Capillary electrochromatography of proteins with polymer-based strong-cation-exchanger microspheres

Monodisperse poly(glycidyl methacrylate-divinylbenzene) microspheres were functionalized with propyl sulfonic acid moieties to obtain beads negatively charged in a wide pH range. They were packed into fused-silica capillary of 50 micro, I.D. in order to separate proteins by capillary electrochromatography (CEC). Baseline separation of four basic proteins as well as three cytochrome c variants with an average column efficiency of 60,000 theoretical plates was obtained under isocratic elution conditions. The high efficiency is attributed to the uniformity of the column packing and the hydrophilic surface coverage of the polymer beads derived from the functionalization process. The effect of pH and salt concentration on protein separations was investigated and the results showed that the CEC separation mechanism is the combination of chromatographic retention and electrophoretic migration. Moreover, the column packed with the strongly acidic poly(glycidyl methacrylate-divinylbenzene) beads was also suitable for protein separations by micro-HPLC with a salt gradient. The comparison between the two kinds of elution modes shows that the column described here exhibited higher peak efficiency with isocratic elution in CEC than with gradient elution in micro-HPLC.     

Enantiomer separation by CEC

Three chiral compounds were successfully separated in a short time with two enantiomer separation models on packed-capillary electrochromatography (CEC). (i) 75 μm I.D. capillaries were packed with 5 μm β-cyclodextrin (βCD) chiral stationary phase (CSP). Effects of voltage, pH and concentration of organic modifier on electroosmotic flow (EOF) and chiral separations were investigated systematically. Enantiomers of a neutral compound (benzoin) and a neutral drug (mephenytoin) were separated within a short time with high efficiency. Efficiency of 32 000 theoretical plates per meter and resolution (R8) of 1.42 were achieved for enantiomers of benzoin using a βCD packed column with 6.2crn packed length. Efficiency of 45 000 theoretical plates per meter andR8 of 3.40 were obtained for enantiomers of mephenytoin. Especially, the enantiomer separation of mephenytion was performed in just 3.4 min with R₈ of 2.60. (ii) 75 μm I.D. capillary was packed with octadecylsilica particles (ODs). Chiral separation of a basic drug, propranolol, was studied with chiral agent, via addition of the dimethyl-β-cyclodextrin (DM β-CD) directly into the mobile phase on this column. Baseline separation and efficiency of 81 000 theoretical plates per meter were achieved for propranolol. Project supported by the Natural Science Foundation of Liaoning Province, China, the National Natural Science Foundation of China (Grant No.29875030), and the Excellent Young Scientist Award from the National Natural Science Foundation of China. (Grant No.29725512).     

Synthesis of task-specific ionic liquids with grafted diglycolamide moiety. Complexation and stripping of lanthanides

Task-specific ionic liquids (TSILs) of a novel class, with the diglycolamide moietity grafted in the alkyl chain of imidazolium cation, were synthesized and characterized. Lanthanide complexation capabilities of TSILs as active components of solid phase extractants were evaluated by studies on the adsorption of lanthanides from aqueous solutions. The TSIL-based solid adsorbents prepared by immobilization of long-alkyl-chain TSILs in siliceous mesostructured cellular foams adsorb trivalent lanthanides. No extraction of lanthanides from aqueous solution into 1-hexyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide ([C₆mim][Nf₂T]) was observed at any acidity in the presence of these TSILs in the aqueous phase. This implies that TSILs suppress the extraction of lanthanides by formation of water-soluble complexes. The TSILs added to solvent extraction systems consisting of N,N,N′,N′-tetraoctyl-3-oxapentanediamide (TODGA) in [C₆mim][Nf₂T] and aqueous HNO₃ solutions show very good stripping properties for lanthanides.     

Capillary electrochromatographic separation of peptides using a macrocyclic polyamine for molecular recognition

A macrocyclic polyamine, 1,5,9,13,17,21,25,29-octaazacyclodotriacontane ([32]ane-N(8)), in the bonded phase was employed as a molecular receptor for CEC separation of oligopeptides. Parameters affecting the performance of the separations were considered. Baseline separation for the mixture of angiotensin I, angiotensin II, [Sar(1), Thr(8)]-angiotensin II, beta-casomorphin bovine, beta-casomorphin human, oxytocin acetate, tocinoic acid, vasopressin, and FMRF amide could be achieved using phosphate buffer (30 mM, pH 7) as the mobile phase. Column efficiency with average theoretical plate numbers of 69 000 plates/m and RSDs of <1% (n = 6) was achieved. [Met(5)]-enkephalin and [Leu(5)]-enkephalin, which have identical pI values and similar masses could be completely separated using acetate buffer (30 mM) with pH gradient (pH 3 inlet side and pH 4 outlet side). The results suggest that the mechanism for the peptide separation was mediated by a combination of electrophoretic migration and chromatographic retention involving anion coordination and anion exchange. After long-term use, the deviation of the EOF of the column after more than 600 injections was still within 6.0% of that for a freshly prepared column.     

