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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 (SIR) techniques.

Careful choice of the solvent, and solvent additives or modifiers may also prove
important.

Electrospray
Page 158

Quattro Ultima
User's Guide

Содержание Quattro Ultima

Страница 1: ...to the instrument Warnings are given throughout this manual where care is required to avoid personal injury If the instrument is used in a manner not specified by the manufacturer the protection prov...

Страница 2: ...Quattro Ultima User s Guide...

Страница 3: ...ray 17 Nanoflow Electrospray 17 Sample Inlet 17 MS Operating Modes 18 MS MS Operating Modes 18 The Daughter Ion Spectrum 19 The Parent Ion Spectrum 20 MRM Multiple Reaction Monitoring 21 The Constant...

Страница 4: ...uring the Analyser Pressure 37 Using the Instrument 37 Start Up Following Overnight Shutdown 37 Preparation for Electrospray Operation 38 Preparation for APcI Operation 40 Operate 42 Automatic Pumping...

Страница 5: ...Modifying the Peak Display 58 Changing the Display 60 Customise Plot Appearance 60 Trace 61 Intensity 61 Grid 61 AutoTune 62 Ion Mode 63 Scope Parameters 64 Gas Controls 64 Ramp Controls 64 Resetting...

Страница 6: ...4 Analog Channels 84 Saving and Restoring a Function List 85 Setting up a Full Scan Function 86 Mass m z 86 Cone Voltage 86 Method 86 Scan Duration secs 87 APcI Probe 87 Setting up a SIR Function 88 C...

Страница 7: ...atching 121 Saving the Calibration 121 Verification 122 Electrospray Calibration with PEG 124 Atmospheric Pressure Chemical Ionisation 125 Introduction 125 Preparing for Calibration 126 Reference Comp...

Страница 8: ...63 Using the Microscope 163 Glass Capillary Option 164 Restarting the Spray 165 Nano LC Option 166 Installation 166 Operation 167 Changing Options 168 Atmospheric Pressure Chemical Ionisation Introduc...

Страница 9: ...eel Sample Capillary 198 The APcI Probe 200 Cleaning the Probe Tip 200 Replacing the Probe Tip Heater 201 Replacing the Fused Silica Capillary 202 The Analyser 204 The Detector 204 Electronics 205 Fus...

Страница 10: ...yoglobin 218 Mixture of Sugars 218 Sodium Iodide and Caesium Iodide or Rubidium Iodide Mixture 219 Preparation of Calibration Solutions 220 PEG Ammonium Acetate for Positive Ion Electrospray and APcI...

Страница 11: ...330lb Data system computer monitor and printer 60kg 130lb Rotary pumps E2M28 40kg 90lb E1M18 32kg 72lb Transformer optional 100kg 220lb Hardware Specifications Page 11 Quattro Ultima User s Guide 535...

Страница 12: ...ed personnel are 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...

Страница 13: ...to the water 200W Exhausts Rotary Pumps The rotary pumps 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 t...

Страница 14: ...be made of Teflon The use of other types of plastic leads to contamination of the instrument CID Gas Argon is required as collision gas The supplied gas should be dry of high purity 99 9 and at a pre...

Страница 15: ...uadrupole Finally the transmitted ions are detected by a conversion dynode phosphor and photomultiplier detection system The output signal is amplified digitised and presented to the data system Instr...

Страница 16: ...pump oil provided that the manual gas ballast valve on the pump is left open The turbomolecular pumps evacuate the analyser and ion transfer region These pumps are both water cooled Vacuum measuremen...

Страница 17: ...c 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...

Страница 18: ...useful tool for instrument tuning and calibration prior to MS MS analysis and for fault diagnosis MS MS Operating Modes The basic features of the four common MS MS scan functions are summarised below...

Страница 19: ...r 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 m 609 fr...

Страница 20: ...on for daughter scan data Example Parents of the specific daughter ion at m 195 from reserpine in electrospray positive ion mode The result Instrument Description Page 20 Quattro Ultima User s Guide M...

Страница 21: ...and herbicide analysis Forensic or toxicology for example screening for target drugs in sport Example Monitor the transition specific fragmentation reaction m 609 195 for reserpine in electrospray pos...

