Thermo Fisher Scientific μCarrier Liquid Handling Application Owner's manual

M5200R0

µCarrier™

Liquid Handling

Application Designer V2.0

Software Manual

P/N 163801

www.lcpackings.nl

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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

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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.

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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 un-

paused 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).

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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)..

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

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,

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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.

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.

Folder list

Sample carrier

Preview

File list, single

click to load the

sample carrier

Sample carrier

settings

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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. 60o 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.

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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’

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

Folder list

Tray Preview

File list, single

click to load the

tray

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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.

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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.

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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.

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.

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.

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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.

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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.

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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

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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.

Timecollection = Timemovement + 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.

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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] * radius2 [mm2] * 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

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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.

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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.

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Use vacuum waste removal. If the hardware setup

enables vacuum waste removal [special kit] the inter-

being 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 15th 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.

Relays Table

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

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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

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Thermo Fisher Scientific μCarrier Liquid Handling Application Owner's manual

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Thermo Fisher Scientific μCarrier Liquid Handling Application Owner's manual

Thermo Fisher Scientific μCarrier Liquid Handling Application Owner's manual