repetitive mesurement

[fredj]

Dear All,
I am trying to make the separation of porphyrins extracted from biological samples. Using the same conditions I can't succeed to have the same chromatogram, there is a difference in the peaks intensities and areas...knowing that I am using the same buffer, the same oven temperature and the same method...
Please if anyone have an idea about this problems or any suggestions let me know I'll be so grateful
cheers

[tom jupille]

Some further questions:

1. If you do repetitive injections of the same sample (from the same vial), do you also see the variations? (if not, then you may be looking at sample to sample differences or sample prep issues)

2. Are the variations over a series of injections systematic (i.e., all trending in one direction) or random?

3. Do the retention times of key peaks also vary or is it just the areas?

[fredj]

concerning the first question: yes I see a variation from the same sample injection. I votex the sample before the injection
2. the variations are rondom
3.the retention time of the front peak dosen't change over the different injection but my key peaks change the retention time and the areas...

[prepcolumn]

may it be because of the pKa of the porphyrins and the pH of your mobile phase? If they are so close to each other this may cause rapid differences in retention.

[tom jupille]

prepcolumn beat me to it, but it's not a sample prep issue. The fact that the retention varies points the finger at something chemical. Temperature can't be ruled out entirely, but that would usually have a diurnal pattern to it (e.g., changing gradually during the day). The most likely culprit is lack of robustness with respect to pH. Sometimes increasing the buffer concentration can help.