ORIGINAL PAPER Retention modeling under organic modifier gradient conditions in ion-pair reversed-phase chromatography. Application to the separation of a set of underivatized amino acids A. Pappa-Louisi & P. Agrafiotou & K. Papachristos Received: 18 September 2009 / Revised: 4 December 2009 / Accepted: 4 December 2009 / Published online: 26 January 2010 # Springer-Verlag 2010 Abstract The combined effect of the ion-pairing reagent concentration, C ipr , and organic modifier content, 8, on the retention under 8-gradient conditions at different constant C ipr was treated in this study by using two approaches. In the first approach, the prediction of the retention time of a sample solute is based on a direct fitting procedure of a proper retention model to 3-D 8-gradient retention data obtained under the same 8-linear variation but with different slope and time duration of the initial isocratic part and in the presence of various constant C ipr values in the eluent. The second approach is based on a retention model describing the combined effect of C ipr and 8 on the retention of solutes in isocratic mode and consequently analyzes isocratic data obtained in mobile phases containing different C ipr values. The effectiveness of the above approaches was tested in the retention prediction of a mixture of 16 underivatized amino acids using mobile phases containing acetonitrile as organic modifier and sodium dodecyl sulfate as ion-pairing reagent. From these approaches, only the first one gives satisfactory predictions and can be successfully used in optimization of ion-pair chromatographic separations under gradient con- ditions. The failure of the second approach to predict the retention of solutes in the gradient elution mode in the presence of different C ipr values was attributed to slow changes in the distribution equilibrium of ion-pairing reagents caused by 8-variation. Keywords Ion-pair reversed-phase chromatography . Isocratic-gradient retention prediction . Combined organic modifier and ion-pair concentration effect . Underivatized amino acids separation optimization Introduction The analytical strategy that uses lipophilic ions to perform the separation of organic and inorganic ionic and/or ionizable solutes with adequate resolution and reasonable run time on traditional reversed-phase, RP, stationary phase is usually referred to as ion-pair chromatography, IPC, whereas the mobile phase additive is usually termed ion- pairing reagent, IPR. The eluent pH is a key factor to control analyte charge status and selectivity may be basically regulated by both IPR concentration, C ipr , and organic modifier volume fraction, 8, in the eluent. IPC is a valuable separation technique and the practical potential of this technique was increased during the last decade by using newly introduced IPRs, such as the compatible with LC-MS hyphenation perfluorinated car- boxylic acids [1] or chaotropic salts [2]. The mechanism, however, of IPC has been a matter of considerable debate during the last three decades due to the multiplicity of interactions involved in an IPC system [3, 4]. Few retention models are thermodynamic, consistent with fundamental physics that highlights the mechanism governing solute retention in IPC. Instead, conceptually simpler and empir- ical models are widely used in practical work of IPC. The principles of different IPC retention models that have been proposed can be found in two representative review articles [3, 4] and references therein. Presented at the Sixth Instrumental Methods of Analysis Conference (IMA 2009) held in Athens, Greece from 4th to 8th October 2009. A. Pappa-Louisi (*) : P. Agrafiotou : K. Papachristos Laboratory of Physical Chemistry, Department of Chemistry, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece e-mail: apappa@chem.auth.gr Anal Bioanal Chem (2010) 397:2151–2159 DOI 10.1007/s00216-009-3381-9