Study of the feasibility of using a pellicular anion-exchange column for separation of transferrin isoforms in human serum by HPLC with UV detection

A method has been optimised for the separation of glycoforms of human serum transferrin, using a high-performance pellicular anion-exchange chromatographic column. The effect of the eluent pH and of the column temperature on the separation of transferrin glycoforms was studied using a standard solution of commercially available human serum transferrin. An HPLC system equipped with an ultraviolet detector was used for the analysis. No immunoassay was used after the anion-exchange chromatographic separation of the glycoforms, in contrast with most currently used methods. The method was applied to the separation and quantification of transferrin glycoforms in serum from a healthy, non-pregnant woman, after saturation of transferrin with iron and further precipitation of lipoproteins. The whole chromatographic run, including re-equilibration of the column, took 35 min.     

Study of the binding of 114mIn radiotracer to human serum components by ultrafiltration and chromatography

The chemical speciation of indium in serum was studied. Ultrafiltration was used to investigate the influence of several buffer systems on the binding characteristics of indium in serum and to study the association of indium with transferrin and albumin. This was performed by means of batch incubation experiments with a 114mIn tracer. Different buffer systems were investigated. A series of bicarbonate, Tris:HCl and HEPES buffers were found to fit for this purpose. Phosphate buffer was not suitable, as it is capable of disrupting the binding between indium and transferrin. Batch ultrafiltration experiments with 114mIn incubated solutions of transferrin and albumin showed that both proteins are capable of binding indium to a high degree. Three chromatographic techniques (SEC, AEC, AC) were used to study the different chemically active species of indium in serum. It is concluded that next to transferrin, albumin is also responsible for the binding and transport of indium in serum.     

Purification of monoclonal antibodies from cell culture supernatants using a modified zirconia based cation-exchange support

A method suitable for the isolation of monoclonal antibodies (Mabs) is described. The protocol utilizes a zirconia based column modified with ethylenediamine-N,N'-tetra(methylenephosphonic) acid to create a novel cation-exchange chromatographic support. Initial experiments using a linear salt gradient demonstrate the ability of this support to efficiently separate Mab from transferrin and bovine serum albumin in a model matrix. Results of the purification of Mab from an actual cell culture supernatant over a range in protein concentrations are also shown. Analyses by enzyme-linked immunosorbent assay and gel electrophoresis demonstrate that Mabs can be recovered from a cell culture supernatant at high yield (92-98%) and high purity (> 95%) in a single chromatographic step.     

High-performance liquid chromatographic separation of human apotransferrin and monoferric and diferric transferrins

A simple method for the chromatographic separation of the different molecular forms of human transferrin according to their respective iron contents is described. The appropriate conditions were developed with a Mono-S cation-exchange column.     

Application of conjoint liquid chromatography with monolithic disks for the simultaneous determination of immunoglobulin G and other proteins present in a cell culture medium

The aim of this study was to develop a chromatographic method, as a substitute for enzyme-linked immunosorbent assays, for the rapid and simultaneous detection of IgG, insulin, and transferrin present in a cell culture medium. Conjoint liquid chromatography (conjoint LC) using monolithic disks was applied for this purpose. An anion-exchange disk was combined with a Protein G affinity disk in a preparative HPLC system. IgG bound to the Protein G disk, whereas transferrin and insulin were captured on the quaternary ammonium (QA) disk. Using this method, it was possible to simultaneously determine the concentrations of IgG, transferrin, and insulin in the cell culture medium. Thus, conjoint LC could be used for the rapid and simultaneous detection of different proteins present in a cell culture medium.     

High-performance liquid chromatographic method for the determination of free gossypol in chicken liver.

A high-performance liquid chromatographic method for the determination of free gossypol in chicken liver at levels down to 0.5 ppm has been developed. Tissue was deproteinized with acetonitrile in presence of ascorbic acid and the filtrate was subjected to hydrolysis with hydrochloric acid. The liberated pure gossypol was partitioned into chloroform and analysed by gradient elution on a 10-microns C18 column. The overall recovery was 83.5 +/- 2.6%, with an overall relative standard deviation of 9%.     

