ICS 5000+ Trouble-shooting guide

ICS 5000+ Troubleshooting Guide
Contents
A Problem Tree (Page 2)
An Overview (Pages 3-7)
An Overview of the Anion-Exchange Chromatography system
Problems Section (pages 8-14)
• Trapped air in syringe/flushing the system (Page 8)
• Changing/Removing air from syringe (Page 9)
• Problems with the pump; low pressure, (signals alarm and red
light) (Page 10)
• Background noise and eluent concentration (Page 11-12)
• High pressure build up in the Halide trap (Page 12-13)
• Blockage in the needle or injection diverter valve (Page 14)
Running the Ion Chromatography System (Pages 15-27)
• Creating a New sequence (Page 15)
• Queuing a sequence/Setting up a countdown sequence (Page 16-
17)
• Changing, Modifying or Creating New Instrument methods
(Pages 17-19)
• Creating an Eluent Gradient (Pages 19-21)
• Creating a Stop Sequence programme (Page 21-22)
• Using the Virtual Column Separation Simulator (Page 22-23)
• How to Analyse Chromatogram Results (Page 23-24)
• Creating a Calibration Curve (Page 24-26)
• Changing the System configuration (Page 26-27)

Problem Tree
2
Are there visible air bubbles
or fluctuation in pressure dips
in the chromatogram?
Trapped air in Syringe/
Flushing the System
(Page 8)
Ye
s
N
o
Is there a problem with the
syringe?
Y
es N
o
Changing/ removing air from
the syringe? (Page 9)
Is the pressure too
low/high, is the alarm
going off?
Is there very high background
Noise?
(Pages 11-12)
Pump Problems
(Page 10)
N
o
Y
es
Is the syringe leaking,
suggesting a
blockage?
N
o
Y
es
Blockage in Needle,
Injection or Diverter
Valve (Page 13)
Blockage of the
Halide Trap (Page
12)
O
r

ICS 5000+ Trouble-shooting guide
An Overview of the Anion-Exchange Chromatography system
This section is a brief overview of the entire chromatography
system; it will section off each stage individually and explain
what each part does in a little bit more detail, making it a lot
easier to understand how the system works. It will also allow
a better understanding for when a problem may occur in the
system, as you will generally have a better understanding of
the system, you’ll be able to tell where the problem may be
occurring and be able to solve it.
Section 1: AS-AP Auto sampler
This is the first section of the
chromatography system and the one
that your samples will first interact
with the system. From (Figure 2),
you can see that the glass vials/ uni-
preps are placed with a shielded
compartment that can be closed, this
section is of a carousel design, with
separate sections for both 2ml and a
separate section for 10ml tubes that
can be used for dilutions and mixing
of the samples being run, this greatly
increases the adaptability of the
system for a variety of testing.
The carousel is divided into three sections labelled with different colours (Red, Green
and Blue); this allows for easy distinction of the positions for each vial, especially
when creating commands for dilutions. The carousel can be shut for protection against
possible air contaminants and particularly useful when running samples that are
sensitive to light. The sections are removable and the carousel can be moved by hand
making it easy for exchanges and maintenance if anything breaks or needs changing.
Through Chromeleon, another command can come in use for mixing, which allows
the carousel to shake and mix the samples for you, saving time and increasing
efficiency. This command can be found on the Sampler section of the Instruments
Tab.
3
Figure 1: ICS 5000+ system
Figure 2: ASAP Auto-Sampler

On the left hand side of the carousel, the syringe is located, this instrument is linked to
the needle and they work in tandem with one another if either of them are broken or
are not working properly the other one is useless on it’s own. The needle injects the
vials and the syringe takes up the sample and pushes it back through the needle into
the system. Underneath the needle there are two ports the injection port on the right
hand side, which is where the sample is injected into the system to be analysed and
the waste port, used for flushing the system and good for removing carry over in the
column, sample loop, suppressor or conductivity detector. Just like the carousel
commands for mixing, the commands for flushing/priming the syringe and needle are
found in the sampler section of the instruments tab on the Chromeleon programme.
Section 2: ICS 5000+ DP
The dual pump section may look confusing, but in
practice is very simple to maintain and its role is
essential to the chromatography system. The pumps
seen in (Figure 3) control the flow rate running
through the system all of the time, including when
it’s on standby there will still be a flow rate which
will be keeping the system stable and allow any
carry over to be run off the system. The section
contains two systems allowing for two simultaneous
tests to be running at the same time, explaining the
pump 1 and pump 2 labelled in (figure 3). One is
for an isocratic system, for consistent Eluent
concentration preventing any changes and the other
being a gradient system that has 4 input tubes
labelled from A-D. These allow for multiple eluents
at different concentrations to be run through the
system for the same test, which allows it to be very
adaptable, as different eluents may react with the
samples in different ways, for example
tetraborate can be used to remove chloride from
a sample by reacting with it in the system.
The pumps then send the eluent or deionised water through diverter valves all across
the system to push the sample along the column, suppressor and conductivity detector
so that it can be analysed. But it can also have a great affect on the overall
chromatogram as changing the flow rates in the system will change run time and in
turn the retention times of all the peaks and the final picture of the sample at the end
of the run.
Underneath the pumps lies a gradient mixer, this contains a small filter that the
eluent will go through before it is run over the suppressor, it reduced the ripple effect
of the changing gradient so that the chromatogram is smoother and easier to read,
increasing reliability.
Section 3: ICS 5000+ EG
4
Figure 3: ICS 5000+ Dual Pump

