Building a Calibration Table in Ezchrom (with screenshots)

This is a quick guide on how to build a calibration table in Ezchrom.  This should work in both Ezchrom Elite as well as Openlab Ezchrom.


1.  Have your method(s) that you would like to use saved and ready.  This first step is pretty straightforward.  You want to have a good idea of what your method parameters will be before you start building your calibration table.

2.  Set up a sequence of runs with your calibration standard(s).  In my example, I am using three levels of calibration.  If you expect your samples to be within a certain range, you want to select standard concentrations that will cover that range.  So, if my samples will have a concentration in a range of 50-75 ng/ul for example.  Then I would want to choose standards at 30, 60, and 90 ng/ul, perhaps.   Whichever calibration levels you choose to have, make sure you have them ready and know which vials they are in, and designate that in your sequence table.


3. Integrate your peaks.  Make sure you have the proper channel selected at the top, for the chromatogram that you want to adjust.  Next select integration events, the two values you want to focus on at first are threshold and width.  You can keep clicking analyze to see how your changes affect the integration.  It’s more or less a trial and error approach.  There are several other options you can continually add to your integration table to fine tune your chromatogram.


In this example, I would like to remove some non peaks at the start of the chromatogram.  I can click on integration off, on the bottom of the screen.  It then wants you to click on the chromatogram where you want to start the cut off:



Next it wants you to click where you want the integration off event to stop:


After you analyze, you get the chromatogram with the unwanted peaks removed:


4.  Calibration.  After integrating your signals, the next step is calibrating.  Peak selection can be done graphically on the chromatogram.  You can click on the icon to define peaks, click at the start of the peaks, then click at the end of the peaks.  The peak table will come up, and you can name the peaks, and designate their concentrations.


Save these changes to your method, and the software will now calculate the amounts of your unknowns based on your calibration table!

Can’t Communicate with my Instrument (GC or LC)!



There’s nothing like  preparing your samples and starting chemstation, only to see “instrument offline”.  There are some basics that can be checked that may be the solution if you are having this problem.

IP addresses:

You always want to start here.  A 68/7890 GC, as well as 1100-1290 LCs use LAN communication for the most part.  The main idea being, is the ip address of the instrument and the ip address of the pc compatible?  You want the ip address to match up until the last set of numbers.  For example:  if the gc is, the pc needs to be 10.1.1.x.  If the LC is, the pc needs to be 192.168.254.x.  With “x” being any digit from 1 to 255.

How can I check the ip address of the GC?  This is pretty simple, on the majority of these GCs you can find out by hitting options, then communication on the gc front panel.  From here you can see what the address currently is, and you can also change it as well.

The LC is not as straight forward, but the default is  You can also connect the gameboy to the module and view/edit the ip address.  If it’s not the default, and you are not sure what the address is, then you will have to go through a process of changing switches on the LAN card and setting it to default.  From there you can keep the default or change to a new address using Telnet.  I may write an article on that procedure in the future.

How can I check the ip address on the pc?  You can do this through the control panel of windows, then go to network and internet, then network connections.  You can see all your network connections on this screen.   Most people will have two connections, a “house” or company network, and a local area connection.  The local are connection is usually the one that is connected to the instrument.  Right-click on that connection, then double click on IPv4.  You will see a screen like this:


From here you can view and change the IP address.  Make sure to keep the subnet mask:  Hit OK to save the changes.

After determining that the addresses are compatible, the next thing you want to do is ping the instrument.  To do this you need to launch the command prompt.  In Windows 7, click on the Start key for windows, in the search bar type CMD and hit enter.  In Windows XP, hit start, and select run, type CMD and hit enter.

Once in the command prompt type: ping (or whatever the ip address of the instrument is).  You should get 4 replies back, 100% packets sent and received.  If not, then check that the cable from the pc to the insturment is connected and good, maybe try swapping it out.  If there is a switch or hub in between make sure that port is operational.  If there are 2 LAN cards on the pc, its possible that you are connecting to the wrong one, or changed the ip address on the wrong one.  Does the LAN card on both the pc and instrument show flashing LED lights on the port?  If not it may be a hardware problem with one of the cards.



