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5.5 Connect a column

Introduction

This section describes how to connect a column to the instrument using a column holder and without
introducing air into the flow path. Several types of column holders are available for ÄKTA pure.

WARNING

To avoid exposing the column to excessive pressure, make sure that the pressure
limit is set to the specified maximum pressure of the column. Before connecting a
column to the ÄKTA pure instrument, read the instructions for use of the column.

Methods automatically include a pressure alarm 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 Pressure alarms, on page 177 for more
information on pressure alarms.

Do not overtighten when connecting columns. Overtightening might rupture the connectors
or squeeze the tubing and thereby result in high back pressure.

Note:

If no column valve is used, remove the column from the system before running a system wash.
The pressure during a system wash may become too high for the column.

Note:

Attach a column holder and connect a column

Follow the instructions below to connect a column to the instrument. Always use a column holder. If
a column valve is used, connect the column to the appropriate A and B ports on the valve. If no column
valve is used, connect the column directly to the flow path tubing. Use appropriate tubing and connec-
tors. The instructions below show a system configured with Column valve V9-Cs.

Action

Step

Attach an appropriate column holder to the rail on the instrument.

1

ÄKTA pure User Manual 29119969 AB

173

5 Operation

5.5 Connect a column

Summary of Contents for AKTApure

Page 1: ...ÄKTA pure User Manual ...

Page 2: ...Page intentionally left blank ...

Page 3: ...les 80 2 6 Accessories 87 3 ÄKTA pure external modules 88 3 1 External air sensors 90 3 2 Fraction collector F9 C 91 3 2 1 Function 93 3 2 2 Fraction collector F9 C illustrations 97 3 2 3 Cassettes Cassette tray and racks 103 3 2 4 Connect tubing to the ÄKTA pure instrument 104 3 3 Fraction collector F9 R 108 3 4 Sample pump S9 and S9H 112 3 5 I O box E9 113 3 5 1 Overview of the I O box 115 3 5 2...

Page 4: ...ple loop 193 5 8 Fractionation 194 5 8 1 Prepare Fraction collector F9 C 200 5 8 2 Prepare Fraction collector F9 R 207 5 8 3 Fractionation overview 211 5 9 Create a method and perform a run 212 5 9 1 Create a method 217 5 9 2 Prepare and perform a run 219 5 9 3 Monitor a run 221 5 9 4 After run procedures 225 6 Performance tests 226 6 1 General performance test actions 229 6 2 Air sensor A and Inl...

Page 5: ...low cell 328 7 8 6 Replace the UV monitor U9 L flow cell 330 7 8 7 Replace Flow restrictor 331 7 8 8 Replace the inlet filters 332 7 8 9 Replace the pump head check valves 335 7 8 10 Replace pump piston seals of Pump P9 or P9H 344 7 8 11 Replace pump piston seals of Pump P9 S 351 7 8 12 Replace pump pistons 352 7 8 13 Replace pump rinsing system tubing 355 8 Troubleshooting 356 8 1 Introduction to...

Page 6: ... settings Advanced 465 9 8 12 System settings Data collection 466 9 9 Manual instructions 467 9 9 1 Manual instructions Pumps 471 9 9 2 Manual instructions Flow path 473 9 9 3 Manual instructions Monitors 474 9 9 4 Manual instructions Fraction collection 478 9 9 5 Manual instructions I O box 479 9 9 6 Manual instructions Alarms 482 9 9 7 Manual instructions Wash settings 483 9 9 8 Manual instructi...

Page 7: ...ystem 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 pure overview 12 1 3 ÄKTA pure user documentation ÄKTA pure User Manual 29119969 AB 7 1 Introduction ...

Page 8: ...s or for diagnostic purposes Prerequisites In order to operate the system according to the intended purpose it is important that you have a general understanding of how the computer and the Microsoft Windows operating system work you understand the concepts of liquid chromatography you have read and understood the Safety instructions chapter in ÄKTA pure Operating Instructions a user account has b...

Page 9: ...d 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 product Note A tip contains useful information that can improve or optimize your procedures Tip ÄKTA pure User Manual 29119969 AB 9 1 Introduction 1 1 Important user information...

Page 10: ... inlets outlets and detectors and choose between different ways to apply and collect samples Purification and maintenance methods are easily created using predefined methods and method phases A method phase refers to a specific step part in the method such as column wash or elution Method phases are described in Phase Properties and are displayed graphically in a method outline This makes methods ...

Page 11: ...es 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 and work with peaks and fractions To perform operations like Design of Experiments users can easily switch to Evaluation Classic When working with the modules Administration Method...

Page 12: ...e in a safe way ÄKTA pure Operating Instructions Instructions neededto install operateand maintainFraction collectors F9 C and F9 R in a safe way Fraction collector F9 C and F9 R Operating Instructions Instructions needed to install operate and maintain Sample pump S9 and S9H in a safe way Sample pump S9 and S9H Operating Instructions Detailed instrument and module descriptions and instruc tions o...

Page 13: ...hod Manual1 Overview and detailed description of network setup and complete software installation Administration of UNICORN and the UNICORN database UNICORN Administration and Technical Manual1 Overview and detailed descriptions of the Evaluation Classic module in UNICORN Description of the evaluation algorithms used in UNICORN UNICORN Evaluation Manual1 Overview and detailed description of the sy...

Page 14: ...s and how these are installed in the instrument In this chapter This chapter contains the following sections See page Section 15 2 1 Overview illustrations 26 2 2 Liquid flow path 28 2 3 Instrument control panel 33 2 4 Instrument modules 77 2 5 Installation of internal modules 80 2 6 Accessories 14 ÄKTA pure User Manual 29119969 AB 2 The ÄKTA pure instrument ...

Page 15: ...s up to 150 ml min In this manual they are referred to as ÄKTA pure 25 25 ml min and ÄKTA pure 150 150 ml min Illustrations of the main parts of the instrument The illustrations below show the location of the main parts of the instrument 1 2 4 3 5 6 Function Part Wet sides 1 Buffer tray 2 Holder rails 3 Instrument control panel 4 Power switch 5 Ventilation panel 6 ÄKTA pure User Manual 29119969 AB...

Page 16: ...1 12 2 3 4 1 Function Part Multi module panel 1 Inlet valve 2 Pump rinsing liquid tube 3 System pump B 4 Pressure monitor 5 System pump A 6 Mixer 7 Outlet valve 8 Injection valve 9 Conductivity monitor 10 Column valve 11 UV monitor 12 16 ÄKTA pure User Manual 29119969 AB 2 The ÄKTA pure instrument 2 1 Overview illustrations ...

Page 17: ...back pressure if used above 50 ml min The larger volumes in the H valves for ÄKTA pure 150 may decrease resolution and increase peak broadening if used in ÄKTA pure 25 Note Label in Module ÄKTA pure 150 ÄKTA pure 25 P9H A P9 A System pump A P9H B P9 B System pump B R9 R9 Pressure monitor M9 M9 Mixer V9H Inj V9 Inj Injection valve V9H IA V9 IA Inlet valve A V9H IB V9 IB Inlet valve B V9H IAB V9 IAB...

Page 18: ...E 5 E 5 F 4F 2F 1 F E F Ports located on the valve head rim are indicated outside the black ring e g 1E 2E etc Ports located on the pivot part of the valve head are indicated 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 Core modules Description Core module A high precision pump which delivers buffer or sample in purificat...

Page 19: ...e 25 their volumes are 0 6 ml 1 4 ml mounted at delivery and 5 ml Three Mixer chambers are available for ÄKTA pure 150 Their volumes are 1 4 ml mounted at delivery 5 ml included in delivery and 15 ml CAUTION Risk of explosion Do not use Mixer chamber 15 ml with an ÄKTA pure 25 system configuration The maximum pressure for Mixer chamber 15 ml is 5 MPa For further information refer to Section 2 4 2 ...

Page 20: ... B 5 B 4 B3 B2 B 1 B 6 B 7 Q Out Inlet valve with two A inlet ports and two B inlet ports No integrated air sensor Inlet valve V9 IAB or V9H IAB A B A1 A2 B1 B2 For further information refer to Section 2 4 4 Inlet valves on page 40 Inlet valve with eight inlet ports seven sample inlets and one buffer inlet and integrated air sensor The Sample inlet valve requires an external Sample pump module Sam...

Page 21: ... Mixer valve on page 47 Enables the use of up to five loops connected to the instrument Loop valve V9 L or V9H L For further information refer to Section 2 4 7 Loop valve on page 53 Connects up to five columns to the instrument and directs the flow to one column at a time The Column valve features two integratedpressure sensors Column valve V9 C or V9H C Allows the user to choose flow direction th...

Page 22: ...er information refer to Section 2 4 10 pH valve on page 61 Directs the flow to the Fraction collector Fraction collector 2 out 10 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 2 4 11 Outlet valves on page 65 Directs the flow to the Fraction collector Fraction co...

Page 23: ... further information refer to Section 2 4 13 UV monitors on page 70 Measures the UV Vis absorbance at up to three wavelengths simultaneously in the range 190 700 nm UV monitor U9 M For further information refer to Section 2 4 13 UV monitors on page 70 Measures the conductivity of buffers and eluted proteins Conductivity monitor C9 For further information refer to Section 2 4 14 Conductivity monito...

Page 24: ...er information refer to Section 3 2 Fraction collector F9 C on page90 Round fraction collector that can collect up to 175 fractions Fraction collector F9 R Up to two fraction collectors can be connected at the same time For further information refer to Section 3 3 Fraction collector F9 R on page 104 Receives analog or digital signals from or transfers analog or digital signals to external equipmen...

Page 25: ...ure monitor The sample pump delivers buffer or sample in purification runs Sample pump S9 or S9H For further information refer to Section 3 4 Sample pump S9 and S9H on page 108 ÄKTA pure User Manual 29119969 AB 25 2 The ÄKTA pure instrument 2 1 Overview illustrations ...

Page 26: ...low path The illustration below shows the flow path for a typical system configuration The individual instrument modules are presented in the table below The configuration of the system is defined by the user 4 5 6 7 8 9 10 11 12 13 14 15 16 17 W1 W2 W 3 2 1 Description Part Pressure monitor 1 Sample pump 2 Sample inlet valve 3 26 ÄKTA pure User Manual 29119969 AB 2 The ÄKTA pure instrument 2 2 Li...

Page 27: ...or 7 Mixer 8 Injection valve 9 Sample loop or Superloop 10 Column valve 11 Column 12 UV monitor 13 Conductivity monitor 14 Flow restrictor 15 Outlet valve 16 Fraction collector 17 Waste W W1 W2 ÄKTA pure User Manual 29119969 AB 27 2 The ÄKTA pure instrument 2 2 Liquid flow path ...

Page 28: ...l an ongoing run It is possible to lock and unlock the Instrument control buttons from UNICORN Location and illustration The illustration below shows the location and detailed view of the Instrument control panel 1 2 3 4 Function Part Power Communication indicator white 1 Continue button with a green light indicator 2 Pause button with an orange light indicator 3 Alarm and error indicator red 4 28...

Page 29: ...tem Settings dialog opens In the System Settings dialog 2 Select Advanced Instrument control panel Select Locked or Unlocked Click OK Buttons The Instrument control panel includes the following buttons Function Part Resumes instrument operation from the following states Pause Wash Hold Pauses the run and stops all pumps ÄKTA pure User Manual 29119969 AB 29 2 The ÄKTA pure instrument 2 3 Instrument...

Page 30: ...ght indicators are off Off The instrumenthas no communication with the Instrument server Power on The Power Communication indicator flashes slowly Power on The system is starting up Connecting The Power Communication indicator flashes quickly Connecting The instrument is ready to use Ready The Power Communication indicator displays a constant light Ready 30 ÄKTA pure User Manual 29119969 AB 2 The ...

Page 31: ...shes slowly A run has been put on hold Hold A run has been paused Pause Both the Power Communication indi cator and Pause button display a constant light The system has been paused due to an alarm To resume the run acknowl edge the alarm and continue the run in UNICORN Alarms and er rors The Power Communication indicator displays a constant light and the Alarm and error indicator flashes ÄKTA pure...

Page 32: ...unication indicator displays a pulsating light A module is being re programmed to be compatible with the current instru ment configuration Re programming All indicators are lit in a wave pattern 32 ÄKTA pure User Manual 29119969 AB 2 The ÄKTA pure instrument 2 3 Instrument control panel ...

Page 33: ...mps 38 2 4 2 Mixer 39 2 4 3 Valves overview 40 2 4 4 Inlet valves 47 2 4 5 Mixer valve 50 2 4 6 Injection valve 53 2 4 7 Loop valve 56 2 4 8 Column valves 60 2 4 9 Versatile valve 61 2 4 10 pH valve 65 2 4 11 Outlet valves 68 2 4 12 Pressure monitors 70 2 4 13 UV monitors 73 2 4 14 Conductivity monitor 75 2 4 15 Flow restrictor ÄKTA pure User Manual 29119969 AB 33 2 The ÄKTA pure instrument 2 4 In...

Page 34: ...low pulsation liquid delivery To ensure delivery of correct liquid volume the pumps must be free from air Each pump head is equipped with a purge valve that is used for this purpose See Section 5 4 Prime inlets and purge pump heads on page 161 The table below contains the operating limits and labels of the system pumps of ÄKTA pure 25 and ÄKTA pure 150 respectively Max pressure Flow rate Pump type...

Page 35: ... pump A 8 The pump piston rinsing system A seal prevents leakage between the pump chamber and the drive mechanism The seal is continuously lubricated by the presence of liquid The pump piston rinsing system continuously flushes the low pressure chamber behind the piston with a low flow of 20 ethanol This prevents any deposition of salts from aqueous buffers on the pistons and prolongs the working ...

Page 36: ...e 2 Rinsing system tube holder bottom 3 Outlet tubing 4 Inlet tubing 5 System pump rinsing systems flow path The illustration below shows the tubing configuration of the pump piston rinsing system of the system pumps 1 2 36 ÄKTA pure User Manual 29119969 AB 2 The ÄKTA pure instrument 2 4 Instrument modules 2 4 1 System pumps ...

Page 37: ...Description Part System pump A 1 System pump B 2 ÄKTA pure User Manual 29119969 AB 37 2 The ÄKTA pure instrument 2 4 Instrument modules 2 4 1 System pumps ...

Page 38: ...he system is going to be exposed to organic solvents or high concentrations of organic acids for longer periods of time Refer to Section 7 8 4 Replace the O ring inside the Mixer on page 323 for how to change the ring Note Location and illustration The illustration below shows the location together with a detailed view of Mixer M9 1 2 3 Description Part Outlet 1 Mixer chamber 0 6 1 4 5 or 15 ml 2 ...

Page 39: ...block define the flow path and function of each type of valve When the Rotary disc turns the flow path in the valve changes Illustration of inlet valve components The illustration below shows the components of a disassembled Inlet valve A or a disassembled Inlet valve B Inlet valve AB is built up by the same parts but have another configuration of defined bores Description Part Valve connection bl...

Page 40: ...ange between different buffers and wash solutions two A and two B inlet ports V9H IAB V9 IAB Inlet valve AB Can be used to generate gradients by mixing buffer from System pump A and buffer from System pump B Enables automatic loading of up to seven samples when used together with a sample pump V9H IS V9 IS Sample inlet valve Increases the total number of inlets to the system V9H IX V9 IX Inlet val...

Page 41: ...Location Inlet valve Before System pump A Inlet valve A Before System pump B Inlet valve B Before both System pump A and System pump B Inlet valve AB Before the sample pump Sample inlet valve For example before another inlet valve Inlet valve IX Illustration of Inlet valve A and Inlet valve B The illustration below shows a detailed view of Inlet valve A and Inlet valve B in this example with label...

Page 42: ...t Out Description Port Buffer inlets of Inlet valve A A1 A7 Buffer inlets of Inlet valve B B1 B7 Not used for ÄKTA pure Q To the respective System pump Out Illustration of Inlet valve AB The illustration below shows a detailed view of Inlet valve AB 1 2 3 4 42 ÄKTA pure User Manual 29119969 AB 2 The ÄKTA pure instrument 2 4 Instrument modules 2 4 4 Inlet valves ...

Page 43: ...tion Port Inlet ports A1 and A2 of Inlet valve AB are used when buffers or samples should be delivered to System pump A A1 A2 Inlet ports B1 and B2 of Inlet valve AB are used when buffers or samples should be delivered to System pump B B1 B2 Outlet port A of Inlet valve AB leads to System pump A A Outlet port B of Inlet valve AB leads to System pump B B ÄKTA pure User Manual 29119969 AB 43 2 The Ä...

Page 44: ...d air sensor located under the plug 1 Ports of Sample inlet valve The illustration below shows the ports of Sample inlet valve in this example labeled V9 IS Description Port Sample inlets S1 S7 Buffer inlet Buff To Sample pump Out 44 ÄKTA pure User Manual 29119969 AB 2 The ÄKTA pure instrument 2 4 Instrument modules 2 4 4 Inlet valves ...

Page 45: ...1500 Tubing connec tor 5 16 with Ferrule blue 3 16 Tubing connec tor 5 16 with Ferrule yellow 1 8 FEP o d 3 16 i d 2 9 mm FEP o d 1 8 i d 1 6 mm Inlets to Inlet valve AB A1 A2 and B1 B2 1500 Tubing connec tor 5 16 with Ferrule blue 3 16 Tubing connec tor 5 16 with Ferrule yellow 1 8 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 A7 1500 Tubing connec tor 5 16 with Ferrul...

Page 46: ...Ferrule blue 3 16 Tubing connec tor 5 16 with Ferrule yellow 1 8 FEP o d 3 16 i d 2 9 mm FEP o d 1 8 i d 1 6 mm Sample inlets to Sample inlet valve S1 S7 1000 Tubing connec tor 5 16 with Ferrule blue 3 16 Tubing connec tor 5 16 with Ferrule yellow 1 8 FEP o d 3 16 i d 2 9 mm FEP o d 1 8 i d 1 6 mm Buffer inlet to Sample inlet valve Buff 580 Tubing connec tor 5 16 with Ferrule blue 3 16 Tubing conn...

Page 47: ...below shows the recommended location together with a detailed view of the Mixer valve Ports and flow paths of the mixer valve The illustration and tables below describe the ports of and different flow paths through the Mixer valve The Mixer valve has two available flow paths By pass and Mixer default If the Mixer valve is installed in the recommended location before the Mixer By pass allows the fl...

Page 48: ...ow shows recommended tubing and connectors Tubing length mm Connector Tubing Connection Tubing label ÄKTA pure 150 ÄKTA pure 25 160 Fingertight connector 1 16 PEEK o d 1 16 i d 1 00 mm PEEK o d 1 16 i d 0 75 mm Pressure monitor R9 to 3 1 Mixer valve V9 M port In 330 Fingertight connector 1 16 PEEK o d 1 16 i d 1 00 mm PEEK o d 1 16 i d 0 75 mm Mixer valve V9 M port Mixer to Mixer M9 3 2 260 Finger...

Page 49: ...xer directs the flow to the Mixer The illustration below shows the different flow paths through the Mixer valve V9 M Primary flow path Alternative flow path Description Flow path The flow from the system pumps bypasses the mixer By pass The flow from the system pumps is directed to the Mixer Mixer is the default flow path Mixer ÄKTA pure User Manual 29119969 AB 49 2 The ÄKTA pure instrument 2 4 In...

Page 50: ... using a Sample pump or System pump A together with the mixer valve For instructions on how to connect and use loops see Section 5 7 Sample application on page 180 Location and illustration of the Injection valve The illustration below shows the location together with a detailed view of the Injection valve in this exampel labeled V9 Inj Ports and flow paths of the Injection valve The illustration ...

Page 51: ...mps via the Mixer SyP Syringe connection Syr Outlet to one of the Column valves or to the column Col Port for connection of a loop Used to fill the loop LoopF Port for connection of a loop Used to empty the loop into the flow path LoopE Loop and System pump waste W1 Sample flow waste W2 ÄKTA pure User Manual 29119969 AB 51 2 The ÄKTA pure instrument 2 4 Instrument modules 2 4 6 Injection valve ...

Page 52: ... flow entering the SaP port is directed to the loop This position is used with Sample pump load the sample pump or a mixer valve and System pump A bypassing the mixer Excess sample leaves the valve through waste port W1 The flow entering the SyP port is directed to the column or the column valve The flow entering the SaP port is directed to waste port W2 This position is used with Sample pump wast...

Page 53: ...on that enables bypassing 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 Ports and flow paths of the Loop valve The illustration and tables below describe the ports and different flow paths through the Loop valve In the Position 4 example the loop is con...

Page 54: ...h The flow direction depends on the Injection valve position Position 1 5 The flow bypasses the loop s By pass is the default flow path By pass Connect tubing The table below shows recommended tubing and connectors Tubing length mm Connector Tubing Connection Tubing label ÄKTA pure 150 ÄKTA pure 25 160 Fingertight connector 1 16 PEEK o d 1 16 i d 0 75 mm PEEK o d 1 16 i d 0 50 mm Injection valve p...

Page 55: ...tion on page 180 Note Always use the the first positions of the valve for the connected loops e g if three loops will be used use port 1F 3F and the corresponding ports 1E 3E to avoid cross contamina tion 3 It is possible to place the Loop valve in other positions in the flow path than the one described above However the volume used for washes will then be incorrect just as the system config urati...

