Genevac Evaporators and Concentor Applications - LABRepCo

ApplicationsThere are many applications for Genevac evaporators and miVacconcentrators. Details of the methods developed by some users and thebenefits they have derived from working with Genevac systems are found inthis section. Details of various techniques used in these applications, such asconcentration technology are found in the Technology section.If you can't find what you are looking for, would like more information or wantto discuss how a Genevac system can help you with your application, pleasecontact us. Applications support is normally provided free of charge for thelifetime of the system.Solvent Removal On-lineGenevac publishes Solvent Removal On-line - an applications newsletter fivetimes each year. The content is purely technical and focuses on differentaspects of evaporation technology and applications issues in each edition.The most recent edition is freely downloaded and back copies are archived inthe applications downloads section. Registration for the newsletter is open toall.Clinical & Diagnostic TestingEvaporative sample preparation fordiagnostic clinical tests must beperformed scrupulously. Genevacsystems are used by clinicalbiochemists for a number of testingmethodologies, such as vitamin D orK analysis and in neonatal screening.Mass Spectrometry (MS) is replacingradio-immunoassay (RIA) in Clinicalbiochemistry analysis thus removingthe need to use radioactive reagents.One of the Steroid assays performed

y Hospital clinical labs is for Vitamin D analysis where there is a need toidentify the relative abundance of vitamin D2 and D3. Samples are typicallyextracted into Hexane, concentrated (e.g. using a miVac) and analysed byMS, which avoids ion suppression problems seen with RIA.miVac concentrators have also been used by the Australian Sports DrugTesting Laboratory to improve the testing for synthetic insulin analogues inathletes' blood samples. Using the miVac’s ability to control of conditionduring the concentration step the analyist has more confidence that they areseeing the true blood concentration of potential drugs of abuse in the athlete’ssampleTandem MS is used to identify metabolic disorders which includeaminoacidemias, urea cycle disorders, organic acidurias, and fatty acidoxidation disorders. Samples can be presented as plasma, urine, blood spotsor urine on filter paper. Sample preparation techniques can include solventextraction, acid derivitisation and evaporation prior to reconstitution in asuitable medium for injection to MS. Evaporation of butanolic HCl used forderivatisation using blowdown techniques results in corrosion of the systemespecially pins/needles/jets which can lead to contamination of samples anderroneous results. The EZ-2 with HCl resistance commonly used for thesesample preparation stages.One example would be the test for CAT (carnitine acyl carnitine translocasedeficiency) in neonates, an inborn error of metabolism. Lack of this enzymeprevents the body from converting fats into energy. Labs do the initial test onGuthrie cards (dried blood spots). The cards are punched and extracted with200ul of Methanol. The supernatant is placed into microtitre plates and thendried, typically using Nitrogen blowdown. Samples are then derivatised usingapprox 100ul 3N HCl in anhydrous Butanol and dried. Finally, samples areresuspended in Water & acetonitrile for analysis by MS.Useful Links:Inborn Errors of Metabolism in Infancy and Early Childhood - updatefrom the American Association of Family PhysiciansCalifornia's Experience Implementing a Pilot Newborn SupplementalScreening Program Using Tandem Mass Spectrometry(method published in Pediatrics)Improved Specificity of Newborn Screening for Congenital AdrenalHyperplasia by Second-Tier Steroid Profiling Using Tandem MassSpectrometry (method published in Clinical Chemistry)

DNA & OligonucleotidesGenevac systems facilitate a wide range ofDNA sample preparation and processingactivities, ranging from straightforward smallvolume concentration of DNA pellets prior toPCR, through to high throughput processing ofmany purified DNA or oligonucleotide samples.Where a dry sample of DNA is required,concentration to dryness in a centrifugalevaporator is the ideal method, presenting theDNA as a dry film in the tube of plate well.Freeze drying of DNA samples is notrecommended because the resultant DNApowder is easily blown about or moved byelectrostatic charges.DNA ConcentrationConcentration of DNA from samples containing water or a mixture of waterand alcohols is a simple process, which for most researchers requires themiVac DNA concentrator. Concentration to remove a few hundred microlitresof alcohol from a DNA pellet takes approximately 10 minutes, or less. This isone of the simplest applications of a Genevac system – in a miVac DNAconcentrator, set the temperature, select the method for alcohols or water,and press start.Oligonucleotides & DNA PurificationThe manufacture of oligonucleotides commonly has two steps whereevaporation is required, evaporation of ammonia solutions following synthesis,and then subsequent evaporation of purified samples. Protection ofoligonucleotides from damage during drying is critical, especially where a tagor label has been attached to the DNA. Two studies by Genevac users showthat this can safely be achieved in a Genevac evaporator:Comparing Evaporators for Drying Oligonucleotides - CatherineMcKeen, Eurogentec SAA Rapid, Safe Technique for Drying Oligonucleotides - Dr Tim Watts,Wellcome Trust Human Genome CentreSamples containing ammonia solution should be evaporated using Dri-Pure®to prevent sample loss and cross contamination. When ammonia solution issubjected to a vacuum the ammonia degasses. Degassing must becontrolled or the resultant foam overflows the tube causing sample loss andcross contamination. In the same way that you control degassing of a bottleof soda by opening it slowly, gentle application of vacuum is required, thiscoupled with the high rotors speeds of Dri-Pure (500g) keep every samplewhere it should be. The latest versions of the EZ-2 come with such a methodpre-programmed, and the method is simple to program on the HT-series.Full details are in this application note on drying ammonia solutions.