Страница 22: ...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 int...

Страница 23: ...voltage connector ESI APcI 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 plu...

Страница 24: ...the instrument is indicated as follows Vacuum LED State Vacuum LED State Vacuum LED Vented No indication Vacuum OK Steady green Pumping Steady amber Pump fault Flashing red Operate LED State Operate...

Страница 25: ...loop fitted As a divert valve to switch the flow of solvent during a LC run As a switching valve to switch for example between a LC system and a syringe pump containing calibrant This valve is pneumat...

Страница 26: ...three remaining outputs are reserved for future developments Contact Closure In In 1 and In 2 inputs are provided to allow external device to start sample acquisition once the device has performed it...

Страница 27: ...ctor connects the instrument to the MUX control base Data System This RJ45 connector connects the instrument to the data system using the network cable supplied Instrument Description Page 27 Quattro...

Страница 28: ...ween the nitrogen supply and the instrument The use of other types of plastic tubing results in chemical contamination of the source Instrument Description Page 28 Quattro Ultima User s Guide ESD EART...

Страница 29: ...s 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 swi...

Страница 30: ...Analyser Turbomolecular Pump RF Generators Turbomolecular Pumps Power Supply High Voltage Power Supplies 4 Low Voltage Power Supplies 2 Pumping Logic PCB Transputer Processor Card TPC Analogue PCB Con...

Страница 31: ...plies 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 a...

Страница 32: ...lyser 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 ana...

Страница 33: ...nstrument 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...

Страница 34: ...connected to a separate vent Caution Do not connect the two exhaust lines together In the event of an instrument failure rotary pump exhaust could be admitted into the source chamber producing severe...

Страница 35: ...m 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...

Страница 36: ...en indicating that the instrument is ready for use Ensure that the gas ballast valve on the E1M18 rotary pump is open The E1M18 rotary pump is operated with its gas ballast valve open at all times If...

Страница 37: ...delay of 10 seconds before the pressure is displayed To switch off the gauge Press or select Options Peak Editor from the tune page The analyser pressure is not recorded in the experimental record fil...

Страница 38: ...source flange Remove the corona discharge pin from its mounting contact and fit the blanking plug Replace the glass tube and adjustment flange Ensure that the source enclosure consisting of the glass...

Страница 39: ...ode If necessary change the ionisation mode using the Ion Mode command Set Source Temp to 100 C and Desolvation Temp to 20 C Warning Operating the source without the source enclosure results in solven...

Страница 40: ...sample cone Connect the APcI high voltage cable between Capillary Corona and the socket positioned at the lower right edge of the source flange Replace the glass tube and adjustment flange Ensure tha...

Страница 41: ...ource without the source enclosure results 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...

Страница 42: ...or operation To obtain an ion beam refer to Obtaining an Ion Beam in either the Electrospray or the Atmospheric Pressure Chemical Ionisation section Automatic Pumping and Vacuum Protection Overview Th...

Страница 43: ...e pumps do 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 swi...

Страница 44: ...p for MS MS Operation The following notes provide a worked example for the acquisition of daughter ion data The experiment is performed in the ESI positive mode using reserpine as a model analyte Rese...

Страница 45: ...s 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 col...

Страница 46: ...vel window click on to launch the tune page Click on This changes from green to grey indicating that the instrument is no longer in operate mode Undo the finger tight connector on the probe to release...

Страница 47: ...the APcI probe hot with no gas or liquid flow shortens the lifetime of the probe heater Select Gas Gas to turn off the supply of nitrogen gas Select Other from the menu bar at the top of the tune pag...

Страница 48: ...or allows the automatic startup and shutdown procedures to be modified or new procedures to be created To access the editor Select Edit Shutdown from the MassLynx Run menu Check the Enable Startup bef...

Страница 49: ...rayed out if this option is selected but the shutdown time can still be changed as this applies to the Enable Shutdown after batch option The Auto Control Tasks Page Task is a dropdown list box with a...

Страница 50: ...the left mouse button below the last entry in the timetable and then add the new task To insert a task Click on the entry in the task timetable before which the new task is to be inserted Select a ta...

