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Quattro LC User's Guide, issue 2. 6666525

1. Hardware Specifications Page 14 Quattro LC User s Guide Overview Sample Inlet Sampling Cone D Extraction Cone Samples SS from the liquid C3 introduction system are introduced at atmospheric pressure into the o IUE ionisation source SG Detector Ions are sampled through a series of orifices The ions are filtered according to their mass to charge B ratio HYz o The mass separated ions undergo collision induced decomposition OF 5 The fragment ions are filtered according to their mass to charge ratio O The transmitted ions are detected by the photomultiplier detection system D The signal is amplified digitised and presented to the MassLynx NT data system The Micromass Quattro LC is a high performance benchtop triple quadrupole mass spectrometer designed for routine LC MS MS operation Quattro LC may be coupled to e a HPLC system with or without an autosampler an infusion pump asyringe pump Ionisation takes place in the source at atmospheric pressure These ions are sampled through a series of orifices into the first quadrupole where they are filtered according to their mass to charge ratio Yz The mass separated ions then pass into the hexapole collision cell where they either undergo collision induced decomposition CID or pass unhindered to the second quadrupole The fragment ions are then mass analysed by the second quadrupole Finally the transmitted ions are detected
2. Maintenance and Fault Finding Page 138 Quattro LC User s Guide Blockage of the stainless steel sample capillary may occur if the desolvation heater is left on without liquid flow This is particularly relevant for samples contained in involatile solvents or high analyte concentrations To avoid this problem it is good practice to switch off the heater before stopping the liquid flow and flush the capillary with solvent A blocked stainless steel sample capillary can often be cleared by removing it and reconnecting it in the reverse direction thus flushing out the blockage Gas flow problems Check all gas connections for leaks using soap solution or a suitable leak searching agent such as Snoop Maintenance and Fault Finding Page 139 Quattro LC User s Guide Replacement of the Stainless Steel Sample Capillary Stainless Steel Capillary Adapter i Nut LC E Union gt GVF 003 Ferrule Ferrule Fused Silica Capillary Liner Tube lt gt Rheodyne Nut amp Ferrule If the stainless steel sample capillary cannot be cleared or if it is contaminated or damaged replace it as follows Remove the probe form the source Disconnect the LC line from the probe and remove the finger tight nut Loosen the grub screw retaining the LC union Remove the two probe end cover retaining screws and remove the probe end cover Unscrew and remove the probe tip Remove the LC union and adapte
3. When changing between megaflow and standard electrospray operation it is essential that the correct tubing is used to connect the probe to the sample injector For megaflow operation 16 o d 0 007 i d peek tubing easily identified by its yellow stripe is used This replaces the standard fused silica tube together with the PTFE sleeves Normal Flow Electrospray PTFE Sleeve Fused Silica Tube PTFE Sleeve e Probe Injector gt Megaflow Electrospray 1 16 o d 0 007 i d Peek Tube Electrospray Page 54 Quattro LC User s Guide Operation m E Corona io een Discharge Pin Mounting Contact High Voltage Socket Exhaust Ensure that the source is assembled as described in Maintenance and Fault Finding and that the instrument is pumped down and prepared for electrospray operation as described in Routine Procedures Ensure that a supply of nitrogen has been connected to the gas inlet at the rear of the instrument and that the head pressure is between 6 and 7 bar 90 100 psi Ensure that the exhaust liner and the cleanable baffle are fitted to the source This is important for optimum electrospray intensity and stability when operating at low flow rates Checking the ESI Probe Connect the electrospray probe to a pulse free pump Solvent should be degassed to prevent beam instabilities caused by bubbles Connect the PTFE tubing of the electrosp
4. gt Collision Cell MS1 RPF only MS2 scanning from pass all masses static at m z 195 m z 50 to 650 daughter mass The result RESERP2 1 0 468 Sb 1 50 00 Sm SG 2x1 00 Parents of 195ES 1005 6 4 84e5 je 0 50 75 100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 500 525 550 575 600 625 50 Instrument Description Page 20 Quattro LC User s Guide MRM Multiple Reaction Monitoring This mode is the MS MS equivalent of SIR Selected Ion Recording As both MSI and MS2 are static this allows greater dwell time on the ions of interest and therefore better sensitivity 100x compared to scanning MS MS Typical application Rapid screening of dirty samples for known analytes Drug metabolite and pharmacokinetic studies Environmental for example pesticide and herbicide analysis Forensic or toxicology for example screening for target drugs in sport Example Monitor the transition specific fragmentation reaction Yz 609 195 for reserpine in electrospray positive ion LC MS MS mode MS1 bie MS2 static at m z 609 pass all masses static at m z 195 parent mass daughter mass The result MRM does not produce a spectrum as only one transition is monitored As in SIR a chromatogram is produced Time Time LC MRM LC MS e High specificity Low specificity e Good signal noise Poor signal noise Inst
5. Drugs and their metabolites Environmental contaminants e g pesticides pollutants Dye compounds Some organometallics Small saccharides Typical ES Negative lon Samples Some proteins Some drug metabolites e g glucuronide conjugates Oligonucleotides Some saccharides and polysaccharides Electrospray Page 63 Quattro LC User s Guide Chromatographic Interfacing Electrospray ionisation can be routinely interfaced to reversed phase and normal phase chromatographic separations Depending on the LC pumping system chromatography column and setup there are some basic options Microbore and capillary chromatography separations employing 1 mm diameter and smaller columns can be interfaced directly to the electrospray probe Typical flow rates for such columns may be in the region of 3 50 l min It is suggested that a syringe pump is used to deliver these constant low flow rates through a capillary column Alternatively accurate pre column splitting of higher flow rates from reciprocating pumps can be investigated In all cases efficient solvent mixing is necessary for gradient elution separations This is of paramount importance with regard to low flow rates encountered with capillary columns HPLC pump manufacturers recommendations should be heeded 2 1mm diameter reversed phase columns are gaining popularity for many separations previously addressed by 4 6mm columns Typically flow rates of
6. Instrument Description Page 28 Quattro LC User s Guide Internal Layout Mechanical Components mE Source Analyser Air Filter Housing Housing behind cover Detector l Pirani auge Active Inverted Magnetron Penning Gauge Source Turbomolecular Pump The main internal mechanical components of the instrument are The source housing inside which is the hexapole transfer lens The hexapole transfer lens is sometimes referred to as the RF lens The analyser housing containing the two quadrupoles and the gas cell The detector attached to the rear of the analyser housing Two 250 litre second turbomolecular pumps one pumping each of the above housings The active inverted magnetron Penning gauge and the Pirani gauge both clamped to the underside of the analyser housing The air filter held in the louvered cover at the left side of the front of the instrument Instrument Description Page 29 Quattro LC User s Guide Electronics High Voltage Power Supplies 4 Transputer Processor Card TPC Analogue PCB a Spare L ee Scan Control PCB luas RF Control Upper PCB Pumping RF Control Lower PCB Logic PCB Low Voltage ower Supplies 2 Analyser Turbomolecular RF Generators ump Turbomolecular mps Power Supply Instrument Description Page 30 Quattro LC User s Guide The main electronics modules of the system are Two low voltage pow
7. Mass Calibration Page 115 Quattro LC User s Guide The recommended scan speed for the scan speed compensation is 1000 amu sec This is the maximum scan speed permissible when using thresholded continuum data Although continuum is recommended centroided data may be used It is possible to scan more quickly in centroided mode but it is unlikely that a faster acquisition rate would be needed for general use Set Scan From to 80 amu and Scan To to 1000 amu Set Scan Time to 0 92 sec and Inter Scan Delay to 0 1 sec Select Continuum as the Data Type Set Run Duration to 2 0 minutes Choose any filename for the data The filename FASTMS1 the name used during an automatic calibration is valid Start the acquisition and inject the reference solution Manual Calibration Find the start and end scans of the reference data for each file in the same way as for the static calibration file From the Calibration dialog box select Process Calibration From File Select Scanning calibration type and MS1 In the lower area the data filename SCNMS1 should be selected automatically If this is not the case or if an alternative filename has been used for the slow scanning acquisition then the correct file can be selected by clicking on the Browse button Enter the start and end scans of the reference data in the From and To boxes Select the OK button to perform the calibration and display the calibration report on the screen in a
8. Routine Procedures Page 33 Quattro LC User s Guide PC Link ADS 7 Mains Switch Rotary Pump Control CID Gas Nitrogen Gas In ESD EARTH FACILITY Source Backing Pumping Line Line Water To Rotary Pump G Connections Switch on the mains to the mass spectrometer using the switch situated on the service panel at the rear of the instrument Switch on the data system As supplied Windows NT is automatically activated following the start up sequence whenever the data system is switched on Windows NT and MassLynx NT can be configured to prevent unauthorised access Consult the system administrator for any passwords that may be requested When the data system has booted up double click on the MassLynx icon in the Windows desktop AA Select Run and Control Panel or press to bring up the acquisition control panel Routine Procedures Page 34 Quattro LC User s Guide Acquisition Control Panel Methods Instrument Configure Status Help A picture of the instrument is displayed To display the tune page either Double click on the mass spectrometer section of the instrument picture or Select Instrument then Tune Mass Spectrometer from the acquisition control panel Alternatively by simply clicking on e the tune page can be launched directly from the MassLynx top level window Routine Procedures Page 35 Quattro
9. 1129 091 1694 140 Mixture of Sugars Reference File sugneg ref Calculated z Value 665 21 1151 37 827 27 1313 42 989 32 1475 48 Reference Information Page 157 Quattro LC User s Guide Sodium lodide and Caesium lodide or Rubidium lodide Mixture Reference File naineg ref Calculated z Value Reference Information Page 158 126 9045 1026 2699 1925 6353 2825 0008 3724 3662 276 7987 1176 1641 2075 5296 2974 8950 3874 2604 426 6929 1326 0584 2225 4238 3124 7892 576 5872 1475 9526 2375 3180 3274 6835 726 4814 1625 8469 2525 2123 3424 577717 876 3757 1775 7411 2675 1065 3574 4719 Quattro LC User s Guide Preparation of Calibration Solutions PEG Ammonium Acetate for Positive lon Electrospray and APcl Prepare a solution of polyethylene glycols at the following concentrations PEG 200 25 ng yl PEG 400 50 ng yl PEG 600 75 ng ul PEG 1000 250 ng ul Use 50 acetonitrile and 50 water containing 2 mmol ammonium nitrate Use reference file PEGH1000 PEG Ammonium Acetate for Positive lon Electrospray Extended Mass Range Prepare a solution of polyethylene glycols at the following concentrations PEG 200 25 ng ul PEG 400 50 ng ul PEG 600 75 ng ul PEG 1000 250 ng ul PEG 1450 250 ng l Use 50 acetonitrile and 50 water containing 2 mmol
10. 1000 APcI mixture alibration PEG 200 400 600 1000 5 ii m z Uses UBQ 8564 85 650 1500 General HBA 15126 36 700 1500 Hb analysis SOD HBB 15867 22 800 1500 Hb analysis MYO 16951 48 700 1600 General Polyethylene glycol ammonium acetate PEGH2000 mixture 80 2000 PEG 200 400 600 1000 1450 ES calibration General 20 4000 ES calibration Sodium iodide Rubidium ES Iodide mixture n calibration Sodium Iodide Caesium Iodide mixture Reference Information Page 154 Horse Heart Myoglobin Reference File myo ref Molecular Weight 16951 48 Calculated Calculated m z Value m z Value Quattro LC User s Guide Calculated m z Value 606 419 808 222 1304 969 616 177 848 583 1413 633 628 841 893 192 1542 053 652 989 942 758 1696 158 679 068 998 155 1884 508 707 320 1060 477 2119 945 738 030 1131 108 2422 651 711 531 1211 829 Polyethylene Glycol PEG NH4 Reference Files PEGH1000 PEGH2000 Calculated z Value Reference Information Page 155 Quattro LC User s Guide Sodium lodide and Caesium lodide Mixture Reference File NAICS Calculated z Value 22 9898 772 4610 1671 8264 2571 1918 3470 5572 132 9054 922 3352 1821 7206 2721 0861 3620 4515 172 884
11. 200 yl min are used allowing direct coupling to the electrospray source The increased sample flow rate requires increased source temperature and drying gas flow rate A UV detector may be placed in line to the Quattro LC probe However ensure that the volume of the detector will not significantly reduce the chromatographic resolution Whenever a UV detector is used the analog output may be input to MassLynx NT for chromatographic processing The interfacing of 4 6mm columns to the electrospray source can be achieved either by flow splitting or by direct coupling In both cases an elevated source temperature and drying gas flow rate are required In general the best results are obtained by splitting after the column using a zero dead volume tee piece so that 200 300 yl min is transferred to the source Caution The maximum operating temperature for the source heater is 150 C Do not set Source Block Temp higher than 150 C Conventional reverse phase and normal phase solvent systems are appropriate for LC electrospray Involatile buffers may be used but prolonged periods of operation are not recommended When using involatile buffers the probe should be moved as far away from the sample cone as possible This may reduce sensitivity slightly but will reduce the rate at which involatile material will be deposited on the sample cone Trifluoroacetic acid TFA and triethylamine TEA may be used up to a level of 0 05 If solvents of high a
12. HV cabling Nanoflow Electrospray Page 76 Quattro LC User s Guide Atmospheric Pressure Chemical lonisation Corona Discharge WEM Exhaust Introduction Cleanable Exhaust Isolation Valve a Sampie LN one pra j Nebuliser Desolvation Gas Gas Analyser Source Enclosure Rotary Turbomolecular Pump Pumps Atmospheric Pressure Chemical Ionisation APcI is an easy to use LC MS interface that produces singly charged protonated or deprotonated molecules for a broad range of involatile analytes The ability to operate with 100 organic or 100 aqueous mobile phases at flow rates up to 2 ml min makes APcI an ideal technique for standard analytical column 4 6mm i d normal phase and reverse phase LC MS The APcI interface consists of the standard Z spray source fitted with a corona discharge pin and a heated nebuliser probe Mobile phase from the LC column enters the probe where it is pneumatically converted into an aerosol and is rapidly heated and converted to a vapour gas at the probe tip Hot gas from the probe passes between the sample cone and the corona discharge pin which is typically maintained at 2 5kV Mobile phase molecules rapidly react with ions generated by the corona discharge to produce stable reagents ions Analyte molecules introduced into the mobile phase react with the reagent ions at atmospheric pressure and typically become protonated in positive ion mode or dep
13. LC User s Guide Pumping A Caution To minimise wear to the lubricated components of the rotary pump the manufacturers recommend that the pump is not started when the oil temperature is below 12 C Pump down time may be decreased Isolation by closing the isolation valve of the source during pump down Select Other from the menu bar at the top of the tune page Click on Pump The rotary pump and the turbomolecular pumps start simultaneously The Vacuum LED on the front of the instrument shows amber as the system pumps down When the system has reached operating vacuum the LED changes to a steady green indicating that the instrument is ready for use If the rotary pump oil has been changed or replenished open the gas ballast valve on the rotary pump See the pump manufacturer s literature for details Rotary pumps are normally noticeably louder when running under gas ballast If opened close the gas ballast valve when the rotary pump has run under gas ballast for 30 minutes Using the Instrument Quattro LC is now almost ready to use To complete the start up procedure and prepare for running samples follow the instructions in Start Up Following Overnight Shutdown in the following pages Routine Procedures Page 36 Quattro LC User s Guide Start Up Following Overnight Shutdown The instrument will have been left in standby mode under vacuum It is recommended that the data system is
14. Multiplier lt 5 LA m 5 50 75 100 125 150 1 5 200 2 5 250 2 Source Analyser Acquire OPERATE Atmospheric Pressure Chemical lonisation Page 81 Quattro LC User s Guide Hints for Sample Analysis Tuning Start by tuning on the solvent ions t is generally found that the most significant analyte tuning parameter to adjust following tuning on the solvent ions is Cone Fine tuning on the analyte of interest can be performed either by large loop injections 1001 or by constant infusion in the mobile phase typically at analyte concentrations of a few ng l e 10y1 loop injections can be monitored using real time chromatogram updates Mobile Phase The choice of mobile phase is an important compound specific factor in APcI For example steroids prefer methanol water mixtures as opposed to acetonitrile water Analyte sensitivity is also dependent on mobile phase composition which can be varied from 100 aqueous to 100 organic for any particular mixture Probe Temperature This can be a critical factor for some analytes nvolatile samples for example steroids generally require high probe temperatures 2400 C Volatile samples for example pesticides can be analysed with low probe temperatures 400 C n some cases too high a probe temperature can lead to thermal degradation of labile samples Desolvation Gas Although a Desolvation Gas flow of approximately 150 litres hour is typical f
15. Nitrogen Select oPERATE Set Capillary to approximately 3kV An ion beam should be visible Tune the source parameters for maximum signal Nanoflow Electrospray Page 74 Quattro LC User s Guide Optimise the position of the needle using the three axis manipulator In the fused silica option the needle tends to optimise further away from the cone than in the borosilicate option The spray from the needle should be easily visible by the microscope or camera and the stability of the spray can be seen using either system A column can be attached directly to the back of the union to reduce any dead volumes to a minimum but care must be taken to match the external diameter of the column with the internal diameter of any is inch sleeve Nanoflow Electrospray Page 75 Quattro LC User s Guide Changing Options To change between the glass capillary and the fused silica options Rotate the stage outwards A Caution Failure to use the stop and handle to rotate the stage can result in permanent damage to the three axis manipulator Remove the protective cover and release the captive screw located underneath the stage Lift off the holder and replace it with the alternative holder securing it with the captive screw Replace the protective cover ensuring that either the PTFE back pressure tubing glass capillary option or the fused silica transfer line is fed through the slot in the back of the protective cover along with the
16. Scanning Calibration x calibration procedure for that particular file and display a calibration report on the screen This calibration report opposite C Static MS1 upper contains four displays re PCS Select Calibration Type Scanning C M52 the acquired spectrum C Scan Speed Compensation the reference spectrum a plot of mass difference Select Calibration File against mass the calibration Combine scans in data file SCNMS1 curve a plot of residual against mass From i To 2 An expanded region can be displayed Browse opposite lower by clicking and dragging with the left mouse button In this way the less intense peaks in the spectrum can be __ Cancel examined to check that the correct peaks have been matched The peaks in the acquired spectrum which have been matched with a peak in the reference spectrum are highlighted in a different colour Mass Calibration Page 98 Print Edi Display Data file SCNMS1 Uncalibrated 1004 1 308 47288 gyjgg 92237 1072 22 122209 Reference file NAIRB 22 99 172 88 472 67 1972 25 522 57 922 36 Mass difference Raw Ref mass 0 35 amu 1372 04 d 1521 83 4 1671 83 rd 1971 42 2121 34 1971 61 id 71 04 2421 15 25710 2271 40 F Quattro LC User s Guide Oo x 26 matches of 28 tested references 2 005 3320 59 3770 48 Print Edi Display Data
17. Take care to ensure that no other reference peak is excluded If the reference peak is not closer to the centre of the peak window or if by reducing the window other reference peaks are excluded then the calibration can be edited manually Manual Editing of Peak Matching If an incorrect peak has been matched in the calibration process this peak can be excluded manually from within the on screen calibration report Using the mouse place the cursor over the peak in the acquired spectrum and click with the right mouse button The peak is excluded and is no longer highlighted If the true reference peak is present then this can be included in the calibration by the same procedure Place the cursor over the required peak and click with the right mouse button The peak is matched with the closest peak in the reference spectrum Manually editing one peak will not affect the other matched peaks in the calibration Saving the Calibration When the instrument is fully calibrated the calibration can be saved under a filename so that it can be recalled for future use For example it is possible to save calibrations for use with different ionisation modes so that when an ionisation source is switched the corresponding calibration is recalled The recalled calibration has the same constraints of mass range and scan speed The ion energy and resolution settings used for the calibration acquisition are also recorded as these can have an eff
18. automated a command from the data system switching on the rotary pump and turbomolecular pumps simultaneously Protection Transient Pressure Trip If the vacuum gauge detects a pressure surge above the factory set trip level of 10 mbar and if the instrument is in the operate mode the following events occur The critical source analyser and detector voltages are switched off The Operate LED shows a steady amber The Vacuum LED shows a steady amber Acquisition will continue although no mass spectral data are recorded When the pressure recovers the voltages are restored and the Vacuum and Operate LED s are steady green Any further deterioration of the system vacuum results in a pump fault and the system is shut down Routine Procedures Page 42 Quattro LC User s Guide Pump Fault A pump fault causes the following to occur The turbomolecular pumps stop pumping On the display the Vacuum LED flashes red The Operate LED is extinguished As the turbos slow down the vent valve opens the rotary pump switches off and the system is vented The pumps will not switch on again unless requested to do so A pump fault can occur as a result of Over temperature of the turbomolecular pumps If the water cooling fails then the turbomolecular pumps switch off when their temperature becomes too high Vacuum leak Refer to Maintenance and Fault Finding later in this manual Malfunction of the turbomolecular p
