LCT User's Guide Micromass UK Limited Floats Road ... manual is a companion to the MassLynx NT User's Guide supplied with the instrument. All information contained in these manuals

LCTUser's Guide

Micromass UK Limited

Floats RoadWythenshawe

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

Tudor RoadAltrinchamWA14 5RZ

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

http://www.micromass.co.uk

The instrument is marked with this symbol wherehigh voltagesarepresent.

The instrument is marked with this symbol wherehot surfacesarepresent.

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

instrument.

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

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

This manual is a companion to the MassLynx NT User's Guide supplied with theinstrument. All information contained in these manuals is believed to be correct at the

time of publication. The publishers and their agents shall not be liable for errorscontained herein nor for incidental or consequential damages in connection with thefurnishing, performance or use of this material. All product specifications, as well as

the information contained in this manual, are subject to change without notice.

Micromass ® is a registered trade mark of Micromass Limited(Reg. U.S. Pat. & T.M. Off.).

Issue 1© Micromass Ltd.

http://www.micromass.co.uk

Table of Contents

LCTUser's Guide

ContentsInstrument Description

Introduction 9Ionisation Techniques 10

Atmospheric Pressure Chemical Ionisation 10Electrospray 10

Nanoflow Electrospray 10Ion Optics 11Internal Layout 12

Mechanical Components 12Electronics 14

The Vacuum System 16Fine Pumping 16Rotary Pumping 16Pressure Measurement 17Vacuum Protection 17

Front Panel Connections 18Desolvation Gas and Probe Nebuliser Gas 18High Voltage 18Heaters 18

Front Panel Controls and Indicators 19Status Display 19Flow Control Valves 19Divert / Injection Valve 19

Rear Panel Connections 20Water 20Nitrogen Gas In 20Exhausts 20Supply Inlet 21System Power 21Electronics 21Rotary Pump 21ESD Earth Facility 21SIP 21Optical Communications Link 21Trigger 21Input 21Event Out 22Contact Closure In 22Analog Channels 22

MassLynx NT Data System 23Software 23

Table of Contents

LCTUser's Guide

Routine ProceduresStart Up Following a Complete Shutdown 25

Preparation 25Pumping 26MCP Detector Conditioning 26Instrument Warm-up 27Using the Instrument 28

Start Up Following Overnight Shutdown 28Preparation for Electrospray Operation 28Preparation for APcI Operation 30Instrument Operation 31

Automatic Pumping and Vacuum Protection 32Transient Pressure Trip 34Water Failure 34Vacuum Fault 34Power Failure 35Nitrogen Supply 35

Tuning 36Source Tuning Menu 36Analyser Tuning Menu 37

RF Lens 37RF DC Offset 1 37RF DC Offset 2, Aperture, Acceleration, Focus, Steering 37MCP Detector 37

Engineer Tuning Menu 38Manual Pusher, Max. Flight Time 38

Other Tune Page Settings 40TDC Settings 40

Start (mV) 40Stop (mV) 40Threshold 40Veff Factor 41Lteff 41

Tuning Setup 42Tune Page Acquisition 42Real Time Tune Page Display 43

Calibration 43Data Acquisition 43Data Processing 43Shutdown Procedures 44

Emergency Shutdown 44Overnight Shutdown 44Complete Shutdown 44

Table of Contents

LCTUser's Guide

ElectrosprayIntroduction 47

Post-column Splitting 49Megaflow 50

Changing Between Flow Modes 50Operation 51

Checking the ESI Probe 51Obtaining an Ion Beam 52

Tuning and Optimisation 52Probe Position 53Nebuliser Gas 53Desolvation Gas 54Cone Gas 55Purge Gas 55Source temperature 57Capillary Voltage 57Sample Cone Voltage 57Extraction Cone Voltage 57

Megaflow Hints 58Removing the Probe 59

Sample Analysis and Calibration 60General Information 60

Typical ES Positive Ion Samples 61Typical ES Negative Ion Samples 61

Chromatographic Interfacing 62LC-MS Sensitivity Enhancement 63

Nanoflow ElectrosprayOverview 65Installing the Interface 66Operation of the Camera System 69Using the Microscope 69Glass Capillary Option 70

Installation 70Operation 70Restarting the Spray 71

Nano-LC Option 72Installation 72Operation 73

Fused Silica Option 74Installation 74Operation 75

Changing Options 76

Table of Contents

LCTUser's Guide

Atmospheric Pressure Chemical IonisationIntroduction 77Preparation 79

Checking the Probe 79Obtaining an Ion Beam 80Hints for Sample Analysis 83

Tuning 83Mobile Phase 83Probe Temperature 83Desolvation Gas 83

Removing the Probe 84

Calibration and Accurate MassIntroduction 85

Nominal Mass Accuracy 85Calibration 86Lock Mass 86Deadtime Correction 87

Generation of an Instrument Calibration 88Lock Mass Correction 93Deadtime Correction 94Exact Mass Measurement: Additional Hints 98

Table of Contents

LCTUser's Guide

Maintenance and Fault FindingIntroduction 101Removal and Replacement of Panels and Cover 102

Side Panel 102Top Panel 102Front Panel 103Moulded Side Cover 103

Electronics Maintenance 103Cooling Fans and Air Filters 104The Vacuum System 104

Vacuum Leaks 104Pirani Gauge 105Active Inverted Magnetron Gauge 105Gas Ballasting 105Oil Mist Filter 106Rotary Pump Oil 106

The Source 107Overview 107Cleaning the Sample Cone in Situ 108Removing and Cleaning the Sample Cone 110Removing and Cleaning the Source Block and Extraction Cone 112Removing and Cleaning the RF Lens Assembly 114Reassembling and Checking the Source 116The Corona Discharge Pin 117

The Electrospray Probe 118Overview 118Replacement of the Stainless Steel Sample Capillary 120

The APcI Probe 122Cleaning the Probe Tip 122Replacing the Probe Tip Heater 123Replacing the Fused Silica Capillary 124

Analyser and Detector 125Fault Finding 126

Introduction 126No Beam 126Unsteady Beam 127High Back Pressure 128Loss of Sensitivity 129Incorrect Isotope Distributions 129High Noise Levels 130

Chemical Noise 130Electronic Noise 130

Poor Analyser Vacuum 130Cleaning Materials 131Preventive Maintenance Check List 132

Daily 132Weekly 132Monthly 132Four-Monthly 132

Table of Contents

LCTUser's Guide

Reference InformationOverview 133Positive Ion 134

Horse Heart Myoglobin 135Polyethylene Glycol 135

PEG + NH4+ 135Sodium Iodide and Caesium Iodide Mixture 136Sodium Iodide and Rubidium Iodide Mixture 136

Negative Ion 137Horse Heart Myoglobin 137Mixture of Sugars 137Sodium Iodide and Caesium Iodide (or Rubidium Iodide) Mixture 138

Preparation of Calibration Solutions 139PEG + Ammonium Acetate for Positive Ion Electrospray and APcI 139PEG + Ammonium Acetate for Positive Ion Electrospray(Extended Mass Range) 139Sodium Iodide Solution for Positive Ion Electrospray 140

Method 1 140Method 2 140

Sodium Iodide Solution for Negative Ion Electrospray 140

Index

Table of Contents

LCTUser's Guide

Instrument DescriptionIntroduction

The LCT time of flight mass spectrometer is available with electrospray ionisation(ESI) and atmospheric pressure chemical ionisation (APcI). LCT utilises two hexapoleRF lenses to transfer ions from the source to the orthogonal acceleration time of flight(TOF) mass analyser. Ions emerging from the analyser are detected by a dualmicrochannel plate detector and ion counting system.

A PC-compatible computer runs the MassLynx NT software system to control theLCT and to acquire and process data.

Instrument DescriptionPage 9

LCTUser's Guide

Ionisation TechniquesUsing the Micromass Z-spray atmospheric pressure ionisation (API) source, twotechniques are available.

Atmospheric Pressure Chemical Ionisation

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

Electrospray

Electrospray ionisation (ESI) takes place as a result of imparting a strong electricalcharge to the eluent as it emerges from the nebuliser. An aerosol of charged dropletsemerges from the nebuliser. These undergo a reduction in size by solvent evaporationuntil they have attained a sufficient charge density to allow sample ions to be ejectedfrom the surface of the droplet (“ion evaporation”).

A characteristic of ESI spectra is that ions may be singly or multiply charged. Sincethe mass spectrometer filters ions according to their mass-to-charge ratio (m),compounds of high molecular weight can be determined if multiply charged ions areformed.

Eluent flows up to 1 ml/min can be accommodated although it is often preferable withelectrospray ionisation to split the flow such that 100 to 200 µl/min of eluent entersthe mass spectrometer.

Nanoflow Electrospray

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

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


LCTUser's Guide

Ion Optics

The principle components of the ion opticalsystem are shown here in schematic form.

Ions generated in the Z-spray source aretransferred to the orthogonal time of flightanalyser via the two independently pumped RFlenses. After entering the analyser the ion beamis focused into the pusher by the acceleration,focus, steer and tube lenses. The pusher thenpulses a section of the beam towards thereflectron, which then reflects ions back to thedetector.

As ions travel from the pusher to the detectorthey are separated in mass according to theirflight times, with ions of the highest mass tocharge ratio (m) arriving later in thespectrum.

The pusher may be operated at repetitionfrequencies of up to 20 kHz, resulting in a fullspectrum being recorded by the detector every 50 microseconds. Each spectrum issummed in the histogram memory of the time to digital converter until thehistogrammed spectra is transferred to the host PC.

If the user has requested an acquisition rate of 1 spectrum/second, each spectrumviewed on the host PC will be the result of summing up to 20,000 individual spectrarecorded at the detector.

Unlike scanning instruments, the TOF performs parallel detection of all masses withinthe spectrum at very high sensitivity and acquisition rates. This characteristic is ofparticular advantage when the instrument is coupled to fast chromatography, sinceeach spectrum is representative of the sample composition at that point in time,irrespective of how rapidly the sample composition is changing.


LCTUser's Guide

MCPDetectorPusher

Reflectron

TOFAnalyser

Z-sprayIon Source

Probe

RF Lens 1 RF Lens 2

Internal LayoutCaution: The internal layout is shown in the following diagrams for informationonly, and does not imply that labelled components are user-serviceable.

Warning: The covers should not be removed without first isolating theinstrument at the electricity supply.

Mechanical Components


LCTUser's Guide

PenningGauge

AnalyserHousing

RF Lens 2Housing

NitrogenRegulator

(for Dry Nitrogen Vent)

CoolingFans

PiraniGauge

PumpingTube

PumpingTube

(AlternativeBottom Exit)

RF Lens 1Housing

Z-spraySource

TurbomolecularPump

WaterConnections

The main internal mechanical components of the instrument are:

• The transfer lens housings, containing the two RF (hexapole) lenses.

• The analyser housing, containing the pusher, detector and reflectron assemblies.

• One 250 litre/second turbomolecular pump, and two 70 litre/secondturbomolecular pumps.

• The active inverted magnetron (Penning) gauge and the Pirani gauges.

Also shown in the above diagram:

• The nitrogen regulator, normally set to 5 psi to control the flow of nitrogen intothe analyser during venting.

• The water connections.

• The connection of the rotary pump lines.

The rotary pump lines can be connected so that they either exit through the rearpanel of the instrument, or vertically downwards through the base of theinstrument. The latter configuration allows the rear of the instrument to bepositioned closer to a wall.

• The air filters, positioned on the rear panel of the instrument.


LCTUser's Guide

Electronics


LCTUser's Guide

Pre-amplifier

Attenuator

DetectorOutput

RF Generator

PusherControl

MainElectronics

PCB

High VoltagePower Supplies

The main electronics modules of the system are:

• Four high voltage power supplies, supplying the probe or corona, reflectron,TOF flight tube and lens circuits.

• Main electronics PCB.

This controls the pumping system, and contains the low voltage lens circuits andsource heaters.

• Pusher control.

This controls the high frequency pusher voltage.

• RF Generator Control.

This controls the RF voltage applied to the RF transfer lenses.

• Attenuator and Preamplifier.

These components condition the output of the detector before the signal travelsto the TDC.


LCTUser's Guide

The Vacuum System

Fine Pumping

LCT is equipped with three water cooled turbomolecular pumps, providingindependent fine pumping of the two RF hexapole and analyser regions. Details of theoperation and maintenance of the pumps can be found in the manufacturer’s manualsprovided.

Rotary Pumping

Source pumping and turbomolecular pump backing is by a direct drive rotary pump.The rotary pump is usually situated on the floor adjacent to LCT. Details of theoperation and maintenance of the pump can be found in the manufacturer’s manualprovided.


LCTUser's Guide

TOFAnalyser

RF Lens 2RF Lens 1

Source

Active InvertedMagnetron Gauge

AutomaticVent

(to N )2Exhaust

RotaryPump

PiraniGauge

IsolationValve

TurbomolecularPumps

Speedivalve

Pressure Measurement

The source pressure is monitored by an active Pirani gauge. The analyser pressure ismonitored by an active inverted magnetron (Penning) gauge. This gauge acts as avacuum switch, switching the instrument out ofOperate mode if the pressure is toohigh. Pressure readings may be displayed on the MassLynx NT tune page.

Vacuum Protection

The vacuum system is fully interlocked to provide adequate protection in the event of:

• a fault in the vacuum system.

• a failure of the power supply.

• a failure of the water supply.

• a vacuum leak.


LCTUser's Guide

Front Panel Connections

Desolvation Gas and Probe Nebuliser Gas

The PTFE gas lines for theDesolvation Gas and probeNebuliser gas areconnected to the front of the instrument using threaded metal fittings.

High Voltage

The electrical connection for the ESI capillary or the APcI corona discharge pin is viathe coaxial high voltage connector. This socket is labeledCapillary / Corona.

Heaters

The electrical connection for the APcI probe or the ESI desolvation heater is via themulti-way connector labeledProbes . This is removed from the front panel by pullingon the metal sleeve of the plug. Both the electrospray desolvation heater and the APcIprobe heater use this connector.

The power for the source block heater is permanently connected.


LCTUser's Guide

INJECTOR

LOAD

NANOFLOW

NEBULISER

DESOLVATION

GAS

OPERATE PUMP STANDBY

INJECT

PROBES

CAPILLARY / CORONA

Front Panel Controls and Indicators

Status Display

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

The display generated by thePump LED is dependent on the vacuum status of theinstrument. TheOperate LED depends on both the vacuum status and whether theoperate mode has been selected from the Data System. Further information is includedin Automatic Pumping and Vacuum Protection(seeRoutine Procedures).

Flow Control Valves

TheDesolvation Gas andNebuliser needle valves are five-turn valves. The flowincreases as the valve is turned counterclockwise.

Divert / Injection Valve

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

• As an injection valve, with the needle port and sample loop fitted.

• As a divert valve, to switch the flow of solvent during a LC run.

• As a switching valve to switch, for example, between a LC system and a syringepump containing calibrant.

This valve is pneumatically operated, using the same nitrogen supply as the rest of theinstrument.

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


LCTUser's Guide

Rear Panel Connections

Water

Water is used to cool the turbomolecular pumps.

