Quattro Micro

User's Guide

Safety Sécurité
The instrument is marked with this Ce pictogramme indique la presence de
symbol where high voltages are present. heute tension.

The instrument is marked with this Ce pictogramme indique la presence de

symbol where hot surfaces are present. surfaces chaudes.

The instrument is marked with this

Ce pictogramme indique la necessite de se
symbol where the user should refer to this
réferer au manuel d'utilisation.
User's Guide for further instructions.

Des avertissements sont donnés dans ce

Warnings are given throughout this
manuel aux endroits où l'utilisateur doit
manual where care is required to avoid
être particulierement prudent pour eviter
personal injury.
les blessures.
High voltages Hot surfaces Poisonous hazard Chemical hazard Flammable material General hazard

Heute tension Surfaces chaudes Risques Chimiques dangereux Produits Hazard général
d’empoisonement inflammables

To maintain the safety integrity of the Afin de garantir la sécurité de l'appareil il

instrument it should be used in a Pollution doit être utilisé dans un environment de
Degree 1 environment. degré 1 de pollution.
The power circuits are designed for a Les circuits électriques sont fabriqués
classification of Installation Category 1 pour une classification d'installation de
(over voltage category). Catégorie 1 (survoltage).
Afin de garantir la sécurité de l'appareil ne
pas enlever les panneaux. Il n'y a pas de
To maintain the safety integrity of the pièces nécessitant de la maintenance a
instrument do not remove any panels. l'interieur.
There are no user serviceable parts inside. Pour toutes questions regardant la
For all questions concerning instrument maintenance de cet appareil qui ne serait
repair, contact Micromass. pas couvert par ce manuel d'utilisation il
conrient de contacter le bureau de service
de Micromass.

If the instrument is used in a manner not Dans le cas où l'appareil serait utilisé de
specified by the manufacturer, the maniere non specificé par le fabricant le
protection provided by the equipment may niveau de protection de l'appareil pourrait
be impaired. altèré

Code: 6666631
Issue 1
© Micromass Ltd.
Quattro Micro
User's Guide
 Quattro Micro
User's Guide

Contents
Instrument Description
Overview 11
Sample Inlet 11
Vacuum System 12
Data System 12
MassLynx Software 12
Theory and Principles of Operation 13
Electrospray Ionisation (ESI) 13
Atmospheric Pressure Chemical Ionisation (APcI) 13
MS Operating Modes 14
MS-MS Operating Modes 14
The Daughter Ion Spectrum 15
The Parent Ion Spectrum 16
MRM: Multiple Reaction Monitoring 17
The Constant Neutral Loss Spectrum 18
Front Panel Controls, Indicators and Connections 19
Cone Gas, Desolvation Gas and Nebuliser Gas 19
Mass Flow Controllers 19
Electrical Connections 20
CID Valve 20
Divert / Injection Valve 20
Status Display 21
Vacuum LED 21
Operate LED 21
Rear Panel Connections 22
Analog Channels 22
Contact Closure 22
Mux Interface 22
Events 23
CE Int (Capillary Electrophoresis Interlock) 23
GF (Gas Fail) 23
FC (FractionLynx Control) 23
PC Link 23

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Quattro Micro
User's Guide

Routine Procedures
Starting Quattro Micro 25
Installing the ESI Probe 27
Setting Up the Syringe Pump 28
Setting Up the Quattro Micro 29
Preparing for Electrospray Operation 29
Obtaining an Ion Beam in ESI Mode 33
Preparing for APcI Operation when in ESI Mode 34
Obtaining an Ion Beam and Tuning in APcI Mode 35
Performing a Sample Analysis 37
Specific Tuning for Maximum Sensitivity 37
Probe Position 37
Corona Current 38
Probe Temperature 38
Desolvation Gas 38
Cone Gas 38

Tuning
Overview 39
The Tune Page 40
Printing Tune Information 40
Experimental Record 40
Saving and Restoring Parameter Settings 40
Modifying the Peak Display 42
Changing the Display 44
Customise Plot Appearance 44
Trace 45
Intensity 45
Grid 45
AutoTune 46
Ion Mode 47
Scope Parameters 48
Gas Controls 48
Ramp Controls 48
Resetting the Zero Level 49
Controlling Readbacks 50
Changing Tune Parameter Settings 51
Source Voltages 51

Data Acquisition
Starting an Acquisition 53
Starting an Acquisition from the Tune Page 53
Parameters 53
Multiple Samples 55
Process 56
Automated Analysis of Sample List 56

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Monitoring an Acquisition 58
The Acquisition Status Window 58
Chromatogram Real-Time Update 58
Spectrum Real-Time Update 58
Instrument Data Thresholds 59
MaxEnt 60
Profile Data 60
Centroid Data 60
SIR Data 60
Ion Counting Threshold 61
Profile Data - Spike Removal 62
Analog Data 63
System Manager 63
Stopping an Acquisition 64
The Function List Editor 64
Introduction 64
The Function List Editor Toolbar 66
Adding a New Function 66
Modifying an Existing Function 67
Copying an Existing Function 67
Removing a Function 67
Changing the Order of Functions 67
Setting a Solvent Delay 68
Analog Channels 68
Saving and Restoring a Function List 69
Setting up a Full Scan Function 70
Mass (m/z) 70
Cone Voltage 70
Method 70
Scan Duration (secs) 71
APcI Probe 71
Setting up a SIR Function 72
Channels 72
Method 73
Retention Window 73
Setting up MS-MS Scanning Functions 74
Mass 74
Collision Energy 76
Setting up a MRM Function 77
Setting up a Survey Function 77
Survey and MSMS Template Pages 78
MS to MSMS Switching 79
MSMS to MS Switching 81
Including and Excluding Masses 82
Monitoring Acquisitions 83

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User's Guide

Mass Calibration
Introduction 85
Overview 86
Calibration Types 86
The Calibration Process 87
Electrospray 87
Introduction 87
Preparing for Calibration 88
Reference Compound Introduction 88
Tuning 88
Instrument Threshold Parameters 89
Calibration Options 90
Selecting the Reference File 90
Removing Current Calibrations 90
Selecting Parameters 91
Automatic Calibration Check 91
Calibration Parameters 92
Mass Measure Parameters 93
Performing a Calibration 94
Acquisition Parameters 96
Starting the Calibration Process 98
Checking the Calibration 100
Calibration Failure 102
Incorrect Calibration 104
Manual Editing of Peak Matching 105
Saving the Calibration 105
Verification 106
Electrospray Calibration with PEG 108
Atmospheric Pressure Chemical Ionisation 109
Introduction 109
Preparing for Calibration 110
Reference Compound Introduction 110
Tuning 110
Calibration Options 110
Selecting Reference File 110
Removing Current Calibrations 110
Selecting Calibration Parameters 110
Performing a Calibration 111
Static Calibration 111
Scanning Calibration and Scan Speed Compensation 117
Calibration Failure 120
Incorrect Calibration 121
Manual Editing of Peak Matching 122
Saving the Calibration 122
Manual Verification 123

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Maintenance Procedures
Maintenance Schedule 125
Safety and Handling 126
Proper Operating Procedures 126
Maintenance Equipment 126
Routine Maintenance 127
Gas-Ballasting the Rotary Pump 127
Checking the Rotary Pump Oil 128
Changing the Rotary Pump Oil 128
Required Materials 128
Procedure 128
Cleaning the Source Assembly 130
Overview 130
Required Materials 130
Spare Parts 131
Disassembling the Source Components 131
Cleaning the Source Components 135
Removing and Cleaning the Hexapole Assembly 136
Reassembling the Source Components 138
Cleaning and Replacing the Corona Discharge Needle 138
Cleaning the APcI Probe Tip 141
Replacing Parts 142
Replacing the Ion Block Cartridge Heater 142
Required Materials 142
Procedure 142
Replacing the ESI Probe Stainless Steel Capillary 144
Required Materials 144
Spare Part 144
Procedure 144
Replacing the ESI Probe Tip 146
Required Materials 146
Spare Part 146
Procedure 146
Replacing the APcI Fused Silica Capillary and Filter Pad 147
Required Materials 147
Spare Parts 147
Procedure 147
Replacing the APcI Probe Heater 149
Required Materials 149
Spare Part 149
Procedure 149

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Troubleshooting
Spare Parts 151
Safety and Handling 151
System Troubleshooting 152
Component Hardware Troubleshooting 153
No Peaks on the Tune Page (No Ion Beam) 153
Unsteady or Low Intensity Peaks (Ion Beam) 154
Unusually High LC Back Pressure 155
Unusually Low LC Back Pressure 155
Insufficient Vacuum 156
Leaking Nitrogen 156
Vacuum Oil Accumulated in the Exhaust Tubing 156
Source Heater and Desolvation Heater Not Heating 156
APcI Heater Not Heating 157
Roughing Pump Fuse Fails 157
Ion Mode Fault 158
Failure to Recognise One Particular Probe Type. 158
Ripple 158
Loss of Communication with Instrument 158
IEEE Communication Errors 159
High Noise Levels in MRM Analyses 159
Chemical Noise 160
Electronic Noise 160
Calling Micromass 160

Reference Information
Overview 161
Editing a Reference File 162
Positive Ion 163
Horse Heart Myoglobin 164
Polyethylene Glycol 164
PEG + NH4+ 164
Sodium Iodide and Caesium Iodide Mixture 165
Sodium Iodide and Rubidium Iodide Mixture 165
Negative Ion 166
Horse Heart Myoglobin 166
Mixture of Sugars 166
Sodium Iodide and Caesium Iodide (or Rubidium Iodide) Mixture 167
Preparation of Calibration Solutions 168
PEG + Ammonium Acetate for Positive Ion Electrospray and APcI 168
PEG + Ammonium Acetate for Positive Ion Electrospray
(Extended Mass Range) 168
Sodium Iodide Solution for Positive Ion Electrospray 169
Method 1 169
Method 2 169
Sodium Iodide Solution for Negative Ion Electrospray 169

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User's Guide

Appendix
Environmental Specifications 171
Operating Temperature 171
Operating Humidity 171
Shipping and Storage Temperature 171
Dimensions 171
Height 171
Length 171
Width 171
Weight 171
Electrical Specifications 172
Line Frequency 172
Fuse Rating 172
Sensitivity Specifications 172
Electrospray Positive Ion 172
Electrospray Negative Ion 172
APcI Positive Ion 172

Index

Table of Contents
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User's Guide

Table of Contents
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User's Guide

Instrument Description
Overview
Quattro Micro is a high
performance triple quadrupole
mass spectrometer designed for
routine LC-MS-MS operation.

The instrument may be coupled

to the following liquid
introduction systems:

· HPLC system, to provide

molecular weight
information from an LC
run or to perform target
analysis and quantification.

· Syringe pump, for analysis

of precious,
low-concentration
compounds.

Sample 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.

The transmitted ions are finally detected by a conversion dynode, phosphor and
photomultiplier detection system. The output signal is amplified, digitised and
presented to the data system.

Sample Inlet
An HPLC system or an infusion pump delivers sample to either an electrospray
ionisation (ESI) probe or atmospheric pressure chemical ionisation (APcI) probe.

The ionisation mode can be changed by changing probes. Recognition pins on the
probes identify the ionisation method to the system.

Instrument Description
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Vacuum System
An external rotary pump and an internal split-flow turbomolecular pump combine to
create a vacuum. The turbomolecular pump evacuates the analyser and ion transfer
region.

The system monitors turbomolecular pump speed and continuously measures the
vacuum with a built-in Pirani gauge. In the event of leaks, electrical failure, or
vacuum pump failure a loss of vacuum will occur. The Pirani gauge also acts as a
switch, discontinuing instrument operation if it senses a vacuum loss.

An easy-access vacuum isolation valve enables routine source maintenance to be

performed without breaking vacuum.

Data System
The data system collects information from the mass analyser. The data system consists
of:

· An embedded PC
· An external workstation
· The MassLynx™ software
The workstation-based data system, incorporating MassLynx 3.5 software, controls the
mass spectrometer and, if applicable, the HPLC system, autosampler, divert valve or
injector valve. The workstation uses the Windows NT® graphical environment with
color graphics, and provides for full user interaction with either the keyboard or
mouse. MassLynx provides full control of the system including setting up and running
selected HPLC systems, tuning, acquiring data, and data processing. MassLynx
instrument control uses an embedded PC to process all data. A network link enables
communication between the workstation and the embedded PC.

The data system can sample analog inputs and thus store data from conventional LC
detectors like UV or ELSD simultaneously with acquired mass spectral data. It can
also acquire UV photodiode array detector data for selected systems such as the
Waters 996 PDA. Comprehensive information detailing the operation of MassLynx is
in the MassLynx User's Guide.

MassLynx Software
MassLynx software, a Windows NT-based application, enables the following
operations:

· Configuring Quattro Micro.

· Creating inlet and MS methods that define operating parameters for a run.
· Tuning and calibrating Quattro Micro.

Instrument Description
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User's Guide

· Running samples.
· Monitoring the run.
· Acquiring data.
Refer to the MassLynx User's Guide and Help for more information on installing and
using MassLynx software.

Theory and Principles of Operation

Electrospray Ionisation (ESI)
In electrospray ionisation (ESI), a strong electrical charge is applied to the eluent as it
emerges from a nebuliser, producing an aerosol of charged droplets. Solvent
evaporation reduces the size of the droplets until a sufficient charge density makes the
ejection of sample ions from the surface of the droplets possible (ion evaporation).
Characteristically, ions are singly or multiply charged, and the mass analyser sorts
them by mass-to-charge (m) ratio. High molecular weight compounds are typically
measured as ions with multiple charges. Eluent flows up to 1 ml/min can be
accommodated, though it is often preferable with electrospray ionisation to split the
flow so that 100 to 200 µl/min of eluent enters the mass spectrometer source.

Atmospheric Pressure Chemical Ionisation (APcI)

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 flowing into a plasma consisting
of solvent ions formed within the atmospheric source by a corona discharge. Proton
transfer then takes place between the solvent ions and the sample. Eluent flows up to
2 ml/min can be accommodated without splitting the flow.

Instrument Description
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MS Operating Modes

Source MS1 Collision Cell MS2 Detector

MS1 Collision Cell MS2

MS Resolving RF Only (Pass all masses)
MS2 RF Only (Pass all masses) Resolving

The MS1 mode, in which MS1 is used as the mass filter, is the most common 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.

MS-MS Operating Modes

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

Collision
MS1 MS2
Cell
Daughter Ion Static
Scanning
Spectrum (parent mass selection)
Static
Parent Ion
Scanning (daughter mass
Spectrum RF only selection)
(pass all
masses) Static
Multiple Reaction Static
(daughter mass
Monitoring (parent mass selection)
selection)
Constant Neutral Scanning (synchronised Scanning (synchronised
Loss Spectrum with MS2) with MS1)

Instrument Description
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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.

Collision Cell
MS1 RF only MS2
(pass all masses)
static at m/z 609 scanning from
(parent mass) m/z 100 to 650

The result:

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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.

Collision Cell
MS1 RF only MS2
(pass all masses)
scanning from static at m/z 195
m/z 50 to 650 (daughter mass)

The result:

Instrument Description
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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 Ý 195 for

reserpine in electrospray positive ion LC-MS-MS mode.

