Thermo Scientific UltiMate 3000 Standard Applications Manual

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

Contacting Us

Page 4 UltiMate 3000 RSLCnano Standard Applications Guide

Contents

1 Using this Manual ............................................................................ 9

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

Contents

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

2.1 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

UltiMate 3000 RSLCnano Standard Applications Guide Page 5

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

2.7.3 Installation Tips ....................................................................................... 49

2.7.4 Testing the Application ........................................................................... 50

2.8 Pre-concentration onto a 200 µm Monolithic Column ........................................ 51

2.8.1 Hardware Layout ..................................................................................... 51

2.8.2 Fluidic Setup ............................................................................................ 52

2.8.3 Installation Tips ....................................................................................... 53

2.8.4 Testing the Application ........................................................................... 53

2.9 Pre-concentration onto a Capillary Column ........................................................ 55

2.9.1 Hardware Layout ..................................................................................... 55

2.9.2 Fluidic Setup ............................................................................................ 56

2.9.3 Installation Tips ....................................................................................... 57

2.9.4 Testing the Application ........................................................................... 58

2.10 EASY-Spray Columns with the RSLCnano ............................................................. 59

2.10.1 EASY-Spray Concept ................................................................................ 59

2.10.2 Example Separation Performance of an EASY-Spray Column ................. 61

2.10.3 Hardware Layout Direct Injection ........................................................... 62

2.10.4 Fluidic Setup using EASY-Spray Columns ................................................ 63

2.10.5 EASY-Spray Transfer lines ........................................................................ 65

2.11 2D Salt Plugs with Nano Column .......................................................................... 66

2.11.1 Hardware-Layout..................................................................................... 66

2.11.2 Fluidic Setup ............................................................................................ 67

2.11.3 Installation Tips ....................................................................................... 68

2.11.4 Testing the Application ........................................................................... 69

2.11.5 Salt Solution Preparation ........................................................................ 70

2.12 Tandem Nano LC .................................................................................................. 71

2.12.1 Hardware Layout ..................................................................................... 71

2.12.2 Fluidic Setup ............................................................................................ 72

2.12.3 Installation Tips ....................................................................................... 73

2.12.4 Testing the Application ........................................................................... 74

2.12.5 Tandem NanoLC-MS ................................................................................ 75

Page 6 UltiMate 3000 RSLCnano Standard Applications Guide

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

UltiMate 3000 RSLCnano Standard Applications Guide Page 7

Contents

Page 8 UltiMate 3000 RSLCnano Standard Applications Guide

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.

UltiMate 3000 RSLCnano Standard Applications Guide Page 9

1  Using 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.

Page 10 UltiMate 3000 RSLCnano Standard Applications Guide

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.

1  Using this Manual

TIP Highlights information of general interest or helpful information that
can make a task easier or optimize the performance of the instrument

UltiMate 3000 RSLCnano Standard Applications Guide Page 11

1  Using this Manual

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

Page 12 UltiMate 3000 RSLCnano Standard Applications Guide

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)

1  Using this Manual

 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

UltiMate 3000 RSLCnano Standard Applications Guide Page 13

2  Application Setup

2 Application Setup

This chapter provides details on each of the application kits available for

the UltiMate 3000 RSLCnano system.

Page 14 UltiMate 3000 RSLCnano Standard Applications Guide

2  Application Setup

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.

UltiMate 3000 RSLCnano Standard Applications Guide Page 15

2  Application Setup

Nano connector sleeve (1.1 cm)

PTFE sleeve (1.8 cm)

Figure 3: Nano connector (top) and PTFE (bottom) sleeve comparison

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.

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).

Page 16 UltiMate 3000 RSLCnano Standard Applications Guide

2  Application Setup

3. Tighten both sides of the black nut equally to ensure that the nano
connector sleeve is in the center of the transparent union Figure

4c.

a

b

c

Figure 4: Nano connector assembly. (a) The two black nuts and
transparent union are mounted on the two outlets to be connected.
(b) A dead volume free connection with the nano connector. (c) The
complete fitting with the black nuts and union housing the nano
connector.

2.1.3 Installing and Configuring the Application Fluidics

Each capillary must be installed sequentially starting from the pump
outlet. Please flush each capillary using the respective pump and ensure
that a droplet is visible at the capillary outlet in question before making
the next connection. This will ensure that all air is removed from the
capillaries and connections and that no air is passed through the
column.

2.1.4 Interfacing the UltiMate 3000 RSLCnano with the Nanospray Flex™ Ion

Source

LC−MS based applications using linear columns commonly use the
Nanospray Flex Ion source (see Figure 41) to interface with Thermo
Scientific Mass Spectrometers. As of January 2019, 1.5 m of fused silica
(20 µm and 50 µm) and a tile for cutting fused silica capillaries have
been included in the pump (NCS-3500RS and NCP-3200RS) accessory
kits. This capillary should be used to connect the outlet of the column or
UV flow cell, if included in the setup, with the emitter installed in the ion
source. The connection between the capillary outlet and the emitter is
realized using a 1/32” micro tight® union assembly included with the
Nanospray Flex Ion Source. For capillaries and columns with 280 µm O.D.
a black sleeve (P/N SC903) should be used with the microtight fitting.

