User's Guide Waters Corporation USA Fax: - 508-482 …...ZMD User's Guide Safety Sécurité The instrument is marked with this symbol where high voltages are present. Ce pictogramme

ZMDUser's Guide

Waters CorporationCorporate Headquarters,

34 Maple Street,Milford,

Massachusetts 01757USA

Tel: - 508-478-20001-800-252-HPLC (4752)

Fax: - 508-482-2674e-mail: [email protected]

http://www.waters.com

This manual provides operational, maintenance and troubleshooting instructions forthe ZMD 2000 and ZMD 4000 mass detectors. This manual is acompanion to the

MassLynx NT User’s Guide supplied with the instrument.

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

herein nor for incidental or consequential damages in connection with the furnishing,performance or use of this material. Al l product specifications, as well as theinformation contained in this manual, are subject to change without notice.

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

Micromass Code 6666473Issue 2,© Micromass Ltd.

http://www.waters.com

ZMDUser's Guide

ZMDUser's Guide

Safety Sécurité

The instrument is marked with this

symbol wherehigh voltagesare present.

Ce pictogramme indique la presence de

heute tension.


symbol wherehot surfacesare present.

Ce pictogramme indique la presence de

surfaces chaudes.


symbol where the user should refer to this

User's Guidefor further instructions.

Ce pictogramme indique la necessite de se

réferer au manuel d'utilisation.

Warnings are given throughout this

manual where care is required to avoid

personal injury.

Des avertissements sont donnés dans ce

manuel aux endroits où l'utilisateur doit

être particulierement prudent pour eviter

les blessures.

High voltages

Heute tension

Hot surfaces

Surfaces chaudes

Poisonous hazard

Risquesd’empoisonement

Chemical hazard

Chimiques dangereux

Flammable material

Produitsinflammables

General hazard

Hazard général

To maintain the safety integrity of the

instrument it should be used in a Pollution

Degree 1 environment.

The power circuits are designed for a

classification of Installation Category 1

(over voltage category).

Afin de garantir la sécurité de l'appareil il

doit être utilisé dans un environment de

degré 1 de pollution.

Les circuits électriques sont fabriqués

pour une classification d'installation de

Catégorie 1 (survoltage).

To maintain the safety integrity of the

instrument do not remove any panels.

There are no user serviceable parts inside.

For all questions concerning instrument

repair, contact the Waters Corporation

service desk.

Afin de garantir la sécurité de l'appareil ne

pas enlever les panneaux. Il n'y a pas de

pièces nécessitant de la maintenance a

l'interieur.

Pour toutes questions regardant la

maintenance de cet appareil qui ne serait

pas couvert par ce manuel d'utilisation il

conrient de contacter le bureau de service

de Waters Corporation.

If the instrument is used in a manner not

specified by the manufacturer, the

protection provided by the equipment may

be impaired.

Dans le cas où l'appareil serait utilisé de

maniere non specificé par le fabricant le

niveau de protection de l'appareil pourrait

altèré

ZMDUser's Guide

ContentsHardware Specifications

Dimensions 13Weights 13

Lifting and Carrying 14Power 15Environment 15Water Cooling 15Exhausts 16

Rotary Pump 16API Gas Exhaust 16

Nitrogen 16

Table of Contents

ZMDUser's Guide

Instrument DescriptionOverview 17Vacuum System 18Ionisation Techniques 18

Atmospheric Pressure Chemical Ionisation 18Electrospray 18

Sample Inlet 19Data System 19Rear Panel Connections 20

Water 20Nitrogen Gas In 20Exhausts 21Waste 21Power Cord 21Electronics Breaker 21Rotary Pump Breaker 21Rotary Pump Power 21PC Link 21

User I/O 22Analog Inputs 22Contact Closure Inputs 23Event Out 23Analog Out 23

Front Panel Controls and Indicators 24Status Display 24

Vent LED 25Vacuum LED 25Operate LED 25

Flow Control Valves 25Divert/Injection Valve 26

Front Panel Connections 27Desolvation Gas and Probe Nebuliser Gas 27Capillary/Corona Pin Voltage 27Electrospray/APcI Heaters 27

Internal Layout 28

Table of Contents

ZMDUser's Guide

Routin e ProceduresStart Up Following aComplete Shutdown 31

Preparation 31Pumping 33Using the Instrument 33

Start Up Following Overnight Shutdown 33Preparation for Electrospray Operation 34Preparation for APcI Operation 36Operate 38Tuning and Calibration 38Source Voltages 39Data Acquisition and Processing 39

Automatic Pumping and Vacuum Protection 40Overview 40Protection 40

Transient Pressure Trip 40Pump Fault 41Power Failure 41

Shutdown Procedures 42Emergency Shutdown 42Overnight Shutdown 42Complete Shutdown 43

Table of Contents

ZMDUser's Guide

ElectrosprayIntroduction 45

Post-column Splitting 47Megaflow 48

Changing Between Flow Modes 48Operation 49

Checking the ESI Probe 50Obtaining an Ion Beam 51

Tuning and Optimisation 51Probe Position 51Nebuliser Gas 52Desolvation Gas 52Cone Gas 53Purge Gas 53Source Temperature 54Capillary Voltage 54Sample Cone Voltage 54Extraction Cone Voltage 55Low Mass Resolution and High Mass Resolution 55Ion Energy 55

Megaflow Hints 56Removing the Probe 57

Sample Analysis and Calibration 58General Information 58

Typical ES Positive Ion Samples 59Typical ES Negative Ion Samples 59

Chromatographic Interfacing 60LC-MS Sensitivity Enhancement 61

Nanoflow ElectrosprayOverview 63Installing the Interface 64Operation of the Camera System 67Using the Microscope 67Glass Capillary Option 68

Restarting the Spray 69Nano-LC Option 70

Installation 70Operation 71

Changing Options 72

Table of Contents

ZMDUser's Guide

Atmospheric Pressure Chemical IonisationIntroduction 73Preparation 74

Checking the Probe 75Obtaining a Beam 76Hints for Sample Analysis 78

Tuning for General Qualitative Analysis 78Specific Tuning for Maximum Sensitivity 78

Corona Voltage 78Probe Position 79Probe Temperature 79Desolvation Gas 79Cone Gas 79

Removing the Probe 80

Table of Contents

ZMDUser's Guide

Mass CalibrationIntroduction 81Electrospray 81

Overview 81Preparing for Calibration 82

Reference Compound Introduction 82Tuning 82

Instrument Threshold Parameters 83Calibration Options 84

Selecting the Reference File 84Removing Current Calibrations 84

Selecting Parameters 85Automatic Calibration Check 85Calibration Parameters 86Mass Measure Parameters 87

Performing a Calibration 88Acquisition Parameters 90Starting the Calibration Process 92

Checking the Calibration 94Calibration Failure 96Incorrect Calibration 98Manual Editing of Peak Matching 99Saving the Calibration 99Verification 100

Electrospray Calibration with PEG 102Atmospheric Pressure Chemical Ionisation 103

Introduction 103Preparing for Calibration 104

Reference Compound Introduction 104Tuning 104

Calibration Options 104Selecting Reference File 104Removing Current Calibrations 104

Selecting Calibration Parameters 105Performing a Calibration 105

Static Calibration 105Acquisition Parameters 105Acquiring Data 107Manual Calibration 108

Scanning Calibration and Scan Speed Compensation 111Acquiring Data 111Manual Calibration 112

Calibration Failure 114Incorrect Calibration 115Manual Editing of Peak Matching 116

Saving the Calibration 116Manual Verification 117

Table of Contents

ZMDUser's Guide

Maintenance and Fault FindingIntroduction 119Cooling Fans and Filters 119The Vacuum System 120

Vacuum Leaks 120Gas Ballasting 121Oil Mist Filter 122Rotary Pump Oil 122Pirani Gauge 122

The Source 123Overview 123Cleaning the Cone Gas Nozzle and Sample Cone 124Removing and Cleaning the Ion Block and Extraction Cone 129Removing and Cleaning the RF Lens Assembly 132Reassembling and Checking the Source 134The Corona Discharge Pin 135

The Electrospray Probe 136Overview 136Replacement of the Stainless Steel Sample Capillary 138

The APcI Probe 140Cleaning the Probe Tip 140Replacing the Probe Tip Heater 141Replacing the Fused Silica Capillary 142

The Analyser 143The Detector 144Electronics 144Fault Finding Check List 145

No Beam 145Unsteady or Low Intensity Beam 145Ripple 145High Back Pressure 146General Loss of Performance 146

Cleaning Materials 147Preventive Maintenance Check List 148

Daily 148Weekly 148Monthly 148Four-Monthly 148

Table of Contents

ZMDUser's Guide

Reference InformationOverview 149Positive Ion 150

Horse Heart Myoglobin 151Polyethylene Glycol 151

PEG + NH4+ 151Sodium Iodide and Caesium Iodide Mixture 152Sodium Iodide and Rubidium Iodide Mixture 152

Negative Ion 153Horse Heart Myoglobin 153Mixture of Sugars 153Sodium Iodide and Caesium Iodide (or Rubidium Iodide) Mixture 154

Preparation of Calibration Solutions 155PEG + Ammonium Acetate for Positive Ion Electrospray and APcI 155PEG + Ammonium Acetate for Positive Ion Electrospray(Extended Mass Range) 155Sodium Iodide Solution for Positive Ion Electrospray 156

Method 1 156Method 2 156

Sodium Iodide Solution for Negative Ion Electrospray 156

Index

Table of Contents

ZMDUser's Guide

Hardware SpecificationsDimensions

WeightsInstrument: 105kg (230lb)

Data system(computer, monitor and printer): 50kg (110lb)

Rotary pump: 40kg (90lb)

Transformer (optional): 45kg (100lb)

Hardware SpecificationsPage 13

ZMDUser's Guide

720mm590mm

540mm

Lifting and Carrying

Warning: Persons with a medical condition, for example a back injury, whichprevents them from handling heavy loads should not attempt to lift theinstrument. Waters Corporation accept no responsibility for any injuries ordamage sustained while lifting the instrument.

Caution: Under no circumstances should the instrument be lifted by the frontmoulded cover, the probe or the source housing.

Before lifting the instrument proceed as follows:

Vent, power down and disconnect the instrument from the power supply.

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

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

Disconnect all connections to LC equipment.

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

The weight of the instrument is 105kg (230lb). Lifting equipment or suitably trainedpersonnel will be required to lift or lower the instrument.

UK Health and Safety guidelinesrecommend that a minimum of six trainedand suitable personnel are required to lift aunit of this weight, positioned for equalweight distribution.

Before undertaking any lifting, lowering ormoving of the instrument:

• Assess the risk of injury.

• Take action to eliminate risk.

If some risk still exists:

• Plan the operation.

• Use trained people.

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

The instrument should be lifted from underneath the frame at either side of theinstrument and should be supported as shown in line with, or close to, the feet uponwhich the instrument stands.


ZMDUser's Guide

PowerInstrument: Class 1device,

230V (+8%, -14%), 50/60 Hz

Data system: 100-120V or 220-240V

Power consumption: 3.0kW max.

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

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

Overall heat dissipation: 2.5kW maximum(excluding LC and optional water chiller)

Humidity: Relative humidity <70%

The instrument complies to the European directive on electrical safety as defined byIEC 1010 part 1, amendment 2.

