Agilent Technologies Agilent 1260 Infinity System User's Manual
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Agilent Technologies Agilent 1260 Infinity System User's Manual

Agilent Technologies Agilent 1260 Infinity System User's Manual

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Summary of Contents for Agilent Technologies Agilent 1260 Infinity

  • Page 1 Agilent 1260 Infinity Binary LC System User Guide Agilent Technologies...
  • Page 2 Notices Warranty © Agilent Technologies, Inc. 2006-2014, receive no greater than Restricted Rights as 2015 defined in FAR 52.227-19(c)(1-2) (June The material contained in this docu- 1987). U.S. Government users will receive No part of this manual may be reproduced ment is provided “as is,”...
  • Page 3 1 The Agilent 1260 Infinity Binary LC - Product Description This chapter discusses the features of the 1260 Infinity Binary LC. 2 Introduction This chapter gives an introduction to the Agilent 1260 Infinity Binary LC and the underlying concepts. 3 Optimization of the Agilent 1260 Infinity Binary LC This chapter considers how to apply the theory and use the features of the LC system to develop optimized separations.

  • Page 4: Table Of Contents

    Contents Contents 1 The Agilent 1260 Infinity Binary LC - Product Description Features of the Agilent 1260 Infinity Binary LC System Components Specifications 2 Introduction Theory of Using Smaller Particles in Liquid Chromatography Benefits of small particle size columns Frictional Heating...
  • Page 5 Contents 6 Appendix Safety Information Solvent Information Agilent Technologies on Internet Setting Up a Method using Edit Entire Method 1260 Infinity Binary LC - System User Guide...
  • Page 6 Contents 1260 Infinity Binary LC - System User Guide...
  • Page 7 1260 Infinity Binary LC - System User Guide The Agilent 1260 Infinity Binary LC - Product Description Features of the Agilent 1260 Infinity Binary LC System Components 1260 Infinity Binary Pump (G1312B) 1260 Infinity High Performance Degasser (G4225A) 1260 Infinity High Performance Autosampler (G1367E)

  • Page 8: The Agilent 1260 Infinity Binary Lc - Product Description

    ZORBAX RRHT columns have an engineered particle size distribution that produces significantly less backpressure than other STM columns. The design features and benefits of the Agilent 1260 Infinity Binary LC are: • The configurable delay volume down to 120 µL in the 1260 Infinity Binary Pump combined with a flow range from 0.05 up to 5 mL/min at...
  • Page 9 The Agilent 1260 Infinity Binary LC - Product Description Features of the Agilent 1260 Infinity Binary LC • The new pull- out valve drive design and user- exchangeable Quick- Change valves in the 1290 Infinity Thermostatted Column Compartment boosts usability and paves the way for ultra high- throughput, multi- method and automated method development solutions.