Collision Cell
MS1 RF only MS2
(pass all masses)
static at m/z 609 static at m/z 195
(parent mass) (daughter mass)

The result:

MRM does not produce a spectrum as only one transition is monitored. As in

SIR, a chromatogram is produced.

Time Time

LC-MRM LC-MS
! High specificity ! Low specificity
! Good signal / noise ! Poor signal / noise

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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.

Collision Cell
RF only MS2
MS1 (pass all masses)
scanning 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.

Instrument Description
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Front Panel Controls, Indicators and Connections

Desolvation Gas Fused Silica
Guide

Nebuliser Gas
Injector / Divert
CID Valve Valve

Fused Silica
Guide

Reset Switch
Syringe Pump

Cone Gas, Desolvation Gas and Nebuliser Gas

The PTFE gas lines for the desolvation gas and nebuliser gas are connected to the
front of the instrument using push-in Legris fittings. The connection for the cone gas
is within the source and uses PTFE tubing.

Mass Flow Controllers

The cone gas and desolvation gas are regulated by electronic mass flow controllers
over the ranges 0-500 litres/hour and 0-1200 litres/hour respectively, and are
controlled by MassLynx from the instrument tune page.

In the event that the desolvation gas decreases to less than 4% of its full scale range,
the instrument generates a signal that enables mechanisms to prevent the accumulation
of solvent in the source enclosure. Any solvent will drain from a port at the right hand
side of the front of the instrument.

For nanoflow applications where very low gas flow rates are required, this signal can
be overridden using Select Gas and Gas Fail Override on the tune page.

Instrument Description
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Electrical Connections
The electrical connection for the APcI probe or the ESI heater is via the ESI / APcI
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.

The high voltage connection for the ESI probe is via the front panel ESI connection.

The high voltage connection for the corona discharge pin is internal to the source.

Warning: Ensure that the instrument is in Standby when fitting the corona
discharge pin.

CID Valve
The CID Gas valve is a fifteen-turn valve. The flow increases as the valve is turned
anticlockwise.

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

Divert / Injection Valve

The divert / injection valve is an Probe
HPLC
electrically driven Rheodyne injector
Pump
that may be used in several ways Sample
depending on the plumbing arrangement: Loop

· 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.

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. Sample Waste
Syringe
For details of the use of the valve as a
divert valve see Setting a Solvent Delay,
page 68.

Instrument Description
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Status Display

Vacuum

Operate

The status of the instrument is indicated as follows:

Vacuum LED

State Vacuum LED

Pumping Flashing green
Below trip level Steady green
Pumped
Above trip level Steady amber
Pump fault Flashing red

Operate LED

State Operate LED

Standby No indication
Operate, above trip level Steady amber
Operate, below trip level Steady green
RF error Flashing red

Instrument Description
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Rear Panel Connections

ANALOGUE CHANNELS CONTACT CLOSURE EVENTS

Ch1 Ch2 EVENT 1 EVENT 2 CE INT GF !

CAUTION!

REFER TO
MANUAL BEFORE
CH3 CH4 EVENT 1 EVENT 2 FC CONNECTING TO
THESE PORTS

CONSULTER
LE MANUEL
AVANT DE
BRANCHER
MUX INTERFACE LES
CONNECTTEURS

PC LINK

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. Analogue data is processed by
a 12 bit ADC with a gain ranging up to 2×1020 counts.

If the input cable is only a two-wire assembly, then the negative pole of each channel
may need to be grounded.

Contact Closure
Two types of contact closure are available:

· In. Two inputs, Event 1 and Event 2, are provided, allowing external devices
to start acquisition. The Event In signal can be TTL or contact closure, 5V
maximum voltage.

· Out. Two outputs, Event 1 and Event 2, are provided whereby the mass
spectrometer is able to trigger an external event.

Mux Interface
This 9-way "D" type connector enables interfacing to the MUX control unit.

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Events
CE Int (Capillary Electrophoresis Interlock)
This connector enables interfacing with a capillary electrophoresis power supply so
that the instrument is safely interlocked against high voltages.

GF (Gas Fail)
In the event that the desolvation gas decreases to less than 4% of its full scale range,
the instrument generates a signal that enables mechanisms to prevent the accumulation
of solvent in the source enclosure. Any solvent will drain from a port at the right hand
side of the front of the instrument.

Additionally, this signal can be utilised to stop solvent flowing into the source by
connecting it to the Stop Flow of the HPLC system.

For nanoflow applications where very low gas flow rates are required, this signal can
be overridden using Select Gas and Gas Fail Override on the tune page.

FC (FractionLynx Control)
A 100mV analog output signal is provided to allow a trigger signal for an external
fraction collection device. The optional FractionLynx software must be purchased for
this.

PC Link
This RJ45 connector links the instrument to the data system using the network cable
supplied.

Instrument Description
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Instrument Description
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User's Guide

Routine Procedures
Starting Quattro Micro
To start Quattro Micro proceed as
follows:

Switch On the switch located

under the left hand side of the
front panel.

Allow 3 minutes for the

embedded PC to initialise.

An audible alert is given when

the PC is ready.

Start the MassLynx software. On/Off

Switch
The default page appears, and the
word Ready appears in the status bar
at the bottom.

Click on the default page to access the tune page

Routine Procedures
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Select Pump from the tune page Options menu.

Click the Diagnostics tab.

Monitor Turbo Speed.

This parameter should reach 98 to 100% within 5 minutes of Pump being

selected.

Before proceeding:

Ensure that the instrument has pumped sufficiently such that the Vacuum LED
on the front panel is steady green (see Status Display, page 21).

The mass spectrometer is sufficiently evacuated to enable operation in 20

minutes.

Routine Procedures
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Installing the ESI Probe

Probe
High Voltage
Desolvation Lead
Lead
Gas
Knurled
Thumbscrews
(2)

Probe
Nebuliser
Gas Flange

CID
Valve Probe Adjuster
Plate

Probe
Adjuster

To install the ESI probe:

Ensure that the isolation valve lever is fully to the left, indicating the valve is
open.

Insert the probe adjustment flange electrical cable in the lower (and larger) of
the two electrical ports on the front panel.

Connect the PTFE tubing from the probe adjustment flange to the desolvation
gas port on the front panel.

Remove the protective sleeve, if fitted, from the electrospray probe tip.

Slide the electrospray probe into the hole in the probe adjustment flange until the
probe body rests on the probe adjustment flange. Ensure the probe identification
contacts touch the screws on the probe adjustment flange.

Secure the probe with the two thumbscrews.

Connect the 4mm PTFE tubing from the probe to the port labelled Neb
(nebuliser gas).

Connect the electrical lead from the probe to the capillary connector on the front
panel.

Routine Procedures
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Setting Up the Syringe Pump

To set up the syringe pump:

Clip the ground cable (with plug-in clip), located at the front panel, lower right,
onto the syringe needle.

Mount the syringe onto the pump, and set the syringe stop appropriately.

Caution: Micromass has incorporated into the syringe pump design a positive
syringe stop to prevent certain syringe types from breaking. Nevertheless, as
added protection against syringe breakage, setting the syringe stop adjuster is
recommended. This prevents the syringe plunger from travelling its full stroke
inside the syringe barrel, thereby reducing the likelihood of breakage.

Syringe
Needle Port

Capillary

Syringe Stop
Adjuster

Screw the Rheodyne 9013 needle port fitting into the peek union, and tighten it
so that it does not leak.

Feed the capillary (ESI probe installation kit) from the top of the moulding to
the syringe area. Connect the capillary to the peek union, using an Upchurch®
Scientific nut, ferrule, and PTFE tubing.

Make a square, even cut on both ends of the capillary before installing, using a
ceramic silica cutter. Examine new cuts for squareness using an eye glass. When
cutting the capillary, allow enough length to form loops at angles and corners.
Never kink the capillary or stretch it tightly from one point to another.

Connect the other end of the capillary to the inlet on the ESI probe with an
Upchurch Scientific nut, ferrule, and PTFE tubing.

Warning: Clip the ground cable (with plug-in clip), located at the front panel,
lower right, onto the syringe needle.

Click on the default page to access the tune page.

Choose a suitable syringe type from the syringe selection editor by selecting
Options, then Syringe Type from the tune page.

Routine Procedures
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Setting Up the Quattro Micro

Preparing for Electrospray Operation
Connect one end of the fused silica capillary tubing to the syringe, and connect
the other end to the ESI probe.

Fill the syringe with a reference solution, and mount it on the syringe pump.

Warning: Be sure to ground the syringe needle with the ground cable provided.

From the MassLynx default page:

Click to open the tune page.

The example shown below uses ions from a solution of PPG1000, reserpine and
PA ß Cyclodextrin.

Routine Procedures
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Select Options from the menu bar, then Pump.

The rotary pump starts to evacuate the detector. In about 20 minutes, the
instrument is sufficiently evacuated to enable operation, and the Vacuum
indicator on the front panel shows green.

To view the actual values for instrument parameters select Readbacks from the
Options menu, then Always On.

Enter the suggested initial reference solution values from the table below in the
corresponding tune page fields.

Mass Span Gain

175.1 5 8
609.2 5 20
1080.8 5 40
2034.6 5 50

These settings are intended as starting points only. Optimum values may vary
between instruments.

Enter the suggested parameter values from the table below in the corresponding
fields of the ES+ Source tab.

Routine Procedures
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 Quattro Micro
User's Guide

Parameter Suggested Value

Capillary (kV) 3.0
Cone (V) 60
Extractor (V) 3
RF Lens (V) 0.2
Source Temperature (°C) 120
Desolvation Gas (litres / hour) 150
Cone Gas (litres / hour) 0

Caution: Failure to flow desolvation gas during ESI operation can cause heat
damage to the source.

Click the Analyser tab.

Enter the parameter values listed below, dependent on whether tuning for MS1
or MS2.

Routine Procedures
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User's Guide

Suggested Value Suggested Value

Parameter
MS1 MS2
LM Resolution 15
HM Resolution 15
Ion Energy (V) 0.5
Entrance 50 2
Collision 0 0
Exit 50 2
LM Resolution 2 15 15
HM Resolution 2 15 15
Ion Energy 2 3 0.5
Multiplier (V) 650 650

Suitable resolution can be obtained by adjusting LM Resolution and

HM Resolution.

Click to start the nitrogen flow.

Routine Procedures
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Obtaining an Ion Beam in ESI Mode

Make sure the ESI probe is installed as described in Installing the ESI Probe.

Change the ionisation mode to ESI, if necessary. Select Ion Mode from the
tune page menu. The current tune page tab indicates ionisation mode.

Keep the tune page ES+ Source tab open for the remaining steps in this
section.

Set Source Temp to 120 °C.

When the source temperature reaches 120 °C:

Click on the tune page to start nitrogen flowing.

From the Options menu, select the type of syringe to be used. For example,
select the Hamilton 250µl gastight syringe from the startup kit.

Click Press For Operate to switch on the instrument high voltages.

Set the syringe flow rate to 10 µl/min., and click on the tune page menu bar.

On the tune page set Desolvation Gas to 150 l/hour.

Check for leaks at the probe and syringe fittings.

Monitor for mass peaks. The peaks should appear at approximately the mass
values entered on the ES+ Source tab.

Increase values in the Gain fields until mass peaks become clearly visible.

Caution: An optimum signal must be obtained before the instrument can

successfully be calibrated.

If the signal is relatively weak and noisy, enhance it by turning the probe
adjuster knob to adjust the orientation of the probe relative to the sample cone
orifice. The signal can also be enhanced by adjusting the desolvation gas flow
from the ES+ Source tab on the tune page.

Caution: If the nitrogen supply to the instrument is turned off overnight, be sure
the API Gas parameter on the tune page is set to Off before restarting nitrogen
flow. Failure to do this may damage the flow meter.

The source is now ready for electrospray use. Refer to Tuning, page 39, for further
information.

Routine Procedures
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Preparing for APcI Operation when in ESI Mode

When in ESI mode, follow these steps to prepare for APcI operation:

Switch the instrument into standby mode by clicking Press for Standby on
the lower right of the tune page.

Disconnect the nebuliser and both electrical connections from the front panel.

Remove the ESI probe by unscrewing the two thumb nuts on the probe.

Remove the middle moulding section and loosen the four thumbscrews to
remove the source enclosure cover.

Warning: The ion source block, which can reach temperatures of 150°C,
maintains the set temperature, even when the source enclosure is removed.

Remove the blanking plug from the corona pin mounting contact, and fit the
corona discharge pin. Ensure the tip of the corona discharge pin aligns with the
tip of the sample cone.

Replace the source enclosure cover and the middle moulding section.

With the corona discharge pin in place, proceed as follows:

Insert the APcI probe into the source and tighten the two thumbscrews.

Connect the 6mm nebuliser gas tube from the probe to the instrument port
marked Neb.

Remove the probe adjustment flange cable from the front panel and seat it in the
rest hole provided just below its electrical socket.

Connect the APcI probe electrical lead to the Source/Probe receptacle on the
front panel.

Connect the LC pump tubing to the APcI probe.

Set the Source Temp to 130°C.

Set APCI Probe Temp to 20°C with zero liquid and nitrogen flow.

Switch the instrument to Operate.

The source is now ready for APcI operation.

Caution: Before restarting nitrogen flow following its interruption, the API gas
flow must be stopped from the tune page. Restarting the nitrogen while the API
gas is flowing can damage the flow meter.

Caution: Do not start the liquid flow until the gas flow and probe heater are
switched on with the probe inserted.

Routine Procedures
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Obtaining an Ion Beam and Tuning in APcI Mode

To obtain an ion beam:

Make sure the corona discharge pin is in place, and the APcI probe is installed
as described above (Preparing for APcI Operation When in ESI Mode).

Change the ionisation mode to APcI, if necessary. Select Ion Mode from the
tune page menu. The current tune page tab indicates ionisation mode.

Keep the tune page APCI+ Source tab open.

Make sure the desolvation gas tube is connected at the front panel.

Set Source Temp to 130°C.

Set Corona to 2µA and Sample Cone to 50V.

Routine Procedures
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When the source temperature reaches 130°C:

Click to start the nitrogen gas flowing.

Set Desolvation Gas to 300 l/hour on the APCI+ source tune page.

Select one of the peak display boxes, and set Mass to 50 and Span to 90.

Click Press to Operate.

Set APCI Probe Temp to 500°C for acetonitrile:water 1:1 flowing at

1 ml/min.

Lower temperatures are required for higher proportions of organic mobile

phase.

When the APcI probe temperature reaches 500°C:

Start the LC pump flowing at 1.0 ml/min.

Adjust the spray approximately to midway between the corona pin and the
sample cone with the probe adjuster.

Refer to Performing a Sample Analysis below, for more information on source tuning.

Warning: The source enclosure and parts of the probe adjustment flange may
reach high temperatures when in use.

Warning: Switch off the liquid flow and allow the probe to cool to less than
100°C before removing it from the source.

Caution: Failure to flow desolvation gas during APcI operation may cause heat
damage to the source.

Routine Procedures
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Performing a Sample Analysis

The following parameters are typical for general qualitative analysis of mixtures.

