I am currently testing a new method to extract a drug from blood using MTBE (ether based).
Setup:
-96-deep-well plate
-300 µL of blood per well
-1 mL of MTBE layered on top of the blood in each well
-pick up tips, go down into the ether, gently pipet and dispense to gently mix both blood and ether. Do it 20 times.
At the moment, I am using the 8-channel to optimize my liquid class for the mixing step.
Once that is complete, I plan to switch to the 96-head.
I performed a quick initial test at a very low speed (40 µL/s) with 20 mixing cycles using high-volume filtered tips.
I chose such a low speed because I was concerned about vapor buildup in the channels.
I was wondering whether any of you have done something similar (got good liquid class parameters?) and whether 96mL of ether could cause any issues (pressure build up, explosion etc.) with the channels, the head or the LHS itself.
I found a few papers but I also wanted your opinion on the topic.
I have worked with MTBE before and I went the opposite route in terms of speed, granted I wasn’t also trying to mix it into another liquid. MTBE has an incredibly high vapor pressure - compared to ethanol it’s 4 to 5 times higher. Combine that with its rather low heat of vaporization and this is why it evaporates/vaporizes so unbelievably fast.
I would imagine you might be seeing the MTBE drip out of the end of the tips if you tried any normal or default liquid class. This dripping is caused by the increase in pressure in the entire air gap that is above the liquid inside the tip and below the plunger of the pipetting channel. As that air gap sees an increase in pressure it pushes the liquid down and out of the tip since the plunger won’t mechanically move up. So MTBE absolutely falls into the liquid group where we would use anti-droplet control (ADC) on our systems - there is a pressure sensor inside the channel in that air gap and when it detects an increase in pressure it will move the plunger up to stabilize the pressure, thus assisting in the prevention of liquid from dripping out the end of the tip.
Take a look at this post where I talked about pre-wetting the tip effectively: Volume Accuracy: Multiple aliquots with the same tips - #7 by MatthewSmith_Hamilton This pre-wetting will allow you to start with an increased vapor pressure inside the tip, preventing a large amount of MTBE that you subsequently aspirate from being capable of vaporizing. Then if you utilize ADC you should be able to find comfortable liquid class parameters to work with.
Unfortunately the key here for the liquid handling of MTBE is speed. Since you can literally watch it evaporate you have to move fast enough to limit this effect. So while I have spoken about MTBE in tips and the channels above you may also want to look at a lidded reservoir or a reservoir with a septum to prevent evaporation from its source location.
Great, thank you for your detailed answer.
Indeed, when I read papers, the recommend to do not go above 150uL/sec.
I will probably go that route as well.
The thing that confuses me is mixing:
I am using an aspiration step, with volume = 0 but I setup the mixing in the advanced section, Prerinsing/Mix settings, to the desired amount. Is it what you mean by pre-wet?
If this is the pre-wet, then, how do you do your mixing? Using Aspirate, dispense steps?