On-line light-scattering detectors

Simple homopolymers, where monodisperse standards and suitable solvents are available, are easily characterized by SEC. Homopolymers for which no monodisperse standards are available additionally require some more elaborate detection system for transformation of the retention time into molecular weight. This can be done by, e.g., universal calibration. Alternatively, an absolute molar mass detector, like an on-line light scattering detector or mass spectrometer, can be used. 

Pille and Solomon investigated the formation of the above mentioned microgels using gel-permeation chromatography with an on-line light scattering detector [249]. They showed that the primary particles appear very early in the reaction and microgels are formed by interparticular reactions of the primary particles. 

For protein characterization, SEC-LS has been used as an analytical procedure for determining the molecular weights of unknown samples and also for studying protein association. In using an on-line light-scattering detector for SEC... 

The main purpose of the detector in a field-flow fractionation (FFF) system is to quantitatively determine particle number, volume, or mass concentrations in the FFF size-sorted fractions. Consequently, a number, volume, or mass dependent size distribution of the sample can be derived from detection systems applied to FFF [e.g., (UV-Vis) fluorescence, refractive index, inductively coupled plasma ionization mass spectrometry (ICPMS)]. Further, on-line light scattering detectors can provide additional size and molecular weight distributions of the sample. 

Technological developments of various sorts are presented. Chapters deals with new packings based on derivatized silica. Modern polymer-based stationary phases especially designed for biopolymer separations are described in Chapter 9. The application of on-line light-scattering detectors to such separations is described in Chapter 12. 

J. Wen, T. Arakawa, and J. S. Philo, Review Size exclusion chromatography with on-line light scattering, absorbance, and refractive index detectors for studying proteins and their interactions, A a/. Biochem. 240 (1996), 155. 

A multidetector approach is often applied in FFF since all detection systems have some advantages and limitations over some size ranges, sample types, and detection limits. For example, the most common detector, the UV-VIS, has limitations that it is selective for both absorption and turbidity but it is still widely used. Elemental analysis of FFF fractions with ICPMS has been successfully developed during recent years, but other mass spectrometric hyphenations are stiU very few. On-line light scattering has proven to be a very valuable system to determine molecular weight and RMS radius distributions (MALS) as well as diffusion coefficients and hydrodynamic radius (DLS). 

Detectors such as those based on on-line light-scattering, UV absorbance, or refractive index measurements are employed to characterize the molecular weights of polypeptides, simple proteins, and glycoproteins or to determine the stoichiometry of protein complexes [22],... 

The latest trend is to smaller beads in smaller columns, as this saves eluent and shortens the time for a chromatographic analysis. This argument can be correct if only one suitable detector is used. However, these modern small columns are not optimal for a combination of detectors. So-called multiple detection is a combination of some detectors with different measurement principles (differential refractometer, spectral photometer, light-scattering detector, on-line viscometer) behind the last column, mostly in series, seldom in a branched ( parallel ) order. In this way, the tedious preparative fractionation of a polymer sample can often be avoided. 

Lesec, J. and Volet, G., Data treatment in aqueous GPC with on-line viscometer and light-scattering detectors, /. Liq. Chromatogr.,13, 831, 1990. 

Multiangle light-scattering detectors are increasingly used to obtain on-line information on protein molecular weight. However, they must be used in combination with refractive index detectors, and so this technique is not compatible with reversed-phase gradient elution. 

An alternative is to determine the polymers molecular weight/mass in the SEC eluent in situ, by use of on-line molecular mass sensitive detectors. Two such detectors are commercially available, the light scattering detector and the viscosity detector. These detectors are usually used in series with a mass concentration detector and require specialised data handling/software to compute the outputs from the twin detectors and to produce molecular weight/masses and distributions. 

In addition to refractive index detectors, specialized detectors such as on-line SEC detectors and low angle laser light scattering detectors are used for determining the distribution of molecular weights by SEC. 

L. J. Rose and F. Beer, Characterization of long chain branching in LDPE s using SEC with on-line viscosity and light scattering detectors, MolMass International Conference Proceedings, 1999 (www.chem.leeds.ac.uk/molmass 99). 

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