Extractive separation of penicillin G by facilitated transport via carrier supported liquid membranes

The facilitated transport of penicillin G (Pen G), through a supported liquid membrane with Amberlite LA-2 dissolved in 1-decanol, supported on a microporous polypropylene membrane, were studied. The distribution coefficient was obtained from a batch extraction experiment. The effects of flow rate, carrier concentration, initial concentration of Pen G, and the pH of feed and stripping phases on the transport rate of Pen G through the supported liquid membrane were also investigated. The results are in agreement with theoretical predictions, and it is demonstrated that the transport of Pen G through the supported liquid membrane is controlled simultaneously by mass transfer across both aqueous and liquid membranes. (c) 1993 John Wiley & Sons, Inc.     

Separation and concentration of L-phenylalanine using a supported liquid membrane

The separation and concentration of L-phenylalanine (L-Phe) using a supported liquid membrane (SLM) is investigated. A cation complex agent, di-2-ethylhexyl phosphoric acid (D₂EHPA), is used as a carrier in the SLM with n-Heptane as a solvent. The reaction order and equilibrium constant in the formation reaction of L-Phe-carrier complex are obtained from the extraction experment. A mathematical model for a carrier mediated counter transport process is proposed to estimate the diffusion coefficient of L-Phe-carrier complex in the liquid membrane. Permeation experiments of L-Phe using a SLM are performed under various operating conditions and optimum conditions for the transport of L-Phe are obtained. Concentration of L-Phe in the strip phase against its concentration is observed. Transport rate of glucose through liquid membrane is less than that of L-Phe in the competitive transport of L-Phe and glucose. And the existence of glucose reduced the transport rate of L-Phe. The performance of separation with continuous strip phase is increased due to the dilution effect in the strip phase.     

Extraction of volatile fatty acids from diluted aqueous solution using a supported liquid membrane

Experimental and analytical studies on the extraction of volatile fatty acids (VFAs) from an aqueous solution using a supported liquid membrane (SLM) were carried out. Teflon and 20% (w/w) tri-n-octyl phosphine oxide (TOPO) in kerosene were used as the supporting membrane and liquid, respectively. The extraction rate of VFAs transferred from the source across the SLM to the sink was measured. It was observed that only undissociated forms of VFAs can penetrate into the SLM and that the complex formation between TOPO and VFA (1/1 molar ratio) inside the membrane enhanced the transfer rate of VFA across the membrane. This phenomenon was explained by mathematical models based on mass transfer and the chemical reactions occurring inside the membrane, suggesting that diffusion of the VFA-TOPO complex inside the membrane may be the rate-limiting step in this experiment.     

Extraction of organic acids from kiwifruit juice using a supported liquid membrane process

In order to extract or remove organic acids from kiwifruit juice, we evaluated their separation and transport rates through supported liquid membranes (SLMs). The liquid membrane consisted of an organic solution composed of a carrier (Aliquat 336/Alamine 336) and a linear alcohol (oleyl alcohol) and was loaded on a microporous polypropylene support (commercial grade Celgard 2500/2400). These SLMs were evaluated (i) in a batch cell to determine the permeability and (ii) in a continuous spiral membrane module to study the effects of various process parameters – flow of feed and strip solutions, membrane composition, recycling mode of operation and kiwifruit juice at natural pH. It was observed that there exists an optimum for each system: pH?2.5–?3.0 for Alamine 336/oleyl alcohol and pH?4.5 for Aliquat 336/oleyl alcohol. At this pH?the flux rates of citric acid and malic acid was greater (6–8 times) than that of quinic acid. The flux rates decreased (greatly for citric acid) with the flow rate of feed and strip solutions and increased (considerably for citric acid) with the SLM composition . The recycling of feed and strip solutions significantly improved the removal efficiency. The SLM system retained its performance over a period of a few days. The SLM process allowed extraction of the above three organic acids (ascorbic acid was removed in trace amounts) from kiwifruit juice at a rate of a few percent (5%) in a single-pass processing.     

