Agilent Technologies 1290 Infinity II 2D-LC Solution User manual

Agilent 1290 Infinity II 2D-LC Solution
User Guide
2D-LC - User Guide

2D-LC - User Guide
Notices
Document Information
Document No: SD-29000383 Rev. B
Part No: G2198-90001 Rev. B
EDITION 02/2020
Copyright
© Agilent Technologies, Inc. 2012-2020
No part of this manual may be repro-
duced in any form or by any means
(including electronic storage and retrieval
or translation into a foreign language)
without prior agreement and written con-
sent from Agilent Technologies, Inc. as
governed by United States and interna-
tional copyright laws.
Agilent Technologies
Hewlett-Packard-Strasse 8
76337 Waldbronn
Warranty
The material contained in this document
is provided “as is,” and is subject to being
changed, without notice, in future edi-
tions. Further, to the maximum extent
permitted by applicable law, Agilent dis-
claims all warranties, either express or
implied, with regard to this manual and
any information contained herein, includ-
ing but not limited to the implied warran-
ties of merchantability and fitness for a
particular purpose. Agilent shall not be lia-
ble for errors or for incidental or conse-
quential damages in connection with the
furnishing, use, or performance of this
document or of any information con-
tained herein. Should Agilent and the user
have a separate written agreement with
warranty terms covering the material in
this document that conflict with these
terms, the warranty terms in the separate
agreement shall control.
Technology Licenses
The hardware and/or software described
in this document are furnished under a
license and may be used or copied only in
accordance with the terms of such
license.
Restricted Rights Legend
U.S. Government Restricted Rights. Soft-
ware and technical data rights granted to
the federal government include only
those rights customarily provided to end
user customers. Agilent provides this
customary commercial license in Soft-
ware and technical data pursuant to FAR
12.211 (Technical Data) and 12.212
(Computer Software) and, for the Depart-
ment of Defense, DFARS 252.227-7015
(Technical Data - Commercial Items) and
DFARS 227.7202-3 (Rights in Commer-
cial Computer Software or Computer
Software Documentation).
Safety Notices
CAUTION
A CAUTION notice denotes a
hazard. It calls attention to an
operating procedure, practice, or
the like that, if not correctly per-
formed or adhered to, could result
in damage to the product or loss
of important data. Do not proceed
beyond a
CAUTION notice until
the indicated conditions are fully
understood and met.
WARNING
A WARNING notice denotes a
hazard. It calls attention to an
operating procedure, practice, or
the like that, if not correctly per-
formed or adhered to, could
result in personal injury or death.
Do not proceed beyond a WARN-
ING notice until the indicated
conditions are fully understood
and met.

2D-LC - User Guide 3
In This Book
This manual covers the Agilent 1290 Infinity II 2D-LC Solution and 2D-LC
Acquisition software (G2198AA).
1Introduction
This chapter describes the product of Agilent 1290 Infinity II 2D-LC Solution.
2Conceptsof2D-LC
This chapter describes the concepts of Agilent 1290 Infinity II 2D-LC Solution.
3 Compatibility Matrix
The compatibility matrix provides information about installation and execution
prerequisites with respect to hardware, firmware and the operating system. Agilent
2D-LC Software is an OpenLAB CDS ChemStation Edition plug-in.
4Installation
This chapter describes the installation of the Agilent 1290 Infinity II 2D-LC Solution.
These installation instructions are valid for the modes standard heart-cutting,
multiple heart-cutting, high resolution sampling and comprehensive 2D-LC except
the connections to the 2D-LC valve. These connections depend on the method, see
chapter "Run the System for detailed information.
5 Method Parameters
This chapter provides background information on method parameters. It helps to
optimize methods in Agilent 1290 Infinity II 2D-LC Solution in the modes standard
heart-cutting, multiple heart-cutting, high resolution sampling and comprehensive
2D-LC.
6 Run the System
This chapter describes how to run the Agilent 1290 Infinity II 2D-LC Solution in the
modes standard heart-cutting, multiple heart-cutting, high resolution sampling and
comprehensive 2D-LC.

