Biotage Extrahera LV-200 User manual

Biotage®Extrahera™LV-200
User Manual

Biotage®Extrahera™LV-200 User Manual | © Biotage 2021
Contents
Biotage®Extrahera™LV-200
User Manual
CONTENTS
2Biotage®Extrahera™LV-200
1System Overview
1 Liquid Handling
2 Working Area
2 Carousel and Lifter
2 Dual Flow Extraction (DFE)
3 Columns, Plates, and Sample Tubes
3 Pressure Unit
3 Waste Kit and Vacuum
3 Safety
4 Visual and Audible Alarm
4 Lighting
4 Adjustable Touch Screen
4 Barcode and QR Scanner (Optional)
4 Biotage®Extrahera™GLP Software License (Optional)
5Power Failure
6Manage Solvents, Sample Types,
Plates, Racks, and Tips
6 Predefined Entries
6 Custom Entries
6 Manage Solvents
8 Manage Sample Types
9 Manage Extraction Media
10 Manage Sample Plates
11 Manage Pipette Tips
11 Manage Dual Flow Extraction Consumables
12 Biotage®Extrahera™GLP Features
13 Set Up a Method
13 Predefined Methods
13 New, Copy, and Edit a Method
14 Method Operations
15 Method Parameters
17 DFE Method Parameters
18 Biotage®Extrahera™GLP Features
19 Export, Import, and Delete Methods
19 Export Methods
19 Import Methods
19 Delete Methods
19 Biotage®Extrahera™GLP Features
20 Set Up and Start a Run
20 Verify the System Setup
20 Select the Method
20 Load the Extraction Media
21 Adjust the Air Pressure
21 Load Solvent Pipette Tips
22 Load Sample Pipette Tips
23 Assign Solvent Bottles and Prime
24 Assign Manually Loaded Solvents (Optional)
24 Load Carousel Plate(s)
25 Enter the Report Name and a Comment (Optional)
25 Load the Sample Plate and Enter the Plate ID
25 Start the Run
25 Biotage®Extrahera™GLP Features
26 Monitor and Control a Run
26 Monitor the Run
26 Pause or Abort the Run
27 Post-Run Activities
27 Biotage®Extrahera™GLP Features
28 View, Export, and Delete Reports
28 View a Report
28 Export Reports
28 Delete Reports
28 Biotage®Extrahera™GLP Features
29 Enable/Disable the DFE Mode
29 Enable the DFE Mode
29 Disable the DFE Mode
30 Maintenance
30 Change the System Settings
30 Export the System Logs
30 Clean the Exterior of the System
31 Clean the Interior of the System
32 Replace the Solvent Tubing
33 Clean or Replace the Waste Tubing
34 Pipette Pump Calibration and Adjustment
35 Replace the Pressure Head Seal
36 Replace the Fuses
36 Drain the Filter Unit
36 Replace the Filters in the Filter Unit

Contents
37 Manual Control
37 Door
37 Maintenance
37 Method Development
38 Set Up Reminders for Pipette Pump Calibrations
39 Troubleshooting
39 Dual Flow Extraction Tips
39 Pipette Pump
39 Pipetting Tips
39 Power Failure
40 Pressure Head
40 Pressurized Air and Nitrogen
40 Retaining Clips
40 Robot
40 Sample Cross-Contamination
40 Solvent Contamination
41 Solvent Pumps
41 Ventilation Fan
41 Waste and Lifter
41 Biotage®Extrahera™GLP Features
42 General Information
42 Consumables and Accessories
42 Manufacturer
42 Contact Us

Biotage®Extrahera™LV-200 User Manual | © Biotage 2021
System Overview
System Overview
Biotage®Extrahera™LV-200 is an automation system for sample
preparation methods for 96 positions plates and column racks.
Extrahera LV-200 can automate:
» Supported Liquid Extraction (SLE),
» Solid Phase Extraction (SPE),
» Phospholipid Depletion (PLD),
» Protein Precipitation (PPT),
» Dual Mode Extraction with in-situ
hydrolysis (Hydro DME+), and
» Dual Flow Extraction (DFE).
The system also offers two filtration methods, Filtration
(extraction media already containing sample) and Filtration+
(with sample load and optional pretreatment).
Liquid Handling
Extrahera LV-200 is equipped with five solvent inlets found on
the right hand side of the system (S1-S5). Solvents are pumped
into five 25 mL disposable solvent reservoirs inside the system
(in position 5 on the working area) where the robot can aspirate
solvent using up to eight disposable pipette tips; see Figure 1.
The system scans the solvent level in the reservoirs (S1-S5), and
fills/refills when necessary, each time a run is started and every
30 minutes during the run. Reservoirs containing solvents that
are defined as highly volatile are filled just before the solvent is
used for the first time in the run.
Figure 1.The robot is aspirating solvent from the solvent reservoirs inside
the system using disposable pipette tips.
It is possible to have an extra solvent rack with five 25 mL,
40 mL, or 100 mL solvent reservoirs in position 6 on the
working area. These reservoirs have to be filled manually by
the user. Note that when running a DFE method, wash and
elution solvents are loaded into the carousel (automatically or
manually) and position 6is used for the DFE column tips.
To ensure that the system cannot be set to aspirate more liquid
into a pipette tip than it can accommodate, the maximum
aspiration volume has to be specified for each pipette tip that is
configured in the software.
After completion of a pump operation, the robot can be
programmed to move the pipette tips either back to the pipette
rack (to be reused) or to the pipette tip waste bin. Note that
it is only possible to reuse a sample pipette tip when mixing
following pretreatment and when loading a sample in aliquots;
see "Large Volumes" below.
The system contains corrosion sensitive parts. Best practice
is to avoid sustained continuous exposure to acidic and basic
vapors by always removing the solvent reservoirs when the
system is not in use, and cleaning the system following usage.
Usage of concentrated strong acids (e.g. TFA and TCA) and
strong inorganic acids (e.g. nitric, sulfuric, hydrochloric, and
perchloric acids) in the solvent pumps is not supported.
Large Volumes
When a sample/solvent volume exceeds the specified capacity
of the selected extraction plate or columns, the sample/solvent
will be dispensed in aliquots, with application of positive
pressure in between aliquots.
A user can choose to either reuse the sample pipette tip or use
a new sample pipette tip for each aliquot. This is defined when
setting up a method in the software’s Manage Methods view.
If the run requires more than the available quantity of sample
pipette tips, the system will automatically pause when it runs
out of tips and prompt the user to load more.
Smart Sample Loading
The Smart Sample Loading feature allows the pipette tips to
move down into the sample plate/rack at a speed that enables
the tips to continuously aspirate slightly below the sample
surface without aspirating air. When the pipette tips have
aspirated the required sample volume or are full, they stop.
To get the optimal Smart Sample Loading performance, set up the
sample plate according to the instructions on page 10.


