EASY-nLC 1000 User Guide Version C

EASY-nLC 1000 User Guide Version C

Thermo

EASY-nLC 1000

User Guide

(Touch-screen software version 3.0)

60053-97227 Revision C January 2012

© 2012 Thermo Fisher Scientific Inc. All rights reserved.

EASY-nLC, AFC, IFC, LCQ Fleet, LXQ, LTQ, nanoViper, and PepMap are trademarks, and Acclaim, Dionex, and Xcalibur are registered trademarks of Thermo Fisher Scientific Inc.

PEEK and PEEKsil are trademarks of SGE International Pty Ltd Corp.

This product uses FreeRTOS from www.freertos.org

(available upon request). FreeRTOS is a registered trademark of Real Time Engineers Ltd.

The following are registered trademarks in the United States: Advion and RePlay are registered trademarks of

Advion Biosystems, Inc. Agilent and Varian are registered trademarks of Agilent Technologies Inc. Bruker is a registered trademark of Bruker Corporation. CMOSens is a registered trademark of Sensirion AG Corp.

Duran is a registered trademark of Schott AG. Micromass and Waters are registered trademarks of Waters

Corporation. PhotoMOS is a registered trademark of Panasonic Electric Works, Co., Ltd.

Sciex is a registered trademark of Applied Biosystems Life Technologies. Teflon is a registered trademark of E.I. du Pont de Nemours and Company. VICI and Valco are registered trademarks of Valco Instruments Co. Inc.

Microsoft and Windows are registered trademarks of the Microsoft Corporation in the United States and other countries.

All other trademarks are the property of Thermo Fisher Scientific and its subsidiaries.

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.

Thermo Fisher Scientific Inc. makes no representations that this document is complete, accurate or errorfree and assumes no responsibility and will not be liable for any errors, omissions, damage or loss that might result from any use of this document, even if the information in the document is followed properly.

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, October 2011; Revision B, October 2011; Revision C, January 2012.

Revision C contains additional information about connecting to the support server.

Software version: Touch-screen software Thermo EASY-nLC version 3.0

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

CAUTION Symbol CAUTION VORSICHT ATTENTION PRECAUCION AVVERTENZA

Electric Shock: This instrument uses high voltages that can cause personal injury. Before servicing, shut down the instrument and disconnect the instrument from line power. Keep the top cover on while operating the instrument. Do not remove protective covers from PCBs.

Elektroschock: In diesem Gerät werden

Hochspannungen verwendet, die

Verletzungen verursachen können. Vor

Wartungsarbeiten muß das Gerät abgeschaltet und vom Netz getrennt werden. Betreiben Sie das Gerät nicht mit abgenommenem Deckel. Nehmen Sie die

Schutzabdeckung von Leiterplatten nicht ab.

Choc électrique: L’instrument utilise des tensions capables d’infliger des blessures corporelles. L’instrument doit être arrêté et débranché de la source de courant avant tout intervention. Ne pas utiliser l’instrument sans son couvercle. Ne pas enlever les étuis protecteurs des cartes de circuits imprimés.

Descarga eléctrica: Este instrumento utiliza altas tensiones, capaces de producir lesiones personales. Antes de dar servicio de mantenimiento al instrumento, éste debera apagarse y desconectarse de la línea de alimentacion eléctrica. No opere el instrumento sin sus cubiertas exteriores quitadas. No remueva las cubiertas protectoras de las tarjetas de circuito impreso.

Shock da folgorazione. L’apparecchio è alimentato da corrente ad alta tensione che puo provocare lesioni fisiche. Prima di effettuare qualsiasi intervento di manutenzione occorre spegnere ed isolare l’apparecchio dalla linea elettrica. Non attivare lo strumento senza lo schermo superiore. Non togliere i coperchi a protezione dalle schede di circuito stampato (PCB).

Chemical: This instrument might contain hazardous chemicals. Wear gloves when handling toxic, carcinogenic, mutagenic, or corrosive or irritant chemicals. Use approved containers and proper procedures to dispose waste oil.

Chemikalien: Dieses Gerät kann gefährliche Chemikalien enthalten. Tragen

Sie Schutzhandschuhe beim Umgang mit toxischen, karzinogenen, mutagenen oder

ätzenden/reizenden Chemikalien.

Entsorgen Sie verbrauchtes Öl entsprechend den Vorschriften in den vorgeschriebenen Behältern.

Chimique: Des produits chimiques dangereux peuvent se trouver dans l’instrument. Portez des gants pour manipuler tous produits chimiques toxiques, cancérigènes, mutagènes, ou corrosifs/irritants. Utiliser des récipients et des procédures homologuées pour se débarrasser des déchets d’huile.

Heat: Before servicing the instrument, allow any heated components to cool.

Hitze: Warten Sie erhitzte Komponenten erst nachdem diese sich abgekühlt haben.

Haute Temperature: Permettre aux composants chauffés de refroidir avant tout intervention.

Química: El instrumento puede contener productos quimicos peligrosos. Utilice guantes al manejar productos quimicos tóxicos, carcinogenos, mutagenos o corrosivos/irritantes. Utilice recipientes y procedimientos aprobados para deshacerse del aceite usado.

Prodotti chimici. Possibile presenza di sostanze chimiche pericolose nell’apparecchio. Indossare dei guanti per maneggiare prodotti chimici tossici, cancerogeni, mutageni, o corrosivi/irritanti. Utilizzare contenitori aprovo e seguire la procedura indicata per lo smaltimento dei residui di olio.

Altas temperaturas: Permita que los componentes se enfríen, ante de efectuar servicio de mantenimiento.

Calore. Attendere che i componenti riscaldati si raffreddino prima di effetturare l’intervento di manutenzione.

Fire: Use care when operating the system in the presence of flammable gases.

Feuer: Beachten Sie die einschlägigen

Vorsichtsmaßnahmen, wenn Sie das

System in Gegenwart von entzündbaren

Gasen betreiben.

Eye Hazard: Eye damage could occur from splattered chemicals or flying particles. Wear safety glasses when handling chemicals or servicing the instrument.

Verletzungsgefahr der Augen:

Verspritzte Chemikalien oder kleine

Partikel können Augenverletzungen verursachen. Tragen Sie beim Umgang mit

Chemikalien oder bei der Wartung des

Gerätes eine Schutzbrille.

Incendie: Agir avec précaution lors de l’utilisation du système en présence de gaz inflammables.

Fuego: Tenga cuidado al operar el sistema en presencia de gases inflamables.

Danger pour les yeux: Des projections chimiques, liquides, ou solides peuvent

être dangereuses pour les yeux. Porter des lunettes de protection lors de toute manipulation de produit chimique ou pour toute intervention sur l’instrument.

Peligro par los ojos: Las salicaduras de productos químicos o particulas que salten bruscamente pueden causar lesiones en los ojos. Utilice anteojos protectores al manipular productos químicos o al darle servicio de mantenimiento al instrumento.

Incendio. Adottare le dovute precauzioni quando si usa il sistema in presenza di gas infiammabili.

Pericolo per la vista. Gli schizzi di prodotti chimici o delle particelle presenti nell’aria potrebbero causare danni alla vista. Indossare occhiali protettivi quando si maneggiano prodotti chimici o si effettuano interventi di manutenzione sull’apparecchio.

General Hazard: A hazard is present that is not included in the above categories.

Also, this symbol appears on the instrument to refer the user to instructions in this manual.

When the safety of a procedure is questionable, contact your local Technical

Support organization for Thermo Fisher

Scientific San Jose Products.

Allgemeine Gefahr: Es besteht eine weitere Gefahr, die nicht in den vorstehenden Kategorien beschrieben ist.

Dieses Symbol wird im Handbuch außerdem dazu verwendet, um den

Benutzer auf Anweisungen hinzuweisen.

Wenn Sie sich über die Sicherheit eines

Verfahrens im unklaren sind, setzen Sie sich, bevor Sie fortfahren, mit Ihrer lokalen technischen

Unterstützungsorganisation für Thermo

Fisher Scientific San Jose Produkte in

Verbindung.

Danger général: Indique la présence d’un risque n’appartenant pas aux catégories citées plus haut. Ce symbole figure également sur l’instrument pour renvoyer l’utilisateur aux instructions du présent manuel.

Si la sûreté d’une procédure est incertaine, avant de continuer, contacter le plus proche Service Clientèle pour les produits de Thermo Fisher Scientific San

Jose.

Peligro general: Significa que existe un peligro no incluido en las categorias anteriores. Este simbolo también se utiliza en el instrumento par referir al usuario a las instrucciones contenidas en este manual.

Pericolo generico. Pericolo non compreso tra le precedenti categorie.

Questo simbolo è utilizzato inoltre sull’apparecchio per segnalare all’utente di consultare le istruzioni descritte nel presente manuale.

Cuando la certidumbre acerca de un procedimiento sea dudosa, antes de proseguir, pongase en contacto con la

Oficina de Asistencia Tecnica local para los productos de Thermo Fisher Scientific

San Jose.

Quando e in dubbio la misura di sicurezza per una procedura, prima di continuare, si prega di mettersi in contatto con il

Servizio di Assistenza Tecnica locale per i prodotti di Thermo Fisher Scientific San

Jose.

CAUTION Symbol CAUTION

Electric Shock: This instrument uses high voltages that can cause personal injury. Before servicing, shut down the instrument and disconnect the instrument from line power. Keep the top cover on while operating the instrument. Do not remove protective covers from PCBs.

Chemical: This instrument might contain hazardous chemicals. Wear gloves when handling toxic, carcinogenic, mutagenic, or corrosive or irritant chemicals. Use approved containers and proper procedures to dispose waste oil.

Heat: Before servicing the instrument, allow any heated components to cool.

Fire: Use care when operating the system in the presence of flammable gases.

Eye Hazard: Eye damage could occur from splattered chemicals or flying particles. Wear safety glasses when handling chemicals or servicing the instrument.

General Hazard: A hazard is present that is not included in the above categories.

Also, this symbol appears on the instrument to refer the user to instructions in this manual.

When the safety of a procedure is questionable, contact your local Technical

Support organization for Thermo Fisher

Scientific San Jose Products.

C

Contents

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

Related Documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .xi

Downloading Manuals from the Customer Manuals Web Site . . . . . . . . . . . . . xii

Safety and Special Notices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .xiii

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

Declaration of Conformity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .xxi

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

System Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

System Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

External System Connections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Hardware Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

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

Components Behind the Right Panel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Components Behind the Left Panel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Back Panel. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Column Setup Options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

Two-column Setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

One-Column Setup. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

Solvent Delivery and Sample Pickup Systems . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Syringe Pumps. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

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

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

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

Pump A and Pump B Solvent Delivery System . . . . . . . . . . . . . . . . . . . . . . . 13

Sample Pickup and Autosampler Wash System . . . . . . . . . . . . . . . . . . . . . . . 21

Predefined Steps for Sample Runs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

Pump Flow Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

Automatic Flow Control System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

Intelligent Flow Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

Load Speed Protection. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

Specifications. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

Performance Specifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

Technical Specifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

Thermo Scientific EASY-nLC 1000 User Guide

v

Contents

Chapter 2 Installing the EASY-nLC Instrument. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .33

Lifting Instructions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

Laboratory Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

Benchtop Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

Internet Access . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

Power and Fuse Requirements. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

Temperature and Humidity Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . 36

Back Panel Connections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

Connecting to Line Power. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

Connecting to the Mass Spectrometer through Contact Closure . . . . . . . . . . 39

Connecting the Ethernet Communication Cables . . . . . . . . . . . . . . . . . . . . . 40

Attaching a Mouse and Keyboard to the USB Connections. . . . . . . . . . . . . . 44

Attaching Add-on Devices through the P-Bus and RS-232 . . . . . . . . . . . . . . 44

Chapter 3 Integrated Instrument Control Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .45

Turning On the EASY-nLC Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

Using the Touch-Sensitive Screen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

Using the Buttons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

Inputting Alphanumeric Text . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

Using Tables and Input Fields . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

Interactive Graphical Controls. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

User Interface Layout . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

Viewing the System Status and Using the Manual Controls . . . . . . . . . . . . . . . 50

Application Menu Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52

Home Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53

Batch Setup Menu. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53

Method Editor Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54

Maintenance Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

Configuration Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56

Logging In to the EASY-nLC System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

Closing Down the EASY-nLC System. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

Chapter 4 Configuring the EASY-nLC System. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .59

Setting Up Communication with the Mass Spectrometer . . . . . . . . . . . . . . . . . 59

Setting Up the Local Area Network Connection . . . . . . . . . . . . . . . . . . . . . . . . 61

Modifying User Permissions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

Changing the Administrator Password. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64

Creating New User Accounts. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

Changing Your Password from the Exit Menu . . . . . . . . . . . . . . . . . . . . . . . 66

Modifying the Time Settings. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

Making System Backups . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68

Setting Up the Properties for the HPLC Device . . . . . . . . . . . . . . . . . . . . . . . . 70

vi

EASY-nLC 1000 User Guide Thermo Scientific

Thermo Scientific

Contents

Chapter 5 Preparing the EASY-nLC Instrument for Use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .73

Preparing Solvent Bottles and Waste Containers . . . . . . . . . . . . . . . . . . . . . . . . 73

Solvent Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74

Solvent A and B Bottles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75

Autosampler Wash Bottles and Waste Beaker . . . . . . . . . . . . . . . . . . . . . . . . 76

Executing Maintenance Scripts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78

Purging and Flushing the Pumps. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79

Installing Columns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83

Connecting the Column Assembly to the Column Out and Waste In

Lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84

Connecting an Emitter to the Analytical Column . . . . . . . . . . . . . . . . . . . . . 86

Using nanoViper Fittings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87

Equilibrating Columns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88

Chapter 6 Running Samples Using the Integrated Software. . . . . . . . . . . . . . . . . . . . . . . . . .91

Preparing a Sample Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91

Creating a Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92

Browsing for a Stored Method or Creating a New Method File. . . . . . . . . . . 92

Starting the Method Editor Wizard and Selecting the Method to be

Edited . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93

Setting Up the Sample Pickup and Loading Steps . . . . . . . . . . . . . . . . . . . . . 94

Building the Gradient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96

Setting Up the Column Equilibration Steps . . . . . . . . . . . . . . . . . . . . . . . . . 98

Setting Up the Autosampler Wash Step. . . . . . . . . . . . . . . . . . . . . . . . . . . . 100

Saving the Method Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102

Creating a Batch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103

Viewing the Batch List and Setting Up a Batch Record . . . . . . . . . . . . . . . . 103

Specifying the Method, Sample Positions, and Number of Injections

Per Sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104

Starting Sample Acquisition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108

Monitoring the Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109

Using the Overview Page in the Home Menu . . . . . . . . . . . . . . . . . . . . . . . 109

Using the Graphs Page in the Home Menu . . . . . . . . . . . . . . . . . . . . . . . . . 111

Stopping Sample Acquisition. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113

Editing the Running Batch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114

Troubleshooting a Sample Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117

Chapter 7 Calibrating the Autosampler’s XYZ Robot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .119

Replacing the Autosampler Adapter Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120

Managing Plate Formats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121

Preparing the Autosampler for the Calibration Routines . . . . . . . . . . . . . . . . . 124

Calibrating Plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125

Calibrating the Wash Bottle and Extra Vial Positions . . . . . . . . . . . . . . . . . . . 129

Checking Sample Pickup. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131

EASY-nLC 1000 User Guide

vii

Contents

Chapter 8 Troubleshooting. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .133

System Startup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134

Batch Runs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134

Autosampler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136

Contact Closure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137

Network Access . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138

System Component States (Valves, Pumps, Autosampler, and Plate

Cooler) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138

Miscellaneous . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139

Manual Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141

Using the Valve Controls. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141

Using the Pump Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142

Using the Autosampler Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146

Chapter 9 Remote Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .149

Connecting the EASY-nLC System to the Support Server . . . . . . . . . . . . . . . . 150

Saving System Files on a USB Removable Storage Device . . . . . . . . . . . . . . . . 153

Chapter 10 Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .157

General Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157

Cleaning Materials. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157

Replacing the Main Power Fuse. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158

Daily Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159

Weekly Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160

Quarterly Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160

Yearly Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161

Maintenance Scripts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161

Prepare – Purge Solvent. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162

Prepare – Flush Air . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163

Prepare – Precolumn Equilibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164

Prepare – Analytical Col Equilibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166

Prepare – Isocratic Flow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167

Test – MS Connection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169

Test – Sample Pickup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170

Test – Leaks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171

Test – Valve Check . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174

Test – Back Pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175

Test – Autosampler Torque. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176

Test – Pump Torque . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177

Calibrate – Valve Tune . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177

Calibrate – Flow Sensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178

Calibrate – Reset Pressure Sensor. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179

Keeping Service Records . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180

Checking and Resetting the Device Usage Counters . . . . . . . . . . . . . . . . . . . . 181

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EASY-nLC 1000 User Guide Thermo Scientific

Contents

Appendix A External Devices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .183

Connecting the RePlay Device to the EASY-nLC Instrument . . . . . . . . . . . . . 184

Adding the RePlay Device to the EASY-nLC Device List . . . . . . . . . . . . . . . . 186

Setting Up a Method with the RePlay Device . . . . . . . . . . . . . . . . . . . . . . . . . 187

Monitoring the Run of a RePlay Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189

Additional Run Time for a RePlay Experiment . . . . . . . . . . . . . . . . . . . . . . . . 190

Appendix B Returning the EASY-nLC Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .191

Transport Instructions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191

Declaration of Contamination. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192

Appendix C Consumables and Replacement Parts. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .193

Ordering Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193

Solvent System Schematic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .197

Thermo Scientific EASY-nLC 1000 User Guide

ix

P

Preface

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

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

Downloading Manuals from the Customer Manuals Web Site

Safety and Special Notices

Contacting Us

Declaration of Conformity

Y

To suggest changes to documentation or to Help

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

Thank you in advance for your help.

Complete a brief survey about this document by going to the following URL.

Thank you in advance for your help.

Related Documentation

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

EASY-nLC system:

• EASY-nLC Virtual Instrument for Xcalibur 2.1.x Quick Reference Guide

• EASY-nLC Preinstallation Requirements Guide

Thermo Scientific EASY-nLC 1000 User Guide

xi

Preface

Downloading Manuals from the Customer Manuals Web Site

For Thermo Scientific software and hardware products, Thermo Fisher Scientific provides instructional guides as PDF files on its Customer Manuals Web site.

Note If you are using a Thermo Scientific software application such as the Xcalibur™ data system (with LC Devices 2.6 or later) to control your EASY-nLC system, the

EASY-nLC 1000 manuals are also available from the data system computer’s Start menu.

Y

To download the EASY-nLC manuals from the Customer Manuals Web site

1. Go to mssupport.thermo.com

.

2. On the Terms and Conditions page, click I Agree.

3. Click Customer Manuals in the left margin of the window.

4. On the right side of the window, click Search.

The Documents - Advanced Search page opens.

xii

EASY-nLC 1000 User Guide

5. Open a list of linked PDF documents for the EASY-nLC as follows: a. In the Model list, select EASY-nLC.

b. In the Document Type list, select Manual.

c.

Click Submit Search.

6. On the Documents page, click the link to the right of Download to view the PDF.

7. Click the Save a Copy icon, , browse to an appropriate folder, and then click Save to save a copy of the document.

Thermo Scientific

Preface

Safety 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 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 1000 User Guide

xiii

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.

xiv

EASY-nLC 1000 User Guide Thermo Scientific

Preface

Contacting Us

There are several ways to contact Thermo Fisher Scientific for the information you need.

Y

To contact Technical Support for the EASY-nLC system

Phone

Fax

Web site

E-mail

Address

800-532-4752

561-688-8736 www.proxeon.com

(North and South America) [email protected]

(Other continents) [email protected]

Thermo Fisher Scientific

Edisonvej 4

DK-5000 Odense C

Find software updates and utilities to download at mssupport.thermo.com

.

Y

To order consumable and spare parts for the EASY-nLC 1000 system

Go to www.proxeon.com/productrange/nano_lc_easy-nlc_1000/accessories_spares/index.html

Contacting Customer Service for Ordering Information

This guide includes the contact information for Thermo Scientific offices globally. Depending on your location, follow these guidelines in making international calls to the appropriate

Customer Service office.

Y

To make an international phone call to a Thermo Fisher Scientific office

Dial the following sequence of numbers: international access code + [country code] + (city or area code) + local phone number

The international access code depends on your location and the location you are calling.

International access code

011

From

United States, Canada, or the Caribbean

Countries other than the

United States, Canada, or the Caribbean

United States, Canada, or the Caribbean

To

Other countries

Other countries

United States, Canada, or the Caribbean

00

1

Thermo Scientific EASY-nLC 1000 User Guide

xv

Preface

Whether you dial the area or city code depends on your location and the location you are calling.

From

United States

To

Location in the

United States with a different area code

Countries other than the United States

Location within the same country with a different area code

Dialing sequence

Dial 1 + area code + local phone number

Dial 0 + area code + local phone number

Y

To contact Customer Service for ordering information

• For North America, see

“Customer Service Information for North America” on page xvi

.

• For Europe, see

“Customer Service Information for Europe” on page xvii

.

• For Australia and Asia, see “Customer Service Information for Australia and Asia” on page xx

.

Customer Service Information for North America

Table 1 lists the Customer Service information for North America.

