Enhanced 2,3-Butanediol Purification Using a Hybrid Extraction Process

Enhanced 2,3-Butanediol Purification Using a Hybrid Extraction Process | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Enhanced 2,3-Butanediol Purification Using a Hybrid Extraction Process Pramod Gawal, Sweta Lataye This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8416826/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Recovering 2,3-butanediol (2,3-BD) from fermentation broth is challenging due to its complex composition and impurities. This work develops an integrated aqueous two-phase extraction–distillation method to improve recovery efficiency. Screening of multiple aqueous two-phase systems identified isobutanol/K 2 HPO 4 as the most effective based on distribution behavior. A CCD-RSM design was used to optimize salt concentration, temperature, and solvent content, yielding ideal conditions of 25% (w/v) K 2 HPO 4 , 40 °C, and 30% (v/v) isobutanol. Under these settings, the system achieved a distribution coefficient of 60.47 and an extraction efficiency of 98.23%, slightly higher (0.42%) than model predictions. The enriched extract was subsequently concentrated by distillation, and scale-up runs delivered 98.31% extraction and 98.02% recovery with product purity exceeding 99% in a single cycle. Methanol-assisted crystallization enabled 96.85% recovery of K 2 HPO 4 from the aqueous stream. Overall, this cost-efficient and scalable process enhances 2,3-BD separation while facilitating solvent and salt reuse for industrial applications. 2 3-Butanediol recovery Bioprocess downstream purification Isobutanol/K2HPO4 system Extraction-distillation integration Process optimization Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction The growing global energy demand—driven by rising living standards and a projected 48% population increase between 2012 and 2040—has intensified interest in sustainable energy options [ 1 , 2 ]. Bioenergy, particularly biomass fermentation, has emerged as a viable alternative to fossil fuels, enabling the microbial and enzymatic production of diverse biofuels and chemicals [ 3 , 4 ]. Among these, 2,3-butanediol (2,3-BD) is a valuable platform molecule. Its high octane rating and substantial heating value make it suitable as a drop-in fuel [ 5 , 6 ], while it also serves as a precursor for chemicals such as methyl ethyl ketone, γ-butyrolactone, and 1,3-butadiene. In addition, 2,3-BD is widely used as a solvent and extraction agent in sectors including pharmaceuticals, antifreeze, fragrances, and printing [ 6 – 8 ]. The rising adoption of microbial pathways for producing 2,3-BD highlights their sustainability advantages over traditional petrochemical processes. Currently, global production is estimated at 32 million tons annually, with a market value of around US $ 43 billion [ 1 , 2 ]. The production of 2,3-BD is achievable by biological fermentation processes as well as chemical methods. The chemical route converts butane from cracked natural gas into butene oxide isomers, which are hydrolyzed under harsh conditions (50 bar, 160–220°C) to yield 2,3-BD [ 1 ]. While established, this method is energy-intensive, costly, and environmentally harmful due to greenhouse gas emissions and toxic by-products. In contrast, microbial fermentation offers a sustainable and eco-friendly alternative, using renewable biomass as feedstock. The process involves enzymatic hydrolysis of biomass to release sugars (e.g., glucose, xylose), followed by fermentation by strains like Klebsiella pneumoniae , Bacillus licheniformis , or Enterobacter aerogenes , which convert sugars into 2,3-BD via specific biosynthetic enzymes [ 9 , 10 ]. Fermentation operates under mild conditions, avoids harmful reagents, and utilizes low-cost feedstocks, reducing both carbon footprint and production costs. These advantages make microbial production a promising and scalable alternative for future 2,3-BD manufacturing. The industrial recovery of 2,3-BD from fermentation broth (FB) is hindered by its elevated boiling temperature (184°C), high water solubility, and low product level (< 10%), combined with the presence of impurities like organic acids, sugars, and proteins [ 5 , 11 ]. These challenges impede the development of a separation process that minimizes both cost and energy consumption. Various techniques, distillation [ 12 ], salting-out [ 13 ], reactive extraction [ 14 ], solvent extraction [ 1 ], aqueous two-phase extraction (ATPE) [ 15 ], sugaring-out [ 16 ], and membrane separation [ 17 ], have been explored, but each has limitations. Distillation, while achieving > 99 wt% purity, is energy-intensive. Solvent extraction is constrained by 2,3-BD’s hydrophilicity, requiring large solvent volumes. Salting-out is ineffective at low concentrations without prior water removal. Reactive extraction faces scalability and corrosion issues due to acidic conditions. Polymer-driven aqueous two-phase system (ATPS), like the dextran/PEG system explored by Ghosh and Swaminathan [ 18 ], showed low partitioning (distribution coefficient ( D ) = 1.15), high polymer costs, and product recovery issues, making it unsuitable for industrial use. Overall, these limitations highlight the urgent need for a scalable, energy-efficient, and economically viable separation strategy that can operate under mild conditions while ensuring high product purity and easy recovery. Despite extensive research, most separation methods for 2,3-BD remain restricted by high energy demands, complex operations, and elevated costs, hindering their large-scale application. A promising alternative is ATPE using short-chain alcohol/inorganic salt systems, which offer lower extractant costs, simplify solvent recovery via evaporation, and eliminate back-extraction steps [ 2 , 8 , 19 ]. Operating under mild conditions, this approach reduces energy use and is suitable for heat-sensitive compounds. While ATPE is well-established for purifying proteins and natural compounds [ 20 , 21 ], its use for separating bulk chemicals such as 2,3-BD remains largely unexplored. The integrated ATPE and distillation (IATPED) method effectively separates 2,3-BD from FB while allowing solvent recovery and recycling. In this approach, 2,3-BD is first transferred into the organic phase through ATPE, and the solvent is then separated by distillation, leaving high-purity 2,3-BD as the bottom fraction. Various studies have studied this approach using different solvent-salt systems. For instance, an (NH 4 ) 2 SO 4 /isopropanol system achieved a D of 45.5 and extraction yield ( Y ) of 97.9% [ 15 ], while an ethanol/K 2 HPO 4 system reached D = 28.34 and Y = 98.13% [ 22 ]. Other reported results include D = 9.9, Y = 93.7% for isopropanol/(NH 4 ) 2 SO 4 [ 23 ]; D = 7.10, Y = 91.7% for ethanol/(NH 4 ) 2 SO 4 [ 24 ]; and D = 5.9, Y = 97.08% for butanol/sodium chloride [ 2 ]. This study examined the partitioning of 2,3-BD in different alcohol–salt ATPS and identified isobutanol (IBA)/K 2 HPO 4 as the most suitable system due to its low cost, high extraction performance, and easy solvent–salt recovery. CCD-RSM optimization of solvent ratio, temperature, and salt concentration yielded optimum conditions of 25% (w/v) K 2 HPO 4 , 40°C, and 30% (v/v) IBA, giving a distribution coefficient of 60.47 and an extraction yield of 98.23%. Distillation of the enriched phase enabled efficient product recovery. Scale-up tests confirmed process reliability, reaching 98.31% extraction, 98.02% recovery, and > 99% purity in one cycle. K 2 HPO 4 was regenerated through methanol-induced crystallization, supporting material reuse. Overall, the integrated ATPE–distillation approach offers a high-purity, scalable, and sustainable route for industrial 2,3-BD recovery from FB. 2. Materials and Methods 2.1. Reagents Glucose (99.5%), K 2 HPO 4 (99%), MgSO 4 ·7H 2 O (99–102%), and (NH 4 ) 2 SO 4 (99.5%) were obtained from Merck, India. D 2 O (99%) and 2,3-BD (99%) were procured from Sigma-Aldrich, USA. MgCl 2 ·6H 2 O (99–102%) and CaCl 2 ·6H 2 O (97%) were procured from Fisher Scientific, USA. Cyclohexane, butyl acetate, methanol, 2-ethyl-1-hexanol, IBA, and ethyl acetate (all ≥ 99%) were obtained from SRL, India. The Bacillus licheniformis strain was acquired from MTCC, Chandigarh, India. All experiments used Millipore-purified distilled water. 2.2. Fermentation Studies In this study, Bacillus licheniformis was used to produce 2,3-BD in a FB prepared with glucose and basal salt medium as per [ 25 ]. Fermentation was carried out at 37°C, pH 6, with agitation at 250 rpm, using a 5% (v/v) inoculum at OD 1, for 48 hr. After centrifugation, the supernatant contained 76.5 g/L of 2,3-BD. The corresponding chromatogram is shown in Fig. S1 . 2.3. Analytical Techniques Gas chromatography (450GC, Bruker, USA) fitted with a CP Wax 57CB column, and FID was used to analyze the FB. The oven was initially set to 60°C for 1 min before being heated to 220°C at a rate of 20°C/min. Nitrogen at 30 mL/min was used as the carrier gas, and the injector and detector temperatures were set to 220°C and 250°C. Quantification used a 2,3-BD calibration curve (0.2–1 g/L) showing excellent linearity (R 2 ≈ 0.999) ( Fig. S2) . The purity of recovered 2,3-BD was verified by NMR spectroscopy (Bruker ASCEND 600 MHz) and GC. For NMR, a 250 µL sample was mixed with 250 µL D 2 O, then analyzed by 1 H NMR (16 scans, 1 s relaxation) and 13 C NMR (1000 scans, 2 s relaxation) to ensure accurate structural characterization. 2.4. ATPE-based Extraction of 2,3-BD from FB A measured amount of salt was first dissolved in the FB to form a uniform aqueous phase, after which the solvent was added at a 1:1 ratio and mixed for 10 min. The mixture was then transferred to a separating funnel, the phases were allowed to settle, and the organic layer was collected. This solvent-rich phase containing 2,3-BD was distilled or evaporated to recover the solvent and concentrate the product. GC analysis was performed at each stage to monitor 2,3-BD content and purity. For distribution studies, the same procedure was repeated using different organic solvents (butyl acetate, 1-octanol, butanol, ethyl acetate, isoamyl alcohol, methanol, IBA, cyclohexane) and FB pre-saturated with 20% (w/v) of various salts (K 2 HPO 4 , (NH 4 ) 2 SO 4 , MgSO 4 ·7H 2 O, MgCl 2 ·6H 2 O, CaCl 2 ·6H 2 O). Extraction performance was evaluated using extraction yield ( Y ) (Eq. 1 ) and distribution coefficient ( D ) (Eq. 2 ) to identify the most effective solvent–salt system. $$\:Y=\frac{{C}_{O}\times\:{V}_{O}}{{C}_{FB}\times\:{V}_{FB}}\times\:100$$ 1 $$\:D=\frac{{C}_{O}\times\:{V}_{O}}{{C}_{W}\times\:{V}_{W}}$$ 2 Here, C O, C W, and C FB denote concentrations of 2,3-BD in the organic and aqueous phase, and FB, while V FB, V W, and V O represent their corresponding volumes. 