Construction Of Deep Eutectic Solvent-based Aqueous Biphasic Systems And Its Application In The Extraction Of Biomacromolecules And Dyes

Deep Eutectic Solvent?DES?is a eutectic mixture composed of a hydrogen bond donor?quaternary ammonium/phosphorus salt?and hydrogen bond acceptor?amine,alcohol,acid?.As a new generation of green solvent,DES has been widely used in many fields due to its obvious advantages,such as simple preparation and wide source of raw materials etc.Aqueous Biphasic Systems?ABSs?are two-phase systems formed by two water-soluble substances mixed under critical conditions,which is a highly efficient and gentle separation technique.The introduction of DES into ABSs for the separation and analysis of complex sample has provided many new opportunities for the development of modern separation technology.The development of molecular biology and gene therapy relies on biological macromolecules,such as DNA and proteins.The efficient and economical separation between them and other biological substrates is the key to life science research.The DES-based ABSs effectively combine the advantages of DES and ABSs,and showing multiple advantages such as low cost,easy large-scale application and environmentally friendly.It is an excellent platform for separating complex samples,which is not only exhibits superior performance in the separation of biological molecules?such as proteins,nucleic acid and amino acids?,and are being extended to environmental restoration in various ways.In this paper,a variety of DESs were synthesized and applied to construct ABSs.The performance of these systems in the extraction of DNA,proteins and dyes was further explored.The main work is summarized as follows:?1?Deep eutectic solvent and ionic liquid based aqueous biphasic system for the extraction of DNAAqueous biphasic systems based on ionic liquids?ILs?and deep eutectic solvents were developed and utilized to efficiently extract DNA.Four kinds of ILs/DESs were mainly constituted by betaine/carboxylic acid and betaine/carbohydrates respectively.While another DES?[TBAB][PPG 400]?was formed by polypropylene glycol 400?PPG 400?and tetrabutylammonium bromide?TBAB?.The phase-formation ability of the studied ABSs was evaluated by using[TBAB][PPG 400]/ILs,[TBAB][PPG400]/DESs and[TBAB][PPG 400]/inorganic salts.The results revealed that the phase forming ability of ABSs involved with the size of anion alky chain of ILs,the viscosity,the density and the hydrophilicity of DESs,ionic radius and ionic valence of inorganic salts.Then the system comprising[TBAB][PPG 400]/IL was selected to ascertain the optimum extraction conditions for the extraction of DNA by the influence factor experiments.Meanwhile the maximum extraction efficiency could be attained 99.60%.Mixed sample experiments were implemented to separate DNA/cytochrome C?Cyt-c?and DNA/bovine hemoglobin?BHb?,where the DNA mainly partitioned to IL-rich bottom phase.It turned out that the relationship between the isoelectric point of analytes and the pH of the system played an important role in the separation process.The result also showed that the studied system can be applied to selectively separate mixtures of nucleic acids and proteins in a single-step.Moreover,the developed system was successfully applied to the extraction of DNA from bovine whole blood with satisfactory result.Finally,the extraction mechanisms associated with the separation process were explored by FT-IR spectra,circular dichroism spectra?CD?,dynamic light scattering?DLS?,transmission electron microscopy?TEM?.The proposed method provides further possibilities for the selective separation of biomolecules.?2?Carboxylic acid-based deep eutectic solvent aqueous biphasic system for the extraction of lysozymeAqueous Biphasic Systems based on deep eutectic solvents were determined and applied in the extraction of lysozyme from chicken egg white.Tetrabutylammonium bromide and benzyltributylammonium bromide were utilized a s hydrogen-bond acceptors to synthesize six kinds of DESs with different carboxylic acids?such as glycolic acid,Gly?.The phase-formation ability of these DESs was evaluated by combining several salts.The results revealed that the content of hydrophilic group and the alkyl side chain length of the carboxylic acids played a crucial role in phase separation process,and the introduce of the benzyl group for quaternary ammonium salt had an aptitude to promote two-phase splitting.Then the system comprising[TBAB][Gly]and Na₂SO₄ was used to appraise the effect of different experimental parameters on the extraction efficiency,including the amount of DES and salt,the temperature,the values of pH and the ionic strength.More than 98%of lysozyme was transferred into the DES-rich phase at the optimum condition.The activity of lysozyme after the process of extraction still retained 91.73%of initial activity,demonstrating high biocompatibility of the studied system.What's more,the proposed method was successfully utilized for the real sample analysis.Finally,UV-vis,FT-IR,CD,DLS and TEM were employed to investigate the extraction mechanism.The results verify the excellent feasibility of the proposed system in the analysis of biological samples.?3?Hydrophilic and hydrophobic deep eutectic solvents based aqueous biphasic system for the extraction of dyeAqueous Biphasic Systems based on hydrophobic and hydrophilic deep eutectic solvents were established and applied to extract dyes.Decyltrimethylammonium chloride-hexafluoroisopropanol?DeTAC-HFIP?ortetrabutylammonium bromide-polypropylene glycol 400?TBAB-PPG 400?were chosen to formed ABSs with four hydrophilic deep eutectic solvents,where the distribution behavior of lemon yellow,methylene blue and Sudan III in these systems was investigated.The results showed that the difference between the top and bottom phases of the ABSs and the hydrophobicity of dyes played a decisive role in the extraction process.Two groups of systems formed by DeTAC-HFIP/TBAB-PPG 400 and choline chloride-glycerol?ChCl-G?were selected to evaluate the effect of different experimental parameters on the extraction behavior of methylene blue,including the amount of dye,ionic strength,temperature and pH.The results revealed that the extraction efficiency for the system of DeTAC-HFIP/ChCl-G was almost unaffected.However,the system of TBAB-PPG400/ChCl-G was the opposite,where pH greatly changed the distribution behavior of methylene blue.Mixed sample experiments showed that the mixtures of tartrazine/Sudan III and Methylene blue/Sudan III were successfully separated selectively in the system of TBAB-PPG400/ChCl-G.The extraction mechanism was further investigated by conductivity measurement,DLS and TEM.It turned out that the hydrophobic interaction was the main driving force in the extraction of dyes.The proposed method provided a new idea for designing a switchable separation system.