This article is from Journal of Chromatography. a, volume 1340. Abstract •An in investigation in to the use of membrane chromatography for the purification of a γ-retrovirus was undertaken.•The first report of a capacity for γ-retrovirus binding to a membrane chromatography device is presented.•A process that produces a large increase in concentration and purity of the studied γ-retrovirus was identified.•Proteomic techniques were used to identify the protein impurities removed and co-purified with the virus containing eluate.
DOI : doi:10.1016/j.chroma.2014.03.023
Cilt :1340 ISSN :0021-9673
This article is from Journal of Chromatography. a, volume 1349. Abstract •Size exclusion chromatography is a general method for nanoparticles separation.•The method is suited for purifying large quantities of protein covered nanoparticles.•Nanoparticles and proteins can be continuously separated by simulated moving bed chromatography.•Particle and protein fractions reached a purity of > 90%.•Reuse of buffer and unbound protein was possible using tangential flow filtration.
DOI : doi:10.1016/j.chroma.2014.04.093
Cilt :1349 ISSN :0021-9673
Various parameters have been evaluated to develop a process for optimization of column manufacture for packed capillary electrochromatography (CEC). Spherisorb ODS-1 was packed into 75 μm I.D. capillaries to establish a standard set of packing conditions to afford high-performance columns free of voids. Numerous silica-based packing materials including porous and non-porous reversed-phase and ion-exchange phases were employed to evaluate the applicability of the standard conditions. Success of column manufacture and performance demonstrate a relationship to the colligative properties of the packing materials under the applied conditions. Frequently encountered difficulties arising from inadequate column conditioning and void formation in the packed bed are identified and discussed.
Solid-phase microextraction (SPME)–gas chromatography–mass spectrometry was used to identify the cuticular hydrocarbons of the subterranean termite Coptotermes formosanus Shiraki. Headspace SPME and direct contact SPME methods were evaluated and compared to the hexane extraction method. Variables, such as temperature, time, number of termites, condition of the termites, and the type of SPME fiber were evaluated. Methods were refined to increase the reproducibility as well as the sensitivity. Both SPME methods were successfully used for the identification of all the major termite cuticular hydrocarbons. Using the headspace SPME method, other compounds of interest could also be identified, such as fatty acids. Using the direct contact SPME method, termites could be repeatedly studied over time to monitor chemical changes.
A hollow fiber supported liquid membrane extraction method for the liquid chromatographic determination of dinitrophenolic compounds at ppt levels has been developed. Different variables affecting the extraction process, such as extraction time, shaking speed, acceptor pH, acceptor buffer concentration, salt content and humic acids have been studied. Enrichment factors up to 7000 times were obtained. Validation of the method included calibration experiments and studies of the linearity of the responses in different matrices. Good linearity was obtained in the environmental matrices evaluated. Detection limits range from 6.0 to 8.0 ng/L, and the relative standard deviations do not exceed 7% in terms of repeatability.
A mixture of sodium fluoresceinate and silver nitrate in aqueous solution is described as a spraying reagent which can be used to detect many of the common anions—in particular those which form insoluble silver salts.
The lipids of gymnosperms frequently feature unusual polyunsaturated fatty acids (PUFAs) such as sciadonic acid (20:3Δ5,11,14) and juniperonic acid (20:4Δ5,11,14,17) showing a first double bond on C-5 which is separated from the next double bond by five methylene units. Compared to “classic” fatty acids, these fatty acids are not easily commercially available and their prices are quite high. For this reason, we wished to isolate those fatty acids from the seed oil of Podocarpus falcatus by countercurrent chromatography (CCC) after conversion of the fatty acids to methyl esters (FAMEs). The contribution of sciadonic acid (20:3Δ5,11,14) and juniperonic acid (20:4Δ5,11,14,17) in the unfractionated sample was 10% and 6% respectively, while oleic acid (18:1Δ9) and linoleic acid (18:2Δ9,12) were the major fatty acids. After a first CCC run with FAMEs from Podocarpus falcatus, fractions enriched in the target compounds were chosen for subsequent isolation by means of two subsequent CCC runs. Initially, 13 mg of juniperonic acid was recovered with a purity of 92% according to analysis by gas chromatography with mass spectrometry (GC/MS). Further purification of this fraction yielded 2.7 mg with a purity of 99% according to GC/MS. The isolation of sciadonic acid was hampered by high amounts of linoleic acid with the same equivalent chain length in suitable fractions of the first CCC separation. After an enrichment step by CCC, the critical pair sciadonic acid and linoleic acid was finally separated as free fatty acids. After this step, 4.4 mg of sciadonic acid was recovered with 99% purity. The methodology could also be applied to isolate larger amounts of those fatty acids or for the isolation of other minor fatty acids.