Страница 51: ...tton Menu Equivalent Purpose File New Create a new startup or shutdown file File Open Open an existing startup or shutdown file File Save or File Save As Save a startup or shutdown file File Print Pri...

Страница 52: ...t a data file and press Open Saving a Startup or Shutdown File To save a startup or shutdown file Press or select Save or Save As from the File menu If this is a new file or if the Save As option has...

Страница 53: ...les To print a startup or shutdown file Press or select Print from the File menu This displays the print dialog Select the printer print range and number of copies and press OK Routine Procedures Page...

Страница 54: ...m the shutdown editor control list menu then the automatic startup and shutdown files are run To run a different startup or shutdown file Open the required file in the shutdown editor and press or sel...

Страница 55: ...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 res...

Страница 56: ...MassLynx NT User Guide Selecting and Viewing Data for more information Saving and Restoring Parameter Settings Whole sets of instrument tuning parameters can be saved to disk as a named file and then...

Страница 57: ...ng information or No to enter a different file name To restore a saved set of parameters Press or choose Open from the tune page File menu Select the required tuning parameter file either by typing it...

Страница 58: ...is range is redisplayed to fill the window The mass displayed in the Mass box is the mass at the centre of the window This operation can be repeated as often as required Pressing once displays the pre...

Страница 59: ...required peak and press Return This becomes the default so if the range is altered with the mouse and is pressed twice Span returns to this value To change the gain of a peak Double click on the line...

Страница 60: ...ppearance The Customise Plot Appearance dialog is displayed To change the colours on the display Press Newest Trace Background or Trace Fill and select a new colour from the dialog displayed To change...

Страница 61: ...ted has a tick next to it Intensity Select either Intensity Relative Intensity or Intensity Absolute Intensity as required Select Intensity Normalise Data to display normalised data The options select...

Страница 62: ...ing parameters A maintenance AutoTune starts from the current tuning parameters set in the tune page and can be quicker than a full AutoTune A maintenance AutoTune can only be performed if the instrum...

Страница 63: ...changing key parameters one at a time to maximise the intensity of a reference peak with respect to that parameter At present ESP APcI Autotuning is carried out with respect to a single user specifie...

Страница 64: ...nu If the gas was previously turned off it is now turned on A tick mark appears next to a gas if it is turned on Ramp Controls To set up a cone voltage ramp Choose Cone Ramp Gradient from the tune pag...

Страница 65: ...n energy voltage ramp Press or choose Use Collision Energy Ramp from the tune page Ramps menu Resetting the Zero Level The zero level or baseline can be repositioned by pressing or by choosing Reiniti...

Страница 66: ...acks are for diagnostic purposes only their function being to confirm a voltage is present The acceptable variation between the set value and the readback value varies depending on the particular tune...

Страница 67: ...Nam e ESI ve ESI ve APcI ve APcI ve trospray Probe Capillary 3 0 kV 3 0 kV Not applicable Discharge Pin Corona Not applicable 2 A 2 A ample Cone Cone 50 V 50 V 50 V 50 V st Ion Tunnel RF Lens 1 0 V 0...

Страница 68: ......

Страница 69: ...of the scanning parameters can be controlled 6 Inlet programs cannot be used 6 Analog data cannot be acquired 6 Multiple sample sequences cannot be acquired To start a single sample acquisition Press...

Страница 70: ...ne press CTRL Return at the end of a line The type of acquisition Function used to collect the data can be any of the following MS MS2 Daughter Parent Neutral Loss Neutral Gain More information is giv...

Страница 71: ...nts To start a multi sample acquisition Set up a sample list see MassLynx NT User Guide Sample Lists for details Choose Start from the top level Run menu or press This displays the start sample list r...

Страница 72: ...s For the process to automatically operate on the data file which has just been acquired Leave unchecked Use Acquired File as Default on the System tab of the MassLynx Options dialog The MassLynx Opti...

Страница 73: ...ication results details for use with LIMS systems If this box is checked the LIMS Export Browse button becomes enabled Press Browse select a file or enter the name of a new one and press Save The Proj...

Страница 74: ...currently being acquired Chromatogram Real Time Update To view in real time the chromatogram that is currently being acquired Open the data file using the MassLynx data browser Press or select Real Ti...

