Investigation of the adsorption mechanism of a peptide in reversed phase liquid chromatography, from pH controlled and uncontrolled solutions.

The single-component equilibrium adsorption of the tripeptide Leucyl-Leucyl-Leucine (LLL) on a high-efficiency Jupiter Proteo column (C(12)) was investigated experimentally and modeled theoretically. The experimental equilibrium isotherms of LLL for adsorption on a C(12) packing material from an aqueous solution of methanol (48%) and trifluoroacetic acid (0.1%) were measured by frontal analysis (FA). The FA measurements were done with two solutions, one in which the pH was controlled, the other in which it was not. Two solutions of LLL in the mobile phase were prepared (4.3 and 5.4 g/L) and their pH measured (2.94 and 2.88), respectively. The first solution was titrated with TFA to match the pH of the mobile phase (2.03), so its pH was controlled. The pH of the other solution was left uncontrolled. In both cases the isotherms could be modeled by a bi-Langmuir equation, a choice consistent with the bimodal affinity energy distribution (AED) obtained for LLL. The isotherm parameters derived from the inverse method (IM) of isotherm determination under controlled pH conditions (by fitting calculated profiles to experimental breakthrough profiles) are in a good agreement with those derived from the FA data. Under uncontrolled pH conditions, the application of IM suggests the coexistence of two different adsorption mechanisms. According to the isotherm parameters found by these three methods (FA, AED and IM), the C(12)-bonded silica can adsorb around 500 and 70 g/L of LLL under controlled and uncontrolled pH conditions, respectively. The adsorption of LLL on the C(12) material strongly depends on the pH of the mobile phase and on the quantity of TFA added, which plays the role of an ion-pairing agent.