Micromass Quattro Ultima User Manual

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Quattro Ultima

User's Guide

Micromass UK Limited

Floats Road

Wythenshawe

M23 9LZ

Tel: +44 161 945 4170 Fax: +44 161 998 8915

Tudor Road

Altrincham

WA14 5RZ

Tel: +44 161 282 9666 Fax: +44 161 282 4400

http://www.micromass.co.uk

The instrument is marked with this symbol where high voltages are

present.

The instrument is marked with this symbol where hot surfaces are

present.

The instrument is marked with this symbol where the user should refer to

this User's Guide for instructions which may prevent damage to the

instrument.

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

injury.

If the instrument is used in a manner not specified by the manufacturer, the protection

provided by the equipment may be impaired.

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

instrument.

All information contained in these manuals is believed to be correct at the time of

publication. The publishers and their agents shall not be liable for errors

contained herein nor for incidental or consequential damages in connection with

the furnishing, performance or use of this material. All product specifications, as

well as the information contained in this manual, are subject to change without

notice.

Code: 6666511 Issue 3 © Micromass Ltd.

Quattro Ultima User's Guide

Hardware Specifications

Dimensions 11 Weights 11

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

Rotary Pumps 13

API Gas Exhaust 13 Nitrogen 14 CID Gas 14

Quattro Ultima

User's Guide

Contents

Instrument Description

Overview 15 Vacuum System 16 Ionisation Techniques 17

Atmospheric Pressure Chemical Ionisation 17

Electrospray 17

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

The Daughter Ion Spectrum 19 The Parent Ion Spectrum 20 MRM: Multiple Reaction Monitoring 21

The Constant Neutral Loss Spectrum 22 Data System 22 Front Panel Connections 23

Desolvation Gas and Probe Nebuliser Gas 23

Capillary / Corona 23

ESI / APcI 23 Front Panel Controls and Indicators 24

Status Display 24

Vacuum LED 24

Operate LED 24 Flow Control Valves 25 Divert / Injection Valve 25

Table of Contents

Quattro Ultima User's Guide

Rear Panel Connections 26

Internal Layout 30

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

Electronics 30 Mechanical Components 32

Routine Procedures

Start Up Following a Complete Shutdown 33

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

Start Up Following Overnight Shutdown 37

Preparation for Electrospray Operation 38 Preparation for APcI Operation 40 Operate 42

Automatic Pumping and Vacuum Protection 42

Overview 42 Protection 42

Transient Pressure Trip 42 Pump Fault 43

Power Failure 43 Tuning 44 Calibration 44 Data Acquisition 44 Data Processing 44 Setting Up for MS-MS Operation 44

Parent Ion Selection 44 Fragmentation 45

Shutdown Procedures 46

Emergency Shutdown 46 Overnight Shutdown 46 Complete Shutdown 47

Table of Contents

Tuning

Quattro Ultima

User's Guide

Automatic Start up and Shutdown 48

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

Overview 55

The Tune Page 56

Printing Tune Information 56

Experimental Record 56

Saving and Restoring Parameter Settings 56

Modifying the Peak Display 58

Changing the Display 60

Customise Plot Appearance 60 Trace 61 Intensity 61

Grid 61 AutoTune 62 Ion Mode 63 Scope Parameters 64 Gas Controls 64 Ramp Controls 64 Resetting the Zero Level 65 Controlling Readbacks 66 Changing Tune Parameter Settings 67 Source Voltages 67

Data Acquisition

Starting an Acquisition 69

Starting an Acquisition from the Tune Page 69

Parameters 70

Multiple Samples 71

Process 72 Automated Analysis of Sample List 72

Monitoring an Acquisition 74

The Acquisition Status Window 74

Chromatogram Real-Time Update 74

Spectrum Real-Time Update 74

Table of Contents

Quattro Ultima User's Guide

Instrument Data Thresholds 75

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

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

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

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

Setting up a SIR Function 88

Channels 88 Method 89 Retention Window 89

Setting up MS-MS Scanning Functions 90

Mass 90 Collision Energy 92

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

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

Mass Calibration

Introduction 101 Overview 102

Calibration Types 102 The Calibration Process 103

Table of Contents

Quattro Ultima

User's Guide

Electrospray 103

Introduction 103

Preparing for Calibration 104

Reference Compound Introduction 104 Tuning 104 Instrument Threshold Parameters 105

Calibration Options 106

Selecting the Reference File 106 Removing Current Calibrations 106

Selecting Parameters 107

Automatic Calibration Check 107 Calibration Parameters 108 Mass Measure Parameters 109

Performing a Calibration 110

Acquisition Parameters 112 Starting the Calibration Process 114

Checking the Calibration 116

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

Electrospray Calibration with PEG 124 Atmospheric Pressure Chemical Ionisation 125

Introduction 125

Preparing for Calibration 126

Reference Compound Introduction 126 Tuning 126

Calibration Options 126

Selecting Reference File 126

Removing Current Calibrations 126 Selecting Calibration Parameters 126 Performing a Calibration 127

Static Calibration 127

Scanning Calibration and Scan Speed Compensation 132 Calibration Failure 135 Incorrect Calibration 136 Manual Editing of Peak Matching 137 Saving the Calibration 137 Manual Verification 138

Electrospray

Introduction 141

Post-column Splitting 144 Megaflow 145

Changing Between Flow Modes 145

Table of Contents

Quattro Ultima User's Guide

Operation 146

Checking the ESI Probe 147 Obtaining an Ion Beam 148

Tuning and Optimisation 148 Megaflow Hints 154 Removing the Probe 154

Sample Analysis and Calibration 155

General Information 155

Typical ES Positive Ion Samples 156

Typical ES Negative Ion Samples 156

Chromatographic Interfacing 157

LC-MS Sensitivity Enhancement 158

Nanoflow Electrospray

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

Restarting the Spray 165

Nano-LC Option 166

Installation 166 Operation 167

Changing Options 168

Atmospheric Pressure Chemical Ionisation

Introduction 169 Preparation 170

Checking the Probe 171

Obtaining a Beam 172 Calibration 173 Hints for Sample Analysis 174

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

Corona Current 175

Probe Position 175

Probe Temperature 175

Desolvation Gas 175

Removing the Probe 176

Maintenance and Fault Finding

Introduction 177 Cooling Fans and Air Filters 177

Table of Contents

Quattro Ultima

User's Guide

The Vacuum System 178

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

The Source 183

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

The Electrospray Probe 196

Overview 196 Replacement of the Stainless Steel Sample Capillary 198

The APcI Probe 200

Cleaning the Probe Tip 200 Replacing the Probe Tip Heater 201

Replacing the Fused Silica Capillary 202 The Analyser 204 The Detector 204 Electronics 205