DOI : 10.1016/j.chroma.2015.03.042 Anahtar Kelimeler :
Countercurrent chromatography, Podocarpus falcatus, Polyunsaturated fatty acid, Gas chromatography, Mass spectrometry
Cilt: 1394 Sayı: 0 Sayfa: 89-94 ISSN: 0021-9673
Triacylglycerols (TAGs) containing less common fatty acids (FAs) were isolated from the seeds of three plants (Santalum album, Crepis foetida, and Leucas aspera). These FAs had allenic (laballenic acid, Lb) and acetylenic (crepenynic, C; ximenynic acids, Xi) bonds. TAGs were analyzed on reversed-phase and chiral columns. High-resolution tandem mass spectrometry identified TAGs by positive electrospray ionization (ESI+). Twenty-two molecular species of TAGs isolated from the seed oil of Santalum album were separated by RP-HPLC and chiral HPLC methods and identified by positive electrospray ionization tandem MS detection (ESI+-MS). Two major enantiomers, i.e., sn-OOLb and sn-LLLb (O represents oleic acid; and L represents linoleic acid), were synthesized from the appropriate phosphatidylcholines. This allowed the identification of enantiomers after separation by chiral chromatography by tandem mass spectrometry. Similarly, TAGs from the seeds of Crepis foetida, and Leucas aspera were analyzed by reversed-phase chromatography and identified by mass spectrometry. Four enantiomers (sn-OOC, sn-LLC, sn-OOXi, and sn-LLXi) were synthesized. A total of six and three enantiomers of TAGs containing crepenynic and ximenynic acids, respectively, were identified by chiral column analysis. The retention times of TAGs containing allenic and acetylenic bonds were always greater on the reversed-phase column than TAGs with the same number of carbon atoms and the same unsaturation (e.g., LLL versus LLLb). From the chiral column, the regioisomers and enantiomers were eluted in the order of symmetric-asymmetric-asymmetric (i.e., sn-OCO, sn-COO, and sn-OOC). Through tandem mass spectrometry, we were able to identify and distinguish regioisomer [DAG]+-type ions, i.e., [MNH4NH3RCOOH]+, that can be considered diagnostic. Unfortunately, enantiomers and TAGs with the same numbers of carbon atoms and the same unsaturation levels have identical mass spectra, such as LLL and LLLb.
The simulated moving bed (SMB) technology is receiving more and more attention as a convenient technique for the production scale continuous chromatographic separation of fine chemicals. Characteristic features of SMBs are improved performances with respect to preparative chromatography and nonlinear competitive adsorption behavior. The selection of the operating conditions to achieve high separation performances under nonlinear conditions is acknowledged to be the major problem in running a SMB unit for a new application. This problem is solved in this paper, where a general theory is developed which provides explicit criteria for the choice of the operating conditions of SMB units to achieve the prescribed separation of a mixture characterized by both constant selectivity Langmuir isotherms and variable selectivity modified Langmuir isotherms. The space of the operating parameters, i.e. the fluid to solid flow-rate ratios, is divided in regions with different separation regimes. The effect of increasing nonlinearity of the system on the operating conditions and the separation performances, namely desorbent requirement, enrichment, productivity and robustness of the separation, is thoroughly analyzed. The obtained results are shown to provide a very convenient tool to find both optimal and robust operating conditions of SMB units. Finally, a comparison between model predictions and experimental data dealing with the resolution of different racemic mixtures assesses the reliability and accuracy of the obtained theoretical findings.