Страница 75: ...ds MassLynx has several parameters that allow control over how the system pre processes data before it is sent to the host computer These parameters are contained in the instrument data thresholding d...

Страница 76: ...re noise use a positive value Do not use a positive value for the baseline level if using ion counting thresholding Points per Dalton can have one of three values 4 8 or 16 Selecting 8 points instead...

Страница 77: ...If a value of zero is entered the intensity level is set so that it sits in the middle of the noise which means that roughly half of the noise data is acquired A value of 10 places the threshold just...

Страница 78: ...i z e 0 0 0 0 1 0 0 2 0 0 5 0 0 0 10 4 8 0 20 11 10 0 40 37 61 0 60 66 69 0 250 100 83 Profile Data Spike Removal Spikes are distinguished from real data by the fact that they are very narrow and when...

Страница 79: ...h that these single ion events are not discarded as spikes Set a suitable value for Spike Percentage Ratio This ratio is used to determine if a data point is a spike by comparing the data point to its...

Страница 80: ...mass spectrometer uses to scan the instrument during an acquisition A function list can be a mixture of different scanning techniques that can be arranged to run either sequentially or concurrently du...

Страница 81: ...r 35 minutes terminating after a total elapsed time of 40 minutes To access this dialog Press on the MS panel of the MassLynx screen A more complicated function list with four SIR functions each runni...

Страница 82: ...tion down the list of functions Create a new function of the indicated type Adding a New Function To add a new function to the list Click one of the toolbar buttons or select the required function fro...

Страница 83: ...runs from 0 to 10 minutes and the second from 10 to 20 minutes Copying an Existing Function To copy an existing function Select the function in the function list Select Copy and then Paste from the Ed...

Страница 84: ...red which are stored with the data acquired from the mass spectrometer Analog channels are typically used to collect data from external units such as UV detectors or FID detectors A reading is made fr...

Страница 85: ...an existing file from the list displayed Press Save If the file already exists on disk confirmation is requested to overwrite the existing information Press Yes to overwrite the file or No to select a...

Страница 86: ...the tune page To apply a ramp to the cone voltage Check Use Cone Voltage Ramp and press CV Ramp to load the cone ramp dialog The four parameters define a gradient for the cone voltage which is then ex...

Страница 87: ...This emphasises the real data and improves the signal to noise ratio A further advantage of MCA is that because data is written to disk only at the end of an experiment scanning speeds can be increase...

Страница 88: ...ng with their dwell times spans and inter channel delay times To set up a SIR function Press or select SIR from the functions menu Many of the fields are described above for the full scan editor Only...

Страница 89: ...tarting monitoring the next mass in the function Repeats is only relevant for experiments having more than one function and specifies the number of repeats of the function Span specifies a small mass...

Страница 90: ...ed MS MS mode and is used to look at fragmentations of a particular ion MS1 is set to the parent mass using Daughters of and is not scanned The resolution of MS1 can be lowered until the peak width at...

Страница 91: ...rent peaks MS2 In this mode MS2 is resolving while MS1 transmits ions over a wide mass range While this scanning mode can be used for acquiring data it is mostly used in the tune window for setting an...

Страница 92: ...setting during an acquisition then the acquisition must be started from the tune page To apply a ramp to the collision energy Check Use Collision Energy Ramp Press CE Ramp to load the collision energy...

Страница 93: ...d All fields in the MRM editor are similar to those already described Setting up a Survey Function Survey scans are used to search for precursor ions To access the dialog Press or select Survey Scan f...

Страница 94: ...d MSMS Template Pages These pages allow the parameters to be set for MS and MS MS scanning during the survey and are similar to normal function editor pages Data Acquisition Page 94 Quattro Ultima Use...

Страница 95: ...changed The number of non coeluting precursors in a single run is not limited Precursor Selection If Automatic is selected all valid masses satisfying selection criteria are monitored If Include Mass...

Страница 96: ...ted allows a delay to be incorporated before precursors are included Data Discard uninteresting survey scans allows only the survey scans that detect precursor ions to be stored This saves on disk spa...

Страница 97: ...f the MSMS to MS switch method is Default the MSMS function stops when the MSMS to MS switch criteria are met If the MSMS to MS switch method is After Time the MSMS function stops when the MSMS to MS...

