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.

1Using this Manual
UltiMate 3000 RSLCnano Standard Applications Guide Page 11
1.2 Conventions
This section describes the conventions used throughout this manual
1.2.1 Special Notices and Informational Notes
Special notices and informational notes in this manual appear different
from the main flow of text. They appear in boxes and a note label
identifies them. The label text appears in uppercase letters and in bold
type.
NOTICE Highlights information necessary to prevent damage to the
instrument or invalid test results.
TIP Highlights information of general interest or helpful information that
can make a task easier or optimize the performance of the instrument

1Using this Manual
Page 12 UltiMate 3000 RSLCnano Standard Applications Guide
1.2.2 Typographical Conventions
These typographical conventions apply to the descriptions in this
manual:
References and Messages
References to figures and tables appear italicized.
Viewpoint
If not otherwise stated, the expressions left and right in this manual
always refer to the viewpoint of a person that is facing the instrument
from the front.
Particularly Important Words
Particularly important words in the main flow of text appear in bold.
Electronic Manual Version (PDF)
The electronic version (PDF) of the manual contains numerous links that
you can click to go to other locations within the manual. These include:
Table of contents entries
Index entries
Cross-references (in blue text), for example, to sections, figures or
online reference materials

1Using this Manual
UltiMate 3000 RSLCnano Standard Applications Guide Page 13
1.3 Reference Documentation
Further information relating to the UltiMate 3000 RSLCnano systems
and associated applications is available as follows:
The UltiMate 3000 RSLCnano system webpage:
https://www.thermofisher.com/order/catalog/product/ULTIM3000
RSLCNANO
Module operating instructions
NCS-3500RS / NCP-3200RS pump modules
VWD-3100 and VWD-3400RS variable wavelength detectors
WPS-3000TPL RS and WPS-3000FC autosamplers
Additionally, you can also find these operating instructions in the
following installation folder (Chromeleon 7/ SII):
“C:\Program Files (x86)\Thermo\Chromeleon\bin\Troubleshooting
Guides”(or “C:\Chromel\Bin\Troubleshooting Guides” when using
Chromeleon 6.80)
Upgrading the UltiMate 3000 RSLCnano System with ProFlow
Technology –Quick Installation Guide
ViperTM and nanoViperTM EASY-SprayTM Column tips and tricks
document
Details on Viper and nanoViper capillaries and application kits
Thermo Scientific Viper and nanoViper Fingertight Fitting System -
brochure
Viper and nanoViper Fingertight Fitting Systems - specifications
The complete and easy guide to configuring your Thermo Scientific
nano LC
Nano, Capillary and Micro LC Columns detailed in the
Chromatography Columns and Consumables catalogue

2Application Setup
Page 14 UltiMate 3000 RSLCnano Standard Applications Guide
2Application Setup
This chapter provides details on each of the application kits available for
the UltiMate 3000 RSLCnano system.

2Application Setup
UltiMate 3000 RSLCnano Standard Applications Guide Page 15
2.1 General Recommendations for Applications
The experimental conditions for each application are described together
with schematics, installation tips, examples and results.
2.1.1 nanoViper Connections
NanoViper (Figure 1) is a fingertight high-pressure fitting that is virtually
dead volume free by design and rated for backpressures up to 1200 bar.
All high-pressure fittings used in the applications on the UltiMate 3000
RSLCnano system use nanoViper. The fittings are factory assembled to
ensure quality and prevent experimental failure due to bad connections.
Figure 1: Internal and external view of a nanoViper fitting
1. Install nanoViper using the removable knurled black nut.
2. Do not overtighten connections (the general guideline is fingertight
plus an additional one eighth of a turn).
3. Remove the knurled black nut once the fitting is tight.

2Application Setup
Page 16 UltiMate 3000 RSLCnano Standard Applications Guide
2.1.2 Making Connections using the Nano Connector
The outlet of the linear nano columns are fitted with a nano connector.
This zero dead volume connection is designed to interface the linear
column outlet with 280 µm fused silica capillaries and is pressure stabile
up to 300 bars. The nano connector uses a special sleeve to ensure
pressure tightness. The assembly of a nano connector is described step
by step in Figure 2 below.
Figure 2: The components of the nano connector
Use a new nano connector sleeve (P/N 6720.0391) each time the
connection is made.
NOTICE: Do NOT use a PTFE sleeve (P/N 160486; supplied with the
columns). The size does not match the nano connector union (Figure 3)
and the pressure resistance is much lower.
Nano connector sleeve (1.1 cm)
PTFE sleeve (1.8 cm)
Figure 3: Nano connector (top) and PTFE (bottom) sleeve comparison
1. Slide the black nut and transparent union onto one of the ends of
the fused silica, and the other black nut onto the other fused silica
end Figure 4a.
2. Slide the nano connector sleeve onto one end of the fused silica
until it reaches the middle of the sleeve. Slide the other end of the
fused silica into the connector sleeve. Make sure that the
connection is dead volume free (i.e. that the ends meet in the
middle Figure 4b).