Herein, a novel strategy was developed to separate and prepare target protein from complex sample by free-flow electrophoresis (FFE), which mainly based on the charge-to-mass ratio (C/M) analysis of proteins. The C/M values of three model proteins, namely Cytochrome C (Cyt C), myoglobin (Mb) and bovine serum albumin (BSA) were analyzed under different pH and the separation of these proteins was predicted by CLC Protein Workbench software. Series of experiments were performed to validate the proposed method. The obtained data showed high accordance with our prediction. In addition, the chamber buffer (CB) of FFE system was optimized to improve the resolution of separation. Meanwhile, in order to evaluate the analytical performance of the proposed method, Cyt C was extracted from swine heart and further separated by FFE based on C/M analysis. Results showed that Cyt C was completely separated from the crude sample and a purity of 96.9% was achieved. The activity of prepared Cyt C was 98.3%, which indicate that the proposed method is promising in a wide variety of research areas where the native properties of proteins should be maintained for downstream analysis.
A method using capillary gel electrophoresis with laser-induced fluorescence detection is described which permits complete sequence determination of antisense DNA analogues of unknown sequence. This method, originally created as a tool to confirm the sequence of antisense oligonucleotides being developed as therapeutic drugs, utilizes data collected under a range of experimental conditions described by the Ogston model as applied to gel electrophoresis. A linear relationship independent of experimental conditions between the relative electrophoretic migration time and the oligonucleotide base number was observed and is shown to be consistent with a simplified version of this model and can be used to facilitate the sequence determination.
On-column complexation of metal ions with 2,6-pyridinedicarboxylate (2,6-PDC) to form anionic complexes enabled their separation by capillary zone electrophoresis with direct UV detection at 214 nm. Nine metal ions, Cu2+, Zn2+, Ni2+, Cd2+, Mn2+, Pb2+, Fe3+, Al3+ and Ca2+, were determined in less than 7 min using10 mM 2,6-PDC solution containing 0.75 mM tetradecyltrimethylammonium bromide at pH 4.0. Satisfactory working ranges (20–300 μM), detection limits (3–10 μM) and good repeatability of the peak areas (RSD 2.1–4.2%, n=5) were obtained using hydrodynamic injection (30 s). The proposed method was used successfully for the determination of Mn2+, Fe3+, Al3+ and Ca2+ in groundwaters.
DOI : 10.1016/S0021-9673(02)00741-0 Anahtar Kelimeler :
Complexation, Derivatization, electrophoresis, Water analysis, Environmental analysis, Metal cations, Pyridinedicarboxylic acid
Cilt: 966 Sayı: 1-2 Sayfa: 245-251 ISSN: 0021-9673
This paper focuses on the application of RPLC × RPLC to pharmaceutical analysis and addresses the specific problem of separating co-eluting impurities/degradation products that maybe “hidden” within the peak envelope of the active pharmaceutical ingredient (API) and thus may escape detection by conventional methods. A comprehensive two-dimensional liquid chromatograph (LC × LC) was constructed from commercially available HPLC equipment. This system utilizes two independently configurable 2nd dimension binary pumping systems to deliver independent flow rates, gradient profiles and mobile phase compositions to dual Fused-Core secondary columns. Very fast gradient separations (30 s total cycle time) were achieved at ambient temperature without excessive backpressure and without compromising optimal 1st dimension sampling rates. The operation of the interface is demonstrated for the analysis of a 1 mg/ml standard mixture containing 0.05% of a minor component. The practicality of using RPLC × RPLC for the analysis of actual co-eluting pharmaceutical degradation products, by exploiting pH-induced changes in selectivity, is also demonstrated using a three component mixture. This mixture (an API, an oxidation product of the API at 1.0%, w/w, and a photo degradant of the API at 0.5%, w/w) was used to assess the stability indicating nature of an established LC method for analysis of the API.
