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The purpose of this study is to separate impurities from dyes using a simple separatory bottle in order to detect to allow for the characterization of these impurities using analytical separation techniques. Foaming agents were used in a glass bottle with a modified cap, to separate a target impurity using the appropriately charged ligand. By running N(2) gas through the solution, the cationic surfactant cetylpyridinium chloride (CPC) and anionic surfactant sodium dodecyl sulfate (SDS) generated foams that separated the anionic dye Orange G and cationic dye Methylene blue, respectively, from a solution containing both dyes. It was found that SDS was able to condense minute quantities of methylene blue from the solution, with high purity. CPC was also able to condense minute quantities of Orange G from the solution, however with less purity. A range of 100nmol to 50μmol of each target dye was tested. For both surfactants, the condensability was shown to increase exponentially as concentration of the target dye decreased. This novel separation method could potentially prove to be a simple, cheap, and effective way to prepare samples and identify components of impurities using HPLC, NMR, and mass spec as well as be used to purify difficult to separate charged compounds.

We have developed a novel continuous flow-through cell separation method using a Percoll density gradient. This method can continuously separate a large number of cells into five fractions according to their densities. To apply this method to the separation of basophils, Percoll density gradients were modified to improve basophil enrichment. When a set of Percoll density gradients was prepared (1.071, 1.075, 1.080, 1.084, and 1.090 g/mL) the basophils in a healthy volunteer were enriched by an average of 23.1 and 63.5% at Percoll densities of 1.075 (fraction 3) and 1.080 g/mL (fraction 4), respectively. On average, the yield of basophils was 1.66 × 10(5) cells in fraction 3 and 1.61 × 10(5) cells in fraction 4 from 9 mL of peripheral blood. The expression of CD203c (cluster of differentiation 203c) on separated basophils was upregulated by anti-immunoglobulin E stimulation similar to basophils in whole blood. Histamine release induced by calcium ionophore was also observed in the separated basophils. The present method will be useful for basophil enrichment since it preserves their function without using counterflow elutriation and immunological reagents, and this method will be effective as a preparative separation for cell purification by flow cytometry.

Recombinant yeast strains displaying aheterologous cellulolytic enzymes on their cell surfaces using a glycosylphosphatidylinositol (GPI) anchoring system are a promising strategy for bioethanol production from lignocellulosic materials. A crucial step for cell wall localization of the enzymes is the intracellular transport of proteins in yeast cells. Therefore, the addition of a highly efficient secretion signal sequence is important to increase the amount of the enzymes on the yeast cell surface. In this study, we demonstrated the effectiveness of a novel signal peptide (SP) sequence derived from the Saccharomyces cerevisiae SED1 gene for cell-surface display and secretory production of cellulolytic enzymes. Gene cassettes with SP sequences derived from S. cerevisiae SED1 (SED1SP), Rhizopus oryzae glucoamylase (GLUASP), and S. cerevisiae α-mating pheromone (MFα1SP) were constructed for cell-surface display of Aspergillus aculeatus β-glucosidase (BGL1) and Trichoderma reesei endoglucanase II (EGII). These gene cassettes were integrated into the S. cerevisiae genome. The recombinant strains with the SED1SP showed higher cell-surface BGL and EG activities than those with the conventional SP sequences (GLUASP and MFα1SP). The novel SP sequence also improved the secretory production of BGL and EG in S. cerevisiae. The extracellular BGL activity of the recombinant strains with the SED1SP was 1.3- and 1.9-fold higher than the GLUASP and MFα1SP strains, respectively. Moreover, the utilization of SED1SP successfully enhanced the secretory production of BGL in Pichia pastoris. The utilization of the novel SP sequence is a promising option for highly efficient cell-surface display and secretory production of heterologous proteins in various yeast species. Biotechnol. Bioeng. 2016;113: 2358-2366. © 2016 Wiley Periodicals, Inc.

