EASY-nLC Series Xcalibur Data System 2.1 or Later User Guide

EASY-nLC Series Xcalibur Data System 2.1 or Later User Guide

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Creating Xcalibur Instrument Methods

This chapter describes how to set up the instrument method parameters for the EASY-nLC instrument.

An Xcalibur instrument method (.meth) contains the chromatographic and data acquisition parameters for your LC/MS system. When creating new instrument methods, set up the parameters for all of the configured devices.

Contents

Setting Up the EASY-nLC Instrument Method Parameters

Saving Instrument Methods

Note For information about setting up the instrument method parameters for the mass spectrometer, refer to the Help for the mass spectrometer.

Setting Up the EASY-nLC Instrument Method Parameters

Use the Instrument Setup window of the Xcalibur data system to create instrument methods for your LC/MS system. To create an instrument method, enter the data acquisition settings for the mass spectrometer and the injection and chromatographic settings for the LC system.

To open the Instrument Setup window

From the Xcalibur Home Page, do one of the following:

• Choose GoTo > Instrument Setup.

–or–

• Click the Instrument Setup icon in the Roadmap view of the Home Page.

The Instrument Setup window opens. The view bar contains icons for the configured devices.

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Creating Xcalibur Instrument Methods

Setting Up the EASY-nLC Instrument Method Parameters

To set up the instrument method parameters for the EASY-nLC instrument

1. In the view bar of the Instrument Setup window, click the Thermo EASY-nLC icon.

The EASY-nLC view opens in the Instrument Setup window with the Sample Pickup and

Loading page displayed (see Figure 9

). At the bottom of the view, the Solvents area lists the solvents specified in the instrument configuration. To change the names of the mobile

phase solvents, see “Setting Up the EASY-nLC Configuration” on page 10 .

2. Enter the appropriate instrument method settings for the EASY-nLC instrument. Click

Next to open the remaining three pages of the EASY-nLC view page by page.

Figure 9

shows the Sample Pickup and Loading page of the EASY-nLC view with typical sample pickup and loading settings for the EASY-nLC 1000 instrument and a sample in an aqueous matrix.

Figure 9.

EASY-nLC view of the Instrument Setup window

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Creating Xcalibur Instrument Methods

Setting Up the EASY-nLC Instrument Method Parameters

For information about the pages of the EASY-nLC view, see these topics:

“Sample Pickup and Loading Page” on page 23

“Gradient Page” on page 25

“Pre-Column and Analytical Column Page” on page 28

“Auto-Sampler Page” on page 31

Sample Pickup and Loading Page

Use the Sample Pickup and Loading page to specify the following:

• The sample volume that the autosampler withdraws (injection volume) from the vial or well position in the Xcalibur injection sequence and the flow rate at which the autosampler draws the sample into the sample loop.

• The solvent volume that pump A uses to push the sample out of the sample loop and onto the column assembly and the flow rate and maximum pressure for the sample loading step.

To open the Sample Pickup and Loading page

1. Open the EASY-nLC view of the Instrument Setup window.

2. If the Sample Pickup and Loading page is not displayed, click the Sample Pickup and

Loading tab.

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Creating Xcalibur Instrument Methods

Setting Up the EASY-nLC Instrument Method Parameters

Sample Pickup and Loading Page Parameters

Table 6 describes the parameters on the Sample Pickup and Loading page of the EASY-nLC

view of the Instrument Setup window.

Table 6. Sample Pickup and Loading page parameters

Parameter

Sample Pickup

Volume (μL)

Description

Flow (μL/min)

Specifies the sample volume that the autosampler withdraws from the vial or well position specified in the Xcalibur sequence.

Maximum volume = sample loop volume – 2 μL

The graphic on the Home > Overview page (touch screen) displays the sample loop size.

Specifies the flow rate at which pump S draws the sample into the sample loop.

Range: 0 to 40 μL/min. The flow must be greater than 0 when the sample pickup volume is greater than 0.

