10 Agilent NanoLC System

Using the Agilent nanoLC System 1

Using the Agilent NanoLC System

This tutorial will describe how to connect the Agilent nanoflow HPLC system for use with Analyst Software 1.4. More detail in setting up the LC system can be found in the Analyst Software LC Devices Setup Manual.

The Basic Agilent 1100 Series nanoflow LC system includes: The 1100 nanoflow pump, the 1100 micro vacuum degasser, the 1100 micro well-plate autosampler, and the handheld controller. The system as shown also includes a thermostatted column oven with switching valve for trapping/sample clean-up workflows, a second 1100 series capillary pump, and a micro well-plate autosampler tray cooler.

The two loop sizes available for the autosampler include an 8 and 40µl loop, which is utilized in MDLC experiments requiring large injections or injections of salt solutions.


10.1 Connecting the Autosampler to the Mass Spectrometer.

The AB/MDS Sciex Aux I/O cable (P/N 014474) must be connected as shown to establish communication between the autosampler and the mass spectrometer.

On the free end of the Aux I/O cable, short together wire 9 (red with black stripe) and wire 10 (orange with black stripe). Isolate these wires (wrap with electrician’s tape) to prevent them from contacting any other wire or metal. Connect the AUX I/O wire 22 (white/black stripes) to pin 3 of the male DB9 connector. Next, connect the AUX I/O wire 21 (green/black stripes) to pin 1. If using the universal cable from Agilent (Agilent p/n 01046-60201), connect the white wire (pin1) to the AUX I/O wire 21 (green/black stripes) and connect the gray wire (pin3) to AUX I/O wire 22 (white/black stripes). Insert the connector into the Autosampler Remote Port. Insert the other end of the AUX I/O cable into the Mass Spectrometer AUX I/O connector.


10.2 Connecting the LC System to the Computer. The Agilent LC system can be controlled using a single communication cable connected to the computer. Connect a RS-232 cable (P/N WC024736) from the serial port on the autosampler to the computer (COM1).

Ensure the dip switches on the back of the autosampler and pump are set as follows:

After adjusting the dip switches on the back of these components, the power should be switched off, then back on again for the changes to take affect.

The autosampler tray cooler (G1330A) does not require an additional serial cable as it plugs directly into the autosampler through a special cable included with the tray cooler.

Connect CAN cables from the capLC pump to the autosampler, from the autosampler to the column oven, and from the column oven to the nanoLC pump.

For real-time monitoring of the system and offline control of the pump, plug the hand held Agilent Series Control Module into a free CAN connector.


10.3 Using the NanoSprayTM Source for NanoLC Applications

⇒ For more information on mounting the NanoSprayTM source on the Q TRAPTM System instrument, refer to the Q TRAPTM System NanoSprayTM Source manual.

⇒ For more information on mounting the NanoSprayTM Source and Interface on the 4000 Q TRAPTM System instrument, refer to the 4000 Q TRAPTM System NanoSprayTM Source manual.

The flow from the LC system can be connected to the input of the analytical column using a length of Agilent 25µm tubing. Fused silica capillary can also be used for connection by using different sized sleeves, nuts and ferrules on each end; use 1/32” fittings to connect to the Agilent and 1/16” fittings to connect to the analytical column.


To use an LC Packings column and a nanoflow adaptor:

Pass the input end of the column into a 300µm ID PEEK sleeve and insert into a nut and ferrule. This now screws into a low dead volume union; the other end of the union takes the tubing from the LC system.

A second low dead volume union is used to connect the output of the column to the fused silica spray tip. Pass the output end of the column into a 300µm ID PEEK sleeve and insert into a nut and ferrule. This now screws into one end of a low dead volume union. Using a 380-400µm ID PEEK sleeve, a nut and ferrule, securely seat the fused silica emitter tip in the reducing union. A 1 cm length can be trimmed off the distal end (opposite end to tip) of the emitter tip to shorten it.

