Measuring and testing – Liquid analysis or analysis of the suspension of solids in a... – Content or effect of a constituent of a liquid mixture
Reexamination Certificate
2001-08-07
2002-08-20
Williams, Hezron (Department: 2856)
Measuring and testing
Liquid analysis or analysis of the suspension of solids in a...
Content or effect of a constituent of a liquid mixture
C073S061630, C210S198200
Reexamination Certificate
active
06435012
ABSTRACT:
BACKGROUND
This invention relates to an improved method for measuring chromatographic media parameters, such as excluded volume and pore volume, of hydrophobic polymer particles in packed beds. Excluded volumes are useful in determining the performance characteristics of hydrophobic polymer substrates (organic or inorganic polymers) used as stationary phases in medium and high pressure reversed phase liquid chromatography (RPC). Chromatographic media properties, such as porosity, compressibility and permeability (flow resistance), are important in selecting polymers for use in high performance chromatographic preparative modes, such as is required in the separation and purification of biomolecules.
Current methods for measuring excluded volumes of chromatographic media in packed beds involves the use of conventional large-molecule “probe” or “marker” materials, such as linear polystyrene or polyethylene glycol. These probe compounds require the use of non-polar mobile phases (such as tetrahydrofuran and toluene) in chromatographic operations; however, the use of non-polar solvents often presents operational problems, such as toxicity, flammability and incompatibility (with aqueous systems). A discussion of performance evaluation (including void-volume determinations) of hydrophobic polymer supports in size-exclusion chromatography is given by E. Meehan, Size Exclusion Chromatography Columns from Polymer Laboratories in
Column Handbook for Size Exclusion Chromatography
, pp 349-366, Academic Press (1999).
The problem addressed by the present invention is to provide an improved method for determining chromatographic media parameters that does not require the use of non-polar solvents and that can be used in aqueous-based solvent systems, for example, water-alcohol mixtures, such as 20% aqueous ethanol.
SUMMARY OF INVENTION
The present invention provides a method for measuring chromatographic media parameters in a packed bed of hydrophobic polymer particles comprising (a) packing a column with hydrophobic polymer particles and a first solvent to provide a packed bed; (b) introducing a large-molecule probe mixture, comprising a crosslinked ionically-charged polymer particle probe and a second solvent, onto the packed bed; (c) eluting the crosslinked ionically-charged polymer particle probe from the packed bed by passing a third solvent through the packed bed, wherein the third solvent is selected from one or more of polar organic solvent and water; and (d) determining an elution volume for the crosslinked ionically-charged polymer particle probe; wherein the chromatographic media parameter is excluded volume, corresponding to the elution volume for the crosslinked ionically-charged polymer particle probe.
In a preferred embodiment, the crosslinked ionically-charged polymer particle probe of the aforementioned method is selected from one or more of anionically-charged emulsion-form polymer particles, cationically-charged emulsion-form polymer particles, anionically-charged ground polymer particles and cationically-charged ground polymer particles, having a particle size from 0.05 to 1 micron.
In another embodiment, the aforementioned method further comprises (i) introducing a small-molecule probe mixture, comprising a small-molecule probe and the second solvent, onto the packed bed; (ii) eluting the small-molecule probe from the packed bed by passing the third solvent through the packed bed; (iii) determining an elution volume for the small-molecule probe; and (iv) determining a pore volume for the hydrophobic polymer particles by subtracting the elution volume for the crosslinked ionically-charged polymer particle probe from the elution volume for the small-molecule probe; wherein the chromatographic media parameter is pore volume.
DETAILED DESCRIPTION
In the determination of the interparticle void volume (“excluded volume”) and other chromatographic media performance parameters, we have discovered that the use of a large-molecule probe (or marker) based on crosslinked ionically-charged polymer particles allows for quick and efficient measurements using polar organic solvents and aqueous-based solvent systems that are environmentally friendly. Use of the aforementioned large-molecule probe particles in place of conventional probe materials provides a probe material that (1) is totally excluded from entering the polymer matrix being evaluated, (2) allows the use of polar solvents during the chromatographic process; and (3) does not contribute to hydrophobic interactions between the probe material and the polymer matrix of the packed chromatography column.
In particular, we have discovered that emulsion-form or ground crosslinked polystyrene particles containing ionizable functional groups (such carboxylate, sulfonate or quaternary ammonium chloride) are particularly useful as large-molecule probe materials in the evaluation of various hydrophobic solid media, particularly macroporous polyvinylaromatic polymers, for chromatographic applications. Carboxylate and sulfonate functional groups are representative of “anionically-charged” probe particles and quaternary ammonium functional groups are representative of “cationically-charged” probe particles.
As used throughout the specification, the following terms shall have the designated meanings, unless the context clearly indicates otherwise.
All percentages referred to will be expressed in weight percent (%), based on total weight of polymer or composition involved, unless specified otherwise. The term “(meth)acrylate” or “(meth)acrylic” refers to either the corresponding acrylate or methacrylate derivatives: such as the corresponding acids, esters, amides, substituted esters or substituted amides. The term “copolymer” refers to polymer compositions containing units of two or more different monomers, including positional isomers. The following abbreviations are used herein: g=gram; ppm=parts per million by weight/volume, cm=centimeter, cc=cubic centimeter, mm=millimeter, ml=milliliter, &mgr;m=microns, min=minute. Unless otherwise specified, ranges listed are to be read as inclusive and combinable and temperatures are in degrees centigrade (° C.).
As used herein, chromatographic media parameters include those properties that are typically used to characterize hydrophobic polymer substrates for suitability in specific end use applications, such as the separation and purification of biomolecules using size exclusion chromatography, gel filtration chromatography, gel permeation chromatography, hydrophobic interaction chromatography or reversed phase chromatography. Typical chromatographic media performance parameters of interest to the chromatography practitioner include, for example, compressibility, permeability (flow resistance), % polymer pore volume (% porosity of polymer bed), pore volume, interparticle void volume (% interstitial volume or excluded volume), % polymer solids (volume), polymer porosity (volume pores/volume polymer).
In general, to determine various chromatographic media parameters of polymer substrates, mobile phases (solvents used to transport molecules through the polymer matrix) must be selected for compatibility with the probe molecules such that interactions of the probe molecule with the hydrophobic surface of the polymer particles is minimized and preferably eliminated, otherwise chromatographic performance parameter measurements may be inaccurate and imprecise. Conventional large-molecule probe materials, such as linear polystyrene and polyethylene glycol, may be used for interparticle void volume determinations; however these types of probe materials require the use of nonpolar mobile phases (such as tetrahydrofuran, dichloromethane and toluene) to minimize hydrophobic interactions between the probe material and the polymer surfaces. The probe materials used in the method of the present invention, however, do not require the use of non-polar solvents and can be used in a wide range of polar organic solvents and aqueous-based solvent systems, for exampl
Howell Thomas J.
Politzer Jay L.
Rohm and Haas Company
Williams Hezron
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