February 25, 2014 at 9:50 pm #1528Milad NazariParticipant
I have recently started working on a project that involves imaging tissues using MALDI MSI. I have been trying to use a LEAP Technologies TM Sprayer to coat the tissues with matrix, but I have not had any luck replicating any of the protocols found in literature. I am mostly having trouble with the nozzle clogging and matrix sputtering.
I am using a-CHCA as matrix, and have tried to optimize the conditions by changing the matrix concentration, solvent composition, nozzle temperature, and flow rate. I was wondering if anyone in this community works with a TM Sprayer, and they have had the same issues.August 7, 2014 at 12:24 am #1542Junhai YangParticipant
you can try to lower the nozzle heating temperature. in my experience, the heating most of time is not necessary.
I generally spray any matrix at 30°C, and never have clogging and sputtering issue.
the heating at nozzle causes matrix crystals build up slowly, and then the pressure at the nozzle also slowly build up, you can actually monitor the pressure with the sprayer’s pressure monitor on the front panel, the pressure will go through a “up-down-up’ circle if you have a too high temperature at the nozzle.
another thing you can do is to make the matrix solution a bit dilute, not so high concentration, say around 10-15mg/mL in mostly organic solvents (90% acetonitrile or even acetone). that way, you have less chance of matrix crushing out during the spray and clog up or spatter.August 24, 2017 at 6:23 pm #2992rob.angeliniParticipant
I am also using the TM sprayer to spray CHCA on brain tissues. I am doing this in a facility so I have limited access to the instrument. The director wants to use the factory specs which employ a flow rate of 200uL/min. This is too high IMHO and it is causing extensive delocalization of analytes. Is there anyone out there that is using the TM sprayer with a much lower flowrate and did not encounter any issue? The trick of lowering the temperature to limit clogging described above looks valid to me but, I am wondering if it works fine also with a lower flow rate than 200 uL/min without clogging the lines. The matrix solution that I am using is CHCA 5 mg/ml + 10mM Ammonium Phosphate Monobasic in 50% ACN. If someone is wondering, the reason for the use of the additive buffer is that reduces matrix-salts clusters as shown in the following publication:
RobMarch 16, 2018 at 12:16 am #3126Alain CREISSENParticipant
Dear Milad, Rob and Junhai
Great conversation. I just started being active in this forum and noticed your conversation. We can certainly help you with these questions if they are still of interest.
I agree with Rob that 0.200ml/min can be too much flow and results in a very wet spray, especially with 50% H20.
My starting point with CHCA sprayed on tissues, is:
<Solvent>70% ACN + 0.1%TFA</Solvent>
Note that this protocol has 70% ACN (instead of 50%). Higher organic content makes the spray evaporate faster. The temperature of 75 Deg C is close to the boiling point of the mixture but should be stable and will evaporate quickly.
Feel free to email me at acreissen(at)htximaging.com if you need more information
AlainMarch 16, 2018 at 11:54 am #3127rob.angeliniParticipant
thank you so much for answering. I have seen your contact on the TM sprayer manual and was thinking to contact you. Good you are here and the discussion is available to all of us. Let’s keep the discussion as open as possible.
I have changed a bit the protocol and it is quite similar to what you are suggesting.
<Solvent>30% ACN + 40% IPA + 30% H2O</Solvent> (I am not using TFA cause I am analyzing lipids)
<NumPasses>10</NumPasses> (do you think 4 passes are enough? and maybe 10 are too much?)
<Concentration>5</Concentration> (I am using 5mg/ml and you are using 10, should I go for 10?)
<FlowRate>0.08</FlowRate> (80 uL/min, you are going 120 if I understood correctly)
<Velocity>1100</Velocity> (very similar to you)
<TrackSpacing>2</TrackSpacing> (also I am going 1 vert, 1 horiz. with an offset of 1mm every two steps)
<Pattern>HH</Pattern> (what’s this?)
<DryTime>5</DryTime> (should I go for 0?, it actually looks that it evaporates instantaneously)
As regard the delivery solvent I am using that mixture (30% ACN + 40% IPA + 30% H2O) instead of 70% ACN cause if you try to spot 1uL of both mixtures on a piece of glass you will see that mine moves much less than yours. I had this experience from my previous MALDI project were I have seen that a mixture of IPA and ACN spreads much less than ACN, if there is water is even better. You can try it out if you want but i think it does not make a major difference, as you say it evaporates very fast.
My project is about analyzing cholesterol and oxysterols after on-tissue (mouse brain) enzymatic assisted derivatization.
The major problem in my case is that with so many treatments it’s hard to pin point which one to optimize first. But maybe this is offtopic and I will send you a specific email about the whole thing. I definitely would need some advice.
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