Agilent 1290 Infinity LC System System Manual and Quick Reference Agilent Technologies
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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.
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 New Features of the Agilent 1290 Infinity LC System System Components 25 22 3 Optimization of the Agilent 1290 Infinity LC System 39 21 Delay Volume and Extra-Column Volume 40 How to Configure the Optimum Delay Volume 4
Contents 6 Appendix 109 Safety Information 110 Solvent Information 113 Agilent Technologies on Internet 114 Setting Up a Method using Edit Entire Method Agilent 1290 Infinity LC System Manual and Quick Reference 115 5
Contents 6 Agilent 1290 Infinity LC System Manual and Quick Reference
Agilent 1290 Infinity LC System Manual and Quick Reference 1 Introduction to Ultra-High Performance Liquid Chromatography Theory of Using Smaller Particles in Liquid Chromatography Benefits of Columns Packed With Sub-2-micron Particles Frictional Heating 8 14 18 This chapter gives an introduction to the Agilent 1290 Infinity LC System and the underlying concepts.
1 Introduction 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.
Introduction to Ultra-High Performance Liquid Chromatography Theory of Using Smaller Particles in Liquid Chromatography 1 The Theory I]ZdgZi^XVa EaViZ =Z^\]i = 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.
1 Introduction 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.
Introduction to Ultra-High Performance Liquid Chromatography Theory of Using Smaller Particles in Liquid Chromatography 1 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.
1 Introduction 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.
Introduction to Ultra-High Performance Liquid Chromatography Theory of Using Smaller Particles in Liquid Chromatography 1 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.
1 Introduction 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.
Introduction to Ultra-High Performance Liquid Chromatography Benefits of Columns Packed With Sub-2-micron Particles Figure 6 1 Cost savings calculator Agilent 1290 Infinity LC System Manual and Quick Reference 15
1 Introduction to Ultra-High Performance Liquid Chromatography Benefits of Columns Packed With Sub-2-micron Particles 8dajbc AZc\i] bb 8dajbc :[[^X^ZcXn C * ¥b 8dajbc :[[^X^ZcXn C (#* ¥b 8dajbc :[[^X^ZcXn C &#- ¥b &*% &'!*%% '&!%%% (*!%%% &%% -!*%% &)!%%% '(!'*% ,* +%%% &%!*%% &,!*%% ,!%%% &'!%%% 6cVanh^h I^bZ GZYjXi^dc :[[^X^ZcXn 6cVanh^h C I^bZ "(( "*% EgZhhjgZ *% )!'%% EZV` "+, KdajbZ (% C#6# )!'%% +!*%% HdakZci "-% JhV\Z &* C#6# Figure 7 16 '!&%% '!*%
Introduction to Ultra-High Performance Liquid Chromatography Benefits of Columns Packed With Sub-2-micron Particles 1 Higher Resolution 6WhdgWVcXZ Pb6JR 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.
1 Introduction 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.
Introduction to Ultra-High Performance Liquid Chromatography Frictional Heating Figure 9 1 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.
1 20 Introduction to Ultra-High Performance Liquid Chromatography Frictional Heating Agilent 1290 Infinity LC System Manual and Quick Reference
Agilent 1290 Infinity LC System Manual and Quick Reference 2 The Agilent 1290 Infinity LC System Product Description New Features of the Agilent 1290 Infinity LC System System Components 22 25 This chapter discusses the features of the 1290 Infinity LC System.
2 The Agilent 1290 Infinity LC System - Product Description New Features of the Agilent 1290 Infinity LC System New Features of the Agilent 1290 Infinity LC System The Agilent 1290 Infinity LC System is designed to offer the greatest flexibility for performing analytical liquid chromatography using all types of current and emergent column technologies.
The Agilent 1290 Infinity LC System - Product Description New Features of the Agilent 1290 Infinity LC System 2 • Data collection rates up to 160 Hz with full spectral information. • New range of ZORBAX RRHD sub-two micron particle size columns for operation at pressures up to 1200 bar. • Blend Assist for automatic buffering and additive blending in the 1290 Infinity Quaternary Pump. The most significant advance is the range of pressures and flow rates that the system can use.
2 The Agilent 1290 Infinity LC System - Product Description New Features of the Agilent 1290 Infinity LC System The pressure range offers the ability to work with the latest sub-two micron particles in long columns for high resolution and short columns for fast separation at increased flow rates. The flow rate range allows not only traditional methods to be used but also superficially porous (or pellicular) packing materials (for example, Poroshell) at high flow rates.
2 The Agilent 1290 Infinity LC System - Product Description System Components System Components The Agilent 1290 Infinity Binary Pump The Agilent 1290 Infinity Binary Pump contains new technology to overcome the problems of pumping LC solvents at ultra-high pressure and high flow rates: heavy duty drive motors on the pistons; new material for the pistons themselves not only to withstand the workload but also to actively transfer heat from the seals; microfluidic heat exchangers and the Jet Weaver, a micr
2 The Agilent 1290 Infinity LC System - Product Description System Components Id VjidhVbeaZg ;gdb hdakZci gZhZgkd^gh HdakZci hl^iX]^c\ kVakZ B^mZg 9Z\VhhZg Eg^bVgn ejbe ]ZVY EgZhhjgZ hZchdg Eg^bVgn ejbe ]ZVY 8]VccZa 6 Figure 12 HZXdcYVgn 8]VccZa 7 ejbe ]ZVY HZXdcYVgn ejbe ]ZVY Id lVhiZ Parts identification and schematic of the 1290 Infinity Binary Pump Each pump head is a dual piston in-series design utilizing novel firmware control and novel piston material, silicon carbide, which efficiently re
2 The Agilent 1290 Infinity LC System - Product Description System Components chromatographic performance these features makes the pump very quiet in operation. When concentrated buffer solutions are used as mobile phase, the active seal wash option is available for use to extend the life of the pump seals. A solvent selection valve allows binary mixtures (isocratic or gradient) to be formed from one of two solvents per channel.