Nitrogen Gas In

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

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

Exhausts

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

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

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

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


LCTUser's Guide

SUPPLY INLET

SYSTEM POWER

ESD EARTH

IN

TRIGGER INPUT

GAS INEXHAUST

OUT WATER

TDC

SIP

IN OUT

ROTARY PUMP12A MAX

ELECTRONICS

Supply Inlet

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

System Power

This switch switches mains power to the instrument. In the event of the instrumentdrawing more than the rated current, the circuit breaker will trip.

Electronics

This switch switches power to the electronics without interrupting power to vacuumpumps (not available on early instruments).

Rotary Pump

Mains power to the rotary pump is controlled by the data system using this socket.

ESD Earth Facility

To prevent damage by electrostatic discharge when handling sensitive electroniccomponents, a suitable wrist band should be connected to this point (not available onearly instruments).

SIP

The TDC (time to digital converter), located in the control PC, receives the SIP (scanin progress) signal via this connector.

Optical Communications Link

Communications between the control PC and the instrument are via the two opticalconnectors labeledIN andOUT.

Trigger

The TDC, located in the control PC, receives the trigger signal via this cable.

Input

The TDC, located in the control PC, receives the input signal via this cable.


LCTUser's Guide

Event Out

Four outputs,Out 1 to Out 4 , are provided to allow various peripherals to beconnected to the instrument. SwitchesS1 to S4 allow each output to be set to beeither a contact closure (upper position) or a voltage output (lower position).

Out 1 andOut 2 , when set to voltage output, each have an output of 5 volts. Thevoltage output of bothOut 3 andOut 4 is 24 volts.

During a sample run an event output may be configured to close between acquisitionsand is used typically to enable an external device to inject the next sample.

Contact Closure In

In 1 andIn 2 inputs are provided to allow an external device to start sampleacquisition once the device has performed its function (typically sample injection).

Analog Channels

Four analog channel inputs are available, for acquiring simultaneous data such as aUV detector output. The input differential voltage must not exceed one volt.


LCTUser's Guide

CONTACTCLOSUREINPUTS

ANALOG INPUTS

EVENT OUT

1

1Vmax

1 3

42

-+

-+

-+

-+

1Vmax

1Vmax

1Vmax

IN 1

IN 2

2

3

4

MassLynx NT Data SystemA PC computer runs the MassLynx NT software system to control LCT, and toacquire and manipulate data from it. A high resolution colour monitor is also supplied.

Interaction with MassLynx NT is via the mouse and keyboard using menu-drivencommands. Printing, file management and other routine procedures are performedusing the appropriate Windows NT modules.

Software

The following software packages are supplied with LCT:

• MassLynx NT

• Screen Capture, a utility for copying user selected areas of any Windowsdisplay. The selected area can be printed directly, or saved as a bitmap file forimporting into other Windows NT applications.

• DataBridge, a utility to convert other format data files into MassLynx format.

• Microsoft Windows NT graphical environment.

• Mouse configuration.

• MaxEnt maximum entropy data enhancement algorithm (option).

• BioLynx (option).

• Visual Basic (option). This software development system is used to createmacros for use with MassLynx NT and other Windows NT applications.

The MassLynx NT User’s Guide describes the many facilities of the Micromasssoftware. Documentation for the other software is also supplied.


LCTUser's Guide


LCTUser's Guide

Routine ProceduresStart Up Following a Complete Shutdown

Preparation

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

Check the level of oil in the rotary pump sight glass. Refill or replenish asnecessary as described in the pump manufacturer’s literature.

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

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

Check that the rotary pump exhaust is connected to a suitable vent.

Check that the exhaust gas from the instrument is connected to a suitable vent.

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

Check that the rotary pump is connected to the rear of the instrument, and thatthe pump is switched off at the pump.

Check that the instrument, data system and other peripheral devices (LCequipment, printer etc.) are connected to suitable mains supplies.

Check that the optical communication cables, and the TDC trigger, input andSIP cables are connected from the instrument to the control PC.

Check that the etherlink connection is made between the control PC and the hostPC.

Switch on the mains to the mass spectrometer using theSystem Power switchsituated on the service panel at the rear of the instrument.

Switch on the electronics using the switch situated on the service panel at therear of the instrument (later instruments only).

Switch on the host PC.

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

Log on to Micromass account (password analysis).

Windows NT and MassLynx NT are configured to prevent unauthorised access.

Routine ProceduresPage 25

LCTUser's Guide

Ensure that the VxWorks disk is inserted into the drive of the control PC.

Switch on the control PC and wait approximately 2 minutes for the program toboot up.

On the host PC, double-click on the MassLynx icon in the Windows desktop anddisplay the tune page.

Pumping

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

Switch the rotary pump on at the pump.

SelectVacuum from the menu bar at the top of the tune page.

Click on Pump .

The rotary pump and the turbomolecular pumps start simultaneously.

TheVacuum LED on the front of the instrument shows amber as the systempumps down.

When the system has reached operating vacuum theVacuum LED changes to asteady green, indicating that the instrument is ready for MCP detector conditioning.

If the rotary pump oil has been changed or replenished, open the gas ballastvalve on the rotary pump. See the pump manufacturer's literature for details.

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

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

MCP Detector Conditioning

The MCP detector must be conditioned beforeuse, by gradually increasing the appliedvoltage over a long time period. This isnecessary to allow escape of all absorbedwater from within the microchannels.


LCTUser's Guide

Under normal operation the analyser automatically vents to dry nitrogen. However, ifthe nitrogen supply was not connected to the instrument when last vented, or if theinstrument has been left vented for more than one day, a significant amount of watervapour may have entered the analyser. Under these circumstances it is good practice toallow the instrument to pump for 12 hours before commencing the conditioningprocess.

In all cases, the analyser pressure must be lower than 4e-6 mbar prior to commencingMCP conditioning.

MCP conditioning should be repeated after every instrument venting.

It is not necessary to recondition the detector if the instrument has been left outof the operate mode while still under vacuum.

During routine cleaning of the source sample cone, the source isolation valve isclosed in order to maintain analyser vacuum. It is not, therefore, necessary torecondition the detector after this procedure.

The procedure for MCP conditioning is as follows:

Ensure that the analyser pressure is below 4e-6 mbar.

Check that theMCP Detector voltage is set to zero on the tune page.

SelectOther , MCP Conditioning to access the MCP conditioning program.

SetStart to 100V,Stop to 2700V,Duration to 600 minutes andStep to5 minutes.

The conditioning time may be reduced to a minimum of 120 minutes, but only ifthe analyser pressure has been less than 4e-6 mbar for at least 2 hours and theinstrument has been previously vented with dry nitrogen.

Caution: Failure to follow the recommended MCP conditioning procedure canseverely reduce detector lifetime.

Instrument Warm-up

Switch the instrument into the operate mode by selectingOperate on theMassLynx tune page.

It is advisable to let the instrument's temperature stabilise for approximately onehour after switching into operate if the best mass accuracy is required.

Leaving the instrument continuously in operate does not shorten the detectorlifetime. It is recommended that the instrument is left in operate at all times(except of course during maintenance procedures) in order to reduce mass scaledrifts due to temperature changes. Switching the instrument out of operate modeovernight is not recommended.


LCTUser's Guide

Using the Instrument

The LCT is now almost ready to use. To complete the start up procedure and preparefor running samples, follow the instructions inStart Up Following OvernightShutdownin the following pages.

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

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

Preparation for Electrospray Operation

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

SelectStandby from the MassLynx tune page

Disconnect the gas and electrical connections from the front panel.

Unscrew the probe thumb nuts and remove the probe

Undo the three thumb screws and remove the probe adjustment flange and glasstube.

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

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

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

Replace the glass tube, adjustment flange and moulded cover.

Ensure that the source enclosure is in place.

The Z-spray source enclosure consists of the glass tube and the probeadjustment flange.

Warning: Operating the source without the source enclosure willresult in solvent vapour escape and the exposure of hot surfaces andhigh voltages.

With the corona discharge pin removed, the plug fitted and the source enclosure inplace, proceed as follows:


LCTUser's Guide

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

Check that the lead of the probe adjustment flange is plugged into the socketlabelledProbes on the front panel.

Connect the electrospray probe's gas line toNebuliser on the front panel.

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

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

Plug the probe lead intoCapillary / Corona on the front panel.

If necessary, change the ionisation mode using theIon Mode command.

SetSource Temp to 100°C andDesolvation Temp to 120°C.

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


LCTUser's Guide

Preparation for APcI Operation

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

SelectStandby from the MassLynx tune page.

Disconnect the gas and electrical connections from the front panel.

Unscrew the probe thumb nuts and remove the probe.

After a period of operation at high flow rates, allow the glass source enclosure tocool before removal.

Undo the three thumb screws and remove the probe adjustment flange and glasstube.

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

Remove the blanking plug from the discharge pin mounting contact and fit thecorona discharge pin, ensuring that the tip is in-line with the tip of the samplecone.

Connect the APcI high voltage cable betweenCapillary / Corona and thesocket positioned at the bottom left corner of the source flange.

Replace the glass tube, adjustment flange and moulded cover.

Warning: Operating the source without the source enclosure willresult in solvent vapour escape and the exposure of hot surfaces andhigh voltages.

With the corona discharge pin fitted and the source enclosure in place, proceed asfollows:

Insert the APcI probe into the source and tighten up the two thumb screws.


SetSource Temp to 150°C.


Do not start the liquid flow until the gas flow and probe heater are switched onwith the probe inserted.


LCTUser's Guide

Instrument Operation

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

Ensure the instrument is in the operate mode.

The instrument will go into the operate mode only if the probe adjustment flangeis in place and a probe is inserted.

On the tune page, selectInlet andGas to turn on the source and probe gases.

SetDesolvation Gas to a flow of 150 litres/hour and adjustNebuliser tomaximum.

The system is now ready for operation. To obtain an ion beam refer toObtaining anIon Beamin either theElectrosprayor theAtmospheric Pressure Chemical Ionisationsection.


LCTUser's Guide

Automatic Pumping and Vacuum Protection


LCTUser's Guide

The pressure falls below the Pirani trip level.The Penning gauge is enabled.

is amber.Vacuum status: .

PUMPPumped Penning trip

The pressure falls below the Penning trip level.

Shutdown.(Power off, cooling water on).

PUMPOK

is green.Vacuum status:

The rotarypump and theturbo pumps

start.is

amber.Vacuum status:

PUMP

Pumping.

The rotary pumpis switched off

after 30 seconds.flashes

red.Vacuum status:

PUMP

Vented afterpump # run up

trip.


after 30 seconds.flashes

red.Vacuum status:

PUMP

Vented afterpump #

speed < 50%trip.


after 30 seconds.is off.

Vacuum status:.

PUMP

Vented

PUMP

Vented

is off.Vacuum status:

The turbopumps reachoperational

speed withinthe run-up

period.is

amber.Vacuum status:

.

PUMP

Pumped Piranitrip

The turbo pumpsdo not reachoperational

speed within therun-up period.Turbo pumpsswitched off.

flashesamber every two

seconds.Vacuum status:

.

PUMP

Venting

The turbopump(s) speed isless than 50% for

more than 5seconds.

Turbo pumpsswitched off.

flashesamber every 2

seconds.Vacuum status:

.

PUMP

Venting

The turbo pumpsspeeds falls to

50%.The vent valve

opens.


50%.The vent valve

opens.


50%.The vent valve

opens.

Select andconfirm

.VENT

Switch off the power.Locate and correct any faults.

Switch onthe power.

Switch offthe power.

SelectPUMP fromthe vacuum

menu.

The turbo pumpsswitch off.

flashesamber every

second.Vacuum status:

.

PUMP

Venting

The status of the system is indicated at all times by the front panel lamps:


LCTUser's Guide

Operate

Pump Rough

Turbo Shutdown

Venting

Vented

Transient Pressure Trip(Operate)

Transient Pressure Trip(Standby)

Pump High (Standby)

Off

PUMP

Amber

Amber

Amber

FlashRed

Green

Green

FlashAmber

Off

Off

Off

Off

Off

OPERATE

Amber

Green

Off

Transient Pressure Trip

The transient trip is designed to protect the instrument from potentially damagingpressure surges and operates routinely whenever the pressure rises.

Should the vacuum gauge(s) detect a pressure surge above the preset trip level(normally set at 1 × 10-5 mbar by software) the following events occur:

• The greenPUMP lamp becomes amber.

• If in the operate mode, the system turns off the critical source, analyser anddetector voltages, and the greenOPERATE lamp becomes amber.

• Acquisition continues though, of course, no real data are recorded.

When the vacuum recovers:

• The amberPUMP lamp becomes green.

• If previously in the operate mode, voltages are restored andOPERATE revertsto green.

The period during which the trip was operative will appear in a raw total ionchromatogram as a period of reduced baseline noise.

Further deterioration of the system pressures results in a “vacuum fault” condition andthe system is shut down (see below).

Water Failure

A failure of the water supply for more than a brief period results in a “vacuum fault”condition and the system is shut down (see below).

Vacuum Fault

A vacuum fault can occur as a result of:

• a failure of the water supply.

• a vacuum leak.

• a malfunction of the turbomolecular or rotary pumps.

A vacuum fault causes the system to vent and is signaled by thePUMP lamp flashingred. Proceed as follows:

Switch off the power to the rotary pumps.

Investigate and rectify the cause of the fault.

Pump down the instrument as described earlier in this chapter.


LCTUser's Guide

Power Failure

In the event of an unexpected failure of the electrical supply the instrument is ventedsafely. If power is unlikely to be restored quickly, follow the shutdown proceduredescribed later in this chapter. When power is restored follow the startup procedure.

Should the power fail and then be restored while the instrument is unattended, thesystem will advance to the “SelectPUMP” position in the left hand column of theflow diagram earlier in this chapter.

Nitrogen Supply

Replacement of nitrogen cylinders should be conducted in accordance with theoperation, handling and storage instructions provided by the local gas supplier.

SelectInlet andGas on the tune page, to close the nitrogen inlet valve prior todisconnecting the supply.

Set the nitrogen inlet pressure to 7 bar (100 psi).

Under no circumstancesshould the nitrogen pressure exceed 10 bar (140 psi).


LCTUser's Guide

TuningBefore sample data are acquired, the instrument should be tuned and calibrated usingsuitable reference compounds.

Tuning parameters have been grouped into 3 menus as described below. Full details ofsource tuning procedures for electrospray, APcI and nanoflow electrospray are givenin the relevant chapter of this document.

Source Tuning Menu

The positive ion electrospray (ES+) source menu is shown.

Sample Cone is sample dependent.Extraction Cone , not normally sampledependent, is normally set within the range 5 to 10V (typically 10V).


LCTUser's Guide

Analyser Tuning Menu

RF Lens

RF Lens controls the amplitude of the RF voltage applied to both RF Lens 1 andRF lens 2.

For work at higherm a value of 500V is typical. However, for optimumtransmission at lowm (<200m) this voltage should be reduced.

RF DC Offset 1

The DC offset of the first RF lens affects the ion energy of the beam as it enters theanalyser. A typical value for theRF DC Offset 1 is 3V. It is recommended that thisvoltage is not varied during routine tuning.

RF DC Offset 2, Aperture, Acceleration, Focus, Steering

Normally these voltages are tuned for maximum resolution whilst observing a suitabletest compound such as melittin. A sensitivity increase can sometimes be achieved bytuning for sensitivity at the expense of resolution.

