Instrumental operation: ICS 3000 Standard Operational Procedure Jagger Instrumental operation: ICS 3000 Scope The scope of this document is to present an overview of the Dionex Ion Chromatography System (ICS) 3000 and to provide the reader with guidelines for standard operations, routine maintenance and simple trouble shooting. Detailed instrument description can be found in Dionex Reference Library (CD- ROM) which contains all relevant and specific manuals. Instrument overview The Dionex ICS 3000 system is designed for ion chromatography (IC). It is compatible with high salt and alkaline solvents and therefore fittings, tubings and interior of the system is made of PEEK (polyether ether ketone) to prevent corrosion. It consists of a dual pump system with four channels on each pump. It is capable of operating two columns with two different detectors at the same time and is equipped with a pulsed amperometry detector (PAD) and a conductivity detector. In practice, the common use currently will be one column at a time and primarily on Pump 1 and the PAD detector which will be described here. The PAD is based on electrochemical detection and is in general superior for detection of carbohydrates that are traditionally difficult to detect. The system also includes an eluent generator, which can be used when ultra pure eluents are needed. In practice, the eluent generator has limitations that make it difficult to use for many applications and it will therefore be bypassed for most applications. The principle of the conductivity detector is that changes in conductivity is measured and interpreted as analytes. This requires the installation of an ion suppressor prior to the conductivity detector, which will suppress the inherently high conductivity coming from the eluents. Figure 1 shows an instrument overview. As opposed to many other HPLC systems, the system should be operated with more or less constant flow to avoid detrimental salt precipitations in the pump, column and detector. If the flow will be stopped for a prolonged time period make sure to replace salt containing eluents in all lines with e.g. 50% methanol (also to avoid microbial growth). HPLC systems are delicate equipment and should be handled accordingly. Regularly check the status of the instrument, preferably on a daily basis and always be careful when operating. Eluents should ALWAYS be prepared from fresh top quality water (Milli-Q/18.2 MΩ·cm) and preferably sample preparation should also be made based on this water quality. The chromatographic system is controlled by a computer installed with Chromeleon™ 7 software (hereafter CM) version 7.1.1.1127 for instrument control and data handling. All commands and instrument handlings goes through this software.
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Instrumental operation: ICS 3000
Standard Operational Procedure
Jagger
Instrumental operation: ICS 3000
Scope
The scope of this document is to present an overview of the Dionex Ion Chromatography System (ICS)
3000 and to provide the reader with guidelines for standard operations, routine maintenance and
simple trouble shooting. Detailed instrument description can be found in Dionex Reference Library (CD-
ROM) which contains all relevant and specific manuals.
Instrument overview
The Dionex ICS 3000 system is designed for ion chromatography (IC). It is compatible with high salt and
alkaline solvents and therefore fittings, tubings and interior of the system is made of PEEK (polyether
ether ketone) to prevent corrosion. It consists of a dual pump system with four channels on each pump.
It is capable of operating two columns with two different detectors at the same time and is equipped
with a pulsed amperometry detector (PAD) and a conductivity detector. In practice, the common use
currently will be one column at a time and primarily on Pump 1 and the PAD detector which will be
described here. The PAD is based on electrochemical detection and is in general superior for detection
of carbohydrates that are traditionally difficult to detect. The system also includes an eluent generator,
which can be used when ultra pure eluents are needed. In practice, the eluent generator has limitations
that make it difficult to use for many applications and it will therefore be bypassed for most
applications. The principle of the conductivity detector is that changes in conductivity is measured and
interpreted as analytes. This requires the installation of an ion suppressor prior to the conductivity
detector, which will suppress the inherently high conductivity coming from the eluents. Figure 1 shows
an instrument overview. As opposed to many other HPLC systems, the system should be operated with
more or less constant flow to avoid detrimental salt precipitations in the pump, column and detector. If
the flow will be stopped for a prolonged time period make sure to replace salt containing eluents in all
lines with e.g. 50% methanol (also to avoid microbial growth).
