Man 4820 4103 UltiMate 3000 RSLCnano Man48204103 EN

thermoscientific
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
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
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
2.8 Pre-concentration onto a 200 µm Monolithic Column........................................ 51
2.8.1 Hardware Layout ..................................................................................... 51
2.8.2 Fluidic Setup ............................................................................................ 52
2.9 Pre-concentration onto a Capillary Column ........................................................ 55
2.9.1 Hardware Layout ..................................................................................... 55
2.9.2 Fluidic Setup ............................................................................................ 56
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
2.11.5 Salt Solution Preparation ........................................................................ 70
2.12 Tandem Nano LC .................................................................................................. 71
2.12.1 Hardware Layout ..................................................................................... 71
2.12.2 Fluidic Setup ............................................................................................ 72
2.12.5 Tandem NanoLC-MS................................................................................ 75

Contents
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

1  Using this Manual
1 Using 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.

1.1 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.

1.2 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

1.2.2 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

1.3 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
2 Application Setup
This chapter provides details on each of the application kits available for
the UltiMate 3000 RSLCnano system.

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.

2.1.2 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).

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.
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.

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.

 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).

2.1.6 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.

2.2 Available Trapping Columns

2.2.1 Available Formats
Trap columns are available in two formats, which are a cartridge-based
µ-precolumn (Figure 5) and a nano trap column (Figure 6). Both types of
trapping columns are UHPLC compatible due to the nanoViper fittings
employed. The choice between a µ-precolumn or a nano trap depends
on application needs such as flexibility, sample loading flow rate and
robustness as well as sample quality, desired loading capacity and
personal preference.
Figure 5: µ-Precolumn (cartridge-based)
TIP P/N 164648 contains two 30 µm ID x 100 mm nanoViper capillaries

and can be used to order replacement capillaries
µ-precolumns are small trap cartridges that are inserted into a cartridge
holder, connected to the switching valve by two 30 µm ID x 100mm
nanoViper capillaries. The stationary phase is retained by a frit at both
ends of the cartridge allowing the mobile phase to flow through it in
both directions without disrupting the column packing. Therefore,
µ-precolumns can be used in both forward- and back-flush operation
(see section 2.2.2 for details). The bed volume is large, but short, giving
it higher absolute loadability compared to nano traps, but the short bed
could result in earlier sample breakthrough for hydrophilic components.
Backpressure is lower compared to nano trap columns and therefore µ-
precolumns can accommodate higher loading flows and are often
preferred when large sample volumes need to be injected.

Figure 6: Nano trap
Nano trap columns consist of a single 15-cm-long nanoViper capillary

containing 1 or 2 cm of stationary phase at one end of the capillary.
Nano traps must be operated exclusively in forward-flush mode. The
chromatographic bed volume is lower than that of µ-precolumns, but
the longer bed length minimizes sample breakthrough. Nano traps give a
higher backpressure than µ-precolumns and are thus operated at lower
flow rates.
TIP: Note that for Acclaim PepMap RSLCnano columns, the difference
between the pressures on the nano trap column and analytical column is
smaller than with the combination of the µ-precolumn and the analytical
column.
2.2.2 The Difference between Forward Flush and Back Flush

The terms forward-flush and back-flush are used to indicate whether the
mobile phase from the NC pump during gradient elution flows in the
same or opposite direction compared to the mobile phase flow during
sample loading. Figure 7 shows the different fluidic setups for a forward-
and a back-flush fluidic pathway.
Figure 7: The different fluidic configurations for forward-flush (left) and

back-flush (right)

For nano trap columns, the packing material is only retained by the frit
at one end of the trap column. In order not to damage nano traps, only
forward-flush can be used.
In the µ-precolumn design, the stationary phase is retained by a frit at

both ends of the column packing. This means that the mobile phase can
flow through the cartridge in either direction without disrupting the
column packing, i.e. forward-flush and back-flush operation.
The choice between forward- and back-flush for the µ-precolumn design
is made on the following criteria.
 In forward-flush, the trap column also acts as a guard column to

protect the separation column.
 In back-flush, better separation is obtained, but any particulates or

insoluble debris from the sample could end up on the separation
column.
NOTICE For pre-concentration applications, better chromatographic

resolution (narrower chromatographic peaks) are produced when the
µ-precolumn is installed in back-flush mode.

2.3 Installing the UltiMate 3000 RSLCnano System

2.3.1 UltiMate 3000 RSLCnano System Components
SRD-3400, (Optional):
SRD-3200 with degassing
or SR-3000 without degassing.
NCS-3500RS module featuring

- NC pump, up to 900 bar
- Loading pump, micro Titanium up to 620 bar
- Column compartment with up to two 860
bar switching valves
Optional:- NCP-3200RS, - PAEK valve
VWD-3400RS with flow cells for

- nano (3nL)
- capillary and micro (45 nL) LC
WPS-3000TPL RS
- Temperature controlled autosampler
equipped with a
860 bar switching valve
- Optional:
8-port valve (350 bar) for micro-
fractionation applications
Figure 8: RSLCnano system overview

2.3.2 NC Pump Configurations
2.3.2.1 ProFlow™ and Classic Flow Meters

Flow meters are used to actively regulate NC pump flow on the
instrument in order to deliver very precise low-flow gradients. There are
two types of flow meter available:
 ProFlow flow meter
The ProFlow flow meter controls pump flow using thermal flow
sensors built into the flow meter. It is a unit dedicated to nano and
low capillary flow rates (50 nL / min – 1500 nL / min) and allows a
pump pressure rating of 900 bar at the full flow rate range for all
common solvents used for reversed phased LC applications.
 Classic flow meter
The classic flow meter determines the flow rate indirectly by

measuring the pressure drop across a restriction capillary contained
within the flow meter itself. The pressure rating of the pump using
the classic flow meter is 800 bar for flow rates ≤ the nominal flow
rate (see Table 1).
2.3.2.2 Flow Selectors for the Classic Flow Meter

Each classic flow meter contains a flow selector that defines the flow
rate range of the flow meter. These flow selectors are interchangeable.
The flow rate ranges of the respective flow selectors and the nominal
flow rates are given in Table 1 below.
Flow Selector Type Total Flow Rate (Sum of Channels A and B)
Nominal Minimum Maximum
Nano (Nan) 500 nL/min 50 nL/min 1000 nL/min
Capillary (Cap) 5 µL/min 500 nL/min 10 µL/min
Micro (Mic) 25 µL/min 2.5 µL/min 50 µL/min

Table 1: Properties of the different flow selectors

2.3.2.3 NCS-3500RS with the ProFlow Flow Meter

High pressure gradient NC_Pump Low pressure gradient Micro pump
Solvent
Rear seal Purge Purge
Flow meter shutoff Inline filter
wash system screws screw
valves
Tubing guides Snap-in valves
Column compartment
Figure 9: NCS-3500RS with ProFlow flow meter
NOTICE The maximum pump pressure available for the column is 900
bar if a ProFlow flow meter is installed.

2.3.2.4 NCS-3500RS with Classic Flow Meter

HHigh pressure gradient NC_Pump Low pressure gradient Micro pump
Rear seal wash
system Flow meter Purge screws Inline filter Purge screw
Tubing guides Snap-in valves
Column compartment
Figure 10: NCS-3500RS with classic flow meter
NOTICE The maximum pump pressure available for the column is 800
bar with a classic flow meter installed.

2.3.2.5 NCP-3200RS
NCP-3200RS module featuring
- NC pump
Figure 11: NCP 3200RS pump
Figure 12: NCP 3200RS pump with ProFlow flow meter installed

Figure 13: NCP-3200RS with a classic flow meter installed
TIP The NCS-3500RS and NCP-3200RS are both compatible with the
ProFlow and classic flow meters.
TIP An upgrade kit from classic nano to ProFlow is available for both
NCS-3500RS and NCP-3200RS. Please order P/N 6041.7850 (ProFlow
flow meter) and P/N 6041.3003 (Upgrade Kit for ProFlow flow meter).
Please see the ProFlow quick installation guide for more details.
2.3.3 Software Compatibility for NCx-3x00RS Operation with ProFlow and

Classic Flow Meters
 ProFlow technology is fully compatible with all previous NCx-3x00RS

modules (mandatory firmware upgrade to version ≥ 1.40 required).
 For LC control via Xcalibur, ProFlow technology requires SII for

Xcalibur ≥ 1.2 with Chromeleon 7.2 SR4 (or later) driver updates.
 ProFlow technology is NOT supported by DCMSLink (based on

Chromeleon 6.8) for Xcalibur.
 For LC control using Chromeleon, ProFlow technology requires:
≥ SR 15b for Chromeleon 6.80

≥ SR4 for Chromeleon 7.2
NOTICE The Chromeleon and SII software versions show in Figure 14 are
the minimum requirements. Later versions are fully compatible.

Figure 14: UltiMate 3000 RSLCnano with ProFlow flow meter –

compatibility matrix

2.3.4 Preparing the RSLCnano for Use

The UltiMate 3000 RSLCnano system must be prepared and primed prior
to use. A brief description of each step is given below. For detailed
information on configuring the system, please refer to the respective
operating instructions for each module.
2.3.4.1 Hardware Installation

 Install the power, SRD and USB cables but do not connect the PC.
 The real seal wash solvent should be installed and primed prior to
powering up the modules (see section 2.3.4.5 for details on how to
prepare the rear seal wash solvent).
 Use the PEEK solvent inlet filter frits for both the NC pump and the
loading pump solvent lines. Do not use metal filters.
 The online degasser for the loading pump must be employed when:
The loading pump is used for gradient formation.