  • Page 10: System Components

    The Agilent 1260 Infinity Binary LC - Product Description System Components System Components Numerous system configurations of the 1260 Infinity Binary LC are possible, tailored to the needs of your individual application requirements. A few configurations are described in more detail in this manual (see “System Setup and Installation”...
  • Page 11 The Agilent 1260 Infinity Binary LC - Product Description System Components 1260 Infinity Binary Pump (G1312B) The binary pump comprises two identical pumps integrated into one housing. Binary gradients are created by high- pressure mixing. Pulse damper and mixer can be bypassed for low flowrate applications or whenever a minimal transient volume is desirable.
  • Page 12 The Agilent 1260 Infinity Binary LC - Product Description System Components Figure 1 The Hydraulic Path of the Binary Pump with Damper and Mixer 1260 Infinity Binary LC - System User Guide...
  • Page 13 The Agilent 1260 Infinity Binary LC - Product Description System Components Damper and mixer can be bypassed for lowest delay volume of the binary pump. This configuration is recommended for low flow rate applications with steep gradients. Figure 2 on page 13 illustrates the flow path in low delay volume mode.
  • Page 14 The Agilent 1260 Infinity Binary LC - Product Description System Components 1260 Infinity High Performance Degasser (G4225A) The Agilent 1260 Infinity High Performance Degasser , model G4225A, comprises four separate vacuum chambers with semipermeable tubings, a vacuum pump and control assembly. When the vacuum degasser is switched on, the control assembly turns on the vacuum pump, which generates a low pressure in the vacuum chambers.
  • Page 15 The Agilent 1260 Infinity Binary LC - Product Description System Components 1260 Infinity High Performance Autosampler (G1367E) Features The 1260 Infinity High Performance Autosampler features an increased pressure range (up to 600 bar) enabling the use of today’s column technology (sub- two- micron narrow bore columns) with the Agilent 1260 Infinity Binary LC.
  • Page 16 The Agilent 1260 Infinity Binary LC - Product Description System Components Injection Sequence Before the start of the injection sequence, and during an analysis, the injection valve is in the mainpass position. In this position, the mobile phase flows through the autosampler metering device, sample loop, and needle, ensuring all parts in contact with sample are flushed during the run, thus minimizing carry- over.
  • Page 17 The Agilent 1260 Infinity Binary LC - Product Description System Components 1290 Infinity Thermostatted Column Compartment (G1316C) 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...
  • Page 18 The Agilent 1260 Infinity Binary LC - Product Description System Components Figure 4 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.
  • Page 19 The Agilent 1260 Infinity Binary LC - Product Description System Components 1260 Infinity Diode Array Detector (G4212B) The 1260 Infinity Diode- Array Detector (DAD) 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.
  • Page 20 The Agilent 1260 Infinity Binary LC - Product Description System Components The chromatographic signals are extracted from the diode- array data within the firmware of the module. Up to eight individual signals can be defined. Each of them comprises a signal wavelength, a diode- bunching bandwidth and - if required - a reference wavelength and bandwidth.
  • Page 21 The Agilent 1260 Infinity Binary LC - Product Description System Components 1200 Infinity Series Quick Change Valves Agilent 1200 Infinity Quick Change Valves support a variety of challenging valve applications. Each valve head comes as a complete kit containing all required capillaries, additional low dispersion heat exchangers and other parts.

  • Page 22: Specifications

    The Agilent 1260 Infinity Binary LC - Product Description Specifications Specifications The modular design of the 1260 Infinity Binary LC allows you to configure a system that exactly meets your individual application requirements. This individual configuration can be different from the standard configuration which is described in this System User Guide.
  • Page 23 The Agilent 1260 Infinity Binary LC - Product Description Specifications Physical Specifications 1260 Infinity Binary Pump (G1312B) Table 1 Physical Specifications Type Specification Comments Weight 15.5 kg (34 lbs) Dimensions 180 x 345 x 435 mm (height × width × depth) (7 x 13.5 x 17 inches)
  • Page 24 The Agilent 1260 Infinity Binary LC - Product Description Specifications Performance Specifications Table 2 Performance Specifications of the Agilent 1260 Infinity Binary Pump (G1312B) Type Specification Comments Hydraulic system Two dual piston in series pumps with servo-controlled variable stroke drive,...
  • Page 25 The Agilent 1260 Infinity Binary LC - Product Description Specifications Table 2 Performance Specifications of the Agilent 1260 Infinity Binary Pump (G1312B) Type Specification Comments Composition < 0.15 % RSD or < 0.04 min SD whatever at 0.2 and 1 mL/min; based...
  • Page 26 The Agilent 1260 Infinity Binary LC - Product Description Specifications 1260 Infinity Binary LC - System User Guide...

  • Page 27: Introduction

    1260 Infinity Binary LC - System User Guide Introduction Theory of Using Smaller Particles in Liquid Chromatography Benefits of small particle size columns Frictional Heating This chapter gives an introduction to the Agilent 1260 Infinity Binary LC and the underlying concepts. Agilent Technologies...

  • Page 28: Theory Of Using Smaller Particles In Liquid Chromatography

    In 2003, Agilent introduced the first commercially available, totally porous silica columns with 1.8 µm particles. In combination with the Agilent 1260 Infinity Binary LC the sub- two micron (1.8 µm) particle size columns can be used in pursuit of two main...
  • Page 29 Introduction Theory of Using Smaller Particles in Liquid Chromatography The Theory Separation efficiency in HPLC can be described by the van Deemter equation (Figure 6 on page 29). This results from the plate- height model that is used to measure the dispersion of analytes as they move down the column.
  • Page 30 Introduction Theory of Using Smaller Particles in Liquid Chromatography The van Deemter plots in Figure 7 on page 30 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.
  • Page 31 Introduction 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.
  • Page 32 Introduction Theory of Using Smaller Particles in Liquid Chromatography Resolution can be described as a function of three parameters: • column efficiency or theoretical plates (N), • selectivity (α), • retention factor (k). According to the resolution equation ( Figure 9 on page 32 ), the selectivity has the biggest impact on resolution (Figure 10...
  • Page 33 2.1, 3.0 and 4.6 mm internal diameters). Additionally to the ZORBAX columns, PoroShell columns with nine selectivity choices are available for use with the Agilent 1260 Infinity Binary LC. This enables the optimum stationary phase to be selected so that the selectivity is maximized.

