quattro ultima user's guide micromass uk limited floats ... · water cooling 13 exhausts 13...

Quattro UltimaUser's Guide

Code: 6666511Issue 3© Micromass Ltd.

Micromass UK Limited

Floats RoadWythenshawe

M23 9LZTel: +44 161 945 4170 Fax: +44 161 998 8915

Tudor RoadAltrinchamWA14 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 arepresent.

The instrument is marked with this symbol where hot surfaces arepresent.

The instrument is marked with this symbol where the user should refer tothis User's Guide for instructions which may prevent damage to the

instrument.

Warnings are given throughout this manual where care is required to avoid personalinjury.

If the instrument is used in a manner not specified by the manufacturer, the protectionprovided by the equipment may be impaired.

This manual is a companion to the MassLynx NT User's Guide supplied with theinstrument.

All information contained in these manuals is believed to be correct at the time ofpublication. The publishers and their agents shall not be liable for errors

contained herein nor for incidental or consequential damages in connection withthe furnishing, performance or use of this material. All product specifications, aswell as the information contained in this manual, are subject to change without

notice.

ContentsHardware Specifications

Dimensions 11Weights 11

Lifting and Carrying 12Power 13Environment 13Water Cooling 13Exhausts 13

Rotary Pumps 13API Gas Exhaust 13

Nitrogen 14CID Gas 14

Instrument DescriptionOverview 15Vacuum System 16Ionisation Techniques 17

Atmospheric Pressure Chemical Ionisation 17Electrospray 17

Nanoflow Electrospray 17Sample Inlet 17MS Operating Modes 18MS-MS Operating Modes 18

The Daughter Ion Spectrum 19The Parent Ion Spectrum 20MRM: Multiple Reaction Monitoring 21The Constant Neutral Loss Spectrum 22

Data System 22Front Panel Connections 23

Desolvation Gas and Probe Nebuliser Gas 23Capillary / Corona 23ESI / APcI 23

Front Panel Controls and Indicators 24Status Display 24

Vacuum LED 24Operate LED 24

Flow Control Valves 25Divert / Injection Valve 25

Table of Contents


Rear Panel Connections 26Event Out 26Contact Closure In 26Analog Channels 26MUX 27Data System 27Water 28Nitrogen Gas In 28Exhausts 29CID Gas 29Power Cord 29Mains Switch 29Fuses 29Rotary Control 29ESD Earth Facility 29

Internal Layout 30Electronics 30Mechanical Components 32

Routine ProceduresStart Up Following a Complete Shutdown 33

Preparation 33Pumping 36Measuring the Analyser Pressure 37Using the Instrument 37

Start Up Following Overnight Shutdown 37Preparation for Electrospray Operation 38Preparation for APcI Operation 40Operate 42

Automatic Pumping and Vacuum Protection 42Overview 42Protection 42

Transient Pressure Trip 42Pump Fault 43Power Failure 43

Tuning 44Calibration 44Data Acquisition 44Data Processing 44Setting Up for MS-MS Operation 44

Parent Ion Selection 44Fragmentation 45

Shutdown Procedures 46Emergency Shutdown 46Overnight Shutdown 46Complete Shutdown 47

Table of Contents


Automatic Start up and Shutdown 48The Shutdown Editor 48The Auto Control Tasks Page 49The Shutdown Editor Toolbar 51Loading Startup and Shutdown Files 52Saving a Startup or Shutdown File 52Printing Startup and Shutdown Files 53Creating Startup and Shutdown Files 54Running Startup and Shutdown Files 54

TuningOverview 55The Tune 6Printing Tune Information 56Experimental Record 56Saving and Restoring Parameter Settings 56Modifying the Peak Display 58Changing the Display 60

Customise Plot Appearance 60Trace 61Intensity 61Grid 61

AutoTune 62Ion Mode 63Scope Parameters 64Gas Controls 64Ramp Controls 64Resetting the Zero Level 65Controlling Readbacks 66Changing Tune Parameter Settings 67Source Voltages 67

Data AcquisitionStarting an Acquisition 69

Starting an Acquisition from the Tune Page 69Parameters 70

Multiple Samples 71Process 72Automated Analysis of Sample List 72

Monitoring an Acquisition 74The Acquisition Status Window 74Chromatogram Real-Time Update 74Spectrum Real-Time Update 74

Table of Contents


Instrument Data Thresholds 75MaxEnt 76Profile Data 76Centroid Data 76SIR Data 76Ion Counting Threshold 77Profile Data - Spike Removal 78Analog Data 79

System Manager 79Stopping an Acquisition 80The Function List Editor 80

Introduction 80The Function List Editor Toolbar 82Adding a New Function 82Modifying an Existing Function 83Copying an Existing Function 83Removing a Function 83Changing the Order of Functions 83Setting a Solvent Delay 84Analog Channels 84Saving and Restoring a Function List 85Setting up a Full Scan Function 86

Mass (m/z) 86Cone Voltage 86Method 86Scan Duration (secs) 87APcI Probe 87

Setting up a SIR Function 88Channels 88Method 89Retention Window 89

Setting up MS-MS Scanning Functions 90Mass 90Collision Energy 92

Setting up a MRM Function 93Setting up a Survey Function 93

Survey and MSMS Template Pages 94MS to MSMS Switching 95MSMS to MS Switching 97Including and Excluding Masses 98Monitoring Acquisitions 99

Mass CalibrationIntroduction 101Overview 102

Calibration Types 102The Calibration Process 103

Table of Contents


Electrospray 103Introduction 103Preparing for Calibration 104

Reference Compound Introduction 104Tuning 104Instrument Threshold Parameters 105

Calibration Options 106Selecting the Reference File 106Removing Current Calibrations 106

Selecting Parameters 107Automatic Calibration Check 107Calibration Parameters 108Mass Measure Parameters 109

Performing a Calibration 110Acquisition Parameters 112Starting the Calibration Process 114

Checking the Calibration 116Calibration Failure 118Incorrect Calibration 120Manual Editing of Peak Matching 121Saving the Calibration 121Verification 122

Electrospray Calibration with PEG 124Atmospheric Pressure Chemical Ionisation 125

Introduction 125Preparing for Calibration 126

Reference Compound Introduction 126Tuning 126

Calibration Options 126Selecting Reference File 126Removing Current Calibrations 126

Selecting Calibration Parameters 126Performing a Calibration 127

Static Calibration 127Scanning Calibration and Scan Speed Compensation 132

Calibration Failure 135Incorrect Calibration 136Manual Editing of Peak Matching 137Saving the Calibration 137Manual Verification 138

ElectrosprayIntroduction 141

Post-column Splitting 144Megaflow 145

Changing Between Flow Modes 145

Table of Contents


Operation 146Checking the ESI Probe 147Obtaining an Ion Beam 148

Tuning and Optimisation 148Megaflow Hints 154Removing the Probe 154

Sample Analysis and Calibration 155General Information 155

Typical ES Positive Ion Samples 156Typical ES Negative Ion Samples 156

Chromatographic Interfacing 157LC-MS Sensitivity Enhancement 158

Nanoflow ElectrosprayOverview 159Installing the Interface 160Operation of the Camera System 163Using the Microscope 163Glass Capillary Option 164

Restarting the Spray 165Nano-LC Option 166

Installation 166Operation 167

Changing Options 168

Atmospheric Pressure Chemical IonisationIntroduction 169Preparation 170

Checking the Probe 171Obtaining a Beam 172Calibration 173Hints for Sample Analysis 174

Tuning for General Qualitative Analysis 174Specific Tuning for Maximum Sensitivity 174

Corona Current 175Probe Position 175Probe Temperature 175Desolvation Gas 175

Removing the Probe 176

Maintenance and Fault FindingIntroduction 177Cooling Fans and Air Filters 177

Table of Contents


The Vacuum System 178Vacuum Leaks 179Pirani Gauge 179Active Inverted Magnetron Gauge 179Gas Ballasting and Rotary Pump Oil Recirculation 180Oil Mist Filter 181Foreline Trap 181Rotary Pump Oil 182

The Source 183Overview 183Cleaning the Cone Gas Nozzle and Sample Cone 184Removing and Cleaning the Ion Block 188Removing and Cleaning the Ion Tunnel Assembly 192Reassembling and Checking the Source 194The Discharge Pin 195

The Electrospray Probe 196Overview 196Replacement of the Stainless Steel Sample Capillary 198

The APcI Probe 200Cleaning the Probe Tip 200Replacing the Probe Tip Heater 201Replacing the Fused Silica Capillary 202

The Analyser 204The Detector 204Electronics 205

Fuses 205Analog PCB 205RF Power PCB 205Power Backplane #2 205Pumping Logic PCB 205Power Sequence PCB 205Rear Panel 205

Fault Finding Check List 206No Beam 206Unsteady or Low Intensity Beam 206Ripple 206High Noise Level in MRM Analyses 207

Chemical Noise 207Electronic Noise 208

High Back Pressure 208General Loss of Performance 209

Cleaning Materials 210Preventive Maintenance Check List 211

Weekly 211Monthly 211Three-Monthly 211Four-Monthly 211

Table of Contents


Reference InformationOverview 213Editing a Reference File 214Positive Ion 215

Horse Heart Myoglobin 216Polyethylene Glycol 216

PEG + NH4+ 216Sodium Iodide and Caesium Iodide Mixture 217Sodium Iodide and Rubidium Iodide Mixture 217

Negative Ion 218Horse Heart Myoglobin 218Mixture of Sugars 218Sodium Iodide and Caesium Iodide (or Rubidium Iodide) Mixture 219

Preparation of Calibration Solutions 220PEG + Ammonium Acetate for Positive Ion Electrospray and APcI 220PEG + Ammonium Acetate for Positive Ion Electrospray(Extended Mass Range) 220Sodium Iodide Solution for Positive Ion Electrospray 221

Method 1 221Method 2 221

Sodium Iodide Solution for Negative Ion Electrospray 221

Table of Contents


Hardware SpecificationsDimensions

WeightsInstrument: 150kg (330lb)

Data system(computer, monitor and printer): 60kg (130lb)

Rotary pumpsE2M28: 40kg (90lb)E1M18: 32kg (72lb)

Transformer (optional): 100kg (220lb)

Hardware Specifications


535mm

700mm

120mm(ventilation)

1325mm180mm

200mm(pumping line)

Lifting and Carrying

Warning: Persons with a medical condition, for example a back injury, whichprevents them from handling heavy loads should not attempt to lift theinstrument.

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 from the rear ofthe instrument.

Disconnect the API gas inlet and the exhaust lines from the rear of theinstrument.

Disconnect all connections to LC equipment.

If the instrument is to be moved over a large distance or in a confined space it isrecommended that any probes are removed from the API source.

The weight of the instrument is 150kg (330lb). Lifting equipment or suitably trainedpersonnel are required to lift or lower the instrument.

UK Health and Safety guidelines recommend that a minimum of six trained andsuitable personnel are required to lift a unit of this weight. The instrument should belifted from underneath the frame with one person at each corner of the instrumentsupporting the instrument in line with, or close to, the feet upon which the instrumentstands. Two further people should support the instrument centrally.

Caution: Under no circumstances should the instrument be lifted by the probe orthe 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 liftingthe instrument.



PowerInstrument: 230V (+10%, -14%), 13A

Data system: 100-120V or 200-240V, 13A

Pumps: 230V (+10%, -14%), 13A

EnvironmentAmbient temperature: 15-28°C (59-82°F)

Short term variance (1.5 hours): ≤2°C (≤4°F)

Overall heat dissipation(excluding LC

and optional water chiller): 4.2kW maximum

Humidity: Relative humidity ≤70%

Water CoolingHeat dissipation into the water: 200W

Exhausts

Rotary Pumps

The rotary pumps must be vented to atmosphere (external to the laboratory) via afume hood or industrial vent.

API Gas Exhaust

The API gas exhaust must be vented to atmosphere (external to the laboratory).

Caution: The API gas exhaust line must not be connected to the rotary pumpexhaust line. In the event of an instrument failure, rotary pump exhaust could beadmitted into the source chamber producing severe contamination.



NitrogenA supply of dry, oil-free nitrogen at 6-7 bar (90-100 psi) is required.

Caution: The lines supplying nitrogen to the instrument must be clean and dry.If plastic tubing is used it must be made of Teflon. The use of other types ofplastic leads to contamination of the instrument.

CID GasArgon is required as collision gas. The supplied gas should be dry, 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 results ina fault.



Instrument DescriptionOverview

The Micromass Quattro Ultima is a high performance benchtop triple quadrupole massspectrometer designed for routine LC-MS-MS operation. Quattro Ultima may becoupled to:

• a HPLC system with or without an autosampler.

• an infusion pump.

• a syringe pump.

Ionisation takes place in the source at atmospheric pressure. These ions are sampledthrough a series of orifices into the first quadrupole where they are filtered accordingto their mass to charge ratio (�).

The mass separated ions then pass into the hexapole collision cell where they eitherundergo collision induced decomposition (CID) or pass unhindered to the secondquadrupole. The fragment ions are then mass analysed by the second quadrupole.Finally the transmitted ions are detected by a conversion dynode, phosphor andphotomultiplier detection system. The output signal is amplified, digitised andpresented to the data system.

Instrument Description


Samplesfrom the liquidintroduction systemare introduced atatmospheric pressure into theionisation source.

Ions are sampled through a series of orifices.

The ions are filtered according to their mass to chargeratio ( ).

The mass separated ions undergo collision induced decomposition.

The fragment ions are filtered according to their mass to charge ratio.

The transmitted ions are detected by the photomultiplier detection system.

The signal is amplified, digitised and presented to the MassLynx NT™ data system.

�

MassLynx NTData System

Sample Inlet

Sampling Coneand Ion Block

Ion Tunnels

Prefilter 1

Quadrupole 1

Collision Cell

Prefilter 2

Quadrupole 2

Detector

Vacuum SystemVacuum is achieved using two direct drive rotary pumps, and two turbomolecularpumps.

The rotary pumps are mounted on the floor external to the instrument. The E1M18pumps the ion source block, while the E2M28 pumps the first ion tunnel and alsobacks the turbomolecular pumps. The E1M18 has an automatic gas ballast controlvalve mounted in the oil return line from the mist filter. This solenoid valve is openedwhenever the E1M18 is switched on, allowing continuous recirculation of the pumpoil provided that the manual gas ballast valve on the pump is left open.

The turbomolecular pumps evacuate the analyser and ion transfer region. These pumpsare both water cooled.

Vacuum measurement is by an active inverted magnetron (Penning) gauge for theanalyser and a Pirani gauge for the gas cell. The Penning gauge acts as a vacuumswitch, 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 fullyinterlocked to provide adequate protection in the event of a fault in the vacuumsystem, a failure of the power supply or vacuum leaks.



Ionisation TechniquesTwo atmospheric pressure ionisation techniques are available.

Atmospheric Pressure Chemical Ionisation

Atmospheric pressure chemical ionisation (APcI) generally produces protonated ordeprotonated molecular ions from the sample via a proton transfer (positive ions) orproton abstraction (negative ions) mechanism. The sample is vaporised in a heatednebuliser before emerging into a plasma consisting of solvent ions formed within theatmospheric source by a corona discharge. Proton transfer or abstraction then takesplace between the solvent ions and the sample. Eluent flows up to 2 ml/min can beaccommodated without splitting the flow.

Electrospray

Electrospray (ESI) ionisation takes place as a result of imparting a strong electricalfield to the eluent flow as it emerges from the nebuliser. producing an aerosol ofcharged droplets. These undergo a reduction in size by solvent evaporation until theyhave attained a sufficient charge density to allow sample ions to be ejected from thesurface of the droplet (“ion evaporation”).

A characteristic of ESI spectra is that ions may be singly or multiply charged. Sincethe mass spectrometer filters ions according to their mass-to-charge ratio, compoundsof 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 withelectrospray ionisation to split the flow such that 100 to 200 µl/min of eluent entersthe mass spectrometer.

Nanoflow Electrospray

The optional nanoflow interface allows electrospray ionisation to be performed in theflow rate range 5 to 1000 nanolitres per minute.

For a given sample concentration, the ion currents observed in nanoflow arecomparable to those seen in normal flow rate electrospray. Great sensitivity gains aretherefore observed when similar scan parameters are used, due to the great reductionsin sample consumption.

Sample InletSample is introduced from a suitable liquid pumping system along with the nebulisinggas 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 fusedsilica capillaries are used for flow injection analyses or for coupling to nano-HPLC.



MS Operating Modes

MS1 Collision Cell MS2

MS Resolving RF Only (Pass all masses)

MS2 RF Only (Pass all masses) Resolving

The MS1 mode, in which MS1 is used as the mass filter, is the most common andmost sensitive method of performing MS analysis. This is directly analogous to usinga single quadrupole mass spectrometer.

The MS2 mode of operation is used, with collision gas present, when switchingrapidly between MS and MS-MS operation. It also provides a useful tool forinstrument tuning and calibration prior to MS-MS analysis, and for fault diagnosis.

MS-MS Operating ModesThe basic features of the four common MS-MS scan functions are summarised below.

MS1Collision

CellMS2

Daughter IonSpectrum

Static(parent mass selection)

RF only(pass allmasses)

Scanning

Parent IonSpectrum

ScanningStatic

(daughter massselection)

Multiple ReactionMonitoring

Static(parent mass selection)

Static(daughter mass

selection)

Constant NeutralLoss Spectrum

Scanning (synchronisedwith MS2)

Scanning (synchronisedwith MS1)



Source MS1 Collision Cell MS2 Detector

The Daughter Ion 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 specificdaughter ion to be used in MRM analysis.

Example:

Daughters of the specific parent at� 609 from reserpine in electrospraypositive ion mode.

The result:



MS1static at m/z 609

(parent mass)

MS2scanning fromm/z 100 to 650

Collision CellRF only

(pass all masses)

The Parent Ion Spectrum

Typical application:

• Structural elucidation.

Complementary or confirmatory information (for daughter scan data).

Example:

Parents of the specific daughter ion at� 195 from reserpine in electrospraypositive ion mode.

The result:



MS1scanning fromm/z 50 to 650

MS2static at m/z 195(daughter mass)


(pass all masses)

MRM: Multiple Reaction Monitoring

This mode is the MS-MS equivalent of SIR (Selected Ion Recording). As both MS1and MS2 are static, this allows greater “dwell time” on the ions of interest andtherefore better sensitivity (~100×) compared to scanning MS-MS.

Typical application:

• Rapid screening of “dirty” samples for known analytes.

Drug metabolite and pharmacokinetic studiesEnvironmental, for example pesticide and herbicide analysis.Forensic or toxicology, for example screening for target drugs in sport.

Example:

Monitor the transition (specific fragmentation reaction)� 609 → 195 forreserpine in electrospray positive ion LC-MS-MS mode.

The result:

MRM does not produce a spectrum as only one transition is monitored. As inSIR, a chromatogram is produced.



MS1static at m/z 609

(parent mass)

MS2static at m/z 195(daughter mass)


(pass all masses)

LC-MRM�

�

High specificityGood signal / noise

LC-MS�

�

Low specificityPoor signal / noise

Time Time

The Constant Neutral Loss Spectrum

The loss of a specific neutral fragment or functional group from an unspecified parentor parents.

Typical applications:

• Screening mixtures, for example during neonatal screening, for a specific classof compound that is characterised by a common fragmentation pathway.

The scans of MS1 and MS2 are synchronised. When MS1 transmits a specificparent ion, MS2 “looks” to see if that parent loses a fragment of a certain mass.If it does it registers at the detector.

The result:

The “spectrum” shows the masses of all parents that actually lost a fragment of acertain mass.

Data SystemThe PC based data system, incorporating MassLynx NT™ software, controls the massspectrometer detector and, if applicable, the HPLC system, autosampler, syringepump, divert valve or injector valve.

The PC uses the Microsoft Windows NT graphical environment with colour graphicsand provides for full user interaction with either the keyboard or mouse.

MassLynx NT provides full control of the system including setting up and runningselected HPLC systems, tuning, acquiring data and data processing.

Analog inputs can be read by the data system so that, where applicable, a trace from aconventional LC detector (for example UV or fluorescence) can be storedsimultaneously with the acquired mass spectral data. A further option is the ability toacquire UV photodiode array detector data.

Comprehensive information detailing the operation of MassLynx NT is contained inthe MassLynx NT User’s Guide.



MS1scanning

MS2scanning


(pass all masses)

Front Panel Connections

Desolvation Gas and Probe Nebuliser Gas

The PTFE gas lines for the desolvation gas and probe nebuliser gas are connected tothe front of the instrument using threaded metal fittings.

Capillary / Corona

The electrical connection for the ESI capillary or the APcI discharge pin is via thecoaxial high voltage connector.

ESI / APcI

The electrical connection for the APcI probe or the ESI heater is via the multi-wayconnector. This is removed from the front panel by pulling on the metal sleeve of theplug. Both the electrospray and APcI heaters use this connector.



CAPILLARY / CORONA

C.I.D. GAS

STANDBY

DESOLVATIONGAS

NEBULISER

CONE GAS

INJECT

INJECTORLOAD

OPERATE

VACUUM

ESI / APci

LC Connection

DesolvationGas

NebuliserGas

SourceConnection

High VoltageConnection

Front Panel Controls and Indicators

Status Display

The display on the front panel of the instrument consists of two 3-colour light emittingdiodes (LEDs).

The display generated by the Vacuum LED is dependent on the vacuum status of theinstrument. The Operate LED depends on both the vacuum status and whether theOperate 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 Steady amber Pump fault Flashing red

Operate LED

State Operate LED State Operate LED

Standby No indicationTransient pressure

tripSteady amber

Operate Steady green RF trip Flashing red



CAPILLARY / CORONA

C.I.D. GAS

STANDBY

DESOLVATIONGAS

NEBULISER

CONE GAS

INJECT

INJECTORLOAD

OPERATE

VACUUM

ESI / APci

DesolvationGas Control

CID GasControl

Cone GasControl

NebuliserGas Control

Flow Control Valves

The Desolvation Gas, Cone Gas and Nebuliser needle valves are five-turnvalves. The CID Gas valve is a fifteen-turn valve. The flow increases as the valve isturned counterclockwise.

Caution: To prevent damage to the CID Gas valve, take care not toover-tighten when turning the supply off.

Divert / Injection Valve

The optional divert / injection valve may be used in several ways depending on theplumbing arrangement:

• As an injection valve, with the needle port and sample 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 syringepump containing calibrant.

This valve is pneumatically operated, using the instrument’s nitrogen supply.

Note that the valve is connected such that the nitrogen supply is alwaysconnected to the valve, irrespective of the flow to the source and probe.

Control of the valve is primarily from the data system. The two switches markedLoad and Inject enable the user to override control of the valve when making loopinjections at the instrument.



Divert / InjectionValve

Load

Inject

Rear Panel Connections

Event Out

Four outputs, Out 1 to Out 4, are provided to allow various peripherals to beconnected to the instrument. Switches S1 to S4 allow each output to be set to beeither 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. Thevoltage output of both Out 3 and Out 4 is 24 volts.