Page 56: ...ports of Column valves V9 C and V9H C have built in pressure sensors that measures the actual pressure over the column For further information on the pressure sensors see Function of pressure monitors integrated in Column valves V9 C or V9H C on page 69 Column valves V9 Cs and V9H Cs lack built in pressure sensors See Section 5 5 Connect a column on page 173 for information on how to set pressure ...

Page 57: ...ion 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 Description Flow path The flow bypasses the column s By pass is the default flow path By pass The flow direction is from the top of the column to the bottom of the column ...

Page 58: ...of the column to the bottom of the column Down flow is the default flow direction Down flow The flow direction is from the bottom of the column to the top of the column Up flow Connect tubing The table below shows recommended tubing and connectors Tubing length mm Connector Tubing Connection Tubing label ÄKTA pure 150 ÄKTA pure 25 100 Fingertight connector 1 16 PEEK o d 1 16 i d 0 75 mm PEEK o d 1...

Page 59: ...ng 6 1 Connect tubing between Injection valve Column valve and UV monitor according to the table above 2 The built in pressure sensors for Column valve V9 C and V9H C have to be re calibrated after installation See Calibrate the monitors on page 305 Note ÄKTA pure User Manual 29119969 AB 59 2 The ÄKTA pure instrument 2 4 Instrument modules 2 4 8 Column valves ...

Page 60: ...nd flow paths through the Versatile valve The valve has four ports 1 4 The Versatile valve has four available sets of flow paths two where a single flow channel is used and two where the flow can be directed through two different channels simultaneously Description Flow path A single flow channel where the flow is directed between port 1 and port 3 1 3 A single flow channel where the flow is direc...

Page 61: ... the UV flow cell The valve directs the flow to the pH electrode and to the flow restrictor or bypasses one or both The Flow restrictor is normally included in the flow path after the Conductivity monitor When installing the pH valve on ÄKTA pure the flow restrictor should be moved from its normal po sition on the conductivity monitor to the pH valve Note Location and illustration of the pH valve ...

Page 62: ...is example labeled V9 pH By pass Primary flow path Flow path for calibrations 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 62 ÄKTA pure User Manual 29119969 AB 2 The ÄKTA pure instrument 2 4 Instrument modules 2 4 10 pH valve ...

Page 63: ... monitor The pH monitor continuously measures the pH of the buffer and eluted proteins when the pH electrode is inline A pH electrode can be installed in the pH flow cell For instruction on how to install the pH electrode see Section 7 5 2 Replace the pH electrode on page 275 The pH electrode should not be ex posed to more than 0 5 MPa during a normal run but can withstand pressure spikes of 0 8 M...

Page 64: ... connector 1 16 PEEK o d 1 16 i d 0 75 mm PEEK o d 1 16 i d 0 50 mm Flow restrictor to port FrR 2R 160 Fingertight connector 1 16 PEEK o d 1 16 i d 0 75 mm PEEK o d 1 16 i d 0 50 mm Port Out to Outlet valve 9pH N A N A N A N A to Calibration port Cal N A 1500 Fingertight connector 1 16 ETFE I D 1 mm ETFE I D 1 mm Port W3 to Waste W3 The dimensions of the PEEK tubing depends on which tubing kit tha...

Page 65: ...O V9 O 1 outlet ports V9H Os V9 Os Location and illustration of outlet valves The illustration below shows the recommended location together with a detailed view of the outlet valves In this example the valves are labeled V9 Os and V9 O Ports of Outlet valves V9 O and V9H O The illustration below shows the ports of Outlet valve V9 O and V9H O in this example labeled V9 O Description Port Inlet por...

Page 66: ...t valves V9 Os and V9H Os The illustration below shows the ports of Outlet valve V9 Os In Out1 Frac W V9 Os Description Port Inlet port In Outlet port Out1 Port to fraction collector Note If a secondary Fraction collector F9 R is used it should be connected to port Out1 Frac Waste port W 66 ÄKTA pure User Manual 29119969 AB 2 The ÄKTA pure instrument 2 4 Instrument modules 2 4 11 Outlet valves ...

Page 67: ... i d 0 50 mm pH valve to Outlet valve 9pH 1500 Fingertight connector 1 16 ETFE o d 1 8 i d 1 mm ETFE o d 1 8 i d 1 mm Outlets from the outlet valves Out1 Out10 If ÄKTA pure previously has been configured without an Outlet valve remove the Union F F between tubing 9 and tubing W before installation of the Outlet valve Then connect tubing 9 to the In port on the Outlet valve and the waste tubing W t...

Page 68: ...nitor of the sample pump measures the pressure after the sample pump the sample pressure To provide a reading of the highest pressure in the system there are connections to all pump cylinders The pressure monitor also contains pump flow restrictors in order to stop siphoning effects Siphoning effects can occur if tubing in the system is disconnected while the inlet tubing is immersed in buffers th...

Page 69: ...pressure before the column Pre column pressure A and the pressure 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 pressure and the Delta column pressure Pressure control of the flow may be base...

Page 70: ...rvice personnel Note The resolution is decreased when more than one wavelength is used simultaneously due to lower sampling frequency per wavelength Do not use more wavelengths than necessary Note Location and illustration of UV monitor U9 M The illustration below shows the location of UV monitor U9 M together with a detailed view of the monitor unit and detector The monitor unit is labelled U9 M ...

Page 71: ...module panel is installed see Example of a typical configuration of the wet side on page16 The UV monitor can also be installed in an Extension box see Extension box on page 86 1 2 3 4 5 Description Part Inlet 1 UV monitor U9 L 2 UV flow cell Two different path lengths are available 2 mm default and 5 mm 3 Outlet 4 UV detector 5 Using two UV monitors It is possible to use two UV monitors in ÄKTA p...

Page 72: ...sure side of the column pressure limits have to be con sidered See UV monitor options on page 416 Note Connect tubing The table below shows the tubing and connectors to be used with UV monitor U9 L If a second UV monitor is used the tubing for this has to be cut manually Note Tubing length mm Connector Tubing Connection Tubing label ÄKTA pure 150 ÄKTA pure 25 160 Fingertight connector 1 16 PEEK o ...

Page 73: ...ated on delivery but can be re calibrated if needed see Section 7 7 3 Calibrate the Conductivity monitor on page 308 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 temperature compensation factor is used to report the conductivity in relation to a set reference temperature Loc...

Page 74: ...ngertight connector 1 16 PEEK o d 1 16 i d 0 75 mm PEEK o d 1 16 i d 0 50 mm UV monitor U9 L to Conductivity monitor C9 7 95 Fingertight connector 1 16 PEEK o d 1 16 i d 0 75 mm PEEK o d 1 16 i d 0 50 mm Conductivity monitor C9 to Flow restrictor 8 74 ÄKTA pure User Manual 29119969 AB 2 The ÄKTA pure instrument 2 4 Instrument modules 2 4 14 Conductivity monitor ...

Page 75: ...ure control parameters on page502 Note Location and illustration of Flow restrictor FR 902 The Flow restrictor is normally included in the flow path after the Conductivity monitor The Conductiv ity monitor is equipped with a special holder for the Flow restrictor The illustration below shows Flow restrictor FR 902 fitted on the Conductivity monitor 1 4 3 2 Function Part Flow restrictor 1 Holder 2 ...

Page 76: ... valve 1 2 3 Function Part Flow restrictor 1 Flow restrictor inlet connection from pH valve ToR port 2 Flow restrictor outlet connection to pH valve FrR port 3 76 ÄKTA pure User Manual 29119969 AB 2 The ÄKTA pure instrument 2 4 Instrument modules 2 4 15 Flow restrictor ...

Page 77: ...de IDs for valves and modules The function of a valve or module is defined by its Node ID not by its physical position Note The Node ID is set by positioning the arrows of the one or two rotating switches at the back of the valve Use a screwdriver to set the arrows of the switches to the desired number A B The illustration shows an example of a valve module with two rotating switches The first rot...

Page 78: ...s and remove the tubing from the existing module Note This step does not apply for a Module Panel 2 Loosen the module with a Torx T20 screwdriver 3 Remove the module 4 Disconnect the cable and secure it in the slit 5 78 ÄKTA pure User Manual 29119969 AB 2 The ÄKTA pure instrument 2 5 Installation of internal modules ...

Page 79: ...s been installed in the instrument but not added to the current system configuration in UNICORN Note Install internal modules in the Extension box The Extension box can be used to install extra modules on the ÄKTA pure instrument See Extension box on page 86 for more information ÄKTA pure User Manual 29119969 AB 79 2 The ÄKTA pure instrument 2 5 Installation of internal modules ...

Page 80: ...e holder The rod has extra rails on both sides Push the button of the rod to attach it to a holder rail The illustration below shows the Rail extension rod 1 2 Function Part Button 1 Extension rod 2 Multi purpose holder The Multi purpose holder can be used to attach accessories eg a Loop holder or a cassette Attach the holder to a holder rail The illustration below shows the Multi purpose holder 1...

Page 81: ...urpose holder 2 Column holder The Column holder has one position for medium sized columns and one position for small sized columns The Column holder can also be used for bottles Use two holders to attach long columns The illustration below shows the Column holder 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 1 2 ...

Page 82: ... holder rod The Column holder rod can be used to attach several HiTrap columns The holder has threaded ports for HiTrap columns and tubing connectors Push the button of the holder to attach the holder to a holder rail The illustration below shows the Column holder rod 1 2 3 Description Part Snap in to holder rails 1 Button 2 Column holder rod 3 82 ÄKTA pure User Manual 29119969 AB 2 The ÄKTA pure ...

Page 83: ...wer part 2 Snap in strips 3 Attachment part 4 Upper tubing 5 Lever 6 Upper part 7 Upper connector 8 Lower connector 9 Tubing holder spool The Tubing holder spool is used to hold and arrange tubing The illustration below shows the Tubing holder spool Description Part 1 2 3 Positions for tubing 1 Tab 2 Snap in to holder rails 3 ÄKTA pure User Manual 29119969 AB 83 2 The ÄKTA pure instrument 2 6 Acce...

Page 84: ...n be attached to the holder rails to hold a sample bottle The illustration below shows the Bottle holder Description Part 1 2 Position for bottle 1 Snap in to holder rails 2 Adapter for air sensor The adapter for air sensor is used to hold an optional air sensor The air sensor with adapter is connected to the Bottle holder see illustrations below Description Part 1 2 3 Air sensor 1 Air sensor adap...

Page 85: ...de must be connected to the Module Panel see Hardware installation of a module on page 77 When an optional module is replaced by a Module panel the removed module has to be deselected in the software configuration see Edit system properties on page 128 Illustration The illustration shows the Module Panels installed in the example configuration used in this manual ÄKTA pure User Manual 29119969 AB ...

Page 86: ...ith extra modules when using ÄKTA pure See the Extension box instruction for more information Location The illustration below shows an Extension box with a module mounted on the side of ÄKTA pure The Extension box can be mounted in three ways On the side of ÄKTA pure Standing on top of or next to ÄKTA pure On a Rail extension rod 29 0113 52 on ÄKTA pure 86 ÄKTA pure User Manual 29119969 AB 2 The Ä...

Page 87: ... of how to connect external modules is also provided In this chapter This chapter contains the following sections See page Section 88 3 1 External air sensors 90 3 2 Fraction collector F9 C 104 3 3 Fraction collector F9 R 108 3 4 Sample pump S9 and S9H 112 3 5 I O box E9 122 3 6 Connection of external modules ÄKTA pure User Manual 29119969 AB 87 3 ÄKTA pure external modules ...

Page 88: ...ned for o d 1 16 tubing at the high pressure side after the pumps It is installed after the injection valve and is used to prevent air entering the column L9 1 2 can be used in the same fashion as L9 1 5 if O D 1 16 ETFE tubing is used between the air sensor and the inlet valve Note Tubing connections Air sensor L9 1 5 Tubing length mm Connector Tubing Connection between ÄKTA pure 150 ÄKTA pure 25...

Page 89: ...n between 100 Fingertight connector 1 16 PEEK o d 1 16 Injection valve and L9 1 2 100 Fingertight connector 1 16 PEEK o d 1 16 L9 1 2 and Column valve the connected column ÄKTA pure User Manual 29119969 AB 89 3 ÄKTA pure external modules 3 1 External air sensors ...

Page 90: ...e User manual In this section This section contains the following subsections See page Section 91 3 2 1 Function 93 3 2 2 Fraction collector F9 C illustrations 97 3 2 3 Cassettes Cassette tray and racks 103 3 2 4 Connect tubing to the ÄKTA pure instrument 90 ÄKTA pure User Manual 29119969 AB 3 ÄKTA pure external modules 3 2 Fraction collector F9 C ...

Page 91: ...8 or 96 wells Full scan is performed only when the system is in state Ready Quick scan Scanning of Cassette type codes to determine which type of Cassettes are used Quick scan is performed during the run to ensure that correct Cassettes are placed in the Fraction collector Fractionation modes to avoid spillage Three fractionation modes are available all of which avoid spillage between wells or tub...

Page 92: ...e waste funnel This position is called Waste Frac in UNICORN Frac cleaning position The Frac cleaning position is used for convenient cleaning of the Dispenser head The Fractionation arm is positioned in the front of the interior of the fractionation collector and the Dispenser head is moved to the center of the Fractionation arm 92 ÄKTA pure User Manual 29119969 AB 3 ÄKTA pure external modules 3 ...

Page 93: ...parts of the exterior of Fraction collector F9 C 1 2 3 5 6 4 Description Part Fractionation indicator 1 Symbol indicating that fractionation is ongoing Do not open the door while the indicator is lit Door 2 Window 3 Door handle 4 Tubing connector for outlet valve tubing 5 Vents 6 ÄKTA pure User Manual 29119969 AB 93 3 ÄKTA pure external modules 3 2 Fraction collector F9 C 3 2 2 Fraction collector ...

Page 94: ...on collector F9 C 1 1 2 3 Description Part Vents 1 UniNet 9 D type connector for communication and power supply 2 Waste tube 3 94 ÄKTA pure User Manual 29119969 AB 3 ÄKTA pure external modules 3 2 Fraction collector F9 C 3 2 2 Fraction collector F9 C illustrations ...

Page 95: ...Fractionation arm main rail 2 Lamp 3 Tubing guide 4 Tubing connection 5 Dispenser head 6 Fractionation arm 7 Tubing guide 8 Height exclusion bar 9 Waste funnel 10 Waste tube 11 Tray catch 12 Waste groove in case of overflow 13 Tray guides 14 ÄKTA pure User Manual 29119969 AB 95 3 ÄKTA pure external modules 3 2 Fraction collector F9 C 3 2 2 Fraction collector F9 C illustrations ...

Page 96: ...2 3 4 5 6 Description Part Dispenser head 1 Nozzle 2 Dispenser head cover 3 Accumulator back part of Dispenser head 4 Drop sync sensor 5 Type code reader 6 96 ÄKTA pure User Manual 29119969 AB 3 ÄKTA pure external modules 3 2 Fraction collector F9 C 3 2 2 Fraction collector F9 C illustrations ...

Page 97: ...ette 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 page100 and Deep well plates on page101 respec tively Illustrations of Fraction collector F9 C tray a...

Page 98: ...tor 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 98 ÄKTA pure User Manual 29119969 AB 3 ÄKTA pure external modules 3 2 Fraction collector F9 C 3 2 3 Cassettes Cassette tray and racks ...

Page 99: ...easily 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 con...

Page 100: ...ments listed in the table below Examples of manufacturers are also listed in the table Examples of manufac turers Height mm Diameter mm Tube or bottle size ml Max Min Max Min NUNC 56 50 11 5 10 5 3 VWR 76 70 12 10 5 5 BD Biosciences VWR 102 96 13 3 12 8 BD Biosciences 120 114 17 16 15 BD 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...

Page 101: ...on Property 24 48 or 96 No of wells Square not cylindrical Shape of wells 10 5 or 2 ml Well volume Approved deep well plates The plates listed in the table below are tested and approved by GE to be used with Fraction collector F9 C 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 ...

Page 102: ...low rates depending on what type of deep well plates that are used The table below lists the maximum flow rates for the different plate types Maximum flow rate ml min Plate type 10 96 deep well plate 15 48 deep well plate 25 24 deep well plate 102 ÄKTA pure User Manual 29119969 AB 3 ÄKTA pure external modules 3 2 Fraction collector F9 C 3 2 3 Cassettes Cassette tray and racks ...

Page 103: ...ure instrument to the fraction collector Action Step Connect the tubing Frac to the Frac port on the outlet valve on the ÄKTA pure instrument 1 Connect the other end of the Frac tubing to the inlet port on the fraction collector 2 Adjust the delay volume setting in UNICORN see Section 9 8 8 System settings Tubing and Delay volumes on page 460 for more details 3 ÄKTA pure User Manual 29119969 AB 10...

Page 104: ...ion runs The fraction collector can be used for Fixed volume fractionation Peak fractionation Combined fixed volume fractionation and peak fractionation Fraction collector F9 R has the following function for reducing sample spill during fractionation Drop Sync Front view illustration The illustration below shows the main parts of the Fraction collector 1 3 4 5 7 6 8 2 104 ÄKTA pure User Manual 291...

Page 105: ...ack 7 Base unit 8 Connector panel illustration The illustration below shows the main parts of the connector panel on the fraction collector 1 2 2 Function Part Node ID switch 1 UniNet 9 F type connector for communication and power supply 2 ÄKTA pure User Manual 29119969 AB 105 3 ÄKTA pure external modules 3 3 Fraction collector F9 R ...

Page 106: ...uide and Tube holder For more information on the assembly of the tube rack see Assemble the Tube rack on page200 For infor mation on which Tube rack to use see Tube rack inserts on page 201 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 c...

Page 107: ... This ensures the correct length of the exposed tubing end 6 4 Re install the tubing holder in the Delivery arm 5 For Fraction collector F9 R Connect the tubing from the fraction collector to the port Frac on the outlet valve 6 For Fraction collector F9 R 2nd Connect the tubing from the second fraction collector to port Out 10 on Outlet valve V9 O or V9H O port Out 1 on Outlet valve V9 Os or V9H O...

Page 108: ...e 150 and one for ÄKTA pure 25 To ensure delivery of correct liquid volume the pumps must be free from air Each pump head is equipped with a purge valve that is used for this purpose The Sample pump also comprises a pressure monitor The table below contains the operating limits and labels of the sample pump configurations Max pressure Flow rate Pump type Pump label Configuration 10 MPa 0 001 to 50...

Page 109: ...Used to remove air from the pump 4 Sample pump outlet port 5 Sample pressure monitor 6 Vents 7 Sample pump inlet port 8 Inlet manifold 9 Rear view 2 1 3 Function Part UniNet 9 type D port 1 IP cover Protects pump electronics from liquid spill 2 Vents 3 ÄKTA pure User Manual 29119969 AB 109 3 ÄKTA pure external modules 3 4 Sample pump S9 and S9H ...

Page 110: ... on page 262 Illustrations of the sample pump piston rinsing system The illustrations below show the parts tubing and flow path of the sample pump piston rinsing system 1 2 3 4 5 Rinsing system flow path Parts and tubing Function Part Pump rinsing liquid tube 1 Pump rinsing liquid tube holder 2 Inlet tubing from the rinsing liquid tube to the left pump head lower rinsing system connection 3 Tubing...

Page 111: ...nector 5 16 with Ferrule blue 3 16 Tubing connector 5 16 with Ferrule yellow 1 8 FEP o d 3 16 i d 2 9 mm FEP o d 1 8 i d 1 6 mm Sample container to samplepumpinletmani fold sample inlet valve not used No label 530 Fingertight con nector 1 16 Fingertight con nector 1 16 PEEK o d 1 16 i d 1 mm PEEK o d 1 16 i d 0 75 mm Sample pump pressure monitor to Inlet valve port SaP 3S The recommended location ...

Page 112: ...tion This section contains the following subsections See page Section 113 3 5 1 Overview of the I O box 115 3 5 2 Analog connector and signals 117 3 5 3 Digital connector and signals 119 3 5 4 Connect external equipment to the I O box 112 ÄKTA pure User Manual 29119969 AB 3 ÄKTA pure external modules 3 5 I O box E9 ...

Page 113: ...nals to external equipment Using two I O boxes It is possible to install up to two I O boxes when using ÄKTA pure If two I O boxes are 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 114 and Section 9 ...

Page 114: ...the ÄKTA pure instru ment 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 114 ÄKTA pure User Manual 29119969 AB 3 ÄKTA pure external modules 3 5 I O box E9 3 5 1 Overview of the I O box ...

Page 115: ...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 ÄKTA pure User Manual 29119969 AB 115 3 ÄKTA pure external modules 3 5 I O box E9 3 5 2 Analog conn...

Page 116: ...selected 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 automatically 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 v...

Page 117: ...ed synchronously and the outputs will be set synchronously Digital input signals The digital in signal can be used to monitor external equipment by registering for example error signals or event marks An event mark can be used as a trigger for watches The measured digital signals can be shown as a curve in UNICORN The unit will handle both open closed circuit and TTL type voltage signals An open c...

Page 118: ...ls 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 the user The level can be changed by instructions either manually in System Settings or by a method It is possible to send pulses from the current level with a pulse length of 0 1 s to 10 s A closed circuit is always closed against signa...

Page 119: ...uracy 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 Required material The following material is required Flat blade screwdriver 2 mm Shielded cable with 9 conductors 4 to 8 mm diameter ÄKTA pure ...

