Connect Alias autosampler to ÄKTA pure or ÄKTA avant Instructions
Table of Contents Table of Contents 1 About this Instruction... 2 Description... 3 Hardware installation... 3.1 Prepare Cable interface... 3.2 Connect the autosampler... 3.3 Connect and fill the flow path... 3.4 Optimize the tubing diameter... 4 Software installation... 5 Preparation and configuration... 5.1 Introduction... 5.2 Configure Alias Service Manager (ASM)... 5.3 Configure SparkLink... 5.4 Configure I/O-box E9... 6 Operation overview... 6.1 Introduction... 6.2 Start a run... 6.3 Monitoring and evaluation... 7 Method setup... 7.1 Workflow... 7.2 Create an autosampler project... 7.3 Create an autosampler method... 7.4 Create an autosampler runtable... 7.5 Update UNICORN method... 7.6 Submit and start an autosampler runtable... 8 Advanced operation... 8.1 Introduction... 8.2 Run states... 8.3 Set up the methods for advanced operation... 8.3.1 Set up the autosampler method... 8.3.2 Set up the UNICORN method... 8.3.3 Additional information and tips for the UNICORN method... 9 Direct control and maintenance... 3 5 10 10 11 13 14 15 18 18 19 23 28 29 29 29 30 31 31 31 33 36 40 45 47 47 47 49 49 49 52 55 2 Connect Alias autosampler to ÄKTA pure or ÄKTA avant Instructions 29040427 AC
1 About this Instruction 1 About this Instruction Scope This document provides instructions on how to connect the Alias autosampler to ÄKTA pure or ÄKTA avant, configure the system, and use the Alias autosampler in a method. The following components are required to connect the Alias autosampler to ÄKTA pure or ÄKTA avant: ÄKTA pure, or ÄKTA avant I/O-box E9 UNICORN software Alias autosampler manufactured by Spark Holland BV Alias Service Manager (ASM) software supplied by Spark Holland BV SparkLink software supplied by Spark Holland BV Prerequisites In order to connect the Alias autosampler to ÄKTA pure or ÄKTA avant and operate the system according to the intended purpose the following prerequisites must be met: You should be familiar with the ÄKTA pure, or ÄKTA avant instrument and UNICORN software and have read the respective Operating Instructions (ÄKTA pure: 29022997, ÄKTA avant: 29101556). You have read the Install I/O-box E9 Instructions (29021463). You have read the documentation supplied with the Alias autosampler. Notes and tips Note: Tip: A Note is used to indicate information that is important for trouble-free and optimal use of the product. A tip contains useful information that can improve or optimize your procedures. Online Help Online help is integrated in both Alias Service Manager and SparkLink software. Click Help in the menu bar, or click the? button when shown in dialogs. Connect Alias autosampler to ÄKTA pure or ÄKTA avant Instructions 29040427 AC 3
1 About this Instruction Typographical conventions Software items are identified in the text by bold italic text. A colon separates menu levels, thus File:Open refers to the Open command in the File menu. Hardware items are identified in the text by bold text (e.g., Power switch). 4 Connect Alias autosampler to ÄKTA pure or ÄKTA avant Instructions 29040427 AC
2 Description 2 Description Introduction The Alias autosampler is a high throughput sample injector intended for use with liquid chromatography systems. The autosampler cannot be used as a stand-alone unit. Connect Alias autosampler to ÄKTA pure or ÄKTA avant Instructions 29040427 AC 5
2 Description Setup of ÄKTA pure The illustration below shows the Alias autosampler connected to ÄKTA pure. Component Alias autosampler I/O-box E9 Fraction collector F9-R (if used) Fraction collector F9-C (if used) Location On the bench to the left of the ÄKTA pure instrument. On the top rail on the left side of ÄKTA pure or on the bench, behind the ÄKTA pure instrument. To the left of the ÄKTA pure instrument, on top of the Alias autosampler. On the bench to the left of the ÄKTA pure instrument. It is recommended to place the autosampler on top of the fraction collector. Note: If the tubing to the Fraction collector is replaced with tubing of different lengths, the Delay volume has to be updated in the System settings. For instructions, refer to "ÄKTA pure User Manual" ( 29-1199-69). 6 Connect Alias autosampler to ÄKTA pure or ÄKTA avant Instructions 29040427 AC
2 Description Setup of ÄKTA avant For ÄKTA avant the most common placement of the I/O-box E9, with the adhesive feet attached, is on the laboratory bench on the right-hand side of the instrument, close to the UniNet ports. Place external equipment, such as the Alias autosampler, on the right hand side of the ÄKTA avant instrument, close to the I/O-box and the wet side of the instrument. I/O-box E9 connectors See instruction Install I/O-box E9 for how to install the box, how to add the module to system properties in UNICORN and how to check Node ID. The illustration below shows the I/O-box E9 connectors needed to connect the Alias autosampler to ÄKTA pure or ÄKTA avant. Part UniNet-9 Node ID Digital in/out Description Connector used to connect I/O-box E9 to ÄKTA pure or ÄKTA avant. Switches used to configure the I/O-box to the ÄKTA pure or ÄKTA avant instrument. Signal connector for digital input and output signals. The Digital in/out connector is used to connect the I/O-box E9 to the Alias autosampler. Connect Alias autosampler to ÄKTA pure or ÄKTA avant Instructions 29040427 AC 7
2 Description I/O-box E9 digital connector pins The digital connector pins are identified according to the illustration below. 9 8 7 6 5 4 3 2 1 Part 1 2 3 4 5 6 7 8 9 Function Digital in signal 1 Digital in signal 2 Digital in signal 3 Digital in signal 4 Signal ground Digital out signal 1 Digital out signal 2 Digital out signal 3 Digital out signal 4 8 Connect Alias autosampler to ÄKTA pure or ÄKTA avant Instructions 29040427 AC
