EASY-nLC 1200 Getting Started Guide Revision A

EASY-nLC 1200 Getting Started Guide Revision A

Thermo

EASY-nLC 1200

Getting Started Guide

Touch-screen application version 4.0

60053-97272 Revision A September 2015

© 2015 Thermo Fisher Scientific Inc. All rights reserved.

AFC, EASY-nLC, EASY-Spray, EASY-column, IFC, Nanospray Flex, nanoViper, Viper, and PepMap are trademarks, and Acclaim, Dionex, LCQ Fleet, LXQ, LTQ, TSQ, and Xcalibur are registered trademarks of

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Thermo Fisher Scientific Inc. provides this document to its customers with a product purchase to use in the product operation. This document is copyright protected and any reproduction of the whole or any part of this document is strictly prohibited, except with the written authorization of Thermo Fisher Scientific Inc.

The contents of this document are subject to change without notice. All technical information in this document is for reference purposes only. System configurations and specifications in this document supersede all previous information received by the purchaser.

This document is not part of any sales contract between Thermo Fisher Scientific Inc. and a purchaser. This document shall in no way govern or modify any Terms and Conditions of Sale, which Terms and Conditions of

Sale shall govern all conflicting information between the two documents.

Release history: Revision A, September 2015

Hardware versions: EASY-nLC 1200 instrument

Software version: Touch-screen software version 4.0

For Research Use Only. Not for use in diagnostic procedures.

Regulatory Compliance

Thermo Fisher Scientific performs complete testing and evaluation of its products to ensure full compliance with applicable domestic and international regulations. When the instrument is delivered to you, it meets all pertinent electromagnetic compatibility (EMC) and safety standards.

Changes that you make to the instrument might void compliance with one or more of these EMC and safety standards.

Changes to the instrument include replacing a part or adding components, options, or peripherals not specifically authorized and qualified by Thermo Fisher Scientific. To ensure continued compliance with EMC and safety standards, replacement parts and additional components, options, and peripherals must be ordered from Thermo Fisher Scientific or one of its authorized representatives.

FCC Compliance Statement

THIS DEVICE COMPLIES WITH PART 15 OF THE FCC RULES. OPERATION IS SUBJECT TO

THE FOLLOWING TWO CONDITIONS: (1) THIS DEVICE MAY NOT CAUSE HARMFUL

INTERFERENCE, AND (2) THIS DEVICE MUST ACCEPT ANY INTERFERENCE RECEIVED,

INCLUDING INTERFERENCE THAT MAY CAUSE UNDESIRED OPERATION.

CAUTION Read and understand the various precautionary notes, signs, and symbols contained inside this manual pertaining to the safe use and operation of this product before using the device.

Notice on Lifting and Handling of

Thermo Scientific Instruments

For your safety, and in compliance with international regulations, the physical handling of this Thermo Fisher

Scientific instrument requires a team effort to lift and/or move the instrument. This instrument is too heavy and/or bulky for one person alone to handle safely.

Notice on the Proper Use of

Thermo Scientific Instruments

In compliance with international regulations: This instrument must be used in the manner specified by Thermo Fisher

Scientific to ensure protections provided by the instrument are not impaired. Deviations from specified instructions on the proper use of the instrument include changes to the system and part replacement. Accordingly, order replacement parts from Thermo Fisher Scientific or one of its authorized representatives.

For manufacturing location, see the label on the instrument.

WEEE Compliance

This product complies with the European Union’s Waste Electrical & Electronic Equipment (WEEE) Directive

2002/96/EC. It is marked with the following symbol:

Thermo Fisher Scientific is registered with B2B Compliance ( B2Bcompliance.org.uk

) in the UK and with the

European Recycling Platform ( ERP-recycling.org

) in all other countries of the European Union and in Norway.

If this product is located in Europe and you want to participate in the Thermo Fisher Scientific Business-to-Business

(B2B) Recycling Program, send an email request to [email protected]

with the following information:

• WEEE product class

• Name of the manufacturer or distributor (where you purchased the product)

• Number of product pieces, and the estimated total weight and volume

• Pick-up address and contact person (include contact information)

• Appropriate pick-up time

• Declaration of decontamination, stating that all hazardous fluids or material have been removed from the product

For additional information about the Restriction on Hazardous Substances (RoHS) Directive for the European Union, search for RoHS on the Thermo Fisher Scientific European language websites.

IMPORTANT This recycling program is not for biological hazard products or for products that have been medically contaminated. You must treat these types of products as biohazard waste and dispose of them in accordance with your local regulations.

Conformité DEEE

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Dieses Produkt entspricht der EU Waste Electrical & Electronic Equipment (WEEE) Richtlinie 2002/96/EC. Es ist mit dem folgenden Symbol gekennzeichnet:

Thermo Fisher Scientific hat Vereinbarungen mit Verwertungs-/Entsorgungsfirmen in allen EU-Mitgliedsstaaten getroffen, damit dieses Produkt durch diese Firmen wiederverwertet oder entsorgt werden kann. Weitere Informationen finden Sie unter www.thermoscientific.de/rohs .

C

Contents

Thermo Scientific

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi

Related Documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xii

Cautions and Special Notices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .xiii

Contacting Us . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xv

Chapter 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1

Instrument Hardware and Software Components and Connections . . . . . . . . . . 2

Autosampler Tray Compartment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

External Solvent Lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

Back Panel. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Column Setup Options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Two-Column Setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

One-Column Setup. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

Solvent System Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

Syringe Pumps. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

Pressure Sensors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Flow Sensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Six-Port Rotary Valves. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

Check Valve Assemblies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

Mixing Tee . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

Predefined Steps for Sample Runs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Overview of a Sample Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Sequence of Events for Each Step of a Sample Run . . . . . . . . . . . . . . . . . . . . 18

Pump Flow Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

Automatic Flow Control System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

Intelligent Flow Control System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

Load Speed Protection System. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

Chapter 2 Installing the EASY-nLC Instrument. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .35

Lifting Instructions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

Laboratory Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

Benchtop Dimensions and Weight Capacity . . . . . . . . . . . . . . . . . . . . . . . . . 37

Internet Access . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

Power and Fuses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

Temperature and Humidity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

EASY-nLC 1200 Getting Started Guide

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Contents

Back Panel Connections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

Connecting to Line Power. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

Connecting to the Mass Spectrometer through Contact Closure . . . . . . . . . . 42

Connecting the Ethernet Communication Cables . . . . . . . . . . . . . . . . . . . . . 44

Attaching a Mouse and Keyboard to the USB Connections. . . . . . . . . . . . . . 48

Attaching Add-on Devices through the P-Bus and RS-232 Ports. . . . . . . . . . 48

Chapter 3 Integrated Instrument Control Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .51

Turning On the EASY-nLC Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52

Using the Touch Screen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53

Using the Buttons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54

Entering Alphanumeric Text . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54

Using Tables and Input Fields . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54

Using the Interactive Graphical Controls. . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

Viewing the System Status. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

User Interface Layout and Application Menu Structure. . . . . . . . . . . . . . . . . . . 59

Home Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61

Batch Setup Menu. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62

Method Setup Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

Maintenance Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64

Configuration Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

Logging In to the EASY-nLC Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66

Closing Down the EASY-nLC Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

Chapter 4 Configuring the EASY-nLC System. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .69

Setting Up Communication with the Mass Spectrometer . . . . . . . . . . . . . . . . . 69

Setting Up the Local Area Network Connection . . . . . . . . . . . . . . . . . . . . . . . . 72

Modifying User Permissions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74

Changing the Administrator Password. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75

Creating New User Accounts. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76

Changing Your Password from the Exit Menu . . . . . . . . . . . . . . . . . . . . . . . 77

Modifying the Time Settings. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78

Purging the Computer Hard Drive . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79

Setting Up the Properties for the HPLC Device . . . . . . . . . . . . . . . . . . . . . . . . 80

Chapter 5 Preparing the EASY-nLC Instrument for Use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .83

Daily and Weekly Maintenance Tasks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83

Preparing the Solvent Bottles and Waste Containers . . . . . . . . . . . . . . . . . . . . . 84

Preparing the Solvents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85

Preparing the Solvent A and B Bottles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86

Installing the Autosampler Wash Bottles and Waste Beaker. . . . . . . . . . . . . . 88

Executing Maintenance Scripts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90

Purging and Flushing the Pumps. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91

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EASY-nLC 1200 Getting Started Guide Thermo Scientific

Thermo Scientific

Contents

Setting Up the Column Assembly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95

Setting Up the Column Assembly for the Nanospray Flex Source . . . . . . . . . 95

Setting Up the Column Assembly for the EASY-Spray Source. . . . . . . . . . . 112

Using nanoViper Fittings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115

Using the Viper Union . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117

Equilibrating Columns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118

Equilibrating the Precolumn . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118

Equilibrating the Analytical Column . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120

Chapter 6 Running Samples Using the Integrated Software. . . . . . . . . . . . . . . . . . . . . . . . .123

Preparing and Loading Samples. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123

Creating a Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124

Browsing for a Stored Method or Creating a New Method File. . . . . . . . . . 124

Starting the Method Setup Wizard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126

Setting Up the Sample Pickup and Loading Steps . . . . . . . . . . . . . . . . . . . . 127

Building the Gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130

Setting Up the Column Equilibration Steps . . . . . . . . . . . . . . . . . . . . . . . . 132

Setting Up the Autosampler Wash Step. . . . . . . . . . . . . . . . . . . . . . . . . . . . 134

Specifying the Temperature of the EASY-Spray Column Heater . . . . . . . . . 137

Saving the Method Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138

Creating a Batch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140

Viewing the Batch List and Setting Up a Batch Record . . . . . . . . . . . . . . . . 140

Specifying the Method, Sample Positions, and Number of Injections . . . . . 141

Starting Sample Acquisition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146

Monitoring the Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147

Using the Overview Page in the Home Menu . . . . . . . . . . . . . . . . . . . . . . . 147

Using the Graphs Page in the Home Menu . . . . . . . . . . . . . . . . . . . . . . . . . 149

Stopping Sample Acquisition. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150

Editing the Running Batch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151

Troubleshooting a Sample Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154

Chapter 7 Installing and Controlling External Devices . . . . . . . . . . . . . . . . . . . . . . . . . . . . .155

Adding Devices to the EASY-nLC Devices List . . . . . . . . . . . . . . . . . . . . . . . . 156

Connecting the RePlay Device to the EASY-nLC Instrument . . . . . . . . . . . . . 158

Setting Up a Method with the RePlay Device . . . . . . . . . . . . . . . . . . . . . . . . . 160

Monitoring the Run of a RePlay Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162

Effect of Additional Run Time for a RePlay Experiment . . . . . . . . . . . . . . . . . 163

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .165

EASY-nLC 1200 Getting Started Guide

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P

Preface

This guide describes how to operate and maintain the Thermo Scientific™ EASY-nLC™ nanoflow liquid chromatography (LC) instrument by using the integrated touch-screen application.

Before you turn on the instrument for the first time, read the first three chapters in this guide. Before you run your first sample, review the chapters that describe how to configure the software, prepare the system for operation, and submit your first sample set (batch).

Contents

Related Documentation

Cautions and Special Notices

Contacting Us

To suggest changes to the documentation or to the Help

Complete a brief survey about this document by clicking the button below.

Thank you in advance for your help.

Thermo Scientific EASY-nLC 1200 Getting Started Guide

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Preface

Related Documentation

In addition to this guide, Thermo Fisher Scientific provides the following documents for the

EASY-nLC instrument as PDF files:

• EASY-nLC 1200 User Guide for the Xcalibur Data System (version 2.1 or later)

• EASY-nLC 1200 Preinstallation Requirements Guide

• EASY-nLC 1200 Troubleshooting and Maintenance Guide

Safety and Regulatory Guide

The EASY-nLC 1200 also ships with a printed copy of the Safety and Regulatory Guide.

This guide contains important safety information about Thermo Scientific liquid chromatography (LC) and mass spectrometry (MS) systems. Make sure that all lab personnel have read and have access to this document.

You can find the EASY-nLC Series manuals in the following locations:

• The USB flash drive provided in the EASY-nLC accessory kit

• The data system computer

To view the product manuals on the data system computer

Choose Start > All Programs > Thermo Instruments > Manuals > LC Devices >

Thermo.

To view the EASY-nLC Help

• From the EASY-nLC view of the Instrument Setup window, choose Help >

Thermo EASY-nLC Help to open the Help to the Welcome topic.

• To view the Help topic for the current view, page, or dialog box, press the F1 key for information about setting parameters. Or, from the EASY-nLC view of the

Instrument Setup window, choose Help > Help On Current Item.

For more information, visit www.thermoscientific.com

.

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EASY-nLC 1200 Getting Started Guide Thermo Scientific

Preface

Cautions and Special Notices

Make sure to follow the safety practices presented in this guide, and observe the safety and special notices that appear in boxes.

Observe all written safety precautions during all phases of operation, service, and repair of this instrument. Failure to comply with these precautions or with specific warnings elsewhere in this manual violates safety standards of design, manufacture, and intended use of the instrument and might result in damage to the instrument, personal injury, or loss of life.

Special Notices

The following two stickers appear on the EASY-nLC instrument:

This sticker warns you that the instrument includes a sharp needle and moving parts that are accessible to the operator. To prevent personal injury or damage to parts of the EASY-nLC instrument, take care when loading samples into the instrument’s tray compartment.

WARNING:

AVERTISSEMENT:

SHARP NEEDLE AND MOVING PARTS.

KEEP HANDS CLEAR.

AIGUILLE POINTURE ET PARTIES MOUVANTES.

This sticker alerts you to consult this manual for instructions on how to operate the instrument.

The safety and special notices in the documentation include the following:

CAUTION Highlights hazards to humans, property, or the environment. Each CAUTION notice is accompanied by an appropriate CAUTION symbol.

CAUTION Highlights electrical hazards to humans or property.

CAUTION Highlights a sharp object hazard to humans.

Thermo Scientific EASY-nLC 1200 Getting Started Guide

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Preface

CAUTION Highlights an eye hazard to humans.

CAUTION Highlights a chemical hazard to humans, property, or the environment.

CAUTION Highlights lifting hazards.

IMPORTANT Highlights information necessary to prevent damage to software, loss of data, or invalid test results; or might contain information that is critical for optimal performance of the system.

Note Highlights information of general interest.

Tip Highlights helpful information that can make a task easier.

IMPORTANT General Residual Hazards

WARNING—Biohazard Human-sourced material, for example microorganisms, cell cultures, tissues, body fluids, and other biological agents can transmit infectious diseases.

To avoid infections with these agents:

• Assume that all biological substances are at least potentially infectious.

• Wear personal protective equipment as required by the hazard and follow good laboratory practice.

• Dispose of bio-hazardous waste in an environmentally safe manner that is consistent with local regulations. Follow a regulated, approved waste disposal program.

xiv

EASY-nLC 1200 Getting Started Guide Thermo Scientific

Preface

Contacting Us

There are several ways to contact Thermo Fisher Scientific for the information you need. You can use your smartphone to scan a QR code, which opens your email application or browser.

Contact us Customer Service and Sales Technical Support

(U.S.) 1 (800) 532-4752 (U.S.) 1 (800) 532-4752

(U.S.) 1 (561) 688-8731 us.customer-support.analyze

@thermofisher.com

(U.S.) 1 (561) 688-8736 us.techsupport.analyze

@thermofisher.com

To find global contact information or customize your request

1. Go to www.thermoscientific.com

.

2. Click Contact Us, select the Using/Servicing a Product option, and then type the product name.

3. Use the phone number, email address, or online form.

To find product support, knowledge bases, and resources

Go to www.thermoscientific.com/support .

To find product information

Go to www.thermoscientific.com/lc-ms .

Note To provide feedback for this document:

• Send an email message to Technical Publications ( [email protected]

).

• Complete a survey at www.surveymonkey.com/s/PQM6P62 .

To order consumable and spare parts for the EASY-nLC instrument

For the EASY-nLC 1200 instrument, go to www.thermoscientific.com/nanoLCMS

Thermo Scientific EASY-nLC 1200 Getting Started Guide

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1

Introduction

Introduction

The EASY-nLC instrument consists of a compact binary liquid chromatography instrument that provides split-free flows down to the low nanoLiter/min range, an autosampler that can hold standard sample vials and microtiter plates, and a built-in instrument control application with a touch-screen interface.

The EASY-nLC 1200 instrument operates in the ultra-high-performance pressure range of 0 to 1200 bar (see

Figure 1

).

Figure 1.

EASY-nLC 1200

Thermo Scientific

To familiarize yourself with the EASY-nLC instrument, read this chapter.

Contents

Instrument Hardware and Software Components and Connections

Autosampler Tray Compartment

External Solvent Lines

Back Panel

Column Setup Options

Solvent System Components

Predefined Steps for Sample Runs

Pump Flow Control

EASY-nLC 1200 Getting Started Guide

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Instrument Hardware and Software Components and Connections

Instrument Hardware and Software Components and Connections

The EASY-nLC instrument consists of the following hardware and software components

(see

Table 1 ).

Table 1. EASY-nLC instrument components

Hardware

Operating pressure range 1 to 1200 bar

Pumps

Pressure sensors

Valves

Flow sensors

Autosampler:

3 high-pressure pumps

3 pressure sensors, one for each pump

4 high-pressure rotary valves

4 check valves

2 nano-flow sensors

• Holds one microtiter plate (96 or 384 wells) plus an additional 6 vials or one 6 × 8 vial adapter plus an additional 6 vials.

• Holds four liquid containers for solvents, rinse, and waste.

Computer

Monitor

Mains power

• Can be cooled to a maximum of 20 °C [68 °F] below ambient temperature.

Embedded computer

Pressure sensitive screen that you can use with gloves

Screen size: 12.1 in. (30.7 cm)

Resolution: 800 × 600 pixels

120/230 Vac, 50/60 Hz,

250 W

Software

EASY-nLC stand-alone, touch-screen application (version 4.0)

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Autosampler Tray Compartment

Installing the EASY-nLC instrument as part of an LC/MS system requires only four connections:

• Solvent outlet connection to the mass spectrometer’s ion source

• Power cable connection to a grounded line power source

• Contact closure connection between the EASY-nLC instrument and the mass spectrometer (see

“Connecting to the Mass Spectrometer through Contact Closure” on page 42 )

• Ethernet connection between the EASY-nLC instrument’s built-in computer and an

Internet access port (see

“Setting Up the Ethernet Connection to the Support Server” on page 47 )

To control the EASY-nLC instrument with the Thermo Xcalibur™ data system or Thermo

Scientific application, you must also connect the EASY-nLC instrument to the data system computer.

Autosampler Tray Compartment

Figure 2

shows the tray compartment and the waste beaker for the ASC autosampler model.

You can open the tray compartment door manually or by using the touch-screen controls.

Figure 2.

Tray compartment for the current ASC model (viewed from above)

Waste beaker

Thermo Scientific

Well plate (96 or 384) or vial (6 × 8) adapter

Three (3) positions for wash solvents

Waste bottle with wash insert for cleaning the outside of the needle

Six (6) extra vial positions for calibration standards or regular samples

The tray compartment holds the sample tray and the wash bottles. The tray compartment for the ASC model holds an additional six vials. The compartment to the right of the tray compartment holds the waste beaker.

The autosampler draws solvent from wash bottle W3 to perform a standard wash and ejects the solvent into wash bottle W4, which contains the needle wash insert. The autosampler can also perform a custom wash using the solvents from wash bottles W1–W3.

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External Solvent Lines

External Solvent Lines

Two PEEKsil™ or nanoViper™ solvent lines exit from the slot in the right side instrument panel, and two low-pressure Teflon™ solvent lines exit through the solvent bottle holder on the top of the instrument. The Column Out line connects to the column assembly’s inlet, and the Waste In line connects to the column assembly’s venting Tee. The low-pressure solvent lines connect to the solvent A and solvent B reservoir bottles.

Note To use LC columns that have bare fused-silica ends with the EASY-nLC 1200 instrument, order the UHPLC Liquid Junction Kit. This kit contains modified Column

Out and Waste In lines as well as the appropriate fittings and unions for 360 μm OD tubing. For information about installing the modified Column Out and Waste In lines, refer to the EASY-nLC Series Troubleshooting and Maintenance Guide. For information about the column setups for these types of LC columns, see

“Setting Up a Column

Assembly with Fused-Silica Columns” on page 98

.

Figure 3

shows the right side panel of the EASY-nLC 1200 instrument.

Figure 3.

Column Out and Waste In solvent lines and low-pressure, Teflon solvent lines

Low-pressure,

Teflon solvent lines

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EASY-nLC Series Getting Started Guide

Column Out Waste In nanoViper solvent lines

Thermo Scientific

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Introduction

Back Panel

Back Panel

The connections for system communication (as part of an LC/MS system), contact closure, and line power are on the instrument’s back panel (see

Figure 4 ).

Figure 4.

Back panel of the EASY-nLC 1200 instrument shipped from the factory in San Jose, California, U.S.A.

Ethernet connection

USB LAN

LAN MAC ADDRESS: 00-6--E0-45-4E-28

RS-232

HARD

DRIVE

MONITOR

P-BUS

IN3 OUT3 IN2 OUT2 IN1 OUT1

I

O

0-120V▼ 11

220-240▼

120/230 V : 50/60 Hz: 250 W

Fuse at 120 V– : T5 AL 250 V

Fuse at 230 V– : T2.5 AL 250 V

Contact closure pins

Power entry module

Serial number and part number

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Back Panel

Table 2 lists the function of each back panel element.

Table 2. Back panel elements from top to bottom and left to right

Element

USB

LAN

Description

Port for keyboard or mouse or removable USB storage device, two (2) provided

For 10/100/1000 MB/sec Ethernet connection

RS-232

MONITOR

For communication with add-on devices, for example a syringe drive

Output for connection of external display

P-BUS For communication with add-on devices

IN/OUT pins Contact closure (primarily for communication with the mass spectrometer)

LAN MAC ADDRESS MAC address of the embedded computer (Gives the EASY-nLC instrument a unique network identifier on your local network.)

I|O Power switch

Fuse holder below

I|O switch

S/N

P/N

Replaceable fuse ratings:

• For 120 V, T 5 AL, 250 V

• For 230 V, T 2.5 AL, 250 V

Instrument serial number

Product order code

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Column Setup Options

Column Setup Options

Two solvent lines exit from the right side of the instrument. Behind the instrument’s right panel, the solvent line labeled Column Out connects to port 4 of valve S, and the solvent line labeled Waste In connects to port 2 of valve W.

The fittings and columns that ship with the EASY-nLC instruments are suitable for the

Nanospray Flex™ ion source. If your LC/MS system has an EASY-Spray™ ion source, use an

EASY-Spray column and a precolumn with nanoViper fittings to create a two-column

assembly (see Figure 80 on page 115 ). For information about setting up the column

assemblies for the Nanospray Flex and EASY-Spray ion sources, see “Setting Up the Column

Assembly” on page 95

.

You can connect the EASY-nLC instrument and enable the EASY-nLC software for these column configurations:

“Two-Column Setup,”

next section

“One-Column Setup” on page 8

Two-Column Setup

A two-column setup consists of a precolumn and an analytical column.

Tip When you install the two-column assembly, make sure that the One Column Setup check box is clear in the EASY-nLC device section on the Devices page of the

Maintenance menu (see

“Setting Up the Properties for the HPLC Device” on page 80

).

When the One Column Setup check box is clear (not selected), valve W moves to position 1–6 during the sample loading step. Excess solvent A exits the venting Tee and passes through valve W to the waste beaker.

Figure 5

shows the two-column assemblies for the Nanospray Flex ion source and the

EASY-nLC 1200 instrument, respectively.

Note The EASY-nLC 1200 instrument ships with nanoflow LC columns for both ion sources. When installing the EASY-nLC instrument, a Thermo Fisher Scientific field service engineer uses the appropriate column to test the instrument performance.

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Column Setup Options

Figure 5.

Two-column assembly for the EASY-nLC 1200 instrument with a Nanospray Flex ion source

Stainless steel union

Venting Tee PEEK™ union

VICI

Flow Flow

Precolumn

Analytical column

One-Column Setup

A one-column setup consists of an analytical column, the venting Tee, and a union and sleeve connection to an emitter. The Column Out solvent line and the analytical column connect to parallel legs of the venting Tee. The Waste In solvent line connects to the perpendicular leg of the Tee.