UltiMate 3000 RSLCnano Standard Applications Guide Page 17

2  Application Setup

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.

Page 18 UltiMate 3000 RSLCnano Standard Applications Guide

2  Application Setup

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).

 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.

UltiMate 3000 RSLCnano Standard Applications Guide Page 19

2  Application Setup

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.

2.1.6 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.

Page 20 UltiMate 3000 RSLCnano Standard Applications Guide

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.

2  Application Setup

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 µ­precolumns can accommodate higher loading flows and are often
preferred when large sample volumes need to be injected.

UltiMate 3000 RSLCnano Standard Applications Guide Page 21

2  Application Setup

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.

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.

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 forward­and a back-flush fluidic pathway.

Figure 7: The different fluidic configurations for forward-flush (left) and
back-flush (right)

Page 22 UltiMate 3000 RSLCnano Standard Applications Guide

2  Application Setup

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.

UltiMate 3000 RSLCnano Standard Applications Guide Page 23

2  Application Setup

Figure 8: RSLCnano system overview

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 micro­fractionation applications

2.3 Installing the UltiMate 3000 RSLCnano System

2.3.1 UltiMate 3000 RSLCnano System Components

Page 24 UltiMate 3000 RSLCnano Standard Applications Guide

2.3.2 NC Pump Configurations

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

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

2  Application Setup

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.

Table 1: Properties of the different flow selectors

UltiMate 3000 RSLCnano Standard Applications Guide Page 25

2  Application Setup

High pressure gradient NC_Pump

Low pressure gradient Micro pump

Rear seal

wash system

Flow meter

Purge

screws

Solvent

shutoff

valves

Inline filter

Purge
screw

Tubing guides

Snap-in valves

Column compartment

2.3.2.3 NCS-3500RS with the ProFlow Flow Meter

Page 26 UltiMate 3000 RSLCnano Standard Applications Guide

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

Low pressure gradient Micro pump

Rear seal wash

system

Flow meter

Purge screws

Inline filter

Purge screw

Column compartment

2  Application Setup

UltiMate 3000 RSLCnano Standard Applications Guide Page 27

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  Application Setup

Figure 11: NCP 3200RS pump

NCP-3200RS module featuring

- NC pump

2.3.2.5 NCP-3200RS

Figure 12: NCP 3200RS pump with ProFlow flow meter installed

Page 28 UltiMate 3000 RSLCnano Standard Applications Guide

2  Application Setup

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.

UltiMate 3000 RSLCnano Standard Applications Guide Page 29

2  Application Setup

Figure 14: UltiMate 3000 RSLCnano with ProFlow flow meter –
compatibility matrix

Page 30 UltiMate 3000 RSLCnano Standard Applications Guide

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.

2  Application Setup

 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.

UltiMate 3000 RSLCnano Standard Applications Guide Page 31

2  Application Setup

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

 For best results, use premixed Fisher Chemical Optima LC-MS grade

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

in solvent composition

solvents:

2.3.4.5 Auxiliary Solvents:

The following solvents are recommendations to ensure robust operation

Page 32 UltiMate 3000 RSLCnano Standard Applications Guide

2  Application Setup

Flow Meter Type

Refresh Solvent

Change Solvent

ProFlow

5 min

15 min

Classic

10 min

30 min

 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:

Table 2: NC Pump block purge times

UltiMate 3000 RSLCnano Standard Applications Guide Page 33

2  Application Setup

Application

Recommended Purge Time

Nano LC (ProFlow)

10 min

Nano LC (classic)

30 min

Capillary LC (classic)

5 min

Micro LC (classic)

5 min

 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):

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 / DCMS
to the NCx-3x00 operating instructions.

Link

please refer

Page 34 UltiMate 3000 RSLCnano Standard Applications Guide

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 pre­calibrated 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%.

2  Application Setup

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

UltiMate 3000 RSLCnano Standard Applications Guide Page 35

2  Application Setup

Solvent

Viscosity %

Water

100

80 / 20 (v/v) Acetonitrile / Water

66

50 % ACN / 50% Water

100

Acetonitrile

50

MeOH

75

IPA

220

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:

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).

Page 36 UltiMate 3000 RSLCnano Standard Applications Guide

2.4 Application Overview

2  Application Setup

Figure 15: Overview of the Applications available for the RSLCnano

UltiMate 3000 RSLCnano Standard Applications Guide Page 37

2  Application Setup

Figure 16: Setup for a Direct Injection experiment onto a
nano column including the optional UV detector

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.

