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ACQUITY UPLC H-Class System Guide

1. Sample Manager FTN s Quaternary Solvent Manager S ACQUITY TUY Detector i hd un ow amp Waiting for QSM Waiting for gradient Ready 4527 psi 0 400 mL min 27 8 C 40 0 C A 900 E 00 0 0017 AU Sample Column A g gt Off 40 0 A B 10 0 D 0 0 273 nm When Empower software controls the system the control panels appear at the bottom of the Run Samples window When MassLynx software controls the system the control panels appear on the Additional Status tab of the Inlet Editor window Quaternary solvent manager control panel 3 8 The quaternary solvent manager control panel displays flow status system pressure total flow rate and solvent composition parameters Rule You can edit these parameters when the system is idle by clicking on the underlined value You cannot edit quaternary solvent manager parameters while the system is running samples Preparing the System Quaternary solvent manager control panel Quaternary Solvent Manager S 9 Flow LED Low Status Waiting for gradient System pressure 4527 psi 0 400 mLimin Flow rate iga _ BA LO E 0 0 Solvent composition 5 o a E R al Stop flow The following table describes the items in the quaternary solvent manager control panel Quaternary solvent manager control panel items Control panel item Description Flow LED Displays the actual flow LED on the front panel of the quaternary solvent manager unless communications with the
2. When operating this device follow standard quality control procedures and the equipment guidelines in this section Caution symbol A 5 A A 6 Attention Changes or modifications to this unit not expressly approved by the party responsible for compliance could void the user s authority to operate the equipment Important Toute modification sur cette unit n ayant pas t express ment approuv e par l autorit responsable de la conformit la r glementation peut annuler le droit de l utilisateur exploiter l quipement Achtung Jedwede Anderungen oder Modifikationen an dem Ger t ohne die ausdr ckliche Genehmigung der f r die ordnungsgem e Funktionst chtigkeit verantwortlichen Personen kann zum Entzug der Bedienungsbefugnis des Systems f hren Avvertenza qualsiasi modifica o alterazione apportata a questa unit e non espressamente autorizzata dai responsabili per la conformit fa decadere il diritto all utilizzo dell apparecchiatura da parte dell utente Atencion cualquier cambio o modificaci n efectuado en esta unidad que no haya sido expresamente aprobado por la parte responsable del cumplimiento puede anular la autorizaci n del usuario para utilizar el equipo ES AREA TACA A TA FY BE AO A ER E AA IRER o YER A n IGE LUTE RIT TE VARTA RATES RESTA KR BRAVE UE A HT AAN A
3. Best combination of efficiency amp speed l 2004 T T 3 17 6 8 Linear Velocity U mm sec gt It is apparent from the figure above that using 1 7 um particles achieves higher efficiency that persists as flow rate increases lower HETP indicates higher efficiency When operating in this area of the plot the peak capacity and the speed of a separation can set limits well beyond those of conventional ACQUITY UPLC H Class System HPLC technology Waters has defined this new level of performance as UltraPerformance chromatography Comparison of chromatographic separations using 5 0 um and 1 7 um particles 0 050 1 7 pm UPLC Column 0 040 0 030 AU 0 020 0 010 0 000 0 1 2 3 4 5 6 min 5 pm HPLC Column 0 040 0 030 AU 0 020 0 010 0 000 0 2 8 4 6 8 10 12 14 15 min Note Each separation was performed on a 2 1 X 50 mm column Chromatographic conditions for the separations were identical except for the flow rate which was scaled based on particle size Features of the ACQUITY UPLC H Class system The ACQUITY UPLC H Class system combines the speed and performance of UPLC with the ability to run HPLC separations This combination provides many benefits including the following High pressure small particle chromatography allowing faster higher resolution analyses compared to traditional HPLC Low solvent consumption significantly less than HPLC
4. Features of the ACQUITY UPLC H Class system 1 3 Flexibility in solvent mixing by using a quaternary solvent manager lt A flow through needle sample manager that facilitates the transfer of HPLC methods to UPLC e Pump and sample manager design enhancements to minimize dispersion and reduce cycle time Flow through needle injector The sample manager in the ACQUITY UPLC H Class system uses a flow through needle mechanism that differs radically from the loop based injector used by the ACQUITY UPLC system The flow through needle mechanism aspirates a sample and holds it in the sample needle in preparation for injecting the sample onto the column The needle serves as part of the injection flow path when the sample is pushed onto the column Using the flow through needle mechanism the system operates similarly to most traditional HPLC systems facilitating the transfer of HPLC methods with its familiar operation The flow through needle mechanism also does not require you to learn new injection modes improves injection accuracy and decreases cycle time for small volume injections Gradients pass through the needle during injection ensuring complete sample recovery Wash solvent The wash system uses a single solvent to clean the outside of the sample needle and prime the wash system The wash solvent does not enter the injection flow path Purge solvent The primary function of the purge solvent is to move sample along the inje
5. See Priming the SM FTN in the Sample Manager Flow Through Needle Operator s Overview and Maintenance Information Wash needle Displays the Wash Needle dialog box See Washing the SM FTN needle in the Sample Manager Flow Through Needle Operator s Overview and Maintenance Information Reset SM Resets the sample manager following an error condition Help Displays the ACQUITY UPLC Console online Help TUV detector control panel The TUV detector s control panel displays absorbance units and wavelength values which you can edit when the system is idle by clicking the underlined value However you cannot edit detector parameters when the system is running samples 3 12 Preparing the System Control panels for other detectors function similarly If your system includes a PDA detector see the ACQUITY UPLC Photodiode Array Detector Getting Started Guide If your system includes an ELS detector see the ACQUITY UPLC Evaporative Light Scattering Detector Getting Started Guide s If your system includes an FLR detector see the ACQUITY UPLC Fluorescence Detector Getting Started Guide for information on the control panel Control panel ACQUITY TUY Detector Lamp On Off LED Status Lamp wn Absorbance 0 0017 AU units A 2233 nm gt Turn detector lamp On Off Value of wavelength A The following table describes the controls and indicators in the TUV detector s
6. lt 0 01 0 02 0 02 Unstablized tetrahydro furan THF fresh 2 44 2 57 2 31 1 80 1 54 0 94 0 42 0 21 0 09 0 05 Unstablized tetrahydro furan THF old gt 2 5 gt 2 5 gt 2 5 gt 2 5 gt 2 5 gt 2 5 gt 2 5 gt 2 5 2 5 1 45 Acids and bases Acetic acid 1 2 61 2 63 2 61 2 43 2 17 0 87 0 14 0 01 lt 0 01 Hydro chloric acid 0 1 0 11 0 02 lt 0 01 Phosphoric acid 0 1 lt 0 01 Trifluoro acetic acid 1 20 0 78 0 54 0 34 0 22 0 06 lt 0 02 lt 0 01 Diammo nium phosphate 50 mM 1 85 0 67 0 15 0 02 lt 0 01 Wavelength selection C 15 Mobile phase absorbance measured against air or water Continued Absorbance at specified wavelength nm 200 205 1210 215 220 1230 240 250 260 280 Triethy 2 33 2 42 12 50 2 45 2 87 1 96 0 50 10 12 10 04 lt 0 01 lamine 1 Buffers and salts Ammonium 1 88 10 94 10 53 10 29 10 15 10 02 lt 0 01 acetate 10 mM Ammonium 0 41 10 10 10 01 lt 0 01 bicarbonate 10 mM Ethylene 0 11 10 07 0 06 10 04 10 03 10 03 10 02 10 02 10 02 10 02 dinitrilo tetraacetic acid diso dium salt disodium EDTA 1 mM 4 2 hydrox 2 45 12 50 12 87 2 08 1 50 10 29 0 03 lt 0 01 yethyl 1 pi pera zineethanes ulfonic a
7. Connecting to the electricity source B 9 System tubing connections The system s external tubing connections for solvent flow and drainage are shown below System tubing connections B 1 Solvent flow and drainage Bottle tray Detector SM FTN 1 8 inch OD Location of back PTFE tubing pressure regulator Location of flow cell Column heater Location of active Sample manager pre heater assembly flow through needle 1 8 inch OD PTFE tubing 1 purge line Stainless steel tubing 1 8 inch OD PTFE from QSM to SM FTN tubing 4 lines for solvent A B C D and 1 line for seal wash Teflon clear tubing to waste valve PEEK to Quaternary waste solvent manager Teflon corrugated tubing PEEK SM FTN syringe tubing from QSM System flow mam System drainage Leak detector Waste B 2 External Connections Instrument external wiring connections ACQUITY UPLC H Class instrument external wiring connections The rear panel connections for ACQUITY UPLC H Class instruments are shown below External power and communications cable Ethernet switch Ethernet cables Solvent tray EN Detector o Column heater WEIER Sample manager Quaternary solvent manager Ethernet cable to PC Power cords Instrument ex
8. control panel TUV detector control panel items Control panel item Description Lamp On Off LED Displays the actual lamp on off LED on the front panel of the detector unless communications with the detector are lost Status Displays the status of the current operation AU Displays the absorbance units nm Displays the value of wavelength A in nm If the detector is in dual wavelength mode the value of wavelength B also appears m i Ignites the detector lamp Pa Lamp Off Extinguishes the detector lamp ACQUITY control panels 3 13 You can access additional functions described in the table below by right clicking anywhere in the detector control panel Additional functions in the detector control panel Control panel function Description Autozero Resets the absorbance value to 0 Reset TUV Resets the detector when present after an error condition Help Displays the ACQUITY UPLC Console online Help Starting the ACQUITY UPLC Console The ACQUITY UPLC Console is a software application that provides a convenient way to configure settings monitor performance run diagnostic tests and maintain the system and its modules It replaces the keypads and small screen displays traditionally found on the fronts of system instruments The ACQUITY UPLC Console functions independently of data applications and does not recognize or control them From the ACQUITY UPLC Console s
9. Before each sample injection a sample manager typically performs wash sequences and aspirates the appropriate sample volume When these tasks are completed the solvent manager begins to deliver the gradient to the injection valve The dwell volume of the system affects the amount of time it takes for this gradient to reach the column and can be a significant component of the overall cycle time The Gradient Smart Start feature coordinates pre injection operations and reduces the impact of the solvent manager s dwell volume on cycle time The gradient starts before or during the sample manager s pre injection tasks resulting in significant time savings Wash Plungers Precipitated material that remains on the solvent manager s pump plungers can damage the high pressure seals The Wash Plungers function washes the seals with solvent to remove any precipitated material You can use the Wash Plungers function as needed or run it as part of the No flow Shutdown feature Features of the ACQUITY UPLC H Class system 1 5 1 6 No Flow Shutdown The No Flow Shutdown feature runs the Wash Plungers function after the solvent manager remains idle for a specified time interval This feature prevents precipitated material from depositing on the pump plungers while the system is idle Automatic Prime When you enable this function of the solvent manager the system primes the lines of the optional solvent selection valve when a new one is sele