The loading pump flow rate is above 20 µL / min.
 Connect the contact closure cable between relay 4 of the
autosampler and mass spectrometer I/O.
 The WPS-3000TPL RS as well as the WPS-3000 T(B)FC is delivered

with the buffer tubing, needle and sample loop preconfigured on the
6-port injection valve. The positions of each of these components is
essential to the normal operation of the sampler. The correct
configuration is depicted in each of the applications respective
hardware layout.
 Power up the modules.
Notice: 60 minutes are required after pump power up for the flow
meter to reach a stable operating temperature. It is recommended to
execute pump and flow meter purges during this time. Flow meter zero
offset adjustments / calibration routines can only be started after the
module has been powered on for 60 minutes.

2.3.4.2 Software Installation

 For LC-MS control using Xcalibur with SII, the order of software
installation should be Foundation -> Xcalibur -> MS Driver -> SII.
 Once the software is installed, connect the USB cable(s) to the PC.
2.3.4.3 Instrument Configuration

 LC-(UV)-MS configuration is done through the instrument
configuration panel accessed via Xcalibur Foundation Instrument
Configuration.
 Verify that the correct flow meter type is displayed under the flow
meter tab within the instrument configuration panel and that the
valve(s) are correctly configured (oven/valves tab of the NC module).
 When configuring the WPS-3000TPL RS or FC autosampler, ensure

that the settings in the instrument configuration match the fluidic
components installed in the autosampler.
NOTICE The sample loop volume will vary according to the application.
Ensure that the sample loop volume in the instrument configuration
matches the hardware.
2.3.4.4 Solvent Preparation

 Only use fresh LC-MS grade solvents
 Degas (sonicate) 10 minutes before installing
 Refresh every 2 weeks to eliminate bacterial growth and/or changes

in solvent composition
 For best results, use premixed Fisher Chemical Optima LC-MS grade
solvents:
NC Pump solvent A - Water with 0.1% Formic Acid (FA) P/N LS118-500
NC Pump solvent B - 80/20 (v/v) Acetonitrile / Water with 0.1% FA P/N
LS122-500
Alternative for NC Pump solvent B (ProFlow only) - Acetonitrile with
0.1% FA P/N LS120-500
2.3.4.5 Auxiliary Solvents:

The following solvents are recommendations to ensure robust operation

 Rear Seal Wash:

10% methanol in water + 0.1% Formic Acid (FA)
 Loading Pump solvent:

Water with 0.1% FA P/N LS118-500
 Autosampler needle wash:

80/20 (v/v) Acetonitrile / Water with 0.1% FA P/N LS122-500
 Transport liquid for µLiter pick up injection:

application dependent, should be the same as mobile phase A
(direct injection) or the loading buffer (pre-concentration).
NOTICE The autosampler needle wash example is a typical strong wash

solvent. Depending on the application, this may need to be adjusted to
prevent carry-over e.g. to 100% Acetonitrile with 0.1% FA.
2.3.4.6 Purging the Pumps and Flow Meter

The NC pumps and flow meter (NCS-3500RS and NCP-3200RS) and the
loading pump (NCS-3500RS only) require purging each time solvents are
refreshed or changed.
 Purging the NC Pump blocks
The pump block purge time (Table 2) depends on the flow meter type
and whether the solvents are being refreshed or changed:
Flow Meter Type Refresh Solvent Change Solvent
ProFlow 5 min 15 min
Classic 10 min 30 min

Table 2: NC Pump block purge times

 Purging the flow meter
The flow meter purge time (Table 3) depends on the flow meter type
and the application scale (nano, capillary or micro):
Application Recommended Purge Time
Nano LC (ProFlow) 10 min
Nano LC (classic) 30 min
Capillary LC (classic) 5 min
Micro LC (classic) 5 min

Table 3: Flow meter purge times
 Purging the loading pump
It is important that all three solvent lines are purged, irrespective of

whether all three channels are used for an application or just one.
Unused solvent channels should be purged with 10 – 50% isopropanol in
water.
Purge each channel for at least 5 minutes.
2.3.4.7 Performing the Adjust Zero Balance Test / Pressure Transducer Test
The type of test required will depend on the flow meter installed.
 ProFlow flow meter -> adjust zero balance test
The test “Adjust Zero Balance” is located under the “Wellness” button
on the NC pump tab on the ePanel. A wizard will guide you through the
procedure.
 Classic flow meter -> pressure transducer test
The test is located under the “NC_Pump_Diagnostics” tab of the ePanel.
TIP The descriptions above refer to the test locations in SII / CM 7.2. For
information on where to find the test in CM 6.8 / DCMSLink please refer
to the NCx-3x00 operating instructions.

2.3.5 Flow meter calibration

Solvent calibration routines differ for the two types of flow meter.
2.3.5.1 ProFlow flow meter solvent calibration

The ProFlow flow meter uses thermal flow sensors for direct
measurement of solvent flow. The ProFlow flow meter comes pre-
calibrated with four solvent types: Water, 100% Acetonitrile, 80%
Acetonitrile and 100% Methanol. The calibration values for these solvent
types are stored locally on the flow meter and are valid for the life of the
flow meter. They are also valid when the flow meter is transferred from
one UltiMate 3000 RSLCnano system to another.
NOTICE The solvent types are valid for a variation in composition ≤ 2%

i.e. a solvent consisting of 98% Water / 2% Acetonitrile can be run using
the pre-calibrated solvent type Water. This is also applicable if the added
modifier changes >2%.
For all other solvents, a custom solvent calibration is required.
Custom solvent calibrations are only valid for the channel on which they
are carried out. To perform a custom solvent calibration, click on the
“Wellness” button on the NC_Pump panel and then”Calibrate Solvent”.
A wizard guides the user through the calibration procedure.
TIP Custom solvent calibrations are also stored locally on the flow meter
hardware and are valid for the life of the flow meter.
The solvent type for the A and B channels should match the solvents
used in the application. To select the solvent type, go to the
PumpModule tab of the ePanel. Under NC Pump -> More Options select
the desired solvent type for each channel from the drop down menu
under ‘solvents’.
2.3.5.2 Viscosity Measurement Test using the Classic Flow Meter

The classic flow meter uses the pressure drop across a restriction
capillary (integrated in the flow selector) to determine solvent flow rate.
During the viscosity measurement calibration, the resistance of the
solvent is measured relative to the factory calibration value for water

and is reported as a percentage of that value. The viscosity

measurement test is located under the “NC_Pump_Diagnostics” tab in
the ePanel. Typical viscosity values for a number of common solvents
are given in Table 4 below:
Solvent Viscosity %
Water 100
80 / 20 (v/v) Acetonitrile / Water 66
50 % ACN / 50% Water 100
Acetonitrile 50
MeOH 75
IPA 220
Table 4: Viscosity values relative to water for common solvents
TIP The viscosity measurement test takes about 15 minutes. The user
should remain present throughout the test, as prompt user intervention
is required part way through. Once the test is finished, the measured
viscosity values can be stored by selecting the ‘apply’ button for the
respective channel. The results should be examined for plausibility (see
Table 4 for reference values).

2.4 Application Overview
Figure 15: Overview of the Applications available for the RSLCnano

2.5 Direct Injection onto a Nano Column

2.5.1 Hardware Layout
The instrument setup presented in

Figure 16 consists of:
SRD-3400 5035.9245
NCS-3500RS 5041.0010A
VWD-3400RS (optional) 5074.0010
3 nL flow cell 6074.0270
WPS-3000TPL RS 5826.0020
Application kit: 6720.0300
NOTICE The NCS-3500RS in this setup

can be exchanged for an NCP-3200RS
(P/N 5041.0030A)
TIP If no valve is available, a Viper union

(P/N 6040.2304, ordered separately) can
be used to connect the capillary from
the sampler valve directly to the column
positioned here in the column oven.
Figure 16: Setup for a Direct Injection experiment onto a

nano column including the optional UV detector

2.5.2 Fluidic Setup

Figure 16 and Figure 17 show the setup using the parts provided in the
Direct Injection nano application kit. Columns are marked with letters
and the tubing with digits. The sample loop is installed in the WPS-
3000TPL RS autosampler. The correct configuration for the sample loop,
needle and buffer tubing is shown in Figure 16.
Figure 17 Fluidic connections for a direct injection experiment onto a

nano column
TIP If no valve is available, a Viper union (P/N 6040.2304, ordered

separately) can be used to connect the capillary (2) directly to the
column (a).
# Item P/N
a 75 µm I.D. x 15 cm, packed with Acclaim PepMap RSLC C18, 2 µm, 100Å, nanoViper 164534
1 nanoViper capillary FS/PEEK sheathed 1/32" I.D. x L 20 µm x 750 mm 6041.5280
nanoViper sample loop 1 µL, FS/PEEK sheathed I.D. x L 100 µm x 127 mm 6826.2401
Polypropylene vials for WPS with glass insert, 250 µL, 25 pcs 6820.0027
Polypropylene caps for WPS vials, 25 pcs 6820.0028
Cytochrome C digest, 1.6 nmol, Lyophilized 161089