2.5 Direct Injection onto a Nano Column

2.5.1 Hardware Layout

Page 38 UltiMate 3000 RSLCnano Standard Applications Guide

2.5.2 Fluidic Setup

#

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

2

nanoViper capillary FS/PEEK sheathed 1/32" I.D. x L 20 µm x 550 mm

6041.5260

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

2  Application 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 WPS­3000TPL 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).

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

UltiMate 3000 RSLCnano Standard Applications Guide Page 39

2  Application Setup

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

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:

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

Page 40 UltiMate 3000 RSLCnano Standard Applications Guide

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 WPS­3000TPL 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 pre­concentration 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  Application Setup

UltiMate 3000 RSLCnano Standard Applications Guide Page 41

2  Application Setup

Figure 19: Setup for a Direct Injection experiment onto
a capillary column including the optional UV detector

The instrument setup presented in

Figure 19 consists of:

SRD-3400 5035.9245
NCS-3500RS 5041.0020
VWD-3400RS (optional) 5074.0010
45 nL flow cell 6074.0280
WPS-3000TPL RS 5826.0020
Application kit: 6720.0305

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.

2.6 Direct Injection onto a Capillary Column

2.6.1 Hardware Layout

Page 42 UltiMate 3000 RSLCnano Standard Applications Guide

2.6.2 Fluidic Setup

2  Application 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 WPS­3000TPL RS autosampler. The correct configuration for the sample loop,
needle and buffer tubing is shown in Figure 19.

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).

UltiMate 3000 RSLCnano Standard Applications Guide Page 43

2  Application Setup

#

Item

P/N

a

300 µm I.D. x 15 cm, packed with Acclaim PepMap RSLC C18, 2 µm,
100Å, nanoViper

164537
1

nanoViper capillary FS/PEEK sheathed 1/32" I.D. x L 50 µm x 750 mm

6041.5580

2

nanoViper capillary FS/PEEK sheathed 1/32" I.D. x L 50 µm x 550 mm

6041.5560

nanoViper sample loop 5 µL, FS/PEEK sheathed I.D. x L 200 µm x 159 mm

6826.2405

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

Page 44 UltiMate 3000 RSLCnano Standard Applications Guide

2.6.3 Installation Tips

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, 200 fmol / µL

Injection Volume

5 µL (Full Loop)

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

Oven temperature

40 °C

WPS temperature

5 °C

Flow Rate

5 µL / min during gradient, 10 µL / min during wash and equilibration

 Follow the “General Recommendations for Applications” (section 2.1

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.

2.6.4 Testing the Application

Test the direct injection application using the following conditions:

2  Application Setup

Table 8: Test conditions for direct injection of 1 pmol Cytochrome C onto a capillary column

UltiMate 3000 RSLCnano Standard Applications Guide Page 45

2  Application Setup

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

Page 46 UltiMate 3000 RSLCnano Standard Applications Guide

2.7 Pre-concentration onto a Nano Column

Figure 22: Setup for a pre-concentration experiment
onto a nano column including the optional UV
detector

The instrument setup presented in

Figure 22 consists of:

SRD-3400 5035.9245
NCS-3500RS 5041.0010A
1x 10-port sw. valve 6041.0001A
VWD-3400RS (optional) 5074.0010
3 nL flow cell 6074.0270
WPS-3000TPL RS 5826.0020
Application kit: 6720.0310

2.7.1 Hardware Layout

2  Application Setup

UltiMate 3000 RSLCnano Standard Applications Guide Page 47

2  Application Setup

#

Item

P/N

a

75 µm I.D. x 15 cm, packed with Acclaim PepMap RSLC C18, 2 µm, 100Å, nanoViper

164534

b

300 µm I.D. x 5 mm, packed with Acclaim PepMap100 C18, 5 µm, 100Å (set of 5
cartridges)

160454

µ-Precolumn holder, 5 mm, with 30 µm I.D. connecting tubing, nanoViper fittings

164649

1

nanoViper capillary FS/PEEK sheathed 1/32" I.D. x L 20 µm x 350 mm

6041.5240

2

nanoViper capillary FS/PEEK sheathed 1/32" I.D. x L 75 µm x 650 mm

6041.5775

3

nanoViper capillary FS/PEEK sheathed 1/32" I.D. x L 75 µm x 550 mm

6041.5760

nanoViper capillary FS/PEEK sheathed 1/32" I.D. x L 20 µm x 750 mm

6041.5280

2.7.2 Fluidic Setup

Figure 22 and Figure 23 shows the setup using the parts provided in the

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.

Page 48 UltiMate 3000 RSLCnano Standard Applications Guide

#

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

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

2.7.3 Installation Tips

2  Application Setup

 Follow the “General Recommendations for Applications” (section 2.1

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.