10. Preventing contamination For information on preventing contamination refer to Controlling Contamination in Ultra Performance LC MS and HPLC MS Systems part number 715001307 on the Waters Web site Visit www waters com Clean solvents Clean solvents ensure reproducible results and permit you to operate with minimal instrument maintenance Dirty solvents can cause baseline noise and drift and they can clog solvent reservoir filters inlet filters and capillary lines Solvent quality Use MS grade solvents for the best possible results the minimum requirement is HPLC grade Filter solvents through an appropriate membrane filter Recommendation Ensure your solvent choices are consistent with the recommendations of the membrane filter manufacturer or supplier Solvent preparation Proper solvent preparation primarily filtration can prevent many pumping problems Recommendation Use brown tinted glassware to inhibit microbial growth Water Use water only from a high quality water purification system If the water system does not deliver filtered water filter the water through a 0 2 um membrane filter Caution Using 100 water can cause microbial growth Waters recommends changing 100 water solutions daily Adding a small amount of an organic solvent 10 prevents microbial growth C 2 Solvent Considerations Using buffers Adjust the pH of aqueous buffers Filter them to remove insoluble material and then blen
11. UPLC system driver CD or from the Method Assistance Kit CD An icon for the ACQUITY UPLC Columns Calculator appears on your computer s desktop after installation Dispersion UPLC systems and autosamplers exhibit low dispersion a fixed instrument characteristic measured by looking at how much peak broadening occurs because of the system design Small particle chromatography uses small high efficiency columns A typical 2 1 x 50 mm UPLC column has an approximate 174 uL volume compared to 2 5 mL for a typical 4 6 x 150 mm HPLC column The smaller column and particle size require a system whose low dispersion reduces dilution and band broadening thus maintaining the peak shape height and sensitivity produced by the high efficiency column An ACQUITY UPLC H Class system typically exhibits a bandspread between 10 and 12 uL value depends on system configuration An Alliance HPLC system can exhibit a bandspread between 35 uL and 50 uL Because of the dispersion differences a band on an Alliance system experiences a three fold increase in dilution compared to an ACQUITY UPLC H Class system As a result UPLC peak concentrations are higher than HPLC concentrations Because solubility effects are more apparent in low dispersion systems that use high pressure 1t 1s important to adjust column load appropriately Carryover 2 4 You observe carryover in chromatographic systems when a previously injected analyte appears as a peak in
12. beeps three times and runs a series of startup tests See Status LEDs on page 3 3 and Power LED on page 3 2 for information on how to interpret indicator LED modes for device or instrument flow status and whether the units are powered on Tip If your system contains a column heater it is automatically powered on when you power on the sample manager To power on the system 1 Power on the ACQUITY UPLC H Class system s workstation 2 Power on the quaternary solvent manager and the sample manager by pressing the power switch on the top left hand side of each devices door See also Status LEDs on page 3 3 and Power LED on page 3 2 for information on how to interpret LED modes for device or instrument flow status and whether the units are powered on Preparing system hardware 3 1 3 After power LEDs on the quaternary solvent manager and on the sample manager show steady green press the power switch on the top left hand side of the detector or detectors Tip To prevent initialization errors only power on the detector or detectors when the flow cell is wet 4 Start the chromatography data system software Tip You can monitor the ACQUITY UPLC Console for messages and LED indications Monitoring startup tests These startup tests run when you power on the ACQUITY UPLC H Class system s workstation CPU board Memory RAM and ROM External communication system Ethernet Clock If the
13. cord to a suitable wall outlet Alternative If your system includes the optional FlexCart connect the female end of the Flexcart s electrical cables included in the startup kit to the receptacle on the rear panel of the each instrument Connect the hooded male end of the Flexcart s electrical cables to the power strips on the back of the cart Finally connect each power strip s cable to a wall outlet operating on its own circuit Connecting to the electricity source B 9 FlexCart power connections AC line F Network switch To circuit A 1 meter Universal TEC Coupler d Meter BEE solvent manager FlexCart power strips To circuit B AC line B 10 External Connections C Solvent Considerations Warning To avoid chemical hazards always observe Good Laboratory Practices when operating your system handling solvents or changing tubing See the Material Safety Data Sheets for the solvents you use The information in this appendix applies only to the following instruments ACQUITY UPLC H Class system modules ACQUITY UPLC PDA detector ACQUITY UPLC PDA el detector ACQUITY UPLC TUV detector ACQUITY UPLC ELS detector ACQUITY UPLC FLR detector Contents Topic Page Introduction C 2 Solvent recommendations C 3 Common solvent properties C 8 Solvent miscibility C 10 Solvent stabilizers C 12 Solvent viscosity C 12 Wavelength selection C 12 C 1 Introduction
14. filter them through dry aluminium oxide to adsorb formed peroxides Solutions that contain strong concentrations of complexing agents like ethylene diaminetetraacetic acid EDTA ACQUITY UPLC H Class system recommendations Contact Waters for recommended system cleaning and flushing procedures See also Controlling Contamination in Ultra Performance LC MS and HPLC MS Systems part number 715001307 on the Waters Web site Visit www waters com Warning Explosion hazard Peroxide contaminants in THF can spontaneously and destructively explode when you partially or completely evaporate the THF C 6 Solvent Considerations Warning Health hazard Hexane 1s a neurotoxin and THF can irritate eyes skin and mucous membranes and cause harmful neurologic effects If you use either or both of these volatile solvents locate your ACQUITY UPLC H Class system inside a fume hood or walk in chamber to minimize exposure to harmful solvent vapors THF hexane ethyl acetate and acetone can be used as the mobile phase in ACQUITY UPLC H Class systems However as with many nonaqueous solvents they can shorten system and instrument life compared with equipment running typical reversed phase solvents If you routinely use THF hexane ethyl acetate or acetone Waters recommends you install the Hexane THF Compatibility Kit When using unstabilized THF ensure that your solvent is freshly prepared Previously opened bottles contain peroxide conta
15. interface you can quickly navigate to visual representations of each module and its components You can also navigate to interactive diagrams which show interconnections and provide diagnostic tools for troubleshooting problems To start the ACQUITY UPLC Console from Empower software In the Run samples window click Display console A in the Sample Manager control panel 3 14 Preparing the System ACQUITY UPLC Console window ACQUITY UPLC Console Local System El ACQUITY UPLC System E Quaternary Solvent Manag Sample Manager FTN Column PDA Detector A x Samples Methods Plots Maintenance Counters Logs 0 System Status Control Configure Maintain Troubleshoot Help Quaternary Solvent Manager QSMBETAO 1 psi B 0 000 mL min top Fic D Lamp Sample Manager PDA Detector ALAC Off 27 0 c Off Column Shutter Erbium filter Sample Room 26 7 C To start the ACQUITY UPLC Console from Masslynx software 1 In the MassLynx window click Inlet Method 2 Inthe Inlet Method window click the ACQUITY Additional Status tab 3 Click Display console AI Starting the ACQUITY UPLC Console 3 15 3 16 Preparing the System Safety Advisories Waters instruments display hazard symbols designed to alert you to the hidden dangers of operating and maintaining the instruments Their corresponding user guides also include the hazard symbols with accompanying text statements describing the hazar
16. on signal connectors PDA detector signal connectors If your system includes a PDA detector see the ACQUITY UPLC Photodiode Array Detector Getting Started Guide for information on signal connectors ELS detector signal connectors If your system includes an ELS detector see the ACQUITY UPLC Evaporative Light Scattering Detector Getting Started Guide for information on signal connectors FLR detector signal connectors If your system includes an FLR detector see the ACQUITY UPLC Fluorescence Detector Getting Started Guide for information on signal connectors External Connections Connecting to the electricity source Each system instrument requires a separate grounded power source The ground connection in all power outlets must be common and physically close to the system Warning Avoid electrical shock Use power cord SVT type in the United States and HAR type or better in Europe For other countries requirements contact your local Waters distributor Power off and unplug each system instrument before performing any maintenance operation on the instrument Connect each system instrument to a common ground To connect to the electricity source Recommendation Use a line conditioner and uninterruptible power supply UPS for optimum long term input voltage stability 1 Connect the female end of the power cord to the receptacle on the rear panel of each instrument 2 Connect the male end of the power
17. panel indicates the lamp status A steady green lamp LED indicates that the lamp is ignited Status LED indications LED mode and color Description Unlit Quaternary solvent manager and sample manager Indicates the instrument is currently idle Detector Indicates the detector lamp is extinguished Steady green Quaternary solvent manager Indicates solvent is flowing Sample manager Indicates the sample manager is operating normally attempting to complete any outstanding samples or diagnostic function requests When sample and diagnostic function requests are finished the LED reverts to the unlit mode Detector Indicates the detector lamp is ignited Preparing system hardware 3 3 Status LED indications Continued LED mode and color Description Flashing green Quaternary solvent manager and sample manager Indicates that the instrument is initializing e Detector Indicates the detector is initializing or calibrating Flashing red Indicates that an error stopped the instrument Refer to the ACQUITY UPLC Console for information regarding the error Steady red Indicates an instrument failure that prevents further operation Power off the instrument and then power on If the LED is still steady red contact your Waters service representative 3 4 Preparing the System Enabling the leak sensors Rule When you power on the system the leak se
18. provides approximate wavelength cutoffs for some other solvents buffers detergents and mobile phases The solvent concentrations represented are those most commonly used If you want to use a different concentration you can determine approximate absorbance using Beer s law because absorbance is proportional to concentration Wavelength cutoffs for different mobile phases BRIJ 35 0 1 UV UV Mobile phase cutoff Mobile phase cutoff nm nm Acetic acid 1 230 Sodium chloride 1 M 207 Ammonium acetate 10 mM 205 Sodium citrate 10 mM 225 Ammonium bicarbonate 10 mM 190 Sodium dodecyl sulfate 190 Polyoxyethylene 35 lauryl ether 190 Sodium formate 10 mM 200 Wavelength selection C 13 Wavelength cutoffs for different mobile phases Continued 1 vial liter UV UV Mobile phase cutoff Mobile phase cutoff nm nm 3 8 cholamidopropyl dimethy 215 Triethyl amine 1 235 lammonio 1 propanesulfonate CHAPS 0 1 Diammonium phosphate 50 mM 205 Trifluoracetic acid 0 1 190 Ethylenedinitrilo tetraacetic 190 TRIS HCl 20 mM pH 7 0 202 acid disodium salt disodium pH 8 0 212 EDTA 1 mM 4 2 hydroxyethyl 1 pipera 225 Triton X 100 0 1 240 zineethanesulfonic acid HEPES 10 mM pH 7 6 Hydrochloric acid 0 1 190 Waters PIC Reagent A 200 1 vial liter Morpholinoethanesulfonic acid 215 Waters PIC Reagent B 6 225 MES 10 mM pH 6 0