Typical voltages are as shown in the tune page above.

MCP Detector

This voltage determines the gain of the detector and is normally set to 2700V. Thisvoltage should be set in conjunction withTDC Stop Threshold (see later).


LCTUser's Guide

Engineer Tuning Menu

The voltages controlled from the engineer menu should be set for optimum resolutionon instrument installation. These voltages should not be varied during routineoperation of the instrument.

It is recommended that a record of these values is kept for future reference.

Manual Pusher, Max. Flight Time

If the Manual Pusher box is checked then the repetition frequency of the pusherpulse is determined by theMax. Flight Time entered.

Max. Flight Time may be set between 50 µsec and 250 µsec .

A Max. Flight Time of 50 µsec permits ions to be analysed up to 1700m. Themass range is proportional to the square of the flight time, so a flight time of, forexample, 100 µsec permits ions to be analysed up to 4 × 1700 = 6800m.

Increasing the flight time reduces the duty cycle (sampling efficiency) of the TOFanalyser resulting in decreased sensitivity.

For the best mass accuracy the instrument should be re-calibrated if the flight time ischanged.


LCTUser's Guide

If Manual Pusher is not checked then the repetition frequency of the pusher pulse isdetermined automatically according to the highestm requested in the acquisitionrange, as shown in the table below.

Highest/ Maximum Flight Time1650 50 µsec3800 75 µsec6800 100 µsec13000 140 µsec


LCTUser's Guide

Other Tune Page Settings

TDC Settings

To access the TDC (time to digital converter) settings:

SelectOptions , TDC settings

Start (mV)

This is the size of the trigger signalthat is necessary to trigger the TDC(start the clock). A typical value is700. This voltage should not need tobe changed once set.

Stop (mV)

This is the size of pulse needed toregister as being an ion, so stoppingthe clock. It should be set to a valuehigh enough to prevent electronicnoise being detected as ions. This may be achieved as follows:

Acquire data with the detector turned off (MCP voltage = 0) and theinstrument in the operate mode.

Use anIntegration Time of about 2 seconds.

At a Stop value of 50mV a signal equivalent to hundreds of ion counts will bedetected as noise.

Increase theStop value in 20mV increments during the acquisition.

The number of ions will fall until a value is reached where only a few (<5)counts are recorded per integration.

Use this value in all subsequent acquisitions.

A typical value is 150mV.

A Stop value that is set too low will result in high background noise. A valuewhich is set too high will result in loss of signal and isotopic distortions.

Threshold

This parameter should normally be set to zero. Setting to 1 will cause all peaks in thespectrum with 1 count to be thresholded out.


LCTUser's Guide

Veff Factor

This should be set to 1.096

Lteff

This is used to make the TOF mass measurement nominally correct without acalibration. The default value is 1195.

To adjust the nominal mass scale (as displayed on the tune page without calibration):

Acquire a TOF spectrum of a standard compound withLteff set to 1195.

Calculate the new value ofLteff from the relation:

( ) ( )Lteff 1195 m mind act= ÷

where:

mind = indicatedm.

mact= actualm.

Enter the newLteff value in the tune page.

Turn off the real time display then turn it on again for the new value to takeeffect.

Once the newLteff value has been entered all subsequent acquisitions should becorrectly mass measured.


LCTUser's Guide

Tuning Setup

This menu enables the real time peak displayparameters to be set:

SelectPeak Display , Setup Scope

Tune Page Acquisition

To access this menu:

SelectAcquire .

This menu allows single acquisitions to be made from the tune page. A savedcalibration file may be selected (selectCalibration ).


LCTUser's Guide

Real Time Tune Page Display

To change the mass range and normalisation ofthe real time display:

Right click on the real time display.

TheSetup box is displayed.

Enter theMin. Mass andMax. Mass asrequired.

Select either:

Normalise to Maximum to normalise the display to the largest peakdisplayed.

or:

Normalise to Value to normalise the display to the indicated number ofcounts.

CalibrationInformation concerning the calibration of LCT is provided inCalibration andAccurate Masslater in this document and in theGuide to Data Acquisition.

Data AcquisitionThe mechanics of the acquisition of sample data are comprehensively described in theGuide to Data Acquisition. Refer to that publication for full details.

Data ProcessingThe processing of sample data is comprehensively described in theMassLynx NT User’s Guide. Refer to that publication for full details.


LCTUser's Guide

Shutdown Procedures

Emergency Shutdown

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

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

A loss of data is likely.

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

Overnight Shutdown

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

Switch off the LC pumps.

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

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

If APcI is being used, reduceAPCI Probe Temp to ambient temperature.

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

SelectInlet followed byGas to turn off the supply of nitrogen gas.

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

ReduceSource Temp to 60°C.

It is not necessary to turn the instrument out of the operate mode.

Complete Shutdown

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


On the tune page, click onPress for Standby .

The tune page indicator changes to red, indicating that the instrument is nolonger in the operate mode.


LCTUser's Guide

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

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

If APcI is being used, reduceAPCI Probe Temp to ambient temperature.

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

SelectInlet followed byGas to turn off the supply of nitrogen gas.

SelectVacuum from the menu bar at the top of the tune page. Click onVent .

The rotary pump and the turbomolecular pumps switch off.

When the turbomolecular pumps have run down to half of their normaloperating speed the vent valve opens and the instrument is automatically ventedto dry nitrogen.

Exit MassLynx.

Shut down the host PC.

Switch off the control PC.

Switch off all peripherals.

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

Switch off power at the wall mounted isolation switches.

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

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

Refill the rotary pump with new oil.


LCTUser's Guide


LCTUser's Guide

ElectrosprayIntroduction

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

Mobile phase from the LC column or infusion pump enters through the probe and ispneumatically converted to an electrostatically charged aerosol spray. The solvent isevaporated from the spray by means of the desolvation heater. The resulting analyteand solvent ions are then drawn through the sample cone aperture into the ion block,from where they are then extracted into the analyser.

The electrospray ionisation technique allows rapid, accurate and sensitive analysis of awide range of analytes from low molecular weight (less than 200 Da) polarcompounds to biopolymers larger than 100 kDa.

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

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

ElectrosprayPage 47

LCTUser's Guide

ProbeExhaust

ExhaustLiner

TurbomolecularPumps

RotaryPump

NebuliserGas

Purge Gas(megaflow only)

Sample

Analyser

DesolvationGas

SampleCone

ExtractionCone

IsolationValve

SourceEnclosure

RFLens

CleanableBaffle

The source can be tuned to fragment ions within the ion block. This can providevaluable structural information for low molecular weight analytes.

The most common methods of delivering sample to the electrospray source are:

• Syringe pump and injection valve.

A flow of mobile phase solvent passes through an injection valve to theelectrospray source. This is continuous until the pump syringes empty and needto be refilled. Sample is introduced through the valve injection loop (usually 10or 20µl capacity) switching the sample plug into the mobile phase flow. Tuningand acquisition are carried out as the sample plug enters the source. (At a flowrate of 10 µl/min a 20µl injection lasts 2 minutes.)

• Reciprocating pump and injection valve.

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

• Infusion pump.

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

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

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

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

These additives should not be used in the mobile phase for flow injection analysis(FIA) studies, to allow easy change over between positive and negative ion analysis.

Degassed solvents are recommended for the syringe and reciprocating pumps.Degassing can be achieved by sonification or helium sparging. The solvents should befiltered, and stored under cover at all times.

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LCTUser's Guide

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

Post-column Splitting

Although the electrospray source can accommodate flow rates up to 1 ml/min, it isrecommended that the flow is split post-column to approximately 200 µl/min. Also,even at lower flow rates, a split may be required to save valuable samples.

The post-column split consists of a zero dead-volume tee piece connected as shown.

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

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

ElectrosprayPage 49

LCTUser's Guide

To Wasteor

UV Cell

LCColumn

Megaflow

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

Changing Between Flow Modes

When changing between megaflow and standard electrospray operation, it is essentialthat the correct tubing is used to connect the probe to the sample injector. Formegaflow operation1/16" o.d., 0.007" i.d. peek tubing, easily identified by its yellowstripe, is used. This replaces the standard fused silica tube, together with the PTFEsleeves.

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LCTUser's Guide

Normal Flow Electrospray

Megaflow Electrospray

OperationWarning: The probe tip is sharp, and may be contaminated withharmful and toxic substances. Always take great care when handlingthe electrospray probe.

Ensure that the source is assembled as described inMaintenance and FaultFinding, and that the instrument is pumped down and prepared for electrosprayoperation as described inRoutine Procedures.

Ensure that a supply of nitrogen has been connected to the gas inlet at the rear ofthe instrument and that the head pressure is between 6 and 7 bar (90-100 psi).

Ensure that the exhaust liner and the cleanable baffle are fitted to the source.

This is important for optimum electrospray intensity and stability whenoperating at low flow rates.

Checking the ESI Probe

Connect the electrospray probe to a pulse free pump.

Solvent should be degassed to prevent beam instabilities caused by bubbles.

Connect the PTFE tubing of the electrospray probe toNebuliser Gas on thefront panel. Secure with the nut provided.

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

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

Turn onNitrogen by selectingInlet thenGas, and check that a nebuliser flowof less than 100 litres/hour is registered.

To monitor the flow rate, selectWindow thenGas Flow on the tune page andobserve the readback window.

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

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LCTUser's Guide

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

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

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

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

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

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

Insert the probe into the source and tighten the two thumb screws.

Plug the probe high voltage cable intoCapillary / Corona on the front panel.

Obtaining an Ion Beam


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

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

Tuning and Optimisation

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

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LCTUser's Guide

0.5mmSample

Capillary

NebulisingCapillary

Probe Position

The position of the probe is adjustedusing the probe adjustment collar(in and out) and the adjustment knob(sideways) located to the left of theprobe. The two screws can beadjusted singly or simultaneously tooptimise the beam. The position foroptimum sensitivity and stability forlow flow rate work (10 µl/min) isshown.

Small improvements may be gained byvarying the position using the sample and

solvent system under investigation. The following information should be consideredwhen setting the probe position:

• 10mm of movement is provided in each direction, with 1.25mm of travel perrevolution of the probe positioning controls.

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

Nebuliser Gas

Optimum nebulisation for electrospray performance is achieved with a nitrogen flowbetween 70 and 90 litres per hour. This can be achieved by fully opening theNebuliser flow control valve, which is situated on the instrument’s front panel.

ElectrosprayPage 53

LCTUser's Guide

8mm

4mm

SampleCone

ProbeTip

In / OutProbe

Adjustment

SidewaysProbe

Adjustment

Desolvation Gas

The desolvation gas, also nitrogen, is heated and delivered as a coaxial sheath to thenebulised liquid spray by the desolvation nozzle.

The position of the desolvation nozzle heater is fixed relative to the probe tipand requires no adjustment.

TheDesolvation Gas flow rate is adjusted by the control value situated on theinstrument’s front panel. The optimumDesolvation Temp and flow rate isdependent on mobile phase composition and flow rate. A guide to suitable settings isgiven below.

TheDesolvation Gas flow rate indicated on the MassLynx tune pagerepresents total drying flow, that is desolvation gas + cone gas (nanoflow only)+ purge gas (if enabled).

Solvent Flow Rateµl/min

Desolvation Temp°C

Desolvation Gas FlowRate

litres/hour

<10 100 to 120 200 to 250

10 to 20 120 to 250 200 to 400

20 to 50 250 to 350 200 to 400

>50 350 to 400 500 to 750

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

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

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LCTUser's Guide

Cone Gas

The cone gas should be used only in thenanoflow mode (see the following chapter).Ensure that the cone gas outlet, situatedinside the source enclosure, is blanked fornormal ESI operation.

Purge Gas

The purge gas is not necessary for mostESI applications. It may be useful formegaflow operation where an analyte issusceptible to acetonitrile adducting.

Purge gas is enabled simply by removingthe blanking plug from the outlet situatedwithin the source enclosure.

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

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LCTUser's Guide

SampleCone

PurgeGas Plug

Cone GasPlug

ElectrosprayPage 56

LCTUser's Guide

Source temperature

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


Capillary Voltage

Capillary usually optimises at 3.0kV, although some samples may tune at valuesabove or below this, within the range 2.5 to 4.0kV for positive electrospray. Fornegative ion operation a lower voltage is necessary, typically between 2.0 and 3.5kV.

At high flow rates this parameter may optimise at a value as low as 1kV.

Sample Cone Voltage

A Cone setting between 25V and 70V will produce ions for most samples, althoughsolvent ions prefer the lower end and proteins the higher end of this range. Wheneversample quantity and time permit,Cone should be optimised for maximum sensitivity,within the range 15V to 150V. IncreasingCone will increase ion fragmentationwithin the source.

Extraction Cone Voltage

Extraction optimises at 5 to 10V. Higher values may induce ion fragmentation oflow molecular weight samples.

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LCTUser's Guide

Megaflow Hints

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

• IncreaseDrying Gas flow to approximately 750 litres/hour.

• IncreaseDesolvation Temp to 400°C.

• IncreaseSource Temp to 150°C.

• Move the probe further away from the sample cone.

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


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

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

Purge gas can be used during megaflow operation to stop the source enclosure fromoverheating. This is also beneficial when the analyte is susceptible to acetonitrileadducting. Purge gas is enabled by removing the blanking plug from the outlet situatedwithin the source enclosure.

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

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

The source enclosure will run cooler if purge gas is used.

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

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

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LCTUser's Guide

Removing the Probe

To remove the probe from the source proceed as follows:

On the tune page selectStandby .

Switch off the liquid flow and disconnect from the probe.

SelectInlet thenGas and turn offNitrogen .

Disconnect the probe cable from the instrument.

Disconnect the nebulising gas supply from the instrument.

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LCTUser's Guide

Sample Analysis and Calibration

General Information

Care should be taken to ensure that samples are fully dissolved in a suitable solvent.Any particulates must be filtered to avoid blockage of the transfer line or the probe’scapillary. A centrifuge can often be used to separate solid particles from the sampleliquid.

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

Higher concentrations will not usually improve analytical performance. Conversely,for biopolymers, lower concentrations often yield better electrospray results. Higherlevels require more frequent source cleaning and risk blocking the transfer capillary.

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

Samples with phosphate buffers and high levels of salts should be avoided.Alternatively, at the expense of a small drop in sensitivity, the probe can be pulledaway from the sample cone to minimise the deposit of involatile material on the cone.

To gain experience in sample analysis, it is advisable to start with the qualitativeanalysis of known standards. A good example of a high molecular weight sample ishorse heart myoglobin (molecular weight 16951.48) which produces a series ofmultiply charged ions that can be used to calibrate them scale from 800-1600 ineither positive ion or negative ion mode.

Polyethylene glycol mixtures, for example 300 / 600 / 1000, are low molecular weightsamples suitable for calibrating them scale from approximately 100 to 1200 inpositive ion mode. A mixture of sugars covers the same range in negative ion mode.

Alternatively, a mixture of sodium iodide and caesium iodide (or a mixture of sodiumiodide and rubidium iodide) can be used for calibration.

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Typical ES Positive Ion Samples

• Peptides and proteins.

• Small polar compounds.

• Drugs and their metabolites.

• Environmental contaminants (e.g. pesticides, pollutants).

• Dye compounds.

• Some organometallics.