UltiMate 3000 RSLCnano Standard Applications Guide Page 49

2  Application Setup

Property

Setting

Mobile Phase A

Water + 0.1% FA

Mobile Phase B

80/20 (v/v) Acetonitrile / Water with 0.1% FA

Loading solvent

Water + 0.1% FA

Sample

Cytochrome C digest, 500 fmol / µL

Injection Volume

1 µL (partial loop fill or µL pickup)

UV detection (Optional)

214 nm

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

Oven temperature

35 °C

WPS temperature

5 °C

Loading flow rate

30 µL / min

NC Flow Rate

300 nL / min (ProFlow or classic flow meter with nano flow selector)

2.7.4 Testing the Application

Test the pre-concentration setup using the following conditions:

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

Page 50 UltiMate 3000 RSLCnano Standard Applications Guide

2.8 Pre-concentration onto a 200 µm Monolithic

Figure 25: Setup for a Pre-concentration experiment
onto a monolithic column including the optional UV
detector

The instrument setup presented in

Figure 25 consists of:

SRD-3400 5035.9245
NCS-3500RS 5041.0020
1x.10 port sw valve 6041.0001A
VWD-3400RS (optional) 5074.0010
3 nL flow cell 6074.0270
WPS-3000TPL RS 5826.0020
Application kit: 6720.0320

Column

2.8.1 Hardware Layout

2  Application Setup

UltiMate 3000 RSLCnano Standard Applications Guide Page 51

2  Application Setup

2.8.2 Fluidic Setup

Figure 25 and Figure 26 shows the setup using the parts provided in the

pre-concentration monolithic application kit. Columns are marked with
letters and tubing with digits. The sample loop is installed in the WPS­3000TPL RS autosampler. The correct configuration for the sample loop,
needle and buffer tubing is shown in Figure 25.

Figure 26: Fluidic connections for a pre-concentration onto a monolithic
column

TIP The schematic shows a 10-port switching valve. This application can
also be performed using a 6-port valve.

Page 52 UltiMate 3000 RSLCnano Standard Applications Guide

2  Application Setup

#

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

1

nanoViper capillary FS/PEEK sheathed 1/32" I.D. x L 50 µm x 350 mm

6041.5540

2

nanoViper capillary FS/PEEK sheathed 1/32" I.D. x L 75 µm x 650 mm

6041.5775

3

nanoViper capillary FS/PEEK sheathed 1/32" I.D. x L 75 µm x 550 mm

6041.5760

nanoViper capillary FS/PEEK sheathed 1/32" I.D. x L 50 µm x 750 mm

6041.5580

nanoViper sample loop 20 µL, FS/PEEK sheathed

6826.2420

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

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

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

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

2.8.3 Installation Tips

 Follow the “General Recommendations for Applications” (section 2.1

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%.

2.8.4 Testing the Application

Test the pre-concentration setup using the following conditions:

UltiMate 3000 RSLCnano Standard Applications Guide Page 53

2  Application Setup

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)

UV detection (Optional)

214 nm

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

Oven temperature

60 °C

WPS temperature

5 °C

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.

Page 54 UltiMate 3000 RSLCnano Standard Applications Guide

2.9 Pre-concentration onto a Capillary Column

Figure 28: Setup for a pre-concentration experiment
onto a capillary column including the optional UV
detector

The instrument setup presented in

Figure 28 consists of:

SRD-3400 5035.9245
NCS-3500RS 5041.0020
1x.10 port sw. valve 6041.0001A
VWD-3400RS 5074.0010
45 nL flow cell 6074.0280
WPS-3000TPL RS 5826.0020
Application kit: 6720.0315

2.9.1 Hardware Layout

2  Application Setup

UltiMate 3000 RSLCnano Standard Applications Guide Page 55

2  Application Setup

#

Item

P/N

a

300 µm I.D. x 15 cm, packed with Acclaim PepMap RSLC C18, 2 µm, 100Å, nanoViper

164537

b

300 µm I.D. x 5 mm, packed with Acclaim PepMap100 C18, 5 µm, 100Å (set of 5
cartridges)

160454

µ-Precolumn holder, 5 mm, with 30 µm I.D. connecting tubing, nanoViper fittings

164649

1

nanoViper capillary FS/PEEK sheathed 1/32" I.D. x L 50 µm x 350 mm

6041.5540

2

nanoViper capillary FS/PEEK sheathed 1/32" I.D. x L 75 µm x 650 mm

6041.5775

3

nanoViper capillary FS/PEEK sheathed 1/32" I.D. x L 75 µm x 550 mm

6041.5760

nanoViper capillary FS/PEEK sheathed 1/32" I.D. x L 50 µm x 750 mm

6041.5580

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 WPS­3000TPL RS autosampler. The correct configuration for the sample loop,
needle and buffer tubing is shown in Figure 28.

Figure 29: Fluidic connections for a pre-concentration onto a capillary
column

TIP The schematic shows a 10-port switching valve. This application can
also be performed using a 6-port valve.

