| PATENT ASSIGNEE'S COUNTRY | USA |
| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | 21.04.2000 |
| PATENT TITLE |
Methods of improving peak resolution in reversed-phase electrochromatography |
| PATENT ABSTRACT |
Peak resolution in reversed-phase electrochromatography is improved in either of two alternative ways. The first is by a stepwise increase in the concentration of acetonitrile or other equivalent buffer constituent or modifier after the sample has been loaded, and the second is the inclusion of a surfactant in the mobile phase. |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | May 18, 1999 |
| PATENT PARENT CASE TEXT | This data is not available for free |
| PATENT CLAIMS |
1. A method for detecting individual solutes in a sample comprising of mixture of solutes by reversed-phase electrochromatography, said method comprising: (a) passing said sample through a stationary solid reversed-phase separation medium by electrokinetic means using a mobile phase having a surfactant dissolved therein at a concentration below its critical micelle concentration yet sufficiently high to cause an improvement in solute band resolution relative to the use of a mobile phase identical thereto but lacking said surfactant, to effect separation of said solutes into individual solute bands; and (b) detecting said individual solute bands. 2. A method in accordance with claim 1 in which said surfactant is an anionic surfactant selected from the group consisting of sodium dodecyl sulfate, sodium decyl sulfate, sodium pentanesulfonate, sodium octanesulfonate, and N-lauroyl-N-methyltaurate. 3. A method in accordance with claim 1 in which said surfactant is a cationic surfactant selected from the group consisting of cetyltrimethylammonium chloride, cetyltrimethylammonium bromide, tetradecyltrimethylammonium bromide, dodecyltrimethylammonium chloride, dodecyltrimethylammonium bromide, hexyltrimethylammonium bromide, and propyltrimethylammonium bromide. 4. A method in accordance with claim 1 in which said surfactant is a nonionic surfactant selected from the group consisting of octyl glucoside, polyoxyethylene-23-lauryl ether, polyoxyethylene-20-cetyl ether, polyoxyethylene-2-stearyl ether, and polyoxyethylene-2-oleyl ether. 5. A method in accordance with claim 1 in which said surfactant is sodium dodecyl sulfate. 6. A method in accordance with claim 5 in which the concentration of said sodium dodecyl sulfate is from about 0.05 mM to about 7.0 mM. 7. A method in accordance with claim 5 in which the concentration of said sodium dodecyl sulfate is from about 0.5 mM to about 3.0 mM. -------------------------------------------------------------------------------- |
| PATENT DESCRIPTION |
This invention relates to reversed-phase electrochromatography. BACKGROUND AND SUMMARY OF THE INVENTION Reversed-phase electrochromatography, including reversed-phase ion exchange chromatography and reversed-phase electrophoresis, is a method of choice for the separation and detection of many types of solutes in multi-solute sample mixtures. The term "electrochromatography" is used herein to denote chromatography in which electroendosmotic flow is used to drive the mobile phase through a bed of separation media. This is distinct from chromatographic systems in which the mobile phase flow is driven by a pump or other pressure-applying device. Like any chromatography, however, peak resolution in reversed-phase electrochromatography is continually sought to be improved. The present invention resides in two methods of improving peak resolution in reversed-phase electrochromatography. The first is by the use of a run buffer that contains a desorbing constituent whose concentration is raised after the sample has been loaded, and the second is by the use of a mobile phase containing an ionic or non-ionic surfactant at a concentration below its critical micelle concentration (CMC). The first method has a dual effect--it narrows the sample zone near the entry point of the sample into the separation medium, and it produces a gradient elution due to the gradual equilibration of the mobile phase to the higher concentration. Both effects result in a narrowing of the solute peaks as they emerge from the separation medium and/or are detected. The second method (the use of the surfactant) improves the partitioning of the solutes by increasing the ligand density and/or charge on the stationary phase. BRIEF DESCRIPTION OF THE FIGURES FIGS. 1a and 1b are chromatograms of a sample mixture obtained on a continuous