Alcohol/salt Aqueous Two-phase System Research Articles

Aqueous two-phase systems coupled with chemometrics-enhanced HPLC-DAD for simultaneous extraction and determination of flavonoids in honey.

In this study, an accurate, rapid, green, and environment friendly method for the extraction and quantitative analysis of flavonoids in honey was established by using the aqueous two-phase extraction combined with the chemometrics-assisted HPLC-DAD. The first purpose of this study was to extract seven flavonoids in five different types of honey using alcohol/salt aqueous two-phase system (ATPS). The system with 2.82mL sodium citrate (30%), 1.58mL water, and 3.10mL isopropanol, showed the highest flavonoids extraction yields in the top phase (87.66-101.50%). Additionally, the three-way array of honey samples based on HPLC-DAD was decomposed mathematically by the alternating trilinear decomposition (ATLD) algorithm to obtain reasonable chromatograms, spectra, and concentration profiles for each analyte. Compared with the traditional solid-phase extraction method, the ATPS-ATLD-based method showed satisfactory spiked recoveries, lower limit of detection, and higher sensitivity, further verifying its accuracy and stability.

Purification of the recombinant green fluorescent protein from tobacco plants using alcohol/salt aqueous two-phase system and hydrophobic interaction chromatography

BackgroundThe green fluorescent protein (GFP) has been regarded as a valuable tool and widely applied as a biomarker in medical applications and diagnostics. A cost-efficient upstream expression system and an inexpensive downstream purification process will meet the demands of the GFP protein with high-purity.ResultsThe recombinant GFP was transiently expressed in an active form in agoinoculated Nicotiana benthamiana leaves by using Tobacco mosaic virus (TMV) RNA-based overexpression vector (TRBO). The yield of recombinant GFP was up to ~ 60% of total soluble proteins (TSP). Purification of recombinant GFP from the clarified lysate of N. benthaniana leaves was achieved by using an alcohol/salt aqueous two-phase system (ATPS) and following with a further hydrophobic interaction chromatography (HIC). The purification process takes only ~ 4 h and can recover 34.1% of the protein. The purity of purified GFP was more than 95% and there were no changes in its spectroscopic characteristics.ConclusionsThe strategy described here combines the advantages of both the economy and efficiency of plant virus-based expression platform and the simplicity and rapidity of environmentally friendly alcohol/salt ATPS. It has a considerable potential for the development of a cost-efficient alternative for production of recombinant GFP.

Separation and determination of tetracycline hydrochloride in real water samples using binary small molecule alcohol-salt aqueous two-phase system coupled with high-performance liquid chromatography.

An environmental and sensitive sample pretreatment method was established and combined with high-performance liquid chromatography (HPLC) for the analysis of tetracycline hydrochloride (TC) in feed water and lake water. One element small molecule alcohol-salt aqueous two-phase system (ATPS) cannot effectively adjust the polarity of the system, but binary small molecule alcohol-salt ATPS can adjust the polarity and improve the extraction efficiency of antibiotics. In this work, a binary ATPS based on ethanol +2-propanol+(NH4 )2 SO4 system was formed and applied to the separation and purification of TC in real water samples. The influence factors on partition behaviors of TC were discussed, including the types and the concentration of phase salts, the volume ratio of alcohol, the pH value, extraction temperature, and the standing time. The response surface methodology was used to determine the best experimental conditions for multi-factor experiments. Under this optimal condition, the extraction efficiency of TC reached 95.7%. This new method is considered to have significant application in the divorce of antibiotics.

Separation/Extraction/Detection of Chloramphenicol Using Binary Small Molecule Alcohol-Salt Aqueous Two-phase System Coupled with High-performance Liquid Chromatography

A green and sensitive sample separation and purification method coupled with high-performance liquid chromatography(HPLC) was developed for the analysis of chloramphenicol(CAP). One element small molecule alcohol-salt aqueous two-phase system(ATPS) can’t effectively adjust the polarity of the system, but binary small molecule alcohol-salt ATPS can adjust the polarity and improve the extraction efficiency of antibiotics. A binary aqueous two-phase system based on 1-propanol+2-propanol and NaH2PO4 system was formed and applied to the separation and purification of trace CAP in real samples. The influence factors on partition behaviors of CAP were discussed, including the types and mass of salts, the volume ratio of alcohol, the pH, temperature and the standing time. The optimal condition was found at pH=5.0, 2.5 g of NaH2PO4, 3.0 mL of 1-propanol and 2-propanol(volume ratio 1:1) and 30 °C by using response surface methodology. Under this optimal condition, the extraction efficiency of CAP reached 98.91%, and partition coefficient of CAP was 17.31.