  • Page 34: Benefits Of Small Particle Size Columns

    Introduction Benefits of small particle size columns Benefits of small particle size columns 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 3...
  • Page 35 Introduction Benefits of small particle size columns Figure 11 Relation between particle size, efficiency and analysis time Analysis time can be shortened without sacrificing column efficiency by optimizing particle size and pressure. 1260 Infinity Binary LC - System User Guide...
  • Page 36 Introduction Benefits of small particle size columns 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.

  • Page 37: Frictional Heating

    In summary, the use of sub- two- micron packing material offers benefits of increased efficiency, higher resolution and faster separations. The features of the Agilent 1260 Infinity Binary LC are discussed in the chapter Product Description. The chapter Optimization considers how to apply the theory and use these features to develop optimized separations.
  • Page 38 Introduction Frictional Heating 1260 Infinity Binary LC - System User Guide...
  • Page 39 Binary LC How to Configure the Optimum Delay Volume Delay Volume and Extra-Column Volume Delay Volumes in the Agilent 1260 Infinity Binary LC Optimum Instrument Configuration for 2.1 mm i.d. Columns Optimum Instrument Configuration for 3 and 4.6 mm i.d. Columns...

  • Page 40: Optimization Of The Agilent 1260 Infinity Binary Lc

    This is achieved with the Agilent 1260 Infinity Binary LC due to the low delay volume of the pump flow path and low- volume of the flow path through the autosampler.
  • Page 41 The extra- column volume is minimized with the Agilent 1260 Infinity Binary LC due to the narrow- bore (0.12 mm i.d.) tubing, the low- volume heat exchangers in the column compartment and the Max- Light cartridge cell in the detector.

  • Page 42: Delay Volumes In The Agilent 1260 Infinity Binary Lc

    Table 4 on page 42 and Table 5 on page 42show the component volumes which contribute to system delay volume in the Agilent 1260 Infinity Binary LC System . Table 4 Delay volumes of the 1260 Infinity Binary LC modules Components Delay Volume (µL)

  • Page 43: Optimum Instrument Configuration For 2.1 Mm I.d. Columns

    Low delay volume configuration to achieve shortest gradient delay for ultra-fast gradient separations In the low delay volume configuration of the Agilent 1260 Infinity Binary Pump the damper and mixer are bypassed to reduce the pump delay volume to about 120 µL.
  • Page 44 Optimization of the Agilent 1260 Infinity Binary LC How to Configure the Optimum Delay Volume Medium delay volume configuration to achieve highest UV sensitivity For high sensitivity UV applications an additional 200 µL mixer ( Low volume mixer ( 200 µL) (5067- 1565)) can be installed to reduce any residual mixing noise.

  • Page 45: Optimum Instrument Configuration For 3 And 4.6 Mm I.d. Columns

    Optimization of the Agilent 1260 Infinity Binary LC How to Configure the Optimum Delay Volume Optimum Instrument Configuration for 3 and 4.6 mm i.d. Columns Standard delay volume configuration for highest UV sensitivity and direct method transferability The relative column volumes for 3 mm and 4.6 mm inner diameter columns are about two and five times larger respectively than for the same length 2.1 mm i.d.
  • Page 46 As can be seen, a seamless method transfer is possible. Figure 16 Comparison Agilent 1200 Series and Agilent 1260 Infinity LC system on one Configuration 1260 Infinity Binary LC - System User Guide...

  • Page 47: How To Achieve Higher Injection Volumes

    How to Achieve Higher Injection Volumes How to Achieve Higher Injection Volumes The standard configuration of the Agilent 1260 Infinity Autosampler can inject a maximum volume of 100 µL with the standard loop capillary. To increase the injection volume, the Multidraw upgrade kit (G1313- 68711) can be installed.

