The article you mention really just gives best practices in general. I don't agree with the assertion that a PDI > 0.1 is indicative of more than one species present. It could be because of partial aggregation of single species or, in the case of polymers, simply a reflection of the inherent broadness of the molecular weight distribution.

What medium are you using for your measurements? If you are using just water, that can lead to artificially high sizes (the reasons are explained in the same article).

John Francis Miller : Hello John, Thank you for your response! I am measuring them undiluted but the sample is in formulation buffer (Sample medium is something I cannot change) I'm measuring them for 5 seconds at 25 deg. I usually thaw the samples about 10-15 min before measuring.

I have been getting consistent radii of about like in the ~77 nm range. Will measuring them for 1 sec help? or measure as soon as the sample thaws (which i do), maybe 5-10mins?

Your advice or any other technical tips would be appreciated! Thank you so much for your help.

She S. Are you confident that freezing the samples doesn't affect them?

Elbethel Damtae : Hi Elbethel. Thank you for your response. No, I'm just measuring samples with DLS to determine aggregation. I'm using samples that have been frozen (for storage) and then thawing them at room temp, maybe for 10-15min before measuring. The sample is in a formulation buffer medium (which I cannot change). I'm measuring them at 25deg for 5 secs with 5 acquisitions. I was using a preset method from the instrument (didn't really change any of the parameters there). Do i need to take account of the medium viscosity, like a viscosity calculation as a internal standard? (like the formulation buffer that the virus is in?)

John Francis Miller : The samples are kept frozen at -80deg for storage. I thaw them before i measure them. These aren't fresh samples from a process, rather frozen samples that i'm trying to analyze. How would we store if we didn't freeze them? Also, note these are adenovirus samples.

She S.

I am sending you excerpts from my article

Article Structure of Micelles of Sodium Dodecyl Sulphate in Water: a...

You have a large segment y of approximately 1.7 correlation functions.

The parameters of settings, which are selected by the device automatic system, are the important criterion of reliability of measurements. In the present study, the setting parameters of the measurements were changed in the following ranges: time of the measurement 50-80 s, the count rate 181.8-549.6 of kilophotones per second (kp/s), the segments on the ordinate axis (y) of the correlogram 0.842-0.923, the index of polydispersity (PdI) from 0.278 up to 0.591. These values were optimal for the equipment. The decrease in count rate during the measurement indicates the loss of particles in the monitoring area due to sedimentation. The value of the segment y, which is used to estimate the ratio of signal/noise of light scattered by studied sample, is optimal in the range 0.6-1.0. If it is greater than 1, it means that there are very large fluctuations of the particles in a dispersed system, the sample absorbs light or fluoresces. The values of the PdI index were optimal also. When PdI 7 the system contains particles with very broad size distribution. In this case the DLS method cannot be applied to study the system. The baseline for all correlation functions was zero. Another its position means that there are very large fluctuations in the system (the presence of air bubbles). When the particles move free, the correlation function is always damped. The correlation function for large particles has a flatter slope than that for small particles. Analysis of the correlation function using the software provides the average particle size (Z) and the average distribution (PdI). After confirming the reliability of our measurements, the hydrodynamic spheres can be identified.

Hello She S.

please make sure you have the correct viscosity as this can influence the result, especially if there are a few percent of sugars, glycerol, etc in the buffer. Otherwise, your polydispersity is good, keeping in mind that %Pd and PDI are not the same, i.e. PDI = square of (relative polydispersity)

https://www.materials-talks.com/polydispersity-from-a-gold-standard/

Ulf Nobbmann : Thank you for your comment! How would i accurately find the viscosity of the buffer medium without the use of a viscometer? (if we don't have one at the moment).

This article : https://www.materials-talks.com/what-is-the-maximum-viscosity-for-dls/ (at the end mentions how to find the viscosity of an unknown dispersant). Is that the correct way to find it, just with beads and the buffer itself? How would we find the Refractive Index of the dispersant buffer?

Thank you again for your comment, really appreciate it!

Hello She S,

What is the concentration (or volume fraction) of adenovirus of your thawed samples? There is a possibility that the somewhat larger size you observe is due to residual attractive (sticky) interactions. In other words, do you see smaller size if you dilute further in buffer? If so, it could indicate sticky interactions, just as can happen in 'sticky' proteins, where the collective diffusion coefficient which is measured by DLS also decreases (apparent larger size) on increasing concentration. Since AV's have very low contrast/scattering (Refr.Ind.~1.36-1.37) samples may look very transparent even if the concentration could be quite high....

Representative viruses are charged. Interviral interactions may then have a significant effect on the relaxation of concentration fluctuations. Did you mean to say that you measured a single spectrum for five seconds?