Tip When you install a one-column setup, select the One Column Setup check box in the EASY-nLC device section on the Devices page of the Maintenance menu (see

“Setting

Up the Properties for the HPLC Device” on page 80 ).

When the One Column Setup check box is selected, valve W remains in the 1–2 position during the sample loading step. Excess solvent A exits through the emitter tip.

IMPORTANT Make sure that you use the venting Tee for the one-column setup as well as for the two-column setup. Inside the instrument housing, the other end of the Waste In line connects to port 2 of valve W. During the Prepare Gradient step of a sample run, valve W provides pressurized venting to the solvent system through the venting Tee.

When the Waste In line is not connected to the venting Tee, the system pressure can only vent through the analytical column. Because venting through the column takes longer than venting through the Tee, the total run time is significantly longer than if you used the venting Tee for a one-column setup.

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Column Setup Options

Figure 6

shows the one-column assembly for the Nanospray Flex source and the EASY-nLC

1200 instrument, respectively.

Figure 6.

One-column setup for the EASY-nLC 1200 instrument and Nanospray Flex source

Column Out line

Venting Tee Analytical column

Union Emitter

VICI

Flow

Waste In line

Sleeves

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Solvent System Components

Solvent System Components

The EASY-nLC solvent system has three syringe pumps (A, B, and S); three pressure sensors, one for each pump; two flow sensors, one for pump A and one for pump B; four rotary valves

(A, B, S, and W); two check valve assemblies, one for solvent A and one for solvent B; and a mixing Tee where solvents A and B are mixed under high pressure.

For more information about the solvent system’s hardware components, see the following descriptions:

Syringe Pumps

(next section)

Pressure Sensors

Flow Sensors

“Six-Port Rotary Valves” on page 12

“Check Valve Assemblies” on page 14

“Mixing Tee” on page 14

Syringe Pumps

The syringe pumps perform these functions (see

Table 3 ).

Table 3. Syringe pump functions

Component

Pumps A and B

Pump S

Function

Draw solvent from the solvent bottles on top of the instrument, empty solvent to the waste beaker in the compartment to the right of the autosampler tray compartment, and deliver solvent to the mixing Tee.

Draws solvent from the W3 bottle, draws sample from the specified sample location into the needle, and empties solvent to the W4 bottle in the autosampler tray compartment.

CAUTION Solvent B acetonitrile concentration >95% can damage the instrument!

For maintenance purposes, the built-in instrument control software tracks the volume pumped by each pump. For information about recording maintenance for the pumps, refer to the EASY-nLC 1200 Troubleshooting and Maintenance Guide.

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Solvent System Components

Pressure Sensors

The three pressure sensors are connected to the solvent path between the syringe pump outlets and the six-port, three-position valves.

Using the strain gauge principle to measure pressure accurately, the pressure sensors are flow-through, zero-dead-volume (ZDV) devices with a wetted path that contains 12 μL of swept volume. The pressure sensors for the EASY-nLC 1200 instrument have an operating range of 0 to 20 000 psi (1379 bar).

Flow Sensors

Two flow sensors provide feedback control for pumps A and B. The two flow sensors are liquid mass flow meters with a patented technology. Each flow meter consists of a length of fused silica capillary, a sensor chip, a heating resistor, and two temperature sensors

(see

Figure 7

).

Figure 7.

Flow sensor schematic

Flow

Tubing

Heater

Sensor chip

Temperature sensors

Based on CMOSens™ Technology

Principle patented: US Pat. 6 813 944

The flow sensors determine the flow rate of solvents passing through the solvent system by measuring the heat transfer through the fused silica capillary. Outside the capillary, a heating resistor on a thermally optimized membrane is maintained above ambient temperature. When liquid flows through the capillary, the temperature distribution upstream and downstream of the heating resistor is disturbed. The two temperature sensors measure this temperature asymmetry.

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Solvent System Components

Because the heat transfer depends on both the solvent flow rate and the solvent mixture, you must recalibrate the flow sensors whenever you change the solvent types for solvents A and B.

For example, if you use methanol instead of acetonitrile for the solvent B mixture, recalibrate the flow sensors as described in the EASY-nLC 1200 Troubleshooting and Maintenance Guide.

CAUTION Solvent B acetonitrile concentration >95% can damage the instrument!

The EASY-nLC 1200 instrument uses two flow sensors with a maximum measuring range of 4.5 μL/min.

Tip When the flow rate produced by pump A or B exceeds the maximum measuring range of the respective flow sensor, use the pump position readback (x.x /140 μL) above the pump graphic to monitor the flow rate.

Six-Port Rotary Valves

The six-port rotary valves can be in three different positions: 1–2 position, 1–6 position, and centered (see

Figure 8 ).

Figure 8.

Valve positions

1–2 position 1–6 position Centered

2

3

1

4

6

5

2

3

1

4

6

5

2

3

1

4

6

5

For maintenance purposes, the built-in instrument control software tracks the number of valve shifts for each valve. For information about checking the devices usage counters, refer to the EASY-nLC 1200 Troubleshooting and Maintenance Guide.

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Solvent System Components

Depending on the valve position, the rotary valves perform these functions (see

Table 4 ).

Note The valve ports are only compatible with nanoViper fittings. Use of other connections may damage the valve.

Table 4. Rotary valve functions

Valve

A and B

S

Function

Direct the solvent flow from syringe pump A and B, respectively.

1–2 position Directs the solvent flow toward the check valve. When the rotary valve is in the 1–2 position, the pump draws solvent from the solvent bottle on top of the instrument or empties solvent to the waste beaker.

1–6 position Directs the solvent flow toward the mixing Tee.

Centered • Prevents backflow into the solvent line upstream of the mixing Tee.

• Allows the backpressure for the solvent A and B lines to equalize before the gradient step of a sample run.

• Provides backpressure for several maintenance scripts.

Directs the solvent flow from syringe pump S and the solvent flow from the mixing Tee.

1–2 position The solvent flow bypasses the sample loop.

1–6 position

Centered

The solvent flow passes through the sample loop.

• Provides backpressure for the Flush Air script for pump S.

W

• Provides backpressure for the system leak test.

• Shuts off the flow to the Column Out line and provides backpressure during the Flow Sensor

Calibration script.

Directs the solvent flow through the venting Tee to the waste beaker or provides pressure venting to the solvent system.

1–2 position

1–6 position

Centered

Provides backpressure to direct flow through the column.

Directs the solvent flow from the venting Tee to the waste beaker.

Blocks the solvent flow through the valve to the waste container.

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Solvent System Components

Check Valve Assemblies

When the corresponding high-pressure (6-port rotary) valves for pumps A or B are in position 1–2, the check valves perform these functions:

• The solvent-side check valve prevents backflow into the associated solvent inlet line when the associated pump is emptying solvent to the waste beaker.

• The waste-side check valve prevents backflow from the waste beaker when the associated pump is filling and drawing solvent from the associated solvent bottle.

Figure 9

shows the check valve connections.

Figure 9.

Check valve connections

From the solvent reservoir bottle on the top of the instrument

Solvent-side check valve

Connected to port 2 of valve A or B

Waste-side check valve

Routed to the waste beaker

Mixing Tee

The mixing Tee mixes solvents A and B before the solvent stream reaches valve S. When connecting the solvent lines to the mixing Tee, connect the solvent A and solvent B lines to perpendicular ports of the mixing Tee.

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Predefined Steps for Sample Runs

Predefined Steps for Sample Runs

During sample runs, the EASY-nLC instrument goes through a series of predefined steps.

“Overview of a Sample Run,” next section, provides a brief overview of the sample steps.

“Sequence of Events for Each Step of a Sample Run” on page 18

provides a more illustrative explanation of each step.

Overview of a Sample Run

During sample runs, the active steps are highlighted in bold on the Home > Overview page

(see

Figure 10 ).

Figure 10. Current step area of the Home > Overview page

Pickup sample

Load sample

Initialize system

Equilibrate precolumn

Prepare gradient Equilibrate analytical column

Gradient Autosampler wash + refill S

Refill AB

To minimize the cycle time, the instrument runs these steps in parallel:

• Refill pumps A, B, and S

• Column equilibration steps and Pickup Sample step

• Gradient step and Autosampler Wash + Refill S step

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Predefined Steps for Sample Runs

Table 5 summarizes the steps of a sample run.

Table 5. Sample run steps

Step

Refill A B

Precolumn and analytical column equilibration

Pickup sample

Load sample

Prepare gradient

Gradient

Autosampler wash

+ refill S

Action

Pumps A and B draw solvent from the solvent bottles on top of the instrument. The instrument then zeros the pressure sensors. At beginning of a sample run, the pumps are checked for drawn air after refilling.

Pump A delivers solvent to the system. For a two-column system, solvent A passes through the precolumn and then out to the waste beaker. After the precolumn equilibration finishes, valve W switches to position 1–2 to direct the solvent flow through the analytical column.

The autosampler moves the needle to the sample position specified in the batch table (injection sequence). Then pump S aspirates sample from the specified vial or well position into the sample loop attached to valve S.

With valve S in position 1–6, pump A delivers solvent to the system, flushing sample out of the sample loop and onto the precolumn or the analytical column. Valve W is in position 1–6 for a two-column system and position 1–2 for a one-column system.

The system vents through the Waste In line or builds up pressure to a precalculated pressure. Pump B builds up pressure to match the precalculated pressure of pump A. Pump B pumps a fixed volume of solvent B into the loop to ensure the alignment of solvents at the mixing Tee. Then valves S and W switch to position 1–2. With valves S and W in position 1–2, the solvent flow bypasses the sample loop and is directed through the column.

Pumps A and B deliver solvent to the system. As the percent composition of solvent B increases, analytes elute off the column or columns.

Pump S draws solvent from one or more wash bottles, washes the autosampler needle and the sample loop, and then refills. At beginning of a sample run, the pump is checked for drawn air after refilling.

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Predefined Steps for Sample Runs

Figure 11 shows a flow chart of the sample run steps.

Figure 11. Flow chart of the sample run steps

Initialize system—check that the hardware is ready.

Refill pump A.

Refill pump B.

Equilibrate precolumn

(for a two-column setup).

Refill pump S.

Pick up sample.

Equilibrate analytical column.

Load sample.

Prepare gradient.

Refill pump A.

Run gradient.

Refill pump B.

Wash autosampler needle and refill pump S.

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Predefined Steps for Sample Runs

Sequence of Events for Each Step of a Sample Run

To understand what happens during a sample run, see the following step descriptions:

“Refill Pumps A, B, and S Step,” next section

“Pickup Sample Step” on page 20

“Column Equilibration Steps” on page 22

“Load Sample Step” on page 24

“Prepare Gradient Step” on page 26

“Gradient Step” on page 29

“Autosampler Wash Step” on page 30

Refill Pumps A, B, and S Step

The instrument refills pumps A and B. With valves A and B in the 1–2 position, the corresponding check valves control the direction of the solvent flow (see

Figure 12 ). When the

pump begins to draw solvent from the solvent bottle, the corresponding check valve assembly opens to the solvent bottle and closes to the waste beaker. After a brief waiting period, the instrument zeros pressure sensors A and B.

As pumps A and B are refilling, the autosampler needle moves to the refill location (w3) and pump S draws solvent, if not already full. Once a pump is refilled, the pressure sensor of this pump is zeroed and a check for trapped air inside this pump is performed. During this check the respective valve moves to CENTER position.

During the air check of pump S, the autosampler needle moves to the W4 location.

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Predefined Steps for Sample Runs

Figure 12. Valves A and B in position 1–2 and pumps A and B in fully retracted position

Autosampler needle

Waste In line

Solvent A

Check valve assembly A

Solvent B

Check valve assembly B

Column

Out line

2

3

1

S

4

6

5

1

2

3

W

4

6

5

2

3

1

A

4

5

6

Flow sensor A

2

3

1

B

4

6

5

Flow sensor B

Mixing Tee

Waste beaker

Thermo Scientific

Pump S Pump A Pump B

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Predefined Steps for Sample Runs

Pickup Sample Step

The Pickup Sample step includes this sequence of actions.

Action

1

2

3

4

5

6

7

Pump S moves to a fixed position (filled with approximately 100 μL of solvent for an instrument with a 20 μL sample loop) by either dispensing excess solvent to the W4 bottle or drawing solvent from the W3 bottle.

Pump S draws 1 μL of air (the first air segment) into the needle.

The XYZ robot moves the needle holder to the specified sample vial or microplate well location.

The needle descends into the sample solution.

Pump S draws the requested sample pickup volume into the needle.

Note The EASY-nLC instrument performs only partial loop injections.

To avoid drawing sample into pump S, the application prevents you from entering a sample pickup volume larger than the sample loop size minus two microliters. The extra two microliters are required for the two air segments that bracket the sample.

The XYZ robot raises the needle out of the sample solution.

To transfer the sample into the loop, pump S draws a fixed volume of air into the needle. This fixed volume of air (~10.25 μL) is equal to the needle volume plus the valve port-to-port volume plus an additional 1 μL of air (the second air segment) to bracket the sample solution inside the loop.

Figure 13 shows pump S drawing the first air segment into the needle. Figure 14

shows pump S drawing the first air segment into the sample loop, while drawing the requested

sample volume into the needle. Figure 15

shows pump S drawing the second air segment through the needle and into the sample loop. At the end of the Pickup Sample step, the sample loop holds the requested sample volume of 10 μL, two 1 μL air segments, and approximately 8 μL of solvent A.

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Predefined Steps for Sample Runs

Figure 13. Pickup Sample step—with pump S drawing the first air segment into the needle

First air segment

120 80 40 0

Pressure sensor

Pump S

2

3

1

S

4

6

5

W3 bottle

Column Out line

From the mixing Tee

Figure 14. Pickup Sample step—with pump S drawing 10 μL of sample from a sample vial

W4 bottle Sample vial

120 80 40 0

Pressure sensor

Pump S

First air segment

2

3

1

S

4

6

5

W3 bottle W4 bottle Sample vial

Column Out line

From the mixing Tee

Figure 15. Pickup Sample step—with the requested sample volume loaded into the loop and bracketed by two air segments

First air segment

120 80 40 0

Pump S

Pressure sensor

Second air segment

From the mixing Tee

Thermo Scientific

2

3

1

S

4

6

5

W3 bottle

Column Out line

W4 bottle Sample vial

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Predefined Steps for Sample Runs

Column Equilibration Steps

For a one-column setup, the sample run includes the analytical column equilibration step. For a two-column setup, the sample run includes both a precolumn equilibration step and an analytical column equilibration step.

The Equilibrate Precolumn step (see Figure 16 ) includes this sequence of actions.

Action

1

2

Valve A and valve W switch to position 1–6. Valve B switches to the centered position. Valve S switches to position 1–2.

Pump A pushes the specified solvent volume through the precolumn at the specified pressure or flow rate. Excess solvent A exits the venting Tee and passes through valve W to the waste beaker.

The analytical column equilibration step includes this sequence of actions.

Action

1

2

For a two-column setup, valve A remains in position 1–6, valve B remains in the centered position, and valve S remains in position 1–2. Valve W switches to position 1–2.

For a one-column setup, valve A switches to position 1–6. Valve B switches to the centered position. Valve S switches to position 1–2. Valve W switches to position 1–2.

Pump A pushes the specified solvent volume through the analytical column at the specified pressure or specified flow rate.

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EASY-nLC Series Getting Started Guide Thermo Scientific

Figure 16. Equilibrate Precolumn step

Pressure sensor

2

3

1

A

4

6

5

Pressure sensor

2

3

1

B

4

6

5

To the autosampler needle

Flow sensor

Flow sensor

Mixing Tee

2

3

1

S

4

5

6

Column Out line

Waste In line

1

2

3

W

4

6

5

1

Introduction

Predefined Steps for Sample Runs

Precolumn

Venting

Tee

Analytical column

Waste beaker

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Predefined Steps for Sample Runs

Load Sample Step

After pump S draws the sample into the sample loop (see “Pickup Sample Step” on page 20 ),

valve S switches to position 1–6 and pump A pushes solvent A through the sample loop, flushing the sample out of the sample loop into the Column Out line and onto the precolumn for a two-column setup or the analytical column for a one-column setup.

The Load Sample step includes this sequence of actions.

Action

1

2

Valve S switches to position 1–6.

Valve W does one of the following:

• Switches to position 1–6 for a two-column setup allowing excess solvent A to flow to the waste beaker.

• Remains in the 1–2 position for a one-column setup, directing the solvent flow through the analytical column.

Pump A pushes the specified solvent volume through the sample loop at a specified pressure or the specified flow rate. In a two-column system, solvent A vents to the waste beaker through valve W.

Figure 17 shows the Load Sample step of a sample run for a two-column setup. Valves A, B, S,

and W are in the 1–6 position.

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Predefined Steps for Sample Runs

Figure 17. Load Sample step (two-column setup)

Pressure sensor

2

3

1

A

4

6

5

Pressure sensor

Solvent A

Solvent B

Sample

2

3

1

B

4

6

5

Centered

Second air segment

Flow sensor

Flow sensor

Mixing Tee

2

3

1

S

4

5

6

Column Out line

Waste In line

1

2

3

W

4

6

5

Waste beaker

Sample

Precolumn

Venting

Tee

Analytical column

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Predefined Steps for Sample Runs

Prepare Gradient Step

Before the gradient begins, this sequence of actions occurs.

Action

1

2

3

4

5

The built-in instrument control software calculates the volume of solvent A required to complete the gradient program. Pump A refills if it does not contain a sufficient volume of solvent to complete the gradient program.

With valves A, W, and S in position 1–6 and valve B in the centered position, the system vents through the Waste In line to a precalculated pressure, or increases the pressure to the precalculated pressure with valve W in position 1–2.

Pump B builds up pressure to match the precalculated pressure of pump A.

Valve B switches to position 1–6. If valve W was in position 1–2 in the previous step, it also switches to position 1–6. The Intelligent Flow Control

(IFC™) system now controls pumps A and B at the precalculated pressure.

With all of the valves in position 1–6, pump B pumps a fixed volume of 0.5 μL of solvent B toward valve S to ensure correct alignment of solvents A and B up

to valve S. See Figure 18 .

Valve W switches to position 1–2. The Automatic Flow Control (AFC™) system now controls pumps A and B.

To align solvents A and B up to valve S, the system delivers a gradient consisting of a minimum of 2% solvent B or the gradient start solvent B%, whichever is higher, until it pumps 0.4 μL of solvent A into the sample loop

(see

Figure 19 ).

Valve S moves to position 1–2, which allows the mobile phase to bypass the sample loop, and the instrument sends the closure signal to the mass spectrometer.

Note During the Prepare Gradient step, a slight amount of solvent B enters the sample loop. This does not affect the next run because the Autosampler Wash step flushes the sample loop before the next run starts.

Figure 18 shows 0.5 μL of solvent B being pumped toward valve S during the Prepare

Gradient step. Figure 19 on page 28

shows 0.4 μL of solvent A being pumped toward valve S during the Prepare Gradient step.

26

EASY-nLC Series Getting Started Guide Thermo Scientific

Figure 18. Excess 0.5 μL of solvent B pumped toward valve S during the Prealign Gradient step

Pump A

Pressure sensor

2

3

1

A

4

5

6

Flow sensor

Mixing Tee

2

3

1

S

4

5

6

Pump B

Pressure sensor

Flow sensor

2

3

1

B

4

5

6

1

2

3

W

4

6

5

1

Introduction

Predefined Steps for Sample Runs

Waste beaker

Solvent A

Solvent B

Thermo Scientific EASY-nLC 1200 Getting Started Guide

27

1

Introduction

Predefined Steps for Sample Runs

Figure 19. The system pumps a mixture of 2% solvent B or higher until it pumps 0.4 μL of solvent A.

Pump A

Pressure sensor

2

3

1

A

4

5

6

Flow sensor

Mixing Tee

2

3

1

S

4

5

6

Pump B

Pressure sensor

Flow sensor

2

3

1

B

4

5

6

1

2

3

W

4

6

5

Waste beaker

Solvent A

Solvent B

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EASY-nLC Series Getting Started Guide Thermo Scientific

1

Introduction

Predefined Steps for Sample Runs

Gradient Step

The gradient step includes this sequence of actions.

Action

1

2

The instrument runs the gradient program.

Note With valve S in position 1–2, the mobile phase bypasses the sample loop during the gradient program. Therefore, the sample loop does not add any gradient delay volume to the system.

If either pump runs out of solvent during the gradient run, both pumps refill.

The refilling process takes approximately 2 minutes.

Figure 20 shows the gradient step of a sample run. Mobile phase from the mixing Tee bypasses

the sample loop as it enters and exits valve S through ports 4 and 3, respectively.

Figure 20. Gradient step with valve A and valve B in position 1–6 and autosampler wash step with valve S in position 1–2

Pump A

Pressure sensor

Column Out line

Sample loop

2

3

1

A

4

6

5

Flow sensor

Mixing Tee

2

3

1

S

4

5

6

Pump B

Pressure sensor

Flow sensor

Waste In line

2

3

1

B

4

6

5

1

2

3

W

4

6

5

Thermo Scientific

Solvent A

Solvent B

EASY-nLC 1200 Getting Started Guide

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1

Introduction

Predefined Steps for Sample Runs

Autosampler Wash Step

The standard autosampler wash step includes this sequence of actions.

Action

1

2

3

The autosampler needle moves to the W4 bottle.

Pump S pushes the specified volume of solvent through the sample loop and needle. As the wash solvent exits the needle, it overfills the needle insert and

washes the exterior surface of the needle. See Figure 21 .

Pump S refills with solvent A from the W3 bottle.

The Autosampler Wash and Refill S step occurs in parallel with the Gradient step.

Figure 21. Autosampler wash step with valve S in position 1–2

Autosampler needle

Pump S

Pressure sensor

W3 bottle

W4 bottle

30

EASY-nLC Series Getting Started Guide

Solvent A

Solvent B

Mixing

Tee

Sample loop

2

3

1

S

4

6

5

Column Out line

Thermo Scientific

1

Introduction

Pump Flow Control

Pump Flow Control

The EASY-nLC instrument contains split-free, high-pressure syringe pumps capable of delivering flows from 1 nL/min to 300 μL/min.

These flow control systems optimize instrument performance:

“Automatic Flow Control System,”

next section

“Intelligent Flow Control System” on page 32

“Load Speed Protection System” on page 33

Automatic Flow Control System

The AFC system (see Figure 22 ) is active during these processes:

• The gradient step of a sample run

• The execution of the isocratic flow maintenance script

By using the flow sensor feedback to regulate pumps A and B, the AFC system accurately maintains the programmed flow rate and solvent composition, even during sudden pressure changes caused by solvent composition changes. For accuracy, the AFC system requires a minimum pressure of 20 bar.

Figure 22. Schematic diagram of the AFC system

Pump A

Pressure sensor A

Flow sensor A

2

3

1

A

4

6

5

A/B mixing

Tee

AFC

TM

Pump B

Pressure sensor B

2

3

1

B

4

6

5

Flow sensor B

Thermo Scientific EASY-nLC 1200 Getting Started Guide

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1

Introduction

Pump Flow Control

Intelligent Flow Control System

The IFC system (see Figure 23

) is active during these processes:

• The column equilibration and sample loading steps of a sample run

• The first part of the prepare gradient step for pumps A and B

• The execution of the precolumn equilibration, analytical column equilibration, leak, and backpressure scripts

By using both the pressure sensor and flow sensor feedback, the IFC system continually regulates pumps A and B. By regulating the pumps A and B with both a flow and pressure feedback system, the IFC system enhances system performance as follows:

• Reduces the loss of productivity caused by instrument stoppage related to overpressure

(for instance, stoppage caused by viscous samples during the sample loading step).

• Uses the pressure range of the instrument more effectively. For example, when you set a maximum pressure for the sample loading and column equilibration steps instead of a flow rate, the system automatically adjusts the flow rate to the maximum acceptable value.

• Builds the pressure rapidly during the prepare gradient step.