2 Description Connections between Alias autosampler and I/O-box E9 The Alias autosampler includes a connection cable with an open end intended for I/O connections. The user should make the connections to the connector supplied with the I/O-box E9 by matching the pins as presented in the table below. For more information see Alias autosampler User Manual and I/O-box E9 instruction. Autosampler Signal Autosampler cable ID I/O-box E9 Digital in/out connector pin UNICORN instruction Function Input 1 RED (4-wired) Pin 6 Pulse digital out 1 or Digital out 1 Set autosampler to inject Prepare for next inject Output 1 BROWN (3-wired) Pin 1 Digital in 1 Autosampler error handling (watch) Signal ground Common ORANGE (4-wired) RED (3-wired) Pin 5 - Signal ground Connect Alias autosampler to ÄKTA pure or ÄKTA avant Instructions 29040427 AC 9
3 Hardware installation 3 Hardware installation 3.1 Prepare Cable interface Follow the instructions below to prepare the remote cable interface. 1 Sort out the different wires of the autosampler cable. 1 2 3 4 Part 1 2 3 4 Description RED (4-wired) ORANGE (4-wired) RED (3-wired) BROWN (3-wired) 2 Open the housing of the cable interface. 10 Connect Alias autosampler to ÄKTA pure or ÄKTA avant Instructions 29040427 AC
3 Hardware installation 3.1 Prepare Cable interface 3 Connect the correct wire to the correct position in the cable interface (see table below) and tighten the screws to get good contact between wires and pins. Wire RED (4-wired) BROWN (3-wired) ORANGE (4-wired) RED (3-wired) Cable interface pin Pin 6 Pin 1 Pin 5 Pin 5 Attach the autosampler cable to the housing. 4 5 Isolate the unused wires with a piece of insulating tape. Close the cable interface housing. 3.2 Connect the autosampler Follow the instructions below to connect the Alias autosampler to ÄKTA pure or ÄKTA avant via the I/O-box E9. 1 Connect the Alias autosampler to mains power using the supplied power cord. Connect Alias autosampler to ÄKTA pure or ÄKTA avant Instructions 29040427 AC 11
3 Hardware installation 3.2 Connect the autosampler 2 Connect the autosampler to a COM-port on the computer using the cable supplied with the autosampler. Use the right 9-p D-sub (female) connector on the back of the autosampler (indicated in the figure below). Computer COM-port 3 Connect the remote cable, with the prepared cable interface, between the left 9-p D-sub (male) connector on the autosampler and the port Digital in/out on the I/O-box E9 (see illustration). Note: The connector has to be fastened to the I/O box with the long screws to achieve good contact and to prevent it from falling out. 12 Connect Alias autosampler to ÄKTA pure or ÄKTA avant Instructions 29040427 AC
3 Hardware installation 3.2 Connect the autosampler 4 Connect the I/O-box E9 to the ÄKTA instrument. Connect the cable (UniNet-9 F-type cable) between the UniNet-9 connector on the I/Obox and the UniNet-9 F-type connector on the instrument. See Install I/O-box E9 Instructions. 3.3 Connect and fill the flow path ÄKTA pure Follow the instructions below to connect the autosampler to the flow path of the ÄKTA pure. See also Section 3.4 Optimize the tubing diameter, on page 14. 1 2 3 4 5 Connect a piece of PEEK tubing i.d. 0.5 mm (about 500 mm long) between port LoopE on the injection valve V9-Inj of the ÄKTA instrument and port 1 (pump) on the autosampler valve. Connect a piece of PEEK tubing i.d. 0.5 mm (about 450 mm long) between port LoopF on the injection valve V9-Inj of the ÄKTA instrument and port 6 (column) on the autosampler valve. Note: Use the tubing connectors that are supplied for each instrument. The connectors used for the autosampler valve differ from the fingertight connectors used for ÄKTA instruments. Autosampler should use Valco connectors. To avoid introducing air in the flow path, during a run, connect a stop plug in the Syr port on the injection valve of the ÄKTA instrument. Turn on the power to the autosampler and ÄKTA instrument. To fill loop in the autosampler and the connected tubing with buffer, perform a manual run in UNICORN. Start a low flow (e.g., 1 ml/min). Turn injection valve on the ÄKTA instrument to Inject. Let the flow run long enough to fill the autosampler loop and connected tubing. Stop the run. Connect Alias autosampler to ÄKTA pure or ÄKTA avant Instructions 29040427 AC 13
3 Hardware installation 3.3 Connect and fill the flow path ÄKTA avant Follow the instructions above for ÄKTA pure, but change the length of the PEEK tubing to: about 750 mm long between port LoopE on the injection valve V9-Inj of the ÄKTA instrument and port 1 (pump) on the autosampler valve. about 700 mm long between port LoopF on the injection valve V9-Inj of the ÄKTA instrument and port 6 (column) on the autosampler valve. Note: The length of the tubing shall always be as short as possible. 3.4 Optimize the tubing diameter Note: Depending on the placement of the autosampler the length of the tubing will differ. Use tubing as short as possible to prevent band broadening. Choose tubing i.d. based on flow rate and viscosity of the liquid used. Follow the instructions below to choose the optimal tubing i.d. when connecting the Alias autosampler to ÄKTA pure or ÄKTA avant 1 2 3 Use PEEK tubing, i.d. 0.50 mm initially. Perform a test run. Check resolution and system pressure. If the resolution is not as expected, change to PEEK tubing i.d. 0.25 mm to increase the resolution. However, be aware that the system pressure will then increase. or If the system pressure is too high, change to PEEK tubing i.d. 0.75 mm to decrease the pressure. E.g., when running at a high flow rate and/or with high viscosity samples. However, be aware that the resolution will then decrease. 14 Connect Alias autosampler to ÄKTA pure or ÄKTA avant Instructions 29040427 AC