The Eluent generator regulates the eluent concentration that is sent through the
system, this works in tandem with the dual pumps. As the eluent types, ratios and
concentrations are changed on Chromeleon, the EG section of the IC sends the right
type of eluent, at the right concentration so that the chromatogram comes out under
the specified conditions. The section when opened contains various parts that can be
changed or replaced, as seen in (figure 4).
The eluent cartridge contains a specific eluent, this
eluent will be specific to the testing being done and
different eluents will act differently with the
samples being run. The eluent from the cartridge
will then travel to the CR-TC which acts as another
form of filter to reduce the background noise in the
final chromatogram, the CRTC is connected to the
eluent cartridge and the suppressor, so once the
eluent has been sent through the system it will come
back and be run through the CRTC before going
into the waste.
Above the CRTC is a degasser, the eluent that is
filtered through the CRTC then travels into the Degasser. The
gasser will remove any excess oxygen from the eluent, which
could create phantom peaks or high background noise issues, so
it is very important to have this in the system otherwise it is a lot
harder to make sure that your data is reliable.
Just like the dual pump section the rest of the system it is ready
for two separate systems, running two entirely different
programmes, both sides can fit a different eluent cartridge, a
degasser and a CR-TC and still be completely separate without
interfering with one another.
Section 4: ICS 5000+ DC
This is the main section of the Ion chromatography
machine, the largest and most important section, as
this is where everything comes together and is
analysed using a combination of the column,
suppressor and the conductivity detector. As the
samples come in through the dual pump section
they arrive at the bottom section of the DC running
along the blue line into the injection valve input and
then coming out though the red line onto the
column. It is then sent up to the suppressor, the
suppressor (figure 8), is used to reduce the
background eluent noise and helps increase the
analytes conductivity before going through the
detector. The suppressor contains a catalyst with a
current that passes through the solution going over it, this current can be changed to
accommodate for different analytes and samples being tested.
5
Figure 4: ICS 5000+ EGC
Figure 6: ICS 5000+ DC
Figure 5: Eluent Degasser

Section 5: (specific to the current Ion chromatography) Halide Trap and
Sample Cartridges
The Ion Chromatographer currently contains a set of a trap column,
sample cartridges and a halide trap, these four attachments work
together to help reduce the overpowering chloride of the samples. The
chloride easily interferes with the results especially for nitrites, where
the peaks are close. The sample once injected goes through a trap
column which is used to remove any onions that may be in either the
eluent or the deionised water which is carrying the sample, these
anions are removed to stop interference in the chromatography and to
help keep the baseline clean. The sample then travels through the
InGuard cartridges which work together to remove the chloride from
the sample, the first cartridge contains a styrene based- sulfonic acid in the
silver form. This resin inside the guard has a very high selectivity for halides
including chloride, so when the sample goes through the cartridge the resin
reacts with the chloride in the sample and creates a silver chloride complex,
which then reacts with the resin surrounding the cartridge, so that it remains
in the cartridge. It then goes through a second cartridge containing the same
resin in a sodium form, which binds to alkali metals; this is used to remove
any residue silver from the system.
The final stage is the concentrator, the concentrator allows the sample to be retained
and concentrated, so that the peaks stand out and samples of far lower concentrations
can be run (ppb/ppt).The concentrator reduces the Limit of detection on samples so
lower concentrated samples can seen, which also improves the range of samples that
the IC can test.
@?
6
Figure 9: Reaction of Ion Chromatography
Figure 7: InGuard Cartridge
Figure 8: Dionex™ IonPac™
ATC-HC Trap Column

The conductivity detector is the last stage of the cycle, the samples are run over after
going through the suppressor which after increasing the conductivity of the analytes in
the sample are picked up and the data is sent to the computer where Chromeleon
visually represents the data in the form of a chromatogram, just like the one presented
in (figure 9).
Problems Section
7
Figure 9: an example of a Chromatogram
Figure 10: Suppressor Method
Figure 11: an example of a Chromatogram

1.0 ICS 5000+ Troubleshooting Guide
Trapped Air in Syringe/Flushing the system:
1.1 On Chromeleon go to the Instruments tab. At the top of the page, there will
be a series of tabs for an overview of the IC and each individual component,
e.g. (Sampler, Pump 1, and DC etc.).
1.2 Got to the tab labelled Sampler.
1.3 Click the button named Prime Syringe, underneath there is the ability to
modify the amount of cleaning cycles made by the syringe (Increase/decrease
the number of cycles to what you think is appropriate, recommended 6-10
cycles for a complete flush of the system and optimum priming of the
syringe).
1.4 Check the clear tube connected to the top of the syringe for air bubbles, check
the whole length of the tube all the way to the junction,(this is located in the
ICS 5000+ Dual Pump section of the IC).
1.5 Flush as many cycles as is needed for all the air to be taken out of the system
via the syringe.
1.6 If the Air is still caught in the syringe see, (Changing/Removing air from
syringe). Otherwise move onto the next step.
1.7 Under the cycle’s button, click the Wash needle and fluidics, underneath is
the volume of water that will be flushed through the system, (recommended
setting 1000.0µl).
1.8 Once the needle and fluidics wash has finished, click the Wash sample loop
at the top of the page, like the Wash needle and fluidics the same volume is
applied.
1.9 Wait until the parameters flash green and say ready, you should be able to
resume with your sequence.
8
1.3
1.7
1.8
1.9
Figure 12: Sampler page; flush routine

