Biotage Selekt Manual de usuario

Biotage®Selekt

User Manual

Biotage®Selekt User Manual | © Biotage 2018

Biotage®Selekt

User Manual

CONTENTS

System Overview

1 Columns

1 Racks and Vessels

2 Solvent Supply

2 Internal UV Detector

2 Collection and Fractionation Methods

2 Audible Alarm

2 Lighting

Prepare the System

3 Inspect the Tubing

3 Mount Column Holders

3 Start Up the System

3 Assign Solvents

4 Prime the Solvent Inlets

Set Up a Purification

5 Specify the Gradient

6 Specify the Information Parameters

6 Specify the Column Parameters

7 Specify the Collection Parameters

7 Specify the Rack Parameters

Start, Monitor, and Control a Purification

8 Start a Purification

8 Monitor the Purification in Progress

9 Replace and Allocate Racks During the Run

9 Line Flush, Decompression, and Detector Flush

9 Auto Extend

9 Pause, End, or Abort a Purification

9 Edit and Manually Extend a Purification

 Unload a Purification

10 Flush the Column with Air

10 Purge the Column

10 Unload the Run

10 Flush the System

10 Shut Down the System

 Results

11 Export a Report to USB

11 Base a New Run on a Previous Run

 Flushes and Purge

12 Flush

12 Air Flush

12 Purge

 Data Administration

13 Manage Column Types

13 Manage Solvents

14 Manage User Accounts

 System Settings

15 System

15 Purification

 Maintenance

16 Calibrate the Internal UV Detector

16 Clean the Exterior of the System

16 Clean the Flow Cell of the Internal UV Detector

17 Clean or Release Check Valves

18 Leaks

19 Replace the Fuses

19 Replace the Needle

19 Sonicate Solvent Inlet Filters

 Troubleshooting

20 Fraction Collector-Related Problems

20 Gradient Problems

20 Internal UV Detector-Related Problems

20 Leak Detected

21 Overpressure Detected

21 Pump-Related Problems

22 QR Reader Problems

22 Contact Biotage®1-Point Support™

 Solvent Specifications

 General Information

24 Consumables and Accessories

24 Manufacturer

24 Contact Us

Contents



System Overview

Biotage®Selekt is an automated flash purification system

with a built-in QR reader for Biotage Sfär columns, RFID reader

for Selekt racks, UV detector, pump, fraction collector, and

touch screen.

Columns

The system has two column channels. Columns can be placed

both on the right side of the system and on the front (up to 50g);

see Figure 1.

Figure 1.Biotage®Selekt with two columns.

QR Reader

All Biotage®Sfär columns have a QR code that can be read by

the QR reader underneath the touch screen; see Figure 2.

Figure 2.Scanning a Biotage®Sfär column.

Racks and Vessels

The system collects fractions into a variety of collection racks

and vessels. The following Selekt racks are available:

48 positions for 13 x 100 mm tubes

with a vessel volume of 9 mL.

35 positions for 16 x 100 mm tubes

with a vessel volume of 14 mL.

35 positions for 16 x 150 mm tubes with

a vessel volume of 22 mL.

28 positions for 18 x 150 mm tubes with

a vessel volume of 27 mL.

15 positions for 25 x 150 mm tubes with

a vessel volume of 45 mL.

6 positions for 120 mL flasks.

10 positions for 240 mL flasks.

8 positions for 480 mL flasks.

The collection bed can either hold a collection tray with up to

three of the racks with vessel volumes up to 120 mL (Figure 3),

or one of the 240 or 480 mL flask racks.

One collection tray is standard, but by extending the fraction

collector, it is possible to use two collection trays at the same

time. The maximum collection volume with no rack change is

3840 mL for standard bed and 7680 mL for extended bed.

RFID Tags

All Selekt racks have an RFID (radio-frequency identification)

tag that can be automatically identified by the system when the

rack is placed on the collection bed.

Figure 3.Collection tray with three racks.

Biotage®Selekt User Manual | © Biotage 2018

System Overview

Solvent Supply

The system is equipped with four solvent inlets found on the

right side of the system (S1-S4).

Optional Secondary Solvent Containment

A maximum of four 5-liter reservoirs can be placed on the

optional secondary solvent containment; see Figure 4.

Larger reservoirs than 5 liters must be placed elsewhere.

Note: To be compliant with the secondary containment

regulations, do not use reservoirs larger than 4 liters.

Figure 4.The optional secondary solvent containment.

Internal UV Detector

The system includes either a UV detector with the range 200 to

400 nm or a UV-VIS detector with the range 198 to 810 nm.

