Solvatochromic studies in polyethylene glycol–salt aqueous biphasic systems

The polarities of the co-existing phases of a polyethylene glycol (PEG)-2000–K₃PO₄ aqueous biphasic system (ABS) have been examined using Reichardt’s carboxylated pyridinium-N-phenoxybetaine dye as a probe. Using this probe, the polarities of these phases have been compared to those of conventional solvent extraction systems and micellar systems using values obtained from the literature. In general, these extraction systems are comparable in polarity to rather polar solvents. Data on the free energy of transfer of solvents suggests that this may be due to the failure of the probe to account for the real polarity of the salt-rich phase compared to the polymer-rich phase. Examination of the monophasic region of these systems suggests that the reason for this is that the probe is partitioned to a discreet solvent domain dominated by PEG, even though phase separation of the solution is not observed. The use of linear free energy relationships for the characterization of ABS is briefly discussed.     

Synthesis and characterization of chondroitin sulfate–methacrylate hydrogels

The various degree of methacrylate (MA) substitution on chondroitin sulfate (CS) was prepared by reacting chondroitin sulfate with methacrylic anhydride (MAA) in the presence of sodium hydroxide (NaOH) as a base. The effects of reaction time, reaction temperature, MAA concentration, and NaOH amount on the substitution degree of CS-MA were tested. The confirmation of the CS-MA chemical structure was carried out by ¹H-NMR, ¹³C-NMR, FTIR and the degree of MA substituent on CS was calculated from the ratios of two peak intensities corresponding to methyl groups on methacrylate and chondroitin sulfate, respectively. Hydrogels were prepared by free radical polymerization of CS-MA precursors with or without acrylic acid (AA). CS-MA hydrogels were easily broken into small pieces during swelling study. However, CS-MA-AA hydrogels remained completely and showed a range of swelling ratio from 200 to 390% and exhibited an increase in swelling ratio with a decreasing degree of MA substitution. The thermal degradability observed with a TGA explained the unstableness of these hydrogels in comparison with the pure CS. The surface morphology conducted by SEM exhibited a porous structure after swelling.     

Stability and thermodynamics of polyuridylic acid-deoxyadenosine complexes in aqueous neutral salt solutions

Using the thermodynamic analysis and methodology of Hill (Biopolymers, 12 , 257 (1973)) for the treatment of optical thermal transition data the effects of various neutral salt additives on the stability and thermodynamics of the poly U–deoxyadenosine interactions that lead to the formation of triple-stranded helical polymer–monomer complexes have been studied. In order of increasing molar effectiveness as polyU–deoxyadenosine complex stability perturbants (pH 7 and in the presence of 1 M NaCl), the various ions may be ranked: SO₄^?2 < Cl^? < Br^? < ClO₄^?; and (CH₃)₄ N⁺ < Li⁺ < Rb⁺ ～ Na⁺ < K⁺ < (CH₃ CH₂)₄ N⁺ < urea < Guan⁺ ～ (CH₃(CH₂)₂)₄ N⁺. Destabilizing neutral salt additives (e.g., NaClO₄) caused a decrease in the absolute magnitude of the apparent enthalpy and entropy of binding relative to the values determined in the presence of NaCl. By contrast, stabilizing additives (e.g., Na₂ SO₄) had the opposite effect on these parameters. Along a melting curve the apparent differential heat of complex formation calculated for the binding of deoxyadenosine to poly U in 1 M NaCl appeared to vary linearly with θ, the extent of fractional binding. For such a linear dependence it can be shown that the integral heat (usually determined calorimetrically) equals the differential heat at θ = 0.5. Correcting the apparent differential heat calculated at θ = 0.5 for ligand activity resulted in values for the integral heat of binding of deoxyadenosine to poly U in 1 M NaCl of ?13 to ?16 kcal/mol. Binding isotherms determined in the presence of different inorganic electrolytes could be superimposed provided that different temperatures were compared. However, the additive (CH₃)₄NCl, which has been shown to interact preferentially with A-T rich regions of DNA (Shapiro, Stannard, and Felsenfeld, Biochemistry, 8 , 3233 (1969)) resulted in a considerably broadened binding isotherm indicating less cooperativity.     

