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.     

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.     

Colour pigments of Trichoderma harzianum. Preliminary investigations with thin-layer chromatography-Fourier transform infrared spectroscopy and high-performance liquid chromatography with diode array and mass spectrometric detection

The colour pigments of Trichoderma harzianum fermentation broth were separated and the main fractions were tentatively identified by reversed-phase thin-layer chromatography-Fourier transform infrared spectroscopy (RP-TLC-FT-IR), RP-HPLC-diode array detection and RP-HPLC-MS. It was established that the multistep gradient elution developed for RP-TLC separation of pigments can be successfully used as a pilot method for the rational design of gradient elution in RP-HPLC for the separation of the same pigments. FT-IR and MS measurements were unable to identify the exact chemical structures of the main pigment fractions, the presence of OH, =CH and C=O (RP-TLC-FT-IR) and OH and NH, substructures (RP-HPLC-MS) was confirmed. It was assumed that the main pigment fractions are oxidation polymers originating from monomer molecules containing polar substructures and double bonds in the alkyl chain which are liable for oxidation during the aerobic fermentation process.     

Lipophilicity characterization by reversed-phase liquid chromatography of some furan derivatives

Lipophilicity is one of the properties which influences the partition of a substance in biological media. The present study reports on the chromatographic behaviour of a newly synthesised series of furan derivatives by RP-HPLC and RP-TLC, with methanol-water and acetonitrile-water as mobile phases, in order to establish if the linear relationships between the retention parameters (log k, R(M)) and the concentration of organic modifier in the mobile phase, phi, allows the extrapolation procedure. Good correlations between the retention parameters were obtained by RP-HPLC and RP-TLC, and the concentration of organic modifier (methanol, acetonitrile) in the mobile phase was established for the studied furan derivatives. However, for the discussed compounds, acetonitrile has a lower sensitivity to changes in the structures. A good correspondence was obtained between the extrapolated parameters for the methanol-water mobile phase when using RP-HPLC and RP-TLC. However, stronger interactions occur in RP-TLC between the compounds and the residual silanol groups than in RP-HPLC.     

Determination of lipophilicity of alpha-(4-phenylpiperazine) derivatives of N-benzylamides using chromatographic and computational methods

This paper describes the evaluation of lipophilicity of alpha-(4-phenylpiperazine) derivatives of N-benzylamides. We employed reversed-phase thin-layer chromatography (RP-TLC) and reversed-phase high performance liquid chromatography (RP-HPLC) as experimental methods, using mixtures of acetonitrile and water as the mobile phases with addition of 0.1%TFA in the HPLC experiments. Retention parameters (R(M)) and capacity factors (log k) determined by applying these methods were linearly dependent on the acetonitrile concentration and enabled us to estimate the relative lipophilicity factors: R(M0) and log k(0). These factors were compared with the calculated partition coefficients C log P obtained using several software packages. The results indicate that both experimental methods (RP-TLC and RP-HPLC) yielded similar results, and these methods enable determining the lipophilicity of alpha-(4-phenylpiperazine) derivatives of N-benzylamides. Significant correlations were found between log P values calculated by Pallas, ALOGPS and C log P Chem3D programs and the experimental data.     

Investigation of lipophilicity of anticancer-active thioquinoline derivatives

The lipophilicity of a series of anticancer propargylthioquinoline derivatives has been investigated using chromatographic and computational methods. The parameters of relative lipophilicity (R(MO) and logk0) of the tested compounds were determined experimentally both by reversed-phase thin layer (RP-TLC), and high-performance liquid chromatographic methods (RP-HPLC, LiChrospher RP18 column), with mixtures of acetonitrile and water as mobile phases. Their phospholipophilicity (logk(0IAM)) was determined using immobilized artificial membrane HPLC (IAM. PC DD2 Regis column). Mobile phase acetonitrile concentrations were in the ranges 50-90% (RP-TLC), 55-90% (RP-HPLC) and 35-60% (IAM-HPLC). The R(M), logk and logk(IAM) values of the compounds investigated were linearly dependent on acetonitrile concentration. The analysis led to the calculation of R(MO), logk0 and logk(0IAM) parameter values for each of the tested compounds. Their partition coefficients (logP) were also calculated with the Pallas and CAChe programs. The obtained results indicated that, among experimental methods, both RP-TLC and RP-HPLC gave similar results, and these methods enable the determination of lipophilicity of derivatives of thioquinolines. Using the IAM-HPLC technique a simple method of estimation of phospholipoplilicity was described. The CAChe program might better predict calculated lipophilicity logP values, and therefore is a useful tool for the early stage of design of new propargyl thioquinolines.     

