Optimum operational conditions for chiral separation of tryptophan enatiomers using ligand exchange liquid chromatography

A simple and precise method for chiral separation of tryptophan enantiomers using high performance liquid chromatography with aligand exchange mobile phase was developed. Chiral separation was performed on a conventional C₁₈ column, using a mobile phase that consisted of a water-methanol solution (88∶12, v/v) containing 10 mmol/Ll-leucine and 5 mmol/L copper sulfate as a chiral ligand additive at a flow rate of 1.0 mL/min. This method allowed baseline separation of two enantiomers with a resolution of 1.84 in less than 30 min. The effect of various conditions, including concentration, type of ligand, organic modifier, pH, flow rate, and temperature, on enantioseparation were evaluated and chiral recognition mechanisms were investigated. Thermodynamic data (ΔΔH and ΔΔS) obtained by van't Hoff plots revealed that enantioseparation is an enthalpy-controlled process.     

Separation of gentamicin by foaming

In order to develop a more economical separation method and to determine the feasibility of such a method for the recovery of gentamicin from fermentation broth, a foam separation process was evaluated using a batch system. The effects of such process variable as surface-active agent, pH, collector–colligent ratio, and the gas flow rate on the separation efficiency of gentamicin were studied. Form the experimental results, the optimal operating conditions selected were: pH below 9.0, collector-to-colligent ratio of 3, and airflow rate at 0.5 vol air/vol liquid/min. Under these operation conditions, the average recovery efficiency of gentamicin was 73% when sodium dodecyl sylfate was used as a collector. A mathematical expression for the foam separation rate was derived. The theoretically predicted values of separation efficiencies agreed reasonably well with the experimental results.     

Enrichment in axial direction of aqueous foam in continuous foam separation

Estimation of overhead production enrichment in continuous foam separation was conducted with a surfactant: sodium n-dodecylbenzenesulfonate (SDBS) and soluble proteins: ovalbumin (OA) and hemoglobin (HB). Axial profiles of the volumetric flow rate and the concentration of the collapsed foam liquid within the column were measured, and the enrichment ratio and the liquid holdup in axial direction were determined experimentally. The proposed model was fitted to the experimental results obtained with various experimental conditions (superficial gas velocity, feed concentration and pH) and was in reasonable agreement with the experimental data by using the least square regression. The present model makes it possible to estimate the foamate concentration at a desired foam height.     

Mixed anionic detergent/aliphatic alcohol-polyacrylamide gel electrophoresis alters the separation of proteins relative to conventional sodium dodecyl sulfate-polyacrylamide gel electrophoresis

The order and relative mobility of proteins on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is affected by unknown components that are differentially present in SDS preparations obtained from different sources [J.B. Swaney, G.F. Vande Woude, and H.L. Bachrach (1974) Anal. Biochem. 58, 337-346]. The modified separation capabilities of such SDS preparations are useful but the use of this phenomenon in a controlled manner requires that the components responsible for the altered separation be identified. Accordingly, this paper describes a polyacrylamide gel electrophoresis system [mixed alcohol/detergent-polyacrylamide gel electrophoresis (MAD-PAGE)] that employs a mixture of alcohol and detergent instead of SDS alone to modify and enhance protein separation relative to conventional SDS-PAGE. A defined mixture consisting of four sulfated alkyl detergents (dodecyl sulfate, tetradecyl sulfate, hexadecyl sulfate, octadecyl sulfate) as well as the four alcohols of corresponding aliphatic chain length was found to be effective at duplicating the electrophoretic effect of USP-grade SDS and thus changed the relative order and position of polypeptides on electrophoresis relative to conventional SDS-PAGE. This method serves as an adjunct to conventional SDS-PAGE by providing another means of resolving proteins that are not normally resolved by SDS-PAGE. Further, it was found that MAD-PAGE is capable of resolving the NS1 protein of influenza virus into three fractions, whereas conventional SDS-PAGE yields one electrophoretic species. Reelectrophoresis of these novel NS1 bands by conventional SDS-PAGE indicated that they were not modified during MAD-PAGE and probably represented distinct molecular forms present in infected cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Performance of a continuous foam separation column as a function of process variables