Table 5: RSLCnano Direct Injection nano LC kit (P/N 6720.0300) contents

2.5.3 Installation Tips

 Follow the “General Recommendations for Applications” (section 2.1
on page 15).
 The impact of dwell/dead volumes on reproducibility is highly

significant. Improper connections of the different elements are the
most likely cause of failure for this application.
2.5.4 Testing the Application

Test the direct injection application using the following conditions:
Property Setting
Mobile Phase A Water + 0.1% FA
Mobile Phase B 80/20 (v/v) Acetonitrile / Water with 0.1% FA
Sample Cytochrome C digest, 500 fmol / µL
Injection Volume 1 µL (Full Loop)
UV detection (optional) 214 nm
Gradient 4% to 50% B in 30 minutes, 90% B for 5 minutes, 25 minutes equilibration
Oven temperature 35 °C
WPS temperature 5 °C
Flow Rate 300 nL / min

Table 6: Test conditions for direct injection of 500 fmol Cytochrome C onto a nano column
Figure 18: Typical MS TIC for a direct injection of 500fmol Cytochrome C

onto a nano column

2.5.5 Large Volume Injections

Direct injection applications in nano LC are typically performed with 1 µL
loops to minimize the gradient delay. Larger volume injections are most
commonly performed using a pre-concentration setup. The WPS-
3000TPL RS and FC autosamplers support custom injection programs
(User-Defined Program (UDP)) which switch the injection valve offline
after sample loading to bypass the loop and thereby reduce gradient
delay (See TN 72277 for details). This way, a larger sample volume can
be injected directly onto the nano column, without using a pre-
concentration setup.
The advantage of such a setup is the ease of use and a minimum loss of
peptides, especially hydrophilic ones. The prerequisites of this setup are
i) desalted samples, since all the sample that is injected will enter the
MS, and ii) an investment of extra analysis time to accommodate the
complete loading of sample at low flow rates.

2.6 Direct Injection onto a Capillary Column


SRD-3400 5035.9245
NCS-3500RS 5041.0020
WPS-3000TPL RS 5826.0020
NOTICE The NCS-3500RS in this setup

can be exchanged for an NCP-3200RS
TIP If no valve is available, a Viper union

(P/N 6040.2304, ordered separately)
can be used to connect the capillary
from the sampler valve directly to the
column positioned here in the column
oven.
Figure 19: Setup for a Direct Injection experiment onto

a capillary column including the optional UV detector

2.6.2 Fluidic Setup

Figure 19 and Figure 20 shows the setup using the parts provided in the
Direct Injection capillary application kit. Columns are marked with letters
and the tubing with digits. The sample loop is installed in the WPS-
Figure 20: Fluidic connections for a direct injection onto a capillary

column
TIP If no valve is available, a Viper union (P/N 6040.2304, ordered

separately) can be used to connect the capillary (2) directly to the
column (a).

# Item P/N
a 300 µm I.D. x 15 cm, packed with Acclaim PepMap RSLC C18, 2 µm, 164537
100Å, nanoViper
nanoViper sample loop 5 µL, FS/PEEK sheathed I.D. x L 200 µm x 159 mm 6826.2405

Table 7: RSLCnano Direct Injection capillary LC kit (P/N 6720.0305) contents


on page 15).
 A detailed description of how to set up and run this application

together with optimized measurement parameters for both LC and
MS is also available in TN72277.

Test the direct injection application using the following conditions:
Property Setting
UV detection (Optional) 214 nm
Gradient 1 min 5% B, then 5% to 35% B in 10 minutes, then to 90% B in 1 min. Ramp to 10

µL / min in 0.1 min. Hold at 90% B for 1 min (10 µL / min), then to 5% B in 0.1 min
(10 µL / min). Hold at 5% B for 2.4 min then ramp to 5µL/min in 0.1 min
Flow Rate 5 µL / min during gradient, 10 µL / min during wash and equilibration
Table 8: Test conditions for direct injection of 1 pmol Cytochrome C onto a capillary column

Figure 21: Typical chromatogram for a direct injection of 1pmol Cytochrome C

onto a capillary column

2.7 Pre-concentration onto a Nano Column


SRD-3400 5035.9245
NCS-3500RS 5041.0010A
1x 10-port sw. valve 6041.0001A
WPS-3000TPL RS 5826.0020
Figure 22: Setup for a pre-concentration experiment

onto a nano column including the optional UV
detector

2.7.2 Fluidic Setup

pre-concentration nano application kit. Columns are marked with letters
and tubing with digits. The sample loop is installed in the WPS-3000TPL
RS autosampler. The correct configuration for the sample loop, needle
and buffer tubing is shown in Figure 23.
Figure 23: Fluidic connections for a pre-concentration experiment onto a

nano column
TIP The schematic shows a 10-port switching valve. This application can
also be performed using a 6-port valve.
# Item P/N
b 300 µm I.D. x 5 mm, packed with Acclaim PepMap100 C18, 5 µm, 100Å (set of 5
160454
cartridges)
µ-Precolumn holder, 5 mm, with 30 µm I.D. connecting tubing, nanoViper fittings 164649
nanoViper capillary FS/PEEK sheathed 1/32" I.D. x L 20 µm x 750 mm 6041.5280

# Item P/N
nanoViper sample loop 20 µL, FS/PEEK sheathed 6826.2420
4 PTFE tubing, 500 µm I.D. 100 cm, used in waste tubing 6720.0077
1/16" Universal Fingertight Fitting, one-piece design, extra long thread (4 pieces) 6720.0015

Table 9: RSLCnano pre-concentration nano LC kit (P/N 6720.0310) contents

on page 15).
 For details on installing the trap column, refer to section 2.2–

“available trapping columns” on page 21.
 If a loss of hydrophilic peptides is observed, adding a stronger ion-

pairing agent such as Trifluoroacetic acid (TFA, up to 0.1%) or
heptafluorobutyric acid (HFBA (0.05%) to the loading solvent can be
considered.
 The 20 µm x 750 mm capillary (P/N 6041.5280) can be used to

convert the pre concentration setup to a direct injection
configuration (see capillary 1 in Figure 17). One further capillary is
required (P/N 6041.5260, capillary 2 in Figure 17) which must be
ordered separately.


Test the pre-concentration setup using the following conditions:
Property Setting
Loading solvent Water + 0.1% FA
Injection Volume 1 µL (partial loop fill or µL pickup)
Loading time 0.5 minutes (may vary according to required injection volume / routine)
Gradient 4% to 55% B in 30 minutes, 90% B for 5 minutes, 8.5 minutes equilibration
Loading flow rate 30 µL / min
NC Flow Rate 300 nL / min (ProFlow or classic flow meter with nano flow selector)
Table 10 Test conditions for pre-concentration injection of 1 pmol Cytochrome C onto a
nano column
Figure 24: Typical chromatogram for a pre-concentration of 500 fmol

Cytochrome C onto a nano column

2.8 Pre-concentration onto a 200 µm Monolithic

Column

SRD-3400 5035.9245
NCS-3500RS 5041.0020
1x.10 port sw valve 6041.0001A
WPS-3000TPL RS 5826.0020
Figure 25: Setup for a Pre-concentration experiment

onto a monolithic column including the optional UV
detector

2.8.2 Fluidic Setup

pre-concentration monolithic application kit. Columns are marked with
letters and tubing with digits. The sample loop is installed in the WPS-
Figure 26: Fluidic connections for a pre-concentration onto a monolithic

column

# Item P/N
a PepSwift Monolithic Capillary Column 200 µm I.D. x 5 cm, (PS-DVB), nanoViper 164557
b PepSwift Monolithic Trap Column 164558
PTFE tubing, 500 µm I.D. x 100 cm, used as waste tubing 6720.0077
1/16" Universal Fingertight Fitting, one-piece design, extra long thread, 4 pieces 6720.0015

Table 11: RSLCnano Pre-concentration monolithic LC kit (P/N 6720.0320) contents

on page 15).
 With column oven temperatures below 45°C, TFA can be used

instead of HFBA as a loading solvent modifier.
 If a UV detector is used in the setup, the detectors time constant

should be reduced to 0.1 seconds due to the speed of the
separation.
 If a loss of hydrophilic components is observed, the HFBA solvent

concentration in the loading solvent can be increased to 0.1%.

Test the pre-concentration setup using the following conditions:
Property Setting
Mobile Phase A 100% Water + 0.05% TFA
Mobile Phase B 50 %/ 50% (v/v) Acetonitrile / Water + 0.04% TFA
Loading Solvent Water + 0.05% HFBA

Property Setting
Sample Cytochrome C digest, 1 pmol / µL, in 98% mobile phase A, 2% mobile phase B.
Note: The sample must be diluted in the loading solvent
Injection Volume 0.5 µL (partial loop or µL pickup)
Loading time 3 minutes (may vary depending on the required injection volume / routine)
Gradient 1% to 70% B in 8 minutes, 90% B for 2 minutes, 8.5 minutes equilibration
Loading flow 10 µL / min
Flow Rate 3 µL / min (capillary flow selector)

Table 12 Test conditions for pre-concentration injection of 500 fmol Cytochrome C onto a
monolithic column
Figure 27: Typical chromatogram for a pre-concentration of 500 fmol of

Cytochrome C onto a monolithic column
TIP When the trap column is switched in line with the analytical column,
a large absorption (injection) peak is detected at 214 nm. This is due to
the different UV absorbance of the ion-pairing agents (e.g. HFBA vs. TFA)
in the loading and analytical solvent.

