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1.1

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.
If you do, you may be exposed to hazards that can lead to personal injury and you may
cause damage to the equipment.

Intended use of the product

ÄKTA avant is a liquid chromatography system intended for method and process devel-
opment in purification of biomolecules. The system can be used to screen for optimal
choice of columns, media and running parameters to purify selected proteins.

The ÄKTA avant system is intended for research use only, and shall not be used in any
clinical procedures, or for diagnostic procedures.

Prerequisites

In order to follow this manual and use the system in the manner it is intended, it is im-
portant that:

•

You have a general understanding of how the computer and Microsoft® Windows®
work.

•

You understand the concepts of liquid chromatography.

•

You have read and understood the Safety instructions chapter in ÄKTA avant
Operating Instructions.

•

A user account has been created according to the UNICORN™ Administration and
Technical Manual.

8

ÄKTA avant User Manual 29035184 AE

1 Introduction

1.1 Important user information

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Summary of Contents for AKTA avant

Page 1: ...ÄKTA avant User Manual ...

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Page 3: ...nlet valves 70 3 5 Injection valve 73 3 6 Column valve 76 3 7 Outlet valve 78 3 8 Pressure monitors 80 3 9 pH valve and pH monitor 83 3 10 Built in fraction collector 84 3 10 1 Function 86 3 10 2 Illustrations of the built in fraction collector 89 3 10 3 Cassettes cassette tray and racks 96 3 11 UV monitor 98 3 12 Conductivity monitor 100 4 Optional instrument modules 101 4 1 System extension over...

Page 4: ...ample application using a Superloop 199 5 7 3 Sample application using a sample loop 202 5 8 Fractionation 203 5 8 1 Prepare the built in fraction collector 210 5 8 2 Prepare Fraction collector F9 R 218 5 8 3 Fractionation overview 225 5 9 Create a method and perform a run 226 5 9 1 Create a method 230 5 9 2 Perform a run 233 5 9 3 Monitor the run 236 5 9 4 After run procedures 239 6 Performance t...

Page 5: ...te the pressure monitors 327 7 7 3 Calibrate the Conductivity Monitor 331 7 7 4 Calibrate the UV monitors 335 7 8 Replacement procedures 336 7 8 1 Replace tubing and connectors 338 7 8 2 Replace the Mixer 340 7 8 3 Replace the O ring inside the Mixer 342 7 8 4 Replace the UV monitor U9 M flow cell 345 7 8 5 Replace UV monitor U9 L flow cell 347 7 8 6 Replace Flow restrictor 348 7 8 7 Replace the i...

Page 6: ...tem settings Watch parameters 472 9 7 9 System settings Advanced 474 9 7 10 System settings Data collection 475 9 8 Using Manual instructions 476 9 8 1 Manual instructions Pumps and pressures 479 9 8 2 Manual instructions Flow path 481 9 8 3 Manual instructions Monitors 483 9 8 4 Manual instructions Fraction collection 487 9 8 5 Manual instructions I O box 488 9 8 6 Manual instructions Alarms 492 ...

Page 7: ... system It also includes relevant guidance for practical handling and maintenance of instrument components In this chapter This chapter contains the following sections See page Section 8 1 1 Important user information 10 1 2 ÄKTA avant overview 12 1 3 Associated Documentation ÄKTA avant User Manual 29035184 AE 7 1 Introduction ...

Page 8: ...f biomolecules The system can be used to screen for optimal choice of columns media and running parameters to purify selected proteins The ÄKTA avant system is intended for research use only and shall not be used in any clinical procedures or for diagnostic procedures Prerequisites In order to follow this manual and use the system in the manner it is intended it is im portant that You have a gener...

Page 9: ...s a hazardous situation which if not avoided could result in minor or moderate injury It is important not to pro ceed until all stated conditions are met and clearly understood NOTICE NOTICE indicates instructions that must be followed to avoid damage to the product or other equipment Notes and tips A note is used to indicate information that is important for trouble free and optimal use of the pr...

Page 10: ...tlets columns and integration of optional components The ÄKTA avant instrument is compact with a built in fraction collector Cooling of the built in fraction collector enables secure storage of the purified samples and unattended runs over night The built in Instrument display shows instrument status and method state ÄKTA avant is controlled by the UNICORN software a complete package for control e...

Page 11: ... and editing of run properties Start monitor and control runs The current flow path is illustrated in the Process Picture which allows manual interactions with the system and provides feedback on run parameters System Control Open results evaluate runs and create reports Evaluation The default Evaluation module includes a user interface optimized for workflows like quick evaluation compare results...

Page 12: ...nstructions for handling the system Descrip tions of components Information about how to run and maintain the system ÄKTA avant User Manual 29035184 System specification and declaration of ma terial conformity ÄKTA avant 25 Product Documentation 28991000 OR ÄKTA avant 150 Product Documentation 28984249 1 1 The instrument is delivered with the relevant document UNICORN user documentation The user d...

Page 13: ...ns of the Evaluation Classic module in UNICORN Description of the evaluation algorithms used in UNICORN UNICORN Evaluation Manual1 Overview and detailed description of the system control features in UNICORN Includes general operation system settings and instructions on how to perform a run UNICORN System Control Manual1 1 Current UNICORN version is added to the title of the manual Data files appli...

Page 14: ...or practical tips on chromatography refer to ÄKTA Laboratory scale Chromatography Systems Instrument Management Handbook product code 29010831 14 ÄKTA avant User Manual 29035184 AE 1 Introduction 1 3 Associated Documentation ...

Page 15: ...nstrument modules In this chapter This chapter contains the following sections See page Section 16 2 1 Overview illustrations 30 2 2 Runs in a cold environment 31 2 3 Removing the foldable door and pump cover 35 2 4 Liquid flow path 37 2 5 Instrument display 43 2 6 Accessories ÄKTA avant User Manual 29035184 AE 15 2 The ÄKTA avant instrument ...

Page 16: ...es 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 Illustration of the main parts of the instrument The following illustration shows the location of the main parts of the instrument Function Part Function Part Buffer tray 2 Fraction collector 1 Wet side 4 Instrument display 3 Pump cover 6 Foldable door 5 16 ÄKTA avant User Manual 290351...

Page 17: ...wet side of the instrument 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Function Part Function Part Flow restrictor 2 Injection valve 1 Column valve 4 pH valve 3 Conductivity monitor 6 UV monitor 5 Quaternary valve 8 Inlet valve B 7 Pressure monitor of system pumps 10 System pump B 9 Inlet valve A 12 System pump flow restrictor 11 ÄKTA avant User Manual 29035184 AE 17 2 The ÄKTA avant ins...

Page 18: ...e Label in Module ÄKTA avant 150 ÄKTA avant 25 P9H A P9 A System pump A P9H B P9 B System pump B P9H S P9 S Sample pump R9 R9 Pressure monitor M9 M9 Mixer V9H Inj V9 Inj Injection valve Q9 Q9 Quarternary valve V9H IA V9 IA Inlet valve A V9H IB V9 IB Inlet valve B V9H IS V9 IS Sample inlet valve V9H C V9 C Column valve V9H pH V9 pH pH valve V9H O V9 O Outlet valve U9 M U9 M UV monitor C9 C9 Conduct...

Page 19: ... Inlet valve X1 V9H IX V9 IX Extra Inlet valve X2 V9H S2 V9 S2 Second Sample inlet valve V9H V V9 V Versatile valve V9H L V9 L Loop valve V9H C2 V9 C2 Second Column valve V9H O2 V9 O2 Second Outlet valve V9H O3 V9 O3 Third Outlet valve L9 1 5 L9 1 5 External air sensor L9 1 5 L9 1 2 L9 1 2 External air sensor L9 1 2 E9 E9 I O box U9 L U9 L Second UV monitor C9 C9 Second Conductivity monitor F9 R F...

Page 20: ...e pivot part of the valve head are in dicated on the inside of the black ring e g 3E and 3F Ports located on the valve head front are indicated by an arrow e g E and F Description of standard modules The following modules are installed in the instrument when delivered Description Module A high precision pump which delivers buffer in purification runs System pump A P9 A or P9H A For further informa...

Page 21: ...ivers sample or buffer in purification runs Sample pump P9 S or P9H S For further information refer to Section 3 1 Pumps on page 56 Pressure monitor which reads the system pressure after System Pump A and System Pump B Pressure monitor R9 For further information refer to Section 3 8 Pres sure monitors on page 78 ÄKTA avant User Manual 29035184 AE 21 2 The ÄKTA avant instrument 2 1 Overview illustr...

Page 22: ...er M9 Three mixer chambers are available for ÄKTA avant 25 Available volumes are 0 6 ml 1 4 ml mounted at delivery and 5 ml Three mixer chambers are available for ÄKTA avant 150 Available volumes are 1 4 ml 5 ml mounted at delivery and 15 ml CAUTION Risk of explosion Do not use Mixer chamber 15 ml with an ÄKTA avant 25 sys tem configuration The maximum pressure for Mix er chamber 15 ml is 5 MPa 50...

Page 23: ...or System Pump B with seven inlet ports and integrated air sensor Inlet valve B V9 IB or V9H IB V9 IB B 5 B 4 B3 B2 B 1 B 6 B 7 Q Out For further information refer to Section 3 4 Inlet valves on page 64 Valve which allows automatic mixing of four dif ferent solutions Quaternary valve Q9 For further information refer to Section 3 4 Inlet valves on page 64 ÄKTA avant User Manual 29035184 AE 23 2 The...

Page 24: ...H Inj For further information refer to Section 3 5 Injec tion valve on page 70 LoopE L o o p F S y r W 1 P a S 2 W V9 Inj Column valve which connects up to five columns to the instrument and directs the flow to one column at a time The column valve features two integrated pressure sensors Column valve V9 C or V9H C Allows the user to choose flow direction through the column or to bypass the column...

Page 25: ... monitor on page 80 Valve which directs the flow to the fraction collec tor any of the ten outlet ports or waste Outlet valve V9 O or V9H O V9 O O u t 2 Out1 Frac O u t 1 0 In O u t 4 W Out6 Out3 Out9 Out5 Out7 O u t 8 For further information refer to Section 3 7 Outlet valve on page 76 Monitor which measures the UV Vis absorbance at up to three wavelengths simultaneously in the range 190 to 700 n...

Page 26: ...ector on page 83 Description of optional modules The following modules may be added to the flow path Description Module Second inlet valves for System pump A to extend the number of inlets up to 14 Second Inlet valve A V9 A2 or V9H A2 For further information refer to Section 4 3 Extra inlet valves on page 110 Second inlet valves for System pump B to extend the number of inlets up to 14 Second Inle...

Page 27: ...on refer to Section 4 3 Extra inlet valves on page 110 A 4 port 4 position valve which can be used to cus tomize the flow path Versatile valve V9 V or V9H V For further information refer to Section 4 8 Versatile valve on page 128 V9 V 1 4 2 3 Valve which enables automatic sample applicationfrom up to five sample loops or to collect intermediate frac tions in automated two step purification Loop va...

Page 28: ...s on page 117 Valve which adds 12 outlet ports to the system giving a total of 32 outlets Third Outlet valve V9 O3 or V9H O3 For further information refer to Section 4 5 Extra Outlet valves on page 117 Sensor which prevents air from being introduced into the flow path External air sensor L9 1 5 or L9 1 2 For further information refer to Section 4 6 External air sensors on page 120 Module which rec...

Page 29: ...uctivity of buffers and sample solutions Second Conductivity monitor C9 For further information refer to Section 4 10 Second Conductivity monitor on page 137 Round fraction collector that can collect up to 175 fractions Second Fraction collector F9 R For further information refer to Section 4 9 Fraction collector F9 R on page 130 ÄKTA avant User Manual 29035184 AE 29 2 The ÄKTA avant instrument 2 ...

Page 30: ...instrument is placed in a cold room use a cold room compat ible computer or place the computer outside the cold room and use the Ethernet cable delivered with the instrument to connect to the computer When the instrument is kept in a cold cabinet or room it is important to tighten all tubing connectors also the inlet manifold connectors Otherwise air might get into the flow path Note When runs are...

Page 31: ...or cold cabinet make sure to follow the precautions listed below Remove the foldable door While holding on to the foldable door unscrew the four screws marked in the following illustration The door can then be removed Remove the pump cover Follow the instruction to remove the pump cover Action Step Open the pump cover 1 ÄKTA avant User Manual 29035184 AE 31 2 The ÄKTA avant instrument 2 3 Removing...

Page 32: ...off the tray 2 Unscrew the two countersinked screws marked in the illustration 3 Pull out the two shafts 4 32 ÄKTA avant User Manual 29035184 AE 2 The ÄKTA avant instrument 2 3 Removing the foldable door and pump cover ...

Page 33: ...ews holding the hinge to the pump cover and remove the hinge 6 Screw the three screws back to the pump cover and put the pump cover in a safe place 7 ÄKTA avant User Manual 29035184 AE 33 2 The ÄKTA avant instrument 2 3 Removing the foldable door and pump cover ...

Page 34: ...the hinge and damper and push the hinge into place 8 Put the outer shaft back into place and screw the two countersinked screws back 9 34 ÄKTA avant User Manual 29035184 AE 2 The ÄKTA avant instrument 2 3 Removing the foldable door and pump cover ...

Page 35: ... overview of the standard flow path 6 7 8 9 10 11 12 13 14 15 16 17 19 20 W1 W2 W 3 2 1 18 4 5 Description Part Pressure monitor 1 Sample pump 2 Sample inlet valve 3 Quaternary valve 4 Inlet valve A 5 ÄKTA avant User Manual 29035184 AE 35 2 The ÄKTA avant instrument 2 4 Liquid flow path ...

Page 36: ...er 10 Injection valve 11 Sample loop or Superloop 12 Column valve 13 Column 14 UV monitor 15 Conductivity monitor 16 Flow restrictor 17 pH valve with pH monitor 18 Outlet valve 19 Fraction collector 20 36 ÄKTA avant User Manual 29035184 AE 2 The ÄKTA avant instrument 2 4 Liquid flow path ...

Page 37: ...ment display Location and illustration The following illustration shows the location and a detailed view of the instrument display Description Part System name 1 Information area This example shows the State window 2 Continue button Tap to continue the run after for example a pause 3 Pause button Tap to pause a run 4 The name of the current system state The state is shown even if another message o...

Page 38: ...displayed State of the system Details of the run Network settings State window Tap State to view the State window The State window displays the current state of the system Information about the built in fraction collector is displayed in the lower left frame for each state The table below describes the different states that can be displayed Description Display State The instrument has no communica...

Page 39: ...ctor is not selected in the instrument configuration A run and fractionation is ongoing Run with fractiona tion ongoing Do not open the Frac drawer during fractionation A wash instruction or a pump synchro nization is ongoing Wash A run has been paused by the user Manual pause A run has been paused by UNICORN System pause ÄKTA avant User Manual 29035184 AE 39 2 The ÄKTA avant instrument 2 5 Instru...

Page 40: ...Details window shows run data for the ongoing run This window can be convenient to use when the computer is not located beside the instrument The instrument can for example be located in a cold room The Details window has two tabs The Volumetric tab shows the flow in ml min and the Linear tab shows the flow in cm h The other parameters are presented in the same way in both tabs 40 ÄKTA avant User ...

Page 41: ...tton is enabled only when the system is in state Not connected Note The Touchscreen tab shows an instruction on how to calibrate the touchscreen Buttons and indicators The Instrument display includes the following buttons and indicators Description Indicator Button Indicates if the Instrument display buttons are unlocked or locked The buttons can be locked from UNICORN Sys tem Control Pauses the r...

Page 42: ...from UNICORN Action Step In System Control on the System menu click Settings 1 Result The System Settings dialog box box opens In the System Settings dialog box 2 Select Advanced Instrument display Click Locked or Unlocked Click OK 42 ÄKTA avant User Manual 29035184 AE 2 The ÄKTA avant instrument 2 5 Instrument display ...

Page 43: ...t Optional modules can be installed on the instrument outside the system chassis when using an Extension box This section describes the available holders other accessories and the Extension box In this section This section contains the following subsections See page Section 44 2 6 1 Column holders 48 2 6 2 Tubing holders and other accessories 53 2 6 3 Extension box ÄKTA avant User Manual 29035184 ...

Page 44: ...th outer diameter between 10 and 50 mm The Column holder can also be used for bottles Use two holders to attach long columns 1 2 3 4 Description Part Position for a medium sized column or bottle 1 Position for a small sized column 2 Tab 3 Snap in to holder rails 4 44 ÄKTA avant User Manual 29035184 AE 2 The ÄKTA avant instrument 2 6 Accessories 2 6 1 Column holders ...

Page 45: ...sized columns with outer diameters be tween 10 and 21 mm Use two clamps to attach long columns 1 2 Description Part Position for a column 1 Inner end tabs 2 ÄKTA avant User Manual 29035184 AE 45 2 The ÄKTA avant instrument 2 6 Accessories 2 6 1 Column holders ...

Page 46: ...hreaded ports for HiTrap columns and tubing connectors Push the button of the holder to attach the holder to a holder rail 1 2 3 Description Part Snap in to holder rails 1 Button 2 Column holder rod 3 46 ÄKTA avant User Manual 29035184 AE 2 The ÄKTA avant instrument 2 6 Accessories 2 6 1 Column holders ...

Page 47: ...HiScreen columns 2 3 4 5 7 6 9 1 8 Function Part Lower tubing 1 Lower part 2 Snap in strips 3 Attachment part 4 Upper tubing 5 Lever 6 Upper part 7 Upper connector 8 Lower connector 9 ÄKTA avant User Manual 29035184 AE 47 2 The ÄKTA avant instrument 2 6 Accessories 2 6 1 Column holders ...

Page 48: ... large inlet tubing o d 3 16 for AKTA avant 150 The following illustration shows the Tubing holder spool for small tubing 1 2 3 Description Part Positions for tubing 1 Tab 2 Snap in to holder rails 3 Tubing holder comb The Tubing holder comb is used to hold and arrange tubing 1 2 3 48 ÄKTA avant User Manual 29035184 AE 2 The ÄKTA avant instrument 2 6 Accessories 2 6 2 Tubing holders and other acce...

Page 49: ...trument display to hold a sample bottle 1 2 Description Part Position for bottle 1 Snap in to holder rails 2 Adapter for air sensor The air sensor adapter is used to hold an external air sensor The air sensor with adapter is connected to the Bottle holder see the following illustra tions ÄKTA avant User Manual 29035184 AE 49 2 The ÄKTA avant instrument 2 6 Accessories 2 6 2 Tubing holders and othe...

Page 50: ...1 2 3 Description Part Bottle holder 1 Air sensor 2 Adapter for air sensor 3 50 ÄKTA avant User Manual 29035184 AE 2 The ÄKTA avant instrument 2 6 Accessories 2 6 2 Tubing holders and other accessories ...

Page 51: ...ton of the rod to attach it to a holder rail 1 2 Function Part Button 1 Extension rod 2 Multi purpose holder The Multi purpose holder can be used to attach accessories for example a Loop holder or a cassette Attach the holder to a holder rail 1 2 3 4 3 ÄKTA avant User Manual 29035184 AE 51 2 The ÄKTA avant instrument 2 6 Accessories 2 6 2 Tubing holders and other accessories ...

Page 52: ... 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 1 2 Function Part Upper attachment to multi purpose holder 1 Lower attachment to multi purpose holder 2 52 ÄKTA avant User Manual 29035184 AE 2 The ÄKTA avant instrument 2 6 Accessories 2 6 2 Tubing holders and other accessories ...

Page 53: ...Standing on top of or next to ÄKTA avant On a Rail extension rod The hanger of the extension box is located on the right or left side of the box depending on where the removable extension box front and back is attached Note Illustration of Extension box This illustration shows the front and the back of the Extension box 1 2 3 4 5 6 7 Function Part Front 1 Front screw 2 Module screw hole 3 ÄKTA ava...

Page 54: ...e instrument modules The following instrument modules can be placed in the Extension box Any V9 or V9H valve Mixer M9 UV monitor U9 L Conductivity monitor C9n 54 ÄKTA avant User Manual 29035184 AE 2 The ÄKTA avant instrument 2 6 Accessories 2 6 3 Extension box ...

Page 55: ...n contains the following subsections See page Section 56 3 1 Pumps 60 3 2 Mixer 62 3 3 Valves overview 64 3 4 Inlet valves 70 3 5 Injection valve 73 3 6 Column valve 76 3 7 Outlet valve 78 3 8 Pressure monitors 80 3 9 pH valve and pH monitor 83 3 10 Built in fraction collector 96 3 11 UV monitor 98 3 12 Conductivity monitor ÄKTA avant User Manual 29035184 AE 55 3 Standard instrument modules ...

Page 56: ...dividual stepper motors controlled by a microprocessor The two pistons and pump heads work alternately to give a continuous low pulsation liquid delivery To ensure delivery of correct volume the pumps must be free from air The purge valve on each of the pump heads is used to remove air For instruction on how to purge the pumps see Section 5 4 Prime inlets and purge pump heads on page 165 The follo...

Page 57: ...150 ml min P9H P9H S ÄKTA avant 150 Location and illustration of pumps The illustration below shows the location of the Sample pump System pump A and System pump B An enlargement of one of the System pumps is also shown Description Part Purge valve Used to remove air from the pump 1 Check valve Non return check valves at the outlet port 2 ÄKTA avant User Manual 29035184 AE 57 3 Standard instrument...

Page 58: ...anism The seal is continuously lubricated by the presence of eluent The pump piston rinsing system continuously flushes the low pressure chamber behind the piston with a low flow of 20 ethanol prepared in deionized water or equivalent This prevents any deposition of salts from aqueous buffers on the pistons and prolongs the working life of the seals The pump piston rinsing system tubing is connect...

Page 59: ...sample pump piston rinsing system 1 Outlet tubing from the sample pump piston rinsing system 2 Inlet tubing to the system pump piston rinsing system 3 Outlet tubing from the system pump piston rinsing system 4 ÄKTA avant User Manual 29035184 AE 59 3 Standard instrument modules 3 1 Pumps ...

Page 60: ...ilter see Section 7 8 7 Replace the inlet filters on page 348 NOTICE Do not run the system with the mixer attached to the instrument if no tubing is connected to the mixer Running the mixer without liquid in the mixer chamber causes excessive wear of the magnet in the mixer chamber Location and illustration of the Mixer The following illustration shows the location of the Mixer and also an enlarge...

Page 61: ...Description Part Outlet 1 Mixer chamber 2 Inlet 3 ÄKTA avant User Manual 29035184 AE 61 3 Standard instrument modules 3 2 Mixer ...

Page 62: ... motorized rotary valve consists of a valve connection block with a number of defined bores with channels to the inlet and outlet ports of the valve The rotary disc mounted on the motor has a number of defined channels The pattern of channels of the Rotary disc together with the pattern and location of the ports of the valve connection block define the flow path and function of each type of valve ...

Page 63: ...escription Part Valve connection block 1 Rotary disc 2 Defined channel s in the rotary disc 3 Defined bores in the valve connection block 4 Inlet and outlet ports are not visible in the picture They are located on the opposite side of the Valve connection block Note ÄKTA avant User Manual 29035184 AE 63 3 Standard instrument modules 3 3 Valves overview ...

Page 64: ... gradients The number of inlets can be increased by installing extra inlet valves For information on how to install extra valves please refer to Section 4 1 System extension overview on page 101 This section describes the location and function of the inlet valves Location and illustration of inlet valves The following illustration shows the location of the inlet valves and also enlargements of Inl...

Page 65: ...t valve B can be used to prevent intro duction of air into the pumps and columns For information on how to change settings and parameters for the use of air sensors see Chapter 5 Operation on page 155 The Run Data for air sensors shows the status of the air sensor If an alarm is triggered and then acknowledged the Run Data will be reset to No Air even if there still is air in the sensor When the f...

Page 66: ...e Quaternary valve is used for automatic mixing of four different solutions The Qua ternary valve opens one inlet port at a time and the different solutions are mixed in the Mixer to form the desired buffer The opening time in the switching valve is controlled by the system The volume for each inlet port opening increases stepwise when the flow increases To obtain a homogeneous buffer composition ...