2.5. Optimization and Statistical Analysis A three-factor, three-level 3 3 CCD combined with RSM was employed to optimize the extraction of 2,3-BD. The effects of salt concentration (10–40% w/v), temperature (20–60°C), and solvent fraction (10–50% v/v) on the extraction yield (Y) were investigated. Design-Expert 13 software generated 20 experimental runs with distinct combinations of parameters. One-way ANOVA with a significance level of p ≤ 0.05 was employed to analyze the effects of each variable and their combined interactions. Model reliability was assessed through R 2 and adjusted R 2 values. The relationship between process variables and extraction yield was modeled using a second-order polynomial regression (Eq. 3 ) to help determine the best extraction conditions. $$\:Y={\alpha\:}_{0}+\sum\:_{i=1}^{n}{\alpha\:}_{i}{X}_{i}+\sum\:_{i=1}^{n}{\alpha\:}_{ij}{X}_{i}{X}_{j}+\sum\:_{i=1}^{n}{\alpha\:}_{ii}{X}^{2}$$ 3 In this regression equation, α₀ denotes the intercept, whereas α i , α i i , and α i ⱼ correspond to the linear, quadratic, and interaction effects. The response variable Y represents the 2,3-BD Y , and X i and Xⱼ are the coded independent factors (with i ≠ j). Optimal process conditions were identified using a desirability function (d), which ranges from 0 (unacceptable) to 1 (fully meets criteria). A d value of 1 signifies ideal conditions for maximizing 2,3-BD extraction while satisfying all constraints [ 2 , 26 , 27 ]. 2.6. Upscaling the IATPED-based Process for Recovering 2,3-BD For scale-up, a 10 L extraction column (LLU10, Shiva Scientific, India) was connected to a batch distillation setup (Model 9600, BR Instruments, USA). The column used for separation was 21 mm in diameter and 60 cm tall, equipped with 50 theoretical stages and a Teflon spinning band to enhance component separation and facilitate solvent recovery. The system supported boiler capacities of 1 to 50 L and allowed adjustable reflux ratios control range from 0:1 to 999:1. It operated in both manual and automated modes, enabling precise control suitable for large-scale 2,3-BD recovery. In the scaled-up experiment, 25% (w/v) K 2 HPO 4 was added to 1 L of FB and stirred until fully dissolved. Then, 30% (v/v) IBA was introduced, and the mixture was stirred for 10 min at 40°C to facilitate extraction. After natural phase separation, the organic layer containing 2,3-BD was collected. This extract was then distilled under atmospheric conditions using a spinning band column. The chiller was set to 25°C, and heating was applied at 50% power to evaporate IBA, allowing for the efficient recovery and concentration of purified 2,3-BD. 3. Results and Discussion 3.1. Distribution Characteristics of 2,3-BD in ATPS Table 1 highlights the partitioning of 2,3-BD across various organic solvents and salt combinations. Hydrophilic solvent–salt systems showed significantly higher D values (0.38–7.3) and Y values (25.3–87.5%) compared to hydrophobic systems. In ATPS, 2,3-BD tends to migrate predominantly into the solvent-rich layer. In contrast, hydrophobic solvents resulted in poor extraction, with 2,3-BD remaining mostly in the salt-rich aqueous phase, indicating the limited effectiveness of these solvents for selective 2,3-BD recovery. Table 1 Distribution of 2,3-BD across different solvent/salt systems. Organic solvent/Inorganic salt MgSO 4 K 2 HPO 4 (NH 4 ) 2 SO 4 CaCl 2 MgCl 2 Methanol D 1.01 3.23 3.8 1.1 1.03 Y(%) 44.23 72.53 77.5 40.5 39.5 Isobutanol D 1.74 7.68 5.8 1 1.16 Y(%) 66.14 87.5 80.5 42.33 42.25 Butyl acetate D 1.02 2.18 2.21 0.97 1.06 Y(%) 53.25 66.36 56.3 44.23 45.6 Ethyl acetate D 1.08 3.21 3.28 1.12 1.16 Y(%) 46.31 76.6 62.3 49.3 48.2 Butanol D 1.1 4.37 3.64 1.08 1.27 Y(%) 51.58 78.85 72.36 44.8 51.05 2-Ethyl-1-hexanol D 1.12 5.25 2.3 1.15 1.13 Y(%) 54.12 79.23 69.5 48.5 42.3 1-octanol D 0.56 1.72 1.65 0.58 0.51 Y(%) 35.21 46.53 38.3 31.25 30.2 Cyclohexane D 0.5 0.52 0.51 0.42 0.38 Y(%) 34.58 37.56 35.6 35.63 33.56 Isoamyl alcohol D 0.46 0.56 0.51 0.49 0.43 Y(%) 25.3 45.2 38.52 39.5 32.25 Volume of FB: 5 mL; Salts concentration (% w/v):20; Volume of solvent: 5 mL; Concentration of 2,3-BD in FB: 76.5 g/L; Mixing duration: 15 min The distribution of 2,3-BD is highly influenced by the salt composition, with anions like phosphate (PO 4 3− ) and sulfate (SO 4 2− ) having a greater impact than cations such as magnesium (Mg 2+ ) and calcium (Ca 2+ ) [ 2 , 8 ]. These highly charged anions enhance ATPS formation, improving 2,3-BD’s selective partitioning into the solvent phase. CaCl 2 and MgCl 2 effectively formed ATPS with all tested solvents, yielding notable 2,3-BD extraction, especially with hydrophilic solvents and some hydrophobic ones like octanol, cyclohexane and isoamyl alcohol. The maximum Y and D were observed with IBA combined with K 2 HPO 4 and (NH 4 ) 2 SO 4 . Adding salts during extraction significantly increased Y and D compared to using IBA alone, reaching maximum values of D = 7.3 and Y = 87.5% with K 2 HPO 4 , and D = 5.8 and Y = 80.5% with (NH 4 ) 2 SO 4 . This improvement is attributed to the salt’s ability to increase water solubility, thereby decreasing 2,3-BD’s hydrophilicity and facilitating its transfer into the organic phase. Salt addition thus improves extraction efficiency, guiding the optimization of solvent-salt systems for industrial 2,3-BD recovery. Salts enhance phase separation by weakening water–2,3-BD hydrogen bonding, decreasing its solubility in the aqueous phase and driving it into the organic layer. High-charge ions lower water’s dielectric constant and enhance hydration, intensifying the salting-out effect; this depends on ion charge, ionic strength, and salt concentration [ 22 , 28 ]. Using (NH 4 ) 2 SO₄ sometimes caused a hazy interface due to protein and amino acid precipitation, while K 2 HPO 4 produced a clear, rapidly forming phase boundary, indicating cleaner separation. Unlike IBA, which forms azeotropes requiring decanters, salts enable fast, efficient phase separation without azeotrope formation. Thus, salt addition offers a more practical, cost-effective alternative for 2,3-BD extraction by simplifying separation and reducing equipment needs [ 1 ]. The IBA/K 2 HPO 4 ATPS was chosen for subsequent experiments owing to its notably rapid phase formation, which facilitates efficient separation within a shorter time frame. Additionally, this system offers the advantage of low recycling costs, making it economically favorable for repeated use in large-scale operations. These combined benefits, swift phase separation coupled with cost-effectiveness, make the IBA/K 2 HPO 4 ATPS an ideal candidate for further detailed investigation and process optimization in the extraction of 2,3-BD. 3.2. Optimization of Extraction Conditions Table 2 summarizes the observed and predicted Y of 2,3-BD across various experimental conditions during optimization. Yields ranged from 43.09% to 98.23%, averaging 75.4%. The maximum yield (98.23%) was attained at 25% (w/v) salt, 30% (v/v) solvent, and 40°C (Run 12), while the lowest (43.09%) was at 10% salt, 10% solvent, and 25°C (Run 17). The regression model showed excellent accuracy, with an R 2 of 0.9997, predicted R 2 of 0.9985, and adjusted R 2 of 0.9994, indicating strong predictive capability. As shown in Table 3 , ANOVA results validated the model’s significance, indicated by a high F-value of 3673.64 and a p-value < 0.0001. These findings demonstrate the model’s reliability in predicting and optimizing 2,3-BD Y . Table 2 CCD-RSM-based summary of parameters and response data for 2,3-BD extraction from FB. Run Solvent concentration(% v/v) Salt concentration(%w/v) Temperature(°C) Actual yield(%) Predicted yield (%) A B C Experimental 1 50 40 60 72.08 71.87 2 50 10 60 65.2 65.21 3 50 10 25 63.58 63.89 4 10 40 60 52.22 51.93 5 30 25 40 98.23 97.81 6 30 5 40 68.23 67.96 7 10 40 25 54.39 54.31 8 30 25 40 98.23 97.81 9 50 25 40 95.89 95.70 10 10 10 60 43.31 43.21 11 50 40 25 72.79 72.84 12 30 25 40 98.23 97.81 13 5 25 40 65.98 66.16 14 30 25 60 83.03 83.66 15 30 25 40 98.23 97.81 16 30 25 40 98.23 97.81 17 10 10 25 43.09 43.30 18 30 25 20 75.35 75.03 19 30 40 40 88.3 88.89 20 30 25 40 98.23 97.81 Table 3 Best-fit RSM model adequacy and ANOVA summary for 2,3-BD extraction from FB. Source Sum of Squares df Mean Square F-value p-value Model 7108.3 9 789.81 3673.64 < 0.0001 Significant A-Solvent 1063.91 1 1063.91 4948.57 < 0.0001 B-Salt 203.42 1 203.42 946.17 < 0.0001 C-Temp 0.7358 1 0.7358 3.42 0.094 AB 2.12 1 2.12 9.87 0.0105 AC 0.985 1 0.985 4.58 0.058 BC 2.64 1 2.64 12.27 0.0057 A² 661.84 1 661.84 3078.4 < 0.0001 B² 885.82 1 885.82 4120.21 < 0.0001 C² 912.48 1 912.48 4244.22 < 0.0001 Residual 2.15 10 0.215 Lack of Fit 1.32 5 0.2647 1.6 0.309 Not insignificant Pure Error 0.8265 5 0.1653 Cor Total 7110.45 19 Salt concentration, solvent volume, and extraction temperature all significantly affected 2,3-BD yield (p < 0.0001). Solvent had the greatest impact (F = 4948.57), followed by salt (F = 946.17), while temperature had a minor effect (F = 3.42). The model’s “lack of fit” was insignificant (F = 1.6), indicating only a 30.9% chance that variability was due to random error, confirming model reliability. Based on these results, Eq. (4) was developed from Eq. ( 3 ) to predict 2,3-BD yield using the key variables, providing a useful tool for optimizing extraction conditions. Y = 98.06 + 10.13A + 4.42B – 0.2637C – 0.51AB + 0.35AC – 0.5729BC -12.19A 2 – 13.42B 2 – 14.14C 2 (4) The close alignment of the coefficient of variation (CV%) and predicted R 2 values ( Table S1 of Supplementary Information) confirms the model’s robustness. Figure 1 a shows an excellent linear correlation between the predicted and experimental 2,3-BD yields, with an average deviation of just 0.6058%. The normal probability plot in Fig. 1 b shows that the residuals follow a normal distribution, supporting the validity of the regression assumptions. In Fig. 1 c, the residuals are randomly scattered against the predicted