Changing/Removing air from syringe)
2.0 Go to the Instruments tab and click the tab labelled sampler at the top of the
page.
2.1 On the right hand side, there should be a variety of console commands; these
do various calibrations on the individual parts of the IC.
2.2 Click the button called Wellness.
2.3 A small screen will pop up with small areas labelling different parts of the
system e.g. (Injection port, rotor seal etc.)
2.4 Click Start change under Syringe movements, this will cause the syringe to
move into a readied position (i.e.) move down and stop before pushing fluid
into the system. (Make sure it’s stopped moving before attempting these next
steps).
2.5 Unscrew the bolt at the bottom of the needle.
2.6 Unscrew the top of the syringe, making sure you have a secure hold on the
syringe, as this will detach it. (The next few steps are for removing air form
the syringe, if you’re just changing it skip to step 11).
2.7 Once the syringe is safely detached, prepare a pot of DI water and place the tip
of the syringe under the water).
2.8 Push out the water in the syringe and suck up water from the pot. Continue to
repeat this until the air is no longer visible.
2.9 Before re-attaching the syringe apply a small amount of pressure to create a
water bubble on the top of the syringe, (this will prevent an air bubble
forming).
2.10 Place the top of the syringe back into the port and twist to finger tightness.
2.11 Re-attach the bolt with finger tightness
2.12 Make sure everything is secure, before clicking Finish change. Press close
and then click empty syringe to waste.
2.13 Prime syringe before continuing with sequence.
Problems with the Pump; Low pressure, (signals alarm and red
9
2.4
2.2
2.13
2.12
Figure 13: Sampler Page Figure 14: Syringe Change

light).
3.1 If the pump has switched itself off, you can check the Audit trail tab on the
instrument tab homepage. (This will inform you why the pump switched
itself off or why the alarm was triggered). You can also check the pressure on
the Pump tab of the instruments homepage, the pressure will be given in psi
(normal values range between 1800-2300Psi).
3.2 Make sure everything is turned off; check the Millipore water filter is
connected around the back of the IC.
3.3 To allow pressure and air release from the pump, turn the flow rate down to
(0.1ml/min, this is essential; if it was any higher this would be very damaging
for the IC and the internals of the pump). You can then release the black valve
on the top of the pump slightly, not completely. Once you have done this you
should see air bubbles coming out of the pump. (Purge) This can take some
time to remove all the air, up to 30 minutes.
3.4 Once finished and you are sure that there are no air bubbles left inside the
pump, re-tighten the black valve (finger tight).
3.5 Prime syringe to flush main tube line.
3.6 Finally switch back to normal flow rate and run a sample or blank and
monitor the baseline, any negative drops in the chromatogram are an
indication of trapped air in the column, which is ultimately form the pump.
3.7 (If it doesn’t connect, go to the homepage of instruments tab, go to the ICS
5000+ DP section and hold down the power button until the lights have gone
off, ( all the blue lights should turn themselves off. Then switch it back on and
check the pump on the homepage to see if it has connected, (there should be a
little green light in the top left hand corner of the small screen for the pump, if
it’s on it should be connected and if it’s off then the green light should be off).
3.8 To see if your pump has been purged and primed properly, check the psi on the
pump tab in instruments. If it’s jumping 10-20psi, then this is an indication of
air trapped in the system. It should only vary around 1-2psi at most, as this
shows the pump is stable and air free.
Background Noise and Eluent Concentration
10
3.1 3.6
3.3
Figure 15: IC Home Page