Recovery of human interferon alpha-2b from recombinant Escherichia coli using alcohol/salt-based aqueous two-phase systems

The purification of intracellular human recombinant interferon-alpha2b (IFN-α2b) from Escherichia coli (E. coli) was studied using alcohol/salt aqueous two-phase system (ATPS). The influences of nine biphasic systems comprising alcohol-based top phase (ethanol, 1-propanol and 2-propanol) and salt-based bottom phase (ammonium sulfate, di-potassium hydrogen phosphate and monosodium citrate) on IFN-α2b purification were studied. The results showed that the optimum condition for purification of IFN-α2b was achieved in ATPS composed of 18% (w/w) 2-propanol with 22% (w/w) ammonium sulfate in the presence of 1% (w/w) sodium chloride (NaCl). The purified IFN-α2b recorded a purification factor (PF) of 16.24 with the yield of 74.64%.

Extraction and purification of anthraquinones derivatives from Aloe vera L. using alcohol/salt aqueous two-phase system

An alcohol/salt aqueous two-phase system (ATPS) composed of 1-propanol and (NH4)2SO4 was employed to purify anthraquinones (AQs) extracted from Aloe vera L. The main influencing system parameters such as type of alcohol, type and concentration of salt, temperature and pH were investigated in detail. Under the optimal extraction conditions, AQs can be extracted into alcohol-rich phase with high extraction efficiency, meanwhile majority polysaccharides, proteins, mineral substances and other impurities were extracted into salt-rich phase. Partitioning of AQs is dependent on hydrophobic interaction, hydrogen bond interaction, and salting-out effect in ATPS. Temperature also played a great role in the partitioning. After ATPS extraction, alcohol can be recycled by evaporation; moreover, salt can be recycled by dilution crystallization method. Compared with other liquid-liquid extractions, this alcohol/salt system is much simpler, lower in cost with easier recovery of phase-forming components, which has the potential scale-up in down-processing of active ingredients in plant.

Aqueous Two-Phase Extraction of Lactic Acid: Optimization by Response Surface Methodology

In this study a suitable alcohol/salt aqueous two-phase (ATP) system was selected for the recovery of lactic acid from an aqueous solution. From the different ATP systems studied, the ethanol/dipotassium hydrogen phosphate ATP system appeared to be favorable. To examine the potential of this ATP system, the extraction yield of lactic acid in aqueous solutions was optimized with the response surface methodology. The parameters studied were concentrations of ethanol (22.00–38.80%, w/w), dipotassium hydrogen phosphate (15.00–31.80%, w/w) and lactic acid (26.36–93.64 g/L). The optimum conditions were found to be 30.23% w/w ethanol, 18.40% w/w dipotassium hydrogen phosphate, and 80 g/L lactic acid. Under these conditions, a favorable extraction yield of lactic acid was obtained. The maximum partition coefficient of lactic acid and extraction yield was determined as 2.26 and 87%, respectively. The optimum extraction conditions were then used to guide the recovery of lactic acid from a real fermentation broth. As a result, the partition coefficient and extraction yield of lactic acid reached 2.06–80%, respectively.

Partition behavior and partition mechanism of antibiotics in ethanol/2-propanol–ammonium sulfate aqueous two-phase systems

In this paper, the feasibility of applying the water-miscible alcohol–salt aqueous two-phase system to separate small molecular antibiotics in aqueous solution was proved. The partition behavior of tetracycline hydrochloride in the ethanol/2-propanol–(NH 4) 2SO 4 aqueous two-phase system was investigated by determining partition coefficients and extraction efficiency. The partition coefficient and extraction efficiency of tetracycline hydrochloride in the ethanol–(NH 4) 2SO 4 ATPS could reach 12.51% and 75.77% respectively at pH 2.00 and 0.50 g/mL (NH 4) 2SO 4, furthermore, that of tetracycline hydrochloride in the 2-propanol–(NH 4) 2SO 4 ATPS could reach 30.03% and 87.70% respectively at pH 1.52 and 0.50 g/mL (NH 4) 2SO 4. The target was found to partition preferentially into the alcohol-rich phase in its cation form at pH < 4.0. The effects of the concentration of salt, the type of alcohol, pH and temperature were discussed in detail. The partition of target to the top phase was enhanced by decreasing pH from its isoelectric point of 5.4 and increasing the concentration of salt and temperature. Another objective of this study was to discuss the underlying mechanism governing the uneven partition of target and phase-separation. The experimental phenomenon and results both indicate that the salting-out effect of salt is the driving force for the uneven partition of target, so the salting-out effect of salts on the separation of target cannot be neglected in discussing aqueous two-phase extraction mechanism.