A simple end-column amperometric detector, without a porous junction, was designed and attached to a capillary electrophoresis instrument to analyze eight mono-, di- and tri-chlorophenols. The platinum detecting electrode was modified by electrodeposition of tin, to enhance sensitivity as well as reduce electrode fouling owing to phenol oxidation. The modified electrode (0.127 mm in diameter) in combination with a 20 μm I.D. separation capillary yielded sharp peaks and enabled us to detect phenol at levels as low as 0.10 μM. Satisfactory separation of the eight chlorophenols was achieved by using a mixed buffer of phosphate-borate.
Preservation of ionic species within Antarctic ice yields a unique proxy record of the Earths climate history. Studies have been focused until now on two proxies: the ionic components of sea salt aerosol and methanesulfonic acid. Measurement of the all of the major ionic species in ice core samples is typically carried out by ion chromatography. Former methods, whilst providing suitable detection limits, have been based upon off-column preconcentration techniques, requiring larger sample volumes, with potential for sample contamination and/or carryover. Here, a new capillary ion chromatography based analytical method has been developed for quantitative analysis of limited volume Antarctic ice core samples. The developed analytical protocol applies capillary ion chromatography (with suppressed conductivity detection) and direct on-column sample injection and focusing, thus eliminating the requirement for off-column sample preconcentration. This limits the total sample volume needed to 300 μL per analysis, allowing for triplicate sample analysis with <1 mL of sample. This new approach provides a reliable and robust analytical method for the simultaneous determination of organic and inorganic anions, including fluoride, methanesulfonate, chloride, sulfate and nitrate anions. Application to composite ice-core samples is demonstrated, with coupling of the capillary ion chromatograph to high resolution mass spectrometry used to confirm the presence and purity of the observed methanesulfonate peak.
Several l-proline and (4R)-hydroxy-l-proline derivatives were evaluated as chiral selectors (CSs) in the separation of enantiomers by counter-current chromatography (CCC). A variety of biphasic solvent systems, all of organic/aqueous nature, were tested in order to determine the appropriate distribution for CSs and racemates (N-(3,5-dinitrobenzoyl)-(±)-leucine and (±)-ketoprofen). Successful separations of DNB-(±)-leucine in analogous experimental conditions allow the comparative study of the enantioselectivity displayed by the considered CSs. The low solubility of certain CSs limits their applicability for preparative purposes even for improved enantioselectivity. The effect that the nature and pH of the buffer solutions used as a component of the solvent system have on the separation was also studied.
This manuscript summarises the techno-economic feasibility of refined cashew nut shell liquid (CNSL). A simple mass transfer based mathematical model for the yield prediction is presented. The process parameters and extraction time for maximum profit and purity of the product were optimized. The optimum extraction time for maximum profit and purity was found to be 0.9 h at 300 bar and 323 K. The influence of the different costs, such as fixed cost, raw material cost, labor cost, utility cost, etc. on profit and cost of production of the extract is also presented.
A capillary electrophoretic (CE) method was developed for the separation of some common alkali and alkaline-earth metal ions using EDTA as complexing agent and pyridine as UV chromophore for indirect detection at pH 5.00. Effects of pH and concentration of complexing agent on the differences in the effective mobilities between two ions were considered and equations derived were used to deduce the optimum conditions for their separation. Baseline separation of a group of metal ions, including K, Na, Li, Mg, Sr and Ba, was achieved in less than 4 min. The calibration range for magnesium was found to be linear up to 1.00 μg/ml when samples were prepared in the running buffer while a hyperbolic calibration curve was obtained when prepared in water. Application of the method to the analysis of magnesium in river water, urine and a solid sample of calcium carbonate was demonstrated. Magnesium in those samples was determined to be 812 μg/ml, 78.0 μg/ml and 0.023% (w/w), respectively, with reproducibilities between 5–9% R.S.D. in terms of peak height depending on sample matrices.
