Hi everyone,
I know I'm asking a lot but I'm learning HPLC for the first time. I'm working with a simple assay containing three simple compounds that separate well. I've recently inject standards and I'm trying to set up a calibration table. Could anyone point me towards a good guide that would allow me to create a calibration table? Also what is the meaning of "levels" in the calibration table? Do they refer to different concentrations of the same compound or different compounds? I know this isn't an easy question but I'm trying to figure this out on my own and any help would be really appreciated.
Thanks again
Harry
There are good chapters on HPLC written on Analytical Chemistry text books by
Skoog, West and Holler
David Harvey
Harris
You may refer those books. They will guide you through.
To simply answer, there are two ways you can extract concentration related information from peaks. Those are either using the peak area or the peak height. But you got to be consistent and it's better if the software can give those numbers for you.
Dear Harry
When you say ChemStation, I almost assume that it is an older version. Below is the manual, that describes the process in detail
https://www.google.com/url?sa=t&source=web&rct=j&url=https://www.agilent.com/cs/library/usermanuals/Public/G2070-91126_Understanding.pdf&ved=2ahUKEwix597AhYbhAhVF2aYKHcBMAtMQFjACegQIARAB&usg=AOvVaw1RHv2E579tabqcZjgz4dhH&cshid=1552718619115
Chromguy also has a quick tutorial here
https://www.google.com/amp/s/chromguy.com/2015/09/19/how-to-build-a-calibration-table-in-chemstation/amp/
You could also refer to the slides from those Agilent E-seminar
https://www.google.com/url?sa=t&source=web&rct=j&url=https://www.agilent.com/cs/library/slidepresentation/public/ChemStation_How%2520to%2520Set%2520Up%2520a%2520Calibration_011409.pdf&ved=2ahUKEwix597AhYbhAhVF2aYKHcBMAtMQFjADegQICBAB&usg=AOvVaw2PsoQcijXa36McTR6lL70T&cshid=1552718619115
If it's a newer version OpenLAB CDS ChemStation Edition), I would also just look up the software manual. You can check this short video by Agilent.
https://community.agilent.com/videos/1149-how-to-create-a-multi-level-calibration-curve-in-openlab-cds-chemstation
But in short, level does refer to a specific concentration. You will need to define the concentration of each compound in each level. There, you also define internal standard concentration in order to use the relative response factor for quantification.
Please look at the following below links which may help you in your analysis:
https://www.agilent.com/cs/library/Support/Documents/F01038.pdf
https://www.agilent.com/cs/library/slidepresentation/public/ChemStation_How%20to%20Set%20Up%20a%20Calibration_011409.pdf
http://remchowiki.science.oregonstate.edu/lib/exe/fetch.php?media=agilent1100_chemstation_operation.pdf
https://www.csus.edu/indiv/d/dixonr/c231/agilentinst.pdf
Thanks
Thank you so much guys! Once I have the calibration table made how do I save the results and load them onto the next run when I have samples containing the same compound of unknown quantity?
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