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

Agilent 1290 Infinity II 2D-LC Solution
MassHunter Acquisition for TOF and Q-TOF
User Guide
2D-LC User Guide

2D-LC User Guide
Notices
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Document No: D0004828 Rev. A
Edition: 05/2021
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2D-LC User Guide 3
In This Book
In This Book
This manual covers the Agilent 1290 Infinity II 2D-LC Solution for MassHunter
TOF and Q-TOF.
1Introduction
This chapter describes the product of Agilent 1290 Infinity II 2D-LC Solution.
2 Concepts of 2D-LC
This chapter describes the concepts of Agilent 1290 Infinity II 2D-LC Solution.
3 Compatibility Matrix
This chapter provides information about installation and execution prerequisites
regarding hardware, firmware, and the operating system. Agilent 1290 Infinity II
2D-LC Solution.
4 Installation
This chapter describes the hardware and software installation of the Agilent
1290 Infinity II 2D-LC Solution. The 2D-LC instrument can be used with the
software described in this document. The installation instructions are valid for
the modes standard heart-cutting, multiple heart-cutting, high resolution
sampling and comprehensive 2D-LC.
5 2D-LC Data Acquisition in MassHunter Workstation 11
This chapter provides information about 2D-LC data acquisition in MassHunter
Workstation 11.
6MethodParameters
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.

In This Book
2D-LC User Guide 4
7 Method Development of Active Solvent Modulation (ASM)
This chapter provides information on how to develop methods when using Active
Solvent Modulation (ASM).
8 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 with the driver-based 2D-LC Solution.
9 Data Analysis
This chapter provides information on how to analyze 2D-LC data with software.
10 Troubleshooting and Diagnostics
This chapter gives an overview about the troubleshooting and diagnostic
features and the different user interfaces.
11 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.
12 Maintenance
This chapter describes the maintenance of the 2D-LC Solution.
13 Parts for Maintenance
This chapter provides information on parts material required for the solution.
14 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.
15 Appendix
This chapter provides addition information on safety, legal and web.

2D-LC User Guide 5
Contents
1Introduction10
Product Description 11
Features 12
Terms related to 2D-LC 14
2 Concepts of 2D-LC 15
Concepts of 2D-LC 16
Heart-Cutting 2D-LC (LC-LC) 18
Multiple Heart-Cutting and High Resolution Sampling 2D-LC 19
Comprehensive 2D-LC (LCxLC) 30
Triggering of 2D-LC 32
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
Licensing 52
4 Installation 53
Hardware Installation 54
Software Installation 89

2D-LC User Guide 6
5 2D-LC Data Acquisition in MassHunter Workstation 11 103
Start the Data Acquisition Software 104
Overview 2D-LC in MassHunter Acquisition 11 106
Instrument Status 107
2D-LC User Interface 108
2D-LC Valves Online Monitor in the 2D-LC User Interface 114
Method Editor Window 116
Sample Run Window 117
Worklist Window 118
Tune Window 119
Instrument Details 120
Log book in MassHunter Acquisition 11 123
6 Method Parameters 125
Method Editor 2D-LC 126
Set the 2D-LC Method parameters 127
Preview (2D-LC) 154
Set up a Peak-Based Experiment Graphically 168
Setup 2D Gradient Graphically 173
Setup Second Dimension Gradient with the Graphical User Interface 175
Additional Information 177
7 Method Development of Active Solvent Modulation (ASM) 185
Method Development of Active Solvent Modulation (ASM) 186
Method Parameters 187
Optimize the Dilution by Using ASM Capillaries 188
Optimize the Sample Loop Flush 189
Include the ASM Phase to the 2D Gradient 190
Optimize Dilution Through Method Settings 191

2D-LC User Guide 7
8 Run the System 192
Familiarization to Start a System Run 193
Prepare the 2D-LC System 194
Configure the 2D-LC System 195
Checkout Familiarization Procedure 198
Prepare the Experiment 199
Run the Experiment 201
9 Data Analysis 229
2D-LC Data Analysis/Data Evaluation for MassHunter 230
GC Image Basic Information 256
10 Troubleshooting and Diagnostics 275
Overview of the Module’s Indicators and Test Functions 276
User Interfaces 277
Agilent Lab Advisor Software 278
Lab Advisor Instrument Control 280
Lab Advisor Service &Diagnostic 286
The Basic Principle of Troubleshooting 294
Recommended Tests to Conclude Troubleshooting 304
11 Error Information 305
What Are Error Messages 306
General Error Messages 307
Module-Specific Error Messages 313