19. by a conversion dynode phosphor and photomultiplier detection system The output signal is amplified digitised and presented to the data system Instrument Description Page 15 Quattro LC User s Guide Vacuum System Vacuum is achieved using a direct drive rotary pump and two turbomolecular pumps The rotary pump mounted on the floor external to the instrument backs the turbomolecular pumps and also pumps the first vacuum stage of the source The turbomolecular pumps evacuate the analyser and ion transfer region These pumps are both water cooled Vacuum measurement is by an active inverted magnetron Penning gauge for the analyser and a Pirani gauge for the gas cell The Penning gauge acts as a vacuum switch switching the instrument out of the OPERATE mode if the pressure is too high The speed of each turbomolecular pump is also monitored and the system is fully interlocked to provide adequate protection in the event of a fault in the vacuum system a failure of the power supply or vacuum leaks Instrument Description Page 16 Quattro LC User s Guide lonisation Techniques Two atmospheric pressure ionisation techniques are available Atmospheric Pressure Chemical lonisation Atmospheric pressure chemical ionisation APcI generally produces protonated or deprotonated molecular ions from the sample via a proton transfer positive ions or proton abstraction negative ions mechanism The sample is vaporised in a hea
20. calibration data file contains no peaks the calibration will fail This may be due to Lack of reference compound No flow of solvent into the source Multiplier set too low Too many consecutive peaks missed If the number of consecutive peaks which are not found exceeds the Missed Reference Peaks parameter set in the Automatic Calibration Check then the calibration will fail Peaks may be missed for the following reasons The reference solution is running out so that the less intense peaks are not detected Multiplier is too low so that the less intense peaks are not detected An incorrect ionisation mode is selected Check that the data have been acquired with lon Mode set to ES Note that it is possible to calibrate in negative ion mode electrospray using the naineg ref reference file with a suitable reference solution Intensity threshold set in the Calibration Parameters dialog box is too high Peaks are present in the acquired calibration file but are ignored because they are below the threshold level Either Initial error or Peak window set in the Calibration Parameters dialog box is too small The calibration peaks lie outside the limits set by these parameters Maximum Std Deviation set in the Automatic Calibration Check dialog box has been exceeded Mass Calibration Page 100 Quattro LC User s Guide The wrong reference file has been selected Check that the correct file nairb ref in this case is sel
21. can be heated to temperatures of 150 C and will be maintained at the set temperature when the source enclosure is removed Touching the ion block when hot may cause burns to the operator Caution The maximum operating temperature for the source heater is 150 C Do not set Source Block Temp higher than 150 C The liquid flow should not be started until the gas flow and probe heater are switched on with the probe inserted Operate s On the MassLynx top level window click on to launch the tune page The top line of the tune page indicates the current ionisation mode If necessary change the ionisation mode using the lon Mode command Depending on the chosen mode of ionisation set Desolvation Temp or APcl Probe Temp to 20 C Click on on the MassLynx tune page The instrument will go into the operate mode only if the probe adjustment flange is in place and a probe is inserted On the tune page select Gas and Nitrogen to turn on the source and probe gases Set Desolvation Gas to a flow of 150 litres hour and adjust Nebuliser to maximum The system is now ready for operation To obtain an ion beam refer to Obtaining an section Routine Procedures Page 41 Quattro LC User s Guide Automatic Pumping and Vacuum Protection Overview The instrument is protected against vacuum system faults due to malfunction of the vacuum pumps excessive pressure excessive temperature The pump down sequence is fully
22. disturbed Leaks on flanges can usually be cured by further tightening of the flange bolts or by replacing the seal All seals are made using O rings When refitting flanges pay attention to the condition of O rings Any that are cut or marked may cause a leak The O rings should be clean and free from foreign matter A hair across an O ring is sufficient to prevent the instrument pumping down Source components that operate at or slightly above atmospheric pressure are not susceptible to vacuum leaks Maintenance and Fault Finding Page 124 Quattro LC User s Guide In the unlikely event of a leak on a feedthrough then the unit should be replaced or returned to Micromass for repair Pirani Gauge The Pirani gauge head does not require routine maintenance Active Inverted Magnetron Gauge For information on cleaning the active inverted magnetron Penning gauge refer to the Edwards literature supplied with the instrument Gas Ballasting Gas ballasting serves two important Gas purposes Ballast When rotary pumps are used to je pump away solvent vapours the Fr HS solvent vapour can become dissolved in the pump oil causing an increase in backing line pressure Gas ballasting is a method of purging the oil to remove dissolved contaminants Oil mist expelled from the rotary pump exhaust is trapped in the oil mist filter This oil is returned to the rotary pump during gas ballasting Gas balla
23. found in the MassLynx NT User s Guide If o centroided data are used for calibration Peak width Da 0 50 then the mass measure parameters are Number of smooths 2 not used Mean PORN Savi With electrospray calibrations eet cum particularly with sodium iodide which Min peak width at has some low intensity peaks at higher B a Gini o mass it is recommended that ne continuum or MCA data are acquired dii C Centroid top Z5 80 00 Mass Calibration Page 91 Quattro LC User s Guide Performing a Calibration Three types of calibration are available with MassLynx static calibration scanning calibration and scan speed compensation These are selected on the Automatic Calibration dialog box see below which is accessed by selecting the Start button from the Calibrate dialog box It is recommended that all three types of calibration are performed so that any mode of data acquisition can be used and mass ranges Types and scan speeds can be changed whilst 7 Eia c Calibration MSI oe LE enl i maintaining correct mass assignment However it is possible to have any combination of these calibrations I Scan Speed Compensation If only a static calibration is present Acquisition Parameters then the instrument is calibrated for M Scanning Calibration M MS2 acquisitions where the quadrupoles are Process held at a single mass as in SIR or FF Acquire amp Calibrate MRM If only a
24. generated by the Vacuum LED is dependent on the vacuum status of the instrument The Operate LED depends on both the vacuum status and whether the Operate mode has been selected from the Data System The status of the instrument is indicated as follows Vacuum LED State Vacuum LED State Vacuum LED Vented No indication Vacuum OK Steady green Pumping Operate LED State Steady amber Operate LED Pump fault State Flashing red Operate LED Standby No indication Transient pressure trip Steady amber Operate Instrument Description Page 24 Steady green RF trip Flashing red Quattro LC User s Guide Flow Control Valves The Desolvation Gas and Nebuliser needle valves are five turn valves The CID Gas valve is a fifteen turn valve The flow increases as the valve is turned counterclockwise A Caution To prevent damage to the CID Gas valve take care not to over tighten when turning the supply off Divert Injection Valve SSS uu EE The optional divert injection valve may be used in several ways depending on the plumbing arrangement Asan injection valve with the needle port and sample loop fitted e Asa divert valve to switch the flow of solvent during a LC run Asaswitching valve to switch for example between a LC system and a syringe pump containing calibrant This valve is pneumaticall
25. ibrate B x Print Edit Display Data file STATMS1 Uncalibrated 22 matches of 22 tested references 400 MESS 459 50 503 53 T Reference file PEGH1000 100 371 23 415 25 459 28 503 31 547 33 591 36 635 39 679 41 723 44 0 Mass difference Raw Ref mass 0 30 x amu 0 11 Residuals Mean residual 7 514080e 10 0 153838 0 06 x x amu Miz 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700 720 740 Mass Calibration Page 113 Quattro LC User s Guide If the correct peaks have been matched then the report can be printed out Select Print Print from the report display Calibration Report Printed Thu Nov 27 17 02 31 1997 Data file SCNMS1 Uncalibrated 21 matches of 22 tested references 1004 415 55 459 57 503 57 195 84 1 39 39 r 45 LLLI 591 60 LL 62 943 73 0 dap Hal dian NUN A L l dian Reference file PEGH1000 1004 107 07 195 12 283 18 371 28 459 28 47 33 635 39 723 44 811 49 O r c RED EN RUE RI Mass difference Raw Ref mass 0 304 Residuals Mean residual 8 315380e 11 0 023084 0 044 x Fe a a a a E EE pene Vots alor s ees ot 200 300 400 500 600 700 800 To accept the calibration Select OK from the calibration report Mass Calibration Page 114 Quattro LC User s G
26. into the filter Maintenance and Fault Finding Page 144 Quattro LC User s Guide Feed the sample capillary through the probe ensuring that a 0 4mm graphitised vespel ferrule GVF 004 is fitted Using a ceramic capillary cutter cut the capillary at the nebuliser so that between 0 5 and 1 0mm of capillary is protruding from the nebuliser It is important to cut the capillary square This should be examined using a suitable magnifying glass Undo the adapter nut from the probe and withdraw the capillary from the probe Remove 20mm of polyamide coating from the end of the capillary using a flame and clean with a tissue saturated with methanol Carefully re feed the sample capillary through the probe ensuring that the graphitised vespel ferrule is still fitted Using a Rheodyne spanner gently tighten the adapter nut to the probe Replace the probe end cover and retaining screws Using a 1 5mm Allen key tighten the grub screw in the probe end cover to clamp the filter Replace the heater and probe tip assembly Maintenance and Fault Finding Page 145 Quattro LC User s Guide The Analyser Quattro LC is fitted with a pre filter assembly that is designed to protect the main analyser by absorbing contamination from the ion beam As a consequence the analyser quadrupoles should never under normal working conditions require cleaning The hexapole transfer lens also serves to effectively remove contamination and the pre f
27. is suspected that the lens does need cleaning it may be withdrawn from the front of the instrument after removing the ion block support Warning Cleaning the various parts of the source requires the use of solvents and chemicals which may be flammable and hazardous to health The user should take all necessary precautions Maintenance and Fault Finding Page 127 Quattro LC User s Guide Cleaning the Sample Cone in Situ This may be necessary due to lack of sensitivity or fluctuating peak intensity or if deposited material is visible on the outside of the sample cone Proceed as follows On the MassLynx top level window click on to launch the tune page Deselect OPERATE Switch off the LC pumps Disconnect the liquid flow at the rear of the probe Set Source Block Temp and either APcl Probe Temp or Desolvation Temp to 20 C to switch off the heaters Warning Removal of the APcI probe or desolvation nozzle when hot may cause burns Caution Removal of the APcI probe when hot will shorten the probe heater s life The cooling time will be significantly shortened if the API gases are left flowing Source Thumb Nuts uu ie c Probe Probe Adjustment Flange Thumb Nuts When APcl Probe Temp or Desolvation Temp has cooled below 100 C Switch off the nitrogen supply by selecting Gas followed by Nitrogen Disconnect both gas lines from the front panel by undoing the knurled nuts Maintenance an
28. of Contents Quattro LC User s Guide Instrument Description Overview Vacuum System Ionisation Techniques Atmospheric Pressure Chemical Ionisation Electrospray Nanoflow Electrospray Sample Inlet MS Operating Modes MS MS Operating Modes The Daughter Ion Spectrum The Parent Ion Spectrum MRM Multiple Reaction Monitoring The Constant Neutral Loss Spectrum Data System Front Panel Connections Desolvation Gas and Probe Nebuliser Gas Capillary Corona ESI APcI Front Panel Controls and Indicators Status Display Vacuum LED Operate LED Flow Control Valves Divert Injection Valve Rear Panel Connections Event Out Contact Closure In Analog Channels Scope Water Nitrogen Gas In Exhausts CID Gas Power Cord Mains Switch Fuses Rotary Control ESD Earth Facility Coml and Com2 PC Link Internal Layout Mechanical Components Electronics Table of Contents 15 16 17 17 17 17 17 18 18 19 20 21 22 22 23 23 23 23 24 24 24 24 25 25 26 26 26 26 2T 27 27 28 28 28 28 28 28 28 28 28 29 29 30 Routine Procedures Start Up Following a Complete Shutdown Preparation Pumping Using the Instrument Start Up Following Overnight Shutdown Preparation for Electrospray Operation Preparation for APcI Operation Operate Automatic Pumping and Vacuum Protection Overview Protection Transient Pressure Trip Pump Fault Power Failure Tuning Source Voltages Calibration Data Acquisition Data Processing Setting
29. psi Atmospheric Pressure Chemical lonisation Page 78 Quattro LC User s Guide Checking the Probe Ensure that the probe heater is off Unplug the probe from the instrument s front panel and remove the probe from the source Connect the PTFE tube to the Nebuliser outlet on the front panel Remove the probe tip assembly by carefully loosening the two grub screws Disconnect the heater from the probe body by pulling parallel to the axis of the probe Ensure that 0 5 to 1mm of fused silica is protruding from the stainless steel nebuliser tube Connect the LC pump to the probe with a flow of 50 50 acetonitrile water at 1 ml min Check that the liquid jet flows freely from the end of the capillary and that the LC pump back pressure reads 250 to 400 psi Check that the nitrogen supply pressure is 6 to 7 bar 90 to 100 psi Select Gas and turn on Nitrogen Check that the liquid jet converts to a fine uniform aerosol Switch off the liquid flow Select Gas and turn off Nitrogen Reconnect the probe tip assembly Insert the APcI probe into the source and secure it by tightening the two thumb screws Connect the probe cable to APcI ESI on the instrument s front panel The plug labelled ESI must first be unplugged from the front panel Atmospheric Pressure Chemical lonisation Page 79 Quattro LC User s Guide Obtaining a Beam A Ensure that the corona discharge pin is fitted as described in Routine Procedur
30. report Using the mouse place the cursor over the peak in the acquired spectrum and click with the right mouse button The peak is excluded and is no longer highlighted If the true reference peak is present then this can be included in the calibration by the same procedure Place the cursor over the required peak and click with the right mouse button The peak is matched with the closest peak in the reference spectrum Manually editing one peak will not affect the other matched peaks in the calibration Saving the Calibration When the instrument is fully calibrated the calibration can be saved under a file name so that it can be recalled for future use The recalled calibration has the same constraints of mass range and scan speed The ion energy and resolution settings used for the calibration acquisition are also recorded as these can have an effect on mass assignment Mass Calibration Page 103 Quattro LC User s Guide Verification Once a full instrument calibration is in place MM it is not always necessary to repeat the full calibration procedure when the instrument is next used Instead a calibration verification can be performed There is no benefit in verifying each calibration individually M Scanning Calibration M MS2 re calibration is just as quick Types Static Calibration M MS1 Scan Speed Compensation If a scanning acquisition is to be made and Acquisition Parameters the calibration is to be c
31. scanning calibration is present then the instrument is only correctly calibrated for scanning acquisitions over the same mass range and at the same scan speed as those used for the calibration If only a scan speed compensation is present with no scanning calibration having been performed then the scan speed compensation is treated as a scanning calibration and the instrument is only correctly calibrated for scanning acquisitions over the same mass range and at the same scan speed as used for the calibration For the scan speed compensation to be used correctly a scanning calibration should also be performed If static and scanning calibrations are both present then the instrument is calibrated for acquisitions where the quadrupole is held at a single mass and for scanning acquisitions with a mass range which lies within the mass range of the scanning calibration providing that the same scan speed is used For example if the instrument is calibrated from Yz 100 to 900 with a 2 second scan 400 amu sec then data can be acquired from 100 500 amu with a 1 second scan time also 400 amu sec whilst maintaining correct mass assignment In this case the static calibration would be used to determine the start mass of the acquisition and the scanning calibration would be used for mass assignment and scan range Mass Calibration Page 92 Quattro LC User s Guide If scanning calibration and scan speed compensation are present t
32. screws washers spacers etc can be stored V Use tweezers and pliers whenever possible V If nylon or cotton gloves are used take care not to leave fibres in sensitive areas Avoid touching sensitive parts with fingers Do not use rubber gloves V Before reassembling and replacing dismantled components inspect O rings and other vacuum seals for damage Replace with new if in doubt Should a fault occur soon after a particular part of the system has been repaired or otherwise disturbed it is advisable first of all to ensure that this part has been correctly refitted and or adjusted and that adjacent components have not been inadvertently disturbed Warning Many of the procedures described in this chapter involve the removal of possibly toxic contaminating deposits using flammable or caustic agents Personnel performing these operations should be aware of the inherent risks and should take the necessary precautions Cooling Fans and Air Filters Always ensure that none of the cooling fans is obstructed It is essential that the fan filter is checked at regular intervals and replaced if there is any doubt about its effectiveness Maintenance and Fault Finding Page 123 Quattro LC User s Guide The Vacuum System The performance of the mass spectrometer will be severely impaired by the lack of a good vacuum in the ion transfer hexapole region or the analyser e An analyser pressure above 10 mbar results in a gener
33. the lead of the probe adjustment flange is plugged into the socket labelled ESI APcl on the front panel Take the electrospray probe and connect its gas line to Nebuliser on the front panel Connect the liquid flow of a LC system or syringe pump to the probe Insert the probe into the source and tighten the two thumb nuts to secure the probe firmly Plug the probe lead into Capillary Corona on the front panel Routine Procedures Page 38 Quattro LC User s Guide ag On the MassLynx top level window click on to launch the tune page The top line of the tune page indicates the current ionisation mode If necessary change the ionisation mode using the lon Mode command Set Source Block Temp to 100 C and Desolvation Temp to 20 C Warning Operating the source without the source enclosure will result in solvent vapour escape and the exposure of hot surfaces and high voltages Warning The ion source block can be heated to temperatures of 150 C and will be maintained at the set temperature when the source enclosure is removed Touching the ion block when hot may cause burns to the operator A Caution The maximum operating temperature for the source heater is 150 C Do not set Source Block Temp higher than 150 C Preparation for APcl Operation Source Thumb Nuts Moulded Cover Probe Adjustment Flange Thumb Nuts If the corona discharge pin is not fitted proceed as follows Disconnect t
34. to perform the calibration and display the calibration report on the screen opposite upper This report contains four displays the acquired spectrum the reference spectrum a plot of mass difference against mass the calibration curve a plot of residual against mass An expanded region opposite lower can be displayed by clicking and dragging with the left mouse button In this way the less intense peaks in the spectrum can be examined to check that the correct peaks have been matched The peaks in the acquired spectrum which have been matched with a peak in the reference spectrum are highlighted in a different colour Compare the acquired and reference spectra to ensure that the correct peaks have been matched Mass Calibration Page 112 Quattro LC User s Guide Oo x Data file STATMS1 Uncalibrated 22 matches of 22 tested references 100 415 55 459 50 503 53 239 37 547 54 591 55 19535 283 42 327 47 371 51 635 56 679 53 723 55 767 56 811 59 855 62 899 57 943 61 987 63 151 33 2 Reference file PEGH1000 100 107 07 151 10 195 12 239 15 283 18 327 20 371 23 415 25 459 28 503 31 547 33 591 36 635 39 679 41 723 44 767 46 811 49 0 Mass difference Raw Ref mass 0 30 amu 0 09 Residuals Mean residual 7 514080e 10 0 153838 0 25 amu 0 10 Miz 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950
35. 0 1072 2494 1971 6149 2870 9803 3770 3457 322 7782 1222 1437 2121 5091 3020 8745 3920 2400 472 6725 1372 0379 2271 4033 3170 7688 622 5667 1521 9321 2421 2976 Sodium lodide and Rubidium lodide Mixture Reference Information Page 156 Reference File NAIRB Calculated z Value 3320 6630 22 9898 772 4610 1671 8264 2571 1918 3470 5572 84 9118 922 3552 1821 7206 2721 0861 3620 4515 172 8840 1072 2494 1971 6149 2870 9803 3770 3457 322 7782 1222 1437 2121 5091 3020 8745 3920 2400 472 6725 1372 0379 2271 4033 3170 7688 622 5667 1521 9321 2421 2976 3320 6630 Negative lon Ref File Name Molecular Mass Chemical Name Sigma Code m z Quattro LC User s Guide Uses Horse heart myoglobin MYONEG M1882 16951 48 700 2400 General Sugar mixture of maltose M5885 raffinose RO250 maltotetraose M8253 corn syrup M3639 SUGNEG 100 1500 Low mass range Sodium Iodide Caesium Iodide or Rubidium Iodide mixture Horse Heart Myoglobin Reference File myoneg ref Calculated z Value 200 3900 ES calibration 891 175 1209 812 1882 490 940 741 1302 952 2117 927 996 138 1411 615 2420 632 1058 460 1540 036
36. 09 109 109 109 111 112 115 115 116 117 117 118 119 120 120 121 Quattro LC User s Guide Maintenance and Fault Finding Introduction 123 Cooling Fans and Air Filters 123 The Vacuum System 124 Vacuum Leaks 124 Pirani Gauge 125 Active Inverted Magnetron Gauge 125 Gas Ballasting 125 Oil Mist Filter 126 Foreline Trap 126 Rotary Pump Oil 126 The Source 127 Overview 127 Cleaning the Sample Cone in Situ 128 Removing and Cleaning the Sample Cone 130 Removing and Cleaning the Source Block and Extraction Cone 132 Removing and Cleaning the Hexapole Transfer Lens Assembly 134 Reassembling and Checking the Source 136 The Discharge Pin 137 The Electrospray Probe 138 Overview 138 Replacement of the Stainless Steel Sample Capillary 140 The APcI Probe 142 Cleaning the Probe Tip 142 Replacing the Probe Tip Heater 143 Replacing the Fused Silica Capillary 144 The Analyser 146 The Detector 146 Electronics 147 Fuses 147 Analog PCB 147 RF power PCB 147 Power Backplane 2 147 Pumping Logic PCB 147 Power Sequence PCB 147 Rear Panel 147 Fault Finding Check List 148 No Beam 148 Unsteady or Low Intensity Beam 148 Ripple 148 High Back Pressure 149 General Loss of Performance 149 Cleaning Materials 150 Preventive Maintenance Check List 151 Daily 151 Weekly 151 Monthly 151 Three Monthly 151 Four Monthly 151 Table of Contents Quattro LC User s Guide Reference Information Overview Positive Ion Horse Heart Myoglo
