OptiMS Waters MS Adapter
For NanoLockSpray and NanoFlow Ion Sources
Installation Guide

RUO-IDV-05-3315-B | 5302402

December 2023

This document is provided to customers who have purchased SCIEX equipment to use in the operation ofsuch SCIEX equipment. This document is copyright protected and any reproduction of this document or
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Contents

1 Overview.................................................................................................................................... 5
Bill of Materials..........................................................................................................................5
Regulatory Compliance.............................................................................................................5International Standards........................................................................................................ 5
Decommissioning and Disposal................................................................................................ 6
Hazard Symbols........................................................................................................................6
Occupational Health and Safety Symbols............................................................................6
Symbols, Indicators, and Labels.......................................................................................... 7
Documentation Symbols and Conventions.......................................................................... 7
2 Install the Adapter.....................................................................................................................9
Remove the Baffle and the Lockspray Capillary (NanoLockSpray Ion Sources Only)..............9
Install the Adapter................................................................................................................... 10
Connect the Cables.................................................................................................................14
3 Align the System with the Mass Spectrometer.................................................................... 18
4 Prepare the Reagents............................................................................................................. 22
Prepare the 10% HAc Solution............................................................................................... 22
Prepare the 20% HAc Solution............................................................................................... 22
Prepare the 200 mM LE Buffer, pH 4.0................................................................................... 23
Prepare the Beta-Galactosidase Stock Solution..................................................................... 23
Prepare the Beta-Galactosidase Test Sample........................................................................ 24
Prepare the Peptide Test Sample........................................................................................... 24
5 Prepare the Buffer Trays........................................................................................................ 25
6 Prepare the System................................................................................................................ 26
Install the Cartridge................................................................................................................. 26
Remove and Install the Interface Plate.............................................................................. 31
Condition the Capillaries......................................................................................................... 33
Configure the Mass Spectrometer for Use with the CESI 8000 Plus System......................... 35
Create a Tune File.................................................................................................................. 42
Establish a Stable Spray......................................................................................................... 44
Optimize the ESI Voltage........................................................................................................ 47
Fine-Tune the Position of the Sprayer Tip...............................................................................50
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Contents

7 Do a Test Separation...............................................................................................................54
Create the MS Methods and Sample List............................................................................... 54
Create the MS Methods..................................................................................................... 54
Create the Sample List.......................................................................................................56
Manually Create the CESI-MS Separation Method.................................................................57
Load the Sample and Start the Run........................................................................................58
Analyze the Data.....................................................................................................................59
8 Shut Down and Disconnect................................................................................................... 66
Remove the Cartridge............................................................................................................. 66
Store the Cartridge..................................................................................................................70
Disconnect the CESI 8000 Plus System from the Mass Spectrometer...................................70

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Overview

1

Overview

This document includes instructions to install an adapter for the mass spectrometer, connect
the CESI 8000 Plus system to the mass spectrometer, and perform a a test separation to verify
correct system operation.
Note: For instructions about how to use the system safely, refer to the document: User Guide.

Bill of Materials
Part Number Description
B85211

Quantity
1

Adapter kit, containing:• Adapter• High-voltage cable• OptiMS Waters MS Adapter for NanoLockSpray andNanoFlow Ion Sources Installation Guide (this document)• Relay cable

B85396

Adapter stage

1

Regulatory ComplianceThis system complies with the regulations and standards listed in this section. For dated
references, refer to the declaration of conformity included with the system and the individual
system components. Applicable labels have been affixed to the system.

International Standards
The OptiMS Waters MS Adapter for NanoLockSpray and NanoFlow Ion Sources has been
evaluated as part of the CESI 8000 Plus High Performance Separation-ESI Module and foundto be in compliance with the International Electrotechnical Commission standard IEC 61010-1,
2nd edition: 2010 Safety requirements for electrical equipment for measurement, control, andlaboratory use - Part 1: General requirements.

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Overview

Decommissioning and Disposal
WARNING! Environmental Hazard. Obey established procedures for disposalof biohazardous, toxic, radioactive, and electronic waste. The customer is
responsible for the disposal of hazardous substances, including chemicals,
waste oils, and electrical components, in accordance with local laws andregulations.
Before decommissioning, obey local regulations to decontaminate the entire system.
When the system is removed from service, obey national and local environmental regulations todivide and recycle different materials.
Note: SCIEX will not accept any system returns without a completed Decontamination Form.
Contact an FSE to get a copy of the form.
Do not discard system components or subassemblies, including computer parts, as unsorted
municipal waste.

Hazard Symbols
This section lists the hazard symbols and conventions used in the laboratory environment, on
the system, and in the documentation.

Occupational Health and Safety Symbols
This section describes some occupational health and safety symbols found in the documentationand laboratory environment.
Table 1-1 General Hazard Symbols
Safety Symbol

Description
Personal Injury Hazard

Table 1-2 Chemical Hazard Symbols
Safety Symbol

Definition
Biohazard

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Overview

Table 1-2 Chemical Hazard Symbols (continued)
Safety Symbol

Definition
Explosion Hazard

Toxic Chemical Hazard

Fire Hazard

Table 1-3 Electrical Hazard Warning Symbols
Safety Symbol

Definition
Electrical Shock Hazard

Table 1-4 Mechanical Hazard Symbols
Safety Symbol

Definition
Hot Surface Hazard

Symbols, Indicators, and Labels
Note: If any of the labels used to identify a component become detached, then contact a SCIEX
service representative.

Documentation Symbols and Conventions
The following symbols and conventions are used throughout the guide.
DANGER! Danger identifies an action that can cause severe injury or death.

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Overview

WARNING! Warning identifies an action that can cause personal injury if
precautions are not obeyed.
CAUTION: Caution identifies an operation that can cause damage to the system or
corruption or loss of data if precautions are not obeyed.
Note: Notes supply important information in a procedure or description.
Tip! Tips supply information that helps to apply the techniques in a procedure or gives ashortcut, but that is not essential to the completion of a procedure.

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Install the Adapter

2

Install the Adapter

Remove the Baffle and the Lockspray Capillary(NanoLockSpray Ion Sources Only)
Note: This procedure does not apply to mass spectrometers with a NanoFlow ion source.
Required Materials• 2 mm hex key

1.