Fuses 205

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

Fault Finding Check List 206

No Beam 206

Unsteady or Low Intensity Beam 206

Ripple 206

High Noise Level in MRM Analyses 207

Chemical Noise 207

Electronic Noise 208 High Back Pressure 208 General Loss of Performance 209

Cleaning Materials 210 Preventive Maintenance Check List 211

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

Table of Contents

Quattro Ultima User's Guide

Reference Information

Overview 213 Editing a Reference File 214 Positive Ion 215

Horse Heart Myoglobin 216 Polyethylene Glycol 216

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

Negative Ion 218

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

Preparation of Calibration Solutions 220

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

Method 1 221

Method 2 221 Sodium Iodide Solution for Negative Ion Electrospray 221

216

Table of Contents

Dimensions

180mm

Quattro Ultima

User's Guide

Hardware Specifications

200mm

(pumping line)

1325mm

535mm

Weights

120mm

(ventilation)

700mm

Instrument: 150kg (330lb)

Data system

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

Rotary pumps

E2M28: 40kg (90lb) E1M18: 32kg (72lb)

Transformer (optional): 100kg (220lb)

Hardware Specifications

Page 11

Quattro Ultima User's Guide

Lifting and Carrying

Warning: Persons with a medical condition, for example a back injury, which

prevents them from handling heavy loads should not attempt to lift the instrument.

Before lifting the instrument proceed as follows:

Vent and power down the instrument.

Disconnect the instrument from the power and water supplies.

Disconnect power and tubing connections to the rotary pump from the rear of the instrument.

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

Disconnect all connections to LC equipment.

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

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

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

Caution: Under no circumstances should the instrument be lifted by the probe or

the source enclosure.

Before undertaking any lifting, lowering or moving of the instrument:

•

Assess the risk of injury.

•

Take action to eliminate the risk.

•

Plan the operation.

•

Use trained people.

•

Refer to local or company guidelines before attempting to lift the instrument.

Micromass accept no responsibility for any injuries or damage sustained while lifting the instrument.

Hardware Specifications Page 12

Power

Environment

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

Quattro Ultima

User's Guide

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

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

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

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

Overall heat dissipation

(excluding LC

and optional water chiller): 4.2kW maximum

Water Cooling

Heat dissipation into the water: 200W

Exhausts

Rotary Pumps

The rotary pumps must be vented to atmosphere (external to the laboratory) via a

fume hood or industrial vent.

API Gas Exhaust

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

Caution: The API gas exhaust line must not be connected to the rotary pump

exhaust line. In the event of an instrument failure, rotary pump exhaust could be admitted into the source chamber producing severe contamination.

Humidity: Relative humidity ≤70%

Hardware Specifications

Page 13

Quattro Ultima User's Guide

Nitrogen

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

CID Gas

Argon 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: 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 of plastic leads to contamination of the instrument.

Caution: Operating with the CID gas at a significantly higher pressure results in

a fault.

Hardware Specifications Page 14

Overview

Quattro Ultima

User's Guide

Instrument Description

Sample Inlet

Sampling Cone

and Ion Block

Ion Tunnels

Prefilter 1

Samples from the liquid introduction system are introduced at atmospheric pressure into the ionisation source.

Ions are sampled through a series of orifices.

The ions are filtered according to their mass to charge ratio ( ).

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.

Quadrupole 1

MassLynx NT

Data System

Collision Cell

Prefilter 2

Quadrupole 2

Detector

The Micromass Quattro Ultima is a high performance benchtop triple quadrupole mass

spectrometer designed for routine LC-MS-MS operation. Quattro Ultima may be

coupled 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 sampled

through a series of orifices into the first quadrupole where they are filtered according

to their mass to charge ratio (m).

The mass separated ions then pass into the hexapole collision cell where they either

undergo collision induced decomposition (CID) or pass unhindered to the second

quadrupole. The fragment ions are then mass analysed by the second quadrupole.

Finally the transmitted ions are detected by a conversion dynode, phosphor and

photomultiplier detection system. The output signal is amplified, digitised and

presented to the data system.

Instrument Description

Page 15

Quattro Ultima User's Guide

Vacuum System

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

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

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

Vacuum measurement is by an active inverted magnetron (Penning) gauge for the analyser and a Pirani gauge for the gas cell. The Penning gauge acts as a vacuum switch, switching the instrument out of the

OPERATE mode if the pressure is too high.

The speed of each turbomolecular pump is also monitored and the system is fully interlocked to provide adequate protection in the event of a fault in the vacuum system, a failure of the power supply or vacuum leaks.

Instrument Description Page 16

Ionisation Techniques

Two atmospheric pressure ionisation techniques are available.

Atmospheric Pressure Chemical Ionisation

Atmospheric pressure chemical ionisation (APcI) generally produces protonated or

deprotonated molecular ions from the sample via a proton transfer (positive ions) or

proton abstraction (negative ions) mechanism. The sample is vaporised in a heated

nebuliser before emerging into a plasma consisting of solvent ions formed within the

atmospheric source by a corona discharge. Proton transfer or abstraction then takes

place between the solvent ions and the sample. Eluent flows up to 2 ml/min can be

accommodated without splitting the flow.

Electrospray

Electrospray (ESI) ionisation takes place as a result of imparting a strong electrical

field to the eluent flow as it emerges from the nebuliser. producing an aerosol of

charged droplets. These undergo a reduction in size by solvent evaporation until they

have attained a sufficient charge density to allow sample ions to be ejected from the

surface of the droplet (“ion evaporation”).

Quattro Ultima

User's Guide

A characteristic of ESI spectra is that ions may be singly or multiply charged. Since

the mass spectrometer filters ions according to their mass-to-charge ratio, compounds

of high molecular weight can be determined if multiply charged ions are formed.

Eluent flows up to 1 ml/min can be accommodated although it is often preferable with

electrospray ionisation to split the flow such that 100 to 200 µl/min of eluent enters

the mass spectrometer.

Nanoflow Electrospray

The optional nanoflow interface allows electrospray ionisation to be performed in the

flow rate range 5 to 1000 nanolitres per minute.

For a given sample concentration, the ion currents observed in nanoflow are

comparable to those seen in normal flow rate electrospray. Great sensitivity gains are

therefore observed when similar scan parameters are used, due to the great reductions

in sample consumption.

Sample Inlet

Sample is introduced from a suitable liquid pumping system along with the nebulising

gas to either the APcI probe or the electrospray probe. For nanoflow electrospray,

metal coated glass capillaries allow the lowest flow rates to be obtained while fused

silica capillaries are used for flow injection analyses or for coupling to nano-HPLC.