37. 137 Quattro LC User s Guide The Electrospray Probe Overview Indications that maintenance is required to the electrospray probe include An unstable ion beam Nebulising gas may be escaping from the sides of the probe tip Ensure that the probe tip O ring is sealing correctly The probe tip setting may be incorrect Adjust the probe tip setting as described in Electrospray The probe tip may be damaged Replace the probe tip There may be a partial blockage of the sample capillary or the tubing in the solvent flow system Clear the blockage or replace the tubing Excessive broadening of chromatogram peaks This may be due either to inappropriate chromatography conditions or to large dead volumes in the transfer capillaries between the LC column or probe connection Ensure that all connections at the injector the column the splitting device if used and the probe are made correctly High LC pump back pressure With no column in line and the liquid flow set to 300 ul min the back pressure should not exceed 7 bar 100 psi Pressures in excess of this indicate a blockage in the solvent flow system Samples containing particulate matter or those of high concentrations are most likely to cause blockages Check for blockages at the tube connections and couplings to the injector the column and if used the flow splitter Concentrated formic acid can be injected to clear blockages Rinse thoroughly afterwards
38. 500 1000 1500 2000 2500 3000 3500 MSI Scanning Calibration 27 matches of 28 tested references SD 0 0459 0 354 as eae amu Bac oe j me XX x E x HR 0 194 me TX SS A TERT T ry 500 1000 1 500 2000 2500 3000 3500 MSI Scan Speed Compensation Calibration 26 matches of 28 tested references SD 0 0538 LX peg ere as eee I E M 9 994 2000 2500 3000 3500 MS2 Static Calibration 28 matches of 28 tested references SD 0 0606 1 09 mM L5 xe M XX X 30707 0 01 ot rp epe T 2000 2500 3000 3500 MS2 Scanning Calibration 28 matches of 28 tested references SD 0 0832 0 744 4 ye i amuj XE 0 17 mr DE E E RR a propor r a 500 1000 2000 2500 3000 3500 MS2 Scan Speed Calibration 28 matches of 28 tested references SD 0 0748 1 15 ame 0 00 oo XC Vif toT Tob Tor et Pede Ebr ee E Fa Feb 215 T ee eee RALA MARA A 500 2000 2500 3000 3500 Mass Calibration Page 97 Quattro LC User s Guide Checking the Calibration The calibration successful or failed can be viewed in more detail by selecting Process Calibration From File from the Calibrate dialog box The dialog box which is then displayed see below allows the choice of calibration type for viewing With the required calibration selected the correct calibration file is automatically called up Clicking on the OK button repeats the MS1
39. Edit Display Data file SCNMS1V Calibrated 459 30 503 32 100 415 29 21 matches of 22 tested references 547 33 591 35 635 36 83 19 327 25 371 26 679 38 Reference file PEGH1000 100 283 18 327 20 371 23 415 25 459 28 503 31 547 33 591 36 635 39 679 41 96 0 Mass difference Raw Ref mass Mean residual 3 954206e 3 0 025099 0 05 x x x x amu 0 03 Miz 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700 A calibration curve will be produced and displayed on the screen in a similar way to when the original calibration was performed An example is shown above When OK is selected from this report unlike the original calibration procedure the instrument calibration is not changed As the verification procedure uses the same matching parameters as the calibration procedure it is possible to validate the current calibration without re calibrating the instrument The report can be printed out by selecting Print Print from the verify report Mass Calibration Page 122 Quattro LC User s Guide Maintenance and Fault Finding Introduction Cleanliness and care are of the utmost importance whenever internal assemblies are removed from the instrument V Always prepare a clear clean area in which to work V Make sure that any tools or spare parts that may be required are close at hand V Obtain some small containers in which
40. MassLynx tune page represents total drying flow that is desolvation gas cone gas nanoflow only purge gas if enabled Electrospray Page 58 Quattro LC User s Guide Desolvation Gas Flow Rate litres hour 10 100 to 120 200 to 250 10 to 20 120 to 250 200 to 400 20 to 50 250 to 350 200 to 400 gt 50 350 to 400 500 to 750 Solvent Flow Rate Desolvation Temp pd min PE Higher desolvation temperatures gives increased sensitivity However increasing the temperature above the range suggested reduces beam stability Increasing the gas flow rate higher than the quoted values leads to unnecessarily high nitrogen consumption Caution Do not operate the desolvation heater for long periods of time without a gas flow To do so could damage the source Cone Gas The cone gas should be used only in the nanoflow mode see the following chapter Ensure that the cone gas outlet situated inside the source enclosure is blanked for normal ESI operation Purge Gas The purge gas is not necessary for most ESI applications It may be useful for megaflow operation where an analyte is susceptible to acetonitrile adducting Purge gas is enabled simply by removing the blanking plug from the outlet situated within the source enclosure Purge gas flow rate is a constant fraction 30 of the total desolvation gas flow Electrospray Page 59 Quattro LC User s Guide ETune QULC ESP
41. Microscope Focusing is adjusted by rotating the top of the microscope Nanoflow Electrospray Page 71 Quattro LC User s Guide Glass Capillary Option Warning Do not touch the sharp end of the capillary As well as the risk of injury by a sliver of glass the needle will become inoperable A Caution The capillaries are extremely fragile and must be handled with great care Always handle using the square end of the capillary With the stage rotated outwards unscrew the union from the end of the assembly Carefully remove the capillary from its case by lifting vertically while pressing down on the foam with two fingers Over the blunt end of the capillary pass the knurled nut approximately 5mm of conductive elastomer and finally the union Tighten the nut finger tight is sufficient so that 5mm of glass capillary is protruding from the end of it This distance is measured from the end of the nut to the shoulder of the glass capillary Capillary Load sample into the capillary using either a PTFE fused silica syringe needle or a gel loader tip P Nube O Screw the holder back into the assembly finger xs Ferrule tight is sufficient Ensure that Capillary is set to OV on the tune page Rotate the stage back into the interface using the stop and handle N Union Manoeuvre the stage so that the microscope or ie oe Nut camera can view the capillary tip Conductive Using
42. Quattro LC User s Guide Micromass UK Limited Floats Road Wythenshawe M23 9LZ Tel 44 161 945 4170 Fax 44 161 998 8915 Tudor Road Altrincham WA14 5RZ Tel 44 161 282 9666 Fax 44 161 282 4400 http www micromass co uk The instrument is marked with this symbol where high voltages are present The instrument is marked with this symbol where hot surfaces are present The instrument is marked with this symbol where the user should refer to this User s Guide for instructions which may prevent damage to the instrument Warnings are given throughout this manual where care is required to avoid personal injury AMAAAAA This manual is a companion to the MassLynx NT User s Guide supplied with the instrument All information contained in these manuals is believed to be correct at the time of publication The publishers and their agents shall not be liable for errors contained herein nor for incidental or consequential damages in connection with the furnishing performance or use of this material All product specifications as well as the information contained in this manual are subject to change without notice Issue 2 Micromass Ltd Quattro LC User s Guide Quattro LC User s Guide Contents Hardware Specifications Dimensions 11 Weights 11 Lifting and Carrying 12 Power 13 Environment 13 Water Cooling 13 Exhausts 13 Rotary Pump 13 API Gas Exhaust 13 Nitrogen 14 CID Gas 14 Table
43. Tuning 82 Mobile Phase 82 Probe Temperature 82 Desolvation Gas 82 Removing the Probe 83 Table of Contents Quattro LC User s Guide Mass Calibration Introduction Electrospray Overview Preparing for Calibration Reference Compound Introduction Tuning Instrument Threshold Parameters Calibration Options Selecting the Reference File Removing Current Calibrations Selecting Parameters Automatic Calibration Check Calibration Parameters Mass Measure Parameters Performing a Calibration Acquisition Parameters Starting the Calibration Process Checking the Calibration Calibration Failure Incorrect Calibration Manual Editing of Peak Matching Saving the Calibration Verification Electrospray Calibration with PEG Atmospheric Pressure Chemical Ionisation Introduction Preparing for Calibration Reference Compound Introduction Tuning Calibration Options Selecting Reference File Removing Current Calibrations Selecting Calibration Parameters Performing a Calibration Static Calibration Acquisition Parameters Acquiring Data Manual Calibration Scanning Calibration and Scan Speed Compensation Acquiring Data Manual Calibration Calibrating MS2 Using the Instrument Calibration Failure Incorrect Calibration Manual Editing of Peak Matching Saving the Calibration Manual Verification Table of Contents 85 85 85 86 86 86 87 88 89 89 89 89 90 91 92 94 96 98 100 102 103 103 104 106 107 107 108 108 108 108 108 108 1
44. Up for MS MS Operation Parent Ion Selection Fragmentation Shutdown Procedures Emergency Shutdown Overnight Shutdown Complete Shutdown Quattro LC User s Guide 33 33 36 36 37 37 39 41 42 42 42 42 43 43 44 44 45 45 45 46 46 47 48 48 48 49 Table of Contents Quattro LC User s Guide Electrospray Introduction Post column Splitting Megaflow Changing Between Flow Modes Operation Checking the ESI Probe Obtaining an Ion Beam Tuning and Optimisation Probe Position Nebuliser Gas Desolvation Gas Cone Gas Purge Gas Source temperature Capillary Voltage Sample Cone Voltage Extraction Cone Voltage Low Mass Resolution and High Mass Resolution Ion Energy Megaflow Hints Removing the Probe Sample Analysis and Calibration General Information Typical ES Positive Ion Samples Typical ES Negative Ion Samples Chromatographic Interfacing LC MS Sensitivity Enhancement Nanoflow Electrospray Overview Installing the Interface Operation of the Camera System Using the Microscope Glass Capillary Option Restarting the Spray Fused Silica Option Changing Options Table of Contents 51 53 54 54 55 55 56 57 57 58 58 59 59 60 60 60 61 61 61 61 62 62 62 63 63 64 65 67 68 71 71 72 fe 74 76 Quattro LC User s Guide Atmospheric Pressure Chemical lonisation Introduction TT Preparation 78 Checking the Probe 19 Obtaining a Beam 80 Calibration 82 Hints for Sample Analysis 82
45. a 10ml plastic syringe or a regulated gas Elastomer supply apply pressure to the back of the tip until Pas Glass a drop of liquid is seen Remove the back Capillary pressure On the tune page select Gas and turn on Nitrogen Select OPERATE Set Capillary between 1 and 1 5kV Nanoflow Electrospray Page 72 Quattro LC User s Guide Adjust Desolvation Gas using the knob on the front panel of the instrument An ion beam should now be visible on the tune page Tune the source voltages adjust the gas flow and adjust the three axis manipulator for maximum ion current The ion current may change dramatically with very slight changes of position but the high resolution of the threads in the manipulator allows very fine tuning Restarting the Spray Should the spray stop it is possible to restart it by adjusting the three axis manipulator so that viewed under magnification the capillary tip touches the sample cone and a small piece of the glass hair shears off It may also be necessary to apply some back pressure to the holder to force a drop of liquid from the capillary Up to 1 4 bar 20 psi can be applied and with this pressure a drop should be visible unless the capillary is blocked Nanoflow Electrospray Page 73 Quattro LC User s Guide Fused Silica Option Use the plug cap prior to assembly in the low dead volume union to set the pilot depth of the From Injector or Column fe
46. adbacks vary with tune page settings If any of these voltages are absent check that the source and hexapole transfer lens assembly have been correctly reassembled Further investigation which will require the services of a qualified service engineer should be left to Micromass personnel Maintenance and Fault Finding Page 149 Quattro LC User s Guide Cleaning Materials It is important when cleaning internal components to maintain the quality of the surface finish Deep scratches or pits can cause loss of performance Where no specific cleaning procedure is given fine abrasives should be used to remove dirt from metal components Recommended abrasives are 600 and 1200 grade emery paper Lapping paper produced by 3M After cleaning with abrasives it is necessary to wash all metal components in suitable solvents to remove all traces of grease and oil The recommended procedure is to sonicate the components in a clean beaker of solvent and subsequently to blot them dry with lint free tissue Recommended solvents are Isopropyl Alcohol IPA Methanol Acetone Following re assembly components should be blown with oil free nitrogen to remove dust particles Warning Many of the procedures described in this chapter involve the removal of possibly toxic contaminating deposits using flammable or caustic agents Personnel performing these operations should be aware of the inherent risks and should take the necessary p
47. al loss in performance indicated by a loss of resolution and an increase in the background noise Above 10 mbar the Operate and Vacuum LEDs on the instrument change from green to amber indicating that the vacuum is insufficient to maintain the instrument in operate Above 10 mbar the Vacuum LED changes to flashing red indicating that the vacuum pump trips have been activated followed by no indication when the instrument is no longer pumping Before suspecting a leak the following points should be noted The turbomolecular pumps will not operate if the rotary pump has failed If the rotary pump is not maintained the oil may become so contaminated that optimum pumping speed is no longer possible Initially gas ballasting may clean the oil If the oil in the rotary pump has become discoloured then it should be changed according to the pump manufacturer s maintenance manual The turbomolecular pumps switch off if an over temperature is detected This could be due to poor backing vacuum failure of the water supply or a leak in the source or analyser The turbomolecular pumps switch off if full speed is not achieved within a set time following start up This could be due to a leak or too high an ambient temperature Vacuum Leaks If a leak is suspected the following basic points may help to locate it Leaks very rarely develop on an instrument that has been fully operational Suspect components that have recently been
48. alibrate page Check Static Calibration and MS1 in the Types area of the dialog box In the Process area of the dialog box check Acquire amp Calibrate Acquisition Parameters Selecting the Acquisition Parameters button brings forward the default mass ranges scan speeds and acquisition mode relevant to the pegh1000 ref reference file The upper area contains the Acquisition Parameters where mass range run time and data type are set When the instrument is fully calibrated any mass range or scan speed is allowed within the upper and lower limits dictated by the calibrations It is therefore sensible to calibrate over a wide mass range Since the pegh1000 ref reference file has peaks from Yz 63 to z 987 it is possible to calibrate over this mass range which is sufficient for the majority of applications with APcI The following example shows a setup to achieve this Run Duration sets the time spent acquiring data for the static calibration The time set must allow chance to inject a volume of reference solution and acquire several scans Data Type allows a choice of centroided continuum or MCA data to be acquired For APclI while either continuum or centroided data may be used Continuum is recommended Mass Calibration Page 109 Quattro LC User s Guide Calibration Acquisition Setup Acquisition Parameters Scan From B0 amu Scan To E 040 amu Run Duration fo 32 mins Data Type Continuum Scan Parameters Sta
49. ammonium nitrate Use reference file PEGH2000 Reference Information Page 159 Quattro LC User s Guide Sodium lodide Solution for Positive lon Electrospray Method 1 Prepare a solution of sodium iodide at a concentration of 2 ug ul micrograms per microlitre in 50 50 propan 2 ol IPA water with no additional acid or buffer Add caesium iodide to a concentration of 0 05 yg pl The purpose of the caesium iodide is to obtain a peak at z 133 Cs to fill the gap in the calibration file between z 23 Na and the first cluster at z 173 which would lead to poor mass calibration in this mass range Do not add more CsI than suggested as this may result in a more complex spectrum due to the formation of NaCsl clusters Use reference file NAICS REF Method 2 Prepare a solution of sodium iodide at a concentration of 2 ug ul micrograms per microlitre in 50 50 propan 2 ol IPA water with no additional acid or buffer Add rubidium iodide to a concentration of 0 05 pg yl The purpose of the rubidium iodide is to obtain a peak at z 85 P Rb with an intensity of about 10 of the base peak at z 173 Rubidium iodide has the advantage that no rubidium clusters are formed which may complicate the spectrum Note that rubidium has two isotopes Rb and Rb in the ratio 2 59 1 giving peaks at z 85 and 87 Use reference file NAIRB REF Sodium lodide Solution for Negative lon Electrospray Either of the above sol
50. an speed compensation then the scan speed should be set at the same speed to be used for later acquisitions Fast Scan Time determines the scan speed used for the scan speed compensation and the upper limit of scan speed that can be used for subsequent acquisitions When using MCA or thresholded continuum data the scan speed is limited to 400 and 1000 amu sec respectively So for a mass range of 100 to 1600 amu the minimum values are 1 5 seconds for thresholded continuum data and 2 5 seconds for MCA data When using centroided data the maximum acquisition rate is much higher although it is unlikely that scan speeds of greater than 2000 amu sec would be needed for acquiring data Select Default then OK to return to the Automatic Calibration dialog box Alternatively select chosen values if a different calibration range is required Mass Calibration Page 95 Quattro LC User s Guide Starting the Calibration Process To start the calibration process Select OK from the Automatic Calibration dialog box The instrument acquires all of the calibration files in the following order using the data file names shown MSI static calibration data file STATMSI MS1 scanning calibration data file SCNMSI MSI scan speed compensation data file FASTMS1 MS2 static calibration data file STATMS2 MS2 scanning calibration data file SCNMS2 MS2 scan speed compensation data file FASTMS2 Once all of the data have been acquired each data file is com
51. ance and Fault Finding Page 151 Quattro LC User s Guide Maintenance and Fault Finding Page 152 Quattro LC User s Guide Reference Information Overview The reference files listed in this chapter have all ion intensities set to 100 Actual ion intensities are not of course all 100 but the calibration software does not take account of the ion intensities and this is a convenient way to store the reference files in the required format Most samples can be purchased from the Sigma chemical company To order contact Sigma via the internet or by toll free or collect telephone or fax Internet http www sigma sial com This site contains a list of worldwide Sigma offices many with local toll free numbers Toll free telephone USA amp Canada 800 325 3010 Outside USA amp Canada 1 314 771 5750 call collect Toll free fax USA amp Canada 800 325 5052 Outside USA amp Canada 1 314 771 5750 call collect and ask for the fax machine Outside USA amp Canada 1 314 771 5757 this is a toll call direct fax line Reference Information Page 153 Quattro LC User s Guide Positive lon Ref File Chemical Name Molecular Name Sigma Code Mass Bovine Ubiquitin U6253 Human a globin H753 Superoxide dismutase Hb internal S2515 15591 35 900 1500 cal Human f globin H7379 Horse heart myoglobin M1882 Polyethylene glycol tat ES and BECEHOU 3m monum acetate 80
52. anning acquisition is now performed When the acquisition is complete the data are combined to give a single spectrum which is compared against the reference file A calibration curve is drawn and a report printed in a similar way to when the original calibration was performed An example is shown below Unlike the original calibration procedure the instrument calibration is not changed and the report that is printed is a verification report Instrument Calibration Report Mass 20 Da to 3930 Da Res 14 5 14 5 IE 0 5 Calibrated 15 12 on 11 27 97 MS1 Scanning Verification 0 135 VEETEE ES E eee ap er eet eye pe ET T ree ee rey eer pry 500 1000 1500 2000 2500 3000 3500 Mass Calibration Page 105 Quattro LC User s Guide Electrospray Calibration with PEG Caution should be used when calibrating with PEG in electrospray mode due to the number of peaks which are produced Although ammonium acetate is added to the PEG reference solution to produce M NH ions under some conditions it is quite usual to see M H M Na and doubly charged ions The spectrum shown below demonstrates how the PEG spectrum can be dominated by doubly charged ions in this case M 2NH if the wrong conditions are chosen In this case the concentration of ammonium acetate in the reference solution is too high 5mmol ammonium acetate is the maximum that should be used and Cone is too low A low Cone voltage encourages the pr
53. apillary LC 64 Microbore 2 1mm LC 54 64 Com1 and Com2 28 Cone gas 59 69 Constant neutral loss 22 Contact closure 26 Conversion dynode 15 146 Cooling fan 123 Corona 17 Coupled column chromatography 65 Index Page 161 Quattro LC User s Guide Data acquisition See Acquisition Data processing 45 Data system 11 22 33 Daughter ion 19 46 47 Desolvation gas 23 25 38 41 58 82 Desolvation temp 39 41 58 Detector 29 146 Dimensions 11 Discharge pin 23 39 78 127 137 Divert valve 25 Drug metabolite 21 Drugs 63 Dye compounds 63 E B Electronics 30 147 Electrospray 17 37 51 Analysis 62 Calibration 85 Negative ion 63 Operation 55 Positive ion 63 Electrospray probe 17 38 55 57 Maintenance 138 Removal 62 Emergency 48 Environment 13 Environmental analysis 21 Environmental contaminants 63 ESD earth facility 28 Event out 26 Exhaust 13 28 33 Exhaust liner 55 69 127 131 Extraction cone 61 132 F Fault finding 123 148 Filter 29 Flow control valve 25 Flow injection analysis 17 52 67 Fluorescence detector 22 Foreline trap 126 Forensic science 21 Formic acid 52 Fragment ions 15 Fragmentation 46 47 52 Fused silica capillary nanoflow 67 74 Fuses 28 147 Index Page 162 G Gas ballast 36 125 151 Gas cell 29 Glass capillary nanoflow 67 72 Gradient elution 64 H Heater 23 Herbicide 21 Hexapole collision cell 15 Hexapole transfer lens 29 127 134 High mass resolution 61 HM R
54. at the cleanable baffle the exhaust liner and the discharge pin blanking plug are fitted Cone Gas Purge Nozzle Gas Plug Encapsulated O Ring 1 Nanoflow Electrospray Page 69 Quattro LC User s Guide Fit a viton O ring and the three shorter nanoflow pillars Install the perspex cover and the Socket nanoflow end flange securing this Head Screws with socket head screws Perspex Cover Do not attempt to refit the moulded cover If not already in place attach the microscope or camera brackets using the screw hole and dowels at the top of the bracket Insert the flexible light guide into the grommet at the base of the perspex cover Set the light source to its brightest Block the Nebuliser and Desolvation Gas outlets on the instrument s front panel The cone gas is split from the desolvation gas internally Nanoflow End Flange Attach the two cables to the sockets marked Capillary Corona and ESI APcl on the front panel of the instrument Set Source Block Temp to approximately 80 C Caution The maximum operating temperature for the source heater is 150 C Do not set Source Block Temp higher than 150 C Nanoflow Electrospray Page 70 Quattro LC User s Guide Objective Lens Grommet Operation of the Camera System Magnification is controlled by the zoom lens A fine focus can be achieved by rotating the objective lens Using the