2 The Agilent 1290 Infinity LC System - Product Description System Components The flow path of the pump has been optimized for minimal delay of gradients and incorporates an innovative mixing system using microfluidics technology. The mixing device, known as the Jet Weaver, employs a network of multi-layer microfluidic channels (120 µm x 120 µm) to unsure the flow is thoroughly mixed.
The Agilent 1290 Infinity LC System - Product Description System Components 2 The 1290 Infinity Quaternary Pump In contrast, the 1290 Infinity Quaternary Pump is equipped with only one pump head and an additional multi-channel gradient valve (MCGV) to portion the eluents according to the programmed gradient. According to this low pressure mixing principle, the solvents meet in the Inlet Weaver and are therefore already mixed before and in the pump head.
2 The Agilent 1290 Infinity LC System - Product Description System Components Table 2 Valve functionalities Pump Pump Agilent Jet Weaver Agilent Jet Weaver Sampler Sampler Waste Waste Filter Figure 14 Filter Standard application Figure 15 Extra-mixing volume setup Pump Pump Agilent Jet Weaver Agilent Jet Weaver Sampler Sampler Waste Waste Filter Filter Figure 16 30 Automatic purge function Figure 17 Backflushing the in-line filter Agilent 1290 Infinity LC System Manual and Quick
The Agilent 1290 Infinity LC System - Product Description System Components 2 The standard application (1) is used for the majority of analysis, while the extra-mixing volume setup (2) is applied for any kind of baseline-criticial application where the mixing performance and the dependant UV-baseline can be significantly enhanced by the use of the Agilent Jet Weaver mixer. An automatic purge function (3) is installed, as well as the possibility of backflushing the in-line filter (4).
2 The Agilent 1290 Infinity LC System - Product Description System Components The Agilent 1290 Infinity Autosampler The Agilent 1290 Infinity Autosampler offers the well-established Agilent flow-through design with variable volume injection and takes it to new levels of performance. New inert materials in the metering device seal and needle seat help to achieve extremely low carryover.
The Agilent 1290 Infinity LC System - Product Description System Components 2 A totally new optional add-on module, the Flexible Cube, works seamlessly with the autosampler to provide additional capabilities. With the addition of the new Flexible Cube module, which comprises a 500 µl syringe pump, one low pressure valve and two high pressure switching valves further options become possible.
2 The Agilent 1290 Infinity LC System - Product Description System Components The Agilent 1290 Infinity Thermostatted Column Compartment The Agilent 1290 Infinity Thermostatted Column Compartment (TCC) controls the temperature between 10 °C below ambient and up to 100 °C at 2.5 ml/min and 80 °C at up to 5 ml/min, respectively. The temperature stability specification is ± 0.05 °C and the accuracy specification ±0.5 °C (with calibration)1.
2 The Agilent 1290 Infinity LC System - Product Description System Components Figure 19 Quick change valve in TCC Up to three TCC can be “clustered” to allow advanced applications such as switching between eight columns for automated method development or to make additional columns available for different applications. Thus, the column to be used becomes a simple method parameter. This requires two 8 position/9 port valve heads, one each in two of the TCCs.
2 The Agilent 1290 Infinity LC System - Product Description System Components The 1290 Infinity Diode-Array Detector The 1290 Infinity Diode-Array Detector is a new optical design using a cartridge cell with optofluidic waveguide technology offering high sensitivity with low dispersion, a wide linear range and a very stable baseline for standard or ultra-fast LC applications.
The Agilent 1290 Infinity LC System - Product Description System Components 2 The module also incorporates electronic temperature control to further enhance the resistance to ambient temperature effects. Although the hydraulic volume of the Max-Light cartridge cell is very small, the path length is a standard 10 mm. However, for even higher sensitivity the alternative Agilent Max-Light high sensitivity cell is available with a path length of 60 mm.
2 38 The Agilent 1290 Infinity LC System - Product Description System Components Agilent 1290 Infinity LC System Manual and Quick Reference
Agilent 1290 Infinity LC System Manual and Quick Reference 3 Optimization of the Agilent 1290 Infinity LC System Delay Volume and Extra-Column Volume Delay Volume 40 Extra-Column Volume 41 40 How to Configure the Optimum Delay Volume How to Achieve Higher Injection Volumes How to Achieve High Throughput 42 51 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 This chapter considers how to apply the
3 Optimization of the Agilent 1290 Infinity LC System Delay Volume and Extra-Column Volume Delay Volume and Extra-Column Volume The delay volume is defined as the system volume between the point of mixing in the pump and the top of the column. The extra-column volume is defined as the volume between the injection point and the detection point, excluding the volume in the column.
Optimization of the Agilent 1290 Infinity LC System Delay Volume and Extra-Column Volume 3 times of about one minute. However, the delay volume must be reduced further to achieve delay times which are a fraction of the intended run time. This is achieved with the Agilent 1290 Infinity LC System due to the low delay volume of the pump flow path, low volume of the Jet Weaver mixer and low-volume of the flow path through the autosampler.
3 Optimization of the Agilent 1290 Infinity LC System How to Configure the Optimum Delay Volume How to Configure the Optimum Delay Volume Table 3 on page 42 and Table 4 on page 43 show the component volumes which contribute to system delay volume in the Agilent 1290 Infinity LC System. In the standard configuration with the Agilent 1290 Infinity Binary Pump, with the Jet Weaver mixer, the 1290 Infinity Autosampler and Thermostatted Column Compartment the system delay volume is about 125 µl.