To Use an LC Packings Column and a MicroIonSprayTM Source Head

Pass the input end of the column into a 300µm ID PEEK sleeve and insert into a nut and ferrule. This now screws directly into the back of the MicroIonSprayTM Source head.

To Use a Vydac Column and a Nanoflow Adaptor

Remove the column from the silver sleeve by loosening the two pins at either end with an allen key. Pass the input end of the column into a 300µm ID PEEK sleeve and insert into a nut and ferrule. This now screws directly into the input end of the column. Using a 380-400µm ID PEEK sleeve, a nut and ferrule, securely seat the fused silica emitter tip in the other end of the column. A 1 cm length can be trimmed off the distal end (opposite end to tip) of the emitter tip to shorten it.


To Use a Vydac Column and a MicroIonSprayTM Source Head

Remove the column from the silver sleeve by loosening the two pins at either end with an allen key. Pass the input end of the column into a 300µm ID PEEK sleeve and insert into a nut and ferrule. This now screws directly into the input end of the column. Use a small length of fused silica tubing (20µm ID x 280µm OD), a 300µm ID PEEK sleeve and two nuts and ferrules to connect the output end of the column to the back of the MicroIonSprayTM Source head.

Now the column (or column and spray tip) can be mounted in the source head on the nanospray source arm. Before mounting the column, pull the X-Y-Z positioning unit back from the instrument, along the rails. Mount the spray tip union or column end into the support piece on the source arm. Ensure that the high voltage line is connected to the source head to apply the voltage to the liquid junction. Move the tip back as far as it will go using the adjustment knob before sliding the source back along the rails, to ensure the nanoLC tip does not hit the curtain plate. If using the MicroIonSpray head, ensure the GS1 line is connected.

Once the column has been connected to the system, go back and check all junctions for leaks. Sometimes the added back pressure created when the column is put in line can cause junctions to leak.


10.4 Creating a Hardware Profile. Open up the Hardware Configuration Editor from the Navigation bar on the left hand side of the screen. Click the New Profile button to create a new hardware profile, name it ‘MS+Agilent nanoLC, then select Add Device. Select the Mass Spectrometer, and then click OK to make it appear in the Edit Profile window. Next, click Setup Device and ensure the integrated syringe pump is not activated by unchecking the box at the bottom of the pane. Click Add Device and select the LC Pump from the Device Type drop down menu, then choose the Pump Agilent 1100. Click OK to add to the hardware profile. Click Setup Device to reveal the following pane: select Nano Pump and enter an access code of 300. Enter the serial number from the front of the pump.

Under the communications tab, enter a baud rate of 19200 for COM1.


If an additional capillary LC pump is to be used , an additional pump must be built into the hardware profile. Select Add Device, select the Autosampler from the Device Type drop down menu, then choose the Autosampler Agilent 1100 and click OK. In the Link Pump area select Specify Pump and enter the serial number from the front of the CapLC pump.



To add the autosampler, select Add Device select the Autosampler, and select the Agilent 1100 autosampler from the list. In this case, if an additional pump is used, you need to specify to link the pump to the serial number of the nano LC pump.



If the column oven is being used, click Add Device once more and select Column Oven from Device Type. Click Setup Device again and change the COM Port Number to 1 and the Baud Rate to 19200.

Once the Hardware Profile has been built, click OK to return to the Hardware Configuration Editor. Click Activate Profile to establish communication with all the peripherals in the profile. A green ‘check mark’ will appear once proper communication has been established.


10.5 1D-LC with direct loading or high flow autosampler flush

Please refer to the Agilent LC manuals for more information on plumbing the LC system for various modes.

Plumbing the LC system for Direct Loading.

For operation in this mode, the analytical column can be plumbed directly into autosampler valve, port 6.