4.1 To change the Eluent concentration on the programme, double click on the
chosen programme and click the tab for the Eluent generator at the bottom
of the page their will be the standard set concentration being used across the
programme, change this value to the one needed and then save as to create the
new programme.
4.2 Remember, changing the Eluent concentrations will change the pressure,
the background noise on the IC and ultimately the entire chromatogram. This
is why you must make sure that you are using the right programme before
running it as it could create an entirely new chromatogram causing peaks to
merge or appear at completely different retention times.
4.3 If the Conductivity detector on the home page of the instrument tab on
Chromeleon starts to show, huge levels of background noise then, this can
severely affect the results as it will merge peaks and create huge amounts of
distortion; the background noise should be as small as possible to reduce this
affect.
4.4 The general background noise should be around 0.1-0.4µS ±0.5µS (the IC
should be stable at this range when nothing is being run and between peaks in
the chromatogram). The best way to test this stability is to flush the system
and then let the background noise to settle, once it has, change the pressure
and Eluent concentration and watch the background noise until it settles again.
4.5 If the noise is any higher, then it could possibly be caused by a build up of
anions around the suppressor causing a lot of build up in the detector. In order
to decrease the noise, you can do the following;
• Wash the sample loop numerous times, remember to keep an eye on the
waste line, so that you can be sure the solution is coming out, there may be air
pockets in it so flush this line is an essential step.
• Then run the IC on an Eluent concentration of 100mM at a flow rate of
0.1ml/min, (this can be put up to 0.3ml/min if needed). The increased Eluent
concentration will initially cause an increase in the background noise as more
Eluent will be going over the suppressor.
• Another alternative is to unscrew the right hand side of the column and place
into a sample pot, so that the liquid passing over it will go off into the pot and
out of the system, this step will take time due to the very low flow rate so be
patient.
• If the increased flow rate and Eluent concentrations do not work, then this
could be due to the suppressor not being able to cope with all of the Eluent
passing over it, in this case increasing the suppressor current (recommended
30mA) is necessary, as this will allow it to cope with the increased work load.
The build up should be reduced by this and should settle down back to normal.
4.6 Overall programmes using high Eluent concentrations require higher
suppressor currents so that it can cope with the larger amounts of Eluent
passing over it. When this happens the background noise may initially start to
increase dramatically as more of it flows over the suppressor, this is expected,
so wait for it to peak and then begin to fall as the build up passes over and out
of the system, this may happen very slowly so patience is needed, wait for
the system to catch up and then settle down before running any samples or
standards.
11

4.7 One final point is to always check the samples your running to make sure that
they are centrifuged and uniprepped beforehand so that the particles do not get
trapped in the system.
High Pressure Build up in the Halide Trap
5.1 Sometimes the source of the blockage can be the point of contact with the
halide trap, this is a common area of high pressure build, as this is the central
point where the sample will concentrate before being released onto the column
and therefore is highly susceptible to crystal build up and eventual blockage.
In order to remove this build up similar the steps are very similar to those
made for column build up;
5.2 Firstly, go to the sampler tab of Chromeleon and select prime syringe, set to a
lower cycle so that it won’t damage the concentrator. Whilst it’s running the
flush, keep the ICS 5000+ DC running, keeping an eye on it at all times
allowing you to spot any leakage points that may inform you of where the
blockage is. Once you have determined that the concentrator is the source of
blockage (if not see problem tree) stop flushing the system and turn off the
flow.
12
4.4
4.1
Figure 17: Eluent Generator Programme page

5.3 The first test is to apply a high flow rate, to try to flush the blockage out,
combined with a high suppressor current to react with the build up and remove
it from the system, on the home tab of Chromeleon click the ‘monitor
baseline’ button, this will allow you to monitor the background noise, if it’s
really high this is a good sign as the blockage will be going over the system,
wait for the background noise to start lowering again. When the noise remains
at a respectable level, you can lower the flow rate and suppressor settings
again, (the background noise may go up again, but this is natural as the IC is
just readjusting the baseline to the change in conditions, it will steady itself
again).
5.4 If the previous step did not work then detach the concentrator from the side
connecting it to the column, once this is done boil some water and get
a plastic syringe. Detach the halide trap completely and carefully syringe the
hot water through the trap, this should react with the sample or crystal build up
and remove it from the system releasing the blockage. If this doesn’t work
then, replacing the concentrator is the best option, but make sure to get a
second opinion as this should only be a last resort.
Blockage in Needle, Injection or Diverter Valve
6.1 Evidence to suggest that there maybe a blockage in the needle, injection, or
diverter valve, will be if a leak appears at the top of the syringe where the line
towards the injection valve lies, this indicates that something is preventing the
sample to be fully sent through the line and a blockage can occur at several
different points at this line a blockage would also create a variance in pressure
far from the normal level for instance a significant increase in the pressure due
to the blockage, the following steps should be taken when this occurs;
6.2 To check whether the sample or general solution is reaching the last point, the
diverter valve, unscrew the tube from the correct valve (The valves should be
located to the right of the syringe), and flush the sample loop, using the
Chromeleon data, sampler page. This line is the final point before the sample
13
5.3

