Category: evaporation

The Challenge Of Mixed Solvents

By: SP Genevac

What Do We Mean by Mixed Solvents? 

This month, we are discussing a complex problem that is becoming more and more prominent because of the chemistries specified and the ways of working that are being adopted by many users of evaporation equipment. We’re principally concerned with mixtures in which there is one volatile component and one high boiling point solvent. An example might be methanol /DMSO, or THF/pyridine. What’s more, the two solvents don’t have to be within the same sample. A rack of tubes where each tube contains a different single solvent, will also exhibit the effect described below. Frequently a user who wishes to remove methanol/DMSO will not even mention the methanol when specifying the application, because methanol is “easy” and it’s the DMSO that is considered the challenge. However, this overlooks a rather important technical issue that we will deal with here. 

Solvent Behavior

To understand this problem it is necessary to understand solvent behavior. The graph below shows the relationship between pressure (on a log scale) and boiling point:

The aim with centrifugal evaporation is to boil off the solvent while not overheating the samples. The way that this is achieved with a Genevac system is by keeping the sample holder block or swing (bucket) at or below the maximum temperature your samples can safely withstand. If the solvent
boiling point is less than this temperature, heat will flow from the sample
holder into the samples and the solvent will boil. If the boiling point goes above the block temperature, boiling stops completely.

It is not sufficient to have the boiling point of the solvent just below the block temperature, because with only a couple of degrees difference, the heat flow will be so slow as to make the evaporation of all but a tiny amount of solvent unfeasibly slow. For example, suppose that your maximum temperature is 40 o C (and hence you never let the sample holder block exceed that), and the solvent is DMSO. If you set the pressure to 2 mbar, the BP of DMSO is 38.5 o C, so you’ll have a measly 1.5 degrees difference to drive the heat into the solution from the block. You could raise the temperature of the block, but then when any one of the samples dries, it will end up above the maximum 40 o C. So that would defeat the object. Clearly in this case it is necessary to run at a lower pressure. At 1 mbar, the DMSO would boil at approx. 28 o C (giving 12 degrees difference, a clear improvement) and at 0.6mbar it would boil at approx. 20 o C. So far, so good. Better vacuum = faster DMSO removal. Certainly nothing new there. But now lets consider the more complex task of removing methanol/DMSO.

A game of two halves

Before we get onto the main feature today, lets digress momentarily and consider another challenge we face with this particular solvent combination. The methanol is more volatile. It will be removed before the DMSO. (No surprise there.) DMSO freezes at 18 o C. This means that if we don’t watch out, we’ll freeze the DMSO while removing the methanol. If we try to remove the methanol at too low a pressure, the whole solution will drop significantly below 18 o C and some of the solution may freeze. If it does, heat flow through the solution by convection will be interrupted and the drying will slow down. The alternative, however, is to run with a pressure so high that the methanol does not boil significantly below 18 o C. For example, at 110mbar,methanol boils at 18 o C. This would leave only 22 o C temperature difference driving heat into the methanol. Since it has quite a high specific latent heat of vaporisation (meaning it takes a lot of heat per unit mass of methanol to boil it), this could itself result in delays. So what pressure should we pick for removing the methanol? Well, it depends. Whether freezing occurs at all, and whether (if it does) it significantly restricts the heat flow, will depend largely on the sample format (e.g. vial or tube) in question, and so trial and error may be required to choose the fastest setting. (Note of course that the other consideration when choosing a good operating pressure for methanol is that if the BP is too low the condenser will perform poorly and solvent recovery will be lower. 12 mbar is probably a sensible lower limit) But enough of this digression. Back to the main story. 

Removing the 2nd Solvent

We jump forward in time now to when the methanol has been removed from the samples and we now wish to remove the DMSO. Whatever we chose as a good pressure for methanol, we can all agree that, right now, we need the very best level of vacuum our system can achieve. And here is where the real problem becomes apparent. As the system pressure falls, so does the boiling point of all the methanol that is sitting in the condenser. As we approach 2 mbar, the boiling point of the methanol is -42 o C. Much below that and the methanol ends up with a boiling point below the temperature of the condenser. That is to say, the condenser is actually warm enough to re-boil the methanol. This results in a huge volume of vapour being produced, which the pump will have to remove before it can get the pressure in the chamber any lower than this.