55. bin Polyethylene Glycol PEG NH4 Sodium Iodide and Caesium Iodide Mixture Sodium Iodide and Rubidium Iodide Mixture Negative Ion Horse Heart Myoglobin Mixture of Sugars Sodium Iodide and Caesium Iodide or Rubidium Iodide Mixture Preparation of Calibration Solutions PEG Ammonium Acetate for Positive Ion Electrospray and APcI PEG Ammonium Acetate for Positive Ion Electrospray Extended Mass Range Sodium Iodide Solution for Positive Ion Electrospray Method 1 Method 2 Sodium Iodide Solution for Negative Ion Electrospray Index Table of Contents 133 154 155 155 155 156 156 157 157 157 158 159 159 159 160 160 160 160 Quattro LC User s Guide Hardware Specifications Dimensions 70mm pumping line 1325mm SS SS SS SSS SS SS l iili SS ventilation Weights Instrument 150kg 3301b Data system computer monitor and printer 60kg 130Ib Rotary pump 40kg 901b Transformer optional 100kg 2201b Hardware Specifications Page 11 Quattro LC User s Guide Lifting and Carrying Warning Persons with a medical condition for example a back injury which prevents them from handling heavy loads should not attempt to lift the instrument Before lifting the instrument proceed as follows Vent and power down the instrument Disconnect the instrument from the power and water supplies Disconnect power and tubing connections to the rotary pump f
56. bined to give a single spectrum which is then compared against the reference spectrum to form a calibration This process takes place in the same order as above If the full calibration dialog box is open then a constantly updated status message for the calibration is displayed Calibrate manuall CAL File Edit Process Help Reference File Last Calibrated Start NAIRB REF 1512 27 Nov 97 Data Directory C Masslynx sanae PRO Data Calibration Status M51 Static Mass 18 Da to 3825 Da Res 14 5 14 51E 0 5 M51 Scanning Mass 20 Da to 3930 Da Res 14 5 14 5 IE 0 5 MS1 Scan Speed Scan 100 to 494 amu sec Res 14 5 14 5 IE 0 5 Compensation MS2 Static Mass 18 Da to 3925 Da Res 14 0 14 0 IE 1 0 M52 Scanning Mass 20 Da to 3930 Da Res 14 0 14 0 IE 1 0 MS2 Scan Speed Scan 100 to 494 amu sec Res 14 0 14 0 IE 1 0 Compensation If when the process is completed the calibration statistics meet with the requirements specified by the selected calibration parameters then a successful calibration message is displayed A calibration report is then printed showing a calibration curve for each of the calibration processes An example of a calibration report is shown opposite Mass Calibration Page 96 Quattro LC User s Guide Instrument Calibration Report Mass 18 Da to 3925 Da Res 14 5 14 5 IE 0 5 Calibrated 15 12 on 11 27 97 MSI Static Calibration 0 654 amu 4 5i Le eU UOCE X C GCEUCEXCEUXC OU
57. ce Rotary Turbomolecular Pump Pumps Enclosure result in permanent damage to the three axis manipulator Installing the Interface To change from the normal electrospray interface and install the nanoflow interface If fitted remove the probe Remove the moulded cover from around the source Undo the three thumb screws and withdraw the probe adjustment flange assembly and glass tube Place the glass tube end on on a flat surface and place the probe support flange assembly on top of the glass tube Remove the PTFE encapsulated source O ring exposed Ensure that the source block heater has been switched off and has cooled before proceeding Observe the Source Block Temp readback on the tune page Warning When the source enclosure has been removed the ion block heater is Nanoflow Electrospray Page 68 Source Quattro LC User s Guide Probe Flange Mounting Pillar po 5 Probe Thumb Nuts Probe Adjustment Flange Unscrew the three probe flange mounting pillars using the holes to obtain the necessary leverage If the sample cone nozzle is not in place remove the two screws that secure the sample cone and fit the cone gas nozzle Replace the two screws Connect the cone gas outlet to the cone nozzle using the PTFE tubing provided The cone gas flow rate is set at 30 of the total desolvation gas flow Ensure that the purge gas is plugged disabled Ensure th
58. d Select OK from the calibration report window to accept the new calibration or select Cancel to retain the previous calibration Incorrect Calibration If the suggested calibration parameters are used and providing that good calibration data have been acquired then the instrument normally calibrates correctly However in some circumstances it is possible to meet the calibration criteria without matching the correct peaks This situation is unusual but it is always wise to examine the on screen calibration report to check that the correct peaks have been matched These errors may occur when the following parameters are set Intensity threshold set to 0 Initial error too high 22 0 e Peak window too high 21 5 e Maximum Std Deviation too high 20 2 If the acquired spectrum looks like the reference spectrum and all of the expected peaks are highlighted then the calibration is OK Mass Calibration Page 119 Quattro LC User s Guide An alternative cause of calibration failure is from contamination or background peaks If a contamination or background peak lies within one of the peak matching windows and is more intense than the reference peak in that window then the wrong peak will be selected Under some conditions this may happen with PEG There are two ways to counter this If the reference peak is closer to the centre of the peak window then the peak window can be narrowed until the contamination peak is excluded
59. d 609 5 while observing the intensity of the non fragmented parent ion in the tune display Adjust the Set mass in the same manner to optimise the intensity of the parent Routine Procedures Page 46 Quattro LC User s Guide Fragmentation Set up a wide range daughter ion scan by adjusting the Mass and Span parameters for peak 2 At this point with the collision gas off a few daughter ions of low intensity may be visible These are the products of unimolecular dissociations Argon 99 9 pure is recommended as the collision gas Select Gas and turn on Collision Adjust CID Gas on the front panel to admit sufficient gas to attenuate the parent ion peak by about 50 Adjust the Entrance Collision and Exit parameters in the Analyser menu to produce the desired degree of fragmentation These parameters are interactive in MS MS operation In daughter ion analysis maximum transmission sensitivity can be achieved by the following adjustments Increasing RF Lens on the Source tune window e Increasing IEnergy 1 on the Analyser window Optimising Collision Optimising Exit Optimising Entrance Optimising the collision gas pressure using the CID Gas needle valve Additionally transmission can be improved at the expense of specificity by reducing HM Res on the Analyser window In most cases where chemical interference with the parent ion is not acute the loss of specificity is negligible Routine Procedure
60. d Fault Finding Page 128 Quattro LC User s Guide Disconnect both electrical connections by pulling back on the plug sleeves to release the plugs from the sockets on the front panel Undo the two knurled thumb nuts that retain the probe and withdraw it from the source Place it carefully to one side Remove the moulded cover from around the source Undo the three thumb screws and withdraw the probe adjustment flange and glass tube Place the glass tube end on on a flat surface and place the probe adjustment flange on top of the glass tube Warning When the source enclosure has been removed the source block is exposed Ensure that the source block heater has cooled before proceeding If fitted remove the APcI discharge pin The sample cone is now accessible Isolation Valve Using a suitable flat blade screwdriver rotate the isolation valve by 90 into its fully anticlockwise position A small improvement in the analyser vacuum may be observed as a result of this operation Maintenance and Fault Finding Page 129 Quattro LC User s Guide The isolation valve is closed when the slot is perpendicular to the direction of flow Carefully wipe the sample cone with a cotton swab or lint free tissue soaked in 50 50 acetonitrile water or 50 50 methanol water A Caution Do not attempt to remove any obstruction by poking This may result in damage to the sample cone Dry the cone using
61. e An injection of 50ul of reference solution will then last for at least 10 minutes When using an infusion pump Fill the syringe with the reference solution Couple the syringe to the electrospray probe with fused silica tubing Set the pump to a flow rate of 4 5 l min Tuning 2670 228 DEFAULT ESP I Txl File lonMode Gas Ramps Other Stop Window Help siale El Gals lenta v Function Set Mass Span Gain Cone ES t Extractor Je lt a Eie lonMode Gas Ramps Other Stop Window Help sjala mit mlale fica 2 Function Set Mass Span Gain 1 LM Res 1 14 5 Kja 2 PrLens pa A mes f gt lEnergy 1 05 Kja 4 Entrance Ez lt gt 922 00 1972 00 2421 00 le 6 87e6 2 89e6 6 43e5 Colision EJP eeeh SowceBockTemp E 0 st oe Desolvation Temp EI viv v LM Res 2 140 lt gt HMRes2 9 ep Eae ide 0 dl Multiplier amp 50 O gt 84 86 320 922 924 970 1972 197 2420 2422 Source Analyser Acquire OPERATE Viw2MwW3M4 Mass Calibration Page 86 Quattro LC User s Guide Before beginning calibration and with reference solution admitted into the source Set Multiplier to 650V Adjust source parameters to optimise peak intensity and shape Set the resolution and ion energy parameters for unit mass resolution on MS1 and MS2 For a good peak distribution across the full mass range Check the intensity of s
62. e Proteins 51 63 Proton abstraction 17 Proton transfer 17 Pump fault 43 Pumping 36 Pumping logic PCB 31 147 Purge gas 59 62 127 R Rear panel Reciprocating pump Reference compound Reserpine Reverse phase RF generator RF generator control PCB RF lens Quattro LC User s Guide 147 52 64 86 153 19 20 21 46 64 77 31 See Hexapole transfer lens RF power PCB Ripple Rotary control Rotary pump Oil Rubidium iodide Saccharides Sample cone Sample cone nozzle Scan control PCB Scope Selected ion recording Sensitivity LC MS Shutdown Complete Emergency Overnight Single ion recording Sodium iodide Source Housing Source block Source temperature Source voltages Specifications Split post column Start up Status Steroids Structural elucidation Sugar mixture Syringe pump 11 13 16 33 43 124 126 33 36 126 63 156 158 160 60 127 128 130 69 130 63 156 158 160 127 136 15 52 64 Index Page 163 Quattro LC User s Guide T U Target compound analysis 65 UV detector 22 26 53 64 TEA UV photodiode array detector 22 See Triethylamine Tetrahydrofuran 65 V THF See Tetrahydrofuran Vacuum 16 124 Threshold parameters 87 Leak 43 124 Toxicology 21 Protection 42 TPC Vacuum LED 24 42 43 See Transputer processor card Vibration 148 Trace enrichment 65 Transformer 11 Transputer processor card 31 W Triethylamine 64 Trifluoroacetic acid 64 Wn S 19 86599 s Tu
63. e although this is dependent upon compound structure High mass biopolymers for example peptides proteins and oligonucleotides produce a series of multiply charged ions The acquired data can be transformed by the data system to give a molecular weight profile of the biopolymer Electrospray Page 51 Quattro LC User s Guide The source can be tuned to fragment ions within the ion block This can provide valuable structural information for low molecular weight analytes The most common methods of delivering sample to the electrospray source are Syringe pump and injection valve A flow of mobile phase solvent passes through an injection valve to the electrospray source This is continuous until the pump syringes empty and need to be refilled Sample is introduced through the valve injection loop usually 10 or 20g capacity switching the sample plug into the mobile phase flow Tuning and acquisition are carried out as the sample plug enters the source At a flow rate of 10 l min a 201 injection lasts 2 minutes Reciprocating pump and injection valve A flow of mobile phase solvent passes through an injection valve to the electrospray source Sample injection and analysis procedure is the same as for the syringe pump The pump reservoirs are simply topped up for continuous operation The most suitable reciprocating pumps for this purpose are those which are specified to deliver a flow between 1 yl min and 1 ml min A constant fl
64. e Cone Voltage A Cone setting between 25V and 70V will produce ions for most samples although solvent ions prefer the lower end and proteins the higher end of this range Whenever sample quantity and time permit Cone should be optimised for maximum sensitivity within the range 15V to 150V Electrospray Page 60 Quattro LC User s Guide Extraction Cone Voltage Extractor optimises at 3 to 10V for most samples and all samples may be optimised within the range 0 to 20V Higher values than this may be set to induce ion fragmentation of low molecular weight samples Increasing Extractor and Cone together will give rise to more severe fragmentation conditions Low Mass Resolution and High Mass Resolution Peak width is affected by the values of low mass resolution LM Res and high mass resolution HM Res Both values should be set low typically 5 0 at the outset of tuning and only increased for appropriate resolution after all other tuning parameters have been optimised A value of 15 arbitrary units usually gives unit mass resolution on a singly charged peak up to z 1600 lon Energy The ion energy parameter usually optimises in the range OV to 3V It is recommended that the value is kept as low or negative as possible without reducing the height intensity of the peak This will help obtain optimum resolution If in positive ion mode an ion energy value below 1V can be used without reducing the peak intensity then source clea
65. e carried out only by Micromass engineers Fuses In the following list the designation T indicates a time lag fuse Analog PCB Fuse No Fuse type Ref No Fl 10A T 20mm anti surge TDS505 1340143 F2 10A T 20mm anti surge TDS505 1340143 RF power PCB Fuse No Fuse type Ref No F1 5A T 20mm anti surge 1340142 Power Backplane 2 Fuse No Fuse type Ref No F7 2A T 20mm anti surge TDS506 1340161 F8 2A T 20mm anti surge TDS506 1340161 Pumping Logic PCB Fuse No Fuse type Ref No F1 2A T 20mm semi delay 1340137 Power Sequence PCB Fuse No Fuse type Ref No F1 4A T 20mm anti surge ceramic 1340164 F2 2A T 20mm anti surge TDS506 1340161 F3 6 3A T 20mm anti surge TDS506 1340163 Rear Panel Fuse No Fuse type Ref No F1 10A T HBC ceramic anti surge 1340147 F2 10A T HBC ceramic anti surge 1340147 Maintenance and Fault Finding Page 147 Quattro LC User s Guide Fault Finding Check List A No Beam Warning There are high voltages present throughout the mass spectrometer Extreme caution should be taken when taking measurements with a meter or an oscilloscope In the standby mode OPERATE not selected the high voltages are switched off in the source and analyser assemblies but high DC voltages and mains voltages remain in the power supply units Any investigation in the RF generator must be made only by a Micromass engineer Refer to the relevant chapters of this manual and check the followin
66. easing in 50 C intervals to 650 C over a period of 10 minutes A Caution Do not set APcl Heater to 650 C immediately as this may damage the probe heater This procedure should remove any chemical contamination from the probe tip Replacing the Probe Tip Heater Probe Tip Assembly Remove the probe tip assembly by carefully loosening the two grub screws Disconnect the heater from the probe body by pulling parallel to the axis of the probe Fit a new heater assembly Reconnect the probe tip assembly Maintenance and Fault Finding Page 143 Quattro LC User s Guide Replacing the Fused Silica Capillary Fused Silica Capillary m Adapter Nut 9 id Filter JD V GVF 004 7 Ferrule Finger tight Nut amp Ferrule o oy A Qc Rheodyne Nut amp Ferrule With the probe removed from the source proceed as follows Remove the probe tip assembly and the heater as described in the preceding section Remove the probe end cover by removing the two screws and the grub screw that retains the LC filter Loosen the filter from the adapter nut Unscrew the adapter nut from the probe Remove and discard the fused silica capillary Using a ceramic capillary cutter cut a new length of 300um o d x 100yum i d fused silica capillary about 1 centimetre excess in length Using a GVF 004 ferrule and the adapter nut connect the sample capillary to the filter ensuring that the liner tube is fully butted
67. ect on mass assignment Mass Calibration Page 120 Quattro LC User s Guide Manual Verification Once a full instrument calibration is in place it is not always necessary to repeat the full calibration procedure when the instrument is next used Instead a calibration verification can be performed There is no benefit in verifying each calibration individually re calibration is just as quick If a scanning acquisition is to be made and the calibration is to be checked Set up a scanning acquisition over the Scanning Verification x required mass range and at the required scan speed in the normal way Select Verification Type C Static Start the acquisition and inject the Scanning reference solution so that reference data is acquired C Scan Speed Compensation Sto p the ac qu isition Select Verification File Combine scans in data file CALVERT From 5 To 18 Browse Access the calibrate dialog box and set all peak matching parameters to the same values that were used for the calibration Select Process Verification from file and check Scanning Calibration see below Cancel Select Scanning Calibration and either MS1 or MS2 depending on the type of data acquired Clicking on Browse select the acquired file and enter the start and end scans of the reference data Select OK to verify the calibration Mass Calibration Page 121 Quattro LC User s Guide T5 Verify B x Print
68. ected in the Calibrate dialog box In the case of too many consecutive peaks missed Check the data in the on screen calibration report to see if the missed peaks are present in the acquired calibration file If the peaks are not present then the first three reasons above are likely causes If the peaks are present in the data but are not recognised during calibration then the latter four are likely reasons Having taken the necessary action proceed as follows If Intensity threshold Initial error and Peak window are adjusted to obtain a successful calibration check the on screen calibration report to ensure that the correct peaks have been matched With a very low threshold and wide ranges set for the initial error and peak window it may be possible to select the wrong peaks and get a successful calibration This is particularly relevant for calibrations with PEG where there may be peaks due to PEG H PEG NH PEG Na and also doubly charged species Select OK from the calibration report window to accept the new calibration or select Cancel to retain the previous calibration Mass Calibration Page 101 Quattro LC User s Guide Incorrect Calibration If the suggested calibration parameters are used and providing that good calibration data have been acquired then the instrument should be calibrated correctly However in some circumstances it is possible to meet the calibration criteria without matching the correc
69. ectrum below the Intensity threshold value measured as a percentage of the most intense peak in the spectrum will not be used in the calibration procedure Mass Calibration Page 90 Quattro LC User s Guide The process of producing a calibration curve is described in detail in the MassLynx NT User s Guide The Polynomial order of the curve has values from 1 to 5 as the available options A polynomial order of 1 should not be used An order of 2 is suitable for wide mass ranges at the high end of the mass scale and for calibrating with widely spaced reference peaks Sodium iodide in particular has widely spaced peaks 150 amu apart and horse heart myoglobin is used to calibrate higher up the mass scale so this is the recommended polynomial order for these calibrations An order of 3 fits a cubic curve to the calibration A fourth order is used for calibrations which include the lower end of the mass scale with closely spaced reference peaks This is suitable for calibrations with PEG which extend below 300 amu A fifth order fit rarely has any benefit over a fourth order fit Mass Measure Parameters These are accessed through Edit Mass Measure Parameters If didis ai x continuum or MCA data are acquired M Background subtract for calibration then these parameters Polynomial order Cancel need to be set before the calibration is T Below curve 33 00 carried out Information on these parameters can be
70. ed singly or simultaneously to optimise the beam Atmospheric Pressure Chemical lonisation Page 80 Quattro LC User s Guide The position of the probe will affect sensitivity However if the sample is contained in a biological matrix or is contained in an involatile solvent the probe should be moved away from the sample cone and towards the corona discharge pin Warning It is normal for the iazeut source enclosure the glass tube Probe and parts of the probe adjustment ccu flange to reach temperatures of up to 60 C during prolonged APcI operation Care should be exercised when handling source components immediately after operation Warning Switch off the liquid flow and allow the probe to cool lt 100 C before removing it from the source DENEN Sideways Caution Failure to employ a Probe desolvation gas flow during APcI Adjustment operation may lead to heat damage to the source BTune QULC APcl BEI Eile lonMode Gas Ramps Other Stop Window Help StS et SO vs dete m m Function Set Mass Span Gain Corona 300 gt 1 Cone 5 lt gt 2 Extractor 3 BTune QULC APcl ixi Fie lonMode Gas Ramps Other Stop Window Help age s mae i RF Lens 0 50 E Function Set Mass Span Gain a e LM Res 1 HM Res 1 lEnergy 1 Source Block Temp APcI Probe Temp Entrance Collision Exit Source Analyser LM Res 2 15 HM Res 2 15 IEnergy 2 2 0 i 650
71. ed when this discolouration reaches the region of the trap furthest from the pump the vacuum side The manufacturers recommend that the sorbent is replaced routinely at three monthly intervals With the instrument vented and the pump switched off replace the sorbent as described in the manufacturer s literature Rotary Pump Oil The oil in the rotary pump should be maintained at the correct level at all times Check the oil level at weekly intervals topping up if necessary It is important to monitor the condition of the oil regularly Replace the oil when it has changed to a noticeable red colour or routinely at 4 month intervals 3000 hours operation At the same time replace the oil mist filter s mist element see above Change the oil in the rotary pump as follows Gas ballast lightly for 30 to 60 minutes Vent and shut down the instrument as described in Routine Procedures It will be found easier to drain the oil while the pump is still warm Drain the oil through the drain hole situated near the oil level sight glass Flush the pump then replace the drain plug and refill the pump with the correct grade oil to the correct level Gas ballast lightly for 30 to 60 minutes For further servicing information refer to the manufacturer s manual Maintenance and Fault Finding Page 126 Quattro LC User s Guide The Source Overview The Z spray source is a robust assembly requiring little maintenance The source consists