Water CoolingThe turbomolecular pumps require water cooling. If a town water supply is used, theinlet temperature should be between 10-20°C (50-68°F). The flow rate should be 0.5litres/min at 10°C and 1.0 litres/min at 20°C. An in-line filter should be used toremove particulates and prevent blockages. If a water chiller is used, it should have aminimum cooling capacity of 200W at 20°C (68°F) with astability of ±2°C (±4°F).The reservoir volume should be 2 litres (minimum) and the supply pressure should bein the range 0.7 to 3 bar (10-40 psi).

Heat dissipation into the water: 200W

The water flow through the turbomolecular pumps is automatically stopped when thesystem is vented. The output pressure can rise above 40psi in this state and a flowby-pass is required for the chiller.


ZMDUser's Guide

Exhausts

Rotary Pump

The rotary pump must be vented to atmosphere (external to the laboratory) via a fumehood or industrial vent.

API Gas Exhaust

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

Caution: The API gas exhaust line must not be connected to the rotary pumpexhaust line as this may result in damage to the instrument. External to thelaboratory, the two exhaust outlets should be separated by at least one metre.

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

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


ZMDUser's Guide

Instrument DescriptionOverview

The ZMD is a quadrupole mass analyser based detector which provides molecularweight and structural information for a wide variety of analytes. Samples areintroduced into the source of the instrument from a HPLC or other liquid introductionsystem. Ionisation at atmospheric pressure takes place in the source. These ions aresampled through a series of orifices into the analyser of the detector where they arefiltered according to their mass to charge ratio (m). The resultant mass separatedions are then detected via a photomultiplier detection system. The signal is amplified,digitised and presented to the data system.

The detector may be coupled to:

• a HPLC system, to provide molecular weight information from a LC run or toperform target analysis and quantification.

• an autosampler and LC pumping system, to provide automated determination ofmolecular weights with unattended operation.

• an infusion pump or a syringe pump, for analyses of precious low concentrationcompounds.

Instrument DescriptionPage 17

ZMDUser's Guide

Samplesfrom the liquidintroduction systemare introduced atatmospheric pressure into theionisation source.

Ions are sampled through a series of orifices.

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

The transmitted ions are detected by the photomultiplier detection system.

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

Sample Inlet

Sampling Cone

Extraction Cone

RF Lens

MassLynx NTData System

Prefilter 1

Quadrupole

Detector

Vacuum SystemVacuum is achieved using a direct drive rotary pump and two turbomolecular pumps.The rotary pump, mounted on the floor external to the instrument, backs theturbomolecular pumps and also pumps the first vacuum stage of the source.

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

Vacuum measurement is by a Pirani gauge situated on the analyser. This acts as avacuum switch, switching the instrument out of the OPERATE mode in the event ofinadequate vacuum.

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

Ionisation TechniquesTwo atmospheric pressure ionisation techniques are available, these being selectedsimply by choice of probe. A recognition system is incorporated so that the datasystem “knows” which is being used.

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 then takes place betweenthe solvent ions and the sample. Eluent flows up to 2 ml/min can be accommodatedwithout splitting the flow.

Electrospray

Electrospray (ESI) ionisation 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, compoundsof high molecular weight can be determined if multiply charged ions are formed.Eluent flows up to 1 ml/min can be accommodated although it is often preferable withelectrospray ionisation to split the flow such that 100 to 200 µl/min of eluent entersthe mass spectrometer.


ZMDUser's Guide

Sample InletSample is introduced from a suitable liquid pumping system along with the nebulisinggas to either the APcI probe or the ESI probe.

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

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

MassLynx NT provides full control of the mass spectrometer including setting up andrunning selected HPLC systems, tuning, acquiring data and data processing

Analog inputs can be read by the data system so that, where applicable, a trace from aconventional LC detector (for example UV or ELSD) can be stored simultaneouslywith the acquired mass spectral data. A further option is the ability to acquire UVphotodiode array detector data (selected systems only, for example Waters 996 PDA).

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


ZMDUser's Guide

Rear Panel Connections

Water

A water supply is required 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, the other end of this tubingcan be connected 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.

Caution: The API gas should be turned off on the tune page before connectingand turning on the nitrogen supply to the rear panel of the instrument. Failure todo this may result in damage to the flowmeter.


ZMDUser's Guide

NitrogenGas In

ExhaustGas

Waste

PC Link

PowerCord

Rotary PumpPower Outlet

Pumping Linesto Rotary Pump

WaterConnections

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. This should be connected using 10mm plastictubing connected to the push-in fitting.

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

Waste

In the event that the nitrogen gas is switched off, or runs out, and the LC systemcontinues to run, solvent will begin to accumulate in the Z-spray source. The source isfitted with an LC exhaust line to allow solvent to drain via the 6mm fitting labeledWaste .

The waste line should be connected to the nitrogen exhaust trap bottle.

Caution: It is essential that the exit of this waste line is kept above the level ofsolvent in the waste bottle. If it becomes submerged, suck-back may occurresulting in contamination of the source.

Power Cord

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

Electronics Breaker

TheElectronics circuit breaker switches mains power to the instrument. In the eventof the instrument drawing more than the rated current, the circuit breaker will trip.

Rotary Pump Breaker

TheRotary Pump circuit breaker switches mains power to the rotary pump. In theevent of the pump drawing more than the rated current, the circuit breaker will trip.

Rotary Pump Power

The rotary pump outlet provides switched mains power to the rotary pump. The rotarypump is controlled via the data system.

PC Link

The connector markedPC Link connects the instrument to the data system via thesupplied network cable.


ZMDUser's Guide

User I/OA number of connectors are available on the rear panel to allow the user to connect tovarious peripherals to the instrument as follows:

Analog Inputs

There are four analog input channels to allow the display of the output of otherdetectors (for example UV, ELSD). The input range is 0 to 1V full scale, a dynamicrange of 106:1.


ZMDUser's Guide

ANALOG INPUTS

1V

1V

1V

1V

IN2

IN1 1

2

CONTACT

CLOSURE

INPUTS

EVENT

OUT

ANALOG

OUT

REFER TO

MANUAL

max max

max max

-

+

-

+

-

+

-

+

-

+

!

CH1

CH2 CH4

CH3

Contact Closure Inputs

Two contact closure inputs are provided to allow a signal from an external device (forexample an autosampler) to trigger the start of an acquisition.

Event Out

Two event outputs are provided for connection to external devices to provide, forexample, start and stop signals. The outputs are rated at 24V, 250mA.

Analog Out

An analog output is provided to allow a trigger signal for an external fractioncollection device. For operation of this output the optional FractionLynx software isrequired.


ZMDUser's Guide

Front Panel Controls and Indicators

Status Display

The display on the front right of the instrument consists of three dual-colour lightemitting diodes (LED).

The display generated by theVent andVacuum LED’s is dependant on the vacuumstatus of the instrument.

TheOperate LED depends on both the vacuum status and whether the operate modehas been selected from the data system.


ZMDUser's Guide

Vent

Vacuum

Operate

ProbeAdjustment

AxisAdjustment

DesolvationGas Cone

Gas

The status of the instrument is indicated as follows:

Vent LED

State Vacuum Gauge Status Vent LED

Vented Any Steady green

Venting Any Flashing green

Vacuum pump trip Any Flashing amber

Vacuum pump tripped Any Steady amber

Vacuum LED

State Vacuum Gauge Status Vacuum LED

Pumping Any Flashing green

PumpedBelow trip level Steady green

Above trip level Steady amber

Operate LED

State Vacuum Gauge Status Operate LED

Any, Operate not selected Any No indication

Not pumped, Operate selected Any Steady amber

Any, Operate selected Above trip level Steady amber

Pumped, Operate selected Below trip level Steady green

Flow Control Valves

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


ZMDUser's Guide

Divert/Injection Valve

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

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

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

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

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

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

Control of the valve is primarily from the data system. The two switches markedLOAD andINJECT enable the user to manually override the control of the valvefrom the data system. This is used when making loop injections at the instrument.


ZMDUser's Guide

Divert / InjectionValve

Inject

Load

Front Panel Connections

Desolvation Gas and Probe Nebuliser Gas

The gas lines for the desolvation gas and probe nebuliser gas are connected to thefront of the instrument using threaded metal fittings. These enable the PTFE tubing toprovide a seal with the union from the front of the instrument.

Capillary/Corona Pin Voltage

The electrical connection for the electrospray capillary or the APcI discharge pin is viathe coaxial connector. This is removed from the front panel by pulling on the metalsleeve of the plug to release it.

Electrospray/APcI Heaters

The electrical connection for the desolvation heater or APcI probe is via the multi-wayconnector. This is removed from the front panel by pulling on the metal sleeve of theplug to release it. Both the electrospray desolvation heater and APcI probe heater usethis connector.

The electrospray source heater is powered from the internal electrical wiring.


ZMDUser's Guide

DesolvationGas

Capillary / CoronaProbes

NebuliserGas

LCConnection

Internal LayoutThe instrument is divided internally into two compartments. The left hand sidecompartment contains the electronics, mounted into a card frame. There are four manboards as standard:

• Transputer Processing Card (TPC)

• Analog PCB

This provides control and power for the source and probe heaters, and voltagesfor the source components.

• High Mass Generator Control PCB

This provides RF control for the RF generator and DC supplies for thequadrupole mass analyser.

• Digital PCB

This board provides control for the scanning functions and the controldigital-to-analogue convertors (DAC). In addition, it converts user’s analoginputs (for example from a UV detector) into digital signals and it also controlsthe optional divert / injection valve and the pumping sequence.


ZMDUser's Guide

Printed CircuitBoards RF Generator

High VoltageSupplies

Low VoltageSupply

SourceTurbo Pump

The PCBs plug directly into a backplane. Also situated on this are the high voltagemodules that supply the detector system, the high voltages for the source and APcIprobe and the DC supply for the analyser.

Situated at the bottom of the chassis is a tray which contains the low voltage supplyfor the instrument, breakers, rotary pump relay and the mains inlet socket.

The right hand side compartment contains the mass spectrometer, the turbomolecularpumps and the RF generator that produces the necessary RF and DC voltages for thequadrupole analyser. This compartment also contains the gas lines, and flow metersfor the source and probes.


ZMDUser's Guide

SourceTurbo Pump

RF Generator

AnalyserHousing


ZMDUser'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 on therotary pump oil levelindicator. If necessary,refill or replenish usingUltragrade 19 or InlandQ45, ensuring that theinstrument and rotarypump are switched offbefore removing the fillerplug. Recheck the level toensure that the correctamount has been added.

Check for oil in the oilmist filter. See themanufacturer's literature for details.

Connect a supply of dry, high purity nitrogen to the connector at the rear of theinstrument and set the outlet pressure to 7 bar (100 psi).

Caution: The API gas should be turned off on the tune page before connectingand turning on the nitrogen supply to the rear panel of the instrument. Failure todo this may result in damage to the flowmeter.

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

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

Check that the data system is connected to the mass spectrometer.

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.This must not be the same vent as the rotary pump exhaust.

