Page 1 ÄKTA ™ avant User Manual...
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Page 3: Table Of Contents
Table of Contents Table of Contents Introduction ......................Important user information ......................ÄKTA avant overview .......................... Associated Documentation ......................The ÄKTA avant instrument ................Overview illustrations ......................... Runs in a cold environment ......................Removing the foldable door and pump cover ................ Liquid flow path .............................
Page 4 Table of Contents 4.11 Second UV monitor ..........................4.12 I/O-box ............................... 4.12.1 Overview of the I/O-box ......................4.12.2 Analog connector and signals ....................4.12.3 Digital connector and signals ....................4.12.4 Connect external equipment to the I/O-box ..............4.13 General system settings ........................Operation ......................
Page 5 Table of Contents Monthly maintenance ........................7.4.1 Check the flow restrictor ......................7.4.2 Check the function of the pump flow restrictors .............. Semiannual maintenance ........................ 7.5.1 Clean the UV flow cell ........................7.5.2 Replace the pH electrode ......................Maintenance when required ......................7.6.1 Clean the instrument externally ....................
Page 6 Table of Contents Reference information ..................System specifications ......................... Module specifications ......................... Tubing and connectors ........................Chemical resistance guide ....................... 9.4.1 General information about biocompatibility and chemical resistance ....9.4.2 Chemical resistance specifications ..................Wetted materials ..........................Predefined methods and phases ....................System settings .............................
Page 7: Introduction
1 Introduction Introduction Purpose of the User Manual The User Manual provides you with instructions and information to run the ÄKTA avant system. It also includes relevant guidance for practical handling and maintenance of instrument components. In this chapter This chapter contains the following sections: Section See page 1.1 Important user information...
Page 8: Important User Information
1 Introduction 1.1 Important user information Important user information Read this before operating the product All users must read the entire Operating Instructions before installing, operating or maintaining the product. Always keep the Operating Instructions at hand when operating the product. Do not operate the product in any other way than described in the user documentation.
Page 9 1 Introduction 1.1 Important user information Safety notices This user documentation contains safety notices (WARNING, CAUTION, and NOTICE) concerning the safe use of the product. See definitions below. WARNING WARNING indicates a hazardous situation which, if not avoided, could result in death or serious injury. It is important not to proceed until all stated conditions are met and clearly understood.
Page 10: Äkta Avant Overview
1 Introduction 1.2 ÄKTA avant overview ÄKTA avant overview Introduction ÄKTA avant is a preparative liquid chromatography system intended for purification of proteins as well as other bio-molecules and used for method and process development. The system is equipped with valves, detectors and fraction collector, integrated in the flow path, needed for automated control and optimal performance.
Page 11 1 Introduction 1.2 ÄKTA avant overview UNICORN modules overview UNICORN consists of four modules: Administration, Method Editor, System Control and Evaluation. The main functions of each module are described in the following table. Module Main functions Administration Perform user and system setup, system log and database administration.
Page 12: Associated Documentation
1 Introduction 1.3 Associated Documentation Associated Documentation Introduction This section describes the user documentation that is delivered with ÄKTA avant. User documentation on the CD The user documentation listed in the table below is available in printed or PDF format. The complete documentation is also available on the User Documentation CD.
Page 13 1 Introduction 1.3 Associated Documentation Documentation Main contents Getting started with Video clips showing common workflows in the • Evaluation Evaluation module. Note: Overview of features of the Evaluation module. • Available in UNICORN 7.0 and later. UNICORN Method Manual Overview and detailed descriptions of the method •...
Page 14 1 Introduction 1.3 Associated Documentation Additional literature For practical tips on chromatography, refer to ÄKTA Laboratory-scale: Chromatography Systems Instrument Management Handbook (product code 29010831). ÄKTA avant User Manual 29035184 AE...
Page 15: The Äkta Avant Instrument
2 The ÄKTA avant instrument The ÄKTA avant instrument About this chapter This chapter provides an overview of the ÄKTA avant instrument and the standard and optional instrument modules. In this chapter This chapter contains the following sections: Section See page 2.1 Overview illustrations 2.2 Runs in a cold environment 2.3 Removing the foldable door and pump cover...
Page 16: Overview Illustrations
2 The ÄKTA avant instrument 2.1 Overview illustrations Overview illustrations Introduction This section provides an overview of the system and its available modules. Instrument configurations ÄKTA avant is available with two standard module configurations, one for flow rates up to 25 ml/min and one for flow rates up to 150 ml/min. In this manual they are referred to as ÄKTA avant 25 (25 ml/min) and ÄKTA avant 150 (150 ml/min).
Page 17 2 The ÄKTA avant instrument 2.1 Overview illustrations Part Function Part Function Holder rails Swivel foot lock/unlock knob Swivel foot Swing out toolbox Power switch Illustration of the wet side modules of the instrument The following illustration shows the modules of the wet side of the instrument. Part Function Part...
Page 18 2 The ÄKTA avant instrument 2.1 Overview illustrations Part Function Part Function System pump A Mixer Sample pump flow restrictor Pressure monitor of sample pump Sample pump Pump rinsing solution tube Sample inlet valve Outlet valve Holder rails Standard modules ÄKTA avant is delivered with the modules listed in the following table.
Page 19 2 The ÄKTA avant instrument 2.1 Overview illustrations Module Label in ÄKTA avant 25 ÄKTA avant 150 Built-in fraction collector Optional modules One or more of the modules in the following table may be added to the flow path of ÄKTA avant.
Page 20 2 The ÄKTA avant instrument 2.1 Overview illustrations Illustration convention In the valve illustrations below, the following convention is used to point out the location of the ports on the valve head. Loop valve V9-L is used as an example. Ports located on the valve head rim are indicated outside V9-L •...
Page 21 2 The ÄKTA avant instrument 2.1 Overview illustrations Module Description System pump B P9 B or P9H B A high precision pump, which delivers buffer in purification runs. For further information, refer to Section 3.1 Pumps, on page 56. Sample pump P9-S or P9H S A high precision pump which delivers sample or buffer in purification runs.
Page 22 2 The ÄKTA avant instrument 2.1 Overview illustrations Module Description Pump flow restrictor Prevents the system from siphoning when the flow path after the pump is open. Gives a small back pressure to the pump in extreme low pressure applications. For further information, refer to Section 3.8 Pres- sure monitors, on page 78.
Page 23 2 The ÄKTA avant instrument 2.1 Overview illustrations Module Description Inlet valve A V9-IA or V9H-IA Inlet valve for System Pump A with seven inlet ports and integrated air sensor. V9-IA For further information, refer to Section 3.4 Inlet valves, on page 64. Inlet valve B V9-IB or V9H-IB Inlet valve for System Pump B with seven inlet ports and integrated air sensor.
Page 24 2 The ÄKTA avant instrument 2.1 Overview illustrations Module Description Sample inlet valve V9-IS or Inlet valve for sample solution, with eight inlet V9H-IS ports (seven sample inlets and one buffer inlet) and integrated air sensor. V9-IS For further information, refer to Section 3.4 Inlet valves, on page 64.
Page 25 2 The ÄKTA avant instrument 2.1 Overview illustrations Module Description pH valve V9-pH or V9H-pH Valve which enables the pH electrode to be includ- ed in the flow path or by-passed during a run. The pH electrode may be calibrated when installed in the pH Valve.
Page 26 2 The ÄKTA avant instrument 2.1 Overview illustrations Module Description Conductivity monitor C9 Monitor which continuously measures the conduc- tivity of buffers and sample solutions. For further information, refer to Section 3.12 Con- ductivity monitor, on page 98. Built-in fraction collector Built-in fraction collector.
Page 27 2 The ÄKTA avant instrument 2.1 Overview illustrations Module Description Inlet valve X1 and Inlet Inlet valve with eight inlet ports. No integrated air sensor. valve X2 V9-IX or V9H-IX For further information, refer to Section 4.3 Extra inlet valves, on page 110. V9-IX Second Sample inlet valve Second inlet valve for Sample pump to extend the...
Page 28 2 The ÄKTA avant instrument 2.1 Overview illustrations Module Description Second Column valve Valve which connects five additional columns to the in- V9-C2 or V9H-C2 strument, extending the number of columns up to 10. The valve allows the user to choose flow direction through the column, or to by-pass the column.
Page 29 2 The ÄKTA avant instrument 2.1 Overview illustrations Module Description Second UV monitor U9-L Monitor which measures the UV absorbance at a fixed wavelength of 280 nm. For further information, refer to Section 4.11 Second UV monitor, on page 139. Second Conductivity Monitor which measures the conductivity of buffers and monitor C9...
Page 30: Runs In A Cold Environment
2 The ÄKTA avant instrument 2.2 Runs in a cold environment Runs in a cold environment Introduction The instrument can be placed and run in a cold cabinet or room. When running the in- strument in a cold environment make sure to take the precautions listed in this chapter. Precautions concerning runs in a cold cabinet NOTICE...
Page 31: Removing The Foldable Door And Pump Cover
2 The ÄKTA avant instrument 2.3 Removing the foldable door and pump cover Removing the foldable door and pump cover Introduction The foldable door and pump cover can be removed from the instrument, for example to fit the ÄKTA avant instrument in a cold cabinet. When using the instrument in a cold room or cold cabinet, make sure to follow the precautions listed below.
Page 32 2 The ÄKTA avant instrument 2.3 Removing the foldable door and pump cover Step Action Lift off the tray. Unscrew the two countersinked screws marked in the illustration. Pull out the two shafts. ÄKTA avant User Manual 29035184 AE...
Page 33 2 The ÄKTA avant instrument 2.3 Removing the foldable door and pump cover Step Action Lift off the pump cover. Unscrew the three screws holding the hinge to the pump cover and remove the hinge. Screw the three screws back to the pump cover and put the pump cover in a safe place.
Page 34 2 The ÄKTA avant instrument 2.3 Removing the foldable door and pump cover Step Action Put the inner shaft back through the hinge and damper, and push the hinge into place. Put the outer shaft back into place, and screw the two countersinked screws back.
Page 35: Liquid Flow Path
2 The ÄKTA avant instrument 2.4 Liquid flow path Liquid flow path Illustration of the flow path The following illustration shows an overview of the standard flow path. Part Description Pressure monitor Sample pump Sample inlet valve Quaternary valve Inlet valve A ÄKTA avant User Manual 29035184 AE...
Page 36 2 The ÄKTA avant instrument 2.4 Liquid flow path Part Description Inlet valve B System pump B System pump A Pressure monitor Mixer Injection valve Sample loop or Superloop™ Column valve Column UV monitor Conductivity monitor Flow restrictor pH valve with pH monitor Outlet valve Fraction collector ÄKTA avant User Manual 29035184 AE...
Page 37: Instrument Display
2 The ÄKTA avant instrument 2.5 Instrument display Instrument display Introduction The Instrument display is located on the front of the ÄKTA avant instrument. The Instru- ment display shows the current state of the system. The Instrument display can also be used to view detailed information about an ongoing method run and to view network settings.
Page 38 2 The ÄKTA avant instrument 2.5 Instrument display Part Description Settings button. Tap to display the Settings window with system parame- ters. Details button. Tap to display the Details window with parameters and data for the ongoing run. State button. Tap to display the State window with the current system state, both in text and color.
Page 39 2 The ÄKTA avant instrument 2.5 Instrument display State Display Description Ready The instrument is ready to use. A run is ongoing. Idle is displayed when the built-in frac- tion collector is not in use. Deselected is displayed when the built- in fraction collector is not selected in the instrument configuration.
Page 40 2 The ÄKTA avant instrument 2.5 Instrument display State Display Description Hold A method is put on hold by UNICORN, or by the user. Alarms and er- The system is paused due to an alarm. rors To resume the run, acknowledge the alarm and continue the run in UNICORN.
Page 41 2 The ÄKTA avant instrument 2.5 Instrument display Settings window Tap Settings to display the Settings window. The System parameters tab shows the system parameters, for example network settings. This information can be useful in contact with service or local IT support. Default values are set on delivery.
Page 42 2 The ÄKTA avant instrument 2.5 Instrument display Lock/Unlock function Follow the instruction to lock or unlock the Pause and Continue buttons of the Instrument display from UNICORN. Step Action In System Control, on the System menu, click Settings. Result: The System Settings dialog box box opens. In the System Settings dialog box: Select Advanced:Instrument display.
Page 43: Accessories
2 The ÄKTA avant instrument 2.6 Accessories Accessories About this section A number of holders are available for attaching columns, bottles and tubing to the ÄKTA avant instrument. The holders are attached to the instrument using the holder rails on the front and wet side of the instrument.
Page 44: Column Holders
2 The ÄKTA avant instrument 2.6 Accessories 2.6.1 Column holders 2.6.1 Column holders Column holder The Column holder has one position for medium sized columns and one position for small sized columns. It can be used for columns with outer diameter between 10 and 50 mm.The Column holder can also be used for bottles.
Page 45 2 The ÄKTA avant instrument 2.6 Accessories 2.6.1 Column holders Column clamp The column clamp can be used to attach small sized columns with outer diameters be- tween 10 and 21 mm. Use two clamps to attach long columns. Part Description Position for a column Inner end tabs...
Page 46 2 The ÄKTA avant instrument 2.6 Accessories 2.6.1 Column holders Column holder rod The Column holder rod can be used to attach several HiTrap™ columns. The holder has threaded ports for HiTrap columns and tubing connectors. Push the button of the holder to attach the holder to a holder rail.
Page 47 2 The ÄKTA avant instrument 2.6 Accessories 2.6.1 Column holders Flexible column holder The Flexible column holder can be used to attach, for example, HiScreen columns. Part Function Lower tubing Lower part Snap-in-strips Attachment part Upper tubing Lever Upper part Upper connector Lower connector ÄKTA avant User Manual 29035184 AE...
Page 48: Tubing Holders And Other Accessories
The Tubing holder spool is used to hold and arrange tubing. It is available in two versions; one for small tubing (o.d. 1/8” and smaller) and one for large inlet tubing (o.d. 3/16”) for AKTA avant 150. The following illustration shows the Tubing holder spool for small tubing.
Page 49 2 The ÄKTA avant instrument 2.6 Accessories 2.6.2 Tubing holders and other accessories Part Description Positions for tubing Snap-in to holder rails Bottle holder The Bottle holder is used for holding bottles. For example, the Bottle holder can be at- tached to the holder rails below the Instrument display to hold a sample bottle.
Page 50 2 The ÄKTA avant instrument 2.6 Accessories 2.6.2 Tubing holders and other accessories Part Description Bottle holder Air sensor Adapter for air sensor ÄKTA avant User Manual 29035184 AE...
Page 51 2 The ÄKTA avant instrument 2.6 Accessories 2.6.2 Tubing holders and other accessories Rail extension The Rail extension rod can be used to attach accessories, for example column holders or a Multi-purpose holder. The rod has extra rails on both sides. Push the button of the rod to attach it to a holder rail.
Page 52 2 The ÄKTA avant instrument 2.6 Accessories 2.6.2 Tubing holders and other accessories Part Function Attachment point for accessories Snap-in to holder rails Attachment points for tubing holders Loop holder The Loop holder can be used to attach up to five 10 ml sample loops. Use two Multi- purpose holders to attach the holder to a holder rail.
Page 53: Extension Box
2 The ÄKTA avant instrument 2.6 Accessories 2.6.3 Extension box 2.6.3 Extension box Description The Extension box can be used to install extra modules on the ÄKTA avant instrument outside the system chassis. It is possible to install up to three extension boxes with extra modules when using ÄKTA avant.
Page 54 2 The ÄKTA avant instrument 2.6 Accessories 2.6.3 Extension box Part Function Feet Back screw Back Hanger Compatible instrument modules The following instrument modules can be placed in the Extension box: • Any V9 or V9H valve • Mixer (M9) •...
Page 55: Standard Instrument Modules
3 Standard instrument modules Standard instrument modules About this section This section describes the design and main functions of the instrument modules installed in ÄKTA avant at delivery. In this section This section contains the following subsections: Section See page 3.1 Pumps 3.2 Mixer 3.3 Valves, overview...
Page 56: Pumps
3 Standard instrument modules 3.1 Pumps Pumps Introduction The ÄKTA avant instrument is fitted with three high precision pumps. There are two system pumps, System pump A and System pump B, and one Sample pump. The system pumps can be used individually, or in combination to generate isocratic or gradient elution in purification methods.
Page 57 3 Standard instrument modules 3.1 Pumps Configuration Label Pump Flow rate Max. type pressure ÄKTA avant 150 P9H A and P9H B 0.01 to 150 5 MPa ml/min Note: When running the Column packing flow instruction, the maximum flow rate is 300 ml/min.
Page 58 3 Standard instrument modules 3.1 Pumps Part Description Connections to pump piston rinsing system: Tubing is connected between the pumps and the Pump piston rinsing system tubes (9) Check valve: Non-return check valves at the inlet port Pump head: Encapsulates the inner parts of the pump System pump B System pump A Sample pump...
Page 59 3 Standard instrument modules 3.1 Pumps Illustration of the pump piston rinsing systems Part Description Inlet tubing to the sample pump piston rinsing system Outlet tubing from the sample pump piston rinsing system Inlet tubing to the system pump piston rinsing system Outlet tubing from the system pump piston rinsing system ÄKTA avant User Manual 29035184 AE...
Page 60: Mixer
3 Standard instrument modules 3.2 Mixer Mixer Introduction The Mixer is located after System pump A and System pump B and before the Injection valve. The purpose of the Mixer is to make sure that the buffers from the System pumps are mixed to give a homogenous buffer composition.
Page 61 3 Standard instrument modules 3.2 Mixer Part Description Outlet Mixer chamber Inlet ÄKTA avant User Manual 29035184 AE...
Page 62: Valves, Overview
3 Standard instrument modules 3.3 Valves, overview Valves, overview Introduction The valves of the ÄKTA avant system allow flexibility in the liquid flow path. This section provides an overview of the valves included in a standard equipped ÄKTA avant system. General design and function of rotary valves All valves on the ÄKTA avant instrument, except for the Quaternary valve, are rotary...
Page 63 3 Standard instrument modules 3.3 Valves, overview Illustration of inlet valve components The following illustration shows the components of a disassembled Inlet valve A or a disassembled Inlet valve B. Part Description Valve connection block Rotary disc Defined channel(s) in the rotary disc Defined bores in the valve connection block Note: Inlet and outlet ports are not visible in the picture.
Page 64: Inlet Valves
3 Standard instrument modules 3.4 Inlet valves Inlet valves Introduction The inlet valves are used to select which buffers or samples to use in a run. Inlet valve A is located before System pump A, Inlet valve B is located before System pump B, and the Sample inlet valve is located before the Sample pump.
Page 65 3 Standard instrument modules 3.4 Inlet valves Part Description Label in ÄKTA avant 25 ÄKTA avant 150 Quaternary valve Inlet valve B V9-IB V9H-IB Integrated air sensor (behind the plug) Inlet ports of Inlet valve A A1 to A7 A1 to A7 Inlet ports Q1-Q4 of Quaternary Q1 to Q4 Q1 to Q4...
Page 66 3 Standard instrument modules 3.4 Inlet valves Parameter Air volume detected Usage ÄKTA avant 25 ÄKTA avant 150 High 10 μl 30 μl Detect even small air bubbles Ports of Inlet valve A and Inlet valve B The following illustration shows the ports of Inlet valve A and Inlet valve B. Port Description Port...
Page 67 3 Standard instrument modules 3.4 Inlet valves Current mixing percentage for Q1-Q4 are shown in Process Picture in System Control. The curves for Q1-Q4 can be shown in a chromatogram and stored in the result data for documentation and for further studies in Evaluation. Ports of Quaternary valve The following illustration shows the ports of the Quaternary valve.
Page 68 3 Standard instrument modules 3.4 Inlet valves Quaternary gradients The Quaternary valve can be used to create a gradient using four different solutions si- multaneously in any combination. The percentage of each solution is controlled by in- structions in the method. It is possible to form gradients that changes the percentage of two, three or four solutions linearly over time.
Page 69 3 Standard instrument modules 3.4 Inlet valves Ports of Sample inlet valve The following illustration shows the ports of the Sample inlet valve. Port Description S1-S7 Sample inlets Buff Buffer inlet To Sample pump ÄKTA avant User Manual 29035184 AE...
Page 70: Injection Valve
3 Standard instrument modules 3.5 Injection valve Injection valve Function of the Injection valve The Injection valve is used to direct sample onto the column. The valve enables usage of a number of different sample application techniques. The Injection valve is labeled V9-Inj for ÄKTA avant 25 and V9H-Inj for ÄKTA avant 150. A sample loop or a Superloop™...