permeable membrane device (SPMD) developed by the US GeologicalSurvey. This is placed in the river for a period of time, and then any organicchemicals are extracted with solvent, concentrated and analysed as theywould be from a fish.Air quality can be monitored similarly by filtering air for a period of time,extracting the filter, and then concentrating and analysing whatever organicmolecules may be trapped on the filter. This is a key activity in industrialenvironments where PAHs may be a risk factor for workers.When concentrating samples the EZ-2 (small volumes) and Rocket (largevolumes) are ideal tools for concentrating in the analytical laboratory.Coupled with SampleGenie they enhance analyte recovery, reduce intersamplevariability and free workers to do more productive tasks thanmonitoring a concentration system. SampleGenie enables large volumesamples to be concentrated directly into an autosampler vial, eliminating theneed to transfer the sample.Useful Papers:Improving Analysis of Airborne PAHs - Nicolas Falquet, ITGA, St-Etienne,FranceUseful Links:The Prestige oil spill: A laboratory study about the toxicity of the water-solublefraction of the fuel oilNavas et al. published in Mar. Environ. Res., 2006, Vol. 62, Supp. 1, pp S352-S355Food & Beverage AnalysisQuality and safety testing of food andbeverages is an area of growingimportance, be that for exporters,importers or government bodies,accurate analytical results arecritical. In common with many otherapplications, careful samplepreparation is critical, especiallywhen the analyte of interest isvolatile.Genevac systems find application ina number of areas, from testing ofbeverages such as Cognac orWhiskey, to pesticide analysis of fruitand vegetables or vitamin levels incereals. The EZ-2 or Rocket are commonly used, often in conjunction withSampleGenie, because this delivers automation of sample transfer and

gives unparalleled sample recovery and inter-test reproducibility with very lowstandard deviations, as the studies from the Laboratoire départementald'analyses de la Drôme clearly establish.Concentration technology in Genevac systems has been developed withleading analytical laboratories. This, with key technologies like Dri-Pure,ensure that samples are concentrated safely and rapidly. SampleGenie is agreat aid to concentration because the evaporator only evaporates the solventcontained in the large flask, and not the solvent in the vial. The systemdetects the end point of evaporation being when the solvent level enters thevial and, once validated, the method will concentrate the sample to therequired level. If a precise volume is required in the vial, then the samplemust be over concentrated and then made up to the desired level with puresolvent.For protein analysis, e.g. for nutritional studies, the EZ-2 with HCl resistanceoption is ideal. Protein digests often require digestion with 6N HCl which ishighly corrosive to traditional evaporation systems, however, HCl resistantGenevac systems are designed to withstand daily use of concentrated HCl.Useful Papers:Analysis for pesticides: Method development to increase the recovery of volatile compounds byusing EZ-2, a new generation of centrifugal evaporatorEnvironmental Protection Agency of Tuscany (ARPAT), Florence, ItalyEvaluation of the EZ-2 for Pesticide AnalysisLaboratoire départemental d'analyses de la Drôme, Valence, FranceEvaluation of SampleGenie for Environmental AnalysisLeochimica Laboratories, Zoppola, ItalyEvaluation of the Rocket & SampleGenie for Pesticide AnalysisLaboratoire départemental d'analyses de la Drôme, Valence, FranceUseful Links:Determination of Oxygen Heterocyclic Components in Citrus Productsby HPLC with UV Detection. Dugo et al. published in J. Agric. FoodChem., 2009, 57 (15), pp 6543–6551.Enterocin 96, a novel class II bacteriocin produced by Enterococcusfaecalis, isolated from Munster cheese. Izquierdo et. al. published in J.Appl. Environ. Microbiol. doi:10.1128/AEM.02772-08.Metabolism & ToxicologyStudiesMetabolic and Toxicology studies look at thepassage of a drug or chemical into and throughthe body. They range from routine safetyscreening for workers potentially at risk tochemicals through to pharmaceutical drug safety