Separation of L-valine from fermentation broths using a supported liquid membrane

A carrier-mediated counter transport process is proposed to separate and to purify an amino acid produced by microbial fermentation. The case of L-valine permeation through a liquid membrane, constituted by a solution of Aliquat 336 in decanol and supported by a hydrophobic microporous membrane, is reported. A mathematical model was developed to estimate distribution coefficients and permeabilities and to predict the influence of hydrodynamic and pH conditions on supported liquid membrane (SLM) performances. Optimum conditions for the transport and the concentration of valine were achieved with synthetic aqueous valine solutions. Series of experiments on fermentation broths, where molasses and biomass contents were varied, permitted pointing out the role of the broth composition on the kinetics and yields of separation. The selectivity of transport of valine by an Aliquat 336/decanol liquid membrane was about 10 toward molasses dyes, 100 toward glucose, and beyond 1000 toward sucrose. This allowed us to achieve the recovery and one step of purification of the product in a single operation. The stability of the Aliquat 336/decanol liquid membrane was sufficient to ensure a selective transport of valine during a continuous run lasting 18 days.     

On-line extraction of volatile fatty acids in acidogenic chemostat culture using a supported liquid membrane

An on-line extraction of volatile fatty acids (acetic and butyric acids) from acidogenic fermentation in chemostat cultures using a supported liquid membrane (SLM) was investigated in order to overcome end-product inhibition. By using SLM, the high-cell-density retaining dilution rate of the chemostat could be increased, thus enhancing the microbial acidogenesis. To further understand this phenomenon, the growth and extraction kinetics were studied. The effect of substrate concentration was found to obey the Haldane equation. Regarding the inhibition by volatile fatty acids, it turned out that undissociated butyric acid rather than acetic acid severely inhibited the growth, corresponding to non-competitive kinetics. The extraction rates of the acids were proportional to their undissociated concentration as well as to the SLM area/broth volume, and butyric acid extraction was easier than that of acetic acid.     

Molecular recognition of carbohydrates by a resorcinarene. Selective transport of alditols through a supported liquid membrane

A supported liquid membrane (SLM) containing a resorcinarene carrier has been used for the selective transport of erythritol, threitol, ribitol and xylitol from concentrated (1.0-0.01 M) aqueous solutions. The membrane is made of a microporous polytetrafluoroethylene film impregnated with a 0.01 M solution of the carrier in CCl4. The permeabilities of the SLM for all alditols were calculated. On the basis of the flux dependence on the initial concentrations of carrier and alditol, the rate-determining step in the transport mechanism is shown to be the migration of the 1:1 carrier-carbohydrate complex in the immobilized organic phase. The flux of sugar is related to the initial concentration of alditol in the feed phase by a saturation law, which allowed the determination of the apparent diffusion coefficients and the stability constants of the resorcinarene complexes of alditols formed in the liquid membrane.     

Supported liquid membrane extraction of peptides

The application of supported liquid membrane (SLM) extraction for the enrichment of short peptides is presented. The extraction efficiency is dependent on the pH of donor phase and salt concentration in acceptor phase. Moreover, the extraction efficiency is also influenced by the peptide amino-acid sequence and hydrophobicity.     

A micro-immuno supported liquid membrane assay (mu-ISLMA)

A chemiluminescent (CL) based micro-immuno supported liquid membrane assay (mu-ISLMA) has been developed that enables clean up, enrichment and detection of simazine in a single miniaturised cartridge system. The mu-ISLM cartridge contains a supported liquid membrane (SLM) sandwiched between a donor and an acceptor plate (channel volumes 1.65 microL), the latter being covered by a thin layer of gold on to which anti-simazine antibodies were covalently immobilised via a self assembled monolayer (SAM) of either dithiobis(11-aminoundecane, hydrochloride) (DTAU) or beta-mercaptoethylamine (beta-MEA). The mu-ISLMA based on DTAU was characterised by both a high apparent extraction efficiency (E(app) = 136%) and high apparent enrichment factor (E(e)(app) = 544), which resulted in a very high sensitivity for simazine (LOD = 0.1 ng L(-1)). The paper discusses the influence of the different SAMs and three different anti-simazine-antibody preparations (polyclonal, affinity purified polyclonal and monoclonal) on the extraction parameters and assay sensitivity. The influence of the sample matrix (e.g. mineral water, orange juice and milk) on the simazine mu-ISLMA was also investigated.     

