Liquid Handling Application Designer V2.0. Software Manual P/N M5200R0

µcarrier Liquid Handling Application Designer V2.0 Software Manual P/N 163801 www.lcpackings.nl M5200R0

User Interface...2 Main Window... 2 Main menu... 2 Main toolbar... 3 The Browser Pane... 4 Sample Carrier Editor... 5 Rectangular:... 6 Circular:... 6 Irregular:... 6 Tray Editor... 7 The tray editor toolbar... 7 Application Editor... 10 Available tasks are... 11 Task list... 12 The tasks... 13 Fraction collection tasks... 13 Basic Fractionation... 13 Advanced Fractionation... 14 PlanarFrac... 19 Liquid handling Tasks... 21 Aliquot task... 21 Duplicate Task... 27 Pipet Task... 32 Multipurpose Tasks... 36 Aspirate Task... 36 Dispense Task... 37 MoveTo Task... 38 Switch Task... 40 Wait Task... 40 Wash Task... 41 Sequential/Batch Mode... 41 Sequence Task... 41 SequenceEnd Task... 42 ZTool... 42 Manual Device Control... 45 XYZ Robot Module... 45 Dosage module... 48 Drive panel... 49 Video Camera... 50 Device Settings... 51 Settings Window... 53 Main... 53 Directories... 56 Controls... 57 Robot... 57 Dosage Module... 59 RS232 / USB... 61 About... 63 M5200R0 1

User Interface This chapter presents a detailed description of the user interface including the options and buttons of the µcarrier V2.0 software. It should be used as a reference by individuals who use the software. Main Window The µcarrier software is a 32 bit Windows application. It has a main window from which three Editors can be opened (Sample Carrier Editor, Tray Editor and Application Editor) and a window for the manual device control (robot and the syringe pump module). In addition, there are several tool and parameter windows. The following screen appears when starting the software after installation. µcarrier will load and show the last saved application (if available) on all consecutive software starts. Main menu The main menu is located on top of the main window. The main menu offers functions to start a new file or to open and save an existing file under File. The Editors can be accessed (sample carrier, tray and application) via the View menu. The audit trail and the camera view option are also accessible under this option. If desired, the tool tips can be enabled via this menu. In addition the user can exit the program. M5200R0 2

The instrumental settings, the sample carrier alignment and the manual device control are located under Tools. Documentation on the Probot and µcarrier software can be found under Help. Main toolbar The main toolbar is located directly under the main menu and offers buttons for several main functions. / / The name of the buttons is given together with a description of their function is presented below. New: Removes the tray and all tasks in the task list. The wizard corresponding to the active editor is started. Browser: This button shows (hides) the browser pane on the left of the screen to enable (disable) easy file access. Open: Opens an existing sample carrier, tray table or application file. The sample carrier files have the extension.rck, the tray files have the extension.try and the application files have the extension.app. Save: Saves a (new) sample carrier, a tray table or an application. Activates the Sample Carrier Editor to create new or modify existing sample carriers Activates the Tray Editor to create new or modify existing trays. Tray layout and spot grouping are available in this editor. Activates the Application Editor to create new or modify existing applications. Grouping of sample carrier spots is also available. Activates the Manual Device Control to manually control the Probot table and dosage module. Software Drive panel: Opens the manual control panel of the robot drive. Run: Runs the application. All tasks are executed in the order they are placed in the task list. Pause: Pauses the application. The application can be unpaused by a click on the run button or on the pause button (only active during run). Stop: Aborts the application and homes the device (only active during run). M5200R0 3

The Browser Pane The browser pane is visible on the left of the screen, allowing easy file access. The file list includes the file types that can be used in the active editor. Each file type lists the relevant files of that type (e.g. only tray files are displayed via in the tray editor).. Folder browser: Folder structure of the computer. Browsing through the folder is done in the same way as in the Windows Explorer. File browser: A list of available files in the selected folder is given. Only the files that can be used in the selected editor are displayed, When selecting a folder without any tray files (e.g. a folder with sample carriers), the file browser will display a message as shown below (or similar error message) indicating the absence of correct files. Adding sample carriers to the tray is also done with the browser pane, but it is first necessary to press the button for adding a M5200R0 4

sample carrier. Refer to the section about the tray editor for more details (page 1-7). Sample carriers and Trays are loaded with a single click. Applications are loaded with a double click Sample Carrier Editor Sample carriers are created or modified via the sample carrier editor. Folder list Sample carrier settings File list, single click to load the sample carrier Sample carrier Preview Standard sample carriers for various MALDI instruments are included in the Sample Carrier folder (and sub folders) of the software. The browser shows a list of the available sample carries. A preview of the sample carrier is shown via a mouse click on the name. As an alternative, a custom made sample carrier can be created by specifying the size, shape and arrangement of the spots. The shape and size of the spot must be specified (circular or rectangular). The height of the spot is important if overfill control during the fractionation is activated, in this mode the robot will move to the next spot if the volume of the fractionated liquid has reached the spot volume. M5200R0 5

The arrangement of the spots can be rectangular, circular or irregular. Rectangular: In the rectangular mode, the number of columns and rows and the distance between the spots in the X- and Y- direction (mm) are specified. If spots in consecutive rows/columns are shifted, an offset can be used. The A Z character label can be assigned to either columns or rows, depending on the spot identification on the MALDI target (e.g. A1, 1A or 11) Circular: Enter the number of circles that make up the sample carrier and then press Enter. Once you have indicated the number of circles, then fill in the fields for radius [in mm], the number of sample positions on that circle and the angle in 0...360, by which the first position is turned out of the "north" direction. An angle of 30 for instance, puts the first sample position on the one o clock position and all other positions on that circle are determined in that manner (e.g. 60 o is equivalent to two o clock). The numbering direction of the positions on each circle clockwise = "CW" or counter clockwise = "CCW" is specified in the last column. The default is CW = clockwise. The button [Update] updates the preview. The button [Clear List] clears all values in the list. Irregular: Put in the number of spots on the MALDI target and press Enter. Put in the relative coordinates in X, Y,Z [mm] to the origin point for each spot of the sample carrier The button [Update] updates the preview. The button [Clear List] clears all values in the list. M5200R0 6

Tray Editor The Tray Editor is used to define the arrangement of the sample carriers on the MALDI target. In addition, grouping of the spots into desired target areas can also be performed. This last feature is also available in the Application Editor. Predefined trays are supplied with the µcarrier and they can be loaded through the browser pane as described above in Section Sample Carrier Editor Folder list Tray Preview File list, single click to load the tray When clicking the right mouse button on the preview windows a context menu appears. This menu gives the same options as the toolbar, which is explained below. The tray editor toolbar In Edit Mode Zoom-in and zoom-out: These buttons can be used to zoom in or out on the tray. If vial numbering of the sample carrier is not visible, the zoom-in function can be used to obtain this information Toggle the display between alphanumerical vial labels and the group numbering. This feature changes the standard numerical notation into an alphanumerical notation (like a microtiterplate: A1...A12, B1...B12.H12). Group/Edit: Allows switching between edit mode to change M5200R0 7

the tray composition and the group mode to select groups. Add a sample carrier to the tray. This button shows all available Sample Carrier files in the browser. Click once on the sample carrier to indicate that you want to load it. Then double click on the selected sample carrier to add it to the tray. Simply double clicking on the sample carrier is not possible, it is necessary to single click, then double click.. Z-movement of sample carrier: The sample carrier Z- coordinate can be changed by the mouse-wheel. Three different magnitudes can be chosen, from left to right: 0.01 mm, 0.1 mm and 1 mm Move the selected sample carrier to the current X, Y, Z coordinate. This function is used to calibrate the Probot. For more information see Chapter 3: Getting Started Rotate the selected sample carrier by 90 in the clockwise direction. Shows the X, Y, Z position of the sample carrier. Shows a grid in the tray editor to facilitate position adjustments. Lock/unlock function enables or disables the manual positioning of the sample carrier. Undo and redo function for position changes or modifications of the sample carriers and for grouping of sample carriers. Cut, copy and paste functions. A sample carrier must be selected before you can make use of these functions. This is done by drawing a frame around the upper left corner of the sample carrier with the left mouse button. In Group Mode: Add/delete a group: A new group is added to the group list with the name NewGroup. Clear: remove all vials/spots from a group Select the order of the pattern to be followed during fractionation. The group list shows all groups present on a tray. M5200R0 8

The name of a group should correspond to the function of the group and can be changed by a left mouse click on the name. The group count can be changed by selecting the group in the group list and using the mouse to draw a frame around the sample carriers that should be in the group. Groups are not limited by sample carrier boundaries; it is possible to put part of a sample carrier, the complete carrier or several sample carriers in one group. The color can be changed by a left mouse click on the color indication in the group list. The type of group, Reagent or Sample, is changed by a left mouse click. A Reagent group consists of one spot that can be used several times, A Sample group consists of (one or) several spots, but each spot can be used only once. The Users column indicates in how many tasks this group is used. M5200R0 9