Table 1. Customer Service information for North America

North America

United States

Phone

Fax

E-mail

Address

[1] 800-532-4752 (toll free)

[1] 877-373-4006 (toll free) [email protected]

1400 North Point Pkwy #10

West Palm Beach, FL 33407

Canada

Phone

Fax

E-mail

Address

[1] 800 530 8447 (toll free)

[1] (905) 890-9161 [email protected]

2845 Argentia Road, Unit 4

Mississauga, Ontario, L5N 8G6

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EASY-nLC 1000 User Guide Thermo Scientific

Preface

Customer Service Information for Europe

Table 2 lists the Customer Service information for Europe.

Tip When calling from a different city or area code within the same country, the dialing sequence is as follows:

0 + (city or area code) + local phone number

Table 2. Customer Service information for Europe (Sheet 1 of 3)

Europe

Austria

Phone

Fax

E-mail

Address

[43] (1) 333 50 34-0 (The city code for Vienna is 1.)

[43] (1) 333 50 34-26 [email protected]

Wehlistrasse 27b

A-1200 Wien (Vienna)

Belgium

Phone

Fax

E-mail

Address

[32] (2) 482 3030

[32] (53) 73 42 41

(The city codes for Brussels and Aalst are 2 and 53, respectively.)

[32] (2) 482 3031 [email protected]

Clintonpark “Keppekouter”

Ninovesteenweg 198

B-9320 ERMEBODEGEM - AALST

Denmark

Phone

Fax

E-mail

Address

[45] 7023 6267

[45] 7023 6263 [email protected]

Fruebjergvej 3

2100 København Ø

Finland – see Sweden, Norway, and Finland

Thermo Scientific EASY-nLC 1000 User Guide

xvii

Preface

Table 2. Customer Service information for Europe (Sheet 2 of 3)

France

(Also representing French-speaking North Africa, Algeria, Morocco, and Tunisia)

Phone

Fax

[33] (1) 60 92 49 50 (The city code for Paris is 1.)

[33] (1) 1 60 92 48 99

E-mail

Address [email protected]

16 Avenue du Québec

Silic 765

Z.A. de Courtaboeuf

F-91963 Les Ulis Cédex

Germany

Phone

Fax

E-mail

Address

[49] (6103) 408 1050 (The city code for Dreieich is 6103.)

[49] (6103) 408 1213 [email protected]

Im Steingrund 4-6

D-63303 Dreieich

Italy

Phone

Fax

E-mail

Address

Numero Verde (toll free)

800 823 162

[39] (02) 95320 225 (The city code for Milan is 02.) [email protected]

Strada Rivoltana

I-20090 Rodano (Milano)

Netherlands

Phone

Fax

E-mail

Address

[31] (76) 579 55 55 (The city code for Breda is 76.)

[31] (76) 581 09 61 [email protected]

Takkebijsters 1

NL-4817 BL Breda

xviii

EASY-nLC 1000 User Guide Thermo Scientific

Thermo Scientific

Table 2. Customer Service information for Europe (Sheet 3 of 3)

Norway – see Sweden, Norway, and Finland

Spain

Phone

Fax

[34] (91) 484 5965 (The city code for Madrid is 91.)

[34] (91) 484 3598

E-mail

Address [email protected]

C/Valportillo I, n o

22 1a Planta

Edificio Caoba

ES-28108 Alcobendas - Madrid

Sweden, Norway, and Finland

Phone

Fax

E-mail

Address

[46] (8) 556 468 20 (The city code for Stockholm is 8.)

[46] (8) 556 468 08 [email protected]

Pyramidbacken 3

S-141 75 Kungens Kurva (Stockholm)

Sweden

Switzerland

Phone

Fax

E-mail

Address

[41] (61) 716 77 40 (The city code for Basel is 61.)

[41] (61) 716 77 20 [email protected]

Neuhofstrasse 11

4153 Reinach

United Kingdom

Phone

Fax

E-mail

Address

[44] 870 241 1034

(toll-free phone number from outside the United Kingdom)

[44] (1442) 233 667

(The city code for Aldbury, Hertfordshire is 1442.) [email protected]

Stafford House

1 Boundary Park

Boundary Way

Hemel Hempstead

Hertfordshire HP2 7GE

Preface

EASY-nLC 1000 User Guide

xix

Preface

Customer Service Information for Australia and Asia

Table 3 lists the Customer Service information for Australia and Asia.

Tip When calling from a different city or area code within the same country, the dialing sequence is as follows:

0 + (city or area code) + local phone number

Table 3. Customer Service information for Australia and Asia

Australia and Asia

Australia

Phone

Fax

E-mail

Address

[61] (3) 9757 4300

(The area code for the South East region of Australia is 3.)

[61] 9763 1169 [email protected]

PO Box 9092

5 Caribbean Drive

Scoresby, Victoria 3179

Japan

Phone

Fax

E-mail

[81] (45) 453-9100 (The city code for Yokohama is 45.)

[81] (45) 453-9110 [email protected]

Address C-2F

3-9 Moriya-cho, Kanagawa-ku

Yokohama 221-0022

P.R. China—Beijing

Phone

Fax

E-mail

Address

[86] 800 810 5118 (toll free) or [86] 400 650 5118 (toll free)

[86] (10) 88370548 (The city code for Beijing is 10.) [email protected]

7th Floor, 7F Tower West, Younghe Plaza

No. 28, Andingmen East Street

Beijing 100007

P.R. China—Shanghai

Phone

E-mail

Address

[86] 800 810 5118 (toll free) or [86] 400 650 5118 (toll free) [email protected]

Building 6

No. 27 Xin Jin Qiao Road

Shanghai 201206

xx

EASY-nLC 1000 User Guide Thermo Scientific

Preface

Declaration of Conformity

U.S. Safety and EMC (Electromagnetic Compliance) Standards

Safety

This instrument has been reviewed for compliance with standard UL 61010-1, “Electrical

Equipment for Measurement, Control, and Laboratory Use; Part 1: General Requirements.”

EMC

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.

WARNING Changes or modification to this unit not expressly approved by the party responsible for compliance could void the user’s authority to operate the equipment.

Note

This equipment has been tested and found to comply with the limits for a Class A digital device, pursuant to Part 15 of the FCC Rules. These limits are designed to provide reasonable protection against harmful interference when the equipment is operated in a commercial environment. This equipment generates, uses, and can radiate radio frequency energy and, if not installed and used in accordance with the instruction manual, may cause harmful interference to radio communications. Operation of this equipment in a residential area is likely to cause harmful interference in which case the user will be required to correct the interference at his own expense.

Note You must use shielded cables with this unit to ensure compliance with the Class A

FCC limits.

Canadian Safety and EMC (Electromagnetic Compliance) Standards

Safety

This instrument has been reviewed for compliance with standard CAN/CSA-C22.2

No. 61010-1, Second edition - “Safety Requirements for Electrical Equipment for

Measurement, Control, and Laboratory Use; Part 1: General Requirements.”

EMC

This Class A digital apparatus meets all requirements of the Canadian Interference-Causing

Equipment Regulations.

Thermo Scientific EASY-nLC 1000 User Guide

xxi

Preface

European Safety and EMC (Electromagnetic Compliance) Standards

Application of Council

Directive(s)

2004/108/EC “Electromagnetic Compatibility”

2006/95/EC “Low Voltage Directive (LVD)”

Standard(s) to which conformity is declared

IEC 61010-1: Second edition, 2001. Safety Requirements for

Electrical Equipment for Measurement, Control and Laboratory

Use. Also conformity with

• EN 61010-1:2001

• UL 61010-1, second edition

• CAN/CSA-C22.2 No. 61010-1, Second Edition

EN/(IEC) 61326-1:2006. Electrical equipment for measurement, control and laboratory use - EMC requirements - Part 1: General requirements

EN/(IEC) 61000-3-2:2006+A1+A2. Electromagnetic compatibility (EMC) - Part 3-2: Limits - Limits for harmonic current emissions (equipment input current ≤16 A per phase)

EN/(IEC) 61000-3-3:2008. Electromagnetic compatibility

(EMC) - Part 3-3: Limits - Limitation of voltage changes, voltage fluctuations and flicker in public low-voltage supply systems, for equipment with rated current ≤16 A per phase and not subject to conditional connection

FCC CFR 47 Part 15, Class A. Radiated Emissions and

Conducted Emissions

Manufacturer’s Name Proxeon Biosystems A/S

Manufacturer’s Address Edisonsvej 4, DK-5000 Odense, Denmark

Type of Equipment

Model Name

Model Numbers

Serial Number

Year of Manufacture

Laboratory Instrumentation

EASY-nLC™ 1000

LC120

LC-010000 and higher

2011–

I, the undersigned hereby declare that the equipment specified above conforms to the above

Directive(s) and Standard(s).

Ole Vorm, Site Manager July 19, 2011

xxii

EASY-nLC 1000 User Guide Thermo Scientific

1

Introduction

The EASY-nLC 1000 system (

Figure 1

) consists of a compact, ultra-high-performance liquid chromatography instrument designed for split-free flows, down to the low nanoLiter/min range and a built-in instrument control software with a touch-screen interface. The system provides ease of use and ease of service without sacrificing functionality or performance.

Figure 1.

EASY-nLC 1000 system

Thermo Scientific

To familiarize yourself with the EASY-nLC system, review the descriptive information in this chapter.

Contents

System Overview

Hardware Components

Column Setup Options

Solvent Delivery and Sample Pickup Systems

Predefined Steps for Sample Runs

Pump Flow Control

Specifications

EASY-nLC 1000 User Guide

1

1

Introduction

System Overview

System Overview

The EASY-nLC system integrates an autosampler and three nanoflow pumps into one compact enclosure. An embedded computer controls the system and provides an intuitive user interface through an integrated pressure-sensitive touch screen. The built-in method editor simplifies method development, the built-in diagnostic tests minimize troubleshooting time, and the easy access to the solvent delivery system components reduces system maintenance time.

These topics provide an overview of the system components and connections:

System Components

External System Connections

System Components

The EASY-nLC 1000 system consists of the following components (see

Table 4 ).

Table 4. EASY-nLC 1000 system components

Hardware

• Pumps 3 high-pressure pumps (1 to 1000 bar) with associated pressure sensors

• Valves 4 high-pressure valves

• Flow sensor 2 nano-flow sensors

• Can hold six fixed vials, plus either one Microtiter™ plate (96 or

384 wells) or one 6 × 8 vial adapter (for a total of 54 vials)

• 4 liquid containers for solvents, rinse, and waste

• Computer

• Monitor

Embedded computer

Pressure sensitive screen that you can use with gloves

Screen size: 12.1 in. (30.7 cm)

Resolution: 800 × 600 pixels

• Dimensions Width: 36 cm (14 in.)

Depth: 38 cm (15 in.)

Height: 45 cm (18 in.)

• Weight 35 kg (77.2 lb)

• Utilities

Software

Mains power, 120/230 Vac, 50/60 Hz, 250 W

• EASY-nLC stand-alone application (version 3.0)

2

EASY-nLC 1000 User Guide Thermo Scientific

1

Introduction

Hardware Components

External System Connections

Installing the EASY-nLC system 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 system and the mass spectrometer

(see

“Connecting to the Mass Spectrometer through Contact Closure” on page 39

)

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

access port (see “Setting Up the Ethernet Connection to the Support Server” on page 43

)

To control the EASY-nLC system from the Thermo Xcalibur data system, you must also connect the EASY-nLC system to the data system computer.

Hardware Components

These topics show the hardware components of the EASY-nLC system:

Autosampler Tray Compartment

Components Behind the Right Panel

Components Behind the Left Panel

Back Panel

Autosampler Tray Compartment

Figure 2 and Figure 3 show the tray compartment and the waste beaker. You can open the tray

compartment door manually or by using the touch-screen controls.

The tray compartment holds the sample tray, the wash bottles, and six extra 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.

The waste beaker to the right of the tray compartment collects solvent delivered by pumps A and B when these pumps empty solvent to waste and solvent delivered by pump A during the precolumn equilibration and sample loading steps (for a two-column system).

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Hardware Components

Figure 2.

Tray compartment (front view)

Solvent bottles

A and B

Tray compartment with vial adapter and wash solvent bottles

Exit for

Column Out and Waste In solvent lines

Waste beaker

Figure 3.

Tray compartment (viewed from above)

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EASY-nLC 1000 User Guide

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

Three (3) positions for wash solvents

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

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

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Hardware Components

Components Behind the Right Panel

The EASY-nLC nanoflow pump and autosampler system consists of three syringe pumps, three pressure sensors, four 3-position valves, four check valves, and a mixing Tee.

You can access the solvent system components by removing the instrument’s right panel

(see

Figure 4

).

Figure 4.

Hardware components behind the right panel of the EASY-nLC 1000 instrument

Solvent A check valve assembly

Solvent B check valve assembly

Autosampler needle

Four 3-position, 6-port valves

Pressure sensor S

Mixing Tee

Flow sensors

Pressure sensors

A and B

Three syringe pumps

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Hardware Components

Components Behind the Left Panel

You can access the integrated computer and the autosampler needle fitting that connects to

the needle guide by removing the left-side panel of the instrument (see Figure 5

).

Figure 5.

Internal features (behind the left panel)

Integrated computer

Needle tubing

Needle guide

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Autosampler

Enclosed refrigerated sample compartment

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Hardware Components

Back Panel

The system communication, contact closure, and power line connections are on the back panel of the instrument (see

Figure 6

).

Figure 6.

Back panel of the EASY-nLC instrument

Thermo Scientific

P-BUS

IN3 OUT3 IN2 OUT2 IN1 OUT1

RS-232 USB PS/2 LAN

HARD

DRIVE

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 undesired operation.

DEVICE: EASY-NLC TM

S/N:

P/N LC100

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

Proxeon Biosystems A/S:

Web: www.proxeon.com

Phone +45 6557 2300 Fax +45 6557 2301 max 250V-

220-240V▼

110-120V▼

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

Fuse at 120V-:T5AL 250V

Fuse at 230V-:T2.5AL 250V

Table 5 lists the function of each back panel element.

Table 5. Back panel elements (Sheet 1 of 2)

Element

RS-232

USB

PS/2

Description

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

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

Input for connection of keyboard and mouse

LAN

MONITOR

For 10/100 MB/sec Ethernet connection

Output for connection of external display

P-BUS

IN/OUT pins

For communication with add-on devices

Contact closure (primarily for communication with the mass spectrometer)

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Column Setup Options

Table 5. Back panel elements (Sheet 2 of 2)

Element

I|O

Description

Power switch

Fuse holder below

I|O switch

P/N

S/N

Replaceable fuse ratings:

• For 120 V, T 5 AL, 250 V

• For 230 V, T 2.5 AL, 250 V

Product order code

Instrument serial number

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

Column Setup Options

Two solvent lines exit from the right side of the instrument. 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.

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

Two-column Setup

One-Column Setup

Two-column Setup

A two-column setup consists of a precolumn and an analytical column. The EASY-nLC 1000

system ships with a two-column assembly (see Figure 7 ).

Figure 7.

Two-column assembly shipped with the EASY-nLC 1000 system

Stainless steel union

Venting Tee PEEK™ union

VICI

Flow

Precolumn

Flow

Analytical column

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Column Setup Options

The stainless steel union connects to the Column Out tubing, the plugged leg of the venting

Tee connects to the Waste In tubing, and the red PEEK union connects to the emitter.

For information about installing the two-column assembly, see

“Installing Columns” on page 83 .

Figure 8

shows the sample loading step for a two-column setup with a precolumn and an analytical column. Excess solvent A exits the venting Tee through the Waste In tubing.

Tip When you install the two-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

(see

“Setting Up the Properties for the HPLC Device” on page 70

) is clear.

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 (see

Figure 8 ).

Figure 8.

Flow path during the sample loading step for a two-column setup (EASY-nLC 1000 system)

To the autosampler needle

To pump S

2

3

1

S

4 5

6

Column Out tubing

Stainless steel union

Venting Tee PEEK union with sleeves and emitter

VICI

From the mixing Tee

Flow

Precolumn

Flow

Analytical column

2

3

1

W

6

Waste beaker

Waste In tubing

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Column Setup Options

One-Column Setup

A one-column setup consists of an analytical column. The Column Out tubing, the Waste In tubing, and the analytical column connect to the venting Tee.

Figure 9

shows the sample loading step for a one-column setup with an analytical column.

During the sample loading step, valve W is in the 1–2 position and excess solvent A exits through the emitter tip.

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 70 ).

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

(see

Figure 9

).

Figure 9.

Flow path for a one-column setup during the sample loading step

To the autosampler needle

To pump S

2

3

1

S

4 5

6

Column Out tubing

Venting Tee Analytical column

Flow

VICI

PEEK union with sleeves and emitter

From the mixing Tee

2

3

1

W

6

4 5

Waste beaker

Waste In tubing

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Solvent Delivery and Sample Pickup Systems

Solvent Delivery and Sample Pickup Systems

These topics describe the components of the solvent delivery and sample pickup systems:

Syringe Pumps

Pressure Sensors

Flow Sensors

Six-Port Valves

Pump A and Pump B Solvent Delivery System

Sample Pickup and Autosampler Wash System

Syringe Pumps

The syringe pumps perform these functions.

Component

Pumps A and B

Pump S

Function

Draw solvent from solvent bottles on top of the instrument, empty solvent to the waste beaker, 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.

For maintenance purposes, the system tracks the volume pumped by each pump

(see

“Checking and Resetting the Device Usage Counters” on page 181

).

Pressure Sensors

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

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 10 ).

The flow sensors determine the flow rate of solvents passing through the 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 Delivery and Sample Pickup Systems

Because the temperature distribution 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, perform the script, “Calibrate – Flow Sensors” on page 178 .

The flow sensors have a maximum measuring range of 4.5 μL/min.

Figure 10. Flow sensor schematic

Flow

Tubing

Heater

Sensor chip

Temperature sensors

Based on CMOSens™ Technology

Principle patented: US Pat. 6 813 944

Six-Port Valves

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

Figure 11

).

Figure 11. Valve positions

1–2 position 1–6 position Centered

2

3

1

6

4 5

2

3

1

6

4 5

For maintenance purposes, the system tracks the number of valve shifts for each valve (see

“Checking and Resetting the Device Usage Counters” on page 181 .

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Solvent Delivery and Sample Pickup Systems

Depending on the valve position, the valves perform these functions.

Component Function

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

Valve S

1–2 position Directs the solvent flow toward the check valve. When the 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 (see Figure 12 ).

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

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

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.

Valve W

1–6 position The solvent flow passes through the sample loop.

Centered N/A

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

1–2 position Provides backpressure to the venting Tee.

1–6 position Directs the solvent flow from the venting Tee to the waste beaker.

Centered N/A

Pump A and Pump B Solvent Delivery System

Pumps A and B draw solvent from the solvent bottles on top of the EASY-nLC system, empty solvent into the waste beaker to the right of the autosampler compartment, and deliver solvent by way of a pressure sensor and a flow sensor to the mixing Tee. The corresponding six-port valves, low-pressure check valves, and pressure sensors control the solvent flow.

Note Flow sensors provide flow control for pumps A and B. The solvent flow from pump S is not controlled by a flow sensor.

These topics describe the pump A and pump B delivery systems:

Filling and Emptying Pumps A and B

Directing Solvent Flow to the Mixing Tee and through Valve S

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Solvent Delivery and Sample Pickup Systems

Filling and Emptying Pumps A and B

When valve A or B is 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 check valve opens to the solvent bottle and closes to the waste beaker.

• When the pump begins to empty, the check valve opens to the waste beaker and closes to the solvent bottle.

Figure 12. Valve A in position 1–2

Pump A drawing solvent from solvent bottle A Pump A emptying to waste

Pump A

Pump A

Pressure sensor

Pressure sensor

1

1

2 6

2 6

A

A

3 5

3 5

Check valves

4

Check valves

4

Solvent A

Solvent A

Waste beaker

Waste beaker

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Solvent Delivery and Sample Pickup Systems

Directing Solvent Flow to the Mixing Tee and through Valve S

When valve A or B is in the 1–6 position, the solvent flow from the corresponding pump connects to the mixing Tee by way of a pressure sensor and a flow sensor.

Valve S controls the solvent path downstream of the mixing Tee:

• When valve S is in 1–2 position, the solvent flow from the mixing Tee bypasses the sample loop. Valve S is in the 1–2 position during the sample pickup step and during the column equilibration steps.

• When valve S is in the 1–6 position, the solvent flow from the mixing Tee passes through the sample loop. Valve S is in the 1–6 position during the sample loading step.

Pump A or Pumps A and B actively pump solvent toward the mixing Tee during these sample run steps:

Column Equilibration Steps

Load Sample Step

Prealign A + B (Prepare Gradient) Step

Gradient Step

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 13 ) 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.

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Solvent Delivery and Sample Pickup Systems

Figure 13. Equilibrate Precolumn step

Pressure sensor

2

3

1

A

4 5

6

Flow sensor

Mixing Tee

Pressure sensor

2

3

1

B

4 5

6

Flow sensor

To the autosampler needle

2

3

1

S

4 5

6

Column Out line

Waste In line

2

3

1

W

4 5

6

Waste beaker

Precolumn

Venting

Tee

Analytical column

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 1–6. 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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Solvent Delivery and Sample Pickup Systems

Load Sample Step

After pump S draws the sample into the sample loop (see “Pickup Sample Step” on page 21 ),

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 system, allowing excess solvent

A to flow to the waste beaker.