System Overview
Figure 3.The plate is lifted up from the carousel to just underneath the
extraction plate or columns, located in position 3 on the working area.
Dual Flow Extraction (DFE)
In dual flow chromatography, the pressure unit is not used
and the extraction media (the DFE column tips) are loaded into
position 6on the working area.
Wash and elution steps are performed in position 3using plates
that are loaded into the carousel. If the method does not require
a waste position, a maximum of four wash/elution plates can
be used (A-D). Each plate can be filled either manually or by the
system using one of the solvents in position 5 (S1-S5). In other
words, if all the wash/elution plates are filled by the user, the
method can use a total of up to nine different solvents.
To process a DFE method, the plate/rack frame in position 3on
the working area must be removed so that the carousel plates
can be moved up by the lifter into a position where they can be
accessed by pipette and DFE column tips. There is also a DFE
software option that has to be enabled. For more information,
see "Enable/Disable the DFE Mode" on page 29.
Figure 4.When running a DFE method, wash and elution plates are loaded
into the carousel. By removing the plate/rack frame in position 3 on the
working area, the carousel plates can be moved up into a position where
the pipette and DFE column tips can access them.
Working Area
The working area has seven positions (see Figure 2):
» 1: Solvent pipette tips.
» 2: Sample pipette tips.
» 3: Extraction plate or columns.
» 4: Sample plate.
» 5: Solvent reservoirs, 5 x 25 mL.
Filled using the five solvent pumps.
» 6: Optional solvent reservoirs, 5 x 25 mL, 5 x 40
mL, or 5 x 100 mL (manually filled). Note that the
100 mL reservoir has a maximum fill volume of
90mL. Additional solvent tips, 50 L, 200 L.
» WASTE: Waste bin for used pipette tips.
Note: A different setup is used for dual flow chromatography;
see page 2.
Figure 2.The working area has seven positions (1-6 and WASTE) and the
carousel has four positions (A-D).
Carousel and Lifter
The carousel has three positions for collection plates (A-C)
and one for a flow-through plate (D), which is used for guiding
waste into the extraction waste collector eliminating the risk of
cross-contamination.
Before each operation in the method, the carousel moves the
flow-through plate or the specified collection plate to the inner
carousel position. When in position, the plate is moved up by
a lifter into a position just underneath the extraction plate or
columns (see Figure 3), so that the Luer fittings of the plate or
columns cannot splash droplets onto each other or adjacent
wells during the operation.

Biotage®Extrahera™LV-200 User Manual | © Biotage 2021
System Overview
Columns, Plates, and Sample Tubes
The following formats are supported:
Extraction: 96-well extraction plates, 96-array plates for 1and
2 mL wells, 96-position extraction racks for 1 mL (tabless)
columns (A format), and 96-position pipette racks for 200 µL
and 1 mL DFE column tips.
Sample: 1 mL x 96 and 2 mL x 96 sample plates, and 96-position
retaining plate for 1.4 mL matrix sample tubes.
Collection: 1 mL x 96 and 2 mL x 96 collection plates.
Wash/Elution Plates (DFE): 1 mL x 96 and 2 mL x 96 plates,
Flow-through plate: The flow-through plate (P/N 414201SP)
is used for guiding waste into the extraction waste collector
eliminating the risk of cross-contamination. The plate can be
cleaned and reused.
Pressure Unit
The pressure unit is designed to process extraction plates or
columns using pressurized gas.
The gas connected to the AIR port (at the right side of the
system) is used to seal the plate or columns and its pressure
has to be adjusted according to how many positions in the
plate/rack that are populated. Use 6 ± 0.2 bar (0.6 ± 0.02 MPa;
87 ± 3 psi) for a fully populated extraction plate/rack and lower
to approximately 4 bar (0.4 MPa; 58 psi) when 50% of the plate/
rack is populated and 3 bar (0.3 MPa; 44 psi) when 25% of the
plate/rack is populated.
The gas connected to the N2port is used when processing
samples. The processing pressure is adjustable from 0 to
5 bar in the software, while the pressure into the system has
to be set to 6 ± 0.2 bar (0.6 ± 0.02 MPa; 87 ± 3 psi). The gas
flow when processing is between 0 and 10 mL/min during
all operations except for plate dry, where it is approximately
600 mL/min at 5 bar.
The gas applied to the samples must be according to
ISO 8573-1, classification 1-6-1. This is essential to prevent
sample contamination and general fouling of the pressure unit.
The filter unit that is supplied with the system delivers
according to ISO 8573-1, class 1-x-1. If the gas applied is not
according to class x-6-x, an air dryer needs to be ordered
separately (P/N 416441SP).
Note that the pressure unit is not used in dual flow
chromatography.
Pressure Gradients
When setting up a method, it is possible to create pressure
gradients for conditioning, equilibration, sample load, wash,
collection, and elution steps. For more information, see page 16.
Waste Kit and Vacuum
A laboratory vacuum source, or a vacuum pump (sold
separately, P/N 356330SP), is required for collecting the waste
in the
5-liter waste reservoir outside the system . Always ensure
that there is sufficient volume in the waste reservoir before
starting a run.
Figure 5
Connect to the upper port of the
waste valve, which is located on
the left side of your system
Connect to
the vacuum
source
Approx. 7 cm
.The setup of the waste kit.
.
Safety
The ventilated system enclosure protects the user against
mechanical hazards and potentially harmful solvents and/or
vapors. The system cannot be operated when:
» the door is open, and/or
» the integral system ventilation fan is not working.
Figure 6.The outlet of the Extrahera top ventilation.


