HPLC for beginner - (Jan/06/2008 )

Hello Everyone!

I will be doing my 1st HPLC assay..most probably without a guidance. I will use it to analyze the concentration of citric acid resulted from fermentation. But, before that, i have to setup a standard curve e.g. calibration curve for the HPLC right? Can anyone guide me on how to prepare standard samples (in this case, citric acid) for the HPLC and also getting the standard curve?

Thanks in advance to anyone who helps

---

you make a series of dilutions that run below and above the range of the compound (in your case, citric acid) that you expect to find in your samples.

then you run them on the hplc and determine the response for each standard (integrate the peaks). plot the results on graph paper (to determine linearity or range of linearity) or have the software do it for you.

run your samples. compare results to standard curve or have the software do it for you.

---

Thanks for the general guideline! I still have questions though. What do i use to as solvent for citric acid and its composition? ethanol? Btw, i'm using reverse-phase HPLC. What bout the setting for HPLC e.g. temp, ppm, etc? Sorry, it may sound i haven't done any homework but it's really tough to find info about the setting.

---

i would dissolve the citric acid (for standard) in whatever the samples would be in or mobile phase or dw.

i would start at room temperature and/or check the literature about the separation of organic acids. some catalogs give chromatograms and conditions (eg-alltech, agilent, etc).

---