title: Complete on-line determination of biopolymer molecular weight via high-performance liquid chromatography coupled to low-angle laser light scattering, ultraviolet, and differential refractive index detection.
abstract: An optically modified high-performance liquid chromatography refractive index detector was developed to allow complete on-line determinations for biopolymer molecular weights. On-line concentration, refractive index, specific refractive index increment (dn/dc2)mu, and Rayleigh factor were determined under flow injection analysis (FIA) and size exclusion chromatography (SEC) conditions using low-angle laser light scattering, ultraviolet, and modified refractive index detection. This instrumental system is capable of determining absolute on-line molecular weights. The error and time requirements involved in conventional methodologies for proteins have been reduced. Sample quantities have been reduced from 150 to 200 mg, in conventional off-line methods, to less than 2 mg for on-line FIA and 0.5 mg for on-line SEC, if mass absorptivities (a) are known. Otherwise, the determination of a will be the most sample-demanding step, requiring about 3 mg of the pure protein. On-line measurements of (dn/dc2)mu are in good agreement with traditional off-line values established at Donnan equilibrium (usually within 5%). In addition, this technique provides true injected mass as determined by the UV detector, after chromatographic exposure where losses may occur, which is then used in the calculation of biopolymer molecular weight.
authors: Barnett Institute, Northeastern University, Boston, Massachusetts 02115.
authors: H H
authors: HH
authors: Stuting
authors: I S
authors: IS
authors: Krull
mesh_terms: D002138:Calibration; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D008027:Light; D008970:Molecular Weight; D012031:Refractometry; D012542:Scattering, Radiation; D014466:Ultraviolet Rays