HPLC systems are delicate equipment and should be handled accordingly. Regularly check the status of
the instrument, preferably on a daily basis and always be careful when operating. Eluents should
ALWAYS be prepared from fresh top quality water (Milli-Q/18.2 MΩ·cm) and preferably sample
preparation should also be made based on this water quality.
The chromatographic system is controlled by a computer installed with Chromeleon™ 7 software
(hereafter CM) version 7.1.1.1127 for instrument control and data handling. All commands and
instrument handlings goes through this software.
Instrumental operation: ICS 3000
Standard Operational Procedure
Jagger
The system consists of 5 compartments/modules:
1. Autosampler
2. Dual pump
3. Eluent generator
4. Column compartment (lower part)
5. Detector compartment (upper part)
Figure 1: General system appearance of the ICS-3000
Instrumental operation: ICS 3000
Standard Operational Procedure
Jagger
Figure 2: Instrument Controller. Software unit that controls communication between computer and HPLC. Must be running
at all times when system is operated.
Communication between the computer (CM) and the HPLC is controlled by the Instrument Controller
Software found under the Start Menu/Programs/Chromeleon/Service Manager. Alternatively, double
click the Chromeleon Icon in the Windows task bar to open the Instrument Controller (figure 2)or right
click and select start Chromeleon Instrument Controller. When the HPLC is to be controlled/operated
the Instrument Controller must be running. Starting and stopping is done by pressing the command
“Start/Stop Instrument Controller” (figure 2). The window can be closed afterwards. The Instrument
Controller does not start automatically after a computer restart and it should therefore be checked that
it is running, before starting operation.
In the operating software (CM) each module has its own operational screen, which may be found as tabs
in the top of the window (figure 3).
Window for starting and stopping
Instrument Controller. Can be closed
after use.
Icon informing about Instrument
Controller status. Green: Controller
running. White with red cross:
Controller stopped.
Instrumental operation: ICS 3000
Standard Operational Procedure
Jagger
Figure 3: Module and system overview from controlling software Chromeleon™ 7. Unmarked tabs: “DC”; Detector
compartment. “EGC1”; Eluent generator. “Audit”; Audit trail logging every event in the system. “Startup”; Programmed
system startup (not recommended to use unless you know what you’re doing).
Operating the pump
The internals of the dual pump system is depicted in figure 4. The lower half of the compartment is
Pump 1 and the upper half is Pump 2. The pumps are operated at flow rates between 0 and 10ml/min
and max pressure is 5000 psi. Each pump is connected to four eluent channels (A, B, C and D). A Rear
Seal Wash system is installed to periodically flush the backside of the pump heads in order to avoid salt
precipitations and corrosion. Wash setting may be “Interval”, “Automatic” or “Off”. Correct setting is
“Interval”, but the rear seal wash solutions needs to be changed on a weekly basis and consists of 20%
methanol/ethanol (figure 4). Note dates of change in the equipment logbook.
Switching between
Instrument and Data panel
Overview Autosampler PAD detector Queue Pump
System 1 – PAD
electrochemical
detection
Instrumental operation: ICS 3000
Standard Operational Procedure
Jagger
Figure 4: Internals of Dual Pump. Pump 2 is placed in the upper half of the compartment, Pump 1 in the lower half. Note the
screw valve at position 3 is the purge valve on Pump 2. Equivalent purge valve on Pump 1 found in the same position.
Position 11 is the rear seal wash reservoir which should be changed on a weekly basis (20% methanol or ethanol).
Preparing eluents
The eluents for ion chromatography are crucial for good performance and the chromatography (column)
is extremely sensitive to contaminations in the eluents. It is therefore of outmost importance that extra
care is taken when eluents are prepared. Remember that the lifetime of eluents for ion chromatography
is also very short compared to other types of chromatography. The biggest risk of contamination is
dissolution of carbonate in the eluents and this occurs when eluents are exposed to atmospheric air.