Page 56 UltiMate 3000 RSLCnano Standard Applications Guide

#

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

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

2.9.3 Installation Tips

2  Application Setup

 Follow the “General Recommendations for Applications” (section 2.1

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.

UltiMate 3000 RSLCnano Standard Applications Guide Page 57

2  Application Setup

Property

Setting

Mobile Phase A

Water + 0.1% FA

Mobile Phase B

80/20 (v/v) Acetonitrile / Water with 0.1% FA

Loading solvent

Water + 0.1% FA

Sample

Cytochrome C digest, 200 fmol / µL

Injection Volume

1 µL (partial loop fill or µL pickup)

UV detection (Optional)

214 nm

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

Oven temperature

35 °C

WPS temperature

5 °C

Loading flow rate

20 µL / min

NC Flow Rate

4 µL / min (capillary flow selector)

2.9.4 Testing the Application

Test the pre-concentration application using the following conditions:

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

Page 58 UltiMate 3000 RSLCnano Standard Applications Guide

2  Application Setup

Separation
column and
temperature
control

nanoViper
fitting

7 μm internal

diameter
glass emitter

Zero dead volume union

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.

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.

UltiMate 3000 RSLCnano Standard Applications Guide Page 59

2  Application Setup

Camera with LED light

Source body

EASY-Spray column

nanoViper connection

Proximity alignment

Temperature dial and readout

Temperature control cable

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

NOTICE The EASY-Spray Ion Source interface is available in two formats, the EASY­Spray 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).

Table 15: EASY-Spray ion sources and compatible mass spectrometers

Page 60 UltiMate 3000 RSLCnano Standard Applications Guide

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.

2  Application Setup

5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Time (min)

Relative Abundance

0

20

40

60

80

100

0

100

12.00 12.10 12.20
Time (min)

PWHH

2.5 sec

0

100

14.20 14.30 14.40
Time (min)

PWHH

3.0 sec

A

B

A

B

2.10.2 Example Separation Performance of an EASY-Spray Column

An example base peak Chromatogram for a BSA digest run on an Easy­Spray (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).

Figure 33: Base Peak Chromatogram of an EASY-Spray column in pre­concentration mode

UltiMate 3000 RSLCnano Standard Applications Guide Page 61

2  Application Setup

Figure 34: Setup for direct injection with EASY­Spray

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
Application kit: 6720.0395

NOTICE The NCP-3200RS in this setup
can be exchanged for an NCS-3500RS.

Figure 35: Setup for pre-concentration with EASY­Spray

The instrument setup for pre­concentration 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
Application kit: 6720.0395

2.10.3 Hardware Layout Direct Injection

Page 62 UltiMate 3000 RSLCnano Standard Applications Guide

2.10.4 Fluidic Setup using EASY-Spray Columns

Item

P/N>

300 µm I.D. x 5 mm, packed with Acclaim PepMap100 C18, 5 µm, 100Å
(set of 5 cartridges)

160454

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 350 mm

6041.5240

nanoViper Capillary FS/PEEK sheathed 1/32" I.D. x L 20 µm x 550 mm

6041.5260

nanoViper Capillary FS/PEEK sheathed 1/32" I.D. x L 20 µm x 650 mm

6041.5275

nanoViper Capillary FS/PEEK sheathed 1/32" I.D. x L 20 µm x 750 mm

6041.5280

nanoViper Capillary FS/PEEK sheathed 1/32" I.D. x L 75 µm x 550 mm

6041.5760

nanoViper Capillary FS/PEEK sheathed 1/32" I.D. x L 75 µm x 650 mm

6041.5775

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

The fluidic setups for EASY-Spray with an UltiMate 3000 RSLCnano are
essentially identical to those used for the direct injection and pre­concentration 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.

2  Application Setup

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.

UltiMate 3000 RSLCnano Standard Applications Guide Page 63

2  Application Setup

Item

P/N>

1/16" Universal Fingertight Fitting, one-piece design, with extra long
thread, 4 pieces

6720.0015
Polypropylene vials for WPS with glass insert, 250 µL, 25 pieces

6820.0027

Polypropylene caps for WPS vials, 25 pieces

6820.0028

Cytochrome C digest, 1.6 nmol, Lyophilized

161089

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.

Page 64 UltiMate 3000 RSLCnano Standard Applications Guide

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.

2  Application Setup

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.