• Small saccharides.

Typical ES Negative Ion Samples

• Some proteins.

• Some drug metabolites (e.g. glucuronide conjugates).

• Oligonucleotides.

• Some saccharides and polysaccharides.

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Chromatographic InterfacingElectrospray ionisation can be routinely interfaced to reversed phase and normal phasechromatographic separations. Depending on the LC pumping system, chromatographycolumn and setup, there are some basic options:

• Microbore and capillary chromatography separations employing 1mm diameter(and smaller) columns can be interfaced directly to the electrospray probe.Typical flow rates for such columns may be in the region of 3-50 µl/min. It issuggested that a syringe pump is used to deliver these constant low flow ratesthrough a capillary column. Alternatively, accurate pre-column splitting ofhigher flow rates from reciprocating pumps can be investigated.

In all cases, efficient solvent mixing is necessary for gradient elution separations.This is of paramount importance with regard to low flow rates encountered withcapillary columns. HPLC pump manufacturers’ recommendations should beheeded.

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

A UV detector may be placed in-line to the probe, provided that the volume ofthe detector does not significantly reduce the chromatographic resolution.Whenever a UV detector is used, the analog output may be input toMassLynx NT for chromatographic processing.

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


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

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

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

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Higher source temperatures (150°C) are also recommended for high aqueous contentsolvents. Tetrahydrofuran (THF) shouldnot be used with peek tubing.

LC-MS Sensitivity Enhancement

The sensitivity of a LC-MS analysis can be increased or optimised in a number ofways, by alterations to both the LC operation and the MS operation.

In the LC area some examples include the use of high resolution columns and columnswith fully end capped packings. For target compound analysis, techniques such astrace enrichment, coupled column chromatography, or phase system switching canhave enormous benefits.

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

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Nanoflow ElectrosprayOverview

The optional nanoflow interface allows electrospray ionisation to be performed in theflow rate range 5 to 1000 nanolitres per minute. There are two options for the sprayingcapillary, which can be alternately fitted to the interface:

• Borosilicate metal coated glass capillary.

Metal coated glass capillaries allow the lowest flow rates to be obtainedalthough they are used for one sample only and then must be discarded.

• Nano-LC or, on early systems, fused silica capillary.

This option is suitable for flow injection analyses or for coupling to nano-HPLC,and uses a pump to regulate the flow rate down to 100 nl/min. If a syringe pumpis to be used, a gas-tight syringe is necessary to obtain correct flow rateswithout leakage. A volume of 25µl is recommended.

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Regulatorand Injector

(Nano-LC option)

ProtectiveCover

Handle

Stop

Fused SilicaOption

GlassCapillaryOption

Nano-LCOption

Stage

Three-axisManipulator

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

The nanoflow end flangeconsists of a three-axismanipulator, a stage, aprotective cover and a stop /handle arrangement forrotation of the manipulatorand stage.

The manipulator and stageare rotated by 90 degrees tochange option or, in the glasscapillary option, to load anew nanovial.

Caution: Failure to usethe stop and handle torotate the stage canresult in permanentdamage to thethree-axis manipulator.

Installing the InterfaceTo change from the normal electrospray interface and install the nanoflow interface:

If fitted, remove the probe.

Remove the moulded cover from around the source.

Undo the three thumb screws and withdraw the probe adjustment flangeassembly and glass tube.

Place the glass tube, end on, on a flat surface and place the probe support flangeassembly on top of the glass tube.

Remove the PTFE encapsulated source O ring.

Warning: When the source enclosure has been removed the ion block heater isexposed. Ensure that the source block heater has been switched off and hascooled before proceeding. Observe theSource Temp readback on the tunepage.


LCTUser's Guide

Exhaust

ExhaustLiner

TurbomolecularPumps

RotaryPump

Cone Gas

SampleCapillary

Analyser

SampleCone

SampleCone

Nozzle

ExtractionCone

IsolationValve

SourceEnclosure

RFLens

CleanableBaffle

Unscrew the three probe flange mounting pillars, using the holes to obtain thenecessary leverage.

If the cone gas nozzle is not inplace, remove the two screws thatsecure the sample cone and fit thecone gas nozzle.

Replace the two screws.

Connect the cone gas outlet to thecone gas nozzle using the PTFEtubing provided.

The cone gas flow rate is set at30% of the total desolvation gasflow.

Ensure that the purge gas isplugged (disabled).

Ensure that the cleanable baffle, the exhaust liner and the corona discharge pinblanking plug are fitted.


LCTUser's Guide

Cone GasNozzle

PTFETubing PTFE

EncapsulatedO Ring

PurgeGas Plug

SourceThumb Nuts

ProbeThumb Nuts

ProbeAdjustment Flange

GlassTube

Probe FlangeMounting Pillar

Fit a viton O ring and the three shorter nanoflow pillars.

Install the perspex cover and thenanoflow end flange, securing thiswith socket head screws.

Do not attempt to refit the mouldedcover.

If not already in place, attach themicroscope or camera brackets usingthe screw hole and dowels at the topof the bracket.

Insert the flexible light guide into thegrommet at the base of the perspexcover.

Set the light source to its brightest.

Block theNebuliser andDesolvation Gas outlets on theinstrument's front panel.

The cone gas is split from the desolvation gas internally.

Attach the two cables to the sockets markedCapillary / Corona andProbeson the front panel of the instrument.

SetSource Temp to approximately 80°C.



LCTUser's Guide

VitonO Ring

SocketHead Screws

NanoflowEnd Flange

PerspexCover

Operation of the Camera SystemMagnification is controlled by the zoom lens. A fine focus can be achieved by rotatingthe objective lens.

Using the MicroscopeFocusing is adjusted by rotating the top of the microscope.


LCTUser's Guide

Microscope

Camera

ZoomLens

ObjectiveLens

Grommet

Glass Capillary Option

Installation

Warning: Do not touch the sharp end of the capillary. As well as the riskof injury by a sliver of glass, the capillary may contain toxic samples

Caution: The capillaries are extremely fragile and must be handled with greatcare. Always handle using the square end of the capillary. The needle maybecome inoperable if the sharp end is touched.

With the stage rotated outwards, unscrew the union from the end of theassembly.

Carefully remove the capillary from itscase by lifting vertically while pressingdown on the foam with two fingers.

Over the blunt end of the capillary, passthe knurled nut, approximately 5mm ofconductive elastomer and finally the union.

Tighten the nut (finger tight is sufficient)so that 5mm of glass capillary isprotruding from the end of it. This distanceis measured from the end of the nut to theshoulder of the glass capillary.

Load sample into the capillary using either afused silica syringe needle or a gel loader tip.

Screw the holder back into the assembly - fingertight is sufficient.

Ensure thatCapillary is set to 0V on the tunepage.

Rotate the stage back into the interface using thestop and handle.

Operation

Manoeuvre the stage so that the microscope orcamera can view the capillary tip.

Using a 10ml plastic syringe or a regulated gassupply, apply pressure to the back of the tip untila drop of liquid is seen.

On the tune page, selectInlet thenGas and turn onNitrogen .


LCTUser's Guide

Foam

Capillary

GlassCapillary

BlueConductiveElastomer

PTFE"Back Pressure"

Tubing

Ferrule

5mm

Knurled Nut

Union

SelectOperate .

SetCapillary between 1 and 1.5kV.

Adjust Desolvation Gas using the knob on the front panel of the instrument.

An ion beam should now be visible on the tune page.

Tune the source voltages, adjust the gas flow and adjust the three-axismanipulator for maximum ion current.

The ion current may change dramatically with very slight changes of positionbut the high resolution of the threads in the manipulator allows very fine tuning.

Restarting the Spray

Should the spray stop, it is possible to restart it by adjusting the three-axis manipulatorso that, viewed under magnification, the capillary tip touches the sample cone and asmall piece of the glass hair shears off.

It may also be necessary to apply some back pressure to the holder to force a drop ofliquid from the capillary. Up to 1.4 bar (20 psi) can be applied and, with this pressure,a drop should be visible unless the capillary is blocked.


LCTUser's Guide

Nano-LC Option

Installation

With the sprayer assembly removedfrom the stage:

Cut approximately 25mm of thered stripe peek tubing and, usingthe plug cap and a Valco nut, set aferrule to the correct position onthe tubing.

At this stage the ferrule is requiredonly to grip the tubing lightly, andshould not be too tight.

Cut the peek such that 10mm ofthe peek protrudes from the backof the ferrule.

Thread approximately 70mm ofthe 90 micron o.d. fused silicathrough the new fitting.

Ensure that the fused silica is flushwith the peek sleeve.

Again using the plug cap, tightenthe nut further to ensure that thefused silica is gripped. Some forcemay be required to do this.

Remove the sleeved fused silicafrom the plug cap and remove theValco nut.

Place an O ring onto the peek tube,using tweezers if necessary.

The O ring is required to seal the region between the ferrule and the end of thethread on the nano-LC chamber.

Thread the sleeved fused silica through the nano-LC chamber.

Rotate the microvolume union in the body such that the ferrule seat is alignedcorrectly.

Insert the chamber into the nano-LC body and tighten using a pair of spanners.


LCTUser's Guide

1mm

From Injector(or Column

Attached Directly)

Make-upFlowOnly

(3-wayInsert

Required)

NebuliserGas

Nano-LCBody

Chamber

NebulisingTip

Red StripePeek Tubing

O Ring

90µm FusedSilica

ValcoFerrule

MicrovolumeInsert

The capillary can now be checked for flow by connecting the output from aHarvard syringe pump to the other side of the union and setting the flow to1 µL/min, using a micropipette to measure the flow. It is recommended that asyringe with a volume of no more than 50 millilitres is used.

Thread the fused silica through the nebulising tip and screw in the nano-LCchamber such that it is screwed in approximately half way.

Cut the fused silica using a tile cutter and adjust the nebulising tip further, suchthat 1mm of fused silica protrudes from the tip.

Attach the nebulising gas tubing to the sprayer using an O ring and the specialscrew.

Attach the sprayer assembly to the stage.

It may be necessary to alter the position of the thumbscrew underneath thebaseplate to attach the sprayer correctly.

Swing the stage into the interface using the stop and handle.

Operation

For tuning purposes it may be useful to infuse a known sample in 95% water using aHarvard syringe pump.

Set the liquid flow to about 200 nl/min.

Switch onGas at the MassLynx tune page.

Set the pressure of the gas on the regulator to approximately 0.5 bar (7 psi).

Ensure there are no leaks of gas at the sprayer, particularly where the PTFEtubing is connected to it.

By viewing under magnification, the spray emanating from the capillary may beexamined and tuned by altering the nebulising tip such that a fine spray is observed.Altering the gas slightly may also help in this tuning process.

Swing the stage back out of the source and place the cover over the sprayerensuring that the tubing coming from the sprayer is threaded correctly through it.

Lock the cover in place with two screws.

Swing the stage back into the source and alter the translation stage (in / outdirection) such that the capillary is approximately 5mm from the cone.

SelectOperate and setCapillary to approximately 2.5kV.

An ion beam should now be present.


LCTUser's Guide

Optimise the ion beam by altering the position of the spray using the controls ofthe translation stage.

The sprayer can now be connected to the HPLC system. The injection valve isplumbed as follows:

• P from the pump.• C to the column (or to the union).• S is the sample port, attach a VISF sleeve here.• W is a waste port.

A short tail of fused silica, attached to the entrance port of the union, and theuse of low pressure PTFE connectors will remove the need to move the stage.This will prevent accidental alteration of the sprayer's position when changingbetween tuning and HPLC operation.

Fused Silica Option

Installation

Use the plug cap prior to assembly in the lowdead volume union to set the ‘pilot depth’ of theferrule on the tubing.

Plumb the low dead volume union as shown,making sure that each of the fused silica piecesare held firmly by the appropriate sleeving and aValco ferrule.

On one side of the union is 375µm o.d., 25µm i.d.fused silica from the column or injector, sleevedto 1/16 inch with PTFE tubing. On the other is thespraying capillary, 90µm o.d., 20µm i.d. fusedsilica, sleeved to1/16 inch with red stripe peektubing.

Approximately 5mm of 90µm o.d. fused silicashould protrude from the end of the nut asshown. This may be cut to size at the end of the assembly process.

Attach the union to the contact plate using the V-shaped clamps. Attach the plateto the manipulator stage.

Replace the protective cover and swing the stage into the interface using the stopand handle.


LCTUser's Guide

5mm

From Injector(or Column

Attached Directly)

1/16" PTFETubing

SprayingCapillary

Red StripePeek Tubing

ValcoFerrule

ValcoFerrule

The Valco injector is attached to the flange by a bracket and can be used for loopinjections. The injection valve is plumbed as follows:

• P from the pump.• C to the column (or to the union).• S is the sample port, attach a VISF sleeve here.• W is a waste port.

Operation

Raise the flow to 1 µl/min. After a few minutes, reduce it to the desired flowrate.

To avoid a very unstable beam, it is imperative to remove any air bubbles fromthe fused silica lines.

SelectInlet thenGas and switch onNitrogen . SelectOperate .

SetCapillary to approximately 3kV.

An ion beam should be visible.

Tune the source parameters for maximum signal.

Optimise the position of the needle using the three-axis manipulator.

In the fused silica option the needle tends to optimise further away from thecone than in the borosilicate option.

The spray from the needle should be easily visible by the microscope or cameraand the stability of the spray can be seen using either system.

A column can be attached directly to the back of the union to reduce any deadvolumes to a minimum, but care must be taken to match the external diameter of thecolumn with the internal diameter of any1/16 inch sleeve.


LCTUser's Guide

Changing OptionsTo change between the glass capillary and the nano-LC or fused silica options:

Rotate the stage outwards.

Caution: Failure to use the stop and handle to rotate the stage can result inpermanent damage to the three-axis manipulator.

Remove the protective cover and release the captive screw located underneaththe stage.

Lift off the holder and replace it with the alternative holder, securing it with thecaptive screw.

Replace the protective cover, ensuring that either the PTFE back pressure tubing(glass capillary option) or the fused silica transfer line is fed through the slot inthe back of the protective cover along with the HV cabling.


LCTUser's Guide

Atmospheric Pressure ChemicalIonisation

Introduction

Atmospheric Pressure Chemical Ionisation (APcI) is an easy to use LC-MS interfacethat produces singly-charged protonated or deprotonated molecules for a broad rangeof involatile analytes.

The ability to operate with 100% organic or 100% aqueous mobile phases at flowrates up to 2 ml/min makes APcI an ideal technique for standard analytical column(4.6mm i.d.) normal phase and reverse phase LC-MS.

The APcI interface consists of the standard Z-spray source fitted with a coronadischarge pin and a heated nebuliser probe. Mobile phase from the LC column entersthe probe where it is pneumatically converted into an aerosol and is rapidly heated andconverted to a vapour / gas at the probe tip. Hot gas from the probe passes betweenthe sample cone and the corona discharge pin, which is typically maintained at 2.5kV.Mobile phase molecules rapidly react with ions generated by the corona discharge toproduce stable reagents ions. Analyte molecules introduced into the mobile phase reactwith the reagent ions at atmospheric pressure and typically become protonated (inpositive ion mode) or deprotonated (in the negative ion mode). The sample andreagent ions pass through the sample cone into the ion block prior to being extractedinto the hexapole transfer lens through the extraction cone.