Page 120: ...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 ter minals for left side or right side cable entry 4 Insert and fasten the single conductors in the screw terminals 5 Close the housing top shell with the latch and screw the housing together 6 System properties Foll...

Page 121: ...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 user to send one of the pre defined signals UV signal con...

Page 122: ... D type F type Constraints The table below indicates usage constraints for the different external modules Constraints Connector External module I O box E9 has no constraints F type I O box E9 I O box E9 2nd requires I O box E9 F type I O box E9 2nd External air sensor L9 has no constraints F type External air sensor L9 External air sensor L9 2 requires External air sensor L9 F type External air se...

Page 123: ...10 m Note ÄKTA pure connector plate The connector plate with the UniNet 9 connectors is located on the back of the ÄKTA pure instrument 9 Function Connector name Connector Connector point for service Test 9 NOTICE Do not connect any module to the connector Test on the ÄKTA pure instrument Plug all unused UniNet 9 ports ports 1 to 8 on the ÄKTA pure instrument with jumpers Note The connector Test s...

Page 124: ...ure instrument and how to install optional modules and add them in UNICORN In this chapter This chapter contains the following sections See page Section 125 4 1 Configuration overview 131 4 2 Configure modules 146 4 3 General system settings 124 ÄKTA pure User Manual 29119969 AB 4 System configuration ...

Page 125: ... 3 Edit system properties 4 Edit system settings Select modules and positions Optional modules can be placed in any of the available positions in the cabinet Its function is determined by the module type and node ID However to achieve an optimal flow path for a purification run most modules have a dedicated location in the cabinet The dedicated location for all modules and if appli cable any const...

Page 126: ...valve V9 IA or V9H IA 5 Inlet valve V9 IB or V9H IB Cannot be used at the same time as Mixer valve V9 M or V9H M 8 Sample inlet valve V9 IS or V9H IS Inlet valve V9 X1 or V9H X1 has no con straints and has no support in the method phases None Inlet valve V9 X1 or V9H X1 Inlet valve V9 X2 or V9H X2 requires Inlet valve V9 X1 or V9H X1 respectively It has no support in the method phases None Inlet v...

Page 127: ... 2 Versatile valve 4 requires Versatile valve 3 None Versatile valve V9 V or V9H V Versatile valve 2 Versatile valve 3 Versatile valve 4 UV monitor U9 M cannot be used at the same time as UV monitor U9 L Note UV monitor U9 L 2nd can be used at the same time as UV monitor U9 M 10 UV monitor U9 M UV monitor U9 L 11 UV monitor U9 L Cannot be used at the same time as UV monitor U9 M Requires the Multi...

Page 128: ...t node ID See NodeID list 2 Install the module 3 Connect tubing 4 Edit system properties When a new module has been installed the system properties have to be updated in UNICORN The system will restart automatically when the configuration has been changed in System Properties and the system can be reconnected The following instruction gives a general descriptionof how to update the system properti...

Page 129: ...an be edited Result The Edit dialog 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 Click 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 130: ... For example if the change in configuration affects the delay volume following the UV monitor or other monitor connected via the I O box the appropriate system settings for Tubing and 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 modu...

Page 131: ...ction 132 4 2 1 Configuration of inlet valves 133 4 2 2 Configuration of Mixer valves 134 4 2 3 Configuration of Loop valves 135 4 2 4 Configuration of column valves 137 4 2 5 Configuration of Versatile valves 138 4 2 6 Configuration of pH valves 139 4 2 7 Configuration of outlet valves 140 4 2 8 Configuration of UV monitors 141 4 2 9 Configuration of Conductivity monitor 142 4 2 10 Configuration ...

Page 132: ...IAB part A 2 ports or V9H IAB part A 2 ports Inlet A Inlet valve V9 IAB or V9H IAB used for inlet A V9 IAB part B 2 ports or V9H IAB part B 2 ports Inlet B Inlet valve V9 IAB or V9H IAB used for inlet B N A Sample inlet V9 IS Sample inlet valve V9 IS N A Sample inlet V9H IS Sample inlet valve V9H IS N A Inlet valve X1 V9 IX Inlet valve V9 IX N A Inlet valve X1 V9H IX Inlet valve V9H IX N A Inlet v...

Page 133: ... in the Component selection list 3 System settings The flow rate for Mixer by pass wash can be set Description Instruction name Sets the flow rate used during Mixer by pass wash and defines wash volumes for mixer by pass wash options Note The flow rate should not exceed 10 ml min if narrow inlet tubing i d 0 75 mm is used Mixer by pass wash settings ÄKTA pure User Manual 29119969 AB 133 4 System c...

Page 134: ...lect Loop valve V9 L or Loop valve V9H L in the Component selection list 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 134 ÄKTA pure User Manual 29119969 AB 4 System configuration 4 2 Configure modules 4 2 3 Co...

Page 135: ...s of pre column pressure when Column valve V9 C or V9H C 5 columns is NOT mounted onto the system In such cases there is no pre column pressure sensor present The estimated pre column pressure is calculated using the measured system pressure and the length of the tubing between the system pressure sensor and the Column The instructions are available only when the Column valve V9 C or V9H C 5 colum...

Page 136: ...jection valve 4 The built in pressure sensors for Column valves V9 C and V9H C have to be re calibrated after installation See Calibrate the monitors on page 305 Note A Pre Column pressure alarm shall always be set to protect the column see Section 5 6 Pressure alarms on page 177 Column valve V9 Cs and V9H Cs does not contain pressure monitors SeeSection5 6Pressurealarms onpage177forhowtoprotectco...

Page 137: ... 2 Select Versatile valve V9 V Versatile valve 2 V9 V Versatile valve 3 V9 V or Versatile valve 4 V9 V in the Component selection list 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 Set delay volume on page 146 ÄKTA pure User Manual 29119969 AB 137 4 System configuration 4 2 Configure modules 4 2 5 Config...

Page 138: ...ponent selection list 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 Set delay volume on page 146 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 138 ÄKTA pure User Manual 29119969 AB 4 S...

Page 139: ...ve V9 O 10 outlets Outlet valve Outlet fractionation valve V9 O V9H O 10 outlets Outlet valve Outlet fractionation valve V9H O V9 Os 1 outlet Outlet valve Outlet control valve V9 Os V9H Os 1 outlet Outlet valve Outlet control valve V9H Os System settings If the valve is not placed in the recommended position using the standard tubing kit the delay volume must be set See Check Set delay volume on p...

Page 140: ...ve to be con sidered See UV monitor options on page 416 for pressure limits Note System properties Follow the instruction below to update the system properties Action Step Open the system properties Edit dialog 1 Select Monitors and sensors from the Component types list 2 Select components and properties according to the following table 3 Property Component selection Monitor U9 L fixed UV monitor ...

Page 141: ...nsors from the Component types list 2 Select Conductivity monitor C9 in the Component selection list 3 System settings If the monitor is placed in the flow path between the UV monitor and the outlet valve the delay volume must be set See Check Set delay volume on page 146 ÄKTA pure User Manual 29119969 AB 141 4 System configuration 4 2 Configure modules 4 2 9 Configuration of Conductivity monitor ...

Page 142: ... used L9 1 2 or L9 1 5 4 System settings The sensitivity of the air sensor can be set Usage Air volume detected Parameter ÄKTA pure 150 ÄKTA pure 25 Detect empty buffer sample vessels 100 μl 30 μl Normal default Detect even small air bubbles 30 μl 10 μl High The sensitivity should be set to Normal when the air sensor is located before the System pump Air sensor ext Before pump A or Air sensor ext ...

Page 143: ...n page 146 Fraction settings and numbering mode can be set Fraction collector F9 C Description Instruction name Fractionation settings comprises fractionation mode and frac tionation order Fractionation settings Fractionation mode Automatic Accumulator or DropSync Fractionation order Row by row Column by column Serpen tine row Serpentine column For fractionation mode DropSync only the Serpentine o...

Page 144: ...whether the fraction number is reset at the end of a method or not Note The default setting is Reset Fractionation numbering mode Drop sync on or off It is recommended to use this setting for flow rates below 2 ml min Higher flow rates can be however be used depending on the properties e g viscosity of the liquid Fractionation settings frac 2 Determines whether fraction number for the second fract...

Page 145: ...X When enabled it sets 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 Valu...

Page 146: ...ay volumes for modules and standard tubing configurations are found in Section 9 13 Delay volumes on page 494 Follow the instructions below to check set the delay volumes Action Step In the System Control module choose System Connect to Systems or click the Con nect to Systems icon Result The Connect to Systems dialog opens 1 Select a system Select Control mode Click OK Result The selected instrum...

Page 147: ...rol panel Select Locked or Unlocked Click OK Power save ÄKTA pure has a power save mode The instrument enters Power save after having been in the Ready state for a set period of time Power save can be used both in room temperature and in cold room temperature The system enters the Ready state when a method run a method queue or a manual run ends To enable Power save a system must be connected and ...

Page 148: ...em Settings dialog opens Select Advanced and select Power save 2 Select On in the Mode field and type the number of minutes in the Time field Note This is the time the instrument will be in state Ready before power save mode is entered Click OK 3 148 ÄKTA pure User Manual 29119969 AB 4 System configuration 4 3 General system settings ...

Page 149: ...See page Section 150 5 1 Before you prepare the system 151 5 2 Prepare the flow path 158 5 3 Start UNICORN and connect to system 161 5 4 Prime inlets and purge pump heads 173 5 5 Connect a column 177 5 6 Pressure alarms 180 5 7 Sample application 193 5 8 Fractionation 211 5 9 Create a method and perform a run ÄKTA pure User Manual 29119969 AB 149 5 Operation ...

Page 150: ...lable Checklist Make sure the system is prepared in accordance with the settings in the method to be run Depending on configuration remember to check 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 150 ÄKTA pure User ...

Page 151: ... Illustration of the flow path The illustration below shows the flow path for a typical system configuration The individual instrument modules are presented in the table below The configuration of the system is defined by the user 4 5 6 7 8 9 10 11 12 13 14 15 16 17 W1 W2 W 3 2 1 Description Part Pressure monitor 1 Sample pump 2 Sample inlet valve 3 ÄKTA pure User Manual 29119969 AB 151 5 Operatio...

Page 152: ...tables below show what Mixer chambers to use in ÄKTA pure at different flow rates If the liquids are difficult to mix use a larger Mixer chamber to achieve optimal mixing However note that a larger Mixer chamber distorts and delays the gradient The default mixer size is 1 4 ml for both systems ÄKTA pure 150 also includes a 5 ml mixer Other mixer sizes are available as accessories Flow rate ml min ...

Page 153: ...th the same nominal path length to be directly compared UV flow cells for UV monitor U9 L UV flow cells are available with two different path lengths 2 mm default and 5 mm When replacing a UV flow cell the path length must be set in the System Control module in System Calibrate Use the nominal flow cell length if the UV flow cell is replaced but not calibrated See Update the cell path length on pa...

Page 154: ...n collector is to be used make sure that tubing is connected as described in Connect tubing on page103 and Connect tubing to ÄKTA pure on page107 and prepare the fraction collector Otherwise place the outlet tubing in suitable tubes or flasks 154 ÄKTA pure User Manual 29119969 AB 5 Operation 5 2 Prepare the flow path ...

Page 155: ...f the fraction collector Fraction collector waste outlet Fraction collector F9 C optional Prepare waste tubing Follow the instructions below to prepare the waste tubing CAUTION Fasten the waste tubing Duringoperationat high pressure the ÄKTA pure instrument may release bursts of liquid in the waste tubing Securely fasten all waste tubing to the ÄKTA pure instrument and to the waste vessel CAUTION ...

Page 156: ... collector in a vessel 1 Make sure that the tubing is securely fastened to the ÄKTA pure instrument 2 Fasten waste tubing from the valves with the clips on the front of the system Fasten waste tubing from the Buffer tray with the clips on the rear of the system 156 ÄKTA pure User Manual 29119969 AB 5 Operation 5 2 Prepare the flow path ...

Page 157: ...with new tubing Do not lengthen the tubing as this might cause obstruction of the tubing Fasten all waste tubing securely to the waste vessel 4 Plug unused valve ports It is recommended to plug all unused valve ports with stop plugs before starting a run See Tubing connectors on page 421 for information about connectors ÄKTA pure User Manual 29119969 AB 157 5 Operation 5 2 Prepare the flow path ...

Page 158: ...s 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 proceed to the next Log On dialog box In the Log On dialog box 2 Click a user name in the User Name list and enter the password in the Password field Note It is als...

Page 159: ...to UNICORN Action Step In the System Control module 1 Click the Connect to Systems button or click Connect to Systems on the System menu Result The Connect to Systems dialog opens ÄKTA pure User Manual 29119969 AB 159 5 Operation 5 3 Start UNICORN and connect to system ...

Page 160: ... can now be controlled by the software 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 Tip Result UNICORN states Connection Connected in control or Connection Connected in view in the status bar in the lower part of the Sys...

Page 161: ...pump heads of the system pumps and the sample pump the sample pump is an optional module 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 162 5 4 1 System pumps 168 5 4 2 Sample pump ÄKTA pure User Manual 29119969 ...

Page 162: ...purging the pump heads and priming the inlets using the Process Picture are described below It is also possible to perform the procedures from the Manual instructions dialog Tip Prime inlet tubing Follow the instructions below to fill all A and B inlet tubing to be used in the run with appropriate buffer solution Action Step Make sure that all inlet tubing that is to be used during the method run ...

Page 163: ...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 Repeat steps 3 to 6 for each piece of inlet tubing that is to be used during the run 7 Purge System pump B F...

Page 164: ... switches to waste position This is necessary to achieve a low back pressure during the purge procedure In the Process Picture 3 Click on the pumps Set Conc B to 100 B Click Set B Result Only System pump B is active 164 ÄKTA pure User Manual 29119969 AB 5 Operation 5 4 Prime inlets and purge pump heads 5 4 1 System pumps ...

Page 165: ...lt A system flow starts Connect a 25 to 30 ml syringe to the purge valve of the left pump head of System pump B Make sure that the syringe fits tightly into the purge connector 6 Open the purge valve by turning it counter clockwise about three quarters of a turn Draw a small volume of liquid slowly into the syringe with a rate of about 1 ml per second 7 Close the purge valve by turning it clockwis...

Page 166: ... In the Process Picture 1 Click on the Injection valve and select Manual load Result The Injection valve switches to manual load position Make sure that the pump flow is on 2 In the Chromatogram pane Check the PreC pressure curve 3 If the PreC pressure do not stabilize within a few minutes there may be air left in the pump Refer to Section 8 6 Troubleshooting Pumps on page 387 166 ÄKTA pure User M...

Page 167: ...k on the pumps Set Conc B to 0 B Click Set B Result Only System pump A is active Validate purge of pump A Follow the procedure described in Validate purge of pump B on page 166 to check if there is air left in the pump End the run Click the End button in the System Control toolbar to end the run ÄKTA pure User Manual 29119969 AB 167 5 Operation 5 4 Prime inlets and purge pump heads 5 4 1 System pu...

Page 168: ...elow to fill all sample inlet tubing to be used in the run with appropriate buffer or solution Skip steps three and four if no sample inlet valve is used 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 ...

Page 169: ...mp heads of the Sample pump Make sure 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 liquid slowly into the syringe until the liquid reaches the sample pump 6 Close the purge valve by turning it clockwise Disconnect the syringe and discard its contents 7 Repeat steps 2 5 for each sample inlet that is...

Page 170: ...ump waste Result The injection valve switches to waste position This is necessary to achieve a low back pressure during the purge procedure In the Process Picture Click on Sample pump 5 If Sample inlet valve V9 IS or V9H IS is used Click on the Sample inlet valve icon if the valve is not used the icon will not be present Select the Buffer inlet Click on the Sample pump icon Set the Sample flow to ...

Page 171: ...e about three quarters of a turn Draw 5 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 Connect the syringe to the right purge valve on the Sample pump and repeat step 6 to step 8 9 ÄKTA pure User Manual 29119969 AB 171 5 Operation 5 4 Prime inlets and purge pump heads 5 4 2 Sample...

Page 172: ...irect inject position Make sure that the pump flow is on 2 In the Chromatogram pane Check the PreC pressure curve 3 If the PreC pressure does not stabilize within a few minutes there may be air left in the pump Refer to Section 8 6 Troubleshooting Pumps on page 387 End the run Click the End button in the System Control toolbar to end the run 172 ÄKTA pure User Manual 29119969 AB 5 Operation 5 4 Pr...

Page 173: ...rms on page177 for more information on pressure alarms Do not overtighten when connecting columns Overtightening might rupture the connectors or squeeze the tubing and thereby result in high back pressure Note If no column valve is used remove the column from the system before running a system wash The pressure during a system wash may become too high for the column Note Attach a column holder and...

Page 174: ...ubing to a Column valve port in this example port 1A 3 Open the System Control module 4 In the Process Picture 5 Click on the Column Select Column down flow Result The Column valve switches to position 1 174 ÄKTA pure User Manual 29119969 AB 5 Operation 5 5 Connect a column ...

Page 175: ...esult A system flow of 0 2 ml min starts When buffer leaves the tubing in a continuous mode and the top part of the column is filled with buffer connect the tubing to the top of the column 7 Connect a piece of tubing to the bottom of the column 8 ÄKTA pure User Manual 29119969 AB 175 5 Operation 5 5 Connect a column ...

Page 176: ...e Column valve Use the port opposite to the one already con nected to the column in this example port 1B If no column valve is used connect the tubing to the next module in the flow path 9 Click the End icon in the System Control toolbar to end the run 10 176 ÄKTA pure User Manual 29119969 AB 5 Operation 5 5 Connect a column ...

Page 177: ...g dimensions See the instructions below to set the pressure alarm for the column to be used in the run and if appli cable to set the parameters for the tubing dimensions Set tubing dimension parameters to calculate pre column pressure For instruments where there is no pre column pressure sensor i e the column is connected without a Column valve or to Column valve V9 Cs or V9H Cs the pre column pre...

Page 178: ...lumns the max delta column pressure media is significantly lower than the max pre column pressure hardware To protect the media if a delta column pressure measurement is not available that is when column valve V9 C or V9H C is not used the pre column pressure alarm must be manually set to the value in the column list that is the lowest of the max pre column pressure and the max delta column pressu...

Page 179: ... Alarms and select Alarm pre column pressure 2 Select Enabled in the Mode field 3 Type the high pressure limit in the High alarm field Click Execute 4 ÄKTA pure User Manual 29119969 AB 179 5 Operation 5 6 Pressure alarms ...

Page 180: ...lumn 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 load the sample Sample pump see further instructions in Section 5 4 2 Sample pump on page 168 System pump A see further instructions in Section 5 4 1 System pumps on page 162 When loading sample using System pump A and an external a...

Page 181: ...o Section 5 9 Create a method and perform a run on page211 for more information on methods and phases Then When preparing to inject manually prime the sample inlet tubing with sample see Prime sample inlets on page 168 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 volu...

Page 182: ... phase of the method to be run refer to Sec tion 5 9 Create a method and perform a run on page211 for more information on methods and phases select Inject sample directly onto the column select Inject 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 ...

Page 183: ...p plug 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 Follow the instruction below to connect the Superloop...

Page 184: ...e 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 In the Process Picture 2 Click the Injection valve and select Manual Load Result The Injection valve switches to Manual Load position 184 ÄKTA pure User Manual 29119969 AB 5 Operation 5 7 ...

Page 185: ...r port with a Stop plug 8 Fill the Superloop using the Sample pump Follow the instruction below to fill the Superloop using the Sample pump NOTICE Glass tube splinter Make sure to set the sample pressure below the max pressure of the Superloop before executing a flow in the Manual instructions dialog when the Superloop is connected The Superloop can also be filled as part of a method run as set in...

Page 186: ...t a High alarm level that is below the maximum pressure of the Superloop Click In the Manual instructions dialog 4 Select Pumps and pressures Sample flow Set Flow rate to an appropriate value for the Superloop size in this example 10 ml min Click 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 C...

Page 187: ... the Sample Application phase in the Method Editor For multiple injections it may be more convenient to fill the Super loop once as described in the instruction below Tip Action Step In the Manual instructions dialog 1 Select Alarms Alarm system pressure Set Mode as Enabled Set a High alarm level that is below the maximum pressure of the Superloop Click In the Process Picture 2 Click the Injection...

Page 188: ...cture 5 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 In the Process Picture 6 Click on the buffer inlet valve A Select the inlet position to be used for sample injection for example A2 Result Inlet valve A switches to the selected port 188 ÄKTA pure User Manual 29119969 AB 5 Operation 5 7 Sample app...

Page 189: ...rate Result A system 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 8 Plug the Syr port on the Injection valve with a Stop plug 9 ÄKTA pure User Manual 29119969 AB 189 5 Operation 5 7 Sample application 5 7 2 Sample application using a Superloop ...

Page 190: ...be connected simultaneously Sample loop is called capillary loop in UNICORN Note How to fill a sample loop Follow the instructions below 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 3 190 ÄKTA pure User Manual 29119969 AB 5 Operati...

Page 191: ... 5 the Injection valve continue to step 7 the Loop valve continue to step 6 In the Process Picture 6 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 ÄKTA pure User Manual 29119969 AB 191 5 Operation 5 7 Sample application 5 7 3 Sample application using a sample loop ...

Page 192: ...ically injected onto the column The loop is emptied and washed out using buffer from the system pumps The total buffer volume to be used for 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 ...

