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

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Abstract
An optically modified high-performance liquid chomatography 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 infection analysis (FIA) and size exclusion chromatography (SEC) conditions using low-angle laser light scattering, ultraviolet, and modified refractive index detection. This instrument 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.

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