Instrument Description

Page 17

Quattro Ultima User's Guide

MS Operating Modes

Source MS1 Collision Cell MS2 Detector

MS1 Collision Cell MS2

MS2

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

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

Resolving RF Only (Pass all masses)

RF Only (Pass all masses) Resolving

MS-MS Operating Modes

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

Daughter Ion

Spectrum

(parent mass selection)

MS1

Static

Collision

Cell

MS2

Scanning

Parent Ion

Spectrum

Multiple Reaction

Monitoring

Constant Neutral

Loss Spectrum

Instrument Description Page 18

Scanning

Static

(parent mass selection)

Scanning (synchronised

with MS2)

RF only (pass all

masses)

Static

(daughter mass

selection)

Static

(daughter mass

selection)

Scanning (synchronised

with MS1)

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 specific daughter ion to be used in MRM analysis.

Example:

Daughters of the specific parent at m 609 from reserpine in electrospray positive ion mode.

Quattro Ultima

User's Guide

The result:

MS1

static at m/z 609

(parent mass)

Collision Cell

RF only

(pass all masses)

MS2

scanning from

m/z 100 to 650

Instrument Description

Page 19

Quattro Ultima User's Guide

The Parent Ion Spectrum

Typical application:

Structural elucidation.

•

Complementary or confirmatory information (for daughter scan data).

Example:

Parents of the specific daughter ion at m 195 from reserpine in electrospray positive ion mode.

The result:

MS1

scanning from

m/z 50 to 650

Collision Cell

RF only

(pass all masses)

MS2

static at m/z 195 (daughter mass)

Instrument Description Page 20

MRM: Multiple Reaction Monitoring

This mode is the MS-MS equivalent of SIR (Selected Ion Recording). As both MS1

and MS2 are static, this allows greater “dwell time” on the ions of interest and

therefore better sensitivity (~100×) compared to scanning MS-MS.

Typical application:

Rapid screening of “dirty” samples for known analytes.

•

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

Example:

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

Quattro Ultima

User's Guide

Collision Cell

MS1

static at m/z 609

(parent mass)

The result:

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

Time

LC-MRM

High specificity

Good signal / noise

RF only

(pass all masses)

Poor signal / noise

static at m/z 195 (daughter mass)

Time

LC-MS

Low specificity

MS2

Instrument Description

Page 21

Quattro Ultima User's Guide

The Constant Neutral Loss Spectrum

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

Typical applications:

Screening mixtures, for example during neonatal screening, for a specific class

•

of compound that is characterised by a common fragmentation pathway.

MS1

scanning

Collision Cell

RF only

(pass all masses)

MS2

scanning

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

The result:

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

Data System

The PC based data system, incorporating MassLynx NT™ software, controls the mass spectrometer detector and, if applicable, the HPLC system, autosampler, syringe pump, divert valve or injector valve.

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

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

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

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

Instrument Description Page 22

Front Panel Connections

Quattro Ultima

User's Guide

CAPILLARY / CORONA

ESI / APci

VACUUM

OPERATE

C.I.D. GAS

INJECTOR

STANDBY

DESOLVATION

GAS

NEBULISER

CONE GAS

LOAD

INJECT

LC Connection

High Voltage

Connection

Source

Connection

Desolvation Gas and Probe Nebuliser Gas

The PTFE gas lines for the desolvation gas and probe nebuliser gas are connected to

the front of the instrument using threaded metal fittings.

Capillary / Corona

The electrical connection for the ESI capillary or the APcI discharge pin is via the

coaxial high voltage connector.

ESI / APcI

The electrical connection for the APcI probe or the ESI heater is via the multi-way

connector. This is removed from the front panel by pulling on the metal sleeve of the

plug. Both the electrospray and APcI heaters use this connector.

Desolvation

Gas

Nebuliser

Gas

Instrument Description

Page 23

Quattro Ultima

User's Guide

Front Panel Controls and Indicators

CID Gas

ontrol

CAPILLARY / CORONA

ESI / APci

VACUUM

OPERATE

Cone Gas

Control

STANDBY

DESOLVATION

GAS

NEBULISER

C.I.D. GAS

INJECTOR

Desolvation Gas Control

Nebuliser

Gas Control

CONE GAS

LOAD

INJECT

Status Display

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

The display generated by the Vacuum LED is dependent on the vacuum status of the instrument. The Operate LED depends on both the vacuum status and whether the Operate mode has been selected from the Data System.

The status of the instrument is indicated as follows:

Vacuum LED

Pumping Steady amber Pump fault Flashing red

Operate LED

Standby No indication

State Vacuum LED State Vacuum LED

Vented No indication Vacuum OK Steady green

State Operate LED State Operate LED

Transient pressure

trip

Steady amber

Operate Steady green RF trip Flashing red

Instrument Description Page 24

Flow Control Valves

The Desolvation Gas, Cone Gas and Nebuliser needle valves are five-turn

valves. The CID Gas valve is a fifteen-turn valve. The flow increases as the valve is

turned counterclockwise.

Caution: To prevent damage to the CID Gas valve, take care not to

over-tighten when turning the supply off.

Divert / Injection Valve

Quattro Ultima

User's Guide

Load

Divert / Injection

Valve

The optional divert / injection valve may be used in several ways depending on the

plumbing 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 syringe pump 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 always connected 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 marked

Load and Inject enable the user to override control of the valve when making loop

injections at the instrument.

Inject

Instrument Description

Page 25

Quattro Ultima User's Guide

Rear Panel Connections

Event Out

Four outputs, Out 1 to Out 4, are provided to allow various peripherals to be connected to the instrument. Switches S1 to S4 allow each output to be set to be either a contact closure (upper position) or a voltage output (lower position).

Out 1 and Out 2, when set to voltage output, each have an output of 5 volts. The voltage output of both Out 3 and Out 4 is 24 volts.

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

Contact Closure In

In 1 and In 2 inputs are provided to allow external device to start sample acquisition

once the device has performed its function (typically sample injection).

SCOPE

DATA SYSTEM

+- +-

CH3

MUX

ANALOG CHANNELS EVENT OUT

CLOSURE

IN 2IN 1

CH4 1V

CONTACT

CH2

CH1

REFER TO MANUAL BEFORE CONNECTING TO

THESE PORTS

CAUTION !

OUT 4OUT 3OUT 2OUT 1

S4S3S2S1

Analog Channels

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

Instrument Description Page 26

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 network cable supplied.