UltiMate 3000 RSLCnano Standard Applications Guide Page 65

2  Application Setup

Figure 37: Setup for a 2D Salt Plugs experiment
including the optional UV detector

The Instrument setup presented in

Figure 37 consists of:

SRD-3400 5035.9245
NCS-3500RS 5041.0010A
2x 10-port sw.valve 6041.0001A
VWD-3400RS 5074.0010
3 nL flow cell 6074.0270
WPS-3000TPL RS 5826.0020
Application kit: 6720.0325

2.11 2D Salt Plugs with Nano Column

2.11.1 Hardware-Layout

Page 66 UltiMate 3000 RSLCnano Standard Applications Guide

2.11.2 Fluidic Setup

#

Item

P/N

a

300 µm I.D. x 10 cm, packed with Poros 10 S with connections, 130 µm I.D.
FS sheathed inlet and outlet, nanoViper

164565

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
cartridges)

160454

µ-Precolumn holder, 5 mm, with 30 µm I.D. connecting tubing, nanoViper fittings

164649

1

nanoViper capillary FS/PEEK sheathed 1/32" I.D. x L 20 µm x 350 mm

6041.5240

2  Application 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

UltiMate 3000 RSLCnano Standard Applications Guide Page 67

2  Application Setup

#

Item

P/N

2

nanoViper capillary FS/PEEK sheathed 1/32" I.D. x L 75 µm x 650 mm

6041.5775

3

nanoViper capillary FS/PEEK sheathed 1/32" I.D. x L 75 µm x 550 mm

6041.5760

4

nanoViper capillary FS/PEEK sheathed 1/32" I.D. x L 75 µm x 250 mm

6041.5730

nanoViper capillary FS/PEEK sheathed 1/32" I.D. x L 20 µm x 750 mm

6041.5280

nanoViper sample loop 20 µL, FS/PEEK sheathed

6826.2420

5

PTFE tubing, 500 µm I.D. 100 cm, used in waste tubing

6720.0077

1/16” Universal Fingertight Fitting, one piece design, long thread, 4 pieces

6720.0015

Polypropylene vials for WPS with glass insert, 250 µL, 25 pcs

6820.0027

Polypropylene caps for WPS vials, 25 pcs

6820.0028

6 Protein Digest Standard, 100 pmol, Lyophilized

88342

Cytochrome C digest, 1.6 nmol, Lyophilized

161089

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

2.11.3 Installation Tips

 Follow the “General Recommendations for Applications” (section 2.1

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.

Page 68 UltiMate 3000 RSLCnano Standard Applications Guide

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

Property

Setting

Mobile Phase A

Water + 0.1% FA

Mobile Phase B

80/20 (v/v) Acetonitrile / Water with 0.1% FA

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
(mol / L)

1 mmol NaCl
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

UV detection (Optional)

214 nm

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

WPS temperature

5 °C

Loading flow rate

10 µL / min

NC Flow Rate

300 nL / min (capillary flow selector)

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.

2.11.4 Testing the Application

Test the 2D salt plug setup using the following conditions.

2  Application Setup

UltiMate 3000 RSLCnano Standard Applications Guide Page 69

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

2  Application Setup

Concentration of
NaCl

Volume of 2000mM NaCl
stock solution

Volume of loading solvent

Total Volume

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

Concentration of
NaCl

Volume of 100mM NaCl
stock solution

Volume of loading solvent

Total Volume

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

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).

Table 19: Guide for the preparation of the Salt Plugs (dilutions from 2000 mM NaCl)

Page 70 UltiMate 3000 RSLCnano Standard Applications Guide

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
consists of:

SRD-3400 5035.9245
NCS-3500RS 5041.0010A
NCP-3200RS 5041.0030A
2x 10-port sw.valve 6041.0001A
VWD-3400RS 5074.0010
3 nL flow cell 6074.0270
WPS-3000FC 5824.0020
Injection valve 6826.0011A
Application kit: 6720.0335

TIP All the components required to convert
the WPS-3000FC for tandem nano LC use
are included in the application kit, except
the WPS injection valve (P/N 6826.0011A)
which must be ordered separately.

The NCP 3200RS accessory kit has two
130 cm long solvent inlet tubings, which
supply solvent from the bottles placed on
the solvent rack to the NCP at the bottom of
the stack.

2.12.1 Hardware Layout

2  Application Setup

UltiMate 3000 RSLCnano Standard Applications Guide Page 71

2  Application Setup

2.12.2 Fluidic Setup

Figure 39 and Figure 40 shows the setup using the parts provided in the

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 WPS­3000FC 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.

Page 72 UltiMate 3000 RSLCnano Standard Applications Guide

2  Application Setup

#

Item

P/N

a

75 µm I.D. x 15 cm, packed with Acclaim PepMap RSLC C18, 2 µm, 100Å, nanoViper

164534

b

300 µm I.D. x 5 mm, packed with Acclaim PepMap100 C18, 5 µm, 100Å (set of 5
cartridges)

160454

µ-Precolumn holder, 5 mm, with 30 µm I.D. connecting tubing, nanoViper fittings

164649

1

nanoViper capillary FS/PEEK sheathed 1/32" I.D. x L 20 µm x 350 mm

6041.5240

2,3

nanoViper capillary FS/PEEK sheathed 1/32" I.D. x L 75 µm x 650 mm

6041.5775

4

nanoViper capillary FS/PEEK sheathed 1/32" I.D. x L 75 µm x 250 mm

6041.5730

nanoViper capillary FS/PEEK sheathed 1/32" I.D. x L 20 µm x 750 mm

6041.5280

nanoViper sample loop 20 µL, FS/PEEK sheathed

6826.2420

5

PTFE tubing, 500 µm I.D. 100 cm, used in waste tubing

6720.0077

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

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

2.12.3 Installation Tips

 Follow the “General Recommendations for Applications” (section 2.1

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.