Optimization of the Agilent 1290 Infinity LC System How to Configure the Optimum Delay Volume Table 4 Delay volumes of 1290 Infinity Binary LC system configurations System Configurations1 Delay Volume (µl) Binary Pump + Fixed Loop Autosampler (MS only) 20 Binary Pump + Jet Weaver + Fixed Loop 55 Binary Pump + standard Autosampler (MS only) 90 Binary Pump + Jet Weaver + Autosampler 125 1 added 5 µl to allow for connections in system configurations Table 5 Delay volumes of 1290 Infinity Quatern
3 Optimization of the Agilent 1290 Infinity LC System How to Configure the Optimum Delay Volume Table 6 System delay times for gradient to reach head of column Flow Rate (ml/min) System Delay Volume (microliters) 1.5 0.01 0.04 0.06 0.08 0.24 0.29 0.34 0.39 2.0 0.01 0.03 0.04 0.06 0.18 0.22 0.26 0.29 3.0 0.01 0.02 0.03 0.04 0.12 0.14 0.17 0.19 4.0 0.01 0.01 0.02 0.03 0.09 0.11 0.13 0.15 5.0 0.00 0.01 0.02 0.02 0.07 0.09 0.10 0.
Optimization of the Agilent 1290 Infinity LC System How to Configure the Optimum Delay Volume 3 injection is made when the valve switches back to mainpass and the sample is flushed onto the column. The valve remains in this position during analysis so that the autosampler is continually flushed and hence the gradient has to flow through this delay volume to reach the column.
3 Optimization of the Agilent 1290 Infinity LC System How to Configure the Optimum Delay Volume volume is optimized for the use with 3 mm and 4.6 mm inner diameter columns. For pump operation it is recommended to set the correct solvent in the pump setup screen. Even though the intelligent control will automatically tune the pressure ripple to a minimum the solvent compressibility can have an effect on maintaining absolutely correct flow rate at high pressure.
Optimization of the Agilent 1290 Infinity LC System How to Configure the Optimum Delay Volume 3 applications there is no need for an additional Jet Weaver mixer. But it is still possible to install an optional 380 µL Jet Weaver mixer for baseline critical applications, such as TFA applications. The optional Jet Weaver mixer has a different housing, which is adapted to the design of the 1290 Infinity Quaternary Pump.
3 Optimization of the Agilent 1290 Infinity LC System How to Configure the Optimum Delay Volume Replacing the Jet Weaver in the 1290 Infinity Binary Pump 1 Remove capillary connections from the Jet Weaver. 2 Remove the hex screws that fix the Jet Weaver to the pump housing. 3 Install the new Jet Weaver. 4 Reinstall the capillary connections.
Optimization of the Agilent 1290 Infinity LC System How to Configure the Optimum Delay Volume 3 Installing the V380 Jet Weaver in the 1290 Infinity Quaternary Pump 1 Open the screw of the Jet Weaver metal lid. 2 Remove the metal lid by lifting it up (1) and pulling it out of the front panel (2). 2 1 3 Insert the Jet Weaver to the opening in the front panel (1) 4 Mount both capillary connections to the Jet Weaver and push it down (2). observing the correct orientation.
3 Optimization of the Agilent 1290 Infinity LC System How to Configure the Optimum Delay Volume 5 Connect the inlet capillary of the Jet Weaver to port 2 of the Multi Purpose Valve. Connect the outlet capillary to port 1.
Optimization of the Agilent 1290 Infinity LC System How to Achieve Higher Injection Volumes 3 How to Achieve Higher Injection Volumes The standard configuration of the Agilent 1290 Infinity Autosampler includes a variable volume sample loop for up to 20 µl injections. The metering device can inject a maximum volume of 40 µl and the sample loop cartridge can be exchanged to allow this (refer to the 1290 Infinity Autosampler manual for details).
3 Optimization of the Agilent 1290 Infinity LC System How to Achieve Higher Injection Volumes One way to achieve larger injections is to use a trapping column selected by a switching valve to capture and concentrate the injection before switching it, i.e. injecting it, onto an analytical column, see Figure 21 on page 52. The valve can be conveniently located in the Thermostatted Column Compartment.
Optimization of the Agilent 1290 Infinity LC System How to Achieve High Throughput 3 How to Achieve High Throughput Some laboratories operate in a high throughput (HT) environment where the workload requires sequences of hundreds or even thousands of injections to be made to complete a body of work. In these situations it is highly desirable to minimize the cycle times as even a few seconds saved per injection will reduce the overall time to complete the work by a significant and useful amount.
3 Optimization of the Agilent 1290 Infinity LC System How to Achieve High Throughput The injection can be optimized for speed remembering that drawing the sample too fast can reduce the reproducibility. Marginal gains are to be made here as the sample volumes used tend towards the smaller end of the range in any case. A significant portion of the injection time is the time taken with the needle movements to and from the vial and into the flush port.
Optimization of the Agilent 1290 Infinity LC System How to Achieve High Throughput Figure 22 3 Alternating Column Regeneration Agilent 1290 Infinity LC System Manual and Quick Reference 55
3 Optimization of the Agilent 1290 Infinity LC System How to Achieve Higher Resolution How to Achieve Higher Resolution Increased resolution in a separation will improve the qualitative and quantitative data analysis, allow more peaks to be separated or offer further scope for speeding up the separation.
Optimization of the Agilent 1290 Infinity LC System How to Achieve Higher Resolution 3 the decision on phases it is likely that short columns were used for fast analysis in each step of the scouting. The resolution equation shows that the next most significant term is the plate count or efficiency, N, and this can be optimized in a number of ways.
3 Optimization of the Agilent 1290 Infinity LC System How to Achieve Higher Resolution The van Deemter curve shows that the optimum flow rate through an STM column is higher than for larger particles and is fairly flat as the flow rate increases. Typical, close to optimum, flow rates for STM columns are: 2 ml/min for 4.6 mm i.d.; and 0.4 ml/min for 2.1 mm i.d. columns. In isocratic separations, increasing the retention factor, k, results in better resolution because the solute is retained longer.
3 Optimization of the Agilent 1290 Infinity LC System How to Achieve Higher Sensitivity How to Achieve Higher Sensitivity The sensitivity of a separation method is linked to the choice of stationary and mobile phases as good separation with narrow peaks and a stable baseline with minimal noise are desirable. The choice of instrument configuration will have an effect and a major impact is the setup of the detector.