System Overview
Visual and Audible Alarm
The system’s visual and audible alarm is triggered by non-user-
initiated pauses, scheduled pauses (defined in the method),
and system errors:
» When a non-user-initiated or scheduled pause occurs,
the software background flashes in blue and an
alarm with short beeps sounds (see Figure 7).
» If a system error occurs, the software background flashes in
red and an alarm with longer beeps sounds (see Figure 7).
The alarm stops when the touch screen is pressed. If desired,
the audible alarm can be disabled in the software’s
Maintenance view (see page 30).
Figure 7.When a non-user-initiated or scheduled pause occurs, the
software background flashes in blue. If a system error occurs, the software
background flashes in red.
Lighting
To provide better visibility for the user, there are lamps
available inside the system. These can be turned on and off in
the software’s Maintenance view (see page 30). There is one
control for the upper lamps and one for the lower.
Adjustable Touch Screen
The system is operated through a 12" touch screen that is
adjustable for user height, and can be front facing or side
facing. There are two ports for connecting USB memory devices
on the left side of the touch screen frame.
Barcode and QR Scanner (Optional)
The barcode and QR scanner (sold separately) can be connected
to one of the two USB ports on the touch screen frame and be
used instead of the touch keyboard to enter information.
Biotage®Extrahera™GLP Software
License (Optional)
With the Extrahera GLP software license, you can combine
the utility of the system’s advanced lab automation with the
organizational features used under Good Laboratory Practices.
Highlighted GLP Features
» Manage
» Oversee system control and access
with five distinct User Roles.
» User roles designed to match lab personnel
with their training and expertise.
» Secure
» Maintain system and data security.
» Incorporated password protection, timed
sessions, and restricted USB port access.
» Network
» Network integration allows for backup/
restore or export of reports and audit trails to
your local area network or LIMS system.
» Receive e-mail alerts for system pauses or errors and view
your runs on your laptop using the remote viewing option.
» Audit
» Automatically monitor all system
operations with precision tracking.
» Generate a clear audit trail documenting all
activities, including user credentials and
reasons for making any method changes.
Note: This manual mentions the GLP features briefly. For more
information and detailed instructions, see the Extrahera GLP
User Manual (P/N 417250).

Biotage®Extrahera™LV-200 User Manual | © Biotage 2021
Power Failure
Power Failure
Warning
» If the system is found with the door closed and the power off,
ensure to ventilate the system properly before turning the
system back on.
The system has open solvent reservoirs. If the ventilation fails
and solvent vapors are not removed, an explosive environment
could be generated. If the system is found with the door closed
and the power off, you must:
1. Ventilate the system properly by opening the door
manually using the T25 Torx screwdriver supplied with
the system; see Figure 8. Ensure to take the necessary
precautions to avoid exposure to potentially harmful
solvents and/or vapors.
2. Remove all solvent reservoirs and remove any spillage
before turning the system back on.
We do not recommend that the system is left unattended for
an extended period of time when using flammable solvents.
Figure 8.Open the door manually by turning the screw counterclockwise
using the T25 Torx screwdriver supplied with the system.


Manage Solvents, Sample Types, Plates, Racks, and Tips
This bell appears in the lower right corner when
important information reardin the setup is available.
View the information by pressing the bell.
Solvents, sample types, extraction media (including DFE
column tips), sample plates, DFE wash and elution plates, and
pipette tips can be created, copied, edited, and deleted in the
Data Administration view. It is also possible to lock a solvent
to a specific reservoir in the solvent rack in position 5on the
working area.
Note: The robot is a precision instrument, i.e. it is important that
you enter the correct values when setting up solvents, sample
types, plates, racks, and tips in the Data Administration view
(see Figure 9).
Figure 9.The Data Administration view.
Predefined Entries
The system comes with a number of predefined entries in the
Data Administration view that cannot be edited or deleted.
If desired, these can be copied and edited to your preferences.
Custom Entries
Custom entries cannot be deleted when used in a method or
on the system, e.g. when a solvent is locked to a specific
solvent reservoir, or when there are dependencies between
entries, e.g. a pipette tip cannot be deleted when it is included
in a solvent or sample type.
Manage Solvents, Sample Types,
Plates, Racks, and Tips
Data Administration
Manage Solvents
To add, copy, edit, view, and delete solvents, and lock a solvent
to a specific solvent reservoir, press Data Administration in
the main menu and then Manage Solvents (see Figure 9).
The Manage Solvents view opens; see Figure 10.
Figure 10.The Manage Solvents view.
Note: Usage of concentrated strong acids (e.g. TFA and TCA) and
strong inorganic acids (e.g. nitric, sulfuric, hydrochloric, and
perchloric acids) in the solvent pumps is not supported.
General Parameters
The following general parameters are available for solvents (see
Figure 11):
» Solvent name: The name that will be
displayed in the software.
» Solvent description: The solvent including all additives.
» Serial dispensing allowed?: Whether serial dispensing is
allowed (Yes) or not (No). Note that serial dispensing is
faster, while individual dispensing has a higher precision.
» Highly volatile?: Whether the solvent is highly volatile
(Yes) or not (No). When a highly volatile solvent is assigned
to the solvent rack in position 5 on the working area, the
reservoir is filled just before the solvent is used for the first
time in the run. The user will be prompted to ensure that
the solvent reservoir is empty before the run is started.
» Requires conditioning?: Whether the pipette
tip requires conditioning (Yes) or not (No).