Quantitative Determination of Digitalis Glycosides in Digitalis Purpurea Leaves by Reversed-Phase Thin-Layer Chromatography

Abstract

A densitometric reversed-phase thin-layer chromatographic(RP-TLC) method for the determination of digitalis glycosides in Digitalis purpurea leaves has been developed. The procedure involves extraction of dry leaf powder with ethanol/chloroform (2:1) and clean-up by Sep-Pak cartridges prior to RP-TLC analysis. RP-TLC was performed on an octadecylsilyl bonded silica gel plate, using a developing solvent of acetonitrile/0.5 M NaCl(1 : 1) for secondary glycosides and acetonitrile/0.5 M NaCl(2 : 3)for primary glycosides. The plate was scanned with a reflectance densitometer at 222 nm. Quantitation of these glycosides was carried out by the internal standard method. The amounts of digitoxin, gitoxin, gitaloxin, purpurea glycoside A, purpurea glycoside B, and glucogitaloxin in a leaf powder sample of Digitalis purpurea were estimated from the calibration graphs for pure glycosides.     

Application of RP-TLC technique for the determination of dissociation constants of 1-substituted pyrrolidin-2-one derivatives

The procedures for measuring dissociation constants (pK(a)) of twenty 1-substituted pyrrolidin-2-one derivatives are described. The dissociation constants of the compounds tested were determined using potentiometric titration, reversed-phase thin-layer chromatography (RP-TLC) and calculated using Pallas and Marvin programs. It was found that the RP-TLC method of determination of pK(a) could be considered as a feasible alternative to potentiometric titration. The Marvin program is a better tool for preliminary estimation of dissociation constant than the Pallas one.     

HPLC characterization of betalains from plants in the amaranthaceae

HPLC characterization of reversed-phase (RP) high-performance liquid chromatography (HPLC) has been widely used in separation and identification of plant pigments. An effective RP-HPLC-based method is established to systematically isolate, identify, and quantitate the betalain pigments in the plants of 37 species of eight genera in the Amaranthaceae. A total of 16 betacyanins and three betaxanthins are characterized mainly using the RP-HPLC method and also with the aid of mass spectroscopy. The identified betacyanins include eight amaranthine-types, six gomphrenin-types, and two betanin-types. They are also divided into six simple (nonacylated) betacyanins and 10 acylated betacyanins. Acylated betacyanins are identified as betanidin 5-O-beta-glucuronosylglucoside or betanidin 6-O-beta-glucoside acylated with ferulic, p-coumaric, or 3-hydroxy-3-methylglutaric acids. Three betaxanthins were separated from Celosia species in the Amaranthaceae and identified to be immonium conjugates of betalamic acid with dopamine, 3-methoxytyramine, and (S)-tryptophan; the latter two are found to be new betaxanthins from plants.     

The study of the lipophilicity of alpha-(4-phenylpiperazin-1-yl)-gamma-phthalimidobutyramides using chromatographic and computational methods

The lipophilicity of the series of alpha-(4-phenylpiperazin-1-yl)-gamma-phthalimido-butyramides (1-8) has been investigated. Several methods, like reversed-phase thin-layer chromatography and high-performance liquid chromatography using reversed-phase RP18 and IAM.DD2 columns, were applied to determine RMO, log k0 and log k(0IAM) factors. The RP-TLC investigations were performed in mixtures of acetone-water and acetonitrile-water. For RP-HPLC method mixtures of acetonitrile, water and 0.01% TFA were used as the mobile phases while for IAM.DD2 investigations mixtures of acetonitrile and water were applied. The partition coefficients of compounds (1-8) were also calculated with the Pallas and CAChe programs. All the obtained data, both from experimental methods and computational calculations, were compared and a suitable conclusion was reached.     

Determination of boron at sub-ppm levels in uranium oxide and aluminum by hyphenated system of complex formation reaction and high-performance liquid chromatography (HPLC)

Boron, at sub-ppm levels, in U3O8 powder and aluminum metal, was determined using complex formation and dynamically modified reversed-phase high-performance liquid chromatography (RP-HPLC). Curcumin was used for complexing boron extracted with 2-ethyl-1,3-hexane diol (EHD). Separation of complex from excess reagent and thereafter its determination using the online diode array detector (DAD) was carried out by HPLC. Calibration curve was found to be linear for boron amounts in the sample ranging from 0.02 microg to 0.5 microg. Precision of about 10% was achieved for B determination in samples containing less than 1 ppmw of boron. The values obtained by HPLC were in good agreement with the data available from other analytical techniques. The precision in the data obtained by HPLC was much better compared to that reported by other techniques. The present hyphenated methodology of HPLC and complex formation reaction is interesting because of cost performance, simplicity, versatility and availability when compared to other spectroscopic techniques like ICP-MS and ICP-AES.     