During a sample run Out 1 closes between acquisitions, and is used typically toenable an external device to inject the next sample. The three remaining outputs arereserved for future developments.

Contact Closure In

In 1 and In 2 inputs are provided to allow external device to start sample acquisitiononce the device has performed its function (typically sample injection).

Analog Channels

Four analog channel inputs are available, for acquiring simultaneous data such as aUV detector output. The input differential voltage must not exceed one volt, thoughfull scale automatically adjusts from 1mV to 1V.



REFER TOMANUAL BEFORECONNECTING TO

THESE PORTS

CONTACTCLOSURE

IN

SCOPE

MUX

DATA SYSTEM

X

Y

ANALOG CHANNELS EVENT OUT

S4S3S2S1

OUT 4OUT 3OUT 2OUT 1

IN 2IN 1

CH1

!

1VCH21V

+ - + -

CH41V

CH31V

CAUTION !

MUX

This 6-way connector connects the instrument to the MUX control base.

Data System

This RJ45 connector connects the instrument to the data system using the networkcable supplied.



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 Inpush-in connector using 6mm PTFE tubing. If necessary this tubing can be connectedto ¼ inch tubing using standard ¼ inch fittings.

Caution: Use only PTFE tubing or clean metal tubing to connect between thenitrogen supply and the instrument. The use of other types of plastic tubingresults in chemical contamination of the source.



ESDEARTH

FACILITY

OUTLET INLET

10 AMP (T)

10 AMP (T)

NITROGEN GAS

WATER

CIDGAS

ROTARY PUMPCONTROL

ROTARY PUMPCONTROL

OUT IN

1

0

MainsSwitch

ElectrostaticDischarge

Earth (Ground)PointPower

Cord

Fuses

SourcePumping Line

BackingLine

To Rotary Pumps

HexapolePumping Line

Rotary PumpControl

NitrogenGas In

CID Gas

WaterConnections

ExhaustGas

Exhausts

The exhaust from the rotary pump should be vented to atmosphere outside thelaboratory.

The gas exhaust, which also contains solvent vapours, should be vented via a separatefume hood, industrial vent or cold trap.

The gas exhaust should be connected using 10mm plastic tubing connected tothe push-in fitting.

Caution: Do not connect these two exhaust lines together as, in the event of aninstrument failure, rotary pump exhaust could be admitted into the sourcechamber 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 standardplug. 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

Refer to Maintenance and Fault Finding for details of rear panel fuses, and all otherinstrument fuses.

Rotary Control

Mains power to the two rotary pumps is controlled by the data system using one ofthese two sockets. The other socket is connected to the solenoid valve situated in theoil return tube on the E1M18 pump.

ESD Earth Facility

A suitable wrist band should be connected to this point when handling sensitiveelectronic components, to prevent damage by electrostatic discharge.



Internal Layout

Electronics



AnalyserTurbomolecular

PumpRF Generators

TurbomolecularPumps

Power Supply

High VoltagePower Supplies (4)

Low VoltagePower

Supplies (2)

PumpingLogic PCB

Transputer ProcessorCard (TPC)

Analogue PCB

Control PCB

Scan Control PCB

RF Control (Upper) PCB

RF Control (Lower) PCB

PowerSequence

PCB

The main electronics modules of the system are:

• Two low voltage power supplies.

• Four high voltage power supplies, plugged into the backplane below the analyserhousing.

These supply the detector system and the high voltages for the source andelectrospray 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 valvesand the solenoids. It also controls the phosphor and dynode voltages.

• Power Sequence PCB.

This PCB examines the vacuum, operate and interlock signals in order tocontrol the switching of various supplies. Also on this PCB is a moduledelivering the photomultiplier voltage.

• Transputer Processor Card (TPC).

This contains the transputer array and controls data acquisition and controlfunctions, as well as interfacing to the PC.

• Analog PCB

This PCB controls the source heater and focussing voltages.

• Control PCB

This supplies various lens voltages to the source and first hexapole.

• Scan Control PCB

This PCB produces control signals for mass, resolution, function energy,collision energy and pre-filter energy.

• RF Generator Control (Upper) PCB

This controls the RF and DC voltages applied to the first quadrupole. It alsosupplies the collision cell voltages.

• RF Generator Control (Lower) PCB

This controls the RF and DC voltages applied to the second quadrupole.



Mechanical Components

The main internal mechanical components of the instrument are:

• The source housing, inside which are the ion tunnels.

The ion tunnels are sometimes referred to as the “RF lenses”.

• 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 abovehousings.

• The active inverted magnetron (Penning) gauge and the Pirani gauge, bothclamped to the underside of the analyser housing.

• The air filter, held in the louvered cover at the left side of the front of theinstrument.



AnalyserHousing Detector

SourceHousing

Air Filter(behind cover)

SourceTurbomolecular

Pump

Active InvertedMagnetron

(Penning) Gauge

PiraniGauge

Routine ProceduresStart Up Following a Complete Shutdown

Preparation

If the instrument has been unused for a lengthy period of time, proceed as follows:

Check the level of oil inthe rotary pump sightglass. Refill or replenish asnecessary as described inthe pump manufacturer’sliterature.

Connect a supply of dry,high purity nitrogen to theconnector on the servicepanel at the rear of theinstrument. Adjust theoutlet pressure to 7 bar(100 psi).

Connect a supply of argonto 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.

Routine Procedures


GasBallast

DrainPlug

Exhaust

FillerPlug

Oil LevelIndicator

Oil MistFilter

ESDEARTH

FACILITY

OUTLET INLET

10 AMP (T)

10 AMP (T)

NITROGEN GAS

WATER

CIDGAS

ROTARY PUMPCONTROL

ROTARY PUMPCONTROL

OUT IN

1

0

NitrogenGas In

CID Gas

WaterConnections

Check that the rotary pump control box is connected to Rotary Control at therear of the instrument, and to the rotary pumps. Check that the solenoid valve onthe E1M18 rotary pump is connected to the other Rotary Control socket.

Check that the instrument, rotary pump control box, data system and otherperipheral devices (LC equipment, printer etc.) are connected to suitable mainssupplies.

Check that the data system is connected to the mass spectrometer via thenetwork 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 separate vent.

Caution: Do not connect the two exhaust lines together. In the event of aninstrument failure, rotary pump exhaust could be admitted into the sourcechamber, producing severe contamination.

Switch on the mains to the mass spectrometer using the switch situated on theservice panel at the rear of the instrument.

Routine Procedures


REFER TOMANUAL BEFORECONNECTING TO

THESE PORTS

CONTACTCLOSURE

IN

SCOPE

MUX

DATA SYSTEM

X

Y

ANALOG CHANNELS EVENT OUT

S4S3S2S1

OUT 4OUT 3OUT 2OUT 1

IN 2IN 1

CH1

!

1VCH21V

+ - + -

CH41V

CH31V

CAUTION !

ESDEARTH

FACILITY

OUTLET INLET

10 AMP (T)

10 AMP (T)

NITROGEN GAS

WATER

CIDGAS

ROTARY PUMPCONTROL

ROTARY PUMPCONTROL

OUT IN

1

0

MainsSwitch

Rotary PumpControl

Data SystemConnection

ExhaustGas

Switch on the data system.

As supplied Windows NT is automatically activated following the start-upsequence whenever the data system is switched on.

Windows NT and MassLynx NT can be configured to prevent unauthorisedaccess. Consult the system administrator for any passwords that may berequested.

When the data system has booted up, double-click on on the Windowsdesktop.

Launch the tune page by clicking on .

Routine Procedures


Pumping

Caution: To minimise wear to the lubricated components of the rotary pump, themanufacturers recommend that the pump is not started when the oil temperatureis below 12°C.

Pump down time may be decreasedby closing the isolation valve ofthe source during pump down.

Select Other from the menu bar atthe top of the tune page.

Click on Pump.

The rotary pump and theturbomolecular pumps startsimultaneously.

The Vacuum LED on the front ofthe instrument shows amber as thesystem pumps down.

When the system has reachedoperating vacuum the LED changes to a steady green, indicating that theinstrument 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 the E2M28 rotary pump oil has been changed or replenished, open the gasballast valve on this pump. See the pump manufacturer’s literature for details.

Rotary pumps are normally noticeably louder when running under gas ballast.

Caution: The instrument should not be vented while the E2M28 rotary pump isrunning under gas ballast. See Maintenance and Fault Finding for moreinformation.

If opened, close the gas ballast valve on the E2M28 rotary pump when the pumphas run under gas ballast for 30 minutes.

Routine Procedures


IsolationValve

Measuring the Analyser Pressure

The analyser pressure may be monitored via the active inverted magnetron (Penning)gauge.

Caution: To maximise the life of the gauge it is recommended that the gauge isswitched on only when the pressure needs to be monitored. Leaving the analyserpressure displayed for long periods necessitates frequent (every 2 to 4 months)cleaning of the Penning gauge.

This gauge operates by generating a high voltage discharge within the vacuumchamber. The magnitude of the discharge current is then measured and used tocalculate the analyser pressure. An undesirable characteristic of this type ofgauge is the slow build up of sputtered material in the discharge region,eventually leading to failure of the gauge.

To switch on the gauge:

Press or select Options, Pressure Monitor from the tune page.

There is a 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 file unless thepressure window is displayed prior to starting the acquisition. However, the gascell pressure (monitored by the gas cell Pirani gauge) is always recorded.

Using the Instrument

Quattro Ultima is now almost ready to use. To complete the start up procedure andprepare for running samples, follow the instructions in Start Up Following OvernightShutdown in the following pages.

Start Up Following Overnight ShutdownThe instrument will have been left in standby mode under vacuum.

It is recommended that the data system is left on overnight. However, if the datasystem 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 VacuumLED indicating that the instrument is ready for use.

Routine Procedures


Preparation for Electrospray Operation

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

Undo the three thumb screws and remove the probe adjustment flange andglass tube.

Disconnect the APcI high voltage cable from the socket positioned at thebottom right corner of the source flange.

Remove the corona discharge pin from its mounting contact, and fit theblanking plug.

Replace the glass tube and adjustment flange.

Ensure that the source enclosure (consisting of the glass tube and the probeadjustment flange) is in place.

Connect the source’s gas line to Desolvation Gas on the front panel. Tightenthe nut to ensure a good seal.

Check that the lead of the probe adjustment flange is plugged into the socketlabelled ESI / APcI on the front panel.

Routine Procedures


SourceThumb Nuts

ProbeThumb Nuts Probe

Adjustment Flange

GlassTube

Take the electrospray probe and connect its gas line to Nebuliser on the frontpanel.

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 theprobe firmly.

Plug the probe lead into Capillary / Corona on the front panel.

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 Ion Mode command.

Set Source Temp to 100°C and Desolvation Temp to 20°C.

Warning: Operating the source without the source enclosure resultsin solvent vapour escape and the exposure of hot surfaces and highvoltages.

Warning: The ion source block can be heated to temperatures of 150°C, and ismaintained at the set temperature when the source enclosure is removed.Touching the ion block when hot may cause burns to the operator.

Routine Procedures


BlankingPlug

CoronaDischarge

Pin

MountingContact

ExhaustLiner

High VoltageSocket

CleanableBaffle

Preparation for APcI Operation

If the corona discharge pin is not fitted, proceed as follows:

Disconnect the gas and electrical connections from the front panel.

Unscrew the probe thumb nuts and remove the probe.

Undo the three thumb screws and remove the probe adjustment flange andglass tube.

Remove the blanking plug from the discharge pin mounting contact and fitthe corona discharge pin, ensuring that the tip is in-line with the tip of thesample cone.Connect the APcI high voltage cable betweenCapillary / Corona and the socket positioned at the lower right edge ofthe source flange.

Replace the glass tube and adjustment flange.

Ensure that the source enclosure (consisting of the glass tube and the probeadjustment flange) is in place.

Connect the source’s gas line to Desolvation Gas on the front panel. Tightenthe nut to ensure a good seal.

Check that the lead of the probe adjustment flange is plugged into the socketlabelled ESI / APcI on the front panel.

Routine Procedures


SourceThumb Nuts


Adjustment Flange

GlassTube

Take the APcI 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 theprobe firmly.




Set Source Temp to 150°C.

Warning: Operating the source without the source enclosure resultsin solvent vapour escape and the exposure of hot surfaces and highvoltages. Allow the glass source enclosure to cool after a period ofoperation at high flow rates before removal.

Warning: The ion source block can be heated to temperatures of 150°C, and ismaintained at the set temperature when the source enclosure is removed.Touching the ion block when hot may cause burns to the operator.

The liquid flow should not be started until the gas flow and probe heater areswitched on with the probe inserted.

Routine Procedures


CoronaDischarge

Pin

MountingContact

SampleCone

High VoltageSocket

BlankingPlug

Operate




Depending on the chosen mode of ionisation, set Desolvation Temp orAPcI Probe Temp to 20°C.

Click on on the MassLynx tune page.

The instrument goes into the operate mode only if the probe adjustment flange isin place and a probe is inserted.

On the tune page, press or select Gas, Gas to turn on the source and probegases.

Set Desolvation Gas to a flow of 150 litres/hour and adjust Nebuliser tomaximum.

The system is now ready for operation. To obtain an ion beam refer to Obtaining anIon Beam in either the Electrospray or the Atmospheric Pressure Chemical Ionisationsection.

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 automated, a command from the data systemswitching 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 of10-3 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.

Routine Procedures


The Vacuum LED shows a steady amber.

Acquisition continues, although no mass spectral data are recorded.

When the pressure recovers, the voltages are restored and the Vacuum and OperateLED’s are steady green.

Any further deterioration of the system vacuum results in a pump fault and the systemis shut down.

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 andthe system is vented.

The 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 switch off when theirtemperature becomes too high.

• Vacuum leak.

Refer to “Maintenance and Fault Finding” later in this manual.

• Malfunction of the turbomolecular pumps.

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 thischapter.

Routine Procedures


TuningInformation concerning the tuning of Quattro Ultima is provided in Tuning later in thisdocument. Refer also to Electrospray, Nanoflow Electrospray and AtmosphericPressure Chemical Ionisation for tuning information specific to those techniques.

CalibrationInformation concerning the calibration of Quattro Ultima is provided in MassCalibration later in this document.

Data AcquisitionThe mechanics of the acquisition of sample data are comprehensively described inData Acquisition later in this document.

Data ProcessingThe processing of sample data is comprehensively described in the MassLynx NTUser’s Guide. Refer to that publication for full details.

Setting Up for MS-MS OperationThe following notes provide a worked example for the acquisition of daughter iondata. The experiment is performed in the ESI positive mode using reserpine as amodel analyte. Reserpine, admitted by constant infusion at a concentration of 50 pg/µl,provides a stable and persistent source of ions for instrument tuning in both the MSand MS-MS modes of operation.

The basic sequence of events is as follows:

• Tuning MS1.

• Tuning MS2.

• Parent ion selection.

• Fragmentation.

More detailed information on these processes can be found in Tuning and DataAcquisition later in this manual.

Parent Ion Selection

For maximum sensitivity in daughter ion analysis the centre of the parent ion selectedby MS1 must be accurately found. The nominal mass of the parent is first determined(if unknown) by viewing it in MS mode:

Routine Procedures


Set up a 1 box display on the tune page and set Function to MS Scan.Observe the candidate parent ion in the display and determine its nominal mass.

In this example the reserpine ion at� 609 is 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 set Function for peak 2 to Daughter Scan.

Place the mouse cursor on the Set mass for peak 2 and type in the nominal massof the parent ion selected by MS1, in this case 609.

Vary the Set value between 608.5 and 609.5 while optimising the intensity ofthe non-fragmented parent ion in the tune display.

The Set mass giving maximum intensity is used for future MS-MS experiments.

Fragmentation

Set up a wide range daughter ion scan by adjusting the Mass and Spanparameters for peak 2.

At this point, with the collision gas off, a few daughter ions of low intensity may bevisible. These are the products of unimolecular dissociations.

Argon (99.9% pure) is recommended as the collision gas.

Select Gas, Collision Gas.

Adjust CID Gas on the front panel to admit sufficient gas to attenuate theparent ion peak by about 50%.

Adjust the Entrance, Collision and Exit parameters in the Analyser menu toproduce the desired degree of fragmentation. (These parameters are interactive inMS-MS operation.)

In daughter ion analysis maximum transmission (sensitivity) can be achieved by thefollowing adjustments:

• 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 reducingHM Resolution 1 and increasing Ion Energy 1 on the Analyser window. In mostcases, where chemical interference with the parent ion is not acute, the loss ofspecificity is negligible.

Routine Procedures


Shutdown Procedures

Emergency Shutdown

In the event of having to shut down the instrument in an emergency, proceed asfollows:

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 exampleovernight or at weekends, proceed as follows:

Switch off the LC pumps.


Click on .

This changes from green to grey indicating that the instrument is no longer inoperate mode.

Undo the finger-tight connector on the probe to release the tubing leading fromthe LC system.

Before disconnecting the probe, it is good practice to temporarily remove theprobe and flush it of any salts, buffers or acids.

If APcI is being used, switch off the probe heater or reduce it to ambienttemperature.

Caution: Leaving the APcI probe hot with no gas or liquid flow shortens thelifetime of the probe heater.

Select Gas followed by Gas to turn off the supply of nitrogen gas.

If the instrument is not to be used for a long period of time:

Reduce Source Temp to 60°C.

Routine Procedures


Complete Shutdown

If the instrument is to be left unattended for extended periods, proceed as follows:



Click on on the tune page.

This changes from green to grey indicating that the instrument is no longer inoperate mode.

Undo the finger-tight connector on the probe to release the tubing leading fromthe LC system.

Before disconnecting the probe, it is good practice to temporarily remove theprobe and flush it of any salts, buffers or acids.

If APcI is being used, switch off the probe heater or reduce it to ambienttemperature.

Caution: Leaving the APcI probe hot with no gas or liquid flow shortens thelifetime 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 page. Click on Vent.

The rotary pump and the turbomolecular pumps switch off. When theturbomolecular pumps have run down to half of their normal operating speedthe 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 theinstrument.

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 manufacturer’s instructions.

Routine Procedures


Automatic Start up and ShutdownMassLynx comes with automatic startup and shutdown files which are run whenStartup or Shutdown is selected from the Run menu.

These are found in the C:\Masslynx\Shutdown directory and are calledShutDownxxx.acl and StartUpxxx.acl where xxx refers to the instrumentconfiguration.

The Shutdown Editor

The shutdown editor allows the automatic startup and shutdown procedures to bemodified or new procedures to be created. To access the editor:

Select Edit Shutdown from the MassLynx Run menu.

Check the Enable Startup before batch box to perform the startup taskswhen a sample list is submitted.

Check the Enable Shutdown after batch box to perform the shutdown tasksafter a batch of samples has completed.

Enter a time in the Shutdown XX.XX minutes after batch or error box atwhich to perform the shutdown tasks.

Routine Procedures


There is an option to perform the shutdown tasks immediately after an error occurs orafter the time defined in the Shutdown XX.XX minutes after batch or errorbox. If the Enable Shutdown on error box is checked then the shutdown tasks areperformed after the defined time.

The Enable immediate Shutdown on error box is grayed out if this optionis selected.

If the Enable immediate Shutdown on error box is checked then the shutdowntasks are performed as soon as the error is detected.

The Enable immediate Shutdown on error box is grayed out if this optionis selected, but the shutdown time can still be changed as this applies to theEnable Shutdown after batch option.

The Auto Control Tasks Page

Task is a dropdown list box with all the available tasks.

Pre Delay is the length of time that elapses before the current task is performed.

Post Delay is the length of time that elapses after the current task has beencompleted and before the next task is started. For example. a Post Delay of60 seconds, in the Vent Instrument task, means that there is a delay of 60 secondsbefore the next task is started, to allow the machine to vent fully.

Ion Mode is a dropdown list box with all the available ionisation modes.

Routine Procedures


File Name is selected from the browser displayed when is pressed, or is typed ingiving the full path name.

To add a task:

Select a task from the dropdown task list box.

Enter the required parameters.

Press .

If this is a new task timetable the task is added to the end of the list. If a taskhas been inserted into the task timetable then all subsequent tasks are addedafter the inserted task. To add a task to the end of the timetable after inserting atask, click twice with the left mouse button below the last entry in the timetableand then add the new task.

To insert a task:

Click on the entry in the task timetable before which the new task is to beinserted.

Select a task from the dropdown task list box.

Enter the required parameters.

Press .

To modify a task:

Click on the entry in the task timetable.

The details for the task are displayed in the fields on the left of the screen.

Change the required parameters.

Press .

To delete a task:

Click on the entry in the task timetable.

The details for the task are displayed in the fields on the left of the screen.

Press .

To delete all tasks:

Press .

Routine Procedures


To change the width of a column:

Position the mouse pointer on the heading between two columns untilappears.

Click the left mouse button and drag until the column is the required width.

The Shutdown Editor Toolbar

Toolbar Button Menu Equivalent Purpose

File… NewCreate a new startup or shutdownfile

File… OpenOpen an existing startup or shutdownfile

File… Saveor

File… Save AsSave a startup or shutdown file

File… Print Print a startup or shutdown file

Control List… Run List Run a startup or shutdown file

Control List… Stop List Stop a startup or shutdown file

Help… Help Topics Invoke help


Routine Procedures

Loading Startup and Shutdown Files

To open a startup or shutdown file:

Press or select Open from the File menu.

This displays the open file dialog.

Select 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 been selected, the Save Asdialog is displayed.

Type a name into the File Name box and press Save.

Routine Procedures


Printing Startup and Shutdown Files

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


Creating Startup and Shutdown Files

To create a startup or shutdown file

Press , or select New from the File menu.

Running Startup and Shutdown Files

If Startup or Shutdown is selected from the Run menu, or from the shutdowneditor 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 select Run Listfrom the shutdown editor control list menu.

To stop running this file:

Press , or select Stop List from the shutdown editor control list menu.

Routine Procedures


TuningOverview

Before sample data are acquired, the instrument should be tuned and, for the highestmass accuracy, calibrated using a suitable reference compound.

• Consult the relevant section of this manual for information concerning sourcetuning procedures in the chosen mode of operation.

• Adjust the tuning parameters in the Source and Analyser menus to optimisepeak shape and intensity at unit mass resolution.

• Care should be taken to optimise the value of the collision energy. Note that, inDaughter and Parent modes, Collision and Exit are interactiveparameters.

Tuning


The Tune PageTo display the tune page:

Press on the MassLynx screen MS panel.

Refer to the fold-out page at the end of this section for details of the tune page layout.

Printing Tune InformationTo print a report, containing a copy of the tune peak information displayed on thescreen along with a record of each parameter setting:

Press , or select Print from the tune page File menu.

This report is not configurable by the user.