The vacuum pump on a system like this is predominantly there to remove the air from the chamber at the start of the run, and then hold pretty good vacuum with fairly low flow. It is certainly not made to pump out hundreds of thousands of litres of methanol vapour in a hurry. The flow rates required would be enormous. Some manufacturers’ pumps would actually be damaged by this much solvent What this means is that there will be a substantial delay (which could be many hours) before the methanol has finally all been removed from the condenser, and before that time, the pressure in the chamber will simply not fall much below 2 mbar. And as we have already established, 2 mbar is not much use for removing DMSO at a sensible speed. This is clearly not ideal. Why not use a colder condenser? Cascade condensers designed to operate over the range -70o C to – 90o C are relatively common. However, a cascade condenser capable of providing the condensing power of the equivalent Genevac system would absorb 4 times the input power and occupy 2-3 times the bench space. Also, a -90 C condenser would not be suitable for the large range of solvents you use. Solvents like Water and DMSO would freeze within the condenser inlet aperture, eventually forming a plug.

Finding Another Way Out

We are now faced with a few choices. One, as we have already mentioned, is to raise the temperature so that the DMSO can be removed at the 2mbar level we are currently stuck with. But if you don’t want to take the solution above 40 o C, this is not an option at all. The second one is to simply wait. Eventually, the methanol will all be gone, and then the pressure will drop and we’ll get the DMSO remaining in the samples to boil. But it will be a long wait. The third one is to intervene. That is, when the methanol has all been removed, when the problem is just beginning, stop the run and drain the condenser. Then restart the run and the vacuum level will no longer be affected by the methanol. DMSO should be removed just as rapidly as if it had been the only solvent. This is pretty inconvenient, although it’s the best option so far. But what if the run is an overnight run ? This could be a real pain. There is a fourth choice. Namely a system that can remove the volatile solvent from the condenser automatically, partway through the run, without you having to intervene.

The TA

The system in question is the Genevac “Triple pot Automatic” condenser (known as the TA for short), This is available for the HT8, HT12 and the larger Mega range of evaporators. The principle is simple. There are two primary pots (before the pump) and the system alternates between them. First, pot 1 is active and solvent leaving the chamber condenses in it. Pot 2, meanwhile is “offline”, defrosting and draining as required. Then, when pot 2 is ready for use, the pots exchange tasks, and pot 1 takes some time out to defrost and drain while pot 2 starts catching the solvent. The TA will alternate between pots many times during a typical run, so not long after the last of the methanol is all captured, the next switchover will happen, the volatile solvent will be removed from the system entirely, and rapid DMSO removal can start. The bonus with this system (and this is actually the reason it was initially developed) is that you never have to lose valuable evaporation time waiting for a defrost. The system can be unloaded, reloaded, and started again immediately.

In Summary

The issues outlined in this article apply equally to all the following situations: 

-When a volatile solvent and a high BP solvent are mixed in the same solution 

-When the sample load is made up of many tubes with different solvents, and some tubes may be volatiles while others may contain high BP solvents. 

-When a sample solution which contains a volatile solvent component needs to be taken to full vacuum at the end of the run to achieve good dryness, even though the last solvent removed is not theoretically a high BP solvent (for example some HPLC fractions containing ACN/Water)

The TA condenser, meanwhile, is also useful when: 

-The time lost defrosting would seriously impact throughput

-The total solvent load is such that it exceeds the volume capacity of conventional condensers.

-The evaporator is integrated into a robotic system; in these applications the condenser must be constantly available.

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Surviving TFA

By: SP Genevac

What’s So Special About TFA?

TFA (Trifluoroacetic acid) has several properties that make it very important that you take the right precautions when evaporating it, in order to get optimum performance and prevent damage.