Application Editor The Application Editor is used to create and modify Fractionation or other liquid handling applications and can be accessed from the main menu. List of available tasks, Single click to get a description. Double click to add the task to the task list. Task list with all tasks that are performed during run. Task description or task parameters. Group mode of the tray editor. The available tasks are listed on the left part of the Application Editor window. A left mouse click on the task gives the explanation; a double left mouse click includes the task in the application task list. The group mode from the tray editor is also available on the bottom part of the screen. The tasks are grouped by similar function and they are listed below with a short description: A more detailed explanation about the task parameters follows. M5200R0 10

Available tasks are Fraction collection Basic Fractionation: This task performs a fractionation based on a timed collection. The settings to be specified have been minimized to the settings required for 90% of all nano spotting. Advanced Fractionation: This task performs the fractionation of the eluate from the Ultimate system with all settings to be specified. Collection is not limited to time, but external or manual control is also available to collect only the peaks. Dual spotting on two different targets can be done and a customizable report is available. Planar Frac: This task performs a planar fractionation, also known as streaking. In this mode the eluate is collected as a single line. Different patterns in which the line should be drawn on the planar target can be selected. Liquid handling Aliquot: Aspirate from a vial on the tray or from a syringe valve channel and distribute equal portions of the liquid into the vials of a group on the tray. Optionally perform a built-in washing step afterwards. Duplicate: Duplicate a group of samples to one or multiple destination groups. The Duplicate task is used for microplate reformatting and microarray spotting. Pipet: Aspirate from a vial or group of vials on the tray or a syringe valve channel, dispense into a group of vials on the tray or into a syringe valve channel. Pipet normally is used to perform a 1 to 1 -transfer of samples from one group of vials into another group of vials. Multipurpose Aspirate: Aspirate liquid into the syringe from a channel of the syringe valve. Dispense: Dispense liquid into a channel of the syringe valve. MoveTo: move the needle in up to three axis directions (X, Y, Z) into specified positions, relative to the current position into the vials of a group with a specified speed. Switch: Switches relay contacts of the robotic device. Wait: Waits on a user keystroke, time to elapse or a contact closure signal Wash: Performs a washing step. M5200R0 11

Sequential/Batch Mode Sequence: Starts the sequential execution of tasks. In sequential mode all tasks in the sequence are performed on one spot before moving to the next spot. In batch mode a task is performed for all its spots before moving to the next task SequenceEnd: Ends a sequentially working task group. Task list After a task is selected by double clicking on it in the list of available tasks, it is added to the task list When a task is selected in this list, the task parameters are displayed on the right and all conditions of the fractionation can be specified. A task can be moved into the task list by dragging and dropping. Select the task by clicking on the first column (Nr.) and drag the number to the desired location in the task list. Tasks can be copied by right clicking and selecting copy or pressing CTRL + C. Pasting is done by right clicking and selecting paste or CTRL + V. Multiple tasks can be pasted at once by copying each task to the clipboard without intermediate pasting. The tasks will be pasted in the order they were copied to the clipboard. Deleting tasks is done by right clicking on the task and selecting delete or select the task and press DEL. M5200R0 12

The tasks In the following section a description is given of every task. The detail of the various descriptions that is provided is dependent on the importance and the complexity of the task. Fraction collection tasks The fraction collection tasks are used for the collection of fractions from a liquid chromatography system. The fraction collection tasks can only work in batch mode (normally active). This means that a fraction collection task can not be embedded in a sequence. See 'Sequential/Batch mode' explanation for the difference between batch and sequence mode. Basic Fractionation General The Basic Fractionation task is used to collect LC-fractions on a MALDI-target with a minimum of variables to be set. This task is intended to cover 90% of all timed fractionations and is much easier to setup than the advanced fractionation task which provides complete flexibility. In the basic fractionation task, a timed collection with a start delay of 0 seconds is used and a start signal should be given. This means that the fractionation will start immediately after the start signal is received. Matrix addition speeds are based on the column flow and are set in a ratio ranging from 1:1 until 1:5. Commands Flow of the chromatographic system [µl/min]. Specify the column flow. This value will be used to calculate the matrix flow according to the set ratio if matrix addition is enabled. Add matrix during collection 1-5x flow of the LC system. Enable the matrix addition and set the flow here. The matrix can be added in a 1:1 ratio up to five times the column flow (1:5) Dispense 10 µl prior to injection. During needle rest in the wash vial, dilution of the matrix in the needle occurs. Enable the dispense step to compensate for this dilution. Collection time [min]. The time elapsed after the start signal at which the collection should stop, regardless the remaining number of available spots. If no more spots are available prior M5200R0 13

to this time, the run will stop. Time per spot [s]. The fractionation time and the movement of the needle towards a spot are counted as the collection time for that spot. Time collection = Time movement + Time _on_spot Collect group. The target area for the spotting. Standby position. The group (single spot, reagent type) at which the needle should wait for the start signal between runs and should rest at the end of the run. Z-coordinate for in-task movement [mm]. The height to be used for spot to spot movement within a task. The movement between tasks occurs at Z = 0 mm. The height is an absolute value, counted from the lowest position of the robot. Advanced Fractionation General The Advanced Fractionation task is a complete program within the µcarrier software, due to the large number of parameters and different modes of operation. This task changes the liquid handler into a high precision multi purpose fraction collector. Time, manual or contact signal control are possible control modes for the fractionation. When using one of the last two control modes, it is possible to discard the waste and collect only the peaks. When a dosage module is installed, a reagent can be added during the fraction collection. One of the most used applications is the addition of matrix solution for MALDI MS during the fractionation. The choice for a second collection group can be made, so one run is collected on two different (types of) sample carriers. The Advanced Fractionation task is designed for versatility and flexibility in contrast to the Basic Fractionation task, which is designed for simplicity. Four different tabs General, Destination, Relays table and Collection Report are used to set up a fractionation with the advanced fractionation task. All tabs will be described in detail. M5200R0 14

General tab Column Flow [µl/min]. Specify the flow of the liquid delivering system. This value, together with the dead volume is used to calculate the delay between a collection event and the action of the fraction collector. Dead volume between detector and needle outlet [µl]. Specify the dead volume of the capillary between the needle outlet and the detector cell delivering the collection controlling signal. Remember: The volume of a tube in [µl] is: length [mm] * radius 2 [mm 2 ] * 3.14. Repetitions of the collection cycle into the same spots. Specify the number of repetitions of the collection cycle for repetitive purification of the chromatographic sample. The task will be repeated until the number of repetitions is over. Collection start delay after sample injection [s]., The start of the spotting can be delayed via this waiting period until the so called "solvent peak" has passed. When the start signal can not be delayed in the chromatogram program, this wait time can be used to introduce the desired delay. Multiple injection tracking. This option is checked to collect multiple injections in one task with the same parameters. The collection cycle for one sample is ended, when a contact closure on input 4 occurs or when the chromatography time [min] is overstepped. Multiple LC sample injections are collected in consecutive spots in the same group. The task will be executed for consecutive injections, until there are no more available spots. Chromatogram [min]. The time past the start signal at which the collection should stop, regardless the remaining number of available spots. If there are no more spots are available, the run will stop before this time is reached. Add reagent to all primary fractions [µl]. This checkbox enables the settings for reagent addition. The addition of reagent can be done Continuously or in a Discrete volume to every fraction. Dosage speed [µl/min]. The speed at which the reagent should be added. The speed also applies to the discrete volume addition and there is no check to M5200R0 15

see if the volume can be applied in the set spotting time. As an example, a 10 µl/min, 20 second spot time and 5 µl reagent addition will result in 3.3 µl reagent addition to each spot. Refill syringe after each discrete dispense step. This option enables the addition of larger volumes without emptying the needle. However, filling the needle takes time which should be taken into account in setting the dosage speed with discreet volume addition. Dispense 10 µl prior to injection. During needle rest in the wash vial, dilution of the matrix in the needle occurs. Enable this dispense step to compensate for this dilution. Collection control mode: Specifies the collection control mode. This can be - Time - Manual - External In the time mode, the entire chromatogram is divided into equal time segments, which are collected sequentially. In the manual mode, the task parameter window is shown and enlarged when the task starts. The user toggles between "collecting / waste" or "advance to next fraction" by clicking the mouse in the enlarged task panel or by pressing any key of the computer keyboard. This allows the user to decide if a peak should be collected or not while keeping ones eyes on the monitor to watch the chromatographic data acquisition In the external mode, the task reacts to contact closure signals at the liquid handlers inputs that indicate the start of a peak (input nr. 1), the end of a peak (input nr. 2), and the top of a peak (input nr. 3). Destination tab Continue fraction counters from previous fractionation task. With this option enabled, it is possible to use the same collect group in different advanced fractionation tasks in order to collect multiple injections in that group. The same collect group has to be specified in each task and the collection will continue behind the previous task. M5200R0 16

