Cosolvent Concentration Research Articles

Co-solvent concentration influence of two- and three-component systems on the high pressure cloud-point behavior for the poly(vinyl stearate) under supercritical CO2

In this research, the experiment in high pressure cloud-point behavior at T (temperature) ≤484.7 K and at p (pressure) ≤207.70 MPa is carried out for the poly(vinyl stearate) [P(VS)] + vinyl stearate (VS) [or C3H8(propane), C4H10(butane), C3H6O(acetone) and C2H5OH(ethanol)] containing supercritical CO2(carbon dioxide). The phase equilibria data for the P(VS) + (10.6 to 54.6) wt% VS mixtures in CO2 are reported for a variety of curves from [UCST(upper critical solution temperature) to LCST(lower critical solution temperature)] in the pressure-temperature space in accordance with the increase of the VS weight fraction. Curves for P(VS) + CO2 + 63.0 wt% VS phase behavior intersect a phase (fluid) → LV (liquid + vapor) curve at ∼383 K (at ∼19.00 MPa). The position of the cloud-point for P(VS) + CO2 curve shifts to lower pressures and temperatures as soon as VS is added. The solubility curves for the P(VS) + (21.9 to 47.9) wt% propane and P(VS) + (27.7 to 46.0) wt% butane mixture under supercritical CO2 vary from UCST to LCST with a decrease in pressure and a co-solvent increase. And also, co-solvent effect of (25.8–95.0) wt% acetone and (13.9–95.0) wt% ethanol in P(VS) + CO2 mixture is shown in the UCST region at temperature range of (428.3 ≤ T ≤ 484.7) K.Phase equilibria data at high pressures was presented for the VS + CO2 system at p ≤ 27.40 MPa and at (313.2 ≤ T ≤ 393.2) K. The VS + CO2 system shows type-I phase curve with a consecutive mixture critical line and was properly correlated with the Peng-Robinson equation.

Formation of chitosan—surfactant complexes in aqueous-alcohol media

The complexation of chitosan and anionic surfactant, sodium dodecyl sulfate (DDS), in aqueous-alcohol media with a variable content of an organic cosolvent (methanol, ethanol, and propan-1-ol) was studied. A complicated effect of the composition of mixed solvents on the binding parameters of DDS by chitosan was observed. The introduction of 10–20 vol.% ethanol and propan-1-ol is accompanied by an increase in the degree of cooperative binding of DDS compared to that found in an aqueous solution, which leads to a decrease in the critical concentration of association and an increase in the cooperativity parameters.

Determination of Protein-Protein Interactions at High Co-Solvent Concentrations Using Static and Dynamic Light Scattering

Protein-protein interactions are commonly measured in terms of the second osmotic virial coefficient, B22 from static light scattering (SLS) or the interaction parameter, kD from dynamic light scattering (DLS). Often these measurements are carried out at high co-solvent compositions, where correction factors are required for the light scattering analysis. For lysozyme in aqueous solutions containing the co-solvents NaCl, arginine chloride, urea, sucrose or guanidine chloride, we show that B22 determination requires using in the light scattering equation the refractive index increment of the protein measured at constant solvent chemical potential. Because the increment decreases with increasing co-solvent composition, using a constant value can lead to mis-interpretation of protein-protein interaction trends deduced from the B22 measurements. Furthermore, there is a contribution to the intensity auto-correlation function measured by dynamic light scattering due to co-solvents. This effect is removed by including longer delay times when fitting the cumulant analysis to determine the diffusion coefficients. We show that an experimentally observed correlation between B22 and kD is recovered once these correction factors have been applied. The findings are particularly relevant to biopharmaceutical industry, where B22 and kD measurements are used for screening excipient effects in liquid formulations.

Gradient supercritical fluid chromatography coupled to mass spectrometry with a gradient flow of make-up solvent for enantioseparation of cathinones

