Buffer. Ensure that the buffer (pK a ) is appropriate for the pH of the solution.

In general, to provide appropriate buffering capacity, the pH of the solution

should be within ± 1 pH unit of the pK a value of the buffer.

žIsocratic method. Whenever possible, use isocratic HPLC condition, as this

is affected less by the variation in flow rate, temperature, and dwell vol-

ume. If gradient HPLC conditions have to be used, a simple linear gradient

is preferred over multistep gradients. Complicated gradient conditions are

more susceptible to differences between HPLC instruments (e.g., flow rate,

dwell volume).