values, indicating uniform variance. Figure 1 d shows no trend between residuals and run order, confirming the absence of systematic errors. Overall, these diagnostic evaluations, carried out in Design Expert using CCD data, demonstrate that the model satisfies all statistical requirements and can reliably estimate and optimize 2,3-BD extraction. Figure 2 shows 3D surface and contour plots illustrating the regression model’s predictions for 2,3-BD extraction yield respond when two factors are varied simultaneously while the third parameter remains fixed. These plots reveal how the key parameters interact and influence extraction efficiency. Figures 2 a & 2 d illustrate how solvent concentration (B) and salt concentration (A) interact, with the maximum yield occurring at 30% solvent and 25% salt (Run 5). This trend indicates a cooperative effect between the two factors that enhances the partitioning of 2,3-BD. Figures 2 b & 2 e illustrate the effect of solvent concentration (A) and temperature (C), with the maximum yield at 30% salt and 40°C (Run 12), emphasizing the role of temperature alongside salt in extraction performance. Figures 2 c & 2 f depict salt concentration (B) and temperature (C) interaction, showing the best extraction at 25% salt and 40°C (Run 15), suggesting that higher solvent levels and temperature enhance 2,3-BD recovery. Overall, these plots provide a clear visualization of variable interactions, aiding in identifying optimal conditions for improved 2,3-BD extraction in the ATPS. Design Expert determined that the best extraction conditions were 25% (w/v) salt, 30% solvent, and a temperature of 40°C. Under these settings, the model projected a maximum 2,3-BD yield of 98.23%, which is 0.42% higher than the theoretical estimate. Validation experiments confirmed an actual yield of 97.81%, closely matching predictions and demonstrating the model’s accuracy and reliability. These findings confirm the effectiveness of the optimized conditions and offer a solid foundation for scaling up 2,3-BD recovery from FB, enhancing both process efficiency and industrial viability. 3.3. 2,3-BD purification from FB Comprehensive optimization of the ATPE system was conducted to identify the most effective conditions for maximizing the 2,3-BD Y from the FB. After extraction, the organic-rich phase containing 2,3-BD was purified through a distillation process, as shown in Fig. 3 . Material balance calculations, presented in Table S2 , revealed that the IATPED approach obtained an impressive 2,3-BD Y of 98.31% and a recovery rate of 98.02%, with the final product exhibiting a purity greater than 99% in a single extraction–distillation cycle. In addition to the high product recovery, the process also demonstrated excellent solvent recyclability, achieving 97% solvent recovery with a purity exceeding 97%. Fig. S3 shows the chromatogram of the purified 2,3-BD, confirming the effectiveness of the purification process. While distillation offers a relatively straightforward method for recovering IBA, the recycling of salts within this ATPS presents a significant challenge. The study focused on the recovery and reuse of K 2 HPO 4 , which predominantly accumulates in the aqueous (bottom) phase. During the distillation of the organic phase, there exists a potential risk of K 2 HPO 4 crystallizing out, which may interfere with phase separation and system efficiency. To address this, a recovery strategy based on methanol-induced crystallization was explored, as illustrated in Fig. 4 . This method involves the dilution of the salt-rich aqueous phase with methanol to promote selective crystallization of K 2 HPO 4 . Experimental results demonstrated that increasing the methanol-to-water phase volume ratio from 0.5 to 2 led to a significant improvement in salt recovery, from 74.75% up to a maximum of 96.85%. However, a further increase in the volume ratio beyond 2.5 resulted in a decline in recovery efficiency, likely due to solubility limitations and co-dissolution effects [ 29 ]. Consequently, a methanol/water volume ratio of 2 was determined as the optimal condition for efficient and reproducible salt recovery, balancing both crystallization yield and process feasibility. Earlier studies report ATPE-based 2,3-BD recovery efficiencies typically between 94.5% and 98.18%. For instance, Li et al. achieved 91.7% using 16% (NH 4 ) 2 SO 4 and 32% EtOH [ 28 ]. Narisetty et al. reported a 97% yield with a D of 45 using 50% isopropanol and 30% (NH 4 ) 2 SO 4 [ 15 ], while Sun et al. obtained 93.7% with 20% (NH 4 ) 2 SO 4 and 34% 2-propanol [ 23 ]. This study’s IBA/K 2 HPO 4 -based ATPS showed superior extraction yield, recovery, and purity compared to these methods ( Table S3 ), demonstrating its potential as a more efficient, scalable, and cost-effective approach for industrial 2,3-BD recovery. This work demonstrates that an IBA/K 2 HPO 4 ATPS is a feasible option for efficient extraction of 2,3-BD from FB. The method achieves high recovery and supports straightforward reuse of IBA through distillation and K 2 HPO 4 through methanol-triggered crystallization, minimizing material usage. Owing to its simplicity and scalability, the system offers strong potential for industrial production of bio-derived 2,3-BD. 3.4. Recovered 2,3-BD: Structural and Purity Characterization The purity and molecular structure of the purified 2,3-BD were thoroughly analyzed by NMR, with the results presented in Fig. S4 . Both carbon-13 ( 13 C) and proton ( 1 H) NMR techniques were employed for comprehensive structural characterization, using commercially available 2,3-BD as the reference standard for comparison. The 13 C NMR spectra of the purified sample and commercial standard exhibited two distinct and well-defined peaks at chemical shifts of 17.3 ppm and 71.2 ppm, which were attributed to the methyl (–CH 3 ) and methine (–CH) carbons, respectively, confirming the expected carbon environment of 2,3-BD. In the 1 H NMR spectrum, the methine proton (–CH) appeared as a quartet at 2.52 ppm, whereas the methyl protons (–CH 3 ) were detected as a doublet at 1.01 ppm. Additionally, a prominent peak at 4.79 ppm is attributed to the residual signal of D 2 O, the solvent employed in the NMR measurement. These spectral features collectively confirm both the high purity and the correct molecular structure of the purified 2,3-BD. 4. Conclusions This work explored the extraction of 2,3-BD using various organic solvents and inorganic salt-based ATPS, ultimately identifying the IBA/K 2 HPO 4 system as the most advantageous due to its low cost and straightforward solvent and salt recyclability. Through CCD and RSM, the key operating factors—salt level, solvent percentage, and temperature—were optimized, each showing a significant influence on 2,3-BD recovery from the FB. The resulting second-order polynomial models reliably captured the interactions between these parameters and the extraction yield. Under the optimal conditions of 30% (v/v) IBA, 40 °C, and 25% (w/v) K 2 HPO 4 , the system achieved a maximum distribution coefficient of 60.47 and an extraction efficiency of 98.31%. The organic-rich layer was subsequently distilled to concentrate the product. Scale-up trials confirmed the robustness of the process, delivering extraction and recovery efficiencies of 98.31% and 98.04%, respectively, and achieving over 99% purity in a single purification step. Furthermore, 96.85% of K 2 HPO 4 was recovered from the aqueous phase through methanol-induced crystallization, contributing to reduced resource consumption and operational costs. Overall, this study demonstrates an efficient, scalable, and economical method for 2,3-BD recovery, suitable for industrial use with reduced waste and lower costs. Declarations CRediT authorship contribution statement Pramod Madhukar Gawal :Conceptualization, Investigation, Formal analysis, Data curation, software, Writing- original draft, review & editing. Sweta Lataye : Conceptualization, Investigation, Formal analysis, Software. Data availability : The authors declare that the data supporting the findings presented are available within the manuscript and its supporting information file. Additional data, or raw data files, will be available from the corresponding author upon reasonable request. Acknowledgments The authors acknowledge the Central Instruments Facility and the Analytical Laboratory of the Department of Chemical Engineering at IIT Guwahati. Funding: No other funding has been consumed for the compilation of this manuscript. Ethical Approval: Not applicable. Consent to Participate: There is no involvement of human subjects in the manuscript. Consent for Publication: Not applicable. Competing Interests: The authors declare no competing interests. References Haider, J., Harvianto, G. R., Qyyum, M. A., & Lee, M. (2018). Cost- and Energy-Efficient Butanol-Based Extraction-Assisted Distillation Designs for Purification of 2,3-Butanediol for Use as a Drop-in Fuel. ACS Sustainable Chemistry & Engineering , 6 (11), 14901–14910. https://doi.org/10.1021/acssuschemeng.8b03414 Gawal, P. M. (2025). 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ADMET and DMPK , 7 (2), 106–130. https://doi.org/10.5599/admet.661 Supplementary Files SupplementoryInformation.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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06:46:23","extension":"html","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":132259,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8416826/v1/a7f80d7a95c0c70490bf5ea0.html"},{"id":99582463,"identity":"a6adc2fb-3047-42e5-a741-38d070562436","added_by":"auto","created_at":"2026-01-06 06:46:22","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":161461,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Actual Vs. Predicted plots for 2,3-BD extraction yield, (b) Externally studentized Residual Vs. Normal % probability plot of 2,3-BD extraction yield, (c) Externally studentized Residual Vs. Predicted 2,3-BD yield, and (d) Externally studentized Residual Vs. Run number.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8416826/v1/de7c0ecc257655cfd6d0eec8.png"},{"id":99582462,"identity":"ce7aa033-0784-4bbc-99e6-b76866120d42","added_by":"auto","created_at":"2026-01-06 06:46:22","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":376811,"visible":true,"origin":"","legend":"\u003cp\u003eResponse surface plot and contour plot of 2,3-BD extraction yield variation with two variables: (a \u0026amp; d) AB, (b \u0026amp; e) AC, and (c \u0026amp; f) BC [ A: Salt (%); B: Solvent (%); C: Temperature (°C)].