19. solvent loss and air introduction during the intake cycle Leaks at high pressure fittings downstream of the intelligent intake valves can leak solvent but do not introduce air To prevent leaks follow Waters recommendations for the proper tightening of system fittings Note specifically whether you are retightening the fitting or installing it for the first time Sample preparation UPLC analysis places some additional restrictions on sample preparation Particulates The small column frit size 0 2 um can become blocked more easily than larger HPLC column frits 2 0 um As a result particle free mobile phase solvents and sample solutions are essential for UPLC analysis See General guidelines on page 2 2 for recommendations on choosing and handling solvents Matching sample diluents When you use the auto dilution option on the SM FTN the purge solvent is used as the sample diluent Ensure that your sample solution is soluble and miscible in your chosen purge solvent 2 6 Optimizing Performance 3 Preparing the System Contents Topic Page Preparing system hardware 3 1 Configuring chromatography data software 3 8 ACQUITY control panels 3 8 Starting the ACQUITY UPLC Console 3 14 Preparing system hardware Powering on the system Powering on the system entails starting the ACQUITY UPLC H Class system workstation system instruments and chromatography data system software Each device or instrument
20. startup tests indicate a malfunction consult the ACQUITY UPLC Console s online Help Monitoring system instrument LEDs Light emitting diodes on each system instrument indicate the instrument s state of functioning The LEDs are specific to their instruments so the significance of their various colors and modes can differ from one instrument to another Power LED 3 2 The power LED on the left hand side of a device or instrument s front panel indicates the power on or power off status of the instrument This LED is green when power is applied to the unit and unlit when power is not applied Tip To provide adequate ventilation the sample manager fans run continuously even when the power switch is in the off position These fans switch off only when you disconnect the power cable from the back of the instrument Preparing the System Status LEDs Flow LED quaternary solvent manager The flow LED on the right hand side of the power LED on the quaternary solvent manager s front panel indicates the flow status A steady green flow LED indicates that there is a flow through the quaternary solvent manager Run LED sample manager The run LED on the right hand side of the power LED on the sample manager s front panel indicates the run status A steady green run LED indicates that injections are being run Lamp LED detector The lamp LED on the right hand side of the power LED on the detector s front
21. the A B C D Solvents area you can select or clear any or all of the solvents A B C D You can change the duration of priming for solvents A through D by entering a different number in the Duration of Prime field All selected solvents are primed for the same duration Priming parameter values Range 0 1 to 60 0 minutes All solvents prime for 2 0 minute Default SE Prime for 3 minutes Prime for Recommendation 7 minutes after changing solvents Tip If you want to return settings to their original values on any tab click Set Defaults Select or clear priming of the seal wash wash solvent and purge solvent If necessary change the duration specified to prime the seal wash and wash solvent and the number of cycles specified to prime the purge solvent Default The seal wash primes for 2 0 minutes the wash solvent for 15 seconds and the purge solvent for 5 cycles Select the Equilibrate to Method tab to review the settings for the final flow rate mobile phases composition temperatures and lamp state Preparing the System 6 On the Equilibrate to Method tab change the values as needed to match your requirements at equilibration Equilibrate to Method tab values System startup parameters Method initial flow rate Default 0 500 mL min Allowed values 0 1 to 2 0 mL min Composition of A B C A 100 A 0 to 100 and D sum must be B C D 0 B 0 t
22. the chromatogram of subsequent samples Carryover tends to occur when a small amount of analyte remains in the system after a sample is injected You can measure carryover by observing analyte peaks that appear when you run a blank sample immediately after an analytical sample Waters measures carryover on the ACQUITY UPLC Optimizing Performance H Class system at less than 0 004 See ACQUITY UPLC H Class System Specifications for details A common cause of carryover is inadequately washing of the system Choosing an appropriate wash solvent can minimize carryover for a particular analysis The wash solvent must be strong enough to dissolve any remaining sample and the wash duration must be long enough to remove the residue from the system Method conditions also affect carryover Too short a hold time at the final conditions of a gradient especially if the gradient is steep can fail to remove all analytes from the system It is important to completely flush the system and reequilibrate the column before proceeding to a subsequent analysis Use caution when choosing the load ahead and loop offline options Initiating these options before the highly organic part of the gradient reaches the needle can leave sample residue in the system The time savings you gain can lead to inadequate system cleaning The hydrophobicity and solubility of your samples as well as cleanliness during sample preparation are additional factors to consider when try
23. tubos de metal cuando se trabaje con tetrahidrofurano THF o cidos n trico o sulf rico concentrados Hay que tener en cuenta que el cloruro de metileno y el sulf xido de dimetilo dilatan los tubos no met licos lo que reduce la presi n de ruptura de los tubos Ba SACH Dn FERR WER MERA F R EP E VI ARI TR BE RENE IPA HT KK o A IN ROUE BCE E NEETER Be PA ME ETA sat Foe HER Hel BYRTA NR KIRIM MR o Tel BB ES IE Se RUE XX ARE AR IN REJ o i 3 Safety Advisories Ba JAKA MEHER Grott SRA EJ AIR R RTE EG R FEUX HSE PT A HI CHA o EA ARR Pe ES HA AU ATACA E R PEH DU SUR ER DEE Kr ET PP ASE KR RER EA TEER E RK KAARE RAII KR RE As WHY Ed FREE AGS AGS 5 gt hei agy FE AA FV HS ARE Al 9 37 ELT IAE JSA 4357104 are REE AF S SA nf he Cone T FHS uEHS E ice dina THF HE EE AM UE SAT a ALA ut A 493 4 Y dl Methylene chloride 2 Y AY ENE Dimethyl sulfoxide Has FHS Sq FHS 89 942 27 4219 TF ALE ZE FAA LS E EHDONMASEKUYUS Fa T R9 LAN AELTIE MESNEKY 7 F Ota DLPRBA DIA AAN LOIRA MX ICH SKEIBLTC TES ELSZELK Eso ke F 2 TAREA LATE KZ EL HET a TZIA FIFE Fe 77 THE SAR OG E To Re LE He OT E EUX HAER FUV d Tt zb FEI HET 2 TOMES a ELI YG HV LOGE Fa T
24. 0 psi and up to 2 mL min at reduced pressures to 62 053 kPa 621 bar 9000 psi The quaternary solvent manager can pump four degassed solvents simultaneously using a gradient proportioning valve GPV to dynamically create a specified composition Sample manager flow through needle The sample manager flow through needle SM FTN uses a direct injection mechanism to inject samples drawn from plates and vials onto a chromatographic column Optional extension loops installed between the sample needle and the injection valve can increase the injection volume beyond that of the sample needle The sample manager flow through needle can also dilute samples using the auto dilution option Column heater Column temperature variations can shift peak retention times and alter peak shapes increasing the difficulty of achieving precise results The ACQUITY UPLC H Class system s column heater helps to ensure precise reproducible separations by controlling the column temperature The column heater heats the column compartment to any temperature from 20 C 68 F above ambient to 90 C 194 F An active preheating device is used to heat the incoming solvent before it enters the column The column compartment can accommodate columns of 2 1 mm to 4 6 mm I D and 50 to 150 mm length Tip Active preheating is the default configuration for the ACQUITY UPLC H Class system An optional passive column stabilizer is available for existing chromato
25. 0 volt input Stop Flow Allows you to stop the flow from the quaternary solvent manager when it receives a contact closure input or 0 volt input an error condition or hardware failure on another instrument for example Sample manager UO signal connectors The rear panel of the sample manager includes a removable connector that holds the screw terminals for I O signal cables This connector is keyed so that it can receive a signal cable inserted only one way Requirement A contact closure output connection Inject Start Out from the sample manager is required to trigger a mass spectrometer an ACQUITY 2996 PDA detector or an ACQUITY ELS detector running under MassLynx software control to start Sample manager I O signal connectors 123456 1 ES a OO E E 53 33 oo HOII wee ES wz OO 5 O 2200022 EE OO EE For electrical specifications see the ACQUITY UPLC H Class System Specifications Signal connections B 7 B 8 Sample manager event out event in connections Signal connections Description Inject Start Indicates with a contact closure output that an injection has started Inject Hold Delays the next injection when the sample manager receives a contact closure input from another system instrument for example TUV detector signal connectors If your system includes a TUV detector see the ACQUITY UPLC Tunable Ultraviolet Detector Getting Started Guide for information
26. 1 vial liter Potassium phosphate Waters PIC Reagent B 6 190 monobasic 10 mM 190 low UV 1 vial liter dibasic 10 mM 190 Sodium acetate 10 mM 205 Waters PIC Reagent D 4 190 Mobile phase absorbance This section lists the absorbances at several wavelengths for frequently used mobile phases Choose the mobile phase carefully to reduce baseline noise The best mobile phase for your application is one that is transparent at the chosen detection wavelengths With such a mobile phase ensure that any absorbance is due only to the sample Absorbance by the mobile phase also reduces the linear dynamic range of the detector by the amount of absorbance the autozero function cancels or autozeroes out Wavelength pH and concentration of the mobile phase affects its absorbance Examples of several mobile phases are given in the table below C 14 Solvent Considerations The absorbances in the table below are based on a 10 mm pathlength Mobile phase absorbance measured against air or water Solvents Absorbance at specified wavelength nm 200 205 210 215 220 230 240 250 260 280 Acetonitrile 0 05 0 03 0 02 0 01 0 01 lt 0 01 Methanol not degassed Methanol degassed Isopropanol 2 06 1 91 1 80 1 00 0 76 0 68 0 53 0 35 0 34 0 37 0 21 0 24 0 24 0 19 0 11 0 06 0 08 0 05 0 02 0 04 0 02 lt 0 01 0 03
27. 210 1215 220 230 1240 1250 260 280 PIC A 0 67 10 29 10 13 10 05 10 03 0 02 10 02 10 02 0 02 lt 0 01 1 vial L PIC B6 1 2 46 2 50 12 42 12 25 1 83 0 63 10 07 lt 0 01 vial L PIC B6 low 0 01 lt 0 01 UV 1 vial L PIC D4 1 0 03 10 03 10 03 10 03 0 02 0 02 10 02 10 02 0 02 0 01 vial L C 18 Solvent Considerations Mobile phase absorbance measured against air or water Continued Detergents Absorbance at specified wavelength nm 200 205 210 215 220 230 240 250 260 280 BRIJ 35 1 0 06 0 03 0 02 0 02 0 02 0 01 lt 0 01 3 3 chola midopropyl dimethy lammonio 1 propane sulfonate CHAPS 0 1 2 40 2 32 1 48 0 80 0 40 0 08 0 04 0 02 0 02 0 01 Sodium dodecyl sulfate SDS 0 1 0 02 0 01 lt 0 01 4 octylphen ol poly ethoxylate Triton X 100 0 1 2 48 2 50 2 43 2 42 2 37 2 37 0 50 0 25 0 67 1 42 Polyoxyeth ylene sorbitan monolau rate Tween 20 0 1 0 21 0 14 0 11 0 10 0 09 0 06 0 05 0 04 0 04 0 03 Wavelength selection C 19 C 20 Solvent Considerations
