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 40l loop, which is utilized in
MDLC experiments requiring large injections or injections of salt solutions.

Using the Agilent nanoLC System

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 electricians 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.

Using the Agilent nanoLC System

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.

Using the Agilent nanoLC System

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 25m 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.

Using the Agilent nanoLC System

To use an LC Packings column and a nanoflow adaptor:

Pass the input end of the column into a 300m 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 300m ID PEEK sleeve and insert into a nut
and ferrule. This now screws into one end of a low dead volume union. Using a 380-400m 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 300m 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 300m ID PEEK sleeve and insert into a nut and
ferrule. This now screws directly into the input end of the column. Using a 380-400m 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.

Using the Agilent nanoLC System

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 300m 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 (20m ID x 280m OD), a 300m 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.

Using the Agilent nanoLC System

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.

Using the Agilent nanoLC System

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.

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

Using the Agilent nanoLC System

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.

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

Using the Agilent nanoLC System

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.

Using the Agilent nanoLC System

10

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 8l 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 1l injection
volume with a 3 Sample Flush Out Factor will flush the loop with 3l 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.

Using the Agilent nanoLC System

11

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.

A) Load sample on column

Analytical Column

MS Detection

3
2

6
3Plug 5
4

Nanoflow pump

1
5

50 um x150 mm

Waste

Waste

1100 Micro Wellplate Sampler

2nd Pump

Agilent micro switching valve

B) Analyze Sample:
Analytical Column

MS Detection

3
2

6
3Plug 5
4
50 um x150 mm

Waste

Waste

1100 Micro Wellplate Sampler

4
6

Nanoflow pump

1 1

2nd Pump

Agilent micro switching valve

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

Using the Agilent nanoLC System

12

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.

Using the Agilent nanoLC System

13

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.

Using the Agilent nanoLC System

14

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 1-2l injection volumes if you have
replaced the 100m tubing between the needle seat and the valve with 20-50m tubing.
The gradient shown below ramps from 5-40% B over 30 minutes, then flushes at 80% B before reequilibrating 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).

Using the Agilent nanoLC System

15

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.

Using the Agilent nanoLC System

16

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.

Using the Agilent nanoLC System

17

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.

Using the Agilent nanoLC System

18

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:
A) Load sample on enrichment column:
2nd Pump

Waste

3Plug 5
4

3
6

Enrichment-Column
Nanoflow pump

Analytical Column

MS Detection

Waste

1100 Micro Wellplate Sampler

Agilent micro switching valve

B) Analyze Sample:2nd Pump

Waste

3Plug 5
4

Enrichment-Column

1
6 Analytical Column

Nanoflow pump

MS Detection

Waste

1100 Micro Wellplate Sampler

Agilent micro switching valve

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.

Using the Agilent nanoLC System

19

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.

Using the Agilent nanoLC System

20

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.

Using the Agilent nanoLC System

21

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 L/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)

Using the Agilent nanoLC System

22

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.

Using the Agilent nanoLC System

23

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.

Using the Agilent nanoLC System

24

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.

Using the Agilent nanoLC System

25

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 m 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.

Using the Agilent nanoLC System

26

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
L/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.

Using the Agilent nanoLC System

27

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 doesnt 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 20m x 10m tips or the 75mID x15m, 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.

Using the Agilent nanoLC System

28

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

Using the Agilent nanoLC System

29

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

H2O H2O

Using the Agilent nanoLC System

30