4 Software installation 4 Software installation Introduction Install the Alias autosampler software on the computer connected to the ÄKTA instrument and Alias autosampler. The software is supplied with the autosampler. Installation is performed in three steps. Installation of: Alias Service Manager (ASM), and SparkLink core, and SparkLink Alias driver Alias Service Manager 1 2 3 Insert the Alias Service Manager CD. Locate and click the Setup file. Follow the instructions in the installation wizard. Result: Connect Alias autosampler to ÄKTA pure or ÄKTA avant Instructions 29040427 AC 15
4 Software installation SparkLink core 1 2 Locate and click the SetupSparkLink file (Windows Installer). Follow the instructions in the installation wizard. Result: SparkLink core consists of two parts: SparkLink configuration, and SparkLink software application To have easy access to these programs it is recommended to create shortcuts on the desktop when installation is complete. 16 Connect Alias autosampler to ÄKTA pure or ÄKTA avant Instructions 29040427 AC
4 Software installation SparkLink Alias driver 1 2 3 Insert the SparkLink software CD and locate the Driver folder. Click the SetupAlias file (Windows Installer). Follow the instructions in the installation wizard. Result: Connect Alias autosampler to ÄKTA pure or ÄKTA avant Instructions 29040427 AC 17
4 Software installation 5 Preparation and configuration 5.1 Introduction Autosampler preparation Prepare the autosampler according to the supplied user documentation for the Alias autosampler. Configure autosampler I/O Note: The I/O configuration is performed differently, depending on the version of the Alias autosampler: Standard version (designed for analytical applications) Prep version (designed for large sample volumes) The autosampler is configured using: ASM, and SparkLink configuration Configure I/O-box E9 The I/O-box E9 is configured using UNICORN software. 18 Connect Alias autosampler to ÄKTA pure or ÄKTA avant Instructions 29040427 AC
5 Preparation and configuration 5.2 Configure Alias Service Manager (ASM) 5.2 Configure Alias Service Manager (ASM) Configure ASM Communication To configure ASM communication, follow the instruction below: 1 2 Turn on the power of the autosampler. Start the ASM software. 3 Select Settings:Communication Connect Alias autosampler to ÄKTA pure or ÄKTA avant Instructions 29040427 AC 19
5 Preparation and configuration 5.2 Configure Alias Service Manager (ASM) 4 Select Com port: COM1 Select Protocol: RS232 Note: Ethernet and USB tabs are not used when running the autosampler together with an ÄKTA system. 5 To check that the chosen communication port is active: Select Instrument:Maintenance:Direct control Check that system status is idle. 20 Connect Alias autosampler to ÄKTA pure or ÄKTA avant Instructions 29040427 AC
5 Preparation and configuration 5.2 Configure Alias Service Manager (ASM) Configure ASM Instrument settings To configure ASM instrument settings, follow the instruction below: 1 2 Start ASM software. Select Settings:Instrument settings. Result: The Instrument settings dialog opens. Connect Alias autosampler to ÄKTA pure or ÄKTA avant Instructions 29040427 AC 21
5 Preparation and configuration 5.2 Configure Alias Service Manager (ASM) 3 If you have Alias - Standard version (Analytic), then In the Instrument settings dialog, enter the intended settings as shown in the table below. Click Change to change the Tray settings. Select the type of plates for the left and right position. If you have Alias - Prep version, then In the Instrument settings dialog, check the Prep mode checkbox and enter the intended settings as shown in the table below. For Prep version, the type of plates are fixed to 12 positions left and right. 4 Click Save settings to save the settings. Click Close to close the dialog. Click File:Exit to close the program. 22 Connect Alias autosampler to ÄKTA pure or ÄKTA avant Instructions 29040427 AC
5 Preparation and configuration 5.2 Configure Alias Service Manager (ASM) Only listed items are relevant when connected to an ÄKTA instrument. Item Tray settings Syringe volume Loop volume Needle volume Input 1 Output Freeze level Standard version Value as required as required (500 μl) 1 as required (100 µl) 1 as required (15 µl) 1 Freeze Alarm Low Prep version Value Fixed, 12 position plates 2500 μl 2 10 ml 2 60 μl 2 Freeze Alarm Low 1 Default value 2 The settings are automatically selected. Note: The Communication and Instrument settings in the ASM software is only performed once, when the autosampler is installed. All other communication with the autosampler is handled via the SparkLink software. 5.3 Configure SparkLink Follow the instructions below to configure SparkLink. 1 Start the SparkLink Configuration software. Connect Alias autosampler to ÄKTA pure or ÄKTA avant Instructions 29040427 AC 23
5 Preparation and configuration 5.3 Configure SparkLink 2 Click New to create a new system configuration or select to open an existing configuration to edit. 3 When creating a new configuration, select the Alias device in the Available devices box and click the arrow button. 24 Connect Alias autosampler to ÄKTA pure or ÄKTA avant Instructions 29040427 AC
5 Preparation and configuration 5.3 Configure SparkLink 4 Click the Configure button. Connect Alias autosampler to ÄKTA pure or ÄKTA avant Instructions 29040427 AC 25
