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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.
10.1
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.
10.2
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
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.
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.
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
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.
Under the communications tab, enter a baud rate of 19200 for COM1.
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.
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
10.5
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.
11
Analytical Column
MS Detection
3
2
6
3Plug 5
4
Nanoflow pump
1
5
50 um x150 mm
Waste
Waste
2nd Pump
B) Analyze Sample:
Analytical Column
MS Detection
3
2
6
3Plug 5
4
50 um x150 mm
Waste
Waste
4
6
Nanoflow pump
1 1
2nd Pump
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
Buffer B
12
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.
14
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).
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.
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.
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.
18
10.6
Waste
3Plug 5
4
3
6
Enrichment-Column
Nanoflow pump
Analytical Column
MS Detection
Waste
3Plug 5
4
Enrichment-Column
1
6 Analytical Column
Nanoflow pump
MS Detection
Waste
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.
19
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.
21
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)
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.
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.
24
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.
26
10.6
27
10.7
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.
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
29
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
30