  • Page 48: How To Achieve Shorter Cycle Times

    Optimization of the Agilent 1260 Infinity Binary LC How to Achieve Shorter Cycle Times How to Achieve Shorter Cycle Times Shorter cycle times can be achieved by selecting a short column with good selectivity. The column dimensions are also determined by the detection system that is used.
  • Page 49 How to Achieve Shorter Cycle Times Automatic delay volume reduction (ADVR) The Agilent 1260 Infinity High Performance Autosampler offers the possibility of performing overlapped injections (OI) and/or automatic delay volume reduction (ADVR). This means that the injection valve is switched out of the flow path after the sample has reached the top of the column.

  • Page 50: How To Achieve High Throughput

    Optimization of the Agilent 1260 Infinity Binary LC How to Achieve Shorter Cycle Times Carry- over is the percentage of compound that remains in the parts of the instrument that come into contact with the sample, and is not flushed onto the column for analysis.

  • Page 51: How To Achieve Lowest Carry-Over

    The level of carryover is reported as the area of the peak in the blank solution that is expressed as a percentage of the area in the previous active injection. The Agilent 1260 Infinity Autosampler is optimized for lowest carryover by careful design of the flow path and use of materials in which sample adsorption is minimized.
  • Page 52 Optimization of the Agilent 1260 Infinity Binary LC How to Achieve Lowest Carry-over Flushing and cleaning of the autosampler to achieve near zero carry-over During the injection routine, the sample loop, the inside of the needle, the seat capillary, and the main channel of the injection valve are in the flow path, and remain there throughout the duration of the run.

  • Page 53: How To Achieve Higher Resolution

    Optimization of the Agilent 1260 Infinity Binary LC 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. This section explains how resolution can be increased by examining the following points: •...
  • Page 54 Optimization of the Agilent 1260 Infinity Binary LC How to Achieve Higher Resolution was used in 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, which can be optimized in a number of ways.
  • Page 55 Optimization of the Agilent 1260 Infinity Binary LC How to Achieve Higher Resolution In isocratic separations, increasing the retention factor, k, results in better resolution because the solute is retained longer. In gradient separations, the retention is described by k...

  • Page 56: Optimum Instrument Configuration For High Resolution

    Optimization of the Agilent 1260 Infinity Binary LC How to Achieve Higher Resolution Optimum Instrument Configuration for High Resolution Column Compartment The column compartment can be used in its standard version for 4.6 mm inner diameter columns. At flow rates above 2 mL/min, and temperatures above 60 °C, the column effluent should be cooled down to the...
  • Page 57 Optimization of the Agilent 1260 Infinity Binary LC How to Achieve Higher Resolution Extra-column Volume To maintain the high resolution achieved on the column, the extra- column volume, especially after the column, should be as low as possible. • For 4.6 mm inner diameter columns the standard delay configuration...
  • Page 58 Optimization of the Agilent 1260 Infinity Binary LC How to Achieve Higher Resolution Data Rate The data rate setting for the UV detector must be selected appropriately. Selecting a data rate that is too low results in increased peak width and...
  • Page 59 Optimization of the Agilent 1260 Infinity Binary LC How to Achieve Higher Resolution Table 6 Relation between data rate and chromatographic performance Data Rate Peak Width Resolution Peak Capacity 80 Hz 0.300 2.25 40 Hz 0.329 2.05 20 Hz 0.416 1.71...

  • Page 60: How To Achieve Higher Sensitivity

    Optimization of the Agilent 1260 Infinity Binary LC 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.

  • Page 61: Optimum Instrument Configuration For High Sensitivity

    Higher than necessary data rates should be avoided because of higher noise levels. • Available UV detectors are the Agilent 1260 Infinity Diode Array Detector (G4212B) and the Agilent 1260 Infinity Variable Wavelength Detector (G1314F), with data rates of 80 Hz and significantly lower noise and drift levels.
  • Page 62 Optimization of the Agilent 1260 Infinity Binary LC How to Achieve Higher Sensitivity Columns Sensitivity is specified as a signal- to- noise ratio (S/N) and hence the need to maximize peak height and minimize baseline noise. Any reduction in peak dispersion will help to maintain peak height and so extra- column volume should be minimized by use of short, narrow internal diameter, connection capillaries and correctly installed fittings.