3 Optimization of the Agilent 1290 Infinity LC System How to Achieve Higher Sensitivity How to Achieve Higher Sensitivity for Detector The detector has a number of parameters that are used to optimize performance.
Optimization of the Agilent 1290 Infinity LC System How to Achieve Higher Sensitivity 3 For example, a signal at wavelength 250 nm with a bandwidth of 16 nm will be an average of the absorbance data from 242 nm to 258 nm. Additionally, a reference wavelength and reference bandwidth can be defined for each signal. The average absorbance from the reference bandwidth centered on the reference wavelength will be subtracted from its equivalent value at the signal wavelength to produce the output chromatogram.
3 Optimization of the Agilent 1290 Infinity LC System How to Achieve Higher Sensitivity but for reasons of convention maxima and minima are chosen in preference to other parts of the spectrum. The reference bandwidth is normally set on a region of the UV spectrum in which the analyte has no absorbance. This is shown in the spectrum for anisic acid in Figure 23 on page 62. This spectrum is typical of many small molecules containing a UV chromophore.
Optimization of the Agilent 1290 Infinity LC System How to Achieve Higher Sensitivity 3 Slit Width Light transmission into the spectrograph and the optical bandwidth are controlled by the variable aperture entrance slit. The default setting for the slit width is 4 nm which is appropriate for most applications as it gives good all-round performance. The performance characteristics affected are sensitivity, spectral resolution and linearity.
3 Optimization of the Agilent 1290 Infinity LC System How to Achieve Higher Sensitivity The injection volume and the sample dissolution solvent are important in controlling dispersion. Care must be taken that the compounds are focused at the top of the column, to avoid peak dispersion due to the injection, which would cause a reduced peak height. To achieve this, the sample should be dissolved in a solvent composition of lower elution strength than the mobile phase.
Optimization of the Agilent 1290 Infinity LC System How to Achieve Higher Sensitivity 3 The peak width setting in the detector allows the user to correctly set these parameters without needing any knowledge other than sight of the chromatogram integration results to see how wide the peaks are. The peak width setting should be set for the narrowest peak width observed in the chromatogram.
3 Optimization of the Agilent 1290 Infinity LC System How to Achieve Lowest Carry Over How to Achieve Lowest Carry Over Carryover is measured when residual peaks from a previous active-containing injection appear in a subsequent blank solvent injection. There will be carry over between active injections which may lead to erroneous results. The level of carryover is reported as the area of the peak in the blank solution expressed as a percentage of the area in the previous active injection.
3 Optimization of the Agilent 1290 Infinity LC System How to Achieve Lowest Carry Over The flush port is located above and behind the needle seat and a peristaltic pump delivers the wash solvent. It has a volume of 0.68 ml and the peristaltic pump delivers 6 ml/min, which means the flush port volume is completely refilled with fresh solvent in 7 s. If the flush port is selected, the user can set how long the outside of the needle is to be washed with fresh solvent.
3 Optimization of the Agilent 1290 Infinity LC System How to Prevent Column Blockages How to Prevent Column Blockages As with any HPLC system, care must be taken to avoid partially or completely blocking the column or the system tubing through inadvertent use.
Optimization of the Agilent 1290 Infinity LC System How to Prevent Column Blockages 3 9 Purge the pumps (the connections up to the column) of any buffer containing mobile phases and flush through 5 ml of solvent before attaching the column to the instrument. 10 Flush the column with compatible mobile phase starting slowly at 0.1 ml/min for a 2.1 mm inner diameter column, 0.2 ml/min for a 3.0 mm inner diameter column, and 0.4 ml/min for 4.6 mm inner diameter.
3 70 Optimization of the Agilent 1290 Infinity LC System How to Prevent Column Blockages Agilent 1290 Infinity LC System Manual and Quick Reference
Agilent 1290 Infinity LC System Manual and Quick Reference 4 System Setup and Installation Installing Software 72 Installing the Module 73 Optimizing the Stack Configuration (Binary LC System) 73 Optimizing the Stack Configuration (Quaternary LC System) 78 Priming the Pump 83 Purging the Pump 85 Flow Connections Between Modules 88 Integration Into the Network 88 This chapter includes information on software installation, stack configurations and preparing the system for operation.
4 System Setup and Installation Installing Software Installing Software Installing the Software Controller and Data System For details of installation procedures for the software, refer to the 1290 Infinity Diode Array Detector Manual and the software manuals. Installing the Agilent Lab Advisor Software For details of installation procedures for the Agilent Lab Advisor software, refer to the software documentation on the Lab Advisor DVD.
4 System Setup and Installation Installing the Module Installing the Module For details of installation procedures for the modules, refer to the individual module manuals. These manuals also contain information on specifications, maintenance and parts.
4 System Setup and Installation Installing the Module >chiVci E^adi HdakZci XVW^cZi 9ZiZXidg 8dajbc XdbeVgibZci 6jidhVbeaZg Ejbe Figure 25 74 Recommended stack configuration for 1290 Infinity with binary pump (front view) Agilent 1290 Infinity LC System Manual and Quick Reference
System Setup and Installation Installing the Module 4 A6C id A8 8]ZbHiVi^dc 86C 7jh XVWaZ id >chiVci E^adi 6cVad\ YZiZXidg h^\cVa dei^dcVa 68 EdlZg 86C 7jh XVWaZ Figure 26 Recommended stack configuration 1290 Infinity with binary pump (rear view) Agilent 1290 Infinity LC System Manual and Quick Reference 75
4 System Setup and Installation Installing the Module Two Stack Configuration In case the autosampler thermostat is added to the system, a two-stack configuration is recommended, which places both heavy modules (1290 Infinity pump and thermostat) at the bottom of each stack and avoids high stacks. Some users prefer the lower height of this arrangement even without the autosampler thermostat. A slightly longer capillary is required between the pump and autosampler.