Page 71 3 Standard instrument modules 3.5 Injection valve Ports and flow paths of the Injection valve The illustration and tables below describe the ports of and different flow paths through the Injection valve. The Injection valve can be set to different positions that give different flow paths through the valve.
Page 72 3 Standard instrument modules 3.5 Injection valve Port Description LoopE Port for connection of a loop or a Loop valve. Used to empty a loop into the flow path. Loop and System pump waste Sample flow waste For larger sample volumes connect the syringe with the help of a union Luer female to 1/16″ male (default).
Page 73: Column Valve
3 Standard instrument modules 3.6 Column valve Column valve Introduction The Column valve is used for connection of columns to the system, and to direct the flow onto the column. Up to five columns can be connected to the Column valve simultane- ously.
Page 74 3 Standard instrument modules 3.6 Column valve Part Function HiPrep™ column connected to ports 1A and 1B Ports and flow paths of the Column valve The following illustration and tables describe the ports of and flow paths through Column valves V9-C and V9H-C. By-pass Port Description...
Page 75 3 Standard instrument modules 3.6 Column valve Flow Description path The flow by-passes the column(s). By-pass is the default flow path. pass Down The flow direction is from the top of the column to the bottom of the col- flow umn.
Page 76: Outlet Valve
3 Standard instrument modules 3.7 Outlet valve Outlet valve Function of the Outlet valve The Outlet valve is used to direct the flow to the fraction collector, to an outlet port, or to waste. The Outlet valve enables collection of fractions. Fractions can be collected by using a fraction collector connected to the Outlet valve, or by using the 10 outlet ports.
Page 77 3 Standard instrument modules 3.7 Outlet valve Ports of the Outlet valve The following illustration shows the ports of the Outlet valve. Port Description Inlet port Frac Port to fraction collector Out1 - Out10 Outlet ports 1 - 10 Waste port ÄKTA avant User Manual 29035184 AE...
Page 78: Pressure Monitors
3 Standard instrument modules 3.8 Pressure monitors Pressure monitors Introduction Four pressure monitors are included in the ÄKTA avant system. Two pressure monitors are integrated in the Column valve, and two pressure monitors are connected to the pumps. This section describes the location and function of the pressure monitors. Location and illustration of the pressure monitors The following illustration shows the location of the pressure monitors on the instrument,...
Page 79 3 Standard instrument modules 3.8 Pressure monitors Function of system and sample pump pressure monitors The pressure monitor of the System pumps measures the system pressure after these pumps, called System pressure in UNICORN. The pressure monitor of the Sample pump measures the system pressure after this pump, called Sample pressure in UNICORN.
Page 80: Ph Valve And Ph Monitor
3 Standard instrument modules 3.9 pH valve and pH monitor pH valve and pH monitor Function of the pH valve The pH valve is used to direct the flow to a pH electrode installed in the integrated flow cell when inline monitoring of pH is desired during a run. The pH valve is labeled V9-pH for ÄKTA avant 25 and V9H-pH for ÄKTA avant 150.
Page 81 3 Standard instrument modules 3.9 pH valve and pH monitor Ports and flow paths of the pH valve The illustration and table below describe the different ports of and flow paths through the pH valve, in this example labeled V9-pH. By-pass Primary flow path Flow path for calibrations...
Page 82 3 Standard instrument modules 3.9 pH valve and pH monitor Flow path Description Restrictor Both pH electrode and Flow restrictor are in use. and pH pH electrode is in use and Flow restrictor is by-passed. Calibration Flow path used when calibrating the pH monitor and when filling the pH flow cell with storage solution.
Page 83: 3.10 Built-In Fraction Collector
3 Standard instrument modules 3.10 Built-in fraction collector 3.10 Built-in fraction collector About this section This section describes the design and function of the built-in fraction collector. In this section This section contains the following sub sections: Section See page 3.10.1 Function 3.10.2 Illustrations of the built-in fraction collector 3.10.3 Cassettes, cassette tray and racks...
Page 84: Function
3 Standard instrument modules 3.10 Built-in fraction collector 3.10.1 Function 3.10.1 Function Introduction ÄKTA avant has a built-in fraction collector for safe and clean handling of fractions. A cooling function protects the fractions from heat degradation. Fractions can be collected in deep well plates and in tubes or bottles of different sizes. Up to six cassettes for deep well plates and tubes can be used.
Page 85 3 Standard instrument modules 3.10 Built-in fraction collector 3.10.1 Function Scanning of cassettes When the fraction collector drawer is closed automatic scanning is performed. There are two types of scanning procedures: • Full scan: Scanning of cassette type codes to identify which cassette types are used, and scanning of rows and columns in deep well plates to identify which plate types are used (24, 48, or 96 wells).
Page 86: Illustrations Of The Built-In Fraction Collector
3 Standard instrument modules 3.10 Built-in fraction collector 3.10.2 Illustrations of the built-in fraction collector 3.10.2 Illustrations of the built-in fraction collector Illustration of the built-in fraction collector exterior The illustration below shows the main parts of the built-in fraction collector. A cassette tray with cassettes is placed in the fraction collector drawer.
Page 87 3 Standard instrument modules 3.10 Built-in fraction collector 3.10.2 Illustrations of the built-in fraction collector Illustration of the built-in fraction collector interior The illustration below shows the main parts of the fractionation chamber inside the fraction collector. Part Description Fractionation arm main rail Button cover Fractionation arm Dispenser head...
Page 88 3 Standard instrument modules 3.10 Built-in fraction collector 3.10.2 Illustrations of the built-in fraction collector Illustration of the dispenser head The illustration below shows the dispenser head of the fraction collector. Part Description Dispenser head Dispenser head cover Nozzle Accumulator (back part of Dispenser head) DropSync sensor (only in ÄKTA avant 25) Type code reader ÄKTA avant User Manual 29035184 AE...
Page 89: Cassettes, Cassette Tray And Racks
The illustrations below show the Cassette tray (for six cassettes), the Rack for 50 ml tubes and the Rack for 250 ml bottles. The fronts of the tray and the racks are marked with the GE monogram. In the cassette tray, the cassette positions are marked 1 to 6.
Page 90 Rack for 50 ml tubes Rack for 250 ml bottles Note: The tray and racks are inserted into the fraction collector with the GE mono- gram facing outwards. Note: Do not use the cassette tray when a rack for tubes or bottles is placed in the fraction collector.
Page 91 3 Standard instrument modules 3.10 Built-in fraction collector 3.10.3 Cassettes, cassette tray and racks QuickRelease function The cassettes for the smaller tube sizes (3, 5, 8, and 15 ml) have a built-in QuickRelease function. The QuickRelease function enables easy handling of tubes in the cassettes. With the QuickRelease device in lock position the tubes are fastened in the cassette and can easily be emptied.
Page 92 3 Standard instrument modules 3.10 Built-in fraction collector 3.10.3 Cassettes, cassette tray and racks Step Action Empty and discard the remaining tubes: Press the QuickRelease device to the lock position, and empty the remain- • ing tubes. Pull the QuickRelease device to the release position, and discard the •...
Page 93 3 Standard instrument modules 3.10 Built-in fraction collector 3.10.3 Cassettes, cassette tray and racks Fraction collector tubes and bottles The tubes and bottles used in the built-in fraction collector must fulfill the requirements listed in the following table. Examples of manufacturers are also listed in the table. Tube or bottle Diameter (mm) Height (mm)
Page 94 Shape of wells Square, not cylindrical Well volume 10, 5, or 2 ml Approved deep well plates The plates listed in the table below are tested and approved by GE to be used with ÄKTA avant. Plate type Manufacturer Part no.
Page 95 3 Standard instrument modules 3.10 Built-in fraction collector 3.10.3 Cassettes, cassette tray and racks Maximum flow rate Fraction collection can be performed at different maximum flow rates depending on what type of deep well plates that are used. The table below lists the maximum flow rates for the different plate types.
Page 96: 3.11 Uv Monitor
3 Standard instrument modules 3.11 UV monitor 3.11 UV monitor Introduction This section describes the design and function of UV monitor U9-M. The module includes a monitor unit and a detector with a UV flow cell. Function of the UV monitor UV monitor U9-M measures the UV absorbance at a wavelength range of 190 to 700 The UV monitor is automatically calibrated every time the instrument is switched on.
Page 97 3 Standard instrument modules 3.11 UV monitor Part Description Part Description Inlet UV detector UV flow cell Outlet UV flow cells NOTICE UV and conductivity flow cells on the high pressure side. When placing UV and/or conductivity flow cells on the high pressure side of the column, the UV flow cell has a maximum pressure limit of 2 MPa (20 bar) and the conductivity flow cell has a maximum pressure limit of 5 MPa (50 bar).
Page 98: 3.12 Conductivity Monitor
3 Standard instrument modules 3.12 Conductivity monitor 3.12 Conductivity monitor Function of the Conductivity monitor The Conductivity monitor continuously measures the conductivity of buffers and eluted proteins. The Conductivity flow cell has two electrodes positioned in the flow path of the cell. An alternating voltage is applied between the electrodes and the resulting current is mea- sured and used to calculate the conductivity of the eluent.
Page 99 3 Standard instrument modules 3.12 Conductivity monitor Location and illustration of Conductivity monitor The following illustration shows the location of the Conductivity monitor and also an enlargement of the monitor. In both ÄKTA avant 25 and ÄKTA avant 150 the monitor is labelled C9.
Page 100: Optional Instrument Modules
4 Optional instrument modules Optional instrument modules About this chapter This chapter contains detailed instructions on the optional instrument modules that can be connected to the ÄKTA avant instrument. A brief description of how to install the modules is also provided. In this chapter This chapter contains the following sections Section...
Page 101: System Extension Overview
4 Optional instrument modules 4.1 System extension overview System extension overview Introduction The ÄKTA avant instrument can easily be extended with additional valves, detectors and a second fraction collector. There are a large number of different hardware modules to choose from in order to customize the number of columns, inlets, outlets, detectors and ways to apply and collect samples.
Page 102: Installation Of Optional Modules
4 Optional instrument modules 4.2 Installation of optional modules Installation of optional modules Introduction Up to six extra modules can be installed in the system: three modules can be installed on the system wet side using the free module positions (module panels are installed in these positions at delivery), and three modules can be connected to the back of the in- strument.
Page 103 4 Optional instrument modules 4.2 Installation of optional modules Install a module in the instrument The instruction below describes how to install the module hardware in the instrument. Note: The illustrations show the principle how to install an optional module. The position of the module on the instrument and the used type of module will depend on the module being installed.
Page 104 4 Optional instrument modules 4.2 Installation of optional modules Step Action Remove the module. Disconnect the cable and secure it in the slit. Connect the cable to the module to be installed. ÄKTA avant User Manual 29035184 AE...
Page 105 4 Optional instrument modules 4.2 Installation of optional modules Step Action Insert the module. Fasten it with a Torx T20 screwdriver. Note: A warning message is displayed at start up if a module has been installed in the instrument but not added to the System Properties dialog box in UNICORN. ÄKTA avant connector plate Modules not installed in the instrument cabinet are connected via a UniNet-9 cable at the back of the system.
Page 106 4 Optional instrument modules 4.2 Installation of optional modules Part Function UniNet-9 connectors (one connector is occupied by the Conductivity monitor) Network connector (Ethernet) Test point for service. Power input connector NOTICE Do not connect any module to the connector Test on the ÄKTA avant instrument.
Page 107 4 Optional instrument modules 4.2 Installation of optional modules Constraints on optional modules The table below indicates usage constraints for the different external modules. External module Constraints I/O-box E9 I/O-box E9 has no constraints. I/O-box E9, 2nd I/O-box E9, 2nd requires I/O-box E9. Fraction collector 2 Fraction collector 2 requires Fraction collector and can not be installed at the same time as...
Page 108 4 Optional instrument modules 4.2 Installation of optional modules Step Action In the Administration module. On the Tools menu, click System Proper- • ties or click the System Properties button to open the dialog box. Result: The System Properties dialog box is displayed. Select the system of interest in the System Properties dialog box.
Page 109 4 Optional instrument modules 4.2 Installation of optional modules Edit system settings It may be necessary to edit the System Settings when the configuration of the system is changed. For example, if the change in configuration affects the delay volume following the UV monitor (or other monitor connected via the I/O-box) the appropriate system settings for Delay volumes have to be updated.
Page 110: Extra Inlet Valves
4 Optional instrument modules 4.3 Extra inlet valves Extra inlet valves Introduction In the ÄKTA avant standard configuration, 7 inlets are available for each inlet valve. To increase the number of inlets, a second inlet valve can be installed that increases the number of inlets to 14 for one of the valves.
Page 111 4 Optional instrument modules 4.3 Extra inlet valves Flow paths in ÄKTA avant Second Sample inlet valve The illustration below shows an optional flow path when a second Sample inlet valve is installed. The flow is directed from the Out port of the second Sample inlet valve (V9-S2 or V9H-S2) to the buffer port BUFF of the Sample inlet valve (V9-IS or V9H-IS) and leaves the valve through the Out port to the Sample pump.
Page 112 4 Optional instrument modules 4.3 Extra inlet valves Connect tubing • Connect tubing from port 7 of one of the standard inlet valves (Inlet valve A or Inlet valve B) to port Out of the second Inlet valve.. and/or • Connect tubing from port BUFF of the standard Sample inlet valve to port Out of the second Sample inlet valve.
Page 113 4 Optional instrument modules 4.3 Extra inlet valves The Inlet valves X1 and X2 can for example be used to increase the number of inlets to the Quaternary valve. For ÄKTA avant 25, the recommended tubing is FEP, o.d. 1/8", i.d. 1.6 mm with Tubing connector, 5/16"...
Page 114: Second Column Valve
4 Optional instrument modules 4.4 Second Column valve Second Column valve Introduction When using the ÄKTA avant standard configuration with one column valve, 5 column positions are available. To increase the number of column positions to 10, an additional column valve can be installed in the instrument. One application is for evaluation of a number of different columns during method optimization.
Page 115 4 Optional instrument modules 4.4 Second Column valve Flow path The second Column valve (V9-C2 or V9H-C2) is connected after the standard Column valve (V9-C or V9H-C). Port Out of the standard Column valve is connected with port In of the second Column valve. Ten columns can now be selected in UNICORN. Note: The standard Column valve (V9-C or V9H-C) must be connected before the second Column valve (V9-C2 or V9H-C2).
Page 116 4 Optional instrument modules 4.4 Second Column valve Connection Tubing Tubing Connector Tubing between... label length (mm) Injection valve and For ÄKTA avant For ÄKTA avant Column valve 25: PEEK, o.d. 25: Fingertight 1/16", i.d. 0.50 mm connector, 1/16" standard Column For ÄKTA avant For ÄKTA avant valve and second...
Page 117: Extra Outlet Valves
4 Optional instrument modules 4.5 Extra Outlet valves Extra Outlet valves Introduction When using the standard configuration with one Outlet valve, 10 outlet positions are available. To increase the number of outlets, one or two extra Outlet valves can be con- nected, adding up to a total of 21 or 32 outlet positions.
Page 118 4 Optional instrument modules 4.5 Extra Outlet valves V9-pH Note: The second or third Outlet valve can not be installed together with the second Fraction collector F9-R. Connect tubing The table below shows recommended connectors and tubing. For an illustration of standard tubing labels see Tubing labels, on page 432.
Page 119 4 Optional instrument modules 4.5 Extra Outlet valves System properties Follow the instruction to update the system properties. Step Action Open the system properties Edit dialog box. In the Component types list, click Valves and pumps. In the Component selection list, select the check boxes corresponding to the installed modules.
Page 120: External Air Sensors
4 Optional instrument modules 4.6 External air sensors External air sensors Introduction Two external air sensors are available as options. The difference is their size and where they can be installed. ÄKTA avant supports installation of one external air sensor at a time.
Page 121 4 Optional instrument modules 4.6 External air sensors Location and illustration The following illustration shows the recommended positions for the external air sensors. Part Function L9-1.5 external air sensor before Sample inlet valve L9-1.2 external air sensor after Injection valve Note: Only one external air sensor can be installed at a time.
Page 122 4 Optional instrument modules 4.6 External air sensors L9-1.2 Connection Tubing Tubing Connector Tubing between... label length (mm) V9-Inj or V9H-Inj For ÄKTA avant For ÄKTA avant and L9-1.2 25: PEEK, o.d. 25: Fingertight 1/16", i.d. 0.50 mm connector, 1/16" For ÄKTA avant For ÄKTA avant 150 : PEEK, o.d.
Page 123 4 Optional instrument modules 4.6 External air sensors System settings The sensitivity of the external air sensor can be set. Parameter Air volume detected Usage ÄKTA avant 25 ÄKTA avant 150 Normal (default) 30 μl 100 μl Detect empty buffer/sample ves- sels High 10 μl...
Page 124: Loop Valve
4 Optional instrument modules 4.7 Loop valve Loop valve Function of the Loop valve The Loop valve allows the user to connect several loops simultaneously to the instrument. It can for example be used for storing intermediate fractions in multi-step purifications, for storing samples to be used in scouting runs, or for storing eluents needed in low volumes.
Page 125 4 Optional instrument modules 4.7 Loop valve Ports and flow paths of the Loop valve The illustration and tables below describe the ports and different flow paths through the Loop valve. In the Position 4 example, the loop is connected to loop position 4 and the loop is being emptied.
Page 126 4 Optional instrument modules 4.7 Loop valve Connect tubing The table below shows recommended tubing and connectors. For an illustration of tubing labels see Tubing labels, on page 432. Tub- Connection Tubing Connector Tubing length ÄKTA avant ÄKTA avant label (mm) Injection valve posi- PEEK,...
Page 127 4 Optional instrument modules 4.7 Loop valve System properties Follow the instruction to update the system properties. Step Action Open the system properties Edit dialog box. In the Component types list, click Valves and pumps Select the Loop valve (V9-L) or Loop valve (V9H-L) check box in the Compo- nent selection list.
Page 128: Versatile Valve
4 Optional instrument modules 4.8 Versatile valve Versatile valve Function of the Versatile valve The Versatile valve is a 4-port, 4-position valve, which can be used to add extra features to the flow path. For example, the valve can be used to connect external equipment to the flow path during parts of a run.
Page 129 4 Optional instrument modules 4.8 Versatile valve Flow Description path Two simultaneously used flow channels where the flow is directed between and 2- port 1 and port 4 and between port 2 and port 3. Two simultaneously used flow channels where the flow is directed between and 3- port 1 and port 2 and between port 3 and port 4.
Page 130: Fraction Collector F9-R
4 Optional instrument modules 4.9 Fraction collector F9-R Fraction collector F9-R Introduction The external Fraction collector F9-R is installed as the second fraction collector in the ÄKTA avant system. It is used to expand the fractionation capabilities of the ÄKTA avant or used to fractionate volatile solutions for example in reversed phase separations.
Page 131 4 Optional instrument modules 4.9 Fraction collector F9-R Front view illustration The illustration below shows the main parts of the Fraction collector. Part Function Lock knob Stationary part of delivery arm Delivery arm Tubing connector Tube sensor Collection tubes Tube rack Base unit ÄKTA avant User Manual 29035184 AE...
Page 132 4 Optional instrument modules 4.9 Fraction collector F9-R Connector panel illustration The illustration below shows the main parts of the connector panel on the fraction col- lector. Part Function Node ID switch UniNet-9 F-type connector (for communication and power supply) Available tubes For Fraction collector F9-R the fractions are collected in tubes of different sizes.
Page 133 4 Optional instrument modules 4.9 Fraction collector F9-R Illustration of the Fraction collector F9-R tube rack Each tube rack is made up of a combination of a Bowl, Tube support, Tube guide and Tube holder. For more information on the assembly of the tube rack, see Assembly in- structions, on page 212.
Page 134 4 Optional instrument modules 4.9 Fraction collector F9-R Standard tubing dimensions The table below shows recommended standard tubing dimensions to connect Fraction collector F9-R. System Tubing length Tubing diameter (mm) (mm) ÄKTA avant 25 0.50 ÄKTA avant 150 1.00 Note: Cut the tubing according to the table above from the spare tubing delivered with the ÄKTA avant instrument.
Page 135 4 Optional instrument modules 4.9 Fraction collector F9-R Step Action Re-install the tubing holder in the delivery arm. Connect the tubing from the fraction collector to port Out 10 on the Outlet valve. Adjust the delay volume setting in UNICORN to the volume of the tubing, see Set the delay volume in UNICORN, on page 506 for more details.