2.9 Pre-concentration onto a Capillary Column


SRD-3400 5035.9245
NCS-3500RS 5041.0020
1x.10 port sw. valve 6041.0001A
VWD-3400RS 5074.0010
WPS-3000TPL RS 5826.0020
Figure 28: Setup for a pre-concentration experiment

onto a capillary column including the optional UV
detector

2.9.2 Fluidic Setup

Figure 28 and Figure 29 presents the setup using the parts provided in
the pre-concentration capillary application kit. Columns are marked with
letters and tubing with digits. The sample loop is installed in the WPS-
Figure 29: Fluidic connections for a pre-concentration onto a capillary

column
# Item P/N
160454
cartridges)

# Item P/N
1/16" Universal Fingertight Fitting, one-piece design, extra long thread (4 pieces) 6720.0015

Table 13: RSLCnano pre-concentration capillary LC kit (P/N 6720.0315) contents

on page 15).
 A detailed description of how to set up this application for LC-MS is

available in TN72277.
 For details on trap column selection and installation, please refer to

section 2.2 - available trapping columns on page 21.
 If a loss of hydrophilic peptides is observed, the concentration of

acetonitrile in the loading solvent can be reduced to ‘1’ % or be
completely removed. The ion paring agent concentration can also be
raised or a strong ion-pairing agent such as trifluoroacetic acid (TFA)
or heptafluorobutyric acid (HFBA) can considered.
 The 50 µm x 750 mm capillary (P/N 6041.5580) can be used to

convert the pre concentration setup to a direct injection
configuration (see capillary 1 in Figure 20). One further capillary is
required (P/N 6041.5560, capillary 2 in Figure 20) which must be
ordered separately.


Test the pre-concentration application using the following conditions:
Property Setting
Loading solvent Water + 0.1% FA
Injection Volume 1 µL (partial loop fill or µL pickup)
Loading time 3 min (may vary according to required injection volume / routine)
Gradient 5% for 1 minute, 5 to 35% B in 10 minutes, 35% B to 90% B in 1 minute, for further
details see TN-72277
NC Flow Rate 4 µL / min (capillary flow selector)

Table 14: Test conditions for pre-concentration injection of 500 fmol Cytochrome C onto a
capillary column
Figure 30: Typical chromatogram for a pre-concentration of 1 pmol

Cytochrome C onto a capillary column

2.10 EASY-Spray Columns with the RSLCnano

2.10.1 EASY-Spray Concept
EASY-Spray is an integrated separation column and emitter concept
designed for robust plug and play low-flow LC-MS analysis. EASY-Spray
columns consist of an integrated separation column and emitter
connected via a zero dead volume union, minimizing post column
volumes and dispensing of the need for tricky post column emitter
connections.
Figure 31 shows a schematic of the EASY-Spray column hardware. The

integrated emitter is protected by a spring-loaded cover, which shields it
when it is not installed. The cover is retracted when the EASY-Spray
column is inserted into the EASY-Spray source.
Separation
column and
temperature 7 μm internal
control diameter
glass emitter
nanoViper
fitting
Zero dead volume union
Figure 31: Schematic overview of an EASY-Spray column
The EASY-Spray column simply slots into the EASY-Spray source and is
connected to the LC outlet using a viper union. In-built column
temperature control ensures optimal retention stability and consistent
chromatographic performance. Figure 32 shows the EASY-Spray source
with an EASY-Spray column installed.

Camera with LED light
Source body
EASY-Spray column
nanoViper connection
Proximity alignment
Temperature dial and readout
Temperature control cable
Figure 32: EASY-Spray source with EASY-Spray column installed
Alignment of the EASY-Spray column is only required when the source is

installed for the very first time on the Mass Spectrometer.
NOTICE The EASY-Spray Ion Source interface is available in two formats, the EASY-
Spray Ion Source (P/N ES081) and EASY-Spray Ion Source NG (P/N ES082). The source
required will depend on the mass spectrometer type (see Table 15 below).
Ion Source Model Thermo Scientific Mass Spectrometer
EASY Spray NG (ES082) TSQ Series

Orbitrap Fusion™ Series
Endura MD™
EASY-Spray (ES081) Exactive™ Series

Orbitrap™ Series
LTQ™ Series
LCQ™ Dec XP Max
Table 15: EASY-Spray ion sources and compatible mass spectrometers

2.10.2 Example Separation Performance of an EASY-Spray Column

An example base peak Chromatogram for a BSA digest run on an Easy-
Spray (ES801) column is shown in Figure 33. The direct connection
between the column outlet and the column emitter affords highly
resolved peaks with virtually zero post column band broadening (Figure
33 A and B).
100 A PWHH
B
2.5 sec
100 A
80
Relative Abundance
0
60 12.00 12.10 12.20
Time (min)
100 B PWHH
40 3.0 sec
20
0 0
14.20 14.30 14.40
5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Time (min)
Time (min)
Figure 33: Base Peak Chromatogram of an EASY-Spray column in pre-

concentration mode

2.10.3 Hardware Layout Direct Injection

The instrument setup for EASY-Spray
direct injection presented in Figure 34
consists of:
SR-3000 5035.9200
NCP-3200RS 5041.0030A
WPS-3000TPL RS 5826.0020
NOTICE The NCP-3200RS in this setup

can be exchanged for an NCS-3500RS.
Figure 34: Setup for direct injection with EASY-

Spray
The instrument setup for pre-

concentration with EASY-Spray
presented in Figure 35 consists of:
SRD-3400 5035.9245
NCS-3500RS 5041.0010A
1x 10-port sw.valve 6041.0001A
WPS-3000TPL RS 5826.0020
Figure 35: Setup for pre-concentration with EASY-

Spray

2.10.4 Fluidic Setup using EASY-Spray Columns

The fluidic setups for EASY-Spray with an UltiMate 3000 RSLCnano are
essentially identical to those used for the direct injection and pre-
concentration applications already described.
The only difference is that instead of connecting the separation column

to the valve (injection or 10-port) directly, it is connected using a Viper
union and nanoViper capillary running from the valve to the EASY-Spray
column. Because the connection tubing is placed before the separation
column, it has no negative impact on separation performance. (i.e. band
dispersion).
TIP The gradient delay resulting from the connecting capillaries varies
according to the length and inner diameter of the tubing. As a rule of
thumb, every 10 cm of 20 µm I.D. tubing contributes 30 nL or 6 seconds
delay at 300 nL/ minute.
Various lengths of connecting capillaries are provided in the UltiMate

3000 RSLCnano EASY-Spray connection kit (see Table 16 below) to offer
maximum user flexibility when connecting the UltiMate 3000 RSLCnano
to the EASY-Spray column. To minimize gradient delay volumes, the
shortest possible connecting capillary is recommended.
Item P/N>
300 µm I.D. x 5 mm, packed with Acclaim PepMap100 C18, 5 µm, 100Å 160454
(set of 5 cartridges)
µ-Precolumn holder, 5 mm, with 30 µm I.D. connecting tubing, 164649

nanoViper fittings
nanoViper Capillary FS/PEEK sheathed 1/32" I.D. x L 20 µm x 350 mm 6041.5240
nanoViper Sample Loop 20 µL, FS/PEEK sheathed 6826.2420
Union Viper 2261.5061
PTFE tubing, 500 µm I.D. x 100 cm, used as waste tubing 6720.0077

Item P/N>
1/16" Universal Fingertight Fitting, one-piece design, with extra long 6720.0015
thread, 4 pieces
Polypropylene vials for WPS with glass insert, 250 µL, 25 pieces 6820.0027
Polypropylene caps for WPS vials, 25 pieces 6820.0028

Table 16: UltiMate 3000 RSLCnano EASY-Spray connection kit (P/N
6720.0395) contents
For a list of available EASY-Spray columns, see – Table 28: EASY-Spray

columns in the Appendix.

2.10.5 EASY-Spray Transfer lines

The EASY-Spray source can also be used in conjunction with linear
columns by adopting an EASY-Spray transfer line (Figure 36: EASY-Spray
Transfer Lines) consist of an emitter with a form factor compatible with
the EASY-Spray source, attached to a nanoViper fitting via a fused silica
capillary.
Figure 36: EASY-Spray Transfer Lines
EASY-Spray transfer lines are available in both nano fIow (P/N ES791A)
and capillary /micro flow (P/N ES792A) compatible formats.

