Importance of Mobile Phase Buffer Selection for HPLC to LC-MS

Background:

Considering the more complex products analytical scientist needs to develop the specific, accurate, robust stability indicating analytical method. At the time of method development analytical scientist need to be focus on the development of mass compatible method. At the start of method development, it is important to have systematic information about sample.

Selection of Buffers for HPLC:

It is essential to separate all probable impurities from the degradation sample with minimum run time. Samples are categorized as neutral and ionic while ionic includes acid, base, ampholytic and organic salts. Acidic and basic nature of samples requires buffer content mobile phase and for neutral buffer usually not required. If there is no resolution between 2 closely eluted peak then need to vary solvent strength of mobile phase. Solvent strength and solvent type impact the selectivity of sample peaks. Following are the solvents which control the retention.

During HPLC method development selection of proper buffer is important for the separation of peaks and its symmetry. Following are the common different types of organic/inorganic buffers:

1.    Phosphate Buffers: Potassium phosphate, Di-Potassium phosphate, Monosodium phosphate, Disodium phosphate, Phosphoric acid etc.

2.    Acetate Buffers: Ammonium acetate, Sodium acetate, etc. (Phosphate and acetate buffers are most common because it can be used at wavelengths below 220 nm)

3.    Di-ethyl amine/Tri-ethyl amine buffers

Ion Pairing reagents buffers like tetrabutyl ammonium hydrogen sulfate, Butane sulfonic acid, Pentane sulfonic acid, Hexane sulfonic acid, Heptane sulfonic acids, etc. Ideally 0.0005 M to 0.02 M conc. is recommended with dedicated HPLC column with proper cleaning method after every usage to improve the life of column