Page 67: ...ufferPro tool allows automatic mixing of buffers during a run Buffer preparation can be performed using corresponding acid base buffer systems Buffer system concen tration pH and salt gradient are defined in the Method Editor In ÄKTA avant 25 BufferPro can be run at flow rates up to 25 ml min In ÄKTA avant 150 BufferPro can be run at flow rates up to 40 ml min Note When using BufferPro immerse the...

Page 68: ...e Sample inlet valve enables automatic loading of different samples when using the Sample pump to inject sample directly onto the column or to fill a sample loop The Sample inlet valve has an inlet dedicated for buffer This Buff inlet is used in methods to fill the Sample pump with solution before sample is introduced The Buff inlet is also used to wash the Sample pump with buffer between runs The...

Page 69: ... The following illustration shows the ports of the Sample inlet valve Description Port Sample inlets S1 S7 Buffer inlet Buff To Sample pump Out ÄKTA avant User Manual 29035184 AE 69 3 Standard instrument modules 3 4 Inlet valves ...

Page 70: ...d to the Injection valve and filled either automatically using the Sample pump or manually using a syringe The sample can also be injected directly onto the column using the Sample pump For instructions on how to connect and use sample loops see Section 5 7 Sample appli cation on page 189 Location and illustration of Injection valve The following illustration shows the location together with a det...

Page 71: ...erent flow paths through the valve Primary flow path Flow path for manual load Closed flow path Alternative flow path Description Port Inlet from sample pump SaP Inlet from the System pumps via the Mixer SyP Syringe connection Syr1 Outlet to the Column valve Col Port for connection of a loop or a Loop valve Used to fill a loop LoopF ÄKTA avant User Manual 29035184 AE 71 3 Standard instrument modul...

Page 72: ...m pump waste The flow entering the SaP port is directed to the column valve This position is used with the sample pump Flow entering the SyP port is directed to waste port W1 Direct inject The flow entering the SaP port is directed to the loop This position is used with the sample pump Excess sample leaves the valve through waste port W1 The flow entering the SyP port is directed to the column or ...

Page 73: ...The number of column positions can be increased by installing an extra Column valve For information on how to install extra valves please refer to Section 4 1 System extension overview on page 101 Location and illustration of the Column valve The following illustration shows the location of the Column valve and also an enlargement of the Column valve with connected columns In ÄKTA avant 25 the Col...

Page 74: ...aths through Column valves V9 C and V9H C By pass Description Port Inlet from Injection valve via a built in pressure monitor In Ports for connection to the top of columns 1A 5A Ports for connection to the bottom of columns 1B 5B Outlet to UV monitor via a built in pressure monitor Out 74 ÄKTA avant User Manual 29035184 AE 3 Standard instrument modules 3 6 Column valve ...

Page 75: ...n valve Both top and bottom of each column shall be connected to the Column valve The top of the column shall be connected to an A port e g 1A and the bottom of the column shall be connected to the corresponding B port e g 1B The flow direction can be set either from the top of the column to the bottom of the column Down flow or from the bottom of the column to the top of the column Up flow In the...

Page 76: ...outlet ports can be convenient when for example a limited number of large fractions is to be collected If no fraction collection is needed the Outlet valve can be set to direct the flow to waste Location and illustration of Outlet valve The following illustration shows the location of the Outlet valve and also an enlargement of the valve In ÄKTA avant 25 the Outlet valve is labelled V9 O In ÄKTA a...

Page 77: ...g illustration shows the ports of the Outlet valve Description Port Inlet port In Port to fraction collector Frac Outlet ports 1 10 Out1 Out10 Waste port W ÄKTA avant User Manual 29035184 AE 77 3 Standard instrument modules 3 7 Outlet valve ...

Page 78: ...s The following illustration shows the location of the pressure monitors on the instrument as well as a detailed view of the pressure monitors of the pumps In both ÄKTA avant 25 and ÄKTA avant 150 the pressure monitors for the System pumps and Sample pump are labelled R9 Description Part Column valve with built in pre column pressure monitor and post column pressure monitor 1 Sample pump pressure ...

Page 79: ...nd outlet ports of the Column valves V9 C and V9H C measure the pressure just before the column pre column pressure A and the pres sure just after the column post column pressure B The delta column pressure C which is the pressure drop over the column is calculated as the difference between the pre column pressure and the post column pressure Pressure alarms can be set for both the pre column pres...

Page 80: ...rmation in the detector flow cells It is recommended to have it selected inline The valve directs the flow to the pH electrode and to the Flow restrictor or by passes one or both Location and illustration of pH valve The following illustration shows the location of the pH valve and also an enlargement of the valve In ÄKTA avant 25 the pH valve is labelled V9 pH In ÄKTA avant 150 the pH valve is la...

Page 81: ...ations Closed flow path Description Port From Conductivity monitor In To Flow restrictor ToR From Flow restrictor FrR To Outlet valve Out Calibration port Cal To Waste W3 Description Flow path Both pH electrode and flow restrictor are by passed By pass Flow restrictor is in use and pH electrode is by passed Restrictor ÄKTA avant User Manual 29035184 AE 81 3 Standard instrument modules 3 9 pH valve...

Page 82: ...uously measures the pH of the buffer and eluted proteins when the pH electrode is in line When pH monitoring is used the pH monitor should be cali brated every day The pH monitor can be calibrated with the pH electrode installed in the pH valve For instruction on how to calibrate the pH monitor please refer to Section 7 7 2 Calibrate the pressure monitors on page 323 The following illustration sho...

Page 83: ...ction collector In this section This section contains the following sub sections See page Section 84 3 10 1 Function 86 3 10 2 Illustrations of the built in fraction collector 89 3 10 3 Cassettes cassette tray and racks ÄKTA avant User Manual 29035184 AE 83 3 Standard instrument modules 3 10 Built in fraction collector ...

Page 84: ...that provides an option to set a desired fraction collector chamber temperature The target temperature can be set to any whole value between 6 C and 20 C The default value is 20 C Settings for the temperature control function can be edited in the System Settings dialog box in System Control or in the Text Instructions pane in Method Editor If the difference between the ambient temperature and the ...

Page 85: ...he sensors in the dispenser head detect when a drop is released from the nozzle The dispenser head moves to the next well or tube just after a drop is released Fractionation with DropSync can be used at flow rates up to 2 ml min Solutions with low surface tension may require a lower flow DropSync is used in ÄKTA avant 25 only Note If using the accumulator with buffers with high salt concentration ...

Page 86: ...llector A cassette tray with cassettes is placed in the fraction collector drawer Description Part Fractionation drawer 1 Fractionation window 2 Cassette tray with cassettes 3 Height exclusion bar 4 Fractionation drawer handle 5 86 ÄKTA avant User Manual 29035184 AE 3 Standard instrument modules 3 10 Built in fraction collector 3 10 2 Illustrations of the built in fraction collector ...

Page 87: ...1 Button cover 2 Fractionation arm 3 Dispenser head 4 Lamp 1 of 2 5 Tubing from the Outlet valve 6 Cooling unit including circulation fan 7 Waste funnel 8 Tray aligning pins 9 Cassette tray positioning discs 2 of 3 10 Fractionation arm guide rail 11 ÄKTA avant User Manual 29035184 AE 87 3 Standard instrument modules 3 10 Built in fraction collector 3 10 2 Illustrations of the built in fraction col...

Page 88: ...ption Part Dispenser head 1 Dispenser head cover 2 Nozzle 3 Accumulator back part of Dispenser head 4 DropSync sensor only in ÄKTA avant 25 5 Type code reader 6 88 ÄKTA avant User Manual 29035184 AE 3 Standard instrument modules 3 10 Built in fraction collector 3 10 2 Illustrations of the built in fraction collector ...

Page 89: ... 6 tubes Cassette for deep well plate 24 48 96 wells Cassette tray for six cassettes Rack for 50 ml tubes for 55 tubes Rack for 250 ml bottles for 18 bottles For information on dimension requirements for tubes and deep well plates to be used in the fraction collector see Fraction collector tubes and bottles on page 93 and Deep well plates on page 94 respectively Illustrations of built in fraction ...

Page 90: ...ced in the fraction collector Note Illustration of cassettes on the cassette tray The illustration below shows cassettes placed on the cassette tray Description Part Cassette tray 1 Cassette position number 2 Cassette 3 Cassette type code 4 Tubes or deep well plates placed in a cassette 5 90 ÄKTA avant User Manual 29035184 AE 3 Standard instrument modules 3 10 Built in fraction collector 3 10 3 Ca...

Page 91: ...asily be loaded with tubes and used tubes can easily be discarded Action Step Load the cassette with tubes before fractionation 1 Pull the QuickRelease device to the release position Load the cassette with tubes and press the QuickRelease device to the locked position Locked position Release position After fractionation pull the QuickRelease device to the release position and remove the tubes cont...

Page 92: ...ck position and empty the remain ing tubes Pull the QuickRelease device to the release position and discard the tubes Discard the tubes Empty the tubes 92 ÄKTA avant User Manual 29035184 AE 3 Standard instrument modules 3 10 Built in fraction collector 3 10 3 Cassettes cassette tray and racks ...

Page 93: ...D Biosciences 116 110 30 28 50 Nalgene Kautex 121 L 64 5 W 641 L 55 W 551 250 ml bottle 1 Length and width of the rectangular bottle base Maximum flow rate Fraction collection can be performed at different maximum flow rates depending on the size of the tubes and bottles that are used The following table lists the maximum flow rates for the fraction collector tubes and bottles Maximum flow rate ml...

Page 94: ...e tested and approved by GE to be used with ÄKTA avant Part no Manufacturer Plate type 7701 5200 Whatman GE 96 deep well plate 353966 BD Biosciences 780270 Greiner Bio One 219009 Porvair Sciences S30009 Seahorse Bioscience 951033405 0030501 306 Eppendorf 7701 5500 Whatman GE 48 deep well plate S30004 Seahorse Bioscience 7701 5102 Whatman GE 24 deep well plate S30024 Seahorse Bioscience 94 ÄKTA ava...

Page 95: ...that are used The table below lists the maximum flow rates for the different plate types Maximum flow rate ml min Plate type 10 96 deep well plate 15 48 deep well plate 25 24 deep well plate ÄKTA avant User Manual 29035184 AE 95 3 Standard instrument modules 3 10 Built in fraction collector 3 10 3 Cassettes cassette tray and racks ...

Page 96: ... a run The second and third wavelength can be turned off or on in method phase properties by manual instructions or in system settings The resolution is decreased when more than one wavelength is used simulta neously due to lower sampling frequency per wavelength Do not use more wavelengths than necessary Note Location and illustration of UV monitor The following illustration shows the location of...

Page 97: ... suitable to use for low protein concentrations If the UV flow cell with 10 mm path length is used do not run at flow rates higher than 50 ml min as this flow cell increases the back pressure and may result in a pressure higher than 2 MPa 20 bar Note The real cell path length of the UV cell is automatically recognized by the monitor when a cell is fitted The UV data is normalized to the nominal pa...

Page 98: ... by multiplying the measured conductance by the cell constant of the flow cell The cell constant is factory calibrated on delivery but can be re calibrated if needed see Section 7 7 3 Calibrate the Conductivity Monitor on page 327 As variation in temperature influences conductivity readings the conductivity flow cell is fitted with a temperature sensor that measures the temperature of the eluent A...

Page 99: ...nd also an enlargement of the monitor In both ÄKTA avant 25 and ÄKTA avant 150 the monitor is labelled C9 Description Part Description Part Conductivity flow cell 2 Inlet 1 Conductivity monitor cable 4 Conductivity monitor 3 Outlet 5 ÄKTA avant User Manual 29035184 AE 99 3 Standard instrument modules 3 12 Conductivity monitor ...

Page 100: ... the following sections See page Section 101 4 1 System extension overview 102 4 2 Installation of optional modules 110 4 3 Extra inlet valves 114 4 4 Second Column valve 117 4 5 Extra Outlet valves 120 4 6 External air sensors 124 4 7 Loop valve 128 4 8 Versatile valve 130 4 9 Fraction collector F9 R 137 4 10 Second Conductivity monitor 139 4 11 Second UV monitor 143 4 12 I O box 153 4 13 General...

Page 101: ...number of columns inlets outlets detectors and ways to apply and collect samples For a list of optional modules see Description of op tional modules on page 26 System extension stages Extension of the system consists of four main stages Description Stage Select modules and positions 1 Install the module s 2 Edit system properties 3 Edit system settings 4 ÄKTA avant User Manual 29035184 AE 101 4 Op...

Page 102: ...stration below shows the free module positions marked in orange The Extension box can be used to hold modules connected to the back of the instrument See Section 2 6 3 Extension box on page 53 for more information Node ID Most of the available optional modules are preconfigured to give the desired function However the function of a module or valve can be changed by changing its Node ID Node ID is ...

Page 103: ...onnect power Always switch off power to the ÄKTA avant in strument before replacing any of its components unless stated otherwise in the user documentation Action Step Disconnect power from the instrument by switching off the instrument power switch 1 Loosen the connectors and remove the tubing from the existing module Note This step does not apply for a Module Panel 2 Loosen the module with a Tor...

Page 104: ...e the module 4 Disconnect the cable and secure it in the slit 5 Connect the cable to the module to be installed 6 104 ÄKTA avant User Manual 29035184 AE 4 Optional instrument modules 4 2 Installation of optional modules ...

Page 105: ...e ÄKTA avant connector plate Modules not installed in the instrument cabinet are connected via a UniNet 9 cable at the back of the system It is possible to connect up to three external modules The following illustration shows the connector plate with the UniNet 9 connectors located on the back of the ÄKTA avant instrument ÄKTA avant User Manual 29035184 AE 105 4 Optional instrument modules 4 2 Ins...

Page 106: ...r 4 NOTICE Do not connect any module to the connector Test on the ÄKTA avant instrument Plug all unused UniNet 9 ports on the ÄKTA avant instrument with jumpers Note The connector Test should be protected by a plastic lid Do not plug the con nector with a jumper Note 106 ÄKTA avant User Manual 29035184 AE 4 Optional instrument modules 4 2 Installation of optional modules ...

Page 107: ...not exceed 10 m Note Edit System Properties When a new module has been installed the system properties have to be updated in UNICORN The systemwillrestart automaticallywhentheconfigurationhas beenchanged in the System Properties dialog box and then the system can be reconnected There are four main types of modules named components in UNICORN to select from Valves and pumps Monitors and sensors Fra...

Page 108: ...e edited Result The Edit dialog box is displayed 1 Select the component type of interest from the Component types list 2 Result All available components are shown in the Component selection list Select the checkbox to select the added component When applicable choose the appropriate Property Note Instrument modules are referred to as Components in UNICORN Click the OK button to apply the changes 3...

Page 109: ...e appropriate system settings for Delay volumes have to be updated This is to ensure that the fractions marked in the chromatogram corresponds to the actual collected fractions Other system settings might also need to be edited for some optional modules All system settings available for ÄKTA avant are found in Section 9 7 System settings on page 459 ÄKTA avant User Manual 29035184 AE 109 4 Optiona...

Page 110: ...also general inlet valves which can be used to increase the number of inlets to for example the Quaternary valve The general inlet valves are delivered without built in air sensors Note Location of ÄKTA avant inlet valves Second Inlet valves V9 A2 V9 B2 or V9H A2 V9H B2 or second Sample inlet valve V9 S2 or V9H S2 can be installed in the lower free position as illustrated below To obtain an option...

Page 111: ...ted V9 IS V9 S2 Out B U F F S1 S2 S3 S4 S5 S6 S7 BUFF S8 S9 S10 S11 S12 S13 S14 Out Second Inlet valve The illustration below shows an optional flow path when a second Inlet valve is installed connected to Inlet valve A The flow is directed from the Out port of the Second Inlet valve V9 A2 or V9H A2 to port A7 of the Inlet valve V9 IA or V9H IA and leaves the valve through the Out port to the Syst...

Page 112: ...or ÄKTA avant 150 FEP o d 3 16 i d 2 9 mm For runs with small volumes of sample the sample inlet tubing can be changed to narrow inlet tubing ETFE o d 1 16 i d 0 75 mm 28 9572 17 Note Inlet valves X1 and X2 Inlet valve X1 and Inlet valve X2 are inlet valves that can be used to create different flow paths They can be combined with any of the other inlet valves or be used on their own The valves can...

Page 113: ... 5 16 Ferrule blue 3 16 Tubing length depends on location System properties Follow the instruction to update the system properties Action Step Open the system properties Edit dialog box 1 In the Component types list click Valves and pumps 2 In the Component selection list select the check boxes corresponding to the installed modules Then click OK 3 System settings There are no system settings avai...

Page 114: ...instrument One application is for evaluation of a number of different columns during method optimization Location and illustration A recommended flow path is to move the Column valve V9 C or V9H C to the free upper position Fig A and to install the second Column valve V9 C2 or V9H C2 in the previous position of Column valve V9 C or V9H C Fig B A B 114 ÄKTA avant User Manual 29035184 AE 4 Optional ...

Page 115: ...mn valve V9 C2 or V9H C2 Otherwise the pressure monitors will not function correctly Note Connect tubing For an illustration of standard tubing labels see Tubing labels on page 432 Action Step Remove the piece of standard tubing between the Injection valve V9 Inj or V9H Inj and the standard Column valve V9 C or V9H C 1 Connect tubing between the Injection valve column valves and the UV monitor acc...

Page 116: ...or can be used System properties Follow the instruction to update the system properties Action Step Open the system properties Edit dialog box 1 In the Component types list click Valves and pumps 2 Select the Column valve 2nd V9 C2 or Column valve 2nd V9H C2 check box in the Component selection list Then click OK 3 System settings The built in pressure sensors for Column valves V9 C and V9H C have...

Page 117: ...l the extra outlet valves in the middle and in the lower position The illustration below shows the recommended positions Flow path The tubing from the pH monitor is connected to the In port of the Outlet valve V9 O or V9H O The Out 10 port of the Outlet valve V9 O or V9H O is connected to the In port of the second Outlet valve V9 O2 or V9H O2 The Out 21 port of the second Outlet valve is connected...

Page 118: ...Tubing Tubing label Connection between 220 For ÄKTA avant 25 Fingertight connector 1 16 For ÄKTA avant 25 PEEK o d 1 16 i d 0 50 mm 101 standard Outlet valve and second Outlet valve For ÄKTA avant 150 Fingertight connector 1 16 For ÄKTA avant 150 PEEK o d 1 16 i d 1 mm 220 111 second Outlet valve and third Outlet valve 1 Cut the spare piece of tubing delivered with the system to correct length 118...

Page 119: ...stalled modules Then click OK 3 System settings If the valve 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 It is recommended not to alter the default values for restrictor and pH cell delay volumes when standard modules and standard tubing for flow restrictor are used Note ÄKTA avant User Manual 290351...

Page 120: ...ternal air sensor L9 1 2 has a 1 2 mm i d and is designed for 1 16 tubing outer diameter at the high pressure side before the column It is installed after the Injection valve and is used to prevent air from entering the Column valve The air sensors can be attached to the instrument using the rails No module panels need to be removed The Run Data for air sensors shows the status of the air sensor I...

Page 121: ...9 1 2 external air sensor after Injection valve 2 Only one external air sensor can be installed at a time Note Connect tubing L9 1 5 Tubing length mm Connector Tubing Tubing label Connection between 200 Tubing connector 5 16 Ferrule yellow 1 8 FEP o d 1 8 i d 1 6 mm L L9 1 5 and V9 IS or V9H IS ÄKTA avant User Manual 29035184 AE 121 4 Optional instrument modules 4 6 External air sensors ...

Page 122: ...16 i d 1 mm 1 Cut the spare piece of tubing delivered with the system to correct length System properties Follow the instruction to update the system properties Action Step Open the system properties Edit dialog box 1 In the Component types list click Monitors and sensors 2 Select the External air sensor L9 check box in the Component selection list 3 In the Property list click Before sample inlet ...

Page 123: ...all air bubbles 30 μl 10 μl High The sensitivity should be set to Normal when the air sensor is located before the Sample pump Before sample pump Due to higher pressure and risk of small air bubbles the sensitivity should be set to High when the air sensor is located after the Injection valve After injection valve Note ÄKTA avant User Manual 29035184 AE 123 4 Optional instrument modules 4 6 Extern...

Page 124: ...ng samples to be used in scouting runs or for storing eluents needed in low volumes The valve also has a built in by pass function that enables by passing all loops The Loop valve is labeled V9 L or V9H L Location and illustration of Loop valve The illustration below shows the recommended location together with a detailed view of Loop valve V9 L 124 ÄKTA avant User Manual 29035184 AE 4 Optional in...

Page 125: ...alve F Ports for connection of loop 1 to loop 5 1F and 1E to 5F and 5E Port connected to the LoopE port of the Injection valve E Ports denoted by the letter F are used for filling the loop and ports denoted by the letter E are used for emptying the loop Note Description Flow path The flow direction depends on the Injection valve position Position 1 5 The flow by passes the loop s By pass is the de...

Page 126: ...tead of a loop as described below Action Step Connect port E on the Loop valve to port LoopE on the Injection valve 1 Connect port F on the Loop valve to port LoopF on the Injection valve 2 Connect one or many loops to the Loop valve See Section 5 7 Sample appli cation on page 189 Note Always use the the first positions of the valve for the connected loops e g if three loops will be used use port ...

Page 127: ...ve V9 L or Loop valve V9H L check box in the Compo nent selection list Then click OK 3 System settings The flow rate for Loop wash can be set Description Instruction name Sets the flow rate used during Loop wash Note The flow rate should not exceed 10 ml min if narrow inlet tubing i d 0 75 mm is used Loop wash settings ÄKTA avant User Manual 29035184 AE 127 4 Optional instrument modules 4 7 Loop v...

Page 128: ...age 515 The valve can be installed in any free module position It is also possible to install the valve in an Extension box See Section 2 6 3 Extension box on page 53 Ports and flow paths of the Versatile valve The following illustration and table describe the different ports of and flow paths through the Versatile valve The valve has four ports 1 4 The Versatile valve has four available sets of f...

Page 129: ...truction to update the system properties Action Step Open the system properties Edit dialog box 1 In the Component types list click Valves and pumps 2 In the Component selection list select the check boxes corresponding to the installed modules Then click OK 3 System settings If the valve is placed in the flow path between the UV monitor and the outlet valve the delay volume must be set See Check ...

Page 130: ... is set by positioning the arrow of the rotating switch at the back of the fraction collector See Connector panel illustration on page132 and Section 9 15 Check and change the Node ID of a module on page 515 Function The external fraction collector can be used for Fixed volume fractionation Peak fractionation Combined fixed volume fractionation and peak fractionation Collecting fractions in revers...

Page 131: ...ction collector 1 3 4 5 7 6 8 2 Function Part Lock knob 1 Stationary part of delivery arm 2 Delivery arm 3 Tubing connector 4 Tube sensor 5 Collection tubes 6 Tube rack 7 Base unit 8 ÄKTA avant User Manual 29035184 AE 131 4 Optional instrument modules 4 9 Fraction collector F9 R ...

Page 132: ...tor for communication and power supply 2 Available tubes For Fraction collector F9 R the fractions are collected in tubes of different sizes Tubes with the following diameter can be used with Fraction collector F9 R 12 mm 18 mm 30 mm The tubes can have a tube length between 50 to 180 mm 132 ÄKTA avant User Manual 29035184 AE 4 Optional instrument modules 4 9 Fraction collector F9 R ...

Page 133: ...mation on which Tube rack to use see Tube rack inserts on page 211 1 2 3 4 5 6 Function Part Single cutout 1 L shaped cutout 2 Bowl 3 Tube support 4 Tube guide 5 Tube holder 6 Note that the tube guide has both single and L shaped cutouts while the tube holder only has single cutouts See Single and L shaped cutouts on page211 for more information Note ÄKTA avant User Manual 29035184 AE 133 4 Option...

Page 134: ...e fraction collector to the ÄKTA avant instrument Action Step Lift out the tubing holder 4 from the delivery arm 1 1 3 4 5 6 Loosen the nut of the tubing holder Do not remove the tubing holder nut 5 from the tubing holder 2 Insert the tubing through the tubing holder 3 Place the tubing holder with the tubing over the tube adjustment cavity 2 of the delivery arm Push the tubing down against the bot...

Page 135: ...tem properties Action Step Open the system properties Edit dialog box 1 In the Component types list click Fraction collectors 2 Select the Fraction collector 2 F9 R check box in the Component selection list Then click OK 3 The Fraction collector check box must be selected for the Fraction collector 2 F9 R check box to be available Note the Fraction collector 2 F9 R check box is not available if th...