Collection and Fractionation Methods

Available detection signals are:

λ-All: The system uses average absorbance

within a user-defined wavelength range for

collection and fractionation. Possible collection

and fractionation parameters are Collect All,

Threshold, and Valley Fractionation. Only available

on systems with a Spektra software license.

UV1 and UV2: The system uses one or two wavelength

signals for collection and fractionation. Possible

collection and fractionation parameters are Collect

All, Threshold, and Valley Fractionation.

All collection methods fractionate on volume, which is when

a test tube or bottle is full based on the specified vessel volume

for the rack type or a user-defined maximum fraction volume

defined for the run.

Signals that are not used for collection and fractionation can be

used for monitoring.

Audible Alarm

If the audible alarm is enabled in the system settings, an

audible warning will sound whenever user interaction is

required. Examples of situations are: 1) rack change is needed,

2) high pressure, 3) misalignment of the collection arm, and

4) pump failure.

Lighting

To provide better visibility for the user, there is a light

strip underneath the touch screen and a mirror behind the

collection racks.



Prepare the System

Inspect the Tubing

Warning

Shut down the system before replacing any tubing. Use only

tubing designed for the Selekt system and supplied by Biotage.

Inspect all tubes before each run to ensure that they do not

show signs of wear or damage and that they are properly

connected and tightened. Use caution when finger tightening

fittings to prevent stripped threads or crushed ferrules.

All external tubing on the system except for the tubing on the

collection arm can be replaced by the user.

Note: All tube types, dimensions, and lengths are essential

for the performance of the system. Only replace tubes with the

equivalent tubes designed for the Selekt system and supplied

by Biotage.

Mount Column Holders

The system is shipped with column holders of different sizes.

Ensure that they are mounted in the desired positions on the

system. Figure 5 shows how to mount a column holder on

the right side of the system. There are also two positions for

column holders on the front of the system that can be used for

Biotage columns up to 50g; see Figure 6.

Figure 5.Slide the column holder screw into the desired position on

the right side of the system and then place the holder over the screw

and fasten.

Figure 6.Biotage columns up to 50g can be placed on the front of

the system.

Prepare the System

Start Up the System

Turn on the system using the power switch located on the left

side of the system; see Figure 7.

Figure 7.The power switch is located on the left side of the system.

Assign Solvents

When a purification is run, the software determines which

solvent inlets are connected to the chromatography solvents

used in the run.

Note: For reversed-phase purification with methanol and

water, it is strongly recommended to premix the water with

5% of methanol and degass either through vacuum or

sonication. Degassing of protic solvent blends decreases

out-gassing of entrapped air during gradient elution, which

will impact gradient performance and flow rates.

Note: All four solvent inlets must be primed with solvent to

achieve the specified pump performance.

1. In the software, select Solvent Setup from one of the

solvent drop-down lists in the gradient view. The Solvent

Setup window opens; see Figure 8.

2. Assign a solvent to each solvent inlet using the solvent

drop-down lists.

To add solvents to the list, select Solvent Administration.

For more information, see “Manage Solvents” on page 13.

3. Prime the solvent inlets that have been assigned a new

solvent; see page 4.

Biotage®Selekt User Manual | © Biotage 2018

Prepare the System

Figure 8.The Solvent Setup window.

Prime the Solvent Inlets

Before you start a purification on your system, you might need

to prime the solvent inlets to:

Remove any air bubbles from the pump and the

solvent inlets by flushing them with solvents.

Empty the solvent inlets of solvents used in the previous

purification and fill them with new solvents.

Note: All four solvent inlets must be primed with solvent to

achieve the specified pump performance.

Note: We recommend to have all solvent reservoirs on the same

physical height to improve accuracy in the solvent mixing.

1. In the software, select Solvent Setup from one of the solvent

drop-down lists in the gradient view. The Solvent Setup

window opens; see Figure 8.

2. Prime the solvent inlets by pressing the corresponding

Prime buttons. Note that 27 mL of the assigned solvent is

used for each prime.



Set Up a Purification

Warning

Never exceed the maximum pressure or flow rate for the

used column.

1. In the software, select the column channel to be used by

pressing Channel 1 or Channel 2 in the lower right corner,

or by swiping left or right on the channel tab along the edge.

2. To base the purification on a previous run, press Previous

Runs and then see “Base a New Run on a Previous Run”

on page 11.

3. Either scan your column using the QR reader underneath

the touch screen (see Figure 9) or select the column type

from the Column Type drop-down list in the Column panel.

Figure 9.The QR reader is located underneath the touch screen.

4. Place the column in a column holder on the right side of the

system or on the front (up to 50g).

