tanong po ulit

1. We are analyzing antioxidants from fats and oil, we are having a difficulity in separating the peak of oxalic acid and ascorbyl palmitate AP target analyte. The probrem is we to inject the standard more or less 10 to 20 timkes to obtain almost a good separation. We do SOP for system stability. What could be the probable cause of the problem?


2. Suggetion for the next seminar, we really need to undestand how to do method validation especially for HPLC and GC, because our employer wants to validate method ASAP. Really need it.


Thanks

Hi Biokemist89,

There could be lots of factors. Cleaning of equipment and column before and after use could be one of the probable causes. We could discuss more your SOP for system stability. We might be missing important facts.

Thanks for the suggestion. We'll do method validation soon.

What we usually do, we filter the solvents, sonicate, purge the system, wash the column with water for 15min at 1mL/min, wash the column with mobile phase for 15 min at 1mL/min, do baseline stability and then if the baseline is passed we proceed to the analysis. Except for that we dont have on line degasser.

Thanks

Hi Biokemist89,
Please let me understand your problem completely. Could you please provide me with the following:
1. Mobile use and composition - isocratic or gradient?
2. Column use - phase, lenght, particle size?
3. Detection use? what wavelenght?
4. flowrate?
5. Injection volume?
It will also help if you could send me copy of chromatogram.

Ive used HPLC before in our sister company, and i also experienced that kind of problem. Please try to clean your column with isopropyl alcohol, use deionized water instead of distilled water, and maintain the temperature of your culumn. I knew these things during the LabTec seminar here in Cebu.

As for the method validation, there are guides on how to validate your the method. You can find it in the internet. my suggestion, run your method as many times as you can. And try to evaluate your result, if it can be repeated (repeatability) and can be reproduced (reproducibility).

tnx

for how long will i wash the column with IPA?



thanx

30 mins of ipa is good enough, right after that, wash it with deionized water, 30 mins also. Pls use only deionized water in washing and in diluting the sample if you are going to dilute it.