  • Page 63: Choosing A Flow Cell

    Optimization of the Agilent 1260 Infinity Binary LC How to Achieve Higher Sensitivity Choosing a Flow Cell Several flavors of the Max- Light Cartridge Flow Cell are available, see (Table 7 on page 63). Table 7 Specifications for Max-Light Cartridge Flow Cells Cartridge Cells •...
  • Page 64 Optimization of the Agilent 1260 Infinity Binary LC How to Achieve Higher Sensitivity Ultra-Low Dispersion The Max- Light Cartridge ULD cell can be used with the G4212A DAD and G4212B DAD . The cell is a requirement for the Ultra- Low Dispersion Kit solution which currently exists as 1290 Infinity Ultra- Low Dispersion Kit (5067- 5189).

  • Page 65: How To Prevent Column Blockages

    Optimization of the Agilent 1260 Infinity Binary LC How to Prevent Column Blockages How to Prevent Column Blockages Columns packed with sub- 2- micron particles also need frits with small pore size to prevent packing material being swept out. This immediately brings increased risk of blocking these frits with particulates from the sample, mobile phase or/and from the instrument itself.
  • Page 66 Optimization of the Agilent 1260 Infinity Binary LC How to Prevent Column Blockages Figure 20 Protection for 4.6 and 2.1 mm id columns packed with 1.8 µm particles, inlet frit with 0.2 µm pore size 1260 Infinity Binary LC - System User Guide...
  • Page 67 Installing the Modules Installing the System Modules Integration Into the Network Capillary and Tubing Connections in Flow Path Priming the System This chapter includes information on software installation, installation of the modules, and preparing the system for operation. Agilent Technologies...

  • Page 68: System Setup And Installation

    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 detector manual and the software manuals. 1260 Infinity Binary LC - System User Guide...
  • Page 69 System Setup and Installation Installing Software 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. Agilent Lab Advisor replaces and extends the diagnostic functions that were formerly available only in the ChemStation software.

  • Page 70: Installing The Modules

    System Setup and Installation Installing the Modules Installing the Modules Installing the System Modules For details of installation procedures for the modules, refer to the individual module manuals. These manuals also contain information on specifications, maintenance and parts. Integration Into the Network For network integration of your system refer to user manuals of your modules (chapter LAN Configuration).

  • Page 71: Capillary And Tubing Connections In Flow Path

    System Setup and Installation Installing the Modules Capillary and Tubing Connections in Flow Path Depending on the system configuration, capillaries of different lengths and diameters are used. These are described below. See module manuals for module- internal capillary and tubing connections. 1260 Infinity Binary LC - System User Guide...
  • Page 72 System Setup and Installation Installing the Modules Connections for Standard Delay Volume Configuration Figure 21 on page 72 shows capillary and tubing connections in the flow path for the standard delay volume configuration of the 1260 Infinity Binary LC. Figure 21 Capillary and tubing connections in flow path (standard delay volume configuration) 1260 Infinity Binary LC - System User Guide...
  • Page 73 System Setup and Installation Installing the Modules Item Description G1312-87303 Capillary ST 0.17 mm x 400 mm S/S (Pump to autosampler) G1312-87304 Capillary ST 0.17 mm x 700 mm S/S (Pump to cooled Autosampler) G1367-87304 Capillary ST 0.17 mm x 250 mm S/S (Autosampler to TCC) 01090-87306 Capillary ST 0.17 mm x 380 nm S/S...
  • Page 74 System Setup and Installation Installing the Modules Connections for Medium and Low Delay Volume Configuration Figure 22 on page 74 shows capillary and tubing connections in the flow path for medium and low delay volume configurations of the 1260 Infinity Binary LC. Figure 22 Capillary and tubing connections in flow path (medium and low delay volume configurations)
  • Page 75 System Setup and Installation Installing the Modules Item Description G1312-87303 Capillary ST 0.17 mm x 400 mm S/S (Pump to autosampler) G1312-87304 Capillary ST 0.17 mm x 700 mm S/S (Pump to cooled Autosampler) G1313-87304 Capillary ST 0.12 mm x 180 mm S/S (Autosampler to TCC) 01090-87610 Capillary ST 0.12 mmx 280 mm S/S...