Figure 23. Schematic diagram of the IFC system

Pump A

Pressure sensor A

Flow sensor A

2

3

1

A

4

5

6

A/B mixing

Tee

IFC TM

Pump B

Pressure sensor B

2

3

1

B

4

5

6

Flow sensor B

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EASY-nLC Series Getting Started Guide Thermo Scientific

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Introduction

Pump Flow Control

You can program the column equilibration and sample loading steps as follows:

• Specify both a set flow and a maximum pressure. The setting reached first (which becomes the limiting parameter) depends on the dimensions of the attached column or columns. If the system reaches the set pressure before it reaches the set flow rate, the IFC system reduces the flow rate to maintain the pressure. If the system reaches the set flow rate before the pressure limit, the pump maintains the set flow rate.

• Specify only a set flow. The IFC system maintains the set flow rate unless the pressure exceeds the maximum system pressure.

• Specify only a maximum pressure. The IFC system adjusts the flow rate to maintain the set pressure.

The duration of both the equilibration and loading steps depends on the total volume of solvent specified and the actual flow rate. When the set pressure is the limiting factor, the system adjusts the actual flow rate, which varies the duration of these steps.

Tip If the duration of the equilibration and loading steps must be fixed, limit these steps by flow rather than by pressure. Use the appropriate precolumn and analytical column maintenance scripts to determine the appropriate flow rates for your LC columns.

Load Speed Protection System

When the Load Speed Protection system (LSP) is on, the batch run stops if the system reaches the set max pressure before it reaches the set flow during the sample loading step. Stopping the batch run when the system pressure rises above an acceptable level prevents the system from running the remaining samples when the column or tubing has become clogged.

For instructions about how to turn on the Load Speed Protection system, see “Setting Up the

Properties for the HPLC Device” on page 80

.

Thermo Scientific EASY-nLC 1200 Getting Started Guide

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2

Installing the EASY-nLC Instrument

To install the EASY-nLC instrument or move the instrument from one laboratory benchtop to another, follow the installation instructions in this chapter after reviewing the

“Cautions and Special Notices” on page xiii .

Note This chapter describes the laboratory requirements and the back panel connections for the EASY-nLC instrument as a stand-alone LC system or as an inlet to a mass spectrometer.

• For information about installing the LC columns, the sample plates, the solvent

bottles, and the wash bottles, see Chapter 5, “Preparing the EASY-nLC Instrument for Use.”

• For information about connecting the column assembly to the mass spectrometer’s ion source, refer to the manual provided with the ion source.

• For information about the internal solvent lines, refer to the EASY-nLC 1200

Troubleshooting and Maintenance Guide.

Contents

Lifting Instructions

Laboratory Requirements

Back Panel Connections

Thermo Scientific EASY-nLC 1200 Getting Started Guide

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2

Installing the EASY-nLC Instrument

Lifting Instructions

Lifting Instructions

The EASY-nLC 1200 instrument weighs 35 kg (77.2 lb). For safety reasons, use two people to move the instrument to a table cart for transport and wear gloves.

CAUTION At more than 30 kg (66 lbs), the EASY-nLC instrument is too heavy for one person alone to lift. When moving the instrument, use two people to place it on a table cart for transport.

CAUTION Before you move the EASY-nLC instrument from one benchtop to another, close down the EASY-nLC instrument (see

“Closing Down the EASY-nLC Instrument” on page 67 ), disconnect the EASY-nLC instrument from line power, the detector, and the

data system computer (if applicable). For information about installing or removing the column assembly from the mass spectrometer’s ion source, refer to the manual provided with the ion source.

Figure 24 shows the lift points for the EASY-nLC instrument. With one person on each side

of the instrument, lift the instrument from the bottom with one hand while stabilizing the top portion of the instrument with the other hand.

Figure 24. Lifting the EASY-nLC 1200 instrument by hand

EASY - nLC 1200

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EASY-nLC 1200 Getting Started Guide Thermo Scientific

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Installing the EASY-nLC Instrument

Laboratory Requirements

Laboratory Requirements

This section describes the laboratory requirements for the EASY-nLC instrument. For more information about the requirements for the EASY-nLC instrument, refer to the EASY-nLC

Series Preinstallation Requirements Guide. For information about the preinstallation requirements for a Thermo Scientific mass spectrometer, refer to its preinstallation requirements guide.

Requirements:

“Benchtop Dimensions and Weight Capacity,” next section

“Internet Access” on page 39

“Power and Fuses” on page 39

“Temperature and Humidity” on page 39

Benchtop Dimensions and Weight Capacity

Place the EASY-nLC instrument on a benchtop or movable table that can support a minimum of two times the instrument weight. For the EASY-nLC instrument, make sure that the table can support a minimum of 70 kg (154 lb).

Table 6 lists the dimensions of the EASY-nLC 1200 instruments. Allow for at least 15 cm

(6 in.) of free space at the back of the instrument for proper air circulation.

Table 6. EASY-nLC instrument dimensions

Dimension

Width

Depth

EASY-nLC 1200

36 cm (14.2 in.)

38 cm (15.1 in.)

Thermo Scientific

To minimize extra column volume between the LC column outlet and the detector inlet, place the EASY-nLC instrument as close as possible to the high-performance liquid chromatography (HPLC) detector, mass spectrometer, or both. However, to avoid exposure to direct heat, place the EASY-nLC instrument away from any detector or mass spectrometer vents.

Figure 25 shows a typical benchtop setup for an EASY-nLC instrument connected to a

Thermo Scientific mass spectrometer. Connecting the data system computer to your local area network (intranet) requires an additional network card.

Using the setup shown in

Figure 25 , you can control the EASY-nLC instrument from the

local data system computer or from another data system computer on the intranet; however, the EASY-nLC instrument might not be able to communicate with the remote support server.

For information about connecting the EASY-nLC instrument to the remote support server, see

“Setting Up the Ethernet Connection to the Support Server” on page 47 .

EASY-nLC 1200 Getting Started Guide

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Installing the EASY-nLC Instrument

Laboratory Requirements

CAUTION Before you move the EASY-nLC instrument from one benchtop to another, close down the EASY-nLC instrument (see

“Closing Down the EASY-nLC Instrument” on page 67 ), disconnect the EASY-nLC instrument from line power, the detector, and the

data system computer (if applicable). For information about installing or removing the column assembly from the mass spectrometer’s ion source, refer to the manual provided with the ion source.

Note

Figure 25

shows the EASY-Spray source, which gets its power through the P-bus cable connection to the EASY-nLC instrument.

Figure 25. EASY-nLC instrument and LTQ™ Series MS detector setup

Solvent lines

Ion source monitor

LTQ Series

MS detector

Data system computer

EASY-nLC 1200 instrument

P-bus cable

Load

Inject

Detector

Waste

Power Vacuum Communication System Scanning

Ethernet switch

Pump On

Ethernet cable from the Ethernet switch to the data system computer

Intranet connection

(Ethernet jack)

Power line connection

Ethernet cable from the EASY-nLC instrument to the Ethernet switch

Contact closure cable from the

EASY-nLC instrument to the MS detector

Ethernet cable from the MS detector to the Ethernet switch

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EASY-nLC 1200 Getting Started Guide Thermo Scientific

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Installing the EASY-nLC Instrument

Laboratory Requirements

Internet Access

For online support and monitoring (remote support), provide Internet access for the

EASY-nLC instrument within reach of the laboratory benchtop.

IMPORTANT The EASY-nLC instrument uses Secure Shell (SSH™), a network protocol for secure data communication, to connect to the support server, which uses the Linux™ operating system. Port 22 is the standard TCP port for SSH programs.

To use the remote support feature, the firewalls for your local network must allow outgoing TCP/IP traffic from the EASY-nLC instrument to the support server at

IP address 46.226.219.10, port 22.

Power and Fuses

Use the EASY-nLC instrument only with properly grounded appliances and power sources.

You can plug the EASY-nLC instrument into these grounded line power sources:

• 120 Vac, 50/60 Hz, 250 W

• 230 Vac, 50/60 Hz, 250 W

Note To determine the appropriate rating for an uninterruptible power supply (UPS), assume it is 250 W.

The fuse requirements for the EASY-nLC instrument are as follows:

• For 120 Vac, one T 5 AL 250 V fuse (5 × 20 mm, IEC 60127)

• For 230 Vac, one T 2.5 AL 250 V fuse

All fuses supplied with the instrument are UL Listed and CSA certified.

Note Depending on the nanospray ion source, your nanospray system might need an additional power receptacle. The EASY-Spray ion source requires an additional power receptacle, whereas the Nanospray Flex ion source draws its line power from the mass spectrometer.

Temperature and Humidity

Avoid locations with high air humidity or extreme changes in temperature (such as direct sunlight, drafts, directly below an air conditioning or heating vent, or directly next to the mass spectrometer or HPLC detector vent).

For optimal autosampler plate cooling, place the unit in an area where the working temperature is within 5 to 30 °C (41 to 86 °F).

The optimal humidity range is between 20 to 80% RH. Avoid condensing humidity.

Thermo Scientific EASY-nLC 1200 Getting Started Guide

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Installing the EASY-nLC Instrument

Back Panel Connections

Back Panel Connections

To connect the EASY-nLC instrument to a mass spectrometer, the intranet within your facility, the Internet for remote support, and other peripheral hardware, follow these procedures:

“Connecting to Line Power” on page 41

“Connecting to the Mass Spectrometer through Contact Closure” on page 42

“Connecting the Ethernet Communication Cables” on page 44

“Attaching a Mouse and Keyboard to the USB Connections” on page 48

“Attaching Add-on Devices through the P-Bus and RS-232 Ports” on page 48

Figure 26 shows an LC/MS system with an EASY-nLC instrument, an LTQ Series mass

spectrometer, and a data system computer. The LC system, mass spectrometer, and data system hardware are connected to three separate fourplex outlets that are hardwired to a common protective earth ground. This figure does not show the monitoring system for the nanoflow ion source or the external rotary pumps for the mass spectrometer.

Figure 26. LC/MS system with an EASY-nLC instrument, an LTQ Series mass spectrometer, and a data system computer

Data system computer and Ethernet switch

EASY-nLC instrument

(back panel)

LTQ Series mass spectrometer

(right panel)

Fourplex outlet for the data system hardware

PRO EON

P-BUS

P-BUS

IN3 OUT3 IN2 OUT2 IN1 OUT1

IN3 OUT3 IN2 OUT2 IN1 OUT1

USB

LAN RS-232

PS/2 LAN

HARD

HARD

DRIVE

MONITOR

MONITOR

This device complies with Part 15 of the FCC Rules.

Operation is subject to the following two conditions:

(1) This device may not cause harmful interference, and

(2) This device must accept any interference received, including

interference that may cause undersired operation.

DEVICE: EASY-NLC

DEVICE: EASY-NLC

P/N LC100

P/N LC100

LAN MAC ADDRESS: 00-6--E0-45-4E-28

Proxeon Biosystems A/S

Thermo Fisher Scientific A/S

Web: www.proxeon.com

Phone +45 6557 2300 Fax +45 6557 2301

0-120V▼

O

O

120/230 V : 50/60 Hz: 250 W

Fuse at 120 V– : T5 AL 250 V

Fuse at 230 V– : T2.5 AL 250 V

Contact closure cable

Peripheral Control

Start

In

Ready

Out

Start

Out

Ethernet

100 Base T

Electronics Normal

Reset

1V MAX 10V MAX

Analog Input

Service Mode

Main Power

On

Mech. Pumps/Accessory

A 5.5 MAX

Power In

230 V AC

15 A

A 5.5 MAX

Off

Laboratory

LAN port

Ethernet cables

40

EASY-nLC 1200 Getting Started Guide

Fourplex outlet for the

EASY-nLC system

Fourplex outlet for the mass spectrometer

Thermo Scientific

2

Installing the EASY-nLC Instrument

Back Panel Connections

Connecting to Line Power

Each EASY-nLC instrument comes with a 2.5 m (8.2 ft) long power cord.

CAUTION Power cords can become frayed and damaged with use. If the power cord is frayed or damaged, replace it with an equivalent power cord.

To meet compliance and safety requirements, the replacement power cord must be no longer than 2.5 m (8.2 ft) and be certified by recognized organizations for your country

(for example, UL, CSA, SEMKO, VDE, or TÜV).

Installing an EASY-nLC instrument requires a minimum of one outlet. The electrical outlet must be earth ground hard-wired to the main panel.

If you are using the EASY-nLC instrument as an inlet to an MS detector, the LC/MS system might require two or more fourplex outlets. The interconnected electrical outlets for the

LC/MS system and the data system computer must have a common point to one ground

connector (see Figure 26 on page 40 ).

CAUTION When using the EASY-nLC instrument as an inlet to an LC/MS system, the

EASY-nLC instrument, the mass spectrometer, the nanospray ion source (if it connects to line power) and the (optional) data system hardware must have a common ground.

Connecting the hardware to external grounds at different potentials can do the following:

• Create a ground loop that causes noise and interference.

• Damage the EASY-nLC instrument’s built-in computer.

CAUTION Improper grounding of the instrument creates an electrical safety hazard.

To connect the EASY-nLC instrument to line power

1. If you are moving an existing instrument from one benchtop to another, make sure that the fuse is installed correctly.

For information about installing the fuse, refer to the EASY-nLC 1200 Troubleshooting

and Maintenance Guide.

2. Connect the supplied power cord to the power entry module on the back panel of the

EASY-nLC instrument and to a properly grounded electrical outlet.

Thermo Scientific EASY-nLC 1200 Getting Started Guide

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Installing the EASY-nLC Instrument

Back Panel Connections

Connecting to the Mass Spectrometer through Contact Closure

The EASY-nLC touch-screen application contains preconfigured values for most of the standard mass spectrometers used in proteomics. For instructions about setting up the software configuration for the mass spectrometer connection, see

“Setting Up

Communication with the Mass Spectrometer” on page 69

.

IMPORTANT Occasionally, Thermo Fisher Scientific releases new software for the

EASY-nLC instrument. Downloading new software files to the EASY-nLC computer erases the mass spectrometer selection.

Figure 27 and Figure 28

show the hardwire connections between the EASY-nLC instrument and an LTQ Series mass spectrometer or Orbitrap Fusion™ mass spectrometer, respectively.

Figure 27. Two-way contact closure connection for an LTQ Series mass spectrometer

EASY-nLC 12-pin connector Mass spectrometer input and output pins

Figure 28. Two-way contact closure connection for an Orbitrap Fusion mass spectrometer

EASY-nLC 12-pin connector Mass spectrometer input and output pins

To make contact closure between the EASY-nLC instrument and the Orbitrap Fusion mass spectrometer, you must modify the Thermo Fisher contact closure cable by replacing one of the two-pin terminal blocks with the 8-pin terminal block provided in the MS Setup Kit.

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EASY-nLC 1200 Getting Started Guide Thermo Scientific

Thermo Scientific

2

Installing the EASY-nLC Instrument

Back Panel Connections

To modify the contact closure cable for the Orbitrap Fusion mass spectrometer

1. Using a small flat-blade screwdriver, loosen the screws for pins 1 and 2. Then pull the wires out of the terminals.

2. Using a small flat-blade screwdriver, connect the cable wires to the 8-pin terminal block provided in the Orbitrap Fusion MS Setup Kit as follows:

• Connect the yellow wire to terminal 1.

• Connect the red wire to terminal 4.

Figure 29 shows the pin outs for the modified MS connector.

Figure 29. 8-pin terminal block with wires connected

To connect the contact closure cable

Using a contact closure cable provided by Thermo Fisher Scientific (see

Table 7

), connect the cable to the mass spectrometer and the EASY-nLC instrument.

For more information, refer to the graphic for the selected mass spectrometer in the

Contact Closure area of the Configuration > Connections page.

Table 7. Contact closure cables

Description

Thermo Fisher contact closure cable

ABI/MDS Sciex™ contact closure cable

Bruker™/Agilent™ contact closure cable

Waters™/Micromass™ contact closure cable

Varian™ contact closure cable

(Varian is now a part of Agilent Technologies Inc.)

Part number

LC160

LC161

LC162

LC163

LC164

EASY-nLC 1200 Getting Started Guide

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Installing the EASY-nLC Instrument

Back Panel Connections

Connecting the Ethernet Communication Cables

How you set up the Ethernet communication cables depends on the type of communication you are trying to establish.

Follow the appropriate procedure to set up communication between the EASY-nLC computer and the data system computer, the intranet for data exchange, or the remote support server.

Setting Up the Ethernet Connections to the Data System Computer

Setting Up the Ethernet Connection to a Local Network for Data Exchange

Setting Up the Ethernet Connection to the Support Server

Setting Up the Ethernet Connections to the Data System Computer

For an LC/MS system with an EASY-nLC instrument and a Thermo Scientific mass spectrometer, you operate the system from a data system computer with the Xcalibur data system and the device drivers for the EASY-nLC instrument and a Thermo Scientific mass spectrometer.

Note You can create instrument methods and injection sequences, submit and process injection sequences, and monitor the instrument status and injection progress with the external data system. The external data system does not include maintenance scripts or direct controls for the EASY-nLC instrument. To perform tasks such as flushing air from the LC system, you must use the touch-screen application.

Figure 25 on page 38 and

Figure 26 on page 40

show a typical benchtop setup for an LC/MS system with an EASY-nLC instrument, a Thermo Scientific mass spectrometer, a data system computer, and an Ethernet switch.

The EASY-nLC computer and the Thermo Scientific mass spectrometer communicate with the data system computer through an Ethernet connection routed through an Ethernet switch connected to the data system computer. To make the Ethernet connections, use the shielded

Ethernet cables supplied in the instrument accessory kits.

When you order the EASY-nLC instrument with a Thermo Scientific mass spectrometer, the

Ethernet card for the LC/MS system is preset at the factory to this IP address: 172.16.0.101.

While installing the LC or LC/MS system, your local Thermo Fisher Scientific field service engineer checks the IP address setting.

If the data system computer has a second network card, you can connect the data system computer to the intranet. After you connect the data system computer to the intranet, you can view the files on the EASY-nLC instrument’s computer hard drive from other computers on the intranet. When you also set up a remote desktop connection, you can monitor and control the LC/MS system from other computers on the intranet.

For information about setting up a remote desktop connection, refer to the Microsoft™

Windows™ operating system Help.

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EASY-nLC 1200 Getting Started Guide Thermo Scientific

Thermo Scientific

2

Installing the EASY-nLC Instrument

Back Panel Connections

To connect the Ethernet cables for an LC/MS system controlled from a data system computer

1. Connect the EASY-nLC instrument and the Thermo Scientific mass spectrometer to the

Ethernet switch as follows: a. Plug a shielded Ethernet cable into the LAN port on the instrument’s back panel.

b. Plug the other end of the cable into the Ethernet switch.

2. Connect the Ethernet switch to the data system computer as follows: a. Verify that the IP address for the dedicated Ethernet card is set to 172.16.0.101.

b. Plug a shielded Ethernet cable into the appropriate Ethernet port in the data system computer. c.

Plug the other end of the cable into the Ethernet switch.

3. If the Ethernet switch has a power save button, make sure that the button is in the Off position.

The current model of the Ethernet switch that ships with Thermo Scientific mass spectrometers has a power save button labeled ECO friendly.

IMPORTANT When the power-save feature is on, the instrument can lose communication with the data system computer.

4. To connect the data system computer to the intranet, go to the next procedure, “To connect the data system computer to the intranet.”

To connect the data system computer to the intranet

1. If the data system computer does not already have a second network card, install one.

2. Using a shielded Ethernet cable, connect the network card’s LAN port to a laboratory

LAN port with intranet access.

When the data system computer is connected to a laboratory LAN port, you can access the admin folder on the EASY-nLC instrument’s computer hard drive.

To control the LC/MS system remotely from another computer on the intranet

1. Connect the hardware as described in these procedures:

To connect the Ethernet cables for an LC/MS system controlled from a data system computer

To connect the data system computer to the intranet

2. Set up a remote desktop connection between the data system computer and the remote local area computer.

For information about setting up a remote desktop connection, refer to the Windows operating system Help.

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Installing the EASY-nLC Instrument

Back Panel Connections

Setting Up the Ethernet Connection to a Local Network for Data Exchange

You can access the chromatographic methods and injection batches stored on the EASY-nLC instrument’s computer hard drive after you make the following connections:

• When you connect a stand-alone EASY-nLC instrument to a laboratory LAN port with intranet access, you can access these files from other computers on the intranet.

• When you connect the EASY-nLC instrument to a data system computer through an

Ethernet switch, you can access these files from the data system computer.

• When you connect the EASY-nLC instrument to a data system computer through an

Ethernet switch and connect the data system computer to a laboratory LAN port with intranet access, you can access these files from other computers on the intranet.

Note When you control the EASY-nLC instrument in the stand-alone mode, the chromatographic methods and batches reside on the integrated computer hard drive.

When you control the EASY-nLC instrument from a data system computer, your instrument methods and sequences created with the data system reside on the data system computer.

To access the files on the EASY-nLC instrument’s computer hard drive from another computer on the intranet

1. Do one of the following:

• For a stand-alone EASY-nLC instrument, use a shielded Ethernet cable to connect the LAN port on the back panel of the EASY-nLC instrument to a laboratory LAN port with intranet access.

• For an EASY-nLC instrument that is part of a Thermo Scientific LC/MS system,

connect the Ethernet cables as described in “Setting Up the Ethernet Connections to the Data System Computer” on page 44 .

2. Set up the software connection for the EASY-nLC instrument as described in “Setting Up the Local Area Network Connection” on page 72 .

With a Thermo Scientific LC/MS system, you can access the files on the EASY-nLC system from the data system computer. If you are already logged in to the EASY-nLC instrument’s computer, you do not need to reenter the user login and password.

To access the files on the EASY-nLC instrument’s computer hard drive from a computer on the intranet (other than the local data system computer for an LC/MS system), you must know the IP address and the user login and password for the EASY-nLC instrument.

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Installing the EASY-nLC Instrument

Back Panel Connections

Setting Up the Ethernet Connection to the Support Server

You can have Thermo Fisher Scientific Technical Support remotely diagnose problems with your EASY-nLC instrument.

Thermo Fisher Scientific recommends the following hardware setup when you want to establish a communication link between the EASY-nLC instrument and the remote support server.

For information about initiating the communication link with the support server, refer to the

Remote Support chapter in the EASY-nLC Series Troubleshooting and Maintenance Guide.

To connect the EASY-nLC to the local network that has Internet capability

1. If the EASY-nLC instrument is connected to a data system computer or an Ethernet switch as shown in

Figure 25 on page 38

, disconnect the Ethernet cable from the LAN port of the EASY-nLC instrument.

2. Using a Category 6 network cable, connect the EASY-nLC instrument directly to the local network that has Internet capability to access the support server (see

Figure 30

).

IMPORTANT For remote diagnostics, connect the EASY-nLC instrument directly to the Internet access port using the supplied Category 6 network cable.

To use the remote support feature, the firewalls for your local network must allow outgoing TCP/IP traffic from the EASY-nLC instrument’s computer to the support server at IP address 46.226.219.10, port 22.

Figure 30. EASY-nLC instrument connected to a local network port with Internet capability

EASY - nLC 1200

Network port with

Internet capability that allows outgoing traffic to port 22

Supplied Ethernet cable

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Installing the EASY-nLC Instrument

Back Panel Connections

Attaching a Mouse and Keyboard to the USB Connections

You can use a mouse and keyboard instead of the built-in touch screen, but Thermo Fisher

Scientific recommends using the touch-screen interface. For more information, see

“Using the

Touch Screen” on page 53

.

Connect the USB mouse or keyboard by using the USB connectors on the back panel

(see

Figure 4 on page 5

) of the instrument. The EASY-nLC application detects the mouse or keyboard within 30 seconds. The application does not recognize all USB-based keyboards or mice.

Attaching Add-on Devices through the P-Bus and RS-232 Ports

The EASY-nLC instrument can control and power add-on devices through the P-bus port, the RS-232 interface, or both. These ports are on the back panel of the instrument (see

Figure 4 on page 5 ).

To control the temperature of the EASY-Spray ion source’s column heater

1. Using the P-bus cable shown in

Figure 31

, connect the EASY-nLC instrument to the

EASY-Spray ion source as follows:

• Connect the P-BUS NLC end of the cable to the P-Bus port on the back panel of the

EASY-nLC 1200 instrument.

• Connect the P-BUS EASY-SPRAY SOURCE end of the cable to the P-Bus port on the bottom of the EASY-Spray ion source.

