Understanding Light Scattering and Chromatography Mode
In a chromatography mode light scattering experiment, the classical or QELS light scattering detector is coupled with a fractionation technique that can separate macromolecules based on their physical properties. For example, HPLC size exclusion chromatography (SEC) and field flow fractionation are two powerful separation techniques. Fractionation avoids ambiguities that might result from a batch mode experiment, where the measured quantities are averaged over the distribution of masses and sizes present in the sample. Moreover, a chromatography mode experiment makes it possible to completely quantify the molar mass and size distribution for the sample.
In a typical chromatography measurement employing light scattering, the light scattering detector and a concentration detector are connected in series after the fractionation device. The concentration detector is usually a refractive index (RI) or ultraviolet absorption (UV) detector.
This type of arrangement has many advantages over traditional column calibration methods. Since the light scattering and concentration are measured for each eluting fraction, the molar mass and size can be determined independently of the elution position. This is particularly important for species with non-globular shapes or that interact with a SEC column; such species typically do not elute in a manner that might be described by a set of column calibration standards.
For light scattering in the chromatography mode, however, this is no problem, since the absolute molar mass and size are determined for each eluting fraction. Since it is not necessary to make any assumptions inherent with the use of calibration standards, it is possible to characterize completely the distributions derived from separation techniques such as FFF or reverse phase chromatography. There are no means to calibrate these techniques by traditional methods.