loop, so if liquid is not getting to this point then this means that the block is
either at the needle or the injection valve or both. If it isn’t then on the sampler
page of Chromeleon, there should be a button to switch the diverter valve
position, click this and it will change the valve that the liquid will run through,
this can be used to check whether it’s the line that’s blocked or whether it’s
the needle.
6.3 To check the needle, make sure that the IC is not running and that it is safe to
move the needle, on the data sampler page, there should be a page for the
ASAP on the right hand side of the page, this should allow you to execute
commands for the needle including moving it to the wash port or injector port.
6.4 Move the needle to the wash port, as this makes it easier to get to, once this is
done, unscrew the line connecting the syringe to the needle. Perform a flush
command and make sure that this line is not blocked, once this is done take a
beaker and fill with hot water, plunge the needle into the water, this will help
warm up the needle and dissolve the possible blockage, inject some hot water
through the needle and make sure that it is going through with ease before
putting it back into the system.
6.5 Perform a further ‘Wash Sample loop’ command, if a leak still persists, then
the problem will lie with the injection port and there may be some sample
build up. There are several steps that can be taken at this stage, you can
perform a blank run at a higher flow rate (not too high though as this could
cause more damage), to try and push the blockage free and out of the system.
6.6 You could also apply the hot water technique and using a plastic syringe, push
hot water directly into the injection port, making sure the needle has been
moved out of the way, and one final step is to unscrew the injection line and
re-adjust the feral, this releases the built up pressure in the line and can often
allow the build up of crystals or sample to be released. Once all these have
been done, screw the line back in and perform one last flush command.
6.7 If the problem continues then the blockage will be from a point further in the
system and you will have to check each point of contact to see where the
sample is being stopped.
Running the Ion Chromatography System
Creating a new sequence
7.1 Once a sequence has already been created you can use the ‘save as’ function to
create a duplicate of the sequence and rename it as your new one.
The data can be removed and replaced, but it is a good way to compare
standards, methods and data in general without disrupting the original data.
But if you’re creating a sequence from scratch, use the following steps;
7.2 Go to Create and select Sequence, the new sequence wizard will pop up and
you will be able to select an instrument to make the sequence on (Anions or
Carbohydrates). Select the one you need and then click next.
7.3 You will then be able to select the number of samples you would like to have
in the sequence when it’s first created. You can also select the number of
injections per samples, the volume of the injections and the positions of the
vials in the sequence. (These can all be changed later).
7.4 The next page allows you to either create a new instrument method and
14

processing method or browse and use one that is already being/been used. The
same can be done for a Report Template and View Settings. (Recommended
channel setting CD_1_Total) click Next.
7.5 Before Finishing, you may want to add a small description of your sequence
and what it’s trying to achieve. Click Finish. (Name it, recommended as date
then purpose e.g. 07/08/2015 Standards).
8.0 ICS 5000+
Troubleshooting Guide
Queuing a sequence/ Setting up a countdown sequence
8.1 Setting up sequences is relatively straight-forward, but you can also queue
several sequences to run after one another, or set a sequence to run at a
specific date or time.
8.2 The following steps will show you how to do this, so that you are able to run
sequences over night, this reduces restrictions on the number of samples that
you can do in one day. It can also be used to run a start up programme in the
morning, so whilst your IC is on standby you can set it up to run an hour
before you get in, to raise the flow rate and/or eluent to the appropriate level
for running your programme, so that the system is all ready for when you start,
reducing your set up time.
8.3 Create a sequence (If you don’t know how to do this go to page 14) that you
wish to run overnight or as a start up programme in the morning.
8.4 Go to the Instrument tab of Chromeleon and at the top of the page, you will
15
7.2 7.3
7.4 7.5
Figure 19: New Sequence Wizard

see the queue tab, click on this to see the queue for all the sequences that are
currently running and those that are pending.
8.5 On the right-hand side of the page there should be a button to add and remove
sequences, select add and then find the sequence you would like to add to the
list. This will automatically queue the sequence to run after the currently
running sequence.
8.6 You may also do a ready check, by selecting the ‘ready check’ button on the
bottom right-hand side of the screen, this will show you any conditions that
need to be accepted or changed before the sequence can run, make sure that
these are met before you queue the sequence up as it will not run without your
permission, which defeats the purpose of setting it up in the first place.
N.B Remember to turn off any injections that are labelled as blanks, as these will
stop the sequence running, which will also prevent anything further in the
sequence from running too.
8.7 To set up a countdown sequence, add a sequence to the queue and click the
box next to the sequence under the heading ‘Start after’, once clicked the
current time and date will come up. This date and time can be changed, so
you could have a sequence run overnight, in the morning or consecutively
over several days, starting at the same time, which could be used for testing
depreciation of a sample over a specified time.
8.8 Overall the possibilities for start up programmes and running sequences is
immense, you could set up a programme to start up the machine every
morning so you don’t have to do it yourself, saving you time to do more
samples in the day, and then due to being able to run samples over night
without supervision, this also increases the workload that you can prepare for
the machine. The queuing and countdown sequence system can make
preparation and analysis far more efficient and easier in the long run.
16
8.4
8.5
8.6
8.7
Figure 21: Sequence Queue Page