Use the manufacturer's instructions to remove the ion source from the mass spectrometer.
Put the ion source on the lab bench.

2.

Remove the baffle and the lockspray capillary from the ion source.
a.

Turn the ion source so that the back of the ion source points forward.

b.

Use a 2 mm hex key to loosen the set screw on the baffle sufficiently to remove thebaffle. Put the baffle in a safe place.
Figure 2-1 Set Screw for the Ion Source Bafflec.

Loosen the fitting at the top of the lockspray capillary to remove it. Put the capillary in asafe place.

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Install the Adapter
Figure 2-2 Fitting for the Lockspray Capillary

3.

Install the ion source on the mass spectrometer.

4.

Connect the top two Fischer connections (green and blue).

Install the Adapter
Remove the existing stage from the ion source and install the adapter stage provided in theadapter kit.
Required Materials• Adapter stage• Adapter• 1/16 inch hex key

1.

Remove the ion source stage from the ion source.

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Install the Adapter
Figure 2-3 Ion Source Stage Installed on the Ion Source

Item

Description

1

High-voltage port

2

Probe cover3

Retaining screw

4

End stopa.

Disconnect the high-voltage cable from the high-voltage port (Figure 2-3, item 1) on themass spectrometer.

b.

Remove the probe cover (Figure 2-3, item 2) and put it in a safe place.

c.

Use a 1/16 inch hex key to loosen the set screws on the end stop only far enough sothat the end stop can be removed.

d.

Remove the end stop (Figure 2-3, item 4) and put it in a safe place.

e.

Loosen the retaining screw (Figure 2-3, item 3).

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Install the Adapterf.

Pull the stop screw out (circled in Figure 2-4) and continue to hold it out, then pull theion source stage away from the ion source and remove the stage.

Figure 2-4 Ion Source Stage Removed from the Rails

2.

Use the thumbscrews to adjust the position of the adapter stage. Refer to the figure: Figure
2-5.
a.

Use thumbscrew 1 to move the vertical position to 4.5.

b.

Use thumbscrew 3 to move the horizontal position to 13.5.

c.

Use thumbscrew 5 to move the forward and backward position to 0, so that the adapter
stage is as far back as possible.

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Install the Adapter
Figure 2-5 Adapter Stage

Item

3.

4.

Description

1

Thumbscrew for vertical position

2

Adapter bracket

3

Thumbscrew for horizontal position

4

Retaining screw

5

Thumbscrew for forward and backward position

Install the adapter stage on the ion source.
a.

Install the adapter stage on the rails.

b.

Pull the stop screw out (circled in Figure 2-4) and continue to hold it out, then push theadapter stage to the first detent position.

c.

Install the end stop on the rails.

d.

Use the 1/16 inch hex key to tighten the set screws. Do not overtighten the set screws.

Turn the adapter so that the lock icons are on top, and then push the adapter into thebracket until the adapter moves fully into position with a snap.

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Install the Adapter
Figure 2-6 Adapter in the Bracket

5.

Pull the stop screw out (circled in Figure 2-4) and continue to hold it out, then push theadapter stage toward the mass spectrometer until the adapter stage locks in position.

6.

Tighten the retaining screw (Figure 2-5, item 4).

Connect the Cables
1.

Put the CESI 8000 Plus system approximately 18 cm to 20 cm (7.1 inches to 7.9 inches)away from the ion source.

2.

Connect the single connector on the end of the relay cable (Figure 2-8, item 2) to the relay
port on the CESI 8000 Plus system (labeled I/O in the following figure). Make sure that theconnector is as far to the right as possible.

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Install the Adapter
Figure 2-7 Relay Cable Connection

3.

If required, then remove the green cap from the External Connections 1 port on the backof the mass spectrometer. Connect the other end of the relay cable (Figure 2-8, item 2) tothe port.
WARNING! Personal Injury Hazard. Route the relay cable where users cannot
trip on it.
Figure 2-8 Relay Cable

Item

4.

Description

1

To External Connections 1 port on mass spectrometer2

To CESI 8000 Plus system

Connect the input cable between the mass spectrometer and the CESI 8000 Plus system.
a.

Connect the high-voltage input cable to the port on the mass spectrometer, to the left of
the ion source.

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Install the Adapter
Figure 2-9 High-Voltage Cable Connectedb.

On the right side panel of the CESI 8000 Plus system, open the door to the high-voltage
connections panel (Figure 2-10).

c.

Connect the white high-voltage cable to the Input port on the connections panel, andthen turn the cable clockwise to lock it in position.

d.

Connect the red banana plug to the red port on the connections panel.

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Install the Adapter
Figure 2-10 High-Voltage Connections Panel

Item

5.

Description

1

Input (red) port on connections panel

2

Red banana plug3

Output (black) port on connections panel

4Black banana plug

5

High-voltage input cable from mass spectrometer

6

High-voltage output cable to adapter

Connect the high-voltage output cable (attached to the adapter) to the CESI 8000 Plus
system.
a.

Connect the white high-voltage cable to the Output port on the connections panel, andthen turn the cable clockwise to lock it in position.

b.

Connect the black banana plug to the black port on the connections panel.

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Align the System with the Mass Spectrometer

Align the System with the Mass
Spectrometer3

To prevent hydrodynamic (siphoning) flow in the capillaries and to let the coolant drain from the
cartridge before the cartridge assembly is removed, align the sprayer tip installed in the mass
spectrometer adapter with the height indicator on the CESI 8000 Plus system.
CAUTION: Potential System Damage. Move the adapter mounting stage as far away as
possible from the inlet on the mass spectrometer. If the sprayer is too close to the inlet,
then damage to the sprayer can occur.
1.

Make sure that the CESI 8000 Plus system is approximately 18 cm to 20 cm (7.1 inches to
7.9 inches) away from the ion source so that the coolant tubes can be extended fully.

2.

To adjust the height of the cart, press the buttons on the front of the cart until the indicatorline on the CESI 8000 Plus system is aligned with the inlet on the mass spectrometer.
WARNING! Personal Injury Hazard. Remove consumables from the mobile cart
before the height is adjusted.
Note: Do not press both the Up and Down buttons at the same time.