Figure 31. EASY-Spray source to EASY-nLC 1200 cable (P/N 70005-63055)

Connects to the P-Bus port on the back panel of the

EASY-nLC instrument.

P-BUS EASY

SPRAY SOURCE

70005-63055 P-BUS nLC 1000

Connects to the P-Bus port on bottom of the

EASY-Spray ion source.

The EASY-nLC instrument provides power to the EASY-Spray ion source through the

P-Bus cable.

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Installing the EASY-nLC Instrument

Back Panel Connections

2. Add the EASY-Spray ion source to the Devices list on the Maintenance > Devices page as described in

“Adding Devices to the EASY-nLC Devices List” on page 156

.

Tip If the temperature readout on the EASY-Spray ion source is blank, make sure that the P-Bus cable is properly connected and that the EASY-nLC instrument is turned on.

If the temperature readout in the EASY-Spray dialog box (available from the Home >

Overview page of the touch-screen application) displays a question mark and the temperature readout on the EASY-Spray ion source displays two vertical bars, make sure that the EASY-Spray column is plugged into the EASY-Spray ion source’s Heater port.

For more information about the EASY-Spray ion source, refer to the EASY-Spray

Series Ion Source User Guide.

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Integrated Instrument Control Software

Integrated Instrument Control Software

The EASY-nLC instrument is controlled by software that runs on an integrated computer; this means you can operate the instrument without the aid of a data system computer.

Note When you are using the EASY-nLC instrument as part of a Thermo Scientific

LC/MS system, you can use the Xcalibur data system to create chromatographic methods and batches and to acquire and process mass spectral data from the mass spectrometer.

For information about controlling the EASY-nLC instrument from the Xcalibur data system, refer to the data system Help. You can access Help from most of the user-interface pages by pressing the F1 key on the keyboard connected to the data system computer.

You can use the built-in instrument control application for the EASY-nLC instrument to set up instrument methods (sample processing protocols), schedule batches (injection sequences), view the run progress and instrument status, manage users, and carry out maintenance and repair work.

To familiarize yourself with the touch-screen application, review this chapter.

Contents

Turning On the EASY-nLC Instrument

Using the Touch Screen

Viewing the System Status

User Interface Layout and Application Menu Structure

Logging In to the EASY-nLC Instrument

Closing Down the EASY-nLC Instrument

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Turning On the EASY-nLC Instrument

Turning On the EASY-nLC Instrument

To turn on the instrument

Press the On/Off (I/O) switch on the back panel of the instrument ( Figure 32

).

Figure 32. On/Off switch on the back panel

The startup procedure takes approximately 5 to 10 minutes while the instrument does the following:

1. Starts the internal computer.

2. Initializes the software components.

3. Checks the hardware components (pumps, valves, autosampler, plate-cooler, flow sensor, and so on).

4. Starts the Linux application.

During this process, a status bar indicates progress on the startup screen.

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Using the Touch Screen

Using the Touch Screen

You control the EASY-nLC instrument directly from the touch screen.

Note You can attach a USB-based mouse and keyboard to the instrument’s back panel

(see

“Attaching a Mouse and Keyboard to the USB Connections” on page 48 ), but the

user interface has been optimized for touch-screen interaction.

The instrument monitor is a transparent layer of glass ( Figure 33

) that detects finger pressure

(even when the operator is wearing gloves) and sends the corresponding commands to the computer.

Figure 33. Touch-screen control for the EASY-nLC instrument

Home > Overview page

Thermo Scientific

The touch-screen controls consist of buttons, tables, input fields, and interactive graphical instrument components. To use the touch-screen controls, see these procedures:

Using the Buttons

Entering Alphanumeric Text

Using Tables and Input Fields

Using the Interactive Graphical Controls

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Integrated Instrument Control Software

Using the Touch Screen

Using the Buttons

Press buttons only one time for a specific action. If the action is irreversible or could cause a system error if the instrument is not prepared for the action, the EASY-nLC application displays a confirmation box where you can cancel or accept the action. For example, when you press Eject/Insert Tray on the Home > Overview page, a confirmation box appears to remind you to remove obstacles from the area in front of the tray compartment before opening the tray compartment door. To continue the action, you must press Accept.

Entering Alphanumeric Text

In most cases, pressing a button on the touch screen causes the instrument to carry out a certain operation. However, when the application requires text or numeric input, the application displays a keyboard or numeric keypad (

Figure 34 ) in front of the main window

so that you can enter values by pressing the appropriate keys. When you press a key, it is highlighted in blue until you press another key.

Figure 34. Touch-screen keyboard and numeric keypad

Using Tables and Input Fields

You can edit many tables (usually when you have entered the data yourself ). You can also access single cells by touching the cell twice to open a keyboard/keypad display for changing the entry.

Long tables have vertical scroll bars that you move either by pressing the up or down arrow symbols, or by dragging the scroll bar and moving it explicitly.

In some tables you can select multiple rows by pressing check boxes on several rows or complete columns by pressing the table headings.

If a screen has blank input fields, press inside the field to enter data. The application displays a keyboard/keypad.

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Integrated Instrument Control Software

Using the Touch Screen

Using the Interactive Graphical Controls

The four valves, three pumps, autosampler, and optional EASY-Spray ion source appear as interactive graphical components on the Home > Overview page (see

Figure 37 on page 57 ).

When you press a graphical component, a dialog box with direct controls for that component opens.

For information about using the manual controls for the valves, pumps, and autosampler, refer to the EASY-nLC 1200 Troubleshooting and Maintenance Guide.

To control the temperature of the EASY-Spray ion source when the instrument is not running a method, follow these procedures:

To open the EASY-Spray (direct control) dialog box

To modify the temperature setting of EASY-Spray ion source

To turn off temperature control and return the ion source to ambient room temperature

To open the EASY-Spray (direct control) dialog box

On the Home > Overview page, press the EASY-Spray icon,

The EASY-Spray dialog box opens ( Figure 35

).

Figure 35. EASY-Spray dialog box

.

Thermo Scientific

To modify the temperature setting of EASY-Spray ion source

1. Open the EASY-Spray dialog box as described in the previous procedure.

2. Press the temperature box.

The Set Value dialog box opens (

Figure 36

).

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Integrated Instrument Control Software

Using the Touch Screen

Figure 36. Set Value dialog box

3. Press the number pad to enter a temperature from 30 to 60 °C.

The application does not accept temperatures below 30 °C or above 60 °C. The application displays temperatures outside this range in red.

4. After you enter an appropriate temperature, press Accept.

The Set Value dialog box closes and the application downloads the new temperature setting to the EASY-Spray ion source.

5. To close the EASY-Spray dialog box, press Close (see Figure 35 on page 55

).

To turn off temperature control and return the ion source to ambient room temperature

1. Open the EASY-Spray dialog box as described in “To open the EASY-Spray (direct control) dialog box” on page 55 .

2. Press Disable (see

Figure 35 on page 55

).

The application turns off the EASY-Spray ion source’s temperature control and the ion source’s temperature returns to the ambient laboratory temperature.

3. To close the EASY-Spray dialog box, press Close (see Figure 35 on page 55

).

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Integrated Instrument Control Software

Viewing the System Status

Viewing the System Status

You can view the system status of the system hardware components and the run status on the

Home > Overview page (

Figure 37

).

Figure 37. Overview page of the Home menu

Pump A status

Valve A icon

Flow sensor A readback

Current sample and status

Autosampler icon

EASY-Spray temperature control icon

Reminder area

Pump B icon

ACN concentration

Mixing Tee readback

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Integrated Instrument Control Software

Viewing the System Status

Table 8 describes the system readbacks.

Table 8. System readbacks

Readback

Pump

Flow sensor

Mixing Tee

Autosampler

ACN concentration

Description

Displays the backpressure, flow rate, and position for the associated pump. The flow rate readback is negative when the pump is refilling.

Displays the measured flow rate from the associated pump (see

“Flow Sensors” on page 11 ).

Displays the %B solvent composition.

Displays the active, pending, and finished samples for a batch run and the temperature of the cooling unit.

Displays the current ACN concentration

The valve icon shows the position of the associated valve (see “Six-Port Rotary Valves” on page 12 ).

For more information about viewing the system status, see

“Monitoring the Run” on page 147 .

In the reminder area dues are displayed:

“Flow sensor calibration due”

Shows if the last successful calibration was more than 6 months ago.

“Solvent refresh due” Shows if the last acknowledged solvent refresh was more than 14 days ago.

“Solvent refreshed” acknowledge button

Shows if the last acknowledged solvent refresh was less than 14 days ago.

Click on the reminder for further actions:

The “Flow sensor calibration due” button is linked to the flow sensor calibration script.

Clicking on “Solvent refresh due” and “Solvents refreshed” shows a solvent refresh confirmation.

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Integrated Instrument Control Software

User Interface Layout and Application Menu Structure

User Interface Layout and Application Menu Structure

The application has five menu tabs at the top of the touch screen (see

Figure 38

) and each menu has two or more pages (vertical tabs at the left).

Figure 38. EASY-nLC user interface layout with menus

Top menu tabs

Logo icon

Vertical tabs

Current user

Login and Exit button

In this guide, the term menus refers to the tabs at the top of the touch screen and the term page refers to the screens opened by pressing the vertical tabs.

During a session, the application tracks which page (vertical tab) you selected inside each menu (top tab) and shows that page when you next choose that menu.

The Thermo Scientific logo icon in the upper left corner and the Login button in the lower left corner are always available.

To log in or log off, press Login (see “Logging In to the EASY-nLC Instrument” on page 66

and

“Closing Down the EASY-nLC Instrument” on page 67

).

To view version information for the application, press the Logo icon.

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Integrated Instrument Control Software

User Interface Layout and Application Menu Structure

To access a specific page of the application, first press the appropriate menu tab and then press the appropriate page tab. In this guide, the following nomenclature describes the navigation to a specific page of the user interface: Menu > Page.

Some of the pages have two or more views. For example, the Batch Setup > Edit page has two views: View Rack and View List. You access these views by pressing View Rack or View List on the page.

The Method Setup > Edit page contains a five-page wizard. You access the wizard pages by pressing the left and right arrows at the bottom of the touch screen.

Figure 39 shows a schematic of the user-interface structure. When you navigate to a Menu >

Page, the menu and page tabs are highlighted in light blue.

Figure 39. Menu and page structure

Home

Batch Setup Method Setup Maintenance Configuration

Overview

Graphs

Queue

File

Edit

File

Edit

Scripts

Log Book

Support

Devices

Users

Connections

Network

Time

Data

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EASY-nLC 1200 Series Getting Started Thermo Scientific

Home Menu

Page

Overview

Graphs

Queue

3

Integrated Instrument Control Software

User Interface Layout and Application Menu Structure

See the following menu descriptions (on pages

61

– 65 ):

Home Menu

Batch Setup Menu

Method Setup Menu

Maintenance Menu

Configuration Menu

Use the three pages of the Home menu to view the status of your sample runs and the instrument components; edit batch jobs in the queue; and take direct control of the valves, the pumps, the autosampler’s XYZ robot, and the cooler temperature.

Function Reference

Manually control the valves, the pumps, and the autosampler components.

Refer to the EASY-nLC 1200 Troubleshooting

and Maintenance Guide.

“Preparing and Loading Samples” on page 123

Monitor the system status.

Display up to four graphs, each selectable from a list, with time-dependent data for the active run. The selection list includes

Pump A, Pump B, Pump S, Valve A,

Valve B, Valve S, Valve W, Sample Cooling, and Gradient.

View a list of the batch jobs, currently running or waiting.

“Viewing the System Status” on page 57 and

“Monitoring the Run” on page 147

“Using the Graphs Page in the Home Menu” on page 149

“Editing the Running Batch” on page 151

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User Interface Layout and Application Menu Structure

Batch Setup Menu

Use the two pages of the Batch Setup menu to set up sample processing jobs. For information about setting up and submitting a batch run, see these procedures:

“Creating a Batch” on page 140 and

“Starting Sample Acquisition” on page 146

.

Page

File

Function

View or change a directory tree of saved batch jobs. You can create or import new jobs and create or delete directories on this page.

Set up new batch jobs.

Reference

“Viewing the Batch List and Setting Up a

Batch Record” on page 140

Edit

View Racks view Select the vial or well positions for the batch job from a graphical view of the configured plate format.

“Specifying the Method, Sample Positions, and Number of Injections” on page 141

View List view View the batch job as a list of samples in the order of processing. Each row in the table holds information about the position, name, and method for that position.

“Editing the Running Batch” on page 117

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User Interface Layout and Application Menu Structure

2/6

3/6

4/6

5/6

6/6

Method Setup Menu

Use the pages of the Method Setup menu to set up the chromatographic methods for your

samples (see “Creating a Method” on page 124 ).

Note If the method does not control an external device, the Method Setup wizard contains five pages. If the method controls external devices, such as the Advion™ RePlay™ device or the EASY-Spray ion source, the Method Setup wizard includes additional pages.

For information about setting up a method that includes the RePlay device, see Chapter 7,

“Installing and Controlling External Devices.”

Page

File

Edit

1/6

Function

View a directory tree of saved methods. You can create or import new methods or create or delete directories from this page.

Method Setup wizard with six pages—

Reference

“Browsing for a Stored Method or Creating a

New Method File” on page 124

Select the method that you want to edit and enter a description of the method. If you installed external devices, select the external devices that you want the method to control.

“Starting the Method Setup Wizard” on page 126

Set up the sample pickup and sample loading steps of the method.

Control the liquid chromatography gradient.

“Building the Gradient” on page 130

Control the equilibration of the precolumn, the analytical column, or both.

“Setting Up the Column Equilibration

Steps” on page 132

Control the autosampler wash step.

“Setting Up the Sample Pickup and Loading

Steps” on page 127

Control the column temperature of the

EASY-Spray ion source.

“Setting Up the Autosampler Wash Step” on page 134

“Specifying the Temperature of the

EASY-Spray Column Heater” on page 137

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User Interface Layout and Application Menu Structure

Maintenance Menu

Use the pages of the Maintenance menu to perform a number of functions designed to provide and improve system status.

Page

Scripts

Log Book

Support

Devices

Function

View a collection of useful maintenance procedures for keeping the instrument functioning properly and to help diagnose and resolve possible problems.

Reference

For information about purging and flushing the pump, see

“Purging and Flushing the

Pumps” on page 91

.

For information about equilibrating the columns and determining the appropriate flow rate for your chromatographic method, see

“Equilibrating Columns” on page 118

.

Electronically enter all the service actions that you perform on the instrument.

Set up support from Thermo Fisher

Scientific.

Add an external device to the system.

For information about other maintenance scripts, refer to the EASY-nLC 1200

Troubleshooting and Maintenance Guide.

For information about the log book, refer to the EASY-nLC 1200 Troubleshooting and

Maintenance Guide.

Refer to the EASY-nLC 1200 Troubleshooting

and Maintenance Guide.

Chapter 7, “Installing and Controlling

External Devices.”

Check and reset the device usage counters.

For information about the device usage counters, refer to the EASY-nLC 1200

Troubleshooting and Maintenance Guide.

Set up the global configuration for the

HPLC.

Calibrate the autosampler.

“Setting Up the Properties for the HPLC

Device” on page 80

Refer to the EASY-nLC 1200 Troubleshooting

and Maintenance Guide.

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Integrated Instrument Control Software

User Interface Layout and Application Menu Structure

Configuration Menu

Use the pages of the Configuration menu to control the instrument setup and to manage the system configuration.

Page

Users

Connections

Network

Time

Data

Function Reference

Manage user accounts (create, edit, and delete).

Select the mass spectrometer that is connected to the EASY-nLC instrument, set up the communications protocol, and view a contact closure wiring diagram for the selected mass spectrometer.

“Creating New User Accounts” on page 76

“Setting Up Communication with the Mass

Spectrometer” on page 69

Set up or view how the EASY-nLC instrument’s computer is identified in a connected computer network. Consult your

IT administrator before making changes to the system.

Set the internal clock in the EASY-nLC instrument and the display format used throughout the application to print date and time information.

“Setting Up the Local Area Network

Connection” on page 72

“Modifying the Time Settings” on page 78

Purges excess data on the hard drive of the

EASY-nLC instrument’s computer.

Removing unnecessary data improves the computer’s performance.

“Purging the Computer Hard Drive” on page 79

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Integrated Instrument Control Software

Logging In to the EASY-nLC Instrument

Logging In to the EASY-nLC Instrument

The EASY-nLC instrument’s built-in software has a user management system that assigns specific access rights to each user. When the instrument ships, it contains two predefined users: guest and admin. The default user account is guest (and does not require a password).

With this account you can see and monitor the system, but you cannot enter any new information.

To start using or configuring the EASY-nLC instrument’s built-in software

1. In the User list, select admin (see

Figure 40 ).

2. In the Password box, enter admin.

3. Press Accept (the factory password for the admin account is also admin).

The first time you start the application, it automatically logs you in as a guest. “Guest” appears above the Login button. The user “Production” is for internal purposes only and therefore not accessible.

Figure 40. EASY-nLC login dialog box

IMPORTANT Change the admin password as soon as possible for security reasons.

4. If security is an issue, change the admin password (see Figure 41 ).

To log out of the EASY-nLC instrument

1. Press Exit.

A confirmation box opens (see

Figure 41 ).

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Integrated Instrument Control Software

Closing Down the EASY-nLC Instrument

Figure 41. Confirmation box from pressing Exit in the lower left corner of the touch screen

2. Press Logout.

An administrator can start a controlled power-down or exit the application from this dialog box. If you are not an administrator, you can only log out or change your password.

Closing Down the EASY-nLC Instrument

With the exception of an emergency shutdown, closing down the instrument in a controlled manner is important to allow all the components to shut down in an orderly sequence. Using this controlled method saves important data so that the instrument starts with the correct information the next time you use it.

CAUTION With the exception of an emergency shutdown, closing down the instrument in a controlled manner is important to allow all the components to shut down in an orderly sequence. If you turn off the power switch during normal operation, you risk damaging essential system components. Follow the procedure outlined here whenever possible.

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Integrated Instrument Control Software

Closing Down the EASY-nLC Instrument

To turn off the EASY-nLC instrument

1. Press Exit (lower left corner of the touch screen).

A confirmation box opens (see

Figure 41 on page 67

).

2. Press Power Down.

The EASY-nLC application displays a white screen with a small progress bar. When the progress bar fills completely and the message prompts you to turn off the instrument, go to the next step.

3. After receiving the message that you can safely turn off the instrument, turn off the power switch on the back panel of the instrument.

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Configuring the EASY-nLC System

After turning on the instrument and logging in as a system administrator, you must configure the instrument’s integrated software application for use and define the network connection for the instrument.

To set up the application configuration, follow the configuration instructions in this chapter.

Contents

Setting Up Communication with the Mass Spectrometer

Setting Up the Local Area Network Connection

Modifying User Permissions

Modifying the Time Settings

Purging the Computer Hard Drive

Setting Up the Properties for the HPLC Device

Setting Up Communication with the Mass Spectrometer

After the EASY-nLC injects sample onto the analytical column, it signals the mass spectrometer to start data acquisition.

IMPORTANT Before you connect the EASY-nLC instrument to a mass spectrometer, make sure that the protective grounding is shared between the instruments.

To set up the mass spectrometer connection

1. Press Configuration > Connections.

The Configuration > Connections page opens.

Figure 42 shows the contact closure

graphic for an Orbitrap Fusion mass spectrometer. For information about connecting the

contact closure cable for a Thermo Scientific mass spectrometer, see “Connecting to the

Mass Spectrometer through Contact Closure” on page 42

.

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Configuring the EASY-nLC System

Setting Up Communication with the Mass Spectrometer

Figure 42. Connections page of the Configuration menu

2. From the Instrument (cable no.) list, select the instrument by manufacturer.

Tip The Instrument (cable no.) list contains the most common mass spectrometers

(by manufacturer) used in proteomics, but if you cannot find your particular mass spectrometer, select Generic.

If your mass spectrometer does not appear on the list and you would like to see it supported in a future release of the EASY-nLC application, provide this information to Thermo Fisher Scientific Customer Service (see

“Contacting Us” on page xv

).

When you select the instrument, a graphic of the contact closure connections appears in the Contact Closure Settings area, but the default selections remain the same.

3. In the Contact Closure Settings area, do the following: a. Check the connections on the graphic for the selected mass spectrometer.

b. From the Protocol list select One-way or Two-way synchronization (see Figure 42

).

• If the graphic for the selected mass spectrometer (contact closure cable) shows only Start In signals, select One-way synchronization. With a one-way connection, the EASY-nLC instrument sends a start signal to the mass spectrometer.

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4

Configuring the EASY-nLC System

Setting Up Communication with the Mass Spectrometer

• If the graphic for the selected mass spectrometer shows two-way communication between the mass spectrometer and the EASY-nLC instrument, select Two-way synchronization. With a two-way connection, the EASY-nLC instrument waits for a ready signal from the mass spectrometer. c.

In the State at Start list, select Open or Closed.

For a Thermo Scientific mass spectrometer, select Open. For other mass spectrometers, refer to the documentation provided with the mass spectrometer or contact Thermo Fisher Scientific Customer Service.

d. In the Start At list, select Gradient or Sample Loading.

• If you want data acquisition to start when the mobile phase gradient starts, select

Gradient.

• If you are running the instrument in the one-column mode and you want data acquisition to start when the instrument loads the sample onto the analytical column, select Sample Loading.

Note By setting the Start At value to be the sample loading start, you might be able to acquire data for early-eluting peptides that would not ordinarily bind to the column material and elute in the solvent front.

Moving the Start At point to the start of the sample loading step can lead to small retention time variations, due to possible differences in pressure buildup before the A/B mixing starts in the actual gradient.

e.

In the Signal Width box, enter an appropriate signal width.

The default signal width is 1 second, which is sufficient for a Thermo Scientific mass spectrometer. For other mass spectrometers, refer to the documentation provided with the mass spectrometer or contact Thermo Fisher Scientific Customer Service.

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Configuring the EASY-nLC System

Setting Up the Local Area Network Connection

Setting Up the Local Area Network Connection

If you connect the EASY-nLC instrument to the local network (see

“Setting Up the Ethernet

Connection to a Local Network for Data Exchange” on page 46 ) and set up the network

configuration for the application, you can access the EASY-nLC instrument’s computer from other computers on the network.

IMPORTANT Ask your local IT administrator to help you configure the EASY-nLC instrument on the local network. The following information will help you through the process.

To connect the EASY-nLC instrument to a local network

1. Connect the Ethernet cables as described in (see

“Setting Up the Ethernet Connection to a Local Network for Data Exchange” on page 46 ).

2. Press Configuration > Network.

The Network page (

Figure 43

) contains the configuration address information.

Figure 43. Network page of the Configuration menu

3. In the Profile list, select one of the following:

• To connect to a local area network, select MS LAN.

• To connect to the remote support server, select Internet.

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Setting Up the Local Area Network Connection

4. For Configuration, select the Dynamic (addressing DHCP) option or the Fixed

(IP address) option.

Your IT administrator knows which option to select.

If IT asks about a MAC address for the instrument—that is, the instrument’s unique network identifier—it is labeled on the connector panel on the back of the instrument.

The identifier is a series of numbers and letters and might look like this:

MAC address: 00:E0:4B:07:89:65

External users can now access the file server in the EASY-nLC instrument’s computer to look at log files, for example. Microsoft Windows Explorer might display the file server as

shown in Figure 44

.

Figure 44. File server access through Windows Explorer

Type the

IP address here.

To access the admin folder on the EASY-nLC instrument’s computer hard drive from another computer on the network

1. Type the IP address of the instrument (in the Address list for Windows Explorer), for example, file:\\172.22.50.18, and then press ENTER.

A login dialog box appears.

You need a valid user name and password to enter the file space. The user name and password are the same as those on the EASY-nLC instrument. For example, if you have not changed the default user name and password, the user name is admin and the password is admin.

2. Enter the same user name and password that you use to log in locally to the EASY-nLC instrument.

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Modifying User Permissions

Modifying User Permissions

The four types of users are Guest (default), Normal, Super User, and Administrator.

Each user type has access to different levels of functionality in the EASY-nLC application

( Table 9

).

Table 9. User types and permission levels for each menu

Menu

Home

Batch Setup

Method Setup

Maintenance N/A

Configuration a

User has read-only access.

b

User has read-write access.

