Information on HPLC method development required

[josephkj]

I have only little idea regarding hplc method development can any body explain me in detail.Considering the following aspects- if the structure of the molecule is known then how to proceed?
1. Which mobile phase I have to select?

2. What buffer I have to use?

3. Which column I have to use?

4. Which wavelength I have to use?

5. In what concentration I have to inject the sample(do I have to do
linearity study)?

6. If HPLC is not having PDA how to fix the wavelength should we
use UV-VIS spectrophotometer?If yes in which solvent I have to
dissolve the compund and find its lamda maximum?

7. If there is a variety of columns to select which one I will
choose?

8. During method development if peak tailing is observed how to
improve the peak shape ? can this be improved by changing the
buffer concentration or changing the buffer?What is the
role of buffer for the improvement in peak shape?

9. Do I have to look for pKa values if so how? what is the
influence ? explain in detail

10.Why buffers are used in hplc mobile phase?

[Uwe Neue]

Contact me, I can send you an article that was written for a textbook. Alternatively, you can get it from your library:

U. D. Neue, E. S. Grumbach, J. R. Mazzeo, K. Tran, D. M. Wagrowski-Diehl, “Method development in reversed-phase chromatographyâ€