Page 193: ...e and controlled by UNICORN Control of the fraction collector can be achieved automatically in a method run or manually In this section This section contains the following subsections See page Section 194 5 8 1 Prepare Fraction collector F9 C 200 5 8 2 Prepare Fraction collector F9 R 207 5 8 3 Fractionation overview ÄKTA pure User Manual 29119969 AB 193 5 Operation 5 8 Fractionation ...

Page 194: ...he height difference between the door and the floor of the fraction collector The tendency to tilt is affected by the placement and weight of the cassettes tubes or bottles Note Prepare the fraction collector Before starting to prepare Fraction collector F9 C check the fractionation settings in the method to be run Perform the steps described below according to the settings in the method Insert th...

Page 195: ...lease function open the cassettes For more information on the QuickRelease function see QuickRelease function on page 99 1 Place the tubes and deep well plates in the cassettes Make sure that the deep well plates are rotated so that the well marked A1 is positioned above the A1 marking on the cassette 2 ÄKTA pure User Manual 29119969 AB 195 5 Operation 5 8 Fractionation 5 8 1 Prepare Fraction coll...

Page 196: ...e Cassette tray Make sure that the cassette type code see illus tration below faces the front of the tray marked with the GE logo 4 Open the door of the fraction collector using the handle 5 196 ÄKTA pure User Manual 29119969 AB 5 Operation 5 8 Fractionation 5 8 1 Prepare Fraction collector F9 C ...

Page 197: ...r The tendency to tilt is affected by the placement and weight of the cassettes A height exclusion bar ensures that the tubes or deep well plates are correctly positioned and cannot damage the Dispenser head Close the door Make sure that it closes properly 7 Result After the door has been closed the fractionation arm scans the cassette type code of each cassette to identify the cassette types If d...

Page 198: ...h snaps into closed position as shown below Note The rack can tilt slightly when not fully inserted into the fraction collector and may harm the fractionation arm The tilt is due to the height difference between the door and the floor of the fraction collector The tendency to tilt is affected by the placement and weight of the tubes or bottles A height exclusion bar ensures that the tubes or bottl...

Page 199: ...t closes properly Note If the rack is inserted with the front of the rack facing the wrong way it will not be possible to close the door 4 ÄKTA pure User Manual 29119969 AB 199 5 Operation 5 8 Fractionation 5 8 1 Prepare Fraction collector F9 C ...

Page 200: ...ractionation 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 Assemble the Tube rack 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...

Page 201: ...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 85 180 mm tubes 50 85 mm tubes Inserts Not required L shaped cutout Tube support L shaped cutout Single cutout Tube guide Single cutout Single cutout Tube holder 30 m...

Page 202: ... inserts depending on the length of the collection tubes See Single and L shaped cutouts on page 201 for detailed information Insert the Tube guide 5 with the tube position numbers upwards The Tube guide should rest about 1 cm above the Tube support 2 Insert the Tube holder 6 with the tube position numbers upwards 3 Check that tube position 1 7 is directly above tube position 1 7 of the Tube guide...

Page 203: ...amage 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 Place the Tube rack 9 over the Central spindle 11 and pull the spring loaded Drive sleeve 12 out so the Tube rack comes to rest 6 9 10 12 11 ÄKTA pure User Manual 29119969 AB 203 5 Operation 5 8 Fractionation 5 8 2 Prepare Fraction collector F9 R ...

Page 204: ...hould be no spaces in the sequence Adjust the Delivery arm Follow the instructions in the table below to adjust the height of the Delivery arm Action Step Lift and then lower the Delivery arm 1 and allow it to move in so the Tube sensor 2 touches the collection tubes of the outer track 1 1 2 Loosen the lock knob 3 2 3 204 ÄKTA pure User Manual 29119969 AB 5 Operation 5 8 Fractionation 5 8 2 Prepar...

Page 205: ...k 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 NOTICE Never lift Fraction collector F9 R by the Delivery arm This may damage the Fraction collector ÄKTA pure User Manual 29119969 AB 205...

Page 206: ... 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 206 ÄKTA pure User Manual 29119969 AB 5 Operation 5 8 Fractionation 5 8 2 Prepare Fraction collector F9 R ...

Page 207: ...r information about different watch options Peak fractionation The two fractionation types listed above can be used in combination Combination of fixed volume and peak fractionation allows fractions col lected by fixed volume fractionation and fractions collected by peak frac tionation to be directed to different collection tubes Combined fixed vol ume fractionation and peak fractionation To be ab...

Page 208: ...ration below shows the fractions collected and the numerical marking of fractions when fixed volume straight fractionation is used with Fraction collector F9 R Volume Absorbance 2 3 4 6 7 8 9 5 10 11 12 13 14 15 16 17 18 19 Fractionation marks shown in chromatogram When fixed volume fractionation is used the delay volume is collected at the beginning of fractionation Fraction collector F9 R saves ...

Page 209: ...etween collection vessels during a run is avoided with all three fractionation modes Automatic The fraction collector uses the Drop Sync mode for flow rates up to 2 ml min and auto matically switches to Accumulator mode for higher flow rates Accumulator Liquid is collected during movement between tubes or wells The liquid is then dis pensed in the next well or tube Fractionation with accumulator c...

Page 210: ... 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 in the instruction Last tube filled in the system settings The flow can be directed to waste or to any of the outlets or the run can be paused If the action is set to Pause the system automa...

Page 211: ... on ÄKTA pure It also contains advice on things to be considered during a run In this section This section contains the following sub sections See page Section 212 5 9 1 Create a method 217 5 9 2 Prepare and perform a run 219 5 9 3 Monitor a run 221 5 9 4 After run procedures ÄKTA pure User Manual 29119969 AB 211 5 Operation 5 9 Create a method and perform a run ...

Page 212: ... and phases on page438 for more information about each method Purification methods Affinity Chromatography AC Affinity Chromatography AC with Tag Removal Anion Exchange Chromatography AIEX Cation Exchange Chromatography CIEX Chromatofocusing CF Desalting Gel filtration GF Hydrophobic Interaction Chromatography HIC Manual Loop Fill NHS coupling Reversed Phase Chromatography RPC Maintenance methods ...

Page 213: ...propriate phases 3 Save the method Create a new method Follow the instructions below to create a new method Action Step In the Method Editor 1 click the Create a new method icon in the Toolbar or select File New Method Result The New Method dialog opens ÄKTA pure User Manual 29119969 AB 213 5 Operation 5 9 Create a method and perform a run 5 9 1 Create a method ...

Page 214: ...most cases methods can be edited using the Phase Properties pane in the Method Editor module However modules without a recommended position are not supported by Phase Properties and have to be edited in the Text Instruction mode The following modules require that the method is created using the Text Instruction mode Inlet valve IX When using Inlet valve IX V9 IX or V9H IX connected to another inle...

Page 215: ...re methods are always created for the set of components of the current system Methods can be shared between systems with the restriction that a method created for an ÄKTA pure 150 configured system cannot be used with an ÄKTA pure 25 configured system and vice versa When a method is opened for a system that has been changed or if a method is saved for a system with a different flow configuration a...

Page 216: ...h are displayed after the method has been adapted Adapt method Change the settings in Phase Properties if required Note Some predefined methods require certain components to be functional Adapting these methods to systems that do not include the required components is not possible the text method will be kept and nothing will be changed Non compatible settings will still be present in the method b...

Page 217: ...Start Protocol dialog opens Step through the displayed pages in the Start Protocol add requested input and make appropriate changes 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 opens For further information on colu...

Page 218: ... instructions performed at the same breakpoint Note Manual runs are only stored temporarily However you can choose to store them perma nently 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 5 218 ÄKTA pur...

Page 219: ...g the Continue icon See the instructions in the table below 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 pumps click the button Note An ended method cannot be resumed resume for example a held or paused method run click the button permanently end the run When ending a...

Page 220: ... pressure in the system Narrow tubing after the column will increase the pressure in the column at a given flow rate Make sure that the sensor limits for the pre column pressure are set so that the maxi mum pressure for the column in use is not exceeded If Column valve V9 C or V9H C is mounted the integrated pressure sensor of the valve allows the system to monitor the post column pressure The lim...

Page 221: ...system 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 solution or distilled water using System CIP If Column valve V9 C or V9H C is mounted the integrated pressure sensor of the valve allows the system to monitor the post column pressure The limit for the pressure sensor in Column valve V9 C or V9H ...

Page 222: ...ing solution s using Column CIP Column storage If the column is not going to be used for a couple of days or longer also perform the following Fill the column with storage solution e g 20 ethanol using Column CIP pH electrode storage If pH monitoring will not be used for a week or longer perform one of the following actions Inject new storage solution into the pH flow cell Refer to Section 7 6 6 S...

Page 223: ...lose and the Log On dialog box opens on the File 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 ÄKTA pure User Manual 29119969 AB 223 5 Operation 5 9 Create a method and perform a ...

Page 224: ...down the instrument Switch off the instrument by pressing the power switch to the O position 224 ÄKTA pure User Manual 29119969 AB 5 Operation 5 9 Create a method and perform a run 5 9 4 After run procedures ...

Page 225: ...is chapter describes how to prepare run and evaluate the different perfor mance tests available In this chapter This chapter contains the following sections See page Section 226 6 1 General performance test actions 229 6 2 Air sensor A and Inlet valve A tests 231 6 3 Air sensor B and Inlet valve B tests 233 6 4 Air sensor S and Sample inlet valve tests 235 6 5 Column valve C tests 237 6 6 Fraction...

Page 226: ... V9 IB test Air sensor S and Sample inlet valve V9H IS test Air sensor S and Sample inlet valve V9 IS test Column valve V9H C test Column valve V9 C test Fraction Collector F9 R Test Fraction Collector F9 R Test Fraction Collector F9 C Test Fraction Collector F9 C Test Sample pump S9H test Sample pump S9 test System Test UV U9 L fixed System Test UV U9 L fixed System Test UV U9 M variable System T...

Page 227: ... report Evaluation Procedures Allows the user to change result name and result location Result Name and Location 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 Alternati...

Page 228: ...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 229: ... installed Inlet valve A is required to run the test Required material The following materials are required Syringe 25 30 ml Distilled water Prepare the test Follow the instructions below to prepare the system before method start Action Step Direct the outlet tubing marked W to a waste container 1 Place inlet tubing A1 into distilled water 2 Prime the A1 inlet and purge the pump See Section 5 4 1 ...

Page 230: ...cked and corrected repeat the test Faulty air sensor A test Action Cause For further information see Section 8 4 Troubleshooting Valves on page 375 Faulty air sensor Make sure that the tubing was correctly prepared see Prepare the test on page 229 Incorrect preparation of tubing Faulty inlet valve A test Action Cause For further information see Section 8 4 Troubleshooting Valves on page 375 The va...

Page 231: ...tly installed Inlet valve B is required to run the test Required material The following materials are required Syringe 25 30 ml Distilled water Prepare the test Follow the instructions below to prepare the system before method start Action Step Direct the outlet tubing marked W to a waste container 1 Place inlet tubing B1 into distilled water 2 Prime the B1 inlet and purge the pump See Section 5 4...

Page 232: ...cked and corrected repeat the test Faulty air sensor B test Action Cause For further information see Section 8 4 Troubleshooting Valves on page 375 Faulty air sensor Make sure that the tubing was correctly prepared see Prepare the test on page 231 Incorrect preparation of tubing Faulty inlet valve B test Action Cause For further information see Section 8 4 Troubleshooting Valves on page 375 The va...

Page 233: ...ed to run the test Required material The following materials are required Syringe 25 30 ml Distilled water Prepare the test Follow the instructions below to prepare the system before method start Action Step Open System Control and update the system setting Tubing Sample pump to injection valve found in System Settings Tubing and Delay volumes Note The system setting Tubing Sample pump to injectio...

Page 234: ...st The tables below describe possible causes of a failed test When possible sources of error have been checked and corrected repeat the test Faulty air sensor S test Action Cause For further information see Section 8 4 Troubleshooting Valves on page 375 Faulty air sensor Make sure that the tubing was correctly prepared see Prepare the test on page 231 Incorrect preparation of tubing Faulty Sample ...

Page 235: ...ectly installed Column valve C is required to run the test Required material The following materials are required Syringe 25 30 ml Distilled water Ref 1 tubing see Reference capillary on page 426 Prepare the test Follow the instructions below to prepare the system before method start Action Step Connect the Ref 1 tubing between Column valve C ports 1A and 1B 1 Make sure the Flow restrictor is inli...

Page 236: ...and corrected repeat the test Action Cause For further information see Section 8 4 Troubleshooting Valves on page 375 Faulty Column valve Calibrate the pressure monitors see Section 7 7 2 Calibrate the pressure monitors on page 305 The integrated pressure monitors are not calibrated Make sure that the reference tubing is connected between column ports 1A and 1B and that the flow restrictor is in l...

Page 237: ... Action Step Immerse waste tubing W in a waste container 1 Immerse inlet tubing A1 in distilled water 2 Prime inlet tubing A1 and purge System pump A See Section 5 4 1 System pumps on page 162 No air shall be left in the pump or tubing 3 Place two 96 deep well plates in positions 1 and 6 in the fraction collector Make sure that no other cassettes are present in the fraction collector during the ru...

Page 238: ...layed on the screen during the startup of the run Read the messages in the dialog and make sure that necessary preparations have been performed Click the Confirm and Continue button in the message dialog to proceed with the test Evaluate the result To evaluate the results do the following Check that correct volumes 2 ml well are collected in the three first wells in row A A1 A3 of both plates Chec...

Page 239: ...thod start see Section 5 4 1 System pumps on page 162 Incorrect volumes collected in the tubes and distur bances of system pressure curves Faulty pump See Section 8 6 Troubleshooting Pumps on page 387 Air trapped in System pump A Faulty System pump A Make sure that the system was correctly prepared see Prepare the test on page 237 Incorrect preparation of buffer and tubing ÄKTA pure User Manual 29...

Page 240: ...thod start Action Step Direct outlet tubing W to a waste container 1 Place inlet tubing A1 into distilled water 2 Prime inlet A1 and purge System pump A See Section 5 4 1 System pumps on page162 3 Place 7 tubes in the Fraction collector in positions 1 to 7 4 In the System Control module select System Settings 5 Result The System Settings dialog opens In the System Settings dialog 6 Select Fraction...

Page 241: ...checked and corrected repeat the test Action Cause Air in pumps Make sure to prime inlet tubing A1 and purge System pump A before method start see Section 5 4 1 System pumps on page 162 Incorrect volumes collected in the tubes and distur bances of system pressure curves Faulty pump See Section 8 6 Troubleshooting Pumps on page 387 Air trapped in System pump A Faulty System pump A Make sure the fra...

Page 242: ...n distilled water Syringe 25 30 ml Ref 1 tubing see Reference capillary on page 426 Prepare the test Follow the instructions below to prepare the system before method start Action Step Connect a tubing between Injection valve port column and the UV inlet preferably the Ref 1 tubing included with the system at delivery 1 Immerse waste tubing W W1 and W2 in a waste container 2 Immerse inlet tubing A...

Page 243: ...oting Pumps on page 387 Air in pump Make sure to prime inlet tubing Buffer and purge sample pump before method start see Section 5 4 2 Sample pump on page 168 Thedelivery of liquidis pulsat ing unstable pump pressure Faulty pump See Section 8 6 Troubleshooting Pumps on page 387 Folded twisted or blocked tubing Check the tubing The pump pressure is too high at a flow rate of 25 ml min or 150 ml min...

Page 244: ... 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 and 1 00 M NaCl in distilled water 3 Connect the Ref 1 tubing between Injection valve port Col and the UV monitor U9 L inlet to generate a back pressure 4 Make sure that the Mixer with a chamber volume of 1 ...

Page 245: ...eat the test Faulty Gradient Test Result Action Cause Make sure to prime the buffer inlets and to purge the System pumps before method start See Section 5 4 1 System pumps on page162 Disturbances caused by air trapped in any of the pumps Replace piston seals See Chapter 7 Maintenance on page 252 Disturbances caused by damaged pump piston seals See Section 8 3 Troubleshooting Monitors on page 360 U...

Page 246: ...has evaporated Incorrectly prepared acetone solution See Calibration of the UV monitor U9 L flow cell length on page312 Wrong UV cell path length set in UNICORN Faulty Pulsation Test Action Cause Make sure to prime and purge the system pumps before starting the test see Section 5 4 1 System pumps on page 162 Air trapped in the pumps Faulty Conductivity Test Result Action Cause See Conductivity mon...

Page 247: ...est 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 266 The inline filter is dirty Calibrate the pressure monitor see Section 7 7 2 Calibrate the pres sure monitors on page 305 The System pressure monitor is not calibrated ÄKTA pure User Manual 29119969 AB 247 6 Performance tests 6 9 System Test UV U9 L fi...

Page 248: ... 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 and 1 00 M NaCl in distilled water 3 Connect the Ref 1 tubing between Injection valve port Col and the UV monitor U9 M inlet to generate a back pressure 4 Make sure that the Mixer with a chamber volume of 1 ...

Page 249: ...t the test Faulty Gradient Test Result Action Cause Make sure to prime the buffer inlets and to purge the System pumps before method start See Section 5 4 1 System pumps on page162 Disturbances caused by air trapped in any of the pumps Replace piston seals See Chapter 7 Maintenance on page 252 Disturbances caused by damaged pump piston seals See Section 8 3 Troubleshooting Monitors on page 360 Uns...

Page 250: ... on page 252 Faulty values at 95 Damaged pump piston seal in System pump A Faulty UV Response Test Result Action Cause Restart the instrument to calibrate the UV monitor Faulty UV monitor See Section 8 3 Troubleshooting Monitors on page 360 Faulty UV Absorbance Test Action Cause Make sure that the acetone solution is 1 and that no solution has evaporated Incorrectly prepared acetone solution Fault...

Page 251: ...ptimal Faulty UV Noise Test Action Cause Flush or clean the UV cell see Section 7 5 1 Clean the UV flow cell on page 273 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 266 Dirt in inline filter Calibrate ...

Page 252: ... In this chapter This chapter contains the following sections See page Section 253 7 1 Maintenance Manager 258 7 2 Maintenance program 261 7 3 Weekly maintenance 269 7 4 Monthly maintenance 272 7 5 Semiannual maintenance 276 7 6 Maintenance when required 302 7 7 Calibration procedures 316 7 8 Replacement procedures 252 ÄKTA pure User Manual 29119969 AB 7 Maintenance ...

Page 253: ...odules are predefined The user can add automated maintenance notifications for the system Maintenance notifications are based on calender periods of system use and for some systems also on operational statistics for the modules Open Maintenance Manager In the System Control module on the System menu click Maintenance Manager to open the Mainte nance Manager dialog box ÄKTA pure User Manual 2911996...

Page 254: ...ected 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 For some systems it is possible to view maintenance notifications for a module of interest Select a notification to view notification details Modules with no plus symbol have no related maintenance notifications Note ...

Page 255: ...d 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 notifi cation 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 notif...

Page 256: ...d system notification select the notification in the Maintenance Manager dialog box and press the Delete button Module notifications are predefined and cannot be deleted If desired they can be disabled Note Handle a maintenance notification Each maintenance notification has a time interval after which the notification is issued When this time interval has been reached a Maintenance Notification di...

Page 257: ...eriorate Acknowledge close the dialog box without action Note The Maintenance Notificationwill be displayed each time the System Control module is opened until the notification is acknowledged Ignore The predefined maintenance notifcation periods use average values The actual service interval for a specific module may be shorter or longer Note ÄKTA pure User Manual 29119969 AB 257 7 Maintenance 7 ...

Page 258: ...eutral buffer and finally distilled water before service and maintenance CAUTION 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 CAUTION Always use appropriate personal protective equipment when decommissioning the equipment Periodic maintenance program The following pe...

Page 259: ...on page 284 Perform Column CIP Section 7 6 4 Clean Fraction collector F9 C on page 288 Clean Fraction collector F9 C Section 7 6 5 Clean Fraction collector F9 R on page 290 Clean Fraction collector F9 R Section 7 8 1 Replace tubing and connectors on page317 Replace tubing and connectors Section 7 6 6 Storage of the pH electrode on page 291 Storage of pH electrode Section 7 6 7 Clean the pH electro...

Page 260: ...he pump head Section 7 6 8 Clean the pump head check valves on page 296 Clean the check valves Section 7 8 9 Replace the pump head check valves on page 332 Replace check valves Section 7 8 10 Replace pump piston seals of Pump P9 or P9H on page 335 Replacepumppistonseals pumpP9 and P9H Section 7 8 11 Replace pump piston seals of Pump P9 S on page 344 Replace pump piston seals pump P9 S Section 7 8 ...

Page 261: ... activities In this section This section contains the following subsections See page Section 262 7 3 1 Change pump rinsing solution 266 7 3 2 Replace the inline filter 267 7 3 3 Clean the Fraction collector F9 C sensors ÄKTA pure User Manual 29119969 AB 261 7 Maintenance 7 3 Weekly maintenance ...

Page 262: ...ired 20 ethanol Syringe 25 30 ml Illustration of the system pump piston rinsing system The illustrations below show the parts and tubing of the system pump piston rinsing system 1 2 3 4 5 Description Part Rinsing system tube holder top 1 Rinsing system tube 2 Rinsing system tube holder bottom 3 Outlet tubing 4 Inlet tubing 5 262 ÄKTA pure User Manual 29119969 AB 7 Maintenance 7 3 Weekly maintenanc...