Biotage®Extrahera™LV-200 User Manual | © Biotage 2021
Manage Solvents, Sample Types, Plates, Racks, and Tips
» Conditioning frequency: How often the pipette tips are
conditioned; only before the first aspiration, before
each step, or before every aspiration. This field is only
enabled when Requires conditioning is set to Yes.
» Conditioning, number of times: The number of
conditioning iterations. This field is only enabled
when Requires conditioning is set to Yes.
» Conditioning flow rate (mL/min): The flow rate that will
be used when conditioning the pipette tip. This field is
only enabled when Requires conditioning is set to Yes.
» Conditioning volume (% of tip capacity): The percentage
of the pipette tip capacity that will be filled with
solvent when conditioning the tip. This field is only
enabled when Requires conditioning is set to Yes.
Figure 11.The general solvent parameters and the tips that can be used
with the solvent. Add a tip by selecting it from the Add tip drop-down list
and pressing the + button.
Tip Parameters
Note: A method cannot be run if it uses a solvent that is missing
settings for the used tip.
The system allows you to have individual flow rate, post
dispense, and air gap settings for each tip that the solvent will
be used with. The predefined solvents include settings for the
50 µL, 200 µL, and 1000 µL pipette tips from Biotage.
To add a tip to a custom solvent, select the tip from the Add tip
drop-down list at the General tab and press the +button (see
Figure 11).
The following solvent parameters are available for each tip
assigned to the solvent (see Figure 12):
» Aspiration flow rate (mL/min): The flow rate that
will be used when aspirating the solvent.
» Dispense flow rate (mL/min): The flow rate that
will be used when dispensing the solvent.
» Aspirate post dispense?: Whether air is aspirated into
the pipette tip after dispensing solvent (Yes) or not (No).
» Aspirate post dispense flow rate (mL/min): The flow
rate that will be used when aspirating post dispense.
» Aspirate post dispense volume (µL):
The volume of the post dispense aspiration.
» Lower air gap flow rate (mL/min):
The flow rate that will be used when aspirating
and dispensing the lower air gap.
» Lower air gap volume (µL):
The volume of the lower air gap.
» Upper air gap flow rate (mL/min):
The flow rate that will be used when aspirating
and dispensing the upper air gap.
» Upper air gap volume (µL):
The volume of the upper air gap.
» Upper air gap dispense pause (ms):
The amount of time that the system will
pause between the solvent dispensation
and the upper air gap dispensation. The
default value of 300 ms can be adjusted
when dealing with viscous, volatile,
or low surface tension solvents.
Figure 12.The parameters than can be set up for each tip that is assigned
to the solvent.
Lock and Unlock a Solvent
To lock a solvent to a specific solvent reservoir in position 5on
the working area:
1. Press Lock in the Manage Solvents view (see Figure 10).
2. In the Lock Solvents to Reservoir Positions view, select the
solvent to be locked from the desired reservoir drop-down
list (see Figure 13) and select the corresponding check box.
As long as the solvent is locked to the reservoir, no other
solvent can be used in that position. To unlock, clear the check
box and press Save.
Upper
air gap
Lower
air gap
Liquid


Manage Solvents, Sample Types, Plates, Racks, and Tips
Figure 13.The Lock Solvents to Reservoir Positions view with methanol
locked to reservoir S1.
Manage Sample Types
To add, copy, edit, view, and delete sample types, press Data
Administration in the main menu and then Manage Sample
Types (see Figure 9 on page 6).The Manage Sample
Types view opens; see Figure 14. The sample type settings are
important if you work with a wide variety of different sample
matrices whose liquid handling properties can vary.
Figure 14.The Manage Sample Types view.
General Parameters
The following general parameters are available for sample
types (see Figure 15):
» Sample name: The name that will be
displayed in the software.
» Sample description: A description of the sample.
Figure 15.The general sample parameters and the tips that can be used
with the sample type. Add a tip by selecting it from the Add tip drop-down
list and pressing the + button.
Tip Parameters
Note: A method cannot be run if it uses a solvent that is missing
settings for the used tip.
The system allows you to have individual flow rate, post
dispense, and air gap settings for each tip that the sample will
be used with. The predefined sample types include settings for
the 50 µL, 200 µL, and 1000 µL pipette tips from Biotage.
To add a tip to a custom sample type, select the tip from the
Add tip drop-down list at the General tab and press the +button
(see Figure 15).
The following sample parameters are available for each tip
assigned to the sample type (see Figure 16):
» Aspiration flow rate (mL/min): The flow rate that
will be used when aspirating samples.
» Dispense flow rate (mL/min): The flow rate that
will be used when dispensing samples.
» Aspirate post dispense?: Whether air is aspirated into the
pipette tip after dispensing sample (Yes) or not (No).
» Aspirate post dispense flow rate (mL/min): The flow rate
that will be used when aspirating air post dispense.
» Aspirate post dispense volume (µL):
The volume of the post dispense aspiration.
» Lower air gap flow rate (mL/min): The flow rate that will be
used when aspirating and dispensing the lower air gap.
» Lower air gap volume (µL):
The volume of the lower air gap.

Biotage®Extrahera™LV-200 User Manual | © Biotage 2021
Manage Solvents, Sample Types, Plates, Racks, and Tips
» Upper air gap flow rate (mL/min): The flow
rate that will be used when aspirating
and dispensing the upper air gap.
» Upper air gap volume (µL): The
volume of the upper air gap.
» Upper air gap dispense pause (ms):
The amount of time that the system will pause
between the sample dispensation and the upper
air gap dispensation. The default value of 1000
ms can be adjusted when dealing with viscous
or pretreated samples that behave differently.
Figure 16.The parameters than can be set up for each tip
that is assigned to the sample type.
Manage Extraction Media
To add, copy, edit, view, and delete extraction plates and
column racks, press Data Administration in the main menu and
then Manage Extraction Media (see Figure 9 on page 6).
The following parameters are available for extraction media:
» Name: The extraction plate or column rack name
that will be displayed in the software.
» Manufacturer: The manufacturer’s name.
» Part number: The manufacturer’s part number.
» Capacity volume (µL): The amount of liquid that each
well or column can accommodate. If the sample load/
solvent volume (in the SPE method) exceeds this, the
sample/solvent will be dispensed in aliquots, with
application of positive pressure in between aliquots.
Note that this aliquot feature is disabled when the capacity
volume is set to zero, which is the default value.
» Format: The number of wells or columns.
» Comment: Optional information about
the plate or column rack.
» Solvent/Sample dispensation height (mm):
The height where the solvent/sample is dispensed by
the pipette tip. See “Tune Pipetting Heights” below.
» Aspiration height (mm): The height where the sample
mixture is aspirated by the pipette tip during mixing in the
extraction media. See “Tune Pipetting Heights” below.
Figure 17.The Manage Extraction Media view.
Tune Pipetting Heights
The pipetting heights for plates and racks can either be entered
manually or set using a wizard (see Figure 18). To open the
software wizard, press Tune Pipetting Heights... when setting
up the new plate or rack. Follow the instructions in the wizard.
Figure 18.Wizard for setting the pipetting heights for plates and racks.
Upper
air gap
Lower
air gap
Liquid