testing and drug prescription compliance and monitoring in the clinical environment.Genevac systems provide an invaluable tool to researchers in these environments,providing fast, safe evaporative sample preparation free from artefact, contaminationand sample loss.Applications range from the relatively simple concentration or drying of solventextracts from tissue samples to qualitatively analyse for the presence of a drug. Twostudies using a miVac concentrator focus on this work:Better Analysis of Insulin Analogues - Dr Catrin Goebel, AustralianSports Drug Testing LabDetection of Diclofenac Residues in Wildlife - Dr Ngaio Richards,Anglia Ruskin UniversityMore complex metabolic studies utilise labelled drug molecules which are fed tosubjects. Samples are taken, extracted and separated via HPLC. Multiple samples areoften collected in scintillant containing microtitre plates, such as the LumaPlate.These must be carefully dried before analysis via a luminescence reader such as aTopCount. If the plates are crudely dried the labelled molecule does not come intointimate contact with the scintillant providing a false reading. The Genevac EZ-2 andDD-4X have been shown to be excellent for this purpose.Customers working with larger volumes have shown that SampleGenie is auseful tool to help automate their sample transfer process without adverseeffect on sample recovery.Useful Papers:Increasing Metabolite Recoveries in ADMET Studies - Dr SophieMcdougall, Sanofi AventisStudies showed that by using a Genevac HT-4 evaporator and Lumaplates can increase the sensitivity and hence the recoveries as theextra gravitational force applied helps the sample mixture to dry as atight pellet at the bottom of the well.Automation without Robotics - Peter Bennett, Drug Metabolism groupat Servier ResearchEvaluation of SampleGenie in their laboratoryAdvances in Sample-prep for Metabolite Profiling - Dr Flavio Cinato,Nerviano MSTrials with EZ-2 showed that the optimal balance between speed andminimal compound degradation.Improving Productivity in Toxicology Screening - Rob Darrington,GenevacUsing a modern centrifugal evaporator like Genevac EZ-2 providesautomation, user safety, reliability of the analysis and productivityUseful Links:

The use of 96-well Scintiplates to facilitate definitive metabolismstudies for drug candidatesNedderman et al. published in J. Pharm Biomed Anal 34, Issue 3,2004, pp607-617In Vitro Metabolism and Covalent Binding of Enol-CarboxamideDerivatives and Anti-Inflammatory Agents Sudoxicam and Meloxicam:Insights into the Hepatotoxicity of SudoxicamObach et al. published in Chem. Res. Toxicol., 2008, 21 (9), pp 1890–1899Glycerolysis of Acyl Glucuronides as an Artifact of in Vitro DrugMetabolism IncubationsR. Scott Obach published in Drug Metab Dispos August 2009 vol. 37no. 8 1581-1586Pharmacokinetics in Animals and Humans of a First-in-Class PeptideDeformylase InhibitorRamanathan-Girish et al. published in Antimicrob Agents Chemother,December 2004, vol. 48 No. 12 pp 4835-4842A High-Throughput Screening Method to Identify Small Molecule Inhibitorsof Thyroid Hormone Receptor Coactivator BindingArnold et al. published in Sci. STKE, June 2006 Vol. 2006, Issue 341, p. pl3Natural Products ResearchNatural products are researched formany reasons, from biodiversitypreservation through flavour orfragrance research, to finding a curefor disease. Whatever your reason,Genevac systems are used byresearchers who are investigatingthe properties of the naturalenvironment.For basic botanical and biologicalresearch, the miVac concentrator rangeare the mainstay for those extracting andpurifying DNA or proteins for genome and proteome research.Genevac systems are widely used by researchers seeking to extractfunctional molecules from natural sources. A typical process would be to takea tissue (or micro-organism broth) sample and then perform a primary solventextraction resulting in a large volume of crude extract. The crude extract isoften concentrated, for which the Rocket Evaporator is ideal, before beingfractionated to isolate each component. Fractionation may use a system suchat the Sepbox, one or two dimensional chromatography which produces manysamples from one extract. These samples need to be evaporated beforestorage or analysis and may require lyophilisation. Genevac HT seriesevaporators are used for this purpose.