High-temperature gas chromatography was applied to assay the activity and selectivity of lipases, and to test various reaction conditions. This technique is capable to analyze mixtures of fatty acids and acylglycerols simultaneously and, therefore, to monitor the hydrolysis of acylglycerols. Lipases can catalyze the hydrolysis of acylglycerols ini aqueous solution. The reaction conditions employed are milder than those of conventional chemical hydrolysis. In practice, this method was used in the sample preparation procedure for the quantitative analysis of γ-linolenic acid in triacylglycerols.
Planar electrochromatography (PEC) is a new technology for thin layer chromatography (TLC) where the separation is driven by electroosmotic forces, not capillary action. This allows for much faster and more efficient chromatography in a planar format. Care needs to be taken when performing these experiments because voltage and flow characteristics can change through a single run, due to buffer gradients, temperature changes (Joule heating) and localized plate heterogeneity. We have designed a PEC instrument and cover grid to allow investigation of flow and voltage characteristics as solvent moves across a TLC plate. Our unique cover grid allows monitoring voltage at eight discrete points between the positive and negative reservoirs. A linear relationship between voltage and distance should be seen, giving a constant voltage drop across a plate, but this did not occur. This non-linear function changes over time, following the plate equilibration. Once a plate is equilibrated, voltage and flow characteristics remain fairly constant. Theoretical calculations support the physical observations. Larger plate widths (5 cm) were also briefly investigated and it is concluded that large width plates could be easily implemented to maintain multiple sample capability.
The principal driving force for the explosive renaissance in liquid chromatography in the last decade can be found in the failure of gas chromatography to accommodate compounds of medium and high molecular weight. However, even liquid chromatography, including its subclass of exclusion chromatography, weakens at very high molecular weights and eventually becomes inapplicable. The primary difficulties stem from unbalanced phase distribution and interfacial adsorption. These problems are largely avoided in field-flow fractionation (FFF).
The review passes through nearly 15 years of development of LC-GC transfer techniques, listing the concepts proposed and discussing the reasons why many of them were not followed up. On the one hand, a number of ideas should be re-evaluated in order to check whether the best choices were made, further elongating the list of techniques in use. On the other, success of LC-GC requires that a minimum number of transfer techniques are selected and promoted in order to get them implemented into standardized methods and bring more LC-GC into routine laboratories.
Traditional Chinese medicines (TCMs) have attracted much attention in recent years. Elution-extrusion and/or back-extrusion counter-current chromatography (EECCC/BECCC) both take full advantage of the liquid nature of the stationary phase. They effectively extend the solute hydrophobicity window that can be studied and rendered the CCC technique particularly suitable for rapid analysis of complex samples. In this paper, a popular traditional Chinese medicine, Evodia rutaecarpa, was used as the target complex mixture for extrusion CCC separations. With a carefully selected biphasic liquid system (n-hexane/ethyl acetate/methanol/water, 3/2/3/2, v/v) and optimized conditions (VCM = VC, mobile phase flow rate: 3 mL/min in descending mode, sample loading: 100 mg), five fractions could be obtained in only 100 min on a 140-mL capacity CCC instrument using both elution- and back-extrusion methods. Each fraction was analyzed and identified compared with the data of major standards using LC/MS. Moreover, the performance of both extrusion protocols was systematically compared and summarized. EECCC could be operated continuously and was found extremely suitable for high-throughput separation; however, post-column addition of a clarifying reagent is recommended to smooth the UV-signal during the extrusion process. Considering BECCC, the practical operation is very simple by just switching a 4-port valve to change the flow direction. The change of flowing direction should be done after a sufficient amount of mobile phase has flushed the column in the classical mode so that solutes with small and medium distribution constants have been eluted. Otherwise, a significant portion of the solutes will stay in the mobile phase inside the column, mix together and produce a broad peak showing in the mobile phase eluting after the stationary phase extrusion. Compared with classical CCC or other preparative separation tools, extrusion CCC approaches exhibit distinguished superiority in the modernization process of traditional Chinese medicines.
