Use of Centrifugal Evaporation in Polymer De-Formulation Chemistry

By: David Clayton, Stephen Knight

Scientists working at the Great Lakes chemical company site in Manchester, UK have an interestingapproach to new product development. As well as developing their own new products, they are also responsible for analyzing their competitor’s products too. This branch of the giant US-owned company specialises in the manufacture of polymer additives and the analytical laboratory in Trafford Park, as well asproviding analytical services to R&D and manufacturing, specialises in de-formulating their rivals’ products. 

Manufacturers use polymer stabilizers to protect a variety of products from premature fading— from outdoor seats to steering wheels to the colourful fibres used in carpets and upholstery, clothing and ropes. Theyare also incorporated into polyethylene pipes used for water and natural gas transmission to assurelong-term stability. Modern complex additives can help  manufacturers keep their plants cleaner and safer, simplify processes, and boost efficiency by compressing several additives into precisely calibrated granular mixtures. 

Plastics are now replacing metals at a faster pace than ever before. One reason is that special polymerstabilizer additives such as the antioxidants and UV stabilizers produced by Great Lakes can give newpolymer compounds many of their important higher performance characteristics. Such additives can extend the life of plastics and increase their range of use by helping plastic maintain its strength and colour consistency. 

Every day we come into contact with many products that include polymers as they are the basis of all plasticarticles, including laboratory microplates, pipette tips, casings and housings, computers, and packaging. Polymer stabilizers, including antioxidants and UV stabilizers, are an integral part in the success plastic plays in our daily lives. They are added in small amounts to protect plastic performance by preventing loss ofmechanical strength, cracking, crazing, yellowing, and loss of gloss due to day-to-day exposure to air, heatand light.  

The two main groups of stabilizers are Antioxidants and UV Stabilizers and these can sometimes be combined in to one additive. In every case the properties of each additive are closely matched to the stability and performance characteristics desired from a specific polymer. For example, phthalate esters are often used as plasticizer in laboratory plastic ware, but can leach from the surface if exposed to certain solvents, so materials must be carefully selected for each application. 

The antioxident additives are a family based on phenolic resins with long chain aliphatic side chains. Theseare known as “hindered” phenolic resins Applications for hindered phenolic antioxidants include, polyethylene, polypropylene, ABS, polyester, polyamide, rubber, PVC, and styrenes. Within this broad range there are products suitable for specialist applications such as a metal deactivator for wire and cable and aphenolic antioxidant for polyurethane fibre.

The job of the analytical unit at Great Lakes is to find out what type of compounds their competition are using and to compare them with their own products. The problem with these polymer additives is that they are resistant solid granules that do not easily lend themselves to analysis. The process starts with treating the granules with toluene or another suitable solvent. After agitation on a heater/stirrer the additives should all be in suspension or dissolved in the toluene. The solids are separated and the supernatant is then treated according to which additive is being studied. An evaporation step then occurs which was formerly done on a rotary evaporator, but is now performed on a Genevac EZ-2 Plus Personal Evaporator. TheEZ-2 allows the speedy and safe removal of the toluene in a closed chamber. Thanks to the built inrefrigerated cold trap, the EZ-2 captures 98% of the toluene vapour in the condenser and the rest in the catch pot, which is attached to the vapour output. This is a big advantage of the EZ-2 over the old way of working where toluene vapour was continually exhausted into the fume extraction system. In addition, because the EZ-2 is a closed system under vacuum, it presents little or no inflammability risk.

The dry residue is then prepared for reverse-phase chromatography using acetonitrile / formic acid, sometimes with TFA as a modifier, leading to analysis by UV or mass spectrometry. The chromatograph collects fractions into tubes for subsequent analysis. The solvent can again be removed using the EZ-2, which will accept blocks from the fraction collector rack or individual tubes according to preference.

David Clayton, senior analytical chemist in the polymer stabilizers laboratory, says: “What I can now do in 2 ½ days would have taken me seven weeks on a rotary evaporator. This lab is low on manpower, but high on equipment so the EZ-2 fits in well. I particularly like the fact that all the waste solvent is contained and I can supervise its safe disposal. The automatic nature of the EZ- 2 is also nice, the way you can place your samples, start the machine and leave it unattended knowing your samples will be safely dried when you come back. I don’t have to stand there and supervise the evaporation process, so I can get on and do moreproductive work in the instrument suite. As we are only a small team, that’s really important”

This combination of enhanced productivity and increased safety with toxic or flammable solvents has madethe EZ-2 the solvent removal system of choice for many chemistry laboratories around the world sinceit was launched two years ago. The application of the centrifugal evaporation technique to polymer chemistry is a particularly good fit due to the large volumes of organic solvents used in the study of these useful andfascinating materials.