Tip For aqueous sample matrixes, set the flow rate to 20 μL/min.

Sample Loading

Volume (μL) Specifies the volume of solvent A that the instrument uses to push the sample out of the sample loop and onto the precolumn (two-column setup) or the analytical column

(one-column setup).

Typically, two times the sample pickup volume plus 2 μL is sufficient.

Range: 0 to 137 μL

IMPORTANT The maximum allowable entry is 137 μL; however, for Xcalibur sequence runs, the sample loading volume plus the precolumn equilibration volume plus the analytical

column equilibration volume must be less than 120 μL.

Flow (μL/min)

See “Taking Corrective Action for Run-Time Errors” on page 45 .

Specifies the flow rate at which pump A pumps solvent A through the instrument to push the sample out of the loop and onto the precolumn or analytical column.

If the Flow box is empty, the instrument runs at the set pressure.

Range: 0 to 100 μL/min

Max Pressure (bar) Specifies the maximum pressure for the sample loading step.

If the Max Pressure box is empty, the instrument runs at the set flow rate.

EASY-nLC II range: 0 to 280 bar

EASY-nLC 1000 range: 0 to 980 bar

Tip If both the Max Pressure and Flow boxes are empty, the instrument runs at the maximum instrument pressure. Leave these boxes empty if you want the instrument to run at the highest possible flow rate that is allowed under the maximum instrument pressure limit.

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Creating Xcalibur Instrument Methods

Setting Up the EASY-nLC Instrument Method Parameters

Gradient Page

Use the Gradient page to set up the gradient program (see

Figure 10 ).

To open the Gradient page

1. Open the EASY-nLC view of the Instrument Setup window.

2. Click the Gradient tab. If you do not see the Gradient tab, click Next.

Figure 10. Gradient page

Thermo Scientific

To set up the gradient program

1. In the Time = 0.00 row, do the following:

• In the Flow (nL/min) column, type a value from 1 to 2000.

• In the %B column, type a value from 0 to 100.

2. To add an additional time point (table row), click Add.

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Creating Xcalibur Instrument Methods

Setting Up the EASY-nLC Instrument Method Parameters

3. In rows 2 or higher of the gradient table, do the following: a. In the Duration column, type a value from 0.01 to 9999.49.

The value in the Duration column is the length of time that you want the instrument to take to reach the specified flow and %B conditions. When you type a value in the

Duration column, the value in the Time column changes to the current Duration value plus the value in the previous row. b. In the Flow (nL/min) column, type a value from 1 to 2000.

c.

In the %B column, type a value from 0 to 100.

Both the flow rate and %B composition change linearly between time points.

4. Edit the table as follows:

• To delete a row, select the row and click Del.

• To reorder the rows as applicable, select the row and click Up or Down.

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Creating Xcalibur Instrument Methods

Setting Up the EASY-nLC Instrument Method Parameters

Gradient Page Parameters

Table 7 describes the parameters on the Gradient page of the EASY-nLC view in the

Instrument Setup window.

Table 7. Gradient page parameters

Parameter

Table

Time

Description

Duration

Specifies the run time when the instrument reaches the conditions specified in the flow and %B columns.

Specifies the length of time between the previous and current time points.

Flow (nL/min)

%B

Range: 0.01 to 9999.59 minutes

Specifies the mobile phase flow rate for the current time point.

Range: 1 to 2000 nL/min

Specifies the percent composition of solvent B in the mobile phase for the current time point.

Buttons

Add

Del

Up

Down

Graph

Flow (nL/min)

Adds a row to the gradient program.

Deletes the selected row from the gradient program.

Moves the selected row up.

Moves the selected row down.

AB mixture (%B)

Time (min)

The y axis on the left side of the graph displays the flow scale.

A black line represents the change in flow rate with time.

The y axis on the right side of the graph displays the percentage of solvent B.

A red line represents change in %B with time.

The x axis displays the run time of the gradient program.