Enrichment and recovery of bovine serum albumin has been examined in a continuous foam separation column. The effects of the operating factors, superficial air velocity, feed flow rate, feed concentration and pH on the above characteristics was investigated. The protein enrichment decreased with the increase in the value of each of these parameters. Protein recovery increased with increasing air velocity, decreased with increasing feed flow rate and did not change very much with increasing feed concentration. Maximum protein recovery was obtained at the isoelectric point (pH 4.8) of the protein. Maximum protein recovery was found to be a strong function of the air velocity in the range 0.05-0.15 cm/s. Further increase in air velocity did not have much effect on recovery because of very large bubbles formed as a result of coalescence. Bubble size was determined as a function of the above factors in the liquid and foam sections of the column. It was found to be dependent on protein concentration, feed flow rate and solution pH. The effect was more significant in the foam section of the column. The bubbles in the foam section were significantly larger (about 3-10 times) than those in the liquid, with a sharp change at the foam-liquid interface. The bubble size measurements were used to calculate the interfacial area and it was shown that the rate of protein removal increases with increasing interfacial area.     

Homologous human blood protein separation using immobilized metal affinity chromatography: protein C separation from prothrombin with application to the separation of factor IX and prothrombin.

Protein C (PC) is a natural anticoagulant and antithrombotic present in human blood at a concentration of 4 microg/mL. Its deficiency can result in excessive clotting and thrombosis. Protein C can be obtained from human blood plasma; however, there are other coagulant proteins in blood, including prothrombin (factor II), which is present in relatively large amounts and is one of the most active components. Protein C and prothrombin are homologous proteins with similar biochemical features; therefore, immunoaffinity chromatography is used for their separation. However, this technology is very expensive, protein C recovery and activity is low, and contamination problems with mouse antibody are likely. Immobilized metal affinity chromatography (IMAC) utilizes the protein metal-binding properties for protein separation. Protein C has twelve surface-accessible histidines, which are the major metal-binding groups for IMAC separation. After investigating metal ion-binding properties of protein C, we used an IDA-Cu column to separate protein C and prothrombin. Following protein adsorption to the column, prothrombin was washed out using a sodium phosphate buffer containing 2 mM imidazole and protein C was recovered with 15 mM imidazole in the buffer. The mild elution condition allows a high protein C activity and a high recovery. Also, this technology introduces no immunoglobulins, and it is relatively inexpensive. IMAC could replace the immunoaffinity technology for the large-scale separation of protein C from blood plasma Cohn Fraction IV-1. In addition, this work demonstrates a significant application of this technology for the separation of factor IX from prothrombin. Prothrombin has proven to be a harmful contaminant in factor IX cocktails that have been administered to humans in the treatment of hemophilia B.     

Chiral separation of vinpocetine using cyclodextrin-modified micellar electrokinetic chromatography

A cyclodextrin-modified micellar electrokinetic chromatography (CD-MEKC) technique has been developed for enantioseparation of vinpocetine using an inexpensive 2-hydroxypropyl-β-CD (HP-β-CD) as the chiral selector (CS). The best chiral separation was achieved using 40 mM HP-β-CD as the CS in 50 mM phosphate buffer (pH 7.0) consisting of 40 mM sodium dodecyl sulfate (SDS) at a separation temperature and separation voltage of 25°C and 25 kV, respectively. To the author's best knowledge, this is the first CD-MEKC study able to successfully separate the four stereoisomer of vinpocetine in separation time of 9.5 min and resolution of 1.04-3.87.     

Macroporous chitin affinity membranes for lysozyme separation

Macroporous chitin membranes with high, controlled porosity and good mechanical properties have been prepared using a technique developed in this laboratory based on silica particles as porogen. They were employed for the affinity separation of lysozyme. Chitin membranes (1 mm thickness) can be operated at high fluxes (>/=1.1 mL/min/cm(2)) corresponding to pressure drops >/=2 psi. Their adsorption capacity for lysozyme ( approximately 50 mg/mL membrane) is by an order of magnitude higher than that of the chitin beads employed in column separation. In a binary mixture of lysozyme and ovalbumin, the membranes showed very high selectivity towards lysozyme. The effect of some important operation parameters, such as the flow rates during loading and elution were investigated. Lysozyme of very high purity (>98%) was obtained from a mixture of lysozyme and ovalbumin, and from egg white. The results indicate that the macroporous chitin membranes can be used for the separation, purification, and recovery of lysozyme at large scale. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 610-617, 1997.     