Firstly, it’s an acid. This means that if not handled with care, it will corrode components of a system over time. Next, it is quite volatile (with a boiling point / pressure characteristic pretty similar to methanol). In combination with other solvents it can present a quite “bump-prone” mixture. Thirdly, (and perhaps most infamously), it exhibits “creep”. In simple terms, this means that liquid TFA can climb the sides of a vessel to the height that any TFA vapor reaches. What is more, it can carry with it dissolved compounds, which may then end up actually leaving the vessel. Fourthly, and particularly worryingly for users who run into this problem, it can seep through polypropylene (including some brands of 96 well microtiter plates). This is not evidence of a failure to seal the bottom of the well during molding of the plate – it actually goes through the plastic itself, due to it having an extremely low surface tension. TFA in sufficient concentration will also attack silicone (both silicone rubber seals and the special oil in a standard Genevac Cole pump). Finally, it can soften a PTFE coating on an item that is exposed to liquid TFA for any great length of time.

So how can we deal with all this?

It’s quite simple when you know how. All you need to do (for an HT system) is:

  1. choose a sample vessel it will not leak through
  2. prevent creep occurring before evaporation begins
  3. evaporate it without bumping, without forming any condensation anywhere and without any liquid being thrown or sprayed onto the PFTE coated chamber
  4. ensure that the TFA vapor is always re-captured in the first condenser pot and not sent through the pump in any great quantity
  5. ensure that when defrosting the condenser the TFA does not creep back into the evaporator

Choose The Right Hardware

Polyprop plates

It has been conclusively demonstrated at a number of user sites that TFA travels through the 96 well polypropylene plates. The simple test to prove this goes as follows:

Load the plates into a suitable Genevac swing (i.e. with a metal base beneath the plate*). Sandwich a piece of filter paper between the base of the plate and the metal swing. Run the machine for a short while (long enough to get up to full rotational speed for 30 minutes) with just TFA in each well of the MTP. If when you open the system up there are 96 telltale marks on the filter paper, this indicates TFA has passed through the bases of the wells. There is no other mechanism by which this pattern could be made on the filter paper.

Experience so far seems to suggest that conical wells that taper to a point are most vulnerable to this effect. To date an official list of good / bad plates has not been compiled though Genevac would welcome the feedback of any users who have experience with particular brands.

Sometimes this effect comes to light in a different way – the user is running the plates in Genevac “open swings” (not advised*) with the lamps shining directly on the base of the plates. When the plates leak, a set of 8 lines can be seen all around the chamber for each level on the rotor (i.e. 3 sets of 8 lines in an HT12). What is actually happening is that liquid TFA is leaking through the wells, flying off at a tangent, hitting the chamber, and (over time) softening the PTFE coating. Though this softening is to some effect temporary, the continuing impingement by drops of liquid TFA at 60 mph (while the coating is soft) starts to damage the chamber coating.

The Right Swings

The effect known as creep results in TFA climbing up the walls of a vessel. This will happen in time if samples are just left in a microtiter plate. Therefore, one of the best things you can do to prevent problems is not to leave samples sitting in plates any longer than you have to. For example, when loading samples including a lot of TFA into an evaporator, make sure to start the run as soon as possible afterwards.

If creep still occurs (i.e. TFA in small quantities climbs out of the wells) there are special swings available from Genevac. These are designed to catch small amounts of TFA as it leaves the plate (or other vessel) and retains it until it turns to vapor, rather than let it be thrown against the chamber wall as liquid droplets. Both the Fast-Stack deep well (used for carrying two plates per rotor position) and the SideBridge swing are available with this modification. Look for “anti-creep” variants in the Accessories Brochure.