5. Connect the correct inlet and outlet tubing to the column.

The tubes for channel 1 are labeled C1 with orange tags and

the tubes for channel 2 are labeled C2 with blue tags.

6. Place the collection rack(s) that you want to use on the

collection bed. Racks with vessel volumes up to 120 mL

require the collection tray. Selekt racks are automatically

identified by the system when placed on the collection

bed. If automatic rack detection has been disabled (see

page 15), select the collection rack manually; see

“Specify the Rack Parameters” on page 7.

7. Set up the run parameters as described in the following

sections.

Note: An invalid setting is highlighted with a red frame

(see Figure 10), press in the bottom right corner for more

information.

Figure 10.The run setup view.

Specify the Gradient

The gradient can be set up using the gradient table or the

gradient graph. It is also possible to calculate a gradient from

Rfvalues obtained from a TLC analysis (see page 6).

Gradient Graph and Table

To add a gradient segment, press-and-hold a segment or

node in the gradient graph and select Add Segment from the

appearing menu. The new segment will be added after the

selected segment or node.

To delete a node or segment, press-and-hold and select Delete

Selected from the appearing menu (see Figure 11).

Figure 11.The press-and-hold menu in the gradient view.

To increase or decrease the length of a gradient segment or

change the solvent mix, either 1) drag the segment or node to

the desired position in the gradient graph, or 2) change the

value in the gradient table.

Note that it is possible to zoom in and out of the gradient using

the pinch-to-zoom feature.

Set Up a Purification

Biotage®Selekt User Manual | © Biotage 2018

Set Up a Purification

Gradient Parameters

CV/mL/mm:ss: The gradient length unit. CV (column

volumes), milliliters, or minutes and seconds; see Figure 12.

Start: The percentage of Solvent B at the

beginning of the gradient segment.

End: The percentage of Solvent B at the

end of the gradient segment.

Length: The length of the gradient segment.

Solvent A and B: The solvents to be used.

Equil.: The column is equilibrated before the run, unless

this options is turned off. The equilibration volume and

flow rate are set automatically and have been optimized

for the selected column type. To see the settings,

expand the Column panel (see Figure 14). We strongly

recommend that the equilibration is not turned off.

Figure 12.The gradient length unit can be selected in the gradient table.

Calculate a Gradient from TLC Data

Note the following when using the TLC to Gradient editor:

Accuracy of prediction can be reduced if using

alcohols, such as methanol and ethanol, very volatile

modifiers such as diethyl ether, or modifiers that

permanently alter the properties of the silica.

Reversed-phase TLC is not supported.

Slight variations in migration rates may occur if samples are

applied too close to the edge of the TLC plate. Apply samples

at least 10 mm from the edge to avoid the edge effect.

To calculate a purification gradient from the Rfvalues obtained

from a TLC analysis:

1. Open the TLC to Gradient editor (see Figure 13) by

pressing TLC.

2. Select the Strong Solvent text box and enter the percentage

of the strongest (most polar) solvent in the TLC analysis.

3. Mark the solvent front of the TLC plate by sliding the Front

line to the correct position.

4. Enter the Rfvalue of the product of interest in the RfProduct

text box or by sliding the Product line to the correct position

on the TLC plate.

5. Enter the Rfvalue of the impurity closest below and above

the product in the RfImpurity 1 and RfImpurity 2 text boxes

or by sliding the corresponding lines to the correct positions

on the TLC plate. A value for RfImpurity 1 is required.

6. If you have two plates, press Add Plate and repeat steps

2 through 5 for the second plate. One plate gives a linear

gradient and two plates give a step gradient.

7. To calculate a purification gradient and the column load

capacity, press Create.

Figure 13.The TLC to Gradient editor.

Specify the Information Parameters

User: The name of the user. If you have not been assigned

a user name, please contact your system supervisor.

Run Name: The name of the run. If left blank, the

name will be auto-generated based on date and

time when the purification is performed.

Comments: Comments on the purification run. (Optional)

Specify the Column Parameters

Sample Mass: The crude sample mass.

Column Type: The column type. Either scan the

column using the QR reader underneath the touch

screen or select a column type from the drop-down

list. If the column name is orange or red, its load

capacity is smaller than the entered sample mass.

Flow Rate: The default (recommended) flow rate

for the selected column type is preselected. If you

want to change it, consider that the maximum flow

rate that can be used depends on the following:

The column and any other accessories used in the setup.

The maximum aspiration rate(s)

defined for the used solvent(s).

Note: If you expand the Column panel, all of the settings for the

selected column type are displayed (see Figure 14). For more

information, see “Manage Column Types” on page 13.



