PRACTICAL ONLINE DETERMINATION OF BIOPOLYMER MOLECULAR-WEIGHTS BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY WITH CLASSICAL LIGHT-SCATTERING DETECTION

Most biopolymer molecules are much smaller than the wavelength of light used in classical light-scattering experiments (ca. 500 nm), and thus the simple Rayleigh equation and a 90-degrees light-scattering photometer are sufficient to determine their molecular weight. In combination with high-performance liquid chromatography (HPLC), it is demonstrated that a simple HPLC fluorimeter can be used as a 90-degrees light-scattering detector for biopolymer molecular weight determinations. To simplify data handling, only relative molecular weights are measured. Three mathematical assumptions are adopted, and their validity for proteins is shown. To place the work in perspective, the relative advantages and limitations of this 90-degrees light-scattering detector are compared with the more commonly used low-angle laser light-scattering detector. Two examples of protein molecular weight determinations are given to illustrate the broad utility of 90-degrees classical light scattering to the study of biopolymer structures and interactions.