You can find more information on Genevac, The EZ-2 Evaporation system and other applications on thewebsite

Increasing Metabolite Recoveries in ADMET Studies of Animal Models Using a Centrifugal Evaporator

By: Sophie Mcdougall

The effectiveness of ADME/Toxicology studies is, now more than ever, critical to the shortening the time-to-market of novel drugs. An ability to obtain rapidly accurate and reliable data from animal models in order to track the metabolic fate of a novel chemical entity in vivo is now a key skill in the drug discovery industry.

Traditionally, the metabolites are tracked through labelling, whether radioactive or dye-labelled, with percentage recoveries calculated after harvesting of key tissues from the animal model. Whilst there have been moves away from radio-labelling in recent years because of the cost and inconvenience associated with working with “hot” materials, it still has much to offer in metabolite tracking. Large fluorescent dye molecules it is now thought, may act as artefacts and actually alter the metabolic fate of certain compounds by interacting in unexpected ways at the cellular chemistry level. Radionuclides, on the other hand, share the same stereochemistry as their stable isotopes and thus behave in exactly the same manner as far as their pharmacokinetics are concerned. It is thought, therefore, that we are still some way off from the time that we can dispense with radio-labelling for such studies entirely.

One of the traditional problems associated with radio-labelling is the accurate calculation of recoveries. This is essential information for the biochemist attempting to ascertain the metabolic pathways for a new drug and the acceptability of low levels of by-products of metabolism in certain tissues. Thus some attention must be focussed on the methods used to ensure the most accurate and timely calculation of recoveries.

The use of microtitre plates which are coated or impregnated with a scintillator allows the use of automated photometric detection platforms such as the Packard TopCount or Wallac MicroBeta to visualise and count the radioactive decays occurring in each well of the microplate over unit time. Emitted alpha or beta particles from the isotopic label strike a solid scintillant molecule. The particle is absorbed and through a molecular cascade mechanism, a photon is emitted. For each collision event, one photon is produced, so the direct correlation enables quantitative measurements to be made. This is now the standard method for calculating recoveries, as the observed counts per minute (cpm) values from the sensitive photomultiplier detectors can be directly correlated through software to provide concentration data or percentage recovery based on known initial doses.

There are two plate types in use for these studies. The solid scintillant type plates have the scintillator impregnated into the plastic matrix itself. These plates, such as the Scintiplate from Perkin Elmer, are easy to use and give consistent results, but sensitivity is sacrificed, as emitted particles must penetrate the plastic wall of the well before a collision with a scintillant molecule is possible. In addition, scintillant is spread evenly throughout the plastic plate, whereas the sample is typically concentrated at the bottom of each well. In the alternative plate type, a slurry which contains the scintillant and a binding compound is coated onto the inner surface of each well. Perkin Elmer Luma Plates are an example of this type of technology.

Because the sample is now in very close proximity to the scintillant, sensitivity is vastly increased, thus giving more accurate recovery data and reducing the necessary counting time. In addition, it has been found that scintillant can become detached from the well wall and closely mingles with the sample. By drying down the liquid sample it is possible to concentrate the sample/scintillant mixture in a very small spatial area.

The studies we have carried out here at Sanofi-Aventis show that by using a centrifugal evaporator of the type supplied by Genevac Ltd, and using Lumaplates, it is possible to increase the sensitivity and hence recoveries considerably. The Genevac HT-4 Centrifugal Evaporator combines a centrifuge with a vacuum assisted evaporation system to quickly dry down the plates whilst preventing crosscontamination from well to well. We have found in our studies that the extra gravitational force applied to the sample slurry in each well causes the scintillantsample mixture to dry as a tight pellet at the bottom of the well.