\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8416826/v1/844f48a51112b80966a1a7b2.png"},{"id":99792490,"identity":"0e99da27-ad02-40c7-8386-4be6950e1e26","added_by":"auto","created_at":"2026-01-08 13:21:00","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":193527,"visible":true,"origin":"","legend":"\u003cp\u003eIATPED using an isobutanol/dipotassium phosphate system for the separation of 2,3-BD from FB [CC: crystallization column; DC: distillation column; EC: extraction column; EP: extract phase; AP: aqueous phase; FB: fermentation broth; IB: isobutanol; MeOH: methanol; 2,3-BD: 2,3-butanediol].\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8416826/v1/83c4111f858313ad01e471e6.png"},{"id":99792435,"identity":"53473a8d-562d-4327-bdaa-4ec84275e2f2","added_by":"auto","created_at":"2026-01-08 13:19:48","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":53546,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of methanol on the recovery of K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8416826/v1/93b73215dca638715d99a3f3.png"},{"id":99804039,"identity":"6c3b8177-a3ef-48a3-9052-456ad6103ea0","added_by":"auto","created_at":"2026-01-08 14:11:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1838660,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8416826/v1/d23988fd-d6a5-49ea-b34c-5a115c050da6.pdf"},{"id":99792601,"identity":"ed56e751-f534-4dd6-8564-c6e6016c20bf","added_by":"auto","created_at":"2026-01-08 13:22:53","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":248274,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementoryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-8416826/v1/ac3a13e71760faf3f7dabdae.docx"}],"financialInterests":"","formattedTitle":"Enhanced 2,3-Butanediol Purification Using a Hybrid Extraction Process","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe growing global energy demand\u0026mdash;driven by rising living standards and a projected 48% population increase between 2012 and 2040\u0026mdash;has intensified interest in sustainable energy options [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Bioenergy, particularly biomass fermentation, has emerged as a viable alternative to fossil fuels, enabling the microbial and enzymatic production of diverse biofuels and chemicals [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Among these, 2,3-butanediol (2,3-BD) is a valuable platform molecule. Its high octane rating and substantial heating value make it suitable as a drop-in fuel [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], while it also serves as a precursor for chemicals such as methyl ethyl ketone, γ-butyrolactone, and 1,3-butadiene. In addition, 2,3-BD is widely used as a solvent and extraction agent in sectors including pharmaceuticals, antifreeze, fragrances, and printing [\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The rising adoption of microbial pathways for producing 2,3-BD highlights their sustainability advantages over traditional petrochemical processes. Currently, global production is estimated at 32\u0026nbsp;million tons annually, with a market value of around US\u003cspan\u003e$\u003c/span\u003e43\u0026nbsp;billion [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe production of 2,3-BD is achievable by biological fermentation processes as well as chemical methods. The chemical route converts butane from cracked natural gas into butene oxide isomers, which are hydrolyzed under harsh conditions (50 bar, 160\u0026ndash;220\u0026deg;C) to yield 2,3-BD [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. While established, this method is energy-intensive, costly, and environmentally harmful due to greenhouse gas emissions and toxic by-products. In contrast, microbial fermentation offers a sustainable and eco-friendly alternative, using renewable biomass as feedstock. The process involves enzymatic hydrolysis of biomass to release sugars (e.g., glucose, xylose), followed by fermentation by strains like \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e, \u003cem\u003eBacillus licheniformis\u003c/em\u003e, or \u003cem\u003eEnterobacter aerogenes\u003c/em\u003e, which convert sugars into 2,3-BD via specific biosynthetic enzymes [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Fermentation operates under mild conditions, avoids harmful reagents, and utilizes low-cost feedstocks, reducing both carbon footprint and production costs. These advantages make microbial production a promising and scalable alternative for future 2,3-BD manufacturing.\u003c/p\u003e \u003cp\u003eThe industrial recovery of 2,3-BD from fermentation broth (FB) is hindered by its elevated boiling temperature (184\u0026deg;C), high water solubility, and low product level (\u0026lt;\u0026thinsp;10%), combined with the presence of impurities like organic acids, sugars, and proteins [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. These challenges impede the development of a separation process that minimizes both cost and energy consumption. Various techniques, distillation [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], salting-out [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], reactive extraction [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], solvent extraction [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], aqueous two-phase extraction (ATPE) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], sugaring-out [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], and membrane separation [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], have been explored, but each has limitations. Distillation, while achieving\u0026thinsp;\u0026gt;\u0026thinsp;99 wt% purity, is energy-intensive. Solvent extraction is constrained by 2,3-BD\u0026rsquo;s hydrophilicity, requiring large solvent volumes. Salting-out is ineffective at low concentrations without prior water removal. Reactive extraction faces scalability and corrosion issues due to acidic conditions. Polymer-driven aqueous two-phase system (ATPS), like the dextran/PEG system explored by Ghosh and Swaminathan [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], showed low partitioning (distribution coefficient (\u003cem\u003eD\u003c/em\u003e)\u0026thinsp;=\u0026thinsp;1.15), high polymer costs, and product recovery issues, making it unsuitable for industrial use. Overall, these limitations highlight the urgent need for a scalable, energy-efficient, and economically viable separation strategy that can operate under mild conditions while ensuring high product purity and easy recovery.\u003c/p\u003e \u003cp\u003eDespite extensive research, most separation methods for 2,3-BD remain restricted by high energy demands, complex operations, and elevated costs, hindering their large-scale application. A promising alternative is ATPE using short-chain alcohol/inorganic salt systems, which offer lower extractant costs, simplify solvent recovery via evaporation, and eliminate back-extraction steps [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Operating under mild conditions, this approach reduces energy use and is suitable for heat-sensitive compounds. While ATPE is well-established for purifying proteins and natural compounds [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], its use for separating bulk chemicals such as 2,3-BD remains largely unexplored.\u003c/p\u003e \u003cp\u003eThe integrated ATPE and distillation (IATPED) method effectively separates 2,3-BD from FB while allowing solvent recovery and recycling. In this approach, 2,3-BD is first transferred into the organic phase through ATPE, and the solvent is then separated by distillation, leaving high-purity 2,3-BD as the bottom fraction. Various studies have studied this approach using different solvent-salt systems. For instance, an (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e/isopropanol system achieved a \u003cem\u003eD\u003c/em\u003e of 45.5 and extraction yield (\u003cem\u003eY\u003c/em\u003e) of 97.9% [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], while an ethanol/K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e system reached \u003cem\u003eD\u003c/em\u003e\u0026thinsp;=\u0026thinsp;28.34 and \u003cem\u003eY\u003c/em\u003e\u0026thinsp;=\u0026thinsp;98.13% [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Other reported results include \u003cem\u003eD\u003c/em\u003e\u0026thinsp;=\u0026thinsp;9.9, \u003cem\u003eY\u003c/em\u003e\u0026thinsp;=\u0026thinsp;93.7% for isopropanol/(NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]; \u003cem\u003eD\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.10, \u003cem\u003eY\u003c/em\u003e\u0026thinsp;=\u0026thinsp;91.7% for ethanol/(NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]; and \u003cem\u003eD\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.9, \u003cem\u003eY\u003c/em\u003e\u0026thinsp;=\u0026thinsp;97.08% for butanol/sodium chloride [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis study examined the partitioning of 2,3-BD in different alcohol\u0026ndash;salt ATPS and identified isobutanol (IBA)/K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003eas the most suitable system due to its low cost, high extraction performance, and easy solvent\u0026ndash;salt recovery. CCD-RSM optimization of solvent ratio, temperature, and salt concentration yielded optimum conditions of 25% (w/v) K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, 40\u0026deg;C, and 30% (v/v) IBA, giving a distribution coefficient of 60.47 and an extraction yield of 98.23%. Distillation of the enriched phase enabled efficient product recovery. Scale-up tests confirmed process reliability, reaching 98.31% extraction, 98.02% recovery, and \u0026gt;\u0026thinsp;99% purity in one cycle. K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e was regenerated through methanol-induced crystallization, supporting material reuse. Overall, the integrated ATPE\u0026ndash;distillation approach offers a high-purity, scalable, and sustainable route for industrial 2,3-BD recovery from FB.