Page 136 4 Optional instrument modules 4.9 Fraction collector F9-R System settings If non-standard tubing is used between the outlet valve and the fraction collector, the delay volume must be set. See Check or set delay volumes, on page 153. Fraction settings and numbering mode can also be set.
Page 137: 4.10 Second Conductivity Monitor
4 Optional instrument modules 4.10 Second Conductivity monitor 4.10 Second Conductivity monitor Introduction It is possible to use two conductivity monitors. The second Conductivity monitor could for example be placed before the column for extended gradient control or on a sample inlet line for control of ionic strength in the sample.
Page 138 4 Optional instrument modules 4.10 Second Conductivity monitor System settings If the monitor is placed in the flow path between the UV monitor and the outlet valve, the delay volume must be set. See Check or set delay volumes, on page 153. ÄKTA avant User Manual 29035184 AE...
Page 139: 4.11 Second Uv Monitor
4 Optional instrument modules 4.11 Second UV monitor 4.11 Second UV monitor Introduction It is possible to use up to two UV monitors simultaneously, a UV monitor U9-L can be installed alongside the standard UV monitor U9-M. The second UV monitor could for example be used to enable the user to cover a larger dynamic range in UV absorbance measurements by using different cell lengths.
Page 140 4 Optional instrument modules 4.11 Second UV monitor Illustration of UV monitor U9-L The following illustration show a detailed view of the monitor and detector. Part Description Inlet UV monitor U9-L UV flow cell. Two different path lengths are available: 2 mm (default) and 5 mm Outlet UV detector...
Page 141 4 Optional instrument modules 4.11 Second UV monitor NOTICE UV and conductivity flow cells on the high pressure side. When placing UV and/or conductivity flow cells on the high pressure side of the column, the UV flow cell has a maximum pressure limit of 2 MPa (20 bar) and the conductivity flow cell has a maximum pressure limit of 5 MPa (50 bar).
Page 142 4 Optional instrument modules 4.11 Second UV monitor System settings If the UV monitor U9-L is placed between the UV monitor U9-M and the outlet valve , or if the UV monitor U9-L is the peak fractionation controlling UV monitor the delay volume has to be updated.
Page 143: 4.12 I/O-Box
4 Optional instrument modules 4.12 I/O-box 4.12 I/O-box About this section This section provides an overview of the function, connectors and signals of the I/O-box E9. In this section This section contains the following subsections: Section See page 4.12.1 Overview of the I/O-box 4.12.2 Analog connector and signals 4.12.3 Digital connector and signals 4.12.4 Connect external equipment to the I/O-box...
Page 144: Overview Of The I/O-Box
4 Optional instrument modules 4.12 I/O-box 4.12.1 Overview of the I/O-box 4.12.1 Overview of the I/O-box Function of the I/O-box The I/O-box E9 is used to interface other equipment in order to measure parameters such as refractive index, light scattering and fluorescence. See Requirements on connected equipment, on page 150 for information on requirements of the equipment that can be connected to ÄKTA avant.
Page 145 4 Optional instrument modules 4.12 I/O-box 4.12.1 Overview of the I/O-box Connectors Part Description Analog in/out Signal connector for analog input and output signals. UniNet-9 Connector used to connect the I/O-box to the ÄKTA avant instrument. Status Status indicator for service purposes. Node ID Switches used to configure I/O-box E9 as I/O-box E9 or I/O-box E9, 2nd.
Page 146: Analog Connector And Signals
4 Optional instrument modules 4.12 I/O-box 4.12.2 Analog connector and signals 4.12.2 Analog connector and signals Analog connector pins Part Function Analog in signal 1 + Analog in signal 1 - (or signal ground) Shield, analog in (both ports) Analog in signal 2 + Analog in signal 2 - (or signal ground) Calibration pin for service purposes Analog out signal (1.9 V)
Page 147 4 Optional instrument modules 4.12 I/O-box 4.12.2 Analog connector and signals Analog input signals There are two analog input channels from which analog input signals can be used for peak detection, or data collection in UNICORN. It is possible to auto-zero the input signals, which means that the current value will be displayed as 0 V in UNICORN.
Page 148: Digital Connector And Signals
4 Optional instrument modules 4.12 I/O-box 4.12.3 Digital connector and signals 4.12.3 Digital connector and signals Digital connector pins Part Function Digital in signal 1 Digital in signal 2 Digital in signal 3 Digital in signal 4 Signal ground Digital out signal 1 Digital out signal 2 Digital out signal 3 Digital out signal 4...
Page 149 4 Optional instrument modules 4.12 I/O-box 4.12.3 Digital connector and signals Digital input signals The digital in-signal can be used to monitor external equipment by registering, for exam- ple, error signals or event marks. An event mark can be used as a trigger for watches. The measured digital signals can be shown as a curve in UNICORN.
Page 150: Connect External Equipment To The I/O-Box
4 Optional instrument modules 4.12 I/O-box 4.12.4 Connect external equipment to the I/O-box 4.12.4 Connect external equipment to the I/O-box Requirements on connected equipment The physical requirements for the connected equipment is described in the following tables. All connected equipment must have a common grounding. Analog input Parameter Value...
Page 151 4 Optional instrument modules 4.12 I/O-box 4.12.4 Connect external equipment to the I/O-box Required material The following material is required: • Flat-blade screwdriver, 2 mm • Shielded cable with 9 conductors, 4 to 8 mm diameter • Wire stripping tool Instruction Follow the instructions to connect one or two external cables to the supplied D-sub connectors.
Page 152 4 Optional instrument modules 4.12 I/O-box 4.12.4 Connect external equipment to the I/O-box System properties Follow the instruction to update the system properties. Step Action Open the system properties Edit dialog box. In the Component types list, click Other. Select the I/O-box (E9) or I/O-box 2 (E9) check box in the Component selec- tion list.
Page 153: 4.13 General System Settings
4 Optional instrument modules 4.13 General system settings 4.13 General system settings Check or set delay volumes When a module has been installed after the UV monitor in the flow path, the delay volume has to be adjusted in the System Setting dialog box in UNICORN, to make sure that the collected fractions correspond to the fractions indicated in the chromatogram.
Page 154 4 Optional instrument modules 4.13 General system settings Step Action In the System Settings dialog box: Select Advanced:Instrument display. • Click Locked or Unlocked. • Click OK. • ÄKTA avant User Manual 29035184 AE...
Page 155: Operation
5 Operation Operation About this chapter This chapter describes the steps involved when operating ÄKTA avant. In this chapter This chapter contains the following sections: Section See page 5.1 Before you prepare the system 5.2 Prepare the flow path 5.3 Start UNICORN and connect to the system 5.4 Prime inlets and purge pump heads 5.5 Connect a column 5.6 Set pressure alarms...
Page 156: Before You Prepare The System
5 Operation 5.1 Before you prepare the system Before you prepare the system Introduction It is important to prepare the standard system in accordance with the settings in the method to be run. Before preparing the system, check the settings in the Method Editor and make sure that all accessories to be used are available.
Page 157: Prepare The Flow Path
5 Operation 5.2 Prepare the flow path Prepare the flow path Introduction This section describes the preparation of inlet tubing, outlet tubing, waste tubing and mixer. For an illustration of the standard typical flow path, see Section 2.4 Liquid flow path, on page 35.
Page 158 5 Operation 5.2 Prepare the flow path ÄKTA avant 150 Three different Mixer chambers are delivered with ÄKTA avant 150. Their volumes are: 1.4 ml, 5 ml (mounted at delivery), and 15 ml. Mixer chamber Flow rate (ml/min), Flow rate (ml/min), volume (ml) Binary gradient Quaternary and BufferPro...
Page 159 5 Operation 5.2 Prepare the flow path The path length of the UV flow cell might differ from the nominal length, which leads to inexact results in the calculation of protein concentration in the eluate. To achieve nor- malized absorbance, the path length of the UV flow cell must be calibrated and the cal- culated flow cell path length set manually.
Page 160 5 Operation 5.2 Prepare the flow path Part Description Waste tubing from the Injection valve, the pH valve and the Outlet valve (W, W1, W2 and W3). Waste tubing from the Fraction collector and the Buffer tray. Instructions The following steps describe how to prepare the waste tubing. Step Action Place the four pieces of waste tubing from the Injection valve, the pH valve...
Page 161 5 Operation 5.2 Prepare the flow path Step Action Cut the waste tubing from the Fraction collector and the Buffer tray to ap- propriate length. It is important that the tubing is not bent and will not be submerged in liquid during the run. Note: If the tubing is too short, replace it with new tubing.
Page 162: Start Unicorn And Connect To The System
5 Operation 5.3 Start UNICORN and connect to the system Start UNICORN and connect to the system Introduction This section describes how to start and log on to UNICORN and how to connect the in- strument to UNICORN. Start UNICORN and log on Follow the instructions to start UNICORN and log on to the program.
Page 163 5 Operation 5.3 Start UNICORN and connect to the system Step Action In the Log On dialog box: Click a user name in the User Name list • enter the password in the Password field. • Note: It is also possible to select the Use Windows Authentication check box and enter a network ID in the User Name box.
Page 164 5 Operation 5.3 Start UNICORN and connect to the system Connect to system Follow the instructions to connect the instrument to UNICORN. Step Action In the System Control module, Click the Connect to Systems button, • click Connect to Systems on the System menu. •...
Page 165: Prime Inlets And Purge Pump Heads
5 Operation 5.4 Prime inlets and purge pump heads Prime inlets and purge pump heads About this section Before usage of a pump, it is important to: • Prime the inlets (fill the buffer inlets with liquid). • Purge the pump (remove air from the pump heads). This section describes how to prime inlets and purge the pump heads of the system pumps and the sample pump.
Page 166: Prime Buffer Inlets And Purge System Pumps
5 Operation 5.4 Prime inlets and purge pump heads 5.4.1 Prime buffer inlets and purge system pumps 5.4.1 Prime buffer inlets and purge system pumps Overview The procedure consists of the following stages: Stage Description Prime all inlet tubing to be used during the run. Validate priming of inlet tubing.
Page 167 5 Operation 5.4 Prime inlets and purge pump heads 5.4.1 Prime buffer inlets and purge system pumps Step Action In the Process Picture: Click the inlet valve icons. (Click both the Inlet A and Inlet B icons, one • at a time, if both inlets are to be primed.) Click the position of the inlet to be filled.
Page 168 5 Operation 5.4 Prime inlets and purge pump heads 5.4.1 Prime buffer inlets and purge system pumps Step Action Check that there is no air left in the pump by following the instructions in Validate prime or purge of System Pump A or B or Sample Pump, on page 172. If air bubbles are indicated, follow the instructions in Purge System Pump B, on page 168 Purge System Pump B...
Page 169 5 Operation 5.4 Prime inlets and purge pump heads 5.4.1 Prime buffer inlets and purge system pumps Step Action In the Process Picture: Click the Inlet valve B icon. • Click the position of one of the inlets that will be used at the beginning •...
Page 170 5 Operation 5.4 Prime inlets and purge pump heads 5.4.1 Prime buffer inlets and purge system pumps Step Action Connect a 25 to 30 ml syringe to the purge valve of the left pump head of System Pump B. Make sure that the syringe fits tightly into the purge con- nector.
Page 171 5 Operation 5.4 Prime inlets and purge pump heads 5.4.1 Prime buffer inlets and purge system pumps Purge System Pump A Purge both pump heads of System Pump A by following the same procedure as in Purge System Pump B, on page 168, but replace step 3 and 4 with the following: Step Action In the Process Picture:...
Page 172 5 Operation 5.4 Prime inlets and purge pump heads 5.4.1 Prime buffer inlets and purge system pumps Validate prime or purge of System Pump A or B or Sample Pump Follow these instructions to check that there is no air left in the pump after performing a prime or a purge.
Page 173: Prime Sample Inlets And Purge Sample Pump
5 Operation 5.4 Prime inlets and purge pump heads 5.4.2 Prime sample inlets and purge Sample Pump 5.4.2 Prime sample inlets and purge Sample Pump Overview The procedure consists of the following stages: Stage Description Prime all sample inlet tubing to be used during the run. Validate priming of inlet tubing.
Page 174 5 Operation 5.4 Prime inlets and purge pump heads 5.4.2 Prime sample inlets and purge Sample Pump Step Action In the Process Picture Click the Sample inlet valve icon. • Select the position of the inlet to be filled. Start at the inlet position with •...
Page 175 5 Operation 5.4 Prime inlets and purge pump heads 5.4.2 Prime sample inlets and purge Sample Pump Step Action Check that there is no air left in the pump by following the instructions in Validate prime or purge of System Pump A or B or Sample Pump, on page 172. If air bubbles are indicated, follow the instructions in Purge Sample Pump, on page 175.
Page 176 5 Operation 5.4 Prime inlets and purge pump heads 5.4.2 Prime sample inlets and purge Sample Pump Step Action In the Process Picture: Click the Sample inlet icon, then click Buffer. • Click the Sample pump icon: Set the Sample flow to 1.0 ml/min for ÄKTA •...
Page 177 5 Operation 5.4 Prime inlets and purge pump heads 5.4.2 Prime sample inlets and purge Sample Pump Step Action Connect the syringe to the right purge valve on the sample pump, and repeat step 6 to 8. Check that there is no air left in the pump by following the instructions in Validate prime or purge of System Pump A or B or Sample Pump, on page 172.
Page 178: Prime Q Inlets
5 Operation 5.4 Prime inlets and purge pump heads 5.4.3 Prime Q inlets 5.4.3 Prime Q inlets Overview The procedure consists of the following stages: Stage Description Prime all Q inlet tubing. Validate priming of Q inlet tubing. Purge Quaternary Valve and the system pumps if pressure signal indicates air bubbles.
Page 179 5 Operation 5.4 Prime inlets and purge pump heads 5.4.3 Prime Q inlets Step Action Connect a 25 to 30 ml syringe to one of the purge valves of either of the system pumps. Make sure that the syringe fits tightly into the purge connec- tor.
Page 180 5 Operation 5.4 Prime inlets and purge pump heads 5.4.3 Prime Q inlets Purge Quaternary Valve and the system pumps If the priming was done thoroughly and the final buffer was drawn all the way into the syringe and the validation of the priming showed that there was no air left in the pump it is not necessary to purge Quaternary Valve and the system pumps.
Page 181 5 Operation 5.4 Prime inlets and purge pump heads 5.4.3 Prime Q inlets Step Action Connect a 25 to 30 ml syringe to the left purge valve of the selected system pump. Make sure that the syringe fits tightly into the purge connector. Open the purge valve by turning it counterclockwise about 3 quarters of a turn.
Page 182: Connect A Column
5 Operation 5.5 Connect a column Connect a column Introduction This section describes how to connect a column to the instrument using a column holder and without introducing air into the flow path. Several types of column holders are available for the ÄKTA avant instrumen, see Section 2.6 Accessories, on page 43. WARNING Before connecting a column, read the instructions for use of the column.
Page 183 5 Operation 5.5 Connect a column Attach a column holder and connect a column Follow the instructions to connect a column to the instrument. Always use a column holder. The column is connected to two opposite parts of the column valve, using appro- priate tubing and connectors.
Page 184 5 Operation 5.5 Connect a column Step Action Connect a suitable tubing to a column valve port, for example port 1A if column position 1 was chosen in the method to be run. In the Process Picture: Click the Column valve icon. •...
Page 185 5 Operation 5.5 Connect a column Step Action In the Process Picture: Click the System pumps icon. • Enter a low System flow (e.g., 0.2 ml/min). • Click Set flow rate. • Result: A system flow of 0.2 ml/min starts. When buffer leaves the tubing on port 1A (if port 1A was chosen in the method to be run) in a continuous mode and the top part of the column is filled with buffer, connect the tubing to the top of the column.
Page 186 5 Operation 5.5 Connect a column Step Action Connect a piece of tubing to the bottom of the column. When buffer leaves the tubing at the bottom of the column in a continuous mode, connect this piece of tubing to the column valve. Use the port opposite to the one already connected to the column, in this example port 2B.
Page 187: Set Pressure Alarms
5 Operation 5.6 Set pressure alarms Set pressure alarms Introduction The columns can be protected by two different types of pressure alarms: • The pre-column pressure alarm protects the column hardware. • The delta-column pressure alarm protects the column media. The column valves V9-C and V9H-C have built-in pressure sensors that automatically measure the pre-column and delta-column pressure.
Page 188 5 Operation 5.6 Set pressure alarms Step Action In the System Control module, on the Manual menu, click Execute Manual Instructions . Result: The Manual instructions dialog box opens. In the Instructions box, select Alarms:Alarm pre column pressure. Click Enabled in the Mode field. Enter the high pressure limit in the High alarm box.
Page 189: Sample Application
5 Operation 5.7 Sample application Sample application Introduction This section describes the different sample application techniques that can be used with ÄKTA avant. The table below shows the alternatives for sample application available in the Sample application phase of a method. Sample applica- Compatible loops tion...
Page 190 5 Operation 5.7 Sample application In this section This section contains the following subsections: Section See page 5.7.1 Sample application using direct injection into the column 5.7.2 Sample application using a Superloop 5.7.3 Sample application using a sample loop ÄKTA avant User Manual 29035184 AE...
Page 191: Sample Application Using Direct Injection Into The Column
5 Operation 5.7 Sample application 5.7.1 Sample application using direct injection into the column 5.7.1 Sample application using direct injection into the column Introduction There are two ways to load sample directly onto a column: • a fixed volume is loaded, or •...
Page 192 5 Operation 5.7 Sample application 5.7.1 Sample application using direct injection into the column When preparing to Then... inject... all the sample in the Method editor, make the following selections • for the Sample Application phase of the method to be run: select Inject sample directly onto column, and select Inject all sample using air sensor.
Page 193 5 Operation 5.7 Sample application 5.7.1 Sample application using direct injection into the column Maximize precision and accuracy To achieve full precision and accuracy when a volume of sample is injected directly onto the column, make the following selections in the Sample Application phase of the method to be run (refer to Section 5.9 Create a method and perform a run, on page 225 for more information on methods and phases): •...
Page 194: Sample Application Using A Superloop
5 Operation 5.7 Sample application 5.7.2 Sample application using a Superloop 5.7.2 Sample application using a Superloop Introduction A Superloop allows injection of large sample volumes onto the column. A Superloop can also be used for multiple injections, for example in a scouting experiment when the same application conditions are required.
Page 195 5 Operation 5.7 Sample application 5.7.2 Sample application using a Superloop Step Action Connect a piece of tubing from the bottom of the Superloop to: port LoopF on the Injection valve • the F port corresponding to the connected E port, e.g., 1F, on the Loop •...
Page 196 5 Operation 5.7 Sample application 5.7.2 Sample application using a Superloop Step Action In the Process Picture: Click the Injection valve and select Manual Load. • Result: The Injection valve switches to Manual Load position. If the loop is connected to: the Injection valve, continue to step 5.
Page 197 5 Operation 5.7 Sample application 5.7.2 Sample application using a Superloop Step Action Load sample into the Superloop by emptying the syringe into the Injection valve. Disconnect the syringe and plug the Syr port with a Stop plug. Fill the Superloop using the Sample pump Follow the instruction below to fill the Superloop using the Sample pump.
Page 198 5 Operation 5.7 Sample application 5.7.2 Sample application using a Superloop Step Action In the Manual instructions dialog box: Select Pumps and pressures:Sample flow. • Set Flow rate to an appropriate value for the Superloop size, in this ex- • ample 10 ml/min.
Page 199: Sample Application Using A Sample Loop
5 Operation 5.7 Sample application 5.7.3 Sample application using a sample loop 5.7.3 Sample application using a sample loop Introduction A sample loop is recommended for injection of smaller sample volumes onto the column. A sample loop can be connected to either the Injection valve or the Loop valve. When using the Loop valve, up to five loops can be connected simultaneously.
Page 200 5 Operation 5.7 Sample application 5.7.3 Sample application using a sample loop Step Action In the Process Picture: Click on the Injection valve and select Manual load. • Result: Injection valve is set to manual load. If the loop is connected to: the Injection valve, continue to step 7.
Page 201 5 Operation 5.7 Sample application 5.7.3 Sample application using a sample loop Tip: By default a union Luer female to 1/16 ″ male is used for connection of the sample syringe to the injection valve. For loading of smaller volumes with higher precision there is an alternative accessory, Fill port INV-907 (18-1127- 66) or the complete kit Injection Valve Kit (18-1110-89).
Page 202: Fractionation
5 Operation 5.8 Fractionation Fractionation Introduction The built-in fraction collector and the optional Fraction collector F9-R collect fractions from ÄKTA avant purification runs. The built-in fraction collector has integrated cooling. The fraction collectors are connected to ÄKTA avant and controlled by UNICORN. The fraction collectors can be automatically controlled in a method run or manually controlled.
Page 203: Prepare The Built-In Fraction Collector