Use special Z-coordinate for X,Y-task movements [mm]. The speed of the liquid handler movements inside the task operations can be optimized, when the Z-coordinate is set to value bigger than 0. In this mode, the table is not completely lowered before the X, Y-move. Caution: make sure, that there is nothing on this level in the way of the needle! Primary collect group. Use the up/down control to select a group for the collection of the fractions. Make sure that the type is sample by checking the group in the GroupEditor,. If this is not done, the task will end after the first collected fraction. In the group color field, the number of vials is given. This is equal to the maximum number of fractions in one collection cycle. Lift needle. This option raises the needle slowly out of the vial during collection in order to avoid outer contamination of the collection capillary. Overfill control. When this option is checked, the table automatically changes to the next vial of the group when the maximum volume of the vial has been reached during collection. The volume is known by the vial description of the sample carrier. Overfill control will not work properly with sample carriers that have zero volumes (e.g. most MALDI targets). Use the sample carrier editor to verify if the sample carrier is zero volume. Secondary collect group: This is a group,where a small portion of each primary fraction can optionally be collected. In practice, this is done to collect peptide fractions on MALDI-MS targets and, parallel to that, on a PVDF membrane for sequencing analysis. Collect time at secondary group [s]. The time the needle fractionates on the second group, after that time it will move back to the first group and finish the remaining collection time on that spot. Change to secondary group at peak top. With external control mode input nr 3 is used as the peak top signal. When this signal is received the needle will move to the second group and will collect for the time specified at Collect time at secondary group [min]. Change to secondary group after time [s]. When time control mode is used, this field specifies the time to move to the next group Collect all / Collect peaks only. With this option the inter-peak fractions can be discarded or collected in a different group for separate analysis. This option is not enabled in time collection. M5200R0 17

Relays Table Use vacuum waste removal. If the hardware setup enables vacuum waste removal [special kit] the interbeing spotted or discarded. When this option is enabled, the go to waste position signals are used to start or stop vacuum removal. Use waste vial counter. When checked, the waste group uses its own vial counter independently from the number of the collected fractions. If unchecked, the waste vials are used parallel to the primary collection positions. That means, when the collection is just in the 15 th vial of the primary group, the waste after that peak is then done into the waste vial number 15 no matter whether the waste vials before have been used or not. Waste group. To specify where the inter-peak fractions shall be collected, use the up/down control of the waste group selector. The waste group is used only when the "Collect peak only" option is selected. Standby position before and after collection cycle. When you have selected a group, the first vial of that group will be used as a park position, when the collection cycle is over. This can be used to collect the HPLC medium separated from the other waste in the time after a collection cycle. Wait for contact start signal at input nr. 0=START. This option will pause the fractionation at the start until a signal is received on the start cable. Usually this signal is given by the Ultimate pump. Use relay no 1 as READY FOR INJECTION output. The specified relay can be used to send a signal to the Ultimate system when the Probot is ready. Use relay no. 4 as marker for collection events. Pulse [s]. This option offers the selection if the liquid handler contact output nr 4 is to be used as a event marker for the fraction collection events and to indicate how long it is pulsed for this purpose The relays table can be used to selectively turn on and off the relay outputs of the Probot to control other devices. The collect column can be used to collect only parts of the chromatogram. There are three options On: Go to the next collection spot Off: Go to the (next) waste spot End: Stop the collection M5200R0 18

Collection Report Sample Identifier. The inputs in this field are used in the collection report to specify the sample, by name or number. The collection report filename will be based on the sample identifier; otherwise a name will be used, based on the application filename. Make report file for a batch of LC samples / Make a separate report file for each LC Sample. This button allows the user to choose to make one report file for all tasks in one application or to generate a report file for each task. The columns to be recorded in the report file can be selected. by right clicking in the bottom field PlanarFrac The PlanarFrac task is used to collect a fraction from a LC system on a planar surface. No vertical movement occurs with this task. The result is one long 'line' of sample in which every region represents a certain part of the chromatogram. General tab Duration of the collection time [min]. This is the time measured from the start of the run (start signal when enabled) that the collection should take. The command i can be used to limit the collection (but not spots) and the movement speed will be adapted to the time and distance. Calculated movement speed [mm/min].this indicates the speed of the needle across the target surface. Calculated travel path length [mm]. This indicates the total distance the needle will travel. The distance is based on the type of collection (chosen in Mode) and the dimensions specified on the second tab (either Rectangular or Circular) Wait for a contact start signal at input nr. 0=START. This option will pause the fractionation at the start until a signal is received via the start cable. The signal is usually given by the Ultimate pump. Move to the robot park position at the end of collection. The park position is specified in the manual device control. Enable this option to move M5200R0 19

the needle away from the end point of the collection. If this command is not enabled, the continued LC flow could flood the target, ruining the fractionation Continuously add reagent [µl/min]. Enables and sets the speed for reagent addition during collection Start position. This is the group (single spot, sample type) where the collection should start. Specify both this group and the Mode to make sure the needle will move the right direction. Mode. This specifies the pattern of the collection., Four different patterns are available. Rectangular snail Circular snail Rectangular meander Circular single Depending on the chosen pattern, either the Rectangular or Circular tab will be displayed Rectangular tab Height of the rectangle (dimension in Y- direction)[mm]. This field indicates the outer area in Y, measured from the starting position, in which the rectangular pattern will be drawn. Width of the rectangle (dimension in X- direction)[mm]. This field indicates the outer area in X, measured from the starting position, in which the rectangular pattern will be drawn. Windings of rectangular snail trail. This field indicates the number of rings in the continuous snail trail. The rings will become smaller and smaller. Number of meander loops from rear to front. The number of lines that make up the meander pattern. Snail direction. The streaking can be done clockwise or counterclockwise Meander direction. The lines of the meander pattern can be drawn horizontal (Rear to front) or vertical (Right to left). M5200R0 20

Circular tab Diameter of the outer circle [mm], which is the maximum dimension of the streaking area. The starting position is on this outer circle and not in the centre. Number of snail windings on a radius. The number of rings in the continuous snail trail. The rings will become smaller and smaller. Circle direction. The streaking can be done clockwise or counterclockwise Liquid handling Tasks The liquid handling tasks are used for common liquid handling steps for sample preparation. These tasks can be used to divide, duplicate or move the content of one sample carrier to another sample carrier or several other sample carriers. This operation can be done for one or several spots on the sample carrier. Aliquot task The Aliquot task is used for the distribution of equal aliquots of a reagent in all vials of a sample destination group. Example: Preparation of a MALDI-MS-target with matrix solution. M5200R0 21

Source, Destination The reagent can be taken up either from a vial on the tray or from one of the valve connectors of the syringe. The task has two tabbed pages, one is called "Source, Destination", the other one is called "Air segment, Wash". Procedure: The task aspirates the specified number of air segments plus an extra-aspiration volume from the reagent source group. This is done only when the reagent is taken from the robotic tray and not from one of the other syringe valve connectors. The number of aliquots which are in the syringe is determined from the reagent source group. Those aliquots are dispensed one after the other into the vials of the destination sample group. After that, the air segments and the extra-aspiration volume are discarded in the wash group vial and then the optional wash step is executed. This procedure is repeated until all of the destination sample group vials are completed. Batch & Sequential Working Mode The Aliquot task is designed to work in batch mode, i.e. outside a sequentially working group of tasks (See: Sequence & SequenceEnd tasks) and it can also be used inside such a group of tasks. When the Aliquot task is used inside a sequence, it works like a pipet task, doing one transfer of liquid to one the destination positions, then executing the next task within the sequence. M5200R0 22