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Reagents\u003c/h2\u003e \u003cp\u003eGlucose (99.5%), K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e (99%), MgSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO (99\u0026ndash;102%), and (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e (99.5%) were obtained from Merck, India. D\u003csub\u003e2\u003c/sub\u003eO (99%) and 2,3-BD (99%) were procured from Sigma-Aldrich, USA. MgCl\u003csub\u003e2\u003c/sub\u003e\u0026middot;6H\u003csub\u003e2\u003c/sub\u003eO (99\u0026ndash;102%) and CaCl\u003csub\u003e2\u003c/sub\u003e\u0026middot;6H\u003csub\u003e2\u003c/sub\u003eO (97%) were procured from Fisher Scientific, USA. Cyclohexane, butyl acetate, methanol, 2-ethyl-1-hexanol, IBA, and ethyl acetate (all \u0026ge;\u0026thinsp;99%) were obtained from SRL, India. The \u003cem\u003eBacillus licheniformis\u003c/em\u003e strain was acquired from MTCC, Chandigarh, India. All experiments used Millipore-purified distilled water.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Fermentation Studies\u003c/h2\u003e \u003cp\u003eIn this study, \u003cem\u003eBacillus licheniformis\u003c/em\u003e was used to produce 2,3-BD in a FB prepared with glucose and basal salt medium as per [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Fermentation was carried out at 37\u0026deg;C, pH 6, with agitation at 250 rpm, using a 5% (v/v) inoculum at OD 1, for 48 hr. After centrifugation, the supernatant contained 76.5 g/L of 2,3-BD. The corresponding chromatogram is shown in \u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Analytical Techniques\u003c/h2\u003e \u003cp\u003eGas chromatography (450GC, Bruker, USA) fitted with a CP Wax 57CB column, and FID was used to analyze the FB. The oven was initially set to 60\u0026deg;C for 1 min before being heated to 220\u0026deg;C at a rate of 20\u0026deg;C/min. Nitrogen at 30 mL/min was used as the carrier gas, and the injector and detector temperatures were set to 220\u0026deg;C and 250\u0026deg;C. Quantification used a 2,3-BD calibration curve (0.2\u0026ndash;1 g/L) showing excellent linearity (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;\u0026asymp;\u0026thinsp;0.999) (\u003cb\u003eFig. S2)\u003c/b\u003e. The purity of recovered 2,3-BD was verified by NMR spectroscopy (Bruker ASCEND 600 MHz) and GC. For NMR, a 250 \u0026micro;L sample was mixed with 250 \u0026micro;L D\u003csub\u003e2\u003c/sub\u003eO, then analyzed by \u003csup\u003e1\u003c/sup\u003eH NMR (16 scans, 1 s relaxation) and \u003csup\u003e13\u003c/sup\u003eC NMR (1000 scans, 2 s relaxation) to ensure accurate structural characterization.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. ATPE-based Extraction of 2,3-BD from FB\u003c/h2\u003e \u003cp\u003eA measured amount of salt was first dissolved in the FB to form a uniform aqueous phase, after which the solvent was added at a 1:1 ratio and mixed for 10 min. The mixture was then transferred to a separating funnel, the phases were allowed to settle, and the organic layer was collected. This solvent-rich phase containing 2,3-BD was distilled or evaporated to recover the solvent and concentrate the product. GC analysis was performed at each stage to monitor 2,3-BD content and purity. For distribution studies, the same procedure was repeated using different organic solvents (butyl acetate, 1-octanol, butanol, ethyl acetate, isoamyl alcohol, methanol, IBA, cyclohexane) and FB pre-saturated with 20% (w/v) of various salts (K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, MgSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO, MgCl\u003csub\u003e2\u003c/sub\u003e\u0026middot;6H\u003csub\u003e2\u003c/sub\u003eO, CaCl\u003csub\u003e2\u003c/sub\u003e\u0026middot;6H\u003csub\u003e2\u003c/sub\u003eO). Extraction performance was evaluated using extraction yield (\u003cem\u003eY\u003c/em\u003e) (Eq.\u0026nbsp;\u003cspan refid=\"Equ1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and distribution coefficient (\u003cem\u003eD\u003c/em\u003e) (Eq.\u0026nbsp;\u003cspan refid=\"Equ2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) to identify the most effective solvent\u0026ndash;salt system.\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$\\:Y=\\frac{{C}_{O}\\times\\:{V}_{O}}{{C}_{FB}\\times\\:{V}_{FB}}\\times\\:100$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Equ2\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ2\" name=\"EquationSource\"\u003e\n$$\\:D=\\frac{{C}_{O}\\times\\:{V}_{O}}{{C}_{W}\\times\\:{V}_{W}}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e2\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eHere, C\u003csub\u003eO,\u003c/sub\u003e C\u003csub\u003eW,\u003c/sub\u003e and C\u003csub\u003eFB\u003c/sub\u003e denote concentrations of 2,3-BD in the organic and aqueous phase, and FB, while V\u003csub\u003eFB,\u003c/sub\u003e V\u003csub\u003eW,\u003c/sub\u003e and V\u003csub\u003eO\u003c/sub\u003e represent their corresponding volumes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Optimization and Statistical Analysis\u003c/h2\u003e \u003cp\u003eA three-factor, three-level 3\u003csup\u003e3\u003c/sup\u003e CCD combined with RSM was employed to optimize the extraction of 2,3-BD. The effects of salt concentration (10\u0026ndash;40% w/v), temperature (20\u0026ndash;60\u0026deg;C), and solvent fraction (10\u0026ndash;50% v/v) on the extraction yield (Y) were investigated. Design-Expert 13 software generated 20 experimental runs with distinct combinations of parameters. One-way ANOVA with a significance level of p\u0026thinsp;\u0026le;\u0026thinsp;0.05 was employed to analyze the effects of each variable and their combined interactions. Model reliability was assessed through R\u003csup\u003e2\u003c/sup\u003e and adjusted R\u003csup\u003e2\u003c/sup\u003e values. The relationship between process variables and extraction yield was modeled using a second-order polynomial regression (Eq.\u0026nbsp;\u003cspan refid=\"Equ3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) to help determine the best extraction conditions.\u003cdiv id=\"Equ3\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ3\" name=\"EquationSource\"\u003e\n$$\\:Y={\\alpha\\:}_{0}+\\sum\\:_{i=1}^{n}{\\alpha\\:}_{i}{X}_{i}+\\sum\\:_{i=1}^{n}{\\alpha\\:}_{ij}{X}_{i}{X}_{j}+\\sum\\:_{i=1}^{n}{\\alpha\\:}_{ii}{X}^{2}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e3\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eIn this regression equation, α₀ denotes the intercept, whereas α\u003csub\u003ei\u003c/sub\u003e, α\u003csub\u003ei\u003c/sub\u003e\u003csub\u003ei\u003c/sub\u003e, and α\u003csub\u003ei\u003c/sub\u003eⱼ correspond to the linear, quadratic, and interaction effects. The response variable Y represents the 2,3-BD \u003cem\u003eY\u003c/em\u003e, and X\u003csub\u003ei\u003c/sub\u003e and Xⱼ are the coded independent factors (with i\u0026thinsp;\u0026ne;\u0026thinsp;j). Optimal process conditions were identified using a desirability function (d), which ranges from 0 (unacceptable) to 1 (fully meets criteria). A d value of 1 signifies ideal conditions for maximizing 2,3-BD extraction while satisfying all constraints [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Upscaling the IATPED-based Process for Recovering 2,3-BD\u003c/h2\u003e \u003cp\u003eFor scale-up, a 10 L extraction column (LLU10, Shiva Scientific, India) was connected to a batch distillation setup (Model 9600, BR Instruments, USA). The column used for separation was 21 mm in diameter and 60 cm tall, equipped with 50 theoretical stages and a Teflon spinning band to enhance component separation and facilitate solvent recovery. The system supported boiler capacities of 1 to 50 L and allowed adjustable reflux ratios control range from 0:1 to 999:1. It operated in both manual and automated modes, enabling precise control suitable for large-scale 2,3-BD recovery.\u003c/p\u003e \u003cp\u003eIn the scaled-up experiment, 25% (w/v) K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e was added to 1 L of FB and stirred until fully dissolved. Then, 30% (v/v) IBA was introduced, and the mixture was stirred for 10 min at 40\u0026deg;C to facilitate extraction. After natural phase separation, the organic layer containing 2,3-BD was collected. This extract was then distilled under atmospheric conditions using a spinning band column. The chiller was set to 25\u0026deg;C, and heating was applied at 50% power to evaporate IBA, allowing for the efficient recovery and concentration of purified 2,3-BD.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Distribution Characteristics of 2,3-BD in ATPS\u003c/h2\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e highlights the partitioning of 2,3-BD across various organic solvents and salt combinations. Hydrophilic solvent\u0026ndash;salt systems showed significantly higher \u003cem\u003eD\u003c/em\u003e values (0.38\u0026ndash;7.3) and \u003cem\u003eY\u003c/em\u003e values (25.3\u0026ndash;87.5%) compared to hydrophobic systems. In ATPS, 2,3-BD tends to migrate predominantly into the solvent-rich layer. In contrast, hydrophobic solvents resulted in poor extraction, with 2,3-BD remaining mostly in the salt-rich aqueous phase, indicating the limited effectiveness of these solvents for selective 2,3-BD recovery.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDistribution of 2,3-BD across different solvent/salt systems.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOrganic solvent/Inorganic salt\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMgSO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eK\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e(NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCaCl\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eMgCl\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eMethanol\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eD\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1.03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eY(%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e44.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e72.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e77.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e40.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e39.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eIsobutanol\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eD\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e7.