28. A RE E V ESN TU ANNE T ETS E M r HEL CVS AEREO EED HH LITIER LT FSU A 10 Safety Advisories Warning To protect against fire replace fuses with those of the A type and rating printed on panels adjacent to instrument fuse covers Attention pour viter tout risque d incendie remplacez toujours les fusibles par d autres du type et de la puissance indiqu s sur le panneau proximit du couvercle de la boite fusible de l instrument Vorsicht Zum Schutz gegen Feuer die Sicherungen nur mit Sicherungen ersetzen deren Typ und Nennwert auf den Tafeln neben den Sicherungsabdeckungen des Ger ts gedruckt sind Attenzione per garantire protezione contro gli incendi sostituire 1 fusibili con altri dello stesso tipo aventi le caratteristiche indicate sui pannelli adiacenti alla copertura fusibili dello strumento aquellos del tipo y caracteristicas impresos en los paneles Advertencia Para evitar incendios sustituir los fusibles por A adyacentes a las cubiertas de los fusibles del instrumento Bf TK AAA EN D A o BVK EJ ENNI L FH EAA DE AR o PE TR MERA UN A A E ER IR AA EAEE HIRERE o A Aa JAA HFE 729 8 717 2 AM Latz do 44 23 SYA KV 4249 ASE FSE A SAA 2 SH KL PWORWIC ta RAM TIRE 2 AW NMOS AMV CAR SIV CWS FA TRENERDE aR E CEH Ei Warnings that apply to all Waters instruments A 11 Electrical and han
29. ACQUITY UPLC H Class System Guide Revision A Waters THE SCIENCE OF WHAT S POSSIBLE Copyright Waters Corporation 2010 All rights reserved Copyright notice 2010 WATERS CORPORATION PRINTED IN THE UNITED STATES OF AMERICA AND IN IRELAND ALL RIGHTS RESERVED THIS DOCUMENT OR PARTS THEREOF MAY NOT BE REPRODUCED IN ANY FORM WITHOUT THE WRITTEN PERMISSION OF THE PUBLISHER The information in this document is subject to change without notice and should not be construed as a commitment by Waters Corporation Waters Corporation assumes no responsibility for any errors that may appear in this document This document is believed to be complete and accurate at the time of publication In no event shall Waters Corporation be liable for incidental or consequential damages in connection with or arising from its use Trademarks ACQUITY ACQUITY UPLC Connections INSIGHT Millennium UPLC and Waters are registered trademarks and Auto Blend eCord Empower LAC E MassLynx nanoACQUITY UPLC and THE SCIENCE OF WHAT S POSSIBLE are trademarks of Waters Corporation PEEK is a trademark of Victrex Corporation PharMed is a registered trademark of Saint Gobain Ceramics amp Plastics Inc Phillips is a registered trademark of Phillips Screw Company Teflon and Tefzel are registered trademarks of E I du Pont de Nemours and Company TORX is a registered trademark of Textron Corporation TRITON is a trademark of Union C
30. ER ad TA FFE ANAS Bats ga Sel e OL EU T KK ER HUE CDI ZR RGB TTT Au er rr AERO A BE TE TREED a D ES Safety Advisories Warning Use caution when working with any polymer tubing under A pressure Always wear eye protection when near pressurized polymer tubing Extinguish all nearby flames Do not use tubing that has been severely stressed or kinked Do not use nonmetallic tubing with tetrahydrofuran THF or concentrated nitric or sulfuric acids Be aware that methylene chloride and dimethyl sulfoxide cause nonmetallic tubing to swell which greatly reduces the rupture pressure of the tubing Attention Manipulez les tubes en polym re sous pression avec precaution Portez syst matiquement des lunettes de protection lorsque vous vous trouvez proximit de tubes en polym re pressuris s Eteignez toute flamme se trouvant proximit de l instrument Evitez d utiliser des tubes s v rement d form s ou endommag s Evitez d utiliser des tubes non m talliques avec du t trahydrofurane THF ou de l acide sulfurique ou nitrique concentr Sachez que le chlorure de m thyl ne et le dim thylesulfoxyde entra nent le gonflement des tuyaux non m talliques ce qui r duit consid rablement leur pression de rupture Vorsicht Bei der Arbeit mit Polymerschl uchen unter Druck ist besondere Vorsicht angebracht In der N he von unter Druck ste
31. Ethyl alcohol 43 9 at 20 C 78 32 15 Ethyl ether 442 at 20 C 34 55 45 Ethylene dichloride 83 35 at 20 C 83 48 13 Heptane 35 5 at 20 C 98 43 4 Hexane 124 at 20 C 68 7 22 Iso octane 41 at 20 C 99 24 12 Isobutyl alcohol 8 8 at 20 C 107 7 28 Isopropyl alcohol 32 4 at 20 C 82 26 12 Isopropyl myristate lt 1 at 20 C 192 6 164 Methanol 97 at 20 C 64 7 11 Methyl t butyl ether 240 at 20 C 55 2 28 Methyl ethyl ketone 74 at 20 C 79 64 9 Methyl isobutyl ketone 16 at 20 C 117 4 18 N Methylpyrrolidone 0 33 at 25 C 202 0 86 Pentane 420 at 20 C 36 07 49 n Propyl alcohol 15 at 20 C 97 2 23 Propylene carbonate 241 7 135 Pyridine 18 at 25 C 115 25 20 Tetrahydrofuran 142 at 20 C 66 0 14 Toluene 28 5 at 20 C 110 62 4 1 2 4 Trichlorobenzene 1 at 20 C 213 5 106 Triethylamine 57 at 25 C 89 5 9 Trifluoroacetic acid 97 5 at 20 C 71 8 3 Water 17 54 at 20 C 100 0 Common solvent properties C 9 Properties of common solvents Continued Solvent Vapor Pressure Boiling Flash mm Hg Torr Point C Point C o xylene 6 at 20 C 144 41 17 Solvent miscibility Before you change solvents refer to the table below to determine solvent miscibility Be aware of these effects Changes involving two miscible solvents can be made directly Changes involving two solvents that are not totally miscible for example from chloroform to water require an intermediate solvent like n p
32. F at high concentrations and high pressure and is recommended for many ELS detector based applications where THF is used in the mobile phase at high concentrations Additives Modifiers 0 1 ethylene diaminetetraacetic acid EDTA 0 1 heptafluorobutyric acid 0 1 triethyl amine TEA 0 1 trifluoracetic acid TFA 0 2 formic acid 0 3 acetic acid 10 mM ammonium bicarbonate 10 mM phosphate buffer 50 mM ammonium acetate 50 mM ammonium hydroxide Sample diluents Acetonitrile Acetonitrile water mixtures Chloroform Dimethylformamide DMF Dimethyl sulfoxide DMSO Isooctane Isopropanol Methanol Methanol water mixtures Methylene chloride Water Recommendation Do not use buffers as needle wash Solvent recommendations C 5 Cleaning agents Recommendation See the cleaning procedures in Controlling Contamination in Ultra Performance LC MS and HPLC MS Systems part number 715001307 on the Waters Web site Visit www waters com Phosphoric acid lt 30 Sodium hydroxide lt 1M Formic acid lt 10 Solvents to avoid Avoid these solvents Solvents that contain halogens fluorine bromine or iodine Strong acids Use them only in weak concentration lt 5 unless as cleaning agents Avoid using acids as mobile phases when their pH lt 1 0 e Peroxidizable compounds such as UV grade ethers non stabilized THF dioxane and diisopropylether If you must use peroxidizable compounds be sure to
33. HB CRUE CRALET o Warnings that apply to all Waters instruments A 9 Warning The user shall be made aware that if the equipment is used in a manner not specified by the manufacturer the protection provided by the equipment may be impaired Attention L utilisateur doit tre inform que si le mat riel est utilis d une fa on non sp cifi e par le fabricant la protection assur e par le mat riel risque d tre d fectueuses Vorsicht Der Benutzer wird darauf aufmerksam gemacht dass bei unsachgem Ber Verwenddung des Ger tes die eingebauten Sicherheitseinrichtungen unter Umst nden nicht ordnungsgem funktionieren Attenzione si rende noto all utente che l eventuale utilizzo dell apparecchiatura secondo modalit non previste dal produttore puo compromettere la protezione offerta dall apparecchiatura Advertencia el usuario debera saber que si el equipo se utiliza de forma distinta a la especificada por el fabricante las medidas de protecci n del equipo podr an ser insuficientes ZH EHA ARA AA AV a a E REE S SE A AE GEIS PR ARE SS o IS BE A TEE ao F BEA DE EMR eS IR ee AIR TT AER ASA A THE K ARI EBENIS AI AZAA BAFA PS YA Ow JE ASU AS MAA DARE BS Fo AIS FEA GS F E AL BA EA AA AF HUN EB P
34. QC samples are out of range might not be valid Do not report these data until you are certain that the instrument performs satisfactorily When analyzing samples from a complex matrix such as soil tissue serum plasma whole blood and other sources note that the matrix components can adversely affect LC MS results enhancing or suppressing ionization To minimize these matrix effects Waters recommends you adopt the following measures Prior to the instrumental analysis use appropriate sample pretreatment such as protein precipitation liquid liquid extraction LLE or solid phase extraction SPE to remove matrix interferences Whenever possible verify method accuracy and precision using matrix matched calibrators and QC samples Use one or more internal standard compounds preferably isotopically labeled analytes vii Authorized representative information Authorized representative Waters Corporation Micromass UK Limited is registered in the United Kingdom with the Medicines and Healthcare Products Regulatory Agency MHRA at Market Towers 1 Nine Elms Lane London SW8 5NQ Waters Corporation Micromass UK Ltd Floats Road Wythenshawe Manchester M23 9LZ United Kingdom Telephone 44 161 946 2400 Fax 44 161 946 2480 Contact Quality manager ISM classification ISM Classification ISM Group 1 Class B viii This classification has been assigned in accordance with CISPR 11 Industrial Scie
35. TQ mass spectrometers collect data at sufficiently fast rates to describe these peaks without affecting the sensitivity or accuracy of the peak measurement These specially matched detectors employ lower flow cell volume minimized tubing volumes and specialized fittings to control bandspreading and maintain these narrow peaks For additional information You can find additional information about the ACQUITY H Class system on the system documention CD including e ACQUITY UPLC H Class Quaternary Solvent Manager Operator s Overview and Maintenance Information e ACQUITY UPLC H Class Sample Manager Flow Through Needle Operator s Overview and Maintenance Information e ACQUITY UPLC H Class Column Heater Operator s Overview and Maintenance Information System specifications ACQUITY UPLC H Class System Visit waters com to find more information and to join the ACQUITY UPLC online community where you can do these things Share ask and interact with ACQUITY UPLC experts and scientists in dynamic discussions Access ACQUITY UPLC publications and user experiences from around the globe Review exclusive FAQs tips and tricks and tutorials Explore the latest ACQUITY UPLC applications and information System components 1 11 1 12 ACQUITY UPLC H Class System Optimizing Performance Follow these tips and guidelines to help ensure optimum performance from your ACQUITY system Contents Topic Page General guidelin
36. arbide Corporation TWEEN is a trademark of ICI Americas Inc Windows is a registered trademark of Microsoft Corporation Other registered trademarks or trademarks are the sole property of their owners Customer comments Waters Technical Communications department invites you to tell us of any errors you encounter in this document or to suggest ideas for otherwise improving it Please help us better understand what you expect from our documentation so that we can continuously improve its accuracy and usability We seriously consider every customer comment we receive You can reach us at tech_comm waters com Contacting Waters Contact Waters with enhancement requests or technical questions regarding the use transportation removal or disposal of any Waters product You can reach us via the Internet telephone or conventional mail Waters contact information Contacting medium Information Internet The Waters Web site includes contact information for Waters locations worldwide Visit www waters com Telephone and fax From the USA or Canada phone 800 252 HPLC or fax 508 872 1990 For other locations worldwide phone and fax numbers appear in the Waters Web site Conventional mail Waters Corporation 34 Maple Street Milford MA 01757 USA Safety considerations Some reagents and samples used with Waters instruments and devices can pose chemical biological and radiological hazards Y
