Dear Mark, Thank you so much for your reply. Interestingly, after several days I posted this question, I found both my previous crystals and precipitant completely dissolved, but instead much larger crystals start to appear. However, most of them are intergrown together, only few are single. And I think I have to get the right timing and freeze them soon, otherwise it may dissolve again.
For cryoprotectant, I am also thinking of starting from low molecular weight PEG. Hopefully at least I could manage to get one or two crystals out of a drop. Finger crossed... Best, Chen On Fri, Jan 24, 2014 at 6:31 PM, Mark van der Woerd <mjvdwo...@netscape.net>wrote: > Chen, > > Dioxane is not easy to work with, exactly for the reasons you describe. > > There is one thing you did not mention, which I know to be an additional > issue: the quality of the dioxane. I do not know if you need good quality > (whatever that is) but it is a fact that crystallization works with dioxane > from some manufacturer/lots and not with others. I have never figured out > why this is so. > > For a paper on kinetics and reservoir volume discussion, I would read the > work by Forsythe et al ( > http://journals.iucr.org/d/issues/2002/10/01/ic0013/vidsup.html, > doi:10.1107/S0907444902014208)<http://dx.doi.org/10.1107/S0907444902014208> > Basically you will find that this paper says that well volume does not > matter much. Content of your drop matters a lot. In your case, having a > very volatile component, equilibration would be really fast. > > One way to get away from all that is to do batch experiments, where there > is no active transport from a drop into a well. But your question remains > very good: you need an "oil" (?) that will not permit water or a volatile > organic substance to escape. I am not sure what would do the trick. > > Dioxane is a decent cryo-protectant by itself. I would think about adding > "gooey" things like low molecular weight PEG to try to get the drops better > behaved. Evaporation of the dioxane will still be an issue, but with a > higher viscosity you may not have to "chase your crystals" through the > drop. > > Hope this helps a little. > > Mark > > > > -----Original Message----- > From: Chen Zhao <c.z...@yale.edu> > To: CCP4BB <CCP4BB@JISCMAIL.AC.UK> > Sent: Wed, Jan 22, 2014 11:00 pm > Subject: [ccp4bb] Control the crystallization process in the presence of > small volatile organic molecules > > Dear all, > > I am now optimizing a hit which contains about 30% 1,4-dioxane using > hanging-drop vapor diffusion at 25 degree. I am having a hard time to > reproduce the results: most of the times the drops are either dry in one > day or full of precipitate, and only occasionally could I get small > crystals. Is there a way to control the vapor diffusion process, like using > oil to seal the reservoir? (I know paraffin is permeable to dioxane) Also > if someone could refer me to studies on the effects of reservoir volume and > surface area to the crystallization kinetics, that would be very helpful. > > I am also seeking for recommendations for freezing crystals in this > condition. What kind of cryoprotectant has a higher chance? Another problem > is that when I tried to freeze the crystals, the drop dries super rapidly, > and the crystals will dissolve if I add reservoir buffer. But I would > assume good cryoprotectant could do the job. On the other hand, this points > back to my previous question on "dioxane-impermeable" oil. If this magic > oil exists, I could use it to seal the drop when freezing. > > Thank you for help! > > Sincerely, > Chen >