In an experiment utilizing direct sample loading without a secondary pump, the most important factor is to include enough of a gradient delay in your LC method to ensure that a.) all of the sample is loaded onto the column at low organic, and b.) if you switch the valve to bypass, this does not occur after the gradient has begun or small volumes of organic will be caught in the loop and can ruin subsequent runs. The gradient delay to clean out the 8µl loop 1X at 250nl/min is 32 min.

One method to accomplish this is the automatic Flush-Out Factor check box located in the Advanced Properties tab of the autosampler. Selecting this will flush the loop with a volume of mobile phase equal to the factor times (x) your injection volume. For example, a 1µl injection volume with a 3 Sample Flush Out Factor will flush the loop with 3µl of mobile phase. After this, the valve on the autosampler will switch from Mainpass to Bypass, which takes the sample loop off-line. At 250nl/min, this example would create a hypothetical delay of approximately 12 minutes.

Important: To minimize the time to load a sample, the line from the needle seat to the autosampler valve has been replaced with a 50 um x 150 mm capillary.


Plumbing the LC System for the High Flow Autosampler Flush.

There are a variety of configurations suitable for direct loading. The example shown here makes use of the a 2nd pump and a switching valve to flush the autosampler, while minimizing volumes in the analytical pathway.

1100 Micro WellplateSampler Agilent micro switching valve

:

1100 Micro Wellplate Sampler Agilent micro switching valve

1100 Micro WellplateSampler Agilent micro switching valveB) Analyze Sample:

A) Load sample on column :

Waste

16

534

2Plug 1

6

4

Waste 2nd Pump

16

534

2Plug 1

6

MS Detection

Nanoflow pump

3

4

5

2

Waste

Analytical Column


16

534

2Plug

Waste 2nd Pump

16

534

2Plug 1

6

MS Detection

Nanoflow pump

3

4

5

2

Waste

Analytical Column

50 um x150 mm

50 um x150 mm

1100 Micro WellplateSampler Agilent micro switching valve

:


1100 Micro WellplateSampler Agilent micro switching valveB) Analyze Sample:

A) Load sample on column :

Waste

16

534

2Plug 1

6

4

Waste 2nd Pump

16

534

2Plug 1

6

MS Detection

Nanoflow pump

3

4

5

2

Waste

Analytical Column


16

534

2Plug

Waste 2nd Pump

16

534

2Plug 1

6

MS Detection

Nanoflow pump

3

4

5

2

Waste

Analytical Column

50 um x150 mm

50 um x150 mm

The pumps, autosampler, and switching valve were plumbed according to the above diagram. Yellow indicates a 25 um ID capillary, while green indicates a 50 um ID capillary.

Recommended buffers for nanoLC pump. Premixing of acetonitrile with water will prevent any solvent mixing artifacts or generation of bubbles.

Buffer A 98% water / 2 % acetonitrile / 0.1% formic acid

Buffer B 2% water / 98 % acetonitrile / 0.1% formic acid


Setting Up the Acquisition Method.

Under Acquire in the Navigation bar, select Build Acquisition Method. The method will open with all elements of the hardware configuration appearing on the left hand side. For setup of the mass spectrometer section of the hardware profile and an IDA experiment, refer to the Information Dependent Acquisition tutorial. The Synchronization Mode should be in LC Sync, the method will start with the injection signal from the autosampler. To build the methods for each of the components, click on the component on the left hand side of the pane.


Autosampler Method.

Basic autosampler parameters are set under the Agilent 1100 Micro Autosampler Properties tab. The syringe size is automatically determined during communication with the device, and therefore is not an editable field. The Draw Speed can be set. To use the Needle wash capability, click Enabled on the right hand side of the pane. Set the Wash Location to Flush Port and set the desired wash time. If using the Temperature Control, click Enabled and set the desired temperature.

Note: the eject speed is only used during Custom Injector Programs where samples are pre-mixed by the needle in designated well position.

Optional: Under the Advanced Properties tab select Bottom Sensing. When this feature is activated, the bottom of the vial will automatically be sensed and the autosampler needle will stop just before the bottom of the vial.