37. at all biological fluids are infectious observe Good Laboratory Practices and consult your organization s biohazard safety representative regarding their proper use and handling Specific precautions appear in the latest edition of the US National Institutes of Health NIH publication Biosafety in Microbiological and Biomedical Laboratories BMBL Safety Advisories Chemical hazard warning This warning applies to Waters instruments that can process corrosive toxic flammable or other types of hazardous material Warning Waters instruments can be used to analyze or process potentially hazardous substances To avoid injury with any of these materials familiarize yourself with the materials and their hazards observe Good Laboratory Practices GLP and consult your organization s safety representative regarding proper use and handling Guidelines are provided in the latest edition of the National Research Council s publication Prudent Practices in the Laboratory Handling and Disposal of Chemicals Caution symbol The caution symbol signifies that an instrument s use or misuse can damage the instrument or compromise a sample s integrity The following symbol and its associated statement are typical of the kind that alert you to the risk of damaging the instrument or sample Caution To avoid damage do not use abrasives or solvents to clean the instrument s case Warnings that apply to all Waters instruments
38. cid HEPES 10 mM pH 7 6 Morpholino 2 42 2 38 1 89 10 90 0 45 0 06 lt 0 01 ethanesulfo nic acid MES 10 mM pH 6 0 C 16 Solvent Considerations Mobile phase absorbance measured against air or water Continued Potassium phosphate monobasic KH PO 10 mM Absorbance at specified wavelength nm 200 0 03 205 lt 0 01 210 215 220 230 240 250 260 280 Potassium phosphate dibasic K HPO 10 mM 0 53 0 16 0 05 0 01 lt 0 01 Sodium acetate 10 mM 1 85 0 96 0 52 0 30 0 03 lt 0 01 Sodium chloride 1M 2 00 1 67 0 40 0 10 lt 0 01 Sodium citrate 10 mM 2 48 2 84 2 31 2 02 1 49 0 54 0 12 0 03 0 02 0 01 Sodium formate 10 mM 1 00 0 73 0 53 0 33 0 20 0 03 lt 0 01 Sodium phosphate 100 mM pH 6 8 1 99 0 75 0 19 0 06 0 02 0 01 0 01 0 01 0 01 lt 0 01 Tris HCI 20 mM pH 7 0 1 40 0 77 0 28 0 10 0 04 lt 0 01 Tris HCI 20 mM pH 8 0 1 80 1 90 0 43 0 13 lt 0 01 Waters PIC reagents Wavelength selection C 17 Mobile phase absorbance measured against air or water Continued Absorbance at specified wavelength nm 200 1205 1
39. cted You can specify the flow rate and duration of the prime for the new solvent line Example If a first injection uses line D1 and a second injection uses line D2 the solvent manager primes line D2 between the first and second injections Flow Ramping This feature allows you to specify the rate at which the solvent manager increases or decreases its flow ACQUITY UPLC H Class System System components Instrument modules FRONT REAR Bottle tray O OO D ro O Detector Column eer Lo Sample manager Ova flow through needle poc Quaternary solvent manager The ACQUITY UPLC H Class core system includes a quaternary solvent manager sample manager flow through needle column heater detectors tunable ultraviolet photodiode array evaporative light scattering fluorescent or mass spectrometry and an ACQUITY UPLC column Waters Empower chromatography software or MassLynx mass spectrometry software controls the ACQUITY UPLC H Class system System components 1 7 1 8 Quaternary solvent manager The quaternary solvent manager is a low pressure mixing high pressure pump that provides steady pulse free solvent flow at analytical flow rates up to 1 mL min at 103 421 kPa 1034 bar 15 00
40. ction pathway The purge solvent also primes the sample syringe and injection pathway It is injected onto the column only during auto dilution when it is used as the dilution solvent Active solvent conditioning HPLC and UPLC applications benefit from additional pre column mobile phase heating to improve chromatographic separations The ACQUITY UPLC H Class column heater uses an active preheater to condition solvent as it enters the column The active preheater is a heat source that 1 4 ACQUITY UPLC H Class System raises the temperature of the incoming mobile phase and injected sample to the same set point as the column compartment Tip Active preheating is the default configuration for the ACQUITY UPLC H Class system An optional passive column stabilizer is available for existing chromatographic methods that are not suited for active preheating Software enhancements Quantum Synchronization Introducing a low pressure sample into the high pressure fluid stream during injection causes a pressure pulse that can affect chromatographic results The Quantum Synchronization feature reduces the impact of this pressure pulse The sample manager and solvent manager communicate to automatically coordinate the injection sequence enabling the solvent manager to provide additional pressure at the exact moment the sample manager switches its injector valve to the inject position to introduce the low pressure sample Gradient Smart Start
41. d them with appropriate organic modifiers After you use a buffer flush it from the pump by running a wet prime with at least five system volumes of HPLC grade distilled or deionized water For shutdowns of more than a day flush the pump with a 20 methanol water solution to prevent microbial growth Caution Some buffers can be incompatible with mass spectrometers Consult the documentation that accompanies your instrument for compatible buffers Tip To avoid salt precipitation nonvolatile buffer concentrations must not exceed 100 mM Buffered solvents When using a buffer choose good quality reagents filtering them through a 0 2 um membrane filter Recommendation To discourage microbial growth replace 100 mobile aqueous phase daily See also For information on preventing contamination refer to Controlling Contamination in Ultra Performance LC MS and HPLC MS Systems part number 715001307 on the Waters Web site Visit www waters com Solvent recommendations The ACQUITY UPLC H Class system was designed for reversed phase chromatography and ACQUITY UPLC BEH column chemistries Waters evaluated the system s reliability using traditional reversed phase solvents This section lists solvents recommended for the ACQUITY UPLC H Class system Contact Waters Customer Service to determine whether you can use solvents that do not appear in the list without adversely affecting instrument or system performance General solven
42. detector flow cell becomes contaminated with residual stabilizer and a substantial cleaning effort is needed to restore the flow path to its initial condition Solvent viscosity Generally viscosity is not a consideration when you operate with a single solvent or under low pressure However with gradient chromatography the viscosity changes that occur as the solvents are mixed in different proportions can effect pressure changes during the run For example a 1 1 water methanol mixture produces twice the pressure of either water or methanol alone If you do not know the extent to which pressure changes affect the analysis monitor the pressure during the run Wavelength selection The tables in this section provide UV cutoff values for these items Common solvents Common mixed mobile phases Chromophores UV cutoffs for common solvents The table below shows the UV cutoff the wavelength at which the absorbance of the solvent equals 1 AU for some common chromatographic solvents C 12 Solvent Considerations Operating at a wavelength near or below the cutoff increases baseline noise because of solvent absorbance UV cutoff wavelengths for common chromatographic solvents Solvent aa Acetone 330 Acetonitrile 190 Diethyl amine 275 Ethanol 210 Isopropanol 205 Isopropyl ether 220 Methanol 205 n Propanol 210 Tetrahydrofuran THF 230 Mixed mobile phases The following table
43. dling symbols Electrical symbols These can appear in instrument user manuals and on the instruments front or rear panels Electrical power on Electrical power off Standby C O Direct current Alternating current Protective conductor terminal Frame or chassis terminal Fuse Recycle symbol Do not dispose in municipal waste B i M A 12 Safety Advisories Handling symbols These handling symbols and their associated text can appear on labels affixed to the outer packaging of Waters instrument and component shipments 11 Keep upright Keep dry Fragile CR I X Use no hooks Electrical and handling symbols A 13 A 14 Safety Advisories B External Connections This section describes the ACQUITY UPLC H Class system s external connections Note A Waters Technical Service representative unpacks and installs your ACQUITY UPLC H Class instruments Warning To avoid back injuries do not attempt to lift the instruments without assistance Caution Contact Waters Technical Service before moving the ACQUITY UPLC H Class instruments Ifyou must transport an instrument or remove it from service contact Waters Technical Service for recommended cleaning flushing and packaging procedures Contents Topic Page System tubing connections B 1 Instrument external wiring connections B 3 Signal connections B 4
44. ds and telling you how to avoid them This appendix presents all the safety symbols and statements that apply to the entire line of Waters products Contents Topic Page Warning symbols A 2 Caution symbol A 5 Warnings that apply to all Waters instruments A 5 Electrical and handling symbols A 12 A 1 Warning symbols Warning symbols alert you to the risk of death injury or seriously adverse physiological reactions associated with an instruments use or misuse Heed all warnings when you install repair and operate Waters instruments Waters assumes no liability for the failure of those who install repair or operate its instruments to comply with any safety precaution Task specific hazard warnings The following warning symbols alert you to risks that can arise when you operate or maintain an instrument or instrument component Such risks include burn injuries electric shocks ultraviolet radiation exposures and others When the following symbols appear in a manuals narratives or procedures their accompanying text identifies the specific risk and explains how to avoid it Warning General risk of danger When this symbol appears on an instrument consult the instruments user documentation for important safety related information before you use the instrument A Warning Risk of burn injury from contacting hot surfaces A Warning Risk of electric shock A Warning Risk of fire A Warning Risk of needle pu
45. es 2 2 ACQUITY UPLC columns calculator 2 3 Dispersion 2 4 Carryover 2 4 Reproducibility 2 5 Cycle time between injections 2 5 Preventing leaks 2 6 Sample preparation 2 6 2 1 General guidelines 2 2 ACQUITY UPLC H Class system guidelines differ from standard HPLC practices primarily due to the constraints that the chromatography of small less than 2 um particles places on the system Chromatography on a UPLC system 1s a much smaller scale higher resolution separation than when using HPLC Analysis time is shorter with UPLC and solvent and sample consumption are significantly reduced The ACQUITY UPLC H Class chromatograph requires optimum performance from the sample manager because sample dispersion is more evident when using smaller columns The reduction in chromatographic run time also makes efficient management of cycle time essential When performing fast UPLC analyses note that a peak of interest can be less than 0 5 seconds Waters recommends a sampling rate of 25 to 50 points across the peak which provides good quantitation and peak representation Sampling rates faster than 20 points per second yield higher baseline noise and you should adjust filter time constants accordingly The optimal ACQUITY UPLC flow rate differs from that of a typical HPLC column The table below offers operating guidelines for ACQUITY UPLC columns under both 1socratic and gradient conditions Note that the values provided are approximati