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

Routine ProceduresPage 31

ZMDUser's Guide

GasBallast

DrainPlug

Exhaust

FillerPlug

Oil LevelIndicator

Oil MistFilter

Switch on the mains to the mass spectrometer using the two mains breakerssituated at the rear of the instrument (lower left side as viewed from the front).

Switch on the data system.

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

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

When the data system has booted up, double-click on the MassLynx icon in theWindows desktop.

After a few seconds the MassLynx window will appear.

To display the tune page click on .


ZMDUser's Guide

Pumping

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

Click on Pump .

The rotary pump will now start and simultaneously the turbomolecular pumpswill start.

TheVacuum LED on the front of the instrument will flash as the systempumps down.

When the system has reached operating vacuum the LED will change to a steadygreen, indicating that the instrument is ready for use.

If the rotary pump oil has been changed or replenished, open the gas ballastvalve on the rotary pump. Refer toRoutine Maintenancefor 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.

Using the Instrument

ZMD is now almost ready to use. To complete the start up procedure and prepare forrunning samples, follow the instructions in the following sections.

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

If the data system has been switched off, switch it on as described in thepreceding section.

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


ZMDUser's Guide

Preparatio n for Electrospra y Operation

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

If necessary, switch the instrument into standby by selecting Press forStandby .

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 andsource enclosure.

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 fi t the blankingplug.

Replace the source enclosure and adjustment flange.


ZMDUser's Guide

BlankingPlug

CoronaDischar ge

Pin

MountingContact

ExhaustLiner

High VoltageSocket

CleanableBaffle

With the discharge pin removed and the blanking plug fitted:

Ensure that the source enclosureand adjustment flange are in place.

Connect the desolvation gas to thefront panel. Tighten the nut toensure a good seal

Check that lead of the probeadjustment flange is plugged intothe socket labelledProbes on thefront panel.

Take the electrospray probe andconnect the nebuliser gas line tothe front panel.

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

Insert the probe into the source and tighten up the two thumb nuts to firmlysecure the probe.

Plug the probe lead into the socket on the front panel labelledCapillary/Corona .

From the MassLynx window select to go to the tune page.

Set the source temperature to 100°C and the desolvation temperature to 150°C.

The source is now ready for electrospray use. To obtain an ion beam follow theinstructions given in the section entitled ‘obtaining an ion beam’.

The ionisation mode used for tuning and acquisition is set automatically whenthe probe (electrospray or APcI) is inserted into the source.

Warning: Operating the source in ESI mode without the sourceenclosure will result in solvent vapour escape and the exposure ofhot surfaces and high voltages.

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.

Caution: If the nitrogen supply to the rear of the instrument is turned offovernight then the API gas should be turned off on the tune page before turningon the nitrogen supply. Failure to do this may result in damage to the flowmeter.


ZMDUser's Guide

SourceThumb Nuts

ProbeThumb Nuts

ProbeAdjustment Flange

SourceEnclosure

Preparation for APcI Operation

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

If necessary, switch the instrument into standby by selectingPress forStandby on the tune page.

Disconnect the gas and electrical connections from the front panel.

Undo the three thumb screws on the source and remove the probe flange andsource enclosure.

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

Replace the adjustment flange and source enclosure.


ZMDUser's Guide

BlankingPlug

CoronaDischarge

Pin

MountingContact

ExhaustLiner

High VoltageSocket

CleanableBaffle

With the discharge pin fitted:

Ensure that the source enclosureand adjustment flange are in place.

Connect the APcI high voltagecable between the socket labelledCorona/Capillary and the socketpositioned at the bottom rightcorner of the source flange.

Insert the APcI probe into thesource and tighten up the twothumb screws.

SetSource Temp to 120°C.

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

The source is now ready for APcI operation.

Warning: Operating the source in APcI mode without the sourceenclosure will result in escape solvent vapour and the exposure ofhot surfaces and high voltages. Allow the glass source enclosure tocool after a period of operation at high flow rates before removal.

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.

Caution: If the nitrogen supply to the rear of the instrument is turned offovernight then the API gas should be turned off on the tune page before turningon the nitrogen supply. Failure to do this may result in damage to the flowmeter.


ZMDUser's Guide

SourceThumb Nuts

ProbeThumb Nuts


SourceEnclosure

Operate

On the MassLynx window, select to open the tune page.

Turn on the API gases and set the desolvation gas flow to 150 litres/hour.

SetDesolvation Temp. to 150°C orAPcI Heater to 600°C.

Click on Press for Operate on the MassLynx tune page.

The instrument will only go into operate if the probe adjustment flange is inplace and the probe is inserted.

Warning: The instrument should not be operated without the source enclosure inplace. Operation of the instrument without the source enclosure allows exposureto high voltages.

Warning: The source enclosure may become hot during operation in APcI orwith high flow rates in electrospray. Do not touch the source enclosure duringoperation or until it has cooled down after operation.

The system is now ready to accept samples and acquire data.

Tuning and Calibration

Before sample data are acquired, the instrument should be tuned and, for the highestmass accuracy, calibrated using a suitable reference compound. Consult the relevantsection of this manual for information regarding sample introduction and tuningprocedures in the chosen mode of operation.


ZMDUser's Guide

Source Voltages

The following illustration shows the various components of ZMD’s ion optic system.The electrode name in the table’s first column is that used throughout this manual todescribed the component. The second column shows the term used in the currentMassLynx release. The voltages shown are typical for an instrument in goodcondition. The polarities given are those actually applied to the electrodes. Onlypositive values need be entered via the tune page.

Data Acquisition and Processing

The acquisition and processing of sample data is comprehensively described in theMassLynx NT Users Guide and theGuide to Data Acquisition. Refer to thosepublications for details.


ZMDUser's Guide

Electrospray Probe

APcI Discharge Needle

Sample Cone

Extraction Cone

RF Lens

Differential Aperture

Prefilter

QuadrupoleAnalyser

Capillary +3.0 -3.0 Not applicable

Corona Not applicable +3.0 -2.0

Cone +60 -60 +60 -60

Extractor +3 -3 +3 -3

RF Lens +0.5 -0.5 +0.5 -0.5

Not adjustable (ground)

Not applicable

Not applicable

(kV) (kV)

(kV) (kV)

(V) (V) (V) (V)

(V) (V) (V) (V)

(V) (V) (V) (V)

Tune Page ESI APcIName +ve -ve +ve -ve

Automatic Pumping and Vacuum Protection

Overview

The instrument is fully protected against vacuum system faults due to:

• malfunction of the vacuum pumps

• excessive pressure

• excessive temperature

The pump down sequence is fully automated, a command from the data systemswitching on the rotary pump and turbomolecular pumps simultaneously. When theinstrument is vented, the rotary pump is switched off once the turbomolecular pumpshave slowed to 50% of operating speed.

Protection

Transient Pressure Trip

If the vacuum gauge detects a pressure surge above the factory set trip level of10-3 mbar, and if the instrument is in the operate mode, the following events occur:

The critical source, analyser and detector voltages are switched off.

TheOperate LED shows a steady amber.

TheVacuum LED shows a steady amber.

Acquisition will continue, although no mass spectral data are recorded.

When the pressure recovers, the voltages are restored and theVacuum andOperateLED’s are steady green.

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


ZMDUser's Guide

Pump Fault

A pump fault causes the following to occur:

The turbomolecular pumps stop pumping.

On the display theOperate LED changes to amber.

TheVent LED shows flashing amber, changing to steady amber as the pumpsstop.

As the pumps slow down the vent valve opens and the system is vented.

The pumps will not switch on again unless requested to do so.

A pump fault can occur as a result of:

• Over temperature of the turbomolecular pumps

If the water cooling fails, then the turbomolecular pumps switch off when theirtemperature becomes too high.

• Vacuum leak

Refer to later sections of this manual.

• Malfunction of the turbomolecular pumps

Contact the Waters Corporation service centre.

• Malfunction of the rotary pump

Contact the Waters Corporation service centre.

Power Failure

In the event of an unexpected power failure, proceed as follows:

Switch OFF the power to the instrument at the wall mounted isolation switch.

When power is restored, follow the start up procedure as described earlier in themanual.


ZMDUser'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.

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

A loss of data is likely.

Overnight Shutdown

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

Switch off the LC pumps.

From the MassLynx window select to open the tune page.

Click on Press for Standby on the tune page.

This will change from green to grey indicating that the instrument is no longerin operate mode.

Undo the connector on the probe to release the tubing leading from the LCsystem.

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

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

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

SelectGas to turn off the supply of nitrogen gas.


ZMDUser's Guide

Complete Shutdown

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


From the MassLynx window select to open the tune page.

Click on Press for Standby on the tune page.

This will change from green to grey indicating that the instrument is no longerin operate mode.

Undo the connector on the probe to release the tubing leading from the LCsystem.

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

If in use, switch off the APcI probe heater or reduce it to ambient temperature.

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

When the APcIProbe Heater readback falls below 100°C:

SelectGas to turn off the supply of nitrogen gas.

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

The turbomolecular pumps switch off. The Vent LED shows flashing amber,changing to steady amber as the pumps stop. As the pumps slow down the ventvalve opens and the system is vented.

Exit MassLynx and shut down the computer.

Switch off all peripherals.

Switch off the power to the instrument using the breakers on the rear panel ofthe instrument.

Switch off power at the wall mounted isolation switches.

Turn off the cooling water supply.

If the instrument is unlikely to be used for more than one month:

Drain the oil from the rotary pump as described inMaintenance and FaultFinding.


ZMDUser's Guide


ZMDUser's Guide

ElectrosprayIntroduction

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

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

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

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

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

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ProbeExhaust

ExhaustLiner

TurbomolecularPumpsRotary Pump

NebuliserGas

Cone Gas

Purge Gas

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 for flow injection analysis (FIA) studies, to alloweasy 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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ZMDUser'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, with approximately 200 µl/minentering the source. Also, even at lower flow rates, a split may be required for savingvaluable 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.

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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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Probe

PTFE Sleeve

PTFE Sleeve

Fused Silica Tube

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

Injector

Normal Flow Electrospray

Megaflow Electrospray

Operation

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

Warning: To avoid the risk of electric shock, the injector or LC column towhich the probe is attached must be grounded. Switch out of operate beforeremoving the probe. Isolate the probe before removing its cover.

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.

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.

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BlankingPlug

CoronaDischarge

Pin

MountingContact

ExhaustLiner

High VoltageSocket

CleanableBaffle

Checking the ESI Probe

Connect the electrospray probe to a pulse free pump.

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

Connect the PTFE tubing of the electrospray probe 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 on the nitrogen supply by selectingGas, and fully open theNebulisergas flow control valve situated on the front panel.

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

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

There should be approximately 0.6 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.

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ZMDUser's Guide

0.6mmSample

Capillary

NebulisingCapillary

Probe TipAssembly

Obtaining an Ion Beam

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

Using the needle valves on the front panel, set theDesolvation Gas flow rateto 300 litres/hour and theCone Gas flow to 50 litres/hour.

Turn on the liquid flow at 10 µl/min and setDesolvation Temp to 150°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.

Probe Position

The position of the probe is adjustedusing the probe adjustment collar(in/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 andsolvent system under investigation. The

following information should be considered when 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 awayfrom the sample cone to achieve optimum stability and sensitivity.