Separation of peptides dissolved in a sodium dodecyl sulfate solution by reversed-phase liquid chromatography: removal of sodium dodecyl sulfate from peptides using an ion-exchange precolumn

Separation of peptides by reversed-phase liquid chromatography is significantly affected by sodium dodecyl sulfate (SDS) in the sample solution. The strongly acidic group of SDS binds to the reversed-phase column where it serves as an ion exchanger and retards the elution of peptides. By using a DEAE precolumn connected in series to a reversed-phase column, the interference of SDS in the separation of peptides by reversed-phase chromatography can be significantly diminished. This simple method is applicable to the separation of peptide mixtures obtained by digestion of proteins extracted from SDS-polyacrylamide gels. Peptide production with some proteases in the presence of SDS was examined using the present method. Lysylendopeptidase was suitable for digestion in the presence of SDS, but V8 protease was not.     

Foam separation of microbial cells

Batch foam separation has been employed to separate Saccharomyces carlsbergensis cells from their broth without the use of any external surface-active agent. A model has been developed to predict the foamate cell concentration as well as the variation of cell concentration in the bulk liquid in the foam column as a function of time. The model assumes a linear equilibrium relation between the cell concentrations at the interface and the bulk. The foam has interface as well as interstitial liquid. The interface is assumed to be in equilibrium with the interstitial liquid, which in turn is assumed to have the same concentration as the bulk. The interfacial area is calculated by assuming the foam bubbles to be pentagonal dodecahedral in shape. The model has been found to explain the results of foam separation of cells quite well, particularly with respect to the effect of bubble size and aeration rate.     

Automated on-line ionic detergent removal from minute protein/peptide samples prior to liquid chromatography-electrospray mass spectrometry

An automated on-line ionic detergent removal pre-column system coupled to capillary liquid chromatography-electrospray mass spectrometry is described. The system involves two micro precolumns, composed of a specific ionic detergent trapping column and a preconcentration column, respectively, and a packed 300 μm I.D. analytical column. Sample loading to the micro precolumns and regeneration of the detergent trapping column were performed at a flow-rate of 50 μl/min, while the flow-rate through the analytical column was set at 5.0 μl/min. Ionic detergent-containing tryptic-digested protein samples were directly applied to the micro precolumns without sample pretreatment and were analysed by UV absorption detection and electrospray mass spectrometry. The presented system allows for the fully automated removal of SDS with virtually no loss in protein/peptides. Maximum SDS load and breakthrough have been determined. Excellent protein recovery and complete removal of SDS is found. The chromatographic separation after SDS removal was completely restored and equalled the reference chromatogram. Mass spectral data confirm these findings. Finally, this technique allows for SDS removal from minute protein samples without the need for any sample handling.     

Evaluation of the performance of protein separation in figure-8 centrifugal counter-current chromatography

The performance of protein separation using the figure-8 column configuration in centrifugal counter-current chromatography was investigated under various flow rates and revolution speeds. The separation was performed with a two-phase solvent system composed of polyethylene glycol 1000/potassium phosphate each at 12.5% (w/w) in water and with lysozyme and myoglobin as test samples. In order to improve tracing of the elution curve, a hollow fiber membrane dialyzer was inserted at the inlet of the UV detector. The results showed that the retention of stationary phase (Sf) and resolution (Rs) increased with decreased flow rate and increased revolution speed. The highest Rs of approximately 1 was obtained at a flow rate of 0.01 mL/min under a revolution speed of 1200 rpm with a 3.4 mL capacity column.     

Integrated SDS removal and peptide separation by strong-cation exchange liquid chromatography for SDS-assisted shotgun proteome analysis