N/A

Guest

Read only a

Read only

Read only

Normal

Read/Write b

User type

Super User

Read/Write

Read/Write

Read/Write

N/A

N/A

Read/Write

Read/Write

Read/Write

N/A

Administrator

Read/ Write

Read/Write

Read/Write

Read/Write

Read/Write

Having a Guest user account means you can access the top menu but not enter data or issue instrument commands.

If a feature is not available, the application grays (blanks) it out, so the user cannot access that function.

Follow these procedures to change passwords and create new user accounts:

“Changing the Administrator Password,” next section

“Creating New User Accounts” on page 76

“Changing Your Password from the Exit Menu” on page 77

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Configuring the EASY-nLC System

Modifying User Permissions

Changing the Administrator Password

The EASY-nLC application supplies a simple administrator password. Consider changing it to a more secure password as soon as possible.

To change the administrator password

1. Log in as admin (otherwise, see

“Logging In to the EASY-nLC Instrument” on page 66

).

2. Press Configuration > Users.

The Configuration > Users page opens ( Figure 45

).

Figure 45. Users page of the Configuration menu

Thermo Scientific

3. Press Open.

The Select User dialog box opens.

4. In the User list, select Admin.

5. Enter the new password as follows: a. Press the Password box.

The Enter Password dialog box opens.

b. Type a new password, and then press OK.

c.

Press the Re enter box.

The Enter Password dialog box opens.

d. Type the password a second time to confirm it, and then press OK.

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Modifying User Permissions

6. Press Save to save the user account information.

Note The user “Production” is a factory default user account that is not accessible and cannot be deleted.

Creating New User Accounts

Use the Users page to create new user accounts for all the people or groups that will use the instrument. Each user has a private file space for batch jobs and methods that other users

(apart from system administrators) cannot see.

In addition, there is a public file space for methods and batch jobs that all users can see and use. Only Super Users and Administrators can copy files into the public spaces and only

Administrators can delete files and directories from the public space.

To create a new user account

1. Log in as admin (otherwise, see

“Logging In to the EASY-nLC Instrument” on page 66

).

2. Press Configuration > Users.

The Configuration > Users page opens (see

Figure 45 on page 75

).

3. Press New.

The User Login, Name, Password, and Re enter boxes also become available for data input.

4. Make entries in the User Login and Name boxes.

5. Select the privileges for the user by level: Normal, Super, or Admin. For user privileges by user type, see

Table 9 on page 74

.

If you want the user to carry out instrument maintenance, you must select Super User as the user type. If you want the user to restore data to the instrument’s computer hard drive, select Admin as the user type.

6. Create an initial password for the defined user by using the Password and Re enter boxes.

Users can change their passwords from the Login/Exit menu.

7. Press Save.

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Modifying User Permissions

Changing Your Password from the Exit Menu

After logging in, all users can change their password from the Exit menu.

To change your password

1. Press Exit in the lower right corner of the touch screen.

The exit confirmation box opens for the current user (see Figure 46

).

Figure 46. Exit confirmation box for the current user

2. Press Change Password.

The Change password for: current user dialog box opens.

3. Make the appropriate entries.

4. Press Accept to accept the new password and close the dialog box.

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Modifying the Time Settings

Modifying the Time Settings

Use the Configuration > Time page (

Figure 47 ) to set both the internal clock in the

EASY-nLC instrument and the display format used throughout the application for date and time information.

To set the time zone

1. Press Configuration > Time.

The Configuration > Time page opens (see Figure 47

).

Figure 47. Time page of the Configuration menu

Continent/city list

2. Select a time zone from the continent/city list.

The Set Time Zone button becomes available.

3. Press Set Time Zone.

To change the display format for the time and date

1. Select a format from the Presets list.

2. Press Set Format.

The time and date format changes immediately on the Maintenance > Log Book page.

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Purging the Computer Hard Drive

Purging the Computer Hard Drive

Use the Configuration > Data page to remove excess data from the computer’s hard drive.

Purge the hard drive when the instrument becomes less responsive or sluggish, as removing excess data improves system performance.

To remove unnecessary files from the instrument’s computer

1. Press Configuration > Data.

The Configuration > Data page opens ( Figure 48 ).

Figure 48. Data page of the Configuration menu

Thermo Scientific

2. Select the check boxes for the data that you want to keep.

3. Press Purge.

The application deletes the excess data. When the purge is complete, the Linux application restarts.

Note Perform the System Purge only when necessary. In the case of errors the system data facilitates troubleshooting.

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Setting Up the Properties for the HPLC Device

Setting Up the Properties for the HPLC Device

Use the Properties page for the EASY-nLC instrument on the Devices page of the

Maintenance menu to set the global instrument configuration settings such as the one- or two-column setup, loop size, idle flow settings, and load speed protection.

To set up the global instrument configuration for the HPLC device

1. Press Maintenance > Devices.

2. In the Devices list, select EASY-nLC (HPLC).

3. Press the Properties tab.

The Properties view for the HPLC device on the Maintenance > Devices page opens

( Figure 49

).

Figure 49. Devices page of the Maintenance menu, showing the HPLC Properties settings

4. Press the corresponding box to open the keypad and enter the appropriate numeric values for these parameters: Loop Volume, Idle Flow Rate, and Idle Mixture.

5. Make the appropriate selections for these check boxes: One Column Setup, Load Speed

Protection (LSP), and Automatic Pre-run Maintenance.

Table 10

describes the parameters in the Properties view for the EASY-nLC instrument on the

Devices page of the Maintenance menu.

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Table 10. HPLC parameter descriptions (Sheet 1 of 2)

Parameter

Loop Volume

Description

Specifies the nominal size of the installed sample loop.

Idle Flow Rate

Take care to specify the correct loop size. If you specify a smaller loop size than the installed loop size, the instrument might draw sample into the sample pump.

Specifies the solvent flow rate when the instrument is idle. This feature activates when the last sample in a batch is done.

Idle Mixture

One Column Setup

If you do not want a solvent flow through the instrument when it is idle, type 0 in the Idle Flow Rate box.

Maintaining long-term emitter stability (with glass emitters) might require constant flow through the system.

Specifies the solvent mixture as a percentage of solvent B when the instrument is idle.

For best results, use a minimum of 70% B to help prevent deposition on the emitter tip, both metal and borosilicate.

Specifies the one-column setup.

The diagram on the Overview page of the Home menu displays the column setup.

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Setting Up the Properties for the HPLC Device

Table 10. HPLC parameter descriptions (Sheet 2 of 2)

Parameter Description

Load Speed Protection When this check box is selected, the instrument uses the flow rate entered in the Sample Loading area on page 2 of the Method

Setup wizard (see

“Setting Up the Sample Pickup and Loading

Steps” on page 127 ) to load the sample onto the precolumn (or

the analytical column for a one-column setup). During a batch run, the sequence stops immediately if the system reaches the maximum pressure limit entered in the Sample Loading area.

Automatic Pre-run

Maintenance

The LSP feature ensures that the solvent delivery system loads each sample onto the column at the same flow rate. Because the flow rate used to load the sample onto the column can affect sample-to-sample reproducibility for quantitation experiments, use the LSP feature to maximize sample-to-sample reproducibility for quantitation experiments.

LSP is only active during the sample loading step of a run.

To use the LSP feature correctly, you must enter both a flow and a maximum pressure limit for sample loading in the method file. To determine an appropriate flow rate for your LC columns, use the precolumn and analytical column equilibration scripts.

Selecting this check box turns on the automatic Flush Air script.

When you start a new batch from the touch-screen application or a new Xcalibur sequence from a data system computer, the instrument runs the automatic Flush Air script if the previous batch (or sequence) started more than 8 hours ago.

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Daily and Weekly Maintenance Tasks

Preparing the EASY-nLC Instrument for Use

To prepare the EASY-nLC instrument for use, log in to the instrument with either Super User

or Administrator privileges (see “Logging In to the EASY-nLC Instrument” on page 66 ), and

then follow these procedures.

Contents

Daily and Weekly Maintenance Tasks

Preparing the Solvent Bottles and Waste Containers

Executing Maintenance Scripts

Purging and Flushing the Pumps

Setting Up the Column Assembly

Using nanoViper Fittings

Using the Viper Union

Equilibrating Columns

Daily and Weekly Maintenance Tasks

For optimal system performance, check the solvent levels, draw fresh solvent through the solvent system, and flush air out of the system on a daily basis.

Because the vapor pressures of formic acid, water, and acetonitrile differ, the solvent composition changes over time. Refill the solvent bottles with fresh solvent to maintain a consistent solvent composition on a weekly basis.

To check the solvent levels

1. Visually inspect solvent bottle A, solvent bottle B, and the autosampler bottle in position W3 (and the bottles in position W1 and W2 if used). Refill the bottles with fresh solvent if necessary.

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Tip You can also inspect the purity of the solvent to ensure no visible precipitates have formed that might lead to blockages in the pump line.

2. Visually inspect the autosampler wash bottle in position W4 and the waste container in the pump compartment, and empty them if necessary.

Note A solvent refresh reminder (“Solvent refresh due”) is shown on the left menu bar, if the last acknowledged solvent refresh is more than 2 weeks ago.

If the solvent refresh was acknowledged within the last 2 weeks, a “Solvents refreshed” button is shown.

To acknowledge that both solvents have been refilled, press either the “Solvent refresh due” or the “Solvents refreshed” button and choose “Yes”.

To draw fresh solvent into the system and to flush air out of the system

1. Run the Purge Solvent with two purge iterations. Then run the Flush Air script with a flush threshold of 12 μL.

2. Repeat

step 1

until the flush volume falls below the threshold.

For more information, see “Purging and Flushing the Pumps” on page 91

.

Preparing the Solvent Bottles and Waste Containers

As you prepare the solvent bottles and empty the waste containers, you work with hazardous solvents and chemicals such as methanol, acetonitrile, formic acid, and so on. Before working with hazardous solvents and chemicals, review the specific hazards for each substance by reading the Material Safety Data Sheets (MSDSs) provided by the manufacturer. When working with solvents and chemicals, wear the appropriate safety gear.

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CAUTION When working with solvents, wear safety glasses and safety gloves. Make sure that the gloves are compatible with the solvents you are using.

When working with volatile hazardous chemicals, use an appropriate fume hood.

Before shipment, the EASY-nLC instrument’s solvent system is flushed with methanol.

CAUTION Methanol (CAS number: 67-56-1) is highly flammable and toxic by inhalation, ingestion, or skin absorption. Take appropriate measures to protect yourself and your equipment. Make sure that the mobile phases are miscible with methanol or flush the A and B solvent lines with an intermediate solvent.

To set up the two bottles that contain the mobile phase solvents, the four autosampler bottles, and the waste beaker, follow these procedures:

“Preparing the Solvents,” next section

“Preparing the Solvent A and B Bottles” on page 86

“Installing the Autosampler Wash Bottles and Waste Beaker” on page 88

Preparing the Solvents

The installation solvents for the EASY-nLC 1200 instrument are as follows:

Solvent A:

Solvent B:

0.1% formic acid in water

0.1% formic acid in a mixture of 80% acetonitrile in water

Wash solvent 3: 0.1% formic acid in water

Note Acetonitrile concentrations greater than 95% in water can shorten the lifetime of components.

To prepare these solutions, order the appropriate UHPLC-MS or LC/MS-grade solvents or solvent blends.

Table 11 lists the solvents and solvent blends that you can order from Thermo

Fisher Scientific.

Table 11. Solvents

Solvent/reagent

Water

Acetonitrile

Formic acid (99.5%)

Water with 0.1% formic acid

Grade Size

Optima™ UHPLC-MS 1 L

Catalog number

A956-1

Optima™ UHPLC-MS 1 L

Optima™ LC/MS 10×1mL

A458-1

A117-10X1AMP

Optima™ LC/MS 1 L LS118-1

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Table 11. Solvents

Solvent/reagent Grade

80% acetonitrile, 20% water with

0.1% formic acid

Optima™ LC/MS

0.1% formic acid in acetonitrile Optima™ LC/MS

Size Catalog number

500mL LS122-500

1 L LS120-1

CAUTION Solvent contaminants can cause system blockages and poor spray stability.

To minimize instrument problems caused by solvent contaminants, follow these guidelines for the EASY-nLC instrument:

• Use only UHPLC-MS or LC/MS-grade solvents.

• Use only commercially manufactured LC/MS-grade solvent blends, as mixing solvents in a typical laboratory environment can introduce contaminants as well as solvent concentration errors.

• Do not filter UHPLC-MS-grade solvents, as filtering solvents can introduce contaminants.

• Do not use HPLC-grade solvents, as HPLC-grade solvents contain more contaminants than LC/MS-grade solvents.

• Do not use water from laboratory purification systems, as laboratory purified water contains more contaminants than UHPLC-MS-grade water.

CAUTION Store and handle all chemicals in accordance with standard safety procedures.

The Optima LC/MS mobile phases can be ordered through www.fishersci.com

(search for

Optima LC/MS)

Preparing the Solvent A and B Bottles

Follow these recommended steps to prepare the two solvent bottles.

To prepare the solvent A and B bottles

1. Fill the 25 ml Schott Duran™ blue cap bottles with the mobile phase solvents.

2. Degas the solvents by sonicating the solvent bottles in a sonication bath or by sparging the solvents with helium gas.

3. Place the bottles in the holder on top of the instrument: solvent A bottle in the front and solvent B bottle in the back position.

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4. Mount the inline filter assembly on the blue cap lids as shown in Figure 50 and Figure 51 .

Figure 50. Inline filter assembly (exploded view)

Figure 51. Assembled inline filter

IMPORTANT To establish proper surface wetting, prime the filters with methanol or acetonitrile.

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Preparing the Solvent Bottles and Waste Containers

Installing the Autosampler Wash Bottles and Waste Beaker

The autosampler compartment holds the sample trays, up to four wash bottles, and a waste

beaker (see Figure 52

). You can operate the instrument after installing the wash bottle that the autosampler uses for needle cleaning (position W4), the wash bottle that contains solvent A for pump S (position W3), and the waste beaker that collects the waste from pumps A and B and from the venting Tee through valve W.

To access the autosampler compartment, you can manually open the tray compartment door or you can press the Eject/Insert Tray button on the Home > Overview page.

Figure 52. Autosampler compartment

Waste beaker

Wash bottles

To install the waste beaker and prepare and install the wash bottles

1. Fill one of the wash bottles with solvent A and degas it.

2. Turn on the instrument and make sure that the area in front of the tray compartment door is free from obstruction.

3. Press Home > Overview. Then press Eject/Insert Tray.

A confirmation box appears to remind you to remove obstacles from the area in front of the tray compartment before commanding the instrument to open the tray compartment door.

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Preparing the Solvent Bottles and Waste Containers

4. Press Accept.

The tray compartment door opens.

5. Put the bottle with the needle wash insert into position W4 (see Figure 53 ).

CAUTION You must place the wash bottle with the needle wash insert in position

W4. Placing this bottle in any other position will cause irreparable damage to the autosampler needle during an injection cycle.

Figure 53. Wash bottle with needle wash insert and view of the four positions on the autosampler bottle holder

W1 W2 W3 W4

Wash bottle with needle wash insert

Autosampler bottle holder

6. Put a wash bottle that contains solvent A into position W3.

Pump S draws solvent from this position. You can place wash solvents for customized washing procedures in positions W1 and W2.

7. Make sure all the bottles are installed with the lids fitting tightly.

8. Place the waste container (a 250 mL plastic beaker) into the compartment on the right side of the autosampler compartment.

9. Press Eject/Insert Tray to move the plate back into the instrument.

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Executing Maintenance Scripts

Executing Maintenance Scripts

The EASY-nLC application has a set of maintenance scripts that you can access from the

Scripts page (

Figure 54

) of the Maintenance menu.

Use the scripts in the Prepare category for common tasks such as drawing fresh solvent through the solvent system, flushing air out of the system, and equilibrating the columns.

Before you start any of the maintenance scripts, prepare the instrument as described in

“Preparing the Solvent Bottles and Waste Containers” on page 84 .

Note You cannot execute maintenance scripts when the EASY-nLC instrument is running batches.

Figure 54. Scripts page of the Maintenance menu

The green START button is active when a job or script is ready to execute. After you press

START, the STOP button replaces the START button and remains active until the job or script ends. During execution you can terminate a job or script by pressing the red STOP button.

When the Schedule button is available, you can automate the start of the Flow Sensor calibration script and all of the scripts in the Prepare category. You cannot schedule the remaining scripts because they require supervision or hardware changes to the instrument.

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Purging and Flushing the Pumps

Purging and Flushing the Pumps

Because the EASY-nLC pumps are purged with methanol before delivery, thoroughly purge the pumps with the mobile phase solvents before doing any analytical runs. In addition, purge the pumps when switching to a new solvent.

The purge pump job fills the selected pump or pumps with solvent and then ejects the solvent into the waste container. You can specify how many iterations of this job you want performed.

After purging the pumps, flush them to remove any air that might be trapped inside. You can specify the maximum number of iterations for this job, the period of time that the pumps are being pressurized, and the success criteria for stopping the operation earlier than scheduled.

To purge and flush pumps A and B

1. Before you purge the pumps with fresh solvent, make sure that the solvent bottles A and B on the top of the instrument are filled with solvent.

2. Press Maintenance > Scripts.

The Scripts page of the Maintenance menu opens.

3. Set up the purge solvents job as follows (see

Figure 55

): a. In the Category list, select Prepare.

b. In the Name list, select Purge Solvent.

c.

Press the Parameters tab.

d. In the Purge Iterations box, enter 10.

This is the number of times that the pumps will completely refill and eject the solvent.

Tip To enter the number of purge iterations, press the empty cell in the Value column. The Set Value dialog box appears. Use the numeric keypad to type the number of purge iterations that you want. Then press Accept. The new value appears in the Value column.

e.

Select the Purge Pump A, Purge Pump B, and Purge Pump S check boxes.

Note The pumps draw and eject solvent as follows:

• Pump S draws solvent from bottle W3 in the autosampler bottle holder and ejects solvent into bottle W4.

• Pump A and pump B draw solvent from the solvent A and B bottles on top of the instrument and eject solvent to the waste cup.

f.

The option “full purge” sets the purge iterations for pumps A & B to 10 iterations and for pump S to 5 iterations. The “full purge” option is recommended for thorough refreshment of solvents in a pump subsystem.

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Figure 55. Purge solvent job parameters

4. To save these settings and continue, press Schedule.

The Queue page of the Home menu appears.

5. Set up the flush air job as follows (see

Figure 56 ):

a. Press Maintenance > Scripts.

b. In the Category list, select Prepare.

c.

In the Name list, select Flush Air. d. Press the Parameters tab.

e.

In the Flush Vol. Threshold [μL] box, enter 12μl.

This values helps to ensure an acceptable amount of solvent compressibility.

Tip To enter the flush volume, press the empty cell in the Value column. The Set

Value dialog box appears. Use the numeric keypad to type the volume that you want. Then press Accept. The new value appears in the Value column.

f.

Select the Flush Pump A, Flush Pump B, and Flush Pump S check boxes.

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Figure 56. Flush air job parameters

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6. Press Schedule.

The Queue page ( Figure 57

) of the Home menu appears.

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Figure 57. Queue page of the Home menu, showing the queued jobs

7. In the Jobs list, make sure the check box in the Auto-Continue column is selected for

Flush Air.

8. Press Overview.

The Overview page of the Home menu opens.

9. Press START.

The default selections appear in the two graph windows on the Scripts page of the

Maintenance menu, but you can change the selection for a window from its corresponding list. These graphs help you to monitor the job.

10. Wait for the jobs to end (the flush air job continues until the flush air volume values are below the specified threshold).

Note While a maintenance job or a script is running, you cannot start other jobs or scripts.

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Setting Up the Column Assembly

Setting Up the Column Assembly

To integrate the EASY-nLC instrument with a Thermo Scientific mass spectrometer, Thermo

Fisher Scientific provides two nanospray ion sources: the Nanospray Flex source and the

EASY-Spray source.

You use different columns for these two sources. The Nanospray Flex source uses standard nanoflow columns for both the one- or two-column setup. The EASY-Spray source uses an

EASY-Spray column as the analytical column in both the one- or two-column setup. To create a two-column setup for the EASY-Spray source, use a standard nanoflow column with nanoViper fittings as the precolumn.

To set up the column assembly for your nanospray source, follow the appropriate procedure:

“Setting Up the Column Assembly for the Nanospray Flex Source,”

next section

“Setting Up the Column Assembly for the EASY-Spray Source” on page 112

Setting Up the Column Assembly for the Nanospray Flex Source

If your LC/MS system has a Nanospray Flex ion source, follow the appropriate procedures to set up the column assembly.

“Setting Up the Column Assembly for an EASY-nLC 1200 Instrument,” next section

“Setting Up the Column Assembly for an EASY-nLC 1200 Instrument” on page 95

“Setting Up a Column Assembly with Fused-Silica Columns” on page 98

“Connecting a Sleeved Emitter to the Analytical Column” on page 111

CAUTION Wear protective gloves and safety glasses when handling the solvent lines. To prevent contamination, use lint-free and powder-free gloves.

Setting Up the Column Assembly for an EASY-nLC 1200 Instrument

For an EASY-nLC Instrument with a Nanospray Flex ion source, you can use standard columns with nanoViper fittings or fused-silica columns.

Depending on the columns you are installing, follow the appropriate procedure:

“Setting Up a Standard Column Assembly,” next section

“Setting Up a Column Assembly with Fused-Silica Columns” on page 98

Setting Up a Standard Column Assembly

Use this procedure for an EASY-nLC 1200 instrument with a Nanospray Flex ion source and standard columns that have nanoViper fittings.

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Table 12

lists the columns, fittings, and unions that make up the two-column assembly. The standard analytical column (P/N 164704) provided with the EASY-nLC 1200 instrument has nanoViper fittings.

Note The EASY-nLC 1200 instrument ships with two analytical columns: an

EASY-Spray column and a standard analytical column with nanoViper fittings. For set up a two-column assembly, you must order the precolumn.

Table 12. Columns and column fittings

Item

Precolumn

(trap column) with nanoViper fittings

Analytical column with nanoViper fittings

PEEK ZDV union with fittings

Stainless steel union, ZDV

(Viper™ union)

Venting Tee

PEEK sleeves (black) for 280 μm fused-silica

Description

Acclaim™ PepMap™ 100, 2 cm length,

75 μm ID, 3 μm particle size, C18 column packing

Acclaim PepMap RSLC, 15 cm length, 50 μm

ID, 2 μm particle size, C18 column packing

0.76 in. length, 0.035 in. thru-port, for 1/32 in. OD tubing

For 1/16 in. OD tubing (HPLC-to-Column connector)

Stainless steel, 0.15 mm bore, for 1/16 in. OD tubing (nanoliter-dead-volume-tee)

330 μm ID, 1/32 in. OD

Part number

Thermo Scientific Dionex™

164705

Thermo Scientific Dionex

164704

IDEX P-771

SC900

SC901

SC903

Make sure that you connect the venting Tee to the Waste In line for both the one-column and two-column setups.

To connect the column assembly for the Nanospray Flex source to the EASY-nLC 1200 instrument

1. Do one of the following:

• For a one-column setup, go to step 2 .

• For a two-column setup, go to step a

.

a. For a one-column setup, connect the Column Out line directly to the venting Tee

( Figure 58

). Then go to

step 3 .

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Figure 58. One-column assembly connected to the EASY-nLC 1200 instrument’s Column Out and Waste In tubing

Column Out tubing

Venting Tee

PEEK union

Analytical column

15 cm length

Valco

Flow

Waste In tubing

PEEK sleeve

2. For a two-column setup, do the following: a. Connect one end of the precolumn to the receiving port of the venting Tee that is parallel to the analytical column. Slowly tighten the fitting until you feel resistance.

b. Connect the Viper union (stainless steel ZDV) to the free end of the precolumn.

Slowly tighten the fitting until you feel resistance.

c.

Insert the nanoViper fitting on the Column Out line into the free end of the Viper union. Slowly tighten the fitting until you feel resistance.

d. Tighten the nanoViper fittings by an additional 45 degrees (1/8-turn). Do not

tighten the fittings by more than a 1/4-turn (see “Using nanoViper Fittings” on page 115 and

“Using the Viper Union” on page 117

).