Selection and optimization of an aqueous two‐phase system for the recovery of 1,3‐propandiol from fermentation broth

AbstractIn this study a suitable alcohol/salt aqueous two‐phase (ATP) system was selected for the recovery of 1,3‐propandiol (1,3‐PD) from fermentation broth. From the different alcohol/salt systems studied the ethanol and dipotassium hydrogen phosphate ATP system appeared to be favorable. To examine the potential of this ATP system the partition coefficient of 1,3‐PD in synthetic solutions was first optimized with the response surface methodology. The parameters studied were concentrations of ethanol (21.99–38.81% w/w), dipotassium hydrogen phosphate (14.99–31.81% w/w) and 1,3‐PD (6.36–73.64 g/L). The optimum conditions were found to be 35.39% w/w for ethanol, 28.40% w/w for dipotassium hydrogen phosphate and 73.6 g/L for 1,3‐PD. Under these conditions the maximum partition coefficient of 1,3‐PD and the extraction yield were determined as 23.14 and 97.82%, respectively. The optimum extraction conditions were then used to guide the recovery of 1,3‐PD from a real fermentation broth. The partition coefficient and extraction yield of 1,3‐PD reached 20.28–97.20% in this case, respectively. A favorable partition of the organic acids lactate, acetate and butyrate in the bottom phase was also achieved. We have also studied the removal of cells and macromolecules from the broth. Removal ratio of cells and proteins were 96.47 and 93.05%, respectively. Thus, the ethanol/dipotassium hydrogen phosphate ATP system appears to be an interesting alternative or can be used as one useful step in the downstream processing of 1,3‐PD from fermentation broth.

In this issue

AbstractMembrane in black for less autofluorescenceWalter et al., Eng. Life Sci. 2010, 10, 103–108.Protein microarrays are an emerging technology for diagnostics and proteomic research. Their performance is highly dependent on the appropriate surface for protein binding. Three‐dimensional substrates like nitrocellulose have a higher binding capacity than planar surfaces and allow immobilization of proteins in a functional manner. Yet, currently used nitrocellulose‐based microarrays suffer from a high background fluorescence which can interfere with the detection of low‐abundance proteins. Therefore, authors from Hanover and Satorius Stedim Biotech in Göttingen (Germany) have developed a black nitrocellulose membrane‐based protein microarray. They measured the limit of detection (LOD) and signal‐to‐noise ratio (SNR) and compared it to several commercially available systems. This new membrane exhibits a lower autofluorescence in combination with increased sensitivity. ……………103http://dx.doi.org/10.1002/elsc.200900078magnified imageDetecting inactivated sporesTalbot et al., Eng. Life Sci. 2010, 10, 109–120.Rapid detection of biological warfare agents (BWA) such as Bacillus anthracis spores is of high importance to prevent widespread contamination and dissemination in case of a terrorist attack. While a wide range of analytical methods are available for the detection and identification of BWAs they are often limited when inactivated samples are analyzed. This is essential when the detection has to take place outside of a bio safety level 3 laboratory. Here, authors from the German Armed Forces in Munster have inactivated spores of the model organism Bacillus atrophaeus by various methods and analysed the detection with RT‐PCR and MALDI‐TOF‐MS. They optimized the inactivation methods that achieve full inactivation while detection of the bacterial spore is still possible. ……………109http://dx.doi.org/10.1002/elsc.200800078magnified imageRecovery of propandiolAydoǧan et al., Eng. Life Sci. 2010, 10, 121–129.1,3‐Propandiol (1,3‐PD) is a bulk chemical used for the fabrication of polymers, cosmetics, foods, lubricants and medicines. The production of 1,3‐PD from renewable resources is an economically attractive process. 1,3‐PD can be produced through fermentation of glycerol by bacteria from several genera including Klebsiella, Citrobacter and Clostridium. Researchers from Ankara, Turkey, and Hamburg, Germany, have studied an alcohol/salt aqueous two‐phase (ATP) system for the recovery of 1,3‐PD from fermentation broth. The ethanol and dipotassium hydrogen phosphate ATP system appeared to be favorable compared with other alcohol/salt systems. The partition coefficient of 1,3‐PD in synthetic solutions was optimized by response surface methodology (RSM) with respect to the concentrations of ethanol, dipotassium hydrogen phosphate and 1,3‐PD. This new method can now be applied as a promising step in downstream processing of 1,3‐PD. ……………121http://dx.doi.org/10.1002/elsc.200900084magnified image