System Setup and Installation Installing the Module 4 A6C id A8 8]ZbHiVi^dc 86C 7jh XVWaZ id >chiVci E^adi 6cVad\ YZiZXidg h^\cVa dei^dcVa 86C 7jh XVWaZ I]Zgbd XVWaZ dei^dcVa 68 EdlZg Figure 28 Recommended two stack configuration for 1290 Infinity with binary pump (rear view) Agilent 1290 Infinity LC System Manual and Quick Reference 77
4 System Setup and Installation Installing the Module Optimizing the Stack Configuration (Quaternary LC System) One Stack Configuration Ensure optimum performance by installing the modules of the Agilent 1290 Infinity Quaternary LC System in the following configuration (see Figure 29 on page 79 and Figure 30 on page 80). This configuration optimizes the flow path for minimum delay volume and minimizes the bench space required.
System Setup and Installation Installing the Module 4 Instant Pilot Solvent cabinet Detector Column compartment Autosampler Pump Figure 29 Recommended stack configuration for 1290 Infinity with quaternary pump (front view) Agilent 1290 Infinity LC System Manual and Quick Reference 79
4 System Setup and Installation Installing the Module LAN to control software CAN Bus cable to Instant Pilot Analog detector signal (optional) AC Power CAN Bus cable Figure 30 80 Recommended stack configuration for 1290 Infinity with quaternary pump (rear view) Agilent 1290 Infinity LC System Manual and Quick Reference
4 System Setup and Installation Installing the Module Two Stack Configuration In case the autosampler thermostat is added to the system, a two-stack configuration is recommended, which places both heavy modules (1290 Infinity pump and thermostat) at the bottom of each stack and avoids high stacks. Some users prefer the lower height of this arrangement even without the autosampler thermostat. A slightly longer capillary is required between the pump and autosampler.
4 System Setup and Installation Installing the Module LAN to control software CAN Bus cable to Instant Pilot Analog detector signal (optional) Thermo cable (optional) CAN Bus cable AC Power Figure 32 82 Recommended two stack configuration for 1290 Infinity with quaternary pump (rear view) Agilent 1290 Infinity LC System Manual and Quick Reference
System Setup and Installation Installing the Module 4 Priming the Pump This procedure is required when... • the pump is used for the first time • whenever one or more of the inlet tubes contains air gaps or is dry for other reasons The purpose of priming is to remove all air bubbles from the pump and its inlet tubes.
4 System Setup and Installation Installing the Module 1 Prepare channel A and pump head A for priming: a Part fill each solvent reservoir with ca. 150 ml of HPLC grade propan-2-ol for priming the pump and place the sintered glass filter ends of the solvent tubing in the reservoirs. b Disconnect the tubing entering the inlet check valve of pump head A. This is the outlet tubing from vacuum degasser channel A. c Attach the priming syringe with threaded adaptor to the tubing.
4 System Setup and Installation Installing the Module Purging the Pump The purging procedure is described for the 1290 Infinity Binary Pump. It can be executed in an equivalent way for the 1290 Infininty Quaternary Pump. • After the pump has been primed for the first time. • When the pump is to be purged with fresh solvent before using the system, or when the solvent is to be exchanged for another.
4 System Setup and Installation Installing the Module 1 To access the setup page for controlling the purge valve, right-click on the pump section, and select Control from the context menu. Alternatively, you can select Instrument > More 1290 Infinity BinPump > Control.
4 System Setup and Installation Installing the Module 2 In the Purge section, set the following parameters: • Duration: 6 min • Flow: 10 ml/min • Composition B: 50 % Composition A will automatically assume 50 %. Leave the On/Off button set to Off. Click OK to exit. 3 Right-click on the pump section, and select Purge On from the context menu. NOTE Do not confuse Purge On with the next item Prime On.
4 System Setup and Installation Installing the Module Flow Connections Between Modules When connecting the modules, always flush each capillary and the column with solvent before connecting to the next component in the flow path. 1 Connect the outlet from the Jet Weaver mixer to the autosampler using a 0.12 mm i.d. stainless steel flexible capillary (color code label is red). This should be connected to port #1 on the autosampler injection valve. 2 Connect a 0.12 mm i.d.
Agilent 1290 Infinity LC System Manual and Quick Reference 5 Quick Start Guide About the Quick Start Guide 90 Preparing the system 91 Turning the System ON 91 Loading the Default Method 92 Configuring the Online Plot 93 Purging the Pump 95 Data Acquisition in Method and Run Control View 96 Method Parameters for Test Mixture and ZORBAX RRHD Column Setting Up the Method 98 Running the Method for a Single Injection 100 Running the method faster 101 96 Data Analysis 103 Data Analysis View 104 Integrating a
5 Quick Start Guide About the Quick Start Guide About the Quick Start Guide This chapter provides information on running the Agilent 1290 Infinity LC system. It can be used as a guide to quickly running a first analysis after installation, serving both as a tutorial example and a check on the overall functioning of the system. It also includes more detailed information about method parameters.
5 Quick Start Guide Preparing the system Preparing the system Turning the System ON If the system is not already fully on with the software showing Ready status, follow these steps: 1 Turn on the computer system and wait for the Windows desktop to appear. 2 Turn on the electrical power to the LC modules using the button at the lower left of each module. A green power on light will be visible in the center of the button. 3 Start the control software on the computer by clicking the icon (if configured).
5 Quick Start Guide Preparing the system Loading the Default Method The ChemStation has a default method named DEF_LC.M which is loaded at first execution or whenever a new blank method template is required. It contains default settings for all modules. With this procedure, you load the method DEF_LC.M. You can use it to set all parameters to default settings, or to get a blank method template before setting up a new method. 1 Go to Method and Run Control view of the ChemStation.
5 Quick Start Guide Preparing the system Configuring the Online Plot 1 If the Online Plot window is not visible: Click View > Online Signals > Signal Window 1 to display the window. 2 To configure the desired signal(s) in the Online Plot window, click Change…. The Edit Signal Plot setup page opens.
