identity of the fragment) in order to determine the eluting concentration and molar mass. The combination of UV and RI detection may even be used to determine the extinction coefficient of each species and aid in their identification.
In addition to quantifying absolute molecular weight, the μDAWN offers all the in-depth analyses available with the miniDAWN TREOS for UHPLC. As with standard HPLC-MALS, changes in molar mass across the UHPLC-MALS peak quantify polydispersity of a biopolymer or assess reversible protein oligomeriza- tion. Triple detection with UV, MALS, and RI enables the application of protein con- jugate analysis to identify the amount of post-translational modification, associated detergent, drug-conjugate, or other additions to a polypeptide background.
Conclusion In order to continue to raise standards in
molecular analysis carried out using UHPLC, researchers need a technique that can deliver
Figure 2 – Light scattering data and measured molar mass for a protein separated by UHPLC (red) overlaid with the separation by standard HPLC (blue). The box indicates a fragment peak that was separable only by UHPLC. The figure at the right is zoomed in to show the fragment and its molar mass as measured by the μDAWN.
efficient and repeatable characterization of bio- molecular samples. This study has demonstrated that with a MALS and RI detector specifically engineered for UHPLC, users can enjoy the ef- ficiency of UHPLC without sacrificing the ability to measure absolute molar mass with MALS. By successfully overcoming the limitations of con- ventional detectors, this capability will enable
unprecedented levels of in-depth analysis to be achieved for UHPLC applications.
Sophia Kenrick, Ph.D., and Aym Berges, Ph.D., are both Application Scientists at Wyatt Tech- nology Corp., 6300 Hollister Ave., Goleta, CA 93117, U.S.A.; tel.: 805-681-9009; e-mail:
skenrick@wyatt.com;
www.wyatt.com
AMERICAN LABORATORY • 13 • JUNE/JULY 2014
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