Experimental RecordTuning parameters are stored with every data file as part of the experimental record.The tuning parameters for a particular data file can be viewed or printed from the databrowser, see the MassLynx NT User Guide, Selecting and Viewing Data, for moreinformation.

Saving and Restoring Parameter SettingsWhole sets of instrument tuning parameters can be saved to disk as a named file andthen recalled at a future date.

A tune parameter file contains the latest settings for the source controls for allsupported ionisation modes not just the ionisation mode currently selected. Tuneparameter files also contain settings for the analyser, inlet set points and peakdisplay.

To save the current tune parameters with the existing file name:

Press , or choose Save from the tune page File menu.

Press Save.

To save the current tune parameters with a new file name:

Select Save As from the tune page File menu.

Enter a new file name or select an existing file from the list displayed.

Press Save.

Tuning


If the selected file already exists on disk a warning is displayed. Press Yes tooverwrite the existing 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 its name or byselecting from the list displayed.

Press Open.

Tuning


Modifying the Peak DisplayThe tune peak display is modified using either the tune peak controls, or the mousedirectly on the display. To select peaks:

Press , or select Options, Peak Editor.

Choose the peaks to be displayed by checking the appropriate boxes.

For each active peak select the Mass, Span and Gain.

To change the function:

Select the function for the peak from the drop down list.

For MS-MS functions, Set is enabled allowing the mass of the parent, daughter,neutral loss or neutral gain ion to be entered.

To change the tune mass:

Click and drag the mouse within the bounds of the axis to draw a “rubber band”around the region of interest.

Release the button.

This range is redisplayed to fill the window. The mass displayed in the Massbox is the mass at the centre of the window.

This operation can be repeated as often as required.

Pressing once displays the previous magnification range and mass, pressingit a second time returns to the default settings.

or:

Enter a value in the Mass box for the required peak and press Return.

This becomes the default, so if the range is altered with the mouse and ispressed twice Mass returns to this value.

or:

Position the cursor at the top of the peak window, just below the line showingthe gain.

When appears, click the left mouse button and drag until the required massis displayed in the Mass box and at the top of the window.

This becomes the default, so if the range is altered with the mouse and ispressed twice Mass returns to this value.

Tuning


To change the span of a peak:

Press the left mouse button at one end of the region of interest and, withoutreleasing the button, drag the mouse horizontally to the other end.

As the mouse is dragged a “rubber band” stretches out to indicate the selectedrange.

Do not go beyond the bounds of the axis.

Release the mouse button to re-display the selected range filling the currentwindow.

This operation can be repeated as often as required.

Pressing once displays the previous magnification range, pressing it asecond time returns to the default settings.

or:

Enter a value in the Span box for the required peak and press Return.

This becomes the default, so if the range is altered with the mouse and ispressed twice Span returns to this value.

To change the gain of a peak

Double click on the line above the peak which shows the gain, to double thegain applied to that peak.

Double click below the peak to half the gain.

or:

Press the left mouse button at one end of the region of interest and, withoutreleasing the button, drag the mouse vertically to the other end.

As the mouse is dragged, a “rubber band” stretches out to indicate the selectedrange.

Do not go beyond the bounds of the axis.

Release the mouse button to re-display the selected range filling the currentwindow.

or:

Enter a value in the Gain box for the required peak and press Return.

Tuning


Changing the DisplayTo change the display using the mouse:

Click in the peak display area with the right mouse button todisplay the pop up menu.

The display area for each peak can be individually changed, e.g.the peak colour for peak 1 can be red and for peak 2 green etc.

Customise Plot Appearance

To change the colour of the background and traces and to change the number of tracesdisplayed:

Select Customise, Plot Appearance.

The Customise Plot Appearance dialog is displayed.

To change the colours on thedisplay:

Press Newest Trace,Background orTrace Fill and select anew colour from thedialog displayed.

To change the number oftraces:

Use to change thenumber, or enter a newvalue in theVisible Traces box,within the range 2 to 20.

If more than one trace is displayed then the older traces can be displayed in a differentshade to the new ones:

Drag the Colour Interpolation slider toward the full position. The colour ofthe old traces is shown in the Trace colour sample (new->old) field.

Tuning


Trace

From the pop-up menu:

Select the Trace, Outline option to display the peak outline only.

or:

Select the Trace, Fill option to fill the trace with the trace fill colour.

or:

Select the Trace, Min/Max option to show the minimum and maximum datapoints only.

The option selected 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 selected each have a tick next to them.

Grid

The options allow vertical and horizontal grid lines to be independently displayed orhidden.

Selected options have ticks next to them. Selecting an option a second timedeselects the option.

Tuning


AutoTuneMassLynx can automaticallytune the mass spectrometer inboth APcI and electrosprayionisation modes. AutoTuneramps the settings for thetuning parameters until theyare optimised to give the bestintensity, resolution and peakshape.

To run AutoTune:

Press on the tunepage to turn on the APIgas, and selectOperate.

Choose AutoTune from the tunepage Options menu.

Press Setup to define theAutoTune setup parameters.

There are two levels of AutoTune:

• A full AutoTune starts from adefault set of tuning parameters.

• A maintenance AutoTune starts from the current tuning parameters set in thetune page and can be quicker than a full AutoTune.

A maintenance AutoTune can only be performed if the instrument is alreadyreasonably well tuned. If the current tuning is too poor AutoTune gives an errorand request a full AutoTune.

The Tune Mass parameter sets the mass to be tuned on. When satisfied with theAutoTune setup parameters:

Press OK to exit.

Press Start.

The AutoTune status bar is updated to show the progress of AutoTune.

The following steps are performed:

• Parameter initialisation and instrument checks

Ensuring that essential status indicators read correctly.

Tuning


Checking that values are defined for all the user controllable instrumentparameters and that these are passed to the data system.

Checking that readbacks for these parameters are within specified tolerances.

• Beam detection

• Focus lens tuning

• Ion energy tuning

• High and low mass resolution tuning

The final four of these steps represent the implementation of the ESP/APcIAutoTune algorithm. This involves changing key parameters, one at a time, tomaximise the intensity of a reference peak with respect to that parameter. Atpresent ESP/APcI Autotuning is carried out with respect to a single userspecified reference peak.

When AutoTune has finished it displays a status dialog to say that AutoTune has beensuccessfully completed.

Press OK to return to the tune page.

The tuning parameters determined by AutoTune are saved to the current tuneparameter file.

Ion ModeSelect the required ionisation mode from the Ion Mode menu. The selected mode hasa tick next to it.

Tuning


Scope ParametersScan Time andInter Scan Delay control thespeed with which the tune peakdisplay is updated.

The tuning system behavesmore responsively if the scantime and inter scan delay areshort.

To change the scope parameters:

Press , or choose Scope Parameters from the tune page Options menu.

Make any required changes to the settings.

Press OK.

Gas ControlsTo turn a gas on or off:

Press or , or choose the required gas from the tune page Gas menu.

If the gas was previously turned off it is now turned on. A tick mark appearsnext to a gas if it is turned on.

Ramp ControlsTo set up a cone voltage ramp:

Choose Cone Ramp Gradientfrom the tune page Ramps menu.

Two values of cone voltage are definedat two particular masses. These valuesdefine a gradient for the cone voltagewhich is then extrapolated to cover thefull mass range.

Make any changes required andpress OK to exit.

To initiate the cone voltage ramp:

Press , or choose Use Cone Ramp from the tune page Ramps menu.

A tick mark appears next to the menu item if the cone voltage ramp is selected.

Tuning


To set up a collision energy ramp:

Choose Collision Energy Ramp Gradient from the tune page Ramps menu.

Two values of collision energy aredefined at two particular masses. Thesevalues define a gradient for the collisionenergy voltage which is thenextrapolated to cover the full massrange.

Make any changes required andpress OK to exit.

To initiate the collision energy voltageramp:

Press , or choose Use Collision Energy Ramp from the tune pageRamps menu.

Resetting the Zero LevelThe zero level (or baseline) can be repositioned by pressing , or by choosingReinitialize from the tune page Options menu.

This command causes the instrument control system to measure the position of thenoise signal so that any baseline offset caused by the electronics or instrumentationcan be compensated for.

It is advisable to reset the zero level whenever one of the multiplier voltages ischanged.

Tuning


Controlling ReadbacksThere are three options for displayingsystem readbacks on the tune page:

• Readbacks displayed continuously.

• Readbacks hidden.

• Readbacks displayed only whendiffering from their defined valuesby more than 10%.

A number of the readbacks are for diagnostic purposes only, their function beingto confirm a voltage is present. The acceptable variation between the set valueand the readback value varies depending on the particular tune parameter. Ifconcerned about any reading, contact your local service office for advice.

To change readback style:

Choose Readbacks from the tune page Options menu.

Select the readback style required.

Press OK.

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Data AcquisitionStarting an Acquisition

There are two ways of starting an acquisition:

• a single sample acquisition from the tune page

• a multiple sample one from the MassLynx top level screen.

Starting an Acquisition from the Tune Page

The easiest way to acquire data is directly from the tune page.

4Acquisitions can be started and stopped.

4Most 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 Acquire on the tune page, or choose Acquire from the tune pageWindow menu.

The dialogshown can beconfigured toautomaticallyset itself toacquire datausing the massrange andfunction typethat is beingused for tuningthe instrument.

Make anyrequiredchanges to thesettings.

Press Start.

Data Acquisition


Parameters

The Data File Name can be up to 128 characters. If the file already exists on disk, aprompt is given to rename the file or to overwrite the existing one. The file is writtento the data directory of the current project.

To change the directory into which data are acquired:

Cancel the acquisition.

Create a new project by choosing Project Wizard, or open an existing one bychoosing Open Project, from the MassLynx top level file menu.

The Text area is used to enter the sample description. The description can bedisplayed on any output of the acquired data and has a maximum length of 74characters. To display text on more than one line press CTRL+Return at the end of aline.

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 given in Function List Editor later in this chapter.

The Data Format that are collected and stored on disk can be any of the following:

• Centroid

• Continuum

• MCA.

More information is given on data formats later on in this chapter.

Set Mass specifies the mass (Daughter Mass, Parent Mass etc.) that is used for theparticular function type. This control is disabled if the function selected does notrequire a set mass.

Start Mass and End Mass specify the masses at which the scan starts and stops.Start Mass must be lower than End Mass.

Run Duration is the length of the acquisition, measured in minutes.

Data Acquisition


Scan Time specifies the duration of each scan in seconds.

Inter Scan Time specifies the time in seconds between a scan finishing and the nextone starting. During this period no data are stored.

Pressing Origin allows additional information about the sample to be analysed to beentered into the following fields:

• Submitter

• Job

• Task

• Conditions

Multiple Samples

The MassLynx top level screen contains a sample list editor for defining multiplesamples which may be used together to perform a quantitative analysis. The list ofsamples is set up using a spreadsheet style editor, which can be tailored to suitdifferent requirements.

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 run dialog.

Check the Acquire Sample Data, Auto Process Samples andAuto Quantify Samples boxes as required.

Enter values in the Run From Sample and To Sample boxes.

The default is all samples in the list.

Check the Priority and/or Night Time Process boxes as required.

See the “Getting Started” chapter of the MassLynx manual for details.

Press OK.

Repeat the above procedure as required.

Sample lists are added to a queue and run sequentially unless Priority orNight Time Process has been checked.

The sample which is currently being acquired has a next to it in the samplelist.

Data Acquisition


Process

The process controls allow processes to be run before and after the acquisition. ThePre-Run control is used to specify the name of a process that is run beforeacquisition of the files in the sample list.

The Post-Run control is used to specify the name of a process which is run afteracquisition of the files in the sample list. This could be used, for example, to switchthe instrument out of operate and to switch off various gases.

To run a process after each sample in the sample list has been acquired:

Format the sample list to display the Process column and enter the name of theprocess to be run for each of the samples.

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 theMassLynx Options dialog.

The MassLynx Options dialog is accessed by choosing Options from theMassLynx Tools menu.

Automated Analysis of Sample List

To display the quantify samples dialog:

Select Process Samples from the Quantify menu. Check the boxes requiredand press OK.

The Quantify Samples dialog allows automatic processing of data files once theyhave been acquired. To perform integration, calibration of standards, quantification ofsamples and printing of quantification reports select the relevant check boxes. SeeQuantify, MassLynx User Guide, for more detailed information about using automatedsample list analysis.

Data Acquisition


Integrate Samples integrates all the sample data files named in the peak list.

Calibrate Standards uses integration results to form quantify calibration curves.

Quantify Samples uses integration results and quantify calibration curves tocalculate compound concentrations.

Print Quantify Reports produces hard copies of the results of integration andquantification.

Export Results to LIMS produces a text file containing the quantification resultsdetails for use with LIMS systems. If this box is checked the LIMS Export Browsebutton becomes enabled. Press Browse, select a file or enter the name of a new oneand press Save.

The Project field displays the project into which data are acquired.

To change the project into which data are acquired, the acquisition should be canceledand a new project created by choosing Project Wizard, or an existing one opened bychoosing Open Project, from the MassLynx top level File menu.

From Sample and To Sample set the range of samples in the sample list which isanalysed.

Data Acquisition


Monitoring an AcquisitionAcquisition status is also shown on the MassLynx screen. The run time is shown onthe MS panel and the scan status, sample number and scan number are shown on theStatus bar at the bottom of the page.

The Acquisition Status Window

The acquisition status window, or scan report,provides a scan by scan statistical report of theprogress of an acquisition.

To display the scan report dialog:

Select Acquisition Status.

This shows details of the scan currently beingacquired.

Chromatogram Real-Time Update

To view in real time the chromatogram that iscurrently being acquired:

Open the data file using the MassLynxdata browser.

Press , or select Real-Time Update from the Display menu. Thechromatogram display is updated as the acquisition proceeds.

Spectrum Real-Time Update

To view in real time the spectrum thatis currently being acquired:

Open the data file using theMassLynx data browser.

Press , or selectReal-Time Update from theDisplay menu.

SelectEnable Real-Time update.Real-time update can also beturned on and off via the Real-Time spectrum toolbar button.

Data Acquisition


When real-time update is on the display is continually updated with spectra from thecurrent acquisition. The actual information displayed is determined by selecting one ofthe following radio buttons.

• Latest scan displays the last acquired scan. This is the default option.

• Average all scans updates the display with spectra formed by averaging allthe spectra that have so far been acquired.

• Average latest scans updates the display with spectra formed by averagingthe last n scans acquired, where n is specified in the associated edit control.

Instrument Data ThresholdsMassLynx has several parameters that allow control over how the systempre-processes data before it is sent to the host computer. These parameters arecontained in the instrument data thresholding dialog.

Instrument data thresholding allows the user to specify the type of data to acquire andwrite to disk, and the type of data to discard and not write to disk. Limiting theamount of data stored on disk can be particularly desirable when acquiring continuumdata and doing long LC runs.

To change data thresholding:

Choose Set Instrument Threshold from the tune page Options menu.

Make the required changes to the information.

Press OK.

These new parameters are downloaded at the start of the next acquisition scan.

Data Acquisition


MaxEnt

The MaxEnt algorithm needs to measure noise accurately within a data file. For thisreason Ion Counting Threshold should be set to zero when acquiring data to beanalysed using MaxEnt.

Profile Data

The controls for profile data allow control of the amount of data collected during acontinuum data acquisition.

Baseline Level is used to lift or drop the baseline to see more or less of the noise. Itis used when Ion Counting Thresholding is disabled (set to zero) to set theposition of the baseline above zero. The baseline level is typically set to a value of 0.

It is possible to use a negative baseline. This reduces the noise seen and acts as a formof thresholding to be applied to 1/16 amu type samples. This takes place after ioncounting and therefore has a less significant effect.

To see more noise use a positive value. Do not use a positive value for the baselinelevel if using ion counting thresholding.

Points per Dalton can have one of three values, 4, 8 or 16.

• Selecting 8 points instead of 16 results in data files approximately half as big.

• Acquiring data at 16 points per Dalton gives the greatest possible resolution.

• Acquiring data at 4 points per Dalton gives data with a smoothed appearance.

Centroid Data

Minimum centroid height sets a height below which detected peaks are ignored.This reduces the size of acquired data files and is useful when concentrating on largerpeaks of interest.

Minimum points per peak is the minimum number of points that a continuumpeak must have to be centroided.

SIR Data

SIR Baseline Level sets the position of the SIR baseline above zero whenIon Counting Threshold is not enabled (i.e. set to zero). The baseline level istypically set to 0. Increasing the value causes the baseline to appear higher.

Data Acquisition


Ion Counting Threshold

Ion Counting Threshold sets the intensity level below which a data point isignored. This threshold is applied to all acquisitions, regardless of scanning mode. It isalso the most significant of all of the data manipulation variables as it is applied to theraw data first.

When an acquisition is started the instrument performs a ‘prescan’ with the ion beamswitched off so that the electronic noise level of the acquisition system and itsstandard deviation can be measured.

The Ion Counting Threshold level entered is multiplied by the standard deviationof the noise to determine the intensity level to be used.

• Values can be set between 0 and 1000, the higher the number the more data isdiscarded.

• If a value of zero is entered the intensity level is set so that it sits in the middleof the noise which means that roughly half of the noise data is acquired.

• A value of 10 places the threshold just above the noise so almost all of the datais acquired.

• If a value of 60 is entered the threshold sits well above the noise level, so verylittle noise data is acquired.

• A value of 30 is suitable for most data. A value of zero disables the facility.

When using an Ion Counting Threshold other than zero, both Profile Data,Baseline Level and SIR Baseline Level should be set to zero.

Ion Counting Threshold should be set so that background noise is removedwithout significantly reducing the intensity of the smallest peaks of interest.

The following table shows the effects of changing baseline noise and ion countingthreshold on background noise and low intensity peaks.

Data Acquisition


BaselineLevel

IonC

ount

Threshold

Typical

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Noise

Typical

Peak

Profile

Typical Intensity

Reduction

Typical Saving

on.D

AT

FileSize

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 compared to their immediate neighbours, very intense. Data points determined tobe spikes are removed by setting the value of this data point to the average of itsimmediate neighbours.

Data Acquisition


Spike removal involves some additional processing while acquiring and reducesthe maximum achievable acquisition rates by approximately 30%.

To perform spike removal during an acquisition:

Check Use Spike Removal.

This is not reflected in the tune page.

Refer to the tune page intensities to assess a suitable value for the intensitythreshold below which spikes are ignored. Set Minimum Spike Intensity tothis value.

A very low intensity signal may include single ion events that can be combinedto produce significant peaks. For this type of data Minimum Spike Intensityshould be set to a suitable value such that these single ion events are notdiscarded 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 datapoint to its immediate neighbours. For example, withSpike Percentage Ratio set to 33%, a data point is regarded as a spike if itsintensity is 3 times (or more) greater than both its immediate neighbours. Asetting of 20% requires an intensity ratio of 5:1 to identify a spike.

Press OK to accept any changes.

Any changes are not downloaded if Cancel is pressed.

Analog Data

Select the number of samples to acquire per second from the drop down list.

System ManagerTo check the communications betweenthe MassLynx software and theembedded PC:

Select Communications Status.

Data Acquisition


Stopping an AcquisitionTo halt the acquisition:

From the tune page, press Stop.

From the MassLynx screen choose Stop from the Run menu, or press .

Data acquired up to this point is saved.

The Function List Editor

Introduction

The function list editor is used to set up the function(s) that the mass spectrometeruses to scan the instrument during an acquisition. A function list can be a mixture ofdifferent scanning techniques that can be arranged to run either sequentially orconcurrently during an acquisition.

Typical uses for mixed function acquisitions are to acquire different SIR groups overdifferent retention windows.

A function list is produced, saved on disk and then referenced by name when anacquisition is started.

Data Acquisition


A simple function list is shown above, containing only one function: a centroidedmode full scan, between 500 and 1500 amu using ES+ ionisation. Immediately abovethe function bar display is a time scale that shows from when the function is active,and for how long it runs. In this case the function starts after 5 minutes and then runsfor 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 running sequentiallyfor 5 minutes, is shown below.

The currently selected function is highlighted and enclosed in a rectangular frame. Ifthe display shows more than one function a new function can be selected either byclicking with the mouse, or by using the arrow keys on the keyboard.

Data Acquisition


The Function List Editor Toolbar

The toolbar is displayed at the top of the tune window and allows some commonoperations to be performed with a single click.

Create a new function list. Edit the selected function.

Open an existing function list. Delete the selected function.

Save the current function list todisk.

Move the selected function up thelist of functions.

Print the current window in portraitformat.

Move the selected function downthe 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 from theFunction menu.

MS2 and Neutral Gain do not have toolbar buttons and can only be created byselection from the Function menu.

The editor for the function type selected is displayed showing default values.

Make any changes required to the parameters and press OK to add the newfunction.

The function editors for each scan type is discussed in detail later on in thischapter.

Data Acquisition


Modifying an Existing Function

To modify an existing function:

Select the function in the function list.

Press , or double click on the function.

This displays the appropriate editor for the function type and allows changes tobe made.

The function list display is updated to show any changes.

Entering a new a value in Total Run Time and pressing sets the maximumretention time for the experiment. The ratio of the functions defined ismaintained. For example, if two functions are defined one from 0 to 5 minutesand the other 5 to 10 minutes then a Total Run Time of 10 minutes isdisplayed. If this value is changed to 20 then the first function now runs from 0to 10 minutes and the second from 10 to 20 minutes.

Copying an Existing Function

To copy an existing function:


Select Copy and then Paste from the Edit menu.

Modify the parameters as described above.

Removing a Function

To remove a function:


Press , choose Delete from the Edit menu, or press Del on the keyboard.

When asked to confirm the deletion, select Yes.

Changing the Order of Functions

Functions are displayed in ascending Start Time and End Time order and this ordercannot be changed. For functions that have the same start and end time the order inwhich they are performed can be changed as follows:

Highlight the required function.

Press or repeatedly until the function is in the required position.

Data Acquisition


Setting a Solvent Delay

No data is stored during thesolvent delay period, whichmeans that solvent peaks thatwould normally be seen elutingon the TIC chromatogram areno longer seen.

For APcI functions the APcIprobe temperature is set to thevalue specified in theAPcI Probe Temp control forthe period of the solvent delay.

To set a solvent delay for afunction list:

Select Solvent Delay from the Options menu.

Analog Channels

If an analog channels hardware option is fitted, up to 4 channels of analog data can beacquired, which are stored with the data acquired from the mass spectrometer. Analogchannels are typically used to collect data from external units such as UV detectors orFID detectors. A reading is made from the external channel at the end of each scanand stored with the data for that scan. The resolution of the chromatography for ananalog channel is therefore dependent on the scan speed used to acquire the massspectrometry data.

To access this dialog:

Select Analog Data from the Options menu on the Scan Functions dialog.

To store data for an analog channel:

Check the box(es) for the channel required.

Enter a textual description for each of the selected analog channels.

Data Acquisition


This description is used on the analog chromatogram dialog as the channeldescription. See “Chromatogram” in the MassLynx User’s Guide.

