Sample evaporation puzzle

[tayzyboy]

This is not strictly LC , but is a part of our sample prep for LC samples.
But an interesting puzzle we've been stuck on for a while I thought I'd throw out for ideas...

We are cleaning up plasma samples which contain a very reactive drug, therefore the sample prep method is a bit odd, but seems to work.

ZnSO4 is added to plasma, this crashes out the proteins with drug bound.
(centrifuged) The aqueous is removed and replaced with methanol. Mixing causes the drug to become free. (centrifuged)
The methanol is then transfered to a new vial and evaporated.

The trouble comes here....
in our rotary evaporator...
1ml of pure methanol evaporates in about an hour
1ml of pure water evaporates in 6-7hours
1ml of extracted sample takes 12+ hours.

Why is our sample taking so long to evaporate??? , The only answer we have come up with is that there may be an unobservable fatty layer on the top of the sample preventing evaporation?

We're thinking of adding some surfactant to the sample to see if this helps.

Any ideas guys and gals?

James

[Peter Apps]

Hi James

It looks as if you have something wrong with your rotary evaporator - it should not take any more than a few minutes (not hours) to evaporate 1 ml of methanol.

Check that you actually have a vacuum - vacuum pump sucking, no ports accidentally open, no taps open.

Have your water bath at 40 - 50 degrees C to speed things up a bit.

For small volumes, evaporation under a stream of nitrogen is often handier than a rotary evaporator, becuase it can be done in a test tube rather than a round bottom flask, and with an evaporation manifold you can do several samples at once.

Peter

[chhubert]

I agree with Peter that evaporation under nitrogen is easier to handle than rotary evaporation. To my experience, evaporating 3mL of methanol under a gentle stream of nitrogen at 40-50 degree celcius takes about 45 minutes. Rotary evaporation is always faster than nitrogen purge. Thus evaporating 1mL of methanol should be less than 15 minutes. I guess the plasma extract that you are handling probably contains mixture of water and methanol. It is always difficult to evaporate last trace of water and people usually do is to add small amount of ethanol into it to facilitate the removal of water 'cos water-ethanol forms azeotrope. Notwithstanding this, you should also check your rotary evaporator for vacuum and water bath as these two are the controlling factors for evaporation. Hope this helps.

[tayzyboy]

Thanks for the tips, I like the idea of trying ethanol, might give that a spin (pun intended)
yes, our evaporator is rather slow. . That is something we're looking into for the future. - though it freezes water quite happily.

Todays efforts have however shown that 50:50 Methanol:Water is evaporating much slower than either 100% H2O or 100% MeOH. As there is going to be water traces in our plasma sample, we think this effect is happening here too.

Anyone know why this might be? some chemical interaction?

James

[Uwe Neue]

Contact me if you want some suggestions on how to improve your sample preparation method.

[Bruce Hamilton]

I'd guess that you probably do have an oil film given your sample evaporates slower than water, but the times you suggest for all solvents as so slow that your Rovap or protocol needs some loving attention.

As others have noted, the vacuum should be checked, as should temperature. In my experience, the main cause of slow evaporations is because the flask, vial, whatever, is too small. You need a large surface area combined with a thin film to remove solvent quickly on any rovap.

The best solution, once you have fixed any vacuum problems, is to quickly apply the highest vacuum possible ( avoiding foaming ) with the fastest speed ( thinnest film ) then slowly introduce heat. However, there are almost as many "best" ways of using rovaps as there are rovaps.

If you do have an oil layer or solids that hold water, evaporation under nitrogen may be even slower, and the best solution for samples with such problems would be to revisit the prep process, rather than introduce other solvents.

Please keep having fun,

Bruce Hamilton

[HW Mueller]

One would presume that this is another case of requiring extraordinary skill to have an oil film on a rotating MeOH mix. Also, it sounds like your roti puts on pressure rather than a vacuum at the relevant site. Plasma extracts have all kinds of small molecules salts, etc. in them which increase the boiling point.
Quite often we have seen interfering gas peaks when evaporating (partially) samples with N2, thus we usually use test tubes with ground glass joints on the rotaries when small volumes are involved.

[Peter Apps]

Hi James

Now I'm puzzled - you have a rotary evaporator that "freezes water quite happily" !? That sounds more like a freeze dryer, in which case you "drying" (aka sublimation) times are not entirely unreasonable. What does this machine of yours look like ??

On the slower evaporation of water - methanol mixtures; there is probably a mixture that gives a lower vapour pressure than either of the pure components.

Peter

[tayzyboy]

Our freeze dryer / evaporator.

Sorry for the confusion. Im not sure what our equipment is classed as.
Basically its a centrifuge which operates under a vacuum, this is coupled to a -90 degree cold trap.

its a Jouan RC10.10.

[Peter Apps]

Hi James

From confusion to understanding - what you have is not a rotary evaporator, and that it why it takes so long to dry down the samples.

If you have the option, a "real" rotary evaporator (aka a Rotovap) will do the job a lot quicker, but for small volumes I still favour warming the samples under a gentle stream of nitrogen - use high purity to avoid possible contamination. Without knowing what you have in your lab it's difficult to make concrete suggestions.

Peter

[chhubert]

James,

I guess the instrument that you have been using is so called centrifugal evaporator. Centrifugation is only used for reducing chance of foaming during evaporation. Definitely, evaporation by this type is barely solvent vaporization under low pressure and high temperature. The evaporation rate, to my experience, depends very much on vacuum pressure, temperature, and no. of samples being evaporated. Too many samples evaporating at the same time reduce evaporation rate significantly. Also as the samples are pretty static during evaporation, oily film may easily form on surface, which hinders further evaporation for solvents underneath it. I suggest you should better choose other evaporation method for your plasma samples, say nitrogen purge, Turbovap, etc.