⇒ For direct loading onto a column, ensure that the Sample Flush Out Factor is enabled.

⇒ For High Flow Autosampler flush, this feature should not be enabled.


NanoLC Pump Method.

Basic pump parameters are set under the LC Pump Gradient tab. Construct a gradient profile in the spreadsheet under this tab, entering the time profile, the flow rates and the percentages of each buffer to be used. Show Graph/Hide Graph can be used to visualize the gradient profile. The first 10 minutes are used for loading the sample onto the column.

Note: This gradient delay (10min.) is usually suitable for 1-2µl injection volumes if you have replaced the 100µm tubing between the needle seat and the valve with 20-50µm tubing.

The gradient shown below ramps from 5-40% B over 30 minutes, then flushes at 80% B before re-equilibrating at 5% B. Fix picture

Note: Remember to adjust the Duration time in the Mass Spectrometer section of this acquisition method according to the length of the gradient. Acquisition should always run longer than the end of the gradient section of the method to take into account the amount of dead volume between the pump and the sprayer. Also, the duration time for the mass spectrometer acquisition should be shorter than the end of the LC Pump gradient. (The MS acquisition does not have to continue into the column re-equilibration time, as there is usually no useful data during this time).


Specific pump parameters are set under the Limits (Advanced) tab. Enter the appropriate numbers for Left and Right Compressibility for the solvent system being used in the LC gradient. In the example shown above, Solvent A is in the left pump head which contains mainly water and Solvent B (right pump head) is mainly acetonitrile. (See Solvent Compressibility for table of Solvent Compressibility values). Because solvents compress under pressure, this compressibility factor compensates for changes in flow rate as pressure changes in the pump drives. Changing the compression rate maintains a uniform and accurate solvent flow through the drive. For a mixture of solvents, it is suggested by Agilent that the compressibility value of the solvent that is present in the highest amount is used.

The Left and Right Dead Volume values are not required for proper functionality.

The Left and Right Stroke Volumes can be set automatically (enter –1.0) or manually. Long strokes are used at high flow rates and shorter strokes are used at low flow rates. This can improve the mixing performance and gradient linearity of the pump. Decreasing the stroke volume decreases the pressure pulsation amplitude, which gives better signal-to-noise ratios and peak area reproducibility at low flow rates. Notice when individual parameters are set under the Advanced tab, the values are automatically updated under the Limits tab. Entering the values under the Advanced tab allows individual settings to be used for each pump head.


Further parameters are available under the Micro Mode tab. Ensure the Pump Mode is set to Micro when using the Agilent LC system.

The Primary Flow Rate sets the approximate pre-split flow rate; this flow range is from 200 - 1500 uL/min. This will affect the solvent consumption for the LC method. For slow gradients, a small primary flow is fine; however, when faster gradients are required, higher primary flows will allow faster gradient formation. The Flow Sensor Calibration Table Index must be set for the solvent system being used (see Flow Sensor Calibration Table Indices for a table of values). This value is used in the active monitoring of the post-split flow rate.

CapLC Pump Method for High Flow Autosampler Flush.

In this example the CapLC pump is used for flushing the autosampler, and ensuring no carry-over. Basic pump parameters are set under the LC Pump Gradient tab. Construct a gradient profile in the spreadsheet under this tab, entering the time profile, the flow rates and the percentages of each buffer to be used. Show Graph/Hide Graph can be used to visualize the gradient profile. Flow from the CapLC pump is initially directed to waste. After the column oven valve switches (at 10 minutes) the CapLC pump is then used to wash the autosampler lines with high percentage of organic solvent.


Column Oven.

Click on the Agilent 1100 Column Oven on the Navigation Bar to reveal the following column oven parameters. For Nanoflow LC, the column oven is primarily used for its integrated valve. After a 10 minute Load/Wash period, the valve is switched so that the NanoLC pump is in-line with the nanobore column. The temperature values should be set to close to room temperature.