Enter an Offset to align the external unit with the mass spectrometer.

Press OK.

Saving and Restoring a Function List

To save a function list:

Choose Save As from the function list File menu.

Enter a new file name, or select an existing file from the list displayed.

Press Save.

If the file already exists on disk, confirmation is requested to overwrite the existinginformation.

Press Yes to overwrite the file, or No to select a different name.

When the editor is closed a prompt is issued to save any changed function lists.

To restore a saved function list:

Choose Open from the function list File menu.

Select the name of the function list to open, either by typing its name or byselecting it from the displayed list.

Press Open.

Data Acquisition


Setting up a Full Scan Function

The full scan function editor, activated by pressing or by selectingMS Scan from the Functions menu, is used to set up centroid, continuum andMCA functions.

Mass (m/z)

Start Mass and End Mass specify the masses at which the scan starts and stops.Start Mass must be lower than End Mass.

Start Time and End Time specify the retention time in minutes during which thisfunction becomes active, and data are acquired.

Cone Voltage

When Use Tune Page is checked, the cone voltage set on the tune page at the startof the acquisition is used.

The cone voltage value cannot be altered during acquisition by typing newvalues into the tune page, since the new values are not downloaded duringacquisition. This can only be done by acquiring from the tune page.

To apply a ramp to the cone voltage:

CheckUse Cone Voltage Ramp andpress CV Ramp to load thecone ramp dialog.

The four parameters define a gradientfor the cone voltage which is thenextrapolated to cover the full massrange of the function.

Method

Ionization Mode specifies theionization mode and polarity to be used during acquisition.

Data specifies the type of data to be collected and stored on disk. There are threeoptions:

• Centroid stores data as centroided, intensity and mass assigned peaks. Data arestored for every scan.

• Continuum. The signal received by the interface electronics is stored regularlyto give an analog intensity picture of the data being acquired. Data are notcentroided into peaks, but are stored for every scan.

Due to the fact that data are acquired to disk at all times, even when no peaksare being acquired, continuum data acquisition places some extra burden on the

Data Acquisition


acquisition system as compared to centroided acquisition. Data files tend to besignificantly larger than centroided ones and the absolute scanning speed(amu/sec) is slower.

It is possible, however, to set a threshold below which the data are not stored.Depending on the nature of the data acquired, this can greatly reduce theseeffects. The threshold can be set so that data considered to be ‘noise’ can bediscarded, thus improving data acquisition speed and reducing data file sizes. Formore information about setting instrument data thresholds see Setting InstrumentData Thresholds earlier in this chapter.

• Multi Channel Analysis (MCA). MCA data can be thought of as ‘summedcontinuum’, with only one intensity accumulated scan being stored for a givenexperiment. As each scan is acquired, its intensity data is added to theaccumulated summed data of previous scans.

An advantage of MCA is that random noise does not accumulate as rapidly asreal data and therefore effectively averages out over a number of scans. Thisemphasises 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 theend of an experiment, scanning speeds can be increased and significantly lessstorage space is required.

The disadvantage of MCA is that, as there is only one scan, it cannot be used fortime resolved data.

For MCA, Scans to Sum defines the number of scans to sum to create aspectrum.

Scan Duration (secs)

Scan Time specifies the duration of each scan in seconds while Inter-Scan Delayspecifies the time in seconds between a scan finishing and the next onestarting. During this period no data are stored.

APcI Probe

Probe Temp, in degrees centigrade, is enabled when Ionization Mode is set toAPcI.

When Use Tune Page Settings is selected the APcI probe temperature set on thetune page at the start of the acquisition is used. This control is enabled when theionization mode is set to APcI.

The APcI probe temperature value cannot be altered by typing new values into tunepage during the acquisition since the new values are not downloaded during theacquisition. This can be done by acquiring from the tune page.

Data Acquisition


Setting up a SIR Function

The SIR (Selected Ion Recording) technique is typically used in situations where onlya few specific masses are to be monitored. Since most of the data acquisition time isspent on these masses, the technique is far more sensitive than full scanning.

The SIR editor is used to enter the masses to be monitored, along with their dwelltimes, 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 those whichdiffer are described below.

Channels

Up to 32 masses can be monitored. To enter a mass:

Type suitable values into the Mass, Dwell and Cone boxes.

Press Add.

Dwell specifies the length of time in seconds for which the highlighted mass ismonitored.

To modify existing settings:

Data Acquisition


Double click on a mass in the list.

This displays the values for the selected mass in the edit fields.

Change Mass, Dwell or Cone as required.

Press Change to update the values in the list.

To sort the list in order of ascending mass:

Press Sort.

Method

Inter Channel Delay specifies the time in seconds between finishing monitoring thehighlighted mass and starting monitoring the next mass in the function.

Repeats is only relevant for experiments having more than one function and specifiesthe number of repeats of the function.

Span specifies a small mass window applied centrally about the highlighted mass.During acquisition this range is scanned over the specified Dwell time. A span of zerocan be set to simply ‘sit on’ the specified mass.

Retention Window

Start and End together specify the retention time in minutes during which thisfunction is active.

Data Acquisition


Setting up MS-MS Scanning Functions

Many of the fields in the MS-MS editors are similar to those in the full scan editor.Only fields which differ significantly are described below.

Mass

Daughter

This is the most commonly used MS-MS mode and is used to look at fragmentationsof a particular ion. MS1 is set to the parent mass using Daughters of, and is notscanned.

The resolution of MS1 can be lowered until the peak width at the base is twomasses wide without the daughter spectrum containing any ions from theadjacent parent masses.

Start and End specify the mass range to be scanned by MS2.

Data Acquisition


It is possible to select the daughter mass to be greater than the parent(precursor) mass. In this case ions which have gained mass in the collision cell,or are of higher mass to charge ratio, are detected. This can occur when amultiply charged ion fragments and loses a charge.

Parent

This mode is used to look for the parent of a particular fragment.

MS2 is set to the mass of the fragment, using Parents of, and is not scanned.

Start and End specify the mass range over which MS1 is scanned. Start is normallyset just below Parents of, and End to a value above the highest expected parentmass.

There are often several masses from which a daughter may come, so that anyone fragment is derived from a number of different 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 tunewindow, for setting and optimising the acquisition conditions.

Neutral Loss

In this mode, the peak in a spectrum that gives the neutral loss specified in Loss of isdetected. The precursor mass is scanned in MS1, and MS2 is scanned at this mass lessthe neutral loss mass. Starting masses are therefore detected on the mass scale of MS1.Start (for MS1) should be greater than Loss of to give MS2 a valid start mass.

Neutral Gain

This is an infrequently used mode, since the mass selected by MS2 is higher than thatof MS1. It is applicable to studies where a precursor ion gains mass by ion moleculereaction or where multiply charged ions fragment into particles with a higher�value.

Data Acquisition


Collision Energy

This specifies the collision energy in electron volts to be used for the collision cellduring the scan.

When Use Tune Page Settings is selected the collision energy set on the tunepage is used. If it is required to adjust the setting during an acquisition then theacquisition 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 ramp dialog.

The four parameters define values ofcollision energy for two particularmasses. This collision energy gradient isthen extrapolated to cover the full massrange of the function.

Data Acquisition


Setting up a MRM Function

Multiple reaction monitoring (MRM) functions are set up in much the same way asSIR functions, but allow a number of MS-MS transitions (fragmentations) betweenMS1 and MS2 to be monitored.

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 from the Functions menu in thescan functions editor.

The function list editor does not add survey functions to the list if non-surveyfunctions are present.

Data Acquisition


Survey and MSMS Template Pages

These pages allow the parameters to be set for MS and MS-MS scanning during thesurvey, and are similar to normal function editor pages.

Data Acquisition


MS to MSMS Switching

Switch Criteria

MSMS scanningcommences:

• If TIC isselected,and the TICof thespectrumrises abovethe specifiedThreshold.

• If Intensityis selected,and theintensity ofthe largestpeak risesabove thespecifiedThreshold.

When a peak topis found, no otherpeaks are lookedfor within thespecifiedDetection Window.

Currently Number of Components is set to 1 and can not be changed. Thenumber 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 Masses Only is selected only masses in the include list (see below) aremonitored.

If Include Masses and Automatic is selected masses on the include list are givenpriority. If no precursors are found then other valid masses are monitored.

A mass is valid if it is not on the exclude list (see below), and it satisfies theprecursor selection criteria.

Data Acquisition


Detected Precursor Inclusion

Auto exclude and Always include are not currently available.

Include after time, if selected, allows a delay to be incorporated before precursorsare included.

Data

Discard uninteresting survey scans allows only the survey scans that detectprecursor ions to be stored. This saves on disk space as survey scans which contain norelevant data are rejected.

Data Acquisition


MSMS to MS Switching

When MSMSfunctions havebeen generated,they are carriedout in paralleluntil theconditions forswitching to MSare satisfied.

When all MSMSfunctions havestopped, the MSsurvey function isagain carried out.

Switch Method

If the MSMS toMS switchmethod isDefault, theMSMS functionstops when theMSMS to MSswitch criteria aremet.

If the MSMS toMS switch method is After Time, the MSMS function stops when the MSMS to MSswitch criteria are met, or otherwise when the specified time has elapsed.

Switch Criteria

To define when MS scanning resumes:

Select one of the three conditions.

Set Threshold to a suitable value.

Data Acquisition


Including and Excluding Masses

Mass ranges andindividual massesto be included orexcluded from theMS-MS scans areentered in therelevant Rangeboxes.

Masses on theExclude list arenot considered fordetection.

Ranges take theformmassX_massY.

Masses andranges in a list arecomma delimited,for example100_200,202,236,250_300.

Data Acquisition


Monitoring Acquisitions

When an acquisition isstarted the automaticswitching status dialog isdisplayed showing theprecursors currently running.

Data Acquisition


Data Acquisition


Mass Calibration

IntroductionThis chapter of the manual is divided into three sections:

• A brief general overview of the calibration process.

• A complete mass calibration of Quattro Ultima using electrospray ionisationwith a mixture of sodium iodide and rubidium iodide as the reference compound.

• A complete mass calibration of Quattro Ultima using atmospheric pressurechemical ionisation (APcI) with PEG as the reference compound.

See Reference Information for details of calibration solutions and their preparation.

Mass Calibration


OverviewMassLynx NT allows a fully automated mass calibration to be performed, whichcovers the instrument for static and scanning modes of acquisition over a variety ofmass ranges and scanning speeds.

A mass spectrum of a reference compound (a calibration file) is acquired and matchedagainst a table of the expected masses of the peaks in the reference compound whichare stored as a reference file. The mass differences between the reference peaks andcalibration peaks are the calibration points. A calibration curve is fitted through thecalibration points.

The vertical distance of each calibration point from the curve is calculated. Thisdistance represents the remaining (or residual) mass difference after calibration.

The standard deviation of the residuals is also calculated. This number is the bestsingle indication of the accuracy of the calibration.

Calibration Types

Each quadrupole analyser requires up to three calibration curves:

• A static calibration is used to ‘park’ the analyser accurately on a specific massof interest (in tuning and SIR for example).

• A scanning calibration enables peaks acquired in a scanning acquisition to bemass measured accurately.

• A scan speed compensation calibration compensates for ‘lag time’ in the systemwhen the instrument is scanned rapidly.

A separate mass spectrum of the reference compound is acquired for each selectedcalibration type.

Quattro Ultima requires these three calibrations for both MS1 and MS2, for amaximum of six calibration curves. The table below show which types of calibrationare necessary for particular types of experiment.

ExperimentCalibration Required

MS1 MS2

MS All -

SIR Static -

MSMS All All

MRM Static Static

Mass Calibration


The Calibration Process

• Tuning the instrument.

• Selecting the appropriate reference file for the reference sample to be used.

• Starting an automatic calibration.

• Checking the calibration report.

Electrospray

Introduction

When a calibration is completed it is possible to acquire data over any mass rangewithin 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’sfull mass range is achievable.

Mass Calibration


Preparing for Calibration

Reference Compound Introduction

The example given here describes an automatic calibration which requires referencecompound to be present for several minutes. The introduction of the referencecompound is best achieved using a large volume Rheodyne injector loop (50 or 100µl)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 µl/min of 50:50 acetonitrile:wateror 50:50 methanol:water through the injector into the source.

An injection of 50µl of reference solution then lasts 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 µl/min.

Tuning

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 MS1and MS2.

For a good peak distribution across the full mass range:

Check the intensity of some of the reference peaks above 1000 amu.

Check also the intensity of the peak at� 173.

Ensure that no peaks are saturated. If necessary, reduce Multiplier.

A cone voltage in the region of 90 is usually suitable.

Mass Calibration


Instrument Threshold Parameters

Before beginning the calibration procedure, some instrument parameters need to bechecked.

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 tobe set correctly:

From the instrument control panel select Instrument thenSet Instrument Threshold to display the instrument data thresholdingwindow.

In the Profile Data section select Compressed (Discard zero intensities)for Data Storage and 16 Points per Dalton.

Select to save the parameters.

Mass Calibration


Calibration Options

To access the calibration options:

Select Calibration then Calibrate Instrument... on the tune page.

Selecting the Reference File

To select the appropriate reference file:

Click on the arrow in the Reference File box and scroll through the files.

Select nairb.ref for a sodium iodide and rubidium iodide reference solution.

Removing Current Calibrations

Select the uncal.cal calibration file from the File, Load Calibration... menuoption.

Select Process, Delete all calibration... followed by File,Save Calibration.

This ensures that a file with no calibration is currently active on the instrumentand prevents any previously saved calibrations from being modified oroverwritten.

Mass Calibration


Selecting Parameters

A number of parameters needs to be set before a calibration is started. Defaultparameters are set when the software is initially loaded which usually give a suitablecalibration, but under some conditions these may need to be adjusted.

Automatic Calibration Check

This is accessed from Edit, AutoCal Check Parameters....

It is here that limits are setwhich the calibration must attainbefore the instrument issuccessfully calibrated. Twouser parameters can be set.

Missed Reference Peakssets the maximum number ofconsecutive peaks which are notmatched when comparing thereference spectrum and the acquired calibration spectrum. If this number is exceededthen the calibration fails. The default value for this parameter, 2, is suitable in mostcases.

Maximum Std Deviation is set to a default of 0.20. During calibration thedifference between the measured mass in the acquired calibration fileand the true mass in the reference file is taken for each pair of matchedpeaks. If the standard deviation of the set of mass differences exceedsthe set value then the calibration fails. Reducing the value of thestandard deviation gives a more stringent limit. Increasing the standarddeviation means that the requirement is easier to meet, but this mayallow incorrect peak matching. Values greater than 0.20 should not beused unless exceptional conditions are found.

Apply Span Correction should always be left on. This allows different mass rangesto be scanned, within the calibrated range, without affecting mass assignment.

Check Acquisition Calibration Ranges causes warning messages to be displayedif an attempt is made to acquire data outside of the calibrated range for mass and scanspeed. It is advisable to leave this on.

Mass Calibration


Calibration Parameters

These are accessed from Edit, Calibration Parameters....

The Peak Match parameters determine the limits within which the acquired datamust lie for the software to recognise the calibration masses and result in a successfulcalibration. The default values are shown.

Increasing thePeak window andInitial error gives agreater chance of incorrectpeak matching. All peaks inthe acquired spectrumbelow theIntensity threshold value(measured as a percentageof the most intense peak inthe spectrum) are not usedin the calibration procedure.

The Polynomial order ofthe curve has values from 1to 5 as the availableoptions:

A polynomial order of1 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 inparticular has widely spaced peaks (150 amu apart), and horse heart myoglobinis used to calibrate higher up the mass scale, so this is the recommendedpolynomial 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 massscale, with closely spaced reference peaks. This is suitable for calibrations withPEG which extend below 300 amu..

A fifth order fit rarely has any benefit over a fourth order fit.

Mass Calibration


Mass Measure Parameters

These are accessed through Edit,Mass Measure Parameters.... Ifcontinuum or MCA data are acquiredfor calibration then these parametersneed to be set before the calibration iscarried out. If centroided data are usedfor calibration then the mass measureparameters are not used.

With electrospray calibrations,particularly with sodium iodide whichhas some low intensity peaks at highermass, it is recommended that continuumor MCA data are acquired.

At high scan speeds instrumentresolution may decrease. Ensure that thecentroiding parameters are set to use thetop 80% (or lower, if appropriate) ofthe peak. Use Centoid top (%), notTop.

Mass Calibration


Performing a Calibration

Three types of calibration are available withMassLynx: static calibration, scanningcalibration and scan speed compensation.These are selected on the AutomaticCalibration dialog box (see below) which isaccessed by selecting Start... from theCalibrate dialog box.

It is recommended that all three types ofcalibration are performed so that any modeof data acquisition can be used and massranges and scan speeds can be changedwhilst maintaining correct mass assignment.However, it is possible to have anycombination of these calibrations:

• If only a static calibration is presentthen the instrument is calibrated foracquisitions where the quadrupoles areheld at a single mass as in SIR orMRM.

• If only a scanning calibration is present then the instrument is only correctlycalibrated for scanning acquisitions over the same mass range and at the samescan speed as those used for the calibration.

• If only a scan speed compensation is present (with no scanning calibrationhaving been performed) then the scan speed compensation is treated as ascanning calibration and the instrument is only correctly calibrated for scanningacquisitions over the same mass range and at the same scan speed as used forthe calibration.

For the scan speed compensation to be used correctly a scanning calibrationshould also be performed.

• If static and scanning calibrations are both present, then the instrument iscalibrated for acquisitions where the quadrupole is held at a single mass and forscanning acquisitions with a mass range which lies within the mass range of thescanning calibration providing that the same scan speed is used.

For example, if the instrument is calibrated from� 100 to 900 with a 2 secondscan (400 amu/sec) then data can be acquired from 100 - 500 amu with a 1second scan time (also 400 amu/sec) whilst maintaining correct massassignment. In this case the static calibration would be used to determine thestart mass of the acquisition and the scanning calibration would be used for massassignment and scan range.

Mass Calibration


• If scanning calibration and scan speed compensation are present then theinstrument is only calibrated for scanning acquisitions over the same mass rangeas that used for the calibration, but the scan speed can be changed provided thatit remains within the scan speeds used for the two calibrations. The mass rangeshould 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 beused providing that the mass range and scan speed are between the lower andupper 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 toStatic Calibration, Scanning Calibration andScan Speed Compensation. Check also the MS1 and MS2 boxes.

In the Process area of the dialog box check Acquire & Calibrate andPrint Report.

Mass Calibration


Acquisition Parameters

Selecting Acquisition Parameters... in the Automatic Calibration dialog box bringsforward a second box, shown below, where the mass ranges, scan speeds andacquisition mode are set. When this box is first accessed it contains default parametersrelevant to the chosen reference file. These default parameters show the limits of scanrange and scan speed for the currently selected instrument and calibration parameters.

The upper area contains theAcquisition Parameterswhere mass range, run time anddata type are set.

When the instrument is fullycalibrated any mass range or scanspeed is allowed within the upperand lower limits dictated by thecalibrations.

If the nairb.ref file is selected,Default gives the parametersshown above. The solutiondescribed in ReferenceInformation is suitable for usewith this reference file.

If compatible reference solutionsand reference files are used, thensimply selecting Default issufficient action - no parameters need be entered manually.

Run Duration sets the time spent acquiring data for each part of the calibration. Thetime set must allow a minimum of three scans to be acquired at the slowest scan speedused. If the run duration is too short then data are not acquired. The slowest scanspeed generally used is 100 amu/sec. With Scan From set to 20 amu and Scan Toset 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 durationmust be greater than 59.6 seconds (3 scans + 2 inter scan delays). A Run Durationof 1.00 minutes is suitable.

The lower area in the Calibration Acquisition Setup dialog box contains theScan Parameters.

When an instrument acquires data for a static calibration it examines thereference file to find the expected reference masses, and then acquires data overa small mass span around each peak’s expected position. Thus the acquired datado not contain continuous scans. Each spectrum comprises small regions ofacquired data around each peak, separated by regions where no data areacquired.

Mass Calibration


Static Span sets the size of this small region around each reference peak. A span of4.0 amu is typical.

Static Dwell determines how much time is spent acquiring data across the span. Avalue of 0.1 second is suitable.

Slow Scan Time determines the scan speed used for the scanning calibration. If botha scanning calibration and a scan speed compensation are to be performed then thescan speed should be set to approximately 100 amu/sec (a scan time of 19.8 secondsover a mass range of 20 to 2000 amu). If only a scanning calibration is to beperformed (without scan speed compensation) then the scan speed should be set at thesame 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.

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


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 thedata file names shown:

MS1 static calibration data file: STATMS1MS1 scanning calibration data file: SCNMS1

MS1 scan speed compensation data file: FASTMS1MS2 static calibration data file: STATMS2

MS2 scanning calibration data file: SCNMS2MS2 scan speed compensation data file: FASTMS2

Once all of the data have been acquired each data file is combined to give a singlespectrum 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 boxis open then a constantly updated status message for the calibration is displayed.

If, when the process is completed, the calibration statistics meet with the requirementsspecified by the selected calibration parameters then a successful calibration messageis displayed. A calibration report is then printed showing a calibration curve for eachof the calibration processes. An example of a calibration report is shown on thefollowing page.

Mass Calibration


Mass Calibration


___________________________________________________________________________________

___________________________________________________________________________________

500 1000 1500 2000 2500 3000 3500M/z-0.15

0.65

amu

MS1 Static Calibration 28 matches of 28 tested references. SD = 0.0465

500 1000 1500 2000 2500 3000 3500M/z-0.19

0.35

amu

MS1 Scanning Calibration 27 matches of 28 tested references. SD = 0.0459

500 1000 1500 2000 2500 3000 3500M/z0.42

1.10

amu

MS1 Scan Speed Compensation Calibration 26 matches of 28 tested references. SD = 0.0538

500 1000 1500 2000 2500 3000 3500M/z0.01

1.09

amu

MS2 Static Calibration 28 matches of 28 tested references. SD = 0.0606

500 1000 1500 2000 2500 3000 3500M/z-0.17

0.74

amu

MS2 Scanning Calibration 28 matches of 28 tested references. SD = 0.0832

500 1000 1500 2000 2500 3000 3500M/z0.00

1.15

amu

28 matches of 28 tested references. SD = 0.0748MS2 Scan Speed Calibration

Checking the Calibration

The calibration (successful or failed) can be viewed in more detail by selectingProcess, Calibration From File... from the Calibrate dialog box. The dialog boxwhich is then displayed (see below) allows the choice of calibration type for viewing.With the required calibration selected the correct calibration file is automaticallycalled up.

Click Browse.. to select the calibration data file (for example STATMS1,SCNMS1, FASTMS!, STATMS2 etc.).

