Chromatography Forum

I know this is can be a foolish question, but I do want to know in order to solve my problem.
I have read a book that if I want an acid to increase its RT just low the buffer's pH less than its pKa 2 unit so that it is in the neutral form and on contrast I will increase the buffer's pH more than the pKa of a base to make sure it become neutral.
1. So firstly, I would like to ask if my knowledge is right and are there exceptions?
2. My next question is about pKa, many substances have more than one pKa. Acetylcysteine is an example, It has the pKa 4 and 9.5, if I want to use it with the pKa 4 or 9.5, how can I control that. Please help me!!!

1. So firstly, I would like to ask if my knowledge is right and are there exceptions?

There are exceptions to *everything*, but your explanation is quite generally correct.

2. My next question is about pKa, many substances have more than one pKa. Acetylcysteine is an example, It has the pKa 4 and 9.5, if I want to use it with the pKa 4 or 9.5, how can I control that. Please help me!!!

N-acetylcysteine has two ionizable groups: a carboxyl (pKa 3.2) and a thiol (pKa 9.5). So:

• below 3.2, the molecule is neutral
  between 3.2 and 9.5 the molecule is -1 charge
  above 9.5 the molecule is -2 charge

The transitions occur over a +/- 1-2 pH unit range on either side of the pKa (+/- 1 pH unit goes from 10% to 90% ionization; +/- 2 pH units goes from 1% to 99% ionization).

You pick the pH depending on what charge you want on the molecule.

Are you trying to lower pH of the mobile phase to obtain retention on reversed-phase column?

I think lower pH incase of acid compoumds will increase the RT,if we use the low pH with the base,we will have the opposite effect.
And I kind of don't understand Mr.Tom Jupile's explanation of Ibuprofen since according to my above post: the pH of buffer(<3,5) lower than pKa of COOH will make it neutral (I got it),but how about the Thiol with pKa 9,2.How can it be neutral with that buffer?

For acids, the pKa is the pH below which the protonated form (free acid; neutral) predominates and above which the deprotonated form (anion; negative) form predominates.

The carboxyl and the thiol are both acids (proton donors). Below pH 3.2, both of them are protonated, so the overall molecule has no charge. At pH 3.2, the carboxyl gives away its proton, but the thiol still retains *its* proton, so the overall molecule has a charge of -1. Above pH 9.2, the thiol also gives up its proton, so the overall molecule has a charge of -2.