In the past two decades, supercritical fluid chromatography has evolved from a niche application to a comprehensive technology and a fully-fledged alternative to conventional high-performance liquid chromatography.In this study, we have focused on chiral separation of synthetic cathinones in gradient supercritical fluid chromatography coupled to mass spectrometry using an inverse gradient of a make-up solvent. Synthetic cathinones possess an amphetamine-like effect and, therefore, are frequently being offered on the Internet as a replacement for illicit drugs. Cathinones are chiral compounds, however, they are usually marketed and used as racemic mixtures. Since the effect of individual enantiomers can significantly vary, there is a need for the development of enantioseparation methods enabling to study the biological effects of individual enantiomers. Since cathinones are basic molecules, they are easily protonated (positively charged) under weakly acidic mobile phase conditions, which is a typical feature of supercritical mobile phases with an alcohol as an organic modifier. The positively charged species represent ideal analytes for ion exchangers, such as chiral zwitterion ion exchangers Chiralpak ZWIX (+) and Chiralpak ZWIX (−), which possess a positively and negatively charged unit in the molecular structure of the selectors. The presence of the positive charge in the selector's structure, functioning as a counter-ion for the positively charged analytes, significantly reduces the required amount of a buffer, which is plausible for hyphenation of such a separation system with mass spectrometry. For mass spectrometry hyphenated to supercritical fluid chromatography, the use of a make-up solvent is required to avoid analyte precipitation when using a low concentration of an organic co-solvent (modifier) in the super-/subcritical mobile phase. Hereby, we introduce a unique approach, which is based on the gradient introduction of the make-up to the post-column effluent. Using this approach, it is possible to keep constant the overall amount of the organic solvent (modifier and make-up) introduced into the mass spectrometer when using a gradient of the organic modifier. We show that the developed gradient elution method facilitates the chiral separation of all employed analytes, while the mobile-phase gradient compensation by the inverse make-up gradient enables their detection with high signal intensities.

Comparative study of the protein denaturing ability of different organic cosolvents

The influence of a wide spectrum of water-miscible organic cosolvents at different concentrations on the denaturation of hen egg-white lysozyme is studied using differential scanning calorimetry (DSC) and circular dichroism (CD). The denaturing ability of cosolvents is characterized with the parameter −∂Td∂x1 reflecting the change in the denaturation temperature with increasing cosolvent concentration. A series of cosolvents according to their denaturing ability is established: glycerol < ethylene glycol < pure water < dimethyl sulfoxide < methanol < ethanol < formamide < acetonitrile, dimethyl formamide, acetone < 2-propanol < 1,4-dioxane < tert-butanol < 1-propanol < tetrahydrofuran < 2-butanol < 1-butanol. The link of the −∂Td∂x1 parameter to the m values obtained in isothermal studies of chemically induced denaturation and to the solvation properties of aqueous-organic mixtures is demonstrated. Near-UV CD measurements indicate that changes in the tertiary structure occur at slightly lower temperature than the DSC peak in some of the mixtures with high organic cosolvent content. Far-UV CD measurements in the mixtures containing alcohols or tetrahydrofuran confirm non-simultaneous disruption of the tertiary and secondary lysozyme structure. Organic cosolvents induce formation of the molten globule state with preserved and even increased secondary structure, which gradually disrupts at higher temperatures.

Non-covalent interactions of imidazolium-based ionic liquids with model pyrrolidones revealed by FTIR spectroscopy and quantum chemical model calculations

Certain ionic liquids (ILs), such as 1-butyl-3-methylimidazolium chloride (bmimCl) and 1-ethyl-3-methylimidazolium acetate (emimAc), are potent cellulose solvents, but the high viscosity of the resulting solutions is problematic in many applications. Organic co-solvents are often employed to alleviate this problem; however, our understanding of the intermolecular interactions determining the IL/co-solvent mixture performance is very limited, hampering the further development of this class of cellulose solvents. Herein, we applied infrared spectroscopy (IR), Raman spectroscopy, and quantum chemical model calculations to investigate the intermolecular interactions in differently concentrated mixtures of bmimCl and emimAc with amidic co-solvents, N-methyl-2-pyrrolidone (NMP) and N-vinyl-2-pyrrolidone (NVP). Based on the detailed analysis of the vibrational spectra of individual mixture components and the improved assignment of the relevant characteristic bands, we determined that bmim+-co-solvent and emim+-co-solvent associates, stabilized by the hydrogen bonds between amidic carbonyl oxygens and the CH groups of the imidazolium rings, are formed in the mixtures. In addition, the data pointed to concomitant disruption of the H-bonds between the IL counterions as the co-solvent concentration was increased, which may indicate partial dissociation of the IL ion pairs. Further, through an extensive deconvolution analysis, we quantified the molar fractions of the co-solvent molecules involved in the associate formation, finding that this fraction is significantly lower for emimAc than for bmimCl mixtures at all the studied concentrations. On the other hand, the influence of the amidic co-solvent structure was negligible, suggesting that also other (aprotic) amides may be applicable as co-solvents. Furthermore, the calculated molar ratio of IL to interacting co-solvent molecules highlighted the possible differences in the associate stoichiometry. The findings indicate that the lower affinity of the IL to co-solvents in emimAc-based mixtures may lead to the retaining of larger IL-rich clusters and, consequently, the comparatively better performance of the system in cellulose dissolution. Finally, increased temperature was found to influence all the studied systems similarly, inflicting about 20% decrease in the fraction of interacting co-solvent molecules at 85 °C as compared to room temperature. In summary, the results of this study provide important implications for the design of new solvent systems for cellulose dissolution.