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

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

Figure 14 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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Solvent Delivery and Sample Pickup Systems

Figure 14. Load Sample step

Rear air segment

Pressure sensor

2

3

1

A

4 5

6

Flow sensor

2

3

1

S

4 5

6

Column Out line

Mixing Tee

Solvent A

Solvent B

Sample

Pressure sensor

2

3

1

B

4 5

6

Centered

Flow sensor

Waste beaker

Waste In line

2

3

1

W

4 5

6

Sample

Precolumn

Venting

Tee

Analytical column

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Solvent Delivery and Sample Pickup Systems

Prealign A + B (Prepare Gradient) Step

Before the gradient begins, this sequence of actions occurs.

Action

1

2

3

4

5

The system 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.

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

Note Because the mobile phase bypasses the sample loop during the gradient program, the sample loop does not add any gradient delay volume to the system.

The AFC™ system takes control of pump A and the solvent flow from pump A stabilizes.

The AFC system takes control of pump B.

Valve B moves to position 1–6 and the system sends the closure signal to the mass spectrometer.

Gradient Step

The gradient step includes this sequence of actions.

Action

1

2

The system runs the gradient program.

Note With valve S in position 1–2, the mobile phase bypasses the sample loop during the gradient program. So 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.

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Solvent Delivery and Sample Pickup Systems

Figure 15 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. The

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

Figure 15. Gradient step with valve A and valve B in position 1–6 and autosampler wash step with valve S in position 1–2

Pump S

Pump A

Pressure

Sensor

Pressure

Sensor

2

3

1

A

4 5

6

Connected to check valves

Flow sensor

Pump B

Solvent A

Solvent B

Sample

Pressure

Sensor

Connected to check valves

2

3

1

B

4 5

6

Mixing Tee

Flow sensor

W3 bottle W4 bottle

Autosampler needle

Sample loop

2

3

1

S

6

4 5

Column Out line

Waste In line

2

3

1

W

6

4 5

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Solvent Delivery and Sample Pickup Systems

Sample Pickup and Autosampler Wash System

Pump S draws and delivers solvent for these sample run steps:

Pickup Sample Step

Autosampler Wash Step

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 a system 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 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 system 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 (~11 μL) is equal to the needle volume plus the valve port-to-port volume plus an additional 1 μL of air to bracket the sample solution inside the loop.

Figure 16 through Figure 18 show pump S drawing the first air segment, the requested sample

pickup volume of 10 μL, and the second air segment. 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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Solvent Delivery and Sample Pickup Systems

Figure 16. Pickup Sample step—with pump S drawing the front air segment into the needle

Air segment

120 80 40 0

Pressure

Sensor

Pump S

2

3

1

S

6

4 5

W3 bottle

Column Out line

From the mixing Tee

Figure 17. Pickup Sample step—with pump S drawing 10 μL of sample from a sample vial

W4 bottle Sample vial

120 80 40 0

Pump S

1 μL front air segment

2

3

1

S

4 5

6

W3 bottle W4 bottle Sample vial

Column Out line

From the mixing Tee

Figure 18. Pickup Sample step—with the requested sample volume loaded into the loop and bracketed by two air segments

1 μL front air segment

120 80 40 0

Pressure

Sensor

Pump S

From the mixing Tee

1 μL rear air segment

2

3

1

S

4 5

6

W3 bottle

Column Out line

W4 bottle Sample vial

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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 (see

Figure 15 on page 20 ).

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

Pump S refills with solvent A from the W3 bottle.

Predefined Steps for Sample Runs

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

Step

Refill A B

Precolumn and analytical column equilibration

Pickup sample

Load sample

Prealign A + B

(Prepare Gradient)

Gradient

Autosampler wash

+ refill S

Pumps A and B draw solvent from the solvent bottles on top of the instrument.

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, the system lowers the flow rate (based on the flow setting, pressure setting, or both of these settings in the method) and equilibrates the analytical column

Pump S draws sample from a vial or well position and loads the sample 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 to a precalculated pressure. Pump B builds up pressure to match the precalculated pressure of pump A. Then valves S and W switch to position 1–2, and the AFC system takes control of pumps A and B.

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.

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

(see

Figure 19 and

Figure 71 on page 109 ).

Figure 19. Current step area of the Home > Overview screen

Pickup sample

Load sample

Prealign A + B

Gradient

Initialize system

Equilibrate precolumn

Equilibrate analytical column

Autosampler wash + refill S

Refill AB

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Predefined Steps for Sample Runs

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

• Refill A and B and Pickup Sample

• Column Equilibration and Pickup Sample

• Prepare Gradient (after valve S switches to position 1-2), Gradient, and

Autosampler Wash + Refill S

Figure 20 on page 24

shows a flow chart of the sample run steps.

Figure 20. Flow chart of the sample run steps

Initialize system—check that the hardware is ready

Refill pump A Refill pump B Pick up sample

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EASY-nLC 1000 User Guide

Equilibrate precolumn

(for a two-column setup)

Equilibrate analytical column

Load sample

Prepare gradient a. Prealign pumps A and B b. Switch valve S to position 1–2

Run gradient

Autosampler wash + refill pump S

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Pump Flow Control

Pump Flow Control

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

These flow control systems optimize the system performance:

Automatic Flow Control System

Intelligent Flow Control

Load Speed Protection

Automatic Flow Control System

The Automatic Flow Control (AFC™) system (see

Figure 21 ) is active during theses processes:

• The gradient step of a sample run

• The execution of the isocratic flow maintenance script

The AFC system uses the output of the flow sensors to regulate the flow and accurately maintain the programmed flow from each channel, even during sudden pressure changes caused by solvent composition changes. For accuracy, the AFC system requires a minimum pressure of 20 bar.

Figure 21. Schematic diagram of the AFC system

Pump A

Pressure sensor A

Flow sensor A

2

3

1

A

4 5

6

AFC

TM

Pump B

A/B mixing

Tee

Pressure sensor B

2

3

1

B

4 5

6

Flow sensor B

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Pump Flow Control

Intelligent Flow Control

The Intelligent Flow Control (IFC™) system (see

Figure 22 ) is active during these processes:

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

• The pressure buildup for pump B during the prepare gradient step

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

The IFC system uses output from both the flow sensors and pressure sensors A and B to regulate the flow from pumps A and B. By controlling the flow rate with both a flow and pressure feedback system, the IFC system enhances system performance as follows:

• Reduces the loss of productivity caused by system stoppage related to overpressure (for instance, stoppage caused by viscous samples during the sample loading step).

• Uses the pressure range of the system (0–1000 bar) 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 22. Schematic diagram of the IFC system

Pump A

Pressure sensor A

Flow sensor A

2

3

1

A

4 5

6

IFC TM

A/B mixing

Tee

Pump B

Pressure sensor B

2

3

1

B

4 5

6

Flow sensor B

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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 of 1000 bar.

• 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

When the LSP system 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 70

.

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1

Introduction

Specifications

Specifications

These topics describe the performance and technical specifications for the EASY-nLC system:

Performance Specifications

Technical Specifications

Performance Specifications

Table 6 lists the performance specifications for the EASY-nLC system.

Table 6. Performance specification

Item Specification

Flow range (gradient) 20 to 2000 nL/min

Recommended: 100 to 1000 nL/min

Up to 25 μL/min Flow while loading and equilibrating

Pressure range

Retention time reproducibility (RT RSD)

Peak widths

0 to 1000 bar

Within the recommended flow range: 0.1 to 0.4%

Outside the recommended flow range: typically better than 1%

Typically 3 to 5 seconds full width at half maximum (fwhm), using PepMap™ nano-bore columns as supplied at delivery

Carryover Typically < 0.05%

Conditions: Injection of 100 fmol tryptic digests of BSA on

PepMap columns as supplied at delivery and by using the standard wash procedure for the autosampler

Autosampler sample pickup volume range

0.1 to 18 μL with the standard 20 μL loop

0.1 to 58 μL with a 60 μL loop

Injection reproducibility

(injection RSD)

Injection linearity

Autosampler formats

Autosampler cooling

0.2% at 5 μL pickup

3.0% at 0.1 μL pickup

0.9985 at 0.5 to 10 μL injection volume

6 × 8 Vials + 6 Vials

1 × 96 well MTP + 6 Vials

1 × 384 well MTP + 6 Vials

2 × 48 PCR strips + 6 Vials

4 × 24 PCR strips + 6 Vials maximum cooling: 20 °C (68 °F) below the ambient room temperature typical setting: 7 °C (45 °F)

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Introduction

Specifications

Technical Specifications

These tables list the technical specifications for the EASY-nLC system:

• General specifications, Table 7

• Physical specifications,

Table 8

• Electrical specifications,

Table 9

• Communication specifications,

Table 10

• Components, Table 11

Table 7. General specifications

Item

Sound pressure level

Operating temperature

Storage temperature

Humidity

Sample viscosity

Solvent A

Solvent B

Safety

Specification

< 70 dBA

5 to 30 °C

–25 to +60 °C

20 to 80% RH, non-condensing

0.1 to 5 cP

Water with 0.1% formic acid

Acetonitrile with 0.1% formic acid

According to IEC 61010 Safety requirements for electrical equipment for measurement, control, and laboratory use

Table 8. Physical specifications

Item

Width

Depth

Height

Weight

Weight in the shipping container

Specification

35 cm (14 in.)

38 cm (15 in.)

45 cm (18 in.)

35 kg (77 lb)

45 kg (99 lb)

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Introduction

Specifications

Table 9. Electrical specifications

Item

Power requirements

Fuses

Specification

120 Vac, 50/60 Hz, 250 W

230 Vac, 50/60 Hz, 250 W

(For uninterruptible power supply (UPS) dimensioning, assume

250 W)

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 are UL Listed and CSA-certified.

Table 10. Communication specifications

Item

Contact closure

IN circuit

OUT circuit

LAN

USB

RS-232

P-Bus

PS/2

Monitor

Specification

3 outputs, 3 inputs, and 6 ground pins

TTL Level

PhotoMOS™ relay protected against high voltages. Continuous switch current from 0.8 to 5 V is 35 mA.

10/100 Mb/s BaseT Ethernet

2 × USB 1.1 for keyboard and mouse

Reserved for hardware add-ons or high-level MS control by using serial communication

Reserved for instrument add-ons, using the internal EASY-nLC instrument protocol bus:

8-wire control and limited power at 9/24 V

Input for connection of keyboard or mouse

Output for connection of external display

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Introduction

Specifications

Table 11. Parts specifications

Item

Pumps

Valves

Autosampler

Specification

• 140 μL volume

(enough for a >10 h, 0–100% B, 300 nL/min gradient)

• 1 nL/min to 300 μL/min flow range

• External pressure sensor

• VICI™/Valco™ rotor/stator

• 6 ports

• 3 positions (1-6, 1-2, or CENTERED)

• Peltier-cooled. Capacity is maximum 20 °C below ambient temperature, measured at 60% RH. Expect less if you remove the side panels, use an adapter plate in the autosampler other than the standard plate, or both.

• Plate holder ejects through spring-mounted autosampler door.

• 4 containers for waste or wash liquids

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Installing the EASY-nLC Instrument

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

“Safety and

Special Notices” on page xiii

.

Note This chapter describes the laboratory requirements and the back panel connections for the EASY-nLC system.

• 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, see “Solvent System Schematic” on page 194 .

Contents

Lifting Instructions

Laboratory Requirements

Back Panel Connections

Lifting Instructions

The 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 35 kg (77.2 lb), the EASY-nLC instrument is too heavy for one person alone to lift. When moving the system, use two people to place it on a table cart for transport.

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Installing the EASY-nLC Instrument

Laboratory Requirements

Figure 23 shows the lift points for the EASY-nLC instrument.

Figure 23. Lifting the EASY-nLC 1000 system by hand

EASY - nLC 1000

Laboratory Requirements

The following topics describe the laboratory requirements for the EASY-nLC system. For more information, refer to the EASY-nLC Preinstallation Requirements Guide.

Benchtop Requirements

Internet Access

Power and Fuse Requirements

Temperature and Humidity Requirements

Benchtop Requirements

Place the EASY-nLC system on a benchtop or movable table that can support a minimum of two times the system weight.

Allow for at least 15 cm (6 in.) of free space at the back of the instrument for proper air circulation.

The dimensions of the EASY-nLC system are as follows:

• Width: 35 cm (14 in.)

• Depth: 38 cm (15 in.)

• Height: 45 cm (18 in.)

To minimize extra column volume between the LC column outlet and the detector inlet, place the EASY-nLC system 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 system away from any detector or mass spectrometer vents.

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Installing the EASY-nLC Instrument

Laboratory Requirements

Figure 24 shows a typical benchtop setup for an EASY-nLC system 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 24 , you can control the EASY-nLC system from the local

data system computer or from another data system computer on the intranet; however, the

EASY-nLC system might not be able to communicate with the remote support server. For information about connecting the EASY-nLC system to the remote support server, see

“Setting Up the Ethernet Connection to the Support Server” on page 43

.

Tip Before you move the EASY-nLC system from one benchtop to another, exit the

EASY-nLC system (see

“Closing Down the EASY-nLC System” on page 58 ), disconnect

the EASY-nLC system 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. EASY-nLC system and LTQ™ Series MS detector setup

Solvent lines

Data system computer

EASY-nLC 1000 system

Ion source monitor

LTQ Series

MS detector

Load Detector

Inject Waste

Power Vacuum Communication System Scanning

EASY - nLC 1000

EASY - nLC II

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 system to the Ethernet switch

Thermo Scientific

Contact closure cable from the EASY-nLC system to the MS detector

Ethernet cable from the MS detector to the Ethernet switch

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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 system within reach of the laboratory benchtop.

IMPORTANT The EASY-nLC system 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 system to the support server at

IP address 195.41.108.93 port 22.

For information about using the remote support feature, see Chapter 9, “Remote Support.”

Power and Fuse Requirements

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

You can plug the EASY-nLC system 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 250 W.

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

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

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

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

Temperature and Humidity Requirements

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.

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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

Connecting to the Mass Spectrometer through Contact Closure

Connecting the Ethernet Communication Cables

Attaching a Mouse and Keyboard to the USB Connections

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

Figure 25 shows an LC/MS system with an EASY-nLC system, 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 25. LC/MS system with an EASY-nLC system, an LTQ Series mass spectrometer, and a data system computer

Data system computer and Ethernet switch

EASY-nLC system

(back panel)

LTQ Series mass spectrometer

(right panel)

Fourplex outlet for the data system hardware

PRO EON

P-BUS

IN3 OUT3 IN2 OUT2 IN1 OUT1

PS/2 LAN

HARD

DRIVE

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.

This instrument is for research use only.

DEVICE: EASY-NLC

P/N LC100

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

Proxeon Biosystems A/S

Web: www.proxeon.com

Phone +45 6557 2300 Fax +45 6557 2301

MONITOR

O

220-240▼

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

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

Thermo Scientific

Fourplex outlet for the

EASY-nLC system

Fourplex outlet for the mass spectrometer

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Installing the EASY-nLC Instrument

Back Panel Connections

Connecting to Line Power

Each EASY-nLC system 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 system 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 system 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 25 on page 37 ).

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

EASY-nLC instrument, the mass spectrometer, 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 system’s built-in computer.

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

Y

To connect the EASY-nLC system to line power

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

“Replacing the Main Power Fuse” on page 158

).

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

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

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Installing the EASY-nLC Instrument

Back Panel Connections

Connecting to the Mass Spectrometer through Contact Closure

The EASY-nLC stand-alone application (touch-screen software) 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 59

.

IMPORTANT Occasionally, Thermo Fisher Scientific releases new software for the

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

Figure 26 shows the hardwire connections between the EASY-nLC system and an LTQ Series

mass spectrometer.

Figure 26. Two-way contact closure connection for an LTQ Series mass spectrometer

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

Peripheral Control

OUT1

IN1

OUT2

IN2

OUT3

TTL

GND

Y

To connect the contact closure cable

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

Table 12

), 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 12. 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

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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 system 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 system 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 system 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 system. To perform tasks such as flushing air from the

LC system, you must use the built-in touch-screen software.

Figure 24 on page 35 and

Figure 25 on page 37

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

The EASY-nLC system 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 system 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 EASY-nLC system’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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Thermo Scientific

2

Installing the EASY-nLC Instrument

Back Panel Connections

Y

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

1. Connect the EASY-nLC system 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 (at the end of 2011 or later) with

Thermo Scientific mass spectrometers has a power save button labeled ECO friendly.

IMPORTANT When the power-save feature is on, the instruments 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.”

Y

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 system’s computer hard drive.

Y

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 system’s computer hard drive after you make the following connections:

• When you connect a stand-alone EASY-nLC system 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 system 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 system 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 system in the stand-alone mode, the chromatographic methods and batches reside on the integrated computer hard drive.

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

Y

To access the files on the EASY-nLC system from another computer on the intranet

1. Do one of the following:

• For a stand-alone EASY-nLC system, 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 system 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 40

.

2. Set up the software connection for the EASY-nLC system as described in “Setting Up the

Local Area Network Connection” on page 61 .

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 system, you do not need to reenter the user login and password.

To access the files on the EASY-nLC system’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 system.

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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 system.

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

For information about initiating the communication link with the support server, see

“Connecting the EASY-nLC System to the Support Server” on page 150

.

Y

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

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

shown in Figure 24 on page 35

, disconnect the Ethernet cable from the LAN port of the

EASY-nLC system.

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

Figure 27

).

IMPORTANT For remote diagnostics, connect the EASY-nLC system 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 system to the support server at

IP address 195.41.108.93, port 22.

Figure 27. EASY-nLC system connected to a local network port with Internet capability

EASY - nLC 1000

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-Sensitive Screen” on page 47 .

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

The EASY-nLC system can control and power add-on devices through the P-bus, the RS-232

interface, or both (see Figure 28 ).

Figure 28. EASY-nLC back panel (top portion)

P-BUS

IN3 OUT3 IN2 OUT2 IN1 OUT1

RS-232 USB

RS-232

PS/2 LAN MONITOR

P-Bus

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EASY-nLC 1000 User Guide Thermo Scientific

3

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 system progress and 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-Sensitive Screen

User Interface Layout

Viewing the System Status and Using the Manual Controls

Application Menu Structure

Logging In to the EASY-nLC System

Closing Down the EASY-nLC System

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Integrated Instrument Control Software

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 29. On/Off switch on the back panel

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

1. Boots 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 application.

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

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Integrated Instrument Control Software

Using the Touch-Sensitive Screen

Using the Touch-Sensitive Screen

You control the EASY-nLC instrument through the touch-sensitive 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 44 ), but the

user interface has been optimized for touch-screen interaction.

The instrument monitor is a transparent layer of glass ( Figure 30

) that detects finger pressure

(even while wearing gloves) and sends the corresponding commands to the computer.

Figure 30. Touch-screen control for the EASY-nLC 1000 instrument

Home > Overview page

The touch-screen controls consist of buttons, tables, input fields, and interactive graphical instrument components as described in these topics:

Using the Buttons

Inputting Alphanumeric Text

Using Tables and Input Fields

Interactive Graphical Controls

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 dialog box where you can cancel or accept the action. For example, when you press Eject Plate on the Home > Overview page, a confirmation dialog 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.

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Integrated Instrument Control Software

Using the Touch-Sensitive Screen

Inputting 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 31 ) 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 31. 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.

Interactive Graphical Controls

The four valves, the three pumps, and the autosampler appear as interactive graphical components on the Home > Overview page. When you press a graphical component, the manual controls for that component opens.

For information about using these manual controls, see

“Manual Controls” on page 141

.

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User Interface Layout

User Interface Layout

The EASY-nLC user interface (

Figure 32

) contains five menu tabs at the top of the touch screen. When you press a tab at the top of the touch screen, a set of side tabs appears on the left.

Figure 32. EASY-nLC user interface layout with menus

Top menu tabs

Logo icon

Side 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 tabs on the left side of the touch screen.

During a session, the application tracks which page (side 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 System” on page 57 and

“Closing Down the EASY-nLC System” on page 58 ).

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

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Viewing the System Status and Using the Manual Controls

Viewing the System Status and Using the Manual Controls

You can view the system status and access manual controls for the system hardware

components on the Home > Overview page ( Figure 33 ).

Figure 33. Overview page of the Home menu

Pump A status Current sample and status

Autosampler icon Valve A icon

Flow sensor A readback

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Pump B icon Mixing Tee readback

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3

Integrated Instrument Control Software

Viewing the System Status and Using the Manual Controls

The system readbacks display the following information.

Readback

Pump

Flow sensor

Mixing Tee

Autosampler

Information

Displays the back pressure, 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

Display the %B solvent composition

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

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

).

For more information about viewing the system status, see

“Monitoring the Run” on page 109 .

You can access the manual controls for the system by pressing the pump, valve, and autosampler icons on the Home > Overview page. Only use these controls for troubleshooting hardware problems. For information about using these controls, see

“Manual Controls” on page 141 .

Interactive component

Pump A, B, or S

Valve A, B, S, or W

Action

Opens the Pump dialog box where you can change the flow rate, refill or empty the pump, and start and stop the pump flow.

