Recently this question was posed where a current dynamic light scattering (DLS) user inquired about detecting large but subvisible particles in a formulation:

“I have been looking at solutions from prefilled syringes which appear in flow microscopy to have significant levels of oligo-micron-sized silicone oil droplets. I don’t see any indication of these in my DLS measurements.”

With specifications for DLS systems like the Zetasizer from sub nanometer to several microns the initial concern is not unreasonable. However, on closer look the situation is not so apparent.  The silicone oil droplets are ‘not seen’ by DLS because there are simply too few of them, because the ones that are detected in the scattering volume are interpreted as dust spikes.

If you have a rough idea of the concentration of the droplets, then you can use the Malvern Zetasizer software under Tools – Calculators – Concentration Utilities
and find that for a very unrealistic 0.1 volume% solution of for example 6 micron oil droplets, there would only be less than 7 of those in the scattering volume. For good DLS results, there should be at least 100 particles in the scattering volume to obtain reliable statistics, and to avoid a non-ideal phenomenon called number fluctuations.
Silicone-oil-droplets-numberFor smaller particles the numbers quickly become much more favorable. That is why the core performance of DLS peaks between 1nm to 1 micron. Outside of this range, certain sample specific conditions have to be met to obtain good data quality.

For subvisible particulates, a counting-based technique is much more suitable like the resonant mass measurement or flow-based imaging, and you can find an application note here on sub-visible particulates with microscopy.  You can also learn in this recorded webinar about how Coriolis Pharma used the Archimedes  (resonant mass measurement instrument), to characterize subvisible particles during biopharmaceutical development.


Further Reading:

App Note: Differentiation and characterization of subvisible particulates in therapeutic protein products

App Note: Analysing sub-visible particles at high resolution using the FPIA-3000 automated image analysis system

If you have any questions, please email me at Thanks!
While opinions expressed are generally those of the author, some parts may have been moderated by our editorial team.