Wyatt Technology

Band Broadening Correction

Band Broadening CorrectionVirtually all online fractionation techniques use detectors in series. As shown in the above animation, however, this creates a fundamental problem. When a narrow "peak" moves along the flow path, each flow cell acts like a small mixing volume that causes the initially sharp peak to broaden with a slight exponential tail. Each detector in the flow path sees an increasingly broadened peak that is different from the initial ideal peak.


Although this broadening might appear slight, it has deleterious effects on any analysis method that relies on data from multiple detectors. Consider the fractionated BSA monomer sample at right detected by a light scattering (LS) and refractive index (RI) detector in series. The calculated molar mass depends on the ratio of the light scattering and refractive index peaks. The RI peak (dashed line) has clearly been broadened with respect to the LS peak (solid line), resulting in a calculated molar mass that has a "grimace", or frown across the peak. For BSA monomer, however, the mass should be constant across the peak.

Wyatt Technology is excited to provide the first real solution to this fundamental problem. Our scientists have developed a patented band broadening correction algorithm that accounts for the physical broadening mechanism. When this band broadening correction is applied, the results are amazing.

BSA band broadening example

Consider the fractionated BSA series at left, where the data in blue is not corrected for band broadening, while the data in red includes the band broadening correction. The uncorrected data exhibits the classic grimace in molar mass across each peak, while the band-broadening-corrected data clearly shows the "staircase" behavior expected for the oligomer sequence.

The fractionated BSA example also shows the effect of the band broadening correction on the LS trace. Applying the correction modifies the LS trace slightly to match the broadened RI trace. This modification results in little, if any real loss in resolution, yet reveals the molar masses accurately.

BSA band broadening example
The band broadening correction revolutionizes chromatography analysis, revealing trends that were never before visible. Moreover, it is critical for such interesting applications as protein conjugate analysis and viscometry, where three detectors in series are needed, and consequently band broadening effects are even more severe. ASTRA can apply the band broadening correction to an arbitrary number of detectors in series, making it possible to perform even these sophisticated analyses to reveal accurate and exciting new information about your system.

 

   

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Wyatt Technology is the recognized leader in light scattering instrumentation and software for determining the absolute molar mass, size, charge and interactions of macromolecules and nanoparticles in solution.

Wyatt's line of multi-angle static light scattering products couple to size exclusion chromatography (SEC-MALS), field-flow fractionation (FFF-MALS), and stop-flow composition-gradient systems (CG-MALS). Our dynamic light scattering (DLS) products operate in traditional cuvette as well as on-line and automated, high-throughput modes. We also offer unique instruments for electrophoretic light scattering (MP-PALS), differential refractometry, and differential viscosity.



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