Choose the right lamp glass

One effect of splattering TFA (and some compound) onto the walls of the chamber is that you will also find it gets splattered onto the small rectangular windows that separate the infra red lamps from the chamber. There is a potential failure sequence that goes like this:

  1. TFA and compound gets splattered on the glass.
  2. Solvent evaporates leaving a dried deposit.
  3. Deposit gets hot, particularly in center of the lamp beam where intensity is greatest.
  4. Compound carburizes and turns black.
  5. Black patches absorb even more lamp heat.
  6. Heat leaves glass by conduction from hot spots to edges. This means there is a temperature gradient.
  7. This means that there is differential expansion.
  8. The glass breaks, causing (at best) downtime or (at worst) an implosion of glass into the chamber.

It is possible to get a different glass arrangement, especially for use in systems suffering from this problem. The system has two different layers of glass. The inner most (facing the chamber) has an extremely low coefficient of thermal expansion. This means that the list above looks more like this:

  1. Heat leaves glass by conduction from hot spots to edges. This means there is a temperature gradient.
  2. So what? (**)

This dual glass system is retrofittable to existing evaporators, or can be specified at manufacture time. There is a third option, however:

  1. TFA and compound gets splattered on the glass
  2. Solvent evaporates leaving a dried deposit
  3. Conscientious operator wipes glass clean before next run.
  4. Problem solved.

Note that the glass can be cleaned with a lint free cloth and some acetone. It is VERY IMPORTANT that you do not press on the glass while doing this. It sits on a sprung seal and pushing against it can push it off the seal and allow dirt to get into the seal.
(**) It is of course good practice to keep the glass clean whatever type you have.

Program Your System Optimally

Prevent Bumping

If you have a mixture of solvents including TFA, you will certainly want to select Dri-Pure™ as an anti-bumping precaution. If TFA is the most volatile of your solvents, and if you have “Variable Dri-Pure™” enabled on your system, you can shorten the vacuum ramp.
(Contact your sales rep for a document explaining how to do this to best effect). If however the TFA is mixed with another, more volatile, solvent (like methylene chloride) then the standard vacuum ramp may be appropriate.

Give the Condenser a Chance

As with all volatile solvents, it is important to choose the correct pressure to optimize the way the condenser deals with the vapor.

For example, if you run TFA with a target pressure of 0.5 mbar, the following happens. At 0.5mbar, the boiling point is –60 deg C. This means it is too cold to condense in the condenser first stage and must go through the pump and be caught in the second stage of the condenser. As vapor is being produced in the evaporator far faster than the pump can pump it out, the pump becomes a serious bottleneck, and a backlog of vapor builds up. This means the target pressure is not achieved, and will probably not go much below 4 or 5 mbar for some time. Since the target is 0.5 mbar, the pump continues pumping in an attempt to lower the pressure, and thus TFA is sent through the pump in great quantity. The pump will suffer over time.

If you instead choose a higher pressure, (12 mbar is a good figure) the boiling point will be –20 deg C, and the condenser will be able to condense the vapor at the same rate at which it is being produced. This means that the target pressure can be achieved, and hence the pump can shut its inlet valve and let the condenser deal with the vapor. This will extend pump life.This means overall that the amount of vapor caught in the first condenser pot is increased, and the amount that travels through the pump to the second pot is decreased.

Of course, there will be some cases where the TFA binds tightly to the compound and so it is impossible to dry the samples completely without dropping the pressure lower than 12mbar. Here’s where some experimentation comes in. Lets say all the TFA that can be removed at 12 mbar is captured in the condenser, and the condenser temperature has dropped back to –45 degrees, but the samples still have some un-evaporated TFA in. It is possible to then drop the pressure to perhaps 4mbar without significant TFA from the first condenser pot migrating through the pump, which may remove the last bits of TFA from the samples. But if you drop the pressure to 2 mbar and below, you will start to see significant amounts of TFA travelling through the pump to the second pot (and beyond).

If dropping this lower is the only way that you can get your samples fully dry, then of course, it has to be done.

Initial Transients

So far the situation we’re describing relates to “steady state” drying once the pressure in the chamber has settled down. But there is more to consider. At the start of a run, there is often a fairly rapid onset of boiling producing a lot of vapor when the working level of vacuum is first approached. At these times, the condenser can be temporarily “overwhelmed”, which means that it cannot condense the vapor at the same rate it is being produced (and so some of that vapor goes through the pump). This situation can be prevented by the use of a gentle vacuum ramp, making the onset of boiling less severe.