Parameter Suggested Value

Corona (µA)* 2
25
Cone (V)
(Monitor ions, slide adjuster up or down to optimise)
Extractor (V) 5
RF Lens (V) 0.2
Source Temp (°C) 130
APCI Probe Temp (°C)* 500
Desolvation Gas (L/hr)* 300
Cone Gas (L/hr)* 100
* See the following section for specific tuning details.

Adjust the values for Corona, Cone, and APCI Probe Temp for optimal
performance.

Specific Tuning for Maximum Sensitivity

For quantitative analysis, optimum APcI conditions should be obtained for each
analyte using standard solutions.

Tuning at high flow rates in APcI may be performed using a tee to introduce a
standard solution (typically 100-1000 pg/µl) at 10 µl/min into the mobile phase stream.

Alternatively, repeat direct loop injections of a standard solution (typically 10-100

pg/µl) into the mobile phase stream may be used to optimise the APcI.

Probe Position
Turn the probe flange adjuster to optimise the signal. Spray should be
approximately midway between the corona pin and the sample cone.

Routine Procedures
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Corona Current
Corona current can have a significant effect on sensitivity. The corona current required
depends upon the polarity of the compound and the polarity of the analytical mobile
phase. Optimisation should be performed in the presence of the analytical mobile
phase.

For polar compounds analysed in a polar mobile phase, the signal may be improved
by reducing the corona current below 2µA.

For compounds of low polarity analysed in a low polarity mobile phase, the signal
may be improved by increasing the corona current above 2µA.

To find the optimum corona current value:

Set Corona Current to 2µA.

Increase Corona Current value in 2µA steps until the optimum value is found.
Allow the current to stabilise before taking a reading.

If the signal continuously decreases, return Corona Current to 2µA, then

reduce the value in 0.5µA steps until the optimum value is found.

Using Corona Current values greater than 0µA will yield the best results for most
samples of this type.

Probe Temperature
For maximum sensitivity, the APcI probe temperature must be optimised as follows,
ensuring that the analytical mobile phase is used during optimisation.

Starting at 650°C, reduce APCI Probe Temp in 50°C decrements, allowing

time for the temperature to stabilise before taking a reading.

It is possible to set APCI Probe Temp too low for the mobile phase. This often
results in significant tailing of chromatographic peaks.

Desolvation Gas
In most circumstances the desolvation gas flow has little effect on signal intensity.
However, in some situations, it can affect chemical background noise levels. Adjusting
desolvation gas can suppress chemical background noise.

Cone Gas
Set the cone gas flow to minimise formation of solvent adducts. The typical
value is about 100 l/hour.

Routine Procedures
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Tuning
Overview

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

· 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
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User's Guide

The Tune Page

To display the tune page:

Press on the MassLynx screen MS panel.

Refer to the fold-out, page 51, 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 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.

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

Select Save As from the tune page File menu.

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

Press Save.

Tuning
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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
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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:

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.

Tuning
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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.

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
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Trace
From the pop-up menu:

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

or:

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

or:

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

The option selected has a tick next to it.

Intensity
Select either Intensity, Relative Intensity or Intensity,
Absolute Intensity as required.

Select Intensity, Normalise Data to display normalised data.

The options selected each have a tick next to them.

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

Press Start.

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

The following steps are performed:

· Parameter initialisation and instrument checks

Ensuring that essential status indicators read correctly.

Tuning
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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.

Press OK to return to the tune page.

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

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

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 (for nebuliser, desolvation and cone gas) or (for collision gas), 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.

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.

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

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 the multiplier voltage 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 the 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
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Changing Tune Parameter Settings

Save current Toggle on / off Toggle on / off Toggle on / off Toggle on / off Edit scope Pause / restart Stop
Most parameters can be modified in the following ways: tune parameters API gas collision gas syringe pump cone ramp settings acquisition acquisition

· Drag the slider bar using the mouse. Create a

· Click on the slider bar and use the left and right arrow keys, to change new tune file Display the
About box
the value by one increment.
Toolbar
The edit window updates as the slider bar is activated. Allows some
routine
utine operations
· Type a new value into the edit window. to
o be performed
with
ith a single click
Other parameters have only an edit window, and are changed by direct typing.

The speed with which the system responds to changes depends on the speed Open an P
Print current window Display tune Display vacuum Toggle on / off Reset zero level and
existing tune file in portrait format peak information OR information collision energy ramp reinitialise the system
with which the peak display refreshes. For the fastest response, set the scope
scan and inter scan times to be as short as possible.

Source Voltages
Enabled for MS-MS
The following illustration shows the various components of Quattro Micro’s ion functions
optical system. The name in the table’s first column is the name
used throughout this manual to describe the component. When Edit window
appropriate, the second column shows the term used in the current Select to
display the
MassLynx NT release. tune
parameters for
that region Slider bar
Tu me

AP

AP
ES

ES
Na
ne

cI

cI
I+

I-v
Pa

+v

-v
ve

Check up to four boxes Click on the arrow to

ge

to display the peaks. select the scan function

Electrospray Probe Capillary +3.0 (kV) -3.0 (kV) Not applicable
Readback
APcI Discharge Pin Corona Not applicable 2 µA 2 µA window
Sample Cone Cone +50 (V) -50 (V) +50 (V) -50 (V)
Peak Display
Extraction Cone Extractor +3 (V) -3 (V) +3 (V) -3 (V)
Up to four masses
Hexapole RF Lens +0.2 (V) -0.2 (V) +0.2 (V) -0.2 (V) can be displayed.
Any one can be
zoomed to occupy
The voltages shown are typical for an instrument in good condition. The
the entire display
polarities given are those actually applied to the electrodes. Only positive values
need be entered via the tune page.

Tuning
Page 51
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User's Guide

Tuning
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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.
✖ 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.

This will
require changes
to the settings
to accommodate
the required
mass range and
scan times.

Press Start.

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.

Data Acquisition
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To change the directory into which data are acquired:

Cancel the acquisition.

Create a new project by choosing Project Wizard, or open an existing one by

choosing Open Project, from the MassLynx top level file menu.

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.

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

· MS
· MS2
· Daughter
· Parent
· Neutral Loss
· Neutral Gain
· Survey Scan
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.

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.

Data Acquisition
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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 default page 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.

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 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.

Data Acquisition
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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:

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

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

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

Data Acquisition
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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.

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

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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.

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.

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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 by
positioning 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 1/16 amu type samples. This takes place after ion
counting and therefore has a less significant effect than Ion Counting Threshold.

To see more noise use a positive value.

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

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

· Acquiring data at 16 points per Dalton gives the greatest possible resolution.
· Acquiring data at 4 points per Dalton gives data with a smoothed appearance.

Centroid Data
Minimum centroid height sets a height below which detected peaks are ignored.
This reduces the size of acquired data files and is useful when concentrating on larger
peaks of interest. A suitable value can be arrived at by inspecting spectral noise levels,
and should be evaluated for each individual system

Minimum points per peak is the minimum number of points that a continuum
peak must have to be centroided. A typical value is 10

SIR Data
SIR Baseline Level sets the position of the SIR baseline above zero. The baseline
level is typically set to 0. Increasing the value causes the baseline to appear higher.

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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. Ion Counting Threshold only effects the
electronic noise level of the system.

The Ion Counting Threshold level entered is multiplied by 1/10 of the standard
deviation of the noise to determine the intensity level to be used, so a value of 10
equates to one standard deviation of the electronic noise level.

· 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.

· A value of 10 places the threshold just above the noise so almost all of the data
is acquired.

· If a value of 200 is entered the threshold sits well above the noise level, so very
little noise data is acquired.

· A value of 20 is suitable for most data.

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.

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on
Ty edu

Ty
Ba

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pic ctio

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Io n ho l
Th

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sel

T y und

Ty

AT
ck

Pr

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No

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res
ine

al
gro
Co d

pic

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ofi

Int n
ak
ise

F
Sa ze
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Le

un

al

al

en

vin
vel

sit

i
g
y
0 0 0 0

1 0 0

2 0 0

5 0 0

0 10 4% 8%

0 20 11% 10%

0 40 37% 61%

0 60 66% 69%

0 250 100% 83%

Profile Data - Spike Removal

Spikes are distinguished from real data by the fact that the peaks 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.

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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.

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.

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.

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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.

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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.

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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 Move the selected function up the
disk. list of functions.

Print the current window in portrait Move the selected function down
format. the list of functions.

Create a new function of the indicated type.

Adding a New Function

To add a new function to the list:

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

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

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

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

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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.

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.

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Setting a Solvent Delay

To set a solvent delay for a function list:

Select Solvent Delay from the Options

menu.

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 enable the divert/injector valve to be used as a

divert valve check Enable Divert Valve. This
diverts the flow of solvent during a solvent delay
period either to or away from the source for the time
period shown in the solvent delay timetable.

Up to four solvent delays can be programmed.

Analog Channels

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, which must be connected to the user
input/output PCB as described in Instrument Description, Rear Panel Connections.

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.

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To access this dialog:

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

To store data for an analog channel:

Check the box(es) for the channel required.

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

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.

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.

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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
ionisation 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:

· 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.

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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
acquisition system as compared to centroided acquisition. Data files tend to be
significantly larger than centroided ones .

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 Instrument Data
Thresholds, page 59.

· 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
ionisation 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 only be done by acquiring from the tune page.

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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.

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.

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

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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 seldom 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:

Check Use Collision Energy Ramp.

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

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.

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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.

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.

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.

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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Mass Calibration

Introduction
This chapter of the manual is divided into three sections:

· A brief general overview of the calibration process.

· A complete mass calibration of Quattro Micro using electrospray ionisation with
a mixture of sodium iodide and rubidium iodide as the reference compound.

· A complete mass calibration of Quattro Micro using atmospheric pressure

chemical ionisation (APcI) with PEG as the reference compound.

See Reference Information, page 161, for details of calibration solutions and their
preparation.

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Overview
MassLynx NT allows a fully automated mass calibration to be performed, which
covers the instrument for static and scanning modes of acquisition over a variety of
mass ranges and scanning speeds.

A mass spectrum of a reference compound (a calibration file) is acquired and matched

against a table of the expected masses of the peaks in the reference compound which
are stored as a reference file. The mass differences between the reference peaks and
calibration peaks are the calibration points. A calibration curve is fitted through the
calibration points.

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

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

Calibration Types
Each quadrupole analyser requires up to three calibration curves:

· A static calibration is used to ‘park’ the analyser accurately on a specific mass

of interest (for example in tuning, SIR and MRM).

· A scanning calibration enables peaks acquired in a scanning acquisition to be

mass measured accurately.

· A scan speed compensation calibration compensates for ‘lag time’ in the system
when the instrument is scanned rapidly.

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

Quattro Micro requires these three calibrations for both MS1 and MS2, thereby
generating a maximum of six calibration curves. The table below show which types of
calibration are necessary for particular types of experiment.

Calibration Required
Experiment
MS1 MS2
MS All -
SIR Static -
MSMS All All
MRM Static Static

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The Calibration Process

· Tuning the instrument.
· Selecting the appropriate reference file for the reference sample to be used.
· Starting an automatic calibration.
· Checking the calibration report.

Electrospray
Introduction
When a calibration is completed it is possible to acquire data over any mass range
within the calibrated range. It is therefore sensible to calibrate over a wide mass range.

With a mixture of sodium iodide and rubidium iodide calibration over the instrument’s
full mass range is achievable.

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Preparing for Calibration

Reference Compound Introduction
The example given here describes an automatic calibration which requires reference
compound to be present for several minutes. The introduction of the reference
compound is best achieved using the instrument's syringe pump:

Fill the syringe with the reference solution. See Setting Up the Syringe Pump,
page 28.

Couple the syringe to the electrospray probe with fused silica tubing.

Set the pump to a flow rate of 10 µl/min.

Tuning
Before beginning calibration, and with reference solution admitted into the source:

Set Multiplier to 650V.

Adjust source parameters to optimise peak intensity and shape.

Set the resolution and ion energy parameters for unit mass resolution on MS1
and MS2.

For a good peak distribution across the full mass range:

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

Check also the intensity of the peak at m 173.

Ensure that no peaks are saturated on the tune page with a Gain of 1. If
necessary, reduce Multiplier.

A cone voltage in the region of 45 is usually suitable.

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Instrument Threshold Parameters

Before beginning the calibration procedure, some instrument parameters need to be

checked.

For most low mass range calibrations, calibration data is acquired in continuum mode.

To allow suitable scanning speeds to be used the continuum data parameters need to
be set correctly:

From the instrument tune page select Options then

Set Instrument Threshold to display the instrument data thresholding
window.

In the Profile Data section select Baseline Level 0 and

16 Points per Dalton.

Select to save the parameters.

Set Ion Counting Threshold and Spike Removal as appropriate, see

Instrument Data Thresholds, page 59.

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Calibration Options

To access the calibration options:

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

Selecting the Reference File

To select the appropriate reference file:

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

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

Removing Current Calibrations

Select default.cal from the Calibrate menu option.

Save the changes to the default.cal file

Check that there is no prior calibration associated with default.cal.

This ensures that a file with no calibration is currently active on the instrument
and prevents any previously saved calibrations from being modified or
overwritten.

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

Automatic Calibration Check

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

It is here that limits are set

which the calibration must attain
before the instrument is
successfully calibrated. Two
user parameters can be set.

Missed Reference Peaks

sets the maximum number of
consecutive peaks which are not
matched when comparing the
reference spectrum and the acquired calibration spectrum. If this number is exceeded
then the calibration fails. The default value for this parameter, 2, is suitable in most
cases.

Maximum Std Deviation is set to a default of 0.20. During calibration the

difference between the measured mass in the acquired calibration file and the true
mass in the reference file is taken for each pair of matched peaks. If the standard
deviation of the set of mass differences exceeds the set value then the calibration fails.
Reducing the value of the standard deviation gives a more stringent limit. Increasing
the standard deviation means that the requirement is easier to meet, but this may allow
incorrect peak matching. Values greater than 0.20 should not be used unless
exceptional conditions are found.

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

Check Acquisition Calibration Ranges causes warning messages to be displayed

if an attempt is made to acquire data outside of the calibrated range for mass and scan
speed. It is advisable to leave this on.

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

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

Increasing the
Peak window and
Initial error gives a
greater chance of incorrect
peak matching. All peaks in
the acquired spectrum
below the
Intensity threshold value
(measured as a percentage
of the most intense peak in
the spectrum) are not used
in the calibration procedure.

The Polynomial order of

the curve has values from 1
to 5 as the available
options:

A polynomial order of
1 should not be used.

An order of 2 is suitable for wide mass ranges at the high end of the mass scale,
and for calibrating with widely spaced reference peaks. Sodium iodide in
particular has widely spaced peaks (150 amu apart), and horse heart myoglobin
is used to calibrate higher up the mass scale, so this is the recommended
polynomial order for these calibrations.

An order of 3 fits a cubic curve to the calibration.

A fourth order is used for calibrations which include the lower end of the mass
scale, with closely spaced reference peaks. This is suitable for calibrations with
PEG which extend below 300 amu..