Thermodynamic Study of Choline Chloride-Based Deep Eutectic Solvents with Water and Methanol

To study the effects of type and content of cosolvent as well as temperature on the properties of two well-known deep eutectic solvents (DESs), i.e., ChCl/EG (choline chloride + ethylene glycol at ...

Liquefied Gas Electrolytes for All-Temperature Lithium Metal Batteries

Among the several challenges to enable next-generation batteries is the development of an electrolyte which compatible with both lithium (Li) metal anode and high-voltage cathode at wide temperature range. Liquefied gas electrolytes with a new cosolvent and higher salt concentration show improved ionic conductivity of > 4 mS/cm at wide temperature range from -80 to +70 °C. With a new solvation structure, the liquefied gas electrolytes demonstrated high-temperature operation of Li-metal batteries at 55°C, which is the operation above the electrolytes’ critical point for the first time. The electrolytes enable improved Li metal stability and coulombic efficiency at aggressive current and capacity of 3 mA·cm-2 and 3 mAh·cm-2 with average coulombic efficiency of 99%. The use of liquefied gas electrolytes presents stable cycling of Li/NMC (4.4 V) cell at all-temperature range between -60 and 55°C. This study opens up a promising avenue toward the applications of all-temperature high energy density Li-metal batteries.

Effect of dimethylsulfoxide, ethanol, α- and β-cyclodextrins and their association on the solubility of natural bioactive compounds

The use of co-solvents to increase drug solubility is limited mainly due to their toxicity. Many solvents are not biodegradable and have significant environmental impact. Furthermore, uncontrolled precipitation of the compound may arise when solvent-based formulation is introduced into an aqueous medium. Cyclodextrins (CD) can form inclusion complexes resulting in an increase in the guest's solubility. Herein, we evaluated the effect of dimethylsulfoxide (DMSO), ethanol (EtOH), CD, and their association, on the solubility of two natural anti-oxidants, trans-anethole (AN) and caffeic acid (CA). The findings revealed that co-solvents or CD enhanced the solubility of these compounds. Nonetheless, if the concomitant presence of co-solvent and CD induced a loss of the solubilizing efficiency of CD it allowed lowering the quantity of solvent needed to achieve the same solubility. Consequently, the concentration of CD and co-solvent in the complexation media should be optimized to solubilize the required amount of the guest.

Thermodynamic framework for switching the lower critical solution temperature of thermo-sensitive particle gels in aqueous solvent

Switching the lower critical solution temperatures (LCSTs) of thermo-sensitive particle gels including poly(N-isopropylacrylamide) (PNIPAM), poly(N-diethylacrylamide) (PNDEAM), and poly(N-vinylcaprolactam) (PNVCL) was investigated in various aqueous solvents. Three prepared particle gels represented a single LCST in a pure water solution near human body temperature. By adding a chaotropic co-solvent, such as dimethylformamide (DMF) or dimethyl sulfoxide (DMSO), to given gels in aqueous solution, the degree of influence on change of LCST was significantly different due to the difference in chemical structures. LCSTs of polymer solutions were affected by changing solvent conditions from pure water to mixed solvents. The LCST behaviors of linear PNIPAM, PNDEAM, and PNVCL in water/DMF or DMSO were determined by thermo-optical analysis (TOA), which supported the switchability of LCSTs in hydrogel systems. We used the photon correlation spectroscopy (PCS) technique to measure the switched LCSTs of hydrogel particles in mixed solvent systems. The switched LCSTs of the investigated systems exhibited a non-linear trend by varying co-solvent contents. A molecular thermodynamic framework, a combination of the modified double lattice with chain length dependence (MDL-CL) model and the Flory-Rehner (F-R) model, was utilized to describe and correlate the liquid-liquid equilibrium (LLE) and thermosensitive swelling behaviors between linear and cross-linked polymer solutions. With the addition of chaotropic co-solvents, a non-linear trend for phase behaviors of the three polymers was described after taking into account temperature and composition dependence on the association fraction (ηij) between the species. We assumed the donor-acceptor concept and competitive hydrogen bonding between polymer/water and water/co-solvent. The association fraction for the secondary lattice model was modified from an arbitrarily determined value to a physically meaningful value. Accordingly, the phase equilibrium of the amphiphilic polymers influenced by the structure-breaker characteristics of the co-solute was properly implemented in both experimental and modeling aspects.

