % by weight of an aqueous buffer solution, the composition having a viscosity of 50 to 600 rnPa-s; to a capillary electrophoretic separation method for mixtures of organic compounds having molecular weights in the range from approximately at least 300 to about 50000, which comprises using the composition as stationary phase in the capillary; to a capillary for an electrophoretic separating device using the composition as stationary phase, as well as to the use of the composition as stationary phase which can be refilled into capillaries of electrophoretic separating devices.
  • Capillary electrophoresis using gels as stationary phase for separating e.g. oligonucleotides is known and described, inter alia, by A. Paulus et al. in Journal of Chromatography 507, (1990), pages 113 to 123.
  • the gel which is based on polyacrylamide, is prepared direct by filling the capillaries with a polymerisable and crosslinkable aqueous buffer solution which is subsequently polymerised.
  • the polymer is covalently bonded to the surface of the capillary with a linker, typically methacryloxypropyltrimethoxysilane. Good separations are achieved in the case of oligonucleotides having about 12 to 50 nucleotides.
  • the capillary system After repeated separating operations the capillary system cannot be used any further because cleaning the capillaries involves far too much expenditure or is impossible and also because the stationary phase in the capillaries cannot be replaced (flushed out). During the separating processes small bubbles often form which impair the separation considerably or render it impossible, therefore making the replacement of the stationary phase necessary.
  • the stationary phase is also rendered impure by the test samples so that a required high reproducibility, such as that of the registry method, cannot be guaranteed if the separation cannot always be carried out with a new stationary phase.
  • WO 95/18813 describes a composition for capillary electrophoresis which consists of at least 12% (weight/volume) of polymer, urea, an organic solvent and, where appropriate, up to 16.2% of water, with which composition oligonucleotides can be separated.
  • the polymer in this case is also prepared in the capillary and bonded covalently so that it cannot be removed anymore.
  • the molecular weight and viscosity are not critical as even crosslinking is possible.
  • the high urea content of at least 5 mol serves for denaturation and is in many cases not required for an efficient separation.
  • the relatively long elution times and low resolution are also felt to be a disadvantage. Better separating performances are only found when higher amounts of organic solvents are used.
  • the invention relates to a substantially urea-free composition consisting of (a) 10 to 30 % by weight of a substantially non-crosslinked water-soluble polymer having an average molecular weight of less than 100000, (b) up to 50 % of at least one organic solvent which is miscible with water, and (c) at least 20 % by weight of an aqueous buffer solution, the composition having a viscosity of 50 to 600 mPa s.
  • the percentages by weight always make up 100 %.
  • the molecular weight is determined by gel permeation chromatography using known standards of e.g polyacrylamide of known molecular weight.
  • the viscosity is determined with a rotary viscometre (Low Shear 30//MS 1/1 ; Gontraves) at 30° C.
  • water-soluble polymers are known.
  • the viscosity is determined by the choice of the polymer and its molecular weight as well as by its concentration in the composition.
  • the required molecular weight can be achieved either by a specific synthesis or, for example, by a hydrolytic degradation of high molecular weight polymers.
  • the polymers must be stable under the chosen separation conditions. It is possible to use synthetic as well as natural polymers.
  • Typical examples are polyvinyl alcohols, which are optionally etherified or esterified, polyvinylpyrrolidone, polyacrylates or polymethacrylates, which are optionally N- monoalkylated or N-dialkylated, polyethylene glycols or copolymers of ethylene glycol and 1 ,2-diols, such as 1 ,2-propylene glycol; polysaccharides or derivatives of polysaccharides, such as cellulose and cellulose ether, starch, dextrans and carragenans. Dendrimers are also suitable as polymers.
  • Preferred water-soluble polymers are polyethylene glycols, polyacrylamides and polyvinyl alcohol.
  • the molecular weight is preferably 5000 to 100 000, more preferably 10 000 to 80 000, particularly preferably 20 000 to 60 000.
  • urea-free means that the aqueous buffer systems conventionally used for separation may contain urea but that no great amount of urea is added to the compositions.
  • the amount of the organic solvent can likewise be used to adjust the viscosity of the composition but also to influence the separation performance.
  • the composition preferably contains 5 to 50 % by weight, preferably 5 to 40 % by weight, more preferably 10 to 40 % by weight and, particularly preferably, 10 to 30 % by weight, of solvent.
  • the solvents can be, for example, polar and protic or, preferably, aprotic solvents.
  • Illustrative examples are unsubstituted or methoxy- or ethoxy-substituted alkanols and polyols, typically methanol, ethanol, propanol and butanol, methoxyethanol and ethoxyethanol; diols, such as ethylene glycol, propanediol, butanediol, cyclohexanediol; triols, such as glycerol or trimethylol- propane; and tetrols, such as pentaerythritol; carboxamides and lactams, the N-atoms of which may contain two or one methyl or ethyl substituent, typically formamide, acetamide, caprolactam, dimethylformamide, dimethylacetamide and N-methylpyrrolidone, ketones and aldehydes, typically acetaldehyde or acetone, sulfones, such as dimethylsulfone, diethyls
  • the composition preferably contains 20 to 85 % by weight, more preferably 20 to 80 % by weight, particularly preferably 25 to 70 % by weight and, most preferably, 30 to 60 % by weight, of an aqueous buffer solution.
  • the pH of the aqueous buffer solution can be in the range from 2 to 12, preferably from 4 to 9.
  • Type, amount and concentration of the buffer are chosen such that the performance of the capillary electrophoretic system is up to about 5 watt, preferably up to 3 watt and, particularly preferably, 1 watt.
  • Suitable buffers are typically those based on phospates and borates, for example 10 to 500 mmol of TRIS (tris(hydroxymethyl)aminomethane) and 10 to 500 mmol of boric acid. It is also possible to use, or additionally use, BIS (bis(2-hydroxy- ethyl)aminotrishydroxymethylmethane).