Useful Papers:High Throughput Natural Product Discovery - a paper by Dr StephenPickrahn, IFFUseful Links:High-Throughput Method for the Production and Analysis of LargeNatural Product Libraries for Drug Discovery. Eldridge et al. publishedin Anal. Chem., 2002, 74 (16), pp 3963–3971.The EZ-2 at work in the Kerr Laboratory for Marine Natural Productresearch at University of Prince Edward Island, CanadaSepbox is a registered trademark of Sepiatech GmbH.Parallel ChemistryGenevac evaporators have been designedfrom the outset to be robust and withstand therigors of working in the chemistry laboratory.Systems were used in the early days forCombinatorial Chemistry where routine use of100% TFA has given systems a tough fieldtesting.Whether working in drug discovery, flavour orfragrence research, liquid chrystal synthesis oracademic reaserch, Genevac systems are theideal tool for use when performing parallelchemistry. When working with a single samplethen a rotary evaporator is ideal, whereas, when you have multiple samplesthis would be a bottleneck. Spending time just watching a rotary evaporator isnot very efficient!The main uses for Genevac systems are:Solvent exchange – removal of reaction solvents between stagesRe-salt formation – removal of strong acids of bases when changingsalt forms. Genevac have a special option for HCl resistance whichenables the use of concentrated HCl – ideal for this purpose.Post Purification Handling – removing purification solvents followingnormal phase, reverse phase or chiral separation. Genevac also offerSampleGenie to enable a large volume to be dried directly into a smallstorage which eliminates the need to reformat samples from a flask intoa vial. Reverse phase HPLC fractions may also be fast lyophilised insome Genevac systems.

Depending on the number and nature of samples then one of severalGenevac systems may be suitable – the EZ-2 and HT series are designed forworking with smaller samples in plates, tubes and vials. When working withlarge volumes in flasks – then the Rocket evaporator is the most suitable,taking up to 6 flasks.Useful Papers:Reactions involving HCl and their Evaporation - Dr Induka Abeysena,GenevacDevelopment of an Evaporator for High Throughput Chemistry - DrTom Smith, GSKSurviving TFA - Application NoteHow to Evaporate High Boiling Point Solvents - Application NoteParallel Dry-Loading of Synthesis Samples onto Silica for Analysis - DrNigel Vicker, Bath University Pharmacy DepartmentUseful Links:Efficient Synthesis of gamma-Lactams by a Tandem ReductiveAmination/Lactamization Sequence. Nöth et al. in J Comb Chem.2008; 10(3): 456–459.Plate-to-Plate Fluorous Solid-Phase Extraction for Solution-PhaseParallel SynthesisWei Zhang, Yimin Lu, & Tadamichi Nagashima in J Comb Chem.2005; 7(6): 893–897.The Application of Noncombinatorial Chemistry to Lead Discovery. DrGraham F Smith available via Slideshare.net.Proteins & PeptidesProtein concentration is an issue that thebiological sciences have been dealing withfor years. Many aspects of the study ofproteins and exploitation of their therapeuticvalue requires concentration before and afterprocessing. Each process step can range inscale from setting up a sitting drop of 100nlin microtitre plate based crystallisation studyto purification of the product from 10,000Lfermentation. All of the currently availabletools have their draw-backs whether it isfrom yield loss on membranes to dilutionfrom capture columns.One of the most commonly used techniquesat all scales is membrane concentration. At