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Align the System with the Mass Spectrometer
Figure 3-1 Alignment of the CESI 8000 Plus System and the Ion Source Inlet

Note: If the cart cannot move to its lowest height or if it becomes locked in position, then the
cart must be programmed again. To set the home position for the mobile cart, refer to the
document: CESI 8000 Plus High Performance Separation-ESI Module User Guide.
3.

When the CESI 8000 Plus system is in visual alignment with the mass spectrometer, stepon the locking lever of each caster to lock each wheel of the mobile cart in position.

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Align the System with the Mass Spectrometer
Figure 3-2 Lock the Wheels of the Mobile Cart

4.

Install the sprayer tip in the adapter.
CAUTION: Potential System Damage. Do not move the sprayer tip too close to theinlet on the mass spectrometer. If the sprayer tip is < 2 mm from the inlet, then theaccidental suction of rinse solution into the mass spectrometer can occur and cause
damage.
a.

Remove the protective sleeve from the sprayer tip.
Figure 3-3 Protective Sleeve and Sprayer Tipb.

While holding the body of the adapter to prevent it from moving towards the inlet, install
the sprayer in the adapter, aligning the arrow on the cartridge with the unlock positionon the adapter.

c.

Push the sprayer into the adapter and turn it counterclockwise to lock it in position. The
triangle on the sprayer should line up with the lock icon on the adapter.

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Align the System with the Mass Spectrometer
Figure 3-4 Sprayer in the Locked Position in the Adapter

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Prepare the Reagents

4

Prepare the ReagentsThe following reagents are used with the silica surface OptiMS cartridge. The sample typecontrols which reagents are required to do a separation.
Note: The examples in this guide use beta-galactosidase stock solution. For information about
how to prepare and use PepCalMix, refer to the document: CESI 8000 Plus High Performance
Separation-ESI Module User Guide.
Table 4-1 Required Reagents for the Silica Surface OptiMS Cartridge
Reagent

Purpose

10% Acetic acid (HAc)

Background electrolyte (BGE) and conductive
liquid

20% HAc

pH adjustment of leading electrolyte (LE)LE buffer, 200 mM ionic strength in ammonium Sample and system suitability preparation
acetate (AmAc), pH 4.0
Beta-galactosidase stock solution, 1 μM

Test sample(Optional) PepCalMix

Test sample

Peptide test sample

Optimize electrospray ionization (ESI) voltage

Prepare the 10% HAc Solution
Use this solution as the background electrolyte (BGE). Prepare new 10% HAc solution everyday.
1.

In a clean 20 mL glass vial, add 18 mL of CE Grade Water.

2.

Inside a fume hood, add 2 mL of HAc to the vial.

3.

Invert the vial three times to mix the contents.

4.

Keep the 10% HAc solution at room temperature.

5.

At the end of the day, discard the solution that was not used.

Prepare the 20% HAc Solution
Only prepare new 20% HAc solution when the 200 mM LE buffer, pH 4.0 is prepared.
1.

In a 100 mL glass bottle, add 80 mL of CE Grade Water.

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Prepare the Reagents
2.

Inside a fume hood, add 20 mL of HAc to the bottle.

3.

Invert the bottle three times to mix the contents.

4.

Attach a label with the name 20% HAc solution and the preparation date to the bottle.

5.

Keep the 20% HAc solution at room temperature.

6.

At the end of the day, discard the solution that was not used.

Prepare the 200 mM LE Buffer, pH 4.0
1.

To prepare 50 mL of 400 mM LE buffer, pH 4.0, do this:a.

In a 50 mL glass volumetric flask, add 20 mL of CE Grade Water.

b.

Inside a fume hood, add 2.7 mL of 7.5 M AmAc to the flask.

c.

To increase the volume to 50 mL, add CE Grade Water.

d.

Invert the flask three times to mix the contents.

2.

Pour the 50 mL of 400 mM AmAc solution into a 100 mL beaker.

3.

Use a calibrated pH meter to measure the initial pH of the solution.

4.

Add aliquots of newly prepared 20% HAc solution until the pH of the solution is 4.0.

5.

Transfer the solution to a 100 mL volumetric flask.

6.

To increase the volume to 100 mL, add CE Grade Water.

7.

Invert the flask three times to mix the contents.

8.

Attach a label with the name 200 mM LE buffer, pH 4.0 and the preparation date to
the flask.

9.

When it is not in use, keep the buffer at 2 °C to 8 °C for as long as 2 years after preparation.

Prepare the Beta-Galactosidase Stock Solution
WARNING! Toxic Chemical Hazard. When handling, storing, and disposing ofchemicals, refer to the chemical product safety data sheets. Obey all of therecommended safety procedures. For health and safety precautions, refer to the
document: Overview Guide.
1.

Add 625 µL of CE Grade Water to the vial with beta-galactosidase stock solution.

2.

Mix the solution in a vortex mixer for at least 30 seconds.

3.

Use a centrifuge to spin the vial and move the liquid to the bottom.

4.

Divide the beta-galactosidase stock solution into aliquots of 50 µL.

5.

Keep the beta-galactosidase stock solution at –35 °C to –15 °C for as long as 1 year.

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Prepare the Reagents

Prepare the Beta-Galactosidase Test Sample
1.

If required, then remove 50 µL of beta-galactosidase stock solution from the freezer and let
it thaw at room temperature.

2.

In a 0.5 mL centrifuge tube, add 50 µL of beta-galactosidase stock solution and 50 µL of
1 μM 200 mM LE buffer, pH 4.0 to make a 1:1 solution.

3.

Mix the solution in a vortex mixer for at least 30 seconds.

4.

To remove any precipitant, use a centrifuge to spin the solution at 12,000 g for 5 minutes.

5.

Use a pipette to put 90 µL of the supernatant in a sample vial.
Keep the sample at 2 °C to 8 °C for as many as 3 days.

Prepare the Peptide Test Sample
1.

In a centrifuge tube, add 100 μL of BGE.

2.

Add 5 μL of a peptide marker, such as pI 9.5.

3.

Put the cap on the tube.

4.

Mix the solution in a vortex mixer for 3 seconds.

5.

Keep the sample at 2 °C to 8 °C.

6.

At the end of the day, discard the solution that was not used.