CAUTION Because the fittings seal against each other inside the union, do not tighten them by more than 90 degrees (1/4-turn). Overtightening the fittings can irreparably damage their sealing surfaces.

3. Connect the Waste In line to the venting Tee as follows: a. Remove the plug from the third port of the venting Tee.

b. Insert the nanoViper fitting at the end of the Waste In line into the free port of the Tee (see

Figure 58 and

Figure 59

). Slowly tighten the fitting until you feel resistance. Then tighten the fitting by an additional 1/8- to 1/4-turn. Do not tighten the fitting by more than a 1/4-turn (see

“Using nanoViper Fittings” on page 115

).

Figure 59 shows the two-column setup. Both the one-column and two-column setups use the

venting Tee.

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Figure 59. Two-column assembly connected to the EASY-nLC 1200 instrument’s Column Out and Waste In tubing

Column Out tubing

Stainless steel, ZDV union for 1/16 in. OD tubing

(Viper union)

Precolumn

2 cm length

Venting Tee

Analytical column

15 cm length

PEEK union

Valco

Flow Flow

Waste In tubing

PEEK sleeve

Setting Up a Column Assembly with Fused-Silica Columns

Use this procedure for an EASY-nLC 1200 instrument with a Nanospray Flex ion source and columns that have exposed fused-silica ends.

When you are using the Nanospray Flex ion source (P/N ES071) and you want to use a pulled fused-silica analytical column, you must modify the EASY-nLC 1200 instrument and install the column using the components in the EASY-nLC UHPLC Liquid Junction Kit.

For information about modifying the EASY-nLC instrument by installing the Column Out and Waste In lines that are supplied in the EASY-nLC UHPLC Liquid Junction Kit, refer to the EASY-nLC 1200 Troubleshooting and Maintenance Guide.

Table 13

lists the items in the UHPLC Liquid Junction Kit (ES269).

Table 13. UHPLC Liquid Junction Kit contents (Sheet 1 of 3)

Description

Column Out line with a nanoViper fitting at one end and bare fused-silica tubing at the other end

Waste In line with a nanoViper fitting on one end and bare fused-silica tubing at the other end

Part number

6041.5290

6041.5289

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Table 13. UHPLC Liquid Junction Kit contents (Sheet 2 of 3)

Description

UHPLC liquid junction cross with a platinum electrode and two-piece protective cover

Protective cover

(bottom)

UHPLC liquid junction cross

Protective cover

(top)

Part number

N/A

Tightening tool used to torque internally-threaded knurled nut fittings (quantity: 2)

Microferrules (quantity: 3)

Blind plug microferrule

(IDEX) P-278

(IDEX) PK-152

(IDEX) P-116

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Table 13. UHPLC Liquid Junction Kit contents (Sheet 3 of 3)

Description

UHPLC fused-silica union, which consists of the following:

• PEEK holder

• Two internally-threaded knurled nuts

• Two microferrules

• Stainless steel cartridge with coned ports and a 280 μm thru-hole

Figure 62 shows an internal view of

this union.

Note The UHPLC fused-silica union has two functions:

Part number

ES272

• For a two-column setup, it connects the Column Out line to the inlet of a

360 μm OD precolumn.

• For system leak testing and backpressure testing, it connects the Column Out line to the Waste In line. For information about leak testing, refer to the EASY-nLC Series

Troubleshooting and Maintenance Guide.

Figure 60 shows a one-column setup with the modified Column Out and Waste In lines and a

fused-silica column with an integrated emitter.

Figure 60. One-column setup with the modified Column Out and Waste In lines

Column Out line with bare fused-silica tubing at one end

(P/N 6041-5290)

Liquid junction with protective cover

Fused-silica analytical column with an integrated emitter

Waste In line with bare fused-silica tubing at one end

(P/N 6041-5289)

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Figure 61 shows a two-column setup with the modified Column Out and Waste In lines and

an analytical column with an integrated emitter. Both the precolumn and analytical column have bare fused-silica ends. The UHPLC fused-silica union connects the Column Out line to the precolumn’s inlet.

Figure 61. Two-column setup with the modified Column Out and Waste In lines and an analytical column with an integrated emitter

Column Out line with bare fused-silica tubing at one end

(P/N 6041-5290)

UHPLC fused-silica union

(P/N ES272)

Precolumn

UHPLC liquid junction cross with protective cover

Pulled fused-silica column with an integrated emitter

Waste In line with bare fused-silica tubing at one end

(P/N 6041-5289)

The UHPLC fused-silica union is designed for a two-column assembly that includes nanoflow columns with fused-silica ends.

Figure 62

shows an enlarged cross section of this union. The ends of the externally-threaded PEEK holder have different internal depths. The stainless steel cartridge, which has coned ports and a 280 μm thru-hole, fits within the deeper end of the PEEK holder. The microferrules are designed for sleeveless use with 360 μm OD fused-silica tubing. The two internally-threaded knurled nuts secure the tubing to the PEEK holder.

Figure 62. UHPLC fused-silica union (enlarged cross section)

Microferrule fitting

(IDEX P/N PK-152)

Internally-threaded knurled nut

Deeper end of the PEEK holder

Stainless steel cartridge with coned ports and a 280 μm thru-hole

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Figure 63 shows a two-column setup with the modified Column Out and Waste In lines and

an analytical column with an external emitter.

Figure 63. Two-column setup with the modified Column Out and Waste In lines and an analytical column with an external emitter

Column Out line with bare fused-silica tubing at one end

(P/N 6041-5290)

UHPLC fused-silica union

(P/N ES272)

Precolumn

Liquid junction assembly

PEEK union

PEEK sleeve for

360 μm OD tubing

Waste In line with bare fused-silica tubing at one end

(P/N 6041-5289)

Use the following tools and materials to set up the column assembly.

Tools

• Small flat-blade screwdriver

• Tightening tool (in kit)

Parts and materials

• UHPLC Liquid Junction Kit (P/N ES269)

• Powder-free safety gloves

To install the column assembly and prepare the system for operation, follow these procedures:

1. To connect the column assembly to the UHPLC liquid junction cross, do one of the following:

“To connect a two-column assembly to the UHPLC liquid junction cross,”

next procedure

–or–

“To connect a one-column assembly to the UHPLC liquid junction cross” on page 105

2.

“To mount the UHPLC liquid junction cross onto the DirectJunction” on page 106

3.

“To prepare a system with the UHPLC liquid junction cross for operation and optimize its performance” on page 108

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To connect a two-column assembly to the UHPLC liquid junction cross

1. Slip an internally-threaded nut and a microferrule (components of the UHPLC fused-silica union) onto the Column Out line (

Figure 64

).

Figure 64. Column Out line with nut and ferrule

Microferrule for 360 μm OD tubing

Internally-threaded knurled nut

Column Out line with bare fused-silica end

2. Insert the stainless steel cartridge into the deeper end of the externally-threaded PEEK holder (see

Figure 62 on page 101 ). Then insert the Column Out line into the deeper end

of the PEEK holder and hand-tighten the nut ( Figure 65 ).

Figure 65. Column Out line connected to the deeper end of the PEEK holder

PEEK holder with stainless steel cartridge

3. Slip an internally-threaded knurled nut and a ferrule onto the inlet end of a precolumn.

Then insert the inlet end of the precolumn into the PEEK holder and hand-tighten the nut (

Figure 66

).

Figure 66. Column Out line and precolumn connected to the PEEK holder

Thermo Scientific

4. Using the tightening tool provided in the UHPLC Liquid Junction Kit, tighten the internally-threaded knurled nuts an additional one-quarter turn (

Figure 67 ).

Figure 67. Using the tightening tools to tighten the nuts an additional one-quarter turn

PEEK holder

Tightening tool

Internally-threaded knurled nuts

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CAUTION Danger Electric Shock Hazard!

Before handling the UHPLC liquid junction cross, make sure that it is disconnected from line power.

5. Remove the internally-threaded knurled nuts and the microferrules from the UHPLC liquid junction cross. Using these nuts and ferrules, connect the precolumn, the analytical column, and the Waste In lines to the UHPLC liquid junction cross as follows:

• Connect the outlet of the precolumn and the inlet of the analytical column to the parallel ends of the cross.

• Connect the Waste In line to the perpendicular end of the cross.

Figure 68 shows the plumbing connections for the UHPLC liquid junction cross. Make

sure that the columns connect to the parallel ends of the cross.

Figure 68. UHPLC liquid junction cross connections for a two-column assembly

Column Out line Precolumn

Waste In line

Analytical column

UHPLC fused-silica union

UHPLC liquid junction cross with 24 in. high-voltage cable

Disconnected from line power

6. Using the tightening tool, tighten the three internally-threaded knurled nuts an additional one-quarter turn.

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To connect a one-column assembly to the UHPLC liquid junction cross

CAUTION Before handling the UHPLC liquid junction cross, make sure that it is disconnected from line power.

1. Remove the externally-threaded knurled nuts and the microferrules from the UHPLC liquid junction cross. Then, using these nuts and ferrules, connect the Column Out line, the analytical column, and the Waste In lines to the UHPLC liquid junction cross as follows:

• Connect the Column Out line and the inlet of the analytical column to the parallel ends of the cross.

• Connect the Waste In line to the perpendicular end of the cross.

Figure 69 shows the plumbing connections for the UHPLC liquid junction cross. Make

sure that the Column Out line and the analytical column connect to the parallel ends of the cross.

Figure 69. UHPLC liquid junction cross connections for a one-column setup

Column Out line

Waste In line

Pulled fused-silica analytical column

UHPLC liquid junction cross with high-voltage cable

24 in. cable

Thermo Scientific

Disconnected from line power

2. Using the tightening extender tool, tighten the three internally-threaded knurled nuts an additional one-quarter turn.

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To mount the UHPLC liquid junction cross onto the DirectJunction

CAUTION During operation, the UHPLC liquid junction cross is at high voltage. To avoid personal injury, make sure that the UHPLC liquid junction cross is securely mounted inside its two-piece protective cover.

1. Using a flat-blade screwdriver, remove the two screws that secure the top of the protective cover to its body.

Figure 70 shows a profile view of the two-piece protective cover.

Figure 70. Protective cover (profile view)

Top

Compression screw

Disconnect before opening

Mounting hole (slides onto the Direct Junction’s metal rod)

2. Slide the protective cover’s body onto the DirectJunction’s metal rod. If necessary, loosen the compression screw in the body until the hole in the body fits over the rod, and then tighten the screw.

3. Mount the UHPLC liquid junction cross onto the body of the protective cover

( Figure 71

). Then, place the top of the protective cover over the liquid junction cross.

Both the top and the body of the cover contain two magnets. When you place the top onto the body, the magnetic attraction holds the two pieces together.

Figure 71. UHPLC liquid junction cross mounted onto the body of the protective cover

Column Out line of a one-column assembly or precolumn of a two-column assembly

Waste In line

Pulled fused-silica analytical column

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Direct Junction’s metal rod

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Setting Up the Column Assembly

4. Using the two screws that you removed in

step 1 on page 106 , secure the top of the

protective cover to its body (

Figure 72

).

Figure 72. UHPLC liquid junction cross secured within the protective cover

Column Out line of a one-column assembly or precolumn of a two-column assembly

Waste In line

Pulled fused-silica analytical column

Direct Junction’s metal rod

5. If necessary, connect an emitter to the outlet of the analytical column as described in

“Connecting a Sleeved Emitter to the Analytical Column” on page 111

.

6. Mount the emitter on the DirectJunction and secure it with the clamp ( Figure 73

).

Figure 73. Column assembly mounted on DirectJunction

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7. If the column is longer than the DirectJunction, use a 1/32 in. PEEK connector (IDEX

P/N P-771) to secure the emitter tip ( Figure 74 ).

Figure 74. PEEK connector used to secure a long analytical column

8. Connect the high voltage cable from the UHPLC liquid junction cross to the socket underneath the ion source (

Figure 75 ).

Figure 75. High-voltage cable connected to the ion source

To prepare a system with the UHPLC liquid junction cross for operation and optimize its performance

1. Turn on the EASY-nLC instrument and log in (see

“Logging In to the EASY-nLC

Instrument” on page 66

).

2. Remove air from the Waste In and Column Out lines by running the Precolumn

Equilibration script. Do the following: a. Open the Precolumn Equilibration script as follows: i.

On the touch screen, press Maintenance > Scripts.

ii. In the Category list, select Prepare.

iii. In the Name list, select Precolumn Equil.

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Setting Up the Column Assembly b. Set up the script parameters as follows: i.

Press the Parameters tab.

ii. Enter the following settings:

– Volume: 30 μL

– Flow: (Leave Empty)

– Max Press: Set to the maximum pressure rating of the installed columns.

c.

Press Start.

IMPORTANT To maintain optimal system performance, be aware of the following:

• Repeatedly connecting and disconnecting the same fused-silica column to the

UHPLC fused-silica union or UHPLC liquid junction cross can damage the polyimide protection layer of the column and cause column blockage.

• The Liquid Junction Kit is designed for 360 μm OD columns. Using the kit with columns of other dimensions can cause leakage, delayed elution, and non-reproducibility of chromatographic performance.

• Use only IDEX brand Very High Pressure (VHP) ferrules for 360 μm OD tubing to connect tubing to the UHPLC fused-silica union and the UHPLC liquid junction cross. Using ferrules that are not specifically designed for ultra-high pressure, 360 μm

OD, fused-silica tubing can cause leaks and add swept volume.

• A poor connection between the analytical column and the emitter can cause broad chromatographic peaks.

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For routine applications with the UHPLC liquid junction cross, follow these guidelines:

• Apply a high voltage potential from 2.0 to 2.5 kV. The actual voltage applied once the spray has been optimized depends on the length and ID of the analytical column.

• Use formic acid rather than acetic acid as a mobile phase modifier to avoid the formation of peptide-Fe cluster ions with acetic acid [(C water clusters of these ions [(C

2

H

4

O

2

) n

2

–nH H

H

2

4

O

2

) n

–nH + 3Fe + O]

O + 3Fe + O]

+ as well as the

+

. You can distinguish ions that contain iron by their isotope distribution (

Figure 76

).

Figure 76. Spectrum of peptide-Fe cluster ions and the theoretical isotope distribution of these ions

When you use the UHPLC liquid junction cross, Thermo Fisher Scientific recommends that you maintain a supply of spare parts (see

Table 14 ).

Table 14. Spare parts for a column assembly with a pulled fused-silica column

Description

VHP sleeveless cross for 360 um OD tubing

VHP microferrule for 360 μm OD tubing

UHPLC spare blind plug

Tightening tool to tighten internally-threaded knurled polymer fittings

Column Out tubing (modified)

Waste In tubing (modified)

PEEK connector (red) for 1/32 in. OD tubing

Part number

UH-752 (IDEX)

PK-152 (IDEX)

P-116 (IDEX)

P-278 (IDEX)

6041.5290

6041.5289

P-771 (IDEX)

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Setting Up the Column Assembly

Connecting a Sleeved Emitter to the Analytical Column

Before you mount the column assembly onto the ion source, connect an emitter to the analytical column. Borosilicate emitters require sleeves. Stainless steel emitters come pre-inserted into microsleeves.

For information about ordering stainless steel emitters sleeves, visit www.proxeon.com/productrange/nano_ES_emitters/online_offline_emitters/index.html

Table 15

lists the sleeves for 1/32 in. fittings that you can order to install a borosilicate emitter.

Table 15. Thermo Fisher Scientific sleeves for 1/32 in. fittings and borosilicate emitters

Color

Natural

Black

Use

For 360 μm OD tubing

For 280 μm OD tubing

Part number

SC603

SC903

CAUTION Because the emitter tip is sharp enough to puncture skin and easily damaged, do not touch it.

To connect an emitter to the PEEK union

1. If you are using the preassembled column assembly, disconnect the analytical column from the PEEK union (IDEX P/N P-771).

2. Connect the white distance-gauge plug to one end of the PEEK union.

3. Connect the emitter to the other end of the PEEK union as follows: a. For a glass emitter, thread the blunt end of the emitter through the adapter sleeve.

Ensure that the blunt end of the emitter does not protrude from the (20 mm length)

sleeve by more than 1 mm ( Figure 77 ).

Figure 77. Sleeved emitter (4x scale)

Adapter sleeve

Blunt end of the glass emitter

Emitter tip

Thermo Scientific

1 mm protrusion b. To ensure proper seating, slide the blunt end of the sleeved emitter through the PEEK nut until it protrudes slightly (approximately 1 to 2 mm) from the front of the fitting.

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Setting Up the Column Assembly c.

As you gently press the sleeved emitter against the union port, screw the nut into the

port until fingertight ( Figure 78

). For a stainless steel emitter, turn the nut an additional half-turn (180 degree) once you feel resistance.

d. Verify that the connection is secure.

Figure 78. Sleeved emitter connected to the PEEK union

Plug Adapter sleeve

Emitter tip

Nut

Union

4. Remove the gauge from the PEEK union and reconnect the analytical column to the union.

Setting Up the Column Assembly for the EASY-Spray Source

If your LC/MS has an EASY-Spray source, follow the appropriate procedure to connect either a one- or two-column setup.

“Connecting an EASY-Spray Column to an EASY-nLC 1200 Instrument” on page 113

The EASY-Spray Ion Source Kit contains the fittings and sleeves for the plumbing

connections (see Table 16

).

Table 16. Fittings and sleeves in the EASY-Spray Ion Source Kit

Image Description

A/B mixing/venting Tee, stainless steel, with three attached ferrules and nuts

Part number

SC901

Sleeves, PEEK, 1/16 in. OD IDEX™, F-233

Viper union

(for the EASY-nLC 1200 instrument)

Thermo Scientific Dionex,

6040.2304

CAUTION Wear protective gloves and safety glasses when handling the solvent lines. To prevent contamination, use lint-free and powder-free gloves.

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Setting Up the Column Assembly

Connecting an EASY-Spray Column to an EASY-nLC 1200 Instrument

Follow the appropriate procedure for a one- or two-column setup. The two-column setup includes a precolumn in the column-out flow path to the venting Tee.

“To connect an EASY-Spray column for a one-column setup to the EASY-nLC 1200 instrument,”

next procedure

“To connect a two-column setup with a precolumn and an EASY-Spray column to an

EASY-nLC 1200 instrument” on page 114

IMPORTANT The venting Tee (P/N SC901) supplied in the EASY-Spray Ion Source Kit has three ports with attached fittings (stainless steel nut and ferrule). The attached fittings must not be used with the EASY-nLC 1200 instrument; however, to keep the internal components of the union aligned, remove only one of the three fittings at a time.

Use the two parallel ports to connect the sample transfer line. Use the perpendicular port to connect the waste line.

To connect an EASY-Spray column for a one-column setup to the EASY-nLC 1200 instrument

Connect the nanoViper fittings at the ends of the Column Out and Waste In solvent lines and the nanoViper fitting at the inlet end of the EASY-Spray column to the venting Tee as follows:

• Remove the fitting (stainless steel nut and ferrule) from one of the venting Tee’s parallel ports. Insert the Column Out solvent line’s nanoViper fitting into the free port. Slowly tighten the fitting until you feel resistance. Then tighten the fitting by an additional 1/8- to 1/4-turn. Do not tighten the fitting by more than a 1/4-turn

(see

“Using nanoViper Fittings” on page 115

).

• Remove the fitting from the venting Tee’s perpendicular port. Insert the Waste In solvent line’s nanoViper fitting into the free port, and then carefully tighten the fitting.

• Remove the fitting from the remaining parallel port. Insert the nanoViper fitting at the inlet end of the EASY-Spray column into the venting Tee’s free parallel port, and then carefully tighten the fitting.

Figure 79 shows the one-column setup for an EASY-Spray column and an

EASY-nLC 1200 instrument.

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Setting Up the Column Assembly

Figure 79. One-column setup with an EASY-Spray column and an EASY-nLC 1200 instruments

Venting Tee

Column Out line

EASY-Spray column

Waste In line

Tip When you install the one-column assembly, make sure that the One Column Setup check box in the EASY-nLC device section on the Devices page of the Maintenance menu is selected (see

“Setting Up the Properties for the HPLC Device” on page 80 ).

To connect a two-column setup with a precolumn and an EASY-Spray column to an

EASY-nLC 1200 instrument

1. Connect the Column Out line to the precolumn inlet with a Viper union as follows: a. Insert the nanoViper fittings on the ends of the tubing into the Viper union.

Note Follow the recommended flow direction stated on the precolumn label. b. Because the nanoViper fittings seal against each other inside the ZDV union, alternate tightening each fitting in increments until you feel resistance (see

“Using the

Viper Union” on page 117

).

c.

Tighten both fittings by an additional 45 degrees (1/8-turn). Do not tighten the fittings by more than a 1/4-turn (see

“Using nanoViper Fittings” on page 115

).

Note The venting Tee has three ports. Use the two parallel ports to connect the sample transfer line. Use the perpendicular port to connect the waste line.

2. Connect the outlet end of the precolumn, the inlet end of the EASY-Spray column, and the Waste In line to the venting Tee as follows:

• Insert the nanoViper fitting on the outlet end of the precolumn into one of the venting Tee’s parallel ports, and then carefully tighten the fitting.

• Connect nanoViper fitting on the inlet end of the EASY-Spray column to the venting Tee’s free parallel port, and then carefully tighten the fitting.

• Insert the fitting on the end of the Waste In line into the venting Tee’s perpendicular port, and then carefully tighten the fitting.

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Using nanoViper Fittings

Figure 80 shows a two-column setup for an EASY-nLC 1200 instrument and an

EASY-Spray source.

Figure 80. Two-column setup for an EASY-nLC 1200 instrument and an EASY-Spray source

Column Out line

EASY-Spray column

Flow

Waste In line

Tip When you install the two-column assembly, clear the One Column Setup check box in the EASY-nLC device section on the Devices page of the Maintenance menu (see

“Setting Up the Properties for the HPLC Device” on page 80

).

Using nanoViper Fittings

IMPORTANT The EASY-nLC 1200 requires the current version of nanoViper fitting systems with a specified pressure range up to 1200 bar. Older versions are only specified up to 1000 bar. The new 1200 bar nanoViper fitting system consists of blue

PEEK tubing, which distinguishes it from the beige 1000 bar version, see Figure 81

.

Figure 81. Visual difference between 1000 bar (top) and 1200 bar nanoViper fitting (bottom)

1200 bar version

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Preparing the EASY-nLC Instrument for Use

Using nanoViper Fittings

For the EASY-nLC 1200 instrument, most of the plumbing connections are made with

nanoViper fittings (see Figure 82 ).

Figure 82. nanoViper fitting

PEEK sealing surface

Screw Removable, black, knurled tightening tool

Even though nanoViper fittings can withstand UHPLC backpressures of up to ~1034 bar

(~15 000 psi), they are fingertight fittings, which require only very small torques to seal. To avoid damage by overtightening, you must follow this procedure.

To connect a nanoViper fitting to a receiving port

1. Insert the nanoViper fitting into the target port and slowly rotate the screw clockwise until you feel resistance.

2. Using the black, knurled tightening tool, turn the screw clockwise to an angle between

0 and 45 degrees (1/8-turn).

IMPORTANT To prevent damage to the sealing surface of the nanoViper fitting, take care not to overtighten the nanoViper fitting.

3. Run the Leaks script for the system as described in the EASY-nLC Series Troubleshooting

and Maintenance Guide.

When the leak test ends, the system is at atmospheric pressure.

IMPORTANT To extend the lifetime of the nanoViper fittings, open and close connections at atmospheric system pressures only. Opening and closing connections at high system pressures can reduce the lifetime of the fitting system.

4. If the Leaks script fails because the new connection is not leak tight, use the black knurled nut to turn the screw up to an additional 45 degrees. Do not turn the screw beyond an angle of 90 degrees from where you felt the initial resistance.

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Using the Viper Union

Using the Viper Union

For an EASY-nLC instrument with the standard Column Out and Waste In lines, use a Viper union to connect the Column Out line to the Waste In line during a system leak test or to connect the Column Out line to the precolumn of a two-column assembly. The Viper union is a true zero-dead-volume (ZDV) union, which means that the fittings connected to its receiving ports seal against each other inside the union.