5 Operation 5.8 Fractionation 5.8.1 Prepare the built-in fraction collector 5.8.1 Prepare the built-in fraction collector Introduction This section describes how to prepare the built-in fraction collector. For detailed informa- tion regarding the types of deep well plates, tubes and cassettes, see Section 3.10.3 Cassettes, cassette tray and racks, on page 89.
Page 204 5 Operation 5.8 Fractionation 5.8.1 Prepare the built-in fraction collector Workflow for preparing the built-in fraction collector Before starting to prepare the built-in fraction collector, check the fractionation settings in the method to be run. Perform the steps described below according to the settings in the method.
Page 205 5 Operation 5.8 Fractionation 5.8.1 Prepare the built-in fraction collector Step Action Place the tubes and deep well plates in the cassettes. Make sure that the deep well plates are rotated so that the well marked A1 is positioned above the A1 marking on the cassette.
Page 206 Action Place the cassettes on the cassette tray. Make sure that the cassette type code (see the illustration) faces the front of the tray marked with the GE monogram. Open the fraction collector drawer by pressing the handle upwards, and pulling out the drawer.
Page 207 Action Place the cassette tray on the tray support of the fraction collector drawer. Make sure that the front of the tray (marked with the GE monogram) faces the front of the drawer and is hooked onto the two pins.
Page 208 Place the rack on the tray support of the fraction collector drawer. Make sure that the front of the rack (marked with the GE monogram) faces the front of the drawer and is hooked onto the two pins.
Page 209 5 Operation 5.8 Fractionation 5.8.1 Prepare the built-in fraction collector Cassette and tray identification When the door of the fraction collector is closed automatic scanning is performed. There are two types of scanning procedures: • Full scan: Scanning of cassette type codes to determine which types of cassettes are used, and scanning of rows and columns in deep well plates to identify which types of plates are used (24, 48, or 96 wells).
Page 210: Prepare Fraction Collector F9-R
5 Operation 5.8 Fractionation 5.8.2 Prepare Fraction collector F9-R 5.8.2 Prepare Fraction collector F9-R Introduction This subsection describes how to prepare and assemble Fraction collector F9-R before a run. Fraction collector F9-R can be connected to ÄKTA avant and controlled by UNICORN. The fraction collector can be automatically controlled in a method run or manually controlled.
Page 211 5 Operation 5.8 Fractionation 5.8.2 Prepare Fraction collector F9-R Tube rack inserts The Fraction collector F9-R is delivered with the 18 mm tube rack mounted. Each tube rack is made up of a combination of a bowl, tube support, tube guide and tube holder. Change the tube holder and the tube guide to collect fractions in 12 mm tubes or 30 mm tubes.
Page 212 5 Operation 5.8 Fractionation 5.8.2 Prepare Fraction collector F9-R Inserts 30 to 50 mm 50 to 85 mm 85 to 180 mm tubes tubes tubes Tube guide Single cutout Single cutout L-shaped cutout Tube holder Single cutout Single cutout Single cutout For 30 to 50 mm tubes, first insert the tube guide from the 18 mm rack using the single cutout, before inserting the tube support for the 30 mm rack.
Page 213 5 Operation 5.8 Fractionation 5.8.2 Prepare Fraction collector F9-R Step Action Insert the tube holder (6) with the tube position numbers upwards: Check that tube position 1 (7) is directly above tube position 1 (7) of the tube guide. Push the flexible bowl out at each rib and snap the tube holder under •...
Page 214 5 Operation 5.8 Fractionation 5.8.2 Prepare Fraction collector F9-R Step Action Place the tube rack (9) over the central spindle (11) and pull the spring loaded drive sleeve (12) out so the tube rack comes to rest. Insert collection tubes Insert a sufficient number of collection tubes in to the tube rack, starting at position 1, pushing each one down as far as they will go.
Page 215 5 Operation 5.8 Fractionation 5.8.2 Prepare Fraction collector F9-R Step Action Lift and then lower the delivery arm (1), and allow it to move in so the tube sensor (2) touches the collection tubes of the outer track. Loosen the lock knob (3) ÄKTA avant User Manual 29035184 AE...
Page 216 5 Operation 5.8 Fractionation 5.8.2 Prepare Fraction collector F9-R Step Action Adjust the height so that the horizontal mark (5) on the tube sensor (6) • is at the same level as the top of the flat collection tubes and approxi- mately 2 mm over the top of the flanged collection tubes (4).
Page 217 5 Operation 5.8 Fractionation 5.8.2 Prepare Fraction collector F9-R Connect tubing Make sure that the tubing to the fraction collector is properly connected. See Connect tubing to the ÄKTA avant instrument, on page 134. Sensor control The sensor control can be switched between the two positions "small tubes" and "large tubes", indicated in the illustration below.
Page 218: Fractionation Overview
5 Operation 5.8 Fractionation 5.8.3 Fractionation overview 5.8.3 Fractionation overview Fractionation types The following table lists the types of fractionation that the fraction collectors can be used for. Type Description Fixed volume frac- During fixed volume fractionation the fraction collector contin- tionation uously switches tubes according to the set volume throughout the entire fractionation.
Page 219 5 Operation 5.8 Fractionation 5.8.3 Fractionation overview Illustration The following illustration show examples of fractionation using fixed volume fractionation and fractionation using peak fractionation. A) Straight/fixed fractionation Volume B) Peak fractionation Volume ÄKTA avant User Manual 29035184 AE...
Page 220 5 Operation 5.8 Fractionation 5.8.3 Fractionation overview Delay volume The delay volume settings are used to make sure that the fractions collected during fractionation, using the outlet valve or the fraction collector, correspond to the fractions indicated in the chromatogram. The delay volume is the volume between the UV monitor, and the fraction collector or outlet that is used, see the following illustration.
Page 221 5 Operation 5.8 Fractionation 5.8.3 Fractionation overview Illustration of fraction marking using fixed volume fractionation with Fraction collector F9-R The illustration below shows the fractions collected, and the numerical marking of frac- tions, when fixed volume (straight) fractionation is used with Fraction collector F9-R. Volume 10 11 12 13 14 15 16 17 18 19 Fractionation marks shown in chromatogram...
Page 222 5 Operation 5.8 Fractionation 5.8.3 Fractionation overview Illustration of fractions and fraction marking using peak fractionation with Fraction collector F9-R The following illustration shows the fractions collected, and the numerical marking of fractions, when peak fractionation is used with Fraction collector F9-R. Volume 2 3 4 9 10 11...
Page 223 5 Operation 5.8 Fractionation 5.8.3 Fractionation overview Fractionation settings for Fraction collector F9-R There are two fractionation settings for Fraction collector F9-R, DropSync off or DropSync on . Dropsync off: No synchronization of collection. DropSync on: When using DropSync the sensors in the tube sensor detect when a drop is released.
Page 224 5 Operation 5.8 Fractionation 5.8.3 Fractionation overview Peak broadening The width of peaks at the fraction collector is influenced by the properties of the column and the dimensions of tubing connecting the components and the components them- selves. Initial sample volume affects the peak width in gel filtration (GF) chromatography. A sample zone is broadened during passage through a GF column so that the sample is diluted and the resolution decreases with increasing sample volume.
Page 225: Create A Method And Perform A Run
5 Operation 5.9 Create a method and perform a run Create a method and perform a run About this section This section provides an overview of how to create a method in UNICORN and how to perform manual and method runs on ÄKTA avant. It also contains advice on things to be considered during and after a run.
Page 226: Create A Method
5 Operation 5.9 Create a method and perform a run 5.9.1 Create a method 5.9.1 Create a method Introduction The predefined methods are built up using phases, where each phase corresponds to a step in a chromatography run with a number of properties associated with that phase. See UNICORN Method Manual for more information about method structure, definitions and concepts of methods in UNICORN.
Page 227 5 Operation 5.9 Create a method and perform a run 5.9.1 Create a method • Intelligent Packing (ÄKTA avant 150 only) • System CIP • System Preparation Main steps when defining a new method The main steps when defining a method are: 1 Create/open a method •...
Page 228 5 Operation 5.9 Create a method and perform a run 5.9.1 Create a method Step Action In the System list, select your system. Click Predefined Method and in the Predefined Method list select your predefined method. Click OK. Result: The phases included in the chosen method is shown in the Method Outline pane, and the default settings for each of the phases is shown in the Phase Properties pane.
Page 229 5 Operation 5.9 Create a method and perform a run 5.9.1 Create a method Text Instruction mode In most cases methods can be edited using the Phase Properties pane in the Method Editor module. However, modules without a recommended position are not supported by Phase Properties and have to be edited in the Text Instruction mode.
Page 230: Perform A Run
5 Operation 5.9 Create a method and perform a run 5.9.2 Perform a run 5.9.2 Perform a run Checklist Make sure that the system is correctly prepared. Check that: • The system is prepared according to Section 5.1 Before you prepare the system, on page 156.
Page 231 5 Operation 5.9 Create a method and perform a run 5.9.2 Perform a run Choose and start a method The following instruction describes how to open a method and start a run. Step Action Open the System Control module and click the Open Method Navigator button.
Page 232 5 Operation 5.9 Create a method and perform a run 5.9.2 Perform a run Perform a manual run Manual runs can be convenient for procedures such as filling tubing with buffer or packing a column with media. Step Action On the Manual menu, click Execute Manual Instructions. Select instruction group and instruction.
Page 233: Monitor The Run
5 Operation 5.9 Create a method and perform a run 5.9.3 Monitor the run 5.9.3 Monitor the run Introduction You may follow the on-going method run in the System Control module. The current system status is shown in the System state panel in the Run Data pane. For example, it may state Run, Wash or Hold.
Page 234 5 Operation 5.9 Create a method and perform a run 5.9.3 Monitor the run Process Picture The Process Picture displays the current flow path, run parameters and real-time data from monitors during a run. It also allows manual interactions with the system. Tubing colors indicate flow path states, as shown in the following illustration and de- scribed in the following table.
Page 235 5 Operation 5.9 Create a method and perform a run 5.9.3 Monitor the run Actions in the Process Picture pane It is possible to interact with the Process Picture pane. • To open a related instruction, click the component icon. The example below shows the pop-up toolbar for the Injection valve icon.
Page 236: After Run Procedures
5 Operation 5.9 Create a method and perform a run 5.9.4 After run procedures 5.9.4 After run procedures Introduction This section describes how to clean the instrument and columns after a chromatographic run, and how to prepare the system for storage. The instrument and the columns should be cleaned between the runs.
Page 237 5 Operation 5.9 Create a method and perform a run 5.9.4 After run procedures System storage If the instrument is not going to be used for a couple of days or longer, also perform the following: • Fill the system and inlets with storage solution (e.g., 20% ethanol) using the System CIP method.
Page 238 5 Operation 5.9 Create a method and perform a run 5.9.4 After run procedures Log off or exit UNICORN Follow the instructions to log off or exit UNICORN. This can be performed from any of the UNICORN modules. If you want to... then...
Page 239: Performance Tests
6 Performance tests Performance tests About this chapter Performance tests should be run after installation to check the function of the ÄKTA avant system. Performance tests can also be used at any time to check the condition of the system, for example, after a prolonged stop. This chapter describes how to prepare, run, and evaluate the available performance tests.
Page 240: General Performance Test Actions
6 Performance tests 6.1 General performance test actions General performance test actions Introduction Some actions are identical for all performance tests. These actions are described in this section. Start the performance tests Follow the instructions to start a performance test. Step Action In the System Control module, on the System menu, click Performance Test...
Page 241 6 Performance tests 6.1 General performance test actions Overview of the Start Protocol dialog The following table describes the pages of the Start Protocol. Page Description Notes Displays the Method Notes of the method. The Method Notes contains a method description and instructions on how to run the method.
Page 242 UNICORN installation folder. For example Temp C:\Program Files\GE Healthcare\UNICORN\UNICORN 7.0\Temp • If the option Print report was selected in the Evaluation Procedures page of the Start Protocol dialog box when starting the test, the report is also automatically printed on the system printer.
Page 243: Air Sensor Tests
6 Performance tests 6.2 Air sensor tests Air sensor tests Method description The Air sensors A, B, and S are integrated in Inlet valve A, Inlet valve B, and Sample inlet valve. The Air sensors test checks if the air sensors detect air. The method run takes approximately 5 minutes.
Page 244 6 Performance tests 6.2 Air sensor tests Possible causes of a failed test The following table describes possible causes of a failed test. When possible sources of error have been checked and taken care of, run the test once again. Cause Action Faulty air sensor...
Page 245: Built-In Fraction Collector Test
6 Performance tests 6.3 Built-in Fraction collector test Built-in Fraction collector test Method description The Fraction collector test checks the functionality of the Fraction collector. For ÄKTA avant 25, fractionation of 2 ml is performed in three sequential wells in each of two 96 deep well plates.
Page 246 Cassette position 6. Make sure that the Cassette type codes (see il- lustration below) faces the front of the tray marked with the GE logo. No other Cassetes must be present in the Fraction collector during the run.
Page 247 Action Place the Cassette tray on the Tray support of the Frac drawer. Make sure that the front of the tray (marked with the GE logo) faces the front of the drawer. Close the Frac drawer. Make sure that it snaps into closed position.
Page 248 6 Performance tests 6.3 Built-in Fraction collector test During the test run Visually check that the fraction collector wash is performed. Evaluate the test To evaluate the result, do the following: • Check that the fractionation marks in the chromatogram correspond to the filled wells and tubes and that there is minimal spillage.
Page 249 6 Performance tests 6.3 Built-in Fraction collector test Possible causes of a failed test The following table describes possible causes of a failed test. When possible sources of error have been checked and taken care of, run the test once again. Cause Action Wrong volumes collect-...
Page 250: Fraction Collector F9-R Test
6 Performance tests 6.4 Fraction collector F9-R test Fraction collector F9-R test Method description The Fraction collector F9-R test checks the functionality of Fraction collector F9-R. The method run takes approximately 3 minutes. Note: The fraction collector test is evaluated manually and no report is generated. Required configuration A correctly installed Outlet valve, and a correctly installed Fraction collector F9-R are required to run the test.
Page 251 6 Performance tests 6.4 Fraction collector F9-R test Step Action In the System Settings dialog box: Select Fraction collection:Fractionation settings frac 2. • In the Drop sync field, click On. • Click OK. • Run and evaluate the test Follow the instructions described in Section 6.1 General performance test actions, on page 240 to start and run the performance test.
Page 252 6 Performance tests 6.4 Fraction collector F9-R test Possible causes of a failed test The table below describes possible causes of a failed test. When possible sources of error have been checked and corrected, repeat the test. Cause Action Incorrect volumes col- Air in pumps: Make sure to prime inlet tubing A1 and purge lected in the tubes, and System pump A before method start, see Section 5.4.1...
Page 253: Quaternary Valve Test
6 Performance tests 6.5 Quaternary valve test Quaternary valve test Method description The Q valve test checks the functionality of the Quaternary valve. Correct gradient for- mation is tested by producing a series of gradient steps. The method run takes approxi- mately 30 minutes for ÄKTA avant 25 and 70 minutes for ÄKTA avant 150.
Page 254 6 Performance tests 6.5 Quaternary valve test Run and evaluate the test Follow the instructions described in Section 6.1 General performance test actions, on page 240 to start, run and automatically evaluate the performance test. Illustration of chromatogram The illustration below shows a chromatogram from a Quaternary valve test. The gradient steps are marked in the illustration.
Page 255 6 Performance tests 6.5 Quaternary valve test Possible causes of a failed test The table below describes possible causes of a failed test. When possible sources of error have been checked and taken care of, run the test once again. Cause Action Disturbances caused by...
Page 256: System Test
6 Performance tests 6.6 System test System test Method description The System test checks the solvent delivery, the functionality of the UV and conductivity monitoring systems, and the valve functionality of the standard system. The method run takes approximately 30 minutes. Required material The following material is required: •...
Page 257 6 Performance tests 6.6 System test Step Action Prime the buffer inlets and purge System pump A and System pump B. See Section 5.4.1 Prime buffer inlets and purge system pumps, on page 166. Run and evaluate the test Follow the instructions described in Section 6.1 General performance test actions, on page 240 to start, run and automatically evaluate the performance test.
Page 258 6 Performance tests 6.6 System test Faulty Step Response Result Cause Action If all values are faulty - Air in pumps: Make sure to prime the buffer inlets and to air in the pump or a purge the System pumps and the Sample pump before faulty pump method start.
Page 259 6 Performance tests 6.6 System test Cause Action The value set for the If the test is performed at cold room temperature, open Cond temp compensa- the System Settings dialog box, select Conductivity:Cond tion factor is not optimal temp compensation and set the Compensation factor to 2.1 Faulty UV Noise Test Cause...
Page 260: Uv Monitor U9-L Test
6 Performance tests 6.7 UV monitor U9-L test UV monitor U9-L test Method description UV U9-L test checks the functionality of the UV monitor U9-L. The method run takes approximately 10 minutes. Required configuration A correctly installed UV monitor U9-L is required to run the test. Required material The following materials are required: •...
Page 261 6 Performance tests 6.7 UV monitor U9-L test Step Action Prime the buffer inlets and purge System pump A and System pump B. See Section 5.4.1 Prime buffer inlets and purge system pumps, on page 166. Run and evaluate the test Follow the instructions described in Section 6.1 General performance test actions, on page 240 to start, run and automatically evaluate the performance test.
Page 262: Maintenance
7 Maintenance Maintenance About this chapter This chapter describes the maintenance program for ÄKTA avant and provides instructions for maintenance and replacement of spare parts. In this chapter This chapter contains the following sections: Section See page 7.1 Maintenance Manager 7.2 Maintenance program 7.3 Weekly maintenance 7.4 Monthly maintenance...
Page 263: Maintenance Manager
7 Maintenance 7.1 Maintenance Manager Maintenance Manager Introduction Maintenance Manager allows the user to display general information about the system and its modules, and also operational statistics of the modules. Notifications for mainte- nance actions of the system and its modules are predefined. The user can add automated maintenance notifications for the system.
Page 264 7 Maintenance 7.1 Maintenance Manager View general information and statistics In the left side of the Maintenance Manager dialog box, select the system of interest to view general information of the selected system. When modules are selected, operational statistics are also displayed. View maintenance notifications Click the plus symbol (+) of the system of interest to expand the list of related maintenance notifications.
Page 265 7 Maintenance 7.1 Maintenance Manager Edit a maintenance notification Follow the instruction to edit a maintenance notification. Step Action In the left side field of the Maintenance Manager dialog box, select a maintenance notification. Result: Details of the selected maintenance notification are displayed in the dialog box.
Page 266 7 Maintenance 7.1 Maintenance Manager Step Action In the Maintenance Manager dialog box, click New System Notification. Result: The NewNotification field appears in the Maintenance Manager dialog box. In the NewNotification field: Enter a name for the new notification. • Select a time interval after which the new notification will be issued.
Page 267 7 Maintenance 7.1 Maintenance Manager Handle a maintenance notification Each maintenance notification has a time interval after which the notification is issued. When this time interval has been reached, a Maintenance Notification message appears. Follow the instruction to handle the notification. Click...
Page 268: Maintenance Program
7 Maintenance 7.2 Maintenance program Maintenance program Introduction This section lists the periodic maintenance activities that should be performed by the user of ÄKTA avant, as well as maintenance activities that should be performed when required. Maintenance is divided into: •...
Page 269 7 Maintenance 7.2 Maintenance program Interval Maintenance action See section Weekly Change pump rinsing solution Section 7.3.1 Change pump rins- ing solution, on page 273 Weekly Check inline filter, replace if Section 7.3.2 Replace the inline necessary filter, on page 275 Weekly Clean built-in fraction collector Section 7.3.3 Clean built-in frac-...