  • the viscosity of the composition is preferably 50 to 500 mPa-s, more preferably 100 to 500 mPa-s and, particularly preferably, 150 to 400 mPa s.
  • the composition of this invention is a clear and viscous solution and is excellently suitable for filling capillary electrophoretic devices, which composition is removed (flushed out) after the separation, allowing the capillaries to be filled again for further measurements.
  • the devices are therefore reusable, which is a considerable economic advantage. This opens up the possibility of making entire systems of capillaries and separators (so-called kits) commercially available.
  • the invention also relates to a capillary for an electrophoretic separating device which is filled with a composition of this invention.
  • Capillary electrophoretic apparatus equipped with detection devices are known and described in the technical literature, and they are commercially available from different producers, e.g. the P/ACE 5000 apparatus, supplied by Beckmann, Fullerton, CA, USA, or 3D CE, supplied by Hewlett Packard, Waldbronn, Germany.
  • the capillary walls are coated with, for example, a thin layer of a polyvinyl alcohol. This layer can be covalently bonded via a linker.
  • the inner diameter of the capillaries can be, for example, from 5 to 200 ⁇ m and, preferably, from 50 to 150 ⁇ m.
  • the length of the capillary may be, for example, from 2 to 100 cm, preferably from 5 to 50 cm.
  • the composition of this invention is conveniently filled into the capillaries under pressure, typically in the range from about 10 5 to 10 7 Pa.
  • the capillaries may consist of a material which is an electric nonconductor, for example glass, quartz, or plastic materials such as teflon.
  • Single capillaries are often provided with a protective layer against damages, typically consisting of polyimide. It is also possible to use capillaries or capillary systems which are obtainable by etching techniques, embossing or micromilling on planar carriers.
  • the novel capillary for an electrophoretic separating device is excellently suitable for separating electrically charged organic compounds of, in particular, bioorganic compounds having molecular weights in the range from approximately at least 300 to about 50000 Da, preferably from 1000 to 30000 Da, particularly preferably from 1500 to 15000 Da and, very particularly preferably, from 1500 to 8000 Da.
  • said device can be used again.
  • the composition can be flushed out in simple manner under pressure using, for example, water, organic solvents or mixtures of water and organic solvents.
  • the organic compounds can be natural or synthetic compound mixtures, for example oligonucleotides having approximately up to 100 nucleotide units, oligomeric RNA or DNA sequences having approximately up to 100 nucleotide units, oligomeric hydrocarbons having approximately up to 100 sugar units, as well as oligomeric peptides and proteins having approximately up to 80 amino-acid units.
  • the invention also relates to a process for separating mixtures of electrically charged organic compounds having molecular weights in the range from approximately at least 300 to about 50 000 Da by capillary electrophoresis, which process comprises charging a capillary electrophoretic device, the capillaries of which are filled with a composition of this invention, with a solution of the organic compounds, establishing an electric field of at least 50 volt/cm and then detecting the organic compounds.
  • the electric field can be, for example, 50 to 2000 volt/cm, preferably 100 to 1500 volt/cm, particularly preferably 200 to 1200 volt/cm and, most preferably, 200 to 600 volt/cm.
  • the detection of the organic compounds is conveniently carried out by optical methods, for example by an abso ⁇ tion measurement at a wave length which is mostly in the UV range, or by a fluorescence detection such as a laser-induced fluorescence; or also by mass spectrometric or NMR-spectroscopic methods.
  • the concentration of the organic compounds in the test sample (solution) can be in the mi- llimolar to the micromolar and up to the femtomolar range.
  • Measurement is usually carried out at a constant temperature, typically in the range from 0 to 100 °C, preferably from 20 bis 50 ° C.
  • the organic compounds can be dissolved in water, aqueous buffer solutions or mixtures of water or buffer solutions with water-miscible organic solvents.
  • one of its ends can be immersed in the test sample and voltage can be applied for a short time (for example for 1 to 10 seconds), the organic compounds being absorbed by electrophoresis.
  • Charging can also be carried out in hydrodynamic manner by creating a difference in pressure between the capillary ends.
  • the amount absorbed can be in the range from a few molecules to the attomol and up to the picomol range.
  • the invention also relates to the use of the novel composition as stationary phase in capillaries of capillary electrophoretic devices.
  • Figures 1 and 2 show the electropherograms for separating a synthetic mixture of a 25-mer of thymidine thiophosphate containing sequences of 5 to 25 units.
  • Figure 1 shows the separation without addition of solvents while figure uses 20 % by weight of acetonitrile. The substantially improved separation performance is clearly visible.
  • Example A1 Preparation of a polyacrylamide.
  • Example B1 Preparation of a capillary with polyethylene glycol in the stationary phase.
  • a glass capillary (fused silica) coated with polyvinyl alcohol and having an inner diameter of 100 ⁇ m, a length of 33.8 cm and a detection window at 25.4 cm (producer Hewlett Packard) is flushed at a pressure of 7-10 5 Pa for 10 minutes with a polymer solution (a mixture consisting of 25 % of polyethylene glycol (PEG 35000 by Fluka), 1.23 g of boric acid and 4.18 g of BIS-TRIS (bis(2-hydroxyethyl)aminotris(hydroxmethyl)methane) dissolved in 55 ml of water, degassed for 1 h and then charged with 20 g of acetonitrile). The weight percentages are based on the entire composition.
  • a capillary is flushed according to Example B1 , but leaving out acetonitrile as solvent.
  • the amount of the polyethylene glycol is also 25 % by weight.
  • Example C1 Separating a synthetic mixture of oliginucleotides.
  • the device according to Example B1 is charged with phosphorothioates of the p(dT)e- 25 thymidine (solution with 10 s molar of in water). Charging (at the cathode side) is carried out hydrodynamically for 24 seconds at a differential pressure of 10 5 Pa.
  • the electrophoretic apparatus is supplied by Hewlett Packard (type 3D CE). The voltage applied is -25kV and the current is 12 ⁇ A.
  • the separation is carried out at 30° C and the detection is carried out via UV absorption at 260 nm.