To connect tubing with nanoViper fittings to the Viper union

1. Insert the nanoViper fittings on the ends of the tubing into the Viper union.

2. Alternate tightening each fitting in increments until you feel resistance.

3. Tighten both fittings by an additional 45 degrees (1/8-turn). Do not tighten the fittings by more than a 1/4-turn (see

“Using nanoViper Fittings” on page 115

).

CAUTION Because the nanoViper fittings seal against each other inside the union, do not tighten them by more than 90 degrees (1/4-turn). Overtightening the fittings can irreparably damage their sealing surfaces.

Figure 83 shows two solvent lines with nanoViper fittings connected to the Viper union.

The Viper union makes a zero-dead-volume connection between the two nanoViper fittings.

Figure 83. Viper union connecting tubing with nanoViper fittings nanoViper fitting

Viper union

(P/N SC900) nanoViper fitting

Figure 84 shows an internal view of the Viper union with nanoViper fittings connected to

both ports. The two nanoViper fittings seal against each other in the center of the union, and a portion of the last thread on each nanoViper fitting is visible.

Figure 84. Viper union with nanoViper fittings connected to both ports (internal view)

Thermo Scientific

Butt joint between the sealing surfaces of the nanoViper fittings

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Preparing the EASY-nLC Instrument for Use

Equilibrating Columns

Equilibrating Columns

After you initially install the column assembly, equilibrate the column or columns and determine an appropriate flow rate for the sample loading step (see

“Setting Up the Sample

Pickup and Loading Steps” on page 127

) of the chromatographic method.

Follow these procedures to equilibrate the columns and to determine the appropriate flow rate for your chromatographic method:

“Equilibrating the Precolumn,”

next section

“Equilibrating the Analytical Column” on page 120

Equilibrating the Precolumn

To equilibrate the precolumn and determine the flow rate for sample loading

1. Open the Parameters view of the Precolumn Equilibration script as follows: a. Press Maintenance > Scripts.

The Maintenance > Scripts page opens.

b. In the Category list, select Prepare.

c.

In the Name list, select Precolumn Equil.

d. Press the Parameters tab.

The Parameters view opens (see

Figure 85 ).

Figure 85. Precolumn equilibration parameters

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2. Set up the script parameters as follows: a. In the Volume [μL] box, enter the volume of solvent A to be used to equilibrate the precolumn.

For best results, use at least 10 column volumes to equilibrate the precolumn.

b. In the Flow [μL/min] box, enter the flow rate for the precolumn equilibration step.

If the flow field is left empty, the pump operates at the set pressure.

c.

In the Max Pressure [bar] box, enter the maximum allowed pressure for the equilibration step.

If the pressure field is left empty, the pump operates at the set flow (as long as it is below the instrument maximum pressure of 1180 bar). If both a flow and a max pressure are specified, the pump flow is limited by whichever parameter is reached first.

CAUTION The maximum pressure rating for the columns is as follows:

• The maximum pressure rating is 1200 bar for the PepMap columns supplied with the EASY-nLC 1200 instrument.

Running the instrument at pressures higher than the column’s maximum pressure rating reduces the column lifespan.

Thermo Fisher Scientific recommends running the equilibration at a set pressure rather than a set flow, to make sure the column pressure limit and the instrument pressure limit are not exceeded (see

Figure 85 on page 118

). If the flow field is left empty, the pump will run at the specified pressure. For best results, use an equilibration volume of 10 column volumes.

Item

EASY-nLC 1200 instrument

PepMap column

EASY-Spray column

Maximum pressure (in bar)

1180

1200

Refer to the specifications provided with the column.

3. Press START.

The default graphs appear in the two graph windows, but you can change the selections from their corresponding lists. These graphs help you to monitor the progress.

4. Monitor the actual flow rate to become familiar with the flow/pressure relationship on your particular column.

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Equilibrating the Analytical Column

To equilibrate the analytical column

1. Open the Parameters view of the Analytical Column Equilibration script as follows: a. Press Maintenance > Scripts.

The Maintenance > Scripts page opens.

b. In the Category list, select Prepare.

c.

In the Name list, select Analytical Col equil.

d. Press the Parameters tab.

The Parameters view of the Analytical Column Equilibration script opens

(see

Figure 86 ).

Figure 86. Analytical column equilibration parameters

2. Set up the script parameters as follows: a. In the Volume [μL] box, enter the volume of solvent A to be used to equilibrate the analytical column.

For best results, use at least 10 column volumes to equilibrate the analytical column.

b. In the Flow [μL/min] box, enter the flow rate for the analytical column equilibration step.

If the flow field is left empty, the pump operates at the set pressure.

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In the Max Pressure [bar] box, enter the maximum allowed pressure for the equilibration step.

If the pressure field is left empty, the pump operates at the set flow (as long as it is below the instrument maximum pressure of 1180 bar).

If both a flow and a maximum pressure are specified, the pump flow is limited by whichever parameter is reached first.

CAUTION The maximum pressure rating is 1200 bar for the PepMap columns supplied with the EASY-nLC 1200 instrument. Running the instrument at pressures higher than the maximum pressure rating for the column reduces the column lifespan.

Thermo Fisher Scientific recommends running the equilibration at a set pressure rather than a set flow, to make sure the column pressure limit and the instrument pressure limit are not exceeded. If the flow field is left empty, the pump will run at the specified pressure. For best results, use an equilibration volume of 10 column volumes.

Item

EASY-nLC 1200 instrument

PepMap column

EASY-Spray column

Maximum pressure (in bar)

1180

1200

Refer to the specifications provided with the column.

3. Press START.

The default graphs appear in the two graph windows, but you may change the selections from their corresponding lists. These graphs help you to monitor the progress.

4. Monitor the actual flow rate to become familiar with the flow/pressure relationship on your particular column or columns.

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Running Samples Using the Integrated Software

To create a method and run your first sample batch by using the integrated instrument control software, follow these procedures.

Contents

Preparing and Loading Samples

Creating a Method

Creating a Batch

Starting Sample Acquisition

Monitoring the Run

Stopping Sample Acquisition

Editing the Running Batch

Troubleshooting a Sample Run

Preparing and Loading Samples

The EASY-nLC instrument ships with adapters for vials (54 vials in a 6 × 8+6 configuration), microplates (96- and 384-well), and PCR strips.

If you are using a new plate format, you must add the plate format to the list of configured plate formats and calibrate the plate as described in the EASY-nLC Series Troubleshooting and

Maintenance Guide.

To prepare the sample vials or microplates

1. Fill some vials or microplate wells with your sample, preferably a known standard.

2. Make sure that no air is trapped in the sample vials or the microplate wells and that the sample is in the bottom of the vials or wells. To do this, tap the vial or microplate gently against a hard surface to have the sample move to the bottom.

3. Put on the vial cap or plate mat.

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To load a microwell plate or vials into the autosampler tray compartment

1. To open the tray compartment door, do one of the following:

• Manually open the tray compartment door.

–or–

• Use the touch-screen controls to open the tray compartment door as follows: i.

On the Home > Overview page, press Eject/Insert Tray.

A confirmation box appears to remind you to remove obstacles from the area in front of the tray compartment before opening the tray compartment door. ii. Make sure that the area in front of the tray compartment is clear, and then press

Accept.

The tray compartment door opens and the plate holder moves forward.

2. Load your microwell plate or sample vials.

3. On the Home > Overview page, press Eject/Insert Tray.

Creating a Method

To create a method, follow these procedures:

1.

“Browsing for a Stored Method or Creating a New Method File,” next section

2.

“Starting the Method Setup Wizard” on page 126

3.

“Setting Up the Sample Pickup and Loading Steps” on page 127

4.

“Building the Gradient” on page 130

5.

“Setting Up the Column Equilibration Steps” on page 132

6.

“Setting Up the Autosampler Wash Step” on page 134

7. (Optional) “Specifying the Temperature of the EASY-Spray Column Heater” on page 137

8.

“Saving the Method Settings” on page 138

Browsing for a Stored Method or Creating a New Method File

Use the Method Setup > File page to browse for stored methods or to select the file folder and enter a name for the new method.

To browse for a stored method

1. Press Method Setup > File.

The Method Setup > File page opens (see Figure 87 ).

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Figure 87. File page of the Method Setup

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2. To view the methods in the folder, press the file folder name in the Path column.

3. To view a description of the method (if available), press the method name.

To create a new method

1. Open the Method Setup > File page (see

Figure 87 ).

2. Press New Method.

The Create New File dialog box opens.

3. In the Folder box, browse to the directory where you want to store the new method.

4. In the New Name box, enter a method name.

5. (Optional) In the Description box, enter a description of the method.

6. Press Save.

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Starting the Method Setup Wizard

The Method Setup wizard consists of five or more pages that guide you through all the parameters required for a working LC method.

Use first page of the Method Setup wizard to select the method that you want to edit and the external devices that want to control.

To start the Method Setup wizard

1. Press Method Setup > Edit.

2. If you are not already on the page 1 (see

Figure 88

), go to page 1 by pressing the appropriate arrow near the bottom of the touch screen.

Figure 88. Method Setup > Edit page 1

3. Press Open.

The Select Method to Open dialog box opens.

4. Select the method that you want to edit.

5. Press Accept.

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To control an external device

In the Method extensions area, select the check box for the device.

• To control the column temperature of the EASY-Spray ion source, select the

EASY-Spray check box.

• To control the Replay device, select the RePlay check box.

By default, the Method Setup wizard contains five pages. For each external device that you add, the wizard increases by one page.

To continue setting up the method, press the right arrow to go to the next page of the Method

Setup wizard (page 2/x, where x equals the total number of pages).

Setting Up the Sample Pickup and Loading Steps

Use the Method Setup > Edit 2/x page (see

Figure 89 ) to set the parameters for picking up the

sample from the well plate and loading it onto the precolumn.

Figure 89. Edit 2/

x page of the Method Setup wizard

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To set up the sample pickup parameters

1. In the Sample Pickup area, press the Volume box and enter the default amount of sample to be picked up.

The autosampler withdraws the requested sample volume; it does not pick up excess sample.

To prevent contamination of pump S, the maximum volume is set to the configured sample loop size minus 2 μL. For example, the maximum sample pickup for an instrument with a 20 μL sample loop is 18 μL.

You can overwrite this setting when you set up the injection sequence from an external

Thermo Scientific data system such as the Xcalibur data system.

2. Press the Flow box to set the flow rate at which the autosampler picks up sample.

Range: 0 to 40 μL/min

Set the speed according to the sample viscosity and accuracy needed. For typical samples,

20 μL/min is optimal. Setting the flow rate too high can draw air bubbles into the sample plug.

To set up the sample loading parameters

1. In the Sample Loading area, press the Volume box and enter the volume of solvent A to be used to load the sample from the sample loop onto the precolumn (or analytical column in a one-column setup).

Range: 0 to 137

Normally it is sufficient to load the precolumn with 2 × sample pickup volume + 2 μL or a minimum of 6 μL, whichever is larger.

2. Press the Flow box and enter the flow rate for sample loading onto the precolumn (or analytical column in a one-column setup).

If you leave the Flow box empty, the pump operates at the set pressure.

Range: 0 to 100μL/min

Tip The application accepts values from 0 to 100 μL/min, but the IFC algorithm (see

“Intelligent Flow Control System” on page 32

) prevents the flow rate for the sample loading step from exceeding 25 μL/min based on the backpressure produced by the system tubing alone without the columns attached.

Use the column equilibration scripts to determine the appropriate flow rate for your application.

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3. Press the Max. Pressure box and set the maximum allowed pressure.

If you leave the Max. Pressure box empty, the pump operates at the set flow (as long as it is below the maximum instrument pressure of 1180 bar).

Tip If you specify both a flow rate and a maximum pressure, the pump flow is limited by the parameter that is reached first. If you leave the flow and max pressure boxes empty, the IFC algorithm allows pump A to run at the maximum system pressure.

IMPORTANT If you enable the LSP feature and specify a maximum pressure, the batch run stops if the system reaches the maximum pressure setting before it reaches the specified flow rate during sample loading.

To continue setting up the method, go to the Edit 3/x page by pressing the right arrow.

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Building the Gradient

Use the Method Setup > Edit 3/x page to build the gradient. This page consists of a schematic view for setting parameters and a graph view showing the gradient as a function of time (see

Figure 90 ).

Use the four buttons at the bottom of the page to add lines to the gradient, move lines up and down, and delete lines. To move or delete lines, first select the line by pressing it.

The rest of this tutorial guides you through building a gradient for analyzing a BSA digest on a C18 column. Because this tutorial is intended to help you set up a method, make sure to set the parameters appropriately for your analyte and column characteristics.

Figure 90. Edit 3/

x page of the Method Setup wizard

Note When you press a cell in the gradient table, the keypad dialog box opens.

To build a gradient

1. In the first row of the gradient table, do the following: a. Press the Flow column cell and enter 300 nL/min. Then press ACCEPT.

b. Press the % B column cell and enter 5 (%). Then press ACCEPT.

The gradient is now set to start at 300 nL/min and 5 %B.

2. Press ADD to add another row.

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3. In the second row of the gradient table, do the following: a. Press the Duration column cell and enter 10 minutes. Then press ACCEPT.

b. Press the Flow column cell and enter 300 nL/min. Then press ACCEPT.

c.

Press the % B column cell and enter 35 (%). Then press ACCEPT.

4. Press ADD to add another row.

5. In this new row, set duration to 2 min, flow to 300 nL/min, and % B to 100.

6. Press ADD to add another row.

7. In this new row, set the duration to 8 min, flow to 300 nL/min, and % B to 100.

Note The Method Setup wizard includes a separate page for setting up the column equilibration parameters, so you do not need to add a column equilibration step to the end of the gradient program. The column equilibration steps run parallel with the sample pickup step at the beginning of a sample run.

To continue setting up the method, go to the Edit 4/x page by pressing the right arrow.

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Setting Up the Column Equilibration Steps

Use the Method Setup > Edit 4/x page to set up the parameters for the column equilibrations

(see

Figure 91 ).

Figure 91. Edit 4/

x page of the Method Setup wizard

To set up the precolumn equilibration

1. In the Precolumn Equilibration area, press the Volume box to set the amount of solvent A to use for equilibration of the precolumn.

For best results, use a least 10 column volumes for to equilibrate the precolumn.

2. Press the Flow box to set the flow rate for performing the equilibration of the precolumn.

If you leave the Flow box empty, the pump operates at the set pressure during a sample run.

3. Press the Max. Pressure box to set the maximum allowed pressure.

If you leave the Max. Pressure box empty, the pump operates at the set flow (as long as it is below the instrument pressure of 1180 bar).

Note If you specify both a flow rate and a maximum pressure, the pump flow is limited by the parameter that is reached first.

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To equilibrate the analytical column

1. In the Analytical Column Equilibration area, press the Volume box to set the amount of solvent A to use for equilibration of the analytical column.

For best results, use at least 10 column volumes to equilibrate the analytical column.

Range: 0 to 137

2. Press the Flow box to set the flow rate for performing the equilibration of the analytical column.

If you leave the Flow box empty, the pump operates at the set pressure during a sample run.

3. Press the Max. Pressure box to set the maximum allowed pressure.

If you leave the Max. Pressure box empty, the pump operates at the set flow (as long as it is below the instrument pressure of 1180 bar).

Note If you specify both a flow rate and a maximum pressure, the pump flow is limited by the parameter that is reached first.

To continue setting up the method, go to the Edit 5/x page by pressing the right arrow.

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Setting Up the Autosampler Wash Step

Use the Method Setup > Edit 5/x page for setting up the autosampler washing procedures (see

Figure 92 ). The EASY-nLC instrument conducts the wash while the gradient is running.

Use the standard wash step or set up a custom wash cycle as described in these procedures:

Setting Up a Standard Wash Step

Setting Up a Custom Wash Program

Setting Up a Standard Wash Step

For most samples in low concentrations, flushing the needle and loop with solvent A is sufficient. You can set the amount of solvent for this action in the Flush Volume box.

Use 100 μL as the default flush volume.

Note This equation defines the minimum flush volume that you can set:

loop volume + needle volume + 1 μL.

Figure 92. Edit 5/

x page of the Method Setup wizard

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To set up a standard wash step

1. Do not select the Custom Wash check box.

2. In the Flush Volume box, type the volume of solvent A (from wash bottle 3) that the autosampler uses to flush the needle and the sample loop.

The range depends on the sample loop size. For the 20 μL sample loop, the range is

30.25 to 100.75 μL.

Setting Up a Custom Wash Program

For a more thorough wash of the needle and loop, select the Custom Wash check box. Use this option to set up a user-definable procedure for washing the loop and needle.

Tip The custom wash program automatically ends with a preset volume of solvent A from bottle W3.

The Custom Wash table consists of three user-editable parameters (see Table 17

). The Order column lists the order in which the autosampler performs the steps. To change the order, use the Up and Down buttons.

Table 17. Custom wash parameters

Parameter

Order

Source

Description

Defines the order of the wash steps. To change the order of the wash steps, use the Up and Down buttons.

Specifies the wash solvent source for the current wash step.

Volume

Selections: Bottle 1, Bottle 2, or Bottle 3

Bottles 1 and 2 contain custom wash solvents. Bottle 3 contains solvent A.

Specifies the volume that pump S draws from the source location for each wash cycle.

Cycles

To avoid drawing solvent into pump S, for a 20 μL sample loop, the maximum volume is 28 μL.

needle volume + loop volume – 2 μL

Specifies the number of wash cycles for the wash step.

For each cycle, the autosampler draws the specified volume of wash solvent from the specified source, and then ejects the wash solvent into wash bottle 4.

When the custom wash has ended, the autosampler automatically empties the pump and refills with solvent from bottle 3 to be ready for the next injection.

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To set up a custom wash program

1. Select the Custom Wash check box.

The custom wash table becomes available.

2. For each row that you want to add to the program, press Add Line.

You can select a different wash solvent for each row.

3. Edit each row as follows:

• In the Source column, select the wash solvent source.

The selections are Bottle 1, Bottle 2, or Bottle 3.

Note The EASY-nLC autosampler holds up to four bottles: three for washing solvents and the fourth for ejecting waste and cleaning the outside of the injection needle.

• Bottles 1 and 2 in positions W1 and W2 contain custom wash solvents.

• Bottle 3 in position W3 contains solvent A.

• Bottle 4 in position W4 contains the needle wash insert.

• In the Volume column, type a volume from 0 to the sample loop size plus 8 μL.

• In the Cycle column, type a value from 0 to 10.

4. To edit an existing wash program, do the following:

• To add a row to the program, press Add Line.

• To remove a row from the program, press Delete Line.

• To copy a selected line, press Copy Line.

• To move a selected row up or down, press Up or Down, respectively.

When you finish setting up the method parameters, save the method as described

“Saving the

Method Settings” on page 138 .

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Specifying the Temperature of the EASY-Spray Column Heater

If your LC/MS system includes the EASY-Spray ion source, use the Method > Edit 6/x page to specify the temperature of the EASY-Spray column heater.

Figure 93. Edit page 6/

x of the Method Setup wizard

Thermo Scientific

To specify the column temperature

1. Press the temperature box.

The Set Value dialog box opens.

2. Press the number pad to enter a temperature from 30 to 60 °C.

The application does not accept temperatures below 30 °C or above 60 °C.

3. After you enter an appropriate temperature, press Accept.

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Saving the Method Settings

After you enter the method settings, save the method.

To save the method

1. Press Save in the top right corner of the Method Setup wizard.

The Save File dialog box opens.

2. Select the folder where you want to store the method as follows: a. Press the Folder box.

The Select Folder dialog box opens ( Figure 94 ).

Figure 94. Select Folder dialog box b. Select the appropriate folder.

c.

Press Accept.

The Select Folder dialog box closes and the folder location appears in the Save As dialog box.

3. Enter the method name as follows: a. Press the Name box.

The virtual keyboard opens.

b. Use the virtual keyboard to type the method name.

c.

Click OK.

The virtual keyboard closes and the new method name appears in the Save File dialog box (

Figure 95

).

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Figure 95. Save File dialog box

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4. (Optional) In the Save As dialog box, enter a description for the method in the

Description box.

5. Click Save to save the method.

The Save File dialog box closes. If the method already exists, the Overwrite Method dialog box opens.

6. If the Overwrite Method dialog box opens, press Yes to overwrite the existing method or press No to close the Save File dialog box without saving the method.

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Creating a Batch

Creating a Batch

Use the Batch Setup > Edit page to set up and schedule batches for sample acquisition on the

EASY-nLC instrument. (See also

“Preparing and Loading Samples” on page 123 and

“Creating a Method” on page 124 .)

To create a batch, follow these procedures:

“Viewing the Batch List and Setting Up a Batch Record,”

next section

“Specifying the Method, Sample Positions, and Number of Injections” on page 141

Viewing the Batch List and Setting Up a Batch Record

Use the Batch Setup > File page to view a list of stored batches, import a batch from another

EASY-nLC instrument, and create an empty batch record (that is, name a new batch and select its folder location).

To view a list of batches

1. Press Batch Setup > File.

The Batch Setup > File page opens (see Figure 96

).

Figure 96. File page of the Batch Setup menu

2. To view your batches, in the Path column, press the folder where you store your batches.

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To specify the batch name and folder location

1. On the Batch Setup > File page, press New Batch.

The Create New File dialog box opens.

2. In the Folder box, browse to the folder where you want to store the batch.

3. In the New Name box, enter a name for the batch.

4. (Optional) In the Description box, enter a description of the batch.

5. Press Save.

The batch name appears under the selected folder.

To set up the batch, go to the next section, “Specifying the Method, Sample Positions, and

Number of Injections.”

Specifying the Method, Sample Positions, and Number of Injections

A batch lists the sample injection order, the vial or well positions where the samples are located, the number of injections per sample, and the method used to run the sample.

To create a batch by using the View Racks view

1. Press Batch Setup > Edit.

2. If you do not see a plate overview, press View Racks.

A plate view for the selected plate format appears. Figure 97

shows the plate view for the

6 × 8 vial plate format.

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Figure 97. View Racks view on the Edit page of the Batch Setup menu

Methods list

Plate button

3. If the plate format is not set to your installed plate, do the following: a. Press the Plate button.

Note The Plate button is the rectangular button imprinted with the plate name on the lower left side. The default plate is 6 × 8 vials.

The Autosampler Rack Configuration dialog box opens.

b. Select a plate format from the list.

c.

Press Accept.

The new plate format appears on the rectangular Plate button.

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4. If you have already created a name and selected a folder location for the batch, do the following: a. Press Open.

b. Select the batch.

c.

Press Accept.

The batch name appears in the batch name box.

5. In the Injections box, enter the number of injections per sample.

Default: 1

6. Browse the methods list next to the Injections box, and select the method that you want to use for the entire batch or for a subset of sample runs in the batch.

If you have not already created a method, create one by following the instructions in

“Creating a Method” on page 124 .

Tip To use different methods in the same batch, select the method, select the vial or

well positions that you want to run with this method (see step 7 ), and then press Add.

7. Select the vials or wells that contain the samples that you want to run in the order that you want the instrument to process them.

• To select an individual vial or well, press the vial or well position in the graphic.

• To select an entire column, press the DOWN ARROW above the column. For example, press the arrow labeled “1” to select all the vials (A1 to F1) or wells

(A1 to H1) from the first column (and first quadrant of a 384-well plate). For the third or fourth quadrant of a 384-well plate, press the UP ARROW below the column.

• To select the well positions on a 384-well plate, use the quadrant selection box to move between quadrants (see

Figure 98

).

• To select wells A1 to P1, do the following: a. In the quadrant selection box, press the first quadrant, b. Press the DOWN ARROW,

1

, above the first column.

c. In the quadrant selection box, press the third quadrant, d. Press the UP ARROW,

1

, below the first column.

.

.

Tip You can clear the vial or well selections by pressing them again.

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From left to right and top to bottom, the four quadrants of a 384-well plate are A1 to

H12, A13 to H24, I1 to P12, I13 to P24.

Figure 98. View Rack view for a 384-well plate

Batch name display

Plate button

Quadrant selection box

8. Press Add to link the selected vials or wells with the selected method.

9. To save the batch, do the following: a. Press Save.

The Save File dialog box opens.

b. If you have not already done so, select the appropriate folder and enter a name for the batch.

c.

Press Save.

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10. Press Schedule (see Figure 97 on page 142 ) to submit the batch to the queue.

The application switches to the Queue page of the Home menu (see Figure 99 ).