Page 270 7 Maintenance 7.2 Maintenance program Maintenance action See section Replace tubing and connectors Section 7.8.1 Replace tubing and connectors, on page 336 Storage of pH electrode Section 7.6.6 Storage of the pH electrode, on page 305 Clean the pH electrode Section 7.6.7 Clean the pH electrode, on page 307 Regenerate dried out pH electrode Section 7.6.8 Regenerate dried out pH electrode,...
Page 271 7 Maintenance 7.2 Maintenance program Maintenance action See section Replace valve modules Section 7.8.10 Replace a valve module, on page 354 Clean pump flow restrictors Section 7.6.10 Clean pump flow restrictors, on page 315 ÄKTA avant User Manual 29035184 AE...
Page 272: Weekly Maintenance
7 Maintenance 7.3 Weekly maintenance Weekly maintenance About this section This section provides instructions for weekly maintenance activities. In this section This section contains the following subsections: Section See page 7.3.1 Change pump rinsing solution 7.3.2 Replace the inline filter 7.3.3 Clean built-in fraction collector sensors ÄKTA avant User Manual 29035184 AE...
Page 273: Change Pump Rinsing Solution
7 Maintenance 7.3 Weekly maintenance 7.3.1 Change pump rinsing solution 7.3.1 Change pump rinsing solution Maintenance interval Replace the pump rinsing solution in the system pumps and the sample pump every week to prevent bacterial growth. Required material The following material are required: •...
Page 274 7 Maintenance 7.3 Weekly maintenance 7.3.1 Change pump rinsing solution Step Action Immerse the inlet tubing to the System pump piston rinsing system in one of the rinsing solution tubes. Note: Make sure that the inlet tubing reaches to the very bottom of the rinsing solu- tion tube.
Page 275: Replace The Inline Filter
7 Maintenance 7.3 Weekly maintenance 7.3.2 Replace the inline filter 7.3.2 Replace the inline filter Maintenance interval Replace the inline filter that is located in the top section of the Mixer every week. When it starts to get clogged the back pressure from the filter will increase as registered by the system pressure sensor.
Page 276: Clean Built-In Fraction Collector Sensors
7 Maintenance 7.3 Weekly maintenance 7.3.3 Clean built-in fraction collector sensors 7.3.3 Clean built-in fraction collector sensors Maintenance interval Clean the fraction collector sensors every week, or when required, for example if the fraction collector fails to read the tray ID or does not collect fractions correctly. Required material The following material is required: •...
Page 277 7 Maintenance 7.3 Weekly maintenance 7.3.3 Clean built-in fraction collector sensors Step Action Wipe off the dispenser head and the DropSync and type code reader sensor windows using a wash bottle with water or 20% ethanol and a cloth. Let the dispenser head dry completely before starting a run. Close the fraction collector door.
Page 278: Monthly Maintenance
7 Maintenance 7.4 Monthly maintenance Monthly maintenance Introduction This section provides instructions for monthly maintenance. In this section This section contains the following subsections Section See page 7.4.1 Check the flow restrictor 7.4.2 Check the function of the pump flow restrictors ÄKTA avant User Manual 29035184 AE...
Page 279: Check The Flow Restrictor
7 Maintenance 7.4 Monthly maintenance 7.4.1 Check the flow restrictor 7.4.1 Check the flow restrictor Maintenance interval Check the back pressure for the flow restrictor every month. Instruction Follow the instruction to check the back pressure of Flow restrictor FR-902. Step Action Immerse the piece of inlet tubing marked A1 in distilled water, and immerse...
Page 280 7 Maintenance 7.4 Monthly maintenance 7.4.1 Check the flow restrictor Step Action Calculate the difference between the two pressure values noted in step 4 and step 6. Check that the pressure difference is within the range 0.2 ± 0.05 MPa. If this is not the case, the Flow restrictor should be replaced, see Section 7.8.6 Replace Flow restrictor, on page 347.
Page 281: Check The Function Of The Pump Flow Restrictors
If there are siphoning, the pump flow restrictors must be replaced at the next service occasion by a GE representative. The piece of tubing marked 2A is connected to pump flow restrictor A, the piece of tubing marked 2B is connected to pump flow restrictor B.
Page 282: Semiannual Maintenance
7 Maintenance 7.5 Semiannual maintenance Semiannual maintenance Introduction This section provides instructions for semiannual (once every six months) maintenance activities. In this section This section contains the following subsections: Section See page 7.5.1 Clean the UV flow cell 7.5.2 Replace the pH electrode ÄKTA avant User Manual 29035184 AE...
Page 283: Clean The Uv Flow Cell
7 Maintenance 7.5 Semiannual maintenance 7.5.1 Clean the UV flow cell 7.5.1 Clean the UV flow cell Maintenance interval Clean the UV flow cell every six months, or when required. NOTICE Keep UV flow cell clean. Do not allow solutions containing dissolved salts, proteins or other solid solutes to dry out in the flow cell.
Page 284 7 Maintenance 7.5 Semiannual maintenance 7.5.1 Clean the UV flow cell Step Action Disconnect the tubing from the top of the UV flow cell, and replace the fin- gertight connector with a Luer connector. Disconnect the tubing from the bottom of the UV flow cell, and connect a piece of waste tubing to the UV flow cell.
Page 285 7 Maintenance 7.5 Semiannual maintenance 7.5.1 Clean the UV flow cell Step Action Squirt the detergent solution through the UV flow cell about five times. Leave the detergent solution in the flow cell for at least 20 minutes. Inject the detergent solution remaining in the syringe into the flow cell. Dis- connect the syringe.
Page 286: Replace The Ph Electrode
7 Maintenance 7.5 Semiannual maintenance 7.5.2 Replace the pH electrode 7.5.2 Replace the pH electrode Maintenance interval Replace the pH electrode every six months, or when required. Required material The following materials are required: pH electrode, deionized water and standard buffer pH 4.
Page 287: Maintenance When Required
7 Maintenance 7.6 Maintenance when required Maintenance when required Introduction This section gives instructions for maintenance activities to be performed when required. In this section This section contains the following subsections: Section See page 7.6.1 Clean the instrument externally 7.6.2 Perform System CIP 7.6.3 Perform Column CIP 7.6.4 Clean built-in fraction collector 7.6.5 Clean Fraction collector F9-R...
Page 288: Clean The Instrument Externally
7 Maintenance 7.6 Maintenance when required 7.6.1 Clean the instrument externally 7.6.1 Clean the instrument externally Maintenance interval Clean the the instrument externally when required. Do not allow spilled liquid to dry on the instrument. Required material The following materials are required: •...
Page 289: Perform System Cip
7 Maintenance 7.6 Maintenance when required 7.6.2 Perform System CIP 7.6.2 Perform System CIP Introduction The System CIP method is used to fill the instrument and the selected inlets and outlets with cleaning solution. WARNING Hazardous substances. When using hazardous chemicals, take all suitable protective measures, such as wearing pro- tective clothing, glasses and gloves resistant to the sub- stances used.
Page 290 7 Maintenance 7.6 Maintenance when required 7.6.2 Perform System CIP Create a System CIP method Follow the instruction to create a System CIP method. Step Action In the Method Editor module, • click the New Method button on the File menu, click New Method. •...
Page 291 7 Maintenance 7.6 Maintenance when required 7.6.2 Perform System CIP Step Action In the New Method dialog. In the System list select the name of the system. • Click Predefined Method, then in the Predefined Method list, click System • CIP.
Page 292 7 Maintenance 7.6 Maintenance when required 7.6.2 Perform System CIP Step Action In the Phase Properties tab of each of the System CIP phases: Enter a note for the first solution (optional). • Enter values for Flow rate, Volume per position and Incubation time. •...
Page 293 7 Maintenance 7.6 Maintenance when required 7.6.2 Perform System CIP Step Action In the Method Editor module, • click the Save the method button on the File menu, click Save As • Result: The Save As dialog opens. In the Save As dialog: Select a target folder to enable the Save button.
Page 294 7 Maintenance 7.6 Maintenance when required 7.6.2 Perform System CIP Clean the manual injection port of the injection valve Follow the instructions to manually clean the manual load position of the Injection valve. Step Action In the System Control module, on the Manual menu, click Execute Manual Instructions.
Page 295 7 Maintenance 7.6 Maintenance when required 7.6.2 Perform System CIP Step Action Fill a syringe with approximately 10 ml of an appropriate cleaning solution (e.g., 1M NaOH). Connect the syringe to Injection valve port Syr, and inject the cleaning solution. Leave the cleaning solution in place for 20 minutes. Fill a syringe with distilled water.
Page 296 7 Maintenance 7.6 Maintenance when required 7.6.2 Perform System CIP Step Action Open the System Control module, on the System menu, click Calibrate. Result: The Calibration dialog opens. In the Calibration dialog, in the Monitor to calibrate list, click pH. Click Prepare for calibration.
Page 297: Perform Column Cip
7 Maintenance 7.6 Maintenance when required 7.6.3 Perform Column CIP 7.6.3 Perform Column CIP Introduction The Column CIP method is used to clean the column after purification runs, to remove non-specifically bound proteins and to minimize the risk for carry-over between different purification runs.
Page 298 7 Maintenance 7.6 Maintenance when required 7.6.3 Perform Column CIP Create a Column CIP method Follow the instructions to create a Column CIP method. Step Action In the Method Editor module, • click the New Method button on the File menu, click New Method. •...
Page 299 7 Maintenance 7.6 Maintenance when required 7.6.3 Perform Column CIP Step Action In the Phase Properties tab of the Column CIP phase: Click Add Step to add a step. • Select the step and click Remove Step to remove a step. •...
Page 300 7 Maintenance 7.6 Maintenance when required 7.6.3 Perform Column CIP Step Action In the Method Editor module, • click the Save the method button on the File menu, click Save As • Result: The Save As dialog opens. In the Save As dialog: Select a target folder to enable the Save button.
Page 301: Clean Built-In Fraction Collector
7 Maintenance 7.6 Maintenance when required 7.6.4 Clean built-in fraction collector 7.6.4 Clean built-in fraction collector Maintenance interval Clean the Fraction collector when required, for example if liquid has been spilled in the Fraction collector chamber. The internal tubing of the fraction collector may need to be replaced for maintenance or for process purposes.
Page 302 7 Maintenance 7.6 Maintenance when required 7.6.4 Clean built-in fraction collector Step Action In the Manual instructions dialog: Select Fraction collection:Frac cleaning position. Click Execute. Result: The dispenser head moves to cleaning position, and the instrument display states System pause. Open the fraction collector drawer and lift off the cassette tray.
Page 303 7 Maintenance 7.6 Maintenance when required 7.6.4 Clean built-in fraction collector Step Action Wipe off the dispenser head and its diode windows using a wash bottle with water or 20% ethanol and a cloth. ÄKTA avant 25 has four diode windows and ÄKTA avant 150 has two diode windows (see the following illustrations).
Page 304: Clean Fraction Collector F9-R
7 Maintenance 7.6 Maintenance when required 7.6.5 Clean Fraction collector F9-R 7.6.5 Clean Fraction collector F9-R Maintenance interval Clean the Fraction collector when required, for example in case of liquid spill. Required material The following materials are required: • Water or 20% ethanol •...
Page 305: Storage Of The Ph Electrode
7 Maintenance 7.6 Maintenance when required 7.6.6 Storage of the pH electrode 7.6.6 Storage of the pH electrode Maintenance interval When pH monitoring is not used, the pH electrode can be stored in storage solution inside the pH flow cell. If pH monitoring is not used for a week or longer, inject new storage solution into the pH flow cell or replace the pH electrode with the dummy electrode that was installed in the pH valve on delivery.
Page 306 7 Maintenance 7.6 Maintenance when required 7.6.6 Storage of the pH electrode Step Action Click Prepare for Calculation. Result: The pH valve switches to the calibration position. Prepare at least 10 ml storage solution by mixing equal volumes of a stan- dard buffer, pH 4, and a 1 M Potassium Nitrate (KNO ) solution.
Page 307: Clean The Ph Electrode
7 Maintenance 7.6 Maintenance when required 7.6.7 Clean the pH electrode 7.6.7 Clean the pH electrode Maintenance interval Clean the pH electrode when required. The pH electrode can be cleaned either when it is installed in the pH valve or when it has been removed. The pH electrode has a limited longevity and should be replaced every six months or when the response time is slow, see Section 7.5.2 Replace the pH electrode, on page286.
Page 308 7 Maintenance 7.6 Maintenance when required 7.6.7 Clean the pH electrode Oil or grease films Wash the electrode tip in liquid detergent and water. If the films are known to be soluble in a particular organic solvent, wash with this solvent. Rinse the electrode tip in distilled water.
Page 309 7 Maintenance 7.6 Maintenance when required 7.6.7 Clean the pH electrode Clean a pH electrode installed in the pH valve Follow the instructions below to clean a pH electrode installed in the pH valve. The cali- bration function is used to switch the position of the pH valve. However, no calibration is performed.
Page 310 7 Maintenance 7.6 Maintenance when required 7.6.7 Clean the pH electrode Step Action Fill a syringe with approximately 10 ml of chosen cleaning solution. Connect the syringe to the pH valve port Cal. Inject the liquid and wait for 5 minutes. Disconnect the syringe.
Page 311: Regenerate Dried Out Ph Electrode
7 Maintenance 7.6 Maintenance when required 7.6.8 Regenerate dried out pH electrode 7.6.8 Regenerate dried out pH electrode Introduction If the glass membrane dries out it is important to restore the hydrated glass layer. Stage Description First try Method 1. If Method 1 does not help, try Method 2.
Page 312: Clean Pump Head Check Valves
7 Maintenance 7.6 Maintenance when required 7.6.9 Clean pump head check valves 7.6.9 Clean pump head check valves Introduction Clean the check valves when required, for example if particles like dust or salt crystals in the check valve cause irregular or low flow. The cleaning procedure is the same for the system pumps and the sample pump.
Page 313 7 Maintenance 7.6 Maintenance when required 7.6.9 Clean pump head check valves Step Action Unscrew the purge valve by turning it counter-clockwise, and lift off the metal ring. Unscrew the plastic nut of the upper check valve using an adjustable wrench, and gently lift off the upper check valve.
Page 314 7 Maintenance 7.6 Maintenance when required 7.6.9 Clean pump head check valves Step Action Unscrew the lower check valve using an adjustable wrench. WARNING Hazardous substances. When using hazardous chemicals, take all suitable protective measures, such as wearing protective clothing, glasses and gloves re- sistant to the substances used.
Page 315: Clean Pump Flow Restrictors
7 Maintenance 7.6 Maintenance when required 7.6.10 Clean pump flow restrictors 7.6.10 Clean pump flow restrictors Introduction Clean the pump flow restrictors when required for example if particles like dust or salt crystals is causing blockage. The cleaning procedure is the same for the system pumps and the sample pump.
Page 316 7 Maintenance 7.6 Maintenance when required 7.6.10 Clean pump flow restrictors Step Action Refill the syringe with distilled water and press gently to flush the flow path through the flow restrictor. Be careful to avoid spillage if there is a salt blockage creating back pressure.
Page 317: Wipe Off Excess Oil From The Pump Head
7 Maintenance 7.6 Maintenance when required 7.6.11 Wipe off excess oil from the pump head 7.6.11 Wipe off excess oil from the pump head Maintenance interval During the first months of use it is normal that excess oil leaks out of the drain hole below the System pump.
Page 318: Clean The Conductivity Flow Cell
7 Maintenance 7.6 Maintenance when required 7.6.12 Clean the Conductivity flow cell 7.6.12 Clean the Conductivity flow cell Maintenance interval Clean the Conductivity flow cell when required. Required material The following materials are required: • Luer connector • Waste container •...
Page 319 7 Maintenance 7.6 Maintenance when required 7.6.12 Clean the Conductivity flow cell Step Action Fill a syringe with distilled water, and connect the syringe to the Luer con- nector. Squirt distilled water through the conductivity flow cell in small amounts. Disconnect the syringe.
Page 320: Calibration Procedures
7 Maintenance 7.7 Calibration procedures Calibration procedures About this chapter This section provides instructions for calibration procedures that can be performed using the System Control module in UNICORN software. In this section This section contains the following subsections: Section See page 7.7.1 Calibrate the pH monitor 7.7.2 Calibrate the pressure monitors 7.7.3 Calibrate the Conductivity Monitor...
Page 321: Calibrate The Ph Monitor
7 Maintenance 7.7 Calibration procedures 7.7.1 Calibrate the pH monitor 7.7.1 Calibrate the pH monitor Maintenance interval Calibrate the pH monitor once a day, when the pH electrode has been replaced, or if the ambient temperature has changed by more than 5°C. Required material Use two pH calibration buffers with a difference of at least one pH unit.
Page 322 7 Maintenance 7.7 Calibration procedures 7.7.1 Calibrate the pH monitor Step Action Select pH in the Monitor to calibrate list. Click Prepare for calibration. Result: The pH valve switches to the calibration position. Enter the pH of the first pH standard buffer in the pH for buffer 1 box. Fill a syringe with approximately 10 ml of the first pH standard buffer.
Page 323: Calibrate The Pressure Monitors
7 Maintenance 7.7 Calibration procedures 7.7.2 Calibrate the pressure monitors 7.7.2 Calibrate the pressure monitors Maintenance interval ÄKTA avant has four pressure monitors: system pump pressure monitor, sample pump pressure monitor, pre-column pressure monitor and post-column pressure monitor. Check the pressure monitors every week, or when the the ambient temperature has changed more than 5°C.
Page 324 7 Maintenance 7.7 Calibration procedures 7.7.2 Calibrate the pressure monitors Step Action In the Calibration dialog, in the Monitor to calibrate list, select the pressure monitor to calibrate. Click Reset pressure. Result: The atmospheric pressure is defined as zero. Date and time of the most recent calibration, and the current pressure value are displayed.
Page 325 7 Maintenance 7.7 Calibration procedures 7.7.2 Calibrate the pressure monitors Tubing and pressures The following table shows the tubing to disconnect when checking and calibrating the pressure monitors. The UNICORN names of the pressures measured by the monitors are also shown. Pressure monitor Tubing to disconnect Pressure in...
Page 326 7 Maintenance 7.7 Calibration procedures 7.7.2 Calibrate the pressure monitors Pressure monitor Tubing to disconnect Pressure in UNICORN Pre-column pres- Tubing to Column valve V9-C or V9H-C PreC pressure sure monitor port In Post-column pres- Tubing to Column valve V9-C or V9H-C PostC pressure sure monitor port Out...
Page 327: Calibrate The Conductivity Monitor
7 Maintenance 7.7 Calibration procedures 7.7.3 Calibrate the Conductivity Monitor 7.7.3 Calibrate the Conductivity Monitor Introduction Two types of calibrations can be performed: • Conductivity monitor - factory calibration: Restores the conductivity cell constant to the factory default value. • Conductivity monitor - user calibration: Calibrates the conductivity cell constant.
Page 328 7 Maintenance 7.7 Calibration procedures 7.7.3 Calibrate the Conductivity Monitor Conductivity Monitor - user calibration Follow the instruction to calibrate the conductivity flow cell constant. Step Action Make sure that the instrument has been switched on for at least one hour. In the System Control module, on the System menu, click Settings.
Page 329 7 Maintenance 7.7 Calibration procedures 7.7.3 Calibrate the Conductivity Monitor Step Action In the Run Data pane of System Control, read the current Cond temp. Tip: If Cond temp is not showing, click the Customize icon. In the Customize dialog box, under the Run Data Groups tab, select to view Cond temp.
Page 330 7 Maintenance 7.7 Calibration procedures 7.7.3 Calibrate the Conductivity Monitor Graph for conductivity value The graph below shows the conductivity value at the current temperature when 1.00 M NaCl calibration solution is used. Conductivity of 1.00 M NaCl at 20–30°C Temperature (°C) ÄKTA avant User Manual 29035184 AE...
Page 331: Calibrate The Uv Monitors
7 Maintenance 7.7 Calibration procedures 7.7.4 Calibrate the UV monitors 7.7.4 Calibrate the UV monitors Automatic wavelength calibration of the UV Monitor The wavelength is automatically calibrated every time the instrument is switched on. If the instrument has been switched on for a couple of days, and the ambient temperature and/or the humidity has changed, restart the instrument using the power switch to cal- ibrate the UV Monitor U9-M.
Page 332 7 Maintenance 7.7 Calibration procedures 7.7.4 Calibrate the UV monitors Prepare for calibration Follow the instructions to prepare for the calibration of the UV monitor U9-L. Step Action Ensure that the flow restrictor is inline in the flow path after the UV flow cell. Mount the union Luer female/1/16”...
Page 333 7 Maintenance 7.7 Calibration procedures 7.7.4 Calibrate the UV monitors Step Action Fit the syringe in the union Luer connector and inject the solution. DO NOT remove the syringe. Note: Air trapped in the UV cell causes inaccurate measurements. To avoid introduc- ing air into the UV cell, gently fill the union Luer up to the edge with test solu- tion that is to be introduced, using the syringe.
Page 334 7 Maintenance 7.7 Calibration procedures 7.7.4 Calibrate the UV monitors Step Action Enter the measured absorbance values into the table in the column UV-900 Absorbance (AU) in the UV-900 cell calibration Excel-file. Note: The values should be converted from mAU to AU. When all absorbance values have been entered into the table, the real UV flow cell path length is shown at the bottom of the table.
Page 335: Replacement Procedures
7 Maintenance 7.8 Replacement procedures Replacement procedures About this section This section gives instructions for the replacement procedures to be performed by the user of ÄKTA avant. WARNING Disconnect power. Always disconnect power from the instrument before replacing any component on the instrument, unless stated otherwise in the user documentation.
Page 336: Replace Tubing And Connectors