Page 263: ... System pump piston rinsing system into the fluid in the rinsing solution tube Note Make sure that the inlet tubing reaches close to the bottom of the rinsing solution tube 4 Connect a 25 to 30 ml syringe to the outlet tubing of the System pump piston rinsing system Draw liquid slowly into the syringe 5 Disconnect the syringe and discard its contents 6 Insert the outlet tubing into the fluid in th...

Page 264: ...nsing liquid tube holder 2 Inlet tubing from the rinsing liquid tube to the left pump head lower rinsing system connection 3 Tubing between the left pump head upper rinsing system connection and the right pump head lower rinsing system connection 4 Outlet tubing from the right pump head upper rinsing system connection to the rinsing liquid tube 5 264 ÄKTA pure User Manual 29119969 AB 7 Maintenance...

Page 265: ...on rinsing system into the fluid in the rinsing liquid tube Note Make sure that the inlet tubing reaches close to the bottom of the rinsing liquid tube 4 Connect a 25 to 30 ml syringe to the outlet tubing of the System pump piston rinsing system Draw liquid slowly into the syringe Note The outlet tubing is colored orange for clarity in the above illustration 5 Disconnect the syringe and discard it...

Page 266: ...the Mixer Use forceps and gloves during the replacement procedure to avoid contaminating 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 damaged Fit the new filter 2 Check the O ring of the Mixer If the O ring is damaged replace it according to Section 7 8 4 Replace the O ring inside the Mixer on pag...

Page 267: ...w the instructions below to clean the fraction collector diodes See Section 3 2 2 Fraction collector F9 C illustrations on page 93 for the locations of the components of the fraction collector Action Step In System Control select Manual Execute Manual Instructions Fraction collection Frac cleaning position Click Execute 1 Result The Dispenser head moves to cleaning position and the Instrument disp...

Page 268: ...mpletely before starting a run 3 Close the fraction collector door 4 Result Automatic scanning is performed In the System Control module press the End icon in the toolbar 5 Result The Dispenser head moves to home position and the Instrument display states Ready 268 ÄKTA pure User Manual 29119969 AB 7 Maintenance 7 3 Weekly maintenance 7 3 3 Clean the Fraction collector F9 C sensors ...

Page 269: ...let tubing marked A1 in distilled water and insert the piece of tubing from Outlet valve port W into a waste container 1 In the System Control module select Manual Execute Manual Instructions 2 Result The Manual instructions dialog opens In the Manual instructions dialog 3 Select Flowpath Injection valve and select Manual Load Click Insert Select Flowpath Column valve and select By pass Click Inse...

Page 270: ...and insert the piece of tubing from Outlet valve port W into a waste container 1 In the System Control module select Manual Execute Manual Instructions 2 Result The Manual instructions dialog opens In the Manual instructions dialog 3 Select Flowpath Injection valve and then select Manual Load Click Insert Select Flowpath Column valve and then select By pass Click Insert Select Flowpath pH valve an...

Page 271: ...isplayed in the Run Data pane 6 Calculate the difference between the two pressure values noted in step 4 and step 6 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 7 Replace Flow restrictor on page 330 ÄKTA pure User Manual 29119969 AB 271 7 Maintenance 7 4 Monthly maintenance ...

Page 272: ...tions for semiannual maintenance activities In this section This section contains the following subsections See page Section 273 7 5 1 Clean the UV flow cell 275 7 5 2 Replace the pH electrode 272 ÄKTA pure User Manual 29119969 AB 7 Maintenance 7 5 Semiannual maintenance ...

Page 273: ... Instruction Follow the instructions below to clean the UV flow cell for UV monitor U9 M or UV monitor U9 L The UV flow cell can be mounted or not mounted on the instrument during the cleaning procedure The illustrations in the instructions below show UV monitor U9 M UV monitor U9 L is cleaned in a corresponding way Action Step Disconnect the tubing from the top of the UV flow cell and replace the...

Page 274: ... the flow cell for at least 20 minutes 7 Inject the detergent solution remaining in the syringe into the flow cell Disconnect the syringe 8 Fill a syringe with distilled water Connect the syringe to the Luer 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...

Page 275: ...trode 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 the ...

Page 276: ...ent externally 278 7 6 2 Perform System CIP 284 7 6 3 Perform Column CIP 288 7 6 4 Clean Fraction collector F9 C 290 7 6 5 Clean Fraction collector F9 R 291 7 6 6 Storage of the pH electrode 293 7 6 7 Clean the pH electrode 296 7 6 8 Clean the pump head check valves 299 7 6 9 Wipe off excess oil from the pump head 300 7 6 10 Clean the Conductivity flow cell 276 ÄKTA pure User Manual 29119969 AB 7 ...

Page 277: ...0 ethanol Instruction Follow the instructions below 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 ÄKTA pure User Manual 29119969 AB 277 7 Maintenance 7 6 Maintenance whe...

Page 278: ...NaOH followed by a neutral buffer and finally distilled water before service and maintenance CAUTION Hazardous substances When using hazardous chemical and biological agents take all suitable protective measures such as wearing protective glasses and gloves resistant to the substances used Follow local and or national regulations for safe operation maintenance and decommissioning of the equipment ...

Page 279: ...thod dialog select System and then System CIP in the Predefined Method drop down list Click OK 2 Result One Method Settings phase and three System CIP phases show in the Method Outline pane Each System CIP phase uses one cleaning solution If desired add additional System CIP phases to the method using the Phase Library 3 ÄKTA pure User Manual 29119969 AB 279 7 Maintenance 7 6 Maintenance when requ...

Page 280: ...ean the manual injection port of the Injection valve on page 282 Note The pH electrode and the pH valve are not included in the system CIP Refer to Sec tion 7 6 7 Clean the pH electrode on page 293 for instructions on how to clean the pH electrode If the fraction collector is included a lower flow rate might need to be set in the instruc tion Fraction collector wash settings Make sure not to excee...

Page 281: ...ding to the instructions above 1 Connect bypass 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 solutions 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 method an...

Page 282: ...p 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 Fill a syringe with approximately 10 ml of an appropriate cleaning solution e g NaOH or buffer solution Connect the syringe to Injection valve port Syr and inject the cleaning solution 6 Fill a ...

Page 283: ...he Calibration dialog select pH from the Monitor to calibrate drop down list 4 Press the Prepare for calibration button 5 Result The pH valve switches to the calibration position Fill a syringe with approximately 10 ml of 1 M NaOH Connect the syringe to the pH valve port Cal and inject the solution 7 Fill a syringe with distilled water Connect the syringe to the pH valve port Cal and inject the di...

Page 284: ...zardous biological agents run System CIP and Column CIP to flush the entire system tubing with bacteriostatic solution e g NaOH followed by a neutral buffer and finally distilled water before service and maintenance CAUTION Hazardous substances When using hazardous chemical and biological agents take all suitable protective measures such as wearing protective glasses and gloves resistant to the su...

Page 285: ...g opens In the New Method dialog select System and then Predefined Method and Column CIP Click OK 2 Result One Method Settings phase and one Column CIP phase will be displayed in the Method Outline pane In the Phase Properties tab of the Method Settings phase select Column type and Col umn position 3 ÄKTA pure User Manual 29119969 AB 285 7 Maintenance 7 6 Maintenance when required 7 6 3 Perform Co...

Page 286: ...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 6 286 ÄKTA pu...

Page 287: ...in the selected folder Perform a Column CIP 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 instruction above 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 start...

Page 288: ... collector wash 1 In System Control select Manual Execute Manual Instructions Fractioncollection Frac tion collector wash Click Execute NOTICE If no column valve is used make sure to replace any columns in the flow path with tubing before a fraction collector wash is per formed Lower the flow for the fraction collector wash in the instruction Fraction collector wash settings if the system back pre...

Page 289: ...l 6 Wipe off the Dispenser head and its diode windows the Drop sync sensor and the Type code reader using a wash bottle with water or 20 ethanol and a cloth 7 Let the fraction collector dry completely before starting a run 8 Close the door of the fraction collector 9 Result Automatic scanning is performed In the System Control module press the End icon in the toolbar 10 Result The Dispenser head m...

Page 290: ...on 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 page104 with a damp cloth 290 ÄKTA ...

Page 291: ...ollow 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 performed Action Step In the System Control module on the System menu click Calibrate 1 Result The Calibration dialog opens In the Calibration dialog click pH on the Monitor to calibrate drop down list 2 Click Prepare for Calc...

Page 292: ...e 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 292 ÄKTA pure User Manual 29119969 AB 7 Maintenance 7 6 Maintenance when required 7 6 6 Storage of the pH electrode ...

Page 293: ...n 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 following solu tions 0 1 M HCl 0 1 M NaOH 0 1 M HCl Rinse the electrode tip in distilled water between each solution Oil or grease films Wash the electrode tip in liquid detergent and ...

Page 294: ... 2 Replace the pH electrode on page 275 Clean a pH electrode installed in the pH valve Follow the instructions below to clean a pH electrode installed in the pH valve The calibration function is used to switch the position of the pH valve However no calibration is performed Action Step Open the System Control module and select System Calibrate 1 Result The Calibration dialog opens Set Monitor to c...

Page 295: ...ed 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 Press the Close button 7 Result The pH valve switches back to the default position and the Calibration dialog closes No calibration is performed ÄKTA pure User Manual 29119969 AB 295 7 Mainte...

Page 296: ...ench Methanol Distilled water Ultrasonic bath Instruction Follow the instructions below to remove and clean the pump head check valves Action Step Switch off the instrument 1 Disconnect the tubing from the pump head and disconnect the pump inlet tubing Dis connect the tubing of the pump rinsing system 2 Unscrew the purge valve by turning it counter clockwise and lift off the metal ring 3 296 ÄKTA ...

Page 297: ...valve 4 Unscrew the two white plastic screws located below each pump head Pull the plastic connectors to the sides to release the inlet manifold 5 Unscrew the lower check valve using an adjustable wrench 6 ÄKTA pure User Manual 29119969 AB 297 7 Maintenance 7 6 Maintenance when required 7 6 8 Clean the pump head check valves ...

Page 298: ...sioning of the equipment 7 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 8 Tighten the nut until fully finger tight and then use the adjustable wrench to tighten a further 90 degrees 9 Refit the inlet manifold and reconnect the tubing to the pump head 10 298 ÄKTA pure User Manu...

Page 299: ...th 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 pure User Manual 29119969...

Page 300: ... flow cell of the Conductivity monitor Action Step Disconnect the fingertight connector and the piece of tubing from the top of the Conduc tivity 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 2 300 ÄKTA pure User Manual 29119...

Page 301: ...cell about five times 6 Leave the liquid in the flow cell for at least 15 minutes 7 Fill a syringe with distilled water Connect the syringe to the Luer connector 8 Inject the distilled water into the Conductivity flow cell to rinse the flow cell Disconnect the syringe 9 Disconnect the Luer connector from the top of the Conductivity flow cell and reconnect the fingertight connector with tubing 10 Ä...

Page 302: ...n contains the following subsections See page Section 303 7 7 1 Calibrate the pH monitor 305 7 7 2 Calibrate the pressure monitors 308 7 7 3 Calibrate the Conductivity monitor 312 7 7 4 Calibrate the UV monitors Menu command in UNICORN Open the System Control module and select System Calibrate to open the Calibration dialog 302 ÄKTA pure User Manual 29119969 AB 7 Maintenance 7 7 Calibration proced...

Page 303: ...e 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 below to calibrate the pH monitor Action Step In the Calibration dialog select pH from the Monitor to calibrate drop down list 1 Click the Prepare for calibration button 2 Result...

Page 304: ...g the second pH standard buffer 8 Result The calibration date and time are displayed in the dialog along with 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 9 If Yes Click the Close button to switch the pH valve back to the default position and to close the Calibration dialog I...

Page 305: ... relevant pressure to display see table Tubing and pressures on page 306 Click OK to close the Customize dialog 2 In the Run Data pane in the System Control module check what pressure is displayed 3 If the zero pressure reading is outside the range 0 02 MPa calibrate the pressure monitor according to the instruction below 4 Calibrate the monitors Follow the instructions below to calibrate any of t...

Page 306: ...Tubing to disconnect Pressure monitor System pressure Tubing from the System pump pressure monitor Systempumppressure monitor Sample pressure Tubing from the Sample pump pressure monitor Samplepumppressure monitor PreC pressure Tubing to Column valve V9 C or V9H C port In Pre column pressure monitor 306 ÄKTA pure User Manual 29119969 AB 7 Maintenance 7 7 Calibration procedures 7 7 2 Calibrate the ...

Page 307: ...isconnect Pressure monitor PostC pressure Tubing to Column valve V9 C or V9H C port Out Post column pressure monitor ÄKTA pure User Manual 29119969 AB 307 7 Maintenance 7 7 Calibration procedures 7 7 2 Calibrate the pressure monitors ...

Page 308: ... after cleaning Conductivity monitor factory calibration Follow the instruction below to restore the conductivity cell constant to the factory default value Action Step In the Calibration dialog select Conductivity monitor factory calibration from the Monitor to calibrate drop down list 1 Result The time for the new calibration and the current value are displayed Click Restore 2 Result The conduct...

Page 309: ...ne 5 Prime and purge the inlet used in step 4 with the 1 00 M NaCl calibration solution Fill the conductivity cell with the calibration solution at 1 ml min Pump in at least 15 ml of the calibration solution and wait until the conductivity signal and the temperature have stabilized before continuing the calibration See Section 5 4 1 System pumps on page162 6 Under continuous flow read the current ...

Page 310: ...lution is used see the graph for conductivity value at the current temperature on page 311 In the Calibration dialog click Calibrate 11 Result The new conductivity cell constant is displayed in the Conductivity cell 1 con stant 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 Control toolbar click the End icon to end the r...

Page 311: ...rrent 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 ÄKTA pure User Manual 29119969 AB 311 7 Maintenance 7 7 Calibration procedures 7 7 3 Calibrate the Conductivity monitor ...

Page 312: ...th must be registered or updated in UNICORN when the flow cell is re placed Note Equipment needed To perform the calibration a calibration kit containing test solutions syringes and accessories is needed A specified kit is available for each cell length Then use calibration kit If using a UV flow cell with the theoretical path length UV 900 2 mm calibration kit Product Code 18 6324 02 2 mm UV 900 ...

Page 313: ... Pumps System flow and set the Flow rate to 0 0 ml min Click Execute Result The absorbance can now be monitored Fill one of the supplied syringes with 1 5 2 ml of the first solution 0 mg l Ensure that there are no air bubbles in the syringe 2 Fit the syringe in the union Luer connector and inject the solution DO NOT remove the syringe 3 Note Air trapped in the UV cell causes inaccurate measurement...

Page 314: ...lues have been entered into the table the real UV flow cell path length is shown at the bottom of the table Note The regression coefficient R2 should be larger than 0 999 If this is not the case one or more measured values are faulty 10 Update the cell path length Follow the instructions below to define the UV cell path length or the UV 2nd cell path length The flow cell path length should be upda...

Page 315: ...e in the Real length input field and click Set If no calibration has been performed enter the nominal flow cell path length in the Real length input field and click Set Result The UV flow cell path length is updated 4 ÄKTA pure User Manual 29119969 AB 315 7 Maintenance 7 7 Calibration procedures 7 7 4 Calibrate the UV monitors ...

Page 316: ...nd connectors 319 7 8 2 Replace internal tubing in Fraction collector F9 C 322 7 8 3 Replace the Mixer 323 7 8 4 Replace the O ring inside the Mixer 325 7 8 5 Replace the UV monitor U9 M flow cell 328 7 8 6 Replace the UV monitor U9 L flow cell 330 7 8 7 Replace Flow restrictor 331 7 8 8 Replace the inlet filters 332 7 8 9 Replace the pump head check valves 335 7 8 10 Replace pump piston seals of ...

Page 317: ...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 ÄKTA pure User Manual 29119969 AB 317 7 Maintenance 7 8...

Page 318: ...bels 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 th...

Page 319: ...place tubing and connectors See Section 3 2 2 Fraction collector F9 C illustrations on page 93 for the locations of the components of the fraction collector Action Step Switch off the power to the fraction collector by switching off power to the ÄKTA pure instrument 1 Remove the connector for the internal tubing that is attached to the inlet port 2 Unscrew the connector from the inlet port Remove ...

Page 320: ...k Cut the new tubing to the same length as the old tubing Use a tubing cutter to get a straight angle cut 4 CAUTION Cut injuries The tubing cutter is very sharp and must be handled with care to avoid injuries Note Use tubing with the original inner diameter and length to ensure that the correct delay volume is maintained 320 ÄKTA pure User Manual 29119969 AB 7 Maintenance 7 8 Replacement procedure...

Page 321: ...ractionation chamber 6 Position the new tubing 7 Gently move the fractionation arm forward while holding the tubing Thread the tubing through the second tubing guide Attach the new tubing to the dispenser head 8 Slide the fingertight connector onto 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 Tighten the connector ...

Page 322: ...ired Mixer Instruction Follow the instruction below to change the Mixer Action Step Disconnect the tubing from the top and bottom of the Mixer 1 Pull the Mixer away from the instrument 2 Attach the new Mixer 3 Reconnect the tubing to the top and bottom of the new Mixer 4 322 ÄKTA pure User Manual 29119969 AB 7 Maintenance 7 8 Replacement procedures 7 8 3 Replace the Mixer ...

Page 323: ...c acid or formic acid for longer periods of time O ring 22 1 1 6 mm for Mixer chamber 15 ml Instruction Follow the instruction below to replace the O ring inside the Mixer Use a forceps and gloves during the replacement procedure to avoid contaminating 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 hal...

Page 324: ...hanol 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 6 324 ÄKTA pure User Manual 29119969 AB 7 Maintenance 7 8 Replacement procedures 7 8 4 Replace the O ring inside the Mixer ...

Page 325: ...azardous chemicals or biological agents in UV flow cell Make sure that the entire flow cell has been flushed thoroughly with bacteriostatic solution e g NaOH and distilled water 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 Push the latch on the UV detector to disconnect the de...

Page 326: ... you accidentally touch the optical fiber connectors clean them according to Clean the optical fiber connectors on page 327 Pull off the UV flow cell from the detector 5 Pull off the black protective caps from the new UV flow cell and connect the new UV flow cell to the detector 6 Connect the detector with the new flow cell connected to the monochromator Pull the latch upwards to fasten the detect...

Page 327: ...ctive clothing glasses and gloves resistant to the substances used Follow local and or national regulations for safe operation and maintenance of the product Action Step Wipe the optical fiber connectors with isopropanol on lens paper 1 Wipe the optical fiber connectors dry with lens paper 2 ÄKTA pure User Manual 29119969 AB 327 7 Maintenance 7 8 Replacement procedures 7 8 5 Replace the UV monitor...

Page 328: ...he entire flow cell has been flushed thoroughly with bacteriostatic solution e g NaOH and distilled water before service and maintenance Follow the instruction below to replace the UV flow cell Action Step Switch off the instrument 1 Disconnect the tubing from the UV flow cell 2 Unscrew the knurled wheel at the bottom of the UV monitor 1 Press the wheel upwards to release the flow cell 2 3 2 1 328...

Page 329: ...the instrument and log on to UNICORN 8 Update the UV flow cell path length in the Calibrate dialog in System Control Note The flow path cell length can be updated with the nominal value see Update the cell path length on page 314 to set the nominal flow cell path length or calibrated and updated with the calculated value see Calibration of the UV monitor U9 L flow cell length on page 312 to calibr...

Page 330: ... tubing 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 restrictor connector marked IN is connected to the pH valve port ToR To Restrictor 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 331: ...quired 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 ÄKTA pure User Manual 29119969 AB 331 7 Maintenance 7 8 Replacement procedures 7 8 8 Replace the inlet filters ...

Page 332: ...e 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 Unscrew the purge valve by turning it counter clockwise and lift off the metal ring 2 332 ÄKTA...

Page 333: ... 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 Unscrew the two white plastic screws located below each pump head Pull the plastic connectors to the sides to release the inlet manifold 7 Unscrew the lower check valve using an adjustable wrench 8 ÄKTA pure User Manual 29119969 AB 333 7 Maintenance 7 8 Replacement pro...

Page 334: ...l 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 11 334 ÄKTA pure User Manual 29119969 AB 7 Maintenance 7 8 Replacement procedures 7 8 9 Replace the pump head check valves ...

Page 335: ... ÄKTA pure 150 P9H P9H S Sample pump S9H ÄKTA pure 150 NOTICE Advanced maintenance Read the instruction carefully before disassembly of the pump head Required material The following materials are required Adjustable wrench For Pump P9 Star screwdriver T20 For Pump P9H Star screwdriver T10 and T20 Ultrasonic bath Ethanol 20 For Pump P9 Tubing giving a back pressure of 6 to 8 MPa 60 to 80 bar For Pu...

Page 336: ...ts of the pump heads of the pumps P9 and P9H Pump P9 1 2 3 4 5 6 7 12 9 8 10 11 13 Pump P9H 1 2 3 4 5 6 7 12 9 8 10 11 13 336 ÄKTA pure User Manual 29119969 AB 7 Maintenance 7 8 Replacement procedures 7 8 10 Replace pump piston seals of Pump P9 or P9H ...