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ZMDUser's Guide

In / OutProbe

Adjustment

SidewaysProbe

Adjustment8mm

4mmCone GasNozzle

ProbeTip

Nebuliser Gas

Optimum nebulisation for electrospray performance is achieved by fully opening theNebuliser flow control valve, which is situated on the instrument’s front panel.

Desolvation Gas

The desolvation gas is heated and delivered as a coaxial sheath to the nebulised liquidspray 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.

To monitor the flow rate, select Window then Gas Flow on the tune page andobserve the readback window. The Desolvation Gas flow rate indicated on thetune page includes 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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Cone Gas

The cone gas reduces theintensity of solvent clusterions and solvent adduct ions.The cone gas flow rateshould be optimised byincreasing until solventcluster ions and / or adductions are reduced as much aspossible without diminishingthe intensity of the ion ofinterest, normally (M+H)+.

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

Purge Gas

The purge gas is notnecessary for most electrospray applications. It allows purging of the source volume toremove excessive solvent vapour.

Purge gas is enabled simply by removing the 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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ConeGas

Purge GasOutlet (Plugged)

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 Sample Cone setting between 25V and 70V will produce ions for most samples,although solvent ions prefer the lower end and proteins the higher end of this range.Whenever sample quantity and time permit,Sample Cone should be optimised formaximum sensitivity within the range 15V to 200V. Increasing the cone voltage willincrease ion fragmentation within the source.

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ZMDUser's Guide

Extraction Cone Voltage

Extraction Cone optimises at 1 to 5V. Higher values may induce fragmentation oflow molecular weight samples.

Low Mass Resolution and High Mass Resolution

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

Ion Energy

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

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

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ZMDUser's Guide

Megaflow Hints

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

• increaseDesolvation Gas flow to approximately 750 litres/hour.

• increaseDesolvation Temp to 300°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 gas flow rates andsource temperatures.

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 vapour trap bottle is connected in the exhaust line tocollect any condensed solvents.

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Removing the Probe

Warning: Risk of electric shock. The LC column or injector to which the probeis attached must be grounded. Switch the instrument out of operate beforeremoving the probe.

To remove the probe from thesource proceed as follows:

On the tune page, switch tostandby.

Switch off the liquid flow anddisconnect from the probe.

SelectGas to turn off thenitrogen.

Disconnect the probe cablefrom the instrument’s frontpanel.

Disconnect the nebulising gassupply from the instrument’s panel.

Undo the two thumb nuts and withdraw the probe.

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ZMDUser's Guide

ProbeThumb Nuts

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 may require more frequent source cleaning and risk blocking the transfercapillary.

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.

Detailed information on data acquisition and processing can be found in theMassLynx NT User’s GuideandGuide to Data Acquisition. Further information onmass calibration and on reference compounds can be found inMass CalibrationandReference Informationlater in this document.

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

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

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

A UV detector may be placed in-line to the ZMD probe. However, ensure thatthe volume of the detector will not significantly reduce the chromatographicresolution. 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.

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

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LC-MS Sensitivity Enhancement

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

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

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

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:

• Glass capillary.

Metal coated borosilicate glass capillaries allow the lowest flow rates to beobtained, but, after use for one sample only, must be discarded.

• Nano-LC.

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

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Regulatorand Injector

(Nano-LC option)

ProtectiveCover

Handle

Stop

GlassCapillaryOption

Nano-LCOption

Stage

Three-axisManipulator

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

The nanoflow 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.

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.


ZMDUser's Guide

Exhaust

ExhaustLiner


Cone Gas

Purge Gas

Analyser

SampleCone

ExtractionCone

IsolationValve

SourceEnclosure

RFLens

CleanableBaffle

SampleCapillary

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 nozzle using the PTFE tubingprovided.

Ensure that the purge gas isplugged (disabled).

Ensure that the cleanable baffle,the exhaust liner and the dischargepin blanking plug are fitted.


ZMDUser's Guide

Cone GasNozzle

PTFETubing

PTFEEncapsulated

O Ring

PurgeGas Plug

SourceThumb Nuts

ProbeThumb Nuts


SourceEnclosure

Probe FlangeMounting Pillar

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

Install the perspexcover and thenanoflow endflange, securing thiswith socket headscrews.

If not already inplace, attach themicroscope orcamera bracketsusing the screw holeand dowels at thetop of the bracket.

Insert the flexiblelight guide into thegrommet at the baseof the perspexcover.

Set the light source to its brightest.

Block theNebuliser andDesolvation Gas outlets on the instrument’s frontpanel.

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

SetSource Temp to approximately 80°C.


ZMDUser'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.


ZMDUser's Guide

Microscope

Camera

ZoomLens

ObjectiveLens

Grommet

Glass Capillary OptionWarning: Do not touch the sharp end of the capillary. As well as the risk ofinjury by a sliver of glass, the needle will become inoperable.

Caution: The capillaries are extremely fragile and must be handled with greatcare. Always handle using the square end of the capillary.

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

Carefully remove the capillary 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.

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

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

On the tune page, selectGas to turn on the nitrogen.

SelectPress for Operate .

SetCapillary between 1 and 1.5kV.


ZMDUser's Guide

Foam

Capillary

GlassCapillary

BlueConductiveElastomer

PTFE"Back Pressure"

Tubing

Ferrule

5mm

Knurled Nut

Union

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.


ZMDUser'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 theregion between the ferrule and the end of the thread on the nano-LC chamber.

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

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

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


ZMDUser'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.

SelectPress for Operate and setCapillary to approximately 2.5kV.

An ion beam should now be present.


ZMDUser'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.

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

Rotate the stage outwards.

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

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

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

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


ZMDUser'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. Mobile phase molecules rapidly reactwith ions generated by the corona discharge to produce stable reagents ions. Analytemolecules introduced into the mobile phase react with the reagent ions at atmosphericpressure and typically become protonated (in positive ion mode) or deprotonated (inthe negative ion mode). The sample and reagent ions pass through the sample coneinto the ion block prior to being extracted via the extraction cone into the RF lens.

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

Atmospheric Pressure Chemical IonisationPage 73

ZMDUser's Guide

CoronaDischarge Pin

ProbeExhaust

ExhaustLiner


NebuliserGas

Cone Gas

Sample

Analyser

DesolvationGas

SampleCone

ExtractionCone

IsolationValve

SourceEnclosure

RFLens

CleanableBaffle

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 settingof Sample Cone . The main reagent ions for a typical sample voltage of 20V are 83,101 and 142.

Acetonitrile adducting may be minimised by optimisation of the cone gas, as describedin Electrospray.

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).


ZMDUser's Guide

BlankingPlug

CoronaDischarge

Pin

MountingContact

ExhaustLiner

High VoltageSocket

CleanableBaffle

Checking the Probe

Ensure that the instrument is in standby and that the probe heater is off.

Warning: The probe tip may be hot. Switch off the liquid flow and, with theprobe gases flowing, allow the probe to cool (<100°C) before removing it fromthe source.

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 carefully pulling parallel to theaxis of the probe.

Ensure that 0.5 to 1mm of fused silica is protruding from the stainless steelnebuliser tube, and that the polyamide coating has been removed.

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).

SelectGas to turn on the nitrogen flow.

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

Switch off the liquid flow.

SelectGas to turn off the nitrogen 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.


ZMDUser's Guide

Obtaining a BeamEnsure that the corona discharge pin is fitted and connected as described inRoutine Procedures, Preparation for APcI Operation.

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

Ensure that the APcI tune page is showing.

The top line of the tune page indicates the current ionisation mode.

SetSource Temp to 130°C.

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

SetCorona to 3kV andSample Cone to 50V.

WhenSource Temp reaches 130°C:

SelectGas to switch on the nitrogen gas.

Using the valves on the front of the instrument, adjustDesolvation Gas to450 litres/hour and adjustCone Gas to 100 litres/hour.

Select one of the peak display boxes and setMass to 50 andSpan to 90.

SelectPress for Operate .

IncreaseGain on the peak display box until peaks become clearly visible.

SetAPcI Probe Temp to 500°C.


ZMDUser's Guide

WhenAPcI Probe Temp reaches 500°C:

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

Adjust the probe’s in / out position so that it is fully retracted.

Adjust the probe’s sidewaysposition so that the spray is directedapproximately at the midpointbetween the corona pin and thesample cone.

Check that a stable beam of solventions is now apparent.

Refer toHints for Sample Analysislater in this chapter for furtherinformation on source tuning.

Warning: It is normal for thesource enclosure and parts of theprobe adjustment flange to reachtemperatures of up to 60°C duringprolonged APcI operation. Careshould be exercised when handling source components immediately afteroperation.

Warning: Switch off the liquid flow and, with probe gases flowing, allow theprobe 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.


ZMDUser's Guide

In / OutProbe

Adjustment

SidewaysProbe

Adjustment

Hints for Sample Analysis

Tuning for General Qualitative Analysis

Refer toObtaining a Beamabove and tune on solvent ions.

Adjust the in/out position of the probe so that it is fully retracted from thesource.

Using the sideways adjuster ensure that the spray is directed approximately atthe mid-point between the corona pin and the sample cone.

This position occurs five full turns away from the stop closest to the corona pin.

For general qualitative analysis of mixtures, the following parameters are typical:

Corona *: 3kV

Sample Cone : 40V

Extraction Cone : 3V

RF Lens : 0V

Source Temp : 130°C

APcI Probe Temp *: 500°C

Desolvation Gas *: 150 litres / hour

Cone Gas : 100 litres / hour

* See the following section for specific tuning details.

Specific Tuning for Maximum Sensitivity

• For quantitive analysis, optimum APcI conditions should be obtained for eachanalyte using standard solutions.

• Tuning may be performed using a tee to introduce a standard solution (typically100 pg/µl) at 10 µl/min into the mobile phase stream.

• Alternatively, repeat direct loop injections of a standard solution (typically10 pg/µl) into the mobile phase stream may be used to optimize the APcI.

Corona Voltage

The APcI corona voltage can have a significant effect on sensitivity. The coronavoltage required depends upon the polarity of the compound and the polarity of theanalytical mobile phase. As recommended above optimization should be done in thepresence of the analytical mobile phase.


ZMDUser's Guide

• An improvement in signal may be obtained for polar compounds, when analysedin a polar mobile phase, by reducingCorona below 2kV.

• Similarly, an improvement in signal may be obtained for compounds of lowpolarity, when analysed in a low polarity mobile phase, by increasingCoronaabove 2kV.

Probe Position

The in / out position of the APcI probe generally has little effect on sensitivity. Thesideways adjustment can have a significant effect upon sensitivity.

Using the sideways adjuster ensure that the spray is directed approximately atthe mid-point between the corona pin and the sample cone.

This position occurs five full turns away from the stop closest to the corona pin.

Adjust the probe position around this point, one turn at a time, to optimise thesignal.

Probe Temperature

It is important to optimiseProbe Temp for maximum sensitivity, as follows:

Ensure that the analytical mobile phase is used during optimisation.

Starting at 650°C reduce the temperature in 50°C steps, allowing time for thetemperature to stabilise before taking a reading.

It is possible to set APcI Probe Temp too low for the mobile phase. This oftenresults in significant chromatographic peak tailing.