5 Quick Start Guide Preparing the system 3 In the Available Signals box, highlight the required signal(s), and click Add to move them to the Selected Signals box. 4 To configure the individual settings for each signal, highlight the signal in the Selected Signal box and set the required values in the lower half of the page. NOTE In addition to the detector signals, parameter traces such as temperature and pressure can also be plotted.
Quick Start Guide Preparing the system 5 Purging the Pump Purge the pump, if ... • The pump has been primed for the first time. • The pump is to be purged with fresh solvent before using the system, or when the solvent is to be exchanged for another. • The pump has been idle for a few hours or more (air may have diffused into the solvent lines and purging is recommended). • The solvent reservoirs are refilled, and the pump requires purging to fill the system with fresh solvent.
5 Quick Start Guide Data Acquisition in Method and Run Control View Data Acquisition in Method and Run Control View All method procedures and setups are described for the 1290 Infinity Binary Pump. They can be executed in an equivalent way for the 1290 Infininty Quaternary Pump. Method Parameters for Test Mixture and ZORBAX RRHD Column The 1290 Infinity LC System is supplied with a ZORBAX RRHD Eclipse Plus C18 1.8 µm, 2.
Quick Start Guide Data Acquisition in Method and Run Control View Table 9 Method parameters for first separation run Module Parameter Setting Pump Solvent A Water Solvent B Acetonitrile Flow rate 0.4 ml/min Initial Composition 60 % A, 40 % B Gradient Timetable At 4 minutes 20 % A, 80 % B Stop Time 5 minutes Injection 1 µl Needle wash Flush port, 6 s Column ZORBAX Eclipse Plus C18 1.8 µm, 2.1 mm x 50 mm i.d.
5 Quick Start Guide Data Acquisition in Method and Run Control View Setting Up the Method This section shows how to quickly set the method conditions for an analysis using the test mixture conditions. For a more detailed explanation of all the available parameters see Appendix, “Setting Up a Method using Edit Entire Method” on page 115. The default method DEF_LC.M has been loaded ready to prepare the new method. Now the key parameters can be edited to create the new method.
Quick Start Guide Data Acquisition in Method and Run Control View 5 b Other parameters can remain at default settings. Click OK to exit the window. The changes are sent to the autosampler module. 4 Right-click the Thermostatted Column Compartment (TCC) area, and select Method... in the context menu. a In the Method page for the 1290 Infinity TCC, enter the following parameters: • Left Temperature 40 °C • Right Temperature Combined b Other parameters can remain at default settings.
5 Quick Start Guide Data Acquisition in Method and Run Control View Running the Method for a Single Injection This section shows how to run a single injection of the test mix using the conditions entered in the previous section.
5 Quick Start Guide Data Acquisition in Method and Run Control View Running the method faster The first exercise ran at a pressure achievable in a standard system. Now the flow rate is increased and the gradient adjusted for a faster separation. 1 Edit the method conditions in the same way as in the previous section to make the following changes: • Flow rate: 1.6 ml/min • Gradient: Change the gradient so that the gradient slope is unchanged in terms of volume compared to the first run.
5 Quick Start Guide Data Acquisition in Method and Run Control View This separation is actually not optimized with these conditions and the user may like to gain further experience with running the system by trying to optimize the method further. Some changes that may help are: • Reduce the concentration of the sample by diluting 1:10. • Increase the range of the gradient. • Increase the temperature. • Examine the spectra of the peaks and select appropriate narrow band detection.
Quick Start Guide Data Analysis 5 Data Analysis A method in the ChemStation contains all the parameters for data acquisition (controlling the system) and data analysis (processing the data to give quantitative and qualitative results). This section looks briefly at integration and reports in data analysis so that the separations generated earlier in this chapter can be integrated and printed.
5 Quick Start Guide Data Analysis Data Analysis View To open a chromatogram in the Data Analysis view: 1 Launch an offline ChemStation. 2 Click Data Analysis in the bottom left of the screen (see Figure 34 on page 103). 3 In the Navigation Panel, find the data directory containing the data files. All the single injection data are represented as a subset called Single Runs. Double click Single Runs to load these data files into the Navigation Table.
5 Quick Start Guide Data Analysis Integrating a Signal 1 Select the Integration Task Tool (see figure below). The Integrate icon and the Set Integration Events Table icon are highlighted in the figure shown below. 2 Click the Set Integration Events Table icon to open the table as shown. 3 Set Baseline Correction to Advanced for gradient runs. 4 Set Slope Sensitivity to 50. Higher numbers will integrate steeper peaks and ignore less steep peaks.
5 Quick Start Guide Data Analysis 8 Exit from the Events Table using the green tick icon (see figure below).
5 Quick Start Guide Data Analysis Specify the Report 1 On the menu bar click Report > Specify Report to display the window shown in the figure below.
5 Quick Start Guide Data Analysis 2 With the example settings shown in the figures above you can produce an Area Percent report on the screen. 3 In the Destination section, select Printer for a paper copy, and select File and PDF to obtain a useful PDF report file stored into the datafile (the data file with .D suffix is actually a directory. The report file can be viewed directly in ChemStation or it can be found in the directory using the normal Windows File Explorer).
Agilent 1290 Infinity LC System Manual and Quick Reference 6 Appendix Safety Information Solvent Information 110 113 Agilent Technologies on Internet 114 Setting Up a Method using Edit Entire Method Method Information 117 Instrument/Acquisition 118 Data Analysis 133 Run Time Checklist 140 115 This chapter provides additional information on safety, legal and web and about setting up a method.
6 Appendix Safety Information Safety Information General Safety Information The following general safety precautions must be observed during all phases of operation, service, and repair of this instrument. Failure to comply with these precautions or with specific warnings elsewhere in this manual violates safety standards of design, manufacture, and intended use of the instrument. Agilent Technologies assumes no liability for the customer’s failure to comply with these requirements.