Page 136: ... the liquid Fractionation settings frac 2 Determines whether fraction number for the fraction collector 2 is reset at the end of a method or not Note The default setting is Reset Fractionation numbering mode frac 2 Peak fractionation parameters sets the detection pa rameters for peak collection i e it determines when a peak starts and ends This information is used by the instructions Peak fraction...

Page 137: ...lls 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 conductivityflow cell has a maximum pressure limit of 5 MPa 50 bar Connect tubing When connectinga second Conductivitymonitorto ÄKTA avant use the tubingdimensions that are most suitable for the chosen location an...

Page 138: ...ced 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 153 138 ÄKTA avant User Manual 29035184 AE 4 Optional instrument modules 4 10 Second Conductivity monitor ...

Page 139: ...nd monitor could also be used to monitor the absorbance of incoming feed and is then placed in the flow path just before the column valve Function of UV monitor U9 L The UV monitor U9 L continuously measures the UV absorbance at the fixed wavelength of 280 nm It is not possible to vary the wavelength or turn on or off the U9 L monitor This is therefore not shown in the Phase Properties pane in Met...

Page 140: ... flow path for example close to the first UV monitor or before the Column valve Use the nearest empty module position or an Extension box to hold the second UV monitor U9 L See Section 2 6 3 Extension box on page 53 The second UV monitor can be located anywhere in the flow path and is therefore shown in the Process Picture in UNICORN as a component without a fixed place This means that it is possi...

Page 141: ...text instruction Fraction Collection Peak fractionation parameters Signal source and choosing UV 2nd as Signal source Note When using two UV monitors with different cell lengths to increase the UV absorption dynamic range the signal from UV monitor U9 L comes from the real cell length and has to be calibrated for exact calculations The signal from UV monitor U9 M is automatically calibrated to nom...

Page 142: ...r the delay volume has to be updated See Check or set delay volumes on page 153 The UV absorption signal from the UV monitor U9 L is correlated to the real cell length If it is important to have absorption signals correlated to the nominal cell length a cali bration should be done 142 ÄKTA avant User Manual 29035184 AE 4 Optional instrument modules 4 11 Second UV monitor ...

Page 143: ...this section This section contains the following subsections See page Section 144 4 12 1 Overview of the I O box 146 4 12 2 Analog connector and signals 148 4 12 3 Digital connector and signals 150 4 12 4 Connect external equipment to the I O box ÄKTA avant User Manual 29035184 AE 143 4 Optional instrument modules 4 12 I O box ...

Page 144: ...e to be used the second I O box has to be configured as I O box E9 2nd The configuration is defined by the Node ID of the I O box The Node ID is set by positioning the arrow of two rotating switches at the back of the I O box see Connectors on page 145 and Section 9 15 Check and change the Node ID of a module on page 515 Location The most common placement of the I O box with the adhesive feet atta...

Page 145: ...he ÄKTA avant instrument UniNet 9 Status indicator for service purposes Status Switches used to configure I O box E9 as I O box E9 or I O box E9 2nd Node ID Signal connector for digital input and output signals Digital in out ÄKTA avant User Manual 29035184 AE 145 4 Optional instrument modules 4 12 I O box 4 12 1 Overview of the I O box ...

Page 146: ...2 or signal ground 5 Calibration pin for service purposes 6 Analog out signal 1 9 V Note Do not use for other purposes Analog out signal 1 7 Signal ground analog out both ports 8 Analog out signal 2 9 Analog signals All analog input and output signals are confined to the same Analog in out connector 146 ÄKTA avant User Manual 29035184 AE 4 Optional instrument modules 4 12 I O box 4 12 2 Analog con...

Page 147: ...lected signal Default output The user sets the desired output signal value of e g mAU or mS which will corresponds to the full scale output voltage 1000 mV Full scale output The corresponding negative full scale output value is set auto matically For example if the full scale output is set to 2000 mAU a UV value of 500 mAU will give an output voltage of 250 mV and 100 mAU will give 50 mV output vo...

Page 148: ...nal 1 6 Digital out signal 2 7 Digital out signal 3 8 Digital out signal 4 9 Digital signals All digital input and output signals will be confined to the same D sub connector and have a common ground The four input signals will be scanned synchronously and the outputs will be set synchronously 148 ÄKTA avant User Manual 29035184 AE 4 Optional instrument modules 4 12 I O box 4 12 3 Digital connecto...

Page 149: ...gical 0 Closed circuit Applied voltage 0 to 0 8 V Digital output signals The digital output signal can be used to control external equipment that can receive digital signals such as pumps or fraction collectors The digital output signals define an open or closed circuit where a logical 1 will result in an open circuit and a logical 0 will give a closed circuit The default level 1 or 0 is set by th...

Page 150: ...meter 2 Channels 2000 mV Range 1 MΩ Input impedance 0 1 0 2 mV Accuracy Analog output Value Parameter 2 Channels 1000 mV Range 100 kΩ Input impedance 0 3 1 mV Accuracy Digital input Value Parameter 4 Channels TTL open closed circuit Compatibility Digital output Value Parameter 4 Channels Open closed circuit Compatibility 150 ÄKTA avant User Manual 29035184 AE 4 Optional instrument modules 4 12 I O...

Page 151: ... contacts 2 5 4 3 2 1 9 8 7 6 Strip the signal cable 3 Strip off 50 mm of the shield insulator Strip off 4 mm of the single conductor insulation Loosen the strain relief clamp and insert the cable with the shield under the strain relief clamp Fasten the strain relief clamp over the cable shield Tip The connection block can be rotated inside the housing in order to position the screw terminals for ...

Page 152: ...ts the alarm limits for the analog signal If the alarm is enabled and the analog signal falls outside the set limits an alarm will be triggered and the method will be paused Alarm analog 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 If the alarm is enabled and the condition set in Value occurs an alarm wi...

Page 153: ...lumes on page 505 Follow the instructions to check set the delay volumes Action Step In the System Control module click Settings on the System menu 1 Result The System Settings dialog box opens In the System Settings dialog box select Fraction collection Delay volumes 2 Enter new values in the Detector Frac Detector Outet valve Restrictor volume and pH cell volume fields if necessary 3 Click OK Lo...

Page 154: ... Step In the System Settings dialog box 2 Select Advanced Instrument display Click Locked or Unlocked Click OK 154 ÄKTA avant User Manual 29035184 AE 4 Optional instrument modules 4 13 General system settings ...

Page 155: ...page Section 156 5 1 Before you prepare the system 157 5 2 Prepare the flow path 162 5 3 Start UNICORN and connect to the system 165 5 4 Prime inlets and purge pump heads 182 5 5 Connect a column 187 5 6 Set pressure alarms 189 5 7 Sample application 202 5 8 Fractionation 225 5 9 Create a method and perform a run ÄKTA avant User Manual 29035184 AE 155 5 Operation ...

Page 156: ...accordance with the settings in the method to be run Make sure that you understand which valve ports to use for inlets and outlets which column type to use which column position to use which buffers and samples to prepare which sample application technique to use that the pH electrode is connected and calibrated if applicable which cassettes with corresponding deep well plates and or tubes to use ...

Page 157: ...to mix use a larger Mixer chamber to achieve optimal mixing However note that a larger Mixer chamber distorts and delays the gradient Note ÄKTA avant 25 Three different Mixer chambers are delivered with ÄKTA avant 25 Their volumes are 0 6 ml 1 4 ml mounted at delivery and 5 ml Flow rate ml min Quaternary and BufferPro gradients Flow rate ml min Binary gradient Mixer chamber volume ml 1 to 21 0 25 ...

Page 158: ...h protein concentrations Flow cells with longer path lengths are suitable to use for low protein concentrations UV flow cells for UV monitor U9 M UV flow cells are available with three different path lengths 0 5 mm 2 mm default and 10 mm The real cell path length of the UV cell is automatically recognized by the monitor when a cell is fitted The UV data is normalized to the nominal path length Thi...

Page 159: ... all inlet tubing that is to be used during the method run in the correct buffers When using high viscosity buffers or samples in combination with high flow rates it is recommended to change to tubing with larger inner diameter or shorten the length of the tubing Note Prepare the outlet tubing Connect outlet tubing to the outlet ports of Outlet valve that are to be used If the external Fraction co...

Page 160: ...et valve W W1 W2 and W3 in a vessel placed below the bench 1 NOTICE The maximum level of the waste vessel for the waste tubing from the valves must be lower than 30 cm above the lab bench Place the three pieces of waste tubing from the Fraction collector and the Buffer tray in a waste vessel placed below the bench 2 NOTICE The maximum level of the waste vessel for the waste tubing from the Fractio...

Page 161: ...oding in the Frac chamber CAUTION Make sure that the waste vessels will hold all the produced volume of the run For ÄKTA avant 25 a suitable waste vessel should typi cally have a volume of 2 to 10 liters For ÄKTA avant 150 a waste vessel should have a volume of 40 liters Plug unused valve ports It is recommended to plug all unused valve ports with stop plugs before starting a run See Section 9 3 T...

Page 162: ...istrationand Technical Manual for more information about e licenses Action Step Double click the UNICORN icon on the desktop 1 Result The Log On dialog box opens Note If there is no connection to the database it is still possible to log on to UNICORN and control a running system The Log On dialog box will give the option to start System Control without a database Click Start System Control to proc...

Page 163: ...sword field Note It is also possible to select the Use Windows Authentication check box and enter a network ID in the User Name box select which UNICORN modules to start click OK Result The selected UNICORN modules open ÄKTA avant User Manual 29035184 AE 163 5 Operation 5 3 Start UNICORN and connect to the system ...

Page 164: ...the Connect to systems dialog box 2 Select the checkbox in front of the system name To control the selected system click Control Click OK Note Instruments that are turned off or disconnected from the network appear dimmed and cannot be connected Tip To view the users currently connected to systems either in control or view mode click the Connected Users button 164 ÄKTA avant User Manual 29035184 A...

Page 165: ... pumps and the sample pump Note that the procedures described in this section may have to be adapted if your system configuration differs from the one described in this manual Note In this section This section contains the following subsections See page Section 166 5 4 1 Prime buffer inlets and purge system pumps 173 5 4 2 Prime sample inlets and purge Sample Pump 178 5 4 3 Prime Q inlets ÄKTA ava...

Page 166: ...nsing system is filled with fresh rinsing solution Note The procedures for purging the pump heads and priming the inlets using the Process Picture are described in the following topic It is also possible to perform the procedures from the Manual instructions dialog box Tip Prime inlet tubing Follow the instructions to fill all A and B inlet tubing to be used in the run with appropriate buffer solu...

Page 167: ...o 30 ml syringe to the purge valve of one of the pump heads of System Pump B Make sure that the syringe fits tightly into the purge connector 4 Open the purge valve by turning it counter clockwise about three quarters of a turn Draw liquid slowly into the syringe until the liquid reaches the pump 5 Close the purge valve by turning it clockwise Disconnect the syringe and discard its contents 6 Repe...

Page 168: ...ot necessary to purge System Pump B However if the pressure signal indicated air bubbles left in the pump follow these in structions to purge both pump heads of System Pump B Action Step Make sure that the piece of waste tubing connected to the injection valve port W1 is placed in a waste vessel 1 In the Process Picture 2 Click the Injection valve icon and then click System pump waste Result The i...

Page 169: ...rocess Picture 4 Click the System pumps icon Set Conc B to 100 B and click Set B Set the System flow to 1 0 ml min for ÄKTA avant 25 or 5 0 ml min for ÄKTA avant 150 Click Set flow rate Result Only System Pump B is active and a system flow through injection valve waste starts ÄKTA avant User Manual 29035184 AE 169 5 Operation 5 4 Prime inlets and purge pump heads 5 4 1 Prime buffer inlets and purg...

Page 170: ... ml s 6 Close the purge valve by turning it clockwise Disconnect the syringe and discard its contents 7 Connect the syringe to the purge valve on the right pump head of System Pump B and repeat steps 6 to 8 Keep the system flow running 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 page172 9 170 ÄKTA av...

Page 171: ...e Inlet valve A icon Click the position of one of the inlets that will be used at the beginning of the run Result The inlet valve switches to the selected port In the Process Picture 2 Click the System pumps icon Set Conc B to 0 B and click Set B Result Only System Pump A is active ÄKTA avant User Manual 29035184 AE 171 5 Operation 5 4 Prime inlets and purge pump heads 5 4 1 Prime buffer inlets an...

Page 172: ...nual load position Make sure that the pump flow is on 2 In the Chromatogram pane 3 Check the PreC pressure curve If the PreC pressure do not stabilize within a few minutes there may be air left in the pump Purge the pump once more then see Section 8 6 Troubleshooting Pumps on page 397 End the run Click the End button in the System Control toolbar to end the run 172 ÄKTA avant User Manual 29035184 ...

Page 173: ...ng system is filled with fresh rinsing solution Note Prime sample inlets Follow the instructions below to fill all sample inlet tubing to be used in the run with appropriate buffer or sample solution Action Step Make sure that all sample inlet tubing that is to be used during the method run is immersed in the correct samples or buffer 1 Make sure that the waste tubing connected to injection valve ...

Page 174: ...ure that the syringe fits tightly into the purge connector 5 Open the purge valve by turning it counter clockwise about three quarters of a turn Draw slowly with the syringe until the sample just passes the Sample inlet valve 6 Close the purge valve by turning it clockwise Disconnect the syringe and discard its contents 7 Repeat steps 4 to 7 for each sample inlet that is to be used in the method r...

Page 175: ...ed air bubbles left in the pump follow the instruction below to purge both the pump heads of the sample pump Action Step Make sure that all sample inlet tubing that is to be used during the method run is immersed in the correct buffers 1 Make sure that the waste tubing connected to injection valve port W2 is immersed in a waste vessel 2 Open the System Control module 3 In the Process Picture 4 Cli...

Page 176: ...purge valve of the sample pump Make sure that the syringe fits tightly into the purge connector 6 Open the purge valve by turning it counter clockwise about three quarters of a turn Draw 5 to 10 ml of liquid slowly into the syringe with a rate of about 1 ml s 7 Close the purge valve by turning it clockwise Disconnect the syringe and discard its contents 8 176 ÄKTA avant User Manual 29035184 AE 5 O...

Page 177: ...in the pump by following the instructions in Validate prime or purge of System Pump A or B or Sample Pump on page172 10 End the run Click the End button in the System Control toolbar to end the run ÄKTA avant User Manual 29035184 AE 177 5 Operation 5 4 Prime inlets and purge pump heads 5 4 2 Prime sample inlets and purge Sample Pump ...

Page 178: ...ion Step Make sure that the pieces of inlet tubing marked A1 B1 and Q1 Q4 are im mersed in the correct buffers The A1 and B1 positions are used for pump synchronization and these lines should already be primed 1 In the Manual instructions dialog box 2 Select Pumps and pressures Quaternary start concentrations Set Start concentration Q1 to 100 Make sure that the other start concentrations are set t...

Page 179: ... 5 for Q2 Q3 and Q4 respectively by setting the respective Quaternary start concentration to 100 Tip The inlet tubing that is immersed in distilled water should be the last piece of inlet tubing to be primed 6 Tip If you will perform a BufferPro run end with either Q1 or Q2 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...

Page 180: ...h pump heads of each system pump have to be purged Action Step In the Manual instructions dialog box 1 Select Pumps and pressures Pump wash and click All in the BufferPro Q inlets list Result A simultaneous pump wash of all the Q inlets is started This will re move air from Quaternary Valve Wait until the pump wash is completed 2 Select Pumps and pressures Quaternary start concentrations Set Start...

Page 181: ... clockwise Disconnect the syringe and discard its contents 6 Repeat steps 3 to 5 for the other three purge valves of the system pumps to get rid of air in all pump heads Keep the system flow running during this procedure 7 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 page172 8 End the run Click the End ...

Page 182: ...the pressure limit is set to the specified maximum pressure for the column Methods automatically include pressure alarms based on the specifications of the chosen column type However when running manual runs you have to set the pressure limits yourself Also to protect the column media special settings are needed See Section 5 6 Set pressure alarms on page 187 for more information on pressure alarm...

Page 183: ...e a column holder The column is connected to two opposite parts of the column valve using appro priate tubing and connectors Action Step Attach an appropriate column holder to the rail on the instrument 1 Attach the column to the column holder 2 ÄKTA avant User Manual 29035184 AE 183 5 Operation 5 5 Connect a column ...

Page 184: ...if column position 1 was chosen in the method to be run 3 In the Process Picture 4 Click the Column valve icon Click e g Position 1 and Down flow Click Set position Result The column valve switches to position 1 184 ÄKTA avant User Manual 29035184 AE 5 Operation 5 5 Connect a column ...

Page 185: ...te 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 6 ÄKTA avant User Manual 29035184 AE 185 5 Operation 5 5 Connect a column ...

Page 186: ...iece of tubing to the column valve Use the port opposite to the one already connected to the column in this example port 2B 8 Click the End button in the System Control toolbar to end the run 9 Register columns See UNICORN Method Manual for information on how to register columns in UNICORN 186 ÄKTA avant User Manual 29035184 AE 5 Operation 5 5 Connect a column ...

Page 187: ...ity 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 conductivityflow cell has a maximum pressure limit of 5 MPa 50 bar Pre column pressure alarms It is important that the pre column pressure alarm is set during all runs where a column is used The pressur...

Page 188: ...ox select Alarms Alarm pre column pressure 2 Click Enabled in the Mode field 3 Enter the high pressure limit in the High alarm box Click Execute 4 For more information on pressure alarms including the Low Alarm parameter see Section 9 7 4 System settings Pumps and pressures on page 464 Note 188 ÄKTA avant User Manual 29035184 AE 5 Operation 5 6 Set pressure alarms ...

Page 189: ...ple carry over when switching techniques for loading samples wash the valve with buffer between the loading of two different samples For example when switching from loading sample in the loop to loading sample directly onto the column with the valve in Direct inject position Note When using a pump for sample application it is important to prime inlets and purge the pump before using the pump to lo...

Page 190: ...tions See page Section 191 5 7 1 Sample application using direct injection into the column 194 5 7 2 Sample application using a Superloop 199 5 7 3 Sample application using a sample loop 190 ÄKTA avant User Manual 29035184 AE 5 Operation 5 7 Sample application ...

Page 191: ...ds and phases Then When preparing to inject manually prime the sample inlet tubing with sample see Prime sample inlets on page 173 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 select Inject fixed sample volume and set the volume to be injected make sure that the flow path from the sample inlet ...

Page 192: ...h sample or buffer see Prime sample inlets on page 173 and make sure that the tubing is immersed in sample be fore starting the run make sure that the flow path from the sample inlet valve up to the injection valve is filled with sample or an appropriate buffer and that the buffer inlet tubing is immersed in buffer all the sample 192 ÄKTA avant User Manual 29035184 AE 5 Operation 5 7 Sample applic...

Page 193: ...fixed sample volume and set the volume to be injected enable the function Prime sample inlet with and set the volume to be used for priming Result the step Finalize sample injection is automatically deactivated in order to maximize precision and accuracy See Minimize sample loss on page 191 for more information If manual priming of the flow path up to the injection valve is preferred enable Prime ...

Page 194: ...ug With a Superloop connected to the valve an over pressure may be created during injection Note Prepare the Superloop To avoid injecting air into the system flow path the Superloop should be prefilled with buffer manually before fitting the Superloop to the system Read the instruction for the Superloop to be used Note Connect the Superloop The following steps describe how to connect the Superloop...

Page 195: ...e Superloop using a syringe Follow the instruction below to fill the Superloop using a syringe Action Step Check if the system is in state Ready 1 If yes The Injection valve is in position Manual Load per default Continue to step 3 If no Continue to step 2 to position the valve ÄKTA avant User Manual 29035184 AE 195 5 Operation 5 7 Sample application 5 7 2 Sample application using a Superloop ...

Page 196: ...o step 5 the Loop valve continue to step 4 In the Process Picture 4 Click the Loop valve Select the position the loop is connected to for example Position 1 Result The Loop valve switches to the selected position Fill a syringe with sample 5 Connect the syringe to Injection valve port Syr 6 196 ÄKTA avant User Manual 29035184 AE 5 Operation 5 7 Sample application 5 7 2 Sample application using a S...

Page 197: ...in the Method Editor For multiple injections it may be more convenient to fill the Superloop once as described in the instruction be low Tip Action Step In the Manual instructions dialog box 1 Select Flow path Injection valve Select Sample pump load from the Position drop down list Click Execute Result The Injection valve switches to Sample pump load position Make sure that the sample inlet tubing...

Page 198: ...in Click Execute Result A sample flow starts in this example of 10 ml min When the Superloop is filled with as much volume as is needed click the End icon in the System Control toolbar to end the run 5 Plug the Syr port on the Injection valve with a Stop plug 6 198 ÄKTA avant User Manual 29035184 AE 5 Operation 5 7 Sample application 5 7 2 Sample application using a Superloop ...

Page 199: ...an be connected simultaneously Sample loop is called capillary loop in UNICORN Note How to fill a sample loop Follow the instructions to fill the sample loop with sample Action Step Connect a suitable sample loop to Injection valve ports LoopF fill and LoopE empty 1 Fill a syringe with sample 2 Connect the syringe to the Injection valve port Syr 3 ÄKTA avant User Manual 29035184 AE 199 5 Operation...

Page 200: ...ected to for example Position 1 Result The Loop valve switches to the selected position Load sample into the sample loop To avoid sample loss due to siphoning leave the syringe in the port until the sample has been injected onto the column during the run 7 It is recommended to overfill the loop to make sure that the loop is completely filled Excess of sample will leave the valve through port W1 Ti...

Page 201: ...or emptying and washing the loop is set in the Method Editor Using a sample loop connected to Loop valve V9 L or V9H L For maximum reproducibility use complete loop fill when loading the loop that is overfill the loop with a sample volume of up to 3 5 times the volume of the loop For minimum sample loss use partial loop fill that is fill only up to 50 of the loop volume Empty the loop with 3 5 tim...

Page 202: ...ectors are connected to ÄKTA avant and controlled by UNICORN The fraction collectors can be automaticallycontrolled in a method run or manually controlled In this section This section contains the following subsections See page Section 203 5 8 1 Prepare the built in fraction collector 210 5 8 2 Prepare Fraction collector F9 R 218 5 8 3 Fractionation overview 202 ÄKTA avant User Manual 29035184 AE ...

Page 203: ...collect fractions through the outlet valve or the optional external Fraction collector F9 R Available cassettes trays and racks The following Cassettes and racks are available Cassette 3 ml tubes for 40 tubes Cassette 5 ml tubes for 40 tubes Cassette 8 ml tubes for 24 tubes Cassette 15 ml tubes for 15 tubes Cassette 50 ml tubes for 6 tubes Cassette for deep well plate 24 48 96 wells Cassette tray ...

Page 204: ...ttles 1 See UNICORN System Control Manual Change the System Settings in UNICORN to set the fractionation mode and other settings for fraction collection 2 Available System Settings are de scribed in Section 9 7 7 System settings Fraction collection on page 468 Prepare cassettes and insert the cassette tray Follow the instructions to prepare the fraction collector before a run Action Step If you ar...

Page 205: ...he deep well plates are rotated so that the well marked A1 is positioned above the A1 marking on the cassette 2 Close the cassettes that have the QuickRelease function 3 ÄKTA avant User Manual 29035184 AE 205 5 Operation 5 8 Fractionation 5 8 1 Prepare the built in fraction collector ...

Page 206: ...e see the illustration faces the front of the tray marked with the GE monogram 4 Open the fraction collector drawer by pressing the handle upwards and pulling out the drawer 5 206 ÄKTA avant User Manual 29035184 AE 5 Operation 5 8 Fractionation 5 8 1 Prepare the built in fraction collector ...

Page 207: ...sult After the door has been closed the fraction collector performs a Full scan of the cassette type code of each cassette to identify the cassette types see Cassette and tray identification on page 209 Prepare a rack for 50 ml tubes or 250 ml bottles Action Step Place 50 ml tubes or 250 ml bottles in the corresponding rack rack with 50 ml tubes shown in illustration 1 ÄKTA avant User Manual 29035...

Page 208: ...t 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 3 Close the drawer Make sure that it snaps into closed position 4 208 ÄKTA avant User Manual 29035184 AE 5 Operation 5 8 Fractionation 5 8 1 Prepare the built in fraction collector ...