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Prepare the Buffer Trays

5

Prepare the Buffer Trays
1.

Use the following table to prepare the required reagents.
Table 5-1 Required Reagents2.

Content

Quantity

VolumeBGE (such as 10% HAc solution or 50 mM AmAc
buffer, pH 3.0)

3 vials

1.5 mL

Peptide test sample

1 vial

1.5 mLCE Grade Water

4 vials

1.5 mL

0.1 M NaOH

1 vial

1.5 mL

0.1 M HCl

1 vial

1.5 mLUse the following figure to put each reagent vial in the correct position in the buffer inlet andoutlet trays.
Note: This layout is the same for all five methods in the CESI 8000 Plus software.
Figure 5-1 Buffer Tray Layout

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Prepare the System

6

Prepare the System

WARNING! Electrical Shock Hazard. Do not touch the adapter when it is connectedto the CESI 8000 Plus system and the separation voltage is on.

Install the Cartridge
Before a cartridge can be installed, the interface plate must be installed on the CESI 8000 Plus
system. Refer to the section: Remove and Install the Interface Plate.
1.

Open the cartridge cover.
Figure 6-1 Cartridge Cover

2.

Loosen the thumbscrews on the insertion bar, and then lift the insertion bar.

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Prepare the System
Figure 6-2 Insertion Bar

3.

Open the access panel.
Figure 6-3 Access Panel4.

Put the sprayer housing through the side panel, and then put the sprayer in the holster.

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Prepare the System
Figure 6-4 Sprayer in the Holster

Note: To prevent damage, make sure that the protective sleeve stays on the sprayer tip. Do
not let the sprayer tip touch any surfaces.
5.

To install the cartridge body, put the ends of the cartridge on the aligning blocks of theinterface plate at a 45-degree angle, and lightly push the cartridge in.
Figure 6-5 Aligning Blocks

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Prepare the System

Item

Description

1

Aligning blocks

2

T slider6.

Turn the cartridge up until it is parallel with the interface plate.

7.

Put the upper handles in the position shown in the figures: Figure 6-6 and Figure 6-7.

8.

Align the T slider in the groove of the interface plate and move the cartridge down. As the
cartridge moves down, the covers on the inlet and outlet sides retract and the capillaries canbe seen below the interface block within the ejectors.
Figure 6-6 T Slider

Item1

Description
Align the T slider in the groove

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Prepare the System
Figure 6-7 Capillaries Below the Interface Block

Item
1
9.

Description
Capillaries can be seen below the interface block within the ejectors

Put the coolant and capillary tubing through the notched arm.

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Prepare the System
Figure 6-8 Coolant and Capillary Tubing

10. Lower the insertion bar, and then tighten the thumbscrews.
Figure 6-9 Thumbscrews on the Insertion Bar

Remove and Install the Interface Plate
1.

To remove the interface plate, do this:a.

Remove the cartridge. Refer to the section: Remove the Cartridge.

b.

Turn off the power to the CESI 8000 Plus system.

c.

Loosen the thumbscrews on the sides of the interface plate.

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Prepare the System
Figure 6-10 Thumbscrews on the Interface Plate

2.

Item

Description

1

Thumbscrewsd.

Hold the handle on the top of the interface plate and pull it out.

e.

Keep the interface plate in a safe place.

To install the interface plate, do this:a.

Hold the handle on the top of the interface plate and install it. Make sure that theinterface plate is flush with the mounting surface of the CESI 8000 Plus system.

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Figure 6-11 Install the Interface Plateb.

Tighten both thumbscrews.
Figure 6-12 Tighten the Thumbscrews

Condition the Capillaries
Condition the capillaries before a new cartridge is used for the first time and before a cartridgethat has been in storage is used again.
Use the CESI-MS conditioning method to rehydrate the coating of the bare fused-silica
capillaries and to establish the electrical connection in the sprayer tip.

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1.

Remove the protective sleeve from the sprayer tip.

2.

To make sure that the flow of liquid through the separation capillary is sufficient, do this:3.

a.

Do a forward rinse with CE Grade Water at 100 psi for 3 minutes.

b.

When a liquid droplet shows at the end of the sprayer tip, stop the application ofpressure.

To make sure that the flow of liquid through the conductive liquid capillary is sufficient, do
this:
a.

Do a reverse rinse with CE Grade Water at 100 psi for 5 minutes.

b.

When a liquid droplet shows at the end of the stainless steel needle, stop theapplication of pressure.

4.

Install the protective sleeve on the sprayer.

5.

Put 10 mL of MeOH in a 50 mL Falcon tube, and then put the Falcon tube in the holster on
the side of the system.
CAUTION: Potential System Damage. Do not put more than 10 mL of MeOH in the
Falcon tube. If there is more than 10 mL in the tube, then the liquid can splash onto
the metal components of the sprayer and cause damage.

6.

Carefully immerse the sprayer tip in the Falcon tube.
Figure 6-13 Sprayer Immersed in Liquid

7.

Run the CESI-MS conditioning method.

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8.

When the method is complete, remove the sprayer from the Falcon tube.

9.

Use lint-free wipes to dry the sprayer carefully.
Figure 6-14 Dry the Sprayer

CAUTION: Potential System Damage. To prevent the sprayer tip from breaking, do not
move the retractable protective guard during drying.
10. Make sure that the ESI voltage on the mass spectrometer is off.
11. Install the sprayer in the adapter on the mass spectrometer.
The cartridge is ready to set a stable spray.
12. Remove the Falcon tube from the holster, and discard the contents.

Configure the Mass Spectrometer for Use with
the CESI 8000 Plus System
1.

In the MassLynx software, click the Inlet Method button to open the Inlet Method window.
Figure 6-15 Inlet Method Button

2.

Click Tools > Instrument Configuration to open the Inlet Configuration dialog.

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Figure 6-16 Inlet Configuration Dialog

3.

Click Configure to open the Inlet Configuration Wizard.
a.

In the Select Pump page, select None in the list of pumps, and then click Next.

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Figure 6-17 Select Pump Page—Inlet Configuration Wizardb.

In the Select Auto Sampler page, select None in the list of autosamplers, and thenclick Next.

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Figure 6-18 Select Auto Sampler Page—Inlet Configuration Wizardc.