DOI : 10.1016/j.chroma.2008.10.095 Anahtar Kelimeler :
Counter-current chromatography, Elution-extrusion, Back-extrusion, Traditional Chinese medicines, Evodia rutaecarpa
Cilt: 1216 Sayı: 19 Sayfa: 4140-4146 ISSN: 0021-9673
Some results obtained by gas chromatography and high-pressure liquid chromatography in vitamin A analysis are given. In particular, it was demonstrated that high-pressure liquid chromatography surpasses gas chromatography as an analytical tool for this application.
A quick and simple method for identification and semi-quantitative determination of nine antioxidants commonly used in lubricants is presented. A dual step thin-layer chromatography (TLC) separation, removes in a first step the oil matrix whereas in a second step the antioxidants are separated. Cutting the spots out of the TLC-plate in the form of triangles allows direct-spray mass spectrometric (MS) measurements, providing MS and MSn spectra (if an appropriate MS instrument is employed) of the antioxidants, allowing their identification but also giving information about potential oxidation or degradation of these additives. Calibration curves within the concentration range relevant for the analysis of real oil samples (0.2–1.2 g L−1) were constructed with R2 values above 0.98 (when using an appropriate internal standard). This allowed the semi-quantitative determination of the selected antioxidants in real oils samples. Comparison with results from HPLC-UV measurement showed acceptable agreement for all analytes.
DOI : 10.1016/j.chroma.2015.01.048 Anahtar Kelimeler :
Thin-layer chromatography, Mass spectrometry, Lubricants, Antioxidants
Cilt: 1383 Sayı: 0 Sayfa: 169-174 ISSN: 0021-9673
A HPLC method is described for the analysis of ochratoxin A at low-ppb levels in samples of artificially contaminated cocoa beans. The samples are extracted in a mixture of methanol–water containing ascorbic acid, adjusted to pH and evaporated to dryness. Samples in this state are then placed onto a Benchmate sample preparation workstation where C18 solid-phase extraction operations are performed. The resulting materials are evaporated to dryness and analyzed by reversed-phase HPLC with fluorescence detection. The method was evaluated for accuracy and precision with R.S.D.s for multiple injections of sample and standard calculated to be 1.1% and 2.5% for sample and standard, respectively. Recoveries of ochratoxin A added to cocoa beans ranged from 87–106% over the range of the assay.
A novel immobilization method for the preparation of tetraphenylporphyrin–silica stationary phases is investigated. Stationary phases consisting of immobilized hydroxyphenyl-triphenylporphyrin (HPTPP) on silica are shown to exhibit unmatched selectivity (α=kC70′/kC60′) and improved efficiency for the separation of fullerenes, with α=7 using 100% toluene as the mobile phase. The HPTPP species are immobilized via reaction with glycidoxypropyltrimethoxysilane. The high shape selectivity of columns packed with HPTPP–silicas enables the single step separation of higher molecular mass fullerenes and higher fullerene isomers using strong fullerene solvents as mobile phases.
A mixture of polydimethylsilicones (Dow Corning 200), average molecular weight 2000 a.m.u., was separated by simultaneous density and temperature-programmed supercritical fluid chromatography and detected by ion mobility detection. Ion mobility spectra were captured by Fourier transform ion mobility spectrometry. Using information from these spectra it was possible to selectively detect a single compound in the complex mixture. A detector temperature investigation demonstrated that, for the efficient transfer of high-molecular-weight compounds from the column to the detector, the interface to the detector must be heated. Using a 50 μm I.D. column, a Guthrie-type restrictor and a detection temperature of 250°C, as many as 70 oligomers were separated and detected.
Thirteen mono- and dilaterally substituted liquid crystalline compounds were investigated for their gas chromatographic behaviour as stationary phases for the separation of mono- and dimethylnaphthalenes. A normal packed column system was used. These compounds offered a good separation for some pairs of methylnaphthalenes.