Changing, Modifying or Creating New Instrument methods
9.1 To create a new method, on the Data tab of Chromeleon, click Create at the
top left hand corner of the page. Click Create Instrument method. (To
change or modify an existing method they will appear underneath your
sequence at the bottom page under the tab ‘Associated items’ there you can
modify existing methods just by double clicking on the method you want to
change.
9.2 When creating a new method, it will ask you what instrument you would like
to create your method in (Anions/Carbohydrates), once you’ve chosen click
next.
9.3 The set up wizard will ask you what run time the method will have in
minutes; this will depend on what task the method is for. It will also ask you
to select channels for the method, depending on what system you’re using.
Once these have both been chosen, click next.
9.4 This page will allow you to change between Isocratic/Gradient, it will also
allow you to set the flow rate, the pressure limits and the ratio of percentages
of each solvent you want travelling through the four tubes A, B, C and D.
(Recommended A 100%) Click next.
9.5 This page will allow you to change your cartridge type and see a small
description of it (including remaining ion % in your current Eluent
Generator Cartridge). The concentration mode can be changed and the
value of the concentration (recommended 23mM Hydroxide), another
feature is the option to have the CRTC off or on (recommended on), click
next.
9.6 The Inject mode page is next, (recommended PushSeqFull, and accept
recommended values (remember to have the diverter valve position on the
right system your using 1 or 2, this must be on the right one).
9.6.1 General settings, these can be changed if necessary, but usually just accept
recommended values and click next.
9.6.2 Sample preparation, this page allows you to put specific commands into the
method, for example, washes between injections, dilutions for certain
conditions that are met. Click next.
9.6.3 For the Fractionation settings These are not needed for this particular
system, so they can be skipped, click next.
9.6.4 CDet Options This page allows you to change the conductivity detectors;
collection rate and temperature controls of the cell and the compartment, click
next.
9.6.5 Suppressor Options This page allows you to select the suppressor you want
to use, this can change based on the method your using, you can also change
the current (recommended 15mA) and the Eluent concentration that will go
through the suppressor, this can be a mix of eluents so that you can suppress
particular ions in the chromatogram, click next.
9.6.6 Temperature Options Allows the alteration of the temperature for the
column and the compartment, you can even say whether it’s in use and
whether it should be on or off, there is even a feature for delays as the
sequence begins, click next.
9.6.7 Comments can be made on the last page; these will be specific to the method,
Click Finish.
17

9.6.8 To apply your new method, next to your samples volume, a tab for instrument
method should be there, just click the drop down box and select your method.
9.6.9 When modifying an existing method it will bring up a brief overview of the
entire method, with small tabs that will take you to each part of the system
and allow you to change it for what you need, once finished just save as and
create a new name for the method and it will appear with all your existing
methods.
10.0 ICS 5000+ trouble-shooting guide
Creating an Eluent Gradient
10.1 Creating an Eluent Gradient is very simple due to the fact that it allows to
manually make any kind of gradient you want, shows you the outcome on a
graph and is easily made when creating a new method or changing an
existing one.
10.2 First you need to either double click an existing method you would like to
change, or click on create and choose new instrument method.
10.3 The tab that is important for Eluent gradients is the Eluent generator tab; you
can refer to all the other tabs in the guide for creating new or changing
existing methods.
10.3.1 Once you’re on the tab, at the top there are two tabs one saying ‘EGC_1
Concentration’ and another saying ‘EGC_1 Concentration Gradient’ faded
out.
10.3.2 To unlock the gradient tab, you must switch the Eluent generator from
isocratic to multi-step gradient, it is automatically set to isocratic, which
means that the concentration remains constant throughout the run, whereas
the multi-step gradient allows it to change throughout the run, and this is the
one you want.
18
Figure 22: Instrument Method Wizard

10.3.3 Once it has been selected, go to the tab for concentration gradients and a
new page will come up, it will have a table labelled with the time and the
concentration.
10.3.4 Both of these values will change based on the programme being run, so the
amount of steps, when they need to be implemented and how long they will
last will all change based on the programme.
10.3.5 To start put the initial concentration that you want the programme to run e.g.
23mM, the programme will automatically run it at this concentration until it
says you would like to change it.
10.3.6 After that put in what times you would like changes in the concentration
and the concentration you want it to go to, this can be either and increase or
decrease in concentration.
10.3.7 If you want the concentration to be held, then write down the range of time
you want it to be held (e.g. time: 15.000 concentration: 23mM, time: 17.000
concentration: 23mM).
10.3.8 Whenever a new step is added, the graph above the table will change to
show how the concentration will change throughout the run time, a nice
little overview of the programme and some foresight into whether it will
work.
10.3.9 Note: Don’t put too high of a gap between two points of concentration
when there isn’t a big enough time gap, for example going from 10mM to
50mM in half a minute, because the system sometimes can’t keep up and
might not get up to the selected concentration within the time limit,
reducing the validity of your results.
10.3.10 Multi-step gradients are very useful for increasing the resolution
(separation) of peaks in the chromatogram, for instance separating a huge
chloride peak from a nitrite peak so that they don’t merge or separating
sulphite, bromide and sulphate peaks from one another.
10.3.11 A good method for increasing resolution of your desired peaks is to run the
sample at a set concentration and flow rate to see the retention times. This
will give you something to work with when determining the times to
change the concentration, this is essential for getting the right resolution,
without the original retention times the change could merge the peaks
instead or do nothing.
10.3.12 You could increase the concentration around the time a peak occurs,
increasing the speed at which is emerges and is read on the chromatogram,
or decrease the concentration so that the peaks comes out later, either
change will increase the distance between the peaks.
10.3.13 Once you have a nice gradient change occurring, the concentration should
be brought back down gradually to the initial concentration before the run
ends.
10.3.14 Save the method and then run your samples using it to see what changes
occur, change it if the desired effect isn’t reached.
19
10.3.1