Aspiration speed [µl/min]: This parameter can be adjusted with the slide control - a low and a high speed range are available. The optimal aspiration speed depends on the viscosity of the liquid and the hydrodynamic resistance of the flow path, i.e. diameter and length of the capillary connecting the syringe with the source. The aspiration speed never should be so high that degassing occurs. Gas bubbles in the flow path decrease dramatically the volumetric accuracy of the system. Aspirate Liquid from: Select the position of the syringe valve when aspirating the liquid. If you intend to aspirate the reagent from a sample carrier on the tray, the position must be "Needle". Select a group: Click on the up/down control and select one of the groups you defined in the GroupEditor,when you wish to aspirate from a sample carrier on the tray. Liquid Level following: Check this parameter if you want the needle to follow the sinking liquid level during aspiration (this feature not available with all liquid handlers and is only valid when aspirating from the tray). Dispense volume [µl]: Put in the aliquot volume that the task should dispense at each vial of the destination sample group. Dispense speed [µl/min]: Adjust this parameter with the slide control.. Two ranges are available, a low and a high speed range. Adjust the dispense speed so that an overpressure in the system cannot occur and that no liquid is spilled or spilled from the destination vial Select a group: Click on the up/down control and select one of the groups that you defined in the group editor when you wish to dispense to a sample carrier on the tray. Liquid Level following: Check this parameter if you want the needle to follow the rising liquid level while the liquid is being dispensed (this feature is not available with all liquid handlers). M5200R0 23

Air Segment, Wash This page contains parameters and options for the minimization of the carryover of liquid, for the optimization of the volumetric accuracy and the working speed. This dialog box offers two main input fields, one for the air segment(s), which is only used when the source is a group on the tray, and one for the optional washing step after each series of aliquoting. The air segment is aspirated on the way to the source reagent group. If the number of air segments is greater than 1, the liquid handler aspirates between two air segments an air segment volume of the reagent, which is to be aliquoted. Each air segment lowers the carry over of syringe liquid into the last aliquot volume by a certain factor. This factor depends in a complex manner on the surface tension of the syringe liquid, and the wetting properties of the inner surface of the capillaries used for the liquid transfer. The liquid carryover across a moving air segment during aspiration comes from the removal of small droplets from the backward moving liquid surface. Because of this factor, the speed of aspiration can increase the carryover. NOTE: The Teflon tubing should be rinsed from time to time with 15% HNO 3 (for 10 minutes) and, afterwards with 30% NaOH (for again 10 minutes). Then purge water through it for 30 minutes. This procedure to "hydrolyse" micro-organisms is necessary, even if you use antiseptic syringe solvents (e.g. alcohol or acetonitrile). It helps to prevent an increase of carryover due to micro-organism layers at the inner wall, which dramatically may alter the wetting and adsorption properties of the transfer capillary. M5200R0 24

Air segment volume [µl]: Enter the volume of one air segment in [µl]. The optimal air segment volume depends on the inner diameter of the capillary.; it should not be less than 3-5 mm and it should not be shorter than four times the inner diameter in the Teflon capillary. If the volume is too small, the air segment may adhere to the wall and the aspirated liquid will pass around the segment. As the total air volume in the flow path is increased, a longer equilibrium time will be needed after a liquid movement to reach volumetric accuracy. In very narrow capillaries (fused silica etc...) the cohesion power of the air/liquid boundary may become so strong that a complete pressure equilibrium of the system is not reached. It will be necessary to take this into account by empirical correction of the aspirated/ dispensed volumes. Number of air segments: Use the left/right control to increase or decrease the number of air segments. Between the air segments, an air segment volume of the sample to be transferred is aspirated. Extra aspiration volume, will be discarded after aliquot [µl]: Specify the reagent volume which should be aspirated after the air segments to securely separate the syringe liquid from the reagent to be aliquoted. If you need a high degree of accuracy and a very low carryover, two or three air segments and 15-25% of the syringe volume as extra aspiration volume should be enough. Pressure equilibrium time after asp. and dispense [s]: Enter the time (in seconds) that the liquid handler should wait after an aspiration or dispense process to give the liquid transfer system time to equilibrate the pressure. A long pressure equilibrium time will increase volumetric accuracy and is needed with compressible, viscous liquids, large air segment volumes, fast aspiration and dispense speeds as well as with large aspiration and dispense volumes Use special Z-coordinate for task movements [mm]: The speed of the liquid handler movement inside the task operation can be optimized. When the Z-coordinate is set to a value that is > 0, the table will move instead of lowering completely for an X, Y-move. Caution: Make sure, that there is nothing on this level, especially, when you travel to the wash position! M5200R0 25

Repetitions of the wash cycle: Use the left/right control to increase/decrease the repetitions of the wash procedure after a single pipet step. The repetitions should be used if you need to have larger wash volumes than the syringe volume or if you have adsorbed contaminants in the needle tip, which need time to be desorbed. If you set the repetitions = 0, then the wash step is not executed. Volume dispensed at Wash [µl]: Specify the volume which is to be aspirated from the syringe valve fill connection and dispensed in the wash group. Dispense speed at wash group [µl/min]: Specify the speed at which the wash liquid is dispensed in the wash group vial with the slider control. Use vacuum waste removal. If present, vacuum waste removal can be used to remove the wash waste by vacuum extraction. Wash needle at: Select one of the available groups as destination for the wash liquid. If you do not select a group, the wash step is not executed. M5200R0 26

Duplicate Task The Duplicate task is used for reformatting applications such as the spotting of microarrays and other liquid handling procedures. It copies all samples of a source group to all of the samples in one or more destination groups. An extra-aspiration sample volume as well as the multiple air gaps prevent the cross contamination from one sample to the next. Procedure: 1. Emptying the syringe so that all of the aliquots can be hold in the syringe. 2. Aspirating of air segments, which are separated by sample segments. 3. Aspirating of an pre-spot volume and extra-aspiration volume (both are optional). 4. Aspirating of as many of the aliquots of the first sample, which the syringe can hold. 5. Dispensing of the Pre-Spot volume in a Pre-Spot group. This is used to get rid of a droplet at the needle tip from the sample aspiration, which otherwise would spoil the first microarray. 6. Distribution of the sample aliquots on the positions nr 1 of the destination groups, then using the Washing procedure and discarding the air segments and the extra aspiration volume in the wash position. Those steps are repeated, until all of the samples are distributed. Visual Example: Source group (light blue) is to be duplicated to three destination groups (yellow, green-yellow, green). The pre-spot group is colored red, the wash position blue. Each sample number corresponds to the destination position numbers. Only Batch Working Mode The Duplicate task is designed to work only in batch mode, it is not possible to use it within a sequentially working group of tasks (see: Sequence & SequenceEnd tasks). M5200R0 27

Source Aspirate Liquid from: Specify the group from which the samples are taken up. Click on the up/ down control and select one of the groups you defined in the GroupEditor. The Duplicate task can only aspirate the sample from the robotic tray, not from the valve connectors of the syringe Liquid level following. Aspiration speed [µl/min]: Adjust the sample aspiration speed ivia the slide control, the low and high speed ranges are available. The optimal aspiration speed depends on the viscosity of the liquid and the hydrodynamic resistance of the flow path, i.e. diameter and length of the capillary connecting the syringe with the source. The aspiration speed never should never be high enough to cause degassing as gas bubbles in the flow path can decrease dramatically the volumetric accuracy of the system Extra aspiration volume [µl], will be discarded after aliquot procedure into the wash group position. Specify the reagent volume which should be aspirated after the air segments to securely separate the syringe liquid from the rinsing reservoir liquid. If you need excellent accuracy and a very low carryover, two or three air segments and 15-25% of the syringe volume as extra aspiration volume should be enough. Pressure equilibrium time after aspirate [s]: Enter the time (in seconds) that the liquid handler should wait after the aspiration process, to give the liquid transfer system time to equilibrate the pressure. A long pressure equilibrium time increases the volumetric accuracy and is needed with compressible, viscous liquids, large air segment volumes, fast aspiration and when the aspiration and dispense volumes are selected. Droplet removal (pre-spot): You can optionally specify a pre-spot group where a small amount of the sample is spotted before the dispensing on the destination groups (microarrays) Dispense at Droplet removal (pre-spot) [µl]. Z-height during moves over source and prespot [mm]. The height of the needle when moving from spot to spot and the height used to remove the pre-spot droplet. M5200R0 28

Destination Duplicate to groups: Specify the groups to which you wish to duplicate the samples. Add groups with the [+]-Button, delete the last group with the [Backspace]-key of the computer keyboard. Change the highlighted group with the up/down control. Dispense volume [µl] to each target position: Specify the sample volume to be dispensed to each of the destination positions Dispense speed [µl/min]: Adjust the dispense speed in [µl/min] with the slide control; two ranges are available low and a high speed. Adjust the dispense speed so that an overpressure in the system cannot occur. Pressure equilibrium time after dispense [s]: Enter the time (in seconds) that the liquid handler should wait after the dispense process, to give the liquid transfer system sufficient time for pressure equilibration. A long pressure equilibrium time increases volumetric accuracy and is needed with compressible, viscous liquids, large air segment volumes Z-coordinate during movements to destination [mm]: The liquid handler movements inside the task operation can be optimized in speed. If the Z- coordinate is set to values > 0, the table travels during the X-Y move at the defined height, rather than being lowered completely. When the Z-tool with is selected, a suggested value is presented, which can be modified. CAUTION: Make sure that there is no obstruction on this Z-level if this option is selected. Speed optimization ON (dispense during X,Ymovement): When you operate with very low dispense volumes (< 1 µl), you can optimize the processing speed by dispensing during the X,Ymovement from one destination position to the next position. M5200R0 29