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1.16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eY(%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e66.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e87.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e80.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e42.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e42.25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eButyl acetate\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eD\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1.06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eY(%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e53.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e66.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e56.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e44.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e45.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eEthyl acetate\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eD\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1.16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eY(%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e46.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e76.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e62.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e49.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e48.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eButanol\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eD\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1.27\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eY(%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e51.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e78.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e72.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e44.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e51.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003e2-Ethyl-1-hexanol\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eD\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1.13\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eY(%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e54.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e79.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e69.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e48.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e42.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003e1-octanol\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eD\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.51\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eY(%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e35.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e46.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e38.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e31.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e30.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eCyclohexane\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eD\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.38\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eY(%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e34.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e37.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e35.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e35.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e33.56\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eIsoamyl alcohol\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eD\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.43\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eY(%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e25.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e45.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e38.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e39.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e32.25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003eVolume of FB: 5 mL; Salts concentration (% w/v):20; Volume of solvent: 5 mL; Concentration of 2,3-BD in FB: 76.5 g/L; Mixing duration: 15 min\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe distribution of 2,3-BD is highly influenced by the salt composition, with anions like phosphate (PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e3\u0026minus;\u003c/sup\u003e) and sulfate (SO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e) having a greater impact than cations such as magnesium (Mg\u003csup\u003e2+\u003c/sup\u003e) and calcium (Ca\u003csup\u003e2+\u003c/sup\u003e) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. These highly charged anions enhance ATPS formation, improving 2,3-BD\u0026rsquo;s selective partitioning into the solvent phase. CaCl\u003csub\u003e2\u003c/sub\u003e and MgCl\u003csub\u003e2\u003c/sub\u003e effectively formed ATPS with all tested solvents, yielding notable 2,3-BD extraction, especially with hydrophilic solvents and some hydrophobic ones like octanol, cyclohexane and isoamyl alcohol. The maximum \u003cem\u003eY\u003c/em\u003e and \u003cem\u003eD\u003c/em\u003e were observed with IBA combined with K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e and (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e. Adding salts during extraction significantly increased \u003cem\u003eY\u003c/em\u003e and \u003cem\u003eD\u003c/em\u003e compared to using IBA alone, reaching maximum values of \u003cem\u003eD\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.3 and \u003cem\u003eY\u003c/em\u003e\u0026thinsp;=\u0026thinsp;87.5% with K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, and \u003cem\u003eD\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.8 and \u003cem\u003eY\u003c/em\u003e\u0026thinsp;=\u0026thinsp;80.5% with (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e. This improvement is attributed to the salt\u0026rsquo;s ability to increase water solubility, thereby decreasing 2,3-BD\u0026rsquo;s hydrophilicity and facilitating its transfer into the organic phase. Salt addition thus improves extraction efficiency, guiding the optimization of solvent-salt systems for industrial 2,3-BD recovery.\u003c/p\u003e \u003cp\u003eSalts enhance phase separation by weakening water\u0026ndash;2,3-BD hydrogen bonding, decreasing its solubility in the aqueous phase and driving it into the organic layer. High-charge ions lower water\u0026rsquo;s dielectric constant and enhance hydration, intensifying the salting-out effect; this depends on ion charge, ionic strength, and salt concentration [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Using (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO₄ sometimes caused a hazy interface due to protein and amino acid precipitation, while K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e produced a clear, rapidly forming phase boundary, indicating cleaner separation. Unlike IBA, which forms azeotropes requiring decanters, salts enable fast, efficient phase separation without azeotrope formation. Thus, salt addition offers a more practical, cost-effective alternative for 2,3-BD extraction by simplifying separation and reducing equipment needs [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe IBA/K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e ATPS was chosen for subsequent experiments owing to its notably rapid phase formation, which facilitates efficient separation within a shorter time frame. Additionally, this system offers the advantage of low recycling costs, making it economically favorable for repeated use in large-scale operations. These combined benefits, swift phase separation coupled with cost-effectiveness, make the IBA/K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e ATPS an ideal candidate for further detailed investigation and process optimization in the extraction of 2,3-BD.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Optimization of Extraction Conditions\u003c/h2\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e summarizes the observed and predicted \u003cem\u003eY\u003c/em\u003e of 2,3-BD across various experimental conditions during optimization. Yields ranged from 43.09% to 98.23%, averaging 75.4%. The maximum yield (98.23%) was attained at 25% (w/v) salt, 30% (v/v) solvent, and 40\u0026deg;C (Run 12), while the lowest (43.09%) was at 10% salt, 10% solvent, and 25\u0026deg;C (Run 17). The regression model showed excellent accuracy, with an R\u003csup\u003e2\u003c/sup\u003e of 0.9997, predicted R\u003csup\u003e2\u003c/sup\u003e of 0.9985, and adjusted R\u003csup\u003e2\u003c/sup\u003e of 0.9994, indicating strong predictive capability. As shown in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, ANOVA results validated the model\u0026rsquo;s significance, indicated by a high F-value of 3673.64 and a p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.0001. These findings demonstrate the model\u0026rsquo;s reliability in predicting and optimizing 2,3-BD \u003cem\u003eY\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCCD-RSM-based summary of parameters and response data for 2,3-BD extraction from FB.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eRun\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSolvent concentration(% v/v)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSalt concentration(%w/v)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTemperature(\u0026deg;C)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eActual yield(%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ePredicted yield (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eB\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eExperimental\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e72.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e71.87\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e65.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e65.21\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e63.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e63.89\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e52.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e51.93\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e98.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e97.81\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e68.