Page 209: ...ermine which type of cassettes are used Quick scan is performed during the run to ensure that correct cassettes are placed in the fraction collector WARNING Moving parts in fraction collector Do not open the fraction collec tor drawer when the fraction collector is active If you need to ac cess the fraction collector press Pause and make sure that the movement has stopped before opening the drawer...

Page 210: ...F9 R check the fractionation settings in the method to be run Perform the steps described below according to the settings in the method Assemble the Tube rack Insert collection tubes Adjust the Delivery arm Update System Settings in UNICORN Illustration of the tube rack Function Part Single cutout 1 L shaped cutout 2 Bowl 3 Tube support 4 Tube guide 5 Tube holder 6 1 2 3 4 5 6 Note that the tube g...

Page 211: ...guide 50 to 180 mm 18 mm 95 18 mm Tube holder 18 mm Tube guide 50 to 180 mm 30 mm 40 30 mm Tube holder 30 mm Tube guide Single and L shaped cutouts When assembling a tube rack different cutouts are used for the various inserts depending on the length of the collection tubes Which cutouts to use are summarized in the tables below 12 mm and 18 mm tube rack inserts 85 to 180 mm tubes 50 to 85 mm tube...

Page 212: ...ack Action Step Insert the tube support 4 if required into the bowl 3 The circular marks on the tube support should face down 1 1 2 3 4 5 6 Note When assembling a tube rack single cutouts 1 and L shaped cutouts 2 are used for various inserts depending on the length of the collection tubes See Single and L shaped cutouts on page 211 for detailed information Insert the tube guide 5 with the tube pos...

Page 213: ...lexible bowl out at each rib and snap the tube holder under the top lip of the rib 8 8 Note Do not force the tube holder into place as this may damage the lip Check that the surface of the tube holder is level 4 Gently move the delivery arm 10 out to the outer stop 5 ÄKTA avant User Manual 29035184 AE 213 5 Operation 5 8 Fractionation 5 8 2 Prepare Fraction collector F9 R ...

Page 214: ...at position 1 pushing each one down as far as they will go All the tubes must be of the same length and diameter and there should be no spaces in the sequence Adjust the delivery arm Follow the instructions to adjust the height of the delivery arm NOTICE Never lift Fraction collector F9 R by the delivery arm This may damage the fraction collector 214 ÄKTA avant User Manual 29035184 AE 5 Operation ...

Page 215: ...ery arm 1 and allow it to move in so the tube sensor 2 touches the collection tubes of the outer track 1 1 2 Loosen the lock knob 3 2 3 ÄKTA avant User Manual 29035184 AE 215 5 Operation 5 8 Fractionation 5 8 2 Prepare Fraction collector F9 R ...

Page 216: ...6 Lock the delivery arm at this height with the lock knob 3 Check that the tube sensor 9 is in the correct position for the tubes used 10 The eluent tubing 7 should be above the center of the collection tube 4 7 8 9 10 Use the sensor control 8 to position the tube holder over the center of the collection tube 5 216 ÄKTA avant User Manual 29035184 AE 5 Operation 5 8 Fractionation 5 8 2 Prepare Frac...

Page 217: ...tubes and large tubes indicated in the illustration below The position for large tubes is used for tubes of approximately 18 mm i d and larger The position for small tubes is used for tubes smaller than 18 mm i d Note that this is a rough approximation Always check that the eluent tubing is centered above the collection tube ÄKTA avant User Manual 29035184 AE 217 5 Operation 5 8 Fractionation 5 8 ...

Page 218: ...signal is used to determine when to switch the tubes See Section 9 7 8 System settings Watch parameters on page471 for information about different watch options Peak fractionation The two fractionation types listed above can be used in combi nation Combination of fixed volume and peak fractionation allows fractions collected by fixed volume fractionation and fractions collected by peak fractionati...

Page 219: ...ation using fixed volume fractionation and fractionation using peak fractionation A Straight fixed fractionation B Peak fractionation Volume Absorbance Absorbance Volume ÄKTA avant User Manual 29035184 AE 219 5 Operation 5 8 Fractionation 5 8 3 Fractionation overview ...

Page 220: ...outlet that is used see the following illustration UV Vis pH Valve The time when a peak is detected by the UV Vis detector is called T0 The peak reaches the fraction collector at time T1 Fraction collector Conductivity Delay volume As the delay volume is affected by the length and diameter of the tubing it should be set according to the tubing and modules used see Section 9 12 Delay volumes on pag...

Page 221: ...s used the delay volume is collected at the beginning of fractionation Fraction collector F9 R saves the delay volume in the first tube fraction 1 see the pre vious example illustration Delay volumes are not indicated with a numerical fraction mark on the chromatogram The built in fraction collector does not save the delay volume instead the delay volume is disposed of in the waste funnel Note The...

Page 222: ...e prefix 2 i e fractions 2 2 2 3 etc Note Fractionation modes for built in fraction collector There are three fractionation modes for the built in Fraction collector Automatic Accu mulator and DropSync Spillage between collection vessels during a run is avoided with all three fractionation modes Automatic The fraction collector uses the DropSync mode for flow rates up to 2 ml min and automatically...

Page 223: ...ottles before starting a run It is not possible to change the cassette configuration during a run When the system state is set to Pause it is possible to take out cassettes or plates from the fraction collector only if they are replaced by cassettes or plates of the same type and are placed in the same positions The action of the system when the last tube in the fraction collector is filled is set...

Page 224: ...er affect the resolution in adsorption chromatography techniques such as affinity chromatography AC ion exchange chromatography IEX and hydrophobic interaction chromatography HIC The effect of peak broadening in the system from sample injection to peak detection including dilution on the column is apparent in the chromatogram from the UV monitor but broadening from the UV monitor to fraction colle...

Page 225: ...uns on ÄKTA avant It also contains advice on things to be considered during and after a run In this section This section contains the following subsections See page Section 226 5 9 1 Create a method 230 5 9 2 Perform a run 233 5 9 3 Monitor the run 236 5 9 4 After run procedures ÄKTA avant User Manual 29035184 AE 225 5 Operation 5 9 Create a method and perform a run ...

Page 226: ...KTA avant 25 to ÄKTA avant 150 See UNICORN Method Manual Note Predefined methods There are several predefined methods to choose from All the predefined methods are listed in the following topics The predefined methods available for each system are defined by the Instrument Con figuration See Section 9 6 Predefined methods and phases on page449 for more informa tion about each method Purification m...

Page 227: ...ited and saved with a new name or overwritten 2 Build edit the Method Outline and or edit the Phase Properties for the appropriate phases 3 Save the method Create a new method Follow the instructions to create a chromatographic method based on a predefined method Action Step Open the Method Editor module and click the New Method button 1 Result The New Method dialog box opens ÄKTA avant User Manua...

Page 228: ...sen method is shown in the Method Outline pane and the default settings for each of the phases is shown in the Phase Properties pane See UNICORN Method Manual for more information about methods and method creation in UNICORN Sample loop is called capillary loop in UNICORN Note 228 ÄKTA avant User Manual 29035184 AE 5 Operation 5 9 Create a method and perform a run 5 9 1 Create a method ...

Page 229: ...ing the Text Instruction mode Inlet valve IX It is necessary to use instructions for both Inlet valve IX V9 IX or V9H IX and the other inlet valve Note Versatile valve V9 V or V9H V I O box E9 UV monitor U9 L 2nd The UV monitor U9 L 2nd is auto zeroed at the same time as UV monitor U9 M in predefined methods All other UV monitor U9 L 2nd instructions need to be edited in the Text Instruction mode ...

Page 230: ...immersed into appropriate waste vessels Consider vessel size and vessel material No tubing is twisted and the flow path is free from leakage Check the pump piston rinsing system Check that liquid is coming out of the outlet tubing and that each tube contains at least 25 ml If not add liquid Evaporation can decrease volumes To fill and prime the system see Section 7 3 1 Change pump rinsing solution...

Page 231: ...anges if necessary Click Next 3 Click Start on the last page of the Start Protocol 4 Result If column logging was chosen during installation of UNICORN and a column type was selected when the method was created the Select Columns dialog box opens For further information on column handling please refer to UNICORN Method Manual and UNICORN System Control Manual If column logging was not chosen durin...

Page 232: ... the same breakpoint Note Manual runs are only stored temporarily However you can choose to store them permanently in a selected directory To save results in a chosen directory click Browse before the run is started 4 Note If a method run is started during a manual run the results from the manual run are not stored To perform the instructions click Execute 5 Manual runs can also be controlled from...

Page 233: ...l Manual Monitor the run To interrupt a method during a run you may click the Hold Pause or End buttons in System Control A held or paused method run can be resumed by clicking the Continue button See the following table then If you want to click the button temporarily hold the method with current flow rate and valve positions sustained click the button temporarily pause the method and stop all pu...

Page 234: ...tration and de scribed in the following table Modules without a fixed place in the system are shown in a panel below the process picture modules are called components in the process picture Indication Color Open flow path with flow Green Closed flow path or an open path without flow Grey Syringe port in loop open for manual injection Blue 234 ÄKTA avant User Manual 29035184 AE 5 Operation 5 9 Crea...

Page 235: ...shows the pop up toolbar for the Injection valve icon Instructions can be given from the pop up toolbar of each component icon To display a detailed picture with explanations for example for a valve right click the component icon and click Detailed picture ÄKTA avant User Manual 29035184 AE 235 5 Operation 5 9 Create a method and perform a run 5 9 3 Monitor the run ...

Page 236: ...NING Corrosive chemicals during maintenance If the system or column is cleaned with a strong base or acid flush with water afterwards and wash with a weak neutral buffer solution in the last step or phase System cleaning After a method run is completed perform the following Rinse the instrument with one or several cleaning solution s e g NaOH buffer solu tion or distilled water using the System CI...

Page 237: ...monitoring will not be used for a week or longer perform one of the following ac tions Inject new storage solution into the pH flow cell Replace the pH electrode with the dummy electrode that is installed in the pH valve on delivery In the following situations in order to increase the lifetime of the pH electrode use the By pass position and store the electrode in storage solution inside the pH fl...

Page 238: ...menu click Exit UNICORN exit UNICORN Result All open UNICORN modules close If an edited method or result is open and not saved when you try to exit or log off UNICORN you will see a warning Click Yes to save No to exit without saving or Cancel to stay logged on Note Shut down the instrument Switch off the instrument by pressing the Power switch to the O position 238 ÄKTA avant User Manual 29035184...

Page 239: ... stop This chapter describes how to prepare run and evaluate the available performance tests In this chapter This chapter contains the following sections See page Section 240 6 1 General performance test actions 243 6 2 Air sensor tests 245 6 3 Built in Fraction collector test 250 6 4 Fraction collector F9 R test 253 6 5 Quaternary valve test 256 6 6 System test 260 6 7 UV monitor U9 L test ÄKTA a...

Page 240: ...ox click one of the following tests 2 Test Air sensors test Fraction Collector test Built in frac Fraction Collector test F9 R Quaternary valve test System test UV test U9 L Click Run Performance Method 3 Result The Start Protocol of the selected test opens Click Next in the Start Protocol dialog box to open the next dialog box The dialog boxes are described in the table Overview of the Start Prot...

Page 241: ... report Evaluation Procedures Allows the user to change result name and result location Result Name and Loca tion During the run A Message dialog box opens during the run Read the messages in the dialog and make sure that necessary preparations have been performed Click Confirm and Continue in the Message dialog box to change system state from System Pause to Run and proceed with the test Alternat...

Page 242: ...N 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 Refer to UNICORN Administration and Technical Manual for information on how to install a printer Print the report and check the status of the tests For each of the tests the report states The test...

Page 243: ...ked Buff in distilled water 1 Prime the inlets A1 B1 and the sample inlet Buff and purge the pumps See Section 5 4 1 Prime buffer inlets and purge system pumps on page 166 and Section 5 4 2 Prime sample inlets and purge Sample Pump on page 173 2 Disconnect the inlet tubing connected to the inlet valve positions A4 B4 and S4 During the test method air is introduced into the inlet valves through the...

Page 244: ...checked and taken care of run the test once again Action Cause For further information see Inlet valves on page 382 Faulty air sensor Make sure that the tubing was correctly prepared see Prepare the Air sensors test on page 243 Incorrect preparation of tubing 244 ÄKTA avant User Manual 29035184 AE 6 Performance tests 6 2 Air sensor tests ...

Page 245: ...required Distilled water Syringe 25 to 30 ml For ÄKTA avant 25 Two Cassettes for deep well plates Two 96 deep well plates For ÄKTA avant 150 Two Racks for 50 ml tubes Twelve 50 ml tubes Prepare the Fraction collector test Follow the instruction to prepare the system before method start Action Step Immerse the piece of inlet tubing marked A1 in distilled water 1 Prime inlet A1 and purge System pump...

Page 246: ...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 4 Open the Frac drawer by pressing the handle upwards and pulling out the drawer 5 246 ÄKTA avant User Manual 29035184 AE 6 Performance tests 6 3 Built in Fraction collector test ...

Page 247: ...d to show correct tube and cassette positions 8 In the System Control module on the Systemmenu click Settings 9 Result The System Settings dialog box opens In the System Settings dialog box 10 Select Fraction collector Fractionation settings For ÄKTA avant 25 In the Fractionation mode drop down list click Ac cumulator In the Fractionation order drop down list click Row by row Click OK Run the test...

Page 248: ... and that there is minimal spillage For further information on delay volumes and fractionation marks see Delay volume on page 220 ÄKTA avant 25 Check that correct volumes 2 ml per well are collected in wells 1A1 to 1A4 and 6A1 to 6A4 ÄKTA avant 150 Check that correct volumes 20 ml per tube are collected in tubes 1A1 to 1A4 and 6A1 to 6A4 248 ÄKTA avant User Manual 29035184 AE 6 Performance tests 6...

Page 249: ...fractionation settings in the System Settings dialog box Liquid is collected in the wrong wells or tubes Incorrect fractionation settings Make sure that only two Cassettes are placed on the Cassette tray and that the Cassettes are positioned on the cassette positions 1 and 6 Liquid is collected in wrong Cassette Incorrect preparation of the Fraction collector Clean the Fraction collector See Secti...

Page 250: ...or collecting the fractions Prepare the Fraction collector F9 R test Follow the instructions to prepare the system before method start Action Step Make sure that the Fraction collector F9 R is connected to port Out 10 on Outlet valve V9 O or V9H O 1 Direct outlet tubing W to a waste container 2 Place inlet tubing A1 into distilled water 3 Prime inlet A1 and purge System pump A See Section 5 4 1 Pr...

Page 251: ...on how to evaluate the fraction collector test Evaluate the result Check that the correct volumes have been collected in the tubes The tubes should contain the following Tube 1 The delay volume Tube 2 3 and 4 2 ml Tube 5 and 6 1 ml Also check that the fractionation marks in the chromatogram correspond to the filled tubes and that spillage is kept to a minimum For further information on delay volum...

Page 252: ...See Section 8 6 Troubleshooting Pumps on page 397 Incorrect volumes col lected in the tubes and disturbances of system pressure curves Air trapped in Sys tem pump A FaultySystempump A Make sure the fraction collector delivery arm is positioned above tube number 1 before starting the test Liquid collected in wrong tubes Make sure that the system was correctly prepared see Prepare the Fraction colle...

Page 253: ...he system before method start Action Step Immerse the pieces of inlet tubing marked Q1 and Q3 in 1 acetone in dis tilled water 1 Immerse the pieces of inlet tubing marked Q2 and Q4 in distilled water 2 Connect the capillary loop marked Ref 1 between Column valve ports 1A and 1B to generate a back pressure 3 For ÄKTA avant 25 Make sure that the Mixer with a chamber volume of 1 4 ml is installed 4 F...

Page 254: ...hromatogram from a Quaternary valve test The gradient steps are marked in the illustration 100 UV 0 UV 5 Q1 UV 95 Q2 5 Q3 UV 95 Q4 95 Q1 UV 5 Q2 95 Q3 UV 5 Q4 50 UV The illustration shows a chromatogram from a Q valve test on ÄKTA avant 25 A chromatogram from a Q valve test on ÄKTA avant 150 has a similar appear ance but a different scale on the y axis Note 254 ÄKTA avant User Manual 29035184 AE 6...

Page 255: ...ntenance chapter Disturbances caused by damaged pump piston seals See UV monitor U9 M and UV detector unit on page 363 Unstable or incorrect UV signal or drifting base line faulty UV monitor Change mixer chamber or replace the Mixer See Sec tion 7 8 2 Replace the Mixer on page 338 Wrong mixer chamber size or faulty mixer See Quaternary valve on page 383 Faulty Quaternary valve See Section 8 6 Trou...

Page 256: ...re method start Action Step Immerse the piece of inlet tubing marked A1 in distilled water 1 Immerse the piece of inlet tubing marked B1 in 1 acetone and 1 M NaCl in distilled water 2 Immerse the piece of inlet tubing marked Buff in 1 acetone and 1 M NaCl in distilled water 3 Connect the capillary loop marked Ref 1 between Column valve ports 1A and 1B to generate a back pressure 4 For ÄKTA avant 2...

Page 257: ...e sure to prime the buffer inlets and to purge the SystempumpsandtheSamplepumpbeforemethodstart See Section 5 4 1 Prime buffer inlets and purge system pumps on page166 and Section 5 4 2 Prime sample inlets and purge Sample Pump on page 173 Disturbances caused by air trapped in any of the pumps Replace piston seals See ÄKTA avant Operating Instructions Maintenance chapter Disturbances caused by dam...

Page 258: ... pump piston seal in System pump B Replace piston seals See ÄKTA avant Operating Instructions Maintenance chapter Faulty values at 95 damaged pump piston seal in System pump A Faulty UV Absorbance Test Action Cause Make sure that the acetone solution is 1 and that no solution has evaporated Incorrectly prepared acetone solution Faulty Pulsation Test Action Cause Make sure to prime and purge the sy...

Page 259: ...on 7 5 1 Clean the UV flow cell on page 283 Air or dirt in the UV flow cell Check buffers for impurities Impure buffer Faulty Pressure Check Test Action Cause Check the tubing Folded twisted or blocked tubing Replace the inline filter see Section 7 3 2 Replace the inline filter on page 275 Dirt in inline filter Calibrate the pressure monitor see Section 7 7 2 Calibrate the pressure monitors on pag...

Page 260: ...er 1 4 ml Prepare the UV U9 L test Follow the instructions to prepare the system before method start Action Step Direct outlet tubing W and W1 to a waste container 1 Place inlet tubing A1 into distilled water 2 Place inlet tubing B1 into a solution of 1 acetone in distilled water 3 Connect the Ref 1 tubing on the Column valve position 1 to generate a back pressure 4 Make sure that the Mixer with a...

Page 261: ... shows a chromatogram from a function test with UV monitor U9 L Possible causes of a failed test The following tables describe possible causes of a failed test When possible sources of error have been checked and corrected repeat the test Faulty UV Absorbance Test Action Cause Make sure that the acetone solution is 1 and that no solution has evaporated Incorrectly prepared acetone solution See Cal...

Page 262: ...this chapter This chapter contains the following sections See page Section 263 7 1 Maintenance Manager 268 7 2 Maintenance program 272 7 3 Weekly maintenance 278 7 4 Monthly maintenance 282 7 5 Semiannual maintenance 287 7 6 Maintenance when required 320 7 7 Calibration procedures 335 7 8 Replacement procedures 262 ÄKTA avant User Manual 29035184 AE 7 Maintenance ...

Page 263: ...nd its modules are predefined The user can add automated maintenance notifications for the system Maintenance notifications are based on cal ender periods of system use and for some modules also on operational statistics Open Maintenance Manager In the System Control module on the System menu click Maintenance Manager to open the Maintenance Manager dialog box ÄKTA avant User Manual 29035184 AE 26...

Page 264: ...re 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 Select a notification to view notification details Modules with no plus symbol have no related maintenance notifications Note 264 ÄKTA avant User Manual 29035184 AE 7 Maintenance 7 1 Maintenance Manager ...

Page 265: ...ecked the notification will not be issued Enter a new interval after which the new notification will be issued Click Restart to reset the counter and add a complete interval before the next notification Edit the message that will be shown in the maintenance notification Click Default to restore the default settings for maintenance notifications Click Apply to save the changes 3 Add a new system no...

Page 266: ... issued If desired write a message that will be shown for the maintenance noti fication Click Apply to save the changes and apply the notification settings 3 Delete a user defined system notification To delete a user defined system notification select the notification in the Maintenance Manager dialog box and click Delete Module notifications are predefined and cannot be deleted If desired they ca...

Page 267: ... reset the counter for a new maintenance notification period Note Make sure that the maintenance action is performed as instructed after the notification is acknowledged otherwise the system performance may deteriorate close the message without action Note The Maintenance Notification will be displayed each time the System Control module is opened until the notification is acknowledged ÄKTA avant ...

Page 268: ...and finally distilled water before service and maintenance Hazardous chemicals during run When using hazardous chemicals run System CIP and Column CIP to flush the entire system tubing with distilled water before service and maintenance Always use appropriate Personal Protective Equipment PPE during operation and maintenance of this product Periodic maintenance program The following periodic maint...

Page 269: ... Replace the pH electrode on page 286 Replace pH electrode Bi annual Maintenance when required The followingmaintenanceshould be performed by the user of ÄKTA avant when required See section Maintenance action Section 7 6 1 Clean the instrument externally on page 288 Clean the instrument externally Section 7 6 2 Perform System CIP on page 289 Perform System CIP system cleaning in place Section 7 6...

Page 270: ...O ring inside the Mixer on page 340 Replace O ring in Mixer Section 7 8 4 Replace the UV monitor U9 M flow cell on page 342 and Section 7 8 5 Replace UV monitor U9 L flow cell on page 345 Replace UV flow cell Section 7 8 6 Replace Flow restrictor on page347 Replace the flow restrictor Section 7 8 7 Replace the inlet filters on page348 Replace inlet filters Section 7 6 11 Wipe off excess oil from t...

Page 271: ... Section 7 8 10 Replace a valve module on page 354 Replace valve modules Section 7 6 10 Clean pump flow restrictors on page 315 Clean pump flow restrictors ÄKTA avant User Manual 29035184 AE 271 7 Maintenance 7 2 Maintenance program ...

Page 272: ...nce activities In this section This section contains the following subsections See page Section 273 7 3 1 Change pump rinsing solution 275 7 3 2 Replace the inline filter 276 7 3 3 Clean built in fraction collector sensors 272 ÄKTA avant User Manual 29035184 AE 7 Maintenance 7 3 Weekly maintenance ...

Page 273: ...low to change rinsing solution and prime the pump piston rinsing systems See the tubing configuration of the pump piston rinsing systems in Illus tration of the pump piston rinsing systems on page 59 Action Step Unscrew the rinsing system tubes from the holders and discard the used pump rinsing solution 1 Fill each of the rinsing system tubes with 50 ml of 20 C ethanol 2 Screw the rinsing solution...

Page 274: ...into the syringe 6 Disconnect the syringe and discard its contents 7 Immerse the outlet tubing in the rinsing solution tube where the inlet tubing of the System pump piston rinsing system is immersed 8 Connect a 25 to 30 ml syringe to the outlet tubing from the Sample pump piston rinsing system Draw liquid slowly into the syringe 9 Disconnect the syringe and discard its contents 10 Immerse the out...

Page 275: ...ps Gloves Instruction Follow the instructions to replace the inline filter that is located in the top of the Mixer Use forceps and gloves during the replacement procedure to avoid contami nating the mixer components Tip Action Step Unscrew the top section of the Mixer 1 Remove the old filter using forceps Replace the support net if this is dam aged Fit the new filter 2 Check the O ring of the Mixe...

Page 276: ...o clean the fraction collector sensors See Illustration of the dis penser head on page 88 for the location of the fraction collector sensors Action Step In the System Control module on the Manual menu click Execute Manual Instructions In the Manual Instructions dialog select Fraction collec tion Frac cleaning position and then click Execute 1 Result The dispenser head moves to cleaning position an...

Page 277: ...letely before starting a run 3 Close the fraction collector door 4 Result Automatic scanning is performed In the System Control module press the End button in the toolbar 5 Result The dispenser head moves to home position and the instrument display states Ready ÄKTA avant User Manual 29035184 AE 277 7 Maintenance 7 3 Weekly maintenance 7 3 3 Clean built in fraction collector sensors ...