bed prepared in accordance with this invention, the separation performed using a running buffer with a constant acetonitrile content (FIG. 1a) and a running buffer with a stepwise increase in the acetonitrile content (FIG. 1b). FIGS. 2a and 2b are chromatograms of a sample mixture obtained on a continuous bed prepared in accordance with this invention, using running buffers both with (FIG. 2a) and without (FIG. 2b) sodium dodecyl sulfate. DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS In accordance with the first aspect of this invention, common buffer constituents or modifiers such as those commonly used to decrease the solute-bed interaction are used. Typical examples are methanol, 2-propanol, acetonitrile and tetrahydrofuran. The effect is achieved by raising the concentration of the constituent or modifier at the rear of the sample zone, i.e., substituting a run buffer with a higher buffer or modifier concentration shortly after the sample has been loaded into the separation medium. The increase in concentration can vary and is not critical; any increase that will result in an improvement in resolution will suffice. In most cases when the constituent or modifier is a liquid, effective results will be achieved by increasing the concentration of the constituent or modifier by about 10% or more by volume (i.e., adding 10% by volume of concentration, rather than multiplying the concentration by 1.10). In preferred embodiments, the concentration before the increase is from about 35% to about 60%, and the increment of increase is from about 10% to about 30%. Most preferred is a concentration before the increase of from about 40% to about 55% and an increase of from about 15% to about 25%. All percents in this paragraph are volume percents. The increase is preferably a stepwise increase, immediately after the introduction of the sample. In accordance with the second aspect of this invention, a mobile phase is used that contains a surfactant at a concentration below the CMC of the surfactant. The surfactant can be anionic, cationic, or nonionic. Typical anionic surfactants are sodium dodecyl sulfate, sodium decyl sulfate, sodium pentanesulfonate, sodium octanesulfonate, and N-lauroyl-N-methyltaurate. The preferred anionic surfactant is sodium dodecyl sulfate. Typical cationic surfactants are cetyltrimethylammonium chloride, cetyltrimethylammonium bromide, tetradecyltrimethylammonium bromide, dodecyltrimethylammonium chloride, dodecyltrimethylammonium bromide, hexyltrimethylammonium bromide, and propyltrimethylammonium bromide. Typical nonionic surfactants are octyl glucoside, polyoxyethylene-23-lauryl ether, polyoxyethylene-20-cetyl ether, polyoxyethylene-2-stearyl ether, and polyoxyethylene-2-oleyl ether. The CMC's of these surfactants are known to those skilled in the art. The CMC of sodium dodecyl sulfate, for example, is 8.1 mM under specified conditions. For sodium dodecyl sulfate, therefore, preferred concentrations in the mobile phase are within the range of about 0.05 mM to about 7.0 mM, while most preferred concentrations are within the range of about 0.5 mM to about 3.0 mM. The buffer solutions serving as the mobile phase, including equilibration buffers and run buffers, are otherwise conventional, and may be formulated according to any of the known procedures and compositions known in the chromatography or electrophoresis art. The appropriate buffer for any separation will depend on the desired pH range of the separation, and the selection will be routinely made by those skilled in the art. Typical buffers include citrate, acetate, phosphate, and borate buffers, although biological buffers can also be used where appropriate. A particularly preferred buffer is a phosphate buffer. The stationary phase in the reversed-phase separations of this invention can be one of any known configuration or chemical composition, including particles, both porous and nonporous, and continuous porous structures. The stationary phase can be retained in a capillary column or a tube of larger diameter than the typical capillary column. Examples of appropriate configurations are found in U.S. Pat. No. 5,647,979, and references (including other patents and patent applications) cited therein, all of which are incorporated herein by reference. The following examples are offered only for purposes of illustration. |
| PATENT EXAMPLES | This data is not available for free |
| PATENT PHOTOCOPY | Available on request |
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