Hello everyone.
I have a problem with HPLC. A few days ago, to create a calibration curve with HPLC, I injected solutions with known concentrations into the device, which gave good peaks in the expected time. But now I inject the solution with the unknown concentration into the device with the same HPLC conditions as before and the device does not show a peak. The filtered sample contains sodium sulfite, potassium peroxymonosulfate, and chloroquine.
Operating conditions: HPLC Waters with C18 column, PDA detector at a wavelength of 340 nm, water and acetonitrile as solvents with a ratio of 90:10 and isocratic method, flow rate 1 ml/min, and injection volume is 20 microliters.
I have checked all the connections for leaks and flushed the column with water and methanol, the system is free of air, the flow path is completely open and the waste is coming out, but I still did not see the peak in the expected time.
Not enough information provided (e.g. Column dimensions, chromatogram, wavelength bandwidth and spectral settings...). Perhaps the actual concentration of your sample is too low to be detected (try a 10-fold increase)? Maybe the method used does not follow good chromatographjy fundamentals and is not selective for the sample or is still on the column OR eluting at the void volume (have the method reviewed by n experienced chromatographer). The sample's matrix may be interfering with the analysis (perhaps sample prep is needed?).
*So many possible reasons and this is best explored by an experienced chromatographer on-site (where your instrument and method setup can be evaluated), not via the web. For the fastest solution to the problem, please contact an experienced industrial chromatographer at your school or local laboratory for assistance.
Where did anyone get the 'dumb' idea to use Potassium Peroxymonosulfate?It will degrade your Chloroquine through oxidation. See these articles;
https://www.researchgate.net/search.Search.html?query=potassium+peroxymonosulfate+and+chloroquine&type=publication
maybe your analyte is not present in the sample, or you have a matrix interference. try to inject a fortified sample ( add standard to your sample) and see what happens.
Hello spike your sample with know concentration of analyte and see the results with the use calibration curve that you made.
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