Air segment, Wash This page contains parameters and options for the minimization of the carryover, for the optimization of the volumetric accuracy and the selection of the working speed. The Air segment, Wash page offers two main input fields, one for the air segment(s), and one for the optional washing step after each aliquot series. The air segment is aspirated on the way to the source group. If the number of air segments is greater than 1, the liquid handler aspirates between two air segments an air segment volume of the sample, which is to be aliquoted. Each air segment lowers the carryover of syringe liquid into the sample by a certain factor. This factor depends in a complex manner on the surface tension of the syringe liquid, together with the wetting properties of the inner surface of the capillaries used for the liquid transfer. The liquid carryover across a moving air segment during aspirate is coming from the removal of small droplets from the backward moving liquid surface. Due to that, the speed of aspiration can also increase the carryover. NOTE: The Teflon tubing should be rinsed from time to time with 15% HNO 3 (for 10 minutes) and, afterwards with 30% NaOH (for again 10 minutes). Then purge water through it for 30 minutes. This procedure to "hydrolyse" micro-organisms is necessary, even if you use antiseptic syringe solvents (e.g. alcohol or acetonitrile). It helps to prevent an increase of carryover due to micro-organism layers at the inner wall, which dramatically may alter the wetting and adsorption properties of the transfer capillary. M5200R0 30

Air segment volume [µl]: Enter the volume of one air segment in [µl]. The optimal air segment volume depends on the inner diameter of the capillary. The pathway of the entire segment should not be shorter than 1.5-3 mm and should not be shorter than four times the inner diameter in a Teflon capillary, otherwise the air segment may adhere to the wall and the aspirated liquid may pass around the segment. As the total air volume in the flow path is increased, a longer pressure equilibrium time is needed after a liquid movement to reach volumetric accuracy. In very narrow capillaries (fused silica etc...) the cohesion power of the air/liquid boundary may become so strong, that complete pressure equilibrium of the system is not reached. You will have to take this into account by empirical correction of the aspirated/ dispensed volumes. Number of air segments: Use the left/right control to increase or decrease the number of air segments. Between the air segments, an air segment volume of the sample is aspirated. Repetitions of the wash cycle: Use the left/right control to increase or decrease the repetitions of the wash procedure after a single duplication step. The repetitions should only be used if you need to have larger wash volumes than the syringe volume or if you have adsorbed contaminants in the needle tip, which need time to be desorbed. If you set the repetitions=0, then the wash step is not executed. Volume dispensed at Wash [µl]: Specify the volume which is aspirated from the syringe valve fill connection and dispensed in the wash group. Dispense speed at wash group [µl/min]: Specify the speed, at which the wash liquid is dispensed in the wash group via the slider control Use vacuum waste removal. If present, vacuum waste removal can be used to remove the wash waste by vacuum extraction. Wash needle at: Select one of the available groups as destination for the wash liquid M5200R0 31

Pipet Task The pipet task is used for the transfer of liquid from a source to a destination. The source and the destination, can also be the vials that are connected directly to the syringe and it is not necessary that they are grouped positions of sample carriers on the tray. The pipet task has two tabbed pages, one is called "Source, Destination", the other one "Air segment, Wash". Procedure: The task aspirates the aspiration volume from the source and dispenses the volume to the destination. The task is repeated until all vials of the smallest sample type group, either as source or destination group, are completed. If only one sample type group is present, this one is limiting the number of task repetitions. If no sample group is in the task, the task is executed only once. When the dispense volume is larger than the aspiration volume, the task adds the difference from the fill connection of the syringe valve. In this way, a one-step dilution of samples to a sample carrier on the tray can be done. When the aspiration volume is larger than the dispense volume, the remaining volume is discarded after the pipetting step in the wash position, together with the optional air segments which are aspirated before the pipetting step. These are used for low carryover transfer of samples. Optionally, a wash step can be performed after the pipetting step. In this case, the wash volume is aspirated from the syringe fill connection and dispensed in the wash group position. M5200R0 32

Batch & Sequential Working Mode The Pipet task is designed to work in batch mode and in sequential mode (See: Sequence & SequenceEnd tasks). When the Pipet task is used inside a sequence, it is doing a transfer of liquid to one the destination positions, then the next task is executed within the sequence. When it is used in batch mode (i.e. outside a Sequence - SequenceEnd task group), it is completing the liquid transfers for all of its group positions. Source, Destination Aspiration volume [µl]: Enter the volume that the task should aspirate at the source in µl. The task cannot aspirate a volume greater than the syringe volume. Aspiration speed [µl/min]: Adjust the aspiration speed in [µl/min] with the slide control. A low and high speed range are available. The optimal aspiration speed depends on the viscosity of the liquid and the hydrodynamic resistance of the flow path (which is related to the diameter and length of the capillary connecting the syringe with the source). The aspiration speed never should be so great that degassing occurs. Gas bubbles in the flow path will decrease dramatically the volumetric accuracy of the system. Aspirate Liquid from: Select the position of the syringe valve when aspirating the liquid. If you intend to aspirate from a sample carrier on the tray, the position must be "Needle". Select a group: Use the up/down control to select one of the groups that is defined in the group editor, when you wish to aspirate from a sample carrier on the tray. Liquid Level following: If you want the needle to follow the sinking liquid level during aspirate (feature not available with all liquid handlers), check this box. Dispense volume [µl] : Enter in the volume [in µl] that the task should dispense at the destination. The task can dispense volumes larger than the syringe volume; in that case, the syringe will be repeatedly filled from the fill connector of the syringe valve. Dispense speed [µl/min]: Adjust the dispense speed [in µl/min] with the slide control. There are two ranges, a low and a high speed range. The dispense speed should be set so that an M5200R0 33

overpressure in the system is excluded and that no liquid is spilled from the destination position. Dispense liquid to: Select the position of the syringe valve when dispensing the liquid to the destination. If you intend to dispense to grouped vials on a sample carrier on the tray, then the selection must be "needle". Select a group: Click on the up/down control and select one of the groups you defined in the group editor when you wish to dispense to a sample carrier on the tray Liquid Level following: If you want the needle to follow the rising liquid level during dispense (feature not available with all liquid handlers), check this box. Air Segment, Wash This page contains parameters and options for the minimization of the carryover, for the optimization of the volumetric accuracy as well as the working speed. The page offers two main input fields, one for the air segment(s), which is only used when the source is a group on the tray, and one for the optional washing step after each pipetting. The air segment is aspirated on the way to the source group. If the number of air segments is > 1, the liquid handler aspirates an air segmented volume of the sample which is to be transferred between two air segments. Each air segment lowers the carry over of syringe liquid into the destination sample by a certain factor. This factor depends in a complex manner on the surface tension of the syringe liquid, and the wetting properties of the inner surface of the capillaries used for the liquid transfer. The liquid carryover across a moving air segment during aspirate is coming from the removal of small droplets from the backward moving meniscus. The speed of aspiration can increase the carryover. NOTE: The Teflon tubing should be rinsed from time to time with 15% HNO 3 (for 10 minutes) and, afterwards with 30% NaOH (for again 10 minutes). Then purge water through it for 30 minutes. This procedure to "hydrolyse" micro-organisms is necessary, even if you use antiseptic syringe solvents (e.g. alcohol or acetonitrile). It helps to prevent an increase of carryover due to micro-organism layers at the inner wall, which dramatically may alter the wetting and adsorption properties of the transfer capillary. M5200R0 34