Opens the Valve dialog box where you can change the valve position.

Autosampler Opens the Autosampler dialog box where you can work with the XYZ robot, change the cooler temperature, or turn off the temperature control unit.

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Application Menu Structure

Application Menu Structure

Table 13

lists the structure of the touch-screen application’s user interface. The application has

five menu tabs at the top of the screen (see Figure 32 on page 49

) and each menu has two or more pages (tabs along the left side of the screen).

To access a specific page of the application, you 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 Editor > 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.

Table 13. User-interface structure

Home

Overview

Graphs

Batch Setup

File

Edit (with alternate views Rack and List)

Method Editor

File

Maintenance

Scripts

Edit (pages 1/5–5/5) Log Book

Configuration

Users

Connections

Queue Support

Devices

Network

Time

Backup

These topics describe the pages and views that you can access from these menus:

Home Menu

Batch Setup Menu

Method Editor Menu

Maintenance Menu

Configuration Menu

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Application Menu Structure

Home Menu

Page

Overview

Graphs

Queue

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 Go to

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

“Manual Controls” on page 141 .

Monitor the system status.

“Viewing the System Status and Using the

Manual Controls” on page 50 and

“Monitoring the Run” on page 109 .

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.

“Using the Graphs Page in the Home Menu” on page 111 .

“Editing the Running Batch” on page 114 .

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 103 and

“Starting Sample Acquisition” on page 108

.

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.

Go to

“Viewing the Batch List and Setting Up a

Batch Record” on page 103 .

Edit Set up new batch jobs.

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

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.

“Specifying the Method, Sample Positions, and Number of Injections Per Sample” on page 104 .

“Editing the Running Batch” on page 117 .

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Application Menu Structure

Method Editor Menu

Use the pages of the Method Editor Menu to set up the chromatographic methods for your samples (see “Creating a Method” on page 100).

Note If the method controls the Advion™ RePlay™ external device, the Method Editor wizard includes six pages. For information about setting up a method that includes the

RePlay device, see Appendix A, “External Devices.”

Page

File

Edit

1/5

2/5

3/5

4/5

5/5

Function Go to

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

“Browsing for a Stored Method or Creating a

New Method File” on page 92 .

Method Editor wizard with five pages—

Use page 1/5 of the Method Editor wizard to select the method that you want to edit and to enter a description of the method.

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

“Starting the Method Editor Wizard and

Selecting the Method to be Edited” on page 93 .

“Setting Up the Sample Pickup and Loading

Steps” on page 94

.

Control the liquid chromatography gradient.

“Building the Gradient” on page 96 .

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

Control the autosampler wash step.

“Setting Up the Column Equilibration

Steps” on page 98

.

“Setting Up the Autosampler Wash Step” page 100 .

on

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Application Menu Structure

Maintenance Menu

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.

Go to

“Maintenance Scripts” on page 161 .

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.

“Keeping Service Records” on

Chapter 9, “Remote Support.”

page 180

Appendix A, “External Devices.”

.

Check and reset the device usage counters.

“Checking and Resetting the Device Usage

Counters” on page 181 .

Set up the global configuration for the

HPLC.

Calibrate the autosampler.

“Setting Up the Properties for the HPLC

Device” on page 70

.

Chapter 7, “Calibrating the Autosampler’s

XYZ Robot.”

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Application Menu Structure

Configuration Menu

Control the instrument setup and manage system configuration.

Page

Users

Connections

Network

Time

Backup

Function

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

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

Go to

“Creating New User Accounts” on page 65

.

“Setting Up Communication with the Mass

Spectrometer” on page 59

Set up or view how the EASY-nLC system is identified in a connected computer network.

Consult your IT administrator before making changes to the system.

“Setting Up the Local Area Network

Connection” on page 61

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

Save and restore backups of the system database and configuration. A backup includes methods, batches, instrument configuration, and user accounts.

“Modifying the Time Settings” on page 67

.

“Making System Backups” on page 68

.

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Logging In to the EASY-nLC System

Logging In to the EASY-nLC System

The EASY-nLC application 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.

Y

To start using or configuring the EASY-nLC system

1. In the User list, select admin (see Figure 34

).

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.

Figure 34. EASY-nLC login dialog box

Thermo Scientific

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

4. If security is an issue, change the admin password (see Figure 35 ).

Y

To log off the EASY-nLC system

1. Press Exit.

A confirmation dialog box opens (see

Figure 35 ).

Figure 35. Confirmation dialog box that opens when you press Exit on the lower-left corner of the screen

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Closing Down the EASY-nLC System

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 System

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.

WARNING If you turn off the power switch during normal operation, you risk damaging essential system components. Follow the procedure outlined here whenever possible.

Y

To turn off the EASY-nLC system

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

A confirmation dialog box opens (see

Figure 35 on page 57 ).

2. Press Power Down.

The EASY-nLC application displays a white screen with a small progress bar. When the progress bar is filled out and the message appears indicating you can safely turn off the instrument, go to the next step.

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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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4

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

Making System Backups

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.

Y

To set up the mass spectrometer connection

1. Press Configuration > Connections.

The Connections page of the Configuration menu opens (see Figure 36 ).

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Configuring the EASY-nLC System

Setting Up Communication with the Mass Spectrometer

Figure 36. Connections page

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2. From the Instrument list, select the correct mass spectrometer (see

Figure 36 ).

The Instrument list contains the most common mass spectrometers used in proteomics, but if you cannot find your particular mass spectrometer, select Generic.

Tip 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

).

3. If the Use Contact Closure option is not already selected, select it.

4. In the Contact Closure 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 36

).

• If the graphic for the selected mass spectrometer 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.

• 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.

Thermo Scientific

4

Configuring the EASY-nLC System

Setting Up the Local Area Network Connection 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 thus be missed from the actual gradient.

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. This signal width 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.

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 42 ) and set up the network

configuration for the application, you can access the EASY-nLC system from other computers on the network.

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

Y

To connect the EASY-nLC system 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 42 ).

2. Press Configuration > Network.

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Configuring the EASY-nLC System

Setting Up the Local Area Network Connection

The Network page (

Figure 37

) contains the configuration address information.

Figure 37. Network page of the Configuration menu

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3. For Network type, select Dynamic addressing (DHCP) or use a Fixed IP address.

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 system to look at log files, for example. Microsoft Windows Explorer might display the file server as shown in

Figure 38 .

Figure 38. File server access through Windows Explorer

Type

IP address here.

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4

Configuring the EASY-nLC System

Modifying User Permissions

Y

To access the admin folder on the EASY-nLC system’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 system. For example, if you haven’t 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 system.

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 14. User types and permission levels for each menu

Menu

Home

Batch Setup

Method Editor

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 commands to the system.

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

Creating New User Accounts

Changing Your Password from the Exit Menu

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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.

Y

To change the administrator password

1. Log in as admin (otherwise, see

“Logging In to the EASY-nLC System” on page 57 ).

2. Press Configuration > Users.

The Configuration > Users page opens ( Figure 39

).

Figure 39. Users page of the Configuration menu

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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.

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Configuring the EASY-nLC System

Modifying User Permissions 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.

6. Press Save to save the user account information.

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.

Y

To create a new user account

1. Log in as admin (otherwise, see

“Logging In to the EASY-nLC System” on page 57 ).

2. Press Configuration > Users.

The Configuration > Users page opens (see

Figure 40 ).

Figure 40. Users page of the Configuration menu

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Configuring the EASY-nLC System

Modifying User Permissions

3. Press New.

The User box changes to the User Login box and becomes available for data input. The

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 14 on page 63

.

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 system, 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.

Changing Your Password from the Exit Menu

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

Y

To change your password

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

The Exit dialog box for the current user opens (see

Figure 41

).

Figure 41. Exit dialog box for the current user

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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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Configuring the EASY-nLC System

Modifying the Time Settings

Modifying the Time Settings

Use the Configuration > Time page (

Figure 42 ) to set the internal clock in the EASY-nLC

system and the display format used throughout the application for date and time information.

Y

To set the time zone

1. Press Configuration > Time.

The Configuration > Time page opens (see Figure 42

).

Figure 42. Time page of the Configuration menu

Thermo Scientific

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.

Y

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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Configuring the EASY-nLC System

Making System Backups

Making System Backups

Use the Configuration > Backup page to save and restore backups of the system database and configuration. A backup includes methods, batches, instrument configuration, and user accounts.

You can also remove changes you have made to the system, completely or selectively, by restoring the factory default settings.

IMPORTANT Make sure that no one else is using the EASY-nLC system when restoring a backup because this backup also influences user accounts.

Y

To create a system backup

1. Press Configuration > Backup.

The Configuration > Backup page opens (

Figure 43

).

Figure 43. Backup page of the Configuration menu

2. Press Backup.

The application makes a .bak file.

Y

To restore a backup file

1. In the Location list, select the file that you want to restore.

2. Press Restore.

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Configuring the EASY-nLC System

Making System Backups

Y

To reset one or all of the settings to the factory values

1. In the Factory Reset area, press Reset.

The Factory Reset dialog box opens.

2. Select the check boxes for the items that you do not want the system to reset.

The check box options are as follows:

• Keep User Data

• Keep System Log

• Keep Maintenance Log

• Keep Backups

3. Press Reset.

The Factory Reset dialog box closes and the items that you did not select are reset. The

Factory Reset does not reset the device usage counters (see

“Checking and Resetting the

Device Usage Counters” on page 181

).

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Configuring the EASY-nLC System

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.

Y

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 Configuration > Devices page opens

(see

Figure 44 ).

Figure 44. 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 and Load

Speed Protection (LSP).

Table 15

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

Devices page of the Maintenance menu.

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Configuring the EASY-nLC System

Setting Up the Properties for the HPLC Device

Table 15. HPLC parameter descriptions

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 pump.

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

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 one-column setup.

One Column Setup

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

Load Speed Protection When this check box is selected, the system uses the flow rate entered in Sample Loading area on page 2/5 of the Method Editor wizard (see

“Setting Up the Sample Pickup and Loading Steps” on page 94 ) to load the sample onto the precolumn (or the analytical

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

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.

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Preparing the EASY-nLC Instrument for Use

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

Administrator privileges (see

“Logging In to the EASY-nLC System” on page 57 ), and then

follow these procedures.

Contents

Preparing Solvent Bottles and Waste Containers

Executing Maintenance Scripts

Purging and Flushing the Pumps

Installing Columns

Using nanoViper Fittings

Equilibrating Columns

Preparing 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 MSDSs provided by the manufacturer. When working with solvents and chemicals, wear the appropriate safety gear.

CAUTION When working with solvents, wear safety glasses.

CAUTION When working with solvents, wear safety gloves.

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Preparing the EASY-nLC Instrument for Use

Preparing Solvent Bottles and Waste Containers

Before shipment, the EASY-nLC 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 topics:

Solvent Requirements

Solvent A and B Bottles

Autosampler Wash Bottles and Waste Beaker

Solvent Requirements

The installation solvents for the EASY-nLC 1000 system are as follows:

Solvent A: 0.1% formic acid in water

Solvent B: 0.1% formic acid in acetonitrile

Wash solvent 3: 0.1% formic acid in water

To prepare these solutions, order the appropriate LC/MS-grade solvents or the pre-blended

solutions. Table 16 lists the solvents that you can order from Thermo Fisher Scientific.

Table 16. Solvents

Solvent/reagent

Water

Acetonitrile

Formic acid (99.5%)

0.1% formic acid in water

0.1% formic acid in acetonitrile

Specification

LC/MS grade

LC/MS grade

LC/MS grade

LC/MS grade

LC/MS grade

IMPORTANT Use only LC/MS-grade solvent. Do not filter solvents. Filtering solvents can introduce contamination.

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

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Preparing Solvent Bottles and Waste Containers

Y

To order LC/MS-grade solvents from Thermo Fisher Scientific

Click this icon,

–or –

.

Go to: www.FisherLCMS.com

, and click the Solvents tab.

Solvent A and B Bottles

Follow these recommended steps to prepare the two solvent bottles.

Y

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.

4. Mount the inline filter assembly on the blue cap lids as shown in Figure 45 and Figure 46 .

Figure 45. Inline filter assembly (exploded view)

Figure 46. Assembled inline filter

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

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Preparing the EASY-nLC Instrument for Use

Preparing Solvent Bottles and Waste Containers

Autosampler Wash Bottles and Waste Beaker

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

beaker (see Figure 47

). 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 Plate button on the Home > Overview screen.

Figure 47. Autosampler compartment

Vial tray

Waste beaker

Wash bottles (W3 and W4)

Y

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 system and make sure that the area in front of the tray compartment door is free from obstruction.

3. Press Home > Overview. Then press Eject Plate.

A confirmation dialog 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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5

Preparing the EASY-nLC Instrument for Use

Preparing 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 48 ).

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

W4. Any other position could lead to instrument malfunction and possible breakdown.

Figure 48. 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 Insert Plate to move the plate back into the instrument.

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Preparing the EASY-nLC Instrument for Use

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 49

) of the Maintenance menu.

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

“Preparing Solvent Bottles and Waste Containers” on page 73 .

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

Figure 49. 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 system.

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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.

Y

To purge and flush pumps A and B

1. Press Maintenance > Scripts.

The Scripts page of the Maintenance menu opens.

2. Set up the purge solvents job as follows (see

Figure 50

): a. In the Category list, select Prepare.

b. In the Name list, select Purge Solvent.

c.

Press the Parameters tab.

d. Press the Purge Iterations box and type 10 with the numeric keypad.

This is the number of times that the pumps will completely refill and eject the solvent. 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.

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Purging and Flushing the Pumps

Figure 50. Purge solvent job parameters

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3. To save these settings and continue, press Schedule.

The Queue page of the Home menu appears.

4. Set up the flush air job as follows (see

Figure 51 ):

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.

Press the Flush Vol. Threshold [μL] box and type 12 with the numeric keypad.

This values helps to ensure an acceptable amount of solvent compressibility. f.

Select the Flush Pump A, Flush Pump B, and Flush Pump S check boxes.

Thermo Scientific

Thermo Scientific

Figure 51. Flush air job parameters

5

Preparing the EASY-nLC Instrument for Use

Purging and Flushing the Pumps

5. Press Schedule.

The Queue page ( Figure 52

) of the Home menu appears.

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Purging and Flushing the Pumps

Figure 52. Queue page of the Home menu, showing the queued jobs

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6. In the Jobs list, make sure the check box in the Auto-Continue column is selected for

Flush Air.

7. Press Overview.

The Overview page of the Home menu opens.

8. 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.

9. 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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5

Preparing the EASY-nLC Instrument for Use

Installing Columns

Installing Columns

The EASY-nLC 1000 instrument ships with a two-column assembly (see Figure 53 ).

Figure 53. Two-column assembly

Stainless steel union Venting Tee PEEK union

Thermo Scientific

VICI

Flow Flow

Precolumn

2 cm length

Analytical column

15 cm length

Table 17

lists the columns, fittings, and unions that make up the two-column assembly.

Table 17. Columns and column fittings

Item Description

Precolumn

(trap column) with nanoViper™ fittings

Acclaim™ PepMap 100, 2 cm length, 75 μm ID,

3 μm particle size, C18 column packing

Analytical column with nanoViper fittings

(Dionex™ part number 164705)

Acclaim PepMap RSLC, 15 cm length, 50 μm ID,

2 μm particle size, C18 column packing

(Dionex part number 164704)

0.76 in. length, 0.035 in. thru-port, for 1/32 in. OD tubing PEEK union with fittings

Stainless steel union

Venting Tee

PEEK sleeves for fused-silica

For 1/16 in. OD tubing, part number SC904

Stainless steel, 0.15 mm bore, for 1/16 in. OD tubing, part number SC901

330 μm ID, 1/32 in. OD, part number SC903

To install the column assembly, follow these procedures:

Connecting the Column Assembly to the Column Out and Waste In Lines

Connecting an Emitter to the Analytical Column

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Installing Columns

Connecting the Column Assembly to the Column Out and Waste In Lines

Make sure that you connect the venting Tee to the Waste In line for both the one-column and two-column setups.

CAUTION Wear gloves when handling the solvent lines.

Y

To connect the column assembly to the instrument

1. Do one of the following:

• For a two-column setup, go to step 2 .

• For a one-column setup, go to step 3 .

2. For a two-column setup, connect the Column Out line to the stainless steel union as follows: a. Slightly loosen the nanoViper fitting connected to the stainless steel union.

b. Insert the nanoViper fitting on the Column Out line into the other end of the stainless steel union and slowly tighten the fitting until you feel resistance.

c.

Tighten both fittings by an additional 45 degrees (1/8-turn). Do not tighten the fittings by more than a 1/4-turn.

CAUTION Because the fittings are sealing 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.

For more information about working with nanoViper fittings, see “Using nanoViper

Fittings” on page 87 .

d. Go to step 4

.

3. For a one-column setup, if you are working with the column assembly shipped with the instrument, remove the precolumn from the column assembly. Connect the Column Out line directly to the venting Tee.

4. Connect the Waste In line to the venting Tee as follows: a. Remove the plug from the third leg 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 54 and Figure 55

).

c.

Slowly tighten the fitting until you feel resistance.

d. 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 general caution following step 2c ).

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Installing Columns

Figure 54 shows the one-column setup and Figure 55 shows the two-column setup. Both

setups use the venting Tee.

Figure 54. One-column assembly connected to the EASY-nLC system Column Out and Waste In tubing

Venting Tee

PEEK union

VICI

Column Out tubing

Flow

Analytical column

15 cm length

PEEK sleeve

Waste In tubing

Figure 55. Two-column assembly connected to the EASY-nLC system Column Out and Waste In tubing

Stainless steel union Venting Tee PEEK union

VICI

Column Out tubing

Waste In tubing

Flow

Precolumn

2 cm length

Flow

Analytical column

15 cm length

PEEK sleeve

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Installing Columns

Connecting an Emitter to the Analytical Column

Before you mount the column assembly onto the ion source, connect an emitter to the analytical column.

CAUTION Because the emitter tip is sharp enough to puncture skin and easily damaged, do not touch it.

Y

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.

2. Connect the white distance-gauge plug to one end of the PEEK union.

3. Using a sleeve, connect the analytical column to the other end of the PEEK union.

4. Thread the blunt end of the emitter through the adapter sleeve. Ensure that the blunt end of the emitter does not protrude from the sleeve by more than 1 mm (see

Figure 56

).

Figure 56. Sleeved emitter

Blunt end of the emitter

Adapter sleeve Emitter tip

5. 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.

6. As you gently press the sleeved emitter against the union port, screw the nut into the port until fingertight. Be careful not to overtighten the fitting.

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Using nanoViper Fittings

Using nanoViper Fittings

For the EASY-nLC system, most of the plumbing connections are made with nanoViper fittings (see

Figure 57

).

Figure 57. nanoViper fitting

PEEK sealing surface Screw Removable, black, knurled tightening tool

Thermo Scientific

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.

Therefore, it is essential to follow the guidelines below to avoid damage by over-tightening.

Y

To use a nanoViper fitting

1. Insert the nanoViper fitting into the target port and slowly rotate the screw clockwise until you feel the resistance.

2. Using the black knurled fitting, tighten the screw clockwise to an angle between

0 and 45 degrees (1/8-turn).

3. Start operating your system at the desired working pressure and check the back pressure.

4. If the back pressure is too low, return the system to 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.

5. Tighten the screw by as much as an additional 45 degrees. Do not turn the screw beyond an angle of 90 degrees from where you felt the initial resistance.

IMPORTANT To prevent damage to the sealing surface of the nanoViper fitting, take care not to over-tighten the nanoViper fitting.

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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 94

) of the chromatographic method.

Y

To equilibrate the precolumn and determine the flow rate for sample loading

1. Open the Precolumn Equilibration script and set up the script parameters as described in

“Prepare – Precolumn Equilibration” on page 164 .

Thermo Fisher Scientific recommends running the equilibration at a set pressure rather than a set flow, to make sure the column pressure limit (800 bar) and the instrument pressure limit (1000 bar) are not exceeded (see

Figure 58 ). 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.

Figure 58. Precolumn equilibration parameters

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2. 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.

3. Monitor the actual flow rate to become familiar with the flow/pressure relationship on your particular column.

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Preparing the EASY-nLC Instrument for Use

Equilibrating Columns

Y

To equilibrate the analytical column

1. Open the Analytical Column Equilibration script and set up the script parameters as described in

“Prepare – Analytical Col Equilibration” on page 166 .

2. Set the desired flow, volume, and maximum pressure (see

Figure 59

).

Thermo Fisher Scientific recommends running the reequilibration at a set pressure rather than a set flow, to make sure the column pressure limit (800 bar) and the instrument pressure limit (1000 bar) are not exceeded. If the flow field is left empty, the pump will run at the specified pressure. For best results, use a reequilibration volume of 10 column volumes.

Figure 59. Analytical column equilibration parameters

Thermo Scientific

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 a Sample Plate

Creating a Method

Creating a Batch

Starting Sample Acquisition

Monitoring the Run

Stopping Sample Acquisition

Editing the Running Batch

Troubleshooting a Sample Run

Preparing a Sample Plate

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.

Y

To prepare the sample vials or microplates

1. Load 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.

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

Chapter 7, “Calibrating the Autosampler’s

XYZ Robot.”