This carbonate accumulates on the columns stationary phase and reduces separation capacity and
carbonate has a higher push effect as mobile phase then the others regularly applied. It is impossible to
completely avoid carbonate contamination but with the proper precautions it can be minimized and
allow a certain lifetime of the eluents (max. 48 hours for top performance). This includes that
Instrumental operation: ICS 3000
Standard Operational Procedure
Jagger
headspace in the eluent bottles must be saturated with nitrogen to minimize the amount of carbon
dioxide. Nitrogen is delivered by a nitrogen generator via a restriction valve and distributed between
three of the four channels (channel D does not get nitrogen supply). Three different eluents are made to
accommodate the range of analytical methods and all three are changed every time new eluents are
prepared. The eluents are always prepared in the same way and always placed on the following
channels:
Channel A: 0.1 M NaOH
Channel B: 1 M NaOAc in 0.1 M NaOH
Channel C: Milli-Q water
Eluent A and B are most labile for carbonate contamination and should be handled accordingly.The
following is a description for generation of each eluent but should be performed alongside so that all
three eluents are done approximately at the same time. All eluents are sensitive to atmospheric air.
Therefore – don’t leave any of the newly prepared eluents standing without N2 over pressure in the
headspace for more than 15 minutes.
Before any eluents are made, turn off the flow on the system and release the nitrogen pressure on the
restriction valve positioned on the backside of the autosampler. Empty all eluent bottles and rinse them
thoroughly in Milli-Q water. Special pressure stable eluent bottles are used since strong corrosive
eluents are pressurized during running. Therefore, don’t send the bottles off to washing, but rinse them
properly before reuse.
Eluent A: 0.1 M NaOH (2L)
1. Measure out 2 L of Milli-Q water in a measuring flask. Make sure to use clean glassware.
2. Poor the two liter of water into the eluent bottle named Eluent A (0.1 M NaOH).
3. Loosely place a lid on the bottle, but don’t tighten it.
4. Place the eluent bottle in the sonication bath and adjust the water volume in the bath to the
level indicated as working level. The sonication bath will fit two eluent bottles at a time.
5. Sonication for 20 min. DON’T put your hands into the sonicator while it is operating and close
the door to the room when you leave it.
6. When the sonication is done, close the lid securely and move the eluent bottle to the HPLC.
7. Connect channel A to the eluent bottle and start the nitrogen flow. Exchange the air in the head
space by repeatedly opening and closing the lid on the bottle to relieve pressure. A total of three
reliefs are sufficient.
8. Use a sterile plastic pipette with a total volume of 10 ml and a manual “pipette bulb” for
pipetting a concentrated 50% NaOH solution (Fluka, 71868-1L). The solution is stored in the
exhausted waste cupboard underneath the HPLC and is extremely concentrated. ALWAYS wear
Instrumental operation: ICS 3000
Standard Operational Procedure
Jagger
proper personal protection equipment (glasses, gloves and lab coat/acid rubber coat) when
handling this solution. I am not kidding – getting that stuff in your eyes will ruin your eyesight!
9. Check the level of NaOH solution in the bottle – if the content is less than half of the original
replace with new to prevent risk of carbonate contamination in the NaOH solution. Place the
remainder of the solution in the toxic cupboard and mark it with “COMMON USE” for other
purposes than HPLC.
10. Pipette 10.4 mL to the 2 L bottle of eluent. Be careful when pipetting, as the solution is very
viscous. Don’t dip the entire pipette into the solution – only the tip underneath the surface.
Close the lid to the NaOH solution immediately after use and only use the same pipette ONCE in
the solution. Don’t double dip – take a new pipette.
11. Dispense the NaOH solution to the water in the eluent bottle underneath the water surface and
be patient as the liquid will flow very slowly (viscous). Slowly rinse the inside of the pipette by
gently pipetting up and down 3 times.