Evaluation of progesterone content in saliva using magnetic particle-based immuno supported liquid membrane assay (m-ISLMA)

Progesterone in saliva was monitored using a new method called magnetic particle-based immuno supported liquid membrane assay (m-ISLMA) in a sequential injection (SI) setup, allowing automatic sample cleanup, analyte enrichment, and detection in a single analysis unit. Progesterone (Ag) diffuses from a continuous flowing sample - the donor - into a supported organic liquid membrane (SLM), based on analyte partitioning (solubility) between the aqueous donor and the organic phase. The Ag is re-extracted from the SLM into a second stagnant aqueous acceptor, containing antibodies (Ab) immobilized on magnetic beads, held at the bottom of the acceptor by a magnet. Due to the formation of strong Ag-Ab-bead complexes and a large excess of Ab-beads, the Ag is accumulated and selectively enriched in the acceptor. The extracted progesterone was quantified by injecting into the acceptor a horseradish peroxidase (HRP) labeled analyte tracer, the substrate (luminol, H(2)O(2), and p-iodophenol), and finally detection of the generated chemiluminescence by a photomultiplier tube. After optimization of experimental parameters (e.g., sample flow rate, extraction time, type of organic solvent and antibody-bead concentration in the acceptor), a detection limit of 8.50+/-0.17 fgL(-1) and a dynamic range between 35 fgL(-1) and 10 pgL(-1) was reached. The progesterone level of saliva for three subjects (women in different period of ovarian cycle) was investigated, and the corresponding progesterone concentrations detected with m-ISLMA coincided well with the expected values.     

Separation of dilute aqueous butanol and acetone solutions by pervaporation through liquid membranes

A simultaneous extraction-stripping process is proposed for separating volatile products from fermentation broths, it is based on pervaporation through a liquid membrane supported with a hydrophobic porous membrane. The liquid membrane prepared with oleyl alcohol was selected as the most suitable for separating volatile products resulting from acetone-butanol fermentation. The separation performance and stability of the oleyl alcohol liquid membrane were investigated by using dilute aqueous butanol and acetone solutions. The oleyl alcohol liquid membrane was found to be superior by far in both selectivity and permeability of butanol to the better known silicone rubber membrane in its high selectivity for alcohols. Using the oleyl alcohol liquid membrane, the dilute aqueous butanol solutions of around 4 g/L obtained in acetone-butanol fermentation could be concentrated up to 100 times. The stability of this liquid membrane was also quite good as long as the surface tension of the feed solution was less than the critical surface tension of the support membrane. No change in the separation performance was found after the continuous usage in a long period of 100 h.     

Novel reactive perstraction system applied to the hydrolysis of penicillin G

The activity of penicillin acylase has been studied in aqueous and organic solvents, as free enzyme as well as immobilized within the membrane of liquid-core capsules. The activity of the enzyme is inhibited by the accumulation of the products of the hydrolysis reaction, namely phenyl acetic acid (PAA). In order to overcome this inhibition a range of organic solvents were tested for use in in situ product recovery. Of these solvents dibutyl sebacate (DBS) was chosen due to the rapid extraction rate, the high logP and to facilitate capsule production. The extraction efficiency at pH 3.5 for PAA was >80% for phase ratios of >50% free solvent with partition coefficients of 8 and 0.7 for PAA and penicillin G (PenG), respectively, thereby showing that PAA could be selectively extracted at pH 3.5 and 25 degrees C. Liquid-core capsules containing DBS were shown to efficiently remove PAA selectively and the PAA could be effectively back-extracted and the capsules re-used in a three-stage process resulting in high product separation. Immobilization of penicillin acylase onto the capsule membranes resulted in increased operational stability of the enzyme and a very high enzyme activity. Over 53.3% of the PAA formed could be recovered in the capsule core with a concentration over sevenfold higher than in the aqueous phase. Higher extraction efficiencies could be obtained by varying the substrate concentration and number of capsules. The enzyme immobilized on capsules could be stored for over 4 months at pH 8 and 4 degrees C with no loss of activity. Over 80% of the initial activity could be recovered over five repeated batch cycles of the bioconversion process. The importance of capsular perstraction and reactive capsular perstraction has been clearly demonstrated.     

Effects of penicillin on synthesis and excretion of lipid and lipoteichoic acid from Streptococcus mutans BHT.

Cultures of Streptococcus mutans BHT grown for at least eight generations in a chemically defined medium containing [1(3)-14C]glycerol, when treated with growth-inhibitory concentrations (0.2 micrograms/ml) of benzylpenicillin (Pen G), produced and excreted increased amounts of lipid and lipoteichoic acid per unit of cells. Cellular lysis was not observed. Compared with untreated controls, lipid excretion increased 15-fold, and lipoteichoic acid excretion increased 6-fold, 4 h after the addition of Pen G. All lipid species showed increased synthesis and excretion after exposure to Pen G. Although the same lipid types were found in both the Pen G-treated and the untreated cultures, the percent composition was altered after treatment with Pen G. The most dramatic example of this was the percentage of intracellular diphosphatidylglycerol found in the Pen G-treated cultures, 22.6%, in contrast to 5.3% found in the untreated cultures.     