2.11 2D Salt Plugs with Nano Column

2.11.1 Hardware-Layout
The Instrument setup presented in
SRD-3400 5035.9245
NCS-3500RS 5041.0010A
VWD-3400RS 5074.0010
WPS-3000TPL RS 5826.0020
Figure 37: Setup for a 2D Salt Plugs experiment

including the optional UV detector

2.11.2 Fluidic Setup

Figure 37 and Figure 38 presents the setup using the parts provided in
the 2D-LC Salt Plugs application Kit. Columns are marked with letters and
tubing with digits. The sample loop is installed in the WPS-3000TPL RS
autosampler. The correct configuration for the sample loop, needle and
buffer tubing is shown in Figure 37.
TIP The schematic shows 10-port switching valves. This application can
also be performed on 6-port valves. Ensure that the relative positions on
the connections are correct and update the valve switching in the
instrument setup and method as necessary.
Figure 38: Fluidic connections for a 2D-LC Salt-Plugs experiment
# Item P/N
a 300 µm I.D. x 10 cm, packed with Poros 10 S with connections, 130 µm I.D.
164565
FS sheathed inlet and outlet, nanoViper
b 75 µm I.D. x 15 cm, packed with Acclaim PepMap RSLC C18, 2 µm, 100Å, nanoViper 164534
c 300 µm I.D. x 5 mm, packed with Acclaim PepMap100 C18, 5 µm, 100Å (set of 5
160454
cartridges)

# Item P/N
1/16” Universal Fingertight Fitting, one piece design, long thread, 4 pieces 6720.0015
6 Protein Digest Standard, 100 pmol, Lyophilized 88342

Table 17: RSLCnano 2D salt plug Kit NCS-3x00 (P/N 6720.0325) contents

on page 15).
 If a loss in hydrophilic peptides is observed, the concentration of

acetonitrile in the loading solvent can be decreased down to 99/1
water/acetonitrile + 0.025% TFA.
 If too much hydrophobic secondary interaction is observed on the

IEX column, the amount of ACN can be increased up to 5% or 10%.
This will be at the expense of the loading efficiency for hydrophilic
peptides on the RP trap column.
 The loading time and desalting time are highly dependent on the
sample quantity and purity. They can be adjusted to meet customer
needs. However, the desalting step must be kept long enough to
avoid the formation of adducts between salt and sample.
 To limit the breakthrough on the SCX column, the loading solvent

must contain as little TFA as possible (maximum of 0.025%).
Alternatively, FA (~ 0.5%) can be used.
 The salt plugs listed here have been chosen for the separation of the
protein mix digest. The best sequence of plugs will highly depend on
the affinity of the peptides present in the sample with the IEX
column.

 After each series of injections, it is useful to wash the column with

consecutive 2 M salt injections. When the salt is washed out from
the column, a 60/40 water/ACN solution can also be used to wash
out peptides which might be bound to the column due to
hydrophobic interactions.

Test the 2D salt plug setup using the following conditions.
Property Setting
Loading solvent 95%/2% (v/v) water/ acetonitrile + 0.025% TFA
Sample Protein mix digest 100 pmol, lyophilized, prepared according to instruction sheet
Salt plugs concentration 1 mmol NaCl

(mol / L) 2 mmol NaCl
5 mmol NaCl
10 mmol NaCl
20 mmol NaCl
50 mmol NaCl
100 mmol NaCl
200 mmol NaCl
1000 mmol NaCl
2000 mmol NaCl
Injection Volume Sample: 10 µL (partial loop fill or µL pickup)

Salt plugs 20 µL
Loading time 5 min (may vary according to required injection volume / routine)
Desalting time 7 min (started after the loading time has passed)
Gradient Isocratic 4% for 10 minutes 4% to 55% B in 30 minutes, 90% B for 5 minutes, 18

minutes equilibration
Oven Temperature 35 °C
NC Flow Rate 300 nL / min (capillary flow selector)

Table 18: Test conditions for a protein digest separation using 2D Salt Plugs

The successful installation of this application is based on the

following attributes:
 The injection profile should be reproducible.
 The peptides should be equally distributed over and within the

different fractions (orthogonal separation).
2.11.5 Salt Solution Preparation

The following protocol can be used to prepare the salt plugs
 Prepare two stock solutions using the loading solvent:
1. 2000 mM NaCl (e.g., 467.5 mg of NaCl in 4 ml loading solvent
2. 100 mM NaCl (e.g. prepare the 100 mM solution of the first table
two times)
 Dilute the stock according to Table 19 and Table 20 below: Use

standard 1.5 ml vials (do not use vials with inserts).
Concentration of Volume of 2000mM NaCl Volume of loading solvent Total Volume

NaCl stock solution
2000 mM 1000 µL 0 µL 1000 µL
1000 mM 500 µL 500 µL 1000 µL
500 mM 1250 µL 750 µL 1000 µL
200 mM 100 µL 900 µL 1000 µL
100 mM 50 µL 950 µL 1000 µL

Table 19: Guide for the preparation of the Salt Plugs (dilutions from 2000 mM NaCl)
Concentration of Volume of 100mM NaCl Volume of loading solvent Total Volume

NaCl stock solution
50 mM 500 µL 500 µL 1000 µL
20 mM 200 µL 800 µL 1000 µL
10 mM 100 µL 900 µL 1000 µL
5 mM 50 µL 950 µL 1000 µL
2 mM 20 µL 980 µL 1000 µL
1 mM 10 µL 990 µL 1000 µL
Table 20: Guide for the preparation of the Salt Plugs (dilutions from 100mM NaCl)

2.12 Tandem Nano LC

Figure 39: Setup for a Tandem nano LC experiment including the optional UV detector
The instrument setup presented in Figure 39

TIP All the components required to convert
consists of:
the WPS-3000FC for tandem nano LC use
are included in the application kit, except
SRD-3400 5035.9245
the WPS injection valve (P/N 6826.0011A)
NCS-3500RS 5041.0010A
which must be ordered separately.
NCP-3200RS 5041.0030A
The NCP 3200RS accessory kit has two
VWD-3400RS 5074.0010
130 cm long solvent inlet tubings, which
supply solvent from the bottles placed on
WPS-3000FC 5824.0020 the solvent rack to the NCP at the bottom of
Injection valve 6826.0011A the stack.

2.12.2 Fluidic Setup

Tandem nano LC application kit. Columns are marked with letters and
tubing with digits. The sample loop is installed in the WPS-3000TPL RS
autosampler. The correct configuration for the sample loop, needle and
buffer tubing is shown in Figure 39.
TIP The schematic below shows 10-port switching valves. This

application can also be performed using 6-port switching valves. Ensure
that the relative positions of the connections are correct and update the
valve switching in the instrument setup and method as necessary.
Figure 40: Fluidic connections for a Tandem nano experiment
NOTICE Control of the post column nano valve can either be performed
using the WPS-3000FC autosampler or by using an external USB
controlled universal electric actuator (P/N EUHB) and corresponding
mounting hardware (P/N CMH12H) from VICI® Valco Instruments, to
control the valve. If the WPS-3000FC sampler is used, the nano injection
kit (P/N 6824.0030) should be installed and the lower valve on the WPS-
3000FC replaced with the 1/32” nano switching valve (P/N 6820.6232).
All necessary parts are included in the kit. A detailed description of how
to set-up, configure and control both hardware variants is given in
TN72899.

# Item P/N
160454
cartridges)
2,3 nanoViper capillary FS/PEEK sheathed 1/32" I.D. x L 75 µm x 650 mm 6041.5775
1/16” Universal Fingertight Fitting, one piece design, long thread, 4 pieces 6720.0015
Fused silica tubing I.D. 20µm O.D. 280µm, 5 meters for nano LC connections 160475
Cutter for fused silica tubing (cleavage stone) 6720.0016
Upgrade kit nano/cap WPS-3000TFC 6824.0030
1/32” 2 pos 6 port nano switching valve (C2N series) 6820.6232
Fittings 1/32” for C2N series nano valve 6820.1320
1/32" PEEK sleeve, 3 cm, 300 µm I.D. (6 pieces) 6720.0079

Table 21: RSLCnano Tandem nano LC kit (P/N 6720.0335)

on page 15).
 The columns are shipped with I.D. x L. 20 µm x 30 cm attached.

Fused silica capillary tubing is provided in the kit to extend the
column outlets in order to reach the nano valve if necessary.
Replacing the attached fused silica by the appropriate length using
the nano connector on the column will give the best result.
 If a loss in hydrophilic peptides is observed, the acetonitrile in the

loading solvent can be removed and / or the 0.1 % FA can be
replaced with 0.05% TFA or 0.1% TFA as required.

 The WPS-3000FC is typically used for fraction collection. By replacing

the divert valve with the nano valve, the autosampler is compatible
with tandem nano LC. Controlling the divert valve position to switch
between the two analytical columns is performed with the
commands Sampler.Collect and Sampler.Drain.

The tandem nano LC setup consists of two pre-concentration nano
setups that can be operated individually; therefore, the system can be
tested and evaluated using the conditions in the table below, as also
described in section 2.7 Pre-concentration onto a Nano Column shown
on page 47.
TIP Testing both pre-concentration setups individually is recommended

before setting them in combination.
Property> Setting
Loading Pump A Water + 0.1% FA
Loading Time 3 min
Gradient NC Pump 4% to 55% B in 30 minutes, 90% B for 5 minutes, 15 minutes equilibration
Oven Temperature 35 °C
Loading flow Rate 5 µL / min
NC Pump flow rate 300 nL / min

Table 22 Test conditions for running the tandem nanoLC application

2.12.5 Tandem NanoLC-MS

Tandem nanoLC can also be coupled directly to the MS detector (i.e.
without employing an optical UV detector). For this configuration, the
modules are mounted together in a single stack.
A deep proteomics application which employs long shallow gradients on

50 cm columns together with full instructions on how to connect,
configure and program the system is detailed in TN72899. The
customized LC methods describe how the system can be used to
constantly deliver sample to the mass spectrometer, thus enabling the
equivalent sample throughput provided by two single nano LC-MS
systems.
Complete LC method parameters for this set-up are also available for
download from the AppsLab library.

2.13 High throughput tandem capillary-flow LC-MS

Tandem capillary-flow LC workflows combine high sensitivity with high
throughput, permitting the analysis of up to 200 samples per day whilst
yielding 100% MS utilization.
Details on how to connect and configure the UltiMate 3000 RSLCnano in

order to run this application are provided in TN72827 together with
complete LC-MS method parameters.
Example data files containing full LC and MS parameters are also

available for download from the AppsLab library.
TIP There is no tandem capillary-flow application kit available to support

this workflow. However, a full list of all the fluidic components required
together with part numbers and set-up instructions necessary for a
successful installation of the application are provided in TN72827.

