Separation and tentative identification of the main pigment fraction of raisins by thin-layer chromatography-Fourier transform infrared and high-performance liquid chromatography-ultraviolet detection

The soluble color pigments of raisin are separated by reversed-phase thin-layer chromatography (TLC), and the capacity of TLC-Fourier transform infrared (FTIR) with both on-line and off-line coupling is assessed for the identification of the main fraction. TLC has also been used as a pilot technique for the development of a gradient elution method for the separation of pigments by high-performance liquid chromatography (HPLC). On-line TLC-FTIR cannot be used for identification because of the strong adsorbance of the stationary phase. Off-line TLC-FTIR combined with the retention behavior of the main pigment fraction indicates that it is a polymer, caramel-like compound composed of erythrose and fructose monomers. Baseline separation of pigments is achieved by HPLC using TLC as a pilot method.     

Classification of Chili Powders by High Performance Liquid Chromatography‐Diode Array Detection

Abstract

The color pigments of six chili powders of different origins (China, Bali, Pakistan, Malaysia, India, and Thailand) were separated and quantified by reversed‐phase high‐performance liquid chromatography (RP‐HPLC) using a narrow‐bore octadecylsilica column (Purospher, 125×3 mm I.D., Merck, Darmstadt, Germany), gradient elution, and diode array detector (DAD). The similarities and dissimilarities among the pigment composition of chili powders have been elucidated by principal component analysis (PCA). The RP‐HPLC separated 71–111 pigment fractions depending on the detection wavelength and on the origin of chili powder. The pigment composition of chili powders from Malayia and China showed marked similarities while the composition of pigments of other chili powders was different. It was concluded that RP‐HPLC–DAD can be successfully employed for the separation and quantitative determination of pigments of chili powders of various origins and may help the classification of chili powders and facilitate the authenticity test of such food products.     

The RP-HPLC analysis of anthocyanins

Summary Conditions were determined for the separation of a complex set of anthocyanins (free aglycones, mono- and multiglycosides and esterified forms) by HPLC. The optimised gradient elution method was then used to carry out qualitative and quantitative analysis of anthocyanin compounds present in the callus tissue ofRudbeckia hirta L. and the tubular flowers of the soil-based plant. The summary content of anthocyanin pigments and the content of the main pigment was identified in the analysed biomass. The method developed is useful for the purposes of monitoring the process of biosynthesis of anthocyanins in tissues obtained through in vitro cultures. The advantages of the method for anthocyanins and its application to other anthocyanin-rich materials are also discussed.     

Separation of plant membrane lipids by multiple solid-phase extraction

Plant membrane lipids were separated by multiple solid-phase extraction (SPE) in a single run. Elution was performed continuously through the modulated stationary phase employing only non-aqueous solvent systems. At the different stages of the glycerolipid separation the SPE manifold combined arninopropyl, arninopropyl/silica gel and silica gel/aminopropyl weak anion exchanger columns. The glycerolipid extract of pigment-containing plant tissues was cleared from the pigments onto the aminopropyl column. The aminopropyl column with the glycerolipid extract was then connected to a silica gel column from which monogalactosyldiacylglycerol, phosphatidylethanolamine, phosphatidylglycerol and digalactosyldiacylglycerol were eluted as individual fractions. The elution was performed under polarity, pH and temperature gradient conditions. To continue the separation, the aminopropyl column was discarded and the silica gel column containing the remaining glycerolipid extract was connected to an aminopropyl anion exchanger column. Individual fractions of sulfoquinovosyldiacylglycerol, phosphatidylcholine and phosphatidylinositol were now eluted. The separation process was supported by ammonium counter ions and by the polarity gradient of the elution systems used. The membrane lipids were isolated from pigment-containing (rice and maize leaves and rice leafy stems) and pigment-free (rice roots) tissues. The repeatability for a standard glycerolipid mixture was 2-6% (n=7), and for rice leaf lipid extracts, 3-7% (n=5). Glycerolipid recovery was 87-95%.     

