Dionex UltiMate 3000 User guide

UltiMate 3000 RSLCnano
Standard Applications
Revision: V1.0 R3
Date: January 2012
Doc.no. 4820.4103
© 2012 Thermo Fisher Scientific Inc.

UltiMate 3000 RSLCnano – Standard Applications
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UltiMate 3000 RSLCnano – Standard Applications
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Table of Contents
1Introduction................................................................................................................4
1.1 Additional Documents...............................................................................................5
2Application Setup.......................................................................................................6
2.1 General Recommendations for Applications.............................................................6
2.1.1 Connections........................................................................................................6
2.1.2 Sample Preparation.............................................................................................7
2.1.3 Mobile Phases.....................................................................................................8
2.2 Installing the UltiMate 3000 RSLCnano system.......................................................9
2.3 Application Overview .............................................................................................12
2.4 Direct Injection onto a Nano Column .....................................................................13
2.4.1 Hardware Layout..............................................................................................13
2.4.2 Fluidic Setup.....................................................................................................15
2.4.3 Installation Tips................................................................................................15
2.4.4 Testing the Application ....................................................................................16
2.4.5 Large Volume Injections ..................................................................................16
2.5 Direct Injection onto a Capillary Column ...............................................................18
2.5.1 Hardware Layout..............................................................................................18
2.5.2 Fluidic Setup.....................................................................................................19
2.5.3 Installation Tips................................................................................................20
2.5.4 Testing the Application ....................................................................................20
2.6 Pre-concentration onto a Nano Column ..................................................................21
2.6.1 Hardware Layout..............................................................................................21
2.6.2 Fluidic Setup.....................................................................................................22
2.6.3 Installation Tips................................................................................................23
2.6.4 Testing the Application ....................................................................................23
2.7 Pre-concentration onto a 200 µm Monolithic Column............................................25
2.7.1 Hardware Layout..............................................................................................25
2.7.2 Fluidic Setup.....................................................................................................26
2.7.3 Installation Tips................................................................................................27
2.7.4 Testing the Application ....................................................................................27
2.8 Pre-concentration on a Capillary Column...............................................................29
2.8.1 Hardware Layout..............................................................................................29
2.8.2 Fluidic Setup.....................................................................................................30
2.8.3 Installation Tips................................................................................................31
2.8.4 Testing the Application ....................................................................................31
2.9 2D Salt Steps with Nano Column............................................................................32
2.9.1 Hardware Layout..............................................................................................32
2.9.2 Fluidic Setup.....................................................................................................33
2.9.3 Installation Tips................................................................................................34
2.9.4 Testing the Application ....................................................................................35
2.9.5 Salt Solutions Preparation ................................................................................36
2.10 Automated off-line 2D LC of Peptides, micro SCX xnano RP..............................37
2.10.1 Hardware Layout..............................................................................................37
2.10.2 Fluidic Setup.....................................................................................................39
2.10.3 Installation Tips................................................................................................40
2.10.4 Testing the Application ....................................................................................41
2.11 Automated off-line 2D LC, Cap RP (basic) x nan RP (acidic) ...............................42
2.11.1 Hardware Layout..............................................................................................42
2.11.2 Fluidic Setup.....................................................................................................43

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2.11.3 Installation tips .................................................................................................44
2.11.4 Testing the application .....................................................................................44
2.12 Tandem nano LC .....................................................................................................46
2.12.1 Hardware Layout..............................................................................................46
2.12.2 Fluidic Setup.....................................................................................................47
2.12.3 Installation Tips................................................................................................48
2.12.4 Testing the Application ....................................................................................48
3FAQ ...........................................................................................................................50
3.1 Interpreting a Chromatogram ..................................................................................50
3.2 Troubleshooting nano LC peptide Applications......................................................50
3.3 The use of TFA and FA...........................................................................................52
3.4 Minimizing Baseline Noise .....................................................................................52
3.4.1 Drift ..................................................................................................................52
3.4.2 Unstable Baseline.............................................................................................53
4Appendix – Traditional Capillary Connections....................................................54
5Appendix – Common Spare Parts in nano LC......................................................56