Atmospheric Pressure Chemical IonisationPage 77

LCTUser's Guide

Probe CoronaDischarge

PinCleanable

Baffle

Exhaust

ExhaustLiner

TurbomolecularPumps

RotaryPump

NebuliserGas

Sample

Analyser

DesolvationGas

SampleCone

ExtractionCone

IsolationValve

SourceEnclosure

RFLens

Changeover between electrospray and APcI operation is simply accomplished bychanging the probe and installing the corona discharge pin within the sourceenclosure.

For APcI operation, the desolvation gas is not heated in the desolvation nozzle.However, it is important that desolvation gas is used throughout.

The background spectrum for 50:50 acetonitrile:water is dependent upon the settingsof Cone andExtractor . The main reagent ions for typical sample cone andextraction cone voltages of 40V and 10V respectively are 42,56,83, and 101.

The transmission of these ions will be dependent on the setting ofRF Lens . A lowerRF Lens voltage is required for optimum transmission at lowerm.

Acetonitrile adducting may be minimised by optimisation of the probe position.


LCTUser's Guide

PreparationEnsure that the source is assembled as described inMaintenance and FaultFinding, and that the instrument is pumped down and prepared for APcIoperation as described inRoutine Procedures.

APcI may be operated with or without the cleanable baffle fitted.

Ensure that a supply of nitrogen has been connected to the gas inlet at the rear ofthe instrument and that the head pressure is between 6 and 7 bar (90-100 psi).

Checking the Probe

Ensure that the probe heater is off.

Unplug the probe from the instrument’s front panel and remove the probe fromthe source.

Connect the PTFE tube to theNebuliser outlet on the front panel.

Remove the probe tip assembly by carefully loosening the two grub screws.

Disconnect the heater from the probe body by pulling parallel to the axis of theprobe.

Ensure that 0.5 to 1mm of fused silica is protruding from the stainless steelnebuliser tube.

Connect the LC pump to the probe with a flow of 50:50 acetonitrile:water at1 ml/min.

Check that the liquid jet flows freely from the end of the capillary and that theLC pump back pressure reads 250 to 400 psi.

Check that the nitrogen supply pressure is 6 to 7 bar (90 to 100 psi).

SelectInlet thenGas and turn onNitrogen .

Check that the liquid jet converts to a fine uniform aerosol.

Switch off the liquid flow.


Reconnect the probe tip assembly.

Insert the APcI probe into the source and secure it by tightening the two thumbscrews.

Connect the probe cable toProbes on the instrument's front panel.


LCTUser's Guide

Obtaining an Ion Beam


LCTUser's Guide

Ensure that the corona discharge pin is fitted as described inRoutineProcedures, Preparation for APcI Operationand that the pin is connected usingthe APcI HV cable.

Ensure that the APcI probe is fitted as described above, that the desolvation gastube is connected to the front panel, and that the cone gas and purge gas outletsare plugged.


SetSource Temp to 150°C.


SetAPcI Probe Temp to 20°C with no liquid flow andNitrogen off.

Initially set Corona to 2.5 kV andCone to 30V.

WhenSource Temp reaches 150°C:

SelectNitrogen to switch on the nitrogen gas.

Using the valves on the front of the instrument, adjustDesolvation Gas to150 litres/hour and setNebuliser Gas to its maximum setting.

Set one of the peak display boxes to show masses down to at least 100m.

SelectOperate .

SetAPcI Probe Temp to 400°C.


LCTUser's Guide

WhenAPcI Probe Temp reaches 400°C:

Start the LC pump at a flow of 1 ml/min.

OptimiseCorona so that the peaks reach maximum intensity.

Optimise the probe position for intensity and stability.

The two screws can be adjustedsingly or simultaneously to optimisethe beam.

The position of the probe will affectsensitivity. However, if the sampleis contained in a ‘biologicalmatrix’ or is contained in aninvolatile solvent the probe shouldbe moved away from the samplecone and towards the coronadischarge pin.

The tune page shows a typicalreagent ion spectrum for amethanol / water mobile phase.

Warning: It is normal for thesource enclosure, the glass tube andparts of the probe adjustment flange to reach temperatures of up to 60°C duringprolonged APcI operation. Care should be exercised when handling sourcecomponents immediately after operation.

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

Caution: Failure to employ a desolvation gas flow during APcI operation maylead to heat damage to the source.


LCTUser's Guide

In / OutProbe

Adjustment

SidewaysProbe

Adjustment

Hints for Sample Analysis

Tuning

• Start by tuning on the solvent ions.

• It is generally found that the most significant analyte tuning parameter to adjustfollowing tuning on the solvent ions isCone .

• Fine tuning on the analyte of interest can be performed either by large loopinjections (100µl) or by constant infusion in the mobile phase typically at analyteconcentrations of a few ng/µl.

• 10µl loop injections can be monitored using real time chromatogram updates.

Mobile Phase

• The choice of mobile phase is an important compound specific factor in APcI.For example, steroids prefer methanol:water mixtures as opposed toacetonitrile:water.

• Analyte sensitivity is also dependent on mobile phase composition, which can bevaried from 100% aqueous to 100% organic for any particular mixture.

Probe Temperature

This can be a critical factor for some analytes.

• Involatile samples (for example steroids) generally require high probetemperatures (>400°C).

• Volatile samples (for example pesticides) can be analysed with low probetemperatures (<400°C).

• In some cases, too high a probe temperature can lead to thermal degradation oflabile samples.

Desolvation Gas

Although aDesolvation Gas flow of approximately 150 litres/hour is typical formost samples, this flow rate should be tuned for maximum sensitivity while ensuringthat the flow rate is not decreased below 100 litres/hour.


LCTUser's Guide

Removing the ProbeAfter a session of APcI operation:

Turn off the LC flow.

SetAPcI Probe Temp to 20°C.

SelectStandby .

When the probe temperature falls below 100°C:


Undo the two thumb nuts and remove the probe from the source.

Warning: Take care when removing the APcI probe. There is a risk of burns tothe operator.

Caution: Removal of the APcI probe when hot will shorten the life of the probeheater.

If the instrument is not to be used for a long period of time the sourcetemperature should be reduced to 60°C.


LCTUser's Guide

ProbeThumb Nuts

Calibration and Accurate MassIntroduction

The elevated resolution and inherent stability of the calibration law of orthogonal TOFinstruments allow accurate mass measurements to be performed.

The basic time of flight calibration from mass (m) to time (t) is of the form:

m Q Pt= +

where:

the charge state is assumed to be 1.

the term P is a “gain” which comes from the instrument geometry(pathlengths and voltages).

Q is an offset, and comes from propagation delays through the electronics(detector rise time and delays of trigger signals through cables).

If a data file is acquired from the instrument with no calibration applied then it isassumed that the offset is zero and the gain P is calculated from the instrumentgeometry.

Nominal Mass Accuracy

It is important that the pathlengths are set up to give at least nominal mass accuracy.Nominal mass measurement is achieved on the LCT by adjustment of theLteff factor.

Acquire a TOF spectrum of a standard compound withLteff set to its defaultvalue of 1195.

Calculate a new value ofLteff from the relation:

( ) ( )Lteff 1195 m mind act= ÷

where:

mind = indicatedm.

mact= actualm.

Enter this new value underTDC Parameters .

After switching the real time tune display off and on, all subsequent massmeasurements will be nominally correct (within ±0.5z).

Calibration and Accurate MassPage 85

LCTUser's Guide

Calibration

With no calibration applied, then the data displayed on the spectrum in MassLynx isjust a set of mass intensity pairs (Mn, In) based upon instrument geometry.

Because of the inherent relationship between mass and time shown above it is prudentto generate higher order calibration coefficients that are applied to the square root ofthe nominal masses (Mn):

M A B M CM DMc n n n3 2= + + + +…

where:

the terms A, B, C, D… are calculated by fitting a polynomial to acquiredmass spectral data.

Mc is the calibrated displayed mass.

If a polynomial of order 1 is requested the values for A & B are calculated andthe higher terms are set to zero.

For a polynomial of order 5 (the highest supported in MassLynx) there will besix terms generated.

When calibrating over a large mass range (>500Da) it is advisable to use a higherorder polynomial as the deviations from the straight line fit become more appreciable.

Once a calibration has been generated from a reference compound such as PEG itshould be used as an “instrument calibration” to be applied to all subsequentlyacquired data (the procedure for this is described below).

Lock Mass

Temperature variations in the environment and in the power supplies may cause theinstrument to drift hundreds of parts per million (PPM) over the course of a day.

Instrument drift can be compensated for by applying a single point lock masscorrection that recalculates term B in the above equation.

The lock mass reference compound must be internal to the sample and may beintroduced by teeing in post column on a LC system.

When performing accurate mass work it is advisable to keep the instrument inthe operate mode at all times so that the power supplies can stabilise.


LCTUser's Guide

Deadtime Correction

The data acquisition system for the instrument is a time to digital converter (TDC).This is an ion counting type of system that generates a mass spectrum by storingarrival times of ions in a histogram memory.

After the arrival and registration of an ion by the TDC there is a minimum timeinterval before a subsequent ion arrival can be registered. This is called the “deadtime”of the TDC and is of the order of 5 nanoseconds.

The consequence of this deadtime is that at high ion currents a proportion of the ionsgenerated are not registered leading to a shift to lower mass centroids and lower areason reported peaks.

There is deadtime correction software in MassLynx that allows accurate massmeasurements to be achieved at a larger range of ion currents than otherwise wouldhave been possible. The use of this software correction is described below.


LCTUser's Guide

Generation of an Instrument CalibrationMake up an analyte solution consisting of PEG 300 (10 nanolitres/millilitre) andPEG 600 (10 nanolitres/millilitre), in a stock solution of 2mM ammoniumacetate in 50:50 acetonitrile : water.

Dilute this by a factor of ten (with the stock solution) and introduce this to theinstrument using a syringe pump operating at 5 microlitres per minute.

A Cone voltage of 30 volts will give a good distribution of PEG peaks.

Acquire data for one minute over the range 100 to 900Da, with a scan time of1 second. Combine at least 30 scans of data.

Centre the data using the menu as set up below, to ensure that the signal is nottoo intense.

If the data acquired is too intense, deadtime distortion will occur.

SelectTOF... and check that no deadtime correction is employed(Resolution = 0 andNp Multiplier = 0).


LCTUser's Guide

Re-centre the data usingResolution = 5000 andNp Multiplier = 0.8 asbelow:

Check that any differences in the centroided masses of the two centred spectraare less than 1mDa.

If this is not the case repeat the acquisition with less intense peaks either bysample dilution or by de-tuning of the electrospray needle by moving the z-sprayadjuster off axis.

Once a satisfactory centred spectrum has been acquired save it in the spectrumhistory.

As a guide, if the real time display is set to a 1 second scan time (set fromPeakDisplay , Tuning Setup on the tune page), an ion count of 500 for the mostintense PEG peak on the real time display usually gives good calibrations. Thisintensity is sufficient for a good signal-to-noise ratio but not so high thatappreciablez shifts occur due to the TDC deadtimes.

Display the saved centred spectrum just generated.

Go to theTools , Make Calibration menu:


LCTUser's Guide

Select the desired reference file.

File peghnh4.ref will give the correct masses with the sample prepared asdescribed above.

ChooseOK.

Make sure that the residual errors are all less than about 3 mDa. The calibrationparameters can be altered by clicking onEdit to reveal the menu below:


LCTUser's Guide

A fifth order polynomial has been chosen here, as a large mass range is beingcovered by the calibration.

After these parameters have been set click onOK to regenerate the calibrationwith the new parameters if required.

Peaks may be excluded from the calibration curve by deselecting them with aright mouse click in the spectrum shown in the top of the calibration menu.

SelectFile , Save as, Calibration .

Enter a filename to either overwrite a previously saved calibration or to generatea new calibration file. ChooseSave, followed byOK on the calibration page.


LCTUser's Guide

ChoosingOK on the calibration page without first saving the calibration as adifferent file will automatically overwrite the current calibration.

The following message will appear:

ChooseOK.

Future acquisitions may be made using the saved calibration by selecting thecalibration file generated above usingBrowse from theAcquire , Calibration menuon the tune page.

ChooseOK.

All subsequent acquisitions will now be tagged with the calibration just generated.

If acquiring data via the sample list, the calibration file is selected viaOptions ,Experiment Setup , Options , Calibration on the tune page. The experimentfile should be saved with the required calibration file selected.


LCTUser's Guide

Lock Mass CorrectionThe application of a single point lock mass correction will now correct for subsequentinstrument drift and bring masses back to within 5 PPM RMS provided, of course, thatthere is no isobaric chemical interference.

The lock mass is found under the center menu whenTOF is clicked:

Centering the spectrum with these parameters will force the peak at 520 to be 520.333exactly and recalibrate the entire mass spectrum.


LCTUser's Guide

Deadtime CorrectionAcquire a spectrum of PEG at the higher concentration of10 nanolitres/millilitre.

Ensure that the most intense PEG peak, measured on the tune page real timedisplay, is approximately 2500 counts high whenIntegration Time is set toone second.

Ensure that all scans combined are of a similar intensity.

Create a centred spectrum with no lock mass and no correction (all parametersset to zero):

Go toTools , Make Calibration and choose the same peghnh4 reference file:

Click on OK to reveal the residual errors, making sure that a polynomial oforder 1 has been selected.


LCTUser's Guide

In the calibration display shown above it can be seen that alternate calibration pointshave residual errors which are either negative (resulting from high intensity peaksbeing shifted to lowerm) or positive (resulting from lower intensity peaks whichhave not undergone am shift).

The procedure described below aims to minimise the point-to-pointm shifts bysetting the appropriateNp Multiplier value in the deadtime correction algorithm.Once set, theNp Multiplier value need not be changed for future acquisitions.

Do not click onOK as this will erase the current calibration.

This menu is being used only to set up the deadtime correction and not to createa new calibration.

Click on Cancel and re-centre the data using aResolution of 5000 and aNp Multiplier of 1.

The mass deviation between adjacent peaks of high and low intensity shouldreduce.

Repeat the process at increasingly lower values ofNp Multiplier until thedeviation is minimised.

At this point the peaks should lie close to the axis (the line of best fit).


LCTUser's Guide

Typical values forNp Multiplier will be about 0.7 and theResolution used shouldbe as measured on the instrument at mass 500.

Now that the deadtime correction has been set up, the difference in reported areas andmasses between corrected and uncorrected centered data can be seen. In the examplebelow the 371 peak has been shifted by 8.7 mDa.


LCTUser's Guide

The model successfully corrects for deviations of up to about 15 mDa at mass 500. Ifhigher ion currents are generated then the limits of the model are reached and nocorrection will be applied. An appreciation should be developed of the range of ioncurrents over which successful mass measurements can be made.

Once theResolution andNp Multiplier figures have been evaluated they can be leftactive in the TOF menu without adversely affecting centered data.


LCTUser's Guide

Exact Mass Measurement: Additional Hints• Best results are obtained if the lock mass gives an intensity of approximately

400 to 500 counts per second as shown on the real time tune display.

• The lock mass should be chosen to be at the upper end of the mass range used.

• Do not change transfer lens voltages (including theRF Lens voltage) withoutre-calibrating the instrument.