Creating a Stop Sequence programme
11.1 A stop sequence programme is used after running samples or standards at the
end of a sequence to help lower the flow rate, suppressor current and eluent
concentration. This is very important as it will help reduce waste whilst the
IC is on standby.
11.2 To create the stop programme, it uses the same method as creating any new
method, you either click on ‘Create’ in the top right hand corner of
Chromeleon’s Data homepage and click ‘Instrument method’ or underneath
your sequence, double-click on one of the instrument methods that you’ve
made so that you can modify it and then save as the new method.
11.3 The programme will come up and allow you to modify it; for a stop sequence
programme, you will want a low flow rate, so that your water dispenser
doesn’t get too low, for instance over the weekend if it’s running standards.
You want to save as much water as possible as if it’s empty, it can cause
20
10.3.3
10.3.5-
10.3.8
Figure 23: Eluent Concentration Page
Figure 24: Eluent Concentration Page

damage to the system when running samples.
11.4 It will also require a lower eluent concentration, other wise you will be
wasting eluent during the systems standby, which isn’t necessary as no
samples would be running. As the eluent concentration is brought down,
this should automatically lower the suppression current too. The suppressor
doesn’t need to be high is your running any samples either.
11.5 Another feature would be to change the method from a multi-step gradient,
to isocratic so that it stays at a consistently low eluent concentration for the
duration of the run. The run time should be relatively low, maybe around 1
minute, as it’s just so the system gets used to the flow rate and when it
finishes it will stay at that during its standby period.
11.6 Once all of the programmes features have been modified, then it can be
saved, instead of just saving changes, right click on the programmes name
in the bottom left-hand corner and click save as, then save it under a new
name so that it will become a separate method and not overwrite the one
your modifying.
11.7 Once the method has been created, add a sample to the sequence and put a
vial of water in the position stated, when the programme is activated at the
end of the sequence, the system should be used to the low flow rate, eluent
concentration and suppressor current and remain at those values whilst the
system waits for it’s next sequence, thus minimising waste and increasing
efficiency of the machine.
Using the Virtual Column Separation Simulator
12.1 First click ‘Tools’ at the very top left hand corner of the screen and click on
‘Virtual Column Separation Simulator’, this will start up the programme
and bring to the page above.
12.2 On this page you can set up any combination of analytes or even
carbohydrates depending on what system you’re using to see how they may
come out on the column.
12.3 When you have selected the analytes that will be coming up in the column, it
will show you the columns that are available to run them on, these columns
can be selected from standard, micro bore and capillary sizes it will also give
you the option for whether the system will be isocratic or gradient, to increase
the accuracy of its estimation and allow you to select the exact column and
system you have.
12.4 Once your column has been chosen, you can change the settings, such as
temperature, Gradient start concentration (if you chose a gradient
column system), the flow rate and the void volume of the programme, as
you change these the picture on the right hand side of the programme will
change to accommodate for the changes.
12.5 Above the virtual chromatogram is an overview of the changes that occur in
the resolution against changes in eluent concentration. It will display the
eluent concentrations needed for the best resolution, fastest separation and
Column QA conditions.
12.6 At the top of the resolution plot, criteria can be selected to change the
resolution plot and how it acts under certain conditions, for instance the
21

resolution of all the analytes can be measured where it will calculate a mean
resolution for the entire plot, but you can also select the resolution of the
selected analyte and show you how it acts under a changing eluent gradient
individually.
12.7 The programme will show the best resolution, QA conditions and fastest
separation eluent concentration for that particular analyte.
12.8 The automatic setting though is critical pairs, which targets the pair of
peaks with the worst resolution, or the peaks that are the most difficult to
separate, helping you to understand the best conditions needed to separate the
peaks that might usually be very difficult to do so.
12.9 Once you’ve finished changing all of the settings, the chromatogram can be
saved, so that it can be referenced when running the samples.
Figure 25: Virtual Column Separation Simulator
How to Analyse Chromatogram Results
13.1 After running a sequence, the results will show up in small images next to
each individual run, they can be viewed from the data home page.
13.2 To get a larger image that you can analyse and manage yourself, just double
click on the sample that you need to look at and it will open up a new
window, consisting of the chromatogram, with multiple options for labelling
and viewing data on each of the individual peaks.
13.3 At the top of the page there should be a results button and a Calibration
and pm button next to one another. The Results tab when clicked will bring
up a table at the bottom of the page, listing each individual peak, its area and
its relative area compared to all other peaks in the sample. This is the best
way to look at specific peaks you want to read.
13.4 Underneath the table will be a few tabs, one called summary is very
important when wanting to compare the peaks of different chromatograms,
for example comparing nitrite or nitrate peaks to see if they are going up
linearly.
22
12.3
12.4
12.5
12.6