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1 Introduction
This document describes the standard setup, recommended experimental conditions and
testing procedures to run standard applications on the Thermo Scientific Dionex UltiMate
3000 RSLCnano system.
Notes: This document is intended for Thermo Fisher Scientific (or authorized)
service personnel as well as customers to assist in the installation and
system test of UltiMate3000 RSLCnano systems.
It is assumed that the individual using this manual has 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
solvent hazards, exposure to UV radiation and the exposure to pressurized
solvents.
At various points throughout the manual, messages of particular importance are indicated by
the following symbols:
Tip: Indicates general information intended to optimize the installation and
setup steps or the performance of the instrument.
Important: Indicates that failure to take note of the accompanying information may
result in damage to the instrument.
Warning: Indicates that failure to take note of the accompanying information may
result in personal injury.
This manual is provided "as is." Every effort has been made to supply complete and accurate
information and all technical specifications have been developed with the utmost care. The
information contained in this manual should not be construed as a commitment by Thermo
Fisher Scientific. Thermo Fisher Scientific assumes no responsibility for any errors that may
appear in this document. This document is believed to be complete and accurate at the time of
publication. In no event shall Thermo Fisher Scientific be liable for incidental or
consequential damages in connection with or arising from the use of this document.
The information contained in this document is subject to change without notice.

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electronic media. No part of this publication may be copied or distributed, transmitted,
transcribed, stored in a retrieval system, or transmitted into any human or computer language,
in any form or by any means, electronic, mechanical, magnetic, manual, or otherwise, or
disclosed to third parties without the express written permission of Thermo Fisher Scientific
Inc.
Trademarks
Analyst is a registered trademark of AB Sciex.
Compass and Hystar are trademarks of Bruker Daltronics.
Nitronic is a registered trademark of AK Steel Corp.
PEEK is a trademark of Victrex PLC.
PharMed is a registered trademark of Saint -Gobain Performance Plastics.
Windows and Windows Vista are registered trademarks of Microsoft Corp.
All other trademarks are property of Thermo Fisher Scientific Inc. and its subsidiaries
1.1 Additional Documents
In addition to the information provided in this manual, the following documents are available:
•Operating Instructions of the individual modules
•Installation Qualification manual for installation of the UltiMate 3000 RSLCnano
•Micro Fraction Collection Option for the Thermo Scientific Dionex UltiMate WPS-
3000PL Nano/Cap Autosampler
•Application/technical notes to be published on the Dionex website
In addition to these documents, Chromeleon templates are available for these applications.
Please contact your local Thermo Scientific sales or service representative to obtain the
templates.

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2 Application Setup
2.1 General Recommendations for Applications
The experimental conditions for each application are presented together with related
information such as schematics, installation tips and examples, results and interpretation.
2.1.1 Connections
All high-pressure fittings used in the applications on the UltiMate 3000 RSLCnano system are
made with nanoViper. nanoViper is a fingertight high-pressure fitting that is dead volume free
by design and back pressure resistant up to 1000 bar. The fittings are assembled in the factory
to ensure consistent fittings and prevent experimental failure due to bad connections.
Figure 1: Example of a nanoViper fitting
1. Install nanoViper as any fingertight fitting.
2. Do not over-tighten connections (general guide line: finger-tight + maximum an
additional one eighth-turn).
3. Remove the black knurled screw.
The outlet of the nano columns is fitted with a nano connector. This is a dedicated connection
designed to offer maximum flexibility in connecting fused silica capillaries and offering
pressure stability 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 below.
Figure 2: nano connector layout
•Use a new nano connector sleeve (P/N 6720.0391) for each connection
Important: Do NOT use a PTFE sleeve. The size does not match the nano
connector and the pressure resistance is much lower.

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Nano connector sleeve (~1.1 cm)
PTFE (1.8 cm)
Figure 3: nano connector (top) and PTFE (bottom) sleeve comparison
1. Slide the golden nut and silver union onto one of the fused silica outlets; slide the
second golden union on the other fused silica end. (→Figure 4a and Figure 4b)
2. Slide the nano connector sleeve onto the fused silica, slide the other end of the fused
silica into the nano connector sleeve and make sure the connection is dead volume free.
Figure 4c
3. Tighten the golden nuts equally fingertight to ensure the nano connector sleeve is
centered in the silver union. Figure 4d
4. For traditional fittings, for example, PTFE sleeves see Appendix – Traditional Capillary
Connections (page 54)
a b
c d
Figure 4: Using the nano connector
2.1.2 Sample Preparation
•Follow the instructions provided with the test sample and use the solvents as indicated.
•After re-dissolving the sample wait at least 15 min before further diluting it.
oUse water with a minimum of 2 % ACN and appropriate ion-pairing agent to
dissolve.
oDilution can be done in the same solvent or with water with appropriate ion-pairing
agent.
Tip: To limit the risks of peptides or proteins adsorption on the walls of the vials,
Thermo Fischer Scientific recommends using glass inserts (Polypropylene vials
for WPS with glass insert, 250 µL, set of 25, P/N 6820.0027).
Tip: When using IEX columns, make sure that the sample solvent contains a very
limited amount of salt and is at the right pH level (for example, adjust to pH 3
when using a SCX column to separate peptides).