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Molecular Biology (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Electrochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Dispersion Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Immunology (AREA)
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  • Investigating Or Analysing Biological Materials (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Compositions Of Macromolecular Compounds (AREA)

Abstract

L'invention se rapporte à une composition pratiquement exempte d'urée comprenant (a) 10 à 30 % en poids d'un polymère soluble dans l'eau pratiquement non réticulée ayant un poids moléculaire moyen inférieur à 100 000, (b) jusqu'à 50 % en poids d'au moins un solvant organique qui est miscible dans l'eau, et (c) au moins 20 % en poids d'une solution tampon aqueuse, cette solution ayant une viscosité comprise entre 50 et 600 mPa.s. La composition peut être utilisée comme phase fixe dans les capillaires de dispositifs électrophorétiques capillaires destinés à séparer des mélanges de composés organiques chargés électriquement, notamment des oligonucléotides de faible poids moléculaire.
PCT/EP1996/005704 1995-12-28 1996-12-19 Procede de separation electrophoretique capillaire a l'aide d'une solution polymere chargeable utilisee comme agent de separation WO1997024610A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
JP09524006A JP2000502734A (ja) 1995-12-28 1996-12-19 分離剤として充填可能ポリマー溶液を使用するキャピラリー電気泳動分離法
AU13750/97A AU1375097A (en) 1995-12-28 1996-12-19 Capillary electrophoretic separation method using a fillable polymer solution as separating agent
EP96944010A EP0870194A1 (fr) 1995-12-28 1996-12-19 Procede de separation electrophoretique capillaire a l'aide d'une solution polymere chargeable utilisee comme agent de separation