Possibility of analytical application of the partition in aqueous biphasic polymeric systems technique

The partition behaviour of a number of ionic and nonionic surface-active substances in the dextran-polyethylene glycol system was examined. The strictly linear dependence of the logarithm of the partition coefficient on the alkyl chain length for a homologous series of nonionic surfactants provides a measure of the difference in the relative hydrophobicity between the two phases of the system, in terms of the free energy of transfer of a CH₂ group from the bottom phase to the top phase of the system. This difference is found to be altered in the presence of NaCl or KCl depending on the salt concentration. It is concluded that the influence of the salt composition of the system on the distributed solutes' behaviour may be due to the effect of the ions on the hydrophobicity difference between the phases.The partition of ionic amphiphiles is found to be dependent on the relative hydrophobicity of the compounds as well as on their charge. It is shown that at salt concentrations up to about 0.1 M NaCl the charged solute partition is determined by its charge as well as its relative hydrophobicity, in the presence of 0.1–0.2 M NaCl the substance distribution is highly dependent on its charge and slightly on its lipophility. At the salt concentrations above 0.2 M the solute partition is determined just by its hydrophobic character and seems to be totally independent of its charge. It is concluded that the partition technique can be used for analytical purposes. The method seems to be unique in providing quantitative information on the amphiphilic surface properties of the solutes being partitioned.     

Physicochemical studies of amylose and its derivatives in aqueous solutions: Thermodynamics of the iodine–triiodide complex

Thermodynamic properties of the amylose–iodine–triiodide complex have been studied by spectrophotometry and by calorimetry using previously studied samples of amylose ionic derivatives, carboxymethylamylose and diethylaminoethylamylose. The ratio of triiodide to total molecular iodine ([I₃]_b/[I]_b + [I₂]_b) in the complex is ca. 0.3 over a range of iodide concentration from 10^?5 to 10^?4M, and there is no evidence for an increasing charge at slightly higher iodide concentration. Direct calorimetric experiments have been carried out in different conditions of polymer, iodine, and iodide concentration in order to study the dependence of the heat of the complexation as a function of the above parameters. It is shown that the dependence of the measured ΔH on the iodide concentration simply derives from the rearrangement of the triiodide equilibrium because of the uptake of a fixed ratio of iodine and triiodide molecules in the complex.     

Thermodynamics of porphyrin dimerization in aqueous solutions.

The dimerization equilibrium of deuteroporphyrin IX and of mesoporphyrin IX in aqueous solutions were studied by fluorimetric techniques over the 0.01-1 microM concentration range, where dimerization is the dominant aggregation process. Deuteroporphyrin IX was studied at several temperatures over the range 22-37 degrees C, and mesoporphyrin at 25 and 37 degrees C. The magnitudes determined for the dimerization equilibrium constants (25 degrees C, neutral pH, phosphate-buffered saline) are 2.3 X 10(6)M-1 and 5.4 X 10(6)M-1 for the deutero and meso derivatives respectively. The meso, deutero and haemato species tested show a similar temperature effect, namely dimerization decreasing with increasing temperature, indicating the involvement of a negative enthalpy change. Van''t Hoff isochore of the dimerization constants determined for deuteroporphyrin IX was linear within the temperature range of 22-37 degrees C, allowing the calculation of the thermodynamic parameters. For deuteroporphyrin dimerization, those were found to be delta G0 = -36. 4kJ X mol-1; delta H0 = -46. 0kJ X mol-1 and delta S0 = -32.2J X K-1 X mol-1 (at neutral pH, 25 degrees C, phosphate-buffered saline), showing the process to be enthalpy-driven. Similar trends have been found for porphyrin species other than those studied here. Our data fit with a hypothesis giving a major role to the solvent in driving porphyrins to aggregate in aqueous solution. The magnitudes and directions of the energetic changes fit better with the expectation of the '' solvophobic force'' theory predicting enthalpy-driven association, than with the classic hydrophobic bonding, predicting the association to be entropy-driven.     

Induced optical activity of chondroitin sulfate c–acridine orange complexes

Chondroitin sulfate C (CSC) and acrdiine orange (AO) formed two types of complexes at neutral pH, depending upon the order of mixing. The induced optical activity of AO was much more pronounced when the polysaccharide was added to dye than the dye to polymer (final concentration of dye was 5 × 10⁵M). The difference in aggregation of the dye molecules is believed to be responsible for the observed peculiarities. The Cotton effects of the CSC-to-dye solution displayed a sharp inversion near 59°C. and the profile at 76°C. was almost a mirror image of that at room temperature. At pH 1.3, however, the order of mixing became unimportant, suggesting that the carboxylate on the polysaccharide way involved more intimately than were sulfates in the peculiarities of the Cotton effects.     