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2.1.3 Mobile Phases
•Always use fresh solvents.
•When running online multidimensional approaches, make sure that the mobile phase of
the first dimension will allow for trapping the sample on the (trap) column used on the
second dimension.
•For example, do not use phosphate buffers from SCX separations as loading solvent in
RP separations.
•Thermo Fischer Scientific recommends replacing (aqueous) solvents at least once every
two weeks.
Tip: Replace solvents completely; do not ’top up’ to avoid unwanted components
building up in the mobile phases.

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2.2 Installing the UltiMate 3000 RSLCnano System
The general UltiMate 3000 RSLCnano system overview is shown in Figure 5. The modular
nature of the Thermo Scientific Dionex UltiMate 3000 platform does allow modifications to
this layout.
Figure 5: RSLCnano system overview
SRD-3400, SRD-3200 with degassing or
SR-3000 without degassing.
NCS-3500RS module featuring
- NC pump, up to 800 bar
- Loading pump, micro Titanium up to 500 bar
- Column compartment with up to two switching valves
Optional:
- NCP-3200RS
- PAEK valve
VWD-3400RS with flow cells for
- nano (3nL),
- capillary (45 nL)
- micro (180 nL) LC
WPS-3000TPL RS
- Temperature controlled autosampler equipped with a
1000 bar switching valve
- Optional:
8-port valve (350 bar) for automated off-line
applications
Figure 6 shows the interior of the NCS-3500RS module. The module consists of two pumps
and an integrated column compartment. The most important elements required for system
setup are indicated in the picture.
Figure 7 shows the interior of the NCP-3200RS module. This module contains only the
NC_Pump, but is identical in capabilities and performance as the NC_Pump in the NCS-
3500RS.

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NC_Pump
Loading pump
Rear seal wash system
Flow meter
Purge screws
Inline filter
Purge screw
Tubing guides
Snap-in valves
Column compartment
Figure 6: Detailed overview of the NCS-3500RS interior
Figure 7: Detailed overview of the NCP-3200RS interior, components are identical to the NC_Pump in the NCS-
3500RE

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Before the parts of the application kit are installed, the UltiMate 3000 RSLCnano system has
to be prepared and primed. To prepare the system, the following steps are required. For more
details, see the operating instructions for the respective module.
1
To control the module, Thermo Scientific Dionex Chromeleon 6.80 SR8 (or later) or
DCMSLink version 2.8 (or later) and appropriate license are required.
2
Prepare electrical and USB connections, power up modules, and then prepare server
configuration.
3
Prepare solvents and install them according to the application.
-NC pump channels A and B
-Loading pump channels A, B, and C (10-50% isopropanol in water for unused
channels)
-Rear seal wash solvent (~10% isopropanol in water)
-Autosampler wash solvent (~10% isopropanol 0.1% FA in water).
Important
Use the PEEK solvent inlet filter frits in both, the NC pump and loading pump
solvent lines.
Tip
Degassing of the loading pump solvents is required when the loading pump is
used for gradient formation or the flow rate is above 20 µL/min.
4
Purge both blocks of the NC pump (min 30 min.), while assisting with a syringe until
liquid exits the purge lines. Purge all channels of the loading pump (minimum 10 min).
Tip
Make sure that the purge screws of the NC_Pump are entirely opened.
5
Purge flow meter for 30 min.
Tip
The purge time with a nano flow selector is 30 minutes. For capillary and micro
flow selectors purge times may be shorter. Please refer to the NCS-3500RS or
NCP-3200RS manual for details.
6
Perform pressure sensor offset calibration, using Chromeleon Diagnostics. To do so, open
the More options panel of the NC_Pump and click the related button.
7
Perform viscosity calibration using Chromeleon Diagnostics or select default viscosities
from the available viscosity list.
Tip
To see the solvent list for which default viscosities are available, use the
Commands dialog (F8) and scroll to Pump module NC_Pump 
%A_Viscosity (%B_Viscosity).
8
Prepare all fluidics using the provided nanoViper connection tubing.
Tip
Do not overtighten the fingertight connection!