2.14 Micro LC Applications

Micro LC applications typically employ flow rates from between 10 and
50 µL / minute using columns with internal diameters between 500 µm
and 1 mm.
For such applications, Thermo Fisher Scientific recommends the use of

50 µm I.D. nanoViper capillaries. A list of the available
nanoViper capillaries is available in Table 31 on page 99.
If UV detection is required, the cap flow cell (45 nL; P/N 6074.0280) is
recommended for all micro LC applications.

2.15 MS Connection Kit

The UltiMate 3000 RSLCnano MS connection kit (P/N 6720.0345; Table
23) contains a variety of common tubings, unions and connectors
necessary to facilitate coupling of the LC column to a mass spectrometer
interface.

Item P/N
Nano LC column to MS tubing I.D. x O.D. x L 20 µm x 280 µm x 1m 6041.5292
Nano LC column to MS tubing I.D. x O.D. x L 20 µm x 360 µm x 1 m 6041.5293
Capillary LC column to MS tubing I.D. x O.D. x L 50 µm x 280 µm x 1 m 6041.5294
Capillary LC column to MS tubing I.D. x O.D. x L 50 µm x 360 µm x 1 m 6041.5295
Fused silica tubing I.D. 20 µm ±3 µm/O.D. 280 µm ±10 µm, 5 meters 160475
Fused silica tubing I.D. 50 µm ±3 µm/O.D. 280 µm ±10 µm, 5 meters 160477
Cutter for fused silica tubing (cleavage stone) 6720.0016
PTFE tubing, 250 µm I.D., low pressure connection of 280 µm O.D. 6720.0030
fused silica capillaries, 5 pieces
1/16" Valco Ferrule and Nut, stainless steel, 10 pc. (for 10-port valve) 161103
PEEK sleeves, precision cut and polished for connections with fused 6720.0064
silica tubing (280 µm O.D.), 5 pieces
Microtight Union inclusive 2 fittings and 1 gauge plug 6720.0074
PEEK sleeves, precision cut and polished for connections with 6720.0076
Microtight Union (360 µm O.D.), 10 pieces
Nano connector incl. sleeves, dead-volume free, up to 300 bar 6720.0390
Sleeves for nano connector, 5 pieces 6720.0391

Table 23: RSLCnano MS Connection Kit (P/N 6720.0345)
TIP The connection between the LC separation and the MS should have
the lowest volume possible to minimize dispersion. Keep in mind that
internal diameter has a much bigger effect on dispersion then the
length of the connecting tubing. Ensure that tubing with the correct
diameter is used at all times.

2.15.1 Mass Spectrometry Interfaces for Linear columns and / or interfacing

the UV detector with the MS.
The mass spectrometry interface connects the LC outlet to the mass
spectrometer inlet. It is here that the column effluent is ionized and
introduced into the mass spectrometer. The type of interface required is
dictated by the application flow rate and type of MS instrument. The
common interfaces applicable to linear columns and / or the VWD
3400RS UV detector available for Thermo Scientific mass spectrometers
are shown below. For details on the connections, also see section 2.1.4.
The MS interface for EASY-Spray is discussed in section 2.10.
2.15.1.1 Nanospray Flex Series Ion Sources

Nanospray Flex™ Ion Source
P/N: ES071
Nano Flow: 20 -1000 nl/min*

*depending on spray needle
MS compatibility
LTQ™ and VelosTM Series
Orbitrap™ Series
Exactive™ series
Legacy TSQ™ series (Quantum Access
Max, Vantage, Ultra etc.)
Nanospray Flex NG™ Ion Source

P/N: ES072
Nano Flow: 20 -1000 nl/min*

*depending on spray needle
MS compatibility
Orbitrap Fusion™ series

TSQ™ Quantis, Altis, Endura and Quantiva
Figure 41: Nanospray Flex Series ion sources

2.15.1.2 Heated Electrospray Ionization (HESI-II) Probe and H-ESI Spray Insert
For micro- and capillary-flow applications with columns ≥ 300 µm i.d.

and flow rates ≥ 5 µL/min, the Ion Max Source and accompanying
ionization probe can be used when adapted for low flow rates. Two
electrospray ionization source housing types are available for the
Thermo Fisher Mass Spectrometers, the Ion Max and Ion Max NG
source. The source type depends on the mass spectrometer type. Each
has their own electrospray ionization probe (see Figure 42).
Heated Electrospray Ionization

(HESI-II) Probe for the Ion MaX source
P/N OPTON-20037 Kit
MS Compatibility
LTQ™ and Velos™ Series
Orbitrap™ Series
Exactive™ series
Legacy TSQ™ series (Quantum Access
Max, Vantage, Ultra etc.)
H-ESI Spray Insert for Ion Max NG Source
P/N 80000-60321
Figure 42: Ionization probes for the Ion Max and Ion Max NG sources.

A low-flow (50 µm I.D.) metal needle is required for low-flow

experiments to give the best chromatographic performance. Both PEEK
and fused silica capillaries are available to interface the source with the
column outlet. A compatibility matrix for the different low-flow options
is shown in Figure 43.
Figure 43: Ionization probes for the Ion Max and Ion Max NG sources

3  FAQs
3 FAQs

3  FAQs
3.1 NC_Pump Solvent Recalibration – Best Practice

The main reason for performing a solvent calibration is to improve
retention time stability or to correct a retention time drift. The
frequency and type of recalibration differs between the ProFlow and the
classic flow meter. Recommendations for re-calibration routines for the
two types of flow meter are provided below.
NOTICE The pump blocks and flow meter should be purged regularly
with fresh solvents. Purging is especially important, before any
calibration routines are carried out. Please see section 2.3.4.6 for details.
3.1.1 ProFlow Flow Meter
3.1.1.1 Adjust Zero Balance Test

Once every 3 months or when a retention time drift is observed.
3.1.1.2 Solvent Calibration

One time only per solvent per channel, for custom solvents ONLY. No
recalibration required.
3.1.2 Classic Flow Meter
3.1.2.1 Pressure Transducer Test

Every time solvents are refreshed or when a retention time drift is
observed.
3.1.2.2 Viscosity Measurement Test

Every time solvents are exchanged or refreshed.
TIP The test also acts as a diagnostic tool.

3  FAQs
3.2 Interpreting a Chromatogram

An LC-UV example Cytochrome C separation using TFA as ion pairing
agent is shown in Figure 44. The different areas of a chromatographic
separation are marked inside the figure.
Figure 44: Example Cytochrome C separation with different parts of the

run identified
The finite volume of an HPLC system results in time offset between the
formation of a gradient, its delivery onto the column and the detection
of the gradient change by the (UV and / or MS) detector. This so-called
gradient delay can be visualized by comparing the programmed gradient
to the UV signal. Figure 44 shows the gradient delay between pump and
UV detector.
The inject peak corresponds to the injection peak in direct injection

setups; in pre-concentration setups this part of the baseline,
corresponds to the switching of the trapping column in line with the
nano column.
The dwell volume represents the volume between the autosampler and
the nano columns. Since there are usually one (direct injection) or two
(pre-concentration) valve switches involved in the application,
introducing additional capillaries and connections, the dwell volume and
gradient delay are not the same volume in direct injection and pre-
concentration setups.

3  FAQs
3.3 Troubleshooting Nano LC Peptide Applications

The above LC-UV chromatogram Figure 44 shows the separation of a
Cytochrome C digest on a nano column. The Cytochrome C standard is
simple compared to a typical proteomics sample and is, therefore, ideal
for troubleshooting nano LC setups.
TIP When troubleshooting a pre-concentration setup, Thermo Fisher

Scientific recommends switching back to a direct injection setup if the
tips below do not provide the remedy. An important and often
overlooked step in troubleshooting is simplifying the setup to isolate the
problem.
In assessing the separation performance of a system, several factors are

evaluated, which are organized in the flow chart below (Figure 45). The
values in the flowchart are based on a Cytochrome C digest separation;
when working with a different standard use a trusted reference
chromatogram for the expected values for number of peaks, intensity
and elution window.

3  FAQs
Figure 45: Decision Tree for troubleshooting a low-flow

3  FAQs
3.4 The Use of TFA and FA

The separation of peptides by reversed phase is carried out in the
presence of an ion-pairing agent, which serves a double function. First,
these (typically) weak acids bring the pH of the solvents down to pH 2-3,
causing most peptides to have an overall positive charge. Secondly, the
negative counter-ion of the acid will serve as an ion-pairing agent for the
positively charges peptides to create an overall neutral analyte that is
more efficiently separated on the RP column. The double function of the
ion-pairing agent results in an efficient separation with minimal
additives added to the solvents.
Most commonly, trifluoro acetic acid (TFA) and formic acid (FA) are
used. In this manual and in most LC-MS applications, FA is preferred as
its use minimizes ion-suppression effects. TFA is a stronger ion-pairing
agent and results in better chromatography, but can result in ionization
suppression. The use of TFA is generally restricted to the loading buffer
or when increased retention (compared to FA) is necessary. When
performing the applications described in this manual with TFA instead of
FA, use the amounts given in Table 24. Figure 46 demonstrates the
effect of ion pairing agent choice on the chromatographic separation of
Cytochrome C.