Air segment volume [µl]: Enter the volume of one air segment in [µl]. The optimal air segment volume depends on the inner diameter of the capillary. The entire path of the segment should not be less than 3-5 mm and at least not shorter than four times the inner diameter in a Teflon capillary. If the path is too short, it is possible that the air segment will adhere to the wall and the aspirated liquid passes around it. The larger the total air volume in the flow path is, the longer pressure equilibrium time is needed after a liquid movement to reach volumetric accuracy. With very narrow capillaries, (fused silica etc...) the cohesion power of the air/liquid boundary may become so strong, that complete pressure equilibrium of the system is not reached. You will have to take this into account by empirical correction of the aspirated/ dispensed volumes. Number of air segments: Use the left/right control to increase or decrease the number of air segments. Between the air segments, an air segment volume of the sample to be transferred is aspirated. Pressure equilibrium time after asp. and dispense [s]: Enter the time (in seconds) that the liquid handler should wait after an aspiration or dispense process, to give the liquid transfer system time to equilibrate the pressure. A long pressure equilibrium time increases the volumetric accuracy and is needed with compressible, viscous liquids, large air segment volumes, fast aspiration and dispense speeds together with big aspiration and dispense volumes. Use special Z-coordinate for task movements [mm]: The speed of the liquid handler movements inside the task operation can be optimized, if the Z-coordinate is set to a value > 0. In this case, the table travels than instead of lowering completely for the X,Y-move. Caution: Make sure, that there is nothing on this level, especially, when you travel to the wash position! Repetitions of the wash cycle: Use the left/right control to increase or decrease the number of repetitions of the wash procedure after a single pipet step. The repetitions should only be used if you need to have larger wash volumes than the syringe volume or if you have adsorbed contaminants in the needle tip which need time to M5200R0 35

be desorbed. If you set the repetitions=0, then the wash step is not executed. Volume dispensed at Wash [µl]: Specify the volume which is aspirated from the syringe valve fill connection and dispensed in the wash group Dispense speed at wash group [µl/min]: Specify the speed at which the wash liquid is dispensed in the wash group vial with the slider control. Use vacuum waste removal. If present, vacuum waste removal can be used to remove the wash waste by vacuum extraction. Wash needle at: Select one of the available groups as destination for the wash liquid. If you select no group, then the wash step is not executed. Multipurpose Tasks The multipurpose tasks are basic tasks which are used to perform a single, simple operation. Usually these tasks are combined in a sequence to build up complex liquid handling and/or movement tasks that are not provided in any of the other tasks. The entire sequence can be viewed upon as being a User Defined Task <Sequence> <Multipurpose task 1> <Multipurpose task 2> <Multipurpose task 3> <etc> <SequenceEnd> The multipurpose tasks can also be used without a sequence. If a sequence is not used, all spots within the single task are processed before continuing to the next task. Aspirate Task The Aspirate task performs an aspiration of liquid into the syringe at a defined position of the syringe valve. M5200R0 36

The Aspirate task is designed to work together with the MoveTo task and the Dispense task in sequential working mode to build complex liquid handling patterns. It also can be used outside a sequence in batch mode (See Sequence SequenceEnd tasks). Aspirate volume [µl].the volume that is aspirated by the syringe. Aspirate speed [µl/min]. The speed at which the aspiration process takes place. When the task is added, the default syringe speed stored in the settings, is used. You can manually change the speed, however if the speed is too high, it is possible that dangerous backpressures can be created. Wait until aspirate process completed. This action prevents any movement during the aspiration process. This should be checked when aspirating from a vial on the robot table. Valve. The position of the rotary valve. When using the Aspirate & Dispense tasks, ensure that the sum of liquid aspirated and dispensed is equal for one sample cycle. If this is not done and multiple cycles are used, the difference can add up and after a number of samples, the application will fail! Dispense Task The Dispense task is used to dispense liquid from the syringe with a defined position of the syringe valve. The Dispense task is designed to work together with the MoveTo -task and the Aspirate task in sequential working mode to build complex liquid handling patterns. It also can be used outside a sequence in batch mode (See Sequence SequenceEnd tasks). M5200R0 37

Dispense volume [µl].the volume that is dispensed by the syringe. Dispense speed [µl/min]. The speed at which the liquid is dispensed. When the task is added, the default syringe speed stored in the settings is used. You can manually change the speed, however if the speed is too high, dangerous backpressures can be generated. Wait until dispense process completed. This prevents any movement during the process. This should be checked when dispensing into a vial on the robot table Valve. The position of the rotary valve. NOTE: When using the Aspirate and Dispense tasks, ensure that the total of all liquid aspirated and dispensed is equal for one sample cycle. If this is not done and multiple cycles are employed,, the difference can add up, and after a certain number of samples, the application will fail! MoveTo Task The MoveTo task moves the table so that the needle is at a specified group position or coordinate. Also movements relative to the current position can be specified. When a destination group is specified, it overrides the separate destination coordinates specified below. Relative movements are possible only in one of the three coordinates within one MoveTo task. M5200R0 38

Destination. When a group is selected, it will be used for all the positions of the group on a consecutive basis.. When the group is reagent type, it will move only to the first spot and remain there Use special Z-coordinate [mm]. When a group is chosen as the destination, the height can be chosen to move from spot to spot. Direct destination coordinates can be used when no group is selected. The selection of a destination group will overrule these coordinates. X,Y,Z Coordinate. The coordinates are specified in mm. Use X,Y,Z. This check box should be enabled to use the coordinates (one, two or all three coordinates can be used). Rel X,Y,Z move. This enables a relative movement from the current position. Wait until dispense process completed. This check box prevents any movement during the process. This should be checked when dispensing into a vial on the robot table Speed Adjustment. When adding the task to the task list, t the default movement speeds are initialized. If desired the speed settings can be changed. CAUTION: Changing the default speed settings may cause a damage of the Probot. X,Y,Z,-Movement is performed in the order: X, Y, Z. The table will not be lowered before the X movement takes place. The M5200R0 39

user should note that this action can seriously damage the needle. The MoveTo task is designed to work in batch mode and in sequential mode. (See Sequence SequenceEnd tasks) Switch Task The Switch task is available through the Application Editor toolbar. It switches the relay contact and other hardware actuators of the robotic device (if available). It can be used in batch- and sequential mode. Wait Task The Wait task stops the execution of the application until one of the three conditions is fulfilled: - Time - Input contact event (0 = the start signal input) - User keystroke (The message will be displayed) It can be used in batch- and sequential mode. M5200R0 40

Wash Task The Wash task performs a washing step to clean the needle. It aspirates liquid from the fill connector of the valve into the syringe and dispenses the specified volume into the group under "Wash needle at". The Wash task is designed to work in batch mode and in sequential mode. (See Sequence SequenceEnd tasks) Sequential/Batch Mode The tasks <SEQUENCE> and <SEQUENCE END> are used in sequential mode. When these are employed, all tasks inside the sequence are processed as being one complete task. Every task is processed for one vial and at the end of the sequence block, the sequence is restarted for the next vial. This sequence is repeated until all vials are processed. The repetition counter is used to indicate how many times the sequence should be processed for all vials. Setting the counter to 1 means that all vials are used once. When working in normal/batch mode, each task finishes all vials before the next task is executed. Sequence Task The Sequence task starts a sequential working mode of the following tasks. The sequential mode of working is ended by the task SequenceEnd. Only the number of times the sequence should be run is specified. M5200R0 41

SequenceEnd Task The SequenceEnd task ends a sequential working mode of the tasks before. The sequential mode of working is started by a corresponding by the Sequence task. ZTool The ZTool is a tool window to help define the Z-coordinate which is used for horizontal movements during the execution of a task. By keeping the needle slightly above the highest vial(s) in a task, the speed of the application is optimized. The Z-coordinate input tool is opened when the checkbox: Use special Z-coordinate for X, Y-task movements [mm] is checked in the task and when the parameter input field for the Z-coordinate is clicked. The software automatically calculates the tallest of the vials used in a task when the collect groups are specified. Only the distance between the needle and the highest vial needs to be filled in. This distance should be > 0. The absolute Z-coordinate can also be directly specified. When using the relative distance, always check the absolute Z- coordinate resulting from the chosen distance. M5200R0 42

Needle position, when Z equals to: This group contains a sketch illustrating the meaning of the calculated height values. The height values illustrate the positions of the needle tip at the corresponding table Z coordinate. The software automatically searches for the highest vial top used and the uppermost sample carrier bottom, when the ZTool is opened. The values refer only to sample carriers and vials used in the specific task! Uppermost top of the vials used by the task [mm]: This value is the table Z-coordinate where the needle tip reaches the upper edge of the highest of the vials being used in the task. This value is based on the vial height input, which is done during the sample carrier definition and the positioning of the sample carrier on the table. Uppermost bottom of the carriers used by the task [mm]: This value indicates the table Z-coordinate, which is where the needle tip reaches the bottom of the sample carrier at the highest positioning level amongst the ones used in the task. The sample carrier bottom positions are defined during the positioning of the sample carriers in the TrayEditor. The values can be modified by clicking on the "vertical carrier adjustment" button in the TrayEditor toolbar. Maximum Z-coordinate [mm]: This value indicates the needle position when the table is at its Z-coordinate maximum. Relative needle height input This group is used to specify the vertical positioning of the needle tip during the horizontal movements in the task. The value is specified relative to the highest vial top or the uppermost sample carrier bottom used in the task. When a value is changed in one of the input fields, the color of the field changes to white. After pressing the [ENTER]-key or after a mouse click into another input field, the values are recalculated according to the change made, and the color changes back to light-gray. M5200R0 43