5 Preparation and configuration 5.3 Configure SparkLink 5 Select Communication type: Serial Select Serial port: COM1 Check that chosen communication port is active by clicking the Test button. Enter volumes according to installed hardware. If you have Alias Prep version, check the Prep mode box. Loop volume and Syringe volume will be fixed. NOTICE Buffer volume = volume in tubing, see Alias User Manual. For Alias options - valves, see Alias User Manual. Settings made in the SparkLink configuration overrides settings in ASM software, Click OK. 6 Save the configuration by clicking Save or Save as. 26 Connect Alias autosampler to ÄKTA pure or ÄKTA avant Instructions 29040427 AC
5 Preparation and configuration 5.3 Configure SparkLink 7 Click the View icon, select desired configuration and click Activate. Note: When opening the SparkLink configuration, the last activated configuration is used. Connect Alias autosampler to ÄKTA pure or ÄKTA avant Instructions 29040427 AC 27
5 Preparation and configuration 5.4 Configure I/O-box E9 5.4 Configure I/O-box E9 Follow the instructions below to configure I/O-box E9. 1 2 Start the UNICORN software. Select System Control:System settings:i/o-box, and set Digital out 1 to Value 0. Result: This setting will put the autosampler in a default pause (freeze) state. 3 Click OK. 28 Connect Alias autosampler to ÄKTA pure or ÄKTA avant Instructions 29040427 AC
6 Operation overview 6 Operation overview 6.1 Introduction This chapter gives an overview of how to operate the autosampler with an ÄKTA instrument. 6.2 Start a run Below is a description how to start a run. 1 2 3 4 5 6 7 Start the ÄKTA instrument as instructed in Operating Instructions. Start the UNICORN software and prepare the UNICORN method. See Section 7.5 Update UNICORN method, on page 40 or Chapter 8 Advanced operation, on page 47. Start the Alias autosampler SparkLink software. In SparkLink, create an autosampler method and setup a Runtable for the selected project. See Chapter 7 Method setup, on page 31 Prepare samples and load the autosampler. In SparkLink, Select a Runtable for the selected project. Click the submit icon to add the runtable to a Queue. Start the run by clicking the Start icon. Result: The autosampler program will start. The first sample will be placed in the Alias autosampler loop. The autosampler will then freeze and wait for commands from the I/O-box. Note: The autosampler should not be opened once the Queue has been started. Start the UNICORN method, method queue or scouting scheme. Connect Alias autosampler to ÄKTA pure or ÄKTA avant Instructions 29040427 AC 29
6 Operation overview 6.3 Monitoring and evaluation 6.3 Monitoring and evaluation The UNICORN Evaluation module visualizes and evaluates data recorded during a run. The illustration below shows the Chromatogram pane. First un-freeze signal Second unfreeze signal Sample injection Note: The injections marks in UNICORN will correspond to the turning of the ÄKTA instrument injection valve. 30 Connect Alias autosampler to ÄKTA pure or ÄKTA avant Instructions 29040427 AC
7 Method setup 7 Method setup 7.1 Workflow Using the Alias autosampler with ÄKTA pure or ÄKTA avant and I/O-box E9 requires the following stages: Stage 1 2 3 4 Description Set up an autosampler project (will contain Methods, Results and Runtables) Set up the autosampler method (i.e., injection parameters, injection mode, etc.) Set up the autosampler runtable (i.e., which vials/wells to inject from, and the injection volumes) Update the UNICORN method to include the autosampler. 7.2 Create an autosampler project Follow the instructions below to create an autosampler project. 1 Start the SparkLink Application software. 2 3 Select the Project tab and click New to create a new project. Enter a project name and click Create. Connect Alias autosampler to ÄKTA pure or ÄKTA avant Instructions 29040427 AC 31
7 Method setup 7.2 Create an autosampler project 4 Select a project: Click a Project node in the list of project in the left navigation panel. Click the Select icon on the Project tab. The project is now displayed in bold to indicate that it is selected. 32 Connect Alias autosampler to ÄKTA pure or ÄKTA avant Instructions 29040427 AC
7 Method setup 7.3 Create an autosampler method 7.3 Create an autosampler method Follow the instrictions below to create an autosampler method. 1 2 Select the Method tab, click Method Editor to create a new method for the selected project. The bottom right of the status line indicates which project is currently selected. To select the requested injection mode, click the Injection tab. Connect Alias autosampler to ÄKTA pure or ÄKTA avant Instructions 29040427 AC 33
7 Method setup 7.3 Create an autosampler method 3 Injection mode: Select Full loop Partial loop, or ul Pickup Injection volume: Can be selected in Partial loop and ul Pickup. In Partial loop, the maximum injection volume is 50% of the loop volume. In ul Pickup the maximum injection volume is ((loop volume 3 needle volume) / 2). Flush volume: 2 3 x the needle volume is recommended. Sample speed: Depends on the viscosity of the samples and can be selected in three settings: Low, Medium and High. Headspace pressure: Should be selected only if properly capped vials are used. It will assist the syringe in aspirating the programmed amount of sample. Air segment: Can be selected to create a good separation between the wash solvents and the aspirated sample. In the Full loop and Partial loop injection mode, the air segment will be aspirated into the tubing between the injection valve and the syringe valve. Be aware that an air segment in the ul Pickup injection method will be introduced into the sample loop and injected into the system. Needle height: Can be set in a range from 2.0 6.0 mm in steps of 0.5 mm. The 2.0 mm is the minimum value, where the needle will be penetrated as deep as possible into the sample vials. Depending of the shape of vial (bottom) a different height should be selected. Analysis time: Time can be set from 0.00 599.99 min. This is the countdown timer before starting to prepare for the next injection. Note: For the setup with the ÄKTA system, the analysis time should be set to 0.20 min. 34 Connect Alias autosampler to ÄKTA pure or ÄKTA avant Instructions 29040427 AC
