Hello, I’m Christopher George, Lab Systems and Automation Manager at Precision Analytical. I’ve been working with automation applications and integrations for about seven years now, and working with various platforms in the computer sciences as a hobby about twice that long.

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We’ve been using Lynx systems for about five years now. Prior to that, our liquid handlers were Tomtec and Tecan Systems. This year we’ll be transitioning the last of our legacy Tecan processes onto Lynx systems. We’re a human sex hormone testing lab that processes urine and saliva samples. Each patient submits about half a dozen samples or more, depending on what is being tested. And we process them for LCMS analytical review. Our focus today will be on the solid phase extraction (SPE) process, where we run multiple solvents through 96-well SPE columns on LM900 VVP96 systems and ST96 systems.

So in this presentation, we’ll be covering a lot of the variables that impact solvent transfers. And that’s going to include environmental factors, the polarity of solvent transfers, interactions with both 1250 microliter filtered and non-filtered tips, and general solvent pipetting settings in Method Manager 4.

So, when it comes to environmental factors, some considerations are negative airflow, static charge management, and temperature management.

The polarity of the solvent can dictate how you approach transfer speeds and what order of transfer steps you implement. This can be a significant factor. But other properties of the solvent can make two solvents of similar polarity behave a bit differently.

This can also influence interactions with the tips themselves. So chemical compatibility with consumable tips is important as well. An A-level compatibility can cause some unwanted interactions with the inner and outer robotic pipette tip surfaces.

Filming, which is how the residual solvent is retained on the inner and outer tip surfaces, is generally a combination of the polarity, chemical compatibility with the tips, and static.

Regarding the size of the tips, liquid handling properties described in this presentation will utilize 1250 microliter filtered and non-filtered tips on VVP and ST systems. That’s what we exclusively use in our lab, and that’s what I have the most experience with. But if you were using smaller tips, I would expect possible improvements in the micro-droplet formation for polar solvents with smaller tips.

Additionally, VVP and ST systems change the effectiveness of handling the solvent transfers differently. Volatility and working surface area can also impact how well you manage the solvent in the method.

All right, let’s dive into the environmental factors. So, view and extraction, safety first. OSHA requires 60 feet per minute face velocity, but ideally greater. Dynamic Devices requires about 750 to 800 cubic feet per minute air change out in the system itself. Lynx systems should extract all solvent vapors from the main operating chamber. This is safe for the instrument and safe for the end user. My systems operated about 100 feet per minute face velocity with 750 CFM of air change out in the system itself. This can have an impact on low volume transfers, though, below 20 microliters.

Here’s an example of a way to achieve negative airflow in an LM900 system. You can see this is our very early, rudimentary DIY method. Very cheap, not the most pretty looking, but all you have to do is take out that bottom panel on like a 900 system, or even an 1800 system, which is the two types we have in our lab. Take a wood board, slap that sucker on, screw it in. We routed out a hole and we got a fume extractor through an SAS system where we plugged the boot onto the backside, which connected to a six-inch duct, and the fume extractor ran about 750 CFM. Very practical approach, but it worked really well.

We primarily use that for method development. Once we established how we were going to tackle this permanently, we came up with this system that we use today on all of our systems. This is a much more polished, fabricated sheet metal boot with an attached vertical stack that feeds into an eight-inch diameter flex tubing and integrates into the lab HVAC. You can see on the left there how it jumps up. This requires a little bit more infrastructure, but the results is a polished solution that our chemists can safely rely on. This setup also pulls 750 cubic feet a minute and has an open-door face velocity of– at minimum– 100, but generally greater, feet per minute. All right, next on the environmental factor, static, the silent killer.

I recommend using ionization bars or something similar to remove any static charge buildup.

Charge interactions with solvents can be unpredictable and intermittent. From my experience, anything that reads greater than two kilovolts has the potential to disrupt a transfer. This becomes much more problematic with low-volume transfers as well.

Here’s an example of how you can manage some of that. On the left is a KEYENCE static reading device. It can help you identify and isolate the locations of new positive or negative charge buildup in a very localized manner. On the right, we have a picture of the KEYENCE ionization bar installed on LM900, VVP96. Dynamic Devices can help you with that side of it. We run positive pressure nitrogen through our bar, which helps distribute the ions across the deck and neutralize any charge buildup. But you could use a different medium for actually dispersing the charge.

Now polarity is when things get– oh, sorry, I jumped a slide. Temperature management is important. Its significance with solvents is actually about the same as aqueous. So, this is more of just like a good practices slide. You’re gonna wanna make sure that you’re operating in a fairly well-controlled, temperature-managed environment, no huge swings above like several degrees at a time. And generally, you’re gonna be wanting to operate your device in the same temperature environment that you’re validating it in.

Now onto polarity. This is when it gets interesting. Generally, in my experience, I’ve found that non-polar solvents are easier to manage than polar. There’s less stickiness to non-polar solvents, and their behavior is closer to an aqueous solution. As you move up to more polar solvents, the increasing intermolecular forces contributes to a slippery behavior in the tips. Filming is an interesting issue with polar solvents too. It won’t impact the majority of the solvent transfer’s working volume, but that last 2% or less can wick down and drip, pop, or have other unwanted interactions.

