Dear Ioannis Anastasopoulos,

The pH of the mobile phase can affect chromatography in a number of ways.

Depending on the compound being analyzed, pH can impact selectivity, peak shape, and retention. With a fairly nonpolar or neutral compound, the effect of pH will typically be insignificant for resolution and retention. When considering method development with ionizable analytes, it is important to understand that ion-suppressed analytes have better retention than ionized analytes. For acidic analytes, choose a low-mobile-phase buffered pH to keep the analytes from being ionized. Knowing the pKa of the analytes permits an effective choice of mobile phase pH. A buffer is effective at ± one pH units from the pK of the buffering ion, giving some flexibility in optimizing the mobile phase. With basic compounds, the ion-suppressed form may be at a high pH that is not

suitable for the column. However, many basic compounds are adequately retained at low pH. While greater retention can be achieved in an ion-suppressed form, this may not be practical or necessary for all basic compounds. An additional issue impacting retention of acidic and basic compounds is the potential ionization of silanols on the silica surface at mid pH. Typically, these silanols become deprotonated and, hence, negatively charged. This may result in more retention for positively-charged, basic ions, which can result in ion-exchange interactions, a type of secondary interaction. The end result is often peak broadening or peak tailing due to an interaction other than the partitioning that is expected with a reversed-phase column. This does not happen at low pH and is another reason why acidic mobile phases are preferred for the separation of ionizable compounds by reversed-phase chromatography.

A general approach to the separation of the mixtures containing an ionisible components is to suppress their ionization. Suppression of the ionization decreases a power of the molecular solvation and exposes the hydrophobic (organic) part of the molecule to the surface interaction. Ionization suppression is usually made by the adding a buffer into the solvent, which shift a pH to the certain value.

In the absence of buffer, easy ionizible components are eluted from the column as very broad peaks. According to the Le Chatelier principle, dissolved ionizible component is present in the solution as a mixture of ions and nonionized molecules

[AB] == [A+] + [B-]

According to the above equilibrium, about 50% of all molecules are ionized in the solution . But, the chromatographic behavior of ions and neutral molecules are different. Let us assume that neutral molecules will be retained, so during the run ions will move faster, and at the first moment they will be separated from the neutral molecules. But, according to the above equilibrium, in the absence of the neutral molecules ions will tend to form them, and this new neutral molecule will also be absorbed, and so on. This process will lead to the spreading of the component along the column and causes the appearance of the broad peak. It  does not occur if the equilibrium is shifted due to the presence of the buffer with the pH at least two units apart of pK of the component.

For more details, you can use the following links:

http://www.agilent.com/cs/library/technicaloverviews/public/5990-9984EN.pdf

http://www.chromacademy.com/essential_guide_webcast/mobile_phase_optimization_strategies_for_reversed_phase_hplc/mobile_phase_optimization_strategies_for_reversed_phase_hplc.pdf

http://quimica.udea.edu.co/~carlopez/cromatohplc/importancia_ph_fase_movil_hplc.pdf

http://hplc.chem.shu.edu/NEW/HPLC_Book/Theory/th_mobph.html

Hoping this will be helpful,

Rafik

appreciate for your help