UltiMate 3000 RSLCnano Standard Applications Guide Page 73

2  Application Setup

Property>

Setting

Mobile Phase A

Water + 0.1% FA

Mobile Phase B

80/20 (v/v) Acetonitrile / Water with 0.1% FA

Loading Pump A

Water + 0.1% FA

Sample

Cytochrome C digest, 500 fmol / µL

Injection Volume

1 µL (Full Loop)

UV detection (Optional)

214 nm

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

WPS temperature

5 °C

Loading flow Rate

5 µL / min

NC Pump flow rate

300 nL / min

 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.

2.12.4 Testing the Application

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.

Table 22 Test conditions for running the tandem nanoLC application

Page 74 UltiMate 3000 RSLCnano Standard Applications Guide

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  Application Setup

UltiMate 3000 RSLCnano Standard Applications Guide Page 75

2  Application Setup

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.

Page 76 UltiMate 3000 RSLCnano Standard Applications Guide

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  Application Setup

UltiMate 3000 RSLCnano Standard Applications Guide Page 77

2  Application Setup

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.

Page 78 UltiMate 3000 RSLCnano Standard Applications Guide

2  Application Setup

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.
fused silica capillaries, 5 pieces

6720.0030
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
silica tubing (280 µm O.D.), 5 pieces

6720.0064

PEEK sleeves, precision cut and polished for connections with fused
silica tubing (360 µm O.D.), 5 pieces

6720.0078
Microtight Union inclusive 2 fittings and 1 gauge plug

6720.0074

PEEK sleeves, precision cut and polished for connections with fused
silica tubing (280 µm O.D.), 10 pieces

6720.0075

PEEK sleeves, precision cut and polished for connections with
Microtight Union (360 µm O.D.), 10 pieces

6720.0076
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.

UltiMate 3000 RSLCnano Standard Applications Guide Page 79

2  Application Setup

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

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

Page 80 UltiMate 3000 RSLCnano Standard Applications Guide

Figure 41: Nanospray Flex Series ion sources

2  Application Setup

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

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).

UltiMate 3000 RSLCnano Standard Applications Guide Page 81

Figure 42: Ionization probes for the Ion Max and Ion Max NG sources.

2  Application Setup

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

Page 82 UltiMate 3000 RSLCnano Standard Applications Guide

3  FAQs

3 FAQs

UltiMate 3000 RSLCnano Standard Applications Guide Page 83

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.

Page 84 UltiMate 3000 RSLCnano Standard Applications Guide

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.

3  FAQs

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 pre­concentration setups.

UltiMate 3000 RSLCnano Standard Applications Guide Page 85

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.

Page 86 UltiMate 3000 RSLCnano Standard Applications Guide

3  FAQs

Figure 45: Decision Tree for troubleshooting a low-flow LC application

UltiMate 3000 RSLCnano Standard Applications Guide Page 87

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.

Page 88 UltiMate 3000 RSLCnano Standard Applications Guide

3  FAQs

Figure 46: Comparison of Cytochrome C separation with different ion
pairing agents. Top uses 0.05% TFA and bottom 0.1% FA

UltiMate 3000 RSLCnano Standard Applications Guide Page 89

3  FAQs

Solvent A

Solvent B

0.1%

0.08%

0.05%

0.04%

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.

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

Page 90 UltiMate 3000 RSLCnano Standard Applications Guide

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

UltiMate 3000 RSLCnano Standard Applications Guide Page 91

3  FAQs

Parameter

Description

Typical Value

DispSpeed

Sets the speed of the syringe used for
dispensing the sample%

2 µL / sec

DrawSpeed

Sets the speed of the syringe used for drawing
the sample.

0.2 µL / sec

DrawDelay

Sets the time that the needle remains in the
vial after drawing the sample

5 sec

WashSpeed

Sets the speed of the syringe for the wash
cycle.

4 µL / sec

WasteSpeed

Sets the speed of the syringe used for
expelling liquid to the waste.

4 µL / sec
DispDelay

Time needle remains in vial after dispense

2 sec

DrawDelay

Time needle remains in vial before liquid is
drawn

5 sec
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
trigger point for sample vial / well

8 mm

TransVialPunctureD
epth

Depth of puncture needle beyond pusher
trigger point for transport vial

8 mm
WashVolume

Volume used in wash operation

50 µL

FlushVolume

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

5 µL

FlushVolume2

Flush volume used in full loop and partial loop
injections from the same vial.