Evaluation of Analytical Countercurrent Chromatographs: High-Speed Countercurrent Chromatograph-4000 Vs. Analytical Toroidal Coil Centrifuge

Abstract

Performance of two countercurrent chromatographic models, high speed countercurrent chromatograph (HSCCC-4000) and analytical toroidal coil centrifuge (TCC), is evaluated in terms of theoretical plate number, resolution factor and separation time to assess their analytical capability. A series of experiments was conducted to investigate the effects of internal diameter and length of the coiled column, and flow rate of the mobile phase on the separation of indole auxins in two-phase solvent systems composed of n-hexane-ethyl acetate-methanol-water at different volume ratios. The three components of indole auxins were completely resolved in 16 min with a HSCCC system equipped with a multilayer coil of a 0.55 mm I.D. PTFE tube with theoretical plates ranging from 1290 to 829. Similar separation was achieved in 24 min with a TCC systems equipped with a 0.3 mm I.D. PTFE tube with theoretical plates ranging from 1811 to 969. It is concluded that both systems have comparable analytical capability at the present stage of development.     

Effect of Alkyl Chain Length of Bonded Silica Phases on Separation,Resolution and Efficiency in High Performance Liquid Chromatography

Abstract

A comparative study of alkyl bonded phases was carried out under optimum solvent conditions for each phase. Three columns, RP-2, RP-8 and RP-18, were tested for their efficiency and resolving power using three groups of compounds in three binary organic-water mobile phases. The organic solvents were acetonitrile, methanol and tetrahydrofuran which are widely used as solvent modifiers.

The results indicate that each of the three factors, i.e. solvent, solute and bonded alkyl chain length, play an important role, with the solvent being the most significant. When tetrahydrofuran-water was used as the mobile phase, the ratio of THF/H₂O did not vary by much when an RP-2, RP-8 or RP-18 column was used to separate naphthalene from biphenyl, dimethylphthalate from diethylphthalate or anthraquinone from methyl, anthraquinone and ethyl anthraquinone. When acetonitrile-water and methanol-water were used the ratio of organic modefier to water changed so as to accomodate the hydrophobic properties of the columns. The efficiency of the columns, expressed as theoretical plates per meter (TPM) was highest when acetonitrile-water was used as the mobile phase. Although there were variations in TPM and resolution from column to column, the three columns gave good separation of the components of the three groups of compounds.     

Vortex Counter-Current Chromatography: Performance of a New Preparative Column

A new preparative column for the vortex counter-current chromatograph was fabricated by making many (966) cylindrical separation units to a high-density polyethylene disk and then threading them with 6–40 taps. The resulting column had a total capacity of 364 mL. The performance of this vortex column was examined with three different two-phase solvent systems each using a set of suitable test samples: hexane–ethyl acetate–methanol–0.1 M hydrochloric acid (1:1:1:1, v/v) for the separation of DNP-amino acids; 1-butanol–acetic acid–water (4:1:5, v/v) for the separation of dipeptides; and hexane–acetonitrile–water (20:15:2, v/v) for the separation of Sudan dyes. Most of the separations show high partition efficiency of over a thousand theoretical plates, as expected based on the results previously obtained in preliminary separations with a small column. Overall, the results of the present study suggest that further improvement of the partition efficiency can be obtained by the modifying column configuration.

