Waters ACQUITY UPLC BEH Column.pdf

ACQUITY UPLCBEH Column Care and Use InstructionsThank you for choosing a Waters ACQUITY UPLCBEH column.The ACQUITY UPLCBEH packing materials were designedspecifically for use with the Waters ACQUITY UPLCsystemand are manufactured in a cGMP,ISO 9002 certified plantusing ultra pure reagents.Each batch of ACQUITY UPLCBEHmaterial is tested chromatographically with acidic,basic andneutral analytes and the results are held to narrow specifica-tion ranges to assure excellent,reproducible performance.Every column is individually tested and a PerformanceChromatogram and Certificate of Batch Analysis are providedon the eCordintelligent chip.ContentsI.Getting Starteda.Column Connectorsb.Column Installationc.Column Equilibrationd.eCordInstallatione.Initial Column Efficiency DeterminationII.Column Usea.Sample Preparationb.pH Rangec.Solventsd.Pressuree.TemperatureIII.Scaling Isocratic MethodsIV.Column Cleaning,Regenerating and Storagea.Cleaning and Regenerationb.StorageV.Introducing eCordIntelligent Chip TechnologyVI.Additional Information12004 Waters Corporation.I.GETTING STARTEDEach ACQUITY UPLCBEH column comes with Certificate ofAnalysis and a Performance Test Chromatogram embeddedwithin the eCordintelligent chip.The Certificate of Analysis isspecific to each batch of packing material contained in theACQUITY UPLCBEH column and includes the gel batch number,analysis of unbonded particles,analysis of bonded particles,and chromatographic results and conditions.The PerformanceTest Chromatogram is specific to each individual column andcontains such information as:gel batch number,column serialnumber,USP plate count,USP tailing factor,capacity factor,and chromatographic conditions.These data should be storedfor future reference.a.Column Connectors The ACQUITY UPLCsystem utilizes tubing and gold platedcompression screws which have been designed to meet stringenttolerance levels and to minimize extra column volumes.Optimized column inlet tubing(part number 430001084)issupplied with the ACQUITY UPLCsystem.The inject valve endof the tubing is clearly marked with a blue shrink tube marker.Insert the opposite end of the tubing into the ACQUITY UPLCcolumn and tighten the compression fitting using two 5/16-inch wrenches.For information on the correct column outlet tubing,pleaserefer to the relevant detector section in the ACQUITY UPLCSystem Operators Guide(part number 71500082502).bColumn InstallationNote:The flow rates given in the procedure below are for a typical 2.1 mm i.d.by 50 mm length 1.7 m column.For 1.0 mm i.d columns,scale the flow rateusing the calculation shown in section III.Additional flow rate and pressureguides are provided in section VI(Additional Information).1.Purge the pumping system of any buffer-containing mobilephases and connect the inlet end of the column to theinjector outlet.2.Flush column with 100%organic mobile phase(methanolor acetonitrile)by setting the pump flow rate to 0.1 mL/minand increase the flow rate to 0.5 mL/min over 5 minutes.3.When the mobile phase is flowing freely from the columnoutlet,stop the flow and attach the column outlet to thedetector.This prevents entry of air into the detection systemand gives more rapid baseline equilibration.4.Gradually increase the flow rate as described in step 2.5.Once a steady backpressure and baseline have beenachieved,proceed to the next section.Note:If mobile phase additives are present in low concentrations(e.g.,ion-pairingreagents),100 to 200 column volumes may be required for complete equilibration.In addition,mobile phases that contain formate(e.g.,ammonium formate,formicacid,etc.)may also require longer initial column equilibration times.c.Column Equilibration ACQUITY UPLCBEH columns are shipped in 100%acetonitrile.It is important to ensure mobile phase compatibility beforechanging to a different mobile phase system.Equilibrate thecolumn with a minimum of 10 column volumes of the mobilephase to be used(refer to Table 1 for a list of column volumes).Table 1.Empty Column Volumes in mL(multiply by 10 for flushsolvent volumes)Column Length Internal Diameter(mm)1.0 mm2.1 mm200.0160.07300.0240.1500.040.21000.080.41500.120.5ACQUITY UPLCBEH Column Care and Use Instructions22004 Waters Corporation.To avoid precipitating mobile phase buffers on your column orin your system,flush the column with five column volumes of awater/organic solvent mixture,using the same or lower solventcontent as in the desired buffered mobile phase.