46. graphic methods that are not suited for active preheating Local Console Controller optional The ACQUITY UPLC Local Console Controller LCC complements chromatography data system CDS software enabling you to control the systems locally Designed to emulate a simple keypad the LCC s minimal functionality bars it from operating as a standalone controller and its installation in a system does not supplant CDS control Rather Waters designed the LCC to prepare system modules for operation define initial ACQUITY UPLC H Class System conditions and run ACQUITY UPLC system diagnostic tests These basic functions are rapidly performed even when a system is remote from the software control and acquisition workstation or LAC E 32 module or when network control is unavailable FlexCart The optional FlexCart provides for the ACQUITY UPLC H Class system a mobile platform It can hold the system instruments as well as the PC and monitor and provides electrical outlets for system instruments and integrated waste management Used with a mass spectrometer the cart s adjustable height lets you position the column outlet close to the inlet probe minimizing system dead volume Column technology ACQUITY UPLC columns are packed with 1 7 um bridged ethylsiloxane hybrid or 1 8 um high strength silica particles that can mechanically endure high pressure conditions The column hardware and the matched outlet tubing can withstand up to 103 421
47. h system in the Quaternary Solvent Manager Operator s Overview and Maintenance Information Wash plungers Initiates the plunger wash sequence which fills and then slowly empties the primary and accumulator chambers with the current solvent composition while performing a high speed volume seal wash This helps to prevent the build up of precipitates on the pump plungers which can damage the high pressure seals Launch ACQUITY UPLC Console Launches the ACQUITY UPLC Console Reset QSM Resets the quaternary solvent manager after an error condition Help Displays the ACQUITY UPLC Console online Help 3 10 Preparing the System Sample manager control panel The sample manager control panel displays current sample compartment and column heater temperatures and set points You can edit these values when the system is idle by click the underlined value You cannot edit sample manager set points while the system is running samples Tip To keep the sample compartment at a constant temperature open its door only when necessary The sample manager s fans stop circulating air whenever the sample compartment door is open Sample manager control panel Run LED Status Sample Manager FTH gt un Waiting for QSM Current sample M Current column heater compartment sample 27 8 Colina 40 0 temperature temperature 40 0 Off LI Sample Eos Display ACQUITY UPLC compartment set Col
48. he miscibility of two liquids subtract the smaller M number value from the larger M number value Ifthe difference between the two M numbers is 15 or less the two liquids are miscible in all proportions at 15 C A difference of 16 indicates a critical solution temperature from 25 to 75 C with 50 C as the optimal temperature Ifthe difference is 17 or greater the liquids are immiscible or their critical solution temperature is above 75 C Some solvents prove immiscible with solvents at both ends of the lipophilicity scale These solvents receive a dual M number The first number always lower than 16 indicates the degree of miscibility with highly lipophilic solvents The second number applies to the opposite end of the scale A large difference between these two numbers indicates a limited range of miscibility For example some fluorocarbons are immiscible with all the standard solvents and have M numbers of 0 and 32 Two liquids with dual M numbers are usually miscible with each other A liquid is classified in the M number system by testing for miscibility with a sequence of standard solvents A correction term of 15 units is then either added or subtracted from the cutoff point for miscibility Solvent miscibility C 11 Solvent stabilizers Do not leave solvents containing stabilizers like THF with butylated hydroxytoluene BHT to dry in the system s flow path A dry flow path including the
49. henden Polymerschl uchen stets Schutzbrille tragen Alle offenen Flammen in der Nahe l schen Keine Schl uche verwenden die stark geknickt oder berbeansprucht sind Nichtmetallische Schl uche nicht f r Tetrahydrofuran THF oder konzentrierte Salpeter oder Schwefels ure verwenden Durch Methylenchlorid und Dimethylsulfoxid k nnen nichtmetallische Schl uche quellen dadurch wird der Berstdruck des Schlauches erheblich reduziert Warnings that apply to all Waters instruments A 7 A A 8 Attenzione fare attenzione quando si utilizzano tubi in materiale polimerico sotto pressione Indossare sempre occhiali da lavoro protettivi nel pressi di tubi di polimero pressurizzati Spegnere tutte le fiamme vive nell ambiente circostante Non utilizzare tubi eccessivamente logorati o piegati Non utilizzare tubi non metallici con tetraidrofurano THF o acido solforico o nitrico concentrati Tenere presente che il cloruro di metilene e il dimetilsolfossido provocano rigonfiamenti nei tubi non metallici riducendo notevolmente la pressione di rottura dei tubi stessi Advertencia se recomienda precauci n cuando se trabaje con tubos de polimero sometidos a presi n El usuario debera protegerse siempre los ojos cuando trabaje cerca de tubos de pol mero sometidos a presi n Si hubiera alguna llama las proximidades No se debe trabajar con tubos que se hayan doblado o sometido a altas presiones Es necesario utilizar
50. ing to minimize carryover Consider contamination from sample preparation tools when seeking possible sources of carryover peaks Reproducibility The precision area reproducibility when using the SM FTN is less than 0 5 RSD for injection volumes from 2 0 to 10 0 uL See ACQUITY UPLC H Class System Specifications for details Cycle time between injections The short run time of a UPLC separation requires efficient use of the time between analyses The ACQUITY SM FTN has a load ahead option that can help decrease cycle time This option instructs the sample manager to aspirate the next sample while the current sample is running The loop offline option on the SM FTN reduces the impact of delay volume on cycle time by taking the needle and extension loop offline before the gradient reaches the injection valve and after the sample transfers to the injection port Reproducibility 2 5 Setting an appropriate syringe draw rate can also help reduce cycle time By default the system uses feedback information from a pressure transducer to optimize the syringe draw rate for maximum throughput and performance Preventing leaks Preventing leaks ensures that the system maintains adequate pressure and sample integrity throughout the analysis Leaks can potentially occur at any tubing connection gasket or seal but are most common at tubing connections Low pressure leaks on the intake side of the solvent manager s pump cause
51. involved a continuous flow of nitrogen into the ion source is required to prevent possible ignition in that enclosed space Ensure that the nitrogen supply pressure never falls below 690 kPa 6 9 bar 100 psi during an analysis in which flammable solvents are used Also ensure a gas fail connection is connected to the LC system so that the LC solvent flow stops if the nitrogen supply fails Warning symbols A 3 A 4 Mass spectrometer shock hazard This warning applies to all Waters mass spectrometers Warning To avoid electric shock do not remove the mass spectrometer s protective panels The components within are not user serviceable This warning applies to certain instruments when they are in Operate mode Warning High voltages can be present at certain external surfaces of the mass spectrometer when the instrument is in Operate mode To avoid non lethal electric shock make sure the instrument is in Standby mode before touching areas marked with this high voltage warning symbol Biohazard warning This warning applies to Waters instruments that can be used to process material that might contain biohazards substances that contain biological agents capable of producing harmful effects in humans Warning Waters s instruments and software can be used to analyze or process potentially infectious human sourced products inactivated microorganisms and other biological materials To avoid infection with these agents assume th
52. kPa 1034 bar 15 000 psi The column dimensions allow optimal MS compatible flow rates and matched outlet tubing minimizes the effect of extra column volume Although the system works with any analytical HPLC column specially designed ACQUITY UPLC columns maximize its high pressure capabilities Compared with traditional HPLC columns ACQUITY UPLC columns deliver superior resolution and sensitivity in the same run time or equivalent resolution greater sensitivity and faster run times eCord technology ACQUITY UPLC columns include an eCord column chip that tracks the usage history of the column The eCord column chip interacts with the system software recording information for up to 50 sample queues run on the column In regulated environments the eCord column chip provides documentation of the column used in the validation method System components 1 9 In addition to the variable column usage data the eCord column chip also stores fixed column manufacturing data including unique column identification certificate of analysis QC test data When you attach the eCord column chip to the receptacle on the column heater information is automatically recorded by the system No user action is required This information is stored only in the eCord column chip Detectors The small particle chemistries utilized in UPLC system chromatography produce very narrow peaks The ACQUITY TUV PDA ELS and FLR detectors and SQ and
53. l to ensure that it is never too high Start gradients with some organic content 0 1 for example to provide more consistent and predictable gradient formation than when you start with 0 organic Use the Load Ahead option when you desire a shorter cycle time Do not use the Load Ahead or Loop Offline options when you are troubleshooting carryover problems When installing or removing a column always hold the active preheater s reusable compression fitting in place Rotate the column or optional in line filter to install or remove it ACQUITY UPLC columns calculator The ACQUITY UPLC columns calculator is a software tool that helps you transfer methods from an HPLC system to a UPLC system or from a UPLC system to an HPLC system The calculator differentiates between systems with binary and quaternary pumps When you input parameter values from your current separation choose a target column with similar resolving power L dp values are automatically calculated and displayed and input the dwell volumes for your current and ACQUITY UPLC columns calculator 2 3 target systems the calculator recommends chromatographic conditions for the target system You can further optimize these conditions based on your particular requirements See also The ACQUITY UPLC columns calculator documentation and the ACQUITY Console online Help for additional details and methodology Tip You can install the calculator from the ACQUITY