Clicking on OK repeats the calibrationprocedure for that particular file and displaya calibration report on the screen. Thiscalibration report (opposite upper) containsfour displays:

the acquired spectrum

the reference spectrum

a plot of mass difference against mass (thecalibration curve)

a plot of residual against mass

An expanded region can be displayed(opposite lower) by clicking and draggingwith the left mouse button. In this way theless intense peaks in the spectrum can beexamined to check that the correct peaks have been matched. The peaks in theacquired spectrum which have been matched with a peak in the reference spectrum arehighlighted in a different colour.

Mass Calibration


Mass Calibration


Calibration Failure

If the calibration statistics do not meet the requirements then a message is displayeddescribing at what point and why the calibration failed. This message also states wherethe attempted calibration data can be viewed so that the exact cause of failure can bedetermined.

There are anumber of reasonsfor a calibration tofail:

• No peaks. Ifthe acquired calibration data file contains no peaks the calibration fails. Thismay 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 whichare not found exceeds the Missed Reference Peaks parameter set in theAutomatic Calibration Check, then the calibration fails. Peaks may be missed forthe following reasons:

The reference solution is running out so that the less intense peaks are notdetected.

Multiplier is too low so that the less intense peaks are not detected.

An incorrect ionisation mode is selected. Check that the data have beenacquired with Ion Mode set to ES+.

Note that it is possible to calibrate in negative ion mode electrospray usingthe naineg.ref reference file with a suitable reference solution.

Intensity threshold, set in the Calibration Parameters dialog box, is toohigh. Peaks are present in the acquired calibration file but are ignoredbecause they are below the threshold level.

Either Initial error or Peak window, set in the Calibration Parametersdialog box, is too small. The calibration peaks lie outside the limits set bythese parameters.

Maximum Std Deviation, set in the Automatic Calibration Check dialogbox, has been exceeded.

The wrong reference file has been selected. Check that the correct file(nairb.ref in this case) is selected in the Calibrate dialog box.

Mass Calibration


In the case of too many consecutive peaks missed:

Check the data in the on-screen calibration report to see if the missed peaks arepresent 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 calibrationthen the latter four are likely reasons.

Having taken the necessary action, proceed as follows:

If Intensity threshold, Initial error and Peak window are adjusted toobtain a successful calibration, check the on-screen calibration report to ensurethat the correct peaks have been matched.

With a very low threshold and wide ranges set for the initial error and peakwindow it may be possible to select the wrong peaks and get a “successful”calibration. This is particularly relevant for calibrations with PEG where theremay be peaks due to PEG+H+, PEG+NH4

+, PEG+Na+, and also doubly chargedspecies.

Select OK from the calibration report window to accept the new calibration, orselect Cancel to retain the previous calibration.

Mass Calibration


Incorrect Calibration

If the suggested calibration parameters are used, and providing that good calibrationdata have been acquired, then the instrument should be calibrated correctly. Howeverin some circumstances it is possible to meet the calibration criteria without matchingthe correct peaks. This situation is unusual, but it is always sensible to examine theon-screen calibration report to check that the correct peaks have been matched. Theseerrors may occur when the following parameters are set:

Intensity threshold set to 0

Initial error too high (>2.0)

Peak window too high (>1.5)

Maximum Std Deviation too high (>0.2).

If the acquired spectrum looks like the reference spectrum and all of the expectedpeaks are highlighted then the calibration is OK.

An alternative cause of incorrect calibration is from contamination or backgroundpeaks. If a contamination or background peak lies within one of the peak matchingwindows, and is more intense than the reference peak in that window, then the wrongpeak is selected. Under some conditions this may happen with PEG. There are twoways to counter this:

• If the reference peak is closer to the centre of the peak window then the peakwindow can be narrowed until the contamination peak is excluded. Take care toensure that no other reference peak is excluded.

• If the reference peak is not closer to the centre of the peak window, or if byreducing the window other reference peaks are excluded, then the calibration canbe edited manually.

Mass Calibration


Manual Editing of Peak Matching

If an incorrect peak has been matched in the calibration process, this peak can beexcluded manually from within the on-screen calibration report.

Using the mouse place the cursor over the peak in the acquired spectrum andclick 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 thesame 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 does 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 nameso that it can be recalled for future use.

The recalled calibration has the same constraints of mass range and scan speed. Theion energy and resolution settings used for the calibration acquisition are also recordedas these can have an effect on mass assignment.

Mass Calibration


Verification

Once a full instrument calibration is in placeit is not always necessary to repeat the fullcalibration procedure when the instrument isnext used. Instead a calibration verificationcan be performed. (There is no benefit inverifying each calibration individually,re-calibration is just as quick.)

If a scanning acquisition is to be made andthe calibration is to be checked:

Set up the instrument and access thecalibrate dialog box as though a fullcalibration is to be carried out.

Set all peak matching parameters to thevalues that were used for the calibration.

Bring up the Automatic Calibrationdialog box by selecting Start... on theCalibrate dialog box.

SelectScanning Calibrationand deselectStatic Calibration andScan Speed Compensation.

DeselectAcquire & Calibrate andselect Acquire & Verifyand Print Report.

Select either MS1 or MS2,depending on the type ofacquisition to beperformed.

SelectAcquisition Parametersto call up the CalibrationAcquisition Set-up dialogbox, as shown.

The parameters entered should be identical to the parameters originally used forthe calibration being verified.

Mass Calibration


Set Scan From, Scan To, Run Duration, Data Type, Scan Time andInter Scan Delay<$]helv_bold> to agree with the acquisitionparameters that are to be used for data acquisition.

With only the scanning calibration selected all of the other options in this dialogbox are unavailable.

Select OK to return to the previous dialog box and OK again to start theverification procedure.

A scanning acquisition is now performed. When the acquisition is complete the dataare 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 originalcalibration was performed. An example is shown below.

Unlike the original calibration procedure the instrument calibration is not changed andthe report that is printed is a verification report.

Mass Calibration


___________________________________________________________________________________

___________________________________________________________________________________

500 1000 1500 2000 2500 3000 3500M/z-0.05

0.13

amu

MS1 Scanning Verification 28 matches of 28 tested references. SD = 0.0336

Electrospray Calibration with PEG

Caution should be used when calibrating with PEG in electrospray mode due to thenumber of peaks which are produced. Although ammonium acetate is added to thePEG reference solution to produce [M+NH4]+ ions, under some conditions it is quiteusual to see [M+H]+, [M+Na]+ and doubly charged ions.

The spectrum shown below demonstrates how the PEG spectrum can be dominated bydoubly charged ions (in this case [M+2NH4]2+) if the wrong conditions are chosen. Inthis 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 toolow.

A low Cone voltage encourages the production of doubly charged ions. The voltageshould be at least 120V.

Doubly charged peaks can be identified because the 13C isotope peak is separated fromthe 12C isotope by only 0.5 Da/e. If the instrument is set to unit mass and data areacquired in continuum mode the doubly charged peaks appear broader as the isotopesare not resolved.

Mass Calibration



Introduction

This chapter describes a complete mass calibration of Quattro Ultima usingatmospheric pressure chemical ionisation. The procedures described should befollowed only after reading the previous chapter in this manual, describing theautomated calibration with electrospray ionisation.

Due to the high flow rates used with APcI, the residence time of an injection ofreference solution in the source is too short to allow a fully automated calibration, andthe 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 onpreparing the reference solution. See the following illustration for a typicalPEG + NH4

+ spectrum.

With PEG the possible calibration range is dependent upon the molecular weightdistribution of the PEGs used in the reference solution. For this example PEG gradesfrom PEG 200 to PEG 1000 are used.

Mass Calibration


Preparing for Calibration

Reference Compound Introduction

It is best to use a large volume injection loop (50µl) with a solvent delivery system setup to deliver 0.2 ml/min of 50:50 acetonitrile:water or methanol:water through theinjector and into the APcI source. An injection of 50µl of reference solution lasts forapproximately 15 seconds, allowing enough time to perform a slow scanningcalibration.

Tuning

Before beginning calibration:

Set Multiplier to 650V.

Adjust source and lens parameters to optimise peak intensity and shape.

Set the resolution and ion energy parameters for unit mass resolution on MS1and MS2.

When a full calibration is completed it is possible to acquire data over any massrange within the calibrated range. It is therefore sensible to calibrate over awide mass range and in this example the calibration 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 by clicking on the arrow in the referencefile 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 instrumentand prevents any previously saved calibrations from being modified oroverwritten.

Selecting Calibration Parameters

A number of parameters needs to be set before a calibration is started. Most of theseparameters can be set at the same value as for electrospray. However, aPolynomial order of 2 is recommended for the calibration Curve Fit.

Mass Calibration


Performing a Calibration

The three types of calibration (static, scanning and scan speed) must be carried out insingle steps.

Static Calibration

Access the Automatic Calibration dialog box by selecting Start... from theCalibrate page.

Check Static Calibration and MS1 in the Types area of the dialog box.

In the Process area of the dialog box, check Acquire & Calibrate.

Acquisition Parameters

Selecting Acquisition Parameters... brings forward the default mass ranges, scanspeeds and acquisition mode relevant to the pegh1000.ref reference file.

The upper area contains theAcquisition Parameterswhere mass range, run time anddata type are set. When theinstrument is fully calibrated anymass range or scan speed isallowed within the upper andlower limits dictated by thecalibrations. It is thereforesensible to calibrate over a widemass range. Since thepegh1000.ref reference file haspeaks from� 63 to� 987, itis possible to calibrate over thismass range which is sufficientfor the majority of applicationswith APcI. The followingexample shows a setup toachieve this.

Run Duration sets the time spent acquiring data for the static calibration. The timeset must allow chance to inject a volume of reference solution and acquire severalscans.

Data Type allows a choice of centroided, continuum or MCA data to be acquired.For APcI, while either continuum or centroided data may be used, Continuum isrecommended.

The lower area in the Calibration Acquisition Setup dialog box contains theScan Parameters.

Mass Calibration


When an instrument acquires data for a static calibration it first examines theselected reference file for the expected reference masses. It then acquires dataover a small mass span around the expected position of each peak. Thus theacquired data do not contain continuous scans, but each “spectrum” is made upof small regions of acquired data around each peak separated by blank regionswhere no data are acquired.

Static Span sets the size of this small region around each reference peak. A value of4.0 amu is typical.

Static Dwell determines how much time is spent acquiring data across the span. Avalue of 0.1 second is suitable.

Slow Scan Time and Fast Scan Time are not available when a static calibrationalone is selected.

Select OK from the Calibration Acquisition Setup to return to the AutomaticCalibration dialog box.

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 filename STAT. While data are being acquired:

Inject the reference solution.

Once the data have been acquired the instrument attempts to produce a staticcalibration automatically. The data file contains only a few scans of the referencecompound, the remaining scans being of background.

As the automatic calibration procedure combines all of the scans in the data file toproduce a calibration spectrum, the resulting spectrum may be too weak to give asuccessful calibration. Whether the calibration is successful or failed, it is wise tocheck the calibration manually.

Mass Calibration


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 peakfor the reference solution.

This can be achieved using Process, Combine Spectra and using the leftmouse button to drag across the peak. The start and end scans are displayed inthe combine spectra dialog box.

Return to the Calibrate dialog box. Accessthe manual calibration options, as shown,by selecting Process,Calibration From File....

Select Static calibration type and MS1.

In the lower area the data file STATMS1should be selected automatically. If this isnot the case the correct file can beselected by clicking on Browse....

Enter the start and end scans of thereference data in the From and To boxes.

Select OK to perform the calibration anddisplay 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 withthe left mouse button. In this way the less intense peaks in the spectrum can beexamined to check that the correct peaks have been matched. The peaks in theacquired spectrum which have been matched with a peak in the reference spectrum arehighlighted in a different colour.

Compare the acquired and reference spectra to ensure that the correct peaks havebeen matched.

Mass Calibration


If insufficient peaks have been matched, or the wrong peaks have been matched, referto the section on calibration failure later in this manual.

Mass Calibration


If the correct peaks have been matched then the report can be printed out:

Select Print, Print from the report display.

To accept the calibration:

Select OK from the calibration report.

Mass Calibration


________________________________________________________________________________________________________

Ca li b r a ti o n R 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

100 200 300 400 500 600 700 800 900M/z-0.03

0.04

amu

21 matches of 22 tested referencesData file SCNMS1 - Uncalibrated

459.57415.55239.39195.34

151.31

283.45503.57

591.60679.62 899.72855.69 943.73

Reference file PEGH1000107.07 195.12 283.18 371.23 459.28 547.33 635.39 723.44 811.49

Mass difference (Raw - Ref mass)

Residuals Mean residual = -8.315380e-11 ± 0.023084

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 slowestspeed required for scanning acquisitions and one at the fastest speed. Once these fileshave been acquired and used for calibration then data may be acquired anywherewithin the mass range at any scan speed between the values used for the two sets ofdata. These data do not have to be acquired through the calibration dialog box, theycan be acquired using the normal scan setup and then accessed from the calibrationdialog 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 andacquire several scans. With a solvent flow rate of 200 µl/min and a 50 µl loop inline, an injection of reference solution lasts approximately 15 seconds allowingat 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.

Mass Calibration


The recommended scan speed for the scan speed compensation is 1000 amu/sec. Thisis the maximum scan speed permissible when using thresholded continuum data.

Although continuum is recommended centroided data may be used. It is possibleto scan more quickly in centroided mode, but it is unlikely that a fasteracquisition 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, isvalid.

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 wayas 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 slowscanning acquisition, then the correct file can be selected by clicking onBrowse....

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 thescreen in a similar way to the static calibration.

Compare the acquired and reference spectra to ensure that the correct peaks havebeen matched.

Mass Calibration


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 seeCalibration 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 thatScan Speed Compensation is selected in the dialog box, and the fast scanning fileis used. Note that for the scan speed compensation the default file is FASTMS1. If analternative filename has been used then this must be selected using the data browser.

Once all three calibrations (static, scanning and scan speed compensation) have beencompleted then the instrument can be used for any mass range within the limits of thescanning 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 thatdata is acquired in MS2 mode instead of MS1.

Using the Instrument

Once all six calibrations (static, scanning and scan speed compensation, each for bothMS1 and MS2) have been completed then the instrument can be used for any massrange within the limits of the scanning calibrations and at any scan speed from 100 to1000 amu/sec.

Mass Calibration


Calibration Failure

When calibration is performed manually there is no warning message to show that thecalibration has not met the set criteria. This must be judged by viewing the on-screencalibration report and examining the matched peaks and statistics associated with thereport. There are a number of reasons for a calibration to fail:

• No peaks. If the acquired calibration data file contains no peaks the calibrationhas 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 whichare not found exceeds the limit set in the Automatic Calibration Checkparameters then the calibration has failed. Peaks may be missed for thefollowing reasons:

The reference solution is running out causing less intense peaks to not bedetected.

Multiplier is too low and less intense peaks are not detected.

The incorrect ionisation mode is selected. Check that the data has beenacquired with Ion Mode set to APcI+.

Intensity threshold, set in the Calibration Parameters dialog box, is toohigh. Peaks are present in the acquired calibration file but are ignoredbecause they are below the threshold level.

Either Initial error or Peak window, set in the Calibration Parametersdialog box, is too small. The calibration peaks lie outside the limits set bythese parameters.

Maximum Std Deviation (set in the Automatic Calibration Check dialogbox) 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.

Mass Calibration


In the case of too many consecutive peaks missed:

Check the on-screen calibration report to see if the missed peaks are present inthe 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 toobtain a successful calibration, check the on-screen calibration report to ensurethat the correct peaks have been matched.

With a very low threshold and wide ranges set for the initial error and peakwindow it may be possible to select the wrong peaks and get a “successful”calibration. This is particularly relevant for calibrations with PEG where theremay be peaks due to PEG+H+, PEG+NH4

+ and PEG+Na. This situation isunusual, but it is always wise to examine the on-screen calibration report tocheck that the correct peaks have been matched.

Select OK from the calibration report window to accept the new calibration, orselect Cancel to retain the previous calibration.

Incorrect Calibration

If the suggested calibration parameters are used and providing that good calibrationdata have been acquired, then the instrument normally calibrates correctly. However insome circumstances it is possible to meet the calibration criteria without matching thecorrect peaks.

This situation is unusual, but it is always wise to examine the on-screen calibrationreport to check that the correct peaks have been matched. These errors may occurwhen the following parameters are set:

Intensity threshold set to 0

Initial error too high (>2.0)

Peak window too high (>1.5)

Maximum Std Deviation too high (>0.2).

If the acquired spectrum looks like the reference spectrum and all of the expectedpeaks are highlighted then the calibration is OK.

Mass Calibration


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 isselected. Under some conditions this may happen with PEG. There are two ways tocounter this:

• If the reference peak is closer to the centre of the peak window then the peakwindow can be narrowed until the contamination peak is excluded. Take care toensure that no other reference peak is excluded.

• If the reference peak is not closer to the centre of the peak window, or if byreducing the window other reference peaks are excluded, then the calibration canbe edited manually.

Manual Editing of Peak Matching

If an incorrect peak has been matched in the calibration process, this peak can beexcluded manually from within the on-screen calibration report.

Using the mouse place the cursor over the peak in the acquired spectrum andclick 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 thesame 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 does 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 filenameso that it can be recalled for future use. For example, it is possible to save calibrationsfor use with different ionisation modes, so that when an ionisation source is switchedthe corresponding calibration is recalled.

The recalled calibration has the same constraints of mass range and scan speed. Theion energy and resolution settings used for the calibration acquisition are also recordedas these can have an effect on mass assignment.

Mass Calibration


Manual Verification

Once a full instrument calibration is in place it is not always necessary to repeat thefull calibration procedure when the instrument is next used. Instead a calibrationverification can be performed. (There is no benefit in verifying each calibrationindividually, 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 therequired mass range and at the requiredscan speed in the normal way.

Start the acquisition and inject thereference solution so that reference data isacquired.

Stop the acquisition.

Access the calibrate dialog box and set allpeak matching parameters to the samevalues that were used for the calibration.

Select Process,Verification from file... and checkScanning Calibration (see below).

Select Scanning Calibration and either MS1 or MS2 depending on the type ofdata acquired.

Clicking on Browse..., select the acquired file and enter the start and end scansof the reference data.

Select OK to verify the calibration.

Mass Calibration


A calibration curve is produced and displayed on the screen in a similar way to whenthe original calibration was performed. An example is shown above. When OK isselected from this report, unlike the original calibration procedure, the instrumentcalibration is not changed. As the verification procedure uses the same matchingparameters as the calibration procedure, it is possible to validate the current calibrationwithout re-calibrating the instrument.

The report can be printed out by selecting Print, Print from the verify report.

Mass Calibration


Mass Calibration


ElectrosprayIntroduction

The ESI interface consists of the standard Z-spray source fitted with an electrosprayprobe. See the following chapter for information concerning the optional nanoflowinterface.

Mobile phase from the LC column or infusion pump enters through the probe and ispneumatically converted to an electrostatically charged aerosol spray. The solvent isevaporated from the spray by means of the desolvation heater. The resulting analyteand 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 awide range of analytes from low molecular weight (less than 200 Da) polarcompounds to biopolymers larger than 100 kDa.

Generally, compounds of less than 1000 Da produce singly charged protonatedmolecules ([M+H]+) in positive ion mode. Likewise, these low molecular weightanalytes yield ([M-H]-) ions in negative ion mode, although this is dependent uponcompound structure.

High mass biopolymers, for example peptides, proteins and oligonucleotides, producea series of multiply charged ions. The acquired data can be transformed by the datasystem to give a molecular weight profile of the biopolymer.

Electrospray


ProbeExhaust

ExhaustLiner

TurbomolecularPumps

E2M28Rotary Pumps

E1M18

Restrictor

NebuliserGas

Cone Gas

Purge Gas

Sample

Analyser

DesolvationGas

SampleCone

IsolationValve

SourceEnclosure

IonTunnel 2

IonTunnel 1

CleanableBaffle

The source can be tuned to fragment ions within the ion tunnel 1 vacuum housing.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 theelectrospray source. This is continuous until the pump syringes empty and needto be refilled. Sample is introduced through the valve injection loop (usually 10or 20µl capacity) switching the sample plug into the mobile phase flow. Tuningand acquisition are carried out as the sample plug enters the source. (At a flowrate of 10 µl/min a 20µl injection lasts 2 minutes.)

• Reciprocating pump and injection valve.

A flow of mobile phase solvent passes through an injection valve to theelectrospray source. Sample injection and analysis procedure is the same as forthe syringe pump. The pump reservoirs are simply topped up for continuousoperation. The most suitable reciprocating pumps for this purpose are thosewhich are specified to deliver a flow between 1 µl/min and 1 ml/min. A constantflow at such rates is more important than the actual flow rate. The injectionvalve on reciprocating pumps may be replaced by an autosampler forunattended, overnight operation.

• Infusion pump.

The pump syringe is filled with sample in solution. The infusion pump thendelivers the contents of the syringe to the source at a constant flow rate. Thisarrangement allows optimisation and analysis while the sample flows to thesource at typically 5-30 µl/min. Further samples require the syringe to beremoved, washed, refilled with the next sample, and replumbed.

A 50:50 mixture of acetonitrile and water is a suitable mobile phase for the syringepump system and the reciprocating pump systems. This is appropriate for positive andnegative ion operation.

Positive ion operation may be enhanced by 0.1 to 1% formic acid in the samplesolution.

Negative ion operation may be enhanced by 0.1 to 1% ammonia in the samplesolution. Acid should not be added in this mode.

These additives should not be used for flow injection analysis (FIA) studies, toallow 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 befiltered, and stored under cover at all times.

Electrospray


It is wise periodically to check the flow rate from the solvent delivery system. Thiscan be carried out by filling a syringe barrel or a graduated glass capillary with theliquid emerging from the probe tip and timing a known volume, say 10µl. Once therate has been measured and set, a note should be made of the back pressure readout onthe pump as fluctuation of this reading can indicate problems with the solvent flow.

Electrospray


Post-column Splitting

Although the electrospray source can accommodate flow rates up to 1 ml/min, it isrecommended that the flow is split post-column to approximately 200 µl/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.

The split ratio is adjusted by increasing or decreasing the back pressure created in thewaste line, by changing either the length or the diameter of the waste tube. A UV cellmay also be incorporated in the waste line, avoiding the requirement for in-line, lowvolume “Z cells”. As the back pressure is varied, the flow rate at the probe tip shouldbe checked as described above.

These principles apply to splitting for both megaflow and normal flow electrospray.

Electrospray


To Wasteor

UV Cell

LCColumn

Megaflow

Megaflow electrospray enables flow rates from 200 µl/min to 1 ml/min to beaccommodated. This allows microbore (2.1mm) or 4.6mm diameter columns to beinterfaced without splitting.

Changing Between Flow Modes

When changing between megaflow and standard electrospray operation, it is essentialthat the correct tubing is used to connect the probe to the sample injector. Formegaflow operation 1/16“ o.d., 0.007" i.d. peek tubing, easily identified by its yellowstripe, is used. This replaces the standard fused silica tube, together with the PTFEsleeves.