7 Method setup 7.3 Create an autosampler method 4 To enable temperature control, click the Temperature control tab. If a temperature control option is installed, select the tab temperature control and program the required temperature for the samples. 5 To edit the wash parameters, click the Wash tab. Set the required wash volume to clean the flow path and sample needle. In case of a second wash solvent availability, a wash program can be programmed by selecting the wash solvent and the wash volume, with up to nine program lines. Add a washing program by clicking the + button. Delete a washing program, by clicking the - button. 6 Click Save in the Method Editor dialog. In the Save As dialog, enter a method name and click Save. Connect Alias autosampler to ÄKTA pure or ÄKTA avant Instructions 29040427 AC 35
7 Method setup 7.4 Create an autosampler runtable 7.4 Create an autosampler runtable Follow the instructions below to create an autosampler runtable. 1 To create a new Runtable, Select the Runtable tab and click New. Note: Active configuration and currently selected project are shown at the bottom right of the status line. 36 Connect Alias autosampler to ÄKTA pure or ÄKTA avant Instructions 29040427 AC
7 Method setup 7.4 Create an autosampler runtable 2 Run settings: Click the Alias icon and select plate type. Method: Select the desired method from the drop-down list. Number of samples: Enter the number of samples to be processed dependent of the maximum number in the selected type of plate. Injection volume: As default the value of the method will be used. Changing the injection volume in the runtable will overrule the injection volume of the method. Sample position: The first sample position can be selected in this field (e.g., A1) Sample plate: Select plate #1 or plate #2. Result file prefix: Default name is "Data". When completed, click Create. Result: The Runtable will open. Connect Alias autosampler to ÄKTA pure or ÄKTA avant Instructions 29040427 AC 37
7 Method setup 7.4 Create an autosampler runtable 3 In this screen, the selected methods, result files, injection volumes, sample positions, plate positions and sample ID are displayed. These values can be modified and additional samples can be added, if needed. 4 To check the sample positions and selected plate type, click Select samples. 5 Click Apply if plate type and sample positions are OK. To change plate type, click Cancel and then select the Alias icon on the ribbon. 38 Connect Alias autosampler to ÄKTA pure or ÄKTA avant Instructions 29040427 AC
7 Method setup 7.4 Create an autosampler runtable 6 To save the Runtable: Click the Save icon. In the Save as dialog, enter a Runtable name and click Save. Tip: The runtable can also be exported to a Microsoft Excel sheet. Click Export and select Excel file as the file type to save the runtable in Excel format. Connect Alias autosampler to ÄKTA pure or ÄKTA avant Instructions 29040427 AC 39
7 Method setup 7.5 Update UNICORN method 7.5 Update UNICORN method Note: Basic operation If the samples are temperature sensitive, follow the instructions described in Chapter 8 Advanced operation, on page 47. Initially the autosampler injection valve is in INJECT position. During basic operation we have two run states for the autosampler: Stage 1 2 State description Preparing for next injection 1 Needle goes to sample vial 2 Alias injection valve switches to LOAD 3 Loading sample loop with programmed volume Injection freeze 1 Autosampler is paused, waiting for the un-freeze signal (UNICORN digital out 1) to go from 0 (default) to 1 2 When the un-freeze signal is received, the Alias injection valve switches to INJECT and the sample loop is now part of the ÄKTA instrument flow path. The sample will be injected onto the column when the injection valve on the ÄKTA instrument is turned. 3 A wash routine is performed in the autosampler after each injection and a new cycle is started, back to stage 1 (see illustration below). AUTOSAMPLER STATE Preparing for next injection Injection freeze = 0 I/O box - Digital out signal = 1 = 0 UNICORN METHOD Method start Sample application Inject Post Sample application >>> Method END Un-freeze signal 0.5 s Note: To enable communication between the ÄKTA instrument and the autosampler, digital out signal has to be added in UNICORN software 40 Connect Alias autosampler to ÄKTA pure or ÄKTA avant Instructions 29040427 AC
7 Method setup 7.5 Update UNICORN method For UNICORN methods created with phases Create a user defined phase and add the instruction Pulse digital out 1 to activate/un-freeze the autosampler. Follow the instructions below. 1 2 Create/open the UNICORN method to be used. Create a user defined phase to activate the autosampler Place the new phase before the Sample Application phase. 3 Click the Text Instruction tab. In the user defined phase: Set base parameter to time. Insert the instruction Pulse digital out 1, length 0.5 sec Connect Alias autosampler to ÄKTA pure or ÄKTA avant Instructions 29040427 AC 41