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e67.96\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e54.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e54.31\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e98.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e97.81\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e95.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e95.70\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e43.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e43.21\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e72.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e72.84\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e98.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e97.81\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e65.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e66.16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e83.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e83.66\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e98.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e97.81\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e98.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e97.81\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e43.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e43.30\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e75.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e75.03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e88.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e88.89\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e98.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e97.81\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eBest-fit RSM model adequacy and ANOVA summary for 2,3-BD extraction from FB.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSource\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSum of Squares\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003edf\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMean Square\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eF-value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ep-value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eModel\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e7108.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e789.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3673.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eSignificant\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA-Solvent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1063.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1063.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4948.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eB-Salt\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e203.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e203.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e946.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC-Temp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.7358\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.7358\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.094\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e9.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.0105\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.985\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.985\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.058\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e12.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.0057\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA\u0026sup2;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e661.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e661.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3078.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eB\u0026sup2;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e885.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e885.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4120.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC\u0026sup2;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e912.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e912.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4244.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eResidual\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.215\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLack of Fit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.2647\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.309\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNot insignificant\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePure Error\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.8265\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.1653\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCor Total\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e7110.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eSalt concentration, solvent volume, and extraction temperature all significantly affected 2,3-BD yield (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Solvent had the greatest impact (F\u0026thinsp;=\u0026thinsp;4948.57), followed by salt (F\u0026thinsp;=\u0026thinsp;946.17), while temperature had a minor effect (F\u0026thinsp;=\u0026thinsp;3.42). The model\u0026rsquo;s \u0026ldquo;lack of fit\u0026rdquo; was insignificant (F\u0026thinsp;=\u0026thinsp;1.6), indicating only a 30.9% chance that variability was due to random error, confirming model reliability. Based on these results, \u003cb\u003eEq.\u0026nbsp;(4)\u003c/b\u003e was developed from Eq.\u0026nbsp;(\u003cspan refid=\"Equ3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) to predict 2,3-BD yield using the key variables, providing a useful tool for optimizing extraction conditions.\u003c/p\u003e \u003cp\u003eY\u0026thinsp;=\u0026thinsp;98.06\u0026thinsp;+\u0026thinsp;10.13A\u0026thinsp;+\u0026thinsp;4.42B \u0026ndash; 0.2637C \u0026ndash; 0.51AB\u0026thinsp;+\u0026thinsp;0.35AC \u0026ndash; 0.5729BC -12.19A\u003csup\u003e2\u003c/sup\u003e \u0026ndash; 13.42B\u003csup\u003e2\u003c/sup\u003e \u0026ndash; 14.14C\u003csup\u003e2\u003c/sup\u003e (4)\u003c/p\u003e \u003cp\u003eThe close alignment of the coefficient of variation (CV%) and predicted R\u003csup\u003e2\u003c/sup\u003e values (\u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e of Supplementary Information) confirms the model\u0026rsquo;s robustness. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea shows an excellent linear correlation between the predicted and experimental 2,3-BD yields, with an average deviation of just 0.6058%. The normal probability plot in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb shows that the residuals follow a normal distribution, supporting the validity of the regression assumptions. In Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec, the residuals are randomly scattered against the predicted values, indicating uniform variance. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed shows no trend between residuals and run order, confirming the absence of systematic errors. Overall, these diagnostic evaluations, carried out in Design Expert using CCD data, demonstrate that the model satisfies all statistical requirements and can reliably estimate and optimize 2,3-BD extraction.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows 3D surface and contour plots illustrating the regression model\u0026rsquo;s predictions for 2,3-BD extraction yield respond when two factors are varied simultaneously while the third parameter remains fixed. These plots reveal how the key parameters interact and influence extraction efficiency. Figures\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea \u0026amp; \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed illustrate how solvent concentration (B) and salt concentration (A) interact, with the maximum yield occurring at 30% solvent and 25% salt (Run 5). This trend indicates a cooperative effect between the two factors that enhances the partitioning of 2,3-BD. Figures\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb \u0026amp; \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee illustrate the effect of solvent concentration (A) and temperature (C), with the maximum yield at 30% salt and 40\u0026deg;C (Run 12), emphasizing the role of temperature alongside salt in extraction performance. Figures\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec \u0026amp; \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef depict salt concentration (B) and temperature (C) interaction, showing the best extraction at 25% salt and 40\u0026deg;C (Run 15), suggesting that higher solvent levels and temperature enhance 2,3-BD recovery. Overall, these plots provide a clear visualization of variable interactions, aiding in identifying optimal conditions for improved 2,3-BD extraction in the ATPS.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDesign Expert determined that the best extraction conditions were 25% (w/v) salt, 30% solvent, and a temperature of 40\u0026deg;C. Under these settings, the model projected a maximum 2,3-BD yield of 98.23%, which is 0.42% higher than the theoretical estimate. Validation experiments confirmed an actual yield of 97.81%, closely matching predictions and demonstrating the model\u0026rsquo;s accuracy and reliability. These findings confirm the effectiveness of the optimized conditions and offer a solid foundation for scaling up 2,3-BD recovery from FB, enhancing both process efficiency and industrial viability.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.3. 2,3-BD purification from FB\u003c/h2\u003e \u003cp\u003eComprehensive optimization of the ATPE system was conducted to identify the most effective conditions for maximizing the 2,3-BD Y from the FB. After extraction, the organic-rich phase containing 2,3-BD was purified through a distillation process, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Material balance calculations, presented in \u003cb\u003eTable S2\u003c/b\u003e, revealed that the IATPED approach obtained an impressive 2,3-BD \u003cem\u003eY\u003c/em\u003e of 98.31% and a recovery rate of 98.02%, with the final product exhibiting a purity greater than 99% in a single extraction\u0026ndash;distillation cycle. In addition to the high product recovery, the process also demonstrated excellent solvent recyclability, achieving 97% solvent recovery with a purity exceeding 97%. \u003cb\u003eFig. S3\u003c/b\u003e shows the chromatogram of the purified 2,3-BD, confirming the effectiveness of the purification process.