Electrospray


Probe

PTFE Sleeve

PTFE Sleeve

Fused Silica Tube

1/16" o.d. 0.007" i.d. Peek Tube

Injector

Normal Flow Electrospray

Megaflow Electrospray

Operation

Ensure that the source is assembled as described in Maintenance and FaultFinding, and that the instrument is pumped down and prepared for electrosprayoperation as described in Routine Procedures.

Ensure that a supply of nitrogen has been connected to the gas inlet at the rear ofthe 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 whenoperating at low flow rates.

Electrospray


BlankingPlug

CoronaDischarge

Pin

MountingContact

ExhaustLiner

High VoltageSocket

CleanableBaffle

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 electrospray probe to Nebulising Gas on thefront panel. Secure with the nut provided.

With the probe removed from the source turn on the liquid flow at 10 µl/min andcheck that liquid flow is observed at the tip of the capillary.

To avoid unwanted capillary action effects, do not allow liquid to flow to theprobe for long periods without the nitrogen switched on.

Turn on the nitrogen by selecting Gas, Gas and fully open the Nebuliser gasflow control valve situated on the front panel.

Check that there is gas flow at the probe tip and ensure that there is nosignificant leakage of nitrogen elsewhere.

Adjust the probe tip to ensure complete nebulisation of the liquid.

There should be approximately 0.5 mm ofsample capillary protruding from thenebulising capillary.

The tip of the electrospray probe can influencethe intensity and stability of the ion beam. Adamaged or incorrectly adjusted probe tipleads to poor electrospray performance.

Using a magnifying glass ensure that bothinner and outer stainless steel capillaries arestraight and circular in cross-section.

Ensure that the inner stainless steel capillary iscoaxial to the outer capillary.

If the two capillaries are not coaxial, it ispossible to bend the outer capillary slightlyusing thumbnail pressure.

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.

Electrospray


0.5mmSample

Capillary

NebulisingCapillary

Obtaining an Ion Beam



Using the needle valve on the front panel, set the desolvation gas flow rate to300 litres/hour.

To monitor the flow rate, select the Source window on the tune page andobserve the Gas Flows readback window.

Turn on the liquid flow at 10 µl/min and set Desolvation Temp to 100°C.

Tuning and Optimisation

The following parameters, after initial tuning, should be optimised using a samplerepresentative of the analyte to be studied. It is usually found, with the exception ofthe sample cone voltage, that settings vary little from one analyte to another.

Probe Position

Electrospray


SidewaysProbe

Adjustment

In / OutProbe

Adjustment

The position of the probe is adjusted using the probe adjustment collar (in/out) and theadjustment knob (sideways) located to the left of the probe. The two screws can beadjusted singly or simultaneously to optimise the beam. The position for optimumsensitivity and stability for low flow rate work (10 µl/min) is shown.

Small improvements may be gained byvarying the position using the sample andsolvent system under investigation. Thefollowing information should be consideredwhen setting the probe position:

• 10mm of movement is provided in eachdirection, with 1.25mm of travel perrevolution of the probe positioningcontrols.

• At higher liquid flow rates the probe tipshould be positioned further away fromthe sample cone to achieve optimum stability and sensitivity. The position is lesscritical than at lower flow rates.

Nebuliser Gas

Optimum nebulisation for electrosprayperformance is achieved by fully openingthe Nebuliser flow control valve, which issituated on the instrument’s front panel.

Desolvation Gas

The desolvation gas is heated and deliveredas a coaxial sheath to the nebulised liquidspray by the desolvation nozzle.

The position of the desolvation nozzleheater is fixed relative to the probetip and requires no adjustment.

The Desolvation Gas flow rate isadjusted by the control value situated on the instrument’s front panel. The optimumDesolvation Temp and flow rate is dependent on mobile phase composition andflow rate. A guide to suitable settings is given below.

To monitor the flow rate, select the Source window on the tune page andobserve the Gas Flows readback window. The Desolvation Gas flow rateindicated on the tune page includes purge gas (if enabled).

Electrospray


DesolvationGas Control

NebuliserGas Control

Cone GasControl

8mm

4mmCone GasNozzle

ProbeTip

Solvent Flow Rateµl/min

Desolvation Temp°C

Desolvation Gas FlowRate

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

>50 350 to 400 500 to 750

Higher desolvation temperatures give increased sensitivity. However increasing thetemperature above the range suggested reduces beam stability. Increasing the gas flowrate higher than the quoted values leads to unnecessarily high nitrogen consumption.

Caution: Do not operate the desolvation heater for long periods of time withouta gas flow. To do so could damage the source.

Cone Gas

The cone gas reduces the intensity of solvent cluster ions and adduct ions. The conegas flow rate should be increased until solvent cluster ions and/or adduct ions arereduced as much as possible without diminishing the intensity of the ion of interest,normally (M+H)+.

Typical cone gas flow rates are in the range 100 to 300 litres per hour.

To monitor the flow rate,select the Source window onthe tune page and observe theGas Flows readbackwindow.

Purge Gas

The purge gas is not necessary formost electrospray applications. Itallows purging of the sourcevolume to remove excessivesolvent vapour.

Purge gas is enabled simply byremoving the blanking plug fromthe outlet situated within the sourceenclosure.

Purge gas flow rate is a constant fraction (30% ) of the total desolvation gas flow.

Electrospray


Purge GasOutlet (Plugged)

ConeGas

Restrictor

The restrictor is positioned on the opposite side of the source block to the samplecone. Screwing the restrictor into the source block restricts the pumping port andincreases the source block pressure.

The restrictor is factory adjusted for optimum sensitivity on low molecular weightsingly charged species. For higher molecular weight compounds (proteins andpeptides) there is usually an increase in sensitivity if the restrictor is wound furtherinto the source block. If the majority of samples to be analysed are higher molecularweight compounds it is recommended that the restrictor is re-optimised for thesecompounds. Continual adjustment and re-adjustment of the restrictor position is notrecommended.

Warning: The restrictor is held at sample cone potential and must not beadjusted with the instrument in the operate mode.

The source glass must be removed to gain access to the restrictor. It is recommendedthat the original position of the restrictor is noted and subsequent adjustment made inhalf turn increments to obtain the optimum position.

Electrospray


Source Temperature

100°C is typical for 50:50 CH3CN:H2O at solvent flow rates up to 50 µl/min. Highersource temperatures, up to 150°C, are necessary for solvents at higher flow rates andhigher water content.

Caution: The maximum operating temperature for the source heater is 150°C.Do not set Source Temp higher than 150°C.

Capillary Voltage

Capillary usually optimises at 3.0kV, although some samples may tune at valuesabove or below this, within the range 2.5 to 4.0kV for positive electrospray. Fornegative 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.

Sample Cone Voltage

A Cone setting between 50V and 100V produces ions for most samples, althoughsolvent ions prefer the lower end and proteins the higher end of this range. Wheneversample quantity and time permit, Cone should be optimised for maximum sensitivity.

Electrospray


RF Lens 1 Voltage

RF Lens 1 settings between 0 and 50V can be beneficial in removing solvent adductsfrom certain samples. Values of 50V and above can provide efficient fragmentation ofsample ions. As for the Cone, this parameter should be optimised for maximumsensitivity.

Aperture Voltage

This generally optimises at 0V.

RF Lens 2 Voltage

RF Lens 2 should normally be operated between 0.2 and 0.5V. A setting of 0.5V canbe particularly beneficial when using short interscan times and short dwell times (<100ms).

Low Mass Resolution and High Mass Resolution

Peak width is affected by the values of low mass resolution (LM Res) and high massresolution (HM Res). Both values should be set low (typically 5.0) at the outset oftuning and only increased for appropriate resolution after all other tuning parametershave been optimised. A value of 15 (arbitrary units) usually gives unit mass resolutionon a singly charged peak up to� 1600.

Ion Energy

The ion energy parameter usually optimises in the range 0V to 3V. It is recommendedthat the value is kept as low (or negative) as possible without reducing the heightintensity of the peak. This helps obtain optimum resolution.

If, in positive ion mode, an ion energy value below -1V can be used withoutreducing the peak intensity then source cleaning is recommended.

Electrospray


Megaflow Hints

With this high flow rate technique the setup procedure involves making the followingadjustments:

• increase Drying Gas flow to approximately 750 litres/hour.

• increase Desolvation Temp to 400°C.

• increase Source Temp to 150°C.

• move the probe further away from the sample cone.

When changing from electrospray to megaflow operation it is not necessary toadjust any source voltages.

Cluster ions are rarely observed with Z-spray. However solvent droplets may formwithin the source enclosure if the source and desolvation temperatures are too low.

Refer to the previous section on operating parameters for typical desolvation gas flowrates.

If the sample is contained within a ‘dirty matrix’ the probe may be moved away fromthe sample cone to extend time between source cleaning operations. This may incur asmall loss in sensitivity.

Warning: It is normal for the source enclosure, the glass tube and parts of theprobe mounting flange, to get hot during prolonged megaflow operation. Careshould be taken when handling source components during and immediately afteroperation.

The source enclosure runs cooler if purge gas is used.

Warning: For health and safety reasons always ensure the exhaust line is ventedoutside the building or to a fume hood.

Warning: Ensure that a plastic bottle is connected in the exhaust line to collectany condensed solvents.

Removing the Probe

To remove the probe from the source proceed as follows:

On the tune page deselect .

Switch off the liquid flow and disconnect from the probe.

Select Gas, Gas to turn off the nitrogen.

Disconnect the probe cable from the instrument.

Disconnect the nebulising gas supply from the instrument.

Electrospray


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’scapillary. A centrifuge can often be used to separate solid particles from the sampleliquid.

There is usually no benefit in using concentrations greater than 20 pmol/µl forbiopolymers or 10 ng/µl for low molecular weight compounds.

Higher concentrations do not usually improve analytical performance. Conversely, forbiopolymers, lower concentrations often yield better electrospray results. Higher levelsrequire more frequent source cleaning and risk blocking the transfer capillary.

Optimisation for low molecular weight compounds may usually be achieved using aconcentration of 1 ng/µl.

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 pulledaway 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 qualitativeanalysis of known standards. A good example of a high molecular weight sample ishorse heart myoglobin (molecular weight 16951.48) which produces a series ofmultiply charged ions that can be used to calibrate the� scale from 800-1600 ineither positive ion or negative ion mode.

Polyethylene glycol mixtures, for example 300/600/1000, are low molecular weightsamples suitable for calibrating the� scale from approximately 100 to 1200 inpositive 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 sodiumiodide and rubidium iodide) can be used for calibration.

Detailed information on data acquisition and processing can be found in theMassLynx NT User’s Guide. Detailed information on mass calibration can be found inMass Calibration later in this document.

Electrospray


Typical ES Positive Ion Samples

• Peptides and proteins.

• Small polar compounds.

• Drugs and their metabolites.

• 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).

• Oligonucleotides.

• Some saccharides and polysaccharides.

Electrospray


Chromatographic InterfacingElectrospray ionisation can be routinely interfaced to reversed phase and normal phasechromatographic separations. Depending on the LC pumping system, chromatographycolumn 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 µl/min. It issuggested that a syringe pump is used to deliver these constant low flow ratesthrough a capillary column. Alternatively, accurate pre-column splitting ofhigher 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 withcapillary columns. HPLC pump manufacturers’ recommendations should beheeded.

• 2.1mm diameter reversed phase columns are gaining popularity for manyseparations previously addressed by 4.6mm columns. Typically flow rates of200 µl/min are used, allowing direct coupling to the electrospray source. Theincreased sample flow rate requires increased source temperature and drying gasflow rate.

A UV detector may be placed in-line to the Quattro Ultima probe. However,ensure that the volume of the detector does not significantly reduce thechromatographic resolution. Whenever a UV detector is used, the analog outputmay be input to MassLynx NT for chromatographic processing.

• The interfacing of 4.6mm columns to the electrospray source can be achievedeither by flow splitting or by direct coupling. In both cases an elevated sourcetemperature and drying gas flow rate are required. In general, the best results areobtained by splitting after the column using a zero dead volume tee piece so that200-300 µl/min is transferred to the source.

Conventional reverse phase and normal phase solvent systems are appropriate forLC-electrospray.

Involatile buffers may be used but prolonged periods of operation are notrecommended. When using involatile buffers the probe should be moved as far awayfrom the sample cone as possible. This may reduce sensitivity slightly, but alsoreduces the rate at which involatile material is deposited on the sample cone.

Trifluoroacetic acid (TFA) and triethylamine (TEA) may be used up to a level of0.05%. If solvents of high aqueous content are to be used then tuning conditionsshould be appropriate for the solvent composition entering the source.

Higher source temperatures (150°C) are also recommended for high aqueous contentsolvents. Tetrahydrofuran (THF) should not be used with peek tubing.

Electrospray


LC-MS Sensitivity Enhancement

The sensitivity of a LC-MS analysis can be increased or optimised in a number ofways, 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 columnswith fully end capped packings. For target compound analysis, techniques such astrace enrichment, coupled column chromatography, or phase system switching canhave enormous benefits.

Similarly, the mass spectrometer sensitivity can often be significantly increased, forinstance by narrow mass scanning or by single ion recording (SIR) techniques.

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

Electrospray


Nanoflow ElectrosprayOverview

The optional nanoflow interface allows electrospray ionisation to be performed in theflow rate range 5 to 1000 nanolitres per minute. There are two options for the sprayingcapillary, which can be alternately fitted to the interface:

• Glass capillary.

Metal coated borosilicate glass capillaries allow the lowest flow rates to beobtained, 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 down to 100 nl/min. If a syringe pumpis to be used, a gas-tight syringe is necessary to obtain correct flow rateswithout leakage. A volume of 25µl is recommended.



Regulatorand Injector

(Nano-LC option)

ProtectiveCover

Handle

Stop

GlassCapillaryOption

Nano-LCOption

Stage

Three-axisManipulator

For a given sample concentration, the ion currents observed in nanoflow arecomparable to those seen in normal flow rate electrospray. Great sensitivity gains aretherefore observed when similar scan parameters are used, due to the great reductionsin sample consumption.

The nanoflow endflange consists of athree-axis manipulator,a stage, a protectivecover and a stop /handle arrangement forrotation of themanipulator and stage.

The manipulator andstage are rotated by90 degrees to changeoption or, in the glasscapillary option, toload a new nanovial.

Caution: Failureto use the stopand handle torotate the stagecan result in permanent damage to the three-axis manipulator.

Installing the InterfaceTo 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 flangeassembly and glass tube.

Place the glass tube, end on, on a flat surface and place the probe support flangeassembly on top of the glass tube.

Remove the PTFE encapsulated source O ring.

Warning: When the source enclosure has been removed the ion block heater isexposed. Ensure that the source block heater has been switched off and hascooled before proceeding. Observe the Source Temp readback on the tunepage.



Exhaust

ExhaustLiner

TurbomolecularPumps

E2M28Rotary Pumps

E1M18

Restrictor

Cone Gas

Analyser

SampleCone

IsolationValve

SourceEnclosure

IonTunnel 1

IonTunnel 2

CleanableBaffle

SampleCapillary

Unscrew the three probe flange mounting pillars, using the holes to obtain thenecessary leverage.

If the cone gas nozzle is notin place, remove the twoscrews that secure the samplecone and fit the cone gasnozzle.

Replace the two screws.

Connect the cone gas outlet tothe cone nozzle using thePTFE tubing provided.

Ensure that the purge gas isplugged (disabled).

Ensure that the cleanablebaffle, the exhaust liner andthe discharge pin blankingplug are fitted.

Fit a viton O ring and the three shorter nanoflow pillars.

Install the perspex cover and the nanoflow end flange, securing this with sockethead screws.



Cone GasNozzle

PTFETubing

PTFEEncapsulated

O Ring

PurgeGas Plug

Probe FlangeMounting Pillar

SourceThumb Nuts


Adjustment Flange

GlassTube

If not already in place, attach the microscope or camera brackets using the screwhole 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 frontpanel.

Attach the two cables to the sockets marked Capillary / Corona andESI / APcI on the front panel of the instrument.

Set Source Temp to approximately 80°C.



Operation of the Camera SystemMagnification is controlled by the zoom lens. A fine focus can be achieved by rotatingthe objective lens.

Using the MicroscopeFocusing is adjusted by rotating the top of the microscope.



Microscope

Camera

ZoomLens

ObjectiveLens

Grommet

Glass Capillary OptionWarning: Do not touch the sharp end of the capillary. As well as the risk ofinjury by a sliver of glass, the needle becomes inoperable.

Caution: The capillaries are extremely fragile and must be handled with greatcare. Always handle using the square end of the capillary.

With the stage rotated outwards, unscrew the union from the end of theassembly.

Carefully remove the capillary fromits case by lifting vertically whilepressing down on the foam with twofingers.

Over the blunt end of the capillary,pass the knurled nut, approximately5mm of conductive elastomer andfinally the union.

Tighten the nut (finger tight issufficient) so that 5mm of glasscapillary is protruding from the endof it. This distance is measured from the end of the nut to the shoulder of theglass capillary.

Load sample into the capillary using either afused silica syringe needle or a gel loader tip.

Screw the holder back into the assembly - fingertight is sufficient.

Ensure that Capillary is set to 0V on the tunepage.

Rotate the stage back into the interface using thestop and handle.

Manoeuvre the stage so that the microscope orcamera can view the capillary tip.

Using a 10ml plastic syringe or a regulated gassupply, apply pressure to the back of the tip untila drop of liquid is seen. Remove the backpressure.

On the tune page, select Gas, Gas to turn on the nitrogen.

Select .



Foam

Capillary

GlassCapillary

BlueConductiveElastomer

PTFE"Back Pressure"

Tubing

Ferrule

5mm

Knurled Nut

Union

Set Capillary between 1 and 1.5kV.

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-axismanipulator for maximum ion current.

The ion current may change dramatically with very slight changes of positionbut 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 manipulatorso that, viewed under magnification, the capillary tip touches the sample cone and asmall piece of the glass hair shears off.

It may also be necessary to apply some back pressure to the holder to force a drop ofliquid 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.



Nano-LC Option

Installation

With the sprayer assembly removedfrom the stage:

Cut approximately 25mm of thered stripe peek tubing and, usingthe plug cap and a Valco nut, set aferrule to the correct position onthe tubing.

At this stage the ferrule is requiredonly to grip the tubing lightly, andshould not be too tight.

Cut the peek such that 10mm ofthe peek protrudes from the backof the ferrule.

Thread approximately 70mm ofthe 90 micron o.d. fused silicathrough the new fitting.

Ensure that the fused silica is flushwith the peek sleeve.

Again using the plug cap, tightenthe nut further to ensure that thefused silica is gripped. Some forcemay be required to do this.

Remove the sleeved fused silicafrom the plug cap and remove theValco nut.

Place an O ring onto the peek tube,using tweezers if necessary.

The O ring is required to seal theregion between the ferrule and the end of the thread on the nano-LC chamber.

Thread the sleeved fused silica through the nano-LC chamber.

Rotate the microvolume union in the body such that the ferrule seat is alignedcorrectly.

Insert the chamber into the nano-LC body and tighten using a pair of spanners.



1mm

From Injector(or Column

Attached Directly)

Make-upFlowOnly

(3-wayInsert

Required)

NebuliserGas

Nano-LCBody

Chamber

NebulisingTip

Red StripePeek Tubing

O Ring

90µm FusedSilica

ValcoFerrule

MicrovolumeInsert

The capillary can now be checked for flow by connecting the output from aHarvard syringe pump to the other side of the union and setting the flow to1 µl/min, using a micropipette to measure the flow. It is recommended that asyringe with a volume of no more than 50 millilitres is used.

Thread the fused silica through the nebulising tip and screw in the nano-LCchamber such that it is screwed in approximately half way.

Cut the fused silica using a tile cutter and adjust the nebulising tip further, suchthat 1mm of fused silica protrudes from the tip.

Attach the nebulising gas tubing to the sprayer using an O ring and the specialscrew.

Attach the sprayer assembly to the stage.

It may be necessary to alter the position of the thumbscrew underneath thebaseplate to attach the sprayer correctly.

Swing the stage into the interface using the stop and handle.

Operation

For tuning purposes it may be useful to infuse a known sample in 95% water using aHarvard syringe pump.

Set the liquid flow to about 200 nl/min.

Switch on Gas at the MassLynx tune page.

Set the pressure of the gas on the regulator to approximately 0.5 bar (7 psi).

Ensure there are no leaks of gas at the sprayer, particularly where the PTFEtubing is connected to it.

By viewing under magnification, the spray emanating from the capillary may beexamined and tuned by altering the nebulising tip such that a fine spray is observed.Altering the gas slightly may also help in this tuning process.

Swing the stage back out of the source and place the cover over the sprayerensuring that the tubing coming from the sprayer is threaded correctly through it.

Lock the cover in place with two screws.

Swing the stage back into the source and alter the translation stage (in / outdirection) such that the capillary is approximately 5mm from the cone.

Select Operate and set Capillary to approximately 2.5kV.

An ion beam should now be present.



Optimise the ion beam by altering the position of the spray using the controls ofthe translation stage.

The sprayer can now be connected to the HPLC system. The injection valve isplumbed as follows:

P from the pump.

C to the column (or to the union).

S is the sample port, attach a VISF sleeve here.

W is a waste port.

A short tail of fused silica, attached to the entrance port of the union, and theuse of low pressure PTFE connectors removes the need to move the stage. Thisprevents accidental alteration of the sprayer’s position when changing betweentuning and HPLC operation.

Changing OptionsTo 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 stage can result inpermanent damage to the three-axis manipulator.

Remove the protective cover and release the captive screw located underneaththe stage.

Lift off the holder and replace it with the alternative holder, securing it with thecaptive 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 inthe back of the protective cover along with the HV cabling.



Atmospheric Pressure ChemicalIonisation

Introduction

Atmospheric Pressure Chemical Ionisation (APcI) is an easy to use LC-MS interfacethat produces singly-charged protonated or deprotonated molecules for a broad rangeof involatile analytes.

The ability to operate with 100% organic or 100% aqueous mobile phases at flowrates 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 coronadischarge pin and a heated nebuliser probe. Mobile phase from the LC column entersthe probe where it is pneumatically converted into an aerosol and is rapidly heated andconverted to a vapour / gas at the probe tip. Hot gas from the probe passes betweenthe sample cone and the corona discharge pin, which is typically operated with adischarge current of 2µA. Mobile phase molecules rapidly react with ions generated bythe corona discharge to produce stable reagents ions. Analyte molecules introducedinto the mobile phase react with the reagent ions at atmospheric pressure and typicallybecome protonated (in positive ion mode) or deprotonated (in the negative ion mode).The sample and reagent ions pass through the sample cone into the ion block prior tobeing extracted into the hexapole transfer lens.

Changeover between electrospray and APcI operation is simply accomplished bychanging the probe and installing the corona discharge pin within the sourceenclosure.



