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

Journal Article · Thu Jan 01 04:00:00 EST 2009 · Journal of Chromatography A

OSTI ID:1041461

Gritti, Fabrice ^[1]; Guiochon, Georges A ^[2]

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1. University of Tennessee, Knoxville (UTK)
2. ORNL

The single-component equilibrium adsorption of the tripeptide Leucyl-Leucyl-Leucine (LLL) on a high-efficiency Jupiter Proteo column (C{sub 12}) was investigated experimentally and modeled theoretically. The experimental equilibrium isotherms of LLL for adsorption on a C{sub 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{sub 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{sub 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.

• Research Organization: Oak Ridge National Laboratory (ORNL)
• Sponsoring Organization: USDOE
• DOE Contract Number: AC05-00OR22725
• OSTI ID: 1041461
• Journal Information: Journal of Chromatography A, Journal Name: Journal of Chromatography A Journal Issue: 18 Vol. 1216; ISSN 0021-9673; ISSN JOCRAM
• Country of Publication: United States
• Language: English

Related Subjects

10 SYNTHETIC FUELS
ADSORPTION
AFFINITY
AQUEOUS SOLUTIONS
CHROMATOGRAPHY
ENERGY SPECTRA
ISOTHERMS
METHANOL
PEPTIDES
SILICA