Manage Solvents, Sample Types, Plates, Racks, and Tips
Manage Sample Plates
To add, copy, edit, view, and delete sample plates, press Data
Administration in the main menu and then Manage Sample
Plates and Racks (see Figure 9 on page 6).
The following parameters are available for sample plates:
» Name: The sample plate name of the that
will be displayed in the software.
» Capacity volume (µL): The maximum amount of liquid that
each well or test tube can accommodate. To optimize the
Smart Loading feature, see “Smart Sample Loading” below.
» Plate/rack height (mm): The distance between the bottom
of the pipette head (when in its highest position) and the
top of the plate. This setting is used when the system
determines a safe scan height for clog detection (GLP
only). For instruction on how to measure the height,
see “Measure the Sample Plate Height” below.
» Format: The number of wells .
» Aspiration height (mm): The height where the sample
is aspirated and premixed by the pipette tip. See “Tune
Pipetting Heights” on page 9. To optimize the Smart
Loading feature, see “Smart Sample Loading” below.
» Pretreatment dispensation height (mm): The height where
pretreatment solvent is dispensed by the pipette tip. See
“Tune Pipetting Heights” on page 9. To optimize the
Smart Loading feature, see “Smart Sample Loading” below.
Note: The aspiration and dispensation heights used by the DFE
column tip for loading the sample are defined in the method.
Figure 19.The Manage Sample Plates view.
Smart Sample Loading
The Smart Sample Loading feature allows the pipette tips to
move down into the sample plate/rack at a speed that enables
the tips to continuously aspirate slightly below the sample
surface without aspirating air. When the pipette tips have
aspirated the required sample volume or are full, theystop.
When a sample/solvent volume exceeds the specified capacity
of the selected extraction plate or columns, the sample/solvent
will be dispensed in aliquots, with the option to either reuse
the sample pipette tip or use a new sample pipette tip for each
aliquot. See “Large Volumes” on page 1.
To get the optimal Smart Sample Loading performance, refer to
“Manage Sample Plates” and set up the sample plate according
to the following:
1. Set the Aspiration height to 1 mm from bottom of the wells/
tubes.
Figure 20
1 mm
.Figure 20a: Use the Smart Sample Loading feature to prevent
aspiration of air. Figure 20b: Set the aspiration height.
2. Set the Pretreatment dispensation height to approximately
4 mm below the top of the wells/tubes.
3. Set the Capacity volume to a value in µL that corresponds to
a liquid surface at 5 mm below the top of the wells/tubes.
Figure 21
4 mm
5 mm
.Image 1: Set the Pretreatment dispensation height. Image 2:
Set the Capacity volume.
To turn the Smart Sample Loading function off, set the Capacity
volume to  (zero) µL.
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 Biotage®Extrahera™LV-200 User Manual | © Biotage 2021
Manage Solvents, Sample Types, Plates, Racks, and Tips
Measure the Sample Plate Height
The Plate height setting is used when the system determines a
safe scan height for clog detection (GLP only).
1. Place the sample plate that you want to measure in position
4on the working area.
2. Measure the distance from the surface of the working area
to the top of the wells/tubes. As a reference, this value
is between 36 and 61 mm for the default Biotage sample
plates.
3. Set the Plate height to the measured value.
Figure 22.The height for this sample plate is 37 mm.
Manage Pipette Tips
To add, copy, edit, view, and delete pipette tips, press Data
Administration in the main menu and then Manage Pipette Tips
(see Figure 9 on page 6).
The following parameters are available for pipette tips:
» Name: The pipette tip name that will
be displayed in the software.
» Manufacturer: The manufacturer’s name.
» Part number: The manufacturer’s part number.
» Capacity (µL): The maximum amount of liquid that
can be aspirated. This setting ensures that the
system cannot attempt to aspirate more liquid
into the pipette tip than it can accommodate.
» Length (mm): The length of the pipette tip.
Figure 23.The Manage Pipette Tips view.
Manage Dual Flow Extraction Consumables
Figure 24.The Manage Dual Flow Extraction Consumables view.
Manage Carousel Plates
To add, copy, edit, view, and delete DFE carousel plates, press
Data Administration in the main menu and then Manage Dual
Flow Extraction Consumables (see Figure 9 on page 6) and
Manage Carousel Plates.
The following parameters are available for DFE carousel plates:
» Name: The wash/elution plate name that
will be displayed in the software.
» Capacity volume (µL): The maximum amount of
liquid that each well can accommodate.
» Plate height (mm): The height of the plate.
» Format: The number of wells.