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Creating a Method

Creating a Method

To create a method, follow these procedures:

1.

Browsing for a Stored Method or Creating a New Method File

2.

Starting the Method Editor Wizard and Selecting the Method to be Edited

3.

Setting Up the Sample Pickup and Loading Steps

4.

Building the Gradient

5.

Setting Up the Column Equilibration Steps

6.

Setting Up the Autosampler Wash Step

7.

Saving the Method Settings

Browsing for a Stored Method or Creating a New Method File

Use the Method Editor > File page to browse for stored methods or to select the file folder and enter a name for the new method.

Y

To browse for a stored method

1. Press Method Editor > File.

The Method Editor > File page opens (see Figure 60

).

Figure 60. File page of the Method Editor

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Creating a Method

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.

Y

To create a new method

1. Open the Method Editor > File page (see Figure 60 on page 92

).

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.

Starting the Method Editor Wizard and Selecting the Method to be Edited

The Method Editor wizard consists of five pages that guide you through all the parameters required for a working LC method.

Use the Method Editor > Edit 1/5 page to select the method that you want to edit.

Y

To start the Method Editor wizard

1. Press Method Editor > Edit.

2. If you are not already on the Edit 1/5 page (see Figure 61 ), go to page 1/5 by pressing the

appropriate arrow near the bottom of the touch screen.

Figure 61. Method Editor > Edit 1/5 page

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Creating a Method

3. Press Open.

The Select Method to Open dialog box opens.

4. Select the method that you want to edit.

5. Press Accept.

To continue setting up the method, go to the Edit 2/5 page by pressing the right arrow.

Setting Up the Sample Pickup and Loading Steps

Use the Method Editor > Edit 2/5 page (see Figure 62

) to set the parameters for picking up the sample from the well plate and loading it onto the precolumn.

Figure 62. Edit 2/5 page of the Method Editor wizard

Y

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 sample pump S, the maximum volume is set to the configured sample loop size minus 2 μL. For example, the maximum sample pickup for a system 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 Thermo Xcalibur data system.

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Creating a Method

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 aqueous solutions, 20 μL/min is a good starting point.

Y

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 system).

Range: 0 to 137

Normally it is sufficient to load the precolumn with two times the sample pickup volume + 2 μL.

2. Press the Flow box and enter the flow rate for sample loading onto the precolumn (or analytical column in a one-column system).

If you leave the Flow box empty, the pump operates at the set pressure.

Range: 0 to 100

Tip The application accepts values from 0 to 100 μL/min, but the IFC algorithm (see

“Intelligent Flow Control” on page 26

) prevents the flow rate for the sample loading step from exceeding 25 μL/min based on the back pressure produced by the system tubing alone without the columns attached.

Use the column equilibration scripts to determine the appropriate flow rate for your application.

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 instrument pressure of 1000 bar).

Range: 0 to 980

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 980 bar.

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 3/5 page by pressing the right arrow.

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Creating a Method

Building the Gradient

Use the Method Editor > Edit 3/5 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 63

).

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 procedure is intended to help you set up a method, make sure to set the parameters appropriately for your analyte and column characteristics.

Figure 63. Edit 3/5 page of the Method Editor wizard

Note When you press a cell in the gradient table, the keypad dialog box opens.

Y

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 10 (%). Then press ACCEPT.

The gradient is now set to start at 300 nL/min and 10 %B.

2. Press ADD to add another row.

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Creating a Method

3. In the second row of the gradient table, do the following: a. Press the Duration column cell and enter 20 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 40 (%). Then press ACCEPT.

4. Press ADD to add another row.

5. In this new row, set duration to 5 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 15 min, flow to 300 nL/min, and % B to 100.

Note You do not need to reequilibrate the columns at the end of the gradient. The parameters for reequilibration are entered directly and separately as described in the next procedure. The reequilibration runs parallel with the sample pickup at the beginning of a sample execution run.

To continue setting up the method, go to the Edit 4/5 page by pressing the right arrow.

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Creating a Method

Setting Up the Column Equilibration Steps

Use the Method Editor > Edit 4/5 page to set up the parameters for the column

reequilibrations (see Figure 64 ).

Figure 64. Edit 4/5 page of the Method Editor wizard

Y

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 reequilibration of the precolumn.

For best results, use 10 column volumes for reequilibration.

2. Press the Flow box to set the flow rate for performing the reequilibration 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 1000 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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Creating a Method

Y

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 10 column volumes for reequilibration.

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 1000 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 5/5 page by pressing the right arrow.

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Creating a Method

Setting Up the Autosampler Wash Step

Use the Method Editor > Edit 5/5 page for setting up the autosampler washing procedures

(see

Figure 65 ). 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 topics:

Autosampler Wash

Custom Wash

Autosampler Wash

For most samples in low concentrations, flushing the needle and loop with solvent 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 The minimum flush volume that you can set is the

loop volume + needle volume + 1 μL.

Figure 65. Edit 5/5 page of the Method Editor wizard

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Custom Wash

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Running Samples Using the Integrated Software

Creating a Method

Y

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.

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 18

). The order column lists the order in which the autosampler performs the steps. To change the order, use the Up and Down buttons.

Table 18. 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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Creating a Method

Y

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:

• Press Add Line to add a row to the program.

• Press Delete Line to remove a row from the program.

• Press Copy Line to copy a selected line.

• Press Up or Down to move a selected row up or down, respectively.

When you finish setting up the method parameters, save the method as described in the next procedure,

“Saving the Method Settings.”

Saving the Method Settings

After you enter the method settings on page 2/5 to 5/5 of the Method Editor wizard, save the method.

Y

To save the method

Press Save in the top-right corner of the Method Editor wizard.

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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 system. (See also “Preparing a Sample Plate” on page 91 and

“Creating a Method” on page 92

.)

To create a batch, follow these procedures:

Viewing the Batch List and Setting Up a Batch Record

Specifying the Method, Sample Positions, and Number of Injections Per Sample

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 system, and create an empty batch record (name a new batch and select its folder location).

Y

To view a list of batches

1. Press Batch Setup > File.

The Batch Setup > File page opens (see Figure 66

).

Figure 66. File page of the Batch Setup menu

Thermo Scientific

2. To view your batches, in the Path column, press the folder where you store your batches.

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Creating a Batch

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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 procedure, “Specifying the Method, Sample Positions, and

Number of Injections Per Sample.”

Specifying the Method, Sample Positions, and Number of Injections Per Sample

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.

Y

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 67

shows the plate view for the

6 × 8 vial plate format.

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Creating a Batch

Figure 67. View Racks view on the Edit page of the Batch Setup menu

Methods list

Thermo Scientific

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.

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.

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Creating a Batch

5. In the Injections box, enter the number of injections per sample.

The default value is 1 injection per sample.

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 92 .

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 system 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).

• To select the well positions on a 384-well plate, use the quadrant selection box to move between quadrants (see

Figure 68

).

From left to right and top to bottom, the four quadrants are A1 to H12, A13 to H24,

I1 to P12, I13 to P24.

Figure 68. View Rack view for a 384-well plate

Batch name display

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Plate button

Quadrant selection box

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Creating a Batch

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.

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.

10. Press Schedule (see Figure 67 on page 105 ) to submit the batch to the queue.

The application switches to the Queue page of the Home menu (see Figure 69 ).

Figure 69. Queue page of the Home menu, showing the queued jobs

Thermo Scientific

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 69 , the admin-20100824-1714 job does not auto-continue when the 6 × BSA

30min job is done.

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Starting Sample Acquisition

Starting Sample Acquisition

Before starting the sample acquisition, make sure that the system is properly set up. This includes having contact closure (see

“Connecting to the Mass Spectrometer through Contact

Closure” on page 39 ). Also make sure that you have set up the mass spectrometer or any other

detector to acquire data during the sample run.

Before starting a batch run, consider running the maintenance script described in “Test – MS

Connection” on page 169 .

Y

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 70

).

Figure 70. Start Queue message box

4. Press Accept.

The instrument starts processing the batch.

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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

Using the Graphs Page in the Home Menu

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 71 ).

Figure 71. Overview page of the Home menu

Pump A status

Valve A position

Flow sensor A readback

Current sample and status

Current step in the sample run

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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

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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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 72 ).

Figure 72. Graphs page of the Home menu

Thermo Scientific

Table 19

describes the different types of graphs that are available. To change graphs, select from their corresponding lists.

Table 19. 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 pumps

(full red line), plus the actual flow calculated on the feedback values from the pumps.

Shows the measured temperature on the plate cooler over time.

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Monitoring the Run

You can change the graph size as follows:

• Touch the graphs to enlarge them and then touch them again to minimize the view.

Figure 73 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 73. Pump B graph enlarged

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Stopping Sample Acquisition

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 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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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.

Y

To edit the running batch

1. Press Home > Queue.

The Queue page of the Home menu opens (see Figure 74

).

Figure 74. Queue page of the Home menu

2. Select the batch job from the list and press Edit.

The Batch Setup > Edit page opens (see Figure 75 ).

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Figure 75. Edit page of the Batch Setup menu

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Editing the Running Batch

Thermo Scientific

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 74 on page 114

).

c.

Press Show Samples.

The Job Properties box opens (see Figure 76

) and the Show Batch button appears in place of the Show Samples button.

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Editing the Running Batch

Figure 76. Sample list job properties on the Queue page d. Click Close.

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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.

In this situation, the safest approach is to press Cancel Batch and then go to the Scripts page of the Maintenance menu (see

“Executing Maintenance Scripts” on page 78 ). Here you can

try to localize and fix the error by using the maintenance scripts (see “Maintenance Scripts” on page 161

).

After you fix the problem, reschedule the batch (possibly changing the number of samples, their well positions, or both).

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Calibrating the Autosampler’s XYZ Robot

To calibrate the autosampler’s XYZ robot, follow the instructions in this chapter.

The calibration covers the following areas of the autosampler.

Microtiter plate or vial plate

Extra vials

Thermo Scientific

Waste bottle with needle wash insert

Wash solvent bottles

Calibrate the autosampler in the following situations:

• When you want to use a new type of vial or microwell plate.

• When you would like to replace the needle.

• For problems with the sample pickup: The needle is not placed in the center of the well, or not all of the liquid is picked up from one or more of the vials.

• For problems with the wash/waste containers: The needle is not placed in the center of the bottle.

Contents

Replacing the Autosampler Adapter Plate

Managing Plate Formats

Preparing the Autosampler for the Calibration Routines

Calibrating Plates

Calibrating the Wash Bottle and Extra Vial Positions

Checking Sample Pickup

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Replacing the Autosampler Adapter Plate

Replacing the Autosampler Adapter Plate

The EASY-nLC system ships with the vial adapter plate installed. The microtiter adapter plate comes in the accessory kit. To load microtiter (microwell) plates into the tray compartment, you must replace the vial adapter plate with the microtiter adapter plate.

The vial adapter plate holds 48 vials + 6 extra vials. The microtiter adapter plate holds one

96-well plate or one 384-well plate.

Follow this procedure to switch between the two adapter plates as necessary.

Y

To replace the adapter plate on the autosampler

1. Press Home > Overview.

The Overview page of the Home menu opens.

2. Make sure that the area in front of the tray compartment is clear, and then press

Eject Plate.

The tray compartment opens.

Figure 77

shows the vial adapter plate mounted to the tray holder. The four studs on the bottom side of the plate fit snugly into the mounting holes on the tray holder.

Figure 77. Open tray compartment

3. Remove the current adapter plate by pulling it up and away from the tray holder

(see

Figure 78 ).

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Managing Plate Formats

Figure 78. Removing the vial adapter plate before installing the microwell plate adapter plate

Mounting stud

4. Install the other adapter plate in the autosampler.

Managing Plate Formats

Follow the appropriate procedure to use an existing plate format, create your own plate format, or delete a plate format you no longer need.

Y

To create a new plate format

1. Open the Tools view for the autosampler as follows: a. Press Maintenance > Devices. b. In the Devices list, select the autosampler device.

c.

Press the Tools tab.

Figure 79 shows the Tools view for the autosampler.

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Managing Plate Formats

Figure 79. Tools view for the autosampler

Copy To button

Calibrate button

Selected plate format

2. Do one of the following:

• To create a new plate format, go to

step 3

.

• To copy an existing plate format (copies the format selection), go to step 4 .

3. Create a new plate format as follows: a. Press New.

The Create New Plate Format dialog box opens ( Figure 80 ).

Figure 80. Create New Plate Format dialog box

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Calibrating the Autosampler’s XYZ Robot

Managing Plate Formats b. In the Format list, select a plate format: 6 × 8 (vials), 8 × 12 (96-well plates), or

16 × 24 (384-well plates).

c.

In the Name box, enter a name for the new plate format.

d. Press Accept.

The new plate name appears in the plate list with the text [uncalibrated] appended on the right.

e.

Go to “Calibrating Plates” on page 125 to calibrate the vial or well positions.

4. Copy an existing plate format as follows: a. Select an existing plate format from the Plate list. b. Press Copy To.

The Copy Plate Format dialog box opens.

c.

In the Name box, type a name for the new plate format.

d. Press Accept.

e.

Go to “Calibrating Plates” on page 125 to calibrate the vial or well positions.

Y

To select the plate format that you want to use

1. Open the Tools view for the autosampler on the Devices page of the Maintenance menu

(see

step 1 on page 121 ).

2. Select a plate format from the Plate list.

3. Press Use.

Tip If you are using a Thermo Scientific software application to control your liquid chromatograph/mass spectrometer (LC/MS) system, the plate selection for the

EASY-nLC system in your Thermo Scientific instrument control software must match the touch-screen application selection.

Y

To delete a plate format

1. Open the Tools view for the autosampler on the Devices page of the Maintenance menu

(see

step 1 on page 121

).

2. Select a plate format from the Plate list.

Note You cannot delete the plate in use. When you select the plate in use (highlighted in bold text), the Delete button becomes unavailable.

3. Press Delete.

The Delete Plate Data dialog box opens.

4. Press Accept.

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Preparing the Autosampler for the Calibration Routines

Preparing the Autosampler for the Calibration Routines

To prepare the autosampler for the calibration routines, you must remove the left side panel from the instrument and load the appropriate adapter plate, vials or microtiter plate, wash bottle (W4) with insert, and (optional) extra vial into the tray compartment.

CAUTION Calibrating the autosampler requires removal of the side plate and visual inspection of the autosampler needle holder. Because the autosampler compartment contains moving parts and sharp needles, make sure to keep hands clear when operating the autosampler during calibration.

Y

To prepare the autosampler for the plate calibration routine

1. Install the appropriate adapter plate.

2. To calibrate the vial or well positions on a plate, do one of the following:

• To calibrate the 6 × 8 vial positions, remove the vial caps from two vials and load them into the adapter plate in positions A1 and F8.

To calibrate a microwell plate, remove the mat or cover from the microwell plate and load the microwell plate onto the adapter plate.

3. Open the Tools view for the autosampler as follows: a. Press Maintenance > Devices.

b. In the Devices list, select the autosampler.

c.

Press the Tools tab.

4. In the Plate area, do the following: a. Select the appropriate plate format from the list, or create a new plate format

(To create and select plate formats, see “Managing Plate Formats” on page 121

).

b. Press Use.

5. Remove the left side panel so that you can watch the movement of the needle.

To calibrate the plate, go to the next topic “Calibrating Plates.”

Y

To prepare the autosampler for the V1 and W4 calibration routine

1. Place an empty vial in position V1.

The V1 position is not available for the microtiter plate formates.

2. Prepare the bottle for position W4 (used for ejecting waste and cleaning the outside of the injection needle). See

“Autosampler Wash Bottles and Waste Beaker” on page 76

.

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Calibrating Plates

3. Install the wash bottle in position W4.

IMPORTANT For the autosampler to determine the appropriate depth for the needle, the wash bottle in position W4 must contain the needle wash insert.

4. Remove the left side panel so that you can watch the movement of the needle.

To calibrate the wash bottle and extra vial positions, go to “Calibrating the Wash Bottle and

Extra Vial Positions” on page 129 .

Calibrating Plates

For information about selecting or creating a plate format, see “Managing Plate Formats” on page 121 .

IMPORTANT When the calibrating the XYZ robot for vial or microwell plates, make sure to remove the vial caps or plate covers. Keeping the caps or covers on makes the needle susceptible to bending.

Calibrating plates requires determining the xyz positions for the top left and bottom right vial

or well. Table 20

lists the top left and bottom right positions for the three plate formats.

Table 20. Top left and bottom right positions on the plate formats

Plate format

6 × 8

8 × 12

16 × 24

Top left position

A1

A1

A1

Bottom right position

F8

H12

P24

Y

To calibrate plates

1. If you have not already done so, prepare the autosampler for the plate calibration routine

(see

“To prepare the autosampler for the plate calibration routine” on page 124

).

2. Open the Autosampler Plate Calibration dialog box as follows: a. Press Maintenance > Devices.

b. In the Devices list, select the autosampler.

c.

Press the Tools tab.

d. In the Plate area, press Calibrate (see Figure 79 on page 122

).

The Autosampler Plate Calibration dialog box opens.

Figure 81 shows the dialog box at the beginning of the plate calibration routine. The

top left and bottom right positions are highlighted in green. The position readbacks for the xyz axes are unpopulated, .

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Calibrating Plates

Figure 81. Plate view in the Autosampler Plate Calibration dialog box

(3)

(1)

(5)

(2)

(4)

3. Press the green vial or well in the upper left corner of the plate view (1).

The needle moves to the current saved coordinate. The saved coordinates for the x and

y axis appear on the left side of the slash, the needle does not descend into the vial or well, and the z-axis value on the left side of the slash remains at 0.

The values on the left side of the slash change as you adjust the needle position.

4. Center the needle above the vial or well as follows:

• Press the Needle In/Out (3) buttons to adjust the x-axis position.

• Press the Needle Left/Right (4) buttons to adjust the y-axis position.

y axis

1

In

A

x axis

Out

Right

Left

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Calibrating Plates

5. Using the step buttons in the Needle Up/Down area (5), adjust the needle height as follows: a. Using the big step button (↓↓), lower the needle until it reaches a level of 2–3 mm

(0.08–0.12 in.) above the vial or well. b. Using the small step button (↓), slowly lower the needle until it reaches the bottom of the vial or well.

Note The O-ring at the top of the needle holder rises a little when the needle

reaches the bottom of the vial or well (see Figure 82

).

Figure 82. O-ring pushed slightly above the needle holder

Observe when the O-ring moves up a bit, or gently hold needle tubing as you press the step button. c.

Press the small step button (↑) once to raise the needle slightly off the bottom of the vial or well.

The appropriate needle height is one small step higher than the bottom of the vial or well.

6. When you are satisfied with the current xyz-coordinate for the vial or well, press Capture.

The color of the vial or well on the plate graphic changes from green to red and the position readbacks for the xyz axes are unpopulated,

(see Figure 83

).

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Calibrating Plates

Figure 83. Plate calibration captured for the A1 position

7. Press the green vial or well in the lower right corner of the plate view.

The needle moves to the current saved coordinate. The saved coordinates for the x and

y axis appear on the left side of the slash, the needle does not descend into the vial or well, and the z-axis value on the left side of the slash remains at 0.

8. Repeat

step 4

through

step 6

on that vial or well.

9. Press Save and then press Close to close the calibration dialog box.

10. Replace vial caps and plate covers as necessary.

11. Go to the next procedure to calibrate the W4 bottle position or reinstall the EASY-nLC instrument’s left side panel.

IMPORTANT To maintain a stable temperature inside the tray compartment, the left side panel must be installed.

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Calibrating the Wash Bottle and Extra Vial Positions

Calibrating the Wash Bottle and Extra Vial Positions

In addition to holding the sample trays, the EASY-nLC tray compartment holds four wash bottles (W1–W4) and six extra vials (V1–V6).

Y

To calibrate the W4 wash bottle position, the extra vial positions, or both

1. If you have not already done so, prepare the autosampler for the extra vial and wash bottle calibration as described in

“To prepare the autosampler for the V1 and W4 calibration routine” on page 124

.

2. Open the Autosampler Vials/Wash Calibration dialog box as follows: a. Press Maintenance > Devices.

b. In the Devices list, select the autosampler.

c.

Press the Tools tab below the Devices list.

d. In the Vials/Wash area, press Calibrate.

The Autosampler Vials/Wash Calibration dialog box opens (see Figure 84

).

3. Press the bottle container in W4 (1) (see

Figure 84

).

The needle moves to the current saved coordinate. The saved coordinates for the x and

y axis appear on the left side of the slash, the needle does not descend into the bottle, and the z-axis value on the left side of the slash remains at 0

Figure 84. Vials/Wash view in the Autosampler Vials/Wash Calibration dialog box

(2)

(5)

(4)

(1)

(3)

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Calibrating the Wash Bottle and Extra Vial Positions

4. Center the needle above the bottle as follows:

• Press the Needle In/Out (2) buttons to adjust the x-axis position.

• Press the Needle Left/Right (3) buttons to adjust the y-axis position.

y axis

In

x axis

Out

Right

Left

5. Using the step buttons in the Needle Up/Down area (5), adjust the needle height as follows: a. Using the big step button (↓↓), lower the needle until it reaches a level of 2–3 mm

(0.08–0.12 in.) above the bottle. b. Using the small step button (↓), slowly let the needle enter the hole and lower the needle until it reaches the bottom of the wash insert in the bottle.

