TY - JOUR
T1 - High-Resolution Asymmetrical Flow Field-Flow Fractionation Data Evaluation via Richardson-Lucy-Based Fractogram Correction
AU - Schmid, Marius
AU - Häusele, Benedikt
AU - Junk, Michael
AU - Brookes, Emre
AU - Frank, Jürgen
AU - Cölfen, Helmut
N1 - Publisher Copyright:
Copyright © 2018 American Chemical Society.
PY - 2018/12/4
Y1 - 2018/12/4
N2 - Asymmetrical flow field-flow fractionation (AF4) is a chromatographic separation technique that can be used for a broad range of particles or macromolecules. As an orthogonal method to size exclusion chromatography (SEC) with a much broader separation size range (1-800 nm) AF4 is gaining importance. However, the data evaluation capacities are far behind in comparison to other techniques like analytical ultracentrifugation (AUC). A program for evaluation of data from AF4 with a coupled multiangle laser light scattering (MALLS) detector was developed that allows the determination of the distributions of diffusion coefficients (D), hydrodynamic radii (R h ), molecular weights (M w ), and relative concentrations (RC) of the obtained species. In addition, two algorithms to remove broadening effects via deconvolution were implemented and tested for their validity. The first is an extension of the known diffusion broadening correction applying the entire diffusion coefficient distribution instead of a single diffusion coefficient. The second applies the Richardson-Lucy algorithm for the deconvolution of overlapping signals from stars in astronomy. This program allows a reproducible strong enhancement of the fractogram resolution allowing for entire baseline separations of proteins. The comparison of the values for M w determined by a partial Zimm plot from each data point of the original fractogram and the deconvolved results shows that especially the Richardson-Lucy algorithm maintains a high degree of data robustness.
AB - Asymmetrical flow field-flow fractionation (AF4) is a chromatographic separation technique that can be used for a broad range of particles or macromolecules. As an orthogonal method to size exclusion chromatography (SEC) with a much broader separation size range (1-800 nm) AF4 is gaining importance. However, the data evaluation capacities are far behind in comparison to other techniques like analytical ultracentrifugation (AUC). A program for evaluation of data from AF4 with a coupled multiangle laser light scattering (MALLS) detector was developed that allows the determination of the distributions of diffusion coefficients (D), hydrodynamic radii (R h ), molecular weights (M w ), and relative concentrations (RC) of the obtained species. In addition, two algorithms to remove broadening effects via deconvolution were implemented and tested for their validity. The first is an extension of the known diffusion broadening correction applying the entire diffusion coefficient distribution instead of a single diffusion coefficient. The second applies the Richardson-Lucy algorithm for the deconvolution of overlapping signals from stars in astronomy. This program allows a reproducible strong enhancement of the fractogram resolution allowing for entire baseline separations of proteins. The comparison of the values for M w determined by a partial Zimm plot from each data point of the original fractogram and the deconvolved results shows that especially the Richardson-Lucy algorithm maintains a high degree of data robustness.
UR - http://www.scopus.com/inward/record.url?scp=85056747147&partnerID=8YFLogxK
U2 - 10.1021/acs.analchem.8b03483
DO - 10.1021/acs.analchem.8b03483
M3 - Article
C2 - 30376711
AN - SCOPUS:85056747147
SN - 0003-2700
VL - 90
SP - 13978
EP - 13986
JO - Analytical Chemistry
JF - Analytical Chemistry
IS - 23
ER -