I am a beginner at HPLC and I am wondering 1) what structural components of compounds i.e. size and polarity can cause discrepancies in peak area of two different analytes with the same concentration and 2) how exactly they affect peak area. I ran HPLC for succinic and tartaric acid using standards of the same concentrations of each acid, but found that succinic acid overall (less polar than tartaric) has a larger magnitude of peak area. Is peak area also affected by succinic acid's longer retention time?
My standard curve for succinic acid is y = 8.2234x + 77.98 and my standard curve for tartaric acid is y = 3.207x + 25.014. These curves were made using essentially the same concentration standards.
Hi Jamie , peak area is depend on the structure of compounds and the response of detector. What is the kind of detector you used ?
We used a UV detector^ I was hoping to know what specific properties of the structure would impact succinic acid's larger magnitude of peak area.
The detector type, sample solubility, concentration present and METHOD employed (the details of which you did not share with us) determine what type of output signal you will observe.
Why are there difference in the signal for two compounds? It is because you used a UV detector that you saw such wide variations. Both compounds have very weak UV absorption. The wavelength selected also contributes to the overall signal as well and for many samples it is key to use the best wavelength (Lambda max) to detect them (though it will not matter much for your samples as only ~ 210, 4 nm would be useful since both have very poor absorption at any standard UV wavelengths. Just look at their structures!). Yes, size, polarity and bonds contribute to measured signal, but the specific method employed needs to be selective for them in the first place to provide data that is worth comparing.
Hi Jamie, it also depends on the extinction coefficient of your compounds. One compound could be much more sensitive than the other (show a much larger peak area) at the same concentration, if the compounds absorbs UV light more strongly.
However, as William Letter mentioned, these two compounds have pretty low absorbance (I look at these all the time, but use LC-MS instead, UV isn't sensitive enough) the only thing you will see if the absorbance from the carboxylic acid group. You can see them, but it will probably take a fairly high concentration.
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