Note The O-ring at the top of the needle holder rises a little when the needle reaches the bottom of the well.

c.

Press the large step button (↑↑) twice to raise the needle off the bottom of the wash bottle insert.

Two large steps higher than the bottom of the wash bottle insert is the appropriate needle height.

6. Press Capture when you are satisfied with the current xyz-coordinate for position W4.

The color of the bottle container number changes from green to red.

7. (Optional) To calibrate the extra vial positions for the 6 × 8 plate format, do the following: a. Press V1 vial (5) to select it.

b. Repeat

step 4

through

step 6

for vial V1.

8. Press Save and then press Close to close the calibration dialog box.

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Calibrating the Autosampler’s XYZ Robot

Checking Sample Pickup

Checking Sample Pickup

Y

To check sample pickup from a microtiter plate

1. Identify the positions of the wells where you have problems.

2. Fill the wells with a 12 μL of solvent A.

3. Put a plastic film on the Microtiter plate.

4. Insert the tray into the tray compartment.

5. Press Maintenance > Scripts.

6. Run the Sample Pickup script with a pickup volume of 10 μL and your default flow

(see

Figure 85 ). Run the script for each well you pointed out in step 1.

Figure 85. Sample Pickup script under the Maintenance menu tab

Thermo Scientific

7. After the EASY-nLC application finishes the script, do the following: a. Eject the tray.

b. Check all wells one by one with a pipette.

c.

Check that there are 2 μL left in all wells.

You are finished when there are 2 μL left in all wells.

d. If there are more than 2 μL left in some or all of the wells, go to

step 8

.

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Calibrating the Autosampler’s XYZ Robot

Checking Sample Pickup

8. Depending on the volume remaining in the wells, do one of the following:

• If there are more than 2 μL left in all wells, calibrate the autosampler again, and then rerun the sample pickup check.

An excess volume in all wells indicates a problem with the autosampler calibration or the alignment of the autosampler tray/adapter plate.

• If there are more than 2 μL left in some of the wells, run the Flush Air script on

pump S (see Figure 86 ), and then rerun the sample pickup check.

An inconsistent sample pickup from each well indicates that there might be air in the system.

Figure 86. Flush Air script under the Maintenance menu tab

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Troubleshooting

To troubleshoot problems with the EASY-nLC system, follow the troubleshooting tips in this chapter. To use the manual controls accessed from the Home > Overview page, follow the

procedures in “Manual Controls” on page 141 .

Contents

System Startup

Batch Runs

Autosampler

Contact Closure

Network Access

System Component States (Valves, Pumps, Autosampler, and Plate

Cooler)

Miscellaneous

Manual Controls

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Troubleshooting

System Startup

System Startup

Table 21

contains troubleshooting tips for system startup problems.

Table 21. Startup troubleshooting tips

Symptom

Dark monitor

Startup bar is no longer proceeding.

Possible causes

Broken or open main power fuse

Defective monitor

Damaged system

Action

1. Turn on the power to the instrument.

2. Remove the right side panel of the

EASY-nLC instrument.

3. If there is no light on the pumps, turn off the

EASY-nLC instrument, replace the right side panel, and check the fuse.

Attach an external monitor to the VGA connector (labeled MONITOR) on the back panel of the instrument (see

Figure 6 on page 7 ).

Switch off the EASY-nLC instrument and wait five seconds before switching it on again. In some cases this recovers the system. Contact Thermo

Fisher Scientific if the problem recurs: [email protected]

[email protected]

Batch Runs

For troubleshooting tips when running a batch, see Table 22

.

Table 22. Running a batch troubleshooting tips (Sheet 1 of 2)

Symptom

Gradient does not start.

Possible causes

Waiting for contact closure

Action

Check the contact closure connections.

Maximum pressure is exceeded during sample loading or reequilibration, or pressure is maintained but the flow is dramatically decreased.

Possible blocked column

Max pressure is exceeded during gradient.

Back pressure that is too high on the column for the specified flow

Blocked flow paths

1. Remove all columns from the system.

2. Run the maintenance script:

“Test – Back Pressure” on page 175

.

Reduce the flow in the method and schedule a new batch with the changed method.

Contact Thermo Fisher Scientific: [email protected]

[email protected]

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Troubleshooting

Batch Runs

Table 22. Running a batch troubleshooting tips (Sheet 2 of 2)

Symptom Possible causes Action

It takes an extremely long time for the flow rate to stabilize.

XYZ robot error

Time-out on flow sensor

Sample is not being picked up.

Air in the system

Leakage in the system

Possible step loss on one of the motors because of a blockage in the movement of the axis

Remove the obstacle and try again. You might need to calibrate the autosampler for more precise penetration of the microtiter plate or wash/waste containers.

Repetition of the error without any visible reason

Purge air from the system as described in

“Purging and Flushing the Pumps” on page 79

.

Run the maintenance script:

“Test – Leaks” on page 171

Air in the pumps

Air in the pumps

Contact Thermo Fisher Scientific: [email protected]

[email protected]

Run the maintenance script:

“Prepare – Flush Air” on page 163

Run the maintenance script:

“Prepare – Flush Air” on page 163

Valve – unknown position Valve trimming needed

Run the maintenance script:

Sample Pickup. Verify that the autosampler removes sample from the vial.

Run the maintenance script:

“Test – Valve Check” on page 174

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Troubleshooting

Autosampler

Autosampler

See

Table 23

for tips on handling autosampler issues. To access the Autosampler control

window ( Figure 87 ), press Home > Overview, and then press the autosampler (plate) icon.

Figure 87. Autosampler control window

Table 23. Autosampler troubleshooting tips (Sheet 1 of 2)

Symptom

Sample pickup fails.

Possible causes

Incorrect autosampler calibration or air in the sample (S) pump

Action

1. Fill some of the wells with a given volume of solvent A, for example, 12 μL.

2. Put a plastic film on the microtiter plate.

3. Run the maintenance script:

“Test – Sample Pickup” on page 170 , with a

pickup volume of 10 μL and your default flow. Run the script for each well.

4. Eject the tray and check the wells one by one with a pipette.

5. Run the maintenance script

“Prepare – Flush

Air” on page 163 , or calibrate the

autosampler if the problem still appears.

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Troubleshooting

Contact Closure

Table 23. Autosampler troubleshooting tips (Sheet 2 of 2)

Symptom Possible causes

The needle cannot penetrate the plastic film on the microtiter plate.

Damaged or bent needle tip

Action

Do the following:

1. Eject the tray.

2. Remove the left side panel visually inspect the needle tip. The needle tip should not be bent or damaged in any way.

Cannot run a batch. A server warning message about the plate format for the autosampler configuration pops up when you run the batch.

Plate format deleted from your configuration with the Delete button from Maintenance >

Devices > Autosampler > Tools

3. If there is any residue on the needle tip, use tissue with some ethanol to wipe it.

If you want to run the batch, you must create the plate format again.

Contact Closure

Table 24

contains troubleshooting tips for contact closure problems.

Table 24. Contact closure troubleshooting tips

Symptom

The mass spectrometer is waiting for a contact closure signal.

Possible causes Action

Incorrect contact closure setup If you are running two-way contact closure (with feedback from the mass spectrometer), set the contact closure Protocol setting to Two-way on the Configuration > Connections page.

-or-

The EASY-nLC system is waiting for a ready signal from the mass spectrometer.

If you are running one-way contact closure (that is, the EASY-nLC system sends a start signal to the mass spectrometer, but feedback from the mass spectrometer is ignored), set the contact closure Protocol setting to One-way on the

Configuration > Connections page.

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Troubleshooting

Network Access

Network Access

Table 25

contains troubleshooting tips for network access problems.

For information about setting up the network connection, see “Setting Up the Ethernet

Connection to a Local Network for Data Exchange” on page 42 and

“Setting Up the Local

Area Network Connection” on page 61 .

Table 25. Network access problems

Symptom

Cannot access the EASY-nLC system through the network.

Possible causes

Unsaved network information on the Configuration >

Network page

No physical connection to the local area network

Broken Ethernet cable

Incorrect address in Windows

Explorer

Incorrect network information for the EASY-nLC system on the Configuration > Network page

Action

Press Configuration > Network. Check the network settings, and then press Save

Configuration.

Check the Ethernet cable connections (see

“Setting Up the Ethernet Connection to a Local

Network for Data Exchange” on page 42

).

Replace the Ethernet cable.

Verify the IP address of the EASY-nLC system, and then retype file:\\IP address in the Windows

Explorer address list.

Press Configuration > Network. Check the network settings, and then press Save

Configuration.

System Component States (Valves, Pumps, Autosampler, and Plate

Cooler)

Table 26

contains troubleshooting tips for the system component states.

Table 26. LED state troubleshooting tip

Symptom

The LED on the device is blinking red.

Possible causes

Device in bootloader mode.

Action

Contact Thermo Fisher Scientific: us.cust[email protected]

The device stopped working.

This could also block the function of other devices.

[email protected]

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Troubleshooting

Miscellaneous

Miscellaneous

For additional issues that might arise while using the EASY-nLC system, consult these miscellaneous troubleshooting tips (

Table 27 ).

Table 27. Miscellaneous troubleshooting tips (Sheet 1 of 2)

Symptom

Copying log files to a USB memory stick failed.

Possible causes

Memory stick in wrong format

Action

Format the memory stick in FAT/FAT16 format.

Memory stick not recognized by the system

Use one of the memory sticks supplied by Thermo

Fisher Scientific, or try plugging the stick into the other USB port.

Contact Thermo Fisher Scientific if neither action works: [email protected]

[email protected]

Copying to the USB memory stick does not stop.

Too much data to be copied Remove the memory stick from the EASY-nLC system and press Ignore when the error message appears.

Contact Thermo Fisher Scientific for a system check-up: [email protected]

Forgot the admin password.

[email protected]

If you are already logged in to the EASY-nLC system,

Thermo Fisher Scientific can extract the password from the system in two ways:

• If the network is set up for remote access, press

Maintenance > Support. Enter your contact information and a message requesting your password information. Then press Connect.

–or–

• Run a factory reset (see

“To reset one or all of the settings to the factory values” on page 69

)

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Troubleshooting

Miscellaneous

Table 27. Miscellaneous troubleshooting tips (Sheet 2 of 2)

Symptom Possible causes Action

System is running slower and slower.

Memory filled with graph data from long gradient run

Unhandled error or SVG

(scalable vector graphics) error

System overload caused by other reasons

Program error in the software release

Press Home > Graph and disable the graphs by pressing No Graph in each graph window.

Restart the application or power down/power up from

Maintenance > Scheduling.

In most situations you can continue your work by pressing Ignore or OK. More serious error situations might require restarting the HPLC.

To request a software change, do one of the following:

• Send your request to one of these e-mail addresses:

[email protected]

[email protected]

–or–

• Connect to the remote support server and send a message to Thermo Fisher Scientific Technical

Support as described in “Connecting the

EASY-nLC System to the Support Server” on page 150

.

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Troubleshooting

Manual Controls

Manual Controls

You can access the manual controls for the instrument’s hardware components by pressing the component icons on the Home > Overview page.

These topics describe how to use the manual controls:

Using the Valve Controls

Using the Pump Controls

Using the Autosampler Controls

Using the Valve Controls

You can manually change the positions of the instrument’s three-position valves from the

Home > Overview page. For more information about the valve positions, see

“Six-Port Valves” on page 12

.

Y

To change the position of a valve

1. Press the valve icon for the valve that you want to control.

The Valve dialog box opens (see

Figure 88 ). The readback box lists the current valve

position.

Figure 88. Valve dialog box

2. Press the button for the position that you want the valve to switch to: 1–2, Center, or 1–6.

The readback box displays the new position.

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Troubleshooting

Manual Controls

Using the Pump Controls

You can manually refill and empty the pumps from the Home > Overview page.

IMPORTANT Use the pump’s manual controls for troubleshooting.

To avoid contaminating the pumps, take care when you use these controls:

• For pump A or B, make sure that its corresponding valve is in position 1–2 before you refill the pump.

• For pump S, before you empty or fill the pump, use the autosampler’s manual controls to insert the autosampler needle into the W4 or W3 bottle, respectively.

Tip To prepare the EASY-nLC instrument for operation, use the Prepare – Purge Solvent

script to flush the solvent lines (see “Prepare – Purge Solvent” on page 162

).

As it empties and refills the selected pump or pumps, the Prepare – Purge Solvent script automatically places the valves and autosampler needle in the appropriate positions.

To work with the pump’s manual controls, follow these procedures:

Opening the Manual Controls for the Pumps

Using the Manual Controls to Fill or Empty Pump A or B

Using the Manual Controls to Fill or Empty Pump S

Opening the Manual Controls for the Pumps

Each pump is represented by a pump icon on the Home > Overview page.

Y

To open the Pump dialog box

Press the pump icon for the pump that you want to control.

Its dialog box opens (see

Figure 89

). The Position readback displays the current pump position from 0 μL (empty) to 140 μL (full).

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Troubleshooting

Manual Controls

Figure 89. Pump dialog box

Volume box

Flow rate box

Readbacks

Note Because pump S does not have an associated flow sensor, the Pump S dialog box does not include a Real Flow readback.

Using the Manual Controls to Fill or Empty Pump A or B

Before using the manual controls to fill or empty pump A or B, make sure that the valves are in the appropriate position. For information about controlling the position of the valves, see

“Using the Valve Controls” on page 141 .

IMPORTANT Before you fill pump A or B, make sure that the pump’s associated valve is in position 1–2. You can contaminate the pump by attempting to fill the pump when its associated valve is in position 1–6.

Y

To fill pump A or B with solvent

1. Check the position of the pump valve on the Home > Overview page.

2. Make sure that the corresponding valve is in position 1–2.

When valve A or B is in the 1–2 position, the corresponding pump draws solvent from the solvent bottle on top of the instrument (see

Figure 12 on page 14

).

3. Press the pump icon for the pump you want to control.

The Pump dialog box (see Figure 89

) opens.

4. In the flow rate box, enter the filling flow rate: –0.01 to –300 μL/min.

5. In the volume box, enter the volume that you want pump A or B to draw.

6. Press Start.

The pump draws solvent until it draws the requested volume or reaches the full position, whichever occurs first.

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Troubleshooting

Manual Controls

Y

To empty pump A or B to waste

1. Check the position of the pump valve on the Home > Overview page.

2. Do one of the following:

• To empty pump A or B directly to waste, make sure that its valve is in position 1–2.

• To empty pump A or B to waste by way of valve W, place the pump valve in position 1–6 and valve W in position 1–6.

Valve

A or B

S

W

Position

1–2

1–6

1–2

1–6

1–2

1–6

Solvent flow

Empties to the waste beaker.

Passes through valve S and into the Column Out line.

Bypasses the sample loop.

Passes through the sample loop.

Passes through the installed columns.

Exits the Column Out line to waste for a one-column setup, or passes through the precolumn and then exits the system to waste for a two-column setup.

3. Press the pump icon for the pump you want to control.

The Pump dialog box (see Figure 89 on page 143 ) opens.

4. In the flow rate box, enter the flow rate: 0.01 to 300 μL/min.

When valve A or B is in position 1–6, the maximum system pressure limit constrains the maximum flow rate.

5. In the volume box, enter the volume that you want the pump to empty to waste.

6. Press Start.

The pump empties until it pumps the requested volume or reaches the empty position, whichever occurs first.

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Troubleshooting

Manual Controls

Using the Manual Controls to Fill or Empty Pump S

Before using the manual controls to fill or empty pump S, make sure that the autosampler needle is inserted into the appropriate wash or waste bottle.

IMPORTANT To avoid contaminating pump S, take care to place the autosampler needle in the appropriate wash bottle before filling the pump.

Y

To fill pump S

1. Make sure that a wash bottle with the appropriate solvent is installed in position W3.

2. Send the autosampler needle to the appropriate position as follows: a. On the Home > Overview page, press the autosampler icon.

The Autosampler dialog box opens (see

Figure 90 on page 146

).

b. Press W3.

The autosampler needle moves to the W3 position.

c.

Press Needle Down.

The autosampler needle descends into the W3 bottle.

d. Press Close to close the Autosampler dialog box.

3. Draw solvent into pump S as follows: a. Press the pump S icon.

The Pump S dialog box opens. b. In the flow rate box, enter the filling flow rate: –0.01 to –300 μL/min. c.

In the volume box, enter the volume that you want pump S to draw.

d. Press Start.

The pump draws solvent until it draws the requested volume or reaches the full position, whichever occurs first.

Y

To empty pump S to waste

1. Make sure that a wash bottle with needle wash insert is installed in position W4.

2. Send the autosampler needle to the appropriate position as follows: a. On the Home > Overview page, press the autosampler icon.

The Autosampler dialog box opens (see

Figure 90 on page 146

).

b. Press W4.

The autosampler needle moves to the W4 position.

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Troubleshooting

Manual Controls c.

Press Needle Down.

The autosampler needle descends into the W4 bottle.

d. Press Close to close the Autosampler dialog box.

3. Dispense solvent from pump S as follows: a. Press the pump S icon.

The Pump S dialog box opens. b. In the flow rate box, enter the flow rate: 0.01 to 300 μL/min. c.

In the volume box, enter the volume that you want the pump to empty to waste. d. Press Start.

The pump empties until it pumps the requested volume or reaches the empty position, whichever occurs first.

Using the Autosampler Controls

You can manually control the XYZ robot and the cooler temperature from the Home >

Overview page.

Y

To open the Autosampler dialog box

Press the autosampler icon on the Home > Overview page.

The Autosampler dialog box opens (see

Figure 90 ).

Figure 90. Autosampler dialog box

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Vial or well position box

Temperature input box

Temperature readback

Thermo Scientific

8

Troubleshooting

Manual Controls

Y

To calibrate the current plate format

1. Press Goto Calibration.

The Tools view for the autosampler on the Maintenance > Devices page opens.

2. To calibrate the XYZ robot for the plate format, follow the instructions in “Calibrating

Plates” on page 125

.

Y

To move the needle to a vial or well position

1. In the Vial or Well box, enter a vial or well position.

2. Press Goto.

Y

To move the needle to a wash bottle position

Press W1, W2, W3, or W4.

Y

To change the cooler temperature

1. Enter a temperature in the temperature box.

2. Press Set.

3. Monitor the temperature change in the temperature readback box.

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9

Remote Support

The EASY-nLC system includes a remote support feature that enables Thermo Fisher

Scientific Technical Support to diagnose and troubleshoot your EASY-nLC system remotely while the system is connected to the support server.

Technical Support cannot initiate the communication link between your EASY-nLC system and the support server. You must initiate this communication link from your EASY-nLC system.

To connect the EASY-nLC system to the support server or download system files to a removable storage device, follow these procedures.

Contents

Connecting the EASY-nLC System to the Support Server

Saving System Files on a USB Removable Storage Device

IMPORTANT To establish communication with the support server, you must first connect the EASY-nLC system directly to your local network, and then open the communication link for remote support through the touch-screen software. Thermo

Fisher Scientific Technical Support cannot initiate this communication link.

The EASY-nLC system uses Secure Shell (SSH), a network protocol for secure data communication, to connect to the support server, which uses the Linux operating system.

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

IP address 195.41.108.93 port 22.

When you establish the communication link with the support server, the support server triggers the file compression utility on the EASY-nLC system to build a zip file with the appropriate log files, and then retrieves the zip file from the EASY-nLC system.

Meanwhile, the EASY-nLC system sends an e-mail with your supplied message and contact information to Thermo Fisher Scientific Technical Support.

After you send a message through the remote support feature, a member of the Thermo

Fisher Scientific Technical Support team will contact you.

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Remote Support

Connecting the EASY-nLC System to the Support Server

Connecting the EASY-nLC System to the Support Server

Use this procedure to establish a communication link between the EASY-nLC system and the support server.

Y

To open a communication link between the EASY-nLC system and the support server

1. If you have not already set up the support server configuration, set it up as follows: a. Press Configuration > Network.

b. In the Support Server area (see Figure 37 on page 62 ), enter the following IP address:

195.41.108.93.

c.

Press Save Configuration.

2. Using a shielded Ethernet cable, connect the LAN port on the back panel of the

EASY-nLC system (see

Figure 91 ) directly to your local network, as described in

“Setting

Up the Ethernet Connection to the Support Server” on page 43 .

The local network must have access to the Internet and allow outgoing traffic to port 22.

Figure 91. Ethernet connection between the EASY-nLC system and the laboratory LAN port

PRO EON

P-BUS

IN3 OUT3 IN2 OUT2 IN1 OUT1

PS/2 LAN

HARD

DRIVE

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.

This instrument is for research use only.

DEVICE: EASY-NLC

P/N LC100

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

Proxeon Biosystems A/S

Web: www.proxeon.com

Phone +45 6557 2300 Fax +45 6557 2301

MONITOR

I

O

110-120V▼

220-240▼

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

Fuse at 120 V : T5AL 250 V

Fuse at 230 V : T2.5 AL 250 V

LAN port with

Internet capability

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Remote Support

Connecting the EASY-nLC System to the Support Server

3. Send a support request message and a zip file containing the appropriate log files to

Technical Support as follows: a. Press Maintenance > Support.

The Support page of the Maintenance menu appears (see

Figure 92 ).