Page 278: ...or monthly maintenance In this section This section contains the following subsections See page Section 279 7 4 1 Check the flow restrictor 281 7 4 2 Check the function of the pump flow restrictors 278 ÄKTA avant User Manual 29035184 AE 7 Maintenance 7 4 Monthly maintenance ...

Page 279: ...ss Click Insert Select Flowpath pH valve and set the pH electrode to Off line and the Restrictor to In line Click Insert Select Flowpath Outlet valve and select Out Waste Click Insert Select Pumps and Pressures System flow and set the Flow rate to 3 0 ml min Click Insert Click Execute Result A system flow of 3 0 ml min starts Note the PreC pressure displayed in the Run Data pane Tip If PreC pressu...

Page 280: ...7 Check that the pressure difference is within the range 0 2 0 05 MPa 8 If this is not the case the Flow restrictor should be replaced see Section 7 8 6 Replace Flow restrictor on page 347 280 ÄKTA avant User Manual 29035184 AE 7 Maintenance 7 4 Monthly maintenance 7 4 1 Check the flow restrictor ...

Page 281: ...ey connect to the pressure monitor 2 Check if there are siphoning If the pump flow restrictors are working there should not be any siphoning 3 If there are siphoning the pump flow restrictors must be replaced at the next service occasion by a GE representative 4 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: ...miannual once every six months maintenance activities In this section This section contains the following subsections See page Section 283 7 5 1 Clean the UV flow cell 286 7 5 2 Replace the pH electrode 282 ÄKTA avant User Manual 29035184 AE 7 Maintenance 7 5 Semiannual maintenance ...

Page 283: ...llowing materials are required Luer connector Waste container Syringe 25 to 30 ml 10 surfactant detergent solution e g Decon 90 Deconex 11 or RBS 25 Distilled water Instruction Follow the instructions below to clean the UV flow cell of UV Monitor U9 M or UV Monitor U9 L The illustrations show UV Monitor U9 M UV Monitor U9 L is cleaned in the same way The UV flow cell can be mounted or not mounted ...

Page 284: ...ith distilled water and connect the syringe to the Luer con nector 3 Squirt the distilled water through the UV flow cell in small amounts Discon nect the syringe 4 Fill a syringe with a 10 surfactant detergent solution such as Decon 90 Deconex 11 RBS 25 or equivalent and connect the syringe to the Luer connector Tip Heat the 10 surfactant detergent solution to 40 C to increase the cleaning effect ...

Page 285: ...connector 9 Inject the distilled water into the UV flow cell to rinse the flow cell Disconnect the syringe 10 Disconnect the Luer connector from the top of the UV flow cell Reconnect the piece of tubing from the Column valve to the top of the UV flow cell 11 Disconnect the waste tubing from the bottom of the UV flow cell Reconnect the piece of tubing from the Conductivity monitor to the bottom of ...

Page 286: ...ectrode by hand and pull the used electrode away 2 Unpack the new pH electrode Remove the cover from the tip of the new pH electrode Make sure that the electrode is not broken or dry 3 Prior to first use of the electrode immerse the glass bulb in deionized water for 30 minutes and then in a standard buffer pH 4 for 30 minutes 4 Carefully insert the new pH electrode into the pH flow cell Tighten th...

Page 287: ...P 297 7 6 3 Perform Column CIP 301 7 6 4 Clean built in fraction collector 304 7 6 5 Clean Fraction collector F9 R 305 7 6 6 Storage of the pH electrode 307 7 6 7 Clean the pH electrode 311 7 6 8 Regenerate dried out pH electrode 312 7 6 9 Clean pump head check valves 315 7 6 10 Clean pump flow restrictors 317 7 6 11 Wipe off excess oil from the pump head 318 7 6 12 Clean the Conductivity flow cel...

Page 288: ...r 20 ethanol Instruction Follow the instructions to clean the instrument externally Action Step Check that no run is in progress 1 Switch off the instrument 2 Wipe the surface with a damp cloth Wipe off stains using a mild cleaning agent or 20 ethanol Wipe off any excess 3 Let the instrument dry completely before using it 4 288 ÄKTA avant User Manual 29035184 AE 7 Maintenance 7 6 Maintenance when ...

Page 289: ...to flush the entire system tubing with distilled water before service and maintenance If hazardous chemicals are used for system or column cleaning wash the system or columns with a neutral solution in the last phase or step Tip Maintenance interval Perform a System cleaning in place System CIP when required for example between runs where different samples and buffers are used This is important to...

Page 290: ...stem CIP method Action Step In the Method Editor module 1 click the New Method button or on the File menu click New Method Result The New Method dialog opens 290 ÄKTA avant User Manual 29035184 AE 7 Maintenance 7 6 Maintenance when required 7 6 2 Perform System CIP ...

Page 291: ... System CIP Click OK Result One Method Settings phase and three System CIP phases show in the Method Outline pane Each System CIP phase uses one cleaningsolution If desired add additional System CIP phases to the method using the Phase Library 3 ÄKTA avant User Manual 29035184 AE 291 7 Maintenance 7 6 Maintenance when required 7 6 2 Perform System CIP ...

Page 292: ...Injection valve select at least one of the sample inlets and clean the manual injection port using a syringe see Clean the manual injection port of the injection valve on page 294 Note The pH electrode is not included in System CIP but the flow restrictor is in line Refer to Section 7 6 7 Clean the pH electrode on page307 for instructions on how to clean the pH electrode 292 ÄKTA avant User Manual...

Page 293: ...according to the previous instructions 1 Connect by pass tubing to all selected column positions and loop positions if a loop valve is used 2 Prepare cleaning solutions and immerse the selected inlet tubing in the so lutions Note Note that each phase uses one solution All inlets selected in one phase should be immersed in the same cleaning solution 3 In the System Control module select the created...

Page 294: ...ctions dialog opens In the Manual instructions dialog select Flowpath Injection valve and click Manual Load Click Execute 2 Connect a suitable sample loop to Injection valve ports LoopF fill and LoopE empty Note Do not use a Superloop when cleaning the Injection valve 3 Connect tubing to Injection valve port W1 and direct this tubing to a waste container 4 294 ÄKTA avant User Manual 29035184 AE 7 ...

Page 295: ...e port Syr and inject the distilled water 6 Clean the pH valve Follow the instructions to clean the pH valve The calibration function is used to switch the valve position However no calibration is performed Action Step Connect tubing to pH valve port W3 and direct the other end of this tubing to a waste container 1 Unscrew the pH electrode from the pH valve and replace it with the dummy electrode ...

Page 296: ...mately 10 ml of 1 M NaOH Connect the syringe to the pH valve port Cal and inject the solution Leave the cleaning solution in place for 20 minutes 6 Fill a syringe with distilled water Connect the syringe to the pH valve port Cal and inject the distilled water 7 Click Close 8 Result The pH valve switches back to the default position and the Calibration dialog closes No calibration is performed 296 ...

Page 297: ... Hazardous chemicals during run When using hazardous chemicals run System CIP and Column CIP to flush the entire system tubing with distilled water before service and mainte nance If hazardous chemicals are used for system or column cleaning wash the system or columns with a neutral solution in the last phase or step Tip Maintenance interval Perform a Column cleaning in place Column CIP when requi...

Page 298: ...alog 2 In the System list select the name of the system Click Predefined Method In the Predefined Method list click Column CIP Click OK Result One Method Settings phase and one Column CIP phase will be dis played in the Method Outline pane In the Phase Properties tab of the Method Settings phase select Column type and Column position 3 298 ÄKTA avant User Manual 29035184 AE 7 Maintenance 7 6 Maint...

Page 299: ...ps button to get a suggested procedure for the selected column type Note that this function is not available for all column types 5 Result Suggested cleaning steps for the selected column type are displayed If several cleaning solutions are used change settings for Inlet A and or Inlet B Select one inlet for each solution If Inlet B is used remember to edit the values in the B column 6 ÄKTA avant ...

Page 300: ...the selected folder Run a Column CIP method Follow the instructions below to run a Column CIP method Action Step In the Method Editor module create a Column CIP method according to the previous instruction 1 Prepare cleaning solutions and immerse the selected inlets in the solutions 2 Connect the column to the selected column position 3 In the System Control module select the created method and st...

Page 301: ...ectors on page 336 Required material The following materials are required Wash bottle Water or 20 ethanol Cloth Instruction Follow the instruction below to clean the interior of the fraction collector See the locations of the components of the fraction collector in Section 3 10 2 Illustrations of the built in fraction collector on page 86 Action Step In System Control module on the Manual menu cli...

Page 302: ...ver 6 Wipe off the fraction collector chamber interior using a damp cloth Wipe off stains using a mild cleaning agent or 20 ethanol Note Be careful not to damage the fractionation arm flat cable 7 Tip The fraction collector arm can easily be moved to facilitate cleaning of the fraction collector Tip The cassette tray positioning discs can easily be removed to facilitate cleaning of the fraction co...

Page 303: ...KTA avant 25 Note Be careful not to scratch the diode windows Let the fraction collector dry completely before starting a run 9 Close the fraction collector drawer 10 Result Automatic scanning is performed In the System Control module click the End button in the toolbar 11 Result The dispenser head moves to home position and the instrument display states Ready ÄKTA avant User Manual 29035184 AE 30...

Page 304: ...n Step Check that no run is in progress 1 Switch off the instrument 2 Wipe the surface with a damp cloth Wipe off stains using a mild cleaning agent or 20 ethanol Wipe off any excess 3 Let the Fraction collector F9 R dry completely before restart 4 Clean DropSync sensor Clean the drop sensor photocell located above the tube sensor see Front view illustration on page 131 with a damp cloth 304 ÄKTA ...

Page 305: ...re required Syringe 25 to 30 ml Storage solution 1 1 mixture of standard buffer pH 4 and 1 M KNO3 Instruction Follow the instructions below to fill the pH flow cell with storage solution The calibration function is used to switch the position of the pH valve However no calibration is per formed Action Step In the System Control module on the System menu click Calibrate 1 Result The Calibration dia...

Page 306: ...trate KNO3 solution 4 Fill a syringe with approximately 10 ml of the storage solution Connect the syringe to the pH valve port Cal and inject the storage solution 5 Click Close 6 Result The pH valve switches back to the default position and the Calibration dialog closes No calibration is performed 306 ÄKTA avant User Manual 29035184 AE 7 Maintenance 7 6 Maintenance when required 7 6 6 Storage of t...

Page 307: ...7 1 Calibrate the pH monitor on page 321 Required material The following materials are required Syringe 25 30 ml Distilled water 0 1 M HCl and 0 1 M NaOH or Liquid detergent or 1 pepsin solution in 0 1 M HCl or 1 M KNO3 Cleaning agents Clean the pH electrode using one of the following procedures Salt deposits Dissolve the deposits by immersing the electrode for a five minute period in each of the ...

Page 308: ...rocedures fail to rejuvenate the electrode try the following procedure This procedure can be performed only when the pH electrode is not installed in the pH valve Note Action Step Heat a 1 M KNO3 solution to 60 C 80 C 1 Place the electrode tip in the heated KNO3 solution 2 Allow the electrode to cool while immersed in the KNO3 solution before re testing 3 If these steps fail to improve the electro...

Page 309: ... performed Action Step Open the System Control module On the System menu click Calibration 1 Result The Calibration dialog opens Set the pH monitor as the monitor to calibrate by clicking pH on the Monitor to calibrate menu 2 Click Prepare for Calibration 3 Result The pH valve switches to the calibration position ÄKTA avant User Manual 29035184 AE 309 7 Maintenance 7 6 Maintenance when required 7 ...

Page 310: ...stilled water and then with the next solution 5 As the last step in the cleaning procedure 6 Fill a syringe with distilled water Connect the syringe to the pH valve port Cal Inject the water Disconnect the syringe Click Close 7 Result The pH valve switches back to the default position and the Calibration dialog closes No calibration is performed 310 ÄKTA avant User Manual 29035184 AE 7 Maintenance...

Page 311: ...t function properly replace it with a new one 3 Regeneration methods Use one or both of the following methods to regenerate a dried out pH electrode Method 1 Soak the electrode in 1 M KNO3 for 5 hours or overnight Method 2 Soak the electrode in 0 1 M HCl overnight Rinse it carefully with distilled water Soak it in 4 pH buffer for 1 hour ÄKTA avant User Manual 29035184 AE 311 7 Maintenance 7 6 Main...

Page 312: ...asonic bath Instruction Follow the instructions to remove and clean the pump head check valves Action Step Before taking the check valve apart always try to clean the check valves by priming the pump heads first with distilled water then with 100 Methanol and then with distilled water again 1 Switch off the instrument 2 Disconnect the tubing from the pump head and disconnect the pump inlet tubing ...

Page 313: ...heck valve using an adjustable wrench and gently lift off the upper check valve 5 Unscrew the two white plastic screws located below each pump head Pull the plastic connectors to the sides to release the inlet manifold 6 ÄKTA avant User Manual 29035184 AE 313 7 Maintenance 7 6 Maintenance when required 7 6 9 Clean pump head check valves ...

Page 314: ...peration and mainte nance of the product 8 Immerse the valves completely in methanol and place them in an ultrasonic bath for a few minutes Repeat the ultrasonic bath with deionized water Refit the check valves 9 Tighten the nut until fully finger tight and then use the adjustable wrench to tighten a further 90 degrees 10 Refit the inlet manifold and reconnect the tubing to the pump head 11 314 ÄK...

Page 315: ...ece of tubing marked 2A from the flow restrictor block and connect a stop plug 1 16 male to the restrictor block 2 Disconnect the pieces of tubing marked 1A1 and 1A2 from the pump outlet check valves Place the tubing marked 1A1 in a waste beaker Connect a union 1 16 female to 1 16 female to the piece of tubing marked 1A2 Connect a union Luer female to 1 16 male to the other side of the union 3 Con...

Page 316: ...Continue until the flow path is free 6 Reconnect the pieces of tubing marked 2A 1A1 and 1A2 and place the buffer bottles on top of the system again 7 If blockage is flushed away with water stronger cleaning solutions can then be used Pump the cleaning solution through the pump in a standard manner and out to the Injection valve waste oulet Note 316 ÄKTA avant User Manual 29035184 AE 7 Maintenance ...

Page 317: ...h Mild cleaning agent or 20 ethanol Instruction Follow the instructions below to clean the System pumps externally Action Step Check that no run is in progress 1 Switch off the instrument 2 Wipe off the excess oil from the pump head with a damp cloth Wipe off stains using a mild cleaning agent or 20 ethanol 3 Let the pump dry completely before using the instrument 4 ÄKTA avant User Manual 29035184...

Page 318: ...ity monitor Action Step Disconnect the Fingertight connector and the piece of tubing from the top of the Conductivity monitor and attach a Luer connector 1 Disconnect the piece of tubing from the bottom of the Conductivity monitor and connect a piece of waste tubing to the Conductivity monitor Place the waste tubing in a waste container 2 Disconnect the Conductivity monitor from the rails or slide...

Page 319: ... syringe to the Luer connector Squirt 1 M NaOH through the conductivity flow cell about five times 7 Leave the liquid in the flow cell for at least 15 minutes 8 Fill a syringe with distilled water Connect the syringe to the Luer connector 9 Inject the distilled water into the conductivity flow cell to rinse the flow cell Disconnect the syringe 10 Disconnect the Luer connector from the top of the c...

Page 320: ...ins the following subsections See page Section 321 7 7 1 Calibrate the pH monitor 323 7 7 2 Calibrate the pressure monitors 327 7 7 3 Calibrate the Conductivity Monitor 331 7 7 4 Calibrate the UV monitors Open the Calibration dialog box Open the System Control module On the System menu click Calibrate to open the Calibration dialog box 320 ÄKTA avant User Manual 29035184 AE 7 Maintenance 7 7 Calib...

Page 321: ... and a pH standard buffer close to the lowest or highest pH you need to measure as your second point Allow the buffers to equilibrate to ambient temperature before use Instruction CAUTION pH electrode Handle the pH electrode with care The glass tip may break and cause injury Follow the instructions to perform the calibration Action Step Open the System Control module On the System menu click Calib...

Page 322: ...H for buffer 2 box 8 Repeat steps 5 to 6 using the second pH standard buffer 9 Result Calibration date and time are displayed in the dialog box and also values for Calibrated electrode slope and Asymmetry potential at pH 7 Is the Calibrated electrode slope 80 and the Asymmetry potential at pH 7 inside the interval 60 mV 10 If Yes Click Close to switch the pH valve back to the default position and ...

Page 323: ...e Tubing and pressures on page 325 1 Click the Customize button to open the Customize dialog box In the Cus tomize dialog box under the Run Data Groups tab select the relevant pressure to display see table Tubing and pressures on page325 Click OK to close the Customize dialog box 2 In the Run Data pane in the System Control module check what pressure is displayed 3 If the zero pressure reading is ...

Page 324: ...Reset pressure 3 Result The atmospheric pressure is defined as zero Date and time of the most recent calibration and the current pressure value are displayed Reconnect the tubing to the pressure monitor 4 324 ÄKTA avant User Manual 29035184 AE 7 Maintenance 7 7 Calibration procedures 7 7 2 Calibrate the pressure monitors ...

Page 325: ...onitors are also shown Pressure in UNICORN Tubing to disconnect Pressure monitor System pressure Tubing from the System pump pressure monitor System pump pressure monitor Sample pressure Tubing from the Sample pump pressure monitor Sample pump pressure monitor ÄKTA avant User Manual 29035184 AE 325 7 Maintenance 7 7 Calibration procedures 7 7 2 Calibrate the pressure monitors ...

Page 326: ...ng to Column valve V9 C or V9H C port In Pre column pres sure monitor PostC pressure Tubing to Column valve V9 C or V9H C port Out Post column pres sure monitor 326 ÄKTA avant User Manual 29035184 AE 7 Maintenance 7 7 Calibration procedures 7 7 2 Calibrate the pressure monitors ...

Page 327: ...ormal usage Perform calibration when the signal is unstable or you suspect that it is incorrect It is also recommended to recalibrate the Conductivity monitor after cleaning Conductivity Monitor factory calibration Follow the instruction to restore the conductivity cell constant to the factory default value Action Step In the Calibration dialog in the Monitor to calibrate list click Conductivity m...

Page 328: ...tion solution 6 Open the System Control module On the Manual menu click Execute Manual Instructions 7 Result The Manual instructions dialog box opens In the Manual instructions dialog box 8 Select Flow path Injection valve in the Position list click Direct inject Click Insert Select Flow path Column position in the Position list click By pass Click Insert Select Flow path pH valve and set the Rest...

Page 329: ...calibration solution is used see the graph for conductivity value at the current temperature Graph for con ductivity value on page 330 In the Calibration dialog box click Calibrate 12 Result The new conductivity cell constant is displayed in the Conductivity cell 1 constant cm box The new constant should normally be 40 10 cm 1 The date and time for the calibration are also displayed In the System ...

Page 330: ...rent temperature when 1 00 M NaCl calibration solution is used 0 3 5 2 0 2 77 80 85 90 95 97 Temperature C Conductivity mS cm Conductivity of 1 00 M NaCl at 20 30 C 330 ÄKTA avant User Manual 29035184 AE 7 Maintenance 7 7 Calibration procedures 7 7 3 Calibrate the Conductivity Monitor ...

Page 331: ...e normalized absorbance the path length of the second UV flow cell must be calibrated The flow cell path length must be registered or updated in UNICORN when the flow cell is replaced Note Equipment needed To perform the calibration a calibration kit containing test solutions syringes and acces sories is needed A specified kit is available for each cell length Then use calibration kit If using a U...

Page 332: ...est kit Concentration mg l Enter the corresponding absorbance values into the column UV Test kit Absorbance AU cm 4 Perform the calibration Follow the instructions to calibrate the second UV Monitor U9 L Action Step Open the System Control module On the Manual menu click Execute Manual Instructions 1 Result The Manual instructions dialog box opens In the Manual Instructions dialog box Select Pumps...

Page 333: ... the monitored absorbance value has stabilized 4 In the Manual Instructions dialog box 5 1 Select Monitors Auto zero UV 2nd 2 Click Insert then click Execute Result The UV absorbance is set to zero Remove the syringe 6 Repeat the injections with the remaining four test solutions in increasing concentration order Use a new syringe for each solution 7 After each injection wait for a stable absorbanc...

Page 334: ...ld be updated when the flow cell has been replaced or calibrated Action Step In the System Control module on the System menu click Calibration 1 In the Calibration dialog box click UV 2nd cell path length in the Monitor to calibrate list 2 Enter the nominal flow cell path length in the Nominal length input field and click Set 3 If a calibration has been performed enter the calculated flow cell pat...

Page 335: ...sections See page Section 336 7 8 1 Replace tubing and connectors 338 7 8 2 Replace the Mixer 340 7 8 3 Replace the O ring inside the Mixer 342 7 8 4 Replace the UV monitor U9 M flow cell 345 7 8 5 Replace UV monitor U9 L flow cell 347 7 8 6 Replace Flow restrictor 348 7 8 7 Replace the inlet filters 349 7 8 8 Replace pump head check valves 352 7 8 9 Replace pump rinsing system tubing 354 7 8 10 R...

Page 336: ...s Tubing cutter Fingertight wrench Instruction Follow the instruction below to replace tubing and connectors Action Step Make sure that no run is in progress on the instrument 1 Unscrew the connectors and disconnect the tubing 2 If the tubing has labels remove the labels to be used with the new tubing later Discard the tubing and connectors 3 336 ÄKTA avant User Manual 29035184 AE 7 Maintenance 7 ...

Page 337: ...els on the new tubing 5 Mount the connectors on the tubing 6 For fingertight connectors Slide the connector onto the tubing For tubing connectors 1 8 Slide the connector onto the tubing Slide the ferrule onto the tubing with the thick end towards the end of the tubing Insert the tubing with connector into the port Make sure to insert the tubing all the way into the bottom of the port 7 Tighten the...

Page 338: ... when the Mixer is damaged Required material The following is required Mixer Instruction Follow the instruction to change the Mixer Action Step Disconnect the tubing from the top and bottom of the Mixer 1 338 ÄKTA avant User Manual 29035184 AE 7 Maintenance 7 8 Replacement procedures 7 8 2 Replace the Mixer ...

Page 339: ... instrument 2 Attach the new Mixer 3 Reconnect the tubing to the top and bottom of the new Mixer 4 Pump out the air inside the new mixer 5 ÄKTA avant User Manual 29035184 AE 339 7 Maintenance 7 8 Replacement procedures 7 8 2 Replace the Mixer ...

Page 340: ...6 mm for Mixer chamber 15 ml O ring 13 1 x 1 6 mm high resistant product code 29 0113 26 Instruction Follow the instruction to replace the O ring inside the Mixer Use a forceps and gloves during the replacement procedure to avoid contam inating the Mixer components Tip Action Step Loosen the top section of the Mixer 1 Unscrew the top section of the Mixer and pull apart the Mixer in two halves 2 34...

Page 341: ...t the new O ring with 20 ethanol and fit it in position Make sure that the inline filter is still in position 5 Reassemble the Mixer components and while holding the Mixer upright screw the top section back onto the Mixer 6 ÄKTA avant User Manual 29035184 AE 341 7 Maintenance 7 8 Replacement procedures 7 8 3 Replace the O ring inside the Mixer ...

Page 342: ...leaning of the optical fiber connectors Lens paper Isopropanol Replace the flow cell CAUTION Hazardous chemicals or biological agents in UV flow cell Make sure that the entire flow cell has been flushed thoroughly with bacteriostaticsolution e g NaOH and distilledwater before service and maintenance Follow the instruction to replace the UV flow cell Action Step Switch off the instrument 1 Disconne...

Page 343: ... no liquid enters the UV detector or monochromator Note While the UV detector is disconnected protect the fiber connectors from dust or other impurities by mounting the rubber protective caps onto them Note Do not touch the optical fiber connectors as this will result in poor monitor performance If you accidentally touch the optical fiber connectors clean them according to Clean the optical fiber ...

Page 344: ...ean the optical fiber connectors Follow the instruction to clean the optical fiber connectors WARNING Hazardous substances When using hazardous chemicals take all suitable protective measures such as wearing protective clothing glasses and gloves resistant to the substances used Fol low local and or national regulations for safe operation and maintenance of the product Action Step Wipe the optical...