Desolvation Gas

In most circumstances the desolvation gas flow has little effect on signal intensity.However, in some situations, it has been observed to have an effect on chemicalbackground noise levels. AdjustingDesolvation Gas can be used as a check forthis.

Cone Gas

The cone gas will reduce solvent ion adducts often apparent at higher flow rates.

OptimiseCone Gas by maximising the intensity of the ions of interest.


ZMDUser's Guide

Removing the ProbeAfter a session of APcIoperation:

Turn off the LC flow.

SetProbe Temp to20°C.

SelectPress forStandby .

When the probe temperaturefalls below 100°C:

SelectGas and turnoff the nitrogen flow.

Undo the two thumbnuts and remove theprobe from the source.

Warning: Take care when removing the APcI probe. There is a risk of burns tothe operator. Allow the probe to cool before removing or handling the probe.

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 reduce thesource temperature to 60°C.


ZMDUser's Guide

ProbeThumb Nuts

Mass CalibrationIntroduction

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

The first section of this chapter describes a complete mass calibration of ZMD usingelectrospray ionisation with polyethylene glycol (PEG) as the reference compound.The second section describes a similar procedure using atmospheric pressure chemicalionisation (APcI) with PEG as the reference compound.

Electrospray

Overview

When a calibration is completed it is possible to acquire data over any mass rangewithin the calibrated range. It is therefore sensible to calibrate over a wide mass range.With PEG the possible calibration range is dependent upon the molecular weightdistribution of the PEGs used in the reference solution. For this example PEG gradesfrom PEG 200 to PEG 1000 are used and the calibration mass range is up to 1200amu. The following illustration shows a typical PEG + NH4+ spectrum.

Mass CalibrationPage 81

ZMDUser's Guide

Preparing for Calibration

Reference Compound Introduction

The example given here describes an automatic calibration which requires referencecompound to be present for several minutes. The introduction of the referencecompound is best achieved using a large volume Rheodyne injector loop (50 or 100µl)or an infusion pump (for example, a Harvard syringe pump).

When using a large volume injection loop:

Set up a solvent delivery system to deliver 10-20 µl/min of 50:50 acetonitrile :water or 50:50 methanol : water through the injector into the source.

When using an infusion pump:

Fill the syringe with the reference solution.

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

Set the pump to a flow rate of 10-20 µl/min.

SeeReference Informationfor advice on preparing the reference solutions.

Tuning


ZMDUser's Guide

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

SetMultiplier to 650V.

Adjust source parameters to optimise peak intensity and shape.

Set the resolution and ion energy parameters for unit mass resolution.

For a good peak distribution across the full mass range:

Check the intensity of some of the reference peaks above 500 amu.

Check also the intensity of the peaks atm89 and 133.

These lower mass peaks are formed when PEG fragments and increase as thecone voltage is raised. A cone voltage in the region of 45V is usually suitable.

Instrument Threshold Parameters

Before beginning the calibration procedure, some instrument parameters need to bechecked. For most low mass range calibrations, calibration data is acquired incontinuum mode. To allow suitable scanning speeds to be used the continuum dataparameters need to be set correctly.

From the acquisition control panel selectInstrument thenInstrument Threshold Settings to display the Instrument Data Thresholdingwindow.

In theProfile Data section setPoints per Dalton to 8.

SelectOK to save the parameters.


ZMDUser's Guide

Calibration Options

To access the calibration options selectInstrument thenCalibrate from theacquisition control panel. This brings forward the calibration dialog box.

Selecting the Reference File

Click on in the reference file box and scroll through the files until theappropriate file can be selected.

Selectpegnh4.ref for a PEG reference solution covering the range 80 to1200 amu. Ensure that there are less than 32 peaks in the calibration referencefile over the range to be calibrated.

Removing Current Calibrations

SelectProcess andDelete all Calibrations to load the default instrumentcalibration, followed byFile andSave As UNCAL.CAL .

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


ZMDUser's Guide

Selecting Parameters

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

Automatic Calibration Check

This is accessed fromEdit , AutoCal Check Parameters.... It is here that limits areset which the calibration must attain before the instrument is successfully calibrated(see below). Two user parameters can be set.

Missed Reference Peaks sets the maximum number of consecutive peaks whichare not matched when comparing the reference spectrum and the acquired calibrationspectrum. If this number is exceeded then the calibration will fail. The default valuefor this parameter, 2, is suitable in most cases.

Maximum Std Deviation is set to a default of 0.20. During calibration thedifference between the measured mass in the acquired calibration file and the truemass in the reference file is taken for each pair of matched peaks. If the standarddeviation of the set of mass differences exceeds the set value then the calibration willfail. Reducing the value of the standard deviation gives a more stringent limit.

Increasing the standard deviation means that the requirement is easier to meet, but thismay allow incorrect peak matching. Values greater than 0.20 should not be usedunless exceptional conditions are found.

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

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


ZMDUser's Guide

Calibration Parameters

These are accessed fromEdit , Calibration Parameters....

ThePeak Match parameters determine the limits within which the acquired datamust lie for the software to recognise the calibration masses and result in a successfulcalibration. These parameters are described in detail in theMassLynx NT User’sGuide. The default values are shown below.

Increasing thePeak window andInitial error gives a greater chance of incorrectpeak matching. All peaks in the acquired spectrum below theIntensity thresholdvalue (measured as a percentage of the most intense peak in the spectrum) will not beused in the calibration procedure.

The process of producing a calibration curve is described in detail in theMassLynx NTGuide to Data Acquisition. ThePolynomial order of the curve has values from 1 to5 as the available options:

A polynomial order of 1 should not be used.

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

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

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

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


ZMDUser's Guide

Mass Measure Parameters

These are accessed throughEdit ,Mass Measure Parameters . Ifcontinuum or MCA data are acquiredfor calibration then these parametersneed to be set before the calibration iscarried out. Information on theseparameters can be found in theMassLynx NT Guide to DataAcquisition. If centroided data areused for calibration then the massmeasure parameters are not used.

When calibrating in electrosprayusing samples that show low intensitypeaks at higher masses, it isrecommended that continuum orMCA data are acquired.


ZMDUser's Guide

Performing a Calibration

Three types of calibration are available with MassLynx: static calibration, scanningcalibration and scan speed compensation. These are selected on theAutomatic Calibration dialog box (see below) which is accessed by selectingStartfrom the calibrate dialog box.

It is recommended that all three types ofcalibration are performed so that any mode ofdata acquisition can be used and mass ranges andscan speeds can be changed whilst maintainingcorrect mass assignment. However, it is possibleto have any combination of these calibrations:

• If only a static calibration is present thenthe instrument is calibrated for acquisitionswhere the quadrupole is held at a singlemass as in SIR.

• If only a scanning calibration is presentthen the instrument is only correctlycalibrated for scanning acquisitions overthe same mass range and at the same scanspeed as those used for the calibration.

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

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

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

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


ZMDUser's Guide

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

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

For a complete calibration:

Check the boxes in theTypes area of the dialog box adjacent toStatic Calibration , Scanning Calibration andScan Speed Compensation.

In theProcess area of the dialog box checkAcquire & Calibrate andPrint Report .


ZMDUser's Guide

Acquisition Parameters

Selecting theAcquisition Parameters... button in the Automatic Calibration dialogbox brings forward a second box, shown below, where the mass ranges, scan speedsand acquisition mode are set. When this box is first accessed it will contain defaultparameters relevant to the chosen reference file. These default parameters show thelimits of scan range and scan speed for the currently selected instrument andcalibration parameters.

The upper area contains theAcquisition Parameters wheremass range, run time and data typeare set.

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

If the pegnh4.ref file is selected, thedefault button will give theparameters appropriate to the massrange of this reference file. The PEGsolution described inReferenceInformation is suitable for use withthis reference file.

If compatible reference solutions and reference files are used, then simply selectingthe default button is sufficient action - no parameters need be entered manually. If notcompatible, input the mass range to be calibrated, after first considering the details inRun Durationbelow.

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

Data Type allows a choice of centroided, continuum or MCA data to be acquired.Continuum or MCA acquisitions are generally used for electrospray calibrations.When using continuum data with 8 channels per amu (see theMassLynx NT Guide toData Acquisition) the maximum acquisition speed is 1000 amu/sec.


ZMDUser's Guide

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

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

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

Slow Scan Time determines the scan speed used for the scanning calibration. If botha scanning calibration and a scan speed compensation are to be performed then thescan speed should be set to approximately 100 amu/sec. If only a scanning calibrationis to be performed (without scan speed compensation) then the scan speed should beset at the same speed to be used for later acquisitions.

Fast Scan Time determines the scan speed used for the scan speed compensation,and the upper limit of scan speed that can be used for subsequent acquisitions. Whenusing MCA or continuum data the scan speed is limited to 400 and 1000 amu/secrespectively. So, for a mass range of 100 to 1600 amu, the minimum values are 1.5seconds for thresholded continuum data and 2.5 seconds for MCA data.

When using centroided data the maximum acquisition rate is much higher, although itis unlikely that scan speeds of greater than 2000 amu/sec would be needed foracquiring data.


ZMDUser's Guide

Starting the Calibration Process

To start the calibration process:

SelectOK from the Automatic Calibration dialog box.

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

Static calibration data file: STAT

Scanning calibration data file: SCN

Scan speed compensation data file: FAST

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

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

If the calibration statistics do not meet the requirements then a message will bedisplayed describing at what point and why the calibration failed. This message alsostates where the attempted calibration data can be viewed so that the exact cause offailure can be determined.


ZMDUser's Guide


ZMDUser's Guide

___________________________________________________________________________________

Instrument Calibration Report. Page 1

Mass 83 Da to 1231 Da. Res=15.0/13.8 IE=1.0

Calibrated - 12:09 on 10/28/99___________________________________________________________________________________

100 200 300 400 500 600 700 800 900 1000 1100 1200M/z-1.54

-1.03

amu

Static Calibration 28 matches of 28 tested references. SD = 0.0345

100 200 300 400 500 600 700 800 900 1000 1100 1200M/z-1.67

-1.21

amu

Scanning Calibration 28 matches of 28 tested references. SD = 0.0454

100 200 300 400 500 600 700 800 900 1000 1100 1200M/z-1.38

-1.11

amu

Scan Speed Compensation Calibration 28 matches of 28 tested references. SD = 0.0381

Checking the Calibration

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

Clicking on theOK button repeats thecalibration procedure for that particular fileand display a calibration report on thescreen. This calibration report contains fourdisplays:

• the acquired spectrum• the reference spectrum• a plot of mass difference

against mass (the calibrationcurve)

• a plot of residual against mass

An expanded region can be displayed byclicking and dragging with the left mousebutton. In this way the less intense peaks inthe spectrum can be examined to check thatthe correct peaks have been matched. Thepeaks in the acquired spectrum which havebeen matched with a peak in the reference spectrum are highlighted in a differentcolour.


ZMDUser's Guide


ZMDUser's Guide

Calibration Failure

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

• No peaks. If the acquired calibration data file contains no peaks the calibrationwill fail. This may be due to:

Lack of reference compound.

No flow of solvent into the source.

Multiplier set too low.

• Too many consecutive peaks missed. If the number of consecutive peaks whichare not found exceeds theMissed Reference Peaks parameter set in theAutomatic Calibration Check, then the calibration will fail. Peaks may be missedfor the following reasons:

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

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

An incorrect ionisation mode is selected. Check that the data have beenacquired withIon Mode set toES+.