3  FAQs
Figure 46: Comparison of Cytochrome C separation with different ion

pairing agents. Top uses 0.05% TFA and bottom 0.1% FA

3  FAQs
3.5 Minimizing Baseline Effects

The 3 nL flow cell (P/N 6074.0270) and 45 nL flow cell (P/N 6074.0280)
are designed to function in the same way as transfer tubing normally
used to connect a column outlet to a mass spectrometer. This allows UV
detection in nano and capillary LC without introducing detrimental post
column band broadening.
Typically, peptide UV detection is performed at a wavelength of 214 nm,

at which most organic compounds absorb quite strongly. A number of
actions can be taken to minimize baseline drift and noise for optimal use
of the UV detection.
3.5.1 Drift
Ensure that the UV lamp has been switched on for sufficient time in
order that the lamp temperature can stabilize. Chromeleon can detect
this and will give a warning during the ‘Ready Check‘ whenever the UV
lamp temperature is not stable. In such cases, the UV detector can be
used but may not perform optimally.
Gradient RP nano LC typically involves a significant change in solvent

composition. The higher absorption from the organic modifier in the
B solvent will result in a rise of the baseline. Varying the ion-pairing
agent concentration (typically FA or TFA) in the A and B solvent can be
used to compensate the baseline rise. As a rule of thumb, the ion-pairing
agent concentrations indicated in Table 24 can be used to obtain a
straight baseline.
Solvent A Solvent B
0.1% 0.08%
0.05% 0.04%
Table 24: Ion pairing agent addition
The age of both lamp and flow cell can have a significant influence on
baseline drift. New lamps and flow cells may show some drift during the
so-called ‘burn in’ period. Allowing sufficient equilibration time for the
lamp is necessary for obtaining a stable baseline.
Lamps should be replaced after approximately 2000 hours. Older flow

cells can be cleaned by flushing overnight with organic solvent or for a

3  FAQs
short period with a strong acidic solution; see the Variable Wavelength
Detector’s Operating Instructions for more details.
3.5.2 Unstable Baseline

Unstable baselines can have various causes. The UltiMate 3000
RSLCnano pumps are designed to provide the best gradient precision,
but solvent miscibility can present a problem. Therefore, Thermo Fisher
Scientific recommends using a minimum of 5% water in the organic
mobile phase.
Baseline artifacts in pre-concentration applications using low loading

flows (< 10 µL/min) may occur. These artifacts are generally only
observed in the UV signal and have no effect on the performance of the
analysis. If artifacts in the baseline are observed, Thermo Fisher
Scientific recommends bypassing the degasser in pre-concentration
applications where no gradient formation is required and loading flows
are below 20 µL/min. If bypassing the degasser is undesired or
impossible, an alternative is to maintain degassing, but to increase the
loading flow during the elution phase to values between 30 and 100
µL/min.

3  FAQs
3.6 Typical WPS-3000TPL RS Autosampler Settings for

Standard Injection Routines.
The Chromeleon software gives the user the freedom to define multiple
settings for the standard injection routines including sample height,
draw speed and puncture depth. Below are typical WPS-3000TPL RS
settings, which can be adopted for common low-flow workflows.
Parameter Description Typical Value
DispSpeed Sets the speed of the syringe used for 2 µL / sec

dispensing the sample%
DrawSpeed Sets the speed of the syringe used for drawing 0.2 µL / sec
the sample.
DrawDelay Sets the time that the needle remains in the 5 sec
vial after drawing the sample
WashSpeed Sets the speed of the syringe for the wash 4 µL / sec
cycle.
WasteSpeed Sets the speed of the syringe used for 4 µL / sec

expelling liquid to the waste.
DispDelay Time needle remains in vial after dispense 2 sec
DrawDelay Time needle remains in vial before liquid is 5 sec

drawn
Sample Height The height at which sample is drawn 2 mm
TransLiquidHeight The height at which transport liquid is drawn 3 mm
PunctureDepth Depth of puncture needle beyond pusher 8 mm

trigger point for sample vial / well
TransVialPunctureD Depth of puncture needle beyond pusher 8 mm

epth trigger point for transport vial
WashVolume Volume used in wash operation 50 µL
FlushVolume Flush volume used in full loop, partial loop 5 µL

and µL pick up injections
FlushVolume2 Flush volume used in full loop and partial loop 3 µL

injections from the same vial.
Loop Overfill Loop overfill factor used in full loop only 2.0
Table 25: Typical WPS-3000TPL RS autosampler settings

4  Appendix
4 Appendix

4  Appendix
4.1 Customized Sample Injection Routines

4.1.1 Introduction to User Defined Program (UDP) Injection Routines
WPS-3000TPL RS autosamplers are equipped with standard injection
routines for Full-loop, Partial-loop and µL pickup, which are suitable for
the majority of applications. However, for applications requiring special
injection routines, or for users wishing to customize one of the standard
injection methods, a UDP should be used.
UDPs provide the user with full control over every aspect of the injection
routine and enable every type of injection, from simply filling the loop
and injecting the sample to complete liquid handling, such as in-well
digestion.
4.1.2 Important Considerations when Writing a UDP

Although a UDP is flexible, it is also unforgiving: every step of the
injection routine must be programmed manually and missing steps will
lead to erroneous injections. The Chromeleon / SII software will execute
the steps sequentially until it encounters an error after which it will
either continue to the next sample or interrupt the analysis.
4.1.3 The UDP Commands

The general layout of a UDP can be broken down into five different
steps:
1. Sample preparation (only when applying liquid handling)
2. Preflush of the injection needle
3. Filling the sample loop
4. Injecting the sample and starting the acquisition run
5. Washing the syringe and preparing for the next injection
Step ‘1’ is often omitted, as samples are usually ready to inject when
placed in the autosampler.

4  Appendix
4.1.4 Example µL pickup UDP for Maximum Sample Pickup.

This example details the steps required to increase the amount of
sample that can be injected in a µL pickup experiment. This type of
injection routine can be used in a pre-concentration experiment for
example.
NOTICE The standard µL pickup injection routine included in the method

editor contains built in volume restrictions, which limit the volume of
sample that can be taken up into the loop, but are designed to ensure
excellent quantitative properties; therefore they should be used in
preference to the following UDP for absolute quantitation experiments.
In the following example, 10 µL of sample is injected using a 20 µL loop

using transport liquid placed in vial R1.
Figure 47: Example UDP injection routine for maximizing µL pickup

volume

4  Appendix
This injection routine can be considered in the steps (2 to 5) described

above.
Step 2 – Preflushing the injection needle (commands 1 to 4)
Step 3 – Filling the sample loop (commands 5 – 9)
Step 4 – Injecting the sample and starting the run (commands 10 – 11)
Step 5 – Washing the syringe and needle and preparing for the next
sample (commands 12-14)
4.1.5 Variable Injection Volumes

In UDPs such as that described in section 4.1.4 above, the injection
volume is pre-defined within the UDP method itself. As such, the
injection volume will remain the same for all analyses run using such a
method, irrespective of the volume entered in the sample sequence
table.
If flexibility in the injection volume is required, the UPD method can be

altered such that the sample volume is defined by the injection volume
as defined in the sample table.
The following steps describe how to adapt the method given in section
4.1.4 to permit injection volumes according to those defined in the
sample table.
Step 1 – Select the “script Editor” tab in the method file
Step 2 - Navigate to the line where the sample vial volume is defined
Step 3 – Delete the text ‘Volume= 10 [µL]’
Step 4 - Replace with ‘Volume=system.Injection._Volume’ (make to

place a comma at the end of the line)
Step 5 – Save the method with an appropriate name

4  Appendix
4.2 Common Application Related Consumables
The tables below list consumables associated with the applications

outlined in this handbook.
4.2.1 Columns
The most common analytical columns used with the UltiMate 3000
RSLCnano system are listed below. For a comprehensive guide of all
available column formats please refer to the Thermo Scientific
Chromatography columns and consumables catalogue.
Item P/N
Acclaim PepMap RSLC C18, 2 μm, 100 Å, 50 μm I.D. x 15 cm, nanoViper 164562
Table 26 Acclaim PepMap C18 RSLC 2 µm particle size columns
Item P/N
μ-Precolumn™ holder, 5 mm, with 30 μm i.d. connecting tubing, nanoViper

164649
fittings
300 µm i.d. x 5 mm, packed with Acclaim PepMap100 C18, 5 µm, 100Å (set
160454
of 5 cartridges)
Acclaim PepMap, C18 3 μm, 100 Å, 75 μm I.D. x 15 cm (20 mm bed length),

164535
nanoViper
Table 27 Acclaim PepMap C18 trap columns

4  Appendix
Item P/N
EASY-Spray column, 15 cm x 75 µm I.D., PepMap100 C18, 3 µm, 100Å ES800A
EASY-Spray column, 15 cm x 50 µm I.D., PepMap RSLC C18, 2 µm, 100Å ES801A
EASY-Spray column, 15 cm x 75 µm I.D., PepMap100 C18, 2 µm, 100Å ES804A
EASY-Spray column, 25 cm x 200 µm I.D., PepMap Pepswift monolith ES810A
EASY-Spray column, 15 cm x 75 µm I.D., Acclaim PepMap C4, 300Å ES811A
EASY-Spray column, 15 cm x 75 µm I.D., Acclaim PepMap C18, 300Å ES812A
EASY-Spray emitter, nano-flow (20µm I.D. transfer line, 7µm I.D. emitter) ES791A
EASY-Spray emitter, micro-flow (75µm I.D. transfer line, 20µm I.D. emitter) ES792A
Table 28: EASY-Spray columns
/N
Item P/N
PepSwift Monolithic Capillary Column, 200 μm I.D. x 5 cm, nanoViper 164557
PepSwift Monolithic Capillary Column, 200 μm I.D. x 25 cm, nanoViper 164542