7 Maintenance 7.8 Replacement procedures 7.8.1 Replace tubing and connectors 7.8.1 Replace tubing and connectors Maintenance interval Replace tubing and connectors when required, for example when a tubing has clogged or has been bent so that the flow is stopped. Required material The following materials are required: •...
Page 337 7 Maintenance 7.8 Replacement procedures 7.8.1 Replace tubing and connectors Step Action Cut the new tubing to the same length as the old tubing. Use a tubing cutter to get a straight angle cut. CAUTION Cut injuries. The tubing cutter is very sharp and must be handled with care to avoid injuries.
Page 338: Replace The Mixer
7 Maintenance 7.8 Replacement procedures 7.8.2 Replace the Mixer 7.8.2 Replace the Mixer Maintenance interval Replace the Mixer when a different Mixer chamber is desired, or when the Mixer is damaged. Required material The following is required: • Mixer Instruction Follow the instruction to change the Mixer.
Page 339 7 Maintenance 7.8 Replacement procedures 7.8.2 Replace the Mixer Step Action Pull the Mixer away from the instrument. Attach the new Mixer. Reconnect the tubing to the top and bottom of the new Mixer. Pump out the air inside the new mixer. ÄKTA avant User Manual 29035184 AE...
Page 340: Replace The O-Ring Inside The Mixer
7 Maintenance 7.8 Replacement procedures 7.8.3 Replace the O-ring inside the Mixer 7.8.3 Replace the O-ring inside the Mixer Maintenance interval Replace the O-ring inside the Mixer if it is damaged, or if it is being replaced with an high resistant O-ring for RPC runs.
Page 341 7 Maintenance 7.8 Replacement procedures 7.8.3 Replace the O-ring inside the Mixer Step Action Remove the outer locking O-ring from the top section. Lift up the top section of the Mixer and pull away the old O-ring inside. Wet the new O-ring with 20% ethanol and fit it in position. Make sure that the inline filter is still in position.
Page 342: Replace The Uv Monitor U9-M Flow Cell
7 Maintenance 7.8 Replacement procedures 7.8.4 Replace the UV monitor U9-M flow cell 7.8.4 Replace the UV monitor U9-M flow cell Maintenance interval Replace the UV flow cell when it is desired to use a flow cell with a different path length, or if the cell is damaged.
Page 343 7 Maintenance 7.8 Replacement procedures 7.8.4 Replace the UV monitor U9-M flow cell Step Action Push the latch on the UV detector to disconnect the detector. Note: While the UV detector is disconnected, the UV lamp becomes inoperable so no UV light can be emitted from the instrument. Pull off the detector and the flow cell from the monochromator.
Page 344 7 Maintenance 7.8 Replacement procedures 7.8.4 Replace the UV monitor U9-M flow cell Step Action Pull off the black protective caps from the new UV flow cell, and connect the new UV flow cell to the detector. Connect the detector, with the new flow cell connected, to the monochro- mator.
Page 345: Replace Uv Monitor U9-L Flow Cell
7 Maintenance 7.8 Replacement procedures 7.8.5 Replace UV monitor U9-L flow cell 7.8.5 Replace UV monitor U9-L flow cell Maintenance interval Replace the UV flow cell when it is desired to use a flow cell with a different path length, or if the cell is damaged.
Page 346 7 Maintenance 7.8 Replacement procedures 7.8.5 Replace UV monitor U9-L flow cell Step Action Unscrew the knurled wheel at the bottom of the UV monitor. Press the wheel upwards to release the flow cell. Pull the flow cell upwards out of the monitor. Hold the flow cell by the top part with the O-ring: do not touch the optical surfaces of the flow cell.
Page 347: Replace Flow Restrictor
7 Maintenance 7.8 Replacement procedures 7.8.6 Replace Flow restrictor 7.8.6 Replace Flow restrictor Maintenance interval Replace the Flow restrictor when required, for example when the back pressure of the Flow restrictor is outside the range 0.2 ± 0.05 MPa. Required material The following material is required: •...
Page 348: Replace The Inlet Filters
7 Maintenance 7.8 Replacement procedures 7.8.7 Replace the inlet filters 7.8.7 Replace the inlet filters Maintenance interval Replace the inlet filter when required, for example when the filters are clogged. Required material The following material is required: • Inlet filter set Instruction Follow the instruction to replace an inlet filter and a support net.
Page 349: Replace Pump Head Check Valves
7 Maintenance 7.8 Replacement procedures 7.8.8 Replace pump head check valves 7.8.8 Replace pump head check valves Maintenance interval Replace a check valve when required, for example if the check valve is damaged or clogged. Required material The following materials are required: •...
Page 350 7 Maintenance 7.8 Replacement procedures 7.8.8 Replace pump head check valves Step Action Unscrew the purge valve by turning it counter-clockwise, and lift off the metal ring. Unscrew the plastic nut of the upper check valve using an adjustable wrench, and gently lift off the upper check valve.
Page 351 7 Maintenance 7.8 Replacement procedures 7.8.8 Replace pump head check valves Step Action Unscrew the two white plastic screws located below each pump head. Pull the plastic connectors to the sides to release the inlet manifold. Unscrew the lower check valve using an adjustable wrench. Replace the lower check valve with a new one.
Page 352: Replace Pump Rinsing System Tubing
7 Maintenance 7.8 Replacement procedures 7.8.9 Replace pump rinsing system tubing 7.8.9 Replace pump rinsing system tubing Maintenance interval Replace the pump rinsing system tubing when required, for example if the tubing is clogged or damaged. Required material The following material is required: •...
Page 353 7 Maintenance 7.8 Replacement procedures 7.8.9 Replace pump rinsing system tubing Connect new tubing Step Action Disconnect the used tubing. Cut the new tubing to desired length. Connect the new tubing according to the previous illustration. Fit all pieces of tubing into the tubing holders on the pump modules. Prime the rinsing systems Before usage, prime the pump rinsing system tubing.
Page 354: Replace A Valve Module
7 Maintenance 7.8 Replacement procedures 7.8.10 Replace a valve module 7.8.10 Replace a valve module Maintenance interval Replace the valve modules when required, for example if a valve module is damaged. Instruction Follow the instruction below to replace a valve module. WARNING Disconnect power.
Page 355 7 Maintenance 7.8 Replacement procedures 7.8.10 Replace a valve module Step Action Remove the valve module. Disconnect the cable and secure it in the slit. Connect the cable to the new valve module. ÄKTA avant User Manual 29035184 AE...
Page 356 7 Maintenance 7.8 Replacement procedures 7.8.10 Replace a valve module Step Action Insert the valve module. Fasten it with a Torx screwdriver. ÄKTA avant User Manual 29035184 AE...
Page 357: Replace Pump Pistons, Piston Seals, O-Rings And Rinse Membranes
7 Maintenance 7.8 Replacement procedures 7.8.11 Replace pump pistons, piston seals, O-rings and rinse membranes 7.8.11 Replace pump pistons, piston seals, O-rings and rinse membranes Introduction Pump pistons, piston seals, O-rings and rinse membranes are pump parts that can be replaced by the user.
Page 358: Troubleshooting
8 Troubleshooting Troubleshooting About this chapter This chapter describes troubleshooting and corrective actions for ÄKTA avant. In this chapter This chapter contains the following sections: Section See page 8.1 Introduction to troubleshooting 8.2 Troubleshooting: General Checklist 8.3 Troubleshooting: Monitors 8.4 Troubleshooting: Valves 8.5 Troubleshooting: Fraction collector 8.6 Troubleshooting: Pumps 8.7 Troubleshooting: Other components...
Page 359: Introduction To Troubleshooting
In this document, search for solutions in the section corresponding to the problem. Make the recommended corrective actions. If problems remain after corrective actions, generate a System error report and contact your local GE representative. ÄKTA avant User Manual 29035184 AE...
Page 360 Select location for the report and click Finish to generate the report. The • filename of the zip file will be and the default Report_YYYYMMDD.zip folder location is: C:Program Files\GE Healthcare\UNICORN E-mail the report to GE Service department. ÄKTA avant User Manual 29035184 AE...
Page 361: Troubleshooting: General Checklist
8 Troubleshooting 8.2 Troubleshooting: General Checklist Troubleshooting: General Checklist Introduction Check the items in the following topics before starting more in-depth troubleshooting work. System checks • Is the correct system selected in UNICORN System Control? For more details, see Section 5.3 Start UNICORN and connect to the system, on page 162. •...
Page 362 Do the buffers have correct pH? The pH of some buffers changes with the tempera- ture. • Are the UV-wavelengths used by the method appropriate with respect to used buffers and proteins? For more details see GE Method Handbooks. ÄKTA avant User Manual 29035184 AE...
Page 363: Troubleshooting: Monitors
8 Troubleshooting 8.3 Troubleshooting: Monitors Troubleshooting: Monitors In this section • UV monitor and UV detector • Conductivity monitor • pH monitor and pH valve • Pressure monitors UV monitor U9-M and UV detector unit Problem Possible cause and action The UV module is not Communication problem •...
Page 364 8 Troubleshooting 8.3 Troubleshooting: Monitors Problem Possible cause and action Too low UV lamp inten- The detector is not correctly fitted • sity Check that the detector is fitted correctly. See Sec- tion 7.8.4 Replace the UV monitor U9-M flow cell, on page 342 .
Page 365 8 Troubleshooting 8.3 Troubleshooting: Monitors Problem Possible cause and action The internal tempera- The air intake on the rear or on the left side of the • ture of the UV monitor instrument is covered is too high Make sure that none of the air intakes on the instru- ment are covered.
Page 366 8 Troubleshooting 8.3 Troubleshooting: Monitors Problem Possible cause and action Ghost peaks Air in the UV flow cell • Use the Flow restrictor. Use the pH valve instruction to manually set the Flow restrictor inline (Flow path:pH valve:Restrictor in-line), or select the Flow restrictor in the Method Settings phase of a method.
Page 367 8 Troubleshooting 8.3 Troubleshooting: Monitors Problem Possible cause and action Baseline drift or noisy Flow restrictor in off-line position • signal Use the Flow restrictor. Use the pH valve instruction to manually set the Flow restrictor inline (Flow path:pH valve:Restrictor in-line), or select the Flow restrictor in the Method Settings phase of a method.
Page 368 8 Troubleshooting 8.3 Troubleshooting: Monitors Problem Possible cause and action Unstable signal Bad pump function • Check that the pump is operating properly. See Exam- ples of pump pressure curves, on page 400 for example of pump pressure curves. Poor mixing function •...
Page 369 8 Troubleshooting 8.3 Troubleshooting: Monitors Problem Possible cause and action The internal tempera- The air intake on the rear or on the right side of the • ture of the UV monitor instrument is covered is too high Make sure that none of the air intakes on the instru- ment are covered.
Page 370 8 Troubleshooting 8.3 Troubleshooting: Monitors Problem Possible cause and action Ghost peaks Air in the UV flow cell • Use the Flow restrictor. Use the pH valve instruction to manually set the Flow restrictor inline (Flow path:pH valve:Restrictor in-line), or select the Flow restrictor in the Method Settings phase of a method.
Page 371 8 Troubleshooting 8.3 Troubleshooting: Monitors Problem Possible cause and action Distorted protein peaks Rapid changes of the refractive index • in IEX gradients (for ex- The refractive index of the buffer changes rapidly in ample step gradients). quick IEX gradients. The rapid change may cause light spreading effects and disturb the shape of the protein peaks in the U9-L 2 mm flow cell.
Page 372 8 Troubleshooting 8.3 Troubleshooting: Monitors Conductivity monitor Problem Possible cause and action The Conductivity moni- The Conductivity monitor cable has come loose • tor is not found by the Switch off the instrument. instrument Reconnect the Conductivity monitor cable (the black cable in the illustration below).
Page 373 8 Troubleshooting 8.3 Troubleshooting: Monitors Problem Possible cause and action Baseline drift of noisy Air in the Conductivity flow cell • signal Use the Flow restrictor. Remove the air by flushing the flow cell with water or buffer. Leaking tubing connections •...
Page 374 8 Troubleshooting 8.3 Troubleshooting: Monitors Problem Possible cause and action Waves on the gradient Bad pump function • Check that the pump is operating properly. See Exam- ples of pump pressure curves, on page 400 for example of pump pressure curves. Air in the flow path •...
Page 375 8 Troubleshooting 8.3 Troubleshooting: Monitors Problem Possible cause and action Incorrect or unstable The temperature compensation factor is not prop- • reading erly set Use a temperature compensation factor. The temper- ature compensation factor is found in System Con- trol:System Settings:Conductivity. Instruction regard- ing the factor is also found in Section 7.7.3 Calibrate the Conductivity Monitor, on page 327.
Page 376 8 Troubleshooting 8.3 Troubleshooting: Monitors Problem Possible cause and action The internal valve tem- The air intake on the rear or on the left side of the • perature is too high instrument is covered Make sure that none of the air intakes on the instru- ment are covered.
Page 377 8 Troubleshooting 8.3 Troubleshooting: Monitors Problem Possible cause and action Unstable pH signal Calibration time out • Check the connections between pH electrode and pH monitor. Regenerate the pH electrode. Place the electrode in deionized water for 30 minutes followed by 30 minutes in a buffer with pH 4.
Page 378 8 Troubleshooting 8.3 Troubleshooting: Monitors Problem Possible cause and action Drift of pH signal when Decreasing salt concentration in the electrode • the pH electrode has membrane due to osmosis to buffer been removed from Regenerate the pH electrode. Place the electrode in storage solution deionized water for 30 minutes followed by 30 minutes in a buffer with pH 4.
Page 379 8 Troubleshooting 8.3 Troubleshooting: Monitors Pressure monitors Problem Possible cause and action Pressure offset The monitors have lost their calibration • Calibrate the pressure monitors. See Section 7.7.2 Calibrate the pressure monitors, on page 323. The temperature has changed • Wait until the temperature has stabilized and calibrate the pressure monitors.
Page 380 8 Troubleshooting 8.3 Troubleshooting: Monitors Problem Possible cause and action Irregular pressure Air bubbles are passing through or are trapped in • curves the pump Check that there is a sufficient volume of buffer present in the flasks. Check all connections and tubing for leaks and con- strictions.
Page 381: Troubleshooting: Valves
8 Troubleshooting 8.4 Troubleshooting: Valves Troubleshooting: Valves General The following table lists the general problems that may occur for the different valves. Problem Possible cause and action The valve is not found The cable between the valve and the ICU is not •...
Page 382 8 Troubleshooting 8.4 Troubleshooting: Valves Inlet valves Problem Possible cause and action Faulty air sensor in the Hardware error • valve Restart the instrument with the power switch. If this error is recurrent, generate a System error report and contact Service. pH valve Problem Possible cause and action...
Page 383 8 Troubleshooting 8.4 Troubleshooting: Valves Quaternary valve Problem Possible cause and action Wrong mixing ratio for The sum of the flow from inlet Q1-Q4 is not equal • Quaternary valve to 100% of the total flow Make sure that the sum of the flow from inlet Q1-Q4 is equal to 100% of the total flow.
Page 384: Troubleshooting: Fraction Collector
8 Troubleshooting 8.5 Troubleshooting: Fraction collector Troubleshooting: Fraction collector In this section • Built-in Fraction collector • Fraction collector F9-R Built-in fraction collector Note: Only ÄKTA avant 25 has DropSync sensors. Problem Possible cause and action The Fraction collector The cable between the Fraction collector and the •...
Page 385 8 Troubleshooting 8.5 Troubleshooting: Fraction collector Problem Possible cause and action Fraction collection Too many commands are pending in the Fraction • tube or well is over- collector filled and fractionation The reason could be that too many Fraction collector movements are lost.
Page 386 8 Troubleshooting 8.5 Troubleshooting: Fraction collector Problem Possible cause and action The Dispenser head Air in the flow path • failed to detect a drop Check the flow path for air. Fill system and purge pumps according to Section 5.4 Prime inlets and purge pump heads, on page 165.
Page 387 8 Troubleshooting 8.5 Troubleshooting: Fraction collector Problem Possible cause and action The Dispenser head Too high flow rate • failed to detect a drop Decrease the flow rate. and has switched to Air in the flow path tube change without •...
Page 388 Incorrect positioning of the Cassette tray in the Frac • drawer Make sure that the front of the tray (marked with the GE logo) is facing the front of the Frac drawer. See Section 5.8.1 Prepare the built-in fraction collector, on page 203. Dirt under the Cassette tray Remove the dirt.
Page 389 8 Troubleshooting 8.5 Troubleshooting: Fraction collector Problem Possible cause and action The loaded Cassette Some of the tubes or plates are incorrectly placed • tray cannot be inserted in the Cassettes into the Frac chamber Check that all tubes and plates are correctly inserted in the Cassettes.
Page 390 8 Troubleshooting 8.5 Troubleshooting: Fraction collector Problem Possible cause and action The Frac chamber is The light has been turned off in UNICORN • dark Turn on the light in UNICORN. In System Control, select System:Settings. Navigate to Fraction collector:Frac- tion collector lamp and select Lamps On.
Page 391 8 Troubleshooting 8.5 Troubleshooting: Fraction collector Problem Possible cause and action Liquid on the floor to Leakage in the Frac arm • the right of the Cas- Contact service. sette tray Spillage by the Fraction Unclean DropSync sensor diode windows or unclean •...
Page 392 8 Troubleshooting 8.5 Troubleshooting: Fraction collector Problem Possible cause and action The Fraction collector Quick scan has not detected the correct Cassette • fractionates in the Clean the Cassette type code of the Cassette. See wrong well or tube Section 7.6.4 Clean built-in fraction collector, on page 301.
Page 393 Detector - Outlet valve: 260 μl Restrictor volume: 85 μl To change the Fraction collector capillary from i.d. 0.5 to i.d. 0.75 mm, contact GE Service. Note: If the Fraction collector capillary is also changed, the delay volume, Detector - Frac, has to be changed to 950 μl.
Page 394 8 Troubleshooting 8.5 Troubleshooting: Fraction collector Problem Possible cause and action The fractionation The Frac drawer has been opened between two runs. • starts in the first row When the instrument is in state Ready and the again drawer is opened and closed, the Fraction collector content is reset.
Page 395 8 Troubleshooting 8.5 Troubleshooting: Fraction collector Problem Possible cause and action The internal tempera- Hot surroundings • ture of the fraction col- Decrease the room temperature. Maximum operating lector is too high temperature is 35°C. Hardware error • Switch off the instrument and wait until the tempera- ture has decreased.
Page 396 8 Troubleshooting 8.5 Troubleshooting: Fraction collector Problem Possible cause and action Fraction numbering Fraction numbering continues from where it was at the does not start at 1 time of the No tube error when the fractionation Manually reset the fraction number in the System settings is restared after a No menu.
Page 397: Troubleshooting: Pumps
8 Troubleshooting 8.6 Troubleshooting: Pumps Troubleshooting: Pumps In this section • Troubleshooting for System pumps and Sample pumps • Example of pump pressure curves • Remove persistent air bubbles Pumps Problem Possible cause and action Liquid is leaking be- Piston seal or rinsing membrane incorrectly fitted •...
Page 398 8 Troubleshooting 8.6 Troubleshooting: Pumps Problem Possible cause and action Leakage around a con- Leaking connection and/or crystallized material • nector around a connector Unscrew the connector and check if it is worn or incor- rectly fitted. If so, replace the connector. Gently finger tighten the connector.
Page 399 8 Troubleshooting 8.6 Troubleshooting: Pumps Problem Possible cause and action High pressure alarm The pressure has increased due to increased viscos- • The viscosity increases in cold room. Lower the flow when performing runs in a cold room. High pressure alarm The parameter selected for pressure control is not •...
Page 400 8 Troubleshooting 8.6 Troubleshooting: Pumps Problem Possible cause and action Pressure overshoot or Too high I factor in the Pressure control parameters • oscillating pressure instruction when pressure control In the Manual Instructions dialog box, decrease the is active I factor of the Pressure control parameters instruc- tion.
Page 401 8 Troubleshooting 8.6 Troubleshooting: Pumps Incident: A large volume of air enters pump. Both pump heads fails to deliver buffer. Action: Purge pump. See Section 5.4.1 Prime buffer inlets and purge system pumps, on page 166. Incident: Blocked outlet check valve Action: Clean the check valve, See Section 7.6.9 Clean pump head check valves, on page 312.
Page 402 8 Troubleshooting 8.6 Troubleshooting: Pumps Incident: Internal leaking inlet check valve. Action: Action: Try first to clean the check valve, see Section 7.6.9 Clean pump head check valves, on page 312. If that does not help replace the check valve, see Sec- tion 7.8.8 Replace pump head check valves, on page 349.
Page 403 8 Troubleshooting 8.6 Troubleshooting: Pumps Air segment test As shown in the previous pressure curves, very often malfunction in inlet or outlet check valves gives the same increased pressure curve pulsation. Sometimes the "air segment test" can help determine if the problem is on the inlet side or the outlet side. •...
Page 404: Troubleshooting: Other Components
8 Troubleshooting 8.7 Troubleshooting: Other components Troubleshooting: Other components In this section • General Hardware: All modules • Mixer • Superloop • Cabinet • Power and ICU • External air sensors • I/O-box E9 • Instrument display General hardware: All modules Problem Possible cause and action Siphoning occurs...
Page 405 8 Troubleshooting 8.7 Troubleshooting: Other components Problem Possible cause and action An unknown instru- The Node ID for one or more of the modules is incor- • ment module is con- rect nected to the system Check Node ID and change the Node ID according to Section 9.15 Check and change the Node ID of a mod- ule, on page 515.
Page 406 8 Troubleshooting 8.7 Troubleshooting: Other components Problem Possible cause and action The internal Mixer The air intake on the rear or on the left side of the • temperature is too instrument is covered high Make sure that none of the air intakes on the instru- ment are covered.