Page 345: ...hemicals or biological agents in UV flow cell Make sure that the entire flow cell has been flushed thoroughly with bacteriostaticsolution e g NaOH and distilledwater before service and maintenance Follow the instruction to replace the UV flow cell Action Step Switch off the instrument 1 Disconnect the tubing from the UV flow cell 2 ÄKTA avant User Manual 29035184 AE 345 7 Maintenance 7 8 Replaceme...

Page 346: ...re that the flow cell does not come into contact with any liquid and that no liquid enters the monitor Insert a new flow cell into the monitor 5 Tighten the knurled wheel firmly 6 Connect the tubing to the new flow cell 7 Switch on the instrument and log on to UNICORN 8 Update the UV flow cell path length in the Calibrate dialog box in the System Control module 9 346 ÄKTA avant User Manual 2903518...

Page 347: ...ubing connected from the used Flow restrictor and discard the used Flow restrictor 1 Connect the tubing to the new Flow restrictor Make sure that the Flow re strictor connector marked IN is connected to the pH valve port ToR To Re strictor and that the Flow restrictor connector marked OUT is connected to the pH valve port FrR From Restrictor 2 Check the back pressure of the new Flow restrictor see...

Page 348: ...uired Inlet filter set Instruction Follow the instruction to replace an inlet filter and a support net Action Step Pull off the inlet filter and the support net from the inlet filter holder 1 Fit the new support net and inlet filter and press the filter into position 2 348 ÄKTA avant User Manual 29035184 AE 7 Maintenance 7 8 Replacement procedures 7 8 7 Replace the inlet filters ...

Page 349: ...able wrench Instruction NOTICE Handle the check valves with care when they have been removed from the pump heads to prevent loss of any internal components Follow the instruction to replace the check valves of a pump Action Step Disconnect the tubing from the pump head and disconnect the pump inlet tubing 1 ÄKTA avant User Manual 29035184 AE 349 7 Maintenance 7 8 Replacement procedures 7 8 8 Repla...

Page 350: ... off the upper check valve 3 Replace the upper check valve with a new one 4 Tighten the nut until fully finger tight and then use the adjustable wrench to tighten a further 180 degrees 5 Place the new metal ring onto the new upper check valve and screw the new purge valve 6 350 ÄKTA avant User Manual 29035184 AE 7 Maintenance 7 8 Replacement procedures 7 8 8 Replace pump head check valves ...

Page 351: ...lve using an adjustable wrench 8 Replace the lower check valve with a new one 9 Tighten the nut until fully finger tight and then use the adjustable wrench to tighten a further 180 degrees 10 Refit the inlet manifold and reconnect the tubing to the pump head 11 ÄKTA avant User Manual 29035184 AE 351 7 Maintenance 7 8 Replacement procedures 7 8 8 Replace pump head check valves ...

Page 352: ...stem tubing Illustration of the pump piston rinsing systems Description Part Inlet tubing to the sample pump piston rinsing system 1 Outlet tubing from the sample pump piston rinsing system 2 Inlet tubing to the system pump piston rinsing system 3 Outlet tubing from the system pump piston rinsing system 4 352 ÄKTA avant User Manual 29035184 AE 7 Maintenance 7 8 Replacement procedures 7 8 9 Replace...

Page 353: ...n 3 Fit all pieces of tubing into the tubing holders on the pump modules 4 Prime the rinsing systems Before usage prime the pump rinsing system tubing Refer to Section 7 3 1 Change pump rinsing solution on page 273 for detailed instructions ÄKTA avant User Manual 29035184 AE 353 7 Maintenance 7 8 Replacement procedures 7 8 9 Replace pump rinsing system tubing ...

Page 354: ...ect power from the instrument before replacing any component on the instrument unless stated otherwise in the user documentation Action Step Disconnect power from the instrument by switching off the Power switch The Power switch is located on the left hand side of the instrument 1 Loosen the module with a Torx T20 screwdriver 2 354 ÄKTA avant User Manual 29035184 AE 7 Maintenance 7 8 Replacement p...

Page 355: ...the valve module 3 Disconnect the cable and secure it in the slit 4 Connect the cable to the new valve module 5 ÄKTA avant User Manual 29035184 AE 355 7 Maintenance 7 8 Replacement procedures 7 8 10 Replace a valve module ...

Page 356: ...Action Step Insert the valve module 6 Fasten it with a Torx screwdriver 7 356 ÄKTA avant User Manual 29035184 AE 7 Maintenance 7 8 Replacement procedures 7 8 10 Replace a valve module ...

Page 357: ...s good reason to believe that there is an internal leakage A sign of leakage is increasing pump rinsing solution volume Always make sure that sufficient spare components are available before attempt ing to replace a spare part Replacing spare parts Read the instructions carefully For example some individual parts of the pump head can be as sembled incorrectly Check the orientation of each part bef...

Page 358: ...ion 359 8 1 Introduction to troubleshooting 361 8 2 Troubleshooting General Checklist 363 8 3 Troubleshooting Monitors 381 8 4 Troubleshooting Valves 384 8 5 Troubleshooting Fraction collector 397 8 6 Troubleshooting Pumps 404 8 7 Troubleshooting Other components 413 8 8 Troubleshooting Instrument communication 415 8 9 Error codes 358 ÄKTA avant User Manual 29035184 AE 8 Troubleshooting ...

Page 359: ...s font by default Text in the Process Pic ture pane in the System control module looks strange Install the font Calibri True Type or switch to an operating system that includes the font UNICORN Method Manual UNICORN Evaluation Manual and UNICORN Administration and Technical Manual Troubleshooting procedure To troubleshoot ÄKTA avant Action Step Always start by checking the General checklist See Se...

Page 360: ...m menu click Create System Error Report 1 Result The first page of a wizard is displayed Click Next and start to enter information about the problem click Next Choose to enclose methods logs or result files Select location for the report and click Finish to generate the report The filename of the zip file will be Report_YYYYMMDD zip and the default folder location is C Program Files GE Healthcare ...

Page 361: ...anual Is the air sensor sensitivity set to normal in UNICORN System Settings to avoid un necessary stops due to minor air bubbles See Air sensor sensitivity on page 65 Instrument communication Have Node IDs been set correctly for all instrument modules To check and change Node IDs see Section 9 15 Check and change the Node ID of a module on page 515 Flow path checks Is all tubing connected correct...

Page 362: ... change the inline and inlet filters For more details see Section 7 8 7 Replace the inlet filters on page 348 and Section 7 3 2 Replace the inline filter on page 275 Does the positioning of the columns correspond to the selections made in the method See Section 5 5 Connect a column on page 182 Purification checks Have all columns been cleaned and prepared according to the column recommen dations H...

Page 363: ...problem Contact Service The cable between the UV module and the ICU is not connected Remove the UV module and make sure that the cable is connected Wrong Node ID Check the module s Node ID If necessary change the Node ID See Section 9 15 Check and change the Node ID of a module on page 515 The UV module is not found by the instru ment ÄKTA avant User Manual 29035184 AE 363 8 Troubleshooting 8 3 Tr...

Page 364: ... clean and dry lint free lens paper Worn out or broken lamp Contact Service Too low UV lamp inten sity Wrong wavelength for current buffer Change wavelength or buffer Dirt in the UV flow cell Clean the UV cell See Section 7 5 1 Clean the UV flow cell on page 283 Unclean optical fiber connectors Clean the connectors See Section 7 8 4 Replace the UV monitor U9 M flow cell on page 342 Broken UV flow ...

Page 365: ...enerate a System error report and con tact Service The internal tempera ture of the UV monitor is too high No UV flow cell is attached Attach UV cell See Section 7 8 4 Replace the UV moni tor U9 M flow cell on page 342 UV flow cell is not correctly installed Verify that the UV cell is correctly installed See Sec tion 7 8 4 Replace the UV monitor U9 M flow cell on page 342 The UV flow cell is broke...

Page 366: ...ter or buffer If persistent clean the UV cell See Section 7 5 1 Clean the UV flow cell on page 283 Air in buffers De gas if necessary Dirt in the UV flow cell Clean the UV cell See Section 7 5 1 Clean the UV flow cell on page 283 Dirt in the flow path Clean the system in accordance to Section 7 6 2 Per form System CIP on page289 Clean the column in ac cordance to Section 7 6 3 Perform Column CIP o...

Page 367: ...if necessary Make sure that both the instrument and the buffers have reached the ambient temperature Impure buffers Check if the signal is noisy with water Unclean optical fiber connectors Clean the connectors See Section 7 8 4 Replace the UV monitor U9 M flow cell on page 342 Dirt in the UV flow cell Perform a System CIP See Section 7 6 2 Perform Sys tem CIP on page 289 If necessary manually clea...

Page 368: ...V cell the buffer with the highest UV absorption shows the lowest UV absorption in the chromatogram and the buffer with the lowest UV absorption shows the highest UV ab sorption This can occur if there is a large difference in refractive index between buffer A and buffer B and the UV is run at high sensitivity The UV curve shows a gradient that is invert ed compared to the ex pected gradient UV mo...

Page 369: ...or current buffer Change to a shorter UV flow cell or change buffer The UV flow cell is not correctly installed Check that the UV flow cell is fitted correctly see Sec tion 7 8 5 Replace UV monitor U9 L flow cell on page 345 Dirt in the UV flow cell Clean the UV flow cell see Section 7 5 1 Clean the UV flow cell on page 283 Broken UV flow cell Replace the cell see Section 7 8 5 Replace UV monitor ...

Page 370: ...nt clean the UV cell See Section 7 5 1 Clean the UV flow cell on page 283 Air in buffers De gas if necessary Dirt in the UV flow cell Clean the UV cell See Section 7 5 1 Clean the UV flow cell on page 283 Dirt in the flow path Clean the system in accordance to Section 7 6 2 Per form System CIP on page289 Clean the column in ac cordance to Section 7 6 3 Perform Column CIP on page 297 Ghost peaks Co...

Page 371: ...he flow cell Note The standard tubing 7 between the UV monitor and the conductivity monitor is too short 170 mm for mounting the monitor for reversed flow direction Per form the following actions 1 Replace the standard tubing with a tubing that is 210 mm long 2 Update the total delay volume The increase in volume depends on the inner diameter i d of the tubing inner diameter i d 0 25 mm 2 µl i d 0...

Page 372: ...e cell constant mea surement has been aborted Air in the Conductivity flow cell Flush the Conductivity flow cell with water Solids in the Conductivity flow cell Clean the Conductivity cell See Section 7 6 12 Clean the Conductivity flow cell on page 318 Unstable conductivity This error can only occur when the temperature compensation is turned on The error will occur when the temperature is outside...

Page 373: ...lter see Section 7 3 2 Replace the inline filter on page 275 Baseline drift of noisy signal Unclean conductivity flow cell Clean the Conductivity cell See Section 7 6 12 Clean the Conductivity flow cell on page 318 The ambient temperature may have decreased in creased Use a temperature compensation factor The temper ature compensation factor is found in System Con trol System Settings Conductivity...

Page 374: ...ce the inline filter on page 275 Waves on the gradient A charged particle has been detected Prepare the sample so that charged particles are eliminated Air bubbles are passing through the flow cell Check for loose tubing connections Use the Flow restrictor Ghost peaks appear in the gradient profile Dirt in the tubing Make sure that the tubing has been washed properly Bad pump function Make sure th...

Page 375: ...xer chamber size is used See Select Mixer chamber on page157 for recommen dations To change the Mixer see Section 7 8 2 Replace the Mixer on page 338 Check the motor operation of the mixer Place a magnet close to the Mixer chamber during run The magnet should vibrate Check that the Mixer chamber is free from solids To open the Mixer see Section 7 3 2 Replace the inline fil ter on page 275 Incorrec...

Page 376: ...dings Decrease the room temperature Maximum operating temperature is 35 C Hardware error Switch off the instrument and wait until the tempera ture has decreased Restart the instrument If this error is recurrent generate a System error report and con tact Service The internal valve tem perature is too high 376 ÄKTA avant User Manual 29035184 AE 8 Troubleshooting 8 3 Troubleshooting Monitors ...

Page 377: ...trode See Section 7 5 2 Replace the pH electrode on page 286 Wrong mixer size for the used flow rate Use the recommended mixer size for the used flow rate See Select Mixer chamber on page 157 Wrong tubing connected between the Inlet valves and the System pumps If BufferPro is run on ÄKTA avant 150 at a flow rate 10 ml min replace the pieces of tubing marked InA and InB with FEP tubing o d 1 8 i d ...

Page 378: ... tubing See Sec tion 7 6 2 Perform System CIP on page 289 Change the tubing It is not possible to in ject calibration solu tion High pressure in the pH cell Decrease the flow rate By pass the pH electrode see Ports and flow paths of the pH valve on page81 and measure pH in fractions manually If ÄKTA avant 25 is used Replace tubing between Column valve and Outlet valve by tubing with i d 0 75 mm tu...

Page 379: ... path see To clean the column see Section 7 6 2 Perform System CIP on page 289 If persistent replace the column Excessively high pres sure values The cable between the Pressure monitors and the ICU is not connected Remove the monitor and make sure that the cable is connected The pressure monitors are not found by the instrument The air intake on the rear or on the left side of the instrument is co...

Page 380: ...erating Instructions A sign of leakage is that the pump rinsing solution volume starts to in crease Blockage of flow path Use the predefined method Prepare System to flush through to clear blockage If necessary replace tubing See Section 7 8 1 Replace tubing and connectors on page 336 Check the mixer inline filter It can be clogged if unfil tered buffers or samples are applied See Section 7 3 2 Re...

Page 381: ...Make sure that none of the air intakes on the instru ment are covered Hot surroundings Decrease the room temperature Maximum operating temperature is 35 C Hardware error Switch off the instrument and wait until the tempera ture has decreased Restart the instrument If this error is recurrent generate a System error report and con tact Service The internal valve tem perature is too high Hardware err...

Page 382: ...electrode properly with distilled water 3 Insert the dummy electrode into the pH valve 4 Rotate the dummy electrode before securing it with the nut Leaking pH valve Module Panel Possible cause and action Problem A Module Panel is missing and the position is left empty Install the missing Module Panel The cable between the Module Panel and the ICU is not connected Remove the Module Panel and make s...

Page 383: ...w Wrong mixing ratio for Quaternary valve The switch valve is not opened and closed correctly Perform Performance test according to ÄKTA avant Unpacking Instructions Check result and if necessary generate a System error report and contact Service Unexpected values for e g pH curve or con ductivity curve using a quaternary flow ÄKTA avant User Manual 29035184 AE 383 8 Troubleshooting 8 4 Troublesho...

Page 384: ...n page 515 The Fraction collector cannot be found by the instrument Obstruction inside the Fraction collector Switch off the instrument and check for obstruction inside the Fraction collector Try to move the Frac arm by hand Switch on the instrument If this error is recur rent generate a System error report and contact Ser vice Fraction collector arm is blocked or internal fault in the Fraction co...

Page 385: ...tor configuration manually in UNICORN In System Con trol select System Settings Navigate to Fraction collector Cassette configuration and select Manual The Fraction collector failed to detect the code on the Cassette The Cassette tray positioning discs in the Frac chamber are missing Replace the Cassette tray positioning discs in the Frac chamber See Section 7 6 4 Clean built in fraction col lecto...

Page 386: ...w rate The Dispenser head failed to detect a drop Air in the flow path 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 If this error is recurrent generate a System error report and contact Service Unclean DropSync sensor diode windows Clean the DropSync sensor diode windows See Sec tion 7 6 4 Clean built in fraction col...

Page 387: ...Decrease the room temperature The temperature control function is turned off In the System Settings dialog box check the settings for the temperature control function Fraction collec tor Temperature Hardware error Switch off the instrument and wait until the tempera ture has decreased Restart the instrument If this error is recurrent generate a System error report and con tact Service The internal...

Page 388: ...Section 5 8 1 Prepare the built in fraction collector on page 203 for information of how to place the Cas settes Objects or dirt under the Cassette Remove the object or dirt The Cassette does not fit the Cassette tray The Cassette tray positioning discs in the Frac chamber are missing Replace the Cassette tray positioning discs in the Frac chamber See Section 7 6 4 Clean built in fraction col lect...

Page 389: ... unclean Clean the the Cassette type codes The QuickRelease of a Cassette is in open position Close the Cassette See Section 5 8 1 Prepare the built in fraction collector on page 203 The Cassette code reader diode window is unclean Clean the Cassette code reader diode window See Section 7 6 4 Clean built in fraction collector on page 301 The automatic scanning is turned off in UNICORN Make sure th...

Page 390: ...looded Check the position of the waste funnel Refit the waste funnel The liquid leaving the nozzle does not strike the waste funnel Salt residuals in the capillary Perform an Accumulator wash If persistent contact Service The Frac capillary is blocked Leakage on the wet side of the instrument Localize the leakage and take care of the leakage for example by tightening connectors Air in pumps Purge ...

Page 391: ...in tubes or plates The tubes are flooded Make sure that the fraction volume is adapted to the tube volume Fraction tubes have not been replaced by empty tubes when the Fraction collector was opened Spillage by the Fraction collector during frac tionation The Frac drawer is not properly closed Close the Frac drawer It is not possible to fractionate The arm flat cable is stuck under the Cassette tra...

Page 392: ...Dirt on the nozzle or DropSync sensor diode win dows Dirt may effect where the drops fall Clean nozzle and DropSync sensor diode windows See Section 7 6 4 Clean built in fraction collector on page 301 The Fraction collector fractionates in the wrong well or tube Some parts in a method require that the Frac drawer is closed Do not open the Frac drawer when the symbol indicat ing ongoing fractionati...

Page 393: ...the tubing between Column valve and Outlet valve by tubing with i d 0 75 mm tubing 6 7 8 9 1R and 2R Note Delay volumes in System Settings have to be changed to Detector Frac 635 μl 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 D...

Page 394: ... component Before deselecting the Fraction collector as component performed in System properties in the Administration module follow the instruction below to turn off tempera ture control The circulation fan in the Fraction collector is blowing even though the Fraction collector is not in use 1 In the System Control module select System Settings 2 Select Fraction collection Fraction collector temp...

Page 395: ...the drop sensor photocell located above the tube sensor with a damp cloth The tubing is not correctly mounted in the tubing holder nut Check that the tubing is correctly mounted see Con nect tubing to the ÄKTA avant instrument on page134 The fraction collector failed to detect a drop and has changed tube without DropSync The fraction collector movement is blocked Make sure that the fraction collec...

Page 396: ... the time of the No tube error Fraction numbering does not start at 1 when the fractionation is restared after a No tube error Manually reset the fraction number in the System settings menu 396 ÄKTA avant User Manual 29035184 AE 8 Troubleshooting 8 5 Troubleshooting Fraction collector ...

Page 397: ...tem pumps on page 166 Bad piston spring Disassemble the pump head and examine the piston spring If the spring is corroded check piston seal and rinse membrane Make sure that the pump piston rinsing system is always used when working with aqueous buffers containing salt Bad pump piston If the piston is damaged replace it according to ÄKTA avant Operating Instructions Bad pump piston seal Replace th...

Page 398: ...e cable between the pump and the ICU is not connected Contact Service A fuse in the instrument ICU is broken Contact Service The pump is not found by the instrument The air intake on the rear or on the left side of the instrument is covered Make sure that none of the air intakes on the instru ment are covered Hot surroundings Decrease the room temperature Maximum operating temperature is 35 C Hard...

Page 399: ...place the inline filter on page 275 Abnormal difference in system pressure com pared to pre column pressure The flow path is blocked Remove obstructions in the flow path For example remove stop plugs and replace constricted tubing Internal pump error combined with high pressure Blocked pump restrictor Contact Service Internal pump error at normal pressure Too low I factor in the Pressure control p...

Page 400: ...stant pressure flow Examples of pump pressure curves The table below shows some examples of pump system pressure curves obtained when errors have occurred The examples can be useful in troubleshooting of the system pumps and the sample pump The system pressure monitor R9 has higher resolution than the other pressure monitors and is therefore recommended for troubleshooting purposes Incident 30 μl ...

Page 401: ...em 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 Incident Inlet check valve is loose Action Tighten the check valve See Section 7 8 8 Replace pump head check valves on page 349 ÄKTA avant User Manual 29035184 AE 401 8 Troubleshooting 8 6 Troubleshooting Pumps ...

Page 402: ...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 Incident One inlet is blocked Action Try first to clean the check valve see Section 7 6 9 Clean pump head check valves on page312 Try also cleaningof the inlet tubingmanifold For examp...

Page 403: ...re curve indicates that there are still air bubbles present repeat the purging process If the problem persists follow the in structions below to purge the pump with methanol see Section 5 4 1 Prime buffer inlets and purge system pumps on page166 for detailed instructions for the purge procedure 1 Make sure that the pump contains water 2 Use a syringe to draw 100 methanol into the pump see Section ...

Page 404: ...e and the ICU is not connected Remove the module and make sure that the cable is connected Two similar modules have been added to the instru ment for example two Inlet valve A Remove one of the modules with the same Node ID The Node ID for one or more of the modules is incor rect for example an Inlet valve A2 has the same Node ID as Inlet valve A The instrument then con siders them to have the sam...

Page 405: ...amber walls and sealings See Section 7 8 2 Replace the Mixer on page 338 See Mixer on page 522 Leakage Bad mixing of eluents Check the function of the Mixer Place a stirrer bar in the palm of your hand Hold the hand above the Mixer The stirrer should move when the Mixer is activated Check the Mixer chamber size and change chamber if necessary See Section 7 8 2 Replace the Mixer on page 338 Unstabl...

Page 406: ...perloop is filled to the max Pressure is not released anywhere Manually turn Injection valve to Manual load position Overpressure during filling Cabinet Possible cause and action Problem The lock unlock knob is in the unlock position Turn the lock unlock knob to the lock position The rubber behind the lock unlock knob is unclean Clean the swirl foot The rubber behind the lock unlock knob is worn o...

Page 407: ...le Power and ICU Possible cause and action Problem The power cord is not connected Connect the power cord to the wall outlet and to the electrical inlet on the instrument Make sure that the cord is attached using the clip thereby preventing the cord from coming loose No electric current in the wall outlet Make sure that there is electric current in the wall outlet A fuse in the instrument ICU is b...

Page 408: ...les are automatically turned off The network cable is not connected Connect the cable see UNICORN Administration and Technical Manual The instrument cannot be seen in UNICORN The cable between a module and the ICU is not connected Remove valves and check that cables are connected One or more jumpers are loose or missing Check the connections on the back of the instrument Empty positions must have ...

Page 409: ...mperature is too high External air sensors Possible cause and action Problem The cable between the external air sensor and the ICU is not connected Check the back of the instrument and make sure that the cable is connected See illustration below The external air sensor has not been selected in UNICORN Select the external air sensor in UNICORN Refer to the installation instructions delivered togeth...

Page 410: ... Problem Hot surroundings Decrease the room temperature Maximum operating temperature is 35 C Hardware error The internal tempera ture of the I O box is too high Wrong Node ID Make sure that the Node ID is 00 for the primary box and 01 for a potential secondary box The I O box is not de tected by the system Cables incorrectly connected Make sure that the cables are connected correctly earth to ear...

Page 411: ...ection error Make sure that Digital Out is connectedcorrectly earth to earth etc Analog In does not measure expected voltage Too long cable between the external equipment and E9 Use as short cable as possible Use shielded cable Connect the cable shield to the D sub connector s shield Noisy analog signal Analog In is not calibrated Contact Service Analog In does not measure expected voltage Analog ...

Page 412: ...high Touch screen is damaged Generate a System error report and contact Service The cursor on the dis play touch screen does not move Touch screen is not calibrated Calibrate touch screen See Section 2 5 Instrument display on page 37 The cursor on the dis play touch screen does not follow the finger No power Make sure that the power cord is connected Make sure that the instrument is on Display is ...

Page 413: ...dules and connections 3 Switch on the instrument 4 In the displayed dialog box in UNICORN select the option Restart the system only and click OK The UNICORN client has lost connection to the in strument server during a temporary overload of the processor Restart the UNICORN client to regain control The run continues and no data will be lost UNICORN has lost com munication with the in strument serv...