72. em 100 120V or 200 240V 13A Power consumption 3 0kW max Environment Ambient temperature 15 28 C 59 82 F Short term variance 1 5 hours x2 C lt 4 F Overall heat dissipation excluding LC and optional water chiller 2 5kW maximum Humidity Relative humidity lt 70 Water Cooling Heat dissipation into the water 200W Exhausts Rotary Pump The rotary pump must be vented to atmosphere external to the laboratory via a fume hood or industrial vent API Gas Exhaust The API gas exhaust must be vented to atmosphere external to the laboratory exhaust line In the event of an instrument failure rotary pump exhaust could be Caution The API gas exhaust line must not be connected to the rotary pump admitted into the source chamber producing severe contamination Hardware Specifications Page 13 Quattro LC User s Guide Nitrogen A supply of dry oil free nitrogen at 6 7 bar 90 100 psi is required If plastic tubing is used it must be made of Teflon The use of other types of f Caution The lines supplying nitrogen to the instrument must be clean and dry plastic will lead to contamination of the instrument CID Gas Argon is required as collision gas The supplied gas should be dry an in line water trap is required of high purity 99 9 and at a pressure of approximately 350 mbar 5 psi Caution Operating with the CID gas at a significantly higher pressure will result in a fault
73. entroided continuum or MCA data to be acquired Continuum or MCA acquisitions are generally used for electrospray calibrations although calibrating in MCA mode limits the maximum acquisition speed to 400 amu sec When using thresholded continuum data with 8 channels per amu see the MassLynx NT User s Guide the maximum acquisition speed is 1000 amu sec The lower area in the Calibration Acquisition Setup dialog box contains the Scan Parameters Mass Calibration Page 94 Quattro LC User s Guide When an instrument acquires data for a static calibration it examines the reference file to find the expected reference masses and then acquires data over a small mass span around each peak s expected position Thus the acquired data do not contain continuous scans Each spectrum comprises small regions of acquired data around each peak separated by regions where no data are acquired Static Span sets the size of this small region around each reference peak A span of 4 0 amu is typical Static Dwell determines how much time is spent acquiring data across the span A value of 0 1 second is suitable Slow Scan Time determines the scan speed used for the scanning calibration If both a scanning calibration and a scan speed compensation are to be performed then the scan speed should be set to approximately 100 amu sec a scan time of 19 8 seconds over a mass range of 20 to 2000 amu If only a scanning calibration is to be performed without sc
74. er supplies Four high voltage power supplies plugged into the backplane below the analyser housing These supply the detector system and the high voltages for the source and electrospray probe Two RF generators bolted to the side of the analyser housing Pumping Logic PCB This controls the turbomolecular pumps the pumping sequence the gas valves and the solenoids It also controls the phosphor and dynode voltages Power Sequence PCB This PCB examines the vacuum operate and interlock signals in order to control the switching of various supplies Also on this PCB is a module delivering the photomultiplier voltage Transputer Processor Card TPC This contains the transputer array and controls data acquisition and control functions as well as interfacing to the PC Scan Control PCB This PCB produces control signals for mass resolution function energy collision energy and pre filter energy Analog PCB This PCB controls the source heater and focusing voltages RF Generator Control Upper PCB This controls the RF and DC voltages applied to the first quadrupole It also supplies the collision cell voltages RF Generator Control Lower PCB This controls the RF and DC voltages applied to the second quadrupole Instrument Description Page 31 Quattro LC User s Guide Instrument Description Page 32 Quattro LC User s Guide Routine Procedures Start Up Following a Complete Shutdown Preparation If t
75. es Preparation for APcI Operation and that the pin is connected using the APcI HV cable Ensure that the APcI probe is fitted as described above that the desolvation gas tube is connected to the front panel and that the cone gas and purge gas outlets are plugged The top line of the tune page indicates the current ionisation mode If necessary change the ionisation mode using the lon Mode command Set Source Block Temp to 150 C Caution The maximum operating temperature for the source heater is 150 C Do not set Source Block Temp higher than 150 C Set APcl Probe Temp to 20 C with no liquid flow and Nitrogen off Initially set Corona to 2 5 kV and Cone to 30V When Source Block Temp reaches 150 C Select Nitrogen to switch on the nitrogen gas Using the valves on the front of the instrument adjust Desolvation Gas to 150 litres hour and set Nebuliser Gas to its maximum setting To monitor the flow rate select Window then Gas Flow on the tune page and observe the readback window Select one of the peak display boxes and set Mass to 50 and Span to 90 Select OPERATE Increase Gain on the peak display box in the range 1 to 20 until peaks become clearly visible Set APcl Probe Temp to 400 C When APcl Probe Temp reaches 400 C Start the LC pump at a flow of 1 ml min Optimise Corona so that the peaks reach maximum intensity Optimise the probe position for intensity and stability The two screws can be adjust
76. es See High mass resolution Humidity 13 l Infusion pump 15 52 86 Injection loop 86 Injection valve 25 52 lon energy 61 lon evaporation 17 lon mode 39 40 41 56 80 lon source See Source L LC MS interface 64 LM Res See Low mass resolution Low mass resolution 61 M Mains switch 28 Maintenance 123 Mass calibration See Calibration Mass measurement 91 Megaflow 54 59 61 64 Metabolites 63 Microscope 71 Mobile phase 82 MRM 19 21 MS1 mode 18 MS2 mode 18 MS MS 18 46 Multiple reaction monitoring See MRM Multiply charged ions 17 Myoglobin 63 155 157 N Nanoflow electrospray 17 67 127 Nano HPLC 17 67 Narrow mass scanning 65 Nebuliser 17 25 38 41 Nebuliser gas 23 58 Neonatal screening 22 Nitrogen 14 27 33 41 55 58 O Oil mist filter 126 Oligonucleotides 51 63 Operate 41 Operate LED 24 42 43 Organometallics 63 J P Parent ion 20 46 PC link 28 33 Peak matching 103 120 PEG See Polyethylene glycol Penning gauge See Active inverted magnetron gauge Peptides 19 51 63 Pesticides 21 63 82 Pharmacokinetic studies 21 Phase system switching 65 Phosphate 63 Phosphor 15 146 Photomultiplier 15 146 148 Pirani gauge 16 29 125 Plasma 17 Pollutants 63 Polyethylene glycol 63 106 107 155 159 Polysaccharides 63 Power 13 Failure 43 Power backplane 2 147 Power cord 28 Power sequence PCB 31 147 Power supplies 31 Preventive maintenance 151 Probe temperature See APcl probe temperatur
77. es that a file with no calibration is currently active on the instrument and prevents any previously saved calibrations from being modified or overwritten Selecting Parameters A number of parameters needs to be set before a calibration is started Default parameters are set when the software is initially loaded which usually give a suitable calibration but under some conditions these may need to be adjusted Automatic Calibration Check Automatic Calibration Check x pul OK Missed Reference Peaks 2 SEES ancel Maximum Std Deviation D 20 Iv Apply Span Correction M Check Acquisition Calibration Ranges This is accessed from Edit AutoCal Check Parameters It is here that limits are set which the calibration must attain before the instrument is successfully calibrated Two user parameters can be set Missed Reference Peaks sets the maximum number of consecutive peaks which are not matched when comparing the reference spectrum and the acquired calibration spectrum If this number is exceeded then the calibration will fail The default value for this parameter 2 is suitable in most cases Mass Calibration Page 89 Quattro LC User s Guide Maximum Std Deviation is set to a default of 0 20 During calibration the difference between the measured mass in the acquired calibration file and the true mass in the reference file is taken for each pair of matched peaks If the standard deviation of the set of mass difference
78. etonitrile water or 50 50 methanol water Maintenance and Fault Finding Page 130 Quattro LC User s Guide Exhaust Liner Cleanable Baffle Extraction Tool Cone Gas Inlet Line Dry the cone and nozzle using nitrogen To minimise down time fit a spare sample cone obtainable from Micromass at this stage If material has built up on the exhaust liner and cleanable baffle Remove the cleanable baffle and the exhaust liner Clean these components or obtain replacements Fit the cleaned or the replacement exhaust liner and cleanable baffle to the ion block Refitting the sample cone is a reversal of the removal procedure Maintenance and Fault Finding Page 131 Quattro LC User s Guide Removing and Cleaning the Source Block and Extraction Cone On the tune page select Other from the menu bar at the top of the tune page Click on Vent The rotary pump and the turbomolecular pumps switch off The turbomolecular pumps are allowed to run down to 50 speed after which a vent valve opens to atmosphere automatically Remove the source enclosure and the sample cone as described in the previous section Extraction Cone 9 S lon Block Heater When the instrument has vented Remove the two screws which secure the ion block and remove the ion block heater and the ion block Separate the extraction cone and the PTFE insulating ring from the ion block Remove the plug and the PTFE sealing washe
79. ew scans of the reference compound the remaining scans being of background As the automatic calibration procedure combines all of the scans in the data file to produce a calibration spectrum the resulting spectrum may be too weak to give a successful calibration Whether the calibration is successful or failed it is wise to check the calibration manually Mass Calibration Page 111 Quattro LC User s Guide Manual Calibration To perform a manual calibration using the acquired data From the chromatogram window call up the calibration file STATMS1 Determine the scan numbers at the beginning and end of the chromatogram peak for the reference solution This can be achieved using Process Combine Spectra and using the left mouse button to drag across the peak The start and end scans will be displayed in the combine spectra dialog box Return to the Calibrate dialog box Access JESSE TSS x the manual calibration options as shown Select Calibration Type by selecting Process c gg Calibration From File C Scanning Select Static calibration type and MS1 Scan Speed Compensation In the lower area the data file STATMS1 CERIS sccm ries should be selected automatically If this is not the case the correct file can be selected by clicking on the Browse Emm i Io f button e Enter the start and end scans of the reference data in the From and To boxes Combine scans in data file STAT Cancel Select OK
80. file SCNMS1 Uncalibrated 100 1371 95 1521 83 1671 69 0 Reference file NAIRB 100 1372 04 1521 93 1671 83 96 D Mass difference Raw Ref mass 0 07 amu 1821 54 1821 72 1971 42 1971 61 0 19 Residuals Mean residual 8 955025e 11 0 031523 0 06 x x X x X x x x x amu x X Ox Me 9 ue x 0 08 Miz 250 500 750 1000 1250 1500 1750 2000 2250 2750 3000 3260 3500 3750 ibrate Beles 26 matches of 28 tested references 2421 15 2121 34 2271 25 2121 51 2271 40 2421 30 0 19 Residuals 0 02 x x amu Ea 0 03 1300 1400 1500 1600 1700 1800 1900 2000 2100 Mean residual 8 955025e 11 0 031523 x x Miz 2200 2300 2400 Mass Calibration Page 99 Quattro LC User s Guide Calibration Failure If the calibration statistics do not meet the requirements then a message will be displayed describing at what point and why the calibration failed an example of a failed calibration message is shown below This message also states where the attempted calibration data can be viewed so that the exact cause of failure can be determined Instrument Calibration x The number of consecutive missed reference points reported by the Static Calibration exceeds the set maximum value of 2 Use the Calibration from file process to view the calibration graphs There are a number of reasons for a calibration to fail e No peaks If the acquired
81. g Unsteady or Normal tuning parameters are set and where appropriate readback values are acceptable All necessary cables have been correctly attached to the source and probe OPERATE is on check the LED on the front panel The source has been assembled correctly and is clean The source isolation valve is open There are no error messages reported by the electronics see the viewing window at the rear of the instrument Low Intensity Beam Should the preceding checks fail to reveal the cause of the problem check that Ripple Gas and liquid flows are normal The analyser pressure is less than 1x10 mbar Peaks appear to vary cyclically in intensity when there is ripple superimposed on the peak e Possible causes are Unstable power supplies in the source supplies or the RF DC generator Unstable photomultiplier supply Vibration from the rotary pumps or even other equipment in the same building The frequency of the ripple measured using an oscilloscope can often help locate the source Mains frequency ripple for example points towards an unstable power supply or vibration from mains powered machinery Maintenance and Fault Finding Page 148 Quattro LC User s Guide High Back Pressure For electrospray a higher than normal back pressure readout on the HPLC pump together with a slowing of the actual solvent flow at the probe tip can imply that there is a blockage in the capillary transfer line or
82. g with these compounds is most likely to be due to a broken fused silica capillary or probe tip heater assembly Low LC pump back pressure For 50 50 acetonitrile water at a flow rate of 1 ml min a LC pump back pressure less than 14 bar 200 psi is indicative of a broken fused silica capillary or a leaking connector High LC pump back pressure For 50 50 acetonitrile water at a flow rate of 1 ml min a LC pump back pressure above 35 bar 500 psi is indicative of a blockage or partial blockage in the fused silica capillary in a LC connector or in the filter It is advisable to change the inner filter pad see Replacing the Fused Silica Capillary in the following pages on a regular basis Gas flow problems Check all gas connections for leaks using soap solution or a suitable leak searching agent such as Snoop Cleaning the Probe Tip Remove any visible deposits on the inner wall of the probe heater with a micro interdental brush supplied in the spares kit soaked in methanol water Before starting an analysis With the probe out of the instrument connect the nebulising gas supply line Select Gas and turn on Nitrogen Allow the gas to flow for several seconds to clear any debris from the heater Turn off Nitrogen Insert the probe into the source Maintenance and Fault Finding Page 142 Quattro LC User s Guide Select Gas and turn on Nitrogen Raise APcl Heater gradually starting at 100 C and incr
83. he gas and electrical connections from the front panel Unscrew the probe thumb nuts and remove the probe Remove the moulded cover which surrounds the source Routine Procedures Page 39 Quattro LC User s Guide Undo the three thumb screws and remove the probe adjustment flange and glass tube Remove the blanking plug from the discharge pin mounting contact and fit the corona discharge pin ensuring that the tip is in line with the tip of the sample cone Corona Discharge Pin i lt O lt 5 Mounting Contact High Voltage Socket Connect the APcI high voltage cable between Capillary Corona and the socket positioned at the bottom left corner of the source flange Replace the glass tube adjustment flange and moulded cover Insert the APcI probe into the source and tighten up the two thumb screws On the MassLynx top level window click on to launch the tune page The top line of the tune page indicates the current ionisation mode If necessary change the ionisation mode using the lon Mode command Set Source Block Temp to 150 C Warning Operating the source without the source enclosure will result in solvent vapour escape and the exposure of hot surfaces and high voltages Allow the glass source enclosure to cool after a period of operation at high flow rates before removal Routine Procedures Page 40 Quattro LC User s Guide Warning The ion source block
84. he instrument has been unused for a lengthy period of time proceed as follows Check the level of oil in the rotary pump sight glass Refill or replenish as necessary as described in the pump manufacturer s literature Exhaust Gas Filler Plug Ballast Connect a supply of dry high purity nitrogen to the connector on the service panel at the rear of the instrument Adjust the outlet pressure to 7 bar 100 psi Connect a supply of argon to the CID Gas connector on the service panel at the rear of the instrument Adjust the outlet pressure to approximately 350 mbar 5 psi Connect the water supply to the connections at the rear of the instrument Check that the rotary pump control box is connected to Rotary Control at the rear of the instrument and to the rotary pump Check that the instrument rotary pump control box data system and other peripheral devices LC equipment printer etc are connected to suitable mains supplies Check that the data system is connected to the mass spectrometer via the PC Link cable Check that the rotary pump exhaust is connected to a suitable vent Check that the exhaust gas from the instrument is connected to a suitable vent This must not be the same vent as the rotary pump exhaust instrument failure rotary pump exhaust could be admitted into the source f Caution Do not connect the two exhaust lines together In the event of an chamber producing severe contamination
85. he source Instrument Description Page 27 Quattro LC User s Guide Exhausts The exhaust from the rotary pump should be vented to atmosphere outside the laboratory The gas exhaust which also contains solvent vapours should be vented via a separate fume hood industrial vent or cold trap The gas exhaust should be connected using 10mm plastic tubing connected to the push in fitting instrument failure rotary pump exhaust could be admitted into the source f Caution Do not connect these two exhaust lines together as in the event of an chamber producing severe contamination CID Gas Argon is required as collision gas See Hardware Specifications for details Power Cord The mains power cord should be wired to a suitable mains outlet using a standard plug For plugs with an integral fuse the fuse should be rated at 13 amps Mains Switch The mains switch switches mains power to the instrument Fuses Rotary Control Mains power to the rotary pump is controlled by the data system using this socket ESD Earth Facility A suitable wrist band should be connected to this point when handling sensitive electronic components to prevent damage by electrostatic discharge Com1 and Com2 The two connections marked Com1 and Com2 are for communication with external devices and are currently not used PC Link The 15 way connector marked PC Link connects the instrument to the data system via the supplied link cable
86. hecked Process Set up the instrument and access the 0 calibrate dialog box as though a full F Acquire amp Calibrate calibration is to be carried out M Acquire amp Verify Set all peak matching parameters to the values that were used for the a Iv Print Report calibration Bring up the Automatic Calibration Cancel dialog box by selecting Start on the Calibrate dialog box Select Calibration Acquisition Setup Scanning Calibration and deselect Acquisition Parameters Static Calibration and Scan From Ei amu Scan Speed Compensa Cancel tion Scan To 3930 amu Default Run Duration EJE mins Deselect from Acquire amp Calibrate and Data Type Continuum x select Acquire amp Verify and Print Report Scan Parameters Select either MS1 or MS2 depending on the type of acquisition to be Scan Time 39 10 sac performed pz Select the Inter Scan Delay o1 0 sec Acquisition Parameters button to call up the Calibration Acquisition Set up dialog box as shown Mass Calibration Page 104 Quattro LC User s Guide Set Scan From Scan To Run Duration Data Type Scan Time and Inter Scan Delay to agree with the acquisition parameters that are to be used for data acquisition With only the scanning calibration selected all of the other options in this dialog box are unavailable Select OK to return to the previous dialog box and OK again to start the verification procedure A sc
87. hen the instrument is only calibrated for scanning acquisitions over the same mass range as that used for the calibration but the scan speed can be changed provided that it remains within the scan speeds used for the two calibrations The mass range should not be changed as there is no static calibration to locate the start mass If all three types of calibration are present then all types of acquisition can be used providing that the mass range and scan speed are between the lower and upper limits used for the scanning calibration and the scan speed compensation For a complete calibration Check the boxes in the Types area of the dialog box adjacent to Static Calibration Scanning Calibration and Scan Speed Compensation Check also the MS1 and MS2 boxes In the Process area of the dialog box check Acquire amp Calibrate and Print Report Mass Calibration Page 93 Quattro LC User s Guide Acquisition Parameters Selecting the Acquisition Parameters button in the Automatic Calibration dialog box brings forward a second box shown below where the mass ranges scan speeds and acquisition mode are set When this box is first accessed it will contain default parameters relevant to the chosen reference file These default parameters show the limits of scan range and scan speed for the currently selected instrument and calibration parameters The upper area contains the Calibration Acquisition Setup Ed Acquisition Parameters whe
88. ibrations and at any scan speed from 100 to 1000 amu sec Mass Calibration Page 117 Quattro LC User s Guide Calibration Failure When calibration is performed manually there is no warning message to show that the calibration has not met the set criteria This must be judged by viewing the on screen calibration report and examining the matched peaks and statistics associated with the report There are a number of reasons for a calibration to fail No peaks If the acquired calibration data file contains no peaks the calibration has failed This may be due to Lack of reference compound Wrong scans or wrong data file being used for the calibration No flow of solvent into the source Multiplier set too low Too many consecutive peaks missed If the number of consecutive peaks which are not found exceeds the limit set in the Automatic Calibration Check parameters then the calibration has failed Peaks may be missed for the following reasons Mass Calibration Page 118 The reference solution is running out causing less intense peaks to not be detected Multiplier is too low and less intense peaks are not detected The incorrect ionisation mode is selected Check that the data has been acquired with lon Mode set to APcl Intensity threshold set in the Calibration Parameters dialog box is too high Peaks are present in the acquired calibration file but are ignored because they are below the threshold level Either Ini
89. ilter assembly should only require cleaning on an infrequent basis Although training will be given during installation it is strongly recommended that this task is carried out by a Micromass service engineer or by other suitably qualified personnel The quadrupole assemblies of Quattro LC are finely machined and aligned assemblies which under no circumstances should be dismantled The Detector The Quattro LC detector system has been designed for trouble free operation over many years The photomultiplier is encapsulated in its own vacuum envelope and is therefore safe from contamination and pressure surges The conversion dynode and phosphor are also long lasting No routine maintenance is required It is strongly recommended that assistance is sought from Micromass if maintenance to the detector system is thought necessary due to spikes or unacceptably high noise levels Maintenance and Fault Finding Page 146 Quattro LC User s Guide Electronics Warning There are high voltages present throughout the mass spectrometer A Extreme caution should be taken when taking measurements with a meter or an oscilloscope In the standby mode OPERATE not selected the high voltages are switched off in the source and analyser assemblies but high DC voltages and mains voltages remain in the power supply units Caution Quattro LC s electronic systems contain complex and extremely sensitive components Any fault finding procedures should b