We report an improved shotgun method for analyzing proteomic samples containing sodium dodecyl sulfate (SDS). This method is based on the use of strong-cation exchange (SCX) liquid chromatography (LC) for SDS removal that can be integrated with peptide separation as the first dimension of the two-dimensional LC tandem mass spectrometry workflow. To optimize the performance of SDS removal, various experimental conditions, including the concentrations of chemical reagents and salts in the sample, the SDS concentration, and the SCX mobile phase composition, were investigated. It was found that a peptide recovery rate of about 90% could be achieved while removing SDS efficiently. One key finding was that, by increasing the SDS concentration to a certain level (0.5%) in the digested peptide sample, the sample recovery rate could be increased. The peptide recovery rate of BSA digests was found to be 90.6 ± 1.0% (n = 3), and SDS in the SCX fractions collected was not detectable by pyrolysis GC-MS, i.e., below the detection limit of 0.00006% for the undesalted SCX fractions. The peptide recovery rates were found to be 90.9% ± 2.7 (n = 3) and 89.5% ± 0.5% (n = 3) for the digests of the membrane-protein-enriched fractions of E. coli cell lysates and the MCF-7 breast cancer cell line, respectively. Compared to the methods that use acid-labile surfactants, such as RapiGest and PPS, for the MCF-7 membrane fraction sample, the SDS method identified, on average (n = 3), more peptides (～5%) and proteins (～16%) than the RapiGest method, while the RapiGest method identified more peptides (～21%) and proteins (～7%) from the E. coli membrane fraction than the SDS method. In both cases, the two methods identified more peptides and proteins than the PPS method. Since SCX is widely used as the first dimension of 2D-LC MS/MS, integration of SDS removal with peptide separation in SCX does not add any extra steps to the sample handling process. We demonstrated the application of this method for 2D-LC MS/MS profiling of the MCF-7 membrane protein fraction and identified 6889 unique peptides, corresponding to 2258 unique proteins or protein groups from two replicate experiments with a false peptide discovery rate of ～0.8%, compared to 5172 unique peptides and 1847 unique proteins identified by the RapiGest method.     

Development of anaerobic sludge bed (ASB) reactor technologies for domestic wastewater treatment: motives and perspectives

During the treatment of raw domestic wastewater in the upflow anaerobic sludge blanket (UASB) reactor, the suspended solids (SS) present in the wastewater tend to influence negatively the methanogenic activity and the chemical oxygen demand (COD) conversion efficiency. These problems led to the emergence of various anaerobic sludge bed systems such as the expanded granular sludge bed (EGSB), the upflow anaerobic sludge blanket (UASB)-septic tank, the hydrolysis upflow sludge bed (HUSB), the two-stage reactor and the anaerobic hybrid (AH) reactor. However, these systems have, like the UASB reactor, limited performance with regard to complete treatment (e.g., removal of pathogens). In this respect, a new integrated approach for the anaerobic treatment of domestic wastewater is suggested. This approach combines a UASB reactor and a conventional completely stirred tank reactor (CSTR) for the treatment of the wastewater low in SS and sedimented primary sludge, respectively. The principal advantages of the proposed system are energy recovery from organic waste in an environmentally friendly way; lowering the negative effect of suspended solids in the UASB reactor; production of a high quality effluent for irrigation; and prevention of odour problems.     

Separation of soybean saponins from soybean meal by a technology of foam fractionation and resin adsorption

Foam fractionation and resin adsorption were used to recover soybean saponins from the industrial residue of soybean meal. First, a two-stage foam fractionation technology was studied for concentrating soybean saponins from the leaching liquor. Subsequently, resin adsorption was used to purify soybean saponins from the foamate in foam fractionation. The results showed that the enrichment ratio, the recovery percentage, and the purity of soybean saponins by using the two-stage foam fractionation technology could reach 4.45, 74%, and 67%, respectively. After resin adsorption and desorption, the purity of soybean saponins in the freeze-dried powder from the desorption solution was 88.4%.     

Reversed-phase HPLC separation of proteins on chemically bonded silica gel columns

Reversed-phase high-performance liquid chromatographic (RP-HPLC) separation of proteins on chemically bonded silica gel columns is described. Efficiency of nonporous alkylsilyl bonded silica gel is compared with that of a macroporous gel that has been widely used for the purpose. A comparative study of the separation under conventional and fast separation conditions is also given. The fast separation technique on the nonporous reversed-phase column has the advantage of improving the recovery of late-eluting hydrophobic and large proteins, such as ovalbumin and apoferritin.     