Страница 98: ...from the MS MS scans are entered in the relevant Range boxes Masses on the Exclude list are not considered for detection Ranges take the form massX_massY Masses and ranges in a list are comma delimit...

Страница 99: ...Monitoring Acquisitions When an acquisition is started the automatic switching status dialog is displayed showing the precursors currently running Data Acquisition Page 99 Quattro Ultima User s Guide...

Страница 100: ...Data Acquisition Page 100 Quattro Ultima User s Guide...

Страница 101: ...ma using electrospray ionisation with a mixture of sodium iodide and rubidium iodide as the reference compound A complete mass calibration of Quattro Ultima using atmospheric pressure chemical ionisat...

Страница 102: ...also calculated This number is the best single indication of the accuracy of the calibration Calibration Types Each quadrupole analyser requires up to three calibration curves A static calibration is...

Страница 103: ...ion report Electrospray Introduction 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 ma...

Страница 104: ...east 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...

Страница 105: ...table 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...

Страница 106: ...irb ref for a sodium iodide and rubidium iodide reference solution Removing Current Calibrations Select the uncal cal calibration file from the File Load Calibration menu option Select Process Delete...

Страница 107: ...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...

Страница 108: ...s 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 widel...

Страница 109: ...then the mass measure parameters are not used With electrospray calibrations particularly with sodium iodide which has some low intensity peaks at higher mass it is recommended that continuum or MCA...

Страница 110: ...ation 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 a...

Страница 111: ...e 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 th...

Страница 112: ...ameters 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 sp...

Страница 113: ...mately 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 scan speed compensation then the scan speed should be set a...

Страница 114: ...cquired each data file is combined 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...

Страница 115: ...9 0 35 amu MS1 Scanning Calibration 27 matches of 28 tested references SD 0 0459 500 1000 1500 2000 2500 3000 3500 M z 0 42 1 10 amu MS1 Scan Speed Compensation Calibration 26 matches of 28 tested ref...

Страница 116: ...procedure for that particular file and display a calibration report on the screen This calibration report opposite upper contains four displays the acquired spectrum the reference spectrum a plot of...

Страница 117: ...Mass Calibration Page 117 Quattro Ultima User s Guide...

Страница 118: ...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 b...

Страница 119: ...nitial 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 wid...

Страница 120: ...nce 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 back...

Страница 121: ...he 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 does not affect the other matched p...

Страница 122: ...rried out Set all peak matching parameters to the values that were used for the calibration Bring up the Automatic Calibration dialog box by selecting Start on the Calibrate dialog box Select Scanning...

Страница 123: ...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 Unlik...

Страница 124: ...2 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...

Страница 125: ...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 polye...

Страница 126: ...ibration covers up to 1000 amu Calibration Options To access the calibration options click on Calibrate on the acquisition control panel Selecting Reference File Set pegh1000 ref as the reference file...

Страница 127: ...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 ha...

Страница 128: ...ne is selected Select OK from the Calibration Acquisition Setup to return to the Automatic Calibration dialog box Acquiring Data To start the acquisition Select OK from the Automatic Calibration dialo...

Страница 129: ...file can be selected by clicking on Browse Enter the start and end scans of the reference data in the From and To boxes Select OK to perform the calibration and display the calibration report on the s...

Страница 130: ...If insufficient peaks have been matched or the wrong peaks have been matched refer to the section on calibration failure later in this manual Mass Calibration Page 130 Quattro Ultima User s Guide...

Страница 131: ...e p o r t P a g e 1 P r i nte d T h u No v 27 1 7 0 2 31 1 9 9 7 ________________________________________________________________________________________________________ 0 100 0 100 0 13 0 30 amu 100...

Страница 132: ...ation 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...

Страница 133: ...ject 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 selec...

Страница 134: ...een 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 rang...

Страница 135: ...n the calibration has failed Peaks may be missed for the following reasons The reference solution is running out causing less intense peaks to not be detected Multiplier is too low and less intense pe...

Страница 136: ...EG 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 matched Select OK from the calibration report window to acce...

Страница 137: ...t 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...

Страница 138: ...ange and at the required scan speed in the normal way Start the acquisition and inject the reference solution so that reference data is acquired Stop the acquisition Access the calibrate dialog box an...