12. Close the lid to the eluent bottle and exchange the headspace air with nitrogen as done in step
7. Swirl the eluent bottle carefully – DON’T shake it, to distribute NaOH evenly.
Eluent B: 1 M NaOAce in 0.1 M NaOH (1L)
1. Weigh 82.04 g anhydrous sodium acetate (Sigma-Aldrich, 71183-1KG,or Fluka). It is important
that the correct quality of sodium acetate is used.
2. Poor this into a 1 L measuring flask (use clean glass ware) and add about 0.8L Milli-Q water.
3. Add a magnetic stir bar and place the solution on a magnetic stirrer till you have a complete
dissolution.
4. Remove the magnetic stir bar (without dipping the magic stick into the solution!) and fill the
remaining volume to 1 L (with freshly tapped Milli-Q water).
5. Mount a 0.22 µm sterile filter cup on top of the cleaned eluent bottle (f.ex. 0.22 µm stericup GP
Millipore Express PLUS membrane, 250 ml funnel, 45 mm neck size). Apply vacuum to filter the
eluent.
6. Loosely place a lid on top of the eluent bottle. Don’t tighten it.
7. Place the eluent bottle in the sonication bath, make sure water surface in the bath is at the
working level.
8. Sonicatefor 20 min. DON’T put your hands into the sonicator while it is operating and close the
door to the room when you leave it.
9. When the sonication is done, close the lid securely and move the eluent bottle to the HPLC.
10. Connect channel B to the eluent bottle and start the nitrogen flow. Exchange the air in the head
space by repeatedly opening and closing the lid on the bottle to relieve pressure. A total of three
reliefs are sufficient.
Instrumental operation: ICS 3000
Standard Operational Procedure
Jagger
11. Follow the procedures for NaOH addition as described for Eluent A step 8-12. EXCEPT! Only add
5.2 mL NaOH to the 1 L NaOAc-solution (ie. there should be the same NaOH concentration in
both channel A and B).
Eluent C: Milli-Q water
1. Fill the rinsed eluent bottle with Milli-Q water freshly tapped from the system.
2. Loosely place a lid on top of the eluent bottle. Don’t tighten it.
3. Place the eluent bottle in the sonication bath and adjust the water volume in the bath to the
level indicated as working level.
4. Sonicatefor 20 min . DON’T put your hands into the sonicator while it is operating and close the
door to the room when you leave it.
5. When the sonication is done, close the lid securely and move the eluent bottle to the HPLC.
6. Connect channel C to the eluent bottle and start the nitrogen flow. Exchange the air in the head
space by repeatedly opening and closing the lid on the bottle to relieve pressure. A total of three
reliefs are sufficient.
End the preparation procedure by purging all three channels as described below and note in the logbook
date of eluent change.
Purging eluents
When eluents are changed the pump needs to be purged to remove air bubble formation and old eluent
from the tubes. The system is sensitive to air (as all HPLCs) and purging is therefore important. Purging
the eluent channels is done be applying high flow through the specified channels and will create high
back pressure on the system, if the purge valve has not been open (Open the purge valve (figure 4) by
approx. half a turn). This will direct the flow path to a waste tube and relieve the pressure on the system
(and the column in particular).
Once the purge valve has been opened choose the appropriate channels and flow distribution to purge
in CM and activate the purge/prime function (figure 5). It is possible to purge more than one channel at
a time by setting the percentage distribution between the channels as desired (equal distribution, 50:50
when two channels, 33% when three and 25% when all four channels are to be purged). The software is
preprogrammed to perform purging for 500 seconds at 5.7 mL/min. Repeat purging (if necessary) until
air bubbles are no longer visible in the eluent channels from the bottles to the pump.
IMPORTANT: Before purging the software will inform that this operation will generate a
high flow through the system and that the purge valve should be open before
executing. Press “Execute despite warning”, if the purge valve is open. If the system
senses any pressure increase (even minor) during purging, the command will be
terminated. Make sure that the valve has been sufficiently loosened and try again.