After the autosampler, pump, column oven, and mass spectrometer parameters have been entered in the method, save the acquisition method in a project folder.


10.6 1D-LC with precolumn desalting Plumbing the LC System for precolumn desalting.

Please refer to the Agilent LC manuals for the system. A typical setup for nanoflow chromatography with an enrichment column would be as follows:

1100 Micro Wellplate Sampler Agilent micro switching valveWaste

2nd Pump

16

534

2Plug 1

6MS Detection

Nanoflow pump

Enrichment-Column34

5

2

Waste

Analytical Column

B) Analyze Sample:

A) Load sample on enrichment column:


2nd Pump

534

2Plug 1

6

MS Detection

Nanoflow pump

Enrichment-Column34

5

2

Waste

Analytical Column

16


2nd Pump

16

534

2Plug 1

6MS Detection

Nanoflow pump

Enrichment-Column34

5

2

Waste

Analytical Column

B) Analyze Sample:

A) Load sample on enrichment column:


2nd Pump

534

2Plug 1

6

MS Detection

Nanoflow pump

Enrichment-Column34

5

2

Waste

Analytical Column

16

Note: By adding a SCX (or other 1st dimension column) between the autosampler and the micro switching valve of the Column Oven, this setup could also be used for two-dimensional LC. Peptides would be eluted by injecting salt plugs, trapping the released peptides, then switching to the NanoLC pump to analyze the trapped peptides. In addition, the autosampler loop size must be changed to 40uL for injecting larger volume salt plugs.


Setting Up the Acquisition Method.

Under Acquire in the Navigation bar, select Build Acquisition Method. The method will open with all elements of the hardware configuration appearing on the left hand side. For setup of the mass spectrometer section of the hardware profile and an IDA experiment, refer to the IDA tutorial. The Synchronization Mode should be in LC Sync if the method is to start with the injection signal from the autosampler. To build the methods for each of the components, click on the component on the left hand side of the pane.


Autosampler Method.

Basic autosampler parameters are set under the Agilent 1100 Micro Autosampler Properties tab. The syringe size is automatically determined during communication with the device, and therefore is not an editable field. The Draw Speed can be set (the eject speed is only used during Custom Injector Programs where samples are pre-mixed by the needle in designated well position). To use the Needle wash capability, click Enabled on the right hand side of the pane. Set the Wash Location to Flush Port and set the desired wash time. If using the Temperature Control, click Enabled and set the desired temperature.

Optional: Under the Advanced Properties tab select Bottom Sensing. When this feature is activated, the bottom of the vial will automatically be sensed and the autosampler needle will stop just before the bottom of the vial.


NanoLC Pump Method.

Basic pump parameters are set under the LC Pump Gradient tab. Construct a gradient profile in the spreadsheet under this tab, entering the time profile, the flow rates and the percentages of each buffer to be used. Show Graph/Hide Graph can be used to visualize the gradient profile. The capillary LC pump is used to load the sample on to the enrichment column at 10 µL/min during the first 5 minutes. Then the gradient from the nanoLC pump below ramps from 5-40% B over 30 minutes, then flushes at 80% B before re-equilibrating at 5% B.

Note: Remember to adjust the Duration time in the Mass Spectrometer section of this acquisition method according to the length of the gradient. Acquisition should always run longer than the end of the gradient section of the method to take into account the amount of dead volume between the pump and the sprayer. Also, the duration time for the mass spectrometer acquisition should be shorter than the end of the LC Pump gradient. (The MS acquisition does not have to continue into the column re-equilibration time, as there is usually no useful data during this time)


Specific pump parameters are set under the Limits (Advanced) tab. Enter the appropriate numbers for Left and Right Compressibility for the solvent system being used in the LC gradient. In the example shown above, Solvent A is in the Left pump head and is mainly water and Solvent B (right pump head) is mainly acetonitrile. (See Solvent Compressibility for table of Solvent Compressibility values). Because solvents compress under pressure, this compressibility factor compensates for changes in flow rate as pressure changes in the pump drives. Changing the compression rate maintains a uniform and accurate solvent flow through the drive. For a mixture of solvents, it is suggested by Agilent that the compressibility value of the solvent that is present in the highest amount is used.

