I've been using 45C for ~1ml cell extracts, dried overnight in plastic Eppendorfs but it has been suggested to me that this may be too hot and start degrading the Eppendorf, giving contaminant signals in the final spectra. (However I'm not sure I've noticed these on my NMR spectra- probably due to lack of sensitivity- though I'm sure if I would have used mass-spec, I would be able to see these).
I was told 30C may be enough? (Rotary speedvac temperature settings normally are room temp, 30C or 45C). Any thoughts/comments much appreciated.
That sounds way too high.
Methanol tends to leach out and dissolve the components of Eppendorfs and I have seen these contaminants in mass spec.
I would suggest (if you really have no choice but to use SpeedVac) to go with room temp. 1mL should take less than overnight to dry (splitting the volume into two would help cut down that time even more to shorten the drying time and the contact with the Eppendorf).
Also depending on what the components of your extract are you can use a preservative to at least partially prevent the degradation.
Hope it helps.
All depends on what you are trying to measure. I have heard concerns of plasticisers leeching from plastic ware in the past, all I can say is that in my lab this is not generally a concern and I use mass spec. Generally, the levels are far too low to be much of a worry, the plastics used are of food grade and with NMR being less sensitive than many techniques at first pass I'd consider 45C to be absolutely fine as its a compromise between long drying times, which i consider to be less than optimal, and degradation due to oxidation of your samples as they dry (you are not going to get thermal decomposition at 45C). Some compounds are as tough as old boots whilst others easily oxidise e.g. catecholamines are notorious. If you have worries the best way to dry down is in glass in a hot block at 40C under a stream of OFN, but its a pain if you have a lot of samples. Try a few methods and compare whether there are statistically significant differences in your data.
For better precision and accuracy it is recommended to try all cell extracts under freeze dry condition (Lyophilization, temp -40-90 degree Celsius ).. Or use insert gas nitrogen (cheaper) for evaporating organic solvents at room temperature in open conditions.. However if it is pure methanol extracts, no such metabolites are present which will degrade at 45°C.
I am not sure weather your extract is pure methanol or methanol:water.. if pure methanol go for nitrogen gas drying process
If MeOH: H2O.. freeze in -80°C and then put in lyophilizer for overnight.
Most of the Speedvac already have temperature control upto -5°C
Eppendorfs Case: It does not leach out in methanol, if its a eppendorf company. provided you should not put it for very long time (more than 2 weeks).. Never try Chloroform, hexane extracts in eppendorf. these are more problematic.
Good luck for your experiments..
I've just read the answer of Pavel Lorkewicz about the contaminents from Eppendorfs he sees in mass spec dissolved in methanol. Pavel may I ask you what contaminants you see? What instrument do you have and if it is a triple quad what transitions do you observe?
Thanks
For a comparatively degradation-prone sample like a cell extract, I'd be concerned about exposing it to any elevated temperature. Are you really saving that much time by running at 45C?
One more thing: I'd recommend that you test it out. You're certainly leaching some contaminants from the tubes. The better questions is whether the amount is significant for YOUR experiments. I'd suggest running a control experiment in which you speed-vac one sample of methanol at RT and another at 45C, then run the MS and NMR on them and see what LEVEL of contamination you're observing.
( sorry for the CAPS. I'd rather use italics for emphasis. Really, I'm not yelling. )
I wanted to add one more thing. I don't know if it applies to all Speedvacs but if we leave ours to run for more than even 1 hour (we have Eppendorf's Vacufuge) it warms up significantly and, while it's difficult to measure the actual temperature inside I can easily tell that it it gets way warmer than the setting. Once again, I don't know if that is the case with all Speedvacs but I wonder if leaving samples to dry overnight at 45C may result in exposing them to much higher temperature.
Also, to follow up on the above answers, I really think the stability depends on the nature of one's sample - that is the components.
The sensitivity of NMR may not be as high as that of mass spec but at the same time if we end up detecting something that was degraded/hydrolyzed or converted (we can deal with losses of certain moieties like decarboxylation, deamination etc e.g. glutamine->pyroglutamate, ATP->AMP, ketoacid decarboxylation etc; esterification, polymerization, oxidation and so on) how good of a science is it going to be (not to be too philosophical)?
Just like Santosh suggested - freeze-drying is way better for preserving more labile species but not everyone has access to a lyophilizer.
Pawel, have you ever tried pasting one of those liquid crystal thermometers inside a Speed-vac? I bet it would give you a very good idea of the internal temperature. If you adhere it to the bottom, it shouldn't interfere with the rotor. For example: http://www.omega.com/ppt/pptsc.asp?ref=RLC-50&ttID=RLC-50&Nav=
You can use speed back concentrator. It is good technique. You dont use temp. option. You can easily get dried samples within 2-3 hours. You must avoid plastic eppendorf.
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