Fundamentals of Reversed Phase Chromatography: Thermodynamic and Exothermodynamic Treatment

Abstract

Reversed phase chromatography (RPC) is the most popular branch of HPLC for the analysis and purification of a wide variety of substances. Despite significant advances in both our knowledge and understanding of the fundamental principles governing the retention behavior in RPC, there is considerable debate in the literature regarding the mechanism of retention. This review addresses the theoretical foundation of the chromatographic technique, with an emphasis on thermodynamic and exothermodynamic treatment of retention equilibrium, as well as their implication on the mechanistic aspects of RPC retention. A unified and rigorous treatment based on the solvophobic theory is reviewed in terms of its ability to shed light on the physicochemical underpinnings of the retention in RPC, and to quantitatively predict the retention behavior of nonpolar compounds, acids and bases, and peptides and proteins. Also highlighted are areas of future challenges in the theory and practice of RPC, potentially leading to a better quantitative understanding and use of the popular technique. It is undoubtedly the unsung champion of the modern biological sciences, enabling the intricacies of cellular biology to be at last discriminated in detail. [2] Without the recent advances in HPLC, modern biology, functional genomics, and proteomics would not exist. The incredible power of HPLC to discern the molecular diversity of biological phenomena is largely attributed to its ability to distinguish mass differences as little as 1 Da in a macromolecule, such as proteins when coupled with mass spectrometry, [3,4] to separate proteins that differ by only a single amino acid, [5] to separate conformational isomers of a long chain polypeptide, [6] and to resolve the different tertiary conformational structures of DNA fragments. [7] The exquisite sensitivity, the speed, and the impressive resolving power of modern HPLC find application in various fields, such as pharmaceutical, food and clinical analysis, pollution control, downstream processing, measurement of physicochemical properties of drugs as well as the separation of peptides, proteins, and nucleic acids. At the heart of the HPLC revolution is the mode of separation that we are all familiar with-reversed phase chromatography (RPC). The seminal works of Horváth and co-workers [8 -11] have contributed significantly to the theory and practice of RPC, resulting in its wide acceptance by the scientific community as a high resolution separation technique of choice. It is estimated that approximately 80% of HPLC analysis is conducted in this mode. [12] The success of this technique is attributed to the employment of microparticulate alkyl-silica monomeric bonded phases, such as octadecylated silica, which offers high separation efficiency combined with unparalleled convenience, versatility and reproducibility. The use of bonded hydrocarbonaceous stationary phases having a variety of functional groups and a wide choice of hydroorganic eluents to modulate retention offer a broad range of operating con...

Deviation from van't Hoff dependence in RP‐LC induced by tautomeric interconversion observed for four compounds

“…By means of these regression parameters (a 0 , a 1 , and a 2 ), the thermodynamic parameters of the retention process can be computed as functions of 1/T [16]:…”

Rapid determination of the applicability of hydrophilic interaction chromatography utilizing ACD Labs Log D Suite: A bioanalytical application

“…The reverse-phase separation mechanism is analogous to the extraction of compounds from water into an organic solvent, such as n-octanol [16,[18][19][20][21]. The octanol-water extraction system is a standard tool used to gauge the lipophilicity of a compound and understand partitioning within a biological system.…”

“…where t R is the retention time of the analyte and t 0 is the elution time of the void volume or non-retained components [2,[16][17][18][19]. The Center for Drug Evaluation and Research (CDER) recommends a minimum k value of >2 [17].…”

Determination of thermodynamic parameters in reversed phase chromatography for polyethylene glycols and their methyl ethers in different mobile phases

“…The results described in these studies were, however, obtained with different stationary phases, hence it appeared to be necessary to perform a similar study of retention in different mobile phases on the same column. Moreover, different approaches have been used in the determination of the thermodynamic parameters 7–19. These approaches differ in the calculation of the distribution coefficient (or the retention factor, which is typically used instead of the distribution coefficient).…”