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Understanding Light Scattering and Chromatography Mode

chromatography system

A chromatography-mode light scattering experiment couples a Multi-Angle static Light Scattering (MALS) detector, such as the DAWN® HELEOS® II or miniDAWN™ TREOS®, with a fractionation device that separates macromolecules based on their physical properties. The most common fractionation method utilizing MALS is HPLC size exclusion chromatography (SEC). SEC-MALS provides accurate distributions of molar mass and size (root mean square radius rg), as opposed to

1.      Standard SEC, which depends on column calibration by reference standards.

2.      Batch (cuvette) mode MALS, where the measured quantities are averaged over all masses and sizes present in the sample

Another powerful separation technique often used in conjunction with MALS is field flow fractionation (FFF) which performs separations over a very wide dynamic range. FFF works without a packed column, by means of the Eclipse DUALTEC and either a flat asymmetric-flow channel or a hollow-fiber flow channel. FFF-MALS seperates solutes from small macromolecules up to micron-sized nanoparticles.

In a typical SEC-MALS or FFF-MALS measurement, the light scattering detector and a concentration detector are connected in series after the fractionation device. The concentration detector is usually a differential refractometer such as an Optilab T-rEX or an ultraviolet absorption (UV) detector.

It may be desirable to add to a MALS instrument a dynamic light scattering (DLS) detection module such as a WyattQELS™ or the DynaPro NanoStar external fiber for measuring hydrodynamic radii rh. SEC-DLS and FFF-DLS size measurements range from sub-nm up to >1 µm

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 (Field Flow Fractionation) or reverse phase chromatography. There are no means to calibrate these techniques by traditional methods.