Experimental investigation of cosolvent flushing of DNAPL in double-porosity soil using light transmission visualization

This paper investigates the effect of cosolvent content and flushing velocity on the remediation of dense non-aqueous phase liquid (DNAPL) in double-porosity medium using light transmission visualization technique (LTV). Double-porosity was created using local silica sand and sintered spheres of kaolin, arranged in a periodic manner. The flushing solution consisted of ethanol–water mixtures with various ethanol concentrations ranging from 20% to 65% by volume. The effect of contact time was investigated by changing the flow rates of the flushing solution. The accuracy of the LTV technique was examined by comparing an actual injected volume of DNAPL with the calculated volumes generated from image analysis. The LTV results allowed for the visualization of the DNAPL enhanced dissolution as well as its mobilization. While the removal efficiency increased with the increase of the ethanol content, the removal efficiency was negatively affected by the velocity of flushing solution. Image analysis revealed that the highest DNAPL removal efficiency of 92% was obtained at 65% ethanol content and low flushing velocity. This efficiency decreased to 85%, in the case of high flushing velocity. Overall, this study demonstrated that the LTV technique is a viable laboratory tool that can accurately depict enhanced DNAPL dissolution and DNAPL mobilization due to cosolvent flushing.

Effect of Static Extraction Time on Supercritical Fluid Extraction of Bioactive Compounds from Phyllanthus niruri

Supercritical fluid extraction (SFE) is an effective method to extract active components from plants. SFE process can be conducted by using a static or/and dynamic extraction. Static extraction is important for the diffusion of solvated analyte to the matrix surface. Therefore, the main objective of this study is to evaluate the static extraction time influence on SFE extraction of Phyllanthus niruri and the main bioactive compounds. The extraction was conducted by using supercritical carbon dioxide (SC-CO2) with 50% ethanol-water as a co-solvent at selected operating conditions (200 bar, 60 °C, 10% co-solvent content and 3 ml/min solvent total flow rate). The static extraction time was investigated in the range of 15 min–75 min by allowing the plant matrix to immerse in a mixture of SC-CO2 and 50% ethanol-water at the operating condition. The quantification of targeted compounds, which were gallic acid (GA), corilagin (CO) and ellagic acid (EA) were analysed by using high performance liquid chromatography (HPLC). The observation on the physical characteristics of the plant matrix was performed by using the scanning electron microscopy (SEM). The best result for total extraction yield was obtained at 60 min of static extraction time, which was 20.7% g/g sample. It also gave a better result for all three targeted compounds in terms of total content percentage (0.35% g GA/g extract, 3.05% g CO/g extract, and 5.17% g EA/g extract). In conclusion, static extraction time was crucial in extracting bioactive compounds in P. niruri by using SFE method and should be considered as the main parameter other than pressure and temperature.

Robust ω-Transaminases by Computational Stabilization of the Subunit Interface

Transaminases are attractive catalysts for the production of enantiopure amines. However, the poor stability of these enzymes often limits their application in biocatalysis. Here, we used a framework for enzyme stability engineering by computational library design (FRESCO) to stabilize the homodimeric PLP fold type I ω-transaminase from Pseudomonas jessenii. A large number of surface-located point mutations and mutations predicted to stabilize the subunit interface were examined. Experimental screening revealed that 10 surface mutations out of 172 tested were indeed stabilizing (6% success), whereas testing 34 interface mutations gave 19 hits (56% success). Both the extent of stabilization and the spatial distribution of stabilizing mutations showed that the subunit interface was critical for stability. After mutations were combined, 2 very stable variants with 4 and 6 mutations were obtained, which in comparison to wild type (Tmapp = 62 °C) displayed Tmapp values of 80 and 85 °C, respectively. These two variants were also 5-fold more active at their optimum temperatures and tolerated high concentrations of isopropylamine and cosolvents. This allowed conversion of 100 mM acetophenone to (S)-1-phenylethylamine (>99% enantiomeric excess) with high yield (92%, in comparison to 24% with the wild-type transaminase). Crystal structures mostly confirmed the expected structural changes and revealed that the most stabilizing mutation, I154V, featured a rarely described stabilization mechanism: namely, removal of steric strain. The results show that computational interface redesign can be a rapid and powerful strategy for transaminase stabilization.