Preparation of a novel carboxyl stationary phase by “thiol‐ene” click chemistry for hydrophilic interaction chromatography

A novel carboxyl‐bonded silica stationary phase was prepared by “thiol‐ene” click chemistry. The resultant Thiol‐Click‐COOH phase was evaluated under hydrophilic interaction liquid chromatography (HILIC) mobile phase conditions. A comparison of the chromatographic performance of Thiol‐Click‐COOH and pure silica columns was performed according to the retention behaviors of analytes and the charged state of the stationary phases. The results indicated that the newly developed Thiol‐Click‐COOH column has a higher surface charge and stronger hydrophilicity than the pure silica column. Furthermore, the chromatographic behaviors of five nucleosides on the Thiol‐Click‐COOH phase were investigated in detail. Finally, a good separation of 13 nucleosides and bases, and four water‐soluble vitamins was achieved.     

用熵最小算法解析桂郁金挥发油GC-MS重叠谱

采用气相色谱-质谱(GC-MS)联用技术对桂郁金中提取的挥发油成分进行检测,同时用熵最小算法对其中的共流物色谱峰进行解析,并通过质谱库检索和程序升温保留指数相结合的方式对解析得到的各纯组分进行定性分析。将桂郁金挥发油各组分的质谱数据直接与美国国家标准及技术研究所(NIST)数据库进行比对,鉴定出38个化合物;在挥发油的色谱图中,存在一些因分离不完全而导致的共流物色谱峰,以保留时间为1 106.52~1 108.38 s及1 184 s的色谱峰为例,经熵最小算法解析,共发现5个纯组分。采用熵最小算法可以清楚地对共流物色谱峰进行解析并得到所包含的各个纯组分,该法可提高复杂成分定性定量分析结果的准确性。     

Assessing chromatographic peak purity using condition index and singular value evolving profiles

A new method for determining chromatographic peak purity is presented. The method uses condition index evolving profiles (CIEPs) and singular value evolving profiles (SVEPs). To produce a CIEP or a SVEP, singular value decompositions are performed on data matrices containing the analyte's pure-component spectrum and sample spectra as they evolve over a chromatographic time profile. Visual inspection of condition indices and singular values as they evolve over time enables detection of less than 0.5% of a spectrally similar impurity with no chromatographic resolution (R_s = 0). Additionally, the CIEPs and SVEPs allow estimation of chromatographic pure regions. This method represents an extension of CIEPs which have been used in successful library searches of multicomponent mixtures based on gas chromatography-Fourier transform infrared spectra and liquid chromatography-UV-visible spectra.     

Eutectic Thin-Layer Chromatography as a New Possibility for Quantification of Plant Extracts—A Case Study

Deep eutectic solvents (DES), compared to classic ones, have interesting properties, such as the ability to solubilize compounds differing in polarity or increased dissolution of selected chemical compounds. They also offer specific interactions between the mobile and stationary phases. Those features make them promising solvents in chromatographic techniques, including the use in the separation of complicated samples. The first quantitative analysis with eutectic thin-layer chromatography (TLC) is presented in the paper. As a case study, five alkaloids from Chelidonium maius were selected as target compounds. A wide range of terpene-based DESs was investigated to develop the chromatographic system, both pure and after dilution. Moreover, a novel approach was employed to adjust polarity, involving mixing DESs differing in chromatographic properties. This procedure has proved to be effective. The best results were obtained with a 2:1 (wt/wt) mixture of DESs: camphor + phenol and menthol + limonene, with a 20% addition of methanol. The chromatographic system was validated and checked on the real sample, which made it the first applicable and operational quantitative eutectic TLC system.     

Chromatographic separation of papain evaluated by immunochemical methods

The chromatographic separation of crude papain preparations on Sephadex G-50 (fine) enables pure papain to be obtained in a single step. Immunochemical techniques have been found to be very convenient for testing the purity of the individual chromatographic fractions. A general approach is presented that makes it possible to follow the course of the chromatographic purification of any immunogenic compound by simple qualitative immunochemical techniques that can be applied in any laboratory.     