2D-LC - User Guide 4
7 Investigate the effects of using different gradients in the 2Dimension
This chapter describes, how shifted gradients in the second dimension can be
used to enlarge the accessible two-dimensional separation space.
8 Data Analysis
This chapter descibes the analyzation of data in 2D-LC and is separated in a
section heart-cutting 2D-LC and a section comprehensive 2D-LC.
9 Troubleshooting and Diagnostics
This chapter gives an overview about the troubleshooting and diagnostic features
and the different user interfaces.
10 Error Information
This chapter describes the meaning of error messages, and provides information
on probable causes and suggested actions how to recover from error conditions.
11 Maintenance
This chapter describes the maintenance of the 2D-LC Solution.
12 Parts for Maintenance
This chapter provides information on parts material required for the solution.
13 Alternative ways to install the System
This chapter describes alternative ways to install and setup the system.
14 Agilent 2D-LC Solution with the selectivity of mass selective detection
(MSD)
This chapter describes the different options to use the Agilent 2D-LC Solution with
mass selective detection (MSD).

2D-LC - User Guide 5
15 Theoretical Background
This chapter gives the theoratical background of 2D-LC and describes the system
components (soft- and hardware) of the Agilent 1290 Infinity II 2D-LC Solution.
16 Appendix
This chapter provides addition information on safety, legal and web.

2D-LC - User Guide 6
Contents
1Introduction10
Product Description 11
Features 13
2Conceptsof2D-LC14
Concepts of 2D-LC 15
Heart-Cutting 2D-LC (LC-LC) 16
Multiple Heart-Cutting and High Resolution Sampling 2D-LC 17
Comprehensive 2D-LC (LCxLC) 29
Triggering of 2D-LC 31
Active solvent Modulation (ASM) 35
3 Compatibility Matrix 45
Supported Chromatographic Data Systems 46
Supported Drivers 47
Supported Operating Systems 48
Supported Firmware 49
Available Languages 50
PC Requirements 51
4Installation52
Delivery checklist 53
Options 54
Recommendations for instrument setup 58
Installation of the 2D-Valves 75
2D-LC Software Configuration 84
Installation of Active Solvent Modulation (ASM) 86

2D-LC - User Guide 7
5 Method Parameters 87
Method Parameters Standard Heart-Cutting 2D-LC 88
Method Parameters Multiple Heart-Cutting and High Resolution Sampling
2D-LC 104
Method Parameters Comprehensive 2D-LC 127
Method Development of Active Solvent Modulation (ASM) 141
6 Run the System 146
Connect the capillaries to the 2D-LC valve 147
Standard Heart-Cutting 2D-LC 148
Multiple Heart-Cutting and High Resolution Sampling 2D-LC 159
Comprehensive 2D-LC 175
Active Solvent Modulation (ASM) 189
7 Investigate the effects of using different gradients in the
2Dimension 192
Investigate the effects of using different gradients in the 2Dimension 193
8 Data Analysis 200
Data Analysis for Heartcutting 2D-LC (LC-LC) 201
Data Analysis for Comprehensive 2D-LC (LCxLC) 219
9Troubleshootingand Diagnostics 231
Overview of the Module’s Indicators and Test Functions 232
User Interfaces 233
Agilent Lab Advisor Software 234
The Basic Principle of Troubleshooting 235
Recommended Tests to Conclude Troubleshooting 245
10 Error Information 246
What Are Error Messages 247
General Error Messages 248
Module-Specific Error Messages 254

2D-LC - User Guide 8
11 Maintenance 258
Introduction to Maintenance 259
Warnings and Cautions 260
Overview of Maintenance 262
Cleaning the Module 263
Correcting Leaks 264
Replace Valve Heads 266
Replacing Parts of the Valve Head 272
Replacing the Fuses of the Infinity Valve Drive 274
Replace the Module Firmware 276
12 Parts for Maintenance 277
2D-LC Loops 278
2D-LC Capillaries 279
ASM Capillaries 280
Pressure Release Kit 281
2D-LC Easy Starter Kit 282
Valve Drive Parts 283
Valve Driver Parts Infinity II 284
Valve Head Parts 285
Vale Options Overview (for 2D-LC) 287
MS Diverter Valve 291
Valve Options Overview (for G7116B) 293
Additional Heater Devices 295
Accessories and Consumables (for G7116B) 297
InfinityLab Quick Connect and Quick Turn Fittings 299
Capillary Kits 303
Important Customer Web Links 304
13 Alternative ways to install the System 305
Legacy Stack Configuration 306

