Category: genevac

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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Advances in Sample Preparation for Metabolite Profiling

By: Flavio Cinato – Research Scientist, Nerviano Medical Sciences, Milan, Italy Rob Darrington* – Product Manager, Genevac Ltd, Ipswich, UK Steve Knight – Marketing Manager, Genevac Ltd, Ipswich, UK

Project Outline: 

The aim of the project was to set up a procedure, based on a semi-preparative LC-MS-MS system to allow the determination of the biological activity and the definitive structure of metabolites taken from in vitro assays or in vivostudies. Purified metabolites could then be tested in several ways, including:

·       Tested on target enzymes for activity

·       Tested on non-target enzymes for activity

·       Definitively identified with NMR

·       Entered into toxicological studies

·       Assessed for possible drug-drug interaction

·       Tested for reactivity

·       Used as standards for pharmacokinetic determinations 

There is a significant advantage in being able to extract the sample from the original assay, purify and identify it so that it can then be used for further study. Typically, metabolites that require further study are synthesised followingdetermination, which extends the time taken for metabolite evaluation considerably. To enable further studies to take place, the goal of the project was to establish a system to provide 50 to 100ul of a 1mM solution of purified identified metabolite in DMSO solution. This solution can then be screened for activity against a number of different targets. The procedure was established using a series of Tyrosine Kinases targets and a compound with well-established metabolism leading to two main metabolites, one of which is active, and another which is inactiveagainst specific targets.

Process: 

Samples were taken from the assay and an aliquot presented to the LC-MS-MS system, first running an analytical column to determine the optimal conditions for preparative separation. Next, the bulk of the sample is separated using the preparative column, and the fractions collected. LC solvents were water and methanol, containing 0.1% formic acid as a modifier. After separation the samples were dried and then diluted to known concentration and reanalysed by LC-MS-MS and NMR. 

It is at the evaporation stage of the process that problems were initially encountered. 

First, one of the drying methods trialled blew nitrogen onto the samples to hasten evaporation, however this resultedin much of the sample drying and sticking to the sides of the tube which made dissolution in minimal (50 to 100ul) DMSO very difficult. This lead to use of a centrifugal concentration system which evaporates the dried sample into a small area at the base of the tube, which is far better for redissolving in minimal solvent. 

The second drying issue that was encountered was in screening samples post drying. At first, blank samples (containing no compound) were run through the whole process and via both drying methods showed up false positives in the screening trials, this was attributed to residual modifier from the LC solvents. 

Thirdly, with both of the methods tried, some compound degradation was observed, attributable to poor temperature control of the samples in the evaporator. Comparisons between standards and pilot samples showed lower activity of the samples that has been dried. Clearly further method development needed to be done as sample degradation is unacceptable due to the sensitive and exacting nature of the toxicologists work. 

These issues led Nerviano to search for a more efficient evaporation system, one that concentrated the sample to the bottom of the tube leaving little or none on the tube walls, removed all the modifier from the LC solvents thereby eliminating a cause of false positives in the screens, and did not degrade the compounds with excessive heat.   Trials with the Genevac EZ-2 for the evaporation of the purified parent and metabolite fractions proved very successful with the activity results very close to those obtained with the respective analytical standards. False positives were eliminated and a low extent of compound degradation was observed. Minor variations seen were acceptable considering the intrinsic variability of the activity as determined by high throughput screening. Above and beyond the greatly improved screening results, an additional benefit was that the evaporation time was sensibly lower compared with the other centrifugal system tried, or the blow down method.  

Conclusions: 

The novel semi-preparative auto-purification system including on-line LC-MS-MS analysis was successfullyinstalled and setup. The new system allows the isolation and identification of pharmacologically active compounds and their metabolites. The Genevac evaporator provided the optimal balance between speed and minimal compound degradation during the evaporation step, and eliminated interferences with mobile phase buffers from the LC solvents. The validity of this procedure was confirmed by subsequent biological activity tests and by proton NMR. Validation of sample preparation techniques is as important as analytical methods to because they either may be the source of erroneous results.

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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.

SP Genevac EZ-2 4.0 Centrifugal Evaporators

Effortless evaporation and unrivalled versatility; welcome to the fourth generation of our SP Genevac EZ-2 4.0 centrifugal evaporators. Our SP Genevac EZ-2 4.0 centrifugal evaporators streamline your workflow, with an evaporator model for every application – protecting your valuable samples throughout the evaporation process.

  • Compact design – saves valuable lab space without compromising on quality
  • Touchscreen Interface– simple set-up for even the most inexperienced user
  • Dri-Pure® anti-bumping technology – protect samples from foaming and cross-contamination
  • Versatile – choose from SP Genevac EZ-2 4.0 models: Standard, Plus & Elite