TurbomolecularPumps

E2M28

AnalyserIonTunnel 2

IonTunnel 1

ProbeExhaust

ExhaustLiner

Rotary PumpsE1M18

Restrictor

NebuliserGas

Sample

DesolvationGas

SampleCone

IsolationValve

SourceEnclosure

CleanableBaffle

CoronaDischarge Pin

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 setting ofCone. The main reagent ions for a typical sample voltage of 40V are 83, 101 and142.

Acetonitrile adducting may be minimised by optimisation of the cone gas and RF Lens1 voltage, as described in Electrospray.

PreparationEnsure that the source is assembled as described in Maintenance and FaultFinding, and that the instrument is pumped down and prepared for APcIoperation as described in Routine Procedures.

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 ofthe instrument and that the head pressure is between 6 and 7 bar (90-100 psi).



CoronaDischarge

Pin

MountingContact

SampleCone

High VoltageSocket

BlankingPlug

Checking the Probe

Ensure that the probe heater is off.

Unplug the probe from the instrument’s front panel and remove the probe fromthe 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 theprobe.

Ensure that 0.5 to 1mm of fused silica is protruding from the stainless steelnebuliser tube.

Connect the LC pump to the probe with a flow of 50:50 acetonitrile:water at1 ml/min.

Check that the liquid jet flows freely from the end of the capillary and that theLC 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, Gas to turn on the nitrogen.

Check that the liquid jet converts to a fine uniform aerosol.

Switch off the liquid flow.


Reconnect the probe tip assembly.

Insert the APcI probe into the source and secure it by tightening the two thumbscrews.

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.



Obtaining a BeamEnsure that the corona discharge pin is fitted and connected as described inRoutine Procedures, Preparation for APcI Operation.

Ensure that the APcI probe is fitted as described above, that the desolvation gastube is connected to the front panel, and that the purge gas outlet is plugged.



Set Source Temp to 130°C.

Set APcI Probe Temp to 20°C with no liquid flow and the nitrogen off.

Set Corona to 2µA and Cone to 50V.

When Source Temp reaches 130°C:

Select Gas, Gas to switch on the nitrogen gas.

Using the valves on the front of the instrument, adjust Desolvation Gas to150 litres/hour and set Nebuliser Gas to its maximum setting.

To monitor the flow rate, select the Source window on the tune page andobserve the Gas Flows readback window.

Select one of the peak display boxes and set Mass to 50 and Span to 90.

Select .

Increase Gain on the peak displaybox in the range 1 to 20 until peaksbecome clearly visible.

Set APcI Probe Temp to 500°C.

When APcI Probe Temp reaches500°C:

Start the LC pump at a flow of1 ml/min.

Adjust the probe’s in / out positionso that it is fully retracted.

Adjust the probe’s sidewaysposition so that the spray is directedapproximately at the midpoint between the corona pin and the sample cone.



In / OutProbe

Adjustment

SidewaysProbe

Adjustment

Check that a stable beam of solvent ions is now apparent.

Refer to Hints for Sample Analysis later in this chapter for further informationon source tuning.

Warning: It is normal for the source enclosure, the glass tube and parts of theprobe adjustment flange to reach temperatures of up to 60°C during prolongedAPcI operation. Care should be exercised when handling source componentsimmediately after operation.

Warning: Switch off the liquid flow and allow the probe to cool (<100°C)before removing it from the source.

Caution: Failure to employ a desolvation gas flow during APcI operation maylead to heat damage to the source.

CalibrationHaving obtained a stable APcI beam, refer to Mass Calibration earlier in this manual.



Hints for Sample Analysis

Tuning for General Qualitative Analysis

Refer to Obtaining a Beam above and tune on solvent ions.

Adjust the in/out position of the probe so that it is fully retracted from thesource.

Using the sideways adjuster ensure that the spray is directed approximately atthe mid-point between the corona pin and the sample cone.

This position occurs five full turns away from the stop closest to the corona pin.

For general qualitative analysis of mixtures, the following parameters are typical:

Corona*: 2 µA

Cone: 80V

RF Lens 1: 40V

Aperture: 0V

RF Lens 2: 0.5V

Source Temp: 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 MRM analysis optimum APcI conditions should be obtained foreach analyte using standard solutions.

• Tuning may be performed using a tee to introduce a standard solution (typically100 pg/µl) at 10 µl/min into the mobile phase stream.

• Alternatively, repeat direct loop injections of a standard solution (typically10 pg/µl) into the mobile phase stream may be used while acquiring in the MRMacquisition mode to optimize the APcI. During an acquisition the sourceparameters may be adjusted and the effects observed.



Corona Current

Corona current can have a significant effect on sensitivity. The corona current requireddepends upon the polarity of the compound and the polarity of the analytical mobilephase. As recommended above optimization should be done in the presence of theanalytical mobile phase.

• An improvement in signal may be obtained for polar compounds, when analysedin a polar mobile phase, by reducing Corona below 2µA.

• Similarly, an improvement in signal may be obtained for compounds of lowpolarity, when analysed in a low polarity mobile phase, by increasing Coronaabove 2µA.

To find the optimum value:

Start at 2µA and increase Corona in 2µA steps until the optimum is found,allowing the current to stabilise before taking a reading.

If the signal continuously decreases, return the current to 2µA and reduceCorona in 0.1µA steps.

Probe Position

The in / out position of the APcI probe generally has little effect on sensitivity. Thesideways adjustment can have a significant effect upon sensitivity.

Using the sideways adjuster ensure that the spray is directed approximately atthe mid-point between the corona pin and the sample cone.

This position occurs five full turns away from the stop closest to the corona pin.

Adjust the probe position around this point, one turn at a time, to optimise thesignal.

Probe Temperature

It is important to optimise APcI Probe Temp for maximum sensitivity, as follows:

Ensure that the analytical mobile phase is used during optimisation.

Starting at 650°C reduce the temperature in 50°C steps, allowing time for thetemperature to stabilise before taking a reading.

It is possible to set APcI Probe Temp too low for the mobile phase. This oftenresults in significant chromatographic peak tailing.

Desolvation Gas

In most circumstances the desolvation gas flow has little effect on signal intensity.However, in some situations, it has been observed to have an effect on chemicalbackground noise levels. Adjusting Desolvation Gas while acquiring in the MRMmode can be used as a check for this.



Removing the ProbeAfter a session of APcI operation:

Turn off the LC flow.

Set APcI Probe Temp to 20°C.

Deselect .

When the probe temperature falls below 100°C:


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 tothe operator.

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

If the instrument is not to be used for a long period of time reduceSource Temp to 60°C.



ProbeThumb Nuts

Maintenance and Fault FindingIntroduction

Cleanliness and care are of the utmost importance whenever internal assemblies areremoved from the instrument.

4Always prepare a clear clean area in which to work.

4Make sure that any tools or spare parts that may be required are close at hand.

4Obtain some small containers in which screws, washers, spacers etc. can bestored.

4Use tweezers and pliers whenever possible.

4 If nylon or cotton gloves are used take care not to leave fibres in sensitive areas.

6 Avoid touching sensitive parts with fingers.

6 Do not use rubber gloves.

4 Before reassembling and replacing dismantled components, inspect O rings andother 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 orotherwise disturbed, it is advisable first of all to ensure that this part has beencorrectly refitted and/or adjusted and that adjacent components have not beeninadvertently disturbed.

Warning: Many of the procedures described in this chapter involvethe removal of possibly toxic contaminating deposits usingflammable or caustic agents. Personnel performing these operationsshould be aware of the inherent risks, and should take the necessaryprecautions.

Cooling Fans and Air FiltersAlways ensure that none of the cooling fans is obstructed. It is essential that the fanfilter is checked at regular intervals, and replaced if there is any doubt about itseffectiveness.

Maintenance and Fault Finding


The Vacuum SystemThe performance of the mass spectrometer is severely impaired by the lack of a goodvacuum in the ion transfer (hexapole) regions or the analyser.

• An analyser pressure above 10-4 mbar results in a general loss in performanceindicated by a loss of resolution and an increase in the background noise.

• Above 10-3 mbar the Operate and Vacuum LEDs on the instrument changefrom green to amber, indicating that the vacuum is insufficient to maintain theinstrument in operate.

• Above 10-2 mbar the Vacuum LED changes to flashing red, indicating that thevacuum pump trips have been activated, followed by no indication when theinstrument is no longer 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 so contaminated thatoptimum pumping speed is no longer possible. Initially, gas ballasting may cleanthe oil. If the oil in the rotary pump has become discoloured, then it should bechanged according to the pump manufacturer’s maintenance manual.

• The turbomolecular pumps switch off if an over temperature is detected. Thiscould be due to poor backing vacuum, failure of the water supply or a leak inthe source or analyser.

• The turbomolecular pumps switch off if full speed is not achieved within a settime following start-up. This could be due to a leak or too high an ambienttemperature.



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 disturbed.

Leaks on flanges can usually be cured by further tightening of the flange boltsor by replacing the seal.

• All seals are made using O rings. When refitting flanges pay attention to thecondition of O rings. Any that are cut or marked may cause a leak. The O ringsshould 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 arenot susceptible to vacuum leaks.

In the unlikely event of a leak on a feedthrough, then the unit should be replaced orreturned 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 tothe Edwards literature supplied with the instrument.



Gas Ballasting and Rotary Pump Oil Recirculation

Gas ballasting serves two importantpurposes:

• When rotary pumps are usedto pump away solventvapours, the solvent vapourcan become dissolved in thepump oil causing an increasein backing line pressure. Gasballasting is a method ofpurging the oil to removedissolved contaminants.

• Oil mist expelled from therotary pump exhaust istrapped in the oil mist filter.This oil is returned to the rotary pump during gas ballasting.

The Quattro Ultima has two rotary pumps; an E1M18 pumping the source block andan E2M28 pumping the first hexapole. Because the source block is maintained at arelatively high pressure, the rate of oil mist being expelled from the E1M18 pump isconsiderably higher than that from the E2M28 pump. Consequently, it isrecommended that the E1M18 pump is operated continuously in gas ballast mode sothat the pump oil is continuously recirculated.

Continual operation in gas ballast mode is not usually recommended as ventingof the instrument whilst rotary pumps are ballasting can cause oil vapour tomigrate into the vacuum housing. However, the E1M18 has an additionalautomatic gas ballast control valve mounted in the oil return line from the mistfilter. This valve is opened whenever the E1M18 is switched on, allowingcontinuous recirculation of the pump oil provided that the manual gas ballastvalve on the pump is left open.

In the event of a vent command or automatic vent (vacuum fault or powerfailure) the pump is switched off and the automatic gas ballast valve closes thuspreventing any contamination of the vacuum housing.

The E2M28 rotary pump is not equipped with an automatic gas ballast control valve,and this pump should normally be operated with the manual gas ballast valve closed.The valve should only be opened for 30 minutes each week to perform routine gasballasting of the pump. If the source is used in the APcI or megaflow electrospraymodes, more frequent gas ballasting is recommended.

The manual gas ballast valve is opened by rotating it fully counterclockwise.

It is normal for the rotary pump to make more noise when the gas ballast valveis open.



GasBallast

DrainPlug

Exhaust

FillerPlug

Oil LevelIndicator

Oil MistFilter

Caution: Failure to gas ballast the rotary pump frequently leads to shortened oillifetime which in turn may shorten rotary pump lifetime.

Caution: The instrument should not be vented with the E2M28 manual gasvalve open. The E1M18 manual gas ballast valve should remain open at alltimes.

Oil Mist Filter

The rotary pumps are fitted with an Edwards EMF20 oil mist filter which traps oilvapour from the rotary pump exhaust. The trapped oil is then returned to the rotarypump during routine gas ballasting. The oil mist filter contains two elements whichrequire 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 massspectrometer. During normal use, the activated alumina (sorbent) absorbs any oilvapour, becoming brown in colour. The sorbent should be replaced when thisdiscolouration reaches the region of the trap furthest from the pump (the vacuum side).The manufacturers recommend that the sorbent is replaced routinely at three-monthlyintervals.

With the instrument vented and the pump switched off, replace the sorbent asdescribed 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 haschanged to a noticeable red colour, or routinely at 4 month intervals (3000 hoursoperation). 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.

Close the gas ballast valve.

Vent and shut down the instrument as described in Routine Procedures.

It is 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 correctgrade oil to the correct level.

Gas ballast lightly for 30 to 60 minutes.

For further servicing information refer to the manufacturer’s manual.



The Source

Overview

The Z-spray source is a robust assembly requiring little maintenance. The sourceconsists 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, withoutventing the instrument, to gain access to the source block and sample cone. Thisallows the following operations to be performed:

• Removing the cone gas nozzle and 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.

Cleaning of the sample cone and cone gas nozzle may be achieved by removing themfrom the source. This may also be done without venting the instrument, by closing theisolation valve located on the ion block. Less frequently it may be necessary to cleanthe ion block and the hexapole lens, in which case the instrument must be vented. Thisshould only be done when the problem is not rectified by cleaning the sample coneand cone gas nozzle, or when charging effects are apparent.

Charging is evidenced by a noticeable progressive drop in signal intensity, oftenresulting in a complete loss of signal. Switching the instrument out of and backinto operate causes the beam momentarily to return.

The hexapole transfer lens should not require frequent cleaning. If it is suspected thatthe lens does need cleaning it may be withdrawn from the front of the instrument afterremoving the ion block support.

Warning: Cleaning the various parts of the source requires the use ofsolvents and chemicals which may be flammable and hazardous tohealth. The user should take all necessary precautions.



Cleaning the Cone Gas Nozzle and Sample Cone

This may be necessary due to lack of sensitivity or fluctuating peak intensity, or ifdeposited material is visible on the outside of the nozzle or sample cone. Proceed asfollows:


Deselect .


Disconnect the liquid flow at the rear of the probe.

Set Source Temp and either APcI Probe Temp or Desolvation Temp to20°C to switch off the heaters.

Warning: Removal of the APcI probe or desolvation nozzle when hot may causeburns.

Caution: Removal of the APcI probe when hot shortens the probe heater’s life.

The cooling time is significantly shortened if the API gases are left flowing.

When APcI Probe Temp or Desolvation Temp has cooled below 100°C:

Switch off the nitrogen supply by selecting Gas followed by Gas.

Disconnect both gas lines from the front panel by undoing the knurled nuts.



SourceThumb Nuts


Adjustment Flange

GlassTube

Disconnect both electrical connections by pulling back on the plug sleeves torelease the plugs from the sockets on the front panel.

Undo the two knurled thumb nuts that retain the probe and withdraw it from thesource. Place it carefully to one side.

Undo the three thumb screws and withdraw the probe adjustment flange andglass tube. Place the glass tube, end on, on a flat surface and place the probeadjustment flange on top of the glass tube.

Warning: When the source enclosure has been removed the source block isexposed. Ensure that the source block heater has cooled before proceeding.

If fitted, remove the APcI discharge pin.

The sample cone and cone gas nozzle are now accessible.

Using a suitable flat blade screwdriver rotate the isolation valve by 90° into itsfully anticlockwise position.

A small improvement in the analyser vacuum may be observed as a result of thisoperation.

The isolation valve is closed when the slot is perpendicular to the direction offlow.



Cone GasNozzle

IsolationValve

Disconnect the cone gas inlet line.

Take the sample cone extraction tool supplied in the source spares kit and screwit to the flange of the sample cone.

Remove the two sample cone retaining screws using a 1.5mm Allen key andwithdraw the sample cone, gasket and cone gas nozzle from the ion block.

Caution: The sample cone is a delicate and expensive component and should behandled with extreme care.

Remove the extraction tool, and separate the sample cone, the gasket and thecone gas nozzle.

Carefully wipe the sample cone and cone gas nozzle with a cotton swab or lintfree tissue soaked in 50:50 acetonitrile:water or 50:50 methanol:water.

Caution: Do not attempt to remove any obstruction by poking. This may resultin damage to the sample cone.

If the components are still not clean, or if the aperture is partially blocked, placethe components in an ultrasonic bath containing 50:50 acetonitrile:water or 50:50methanol:water.

To minimise down time fit a spare sample cone and cone gas nozzle, obtainablefrom Micromass, at this stage.



Dry the cone and nozzle using nitrogen.

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 ionblock.

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 parallel tothe direction of gas flow. Note that the valve can be rotated past the fully openposition.



SampleCone Cone Gas

Nozzle

Gasket

ExhaustLiner

ExtractionTool

CleanableBaffle

Removing and Cleaning the Ion Block

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 turbomolecularpumps are allowed to run down to 50% speed after which a vent valve opens toatmosphere automatically.

Remove the source enclosure, sample cone and cone gas nozzle as described inthe previous section.



IonBlock

CoverPlate

O Ring

When the instrument has vented:

Remove the four screws, together with the washers, which secure the cover plateto the ion block and remove the cover plate.

Warning: The heater supply remains live until the system is fully vented. Do notremove the cover plate until the system has vented.

Ensure that the O ring remains in position on the ion block.



Remove the two screws from the heater connections on the ion block.

Carefully straighten the heater supply leads, in such a way that the ion block canlater be withdrawn without fouling these leads.

Loosen the screw on the thermocouple’s securing clip and unhook thethermocouple 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 leadsprotruding from the ion block support.

Ensure that the three O rings remain in position on the ion block support.



Ion BlockSupport

O Rings

O RingRestrictorand Bush

Ion Block

Thermocouple

HeaterLeads

Heater SupplyLeads

PTFEWasher

Unscrew the restrictor’s outer bush from the ion block, taking care not to disturbthe setting of the inner restrictor. Collect the PTFE washer, and ensure that theO ring remains in position on the restrictor shaft.

Wipe the inner end of the restrictor with a cotton swab or lint free tissue soakedin 50:50 acetonitrile:water or 50:50 methanol:water, to remove any carbondeposits.

Leaving the heater, valve, thermocouple clip and terminal block in place,immerse the ion block in an ultrasonic bath containing 50:50 acetonitrile:wateror 50:50 methanol:water, followed by 100% methanol.

Dry all components using a flow of nitrogen, or place them in a warm oven.



Removing and Cleaning the Ion Tunnel Assembly

To remove the ion tunnel assembly, proceed as follows:

Remove the ion block, as described above.

Remove the three screws retaining the ion block support and carefully withdrawit from the pumping block.

Ensure that the 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 differential apertureplate.

Caution: Take care not to scratch the internal bore of the pumping block as theion tunnel assembly is withdrawn.



O Rings

View from rear

Ion Tunnel 1

Ion BlockSupport

To clean the ion tunnel proceed as follows:

Immerse the complete assembly in a suitable solvent (100% methanol) andsonicate in an ultrasonic bath.

Thoroughly dry the assembly using a flow of nitrogen.

In severe cases:

Remove the differential aperture plate and clean thoroughly. A glass fibre pencilcan be used to remove burn marks around the aperture.

Clean the component in an ultrasonic bath.

Do not disassemble the ion tunnel. Cleaning the tunnel should be carried outwith great care using the brush provided.

Gently insert the brush into the aperture and use a rotary motion to clean theapertures. Take great care not to bend the plates on the tunnel. A glass fibrepencil can be used to remove burn marks on the entrance and exit plates.

Subsequently clean the tunnel in an ultrasonic bath as above, ensuring that nofibres from the brush remain.

Reassemble the differential aperture.



DifferentialAperture

Plate

Reassembling and Checking the Source

Check the condition of all O rings. Replace them if necessary.

With the two springs in a horizontal plane, feed the hexapole transfer lens intothe instrument. Ensure that the O ring is correctly fitted to the differentialaperture plate, and that the assembly is pushed fully in.

Replace the ion block support, pushing it in against the springs of the ion tunnelassembly.

Replace the three retaining screws.

Replace the restrictor and bush, complete with O ring and PTFE washer, on theion block.

Locate the ion block on the peek ion block support, and secure with the twoscrews taking care not to over-tighten the screws.



Springs

Insert the thermocouple into its location, and secure it with the clip.

Reconnect the heater leads and heater supply leads to the terminal block,carefully bending the supply leads as necessary.

Replace the cover plate.

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, 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 blanking plug, as necessary.

Fit the source enclosure and the probe adjustment flange.

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.



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 thesolvent 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 largedead volumes in the transfer capillaries between the LC column or probeconnection.

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 µl/min the back pressureshould not exceed 7 bar (100 psi). Pressures in excess of this indicate ablockage in the solvent flow system.

Samples containing particulate matter, or those of high concentrations, are mostlikely 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. Rinsethoroughly afterwards.



Blockage of the stainless steel sample capillary may occur if the desolvationheater is left on without liquid flow. This is particularly relevant for samplescontained in involatile solvents or high analyte concentrations. To avoid thisproblem it is good practice to switch off the heater before stopping the liquidflow, and flush the capillary with solvent.

A blocked stainless steel sample capillary can often be cleared byremoving it and reconnecting it in the reverse direction, thus flushing outthe blockage.

• Gas flow problems

Check all gas connections for leaks using soap solution, or a suitable leaksearching agent such as Snoop.



Replacement of the Stainless Steel Sample Capillary

If the stainless steel sample capillary cannot be cleared, or if it is contaminated ordamaged, 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 endcover.

Unscrew and remove the probe tip.

Remove the LC union and adapter nut. Withdraw and discard the stainless steelsample capillary.

Remake the LC connection to the LC union.

Sleeve one end of new sample capillary with the PTFE liner tube.



Stainless SteelCapillary

Fused SilicaCapillary

LCUnion

Finger-tightNut & Ferrule

RheodyneNut & Ferrule

GrubScrew

EndCover

ProbeTip

AdapterNut

LinerTube

LinerTube

LinerTube

GVF/16Ferrule

GVF/003Ferrule 0.5mm

Using a GVF/16 ferrule and the adapter nut, connect the sample capillary to theLC union, ensuring that both the liner tube and sample capillary are fully buttedinto 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 protrudesfrom the probe tip.

Replace the probe end cover and tighten the grub screw to clamp the LC union.



The APcI ProbeIndications that maintenance to the APcI probe is required include:

• The probe tip assembly becomes contaminated, for example by involatilesamples 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 examplereserpine and common pesticides) should display peak half widths of the order0.1 minutes for 10µl loop injections at a flow rate of 1 ml/min. The appearanceof significant peak broadening or tailing with these compounds is most likely tobe 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 backpressure less than 14 bar (200 psi) is indicative of a broken fused silicacapillary 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 backpressure above 35 bar (500 psi) is indicative of a blockage or partial blockagein the fused silica capillary, in a LC connector or in the filter. It is advisable tochange the inner filter pad ( see “Replacing the Fused Silica Capillary” in thefollowing pages) on a regular basis.

• Gas flow problems.

Check all gas connections for leaks using soap solution, or a suitable leaksearching agent such as Snoop.

Cleaning the Probe Tip

Remove any visible deposits on the inner wall of the probe heater with amicro-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.


Allow the gas to flow for several seconds to clear any debris from the heater.


Insert the probe into the source.




Raise APcI Probe Temp gradually, starting at 100°C and increasing in 50°Cintervals to 650°C over a period of 10 minutes.