The influence of electrostatic interactions on partition in aqueous polyethylene glycol/dextran biphasic systems: Part II

In aqueous polyethylene glycol/dextran two-phase systems, the hydrophobicity, free volume, surface tension, and interfacial tension of the phases in equilibrium were measured as a function of pH and ionic strength. These parameters were found to change with pH, but the pattern and magnitude cannot explain the unusual partition of charged macromolecules, observed previously. The electrostatic potential difference was determined by a new experimental approach based on the measurement of the pH difference between the phases at equilibrium. In polyethylene glycol/dextran systems containing sodium chloride as ionized species, the electrostatic potential is not constant in the pH range 2 to 11. The partition behavior of charged macromolecules and its dependence on pH can be explained by the combined action of charge and phase potential. This conclusion was tested with poly-L-glutamate, which partitioned as predicted and in a pattern opposite to positively charged macro- molecules. (c) 1995 John Wiley & Sons, Inc.     

Lipase in biphasic alginate beads as a biocatalyst for esterification of butyric acid and butanol in aqueous media

Esterification of organic acids and alcohols in aqueous media is very inefficient due to thermodynamic constraints. However, fermentation processes used to produce organic acids and alcohols are often conducted in aqueous media. To produce esters in aqueous media, biphasic alginate beads with immobilized lipase are developed for in situ esterification of butanol and butyric acid. The biphasic beads contain a solid matrix of calcium alginate and hexadecane together with 5 mg/mL of lipase as the biocatalyst. Hexadecane in the biphasic beads serves as an organic phase to facilitate the esterification reaction. Under optimized conditions, the beads are able to catalyze the production of 0.16 mmol of butyl butyrate from 0.5 mmol of butyric acid and 1.5 mmol of butanol. In contrast, when monophasic beads (without hexadecane) are used, only trace amount of butyl butyrate is produced. One main application of biphasic beads is in simultaneous fermentation and esterification (SFE) because the organic phase inside the beads is very stable and does not leach out into the culture medium. SFE is successfully conducted with an esterification yield of 6.32% using biphasic beads containing iso-octane even though the solvent is proven toxic to the butanol-producing Clostridium spp.     

Helix–random chain transitions of polyamino acids in organic acid–salt solutions

Polyamino acids which are soluble and helical in acetic acid and dichloroacetic acid (DCA) have been observed to undergo a helix to random chain transition upon the addition of lithium salts of strong acids. The transition can be reversed by diluting the salt. Apparently only lithium cations are able to bring about the polycarbobenzoxy-L -lysine (PCBL) transition in acetic acid, whereas the anions display a varying degree of effectiveness; ClO₄^? > Br^? > TSA^? > Cl^? > NO₃^?. The lithium salts of carboxylate anions such as OAc^? and TFA^? do not cause polymer unwinding in acetic acid. Neither do the acids, TSA, HCl, TFA, or DCA induce the transformation in acetic acid. Poly-L -alanine (PLA) in DCA unfolds as LiBr is added, but does not unfold in the presence of 0.5M (CH₃)₄NBr, 0.25M CsBr, or 0.32M HCl. These results are explained on the basis of a direct interaction of the lithium salt with the polymer amide groups to form an ion-pair complex. The extent to which the union of the ion pair can dissociate from the complex in the low dielectric constant, environment determines the degree of unfolding of the polymer. The anion dissociation equilibrium presumably therefore would lie in the same order as given above. Acids such as HCl and TSA are considered to substantially protonate and ion-pair with the polymer, but do not readily dissociate the anion partner from the complex, and therefore do not produce an unstable positively charged helical structure.     

Vesicle formation in aqueous solutions of bile salt and monoacylglycerol

We have investigated by means of quasielastic light scattering and freeze-fracture electron microscopy the aggregation behavior of aqueous solutions of taurocholate and monoolein. A spontaneous micelle-to-vesicle transition has been observed upon dilution of a mixed micellar solution. The size and stability of the micelles and vesicles present in these solutions has been studied as a function of the concentration and incubation time.     

Improvement of the stability of glucose oxidase via encapsulation in sodium alginate–cellulose sulfate–poly(methylene-co-guanidine) capsules

Encapsulation of glucose oxidase (GOD) in polyelectrolyte complex capsules and its influence on properties of the enzyme is reported. The immobilization of GOD in the capsules made of sodium alginate (SA), cellulose sulfate (CS), poly(methylene-co-guanidine) (PMCG), CaCl₂ and NaCl (GOD–SA–CS/PMCG capsules) was achieved using a one-step highly reproducible encapsulation protocol which was monitored by a Electrospray Ionization-Mass Spectrometry (ESI-MS). A leakage of the enzyme from the capsules was negligible. Encapsulated GOD exhibited higher thermostability, wider range of pH optimum and improved storage stability in comparison with free GOD. The 92% retained activity by the encapsulated GOD after 45 biooxidation cycles was markedly higher than that of the GOD entrapped in calcium pectate gel beads showing no activity after 12 cycles. Optimization of conditions of oxygen supplementation resulted in increased oxygen availability within the GOD–SA–CS/PMCG capsules. Oxygen supplementation was accompanied with a mild decrease in the mechanical resistance of the SA–CS/PMCG capsules.     