Страница 139: ...e 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 t...

Страница 140: ...Mass Calibration Page 140 Quattro Ultima User s Guide...

Страница 141: ...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 molec...

Страница 142: ...ch 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 Infusion pump The pump syringe is f...

Страница 143: ...a graduated glass capillary with the liquid emerging from the probe tip and timing a known volume say 10 l Once the rate has been measured and set a note should be made of the back pressure readout on...

Страница 144: ...wn 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 incorporate...

Страница 145: ...ray operation it is essential that the correct tubing is used to connect the probe to the sample injector For megaflow operation 1 16 o d 0 007 i d peek tubing easily identified by its yellow stripe i...

Страница 146: ...onnected 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 Thi...

Страница 147: ...nificant 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 capill...

Страница 148: ...e tune page and observe the Gas Flows readback window Turn on the liquid flow at 10 l min and set Desolvation Temp to 100 C Tuning and Optimisation The following parameters after initial tuning should...

Страница 149: ...rates Nebuliser Gas Optimum nebulisation for electrospray performance is achieved by fully opening the Nebuliser flow control valve which is situated on the instrument s front panel Desolvation Gas Th...

Страница 150: ...uster ions and adduct ions The cone gas flow rate should be increased until solvent cluster ions and or adduct ions are reduced as much as possible without diminishing the intensity of the ion of inte...

Страница 151: ...into the source block If the majority of samples to be analysed are higher molecular weight compounds it is recommended that the restrictor is re optimised for these compounds Continual adjustment an...

Страница 152: ...h 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...

Страница 153: ...ed 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 a...

Страница 154: ...d 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 probe mounting flange to get hot duri...

Страница 155: ...all 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...

Страница 156: ...Environmental contaminants e g pesticides pollutants Dye compounds Some organometallics Small saccharides Typical ES Negative Ion Samples Some proteins Some drug metabolites e g glucuronide conjugates...

Страница 157: ...placed in line to the Quattro Ultima probe However ensure that the volume of the detector does not significantly reduce the chromatographic resolution Whenever a UV detector is used the analog output...

Страница 158: ...y 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 spectromete...

Страница 159: ...to be obtained but after use for one sample only must be discarded Nano LC This option is suitable for flow injection analyses or for coupling to nano HPLC and uses a pump to regulate the flow rate do...

Страница 160: ...or Installing the Interface To change from the normal electrospray interface and install the nanoflow interface If fitted remove the probe Undo the three thumb screws and withdraw the probe adjustment...

Страница 161: ...sure that the purge gas is plugged disabled Ensure that the cleanable baffle the exhaust liner and the discharge pin blanking plug are fitted Fit a viton O ring and the three shorter nanoflow pillars...

Страница 162: ...rommet 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 Attach the two cables to the sockets marke...

Страница 163: ...he zoom lens A fine focus can be achieved by rotating the objective lens Using the Microscope Focusing is adjusted by rotating the top of the microscope Nanoflow Electrospray Page 163 Quattro Ultima U...

Страница 164: ...illary is protruding from the end of it This distance is measured from the end of the nut to the shoulder of the glass capillary Load sample into the capillary using either a fused silica syringe need...

Страница 165: ...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 t...

Страница 166: ...sed silica is gripped Some force may be required to do this Remove the sleeved fused silica from the plug cap and remove the Valco nut Place an O ring onto the peek tube using tweezers if necessary Th...

Страница 167: ...ning purposes it may be useful to infuse a known sample in 95 water using a Harvard syringe pump Set the liquid flow to about 200 nl min Switch on Gas at the MassLynx tune page Set the pressure of the...

Страница 168: ...ing between tuning and HPLC operation Changing Options To change between the glass capillary and the nano LC options Rotate the stage outwards Caution Failure to use the stop and handle to rotate the...

Страница 169: ...rona discharge pin which is typically operated with a discharge current of 2 A Mobile phase molecules rapidly react with ions generated by the corona discharge to produce stable reagents ions Analyte...

Страница 170: ...scribed in Electrospray 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 describe...

Страница 171: ...onitrile 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...

Страница 172: ...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 the Source window on the tune page and...