Figure 92. Support page of the Maintenance menu

Thermo Scientific b. In the Connect to EASY-nLC Support area, press Connect.

The Message to Support Team dialog box appears (see Figure 93 ).

Figure 93. Message to Support Team dialog box

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Remote Support

Connecting the EASY-nLC System to the Support Server c.

Select a support region, provide valid contact information, and describe the problem that you are experiencing.

Note When you place the cursor in the Phone Number box, the program prompts you for a country code and phone number.

Tip When you place the cursor in the Email Address box, the program prompts you to enter an address in this format: name@company.com.

To enter the @ symbol, press Shift. The numbers row changes to the symbols row. Then press @.

d. Press Accept.

After you enter the requested information, the Status box on the right side of the

Support page should change from Not Connected to Connected. e.

Depending on the connection status, do one of the following:

• If the status changes from Not Connected to Connected, go to step 3f .

• If the connection fails, see “To troubleshoot a failed connection to the support server.”

When the connection fails, the SSH Connection Error dialog box might appear with this message, “Not able to determine connection status. See message.log for details.”

The message.log file is in the admin\systemLog folder on the EASY-nLC system. For information about accessing the admin folder from another

computer on your local network, see “To access the admin folder on the

EASY-nLC system’s computer hard drive from another computer on the network” on page 63 .

While this file might provide useful information to a member of Thermo

Scientific Technical Support, users should follow the suggested troubleshooting procedure,

“To troubleshoot a failed connection to the support server.”

f.

After the status changes to Connected, allow enough time for the EASY-nLC system to create the zip file in the admin folder and for the support server to retrieve the zip

file. Then go to step 4 .

After you send a message with the appropriate contact information through the remote support feature, a member of the Thermo Fisher Scientific Technical Support team will contact you.

4. To close the communication link, press Disconnect.

5. To return the system to normal operation, reconnect the Ethernet cables as appropriate

(see

“Connecting the Ethernet Communication Cables” on page 40 ).

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Remote Support

Saving System Files on a USB Removable Storage Device

Y

To troubleshoot a failed connection to the support server

1. Check the support server configuration (see step 1 on page 150 ).

2. Verify that the Ethernet cable is securely connected to both the LAN port on the back panel of the EASY-nLC system and the laboratory LAN port.

3. Verify that the Ethernet cable is working.

4. Ask your IT administrator if the network firewalls allow outgoing traffic to port 22 through an SSH connection.

Saving System Files on a USB Removable Storage Device

The EASY-nLC system includes network capability. Through the network you can back up your system, export and import batches and methods, analyze your system by examining the log files, and perform many other tasks. However, when you are not connected to the network or the network is broken, or you do not have a computer with network access that is near the

EASY-nLC system, you can use the copy function to copy all the system files on the system to a USB removable storage device, such as a USB flash drive or memory stick.

The EASY-nLC system ships with a USB flash drive for your convenience. But you can use any USB storage device formatted with the file system FAT/FAT16 and at least 128 MB of free space.

Y

To copy system files to a USB storage device

1. Press Maintenance > Support.

The Support page of the Maintenance menu opens (see Figure 92 on page 151 ).

2. Insert the USB storage device into the connector panel on the back of the EASY-nLC instrument. Choose between one of two USB ports on the upper left side of the panel

(see

Figure 94 ).

Figure 94. USB port location on the back panel of the EASY-nLC instrument

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Remote Support

Saving System Files on a USB Removable Storage Device

3. In the Log File Copy area, select the To USB Removable Storage option.

Note When you select the To Home Directory option, the EASY-nLC system creates zip files in the admin folder. For information about accessing the admin folder from

another computer on your local network, see “To access the admin folder on the

EASY-nLC system’s computer hard drive from another computer on the network” on page 63

.

4. Press Copy Log Files.

When the copying is finished, the following confirmation appears.

5. Press Close and remove the USB storage device from the connector panel.

6. Insert the USB storage device into your computer.

Five zipped files appear on the USB storage device (see

Figure 95 ).

Figure 95. Windows XP view of the files on the USB memory stick

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Saving System Files on a USB Removable Storage Device

The files are as follows: batchlog.zip Contains one folder for each batch you have run with information on samples, methods, and EASY-nLC device performance.

etc.zip Contains system configuration files.

export.zip logfiles.zip

Contains exported batches and methods per user and system backup.

Contains system log files with information on overall system performance.

maintlog.zip Contains one folder for each maintenance script you have run with information on EASY-nLC device performance.

If you have problems with your system, forward the files to Thermo Fisher Scientific

Technical Support for assistance.

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Maintenance

To familiarize yourself with the maintenance schedule and maintenance scripts for the

EASY-nLC system, review this chapter. Following the maintenance schedule and using the maintenance scripts will help ensure the longevity of your instrument.

Contents

General Maintenance

Daily Maintenance

Weekly Maintenance

Quarterly Maintenance

Yearly Maintenance

Maintenance Scripts

Keeping Service Records

Checking and Resetting the Device Usage Counters

General Maintenance

Maintain a clean working environment for the EASY-nLC instrument and replace its fuses as necessary.

These topics specify the appropriate cleaning solvents and fuses:

Cleaning Materials

Replacing the Main Power Fuse

Cleaning Materials

When cleaning the outside of the EASY-nLC instrument, use a mild detergent and a clean cloth.

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General Maintenance

Replacing the Main Power Fuse

The EASY-nLC uses these fuses:

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

• For 230 Vac, the instrument uses one T 2.5 AL 250 V fuse.

Use only UL Listed and CSA-certified fuses, All fuses supplied with the system are UL Listed and CSA certified.

CAUTION Before removing the fuses, turn off the instrument and remove the power cable.

Y

To replace the main power fuses

1. Move the EASY-nLC instrument to a benchtop where you can access the back panel.

Figure 96 shows the steps required to remove the fuse holder.

2. Turn off the power and pull out the power plug.

3. Place a flat-blade screwdriver into the slot in the top of the fuse holder, and then twist the screwdriver to loosen the fuse holder.

4. Pull the fuse holder out of the power entry module.

Figure 96. Replacing the main power fuse

Turn off the power and pull the power plug out of the power receptacle.

Using a flat-blade screwdriver, loosen the fuse holder.

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Daily Maintenance

5. Install the appropriate fuse as follows. a. Do one of the following:

• Insert the 5 A fuse into the slot that aligns with the white triangle at the end of the 110–120 V line power text.

• Insert the 2.5 A fuse into the slot that aligns with the white triangle at the end of the 220–240 V text.

b. Make sure that the fuse ends align with the mounting brackets.

Note The fuse holder holds two fuses. One fuse is for a 220–240 V line power and the other is for 110–120 V line power.

6. Insert the fuse holder into the power entry module in the orientation that corresponds to the operating power.

The triangle to the right of the voltage rating points toward the white mark at the bottom of the power entry module (see

Figure 97 ).

Figure 97. Power entry module

Appropriate orientation for

220–240 line voltage

Daily Maintenance

Check the solvent levels on a daily basis.

Y

To check 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 if necessary.

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 if necessary.

3. Run the Purge Solvent and Flush Air scripts with two purge iterations or until flush volumes fall below 12 μL.

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Weekly Maintenance

Weekly Maintenance

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

Quarterly Maintenance

Perform the following procedures quarterly (every three months).

Y

To check the back pressure

Run the Back Pressure script for both solvents (see “Test – Back Pressure” on page 175

).

IMPORTANT The script evaluates the back pressure based on using 99.9% water for solvent A and 99.9% acetonitrile for solvent B.

Y

To check the cooler temperature

Check that the plate temperature is at its set point or approximately 18 to 20 °C

(64 to 68 °F) below ambient temperature (readout on the Home > Overview page).

Y

To check the autosampler pickup function

Run the Sample Pickup script to check the sample pickup function (see

“Test – Sample

Pickup” on page 170 ).

Y

To check the EASY-nLC system for leaks

Run the Leaks script for “A+B” and for “System,” to check for and pinpoint instrument

leaks (see “Test – Leaks” on page 171 ).

IMPORTANT Check the valve rotor shifts regularly. When the valve rotor shifts exceed 6000, Thermo Fisher Scientific recommends checking the system for leaks.

From this point forward, perform leak tests every 1000 shifts and exchange the rotor when the leak test fails.

Y

To check the check valves

Run the Purge Solvent script (see

“Prepare – Purge Solvent” on page 162 ) and do the

following:

• When the pumps are ejecting solvent, make sure that solvent is exiting the A and B waste tubing into the waste beaker.

• When the pumps are refilling, make sure that no solvents are going into the pump through these tubes.

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Maintenance

Yearly Maintenance

Yearly Maintenance

Replace the solvent filters and check the flow sensor calibration once a year.

Y

To exchange low-pressure solvent filters and high-pressure inline filters

Thoroughly clean new solvent filters before use as these can be a contamination source.

Y

To check the flow sensor calibration

Run the Flow Sensor calibration script with the Inspection Only parameter enabled (see

“Calibrate – Flow Sensors” on page 178 ).

This checks that the flow sensor calibration is working.

Maintenance Scripts

Y

To select a maintenance script

1. In the EASY-nLC application, press Maintenance > Scripts.

2. Select a category and then the script for the specific operation you want.

You can also schedule some of the scripts for execution using the job queue.

Each of the following topics combines the category with the actual script name.

Prepare – Purge Solvent

Prepare – Flush Air

Prepare – Precolumn Equilibration

Prepare – Analytical Col Equilibration

Prepare – Isocratic Flow

Test – MS Connection

Test – Sample Pickup

Test – Leaks

Test – Valve Check

Test – Back Pressure

Test – Autosampler Torque

Test – Pump Torque

Calibrate – Valve Tune

Calibrate – Flow Sensors

Calibrate – Reset Pressure Sensor

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Maintenance Scripts

Prepare – Purge Solvent

The Purge Solvent script fills the selected pump or pumps with solvent and then ejects it into the waste container. Use the Purge Solvent script when exchanging solvents, removing air from the solvent lines, or filling the pumps.

Y

To set the Purge Solvent script parameters

1. Press Maintenance > Scripts (see

Figure 98

).

2. In the Category list, select Prepare.

3. In the Name list, select Purge Solvent.

4. Press the Parameters tab.

5. Press Purge Iterations to specify the number of empty/fill cycles to be performed:

• To exchange solvents, use two iterations.

• To remove air, use a minimum of 5 iterations.

6. Select the check boxes for the appropriate pump or pumps.

Note Entering a value of zero (0) for purge iterations will just refill the pumps.

Figure 98. Purge Solvent script parameters

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Maintenance

Maintenance Scripts

Prepare – Flush Air

The Flush Air script pressurizes the pump and then releases that pressure into the flow lines toward the waste container, which is effective for removing air from inside the pump head.

Pressurization time is calculated dynamically while the script is executed and is based on measurements from previous iterations.

The pump pressurizes, releases pressure, and empties for each iteration of the script.

The pressurization is limited to 200 bar and the script measures the amount of pumping required to reach 200 bar. Because the solvents are slightly compressible and the pump itself expands, even an air-free pump allows some pumping before reaching 200 bar. Depending on the solvents, a pumped volume less than 12 μL is acceptable.

Y

To set the Flush Air script parameters

1. Select the Flush Air script as follows: a. Press Maintenance > Scripts. b. In the Category list, select Prepare.

c.

In the Name list, select Flush Air.

2. Set the parameters (see

Figure 99

) for this script as follows: a. Press the Parameters tab.

b. Press Flush Threshold to enter the tolerated threshold for an accepted test result.

Below 12 μL is satisfactory.

The script terminates after the first iteration that gives a flush volume below the set threshold.

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Maintenance

Maintenance Scripts

Figure 99. Flush Air script parameters

Prepare – Precolumn Equilibration

The Precolumn Equilibration script equilibrates the precolumn with solvent from pump A.

Use this script to equilibrate or flush the precolumn and to determine a suitable flow rate for the sample loading step in your method.

Y

To set the Precolumn Equilibration script parameters

1. Select the Precolumn Equilibration script as follows: a. Press Maintenance > Scripts. b. In the Category list, select Prepare. c.

In the Name list, select Precolumn Equilibration.

2. Set the parameters (see

Figure 100 ) for this script as follows:

a. Press the Parameters tab.

b. Press Volume to set how much solvent A to use for reequilibration of the precolumn.

For best results, use 10 column volumes for reequilibration.

c.

Press Flow to set the flow rate for performing the reequilibration of the precolumn. If the flow field is left empty, the pump operates at the set pressure.

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Maintenance

Maintenance Scripts d. Press Max Pressure to set the maximum allowed pressure.

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 1000 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 PepMap columns supplied with the EASY-nLC 1000 system is 800 bar. Running the system at pressures higher than 800 bar reduces the column lifespan.

Figure 100. Precolumn Equilibration script parameters

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Maintenance

Maintenance Scripts

Prepare – Analytical Col Equilibration

The Analytical Col Equilibration script equilibrates the analytical column with solvent from pump A.

Use this script to equilibrate or flush the analytical column, determine a suitable equilibration flow rate for the method, or initialize a new analytical column.

Y

To set the Analytical Col Equilibration script parameters

1. Select the Analytical Col Equilibration script as follows: a. Press Maintenance > Scripts. b. In the Category list, select Prepare. c.

In the Name list, select Analytical Col Equilibration.

2. Set the parameters (see

Figure 101 ) for this script as follows:

a. Press the Parameters tab.

b. Press Volume to set how much solvent A to use for reequilibration of the analytical column. For best results, use 10 column volumes for reequilibration.

c.

Press Flow to set the flow rate for performing the reequilibration of the analytical column. If the flow field is left empty, the pump operates at the set pressure.

d. Press Max Pressure to set the maximum allowed pressure.

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 1000 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 for the PepMap columns supplied with the EASY-nLC 1000 system is 800 bar. Running the system at pressures higher than 800 bar reduces the column lifespan.

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Figure 101. Analytical Col Equilibration script parameters

10

Maintenance

Maintenance Scripts

Prepare – Isocratic Flow

The Isocratic Flow script runs solvent A and B at a fixed mixture ratio. The script utilizes the

AFC system to accurately control the flow.

Use this script to tune the mass spectrometer at a given B percentage or when cleaning the instrument, the columns, or both.

Y

To set the Isocratic Flow script parameters

1. Select the Isocratic Flow script as follows: a. Press Maintenance > Scripts.

b. In the Category list, select Prepare. c.

In the Name list, select Isocratic Flow.

2. Set the parameters (see

Figure 102 ) for this script as follows:

a. Press the Parameters tab.

b. Press Volume to specify the total volume to be delivered by the pumps.

c.

Press Flow to specify the flow rate to be used.

d. Press AB Mix to specify the solvent composition.

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Maintenance Scripts e.

If you want the solvent flow to continue until you press Stop, select the Run

Indefinitely check box.

When either pump runs out of solvent, both pumps refill. The refilling process takes approximately 2 minutes.

Figure 102. Isocratic Flow script parameters

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Maintenance

Maintenance Scripts

Test – MS Connection

The MS Connection script (see Figure 103

) tests the contact closure function between the

EASY-nLC system and the mass spectrometer.

Use this script when testing contact closure.

There are no parameters associated with the MS Connection script. It tests the settings on the

Connection page of the Configuration menu (see “Setting Up Communication with the Mass

Spectrometer” on page 59 ).

Figure 103. MS Connection script

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Maintenance

Maintenance Scripts

Test – Sample Pickup

The Sample Pickup script tests the autosampler pickup function by picking up a set volume from the well plate and ejecting it into waste.

Use this script when testing the sample pickup function and determining sample pickup flow rates for method setup.

Y

To set the Sample Pickup script parameters

1. Select the Sample Pickup script as follows: a. Press Maintenance > Scripts. b. In the Category list, select Test. c.

In the Name list, select Sample Pickup.

2. Set the parameters (see

Figure 104 ) for this script as follows:

a. Press the Parameters tab.

b. Press Volume to specify the volume to be picked up.

c.

Press Flow to specify the flow rate that pump S uses to aspirate the sample.

d. Press Position to specify the position of the sample in the autosampler. See the

Position pop-up box in Figure 104

.

Figure 104. Sample Pickup script parameters

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Maintenance

Maintenance Scripts

Test – Leaks

2

3

4

Step

1

The Leaks script tests for possible leaks at the pump seal and in the flow path from the pump to its associated valve. The Leaks script can also test for system leaks in the flow path downstream of valves A and B.

Use this script when you suspect leak problems in the pumps (pump seals), in the system flow path between the pump outlet and the associated valve, or downstream of the pump valves.

To run a system leak test, you must connect the Waste In solvent line to the Column out solvent line using a stainless steel union (P/N SC904).

During the leak test of pump A or B, the following events occur.

Event

The pump refills.

The associated valve centers, blocking the solvent flow through the valve.

The pump builds pressure to 980 bar.

When the pressure stabilizes at 980 bar, the pump calculates the flow loss based on the pump piston movement required to maintain a pressure of 980 bar. If the flow loss is less than 400 nL/min, the pump passes the leak test.

During a leak test of the system, the script pressurizes the flow paths shown with red dashed lines in

Figure 105 and monitors the flow sensors and pump piston movement.

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Maintenance

Maintenance Scripts

Figure 105. Pressurized lines for a system leak test

Pump S

Pressure

sensor

Pump A

Pressure

sensor

2

3

1

A

6

4 5

Flow sensor

To the autosampler needle

Sample loop

2

3

1

S

6

4 5

Mixing Tee

Pump B

Pressure

sensor

Flow sensor

Column Out line

Waste In line

2

3

1

B

6

4 5

Centered

2

3

1

W

4 5

6

Union

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Maintenance Scripts

Y

To set the Leaks script parameters

1. Select the Leaks script as follows: a. Press Maintenance > Scripts. b. In the Category list, select Test. c.

In the Name list, select Leaks.

2. Set the parameters (see

Figure 106 ) for this script as follows:

a. Press the Parameters tab.

b. To select pump A or B, both pumps, or the entire system flow path for the leak test, press the cell in the Value column and make your selection from the list (see

Figure 106

).

Note You cannot perform the leak test for pump S as it does not operate under pressure during normal batch execution.

Figure 106. Leaks script parameters

Thermo Scientific

3. For a system leak test, connect the Column Out line to the Waste In line using the stainless steel union (P/N SC904) as shown in

Figure 105 on page 172

.

For information about troubleshooting system leaks, contact your local Thermo Fisher

Scientific service representative.

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Maintenance

Maintenance Scripts

Test – Valve Check

The Valve Check script performs a self-test for the selected valves.

Use this script when valves begin to move slowly or cannot reach the required positions and after exchanging valve parts (rotor or stator).

IMPORTANT Only use this script when instructed to by Thermo Fisher Scientific. Make sure to record any numbers that the script generates.

Y

To set the Valve Check script parameters

1. Select the Valve Check script as follows: a. Press Maintenance > Scripts. b. In the Category list, select Test. c.

In the Name list, select Valve Check.

2. Set the parameters (see

Figure 107 ) for this script as follows:

a. Press the Parameters tab.

b. Select the check boxes for the valves that you want to test (

Figure 107 ).

Figure 107. Valve Check script parameters

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Maintenance

Maintenance Scripts

Test – Back Pressure

The Back Pressure script determines instrument back pressure for solvent A and B. The script runs at a preset flow and measures the back pressure on the system.

IMPORTANT Before you perform the Back Pressure script, ensure that the solvent A bottle contains water and the solvent B bottle contains acetonitrile. This test is not valid for other solvents.

Use this script when testing for instrument flow path blockage. Before running the script, connect column output to waste input as shown in

Figure 105 on page 172

.

Y

To set the Back Pressure script parameters

1. Select the Back Pressure script as follows: a. Press Maintenance > Scripts. b. In the Category list, select Test. c.

In the Name list, select Back Pressure.

2. Set the parameters (see

Figure 108 ) for this script as follows:

a. Press the Parameters tab.

b. Test solvent A, solvent B, or both A and B by selecting the corresponding check box

or check boxes ( Figure 108

).

Figure 108. Back Pressure script parameters

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Maintenance

Maintenance Scripts

Test – Autosampler Torque

The Autosampler Torque script ( Figure 109 ) measures the torque required to move the XYZ

robot on each of its axes.

IMPORTANT Performing this script incorrectly will compromise the performance of the instrument. Contact your local Thermo Fisher Scientific representative before running this script.

Figure 109. Autosampler Torque script description

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Maintenance

Maintenance Scripts

Test – Pump Torque

The Pump Torque script ( Figure 110

) measures the actuator’s ability to generate pressure at predefined torque levels.

IMPORTANT Performing this script incorrectly will compromise the performance of the instrument. Contact your local Thermo Fisher Scientific representative before running this script.

Figure 110. Pump Torque script

Calibrate – Valve Tune

The Valve Tune script automatically retunes the valve.

IMPORTANT Performing this script incorrectly will compromise the performance of the instrument. Contact your local Thermo Fisher Scientific representative before running this script.

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Maintenance

Maintenance Scripts

Calibrate – Flow Sensors

The Flow Sensors script calibrates the flow sensors on pumps A and B.

Use this script when you change the type of solvent in solvent bottles A or B, or you suspect the calibration to be wrong.

This calibration does not require any additional plumbing.