Page 407 8 Troubleshooting 8.7 Troubleshooting: Other components Problem Possible cause and action The temperature of the The air intake on the back or on the left side of the • instrument or an instru- instrument is covered ment component is too Make sure that none of the air intakes on the instru- high ment are covered.
Page 408 8 Troubleshooting 8.7 Troubleshooting: Other components Problem Possible cause and action One or more modules One or some of the minor modules use too much • are automatically current. Minor modules include all modules except turned off the Fraction collector, the UV monitor and the pumps.
Page 409 8 Troubleshooting 8.7 Troubleshooting: Other components Problem Possible cause and action The internal instru- The air intake at the rear or on the right side of the • ment temperature is instrument is covered too high Make sure that none of the air intakes on the instru- ment are covered.
Page 410 8 Troubleshooting 8.7 Troubleshooting: Other components Problem Possible cause and action Liquid is leaking from One of the connections is not tight enough • the external air sensor Tighten the connectors. The internal tempera- Hot surroundings • ture of the air sensor is Decrease the room temperature.
Page 411 8 Troubleshooting 8.7 Troubleshooting: Other components Problem Possible cause and action External digital equip- Cables incorrectly connected • ment does not respond Make sure that the cables are connected correctly, to set changes in Digi- earth to earth etc. tal Out To isolate the problem: 1 Connect Digital Out (e.g.
Page 412 8 Troubleshooting 8.7 Troubleshooting: Other components Problem Possible cause and action Weak light Display is worn out • Generate a System error report and contact Service. The internal tempera- The main board for the display is overheated • ture of the instrument Switch off the instrument and wait until the tempera- display is too high ture has decreased.
Page 413: Troubleshooting: Instrument Communication
8 Troubleshooting 8.8 Troubleshooting: Instrument communication Troubleshooting: Instrument communication Scenario Possible cause and action Error message in Cause: Conductivity monitor cable is not connected. UNICORN: (Error) The Action: first conductivity moni- 1 Switch off the instrument. tor has been reported lost by the instrument...
Page 414 8 Troubleshooting 8.8 Troubleshooting: Instrument communication Scenario Possible cause and action Warning messages in Cause: Two or several modules have the same Node ID. UNICORN: (Warning) Action: Two instrument mod- 1 Switch off the instrument. ules have the same Node ID. 2 Check the Node ID for all modules, see Section 9.15 Check and change the Node ID of a module, on page 515.
Page 415: Error Codes
8 Troubleshooting 8.9 Error codes Error codes Introduction This section describes the error codes that can appear for the different modules, together with corrective actions. All modules Error Description Action code 0 - 19 Internal instrument error Restart the instrument. If recurrent please contact Service.
Page 416 8 Troubleshooting 8.9 Error codes Pressure monitor Error Description Action code 20, 21, Internal instrument error Restart the instrument. If recurrent please 24-27 contact Service. High temperature See Pressure monitors, on page 379. Air sensor Error Description Action code High temperature See External air sensors, on page 409.
Page 417 8 Troubleshooting 8.9 Error codes Conductivity monitor Error Description Action code 20-27 Internal instrument error Restart the instrument. If recurrent please contact Service. High temperature See Conductivity monitor, on page 372 Temperature data error Restart the instrument. If recurrent please contact Service.
Page 418 8 Troubleshooting 8.9 Error codes Error Description Action code 28, 29 Lamp error Restart the instrument. If recurrent please contact Service. High temperature See UV monitor U9-M and UV detector unit, on page 363. UV Detector U9-D Error Description Action code 24, 26, 28, Internal instrument error...
Page 419 8 Troubleshooting 8.9 Error codes UV Monitor U9-L Error Description Action code High temperature See UV monitor U9-L, on page 368. 52, 55 Low lamp intensity Contact Service. Autozero out of range Autozero requested when AU value is larger than 2. Too low light intensity, S No light through flow cell.
Page 420 8 Troubleshooting 8.9 Error codes Instrument display Error Description Action code High temperature See Instrument display, on page 411 I/O-box Error Description Action code High temperature See I/O-box, on page 410 Analog in signal below -2V Check the external equipment connected to the I/O-box.
Page 421: Reference Information
9 Reference information Reference information About this chapter This chapter lists reference details for ÄKTA avant. Refer to ÄKTA avant 25 Product Documentation and ÄKTA avant 150 Product Documen- tation for detailed technical specifications. In this chapter This chapter contains the following sections: Section See page 9.1 System specifications...
Page 422: System Specifications
9 Reference information 9.1 System specifications System specifications Technical specifications Parameter Data System configuration Benchtop system, external computer Control system UNICORN™ 6.3.2 or later version Connection between PC and instrument Ethernet Dimensions (Length x Depth x Height) 860 x 710 x 660 mm Weight (excluding computer) 116 kg Power supply...
Page 423 9 Reference information 9.1 System specifications Enviromental requirements Parameter Data Storage and transport temperature range -25°C to 60°C Chemical environment See Section 9.4 Chemical resistance guide, on page 440. Operating temperature range 4°C to 35°C Relative humidity 20% to 95%, noncondensing Equipment noise level Equipment Acoustic noise...
Page 424: Module Specifications
9 Reference information 9.2 Module specifications Module specifications Introduction This section specifies the operating data of the components in ÄKTA avant. For general data for the system see Section 9.1 System specifications, on page 422. System pumps Parameter Data Pump type Piston pump, metering type Flow rate range (normal range) ÄKTA avant 25: 0.001 to 25 ml/min...
Page 425 9 Reference information 9.2 Module specifications Sample pump Parameter Data Pump type Piston pump, metering type Flow rate range ÄKTA avant 25: 0.001 to 50 ml/min ÄKTA avant 150: 0.01 to 150 ml/min Pressure range ÄKTA avant 25:0 to 10 MPa (0 to 100 bar) ÄKTA avant 150: 0 to 5) MPa (0 to 50 bar) Viscosity range 0.7 to 10 cP...
Page 426 9 Reference information 9.2 Module specifications Gradient formation Parameter Data Gradient flow rate range ÄKTA avant 25: Binary: 0.25 to 25 ml/min • Quaternary: 1 to 25 ml/min • ÄKTA avant 150: Binary: 1 to 150 ml/min • Quaternary: 2 to 40 ml/min •...
Page 427 9 Reference information 9.2 Module specifications Number of inlets Parameter Data Inlet A 7, expandable to 14 Inlet B 7, expandable to 14 Sample inlet 7, expandable to 14 Quaternary inlet Pressure monitors Parameter Data Number of sensors Placement of sensors The System pressure monitor is located after the System •...
Page 428 9 Reference information 9.2 Module specifications Item Description Resolution 0.001 mAU Linearity U9-M: within ± 2% at 0 to 2 AU U9-L: within ± 5% at 0 to 2 AU Drift U9-M (2 mm cell at 280 nm): ≤ | 0.2 mAU | AU/h U9-L (2 mm cell): ≤...
Page 429 9 Reference information 9.2 Module specifications Conductivity monitor Item Description Number of monitors Up to 2 Conductivity reading range 0.01 to 999.99 mS/cm Accuracy ±0.01 mS/cm or ±2%, whichever is greater, (within 0.3 to 300 mS/cm) Operating pressure 0 to 5 MPa (0 to 50 bar) Flow cell volume 22 µl Temperature monitor integrated...
Page 430 9 Reference information 9.2 Module specifications Parameter Data Fraction volumes ÄKTA avant 25: 0.1 to 20 000 ml ÄKTA avant 150: 1 to 20 000 ml Delay volume, with pH electrode and ÄKTA avant 25: 142 µl flow restrictor off-line (UV – Outlet valve) ÄKTA avant 150: 535 µl Built-in fraction collector Item...
Page 431 9 Reference information 9.2 Module specifications Parameter Data Spillage-free mode DropSync Fractionate flammable liquids Delay volume, with pH electrode and ÄKTA avant 25: 240 µl flow restrictor off-line ÄKTA avant 150: 928 µl (UV – dispenser head) Dimensions (W x D x H) 320 x 400 x 250 mm Weight 5 kg...
Page 432: Tubing And Connectors
9 Reference information 9.3 Tubing and connectors Tubing and connectors Introduction This section describes the tubing and connectors to use with ÄKTA avant 25 and ÄKTA avant 150. Tubing labels The illustration below shows the tubing labels for for the standard system configuration. ÄKTA avant User Manual 29035184 AE...
Page 433 9 Reference information 9.3 Tubing and connectors Tubing types The table below shows the tubing types used in ÄKTA avant. Description Color Scope of use Volume/cm PEEK, o.d. 1/16", i.d. 0.25 Blue High pressure tubing 0.5 μl Reference capillary 1 PEEK, o.d.
Page 434 9 Reference information 9.3 Tubing and connectors Description Use with tubing... Tubing connector 5/16" + ETFE, o.d. 1/16", i.d. 0.75 mm Ferrule (blue), 1/16" Tubing connector 5/16" + ferrule FEP o.d. 3/16" i.d. 2.9 mm (blue) 3/16" Tubing connector 5/16" + FEP, o.d.
Page 435 9 Reference information 9.3 Tubing and connectors Label Description Tubing Length Volume (ml) (mm) ÄKTA avant ÄKTA avant ÄKTA avant ÄKTA avant S1 to Inlets to Sample FEP, o.d. FEP, o.d. 1000 inlet valve 1/8", i.d. 1.6 3/16", i.d. 2.9 Q1 to Inlets to Quate- FEP, o.d.
Page 436 9 Reference information 9.3 Tubing and connectors Label Description Tubing Length Volume (μl) (mm) ÄKTA avant ÄKTA avant ÄKTA avant ÄKTA avant System pump A PEEK, o.d. PEEK, o.d. right to Restrictor 1/16", i.d. 1/16", i.d. 1.0 0.75 mm Restrictor A to PEEK, o.d.
Page 437 9 Reference information 9.3 Tubing and connectors Label Description Tubing Length Volume (μl) (mm) ÄKTA avant ÄKTA avant ÄKTA avant ÄKTA avant Injection valve to PEEK, o.d. PEEK, o.d. Column valve 1/16", i.d. 1/16", i.d. 1.0 0.50 mm Column valve to PEEK, o.d.
Page 438 9 Reference information 9.3 Tubing and connectors Optional high pressure tubing Label Description Tubing Length Volume (μl) (mm) ÄKTA avant ÄKTA avant ÄKTA avant ÄKTA avant Injection valve PEEK, o.d. PEEK, o.d. port LoopF to 1/16", i.d. 1/16", i.d. 1.0 Loop valve port F 0.50 mm Injection valve...
Page 439 9 Reference information 9.3 Tubing and connectors Reference tubing The table below shows the label, diameter, and standard length of the reference capillary. The capillary is used during the System performance tests. The capillary is not mounted at delivery. Label Description Tubing Length...
Page 440: Chemical Resistance Guide
9 Reference information 9.4 Chemical resistance guide Chemical resistance guide Introduction This section provides general information about biocompatibilty and detailed information about chemical resistance of the ÄKTA avant instrument. In this section Section See page 9.4.1 General information about biocompatibility and chemical resis- tance 9.4.2 Chemical resistance specifications ÄKTA avant User Manual 29035184 AE...
Page 441: General Information About Biocompatibility And Chemical Resistance
9 Reference information 9.4 Chemical resistance guide 9.4.1 General information about biocompatibility and chemical resistance 9.4.1 General information about biocompatibility and chemical resistance Biocompatibility The ÄKTA avant instrument is designed for maximum biocompatibility, with biochemically inert flow paths constructed mainly from titanium, PEEK and highly resistant fluoropoly- mers and fluoroelastomers.
Page 442 9 Reference information 9.4 Chemical resistance guide 9.4.1 General information about biocompatibility and chemical resistance Note: Chemical influences are time and pressure dependent. Unless otherwise stated, all concentrations are 100%. ÄKTA avant User Manual 29035184 AE...
Page 443: Chemical Resistance Specifications
This section provides detailed information about chemical resistance of the ÄKTA avant instrument to some of the most commonly used chemicals in liquid chromatography. Regarding exposure to solutions not covered by this information, contact your GE repre- sentative for recommendations.
Page 444 9 Reference information 9.4 Chemical resistance guide 9.4.2 Chemical resistance specifications Chemical Concentra- CAS no/EC no tion Sodium hydroxide/ethanol 1 M/40% Sodium chloride 7647-14-5/ 231-598-3 Sodium hypochlorite 7681-52-9/231-668-3 If hydrochloric acid, HCl, is used as a cleaning agent when columns are connected to the system, the HCl concentration should not exceed 0.1 M in the pressure sensors.
Page 445 9 Reference information 9.4 Chemical resistance guide 9.4.2 Chemical resistance specifications Chemical Concentra- CAS no/EC no tion Ethanol 100% 75-08-1/ 200-837-3 Isopropanol 100% 67-63-0/ 200-661-7 Methanol 100% 74-93-1/ 200-659-6 Water/organic mobile Max 5% phase/formic acid formic acid Mobile phase system. Note: It is recommended to replace the mixer sealing ring with the highly resistant O-ring (product code 29-0113-26) if the system is to be exposed to organic...
Page 446 9 Reference information 9.4 Chemical resistance guide 9.4.2 Chemical resistance specifications Chemical Concentra- CAS no/EC no tion Benzyl alcohol 100-51-6/ 202-859-9 Dithioerythritol (DTE) 100 mM 3483-12-3 / 222-468-7 Dithiothreitol (DTT) 100 mM 3483-12-3 / 222-468-7 Ethylenediaminetetraacetic 100 mM 60-00-4/ 200-449-4 acid (EDTA) Mercaptoethanol 20 mM...
Page 447: Wetted Materials
9 Reference information 9.5 Wetted materials Wetted materials Introduction The tables below list the materials that come into contact with process fluids in the ÄKTA avant system. Note: For more detailed material information, see ÄKTA avant 25 Product Documen- tation and ÄKTA avant 150 Product Documentation. Primary flow path Material Abbreviation...
Page 448 9 Reference information 9.5 Wetted materials Material Abbreviation Titanium grade 5 Used in pressure sensors only. Pump rinse system Material Abbreviation EthylenePropyleneDiene M-class rubber EPDM PolyEtherEtherKetone PEEK PolyPropylene PolyPhenylene Sulfide PolyVinylidene DiFluoride PVDF Silicone ÄKTA avant User Manual 29035184 AE...
Page 449: Predefined Methods And Phases
9 Reference information 9.6 Predefined methods and phases Predefined methods and phases Introduction A predefined method contains a set of phases, each phase reflecting a specific stage of a chromatography or maintenance run. You can select additional phases from the phase libraries and add these to an existing method, or remove phases that are not required.
Page 450 9 Reference information 9.6 Predefined methods and phases Predefined purification Principle Included phases method Anion Exchange Chro- After equilibration and sample application, matography (AIEX) negatively charged proteins are adsorbed to the column ligand. After a wash, to re- move unbound sample, elution is per- formed using a gradient of increasing salt concentration ( e.g.
Page 451 9 Reference information 9.6 Predefined methods and phases Predefined purification Principle Included phases method Chromatofocusing (CF) After equilibration and sample application, elution is performed using a pH gradient. The proteins separate and elute according to their isoelectric points. Finally, the col- umn is re-equilibrated.
Page 452 9 Reference information 9.6 Predefined methods and phases Predefined purification Principle Included phases method Hydrophobic Interaction After equilibration and sample application Chromatography (HIC) (use a buffer containing a high salt concen- tration, e.g, 2 M Ammonium Sulphate) hy- drophobic proteins are adsorbed to the column ligand.
Page 453 9 Reference information 9.6 Predefined methods and phases Predefined purification Principle Included phases method Reversed Phase Chro- After equilibration and sample application, matography (RPC) hydrophobic proteins adsorb to the column ligand. After a wash to remove unbound sample, elution is performed by generating a gradient of a non-polar, organic solvent such as acetonitrile.
Page 454 9 Reference information 9.6 Predefined methods and phases Predefined maintenance Principle Included phases method Column CIP The column is filled with a cleaning solu- tion. Select inlet positions. Enter the solu- tion identity, volume, flow rate and incuba- tion time. By adding steps, several cleaning solutions can be used.
Page 455 9 Reference information 9.6 Predefined methods and phases Predefined maintenance Principle Included phases method System CIP The system is filled with cleaning solution. Select for example inlets, outlets and col- umn positions to be cleaned. Three System CIP phases are included in the method to facilitate the use of three different cleaning solution.
Page 456 9 Reference information 9.6 Predefined methods and phases Predefined phases The following table describes the predefined phases. Phase Name Description Method Set- The first, and mandatory, phase in any method. Defines common tings parameters used in the subsequent phases. The Method Settings phase defines: 1 Column type Note: The Column type list can be filtered in two steps:...
Page 457 9 Reference information 9.6 Predefined methods and phases Phase Name Description Unit selection for Method base and Flow rate • Monitor settings: • pH monitor Air sensor alarm settings UV monitor Note: The first wavelength of U9-M and the fixed wavelength for U9-L or U9-L 2nd is always turned on.
Page 458 9 Reference information 9.6 Predefined methods and phases Phase Name Description Equilibration Equilibrates the column before purification, or re-equilibrates the column after purification. Manual Loop Is used to manually fill the additional sample application loops Fill mounted on the loop valve. The filling options are: Partial loop fill •...
Page 459: System Settings
9 Reference information 9.7 System settings System settings The System Settings function is used to set the parameters for the available instructions. The Edit dialog box in which to edit the system settings are shown below. The following subsections list the system settings available for ÄKTA avant. Section See page 9.7.1 System settings - UV...
Page 460: System Settings - Uv
9 Reference information 9.7 System settings 9.7.1 System settings - UV 9.7.1 System settings - UV The following table describes the UV related system settings available for ÄKTA avant. Instruction name Description Alarm UV1 Enables or disables the alarm for the UV 1 signal from UV monitor U9-M.
Page 461: System Settings - Conductivity
9 Reference information 9.7 System settings 9.7.2 System settings - Conductivity 9.7.2 System settings - Conductivity The following table describes the conductivity related system settings available for ÄKTA avant. Instruction name Description Alarm conductivity Enables or disables the conductivity alarm. When en- abled, it sets the alarm limits for the conductivity signal.
Page 462 9 Reference information 9.7 System settings 9.7.2 System settings - Conductivity Instruction name Description Cond temp condensation Is used to adjust the conductivity values to a reference temperature in order to compare conductivity values between runs that have been performed at different temperatures.
Page 463: System Settings - Ph
9 Reference information 9.7 System settings 9.7.3 System settings - pH 9.7.3 System settings - pH The table below describes the pH related system settings available for ÄKTA avant. Instruction name Description Alarm pH Enables or disables the pH alarm. When enabled, it sets the alarm limits for the pH signal.
Page 464: System Settings - Pumps And Pressures
9 Reference information 9.7 System settings 9.7.4 System settings - Pumps and pressures 9.7.4 System settings - Pumps and pressures The following table describes the pressure alarm related system settings available for ÄKTA avant. Instruction name Description Alarm system pressure Sets the alarm limits for the system pressure.
Page 465 9 Reference information 9.7 System settings 9.7.4 System settings - Pumps and pressures Instruction name Description Alarm delta column pres- Sets the alarm limits for the delta column pressure (pre- sure column pressure minus post-column pressure). When enabled and the delta column pressure falls outside the set pressure limits, an alarm will be triggered and the method will be paused.
Page 466: System Settings - Air Sensor
9 Reference information 9.7 System settings 9.7.5 System settings - Air sensor 9.7.5 System settings - Air sensor The table below describes the air sensor related system settings available for ÄKTA avant. Instruction name Description Alarm air sensors Enables or disables the air sensor alarm for the built-in air sensor at inlet A, inlet B or the sample inlet.
Page 467: System Settings - I/O-Box
9 Reference information 9.7 System settings 9.7.6 System settings - I/O-box 9.7.6 System settings - I/O-box The following table describes the I/O-box related system settings available for ÄKTA avant. Instruction name Description Digital out X Sets the value of the signal sent out by digital port number X to either 0 or 1.
Page 468: System Settings - Fraction Collection
9 Reference information 9.7 System settings 9.7.7 System settings - Fraction collection 9.7.7 System settings - Fraction collection The following tables describe the fraction collection related system settings available for ÄKTA avant. Instruction name Description Fractionation settings Fractionation settings comprises fractionation mode and fractionation order.
Page 469 9 Reference information 9.7 System settings 9.7.7 System settings - Fraction collection Instruction name Description Peak fractionation param- The Peak fractionation parameters set the detection eters parameters for peak collection, that is they decide when a peak starts and ends. This information is used by the instructions Fraction collection:Peak fractionation and Fraction collection:Peak frac in outlet valve in order to start/end the peak collection.
Page 470 9 Reference information 9.7 System settings 9.7.7 System settings - Fraction collection Parameter for Description Delay volumes Detector - Frac Is used to define the delay volume between the monitor and the second Fraction collector. The instruction is used to make sure that the collected fractions correspond to the fractions indicated in the chromatogram.
Page 471: System Settings - Watch Parameters
9 Reference information 9.7 System settings 9.7.8 System settings - Watch parameters 9.7.8 System settings - Watch parameters The following table describes the watch parameter settings available for ÄKTA avant. Instruction name Description Watch UV parameters Sets the accepted signal fluctuation and Delta peak limit of the UV signal for some of the tests in the Watch:Watch and Watch:Hold until instructions.
Page 472: System Settings - Advanced
9 Reference information 9.7 System settings 9.7.9 System settings - Advanced 9.7.9 System settings - Advanced The following table describes the advanced system settings available for ÄKTA avant. Instruction name Description Constant pressure flow Sets the values for the P and I factors needed to keep parameters a constant pressure by varying the flow rate.