6 Appendix Safety Information the instrument must be made inoperative and be secured against any intended operation. Make sure that only fuses with the required rated current and of the specified type (normal blow, time delay, and so on) are used for replacement. The use of repaired fuses and the short-circuiting of fuse holders must be avoided. Some adjustments described in the manual, are made with power supplied to the instrument, and protective covers removed.
6 Appendix Safety Information Safety Symbols Table 10 Safety Symbols Symbol Description The apparatus is marked with this symbol when the user should refer to the instruction manual in order to protect risk of harm to the operator and to protect the apparatus against damage. Indicates dangerous voltages. Indicates a protected ground terminal. Indicates eye damage may result from directly viewing the light produced by the deuterium lamp used in this product.
6 Appendix Solvent Information Solvent Information Flow Cell To protect optimal functionality of your flow-cell: • Avoid the use of alkaline solutions (pH > 9.5) which can attack quartz and thus impair the optical properties of the flow cell. Use of Solvents Observe the following recommendations on the use of solvents. • Brown glass ware can avoid growth of algae.
6 Appendix Agilent Technologies on Internet Agilent Technologies on Internet For the latest information on products and services visit our worldwide web site on the Internet at: http://www.agilent.com Select Products/Chemical Analysis It will provide also the latest firmware of the modules for download.
6 Appendix Setting Up a Method using Edit Entire Method Setting Up a Method using Edit Entire Method All method procedures and setups are described for the 1290 Infinity Binary Pump. They can be executed in an equivalent way for the 1290 Infininty Quaternary Pump. A method in the ChemStation contains all the parameters for Data Acquisition (controlling the system) and Data Analysis (processing the data to give quantitative and qualitative results).
6 Appendix Setting Up a Method using Edit Entire Method This dialog summarizes the sections that will be viewed, and offers an opportunity to bypass certain parts by deselecting them. Depending on the selected parts, the function sequentially shows several screens: • Method Information comprises a text description about the method. • Instrument/Acquisition comprises: • injector parameters, • pump parameters, • oven parameters, • detector parameters, and • instrument curves.
Appendix Setting Up a Method using Edit Entire Method 6 Method Information The Method Information screen can also be directly accessed through the menu Method > Method Information or by right-clicking on the graphical user interface. This box allows information about the method to be entered. This information will be displayed above the system diagram on the Method and Run Control screen whenever this method is loaded and resident in memory.
6 Appendix Setting Up a Method using Edit Entire Method Instrument/Acquisition Setup Instrument Method The Setup Method screen can be directly accessed through the menu Instrument > Setup Instrument Method..., or by right-clicking on the graphical user interface on any module icon and then selecting Method... in the context menu. This next stage in Edit Entire Method is the Setup Method screen with six tab dividers for different modules or functions .
Appendix Setting Up a Method using Edit Entire Method 6 Autosampler tab (HiP-ALS) Figure 37 Setup Method screen –High Performance Autosampler tab • Injection Mode • Injection volume sets the volume to be injected (example 3 µl), • Standard injection indicates that no external needle wash is done, • Injection with needle wash is used to reduce potential carryover. This is the recommended option and is configured in the next entry. • Needle Wash – if selected above.
6 Appendix Setting Up a Method using Edit Entire Method • Location determines which vial or well plate will be used, if Wash Vial was selected. NOTE Vials should not have a septum i.e., they should be open to avoid transfer of carryover material on the septum. • Repeat determines, if Wash Vial was selected, how many times the needle dips into the vial (default 3, maximum 5). • Stop Time / Post Time are set to No Limit / Off and these values are taken care of in the pump tab.
6 Appendix Setting Up a Method using Edit Entire Method This reduces the delay volume of the system by about 70 µl and allows the gradient changes to reach the column sooner. • Enable overlapped injection also switches the injection valve from mainpass to bypass after the injection has taken place either after the sample has flushed out of the injector or at some specified later time in the run.
6 Appendix Setting Up a Method using Edit Entire Method High Performance Autosampler (Hip_ALS Injector Program) tab Figure 38 Setup Method screen - HiP Autosampler Injector Program tab This allows specialized injection procedures to be constructed which involve the manipulation of aliquots from multiple vials as, for instance, in pre-column derivatization. Reagent chemicals are automatically mixed with the sample to enhance detectability or sensitivity.
Appendix Setting Up a Method using Edit Entire Method 6 Binary Pump (BinPump) tab Figure 39 Setup Method screen – Binary Pump tab • Flow sets the flow rate up to 5 ml/min. For the example separation 0.4 ml/min is used. If the back pressure briefly reaches the maximum pressure setting the flow will be reduced for a few seconds to lower the pressure but if the pressure continues to be limited in this way an error condition will be created and the flow will be stopped.
6 Appendix Setting Up a Method using Edit Entire Method channels, for example A2 and B1; it is not possible to mix A1 with A2 or B1 with B2. The value entered for the proportions of A and B defines the composition of an isocratic method or they define the starting conditions of a gradient method and the equilibration conditions between gradient runs. Only the value of B is entered, A will then update to show 100% minus B when the cursor is moved.
6 Appendix Setting Up a Method using Edit Entire Method • Stop Time defines the overall time for the separation or run and is sometimes referred to as the ‘Run Time’ by some users. This is the time, counted in minutes since the injection was made, that the run ends which means that data acquisition will stop, the flow, composition and other system settings will revert to the initial values for the method and the system will become available to make the next injection.
6 Appendix Setting Up a Method using Edit Entire Method Thermostatted Column Compartment (TCC) tab Figure 40 Setup Method screen – Thermostatted Column Compartment tab • Temperature defines the temperature of the left and right-hand side column holders which can be independently controlled or linked together by clicking the Combined radio button on.
Appendix Setting Up a Method using Edit Entire Method 6 The temperature of each zone can be set from -5 °C to 100 °C and the user should check that the column is suitable for operation at that temperature. (Agilent ZORBAX RRHD and RRHT StableBond phases can be used at the higher end of the range). The temperature is controlled to ± 0.15 °C down to 10 °C below ambient although it should be noted that there are very few applications operating below 12-15 °C.
