Peak shape and dispersion behavior of solutes in counter-current chromatography with a single phase.

The dispersion behavior of solutes was investigated in a rotating flowing coiled tube. Potassium iodide, tartrazine, ascorbic acid, lysozyme, bovine serum albumin (BSA), and silver nanoparticle (AgNPs) samples were eluted in a coiled tube of counter-current chromatography (CCC) apparatus with a single phase. Apparent convection peaks of low-diffusivity solutes appeared in the static CCC tube, while Gaussian-like peaks showed up for the high-diffusivity solutes. When the rotation speed of the CCC apparatus was elevated, all solute peak widths became smaller, and the convection peaks of AgNPs and BSA were minimized and formed Gaussian-like peaks. The axial dispersions of all solutes were reduced owing to the higher radial mass transfer in the rotating CCC column. The same reasoning could also be used to rationalize other special band shapes encountered in two-phase CCC separations.

Size-fractionation of silver nanoparticles using ion-pair extraction in a counter-current chromatograph.

Size separation of silver nanoparticles was investigated in counter-current chromatography (CCC) based on a unique step-gradient extraction process. Carboxylate anions were modified on silver nanoparticles to produce water-dispersible nanoparticles. The aqueous nanoparticles were readily transferred to the organic phase (toluene/hexane=1:1, v/v) together with the phase transfer catalyst, tetraoctylammonium bromide (TOAB), owing to the ion-pair adduct formation between silver nanoparticle anions and tetraoctylammonium cations. Smaller nanoparticles were found to be more readily transferred to the organic phase compared to larger nanoparticles. Various concentrations of TOAB in the organic elution phase were used in the CCC extraction experiments. It appeared that a concentration of 0.02 mM of TOAB was adequate to achieve optimum separation and recovery for the aqueous Ag nanoparticle sample (1.5mg) in the CCC extraction experiments. Samples of 15.8+/-5.3 nm were separated; the distributions of four fractions collected were 13.7+/-1.9, 14.1+/-3.5, 19.2+/-4.3, and 22.2+/-4.9 nm. Compared with the stepwise extraction performed in this study, the step-gradient extractions using CCC provided much better size discrimination.

Liquid-liquid extraction of lysozyme using a dye-modified ionic liquid.

An affinity-dye, Cibacron Blue 3GA (CB), derivatized organic salt [BMIM]3[CB] was synthesized for lysozyme extraction. This compound was formed by mixing an ionic liquid (IL) [BMIM][Cl] and the silver salt of CB. Liquid-liquid extractions of lysozyme from the aqueous and [BMIM]3[CB] in [BMIM][PF6] solutions were examined in this study. The transfer of lysozyme from the aqueous phase to the IL phase decreased while the pH of the aqueous phase increased. An extraction higher than 90% was observed at pH 4. Under a high ionic strength, the lysozyme would transform back from the IL phase into the aqueous phase. Lysozyme molecules were almost quantitatively recovered from the IL phase to the aqueous solutions of 1M KCl under pH 9-11. It appeared that the extraction was specific for lysozyme in contrast to cytochrome c, ovalbumin, and bovine serum albumin. The extraction efficiency of the IL phase remained essentially the same after eight cycles of extraction.

Protein separation and enrichment by counter-current chromatography using reverse micelle solvent systems.

A protein mixture consisting of myoglobin, cytochrome c, and lysozyme was separated by high-speed counter-current chromatography using a two-phase aqueous/reverse micelle-containing organic solvent system. About 50% stationary phase retention ratio was obtained in most chromatographic experiments. Separations were manipulated mainly by pH gradients that controlled the electrostatic interactions between the protein molecules and reverse micelles. Separations were further improved by incorporating an ionic strength gradient along with the pH gradient. Control of ionic strength in the aqueous solution helped fine-tune protein partitioning between the stationary and mobile phases. Although non-specific protein interactions affected baseline resolution, recovery of cytochrome c and lysozyme reached 90% and 82%. Furthermore, concentration or enrichment of these two proteins was achieved from a large-volume sample load. This technique can potentially be employed in the recovery and enrichment of proteins from large-volume aqueous solutions.

Counter-current chromatography using hexane/surfactant-containing water solvent systems.

We developed a n-hexane/surfactant-containing water solvent system in counter-current chromatography (CCC) in order to separate hydrophobic compounds. By using the upper phase as the mobile phase, we have separated steroid samples. Retention times of steroids progesterone and delta4-androstene-3,17-dione increased slightly by increasing the concentration below the critical micellar concentration (CMC) of surfactant sodium 1-heptanesulfonate. However, the retention times increased drastically while the SHS concentrations were above the CMC. The partition of these two steroids in the two phases was significantly dependent on the interaction with micelles. Aromatic hydrocarbons were not retained by the lower phase no matter what the surfactant concentrations were. Their hydrophobic interaction with n-hexane greatly exceeded that with the micellar solution. The retention times of esters, however, were only slightly affected by the surfactant addition even above the CMC. The weaker interaction between esters and the micellar solution was probably due to their higher polarity. The micellar solvent systems provide an alternative way for hydrophobic sample separations in CCC, but the performance is limited.