The Left and Right Dead Volume values are not required for proper functionality.

The Left and Right Stroke Volumes can be set automatically (enter –1.0) or be set manually. Long strokes are used at high flow rates and shorter strokes are used at low flow rates. This can improve the mixing performance and gradient linearity of the pump. Decreasing the stroke volume decreases the pressure pulsation amplitude, which gives better signal-to-noise ratios and peak area reproducibility at low flow rates. Notice when individual parameters are set under the Advanced tab, the values are automatically updated under the Limits tab. Entering the values under the Advanced tab allows individual settings to be used for each pump head.


Further parameters are available under the Micro Mode tab. Ensure the Pump Mode is set to Micro when using the Agilent LC system.

The Primary Flow Rate sets the approximate pre-split flow rate; this flow range is from 200 - 1500 uL/min. This will affect the solvent consumption for the LC method. For slow gradients, a small primary flow is fine; however, when faster gradients are required, higher primary flows will allow faster gradient formation. The Flow Sensor Calibration Table Index must be set for the solvent system being used (see Appendix B for a table of values). This value is used in the active monitoring of the post-split flow rate.


CapLC Pump Method.

In this example the CapLC pump is used for loading and washing the C18 trap column. Basic pump parameters are set under the LC Pump Gradient tab. Construct a gradient profile in the spreadsheet under this tab, entering the time profile, the flow rates and the percentages of each buffer to be used. Show Graph/Hide Graph can be used to visualize the gradient profile. The CapLC pump is used to load the sample and wash the trap column with 5%B. After the column oven valve switches the enrichment column over to the NanoLC pump, the CapLC pump is then used to wash the autosampler lines with high percentage of organic solvent. After the lines are washed, the pump is changed back to high aqueous solvent to be ready for the next injection.


Column Oven.

Click on the Agilent 1100 Column Oven on the Navigation Bar to reveal the following column oven parameters. For Nanoflow LC, the column oven is primarily used for its integrated valve. After a 5 minute Load/Wash period, the valve is switched so that the NanoLC pump is in-line with the enrichment and analytical column (75 µm x 15 cm, C18). The temperature values should be set to close to room temperature.

After the autosampler, pump and mass spectrometer parameters have been entered in the method, save the acquisition method in a project folder.


10.6 Using Handheld Controller for Manual Pump Control.

Plugging the handheld control module into the CAN port at the back of the pump will allow manual control and real-time monitoring of the pumps. To monitor the real-time flow and pressure, choose Views / F5 then select the 2-Status view. (Use the arrow keys and the Enter key to move cursor and select options) This screen will show the flow rate and pressure trace from the pump. See Agilent Manual for more detailed description of control module functions.

Purging the Pump Heads.

Whenever a new buffer has been placed in reservoir or the pumps have been idle for an extended period (overnight), the pumps must be purged. In the main menu of the controller, choose Views / F5 button to show the drop down menu, then choose 3-System. On the next pane, choose the Control / F1 button, then Cap pump from the drop down menu. Finally, choose Purge Task / F1 button here to bring up the purge options. Choose the two buffer reservoirs that will be used (usually A1 and B1), enter a purge time (suggested time is 3 minutes) and a flow rate (2500 µL/min). Turn the pump on by choosing On – Off / F7 button. Once the pump is flowing (the orange light has gone off on the pump), the purge task can be started by pressing Purge / F8 button. After purging is complete, press Done / F6 twice to return to the main menu.

Repeat this procedure for the NanoLC pump.

Manual Control of Pumps.