Enter in the distance [in mm], that the needle should stay above the highest of the vials which are used in the task. This distance should be > 0! Absolute table Z-position input In this group, the table z coordinate is specified directly as an absolute value of the robotic coordinate system. This value is also recalculated according to the inputs in the "relative input" fields. When closing the ZTool with [Ok], this value is inserted in the task parameter by which the ZTool was opened. The robot can also be used to directly teach the current position of the table to set the absolute Z-value. Use the drive panel before opening the ZTool to move the table to the desired height. Test buttons and confirmation This group of buttons is used to move the robot table to the desired Z-coordinate and to confirm or cancel the change of the Z-coordinate task. Moves the table immediately to the specified Z-coordinate. CAUTION: Make sure that there is no obstruction at this Z- level which can cause damage during the move! Moves the table immediately to the "needle scope coordinate" being specified in the manual / robot window. This coordinate should be so small, that the needle tip is above all of the vials and sample carriers on the robotic table. The "needle scope Z- coordinate" is used for the movements to the first of the sample positions in a task and after the completion of a task. Immediately stops the movement of the table. This button is to be used as an emergency halt to stop the table manually (e.g. when it appears that the needle will crash and be damaged). This button enters the absolute Z-coordinate into the parameter of the task from which the ZTool window was opened. The ZTool is closed with this button. This button cancels the changes you made to the Z-coordinate task and leaves the value in the task unchanged. The ZTool is closed by this button. M5200R0 44

Manual Device Control In the manual device control the Probot components (XYZ-robot module and Dosage module) can be controlled without using a task. The manual device control can be used for alignment and dosage module purging. XYZ Robot Module The Robot window is shown on the left of the screen. It consists of three different tabs Manual Contact and Park & Wash. Manual In the manual page, all controls for a manual XYZ movement are present: M5200R0 45

[GoPark] : Moves the needle to the park position (specified in Park&Wash). [GoWash] : Moves the needle to the Wash position (specified in Park&Wash). [Home Z-XY]: First homes the Z-axis, then the Y- and finally X- axis. [External]: Sets the robot in the external control mode. In this mode, you need a remote control device that is connected via a cable to the robot to drive it manually similarly to the drive panel of this software. The stepwidth specifies the stepwidth [in mm] in the one-step mode, driving with the external remote control and the (software) drive panel. [Internal]: Returns the robot to the internal mode after [external] operation. In the internal mode, the computer has the control. A click on the internal mode button presents the current position to which the robot has been driven to by the external remote control. [Read positions and status]: Reads out the positions, the status and the firmware versions of the internal microprocessors. [GoXY]: Moves the table so, that the needle is at the X and Y positions the user has put in the line "Position [mm]" [GoXY-Z]: Moves the table first in X and Y then in Z direction to the positions the user has put in the line "Position [mm]". [GoX], [GoY],[ GoZ]: moves the separate axes according to above. Contact A click on a contact output button switches the corresponding liquid handler output relay. The current contact input status is only read immediately, if the checkbox Scan the contact inputs for changes is checked. M5200R0 46

If the checkbox remains unchecked, the display is automatically updated with the next change of an input from high to low, i.e. from open to closed. [NeedleUp]: Lifts the outer needle with the lifting magnet, so that the liquid coming out of the needle can reach the robotic table. [NeedleDown]: Releases the outer needle from the lifting magnet, so that all solvent coming through the needle, is removed to the vacuum. [VacuPump]: Switches on and off the vacuum pump when present in the system. Park &Wash In the Park&Wash page, the park and wash positions can be defined either by single vial group on the tray of type reagent or by exact X, Y, Z coordinates. Needle scope coordinate during tray move [mm]: This is the coordinate, the table travels along during X,Y-movements, when no other Z-coordinate is specified in a task. The value 0.0 mm means that the table travels at the lowest possible height, thereby positioning the needle at the largest distance to the table. The value 0.0 mm is the most secure value. If you like to speed up your application by saving travel time, you can put in higher values, but heed the warning below: CAUTION: Make sure that there is no obstruction at this Z- level which can cause damage to the needle or the table during the move! M5200R0 47

Dosage module This page contains all of the necessary controls for the syringe pump to manually aliquot volumes, to switch the valve to a defined position, to purge and to home the syringe. It contains a controls field for the valve and one for the syringe itself. The blue bar in the syringe controls field represents the current filling state of the syringe, as does the value right beneath the label "Current volume [µl]". Valve [Fill (Reagent)]: Switches the valve so that the syringe is connected with the fill connector of the valve, which normally is connected with a rinsing liquid or a reagent. [Purge (Waste)]: Switches the valve so that the syringe is connected with the purge connector of the valve, which normally is connected to a waste vial. [Auxiliary (closed)]: Switches the valve so that the syringe is connected with the Auxiliary connector of the valve, which normally is closed. [Pump (Needle)]: Switches the valve so that the syringe is connected with the needle. Syringe Aliquot volume [µl]: Specify the volume [in µl], which can be aspirated / dispensed by the buttons [DispenseAliq.] / [AspirateAliquot]. If the volume to be aspirated or dispensed is too large, then the command simply is rejected. [Empty]: Dispenses the entire volume which is still in the syringe. [Fill]: Fills the syringe. [Purge]: Starts a repeated filling from the "fill" connection of the valve and empties it to the purge connection of the valve. The purging of the syringe either can be stopped by the [Stop] button or by a second click on the [Purge] button. [Home]: Starts a homing sequence for both the valve and the syringe. The valve is in the fill position when the syringe is homed. M5200R0 48

[Stop]: Stops the movement of the syringe immediately. Initiates a homing sequence, first for the valve, and then for the syringe. Drive panel The drive panel is accessible through the main toolbar with the "drive" button. It is used to move the robot manually to a dedicated place, to activate a homing sequence, to access directly to the wash and park positions of the robot and to use the robot as a teaching instrument for sample carrier positions. It can also be used to drive the table / needle to the mouse position in the TrayEditor. The current X,Y-position of the needle over the tray is indicated in the TrayEditor by the crossing point of two blue lines. Home the instrument: This action is necessary after the door is opened during a run, after contact of a security end switch of one of the axis, after the detection of the needle crash security switch and after switching on the device. The home sequence moves all axes to their calibration positions. It is done automatically before the start of an application and after user abort. Move to the robot park position: This button moves the needle directly to the park position, that is defined in the robot hardware window. Move to the robot wash position: The button directly moves the needle to the robot wash position. Move to the mouse position in the TrayEditor: When the "little mouse" button is down and you click the TrayEditor with the mouse, a warning dialog will be presented and the robot will be driven along the X, Y axes, to place it the needle at the position over the tray where you clicked. In this manner, you can check the X, Y-positioning of separate vials in your sample carriers. It is suggested that you use this feature together with the arrow keys of the drive panel and at high magnification of the TrayEditor. Move to the vial position in the TrayEditor: When the "move to vial" button is down, and you click into the TrayEditor over a vial with the mouse, a warning dialog will be presented and the robot will move the needle into the vial. Arrow buttons: the outer four arrows move in the X and Y direction, the inner two arrows move in the Z direction. Using the arrow keys with the left mouse button produces a single step movement, using them with the right mouse button produces a continuous movement, as long as the mouse button is kept down. The step width is adjusted in the small yellow input field, either with a figure input or with the + or - buttons below the input M5200R0 49

field. The magnitude of step width change with each mouse click on +/- is adjusted with the three narrow buttons below the numbers. The buttons correspond to 0.025, 0.1 and 1.0 mm stepwidth change. Video Camera The camera option in µcarrier is accessed through View Show camera window. The [ON/OFF] button starts and stops the display of the camera The [Settings] button opens the settings of the camera image and the recording. The details are discussed below. Rate: This sets the time between the each frame that is to be recorded and displayed with the camera. On computers with shared memory for system and graphics (laptops, older systems), this value should not be lower than 100 ms. Source: This sets the source for the video signal. When only one device is installed, the source should be 0. On typical installations only one capture device is present on the system. The save buttons stores the present camera image as a *.BMP file. The play button starts the recording of 10 seconds of video. Since video files are large, the recording time is limited to 10 seconds M5200R0 50

The stop button can be used to stop the video recording before the 10 seconds are over. Device Settings When Settings is selected, three windows appear. In the first window, the settings related to the device can be set. It is strongly recommended that you leave all settings on the first two tabs as they are presently set. On the device settings tab the saturation should be set to 0 and the other parameters can be set according to personal preference.. The settings on the fourth tab Camera Controls do not affect the camera image. After clicking OK, the Windows Video Compression dialog appears. If any compression codecs (MPEG, etc) are present on the computer, a compression method can be chosen here. However, compressing video data uses a large amount of system resources and this could interfere with the fractionation. The time limit of 10 seconds is not removed. It is therefore recommended to leave this setting to No Recompression. M5200R0 51