Set Up a Purification

Figure 14.The Column panel expanded.

Specify the Collection Parameters

λ-All*:Select whether the average absorbance within

a user-defined wavelength range should be used for

collection and fractionation (Collect), used for monitoring

(Show), or not be shown during the run (Hide).

UV1 and UV2: Select whether the wavelength signals should

be used for collection and fractionation (Collect), used for

monitoring (Show), or not be shown during the run (Hide).

Wavelengths*:The shortest and longest wavelength to

be included in the λ-All signal. The range is 200 to 400

nm (UV detector) or 198 to 810 nm (UV-VIS detector).

Wavelength: The wavelength to be used for

UV1 and UV2. The range is 200 to 400 nm (UV

detector) or 198 to 810 nm (UV-VIS detector).

Threshold: Used to collect samples with an absorbance

exceeding the set absorbance threshold.

Collect All: Used to collect the entire run.

UV Baseline Correction*: When this option is turned on, the

gradient run is preceded by a light absorbance detection

phase. During this phase, the light absorbance of the used

chromatography solvents is measured for all wavelengths

that will be used for collection and fractionation (UV1,

UV2, and/or λ-All). The measurement results in a baseline

containing the maximum absorbance of the solvent. During

the gradient run, the baseline is subtracted from the signal.

*Only available on systems with a Spektra software license.

Specify the Rack Parameters

The Selekt collection racks have RFID tags that are

automatically identified by the system when placed on the

collection bed.

If the RFID rack detection feature is disabled in the system

settings (see page 15), it is possible to select the collection

rack manually by expanding the Rack panel (see Figure 15).

If you want to use a lower fraction volume than the one defined

for the selected rack(s), expand the Rack panel, enable the

Max Fraction Volume option, and enter the desired volume.

Note that this setting will only be applied on racks with a higher

vessel volume.

Figure 15.The Rack panel expanded on a system with extended

collection bed.

Biotage®Selekt User Manual | © Biotage 2018

Start, Monitor, and Control a Purification

Warning

Read and follow the safety precautions against static electricity

in the “Biotage®Selekt Installation and Safety” document

(P/N 416182) that is supplied with the system.

Always ensure that the settings for the column selected in the

software are correct for the column to be used before starting

a run. Never exceed the maximum pressure or flow rate for the

used column.

Keep hands away from the collection arm area until the arm has

returned to its home position (in the inner right corner) and the

system is paused or in standby.

The system may be pressurized when paused.

Monitor the waste reservoir to prevent overflow during operation.

Start a Purification

1. Ensure that a sufficient quantity of the correct solvent

is present in each solvent reservoir and that the waste

reservoir has sufficient capacity for the run.

2. Start the purification by pressing . If the button is

disabled, press in the bottom right corner for more

information.

3. When the Sample Load dialog opens, load your sample.

4. Start the gradient run by pressing the appropriate button in

the Sample Load dialog. We recommend that a gradient run

is not started until the UV lamp is sufficiently warmed up.

Figure 16.The run view.

Monitor the Purification in Progress

While a purification is running, the programmed gradient (both

graph and table), a dynamic chromatogram, and an absorbance

spectrum for the whole detector range are displayed in the run

view (see Figure 16).

Expand a graph or the gradient table by pressing .

Change the length unit on the chromatogram by selecting CV,

mm:ss, or mL from the drop-down menu in the gradient table.

Fractions

Fractions that are already collected can be located by matching

their colors and numbers on the chromatogram with the vessel

colors and numbers in the Rack panel. Fractions from the same

peak have the same color. Fractions only collected due to the

Collect All option are colored gray.

Chromatogram

Light absorbance and the start threshold (if defined) are

displayed in the chromatogram using the following colors:

= Absorption measured by the internal detector for the whole

λ-All range and the threshold in mAU.

= Absorption measured by the internal detector at

wavelength UV1 and the threshold in mAU.

= Absorption measured by the internal detector at

wavelength UV2 and the threshold in mAU.

The defined wavelengths and real-time measurements of the

absorption are displayed in the Collection panel.

Status in the Top Pane

Conversion: When reversed phase is run on one channel

and normal phase on the other, a solvent flush is

performed between runs using different channels.

Equilibration Flush: The system empties the

solvent inlets of solvents used in the previous

purification and fills them with new solvents.

Equilibration: The system is running a column equilibration.

Purge: The system releases the column pressure

before sample loading is enabled.

Sample Load: Sample can be loaded onto the column.

UV Warm-up: The UV lamp is being warmed up.

UV Zero: The system is setting the UV zero level using

the solvent mix used at the start of the gradient.

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