13.5 Sometimes peaks aren’t labelled either because their retention time is
different to the peaks that are normally read, it’s too small compared to other
peaks or it’s just an error in the system, to resolve this just drag your mouse
across the bottom of the peak to create a line, once it’s connected then the
peaks results should appear on the results table.
13.6 If a peak has not been identified yet then just right click it and click manual
peak identification. This should produce a window that allows you to modify
the programme to recognise certain peaks automatically, at the bottom it will
have a drop down box with all your current analytes with their retention
times, but you can just type in the chemical it is and add it to the roster.
(Do not do this unless you are sure what the peak is).
13.7 If you have changed the programme and the retention times for your known
analytes have changed and are no longer being labelled correctly then a
simple way to resolve this is to click on the processing tab at the very top of
the window, and it will change the icons below, where there should be a
section labelled ‘component table’ click on ‘peak windows’ and it should
highlight the windows in which the different peaks should come under before
they’re read, if a peak does not come under any of them it is not labelled. The
ranges can be moved to accommodate for the programme changes. (This
should only be done if you know the retention times for all the peaks).
13.8 If peaks have merged then you can right click them and click ‘split peak’
which will separate the two so they are readable. To move the baseline back
to where it’s meant to be just hover the mouse on the line joining the two
peaks until it comes up with a symbol that has the line being pulled down.
Creating a Calibration Curve
14.1 To create a calibration curve, you must first set your calibration standards,
to do this go to the drop down box next to the standard and click the drop
down box to change it from unknown to calibration standard.
14.2 The box next to it will no longer be greyed out and you can now change it by
a drop down box to select a level. Select ‘01’ for your first calibration
23
13.1
13.3
13.4
Figure 26: Sequence Page
Figure 27: Results Page

standard, ‘02’ for the second and so on and so forth.
14.3 Once you have selected all of your calibration standards with their
appropriate levels, then double click on of the samples to bring up the
enlarged results window.
14.4 In this window select the ‘Calibration and pm’ button at the top left hand
corner of the screen, this should bring up the calibration plot and a table
representing all of the labelled analytes in the chromatogram with their
appropriate levels.
14.5 The table will automatically have every level set to the value of 1, but these
can be changed to the scale appropriate ‘e.g. ppm values scaling from 20-
200’.
14.6 Selecting the values for each level will change the calibration plot seen in the
top right hand corner, once you have put in all the values it will show the
finished plot, it will automatically create a line of best fit too.
14.7 Once you are happy with the results you may save it for future reference. The
importance of this plot is very important as it is used to compare the rest of
your results against it. Using the calibration plot it will compare the results to
the line to see where they align and on the ‘Results’ table the value will
come up for the peak. This is very important if you are retesting a sample as
you can compare the definitive areas of both peaks to make sure they are
consistent.
14.8 When the calibration curve has been made, it may have three lines instead of
one, like the one in (figure…..) these lines are the line of best fit, and two
error lines to correct for any outlying data points in the sample.
24
14.1
14.5
14.6
Figure 28: Calibration Points

15.0 ICS
5000+ Troubleshooting Guide
Changing the system configuration
15.1 The Chromatograph can be used for many different methods, these methods
vary and this can sometimes mean changing the configuration inside the IC.
This can be relatively tricky for someone new to the instrument so a diagram
has been made to illustrate the injection ports of the DC and the positioning of
the attachments within the system.
15.2 The steps taken also change based on the method that you are changing to, for
instance Nitrites and Nitrates include the Halide traps, Trap column and
Concentrator attachments, as well as using both systems and a switch between
the load and inject modes, within the programme. This difference can be seen
in the diagrams above, but the changes made between the configurations is
very simple, to change to the nitrites and nitrates method do the following;
15.3 Firstly, to change the Chromeleon programme configuration, go to the
computer desktop and go into the Chromeleon Instrument controller, which
should be in the bottom right-hand corner of the screen where the time is.
When open click on configure instrument and it will take you to the command
screen (make sure that your are not running anything as you will not be able to
make any changes to the configuration otherwise). Click on file and load up
the configuration named ‘Nitrite and Nitrate IC Configuration’.
15.4 After changing the instrumental side, make sure that the flow is turned off
before changing the tubing inside the DC. To begin with, reattach the Trap
column into Position 3, on the 6-port diverter valve for system 2, re-attach the
halide traps to position 2 on the same 6-port valve. Position 4 and 1 should
have the 10µl sample loop, which shouldn’t be moved between the methods.
25
Figure 29: Calibration curve of Chromatogram Standard

Position 6 should have the waste line and position 5 is the sample line from
the Auto-Sampler.
15.5 As for the System 1 6-port Diverter Valve, the Halide Traps should be
connected to position 5 and the waste to position 6. Like system 2, position 4
and 1 are connected, but for this system it’s by an Analytical concentrator.
This device can also remain attached when changing methods. Position 2 is
connected to two restrictor back pressure coils, a 500 psi and a 1000 psi coil,
which will carry the eluent; this will need to be changed when changing
methods. Position 3 is the column flow, the final line that directs the sample
through the heater and onto the column.
15.6 When changing from Nitrates to Sulphites a few changes have to be made, but
the system becomes far simpler, as only one system is used instead of two
simultaneously.
15.7 Detach the Trap column on one side and cap it off so that it doesn’t dry out,
this also goes for the Halide Traps, they only need to be detached from the
System 2 6-port diverter valve and capped off to prevent drying out.
26
Figure 30: Nitrite and Nitrate Configuration
Figure 31: Sulphite Configuration

ICS 5000+ Trouble-shooting guide

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