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2.3 Application Overview
Table 1: Property overview of different flow selectors
Flow Selector
Type
I.D. P/N Total Flow Rate
(Sum of channel A and B)
Nominal Minimum Maximum
Nano Nan 6041.0002 500 nL/min 50 nL/min* 1000 nL/min
Capillary Cap 6041.0003 5 µL/min 500 nL/min 10 µL/min
Micro Mic 6041.0014 25 µL/min 2.5 µL/min 50 µL/min
*Lower flow rates are available upon request

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2.4 Direct Injection onto a Nano Column
2.4.1 Hardware Layout
Figure 8: Setup for a Direct Injection experiment onto a
nano column
The preferred setup is presented in
Figure 8 and consists of:
SR-3000 5035.9200
NCP-3200RS 5041.0030
VWD-3400RS 5074.0010
3 nL flow cell 6074.0270
WPS-3000TPL RS 5826.0020
Application kit: 6720.0300

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Figure 9: Setup for a Direct Injection experiment onto a
nano column
The alternative setup is presented in
Figure 9 and consists of:
SRD-3400 5035.9245
NCS-3500RS 5041.0010
VWD-3400RS 5074.0010
3 nL flow cell 6074.0270
WPS-3000TPL RS 5826.0020
Application kit: 6720.0300

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2.4.2 Fluidic Setup
Figure 10 presents the setup using the parts of the Direct Injection application kit. Columns
are marked with letters, tubing with digits, and the sample loop is installed in the WPS-
3000PL Autosampler
Figure 10: Fluidic connections for a Direct Injection experiment onto a nano column
Table 2: UltiMate 3000 RSLCnano Direct Injection nano LC kit (P/N 6720.0300) contents
# Item Replacement 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
2.4.3 Installation Tips
•Follow the General Recommendations for Applications (→page 6).
•The impact of dwell (dead) volumes on reproducibility is very important. Improper
connections of the different elements are the most likely cause of failure for this
application.
Tip: When no valve is
available, a union can be used to
connect the capillary column
(P/N 6040.2304)

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2.4.4 Testing the Application
The direct injection can be tested using the following conditions:
Property Setting
Mobile phase A 100% water + 0.05% TFA
Mobile phase B 20%/80% (v/v) water/ACN + 0.04% TFA
Sample Cytochrome C digest 1 pmol/μL, prepared according to the instruction sheet
Injection volume 1 μL
UV detection 214 nm
Gradient 4% to 55% B in 30 min
90% B for 5 min
25 min equilibration
WPS temperature 5°C (WPS-3000(B)T only)
Flow rate 300 nL/min (nano flow selector)
Figure 11: Typical chromatogram for a Direct Injection of 1 pmol Cyt C onto a nano column
For details on interpretation and troubleshooting of the Cytochrome C nano LC separation,
see the FAQ section on page 50.
2.4.5 Large Volume Injections
Typically direct injections in nano LC are performed with 1 µL loop sizes to minimize the
gradient delay. Larger volume injections are performed with a pre-concentration setup. The
WPS-3000PL autosampler series allows for a custom injection program (UDP) to switch back
the injection valve after sample loading to bypass the loop. This way a larger sample volume
can be injected and pre-concentrated directly onto the nano column, without using a pre-
concentration setup.

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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 desalted samples, since all that is injected
will enter the MS, and an investment of extra analysis time to accommodate the loading of
sample with nano flow.

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2.5 Direct Injection onto a Capillary Column
2.5.1 Hardware Layout
Figure 12: Setup for a Direct Injection experiment onto a
capillary column
The recommended setup is
presented in Figure 12 and consists
of:
SRD-3400 5035.9245
NCS-3500RS 5041.0020
VWD-3400RS 5074.0010
45 nL flow cell 6074.0280
WPS-3000TPL RS 5826.0020
Application kit: 6720.0305

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2.5.2 Fluidic Setup
Figure 13 presents the setup using the parts of the Direct Injection application kit. Columns
are marked with letters, tubing with digits, and the sample loop is installed in the
WPS-3000PL Autosampler.
Figure 13: Fluidic connections for a Direct Injection experiment onto a capillary column
Table 3: UltiMate 3000 RSLCnano Direct Injection capillary LC Kit (P/N 6720.0305) contents
# Item Replacement 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
Tip: When no valve is
available, a union can be used to
connect the capillary column
(P/N 6040.2304)