HPLC

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Tirumal Rao
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HPLC

I have recently read an article that recomends to use of mobile phases with an acidic pH for acidic components it was suggested that using acidic mobile phases reduce the ionization of the acidic analysis and the silinols this is supposed to the imrove the peak shape and reduce tailing can u explain the reason ?

Dr. Analytical
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The article is correct. Here

The article is correct. Here is an explanation:

Consider the ionization equilibrium for an acidic compound (HA).

HA + H2O <-> A- + H3O+

In reversed phase chromatography, more polar compounds elute first, followed by less polar compounds. The ionic form (A-) is more polar than the neutral form (HA), so it would elute faster. If A is a smaller molecule, A- may not be retained at all; it would elute in the void volume.

So, if we want more retention, we should try to put the molecule in its neutral form. Since the ionization is an equilibrium, if we add acid to the solution (the H3O+ on the right side of the equation), the equilibrium will be forced to the left, producing mostly HA. If we make the pH of the solution at least two units less than the pKa for the acid, almost all of the acid will be in its neutral form. Usually you just need to make the solution acidic; the exact pH does not matter as long as it is much smaller than the pKa.

If the pH is near the pKa of the acid, then both forms will be present in the column. Since the retention behavior of each form is different, the result is often a broad or tailed peak.

The same situation exists for basic compounds. In this case, the pH must be greater than the pKa to keep the base in its neutral form. However, many LC stationary phases are not stable in basic solution, so we have to try other methods.

If you have any more questions, just post them here.

Dr. Analytical
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I wanted to add another

I wanted to add another comment about silanols.

The surface of silica is covered with these silanols (SiOH). They are weak acids. Thus,

SiOH + H2O <-> SiO- + H3O+

The pKa is around 4 - 6. So at or above this pH, the surface has a negative charge, and will behave a little bit like an ion exchange material. This means there is another separation mechanism on the surface, and this can also cause peak shape problems, especially if the analytes have a positive charge.

Making the mobile phase acidic will eliminate the ionization, putting the silanols in their neutral form.

Tirumal Rao
Tirumal Rao's picture
How should i selective

How should i selective digestive enzymes for peptide mapping ? presently i am using SV8 Proteiase for my protein?