the small scale this is usually based on concentrators that fit in a centrifuge orsmall pressure based filers both of which rely on dead-end filtration to pushthe supernatant through a membrane and retain the protein. The small scalemethods used suffer badly from concentration polarisation a phenomenonobserved in membrane concentrations where the protein concentration at themembrane increases due to the removal of liquid, this is analogous to thecake formed in conventional filtration. In larger applications a cross flowmembrane is commonly used where the pressure potential for filtration isprovided by a tangential flow to the membrane surface. This cross flow helpsto reduce the effect of concentration polarisation by sweeping the membraneto clear the retained protein; however, due to constant solvent removal withinthe system there is still a polarisation gradient setup at steady state. When aprotein reaches ultra-high concentration at the membrane interface then gelformation is likely this can reduce the activity of the protein and, if the regimefor re-suspension is not effective, lead to loss of yield. When we couple thisto the non-specific binding effects of most membrane systems it makes this atechnique that the industry is using for necessity rather than optimisedperformance.One alternative on for laboratory scale concentration is to use CentrifugalVacuum concentration where the solvent is boiled from the supernatant undervacuum so that the temperature of boiling is below the denaturation thresholdof the protein. This can dramatically improve both the yield afterconcentration and also the activity of the protein. This technique would not beused on the large scale but finds a niche in the small scale laboratory work,for example preparation of sample prior to screening against a panel ofprecipitants to determine the optimum conditions for crystallisation prior toXRD. For hanging and sitting drop experiments concentrations greater than10 to 20 mg/ml are often required, this is very difficult to achieve in membranebased concentrators due to the limiting factors that have been highlightedabove. Where the sample has been purified using reverse phasechromatography (typically peptides), leaving the sample in water and organicsolvent, this cannot be concentrated using a membrane because the organicsolvents damage the membrane. In a vacuum concentrator the sampleremains in a micro-centrifuge tube and the volume remaining can beaccurately determined by the time of concentration. One potential limitation ofthis technique is that the salt and buffer components of the solution will alsobe concentrated, hence this is ideally suited to be a polishing step that is usedafter a capture column or membrane concentration step and therefore allowsthe sample to reach the very high concentrations needed forcrystallisation. The most commonly used systems for these applications arethe miVac range of biological concentrators or the EZ-2.Peptide SynthesisPeptide synthesis is normally performed using solid phase supported stubwith the sequential addition of protected amino acids. Between each additionstep, the protected terminal needs to be deprotected so that the next acid canreact and join. When the desired peptide sequence is complete, it is cleavedfrom the solid support, usually using a strong acid such as TFA. The peptide

in cleavage cocktail is then dried and purified. Following purification thepeptide can be fast lyophilised in a Genevac HT series evaporator or driedusing a traditional freeze drier.Useful Papers:Better Analysis of Insulin Analogues - Dr Catrin Goebel, AustralianSports Drug Testing LabExamining the effects of sample preparationon data qualityControlled Concentration for MALDI-TOF MS - Steve Knight, GenevacLooking at the effects of sample preparation on MALDI targets incancer resarchUseful Links:Peptide Library Synthesis - protocol published by Jamie M. R. Moore,Guy Laboratory, UCSFQuantitative Proteomics of the Thyroid Hormone Receptor-CoregulatorInteractionsMoore et al. published in J. Biol. Chem. 2004 vol 279, pp 27584-27590A High-Throughput Screening Method to Identify Small MoleculeInhibitors of Thyroid Hormone Receptor Coactivator BindingArnold et al. published in Sci. STKE, 2006, Issue 341, p. pl3Development of small molecules that mimic the binding of ƒÖconotoxinsat the N-type voltage-gated calcium channelSchroeder, Smythe & Lewis, published in Molecular Diversity 2004, Vol8, No 2, pp127-134Post Purification Sample HandlingDepending on the process, and therefore the nature of the samples, thenGenevac provides a number of solutions to facilitate post purification samplehandling, including reformatting.Normal Phase HPLC, SFC or Flash chromatography – removal oforganic solvents is simple in a Genevac evaporator. Evaporation of

solvent mixtures is safe when using Dri-Pure which eliminates theeffects of bumping.Reverse Phase HPLC– evaporation of water and acetonitrile (ormethanol) is the principal application of many Genevac systems. Amultiple stage method has been developed to remove the organicsolvent without freezing the water, remove the water, and then dry anyremaining stubborn solvent. Alternatively, the LyoSpeed fastlyopilisation method can be used in Genevac HT series evaporators.Post Purification Handling following removal of purification solvents thesample typically needs to be reformatting from the fraction tubes or flask, intoa small vial for compound storage. Genevac developed SampleGenie toenable a large volume to be dried (or lyophilised) directly into a small storagevial which eliminates the need to reformat samples from a flask or multipletubes into a vial.Depending on the number and nature of samples then one of severalGenevac systems may be suitable – the EZ-2 and HT series are designed forworking with smaller samples in plates, tubes and vials. When working withlarge volumes in flasks – then the Rocket evaporator is the most suitable,taking up to 6 flasks.Useful Papers:Drying HPLC Fractions - applications noteFast Lyophilisation for HPLC Fractions - Dr Induka Abeysena & RobDarringtonIntegration for High Throughput HPLC Purification - Paul de Fraine,Syngenta & Dr Mike JonesDrying Solvent Mixtures - applications noteUseful Links:High-Throughput Method for the Production and Analysis of LargeNatural Product Libraries for Drug Discovery. Eldridge et al. publishedin Anal. Chem., 2002, 74 (16), pp 3963–3971.