DOLPA

Sodium bis(2-ethyl-l-hexyl) sulfosuccinate (Aerosol OT, AOT) sodium do-decylbenzene sulfonate (SDBS) sodium di-2-ethyl hexyl phosphate (NaDEHP) dioleyl phosphoric acid (DOLPA) di(tridecyl) phosphoric acid (DTDPA) sodium dodecyl sulfate (SDS) 1,3-dilauroyl glycerol-2-disodium phosphate (2-modified 1,3-diacyl glycerol)... 

Aqueous pH alters the protein charge property and affects the extraction efficiency. Haemoglobin (Mw 64,500, pi 6.8) is a difficult protein in terms of being able to completely extract it into reverse micelles. The representative anionic surfactant, di-2-ethylhexyl sulfosuccinate (AOT), cannot extract it, and gives rise to an interfacial precipitate. In contrast, we succeeded in the complete extraction of haemoglobin using synthetic anionic surfactants, dioleyl phosphoric acid (DOLPA), as seen in... 

Figure 14.1 [6]. Only for pHs below the isoelectric point (pi) of the protein should the protein be extracted with anionic surfactants such as DOLPA or AOT, whereas for pHs above the pi, extraction into the reverse micellar phase should be inhibited because of the unfavourable electric repulsion. This result indicates that electrostatic interactions are required to extract haemoglobin efficiently into reverse micelles.

Although AOT is also an anionic surfactant of the same type as DOLPA, haemoglobin cannot be transferred into the AOT reverse micellar phase, and most haemoglobin can be seen at the oil-water interface as a red precipitate. Adachi and Harada have reported that cytochrome c precipitated as a cytochrome c-AOT complex at low concentrations of AOT [7]. It was found that this precipitate is likewise the AOT-haemoglobin complex (AOT/haemoglobin = 120 1) from the results of elemental analysis [8]. These results indicate that the difference in the extraction ability of DOLPA and AOT might depend on the hydrophobicity of the surfactants provided to the hydrophilic proteins. 

From the results of Figure 14.3, interestingly, the protein extraction behaviour by dilinoleyl phosphoric acid (DLIPA) is distinct from the result obtained by DOLPA, in spite of the similar hydrophobic chain. The oleyl and linoleyl groups are unsaturated C18 chains. The former has one unsaturated cis-double bond at the position between C9 and CIO, while the latter has two at the position between C9 and CIO and between C12 and Cl 3. The two double bonds in the hydrophobic part of the surfactant DLIPA provide some inflexibility in the chain, which can lead to steric hindrance and to solvation by the organic phase (Figure 14.5). 

Carlson and Nagarajan have reported that the addition of alcohol improved the back extraction efficiency [10]. It is well known that alcohol is a representative cosurfactant so far. A long-chain alcohol stabilizes the reverse micelles, while a short-chain alcohol sometimes inhibits the formation of reverse micelles. In haemoglobin back extraction, the addition of isopropyl alcohol or ethanol is significant in facilitating haemoglobin release from DOLPA reverse micelles (Figure 14.6) [6]. 

FIGURE 14.5. Schematic illustration of the difference in the extraction modes of DOLPA and DLIPA [9]. 

FIGURE 14.7. Effect of the addition of alcohol on the lithium ion leakage from DOLPA reverse micelles during back extraction. A back-extraction operation was performed by contacting the reverse micellar solution extracting lithium in advance and the fresh recovery of the aqueous solution. The observed amounts of lithium ion in each phase were measured using an ICP analyser. 

FIGURE 14.8. Schematic diagram of the back-transfer of proteins from the DOLPA reverse micellar phase by the addition of alcohol [6]. 

Figure 14.10 shows the extraction behaviours of N-cytc and G-cytc by DOLPA reverse micelles. 

This shows that the cytochrome c extraction into an organic phase is carried out at a very low DOLPA concentration, in which reverse micelles cannot be formed. Also, the molar ratio required for the complete protein extraction was approximately 20, which corresponds to the number of cationic charged residues available for the electrostatic interaction with an anionic surfactant in one cytochrome c molecule. These results support the concept that proteins are extracted by electrostatic interactions with surfactant molecules, and that the existence of reverse micelles is not necessary for causing protein transfer, as mentioned above. 

In addition. Figure 14.10 indicates that almost all of the G-cytc readily vanished from the aqueous feed solution at a relatively low DOLPA concentration in the organic... 

FIGURE 14.10. Extraction behaviour of N-cytc and G-cytc into the organic phase containing DOLPA. The amount of depleted proteins from aqueous solution (open symbols) and extracted proteins in the surfactant solution (closed symbols) were estimated by the decrease and increase in the protein concentration during the extraction operation, respectively [19]. 

Hence DOLPA reverse micelles recognize the Arg-rich proteins through specific phosphate-guanidinium groups. Such noncovalent bonds of surfactant molecules lead to alterations in the hydrophilic protein surface into sufficiently hydrophobic surfaces to be solubilized in a nonpolar solvent. Molecular recognition on the protein surface facilitates protein transfer, a significant characteristic for the specific separation of biomolecules. 

Dioleyl phosphate (DOLPA) as functional monomer with y-ray irradiation... 

Figure 9.7 shows the pH dependence of the competitive adsorption of Zn(II) and Cu(II) on Zn(II)-imprinted resins which were irradiated with y-rays. DOLPA was used as a functional monomer. No difference was observed between non-irradiated and irradiated polymers with respect to their ability for Zn(II) binding. An irradiated blank polymer, which did not include DOLPA, adsorbed no metal ions. These results clearly showed that irradiation with y-rays did not destroy the Zn(II)-... 

---