Agilent Technologies 1290 Infinity LC System User manual

Agilent Technologies
Agilent 1290 Infinity
LC System
System Manual and
Quick Reference

Agilent 1290 Infinity LC System Manual and Quick Reference
Notices
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2012
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Manual Part Number
G4220-90301
Edition
05/2012
Printed in Germany
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Agilent 1290 Infinity LC System Manual and Quick Reference 3
In This Guide...
In This Guide...
This manual covers the Agilent 1290 Infinity LC System.
1 Introduction to Ultra-High Performance Liquid Chromatography
This chapter gives an introduction to the Agilent 1290 Infinity LC System and
the underlying concepts.
2 The Agilent 1290 Infinity LC System - Product Description
This chapter discusses the features of the 1290 Infinity LC System.
3 Optimization of the Agilent 1290 Infinity LC System
This chapter considers how to apply the theory and use the features of the LC
system to develop optimized separations.
4 System Setup and Installation
This chapter includes information on software installation, stack
configurations and preparing the system for operation.
5QuickStartGuide
This chapter provides information on data acquisition and data analysis with
the 1290 Infinity LC System.
6 Appendix
This chapter provides additional information on safety, legal and web and
about setting up a method.

4 Agilent 1290 Infinity LC System Manual and Quick Reference
Contents
Contents
1 Introduction to Ultra-High Performance Liquid Chromatography 7
Theory of Using Smaller Particles in Liquid Chromatography 8
Benefits of Columns Packed With Sub-2-micron Particles 14
Frictional Heating 18
2 The Agilent 1290 Infinity LC System - Product Description 21
New Features of the Agilent 1290 Infinity LC System 22
System Components 25
3 Optimization of the Agilent 1290 Infinity LC System 39
Delay Volume and Extra-Column Volume 40
How to Configure the Optimum Delay Volume 42
How to Achieve Higher Injection Volumes 51
How to Achieve High Throughput 53
How to Achieve Higher Resolution 56
How to Achieve Higher Sensitivity 59
How to Achieve Lowest Carry Over 66
How to Prevent Column Blockages 68
4 System Setup and Installation 71
Installing Software 72
Installing the Module 73
5QuickStartGuide 89
About the Quick Start Guide 90
Preparing the system 91
Data Acquisition in Method and Run Control View 96
Data Analysis 103

Agilent 1290 Infinity LC System Manual and Quick Reference 5
Contents
6 Appendix 109
Safety Information 110
Solvent Information 113
Agilent Technologies on Internet 114
Setting Up a Method using Edit Entire Method 115

6 Agilent 1290 Infinity LC System Manual and Quick Reference
Contents

7
Agilent 1290 Infinity LC System Manual and Quick Reference
Agilent Technologies
1
Introduction to Ultra-High Performance
Liquid Chromatography
Theory of Using Smaller Particles in Liquid Chromatography 8
Benefits of Columns Packed With Sub-2-micron Particles 14
Frictional Heating 18
This chapter gives an introduction to the Agilent 1290 Infinity LC System and
the underlying concepts.

8 Agilent 1290 Infinity LC System Manual and Quick Reference
1Introduction to Ultra-High Performance Liquid Chromatography
Theory of Using Smaller Particles in Liquid Chromatography
Theory of Using Smaller Particles in Liquid Chromatography
Introduction
In 2003, Agilent introduced the first commercially available porous silica
columns with 1.8 µm particles. These were the first in a class of columns
which has become known as ‘sub-two micron’ or STM columns. For use with
the Agilent 1200 Series Rapid Resolution LC system, launched in 2006, these
packing materials were introduced in ZORBAX RRHT columns capable of
withstanding 600 bar pressure. In 2009 the range was extended to include
RRHD columns capable of routine use at 1200 bar pressure to support the
introduction of the Agilent 1290 Infinity LC System with its extensive
operating power range up to 1200 bar pressure and 5 ml/min flow rate.
These sub-two micron (1.8 µm) particle size columns can be used in pursuit of
two main objectives:
1Faster Chromatography
Short columns with sub-two-micron particles offer the opportunity to
dramatically reduce analysis time by increasing the flow rate without losing
separation performance.
2Higher Resolution
Long columns with sub-two-micron particles provide higher efficiency and
therefore higher resolution, required for the separation of complex samples.
Lower dispersion also means less dilution of the analyte peaks and
consequent sensitivity gains, particularly for LC/MS.
The pressure needed to drive solvent through a column containing STM
particles rises rapidly as flow rate is increased for faster separations and very
rapidly as the length of the column increases for more resolution. Thus the
acceptance of STM columns has been synonymous with the development of
UHPLC systems – that is HPLC systems that offer higher pressures than the
400 bar norm that was extant since the early days of HPLC. Ultra-High
Performance (or Ultra-High Pressure) LC systems also offer low delay volumes
and the fast data collection required for narrow peaks from fast or high
resolution chromatography. The Agilent 1290 Infinity LC is a landmark in
UHPLC as it is the first system that can completely cover and extend upon all
the disparate performance ranges of UHPLCs pre-existing on the market.

Agilent 1290 Infinity LC System Manual and Quick Reference 9
Introduction to Ultra-High Performance Liquid Chromatography 1
Theory of Using Smaller Particles in Liquid Chromatography
The Theory
Separation efficiency in HPLC can be described by the van Deemter equation
(Figure 1 on page 9). This results from the plate-height model used to measure
the dispersion of analytes as they move down the column. H is the Height
Equivalent to a Theoretical Plate (sometimes HETP), dpis the particle size of
the column packing material, u0is the linear velocity of the mobile phase and
A, B and C are constants related to the different dispersive forces. The A term
relates to eddy diffusion or multiple flow paths through the column; B relates
to molecular diffusion along the column axis (longitudinal); C relates to mass
transfer of the analyte between the mobile and stationary phases. The
separation is at its most efficient when H is at a minimum. The effect of each
individual term and the combined equation are shown in Figure 1 on page 9
where the plate height is plotted against the linear flow rate through the
column. This type of plot is known as a Van Deemter Curve and is used to
determine the optimum flow rate (minimum point of the curve) for best
efficiency of separation for a column.
Figure 1 A hypothetical Van Deemter curve
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10 Agilent 1290 Infinity LC System Manual and Quick Reference
1Introduction to Ultra-High Performance Liquid Chromatography
Theory of Using Smaller Particles in Liquid Chromatography
The van Deemter plots in Figure 2 on page 10 show that reducing particle size
increases efficiency. Switching from commonly used 3.5 µm and 5.0 µm
particle sizes to 1.8 µm particles offers significant performance improvements.
The 1.8 µm particles give two to three times lower plate height values and
proportionately higher efficiencies. This allows a shorter column to be used
without sacrificing resolution and hence the analysis time is also reduced by a
factor of two to three. The increased efficiency is derived to a large extent
from the reduction in multiple flow paths as a result of the smaller particles -
this leads to a smaller A term (eddy diffusion). In addition, smaller particles
mean shorter mass transfer times, reducing the C term, and it can be seen that
the overall effect is a much reduced loss of efficiency as the flow rate increases
(the slope of the line is reduced). This means that the separation on smaller
particles can be further accelerated by increasing the flow rates without
significantly reducing efficiency.
Figure 2 Van Deemter curve for different particle sizes
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