     

Extraction separation of rare-earth ions via competitive ligand complexations between aqueous and ionic-liquid phases

The extraction separation of rare earth elements is one of the most challenging separation processes in hydrometallurgy and advanced nuclear fuel cycles. The TALSPEAK process (trivalent actinide lanthanide separations by phosphorus-reagent extraction from aqueous komplexes) is a prime example of these separation processes. The objective of this paper is to explore the use of ionic liquids (ILs) for the TALSPEAK-like process, to further enhance its extraction efficiencies for lanthanides, and to investigate the potential of using this modified TALSPEAK process for separation of lanthanides among themselves. Eight imidazolium ILs ([C(n)mim][NTf(2)] and [C(n)mim][BETI], n = 4,6,8,10) and one pyrrolidinium IL ([C(4)mPy][NTf(2)]) were investigated as diluents using di(2-ethylhexyl)phosphoric acid (HDEHP) as an extractant for the separation of lanthanide ions from aqueous solutions of 50 mM glycolic acid or citric acid and 5 mM diethylenetriamine pentaacetic acid (DTPA). The extraction efficiencies were studied in comparison with diisopropylbenzene (DIPB), an organic solvent used as a diluent for the conventional TALSPEAK extraction system. Excellent extraction efficiencies and selectivities were found for a number of lanthanide ions using HDEHP as an extractant in these ILs. The effects of different alkyl chain lengths in the cations of ILs and of different anions on extraction efficiencies and selectivities of lanthanide ions are also presented in this paper.     

Use of Centrifugal Partition Chromatography and Proteins in The Preparative Separation of Amino Acid Enantiomers

Abstract

The growth of analytical methodologies for the separation of enantiomers has been impressive. Attention is now turning to the large scale separation of enantiomers. Often scaling-up sensitive analytical separations is ineffective and inefficient. Centrifugal partition chromatography (CPC) may be a viable alternative for the preparative separation of racemic mixtures in some cases. The use of proteins as chiral selectors in CPC is examined. Attention was focused on proteins that previously were used as bonded phases in analytical LC columns. The enzymatic properties of α-chymotrypsin allowed it to be used as a bioreactor in conjunction with CPC. When proteins are used as components of the stationary or mobile phase there can be problems with denaturation. However, when used in external incubation processes or as column bioreactors coupled with CPC, effective gram-scale separations can be performed. Tryptophan methyl ester was used as a model compound to evaluate this approach.     

A reinterpretation of C5-BTBP extraction data, performed in various alcohols

The separation of trivalent actinides from trivalent lanthanides present in used nuclear fuel can be achieved by using solvent extraction and the BTBP class of ligands. This separation is relevant for the advanced reprocessing of the used fuel. The choice of diluents in such BTBP based systems has shown to affect the extraction as well as the separation. Long chained alcohols have previously been investigated as such diluents, showing that the americium extraction is higher into alcohols having shorter chains (hexanol, and heptanol) than in longer chained ones (nonanol and decanol). In this work it is shown that not only the distribution ratio, but also the contact time needed before reaching extraction equilibrium is shorter when using shorter chain length of the alcohol diluent. It is also shown that the rate of extraction can be correlated to the interfacial tension between the diluent and the aqueous phase. A low interfacial tension gives a fast extraction while an extraction system with higher interfacial tension needs a longer time of phase contact in order to reach extraction equilibrium.     

Anion exchange silica monolith for capillary liquid chromatography

An anion exchange monolithic silica capillary column was prepared by surface modification of a hybrid monolithic silica capillary column prepared from a mixture of tetramethoxysilane (TMOS) and methyltrimethoxysilane (MTMS). The surface modification was carried out by on-column copolymerization of N-[3-(dimethylamino)propyl]acrylamide methyl chloride-quaternary salt (DMAPAA-Q) with 3-methacryloxypropyl moieties bonded as an anchor to the silica surface to form a strong anion exchange stationary phase. The columns were examined for their performance in liquid chromatography (LC) and capillary electrochromatography (CEC) separations of common anions. The ions were separated using 50 mM phosphate buffer at pH 6.6. Evaluation by LC produced an average of 30,000 theoretical plates (33 cm column length) for the inorganic anions and nucleotides. Evaluation by CEC, using the same buffer, produced enhanced chromatographic performance of up to ca. 90,000 theoretical plates and a theoretical plate height of ca. 4 μm. Although reduced efficiency was observed for inorganic anions that were retained a long time, the results of this study highlight the potential utility of the DMAPAA-Q stationary phase for anion separations. Figure Micro-LC performance evaluation of a strong anion exchange silica monolith column, 100H-MOP-DMAPAA-Q, 33 cm in length, with a mobile phase of 50 mM phosphate buffer, pH 2.8; linear velocity: u = 1.8 mm/s; UV-Vis detection at 254 nm. Sample solution (5 mg/mL of each component, 4 mL) was injected in split flow injection mode at a split ratio of ca. 1:1900 with a pump flow rate of 1.5 mL/min     