Page 473 9 Reference information 9.7 System settings 9.7.9 System settings - Advanced Wash settings The settings for system wash are accessible as parameters to the Advanced:System and pump wash settings and Advanced:Loop wash settings instructions. Parameter for System Description and pump wash settings System flow rate Sets the flow rate used for the Pumps and pressures:Sys- tem wash instruction.
Page 474: System Settings - Data Collection
9 Reference information 9.7 System settings 9.7.10 System settings - Data collection 9.7.10 System settings - Data collection The following table describes the data collection related system settings available for ÄKTA avant. Instruction name Description Name of given curve. E.g., The Maximum number of data points parameter deter- UV1, UV2, Cond mine the maximum number of data points collected for...
Page 475: Using Manual Instructions
9 Reference information 9.8 Using Manual instructions Using Manual instructions It is possible to manually interact with an ongoing method using Manual instructions. Step Action In the System Control module: On the Manual menu. click Execute Manual Instructions • use the shortcut Ctrl +M. •...
Page 476: Manual Instructions - Pumps And Pressures
9 Reference information 9.8 Using Manual instructions 9.8.1 Manual instructions - Pumps and pressures 9.8.1 Manual instructions - Pumps and pressures The following table describes the pump and related manual instructions available for ÄKTA avant. Instruction name Description System flow Defines the system flow rate.
Page 477 9 Reference information 9.8 Using Manual instructions 9.8.1 Manual instructions - Pumps and pressures Instruction name Description Loop wash Is used to wash the Loop valve. It is possible to wash a single sample application loop, all loops or only the by- pass position.
Page 478 9 Reference information 9.8 Using Manual instructions 9.8.1 Manual instructions - Pumps and pressures Instruction name Description System wash BufferPro Is used to fill the system with the selected BufferPro buffer composition and stabilize the pH before the flow is directed to the column. The wash flow can be directed to either the waste position of the injection valve or the outlet valve.
Page 479: Manual Instructions - Flow Path
9 Reference information 9.8 Using Manual instructions 9.8.2 Manual instructions - Flow path 9.8.2 Manual instructions - Flow path The following table describes the flow path related manual instructions available for ÄKTA avant. Instruction name Description Injection valve Sets the Injection valve to the selected position. The in- struction gives an injection mark in the chromatogram when the inlet valve switches to Inject or Direct Inject.
Page 480 9 Reference information 9.8 Using Manual instructions 9.8.2 Manual instructions - Flow path Instruction name Description Versatile valve to Versa- Turns the numbered Versatile valve to the selected po- tile valve 4 sition. Note: Four sets of positions are available. In positions 1-3 and 2-4 only a single flow channel can be used.
Page 481: Manual Instructions - Monitors
9 Reference information 9.8 Using Manual instructions 9.8.3 Manual instructions - Monitors 9.8.3 Manual instructions - Monitors The table below describes the monitor related manual instructions available for ÄKTA avant. Instruction name Description Auto zero UV Sets the UV signals from U9-M to 0 mAU. Auto zero UV 2nd Sets the UV signal from U9-L, 2nd to 0 mAU.
Page 482 9 Reference information 9.8 Using Manual instructions 9.8.3 Manual instructions - Monitors Instruction name Description Relative scale cond 2nd Facilitates monitoring using a second conductivity monitor, of a gradient, for which the user sets the con- ductivity values for 0% and 100%. The Relative scale cond instruction can be set in ascending manner (0% for low and 100% for high conductivity) or in descending manner (0% for high and 100% for low conductivity).
Page 483: Manual Instructions - Fraction Collection
9 Reference information 9.8 Using Manual instructions 9.8.4 Manual instructions - Fraction collection 9.8.4 Manual instructions - Fraction collection The following table describes the fraction collection related manual instructions available for ÄKTA avant. Instruction name Description Fractionation Is used when collecting fractions with a fraction collec- tor.
Page 484 9 Reference information 9.8 Using Manual instructions 9.8.4 Manual instructions - Fraction collection Instruction name Description Accumulator wash Accumulator wash is used to wash the accumulator of built-in the fraction collector with the current solution present in the system. The wash flow rate is set in the instruction Wash settings:Fraction collector wash settings and the current inlet positions are used.
Page 485 9 Reference information 9.8 Using Manual instructions 9.8.4 Manual instructions - Fraction collection Instruction name Description Cassette configuration Only for Fraction collector F9. Cassette configuration is set to either automatic or manual: Automatic: the fraction collector automatically de- • tects the cassette types present in the fraction col- lector.
Page 486 9 Reference information 9.8 Using Manual instructions 9.8.4 Manual instructions - Fraction collection Instruction name Description Feed tube frac 2 During fractionation or peak fractionation the instruction Feed tube frac 2 moves the second Fraction collector tube rack forward one tube after the set delay volume has been collected and a fraction mark is set.
Page 487: Manual Instructions - I/O-Box
9 Reference information 9.8 Using Manual instructions 9.8.5 Manual instructions - I/O-box 9.8.5 Manual instructions - I/O-box The following table describes the I/O-box related manual instructions available for ÄKTA avant. Instruction name Description Auto zero analog in X Sets the value of the analog signal in the analog port number X to 0 mV.
Page 488: Manual Instructions - Alarms
9 Reference information 9.8 Using Manual instructions 9.8.6 Manual instructions - Alarms 9.8.6 Manual instructions - Alarms The following table describes the alarm related manual instructions available for ÄKTA avant. Instruction name Description Alarm system pressure Sets the alarm limits for the system pressure. When enabled and the system pressure falls outside the set pressure limits, an alarm will be triggered and the method will be paused.
Page 489 9 Reference information 9.8 Using Manual instructions 9.8.6 Manual instructions - Alarms Instruction name Description Alarm delta column pres- Sets the alarm limits for the delta column pressure (pre- sure column pressure minus post-column pressure). When enabled and the delta column pressure falls outside the set pressure limits, an alarm will be triggered and the method will be paused.
Page 490 9 Reference information 9.8 Using Manual instructions 9.8.6 Manual instructions - Alarms Instruction name Description Alarm UV 2nd Enables or disables the alarm for the UV signal from UV monitor U9-L, 2nd. When enabled, it sets the alarm limits for the UV signal from U9-L, 2nd. When the UV signal falls outside the set limits, an alarm will be trig- gered and the method will be paused.
Page 491 9 Reference information 9.8 Using Manual instructions 9.8.6 Manual instructions - Alarms Instruction name Description Alarm digital in X Enables or disables the alarm for the signal in digital port number X. The alarm can be triggered by either of the signal values, 0 or 1.
Page 492: Manual Instructions - Advanced
9 Reference information 9.8 Using Manual instructions 9.8.7 Manual instructions - Advanced 9.8.7 Manual instructions - Advanced The following table describes the advanced manual instructions available for ÄKTA avant. Instruction name Description Pressure control parame- By using Pressure control the method can be run with ters the set flow rate without the risk of method stop due to pressure alarm.
Page 493 9 Reference information 9.8 Using Manual instructions 9.8.7 Manual instructions - Advanced Instruction name Description Column packing flow Is used to set flow rates over 25 ml/min and 150 ml/min for ÄKTA avant 25 and ÄKTA avant 150, respectively. Both A and B pumps are used to generate the flow, making it possible to set flow rates up to 50 ml/min and 300 ml/min for ÄKTA avant 25 and ÄKTA avant 150, re- spectively.
Page 494 9 Reference information 9.8 Using Manual instructions 9.8.7 Manual instructions - Advanced Instruction name Description Stop volume count Stops the volume counter function. The counted volume is stored in the memory and can be recalled with the instruction Hold counted volume. The counted volume can also be recalled in following runs and is stored until a new Stop volume count instruction is issued.
Page 495: Manual Instructions - Watch Parameters
9 Reference information 9.8 Using Manual instructions 9.8.8 Manual instructions - Watch parameters 9.8.8 Manual instructions - Watch parameters The following table describes the watch parameter instructions available for ÄKTA avant. Instruction name Description Watch UV parameters Sets the accepted signal fluctuation and Delta peak limit of the UV signal for some of the tests in the Watch and Hold until instructions.
Page 496: Manual Instructions - Other
9 Reference information 9.8 Using Manual instructions 9.8.9 Manual instructions - Other 9.8.9 Manual instructions - Other The following table describes the other manual instructions available for ÄKTA avant. Instruction name Description Set mark Inserts a mark into the current chromatogram with the text entered for the parameter Mark text .
Page 497: Available Run Data
9 Reference information 9.9 Available Run data Available Run data Run data The following table lists all available Run data for ÄKTA avant. Run Data Range/Unit Description System state Status of connection and run. Acc. Volume Total accumulated volume in the cur- rent method or manual run.
Page 498 9 Reference information 9.9 Available Run data Run Data Range/Unit Description Sample flow lin- cm/h The set flow velocity of the sample pump. Only available if a column is se- lected. Sample pressure -1.00 to 10.00 MPa The sample pressure signal (at the (ÄKTA avant 25) sample pump).
Page 499 9 Reference information 9.9 Available Run data Run Data Range/Unit Description Conc Q3 0.0% to 100.0% The set concentration of Q3 or the current value during a quaternary or BufferPro gradient Conc Q4 0.0% to 100.0% The set concentration of Q4 or the current value during a quaternary or BufferPro gradient Cond...
Page 500 9 Reference information 9.9 Available Run data Run Data Range/Unit Description Valve X2 The set position of the X2 valve. Air inlet A No air, Air The current state of the air alarm for the integrated air sensor in inlet valve Air inlet B No air, Air The current state of the air alarm for...
Page 501: 9.10 Available Curves
9 Reference information 9.10 Available Curves 9.10 Available Curves Curves The table lists data available in curves for ÄKTA avant. Curve Range Sam- Description pling frequen- UV 1_280 -6000.000 to 6000.000 10 Hz The first UV/Vis absorbance signal of the U9-M monitor. UV 2_0 -6000.000 to 6000.000 2 Hz...
Page 502 9 Reference information 9.10 Available Curves Curve Range Sam- Description pling frequen- Sample flow 0.001 to 50.000 1 Hz The set flow rate of the sample ml/min (ÄKTA avant pump. 0.01 to 300.00 ml/min (ÄKTA avant 150) Sample flow cm/h 1 Hz The set flow velocity of the linear...
Page 503 9 Reference information 9.10 Available Curves Curve Range Sam- Description pling frequen- Conc Q3 0.0% to 100.0% 0.5 Hz The set concentration of Q3 or the current value during a quaternary or BufferPro gradi- Conc Q4 0.0% to 100.0% 0.5 Hz The set concentration of Q4 or the current value during a quaternary or BufferPro gradi-...
Page 504: 9.11 Injection Volumes And Peak Broadening
9 Reference information 9.11 Injection volumes and peak broadening 9.11 Injection volumes and peak broadening Introduction The width of peaks at the fraction collector is influenced by the following: • the properties of the column, • the dimensions of the tubing, •...
Page 505: 9.12 Delay Volumes
9 Reference information 9.12 Delay volumes 9.12 Delay volumes Introduction A number of methods, both theoretical and experimental, exist for determining the delay volume of a system. The easiest and recommended method is to perform a theoretical determination. Delay volumes for standard configurations are listed in Standard delay volumes in ÄKTA avant 25 and ÄKTA avant 150, on page 507.
Page 506 9 Reference information 9.12 Delay volumes Step Action To obtain the total delay volume, sum up half of the flow cell volume of the monitor used (that is, the UV or UV/Vis monitor) with all volumes of tubing and modules that are located after the monitor in the flow path. Note: For pH-valve V9-pH and V9H-pH always use the volume for the valve in by- pass position.
Page 507 9 Reference information 9.12 Delay volumes Standard delay volumes in ÄKTA avant 25 and ÄKTA avant 150 The table below shows the delay volumes of different sections of the flow path. Flow path Volume (μl) UV detector to built-in fraction collector ÄKTA avant 25: 518 ÄKTA avant 150: 1807 Note:...
Page 508 9 Reference information 9.12 Delay volumes Use of different monitors for peak fractionation in the same method If different monitors or detectors are used for peak fractionation in different parts of the same method, the delay volumes have to be set as method instructions for each of the method parts.
Page 509: 9.13 Component Volumes
9 Reference information 9.13 Component volumes 9.13 Component volumes Introduction This section describes the component volumes and delay volumes of the ÄKTA avant instrument. The table below shows the component volumes of ÄKTA avant. Component Volume (μl) Inlet valve V9-IA, V9-IB, V9-A2, V9-B2 Inlet valve V9H-IA, V9H-IB, V9H-A2, V9H-B2 Sample inlet valve V9-IS, V9-S2 Sample inlet valve V9H-IS, V9H-S2...
Page 510 9 Reference information 9.13 Component volumes Component Volume (μl) Mixer, 5 ml 5000 Mixer, 15 ml 15000 Loop valve V9-L Loop valve V9H-L Versatile valve V9-V Versatile valve V9H-V Injection valve V9-Inj Injection valve V9H-Inj Column valve V9-C,V9-C2 Column valve V9H-C, V9H-C2 UV monitor U9-M: Flow cell 0.5 mm UV monitor U9-M: Flow cell 2 mm UV monitor U9-M: Flow cell 10 mm...
Page 511 9 Reference information 9.13 Component volumes Component Volume (μl) Built-in fraction collector internal tubing ÄKTA avant 25: 31 ÄKTA avant 150: 135 Built-in fraction collector accumulator Built-in fraction collector dispenser head ÄKTA avant 25: 53 ÄKTA avant 150: 68 Fraction collector F9-R tubing ÄKTA avant 25: 98 ÄKTA avant 150: 393 Note:...
Page 512: 9.14 Pressure Control
9 Reference information 9.14 Pressure control 9.14 Pressure control Introduction By using the function Pressure control to regulate the run, the method can be run with the set flow rate without the risk of method stop due to pressure alarm. If the pressure approaches the pressure limit, for example if the sample has higher viscosity than the buffer, the flow rate is automatically lowered.
Page 513 P. Soft media is defined as all GE separation media, except silica and MonoBeads. Rigid media is defined as GE separation media that is based on silica and MonoBeads. Back pressure Using narrow tubing between components will improve resolution but will lead to in- creased back pressure in the system.
Page 514 9 Reference information 9.14 Pressure control Additional instructions for avoiding pressure alarms The instruction Max flow during valve turn sets the maximum flow rate used during the turning of the Injection valve and Outlet valve in order to avoid high pressure alarms. If the flow rate passing through the Injection valve or the Outlet valve is higher than the set max flow rate, the valves will only turn after decreasing the flow to the specified flow rate.
Page 515: Check And Change The Node Id Of A Module
9 Reference information 9.15 Check and change the Node ID of a module 9.15 Check and change the Node ID of a module Introduction Node ID is a unit number designation that is used by the instrument to distinguish be- tween several units of the same type.
Page 516 9 Reference information 9.15 Check and change the Node ID of a module Module Label Node ID pH Valve V9-pH or V9H-pH pH Monitor Note: The pH monitor is included in the pH valve module box. Outlet Valve V9-O or V9H-O UV Monitor U9-M UV detector...
Page 517 9 Reference information 9.15 Check and change the Node ID of a module Module Label Node ID Unused post-column pressure monitor in second Column valve Second Outlet valve V9-O2 or V9H-O2 Third Outlet valve V9-O3 or V9H-O3 External air sensor L9-1.2 or L9-1.5 I/O-box Second I/O-box...
Page 518 9 Reference information 9.15 Check and change the Node ID of a module Check and change the Node ID The Node ID of a module is set by the positions of an arrow of two rotating switches at the back of the module. Follow the instructions to check or change the Node ID. Step Action If applicable, remove the module according to the instruction in Install a...
Page 519: 10 Ordering Information
10 Ordering information 10 Ordering information Introduction This chapter lists accessories and user replaceable spare parts available for ÄKTA avant. Tubing Item Code no. Reference capillary 1 28950749 Reference capillary 2 28950750 ETFE Tubing kit 10×1.0 m, id 1.0 mm, od 1/16″ 28980995 Note: Outlet tubing for ÄKTA avant 25.
Page 520 10 Ordering information Item Code no. Complete tubing marking kit 28956608 Note: Tags for all tubing in ÄKTA avant 25 and ÄKTA avant 150. Inlet/outlet tubing tag kit, ÄKTA avant 25 28981001 Note: Tags for all inlet and outlet tubing in ÄKTA avant 25. Inlet/outlet tubing tag kit, ÄKTA avant 150 28981004 Note:...
Page 521 10 Ordering information Item Product code Union, 5/16" female to M6 male 18112776 Union, 5/16" female to 1/16" male 18114208 Union, M6 female to 1/16" male 18385801 Ferrule for 1/16" tubing connector, blue 18112706 Ferrule for 1/8" tubing connector, yellow 18112118 Ferrule for 3/16"...
Page 522 10 Ordering information Item Code no. Screw lid kit, GL45 11000410 Conversion kits Item Code no. ÄKTA avant Conversion kit, 25 to 150 28980168 Note: Contact Service for installation. ÄKTA avant Conversion kit, 150 to 25 28981861 Note: Contact Service for installation. Pump spare parts Item Code no.
Page 523 10 Ordering information Item Code no. Mixer chamber 5 ml (mounted at delivery in ÄKTA avant 28956246 150) Mixer chamber 15 ml 28980309 O-ring 13.1 × 1.6 mm 28953545 Note: For Mixer chamber 0.6, 1.4, and 5 ml. O-ring 13.1 × 1.6 mm High resistance 29011326 (can be used as an alternative to 28953545) O-ring 22.1 ×...
Page 524 10 Ordering information Item Code no. Inlet Valve V9-X2 28957234 Column Valve V9-C2 28957236 Outlet Valve V9-O2 28957238 Outlet Valve V9-O3 28957240 Versatile Valve V9-V 29011353 Loop Valve V9-L 29011358 ÄKTA avant 150 Item Code no. Column Valve V9H-C 28979241 pH Valve V9H-pH 28979246 Inlet Valve V9H-IA...
Page 525 10 Ordering information Injection valve accessories Item Code no. Sample loop, 10 µl 18112039 Sample loop, 100 µl 18111398 Sample loop, 500 µl (mounted at delivery) 18111399 Sample loop, 1 ml 18111401 Sample loop, 2 ml 18111402 Sample loop, FEP 10 ml 18116124 Superloop, M6 fitting, 10 ml 19758501...
Page 526 10 Ordering information Item Code no. Rack, for 50 ml tubes 28980319 Note: For 55 tubes Rack, for 250 ml bottles 28981873 Fraction Collector F9-R Item Code no. Fraction Collector F9-R 29011362 Tube Rack Complete, 175 × 12 mm 19868403 Tube Rack Complete, 95 ×...
Page 527 10 Ordering information UV monitor Item Code no. UV flow cell U9-0.5, 0.5 mm for U9-M 28979386 UV flow cell U9-2, 2 mm for U9-M 28979380 UV flow cell U9-10, 10 mm for U9-M 28956378 UV Monitor U9-L (fixed wavelength) 29011360 UV flow cell 2 mm for U9-L 29011325...
Page 528 10 Ordering information Module components Item Code no. Module Panel 29011364 Dummy Module 28956493 Extension Box 29110806 Cables Item Code no. Jumper 1 IEC 1394 (F-type) 28956489 External module cable, short (F-type) 29012474 External module cable, long (F-type) 29011366 Flow restrictor Item Code no.
Page 529 10 Ordering information Trays Item Code no. Wet side waste tray 28956487 Front side waste tray 28956485 User Documentation Item Code no. ÄKTA avant User Manual 29035184 Note: Covers ÄKTA avant 25 and ÄKTA avant 150. ÄKTA avant User Manual 29035184 AE...
Page 530: Index
Index Index Cold cabinet, 30 precautions, 30 Advanced system settings, 472 prepare for a run, 31 Affinity chromatography Cold room, 30 predefined method, 449 Column Air sensors cleaning, 237 air sensor adapter, 49 column CIP, 297 description, 65 column holder, 44 error codes, 416 Column valve, 73 external air sensors, 120...
Page 531 Index Elution Hardware installation predefined phase, 457 of a module, 103 Equilibration Holders predefined phase, 458 air sensor adapter, 49 Extension box, 53 bottle holder, 49 column clamp, 45 column holder, 44 column holder rod, 46 Flow cells Extension box, 53 conductivity, 98 flexible column holder, 47 pH, 80...
Page 532 Index Instrument display watch parameters, 495 function, 38 Manual loop fill location, 37 predefined phase, 458 states, 38 Manual Loop Fill Instrument overview predefined method, 452 Fraction collector F9-R, 131 Methods Instrument configura- affinity chromatogra- tions, 16 phy, 449 Intelligent Packing anion exchange, 450 Predefined method, 454 cation exchange, 450...
Page 533 Index UV monitor, 96 column preparation, 457 versatile valve, 128 column wash, 457 Monitors elution, 457 conductivity, 98 equilibration, 458 troubleshooting, 363 Intelligent Packing, 458 UV, 96 manual loop fill, 458 method settings, 456 miscellaneous, 458 predefined phases, 456 Node ID sample application, 458 change Node ID, 518 system CIP, 458...
Page 534 Index Prime inlet tubing B, 166 Prime sample inlets, 173 Safety notices, 9 Process picture, 234 Sample application Pump and pressure set- flow paths, 71 tings, 464 injection valve, 70 Pumps predefined phase, 458 change pump rinsing solu- sample loading, 189 tion, 273 sample loop, 199 clean check valves, 312...
Page 535 Index UV, 460 second, 139 wash settings, 473 UV monitor U9-L watch parameters, 471 description, 139, 158 System storage, 237 error codes, 419 System test system test, 260 UV U9-L, 260 troubleshooting, 368 UV monitor U9-M description, 96, 158 error codes, 417 Technical specifications troubleshooting, 363 component specifica-...
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Page 538 All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them. A copy of these terms and conditions is available on request. Contact your local GE Healthcare repre- sentative for the most current information.

GE AKTA avant User Manual

1 Introduction

2 The ÄKTA Avant Instrument

3 Standard Instrument Modules

4 Optional Instrument Modules

5 Operation

6 Performance Tests

7 Maintenance

8 Troubleshooting

9 Reference Information

10 Ordering Information

Index

Quick Links

Troubleshooting

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Summary of Contents for GE AKTA avant