To start the solvent flow off-line using the handheld controller, choose Views / F5 from the main menu, then the 1-Analysis view. Press Settings / F1 to show the drop down menu, and then select 2-Cap Pump. Set the desired flow rate on this screen. Next, press More / F1 to reveal another drop down menu with detailed pump settings. Choose 1-Solvents to set the Solvent B % and the solvent system to be used. For example, if Solvent A is mostly water and Solvent B is mostly acetonitrile, the H2O – ACN option should be selected from the drop down menu. Press Done when finished.

Press More / F1 to reveal the pump settings menu again. Specify the solvent reservoirs being used under 2-Channel Selection. Under 3-Auxillary, the solvent compressibility and stroke volumes can be set. Select Auto for both, see Agilent manual for detailed explanation of these features.

Specify the primary flow rate to be used under 4-Primary Flow. Ensure Micro pump is selected under 5-Mode. Once the pump settings have been entered, press Done / F6 to return to the main menu. Press On –Off / F7 to start the pump flowing.


10.7 Optimizing Source Conditions. In the Acquire section, launch the queue by clicking on the Queue button or by choosing Sample Queue from the View pull-down menu. Equilibrate the pump, autosampler and ionization source for the method to be run by clicking the button from the Queue toolbar, or choosing Equilibrate from the Acquire pull-down menu. This will open the following dialogue box; choose the acquisition method to be run and an appropriate equilibration time (usually one minute is fine to start flow). Further equilibration after this step is dependent on the column to be used and column conditions.

Now the flow has started, open Manual Tune from the Tune section of the Navigation Bar and enter the Ionspray Voltage (IS) (1000-3200V). Set the Enhanced MS Mass Range to 400-1500. Click Start to start monitoring the spectrum. Ensure MCA is unchecked so the TIC can be monitored for fluctuation. Adjust the IS in 100V increments until a stable spray is achieved. The position of the spray tip relative to the orifice can also be adjusted until a stable spray is observed. Be careful not to spray too close to the orifice. In addition, the Curtain Gas (CUR) can be adjusted to optimize the signal, use the highest curtain gas setting that doesn’t diminish the intensity of the sample ions. Also, ensure the interface heater is turned on in the method.

If the 4000 QTrap is being used, the MicroIonSpray source with a New Objective tip gives optimal results (use the 20µm x 10µm tips or the 75µmID x15µm, depending on the flow rate). Position the tip 1-10 mm back from the heater orifice, 0-2 mm off centre. Set the heater to 100-200 °C and a GS1 flow rate sufficient to prevent boiling of solvent in the tip (5-25). Adjust the IS in 100V increments until a stable spray is achieved (1000-4500 volts)

Once these optimum settings are determined, remember to adjust them in the LC acquisition method that will be used and save the modified method.

Note: For optimal tuning of the sprayer position, it is recommended to tune the spray by infusing a peptide from the syringe pump at a similar flow rate to be used (at 10-15% acetonitrile to mimic the center of the gradient). This must be done before the LC flow is hooked up. Then reconfirm the spray looks stable using the procedure suggested above.

Refer to the Batch Editor tutorial to automatically run the LC method.


Appendix: Solvent Compressibility

See the Agilent manual to optimize compressibility setting for other solvents. For mixtures of solvents, it is suggested by Agilent to use the compressibility value of the solvent that is present in the highest amount.

Solvent (pure) Compressibility (10-6 / bar)

Acetone 126

Acetonitrile 115

Benzene 95

Carbon tetrachloride 110

Chloroform 100

Cyclohexane 118

Ethanol 114

Ethyl acetate 104

Heptane 120

Hexane 150

Isobutanol 100

Isopropanol 100

Methanol 120

1-Propanol 100

Toluene 87

THF 95

Water 46


Flow Sensor Calibration Table Indices

Flow Sensor Calibration Table Indices

Solvent System

-7 H2O – DMF

-6 H2O – Acetone

-5 H2O – THF

-4 H2O – Ethanol

-3 H2O – Acetonitrile

-2 H2O – Methanol

-1 H2O – Isopropanol

0 H2O – H2O

10 Agilent NanoLC System

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