Adsorption at the Water/Hydrophobe Interface versus Partition into the Interior of the Hydrophobe: Quantitative Evaluation of the Solute Retention Selectivity at the Water/Hydrocarbon Interface

Molecular level understanding of the chemistry at the water/hydrophobe interface is crucial to chemical separation processes in aqueous media, such as reversed-phase liquid chromatography and solid-phase extraction. However, direct observation of the behavior of molecules and ions at the interface in reversed-phase separation systems still remains a major challenge, and probing techniques that provide the spatial information of the distribution of molecules and ions are required. In this paper, the molecular distribution between the aqueous solution and alkyl-bonded silica particles is studied by surface-bubble-modulated liquid chromatography (SBMLC). We determine the distribution coefficients of various organic compounds referring to their accumulations onto the water/alkyl chain interface and into the alkyl chain layer from the bulk water by SBMLC. The bulk water-to-alkyl chain layer distribution coefficient is corrected for the contribution of the end-capped silica surface to the solute retention using end-capped C₈- and C₁₈-bonded silica columns. The experimental data provide a picture of the spatial distribution of organic molecules in alkyl-bonded silica particles exposed to water. It has been revealed that the water/alkyl chain interface exhibits an accumulation selectivity for organic compounds which is quite different from that of the interior of the alkyl chain layer and the overall separation selectivity of the reversed-phase systems is determined by the relative sizes of the aqueous/hydrophobe interface and the hydrophobe

New Molecular Motif for Recognizing Sialic Acid Using Emissive Lanthanide–Macrocyclic Polyazacarboxylate Complexes: Deprotonation of a Coordinated Water Molecule Controls Specific Binding

A new molecular motiflanthanide–macrocyclic polyazacarboxylate hexadentate complexes, Ln³⁺-ABNOTAwas found to specifically bind to sialic acid with strong emission enhancement and high affinity. The selectivity toward sialic acid over other monosaccharides was one of the highest among artificial receptors. Also, the novel binding mechanism was investigated in detail; binding selectivity is controlled by interactions between sialic acid and both the central metal and a hydroxyl group produced by deprotonation of a coordinated water molecule in the Ln³⁺ complex

Surface-Bubble-Modulated Liquid Chromatography: A New Approach for Manipulation of Chromatographic Retention and Investigation of Solute Distribution at Water/Hydrophobic Interfaces

In this paper, we present a new chromatographic method termed surface-bubble-modulated liquid chromatography (SBMLC), that has a hybrid separation medium incorporated with surface nanobubbles. Nanobubbles or nanoscale gas phases can be fixed at the interface between water and a hydrophobic material by delivering water into a dry column packed with a nanoporous material. The incorporation of a gas phase at the hydrophobic surface leads to the formation of the hybrid separation system consisting of the gas phase, hydrophobic moieties, and the water/hydrophobic interface or the interfacial water. One can change the volume of the gas phase by pressure applied to the column, which in turn alters the area of water/hydrophobic interface or the volume of the interfacial water, while the amount of the hydrophobic moiety remains constant. Therefore, this strategy provides a novel technique not only for manipulating the separation selectivity by pressure but also for elucidating the mechanism of accumulation or retention of solute compounds in aqueous solutions by a hydrophobic material. We evaluate the contributions of the interfacial water at the surface of an octadecyl bonded silica and the bonded layer itself to the retention of various solute compounds in aqueous solutions on the column packed with the material by SBMLC. The results show that the interfacial water formed at the hydrophobic surface has a key role in retention even though its volume is rather small. The manipulation of the separation selectivity of SBMLC for some organic compounds by pressure is demonstrated

Determination of the <i>cis</i>–<i>trans</i> Isomerization Barriers of l‑Alanyl‑l‑proline in Aqueous Solutions and at Water/Hydrophobic Interfaces by On-Line Temperature-Jump Relaxation HPLC and Dynamic On-Column Reaction HPLC

Proline <i>cis</i>–<i>trans</i> isomerization is known to play a key role in the rate-determining steps of protein folding. It is thus very important to understand the influence of environments, not only bulk solutions but also microenvironments such as interfaces, on the isomerization reaction of proline peptides. Here we present two HPLC methods for measurements of kinetic and equilibrium parameters for the isomerization reactions in bulk solutions and at liquid/solid interfaces. On-line temperature-jump relaxation HPLC (T-jump HPLC) allows the determination of forward and reverse rate constants of the isomerization in a bulk solution by monitoring the whole time course of conversion of pure isomers from both sides of the reaction, in contrast to other HPLC and capillary zone electrophoresis as well as spectrometric and calorimetric methods, which use a mixture of the isomers. We can then determine <i>cis</i>–<i>trans</i> isomerization barriers of the peptide at liquid/solid interfaces from the kinetic data obtained by dynamic on-column reaction HPLC and T-jump HPLC. We observed that the interconversion around the peptide bond for l-alanyl-l-proline (Ala-Pro) in water is accelerated at the surfaces of an alkyl-bonded silica and a poly­(styrene–divinylbenzene) copolymer resin, and this is caused by a remarkable decrease in the enthalpy of activation. The molecular structures of the <i>cis</i> and <i>trans</i> forms of Ala-Pro estimated by quantum mechanics calculation reveal that an equilibrium shift toward the <i>cis</i> form as well as the rapid isomerization of Ala-Pro at the water/hydrophobic interfaces can be attributed to the lower polarity of the interfacial water at the surfaces of the hydrophobic materials compared to that of bulk water