Page 414: ... dialog box in UNICORN select the option Restart the system only and click OK Cause One module has an incorrect Node ID Warning messages in UNICORN Warning Gate 12 Internal in strument error Action 1 Switch off the instrument 2 Check the Node ID for all modules see Section 9 15 Check and change the Node ID of a module on page 515 3 Switch on the instrument 4 In the displayed dialog box in UNICORN ...

Page 415: ...de Restart theinstrument If recurrentplease contact Service Internal instrument error 21 69 Valve Action Description Error code Restart theinstrument If recurrentplease contact Service Internal instrument error 20 24 Restart theinstrument If recurrentplease contact Service Valve not finding position 22 Restart theinstrument If recurrentplease contact Service Faulty air sensor 23 See Section 8 4 Tr...

Page 416: ...on Error code Restart theinstrument If recurrentplease contact Service Mixer motor error 25 Restart theinstrument If recurrentplease contact Service Internal instrument error 26 See Mixer on page 405 High temperature 27 pH monitor Action Description Error code Restart theinstrument If recurrentplease contact Service Internal instrument error 20 21 Contact Service No factory calibration 25 SeepHmon...

Page 417: ...estart the instrument If recurrent please contact Service Internal pump error 51 53 See Section 8 6 Troubleshooting Pumps on page 397 High temperature 54 UV Monitor U9 M Action Description Error code Restart theinstrument If recurrentplease contact Service Grating error 21 25 26 31 Restart theinstrument If recurrentplease contact Service Block filter error 22 23 32 Restart theinstrument If recurre...

Page 418: ...reappears contact Service Too high light intensity 27 Check that the detector is fitted correctly If warning reappears contact Service Too high light intensity R channel 30 Check that the flow cell is fitted correctly If warning reappears contact Service Too high S light intensity S channel 32 Check optical pathway and restart the instrument If recurrent please contact Service No light detected 34...

Page 419: ...act Service Measurement error 61 Fraction Collector F9 R Action Description Error code See Fraction Collector F9 R on page 394 High temperature 20 Clean the sensor and remove air bubbles in the flow path DropSync warning 21 Check that the tube sensor is adjusted properly Tube sensor error 22 Increase the fraction size Fraction size must be greater than 1 10 of the delay volume Delay queue full 23 ...

Page 420: ...on page 410 High temperature 20 Check the externalequipmentconnected to the I O box Analog in signal below 2V 21 Check the externalequipmentconnected to the I O box Analog in signal above 2V 22 Restart theinstrument If recurrentplease contact Service Internal instrument error 23 28 420 ÄKTA avant User Manual 29035184 AE 8 Troubleshooting 8 9 Error codes ...

Page 421: ...ecifications 424 9 2 Module specifications 432 9 3 Tubing and connectors 440 9 4 Chemical resistance guide 447 9 5 Wetted materials 449 9 6 Predefined methods and phases 459 9 7 System settings 475 9 8 Using Manual instructions 497 9 9 Available Run data 501 9 10 Available Curves 504 9 11 Injection volumes and peak broadening 505 9 12 Delay volumes 509 9 13 Component volumes 512 9 14 Pressure cont...

Page 422: ...P tubing inner diameter i d 1 6 mm Tubing connector 5 16 Ferrule yellow 1 8 Pump to Injection valve PEEK tubing i d 0 75 mm Fingertight connector 1 16 After Injection valve PEEK tubing i d 0 50 mm Fingertight connector 1 16 Outlet and waste ETFE tubing i d 1 0 mm Fingertight connector 1 16 ÄKTA avant 150 Inlet FEP tubing i d 2 9 mm Tubing connector 5 16 Ferrule blue 3 16 After pumps PEEK tubing 1 ...

Page 423: ...hemical resistance guide on page 440 Chemical environment 4 C to 35 C Operating temperature range 20 to 95 noncondensing Relative humidity Equipment noise level Acoustic noise level Equipment 70 dBA ÄKTA avant instrument ÄKTA avant User Manual 29035184 AE 423 9 Reference information 9 1 System specifications ...

Page 424: ... ÄKTA avant 25 0 01 to 50 ml min Flow rate range column packing flow ÄKTA avant 150 0 01 to 300 ml min ÄKTA avant 25 0 to 20 MPa 0 to 200 bar Pressure range ÄKTA avant 150 0 to 5 MPa 0 to 50 bar ÄKTA avant 25 0 35 to 10 cP Viscosity range ÄKTA avant 150 0 35 to 5 cP ÄKTA avant 25 Flow rate specifications Accuracy 1 2 Precision RSD 0 5 Conditions 0 25 to 25 ml min 3 MPa 0 8 to 2 cP ÄKTA avant 150 A...

Page 425: ...cP Viscosity range ÄKTA avant 25 Flow rate specifications Accuracy 2 RSD 0 5 Conditions 0 25 to 50 ml min 3 MPa 0 8 to 3 cP ÄKTA avant 150 Accuracy 2 RSD 0 5 Conditions 1 0 to 150 ml min 3 MPa 0 8 to 2 cP Mixer Data Parameter Chamber with magnetic stirrer Mixing principle ÄKTA avant 25 0 6 1 4 default or 5 ml Mixer volume ÄKTA avant 150 1 4 5 default or 15 ml ÄKTA avant User Manual 29035184 AE 425...

Page 426: ...Rotary valves Type 6 standard valves up to 6 optional valves Number of valves Inlet valve Sample Inlet valve Injection valve Column valve pH valve Outlet valve Functions Second Inlet valve Second Sample Inlet valve Loop valve Versatile valve Second Column valve Second and Third Outlet valves Extra Inlet valves Options Quaternary valve Data Parameter 4 port solenoid actuated membrane valve Type Qua...

Page 427: ...valve V9 C or V9H C The Sample pressure monitor is located after the Sample pump Placement of sensors ÄKTA avant 25 0 to 20 MPa 0 to 200 bar Range ÄKTA avant 150 0 to 5 MPa 0 to 50 bar ÄKTA avant 25 0 02 MPa or 2 whichever is greater Accuracy ÄKTA avant 150 0 015 MPa or 1 5 whichever is greater UV monitors Description Item Up to 2 Number of monitors U9 M 190 to 700 nm in steps of 1 nm up to 3 wave...

Page 428: ...9 L 10000 h Standard Optical path length 2 mm Cell volume 2 µl Total volume 11 µl Flow cells U9 M Option Optical path length 10 mm Cell volume 8 µl Total volume 12 µl Optical path length 0 5 mm Cell volume 1 µl Total volume 10 µl Standard Optical path length 2 mm Cell volume 2 µl Total volume 30 µl Flow cells U9 L Option Optical path length 5 mm Cell volume 6 µl Total volume 20 µl 428 ÄKTA avant U...

Page 429: ...tivity monitor Description Item 0 C to 99 C Reading range 1 5 C within 4 C to 45 C Accuracy pH monitor Data Parameter 0 to 14 pH reading range 0 1 pH unit within pH 2 to 12 temp within 3 C from calibration temp Accuracy 0 to 0 5 MPa 0 to 5 bar Operating pressure ÄKTA avant 25 76 µl Flow cell volume ÄKTA avant 150 129 µl Outlet valve fractionation Data Parameter 10 expandable to 32 Number of outlet...

Page 430: ...0 ml Fraction volumes ÄKTA avant 25 Automatic DropSync or accumulator Spillage free modes ÄKTA avant 150 Automatic or accumulator Covered vessels and climate control settable 6 C to 20 C Protection of fractions No Organic solvents ÄKTA avant 25 518 µl Delay volume with pH electrode and flow restrictor off line UV dispenser head ÄKTA avant 150 1807 µl Fraction collector F9 R 2nd Data Parameter Up t...

Page 431: ... and flow restrictor off line UV dispenser head ÄKTA avant 150 928 µl 320 x 400 x 250 mm Dimensions W x D x H 5 kg Weight I O box Data Parameter 2 analog in 2 analog out 4 digital in 4 digital out Number of ports In 2 V Out 1 V Analog range ÄKTA avant User Manual 29035184 AE 431 9 Reference information 9 2 Module specifications ...

Page 432: ... 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 432 ÄKTA avant User Manual 29035184 AE 9 Reference information 9 3 Tubing and connectors ...

Page 433: ... waste tubing Transpar ent ETFE o d 1 16 i d 1 0 mm 35 0 μl Pump rinse solution tubing Transpar ent Silicone o d 4 1 mm i d 2 1 mm 0 5 ml Waste tubing from Buffer tray Transpar ent Silicone o d 12 mm i d 8 mm Many different sizes types of tubing can be connected to a chromatography system Tubing with a smaller inner diameter i d holds less delay volume and will therefore generate less dilution of ...

Page 434: ...The table below shows the labels standard diameters and standard lengths of the inlet tubing Volume ml Length mm Tubing Description Label ÄKTA avant 150 ÄKTA avant 25 ÄKTA avant 150 ÄKTA avant 25 9 9 3 0 1500 FEP o d 3 16 i d 2 9 mm FEP o d 1 8 i d 1 6 mm Inlets to Inlet valve A A1 to A7 9 9 3 0 1500 FEP o d 3 16 i d 2 9 mm FEP o d 1 8 i d 1 6 mm Inlets to Inlet valve B B1 to B7 2 0 0 6 300 FEP o ...

Page 435: ...FEP o d 1 8 i d 1 6 mm Inlet to Sample in let valve Buff 3 8 1 2 580 FEP o d 3 16 i d 2 9 mm FEP o d 1 8 i d 1 6 mm Sample inlet valve to Sample pump InS 1 Narrow inlet tubing ETFE o d 1 16 i d 0 75 mm 28 9572 17 is available for S1 S7 High pressure tubing The tables below shows the labels standard diameters and standard lengths of the standard high pressure tubing and the optional high pressure t...

Page 436: ...PEEK o d 1 16 i d 0 75 mm Restrictor B to Pressure monitor 2B 270 150 340 PEEK o d 1 16 i d 1 0 mm PEEK o d 1 16 i d 0 75 mm Sample pump left to Restrictor S 1S1 270 150 340 PEEK o d 1 16 i d 1 0 mm PEEK o d 1 16 i d 0 75 mm Sample pump right to Restrictor S 1S2 90 50 115 PEEK o d 1 16 i d 1 0 mm PEEK o d 1 16 i d 0 75 mm Restrictor S to Sample pressure monitor 2S 420 230 530 PEEK o d 1 16 i d 1 0...

Page 437: ...o d 1 16 i d 0 50 mm Conductivitymon itor to pH valve 8 60 10 75 PEEK o d 1 16 i d 1 0 mm PEEK o d 1 16 i d 0 50 mm pH valve to flow restrictor 1R 60 10 75 PEEK o d 1 16 i d 1 0 mm PEEK o d 1 16 i d 0 50 mm Flow restrictor to pH valvel 2R 170 40 215 PEEK o d 1 16 i d 1 0 mm PEEK o d 1 16 i d 0 50 mm pH valve to Outlet valve 9 400 100 500 PEEK o d 1 16 i d 1 0 mm PEEK o d 1 16 i d 0 50 mm Outlet va...

Page 438: ...lve to second Column valve 5C21 170 40 220 PEEK o d 1 16 i d 1 0 mm PEEK o d 1 16 i d 0 50 mm Standard Outlet valve to second Outlet valve 101 170 40 220 PEEK o d 1 16 i d 1 0 mm PEEK o d 1 16 i d 0 50 mm Second Outlet valve to third Out let valve 111 440 440 220 FEP o d 1 8 i d 1 6 mm FEP o d 1 8 i d 1 6 mm External air sen sor L9 1 5 to Sample inlet valve L 80 20 100 PEEK o d 1 16 i d 1 0 mm PEE...

Page 439: ...o d 1 16 i d 1 0 mm Outlets from Outlet valve V9 O or V9H O Out1 Out32 3 0 V9H O FEP o d 1 8 i d 1 6 mm Waste tubing The table below shows the labels diameters and standard lengths of the waste tubing The waste tubing is mounted on delivery Volume ml Length mm Tubing Description Label 1 2 1500 ETFE o d 1 16 i d 1 0 mm System pump waste W1 1 2 1500 ETFE o d 1 16 i d 1 0 mm Sample pump waste W2 1 2 ...

Page 440: ...ed information about chemical resistance of the ÄKTA avant instrument In this section See page Section 441 9 4 1 General information about biocompatibility and chemical resis tance 443 9 4 2 Chemical resistance specifications 440 ÄKTA avant User Manual 29035184 AE 9 Reference information 9 4 Chemical resistance guide ...

Page 441: ... acid and make sure that the column is not mounted on the Column Valve V9 C The Column Valve V9 C contains a pressure sensor which can be damaged by 1 M hydrochloric acid If sodium hypochlorite is used as sanitizing agent instead of 2 M sodium hydroxide use a concentration up to 10 Organic solvents Reversed phase chromatography of proteins works well with 100 acetonitrile and ad ditives trifluoroa...

Page 442: ...ssure dependent Unless otherwise stated all concentrations are 100 Note 442 ÄKTA avant User Manual 29035184 AE 9 Reference information 9 4 Chemical resistance guide 9 4 1 General information about biocompatibility and chemical resistance ...

Page 443: ...e sure that you have the MSDS available from your chemical distributor and or databases on the internet Note Aqueous buffers The specified aqueous buffers are suitable for continuous use CAS no EC no Concentra tion Chemical N A N A Aqueous buffers pH 2 12 Strong chemicals and salts for CIP The following chemicals are suitable for up to 2 h contact time at room temperature CAS no EC no Concentra ti...

Page 444: ...ilization and denaturing agents The following chemicals are suitable for continuous use as additives in separation and purification methods CAS no EC no Concentra tion Chemical 50 01 1 200 002 3 6 M Guanidinium hydrochloride 151 21 3 205 788 1 1 Sodium dodecyl sulfate SDS 9005 64 5 500 018 3 1 Tween 20 57 13 6 200 315 5 8 M Urea Chemicals used in reversed phase chromatography RPC The following che...

Page 445: ...c acid for a longer period of time Note Salts and additives for hydrophobic interaction chromatography HIC The following chemicals are suitable for continuous use CAS no EC no Concentra tion Chemical 12125 02 9 235 186 4 2 M Ammonium chloride 7783 20 2 231 984 1 3 M Ammonium sulfate 107 21 1 203 473 3 50 Ethylene glycol 56 81 5 200 289 5 50 Glycerol Reducing agents and other additives The followin...

Page 446: ...0 7 231 211 8 4 M Potassium chloride Other substances The following chemicals are suitable for continuous use CAS no EC no Concentra tion Chemical 67 64 1 200 662 2 10 Acetone 7664 41 7 231 635 3 30 Ammonia 67 68 5 200 664 3 5 Dimethyl sulphoxide DMSO 75 08 1 200 837 3 20 Ethanol for long term stor age 7664 38 2 231 633 2 0 1 M Phosphoric acid 446 ÄKTA avant User Manual 29035184 AE 9 Reference inf...

Page 447: ... Ethylene ChloroTriFluoroEthylene ETFE Ethylene TetraFluoroEthylene FEP Fluorinated Ethylene Propylene FPM FKM Fluorinated Propylene Monomer FFPM FFKM Fully Fluorinated Propylene Monomer PCTFE PolyChloroTriFluoroEthylene PEEK PolyEtherEtherKetone PP PolyPropylene PTFE PolyTetraFluoroEthylene UHMWPE UltraHighMolecularWeightPolyEthylene Alumina Aluminum oxide Elgiloy Hastelloy C 276 Quartz glass Rub...

Page 448: ...ump rinse system Abbreviation Material EPDM EthylenePropyleneDiene M class rubber PEEK PolyEtherEtherKetone PP PolyPropylene PPS PolyPhenylene Sulfide PVDF PolyVinylidene DiFluoride Silicone 448 ÄKTA avant User Manual 29035184 AE 9 Reference information 9 5 Wetted materials ...

Page 449: ...cation methods The Method Editor has predefined methods for different separation techniques The methods include a number of relevant phases The following table describes the available predefined purification methods and which phases that are included Included phases Principle Predefined purification method After equilibration and sample application the protein of interest is adsorbed to the column...

Page 450: ...done to minimize the risk for salt precipitation Anion Exchange Chro matography AIEX After equilibration and sample application positively 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 NaCl After column re equilibration a fraction collector accumu lator wash is done to minimize t...

Page 451: ... the col umn is re equilibrated Chromatofocusing CF After equilibration and sample application the proteins are eluted isocratically This technique is commonly used for buffer ex change Desalting After equilibration and sample application proteins separate and elute according to their size largest first Gel filtration GF ÄKTA avant User Manual 29035184 AE 451 9 Reference information 9 6 Predefined...

Page 452: ...h is done to minimize the risk for salt precipitation Hydrophobic Interaction Chromatography HIC The sample application loops of the loop valve are manually filled with samples The method will guide the user through the process by pausing and displaying on screen instructions Up to five loops can be filled with different samples The loops are filled in descending order Partial or complete loop fil...

Page 453: ...ctor Do not fractionate flammable liquids in the built in fraction collector When running RPC methods collect fractions through the outlet valve or the optional external Fraction collector F9 R Predefined maintenance methods A number of predefined methods for preparation and cleaning are available These maintenance methods are used to prepare the system clean the system and to fill the system with...

Page 454: ...result is logged in the column logbook Column Performance Test The column is filled with buffer solution Select inlet positions Enter the solution identity volume flow rate and incubation time By adding steps several preparation solutions can be used Column Preparation Packs AxiChrom columns with a prede terminedcolumn type by motorizedpower or by a flow of hydraulic liquid that pushes the adaptor...

Page 455: ... solution AdditionalSystem CIP phases can be added from the Phase Library if de sired System CIP The system is filled with preparation solu tion Select for example inlets outlets and column positions to be prepared Two System Preparation phases are included in the method Additional System Prepara tion phases can be added from the Phase Library if desired System Preparation ÄKTA avant User Manual 2...

Page 456: ...technique to be used in the list Show by technique Select Show only suggested columns to show the columns that are suggested for the selected chromatography tech nique 2 Pressure limit 3 Flow rate 4 Option to control the flow to avoid overpressure Note Default values for pressure limits and flow rate are given for the selected column type Column position Flow restrictor use Buffer preparation Manu...

Page 457: ...ion runs by rinsing the column with a cleaning solution to remove non specifically bound proteins By adding steps several cleaning solutions can be used sequen tially Column CIP Tests the efficiency of a packed column in terms of height equiva lent to a theoretical plate HETP and the peak asymmetry factor As ColumnPerfor mance Test Prepares the column before use by removing the storage solution an...

Page 458: ... of flowthrough Sample Appli cation Cleans the system after purification runs by rinsing the system with a cleaning solution One cleaning solution is used per phase System CIP Prepares the system before a run by removing storage solution and filling the system and inlets with buffer solution One prepara tion solution is used per phase System Prepa ration A flow of hydraulic liquid pushes the adapt...

Page 459: ...ection 460 9 7 1 System settings UV 461 9 7 2 System settings Conductivity 463 9 7 3 System settings pH 464 9 7 4 System settings Pumps and pressures 466 9 7 5 System settings Air sensor 467 9 7 6 System settings I O box 468 9 7 7 System settings Fraction collection 471 9 7 8 System settings Watch parameters 472 9 7 9 System settings Advanced 474 9 7 10 System settings Data collection ÄKTA avant U...

Page 460: ...he wavelengths for UV monitor U9 M The corre sponding curves in the chromatogram are marked with the set wavelengths Note The instruction is available when UV monitor U9 M variable is selected in the component list For best performance do not use more wavelengths than necessary for the application At low wavelengths the eluent can have significant absorbance of its own Wavelength Filters the noise...

Page 461: ...or high conductivity or in descending manner 0 for high and 100 for low conductivity Note The Relative scale cond in descending manner is espe cially useful for conductivity visualization in RPC and HIC where the conductivity curve is reversed compared to the concentration curve i e high conductivity at 0 B and low conductivity at 100 B Relative scale cond Facilitates monitoring using the second c...

Page 462: ... factor to 0 turns this func tion off Is used to adjust the conductivity values for the second conductivitymonitor to a reference temperature in order to compare conductivity values between runs that have been performed at different temperatures Cond2ndtempcondensa tion Setting the compensation factor to 0 turns this func tion off 462 ÄKTA avant User Manual 29035184 AE 9 Reference information 9 7 ...

Page 463: ...Instruction name Enables or disables the pH alarm When enabled it sets the alarm limits for the pH signal When the pH falls outside the set limits an alarm will be triggered and the method will be paused Alarm pH ÄKTA avant User Manual 29035184 AE 463 9 Reference information 9 7 System settings 9 7 3 System settings pH ...

Page 464: ...rm limit Note Setting the Low alarm or the system flow rate to 0 deac tivates the low pressure alarm Alarm system pressure Sets the alarm limits for the sample pressure When enabled and the sample pressure falls outside the set pressure limits an alarm will be triggered and the method will be paused Default values for the alarm limits are set by the values in the column list when a column is selec...

Page 465: ...ns is selected in the component list Alarm delta column pres sure Sets the alarm limits for the pre column pressure When enabled and the pre column pressure falls outside the set pressure limits an alarm is issued and the method will be paused Default values for the alarm limits are set by the values in the column list when a column is selected in the method and Alarm pre column pressure is set as...

Page 466: ... when a buffer or sample vessel is empty High 10 µl is used to detect small air bubbles Sets the sensitivity of the optional air sensor number The optional air sensor can be located either before any of the inlets A or B or after the injection valve Air sensor sensitivity ext Normal 30 µl is used to detect when a buffer or sample vessel is empty High 10 µl is used to detect small air bubbles When ...

Page 467: ...g signal falls outside the set limits an alarm will be triggered and the method will be paused Alarm analog 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 If the alarm is enabled and the condition set in Value occurs an alarm will be triggered and the method will be paused Alarm digital in X Enables the us...

Page 468: ...can will be performed when the door of the fraction collector is closed to determine which type of cassettes and plates are used If Manual is selected used plates and tubes in each tray position are entered Sets the fraction collector chamber target temperature Fractioncollectortemper ature Lamps in the fraction collector chamber on or off Fraction collector lamp DropSync synchronises tube change ...

Page 469: ...ctions correspond to the fractions indicated in the chro matogram The delay volume must be changed when changing tubing to another inner diameter or length or when removing or adding components Detector Out let valve Is used to calculate the delay volume between the monitor and the Outlet valve The instruction is used to make sure that the collected fractions correspond to the fractions indicated ...

Page 470: ... used to make sure that the collected fractions correspond to the fractions indicated in the chromatogram The instruction is available only when the second Fraction collector is selected in the component list Detector Frac 2 470 ÄKTA avant User Manual 29035184 AE 9 Reference information 9 7 System settings 9 7 7 System settings Fraction collection ...

Page 471: ...ctivity monitor for some of the tests in the Watch Watch and Watch Hold until instructions Watchcond2ndparame ters Sets the value for the accepted fluctuation of the pH signal used for the test Stable signal in the instructions Watch Watch and Watch Hold until Watch pH parameters Sets the value for the accepted fluctuation of the flow rate signal used for the test Stable signal in the instruc tion...

Page 472: ...ameters provides the P and I factors used in the regulator and can be adjusted for different columns Pressure control parame ters Sets the maximum flow rate used during the turning of the injection and outlet valve in order to avoid high pressure alarms Max flow during valve turn Sets which flow Automatic System flow or Sample flow to use to calculate the progress of the method In automatic mode t...

Page 473: ...ump washes System pump wash vol ume Sets the flow rate used during sample pump washes Note The flow rate should not exceed 10 ml min if narrow inlet tubing i d 0 75 mm is used Sample flow rate Sets the wash volume used during sample pump washes Sample pump wash vol ume Description Parameter for loop wash settings Sets the flow rate used during Loop wash Note The volume for loop wash is set in the ...

Page 474: ...n curve Data reduction occurs if the maximum number of data points is exceeded To avoid data reduc tion set the maximum number of data points to be collected to 180000 or insert a New Chromatogram instruction in the method Note The default setting is 54000 data points which corre sponds to 1 5 h for a signal of 10 Hz Name of given curve E g UV1 UV2 Cond 474 ÄKTA avant User Manual 29035184 AE 9 Ref...

Page 475: ... for the instruction group that you want to modify Select the instruction that you want to modify Enter the new values for the instruction To execute several instructions at the same breakpoint select and edit an instruction and click Insert Repeat for several instructions 3 To update parameter fields during method run select the Auto update checkbox 4 To perform the instructions click Execute 5 A...