Страница 173: ...g 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 100 C before removing it...

Страница 174: ...mp 130 C APcI Probe Temp 500 C Desolvation Gas 150 litres hour Cone Gas 100 litres hour See the following section for specific tuning details Specific Tuning for Maximum Sensitivity For quantitative M...

Страница 175: ...t can have a significant effect upon sensitivity Using the sideways adjuster ensure that the spray is directed approximately at the mid point between the corona pin and the sample cone This position o...

Страница 176: ...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 Caution Removal of the APcI probe when hot shortens the life...

Страница 177: ...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...

Страница 178: ...ger pumping Before suspecting a leak the following points should be noted The turbomolecular pumps do not operate if the rotary pump has failed If the rotary pump is not maintained the oil may become...

Страница 179: ...e 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 pres...

Страница 180: ...can cause oil vapour to migrate into the vacuum housing However the E1M18 has an additional automatic gas ballast control valve mounted in the oil return line from the mist filter This valve is opened...

Страница 181: ...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 Forelin...

Страница 182: ...ve Change the oil in the rotary pump as follows Gas ballast lightly for 30 to 60 minutes Close the gas ballast valve Vent and shut down the instrument as described in Routine Procedures It is easier t...

Страница 183: ...y closing the isolation valve located on the ion block Less frequently it may be necessary to clean the ion block and the hexapole lens in which case the instrument must be vented This should only be...

Страница 184: ...olvation 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 shortens the probe heater s...

Страница 185: ...ass 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 sam...

Страница 186: ...cone the gasket and the cone gas nozzle Carefully wipe the sample cone and cone gas nozzle with a cotton swab or lint free tissue soaked in 50 50 acetonitrile water or 50 50 methanol water Caution Do...

Страница 187: ...ner and cleanable baffle to the ion block Refitting the sample cone and cone gas nozzle is a reversal of the removal procedure The source isolation valve is fully open when the screwdriver slot is par...

Страница 188: ...he 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 sample cone and c...

Страница 189: ...ate to the ion block and remove the cover plate Warning The heater supply remains live until the system is fully vented Do not remove the cover plate until the system has vented Ensure that the O ring...

Страница 190: ...couple from its location Remove the two screws which secure the ion block to the ion block support Withdraw the ion block leaving the thermocouple and heater supply leads protruding from the ion block...

Страница 191: ...tton swab or lint free tissue soaked in 50 50 acetonitrile water or 50 50 methanol water to remove any carbon deposits Leaving the heater valve thermocouple clip and terminal block in place immerse th...

Страница 192: ...three O rings remain in position on the rear face of the support Using a lint free tissue to gently grasp the ion tunnel carefully withdraw it Ensure that the O ring remains correctly located on the d...

Страница 193: ...h Do not disassemble the ion tunnel Cleaning the tunnel should be carried out with great care using the brush provided Gently insert the brush into the aperture and use a rotary motion to clean the ap...

Страница 194: ...re plate and that the assembly is pushed fully in Replace the ion block support pushing it in against the springs of the ion tunnel assembly Replace the three retaining screws Replace the restrictor a...

Страница 195: ...d Replace the sample cone gasket and sample cone nozzle on the ion block Reconnect the cone gas supply When the instrument has pumped down Open the isolation valve Fit the APcI corona discharge pin or...

Страница 196: ...atography 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...

Страница 197: ...ood 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 reconnecti...

Страница 198: ...g screws and remove the probe end cover Unscrew and remove the probe tip Remove the LC union and adapter nut Withdraw and discard the stainless steel sample capillary Remake the LC connection to the L...

Страница 199: ...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 a...

Страница 200: ...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 th...

Страница 201: ...obe heater This procedure should remove any chemical contamination from the probe tip Replacing the Probe Tip Heater Remove the probe tip assembly by carefully loosening the two grub screws Disconnect...

Страница 202: ...e probe Remove and discard the fused silica capillary Using a ceramic capillary cutter cut a new length of 300 m o d 100 m i d fused silica capillary about 1 centimetre excess in length Using a GVF 00...

Страница 203: ...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 capillar...

Страница 204: ...er or by other suitably qualified personnel The quadrupole assemblies of Quattro Ultima are finely machined and aligned assemblies which under no circumstances should be dismantled The Detector The Qu...

