Use of Trialkyl Ammonium Phosphate (TAAP) Buffers in Reverse Phase HPLC for High Resolution and High Recovery of Peptides and Proteins

Abstract

A new buffer system compatible with reverse phase high pressure liquid chromatography (RP -HPLC) column packings and giving high recoveries and high resolution for peptides and proteins is described. The TEAP buffer (triethylammonium phosphate), one of several TAAP buffers (trialkylammonium phosphate) compatible with RP-HPLC, gives high resolution and high recovery for closely related peptides or proteins as well as day to day reproducibility. It is UV transparent to less than 200 nm, extremely versatile, and could probably be used for several other classes of compounds (phosphatidyl cholines and sphingomyelin, for example). Furthermore, this system is compatible with in vitro and in vivo biological systems after removal of the organic solvent. We have applied our system to the purification of radioactive peptides, i.e. the separation and total recovery of tritiated luteinizing hormone releasing factor (LRF) from closely related radioactive impurities. Peptides and proteins as different as LRF, somatostatin, insulin and cytochrome C are easily separated and recovered. Application to peptide mapping is also described. The influence of flow rate, temperature and column packings on resolution is discussed and illustrated. Linearity between load and integrated response is demonstrated over four order of magnitude while retention time for a particular component remains constant. A flow rate of 1–1.5 ml/min and ambient or lower temperatures gives optimal resolution. Furthermore, it was found that the higher the hydrophobicities of the support was, the higher the resolution, but also possible the higher the chances for peptide or protein loss.