ChangingSample Cone will not change the calibration.

• Always be aware of possible chemical interference problems, either on thesample or the lock mass peak.

• When performing a base calibration (using PEG, for example) better results maybe obtained by using a stronger solution and moving the probe off axis to limitthe ion current, rather than using a lower concentration.

This has the effect of minimising any possible chemical interference frombackground ions.

• Always check the stability of the spray (see the instructions for the setting of theprobe tip inMaintenance and Fault Finding).

Short term variations in the spray produce fluctuations in the number of ions perpeak per pusher pulse, giving rise to errors in the deadtime correctioncalculations. The number of ions per peak per pusher pulse is calculated fromthe ion current integrated over a period of time thus only giving an averagevalue.

• To obtain the best deadtime correction, only combine scans of a similarintensity, either at the top of a chromatographic peak or in the tail of achromatographic peak.

This ensures the number of ions per peak per push is calculated correctly.

• The deadtime correction algorithm can only correct for ion intensities andmshifts up to a limit. If the ion current approaches approximately 10,000 countsper peak (without correction) in one second then the limits of the model arebeing reached.

If the limits of the model are exceeded no correction is applied - the same resultwill be obtained by centring the data withResolution andNp Multiplier setto zero.

• If the limits of the deadtime correction algorithm are exceeded it may bepossible to use the C13 isotope instead.

• For the best mass accuracy when mass measuring doubly charged ions, it isadvisable to use a doubly charged lock mass peak.


LCTUser's Guide

• To obtain the true number of ions per peak, areas must be selected on the peakcentre menu.

• The standard deviation in the determination of the mass (strictlym) centroid ofa triangular-shaped peak (σppm) due to ion statistics alone is given by theequation below.

A triangle is assumed to be a close enough approximation to the shape of themass spectrometer peak for the equation to be valid.

σppm = 106∆M / M(24Np)0.5 PPM

where:

∆M is the width (m) of a triangular peak across the base.

M is them value of the peak.

Np is the number of ions per peak.

Using the above equation we can calculate the number of ions per peak requiredto give a standard deviation of 5 PPM when measuring a peak at 500m.

Assuming 5000 (FWHM) resolution, so∆M = 0.2m (width at base = twicewidth at half height), then:

Np = [5×10-6 × 500÷0.2 × (240.5)]-2 = 267 ions per peak.

Thus standard deviations of less than 5 PPM cannot be expected unless thenumber of ions per peak is greater than 267.


LCTUser's Guide


LCTUser's Guide

Maintenance and Fault FindingIntroduction

Cleanliness and care are of the utmost importance whenever internal assemblies areremoved from the instrument.

✔ Always prepare a clear clean area in which to work.

✔ Make sure that any tools or spare parts that may be required are close at hand.

✔ Obtain some small containers in which screws, washers, spacers etc. can bestored.

✔ Use tweezers and pliers whenever possible.

✔ If nylon or cotton gloves are used take care not to leave fibres in sensitive areas.

✖ Avoid touching sensitive parts with fingers.

✖ Do not use rubber gloves.

✔ Before reassembling and replacing dismantled components, inspect O rings andother vacuum seals for damage. Replace with new if in doubt.

Should a fault occur soon after a particular part of the system has been repaired orotherwise disturbed, it is advisable first of all to ensure that this part has beencorrectly refitted and / or adjusted and that adjacent components have not beeninadvertently disturbed.

Warning: Many of the procedures described in this chapter involvethe removal of possibly toxic contaminating deposits usingflammable or caustic agents. Personnel performing these operationsshould be aware of the inherent risks, and should take the necessaryprecautions.

Maintenance and Fault FindingPage 101

LCTUser's Guide

Removal and Replacement of Panels and CoverWarning: There are high voltages and hot surfaces present throughout themass spectrometer. The instrument's panels must not be removed unlessthe instrument has been electrically isolated at the power outlet.

To remove the instrument's panels for maintenance purposes, follow in order thefollowing procedures.

To replace the panels, follow in reverse order the removal procedures.

Side Panel

Caution: Ensure that the panel isadequately supported duringremoval. Damage to internalelectronic components could resultif the panel is allowed to drop.

Loosen the four screws A by 2 to3 turns.

While supporting the underside ofthe panel, slide the panel to theright until the slots in the panelclear the screw heads.

Withdraw the panel from the instrument.

Top Panel

Loosen the two screws B.

Tilt the top panel to clearscrews B.

Slide the top panel horizontallyuntil the two pegs clear theirlocating holes, as shown.

Lift off the top panel.


LCTUser's Guide

A

B

LocatingPegs

Front Panel

Remove the two screws C.

Unscrew by 2 or 3 turns the twoscrews D.

Withdraw the panel as shown.

Moulded Side Cover

Disconnect all gas and electricalconnections to the source.

Remove the probe, if fitted.

Remove the Z-spray adjustmentflange (or the nanoflow end flangeassembly) and the transparentsource enclosure.

Remove the three screws E.

Remove the three screws F.

Remove the moulded cover.

Electronics MaintenanceRemoval of the right side instrumentcover gives access to the electronicssystems. On the top of the electronicsconnector panel is a switch. In normaloperation this switch should be in thedown position.

For electronic maintenance and faultfinding this switch may be set to the upposition which will allow the electronicscircuit breaker to be switched offwithout venting the vacuum system.

Caution: Do not leave the instrument unattended with this switch in the upposition as a reduced level of vacuum protection is in operation.

Return the switch to the down position at the earliest opportunity.


LCTUser's Guide

E

F

C

D

Cooling Fans and Air FiltersAlways ensure that none of the cooling fans is obstructed. It is essential that the fanfilter is checked and cleaned at regular intervals, and replaced if there is any doubtabout its effectiveness.

The Vacuum SystemThe performance of the mass spectrometer will be severely impaired by the lack of agood vacuum in the ion transfer (hexapole) region or the analyser.

• An excessive analyser pressure results in a general loss in performance indicatedby a loss of resolution and an increase in the background noise.

• As the vacuum deteriorates, the vacuum becomes insufficient to maintain theinstrument in the operate mode.

Before suspecting a leak, the following points should be noted:

• The turbomolecular pumps will not operate if the rotary pump has failed.

• If the rotary pump is not maintained, the oil may become so contaminated thatoptimum pumping speed is no longer possible. Initially, gas ballasting may cleanthe oil. If the oil in the rotary pump has become discoloured, then it should bechanged according to the pump manufacturer's maintenance manual.

• The turbomolecular pumps switch off if an over temperature is detected. Thiscould be due to poor backing vacuum, failure of the water supply or a leak inthe source or analyser.

• The turbomolecular pumps switch off if full speed is not achieved within a settime following start-up. This could be due to a leak or too high an ambienttemperature.

Vacuum Leaks

If a leak is suspected, the following basic points may help to locate it:

• Leaks very rarely develop on an instrument that has been fully operational.Suspect components that have recently been disturbed.

Leaks on flanges can usually be cured by further tightening of the flange boltsor by replacing the seal.

• All seals are made using O rings. When refitting flanges pay attention to thecondition of O rings. Any that are cut or marked may cause a leak. The O ringsshould be clean and free from foreign matter.

A hair across an O ring is sufficient to prevent the instrument pumping down.


LCTUser's Guide

• Source components that operate at, or slightly above, atmospheric pressure arenot susceptible to vacuum leaks.

In the unlikely event of a leak on a feedthrough, then the unit should be replaced orreturned to Micromass for repair.

Pirani Gauge

The Pirani gauge head does not require routine maintenance.

Active Inverted Magnetron Gauge

For information on cleaning the active inverted magnetron (Penning) gauge, refer tothe Edwards literature supplied with the instrument.

Gas Ballasting

Gas ballasting serves two importantpurposes:

• When rotary pumps are used topump away solvent vapours, thesolvent vapour can becomedissolved in the pump oilcausing an increase in backingline pressure. Gas ballasting is amethod of purging the oil toremove dissolved contaminants.

• Oil mist expelled from therotary pump exhaust is trappedin the oil mist filter. This oil isreturned to the rotary pumpduring gas ballasting.

Gas ballasting should be performed routinely on a weekly basis for 30 minutes. If thesource is used in the APcI or megaflow electrospray modes, more frequent gasballasting is recommended.

Gas ballasting is performed on the E2M28 pump by rotating the gas ballast valve 5 to6 turns in a counterclockwise direction.

It is normal for the rotary pump to make more noise when the gas ballast valveis open.

Caution: Failure to gas ballast the rotary pump frequently leads to shortened oillifetime which in turn may shorten rotary pump lifetime.

Caution: Under no circumstances should gas ballasting be performed duringoperation.


LCTUser's Guide

GasBallast

DrainPlug

Exhaust

FillerPlug

Oil LevelIndicator

Oil Mist Filter

The E2M28 rotary pump is fitted with an Edwards EMF20 oil mist filter which trapsoil vapour from the rotary pump exhaust. The trapped oil is then returned to the rotarypump during routine gas ballasting. The oil mist filter contains two elements whichrequire the following maintenance:

• Change the odour element monthly or whenever the pump emits an oily odour.

• Change the mist element every time the rotary pump oil is changed.

To change the elements follow the instructions in the Edwards manual.

Rotary Pump Oil

The oil in the rotary pump should be maintained at the correct level at all times.Check the oil level at weekly intervals, topping up if necessary.

It is important to monitor the condition of the oil regularly. Replace the oil when it haschanged to a noticeable reddish brown colour, or routinely at 4 month intervals (3000hours operation). At the same time, replace the oil mist filter's mist element (seeabove).

Change the oil in the rotary pump as follows:

Gas ballast lightly for 30 to 60 minutes.

Vent and shut down the instrument as described inRoutine Procedures.

It will be found easier to drain the oil while the pump is still warm.

Drain the oil through the drain hole situated near the oil level sight glass.

Flush the pump, then replace the drain plug and refill the pump with the correctgrade oil to the correct level.

Gas ballast lightly for 30 to 60 minutes.

For further servicing information refer to the manufacturer’s manual.


LCTUser's Guide

The Source

Overview

The Z-spray source is a robust assembly requiring little maintenance. The sourceconsists of three basic parts:

• The probe adjustment flange.

• The glass tube.

• The source flange assembly.

The probe adjustment flange and the glass tube can be readily removed, withoutventing the instrument, to gain access to the source block and sample cone. Thisallows the following operations to be performed:

• Wiping the sample cone.

• Removing the sample cone.

• Fitting or removing the APcI corona discharge pin.

• Fitting or removing the exhaust liner and cleanable baffle.

• Fitting or removing the nanoflow electrospray interface.

• Enabling or disabling the purge gas.

The sample cone may be cleaned in situ, by gentle wiping with a cotton swab or linttissue soaked with 50:50 acetonitrile:water. More thorough cleaning of the samplecone may be achieved by removing it from the source. This may also be done withoutventing the instrument, by closing the isolation valve located on the ion block. Lessfrequently it may be necessary to clean the ion block, the extraction cone and thehexapole lens, in which case the instrument must be vented. This should only be donewhen the problem is not rectified by cleaning the sample cone or when chargingeffects are apparent.

Charging is evidenced by a noticeable progressive drop in signal intensity, oftenresulting in a complete loss of signal. Switching the instrument out of and backinto operate causes the beam momentarily to return.

The hexapole transfer lens should not require frequent cleaning. If it is suspected thatthe lens does need cleaning it may be withdrawn from the front of the instrument afterremoving the ion block support.

Warning: Cleaning the various parts of the source requires the use ofsolvents and chemicals which may be flammable and hazardous tohealth. The user should take all necessary precautions.


LCTUser's Guide

Cleaning the Sample Cone in Situ

This may be necessary due to lack of sensitivity or fluctuating peak intensity, or ifdeposited material is visible on the outside of the sample cone. Proceed as follows:

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

SelectStandby .


Disconnect the liquid flow at the rear of the probe.

SetSource Block Temp and eitherAPcI Probe Temp orDesolvation Temp to 20°C to switch off the heaters.

Warning: Removal of the APcI probe or desolvation nozzle when hot may causeburns.

Caution: Removal of the APcI probe when hot will shorten the probe heater'slife.

The cooling time will be significantly shortened if the API gases are left flowing.

WhenAPcI Probe Temp or Desolvation Temp has cooled below 100°C:

Switch off the nitrogen supply by selectingGas followed byNitrogen .

Disconnect both gas lines from the front panel by undoing the knurled nuts.


LCTUser's Guide

SourceThumb Nuts

ProbeThumb Nuts

ProbeAdjustment Flange

GlassTube

Disconnect both electrical connections by pulling back on the plug sleeves torelease the plugs from the sockets on the front panel.

Undo the two knurled thumb nuts that retain the probe and withdraw it from thesource. Place it carefully to one side.

Undo the three thumb screws and withdraw the probe adjustment flange andglass tube. Place the glass tube, end on, on a flat surface and place the probeadjustment flange on top of the glass tube.

Warning: When the source enclosure has been removed the source block isexposed. Ensure that the source block heater has cooled before proceeding.

If fitted, remove the APcI corona discharge pin.

The sample cone is now accessible.

Using a suitable flat bladescrewdriver rotate the isolationvalve by 90° into its fullyanticlockwise position.

A small improvement in theanalyser vacuum may be observedas a result of this operation.

The isolation valve is closed whenthe slot is perpendicular to thedirection of flow.

Carefully wipe the sample conewith a cotton swab or lint freetissue soaked in 50:50acetonitrile:water or 50:50methanol:water.

Caution: Do not attempt to remove any obstruction by poking. This may resultin damage to the sample cone.

Dry the cone using nitrogen.

If the sample cone is still not clean, or if the aperture is partially blocked, proceed tothe following section. Otherwise, when the cone is clean and dry:

Open the isolation valve.

Replace all removed components, following in reverse order the removalprocedures.


LCTUser's Guide

SampleCone

IsolationValve

Removing and Cleaning the Sample Cone

Caution: The sample cone is a delicate and expensive component and should behandled with extreme care.

It is not necessary to vent the instrument to remove the sample cone. The source blockincorporates an isolation valve for this purpose. To remove the sample cone proceedas follows:

Follow the procedure in the previous section, to gain access to the sample cone.

Using a suitable flat blade screwdriver rotate the valve by 90° into its fullyanticlockwise position.

A small improvement in the analyser vacuum may be observed as a result of thisoperation.

The isolation valve is in the closed position when the slot is perpendicular to thedirection of flow.

Disconnect the cone gas inlet line (if fitted).

Take the sample cone extraction tool supplied in the source spares kit and screwit to the flange of the sample cone.

Remove the two sample cone retaining screws using a 1.5mm Allen key andwithdraw the sample cone and cone gas nozzle (if fitted) from the ion block.

Remove the extraction tool, and separate the sample cone from the cone gasnozzle. Place both components in an ultrasonic bath containing 50:50acetonitrile:water or 50:50 methanol:water.


LCTUser's Guide

Dry the cone and nozzle using nitrogen.

To minimise down time fit a spare sample cone, obtainable from Micromass, atthis stage.

If material has built up on the exhaust liner and cleanable baffle:

Remove the cleanable baffle and the exhaust liner.

Caution: Do not attempt to remove the baffle without first removing the samplecone.

Clean these components, or obtain replacements.

Fit the cleaned (or the replacement) exhaust liner and cleanable baffle to the ionblock.