Page 476: ... Note Set gradient length value to 0 to perform a step gradient Gradient Is used to change buffers in the inlet tubing pump and mixer Note Pressing End during Pump wash will terminate both the wash and the run immediately Pressing Continue during Pump wash will terminate the wash and the run will continue from the point at which the Pump wash instruction was executed An instruction issued when a P...

Page 477: ...injection valve or the outlet valve The flow is directed to the end of the flow path if outlet valve is not present Note Pressing End during System wash will terminate both the wash and the run immediately Pressing Continue during System wash will termi nate the wash and the run will continue from the point at which the System wash instruction was ex ecuted If System wash is performed during a Gra...

Page 478: ... trations Defines the length and end concentration of the quater nary gradient The start values are set by the instruction Quaternary start concentration The sum of the target concentrations Q1 to Q4 must be 100 Quaternary gradient Is used to set or change the pH when automatic buffer preparation is active during a method run BufferPro pH Is used to set higher isocratic flow rates This can be usef...

Page 479: ...n position This position is only available when performing calibration of the pH monitor In System control select System Calibrate The calibration position can also be used to fill the pH cell with storage solution since the pH valve is in open posi tion Note It is not possible to turn the pH valve during any type of fractionation as it affects the delay volume The pH valve instruction can be give...

Page 480: ...e extra valve is a basic 8 port valve without air sensor to be used for general applications Valve X1 Turns the extra inlet valve to the selected position The extra valve is a basic 8 port valve without air sensor to be used for general applications Valve X2 Sets an injection mark in the chromatogram at the point where this instruction is executed Note The instruction is useful when the sample is ...

Page 481: ... UV Filters the noise in the UV monitor signal from U9 L 2nd A column specific averaging time is set automatically when a column is defined in a method run and Averag ing time is set as a variable Noise reduction UV 2nd Facilitates monitoring using the conductivity monitor of a gradient for which the user sets the conductivity values for 0 and 100 The Relative scale cond in struction can be set in...

Page 482: ...n descending manner 0 for high and 100 for low conductivity Note The Relative scale cond instruction in descending man ner is especially useful for conductivity visualization in RPC and HIC where the conductivity curve is reversed compared to the concentration curve i e high conduc tivity at 0 B and low conductivity at 100 B Relative scale cond 2nd 482 ÄKTA avant User Manual 29035184 AE 9 Referenc...

Page 483: ...on after the set delay volume specifiedin the System Settings Fractioncollection De lay volumes instruction has been collected The outlet valve is then turned to position Waste Stop peak fractionation Last tube filled sets the action to perform after the built in fraction collector fills the last tube of the run pause the fractionation direct the flow to one of the outlet ports of the outlet valve...

Page 484: ...Only for Fraction collector F9 Reset tube type resets all the tube types in the fraction collector Note It is not allowed to execute the Reset tube type instruc tion during fractionation Reset tube type Only for Fraction collector F9 Frac cleaning position enables manual cleaning of the dispenser head The system is paused and the fractionation arm is moved to the middle front of the interior of th...

Page 485: ...een collected The Outlet valve V9 O or V9H O re mains in position Outlet 10 Frac 2 and peak fractiona tion in the second Fraction collector continues Stop fractionation frac 2 Enables collection of only those peaks that fulfill the conditions set in the Peak fractionation parameters instruction Peak fractionation frac 2 Ends the peak fractionation in second Fraction collector after the set delay v...

Page 486: ...d to position Waste Stop frac in outlet valve Applicable if no fraction collector is used Peak frac in outlet valve enables collection of only those peaks that fulfill the conditions set in Peak fractionation parame ters When the set fraction size outlet has been collect ed the outlet valve turns to the next position A fraction mark is set in the chromatogram for each new outlet position Peak frac...

Page 487: ... X Sets the value of the signal sent out by digital port number X to either 0 or 1 The default value is 1 Digital out X Generates a pulsed signal in digital port number X The signal changes from the initial state 0 or 1 to the oppo site state and returns to the initial state after the defined length of time Pulse digital out X Enables the user to send one of the pre defined signals UV signal condu...

Page 488: ...st exceeds the Low alarm limit for ten seconds continuously and then falls below the Low alarm limit Note Setting the Low alarm or the system flow rate to 0 deac tivates the low pressure alarm Alarm system pressure Sets the alarm limits for the sample pressure When enabled and the pressure falls outside the set pressure limits an alarm will be triggered and the method will be paused Low alarm is o...

Page 489: ...lues in the column list when a column is selected in the method and Alarm pre column pressure is set as a variable Low alarm is only triggered if the pressure first exceeds the Low alarm limit for ten sec onds continuously then falls below the Low alarm limit Note Setting the Low alarm to 0 deactivates the low pressure alarm Alarm pre column pres sure Enables or disables the alarm for the UV signa...

Page 490: ...d the method will be paused Alarm inlet A air sensor Enables or disables the air sensor alarm for the built in air sensor at inlet B If the alarm is enabled and air is detected an alarm will be triggered and the method will be paused Alarm inlet B air sensor Enables or disables the air sensor alarm for the built in air sensor at the sample inlet If the alarm is enabled and air is detected an alarm...

Page 491: ...be triggered by either of the signal values 0 or 1 If the alarm is enabled and the condition set in Value occurs an alarm will be triggered and the method will be paused Alarm digital in X ÄKTA avant User Manual 29035184 AE 491 9 Reference information 9 8 Using Manual instructions 9 8 6 Manual instructions Alarms ...

Page 492: ...at the selected Pressure sensor using the P and I factors set in the Constant pressure flow parameters instruction The total volume is continuously updated using the actual flow rate Constant pressure flow Both pressure control flow rates can be set either as volumetric or as linear flow A column type must be se lected before using linear flow Note When Constant pressure flow is used the P and I f...

Page 493: ... in the chromatogram The delay volume must be changed when changing tubing to an other i d or length or when removing or adding compo nents Delay volume Monitor to outlet valve Is used to define the delay volume between the monitor and the Fraction collector The instruction is used to make sure that the collected fractions correspond to thefractionsindicatedinthechromatogram Theinstruc tion is ava...

Page 494: ...ion with Watch instructions Sets the system to Hold The system will remain in the state Hold until the accumulated volume reaches the volume stored by the instructions Start volume count Stop volume count Hold counted volume Defines the flow from which the volume base is calcu lated When set to Automatic the position of the injec tion valve determines if the system flow or the sample flow is used ...

Page 495: ...h cond parameters Sets the value for the accepted fluctuation of the pH signal used for the test Stable signal in the instructions Watch and Hold until Watch pH parameters Sets the value for the accepted fluctuation of the flow rate signal used for the test Stable signal in the instruc tions Watch and Hold until with signal System flow Watch flow parameters Sets the value for the accepted fluctuat...

Page 496: ...xt entered for the parameter Mark text Set mark Sets the system to pause or end after a set volume or time has passed Select base sets the base to either accumulated time or accumulated volume Timeout sets the volume or time Action sets the action to per form pause or end Timer 496 ÄKTA avant User Manual 29035184 AE 9 Reference information 9 8 Using Manual instructions 9 8 9 Manual instructions Ot...

Page 497: ...r in the scouting scheme N A Scouting no The set flow rate of the system pumps 0 001 to 50 000 ml min ÄKTA avant 25 System flow 0 01 to 300 00 ml min ÄKTA avant 150 The set flow velocity of the system pumps Only available if a column is selected cm h System flow lin ear The system pressure signal at the system pumps 1 00 to 20 00 MPa ÄKTA avant 25 System pressure 1 00 to 5 00 MPa ÄKTA avant 150 Th...

Page 498: ...C pressure 1 00 to 5 00 MPa ÄKTA avant 150 The first UV Vis absorbance signal of the U9 M monitor 6000 000to6000 000 mAU UV 1 The 2nd UV Vis absorbance signal of the U9 M monitor 6000 000to6000 000 mAU UV 2 The 3rd UV Vis absorbance signal of the U9 M monitor 6000 000to6000 000 mAU UV 3 The UV absorbance signal of the U9 L monitor 6000 000to6000 000 mAU UV 2nd The pH signal 0 00 to 14 00 pH pH Tar...

Page 499: ... the Column valve N A Column position The set flow direction position of the Column valve V9 C and Column valve V9H C N A Flow direction The set position of the outlet valve N A Outlet The set position of the inlet valve A A1 A7 Inlet A The set position of the inlet valve B B1 B7 Inlet B The set position of the sample inlet valve S1 S7 buff Sample inlet The set position of the pH valve N A pH valv...

Page 500: ...Air sample inlet The current state of the air alarm for the external air sensors No air Air Air external The current tube position of the frac tion collector N A Frac position N A Frac temp The current tube position of the frac tion collector 2 N A Frac 2 position The I O box analog input signals 2000 0 to 2000 0 mV Analog in 1 to Analog in 4 The I O box digital input signals 0 1 Digital in 1 to D...

Page 501: ... of a set range 1 Hz 0 0 to 100 0 Cond The set concentration B or the current value during a gradi ent 1 Hz 0 0 to 100 0 Conc B The pH signal 1 Hz 0 00 pH The set flow rate of the system pumps 1 Hz 0 001 to 50 000 ml min ÄKTA avant 25 System flow 0 01 to 300 00 ml min ÄKTA avant 150 The set flow velocity of the system pumps Only available if a column is selected 1 Hz cm h System flow linear The sy...

Page 502: ... avant 150 The pre column pressure sig nal 1 Hz 1 00 to 20 00 MPa ÄKTA avant 25 PreC pres sure 1 00 to 5 00 MPa ÄKTA avant 150 The delta column pressure signal 1 Hz 1 00 to 20 00 MPa ÄKTA avant 25 DeltaC pres sure 1 00 to 5 00 MPa ÄKTA avant 150 The post column pressure sig nal 1 Hz 1 00 to 20 00 MPa ÄKTA avant 25 PostC pres sure 1 00 to 5 00 MPa ÄKTA avant 150 The set concentration of Q1 or the c...

Page 503: ... chamber 0 5 Hz 0 C to 99 0 C Frac temp The I O box analog input sig nals 10 Hz 2000 0 to 2000 0 mV Analog in 1 to Analog in 4 The nominal cell path length of the U9 M monitor 1 Hz 0 2 0 5 1 0 cm UV cell path length The nominal cell path length of the U9 L monitor 1 Hz 0 2 0 5 cm UV cell path length 2nd The I O box digital input sig nals 10 Hz 0 1 Digital in 1 to Digital in 8 The I O box digital o...

Page 504: ...le volume Sample volume does not however affect the resolution in adsorption chromatography techniques such as affinity chromatography AC ion exchange chro matography IEX and hydrophobic interaction chromatography HIC if the retention factor k is high Broadening from UV monitor to fraction collection The effect of peak broadening in the system from sample injection to peak detection including dilu...

Page 505: ...action collector UV Vis pH Valve The time when a peak is detected by the UV Vis detector is called T0 The peak reaches the fraction collector at time T1 Fraction collector Conductivity Delay volume Theoretical determination of delay volumes A theoretical determination is performed as described in the following steps Action Step Identify all components in the system flow path that contribute to the...

Page 506: ...Fraction collector Action Step In the System Control module on the System menu click Settings 1 Result The System Settings dialog box opens Click Fraction collection Delay volumes Enter the delay volume between the UV monitor and the Fraction collec tor in the Detector Frac field Enter the delay volume between the UV monitor and the Outlet valve in the Detector Outlet valve field and click OK 2 Th...

Page 507: ...0 UV detector to Fraction collector F9 R 2nd With pH electrode and flow restrictor off line ÄKTA avant 150 928 Delay volumes for extended systems If modules and tubing are added to or subtracted from the ÄKTA avant system between the UV monitor and the fraction collector use the standard delay volumes values in Standard delay volumes in ÄKTA avant 25 and ÄKTA avant 150 on page 507 and correct with...

Page 508: ...tionation in different parts of the same method the delay volumes have to be set as method instructions for each of the method parts For example both an external fluorescence detector and the UV monitor module can be used for peak fractionation 508 ÄKTA avant User Manual 29035184 AE 9 Reference information 9 12 Delay volumes ...

Page 509: ...X 20 External air sensor L9 1 2 35 External air sensor L9 1 5 549 Pump P9 P9 A and P9 B total volume for two heads including T connector and check valves 2163 Pump P9H P9H A P9H B P9H S total volume for two heads including T connector and check valves 30 System pump flow restrictor 30 Sample pump flow restrictor 45 System pump pressure monitor R9 45 Sample pump pressure monitor R9 1392 Sample pump...

Page 510: ...V monitor U9 L Flow cell 2 mm 20 Second UV monitor U9 L Flow cell 5 mm 22 Conductivity cell 10 Flow restrictor FR 902 15 pH valve V9 pH in By pass position 35 pH valve V9H pH in By pass position 76 pH flow cell mounted in pH valve V9 pH 129 pH flow cell mounted in pH valve V9H pH 48 Flow restrictor FR 902 and tubing when mounted on pH valve V9 pH 94 Flow restrictor FR 902 and tubing when mounted o...

Page 511: ...lt in fraction collector dispenser head ÄKTA avant 150 68 ÄKTA avant 25 98 Fraction collector F9 R tubing ÄKTA avant 150 393 The given values for the component volumes of the valves are average values Depending on the chosen flow path the actual component volume may differ Note ÄKTA avant User Manual 29035184 AE 511 9 Reference information 9 13 Component volumes ...

Page 512: ... the factors used for pressure regulation Description Parameter Proportional component in PI pressure regulation Reduces the error between actual and requested target pressure but may leave a permanent error P factor Integrating component in PI pressure regulation Eliminates the stationary error from the P factor but introduces a slight instability that may lead to oscillation in the pressure and ...

Page 513: ... needed to speed up pressure ramp up times 300 600 8 Large soft1 media columns A lower I value than the default value is needed to avoid large fluctuations in pressure or flow rate As an alternative try in creasing P 15 8 Small rigid2 media columns 40 20 1 Soft media is defined as all GE separation media except silica and MonoBeads 2 Rigid media is defined as GE separation media that is based on s...

Page 514: ... will be restored The instruction is found under Advanced in the System settings dialog box Constant pressure flow By using the text instruction Constant pressure flow it is possible to continuously adjust the flow rate to keep a certain pressure This could be useful for example in column packing at constant pressure The Advanced Constant pressure flow instruction is found in the Manual Instructio...

Page 515: ...by its physical position Note Node ID for standard modules The following table lists the Node ID for the standard modules Node ID Label Module 0 P9 A or P9H A System Pump A 1 P9 B or P9H B System Pump B 2 P9 S or P9H S Sample Pump 0 R9 Pressure Monitor system pressure 1 R9 Pressure Monitor sample pressure 0 M9 Mixer 4 V9 Inj or V9H Inj Injection Valve 0 Q9 Quaternary Valve 0 V9 IA or V9H IA Inlet ...

Page 516: ...le 12 V9 A2 or V9H A2 Second Inlet valve A 13 V9 B2 or V9H B2 Second Inlet valve B 15 V9 IX or V9H IX Extra Inlet valve X1 16 V9 IX or V9H IX Extra Inlet valve X2 14 V9 S2 or V9H S2 Second Sample inlet valve 20 V9 V or V9H V Versatile valve 21 V9 V or V9H V Versatile valve 2 23 V9 V or V9H V Versatile valve 3 24 V9 V or V9H V Versatile valve 4 17 V9 L or V9H L Loop valve 6 V9 C2 or V9H C2 Second C...

Page 517: ...tlet valve 10 V9 O3 or V9H O3 Third Outlet valve 0 L9 1 2 or L9 1 5 External air sensor 0 E9 I O box 1 E9 Second I O box 1 U9 L Second UV Monitor 1 C9 Second Conductivity Monitor 1 F9 R Fraction collector 2 ÄKTA avant User Manual 29035184 AE 517 9 Reference information 9 15 Check and change the Node ID of a module ...

Page 518: ...w of two rotating switches at the back of the module 2 The first rotating switch labeled A sets the tens The second switch labeled B sets the units For example for Node ID 13 the A switch is set to 1 and the B switch to 3 A B Check the Node ID and compare it with the listed Node IDs in the tables above 3 To change the Node ID use a screwdriver to set the arrows of the switches to the desired numbe...

Page 519: ... FEP i d 1 6 mm o d 1 8 Note Inlet tubing for ÄKTA avant 25 Outlet tubing for ÄKTA avant 150 28980987 Tubing kit 10 1 5m FEP id 2 9 mm o d 3 17 Note Inlet tubing for ÄKTA avant 150 28957217 Sample tubing kit for 7 inlets i d 0 75 mm Note Narrow inlet tubing for ÄKTA avant 25 28956504 Rinse system tubing 28956606 Replacement tubing kit ÄKTA avant 25 28979446 Replacement Tubing Kit ÄKTA avant 150 28...

Page 520: ...t filter holder kit 11000414 Inlet filter set Fittings and connectors Product code Item 18111255 Fingertight connector 1 16 male 18112707 Tubing connector for o d 1 16 tubing 18112117 Tubing connector for o d 1 8 tubing 18111249 Tubing connector for o d 3 16 tubing 11000339 Union 1 16 female to 1 16 female 5 pcs 11000852 Union Fingertight 1 16 female to 1 16 female i d 0 3 mm 4 pcs 18111258 Union ...

Page 521: ...28956342 Adapter for air sensor 28956327 Bottle holder 28956319 Column clamp for columns o d 10 to 21 mm 28956282 Column holder 28956270 Column holder rod 28956295 Flexible column holder 29011350 Loop holder 29011349 Multi purpose holder 29011352 Rail extension 28954329 Tube holder 5 pack 28956286 Tubing holder comb 28956274 Tubing holder spool for small tubing o d 1 8 and smaller 29014283 Tubing ...

Page 522: ...ode no Item 28960250 P9 S Seal kit 65 ml 28952642 P9 Seal kit 25 ml seal kit P9 18111213 Piston kit 100 ml piston kit P9 S 28952640 P9 Piston kit 25 ml 18112866 Check valve kit check valves in out for P9 P9H and P9 S 28979368 P9H Piston kit 150 ml 28979373 P9H Seal kit 150 ml Mixer Code no Item 28956186 Mixer chamber 0 6 ml 28956225 Mixer chamber 1 4 ml mounted at delivery in ÄKTA avant 25 522 ÄKT...

Page 523: ... O ring 22 1 1 6 mm Note For Mixer chamber 15 ml 18102711 Online filter kit Valves ÄKTA avant 25 Code no Item 28956506 Column Valve V9 C 28956508 pH Valve V9 pH 28956510 Inlet Valve V9 IA 28962006 Inlet Valve V9 IB 28962007 Inlet Valve V9 IS 28956512 Outlet Valve V9 O 28956514 Injection Valve V9 Inj 28957221 Inlet Valve V9 A2 28957223 Inlet Valve V9 B2 28957225 Inlet Valve V9 S2 28957227 Inlet Val...

Page 524: ...Valve V9H IA 28979277 Inlet Valve V9H IB 28979279 Inlet Valve V9H IS 28979281 Outlet Valve V9H O 28979283 Injection Valve V9H Inj 28979303 Inlet Valve V9H A2 28979315 Inlet Valve V9H B2 28979320 Inlet Valve V9H S2 28979326 Inlet Valve V9H X1 28979328 Inlet Valve V9H X2 28979330 Column Valve V9H C2 28979332 Outlet Valve V9H O2 28979337 Outlet Valve V9H O3 29090691 Versatile Valve V9H V 29090689 Loo...

Page 525: ...385 Superloop M6 fitting 150 ml 18112766 Fill port INV 907 18111089 Injection kit INV 907 28985812 Connector 1 16 Male Luer Female Built in fraction collector Code no Item 28954209 Cassette tray 28956402 Cassette for 50 ml tubes 2 pack Note For 6 tubes 28956404 Cassette for 15 ml tubes 2 pack 29133422 Cassette for 5 ml tubes 2 pack 28956425 Cassette for 8 ml tubes 2 pack 28956427 Cassette for 3 ml...

Page 526: ... Tube Rack Complete 95 10 18 mm 18112467 Tube Rack Complete 40 x 30 mm 18305103 Bowl 18305402 Tube Support 18646401 Tubing Holder 19724202 Tube Holder and Guide 175 12 mm 19868902 Tube Holder and Guide 95 10 18 mm 18112468 Tube Holder and Guide 40 30 mm 19606702 Drive sleeve pH monitor Code no Item 28954215 pH electrode 28956497 O ring 5 3 2 4 mm 526 ÄKTA avant User Manual 29035184 AE 10 Ordering ...

Page 527: ...29011325 UV flow cell 2 mm for U9 L 18112824 UV flow cell 5 mm for U9 L Conductivity monitors Code no Item 28956495 Conductivity Monitor C9 standard module 29011363 Conductivity monitor C9n optional External air sensors Code no Item 28956502 Air Sensor L9 1 2 mm 28956500 Air Sensor L9 1 5 mm I O box Code no Item 29011361 I O box E9 ÄKTA avant User Manual 29035184 AE 527 10 Ordering information ...

Page 528: ...4 F type 29012474 External module cable short F type 29011366 External module cable long F type Flow restrictor Code no Item 18112135 Flow Restrictor FR 902 Barcode scanner Code no Item 28956452 Barcode Scanner 2 D with USB UniTag Code no Item 28956491 UniTag sheet with 108 labels 528 ÄKTA avant User Manual 29035184 AE 10 Ordering information ...

Page 529: ...487 Wet side waste tray 28956485 Front side waste tray User Documentation Code no Item 29035184 ÄKTA avant User Manual Note Covers ÄKTA avant 25 and ÄKTA avant 150 ÄKTA avant User Manual 29035184 AE 529 10 Ordering information ...

Page 530: ...ance method 455 system predefined phase 458 Cleaning column 237 Cold cabinet 30 precautions 30 prepare for a run 31 Cold room 30 Column cleaning 237 column CIP 297 column holder 44 Column valve 73 pressure alarm 187 storage 237 Column CIP maintenance method 454 predefined phase 457 Column performance test maintenance method 454 predefined phase 457 Column preparation maintenance method 454 predefi...

Page 531: ...ration predefined method 451 H Hardware installation of a module 103 Holders air sensor adapter 49 bottle holder 49 column clamp 45 column holder 44 column holder rod 46 Extension box 53 flexible column holder 47 loop holder 52 multi purpose holder 51 ordering information 522 rail extension 51 tubing holder comb 48 tubing holder spool 48 Hydrophobic interaction chro matography HIC predefined metho...

Page 532: ...other 496 pumps 476 watch parameters 495 Manual loop fill predefined phase 458 Manual Loop Fill predefined method 452 Methods affinity chromatogra phy 449 anion exchange 450 cation exchange 450 chromatofocusing 451 create a method 227 create method 226 desalting 451 gel filtration 451 hydrophobic interaction chromatography HIC 452 Intelligent Packing 454 maintenance methods 226 manual loop fill 45...

Page 533: ...olumn preparation 457 column wash 457 elution 457 equilibration 458 Intelligent Packing 458 manual loop fill 458 method settings 456 miscellaneous 458 predefined phases 456 sample application 458 system CIP 458 system preparation 458 pH monitor calibration 321 clean the pH electrode 307 description 82 error codes 416 replace the pH elec trode 286 storage of the pH elec trode 237 305 troubleshootin...

Page 534: ...ilable run data 497 choose method 231 monitor a run 233 perform a run 230 perform manual run 232 S Safety notices 9 Sample application flow paths 71 injection valve 70 predefined phase 458 sample loading 189 sample loop 199 superloop 194 Sample loop sample loading 199 Scanning of cassettes full scan 85 quick scan 85 Software overview software modules 11 Storage column 237 pH electrode 237 system 2...

Page 535: ...158 331 clean UV flow cell 283 description 96 replace UV flow cell 342 345 second 139 UV monitor U9 L description 139 158 error codes 419 system test 260 troubleshooting 368 UV monitor U9 M description 96 158 error codes 417 troubleshooting 363 UV settings 460 V Valves Column valve 73 error codes 415 extra Column valve 114 extra Inlet valves 110 extra Outlet valves 117 injection valve 70 Inlet val...

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Page 538: ... Software End User License Agreement for Life Sciences Software Products A copy of this Standard Software End User License Agreement is available on request 2013 2017 General Electric Company 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 lo...

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