On-Column Labeling of Gram-Positive Bacteria with a Boronic Acid Functionalized Squarylium Cyanine Dye for Analysis by Polymer-Enhanced Capillary Transient Isotachophoresis

A new asymmetric, squarylium cyanine dye functionalized by boronic acid (“SQ-BA”) was designed and synthesized for on-capillary labeling of gram-positive bacteria to provide for high sensitivity detection by way of a modified form of capillary electrophoresis with laser induced fluorescence detection (CE-LIF). The CE-based separation employed a polymer-enhanced buffer with capillary transient isotachophoresis in a new hybrid method dubbed “PectI.” It was found that the addition of various monosaccharides to SQ-BA in a batch aqueous solution greatly enhanced the emission of the boronic acid functionalized dye by a factor of up to 18.3 at a long wavelength (λ_ex = 630 nm, λ_em = 660 nm) with a high affinity constant (<i>K</i> = ∼10^2.80 M^–1) superior to other sugar probes. Semiempirical quantum mechanics calculations suggest that the mechanism for this high enhancement may involve the dissociation of initially nonemissive dye associates (stabilized by an intramolecular hydrogen bond) upon complex formation with sugars. The fluorescence emission of SQ-BA was also significantly enhanced in the presence of a gram-positive bacterial spore, <i>Bacillus globigii</i> (Bg), which serves as a simulant of <i>B. anthracis</i> (or anthrax) and which possesses a peptidoglycan (sugar)-rich spore coat to provide ample sites for interaction with the dye. Several peaks were observed for a pure Bg sample even with polyethyleneoxide (PEO) present in the CE separation buffer, despite the polymer’s previously demonstrated ability to focus microoorganisms to a single peak during migration. Likewise, several peaks were observed for a Bg sample when capillary transient isotachophoresis (ctITP) alone was employed. However, the new combination of these techniques as “PectI” dramatically and reproducibly focused the bacteria to a single peak with no staining procedure. Using PectI, the trace detection of Bg spores (corresponding to approximately three cells per injection) along with separation efficiency enough to separate Bg from another gram-positive bacteria, <i>Saccharomyces cerevisiae</i> (resolution, <i>R</i>_s = 6.09, and apparent plate number, <i>N</i> = 2.7–3.3 × 10⁵), were successfully achieved

Molecular Design of Boronic Acid-Functionalized Squarylium Cyanine Dyes for Multiple Discriminant Analysis of Sialic Acid in Biological Samples: Selectivity toward Monosaccharides Controlled by Different Alkyl Side Chain Lengths

We designed a new series of boronic acid-functionalized squarylium cyanine dyes (SQ-BA) with different lengths of alkyl chain residues, suitable for multiple discriminant analysis (MDA) of sialic acid (Neu5Ac) in biological samples. The SQ-BA dyes form aggregates based on hydrophobic interactions, which result in quenched fluorescence in aqueous solutions. When the boronic acid binds with saccharides, the fluorescence intensity increases as a result of dissociation to the emissive monomeric complex. We inferred that different dye aggregate structures (<i>H</i>-aggregates and <i>J</i>-aggregates) were induced depending on the alkyl chain length, so that monosaccharides would be recognized in different ways (especially, multipoint interaction with <i>J</i>-aggregates). A distinctive emission enhancement of SQ-BA dyes with shorter-alkyl-chains in the presence of Neu5Ac was observed (2.4-fold fluorescence enhancement; with formation constant 10^1.7 M^–1), with no such enhancement for SQ-BA dyes with longer-alkyl-chain. In addition, various enhancement factors for other monosaccharides were observed depending on the alkyl chain length. Detailed thermodynamic and NMR studies of the SQ-BA complexes revealed the unique recognition mechanism: the dye aggregate with a shorter-alkyl-chain causes the slipped parallel structure and forms a stable 2:1 complex with Neu5Ac, as distinct from longer-alkyl-chain dyes, which form a 1:1 monomeric complex. MDA using the four SQ-BA dyes was performed for human urine samples, resulting in the successful discrimination between normal and abnormal Neu5Ac levels characteristic of disease. Thus, we successfully controlled various responses to similar monosaccharides with a novel approach that chemically modified not the boronic acid moiety itself but the length of the alkyl chain residue attached to the dye in order to generate specificity

Multistep pH-Peak-Focusing Countercurrent Chromatography with a Polyethylene Glycol-Na₂SO₄ Aqueous Two Phase System for Separation and Enrichment of Rare Earth Elements

Multistep pH-peak-focusing countercurrent chromatography was developed for separation and enrichment of rare earth metal ions using a polyethylene glycol-Na₂SO₄ aqueous two phase system (ATPS) and pH stepwise gradient elution. Metal ions in a sample solution are chromatographically extracted in a basic stationary phase (polymer-rich phase of the ATPS) containing a complexation ligand such as acetylacetone at the top of the countercurrent chromatography (CCC) column. After the sample solution is introduced, the mobile phases of which the pH values have been adjusted with buffer reagents are delivered into the column by stepwise gradient elution in order of decreasing pH. Each metal ion is concentrated at a pH border formed between the zones of different pH in the CCC column through extraction with a complexing agent into the stationary phase at the front side of the border (basic region) and back extraction into the mobile phase at the back side of the border (acidic region), moving toward the outlet of the column with the pH border. Mutual separations of La­(III), Ce­(III), Nd­(III), Yb­(III), and Sc­(III) were achieved by the present method using five step pH gradient elution, and each rare earth metal ion was effectively enriched at each of the five pH borders. The mechanism for formation of pH profile of the column effluent and the potential of this technique for preparative scale separation are also discussed