Separation and quantitation of colour pigments of chili powder (Capsicum frutescens) by high-performance liquid chromatography-diode array detection

The performance of reversed-phase thin-layer (RP-TLC) and reversed-phase high-performance liquid chromatography (RP-HPLC) was compared for the separation and determination of the colour pigments of chili (Capsicum frutescens) powder using a wide variety of eluent systems. No separation of pigments was achieved in RP-TLC, however, it was established that tetrahydrofuran shows an unusually high solvent strength. RP-HPLC using water-methanol-acetonitrile gradient elution separated the chili pigments in many fractions. Diode array detection (DAD) indicated that yellow pigments are eluted earlier than the red ones and chili powder contains more yellow pigments than common paprika powders. It was established that the very different absorption spectra of pigments make the use of DAD necessary.     

High-performance liquid chromatography–off line mass spectrometry analysis of anthraquinones produced by Geosmithia lavendula

Lilac coloured species of Geosmithia lavendula produce a mixture of polyhydroxylated anthraquinones under condition of submerged fermentation. Three pigments had been isolated and identified earlier as a 1,3,6,8-tetrahydroxyanthraquinone (compound 7), rhodolamprometrin (1-acetyl 2,4,5,7-tetrahydroxyanthraquinone; compound 5), and 1-acetyl 2,4,5,7,8-penthahydroxyanthraquinone (compound 4). A new HPLC method was developed for the separation of three known and ten new anthraquinone pigments. In addition, five new pigments were determined by FTMS as coeluting impurities. The analyses were performed on a reversed phase column using gradient elution with a mobile phase system consisting of phosphate buffer (50 mM; pH = 2.0) and acetonitrile. The structure evaluation was based namely on FTMS and UV–VIS spectrometry. The developed procedure was used for the determination of individual anthraquinones in fermentation broth of G. lavendula after 14 days of cultivation. The extractable amount and LOQ (both in μg ml⁻¹) for the two main pigments from G. lavendula are 50.02 and 2.15 for compound 4, and 63.77and 2.75, for compound 5, respectively.     

The offline combination of thin-layer chromatography and high-performance liquid chromatography with diode array detection and micrOTOF-Q mass spectrometry for the separation and identification of spinochromes from sea urchin (Strongylocentrotus droebachiensis) shells

Thin-layer chromatography (TLC) with off-line high-performance liquid chromatography coupled to diode array detection and micrOTOF-Q mass spectrometry (HPLC-DAD-MS) resulted in the successful fractionation, separation and identification of spinochrome pigments from sea urchin (Strongylocentrotus droebachiensis) shells. Two fractions of pigments were separated by TLC and eluted with methanol using a TLC-MS interface. HPLC-DAD-MS analysis of the fractions indicated the presence of six sea urchin pigments: spinochrome monomers B and D, three spinochrome dimers (anhydroethylidene-6,6'-bis(2,3,7-trihydroxynaphthazarin) and its isomer and ethylidene-6,6'-bis(2,3,7-trihydroxynaphthazarin)), and one pigment that was preliminary identified as a spinochrome dimer with the structural formula C(22)H(16)O(16).     

梯度洗脱优化-离子色谱-脉冲安培法分析婴幼儿配方乳粉中的糖和糖醇

建立了梯度洗脱优化-离子色谱-脉冲安培检测技术分析婴幼儿配方乳粉中麦芽糖醇、半乳糖、葡萄糖、蔗糖、果糖、乳糖6种与代谢疾病紧密相关的糖和糖醇的方法。该方法对样品的前处理、色谱柱的选择、淋洗液的梯度等色谱条件进行了优化,重点研究了淋洗液梯度对各组分分离效果的影响。将梯度洗脱程序分解为弱保留组分分离、乳糖分离、基质消除、系统平衡4个子程序,并分别对其进行了条件优化,实现了6种组分的完全分离,并有效解决了弱保留组分色谱峰易重叠、乳糖色谱峰易拖尾等现象。6种组分的线性范围为0.5~100 mg/L,相关系数（r）>0.99,方法的检出限（S/N=3）为0.06~0.40 mg/L。该方法具有分离度好、灵敏度高等优点,可满足例行分析的要求,同时也为淋洗液的梯度优化提供了可借鉴的方法。     