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

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

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

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

The wrong reference file has been selected. Check that the correct file(peg1200.ref in this case) is selected in the Calibrate dialog box.


ZMDUser's Guide

In the case of too many consecutive peaks missed:

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

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

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

Having taken the necessary action, proceed as follows:

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

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

SelectOK from the calibration report window to accept the new calibration, orselectCancel to retain the previous calibration.


ZMDUser's Guide

Incorrect Calibration

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

• Intensity threshold set to 0

• Initial error too high (>2.0)

• Peak window too high (>1.5)

• Maximum Std Deviation too high (>0.2).

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

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

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

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


ZMDUser's Guide

Manual Editing of Peak Matching

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

Using the mouse place the cursor over the peak in the acquired spectrum andclick with the right mouse button.

Select the peak in the matched spectrum using the mouse and click again withthe right mouse button.

The peak is excluded and is no longer highlighted.

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

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

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

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

Saving the Calibration

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

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


ZMDUser's Guide

Verification

Once a full instrument calibration is in place it isnot always necessary to repeat the full calibrationprocedure when the instrument is next used. Insteada calibration verification can be performed. (Thereis no benefit in verifying each calibrationindividually, re-calibration is just as quick.)

If a scanning acquisition is to be made and thecalibration is to be checked:

Set up the instrument and access the calibratedialog box as though a full calibration is to becarried out.

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

Bring up the Automatic Calibration dialog boxby selectingStart... on the Calibrate dialogbox.

SelectScanning Calibration and deselectStatic Calibration andScan Speed Compensation .

In Process , deselectAcquire & Calibrate and selectAcquire & Verify andPrint Report .

Select theAcquisition Parameters...button to call up the CalibrationAcquisition Setup dialog box.

SetScan From , Scan To ,Run Duration , Data Type ,Scan Time andInter Scan Dela y to agree withthe acquisition parameters that areto be used for data acquisition.

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

SelectOK to return to the previous dialog box andOK again to start theverification procedure.


ZMDUser's Guide

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

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


ZMDUser's Guide

Electrospray Calibration with PEG

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

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

A low Sample Cone voltage encourages the production of doubly charged ions. Thevoltage should be at least 30V.

Doubly charged peaks can be identified because the13C isotope peak is separated fromthe 12C isotope by only 0.5 Da/e. If the instrument is set to unit mass and data isacquired in continuum mode the doubly charged peaks will appear broader as theisotopes will not be resolved.


ZMDUser's Guide

Atmospheric Pressure Chemical Ionisation

Introduction

This chapter describes a complete mass calibration of ZMD using atmosphericpressure chemical ionisation. The procedures described should be followed only afterreading the previous chapter in this manual, describing the automated calibration withelectrospray ionisation.

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

The recommended reference compound for APcI is a solution of polyethylene glycol(PEG) containing ammonium acetate. SeeReference Informationfor advice onpreparing the reference solution. See the following illustration for a typicalPEG + NH4+ spectrum.

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


ZMDUser's Guide

Preparing for Calibration

Reference Compound Introduction

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

Tuning

Before beginning calibration:

SetMultiplier to 650V.

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

SetSample Cone in the region of 30-35V so that some fragmentation occursto give some of the lower mass peaks in the spectrum (m 89 and 133).

Set the resolution and ion energy parameters for unit mass resolution.

When a full calibration is completed it is possible to acquire data over any massrange within the calibrated range. It is therefore sensible to calibrate over awide mass range and in this example the calibration will cover up to 1000 amu.

Calibration Options

To access the calibration options:

SelectCalibrate Instrument from the tune page.

Selecting Reference File

Setpegnh4.ref as the reference file by clicking on the arrow in the referencefile box and scrolling through the files until the appropriate file can be selected.

Leave theUse Air Refs box blank when calibrating in APcI.

Removing Current Calibrations

SelectYes to the query:Save changes to C:\Masslynx\Default.pro\AcquDB\Default.cal?

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


ZMDUser's Guide

Selecting Calibration Parameters

A number of parameters needs to be set before a calibration is started. Most of theseparameters can be set at the same value as for electrospray. However, aPolynomial order of 4 is recommended for the calibrationCurve Fit .

Performing a Calibration

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

Static Calibration

Access the Automatic Calibration dialog box by selectingCalibrate , Startfrom the Calibrate page.

CheckStatic Calibration in theTypes area of the dialog box.

In theProcess area of the dialog box, checkAcquire & Calibrate .

Acquisition Parameters

Selecting theAcquisition Parameters... button brings forward the default massranges, scan speeds and acquisition mode relevant to the pegnh4.ref reference file.

The upper area contains theAcquisition Parameters where mass range, run timeand data type are set. When the instrument is fully calibrated any mass range or scanspeed is allowed within the upper and lower limits dictated by the calibrations. It istherefore sensible to calibrate over a wide mass range. Since the pegnh4.ref referencefile has peaks fromm 89 tom 1004, it is possible to calibrate over this full massrange which is sufficient for the majority of applications with APcI.

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

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


ZMDUser's Guide

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

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

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

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

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

SelectOK from the Calibration Acquisition Setup to return to the AutomaticCalibration dialog box.


ZMDUser's Guide

Acquiring Data

To start the acquisition:

SelectOK from the Automatic Calibration dialog box.

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

Inject the reference solution.

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

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


ZMDUser's Guide

Manual Calibration

To perform a manual calibration using the acquired data:

From the chromatogram window call up the calibration fileSTAT.

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

This can be achieved using Process, Combine Spectra and using the left mousebutton to drag across the peak. The start and end scans will be displayed in thecombine spectra dialog box.

Return to the Calibrate dialog box. Accessthe manual calibration options, as shown, byselectingCalibrate From File....

SelectStatic calibration type.

In the lower area the data file STAT shouldbe selected automatically. If this is not thecase the correct file can be selected byclicking on the Browse... button.

Enter the start and end scans of thereference data in theFrom andTo boxes.

SelectOK to perform the calibration anddisplay the calibration report on the screen(opposite upper).

This report contains four displays:

• the acquired spectrum• the reference spectrum• a plot of mass difference against mass (the calibration curve)• a plot of residual against mass.

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

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

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


ZMDUser's Guide


ZMDUser's Guide

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

SelectPrint , Print from the report display.

To accept the calibration:

SelectOK from the calibration report.


ZMDUser's Guide

Scanning Calibration and Scan Speed Compensation

Acquiring Data

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

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

SetScan From to 80 amu andScan To to 1000 amu.

SetScan Time to 9.2 sec andInter Scan Delay to 0.1 sec.

SelectContinuum as theData Type .

Although continuum is recommended centroided data may be used.

SetRun Duration to 2.0 minutes.

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

Choose any filename for the data.

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

Start the acquisition and inject the reference solution.


ZMDUser's Guide

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

Although continuum is recommended centroided data may be used. It is possibleto scan more quickly in centroided mode, but it is unlikely that a fasteracquisition rate would be needed for general use.

SetScan From to 80 amu andScan To to 1000 amu.

SetScan Time to 0.92 sec andInter Scan Delay to 0.1 sec.

SelectContinuum as theData Type .

SetRun Duration to 2.0 minutes.

Choose any filename for the data.

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

Start the acquisition and inject the reference solution.

Manual Calibration

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

From the calibration dialog box selectCalibrate From File... .

SelectScanning calibration type.

In the lower area the data filename SCN should be selected automatically. If thisis not the case, or if an alternative filename has been used for the slow scanningacquisition, then the correct file can be selected by clicking on theBrowse...button.

Enter the start and end scans of the reference data in theFrom andTo boxes.

Select theOK button to perform the calibration and display the calibration reporton the screen in a similar way to the static calibration.

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


ZMDUser's Guide

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

SelectPrint from the report display.

If insufficient peaks have been matched or the wrong peaks have been matched seeCalibration Failure later in this chapter. To accept the calibration:

SelectOK from the calibration report.

The same procedure is used for the scan speed compensation except thatScan Speed Compensation is selected in the dialog box, and the fast scanning fileis used. Note that for the scan speed compensation the default file isFAST.RAW . Ifan alternative filename has been used then this must be selected using the databrowser.

Once all three calibrations (static, scanning and scan speed compensation) have beencompleted then the instrument can be used for any mass range within the limits of thescanning calibrations and at any scan speed from 100 to 1000 amu/sec.


ZMDUser's Guide

Calibration Failure

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

• Calibration acquisitions will not proceed if there are more than 32 referencepeaks in the selected reference file.

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

Lack of reference compound.

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

No flow of solvent into the source.

Multiplier set too low.

• Too many consecutive peaks missed. If the number of consecutive peaks whichare not found exceeds the limit set in the Automatic Calibration Checkparameters then the calibration has failed. Peaks may be missed for thefollowing reasons:

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

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

The incorrect ionisation mode is selected. Check that the data has beenacquired withIon Mode set toAPcI+ .

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

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

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

The wrong reference file has been selected. Check that the correct file(pegnh4.ref in this case) is selected in the Calibrate dialog box.


ZMDUser's Guide

In the case of too many consecutive peaks missed:

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

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

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

Having taken the necessary action, proceed as follows:

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

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

SelectOK from the calibration report window to accept the new calibration, orselectCancel to retain the previous calibration.

Incorrect Calibration

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

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

• Intensity threshold set to 0

• Initial error too high (>2.0)

• Peak window too high (>1.5)

• Maximum Std Deviation too high (>0.2).

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


ZMDUser's Guide

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

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

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

Manual Editing of Peak Matching

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

Using the mouse place the cursor over the peak in the acquired spectrum andclick with the right mouse button.

Select the peak in the matched spectrum using the mouse and click again withthe right mouse button.

The peak is excluded and is no longer highlighted.

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

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

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

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

Saving the Calibration

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

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


ZMDUser's Guide

Manual Verification

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

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

Set up a scanning acquisition over therequired mass range and at therequired scan speed in the normalway.

Start the acquisition and inject thereference solution so that referencedata is acquired.

Stop the acquisition.

Access the calibrate dialog box andset all peak matching parameters tothe same values that were used for thecalibration.

SelectProcess ,Verification from file... and checkScanning Calibration .

Click on Browse... , select the acquired file and enter the start and end scans ofthe reference data.

SelectOK to verify the calibration.


ZMDUser's Guide

A calibration curve will be produced and displayed on the screen in a similar way towhen the original calibration was performed. WhenOK is selected from this report,unlike the original calibration procedure, the instrument calibration is not changed. Asthe verification procedure uses the same matching parameters as the calibrationprocedure, it is possible to validate the current calibration without re-calibrating theinstrument.

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


ZMDUser'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, as defined by the substance's manufacturers.

Warning: There are high voltages and hot surfaces present within thesource. Always switch out of operate, disconnect the source plug and allowthe source to cool before handling source components.

Cooling Fans and FiltersAlways ensure that none of the cooling fans are obstructed. It is essential that the fanfilters are checked at regular intervals and cleaned if there is any doubt about theireffectiveness.

Maintenance and Fault FindingPage 119

ZMDUser's Guide

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

• An analyser pressure above 10-4mbar results in a general loss in performanceindicated by a loss of resolution and an increase in the background noise.