  • Page 76: Priming The System

    System Setup and Installation Installing the Modules Priming the System Initial Priming When Before a degasser or solvent tubing can be used, it is necessary to prime the system. Isopropanol is recommended as priming solvent due to its miscibility with nearly all HPLC solvents and its excellent wetting properties.
  • Page 77 System Setup and Installation Installing the Modules 1 Open the purge valve of the pump 2 Set the flow rate to 5 mL/min. 3 Select channel A1 4 Turn the flow on 5 Observe if the solvent in the tubing of channel A1 is advancing towards the pump.
  • Page 78 System Setup and Installation Installing the Modules Regular Priming When When the pumping system has been turned off for a certain time (for example, overnight) air will rediffuse into the solvent channel between the vacuum degasser and the pump. If solvents containing volatile components are left in the degasser without flow for a prolonged period, there will be a slight loss of the volatile components.
  • Page 79 System Setup and Installation Installing the Modules Changing Solvents When When the solvent of a channel is to be replaced by another solvent that is not compatible (solvents are immiscible or one solvent contains a buffer), it is necessary to follow the procedure below to prevent clogging of the pump by salt precipitation or residual liquid droplets in parts of the system.
  • Page 80 System Setup and Installation Installing the Modules Table 8 Choice of Priming Solvents for Different Purposes Activity Solvent Comments After an installation Isopropanol Best solvent to flush air out of When switching between Isopropanol the system reverse phase and normal Miscible with almost all phase (both times) solvents...
  • Page 81 Setting Up the Method Running the Method for a Single Injection Data Analysis Data Analysis View Integrating a Signal Specify the Report This chapter provides information on data acquisition and data analysis with the 1260 Infinity Binary LC. Agilent Technologies...

  • Page 82: Quick Start Guide

    About the Quick Start Guide About the Quick Start Guide This chapter provides information on running the Agilent 1260 Infinity Binary LC. 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.

  • Page 83: Preparing The System

    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.

  • Page 84: Loading The Default Method

    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.

  • Page 85: Configuring The Online Plot

    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….
  • Page 86 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.

  • Page 87: Purging The Pump

    Quick Start Guide Preparing the System 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. •...

  • Page 88: Data Acquisition In Method And Run Control View

    Quick Start Guide Data Acquisition in Method and Run Control View Data Acquisition in Method and Run Control View Method Parameters for System Checkout with Isocratic Checkout Sample For checkout of the 1260 Infinity Binary LC, a test run with an isocratic test mixture (Agilent isocratic checkout sample (01080- 68704)) will be performed on the column ordered and delivered with your system.
  • Page 89 Quick Start Guide Data Acquisition in Method and Run Control View Table 9 Method parameters for first separation run - standard delay volume configuration Module Parameter Setting Pump Solvent A Water Solvent B Acetonitrile Flow rate 4.0 mL/min Solvent 40 % A, 60 % B Composition Stop Time 1 min (1 min post time)
  • Page 90 Quick Start Guide Data Acquisition in Method and Run Control View Table 10 Method parameters for first separation run - low delay volume configuration Module Parameter Setting Pump Solvent A Water Solvent B Acetonitrile Flow rate 0.75 mL/min Solvent 40 % A, 60 % B Composition Stop Time 1.2 min (1 min post time)
  • Page 91 Quick Start Guide Data Acquisition in Method and Run Control View Checkout Preparation 1 Fill solvent bottle A with HPLC grade Water. Fill solvent bottle B with HPLC grade Acetonitrile. 2 If installed, purge each channel of the on- line degasser using the supplied syringe.

  • Page 92: Setting Up The Method

    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. The default method DEF_LC.M has been loaded ready to prepare the new method. Now the Prerequisites key parameters can be edited to create the new method.
  • Page 93 Quick Start Guide Data Acquisition in Method and Run Control View 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: •...

  • Page 94: Running The Method For A Single Injection

    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 isocratic checkout sample using the conditions entered in the previous section. Analyses on the ChemStation can be run in two modes: •...
  • Page 95 Quick Start Guide Data Acquisition in Method and Run Control View 6 The injection is made, and the chromatogram appears in the Online Plot. The data acquisition will stop when the Stop Time is reached. The chromatogram should look similar to the one below: 1260 Infinity Binary LC - System User Guide...

  • Page 96: Data Analysis

    Quick Start Guide Data Analysis 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.

  • Page 97: Data Analysis View

    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. 3 In the Navigation Panel, find the data directory containing the data files.

  • Page 98: Integrating A Signal

    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.