Introduction Myrosinase (thioglucoside glucohydrolase; E.C. 3.2.1.147), is a plant enzyme of increasing interest and importance to the biomedical community. Myrosinase catalyses the formation of isothiocyanates such as sulforaphane (from broccoli) and 4-(α-l-rhamnopyranosyloxy)benzyl isothiocyanate (from moringa), which are potent inducers of the cytoprotective phase-2 response in humans, by hydrolysis of their abundant glucosinolate (β-thioglucoside N-hydroxysulphate) precursors. Objective To develop an aqueous two-phase counter-current chromatography (CCC) system for the rapid, three-step purification of catalytically active myrosinase. Methods A high-concentration potassium phosphate and polyethylene glycol biphasic aqueous two-phase system (ATPS) is used with a newly developed CCC configuration that utilises spiral-wound, flat-twisted tubing (with an ovoid cross-section). Results Making the initial crude plant extract directly in the ATPS and injecting only the lower phase permitted highly selective partitioning of the myrosinase complex before a short chromatography on a spiral disk CCC. Optimum phase retention and separation of myrosinase from other plant proteins afforded a 60-fold purification. Conclusion Catalytically active myrosinase is purified from 3-day broccoli sprouts, 7-day daikon sprouts, mustard seeds and the leaves of field-grown moringa trees, in a CCC system that is predictably scalable. Copyright © 2014 John Wiley & Sons, Ltd.

A rapid method combining microwave-assisted extraction (MAE) and high-speed counter-current chromatography (HSCCC) was applied for preparative separation of six bioactive compounds including loganic acid (I), isoorientin-4'-O-glucoside (II), 6'-O-β-d-glucopyranosyl gentiopicroside (III), swertiamarin (IV), gentiopicroside (V), sweroside (VI) from traditional Tibetan medicine Gentiana crassicaulis Duthie ex Burk. MAE parameters were predicted by central composite design response surface methodology. That is, 5.0 g dried roots of G. crassicaulis were extracted with 50 mL 57.5% aqueous ethanol under 630 W for 3.39 min. The extract (gentian total glycosides) was separated by HSCCC with n-butanol/ethyl acetate/methanol/1% acetic acid water (7.5:0.5:0.5:3.5, v/v/v/v) using upper phase mobile in tail-to-head elution mode. 16.3, 8.8, 12., 25.1, 40.7, and 21.8 mg of compounds I-VI were obtained with high purities in one run from 500 mg of original sample. The purities and identities of separated components were confirmed using HPLC with photo diode array detection and quadrupole TOF-MS and NMR spectroscopy. The study reveals that response surface methodology is convenient and highly predictive for optimizing extraction process, MAE coupled with HSCCC could be an expeditious method for extraction and separation of phytochemicals from ethnomedicine.

Chlorogenic acid, a major compound among phenolic acids of Flaveria bidentis (L.) Kuntze and Flos lonicerae, was successfully separated by pH-zone-refining counter-current chromatography. Caffeic acid and chlorogenic acid were selected as test samples, which represent as phenolic acids for separation by the pH-zone-refining counter-current chromatography. The separation of these test samples was performed with two-phase solvent system composed of methyl-tert.-butyl-ether-acetonitrile-water at a volume ration of 4:1:5 (v/v) where trifluoroaceetic acid (8 mM) was added to the organic stationary phase as a retainer and NH4OH (10 mM) to the aqueous mobile phase as an eluter. For separation of crude extract from Flaveria bidentis (L.) Kuntze and Flos lonicerae. a slightly polar two-phase solvent system composed of methyl-tert.-butyl-ether-acetonitrile- n-butanol-water at a volume ratio of 4:1:1:5 (v/v) was selected where trifluoroacetic acid (3 mM) was added to the organic stationary phase as a retained and NH4OH (3 mM) to the aqueous mobile phase as an eluter. pH-zone-refining counter-current chromatography yielded 16.2 mg amount of chlorogenic acid with the purity of 91% from 1.4 g of Flaveria bidentis (L.) Kuntze, and 134 mg of chlorogenic acid at the purity of 99% from 1.3 g of crude extract of Flos lonicerae. These results suggest that pH-zone-refining counter-current chromatography is suitable for the isolation of the chlorogenic acid from the crude extracts of Flaveria bidentis (L.) Kuntze and Flos lonicerae.

High-speed counter-current chromatography (HSCCC) was investigated as a new sample pretreatment method for the analysis of trace polycyclic aromatic hydrocarbons (PAHs) in water environmental samples. The experiment was performed with a nonaqueous binary two-phase solvent system composed of heptane-acetonitrile. The HSCCC column was first entirely filled with the upper stationary phase of the and a large volume of water sample was pumped into the column while the CCC column was rotated at 1600 rpm. Finally, the trace amount of PAHs extracted and enriched in the stationary phase were eluted out by the lower mobile phase. and analyzed by gas chromatography-flame ionization detector (GC-FID) or gas chromatography-mass spectrometry (GC-MS) after concentration. The enrichment and cleanup of PAHs can be fulfilled online by this methodwithhigh recoveries (84.1%–103.2%) and good reproducibility (RSDs 4.9–12.2%) for 16 EPA PAHs under the optimized HSCCC pretreatment conditions. This method has been successfully applied to determine PAHs in lake waterwhere 8 PAHs were detected in the concentration of 40.9–89.9 ng/L. The present method is extremely suitable in the preparation of large volume of environmental water sample for the determination of a trace amount of organic pollutants including PAHs as studied in this paper.