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWhile distillation offers a relatively straightforward method for recovering IBA, the recycling of salts within this ATPS presents a significant challenge. The study focused on the recovery and reuse of K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, which predominantly accumulates in the aqueous (bottom) phase. During the distillation of the organic phase, there exists a potential risk of K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e crystallizing out, which may interfere with phase separation and system efficiency. To address this, a recovery strategy based on methanol-induced crystallization was explored, as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. This method involves the dilution of the salt-rich aqueous phase with methanol to promote selective crystallization of K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e. Experimental results demonstrated that increasing the methanol-to-water phase volume ratio from 0.5 to 2 led to a significant improvement in salt recovery, from 74.75% up to a maximum of 96.85%. However, a further increase in the volume ratio beyond 2.5 resulted in a decline in recovery efficiency, likely due to solubility limitations and co-dissolution effects [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Consequently, a methanol/water volume ratio of 2 was determined as the optimal condition for efficient and reproducible salt recovery, balancing both crystallization yield and process feasibility.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eEarlier studies report ATPE-based 2,3-BD recovery efficiencies typically between 94.5% and 98.18%. For instance, Li et al. achieved 91.7% using 16% (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e and 32% EtOH [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Narisetty et al. reported a 97% yield with a \u003cem\u003eD\u003c/em\u003e of 45 using 50% isopropanol and 30% (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], while Sun et al. obtained 93.7% with 20% (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e and 34% 2-propanol [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. This study\u0026rsquo;s IBA/K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e-based ATPS showed superior extraction yield, recovery, and purity compared to these methods (\u003cb\u003eTable S3\u003c/b\u003e), demonstrating its potential as a more efficient, scalable, and cost-effective approach for industrial 2,3-BD recovery.\u003c/p\u003e \u003cp\u003eThis work demonstrates that an IBA/K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e ATPS is a feasible option for efficient extraction of 2,3-BD from FB. The method achieves high recovery and supports straightforward reuse of IBA through distillation and K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e through methanol-triggered crystallization, minimizing material usage. Owing to its simplicity and scalability, the system offers strong potential for industrial production of bio-derived 2,3-BD.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Recovered 2,3-BD: Structural and Purity Characterization\u003c/h2\u003e \u003cp\u003eThe purity and molecular structure of the purified 2,3-BD were thoroughly analyzed by NMR, with the results presented in \u003cb\u003eFig. S4\u003c/b\u003e. Both carbon-13 (\u003csup\u003e13\u003c/sup\u003eC) and proton (\u003csup\u003e1\u003c/sup\u003eH) NMR techniques were employed for comprehensive structural characterization, using commercially available 2,3-BD as the reference standard for comparison. The \u003csup\u003e13\u003c/sup\u003eC NMR spectra of the purified sample and commercial standard exhibited two distinct and well-defined peaks at chemical shifts of 17.3 ppm and 71.2 ppm, which were attributed to the methyl (\u0026ndash;CH\u003csub\u003e3\u003c/sub\u003e) and methine (\u0026ndash;CH) carbons, respectively, confirming the expected carbon environment of 2,3-BD. In the \u003csup\u003e1\u003c/sup\u003eH NMR spectrum, the methine proton (\u0026ndash;CH) appeared as a quartet at 2.52 ppm, whereas the methyl protons (\u0026ndash;CH\u003csub\u003e3\u003c/sub\u003e) were detected as a doublet at 1.01 ppm. Additionally, a prominent peak at 4.79 ppm is attributed to the residual signal of D\u003csub\u003e2\u003c/sub\u003eO, the solvent employed in the NMR measurement. These spectral features collectively confirm both the high purity and the correct molecular structure of the purified 2,3-BD.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003eThis work explored the extraction of 2,3-BD using various organic solvents and inorganic salt-based ATPS, ultimately identifying the IBA/K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e system as the most advantageous due to its low cost and straightforward solvent and salt recyclability. Through CCD and RSM, the key operating factors—salt level, solvent percentage, and temperature—were optimized, each showing a significant influence on 2,3-BD recovery from the FB. The resulting second-order polynomial models reliably captured the interactions between these parameters and the extraction yield. Under the optimal conditions of 30% (v/v) IBA, 40 °C, and 25% (w/v) K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, the system achieved a maximum distribution coefficient of 60.47 and an extraction efficiency of 98.31%. The organic-rich layer was subsequently distilled to concentrate the product. Scale-up trials confirmed the robustness of the process, delivering extraction and recovery efficiencies of 98.31% and 98.04%, respectively, and achieving over 99% purity in a single purification step. Furthermore, 96.85% of K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003ewas recovered from the aqueous phase through methanol-induced crystallization, contributing to reduced resource consumption and operational costs. Overall, this study demonstrates an efficient, scalable, and economical method for 2,3-BD recovery, suitable for industrial use with reduced waste and lower costs.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePramod Madhukar Gawal\u003c/strong\u003e:Conceptualization, Investigation, Formal analysis, Data curation, software, Writing- original draft, review \u0026amp; editing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSweta Lataye\u003c/strong\u003e: Conceptualization, Investigation, Formal analysis, Software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e: The authors declare that the\u0026nbsp;data\u0026nbsp;supporting the findings presented are available within the manuscript and its supporting information file. Additional data, or raw data files, will be available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors acknowledge the Central Instruments Facility and the Analytical Laboratory of the Department of Chemical Engineering at IIT Guwahati.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eNo other funding has been consumed for the compilation of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval:\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate:\u0026nbsp;\u003c/strong\u003eThere is no involvement of human subjects in the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for Publication:\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests:\u0026nbsp;\u003c/strong\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHaider, J., Harvianto, G. 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Investigation of possible solubility and dissolution advantages of cocrystals, I: Aqueous solubility and dissolution rates of ketoconazole and its cocrystals as functions of pH. \u003cem\u003eADMET and DMPK\u003c/em\u003e, \u003cem\u003e7\u003c/em\u003e(2), 106\u0026ndash;130. https://doi.org/10.5599/admet.661\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"2,3-Butanediol recovery, Bioprocess downstream purification, Isobutanol/K2HPO4 system, Extraction-distillation integration, Process optimization","lastPublishedDoi":"10.21203/rs.3.rs-8416826/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8416826/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRecovering 2,3-butanediol (2,3-BD) from fermentation broth is challenging due to its complex composition and impurities. This work develops an integrated aqueous two-phase extraction–distillation method to improve recovery efficiency. Screening of multiple aqueous two-phase systems identified isobutanol/K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4 \u003c/sub\u003eas the most effective based on distribution behavior. A CCD-RSM design was used to optimize salt concentration, temperature, and solvent content, yielding ideal conditions of 25% (w/v) K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, 40 °C, and 30% (v/v) isobutanol. Under these settings, the system achieved a distribution coefficient of 60.47 and an extraction efficiency of 98.23%, slightly higher (0.42%) than model predictions. The enriched extract was subsequently concentrated by distillation, and scale-up runs delivered 98.31% extraction and 98.02% recovery with product purity exceeding 99% in a single cycle. Methanol-assisted crystallization enabled 96.85% recovery of K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e from the aqueous stream. Overall, this cost-efficient and scalable process enhances 2,3-BD separation while facilitating solvent and salt reuse for industrial applications.\u003c/p\u003e","manuscriptTitle":"Enhanced 2,3-Butanediol Purification Using a Hybrid Extraction Process","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-06 06:46:17","doi":"10.21203/rs.3.rs-8416826/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0061169c-1e3b-4dbb-8928-e7380b7d93b7","owner":[],"postedDate":"January 6th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-06T06:46:17+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-06 06:46:17","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8416826","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8416826","identity":"rs-8416826","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}