In the Select Detectors page, select the first item in the Configured Detectors list,
and then click the left arrow to remove the detector from the list.

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Figure 6-19 Select Detectors Page—Inlet Configuration Wizardd.

Do step 3.c until the Configured Detectors list is empty. Then click Next.
The Finished Configuring page opens.

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Figure 6-20 Finished Configuring Page—Inlet Configuration Wizarde.
4.

Click Finish to close the wizard.

In the Inlet Configuration window, click Events & Triggering to open the Events and
Triggering Wizard.
a.

In the Choose Events page, in the Event In area, select 2 and then click Next.

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Figure 6-21 Choose Events Page—Events and Triggering Wizardb.

In the Choose Triggering Method page, in the Pump area, click Trigger by Contact
Closure.

c.

In the Detector area, click Trigger by Contact Closure, and then click Next.

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Figure 6-22 Choose Triggering Method Page—Events and Triggering Wizard

The Finished Configuring page opens.
d.

Click Finish to close the wizard.

Create a Tune File
Create a tune file to set the ESI voltage and other parameters for the mass spectrometer. This
file is used as the starting point for two more tune files that are required for the test separation.
The additional files are created in the section: Fine-Tune the Position of the Sprayer Tip.
1.

In the MassLynx software, click the MS Tune button.
Figure 6-23 MS Tune Button

2.

Put the mass spectrometer in Operate mode.
a.

If necessary, manually turn on the gas supplies for the mass spectrometer.

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In the bottom right corner of the Tune window, click Operate.

The system is removed from Standby mode.
Figure 6-24 Operate Button (Far Left) and System Status (Right)

3.

Open the Nanoflow+ tab, and type the values shown in the following figure.
Figure 6-25 Nanoflow+ Tab

4.

To the right of the Nanoflow+ tab, type the values shown in the following figure.
Figure 6-26 Mass Range and Scan Conditions

5.

Click File > Save As, and type the name OptiMS_OFF.

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Establish a Stable Spray
1.

Make sure that the sprayer tip is in the correct position for the inlet on the mass
spectrometer.

2.

In the MassLynx software, make sure that the ESI voltage is zero.

3.

To fill the conductive liquid capillary with BGE, do this:a.

In the 32 Karat software, go to the Direct Control window.

b.

Click the Pressure field.
Figure 6-27 Pressure Settings Dialog to Fill the Conductive Liquid Capillaryc.

In the Pressure field, type 100.

d.

In the Duration field, type 2.

e.

Click Reverse.

f.

Click Pressure.

g.

Click OK.
To identify the direction of the pressure being applied, in the Direct Control window, lookat the Rinse Direction icon (a blowing face identified by a red arrow in the following
figure). The reverse direction refers to rinsing the conductive liquid capillary, and theblowing face shows on the right side of the window.

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Figure 6-28 Rinse Direction (Blowing Face) Icon

When the conductive liquid capillary is filled with BGE, a droplet will show at the end of
the stainless steel needle.
Figure 6-29 Droplet at the End of the Stainless Steel Needle

4.

When the rinse is complete, to fill the separation capillary with BGE, do this:Installation Guide
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Go to the Direct Control window.

b.

Click the Rinse field.
Figure 6-30 Pressure Settings Dialog to Fill the Separation Capillaryc.

In the Pressure field, type 100.

d.

In the Duration field, type 2.

e.

Click Forward.

f.

Click Pressure.

g.

Click OK.
When the separation capillary is filled with BGE, a droplet will show at the end of thesprayer tip.
Figure 6-31 Droplet at the End of the Sprayer Tip

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Note: If the separation capillary is empty, then the first droplet of solution might notshow at the sprayer tip for as long as 7 minutes.
5.

To find the minimum ESI voltage, refer to the section: Optimize the ESI Voltage.

Optimize the ESI Voltage
1.

Make sure that the separation capillary and conductive liquid capillary are filled with BGE.

2.

To set the values for voltage in the 32 Karat software, do this:a.

Go to the Direct Control window, and then double-click the Voltage field.
Figure 6-32 Voltage Settings Dialogb.

In the Voltage field, do this:• For the silica surface OptiMS cartridge, type 20.
• For the neutral OptiMS cartridge, type 30.

c.

In the Duration field, type 60.

d.

In the Ramp time field, type 1.

e.

Click Normal.

f.

Click OK.

Note: If the BGE used is 10% HAc solution, then the electrical current should be 2 μA to
3 μA. If the BGE used is 50 mM AmAc buffer, pH 3.0, then the electrical current should be
0.5 µA to 1 µA.
3.

In the Analyst TF software, in the Navigation bar, under Tune and Calibrate, double-click
Manual Tuning.

4.

On the Source/Gas tab, do this:a.

In the Curtain Gas (CUR) field, type 10.

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Note: In the Analyst TF software, the minimum value for curtain gas is 10. CESI-MS
runs use a value of 5 for curtain gas. If the mass spectrometer is connected to the CESI
8000 Plus system, then the factory setting of 5 for curtain gas is applied.
b.

In the IonSpray Voltage Floating (ISVF) field, type 1.0.
The IonSpray Voltage Floating (ISVF) parameter sets the ESI voltage.

c.
5.

In the Interface Heater Temperature (IHT) field, type 50.0.

On the MS tab, do this:a.

In the Scan type field, select TOF MS.

b.

In the Accumulation time field, type 500.

c.

In the TOF Masses (Da) Min field, type 200.

d.

In the TOF Masses (Da) Max field, type 2000.

e.

In the Period Duration field, type 10.

6.

Click Start.

7.

If a non-SCIEX mass spectrometer is in use, then do this on the mass spectrometer
acquisition computer:8.

a.

Set the ESI voltage to zero.

b.

Set the scan range from 200 m/z to 2000 m/z.

c.

Click Start.

Increase the value in the IonSpray Voltage Floating (ISVF) field in increments of 0.1 kV
until a continuous signal shows in the mass spectrum window.
Note: This is the minimum ESI voltage.
Figure 6-33 Typical Spray Profile of a Cartridge that Uses 10% HAc Solution as the
BGE

9.

In the Direct Control window, click Stop.