An improved type-J counter-current chromatography (CCC) planet centrifuge with two spiral tube columns (volume 2×15 mL, β value 0.3-0.7, tubing 0.8 mm id) was developed and evaluated for its retention ability of four typical different solvent systems including heptane-methanol (1:1, v/v) (A), hexane-ethyl acetate-methanol-water (1:1:1:1, v/v) (B), n-butanol-acetic acid-water (4:1:5, v/v) (C), PEG1000-K(2)HPO(4)-water (12.5:12.5:75, w/w) (D) under eight different operation modes. The results indicated that the spiral tube column could significantly increase the retention of four typical solvent systems compared with a traditional multilayer coil column with similar parameters (volume 35 mL, β value 0.3-0.7, tubing 0.8 mm id). The retention of stationary phase (S(f)) for the less polar system (A) and moderately polar solvent system (B) can be increased by about 10%, and for the polar system (C) and aqueous two-phase system (ATPS) (D) by 30-40%. The preliminary applications of this spiral tube column to the separation of small molecular compounds such as moderately polar theaflavins, polar anthocyanins and dipeptides were successful. Acceptable resolution can be obtained between cytochrome c and myoglobin, lysozyme and myoglobin when it was applied on protein separation; however, it still needs to be improved with regard to its column efficiency.

High-speed counter-current chromatography (HSCCC) was successfully applied for the preparative separation and purification of alkaloids from Corydalis bungeana Turcz. (Kudiding in Chinese) for the first time. After the measurement of partition coefficient of seven target alkaloids in the nine two-phase solvent systems composed of CHCl3‐MeOH‐(0.1 M; 0.2 M; 0.3 M) HCl (4:1.5:2; 4:2:2; 4:3:2, v/v), CHCl3‐ MeOH‐0.2 M HCl (4:2:2, v/v) and CHCl3‐MeOH‐0.3 M HCl (4:3:2, v/v) were finally selected for the HSCCC separation using the first upper phase as the stationary phase and the stepwise elution of the two lower mobile phases. Consequently, sanguinarine (10 mg), corynoline (25 mg), protopine (20 mg), corynoloxine (18 mg), and 12-hydroxycorynoline (8 mg) were obtained from 200 mg of crude alkaloid extracts with purities of 94‐99% as determined by HPLC. Their chemical structures were characterized on the basis of 1 H-NMR, 13 C-NMR, and LC-ESI-Q-TOF-MS/MS analyses.

Fractionation of clarified E. coli lysate components in bench-scale and preparative-scale centrifugal precipitation chromatography (CPC), using a solution of cationic surfactant cetyltrimethylammonium bromide (CTAB) containing 0.5 M NaCl as precipitant, are compared here. Step gradient of CTAB from 0.50% to 0.16% (w/v) gave a successful fractionation in bench-scale CPC; however, a linear gradient of lower CTAB concentration, 0.20-0% (w/v), was used in the preparative scale and resulted in similar fractionation. The preparative-scale CPC has a superior sample loading capacity by the use of tubular dialysis membrane inside convoluted tubing as the separation channel. In this study, the quantity of the sample loaded into the preparative CPC was about 15 times more than that in the bench scale, and in a single run the preparative CPC could prepare approximately 3 mg of plasmid DNA with about 96% of RNA removed. The higher surface area per length of the separation channel in the preparative CPC was believed to benefit mass transfer of CTAB across the membrane, leading to less CTAB being required in the process.