Vacuum ramping is not just for preventing bumping!

You will know if the vacuum ramp is sufficiently gentle if the system manages to pretty much achieve its target pressure all the way through the ramp. If not, then the condenser is not condensing everything as it arrives, and so it is going through the pump.

Pump Life

For small quantities of TFA, run at the right pressure, the pump will not suffer unduly. However, if strong solutions (20% and more) are run regularly, there will inevitably be some effect on the pump. The silicone oil in the Genevac Cole pump will degrade in time with exposure to a lot of TFA, and in these circumstances Genevac recommend that the pump be “upgraded” to the “CPC” specification. This simply means that a different (more expensive) oil is used and the pump rotational speed is changed to reflect the different viscosity of the CPC oil.

This upgraded can be carried out in the field to an existing pump.

Caring for your condenser

Perhaps the most obvious bit of advice about the condenser is “remember to empty it”. Some users do forget and though the system still (eventually) dries the samples by pulling the solvent out through the pump, the drying times are very long. Therefore it is only likely to be on longer than necessary overnight runs that an un-emptied condenser goes unnoticed.

Samples will only get dry in these circumstances if the run is programmed for a period way longer than it really needs to be, and that in itself might be said to be bad practice. Even if you have all night to do a run, you should still aim to program the system to run only long enough to get the samples dry, because needless prolonged running ages a system prematurely. A system that does an 8 hour run every night then switches off for 8 hours will live 50% longer than one that runs right through to morning for no reason.

Full condensers often occur not so much because they were not emptied, but because they were not emptied fully. This might be because the condenser was not fully defrosted before draining, or because some sort of residue or sludge has built up which is obstructing the draining process. Though not that common, this is still something you should take steps to prevent.
On a system running mostly TFA (with not much of any other solvent), it is wise to “flush” the condenser periodically to prevent such build-ups. To do the best job of this, use a flushing kit. This is a factory option for an HT-4/DD-4 but can be retro-fitted to external condensers (i.e. HT-8/12 and Megas). It consists principally of a “probe” which reaches down into the condenser pot and is used to spray methanol onto the walls of the pot as part of a semi-automated cleaning process.

A slightly less effective, more time consuming, but still worthwhile method (on a system without a flushing kit) is to evaporate a full “dummy run” of pure methanol. On an HT-8 or 12, this should amount to a litre or more (300 mls on an HT-4) and the run should be carried out at a pressure of at least 12 mbar to ensure the methanol is all condensed in the first condenser pot, where most rinsing is required.

IMPORTANT – How to defrost

This section applies equally to Series 1 and Series 2 systems.

The process of defrosting a condenser involves heating the contents of it until all the ice is melted. On a standard Genevac condenser this means that heat is applied for a

predetermined length of time. With TFA, however, there is a risk that you may end up producing hot liquid TFA in the condenser before the defrost cycle is over. This potentially leads to accelerated creep, meaning that the TFA that you have already trapped in the condenser can make its way back up the tubing into the evaporator chamber, causing quite a mess.

There are two ways to prevent this. On a new system that is specified in advance to be for TFA use, a special “defrost” temperature sensor is fitted by Genevac which ensures that defrost heating stops when the liquid in the condenser rises above a certain temperature. This means the TFA can never get hot enough to cause a problem.

But this is only available as a factory option, not as a retrofit.

For systems without this extra sensor, the best way to defrost with TFA is to open the drain valves before commencing the defrost. This ensures that as soon as liquid TFA is formed, it drains out of the condenser, and there is never a large body of liquid TFA being heated.

This technique should only be used when TFA is present in the condenser, not with other solvents. If you are using other aqueous solutions you will find that ice in the condenser thaws a lot quicker if surrounded by warm water, than if you have a dry lump of ice touching the warmed condenser walls in a few points. Hence you should keep the drain valves shut during defrost when not using TFA.

