Regulating particle morphology during a spray freeze drying ...

LIGHT‐SCATTERING33samples correctly, a SEC column hasto be calibrated withstandardmolar mass.samples ofknowna)b)Figure 19. Peril of molecular weight prediction over the rT. LS measurementsinitially could lead to misinterpretation of an unfolded antigen [Philo2006].A problem of the “conventional” calibration is that elutionpositions can change, due tocolumn alteration or if themacromolecule has any tendency to interact with the columnmatrix [Philo et al. 1996] . Electrostatic interactions of positively charged proteins withnegative charges on agarose or Sephacryl gelsmay lead to retardation of theelutedproteins and thus to a wrong molar mass [LeMaire et al. 1987]. Philo et al. [1996]reported that LS could detect an unfolded antigen monomer mixed with a dimer(Figure 19) because of its shorter retention time.3.3.2 Asymmetricalflow field‐flow fractionation (AF4/aF‐FFF)For a sample that contains proteins with a largeaggregate fraction or proteins with widemolar mass ranges, different columns will berequired for SEC separation. Giddings[1966] developed a new technique called field‐flow fractionation (FFF) that is able toseparate particles from the lowest nm‐ up tobinary μm‐range with high resolutioninside a narrowchannel. Nowadays asymmetrical flow FFF probably isthe most versatileapplication [Fraunhofer and Winter 2004]. TheAF4 channel is composed of the non‐permeable upper wall made of Plexiglas, a spacer that regulates the channel height, and