(For example,flush the column and system with 60%methanol in water priorto introducing 60%methanol/40%buffer mobile phase).d.eCordInstallationThe eCordbutton should be attached to the side of the columnheater module.The eCordbutton is magnetized and does notrequire specific orientation.e.Initial Column Efficiency Determination 1.Perform an efficiency test on the column before using it.Waters recommends using a suitable solute mixture,as foundin the“Performance Test Chromatogram”,to analyze the columnupon receipt.2.Determine the number of theoretical plates(N)and use thisvalue for periodic comparisons.3.Repeat the test at predetermined intervals to track columnperformance over time.Slight variations may be obtainedon two different UPLC systems due to the quality of the connections,operating environment,system electronics,reagent quality,column condition and operator technique.II.COLUMN USETo ensure the continued high performance of ACQUITY UPLCBEH columns,follow these guidelines:a.Sample Preparation 1.Sample impurities often contribute to column contamination.One option to avoid this is to use Waters Oasissolid-phaseextraction cartridges/columns or Sep-Pakcartridges of theappropriate chemistry to clean up the sample before analysis.2.It is preferable to prepare the sample in the operating mobilephase or a mobile phase that is weaker(less organic modifier)than the mobile phase for the best peak shape and sensitivity.3.If the sample is not dissolved in the mobile phase,ensurethat the sample,solvent and mobile phases are miscible inorder to avoid sample and/or buffer precipitation.4.Filter sample with 0.2 m membranes to remove particulates.If the sample is dissolved in a solvent that contains an organicmodifier(e.g.,acetonitrile,methanol,etc.)ensure that themembrane material does not dissolve in the solvent.Contactthe membrane manufacturer with solvent compatibility questions.Alternatively,centrifugation for 20 minutes at 8,000 rpm,followed by the transfer of the supernatant liquid to anappropriate vial,could be considered.b.pH Range The recommended operating pH range for ACQUITY UPLCBEH columns is 1 to 12.A listing of commonly used buffersand additives is given in Table 2.Additionally,the column lifetimewill vary depending upon the operating temperature,the typeand concentration of buffer used.For example,the use of phosphatebuffer at pH 8 or above in combination with elevated temperatureswill lead to shorter column lifetimes.ACQUITY UPLCBEH Column Care and Use Instructions32004 Waters Corporation.c.Solvents To maintain maximum column performance,use high qualitychromatography grade solvents.Filter all aqueous buffers prior touse.Pall Gelman Laboratory Acrodiscfilters are recommended.Solvents containing suspended particulate materials will generallyclog the outside surface of the inlet distribution frit of the column.This will result in higher operating pressure and poorer performance.Degas all solvents thoroughly before use to prevent bubble formationin the pump and detector.The use of an on-line degassing unitis also recommended.This is especially important when runninglow pressure gradients since bubble formation can occur as aresult of aqueous and organic solvent mixing during the gradient.d.Pressure ACQUITY UPLCBEH columns can tolerate pressures of up to15,000 psi(1034 bar or 103 Mpa).e.Temperature Temperatures between 20 C 55 C are recommended for operatingACQUITY UPLCBEH columns in order to enhance selectivity,lower solvent viscosity and increase mass transfer rates.Operating at the extremes of pH,temperature,and/or pressurewill result in a shortened column lifetime.III.SCALING UP/DOWN ISOCRATIC METHODSThe following formulas will allow scale up or scale down,while maintaining the same linear velocity,and provide newsample loading values:If column i.d.and/or length are altered:F2=F1(r2/r1)2Load2=Load1(r2/r1)2(L2/L1)Injection volume2=Injection volume1(r2/r1)2(L2/L1)Where:r=Radius of the columnF=Flow rateL=Length of column1=Original,or reference column2=New columnACQUITY UPLCBEH Column Care and Use Instructions42004 Waters Corporation.Table 2.Buffer Recommendations for Using ACQUITY UPLC BEH Columns from pH 1 to 12Additive/BufferpKaBuffer range VolatilityUsed for Comments(1 pH unit)Mass SpecTFA0.3VolatileYesIon pair additive,can suppress MS signal,used in the 0.02-0.1%range.Acetic Acid4.76VolatileYesMaximum buffering obtained when used with ammonium acetate salt.Used in 0.1-1.0%range.Formic Acid3.75VolatileYesMaximum buffering obtained when used with ammonium formate salt.Used in 0.1-1.0%range.Acetate(NH4CH2COOH)4.763.76 5.76VolatileYesUsed in the 1-10 mM range.Note that sodium or potassium salts are not volatile.Formate(NH4COOH)3.752.75 4.75VolatileYesUsed in the 1-10 mM range.Note that sodium or potassium salts are not volatile.Phosphate 12.151.15 3.15Non-volatileNoTraditional low pH buffer,good UV transparency.Phosphate 27.26.20 8.20Non-volatileNoAbove pH 7,reduce temperature/concentration and use a guard column to maximize lifetime.4-Methylmorpholine8.47.4 9.4VolatileYesGenerally used at 10 mM or less.Ammonia(NH4OH)9.28.2 10.2VolatileYesKeep concentration below 10 mM and temperatures below 30 C.10.3(HCO3-)Used in the 5-10 mM range(for MS work keep source 150 C).Adjust pH with Ammonium Bicarbonate9.2(NH4+)6.8 11.3VolatileYesammonium hydroxide or acetic acid.Good buffering capacity at pH 10.6.3(H2CO3)Note:use ammonium bicarbonate(NH4HCO3),not ammonium carbonate(NH4)2CO3).Ammonium(Acetate)9.28.2 10.2VolatileYesUsed in the 1-10 mM range.Ammonium(Formate)9.28.2 10.2VolatileYesUsed in the 1-10 mM range.Borate9.28.2 10.2Non-VolatileNoReduce temperature/concentration and use a guard column to maximize lifetime.CAPSO9.78.7 10.7Non-VolatileNoZwitterionic buffer,compatible with acetonitrile,used in the 1-10 mM range.Low odor.Glycine2.4,9.88.8 10.8Non-VolatileNoZwitterionic buffer,can give longer lifetimes than borate buffer.1-Methylpiperidine10.29.3 11.3VolatileYesUsed in the 1-10 mM range.CAPS10.49.5 11.5Non-VolatileNoZwitterionic buffer,compatible with acetonitrile,used in the 1-10 mM range.Low odor.Triethylamine10.79.7 11.7VolatileYesUsed in the 0.1-1.0%range.Volatile only when titrated with acetic acid(not hydrochloric or phosphoric).