why is the peak area of standard decreasing?

[moonchips]

The peak area of my standard (BKF) kept decreasing in the same sequence. I have one standard injection between every three sample injections. The peak area decreased about 5% for every standard injection.

Can any one help me to troubleshoot this problem?

Thanks a lot!

[AdrianF]

I am sure we can help solve your problem but we need more details.
eg did the sample peaks decrease as well?
type of compound ?
volume injected?
make of autosampler?
column?
mobile phase?
Gradient/isocratic? etc

[bartjoosen]

Temperature of sample chamber?
Solvent used? Solubility in the solvent?

[moonchips]

Thanks for your answers.
did the sample peaks decrease as well? Yes, eventhough samples are from different preparation, there was a decreasing trend.

type of compound ?
antioxidant. BKF(might be light sensitive). this is the link to the compound:
http://webbook.nist.gov/cgi/cbook.cgi?Name=BKF&Units=SI

volume injected? 30uL

make of autosampler?
Agilent 1100 series: G1313A

column?
Thermo Hypersil ODS C18, 5 µm, 120 Å 150 mm*4.6 mm

mobile phase? water/ACN
Gradient/isocratic? etc

Gradient:
Time water ACN
0 50 50
11 0 100
25 0 100
30 50 50
35 50 50


Temperature of sample chamber? 60C

Solvent used? Solubility in the solvent? Isopropanol, at least 100ug/mL, my standard is 1ug/mL.

thanks

[bartjoosen]

60C as a temperature for the sample chamber?
Isn't it possible that your samples are decreasing due to degradation?
If you make a new sample prep, after a few hours, is the area back to normal, or still decreased?

[moonchips]

Yes, 60 C is high.
If sample is degrading, would it be the same degree of degradation for all injections?

I thought my sample might be light sensitive. I am going to compare the result using aluminum foil to protect the vial with results without light protection. Also, I am going to compare the difference of capping the vial with not capping the vial to check solubility issue.

[moonchips]

Is it posible that is due to the column or the injector is "dirty". By "dirty", I mean there is small amount of sample left in the injector or inside the column, and the sequential injection would be affected, because my sample is easier to condense at the place where there is trace amount of left over from last injection.
So signal would be smaller and smaller as the injector or the comlumn gets dirtier and dirtier.

[sassman]

I assume you are using UV (or PDA) detection? You said the sample chamber is at 60C. Do you mean that the column is at 60C? By sample chamber, I think they were referring to the tray that holds your sample vials. I am not sure why you would want to keep this at 60C, unless there are solubility issues.

What happens if you shut the instrument down, and try to rerun the sample the next day? Is the response back to normal?

[moonchips]

oh, I misunderstood.
60 C is column temperature.
Sample chamber is room temperature and not controlled. Sample chamber is not light protected either.
yes, it is a nice suggestion to run the same sample the second day. I will try

[danko]

Hi moonchips,

Do you see additional peaks; indicating degradation over time (whether the potential degradation is light induced or otherwise, is not that important at this stage – not until you know that this is the actual issue).

I’ve seen it many times; people examine the chromatogram pretty thoroughly for new peaks, but they forget to check out the injection peak/disturbance. Often the degadants are much more polar and thus elute unobserved with the dead volume.

Best Regards

[max_planck]

Are you using activated hydrolizing HPLC vials? This might be your problem.

[moonchips]

After a long time wash and equilibration, I ran the same sample the second day, and the peak area came back to normal (peak area was 14 yesterday and today it was 21). So it should be some kind of adsorption, right? How to solve this kind of problem?

danko,
I did see a tiny peak today at 7.5 min today, which wasn't there yesterday. There is degradation, but it is not the major problem.

What is activated hydrolizing HPLC vial?

[ym3142]

Moonchips,

I guess you did not see similar decrease in consequtive std inj assuming the std does not have similar matrix to sample. This only happened after sample was injected.

Find a PDA/DAD detector, check UV of all the injections, I predict you will see different UV spectra.

IF SO, wash the UV detector using proper solvent.

Good Luck.

[moonchips]

Sorry, I made confusion.

I injected the same std today as yesterday. I did not inject any samples today.

My detector might be dirty

[ym3142]

did you or didn't you see the decreasing with only std injections?

thanks for clarifying the confusion.