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CH371595 1995-12-28
CH3715/95 1995-12-28

Publications (1)

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WO1997024610A1 true WO1997024610A1 (fr) 1997-07-10

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EP (1) EP0870194A1 (fr)
JP (1) JP2000502734A (fr)
AU (1) AU1375097A (fr)
WO (1) WO1997024610A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001007904A1 (fr) * 1999-07-26 2001-02-01 Pe Corporation (Ny) Procede et appareil permettant de reduire une largeur de pic associe a l'etablissement d'un champ electrique

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989009206A1 (fr) * 1988-03-24 1989-10-05 Pharmacia Ab COMPOSITION DE DERIVES D'ACRYLAMIDE STABLE EN STOCKAGE ET UTILISEE POUR GENERER DES GRADIENTS A pH IMMOBILISE
EP0341577A2 (fr) * 1988-05-09 1989-11-15 SERVA FEINBIOCHEMICA GmbH & Co. Gels d'électrophorèse à teneur élevée en polyols
US5089111A (en) * 1989-01-27 1992-02-18 Bio-Rad Laboratories, Inc. Electrophoretic sieving in gel-free media with dissolved polymers
EP0523982A1 (fr) * 1991-07-17 1993-01-20 The Perkin-Elmer Corporation Solution polymère à viscosité basse pour l'électrophorèse capillaire
WO1993015395A1 (fr) * 1992-01-31 1993-08-05 Beckman Instruments, Inc. Colonne capillaire contenant une composition reticulee dynamiquement et procede d'utilisation
US5264101A (en) * 1989-11-06 1993-11-23 Applied Biosystems, Inc. Capillary electrophoresis molecular weight separation of biomolecules using a polymer-containing solution
WO1995018813A1 (fr) * 1994-01-07 1995-07-13 Hybridon, Inc. Procede d'analyse d'analogues d'oligonucleotides

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989009206A1 (fr) * 1988-03-24 1989-10-05 Pharmacia Ab COMPOSITION DE DERIVES D'ACRYLAMIDE STABLE EN STOCKAGE ET UTILISEE POUR GENERER DES GRADIENTS A pH IMMOBILISE
EP0341577A2 (fr) * 1988-05-09 1989-11-15 SERVA FEINBIOCHEMICA GmbH & Co. Gels d'électrophorèse à teneur élevée en polyols
US5089111A (en) * 1989-01-27 1992-02-18 Bio-Rad Laboratories, Inc. Electrophoretic sieving in gel-free media with dissolved polymers
US5264101A (en) * 1989-11-06 1993-11-23 Applied Biosystems, Inc. Capillary electrophoresis molecular weight separation of biomolecules using a polymer-containing solution
EP0523982A1 (fr) * 1991-07-17 1993-01-20 The Perkin-Elmer Corporation Solution polymère à viscosité basse pour l'électrophorèse capillaire
WO1993015395A1 (fr) * 1992-01-31 1993-08-05 Beckman Instruments, Inc. Colonne capillaire contenant une composition reticulee dynamiquement et procede d'utilisation
WO1995018813A1 (fr) * 1994-01-07 1995-07-13 Hybridon, Inc. Procede d'analyse d'analogues d'oligonucleotides

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001007904A1 (fr) * 1999-07-26 2001-02-01 Pe Corporation (Ny) Procede et appareil permettant de reduire une largeur de pic associe a l'etablissement d'un champ electrique
US6372106B1 (en) 1999-07-26 2002-04-16 Applera Corporation Capillary electrophoresis method and apparatus for reducing peak broadening associated with the establishment of an electric field
US7169276B2 (en) 1999-07-26 2007-01-30 Applera Corporation Capillary electrophoresis method and apparatus for reducing peak broadening associated with the establishment of an electric field

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JP2000502734A (ja) 2000-03-07
AU1375097A (en) 1997-07-28
EP0870194A1 (fr) 1998-10-14

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