On an HT-4 and DD-4, it is a bit more awkward to defrost with the drain open. On an HT- 4, open the lid, open the drain valves, then close the lid far enough to see the screen (and hence navigate the defrost menu) but not far enough that the lid re-closes the drain valve.

On a DD-4, lower the lid so that you can get at the defrost button, start the defrost cycle, then leave it fully open again, ensuring the drain valves are not closed by the lid.

Automatic Shutdown

The Genevac series 2 systems have a feature called “Autoshutdown”, which ensures that after a run has finished, the system goes into a “sleep” mode. This is the same state that the system boots up in, and the same state the system goes into when you hit STOP twice from the main menu.

The “sleep” state means that
• The condenser is no longer refrigerating
• The pump switches off (after 30 mins of purging to ensure no vapours remain which could condense in the pump).

People often ask “why not go straight into defrost automatically, as well?” The answer is that your samples are still in the evaporator chamber, and the last thing you want is for solvent vapour to re-evaporate from the condenser and condense on your samples.

Autoshutdown is a “system option” (not a “program option”), which means that if it is enabled, it is enabled for all programs. It cannot be selected on a program by program basis. It can be found by choosing “Options” on the main menu screen and then choosing “options” again. Hitting the enter key toggles its status.

It doesn’t matter (wear wise) that the evaporation is over at midnight, if the system continues to run till 8am when you return, that’s 8 hrs more wear. Autoshutdown therefore makes your system live longer.

Another advantage of Autoshutdown is that if the condenser has been off all night it will have got most of the way back to room temperature, and you might find that it is almost ready to drain without any defrost being required. Of course, you should verify that this is the case before relying on it, because not fully draining a condenser before restarting will (over a few runs) potentially lead to a cumulative problem. To verify if overnight shutdown is thawing your condenser, simply drain it in the morning, then do a defrost cycle and see if anything else comes out. If not, the overnight shutdown was enough to thaw it.

Under normal circumstances Genevac would always recommend that this option be enabled, particularly if the system is run overnight.
The one exception to this rule is with TFA.
You might enable Autoshutdown, then find that you open the door of the evaporator and are met with TFA fumes (or worse, liquid TFA in the chamber) the morning after the run, because of creep from the condenser.

In this case, you may find it preferable to disable Autoshutdown, so that the condenser continues to hold the TFA safely refrigerated in the condenser until you return to the system the next morning.

Look after the Seals

Don’t dispose of TFA in the Arctic.

But seriously, nitrile seals can swell on prolonged contact with TFA. In extreme cases, a swollen door seal will prevent the door from closing and then the evaporator is unusable until the door seal is replaced. If there are any spillages of solvent onto the door seals, (or lid seals on an HT-4/DD-4, or the window seal on an HT-12 door) clean them up with methanol immediately. Ensure all staff are aware of this, and you should not have a problem.

You might even want to consider obtaining a spare door/lid seal just in case, if you frequently suffer spillages.

Finally, some tricks of the Trade

Often evaporation does not go the same when there are compounds present as when you are “practising” using just solvents. For one thing, it is often slower.

Another example is as follows. Whereas you might expect a volatile solvent like TFA to be evaporated before water (in a TFA / water mixture), it can be the case that significant amounts of TFA remain bound to the compound at the end of the evaporation of aqueous TFA.

In some cases the problem can be resolved by using other additional solvents. Toluene is popular for assisting in the removal of TFA, and any experience gleaned in this area from using a rotary evaporator will be relevant when using a Genevac.

Another lesser known fact is that the whereas pure TFA suffers creep, addition of water to the mixture reduces the creep (up to about 40% water by which time it has pretty much stopped completely). So if it is possible to run with an aqueous mixture, this will be better from the point of view of creep.

