In an HPLC analysis of different analytes (salicylic acid, salicylamide, and other analytes), a mixture of phosphate buffer pH 4.5 and acetonitrile (gradient) using a C18 column, results in usually good separation of the peaks, HOWEVER in some analyses, the peak of salicylic acid appears not sharp with splitting.
Is there any suggestion to get good peak (sharp) for salicylic acid e.g. composition of mobile phase or pH..etc.
Hard to provide constructive suggestions without a chromatogram and detailed method/sample conditions.... Perhaps a pH closer to 3.0 (for Phosphate buffer, use phosphoric acid and settle on a pH of 3.2) would be a better choice. Proper ionization is important to prevent artifacts.
True peak "Splitting" often implies a void at the column head. It can also be caused by column fouling / sample overload (as in you have not developed a proper wash method to clean and re-equil the column in -between runs or simply injected too much material). The other question you must ask is, do I have co-elution? Maybe the peak is not split but another peak is eluting close to or underneath the same peak? This can be worked out using a DAD (if the material absorbs, as most do) to look at their spectral characteristics (a true split peak must have identical spectra). You can also change the injection volume (lower) to see if the peak shape changes to reveal the 'other' peak.
Dear Fuad Al-Rimawi,
Sharpness or tailing of HPLC peaks may not be related only to mobile phase or pH. Column long, particls size or even manufacturer may also have their influence. Any way, you can have alook at files below.
Acids tend to tail and not give sharp peaks in chromatographic separations. Therefore, I think the best way to quantify these organic acids whether for GC or HPLC analyses is to derivatize these acids; converting them to esters for example. There are commercially available, fast, and reliable derivatizing agents. In addition, splitting of a peak, as on colleague has pointed out does not mean that the acid shows up as two peaks. As a away out, try the acid by itself and see if it comes out as one peak or it splets. I am with what Bill letter said, splitting could be due to several things including amount injected, junk on the column among other things.
Keep the pH below 3.0 use hypersil gold column, it might help. Splitting of peaks might be due to high pH which is favouring ionised and unionised drug. Chek the pka values as well. Keep the pH of mobile phae at least 2.5 unit less than pKa to elute the drugs as uniosed drug. Provide details of method and chromatograms for more explaination.
Hello Fuad, we have an LC MSMS Method for determination of NSAID's in Milk. The pH is 4,5 (Pump A 500mg acetic acid+ 385mg Ammonium Acetate in 1L Water, Pump B 500mg acetic acid+ 385mg Ammonium Acetate in 5mL Water than add 995mL acetonitrile) Gradient is from 90%A to 20%A in 8min. Salicylic acid retention time is 4.7min on a phenomenex Aqua C18 150x2mm 3µ column. Earlier there are only Paracetamol and 4-Acetaminophen eluting. Short after the salicylic acid there is acetylsaicylicacid and we get also a Signal with the same MRM'S like the salicylic acid but I think it is an in source fragmentation of the ASS to SA. So we have a good peak shape with the same pH than in your Setup, but maybe a different column.
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