Page 337: ...seals and pump membrane housing of both pump heads of a pump at the same time Note Disassemble the pump head Action Step Make sure that no run is in progress on the instrument 1 Disconnect the tubing from the pump head and disconnect the pump inlet tubing 2 Unscrew the two white plastic screws located below each pump head by hand Pull the plastic connectors to the sides to release the inlet manifo...

Page 338: ...sher using a star screwdriver T20 For Pump P9H un screw the four screws of the support washer using a star screwdriver T10 Discard the O ring 1 on the support washer and the discard the piston seal 2 located in the front section of the pump head Note Be careful not to scratch the metal surfaces 6 Pump P9H 2 1 Pump P9 2 1 338 ÄKTA pure User Manual 29119969 AB 7 Maintenance 7 8 Replacement procedure...

Page 339: ...n spring 8 Inspect the piston and return spring for signs of damage If damaged discard the piston and return spring and use a new piston and return spring when assembling the pump head 9 Clean the pump head and pump membrane housing in an ultrasonic bath If there are particles on any surfaces the check valves should be removed and cleaned separately see Section 7 6 8 Clean the pump head check valv...

Page 340: ...ge valve back into the pump head 1 Note Always use Lubricant 56686700 when exchanging the O ring 3 x 1 mm Wet a new seal with 20 ethanol Place the new seal in the hole in the front section of the pump head and press it into position 2 Pump P9H Pump P9 340 ÄKTA pure User Manual 29119969 AB 7 Maintenance 7 8 Replacement procedures 7 8 10 Replace pump piston seals of Pump P9 or P9H ...

Page 341: ...screws of the support washer Make sure to tighten the screws fully 3 Pump P9H Pump P9 Wet a new O ring 21 4 x 1 6 mm with 20 ethanol Fit the O ring around the support washer 4 Pump P9H Pump P9 ÄKTA pure User Manual 29119969 AB 341 7 Maintenance 7 8 Replacement procedures 7 8 10 Replace pump piston seals of Pump P9 or P9H ...

Page 342: ...fold 7 Reconnect the tubing to the pump head and reconnect the pump inlet tubing 8 Break in the new pump piston seal Follow the instruction below to run in the new pump piston seal of pumps P9 and P9H Action Step Fill a buffer vessel with 20 ethanol in water Immerse the inlet tubing for example A1 for System pump A or B1 for System pump B in the buffer vessel Place the buffer vessel on the Buffer ...

Page 343: ...low rate to 25 0 ml min Click Insert Click Execute Result A system flow starts For a sample pump in the Manual instructions dialog Select Flow path Column valve and select the position of the tubing connected to the Column valve in this example Position 1 Click Insert Select Flow path Sample inlet and select a Position in this example S1 Click Insert Select Flow path Injection valve and select Dir...

Page 344: ...ump type Label Configuration P9 S P9 S Sample pump S9 ÄKTA pure 150 NOTICE Advanced maintenance Read the instruction carefully before disassembly of the pump head Required material The following materials are required Adjustable wrench Star screwdriver T20 Cross headed screwdriver Hex wrench Ultrasonic bath Ethanol 20 Reference capillary Ref 1 P9 S Seal kit 65 ml 344 ÄKTA pure User Manual 29119969...

Page 345: ...ollow the instructions below to replace the o ring piston seal and rinse membrane of pump P9 S Always replace the o rings piston seals and rinse membranes of both pump heads of a pump at the same time Note Disassemble the pump head Action Step Make sure that no run is in progress on the instrument 1 Disconnect the tubing from the pump head and disconnect the pump inlet tubing 2 ÄKTA pure User Manu...

Page 346: ... of the two screws of the pump head using a hex wrench Unscrew the second screw and at the same time push firmly on the front of the rinse chamber to compensate for the pressure of the piston return spring 5 Place the pump head face down on the bench Pull out the piston together with the return spring 6 346 ÄKTA pure User Manual 29119969 AB 7 Maintenance 7 8 Replacement procedures 7 8 11 Replace p...

Page 347: ...rane located between the drain plate and the rinse chamber 8 Lift off the rinse chamber Gently pull off the piston seal Discard the used seal 9 Clean the pump head rinse chamber and drain plate in an ultrasonic bath If there are particles on any surfaces the check valves should be removed and cleaned separately see Section 7 6 8 Clean the pump head check valves on page 296 10 ÄKTA pure User Manual...

Page 348: ... hole in the front section of the pump head and press it into position 2 With the pump head facing downwards on the bench place the rinse chamber onto the front section of the pump head with the rinse ports in line with the check valves The conical depression in the rinse chamber shall be facing upwards Wet a new membrane with 20 ethanol and place the membrane into the rinse chamber with the conic...

Page 349: ...using a hex wrench Screw the second screw of the pump head Make sure to tighten both screws fully 3 Reconnect the tubing of the pump piston rinsing system 4 Reconnect the inlet manifold 5 Reconnect the tubing to the pump head and reconnect the pump inlet tubing 6 Break in the new pump piston seal see instruction below 7 Break in the new pump piston seal Follow the instruction below to break in the...

Page 350: ...is example S1 Click Insert Select Flow path Injection valve and select Direct inject from the Position drop down list Click Insert Select Pumps Sample flow and set the Flow rate to 25 0 ml min Click Insert Click Execute Result A sample flow of 25 ml min starts Run the flow for 2 hours 6 Discard the used buffer 7 350 ÄKTA pure User Manual 29119969 AB 7 Maintenance 7 8 Replacement procedures 7 8 11 ...

Page 351: ...and should also be replaced To replace the piston and the seal of a system pump see Section 7 8 10 Replace pump piston seals of Pump P9 or P9H on page 335 Replace pump pistons of Pump P9 S If a damaged piston has been in operation the piston seal will be destroyed and should also be replaced To replace the piston and the seal of Pump P9 S see Section 7 8 11 Replace pump piston seals of Pump P9 S o...

Page 352: ...uired material The following material is required Rinsing system tubing System pump piston rinsing system The illustrations below show the parts and tubing of the system pump piston rinsing system 1 2 3 4 5 Description Part Rinsing system tube holder top 1 Rinsing system tube 2 Rinsing system tube holder bottom 3 Outlet tubing 4 Inlet tubing 5 352 ÄKTA pure User Manual 29119969 AB 7 Maintenance 7 ...

Page 353: ...between the left pump head upper rinsing system connection and the right pump head lower rinsing system connection 4 Outlet tubing from the right pump head upper rinsing system connection to the rinsing liquid tube 5 Connect new tubing Action Step Disconnect the used tubing 1 Cut the new tubing to desired length 2 Connect the new tubing according to the illustrations above 3 Fit all pieces of tubi...

Page 354: ...me the pump rinsing system tubing Refer to Section 7 3 1 Change pump rinsing solution on page 262 for detailed instructions 354 ÄKTA pure User Manual 29119969 AB 7 Maintenance 7 8 Replacement procedures 7 8 13 Replace pump rinsing system tubing ...

Page 355: ...on to troubleshooting 358 8 2 Troubleshooting General Checklist 360 8 3 Troubleshooting Monitors 375 8 4 Troubleshooting Valves 377 8 5 Troubleshooting Fraction collector 387 8 6 Troubleshooting Pumps 394 8 7 Troubleshooting Other components 401 8 8 Troubleshooting Instrument communication 403 8 9 Troubleshooting Method development 404 8 10 Error codes ÄKTA pure User Manual 29119969 AB 355 8 Troub...

Page 356: ... operating system that includes the font UNICORN Method Manual UNICORN Evaluation Manual and UNICORN Administration and Technical Manual Troubleshooting procedure To troubleshoot ÄKTA pure Action Step Always start by checking the General checklist See Section 8 2 Troubleshooting General Checklist on page 358 1 In this document search for solutions in the section corresponding to the problem 2 Make...

Page 357: ...em 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 UNICORN 2 E mail the report to GE Service department 3 ÄKTA pure User Manual 29119969 AB 357 8 Troubleshooting 8 1 Introduction to troubleshooting...

Page 358: ...ment modules To check and change Node IDs see Node ID on page 77 Flow path checks Is all tubing connected correctly See Section 9 3 Tubing and connectors on page420 and Section 5 2 Prepare the flow path on page 151 Is there leakage at any of the connections Is any tubing folded or twisted Is the inlet tubing correctly immersed in the buffer solution beneath the liquid surface but not too close to ...

Page 359: ...and or filtration prior to sample loading Are the correct buffers used for the chosen columns and proteins Check buffers for precipitations Adjust to room temperature Are the chosen columns suitable for the chosen target proteins Do the buffers have correct pH The pH of some buffers changes with the temperature Are the UV wavelengths used by the method appropriate with respect to used buffers and ...

Page 360: ...is not found by the instrument The detector is not correctly fitted Contact Service Unclean optical fiber connectors Clean the connectors See Section 7 8 5 Replace the UV monitor U9 M flow cell on page 325 Dirt on optical sensors in detector Remove visible dirt on detector photo diodes Use lint free lens paper winded around a thin wood splinter e g a match or toothpick Clean the sensors with Isopr...

Page 361: ...side of the instrument is covered Make sure that none of the air intakes on the instrument are covered Hot surroundings Decrease the room temperature Maximum operating tempera ture is 35 C Hardware error Switch off the instrument and wait until the temperature has decreased Restart the instrument If this error is recurrent gen erate a System error report and contact Service The internal temperatur...

Page 362: ...ater or buffer If persis tent clean the UV cell See Section 7 5 1 Clean the UV flow cell on page 273 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 273 Dirt in the flow path Clean the system in accordance to Section 7 6 2 Perform System CIP on page278 Clean the column in accordance to Section 7 6 3 Perform Column CIP o...

Page 363: ... 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 5 Replace the UV monitor U9 M flow cell on page 325 Dirt in the UV flow cell Perform a System CIP See Section 7 6 2 Perform System CIP on page 278 If necessary manually clea...

Page 364: ... 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 absorption 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 gradi ent that is inverted com paredtotheexpectedgradi ent UV monitor U9 L...

Page 365: ...rrent 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 Section 7 8 6 Replace the UV monitor U9 L flow cell on page 328 Dirt in the UV flow cell Clean the UV flow cell see Section 7 5 1 Clean the UV flow cell on page 273 Broken UV flow cell Replace the cell see Section 7 8 6 Replace the UV monito...

Page 366: ...tent clean the UV cell See Section 7 5 1 Clean the UV flow cell on page 273 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 273 Dirt in the flow path Clean the system in accordance to Section 7 6 2 Perform System CIP on page278 Clean the column in accordance to Section 7 6 3 Perform Column CIP on page 284 Ghost peaks Co...

Page 367: ...25 mm 2 µl i d 0 50 mm 8 µl i d 0 75 mm 18 µl i d 1 0 mm 32 µl Distorted protein peaks in IEX gradients for example step gradients Conductivity monitor Possible cause and action Problem Test was performed at cold room temperature and the conductiv ity temperature compensation factor was not optimized The conductivity part of the system performance test failed In the System Settings dialog select C...

Page 368: ... to the Mixer chamber during run The magnet should vibrate Check that the Mixer chamber is free from solids To replace the inline filter see Section 7 3 2 Replace the inline filter on page266 Baseline drift of noisy signal Unclean conductivity flow cell Clean the Conductivity cell see Section 7 6 10 Clean the Conduc tivity flow cell on page 300 The ambient temperature may have decreased increased ...

Page 369: ...ace the inline filter on page 266 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 t...

Page 370: ...uring 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 filter on page 266 Incorrect or unstable read ing pH monitor and pH valve Possible cause and action Problem The cable between the pH valve and the ICU is not connected Remove the pH valve and make sure that the cable is connected See Hardware installation of a...

Page 371: ...electrode on page 275 Wrong mixer size for the used flow rate Use the recommended mixer size for the used flow rate See Unstable pH signal Decreasing salt concentration in the electrode membrane due to osmosis to buffer Regenerate the pH electrode Place the electrode in deionized water for 30 minutes followed by 30 minutes in a buffer with pH 4 Drift of pH signal when the pH electrode has been re ...

Page 372: ...Mixer Replace the inline filter in the Mixer See Section 7 3 2 Replace the inline filter on page 266 Solids in the flow path To use the predefined method System CIP to clean the flow path see Section 7 6 2 Perform System CIP on page 278 To clean the column see Section 7 6 3 Perform Column CIP on page 284 If persistent replace the column 0 25 mm i d tubing kit is mounted A smaller tubing inner diam...

Page 373: ...t surroundings Decrease the room temperature Maximum operating tempera ture is 35 C Hardware error Switch off the instrument and wait until the temperature has decreased Restart the instrument If this error is recurrent gen erate a System error report and contact Service The internal temperature of the pressure monitor is too high ÄKTA pure User Manual 29119969 AB 373 8 Troubleshooting 8 3 Trouble...

Page 374: ... page 335 Replace the piston seal of the sample pump according to the instructions in Section 7 8 11 Replace pump piston seals of Pump P9 S on page 344 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 317 Check the mixer inline filter It can be clogged if unfiltered ...

Page 375: ...ir intakes on the instrument are covered Hot surroundings Decrease the room temperature Maximum operating tempera ture is 35 C Hardware error Switch off the instrument and wait until the temperature has decreased Restart the instrument If this error is recurrent gen erate a System error report and contact Service The internal valve tempera ture is too high Hardware error Generate a System error re...

Page 376: ...as installed in the valve 1 Remove the dummy electrode 2 Wet the dummy 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 Modul...

Page 377: ...tion arm by hand Switch on the instrument If this error is recurrent generate a System error report and contact Service Fraction collector arm is blocked or internal fault in the fraction collector Salt crystals or protein residuals block the accumulator Restart the instrument and perform an accumulator wash Mechanical error If this error is recurrent generate a System error report and contact Ser...

Page 378: ...ides in the fractionation chamber are missing Replace the Tray guides See Section 7 6 4 Clean Fraction collector F9 C on page 288 The Tray catch is broken Contact service The Cassette tray is loose in the fraction collector Unclean Drop sync sensor diode windows Clean the Drop sync sensor diode windows See Section 7 6 4 Clean Fraction collector F9 C on page288 for location of the Drop sync sensor ...

Page 379: ...ate a System error report and contact Service The Dispenser head failed to detect the flow properly and has switched to tube change with reduced accu mulator functionality Too high flow rate Decrease the flow rate Air in the flow path Check the flow path for air Fill system and purge pumps accord ing to Section 5 4 1 System pumps on page 162 If this error is recurrent generate a System error repor...

Page 380: ...outwards See Prepare and insert the Cassette tray on page 195 Dirt under the Cassette tray Remove the dirt Cassette tray in wrong posi tion The Cassette tray is facing the wrong direction Check that the GE logotype is facing outwards when the tray is inserted into the fraction collector Some of the tubes or plates are incorrectly placed in the Cas settes Check that all tubes and plates are correct...

Page 381: ...g is turned off in UNICORN Make sure that the automatic scanning is turned on in UNICORN software In System Control select System Settings Navigate to Fraction collector Cassette configuration and select Auto matic Wells in deep well plates are prefilled to a volume above 25 of the total well volume Full scan will not work with prefilled wells during these conditions Hardware error Generate a Syst...

Page 382: ...g in the fraction collector 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 pumps See Section 5 4 1 System pumps on page 162 Bad pump function See troubleshooting of pumps in Section 8 6 Leakage inside the Frac chamber Contact Service The fraction volume found in the tubes or wells are sm...

Page 383: ... Section 7 6 4 Clean Fraction collector F9 C on page 288 Unsupported deep well plate is used Make sure that approved deep well plates are used See Deep well plates on page 101 Wells in deep well plates are prefilled to a volume above 25 of the total well volume Full scan will not work with prefilled wells during these conditions The deep well plate is not correctly positioned in the Cassette See P...

Page 384: ...fore starting a method run Whenstartingamethodrun during a manual run the fraction collector generates an error message indicating that the wrong Cassettes are in place even though the correct Cassettes are present in the Fraction col lector Action High pressure alarm when collecting fractions with the fraction collector Decrease the flow rate or use Outlet valve fractionation or replace the follo...

Page 385: ...Spillage between fractions Fraction collector F9 R Possible cause and action Problem The cable between the fraction collector and the ÄKTA pure instrument is not connected Make sure that the cable is connected Wrong node ID Check the Node ID of the fraction collector If necessary change the Node ID See Section 9 16 Node IDs on page 503 The fraction collector can not be found by the instru ment Hot...

Page 386: ...nged tube without drop sync The fraction collector movement is blocked Make sure that the fraction collector can move and is free from obstructions The tube sensor has not de tected a new tube The flow rate is too high Reduce the flow rate The fraction volume is too small Collect larger fractions Too many fraction collector instructions have been sent Wait for a while and try again The delay queue...

Page 387: ... 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 Section 7 8 12 Replace pump pistons on page 351 Bad pump piston seal Replace the piston seal and ...

Page 388: ...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 instrument are covered Hot surroundings Decrease the room temperature Maximum operating tempera ture is 35 C Hardware error Switch off the instrument and wait until the temperature has decreased Restart the instrument If this error is recurrent gen erate...

Page 389: ... pump error at nor mal pressure Too low I factor in the Pressure control parameters instruction In the Manual Instructions dialog increase the I factor of the Pressure control parameters instruction Too slow pressure build up when pressure control is active Too low I factor in the Constant pressure flow parameters in struction In the Manual Instructions dialog increase the I factor of the Constant...

Page 390: ... the other pressure monitors and is therefore recommended for troubleshooting purposes Incident 30 μl of air enters pump Action Purge pump See Section 5 4 Prime inlets and purge pump heads on page161 If the problem is persistent see Remove persistent air bubbles on page 392 Incident A large volume of air enters pump Action Purge pump See Section 5 4 Prime inlets and purge pump heads on page 161 39...

Page 391: ...nt Inlet check valve is loose Action Tighten the check valve See Section 7 8 9 Replace the pump head check valves on page332 Incident Leaking inlet check valve Action Replace the check valve See Section 7 8 9 Replace the pump head check valves on page332 ÄKTA pure User Manual 29119969 AB 391 8 Troubleshooting 8 6 Troubleshooting Pumps ...

Page 392: ...structions below to purge the pump with methanol see Section 5 4 Prime inlets and purge pump heads on page 161 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 5 4 Prime inlets and purge pump heads on page 161 for details 3 Set the pump flow to 2 5 ml min for ÄKTA pure 25 or 10 ml min for ÄKTA ...

Page 393: ...6 Purge the pump again using an appropriate buffer ÄKTA pure User Manual 29119969 AB 393 8 Troubleshooting 8 6 Troubleshooting Pumps ...

Page 394: ...one of the modules with the same Node ID The Node ID for one or more of the modules is incorrect for example an Inlet valve A2 has the same Node ID as Inlet valve A The instrument then considers them to have the same identity Remove Inlet valve A2 and change the Node ID according to Section 9 16 Node IDs on page 503 Modules cannot be found by the instrument The Node ID for one or more of the modul...

Page 395: ...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 Select Mixer chamber on page 152 Noisy UV signal The Mixer chamber is not correctly installed Verify that the Mixer chamber is correctly installed See Sec tion 7 8 3 Replace the Mixer on page322 If the error is recurrent replace the Mixer cham...

Page 396: ...Contact Service Liquid from the Buffer tray is flowing onto the table 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 el...

Page 397: ...d 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 for UniNet 9 connectors 1 to 8 must have connected jumpers See the illustrat...

Page 398: ...is connected The external air sensor has not been selected in UNICORN Select the external air sensor in UNICORN Refer to the installation instructions delivered together with the external air sensor The external air sensor is not found by the instrument One of the connections is not tight enough Tighten the connectors Air is introduced into the flow path One of the connections is not tight enough ...

Page 399: ...ected correctly earth to earth etc To isolate the problem External digital equipment does not respond to set changes in Digital Out 1 Connect Digital Out e g pin 6 to Digital In pin 1 2 3 or 4 2 Change the Digital Out 1 signal between 1 and 0 Verify that Digital out follows 3 If not contact Service If it does the problem probably lies within the connected equip ment Auto zero on the wrong level Re...

Page 400: ...f the air intakes of the instrument are covered Hot surroundings Decrease the room temperature Maximum operating tempera ture is 35 C Hardware error Switch off the instrument and wait until the temperature has decreased Restart the instrument If this error is recurrent gen erate a System error report and contact Service The internal temperature of the instrument control pan el is too high 400 ÄKTA...

Page 401: ...in control The run continues and no data will be lost UNICORNhaslostcommuni cation with the instrument server The module is not functioning properly In the displayed dialog in UNICORN select the option Restart the system only and click OK If the problem still remains replace the module or contact Service Warning message in UNICORN Instrument mod ule is missing Two or several modules have the same ...

Page 402: ...r all modules see Section 9 16 Node IDs on page 503 3 Switch on the instrument 4 In the displayed dialog in UNICORN select the option Restart the system only and click OK Warning messages in UNICORN Warning Gate 12 Internal instrument er ror 402 ÄKTA pure User Manual 29119969 AB 8 Troubleshooting 8 8 Troubleshooting Instrument communication ...

Page 403: ...anually replace the text instructions that are not available for the new configura tion Refer to Open and save methods for different systems on page215 for more information A method has been created for a system that now has a different configuration Action 1 Open the method in the method editor 2 Save as and select the new system OR If the configuration is different on the new system select to ei...

Page 404: ...tion Description Error code Restart the instrument If recurrent please con tact Service Internal instrument error 20 24 Restart the instrument If recurrent please con tact Service Valve not finding position 22 Restart the instrument If recurrent please con tact Service Faulty air sensor 23 See Section 8 4 Troubleshooting Valves on page 375 High temperature 25 Pressure monitor Action Description Er...

