In preparation of standard and sample solutions in HPLC, diluent is used (usually mobile phase), but I have noticed sometimes it is not working to use the same mobile phase (with same pH) as diluent for standards and samples and this is due to pH.
Dear Fuad,
it is not necessary to use the eluent mixture for the preparation of standards. The injected volume (sample) is smaller than the volume of eluent during the running in the column. You should decide the solvent to be used and its pH considering the chemical structure of your analytes. E.g, if you are using a C18 column it is necessary to tranform ionic substances in acidic ones adding the quantity of acid to reach the desired pH value. The acidification of the eluent is needed too, but the value of its pH can result different from the pH used for the preparation of your sample. For this reason you must find the best conditions injecting the standard solution prepared using a range of pH around the pKa of your analyte.
Thanks Dr. Sabino for these valuable information. I believe that acidification of eluent is a good strategy to get sharp peaks.
OK, you are right. An analytical strategy is always requested!
Best wishes
Sabino
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