90. injection loop due to particulate matter from the sample To clear the blockage Remove the probe from the source and increase the solvent flow to 50 yl min to remove the blockage Often injections of neat formic acid help to redissolve any solute which has precipitated out of solution If the blockage cannot be cleared in this fashion Remove the finger tight nut and tubing from the back of the probe If the back pressure remains high replace the tubing with new tube or first try removing both ends of the tube If the back pressure falls replace the stainless steel sample tube inside the probe or try reversing the tube to blow out any blockage Reconnect the tubing to the probe The solvent flow can be readjusted and the probe replaced into the source To check the flow rate from the solvent delivery system fill a syringe barrel or a graduated glass capillary with the liquid emerging from the probe tip and time a known volume say 10yl Once the rate has been measured and set a note should be made of the back pressure readout on the pump as fluctuation of this reading can indicate problems with the solvent flow For APcI a higher than normal back pressure readout on the HPLC pump can imply that after a long period of use the filter pad requires replacement General Loss of Performance Should the preceding checks fail to reveal the source of the problem proceed as follows Check that the source and probe voltage re
91. it is possible to acquire data over any mass range within the calibrated range It is therefore sensible to calibrate over a wide mass range and in this example the calibration will cover up to 1000 amu Calibration Options To access the calibration options click on the Calibrate button of the acquisition control panel Selecting Reference File Set pegh1000 ref as the reference file by clicking on the arrow in the reference file box and scrolling through the files until the appropriate file can be selected Leave the Use Air Refs box blank when calibrating in APcI Removing Current Calibrations Select uncal cal from the File Load Calibration menu option Select Process Delete all calibration followed by File Save Calibration This ensures that a file with no calibration is currently active on the instrument and prevents any previously saved calibrations from being modified or overwritten Mass Calibration Page 108 Quattro LC User s Guide Selecting Calibration Parameters A number of parameters needs to be set before a calibration is started Most of these parameters can be set at the same value as for electrospray However a Polynomial order of 2 is recommended for the calibration Curve Fit Performing a Calibration The three types of calibration static scanning and scan speed must be carried out in single steps Static Calibration Access the Automatic Calibration dialog box by selecting Start from the C
92. ition of daughter ion data The experiment is performed in the ESI positive mode using reserpine as a model analyte Reserpine admitted by constant infusion at a concentration of 50 pg ul provides a stable and persistent source of ions for instrument tuning in both the MS and MS MS modes of operation The basic sequence of events is as follows Tuning MSI described earlier in this chapter Tuning MS2 described earlier in this chapter Parent ion selection Fragmentation Parent lon Selection For maximum sensitivity in daughter ion analysis the centre of the parent ion selected by MSI must be accurately found The nominal mass of the parent is first determined if unknown by viewing it in MS mode Set up a 1 box display in the tune page and set Function to MS Observe the candidate parent ion in the tune display and determine its nominal mass In this example the reserpine ion at z 609 will be used as a model parent The accurate top of the parent ion can be found experimentally by performing a daughter ion scan over a restricted mass range in the absence of collision gas On the tune page place the mouse cursor on the Set mass for peak 2 and type in the nominal mass of the parent ion selected by MS1 in this case 609 Double click on peak 2 to zoom in Select Other followed by Scope Parameters Set Mass Increment to 0 1 z Using the left and right arrow controls vary the set Mass value between 608 5 an
93. ivity gains are therefore observed when similar scan parameters are used due to the great reductions in sample consumption Sample Inlet Sample is introduced from a suitable liquid pumping system along with the nebulising gas to either the APcI probe or the electrospray probe For nanoflow electrospray metal coated glass capillaries allow the lowest flow rates to be obtained while fused silica capillaries are used for flow injection analyses or for coupling to nano HPLC Instrument Description Page 17 Quattro LC User s Guide a c Collision Cell MS2 Resolving RF Only Pass all masses RF Only Pass all masses MS1 Resolving The MS1 mode in which MS1 is used as the mass filter is the most common and most sensitive method of performing MS analysis This is directly analogous to using a single quadrupole mass spectrometer The MS2 mode of operation is used with collision gas present when switching rapidly between MS and MS MS operation It also provides a useful tool for instrument tuning and calibration prior to MS MS analysis and for fault diagnosis The basic features of the four common MS MS scan functions are summarised below Collision Cell bee Daughter Ion Static Scannin Spectrum parent mass selection 8 Static Scanning daughter mass selection Parent Ion Spectrum RF only pass all Multiple Reaction Static masses Monitori
94. left on overnight However if the data system has been switched off switch it on as described in the preceding section The display on the front of the instrument displays a steady green Vacuum LED indicating that the instrument is ready for use Preparation for Electrospray Operation Source Thumb Nuts aa n Moulded Cover Pe ee Probe Adjustment Flange Thumb Nuts If the corona discharge pin is fitted proceed as follows Disconnect the gas and electrical connections from the front panel Unscrew the probe thumb nuts and remove the probe Remove the moulded cover which surrounds the source Undo the three thumb screws and remove the probe adjustment flange and glass tube Disconnect the APcI high voltage cable from the socket positioned at the bottom right corner of the source flange Routine Procedures Page 37 Quattro LC User s Guide Exhaust a Cleanable Blanking Baffle Plug Corona Discharge Pin Mounting Contact High Voltage Socket Remove the corona discharge pin from its mounting contact and fit the blanking plug Replace the glass tube adjustment flange and moulded cover Ensure that the source enclosure is in place The Z spray source enclosure consists of the glass tube and the probe adjustment flange Connect the source s gas line to Desolvation Gas on the front panel Tighten the nut to ensure a good seal Check that
95. low option is to be used the sample cone nozzle on the ion block Reconnect the cone gas supply nanoflow operation only When the instrument has pumped down Open the isolation valve Plug the purge and cone gas outlets and fit the APcI corona discharge pin Fit the source enclosure and the probe adjustment flange Insert the APcI probe and connect the Nebuliser gas line Select Gas and turn on Nitrogen Fully open the Nebuliser gas valve Set Desolvation Gas to read back 400 litres hour monitored on the tune page Maintenance and Fault Finding Page 136 Quattro LC User s Guide Check for gas leaks using soap solution Reduce Desolvation Gas to 150 litres hour Set Source Block Temp to 150 C and APcl Probe Temp to 20 C Caution The maximum operating temperature for the source heater is 150 C Do not set Source Block Temp higher than 150 C Select LUPERATE J on the tune page With Corona set to zero check that the Cone readback is reading the correct set value Set Corona to 4 0kV Check that the Corona readback is 4 0 kV and that the Cone readback is still reading the same set value Check that all other readbacks on the tune page agree with the set values The Discharge Pin If the corona discharge pin becomes dirty or blunt Remove it from the source Clean and sharpen it using 600 grade emery paper If the needle becomes bent or otherwise damaged it should be replaced Maintenance and Fault Finding Page
96. ms The abbreviated form shown below shows the selected reference file and the date the instrument was last calibrated 35 Calibrate DEFAULT CAL Ic Es File Edi Process Help Reference File m Last Calibrated Start 1210 28Jun 97 Status gt gt Data Directory CXMASSLYNXNDEFAULT PROSDatas Selecting the Status button displays the full dialog box which shows the current calibration status Selecting the lt lt Fold button returns to the abbreviated box Calibrate DEFAULT CAL File Edit Process Help Reference File m Last Calibrated nairb ref hd 1210 28Jun 97 Data Directory CNMASSLYNXSDEFALLT PROSData s Calibration Status M51 Static Mo calibration M51 Scanning Mo calibration MS1 Scan Speed No calibration Compensation MS2 Static Mo calibration M52 Scanning No calibration MS2 Scan Speed No calibration Compensation Mass Calibration Page 88 Quattro LC User s Guide Selecting the Reference File Click on the arrow in the Reference File box and scroll through the files until the appropriate file can be selected Select nairb ref for a sodium iodide and rubidium iodide reference solution covering the range 23 to 3920 amu Removing Current Calibrations Select the uncal cal calibration file from the File Load Calibration menu option Select Process Delete all calibration followed by File Save Calibration This ensur
97. n be carried out by filling a syringe barrel or a graduated glass capillary with the liquid emerging from the probe tip and timing a known volume say 101 Once the rate has been measured and set a note should be made of the back pressure readout on the pump as fluctuation of this reading can indicate problems with the solvent flow Post column Splitting Although the electrospray source can accommodate flow rates up to 1 ml min it is recommended that the flow is split post column to approximately 200 yl min Also even at lower flow rates a split may be required for saving valuable samples The post column split consists of a zero dead volume tee piece connected as shown LC Column To Waste or i UV Cell The split ratio is adjusted by increasing or decreasing the back pressure created in the waste line by changing either the length or the diameter of the waste tube A UV cell may also be incorporated in the waste line avoiding the requirement for in line low volume Z cells As the back pressure is varied the flow rate at the probe tip should be checked as described above These principles apply to splitting for both megaflow and normal flow electrospray Electrospray Page 53 Quattro LC User s Guide Megaflow Megaflow electrospray enables flow rates from 200 yl min to 1 ml min to be accommodated This allows Microbore 2 1mm or 4 6mm diameter columns to be interfaced without splitting Changing Between Flow Modes
98. ng parent mass selection Static daughter mass selection Constant Neutral Scanning synchronised Scanning synchronised Loss Spectrum with MS2 with MS1 Instrument Description Page 18 Quattro LC User s Guide The Daughter lon Spectrum This is the most commonly used MS MS scan mode Typical applications are Structural elucidation for example peptide sequencing Method development for MRM screening studies Identification of daughter ions for use in MRM transitions Optimisation of CID tuning conditions to maximise the yield of a specific daughter ion to be used in MRM analysis Example Daughters of the specific parent at z 609 from reserpine in electrospray positive ion mode e L3 x Collision Cell MS1 id MS2 static at m z 609 Paora massas scanning from parent mass m z 100 to 650 The result RESERP1 1 0 989 Sb 1 50 00 Sm SG 2x1 00 Daughters of 6O9ES 1004 609 9 54e5 236 224 365 577 04 r r h 100 125 180 175 200 225 260 275 300 325 350 375 400 425 450 475 500 525 520 5 5 600 G25 Gao Dale Instrument Description Page 19 Quattro LC User s Guide The Parent lon Spectrum Typical application Structural elucidation Complementary or confirmatory information for daughter scan data Example Parents of the specific daughter ion at z 195 from reserpine in electrospray positive ion mode
99. ng the sample and solvent system under investigation The following information should be considered when setting the probe position 10mm of movement is provided in each direction with 1 25mm of travel per revolution of the probe positioning controls At higher liquid flow rates the probe tip should be positioned further away from the sample cone to achieve optimum stability and sensitivity The position is less critical than at lower flow rates Nebuliser Gas Optimum nebulisation for electrospray performance is achieved with a nitrogen flow between 70 and 90 litres per hour This can be achieved by fully opening the Nebuliser flow control valve which is situated on the instrument s front panel Desolvation Gas The desolvation gas also nitrogen is heated and delivered as a coaxial sheath to the nebulised liquid spray by the desolvation nozzle 125 A Nebuliser The position of the desolvation nozzle Gas Control Desolvation heater is fixed relative to the probe Gas Contre tip and requires no adjustment The Desolvation Gas flow rate is adjusted by the control value situated on the instrument s front panel The optimum Desolvation Temp and flow rate is dependent on mobile phase composition and flow rate A guide to suitable settings is given below To monitor the flow rate select Window then Gas Flow on the tune page and observe the readback window The Desolvation Gas flow rate indicated on the
100. nge to get hot during prolonged megaflow operation Care should be taken when handling source components during and immediately after operation The source enclosure will run cooler if purge gas is used Warning For health and safety reasons always ensure the exhaust line is vented outside the building or to a fume hood Warning Ensure that a plastic bottle is connected in the exhaust line to collect any condensed solvents Removing the Probe To remove the probe from the source proceed as follows On the tune page deselect OPERATE Switch off the liquid flow and disconnect from the probe Select Gas and turn off Nitrogen Disconnect the probe cable from the instrument Disconnect the nebulising gas supply from the instrument Sample Analysis and Calibration General Information Care should be taken to ensure that samples are fully dissolved in a suitable solvent Any particulates must be filtered to avoid blockage of the transfer line or the probe s capillary A centrifuge can often be used to separate solid particles from the sample liquid There is usually no benefit in using concentrations greater than 20 pmol l for biopolymers or 10 ng yl for low molecular weight compounds Electrospray Page 62 Quattro LC User s Guide Higher concentrations will not usually improve analytical performance Conversely for biopolymers lower concentrations often yield better electrospray results Higher levels require more f
101. ning 44 APcl 80 82 108 Electrospray 86 X Turbomolecular pump 16 27 29 43 124 X scope 27 Y Y scope 27 Index Page 164
102. ning is recommended Megaflow Hints A With this high flow rate technique the setup procedure involves making the following adjustments increase Drying Gas flow to approximately 750 litres hour increase Desolvation Temp to 400 C increase Source Block Temp to 150 C move the probe further away from the sample cone When changing from electrospray to megaflow operation it is not necessary to adjust any source voltages Caution The maximum operating temperature for the source heater is 150 C Do not set Source Block Temp higher than 150 C Cluster ions are rarely observed with Z spray However solvent droplets may form within the source enclosure if the source and desolvation temperatures are too low Refer to the previous section on operating parameters for typical desolvation gas flow rates Electrospray Page 61 Quattro LC User s Guide Purge gas can be used during megaflow operation to stop the source enclosure from overheating This is also beneficial when the analyte is susceptible to acetonitrile adducting Purge gas is enabled by removing the blanking plug from the outlet situated within the source enclosure If the sample is contained within a dirty matrix the probe may be moved away from the sample cone to extend time between source cleaning operations This may incur a small loss in sensitivity Warning It is normal for the source enclosure the glass tube and parts of the AN probe mounting fla
103. nitrogen If the sample cone is still not clean or if the aperture is partially blocked proceed to the following section Otherwise when the cone is clean and dry Open the isolation valve Replace all removed components following in reverse order the removal procedures Removing and Cleaning the Sample Cone Caution The sample cone is a delicate and expensive component and should be handled with extreme care It is not necessary to vent the instrument to remove the sample cone The source block incorporates an isolation valve for this purpose To remove the sample cone proceed as follows Follow the procedure in the previous section to gain access to the sample cone Using a suitable flat blade screwdriver rotate the valve by 90 into its fully anticlockwise position A small improvement in the analyser vacuum may be observed as a result of this operation The isolation valve is in the closed position when the slot is perpendicular to the direction of flow Disconnect the cone gas inlet line if fitted Take the sample cone extraction tool supplied in the source spares kit and screw it to the flange of the sample cone Remove the two sample cone retaining screws using a 1 5mm Allen key and withdraw the sample cone and sample cone nozzle if fitted from the ion block Remove the extraction tool and separate the sample cone from the sample cone nozzle Place both components in an ultrasonic bath containing 50 50 ac
104. nt upon the molecular weight distribution of the PEGs used in the reference solution For this example PEG grades from PEG 200 to PEG 1000 are used PEG 200 400 6001000 AP cl PEG 27 0 970 Cn Tap 4 Ht Sm Mn 2x0 40 Sb 1 5 00 Cm 25 32 18 20x11 080 Scan AP 105 133 0 3392 7 0426 283 2 41534323 476 3 E 520 4 596 5 740 5 7045 820 6 872 6 916 7 bad 2 960 8 240 2 l E213 bes 3 B53 F634 boza Paa 855 6295 6736 591 3 535 3 679 4 723 4 767 5 811 5 855 6 261 2 305 2 200 250 300 360 400 450 500 550 600 650 700 750 800 850 a00 Mass Calibration Page 107 Quattro LC User s Guide Preparing for Calibration Reference Compound Introduction It is best to use a large volume injection loop S50yl with a solvent delivery system set up to deliver 0 2 ml min of 50 50 acetonitrile water or methanol water through the injector and into the APcI source An injection of 50l of reference solution lasts for approximately 15 seconds allowing enough time to perform a slow scanning calibration Tuning Before beginning calibration Set Multiplier to 650V Adjust source and lens parameters to optimise peak intensity and shape Set Cone in the region of 30 35V so that some fragmentation occurs to give some of the lower mass peaks in the spectrum Set the resolution and ion energy parameters for unit mass resolution on MS1 and MS2 When a full calibration is completed
105. oduction of doubly charged ions The voltage should be at least 30V PEG 1 0 661 Sm Mn 230 20 Sb 1 5 00 Sean ES 100 511 6 2 3608 M 2NH4 2 555 6 M NH4 520 4 Doubly charged peaks can be identified because the C isotope peak is separated from the C isotope by only 0 5 Da e If the instrument is set to unit mass and data is acquired in continuum mode the doubly charged peaks will appear broader as the isotopes will not be resolved Mass Calibration Page 106 Quattro LC User s Guide Atmospheric Pressure Chemical lonisation Introduction This chapter describes a complete mass calibration of Quattro LC using atmospheric pressure chemical ionisation The procedures described should be followed only after reading the previous chapter in this manual describing the automated calibration with electrospray ionisation Due to the high flow rates used with APcI the residence time of an injection of reference solution in the source is too short to allow a fully automated calibration and the procedure therefore has to be carried out in several steps The recommended reference compound for APcI is a solution of polyethylene glycol PEG containing ammonium acetate See Reference Information for advice on preparing the reference solution See the following illustration for a typical PEG NH spectrum With PEG the possible calibration range is depende
106. of three basic parts The probe adjustment flange The glass tube The source flange assembly The probe adjustment flange and the glass tube can be readily removed without venting the instrument to gain access to the source block and sample cone This allows the following operations to be performed Wiping the sample cone Removing the sample cone Fitting or removing the APcI discharge pin Fitting or removing the exhaust liner and cleanable baffle Fitting or removing the nanoflow electrospray interface Enabling or disabling the purge gas The sample cone may be cleaned in situ by gentle wiping with a cotton swab or lint tissue soaked with 50 50 acetonitrile water More thorough cleaning of the sample cone may be achieved by removing it from the source This may also be done without venting the instrument by closing the isolation valve located on the ion block Less frequently it may be necessary to clean the ion block the extraction cone and the hexapole lens in which case the instrument must be vented This should only be done when the problem is not rectified by cleaning the sample cone or when charging effects are apparent Charging is evidenced by a noticeable progressive drop in signal intensity often resulting in a complete loss of signal Switching the instrument out of and back into operate causes the beam momentarily to return The hexapole transfer lens should not require frequent cleaning If it
107. ome of the reference peaks above 1000 amu Check also the intensity of the peaks at z 85 and 173 A cone voltage in the region of 90 is usually suitable The peak at 23 is Na the 85 peak is rubidium and the others are Nal clusters A typical tune page is shown above Instrument Threshold Parameters Before beginning the calibration procedure some instrument parameters need to be checked For most low mass range calibrations calibration data is acquired in continuum mode To allow suitable scanning speeds to be used the continuum data parameters need to be set correctly From the instrument control panel select Instrument then Set Instrument Threshold to display the Instrument Data Thresholding window Instrument Data Thresholding x Profile Data Data Storage Compressed Discard zero intensities Re Baseline Level 1 0 Points per dalton e Centroid Data Dynamic Range Standard z Data Threshold o Peak Acceptance High Save All Peaks m SIR Data SIR Baseline Level o OK Cancel Default Advanced In the Profile Data section select Compressed Discard zero intensities for Data Storage and 8 Points per Dalton Select to save the parameters Mass Calibration Page 87 Quattro LC User s Guide Calibration Options To access the calibration options click on Instrument then Calibrate on the acquisition control panel This brings forward the calibration dialog box which can be displayed in two for