The third dialog is the Windows Video Format dialog which is used to set the video resolution and pixel depth, to decrease the file size. The time limit of 10 seconds is not affected by these settings. M5200R0 52

Settings Window The settings window is accessed through Tools Settings. There are six tabs Main, Directories, Controls, Robot, Dosage module and RS232 / USB. Main The general settings are specified on the main tab of the settings window,. All other tabs deal with one specific part of the Probot control. Always start the software in real device control mode: When this checkbox is checked, the software always expects a device to communicate with at startup. This is the best adjustment for computers on the lab bench which are used exclusively for the control of the liquid handler. If you wish to run the software from time to time without the device M5200R0 53

connected and switched on, leave the checkbox unchecked. Then the standard request at software startup will occur, "Do you wish to run a real device?", which you can answer according to your needs. Remote application start by start.ini file in the ucarrier folder. This option allows the use of remote starting of an application through a Laboratory Information Management System (LIMS). USB / RS232 device detection at startup (unchecked RS232 only). Leave this unchecked, since all Probot systems are RS232 controlled. Home the instrument after opening the device door during run. When the door is opened during run, the run will stop. If you check this option, the Probot will perform a homing procedure before continuing. This takes time and can destroy the spotting. It is recommended that you leave this feature unchecked Drive panel arrow direction = table movement (unchecked=inverse). This check box controls the behavior of the robot under manual control with the drive panel. If the feature is checked, the arrows in the drive panel indicate the movement of the robotic table, if the feature is unchecked, the arrows indicate the direction of the needle relative to the table. Grid width of the drawing grid in the TrayEditor [mm]: Defines the grid width of the grid in the TrayEditor [in mm]. The grid can be used to adjust the positions of sample carriers on the tray. Always show position numbers. This forces the display of position numbers in any zoom level. High process priority: When this feature is checked, the µcarrier software is started with increased process priority, when the feature is unchecked, the software is started in normal process priority. No user message at steploss detection: When this feature is checked, the software corrects a steploss error during an application run without interrupting the run by a user message. Enable idle mode during application. µcarrier is designed to consume only the necessary system resources and leave as much resources as possible available to other programs. However, on older computers the remaining system resources can be insufficient for other programs. Enabling this option will decrease the amount of system resources used by µcarrier, but it could introduce delays in Probot response resulting in incorrect (1-2 seconds) spotting times. This option should only be used in cases of significant problems due to insufficient system resources. M5200R0 54

Set Robot output no 3 in case of hardware error. In case of a hardware error that will stop the correct fractionation in an overnight run, output no 3 can be used to send a signal to be received by equipment that the Probot is interfaced to. An example of this is the connection of output no 3 to an input on the Ultimate system. The moment a hardware error occurs and the signal is sent and received by the Ultimate, the flow will be stopped and the run aborted. Note that with very severe hardware errors, it is possible that all communication has stopped and µcarrier can not send the signal. Generate log file of the device communication (commlog.txt). All communication parameters are stored in this log file. These parameters are useful for diagnostic purposes, but with extensive use of the Probot, the commlog folder can contain megabytes of log data. Re-reference Z-axis after number of z-moves. When the number of vertical movements specified in the textbox is reached, the Probot will move to 0 mm and and then continue the run. During fractionation (especially in time control) this feature should be disabled since it will result in unequal spot sizes. M5200R0 55

Directories Select the folder to be used in the folder structure on the bottom and when the desired folder is selected, clicking the [Change] button will set the selected path as the default folder. M5200R0 56

Controls All hardware components present are specified on the Controls tab,. The vacuum waste removal, external remote control and dosage module can be added or removed from the software configuration by checking or unchecking the corresponding items in the Controls tab Robot Default speeds The motor speeds for the robot table movement are indicated on this tab. These parameters are important for a proper operation of the instrument. In any case of a malfunction of the table drive (e.g. slow movement, noise and position errors) check that all settings are correct. NOTE: NEVER change these parameters if not instructed to do so by the manufacturer! M5200R0 57

Setup The dimensions of the Probot table and the values to calculate movement times are indicated on this tab. These parameters are important for a proper operation of the instrument. In any case of a malfunction of the table drive (e.g. slow movement, noise and position errors) check if all settings are correct. NOTE: NEVER change these parameters if not instructed to do so by the manufacturer! Firmware The firmware of the axis motors can be read in the Firmware tab in the Robot Tab. M5200R0 58

Dosage Module Default speeds The Default speeds have been set to save values for a µtee setup with 30 µm I.D. needle. If the dosage module speeds are above these values in a Probot with µtee and 30 µm needle, the pressure on the rotary valve can increase to dangerous levels. The speeds can be slightly increased, if the system has a larger I.D. needle, however it is recommended that these values are not changed. If in doubt contact the manufacturer for advice. Purge speed [µl/min]: Specify the desired value for the dispense speed during the manual purging process and when the syringe is dispensing liquid into the valve connector "purge" in a task. Reagent fill speed [µl/min]: specify the speed by which the filling process of the syringe shall be done from the fill connection of the valve, when no other value is specified in a task. Default dispense speed [ml/min]: Specify the default dispense speed which is to be used for dispensing processes to the needle connector of the valve, if no other value is specified in a task. Default aspirate speed [µl/min]: See Default dispense speed. Default wait time after aspirate [s]: When the check box "Use asp wait" is checked, the system will be stopped for the specified time is waited after an aspiration process, if no other value is specified in a task. Default wait time after dispense (see default after aspirate). M5200R0 59

Setup The setup values are critical for the volumetric accuracy of the syringe, especially the calibration of the axis and the nominal stroke length. There is a fair degree of flexibility concerning the choice of the syringes. The user should note that if a change is made, care should be taken, if you are in doubt concerning the right values in the syringe setup page, contact the manufacturer. The syringe nominal volume must be changed if a syringe with a nominal volume different than 100µl is used. The nominal stroke length [mm] must be changed if the stroke length of the syringe is different for the nominal volume 60.0 mm (the value which for the standard system). NOTE: NEVER change the Syringe calibration [steps per mm] if not instructed to do so by the manufacturer. The old Probot model needs a value of 4000 steps /mm for the syringe, the new Probot model needs a value of 2000 Steps/mm. The backlash compensation [mm] is the distance that the plunger is moved further than needed to aspirate a certain volume. After the aspiration, the plunger is moved forward by that value again. This feature is used to eliminate all mechanical backlash, especially the backlash due to the elasticity and smoothness of the plunger seal. In this way, all volume measurements are done always under the same stress direction. Normally the value does not have to be changed, except when very large syringes with very smooth and flexible plunger seals are used. Syringe full at distance from home [mm]: This value specifies how far from the homing position (which is a calibration position) the plunger is set at a full stroke fill. This value must NOT be smaller than the backlash compensation M5200R0 60

value. When the value is set too large, it may happen that the syringe security limit switch is activated when completely emptying the syringe. In that case, the value has to be decreased. A syringe [Stop] in the manual page and a homing then should release the problem RS232 / USB The RS232/USB tab offers two tabbed pages, the first one is called serial SerialPort, the second one Communication Monitor. SerialPort The communication between the Probot and the computer can be checked via this window. The available COM ports, the one in use and its status are displayed. Most importantly is the button [Switch Online (Device Power-On-reset)], which enables the manual reinitialization of communications without restarting the software. The USB section is reserved for future use. M5200R0 61

Communication Monitor The second page with the label Communication Monitor is a tool to monitor all of the communication strings which are passed over the serial port. In addition,, the command strings and requests the computer is sending can be monitored as well as the answers and interrupt requests of the connected devices. In addition to the Observation " function, it is possible to enter command and diagnosis strings and send them separately to the device(s). This allows for the rapid detection of hardware errors and distinguishes these errors from errors which may occur for other reasons (software, use etc...). For the command documentation, please refer to the hardware manual. [Clear Monitor]: This button clears the text field under the labels Communication Protocol. Monitor ON: When the check box is checked, all RS232 strings sent are listed continuously together the sending times. M5200R0 62

[Send->]: This button sends the command string, which is in the text line right beneath the button. In the second part of that line, the answer of the device is shown, when there was one. NOTE: For remote diagnostics, it can be helpful to email the contents of the two text fields to your service representative. You can mark the content of the text field when you click in it with the right mouse button and copy it into the windows clipboard. Then open a text Editor (Windows Edit, Windows Notepad, Windows Wordpad or some other text editor), where you can paste the text again with the right mouse button and save it in a file or paste it directly to your Email client. About The about window is accessed via Help / About. It provides information about the software version, the distributor and the owner of the copyright. M5200R0 63