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Creating Xcalibur Instrument Methods

Setting Up the EASY-nLC Instrument Method Parameters

Pre-Column and Analytical Column Page

Use the Pre-column and Analytical Column page to set up the column equilibration steps

(see

Figure 11 ).

To open the Pre-column and Analytical Column page

1. Open the EASY-nLC view of the Instrument Setup window.

2. Click the Pre-column and Analytical Column tab. If you do not see the Pre-column and

Analytical Column tab, click Next.

Figure 11. Pre-column and Analytical Column page

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Creating Xcalibur Instrument Methods

Setting Up the EASY-nLC Instrument Method Parameters

Pre-Column and Analytical Column Page Parameters

Table 8 describes the parameters on the Pre-column and Analytical Column page of the

EASY-nLC view of the Instrument Setup window.

Note For information about determining the appropriate flow rate for the precolumn and analytical column equilibration steps, refer to the EASY-nLC Series Getting Started Guide.

Table 8. Pre-column and Analytical Column page parameters (Sheet 1 of 2)

Parameter

Pre-column Equilibration

Description

Volume (μL) Specifies the volume of solvent A that pump A pushes through the precolumn to waste during the precolumn equilibration step.

Flow (μL/min)

Max Pressure (bar)

Range: 0 to 137 μL

IMPORTANT The maximum allowable entry is 137 μL; however, for Xcalibur sequence runs, the sample loading volume plus the precolumn equilibration volume plus the analytical

column equilibration volume must be less than 120 μL.

Specifies the flow rate for the precolumn equilibration step. If the Flow box is empty, the instrument runs at the set pressure.

Range: 0 to 100 μL/min

Specifies the maximum pressure for the precolumn equilibration step. If the Max Pressure box is empty, the instrument runs at the set flow rate.

EASY-nLC II range: 0 to 280 bar

EASY-nLC 1000 range: 0 to 980 bar

Tip If both the Max Pressure and Flow boxes are empty, the instrument runs at the maximum instrument pressure.

Leave these boxes empty if you want the instrument to run at the highest possible flow rate that is allowed under the maximum instrument pressure limit.

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Creating Xcalibur Instrument Methods

Setting Up the EASY-nLC Instrument Method Parameters

Table 8. Pre-column and Analytical Column page parameters (Sheet 2 of 2)

Parameter Description

Analytical Column Equilibration

Volume (μL) Specifies the volume of solvent A that pump A pushes through the analytical column during the analytical column equilibration step.

Flow (μL/min)

Range: 0 to 137 μL

IMPORTANT The maximum allowable entry is 137 μL; however, for Xcalibur sequence runs, the sample loading volume plus the precolumn equilibration volume plus the analytical

column equilibration volume must be less than 120 μL.

See “Taking Corrective Action for Run-Time Errors” on page 45 .

Specifies the flow rate for the analytical column equilibration step. If the Flow box is empty, the instrument runs at the set pressure.

Range: 0 to 3 μL/min

Specifies the maximum pressure for the analytical column equilibration step.

Max Pressure (bar)

EASY-nLC II range: 0 to 280 bar

EASY-nLC 1000 range: 0 to 980 bar

Note If both the Max Pressure and Flow boxes are empty, the instrument runs at the maximum instrument pressure. Leave these boxes empty if you want the instrument to run at the highest possible flow rate that is allowed under the maximum instrument pressure limit.

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Creating Xcalibur Instrument Methods

Setting Up the EASY-nLC Instrument Method Parameters

Auto-Sampler Page

Use the Auto-sampler page to set up the wash step for the needle and sample loop (see

Figure 12 ).

To open the Auto-sampler page

1. Open the EASY-nLC view of the Instrument Setup window.

2. Click the Auto-sampler tab. If you do not see the Auto-sampler tab, click Next.

Figure 12. Auto-sampler page

Thermo Scientific

To set up the standard autosampler wash step

1. Select the Standard option.

2. In the Flush Volume (μL) box, type the volume of solvent A (from wash bottle 3) that the autosampler uses to flush the needle and the sample loop.