Molecular configuration of amylose and its complexes in aqueous solutions. Part III. Investigation of the DP distribution of helical segments in amylose–iodine complexes

The equilibrium normality [I_f] of free iodine in amylose-iodine complex formation is a function of the length of the polyiodine chains. This length depends on the DP of helical segments of amylose (sDP _n). Values of [I_f] and of the concentration of the bound iodine [I_b] were determined by the continuous photometric titration with automatic recording. Plots of [I_b] versus [I_f] give an integral distribution curve. Since the relation between [I_f] and sDP _n is known, the graphic differentiation of the [I_b] versus [l_f] curve furnishes the differential distribution curve, representing the mass distribution of the helical segments according to their DP . The peak of this curve is characteristic of the percentage and DP of those helical segments, which occur in the largest amount. On the basis of the differential distribution curve the polymolecularity of the investigated sample may be judged. The titration of amylose samples degraded by various methods gives different distribution curves. Titrating mixtures of samples with widely differing average DP values results in differential curves having more than one maximum.     

Long‐lived electrochemiluminescence of ruthenium (II) complexes/tri‐n‐propylamine in aqueous solutions

Although the clinical use of immunoassays based on the oxidative‐reduction electrochemiluminescence (ECL) of tris(2,2′‐bipyridine)ruthenium (II)/tri‐n‐propylamine has been a great success, elucidation of the ECL generation mechanism still remains unsatisfactory. We report here our experimental observations of long‐lived luminescence that remains detectable for several seconds after termination of electrochemical heterogeneous oxidation. Long‐lived luminescence was observed in both a surfactant‐free buffer and a surfactant‐containing broadly used commercial buffer under different conditions. The slow decay of emission seems to have been unnoticed in previous ECL mechanistic studies. Within the frame of the reaction schemes so far proposed, its origin is inconclusively ascribed to the reductive‐oxidation process of ruthenium (II) complex, that is Ru(bpy)₃²⁺ → Ru(bpy)₃¹⁺ → Ru(bpy)₃²⁺* → Ru(bpy)₃²⁺ with the involvement of the tri‐n‐propylamine‐derived radical cation. It is anticipated that long‐lived ECL will suggest a research approach to separate some homogeneous reactions from the complicated reaction system and therefore help to resolve the mechanistic mystery.     

An infrared study on the conformation of poly-L-proline in concentrated aqueous salt solutions

The infrared absorption of poly-L -proline in concentrated aqueous salt solutions was measured in the fundamental region. Of primary interest were the carbonyl absorption of the peptide linkage and the methylene C–H bending absorption of the pyrrolidine ring. These spectral regions each show an additional component in the concentrated salt solutions. Using the position of the absorptions of poly-L -proline I (cis) and II (trans) as models, we conclude that both cis–trans linkages are present in the peptide in salt solutions. Increasing the temperature shifts the equilibrium slightly in favor of cis.     

The effect of poly(ethyleneglycol) esters on the partition of proteins and fragmented membranes in aqueous biphasic systems

The hydroxyl groups of poly(ethyleneglycol) have been esterified (partly) with a number of carboxylic acids. When these esters are included in dextranpoly(ethyleneglycol)-water biphasic systems the partitions of proteins and membranes between the two phases (and the interface) are in some cases strongly affected. The affinity of serum albumin for the poly(ethyleneglycol)-rich phase is strongly increased when the fatty acid group consists of more than 10 carbon atoms. The partition also depends on the number of double bonds in the fatty acid. A corresponding relationship is found for membranes from spinach chloroplasts. The partitions of ovalbumin, lysozyme (EC 3.2.1.17) and ribonuclease (EC 3.1.4.22) are not influenced by the fatty acid esters. Esters of dibasic carboxylic acids show a minute but marked effect on the partition of proteins in general while malate and tartrate esters affect strongly the partition of chloroplast membranes. The partitions of both proteins and membranes are influenced by poly(ethyleneglycol) deoxycholate. Experiments with malate dehydrogenase (EC 1.1.1.37), lactate dehydrogenase (EC 1.1.1.27), fumarase (EC 4.2.1.2), enolase (EC 4.2.1.11) and glutamate-oxaloacetate transaminase (EC 2.6.1.1) show that their partitions, measured on enzymic activity basis, is changed when esters of benzoic, linolenic, tartaric or deoxycholic acid are included in the biphasic system. The mechanism behind the effect of the esterified poly(ethyleneglycol) on the partition of biomaterial, in this type of aqueous biphasic systems, is discussed in terms of a direct binding of the esters to the partitioned material.