Quattro Ultima

User's Guide

Instrument Description

Page 27

Quattro Ultima

User's Guide

Power

Cord

Electrostatic

Discharge

Earth (Ground)

Point

Rotary Pump

Control

Mains

Switch

Fuses

Water

Backing

Line

Source

Pumping Line

Hexapole

Pumping Line

To Rotar

Pumps

Water is used to cool the turbomolecular pumps.

CID Gas

Exhaust

Gas

ROTARY PUMP

CONTROL

ROTARY PUMP

CONTROL

CID

GAS

NITROGEN GAS

OUTLET INLET

Nitrogen

Gas In

ESD

EARTH

FACILITY

OUT IN

10 AMP (T)

WATER

Water

Connections

Nitrogen Gas In

The nitrogen supply (100 psi, 7 bar) should be connected to the Nitrogen Gas In push-in connector using 6mm PTFE tubing. If necessary this tubing can be connected to ¼ inch tubing using standard ¼ inch fittings.

Caution: Use only PTFE tubing or clean metal tubing to connect between the

nitrogen supply and the instrument. The use of other types of plastic tubing results in chemical contamination of the source.

Instrument Description Page 28

Exhausts

CID Gas

Quattro Ultima

User's Guide

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

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

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

Caution: Do not connect these two exhaust lines together as, in the event of an

instrument failure, rotary pump exhaust could be admitted into the source chamber producing severe contamination.

Argon is required as collision gas. See Hardware Specifications for details.

Power Cord

The mains power cord should be wired to a suitable mains outlet using a standard plug. For plugs with an integral fuse, the fuse should be rated at 13 amps.

Mains Switch

The mains switch switches mains power to the instrument.

Fuses

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

Rotary Control

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

ESD Earth Facility

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

Instrument Description

Page 29

Quattro Ultima User's Guide

Internal Layout

Electronics

High Voltage

Power Supplies (4)

Transputer Processor

Card (TPC)

Analogue PCB

Control PCB

Scan Control PCB

RF Control (Upper) PCB

Analyser

Turbomolecular

Pump

Low Voltage

Power

Supplies (2)

Pumping

Logic PCB

Power

Sequence

PCB

RF Control (Lower) PCB

RF Generators

Turbomolecular

Pumps

Power Supply

Instrument Description Page 30

Quattro Ultima

User's Guide

The main electronics modules of the system are:

Two low voltage power supplies.

•

Four high voltage power supplies, plugged into the backplane below the analyser

•

housing.

These supply the detector system and the high voltages for the source and electrospray probe.

Two RF generators, bolted to the side of the analyser housing.

•

Pumping Logic PCB.

•

This controls the turbomolecular pumps, the pumping sequence, the gas valves and the solenoids. It also controls the phosphor and dynode voltages.

Power Sequence PCB.

•

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

Transputer Processor Card (TPC).

•

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

•

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 also supplies the collision cell voltages.

•

RF Generator Control (Lower) PCB

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

Instrument Description

Page 31

Quattro Ultima User's Guide

Mechanical Components

Air Filter

(behind cover)

Source

Housing

Analyser

Housing

Source

Turbomolecular

Pump

Detector

Active Inverted

Magnetron

(Penning) Gauge

Pirani

Gauge

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 above housings.

•

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

•

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

Instrument Description Page 32

Routine Procedures

Start 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 in the rotary pump sight glass. Refill or replenish as necessary as described in the pump manufacturer’s literature.

Connect a supply of dry, high purity nitrogen to the connector on the service panel at the rear of the instrument. Adjust the outlet pressure to 7 bar (100 psi).

Gas

Ballast

Exhaust

Quattro Ultima

User's Guide

Oil Mist

Filter

Filler

Plug

Oil Level Indicator

Drain

Plug

Connect a supply of argon to the CID Gas connector on the service panel at the rear of the instrument. Adjust the outlet pressure to approximately 350 mbar (5 psi).

Connect the water supply to the connections at the rear of the instrument.

CID Gas

Nitrogen

Gas In

ROTARY PUMP

CONTROL

ROTARY PUMP

CONTROL

CID GAS

NITROGEN GAS

OUTLET INLET

ESD

EARTH

FACILITY

WATER

OUT IN

10 AMP (T)

Water

Connections

Routine Procedures

Page 33

Quattro Ultima User's Guide

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

REFER TO

MANUAL BEFORE

Data System

Connection

SCOPE

MUX

DATA SYSTEM

CONNECTING TO

THESE PORTS

CONTACT

CH2

CH1 1V

+- +-

CH3 1V

ANALOG CHANNELS EVENT OUT

CLOSURE

CH4 1V

IN 2IN1

Mains

Switch

Rotary Pump

Control

CAUTION !

S4S3S2S1

OUT 4OUT 3OUT 2OUT 1

ROTARY PUMP

CONTROL

ROTARY PUMP

CONTROL

CID GAS

NITROGEN GAS

OUTLET INLET

ESD

EARTH

FACILITY

OUT IN

10 AMP (T)

WATER

Exhaust

Gas

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

Check that the data system is connected to the mass spectrometer via the network 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 an

instrument failure, rotary pump exhaust could be admitted into the source chamber, producing severe contamination.

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

Routine Procedures Page 34

Quattro Ultima

User's Guide

Switch on the data system.

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

Windows NT and MassLynx NT can be configured to prevent unauthorised access. Consult the system administrator for any passwords that may be requested.

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

Launch the tune page by clicking on .

Routine Procedures

Page 35

Quattro Ultima User's Guide

Pumping

Caution: To minimise wear to the lubricated components of the rotary pump, the

manufacturers recommend that the pump is not started when the oil temperature is below 12°C.

Pump down time may be decreased by closing the isolation valve of the source during pump down.

Isolation

Valve

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

Click on Pump.

The rotary pump and the turbomolecular pumps start simultaneously.

The Vacuum LED on the front of the instrument shows amber as the system pumps down.

When the system has reached operating vacuum the LED changes to a steady green, indicating that the instrument is ready for use.

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 gas ballast 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 is

running under gas ballast. See Maintenance and Fault Finding for more information.

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

Routine Procedures Page 36

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 is

switched on only when the pressure needs to be monitored. Leaving the analyser pressure 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 vacuum chamber. The magnitude of the discharge current is then measured and used to calculate the analyser pressure. An undesirable characteristic of this type of gauge is the slow build up of sputtered material in the discharge region, eventually leading to failure of the gauge.

To switch on the gauge:

Quattro Ultima

User's Guide

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 the pressure window is displayed prior to starting the acquisition. However, the gas cell 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 and prepare for running samples, follow the instructions in Start Up Following Overnight Shutdown in the following pages.

Start Up Following Overnight Shutdown

The instrument will have been left in standby mode under vacuum.