Reversed-phase high-performance liquid chromatography of some ultra-heterogeneous and covalently-modified proteins from human hair

A new procedure for the fractionation of the heterogeneous cystine-rich proteins from human hair, utilizing reversed-phase high-performance liquid chromatography, is described. Of these proteins 27 fractions have been collected and analyzed for amino acid composition. There seems to be little correlation between the elution order and the hydrophobicity of the fraction constituents except for the late-eluting fractions. Based on the elution profiles and amino acid contents, these fractions appear to fall into four families. The effects of alkyl chain length, flow-rate and gradient slope, as well as various additives to the organic modifier on the separation have also been investigated. A low flow-rate (0.4 ml/min) and a shallow gradient were essential for the separation of these proteins as was the use of short alkyl chain (C4) or medium alkyl chain (C8) columns. However, with the C4 column reproducibility and recovery were excellent.     

BIOCHEMICAL ISOLATION AND SPECTROSCOPIC CHARACTERIZATION OF POSSIBLE PHOTORECEPTOR PIGMENTS FOR PHOTOTAXIS IN A FRESHWATER Peridinium

Photoreceptor pigments have been isolated biochemically from the freshwater dinoflagellate Peridinium gatunense, and characterized spectroscopically. At least four different chromoproteins can be detected in the crude extract and the membrane fraction isolated from the cells absorbing at 580, 638, 667 and 710 nm, which correspond with the maxima in the action spectrum for phototaxis in this organism. Light energy absorbed by shorter wavelength pigments is emitted as fluorescence at wavelengths which are absorbed by pigments with maxima at longer wavelengths. Protein separation on a MonoQ anion exchanger column using fast liquid chromatography resulted in a non-bound fraction and four bound fractions eluted by an NaCI gradient, which differed in their pigment composition.     

Development of a rapid and convenient method to separate eight ginsenosides from Panax ginseng by high-speed counter-current chromatography coupled with evaporative light scattering detection

Ginsenosides exhibit diverse biological activities and are major well-known components isolated from the radix of Panax ginseng C.A. Meyer. In the present work, a rapid and facile method for the separation and purification of eight ginsenosides from P. ginseng by high-speed counter-current chromatography coupled with evaporative light scattering detector (HSCCC-ELSD) was successfully developed. The crude samples for HSCCC separation were first purified from ginseng extract using a macroporous resin; the extract was loaded onto a Diaion-HP20 column and fractionated by methanol and water gradient elution. The ginsenosides-protopanaxadiol (PPD) and protopanaxatriol (PPT) fractions were subsequently eluted with 65 and 80% methanol and water gradient elution, respectively. Furthermore, these two fractions were separated by HSCCC-ELSD. The two-phase solvent system used for separation was composed of chloroform/methanol/water/isopropanol at a volume ratio of 4:3:2:1. Each fraction obtained was collected and dried, yielding the following eight ginsenosides: Rg(1), Re, Rf, Rh(1), Rb(1), Rc Rb(2) and Rd. The purity of these ginsenosides was greater than 97% as assessed by HPLC-ELSD, and their structures were characterized by electrospray-ionization mass spectrometry (ESI-MS) and nuclear magnetic resonance spectroscopy. This is the first report regarding the separation of the ginsenosides Rh(1), Rb(2) and Rc from P. ginseng by HSCCC.     

Computer-assisted high-performance liquid chromatography method development with applications to the isolation and analysis of phytoplankton pigments

We used chromatography modeling software to assist in HPLC method development, with the goal of enhancing separations through the exclusive use of gradient time and column temperature. We surveyed nine stationary phases for their utility in pigment purification and natural sample analysis. For purification, a complex algal matrix was separated on an efficient monomeric column, from which partially purified fractions were collected and purified on polymeric columns that exaggerated resolution between pigments of interest. Additionally, we feature an HPLC method that is simple, fast, demonstrates excellent transferability and is ideal for quantitative analysis of pigments in dilute natural water samples.     

