I used c18 and platinum columns and all drugs appeared at the same retention time.
By using different mobile phases, aqueous:organic 90:10 v/v. Acetate buffer pH 4 was used also but no separation was observed.
I tried ion pair reagent and B_CD and they were not effective.
The same retention times are the problem.
The easiest way would be to switch to gradient separations. They are much more powerful and easier to establish. If you do not have a gradient pump, you have to optimize your solvent mixture carefully (different solvent/water ratios). If you find for your analytes an isocratic method in the literature, you may use this.
Hydrophilic interaction chromatography (HILIC) is a liquid chromatography technique when a polar stationary phases — silica or polar bonded phases and aqueous mobile phase containing amount of a less polar solvent (often acetonitrile) is applied. HILIC is important for the separation of highly polar substances including biologically active compounds, such as drugs, amino acids, peptides, proteins, nucleosides, neurotransmitters, etc. HILIC is also the appropriate method for analysis of plant extract polar components such as carbohydrates, amino acids, peptides, phenolic acids, flavonoids and some alkaloids. Plant extracts are usually multicomponent mixtures of compounds of wide polarity range, which often cannot be analyzed by use of a single separation and detection method because of the high chemical diversity. Good results are obtained by use of coupled method, e.g., reversed-phase liquid chromatography (RP-LC) and HILIC mode separation. The elution order in HILIC is usually opposite to that in RP separations. This orthogonality determines one of the advantages of HILIC — the possibility of creating multidimensional separations. An important feature of HILIC is the improved sensitivity with electrospray mass spectrometry. This is significant for the analysis of components existing in small concentration in multicomponent mixtures. The high acetonitrile content also gives additional advantage of HILIC — faster separations due to the lower viscosity of HILIC eluents compared to standard RP ones.
First of all your mobile phase should be strong polar check the composition by changing ratio, and also vary the flow rate.Then check the effect of buffer and examine optimum condition in basic medium there will be better results.
Using a reversed phase column, and mobile phase: Acetonitrile or methanol : Phosphate buffer pH 3.5 or 4, where phosphate buffer is better, but concerning the acidic pH, as the amidic drug may be not stable in alkaline pH.
Kind regards
Thanks alot dr. Abobakr. Your suggestion had been tried before I ask.
Regards.
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