High-Resolution Asymmetrical Flow Field-Flow Fractionation Data Evaluation via Richardson–Lucy-Based Fractogram Correction

Abstract

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 (Rh), molecular weights (Mw), 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 Mw 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.

Asymmetric flow field-flow fractionation as a multifunctional technique for the characterization of polymeric nanocarriers

“…AF4 presents several limitations, though: peak broadening is more accentuated than chromatography, and baseline separation is often unfeasible. Although deconvolution methods have been developed to improve peak resolution [170], more often than not it may not be possible to identify exactly the protein to which the drug is transferred: consider, for example, that albumin and high-density lipoproteins are both acceptors of lipophilic drugs, and being of similar size their peaks have a high chance of overlapping [164,167]. Once acknowledged its limitations, AF4 is a powerful tool in the study of drug release to serum proteins.…”

Progress in Multidimensional Particle Characterization

“…A step towards the determination of nD PSDs was conducted by Cölfen and coworkers. The authors studied asymmetric field flow fractionation (AF4) coupled to a multiple laser light scattering detector (Schmid et al, 2018). AF4 was used to measure diffusion coefficient distributions based on peak broadening corrected by a new algorithm, which uses diffusion coefficient distributions instead of a single mean diffusion coefficient.…”

“…12 Hydrodynamic radius distribution (Ri) and the respective molecular weight (M W ) of the analysed species as retrieved from a FFF measurement after deconvolution with varying number of algorithm iterations (It). Adapted with permission from (Schmid et al, 2018). Copyright 2018 American Chemical Society.…”

Revision of the calibration experiment in asymmetrical flow field-flow fractionation

“…Recently, we used a calibration method [22] that makes use of the volume calculation as reported by Wahlund and Giddings [1] and then adjusts w by a simple bisection accordingly to Eqs. (3) and (5) .…”