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

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Mass Measure Parameters

These are accessed through Edit,
Mass Measure Parameters.... If
continuum or MCA data are acquired
for calibration then these parameters
need to be set before the calibration is
carried out. If centroided data are used
for calibration then the mass measure
parameters are not used.

With electrospray calibrations,

particularly with sodium iodide which
has some low intensity peaks at higher
mass, it is recommended that continuum
or MCA data are acquired.

It is important that the data are

smoothed correctly, and that the PWHH
is entered in the smoothing parameters
as shown.

At high scan speeds instrument

resolution may decrease. Ensure that the centroiding parameters are set to use the top
of the peak so that mass assignment of peaks is accurate.

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Performing a Calibration
Three types of calibration are available with
MassLynx: static calibration, scanning
calibration and scan speed compensation.
These are selected on the Automatic
Calibration dialog box (see below) which is
accessed by selecting Start... from the
Calibrate dialog box.

It is recommended that all three types of

calibration are performed so that mass
ranges and scan speeds can be changed
whilst maintaining correct mass assignment.
However, it is possible to have any
combination of these calibrations:

· If only a static calibration is present

then the instrument is calibrated for
acquisitions where the quadrupoles are
held at a single mass as in SIR or
MRM.

· If only a scanning calibration is

present then the instrument is only correctly calibrated for scanning acquisitions
over the same mass range and at the same scan speed as those used for the
calibration.

· If only a scan speed compensation is present (with no scanning calibration

having been performed) then the scan speed compensation is treated as a
scanning calibration and the instrument is only correctly calibrated for scanning
acquisitions over the same mass range and at the same scan speed as used for
the calibration.

For the scan speed compensation to be used correctly a scanning calibration

should also be performed.

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

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

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· If scanning calibration and scan speed compensation are present then the
instrument is only calibrated for scanning acquisitions over the same mass range
as that used for the calibration, but the scan speed can be changed provided that
it remains within the scan speeds used for the two calibrations. The mass range
should not be changed as there is no static calibration to locate the start mass.

· If all three types of calibration are present then all types of acquisition can be
used providing that the mass range and scan speed are between the lower and
upper limits used for the scanning calibration and the scan speed compensation.

For a complete calibration:

Check the boxes in the Types area of the dialog box adjacent to
Static Calibration, Scanning Calibration and
Scan Speed Compensation. Check also the MS1 and MS2 boxes.

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

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

The upper area contains the

Acquisition Parameters
where mass range, run time and
data type are set.

When the instrument is fully

calibrated any mass range or scan
speed is allowed within the upper
and lower limits dictated by the
calibrations.

Select the nairb.ref file.

The solution described in

Reference Information is suitable
for use with this reference file.

If compatible reference solutions

and reference files are used, then
simply selecting Default is
sufficient action - no parameters
need be entered manually.

Run Duration sets the time spent acquiring data for each part of the calibration. The
time set must allow a minimum of three scans to be acquired at the slowest scan speed
used. If the run duration is too short then data are not acquired. The slowest scan
speed generally used is 100 amu/sec. With Scan From set to 20 amu and Scan To
set to 2000 amu a scan time of 19.8 seconds is required, and an Inter Scan Delay
(in the lower area of the box) of 0.1 second is usually used. Therefore the run duration
must be greater than 59.6 seconds (3 scans + 2 inter scan delays). A Run Duration
of 1.00 minutes is suitable.

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

When an instrument acquires data for a static calibration it examines the

reference file to find the expected reference masses, and then acquires data over
a small mass span around each peak’s expected position. Thus the acquired data
do not contain continuous scans. Each spectrum comprises small regions of
acquired data around each peak, separated by regions where no data are
acquired.

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

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

Slow Scan Time determines the scan speed used for the scanning calibration. If both
a scanning calibration and a scan speed compensation are to be performed then the
scan speed should be set to approximately 100 amu/sec (a scan time of 19.8 seconds
over a mass range of 20 to 2000 amu). If only a scanning calibration is to be
performed (without scan speed compensation) then the scan speed should be set at the
same speed to be used for later acquisitions.

Fast Scan Time determines the scan speed used for the scan speed compensation,
and the upper limit of scan speed that can be used for subsequent acquisitions. A
fast scan time of 4000 amu/sec is adequate for most applications. A scan range from
20 amu to 1100 amu requires a Fast Scan Time of 0.26 sec at an
Inter Scan Delay of 0.1 sec. The Acquisition Setup must be edited to calibrate over
the mass range desired.

Select OK to return to the Automatic Calibration dialog box. Alternatively,

select chosen values if a different calibration range is required.

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Starting the Calibration Process

To start the calibration process:

Select OK from the Automatic Calibration dialog box.

The instrument acquires all of the calibration files in the following order using the
data file names shown:

MS1 static calibration data file: STATMS1

MS1 scanning calibration data file: SCNMS1
MS1 scan speed compensation data file: FASTMS1
MS2 static calibration data file: STATMS2
MS2 scanning calibration data file: SCNMS2
MS2 scan speed compensation data file: FASTMS2

Once all of the data have been acquired each data file is combined to give a single
spectrum which is then compared against the reference spectrum to form a calibration.
This process takes place in the same order as above. If the full calibration dialog box
is open then a constantly updated status message for the calibration is displayed.

If, when the process is completed, the calibration statistics meet with the requirements
specified by the selected calibration parameters then a successful calibration message
is displayed. A calibration report is then printed showing a calibration curve for each
of the calibration processes. Examples of calibration reports are shown on the
following pages.

For the acquisition to be effective, it must be saved under a suitable file name.

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Checking the Calibration

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

Click Browse.. to select the calibration data file (for example STATMS1,
SCNMS1, FASTMS1, STATMS2 etc.). The selected file must be from the
appropriate project.

Clicking on OK repeats the calibration

procedure for that particular file and display
a calibration report on the screen. This
calibration report (opposite) contains four
displays:

· the acquired spectrum

· the reference spectrum
· a plot of mass difference against mass
(the calibration curve)

· a plot of residual against mass

An expanded region can be displayed
(opposite lower) by clicking and dragging
with the left mouse button. In this way the
less intense peaks in the spectrum can be examined to check that the correct peaks
have been matched. The peaks in the acquired spectrum which have been matched
with a peak in the reference spectrum are highlighted in a different colour.

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Calibration Failure
If the calibration statistics do not meet the requirements then a message is displayed
describing at what point and why the calibration failed. This message also states where
the attempted calibration data can be viewed so that the exact cause of failure can be
determined.

There are a
number of reasons
for a calibration to
fail:

· No peaks
If the acquired calibration data file contains no peaks the calibration fails. This
may be due to:

Lack of reference compound.

No flow of solvent into the source.

Multiplier set too low.

· Too many consecutive peaks missed

If the number of consecutive peaks which are not found exceeds the
Missed Reference Peaks parameter set in the Automatic Calibration Check,
then the calibration fails. Peaks may be missed for the following reasons:

The reference solution is running out so that the less intense peaks are not
detected.

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

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

Note that it is possible to calibrate in negative ion mode electrospray using

the naineg.ref reference file with a suitable reference solution.

Intensity threshold, set in the Calibration Parameters dialog box, is too

high. Peaks are present in the acquired calibration file but are ignored
because they are below the threshold level.

Either Initial error or Peak window, set in the Calibration Parameters

dialog box, is too small. The calibration peaks lie outside the limits set by
these parameters.

Maximum Std Deviation, set in the Automatic Calibration Check dialog

box, has been exceeded.

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

In the case of too many consecutive peaks missed:

Check the data in the on-screen calibration report to see if the missed peaks are
present in the acquired calibration file.

If the peaks are not present then the first three reasons (as explained above in
"No peaks") are likely causes.

If the peaks are present in the data but are not recognised during calibration
then the latter four ("Too many consecutive peaks missed") are likely reasons.

Having taken the necessary action, proceed as follows:

If Intensity threshold, Initial error and Peak window are adjusted to

obtain a successful calibration, check the on-screen calibration report to ensure
that the correct peaks have been matched.

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

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

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Incorrect Calibration
If the suggested calibration parameters are used, and providing that good calibration
data have been acquired, then the instrument should be calibrated correctly. However
in some circumstances it is possible to meet the calibration criteria without matching
the correct peaks. This situation is unusual, but it is always sensible to examine the
on-screen calibration report to check that the correct peaks have been matched. These
errors may occur when the following parameters are set:

Intensity threshold set to 0

Initial error too high (>2.0)

Peak window too high (>1.5)

Maximum Std Deviation too high (>0.2).

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

An alternative cause of incorrect calibration is from contamination or background

peaks. If a contamination or background peak lies within one of the peak matching
windows, and is more intense than the reference peak in that window, then the wrong
peak is selected. Under some conditions this may happen with PEG. There are two
ways to counter this:

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

· If the reference peak is not closer to the centre of the peak window, or if by
reducing the window other reference peaks are excluded, then the calibration can
be edited manually.

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Manual Editing of Peak Matching

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

Using the mouse place the cursor over the peak in the reference spectrum and
click with the right mouse button.

Place the cursor over the peak in the acquired spectrum and click with the right
mouse button.

The peak is excluded and is no longer highlighted.

If the true reference peak is present then this can be included in the calibration by the
same procedure.

Place the cursor over the required peak and click with the right mouse button.

The peak is matched with the closest peak in the reference spectrum.

Manually editing one peak does not affect the other matched peaks in the calibration.

Saving the Calibration

When the instrument is fully calibrated the calibration must be saved under a file
name so that it can be applied and recalled for future use.

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

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Verification
Once a full instrument calibration is in place
it is not always necessary to repeat the full
calibration procedure when the instrument is
next used. Instead a calibration verification
can be performed. (There is no benefit in
verifying each calibration individually,
re-calibration is just as quick.)

If a scanning acquisition is to be made and

the calibration is to be checked:

Set up the instrument and access the

calibrate dialog box as though a full
calibration is to be carried out.

Set all peak matching parameters to the

values that were used for the calibration.

Bring up the Automatic Calibration

dialog box by selecting Start... on the
Calibrate dialog box.

Select Scanning Calibration and deselect Static Calibration and

Scan Speed Compensation.

Deselect Acquire & Calibrate and select Acquire & Verify and
Print Report.

Select either MS1 or MS2, depending on the type of acquisition to be

performed.

Select Acquisition Parameters to call up the Calibration Acquisition Set-up

dialog box.

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

Set Scan From, Scan To, Run Duration, Data Type, Scan Time and
Inter Scan Delay to agree with the acquisition parameters that are to be used
for data acquisition.

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

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

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A scanning acquisition is now performed. When the acquisition is complete the data
are combined to give a single spectrum which is compared against the reference file.
A calibration curve is drawn and a report printed in a similar way to when the original
calibration was performed.

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

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Electrospray Calibration with PEG

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

The spectrum shown below demonstrates how the PEG spectrum can be dominated by
doubly charged ions (in this case [M+2NH4]2+) if the wrong conditions are chosen. In
this case the concentration of ammonium acetate in the reference solution is too high
(5mmol ammonium acetate is the maximum that should be used) and Cone is too
low.

A low Cone voltage encourages the production of doubly charged ions. The voltage
should be at least 60V.

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

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Atmospheric Pressure Chemical Ionisation

Introduction
This chapter describes a complete mass calibration of Quattro Micro using
atmospheric pressure chemical ionisation. The procedures described should be
followed only after reading the previous chapter in this manual, describing the
automated calibration with electrospray ionisation.

Due to the high flow rates used with APcI, the residence time of an injection of
reference solution in the source is too short to allow a fully automated calibration, and
the procedure therefore has to be carried out in several steps.

The recommended reference compound for APcI is a solution of polyethylene glycol

(PEG) containing ammonium acetate. See Reference Information, page 161, for advice
on preparing the reference solution. See the following illustration for a typical
PEG + NH4+ spectrum.

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

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Preparing for Calibration

Reference Compound Introduction
It is best to use a large volume injection loop (50µl) with a solvent delivery system set
up to deliver 0.2 ml/min of 50:50 acetonitrile:water or methanol:water through the
injector and into the APcI source. An injection of 50µl of reference solution lasts for
approximately 15 seconds, allowing enough time to perform a slow scanning
calibration.

Tuning
Before beginning calibration:

Set Multiplier to 650V.

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

Set the resolution and ion energy parameters for unit mass resolution on MS1
and MS2.

When a full calibration is completed it is possible to acquire data over any mass
range within the calibrated range. It is therefore sensible to calibrate over a
wide mass range and in this example the calibration covers up to 1050 amu.

Calibration Options
To access the calibration options click on Calibrate from the tune page.

Selecting Reference File

Set pegnh4.ref as the reference file by clicking on the arrow in the reference
file box and scrolling through the files until the appropriate file can be selected.

Leave the Use Air Refs box blank when calibrating in APcI.

Removing Current Calibrations

Select Default from the Calibrate menu option.

Enter Yes to save the calibration into the most suitable project directory.

This ensures that a file with no calibration is currently active on the instrument
and prevents any previously saved calibrations from being modified or
overwritten.

Selecting Calibration Parameters

A number of parameters needs to be set before a calibration is started. Most of these
parameters can be set at the same value as for electrospray. However, a
Polynomial order of 3 is recommended for the calibration Curve Fit.

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Performing a Calibration
The three types of calibration (static, scanning and scan speed) must be carried out in
single steps.

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

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

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

Acquisition Parameters

Selecting Acquisition Parameters... brings forward the mass ranges, scan speeds
and acquisition mode relevant to the pegnh4.ref reference file.

The upper area contains the

Acquisition Parameters
where mass range, run time and
data type are set. When the
instrument is fully calibrated any
mass range or scan speed is
allowed within the upper and
lower limits dictated by the
calibrations. It is therefore
sensible to calibrate over a wide
mass range. Since the pegnh4.ref
reference file has peaks from
m 89 to m 2017, it is possible
to calibrate over this mass range.
A calibration effective up to
1000 amu is sufficient for the
majority of applications with
APcI. The following example
shows a setup to achieve this.

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

Data Type allows a choice of centroided, continuum or MCA data to be acquired.

For APcI, while either continuum or centroided data may be used, Continuum is
recommended.

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

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

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

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

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

Select OK from the Calibration Acquisition Setup to return to the Automatic

Calibration dialog box.

Acquiring Data

To start the acquisition:

Select OK from the Automatic Calibration dialog box.

The instrument acquires a calibration file ready for static calibration using the data file
name STAT. While data are being acquired:

Inject the reference solution.

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

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

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Manual Calibration

To perform a manual calibration using the acquired data:

From the chromatogram window call up the calibration file STATMS1.

Determine the scan numbers at the beginning and end of the chromatogram peak
for the reference solution.

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

Return to the Calibrate dialog box. Access

the manual calibration options, as shown,
by selecting Calibrate From File....

Select Static calibration type and MS1.

In the lower area the data file STATMS1

should be selected automatically. If this is
not the case the correct file can be
selected by clicking on Browse....

Enter the start and end scans of the

reference data in the From and To boxes.

Select OK to perform the calibration and

display the calibration report on the screen
(opposite upper).

This report contains four displays:

the acquired spectrum

the reference spectrum

a plot of mass difference against mass (the calibration curve)

a plot of residual against mass.

