Light scattering detectors online

Stolyhwo et al. (106) described a different analytical methodology, one that circumvents most of the problems encountered so far and permits the analysis of TGs up to and including tribehenin and the quantitative analysis of these complex mixtures with few calibrations, using a laser-light-scattering detector. For the achievement of most separations, a 250 X 4.0-mm ID HIBAR LICHROSPHER1000 CH-18/3 (5-/an particles) was used. For the achievement of some difficult separations, a second, similar column was connected online to the first one. 

Figure 7.15 shows SEC/DRI/LALLSP chromatograms of dextran T150 in water at the mobile phase. It has been demonstrated that an online light-scattering detector can easily detect the effects of protein aggregation whereas the trimer peak is not easily detected by the single DRI or UV detector alone. 

This technique is often used online in conjunction with size-exclusion chromatography. Since the signal from the light-scattering detector is directly proportional to the molecular mass of the protein times the concentration (mg mL ), by combining this signal with that from a concentration detector (refractive index or absorbance) it is possible to measine the molecular mass of each protein coming off the column. 

To avoid these artefacts, it was proposed that 0.3 M acetic acid/0.2 M sodium acetate (pH = 4.5) be used as solvent since there was no evidence for aggregation in this mixture [50]. At that time, it was demonstrated that the value of DA modifies the K and a parameters. In this solvent, the absolute M values were obtained from steric exclusion chromatography (SEC) equipped with online viscometer and light-scattering detector, allowing the Mark-Houwink parameters to be determined without fractionation and also the relation between the radius of gyration and the molecular weight. 

This type of measurements can very elegantly be realized online by coupling several detectors at the end of the SEC column such as a concentration detector (refractive index detector, spectrophotometric detector, etc.) and an absolute detector measuring the molar mass or related property of the separated species such as laser light scattering detector or capillary viscometer detector. These modern sophisticated separation systems allow not only the separation of the analyzed species but also their very detailed analysis and characterization as concerns the MMD or PSD, as well as other structural and compositional characteristics of simple polymers, co-polymers, etc. A schematic representation of a procedure of SEC data treatment from an experimental chromatogram to the final MMD or PSD data is shown in Figure 8. 

Refractive index detectors are used to measure concentration profiles and (if used alone) molar masses based on a calibration. In addition, they are used to measure the slice concentration when working with online light scattering detectors, viscometers, or online mass spectrometry. They are also used to measure the refractive index increment, dnidc, in a single point experiment when light scattering or triple detection is used. In combination with other concentration detec-... 

Online coupling to viscometry and/or light-scattering detectors Calibration by online or offline coupling with mass spectrometry Comprehensive two-dimensional LC (LC x SEC) or LC X SEa/MALDI-MS... 

To address the molecular heterogeneity of complex polymers, fractionation methods quite frequently must be hyphenated with information-rich detectors, as has been highlighted in Section 2.03.4. For SEC, multidetector setups are required to analyze distributed properties like branching or molecular shape in addition to MMD. In such cases, light-scattering detectors or online viscometers are used. These couplings, however, do not provide information on the chemical composition or the microstmcture as a function of molar mass. 

Wen, J., T. Arakawa, and J. S. Philo. 1996. Size-exclusion chromatography with online light-scattering, absorbance, and refractive index detectors for studying proteins and their interactions. Anal Biochem 240 155-66. 

FFF-multiangle light scattering (MALS) MATS detectors enable the MW of eluted macromolecules to be measured online. When combined in series with a mass detector such as a UV or refractive index detector, the MW distribution is generated directly without any calibration with standards. Furthermore, MALS also yields the radius of gyration of the molecules and the variation of this size parameter with MW can be used to deduce molecular conformation information. 

In 1953, Stockmayer and Fixman observed that the ratio, h, of hydrodynamic or Stokes radii (/ h) of the branched and linear macromolecules could also be used as a measure of branching. The relatively recent introduction of online quasielastic light scattering (QELS, also known as dynamic light scattering and as photon correlation spectroscopy) detectors for SEC permits direct determination of h by... 

---