It is recommended that the data system is left on overnight. However, if the data system has been switched off, switch it on as described in the preceding section.

The display on the front of the instrument displays a steady green Vacuum LED indicating that the instrument is ready for use.

Routine Procedures

Page 37

Quattro Ultima User's Guide

Preparation for Electrospray Operation

Source

Thumb Nuts

Probe

Thumb Nuts

If the corona discharge pin is fitted, proceed as follows:

Disconnect the gas and electrical connections from the front panel.

Unscrew the probe thumb nuts and remove the probe

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

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

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

Glass

Tube

Probe

Adjustment Flange

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

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

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

Routine Procedures Page 38

Replace the glass tube and adjustment flange.

Cleanable

Baffle

Exhaust

Liner

Blanking

Plug

Quattro Ultima

User's Guide

Corona

Discharge

Mounting

Contact

High Voltage

Socket

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

Connect the liquid flow of a LC system or syringe pump to the probe.

Insert the probe into the source and tighten the two thumb nuts to secure the probe firmly.

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

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 results

in solvent vapour escape and the exposure of hot surfaces and high voltages.

Warning: The ion source block can be heated to temperatures of 150°C, and is

maintained at the set temperature when the source enclosure is removed. Touching the ion block when hot may cause burns to the operator.

Routine Procedures

Page 39

Quattro Ultima User's Guide

Preparation for APcI Operation

Source

Thumb Nuts

Glass

Tube

Probe

Thumb Nuts

Probe

Adjustment Flange

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 and glass tube.

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

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

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

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

Routine Procedures Page 40

Replace the glass tube and adjustment flange.

Quattro Ultima

User's Guide

Blanking

Plug

Sample

Cone

Corona

Discharge

Mounting

Contact

High Voltage

Socket

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 the probe firmly.

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 150°C.

Warning: Operating the source without the source enclosure results

in solvent vapour escape and the exposure of hot surfaces and high voltages. Allow the glass source enclosure to cool after a period of operation at high flow rates before removal.

Warning: The ion source block can be heated to temperatures of 150°C, and is

maintained 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 are switched on with the probe inserted.

Routine Procedures

Page 41

Quattro Ultima User's Guide

Operate

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.

Depending on the chosen mode of ionisation, set Desolvation Temp or APcI 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 is in place and a probe is inserted.

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

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

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

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 system switching on the rotary pump and turbomolecular pumps simultaneously.

Protection

Transient Pressure Trip

If the vacuum gauge detects a pressure surge above the factory set trip level of

-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 Page 42

Quattro Ultima

User's Guide

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 Operate LED’s are steady green.

Any further deterioration of the system vacuum results in a pump fault and the system is 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 and the 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 their temperature 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 this chapter.

Routine Procedures

Page 43

Quattro Ultima User's Guide

Tuning

Information concerning the tuning of Quattro Ultima is provided in Tuning later in this document. Refer also to Electrospray, Nanoflow Electrospray and Atmospheric Pressure Chemical Ionisation for tuning information specific to those techniques.

Calibration

Information concerning the calibration of Quattro Ultima is provided in Mass Calibration later in this document.

Data Acquisition

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

Data Processing

The processing of sample data is comprehensively described in the MassLynx NT User’s Guide. Refer to that publication for full details.

Setting Up for MS-MS Operation

The following notes provide a worked example for the acquisition of daughter ion data. The experiment is performed in the ESI positive mode using reserpine as a model analyte. 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 MS and 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 Data Acquisition later in this manual.

Parent Ion Selection

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

Routine Procedures Page 44

Quattro Ultima

User's Guide

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 z 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 mass of the parent ion selected by MS1, in this case 609.

Vary the Set value between 608.5 and 609.5 while optimising the intensity of the 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 Span parameters for peak 2.

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

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

Select Gas, Collision Gas.

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

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

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

•

Optimising Collision.

•

Optimising Exit.

•

Optimising Entrance.

•

Optimising the collision gas pressure using the CID Gas needle valve.

Additionally, transmission can be improved at the expense of specificity by reducing HM Resolution 1 and increasing Ion Energy 1 on the Analyser window. In most cases, where chemical interference with the parent ion is not acute, the loss of specificity is negligible.

Routine Procedures

Page 45

Quattro Ultima User's Guide

Shutdown Procedures

Emergency Shutdown

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

Switch OFF the power at the wall mounted isolation switch(es), if fitted. If not, switch the power off at the rear of the instrument and switch off all peripherals.

Isolate any LC systems to prevent solvent flowing into the source.

A loss of data is likely.

Overnight Shutdown

When the instrument is to be left unattended for any length of time, for example overnight or at weekends, proceed as follows:

Switch off the LC pumps.

On the MassLynx top-level window, click on to launch the tune page.

Click on .

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

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

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

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

Caution: Leaving the APcI probe hot with no gas or liquid flow shortens the

lifetime 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 Page 46

Complete Shutdown

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

Switch off the LC pumps.

On the MassLynx top-level window, click on to launch the tune page.

Click on on the tune page.

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

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

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

Quattro Ultima

User's Guide

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

Caution: Leaving the APcI probe hot with no gas or liquid flow shortens the

lifetime of the probe heater.

Select Gas, Gas to turn off the supply of nitrogen gas.

Select Other from the menu bar at the top of the tune page. Click on Vent.

The rotary pump and the turbomolecular pumps switch off. When the turbomolecular pumps have run down to half of their normal operating speed the vent valve opens and the instrument is vented to atmosphere.

Exit MassLynx.

Shut down the computer.

Switch off all peripherals.

Switch off the power to the instrument using the switch on the rear panel of the instrument.

Switch off power at the wall mounted isolation switches.

If the instrument is to be switched off for more than one week:

Drain the oil from the rotary pump according to the manufacturer’s instructions.

Routine Procedures

Page 47

Quattro Ultima User's Guide

Automatic Start up and Shutdown

MassLynx comes with automatic startup and shutdown files which are run when Startup or Shutdown is selected from the Run menu.

These are found in the C:\Masslynx\Shutdown directory and are called ShutDownxxx.acl and StartUpxxx.acl where xxx refers to the instrument configuration.

The Shutdown Editor

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

Select Edit Shutdown from the MassLynx Run menu.

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

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

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

Routine Procedures Page 48

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

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

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

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

Enable Shutdown after batch option.

The Auto Control Tasks Page

Quattro Ultima

User's Guide

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 been

completed and before the next task is started. For example. a Post Delay of 60 seconds, in the Vent Instrument task, means that there is a delay of 60 seconds before 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

Page 49

Quattro Ultima User's Guide

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

To add a task:

To insert 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 task has been inserted into the task timetable then all subsequent tasks are added after the inserted task. To add a task to the end of the timetable after inserting a task, click twice with the left mouse button below the last entry in the timetable and then add the new task.