A new general approach to purify proteins from complex mixtures

The selection of chromatography media and their sequential use represent a major difficulty to isolate a single protein from very crude protein extracts. The process described here consists of two main steps: (i) a rational selection of few media from a relatively large collection and (ii) the definition of the sequence of columns to get the best purity of the target protein. From the first step, one sorbent is selected for its properties to capture the protein to purify, regardless whether other protein impurities are also co-adsorbed; then 5-7 other complementary sorbents are identified to remove impurities but without interacting with the target protein under the same buffering conditions. The second step consists in superimposing sorbents under a cascade manner with the sorbent in charge to capture the target protein located in the last position. Non-adsorbed proteins are eliminated in the flowthrough; other impurities are progressively removed by the sorbent sequence and the target protein is finally desorbed and isolated from the last sorbent using an optimized gradient. All operations are performed with a single adsorption buffer for all columns and all monitoring performed by means of mass spectrometry associated with ProteinChip arrays and polyacrylamide gel electrophoresis. Examples of protein isolation/identification from human serum are described namely thyroxin-binding proteins and transferrin. The first is isolated thanks to a series of dye chromatography media, the second (transferrin) using current chromatographic media. In both cases the target proteins were purified at a level estimated of about 95% and 85%, respectively. Isolated proteins were pure enough for the purpose of formal identification by either peptide fingerprinting or sequencing.     

Large-scale purification of Leuconostoc mesenteriodes NRRL B512F dextransucrase for use in the biosynthesis of dextran by batch and continuous chromatography

The extracellular enzyme dextransucrase was produced from Leuconostoc mesenteriodes NRRL B512F and purified by ultracentrifugation and cross-flow ultrafiltration for use in the biosynthesis of the macromolecule dextran by ion exchange chromatographic reaction—separation techniques. The two-stage purification process yielded over 90% pure dextransucrase with overall enzyme recovery of over 60%. A second stage of centrifugation was required to achieve complete cell removal. The purified enzyme contained 1–2 g l⁻¹ of solute ions, which affected the operation of the chromatographic system. Gel filtration removed over 93% of the remaining ions but resulted in high activity losses. Two-phase separation with polyethylene glycol (PEG) and purification by ion exchange chromatography were less successful in desalting the enzyme. PEG precipitation was successful in concentrating the enzyme, but the ions remained predominantly with the enzyme portion of the two phases. The purified enzyme was found to be unstable during storage.Use of the enzyme in chromatographic reactor—separators for the production of dextran resulted in over 33% more high molecular weight dextran (the desired product) and a useful pure fructose byproduct being obtained than for a conventional reactor. Sodium and potassium ions in the enzyme hampered continuous operation by displacing calcium ions from the resin and thus reducing the separation efficiency of the system. Partial regeneration of the resin with calcium nitrate rather than complete enzyme desalting, which was very expensive and resulted in high activity losses, helped overcome this effect.     

Baclofen impurities: Facile synthesis and novel environmentally benign chromatographic method for their simultaneous determination in baclofen

An efficient, economic and high yielding method was described for the synthesis of baclofen (BAC) pharmacopoeial impurities (impurity A and impurity B) which can be used for gram‐scale synthesis. Furthermore, a novel ecofriendly thin‐layer chromatographic TLC–densitometric method was established and validated for the determination of BAC and its synthesized impurities. The developed TLC–densitometric method is based on the chromatographic separation using TLC plates (60 F₂₅₄) using a green mobile phase of ethyl acetate–methanol–ammonia solution, 33% (8:2:0.1, by volume) with UV scanning at 220 nm. The proposed method was validated with respect to International Conference on Harmonization guidelines. The validated method was successfully applied for determination of BAC in pure form and in its commercial dosage form. Additionally, the greenness profile of the developed method was evaluated and compared with those of the reported chromatographic methods. The developed method was found to be superior to the published methods, being environmentally benign.     

High-performance liquid chromatography using a color-forming agent as a component of the mobile phase. Separation and determination of nickel and zinc wity xylenol orange

Summary A liquid chromatographic procedure for separating nickel and zinc has been developed. Xylenol orange, which is one of the sensitive and commercially available color-forming agents, was used as a component of the mobile phase. The two ions could be separated on weak anion-exchange gels within 10 min. The procedure was suitable for the determination of nickel and zinc in relatively pure solutions with fairly high sensitivity. The described liquid chromatographic analysis would be also potentially applicable for any aqueous sample containing trace levels of metal cations at 1–10 ppm.     