54. ments ss A 5 Electrical and handling symbols aaaaaaaataass sssnnnnnsnnnsnnnnnnonnoannannnnnnnnnnnnnnennn A 12 Ele BEA rra A 12 Handling symbols aa A 13 B External Connections nina i B 1 System tubing CONNECTIONS s cssesssccssassicessesssscsssscsssecvacesssseessseseseesesseceeseasesvass B 1 Instrument external wiring connections ss B 3 ACQUITY UPLC H Class instrument external wiring connections B 3 Ethernet connections A E B 4 Colunn heater OIE aan B 4 Sianal Connections ua B 4 Makne se al connetti ai sme B 4 Connecting to the electricity source ss B 9 C Solvent Considerations ainia C 1 Introduction ns C 2 P la SA de intra C 2 A Eegeregie C 2 Solvent UE EE C 2 Solvent PP A dI daa C 2 WILEY PODA O et ta aris un oo iri re ees C 2 Solvent recommendations scecssesssssossssseosscosecoscasoacossassasesscssacsssssscssssessevssseess C 3 General rent EE nissan C 3 Table of Contents Xi xii Solvents to FOIE A a TT R NETANE C 6 ACQUITY UPLC H Class system recommendations ssssssssessssssssssesreeeess C 6 Quaternary solvent manager recommendations C 7 Sample manager recommendatonsg C 8 Detector recommendations sine C 8 Common solvent properties ss C 8 el E EE e UE EE C 10 Using miscibility numbers OM oumbere nono non nronononononos C 11 Solvent stabilizers asalkan ii C 12 Solyent VISCOSLLY EE C 12 Wavelength s l ction sescisssssssssisvsssentosecsssasau
55. minants which cause baseline drift Chloroform methylene chloride halogenated solvents and toluene are generally not recommended for use in ACQUITY UPLC H Class systems Nevertheless you can use these solvents in weak dilutions lt 10 as additives sample diluents or modifiers Contact your Waters sales representative or local technical support organization to determine whether a specific method is suitable to use with the ACQUITY UPLC H Class system instruments and components When using THF or hexane install stainless steel tubing and minimize the use of PEEK components Aqueous solvents must not remain in a shut down system because they serve as a substrate for microbial colonies Microbes can clog system filters and capillary lines To prevent their proliferation add a small amount 10 of an organic solvent such as acetonitrile or methanol Methanesulfonic acid is not recommended for use in ACQUITY UPLC H Class systems Quaternary solvent manager recommendations The seal wash system must never run dry particularly during separations that use a polar mobile phase Isopropyl alcohol or mixtures of methanol and water like 20 methanol water are effective seal wash solvents for THF solvent mixtures Solvent recommendations C 7 For reversed phase applications use aqueous seal wash solutions with a weak organic component for example 1 9 methanol water Do not use 100 organic seal wash solu
56. ncture A Warning Risk of injury caused by moving machinery A Warning Risk of exposure to ultraviolet radiation Warning Risk of contacting corrosive substances Warning Risk of exposure to a toxic substance Warning Risk of personal exposure to laser radiation A 2 Safety Advisories Warning Risk of exposure to biological agents that can pose a serious health threat Warnings that apply to particular instruments instrument components and sample types The following warnings can appear in the user manuals of particular instruments and on labels affixed to them or their component parts Burst warning This warning applies to Waters instruments fitted with nonmetallic tubing Warning Pressurized nonmetallic or polymer tubing can burst Observe these precautions when working around such tubing e Wear eye protection Extinguish all nearby flames Do not use tubing that is or has been stressed or kinked Do not expose nonmetallic tubing to incompatible compounds like tetrahydrofuran THF and nitric or sulfuric acids e Be aware that some compounds like methylene chloride and dimethyl sulfoxide can cause nonmetallic tubing to swell which significantly reduces the pressure at which the tubing can rupture Mass spectrometer flammable solvents warning This warning applies to instruments operated with flammable solvents Warning Where significant quantities of flammable solvents are
57. nsors default to disabled unless previously enabled To enable the leak sensors 1 Inthe ACQUITY UPLC Console select Control gt Leak Sensors Leak Sensors dialog box Disabled Ge Solvent _Enable All Click to enable Disable All or disable all instrument Disablel ksa leak sensors z E E Ka O Sample Manager TUY Detector h x Click to enable or disable individual instrument leak sensors 2 To enable the leak sensor for an individual instrument click the status on the left hand side of the instrument description Tip To enable all leak sensors click Enable All Starting up the system Use the Start up system function to prime the quaternary solvent manager after changing the mobile phase after changing the sample needle or after the system has been idle a long period of time for example overnight Before you begin this procedure ensure that the system is correctly configured for use Recommendation Prime the quaternary solvent manager for a minimum of 5 minutes 1f you are changing to solvents whose compositions differ from the compositions of solvents already in the system Preparing system hardware 3 5 3 6 To start up the system 1 2 In the ACQUITY UPLC Console click Control gt Start up system In the Prime Solvents tab of the System Startup dialog box review the settings for the A B C D Solvents mobile phase Tip In
58. ntific and Medical ISM instruments requirements Group 1 products apply to intentionally generated and or used conductively coupled radio frequency energy that is necessary for the internal functioning of the equipment Class B products are suitable for use in both commercial and residential locations and can be directly connected to a low voltage power supply network Table of Contents Ada ums laa a aa 11 Trademarks inicia 11 Customer COMMENTS soriana N EKATARINA NEEE Esaa RERA iii Contacting Waters E iv Safety CONSIDERATIONS EE iv Considerations specific to the ACQUITY UPLC H Class instruments v AI anti E AS v Operating the ACQUITY UPLC H Class instruments ss vi Applicable symbols ninia vi Ahente and dd tendue vi Intended use of the ACQUITY UPLC H Class system vi CARDO a Eed vil VV E vii Authorized representative information esse viii Authorized representative viii ISM classification A aan AREA IAE viii ISM Classification ISM Group 1 Class Bo vili 1 ACQUITY UPLC H Class System aaaaaaaasasnt ncnnansnnannnnnnnnnnnnnnnnnnnnnnn 1 1 UltraPerformance Liquid Chromatography sesssesssessoossosssoossosesoossossesossoo 1 2 Features of the ACQUITY UPLC H Class system sessesssesssossosssooseooeecoeeoo 1 3 Flow through needle injector sn 1 4 Wael VAN n cessaire 1 4 PARES SOS nr 1 4 OS solvent COOL tatin aline 1 4 NE SRA OSIRIS ra arr 1 5 System TE tala a aaa a i 1 7 Column technology Ro 1 9 Table of C
59. o 100 i C 0 to 100 D 0 to 100 Column temperature Off Depends on type of column compartment Sample temperature On Off or 4 0 to 40 0 C 39 2 to 104 F Lamp On On or off Note For light guiding flow cells do not power on operate or ignite the lamp of the detector when there is no flow through the cell or when the cell is dry 7 Ifyou changed the sample needle click Change 8 In the Volume Configuration dialog box select the size of the new needle and then click OK 9 Click Start Result The lamp in the optical detector ignites the ACQUITY UPLC H Class system sets the column and sample temperatures and all priming starts After priming finishes the sample manager characterizes the needle and seal if selected and then logs the results of the characterizations into the database Finally the system establishes the method flow rate solvent selections and composition Preparing system hardware 3 7 Configuring chromatography data software Configure the chromatography data system software for use with ACQUITY __ Start the chromatography data system software and log in Select system instruments and name the system see Empower or MassLynx Help for details Open the ACQUITY Console and control panels ACQUITY control panels You can monitor control panels for the quaternary solvent manager sample manager and detector from your chromatography data system Control panels
60. ons and that optimum performance for your molecule or separation can occur at a different flow rate and or pressure Optimal flow rates for molecular weight range Column size Molecular weight Flow rate 2 1 X 50 mm lt 500 600 uL min 2 1 x 50 mm 1000 300 uL min 2 1 X 50 mm 1500 150 uL min 2 1 X 50 mm 2000 100 uL min Follow these general recommendations when running a UPLC analysis Use high quality solvents buffers and additives HPLC or MS grade Use high quality water HPLC or MS grade Always use solvent filters on tubing lines in solvent bottles Filter buffers with a 0 2 um filter membrane Optimizing Performance Keep concentrated stock solutions to use when preparing working solutions Do not top off buffers which can promote microbial growth Do not block the degasser vent line Trim the tubing if necessary Do not submerge the waste or degasser vent lines in liquid See the ACQUITY UPLC H Class Quaternary Solvent Manager Operator s Overview and Maintenance Information for details on how to route the tubing Keep all solvent lines primed Flush buffers out of the system with water immediately after you use them Use 10 20 organic solvent in water as a storage solvent if you keep the system idle for extended periods of time longer than 24 hours Keep the seal wash line primed Prime solvent lines during System Start up e Monitor the waste leve
61. ontents ix For additional M O MATIOM assuusunsbsnna tuas vk i SK KS T L L ones 1 10 2 Optimizing Performance anamnesis 2 1 General guidelines sslsmsvsakasts li a aaa basa Eas Eas 2 2 ACQUITY UPLC columns calculator cccsssessssessesescsssescssessessacessessecsese 2 3 DIS POF SION ainia aaa 2 4 Carryover E 2 4 Reproducibility isis aan 2 5 Cycle time between injections ss 2 5 Preyentino leaks iniciacion usus 2 6 Sample preparation sacra ainia 2 6 Parten krn sil 2 6 Matching sample diluents sisi 2 6 3 Preparino EE 3 1 Preparing system hardware assm st nsm kinufl st ttaslsksslklisikuni 3 1 Powerme n te syster A 3 1 Monitoring startup aia 3 2 Monitoring system metrument LEDS ii ka alar a Ga akan Haka 3 2 Poner LED oasis 3 2 Ss E A 3 3 bios the lenk sensors o 3 5 EEE ES a A A 3 5 Configuring chromatography data software ss 3 8 ACQUITY control panels slbanms nssl sbusnlassaka nar 3 8 Quaternary solvent manager control pare union o 3 8 Sample manager control panel sss ka 3 11 TUV detector control panel aiiiiiiiaiiiiaiiiasaaasaaasananaatnntannnnnntnnnnnnnnnnnniia 3 12 x Table of Contents Starting the ACQUITY UPLC Console aa avesttstttt nninnnnnnannnnnnnnnnnnnnnnnn r 3 14 A Safety AUVISOTISS mono noia A 1 KE e TE A 2 EE ER A 2 Warnings that apply to particular instruments instrument components and sample FBG da ee A 3 a oo A s A 5 Warnings that apply to all Waters instru
62. ou must know the potentially hazardous effects of all substances you work with Always follow Good Laboratory Practice and consult your organization s safety representative for guidance When you develop methods follow the Protocol for the Adoption of Analytical Methods in the Clinical Chemistry Laboratory American Journal of Medical Technology 44 1 pages 30 37 1978 This protocol addresses good operating procedures and the techniques necessary to validate system and method performance Considerations specific to the ACQUITY UPLC H Class instruments High voltage hazard Warning To avoid electric shock do not remove the ACQUITY UPLC H Class instruments protective panels The components within are not user serviceable Safety advisories Consult the Safety Advisories information on the documentation CD for a comprehensive list of warning and caution advisories Operating the ACQUITY UPLC H Class instruments When operating the ACQUITY UPLC H Class instruments follow standard quality control QC procedures and the guidelines presented in this section Applicable symbols A lt 3 A EC REP Definition Authorized representative of the European Community A mo Confirms that a manufactured product complies with all applicable European Community directives amp ABN 49 065 444 751 N Australia C Tick EMC Compliant Ate EM Ken Confirms that a manufactured produc