Refitting the sample cone is a reversal of the removal procedure.


LCTUser's Guide

SampleCone Sample

ConeNozzle

ExhaustLiner

Cone GasInlet Line

ExtractionTool

CleanableBaffle

Removing and Cleaning the Source Block and Extraction Cone

On the tune page selectOther from the menu bar at the top of the tune page.Click on Vent .

The rotary pump and the turbomolecular pumps switch off. The turbomolecularpumps are allowed to run down to 50% speed after which a vent valveautomatically admits dry nitrogen.

Remove the source enclosure and the sample cone as described in the previoussection.

When the instrument has vented:

Remove the two screws which secure the ion block and remove the ion blockheater and the ion block.

Separate the extraction cone and the PTFE insulating ring from the ion block.

Remove the plug and the PTFE sealing washer.

Remove the sample cone as described above.


LCTUser's Guide

PTFERingPlug Washer

IonBlock

Ion BlockHeater

ExtractionCone

Leaving the valve stem in place, immerse the ion block in an ultrasonic bathcontaining 50:50 acetonitrile:water or 50:50 methanol:water, followed by 100%methanol.

Clean the sample cone and the extraction cone using in turn:

• concentrated formic acid.• 50:50 acetonitrile:water or 50:50 methanol:water.• 100% methanol.

Warning: Strong acid causes burns. Carry out this procedure in a fume cupboardusing protective equipment.

Dry all components using a flow of nitrogen, or place them in a warm oven.


LCTUser's Guide

Removing and Cleaning the RF Lens Assembly

To remove the RF hexapole transfer lens assembly, proceed as follows:

Remove the ion block, as described above.

Remove the three screws retaining the ion block support and carefully withdrawit, together with the support liner and O rings, from the pumping block.

Using a lint free tissue to gently grasp the hexapole, carefully withdraw it.

Caution: Take care not to scratch the internal bore of the pumping block as thehexapole lens assembly is withdrawn.


LCTUser's Guide

SupportLlner

HexapoleTransfer Lens

Ion BlockSupport

To clean the hexapole transfer lens proceed as follows:

Immerse the complete assembly in a suitable solvent (100% methanol) andsonicate in an ultrasonic bath.

Thoroughly dry the assembly using a flow of nitrogen.

In severe cases:

Remove, clean, dry and replace each rod separately (one at a time).

Reassemble the assembly with extreme care, checking the assembly against thediagram.


LCTUser's Guide

LocationRecess

DifferentialAperture

PlateRod LocatingScrews & Washers

Reassembling and Checking the Source

Feed the hexapole transfer lens into the instrument, allowing the recesses in thedifferential aperture plate to locate onto the two support rails within the analyserassembly. Ensure that the assembly is pushed fully in.

Check the condition of the O rings on the ion block support. Replace them ifnecessary.

Replace the ion block support, pushing it in against the springs of the hexapoleassembly.

Replace the three retaining screws.

Fit the plug and sealing ring to the ion block.

Fit the insulating ring and extraction cone.

Offer the ion block up to the peek ion block support, locate the two dowels andpush firmly.

Replace the ion block heater.

Replace and firmly tighten the two retaining screws taking care not toover-tighten the screws.

On the tune page selectOther and click onPump .

Replace the PTFE exhaust liner and cleanable baffle, if removed.

Replace the sample cone and, if the nanoflow option is to be used, the cone gasnozzle on the ion block.

Reconnect the cone gas supply (nanoflow operation only).

Plug the purge and cone gas outlets and fit the APcI corona discharge pin.

Fit the source enclosure and the probe adjustment flange.

Insert the APcI probe and connect theNebuliser gas line.

SelectGas and turn onNitrogen . Fully open theNebuliser gas valve.

SetDesolvation Gas to read back 400 litres/hour (monitored on the tunepage).

Check for gas leaks using soap solution.

ReduceDesolvation Gas to 150 litres/hour.

SetSource Block Temp to 150°C, andAPcI Probe Temp to 20°C


LCTUser's Guide

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

SelectOperate on the tune page.

With Corona set to zero, check that theCone readback is reading the correctset value.

SetCorona to 4.0kV.

Check that theCorona readback is 4.0 kV and that theCone readback is stillreading the same set value.

Check that all other readbacks on the tune page agree with the set values.

The Corona Discharge Pin

If the corona discharge pin becomes dirty or blunt:

Remove it from the source.

Clean and sharpen it using 600 grade emery paper.

If the needle becomes bent or otherwise damaged it should be replaced.


LCTUser's Guide

The Electrospray Probe

Overview

Warning: The probe tip is sharp, and may be contaminated withharmful and toxic substances. Always take great care when handlingthe electrospray probe.

Indications that maintenance is required to the electrospray probe include:

• An unstable ion beam.

Nebulising gas may be escaping from the sides of the probe tip.

Ensure that the probe tip O ring is sealing correctly.

The probe tip setting may be incorrect.

Adjust the probe tip setting as described inElectrospray.

The probe tip may be damaged.

Replace the probe tip.

There may be a partial blockage of the sample capillary or the tubing in thesolvent flow system.

Clear the blockage or replace the tubing.

• Excessive broadening of chromatogram peaks.

This may be due either to inappropriate chromatography conditions, or to largedead volumes in the transfer capillaries between the LC column or probeconnection.

Ensure that all connections at the injector, the column, the splitting device(if used) and the probe are made correctly.

• High LC pump back pressure.

With no column in line and the liquid flow set to 300 µl/min the back pressureshould not exceed 7 bar (100 psi). Pressures in excess of this indicate ablockage in the solvent flow system.

Samples containing particulate matter, or those of high concentrations, are mostlikely to cause blockages.

Check for blockages at the tube connections and couplings to the injector,the column and, if used, the flow splitter.


LCTUser's Guide

Concentrated formic acid can be injected to clear blockages. Rinsethoroughly afterwards.

Blockage of the stainless steel sample capillary may occur if the desolvationheater is left on without liquid flow. This is particularly relevant for samplescontained in involatile solvents or high analyte concentrations. To avoid thisproblem it is good practice to switch off the heater before stopping the liquidflow, and flush the capillary with solvent.

A blocked stainless steel sample capillary can often be cleared byremoving it and reconnecting it in the reverse direction, thus flushing outthe blockage.

• Gas flow problems

Check all gas connections for leaks using soap solution, or a suitable leaksearching agent such as Snoop.


LCTUser's Guide

Replacement of the Stainless Steel Sample Capillary

If the stainless steel sample capillary cannot be cleared, or if it is contaminated ordamaged, replace it as follows:

Remove the probe form the source.

Disconnect the LC line from the probe and remove the finger-tight nut.

Loosen the grub screw retaining the LC union.

Remove the two probe end cover retaining screws, and remove the probe endcover.

Unscrew and remove the probe tip.

Remove the LC union and adapter nut. Withdraw and discard the stainless steelsample capillary.

Remake the LC connection to the LC union.

Sleeve one end of new sample capillary with the PTFE liner tube.


LCTUser's Guide

Stainless SteelCapillary

Fused SilicaCapillary

LCUnion

Finger-tightNut & Ferrule

RheodyneNut & Ferrule

GrubScrew

EndCover

ProbeTip

AdapterNut

LinerTube

LinerTube

LinerTube

GVF/16Ferrule

GVF/003Ferrule 0.5mm

Using a GVF/16 ferrule and the adapter nut, connect the sample capillary to theLC union, ensuring that both the liner tube and sample capillary are fully buttedinto the LC union.

Disconnect the LC connection and feed the sample capillary through the probe,ensuring that a 0.3mm graphitised vespel ferrule (GVF/003) is fitted.

Using a Rheodyne spanner, gently tighten the adapter nut onto the probe.

Replace the probe tip and adjust so that 0.5mm of sample capillary protrudesfrom the probe tip.

Replace the probe end cover and tighten the grub screw to clamp the LC union.


LCTUser's Guide

The APcI ProbeIndications that maintenance to the APcI probe is required include:

• The probe tip assembly becomes contaminated, for example by involatilesamples if the probe temperature is too low during operation (300°C).

• The appearance of chromatogram peak broadening or tailing.

Samples that give rise to a good chromatogram peak shape in APcI (for examplereserpine and common pesticides) should display peak half widths of the order0.1 minutes for 10µl loop injections at a flow rate of 1 ml/min. The appearanceof significant peak broadening or tailing with these compounds is most likely tobe due to a broken fused silica capillary or probe tip heater assembly.

• Low LC pump back pressure.

For 50:50 acetonitrile:water at a flow rate of 1 ml/min, a LC pump backpressure less than 14 bar (200 psi) is indicative of a broken fused silicacapillary or a leaking connector.

• High LC pump back pressure.

For 50:50 acetonitrile:water at a flow rate of 1 ml/min, a LC pump backpressure above 35 bar (500 psi) is indicative of a blockage or partial blockagein the fused silica capillary, in a LC connector or in the filter. It is advisable tochange the inner filter pad on a regular basis (see “Replacing the Fused SilicaCapillary” in the following pages).

• Gas flow problems.

Check all gas connections for leaks using soap solution, or a suitable leaksearching agent such as Snoop.

Cleaning the Probe Tip

Remove any visible deposits on the inner wall of the probe heater with amicro-interdental brush (supplied in the spares kit) soaked in methanol:water.

Before starting an analysis:

With the probe out of the instrument, connect the nebulising gas supply line.

SelectGas and turn onNitrogen .

Allow the gas to flow for several seconds to clear any debris from the heater.

Turn off Nitrogen .

Insert the probe into the source.


LCTUser's Guide

SelectGas and turn onNitrogen .

RaiseAPcI Heater gradually, starting at 100°C and increasing in 50°C intervalsto 650°C over a period of 10 minutes.

Caution: Do not setAPcI Heater to 650°C immediately as this may damagethe probe heater.

This procedure should remove any chemical contamination from the probe tip.

Replacing the Probe Tip Heater

Remove the probe tip assembly by carefully loosening the two grub screws.

Disconnect the heater from the probe body by pulling parallel to the axis of theprobe.

Fit a new heater assembly.

Reconnect the probe tip assembly.


LCTUser's Guide

GrubScrews

Heater

Probe TipAssembly

Replacing the Fused Silica Capillary

With the probe removed from the source proceed as follows:

Remove the probe tip assembly and the heater, as described in the precedingsection.

Remove the probe end cover by removing the two screws and the grub screwsthat retain the LC filter.

Loosen the filter from the adapter nut.

Unscrew the adapter nut from the probe.

Remove and discard the fused silica capillary.

Using a ceramic capillary cutter, cut a new length of 300µm o.d. × 100µm i.d.fused silica capillary, about 1 centimetre excess in length.

Using a GVF/004 ferrule and the adapter nut, connect the capillary to the filterensuring that the capillary passes through the ferrule but stops short of the filter.


LCTUser's Guide

0.5 to1mm

Fused SilicaCapillary

Finger-tightNut & Ferrule

RheodyneNut & Ferrule

GVF/004Ferrule

AdapterNut

Filter

PTFETube

GrubScrew

GrubScrew

Feed the sample capillary through the probe, ensuring that a 0.4mm graphitisedvespel ferrule (GVF/004) is fitted.

Using a ceramic capillary cutter, cut the capillary at the nebuliser so thatbetween 0.5 and 1.0mm of capillary is protruding from the nebuliser.

It is important to cut the capillary square. This should be examined using asuitable magnifying glass.

Undo the adapter nut from the probe and withdraw the capillary from the probe.

Remove 20mm of polyamide coating from the end of the capillary using a flameand clean with a tissue saturated with methanol.

Carefully re-feed the sample capillary through the probe ensuring that thegraphitised vespel ferrule is still fitted.

Using a Rheodyne spanner, gently tighten the adapter nut to the probe.

Replace the probe end cover and retaining screws.

Using a 1.5mm Allen key, tighten the grub screw in the probe end cover toclamp the filter.

Replace the heater and probe tip assembly.

Analyser and DetectorThe analyser and detector assemblies do not require routine maintenance.

Caution: These assemblies are finely aligned and should not be removed fromthe instrument under any circumstances.


LCTUser's Guide

Fault Finding

Introduction

The majority of faults that occur can be traced to a malfunction of the ion source orinlet system. On systems equipped with more than one source, this can often beconfirmed by changing sources to see if the fault “moves” with the source.

Should a fault occur soon after a part of the system has been repaired or otherwisedisturbed, it is advisable first of all to ensure that this part has been correctly refittedand adjusted, and that adjacent components have not been inadvertently disturbed.

No Beam

Refer to the relevant chapters of this manual and check the following:

• The tune page real time display is activated by pressing the appropriate buttonon the tool bar of the tune page.

• Normal tuning parameters are set and, where appropriate, readback values areacceptable.

• All necessary cables have been correctly attached to the source and probe.

• Solvent is reaching the probe tip and the solvent flow rate is as required.

For solvent flow rates below 100 µl/min it may be necessary temporarily to turnoff the nebulising gas and remove the probe from the source to allow the solventto be seen at the probe tip.

• The flows of desolvation gas and nebuliser gas are on and are set to the correctflow rates.

• The source has been assembled correctly and is clean.

• The source isolation valve is open.

If, after performing the above checks, the beam is still absent:

Set the real time tune display to show the mass range down tom 10.

Check that there is an interference ‘peak’ at approximatelym 60 due to thepusher pulse being switched off.

If this interference peak is not present, either the pusher is not pulsing or theoutput from the detector is not reaching the TDC (time to digital converter).

The most likely cause of an absent pusher interference pulse is a faultyattenuator. This must only be replaced by trained maintenance personnel.

If the pusher interference peak is not present no data will be acquired.


LCTUser's Guide

Unsteady Beam

Refer to the relevant chapters of this manual and check that:

• Capillary (electrospray) andCone are tuned correctly.

• The capillary is not protruding too far from the end of the probe.

• The probe is not too far into the source.

• The flow of solvent from the HPLC pump is correct and steady.

To do this, remove the probe, degas the solvent, increase the flow rate forseveral minutes to purge any trapped air then reset and re-measure the flowrate.

• Solvents have been adequately degassed.

• The nitrogen flow of desolvation gas and nebuliser gas is steady. The nitrogensupply pressure should be 7 bar (100 psi) ±10%.

• Desolvation Temp is not set too high for the liquid flow rate used.

High temperatures can vapourise solvent within the electrospray probe.

Should the preceding checks fail to reveal the cause of the problem, proceed to thefollowing section.


LCTUser's Guide

High Back Pressure

For electrospray, a higher than normal back pressure readout on the HPLC pump,together with a slowing of the actual solvent flow at the probe tip, can imply that thereis a blockage in the capillary transfer line or injection loop due to particulate matterfrom the sample.

To clear the blockage:

Remove the probe from the source and increase the solvent flow to 50 µl/min toremove the blockage.

Often, injections of neat formic acid help to redissolve any solute which hasprecipitated out of solution.

If the blockage cannot be cleared in this fashion:

Remove the finger-tight nut and tubing from the back of the probe.

If the back pressure remains high:

Replace the tubing with new tube (or first try removing both ends of thetube).

If the back pressure falls:

Replace the stainless steel sample tube inside the probe (or try reversingthe tube to blow out any blockage).

Reconnect the tubing to the probe.