Figure 99. Queue page of the Home menu, showing the queued jobs

11. If necessary, move any scheduled batch in the job queue by selecting it and pressing Up or

Down.

You can also delete batches from the Queue by selecting the batches and pressing Delete.

For a batch run to start automatically after the previous batch is finished, you must select its corresponding check box in the Auto-Continue column. In the case shown in

Figure 99 , the admin-20100824-1714 job does not auto-continue when the

6 × BSA 30 min job is done.

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Starting Sample Acquisition

Starting Sample Acquisition

Before starting the sample acquisition, make sure that the LC/MS system is properly set up.

This includes having contact closure (see “Connecting to the Mass Spectrometer through

Contact Closure” on page 42

). Also make sure that you have set up the mass spectrometer or any other detector to acquire data during the sample run.

To start a batch run

1. Create a batch and submit it to the queue as described in the previous procedure.

2. Press Home > Overview.

The Overview page of the Home menu opens.

3. Press START to start running the batch.

A message box opens, prompting you to check that you have installed the sample plate, filled solvent bottles A and B, installed the W3 bottle with solvent A, installed the W4 bottle with the needle insert, and emptied the waste beaker (see

Figure 100

).

Figure 100. Start Queue message box

4. Press Accept.

If the Automatic Pre-run Maintenance feature (touch-screen application version 3.2 or later) is turned on and more than 8 hours have elapsed since the last sequence began, the instrument automatically runs the Flush Air script. After the Flush Air script finishes, the instrument starts processing the batch. For information about turning on the Automatic

Pre-run Maintenance feature, see

“Setting Up the Properties for the HPLC Device” on page 80 .

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6

Running Samples Using the Integrated Software

Monitoring the Run

Monitoring the Run

You can monitor the run and the system status during analysis from two different pages of the

Home menu: Overview or Graphs.

Follow these procedures to monitor the run and view the system status:

“Using the Overview Page in the Home Menu,” next section

“Using the Graphs Page in the Home Menu” on page 149

Using the Overview Page in the Home Menu

The Overview page provides a full schematic overview of the system, complete with real-time

updating of all critical component data ( Figure 101

).

Figure 101. Overview page of the Home menu

Pump A status

Current sample and status

Valve A position

Flow sensor A readback

Current step in the sample run

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147

6

Running Samples Using the Integrated Software

Monitoring the Run

The system schematic provides information on these items:

• Hardware parts in the system:

– Position, pressure, and flow for pumps A, B, and S

– Valve positions (1–2, 1–6, or centered)

– Percentage of solvent B being delivered

– Actual flow being measured by the flow sensors

Note When pumps A and B are delivering solvent to the mixing Tee, the flow sensors measure the actual flow rate. During the refill A B step, pumps A and B are drawing solvent from the solvent bottles.

• Samples to be analyzed, plus their current status

• Current sample and job, plus the next sample and job

• Current step in the sample run

• If the EASY-nLC instrument controls the temperature of the EASY-Spray ion source’s column heater, the column temperature setting appears (in Centigrade) in the third column of the run step area.

Pickup sample Initialize system

Load sample Equilibrate precolumn

Prepare gradient Equilibrate analytical column

Gradient Autosampler wash + refill S

Refill AB

Column:T:35

Column temperature readback

When the batch starts, the green START button is grayed out and the red STOP button becomes active. When the instrument is running, three counters measure the time for job, sample, and gradient.

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6

Running Samples Using the Integrated Software

Monitoring the Run

Using the Graphs Page in the Home Menu

The Graphs page displays up to four graphs with time-dependent data for the currently active sample (see

Figure 102 ).

Figure 102. Graphs page of the Home menu

Thermo Scientific

Table 18

describes the different types of graphs that are available. To change graphs, select from their corresponding lists.

Table 18. Graphical data types

Graph

Pump A, pump B, and pump S

Valves A, B, S, and W

Gradient

Temperature

Description

Shows the desired flow (black line), real flow (blue line), and pressure

(red line) over time for each of the three pumps.

Shows the valve position over time for each of the four valves.

Shows the theoretical gradient (dotted red line) and the actual gradient dynamically calculated on the feedback values from the pump flow sensors (full red line), plus the actual flow calculated on the feedback values from the pump flow sensors.

Shows the measured temperature on the plate cooler over time.

EASY-nLC 1200 Getting Started Guide

149

6

Running Samples Using the Integrated Software

Stopping Sample Acquisition

You can change the graph size as follows:

• Touch the graphs to enlarge them and then touch them again to minimize the view.

Figure 103

shows the enlarged flow graph for pump B.

• Touch the y axis of each graph to cycle the Flow axis through several preset scales.

Figure 103. Pump B graph enlarged

Stopping Sample Acquisition

You can stop the sample acquisition during the run by pressing the red STOP button on the

Home > Overview page. This halts all actions on the HPLC system and the following buttons become active:

Restart Sample: Runs the method from its beginning again. Only press this button if you are sure you have enough sample in the vial or microtiter plate.

Cancel Sample: Cancels the current sample and skips to the next sample in the batch job.

Cancel Batch: Cancels the entire current batch job.

Eject Plate: Ejects the plate for your inspection to help you determine how to proceed.

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6

Running Samples Using the Integrated Software

Editing the Running Batch

Editing the Running Batch

You can edit the running batch if you want to add samples or remove samples that the instrument has not already run.

To edit the running batch

1. Press Home > Queue.

The Queue page of the Home menu opens (see Figure 104

).

Figure 104. Queue page of the Home menu

Thermo Scientific

2. Select the batch job from the list and press Edit.

The Batch Setup > Edit page opens (see

Figure 105 ).

EASY-nLC 1200 Getting Started Guide

151

6

Running Samples Using the Integrated Software

Editing the Running Batch

Figure 105. Edit page of the Batch Setup menu

3. Edit the batch—that is, add, change, or remove samples that are not yet tested.

4. Press Reschedule.

The batch continues running.

IMPORTANT If you do not reschedule the batch after you complete the batch edit, the instrument stops after the current sample run.

5. (Optional) Check the sample list as follows: a. Press Home > Queue.

b. Press Properties (see Figure 104 on page 151 ).

c.

Press Show Samples.

The Job Properties box opens (see

Figure 106 ) and the Show Batch button appears in

place of the Show Samples button.

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EASY-nLC 1200 Getting Started Guide Thermo Scientific

6

Running Samples Using the Integrated Software

Editing the Running Batch

Figure 106. Sample list job properties on the Queue page d. Click Close.

Thermo Scientific EASY-nLC 1200 Getting Started Guide

153

6

Running Samples Using the Integrated Software

Troubleshooting a Sample Run

Troubleshooting a Sample Run

If a problem occurs during a sample analysis, the EASY-nLC instrument displays the error in a pop-up box and stops the sample processing.

To troubleshoot an error condition that stops the sample processing

1. Press Cancel Batch.

2. Refer to the troubleshooting tips or follow the troubleshooting procedures in the

EASY-nLC Series Troubleshooting and Maintenance Guide.

After you fix the problem, reschedule the batch (possibly changing the number of samples, their well positions, or both).

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EASY-nLC 1200 Getting Started Guide Thermo Scientific

7

Installing and Controlling External Devices

The EASY-nLC instrument (with the 3.2 version of the touch-screen software) can control the Advion Replay device and the EASY-Spray ion source’s column temperature.

To add the EASY-Spray device to the EASY-nLC device list or to install and use the optional

Advion RePlay device, follow the instructions in this appendix.

The RePlay device was specifically developed for nanoflow LC/MS analyses. It splits the sample from one injection, so a second analysis is provided with no additional sample required. The RePlay device consists of a primary column, a six-port valve, a flow sensor, tubing, a control panel, and a secondary column/emitter. It performs a second analysis of one sample without a reinjection, which allows for twice the analytical possibilities from every nanoLC injection. For more information, visit the Advion website: www.advion.com/biosystems/replay/index.php

Thermo Fisher Scientific does not offer support for—or is in any way responsible for—the functionality of the RePlay device itself and the associated tubing and columns that it requires.

For information about connecting the EASY-Spray ion source to the EASY-nLC instrument, refer to the EASY-Spray Series Ion Source User Guide.

Contents

Adding Devices to the EASY-nLC Devices List

Connecting the RePlay Device to the EASY-nLC Instrument

Setting Up a Method with the RePlay Device

Monitoring the Run of a RePlay Method

Effect of Additional Run Time for a RePlay Experiment

Thermo Scientific EASY-nLC 1200 Getting Started Guide

155

7

Installing and Controlling External Devices

Adding Devices to the EASY-nLC Devices List

Adding Devices to the EASY-nLC Devices List

The EASY-nLC instrument (with the 3.2 version of the touch-screen software) can control the Replay device and the EASY-Spray’s column heater.

To add a device to the device list in the EASY-nLC application

1. Press Maintenance > Devices and check that the device is not already present in the

Devices list. If not, add the device as described in the following steps.

2. For each device that you want to add, do the following: a. Press Add Device.

The Select a Device to Add dialog box opens.

Figure 107. Select a Device to Add dialog box b. Do one of the following:

• To add the Replay device, select Advion RePlay and press Accept.

• To add the EASY-Spray device, select EASY-Spray and press Accept.

3. Verify that the Devices list includes the added device.

Selecting the device displays information about the device driver on the About page (see

Figure 108

and

Figure 109 ).

156

EASY-nLC 1200 Getting Started Guide Thermo Scientific

Figure 108. About information for the Replay device

7

Installing and Controlling External Devices

Adding Devices to the EASY-nLC Devices List

Figure 109. About information for the EASY-Spray device

Thermo Scientific EASY-nLC 1200 Getting Started Guide

157

7

Installing and Controlling External Devices

Connecting the RePlay Device to the EASY-nLC Instrument

Connecting the RePlay Device to the EASY-nLC Instrument

Before you can use the RePlay device with the EASY-nLC instrument, you must connect the contact closure cables and the solvent lines.

The Replay device has a remote port on its back panel and a six-port valve (see

Figure 110 ) on

its front panel.

Figure 110. Front panel of the Replay device (courtesy of the Advion Inc. website)

Valve

To connect the RePlay device to the EASY-nLC instrument

1. Turn off the EASY-nLC instrument.

2. Connect the Remote cable port on the back panel of the RePlay device to the Out3 pins

on the back panel of the EASY-nLC instrument (see Figure 111

).

Figure 111. RePlay device connection to the EASY-nLC contact closure port

RePlay device (back panel)

Remote cable connector

EASY-nLC contact closure port

LCQ Fleet™, LXQ™, or

LTQ MS detector

(right side panel)

158

EASY-nLC 1200 Getting Started Guide

Peripheral Control

Start

In

Ready

Out

Start

Out

Ethernet

100 Base T

Electronics Normal

Reset

1V MAX 10V MAX

Analog Input

Service

Main Power

On

Mech. Pumps/Accessory

A 5.5 MAX

Power In

230 V AC

10 A

A 5.5 MAX

Off

Peripheral control port

Thermo Scientific

Thermo Scientific

7

Installing and Controlling External Devices

Connecting the RePlay Device to the EASY-nLC Instrument

3. Connect the solvent lines to the valve on the front panel of the RePlay device

(see

Figure 112 ) as follows:

• Connect the output end of the EASY-nLC analytical column to position 5 of the

RePlay valve.

• Connect position 4 of the RePlay valve to the secondary column/emitter.

Note For more information about connecting and using the RePlay device, refer to the Advion RePlay User’s Manual supplied by Advion Biosystems, Inc.

Figure 112. Valve connections for the standard plumbing configuration

Plugged

Capture

Cartridge

3

4

2

5

1

6

Secondary column/emitter

Flow sensor

EASY-nLC column

Waste

4. Turn on the EASY-nLC instrument.

EASY-nLC 1200 Getting Started Guide

159

7

Installing and Controlling External Devices

Setting Up a Method with the RePlay Device

Setting Up a Method with the RePlay Device

To set up a method with the RePlay device

1. To use the RePlay device, enable the method extension on page 1 of the Method Setup wizard (you can also open an existing method and enable its extension).

An extra RePlay Method Setup page is now available (see

Figure 113 ).

Figure 113. Page 1/7 of the Method Setup wizard

2. On page 3 of the Method Setup wizard, properly set up the gradient. For more

information, see “To build a gradient” on page 130

.

The RePlay device uses a nanoflow splitter and a valve to split the EASY-nLC eluent to a direct online analysis and to a Capture Cartridge in the device. The RePlay valve switches to direct the captured chromatogram to the mass spectrometer for a second analysis of the same injection. To acquire the second analysis, the gradient has to be prolonged.

160

EASY-nLC 1200 Getting Started Guide Thermo Scientific

7

Installing and Controlling External Devices

Setting Up a Method with the RePlay Device

3. On page 6, enter the playback delay relative to the MS START signal (which can be the start of either sample loading onto the columns or the start of the gradient, and set from the Configuration > Connections page).

This value, as shown in Figure 114 , indicates how long into the gradient to switch back to

playback mode. At the end of the gradient, the contact closure control reverts to capture mode.

Figure 114. RePlay Playback Delay setting

Thermo Scientific EASY-nLC 1200 Getting Started Guide

161

7

Installing and Controlling External Devices

Monitoring the Run of a RePlay Method

Monitoring the Run of a RePlay Method

The readback for a RePlay method displays Capture or Replay.

To monitor the run of a RePlay method

1. When running a sample using a method with the RePlay extension, press Home >

Overview.

2. View the RePlay (“RP”) mode readback in the center status box (see Figure 115

).

Figure 115. RePlay method readback

162

EASY-nLC 1200 Getting Started Guide

RePlay method readback

Thermo Scientific

7

Installing and Controlling External Devices

Effect of Additional Run Time for a RePlay Experiment

Effect of Additional Run Time for a RePlay Experiment

For a typical RePlay application—injecting and loading 5 μL of sample with a gradient of

60 minutes + 30 minutes RePlay—the full timing is as follows:

• 2 minutes – Refilling pumps at 100 μL/min

• 2 minutes – Injecting 5 μL at 20 μL/min and waiting for pressures to settle

• 12 minutes – Loading the sample onto the analytical column with 15 μL at 1.25 μL/min

• 60 minutes – Gradient + autosampler wash

• 30 minutes – RePlay while also reequilibrating the analytical column

In total, you can achieve an MS utilization rate of close to 85 percent (90/106), despite the fact that the EASY-nLC/MS system cannot overlap (parts of ) two runs in parallel.

Thermo Scientific EASY-nLC 1200 Getting Started Guide

163

I

Index

A

accessing

EASY-nLC manuals

xii

files on the instrument’s hard drive

46

,

72

support server

47

accounts, adding new users

76

acetonitrile, solvent requirements

85

admin folder, accessing

46 ,

73

admin, changing password

75

air segments, sample pickup

20

air, removing

123

alphanumeric text, entering

54

analytical column column setup options

7

equilibration step, method

22

installing

EASY-Spray column

EASY-nLC 1200 instrument

113

with a Nanospray Flex source

96

pulled, fused-silica columns with a Nanospray Flex source

102

preparation for use

120

application version

59

Automatic Flow Control (AFC) description

31

Automatic Pre-run Maintenance check box

80 ,

146

autosampler sample pickup method parameters

128

tray compartment, accessing

3

wash step, method

30

, 134

B

back panel, instrument

5 ,

40

backpressure analytical column rating

121

precolumn rating

119

backup, system

79

Batch Setup menu

62

batches

Thermo Scientific editing on the Home > Queue page

151

setting up and scheduling

140

starting

146

beaker, plastic waste

88

black knurled nut tool, nanoViper fitting

116

blending solvents

86

bottles for solvents A and B, filling

86

wash

88

C

cables contact closure

43

Ethernet

44

P-Bus

48

power cord

41

carryover, reducing

135

changing the administrator password

75

check valves, description

14

checking, solvent levels

83

cleanup, computer file system

79

clock, internal

78

Column Out line modified with bare fused-silica tubing at one end

98

standard with nanoViper fittings at both ends

4

columns

See analytical column or precolumn

compliance

FCC

iii

regulatory

iii

WEEE

v

components, system

2

computer, embedded cause of damage to

41

purging data from

79

Configuration

78

Configuration menu

65

configuring

EASY-nLC 1200 Getting Started Guide 165

Index: D devices

80

global settings

80

RePlay

156

connecting add-ons

48

column assembly

95

contact closure cable

42

mouse and keyboard

48

nanoViper fittings

116

solvent bottles

86

to line power

41

to the data system computer

45

to the network

72

contact closure cables, ordering

43

configuration setting

69

with the mass spectrometer

42

contacting us

xv

creating a method

126

new user accounts

76

Custom Wash, sample pickup system

135

Cycles parameter, autosampler wash

135

D

daily maintenance

83

data system computer connecting to the Ethernet switch

45

connecting to the laboratory LAN port

45

using to access folders on the system computer

46

data types, graphs

149

data, removing excess

79

date and time format, setting

78

devices, configuring

80

dimensions, EASY-nLC system

37

documentation accessing

xii

additional

xii

E

EASY-Spray source adding to the Devices list

156

column assembly for

112

direct control of

55

Eject

124

Eject/Insert Tray button, using

54

, 88 ,

124

electromagnetic compatibility

iii

emitter, installing

111

equilibrating analytical column

22

,

120

precolumn

22 ,

118

166

EASY-nLC 1200 Getting Started Guide

Ethernet cables, connecting data system computer

45

EASY-nLC system to the intranet

46

EASY-nLC system to the support server

47

Ethernet switch

45

Exit button

68

F

FCC compliance

iii

Fe-containing ions

110

file system, computer cleanup

79

filtering solvents

86

firewalls, laboratory network access

39

flow rate gradient

130

measuring

11

monitoring

148

sample loading

128

sample pickup

128

flow sensors, description

11

formic acid, solvent requirements

85

fused-silica tubing, bare

98

G

global configuration settings

80

gloves, wearing

112

gradient delay volume

29

method step

29

setting up the method

130

graphical data types

149

grounding requirements

41

H

Home > Overview page

57

Home menu

61

HPLC-grade solvents

86

HPLC-to-column connector

See Viper union

humidity specification

39

I

injections number per sample

143

pickup sample step

20

sample pickup volume

128

Insert Plate button, using

89

insert, needle wash

3

, 89

Intelligent Flow Control (IFC) system description

32

Thermo Scientific

interface layout

59

Internet access

39

ion source, power requirements for

39

iron-containing ions

110

K

keyboard connection

48

keyboard, touch-screen software

54

kit, UHPLC Liquid Junction

98

L

LC connections

EASY-nLC 1200, connecting to

113

EASY-nLC II, connecting to

113

tools and supplies

112

LC/MS-grade solvents

86

leak testing the nanoViper connections

116

UHPLC fused-silica union for the

modified solvent lines

100

lifting instructions

36

liquid junction cross, UHPLC

99

Load Speed Protection option

33

, 82

loading samples into the autosampler

124

logging in to the application

66

Logo icon, Thermo Scientific

59

Loop Volume box

81

M

Maintenance menu

64

maintenance, daily

83

manuals, accessing

xii

mass spectrometer, contact closure configuration setting

69

hardware connection

42

menu structure

61

menus

Batch Setup

62

Configuration

65

Home

61

Maintenance

64

Method Setup

63

structure

60

Method Setup menu

63

method, LC accessing from another computer

46 ,

73

analytical column equilibration parameters

133

gradient table

130

method editor, starting

126

precolumn equilibration parameters

132

Thermo Scientific microwell plates, preparing samples

123

mixing solvents

86

mixing Tee connections

14

readback

57 –

58

monitoring the run

147

See also RePlay, monitoring the run

mouse connection

48

MS Setup Kit

42

N

Nanospray Flex source, column assembly for

95

nanoViper fittings connecting to Viper union

117

using

116

needle wash insert

3 ,

89

network card, installing in data system computer

45

network connection

72

noise and interference, cause of

41

numeric keypad

54

O

On/Off switch

52

one-column setup configuration check box

80

description

8

installation

EASY-nLC 1200 instrument

Nanospray Flex source

96

EASY-nLC 1200 instrument

EASY-Spray source

113

Orbitrap Fusion contact closure connection

42

ordering information columns

96

contact closure cable

43

product order code, instrument

6

sleeves, PEEK

112

UHPLC Liquid Junction Kit

98

venting Tee

96 ,

112 –

113

Viper union

112

overtightening nanoViper fittings

116

P

password changing

75

initial

66

peptide-Fe cluster ions

110

port 22, firewall setup for

39 ,

47

power requirements

39

Index: K

EASY-nLC 1200 Getting Started Guide

167

Index: Q turning off

68

power cord, connection and specifications

41

Power Down button

68

power-save feature, Ethernet switch

45

precolumn equilibration step, method

22 ,

132

preparation for use

118

Prepare Gradient step

26

preparing samples

123

solvent bottles and waste containers

85

well plates

123

pressure sensors

11

pulled fused-silica analytical column, installing

98

pump, flow control description for

31

purging and flushing pumps

91

purification system, water

86

Q

quadrant selection box, 384-well plate

144

R

regulatory compliance

iii

remote desktop connections

45

remote support

47

RePlay additional run time

163

configuring

156

connecting to EASY-nLC

155

monitoring the run

162

rotary valves, description

12

run time, excessively long

8

running batch, editing

151

S

safety handling emitters

111

handling the UHPLC liquid junction cross

104

105

precautions for the EASY-nLC system

xiii

safety standards

iii

sample loading, method parameters

128

pickup method parameters

128

sample loop bypassed during gradient program

29

configuring

81

maximum capacity

20

washing

30

sample runs predefined steps

15

168

EASY-nLC 1200 Getting Started Guide troubleshooting

154

sample trays, installing

3 ,

54 ,

124

sealing surface, nanoViper fitting

117

Secure Shell (SSH) network protocol

39

sequence, injection

140

sharp objects, emitter

111

sleeves for 1/16 in. OD tubing

112

for 1/32 in. ID fittings

111

sluggish system performance

79

software, integrated description

51

solvent A and B bottles, preparing

86

solvents checking levels

83

filtering

86

solvent-side check valve

14

Source column, autosampler wash

135

Standard option, autosampler wash

135

Start button, green

90 ,

148

starting the EASY-nLC

52

Stop button, red

90

, 148

, 150

stopping sample acquisition

150

support server, setting up the hardware connections

47

system overview, monitoring

147

T

Tees mixing

10

, 14

UHPLC liquid junction cross

99

venting

4

,

7 –

8

,

13

temperature

EASY-Spray direct control

55

environment specification

39

terms used in this guide

59

Thermo Scientific logo icon

59

tightening tool internally-threaded knurled nut

99

nanoViper fitting

116

time zone, setting

78

tools and supplies, connecting the LC

112

touch screen, using

53

trap column

See precolumn

tray compartment, accessing

3 ,

54 ,

88 ,

124

trays, sample

88

troubleshooting, sample runs

154

turning off the EASY-nLC

68

two-column setup description

7

Thermo Scientific

Index: U installation

EASY-nLC 1200 instrument

Nanospray Flex source

97

EASY-nLC 1200 instrument

EASY-Spray source

114

U

UHPLC-MS grade solvents, ordering

85

unions

UHPLC fused-silica union

100

– 101

Viper

98

,

112

URLs consumable parts

xv

ordering information stainless steel emitters

111

USB flash drive with manuals

xii

USB ports, EASY-nLC

6

user documentation

xii

user permissions

74

V

valves check valves, description

14

rotary valves, description

12

venting Tee connections

4

, 8 ,

96

97

, 112

– 114

function

7 ,

13 ,

88

purpose

22

version, application

59

vials, preparing sample

123

Viper union description and part number

96

in a two-column assembly

98

in the EASY-Spray Ion Source Kit

112

internal view

117

nanoViper fittings, connecting

117

volume, maximum custom wash solvent volume per step

135

sample loading step

128

sample pickup step

128

W

wash bottles description

3

preparing

88

wash step, autosampler

30

, 134

waste beaker, installing

88

Waste In line modified with bare fused-silica at one end

98

standard with nanoViper fittings at both ends

4

Thermo Scientific waste-side check valve

14

water, solvent requirements

85

WEEE compliance

v

well plates, preparing

123

Windows Explorer

73

Z

zero-dead-volume fittings

Viper union

117

EASY-nLC 1200 Getting Started Guide

169

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