An expanded region can be displayed by clicking and dragging with the left mouse
button. In this way the less intense peaks in the spectrum can be examined to check
that the correct peaks have been matched. The peaks in the acquired spectrum which
have been matched with a peak in the reference spectrum are highlighted in a different
colour.

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

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If insufficient peaks have been matched, or the wrong peaks have been matched, refer
to Calibration Failure, page 120.

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

Select Print, Print from the report display.

To accept the calibration:

Select OK from the calibration report.

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Scanning Calibration and Scan Speed Compensation

Acquiring Data

To complete the calibration of the instrument two further data files must be acquired.
Both files are acquired in scanning mode over the same mass range, one at the slowest
speed required for scanning acquisitions and one at the fastest speed. Once these files
have been acquired and used for calibration then data may be acquired anywhere
within the mass range at any scan speed between the values used for the two sets of
data. These data do not have to be acquired through the calibration dialog box, they
can be acquired using the normal scan setup and then accessed from the calibration
dialog box as described below.

The recommended scan speed for the scanning calibration is 100 amu/sec.

Set Scan From to 50 amu and Scan To to 1050 amu.

Set Scan Time to 10 sec and Inter Scan Delay to 0.1 sec.

Select Continuum as the Data Type.

Although Continuum is recommended centroided data may be used.

Set Run Duration to 2.0 minutes.

This allows time to start the acquisition, inject the reference solution and
acquire several scans. With a solvent flow rate of 200 µl/min and a 50 µl loop in
line, an injection of reference solution lasts approximately 15 seconds allowing
at least one full scan of useful data to be acquired.

Choose any filename for the data.

The filename SCNMS1, the name used during an automatic calibration, is valid.

Start the acquisition and inject the reference solution.

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The recommended scan speed for the scan speed compensation is 4000 amu/sec.

Although continuum is recommended centroided data may be used. It is possible

to scan more quickly in centroided mode, but it is unlikely that a faster
acquisition rate would be needed for general use.

Set Scan From to 50 amu and Scan To to 1050 amu.

Set Scan Time to 0.24 sec and Inter Scan Delay to 0.1 sec.

Select Continuum as the Data Type.

Set Run Duration to 2.0 minutes.

Choose any filename for the data.

The filename FASTMS1, the name used during an automatic calibration, is

valid.

Start the acquisition and inject the reference solution.

Manual Calibration

Find the start and end scans of the reference data for each file in the same way
as for the static calibration file.

From the tune page select Calibration.

Select Scanning calibration type and MS1, Calibrate Instrument,

Calibrate From File.

In the lower area the data filename SCNMS1 should be selected automatically.
If this is not the case, or if an alternative filename has been used for the slow
scanning acquisition, then the correct file can be selected by clicking on
Browse....

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

Select OK to perform the calibration and display the calibration report on the
screen in a similar way to the static calibration.

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

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

Select Print, OK from the report display.

If insufficient peaks have been matched or the wrong peaks have been matched see
Calibration Failure, page 120. To accept the calibration:

Select OK from the calibration report.

The same procedure is used for the scan speed compensation except that
Scan Speed Compensation is selected in the dialog box, and the fast scanning file
is used. Note that for the scan speed compensation the default file is FASTMS1. If an
alternative filename has been used then this must be selected using the data browser.

Once all three calibrations (static, scanning and scan speed compensation) have been
completed then the instrument can be used for any mass range within the limits of the
scanning calibrations and at any scan speed from 100 to 4000 amu/sec.

Calibrating MS2

The calibration of MS2 is carried out in exactly the same manner as above, except that
data is acquired in MS2 mode instead of MS1.

Using the Instrument

Once all six calibrations (static, scanning and scan speed compensation, each for both
MS1 and MS2) have been completed then the instrument can be used for any mass
range within the limits of the scanning calibrations and at any scan speed from 100 to
4000 amu/sec.

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

· No peaks. If the acquired calibration data file contains no peaks the calibration
has failed. This may be due to:

Lack of reference compound.

Wrong scans or wrong data file being used for the calibration.

No flow of solvent into the source.

Multiplier set too low.

· Too many consecutive peaks missed. If the number of consecutive peaks which
are not found exceeds the limit set in the Automatic Calibration Check
parameters then the calibration has failed. Peaks may be missed for the
following reasons:

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

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

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

Intensity threshold, set in the Calibration Parameters dialog box, is too

high. Peaks are present in the acquired calibration file but are ignored
because they are below the threshold level.

Either Initial error or Peak window, set in the Calibration Parameters

dialog box, is too small. The calibration peaks lie outside the limits set by
these parameters.

Maximum Std Deviation (set in the Automatic Calibration Check dialog

box) has been exceeded.

The wrong reference file has been selected. Check that the correct file
(pegNH4.ref in this case) is selected in the Calibrate dialog box.

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In the case of too many consecutive peaks missed:

Check the on-screen calibration report to see if the missed peaks are present in
the acquired calibration file.

If the peaks are not present then the first three reasons above are likely causes.

If the peaks are present in the data, but are not recognised during calibration,
then the latter four are likely reasons.

Having taken the necessary action, proceed as follows:

If Intensity threshold, Initial error and Peak window are adjusted to

obtain a successful calibration, check the on-screen calibration report to ensure
that the correct peaks have been matched.

With a very low threshold and wide ranges set for the initial error and peak
window it may be possible to select the wrong peaks and get a “successful”
calibration. This is particularly relevant for calibrations with PEG where there
may be peaks due to PEG+H+, PEG+NH4+ and PEG+Na. This situation is
unusual, but it is always wise to examine the on-screen calibration report to
check that the correct peaks have been matched.

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

Incorrect Calibration
If the suggested calibration parameters are used and providing that good calibration
data have been acquired, then the instrument normally calibrates correctly. However in
some circumstances it is possible to meet the calibration criteria without matching the
correct peaks.

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

Intensity threshold set to 0

Initial error too high (>2.0)

Peak window too high (>1.5)

Maximum Std Deviation too high (>0.2).

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

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An alternative cause of calibration failure is from contamination or background peaks.

If a contamination or background peak lies within one of the peak matching windows,
and is more intense than the reference peak in that window, then the wrong peak is
selected. Under some conditions this may happen with PEG. There are two ways to
counter this:

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

· If the reference peak is not closer to the centre of the peak window, or if by
reducing the window other reference peaks are excluded, then the calibration can
be edited manually.

Manual Editing of Peak Matching

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

Using the mouse place the cursor over the peak in the reference spectrum and
click with the right mouse button.

Place the cursor over the peak in the acquired spectrum and click with the right
mouse button.

The peak is excluded and is no longer highlighted.

If the true reference peak is present then this can be included in the calibration by the
same procedure.

Place the cursor over the required peak and click with the right mouse button.

The peak is matched with the closest peak in the reference spectrum.

Manually editing one peak does not affect the other matched peaks in the calibration.

Saving the Calibration

When the instrument is fully calibrated the calibration, for it to be effective, must be
saved under a filename so that it can be recalled for future use. For example, it is
possible to save calibrations for use with different ionisation modes, so that when an
ionisation source is switched the corresponding calibration is recalled.

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

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Manual Verification
Once a full instrument calibration is in place it is not always necessary to repeat the
full calibration procedure when the instrument is next used. Instead a calibration
verification can be performed. (There is no benefit in verifying each calibration
individually, re-calibration is just as quick.)

If a scanning acquisition is to be made and the calibration is to be checked:

Set up a scanning acquisition over the

required mass range and at the required
scan speed in the normal way.

Start the acquisition and inject the

reference solution so that reference data is
acquired.

Stop the acquisition.

Access the calibrate dialog box and set all

peak matching parameters to the same
values that were used for the calibration.

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

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

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

Select OK to verify the calibration.

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A calibration curve is produced and displayed on the screen in a similar way to when
the original calibration was performed. An example is shown above. When OK is
selected from this report, unlike the original calibration procedure, the instrument
calibration is not changed. As the verification procedure uses the same matching
parameters as the calibration procedure, it is possible to validate the current calibration
without re-calibrating the instrument.

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

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Maintenance Procedures
Maintenance Schedule
The following table lists periodic maintenance schedules to be followed to ensure
optimum performance.

The maintenance frequencies shown apply to instruments that normally receive

moderate use.

Maintenance Procedure Frequency

Daily (APcI)
Gas-ballast the rotary pump
Weekly ( ESI )

Check and adjust the rotary pump oil

Weekly
level

Change the oil in the rotary pump Every 3000 hours of pump operation

Clean the cone gas cone, sample cone, When sensitivity decreases to
and baffle plate unacceptable levels

When sensitivity decreases to

Clean the ESI and APcI probe tip
unacceptable levels

Clean the corona discharge needle (APcI When sensitivity decreases to

mode) unacceptable levels

When it is visibly fouled

Clean the ion block assembly When background or peak contaminants
are unacceptably high

When sensitivity decreases to

unacceptable levels
Clean all source components When cleaning the cone gas cone,
sample cone, and baffle plate fails to
improve analytical results

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Safety and Handling

Bear in mind the following safety considerations when performing maintenance
procedures.

Warning: Never open the instrument’s top or side panels to access power
supplies or other components. The instrument does not contain user-serviceable
parts.

Warning: Observe good laboratory practice when handling solvents, changing

tubing, or operating the Quattro Micro. Know the physical and chemical
properties of the solvents to be used. Refer to their Material Safety Data Sheets.

Caution: Never disconnect an electrical assembly while the system is plugged

in. This could damage electrical parts.

Also, turn off the power and wait 10 seconds before disconnecting an assembly.

Caution: Do not touch integrated circuit chips or other circuit board

components. Static electric charge can damage electronic components.

Proper Operating Procedures

Follow all recommended procedures and guidelines to maintain the detector’s
operating efficiency.

Maintenance Equipment
Routine parts cleaning requires the following equipment:

· An ultrasonic bath with a minimum chamber size of 300mm × 150m × 100mm

(12" × 6" × 4").

· Glass vessels, approximately 100mm (4") in diameter and 120mm(43/4") high.

· A 500ml graduated cylinder (to use when cleaning the hexapole assembly).

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Routine Maintenance
Gas-Ballasting the Rotary Pump
When the rotary pump draws large quantities of solvent vapors, the vapours tend to
condense in the pump oil, which adversely affects pump efficiency. Gas-ballasting
purges condensed contaminants from the oil and also returns any oil to the pump from
the oil mist filter.

Gas-ballast the rotary pump when the following conditions apply:

· With ESI operation, once a week.

· With frequent APcI operation, once a day.
· If the pump oil appears cloudy.
· If the vacuum pressure is higher than normal.
· If condensate forms in the rotary pump exhaust line.
· When changing the rotary pump oil.
· If the level of accumulated oil in the oil mist filter is high.
Caution: Failure to routinely gas-ballast the rotary pump shortens oil life and
consequently shortens pump life.

Caution: Do not vent the instrument when the rotary pump is gas-ballasting.

Caution: Do not gas-ballast the rotary pump while Quattro Micro is in the
Operate mode.

Caution: Never gas-ballast the rotary pump for more than 2 hours.

To gas-ballast the pump:

Shut the vacuum system isolation valve, moving its handle fully to the right.

Turn on the gas-ballast.

When the oil is clear and has drained back to the rotary pump:

Return the gas-ballast control to its normal position.

Open the vacuum system isolation valve.

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Checking the Rotary Pump Oil

The oil level can be checked while the pump is operating. However, the instrument
must be vented and shut down before adding oil.

The rotary pump oil level appears in the oil level sight glass on the rotary pump.

Check the oil level at weekly intervals.

At all times it should be at or near the MAX level as indicated by the markings beside
the sight glass. If oil must be added:

Vent and shut down Quattro Micro before removing the oil filler plug.

Examine the oil each time the oil level is checked. It should be colorless and free of
visible contaminants. If the oil is discolored, change it as described below.

Changing the Rotary Pump Oil

Change the rotary pump oil every 3 to 4 months, or whenever it becomes noticeably
discolored.

Required Materials
· Rubber gloves
· Flat-blade screwdriver
· Container to catch used oil
· Funnel
· Vacuum oil (use only Ultragrade 19 or Inland Q45 (Edwards 45) vacuum pump
oil).

Procedure
To change the rotary pump oil:

Operate the pump to warm the oil before draining.

Gas ballast the rotary pump as described above.

Vent and shut down the instrument, turning the power switch to off.

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Gas-Ballast
Motor
Control
Terminal
NW25
Box
Outlet Inlet Port
Nozzle
Oil Filler
Plug

Power
Switch

MAX

Oil Sight
Glass

MIN

Base
Plate
Oil Drain
Plug

Raise the pump 150 to 200mm (6 to 8 inches) above the floor, if it is not already
elevated.

Place an object under the motor to tilt the pump toward the side on which the oil
drain plug is located.

Remove the oil filler plug to facilitate drainage.

Using the flat-blade screwdriver, remove the oil drain plug.

Let the oil drain completely, then replace the oil drain plug.

Fill the pump until the oil in the sight glass reaches the MAX level.

Allow a few minutes for the oil to drain into the pump.

Recheck the oil level, and add more oil if necessary.

Replace the oil filler plug and, if necessary, lower the pump to the floor.

Turn the rotary pump power switch to On.

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Cleaning the Source Assembly

Overview
The sample cone, cone gas cone, and baffle plate should be cleaned:

· when they are visibly fouled,

or

· when LC and sample-related causes for decreased signal intensity have been
ruled out.

When cleaning these parts fails to increase signal sensitivity, also clean the extraction
lens, hexapole, and ion block.

The source-cleaning procedure is as follows:

· Disassemble the source components, which are fully described later in this
chapter.

· Clean the source components.

· Remove and clean the hexapole assembly.
· Replace the hexapole assembly.
· Reassemble the source components.
Required Materials
The following materials are required to clean the source components:

· Lint-free cotton or powder-free nitrile gloves.

· 150mm (6") forceps or needle-nose pliers.
· 2.5mm hex wrench.
· 6mm hex wrench.
· Small, flat-blade screwdriver.
· Large, flat-blade screwdriver.
· Clean 1000ml beaker.
· Clean 500ml graduated cylinder.
Use only glassware not previously cleaned with surfactants.

· HPLC-grade methanol.
· HPLC-grade water.

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· Formic acid.
· Ultrasonic bath.
· Source of oil-free, inert gas (nitrogen or helium) for drying (air-drying optional).
· Lint-free paper towels.
Spare Parts
The following spare parts may be required when cleaning source components:

Item Reference Number

Ion block D ring (AS035) 5711968

Viton O ring (AS214) 5711294

Extraction cone O ring 5711967

Sample cone O ring 5711961

Disassembling the Source Components

To disassemble the source components:

Set Source Temp and either APcI Probe Temp or Desolvation Temp to
20°C to switch off the heaters.

Caution: The probe should be cooled to below 100°C and the source cooled to
below 50°C before removal. Failing to do so will shorten the life of the probe
heater.

To reduce cooling time significantly, continue flowing API gas.

When the probe has cooled, stop the nitrogen flow by deselecting on the
toolbar or by choosing Gas from the Gas menu.

Remove the APcI or ESI probe.