Surviving TFA – A summary

As we have already seen, correct programming will reduce both the condensation and the amount of TFA that travels through the pump (or travels back into the chamber later). Careful choice of microtitre plates and (if necessary) sample swings will ensure that the chamber is never sprayed with liquid TFA. Defrosting the condenser with the drain valve open and also flushing it on a regular basis will greatly reduce problems. Wiping the lamp window glass clean if anything accumulates on it will prevent build up of hard baked on deposits that could cause the glass to break. And by cleaning up all spillages you can prevent swelling seals that could cause the system to lose vacuum.

But if there’s one overriding thing to remember, it is this.
If you have a problem, contact Genevac.
• If you need assistance setting up the system, email service@scientificproducts.com.
• If you think there is a fault, call the service department.

Don’t suffer in silence.

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Using Innovation to Enhance Revelation: SP Genevac EZ-2 Optimizes Screening for Novel Active Antimicrobial Compounds

By: Dr Jayneil Patel, Head of Metabolite Interactions & Dr Induka Abeysena, Portfolio Manager – SP Genevac

Introduction

Penicillin, the first antibiotic, was discovered over 90 years ago and revolutionized medical potential. Before the advent of antibiotics, bacterial infections were a leading cause of death and the downfall of numerous surgical procedures. The capacity of antibiotics to enable patients to recover from severe infection and reduce the risk of infection of surgical wounds took the medical world by storm, leading to their routine use in both the management of disease and in prophylaxis.

However, the resultant widespread overuse of antibiotics has led to many bacteria acquiring resistance to several of the most potent antibiotics available. This threatens the future of medical success as even the most efficacious antibiotics have been rendered inactive against life-threatening bacterial pathogens. Hundreds of thousands of lives are lost every year because of antibiotic-resistant infections.

For many years, it has been known that the development of novel antibiotics that can kill drug-resistant bacteria is essential to maintaining modern medicine standards and mitigating the risk of returning to a pre-antibiotic era.

So far, traditional approaches to antibiotic discovery have failed to generate the new drugs needed to treat antibiotic-resistant infections. No new classes of antibiotics have been discovered since the 1980s; all antibiotics new to the market in the past three decades have been variations of existing drugs, created using synthetic biology methods to achieve greater efficacy and improve yields. With a renewed sense of urgency, researchers are once again looking to nature for inspiration to inform the development of new antimicrobial agents. 

Discovering and developing genuinely new antibiotics requires challenging science methodologies and is time-consuming and expensive. The start-up company, Bactobio, is not deterred by these obstacles and is dedicated to discovering novel compounds, including antibiotics, from bacteria. Evaporation is a crucial step in their search methodology and has been streamlined using the SP Genevac EZ-2 centrifugal evaporator.

SP Genevac EZ-2 Centrifugal Evaporator

The EZ-2 solvent evaporator (EZ-2) is part of the benchtop range manufactured by SP Industries, an industry leader in biopharma processing and life science equipment including centrifugal evaporation and concentration processes. First launched in 2002, the EZ-2 Series is now in its fourth generation and represents the pinnacle of parallel evaporation. It is compact and has been engineered to be compatible with many common organic solvents, including corrosive acids. In addition, it is easy to use and boasts several clever features that enable efficient solvent removal.

The EZ-2 incorporates various technologies to ensure that temperature and pressure are precisely controlled to protect valuable samples and can be programmed to function without the need for operator observation. Smart evaporation software continually monitors and directly controls sample temperature to avoid overheating.

Furthermore, temperature measurements are taken from the sample holder, so no contact with the sample itself is required, preventing the risk of contamination. In addition, several samples can be dried at once without risk of sample cross-contamination due to the Dri-Pure® anti-bumping technology.

The EZ-2 remains easy to use despite the incorporation of sophisticated technology. Once the samples are loaded and the desired program is selected, the system can be left to operate unattended. The auto stop function ensures that the EZ-2 only runs as long as is needed for evaporation to stop. In the EZ-2 Plus model, the removed solvent is contained in an easy-to-empty jar meaning the sample can be retrieved at a time convenient to the user.