  • Page 99: Specify The Report

    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. 1260 Infinity Binary LC - System User Guide...
  • Page 100 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.
  • Page 101 1260 Infinity Binary LC - System User Guide Appendix Safety Information Solvent Information Material Information Agilent Technologies on Internet Setting Up a Method using Edit Entire Method Method Information Instrument/Acquisition Data Analysis Run Time Checklist This chapter provides additional information on safety, legal and web and about setting up a method.

  • Page 102: Appendix

    Agilent Technologies assumes no liability for the customer’s failure to comply with these requirements. Ensure the proper usage of the equipment.
  • Page 103 Appendix Safety Information Operation Before applying power, comply with the installation section. Additionally the following must be observed. Do not remove instrument covers when operating. Before the instrument is switched on, all protective earth terminals, extension cords, auto- transformers, and devices connected to it must be connected to a protective earth via a ground socket.
  • Page 104 Appendix Safety Information Safety Symbols Table 11 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.

  • Page 105: Solvent Information

    Appendix Solvent Information Solvent Information Observe the following recommendations on the use of solvents. • Follow recommendations for avoiding the growth of algae, see pump manuals. • Small particles can permanently block capillaries and valves. Therefore, always filter solvents through 0.4 µm filters. •...
  • Page 106 Appendix Solvent Information PEEK PEEK (Polyether- Ether Ketones) combines excellent properties with regard to chemical resistance, mechanical and thermal stability. It is stable in a pH range of 1 to 12.5 and inert to many common solvents. There are several known incompatibilities with chemicals such as chloroform, methylene chloride, THF, DMSO >...
  • Page 107 Appendix Solvent Information Tantalum (Ta) Tantalum is inert to most common HPLC solvents and almost all acids except fluoric acid and acids with free sulfur trioxide. It can be corroded by strong bases (e.g. hydroxide solutions > 10 %, diethylamine). It is not recommended for the use with fluoric acid and fluorides.
  • Page 108 Appendix Solvent Information Diamond-Like Carbon (DLC) Diamond- Like Carbon is inert to almost all common acids, bases and solvents. There are no documented incompatibilities for HPLC applications. Fused silica and Quartz (SiO Fused silica is used in 1290 Infinity Flow Cells and capillaries. Quartz is used for classical flow cell windows.
  • Page 109 Appendix Solvent Information Fluorinated polymers (PTFE, PFA, FEP, FFKM) Fluorinated polymers like PTFE (polytetrafluorethylene), PFA (perfluoroalkoxy) and FEP (fluorinated ethylene propylene) are inert to almost all common acids, bases, and solvents. FFKM is perfluorinated rubber, which is also resistant to most chemicals. As an elastomer, it may swell in some organic solvents like halogenated hydrocarbons.

  • Page 110: Agilent Technologies On Internet

    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 1260 Infinity Binary LC - System User Guide...

  • Page 111: Setting Up A Method Using Edit Entire Method

    Appendix Setting Up a Method using Edit Entire Method Setting Up a Method using Edit Entire Method 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). The parameters are accessed through a series of screens which each focus on one module or function.
  • Page 112 Appendix Setting Up a Method using Edit Entire Method 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, •...

  • Page 113: Method Information

    Appendix Setting Up a Method using Edit Entire Method 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.