Manage Solvents, Sample Types, Plates, Racks, and Tips
» Aspiration height (mm): The height where:
» the DFE column tips aspirate during wash,
elution, and posttreatment, and
» the sample pipette tips aspirate
during posttreatment mixing.
» Dispensation height (mm): The height where
» the DFE column tips dispense during
wash, elution, and posttreatment,
» the solvent pipette tips dispense the wash/
elution/posttreatment solvent, and
» the sample pipette tips dispense
during posttreatment mixing.
Set the aspiration and dispensation heights using the Tune
Pipetting Heights wizard; see page 9.
Note: When setting up a DFE method, it is possible to set
individual aspiration and dispensation heights for each
wash and elution step. These will override the aspiration and
dispensation heights set for the carousel plate.
Figure 25.The Manage Carousel Plates view.
Manage Dual Flow Extraction Tips
To add, copy, edit, view, and delete DFE column tips, press
Data Administration in the main menu and then Manage Dual
Flow Extraction Consumables (see Figure 9 on page 6) and
Manage Dual Flow Extraction Tips.
The following parameters are available for DFE column tips:
» Name: The DFE column tip name that will
be displayed in the software.
» Manufacturer: The manufacturer’s name.
» Part number: The manufacturer’s part number.
» Capacity (µL): The maximum amount of liquid that
can be aspirated. This setting ensures that the
system cannot attempt to aspirate more liquid
into the column tip than it can accommodate.
» Length (mm): The length of the DFE column tip.
» Media mass: The mass of the media
used in the DFE column tip.
» Format: The number of positions in the pipette rack.
Figure 26.The New DFE Tip view.
Biotage®Extrahera™GLP Features
» Custom entries cannot be edited.
» Deleting an unused custom entry requires that a reason
for deleting and the user password are entered.
» Locking/unlocking a solvent to a specific solvent reservoir
in position 5on the working area requires that a reason
for the action and the user password are entered.
» All creations and deletions of custom entries in the Data
Administration view and locking and unlocking of solvents
are logged in the Data Administration audit trail.
For more information, see the Extrahera GLP User Manual
(P/N 417250).

 Biotage®Extrahera™LV-200 User Manual | © Biotage 2021
Set Up a Method
Set Up a Method
Manage Methods
This bell appears in the lower right corner when
important information regarding the method setup is
available. View the information by pressing the bell.
Methods can be created, copied, edited, viewed, imported,
exported, and deleted in the Manage Methods view (see
Figure 27).
For instructions on how to import, export, and delete methods,
see “Export, Import, and Delete Methods” on page 19.
Predefined Methods
The system comes with a number of predefined Biotage
methods that cannot be modified or deleted. If desired, they
can be copied and then edited to your preferences.
Figure 27.The Manage Methods view.
New, Copy, and Edit a Method
To create a new method, press Manage Methods in the main
menu, select the User methods folder, press New, and then
select the type of method (see Figure 28):
» SLE (Supported Liquid Extraction)
» SPE (Solid Phase Extraction)
» PPT/PLD (Protein Precipitation/Phospholipid Depletion)
» Filtration (collects filtrate from extraction
media that is preloaded with sample)
» Filtration+ (sample load and filtration preceded
by an optional pretreatment operation)
» Hydro DME+ (Dual Mode Extraction)
» DFE (Dual Flow Extraction)
Note: There are two PPT/PLD modes, Solvent First or Sample
First, where the sample is either added before or after the
solvent. The mode can be changed in the Maintenance view (see
page 30). This has to be done before you set up the method.
Note: The pressure unit is not initialized in the Filtration method
and disabled in DFE method.
To copy or edit a method, select the folder containing the
method, select the method, and then press Copy or Edit (see
Figure 27). Copies are saved in the User methods folder.
Figure 28.The Select Method Type view.
To save changes in a displayed method, press Save in the top
pane. To return to the Manage Methods view without saving,
press Cancel in the top pane.
For information on the available method parameters, see the
following sections in this chapter.
Exclamation Mark
When setting up a method, the exclamation mark ( ) shows
which information is required to run the method (see Figure 29).
Methods that do not contain all the required information
are highlighted with an exclamation mark ( ) in the Manage
Methods view (see Figure 30).


Set Up a Method
Figure 30.User methods highlighted with do not have all the necessary
information to be run and are not displayed in the Run view.
Figure 29.Setting up an SPE method. The exclamation mark ( ) shows
which information is required to run the method. For more information,
press in the lower right corner.
Method Operations
The following operations can be enabled or disabled when
setting up a method:
Operation SLE SPE P P T/
PLD Filtration Filtration+ Hydro DME+ DFE
Pretreatment  
1  
Conditioning  
Equilibration  
Load/Solvent
load
223
422
Wash  
Elution   
Posttreatment 
Collection    
Table 1.Method operations that can be enabled or disabled.
1The pretreatment step can be moved to take place immediately before load.
2Sample load.
3 The sample is either added before or after the solvent depending on a
system setting. To change this setting, press Maintenance in the main
menu and then disable or enable the Add solvent first option in the
PPT/PLD Mode field. This has to be done before you set up the method.
4A load operation is incorporated into the collection operation.
The following operations has the option to use more than one
type of solvent tip. In Pretreatment it is possible to assign
different tips to the different sub-steps.
Operation SLE SPE P P T/
PLD Filtration Filtration+ Hydro DME+ DFE
Pretreatment  
1
Conditioning  
Equilibration  
Load/Solvent
load
Wash  
Elution  
Posttreatment
Collection
Table 2.Operations that can use more than one tip.
Any combination of 50 L, 200 L, 1000 L, and 1000 L wide
boar tips can be used.
Note: While all tips can be placed in position 1, only 50L and
200L tips can be placed in position 6. These tips require
a special short pipette rack, see more information under
Consumables and Accessories page 42.