108. or most samples this flow rate should be tuned for maximum sensitivity while ensuring that the flow rate is not decreased below 100 litres hour Atmospheric Pressure Chemical lonisation Page 82 Quattro LC User s Guide Removing the Probe After a session of APcI operation Turn off the LC flow Set APcl Probe Temp to 20 C Deselect OPERATE When the probe temperature falls below 100 C Select Gas and turn off Nitrogen Undo the two thumb nuts and remove the probe from the source Warning Take care when removing the APcI probe There is a risk of burns to the operator AN Caution Removal of the APcI probe when hot will shorten the life of the probe heater O i EE f J D i S i Probe Thumb Nuts If the instrument is not to be used for a long period of time the source temperature should be reduced to 60 C Atmospheric Pressure Chemical lonisation Page 83 Quattro LC User s Guide Atmospheric Pressure Chemical lonisation Page 84 Quattro LC User s Guide Introduction MassLynx NT allows a fully automated mass calibration to be performed which covers the instrument for static and scanning modes of acquisition over a variety of mass ranges and scanning speeds The first section of this chapter describes a complete mass calibration of Quattro LC using electrospray ionisation with a mixture of sodium iodide and rubidium iodide as the reference compound The second section desc
109. ow at such rates is more important than the actual flow rate The injection valve on reciprocating pumps may be replaced by an autosampler for unattended overnight operation nfusion pump The pump syringe is filled with sample in solution The infusion pump then delivers the contents of the syringe to the source at a constant flow rate This arrangement allows optimisation and analysis while the sample flows to the source at typically 5 30 l min Further samples require the syringe to be removed washed refilled with the next sample and replumbed A 50 50 mixture of acetonitrile and water is a suitable mobile phase for the syringe pump system and the reciprocating pump systems This is appropriate for positive and negative ion operation Positive ion operation may be enhanced by 0 1 to 1 formic acid in the sample solution Negative ion operation may be enhanced by 0 1 to 1 ammonia in the sample solution Acid should not be added in this mode These additives should not be used for flow injection analysis FIA studies to allow easy change over between positive and negative ion analysis Degassed solvents are recommended for the syringe and reciprocating pumps Degassing can be achieved by sonification or helium sparging The solvents should be filtered and stored under cover at all times Electrospray Page 52 Quattro LC User s Guide It is wise periodically to check the flow rate from the solvent delivery system This ca
110. ple UV or fluorescence can be stored simultaneously with the acquired mass spectral data A further option is the ability to acquire UV photodiode array detector data Comprehensive information detailing the operation of MassLynx NT is contained in the MassLynx NT User s Guide Instrument Description Page 22 Quattro LC User s Guide Front Panel Connections CAPILLARY CORONA STANDBY IM Desolvation LC Connection as High Voltage Source oe Connection Connection as Desolvation Gas and Probe Nebuliser Gas The PTFE gas lines for the desolvation gas and probe nebuliser gas are connected to the front of the instrument using threaded metal fittings Capillary Corona The electrical connection for the ESI capillary or the APcI discharge pin is via the coaxial high voltage connector ESI APcl The electrical connection for the APcI probe or the ESI heater is via the multi way connector This is removed from the front panel by pulling on the metal sleeve of the plug Both the electrospray and APcI heaters use this connector Instrument Description Page 23 Quattro LC User s Guide Front Panel Controls and Indicators CAPILLARY CORONA ZZ es E VACUUM OPERATE Pa Desolvation Gas Control Nebuliser Gas Control CID Gas Control Status Display The display on the front panel of the instrument consists of two 3 colour light emitting diodes LEDs The display
111. plete Shutdown If the instrument is to be left unattended for extended periods proceed as follows Switch off the LC pumps On the MassLynx top level window click on to launch the tune page Click on LOPERATE J on the tune page This will change from green to grey indicating that the instrument is no longer in operate mode Undo the finger tight connector on the probe to release the tubing leading from the LC system Before disconnecting the probe it is good practice to temporarily remove the probe and flush it of any salts buffers or acids If APcI is being used switch off the probe heater or reduce it to ambient temperature Caution Leaving the APcI probe hot with no gas or liquid flow will shorten the lifetime of the probe heater Select Gas followed by Nitrogen to turn off the supply of nitrogen gas Select Other from the menu bar at the top of the tune page Click on Vent The rotary pump and the turbomolecular pumps switch off When the turbomolecular pumps have run down to half of their normal operating speed the vent valve opens and the instrument is vented to atmosphere Exit MassLynx Shut down the computer Switch off all peripherals Switch off the power to the instrument using the switch on the rear panel of the instrument Switch off power at the wall mounted isolation switches If the instrument is to be switched off for more than one week Drain the oil from the rotary pump according to the manufact
112. ptions for the spraying capillary which can be alternately fitted to the interface Borosilicate metal coated glass capillary Metal coated glass capillaries allow the lowest flow rates to be obtained although they are used for one sample only and then must be discarded Fused silica capillary This option is suitable for flow injection analyses or for coupling to nano HPLC and uses a pump to regulate the flow rate down to 100 nl min If a syringe pump is to be used a gas tight syringe is necessary to obtain correct flow rates without leakage A volume of 25ul is recommended Nanoflow Electrospray Page 67 Quattro LC User s Guide For a given sample concentration the ion currents observed in nanoflow are comparable to those seen in normal flow rate electrospray Great sensitivity gains are therefore observed when similar scan parameters are used due to the great reductions in sample consumption The nanoflow end flange consists of a three axis manipulator a stage a p gt Exhaust Capillary Cleanable protective cover and a stop lt Baffle Exhaust handle arrangement for Sample Liner one rotation of the manipulator Nozzle TEPEN and stage The manipulator and stage 5E Isolation are rotated by 90 degrees to Valve change option or in the glass capillary option to load a new nanovial Caution Failure to use m the stop and handle to l 4 i n N rotate the stage can Sour
113. queous content are to be used then tuning conditions should be appropriate for the solvent composition entering the source Electrospray Page 64 Quattro LC User s Guide Higher source temperatures 150 C are also recommended for high aqueous content solvents Tetrahydrofuran THF should not be used with peek tubing LC MS Sensitivity Enhancement The sensitivity of a LC MS analysis can be increased or optimised in a number of ways by alterations to both the LC operation and the MS operation In the LC area some examples include the use of high resolution columns and columns with fully end capped packings For target compound analysis techniques such as trace enrichment coupled column chromatography or phase system switching can have enormous benefits Similarly the mass spectrometer sensitivity can often be significantly increased for instance by narrow mass scanning or by single ion recording techniques Careful choice of the solvent and solvent additives or modifiers may also prove important Electrospray Page 65 Quattro LC User s Guide Electrospray Page 66 Quattro LC User s Guide Nanoflow Electrospray Overview Fused Silica Option Glass Capillary Option Injector Three axis Manipulator Handle Stage Protective Cover The optional nanoflow interface allows electrospray ionisation to be performed in the flow rate range 5 to 1000 nanolitres per minute There are two o
114. r Remove the sample cone as described above Maintenance and Fault Finding Page 132 Quattro LC User s Guide Leaving the valve stem in place immerse the ion block in an ultrasonic bath containing 50 50 acetonitrile water or 50 50 methanol water followed by 100 methanol Clean the sample cone and the extraction cone using in turn concentrated formic acid 50 50 acetonitrile water or 50 50 methanol water 100 methanol Warning Strong acid causes burns Carry out this procedure in a fume cupboard using protective equipment Dry all components using a flow of nitrogen or place them in a warm oven Maintenance and Fault Finding Page 133 Quattro LC User s Guide Removing and Cleaning the Hexapole Transfer Lens Assembly Hexapole Transfer Lens lon Block A SOK N e Z D Support Liner To remove the hexapole transfer lens assembly proceed as follows Remove the ion block as described above Remove the three screws retaining the ion block support and carefully withdraw it together with the support liner and O rings from the pumping block Using a lint free tissue to gently grasp the hexapole carefully withdraw it A Caution Take care not to scratch the internal bore of the pumping block as the hexapole lens assembly is withdrawn Maintenance and Fault Finding Page 134 Quattro LC User s Guide Differential Aperture Rod Locating Plate Screws amp Washers Location Reces
115. r nut Withdraw and discard the stainless steel sample capillary Remake the LC connection to the LC union Sleeve one end of new sample capillary with the PTFE liner tube Maintenance and Fault Finding Page 140 Quattro LC User s Guide Using a GVF 16 ferrule and the adapter nut connect the sample capillary to the LC union ensuring that both the liner tube and sample capillary are fully butted into the LC union Disconnect the LC connection and feed the sample capillary through the probe ensuring that a 0 3mm graphitised vespel ferrule GVF 003 is fitted Using a Rheodyne spanner gently tighten the adapter nut onto the probe Replace the probe tip and adjust so that 0 5mm of sample capillary protrudes from the probe tip Replace the probe end cover and tighten the grub screw to clamp the LC union Maintenance and Fault Finding Page 141 Quattro LC User s Guide The APcl Probe Indications that maintenance to the APcI probe is required include The probe tip assembly becomes contaminated for example by involatile samples if the probe temperature is too low during operation 300 C The appearance of chromatogram peak broadening or tailing Samples that give rise to a good chromatogram peak shape in APcI for example reserpine and common pesticides should display peak half widths of the order 0 1 minutes for 10ul loop injections at a flow rate of 1 ml min The appearance of significant peak broadening or tailin
116. ray probe to Nebulising Gas on the front panel Secure with the nut provided With the probe removed from the source turn on the liquid flow at 10 l min and check that liquid flow is observed at the tip of the capillary Electrospray Page 55 Quattro LC User s Guide To avoid unwanted capillary action effects do not allow liquid to flow to the probe for long periods without the nitrogen switched on Turn on Nitrogen by selecting Gas and check that a nebuliser flow of less than 100 litres hour is registered To monitor the flow rate select Window then Gas Flow on the tune page and observe the readback window Check that there is gas flow at the probe tip and ensure that there is no significant leakage of nitrogen elsewhere Adjust the probe tip to ensure complete nebulisation of the liquid There should be approximately 0 5 mm of sample capillary protruding from the nebulising capillary The tip of the electrospray probe can influence a uela the intensity and stability of the ion beam A damaged or incorrectly adjusted probe tip will lead to poor electrospray performance Nebulising Capillary Using a magnifying glass ensure that both inner and outer stainless steel capillaries are straight and circular in cross section Ensure that the inner stainless steel capillary is coaxial to the outer capillary If the two capillaries are not coaxial it is possible to bend the outer capillary slightly using thumbnail pressu
117. re Insert the probe into the source and tighten the two thumb screws Plug the probe high voltage cable into Capillary Corona on the front panel Obtaining an lon Beam Electrospray Page 56 If necessary change the ionisation mode using the lon Mode command The top line of the tune page indicates the current ionisation mode Using the needle valve on the front panel set the desolvation gas flow rate to 300 litres hour To monitor the flow rate select Window then Gas Flow on the tune page and observe the readback window Turn on the liquid flow at 10 l min and set Desolvation Temp to 100 C Quattro LC User s Guide Tuning and Optimisation The following parameters after initial tuning should be optimised using a sample representative of the analyte to be studied It will usually be found with the exception of the sample cone voltage that settings will vary little from one analyte to another Probe Position Probe Adjustment Sideways Probe Adjustment The position of the probe is adjusted using the probe adjustment collar in out and the adjustment knob sideways located to the left of the probe The two screws can be adjusted singly or simultaneously to optimise the beam The position for optimum sensitivity and stability for low flow rate work 10 yl min is shown Electrospray Page 57 Quattro LC User s Guide Small improvements may be gained by varying the position usi
118. re mass range run time and Acquisition Parameters E data type are set NEN Scan From 0 amu Cancel When the instrument is fully Scan To 330 pue T1 calibrated any mass range or scan speed is allowed within the upper and lower limits dictated Data Type Continuum by the calibrations Run Duration 96 mins Scan Parameters If the nairb ref file is selected Mo the default button will give the Seales wt i parameters shown above The Static Dwell 0 10 sec solution described in Reference Soa cai He 3330 x Information is suitable for use with this reference file East Scan Time 7 82 Inter Scan Delay 0 10 sec If compatible reference solutions and reference files are used then simply selecting the default button is sufficient action no parameters need be entered manually Run Duration sets the time spent acquiring data for each part of the calibration The time set must allow a minimum of three scans to be acquired at the slowest scan speed used If the run duration is too short then data will not be acquired The slowest scan speed generally used is 100 amu sec With Scan From set to 20 amu and Scan To set to 2000 amu a scan time of 19 8 seconds is required and an Inter Scan Delay in the lower area of the box of 0 1 second is usually used Therefore the run duration must be greater than 59 6 seconds 3 scans 2 inter scan delays A Run Duration of 1 00 minutes is suitable Data Type allows a choice of c
119. recautions Maintenance and Fault Finding Page 150 Quattro LC User s Guide Preventive Maintenance Check List Daily Weekly Monthly Avoid venting the instrument when the rotary pump is gas ballasting Do not gas ballast the rotary pump for more than 2 hours under any circumstances 3 Under no circumstances should gas ballasting be performed during instrument operation For full details of the following procedures consult the relevant sections of this chapter and or refer to the manufacturer s literature Gas ballast the rotary pump lightly for 20 minutes at the end of a day s electrospray operation Gas ballast the rotary pump for 30 minutes at the end of a day s megaflow or APcI operation It is normal for the rotary pump noise level to increase during gas ballasting Gas ballast for at least 30 minutes by rotating the gas ballast knob anticlockwise by 5 to 6 turns When gas ballast is complete check the rotary pump oil level and colour Oil that has become noticeably red in colour should be replaced Check the water chiller level and temperature if fitted Check all cooling fans and filters Change the odour element in the oil mist filter Three Monthly Change the sorbent in the foreline trap Four Monthly Change the mist element in the oil mist filter Change the oil in the rotary pump Gas ballast lightly for 30 to 60 minutes both before and after changing oil Mainten
120. requent source cleaning and risk blocking the transfer capillary Optimisation for low molecular weight compounds may usually be achieved using a concentration of 1 ng yl Samples with phosphate buffers and high levels of salts should be avoided Alternatively at the expense of a small drop in sensitivity the probe can be pulled away from the sample cone to minimise the deposit of involatile material on the cone To gain experience in sample analysis it is advisable to start with the qualitative analysis of known standards A good example of a high molecular weight sample is horse heart myoglobin molecular weight 16951 48 which produces a series of multiply charged ions that can be used to calibrate the Yz scale from 800 1600 in either positive ion or negative ion mode Polyethylene glycol mixtures for example 300 600 1000 are low molecular weight samples suitable for calibrating the Yz scale from approximately 100 to 1200 in positive ion mode A mixture of sugars covers the same range in negative ion mode Alternatively a mixture of sodium iodide and caesium iodide or a mixture of sodium iodide and rubidium iodide can be used for calibration Detailed information on data acquisition and processing can be found in the MassLynx NT User s Guide Detailed information on mass calibration can be found in Mass Calibration later in this document Typical ES Positive lon Samples Peptides and proteins Small polar compounds
121. ribes a similar procedure using atmospheric pressure chemical ionisation APcI with PEG as the reference compound See Reference Information for details of calibration solutions and their preparation Electrospray Overview When a calibration is completed it is possible to acquire data over any mass range within the calibrated range It is therefore sensible to calibrate over a wide mass range With a mixture of sodium iodide and rubidium iodide calibration over the instrument s full mass range is achievable Spectrum SCNMS1 Blea E File Edit Display Process Tools Window Help 8l x 2 ala wee lalale o Aala lele AIRE calibration 20Da to 3930Da SCNMS1 1 0 678 Sm Mn 2x0 60 Cm 1 2 100 922 4 3021 0 ra 25713 2871 0 250 500 750 1000 1250 1500 1750 2000 2250 2500 2750 3000 3250 3500 Mass Calibration Page 85 Quattro LC User s Guide Preparing for Calibration Reference Compound Introduction The example given here describes an automatic calibration which requires reference compound to be present for several minutes The introduction of the reference compound is best achieved using a large volume Rheodyne injector loop 50 or 1001 or an infusion pump for example a Hamilton syringe pump When using a large volume injection loop Set up a solvent delivery system to deliver 4 5 jl min of 50 50 acetonitrile water or 50 50 methanol water through the injector into the sourc
122. rom the rear of the instrument Disconnect the API gas inlet and the exhaust lines from the rear of the instrument Disconnect all connections to LC equipment If the instrument is to be moved over a large distance or in a confined space it is recommended that any probes are removed from the API source The weight of the instrument is 150kg 330Ib Lifting equipment or suitably trained personnel will be required to lift or lower the instrument UK Health and Safety guidelines recommend that a minimum of six trained and suitable personnel are required to lift a unit of this weight The instrument should be lifted from underneath the frame with one person at each corner of the instrument supporting the instrument in line with or close to the feet upon which the instrument stands Two further people should support the instrument centrally Caution Under no circumstances should the instrument be lifted by the front moulded cover the probe or the source enclosure Before undertaking any lifting lowering or moving of the instrument Assess the risk of injury Take action to eliminate the risk Plan the operation Use trained people Refer to local or company guidelines before attempting to lift the instrument Micromass accept no responsibility for any injuries or damage sustained while lifting the instrument Hardware Specifications Page 12 Quattro LC User s Guide Power Instrument 230V 10 14 13A Data syst
123. rotonated in the negative ion mode The sample and reagent ions pass through the sample cone into the ion block prior to being extracted into the hexapole transfer lens through the extraction cone Atmospheric Pressure Chemical lonisation Page 77 Quattro LC User s Guide Changeover between electrospray and APcI operation is simply accomplished by changing the probe and installing the corona discharge pin within the source enclosure For APcI operation the desolvation gas is not heated in the desolvation nozzle However it is important that desolvation gas is used throughout The background spectrum for 50 50 acetonitrile water is dependent upon the settings of Cone and Extractor The main reagent ions for typical sample and extraction voltages of 40V and 3V respectively are 42 56 83 and 101 Acetonitrile adducting may be minimised by optimisation of the probe position Preparation Ensure that the source is assembled as described in Maintenance and Fault Finding and that the instrument is pumped down and prepared for APcI operation as described in Routine Procedures Corona Discharge Pin ee Mounting D 2 Contact pom eem High Voltage Socket APcI may be operated with or without the cleanable baffle fitted Ensure that a supply of nitrogen has been connected to the gas inlet at the rear of the instrument and that the head pressure is between 6 and 7 bar 90 100
124. rrule on the tubing Attached Directly Plumb the low dead volume union as shown 1 Tubing P P making sure that each of the fused silica pieces RA AN are held firmly by the appropriate sleeving and a Valco SIGs Valco ferrule Petals rf j ion i j Red Stripe 3 On one side of the union is 375um o d 25um i d Peek Tubing fused silica from the column or injector sleeved J to is inch with PTFE tubing On the other is the Valco iN f Ferrule spraying capillary 90um o d 20um i d fused silica sleeved to 16 inch with red stripe peek Spraying 7 tubing Capillary Approximately 5mm of 90um o d fused silica should protrude from the end of the nut as shown This may be cut to size at the end of the assembly process Attach the union to the contact plate using the V shaped clamps Attach the plate to the manipulator stage Replace the protective cover and swing the stage into the interface using the stop and handle The Valco injector is attached to the flange by a bracket and can be used for loop injections The injection valve is plumbed as follows P from the pump Cto the column or to the union e S is the sample port attach a VISF sleeve here W is a waste port Raise the flow to 1 yl min After a few minutes reduce it to the desired flow rate To avoid a very unstable beam it is imperative to remove any air bubbles from the fused silica lines Select Gas and switch on
125. rument Description Page 21 Quattro LC User s Guide The Constant Neutral Loss Spectrum The loss of a specific neutral fragment or functional group from an unspecified parent or parents Typical applications Screening mixtures for example during neonatal screening for a specific class of compound that is characterised by a common fragmentation pathway Qa Collision Cell MS1 RF only MS2 pass all masses scanning scanning The scans of MSI and MS2 are synchronised When MS1 transmits a specific parent ion MS2 looks to see if that parent loses a fragment of a certain mass If it does it will register at the detector The result The spectrum will show the masses of all parents that actually lost a fragment of a certain mass Data System The PC based data system incorporating MassLynx NT software controls the mass spectrometer detector and if applicable the HPLC system autosampler syringe pump divert valve or injector valve The PC uses the Microsoft Windows NT graphical environment with colour graphics and provides for full user interaction with either the keyboard or mouse MassLynx NT provides full control of the system including setting up and running selected HPLC systems tuning acquiring data and data processing Analog inputs can be read by the data system so that where applicable a trace from a conventional LC detector for exam