63. quaternary solvent manager are lost Status Displays the status of the current operation System Pressure Displays system pressure in kPa bar or psi You can customize pressure units via the ACQUITY UPLC Console Flow Rate Displays the flow rate of solvent through all lines of the quaternary solvent manager from 0 000 to 2 000 mL min under normal operation and 0 000 to 4 000 mL min when priming Solvent Composition Displays the percentage of solvent to be drawn from the solvent lines A through D Composition values range from 0 0 to 100 0 Stop Flow Immediately stops all flow from the quaternary solvent manager ACQUITY control panels 3 9 You can access these additional functions by right clicking anywhere in the quaternary solvent manager control panel Additional functions in the quaternary solvent manager control panel Control panel function Start up system Prime solvents Description Brings the system to operational conditions after an extended idle period or when switching to different solvents See Starting up the system in the Quaternary Solvent Manager Operator s Overview and Maintenance Information Displays the Prime Solvents dialog box See Priming the quaternary solvent manager in the Quaternary Solvent Manager Operator s Overview and Maintenance Information Prime seal wash Starts priming the seal wash See Priming the seal was
64. ropanol Temperature affects solvent miscibility If you are running a high temperature application consider the effect of the higher temperature on solvent solubility Buffers dissolved in water can precipitate when mixed with organic solvents When you switch from a strong buffer to an organic solvent thoroughly flush the system with distilled water before you add the organic solvent Solvent miscibility 1 Viscosity Boilin Miscibilit os Solvent cP 20 C point c number i a 1 atm 1 atm M 0 0 N hexane 0 313 68 7 29 1 8 Triethylamine 0 38 89 5 26 4 2 Tetrahydrofuran THF 0 55 66 0 17 220 4 3 1 propanol 2 30 97 2 15 210 4 3 2 propanol 2 35 117 7 15 5 2 Ethanol 1 20 78 3 14 210 5 4 Acetone 0 32 56 3 15 17 330 5 5 Benzyl alcohol 5 80 205 5 13 5 7 Methoxyethanol 1 72 124 6 13 6 2 Acetonitrile 0 37 81 6 11 17 190 C 10 Solvent Considerations Solvent miscibility Continued Viscosity Boilin Miscibilit SE Solvent cP 20 point e number A 1 atm 1 atm M 6 2 Acetic acid 1 26 117 9 14 6 4 Dimethylformamide 0 90 153 0 12 6 5 Dimethylsulfoxide 2 24 189 0 9 6 6 Methanol 0 60 64 7 12 210 9 0 Water 1 00 100 0 Using miscibility numbers M numbers Use miscibility numbers M numbers to predict the miscibility of a liquid with a standard solvent To predict t
65. ssssseusseesecessussessesesecestescesasscesseesss C 12 UV etotte Tor Gono slo a A lan C 12 Mixed mobile Mo aa ti C 13 Mobile phase absorbant ai intrants C 14 Table of Contents 1 ACQUITY UPLC H Class System Waters designed the ACQUITY UPLC H Class system to perform both UPLC and HPLC analyses Hardware and software features facilitate transferring between the two types of chromatography Contents Topic Page UltraPerformance Liquid Chromatography 1 2 Features of the ACQUITY UPLC H Class system 1 3 System components 1 7 1 1 UltraPerformance Liquid Chromatography 1 2 In 2004 Waters made significant advances in instrumentation and column design to introduce UPLC technology to the field of separation science By employing this technology Waters ACQUITY UPLC systems achieve a marked increase in resolution speed and sensitivity in liquid chromatography when compared to conventional systems Ultra Performance liquid chromatography uses columns packed with 1 7 um diameter round particles and operating pressures up to 15 000 psi The van Deemter equation an empirical formula that describes the relationship between linear velocity and column efficiency considers particle size as one of the variables Thus the equation can be used to characterize theoretical performance across a range of particle sizes History of particle size in liquid chromatography Existing HPLC Technology UPLC Technology Operating Range
66. t complies with all applicable United States and Canadian safety requirements C This product has been tested to the requirements of CAN CSA C22 2 No 61010 1 second edition including Amendment 1 or a later version of the same standard incorporating the same level of testing requirements Audience and purpose This guide is intended for personnel who install operate and maintain ACQUITY UPLC H Class instruments It gives an overview of the system s technology and operation Intended use of the ACQUITY UPLC H Class system The Waters ACQUITY UPLC H Class System is for research use only vi Calibrating To calibrate LC systems follow acceptable calibration methods using at least five standards to generate a standard curve The concentration range for standards must include the entire range of QC samples typical specimens and atypical specimens When calibrating mass spectrometers consult the calibration section of the operator s guide for the instrument you are calibrating In cases where an overview and maintenance guide not operator s guide accompanies the instrument consult the instrument s online Help system for calibration instructions Quality control Routinely run three QC samples that represent subnormal normal and above normal levels of a compound Ensure that QC sample results fall within an acceptable range and evaluate precision from day to day and run to run Data collected when
67. t guidelines Always observe the following general solvent guidelines Solvent recommendations C 3 C 4 e Use high quality brown tinted glassware to inhibit microbial growth Filter solvents Small particles can permanently block a system s capillary lines Filtering solvents also improves check valve performance Recommended solvents e Acetonitrile e Acetonitrile water mixtures e Isopropanol Methanol Methanol water mixtures Water Other solvents You can use the following solvents Note however that these solvents can shorten instrument life If you routinely use the solvents on this list Waters recommends you install the Hexane THF Compatibility Kit Tetrahydrofuran THF Hexane Acetone Ethyl acetate Hexafluoroisopropanol HFIP Notes 1 4 aqueous solutions of HFIP for oligonucleotide applications HFIP should never be used in wash solvents For additional information see page C 6 Consider solvent polarity when you change typical reversed phase solvents Flush the system with a solvent of intermediate polarity like isopropanol before introducing nonpolar solvents like THF or hexane Hexane THF Compatibility Kit The ACQUITY UPLC System Hexane THF Compatibility Kit contact Waters for part number can be installed in ACQUITY UPLC systems with closed Solvent Considerations waste management It is designed for users that need to run their systems with hexane or TH
68. terials 9 32 inch nut driver Flat blade screwdriver Connector Signal cable To make signal connections 1 Insert the connector into the connector port on the back of the instrument Connector port Connector Using the flat blade screwdriver attach the positive and negative leads of the signal cable to the connector Screw AD C Connector Signal cable Signal connections B 5 3 Place the grounding cable fork terminal on the grounding stud on the back of the instrument and secure it with the locking nut Use the 9 32 inch nut driver to tighten the locking nut until the fork terminal does not move Fork terminal AFE R c Locking nut Grounding stud radient Start Quaternary solvent manager I O signal connectors The rear panel of the quaternary solvent manager includes a removable connector that holds the screw terminals for I O signal cables This connector is keyed so that it can be inserted only one way Quaternary solvent manager UO signal connections Gradient Start Gradient Start Ground Ground Stop Flow Stop Flow For electrical specifications see the ACQUITY UPLC H Class System Specifications B 6 External Connections Quaternary solvent manager event in connections Signal connection Description Gradient Start Initiates the pumps to begin gradient operation by either contact closure input or
69. ternal wiring connections B 3 Ethernet connections The sample manager incorporates an internal Ethernet switch that accommodates the PC workstation and up to six ACQUITY UPLC H Class modules Connect the shielded Ethernet cables from each module to the electronic connections on the rear panel of the sample manager The sample manager is connected internally to the Ethernet switch Column heater connection The sample manager powers and communicates with the column heater The external communication cable must be connected to the rear of the column heater and the sample manager To make column heater connections Caution To avoid damaging electrical parts never disconnect an electrical assembly while power 1s applied to an instrument To interrupt power to an instrument set the power switch to Off and then unplug the power cord from the AC outlet After power is removed wait 10 seconds thereafter before you disconnect an assembly 1 Make sure the sample manager and the column heater are powered off 2 Connect the external communication cable to the High Density HD port on the rear of the column heater 3 Connect the other end of the external communication cable to the QSPI port on the rear of the sample manager Signal connections Making signal connections Refer to the signal connection location shown on the silk screened label affixed to the rear panel of each instrument B 4 External Connections Required ma
70. tions Sample manager recommendations Do not use concentrations of THF or hexane greater than 10 as purge solvent Typical organic sample diluents such as dimethylsulfoxide DMSO and dimethylformamide DMF are supported Detector recommendations To transport a flow cell while temperatures are below 5 C fill it with alcohol Common solvent properties The following table lists the properties for some common chromatographic solvents Properties of common solvents Solvent Vapor Pressure Boiling Flash mm Hg Torr Point C Point C Acetone 184 5 at 20 C 56 29 20 Acetonitrile 88 8 at 25 C 81 6 6 n butyl acetate 7 8 at 20 C 126 11 22 n butyl alcohol 4 4 at 20 C 117 5 37 n butyl chloride 80 1 at 20 C 78 44 9 Chlorobenzene 8 8 at 20 C 131 69 28 Chloroform 158 4 at 20 C 61 15 Cyclohexane 77 5 at 20 C 80 72 20 Cyclopentane 400 at 20 C 49 26 7 o Dichlorobenzene 1 2 at 20 C 180 48 66 Dichloromethane 350 at 20 C 39 75 Dimethyl acetamide 1 3 at 25 C 166 1 70 C 8 Solvent Considerations Properties of common solvents Continued Solvent Vapor Pressure Boiling Flash mm Hg Torr Point C Point C N N Dimethylformamide 2 7 at 20 C 153 0 58 Dimethyl sulfoxide 0 6 at 25 C 189 0 88 1 4 Dioxane 29 at 20 C 101 32 12 Ethyl acetate 73 at 20 C 77 11 4
71. umn heater set Console point point The following table describes the items in the sample manager s control panel Sample manager control panel items Control panel item Description Run LED Displays the actual run LED on the front panel unless communications are lost Status Displays the status of the current operation Current Sample Displays the current sample compartment Compartment Temperature temperature to 0 1 C resolution even when active temperature control is disabled Sample Compartment Set Displays the current sample compartment Point set point to 0 1 C resolution When active temperature control is disabled this field displays Off ACQUITY control panels 3 11 Sample manager control panel items Continued Control panel item Description Current Column Heater Displays the current column heater Temperature temperature to 0 1 C resolution even when active temperature control is disabled Column Heater Set Point Displays the current column heater set point to 0 1 C resolution When active temperature control is disabled this field displays Off Displays the ACQUITY UPLC Console A Display Console You can access these additional functions by right clicking anywhere in the sample manager control panel Additional functions in the sample manager control panel Control panel function Description Prime Displays the Prime dialog box

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