Ultrasound-assisted supercritical CO2 extraction of cucurbitacin E from Iberis amara seeds

Cucurbitacin E (CuE) is an active compound with heterogeneous biological activity and is abundant in Iberis amara seeds. To extract CuE from the seeds, a novel procedure using ultrasound-assisted supercritical CO2 extraction (UASCE) was exploited in this study. The Taguchi design method was adopted for optimizing the experimental conditions of UASCE using L25 (56) orthogonal array. The highest CuE yield (8.61 ± 0.06 mg/g, dry mass) for UASCE was achieved at 60 min dynamic extraction time, 25 MPa pressure, 50 °C, 2 mL/min CO2 flow rate, 12% co-solvent concentration, and 200 W ultrasonic power. The obtained results revealed that UASCE improved the CuE yield by 26.1% and decreased extraction duration by 34.8% as well as reduced the usage amount of CO2 by 52% compared with orthodox supercritical CO2 extraction. Furthermore, the kinetics study demonstrated that the adopted Sovová models fitted well with the experimental extraction curves. The convective mechanism plays a prominent role in UASCE process and mass transfer coefficients increased with the amplification of ultrasonic power. This hybrid technique can be considered as an efficient and promising extraction procedure to obtain CuE from Iberis amara seeds in industrial scale.

Impact of Co-Solvent and LiTFSI Concentration on Ionic Liquid-Based Electrolytes for Li-S Battery

Various organic compounds were investigated as co-solvents in an ionic liquid (IL), N,N-butylmethyl pyrrolidinium bis(trifluoromethylsulfonyl)imide (Pyr14TFSI), based electrolyte for Li-S batteries. For the Pyr14TFSI/Co-solvent binary system, only 1,3-dioxolane (DOL) afforded stable cycling performance with reasonable Coulombic efficiency (CE). Further increase in DOL concentration leads to a higher initial capacity but deteriorated CE. In an attempt to balance CE and capacity, 2.0 M LiTFSI Pyr14TFSI/DOL (2:1) exhibited optimal performance in terms of initial discharge capacity of 896 mAhg−1 and CE of 98.1% for over 200 cycle. The role of DOL and salt concentration interplay in the binary electrolyte was simulated by ab-initio molecular dynamics (AIMD). The solvation energy with varying co-solvent and LiTFSI salt concentration provides useful information when looking toward implementing other IL-based electrolyte systems for Li-S chemistry.

Hydroformylation of Higher Olefins in Aqueous Biphasic Medium Using Rhodium-Sulfoxantphos Catalyst: Activity and Selectivity Study

Hydroformylation of higher olefins such as 1-hexene, 1-octene, 1-decene and 1-dodecene has been studied in an aqueous biphasic medium using water-soluble Rh-sulfoxantphos complex catalyst. The effect of temperature, presence of various co-solvents and concentration of co-solvent on the reaction rate and chemo and regioselectivity was investigated. N-Methyl pyrrolidone (NMP) was found to be the best co-solvent, which enhances the rate dramatically (4-96 fold) as compared to the reactions in aqueous-organic biphasic medium for hydroformylation of higher olefins. Catalyst recycle study was performed to check the leaching of metal in organic phase.

Formulation of drug-loaded oligodepsipeptide particles with submicron size.

The size of particulate carriers is key to their transport and distribution in biological systems, and needs to be tailored in the higher submicron range to enable follicular uptake for dermal treatment. Oligodepsipeptides are promising nanoparticulate carrier systems as they can be designed to exhibit enhanced interaction with drug molecules. Here, a fabrication scheme for drug-loaded submicron particles from oligo[3-(S)-sec-butylmorpholine-2,5-dione]diol (OBMD) is presented based on an emulsion solvent evaporation method with cosolvent, surfactant, and polymer concentration as variable process parameters. The particle size (300-950 nm) increased with lower surfactant concentration and higher oligomer concentration. The addition of acetone increased the particle size at low surfactant concentration. Particle size remained stable upon the encapsulation of models compounds dexamethasone (DXM) and Nile red (NR), having different physicochemical properties. DXM was released faster compared to NR due to its higher water solubility. Overall, the results indicated that both drug-loading and size control of OBMD submicron particles can be achieved. When applied on porcine ear skin samples, the NR-loaded particles have been shown to allow NR penetration into the hair follicle and the depth reached with the 300 nm particles was comparable to the one reached with the cream formulation. A potential benefit of the particles compared to a cream is their sustained release profile.