Optimization of a liquid-liquid extraction method for HPLC-DAD determination of penicillin-V in human plasma

Optimization of a liquid-liquid extraction procedure used for isolation of penicillin-V in human plasma samples and the subsequent HPLC-DAD analysis are reported. Some aspects related to the stability of penicillin-V in plasma and according to pH are also given. From seven tested solvents, the highest extraction yield was obtained with methyl-t-buthyl ether. Influence of the extraction parameters, such as pH of aqueous phase, sample/solvent volumetric ratio, and extraction duration, is discussed. HPLC separation was achieved on an Eclipse XDB-C8 column, using a mobile phase composed by aqueous phosphate buffer (pH=7.3), methanol and acetonitrile in the ratio 8:1:1. Quantitation limits of 50 ng/ml were obtained. The optimal sample preparation method and HPLC-DAD separations were used for a bioequivalence study, made on 36 volunteers.     

Mixed-ligand chelate extraction of trivalent lanthanides with 4,4,4-trifluoro-1-phenyl-1,3-butanedione and neutral oxo-donors

Mixed-ligand chelate extraction of trivalent lanthanides such as Nd(III), Eu(III) and Lu(III) into benzene with mixtures of 4,4,4-trifluoro-1-phenyl-1,3-butanedione (HBFA) and bis-2-ethylhexyl sulphoxide (B2EHSO) or triphenylphosphine oxide (TPhPO) from thiocyanate solutions was investigated. The results demonstrate that these metal ions are extracted as Ln(BFA)(3) with HBFA alone and in the presence of a neutral oxo-donor, B2EHSO or TPhPO(S), as Ln(BFA)(3).S and Ln(BFA)(3).2S. The equilibrium constants of the above species increase monotonically with decreasing ionic radii of these metals ions. The addition of a neutral donor to the metal chelate system not only enhances the extraction efficiency but also improves the selectivity among these trivalent lanthanides. Hence this mixed-ligand system may be useful for the extraction and separation of individual lanthanides and also for the separation of lanthanides as a group from other metal ions.     

Semimicro Size Exclusion Chromatography For Oligomers. I. Column Packing Procedure

Abstract

Polystyrene gels of a particle diameter 10 ± 2 μm for the use in oligomer separation were packed into 1.5 mm i.d. × 25 cm length columns by the balanced density slurry-packing technique under a constant flow rate of 500 μL/min. The slurry solvent was a mixture of toluene and chloroform (50.5/49.5, v/v). The example of the number of theoretical plates (N) of these columns was 8600 plates/25 cm (HETP = 29.1 μm) at flow rate of 40 μL/min by injecting 1 μL of 0.5% benzene solution. Sixteen columns were connected and the overall value of N was 103000 plates/4 m. A typical example of oligomer separation was demonstrated. A constant-flow technique is preferable to a constant-pressure technique. When two or three column blanks were packed together, the columns located at the outlet of the packer-column assembly had higher values of N. Optimum flow rate of the slurry solvent when three column blanks were packed together lay between 400 and 500 μL/min. The packing efficiency, that is, the probability of getting valid columns was about 60%. Viscous slurry solvents were not effective to get efficient columns. To pack gels in the less swollen state gave sometimes efficient columns. Pressure monitoring in progress of packing was very effective to foresee the column efficiency.     

Theory of Open Capillary Chrcmatography Using Electrical Diffuse Layer as the Virtual Stationary Phase for Charged Macro Mxecular Separations

Abstract

Electrical diffuse layers established by arrangement of ions along a metal capillary column can be used as the virtual “stationary phase” for charged macromolecular separations and determinations. Preliminary studies have indicated that resolution depends on solvent flow rate, pH, column diameter, etc., as well as the applied potential. In this communication we report a theory which underlies the mechanism of retention of charged macro-molecules, e.g., proteins, on the column. The utility and difficulties of this novel fractionation technique will also be discussed.     

Hydrophobic-phase-modified fused-silica columns for capillary electrophoresis

Coated capillaries modified with a hydrophobic layer were developed. Linear hydrocarbons and ethylbenzene modified surfaces greatly improved the electrophoretic performance of the capillaries. The column efficiency for organic compounds reached as high as 327 000 theoretical plate numbers per meter on a 50 microm I.D. linear hydrocarbon (C6) surface treated fused-silica capillary column. This value did not change during 50 repeated analyses and the columns showed strong stability against 0.1 M NaOH and 0.1 M HCl. The relative standard deviation of the run-to-run, day-to-day, and capillary-to-capillary coating with hydrophobic layer showed values of < or =2.5%, and good reproducibility. The separations of four aromatic amines and six pharmacological amines at pH 2.5 is reported.     