 Biotage®Extrahera™LV-200 User Manual | © Biotage 2021
Set Up a Method
Method Parameters
Note: For DFE method parameters, see page 17.
Available method parameters are listed in alphabetical order:
» Advanced pressure settings: Press Edit... to set up a
pressure gradient. For more information, see page 16.
» Air push after last elution?: Whether to apply positive
pressure with a gas flow of approximately 10 mL/min
after the last elution (Yes) or not (No). SLE only.
» Air push time (s): The amount of time, in seconds, that
positive pressure with a gas flow of approximately 10 mL/min
will be applied to the extract
ion plate or columns to trigger
gravity loading or elution. This field is only enabled for
elution
when Air push after last elution? is set to Yes. SLE
only. Note that this field is disabled when a pressure gradient
is enabled; see “Set Up a Pressure Gradient” on page 16.
» Clog detection?: GLP feature. For more information,
see the Extrahera GLP User Manual (P/N 417250).
» Clog settings: GLP feature. For more information,
see the Extrahera GLP User Manual (P/N 417250).
» Collect in position: In which position the
collection platerack or the flow-through plate
(waste) is to be loaded onto the carousel.
» Collection plate height (%): How high, in percentage of the
maximum height, the collection
plate is moved up by the
lifter. See Table 3 on page 16 for guidelines. Filtration only.
» Conditioning solvent: The solvent to be used for
pipette tip conditioning. SPE only. This field is only
visible when Tip conditioning? is set to Yes.
Note: The solvent must include parameters for the pipette tip
it will be used with; see “Manage Solvents” on page 6.
» Dispose solvent tips?: Whether the pipette tips
will be disposed of after the solvent load (Yes)
or reused (No). PPT/PLD (solvent first) only.
» Dispose solvent tips after each step?: Whether the pipette
tips will be disposed of after each step (Yes) or reused (No).
Note that it is not possible to reuse a pipette tip after sample
dispensation. Not available for Filtration and PPT/PLD.
» Extraction media: The type of extraction plate or columns.
» Method comment: Optional information about the method.
» Method name: The name of the method.
» Mix number of times: The number of mixing iterations when
mixing the sample with a solvent following pretreatment
(except for PPT/PLD). Not available for Filtration.
» Mix volume (µL): The aspiration volume when mixing
the sample with a solvent following pretreatment
(except for PPT/PLD). Not available for Filtration.
» Move pretreatment step: Use the arrow buttons to move
the pretreatment operation to the desired position,
before conditioning (default) or before load. SPE only.
» Number of steps: The number of steps the operation
contains. Not available for Filtration and PPT/PLD.
» Number of times: The number of mixing iterations
in the sample plate. This field is only enabled when
Premix? is set to Yes. Not available for Filtration.
» Pause after this step/last step/each load? Whether
to pause after a certain step or operation (Yes) or not
(No). When the system is paused, it is possible to open
the door to, for example, check for clogged wells or
columns. Not available for Filtration and PPT/PLD.
» Plate dry after last elution?: Whether to remove the last
residual solvent from the sorbent bed using positive
pressure at 5 bar with a gas flow of approximately
600 mL/min after the last elution (Yes) or not (No). SPE only.
» Plate dry after last wash?: Whether to remove the last
residual solvent from the sorbent bed using positive
pressure at 5 bar with a gas flow of approximately
600 mL/min (Yes) or not (No). SPE only.
» Plate dry time (s): The amount of time, in seconds, that
positive pressure at 5 bar with a gas flow of approximately
600 mL/min will be applied to remove the last residual
solvent from the sorbent bed. This field is only enabled when
Plate dry after last elution/wash? is set to Yes. SPE only.
» Positive pressure time (s): The amount of time, in seconds,
that positive pressure will be applied to the extraction
plate or columns. Not available for SLE. Note that this
field is disabled when a pressure gradient is enabled;
see “Set Up a Pressure Gradient” on page 16.
» Premix?: Whether the sample will be premixed (Yes)
or not (No) before it is loaded into the extraction
plate or columns. Not available for Filtration.
» Pressure (bar): The pressure, in bar, that will be applied to
the extraction
plate or columns. Not available for SLE. Note
that this setting will not be used when a pressure gradient is
enabled; see “Set Up a Pressure Gradient” on page 16.
» Repeat (number of times): The number of times
this step will be repeated. SPE and SLE only.
» Reuse tips for pretreatment mix and sample load?: Whether
to reuse a sample pipette tip when mixing following
pretreatment, when loading a sample in aliquots, or when
rinsing the wells/tubes in the sample plate (Yes) or not (No).
Not available for Filtration, PPT/PLD, and Hydro DME+.
» Rinse repeat number of times: The number of rinsing
iterations in the sample plate. This field is only
enabled when Premix? is set to Yes. SPE only. This
field is only visible when Rinsing? is set to Yes.
» Rinse solvent: The solvent to be used to rinse the
wells/tubes in the sample plate. SPE only. This field
is only visible when Rinsing? is set to Yes.
Note: The solvent must include parameters for the pipette tip
it will be used with; see “Manage Solvents” on page 6.


Set Up a Method
» Rinse volume (µL): The amount of rinsing solvent to be
used for each well/tube in the sample plate. SPE only.
This field is only visible when Rinsing? is set to Yes.
» Rinsing?: Whether to remove the last residual sample
from the sample plate by rinsing the wells/tubes
with solvent, and then transferring the liquid to the
extraction media (Yes) or not (No). SPE only.
» Sample pipette tip type: The type of pipette
tips to be used when loading and mixing the
sample. Not available for Filtration.
» Sample plate/rack: The type of sample plate
or tubes. Not available for Filtration.
» Sample type: The type of sample. This setting is important
if you work with a wide variety of different sample
matrices whose liquid handling properties can vary.
» Sample volume (µL): The amount of sample to be used
for each well or column in the extraction plate/rack. If the
volume exceeds the specified capacity of the selected
extraction
plate or columns, the sample will be dispensed
in aliquots, with application of positive pressure in
between each aliquot. Not available for Filtration.
Note: If the capacity of the selected extraction
plate or
columns is set to zero, which is the default value, the system
will still dispense the requested sample volume.
Note: When loading a sample in aliquots, the sample pipette
tip can be reused. See Reuse tips for pretreatment mix and
sample load above.
» Solvent: The solvent to be used for this
step. Not available for Filtration.
Note: The solvent must include parameters for the
pipette tip(s) it will be used with; see “Manage Solvents”
on page 6.
» Solvent tips: The tip to be used in a specific step. When
position 6 is used for additional tips it is possible to chose
tips from either position 1 or position 6 for each step.
» Starting sample volume in plate/rack (µL): The
amount of available liquid in each well/tube in the
sample plate, prior to starting the method.
» Tip conditioning?: Whether to condition the pipette
tip with a solvent (Yes) or not (No). SPE only.
» Volume (µL): The amount of solvent to be
used. Not available for Filtration.
» Wait time (min): The amount of time to
wait to complete the process step.
Set Up a Pressure Gradient
When setting up an SPE method, it is possible to create a
pressure gradient for any conditioning, equilibration, load,
wash, or elution step. Pressure gradients are also available
for load and elution steps in SLE methods, and for the
collection step in PPT/PLD, Filtration, Filtration+, and Hydro
DME+ methods. To set up a pressure gradient, press the Edit...
button for the desired step (see Figure 31).
The following pressure gradient parameters are available (see
Figure 31):
» Use advanced pressure settings? Whether to
use a pressure gradient (Yes) or not (No).
» Number of steps: The number of steps
the pressure gradient contains.
» Pressure (bar): The pressure, in bar, that will be
applied to the extraction
plate or columns.
» Positive pressure time (s): The amount of
time, in seconds, that positive pressure will be
applied to the extraction
plate or columns.
» Air push/Plate dry: Whether to apply positive pressure
with a gas flow of approximately 10 mL/min (air push)
or 600 mL/min at 5 bar (plate dry) after the pressure
gradient has been completed (Yes) or not (No).
» Air push time/Plate dry time (s): The amount of time, in
seconds, that positive pressure will be applied to remove
the last residual solvent from the sorbent bed. This field
is only enabled when Air push/Plate dry? is set to Yes.
Figure 31.In the Edit Advanced Pressure Settings view, you can set up a
pressure gradient for a conditioning, equilibration, sample load, wash,
collection, or elution step.
Specify the Collection Plate Height (Filtration Only)
When setting up a Filtration method, you need to specify how
high the collection plate is to be moved up by the lifter toward
the extraction media. The height is specified in percentage of
the maximum height. The table below lists the approximate
settings for the Collection plate height (%) parameter for some
combinations of collection plate and extraction media.
Collection Plate Extraction Media Height
(%)
1/2 mL Collection Plate, 96 Extraction Plate, 96 90
1/2 mL Collection Plate, 96 1 mL Array Plate, 96 73
1/2 mL Collection Plate, 96 1 mL Column Rack, 96 73
Table 3.Examples of settings for the Collection plate height (%)
parameter that is used in the Filtration method.