6 Appendix Setting Up a Method using Edit Entire Method Diode-Array Detector (DAD) tab Figure 41 Setup Method screen – Diode-Array Detector tab • Signals: Up to eight separate signals (chromatograms) can be recorded. To mark a signal for collection the Use Signal box is checked for that signal, the wavelength and bandwidth are defined, and if a reference signal is required that box is checked and defined.
Appendix Setting Up a Method using Edit Entire Method 6 analysis stops before the end of the run defined in the pump. This can be the case when a gradient equilibration ramp has been set at the end of the gradient.
6 Appendix Setting Up a Method using Edit Entire Method • Store controls the spectral collection mode with the following options: None – no spectra stored, Apex+Baselines – 3 spectra taken at start, apex and end of peak, Apex+Slopes+Baselines – 5 spectra taken at start, upslope, apex, downslope and end of peak, All in Peak – all the available spectra within a peak are stored, All – all spectra throughout the run are stored, Every 2nd Spectrum – stores only alternate spectra acquired throughout the run.
6 Appendix Setting Up a Method using Edit Entire Method The entrance slit to the spectrograph controls the spectral resolution and influences the baseline noise and sensitivity. The default setting is 4 nm, which is suitable for most applications. See “How to Achieve Higher Sensitivity” on page 59for further discussion of this parameter.
6 Appendix Setting Up a Method using Edit Entire Method Instrument Curves Tab Figure 42 Setup Method screen – Instrument Curves tab The instrument curves tab allows monitored data streams other than detector signals to be stored with the data by checking the relevant box. These are primarily used for diagnostic purposes. They are: • Pump: • Pressure • Flow • A/B Composition — can be useful for overlaying the gradient profile on a chromatogram.
6 Appendix Setting Up a Method using Edit Entire Method Data Analysis Signal Details The Signal Details screen can also be directly accessed in Method and Run Control view: right-click on the graphical user interface on the Calibration icon, and then select Signal Details in the context menu. In the Data Analysis view, it can be accessed through the menu Calibration > Signal Details.
6 Appendix Setting Up a Method using Edit Entire Method Figure 43 134 Signal details Agilent 1290 Infinity LC System Manual and Quick Reference
6 Appendix Setting Up a Method using Edit Entire Method Edit Integration Events The Edit Integration Events screen can also be directly accessed in Method and Run Control view by right-clicking on the graphical user interface on the Integration Events icon and then clicking Edit Integration Events in the context menu. In the Data Analysis view it can be accessed through the menu Integration > Integration Events... or the Edit Integration Events task icon.
6 Appendix Setting Up a Method using Edit Entire Method The Edit Integration Events screen has two tables: • Initial Events For All Signals contains events (integration parameters) that apply to all signals acquired with the method, • Specific Events For Signal contains events which are specific for one type of detector or specific to different signals from the same detector.
6 Appendix Setting Up a Method using Edit Entire Method Figure 45 Specify Report Screen To setup a simple area% report with Classic Reporting, which prints to the printer and to a PDF file, enter the following settings in these sections of the Specify Report screen: Agilent 1290 Infinity LC System Manual and Quick Reference 137
6 Appendix Setting Up a Method using Edit Entire Method On the Reporting settings tab: • Report mode: Use Classic Reporting • Style • Report Style: Short • Quantitative results sorted by: Signal • Add Chromatogram Output: Checked • Chromatogram Output: Portrait • Size: • Time axis 100 % of page • Response axis 40 % of page • Destination • Printer: Checked • Screen: Unchecked • File: Checked • File Setting: • PDF: Checked • Unique PDF file name: Checked On the Quantitation settings tab: • Calculation mode •
Appendix Setting Up a Method using Edit Entire Method 6 Instrument Curves Figure 46 Instrument Curves screen The Instrument Curves checkboxes allow these recorded parameters to be overlaid as a graph on the chromatogram.
6 Appendix Setting Up a Method using Edit Entire Method Run Time Checklist The Run Time Checklist can also be directly accessed through the menu Method > Run Time Checklist... or by clicking on the Run Time Checklist icon at the top right of the screen. Figure 47 Run Time Checklist Screen The Run Time Checklist selects whether the method should run both data acquisition and data analysis and also offers an opportunity to link macro commands or programs into the work flow at various points.
Appendix Setting Up a Method using Edit Entire Method 6 The access points in the work flow of the method are: • Pre-Run Command / Macro • Customized Data Analysis Macro • Post-Run Command / Macro Save Method with Data saves a copy of the method in the data file and names it RUN.M. This is not needed if the ChemStation is operated in the usual configuration as the software always saves the method in the data file (all versions since B.02.01).
Index Index A Agilent 1290 Infinity LC System new features 22 power range 22 system Components 25 Agilent on internet 114 algae 113 analysis data 103 automated delay volume reduction B bandwidth 62 Binary Pump description 25 C calculator costs 14 carryover 66 column blockage 68 usage guidelines 68 column temperature 18 thermostatting 18 columns Sub-2-micron particles 14 configuration one stack 73, 78 two stack front 76, 81 two stack rear 77, 82 two stack 76, 81 configuring 142 online plot 93 descrip
Index O online plot configuring 93 optimization achieving high throughput 53 achieving higher resolution 56 achieving higher sensitivity 59 achieving lowest carryover 66 chromatographic separation 11 column use 59 conditions for HPLC 11 detector sensitivity 60 injection volumes 51 preventing column blockages 68 pump mixer volume 59 slit width 63 wavelength and bandwidth 60 P peak width 64 pump mixer volume purging pump 85 59 Q quick start guide introduction safety general information 110 symbols 112 se
www.agilent.com In This Book This manual contains technical reference information about the Agilent 1290 Infinity LC System. The manual describes the following: • introduction, • product description, • system optimization, • setup and installation, • quick start guide.