Now chemical compatibility is very important. This is like doing your homework before you actually start tackling what solvents you’re gonna use with your tips.

Once again, I jumped a slide, sorry. Here’s an example of a post– five seconds after a dispense of methanol.

You can’t really see the filming. It’s hard to pick that up with a picture unless you’re getting it at just the right lighting, but you could see some droplet formations over here, and that’s just five seconds after your dispense. That filming’s already happening, pulling down. It builds up around the orifice, and then occasionally you’ll just get larger bubbles or even smaller bubbles, and they can just pop, smaller microbubbles. That’s a problem if the lens is actually moving its head around, so sometimes you have to get a little strategic about pausing, waiting. I also have some dispense options I’m gonna display later that can kinda help manage that a bit.

Now, on to chemical compatibility, I jumped ahead. It’s really interesting stuff. So, polypropylene base of OEM Lynx tips is generally compatible with most solvents to the point of not structurally compromising the tips or the transfer, but not all solvents. Like I said, do your homework.

It’s not hard to look up online and find a chemical compatibility chart. Methanol generally has the best compatibility and works really well for a long period of time. Most chemical compatibility charts will show you the X amount of time submerged or interaction over a length of time, like, for example, 30 days of constant exposure. That seems unlikely for most protocols. In our protocols, we’re only interacting with it for maybe 10 to 15 minutes at a time, but we do retain those tips that are not getting contaminated with actual sample, and we reuse those over and over again. So we use them, in some cases, for as long as a month before we change them out, and even then, that’s kind of a conservative estimate. It could go longer with something like methanol. Hexane, you have to get a little more conservative with. It can start warping plastics over time. Constant interaction with polypropylene does have that effect, but once again, we’re not using it for an extended duration of time, just over many consecutive days, maybe a couple times a day. MTBE gets a little trickier, and you’ll see later when we talk about how some of its filming interactions happen. If it had constant exposure, you get even less out of it. So once again, it’s just a reminder, not all solvents are built the same. Just make sure you do your homework on what kind of compatibility you have.

All right, filter tips versus non-filter tips. As I said, we only work with 1250s for the high-throughput of our production.

It actually gets really interesting with these two. So, filter tips reduce the effective cavity space for volatilization. When you think about a filter tip, let’s just say like I have one this big, and then you have the filter right here going up the top, and then from there, you have your working volume somewhere in there. It could be a large volume or a small volume, but that’s your effective cavity, from the point of your working liquid to the filter itself. It stops it. So, the closer the transfer is to maximizing the total tip volume, the more difficult it will be to maintain equilibrium between pre-volatilized leading tip cavity to the liquid solvent in the tip.

And another note is that you might be limited in, to just how you have your system set up. Filter tips are required for using on the VVP system to prevent any of that volatilization from climbing up into the head of the channels of a VVP system. But you could use it on an ST system, and then you wouldn’t have to worry about some of those issues. Now, this is when we start seeing some advantages of the non-filtered tip.

Because a non-filtered tip being the same size, but not having that filter, actually increases the total cavity space you have to work with in  that tip. Now, if you double down and use that on an ST system, you have the whole head space of the back of the plunger there. So that leads to a lot of volatilized area, which leads to a much more stable equilibrium between a volatilized area with the actual working transfer. Now, if you try to use a non-filtered tip on a VVP, wouldn’t recommend that, because now you’re trying to actually volatilize stuff up to the actual filters that go to the head. So, there’s some limitations there on how you set up your stuff. So, your approach of what system and what filter or non-filter type can impact not only how well your transfer works, but just your productivity.

Okay, so these are just some like basic, general best practices I’ve found when working with solvents. If I’m starting from scratch, I’ll usually just start with this, bare bones, and then tweak it from there.

So, you’ll wanna do a triple tip conditioning step.

The reason you’ll wanna do three is because the first one, maybe two, helps with the actual physical tip conditioning, that interaction with the plastic itself. That’s pretty common, even in aqueous. But then that extra two or three on top of that, allows for extra volatilization to the point that it stabilizes that interaction between the volatilized solvent and the working transfer.

If you don’t do this, or you try to skip ahead and do it quickly, what could end up happening is, as you’re transferring your working volume, especially if it’s a large volume, if it’s not stable up inside that tip, or even in like the head space, if you’re working with an ST, more volatilized solvent can go up and actually displace your liquid. So, you might start getting some dripping or some issues just retaining your sample.

Now, if you’re using unfiltered tips on an ST, they perform better due to the large open volume space of vapor after conditioning.

If you’re using a VVP, the other thing you’d have to worry about is making sure you’re not venting or pushing out that vapor volume. You just spent a lot of work doing a triple condition, it takes a little bit of time, don’t just go and vent that out. I know it’s very common with VVPs because it’s advantageous to just use extra dispense pressure because, why not? It’s like infinite. But in the case of solvents, you have to be a little more sparing on how you use that.