7 Method setup 7.5 Update UNICORN method 4 Click the Phase Properties tab. In the Sample Application phase: Select to inject sample from loop. Set Loop type: Capillary loop. Empty loop with: 3 times the autosampler loop volume plus connecting tubing volume. Note: Connecting tubing volume is calculated for the tubing between port LoopF on the injection valve V9-Inj of the ÄKTA instrument and port 6 (column) on the autosampler valve. For UNICORN methods created as text methods Insert the Pulse digital out 1 instruction in the sample application block. Turn the injection valve to position Inject. Turn the injection valve to position Manual Load after a certain volume, depending on the loop size in the autosampler. 42 Connect Alias autosampler to ÄKTA pure or ÄKTA avant Instructions 29040427 AC
7 Method setup 7.5 Update UNICORN method Handle multiple sample injections When running methods with multiple sample injections this can be handled by creating a Scouting method or starting a Method queue. Scouting scheme: To create a scouting scheme to automatically handle several samples injections. Follow the instructions below. 1 Create a scouting scheme: Click on the Scouting icon and select for example Flow rate or Sample ID as scouting parameters. Add the wanted amount of runs. If desired, enter the Sample ID of the samples to be run. Connect Alias autosampler to ÄKTA pure or ÄKTA avant Instructions 29040427 AC 43
7 Method setup 7.5 Update UNICORN method 2 If sample ID has been chosen as scouting variable it can been used as result name. In the phase properties for Method Settings click on Result Name & Location. Select to use Variable as Result name. Select Sample_ID in the drop-down list. Method queue: As an alternative to scouting scheme, it is possible to create a Method queue. Create your method/methods in UNICORN and add them to a method queue. A method queue can handle up to 10 consecutive methods. 44 Connect Alias autosampler to ÄKTA pure or ÄKTA avant Instructions 29040427 AC
7 Method setup 7.6 Submit and start an autosampler runtable 7.6 Submit and start an autosampler runtable The next steps describes how to submit a runtable to a queue and start the queue. When clicking Start queue in SparkLink software, the first sample will immediately be loaded into the sample loop. Make sure to load the autosampler with samples and prepare the UNICORN method before starting the queue. To update the UNICORN method see Section 7.5 Update UNICORN method, on page 40 or Chapter 8 Advanced operation, on page 47. When the autosampler and UNICORN methods are ready, continue with the instructions below. 1 To add the runtable to the queue, click Submit. 2 The plate type and sample positions are displayed. Click OK to add the runtable to the queue. Connect Alias autosampler to ÄKTA pure or ÄKTA avant Instructions 29040427 AC 45
7 Method setup 7.6 Submit and start an autosampler runtable 3 To start processing the queue immediately, click Start queue. Note: The first sample will be loaded into the sample loop. Result: The autosampler is now ready and is waiting for an instruction from the UNICORN method to proceed to the next injection in the runtable. for an overview Note: Additional runtables can be added to an ongoing queue. Proceed with step 7 in Section 6.2 Start a run, on page 29. 46 Connect Alias autosampler to ÄKTA pure or ÄKTA avant Instructions 29040427 AC
7 Method setup 8 Advanced operation 8.1 Introduction By using the Analysis time setting in the autosampler method and not allowing the timer to finish countdown during the first un-freeze signal, the autosampler is prevented from starting the next injection cycle until the chromatography run has been performed. At the end of the UNICORN method the analysis timer countdown is re-activated and completed, leading to the start of the next injection cycle. In this way the samples are kept in the cold compartment until the next purification run starts. Note: When the autosampler runtable is started (first injection) it will immediately begin to load the first sample into the loop. 8.2 Run states Description During advanced operation three run states exist for the autosampler. Initially the autosampler injection valve is in INJECT position. Stage 1 2 State description Preparing for next injection 1 Needle goes to sample vial. 2 Alias injection valve switches to LOAD. 3 Loading sample loop with programmed volume. Injection freeze 1 Autosampler is paused, waiting for the un-freeze signal (UNICORN digital out 1) to go from 0 (default) to 1. 2 When the un-freeze signal is received, the Alias injection valve switches to INJECT and the sample loop is now part of the ÄKTA instrument flow path. The sample will be injected onto the column when the injection valve on the ÄKTA instrument is turned. 3 A wash routine is performed in the autosampler after each injection. Connect Alias autosampler to ÄKTA pure or ÄKTA avant Instructions 29040427 AC 47
8 Advanced operation 8.2 Run states Stage 3 State description Analysis timer running 1 Analysis time that has been set in the Alias method starts to countdown when the un-freeze signal is received. Analysis time countdown will pause/hold when the unfreeze signal ends. 2 Analysis timer is continued by sending a second un-freeze signal in the end of the UNICORN method. The timer will then continue to countdown remaining time. 3 When the analysis timer has counted down to zero a new cycle starts, back to stage 1. Illustration of the Run states Analysis timer running Tot 0.20 min = 12 s AUTOSAMPLER STATE Preparing for next injection Injection freeze 5 s Pause/Hold 7 s = 0 I/O box - Digital out signal = 1 = 0 = 1 = 0 UNICORN METHOD Method start Sample application Inject Post Sample application >>> >>> Method END First un-freeze signal, 5 s Second un-freeze signal, >7 s Note: Note: The analysis timer will only count down as long as UNICORN digital out 1 is set to 1 (un-freeze signal). When the un-freeze signal is not present (UNICORN digital out 1 set to 0), the countdown will pause/hold but the state for the autosampler will still be Analysis timer running and a new cycle is NOT initiated. To enable communication between the ÄKTA instrument and the autosampler, digital out signals have to be added in UNICORN software. 48 Connect Alias autosampler to ÄKTA pure or ÄKTA avant Instructions 29040427 AC