 Biotage®Extrahera™LV-200 User Manual | © Biotage 2021
Set Up a Method
DFE Method Parameters
Available DFE method parameters are listed in alphabetical order:
» Aspiration flow rate (mL/min): The flow rate to be
used when aspirating liquid. When aspirating solvent,
this setting overrides the flow rate defined for the
solvent. For residual purge, see page 18.
» Aspiration height (mm): The height used when
aspirating from the sample plate using the DFE
column tip. Press Tune Pipetting Heights... to
set the height using a wizard; see Figure 32.
For aspiration height in residual purge, see page 18.
» Cycle volume (µL): The amount of liquid to be used
for each cycle. For residual purge, see page 18.
» DFE tips: The type of dual flow chromatography
column tip to be used.
» Dispensation flow rate (mL/min): The flow rate to
be used when dispensing liquid. When dispensing
solvent, this setting overrides the flow rate defined
for the solvent. For residual purge, see page 18.
» Dispensation height (mm): The height used when
dispensing into the sample plate using the DFE
column tip. Press Tune Pipetting Heights... to
set the height using a wizard; see Figure 32.
For dispensation height in residual purge, see page 18.
» Dispose solvent tips?: Whether the pipette tips will
be disposed of after dispensing the posttreatment
solvent into the elution plate (Yes) or reused (No).
» Fill volume (µL): The amount of solvent filled into the
wash/elution plate on the carousel. The plates can
be filled manually or by the system; see the Filled by
system? setting in the method’s Carousel tab.
» Filled by system?: Whether the wash/elution solvent
is loaded into the carousel plate by the user before the
run is started or by the system. The system can either
load the solvent in the method’s preparation phase
(Yes, before method) or straight before the wash/elution
step is to be performed (Yes, before step). Note that
the latter option requires that the DFE column tips are
temporarily returned to the DFE rack in position 6.
» Hold time after aspiration/dispensation (seconds):
The amount of time that the system will pause
after each aspiration/dispensation.
» Last dispense position: In which position the last
dispensation in the step is to be performed, in the
solvent reservoir or the waste position on the carousel.
» Method comment: Optional information about the method.
» Method name: The name of the method.
» Mix?: Whether the sample/eluate is mixed (Yes) or not
(No) with the pretreatment/posttreatment solvent.
» Mix aspiration/dispensation height (mm):
The height used for aspiration/dispensation when
mixing the sample/eluate with the pretreatment/
posttreatment solvent. Press Tune Pipetting Heights...
to set the heights using a wizard; see Figure 32.
» Mix number of times: The number of mixing
iterations when mixing the sample/eluate with the
pretreatment/posttreatment solvent. This field
is only available when Mix? is set to Yes.
» Mix volume (µL): The aspiration volume when mixing
the sample/eluate with the pretreatment/posttreatment
solvent. This field is only available when Mix? is set to Yes.
» Number of cycles: The number of aspirate-
hold-dispense-hold cycles.
» Number of steps: The number of steps
the operation contains.
» Pause after last cycle/step? Whether to pause after a
certain step or operation (Yes) or not (No). When the
system is paused, it is possible to open the door.
» Plate/rack: The type of plate to be
used in the carousel position.
» Purge settings: See “DFE Method
Parameters” on page 17.
» Residual purge: See “DFE Method
Parameters” on page 17.
» Sample mix tip type: The type of pipette tip to
be used when mixing sample/eluate with the
pretreatment/posttreatment solvent.
» Sample plate/rack: The type of sample plate or tubes.
» Solvent: The solvent to be used for this step.
Note: The solvent must include parameters for the tip(s)
it will be used with (including the DFE column tip, if
applicable); see “Manage Solvents” on page 6.
» Solvent dispensation height (mm): The height used when
dispensing the posttreatment solvent into the (last) elution
plate. If there is no elution step, the system will dispense
into the (last) wash plate. Press Tune Dispensation
Height... to set the height using a wizard; see Figure 32.
» Starting sample volume in plate/rack (µL):
The amount of available liquid in each well/tube in
the sample plate, prior to starting the method.
» Upper air gap volume, DFE tip (µL): The volume of the
upper air gap to be used with the specified DFE column
tip. The air gap is aspirated before the DFE column tip
is picked up. For residual purge, see page 18.
» Used for: What the carousel position is to
be used for; wash, elution, or waste.
» Volume (µL): The amount of solvent to be used.
» Wait time (min): The amount of time to wait to complete
the pretreatment/posttreatment operation.