• Above 10-3mbar the instrument trips into standby, with theVacuum LED on theinstrument changing from green to amber, indicating that the vacuum isinsufficient to maintain the instrument in operate.

• Above 10-2mbar theVent LED changes to flashing amber, indicating that thevacuum pump trips have been activated, followed by a steady amber indicationwhen the instrument is no longer pumping.

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 has not been maintained for some time, the oil may havebecome sufficiently contaminated that optimum pumping speed is no longerpossible. Initially, gas ballasting the rotary pump may clean the oil. If the oil inthe rotary pump has become discoloured then it should be changed.

• The turbomolecular pumps will switch off if an over temperature is detected.This could be due to poor backing vacuum (see above), failure of the watersupply or a leak in the source or analyser.

• The turbomolecular pumps will switch off if they are unable to reach full speedwithin a set time following start-up. This fault could be caused by a leak orfailure of the water supply.

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.

• Undisturbed vacuum flanges rarely start leaking. Suspect flanges that have beenrecently disturbed.

• All seals are made using O rings. When refitting flanges pay attention to thecleanliness 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 issufficient to prevent the instrument pumping down.

Leaks on flanges can usually be cured by further tightening of the flange bolts or byreplacing the seal. In the unlikely event of a leak on a feedthrough, then the unitshould be returned to Waters Corporation for repair.


ZMDUser's Guide

Gas Ballasting

When rotary pumps are used topump away large quantities ofvapours, the solvent or gas candissolve in the vacuum oilcausing an increase in thebacking line pressure. Gasballasting is a method used topurge the oil of thecontaminants, by introducing airinto the low vacuum stage ofthe pump.

Gas ballasting should be carriedout:

• Routinely, once a weekfor approximately 30 minutes.

• With frequent APcI operation, once a day for approximately 15 minutes.

• Whenever the backing pressure is high.

• Following an oil change. Normally 30 minutes are sufficient.

The presence of condensation in the rotary pump exhaust line is an indicationthat gas ballasting is necessary.

Gas ballasting is performed as follows:

Turn the gas ballast control, on top of the pump, six turns anti-clockwise to openit fully.

Caution: The gas ballast control should not be left in the open positionroutinely. To do so leads to an increased rate of oil loss from the pump.

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. Do not vent the instrument while ballast valves are open.


ZMDUser's Guide

GasBallast

DrainPlug

Exhaust

FillerPlug

Oil LevelIndicator

Oil MistFilter

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 proceed as follows:

Vent the instrument and switch off the power to the rotary pump.

Remove the drain plug, drain the oil from the filter and refit the drain plug.

Disconnect the exhaust tubing to the filter.

Remove the four screws that hold the upper and lower parts of the filtertogether. Lift out the used filter element and dispose of it safely.

Fit a new element. Ensure that the foam sealing rings at the top and bottom ofthe element are seated correctly.

Refit the upper and lower parts of the filter. Reconnect the exhaust tubing.

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 also important regularly to monitor the condition of the oil, which must bechanged as soon as it becomes noticeably discoloured. This would typically be atintervals of 2-6 months. The oil in the rotary pump should be changed as follows:

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.

Remove the oil filler plug and place a suitable container under the drain plug.

Allow the oil to drain into the container.

Refit the drain plug and refill the pump with clean oil until the oil level reachestheMAX level on the bezel of the sight glass.

Gas ballast the rotary pump for 30 minutes as described above.

Pirani Gauge

The Pirani gauge head does not require routine maintenance.


ZMDUser'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 source enclosure.

• The source flange assembly.

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

• Removing the cone gas nozzle and sample cone.

• Fitting or removing the APcI discharge pin.

• Fitting or removing the exhaust liner and cleanable baffle.

• Fitting or removing the nanoflow electrospray interface.

• Enabling or disabling the purge gas.

Cleaning of the sample cone and cone gas nozzle may be achieved by removing themfrom the source. This may also be done without venting the instrument, by closing theisolation valve located on the ion block. Less frequently it may be necessary to cleanthe ion block, the extraction cone and the RF lens, in which case the instrument mustbe vented. This should only be done when the problem is not rectified by cleaning thesample cone and cone gas nozzle, or when charging effects are apparent.

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

The RF lens should not require frequent cleaning. If it is suspected that the lens doesneed cleaning it may be withdrawn from the front of the instrument after removing theion block support.

Warning: Cleaning the various parts of the source requires the use ofsolvents and chemicals which may be flammable and hazardous tohealth. Personnel performing these operations should be aware ofthe inherent risks and should take the necessary precautions, asdefined by the manufacturers of the substances.

Warning: There are high voltages and hot surfaces present within thesource. Always switch out of operate, disconnect the source plug and allowthe source to cool before handling source components.


ZMDUser's Guide

Cleaning the Cone Gas Nozzle and Sample Cone

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

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

Check that the Operate button is grey, indicating that the instrument is instandby.


Disconnect the liquid flow at the rear of the probe.

SetSource Temp and eitherAPcI Probe Temp or Desolvation Temp to20°C to switch off the heaters.

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

Caution: Removal of the APcI probe when hot 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.

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

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.


ZMDUser's Guide

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 has cooled before proceeding.

If fitted, remove the APcI discharge pin.

The sample cone and cone gas nozzle are now accessible.


ZMDUser's Guide

SourceThumb Nuts

ProbeThumb Nuts


SourceEnclosure

Using a suitable flat blade screwdriver rotate the isolation valve by 90° into itsfully anticlockwise position.

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

The isolation valve is closed when the slot is parallel with the instrument's frontto rear axis.

Disconnect the cone gas inlet line.

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

Remove the two sample cone retaining screws and withdraw the sample cone,gasket and cone gas nozzle from the ion block.

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

Remove the extraction tool, and separate the sample cone, the gasket and thecone gas nozzle.

Carefully wipe the sample cone and cone gas nozzle with a cotton swab or lintfree tissue soaked in 50:50 acetonitrile : water or 50:50 methanol : water.

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


ZMDUser's Guide

Cone GasNozzle

IsolationValve

If the components are still not clean, or if the aperture is partially blocked, placethe components in an ultrasonic bath containing 50:50 acetonitrile : water or50:50 methanol : water.

Warning: Cleaning the various parts of the source requires the use ofsolvents and chemicals which may be flammable and hazardous tohealth. Personnel performing these operations should be aware ofthe inherent risks and should take the necessary precautions, asdefined by the manufacturers of the substances.

To minimise down time fit a spare sample cone and cone gas nozzle, obtainablefrom Waters Corporation.


ZMDUser's Guide

SampleCone Cone Gas

Nozzle

ExhaustLiner

Cone GasInlet Line

ExtractionTool

CleanableBaffle

Gasket

Dry the cone and nozzle using nitrogen.

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

Remove the cleanable baffle and the exhaust liner.

Clean these components, or obtain replacements.

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

Refitting the sample cone and cone gas nozzle is a reversal of the removal procedure.


ZMDUser's Guide

Removing and Cleaning the Ion 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 valve opens toatmosphere automatically.

Warning: The heater supply remains live until the system is fully vented.Also, the source surfaces may be hot. Do not proceed until the system hasvented and the source has cooled.

When the instrument has vented and cooled:

Remove the source enclosure, sample cone and cone gas nozzle as described inthe previous section.

Remove the four screws, together with the washers, which secure the cover plateto the ion block and remove the cover plate.


ZMDUser's Guide

IonBlock

CoverPlate

O Ring

Ensure that the O ring remains in position on the ion block.

Remove the two screws from the heater connections on the ion block andwithdraw the leads.

Using a pair of needle nose pliers, carefully straighten the heater supply leads insuch a way that the ion block can later be withdrawn without fouling these leads.

Loosen the screw on the thermocouple’s securing clip and unhook thethermocouple from its location.


ZMDUser's Guide

View from Rear

Ion BlockSupport

O Rings

PlugPTFE

Washer

IonBlock

ExtractionCone

Insulator

IonBlock

Heater SupplyLeads

HeaterLeads

HeaterGrub Screws

Thermocouple

ExtractionCone

Insulator

Remove the two screws which secure the ion block to the ion block support.

Withdraw the ion block sufficiently to allow the extraction cone to bedisconnected from its supply lead.

Remove the ion block, leaving the extraction cone lead, thermocouple and heatersupply leads protruding from the ion block support.

Ensure that the three O rings remain in position on the ion block support.

Unscrew the plug (located on the ion block opposite the sample cone) andcollect the PTFE washer.

Remove the extraction cone and insulator from the ion block.

Loosen the two heater grub screws and withdraw the heater cartridges from theion block.

Clean the extraction cone in concentrated formic acid.

Leaving the isolation valve, thermocouple clip and terminal block in place,immerse the ion block and the extraction cone in an ultrasonic bath containing50:50 acetonitrile : water or 50:50 methanol : water, followed by 100%methanol.

Dry all components using a flow of nitrogen.


ZMDUser's Guide

Removing and Cleaning the RF Lens Assembly

To remove the RF lens assembly, proceed as follows:

Remove the ion block, as described above.

Remove the three screws retaining the ion block support and carefully withdrawit from the pumping block.

Ensure that the three O rings remain in position on the rear face of the support.

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

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


ZMDUser's Guide

O Rings

View from Rear

RFLens

Ion BlockSupport

To clean the RF 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.


ZMDUser's Guide

LocationRecess

DifferentialAperture

PlateRod LocatingScrews & Washers

Reassembling and Checking the Source

Check the condition of all O rings. Replace them if necessary.

Feed the RF lens into the instrument, allowing the recesses in the differentialaperture plate to locate onto the two support rails within the analyser assembly.Ensure that the assembly is pushed fully in.

Pass the ion block support over the heater, thermocouple and extraction coneleads. Locate the ion block support, pushing it in against the springs of the RFlens assembly.

Replace the three retaining screws.

Replace the plug, complete with PTFE washer, on the ion block.

Refit the extraction cone into the recess on the ion block support, with the conepointing towards the ion block.


ZMDUser's Guide

Springs

Connect the extraction cone lead to the connector on the extraction cone.

Locate the peek insulator and O ring onto the rear face of the ion block.

Fit the ion block to the ion block support, using the dowels for alignment.Secure with the two screws.

Insert the thermocouple into its location, and secure it with the clip ensuring thatit cannot readily be prised off.

Reconnect the heater leads and heater supply leads to the terminal block.

When assembling the ion block heater and the heater supply leads, ensure thatthe ring tags are flush with each other before fitting the fixing screws.

Replace the cover plate.

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

Replace the sample cone, gasket and cone gas nozzle on the ion block.

Reconnect the cone gas supply.

Fit the APcI corona discharge pin or blanking plug, as necessary.

Fit the source enclosure and the probe adjustment flange.

On the tune page selectOther and click onPump .

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.


ZMDUser'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.

Warning: To avoid the risk of electric shock, the injector or LC column towhich the probe is attached must be grounded. Switch out of operate beforeremoving the probe. Isolate the probe before removing its cover.

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.


ZMDUser's Guide

• High LC pump back pressure.

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

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

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

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

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

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

• Gas flow problems

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


ZMDUser'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 from 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.

Using a piece of conductive elastomer and the coupling, connect the samplecapillary to the LC union, ensuring that the sample capillary is fully butted intothe LC union.