Reversibility of structural rearrangements in the negative vesicular membrane upon electrostatic adsorption/desorption of the polycation

Interaction of small unilamellar vesicles (SUVs), composed of negative diphosphatidylglycerol (cardiolipin, CL(2-)) and neutral dipalmitoylphosphatidylcholine (DPPC), with poly(N-ethyl-4-vinylpyridinium bromide) (PEVP) was studied in water solution above and below the vesicular membrane melting point by means of differential scanning calorimetry, photon correlation spectroscopy, microelectrophoresis, conductometry, and fluorescence techniques. It has been found that CL(2-) species are homogeneously distributed within DPPC-CL(2-) SUV membrane leaflets and between them. Interaction of PEVP with DPPC-CL(2-) SUVs led to drastic structural rearrangements in the membrane if it was in the fluid state (liquid SUVs). Negative CL(2-) molecules migrated from the inner to the outer membrane leaflet and segregated in the vicinity of adsorbed PEVP chains. In addition, PEVP adsorption terminated completely the exchange of lipid molecules between the SUVs. At the same time, the integrity of liquid SUVs contacting PEVP remained unchanged. Since the interaction of PEVP with liquid SUVs was predominantly electrostatic in nature, the polycation could be completely removed from the vesicular membrane by addition of an excess of polyacrylic acid (PAA) polyanions forming a more stable electrostatic complex with PEVP. Removal of PEVP resulted in complete resumption of the original distribution of lipids in lateral and transmembrane directions as well as intervesicular lipid exchange. In contrast, PEVP interacting with DPPC-CL(2-) SUVs formed defects in the vesicular membrane if it was in the gel state (solid SUVs). Such interaction was contributed not only by electrostatic but most likely by hydrophobic interactions involving the defected membrane sites. PEVP kept contacting solid SUVs in the presence of an abundant amount of PAA. The established phenomena may be important for understanding the biological effects of polycations.     

A proapoptotic peptide conjugated to penetratin selectively inhibits tumor cell growth

The peptide KLA (acetyl-(KLAKLAK)₂-NH₂), which is rather non toxic for eukaryotic cell lines, becomes active when coupled to the cell penetrating peptide, penetratin (Pen), by a disulfide bridge. Remarkably, the conjugate KLA–Pen is cytotoxic, at low micromolar concentrations, against a panel of seven human tumor cell lines of various tissue origins, including cells resistant to conventional chemotherapy agents but not to normal human cell lines. Live microscopy on cells possessing fluorescent labeled mitochondria shows that in tumor cells, KLA–Pen had a strong impact on mitochondria tubular organization instantly resulting in their aggregation, while the unconjugated KLA and pen peptides had no effect. But, mitochondria in various normal cells were not affected by KLA–Pen. The interaction with membrane models of KLA–Pen, KLA and penetratin were studied using dynamic light scattering, calorimetry, plasmon resonance, circular dichroism and ATR-FTIR to unveil the mode of action of the conjugate. To understand the selectivity of the conjugate towards tumor cell lines and its action on mitochondria, lipid model systems composed of zwitterionic lipids were used as mimics of normal cell membranes and anionic lipids as mimics of tumor cell and mitochondria membrane. A very distinct mode of interaction with the two model systems was observed. KLA–Pen may exert its deleterious and selective action on cancer cells by the formation of pores with an oblique membrane orientation and establishment of important hydrophobic interactions. These results suggest that KLA–Pen could be a lead compound for the design of cancer therapeutics.     

Integrated fragmentation of human IgG and purification of Fab using a reactant adsorptive membrane bioreactor separator system

This article discusses an integrated separation–reaction–separation scheme for producing Fab fragment directly from human immunoglobulin G (hIgG) present in serum feed. The novel reactant adsorptive membrane bioreactor separator (or RAMBS) system used in the current study consisted of a stack of microporous adsorptive membranes held within a temperature controlled module. The membrane stack, in the presence of salt, selectively and reversibly adsorbed hIgG by hydrophobic interaction while allowing most other serum proteins to flow through. The bound hIgG was then fragmented by pumping a solution of papain through the reactor at controlled temperature and flow rate. The salt concentration and pH for reaction and separation were systematically optimized using pure hIgG as reactant. The Fab fragment was separated from undigested hIgG and other byproducts such as Fc fragment based on their differences in hydrophobicity. Under optimal conditions, Fab was obtained in the reaction flow through while the other proteins remained bound to the membrane, these being subsequently eluted by lowering the salt concentration. The RAMBS system in addition to being convenient from process integration and intensification points of view also showed higher catalytic efficiency of papain in comparison to that in liquid phase reactions. Biotechnol. Bioeng. 2009; 104: 152–161 © 2009 Wiley Periodicals, Inc.     