Profiling of colour pigments of chili powders of different origin by high-performance liquid chromatography

The colour pigments of five chili powders of different origins were separated and quantified by reversed-phase high-performance liquid chromatography (RP-HPLC). The similarities and dissimilarities of pigment composition of chili powders were elucidated by principal component analysis (PCA). RP-HPLC separated 50-100 pigment fractions depending on the detection wavelength and on the origin of chili powder. It was found that the pigment composition of chili powders from Malaysia and China and from India and Pakistan show marked similarities while the composition of colour pigments of chili powder from Thailand was different. It was further established that the chromatograms are similar in the first 5-35 min of development, they are highly different between 35 and 75 min and moderately different at the end of the chromatograms. It was concluded that RP-HPLC followed by PCA can be successfully used for the identification of chili powders according to the composition of their colour pigments.     

Application of displacement chromatography for the proteome analysis of a human plasma protein fraction

It was the aim of this study to compare the performance of displacement chromatography with gradient elution chromatography both applied as the cation-exchange separation step for a proteome analysis in a bottom-up approach using multidimensional chromatography for the separation of tryptic peptides prior to their mass spectrometric analysis. The tryptic digest of the human Cohn fraction IV-4 served as a sample. For both chromatography modes commonly used operating parameters were chosen thus ensuring optimal separation results of equal sample amounts for each mode. All resulting fractions were analyzed with an HPLC-chip–LC–MS system. The eluate of the HPLC-chip column was ionized by electrospray ionization (ESI) and analyzed with an ion-trap mass spectrometer. For guaranteeing high confidence concerning the identity of the peptides, the mass spectrometric data were processed by different bioinformatic tools applying stringent criteria. By the displacement approach the total amount of identified proteins (78) was significantly higher than in the gradient mode (58). The results showed that displacement chromatography is a well suited alternative in comparison to gradient elution separation for analysis of proteomes via the bottom-up approach applying multidimensional chromatography, especially in those cases when larger quantities of proteins are available.     

Ionization of dichlorophenols for their analysis by capillary electrophoresis-mass spectrometry

The qualitative and quantitative betalain pigment content of two cultivars of prickly pear (Opuntia ficus-indica) fruits grown in southeastern Spain was evaluated. After methanolic extraction of crushed fruits, reversed-phase high-performance liquid chromatography and photodiode array detection were applied simultaneously for the separation, identification and quantification of these pigments. Two main pigments were obtained, which were identified as indicaxanthin (lambda(max) 484 nm) and betanin (lambda(max) 535 nm). Spectrophotometric evaluation of both pigments showed a yield of around 20-30 mg per 100 g of fresh pulp. When the influence of temperature (25 to 90 degrees C) on betacyanin pigment stability was investigated, the results revealed a substantial degree of thermodegradation at temperatures higher than 70 degrees C.     

Fractionation and separation of human salivary proteins by pH-gradient ion exchange and reversed phase chromatography coupled to mass spectrometry

In the present work, a 2-D capillary liquid chromatography method for fractionation and separation of human salivary proteins is demonstrated. Fractionation of proteins according to their pI values was performed in the 1-D employing a strong anion exchange (SAX) column subjected to a wide-range descending pH gradient. Polystyrene-divinylbenzene (PS-DVB) RP columns were used for focusing and subsequent separation of the proteins in the 2-D. The SAX column was presaturated with a high pH buffer (A) consisting of 10 mM amine buffering species, pH 9.0, and elution was performed with a low pH elution buffer (B) having the same buffer composition and concentration as buffer A, but pH 3.5. Isoelectric point fractions eluting from the 1-D column were trapped on PS-DVB trap columns prior to back-flushed elution onto the PS-DVB analytical column for separation of the proteins. The 1-D fraction eluting at pH 9.0-8.7 was chosen for further analysis. After separation on the RP analytical column, nine RP protein fractions were collected and tryptic digested for subsequent analyses by MALDI TOF MS and column switching capillary LC coupled to ESI TOF MS and ESI QTOF MS. Eight proteins and two peptides were identified in the pH 9.0-8.7 fraction using peptide mass fingerprinting and uninterpreted MS/MS data.     

