Agilent Technologies 1290 Infinity LC System Bedienungsanleitung

Agilent Technologies

Agilent 1290 Infinity

LC System

System Manual and

Quick Reference

Agilent 1290 Infinity LC System Manual and Quick Reference

Notices

2012

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Manual Part Number

G4220-90301

Edition

05/2012

Printed in Germany

Agilent Technologies

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76337 Waldbronn

This product may be used as a com-

ponent of an in vitro diagnostic sys-

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plies with the relevant regulations.

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Agilent 1290 Infinity LC System Manual and Quick Reference 3

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

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

Agilent 1290 Infinity LC System Manual and Quick Reference

Agilent Technologies

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

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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