Electromembrane extraction of trace amounts of naltrexone and nalmefene from untreated biological fluids

Nalmefene and naltrexone are used to block the effects of narcotics and alcohol. In the present work, for the first time a microextraction technique was presented to reduce matrix interferences and improve detection limits of the drugs in urine and plasma samples. Electromembrane extraction (EME) followed by high performance liquid chromatography (HPLC) coupled with ultraviolet (UV) detection was optimized and validated for quantification of nalmefene and naltrexone from biological fluids. The membrane consists 85% of 2-nitrophenyl octyl ether (NPOE) and 15% di-(2-ethylhexyl) phosphate (DEHP) immobilized in the pores of a hollow fiber. A 100 V electrical field was applied to make the analytes migrate from sample solution with pH 2.0, through the supported liquid membrane (SLM) into an acidic acceptor solution with pH 1.0 which was located inside the lumen of hollow fiber. Extraction recoveries in the range of 54% and 75% were obtained in different biological matrices which resulted in preconcentration factors in the range of 109-149 and satisfactory repeatability (2.0相似文献   

Bioconversion with whole cell penicillin acylase in aqueous two-phase systems

The deacylation of Pen G was carried out by using recombinant E. coli in an aqueous two-phase system consisting of polyethylene glycol and potassium phosphate solution, which partitions the cells to the bottom phase and the products to the top phase. Bioconversion and product separation were carried out in the same reactor. Repeated batch conversion was employed ten times and enzymic activity showed only a slight decline. When pure enzyme was used for bioconversion in an aqueous two-phase system, the decline was fast and bioconversion using whole cell penicillin acylase was better than that obtained using the pure acylase.     

Enhancement of penicillin G hydrolysis using penicillin acylase to 6-aminopenicillanic acid by simultaneous chromatographic separation

Penicillin G (Pen-G) was hydrolyzed to 6-aminopenicillanic acid (6-APA) and phenylacetic acid (PAA) in a chromatographic reactor-separator using the mixture of immobilized Escherichia coli cells and a macroporous adsorbent as stationary phase and a phosphate buffer of pH 7.8 as eluant. Pen-G conversion of 98% was observed without adjustment of the eluant pH due to the effective separation of 6-APA from Pen-G and PAA. At a sample load of 600 mg Pen-G, the volume overload gave higher Pen-G conversion (86%) than the mass overload (68%), while their difference in product resolution (0.9 and 1.0, respectively) was insignificant.     

Separation and recovery of radioactive and non-radioactive toxic trace elements from aqueous industrial effluents

An update is presented on liquid membrane-based processes as viable and relevant alternatives to conventional approaches such as precipitation, solvent extraction, ion exchange processes and electrochemical techniques for the removal and recovery of some toxic and/or valuable trace metal ions including some actinides and fission products e.g. U, Am, Y etc and As, Cd, Co, Cr, Cu, Hg, Ni, Pb, Zn etc from radioactive as well as non-radioactive aqueous waste solutions respectively. In particular, results of experiments aimed at developing supported liquid membrane(SLM)-based process using commercially available porous membranes and indigenously prepared track--etch membranes (TEMs) have been critically examined in laboratory studies to generate basic data needed to evaluate their utility for continuous operation without regeneration. These include effect of pore size, porosity, optimum pore size and their reusability. It is clearly demonstrated that indigenously prepared 10 microm thick TEMs with a porosity in the range of 2-5% give comparable transport rates for metal ions-matching with that of commercial membranes of much higher thickness (160 microm) and higher porosity of 60-85%. The smaller thickness of TEMs more than compensates for their lower porosity. It is shown that because of their well defined pore characteristics TEMs could serve as model supports in SLM studies. By comparing the values of permeability coefficient (P) for TEM and polytetraflouroethylene (PTFE) supports for the transport of Pb2+ chosen as a typical divalent metal ion, and using di-2 ethyl hexyl phosphoric acid (D2EHPA) as the carrier, it is unambiguously proved that diffusion of the metal complex across the membrane is the rate controlling step in metal ion transport in SLM-based processes. An overview of the experimental findings along with future outlook and suggestions for further work are presented in this paper.