聚氨酯固定化热带假丝酵母发酵木糖醇

固定在多孔聚氨酯载体中的热带假丝酵母(Candida tropicalis), 可有效地利用玉米芯半纤维素水解液生产木糖醇。在摇瓶条件下, 采用分批发酵方式, 确立了适宜的发酵工艺参数为: 接种量7%, 聚氨酯加入量1.0 g/100 mL, 温度30°C, 初始pH值6.0, 分段改变摇床转速进行溶氧调节, 其中0~24 h 为200 r/min; 24 h~46 h为140 r/min。聚氨酯固定化提高了菌体对发酵抑制物的耐受力, 固定化细胞密度高, 发酵性能稳定, 发酵产率和体积生产速率都有所提高。水解液未经脱色与离子交换便可转化成木糖醇, 大幅降低了成本, 显示了良好的应用前景。固定化细胞连续重复进行12批次21 d的发酵, 木糖醇得率平均为67.6%, 体积生产速率平均为1.92 g/(L·h)。     

Capillary electrophoresis enantioselective separation of vigabatrin enantiomers by precolumn derivatization with dehydroabietylisothiocyante and UV-vis detection

A simple and reliable capillary electrophoresis (CE) method with UV-vis detection is presented for the enantioselective separation and determination of vigabatrin enantiomers. Dehydroabietylisothiocyante (DHAIC), a novel chiral derivatizing reagent, was used for precolumn derivatization of vigabatrin enantiomers. Optimal separation was obtained with a running buffer consisting of 50 mM Na2HPO4 (pH 9.0), 17 mM sodium dodecyl sulfate (SDS) and 25% acetonitrile. The enantiomeric separation of vigabatrin derivatives was achieved within 25 min, and the resolution was found to be 2.1. Detection was followed by direct UV absorptiometric measurements at 202 nm. A calibration curve ranging from 0.3 to 6.0 microg/ml was shown to be linear, and the limit of detection was 0.15 microg/ml. The developed method has been applied to the determination of vigabatrin enantiomers spiked in human plasma, no interferences were found from endogenous amino acids.     

Modeling the performance of pilot-scale countercurrent chromatography: scale-up predictions and experimental verification of erythromycin separation

Biosynthesis of polyketide antibiotics, such as erythromycin A (EA), can result in the formation of analogues of the main compound that are chemically and structurally extremely similar. The large-scale purification of these antibiotics by conventional high-performance liquid chromatography (HPLC) can be prohibitively expensive due to the large volume of both solvent and adsorbent required. This study examines the feasibility of using a novel pilot-scale countercurrent chromatography (CCC) machine as an alternative to HPLC. CCC is a low-pressure (typically <4000 kN m(-2)) liquid-liquid chromatographic technique that allows the separation of solutes on the basis of their partitioning between two immiscible liquid phases. The effects of mobile phase flow rate, column rotational speed, and sample injection volume on the attainable yield and purity of EA were investigated. Our results show that, at a mobile phase flow rate of 40 mL min(-1), a rotational speed of 1200 rpm, and an injection volume of 100 mL (10 g total erythromycin), EA could be satisfactorily fractionated with a purity of approximately 92% (w/w) and a recovery yield of approximately 100% (w/w). The total solute throughput was estimated to be 0.41 kg day(-1). More importantly, we demonstrated simple and predictive linear scale-up of the CCC separation based on data obtained from a single laboratory-scale CCC chromatogram, and verified this experimentally. The retention time and peak width of the target compound at the pilot scale could be predicted to within 4% for operation at a range of mobile-phase flow rates and injection volumes. This predictable nature of CCC separations, unlike HPLC methods, can greatly reduce process development times and enable a complete process-scale operating scenario to be planned.     

Analysis of alkyl sulfates in protein solutions by isocratic and gradient ion chromatography

An ion-chromatographic analysis for separation and quantitation of long-chain alkyl sulfates in both commercial samples of sodium dodecyl sulfate (SDS) (lauryl sodium sulfate) and protein solutions was developed. The separation was performed on a hydrophobic resin-based column utilizing tetrabutylammonium hydroxide as an ion-pair reagent and acetonitrile as an organic modifier. Sensitive and selective detection of alkyl sulfates was achieved with an anion suppressor and a conductivity detector. Gradient elution with acetonitrile was developed for the detection of a broad range of alkyl chain lengths (C-10--C-20) at high sensitivity. Because of the wide linear range of this method (0.2-700 micrograms/ml), trace levels of C-10, C-14, C-16, C-18, and C-20 alkyl sulfates can be accurately measured in the presence of high concentrations of C-12 alkyl sulfate (SDS). Thus the alkyl sulfate purity of commercial SDS solutions can be accurately and precisely determined without any sample treatment. For analysis of alkyl sulfates from protein solutions, sample treatment consisted of a one-step ion-pair extraction prior to chromatographic resolution and quantitation. Alkyl sulfates from 2-150 micrograms/ml were recovered in high yield from wide variety of protein solutions.