(as acetate salt)Used as ion-pair for DNA analysis at pH 7-9.Pyrrolidine11.310.3 12.3VolatileYesMild buffer,gives long lifetime.IV.COLUMN CLEANING,REGENERATING AND STORAGE a.Cleaning and RegenerationChanges in peak shape,peak splitting,shoulders on the peak,shifts in retention,change in resolution or increasing backpressuremay indicate contamination of the column.Flushing with aneat organic solvent,taking care not to precipitate buffers,isusually sufficient to remove the contaminant.If the flushing proceduredoes not solve the problem,purge the column using the followingcleaning and regeneration procedures.Use the cleaning routine that matches the properties of thesamples and/or what you believe is contaminating the column(see Table 3 below).Flush columns with 20 column volumes ofHPLC-grade solvents.Increasing mobile phase temperature to35-55 C increases cleaning efficiency.If the column performanceis poor after regenerating and cleaning,call your local Watersoffice for additional support.Table 3.Column Cleaning SequencePolar SamplesNon-polar SamplesProteinaceous Samples1.water1.isopropanol(or an Option 1:Inject repeatedappropriate isopropanol/aliquots of dimethylwater mixture*)sulfoxide(DMSO)2.methanol2.tetrahydrofuran(THF)Option 2:gradient of 10%to 90%B where:3.tetrahydrofuran(THF)3.dichloromethaneA=0.1%trifluoroacetic acid(TFA)in water4.methanol4.hexaneB=0.1%trifluoroacetic acid(TFA)in acetonitrile(CH3CN)5.water5.isopropanol(followed byOption 3:Flush column withan appropriate isopropanol/7M guanidine hydrochloride,water mixture*)or 7M urea6.mobile phase6.mobile phase*Use low organic solvent content to avoid precipitating buffers.b.Storage For periods longer than four days at room temperature,storethe column in 100%acetonitrile.For elevated temperatureapplications,store immediately after use in 100%acetonitrilefor the best column lifetime.Do not store columns in bufferedeluents.If the mobile phase contained a buffer salt,flush thecolumn with 10 column volumes of HPLC grade water(seeTable 1 for common column volumes)and replace with 100%acetonitrile for storage.Failure to perform this intermediatestep could result in precipitation of the buffer salt in the columnwhen 100%acetonitrile is introduced.Completely seal columnto avoid evaporation and drying out of the bed.Note:If a column has been run with a mobile phase that contains formate(e.g.,ammonium formate,formic acid,etc.)and is then flushed with 100%acetonitrile,slightly longer equilibration times may be necessary when the column is re-installed and run again with a formate-containing mobile phase.V.eCorda.IntroductionThe eCordintelligent chip is a new technology that will providethe history of a columns performance throughout its lifetime.TheeCordis permanently attached to the column to assure that thecolumns performance history is maintained in the event that thecolumn is moved from one instrument to another.At the time of manufacture,tracking and quality control informationwill be downloaded to the eCord.Storing this information onthe chip will eliminate the need for a paper Certificate ofAnalysis.Once the user installs the column,the software willautomatically download key parameters into a column historyfile stored on the chip.The eCordprovides a solution to easilytrack the history of column usage.ACQUITY UPLCBEH Column Care and Use Instructions52004 Waters Corporation.Waters eCord-intelligent chipeCordinserted into sideof column heaterd.Customer Use InformationThe eCordchip will automatically capturecolumn use data.The top of the screen identifiesthe column including chemistry type,columndimensions and serial number.The overallcolumn usage information includes the totalnumber of samples,total number of injections,total sample sets,date of first injection,dateof last injection,maximum pressure and temperature.The information also details thecolumn history by sample set including datestarted,sample set name,user name,systemname,number of injections in the sample set,number of samples in the sample set,maximumpressure and temperature in the sample setand if the column met basic system suitabilityrequirements.c.Manufacturing Information The eCordchip provides the user with an overview of the bulkmaterial QC test results.ACQUITY UPLCBEH Column Care and Use I