3 µL
Loop Overfill

Loop overfill factor used in full loop only

2.0

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.

Page 92 UltiMate 3000 RSLCnano Standard Applications Guide

Table 25: Typical WPS-3000TPL RS autosampler settings

4  Appendix

4 Appendix

UltiMate 3000 RSLCnano Standard Applications Guide Page 93

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.

Page 94 UltiMate 3000 RSLCnano Standard Applications Guide

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.

4  Appendix

-

Figure 47: Example UDP injection routine for maximizing µL pickup
volume

UltiMate 3000 RSLCnano Standard Applications Guide Page 95

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

Page 96 UltiMate 3000 RSLCnano Standard Applications Guide

4  Appendix

Item

P/N

Acclaim PepMap RSLC C18, 2 μm, 100 Å, 50 μm I.D. x 15 cm, nanoViper

164562

Acclaim PepMap RSLC C18, 2 μm, 100 Å, 50 μm I.D. x 25 cm, nanoViper

164709

Acclaim PepMap RSLC C18, 2 μm, 100 Å, 50 μm I.D. x 50 cm, nanoViper

164710

Acclaim PepMap RSLC C18, 2 μm, 100 Å, 75 μm I.D. x 15 cm, nanoViper

164534

Acclaim PepMap RSLC C18, 2 μm, 100 Å, 75 μm I.D. x 25 cm, nanoViper

164536

Acclaim PepMap RSLC C18, 2 μm, 100 Å, 75 μm I.D. x 50 cm, nanoViper

164540

Acclaim PepMap RSLC C18, 2 μm, 100 Å, 75 μm I.D. x 75 cm, nanoViper

164939

Item

P/N

μ-Precolumn™ holder, 5 mm, with 30 μm i.d. connecting tubing, nanoViper
fittings

164649

300 µm i.d. x 5 mm, packed with Acclaim PepMap100 C18, 5 µm, 100Å (set
of 5 cartridges)

160454

Acclaim PepMap, C18 3 μm, 100 Å, 75 μm I.D. x 15 cm (20 mm bed length),
nanoViper

164535

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.

Table 26 Acclaim PepMap C18 RSLC 2 µm particle size columns

Table 27 Acclaim PepMap C18 trap columns

UltiMate 3000 RSLCnano Standard Applications Guide Page 97

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, 25 cm x 75 µm I.D., PepMap RSLC C18, 2 µm, 100Å

ES802A

EASY-Spray column, 50 cm x 75 µm I.D., PepMap RSLC C18, 2 µm, 100Å

ES803A

EASY-Spray column, 15 cm x 75 µm I.D., PepMap100 C18, 2 µm, 100Å

ES804A

EASY-Spray column, 75 cm x 75 µm I.D., PepMap RSLC C18, 2 µm, 100Å

ES805A

EASY-Spray column, 75 cm x 150 µm I.D., PepMap RSLC C18, 2 µm, 100Å

ES806A

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

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

Item

P/N

PepSwift Monolithic Trap Column, 200 μm x 5 mm, set of 2, nanoViper

164558

Table 28: EASY-Spray columns

/N

Table 29: PepSwift Monolithic linear analytical columns

Table 30: Pepswift Monolithic trap columns

Page 98 UltiMate 3000 RSLCnano Standard Applications Guide

4.2.2 nanoViper Capillaries, Sample Loops and Connectors

Length
(mm)

I.D. [Colour Code]

10 μm
[Green]

20 μm
[Orange]

50 μm
[Brown]

75 μm
[Black]

100 μm
[Red]

150 μm
[Purple]

70 6041.5120

6041.5123

6041.5126

6041.5810

6041.5817

150

6041.5118
(L 180 mm)

6041.5121

6041.5124

6041.5127

6041.5811

6041.5818
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

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

Nano LC column to MS tubing ID x OD x L 20 μm x 360 μm x 1 m

6041.5293

Nano LC column to MS tubing ID x OD x L 50 μm x 280 μm x 1 m

6041.5294

Nano LC column to MS tubing ID x OD x L 50 μm x 360 μm x 1 m

6041.5295

4  Appendix

Table 31: Matrix for connection tubing for the UltiMate 3000 RSLCnano system

Table 32: Custom / One side nanoViper capillaries

UltiMate 3000 RSLCnano Standard Applications Guide Page 99

4  Appendix

Item

P/N

Sample loop 1 μL with nanoViper fittings connections

6826.2401

Sample loop 5 μL with nanoViper fittings connections

6826.2405

Sample loop 10 μL with nanoViper fittings connections

6826.2410

Sample loop 20 μL with nanoViper fittings connections

6826.2420

Sample loop 50 μL with nanoViper fittings connections

6826.2450

Sample loop 125 μL with nanoViper fittings connections

6826.2412

Table 33: Sample loops with nanoViper fittings

Page 100 UltiMate 3000 RSLCnano Standard Applications Guide