2D-LC - User Guide 9
14 Agilent 2D-LC Solution with the selectivity of mass selective detection
(MSD) 311
Agilent 2D-LC Solution with an Agilent Single Quadrupole Detector 312
Agilent 2D-LC Solution with high-end MS detection 318
15 Theoretical Background 322
Theoretical basis of 2D-LC 323
2nd dimension as detector 331
Successful Mode Combinations 333
Solvent Elution Modes 334
Practical Issues 340
16 Appendix 342
General Safety Information 343
Waste Electrical and Electronic Equipment (WEEE) Directive 350
Radio Interference 351
Sound Emission 352
Solvent Information 353
Further Information 354
Agilent Technologies on Internet 355

2D-LC - User Guide 10
1Introduction
Product Description 11
Features 13
This chapter describes the product of Agilent 1290 Infinity II 2D-LC Solution.

2D-LC - User Guide 11
1Introduction
Product Description
Product Description
2D-LC offers great improvements in resolving power over conventional 1D-LC.
The advantages are as follows:
•Further resolution of a complex mixture that cannot be separated on a single
column
•Increased peak capacity
•Sample cleanup by removing matrix or interfering compounds
•Increase sample throughput (two separations going on at once)
Pre-configured systems and a dedicated 2D-LC training allow an easy start into
2D-LC.
The Agilent 1290 Infinity II 2D-LC Solution can switch to 1D-UHPLC and all the
different 2D-LC techniques easily. The easy-to-use software of the Agilent 1290
Infinity II 2D-LC Solution is designed for fasted method setup in all available
modes:
•Comprehensive
•Heart-cutting
•Multiple heart-cutting
•High resolution sampling

2D-LC - User Guide 12
1Introduction
Product Description
Figure 1 Dual Stack Configuration
Autosampler
2D Pump
1D Pump
2D-LC Valve
Parking deck A
Parking deck B
1D Detector
2D Detector
2D Column compartment

2D-LC - User Guide 13
1Introduction
Features
Features
•Boosts performance through ultrahigh peak capacity in excess of 1000 – for
unmatched power to separate most complex samples
•Saves time through trouble-free instrument setup – with easy starter kit to
enable fastest familarization
•Reduces costs through single-vendor solution – higher returns on investment
with one system for both 1D-LC and 2D-LC
•Supports comprehensive, heart-cutting and multiple heart-cutting 2D-LC – for
any kind of 2D-LC operation(“Comprehensive 2D-LC (LCxLC)” on page 29,
“Heart-Cutting 2D-LC (LC-LC)” on page 16, “Multiple Heart-Cutting and High
Resolution Sampling 2D-LC” on page 17)
•Utilizes the powerful 1290 Infinity Binary Pump in the second dimension – for
highest speed, accuracy, precision and resolution
•Highly flexible in the first dimension – even older Agilent LC systems can be
upgraded for a very cost-effective access to the power of 2D-LC
•Allows fully symmetric flow path for comprehensive 2D-LC and matched
loops – using innovative and unique 1290 Infinity Valve Drive
•New Heart-Cut Viewer software – for unmatched usability in multiple
heart-cutting data analysis
•Easy-to-use 2D-LC acquisition software - for fastest and most easy system
and method set-up
•Powerful comprehensive 2D-LC data analysis - using LC Image Software from
GC Image LLC, USA

2D-LC - User Guide 14
2Conceptsof2D-LC
Concepts of 2D-LC 15
Heart-Cutting 2D-LC (LC-LC) 16
Multiple Heart-Cutting and High Resolution Sampling 2D-LC 17
Principles of Heart-cutting 2D-LC 20
Comprehensive 2D-LC (LCxLC) 29
Triggering of 2D-LC 31
Concept of Peak Triggering 31
Concept of Time Triggering 34
Active solvent Modulation (ASM) 35
Introduction to Active Solvent Modulation (ASM) 35
Operating Principle 37
Understanding the ASM factor 41
Comprehensive 2D-LC and Active Solvent Modulation 44
This chapter describes the concepts of Agilent 1290 Infinity II 2D-LC Solution.

2D-LC - User Guide 15
2 Concepts of 2D-LC
Concepts of 2D-LC
Concepts of 2D-LC
In a 2D-LC-System, Pump 1 generates the first dimension gradient. An
autosampler injects the sample and separates it by column 1. A 2D-LC Valve
(Injector) connects the first dimension to the second dimension and stores
sample peaks intermediately. These sample peaks are re-injected to the second
dimension, separated by a second column and measured by Detector 2.
Figure 2 Concept of a 2D-LC-System
In 2D-LC the following concepts exist:
•Comprehensive 2D-LC (LC×LC)
In LC×LC, the total eluent from the first dimension is injected on to the column
in the second dimension.
•Heart-cutting 2D-LC (LC-LC)
In LC-LC only parts of the eluent from the first dimension are injected on to
the column in the second dimension.