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10. On the Source/Gas tab, in the IonSpray Voltage Floating (ISVF) field, type 0.0, and thenclick OK.
Make sure that there is no spray or background mass spectrum.
Note: If there is still spray when IonSpray Voltage Floating (ISVF) is 0.0, then there might
be an issue with the connection. Refer to the section: Fine-Tune the Position of the Sprayer
Tip.
11. In the IonSpray Voltage Floating (ISVF) field, type the minimum ESI voltage, and thenclick OK.
Make sure that electrospray starts again.
12. In the IonSpray Voltage Floating (ISVF) field, increase the value by 100 V.
13. To find the best position for the sprayer in relation to the inlet on the mass spectrometer, use
the XYZ-axes adjustment knobs to get a maximum total ion current signal.
Note: Keep the sprayer tip at least 2 mm away from the curtain gas plate.
14. To check the stability of the spray, continue to apply 20 kV (normal polarity) for 20 minutes.
15. In the Analyst TF software, do this:a.

Open the Advanced MS tab, and then click MCA.

b.

Open the MS tab.

c.

In the Scan type field, select TOF MS.

d.

In the Accumulation time field, type 1.

e.

In the TOF Masses (Da) Min field, type 70.

f.

In the TOF Masses (Da) Max field, type 2000.

g.

In the Period Duration field, type 5.

16. Click Acquire, and then type a file name (for example, Baseline).
17. If a non-SCIEX mass spectrometer is in use, then do this on the mass spectrometer
acquisition computer:a.

Set the ESI voltage to 1.0 kV and continue to acquire data.

b.

Increase the ESI voltage in increments of 0.1 kV until there is a continuous signal in themass spectrum window.

c.

To find the best position for the sprayer in relation to the inlet on the mass spectrometer,maximize the XIC (eXtracted Ion Electropherogram) signal for the pI 7.0 or pl 10 marker
in use while minimizing its fluctuations.

d.

When the position of the sprayer is optimized, decrease the ESI voltage in incrementsof 0.1 kV until the spray stops.

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Increase the ESI voltage in increments of 0.1 kV until continuous spray is detected.

f.

Record this value as the minimum ESI voltage.

g.

Increase the minimum ESI voltage by 0.2 kV.

18. Use the Y-axis alignment knob to maximize the baseline fluctuations.
Figure 6-34 Find the Baseline Fluctuationa.

If the baseline fluctuation is < 40% within 2 minutes to 5 minutes, then make a noteof the ESI voltage for the mass spectrometer methods (ion collection ESI voltage):
Baseline fluctuation (%) = [(highest value - average value)/average value] x 100.

b.

If the baseline fluctuation is > 40%, then do this procedure again until a good baseline isset. If a good baseline cannot be set, then refer to the section: Fine-Tune the Position of
the Sprayer Tip.

19. If a non-SCIEX mass spectrometer is in use, then do this:a.

Monitor the spray stability for 20 minutes to 30 minutes to make sure that the baseline
fluctuation is ≤ 40%.
If the baseline fluctuation is > 40%, then condition the capillaries. Refer to the section:
Condition the Capillaries.

b.

Turn off the electrospray voltage on the mass spectrometer.

Fine-Tune the Position of the Sprayer Tip
CAUTION: Potential System Damage. Do not move the sprayer tip too close to the inlet
on the mass spectrometer. If the sprayer tip is < 2 mm from the inlet, then the accidental
suction of rinse solution into the mass spectrometer can occur and cause damage.

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Before a separation is run, it is critical to optimize the position of the sprayer tip in front of
the inlet on the mass spectrometer to get the correct ESI voltage. If the sprayer tip is too far
away from the inlet on the mass spectrometer, then a high ESI voltage can be required, whichcauses analyte fragmentation. For intact proteins, electrochemical reactions such as oxidation
can occur.
Use this procedure to adjust the position of the sprayer tip and find the required ESI voltage formass spectrometer acquisition:• After installing a cartridge• If the signal separation voltage is changed• If a different BGE is used
For troubleshooting information, refer to the document: CESI 8000 Plus High Performance
Separation-ESI Module User Guide.
1.

Fill the separation capillary and the conductive liquid capillary with BGE.

2.

Put the sprayer tip 2 mm to 5 mm from the inlet on the mass spectrometer.
Tip! If the camera is available, use it to help position the tip. If not, look through the glass
window on the front of the ion source as you move the sprayer tip.

3.

In the 32 Karat software, use the Direct Control window to apply 20 kV voltage.
• Make sure that the cartridge does not spray when the ESI voltage is 0 V during mass
spectrometer scanning.
• Monitor the Direct Control window to make sure that the electrical current is stable.

4.

In the 32 Karat software, use the Direct Control window to apply 20 kV voltage and 1 psipressure in both directions.
• Make sure that the cartridge does not spray when the ESI voltage is 0 V during mass
spectrometer scanning.
• Monitor the Direct Control window to make sure that the electrical current is stable.

5.

Adjust the position of the sprayer tip to maximize the mass spectrometer signal intensity.
a.

In the MassLynx software, open the Nanoflow+ tab and then type 0.5 in the Capillary(kV) field to set the ESI voltage.

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Figure 6-35 Nanoflow+ Tab in the MS Tune Fileb.

Increase the value in the Capillary (kV) field in increments of 0.1 kV until electrosprayionization is detected. Record this voltage.

c.

Set ESI voltage to 0 kV and confirm that there is no electrospray ionization.

d.

Change the value in the Capillary (kV) field to 0.2 kV higher than the voltage found instep 5.b. Electrospray ionization should start. To make sure that the spray is stable, let it
continue for 5 minutes.
Note: If a voltage of 20 kV and pressure of 1 psi are not sufficient to establish a stablesignal, then increase the pressure at both ends of the cartridge, to a maximum of 3 psi.
If pressure > 1.0 psi is required to maintain a stable electrospray ionization, then makesure to change the required pressure in the CESI-MS separation method.

e.

Use the knobs on the adapter stage to adjust the position of the sprayer tip to maximize
the signal intensity while minimizing fluctuations in signal intensity.
Make sure that the sprayer tip is 2 mm to 5 mm from the ion source inlet.

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Figure 6-36 Sprayer Tip and Ion Source Inlet

6.