IMPORTANT Because the flow sensor calibration requires a stable operating temperature, ensure that the instrument’s side panels are installed and that the instrument has been on for a minimum of one-half hour.

Y

To set the Flow Sensors script parameters

1. Select the Flow Sensors script as follows: a. Press Maintenance > Scripts. b. In the Category list, select Calibrate. c.

In the Name list, select Flow Sensors.

2. Set the parameters (see

Figure 111 ) for this script as follows:

a. Press the Parameters tab.

b. Select the check boxes for the flow sensors (A, B, or both) that you would like to either inspect or inspect and calibrate.

Tip When you clear the Inspection only check box, the script inspects the accuracy of the current calibration by performing an exact or fast inspection, performs a two-point calibration, and then stores the new calibration setting. For a more accurate evaluation of the calibration, select the Exact Inspection option when you are calibrating the flow sensors.

c.

Do one of the following:

• To check the accuracy of the current calibration only (perform an inspection only), select the Inspection Only check box.

Note When you select the Inspection Only check box, the program does not recalibrate the flow sensors.

• To inspect and calibrate the sensors, clear the Inspection Only check box.

d. Select the inspection speed as follows:

• When you only want a rough estimate of the flow sensor accuracy, select the

Inspection Only check box and the Fast Inspection option.

• When you are recalibrating the flow sensors, clear the

Inspection Only

check box and select the Exact Inspection option.

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Figure 111. Flow Sensors script parameters

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Maintenance

Maintenance Scripts

Calibrate – Reset Pressure Sensor

This script auto-zeroes the pressure sensor.

IMPORTANT Performing this script incorrectly will compromise the performance of the instrument. Contact your local Thermo Fisher Scientific representative before running this script.

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Maintenance

Keeping Service Records

Keeping Service Records

Use the Maintenance > Log Book page to electronically enter all the service actions that you perform on the instrument.

Y

To record a service action

1. Press Maintenance > Log Book.

The Maintenance > Log Book page opens (see

Figure 112 ).

Figure 112. Log Book page of the Maintenance menu tab

2. Press Enter Log Entry.

The Log Entry dialog box opens.

3. In the box at the top of the dialog box, enter a description of the service action.

4. In the Templates list, select a template.

The Templates list contains the following selections: Comment, New Part, Maintenance,

Property Change, and Precolumn Changed by <User> on <Date>.

5. In the Components list, select a hardware component.

The Components list contains the following selections: None, Gradient, Valve B, Valve A,

Valve S, Valve W, Analytical Column, Precolumn, Autosampler, Pump A, Pump B, and

Pump S.

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Maintenance

Checking and Resetting the Device Usage Counters

6. Do one of the following:

• Press Add to add this entry and keep the Log Entry dialog box open.

• Press Add and Close to add this entry and close the Log Entry dialog box.

• Press Close to close the Log Entry dialog box without saving any entries.

Y

To search the log

1. In the Log Book list, select Queue Job or User.

2. In the User List, select Any, Admin, or Guest.

3. In the Time From list, select a time.

4. In the Time To list, select a time.

5. Press Search.

The search results appear in the Search Result table.

Checking and Resetting the Device Usage Counters

The EASY-nLC system tracks the volume pumped by pumps A, B, and S and the number of valve and rotor shifts for valves A, B, and S.

Y

To view the device usage values

1. Press Maintenance > Devices.

2. Do one the following:

• To view the volume pumped by a pump, select the pump from the list of devices.

• To view the number of valve and rotor shifts for a valve, select the valve from the list of devices.

3. Press the Summary tab.

The Summary view opens.

Y

To reset the volume pumped or reset the rotor shifts to zero

1. Open the appropriate Summary page.

2. Press Reset.

For a pump, the value in the Intermediate Volume box resets to 0. For a valve, the value in the Rotor Shifts box resets to 0.

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A

External Devices

To install and use the Advion RePlay™ device, follow the instructions in this appendix.

Note Currently, the EASY-nLC system supports only the Advion RePlay device. Thermo

Fisher Scientific might choose to support additional external devices for use with the

EASY-nLC instrument in future releases.

The RePlay device was specifically developed for nanoflow LC/MS analysis. 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 Web site: 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.

Contents

Connecting the RePlay Device to the EASY-nLC Instrument

Adding the RePlay Device to the EASY-nLC Device List

Setting Up a Method with the RePlay Device

Monitoring the Run of a RePlay Method

Additional Run Time for a RePlay Experiment

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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 system, 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 113 ) on

its front panel.

Figure 113. Front panel of the Replay device (courtesy of the Advion Inc. Web site)

Valve

Y

To connect the RePlay device to the EASY-nLC system

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 114

).

Figure 114. 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)

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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

A

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 115 ) 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 115. 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.

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External Devices

Adding the RePlay Device to the EASY-nLC Device List

Adding the RePlay Device to the EASY-nLC Device List

Y

To add the RePlay device to the device list in the EASY-nLC application

1. Press Maintenance > Devices and check that the RePlay device is not already present in the Devices list. If not, press Add Device.

2. In the Select a Device to Add dialog box, locate the Advion RePlay device.

3. Select the device and press Accept.

4. Verify that the RePlay device was added to the system by locating it in the Devices list.

Selecting the device shows information about the device driver (see

Figure 116 ).

Figure 116. RePlay About tab under the Devices list

186

EASY-nLC 1000 User Guide Thermo Scientific

A

External Devices

Setting Up a Method with the RePlay Device

Setting Up a Method with the RePlay Device

Y

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 Editor

(you can also open an existing method and enable its extension).

An extra RePlay Method Editor page is now available (see Figure 117

).

Figure 117. Page 1/6 of the Method Editor

Thermo Scientific

2. On page 3 of the Method Editor, properly set up the gradient. For more information, see

“To build a gradient” on page 96

.

RePlay 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 RePlay 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.

EASY-nLC 1000 User Guide

187

A

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 118 , 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 118. RePlay Playback Delay setting

188

EASY-nLC 1000 User Guide Thermo Scientific

A

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.

Y

To monitor the run of a RePlay method

1. When running a sample using a method with the RePlay extension, open the Home >

Overview page.

2. View the RePlay (“RP”) mode readback in the center status box (see Figure 119

).

Figure 119. RePlay method readback

Thermo Scientific

RePlay method readback

EASY-nLC 1000 User Guide

189

A

External Devices

Additional Run Time for a RePlay Experiment

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 system cannot overlap (parts of ) two runs in parallel.

190

EASY-nLC 1000 User Guide Thermo Scientific

B

Returning the EASY-nLC Instrument

If it is necessary to return the EASY-nLC system to the factory, follow the instructions in this appendix.

Contents

Transport Instructions

Declaration of Contamination

Transport Instructions

Y

To prepare the instrument for transport

1. Press Home > Overview.

2. Press Eject Plate.

3. Remove the plates/tubes.

4. Remove the waste container and other bottles (see

“Autosampler Wash Bottles and Waste

Beaker” on page 76 ).

5. Press Insert Plate.

6. Close down the system properly (see “Closing Down the EASY-nLC System” on page 58 ).

7. Remove the cables on the back side.

8. Fill out the Declaration of Contamination of Equipment in

“Declaration of

Contamination” on page 192

.

9. Place the EASY-nLC instrument in the original shipping container.

Thermo Scientific EASY-nLC 1000 User Guide

191

B

Returning the EASY-nLC Instrument

Declaration of Contamination

Declaration of Contamination

To return, service, or repair the EASY-nLC instrument, you must complete and submit a

Declaration of Contamination of Equipment” to Thermo Fisher Scientific with the returned equipment. Qualified personnel must review the declaration. Contact Thermo Fisher

Scientific for additional copies of this form or if you have any questions regarding the contents of this declaration.

Description of equipment

Equipment type/model: ______________________ Serial No.: __________________

Date of receiving/purchasing equipment: _________________________

Reason for return: [ ] Maintenance [ ] Repair [ ] End of test

[ ] ______________________________________

Describe symptoms and problems: ___________________________________________

______________________________________________________________________

Equipment condition

Has the equipment been used?

[ ] Yes [ ] No

Has the equipment been exposed to potentially harmful substances?

[ ] Yes [ ] No

If yes, attach list of all known harmful substances including chemical name and symbol, and precautions associated with the substances.

Were any of the harmful substances

• Radioactive?

• Toxic?

• Corrosive?

[ ] Yes

[ ] Yes

[ ] Yes

[ ] No

[ ] No

[ ] No

• Explosive?

[ ] Yes [ ] No

Has the equipment been properly decontaminated and/or cleaned before being returned?

[ ] Yes [ ] No

Legally binding declaration

I hereby declare that the information supplied on this form is accurate and sufficient to judge any contamination level.

Name: ________________________________________________________________

Job title: ____________________ Organization: _____________________________

Address: _______________________________________________________________

Telephone: ________________ Fax: ________________ E-mail: __________________

Signature: ______________________________________________________________

192

EASY-nLC 1000 User Guide Thermo Scientific

C

Consumables and Replacement Parts

To order consumables and replacement parts for the EASY-nLC system, see the ordering information and solvent system schematics in this appendix.

Contents

Ordering Information

Solvent System Schematic

Ordering Information

To obtain ordering information for the EASY-nLC instrument’s spare parts and accessories, go to the following Web site: http://www.proxeon.com/productrange/nano_lC_easy-nlc_1000/accessories_spares

These EASY-nLC parts are consumable parts and not covered by the normal first year Limited

Warranty offered by Thermo Fisher Scientific, or any other service contract agreement containing extended warranty coverage.

• All flow lines including Tee-pieces and fittings (nuts, ferrules, sleeves, and valve blank stops)

• Check valves

• Solvent filters, inline filters, and all associated filter holders

• Pump piston seals

• Valve rotors and stators

• All bottles and lids

• All sample vials, microtiter plates, and associated lids and mats

• Columns (supplied by Dionex, a part of Thermo Fisher Scientific)

• Fuses

• Autosampler needle

Thermo Scientific EASY-nLC 1000 User Guide

193

C

Consumables and Replacement Parts

Solvent System Schematic

Solvent System Schematic

The pump and valve compartment of the EASY-nLC instrument contains all the flow lines between the components (see

Figure 120 on page 195

).

In addition to pumps and valves, the solvent delivery and waste system includes several filters and four check valves. The two check valves on each solvent line ensure that solvent is drawn from the solvent bottles and ejected to waste without switching valve positions.

2

3

4

ID

1

Table 28

lists the tubing requirements and identifies the tubing components shown in

Figure 120

. The Low-Pressure Solvent Line Kit (P/N LC230) contains the Tees, check valves, and low-pressure solvent lines to connect one pump (A or B) to the waste system and to its corresponding solvent bottle and six-port valve. The EASY-nLC 1000 Flow Line Kit

(P/N LC570) contains all of these flow lines. The stainless steel tubing is prebent.

Table 28. Tubing requirements

5

6

7

8

9

D

E

F

G

Connections

Pump outlet to pressure sensor inlet

Pressure sensor outlet to valve A or B

Pressure sensor outlet to valve S

Mixing Tee to valve S,

Valve A to flow sensor A,

Valve B to flow sensor B,

Flow sensor A to mixing Tee

Flow sensor B to mixing Tee

Autosampler needle connected to port 1 of valve S

Column Out tubing connected to port 3 of valve S

Waste In line, venting Tee to port 2 of valve W

Sample loop, 20 μL

Port 2 of valve A to check valve A

Port 2 of valve B to check valve B

Tubing (2) from check valves to solvent bottles

Tubing (2) from check valves to waste beaker

Tubing from valve W to the waste beaker

Tubing Part number

Stainless steel, 250 μm ID, 150 mm length LC512

Stainless steel, 250 μm ID, 150 mm length LC513

Stainless steel, 250 μm ID, 150 mm length LC514 nanoViper, 20 um ID, 350 mm length LC522 nanoViper, 10 um ID, 180 mm length

PEEKsil™, 150 um ID, 550 mm length nanoViper, 20 um ID, 550 mm length nanoViper, 75 um ID, 550 mm length nanoViper, 250 um ID, 410 mm length

Teflon™, 500 um ID, 150 mm length

Teflon, 500 um ID, 390 mm length

Teflon, 500 um ID, 390 mm length

Teflon, 500 um ID, 330 mm length

LC543

LC302

LC560

LC562

LC472 kit LC230 kit LC230 kit LC230

LC263

194

EASY-nLC 1000 User Guide Thermo Scientific

C

Consumables and Replacement Parts

Solvent System Schematic

Figure 120. Physical layout and schematics of the EASY-nLC 1000 solvent system

E

6

Solvent A

F

F

8

D

9

1

2

S

6

1

2

W

6

1

2

A

6

7

4

D

E

Solvent B

1

2

B

6

4

4

3

Pressure sensor

1

G

4

2

Pressure sensor

1

5

2

Pressure sensor

1

Waste

Thermo Scientific EASY-nLC 1000 User Guide

195

I

Index

A

accessing files on the instrument’s hard drive

42

,

61

support server

43

,

150

accounts, adding new users

65

acetonitrile, solvent requirements

74

adapter plate, replacing

120

admin folder, accessing

42 ,

63

admin, changing password

64

analytical column column setup options

8

equilibration script

166

equilibration step, method

16

installing

83

preparation for use

89

application version

49

Australia and Asia, customer service for

xx

Automatic Flow Control (AFC) description

25

autosampler calibration

119

checking sample pickup

160

manual control

146

sample pickup method parameters

94

Sample Pickup script

131

Torque script

176

tray compartment, accessing

3

troubleshooting

136

wash step, method

23

, 100

B

back panel, instrument

7 ,

37 ,

150

back pressure analytical column rating

166

precolumn rating

165

testing

175

Back Pressure script

175

backup, system

68

Thermo Scientific batches editing on the Home > Queue page

114

setting up and scheduling

103

starting

108

troubleshooting

134

beaker, waste

4

C

calibrating autosampler

119

flow sensors

178

plates

125

carryover reducing

101

specification

28

changing the administrator password

64

checking sample pickup

131

solvent levels

159

valves

160

Column Out tubing

4

,

9 ,

84

columns

See analytical column or precolumn

compliance, electromagnetic

xxi

components system

2

troubleshooting

138

computer damage, cause of

38

configuring devices

70

global settings

70

RePlay

186

connecting add-ons

44

contact closure cable

39

mouse and keyboard

44

to line power

38

to the data system computer

41

to the network

61

consumable parts list

193

EASY-nLC 1000 User Guide 197

Index: D contact closure cables, ordering

39

configuration setting

59

troubleshooting

137

with the mass spectrometer

39

contamination declaration

192

preventing

142

cooler temperature checking

160

manual control

147

creating a method

93

new user accounts

65

plate formats

121

Custom Wash, sample pickup system

101

customer service, contacting

xvi

Cycles, autosampler wash

101

D

daily maintenance

159

data system computer connecting to the Ethernet switch

41

connecting to the laboratory LAN port

41

using to access folders on the EASY-nLC system’s computer

42

data types, graphs

111

Declaration of Conformity

xxi

Declaration of Contamination

192

devices configuring

70

usage counters

181

dimensions, EASY-nLC instrument

29

E

EASY-nLC description

2

Eject Plate button, using

47

, 76

emitter, installing

86

equilibrating analytical column

16

,

89

precolumn

15 ,

88

Ethernet cables, connecting data system computer

41

EASY-nLC system to the intranet

42

EASY-nLC system to the support server

43

Ethernet switch

41

Europe, customer service for

xvii

Exit button

58

198

EASY-nLC 1000 User Guide

F

Fast Inspection option

178

filling a pump with solvent

143

firewalls, laboratory network access

36

fittings and adapters, column assembly

9

flow lines, tubing requirements

194

flow rate determining appropriate

164 ,

166

gradient

96

manual control

142

measuring

11

monitoring

110

sample loading

95

sample pickup

95

flow sensors calibration script

178

description

11

maintenance schedule

161

Flush Air script

163

formic acid, solvent requirements

74

fuse, replacing

158

G

global configuration settings

70

gloves, wearing

84

gradient delay volume

19

method step

19

setting up the method

96

graphical data types

111

grounding requirements

38

H

humidity specification

36

I

injections number per sample

106

pickup sample step

21

sample pickup volume

94

Insert Plate button, using

77

Inspection Only check box

178

Intelligent Flow Control (IFC) system description

26

interface layout

49

international calls, making

xv

Internet access

36

IP address, support server

150

Isocratic Flow script

167

Thermo Scientific

K

keyboard connection

44

L

Leaks script

171

leaks, checking for

160

lifting instructions

33

Load Speed Protection option

27

, 71

log book

180

logging in to the application

57

Logo icon, Thermo Scientific

49

Loop Volume box

71

low-pressure solvent filters

161

M

maintenance daily

159

scripts

161

yearly

161

manuals, EASY-nLC

xii

mass spectrometer, contact closure configuration setting

59

hardware connection

39

troubleshooting

137

, 169

menu structure

52

message.log file

152

method, LC accessing from another computer

42 ,

63

analytical column equilibration parameters

99

gradient table

96

method editor, starting

93

precolumn equilibration parameters

98

microwell plates, preparing samples

91

monitoring the run

109

See also RePlay, monitoring the run

mouse connection

44

MS Connection script

169

N

nanoViper fittings, using

87

needle position, calibrating

124 –

130

network access, troubleshooting

138

connection

61

network card, installing in data system computer

41

noise and interference, cause of

38

North America, customer service for

xvi

Thermo Scientific

O

one-column setup configuration check box

70

description

10

installing

84

P

password changing

64

initial

57

performance specifications

28

plates calibrating

125

managing formats

121

port 22, firewall setup for

36 ,

43 ,

149

power requirements

36

turning off

58

power cord, connection and specifications

38

Power Down button

58

power-save feature, Ethernet switch

41

precolumn equilibration step, method

15

,

98

installing

84

preparation for use

88

preparing autosampler for the calibration routines

124

solvent bottles and waste containers

74

well plates

91

pressure sensors

11

product description

2

pump flow control description

25

manual control

142

volume pumped usage counter

181

Pump dialog box

143

Purge Solvent script

162

purging and flushing pumps

79

Q

quadrant selection box, 384-well plate

106

R

remote desktop connections

41

remote support

43 ,

149

replacement parts

193

Index: K

EASY-nLC 1000 User Guide

199

Index: S

RePlay additional run time

190

configuring

186

connecting to EASY-nLC

183

monitoring the run

189

reset pressure sensor script

179

running batch, editing

114

S

safety calibrating the autosampler

124

handling emitters

86

precautions for the EASY-nLC system

xiii

standards (EMC)

xxi

sample loading, method parameters

95

pickup check

131

pickup method parameters

94

Pickup script

170

sample loop bypassed during gradient program

19

configuring

71

maximum capacity

21

ordering

194

washing

23

sample runs predefined steps

23

troubleshooting

117

scripts

Analytical Col Equilibration

166

Autosampler Torque

176

Back Pressure

175

Flow Sensors

178

Flush Air

163

Isocratic Flow

167

Leaks

171

Maintenance

161

MS Connection

169

Purge Solvent

162

Reset Pressure Sensor

179

Sample Pickup

131

,

170

Valve Check

174

Valve Tune

177

Secure Shell (SSH) network protocol

36

, 149

service log

180

sharp objects autosampler needle

124

emitter

86

solvent A and B bottles, preparing

75

solvent system schematic

194

200

EASY-nLC 1000 User Guide solvents checking levels

159

filtering

74

ordering Fisher Scientific solvents

75

Source column, autosampler wash

101

specifications performance

28

technical

29

SSH Connection Error message box

152

Standard option, autosampler wash

101

Start button, green

78 ,

110

starting the EASY-nLC

46

Stop button, red

78

, 110

, 113

stopping sample acquisition

113

support server establishing communication with

150

IP address

150

support server, setting up the hardware connections

43

system overview, monitoring

109

system startup, troubleshooting

134

T

technical specifications

29

technical support, contacting

xv

temperature maintaining stable

128

manual control

147

specification

36

time setting

67

touch screen, using

47

transport instructions

191

trap column

See precolumn

tray compartment, accessing

3 ,

47

troubleshooting autosampler

136

contact closure

137

network access

138

running a batch

134

sample runs

117

system components

138

system startup

134

tubing requirements

194

turning off the EASY-nLC

58

two-column setup description

8

installing

84

Thermo Scientific

Index: U

U

URLs consumable parts

xv

customer manuals

xii

usage counters, device

181

USB ports, EASY-nLC

7

,

153

USB storage device, copying system files

153

user interface layout

49

user permissions

63

V

Valve Check script

174

valve position description

12

manual control

141

Valve Tune script

177

venting Tee connections

9

– 10 ,

84 –

85

purpose

13 ,

15

version, application

49

vials extra vial positions, calibrating

129

plate format, calibrating

125

preparing sample

91

volume, maximum custom wash solvent volume per step

101

sample loading step

95

sample pickup step

94

W

warranty

193

wash bottles calibrating W4 bottle with insert

129

description

3

preparing

76

wash step, autosampler

23

, 100

waste beaker description

4

installing

76

Waste In tubing

4 ,

9

, 84

water, solvent requirements

74

weight, EASY-nLC instrument

29

well plates calibrating

125

plate formats, managing

121

preparing

91

Windows Explorer

62

, 138

Thermo Scientific

X

XYZ robot calibrating

124

130

manual control

146

EASY-nLC 1000 User Guide

201

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