Caution: Do not set APcI Probe Temp to 650°C immediately as this maydamage the probe 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 the heater from the probe body by pulling parallel to the axis of theprobe.

Fit a new heater assembly.

Reconnect the probe tip assembly.



GrubScrews

Heater

Probe TipAssembly

Replacing the Fused Silica Capillary

With the probe removed from the source proceed as follows:

Remove the probe tip assembly and the heater, as described in the precedingsection.

Remove the probe end cover by removing the two screws and the grub screwthat 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 300µm o.d. × 100µm 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 thefilter ensuring that the liner tube is fully butted into the filter.



0.5 to1mm

Fused SilicaCapillary

Finger-tightNut & Ferrule

RheodyneNut & Ferrule

GVF/004Ferrule

AdapterNut

Filter

PTFETube

GrubScrew

GrubScrew

Feed the sample capillary through the probe, ensuring that a 0.4mm graphitisedvespel ferrule (GVF/004) is fitted.

Using a ceramic capillary cutter, cut the capillary at the nebuliser so thatbetween 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 asuitable 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 flameand clean with a tissue saturated with methanol.

Carefully re-feed the sample capillary through the probe ensuring that thegraphitised 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 toclamp the filter.

Replace the heater and probe tip assembly.



The AnalyserQuattro Ultima is fitted with a pre-filter assembly that is designed to protect the mainanalyser by absorbing contamination from the ion beam. As a consequence theanalyser quadrupoles should never, under normal working conditions, require cleaning.

The hexapole transfer lens also serves to effectively remove contamination, and thepre-filter assembly should only require cleaning on an infrequent basis. Althoughtraining is given during installation, it is strongly recommended that this task is carriedout by a Micromass service engineer or by other suitably qualified personnel.

The quadrupole assemblies of Quattro Ultima are finely machined and alignedassemblies which under no circumstances should be dismantled.

The DetectorThe Quattro Ultima detector system has been designed for trouble-free operation overmany years. The photomultiplier is encapsulated in its own vacuum envelope and istherefore safe from contamination and pressure surges. The conversion dynode andphosphor are also long lasting. No routine maintenance is required.

It is strongly recommended that assistance is sought from Micromass if maintenanceto the detector system is thought necessary due to spikes or unacceptably high noiselevels.



ElectronicsWarning: There are high voltages present throughout the mass spectrometer.Extreme caution should be taken when taking measurements with a meter or anoscilloscope. In the standby mode (Operate not selected) the high voltages areswitched off in the source and analyser assemblies, but high DC voltages andmains voltages remain in the power supply units.

Caution: Quattro Ultima’s electronic systems contain complex and extremelysensitive components. Any fault finding procedures should be carried out onlyby Micromass engineers.

Fuses

In the following list, the designation “T” indicates a time lag fuse.

Analog PCB

Fuse No Fuse Type Ref. No.F1 10A (T) 20mm anti-surge TDS505 1340143F2 10A (T) 20m 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 1340161F8 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 1340164F2 2A (T) 20mm anti-surge TDS506 1340161F3 6.3A (T) 20mm anti-surge TDS506 1340163

Rear Panel

Fuse No Fuse Type Ref. No.F1 10A (T) HBC ceramic anti-surge 1340147F2 10A (T) HBC ceramic anti-surge 1340147



Fault Finding Check ListWarning: There are high voltages present throughout the mass spectrometer.Extreme caution should be taken when taking measurements with a meter or anoscilloscope. In the standby mode (Operate not selected) the high voltages areswitched off in the source and analyser assemblies, but high DC voltages andmains voltages remain in the power supply units.

Any investigation in the RF generator must be made only by a Micromassengineer.

No Beam

Refer to the relevant chapters of this manual and check the following:

• Normal tuning parameters are set and, where appropriate, readback values areacceptable.

• 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 windowat the rear of the instrument).

Unsteady or Low Intensity Beam

Should the preceding checks fail to reveal the cause of the problem check that:

• Gas and liquid flows are normal.

• The analyser pressure is less than 1x10-4 mbar.

Ripple

Peaks appear to vary cyclically in intensity when there is ripple superimposed on thepeak. 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 helplocate the source. Mains frequency ripple, for example, points towards anunstable power supply or vibration from mains powered machinery.



High Noise Level in MRM Analyses

The background noise in MRM analyses can either be electronic or chemical. Todistinguish between chemical noise and electronic noise an acquisition should beperformed with and without ions being transmitted to the detector. Ions are bestprevented from reaching the detector by setting the ion energy 1 (MS1) and ion energy2 (MS2) fully negative. If there is a significant decrease in the background noise withthe ion energies set negative then the major contribution to the overall noise ischemical noise. Any residual noise is electronic noise.

If the dominant source of noise is chemical, a reduction in electronic noise does notyield significant improvements in overall signal to noise ratio.

Chemical Noise

The most common source of noise is chemical noise.

• If the auto injector, probe or connecting tubing have been exposed to a highconcentration of the compound to be analysed then this may be giving a highbackground due to “carry over”. This can occur if concentrations of a few ng/µlare used for tuning prior to attempting sub pg/µl detection levels. If the injectoris contaminated the signal level normally changes when injections of mobilephase are made.

Repetitive injections of 10% formic acid and / or isopropanol may help reducethe noise. If the probe or connecting tubing are contaminated then infusing 10%formic acid and / or isopropanol with a syringe pump may help.

• Check that the LC system is not adding contaminants into the mobile phase.

Using a syringe pump, infuse a syringe of mobile phase taken from the solventreservoir. Compare this with the MRM background when the LC system isdelivering the solvent.

• Try a different MRM transition.

This may reduce the noise level if the compound(s) contributing to the chemicalnoise do not yield the same set of daughter ions as the compound beinganalysed.

• Check the purity of solvents and additives.

Try a different type of solvent or the same type of solvent from a differentmanufacturer. Ensure all solvents and additives are HPLC grade. Check thecleanliness of any glassware used.



Electronic Noise

If the noise has been identified as electronic noise, then the ion counting thresholdlevel should be checked as follows:

Increase Ion Counting Threshold until the valleys of the peak-to-peak noiseare brought down to the zero line. A value of 30 is typical.

It is not possible to give absolute values for typical peak-to-peak electronic noise, asthis is dependent on detector gain and the dwell time used for the MRM experiment.However, the electronic noise should be fairly constant for a particular instrument, soa measurement made previously under the same MRM conditions (with ion energiesnegative) should provide a meaningful comparison to see if the electronic noise levelhas changed.

If, after checking the ion counting threshold as above, electronic noise is considered tobe the dominant source of noise and has become significantly worse since instrumentinstallation then further investigations should be carried out by a qualified Micromassengineer.

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 thereis a blockage in the capillary transfer line or injection loop due to particulate matterfrom the sample. To clear the blockage:

Remove the probe from the source and increase the solvent flow to 50 µl/min toremove the blockage.

Often, injections of neat formic acid help to redissolve any solute which hasprecipitated 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 tryremoving 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 agraduated glass capillary with the liquid emerging from the probe tip, and timea known volume, say 10µl.



Once the rate has been measured and set, a note should be made of the backpressure readout on the pump, as fluctuation of this reading can indicateproblems with the solvent flow.

For APcI a higher than normal back pressure readout on the HPLC pump can implythat, 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 asfollows:

Check that the source and probe voltage readbacks vary with tune page settings.

If any of these voltages are absent check that the source and hexapole transferlens assembly have been correctly reassembled.

Further investigation, which require the services of a qualified service engineer,should be left to Micromass personnel.



Cleaning MaterialsIt is important when cleaning internal components to maintain the quality of thesurface finish. Deep scratches or pits can cause loss of performance. Where nospecific cleaning procedure is given, fine abrasives should be used to remove dirt frommetal 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 suitablesolvents to remove all traces of grease and oil. The recommended procedure is tosonicate the components in a clean beaker of solvent and subsequently to blot themdry with lint-free tissue. Recommended solvents are:

• Isopropyl Alcohol (IPA)

• Methanol

• Acetone

Following re-assembly, components should be blown with oil-free nitrogen to removedust particles.

Warning: Many of the procedures described in this chapter involvethe removal of possibly toxic contaminating deposits usingflammable or caustic agents. Personnel performing these operationsshould be aware of the inherent risks, and should take the necessaryprecautions.



Preventive Maintenance Check List6 Avoid venting the instrument when the rotary pump is gas ballasting.

6 Do not gas ballast the rotary pump for more than 2 hours under anycircumstances.

For full details of the following procedures, consult the relevant sections of thischapter and/or refer to the manufacturer’s literature.

Weekly

• Gas ballast for at least 30 minutes by rotating the gas ballast knobanticlockwise 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).

Monthly

• 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.



Reference InformationOverview

Calibration reference files consist of two columns of numbers separated by anynumber of spaces or TAB characters. The first column contains the reference peakmasses and the second column contains the reference peak intensities.

The reference files listed in this chapter have all ion intensities set to 100%. Actualion intensities are not, of course, all 100%, but the calibration software does not takeaccount of the ion intensities and this is a convenient way to store the reference filesin the required format. However, if required, realistic intensity values can be enteredto improve the appearance of the reference spectra.

Most samples can be purchased from the Sigma chemical company. To order, contactSigma 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-freenumbers.

Toll-free telephone:

USA & Canada 800-325-3010

Outside USA & Canada ++1 314-771-5750 (call collect)

Toll-free fax:

USA & Canada 800-325-5052

Outside USA & Canada ++1 314-771-5750(call collect and ask for the fax machine)

Outside USA & Canada ++1 314-771-5757(this is a toll call) (direct fax line)

Reference Information


Editing a Reference FileCalibration reference files can be created or edited using any Windows text editor. Toread the currently selected reference file into the Notepad text editor:

Press or select Reference File... from the Calibration, Edit menu.

To save the reference file after editing either:

Select Save from the Notepad File menu to save the file under the currentname.

or:

Select Save as from the Notepad File menu to save as a new reference filewith a new name.

Textual information or comments can be stored in the reference file. Lines which aretextual information or comments must start with the semi-colon ( ; ) character.



Positive Ion

Ref. FileName

Chemical Name[Sigma Code #]

MolecularMass

� Uses

UBQBovine Ubiquitin[U6253]

8564.85 650-1500 General

HBAHuman α globin[H753]

15126.36 700-1500 Hb analysis

SODSuperoxide dismutase[S2515]

15591.35 900-1500Hb (internalcal.)

HBBHuman β globin[H7379]

15867.22 800-1500 Hb analysis

MYOHorse heart myoglobin[M1882]

16951.48 700-1600 General

PEGH1000

Polyethylene glycol +ammonium acetatemixturePEG 200+400+600+1000

80-1000ES+ andAPcI+calibration

PEGH2000

Polyethylene glycol +ammonium acetatemixturePEG 200+400+600+1000+1450

80-2000ES+calibration

NAICSSodium Iodide / CaesiumIodide mixture

20-4000General,ES+calibration

NAIRBSodium iodide / RubidiumIodide mixture

20-4000ES+calibration



Horse Heart Myoglobin

Reference File: myo.refMolecular Weight: 16951.48

ChargeState

Calculated� Value

ChargeState

Calculated� Value

ChargeState

Calculated� Value

28+ 606.419 21+ 808.222 13+ 1304.969

616.177 20+ 848.583 12+ 1413.633

27+ 628.841 19+ 893.192 11+ 1542.053

26+ 652.989 18+ 942.758 10+ 1696.158

25+ 679.068 17+ 998.155 9+ 1884.508

24+ 707.320 16+ 1060.477 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

107.07 503.31 899.54 1295.78 1692.01

151.10 547.33 943.57 1339.80 1736.04

195.12 591.36 987.60 1383.83 1780.07

239.15 635.39 1031.62 1427.86 1824.09

283.18 679.41 1075.65 1471.88 1868.12

327.20 723.44 1119.67 1515.91 1912.15

371.23 767.46 1163.70 1559.94 1956.17

415.25 811.49 1207.73 1603.96 2000.20



Sodium Iodide and Caesium Iodide Mixture

Reference File: NAICS

Calculated� Value

22.9898 772.4610 1671.8264 2571.1918 3470.5572

132.9054 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

Sodium Iodide and Rubidium Iodide Mixture

Reference File: NAIRB

Calculated� Value

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 Ion

Ref. FileName

Chemical Name[Sigma Code #]

MolecularMass

� Uses

MYONEGHorse heart myoglobin[M1882]

16951.48 700-2400 General

SUGNEG

Sugar mixture of:maltose [M5885]

raffinose [R0250]maltotetraose

[M8253]corn syrup [M3639]

100-1500Low massrange

NAINEGSodium Iodide / CaesiumIodide (or RubidiumIodide) mixture

200-3900ES-calibration

Horse Heart Myoglobin

Reference File: myoneg.ref

Calculated� Value

891.175 1209.812 1882.490

940.741 1302.952 2117.927

996.138 1411.615 2420.632

1058.460 1540.036

1129.091 1694.140

Mixture of Sugars

Reference File: sugneg.ref

Calculated� Value

179.06 665.21 1151.37

341.11 827.27 1313.42

503.16 989.32 1475.48



Sodium Iodide and Caesium Iodide (or Rubidium Iodide) Mixture

Reference File: naineg.ref

Calculated� Value

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 3424.5777

876.3757 1775.7411 2675.1065 3574.4719



Preparation of Calibration Solutions

PEG + Ammonium Acetate for Positive Ion Electrospray and APcI

Prepare a solution of polyethylene glycols at the following concentrations:

PEG 200 25 ng/µl

PEG 400 50 ng/µl

PEG 600 75 ng/µl

PEG 1000 250 ng/µl

Use 50% acetonitrile and 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 following concentrations:

PEG 200 25 ng/µl

PEG 400 50 ng/µl

PEG 600 75 ng/µl

PEG 1000 250 ng/µl

PEG 1450 250 ng/µl

Use 50% acetonitrile and 50% water containing 2 mmol ammonium nitrate.

Use reference file PEGH2000



Sodium Iodide Solution for Positive Ion Electrospray

Method 1

Prepare a solution of sodium iodide at a concentration of 2 µg/µl (microgramsper microlitre) in 50:50 propan-2-ol (IPA):water with no additional acid orbuffer.

Add caesium iodide to a concentration of 0.05 µg/µl.

The purpose of the caesium iodide is to obtain a peak at� 133 (Cs+) to fill thegap in the calibration file between� 23 (Na+) and the first cluster at� 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 complexspectrum due to the formation of NaCsI clusters.

Use reference file NAICS.REF.

Method 2

Prepare a solution of sodium iodide at a concentration of 2 µg/µl (microgramsper microlitre) in 50:50 propan-2-ol (IPA):water with no additional acid orbuffer.

Add rubidium iodide to a concentration of 0.05 µg/µl.

The purpose of the rubidium iodide is to obtain a peak at� 85 (85Rb+) with anintensity of about 10% of the base peak at� 173. Rubidium iodide has theadvantage that no rubidium clusters are formed which may complicate thespectrum. Note that rubidium has two isotopes (85Rb and 87Rb) in the ratio2.59:1, giving peaks at� 85 and 87.

Use reference file NAIRB.REF.

Sodium Iodide Solution for Negative Ion Electrospray

Either of the above solutions is suitable for calibration in negative ion mode. In bothcases the first negative reference peak appears at� 127 (I-) and the remaining peaksare due to NaI clusters.

Use reference file NAINEG.REF.



Index

AAcetonitrile 144

Adducts 172Acquisition 46, 71, 130

Parameters 114, 129Active inverted magnetron gauge 18, 34, 181Air filter 179Ambient temperature 15Ammonia 144Ammonium acetate 127, 222Analog channels 86Analog data 81Analog input 24, 28Analog PCB 33, 207Analyser 34, 206APcI 19, 42, 171

Analysis 176Calibration 127Tuning 174

APcI probe 19APcI probe 89, 178, 202

Checking 173Filter 202Fused silica capillary 204Maintenance 202Position 177Removal 178Temperature 44, 176, 177, 202Tip heater 25, 203

Aperture 176Argon 16, 31, 35Atmospheric pressure chemical ionisation

See: APcIAuto control 51AutoTune 64

BBack pressure 202, 210Biopolymers 157

CCaesium iodide 157, 219, 221, 223Calibration 46, 103

APcI 127Checking 118Electrospray 157Failure 120, 137Incorrect 122, 138Manual 131, 135Parameters 110, 128Saving 123, 139Scan speed compensation 112, 134Scanning 112, 134Static 112, 129Verification 124, 140

Camera 165Capillary 154Capillary / Corona 42Capillary / Corona 25, 41Centroid data 78, 88Channels 90Charging 185CID

See: Collision induced decompositionCID Gas

See: Collision gasCleanable baffle 148, 163, 172, 185, 189Cleaning 202, 212Cluster ions 156, 159Collision cell

See: Hexapole collision cellCollision energy 94Collision gas 16, 27, 31, 35, 47Collision induced decomposition 17, 21Column

4.6mm LC 147, 159, 171Capillary LC 159Microbore (2.1mm) LC 147, 159

Conditions 73Cone 88, 176Cone gas 152, 163, 176Cone gas nozzle 163, 185, 186Constant neutral loss 24Contact closure 28Continuum data 88Conversion dynode 17Conversion dynode 206Cooling fan 179Corona 19, 176, 177Coupled column chromatography 160

Index


DData acquisition 71

See: AcquisitionData processing 46Data system 13, 24, 36Daughter 92Daughter ion 21, 46, 47Desolvation gas 25, 27, 40, 42, 44, 151, 176,177Desolvation temp 41, 44, 151Detector 34, 206Dimensions 13Discharge pin 25, 42, 171, 185, 197Divert valve 27Drug metabolite 23Drugs 158Dwell 90Dye compounds 158

EElectronics 32, 207Electrospray 19, 40, 143

Analysis 157Calibration 105Negative ion 158Operation 148Positive ion 158

Electrospray probe 19, 41, 149, 151Maintenance 198Removal 156

Emergency 48End mass 72Environment 15Environmental analysis 23Environmental contaminants 158ESD earth facility 31Event out 28Exhaust 15, 31, 36Exhaust liner 148, 163, 185, 189

FFault finding 179, 208Filter 34Flow control valve 27Flow injection analysis 19, 144, 161Fluorescence detector 24Foreline trap 183Forensic science 23Formic acid 144Fragment ions 17Fragmentation 46, 47, 144Full scan function 88Function list editor 82Fuses 31, 207

GGas 66Gas ballast 38, 182Gas cell 34Glass capillary (nanoflow) 161, 166Gradient elution 159Grid 63

HHeater 25Herbicide 23Hex 176Hexapole collision cell 17Hexapole transfer lens 34, 185, 194High mass resolution 155HM Res

See: High mass resolutionHumidity 15

IInfusion pump 17, 106, 144Injection loop 106Injection valve 27, 144Intensity 63Inter scan time 73Ion block 190Ion counting threshold 79Ion energy 155Ion evaporation 19Ion mode 41, 43, 44, 150, 174Ion source

See: Source

JJob 73

LLC-MS interface 159LM Res

See: Low mass resolutionLow mass resolution 155

Index


MMains switch 31Maintenance 179Mass calibration

See: CalibrationMass measurement 111MaxEnt 78MCA 89Megaflow 147, 156, 159Metabolites 158Method 88, 91Microscope 165Mode 65MRM 21, 23, 95, 176MS1 mode 20MS2 mode 20, 93MS-MS 20, 46MS-MS function 92Multi channel analysis

See: MCAMultiple reaction monitoring

See: MRMMultiple samples 73Multiply charged ions 19MUX 29Myoglobin 157, 218, 220

NNanoflow electrospray 19, 161, 185Nano-HPLC 19, 161Nano-LC (nanoflow option) 168Narrow mass scanning 160Nebuliser 19, 27, 41, 43, 44Nebuliser gas 25, 151Neonatal screening 24Neutral gain 93Neutral loss 93Nitrogen 16, 30, 35, 44, 148

OOil mist filter 183Oligonucleotides 143, 158Operate 44Operate LED 26, 44, 45Organometallics 158Origin 73

PParent 93Parent ion 22, 46PC link 36Peak matching 123, 139PEG

See: Polyethylene glycolPenning gauge

See: Active inverted magnetron gaugePeptides 143Peptides 21, 158Pesticides 23, 158Pharmacokinetic studies 23Phase system switching 160Phosphate 157Phosphor 17, 206Photomultiplier 206Photomultiplier 17, 208Pirani gauge 18, 34, 181Plasma 19Pollutants 158Polyethylene glycol 126, 127, 157, 218, 222Polysaccharides 158Post delay 51Power 15

Failure 45Power backplane #2 207Power cord 31Power sequence PCB 33, 207Power supplies 33Pre delay 51Precursor 97Pressure 39Preventive maintenance 213Process 74Profile data 78

Spike removal 80Proteins 143, 158Proton abstraction 19Proton transfer 19Pump fault 45Pumping 38Pumping logic PCB 33, 207Purge gas 152, 185

Index


RRamp 66Readbacks 68Rear panel 28, 207Reciprocating pump 144, 159Reference compound 106, 215Reserpine 21, 22, 23, 46Restrictor 153Retention 91Reverse phase 159, 171RF generator 33RF generator control PCB 33RF lens

See: Hexapole transfer lensRF power PCB 207Ripple 208Rotary control 31, 36Rotary pump 180Rotary pump 13, 15, 18, 36, 45, 183

Oil 35, 38, 184Rubidium iodide 157, 219, 221, 223Run duration 72

SSaccharides 158Sample cone 154, 185, 186Scan control PCB 33Scan duration 89Scan time 73Scope 66Selected ion recording 23

See: SIRSensitivity 208

LC-MS 160Set Mass 72Shutdown 48

Automatic 50Complete 49Editor 50Emergency 48Overnight 48

Single ion recordingSee: SIR

SIR 160SIR data 78SIR function 90Sodium iodide 157, 219, 221, 223Solvent delay 86Source 185, 196

Housing 34Source block

Ion block 190Source temperature 41, 43, 154, 176Source voltages 69Specifications 13Split, post-column 146, 159Start mass 72Start up 35

Automatic 50Status 26Structural elucidation 21, 22Submitter 73Sugar mixture 157, 220Survey 95Syringe pump 17, 144, 159System manager 81

Index


TTarget compound analysis 160Task 51, 73TEA

See: TriethylamineTetrahydrofuran 159THF

See also: TetrahydrofuranThreshold parameters 107Thresholds 77Toxicology 23TPC

See: Transputer processor cardTrace 63Trace enrichment 160Transformer 13Transputer processor card 33Triethylamine 159Trifluoroacetic acid 159Tune page 58Tuning 57

APcI 128, 174Electrospray 106

Turbomolecular pump 18, 30, 34, 45, 180

UUV detector 146UV detector 24, 28, 159UV photodiode array detector 24

VVacuum 18, 180

Leak 45, 181Protection 44

Vacuum LED 26, 45Vibration 208

WWater cooling 15, 30, 35, 45Weights 13

ZZero 67

Index


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