The Use of Water Structure Breakers, Urea and Guanidium Chloride, New Mobile Phase Modifiers in Reversed-phase Partition High-Performance Liquid Chromatography

Abstract

IntroductionToday, RP-HPLC employing a hydrocarboneous bonded stationary phase, typically an n-octadecyl silanized packing surface with a hydro-organic mobile phase, has been quite popular for the separation of a wide variety of mixtures. The RP-HPLC separation processes are thought to be controlled by the so-called hydrophobic interaction 1 at work on phasepartitioning event of solute molecules between the mobile and stationary phases. Because the retention of solutes depends on the solute-solvent interaction, 2,3 manipulation of the organicsolvent content in the mobile phase is reasonably important. In addition, the stationary phase also plays a highly active role in chromatographic processes. It has been speculated that as the content of an organic solvent in a mobile phase decreases, the bending and tangling of alkyl chains on the particle surface take place through a hydrophobic interaction, 4 which doubtless reduces the separation efficiency. Modifiers in the mobile phase, which are employed for the benefit of separation improvement, strongly affect RP-HPLC processes in two ways: partitioning of the solutes and solvation of the stationary phase.The interaction between the nonpolar moiety of a molecule and water, the so-called hydrophobic interaction, is an important factor in many physicochemical processes: for example, protein denaturation, 5-7 surfactant micellization and absorption. 8 More basically, the slight solubility of hydrocarbon liquids in water is easy to observe but unexpectedly difficult to explain. 9 It is generally accepted that the hydrophobic interaction is due to an increased "ordering" or "structuring" of water molecules in the close vicinity of a nonpolar solute. 10Thus a very common conclusion is that the small solubility of nonpolar solutes in water results from this entropy-unfavorable structuring processes. Here, we define an additive which destroys the structure of pure water as a "water structure breaker", 11 such as urea and guanidium chloride (GuCl). In fact, that the longrange order inherent in water solvent is destroyed in urea solution has been demonstrated using nuclear magnetic resonance spectroscopy. 12 Urea and GuCl are also well-known as powerful protein denaturants. [13][14][15][16] In addition, the solubilities of some hydrocarbons in water increase in the presence of urea. 11,17 An explanation for this phenomenon is that the breakup of a strengthened water structure ("iceberg" structure 18 ) around the nonpolar solutes is quite readily achieved by the "hydrophobic interaction breakers". So far, most investigations of water structure-breaking agents have been focused on their effects on some physicochemical phenomena: micelle formation, 19,20 self-association of dyes, 21 protein denaturation, etc. In contrast, there have been few studies that utilized water structure-breaking agents in separation techniques. Urea has often been used only as a denaturant or an unfolding agent in the hydrophobic interaction chromatography of protein. Organic solvent-free mobile-phase sy...

Effect of Urea on the Ion-Association Capillary Electrophoresis Separation of the Anionic Metal Complexes Using Hydrophobic Tetraalkylammonium as Ion-Association Agent

“…the solute partitioning and the solvation of bonded alkyl chain brush. 9 As shown in Figs. 1b -1e, the peak shape and the resolution of the complexes were altered with the addition of urea.…”

“…On the other hand, it was reported that urea reduced the hydrophobic interaction of the solutes in solution, 5 and that of in the liquid chromatographic separation process. 9 The hydrophobic interaction also makes a large contribution to the ion-association. 3 In the solvation of nonpolar solutes and bulky hydrophobic ionic solutes with nonpolar groups, such as tetraalkylammonium, in water, it has been widely recognized that the hydrophobic hydration shells, which consist of ordered and structured water molecules, are formed around the solutes.…”

“…[6][7][8] We previously found that urea can be used as the new mobile phase modifier in the reversed-phase partition high-performance liquid chromatography (RP-HPLC), and demonstrated an organic solvent-free RP-HPLC system using an aqueous urea solution as a mobile phase. 9 A possible explanation of the effect of urea as a mobile phase modifier in the RP-HPLC process is that it leads to a reduction of the hydrophobic 2010 © The Japan Society for Analytical Chemistry † To whom correspondence should be addressed. The effect of urea as an electrophoretic buffer solution modifier on the ion-association (IA) capillary electrophoresis (CE) separation of four anionic metal complexes of Al(III), Co(III), Cr(III), and Fe(III) with 2,2′-dihydroxyazobenezene-5,5′-disulfonate (DHABS) using a hydrophobic ion-association agent, tetrapentylammonium bromide, was studied.…”

Separation of single‐base sequential isomers of single‐stranded DNA by capillary electrophoresis and its application in the discrimination of single‐base DNA mutations

“…The simultaneous separation of a mixture of a 12‐mer oligonucleotide and all 12 of its possible single‐base substitution mutants has been demonstrated by the proposed method. This separation uses an acidic electrophoretic buffer solution containing a resolution‐enhancing modifier, urea . On the other hand, single nucleotide polymorphism (SNP) is substitution of single bases at specific positions in the genome.…”

Anion exchange selectivity: Study of sorbents with various matrices for separation of chlorine oxoanions and chloroacetic acids

“…It was suggested in [15] that the structure of ions is among important reasons for the ion-exchange selectivity. It is possible that the nature of hydrophobic interaction with the sorbent can be changed by controlling the degree of solvation both for ions being separated and for hydrocarbon constituents of the fi xed phase [16].…”