Page 405: ...art the instrument If recurrent please con tact Service Internal instrument error 20 21 Contact Service No factory calibration 25 See pH monitor and pH valve on page 370 High temperature 26 Conductivity monitor Action Description Error code Restart the instrument If recurrent please con tact Service Internal instrument error 20 27 See Conductivity monitor on page 367 High temperature 28 Restart th...

Page 406: ...ase con tact Service Spectrum calibration error 27 Restart the instrument If recurrent please con tact Service Lamp error 28 29 See UV monitor U9 M and UV detector unit on page 360 High temperature 30 UV detector U9 D Action Description Error code Restart the instrument If recurrent please con tact Service Internal instrument error 24 26 28 29 31 33 Check that the detector and flow cell are fitted...

Page 407: ...hen AU value is larger than 2 Autozero out of range 54 No light through flow cell Check solution absorp tion and that the cell is fitted correctly Too low light intensity S channel 58 Restart the instrument If recurrent please con tact Service Internal instrument error 59 60 Restart the instrument If recurrent please con tact Service Measurement error 61 Fraction collector F9 C Action Description ...

Page 408: ...ystem error report and contact ser vice Internal fraction collector error 57 59 Lower the flow rate or increase the fractionation volume Tube change is too fast 63 The instrument restarts If the error is recurrent generate a system error report and contact ser vice Fractioncollectorarm movement is blocked 68 72 Fraction collector F9 R Action Description Error code See Section 8 5 Troubleshooting F...

Page 409: ...Action Description Error code See section Troubleshooting High temperature 20 Check the external equipment connected to the I O box Analog in signal below 2V 21 Check the external equipment connected to the I O box Analog in signal above 2V 22 Restart the instrument If recurrent please con tact Service Internal instrument error 23 28 ÄKTA pure User Manual 29119969 AB 409 8 Troubleshooting 8 10 Err...

Page 410: ... 9 2 Module specifications 420 9 3 Tubing and connectors 428 9 4 Recommended tubing kits for prepacked columns 431 9 5 Chemical resistance guide 436 9 6 Wetted materials 438 9 7 Predefined methods and phases 449 9 8 System settings 466 9 9 Manual instructions 488 9 10 Available Run data 491 9 11 Available Curves 493 9 12 Injection volumes and peak broadening 494 9 13 Delay volumes 499 9 14 Compone...

Page 411: ...ypical Power consumption 25 VA power save IP 21 Enclosure protective class ÄKTA pure 25 Tubing and connectors Inlet FEP tubing i d 1 6 mm Tubing connec tor 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 Optio...

Page 412: ...d 1 0 mm Fingertight connector 1 16 Optional tubing kits i d 0 5 mm i d 1 0 mm Environmental ranges Data Parameter 25 C to 60 C Storage and transport temperature range See the relevant purification instrument User manual Chemical environment Operating ranges Data Parameter 4 C to 35 C Operating temperature range 20 to 95 non condensing Relative humidity Equipment noise level Acoustic noise level E...

Page 413: ... pure 150 0 01 to 150 ml min up to 300 ml min during column packing ÄKTA pure 25 Pressure range 0 to 20 MPa 2900 psi ÄKTA pure 150 0 to 5 MPa 725 psi ÄKTA pure 25 Viscosity range 0 35 to 10 cP 5 cP above 12 5 ml min ÄKTA pure 150 0 35 to 5 cP ÄKTA pure 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 pure 150 Accuracy 1 5 Precision RSD ...

Page 414: ...y range ÄKTA pure 25 Flow rate specifications Accuracy 2 Precision RSD 0 5 Conditions 0 25 50 ml min 3 MPa 0 8 3 cP ÄKTA pure 150 Accuracy 2 Precision RSD 0 5 Conditions 1 0 150 ml min 3 MPa 0 8 3 cP Valves Data Parameter Rotary valves Type Up to 12 Number of valves Standard Injection Functions Options Inlet A Inlet B Sample inlet Extra inlet Mixer by pass Loop selection Column selection pH Outlet...

Page 415: ...ure 25 Mixer volume 0 6 1 4 or 5 ml ÄKTA pure 150 1 4 5 or 15 ml Gradient formation Data Parameter ÄKTA pure 25 Gradient flow rate range 0 1 to 25 ml min ÄKTA pure 150 0 5 to 150 ml min ÄKTA pure 25 Gradient composition accuracy 0 6 Conditions 5 to 95 B 0 5 to 25 ml min 0 2 to 2 MPa 0 8 to 2 cP ÄKTA pure 150 0 8 Conditions 5 to 95 B 2 to 150 ml min 0 2 to 2 MPa 0 8 to 2 cP ÄKTA pure User Manual 29...

Page 416: ...ors Integrated in inlet valve A inlet valve B and sample inlet valve After the injection valve Before the system pumps Before the sample pump Placement Ultrasonic Sensing principle UV monitor options Data Parameter Up to 2 Number of monitors U9 L 280 nm Wavelength range U9 M 190 to 700 nm in steps of 1 nm up to 3 wavelengths 6 to 6 AU Absorbance range 0 001 mAU Resolution U9 L within 5 at 0 to 2 A...

Page 417: ...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 Conductivity monitor options Data Parameter 0 01 to 999 99 mS cm Conductivity reading range 0 01 mS cm or 2 whichever is greater within 0 3 to 300 mS cm Accuracy 0 to 5 MPa Operating pressure 22 μl Flow cell volume 0 C to 99 C Temperature monitor ra...

Page 418: ...meter 10 Number of outlets 0 01 to 20 000 ml Fraction volumes ÄKTA pure 25 Delay volume UV outlet valve 125 µl 66 µl with optional tubing kit i d 0 25 mm ÄKTA pure 150 296 µl 245 µl with optional tubing kit i d 0 5 mm Fraction collector options Data Parameter Up to two Number of fraction collectors The second fraction collector must be an F9 R F9 C Up to 576 Number of fractions F9 R Up to 175 418 ...

Page 419: ... F9 R 205 µl 86 µl with optional tubing kit i d 0 25 mm F9 C 435 µl 214 µl with optional tubing kit i d 0 25 mm ÄKTA pure 150 F9 R 473 µl 278 µl with optional tubing kit i d 0 5 mm F9 C 876 µl 508 µl with optional tubing kit i d 0 5 mm F9 C 390 x 585 x 320 mm F9 R 320 x 400 x 250 mm Dimensions W x D x H F9 C 21 kg F9 R 5 kg Weight I O box Data Parameter 2 analog in 2 analog out 4 digital in 4 digi...

Page 420: ...nd waste tub ing Transparent ETFE o d 1 16 i d 1 0 mm 0 3 ml Waste tubing from Buffer tray Transparent Silicone o d 12 mm i d 8 mm Pump rinse solution tubing Note 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 the protein peak Narrow tubing however increas...

Page 421: ... 16 i d 0 75 mm Tubing connector 5 16 Ferrule blue 1 16 FEP o d 3 16 i d 2 9 mm Tubing connector 5 16 ferrule blue 3 16 FEP o d 1 8 i d 1 6 mm Tubing connector 5 16 Ferrule yellow 1 8 Other connectors The table below shows other connectors used in ÄKTA pure Scope of use Description Stop plug for valve ports Stop plug 1 16 Syringe connector for pH valve and Injection valve Luer female ÄKTA pure Use...

Page 422: ... 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 AB A1 A2 and B1 B2 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 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 B7 2 0 0 6 300 FEP o d 3 16 i d 2 9 mm FEP o d 1 8 i d 1 6 mm From Inlet valve A or Inlet valve AB to Sys tem pump A InA 2 ...

Page 423: ...tandard lengths of the standard high pressure tubing and the optional high pressure tubing Standard high pressure tubing Volume μl Length mm Tubing Description Label ÄKTA pure 150 ÄKTA pure 25 ÄKTA pure 150 ÄKTA pure 25 270 150 340 PEEK o d 1 16 i d 1 0 mm PEEK o d 1 16 i d 0 75 mm System pump A left to Restrictor A 1A1 270 150 340 PEEK o d 1 16 i d 1 0 mm PEEK o d 1 16 i d 0 75 mm System pump A r...

Page 424: ... monitor to injection valve 3S 310 180 400 PEEK o d 1 16 i d 1 0 mm PEEK o d 1 16 i d 0 75 mm Pressure monitor to Mixer 3 160 90 200 PEEK o d 1 16 i d 1 0 mm PEEK o d 1 16 i d 0 75 mm Mixer to Injection valve 4 70 30 160 PEEK o d 1 16 i d 0 75 mm PEEK o d 1 16 i d 0 50 mm Injection valve to Column valve 5 70 30 160 PEEK o d 1 16 i d 0 75 mm PEEK o d 1 16 i d 0 50 mm Column valve to UV monitor 6 80...

Page 425: ...h mm Tubing Description Label ÄKTA pure 150 ÄKTA pure 25 ÄKTA pure 150 ÄKTA pure 25 130 70 160 PEEK o d 1 16 i d 1 0 mm PEEK o d 1 16 i d 0 75 mm Pressure monitor to Mixer valve 3 1 260 150 330 PEEK o d 1 16 i d 1 0 mm PEEK o d 1 16 i d 0 75 mm Mixer valve to Mixer 3 2 200 120 260 PEEK o d 1 16 i d 1 0 mm PEEK o d 1 16 i d 0 75 mm Mixer valve to Injec tion valve 3 3 80 40 180 PEEK o d 1 16 i d 0 7...

Page 426: ...the System performance tests Volume μl Length mm Tubing Description Label 20 400 PEEK o d 1 16 i d 0 25 mm Reference capillary Ref 1 Outlet tubing The table below shows the labels diameters and standard lengths of the outlet tubing The tubing is not mounted on delivery Volume ml Length mm Tubing Description Label 1 2 1500 ETFE o d 1 16 i d 1 0 mm Outlets from Outlet valve V9 Os or V9H Os Out 1 2 1...

Page 427: ...00 ETFE o d 1 16 i d 1 0 mm pH valve waste W3 1 2 1500 ETFE o d 1 16 i d 1 0 mm System waste W 80 1500 Silicone o d 12 mm i d 8 mm Top tray waste N A 70 1350 Silicone o d 12 mm i d 8 mm Fraction collector F9 C waste N A ÄKTA pure User Manual 29119969 AB 427 9 Reference information 9 3 Tubing and connectors ...

Page 428: ...ding HisTrap GSTrap except 4B StrepTrap 0 5 with Pressure controlor 0 75 0 5 or 0 75 GSTrap 4B 5 ml 0 5 with Pressure control or 0 75 0 5 or 0 75 All HiScreen 0 5 0 5 RESOURCE Q S ETH ISO PHE and RPC 1 ml 0 5 0 5 RESOURCE RPC 3 ml 0 75 with Pressure control for maximum flow rate combined with high viscosity 0 75 0 5 or 0 75 RESOURCE Q S 6 ml Avoid combination of column valve V9 C and pH position i...

Page 429: ...5 Avoid combination of column valve V9 C or V9H C and pH posi tion in valve V9 pH or V9H pH 0 5 0 5 Mono Q Mono S Mono P 5 50 GL Resolution may be improved with 0 25 Avoid column valve V9 C and pH position in V9 pH 0 25 0 25 Superdex 200 5 150 GL 0 5 0 5 Mono P 5 200 GL 0 5 or 0 75 0 5 or 0 75 Mono Q Mono S 10 100 GL 0 5 0 5 Superdex Peptide 75 200 Superose 6 12 10 300 GL 0 75 0 5 or 0 75 All HiPr...

Page 430: ...erdex 30 pg 75 pg 200 pg 0 75 0 5 or 0 75 HiLoad 26 10 Phenyl Sepharose HP Q Sepharose HP SP Sepharose HP 0 5 with Pressure control or 0 75 0 5 or 0 75 HiLoad 26 60 Superdex 30 pg 75 pg 200 pg 1 Standard system configuration Normal flow rates Moderate sample and buffer viscosities 2 Complex system configuration for additional tubing use the same tubing dimension as the Tubing Kit Maximum flow rate...

Page 431: ...iled information about chemical resistance of the ÄKTA pure instrument In this section See page Section 432 9 5 1 General information about biocompatibility and chemical resistance 433 9 5 2 Chemical resistance specifications ÄKTA pure User Manual 29119969 AB 431 9 Reference information 9 5 Chemical resistance guide ...

Page 432: ...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 additives trifluo roacetic acid TFA up to 0 2 or formic acid up to 5 Strong...

Page 433: ...ffers 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 Concentration Chemical 75 05 8 200 835 2 70 Acetic acid N A 10 Decon 90 75 08 1 200 837 3 100 Ethanol 67 56 1 200 659 6 100 Methanol 7647 01 0 231 595 7 0 1 M Hydrochloric acid1 67 63 0 200 661 7 100 Isopropanol 1310 73 2 215 185 5 2 M Sodium hydroxide N...

Page 434: ...nol 74 93 1 200 659 6 100 Methanol N A Max 5 formic acid Water organic mobile phase formic acid 1 Organic solvents can penetrate weaknesses in PEEK tubing walls more easily than water based buffers Special care should therefore be taken with prolonged use of organic solvents close to pressure limits Depending on pressure tubing between pump head and pressure monitor needs to be changed See Section...

Page 435: ...nzyl alcohol 3483 12 3 222 468 7 100 mM Dithioerythritol DTE 3483 12 3 222 468 7 100 mM Dithiothreitol DTT 60 00 4 200 449 4 100 mM Ethylenediaminetetraacetic acid EDTA 37482 11 4 253 523 3 20 mM Mercaptoethanol 7447 40 7 231 211 8 4 M Potassium chloride Other substances The following chemicals are suitable for continuous use CAS no EC no Concentration Chemical 67 64 1 200 662 2 10 Acetone 7664 41...

Page 436: ...orinated Ethylene Propylene FPM FKM Fluorinated Propylene Monomer FFPM FFKM Fully Fluorinated Propylene Monomer PCTFE PolyChloroTriFluoroEthylene PEEK PolyEtherEtherKetone PP PolyPropylene PTFE PolyTetraFluoroEthylene PVDF PolyVinylidene DiFluoride UHMWPE UltraHighMolecularWeightPolyEthylene Aluminum oxide Elgiloy Hastelloy C 276 Quartz glass Ruby Sapphire Titanium grade 2 Titanium grade 5 436 ÄKT...

Page 437: ...aterial EPDM EthylenePropyleneDiene M class rubber PEEK PolyEtherEtherKetone PP PolyPropylene PPS PolyPhenylene Sulfide PVDF PolyVinylidene DiFluoride Silicone ÄKTA pure User Manual 29119969 AB 437 9 Reference information 9 6 Wetted materials ...

Page 438: ...ution profile and fractionation are set on the Phase Properties pane in the appropriate phases This section describes the predefined methods and phases A method is built up by a number of phases Each phase represents a major process step in the method for example equilibration or elution Predefined methods that include all the phases necessary to run the system are available for different chromato...

Page 439: ...cation method After equilibration and sample application the protein of interest is adsorbed to the column ligand After a wash to remove unbound sample elution is performed either by using a buffer containing a competitor to displace the protein of interest or by changing the pH or ionic strength Finally the column is re equilibrated with start buffer Affinity Chromatography AC ÄKTA pure User Manu...

Page 440: ...quired solutions The method phases are pre configured to use the following solutions in the following inlets and loop Inlet A1 Equilibration buffer Inlet A2 or sample inlet Sample Inlet B1 Cleavage buffer Inlet B2 Affinity Regeneration buffer Loop Protease Affinity Chromatography AC with Tag Removal Note Select Inject sample directly onto the col umn in the sample application phase The protease ap...

Page 441: ...i tively charged proteins are adsorbed to the col umn ligand After a wash to remove unbound sample elution is performed using a gradient of increasing salt concentration of e g NaCl Final ly the column is washed and re equilibrated with start buffer Cation Exchange Chromatog raphy CIEX After equilibration and sample application elu tion is performed using a pH gradient The pro teins separate and e...

Page 442: ...ng a gradient of decreasing salt concentration Finally the column is washed and re equilibrated with start buffer 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 sam ples The loop...

Page 443: ... in the following inlets and sample loop Inlet A1 Coupling buffer for example 0 2 M NaHCO3 0 5 M NaCl pH 8 3 Inlet A2 Activation solution for example 1 mM HCl Inlet B1 High pH buffer for example 0 5 M ethanolamine 0 5 M NaCl pH 8 3 Inlet B2 Low pH buffer for example 0 1 M Sodi um acetate 0 5 M NaCl pH 4 0 Sample loop Ligand in coupling buffer Note The NHS coupling phase is preconfigured to empty t...

Page 444: ...After a wash to remove unbound sample elution is performed by generating a gradient of a non polar organic solvent such as acetonitrile Final ly the column is washed and re equilibrated ReversedPhaseChromatogra phy RPC 444 ÄKTA pure User Manual 29119969 AB 9 Reference information 9 7 Predefined methods and phases 9 7 1 Predefined purification methods ...

Page 445: ...ght equivalent to a theoretical plate HETP and the peak asymmetry factor As The result is logged in the column logbook Column Performance Test The column is filled with buffer solution Select inlet positions Enter the solution identity vol ume flow rate and incubation time By adding steps several preparation solutions can be used Column Preparation Packs AxiChrom columns with a predeter mined colu...

Page 446: ...ional System CIP phases can be added from the Phase Library if desired System CIP The system is filled with preparation solution Select for example inlets outlets and column positions to be prepared Two System Prepara tionphasesareincludedinthemethod Addition al System Preparation phases can be added from the Phase Library if desired System Preparation 446 ÄKTA pure User Manual 29119969 AB 9 Refer...

Page 447: ...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 Unit selection for Method base and Flow rate Monitor settings pH monitor Air sensor alarm settings UV monitor Note The first wavelength of U9 M and the fixed wavelength for U9 L or U9 L 2nd is always turned on ...

Page 448: ...al cleaning solutions can be used sequentially Column CIP Prepares the system before a run by removing storage solution and filling the system and inlets with buffer solution One preparation solution is used per phase System Prepara tion Cleans the system after purification runs by rinsing the system with a cleaning solution One cleaning solution is used per phase System CIP Tests the efficiency o...

Page 449: ...9 8 2 System settings Conductivity 452 9 8 3 System settings pH 453 9 8 4 System settings Pressure alarms 455 9 8 5 System settings Air sensor 457 9 8 6 System settings I O box 458 9 8 7 System settings Fraction collection 460 9 8 8 System settings Tubing and Delay volumes 462 9 8 9 System settings Wash settings 463 9 8 10 System settings Watch parameters 464 9 8 11 System settings Advanced 465 9 ...

Page 450: ...al falls outside the set limits an alarm will be triggered and the method will be paused Alarm UV 2nd Wavelength sets the wavelengths for UV monitor U9 M The cor responding 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 fo...

Page 451: ...manner 0 for low and 100 for high conductivity or in descending manner 0 for high and 100 for low conductivity Note The Relative scale cond in descending manner is especially useful for conductivity visualization in RPC and HIC where the conductiv ity curve is reversed compared to the concentration curve i e high conductivity at 0 B and low conductivity at 100 B Relative scale cond Cond temp compe...

Page 452: ...ruction name Alarm pH 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 452 ÄKTA pure User Manual 29119969 AB 9 Reference information 9 8 System settings 9 8 3 System settings pH ...

Page 453: ...d 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 sample pressure is set as a variable Low alarm is only triggered if the pressure first 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 deactivates the low pressure a...

Page 454: ...by the values 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 seconds continuously then falls below the Low alarm limit Note Setting the Low alarm to 0 deactivates the low pressure alarm Alarm pre column pressure 454 ÄKTA pure User Manual 29119969 AB 9...

Page 455: ...the alarm for the optional air sensor number X If the alarm is enabled and air is detected an alarm will be triggered and the method will be paused Alarm external air sensor X Sensitivity inlet A air sensor is used together with the Alarm inlet A air sensor instruction and sets the sensitivity of the built in air sensors at inlet A Sensitivity inlet A air sensor Normal 30 µl is used to detect when...

Page 456: ...ample vessel is empty High 10 µl is used to detect small air bubbles When located before an inlet the default sensitivity is Normal When located after the injection valve the default sensitivity is High and the pump currently pumping onto the column is used for calculating the air volume for the external air sensor Note Using an air sensor after the injection valve is only useful when running at l...

Page 457: ...e analog signal falls outside the set limits an alarm will be triggered and the method will be paused Alarm analog in X Alarm digital in X enables or disables the alarm for the signal in digital port number X The alarm can be triggered by either of the signal values 0 or 1 If the alarm is enabled and the condition set in Value occurs an alarm will be triggered and the method will be paused Alarm d...

Page 458: ...ates are used If Manual is selected used plates and tubes in each tray position are entered Fraction collector lamp Lamps in the fraction collector chamber on or off Fraction collector lamp The Peak fractionation parameters set the detection parameters for peak collection that is they decide when a peak starts and ends This information is used by the instructions Peak fractiona tion and Peak frac ...

Page 459: ...nd of a method or not Note The default setting is Reset Fractionation numbering mode frac 2 Peak fractionation parameters sets the detection parameters for peak collection i e it determines when a peak starts and ends This information is used by the instructions Peak fractionation Peak fractionation frac 2 and Peak frac in outlet valve in order to start end the peak collection Peak fractionation p...