All right, moving into the actual solvents we use regularly. The first one being hexane. This one is nonpolar nature. It’s easier to get a clean transfer with hexane. It behaves a little bit more aqueous, but there are some like differences too. So once again, you want a triple tip condition, and we shoot for about 25% higher volume than the transfer volume calls for.

The aspiration, I’ll talk about both VVP and STs, but in some of these it’ll break up a little bit. So, I recommend like about 85 millibars on the aspiration side. And then on ST, you’re gonna wanna do 250 microliters a second. Use hydrostatic pressure correction (HPC) for the functional transfer. Now, that’s optional for the actual conditioning portion. You could speed up the time a little bit, turn off HPC for the conditioning, and then slap it on for the actual transfer itself. And then throw on a 20-microliter trailing air gap too. It just helps with your buildup.

Wicking isn’t as pronounced with hexane, which is really great.

And then getting onto the dispense, you’re gonna wanna kick up the pressure a bit, hit up to 225 millibar, and then on the ST, run 100 microliters a second. Dispense with about 25 to 35% greater volume than transferred– it just helps give a little extra push. And if using an ST system, you can make up the 25 to 30% as a pre-air gap. And then use that as your post push. Additional blowout built in on the VVP can be built in at about 70% of the transfer volume amount.

Now, this is when things start getting a little trickier is when we start moving more polar. So MTBE is polar and much more slick. As with hexane, triple conditioning steps help prepare the functional liquid transfer, so do that first.

Your aspiration settings actually start getting jacked up a bit. You wanna increase that pressure to 225 millibar on the aspiration side. Once again, use HPC on the functional transfer. You don’t have to use it for the conditioning. For the ST, it’s 250 microliters a second.

Optional air gap for smaller volume, I recommend anything greater than 300 microliters. You definitely wanna start throwing in a little bit of extra trailing air gap there.

So wicking is when things start coming into play here. Wicking and tiny drop pops are more of a concern with the solvent and may require some fine tuning on the trailing air gap and travel time holding the liquid. If doing a large volume transfer, you may have to do a second dispense to help push down any accumulated MTBE after the initial dispense, because the minor film pull down can be enough to cause tiny pops while traveling.

Now for the dispense, we’re gonna push that up even, we’re gonna keep that up at the high-pressure point at 225 millibar and then 100 microliter per second on the ST system. You wanna push with 70% greater volume than when transferred on the VVP. Just do a small blowout. A second dispense step can be more beneficial than a quick blowout in the first dispense to deal with filming. I’ll get into more details on the methanol side because it’s kind of optional for an MTBE, and that’s more of just tuning based on your protocol. With methanol, it starts becoming more of a requirement. And you’ll notice the settings for the ST were actually the same for hexane and MTBE. I’ll get into that in a minute, too.

Now, methanol is very polar and slick. It also experiences much more filming. Now aspiration, once again, you wanna run that at a super high 225 millibar and then 250 microliters per second with the ST, with HPC, once again, on the functional transfer. Now the trailing air gap’s optional for smaller volumes, but recommended for greater than 300 again. Now, the reason why the ST systems is using the same push-pull for its aspirations and dispenses is actually because of that stability that’s happening above the tip. Once you do the conditioning step, which isn’t as critical with the pressures going in and out as long as it’s roughly where it should be, once that stability happens, your transfers get much more easier to manage. So you can actually use and reuse the same settings over and over again, versus the VVP where you really have to start tuning those because you have such a small working space for the volatilized area.

Now, wicking and filming are much more problematic with methanol. Avoid as much surface area contact as you can with that tip.

For VVP systems, run dispense number one at about 30 millibars and 50 to 100% more volume than the transfer with no blowout. Go really slow and take that methanol and keep that methanol pulling itself down as it drags. It’ll kind of help remove some of that built-up interior film and drag it off the wall.

Now, we split that up into a second dispense. Once again, going about 30 millibars with the VVP and the same dispense volume as the first. Additionally, though, in the second step, add that same volume in the blowout as well. So, if you know, when you add a blowout step, it defaults to a much higher pressure. What we’re doing is, by adding a second step with its blowout on the inside, once that kind of pulls down a little bit more, it gives it a little bit more time to develop any droplet in the interior there and just punch it out.

This can also help deal with some of the exterior filming up too, which is nice. Now, it’s roughly the same kind of thing with the ST system. It’s just you’re not really changing the pressure as much, and since you’re working with a static set of volume, you can’t just keep adding volume to that blowout. You do have to prep your pre-air gap a little bit to kind of build in that last pushout for a second dispense. The other advantage to doing this in two steps instead of trying to pack it all into one step, I know like coding efficiency, you wanna do it as minimum steps as possible, but it actually, the timing in Method Manager and how it triggers those steps works to our advantage here in that, between the first dispense and the second dispense, there’s an initialization and then ending that step, and it just takes a little bit of time. That time is actually just enough for it to just kind of naturally and organically give you enough time for it to pull down on its own and prep for that second dispense.

Yeah, hopefully you’re able to take some useful information from this presentation and apply it to your applications, either directly or can build off the information as a starting point for any other kinds of solvent transfers and interactions. That’s all I have for you today, thank you.