2Application Setup
UltiMate 3000 RSLCnano Standard Applications Guide Page 17
3. Tighten both sides of the black nut equally to ensure that the nano
connector sleeve is in the center of the transparent union Figure
4c.
a
b
c
Figure 4: Nano connector assembly. (a) The two black nuts and
transparent union are mounted on the two outlets to be connected.
(b) A dead volume free connection with the nano connector. (c) The
complete fitting with the black nuts and union housing the nano
connector.
2.1.3 Installing and Configuring the Application Fluidics
Each capillary must be installed sequentially starting from the pump
outlet. Please flush each capillary using the respective pump and ensure
that a droplet is visible at the capillary outlet in question before making
the next connection. This will ensure that all air is removed from the
capillaries and connections and that no air is passed through the
column.
2.1.4 Interfacing the UltiMate 3000 RSLCnano with the Nanospray Flex™ Ion
Source
LC−MS based applications using linear columns commonly use the
Nanospray Flex Ion source (see Figure 41) to interface with Thermo
Scientific Mass Spectrometers. As of January 2019, 1.5 m of fused silica
(20 µm and 50 µm) and a tile for cutting fused silica capillaries have
been included in the pump (NCS-3500RS and NCP-3200RS) accessory
kits. This capillary should be used to connect the outlet of the column or
UV flow cell, if included in the setup, with the emitter installed in the ion
source. The connection between the capillary outlet and the emitter is
realized using a 1/32” micro tight® union assembly included with the
Nanospray Flex Ion Source. For capillaries and columns with 280 µm O.D.
a black sleeve (P/N SC903) should be used with the microtight fitting.

2Application Setup
Page 18 UltiMate 3000 RSLCnano Standard Applications Guide
For 360 µm O.D. capillaries and columns (e.g. the Acclaim PepMap RSLC
C18 75 cm PepMap RSLC, P/N 164939) a beige sleeve (P/N SC603)
should be used (both types are included with the ion source). For more
details on connecting the UltiMate 3000 RSLCnano with a Thermo
Scientific mass spectrometer, please refer to “The Complete and Easy
Guide to Configuring Your Thermo Scientific Nano LC for Mass
Spectrometric Analysis”.
2.1.5 Sample Preparation for Reversed Phase LC Separation
The following are recommendations for Cytochrome C standard digest
(P/N 161089) preparation. The glass vial contains 1.6 nmol lyophilized
Cytochrome C digest. The sample preparation procedure depends on the
system configuration and application in question (e.g. nano, capillary or
micro).
NOTICE The sample dilution protocol described here differs from the
product sheet and is designed to offer the user a starting point. The
sample concentration required to run a particular application may
deviate from the sample concentrations given below. Sample dilutions
may also need to be prepared in a different buffer to that given below.
Please check the required sample concentration and dilution conditions
for the application and prepare the sample accordingly!
Reconstitution Solvent –(98% Water / 2% Acetonitrile containing
0.1% FA). Prepare by mixing 980 µL Water + 0.1% FA and 20 µL 100%
Acetonitrile + 0.1% FA in a vial. (See section 2.3.4.4 for information
about the individual solvents on page 32.). The use of 2%
acetonitrile is recommended, to ensure complete dissolution of
hydrophobic peptides.
Reconstituted the sample in 200 µL reconstitution solvent to
prepare a stock solution of 8 pmol / µL for nano / cap applications.
Reconstituted the sample in 100 µL reconstitution solvent to
prepare a stock solution of 16 pmol / µL for micro applications.
Vortex briefly and wait at least 10 minutes to ensure reconstitution
of all peptides prior to use / further dilution.

2Application Setup
UltiMate 3000 RSLCnano Standard Applications Guide Page 19
For nano flow applications, dilute the stock solution to 500 fmol /
µL using mobile phase A (direct injection) or loading buffer (pre-
concentration) as follows:
Prepare 150 µL mobile phase A in an autosampler vial (with
insert) and add 10 µL from the 8 pmol / µL Cytochrome C stock
solution.
Mix (on Vortex or with pipette) briefly to homogenate the
solution.
Ensure there are no air bubbles at the bottom of the vial.
TIP: To limit the risk of peptide or protein adsorption on the walls of the
vials, Thermo Fisher Scientific recommends using vials containing glass
inserts (Polypropylene vials for WPS with glass insert, 250 µL, set of 100,
P/N 6820.0027).

2Application Setup
Page 20 UltiMate 3000 RSLCnano Standard Applications Guide
2.1.6 Mobile Phases
Always use fresh LC-MS grade solvents.
Thermo Fisher Scientific recommends replacing your solvents at
least once every two weeks.
Avoid the use of detergents when cleaning glassware. All glassware
used for LC-MS applications (including graduated cylinders) should
be rinsed with LC-MS grade solvents prior to use and should be
labelled and stored separately.
IMPORTANT: When installing fresh mobile phase on the LC system,
replace the mobile phase solvent in the bottle completely. DO NOT “top
up” mobile phases to avoid solvent composition changes or unwanted
components building up in the mobile phase bottles.