Development and validation of reversed-phase column high-performance liquid chromatographic and first-derivative UV spectrophotometric methods for estimation of voriconazole in oral suspension powder

This research paper describes validated reversed-phase high-performance column liquid chromatographic (RP-HPLC) and first-derivative UV spectrophotometric methods for the estimation of voriconazole (VOR) in oral suspension powder. The RP-HPLC separation was achieved on Phenomenex C18 column (250 x 4.6 mm id, 5 microm particle size) using water-acetonitrile (40 + 60, v/v; pH adjusted to 4.5 +/- 0.02 with acetic acid) as the mobile phase at a flow rate of 1.4 mL/min and ambient temperature. Quantification was achieved with photodiode array detection at 255 nm over the concentration range of 0.1-1 microg/mL with mean recovery of 99.49 +/- 0.83% for VOR by the RP-HPLC method. Quantification was achieved with UV detection at 266 nm over the concentration range of 8-20 microg/mL with mean recovery of 99.74 +/- 0.664% for VOR by the first-derivative UV spectrophotometric method. These methods are simple, precise, and sensitive, and they are applicable for the determination of VOR in oral suspension powder.     

Separation and identification of some common isomeric plant triterpenoids by thin-layer chromatography and high-performance liquid chromatography

Chromatographic separation of 10 triterpenoids (α-amyrin, β-amyrin, δ-amyrin, lupeol, lupenon, lupeol acetate, cycloartenol, cycloartenol acetate, ursolic acid, oleanolic acid) and 2 sterols (stigmasterol and β-sitosterol) was studied. The chromatographic techniques included silica gel and reversed-phase (C₁₈ RP) thin-layer chromatography (TLC) and C₁₈ RP high-performance liquid chromatography (HPLC) using UV and mass spectrometric (MS) detection with atmospheric pressure chemical ionization (APCI). The TLC separation of the isomeric triterpenols lupeol, α-amyrin, β-amyrin and cycloartenol was achieved for the first time using C₁₈ RP-HPTLC plates. Cycloartenol could be separated from related compounds only on C₁₈ RP-TLC but not on the C₁₈ RP-HPLC. δ-Amyrin isolated from the tomato fruit surface extract could be separated from other amyrins only by HPLC. Tandem mass spectrometry allowed discrimination between the isomers lupeol, α-amyrin, β-amyrin, δ-amyrin, cycloartenol and between lupeol acetate and cycloartenol acetate. The combination of 3 TLC methods and 2 HPLC methods enables qualitative determination of all 12 compounds and proves to be useful for the analysis of plant extracts. It is recommended that TLC screening on silica gel and C₁₈ RP be performed before HPLC analysis.     

红花中红花黄色素含量的反相高效液相色谱测定

本文分离纯化了红花（Carthamus tinctorius L）中Safflower yellow A，Safflomin A，Safflower yellow B，Safflomin C四种红花黄色素，并用所纯化的红花黄色素做标准品，用反相高效液相色谱法，梯度洗脱，同时测定了不同产地红花中四种红花黄色素的含量。该方的法准确度高，重现性好。     

Interaction of surfactants with homologous series of peptides studied by reversed-phase thin-layer chromatography

The relative strength of interaction between anionic (SDS) and nonionic surfactant (octaethoxylated oleyl alcohol, GEN) and homologous series of peptides was determined by reversed-phase thin-layer chromatography (RP-TLC) carried out on alumina layers impregnated with paraffin oil. The relative strength of interaction was calculated and was correlated with the physicochemical parameters of peptides. It was established that each peptide interacted with both surfactants and with their mixture (1:1, m/m). The relative strength of interaction depended on the number of amino acid units in the peptide, side chain bulk and electronic properties and hydrophobicity of the amino acids. The impact of individual parameters highly depended on the character of surfactant. The data prove that the retention order of peptides can be modified by adding different surfactants and surfactant mixtures to the mobile phase resulting in improved separation.     