2D-LC User Guide 8
12 Maintenance 317
Introduction to Maintenance 318
Warnings and Cautions 319
Overview of Maintenance 321
Cleaning the Module 322
Correcting Leaks 323
Replace Valve Heads 324
Replacing Parts of the Valve Head 330
Replacing the Fuses of the Infinity Valve Drive 332
13 Parts for Maintenance 334
2D-LC Loops 335
2D-LC Capillaries 336
ASM Capillaries 337
Pressure Release Kit 338
2D-LC Easy Starter Kit 339
Valve Drive Parts 340
Valve Driver Parts Infinity II 341
Valve Head Parts 342
Vale Options Overview (for 2D-LC) 344
MS Diverter Valve 348
Valve Options Overview (for G7116B) 350
Additional Heater Devices 352
Accessories and Consumables (for G7116B) 354
InfinityLab Quick Connect and Quick Turn Fittings 356
Capillary Kits 360
Important Customer Web Links 361

2D-LC User Guide 9
14 Theoretical Background 362
Theoretical basis of 2D-LC 363
2D as detector 371
Successful Mode Combinations 373
Solvent Elution Modes 374
Practical Issues 380
15 Appendix 382
General Safety Information 383
Waste Electrical and Electronic Equipment (WEEE) Directive 389
Radio Interference 390
Sound Emission 391
Solvent Information 392
Further Information 393
Agilent Technologies on Internet 394

2D-LC User Guide 10
1Introduction
Product Description 11
Features 12
Terms related to 2D-LC 14
This chapter describes the product of Agilent 1290 Infinity II 2D-LC Solution.

2D-LC User Guide 11
1Introduction
Product Description
Product Description
The 1290 Infinity II 2D-LC System is an innovative solution for solving most
complex separations, analyzing complex samples, and simplifying complex
workflows. From separation of a few co-eluting compounds to mixtures of
highest complexity - Agilent 2D-LC Solutions allow choosing from 2D-LC modes
(multiple) heart-cutting with high-resolution sampling and comprehensive 2D-LC.
A wide range of applications in many industries benefit from orthogonal
separations of samples, that cannot be resolved in one dimension at all or good
enough within a short time. Comprehensive 2D-LC offers unmatched peak
capacity for complex samples or sample matrices. 2D-LC can be used for
desalting samples for salt sensitive separations or for making buffer-based
separations MS compatible. In many cases, 2D-LC can be applied for simplifying
workflows by replacing multiple 1D separations by one 2D analysis or by
replacing offline fractionation through 2D-LC for faster, more reliable, and fully
automated workflows.
The unique Agilent 2D-LC software with excellent ease of use makes 2D-LC
available to everyone – from beginners, who want to create and review 2D-LC
measurements in minutes up to experts using most advanced method
development and data analysis capabilities.

1Introduction
Features
2D-LC User Guide 12
Features
Agilent InfinityLab 2D-LC Solutions offers following key features:
•Agilent 2D-LC is based on 1290 Infinity II Systems with UHPLC performance,
fast gradients, high sensitivity and excellent robustness
•Dedicated 2D-LC valves use completely symmetric flow paths for
reproducible retention times and peak areas.
•A wide range of modules can be used in both dimensions. Third-party
detectors are supported via UIB II including the sue of compatible detectors
for data analysis
•Powerful Agilent 2D-LC software is available for OpenLab CDS, MassHunter
and ChemStation. Measurements can be set up easily with a few mouse
clicks: Starting with a 1D measurement, choose spots where you want to
increase resolution and draw your second dimension gradient.
•Agilent 2D-LC instrument control is fully automated and eliminates the need
for tedious manual valve programming. Separation in the first and second
dimension are completely independent by using Agilent multiple heart-cutting
valves for highest storage capacity for up to 12 cuts at one time, fast and
parallel analysis.
•High-resolution sampling is available for flexibly analyzing short cuts to broad
peaks while retaining first dimension separation. By analyzing complete
peaks, highly reproducible quantitative measurements can be achieved.
•Multiple heart-cutting and High-resolution sampling can be combined
arbitrarily within one run
•Shifted gradients, which can be edited graphically or numerically, maximize
the available 2D separation space for highest peak capacity and fastest
analysis
•Dedicated flush gradients are available for fast analysis and minimum
carryover
•Cuts can be defined in time-based mode for highly reproducible
measurements or peak-based mode for variable retention times or unknown
samples. Even in peak-based mode, both first and second dimension
detectors provide complete chromatograms.
•Dynamic peak parking combines time- and peak-based approaches for
dealing with shifting first dimension retention times e.g. of
biopharmaceuticals by using reference compounds.