The solvent flow can now be readjusted and the probe replaced into the source.

To check the flow rate from the solvent delivery system, fill a syringe barrel or agraduated glass capillary with the liquid emerging from the probe tip, and timea known volume, say 10µl.

Once the rate has been measured and set, a note should be made of the backpressure readout on the pump, as fluctuation of this reading can indicateproblems with the solvent flow.

For APcI a higher than normal back pressure readout on the HPLC pump can implythat, after a long period of use, the filter pad requires replacement.


LCTUser's Guide

Loss of Sensitivity

As the ion source becomes dirty after prolonged use, the performance will degrade.

Unstable or reduced ion currents are indicators that the source needs cleaning. Theusual remedy is to clean the source as described earlier in this chapter.

An increase in the analyser pressure above 4 × 10-6 mbar can also cause loss ofsensitivity, although the pressure at which this occurs will be sample dependent.

Incorrect Isotope Distributions

Incorrect isotope distributions can be caused by:

• The TDCStop (mV) threshold being set too high.

Refer to the tune page settings section ofRoutine Proceduresfor informationregarding the setting of this parameter.

• A faulty attenuator.

Attenuators can fail so that they are open circuit (no beam or pusherinterference ‘peak’ present), or they can fail such that they stop attenuating.Thelatter failure mode gives rise to incorrect isotope distributions.

When the attenuator fails in this way the TDCStop (mV) threshold can beincreased to a significantly higher value than that used previously withoutreducing the beam intensity.

In normal operation setting the TDC threshold above 200 or 250mV will start toreduce the beam intensity. If the attenuator has failed the TDC threshold can beincreased to 500mV or higher before the beam intensity is reduced.


LCTUser's Guide

High Noise Levels

High noise levels can either be chemical or electronic in nature.

Chemical Noise

Chemical noise usually originates from contaminated samples, solvents or sourcegases.

Chemical noise can be distinguished from electronic noise simply by stopping sourceionisation. If no liquid or gases are entering the source and all the source voltages areset to zero then the remaining noise will be electronic in nature.

Electronic Noise

Electronic noise can be caused by setting the TDCStop (mV) threshold too low.Refer to the tune page settings section ofRoutine Proceduresfor informationregarding the setting of this parameter.

The microchannel plate detector can be damaged by failure to properly condition thedetector following venting of the system to atmosphere. If the detector is producingmicrodischarges, excessive noise will be apparent on the baseline of mass spectra inthe absence of any ion beam. Reducing the detector voltage will reduce the number ofdischarges and reduce the noise.

Caution: It is strongly recommended that assistance is sought from Micromass ifmaintenance to the detector system is thought necessary.

Caution: Assistance from Micromass should be sought if, due to symptoms suchas excessive noise, spikes, loss of detector gain or abnormal peak shapes,maintenance to any of the components within the TOF analyser housing isthought to be necessary.

Poor Analyser Vacuum

Before suspecting a pump fault or vacuum leak (seeVacuum Systemearlier in thischapter) it is worth checking the inverted magnetron (Penning) gauge. If this gaugehas become dirty it will indicate a poor vacuum, or even fail to register at all.

For information on cleaning the gauge, refer to the Edwards literature supplied withthe instrument.

Warning: The instrument must be vented and electrically isolated at the supplyoutlet before removing the instrument's covers to gain access to the activeinverted magnetron gauge.

Note that if the instrument has been vented to atmosphere (instead of dry nitrogen) itmay take one to two days before reaching the vacuum levels obtained prior to venting.


LCTUser's Guide

Cleaning MaterialsIt is important when cleaning internal components to maintain the quality of thesurface finish. Deep scratches or pits can cause loss of performance. Where nospecific cleaning procedure is given, fine abrasives should be used to remove dirt frommetal components. Recommended abrasives are:

• 600 and 1200 grade emery paper.

• Lapping paper (produced by 3M).

After cleaning with abrasives it is necessary to wash all metal components in suitablesolvents to remove all traces of grease and oil. The recommended procedure is tosonicate the components in a clean beaker of solvent and subsequently to blot themdry with lint-free tissue. Recommended solvents are:

• Isopropyl Alcohol (IPA)

• Methanol

• Acetone

Following re-assembly, components should be blown with oil-free nitrogen to removedust particles.

Warning: Many of the procedures described in this chapter involvethe removal of possibly toxic contaminating deposits usingflammable or caustic agents. Personnel performing these operationsshould be aware of the inherent risks, and should take the necessaryprecautions.


LCTUser's Guide

Preventive Maintenance Check List✖ Avoid venting the instrument when the rotary pump is gas ballasting.

✖ Do not gas ballast the rotary pump for more than 2 hours under anycircumstances.

✖ Under no circumstances should gas ballasting be performed during instrumentoperation.

For full details of the following procedures, consult the relevant sections of thischapter and / or refer to the manufacturer's literature.

Daily

• Gas ballast the rotary pump lightly for 20 minutes at the end of a day'selectrospray operation.

• Gas ballast the rotary pump for 30 minutes at the end of a day's megaflow orAPcI operation.

It is normal for the rotary pump noise level to increase during gas ballasting.

Weekly

• Gas ballast for at least 30 minutes by rotating the gas ballast knobanticlockwise by 5 to 6 turns.

When gas ballast is complete, check the rotary pump oil level and colour.

Oil that has become noticeably red in colour should be replaced.

• Check the water chiller level and temperature (if fitted).

Monthly

• Check all cooling fans and filters.

• Change the odour element in the oil mist filter.

Four-Monthly

• Change the mist element in the oil mist filter.

• Change the oil in the rotary pump.

Gas ballast lightly for 30 to 60 minutes both before and after changing oil.


LCTUser's Guide

Reference InformationOverview

The reference files listed in this chapter have all ion intensities set to 100%. Actualion intensities are not, of course, all 100%, but the calibration software does not takeaccount of the ion intensities and this is a convenient way to store the reference filesin the required format.

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

Internet:

http://www.sigma.sial.com

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

Toll-free telephone:

USA & Canada 800-325-3010

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

Toll-free fax:

USA & Canada 800-325-5052

Outside USA & Canada ++1 314-771-5750(call collect and ask for the fax machine)

Direct fax:

Outside USA & Canada ++1 314-771-5757 (this is a toll call)

Reference InformationPage 133

LCTUser's Guide

http://www.sigma.sial.com

Positive Ion

Ref. FileName

Chemical Name[Sigma Code #]

MolecularMass

/ Uses

UBQBovine Ubiquitin[U6253]

8564.85 650-1500 General

HBAHumanα globin[H753]

15126.36 700-1500 Hb analysis

SODSuperoxide dismutase[S2515]

15591.35 900-1500Hb (internalcal.)

HBBHumanβ globin[H7379]

15867.22 800-1500 Hb analysis

MYOHorse heart myoglobin[M1882]

16951.48 700-1600 General

PEGH1000

Polyethylene glycol +ammonium acetatemixturePEG 200+400+600+1000

80-1000ES+ andAPcI+calibration

PEGH2000

Polyethylene glycol +ammonium acetatemixturePEG 200+400+600+1000+1450

80-2000ES+calibration

NAICSSodium Iodide / CaesiumIodide mixture

20-4000General,ES+calibration

NAIRBSodium iodide / RubidiumIodide mixture

20-4000ES+calibration


LCTUser's Guide

Horse Heart Myoglobin

Reference File: MYO.REFMolecular Weight: 16951.48

ChargeState

Calculated/ Value

ChargeState

Calculated/ Value

ChargeState

Calculated/ Value

28

+

606.419 21

+

808.222 13

+

1304.969

616.177 20

+

848.583 12

+

1413.633

27

+

628.841 19

+

893.192 11

+

1542.053

26

+

652.989 18

+

942.758 10

+

1696.158

25

+

679.068 17

+

998.155 9

+

1884.508

24

+

707.320 16

+

1060.477 8

+

2119.945

23

+

738.030 15

+

1131.108 7

+

2422.651

22

+

771.531 14

+

1211.829

Polyethylene Glycol

PEG + NH4+

Reference Files: PEGH1000.REF, PEGH2000.REF

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


LCTUser's Guide

Sodium Iodide and Caesium Iodide Mixture

Reference File: NAICS.REF

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.REF

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


LCTUser's Guide

Negative Ion

Ref. FileName

Chemical Name[Sigma Code #]

MolecularMass

/ Uses

MYONEGHorse heart myoglobin[M1882]

16951.48 700-2400 General

SUGNEG

Sugar mixture of:maltose [M5885]raffinose [R0250]maltotetraose

[M8253]corn syrup [M3639]

100-1500Low massrange

NAINEGSodium Iodide / CaesiumIodide (or RubidiumIodide) mixture

200-3900ES-calibration

Horse Heart Myoglobin

Reference File: MYONEG.REF

Calculated/ Value

891.175 1209.812 1882.490

940.741 1302.952 2117.927

996.138 1411.615 2420.632

1058.460 1540.036

1129.091 1694.140

Mixture of Sugars

Reference File: SUGNEG.REF

Calculated/ Value

179.06 665.21 1151.37

341.11 827.27 1313.42

503.16 989.32 1475.48


LCTUser's Guide

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


LCTUser's Guide

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 acetate.

Use reference file PEGH1000.REF.

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 acetate.

Use reference file PEGH2000.REF.


LCTUser's Guide

Sodium Iodide Solution for Positive Ion Electrospray

Method 1

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

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

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

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

Use reference file NAICS.REF.

Method 2

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

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

The purpose of the rubidium iodide is to obtain a peak atz 85 (85Rb+) with anintensity of about 10% of the base peak atz 173. Rubidium iodide has theadvantage that no rubidium clusters are formed which may complicate thespectrum. Note that rubidium has two isotopes (85Rb and87Rb) in the ratio2.59:1, giving peaks atz 85 and 87.

Use reference file NAIRB.REF.

Sodium Iodide Solution for Negative Ion Electrospray

Either of the above solutions is suitable for calibration in negative ion mode. In bothcases the first negative reference peak appears atm 127 (I–) and the remaining peaksare due to NaI clusters.

Use reference file NAINEG.REF.


LCTUser's Guide

Index

AAcceleration 37Accurate mass 85Acetonitrile 48

Adducts 55, 58, 78Acquisition 42, 43Active inverted magnetron gauge 17, 105Air filter 104Ammonia 48Ammonium acetate 139Analog channels 22APcI 10, 77

Analysis 83Preparation 30Tuning 80, 83

APcI probe 18, 84, 122Checking 79Filter 122Fused silica capillary 124Maintenance 122Temperature 83, 122Tip heater 123

Aperture 37Atmospheric pressure chemical ionisation

See: APcIAttenuator 15

BBack pressure

High 122, 128Low 122

Biopolymers 60

CCaesium iodide 60, 136, 138, 140Calibration 85

Electrospray 60Camera 69Capillary 57Charging 107Cleanable baffle 67, 79, 107, 111Cleaning materials 131Cluster ions 58, 62Column

4.6mm LC 50, 62, 77Capillary LC 62Microbore (2.1mm) LC 50, 62

Cone gas 55, 67Cone gas nozzle 67, 116Contact closure in 22Cooling fan 104Corona 10Corona discharge pin 18, 30, 78, 107, 117Coupled column chromatography 63

DData processing 43Deadtime correction 87, 94Desolvation gas 18, 19, 54, 83Desolvation heater 18Desolvation temp 54Discharge pin

See: Corona discharge pinDivert / injection valve 19Drugs 61Dye compounds 61

IndexPage 141

LCTUser's Guide

EElectronics 14, 21, 103Electrospray 10, 47

Analysis 60Negative ion 61Operation 51Positive ion 61Preparation 28

Electrospray probe 51, 53Maintenance 118Removal 59

Electrostatic dischargeSee: ESD

Environmental contaminants 61ESD earth 21Event out 22Exhaust 20Exhaust liner 67, 107, 111Extraction cone 36, 57, 112

FFault finding 101, 126Flow injection analysis 48, 65Focus 37Formic acid 48Fragmentation 48Fused silica capillary (nanoflow) 65, 74

GGas ballast 105, 132Glass capillary (nanoflow) 65, 70Gradient elution 62

HHexapole lens

See: RF lens

IInfusion pump 48Injection valve 48Input 21Ion evaporation 10Ion mode 52, 81Ion optics 11Ion source

See: SourceIsotope distribution

Incorrect 129

LLC-MS interface 62Lock mass 86, 93, 98Lteff 41

MMaintenance 101Manual pusher 38MassLynx 23Max. flight time 38MCP detector 37

Conditioning 26Megaflow 50, 55, 58, 62Metabolites 61Microscope 69Mobile phase 83Multiply charged ions 10Myoglobin 60, 135, 137

NNanoflow electrospray 10, 65, 107Nano-HPLC 65Nano-LC (nanoflow option) 72Nebuliser 10Nebuliser gas 18, 19, 53Nitrogen 20, 25, 35, 51, 53Noise 130

OOil mist filter 106Oligonucleotides 47, 61Optical communications link 21Organometallics 61

IndexPage 142

LCTUser's Guide

PPanels, removal 102PEG

See: Polyethylene glycolPenning gauge

See: Active inverted magnetron gaugePeptides 47, 61Pesticides 61, 83Phase system switching 63Phosphate 60Pirani gauge 17, 105Plasma 10Pollutants 61Polyethylene glycol 60, 135, 139Polysaccharides 61Power 21

Failure 35Preamplifier 15Preventive maintenance 132Probe temperature

See: APcI probe temperatureProteins 47, 61Proton abstraction 10Proton transfer 10Purge gas 55, 58, 107Pusher 11

Control 14

RReciprocating pump 48, 62Reference compound 133Reflectron 11Reverse phase 62, 77RF DC Offset 1 37RF DC Offset 2 37RF Generator 15RF lens 12, 37, 107, 114Rotary pump 16, 21, 34, 104, 106

Oil 106Rubidium iodide 60, 136, 138, 140

SSaccharides 61Sample cone 36, 57, 107, 108, 110Scan in progress 21Sensitivity

LC-MS 63Loss of 129

Shutdown 44SIP

See: Scan in progressSodium iodide 60, 136, 138, 140Software 23Source 101, 107, 116

Block 112Heater 18Temperature 57

Split, post-column 49, 62Start (mV) 40Status display 19Steering 37Steroids 83Stop (mV) 40Sugar mixture 60, 137Supply inlet 21Syringe pump 48, 62System power 21

TTarget compound analysis 63TDC

See: Time to digital converterTEA

See: TriethylamineTetrahydrofuran 63THF

See: TetrahydrofuranThreshold 40Time to digital converter 11, 21, 40, 87Trace enrichment 63Transfer lens

See: RF lensTransient pressure trip 34Triethylamine 62Trifluoroacetic acid 62Trigger 21Tune page display 43Tuning 36

APcI 80, 83Electrospray 52

Turbomolecular pump 16, 34, 104

UUV detector 49, 62

IndexPage 143

LCTUser's Guide

VVacuum 16, 26, 104

Fault 34Leak 34, 104Poor 130Protection 32

Veff factor 41

WWater cooling 20, 34

Failure 34

IndexPage 144

LCTUser's Guide

Documents

LCT User's Guide Micromass UK Limited Floats Road ... manual is a companion to the MassLynx NT User's Guide supplied with the instrument. All information contained in these manuals