Typical Workflow At Bactobio

Bacteria are the most genetically diverse organisms on the planet and produce an equally diverse range of chemicals. Indeed, research in bacteria has led to numerous important discoveries. In order to isolate new chemicals in bacteria, natural bacterial samples must be cultured in a laboratory. However, cultures have only been achieved for <1% of known bacteria. There is, therefore, a vast potential for discovering new compounds in the remaining 99% of bacteria. Bactobio is unlocking that potential using a novel technique to culture previously unculturable bacteria.

The Bacterial Community Cultivation platform (BACCU) from Bactobio directs the evolution of unculturable bacteria, enabling the bacteria to evolve to become culturable in a laboratory setting. Once in the lab, Bactobio screens these bacteria for valuable metabolites. 

To date, Bactobio has boosted cultivation rates from less than 1% to over 15% – allowing for the creation of a library of novel bacterial species for downstream metabolite screening.

Bactobio harnesses evolution by combining synthetic and natural factors to direct the bacteria’s evolution. Through the use of next-generation sequencing, genetic diversity is tracked to study the biomes and minimize diversity loss. Bactobio closes the loop on the iterative process using literal and machine learning to improve the directed evolution, therefore gaining access to more diversity. A key goal is to discover novel antibiotics that are effective against resistant bacteria. Previously uncultured bacteria are isolated from soil samples collected from a diverse range of environments and screened for genetic novelty. These are then cultured in the laboratory and screened for antibiotic activity.

The team use the EZ-2 to dry extracted metabolites from liquid culture as part of an activity-guided screening process. This process, using a combination of media optimization methods, HPLC and mass spectrometry, takes them from cultured species to active compound(s). At each stage of the workflow the samples are dried down from a range of different solvents using the EZ-2.

How The SP Genevac EZ-2 Has Increased Efficiency

Bactobio has been using the EZ-2 since it was formed in 2020 and is confident that it has enabled increased productivity and efficiency.

“The SP Genevac EZ-2 is working very well and is a critical and reliable instrument in our workflow. After 10 months we are already looking to order a second SP Genevac instrument to help meet our increased throughput, which exemplifies our favorability for the instrument and the crucial role it plays in our compound identification pipeline. We also appreciate the ease of use and zero sample prep time which greatly adds to our efficiencies.”

Using the EZ-2, many samples can be dried quickly and in parallel, resulting in a high screening throughput. This is further helped by the lack of sample preparation requirements for EZ-2 drying and the ease of set-up due to the capacity to store pre-set programs. Furthermore, the samples can be left drying overnight, so productivity in the laboratory during the day is maximized.

Evaporation can be a harsh process and maintaining the sample integrity of these novel compounds during evaporation is very important, to avoid losing valuable samples and repeat experiments. Precise sample temperature control plays a key role in this, and the innovative technologies built into the SP Genevac EZ-2 are able to evaporate samples using controlled conditions to protect the integrity of these novel compounds.

References

  1. Wright GD. Nat. Prod. Rep 2017;34:694-701.
  2. Bactobio www.bacto.bio
  3. EZ-2 Series. SP Scientific. (2019). https://www.spscientific. com/Products/Centrifugal_Evaporators___Sample_ Concentrators/Genevac/EZ-2_Series/EZ-2_Series/
  4. Suitor JT, et al. One-Pot Synthesis of Adipic Acid from Guaiacol in Escherichia coli. ACS Synthetic Biology, 2020; DOI: 10.1021/ acssynbio.0c00254
  5. Moore JM, et al. Use and discovery of chemical elicitors that stimulate biosynthetic gene clusters in Streptomyces bacteria. Methods Enzymol. 2012;517:367-85. doi: 10.1016/B978-0-12- 404634-4.00018-8. PMID: 23084948.
  6. Bandyopadhyay S, et al. Discovery of iron-sensing bacterial riboswitches. Nat. Chem. Biol., 2020, DOI: 10.1038/s41589- 020-00665-7

Acknowledgements

SP Industries would like to extend thanks to the team at Bactobio, for their significant contribution to the content and creation of this article.

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