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

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 Page 50

To change the width of a column:

Position the mouse pointer on the heading between two columns until appears.

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

The Shutdown Editor Toolbar

Toolbar Button Menu Equivalent Purpose

Quattro Ultima

User's Guide

File… New

File… Open

File… Save

File… Save As

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

Create a new startup or shutdown file

Open an existing startup or shutdown file

Save a startup or shutdown file

Routine Procedures

Page 51

Quattro Ultima User's Guide

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 As dialog is displayed.

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

Routine Procedures Page 52

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.

Quattro Ultima

User's Guide

Select the printer, print range and number of copies and press OK.

Routine Procedures

Page 53

Quattro Ultima User's Guide

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 shutdown editor control list menu, then the automatic startup and shutdown files are run.

To run a different startup or shutdown file:

Open the required file in the shutdown editor and press , or select Run List from 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 Page 54

Overview

Quattro Ultima

User's Guide

Tuning

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

•

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

•

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

•

Care should be taken to optimise the value of the collision energy. Note that, in

Daughter and Parent modes, Collision and Exit are interactive parameters.

Tuning

Page 55

Quattro Ultima User's Guide

The Tune Page

To 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 Information

To print a report, containing a copy of the tune peak information displayed on the screen 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 Record

Tuning 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 data browser, see the MassLynx NT User Guide, Selecting and Viewing Data, for more information.

Saving and Restoring Parameter Settings

Whole sets of instrument tuning parameters can be saved to disk as a named file and then recalled at a future date.

A tune parameter file contains the latest settings for the source controls for all supported ionisation modes not just the ionisation mode currently selected. Tune parameter files also contain settings for the analyser, inlet set points and peak display.

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

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

Press Save.

Tuning Page 56

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.

Quattro Ultima

User's Guide

If the selected file already exists on disk a warning is displayed. Press Yes to overwrite 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 by selecting from the list displayed.

Press Open.

Tuning

Page 57

Quattro Ultima User's Guide

Modifying the Peak Display

The tune peak display is modified using either the tune peak controls, or the mouse directly 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 Mass box 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, pressing it 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 is pressed twice Mass returns to this value.

or:

Tuning Page 58

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

When appears, click the left mouse button and drag until the required mass is 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 is pressed twice Mass returns to this value.

Quattro Ultima

To change the span of a peak:

Press the left mouse button at one end of the region of interest and, without releasing the button, drag the mouse horizontally to the other end.

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

Do not go beyond the bounds of the axis.

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

This operation can be repeated as often as required.

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

User's Guide

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 is pressed 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 the gain 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, without releasing the button, drag the mouse vertically to the other end.

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

Do not go beyond the bounds of the axis.

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

or:

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

Tuning

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Changing the Display

To change the display using the mouse:

Click in the peak display area with the right mouse button to display 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 traces displayed:

Select Customise, Plot Appearance.

The Customise Plot Appearance dialog is displayed.

To change the colours on the display:

Press Newest Trace,

Background or Trace Fill and select a

new colour from the dialog displayed.

To change the number of traces:

Use to change the number, or enter a new value in the Visible Traces box, within the range 2 to 20.

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

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

Tuning Page 60

Trace

Intensity

Quattro Ultima

User's Guide

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 data points only.

The option selected has a tick next to it.

Grid

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.

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

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

Tuning

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AutoTune

MassLynx can automatically tune the mass spectrometer in both APcI and electrospray ionisation modes. AutoTune ramps the settings for the tuning parameters until they are optimised to give the best intensity, resolution and peak shape.

To run AutoTune:

Press on the tune page to turn on the API gas, and select Operate.

Choose AutoTune from the tune page Options menu.

Press Setup to define the AutoTune setup parameters.

There are two levels of AutoTune:

•

A full AutoTune starts from a default set of tuning parameters.

•

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

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

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

Press OK to exit.

Tuning Page 62

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.

Quattro Ultima

User's Guide

Checking that values are defined for all the user controllable instrument parameters 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/APcI AutoTune algorithm. This involves changing key parameters, one at a time, to maximise the intensity of a reference peak with respect to that parameter. At present ESP/APcI Autotuning is carried out with respect to a single user specified reference peak.

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

Ion Mode

Select the required ionisation mode from the Ion Mode menu. The selected mode has a tick next to it.

Press OK to return to the tune page.

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

Tuning

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Scope Parameters

Scan Time and Inter Scan Delay control the

speed with which the tune peak display is updated.

The tuning system behaves more responsively if the scan time and inter scan delay are short.

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 Controls

To 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 appears next to a gas if it is turned on.

Ramp Controls

To set up a cone voltage ramp:

Choose Cone Ramp Gradient from the tune page Ramps menu.

Two values of cone voltage are defined at two particular masses. These values define a gradient for the cone voltage which is then extrapolated to cover the full mass range.

Tuning Page 64

Make any changes required and press 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.

Quattro Ultima

User's Guide

To set up a collision energy ramp:

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

Two values of collision energy are defined at two particular masses. These values define a gradient for the collision energy voltage which is then extrapolated to cover the full mass range.

Make any changes required and press OK to exit.

To initiate the collision energy voltage ramp:

Press , or choose Use Collision Energy Ramp from the tune page

Ramps menu.

Resetting the Zero Level

The zero level (or baseline) can be repositioned by pressing , or by choosing Reinitialize from the tune page Options menu.

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

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

Tuning

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Controlling Readbacks

There are three options for displaying system readbacks on the tune page:

Readbacks displayed continuously.

•

Readbacks hidden.

•

Readbacks displayed only when

•

differing from their defined values by more than 10%.

A number of the readbacks are for diagnostic purposes only, their function being to confirm a voltage is present. The acceptable variation between the set value and the readback value varies depending on the particular tune parameter. If concerned 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.

Tuning Page 66

Check up to four boxes

to display the peaks.

Select to

display the

tune

parameters for

that region

Create a

new tune file

Open an

existing tune file

Print current window

in portrait format

Display tune

peak information

Display vacuum

information

Toggle on /

collision energ

Click on the arrow to

select the scan function

Edit window

Readback

window

Slider bar

Toolbar

Allows some

routine operations

to be performed

with a single click

Enabled for MS-MS

functions

rag the slider bar using the mouse.