2D-LC - User Guide 16
2 Concepts of 2D-LC
Heart-Cutting 2D-LC (LC-LC)
Heart-Cutting 2D-LC (LC-LC)
The following items are characteristic for LC-LC:
•Only parts of the effluent of the first column - only the peaks of interest eluted
from the 1
st
dimension column - are injected to the second column
•A peak from the 1
st
dimension is sampled as a whole and a method with a
lower flow rate and a gradient typically with a longer run time than the
collection time is used to improve separation efficiency
•Typically longer columns with higher separation efficiency are used in 2
nd
dimension column
There are two modes of LC-LC:
•Time-triggered LC-LC
•Peak-triggered LC-LC
NOTE
Heart-Cutting 2D-LC (LC-LC) is the method of choice if the samples to analyze
are known or to improve confidence of an experiment (pharma, method
development and so on).
NOTE
Multiple peaks eluted from the first dimension column can be sampled and
analyzed in the second dimension but the run time of the second dimension
must match the retention time between two first dimension peaks. A started
second dimension analysis will always be finished! Thus, a second peak being
eluted from the first dimension might be lost, if sampled while the second
dimension analysis is still running.

2D-LC - User Guide 17
2 Concepts of 2D-LC
Multiple Heart-Cutting and High Resolution Sampling 2D-LC
Multiple Heart-Cutting and High Resolution
Sampling 2D-LC
Typically, the gradient time in the second dimension is much longer for
heart-cutting than with the comprehensive technique. The disadvantage of the
standard heart-cutting techniques is that peaks cannot be sampled while a
second dimension gradient is still running. In the examples shown here, the
gradient from the second dimension is analyzing the first peak (purple), while the
second and third peak (gray and yellow) elute from the first dimension column.
The second dimension is ready when the 4th peak (green) elutes from the first
dimension; this peak can be analyzed. As the second dimension is occupied
again, the fifth peak (blue) cannot be analyzed.

2D-LC - User Guide 18
2 Concepts of 2D-LC
Multiple Heart-Cutting and High Resolution Sampling 2D-LC
This problem is addressed using a setup called multiple heart-cutting 2D-LC.
Here, the sampling loops on the 2D-LC valve are exchanged with
6-position/14-port selection valves, which are equipped with six loops each. In
this configuration, a peak can be cut out and stored, then analyzed as soon as the
second dimension is free.

2D-LC - User Guide 19
2 Concepts of 2D-LC
Multiple Heart-Cutting and High Resolution Sampling 2D-LC
Peaks that are cut out and stored during a run are analyzed consecutively in the
second dimension, even when the first dimension is still running. To avoid
carry-over the peaks are analyzed in reverse order of storage in a single Multiple
Heart-Cutting Valve.

2D-LC - User Guide 20
2 Concepts of 2D-LC
Multiple Heart-Cutting and High Resolution Sampling 2D-LC
Principles of Heart-cutting 2D-LC
Multiple Heart-Cutting - Principles
Multiple Heart-Cutting - Principles
Multiple Heart-Cutting 2D-LC is a complex workflow, working on a special
algorithm for filling the sample loops and analyzing the stored cuts, based on
different criteria. “Multiple Heart-Cutting - Principles” on page 20 illustrates the
principles of the Multiple Heart-Cutting algorithm, following these principles:
•
2
D analysis is done as soon as possible. As long as the 2
nd
dimension is free,
any next cut from the 1st dimension will be always directly transferred to the
2
nd
dimension and analysed. This means:
•The first 1D cut will be always directly analysed in the 2
nd
dimension.
•If the 2
nd
dimension is free, when the next
1
D cut is taken, it will also be
directly analysed.
•If the 2
nd
dimension is occupied, the next
1
D cut will be stored in the next
sample loop.
•If all sample loops in the 1st dimension are occupied, the peak is lost.
•A peak parking deck will always be completely analysed, before switching to
the other parking deck.
•Before analyzing a new parking deck, a flush gradient is run to avoid
contamination.
•Stored cuts are analysed in backwards order to avoid contamination.