Thermo Scientific Dionex UltiMate 3000 Series User guide

UltiMate™3000
RSLCnano
Standard Applications Guide
Revision: 3.1
Date: April 2019
Document No.: 4820.4103
Copyright © 2019 Thermo Fisher Scientific Inc. All rights reserved.
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UltiMate 3000 RSLCnano Standard Applications Guide Page 3
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Page 4 UltiMate 3000 RSLCnano Standard Applications Guide

Contents
UltiMate 3000 RSLCnano Standard Applications Guide Page 5
Contents
1 Using this Manual ............................................................................ 9
1.1 About this Manual ................................................................................................10
1.2 Conventions ..........................................................................................................11
1.2.1 Special Notices and Informational Notes.................................................11
1.2.2 Typographical Conventions......................................................................12
1.3 Reference Documentation....................................................................................13
2 Application Setup............................................................................14
2.1 General Recommendations for Applications........................................................15
2.1.1 nanoViper Connections............................................................................15
2.1.2 Making Connections using the Nano Connector .....................................16
2.1.3 Installing and Configuring the Application Fluidics..................................17
2.1.4 Interfacing the UltiMate 3000 RSLCnano with the Nanospray Flex™ Ion
Source 17
2.1.5 Sample Preparation for Reversed Phase LC Separation ..........................18
2.1.6 Mobile Phases..........................................................................................20
2.2 Available Trapping Columns .................................................................................21
2.2.1 Available Formats.....................................................................................21
2.2.2 The Difference between Forward Flush and Back Flush..........................22
2.3 Installing the UltiMate 3000 RSLCnano System....................................................24
2.3.1 UltiMate 3000 RSLCnano System Components .......................................24
2.3.2 NC Pump Configurations..........................................................................25
2.3.3 Software Compatibility for NCx-3x00RS Operation with ProFlow and
Classic Flow Meters ..............................................................................................29
2.3.4 Preparing the RSLCnano for Use..............................................................31
2.3.5 Flow meter calibration.............................................................................35
2.4 Application Overview............................................................................................37
2.5 Direct Injection onto a Nano Column ...................................................................38
2.5.1 Hardware Layout......................................................................................38
2.5.2 Fluidic Setup.............................................................................................39
2.5.3 Installation Tips........................................................................................40
2.5.4 Testing the Application ............................................................................40
2.5.5 Large Volume Injections...........................................................................41
2.6 Direct Injection onto a Capillary Column..............................................................42

Contents
Page 6 UltiMate 3000 RSLCnano Standard Applications Guide
2.6.1 Hardware Layout..................................................................................... 42
2.6.2 Fluidic Setup............................................................................................ 43
2.6.3 Installation Tips ....................................................................................... 45
2.6.4 Testing the Application ........................................................................... 45
2.7 Pre-concentration onto a Nano Column.............................................................. 47
2.7.1 Hardware Layout..................................................................................... 47
2.7.2 Fluidic Setup............................................................................................ 48
2.7.3 Installation Tips ....................................................................................... 49
2.7.4 Testing the Application ........................................................................... 50
2.8 Pre-concentration onto a 200 µm Monolithic Column........................................ 51
2.8.1 Hardware Layout..................................................................................... 51
2.8.2 Fluidic Setup............................................................................................ 52
2.8.3 Installation Tips ....................................................................................... 53
2.8.4 Testing the Application ........................................................................... 53
2.9 Pre-concentration onto a Capillary Column ........................................................ 55
2.9.1 Hardware Layout..................................................................................... 55
2.9.2 Fluidic Setup............................................................................................ 56
2.9.3 Installation Tips ....................................................................................... 57
2.9.4 Testing the Application ........................................................................... 58
2.10 EASY-Spray Columns with the RSLCnano............................................................. 59
2.10.1 EASY-Spray Concept................................................................................ 59
2.10.2 Example Separation Performance of an EASY-Spray Column................. 61
2.10.3 Hardware Layout Direct Injection ........................................................... 62
2.10.4 Fluidic Setup using EASY-Spray Columns ................................................ 63
2.10.5 EASY-Spray Transfer lines........................................................................ 65
2.11 2D Salt Plugs with Nano Column.......................................................................... 66
2.11.1 Hardware-Layout..................................................................................... 66
2.11.2 Fluidic Setup............................................................................................ 67
2.11.3 Installation Tips ....................................................................................... 68
2.11.4 Testing the Application ........................................................................... 69
2.11.5 Salt Solution Preparation ........................................................................ 70
2.12 Tandem Nano LC.................................................................................................. 71
2.12.1 Hardware Layout..................................................................................... 71
2.12.2 Fluidic Setup............................................................................................ 72
2.12.3 Installation Tips ....................................................................................... 73
2.12.4 Testing the Application ........................................................................... 74
2.12.5 Tandem NanoLC-MS................................................................................ 75