APcI-ve

APcI+ve

ESI-ve

Not adjustable

Not applicable

ESI+ve

Tune Page

Name

Capillary +3.0 (kV) -3.0 (kV) Not applicable

Corona Not applicable 2 µA 2 µA

Cone +50 (V) -50 (V) +50 (V) -50 (V)

RF Lens 1 0 (V) 0 (V) 0 (V) 0 (V)

Aperture 0 (V) 0 (V) 0 (V) 0 (V)

RF Lens 2 +0.2 (V) -0.2 (V) +0.2 (V) -0.2 (V)

nent. When appropriate, the second column shows the term

Prefilter

Data Acquisition

Starting 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.

Acquisitions can be started and stopped.

Most of the scanning parameters can be controlled.

Quattro Ultima

User's Guide

Inlet programs cannot be used.

Analog data cannot be acquired.

Multiple sample sequences cannot be acquired.

To start a single sample acquisition:

Press Acquire on the tune page, or choose Acquire from the tune page Window menu.

The dialog shown can be configured to automatically set itself to acquire data using the mass range and function type that is being used for tuning the instrument.

Make any required changes to the settings.

Press Start.

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Parameters

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

To change the directory into which data are acquired:

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

Cancel the acquisition.

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

The type of acquisition Function used to collect the data can be any of the following:

•

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 the particular function type. This control is disabled if the function selected does not require 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 Page 70

Scan Time specifies the duration of each scan in seconds.

Inter Scan Time specifies the time in seconds between a scan finishing and the next

one starting. During this period no data are stored.

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

Submitter

•

Job

•

Task

•

Conditions

•

Multiple Samples

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

Quattro Ultima

User's Guide

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 and Auto 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 or Night Time Process has been checked.

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

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Process

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

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

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

For the process to automatically operate on the data file which has just been acquired:

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

Leave unchecked Use Acquired File as Default on the System tab of the MassLynx Options dialog.

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

Automated Analysis of Sample List

To display the quantify samples dialog:

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

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

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User's Guide

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 to

calculate compound concentrations.

Print Quantify Reports produces hard copies of the results of integration and quantification.

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

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

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

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

Data Acquisition

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Monitoring an Acquisition

Acquisition status is also shown on the MassLynx screen. The run time is shown on the MS panel and the scan status, sample number and scan number are shown on the Status 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 the progress of an acquisition.

To display the scan report dialog:

Select Acquisition Status.

This shows details of the scan currently being acquired.

Chromatogram Real-Time Update

To view in real time the chromatogram that is currently being acquired:

Open the data file using the MassLynx data browser.

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

Spectrum Real-Time Update

To view in real time the spectrum that is currently being acquired:

Open the data file using the MassLynx data browser.

Press , or select

Real-Time Update from the Display menu.

Data Acquisition Page 74

Select Enable Real-Time update. Real-time update can also be turned on and off via the Real-Time spectrum toolbar button.

When real-time update is on the display is continually updated with spectra from the current acquisition. The actual information displayed is determined by selecting one of the 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 all

•

the spectra that have so far been acquired.

Average latest scans updates the display with spectra formed by averaging

•

the last n scans acquired, where n is specified in the associated edit control.

Instrument Data Thresholds

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

Quattro Ultima

User's Guide

Instrument data thresholding allows the user to specify the type of data to acquire and write to disk, and the type of data to discard and not write to disk. Limiting the amount of data stored on disk can be particularly desirable when acquiring continuum data 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

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MaxEnt

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

Profile Data

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

Baseline Level is used to lift or drop the baseline to see more or less of the noise. It is used when Ion Counting Thresholding is disabled (set to zero) to set the position 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 form of thresholding to be applied to counting and therefore has a less significant effect.

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

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

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

•

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 larger peaks of interest.

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

/16 amu type samples. This takes place after ion

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.

SIR Data

SIR Baseline Level sets the position of the SIR baseline above zero when Ion Counting Threshold is not enabled (i.e. set to zero). The baseline level is

typically set to 0. Increasing the value causes the baseline to appear higher.

Data Acquisition Page 76

Ion Counting Threshold

Ion Counting Threshold sets the intensity level below which a data point is

ignored. This threshold is applied to all acquisitions, regardless of scanning mode. It is also the most significant of all of the data manipulation variables as it is applied to the raw data first.

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

The Ion Counting Threshold level entered is multiplied by the standard deviation of 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 is

•

discarded.

If a value of zero is entered the intensity level is set so that it sits in the middle

•

of the noise which means that roughly half of the noise data is acquired.

Quattro Ultima

User's Guide

A value of 10 places the threshold just above the noise so almost all of the data

•

is acquired.

If a value of 60 is entered the threshold sits well above the noise level, so very

•

little 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 removed

without significantly reducing the intensity of the smallest peaks of interest.

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

Data Acquisition

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Baseline Level

Threshold

00 0 0

10 0

20 0

50 0

Ion Count

Background

Noise

Typical

Profile

Peak

Typical

Typical Intensity

Reduction

on .DAT File Size

Typical Saving

010 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 to be spikes are removed by setting the value of this data point to the average of its immediate neighbours.

Data Acquisition Page 78

Quattro Ultima

Spike removal involves some additional processing while acquiring and reduces the 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 intensity threshold below which spikes are ignored. Set Minimum Spike Intensity to this value.

A very low intensity signal may include single ion events that can be combined to produce significant peaks. For this type of data Minimum Spike Intensity should be set to a suitable value such that these single ion events are not discarded as spikes.

User's Guide

Set a suitable value for Spike Percentage Ratio.

This ratio is used to determine if a data point is a spike by comparing the data point to its immediate neighbours. For example, with Spike Percentage Ratio set to 33%, a data point is regarded as a spike if its intensity is 3 times (or more) greater than both its immediate neighbours. A setting 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 Manager

To check the communications between the MassLynx software and the embedded PC:

Select Communications Status.

Data Acquisition

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Stopping an Acquisition

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

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

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

Data Acquisition Page 80

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User's Guide

A simple function list is shown above, containing only one function: a centroided mode full scan, between 500 and 1500 amu using ES+ ionisation. Immediately above the 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 runs for 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 sequentially for 5 minutes, is shown below.

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

Data Acquisition

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The Function List Editor Toolbar

The toolbar is displayed at the top of the tune window and allows some common operations 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 to disk.

Print the current window in portrait format.

Adding a New Function

To add a new function to the list:

Click one of the toolbar buttons, or select the required function from the

Function menu.

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

Move the selected function up the list of functions.

Move the selected function down the list of functions.

Create a new function of the indicated type.

Data Acquisition Page 82

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 new function.

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

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 to be made.

The function list display is updated to show any changes.

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

Quattro Ultima

User's Guide

Copying an Existing Function

To copy an existing function:

Select the function in the function list.

