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
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05/2012
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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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Agilent 1290 Infinity LC System Manual and Quick Reference 11
Introduction to Ultra-High Performance Liquid Chromatography 1
Theory of Using Smaller Particles in Liquid Chromatography
A chromatographic separation can be optimized based on physical parameters
of the HPLC column such as particle size, pore size, morphology of the
particles, the length and diameter of the column, the solvent velocity, and the
temperature. In addition, the thermodynamics of a separation can be
considered and the properties of the solute and the stationary and mobile
phases (percentage of organic solvent, ion strength, and pH) can be
manipulated to achieve the shortest possible retention and highest selectivity.
Figure 3 Selecting optimal conditions for HPLC
Resolution can be described as a function of three parameters:
•column efficiency or theoretical plates (N),
•selectivity (),
•retention factor (k).
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12 Agilent 1290 Infinity LC System Manual and Quick Reference
1Introduction to Ultra-High Performance Liquid Chromatography
Theory of Using Smaller Particles in Liquid Chromatography
According to the resolution equation ( Figure 4 on page 12 ), the selectivity has
the biggest impact on resolution (Figure 5 on page 12). This means that the
selection of appropriate mobile and stationary phase properties and
temperature is critical in achieving a successful separation.
Figure 4 Resolution equation
Figure 5 Effect of plate number, separation factor and retention factor on R
No matter whether the rapid resolution separation method is being newly
developed or simply transferred from an existing conventional method, it is
clearly beneficial to have a wide choice of stationary phase chemistries
available in a range of column formats.

Agilent 1290 Infinity LC System Manual and Quick Reference 13
Introduction to Ultra-High Performance Liquid Chromatography 1
Theory of Using Smaller Particles in Liquid Chromatography
Agilent already offered more than 140 ZORBAX 1.8 µm Rapid Resolution High
Throughput (RRHT) columns (14 selectivity choices; 15 to 150 mm long; 2.1,
3.0 and 4.6 mm internal diameters) and with the launch of the Agilent 1290
Infinity LC the STM range is extended to include the Rapid Resolution High
Definition (RRHD) 1200 bar columns. This enables the optimum stationary
phase to be selected so that the selectivity is maximized. The resolution, flow
rate and analysis time can be optimized by selecting the appropriate column
length and diameter and operation with longer STM columns has become more
accessible than ever before.
Many laboratories perform an extensive screening process to select the best
combination of stationary phase, mobile phase and temperature for their
separations. Agilent offers Method Development Solutions on both the 1200
Series and 1290 Infinity LC systems. These offer complete automation of this
time consuming selection process – making method development and method
transfer an easier and more reliable task.
ZORBAX 1.8 µm RRHD and RRHT columns use the same chemistry as
ZORBAX columns with 3.5 and 5 µm particles. As a result, for any particular
ZORBAX phase, the 5.0, 3.5 and 1.8 µm particles provide identical selectivity,
which allows easy, fast and secure bidirectional method transfer between
conventional LC, UHPLC and preparative LC.

14 Agilent 1290 Infinity LC System Manual and Quick Reference
1Introduction to Ultra-High Performance Liquid Chromatography
Benefits of Columns Packed With Sub-2-micron Particles
Benefits of Columns Packed With Sub-2-micron Particles
Faster Chromatography
There are several advantages of having shorter run times. High Throughput
labs now have higher capacity and can analyze more samples in less time.
More samples in less time also means lower costs. For example, by reducing
the analysis time from 20 min per sample to 5 min, the cost for 700 samples is
reduced by 79 % (Table 1 on page 14).
The Agilent cost savings calculator provides an easy way to calculate the cost
savings by switching from conventional HPLC to UHPLC using 1.8 µm particle
size columns. This calculator is available on the Agilent Technologies web site
along with a method translator calculator – www.chem.agilent.com. The
results are presented graphically and in tabular form.
Shorter run times also deliver faster answers. This is important in process
control and rapid release testing. Instead of waiting hours to release a single
batch of a drug, all the system suitability, calibration and sample analysis can
now be done in less than an hour. Rapid answers are also important for
synthetic chemists using open access LC/MS systems for compound
confirmation and reaction control. Shorter run times can also accelerate the
method development process significantly.
Tab le 1 Time and cost savings over 700 runs
Cycle time 20 min cycle time 5 min cycle time
Runs 700 700
Approx. costs/analysis1
1solvents = $ 27/l, disposal = $ 2/l, labor = $ 30/h
$ 10.58 $ 2.24
Approx. cost/700runs1$ 7400 $ 1570
Cost savings - $ 5830
Time2
224 hours/day
10 days 2.5 days