Stop the liquid flow, and disconnect the LC line from the probe.

Click Press For Standby on the MassLynx tune page.

The icon changes from green to red. This means all high voltages are turned off,
as well as the ESI desolvation /APcI probe heater.

Select Options then Vent from the tune page, and click OK when the message
box appears. If cleaning only the sample and cone gas cones, it is not necessary
to vent the system.

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When the instrument is vented power to the turbomolecular pump is interrupted.

However the pump does not stop immediately. Venting is a controlled and safe
process over several minutes designed to prevent damage to the instrument.
When the turbomolecular pump stops, a vent valve opens automatically and
vents the vacuum chamber to the atmosphere. The rotary pump stops running
about 3 minutes after the vent cycle begins.

Disconnect the front panel gas

and electrical connections.

Unscrew the probe’s two

knurled thumbscrews, and
retract it from the source.

Warning: Handle the probe

carefully. It might still be hot.

Remove the centre section panel

from the front of the instrument, Removable
Centre Panel
pulling it away from the
instrument.

Unscrew the four thumbscrews

and remove the source
enclosure cover.

Probe Lead Knurled

Thumbscrews
(2)

Desolvation
Gas

Nebuliser
Gas

Probe

Probe Adjuster
Plate

Source Enclosure
Cover Thumbscrew
(4)

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Caution: Wear lint-free cotton or powder-free nitrile gloves for the rest of the
cleaning procedure.

If using an APcI probe, carefully remove the corona discharge needle.

6mm Hex
Screw

Corona
Discharge
Needle

Gas
Cone

PTFE
Tubing

Remove the PTFE tube attached to the cone gas cone.

Remove the two screws that

secure the cone retainer, using 6mm Hex
the small, flat-blade Screw Isolation
Valve
screwdriver.
Cone
Retainer

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Remove the cone gas cone, O ring, and sample cone from the ion block.

Carefully separate the sample Isolation

cone from the gas cone, then Valve
Sample
remove the sample cone O ring. Cone

Remove the baffle plate, and set

Cone Gas
all pieces aside. Cone
Caution: Take care not to
scratch the highly polished cone
orifice surfaces.

Remove the two screws that

Cone
secure the ion block, using the Retainer Clip
6mm hex wrench.

Peek Connector
Block

Peek Ion
Block Support

Isolation
Valve

O Rings

O Rings

Extraction
PTFE Cone
Ring
Cone Cone Gas
Retainer Cone

Remove the ion block from the ion block support.

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Place the ion block on a flat surface, and remove any O rings.

Use a screwdriver to remove the Hold-Down

hold-down screw from the peek Screw Peek Extraction
Extraction
extraction cone retainer. Cone Retainer
Cone

Place the shaft of a small,

flat-blade screwdriver in the ion
block relief, and carefully pry
the insulator O ring away from
the ion block.

Take care not to damage the

ion block surface and insulator
O ring.

Grasp the extraction cone pin

with the needle-nose pliers, then
lift the extraction cone from the
ion block.

Insert the small, flat-blade screwdriver under the inner edge of polymeric seal
ring and carefully pry the seal ring and O ring out of the ion block.

Take care not to damage the seal and O ring on the ion block.

Remove the D-shaped O ring from the front of the ion block.

Remove the ion block plug and seal using a flat-blade screwdriver.

Remove the O ring from around the sample cone orifice, taking care not to
scratch the ion block surface.

Cleaning the Source Components

To clean the source components:

Place the ion block in a beaker with methanol : water (1:1).

Place the beaker containing the ion block and methanol : water mixture in an
ultrasonic bath for 20 minutes.

Remove the ion block from the methanol/water mixture, and place it in a beaker
containing 100% methanol.

If the sample cone contains debris, place a drop of formic acid on its orifice.

Warning: Use extreme care when working with formic acid. Use a fume hood
and appropriate protective equipment.

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Place the sample cone, cone gas cone, and extraction cone in a beaker with
methanol : water (1:1).

If the parts are obviously fouled, use a mixture of 45% methanol, 45% water,
and 10% formic acid.

Expose all parts to ultrasound for about 30 minutes.

If formic acid was used in the cleaning solution:

Rinse the parts, immersing them in a beaker of water and setting the
beaker in an ultrasonic bath for about 20 minutes to remove formic acid
from them.

Displace the water by immersing the parts in a beaker of methanol and

setting the beaker in the ultrasonic bath for 10 minutes.

Carefully remove the parts from the beaker and blow-dry them, using inert,
oil-free gas.

Alternatively. the parts may be placed on lint-free towels and allowed to air dry.
Wipe off any water spots with a lint-free cloth.

Removing and Cleaning the Hexapole Assembly

Pumping
Block

Peek Ion
Block Support O Rings

Hexapole
Assembly

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To remove and clean the hexapole assembly:

Remove the screws securing the ion block support with the 3mm hex wrench,
and remove the ion block support from the pumping block.

Remove any O-rings that remain stuck to the surface of the pumping block.

Grasp the hexapole gently by hand, and carefully slide it out.

Caution: Never squeeze the hexapole rods together when removing the
hexapole, as their orientation relative to one another is critical to the Quattro
Micro’s performance. Take care not to scratch the bored surfaces of the pumping
block as the hexapole is withdrawn.

Bend a length of stainless steel Rear

tubing into a hook shape, and Support
insert the hook into one of the Ring
holes in the rear support ring.

Carefully suspend the hexapole

assembly in a graduated Alignment
cylinder, then add methanol to Notch
the cylinder until the assembly
is covered.

Place the graduated cylinder in

an ultrasonic bath for 30 minutes.

Remove the hexapole assembly from the graduated cylinder, and place it on a
lint-free cloth. Allow it to air-dry, or use a nitrogen flow to dry it.

Insert the hexapole assembly by aligning the notches in the differential aperture
at the rear of the hexapole with the two bottom support rails on the analyser
assembly, then carefully slide it into place. Be sure to insert the assembly fully.

Check the condition of the three rear ion block support O rings, replacing them
with new ones if necessary. Ensure that the O rings are properly installed before
reattaching the ion block support. Also make sure the pin in the ion block
support aligns with the notch on the ion block.

Secure by alternately tightening the retaining screws.

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Reassembling the Source Components

To reassemble the source components:

Check the condition of the two front ion block support O rings. Replace them, if
necessary. Be sure they are properly installed before proceeding.

Replace the vespel sealing ring and O ring on the ion block support.

Press the extraction cone into place in the ion block support, and secure it with
the peek retainer and screw.

Replace the sample cone and its O ring, then the cone gas cone, secured with the
retainer spring and two screws as well as the ion block plug and seal.

Replace the ion block assembly onto the peek support block, then the cone gas
cone secured with two 6mm hex screws. Avoid overtightening.

If only the sample and cone gas cone have been cleaned, turn the isolation valve
back to Open.

If using APcI, replace the corona discharge needle (see Cleaning and Replacing
the Corona Discharge Needle below).

Reattach the PTFE tube to the cone gas cone.

Replace the source enclosure and source enclosure cover.

Pump down the instrument and turn on the API gas.

Cleaning and Replacing the Corona Discharge Needle

The corona discharge needle should be cleaned if it looks corroded or black, or when
the signal intensity weakens.

Required Materials

The following materials are needed to clean the corona discharge needle:

· Lint-free cotton or powder-free nitrile gloves.

· Lapping film.
· HPLC-grade methanol.
· Lint-free tissue.
Procedure

To remove, clean and replace the corona discharge needle:

Set Source Temp and APcI Probe Temp to 20°C to switch off the heaters.

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Caution: Do not remove the probe before it cools to below 100°C and the
source heaters cools to below 50°C. Doing so will shorten the life of the probe
heater.

To reduce cooling time significantly, continue flowing API gas.

When the probe has cooled, stop the nitrogen flow by deselecting on the
toolbar or by choosing Gas from the Gas menu.

Click Press For Standby on the MassLynx tune page.

Ensure that the icon changes from green to red.

This means all high voltages are turned off, as well as the APcI probe heater.

Remove the centre panel from the front of the instrument.

Remove the two knurled thumbscrews from the top of the probe.

Disconnect the nebuliser gas and the probe electrical connection.

Remove the APcI probe.

Remove the source enclosure cover.

Probe Lead Knurled

Thumbscrews
(2)

Desolvation
Gas

Nebuliser
Gas

Probe

Probe Adjuster
Plate

Source Enclosure
Cover Thumbscrew
(4)

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Caution: Wear lint-free cotton or powder-free nitrile gloves for the rest of the
cleaning procedure.

Warning: The inner surfaces of the source enclosure and its constituent
components are hot.

Remove the corona discharge needle from the source, pulling it straight out.

6mm Hex
Screw

Corona
Discharge
Needle

Gas
Cone

PTFE
Tubing

Clean and sharpen the tip of the needle with the lapping film, then wipe it clean
with a methanol-saturated tissue. Replace the needle if it is deformed or
otherwise damaged.

Reinstall the needle with the tip pointing toward the sample cone apex.

Replace the source enclosure cover.

Replace the probe, and reconnect the LC line.

Replace the middle panel section.

Reconnect the front panel gas and electrical connections.

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Cleaning the APcI Probe Tip

The APcI probe tip should be cleaned when a buffer build-up is detected on the probe
tip or when the signal intensity weakens.

To clean the APcI probe tip:

Shut off the liquid flow.

Click on the toolbar (or choose Gas from the Gas menu) to start nitrogen
flowing.

Adjust the nitrogen flow to approximately 650 litres per hour, as indicated by
the tune page desolvation gas meter.

Set the APcI probe heater temperature to 650°C, and press Enter.

Click the Operate icon, and wait 10 minutes with the APcI probe heater at
650°C.

This will remove any chemical contamination from the probe tip

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Replacing Parts
Replacing the Ion block cartridge heater
If the cartridge heater fails to heat it must be replaced. See System Troubleshooting.

Required Materials
· 3mm hex wrench.
· 1.5mm hex wrench.
· Flat-blade screwdriver
· Needle-nose pliers
Procedure

Peek Terminal
Ring Block
Tags

Cartridge
Heaters

To replace the ion block cartridge heater:

Follow the procedure for venting the instrument as described in Cleaning the
Source Assembly.

Remove the two screws securing the heater cartridge wires from the peek
terminal block.

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Carefully swing the ring tags out of the terminal block.

Loosen the two set screws that secure the heater cartridges in the ion block,
using the 1.5mm hex wrench.

Slide the heater cartridges out of the ion block gently with the needle-nose
pliers.

Slide the new heater cartridges into the ion block with the needle-nose pliers.
Secure them with two hex-head set screws and the 1.5mm hex wrench.

Position the two heater cartridge ring tags onto the peek block terminals with the
bent portion of their shafts extending into the pumping block

Tighten the two terminal block screws with a flat-blade screwdriver.

Replace the ion block cover plate, and secure with the four hex screws.

Reinstall the source enclosure cover and probe.

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Replacing the ESI Probe Stainless Steel Capillary

The stainless steel sample capillary on the ESI probe must be replaced if it clogs and
cannot be cleared, or if it becomes contaminated or damaged.

Required Materials
· Flat-blade screwdriver.
· 1.5mm hex wrench.
· ¼ inch (6mm) wrench.
· 5/16 inch wrench.
· 7/16 inch wrench.
· Loupe (magnifying glass).
Spare Part

Item Reference Number

Capillary tube M955088AD1

Procedure
Caution: All work done on the
probe should be carried out on a
clean work bench.
Stainless Steel
To replace the stainless steel Capillary
capillary:

Switch the instrument into

standby and remove the probe
from the source.

Remove the two end-cover Probe

retaining screws on the ESI Tip
probe with the flat-blade
screwdriver.

Loosen the set screw on the LC peek union with the 1.5mm hex wrench, and
remove the probe’s end-cover.

Unscrew the probe tip with the ¼ inch (6mm) wrench, and remove it.

Remove the LC union with the 5/16" and 7/16" wrenches.

Remove the capillary from the coupling nut with the 7/16" wrench. Discard the
capillary and the PTFE liner and ferrule assembly.

Maintenance Procedures
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Remove the conductive sleeve from the inner bore of the probe assembly fitting.

Slide a new ferrule onto the liner tube with the needle-nose pliers.

Slide the coupling nut onto the capillary followed by the PTFE liner tube and
ferrule.

Connect a piece of 0.007" peek tubing with finger-tight nut and ferrule into the
opposite side of the LC union, setting the capillary’s depth.

Press the capillary into the union until it seats, and tighten the adapter nut to the
LC union until it is snug but not tight.

Pull on the capillary gently, testing to ensure it stays in place.

Remove the peek tubing from the union.

Slide the conductive sleeve onto the capillary, then feed the capillary through the
probe.

Attach the coupling nut to the probe, and gently tighten it with the 7/16" wrench.

Replace the probe tip, and screw down until a 0.5mm length of the capillary
protrude from its end. Use the loupe, provided in the startup kit, to ascertain the
length of capillary that protrudes from the probe tip.

Reconnect the LC line, turn on the fluid flow, and check the probe for liquid
leaks.

Caution: Check for leaks carefully! Leakage can destroy a probe.

If a leak is found, disassemble the probe and tighten the fittings at the LC union.

Attach the nebuliser gas connection and turn on the nitrogen, by clicking on
the toolbar (or choose Gas from the Gas menu) on the tune page.

Check the probe tip for nitrogen leaks. If a leak is found, replace the probe tip
assembly and its O ring.

Replace the probe end-cover, and secure it with the two slotted screws. Tighten
the set screw to clamp the LC union in place.

Maintenance Procedures
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Replacing the ESI Probe Tip

The probe tip must be replaced if the following problems occur:

· A blockage occurs in the internal metal sheathing through which the stainless
steel capillary passes.

· The threads sustain damage.

Replace the O ring if gas leaks from the O ring.

Required Materials
¼" (6mm) open-end wrench.

Loupe (magnifying glass).

Spare Part

Item Reference Number

ESI probe tip M955016BC1

Procedure
Caution: All work done on the probe should be carried out on a clean work
bench.

To replace the ESI probe tip:

Switch the instrument into standby and remove the probe from the source.

Unscrew and remove the probe tip with the ¼" (6mm) wrench.

Install the new probe tip, and screw down until 0.5mm of the capillary protrudes
from the end. Use the loupe (provided in the startup kit) to check the capillary
position.

Maintenance Procedures
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Replacing the APcI Fused Silica Capillary and Filter Pad

Replace the fused silica capillary and/or filter pad when you note decreased signal
intensity and increased back pressure.

Required Materials
· 1.5mm hex wrench.
· 5/16 open-end wrench.
· 7/16 open-end wrenches (2).
· Ceramic capillary cutter (from the tools kit).
· Butane lighter or match.
· HPLC-grade methanol.
· Flat-blade screwdriver.
· Lint-free paper towels.
· Loupe (magnifying glass).
Spare Parts

Item Reference Number

GVF004 ferrules (2 off) 6070737

Fused silica M955710BD1

Filter pad 6367405

Procedure
Caution: All work done on the probe should be carried out on a clean work
bench.

To replace the fused silica capillary and filter pad:

Switch the instrument into standby and remove the probe from the source.

Slide the probe tip and heater assembly off the probe.