Select Copy and then Paste from the Edit menu.

Modify the parameters as described above.

Removing a Function

To remove a function:

Select the function in the function list.

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 order cannot be changed. For functions that have the same start and end time the order in which 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

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Setting a Solvent Delay

No data is stored during the solvent delay period, which means that solvent peaks that would normally be seen eluting on the TIC chromatogram are no longer seen.

For APcI functions the APcI probe temperature is set to the value specified in the APcI Probe Temp control for the period of the solvent delay.

To set a solvent delay for a function 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 be acquired, which are stored with the data acquired from the mass spectrometer. Analog channels are typically used to collect data from external units such as UV detectors or FID detectors. A reading is made from the external channel at the end of each scan and stored with the data for that scan. The resolution of the chromatography for an analog channel is therefore dependent on the scan speed used to acquire the mass spectrometry data.

To access this dialog:

To store data for an analog channel:

Data Acquisition Page 84

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

Check the box(es) for the channel required.

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

This description is used on the analog chromatogram dialog as the channel description. 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 existing information.

Quattro Ultima

User's Guide

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 by selecting it from the displayed list.

Press Open.

Data Acquisition

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Setting up a Full Scan Function

The full scan function editor, activated by pressing or by selecting MS Scan from the Functions menu, is used to set up centroid, continuum and MCA 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 this

function becomes active, and data are acquired.

Cone Voltage

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

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

To apply a ramp to the cone voltage:

Check Use Cone Voltage Ramp and press CV Ramp to load the cone ramp dialog.

The four parameters define a gradient for the cone voltage which is then extrapolated to cover the full mass range of the function.

Method

Ionization Mode specifies the

ionization mode and polarity to be used during acquisition.

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

•

Data Acquisition Page 86

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

Continuum. The signal received by the interface electronics is stored regularly to give an analog intensity picture of the data being acquired. Data are not centroided into peaks, but are stored for every scan.

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

Quattro Ultima

User's Guide

acquisition system as compared to centroided acquisition. Data files tend to be significantly 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 these effects. The threshold can be set so that data considered to be ‘noise’ can be discarded, thus improving data acquisition speed and reducing data file sizes. For more information about setting instrument data thresholds see Setting Instrument Data Thresholds earlier in this chapter.

Multi Channel Analysis (MCA). MCA data can be thought of as ‘summed

•

continuum’, with only one intensity accumulated scan being stored for a given experiment. As each scan is acquired, its intensity data is added to the accumulated summed data of previous scans.

An advantage of MCA is that random noise does not accumulate as rapidly as real data and therefore effectively averages out over a number of scans. This emphasises the real data and improves the signal to noise ratio.

A further advantage of MCA is that because data is written to disk only at the end of an experiment, scanning speeds can be increased and significantly less storage space is required.

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

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

Scan Duration (secs)

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

APcI Probe

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

APcI.

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

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

Data Acquisition

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Setting up a SIR Function

The SIR (Selected Ion Recording) technique is typically used in situations where only a few specific masses are to be monitored. Since most of the data acquisition time is spent 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 dwell times, spans and inter-channel delay times.

To set up a SIR function:

Press or select SIR from the functions menu.

Many of the fields are described above for the full scan editor. Only those which differ 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 is monitored.

To modify existing settings:

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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 the

highlighted mass and starting monitoring the next mass in the function.

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

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

Retention Window

Start and End together specify the retention time in minutes during which this

function is active.

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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 fragmentations of a particular ion. MS1 is set to the parent mass using Daughters of, and is not scanned.

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

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

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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 a multiply 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 normally set just below Parents of, and End to a value above the highest expected parent mass.

There are often several masses from which a daughter may come, so that any one 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 tune window, 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 is detected. The precursor mass is scanned in MS1, and MS2 is scanned at this mass less the 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 that of MS1. It is applicable to studies where a precursor ion gains mass by ion molecule reaction or where multiply charged ions fragment into particles with a higher m value.

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Collision Energy

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

When Use Tune Page Settings is selected the collision energy set on the tune page is used. If it is required to adjust the setting during an acquisition then the acquisition must be started from the tune page.

To apply a ramp to the collision energy:

The four parameters define values of collision energy for two particular masses. This collision energy gradient is then extrapolated to cover the full mass range of the function.

Check Use Collision Energy Ramp.

Press CE Ramp… to load the collision energy ramp dialog.

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Setting up a MRM Function

Multiple reaction monitoring (MRM) functions are set up in much the same way as SIR functions, but allow a number of MS-MS transitions (fragmentations) between MS1 and MS2 to be monitored.

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User's Guide

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 the scan functions editor.

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

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Survey and MSMS Template Pages

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

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MS to MSMS Switching

Switch Criteria

MSMS scanning commences:

If TIC is

•

selected, and the TIC of the spectrum rises above the specified Threshold.

If Intensity

•

is selected, and the intensity of the largest peak rises above the specified Threshold.

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When a peak top is found, no other peaks are looked for within the specified Detection Window.

Currently Number of Components is set to 1 and can not be changed. The number of non coeluting precursors in a single run is not limited.

Precursor Selection

If Automatic is selected all valid masses satisfying selection criteria are monitored.

If Include Masses Only is selected only masses in the include list (see below) are monitored.

If Include Masses and Automatic is selected masses on the include list are given priority. 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 the precursor selection criteria.

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Detected Precursor Inclusion

Auto exclude and Always include are not currently available.

Include after time, if selected, allows a delay to be incorporated before precursors

are included.

Data

Discard uninteresting survey scans allows only the survey scans that detect

precursor ions to be stored. This saves on disk space as survey scans which contain no relevant data are rejected.

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MSMS to MS Switching

When MSMS functions have been generated, they are carried out in parallel until the conditions for switching to MS are satisfied.

When all MSMS functions have stopped, the MS survey function is again carried out.

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Switch Method

If the MSMS to MS switch method is Default, the MSMS function stops when the MSMS to MS switch criteria are met.

If the MSMS to MS switch method is After Time, the MSMS function stops when the MSMS to MS switch 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.

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Including and Excluding Masses

Mass ranges and individual masses to be included or excluded from the MS-MS scans are entered in the relevant Range boxes.

Masses on the Exclude list are not considered for detection.

Ranges take the form massX_massY.

Masses and ranges in a list are comma delimited, for example 100_200,202,236, 250_300.

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Monitoring Acquisitions

When an acquisition is started the automatic switching status dialog is displayed showing the precursors currently running.

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Micromass Quattro Ultima User Manual

Specifications and Main Features

Frequently Asked Questions

User Manual

Hardware Specifications

Instrument Description

Routine Procedures

Tuning

Data Acquisition