Agilent 1290 Infinity LC System Manual and Quick Reference 15
Introduction to Ultra-High Performance Liquid Chromatography 1
Benefits of Columns Packed With Sub-2-micron Particles
Figure 6 Cost savings calculator

16 Agilent 1290 Infinity LC System Manual and Quick Reference
1Introduction to Ultra-High Performance Liquid Chromatography
Benefits of Columns Packed With Sub-2-micron Particles
Figure 7 Relation between particle size, efficiency and analysis time
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Agilent 1290 Infinity LC System Manual and Quick Reference 17
Introduction to Ultra-High Performance Liquid Chromatography 1
Benefits of Columns Packed With Sub-2-micron Particles
Higher Resolution
Long columns packed with smaller particles result in higher efficiency and
higher resolution. This is important for analysis of complex samples from
metabolomics or proteomics studies. Also, applications such as impurity
profiling can benefit from higher separation power. Even the LC/MS analysis
of drugs in biological fluids can benefit from the higher peak capacity, because
of the reduced interference from ion suppression. In general, higher
separation power provides more confidence in the analytical results.
Figure 8 Peak capacities of more than 700 can be achieved using a ZORBAX RRHT
SB-C18 column (2.1 x 150 mm, 1.8 µm) to analyze a tryptic digest of BSA
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18 Agilent 1290 Infinity LC System Manual and Quick Reference
1Introduction to Ultra-High Performance Liquid Chromatography
Frictional Heating
Frictional Heating
Forcing mobile phase through the column at higher pressure and higher flow
rates generates heat. The resulting temperature gradients (radial and
longitudinal) can have an impact on the column efficiency.
where F is the flow rate and p is the pressure.
Powerful column thermostatting (for example, using a water bath) generates a
strong radial temperature gradient, which leads to significant loss in column
efficiency. Still-air column thermostatting reduces the radial temperature
gradient and therefore reduces the efficiency losses, but a higher column
outlet temperature has to be accepted. The raised temperature may have an
effect on selectivity. At lower back-pressure, performance losses due to
frictional heat are minimized so that 4.6 or 3 mm inner diameter sub-2-micron
columns still deliver superior efficiencies compared with the respective
2.1 mm inner diameter columns.
An example of a gradient method transfer to a 2.1 mm i.d. STM column in
which the separation has been speeded up is shown in Figure 9 on page 19.
The initial run on the 2.1 mm column was at a flow rate of 0.22 ml/min
generating 380 bar pressure at a temperature setting of 37 °C with all peaks
separated in 12.5 min (chromatogram not shown). The flow was increased to
0.66 ml/min and the gradient times adjusted downwards by a factor of three
generating 1020 bar pressure with all peaks eluted in 4.2 min (Figure 9 on
page 19 above). This should give the same separation but a loss of resolution
can be seen between peaks 7 and 8 and between 5 and the main peak and this
is due to heating in the column changing the selectivity for these compounds.
It was found that running the column thermostat at 5 °C lower was sufficient
to offset the heating effect inside the column and restore the separation
(Figure 9 on page 19 below). The pressure increased to 1070 bar which also
indicates that the in-column temperature was lower.

Agilent 1290 Infinity LC System Manual and Quick Reference 19
Introduction to Ultra-High Performance Liquid Chromatography 1
Frictional Heating
Figure 9 Influence of frictional heat generation on selectivity and effect of lowering
column temperature
In summary, the use of sub-two-micron packing material offers benefits of
increased efficiency, higher resolution and faster separations. The Agilent
1290 Infinity LC System and RRHD columns increase the separation space
available and enable more of these benefits to be accessed. The features of the
1290 Infinity LC are discussed in “The Agilent 1290 Infinity LC System -
Product Description” on page 21 and “Optimization of the Agilent 1290
Infinity LC System” on page 39considers how to apply the theory and use
these features to develop optimized separations.

20 Agilent 1290 Infinity LC System Manual and Quick Reference
1Introduction to Ultra-High Performance Liquid Chromatography
Frictional Heating