以脉冲萃取柱利用三异辛胺从高浓度铀溶液中回收铀

利用三异辛胺(TOA)纯化Gattar小型试验工厂的高浓度铀溶液(洗脱液的铀浓度7 g·L^-1),研究了脉冲萃取柱的性能。利用实验室级脉冲萃取柱进行了实验室规模的溶剂萃取实验和后续实验。结果表明,在室温、pH=1和有机相与水相的比例(V_O/V_A)约为1.8：1时,加入二(2-乙基己基)磷酸(D2EHPA)使其与TOA的比例(V_D2EHPA/V_TOA)为2：3,可使萃取克服Cl-的抑制效应,提高效率。将结论用于考察试验工厂级萃取柱的流体力学和传质性能,结果表明用脉冲萃取柱萃取铀可以达到97%的萃取效率,具有可行性。     

Separation of thorium from lanthanides by solvent extraction with ionizable crown ethers

Thorium and the lanthanides are extracted by alpha-(sym-dibenzo-16-crown-5-oxy)acetic acid and its analogues in different pH ranges. At pH 4.5, Th is quantitatively extracted by the crown ether carboxylic acids into chloroform whereas the extraction of the lanthanides is negligible. Separation of Th from the lanthanides can be achieved by solvent extraction under this condition. The extraction does not require specific counteranions and is reversible with respect to pH. Trace amounts of Th in water can be quantitatively recovered using this extraction system for neutron activation analysis. The nature of the extracted Th complex and the mechanism of extraction are discussed.     

Performance comparison of three types of high-speed counter-current chromatographs for the separation of components of hydrophilic and hydrophobic color additives

The performance of three types of high-speed counter-current chromatography (HSCCC) instruments was assessed for their use in separating components in hydrophilic and hydrophobic dye mixtures. The HSCCC instruments compared were: (i) a J-type coil planet centrifuge (CPC) system with a conventional multilayer-coil column, (ii) a J-type CPC system with a spiral-tube assembly-coil column, and (iii) a cross-axis CPC system with a multilayer-coil column. The hydrophilic dye mixture consisted of a sample of FD&C Blue No. 2 that contained mainly two isomeric components, 5,5'- and 5,7'-disulfonated indigo, in the ratio of ～7:1. The hydrophobic dye mixture consisted of a sample of D&C Red No. 17 (mainly Sudan III) and Sudan II in the ratio of ～4:1. The two-phase solvent systems used for these separations were 1-butanol/1.3M HCl and hexane/acetonitrile. Each of the three instruments was used in two experiments for the hydrophilic dye mixture and two for the hydrophobic dye mixture, for a total of 12 experiments. In one set of experiments, the lower phase was used as the mobile phase, and in the second set of experiments, the upper phase was used as the mobile phase. The results suggest that: (a) use of a J-type instrument with either a multilayer-coil column or a spiral-tube assembly column, applying the lower phase as the mobile phase, is preferable for separating the hydrophilic components of FD&C Blue No. 2; and (b) use of a J-type instrument with multilayer-coil column, while applying either the upper phase or the lower phase as the mobile phase, is preferable for separating the hydrophobic dye mixture of D&C Red No. 17 and Sudan II.     

Centrifugal Partition Chromatography. II. Selectivity Efficiency

Abstract

Centrifugal Partition Chromatography (CPC) is a technique that uses two immiscible liquids. One liquid is used as a stationary phase, the second one as the mobile phase. Using the liquid systems methanol-hexane and octanol-water, the selectivity and efficiency of CPC apparatus were tested. It is shown that CPC can be used to determine partition coefficients or to purify compounds. The selectivity can be changed the same way as in liquid chromatography. The efficiency study has shown that the plate count presents a minimum value at a particular flow rate. By plotting the number of channels required to obtain one theoretical plate versus the flow rate, it is possible to obtain a “van Deemter-type” plot. Interestingly, these plots show maxima for CPC. These results are opposite to those found for liquid or gas chromatography which have minima. A flow change, from laminar to nonlaminar is thought to explain these results. Reynolds numbers were determined for all solvent systems. Efficiency in CPC was also found to be solute dependent: the larger the partition coefficient, the lower the efficiency.