Optimization of the separation of chlorophenols with stepwise gradient elution in reversed phase liquid chromatography

An optimization technique based on gradient elution was used to separate eleven chlorophenols by reversed phase liquid chromatography. The separation was based on gradient elution with a stepwise variation pattern of the volume fraction of organic modifier, phi, in the mobile phase. Initially, two-, three-, and four-parameter equations which describe the dependence of ln k' upon phi, were examined for their ability to fit the experimental data. It was found that, among these equations, the four-parameter equation gave the best fit of the experimental data. In addition to separation optimization, a non-linear least squares program with a grid search for initial estimates was used to determine the best variation pattern. The best variation pattern was obtained with phi(1)= 0.27, phi(2)= 0.39, phi(3)= 0.62, t(1) = 33 min, and t(2) = 11 min. This pattern allowed the chromatographic separation of the chlorophenols with a good resolution and a total analysis time of 51 min. Good agreement was observed between predicted and experimental values of the retention times under optimal condition.     

A fully automated 2-D LC-MS method utilizing online continuous pH and RP gradients for global proteome analysis

The conventional 2-D LC-MS/MS setup for global proteome analysis was based on online and offline salt gradients (step and continuous) using strong-cation-exchange chromatography in conjunction with RP chromatography and MS. The use of the online system with step salt elution had the possibility of resulting in peptide overlapping across fractions. The offline mode had the option to operate with continuous salt gradient to decrease peak overlap, but exhibited decreased robustness, lower reproducibility, and sample loss during the process. Due to the extensive washing requirement between the chromatography steps, online continuous gradient was not an option for salt elution. In this report, a fully automated, online, and continuous gradient (pH continuous online gradient, pCOG) 2-D LC-MS/MS system is introduced that provided excellent separation and identification power. The pH gradient-based elution provided more basic peptides than that of salt-based elution. Fraction overlap was significantly minimized by combining pH and continuous gradient elutions. This latter approach also increased sequence coverage and the concomitant confidence level in protein identification. The salt and pH elution-based 2-D LC-MS/MS approaches were compared by analyzing the mouse liver proteome.     

酪蛋白多肽的制备和色谱分离方法

为了得到低成本的多肽,本文利用胰蛋白酶对酪蛋白进行了充分的酶解。采用分析级反相高效液相色谱-电喷雾质谱联用技术(RP-HPLC/ESI-MS)分析了酶解产物各组分的组成,并通过改变流动相的梯度洗脱程序,优化了分析级色谱条件以充分分离相对含量较高的多肽组分;将优化的分析级色谱条件直接放大到制备级RP-HPLC中,在程序控制下通过紫外吸收信号结合ESI-MS信号共同引导实现了多肽的全自动化分离和收集。整个过程方便快捷,经过这样一个单一的分离步骤,得到了多个纯度较高的多肽。除此之外,本文还考察了流动相的酸碱性、柱上样量等因素对该体系制备级分离的影响,并对一次分离中分辨率不好的亲水性多肽混合物进行了二次分离,得到了多个新的多肽。本文建立的多肽制备方法为多肽和多肽材料的广泛应用提供了一种选择。     

Separation of pyranoanthocyanins from red wine by column chromatography

With the aim of monitoring the formation of anthocyanin-derived pigments and contributing to the study of their chromatic properties, stability and relative contribution to the colour of red wines, a method for fractionation of the colouring material was set up. The method was based on the distinct reactivity of the different pigment families towards bisulfite (hydrogen sulfite). The wine, acidified and bleached with NaHSO₃, was placed in a Toyopearl^® HW-40(s) gel column and submitted to elution with ethanol. Two fractions with different pigment compositions were collected and analysed by liquid chromatographay diode array detection-mass spectrometry. Compounds present in each fraction were identified according to their UV-visible and MSⁿ mass spectra, showing that the first one was mostly constituted of pyranoanthocyanins, whereas the second basically contained anthocyanins and anthocyanin-flavanol condensation products. A large variety of new pigments were detected, some of which had not been previously reported in red wines, as far as we know. Characteristic MS² and MS³ fragmentation patterns were observed within each family of compounds, which could be further applied for characterisation of unknown pigments in other wines.