126. s To clean the hexapole transfer lens proceed as follows Immerse the complete assembly in a suitable solvent 100 methanol and sonicate in an ultrasonic bath Thoroughly dry the assembly using a flow of nitrogen In severe cases Remove clean dry and replace each rod separately one at a time Reassemble the assembly with extreme care checking the assembly against the diagram Maintenance and Fault Finding Page 135 Quattro LC User s Guide Reassembling and Checking the Source Feed the hexapole transfer lens into the instrument allowing the recesses in the differential aperture plate to locate onto the two support rails within the analyser assembly Ensure that the assembly is pushed fully in Check the condition of the O rings on the ion block support Replace them if necessary Replace the ion block support pushing it in against the springs of the hexapole assembly Replace the three retaining screws Fit the plug and sealing ring to the ion block Fit the insulating ring and extraction cone Offer the ion block up to the peek ion block support locate the two dowels and push firmly Replace the ion block heater Replace and firmly tighten the two retaining screws taking care not to over tighten the screws Check that the isolation valve is closed On the tune page select Other and click on Pump Replace the PTFE exhaust liner and cleanable baffle if removed Replace the sample cone and if the nanof
127. s Page 47 Quattro LC User s Guide Shutdown Procedures Emergency Shutdown In the event of having to shut down the instrument in an emergency proceed as follows Switch OFF the power at the wall mounted isolation switch es if fitted If not switch the power off at the rear of the instrument and switch off all peripherals Isolate any LC systems to prevent solvent flowing into the source A loss of data is likely Overnight Shutdown When the instrument is to be left unattended for any length of time for example overnight or at weekends proceed as follows Switch off the LC pumps On the MassLynx top level window click on to launch the tune page Click on OPERATE This will change from green to grey indicating that the instrument is no longer in operate mode Undo the finger tight connector on the probe to release the tubing leading from the LC system Before disconnecting the probe it is good practice to temporarily remove the probe and flush it of any salts buffers or acids If APcI is being used switch off the probe heater or reduce it to ambient temperature A Caution Leaving the APcI probe hot with no gas or liquid flow will shorten the lifetime of the probe heater Select Gas followed by Nitrogen to turn off the supply of nitrogen gas If the instrument is not to be used for a long period of time Reduce Source Block Temp to 60 C Routine Procedures Page 48 Quattro LC User s Guide Com
128. s exceeds the set value then the calibration will fail Reducing the value of the standard deviation gives a more stringent limit Increasing the standard deviation means that the requirement is easier to meet but this may allow incorrect peak matching Values greater than 0 20 should not be used unless exceptional conditions are found Apply Span Correction should always be left on This allows different mass ranges to be scanned within the calibrated range without affecting mass assignment Check Acquisition Calibration Ranges causes warning messages to be displayed if an attempt is made to acquire data outside of the calibrated range for mass and scan speed It is advisable to leave this on Calibration Parameters These are accessed from Edit Calibration Parameters The Peak Match parameters determine the limits within which the acquired data must lie for the software to recognise the calibration masses and result in a successful calibration These parameters are described in detail in the MassLynx NT User s Guide The default values are shown below Calibration Parameters Peak Match OK v Perform auto peak matching EL Peak window Da 1 00 Cancel Initial error Da 2 00 Intensity threshold 0 01 Curve Fit Polynomial order Intensity weighting Display Calibrate display Increasing the Peak window and Initial error gives a greater chance of incorrect peak matching All peaks in the acquired sp
129. sample acquisition once the device has performed its function typically sample injection Analog Channels Four analog channel inputs are available for acquiring simultaneous data such as a UV detector output The input differential voltage must not exceed one volt though full scale automatically adjusts from 1mV to 1V Instrument Description Page 26 Quattro LC User s Guide Scope The X and Y Scope outputs are provided as a diagnostic tool for engineer s use enabling peaks to be displayed on an oscilloscope Vertical gain and display response are set via software The X output is a 0 to 5 volts ramp 1kQ output impedance The Y output is 0 to 10 volts full scale 100 impedance Mains Switch Rotary Pump Control CID Gas ROTARY Nitrogen Gas PC Link Hd Communication Ports Currently Not Used Electrostatic Source Backing Discharge Pumping Line Line Earth Ground Y Point Exhaust Water To Rotary Pump Gas Connections Water Water is used to cool the turbomolecular pumps Nitrogen Gas In The nitrogen supply 100 psi 7 bar should be connected to the Nitrogen Gas In push in connector using 6mm PTFE tubing If necessary this tubing can be connected to 4 inch tubing using standard 4 inch fittings nitrogen supply and the instrument The use of other types of plastic tubing will f Caution Use only PTFE tubing or clean metal tubing to connect between the result in chemical contamination of t
130. similar way to the static calibration Compare the acquired and reference spectra to ensure that the correct peaks have been matched Mass Calibration Page 116 Quattro LC User s Guide If the correct peaks have been matched then the calibration report can be printed out Select Print Print from the report display If insufficient peaks have been matched or the wrong peaks have been matched see Calibration Failure later in this chapter To accept the calibration Select OK from the calibration report The same procedure is used for the scan speed compensation except that Scan Speed Compensation is selected in the dialog box and the fast scanning file is used Note that for the scan speed compensation the default file is FASTMS1 If an alternative filename has been used then this must be selected using the data browser Once all three calibrations static scanning and scan speed compensation have been completed then the instrument can be used for any mass range within the limits of the scanning calibrations and at any scan speed from 100 to 1000 amu sec Calibrating MS2 The calibration of MS2 is carried out in exactly the same manner as above except that data is acquired in MS2 mode instead of MS1 Using the Instrument Once all six calibrations static scanning and scan speed compensation each for both MS1 and MS2 have been completed then the instrument can be used for any mass range within the limits of the scanning cal
131. sting should be performed routinely on a weekly basis for 30 minutes If the source is used in the APcI or megaflow electrospray modes more frequent gas ballasting is recommended Gas ballasting is performed on the E2M28 pump by rotating the gas ballast valve 5 to 6 turns in a counterclockwise direction It is normal for the rotary pump to make more noise when the gas ballast valve is open Caution Failure to gas ballast the rotary pump frequently leads to shortened oil lifetime which in turn may shorten rotary pump lifetime Caution Under no circumstances should gas ballasting be performed during operation gt Maintenance and Fault Finding Page 125 Quattro LC User s Guide Oil Mist Filter The E2M28 rotary pump is fitted with an Edwards EMF20 oil mist filter which traps oil vapour from the rotary pump exhaust The trapped oil is then returned to the rotary pump during routine gas ballasting The oil mist filter contains two elements which require the following maintenance Change the odour element monthly or whenever the pump emits an oily odour Change the mist element every time the rotary pump oil is changed To change the elements follow the instructions in the Edwards manual Foreline Trap The foreline trap stops oil vapour migrating from the rotary pump to the mass spectrometer During normal use the activated alumina sorbent will absorb any oil vapour becoming brown in colour The sorbent should be replac
132. t peaks This situation is unusual but it is always sensible to examine the on screen calibration report to check that the correct peaks have been matched These errors may occur when the following parameters are set Intensity threshold set to 0 Initial error too high 22 0 e Peak window too high 21 5 e Maximum Std Deviation too high 20 2 If the acquired spectrum looks like the reference spectrum and all of the expected peaks are highlighted then the calibration is OK An alternative cause of incorrect calibration is from contamination or background peaks If a contamination or background peak lies within one of the peak matching windows and is more intense than the reference peak in that window then the wrong peak will be selected Under some conditions this may happen with PEG There are two ways to counter this If the reference peak is closer to the centre of the peak window then the peak window can be narrowed until the contamination peak is excluded Take care to ensure that no other reference peak is excluded If the reference peak is not closer to the centre of the peak window or if by reducing the window other reference peaks are excluded then the calibration can be edited manually Mass Calibration Page 102 Quattro LC User s Guide Manual Editing of Peak Matching If an incorrect peak has been matched in the calibration process this peak can be excluded manually from within the on screen calibration
133. ted nebuliser before emerging into a plasma consisting of solvent ions and formed within the atmospheric source by a corona discharge Proton transfer or abstraction then takes place between the solvent ions and the sample Eluent flows up to 2 ml min can be accommodated without splitting the flow Electrospray Electrospray ESI ionisation takes place as a result of imparting a strong electrical charge to the eluent as it emerges from the nebuliser An aerosol of charged droplets emerges from the nebuliser These undergo a reduction in size by solvent evaporation until they have attained a sufficient charge density to allow sample ions to be ejected from the surface of the droplet ion evaporation A characteristic of ESI spectra is that ions may be singly or multiply charged Since the mass spectrometer filters ions according to their mass to charge ratio compounds of high molecular weight can be determined if multiply charged ions are formed Eluent flows up to 1 ml min can be accommodated although it is often preferable with electrospray ionisation to split the flow such that 100 to 200 yl min of eluent enters the mass spectrometer Nanoflow Electrospray The optional nanoflow interface allows electrospray ionisation to be performed in the flow rate range 5 to 1000 nanolitres per minute For a given sample concentration the ion currents observed in nanoflow are comparable to those seen in normal flow rate electrospray Great sensit
134. ter and Parent modes Collision and Exit are interactive parameters Source Voltages The following illustration shows the various components of Quattro LC s ion optical system The name in the table s first column is the name used throughout this manual to describe the component When appropriate the second column shows the term used in the current MassLynx NT release The voltages shown are typical for an instrument in good condition The polarities given are those actually applied to the electrodes Only positive values need be entered via the tune page Routine Procedures Page 44 Quattro LC User s Guide Tune Page APcl Name ve ve Electrospray Probe Capillary 3 0 kV 3 0 kV Not applicable Sample Cone 7 Hexapole Transfer Lens RF Lens zu me y d Calibration Information concerning the calibration of Quattro LC is provided irj Mass Calibration later in this document and in the Guide to Data Acquisition 77T TTTTTTTTTTTS Data Acquisition The mechanics of the acquisition of sample data are comprehensively described in the Guide to Data Acquisition Refer to that publication for full details Data Processing The processing of sample data is comprehensively described in the MassLynx NT User s Guide Refer to that publication for full details Routine Procedures Page 45 Quattro LC User s Guide The following notes provide a worked example for the acquis
135. tial error or Peak window set in the Calibration Parameters dialog box is too small The calibration peaks lie outside the limits set by these parameters Maximum Std Deviation set in the Automatic Calibration Check dialog box has been exceeded The wrong reference file has been selected Check that the correct file peg1000 ref in this case is selected in the Calibrate dialog box Quattro LC User s Guide In the case of too many consecutive peaks missed Check the on screen calibration report to see if the missed peaks are present in the acquired calibration file If the peaks are not present then the first three reasons above are likely causes If the peaks are present in the data but are not recognised during calibration then the latter four are likely reasons Having taken the necessary action proceed as follows If Intensity threshold Initial error and Peak window are adjusted to obtain a successful calibration check the on screen calibration report to ensure that the correct peaks have been matched With a very low threshold and wide ranges set for the initial error and peak window it may be possible to select the wrong peaks and get a successful calibration This is particularly relevant for calibrations with PEG where there may be peaks due to PEG H PEG NH and PEG Na This situation is unusual but it is always wise to examine the on screen calibration report to check that the correct peaks have been matche
136. tic Span amu Static Dwell sec mY Inter Scan Delay sec The lower area in the Calibration Acquisition Setup dialog box contains the Scan Parameters When an instrument acquires data for a static calibration it first examines the selected reference file for the expected reference masses It then acquires data over a small mass span around the expected position of each peak Thus the acquired data do not contain continuous scans but each spectrum is made up of small regions of acquired data around each peak separated by blank regions where no data are acquired Static Span sets the size of this small region around each reference peak A value of 4 0 amu is typical Static Dwell determines how much time is spent acquiring data across the span A value of 0 1 second is suitable Slow Scan Time and Fast Scan Time are not available when a static calibration alone is selected Select OK from the Calibration Acquisition Setup to return to the Automatic Calibration dialog box Mass Calibration Page 110 Quattro LC User s Guide Acquiring Data To start the acquisition Select OK from the Automatic Calibration dialog box The instrument acquires a calibration file ready for static calibration using the data file name STAT While data are being acquired Inject the reference solution Once the data have been acquired the instrument attempts to produce a static calibration automatically The data file contains only a f
137. uide Scanning Calibration and Scan Speed Compensation Acquiring Data To complete the calibration of the instrument two further data files must be acquired Both files are acquired in scanning mode over the same mass range one at the slowest speed required for scanning acquisitions and one at the fastest speed Once these files have been acquired and used for calibration then data may be acquired anywhere within the mass range at any scan speed between the values used for the two sets of data These data do not have to be acquired through the calibration dialog box they can be acquired using the normal scan setup and then accessed from the calibration dialog box as described below The recommended scan speed for the scanning calibration is 100 amu sec Set Scan From to 80 amu and Scan To to 1000 amu Set Scan Time to 9 2 sec and Inter Scan Delay to 0 1 sec Select Continuum as the Data Type Although Continuum is recommended centroided data may be used Set Run Duration to 2 0 minutes This allows time to start the acquisition inject the reference solution and acquire several scans With a solvent flow rate of 200 ul min and a 50 yl loop in line an injection of reference solution lasts approximately 15 seconds allowing at least one full scan of useful data to be acquired Choose any filename for the data The filename SCNMS1 the name used during an automatic calibration is valid Start the acquisition and inject the reference solution
138. umps Refer to the pump manufacturer s manuals Malfunction of the rotary pump Refer to the pump manufacturer s manuals Power Failure In the event of an unexpected power failure proceed as follows Switch OFF the power to the instrument at the wall mounted isolation switch When power is restored follow the start up procedure as described earlier in this chapter Routine Procedures Page 43 Quattro LC User s Guide BTune QULC ESP oI x File lonMode Gas Ramps Other Stop Window Help sigeJ l xi mias s Function Set Mass Span Gain p a m p maesi po b 2 s z 59 T E HM Res 1 3e sz s Emmy Bs q b 4 F x 1 Cr EnimPea w lt Pe gt 175 00 609 30 1080 80 4 04e7 3 15e6 4 14e6 Collision pO p Exit mresa ET Hp HM Res 2 Enor Multiplier nem ds 174 176 608 610 1080 X 1082 Source Analyser Acquire OPERATE V1lMwM2M3T 4 Before sample data are acquired the instrument should be tuned and for the highest mass accuracy calibrated using a suitable reference compound Consult the relevant section of this manual for information concerning source tuning procedures in the chosen mode of operation Adjust the tuning parameters in the Source and Analyser menus to optimise peak shape and intensity at unit mass resolution Care should be taken to optimise the value of the collision energy Note that in Daugh
139. urer s instructions Routine Procedures Page 49 Quattro LC User s Guide Routine Procedures Page 50 Quattro LC User s Guide Electrospray Introduction Exhaust Cleanable Exhaust Baffle gt Liner md Purge Gas iMegaflow only Sample Cone RF Isolation Lens Valve Nebuliser Desolvation 1 n Extraction i Analyser Cone E source nclosure B yy mim a Rotary Turbomolecular Pump Pumps The ESI interface consists of the standard Z spray source fitted with an electrospray probe See the following chapter for information concerning the optional nanoflow interface Mobile phase from the LC column or infusion pump enters through the probe and is pneumatically converted to an electrostatically charged aerosol spray The solvent is evaporated from the spray by means of the desolvation heater The resulting analyte and solvent ions are then drawn through the sample cone aperture into the ion block from where they are then extracted into the analyser The electrospray ionisation technique allows rapid accurate and sensitive analysis of a wide range of analytes from low molecular weight less than 200 Da polar compounds to biopolymers larger than 100 kDa Generally compounds of less than 1000 Da produce singly charged protonated molecules M H in positive ion mode Likewise these low molecular weight analytes yield M H ions in negative ion mod
140. utions is suitable for calibration in negative ion mode In both cases the first negative reference peak appears at Yz 127 T and the remaining peaks are due to Nal clusters Use reference file NAINEG REF Reference Information Page 160 Quattro LC User s Guide Index A C Acetonitrile 52 Caesium iodide 63 156 158 160 Adducts 59 62 78 Calibration 45 85 Acquisition 45 111 APcl 107 Control panel 34 Checking 98 Parameters 94 109 Electrospray 62 63 85 Active inverted magnetron gauge 16 29 125 Failure 100 118 Air filter 123 Incorrect 102 119 Ambient temperature 13 Manual 112 116 Ammonia 52 Parameters 90 109 Ammonium acetate 107 159 Saving 103 120 Analog input 22 26 Scan speed compensation 92 115 Analog PCB 31 147 Scanning 92 115 Analyser 29 146 Static 92 109 Ee 17 39 77 Verification 104 121 Analysis 82 Camera 71 Calibration 107 Capillary 60 Tuning 80 82 Capillary Corona 23 38 40 APcl probe 17 83 142 Charging 127 Checking 79 CID Filter 142 See Collision induced decomposition Fused silica capillary 144 CID Gas Maintenance 142 See Collision gas Temperature 41 82 142 Cleanable baffle 55 69 78 127 131 Tip heater 143 Cleaning 142 150 Argon 14 28 33 Cluster ions 61 64 Atmospheric pressure chemical ionisation Collision cell See APcl See Hexapole collision cell Collision gas 14 25 28 33 47 B Collision induced decomposition 15 19 Column Back pressure 142 149 4 6mm LC 54 64 77 Biopolymers 62 C
141. x Eile lonMode Gas Ramps Other Stop Window Help siale min xi la _ 2l Function Set Mass X Span Gain Capillary 300 S v4 Dac zl hm Ta Ton Cone Stress T Tx Extractor 3 lt i File lonMode Gas Ramps Other Stop Window Help siala mn mlae i 2 Function Set Mass X Span Gain 1 LM Res 1 150 2 RF Lens 080 i mres 59 3 lEnergy1 5 lt 4 cas 151 00 195 00 520 00 741 00 Entrance a E Jemm 6536 x20 964e6 x10 1 07e7 xi 45265 Collision 1 a E Source Block Temp 0 Exi 6 d Desolvation Temp LM Res 2 59 kiea HM Res 2 150 kae Source _ Analyser _ IEnergy 2 20 lt Multiplier e e Mul LL 150 152 194 186 8 520 52 740 742 Source Analyser Acquire OPERATE V1I1wM2M3M 4 Source temperature 100 C is typical for 50 50 CH3CN H20 at solvent flow rates up to 50 l min Higher source temperatures up to 150 C are necessary for solvents at higher flow rates and higher water content Caution The maximum operating temperature for the source heater is 150 C Do not set Source Block Temp higher than 150 C Capillary Voltage Capillary usually optimises at 3 0kV although some samples may tune at values above or below this within the range 2 5 to 4 0kV for positive electrospray For negative ion operation a lower voltage is necessary typically between 2 0 and 3 5kV At high flow rates this parameter may optimise at a value as low as 1kV Sampl
142. y operated using the same nitrogen supply as the rest of the instrument Note that the valve is connected such that the nitrogen supply is always connected to the valve irrespective of the flow to the source and probe Instrument Description Page 25 Quattro LC User s Guide Control of the valve is primarily from the data system The two switches marked Load and Inject enable the user to override control of the valve when making loop injections at the instrument Rear Panel Connections Event Out Four outputs Out 1 to Out 4 are provided to allow various peripherals to be connected to the instrument Switches S1 to S4 allow each output to be set to be either a contact closure upper position or a voltage output lower position Out 1 and Out 2 when set to voltage output each have an output of 5 volts The voltage output of both Out 3 and Out 4 is 24 volts During a sample run Out 1 closes between acquisitions and is used typically to enable an external device to inject the next sample The three remaining outputs are reserved for future developments EVENT OUT A S4 our4 CAUTION REFER TO MANUAL BEFORE CONNECTING Ss OUTS TO THESE PORTS S2 OUT 2 1 OUT 1 PCB SUPPORT CONTACT CLOSURE IN ve CH1 CH3 ANALOG CHANNELS Contact Closure In In 1 and In 2 inputs are provided to allow external device to start

Quattro LC User's Guide, issue 2. 6666525

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