ZMDUser's Guide

Stainless SteelCapillary

Fused SilicaCapillary

LCUnion

Finger-tightNut & Ferrule

RheodyneNut & Ferrule

GrubScrew

EndCover

ProbeTip

Coupling

LinerTube

LinerTube

LinerTube

ConductiveSleeve

PSO16GVFFerrule

O Ring0.6mm

Disconnect the LC connection and feed the sample capillary through the probe.

Replace the probe tip and adjust so that 0.6mm of sample capillary protrudesfrom the probe tip.

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


ZMDUser'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.

• 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 to turn on the nitrogen.

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

SelectGas to turn off the nitrogen.

Insert the probe into the source.

SelectGas to turn on the nitrogen.


ZMDUser's Guide

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.


ZMDUser's Guide

Slotted 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 coupling.

Unscrew the coupling 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 coupling, connect the capillary to the filterensuring that the capillary passes through the ferrule but stops short of the filter.


ZMDUser's Guide

0.5 to1mm

PTFETube

GrubScrew

GrubScrew

Fused SilicaCapillary

Finger-tightNut & Ferrule

RheodyneNut & Ferrule

GVF/004Ferrule

O Ring

Coupling

Filter

Feed the sample capillary through the probe, ensuring that the O ring is fitted,and gently tighten the coupling.

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 coupling 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 the O ringis still fitted.

Gently tighten the coupling to the probe.

Replace the probe end cover and retaining screws.

Tighten the grub screws in the probe end cover to clamp the filter.

Replace the heater and probe tip assembly.

The AnalyserZMD is fitted with a pre-filter assembly that is designed to protect the main analyserby absorbing contamination from the ion beam from the source. As a consequence, theanalyser quadrupole should never, under normal working conditions, require cleaning.

The quadrupole assembly of ZMD is a finely machined and aligned assembly whichunder no circumstancesshould be dismantled.


ZMDUser's Guide

The DetectorThe ZMD detector system has been designed for trouble-free operation over manyyears. The photomultiplier is encapsulated in its own vacuum envelope and istherefore safe from contamination and pressure surges. The conversion dynode andphosphor are also long lasting. No routine maintenance is required.

Users should not attempt any repairs to the ZMD detector. This task should only becarried out by a Waters Corporation service engineer.

ElectronicsThe ZMD electronics do not require routine maintenance. In the event of suspectedproblems with the instrument's electronics, contact the Waters Corporation servicedesk. Repairs to instrument electronics must only be undertaken by Waters personnel.

Warning: There are high voltages present throughout the mass spectrometer.Users should not attempt any repairs to the ZMD electronics. This task shouldonly be carried out by Waters Corporation personnel.

Caution: ZMD’s electronic systems contain complex and extremely sensitivecomponents. Any fault finding procedures should be carried out only by WatersCorporation personnel observing the most stringent precautions againstelectrostatic discharge.


ZMDUser's Guide

Fault Finding Check List

No Beam

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

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

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

• Operate is on (check the LED on the front panel).

• The source has been assembled correctly and is clean.

• The isolation valve is open.

Unsteady or Low Intensity Beam

Should the preceding checks fail to reveal the cause of the problem check that:

• Gas and liquid flows are normal.

• The analyser pressure is less than 1x10-4 mbar.

Ripple

Peaks appear to vary cyclically in intensity when there is ripple superimposed on thepeak. Possible causes are:

• Unstable power supplies in the source supplies or the RF/DC generator.

• Unstable photomultiplier supply.

• Vibration from the rotary pumps or from other equipment in the same building.

Contact Waters Corporation for advice.


ZMDUser'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 re-dissolve 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 fused silica tubing with new tube(or first try removing both ends of the tube).

If the back pressure falls, replace the stainless steel sample tube inside the probe(or try reversing the tube to blow out any blockage).

Reconnect the tubing to the probe.

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

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

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

For APcI a higher than normal back pressure readout on the HPLC pump can implythat, after a long period of use, the fused silica tubing inside the probe requiresreplacement.

General Loss of Performance

Should the preceding checks fail to reveal the source of the problem proceed asfollows:

Check that the source and probe voltage readbacks vary with tune page settings.

If any of these voltages are absent check that the source and RF lens assemblyhave been correctly reassembled.

Further investigation, which will require the services of a qualified serviceengineer, should be entrusted to Waters Corporation personnel.


ZMDUser's Guide

Cleaning MaterialsWarning: 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, as defined by the manufacturers of the substances.

Caution: Use only HPLC grade solvents for cleaning

It 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.

• 3 micron lapping paper.

• Tungsten wool

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.


ZMDUser'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.

✖ Do not gas ballast the rotary pump during ESI or APcI operation.

For full details of the following procedures, consult the relevant sections of thischapter.

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.

Weekly

✔ Check the rotary pump oil level and colour.

Gas ballast lightly for 30 to 60 minutes both before and after topping up the oil.

✔ Check the water chiller level and temperature (if fitted).

Monthly

✔ Check all cooling fans and filters.

Four-Monthly

✔ Change the oil in the rotary pump after 3000 hours operation.

Gas ballast lightly for 30 to 60 minutes both before and after changing oil.


ZMDUser'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 149

ZMDUser's Guide

http://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


ZMDUser'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 File: PEGNH4.REF

Calculated/ Value

89.0603 459.2805 872.5430 1268.7790 1665.0149

133.0865 503.3068 916.5692 1312.8052 1709.0411

177.1127 564.3595 960.5955 1356.8314 1753.0673

221.1389 608.3857 1004.6217 1400.8576 1797.0935

239.1495 652.4120 1048.6479 1444.8838 1841.1197

283.1757 696.4382 1092.6741 1488.9100 1885.1460

327.2019 740.4644 1136.7003 1532.9362 1929.1722

371.2281 784.4906 1180.7265 1576.9625 1973.1984

415.2543 828.5168 1224.7527 1620.9887 2017.2246


ZMDUser'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


ZMDUser'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


ZMDUser'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


ZMDUser'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 PEGNH4.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 PEGNH4.REF.


ZMDUser'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.


ZMDUser's Guide

Index

AAcetonitrile 46

Adducts 74Acquisition 39Ambient temperature 15Ammonia 46Ammonium acetate 155Analog input 19, 22Analog out 23Analog PCB 28Analyser 143APcI 18, 36, 73, 103

Analysis 78Tuning 76

APcI probe 19, 80, 140Checking 75Fused silica capillary 142Maintenance 140Position 79Removal 80Temperature 78, 79, 140Tip heater 141

Apply span correction 85Atmospheric pressure chemical ionisation

See: APcIAutomatic pumping 40Autosampler 17, 19

BBack pressure

High 140, 146Low 140

Biopolymers 58

CCaesium iodide 58, 152, 154, 156Calibration 81

Electrospray 58Failure 96, 114Incorrect 98, 115Manual 108, 112Verification 100, 117

Camera 67Capillary 27, 54Charging 123Check acquisition calibration ranges 85Cleanable baffle 49, 65, 74, 123, 128Cleaning materials 147Cluster ions 56, 60Column

4.6mm LC 48, 60, 73Capillary LC 60Microbore (2.1mm) LC 48, 60

Cone gas 53, 65, 78, 79Cone gas nozzle 65Contact closure 23Cooling fan 119Corona 78Corona pin 27, 36, 73, 123, 135Coupled column chromatography 61

DData system 13, 15, 19Data type 90, 105Desolvation gas 25, 27, 52, 78, 79Desolvation temp 52Detector 144Digital PCB 28Dimensions 13Discharge pin

See: Corona pinDivert valve 19, 26Drugs 59Dye compounds 59

IndexPage 157

ZMDUser's Guide

EElectronics 144Electrospray 18, 34, 45, 81

Analysis 58Negative ion 59Operation 49Positive ion 59

Electrospray probe 19, 50, 51Maintenance 136Removal 57

Eluent 18Emergency 42Environment 15Environmental contaminants 59Event out 23Exhaust 16, 21Exhaust liner 49, 65, 123, 128Extraction cone 55, 78

FFast scan time 91Faults 119, 145Filter 119Flow control valve 25Flow injection analysis 46, 63Formic acid 46Fragmentation 46

GGas ballast 121Glass capillary (nanoflow) 63, 68Gradient elution 60

HHeat dissipation 15Heater 27High mass generator control PCB 28High mass resolution 55HM Res

See: High mass resolutionHPLC 17Humidity 15

IInfusion pump 46Initial error 86Inject 26Injection valve 26, 46Injector valve 19Intensity threshold 86Ion energy 55Ion evaporation 18Ion mode 51Ion source

See: Source

LLayout 28LC-MS interface 60Lifting 14LM Res

See: Low mass resolutionLoad 26Low mass resolution 55

MMains breaker 21Maintenance 119Mass calibration 81MassLynx 19Maximum std deviation 85Megaflow 48, 56, 60Metabolites 59Microscope 67Missed reference peaks 85, 96Myoglobin 58, 151, 153

NNanoflow electrospray 63, 123Nano-HPLC 63Nano-LC (nanoflow option) 70Narrow mass scanning 61Nebuliser gas 27, 52Nitrogen 16, 20, 49

OOil mist filter 122Oligonucleotides 45, 59Operate 38Operate LED 25Organometallics 59

IndexPage 158

ZMDUser's Guide

PPC link 21, 31Peak match 86Peak matching 99, 116Peak window 86PEG

See: Polyethylene glycolPeptides 45, 59Pesticides 59Phase system switching 61Phosphate 58Pirani gauge 18, 122Pollutants 59Polyethylene glycol 58, 81, 102, 103, 151,155Polynomial order 86Polysaccharides 59Power 15

Failure 41Processing 39Proteins 45, 59Proton abstraction 18Proton transfer 18Pump fault 41Pumping 33Purge gas 53, 123

QQuadrupole 17

RReciprocating pump 46, 60Reference compound 82, 104, 149Reference file 84Reverse phase 60, 73RF lens 73, 78, 123, 132, 134, 146Ripple 145Rotary pump 13, 16, 21, 122

Oil 122Rubidium iodide 58, 152, 154, 156Run duration 90, 105

SSaccharides 59Sample cone 54, 78, 124Scan speed compensation 88, 111Scanning calibration 88, 111Sensitivity

LC-MS 61Shutdown 42Single ion recording 61Slow scan time 91Sodium iodide 58, 152, 154, 156Source 123, 134

Voltages 39Source temperature 54, 78Span correction 85Split, post-column 47, 60Start up 31Static calibration 88Static dwell 91, 106Static span 91, 106Sugar mixture 58, 153Syringe pump 46, 60

TTarget compound analysis 61TEA

See: TriethylamineTetrahydrofuran 60THF

See: TetrahydrofuranThreshold 83Trace enrichment 61Transformer 13Triethylamine 60Trifluoroacetic acid 60Tuning 104

APcI 76Electrospray 82

Turbomolecular pump 18

IndexPage 159

ZMDUser's Guide

UUser I/O 22UV detector 47, 60

VVacuum 18, 120

Leak 41, 120Protection 40

Vacuum LED 24, 25, 33Vent LED 24, 25

WWaste 21Water 15, 20Water cooling 15Weights 13

IndexPage 160

ZMDUser's Guide

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