8 Advanced operation 8.3 Set up the methods for advanced operation 8.3 Set up the methods for advanced operation 8.3.1 Set up the autosampler method Follow the instructions in Section 7.3 Create an autosampler method, on page 33 and Section 7.4 Create an autosampler runtable, on page 36 to create an autosampler method and runtable. In the method, set Analysis time to 0.20 min. 8.3.2 Set up the UNICORN method Add two un-freeze events UNICORN method, see Section Illustration of the Run states, on page 48 Principle The principle of the method is described below. Stage 1 2 Description The first un-freeze event is located before the sample application phase and should have a duration of 5 seconds. This un-freeze event causes the injection valve of the autosampler to be set to inject position. The analysis timer starts to countdown but pauses/holds after 5 seconds. The autosampler is still in the state Analysis timer running, preventing the autosampler from starting the next injection cycle. The autosampler remains in this state until the second un-freeze event is sent to allow the Analysis timer to continue and finalize the countdown. The second un-freeze event is located at the end of the method and should have a duration of 0.40 min, to allow the analysis timer to continue and count down the remaining time, including delay times. When the analysis timer has counted down to zero a new cycle starts and the next sample is placed in the autosampler loop. Connect Alias autosampler to ÄKTA pure or ÄKTA avant Instructions 29040427 AC 49
8 Advanced operation 8.3 Set up the methods for advanced operation 8.3.2 Set up the UNICORN method Instruction Follow the instructions below to create the method. 1 2 Create two user defined phases. Insert the First "unfreeze event" phase before Sample Application and the Second "unfreeze event" phase at the end of the method. 50 Connect Alias autosampler to ÄKTA pure or ÄKTA avant Instructions 29040427 AC
8 Advanced operation 8.3 Set up the methods for advanced operation 8.3.2 Set up the UNICORN method 3 Open the instruction box and edit the user defined phases as shown and described below: Unfreeze Set autosampler to inject (First unfreeze event) Set base parameter to time. Insert the instruction Pulse digital out 1, length 5 sec. Set Breakpoint for End_Block to 0.20 min. Unfreeze, Prepare for next injection (Second unfreeze event) Set base parameter to time. Insert the instruction Digital out 1 =1. Insert the instruction Digital out 1 =0 at breakpoint 0.40 min. Set Breakpoint for End_Block to 0.50 min. Connect Alias autosampler to ÄKTA pure or ÄKTA avant Instructions 29040427 AC 51
8 Advanced operation 8.3 Set up the methods for advanced operation 8.3.3 Additional information and tips for the UNICORN method 8.3.3 Additional information and tips for the UNICORN method Delay sample application in UNICORN method Miscellaneous phase - Pause timer If the sample loading for the next injection does not finish before the UNICORN Sample Application takes place, a pause needs to be introduced. This is achieved by adding a Miscellaneous phase before Equilibration. In the Phase Properties dialog, check the Pause timer checkbox and set the time as indicated in the table below. Note: The time should be set so that the UNICORN runtime before Sample Application and the pause time are sufficient for the autosampler to finish loading the sample in the loop. The table below shows the time needed for the autosampler to fill the loop depending on the injection volume. Injection volume (ml) 0-1 1-2 2-5 5-10 Time to fill the loop (sec) 30 46 120 240 52 Connect Alias autosampler to ÄKTA pure or ÄKTA avant Instructions 29040427 AC
8 Advanced operation 8.3 Set up the methods for advanced operation 8.3.3 Additional information and tips for the UNICORN method Miscellaneous phase - Method delay Alternatively, the Method delay option can be used. In that case a delay volume has to be set. Note that you must calculate how long time the set delay volume represents, considering the current flow rate. Adapt length of UNICORN method prior to sample application: Another alternative is to omit the Miscellaneous phase and to adapt the length of the phases prior to the sample injection. For example, extend the duration of the equilibration phase so that the time is sufficient for the autosampler to fill the loop. Note: If samples with different volumes are scheduled to run, a scouting table could be created to adapt the pause timer, method delay or other parameter used to ensure that there is sufficient time for the autosampler to fill the loop before the sample injection. Add error handling phase in UNICORN Optional, a user defined phase for autosampler error handling can be added. Insert the error handling user defined phase directly after the Method Settings phase. Connect Alias autosampler to ÄKTA pure or ÄKTA avant Instructions 29040427 AC 53