If pinging the instrument was successful, the last piece of the puzzle is ensuring the configuration in chemstation has the proper ip address for the instrument.  If you are using a classic version of chemstation (pre-Openlab), go to start, all programs, agilent chemstation, and launch the configuration editor.  From here you can view/edit the ip address of what chemstation thinks the instrument is configured to be.  In Openlab chemstation, launch the Openlab control panel, select your instrument on the left hand side, and click on configure instrument on the top of the screen.  From here you can view/edit the ip address of what Openlab is expecting your instrument to be configured to be.



I hope these basic steps of checking/pinging the ip address have been helpful.  Please leave a comment if you have any feedback or questions, thanks!




Forced Cold Restart on HPLC

Sometimes you might get strange errors on the HPLC modules.  This trick is called the forced cold restart, or factory reset.  Basically clears errors and resets set points on the module to the factory settings.  This is useful for strange oddball errors that may appear on the HPLC.  This works on many of the 1100 up to the 1260 modules.  On the back of the module, you will see a row of white dip switches, usually with the label CONFIG.  These switches are 1-8 starting from the left.

Take note of the exact arrangement of the switches currently, so that you can revert them back to the way they were when you are finished.  Turn off the module.  Make switches 1,2, and 8 up, and every other switch down.  Turn on the module for a few seconds, then turn it off.  Return switches to the original configuration.  Forced cold restart complete.

HPLC No Flow!

It happens sometimes that the HPLC pump is on and pumping,  but there is no flow coming out.  If you have an air bubble somewhere in the tubing, you will notice that its either not moving, or just moving back and forth.  The very first thing you can try, is just opening the purge valve, and pumping at 5ml/min to get an air bubbles out.

Also, you want to check your solvent bottles, make sure there is solvent there.  If you are using solvent filters on the ends of your lines, make sure they are not clogged.

After checking that, you can try loosening the line going into the active inlet valve:


Sometimes just relieving the pressure here can solve the issue.  If not, you will want to remove the outlet ball valve.  There is capillary on top of the valve that must be loosened and removed first.  Then you can remove the valve.


Sometimes the pump can get air locked and just needs to be “burped”.  Removing this valve and running the pump will allow it to release any air that may be trapped.  You will see a small well where the valve was sitting.  You want to make sure that the well fills up with solvent with the pump on.  If not, you problem may actually be the Active inlet valve.  That valve has a cartridge that can get clogged, or it could be the actual valve itself.  Replace the cartridge first, then replace the valve if that doesn’t help.

This procedure will work for Agilent 1100\1120\1200\1220\1260 Binary\Quat\Iso pumps. For Quat pumps you also want to make sure that the MCGV isn’t blocking solvent from reaching the active inlet valve.  You can remove the line that goes in the middle of that MCGV and just place it inside a beaker of solvent.


Turn on the pump and see if there is any change.

These procedures assume that the pump is actually moving.  You can usually tell just by hearing the pump mechanism moving when you turn the pump on.

High Pressure in my HPLC!

Ok so no one wants to see the pressure on their HPLC higher than what it should be.  In my experience the number one cause of high pressure is a clog in the autosampler.  Followed by a clog in the purge valve frit, as the second highest cause of HPLC over pressure.  Luckily, there’s an easy way to diagnose whether or not there’s a clog in the autosampler.  This technique works on Agilent model HPLCs and Chemstation, not sure about other brands.

Turn the pump on, once you see the high pressure, right-click on the diagram of the autosampler.  Select the switch valve to bypass option (see below).  See if pressure drops immediately, if so change the needle and needle seat (or backflush).


Note: Older versions of chemstation will only have that option available in the diagnosis tab.  I may make a tutorial on the process in the future.

What if the pressure is still high?  Try removing the column from the flow path.  If that doesn’t help, try removing the autosampler from the flow path.  If that doesn’t work, try opening the purge valve.  If opening the purge valve does not decrease the pressure, try replacing the purge valve frit.

Pro Tip:  When installing a brand new column, make note of the pressure of the system.  This is what your pressure baseline is, and will be a good reference in the future when you are not sure if your pressure is where it should be.

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