In conventional high-performance liquid chromatography, chiral separations are performed by chiral column with a chiral selector (CS) chemically boned to the solid support. In contrast, high-speed counter-current chromatography (HSCCC) performs chiral separations by dissolving CS in the liquid stationary phase. During the past two decades, several CSs were developed to successfully carry out chiral HSCCC which include N-dodecanoyl-L-proline-3,5-dimethylanilide, β-cyclodextrin derivatives, vancomycin, cinchona alkaloid derivatives, cellulose and amylose derivatives, tartaric acid derivatives, etc. Compared to HPLC which uses over hundred different kinds of CSs, the number of CSs effectively used in HSCCC is limited to several compounds. This may be due to the violent molecular movement of CS dissolved in the liquid stationary phase which reduces chiral selectivity based on steric affinity. Future development strategy of CS for HSCC proposed here is to suppress the molecular movement of the CS in the liquid stationary phase by the following three ways: 1) using viscous stationary phase such as aqueous-aqueous polymer phase system; 2) attaching a long hydrophobic chain to the asymmetric carbon, or 3) chemically bonding CS onto hydrophobic small particles such as carbon nanotubes, gold colloidal particles, and submicron silica particles.

A new protein fractionation technique presented transfers ammonium sulfate to create a gradient of ammonium sulfate inside a membrane unit for differential precipitation of proteins called “centrifugal precipitation chromatography.” Because it does not require any solid support, it should provide a better alternative to the conventional chromatography with solid stationary phase. To understand the phenomena and achieve a better separation, a mathematical model explaining the ammonium sulfate gradient formation inside the stationary membrane unit is investigated. The model is extended with empirical correlation to the centrifugal membrane unit—a new approach for protein purification. The model agreed well with the experiments for both stationary and centrifugal units. Upon using the model to calculate the ammonium sulfate gradient formation in a membrane unit, this new technique can be useful in separating a mixture of proteins whose solubility in ammonium sulfate solution differs. To demonstrate the technique experimentally, mixtures of proteins are loaded into the column; the partial resolution between proteins is achieved with the step concentration switch of the inlet ammonium sulfate solution.

Guest Editor's Introduction: Most of the continuous flow centrifuge devices utilized a rotary seal, typically consisting of a pair of discoid structures, one of which moves with the rotor while the other remains stationary. Although the rotary seals had been satisfactorily used in various techniques such as countercurrent elutriation and field-flow fractionation, when applied for blood processing, a sealless structure is more preferable to prevent cellular damage. This paper was concerned with the first flow-through centrifuge without rotating seals, and describes the principle and basic designs of a sealless continuous flow centrifuge. This paper was printed in Science vol. 189, page 999–1000 (1975), and reprinted here with permission.

The analytical capability of the high speed countercurrent chromatograph model 4000 (HSCCC-4000) has been improved by optimizing the dimensions of the multilayer coiled column. Using a two-phase solvent system of n-hexane – ethyl acetate – methanol – water (1:1:1:1) and a set of indole auxins as test samples, a series of studies was conducted to evaluate performance of coiled columns with i.d.s ranging from 0.1 to 0.55 mm. The studies on the stationary phase retention indicated that multilayer coils of 0.55 mm and 0.30 mm i.d. provide satisfactory retention of the stationary phase while the 0.10 mm i.d. column fails to yield reproducible retention. The best partition efficiencies were obtained from the 0.30 mm i.d. multilayer coil with a 6 ml capacity which produced theoretical plate numbers ranging from 5500 to 10500 with a resolution factor of 2.30. The feasibility of interfacing the HSCCC-4000 with a mass spectrometer is briefly discussed.

Commercial digitonin has been separated into its components using high-performance liquid chromatography and centrifugal countercurrent chromatography. The individual glucosides have been identified by californium-252 plasma desorption mass spectrometry, occasionally supplemented by hydrolysis and analysis of the liberated sugars and aglycones. Mass spectra of commercial digitonin that have appeared in the literature are discussed in the light of their complex nature. A new glycoside of molecular weight 1035 is described and a structure proposed.

A simple and accurate micromethod for the determination of erythrocyte osmotic fragility is introduced. The method uses a laminar parabolic flow pattern, together with gravity, to retain cells in a long, small-diameter tube while a solution with decreasing osmolarity is passed through the tube. As the cells hemolyze, hemoglobin released from the cells is quickly removed by the axial flow pattern and monitored with a 547 nm optical detector for recording the hemolysis curve. Consequently, a continuous curve is obtained, with a peak occurring at the salt concentration that produces the maximum hemolysis rate. The advantages of this method are simplicity, accuracy, and small sample size (2 microliters of whole blood). The small sample size is of particular importance for infants. A comparison is made with the Parpart method using samples from 18 normal adults. Results are also given for a few abnormal adults and for a series of 26 normal newborns.