8 Advanced operation 8.3 Set up the methods for advanced operation 8.3.3 Additional information and tips for the UNICORN method In the error handling example shown below, the ÄKTA pure instrument is set to Pause and it displays a message to the user. Because this is done with a Watch command, multiple Watch commands have to be added to manage more then one error event per run. 54 Connect Alias autosampler to ÄKTA pure or ÄKTA avant Instructions 29040427 AC
9 Direct control and maintenance 9 Direct control and maintenance Introduction SparkLink also offers the possibility for: Direct control Maintenance, and Service of the Alias autosampler. Direct control If a queue is still active, Direct control buttons will not be active. Make sure to pause the queue before opening Direct control / Service. 1 2 In the menu bar click Direct Control. Click the arrow below the system picture and select Direct Control. Connect Alias autosampler to ÄKTA pure or ÄKTA avant Instructions 29040427 AC 55
9 Direct control and maintenance 3 In the Alias - Direct control screen, the sample cooling, sample tray and washing can be selected. Sample cooling: Sample cooling option (if installed) can be turned on/off. It displays the actual sample temperature and set point. Sample tray: The sample tray can be moved to the front, to load the sample racks or micro plates and the tray can be moved to the rear position. Tip: By opening/closing the door of the Alias, the same functionality will be achieved. Washing: By clicking the Initial wash button, the Initial wash will be started and will rinse the needle, the buffer tubing and the syringe. If the second wash port is activated in the settings, the second wash solvent can be also used to rinse the needle, the buffer tubing and the syringe. Tip: Perform an initial wash manually before starting a method. SSV priming: In case of an installed Solvent Selection Valve, the SSV priming menu can be used to prime all lines. The Initialize button can be used to initialize the autosampler, where all the modules will be set into the home position. It can also be used to reset an error state. 56 Connect Alias autosampler to ÄKTA pure or ÄKTA avant Instructions 29040427 AC
9 Direct control and maintenance 4 In Alias - Direct control screen, click System info. The System info screen is displaying the autosampler hardware information, like firmware version and serialnumber. This information might be requested by the technical support department of the manufacturer in case of an upgrade or service action. Connect Alias autosampler to ÄKTA pure or ÄKTA avant Instructions 29040427 AC 57
9 Direct control and maintenance Maintenance 1 Select Maintenance. 2 In the Alias Maintenance control, there are four more screens available for controlling the autosampler. 3 I/O screen: In the screen, the Auxiliary #1 can be switched On or Off. This function can be used to check if the hardware connection to another device is properly made. The integrated compressor can be switched On or Off, by selecting the PASA Compressor. 58 Connect Alias autosampler to ÄKTA pure or ÄKTA avant Instructions 29040427 AC
9 Direct control and maintenance 4 Valves screen: The screen, displays the position of the Injection valve, syringe valve and the optional Solvent Selection Valve. Positions of the valves can be changed and will be displayed accordingly. Connect Alias autosampler to ÄKTA pure or ÄKTA avant Instructions 29040427 AC 59
9 Direct control and maintenance 5 Log counters screen: The autosampler Log counters screen provides the details on the movements of the Injector valve, the Syringe valve and the Syringe. A service technician can reset if one of the modules have been replaced. 60 Connect Alias autosampler to ÄKTA pure or ÄKTA avant Instructions 29040427 AC
9 Direct control and maintenance 6 Syringe / needle screen: Syringe and needle can be moved to Home, Exchange and End positions Related positions of needle and tray can be adjusted. Syringe can be adjusted. Connect Alias autosampler to ÄKTA pure or ÄKTA avant Instructions 29040427 AC 61
9 Direct control and maintenance 7 System info: Displays information on Instrument firmware and used Driver. 62 Connect Alias autosampler to ÄKTA pure or ÄKTA avant Instructions 29040427 AC
For local office contact information, visit www.gelifesciences.com/contact GE Healthcare Bio-Sciences AB Björkgatan 30 751 84 Uppsala Sweden www.gelifesciences.com/akta GE, GE monogram, ÄKTA and UNICORN are trademarks of General Electric Company. Alias is a trademark of Spark Holland BV. Excel, Microsoft and Windows are registered trademarks of Microsoft Corporation. Images of the Alias autosampler and dialogs from the SparkLink software are reproduced with permission from Spark Holland BV. The SparkLink software and the Alias autosampler are non GE Healthcare products manufactured and sold by third parties and GE Healthcare assumes no liability whatsoever for the performance or use of these products. Any use of UNICORN 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. UNICORN 6 2009-2016 General Electric Company 2013-2016 General Electric Company First published Apr. 2013 All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them. A copy of these terms and conditions is available on request. Contact your local GE Healthcare representative for the most current information. GE Healthcare Europe GmbH Munzinger Strasse 5, D-79111 Freiburg, Germany GE Healthcare UK Limited Amersham Place, Little Chalfont, Buckinghamshire, HP7 9NA, UK GE Healthcare Bio-Sciences Corp. 100 Results Way, Marlborough, MA 01752, USA GE Healthcare Dharmacon, Inc. 2650 Crescent Dr., Lafayette, CO 80026, USA HyClone Laboratories, Inc. 925 W 1800 S, Logan, UT 84321, USA GE Healthcare Japan Corporation Sanken Bldg. 3-25-1, Hyakunincho Shinjuku-ku, Tokyo 169-0073, Japan 29040427 AC 05/2016 a346