Страница 205: ...designation T indicates a time lag fuse Analog PCB Fuse No Fuse Type Ref No F1 10A T 20mm anti surge TDS505 1340143 F2 10A T 20m anti surge TDS505 1340143 RF Power PCB Fuse No Fuse Type Ref No F1 5A T...

Страница 206: ...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 Unsteady or L...

Страница 207: ...over This can occur if concentrations of a few ng l are used for tuning prior to attempting sub pg l detection levels If the injector is contaminated the signal level normally changes when injections...

Страница 208: ...ay 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...

Страница 209: ...lacement 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 readbacks vary with tune page setti...

Страница 210: ...ve 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 Is...

Страница 211: ...ticlockwise 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 te...

Страница 212: ...Maintenance and Fault Finding Page 212 Quattro Ultima User s Guide...

Страница 213: ...equired format However if required realistic intensity values can be entered to improve the appearance of the reference spectra Most samples can be purchased from the Sigma chemical company To order c...

Страница 214: ...menu To save the reference file after editing either Select Save from the Notepad File menu to save the file under the current name or Select Save as from the Notepad File menu to save as a new refere...

Страница 215: ...Hb analysis MYO Horse heart myoglobin M1882 16951 48 700 1600 General PEGH1000 Polyethylene glycol ammonium acetate mixture PEG 200 400 600 1000 80 1000 ES and APcI calibration PEGH2000 Polyethylene...

Страница 216: ...477 8 2119 945 23 738 030 15 1131 108 7 2422 651 22 771 531 14 1211 829 Polyethylene Glycol PEG NH4 Reference Files PEGH1000 PEGH2000 Calculated Value 63 04 459 28 855 52 1251 75 1647 99 107 07 503 31...

Страница 217: ...5 1372 0379 2271 4033 3170 7688 622 5667 1521 9321 2421 2976 3320 6630 Sodium Iodide and Rubidium Iodide Mixture Reference File NAIRB Calculated Value 22 9898 772 4610 1671 8264 2571 1918 3470 5572 84...

Страница 218: ...INEG Sodium Iodide Caesium Iodide or Rubidium Iodide mixture 200 3900 ES calibration Horse Heart Myoglobin Reference File myoneg ref Calculated Value 891 175 1209 812 1882 490 940 741 1302 952 2117 92...

Страница 219: ...045 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...

Страница 220: ...nd 50 water containing 2 mmol ammonium nitrate Use reference file PEGH1000 PEG Ammonium Acetate for Positive Ion Electrospray Extended Mass Range Prepare a solution of polyethylene glycols at the foll...

Страница 221: ...dide at a concentration of 2 g l 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 g l The purpose of the rubidi...

Страница 222: ...Reference Information Page 222 Quattro Ultima User s Guide...

Страница 223: ...lectrospray 157 Failure 120 137 Incorrect 122 138 Manual 131 135 Parameters 110 128 Saving 123 139 Scan speed compensation 112 134 Scanning 112 134 Static 112 129 Verification 124 140 Camera 165 Capil...

Страница 224: ...lt finding 179 208 Filter 34 Flow control valve 27 Flow injection analysis 19 144 161 Fluorescence detector 24 Foreline trap 183 Forensic science 23 Formic acid 144 Fragment ions 17 Fragmentation 46 4...

Страница 225: ...erate 44 Operate LED 26 44 45 Organometallics 158 Origin 73 P Parent 93 Parent ion 22 46 PC link 36 Peak matching 123 139 PEG See Polyethylene glycol Penning gauge See Active inverted magnetron gauge...

Страница 226: ...PCB 33 Scan duration 89 Scan time 73 Scope 66 Selected ion recording 23 See SIR Sensitivity 208 LC MS 160 Set Mass 72 Shutdown 48 Automatic 50 Complete 49 Editor 50 Emergency 48 Overnight 48 Single io...

Страница 227: ...ment 160 Transformer 13 Transputer processor card 33 Triethylamine 159 Trifluoroacetic acid 159 Tune page 58 Tuning 57 APcI 128 174 Electrospray 106 Turbomolecular pump 18 30 34 45 180 U UV detector 1...

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