Using the flat-blade screwdriver, remove the two slotted screws.

Using the 1.5mm hex wrench, loosen the two set screws that retain the LC filter,
then remove the probe end.

Remove the filter cartridge from the adapter nut with one 5/16" and one 7/16"
wrench. If the ferrule remains inside the cartridge, remove it.

Maintenance Procedures
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Separate the two halves of the filter cartridge with two 7/16" wrenches.

Remove the old filter pad and replace it with a new one.

Unscrew the adapter nut from the probe with a 5/16" wrench. Discard the fused
silica capillary.

Using the ceramic capillary cutter, cut a new length of fused silica 161.5mm
long. Cut the capillary squarely. Examine new cuts for squareness with a loupe.

Remove approximately 20mm of polyamide coating from the capillary end with
a flame, then clean it with a methanol-saturated tissue.

Slide a GVF004 ferrule, followed by the adapter nut and another GVF004
ferrule, onto the capillary.

Position the filter with the flow direction arrow pointing toward the adapter nut,
then connect the adapter nut to the filter. Make sure the capillary seats squarely
against the filter cartridge interior.

Tighten the adapter nut until the capillary is snug in the fitting, then gently pull
on the capillary to ensure it stays in place

Feed the sample capillary through the probe, and gently tighten the probe
adapter nut with the 5/16" wrench.

The capillary must protrude 1.0mm from the nebulising tube.

Replace the probe end-cover and retaining screws.

Caution: Overtightening the fitting can damage the capillary.

Tighten the set screws in the probe end cover with the 1.5mm hex wrench to
clamp the filter in place.

Replace the probe tip and heater assembly.

Maintenance Procedures
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Replacing the APcI Probe Heater

The APcI probe heater must be replaced if it fails to heat.

Required Materials
· 1mm hex wrench or flat-blade screwdriver, depending on the type of screw that
secures the probe tip.

Spare Part

Item Reference Number

APcI probe heater M955315CC1

Procedure
Caution: All work done on the probe should be carried out on a clean work
bench.

To replace the probe heater:

Switch the instrument into standby and remove the probe from the source.

Loosen the two set screws at the base of the probe tip assembly, and slide the
probe tip off.

Separate the heater from the probe body, pulling it parallel to the axis of the
probe.

Carefully install the new heater onto the probe. Take care not to damage the
fused silica capillary.

Replace the probe tip assembly, and secure it with the two set screws.

Maintenance Procedures
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Maintenance Procedures
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Troubleshooting
This chapter describes how to troubleshoot the Quattro Micro with the help of
recommended troubleshooting procedures. This chapter covers:

· Safety and handling.

· General troubleshooting.
· Component hardware troubleshooting.

Spare Parts
Refer to Accessories and Spare Parts, for spare parts information. Parts not included
in that document are not recommended for replacement by the customer.

Safety and Handling

When troubleshooting the Quattro Micro, keep the following safety considerations in
mind.

Warning: To avoid the possibility of electric shock, never disconnect an

electrical assembly while power is applied to the Quattro Micro. Once power is
turned off, wait approximately 10 seconds before disconnecting an assembly.

Warning: To prevent injury, always observe good laboratory practices when

handling solvents, changing tubing, or operating Quattro Micro. Know the
physical and chemical properties of the solvents used. Refer to the Material
Safety Data Sheets for the solvents in use.

Warning: To avoid the possibility of electric shock, do not remove the

instrument panels. There are no user-serviceable items inside.

Caution: To prevent circuit damage due to static charges, do not touch

integrated circuit chips or other components that do not specifically require
manual adjustment.

Troubleshooting
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System Troubleshooting
There are a few basic steps for performing system troubleshooting:

· Examine the system, checking the simple things first. Is something obvious
causing the problem (for example, is the instrument and its cables improperly
connected, is there any leakage of fluid, vacuum or gas?)

· Compare current system operation with the way the system operated before the
problem started. To help identify normal operating conditions:

# Record a map of the LC system (tubing and electrical connections).

# Keep a daily log.

# Run test samples regularly. Check the instrument performance with

known samples, preferably the ones used for instrument acceptance.

This illustrates the importance of keeping track of system parameters and

the performance during normal operation. Troubleshooting is easier if the
typical conditions when the system is operating correctly are known.

For example, are the system tuning parameters similar to those when a test
species was previously run? Are the lens settings required for optimum
sensitivity higher than those previously obtained? If extreme values have
to be used to achieve good results, this implies that some part of the
system requires attention.

· Methodically check and eliminate possible causes to identify the system

parameter that is atypical.

· Refer to the troubleshooting information in the following pages. These tables

enable possible causes of a symptom to be identified and suggested corrective
actions to be taken.

If it is determined that there is a problem related to another system component (for

example HPLC, autosampler, UV detector) refer to the appropriate operator’s manual.

Troubleshooting
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Component Hardware Troubleshooting

The following tables provide suggestions for resolving hardware problems.

No Peaks on the Tune Page (No Ion Beam)

Possible Cause Corrective Action

Operating parameters Optimise parameters. Refer to Obtaining an Ion Beam,
(Capillary/Corona, Cone, Extractor, page 33.
RF Lens, Ion Energy, Gas Nitrogen
and Heaters) on the tune page are Once a beam has been obtained, ensure that all lenses
improperly set. affect the beam in a sensible manner.

Check that all necessary cables have been correctly

Cables are not properly connected.
attached to the source and probe.

Put the instrument into operate by clicking Operate.

Instrument is not in the operate mode. When in the operate mode, this icon is green and the
Operate LED on the front panel is also green.

Re-initialise by going to the tune page and selecting

Communication failure
Options. Reboot the embedded PC.

Check that sample is loaded correctly in the autosampler

No sample is present.
or in the syringe pump syringe.

Isolation valve is closed. Open the isolation valve.

Clean the source components. Refer to Cleaning the

The source components are dirty.
Source Assembly, page 130.

Check that the nitrogen pressure is 6 to 7 bar (90 to 100

psi) and the gas flow rate on the tune page is
Insufficient nitrogen flow >100 l/hour.
The desolvation and probe heaters shut off when the
nitrogen flow rate falls below 50 l/hour.

Check solvent flow from the autosampler or syringe

No LC flow.
pump.

Fluid leak in the HPLC system. Check for leaks in the HPLC system and correct.

Broken fused silica capillary in the APcI Replace the APcI fused silica capillary.
probe. Refer to Replacing the Fused Silica Capillary page 147.

Check that the source and probe voltage readbacks vary

with the tune page settings.
If any of these voltages are absent, disassemble and
Source components have been incorrectly
correctly reassemble the source and hexapole lens
assembled.
assemblies. Refer to Cleaning the Source Assembly,
page 130, and Removing and Cleaning the Hexapole
Assembly, page 136.

Replace capillary.
Refer to Replacing the APcI Fused Silica Capillary and
Blocked ESI or APcI capillary
Filter Pad, page 147, and Replacing the ESI Probe
Stainless Steel Capillary, page 144.

Troubleshooting
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Unsteady or Low Intensity Peaks (Ion Beam)

Possible Cause Corrective Action

Check that the source and desolvation temperature and
gas flow settings are suitable for the flow rate.
Liquid inside the source enclosure is an indication that
the temperature is too low.
Poor nebulisation. The nitrogen pressure should be from 6 to 7 bar ( 90 to
100 psi) and desolvation nitrogen flow rate should be
greater than 100 l/hour.
Check the stability of the nitrogen flow (good quality 2
stage regulator).

Troubleshoot the autosampler.

Problem with sample delivery Check the syringe in the syringe pump for leaks and
(autosampler, syringe pump or HPLC grounding.
system). Check for sufficient sample in the vials.
Look for pressure variation on injection.

Fluid leak in the HPLC system. Check for leaks in the HPLC system and correct.

Clean source components. Refer to Cleaning the Source

Source components require cleaning.
Assembly, page 130.

Check all settings are correct.

Lens settings wrong or atypical. Check readbacks are sensible.
Check that all lens parameters affect the beam.

Cone or collision cell voltage ramp is on. Set the voltage ramp off.

Check that the probe position is correct.

Check that the ESI probe stainless steel capillary
ESI or APcI capillary is not properly
protrudes 0.5mm as described on page 144.
installed.
Check that APcI capillary height is set at 1.0 mm. Refer
to page 147.

Check the alignment as described in Cleaning and

Corona pin is not correctly aligned.
Replacing the Corona Discharge Needle.

Infuse sample and optimise the gas pressure.

CID gas pressure is wrong. Check the CID gas regulator is set to 0,5bar, and is not
leaking.

Check Entrance, Exit and Collision are optimised,

Collision cell parameters incorrect.
with sensible readbacks.

Ensure Multiplier is 650V. Check the ion energy and

Analyser and multiplier parameters
resolution parameters are set correctly for the
incorrect.
acquisition.

Troubleshooting
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Unusually High LC Back Pressure

Possible Cause Corrective Action

Remove the probe from the source, and increase
Blockage in the capillary or injection loop due to
the solvent flow to 500 µl/min to clear the
particulate matter from the sample.
blockage.

Replace the filter pad. Refer to Replacing the

APcI probe filter pad is blocked. APcI Fused Silica Capillary and Filter Pad,
page 147.

Remove the finger-tight nut and tubing from the

back of the probe.
Tubing from LC system is blocked.
If the back pressure remains high, replace the
tubing .

The ESI stainless steel sample capillary inside

Replace the capillary, see page 144.
the probe is blocked.

The ESI capillary is not fully seated in the LC

Remove and disassemble the probe and reseat
union or the APcI capillary is not properly seated
the capillary correctly in the union.
in the filter.

Unusually Low LC Back Pressure

Possible Cause Corrective Action

Leaking connector. Check all fittings and tighten if necessary.

Problem with LC solvent delivery. Troubleshoot the LC system.

Broken flow cell in UV detector. Replace the flow cell.

Troubleshooting
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Insufficient Vacuum
Any reading greater than 5 × 10-4 mbar on the Pirani gauge, when CID gas is off.

Possible Cause Corrective Action

Disassemble source and check condition of ion block
Leaking ion block o-rings. O rings.
Refer to Cleaning the Source Assembly, page 130.

Gas ballast the rotary pump to return accumulated oil

from the oil mist filter.
Roughing pump not operating correctly. Check vacuum pump oil. If the oil is dirty, flush the
pump with clean oil, then fill the pump with oil.
Repeat if necessary.

Leak in vacuum backing line. Check vacuum hose for cracks or vacuum leaks.

Restriction in vacuum pump exhaust

Check exhaust line for restrictions.
tubing.

Check turbo pump speed on the Diagnostics tune

Turbo pump not operating properly.
page.

Leaking Nitrogen
Hissing sound or solvent smell.

Possible Cause Corrective Action

Visually inspect the source enclosure sealing surfaces
Poor seal around the source enclosure. for imperfections or nicks.
Also, check the condition of the encapsulated o-rings.

Vacuum Oil Accumulated in the Exhaust Tubing

Possible Cause Corrective Action

Oil mist filter needs replacement. Replace oil mist filter element and odor filter.

Source Heater and Desolvation Heater Not Heating

Possible Cause Corrective Action

Source heater has blown. Check readback. Replace heater if necessary.

Check Desolvation Temp readback. Call Micromass

Main system PCB fuse blown.
if wrong.

Troubleshooting
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APcI Heater Not Heating

Possible Cause Corrective Action

If desolvation heater is OK when in ESI
Replace the APcI heater. Refer to Replacing the APcI
mode, then the APcI heater may need to
Probe Heater, page 149.
be replaced.

Roughing Pump Fuse Fails

Possible Cause Corrective Action

Oil mist filter element is saturated. Replace oil mist filter element and odour filter.
Vacuum oil may also be accumulating in
exhaust tubing. Replace the fuse.

Ballast the pump for 20 to 30 minutes. Refer to

System needs to be ballasted.
Gas-Ballasting the Rotary Pump, page 127.

The ac line voltage to the instrument must be checked

The ac line voltage is less than 208Vac.
by a qualified electrician.

Change vacuum pump oil. Refer to Changing the Rotary

Vacuum pump oil is very dirty.
Pump Oil, page 128.

Troubleshooting
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Ion Mode Fault

Drop-down menu options are grayed out or instrument spontaneously switches probe
type.

Possible Cause Corrective Action

One or both of the probe contact pins Remove probe cover, free the contact pin, and ensure
jammed inside the probe and are not that both pins and associated springs move freely within
making contact with probe support plate. the bushing.

Failure to Recognise One Particular Probe Type

Possible Cause Corrective Action

Remove and try another probe of the same type.
Problem with the probe. Check that the Source Recognition ID voltage on the
Diagnostics page is <2V for ESI, and >2V for APcI

Ripple
Peaks and baseline appear to vary cyclically in intensity

Possible Cause Corrective Action

Erratic LC solvent flow. Troubleshoot the LC system.

Adjust the temperature and gas flow settings.

Poor nebulisation due to incorrect
Liquid in the source enclosure is an indication that the
temperature and gas flow settings.
temperature is too low.

Vibration from the rotary pumps or even Check for and eliminate excessive bench top and
other equipment in the same building. instrument vibration.

Loss of Communication with Instrument

Possible Cause Corrective Action

Reset the workstation and reboot the embedded PC from
the front panel using a short length of peek tubing to
Instrument to MassLynx host engage the switch, see the diagram on page 19.
communication failed. Wait 3 minutes for the audible signal indicating the
embedded PC has booted from Quattro Micro before
starting MassLynx.

Troubleshooting
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IEEE Communication Errors

Possible Cause Corrective Action

Power down the system components and start up the
system components in correct order:
Workstation
Instruments powered up in the wrong Quattro Micro
sequence. Inlet modules.
Wait 3 minutes for the audible signal indicating the
embedded PC has booted from Quattro Micro before
starting MassLynx.

Wrong IEEE address or conflicting Check system IEEE settings and enter the correct
address. addresses.

Systematically replace IEEE cables until the problem

Faulty IEEE cable in IEEE chain.
cable is located.

Ensure that the network cable for the instrument is

Network cables confused with site connected to the correct network card in the PC.
network. Ensure that the network card with the BNC connector is
configured to the site network.

High Noise Levels in MRM Analyses

The background noise in MRM analysis can be either electronic or chemical. To
distinguish between the two:

Start an acquisition.

During the acquisition set Ion Energy 1 and Ion Energy 2 fully negative on
the tune page.

· A significant decrease in signal when the ion energies are set negative implies
that the major contribution to the overall noise is chemical.

· Any residual noise is electronic.

Troubleshooting
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Chemical Noise

Possible Cause Corrective Action

High background due to carry-over after Repeat injections of 10% formic acid and/or
tuning with strong concentrations. isopropanol.

Contaminated injector. (Signal changes Repeat injections of 10% formic acid and/or
upon injection of mobile phase) isopropanol.

Contaminated tubing Replace tubing

Flush with methanol at 0.5 ml/min until the background

level falls.
Contaminated probe.
Replace the stainless steel capillary, see page 144.
Replace the APcI fused silica capillary, see page 147.

Infuse mobile phase from the solvent reservoir using a

syringe pump. Compare MRM background levels.
Contaminated HPLC system.
Check purity of solvents. Replace if necessary.
Ensure all solvents are HPLC grade.

