Dear Philip Lifwergren

Despite you do not mention the molecular weight of HA, I think that in general you should be able to dissolve HA in PBS at a concentration of 20 mg/ml. The problem might come from the fact that you are using a derivative of HA. It is not possible to be specific without knowing details about you material, but some general suggestions could be:

a) Is your derivative well soluble in PBS? If the hydrophobization of HA by non-polar groups is significant, you will get water insoluble materials (as, e.g., in https://doi.org/10.1016/j.carbpol.2019.115162). You could then be loosing a part of you material by sedimentation or measure viscosity of a suspension.

b) Is your derivative homogeneous? Differences in the degree of HA modification within one batch (inhomogeneous reaction) could be responsible, or maybe even differences in particle size distribution, if you do not dissolve for sufficient time (smaller particles dissolve faster).

c) Were the solutions with different viscosity from the same reaction batch, or were you dissolving products of various reactions? If your samples were from more reactions, then the observed effect in viscosity may just be a result of getting different modification degrees out of different reactions.

d) Are you using elevated temperatures or extreme pH in your processes? If yes, could various samples / solution be subjected to these conditions for different times? This could lead to significant molecular weight differences responsible for the viscosity variations.

One last idea could be insuficient mixing time. Overnight mixing with vigorous stirring is often necessary for HA, and high molecular weights at higher concentrations could even require a day or two (or special stirring equipment for viscous solutions).

Hope at least some of this helps

Regards

Josef

Josef Chmelar

Thanks for your in-depth answer!

The Mw of HA is 125 kDa and the Mw of functionalized HA is 145 kDa, so not that big compared to what I have seen others use.

The inconsistency has mainly been between batches, rather than within batches.

However, NMR shows no difference in the degree of functionality.

Have concluded that the different viscosities have been caused by aggregations rather than different viscosities.

Have used 200 mM HEPES with intense stirring and sonication to remove remaining aggregates.

Best regards /Philip

Dear Philip Lifwergren

Thank you for the specifications. Mw around 150 kDa is quite low and should dissolve reasonably well.

If the problems were between batches, then I presume it was due to differences in the individual reactions. Even when repeating the same protocol, you can have slightly different results in the substitution pattern, which can translate to larger viscosity variations. Your identification of aggregation as the cause seems plausible, since aggregation properties can be very sensitive to the substitution. Did the process you describe at the end of your post (removing of aggregates) eliminate the viscosity differences?

And regarding NMR, if you use just D2O as the solvent it should be fine, but we observed problems with substitution degrees from NMR for insoluble derivatives requiring mixed deuterated solvents, as described in: https://doi.org/10.1039/C6AY03067J

Regards

Josef