The result of such pelleting is that the photomultiplier head captures far more of the emitted photons than is the case when the scintillant is distributed evenly around the well. This extra sensitivity has allowed us to reduce count times, which together with the accelerated drying times given by the Genevac evaporator, has significantly contributed to improved throughput in our department.

Consulting with the supplier and industry colleagues we are aware that similar results have been experienced at our Montpellier, France site and, according to Genevac, at Laboratoires Fournier and Pfizer Global Research in the UK. We decided to further study this effect by comparing results from an older Savant Speedvac 250 machine, which is also in our lab. Our results showed that whilst the Savant unit also increased sensitivity slightly with Lumaplates, the lower g-force meant that the slurry dried more on the walls and less in the bottom of the well. The best results were clearly demonstrated by the higher g-force Genevac machine. The applied force in this case is equivalent to around 500G and this has proved more than adequate for our needs. It is not at all clear that any higher g-force would give better results, as the Genevac seems to apply sufficient force to concentrate the sample adequately central to where the photomultiplier head will have highest read sensitivity.

It can therefore be concluded that the use of Luma plates and Genevac evaporators has therefore contributed to increased efficiency in the ADME analysis laboratory at Sanofi Aventis by shortening both sample preparation times and analysis times. With the direct correlation between early phase critical pathway lengths and overall time to market for new drugs it is easy to see that this system has a relatively short payback time. It also demonstrates that there is still a useful role for radio-labelling in drug discovery.

Comparative Study of Two Different Centrifugal Evaporators in the Commercial Preparation of Dye Labelled Oligonucleotides.

By: K.S.Trevett and C.M.McKeen (Eurogentec S.A)

The first system tested was a 15-year old design known as the SF-60 and supplied by Genevac Ltd, whilst the second system was a modern computer- controlled evaporation system, the Genevac HT-4. The SF-60has been in regular use for routine drying at Eurogentec for many years, but is known to be very simplisticand to have several drawbacks. Chief amongst these is an inability to control the heat input into the samples, protection from direct heating and lack of temperature control. These problems were entirely solved in the sophisticated design of the HT-4 

A variety of oligonucleotide probes were selected for the study, each of which were less than 20 bases in length and were modified at the 5’ and/or 3’ end. Five of each of the following fluorescently labelledoligonucleotides were used : 

5’ FAM

5’ Cy5

5’ Cy3

5’ HEX

5’ JOE

5’ TET

5’ FAM 3’ TAMRA 

Each of the samples were split equally into three alloquots ; control (which underwent no lyophilisation), and the remaining two which were placed in the HT4 and SF-60 respectively. 

The test samples were subsequently evaporated to dryness in their respective instrument. The HT4samples underwent a pre-programmed drying run designed to evaporate water and other solvents, while the SF-60 samples were run until the heat lamp switched off, indicating that all the solvent had evaporated, and thus the run was ended manually.
Following the removal of the probes from the instruments, the oligonucleotides were resuspended in 100µlMilliQ water and vortexed until fully dissolved.

Each of the samples were prepared for mass spectrometry analysis using a Dynamo Maldi-Tof instrumentpowered by a nitrogen laser.
The analyte (3µl) was mixed with cation-exchange resin (3µl), and a hydroxypicolinic acid matrix (3µl), which is necessary for the desorption/ionisation reaction. The analyte-matrix mixture (3µl) was then spotted onto a stainless steel target and allowed to crystallise.

The mass spectra for the Control, HT4 and SF-60 samples were obtained in the positive ion mode, and the acquired molecular weights were compared against the calculated theoretical mass of the test probes toidentify whether the sequence and modification were present and correct.

Following the mass spectrometry analysis, the samples underwent analytical HPLC to verify the presence of the dye and its conjugation to the primer, and to illustrate the sample purity. Each sample (20µg) was suspended in a volume of water (120µl) and filtered before being introduced into a Waters ‘Alliance’separations module coupled to a photodiode array detector. The analytes were subjected to reverse-phasechromatography, with elution facilitated by 95% acetonitrile over a 30 minute gradient.
With the exception of the FAM labelled samples, a significant degradation was observed using the SF60 but no degradation was observed using the HT4. The results of the cy5

Labelled oligos are shown

The results clearly show that the cy5 has degraded to give a product, while the molecular weight of the product matches the structure below, we have not determined the struction of the degradation product.

Cy 5 labelled oligo after drying in the SF60

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