The range depends on the sample loop size (see

Table 9 on page 33

).

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Setting Up the EASY-nLC Instrument Method Parameters

To set up a custom wash program

1. Select the Custom option.

The custom wash table becomes available.

2. For each row that you want to add to the program, click Add.

You can select a different wash solvent for each row.

3. Edit each row as follows:

• In the Source column, select the wash solvent source: Bottle 1, Bottle 2, or Bottle 3.

IMPORTANT The EASY-nLC tray compartment holds up to four wash bottles:

• Bottles 1 and 2 in positions W1 and W2 can contain custom wash solvents.

• Bottle 3 in position W3 must contain solvent A.

• Bottle 4 in position W4 must contain the needle wash insert.

During a custom wash program, the autosampler does the following:

1. Empties the contents of pump S into the needle wash bottle (W4).

2. Performs the steps in the custom wash program. During each cycle, the autosampler draws solvent from the specified bottle, and then ejects the solvent into the needle wash bottle (W4).

3. Performs a final wash cycle with a preset volume of solvent A from bottle 3 (W3).

4. Refills pump S with solvent A from bottle 3 (W3) to be ready for the next injection.

• In the Volume (μL) column, type a volume from 0 to the sample loop size plus 8 μL.

If you enter a volume that is higher than 58 μL, an Invalid Method Parameter message box appears and the program resets the volume to the maximum allowed.

• In the Cycles column, type a value from 0 to 10.

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Creating Xcalibur Instrument Methods

Setting Up the EASY-nLC Instrument Method Parameters

Auto-sampler Page Parameters

Table 9 describes the parameters on the Auto-sampler page of the EASY-nLC view of the

Instrument Setup window.

Table 9. Auto-sampler page parameters (Sheet 1 of 2)

Parameter

Standard

Standard option

Flush Volume (μL)

Description

Selecting this option enables the standard wash option.

Specifies the flush volume that pump S is to push through the sample loop.

Range for the 20 μL sample loop: 30.25 to 100.75 μL

Range for the 50 μL sample loop: 60.26 to 70.75 μL

Custom

Custom option Selecting this option enables the custom wash program.

For information about the steps that the autosampler performs during a custom wash program, see

“To set up a custom wash program” on page 32 .

Custom wash table columns

Order

Source

Defines the order of the custom wash steps.

Specifies the wash solvent source for the current wash step.

Volume (μL)

Cycles

Selections: Bottle 1, Bottle 2, or Bottle 3

Note Bottles 1 and 2 contain custom wash solvents. Bottle 3 contains solvent A.

Specifies the wash solvent volume for each wash cycle.

Maximum allowed volume at run time: Sample loop size plus 8 μL.

Note The Xcalibur method editor for the EASY-nLC instrument does not know the configured sample loop size. When you submit a sequence with the instrument method,

Xcalibur acquisition service validates the custom wash volumes against the configured sample loop size. To prevent solvent from being drawn into pump S, the program limits the volume to the sample loop size plus 8 μL

For information about changing the sample loop size on the Maintenance > Devices page of the touch-screen software, see

“To change the loop volume, column setup, idle mixture, or idle flow rate” on page 13 .

Specifies the number of wash cycles for the wash step.

Range: 1 to 10

Note For each cycle, the autosampler draws the specified volume of wash solvent from the specified source into the needle, and then ejects the wash solvent into wash bottle 4 (W4).

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Saving Instrument Methods

Table 9. Auto-sampler page parameters (Sheet 2 of 2)

Parameter

Buttons

Add

Del

Up

Down

Description

Adds a row to the wash program.

Deletes the selected row from the wash program.

Moves the selected row up.

Moves the selected row down.

Saving Instrument Methods

After you set up the instrument method parameters for the LC/MS system, save the instrument method to an appropriate directory.

To save your current instrument method

1. Choose File > Save As.

The Save As dialog box opens.

2. In the File Name box, type a name for the new instrument method.

3. Click Save.

The File Summary Information dialog box opens.

4. Click OK.

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