Chromatography Forum

I use a relatively new normal phase HPLC column for the analysis of tocopherols and I have observed that progressively the peak areas of samples and standards within a day become lower using both DAD and fluorescence detectors on-line. Due to various system problems we initially though something was happening with autosampler. We replaced it by a manual injection vale and the same thing happended. We measured the outflow volume to see if we had somewhere any leak but the volume corresponded to the expected one for the specified flow. We then used 2 old columns (lack of a new one) with higher pressure and the peak areas of the fresh sample and standard were relatively close to expected values (the new one gives half peak area). Is there a possible explanation for such a behaviour? If somebody could assist I appreciate it.

Hello Nikolas,
Before the readers can provide any explanation, they need to know the following critical information?
1. What is your column material?
2. What is your mobile phase?

Thanks.

the column matterial is Si-100 5u Licrosphere and the mobile phase hexane-isopropanol 99/1 v/v flow rate 1.2 ml/min full loop injection 20 ul

the column matterial is Si-100 5u Licrosphere and the mobile phase hexane-isopropanol 99/1 v/v flow rate 1.2 ml/min full loop injection 20 ul

Out of several reasons, one possibility is indeed the injector, which gets contaminated over a period of time. Do you include a needle wash program? I have really dirty injection needles of autosamplers when samples are in non polar mobile phase (the solvent evaporates and leaves a residue). Couple of ideas:

Try the same experiment with something without many functional groups such as toluene or dinitrobenzene as an internal standard (in a test mix). If you see the same trend, it is possible that injector is getting dirty.

Secondly, can you use a relatively stronger mobile phase? It may be possible that something in your column is irreversibly adsorbing the analyte. Is it a neat tocopherol sample or a real sample?

Check after washing (the column+injector) if you can recover the original peak areas.

Thanks, I will think about it. Nevertheless, I include between injections washing. The observation has been made both in neat tocopherol standards and real sample.

Does the area count return in a new run/next day or is it consistently falling?

Possibilities I can think of without going into details.

1. Sample degradation: Maybe you have the sample standing at room temperature and the sample is degrading in the vial?

2. Enviromental effect: The detector(s) could be negatively effected by enviromental changes in the lab? Temperatur/humidity/direct sun light?

3. Chromatography: Peak shape changing over time causing peak width to increase?

4. Noise increasing between runs causing signal intensity to slowly be drowned out in noise?

Like I said.. Just random thoughts that might lead you onto the cause..

Hello,

more than 10 years ago, I fought against a similar phenomenon with tocopherol.

As tocopherol is an antioxidant, it is easily oxidized and my peak areas did decrease due to this oxidative degradation.

I could fix this by addition of ascorbic acid (to sample and standard solution) which is oxidized more easily than tocopherol and acts as a "oxidation protector". From this time point, I never had any issues with decreasing peak areas.

My sample preparation solvent was organic and the ascorbic acid did not dissolve in a visually noticeable manner. Nevertheless, it worked fine.

Regards

Florian

Thank you for the suggestions

Does the area count return in a new run/next day or is it consistently falling?

Possibilities I can think of without going into details.

1. Sample degradation: Maybe you have the sample standing at room temperature and the sample is degrading in the vial?

2. Enviromental effect: The detector(s) could be negatively effected by enviromental changes in the lab? Temperatur/humidity/direct sun light?

3. Chromatography: Peak shape changing over time causing peak width to increase?

4. Noise increasing between runs causing signal intensity to slowly be drowned out in noise?

Like I said.. Just random thoughts that might lead you onto the cause..

Thanks for the suggestions

Thank you for the suggestions

Does the area count return in a new run/next day or is it consistently falling?

Possibilities I can think of without going into details.

1. Sample degradation: Maybe you have the sample standing at room temperature and the sample is degrading in the vial?

2. Enviromental effect: The detector(s) could be negatively effected by enviromental changes in the lab? Temperatur/humidity/direct sun light?

3. Chromatography: Peak shape changing over time causing peak width to increase?

4. Noise increasing between runs causing signal intensity to slowly be drowned out in noise?

Like I said.. Just random thoughts that might lead you onto the cause..