Table 29: PepSwift Monolithic linear analytical columns
Item P/N
PepSwift Monolithic Trap Column, 200 μm x 5 mm, set of 2, nanoViper 164558

Table 30: Pepswift Monolithic trap columns

4  Appendix
4.2.2 nanoViper Capillaries, Sample Loops and Connectors
Length I.D. [Colour Code]

(mm)
10 μm 20 μm 50 μm 75 μm 100 μm 150 μm
[Green] [Orange] [Brown] [Black] [Red] [Purple]
70 6041.5120 6041.5123 6041.5126 6041.5810 6041.5817
6041.5118 6041.5121 6041.5124 6041.5127 6041.5811 6041.5818

150
(L 180 mm)
250 - - 6041.5730 6041.5812 6041.5819
350 6041.5240 6041.5540 6041.5735 6041.5813 6041.5820
450 - - - 6041.5814 6041.5821
550 6041.5260 6041.5560 6041.5760 6041.5815 6041.5822
650 6041.5275 6041.5575 6041.5775 - -
750 6041.5280 6041.5580 6041.5780 6041.5816 6041.5823
850
950 6041.5122 6041.5125 6041.5128
1100 6041.5711 6041.5828

Table 31: Matrix for connection tubing for the UltiMate 3000 RSLCnano system
Description P/N
nanoViper, ID x L, 20 μm x custom length 6041.5299
nanoViper, ID x L, 50 μm x custom length 6041.5599A
nanoViper, ID x L, 75 μm x custom length 6041.5799
nanoViper, thermally insulated, ID x L, 75 μm x300 mm 6083.2415
nanoViper ID x OD x L 75 μm x 360 μm x 550 mm 6041.5289
Liquid junction capillary interface ID x OD x L 20 μm x 360 μm x 550 mm 6041.5290
Nano LC column to MS tubing ID x OD x L 20 μm x 280 μm x 1 m 6041.5292

Table 32: Custom / One side nanoViper capillaries

4  Appendix
Item P/N
Sample loop 1 μL with nanoViper fittings connections 6826.2401
Table 33: Sample loops with nanoViper fittings

4  Appendix
4.3 UltiMate 3000 RSLCnano convenience bundles, list of contents

Description U3000 Nano RSLCnano with VWD U3000 Nano RSLCnano, no detector U3000 Cap flow RSLCnano, no detector U3000 Nano RSLCnano with EASY-Spray
Item P/N: 5200.0350 P/N: 5200.0355 P/N: 5200.0356 P/N: 5200.0357
Name P/N Module P/N Module P/N Module P/N Module
Solvent Rack 5035.9245 SRD-3400 with degasser 5035.9245 SRD-3400 with degasser 5035.9245 SRD-3400 with degasser 5035.9245 SRD-3400 with degasser
NCS-3500RS nano pump with 5041.0010A NCS-3500RS nano pump with 5041.0020 NCS-3500RS Cap pump with 5041.0010A NCS-3500RS nano pump with
Pump 5041.0010A
integrated column compt. integrated column compt. integrated column compt. integrated column compt.
Autosampler 5826.0020 WPS-3000RPL RS nano/cap 5826.0020 WPS-3000RPL RS nano/cap 5826.0020 WPS-3000RPL RS nano/cap 5826.0020 WPS-3000RPL RS nano/cap
VWD-3400RS 4 channels
Detector 5074.0010 - - - - - -
without flow cell
Accessory 1 6074.0270 UZ-View™ flow cell 3nL(nano) - - - - - -
Accessory 2 6041.0001A 2P-10P valve for NCS-3500 6041.0001A 2P-10P valve for NCS-3500 6041.0001A 2P-10P valve for NCS-3500 6041.0001A 2P-10P valve for NCS-3500
Accessory 3 6720.0310 Preconcentration nano kit 6720.0310 Preconcentration nano kit 6720.0315 Preconcentration cap kit 6720.0395 Easy-Spray connection kit
Accessory 4 6000.1004 Signal cable, 6pol.5m 6000.1004 Signal cable, 6pol.5m 6000.1004 Signal cable, 6pol.5m 6000.1004 Signal cable, 6pol.5m
Column to MS tubing ID x OD 6041.5292 Column to MS tubing ID x OD x 6041.5294 Column to MS tubing ID x OD

Accessory 5 6041.5292 - -
x L, 20µm x 280µm x 1m L, 20µm x 280µm x 1m x L, 50µm x 280µm x 1m

Accessory 6 6041.5293 - -
x L, 20µm x 360µm x 1m L, 20µm x 360µm x 1m x L, 50µm x 360µm x 1m

Accessory 7 6041.5294 - -
x L, 50µm x 280µm x 1m L, 50µm x 280µm x 1m x L, 50µm x 360µm x 550mm
Column to MS tubing ID x OD 6041.5295 Column to MS tubing ID x OD x 6826.2405 nanoViper sampler loop for
Accessory 8 6041.5295 - -
x L, 50µm x 360µm x 1m L, 50µm x 360µm x 1m WPS-TPL RS, 5 µL
Accessory 9 161089 Cytochrome C digest sample 161089 Cytochrome C digest sample 161089 Cytochrome C digest sample - -
Table 34: Convenience Bundles Bill of Material (BOM) list

4  Appendix
4.4 Hardware Accessories

The tables below list the common hardware accessories required for the
applications described in this manual.
Item P/N
Low-dispersion 2 pos 10 port valve high pressure for NCS-3500RS 6041.0001A
Low-dispersion 2 pos 6 port valve high pressure for NCS-3500RS 6041.0004A
Low-dispersion 2 pos 10 port valve, PAEK, bio, NCS-3500RS 6041.0012
Viper blind plug 6040.2303
Purge capillary 6040.2385
Table 35: Valves and accessories
Item P/N
ProFlow flow meter 6041.7850
Upgrade Kit for ProFlow flow meter 6041.3003
Classic flow meter with nano flow selector 6041.7901A
Classic flow meter with capillary flow selector 6041.7902A
Classic flow meter with micro flow selector 6041.7903A
Flow selector, Nano LC (recommended: 50–1,000 nL/min) 6041.0002
Flow selector, Capillary LC (recommended: 0.5–10 µL/min) 6041.0003
Flow selector, Micro LC (recommended: 5–50 µL/min) 6041.0014
Table 36: NC Pump Flow Control

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Date: April 2019

Document No.: 4820.4103

Copyright © 2019 Thermo Fisher Scientific Inc. All rights reserved.

Valco is a trademark of Valco Instruments Co.

Printed manual version only

UltiMate 3000 RSLCnano Standard Applications Guide Page 3

Page 4 UltiMate 3000 RSLCnano Standard Applications Guide

2 Application Setup ............................................................................14

UltiMate 3000 RSLCnano Standard Applications Guide Page 5

2.6.1 Hardware Layout ..................................................................................... 42

Page 6 UltiMate 3000 RSLCnano Standard Applications Guide

2.13 High throughput tandem capillary-flow LC-MS .................................................... 76

UltiMate 3000 RSLCnano Standard Applications Guide Page 7

Page 8 UltiMate 3000 RSLCnano Standard Applications Guide

1 Using this Manual

UltiMate 3000 RSLCnano Standard Applications Guide Page 9

1.1 About this Manual

NOTICE This document is intended for Thermo Fisher Scientific (or

Page 10 UltiMate 3000 RSLCnano Standard Applications Guide

1.2.1 Special Notices and Informational Notes

NOTICE Highlights information necessary to prevent damage to the

TIP Highlights information of general interest or helpful information that

UltiMate 3000 RSLCnano Standard Applications Guide Page 11

1.2.2 Typographical Conventions

References and Messages

Particularly Important Words

Particularly important words in the main flow of text appear in bold.

Electronic Manual Version (PDF)

 Table of contents entries

 Cross-references (in blue text), for example, to sections, figures or

Page 12 UltiMate 3000 RSLCnano Standard Applications Guide

1.3 Reference Documentation

 The UltiMate 3000 RSLCnano system webpage:

 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

 Upgrading the UltiMate 3000 RSLCnano System with ProFlow

 ViperTM and nanoViperTM EASY-SprayTM Column tips and tricks

 Details on Viper and nanoViper capillaries and application kits

Thermo Scientific Viper and nanoViper Fingertight Fitting System -

Viper and nanoViper Fingertight Fitting Systems - specifications

 The complete and easy guide to configuring your Thermo Scientific

 Nano, Capillary and Micro LC Columns detailed in the

UltiMate 3000 RSLCnano Standard Applications Guide Page 13

Page 14 UltiMate 3000 RSLCnano Standard Applications Guide

2.1 General Recommendations for Applications

2.1.1 nanoViper 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

UltiMate 3000 RSLCnano Standard Applications Guide Page 15

2.1.2 Making Connections using the Nano Connector

Figure 2: The components of the nano connector

Nano connector sleeve (1.1 cm)