Page 460: ...the length of the tubing between the system pressure sensor and the column valve or by using the measured sample pressure and the length of the tubing between the sample pressure sensor and the column valve depending on the position of the injection valve Tubing Sample pump to injec tion valve The instruction is available only when Column valve V9 C or V9H C 5 columns is not selected and a sample ...

Page 461: ...ponent list Delay volume Monitor to frac Delay volume Monitor to frac 2 is used to define the delay volume between the monitor and the second Fraction collector The in struction is used to make sure that the collected fractions corre spond to the fractions indicated in the chromatogram The instruc tion is available only when the second Fraction collector is selected in the component list Delay vol...

Page 462: ...ettings sets the flow rate and the wash volume 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 pump wash settings Fraction collector wash settings sets the flow rate during Frac tion collector wash Fraction collector wash settings Mixer by pass wash settings sets the flow rate used during Mixer by pass wash and defines...

Page 463: ...ers Watch pH 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 Watch flow parameters sets the value for the accepted fluctua tion of the flow rate signal used for the test Stable signal in the instructions Watch and Hold until with signal System flow Watch flow parameters Watch pressu...

Page 464: ...pressure alarm Pressure control is enabled in the instruction System flow or Sample flow Pressure control parameters provides the P and I factors used in the regulator and can be adjusted for different columns Pressure control parameters Constant pressure flow parameters sets the values for the P and I factors needed to keep a constant pressure by varying the flow rate The signal used for pressure...

Page 465: ...ven curve Data reduction occurs if the maximum number of data points is exceeded To avoid data reduction 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 corresponds to 1 5 h for a signal of 10 Hz Data collection ÄKTA pure User Manual 29119969 AB 465 9 Reference information 9 8...

Page 466: ...uction and click Insert Repeat for several instructions 3 To update parameter fields during method run check the Auto update box 4 To perform the instructions click Execute 5 All available manual instructions are described in the following subsections See page Section 467 9 9 1 Manual instructions Pumps 471 9 9 2 Manual instructions Flow path 473 9 9 3 Manual instructions Monitors 474 9 9 4 Manual...

Page 467: ...ps A and B Note Set gradient length value to 0 to perform a step gradient Gradient Pump A wash 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 instr...

Page 468: ...wash is used to change buffers in the sample inlet tubing and sample pump Note Pressing End during Sample pump wash will terminate both the wash and the run immediately Pressing Continue during Sample pump wash will terminate the wash and the run will continue from the point at which the Sample pump wash instruction was executed An instruction issued when a Sample pump wash is in progress will not...

Page 469: ...annot be executed when the system is in state HOLD Mixer by pass wash Loop wash is used to wash the Loop valve It is possible to wash a single sample application loop all loops or only the by pass position Loop wash instruction is not available if a Column valve is not mounted on the system Note Pressing End during Loop wash will terminate both the wash and the run immediately Pressing Continue du...

Page 470: ...ontinue from the point at which the System wash instruction was executed If System wash is performed during a Gradient operation the current component B concentration is maintained during the wash An instruction issued during a system wash operation cannot be executed until the wash is completely finished and all valves have turned back to the previous positions System wash cannot be executed when...

Page 471: ...ters the injection valve via SaP port Injection valve Column position turns the Column valve to the position specified in the parameter Position Column position pH valve sets the pH cell and the 0 2 MPa restrictor in positions inline or offline pH valve The pH valve also has a calibration position This position is only available when performing calibration of the pH monitor In Sys tem control sele...

Page 472: ...h two channels simultaneously Versatile valve X Instruction Inlet valve X1 turns the extra valve to the selected position The extra valve is a basic 8 port valve without air sensor to be used for general applications Inlet valve X1 Instruction Inlet valve X2 turns the extra valve to the selected position The extra valve is a basic 8 port valve without air sensor to be used for general applications...

Page 473: ...tically when a column is defined in a method run and Av eraging time is set as a variable Noise reduction UV Noise reduction UV 2nd filters the noise in the UV monitor signal from U9 L 2nd A column specific averaging time is set automat ically when a column is defined in a method run and Averaging time is set as a variable Noise reduction UV 2nd Relative scale cond facilitates monitoring of a grad...

Page 474: ...d The outlet valve is then turned to position Waste Stop peak fractionation Only for Fraction collector F9 C Last tube filled sets the action to perform after the 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 or direct the flow to waste Last tube filled Only for Fraction collector F9 R Sets fraction numbers ...

Page 475: ...ion Reset tube type Only for Fraction collector F9 C 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 the fraction collector It is then possible to open the door of the fraction colelctor and manually clean the dispenser head Note The Frac cleaning position instruction cannot be execu...

Page 476: ...start occurs when the instruction is issued The instruction overrides the continuous numbering mode if Fraction ation numbering mode frac 2 is set to Continue in System Set tings Reset frac number frac 2 During fractionation or peak fractionation the instruction Feed tube frac 2 moves the second Fraction collector tube rack forward one tube after the set delay volume has been collected and a fract...

Page 477: ...n collector is used Stop peak frac in outlet valve ends the peak fractionation in outlet valve after the set delay volume specified in System Settings Tubing and Delay volumes has been collected The outlet valve is then turned to position Waste Stop peak frac in outlet valve Peak fractionation parameters sets the detection parameters for peak collection i e it determines when a peak starts and end...

Page 478: ...on analog in X Digital out 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 Pulse digital out X generates a pulsed signal in digital port number X The signal changes from the initial state 0 or 1 to the opposite state and returns to the initial state after the defined length of time Pulse digital out X Configure analog out X ena...

Page 479: ... outside the set pressure limits an alarm will be triggered and the method will be paused Low alarm is only triggered if the pressure first exceeds the Low alarm limit for ten seconds continuously then falls below the Low alarm limit Note Setting the Low alarm to 0 deactivates the low pressure alarm Alarm sample pressure Alarm delta column pressure sets the alarm limits for the delta column pressu...

Page 480: ...falls out side the set limits an alarm is issued and the method will be paused Note It is not possible to set an alarm signal for the UV 2 or UV 3 signals from UV monitor U9 M Alarm UV1 Alarm UV 2nd enables or disables the alarm for the UV signal from UV monitor U9 L 2nd When enabled it sets the alarm limits for the UV signal from U9 L 2nd When the UV signal falls outside the set limits an alarm w...

Page 481: ... enabled and air is detected an alarm will be triggered and the method will be paused Alarm external air sensor X Alarm analog in X enables or disables the alarm for the analog signal in port number X When enabled it sets 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 a...

Page 482: ... System pump wash settings Sample pump wash settings sets the flow rate and wash volume used during sample pump washes Sample pump wash settings Fraction collector wash settings sets the flow rate during Frac tion collector wash Fraction collector wash settings Mixer by pass wash settings sets the flow rate used during Mixer by pass wash and defines wash volumes for mixer by pass wash options Note...

Page 483: ...ers Watch pH 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 Watch flow parameters sets the value for the accepted fluctua tion of the flow rate signal used for the test Stable signal in the instructions Watch and Hold until with signal System flow Watch flow parameters Watch pressu...

Page 484: ... 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 selected before using linear flow Note When Constant pressure flow is used the P and I fact...

Page 485: ...atogram The delay volume must be changed when changing tubing to another i d or length or when removing or adding components Delay volume Monitor to outlet valve Delay volume Monitor to frac 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 the fractions indicated in the chromatogram The...

Page 486: ...Watch instruc tions Hold counted volume 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 Method progressing flow Method progressing flow defines the flow from which the volume base is calculated When set to Automatic the position of the injection valve...

Page 487: ... text entered for the parameter Mark text Set mark Timer 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 perform pause or end Timer ÄKTA pure User Manual 29119969 AB 487 9 Reference information 9 9 Manual instructions 9 9 10 Manual instructions ...

Page 488: ...pure 150 The set flow velocity of the system pumps Only available if a column is selected cm h System linear flow The set flow rate of the sample pump 0 001 50 000 ml min ÄKTA pure 25 Sample flow 0 01 300 00 ml min ÄKTA pure 150 The set flow velocity of the sample pump Only available if a column is selected cm h Sample linear flow The set position of the inlet valve A A1 A7 Inlet A The set positio...

Page 489: ...ressure signal 1 00 20 00 MPa ÄKTA pure 25 DeltaC pressure 1 00 5 00 MPa ÄKTA pure 150 The post column pressure signal 1 00 20 00 MPa ÄKTA pure 25 PostC pressure 1 00 5 00 MPa ÄKTA pure 150 The UV absorbancesignal of the U9 L monitor 6000 000 6000 000mAu UV The first UV Vis absorbance signal of the U9 M monitor 6000 000 6000 000mAU UV 1 The 2nd UV Vis absorbance signal of the U9 M monitor 6000 000...

Page 490: ...alve 3 N A Versatile valve 3 The set position of the versatile valve 4 N A Versatile valve 4 The current state of the air alarm for the inte grated air sensor in inlet valve A No air Air Air inlet A The current state of the air alarm for the inte grated air sensor in inlet valve B No air Air Air inlet B The current state of the air alarm for the inte grated air sensor in inlet valve IS No air Air ...

Page 491: ... 0 100 0 Conc B The set flow rate of the system pumps 1 Hz 0 001 50 000 ml min ÄKTA pure 25 System flow 0 01 300 00 ml min ÄKTA pure 150 The set flow velocity of the system pumps Only available if a column is selected 1 Hz cm h System linear flow The set flow rate of the sample pump 1 Hz 0 001 50 000 ml min ÄKTA pure 25 Sample flow 0 01 300 00 ml min ÄKTA pure 150 The set flow velocity of the samp...

Page 492: ...20 00 MPa ÄKTA pure 25 PostC pressure 1 00 5 00 MPa ÄKTA pure 150 The temperature signal in the con ductivity flow cell 0 5 Hz 0 0 99 0 C Cond temp The I O box analog input signals 10 Hz 2000 0 2000 0 mV Analog in 1 to Analog in 4 The nominal cell path length of the U9 L monitor 1 Hz 0 2 0 5 cm UV cell path length The nominal cell path length of the U9 M monitor 1 Hz 0 05 0 2 1 0 cm UV cell path l...

Page 493: ... chromatography techniques such as affinity chromatography AC ion exchange chromatography IEX and hydrophobic interaction chromatography HIC if the retention factor k is high 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 collectio...

Page 494: ...y volumes A theoretical determination is performed as described in the steps below Action Step Identify all components in the system flow path that contribute to the delay volume of interest 1 Determine the internal volumes of all hardware modules and tubing see Section 9 14 Component volumes on page499 for information about theoretical module volumes and Section 9 3 Tubing and connectors on page4...

Page 495: ...and click OK 2 In the System Settings dialog 3 Select Delay volume Monitor to outlet valve or Monitor to frac according to the configuration you are using Type in the volume in the Volume field and click OK Note The system will use the delay volume appropriate to the configuration used and ignore other settings e g the value for Monitor to outlet valve will be ignored if you are using a fraction c...

Page 496: ...volume Monitor to frac F9 R Delay volume Monitor to outlet valve 214 µl 86 µl 66 µl N A No 0 25 225 µl 96 µl 77 µl 25 µl Yes 0 25 435 µl 205 µl 125 µl N A No 0 5 stan dard 462 µl 231 µl 152 µl 48 µl Yes 0 5 stan dard 803 µl 400 µl 223 µl N A No 0 75 857 µl 454 µl 277 µl 88 µl Yes 0 75 1319 µl 675 µl 361 µl N A No 1 0 1410 µl 766 µl 452 µl 143 µl Yes 1 0 Delay volumes ÄKTA pure 150 Delay volume UV ...

Page 497: ... flow rate is not correct check if air bubbles may have entered the pump Make sure to correct any problems before continuing 1 Fill a small sample loop for example 20 μl with a 1 to 5 acetone solution 2 Set all components between the monitor and the fraction collector to by pass mode 3 Fill the system with water Run the pump at a flow rate of 1 ml min and inject the acetone solution as a sample 4 ...

Page 498: ... of the volume of the UV flow cell Use of different monitors for peak fractionation in the same method If different monitors or detectors are used for peak fractionation in different parts of the same method the delay volumes have to be set as method instructions for each of the method parts For example both an external fluorescence detector and the UV monitor module can be used for peak fractiona...

Page 499: ... 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 45 System pump pressure monitor R9 45 Sample pump pressure monitor R9 1392 Sample pump S9 600 Mixer 0 6 ml 1400 Mixer 1 4 ml 5000 Mixer 5 ml 15000 Mixer 15 ml 14 Mixer valve V9 M 31 Mixer valve V9H M 17 Loop valve V9 L 76 Loop val...

Page 500: ...H in By pass position 35 pH valve V9H pH in By pass position 76 pH flow cell 48 Flow restrictor FR 902 and tubing when mounted on pH valve V9 pH 9 Outlet valve V9 O 82 Outlet valve V9H O 9 Outlet valve V9 Os 28 Outlet valve V9H Os 147 Fraction collector F9 C internal tubing 0 5 mm i d 94 Fraction collector F9 C Dispenser head The given values for the component volumes of the valves are average val...

Page 501: ... 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 os cillation in the pressure and the actual flow rate Set I 0 to disable the I factor As a general guide use a small I factor for high pressure columns and a large I fact...

Page 502: ...bing between components will improve resolution but will lead to increased back pressure in the system Narrow tubing after the column will increase the pressure in the column at a given flow rate Make sure that the pressure sensor limits in the system are set so that the maximum pressure for the column used is not exceeded Additional instructions for avoiding pressure alarms The instruction Max fl...

Page 503: ... IDs for UV monitor U9 M and UV monitor U9 D should only be changed by service personnel Note Node ID Label Module 0 V9 IA V9H IA Inlet valve A 1 V9 IB V9H IB Inlet valve B 3 V9 IAB V9H IAB Inlet valve AB 15 V9 IX V9H IX Inlet valve X1 16 V9 IX V9H IX Inlet valve X2 22 V9 M V9H M Mixer valve 2 V9 IS V9H IS Sample inlet valve 17 V9 L V9H L Loop valve 5 V9 C V9H C Column valve 5 columns 2 N A Pre co...

Page 504: ...ity monitor 0 L9 External air sensor 1 L9 External air sensor 2 2 L9 External air sensor 3 3 L9 External air sensor 4 0 F9 C Fraction collector cassettes 0 F9 R Fraction collector round 1 F9 R Fraction collector round 2nd 2 P9 S P9H S Sample pump 0 E9 I O box 1 E9 I O box 2nd Check Change Node ID Action Step Remove the module according to the instruction in Section 2 5 Installation of internal mod...

Page 505: ...switch labeled B sets the units For example to set the Node ID to 6 for a valve module switch A is set to 0 and switch B is set to 6 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 screw driver to set the arrows of the switches to the desired number 4 Re install the module in the instrument if applicable 5 ÄKTA pure User Manual 291199...

Page 506: ...ber 0 6 1 4 and 5 ml 29011326 O ring 13 1 1 6 mm highly resistant can be used as an alternative to 28953545 28981857 O ring 22 1 1 6 mm Note For Mixer chamber 15 ml 18102711 Online filter kit Tubing Code no Item 28950749 Reference capillary 1 28950750 Reference capillary 2 29011327 Tubing Kit 0 5 mm standard ÄKTA pure 25 29051669 Tubing Kit 0 5 mm ÄKTA pure 150 29011328 Tubing Kit 0 25 mm ÄKTA pur...

Page 507: ...lve V9H IA 7 ports 29011333 Tubing kit for inlet valve V9 IB 7 ports 29051189 Tubing kit for inlet valve V9H IB 7 ports 29011334 Outlet tubing kit ÄKTA pure 25 29048611 Outlet tubing kit ÄKTA pure 150 29011348 Rinse system tubing 28954326 Union 1 16 male male i d 0 5 mm 5 pack 18111246 Tubing cutter 11000407 Inlet filter holder kit 11000414 Inlet filter set Holders Code no Item 28956342 Adapter fo...

Page 508: ...flow cell U9 0 5 0 5 mm for U9 M 28979380 UV flow cell U9 2 2 mm for U9 M 28956378 UV flow cell U9 10 10 mm for U9 M 29011325 UV flow cell 2 mm for U9 L 18112824 UV flow cell 5 mm for U9 L I O box Code no Item 29011361 I O box E9 Fraction collector F9 C Code no Item 29027743 Fraction collector F9 C 29033632 Tubing kit for F9 C 28 954209 Cassette tray 28954212 Cassette for deepwell plate 2 pack 770...

Page 509: ...k 28956427 Cassette for 3 ml tubes 2 pack 29133422 Cassette for 5 ml tubes 2 pack 28956425 Cassette for 8 ml tubes 2 pack 28956404 Cassette for 15 ml tubes 2 pack 28980319 Rack for 50 ml tubes 28981873 Rack for 250 ml bottles 29032425 Cable 2 5 m UniNet 9 D type ÄKTA pure User Manual 29119969 AB 509 10 Ordering information ...

Page 510: ... 18112468 Tube rack upgrade kit 40 x 30 mm 19606702 Drive sleeve Valves Code no Item 29011367 Column valve kit V9 C 29050951 Column valve kit V9H C 29011355 Column valve V9 Cs 29090693 Column valve V9H Cs 28957227 Inlet valve V9 X1 28979326 Inlet valve V9H X1 28957234 Inlet valve V9 X2 28979328 Inlet valve V9H X2 29012263 Inlet valve kit V9 IA 29050945 Inlet valve kit V9H IA 29012370 Inlet valve k...

Page 511: ...e kit V9H Os 1 outlet 29011359 pH valve kit V9 pH 29051684 pH valve kit V9H pH 29011353 Versatile valve V9 V 29090691 Versatile valve V9H V All valve kits include the necessary tubing Note Injection valve accessories Code no Item 18112039 Sample loop 10 µl 18111398 Sample loop 100 µl 18111399 Sample loop 500 µl mounted at delivery 18111401 Sample loop 1 ml 18111402 Sample loop 2 ml 18116124 Sample...

Page 512: ...5 mm pH monitor Code no Item 28954215 pH electrode 28956497 O ring 5 3 2 4 mm Conductivity monitor Code no Item 29011363 Conductivity monitor C9 Flow restrictor Code no Item 18112135 Flow restrictor FR 902 Module components Code no Item 29011364 Module Panel 29011351 Multi module front 29110806 Extension box 512 ÄKTA pure User Manual 29119969 AB 10 Ordering information ...

Page 513: ...on kit 150 ml 28979364 Check valve kit 29050593 Sample pump S9H Sample Pump S9 Code no Item 29027745 Sample pump S9 28960250 P9 S Seal kit 18111213 P9 S Piston kit 28979364 Check valve kit 29032425 Cable 2 5 m UniNet 9 D type UNICORN There are different UNICORN products and licenses available for different purposes for example licenses for use with a workstation or for working remotely Contact you...

Page 514: ...ning 222 column CIP 284 column holder 81 column valve 56 connect column 173 pressure alarm 177 storage 222 Column CIP maintenance method 445 predefined phase 448 Column performance test maintenance method 445 predefined phase 216 448 Column preparation maintenance method 445 predefined phase 448 Column valve description 56 flow paths V9 C 57 flow paths V9 Cs 58 V9 C test 235 Column wash predefined...

Page 515: ...der rod 82 Extension box 86 flexible column holder 83 loop holder 81 multi purpose holder 80 ordering information 508 rail extension 80 tubing holder comb 84 tubing holder spool 83 Hydrophobic interaction chromatogra phy HIC predefined method 442 I I O box E9 connectors 114 description 113 error codes 409 Fraction collector F9 C installa tion 113 specifications 419 troubleshooting 399 Injection va...

Page 516: ...og raphy HIC 442 Intelligent Packing 445 maintenance methods 212 manual loop fill 442 NHS coupling 443 predefined maintenance meth ods 445 predefined methods 212 439 purification methods 212 reverse phase chromatography RPC 444 Method settings predefined phase 447 Miscellaneous predefined phase 448 Mixer choose mixer chamber 152 description 38 error codes 405 mixer valve 47 replace mixer 322 repla...

Page 517: ... loop fill 448 method settings 447 miscellaneous 448 predefined phases 447 sample application 448 system CIP 216 448 system preparation 448 pH monitor calibration 303 clean the pH electrode 293 description 63 error codes 405 replace the pH electrode 275 storage of the pH electrode 222 291 troubleshooting 370 pH valve description 61 flow paths 62 troubleshooting 370 376 Power and ICU troubleshootin...

Page 518: ...n 217 prepare run 217 S Safety notices 8 Sample application flow paths 50 injection valve 50 predefined phase 448 sample loading 180 sample loop 190 superloop 183 Sample loop sample loading 190 Sample pump description 108 Software overview software modules 10 Storage column 222 pH electrode 222 system 222 Superloop sample loading 183 troubleshooting 396 System CIP maintenance method 446 predefined...

Page 519: ...monitor U9 L description 71 153 error codes 407 replace the flow cell 328 system test 244 troubleshooting 364 UV monitor U9 M description 70 153 error codes 406 system test 248 troubleshooting 360 V Valves column valve 56 error codes 404 extra inlet valves 40 injection valve 50 inlet valves 40 loop valve 53 mixer valve 47 outlet valve 65 overview 39 pH valve 61 specifications 414 versatile valve 6...

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Page 522: ... is subject to GE Healthcare Standard Software End User License Agreement for Life Sciences Software Products A copy of this Standard Software End User License Agreement is available on request 2012 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...

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