  • Page 114: Instrument/Acquisition

    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.
  • Page 115 Appendix Setting Up a Method using Edit Entire Method Autosampler tab Figure 25 Setup Method screen – High Performance Autosampler tab • Injection Mode • Injection volume sets the volume to be injected (example 1 µl), • Standard injection indicates that no external needle wash is done, •...
  • Page 116 Appendix Setting Up a Method using Edit Entire Method • 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.
  • Page 117 Appendix Setting Up a Method using Edit Entire Method • Advanced - High Throughput • Automatic delay volume reduction (ADVR) switches the injection valve from mainpass to bypass after the injection has taken place and a volume defined by the sample flush out factor has swept through the injector.
  • Page 118 A commonly used example is the derivatization of amino acids with OPA and FMOC reagents. For details, please refer to the Agilent 1260 Infinity High Performance Autosampler manual. 1260 Infinity Binary LC - System User Guide...
  • Page 119 Appendix Setting Up a Method using Edit Entire Method Binary Pump Tab Figure 27 Setup Method screen – Binary Pump tab • Flow sets the flow rate up to 5 mL/min. For the example separation 4 mL/minis used. If the back pressure briefly reaches the maximum pressure setting, the flow will be reduced for a few seconds to lower the pressure.
  • Page 120 Appendix Setting Up a Method using Edit Entire Method • Solvents defines the mobile phases that are available and the percentage proportions that are pumped on the two channels, A and B. On each channel, a drop- down box allows the selection of a solvent from a list so that the pump control uses the optimum compressibility settings.
  • Page 121 Appendix Setting Up a Method using Edit Entire Method the settings for time 0.00 min into the timetable, these values are picked up from other setpoints on this screen. However, some users like to see a ‘complete’ list in the timetable and make an entry for 0.00 min. There is no problem with this but if the initial conditions are ever changed then the new settings must be entered in both the timetable and the setpoints in the Solvents section of the screen.
  • Page 122 Appendix Setting Up a Method using Edit Entire Method Thermostatted Column Compartment (TCC) tab Figure 28 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.
  • Page 123 Appendix Setting Up a Method using Edit Entire Method 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 RRHT StableBond phases can be used at the higher end of the range).
  • Page 124 Appendix Setting Up a Method using Edit Entire Method Diode Array Detector Tab Figure 29 Setup Method screen – Diode Array Detector tab • Signals: Up to eight separate signals (chromatograms) can be recorded. To mark a signal for collection, check the Use Signal box for that signal, define the wavelength and bandwidth, and if a reference signal is required also check and define that box.
  • Page 125 Appendix Setting Up a Method using Edit Entire Method • Reference Wavelength sets the central wavelength (nm) of the reference band which is subtracted from the analytical signal, • Reference Bandwidth sets the width (nm) of the reference band. • Peakwidth sets the data collection rate and signal filtering. •...
  • Page 126 Appendix Setting Up a Method using Edit Entire Method • Advanced - Spectrum Spectra can be saved during the run on a continuous or peak- controlled basis (this applies to the ChemStation software. Some software packages, e.g. EZChrom, only support continuous collection of all spectra and the peak- controlled options do not appear).
  • Page 127 Appendix Setting Up a Method using Edit Entire Method • Advanced - Analog Output The 1260 Infinity DAD has one analog signal output connector for use with data systems that do not accept a digital input. The following may be set: •...
  • Page 128 Appendix Setting Up a Method using Edit Entire Method Instrument Curves Tab Figure 30 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.

  • Page 129: Data Analysis

    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 >...
  • Page 130 Appendix Setting Up a Method using Edit Entire Method Figure 31 Signal details 1260 Infinity Binary LC - System User Guide...
  • Page 131 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.
  • Page 132 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.
  • Page 133 Appendix Setting Up a Method using Edit Entire Method Specify Report The Specify Report screen can also be directly accessed in Method and Run Control view by right- clicking on the graphical user interface on the Report icon and then selecting Specify Report in the context menu. In the Data Analysis view it can be accessed through the menu Report >...
  • Page 134 Appendix Setting Up a Method using Edit Entire Method • Chromatogram Output: Portrait • Size: • Time axis 100 % of page • Response axis 40 % of page • Destination • Printer: Checked • Screen: Unchecked • File: Checked •...
  • Page 135 Appendix Setting Up a Method using Edit Entire Method Instrument Curves Figure 34 Instrument Curves screen The Instrument Curves checkboxes allow these recorded parameters to be overlaid as a graph on the chromatogram. 1260 Infinity Binary LC - System User Guide...

  • Page 136: Run Time Checklist

    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 35 Run Time Checklist Screen The Run Time Checklist selects whether the method should run both data...
  • Page 137 Appendix Setting Up a Method using Edit Entire Method 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.
  • Page 138 Index Index compressibility compensation configuration active seal wash flow accuracy low delay volume ADVR flow cell medium delay volume Agilent flow precision standard delay volume on internet flow range configuring alternating column regeneration operational online plot setable ambient non-operating temperature control and data evaluation frequency range ambient operating temperature...
  • Page 139 Index line voltage extra-column volume symbols loading optimizing sensitivity default flow cell instrument configuration optimization low delay volume configuration overlapped injections setable flow range setup instrument method solvent selection valve medium delay volume configuration performance solvents, change specifications Method Information 112, specification pH range...
  • Page 140 In This Book This manual contains information about the Agilent 1260 Infinity Binary LC. The manual describes the following: • Product description, • introduction, • specifications, • system optimization, • system setup and installation, • quick start guide. ©...

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