Supercritical fluid extraction of Arbutus unedo distillate residues – Impact of process conditions on antiproliferative response of extracts

The strawberry tree (Arbutus unedo) is an evergreen plant belonging to the Ericaceae family, an endemic specie of the Mediterranean. In Portugal it grows mostly wild and its fruits are mainly used to produce a distillated alcoholic beverage called “Aguardente de Medronho”. From the process, a residue is obtained which is normally discarded, despite it may figure as biomass for valorization.This work intends to study different conditions of supercritical fluid extraction (SFE), using carbon dioxide as solvent and ethanol as co-solvent for the recovery of bioactive compounds from that residue. After a screening design of experiments varying pressure, temperature, co-solvent, flow rate and time of extraction, an optimization design of experiments (central composite design) was performed. Time (90 min) and flow rate (10 g/min) were established. Variables were pressure (100, 200 and 300 bar), temperature (35, 45 and 55 °C) and co-solvent concentration (0, 10 and 20 %). Extracts were analyzed by GC–MS and LC–MS/MS and cytotoxicity and antiproliferative effect of the extracts was evaluated in Caco2 and HT-29 cell models respectively. Chemical characterization showed an increase in phenolic compounds such as pyrogallol, gallic acid, catechol and protocatechuic acid amongst others, with the increase of the co-solvent. GC–MS analysis revealed a diversity of compounds from fatty acid esters, terpenes and small molecules with recognized bioactivity. The most promising extract was obtained using 300 bar and 55 °C and 20 % of co-solvent. Besides being the extract with the highest yield, it also presented the lowest EC50 (0.569 mg/mL) mainly due to the presence of phenolic compounds.

Comparison of Methods for Determining Dead Times in Supercritical Fluid Chromatography

Abstract—The three most common methods for measuring chromatographic dead time in supercritical fluid chromatography are tested. It is shown that at low concentrations of a polar cosolvent in the mobile phase, the use of the simplest “system peak” method leads to highly inaccurate results. With an increase in the percentage of the cosolvent to 10–15%, this method can be used for a rough estimation of dead times, especially for nonpolar stationary phases. The use of nitrogen(I) oxide as a marker of the dead time of an unretained component, on the contrary, is well suited in the case of using pure carbon dioxide as a mobile phase. The static method, based on other principles, can theoretically yield very accurate values for dead time, but it requires knowledge of the velocity profile of the mobile phase along the path of the entire chromatography.

Urea Stress: Myo-inositol's efficacy to counteract destabilization of TIM-β-globin complex by urea is as good as that of the methylamine

Do kidney osmolytes counteract the deleterious effects of urea on kidney proteins? To answer this question, we measured guanidinium chloride (GdmCl)-induced denaturation of triose phosphate isomerase-β-globin subunit complex (TIM-β-globin) from sheep kidney in the presence of various concentrations of urea and each of kidney osmolytes (glycine betaine, myo-inositol and sorbitol) alone and in combination at pH 7.5 and 25 °C. Analysis of GdmCl-induced denaturation curve at a given osmolyte (or urea) concentration gave ΔGD0 (Gibbs free energy change in the absence of GdmCl). For a co-solute (CO), a linear plot of ΔΔGD0 (difference between values of ΔGD0 in the presence and absence of CO) against [CO] (molar concentration of CO) yielded m-value (=δΔΔGD0/δ[CO]). m-value for urea (murea) and that for the osmolyte (mosm) were used to determine [urea]:[osmolyte], the molar ratio for the perfect counteraction of the denaturing effect of urea on the protein stability by an osmolyte. For each osmolyte, we tested this ratio for the perfect counteraction. It was observed that glycine betaine and myo-inositol provide perfect counteraction at the predicted molar ratio whereas sorbitol fails to do so. Furthermore, urea and GdmCl induced a two-state denaturation, and both TIM and β-globin have identical thermodynamic stability.