Click File > Save As and type the name OptiMS_RUN.
These are the settings used by the mass spectrometer during data acquisition after the
sample has been separated by the CESI 8000 Plus system.

7.

Change the value in the Capillary (kV) field to 0.4 kV higher than the voltage recorded instep 5.b, click File > Save As, and then type the name OptiMS_ON.
These are the settings used to remove any rinse solution that collects on the sprayer tip.

8.

In the MassLynx software, to open the OptiMS_OFF MS tune file, click File > Open.

9.

In the 32 Karat software, go to the Direct Control window, and then set the voltage to 1.0 kV
with a ramp of 5.0 minutes and a duration of 5.0 minutes.

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7

Do a Test Separation

This section gives the instructions to do a test separation with beta-galactosidase test sample to
make sure that the CESI 8000 Plus system and the mass spectrometer operate correctly.
As an alternative, PepCalMix can be used as the test sample. PepCalMix has 20 different
stable isotope-labeled peptides and is available from SCIEX. For instructions about how to use
PepCalMix, refer to the document: CESI 8000 Plus High Performance Separation-ESI Module
User Guide.
Note: Before a test separation is run, make sure that a stable spray has been establishedand the optimum ESI voltage has been set. Refer to the section: Fine-Tune the Position of theSprayer Tip.

Create the MS Methods and Sample List
Use the MassLynx software to create the files that are required for the test separation.

Create the MS Methods
1.

In the MassLynx software, click the MS Method button.
Figure 7-1 MS Method Button

2.

Double-click the first row in the table to open the Function:1 MS dialog.

3.

Type the values shown in the following figure, and then click OK.

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Figure 7-2 Function: 1 MS Dialog for cap_on MS Method

4.

Click File > Save As and type cap_on.
This method applies a high ESI voltage to remove any drops of buffer from the sprayer tip
after the Rinse events.

5.

Do the previous steps twice more to create methods with the time values and file names in
the following table.

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Table 7-1 MS Method Parameters
File Name

Start Time

End Time

Purpose

cap_off0

0.03

Turn off high ESI voltage

bgal_250-2000

030Collect MS data after CE separation

Create the Sample List
CAUTION: Potential System Damage. Always use the OptiMS_OFF tune file for the lastrow in a sample list to turn off the ESI voltage at the end of the run. If voltage is applied
when the capillary is empty, then the sprayer can break.
1.

To open a new sample list, in the MassLynx software, click File > New.

2.

Complete the first row in the sample table.

3.

a.

Click the File Name cell, and then type discard 1.

b.

Double-click the MS Tune File cell, and then select the OptiMS_OFF MS tune file.

c.

Double-click the MS File cell, and then select the cap_off MS file.

To complete the sample list, add the information in the following table to rows 2 through 5.
CAUTION: Potential System Damage. To keep the synchronization between the
CESI 8000 Plus system and the mass spectrometer and prevent the introduction ofsolutions that are not compatible to the mass spectrometer, when multiple CESI-MS
separations are done, always use rows that are equivalent to row 1 to row 4 in thefollowing table.
Table 7-2 Sample List

4.

Row

File NameMS Tune FileMS File1

discard 1

Optims_OFF

cap_off

2

discard 2

OptiMS_ON

cap_on3

discard 3

OptiMS_OFF

cap_off4

bgal_1

OptMS_RUN

bgal_250-2000

5

discard 4

OptiMS_OFF

cap_off

To save the sample list, do this:a.

Click File > Save.

b.

Type a name for the sample list, and then click OK.

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Manually Create the CESI-MS Separation Method
The cesi-ms_Waters-ms.met method file is installed with the 32 Karat software 10.3 or laterin the folder C:\32Karat\projects\CE MS\Method. If the method is missing, then use thisprocedure to create it manually.
1.

In the 32 Karat software, click File > Method > New.

2.

Use the initial conditions in the following figure.
Figure 7-3 Initial Conditions for the CESI-MS Separation Method

3.

Use the time program in the following figure.
Figure 7-4 Time Program for the CESI-MS Separation Method

4.

In row 11 and row 14 of the sequence table, click the Event cell, then select Separate.

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5.

Set the parameters that show in the following figure, and then click OK.
Figure 7-5 Separate Dialog: Pressure Settings for Rows 11 and 14

6.

Click File > Method > Save As, and then save the method as cesi-ms_Waters-ms.met.

Load the Sample and Start the Run
1.

Put the vial with beta-galactosidase test sample in position SI:A1 of the sample inlet tray.

2.

In the 32 Karat software, open the CESI-MS Separation for Waters MS method file.

3.

In the MassLynx software, open the sample list created in the section: Create the MS
Methods and Sample List.

4.

In the sample list, select all the rows in the table, and then click the Start Run icon.
Figure 7-6 Start Run Icon

The Start Sample List Run dialog opens.

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Figure 7-7 Start Sample List Run Dialog

5.

Make sure that the values in the Samples section show the rows in the sample list to be run
and that the Acquire Sample Data check box is selected, and then click OK.
Wait for the message Waiting for Inlet Start to show at the bottom of the
MassLynx software window.

6.

In the 32 Karat software tool bar, click the Single Run icon.
The Single Run Acquisition dialog opens.

7.

Make sure that the CESI-MS Separation for Waters MS method file shows in the
Sequence name field.
If the correct method file does not show, then click the folder icon and browse to the
CESI-MS Separation for Waters MS method file.

8.

In the 32 Karat software, go to the Direct Control window, and then click Load.

9.

When the carriers have moved forward, open the front cover of the CESI 8000 Plus systemand put the inlet and outlet buffer trays in the correct carrier.

10. Put the sample inlet tray in the back of the inlet carrier (left).
11. Click Start.
The run requires approximately 30 minutes to complete.

Analyze the Data
Use the MassLynx software to analyze the data from the test run.
1.

Click File > Open Data File.

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Figure 7-8 Data Browser Dialog

2.

Select the bgal_1 file from the test run, clear the Spectrum check box, and then click OK.
The total ion chromatogram (TIC) opens in the Chromatogram window.

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Figure 7-9 TIC for Test Run in Chromatogram Window

3.

Extract the chromatograms for two of the peptides:a.

Click Display > Mass.

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Figure 7-10 Mass Chromatogram Dialogb.