Ensure glassware is not cleaned with commercial

Contaminated glassware.
surfactants in the cleaning process.

Electronic Noise
Corrective Action
Check that the valleys of peak-peak noise, when ion energies are fully negative, just touch the
baseline. Increase Ion Counting Threshold to suit.

Calling Micromass
Many problems with Quattro Micro can be easily corrected by the user. However, if
this is not the case, it is necessary to contact Micromass.

When calling Micromass, have the following information available:

· Nature of the symptom.

· Quattro Micro serial number.
· Details of flow rate, mobile phases and sample concentrations.
· Details of gas cell operating pressure.
· Tune page settings.
· Software version update reference.

Troubleshooting
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Reference Information
Overview
Calibration reference files consist of two columns of numbers separated by any
number of spaces or TAB characters. The first column contains the reference peak
masses and the second column contains the reference peak intensities.

The reference files listed in this chapter have all ion intensities set to 100%. Actual
ion intensities are not, of course, all 100%, but the calibration software does not take
account of the ion intensities and this is a convenient way to store the reference files
in the required format. However, if required, realistic intensity values can be entered
to improve the appearance of the reference spectra.

Most samples can be purchased from the Sigma chemical company. To order, contact
Sigma via the internet, or by toll-free (or collect) telephone or fax:

Internet:

http://www.sigma.sial.com

This site contains a list of worldwide Sigma offices, many with local toll-free
numbers.

Toll-free telephone:

USA & Canada 800-325-3010

Outside USA & Canada ++1 314-771-5750 (call collect)

Toll-free fax:

USA & Canada 800-325-5052

Outside USA & Canada ++1 314-771-5750

(call collect and ask for the fax machine)

Outside USA & Canada ++1 314-771-5757

(this is a toll call) (direct fax line)

Reference Information
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Editing a Reference File

Calibration reference files can be created or edited using any Windows text editor. To
read the currently selected reference file into the Notepad text editor:

Press or select Reference File... from the Calibration, Edit menu.

To save the reference file after editing either:

Select Save from the Notepad File menu to save the file under the current
name.

or:

Select Save as from the Notepad File menu to save as a new reference file
with a new name.

Textual information or comments can be stored in the reference file. Lines which are
textual information or comments must start with the semi-colon ( ; ) character.

Reference Information
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Positive Ion
Ref. File Chemical Name Molecular
/ Uses
Name [Sigma Code #] Mass
Bovine Ubiquitin
UBQ 8564.85 650-1500 General
[U6253]
Human a globin
HBA 15126.36 700-1500 Hb analysis
[H753]
Superoxide dismutase Hb (internal
SOD 15591.35 900-1500
[S2515] cal.)
Human b globin
HBB 15867.22 800-1500 Hb analysis
[H7379]
Horse heart myoglobin
MYO 16951.48 700-1600 General
[M1882]
Polyethylene glycol +
ES+ and
ammonium acetate
PEGH1000 80-1000 APcI+
mixture
calibration
PEG 200+400+600+1000
Polyethylene glycol +
ammonium acetate
ES+
PEGH2000 mixture 80-2000
calibration
PEG 200+400+600+1000
+1450
General,
Sodium Iodide / Caesium
NAICS 20-4000 ES+
Iodide mixture
calibration
Sodium iodide / Rubidium ES+
NAIRB 20-4000
Iodide mixture calibration

Reference Information
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Horse Heart Myoglobin

Reference File: myo.ref
Molecular Weight: 16951.48

Charge Calculated Charge Calculated Charge Calculated

State / Value State / Value State / Value
28+ 606.419 21+ 808.222 13+ 1304.969
616.177 20+ 848.583 12+ 1413.633
+ + +
27 628.841 19 893.192 11 1542.053
+ + +
26 652.989 18 942.758 10 1696.158
25+ 679.068 17+ 998.155 9+ 1884.508
+ + +
24 707.320 16 1060.477 8 2119.945
+ + +
23 738.030 15 1131.108 7 2422.651
22+ 771.531 14+ 1211.829

Polyethylene Glycol
PEG + NH4+
Reference Files: PEGH1000, PEGH2000 .

Calculated / Value
63.04 459.28 855.52 1251.75 1647.99
107.07 503.31 899.54 1295.78 1692.01
151.10 547.33 943.57 1339.80 1736.04
195.12 591.36 987.60 1383.83 1780.07
239.15 635.39 1031.62 1427.86 1824.09
283.18 679.41 1075.65 1471.88 1868.12
327.20 723.44 1119.67 1515.91 1912.15
371.23 767.46 1163.70 1559.94 1956.17
415.25 811.49 1207.73 1603.96 2000.20

Reference Information
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Sodium Iodide and Caesium Iodide Mixture

Reference File: NAICS

Calculated / Value
22.9898 772.4610 1671.8264 2571.1918 3470.5572
132.9054 922.3552 1821.7206 2721.0861 3620.4515
172.8840 1072.2494 1971.6149 2870.9803 3770.3457
322.7782 1222.1437 2121.5091 3020.8745 3920.2400
472.6725 1372.0379 2271.4033 3170.7688
622.5667 1521.9321 2421.2976 3320.6630

Sodium Iodide and Rubidium Iodide Mixture

Reference File: NAIRB

Calculated / Value
22.9898 772.4610 1671.8264 2571.1918 3470.5572
84.9118 922.3552 1821.7206 2721.0861 3620.4515
172.8840 1072.2494 1971.6149 2870.9803 3770.3457
322.7782 1222.1437 2121.5091 3020.8745 3920.2400
472.6725 1372.0379 2271.4033 3170.7688
622.5667 1521.9321 2421.2976 3320.6630

Reference Information
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Negative Ion
Ref. File Chemical Name Molecular
/ Uses
Name [Sigma Code #] Mass
Horse heart myoglobin
MYONEG 16951.48 700-2400 General
[M1882]
Sugar mixture of:
maltose [M5885]
raffinose [R0250] Low mass
SUGNEG 100-1500
maltotetraose range
[M8253]
corn syrup [M3639]
Sodium Iodide / Caesium
ES-
NAINEG Iodide (or Rubidium 200-3900
calibration
Iodide) mixture

Horse Heart Myoglobin

Reference File: myoneg.ref

Calculated / Value
891.175 1209.812 1882.490
940.741 1302.952 2117.927
996.138 1411.615 2420.632
1058.460 1540.036
1129.091 1694.140

Mixture of Sugars
Reference File: sugneg.ref

Calculated / Value
179.06 665.21 1151.37
341.11 827.27 1313.42
503.16 989.32 1475.48

Reference Information
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Sodium Iodide and Caesium Iodide (or Rubidium Iodide) Mixture

Reference File: naineg.ref

Calculated / Value
126.9045 1026.2699 1925.6353 2825.0008 3724.3662
276.7987 1176.1641 2075.5296 2974.8950 3874.2604
426.6929 1326.0584 2225.4238 3124.7892
576.5872 1475.9526 2375.3180 3274.6835
726.4814 1625.8469 2525.2123 3424.5777
876.3757 1775.7411 2675.1065 3574.4719

Reference Information
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Preparation of Calibration Solutions

PEG + Ammonium Acetate for Positive Ion Electrospray and APcI
Prepare a solution of polyethylene glycols at the following concentrations:

PEG 200 25 ng/µl

PEG 400 50 ng/µl

PEG 600 75 ng/µl

PEG 1000 250 ng/µl

Use 50% acetonitrile and 50% water containing 2 mmol ammonium nitrate.

Use reference file PEGH1000.

PEG + Ammonium Acetate for Positive Ion Electrospray

(Extended Mass Range)
Prepare a solution of polyethylene glycols at the following concentrations:

PEG 200 25 ng/µl

PEG 400 50 ng/µl

PEG 600 75 ng/µl

PEG 1000 250 ng/µl

PEG 1450 250 ng/µl

Use 50% acetonitrile and 50% water containing 2 mmol ammonium nitrate.

Use reference file PEGH2000

Reference Information
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Sodium Iodide Solution for Positive Ion Electrospray

Method 1
Prepare a solution of sodium iodide at a concentration of 2 µg/µl (micrograms
per microlitre) in 50:50 propan-2-ol (IPA):water with no additional acid or
buffer.

Add caesium iodide to a concentration of 0.05 µg/µl.

The purpose of the caesium iodide is to obtain a peak at z 133 (Cs+) to fill the
gap in the calibration file between z 23 (Na+) and the first cluster at z 173,
which would lead to poor mass calibration in this mass range.

Do not add more CsI than suggested as this may result in a more complex
spectrum due to the formation of NaCsI clusters.

Use reference file NAICS.REF.

Method 2
Prepare a solution of sodium iodide at a concentration of 2 µg/µl (micrograms
per microlitre) in 50:50 propan-2-ol (IPA):water with no additional acid or
buffer.

Add rubidium iodide to a concentration of 0.05 µg/µl.

The purpose of the rubidium iodide is to obtain a peak at z 85 (85Rb+) with an

intensity of about 10% of the base peak at z 173. Rubidium iodide has the
advantage that no rubidium clusters are formed which may complicate the
spectrum. Note that rubidium has two isotopes (85Rb and 87Rb) in the ratio
2.59:1, giving peaks at z 85 and 87.

Use reference file NAIRB.REF.

Sodium Iodide Solution for Negative Ion Electrospray

Either of the above solutions is suitable for calibration in negative ion mode. In both
cases the first negative reference peak appears at m 127 (I-) and the remaining peaks
are due to NaI clusters.

Use reference file NAINEG.REF.

Reference Information
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Reference Information
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Appendix
Environmental Specifications
Operating Temperature
15 to 28°C (59 to 82.4°F).

Short term variation of 2°C within 1.5 hours.

Operating Humidity
20 to 80%, noncondensing.

Shipping and Storage Temperature

–20 to 60°C (–4 to 140°F).

Dimensions
Height
572mm (23").

Length
880mm (34.6").

Width
390mm (15.4").

Weight
(excluding data system and rotary pump)

115kg (253lbs).

Appendix
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Electrical Specifications
Line Frequency
50Hz, 47 to 53Hz.

60Hz, 57 to 63Hz.

Fuse Rating
10A, 250V ac.

Sensitivity Specifications
Electrospray Positive Ion
Measured signal/noise ratio obtained from the chromatogram monitoring the transition
m 609 - m 195 on injection of 10pg (16fmol) of reserpine will be >20:1.

10µl of a 1 pg/µl reserpine solution in 50/50 acetonitrile/water (no additives) will be

injected at a flow rate of 200 µl/min, in MRM mode, 1 second dwell, span 0Da.The
resolution of the ion at m 609 will be <1Da peak width at half height (MS1 and
MS2).

Electrospray Negative Ion

The signal/noise ratio measured on the [M-H]- peak at m 503 from a sample
consumption of 10ng raffinose will be >100:1.

A solution of 5 ng/µl Raffinose in 50/50 acetonitrile/water (no additives) will be

introduced at a flow rate of 10 µl/min and the summation of two 6 second scans over
the mass range m 100-600 represents a total sample consumption of 10ng.

APcI Positive Ion

Measured signal/noise ratio obtained from the mass chromatogram of m 609 in SIR
mode on direct injection of 10pg (16fmol) reserpine (10µl injection of a 1pg/µl
solution) at a flow a rate of 1ml/min will be >10:1.

Appendix
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Index
A C
Acquisition 53, 112 Caesium iodide 165, 167, 169
Parameters 96, 111 Calibration 85
Ammonium acetate 109, 168 APcI 109
Analog channels 68 Checking 100
Analog data 63 Failure 102, 120
Analog input 22 Incorrect 104, 121
APcI 13, 34, 35 Manual 113, 118
Calibration 109 Parameters 92, 110
Sensitivity specifications 172 Saving 105, 122
APcI probe 71, 141 Scan speed compensation 94, 117
Capillary 147 Scanning 94, 117
Filter pad 147 Static 94, 111
Position 37 Verification 106, 123
Temperature 38 Centroid data 60, 70
Tip heater 20, 149 Channels 72
Atmospheric Pressure Chemical Ionisation Collision energy 76
See APcI Collision gas 20
AutoTune 46 Collision induced decomposition 15
Conditions 55
Cone 70
B Cone gas 38
Back pressure Constant neutral loss 18
High 155 Continuum data 70
Low 155 Corona 38
Discharge needle 138

D
Data acquisition 53
Daughter 74
Daughter ion 15
Desolvation gas 19, 38
Desolvation heater 156
Dimensions 171
Divert valve 20
Drug metabolite 17
Dwell 72

E
Electrospray 29
Calibration 87
Probe 27, 144
Sensitivity specifications 172
Electrospray ionization 13
End mass 54
Environmental analysis 17
ESI
See Electrospray

Index
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F N
Forensic science 17 Neonatal screening 18
Full scan function 70 Neutral gain 75
Function list editor 64 Neutral loss 75
Fuse 172 Nitrogen
Leaking 156
Noise 160
G
Gas
Gas-ballast
48
127
O
Grid 45 Operate LED 21
Origin 55
H
Heater 20
P
Herbicide 17 Parent 75
Hexapole 136 Parent ion 16
Humidity 171 Peak matching 105, 122
Peptides 15
Pesticides 17
I Pharmacokinetic studies 17
IEEE 159 Pirani gauge 12
Intensity 45 Polyethylene glycol 108, 109, 164, 168
Inter scan time 54 Precursor 79
Ion block cartridge heater 142 Process 56
Ion counting threshold 61 Profile data 60
Ion mode 158 Spike removal 62

J R
Job 55 Ramp 48
Readbacks 50
Reference compound 88, 161
L Reserpine 15, 16, 17
Retention 73
Line frequency 172
Ripple 158
Rotary pump 127, 157
M Oil 128, 156
Rubidium iodide 165, 167, 169
Mass measurement 93 Run duration 54
MaxEnt 60
MCA 71
Method 70, 73
Mode 47
MRM 15, 17, 77, 159
MS1 mode 14
MS2 mode 14, 75
MS-MS 14
MS-MS function 74
Multiple samples 55
Myoglobin 164, 166

Index
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S T
Safety 126, 151 Task 55
Sample inlet 11 Temperature
Scan duration 71 Operating 171
Scan time 54 Shipping and storage 171
Scope 48 Threshold parameters 89
Selected ion recording 17 Thresholds 59
Sensitivity 172 Toxicology 17
Set Mass 54 Trace 45
SIR data 60 Troubleshooting 151
SIR function 72 Tune page 40
Sodium iodide 165, 167, 169 Tuning 37, 39
Software APcI 110
MassLynx 3.5 12 Electrospray 88
Solvent delay 68 Turbomolecular pump 12
Source 130
Cleaning 130
Heater 142, 156 U
Source voltages 51 UV detector 22
Specifications 171
Electrical 172
Environmental 171 V
Sensitivity 172
Vacuum 156
Start mass 54
Vacuum LED 21
Starting 25
Vacuum system 12
Structural elucidation 15, 16
Submitter 55
Sugar mixture 166 W
Survey 77
Syringe pump 28 Weight 171
System manager 63
Z
Zero 49

Index
Page 175