2D-LC User Guide 13
1Introduction
Features
•Multi-inject speeds up such analyses by sequentially injecting cuts from
multiple sample loops.
•Smart peak parking optimizes runs for the highest possible number of cuts
and shortest run time.
•A wide range of first and second dimension solvents and gradients can be
combined with the optional Agilent Active Solvent Modulation Technology for
multiple heart-cutting and high-resolution sampling measurements. By
reducing first dimension solvent effects, second dimension separation is
optimized and sensitivity is increased.
•The 2D-LC system can be prepared for a run by interactively and
automatically flushing both dimensions’ flow paths and all sample loops.
•The progress of a 2D run including parking of cuts in sample loops of deck
valves and their analysis can be monitored in the dashboard.
•The 2D-LC system can be combined with analytical fraction collection.
•With an optional valve, switch easily between 1D and 2D separation
GC Image Software
•GC Image LC x LC Edition Software for UV and single quadrupole or (Q-)TOF
and QQQ detection is available from Agilent.
It is used for visualizing 2D data and offers a sophisticated data analysis for
comprehensive 2D-LC data including qualitative and quantitative results and
statistical analysis
LabAdvisor
•Diagnostic tests help with troubleshooting the 2D-LC system

1Introduction
Terms related to 2D-LC
2D-LC User Guide 14
Terms related to 2D-LC
Term Definition
2D-LC Two-dimensional liquid chromatography
1D One-dimensional
1D-LC is the classical (one dimensional) chromatography, which provides
one-dimensional data. Usually, you would not even think about dimensions in the 1D
world.
1DFirst dimension
For example, a 1D column is the column used in the first dimension, and a 1D
chromatogram is the chromatogram acquired for the first dimension.
2D Two-dimensional
2D-LC is two dimensional chromatography, which provides two-dimensional data.
Two-dimensional data is data that has a first and a second dimension.
For example, 2D results can have chromatograms and peaks in each dimension. A 2D
peak has a retention time in each dimension. 2D peaks are peaks in the
two-dimensional contour plot.
2DSecond dimension
For example, a 2D pump is a pump installed in the second dimension. The 2D
retention time is the retention time in the second dimension chromatogram, or the
weighted averaged retention time for the second dimension in a two-dimensional
peak. 2D peaks are peaks in 2D cut chromatograms.
LCxLC Comprehensive 2D-LC
LC-LC Heart-cutting 2D-LC
MHC Multiple Heart-cutting
HiRes High-Resolution Sampling

2D-LC User Guide 15
2Conceptsof2D-LC
Concepts of 2D-LC 16
Heart-Cutting 2D-LC (LC-LC) 18
Multiple Heart-Cutting and High Resolution Sampling 2D-LC 19
Principles of Heart-cutting 2D-LC 22
Comprehensive 2D-LC (LCxLC) 30
Triggering of 2D-LC 32
Concept of Peak Triggering 32
Concept of Time Triggering 34
Active Solvent Modulation (ASM) 35
Introduction to Active Solvent Modulation (ASM) 35
Operating Principle 37
Understanding the ASM Factor 42
Comprehensive 2D-LC and Active Solvent Modulation 44
This chapter describes the concepts of Agilent 1290 Infinity II 2D-LC Solution.

2 Concepts of 2D-LC
Concepts of 2D-LC
2D-LC User Guide 16
Concepts of 2D-LC
In a 2D-LC-System, 1D pump generates the 1D gradient. An autosampler injects
the sample and separates it by 1D column. 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 2D column and measured by the 2D detector.
Figure 1 Concept of 2D-LC
Figure 2 Conceptual illustration of heart-cutting 2D-LC principle
D
1
D
2

2D-LC User Guide 17
2 Concepts of 2D-LC
Concepts of 2D-LC
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.
Figure 3 Standard 2D-LC valve (G4236A) with two loops (concurrent)

2 Concepts of 2D-LC
Heart-Cutting 2D-LC (LC-LC)
2D-LC User Guide 18
Heart-Cutting 2D-LC (LC-LC)
The following items are characteristic for LC-LC:
•Only parts of the effluent of the 1D column - only the peaks of interest eluted
from the 1D column - are injected to the 2D column
•A peak from the first 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 2D
column
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 19
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.
D
1
D
2

2 Concepts of 2D-LC
Multiple Heart-Cutting and High Resolution Sampling 2D-LC
2D-LC User Guide 20
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.
Figure 4 Standard 2D-LC valve (G4236A) with MHC 1300 bar (counter current)