In the Description (m/z) field, do this:• For a NanoLockSpray ion source, type 714.8649, 729.3652.
• For a NanoFlow ion source, type 714, 729.

c.

In the Abs Window (Da) field, type 5.0.

d.

Click OK.

A pane that shows the extracted ion chromatogram for each m/z opens in the
Chromatogram window.

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Figure 7-11 TIC and XICs in the Chromatogram Window

4.

Calculate the signal-to-noise ratio (S/N) for the 729 Da peak.
a.

Click the pane with the 729 Da peak to select it.
When the pane is selected, a small square shows near the left edge. In the figure:
Figure 7-11, the top pane is selected.

b.

Click Process > Signal To Noise.

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Figure 7-12 Signal To Noise Dialogc.

Click the Signal field, and then click-drag across the peak in the extracted ion
chromatogram to select it.

d.

Click the Noise field, and then click-drag across an area in the extracted ion
chromatogram with noise. The selected noise region should be approximately fourtimes wider than the peak selected before.

e.

Click OK.

A new pane that shows the S/N ratio opens in the Chromatogram window.

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Figure 7-13 TIC, XICs, and S/N in the Chromatogram Window

5.

Examine the results.
• The S/N for the 729 Da peak should be > 50.
• Make sure that the two peaks are separated by at least 0.05 minutes.
If the results are not similar to these values, then set the home position on the mobile cart.
Refer to the document: CESI 8000 Plus High Performance Separation-ESI Module User
Guide.

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Shut Down and Disconnect

8

Shut Down and Disconnect
Remove the Cartridge

If the CESI 8000 Plus system will be shut down and the power turned off, then remove thecartridge.
1.

Make sure that the ESI voltage on the mass spectrometer is off.

2.

Do the applicable procedure in the section: Store the Cartridge.

3.

In the 32 Karat software, go to the Direct Control window, and then click Load.

4.

Open the cartridge cover.
Figure 8-1 Cartridge CoverThe coolant pump for the CESI 8000 Plus system turns on and releases the coolant fromthe cartridge coolant lines. Approximately 30 seconds are required. Wait for the pump toturn off.
5.

Loosen the thumbscrews on the insertion bar.

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Figure 8-2 Thumbscrews on the Insertion Bar

6.

Lift the insertion bar fully.

7.

To prevent damage to the sprayer tip, retract the stage as far away from the inlet on themass spectrometer as possible.

8.

To loosen the end of the sprayer from the adapter, turn the arrow on the sprayer to the
Unlock position.
WARNING! Hot Surface Hazard. The surfaces of the ion source become hot
during operation. Let the ion source cool for at least 30 minutes before thesprayer is removed from the adapter on the mass spectrometer.
Figure 8-3 Unlock the Sprayer from the Cartridge

Item1

Description
Sprayer in the Unlock position

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Shut Down and Disconnect

Item
2
9.

Description
Turn to lock the sprayer from the adapter

Remove the sprayer from the adapter.
Figure 8-4 Sprayer Removal

10. Install the protective sleeve on the sprayer tip.
11. Put the tubing with the sprayer end through the access panel.
Figure 8-5 Sprayer and Tubing Through the Access Panel

12. Remove the coolant tubing from the notched arm.

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Figure 8-6 Coolant and Capillary Tubing

13. Hold the cartridge sprayer with one hand and the midsection of the cartridge in the other,
then lift the cartridge up and pull it out.
Figure 8-7 Remove the Cartridge

Note: As the cartridge is moved up, the sheaths on the inlet and outlet sides go down overthe capillary ends.
Note: Drops of liquid coolant that might fall from the cartridge ends are normal and do notcause damage to the hardware.
14. Refer to the section: Store the Cartridge.

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Shut Down and Disconnect

Store the Cartridge
WARNING! Puncture Hazard. Be careful when handling the cartridge. The tips ofthe capillaries are extremely sharp.
CAUTION: Potential System Damage. To prevent contamination of the internal surface ofthe separation capillary, do not use the pressure rinse function to blow air through thecapillaries. Use the vacuum function to dry the capillaries.
1.

2.

To prepare the cartridge to be stored for less than 3 days, do this:a.

Run the CESI-MS cleaning method.

b.

Store the cartridge in the system for as long as 3 days.

To prepare the cartridge to be stored for more than 3 days, do this:a.

Run the CESI-MS storage method.
Note: The CESI-MS storage method cannot be run as part of a sequence because
methanol is volatile and can evaporate before the sequence is complete. Vials filledwith methanol must be used immediately. The ESI needle can stay in air but not inliquid. The ESI needle can be dried by vacuum.

b.

Disconnect the sprayer from the ion source.

c.

Remove the cartridge from the system.

d.

Install the protective sleeve on the sprayer tip.

e.

Put the sprayer tip in a 2 mL microcentrifuge tube of water.

f.

Loosely seal the tube with Parafilm.

g.

Keep the cartridge in a safe place at room temperature.

Disconnect the CESI 8000 Plus System from theMass Spectrometer
Disconnect the CESI 8000 Plus system from the mass spectrometer when:• The mass spectrometer will be used without the CESI 8000 Plus system.
• The mass spectrometer or the CESI 8000 Plus system will be moved.
1.

Make sure that the cartridge has been removed.
Refer to the section: Remove the Cartridge.

2.

Turn off the CESI 8000 Plus system.

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Shut Down and Disconnect
3.

On the CESI 8000 Plus system high-voltage connections panel, disconnect the input andoutput cables and the black and red banana plugs.
Refer to the figure: Figure 2-10.

4.

Disconnect the relay cable from the CESI 8000 Plus system and the mass spectrometer.

5.

Attach the green cover to the External Connections 1 port on the back of the mass
spectrometer.

6.

Disconnect the high-voltage input cable from the mass spectrometer.

7.

Adjust the height of the mobile cart to the lowest position.

8.

Disconnect the power cables from the CESI 8000 Plus system and the mobile cart.

9.

To unlock the wheels on the mobile cart, lift the lever at each caster.
Figure 8-8 Unlock the Wheels of the Mobile Cart

10. Carefully move the CESI 8000 Plus system away from the mass spectrometer.
11. Remove the adapter from the ion source.
12. Keep the adapter and cables in a safe place.

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