Chromatography Forum

Hello friends-
I am running a two-component matrix. Two analytes. One is Octinoxate (OMC) and the other is Avobenzone (AVB).
Octinoxate comes out in 4.8 minutes and the Avobenzone comes in 6.5 minutes.
Anyway, the first couple of sets of analysis that I have run, my OMC peak had much higher response than the AVB peak. (In assay analysis OMC should be around 6%, and AVB is about 2%).
The last two times it's the opposite!- The AVB peak (The second peak that comes in 6.5 minutes with 2% analyte) is much higher. It is very strange!
The peak shapes are good. No problem whatsoever with the peak shapes-
The area counts do not make a difference and the results are acceptable. They are in range.
My detector intensity is ok, and my lamp intensity is good as well. My system suitability has passed in every single time. - I've checked it.
The lamp has only 130 hours on it.
The detector range changes from 310 nm to 357 nm in 5.5 minutes, so it can detect both analytes at given retention times,
My HPLC is Dionex P680 with ASI 100 and PDA 100 Photodiode Array detector
My column is RP Supelco 15 cm x 4.6mm x 5 mm
My flow rate is about 1 ml/min
My mobile phase composition is 90: 8: 2 (Methanol: Water: 0.5% H3 PO4)
My Chromatography software is Chromeleon
Solvents look good- I filter them on regular basis- But it's been a mystery problem- Thought anyone would have an idea what's happening?

Now that OMC peak is smaller- The software doesn't integrate the first peak properly and my calibration for the first analyte is not perfect. I changed the signal to a smaller value, so it can integrate it properly.
I can run 3-point calibration and the data looks ok for the first analyte. It is perfect for the second one.
However; If I ran 4 point calibration, the highest calibration point for the first analyte comes above the linear regression curve, and henceforth; my results on the first analyte comes lower than it should be. No problem with the second analyte calibration.
I ran recoveries before and they came out ok- Not between 98-102% but consistent-
I have no idea why this is happening-

Any word of wisdom why this is acting weird?
I would appreciate it very much

Thank you

is your curve made of one concentration or is it a multi level curve?
from what i have understood you are doing a multi level calibration.

what are your standard concentration % ratio for each compound compare to the analyte.

as for the difference in the peak response for both compounds; explaining that depends on many factors which are related to your samples and sample preparation.
were those injections of standards or unknown samples?

Dear Unmgvar
The curve is based on 4 point calibration
My Standards for OMC were 6.00 % W/W, and 2.0 % W/W for AVB.
The results for both of these analytes came around 5.898% and 1.93% respectively.
Like I said, near perfect calibration for AVB but not so perfect for OMC
( The first peak)
Samples are made in MeOH and then get sonicated for a while.
Notice- I didn't have this problem before. I kept everything same since beginning of the method development.
Does this have anything to do with the detector shifting the wavelength?
The peaks from injections are all behaving the same way ( Standards and Samples)- Another words- This probelm starts from the beginning of the sequence till the end

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You should be able to tell from the UV spectra if wavelength shifting could lead to the problem that you describe. If you have the flexibility, just use another UV detector or you can put an ELSD in tandem in order to make sure that it is your detector that malfunctioning and not something else...

Or you could try some runs at 310 nm and just check for OMC. This would eliminate the wavelength change as a problem.

mijnadarian,


can you send me the sequence as a backup file so that i will open it with my chromeleon?
pictures are worth 1000's of words
this way it will be easier for me to help you.