CAPILLARY ELECTROPHORETIC SEPARATION METHOD USING A FILLABLE POLYMER SOLUTION AS SEPARATING AGENT

Abstract: 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 100 000, (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 mPa.s. The composition can be used as stationary phase in the capillaries of capillary eletrophoretic devices for separating mixtures of electrically charged organic compounds, in particular of low molecular oligonucleotides.

Description: Capillary electrophoretic separation method using a tillable polymer solution as se p aratin g agent 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. 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. In this composition 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. In Applied and Theoretical Electrophoresis (1994), 4, pages 39-41, C. Heller et al. state that owing to the resulting viscosity it is possible to use ten percent solutions of polyacrylamide in an aqueous buffer as tillable and removable stationary phase in capillary electrophoresis if the linear polyacrylamide has an average molecular weight of 80000. Owing to the reduced mobility of the oligonucleotides, longer elution times are found. The resolution (width and overlapping of the peaks) is also felt to be insufficient, in particular in the case of oligonucleotides having shortish chains. Surprisingly, it has now been found that in the case of mixtures of electrically charged organic compounds having molecular weights preferably in the range from approximately at least 300 to about 50000 Da, a substantially enhanced resolution and faster migration is obtained if tillable and removable polymer solutions of a specific viscosity are used which additionally contain an organic solvent which is miscible with water. In addition, the reproducibility (elution time of the organic compounds) is surprisingly high so that these stationary phases are suited, on the one hand, for automatation and, on the other hand, for a standardisation in the qualitative determination of unknown compound mixtures. Surprisingly, it has also been found that when oligonucleotides are separated, even phosphates are separated from thiophosphates or other modified oligonucleotides, but, which is of great importance when synthetic oligonucleotides are examined. In many cases resolution is even of such a high degree that mixtures of oiigomeric compounds can be separated and determined which differ only in one unit or other structural element. In one of its aspects, 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. Many suitable 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. Essentially 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, diethylsulfone and tetramethylenesulfone; sulfoxides, typically dimethylsulfoxide, diethyl- sulfoxide and tetramethylenesulfoxide; and nitriles, such as acetonitrile, propionitrile, butyronitrile and benzonitrile. 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 (for example UV ab¬ soφtion measurement) 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. In order to prevent an electroosmotic flow at the pH value of the stationary phase, 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 μm and, preferably, from 50 to 150 μm. 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. By replacing the composition of this invention in the capillary, 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 absoφtion 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. For charging the capillary, 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. The following Examples illustrate the invention. 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. A) Working Examples for polymers. Example A1 : Preparation of a polyacrylamide. 5 g of acrylamide are dissolved in 45 mi of water and the solution is degassed with helium for 30 minutes. 300 mg of ammonium persulfate and 300 mg of N,N,N',N'- tetramethylenediamine are then added and the mixture is polymerised overnight at a temperature of 25 °C. The polymer solution is then dialysed to remove low molecular reaction products, ammonium persulfate and N,N,N',N'-tetramethylene diamine. 200 ml of methanol are then added to the purified polymer solution and the precipitated polyacrylamide is dried. The average molecular weight is: M w = 60000; M n = 40000. The polymer can be stored and dissolved in the desired buffer solution before use. B) Preparation of capillaries. 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 μm, 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. Comparison Example: 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. C) Use Examples 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 μA. The separation is carried out at 30° C and the detection is carried out via UV absorption at 260 nm. The same separation is carried out using the device according to the Comparison Example. The results are shown in figures 1 and 2. The electropherograms (y axis absoption, x axis time) show in comparison that the elution times are shortened, that the degree of separation is substantially improved and that even phosphates can be detected in the presence of thiophosphates.

Claims: WHAT IS CLAIMED IS: 1. A composition which is substantially urea-free, consisting of (a) 10 to 30 % by weight of a substantially non-crosslinked water-soluble polymer having an average molecular weight of less than 100 000, (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 mPa-s. 2. A composition according to claim 1 , wherein the polymers (a) are selected from the group consisting of polyethylene glycols, polyacrylamides and polyvinyl alcohol. 3. A composition according to claim 1 , wherein the average molecular weight of the polymer (a) is from 20 000 to 60 000. 4. A composition according to claim 1 , wherein the amount of the organic solvent (b) is from 10 to 40 % by weight. 5. A composition according to claim 1 , wherein the organic solvent (b) is a polar and protic or aprotic solvent. 6. A composition according to claim 1, wherein the organic solvent (b) is acetonitrile. 7. A composition according to claim 1 , wherein the amount of the aqueous buffer solution (c) is from 20 to 85 % by weight. 8. A composition according to claim 1 , wherein the amount of the aqueous buffer solution (c) is from 30 to 60 % by weight. 9. A composition according to claim 1 , wherein the viscosity is from 50 to 500 mPa-s. 10. A capillary for an electrophoretic device which is filled with 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 100 000, (b) up to 0 % by weight of at least one organic solvent which is miscible with water, and (c) at least 0 % by weight of an aqueous buffer solution, the composition having a viscosity of 50 to 600 mPa-s. 11. A capillary according to claim 10, which is 2 to 100 cm long. 12. A process for separating mixtures of electrically charged organic compounds having molecular weights in the range from approximately at least 300 to about 50000 Da by capillary electrophoresis, which process comprises charging a capillary electrophoretic device, the capillaries of which are filled with 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 100 000, (b) up to 50 % by weight of an 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, with a solution of the organic compounds, establishing an electric field of at least 50 volt/cm and then detecting the organic compounds. 13. Use of the composition of this invention according to claim 1 as stationary phase in capillaries for capillary electrophoretic devices.

Inventors: BARME IRIS, BRUIN GERARDUS JACOBUS MARIA, PAULUS ARAN, BARME, IRIS, BRUIN, GERARDUS, JACOBUS, MARIA, PAULUS, ARAN
Applicants: BARME IRIS, BRUIN GERARDUS JACOBUS MARIA, CIBA GEIGY AG, PAULUS ARAN, BARME, IRIS, BRUIN, GERARDUS, JACOBUS, MARIA, NOVARTIS AG, PAULUS, ARAN
Application year: 1996
Main classification: G01N27
IPC codes: C08L29, C08L33, C08L101, G01N27