Chromatography Forum

Dear All,

I would be grateful if you could help me to understand the reason of getting same area at same retention time for different concentrations of nonylphenol.

To begin with, I am using HPLC-UV to identify nonylphenol in soil samples, the method is gradient of ACN and water 50:50 for 1 min then 0:100 for 10 min followed by 50:50 for 10 min. The wave length is 277.

I ran standard concentrations of 1, 10, 100, 1000 and 5000 ng/mL. I got the same peak area at the same retention time (14.58 min) .

Is this a saturation issue or what?

Looking forward to hear from you

Thanks in advance

Please let us know a Little bit about your sample preparation.

The most simple explanation is that the peak your are looking at is not nonylphenol.

The most simple explanation is that the peak your are looking at is not nonylphenol.

Yeah. And are you actually using (and looking to determine) nonylphenol or nonylphenol ethoxylate? Nonylphenol ethoxylate is what has been added to household and laundry products as a nonionic surfactant.

So my guess is that you are trying to determine nonylphenol ethoxylate environmental contamination here.

Dear All,

Thank you so much for your responses, I do agree with all of you that the peak is not for nonylphenol (4-NP). Therefore, today I have tried different (simple) method (ACN 80%: water 20%, wavelength, 230 nm) and I got a peak and the area is quite proportional to the concentration.

Currently, I am testing the standard and the detection limit for this method then I will work with soil samples.

Thanks again and your concern is well appreciated.

We used a little acetic acid in the aqueous, and we used 280nm detection (more specificity).

We used a little acetic acid in the aqueous, and we used 280nm detection (more specificity).

Thanks for sharing your method. I did use 280 nm recently along with 230 nm. However, I found the peak area is higher for 230 compared to 280.
I do not know yet the reason for increasing the response for 230 nm. Anyway, I am using both till I get the limit of detection. I will test both wavelengths in the real soil samples to decide.

We used a little acetic acid in the aqueous, and we used 280nm detection (more specificity).

Thanks for sharing your method. I did use 280 nm recently along with 230 nm. However, I found the peak area is higher for 230 compared to 280.
I do not know yet the reason for increasing the response for 230 nm. Anyway, I am using both till I get the limit of detection. I will test both wavelengths in the real soil samples to decide.

The lower wavelengths will generally provide more absorbance, but also can detect other components in a sample, so the added specificity of the the higher 280nm wavelength typically helps us to detect lower levels because the baseline is better due to added specificity.