Contents
UltiMate 3000 RSLCnano Standard Applications Guide Page 7
2.13 High throughput tandem capillary-flow LC-MS ....................................................76
2.14 Micro LC Applications ...........................................................................................77
2.15 MS Connection Kit ................................................................................................78
2.15.1 Mass Spectrometry Interfaces for Linear columns and / or interfacing the
UV detector with the MS. .....................................................................................80
3 FAQs ...............................................................................................83
3.1 NC_Pump Solvent Recalibration –Best Practice ..................................................84
3.1.1 ProFlow Flow Meter.................................................................................84
3.1.2 Classic Flow Meter ...................................................................................84
3.2 Interpreting a Chromatogram...............................................................................85
3.3 Troubleshooting Nano LC Peptide Applications ...................................................86
3.4 The Use of TFA and FA ..........................................................................................88
3.5 Minimizing Baseline Effects ..................................................................................90
3.5.1 Drift ..........................................................................................................90
3.5.2 Unstable Baseline.....................................................................................91
3.6 Typical WPS-3000TPL RS Autosampler Settings for Standard Injection
Routines. .........................................................................................................................92
4 Appendix.........................................................................................93
4.1 Customized Sample Injection Routines ................................................................94
4.1.1 Introduction to User Defined Program (UDP) Injection Routines ...........94
4.1.2 Important Considerations when Writing a UDP ......................................94
4.1.3 The UDP Commands ................................................................................94
4.1.4 Example µL pickup UDP for Maximum Sample Pickup. ...........................95
4.1.5 Variable Injection Volumes......................................................................96
4.2 Common Application Related Consumables ........................................................97
4.2.1 Columns ...................................................................................................97
4.2.2 nanoViper Capillaries, Sample Loops and Connectors.............................99
4.3 UltiMate 3000 RSLCnano convenience bundles, list of contents .......................101
4.4 Hardware Accessories.........................................................................................102

Contents
Page 8 UltiMate 3000 RSLCnano Standard Applications Guide

1Using this Manual
UltiMate 3000 RSLCnano Standard Applications Guide Page 9
1Using this Manual
This chapter provides information about this manual, the conventions used
throughout the manual, and the reference documentation that is available
in addition to this manual.

1Using this Manual
Page 10 UltiMate 3000 RSLCnano Standard Applications Guide
1.1 About this Manual
This document describes the setups, recommended experimental
conditions and testing procedures required to run standard applications
on the Thermo Scientific Dionex UltiMate3000 RSLCnano system.
NOTICE This document is intended for Thermo Fisher Scientific (or
authorized) service personnel as well as customers to assist in the
installation and application testing of UltiMate 3000 RSLCnano systems.
It does not replace the IQ or OQ procedures. It is assumed that the
individual using this manual has had sufficient training in the installation
and usage of analytical instrumentation and is aware of the potential
hazards including (but not limited to) electrical hazards, chemical
hazards, exposure to UV radiation and exposure to pressurized solvents.
This manual contains important information about the correct care and
use of the UltiMate 3000 RSLCnano. Please read this manual carefully
before installing or running any of the applications described. Keep this
manual close to the UltiMate 3000 RSLCnano for future reference and
pass it on to any subsequent user.