Novel method for high-performance liquid chromatography of azo derivatives of conjugated and unconjugated bilirubin

A method for the separation and quantitation of ethyl anthranilate or p-iodoaniline azo derivatives of bile pigments was developed using reversed-phase high-performance liquid chromatography. A convenient separation was achieved in 15 min, permitting the quantitation of the unconjugated azo-dipyrrole (alpha o) and its glucuronide (delta), xyloside (alpha 2) and glucoside (alpha 3) conjugates. The pathological beta- and gamma-azo pigments, derived from bilirubin glucuronide isomers that occur in cholestatic bile or plasma, are also detected in this system. The results of this method as applied to bile from 25 healthy dogs were in excellent agreement with the values obtained by reversed-phase chromatography of bilirubin and its mono- and dimethyl esters produced from the corresponding conjugates by alkaline methanolysis. This system permits the sensitive and convenient determination of bilirubin and its conjugation pattern in biological fluids.     

Improvement of chemical analysis of antibiotics. X. Determination of eight tetracyclines using thin-layer and high-performance liquid chromatography

Analytical methods for eight tetracyclines (TCs) were established using silica gel high-performance thin-layer chromatography (HPTLC), reversed-phase thin-layer chromatography (RP-TLC) and high-performance liquid chromatography (HPLC). Good separations of eight TCs were obtained using chloroform-methanol-5% disodium ethylenediaminetetraacetate solution (65:20:5) (lower layer) and methanol acetonitrile 0.5 M oxalic acid solution (1:1:4) (pH 3.0) on silica gel HPTLC and C8 TLC plates, respectively. A combination of HPTLC and RP-TLC made possible the identification of the eight TCs. Each calibration graph was linear between 0.1 and 1.0 microgram using UV densitometry except for rolitetracycline. For detection reagents, the diazonium salts including Fast Violet B gave variously coloured spots with the eight TCs and good sensitivities were obtained except with minocycline. In HPLC, the simultaneous analysis of the eight TCs on a C8 column was possible using methanol-acetonitrile-0.01 M oxalic acid solution (1:1.5:7) adjusted to pH 3.0 as the mobile phase. A linear relationship was obtained between 1.0 and 10 ng using the usual sample preparation except for rolitetracycline. The direct determination of rolitetracycline was possible using tetrahydrofuran, dimethyl sulphoxide and the mobile phase as solvents for preparation of the sample. For the determination of residual rolitetracycline, it was effective to measure the amount of rolitetracycline as tetracycline by HPLC, HPTLC and RP-TLC after conversion of rolitetracycline to tetracycline by incubating for 5 min in methanol at 50 degrees C.     

反相高效液相色谱法测定药用植物大红袍中的两个生物活性成分

建立了反相高效液相色谱-二极管阵列检测器(RP-HPLC-DAD)测定药用植物大红袍中具有抗菌活性的异黄酮类化合物3′-geranyl-5,7,4′-trihydroxyisoflavone(化合物1)及具有良好免疫抑制活性的紫檀烯类化合物8,9-dihydroxy-1-methoxy-［6′,6′-dimethylpyrano(2′,3′: 2,3)］pterocarpene(化合物2)含量的方法。采用的色谱柱为Agilent Zorbax SB-C18柱(250 mm×4.6 mm, 5 μm),以乙腈-0.1%甲酸水溶液为流动相进行梯度洗脱,流速为1.0 mL/min;柱温30 ℃。化合物1和化合物2分别在4.4～13.2 μg和0.428～1.284 μg范围内呈线性关系;平均回收率分别为99.65%和99.11%,相对标准偏差分别为1.83%和2.59%(n=5)。该方法快速简便,灵敏度和分离度好,适用于大红袍药材中活性黄酮类成分的测定。     

Effects of fluorescent light and vacuum packaging on the rate of decomposition of pigments in paprika (Capsicum annuum) powder determined by reversed-phase high-performance liquid chromatography.

The effect of storage time, the presence of light and oxygen on the decomposition rate of carotenoid pigments in paprika (Capsicum annuum) powders was determined by reversed-phase high-performance liquid chromatography (RP-HPLC). The similarities and dissimilarities of pigment composition of samples under various storage conditions was elucidated by principal component analysis (PCA) and stepwise regression analysis (SRA). Calculations proved that the overall decomposition rate of pigment sections equally depended on the storage time and on the presence of light and oxygen, the effect of storage time being the most decisive factor while the impact of oxygen was the lowest. The selectivity of decomposition also depended on the storage time and on the presence of oxygen the influence of storage time being the most important. RP-HPLC followed by PCA and SRA can be successfully used for the study of the impact of environmental conditions on the decomposition of carotenoid pigments of paprika powders.