High-performance liquid chromatographic separation of subcomponents of antimycin A

Using a reversed-phase high-performance liquid chromatographic (HPLC) technique, a mixture of antimycins A was separated into eight hitherto unreported subcomponents, A1a, A1b, A2a, A2b, A3a, A3b, A4a, and A4b. Although a base-line resolution of the known four major antimycins A1, A2, A3, and A4 was readily achieved with mobile phases containing acetate buffers, the separation of the new antibiotic subcomponents was highly sensitive to variation in mobile phase conditions. The type and composition of organic modifers, the nature of buffer salts, and the concentration of added electrolytes had profound effects on capacity factors, separation factors, and peak resolution values. Of the numerous chromatographic systems examined, a mobile phase consisting of methanol-water (70:30) and 0.005 M tetrabutylammonium phosphate at pH 3.0 yielded the most satisfactory results for the separation of the subcomponents. Reversed-phase gradient HPLC separation of the dansylated or methylated antibiotic compounds produced superior chromatographic characteristics and the presence of added electrolytes was not a critical factor for achieving separation. Differences in the chromatographic outcome between homologous and structural isomers were interpreted based on a differential solvophobic interaction rationale. Preparative reversed-phase HPLC under optimal conditions enabled isolation of pure samples of the methylated antimycin subcomponents for use in structural studies.     

Correction of retention time shifts for chromatographic fingerprints of herbal medicines

In this study, the combination of chemometric resolution and cubic spline data interpolation was investigated as a method to correct the retention time shifts for chromatographic fingerprints of herbal medicines obtained by high-performance liquid chromatography-diode array detection (HPLC-DAD). With the help of the resolution approaches in chemometrics, it was easy to identify the purity of chromatographic peak clusters and then resolve the two-dimensional response matrix into chromatograms and spectra of pure chemical components so as to select multiple mark compounds involved in chromatographic fingerprints. With these mark components determined, the retention time shifts of chromatographic fingerprints might be then corrected effectively. After this correction, the cubic spline interpolation technique was then used to reconstruct new chromatographic fingerprints. The results in this work showed that, the purity identification of the chromatographic peak clusters together with the resolution of overlapping peaks into pure chromatograms and spectra by means of chemometric approaches could provide the sufficient chromatographic and spectral information for selecting multiple mark compounds to correct the retention time shifts. The cubic spline data interpolation technique was user-friendly to the reconstruction of new chromatographic fingerprints with correction. The successful application to the simulated and real chromatographic fingerprints of two Cortex cinnamomi, fifty Rhizoma chuanxiong, ten Radix angelicae and seventeen Herba menthae samples from different sources demonstrated the reliability and applicability of the approach investigated in this work. Pattern recognition based on principal component analysis for identifying inhomogenity in chromatographic fingerprints from real herbal medicines could further interpret it.     

Estimation of individual ultraviolet spectra in incomplete two-component separations by high-performance liquid chromatography

A method is described for the estimation of spectral features in a two-component chromatographic peak by means of a u.v. diode-array detector. The calculation relies on the assumption that the front of a fused chromatographic peak contains a single pure component. The spectrum of this component is used in calculating the concentration profile of the other component, thus allowing the determination of a solution band for the spectrum of the second component. The boundaries of the solution band are based on non-negativity restrictions of chromatographic and spectral features. The method does not require the use of principal components analysis.     

Liquid chromatographic determination of lysine, methionine, and threonine in pure amino acids (feed grade) and premixes: collaborative study

A total of 17 laboratories (including one author's laboratory) participated in a collaborative study for determination of lysine, methionine, and threonine in trade products or concentrated amino acid premixes. Thirteen samples, 4 pure amino acids and 6 premixes, including 3 Youden matched pairs, were analyzed. The applied liquid chromatographic (LC) method using cation-exchange resin and post-column derivatization with ninhydrin or o-phthaldialdehyde was shown to be accurate and specific for the analytes. Titration procedures, normally used for the assay of pure amino acids, are unspecific and the accuracy of the results can be affected by impurities. Repeatability relative standard deviations, RSDr, ranged from 0.84 to 1.17% for pure amino acids and from 0.50 to 1.68% for premixes; reproducibility relative standard deviations RSDR, ranged from 1.52 to 2.31% for pure amino acids and from 1.48 to 2.59% for premixes. Recoveries were between 97.5 and 102.8% of the expected amino acid assays. The method has been adopted Official First Action status by AOAC INTERNATIONAL.