Separation of abietane-type diterpenoids from Clerodendrum kaichianum Hsu by high-speed counter-current chromatography using stepwise elution

High-speed counter-current chromatography (HSCCC) was successfully used for the separation of abietane-type diterpenoids from the medicinal plant C. kaichianum, which were not separated in our previous study using preparative HPLC. The HSCCC separation employed the lower phases of n-hexane–ethyl acetate–methanol–water (HEMW) 4:5:4:5 and HEMW 4:5:5:4 as the mobile phase for stepwise elution while the upper phase of HEMW 4:5:4:5 was used as the stationary phase. HSCCC separation yielded 90.5 mg of compound 1(kaichianone A), 137.7 mg of compound 2 (kaichianone B), 125.0 mg of compound 3 (teuvincenone E), and 227.6 mg of compound 4 (taxusabietane A) with purities of 95.3%, 97.2%, 97.8%, and 98.6%, respectively, as determined by HPLC. Compounds 1–2 are two new abietane-type diterpenoids while Compounds 3–4 are known abietane-type diterpenoids, analyzed by ESIMS and NMR data. The results demonstrated that HSCCC can be an excellent alternative for other separation methods. The two new compounds showed significant cytotoxicity against ileocecal carcinoma HCT-8 and breast adenocarcinoma MCF-7 cells.     

Comparison of Micellar Electrokinetic Chromatography with HPLC for the Separation of Hematoporphyrin Derivatives

ABSTRACT

A mixed SDS micelle and BSA buffer system was used in the micellar electrokinetic chromatography (MEKC) separation of hematoporphyrin derivatives (HPD) at pH 8.0 with untreated capillaries. The effects of altering the composition of sodium dodecyl sulfate (SDS) electrolyte solution on the separation efficiency of the hematoporphyrin derivatives were presented. The results show that separation efficiencies were enhanced by using a mixture of SDS and BSA. The results demonstrated that CE methodology can compete with well-established techniques such as HPLC for the separation of biomedical and pharmaceutical samples with regard to time and expense of analysis.     

Chromatographic Separation of the Halides

Abstract

Column chromatography with the liquid ion exchanger Aliquat 336 was evaluated for the separation of the halides in aqueous solution. The separation was successfully accomplished by a gradient elution technique using 0.1 M sodium acetate and various concentrations of sodium nitrate as eluants. A comparison of two methods of column loading was made.     

Separation and Identification op Metal Ions by Reversed Phase Then Layer Chromatography

Abstract

Conditions for the simultaneous separation and identification of as many as ten different metal ions have been established using the technique of reversed-phasethin-layer chromatography. Complete resolution of the ten ions in sub-microgram amounts was achieved using a liquid ion exchanger supported on cellulose. The total time for the analysis was 3–4 hours.     

Separation of Prostaglandins on a Hydrophobic Sephadex Derivative

Abstract

Liquid-gel chromatography on a hydrophobic Sephadex derivative has been used for the separation of prostaglandins. Solvent systems are given for separation of PGE and PGF compounds either as free acids or as methyl esters, using a Sephadex derivative containing 12% hydroxyalkyl groups. The columns are simple to prepare and can be used repeatedly over long periods of time. The recovery is high and the technique should be a useful complement to other methods used for the isolation of prostaglandins.     

Chromatographic Behaviour of Amino Sugars on Metal Salt Impregnated Thin Layers

Abstract

Suitable TLC separation scheme for Amino Sugars on silica gel G plates impregnated with metal salt, using the solvent system methanol-acetone (50:10) has been worked out. The visualization of the spots was done by spraying with ninhydrin in 1 - butanol.     

The Separation of Bilirubin,Photobilirubin and their Major Isomers by Ion-Pair High Pressure Liquid Chromatography

Abstract

The separation of the three major isomers and the main photoproducts of bilirubin by Ion-Pair High Pressure Liquid Chromatography is described. Chromatographic procedures found in the literature proved difficult to reproduce, had unacceptably long elution times (over 2 hours), required complex gradients or two or more columns. As these reports provided no data to show why specific chromatographic conditions were chosen an optimization study was carried out. The separation is shown to be dependent on pH, organic content and buffer concentration of the mobile phase, ion-pairing agent concentration and injection solvent. A separation, carried out in less than thirty minutes on octadecyl silica using isocratic conditions with detection at 455 nm is demonstrated. This technique will be used in the elucidation of the mechanism involved in phototherapy treatment of neonatal jaundice.     

Method Development and Validation for the Determination of Five Synthetic Food Colorants in Alcoholic Beverages by Reversed‐Phase High Performance Liquid Chromatography Coupled with Diode‐Array Detector

Abstract

An accurate method based on the use of reversed‐phase high performance liquid chromatography coupled with diode‐array detection was devised for the determination of five synthetic food colorants added to alcoholic beverages with natural colors. A C18 stationary phase was used and the mobile phase contained methanol and 40 mM ammonium acetate buffer solution. The synthetic food colorants were detected at their corresponding individual characteristic maxima of absorbance wavelength. Successful separation was achieved within 11 min for all the analytes using an optimized gradient elution, column temperature, buffer concentration and flow rate. Accurate sample quantification was feasible using matrix‐matched calibration curves. The method was successfully validated by determination of linearity ranges, the limits of quantification and detection, precision and recovery for all colorants tested. The proposed and validated method was used to analyze some alcoholic beverage samples, consisting of eight red wines, six coolers, four aromatized spirits, five bitters, three cocktails and four liquors from different Chinese manufacturers. The results showed the bitters and red wines did not have synthetic colorants, but colorants were found in all the samples of other kinds of alcoholic beverages. No analyzed sample exceeded the limit established by Chinese legislation.     

A Fluorometric Enzyme Inhibition Detector for Carbamate Pesticide Analysis by High Speed Liquid Chromatography

Abstract

A fluorometric enzyme inhibition detector has been developed for the detection of carbamate pesticides after separation using high speed liquid chromatography.

Housefly head cholinesterase was found to have the greatest sensitivity toward the carbamates, together with horse plasma and bovine erythrocyte cholinesterases. The substrate N-methyl indoxyl acetate is used for monitoring of enzyme activity.     

Simultaneous Determination of Preservatives,Artificial Sweeteners,and Synthetic Dyes in Kimchi by Ultra-Performance Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometry (UPLC-ESI-MS/MS)

Abstract

A simple and rapid method was developed for the simultaneous determination of five preservatives, six artificial sweeteners, and nine synthetic dyes in kimchi using ultra-performance liquid chromatography electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS) with multiple reaction monitoring (MRM). The chromatographic separation was performed in 5.5?min using an Acquity UPLC BEH C18 column (100?mm × 2.1?mm, 1.7?µm) with a mobile phase composed of 0.002% trifluoroacetic acid and 10?mM aqueous ammonium acetate or 9:1 (v/v) methanol:acetonitrile. Linear calibration curves were obtained with correlation coefficients above 0.98. The limits of quantification ranged between 0.227 and 8.569?ng/mL, while the recovery values in kimchi samples were from 83.1 to 113.5%. Forty kimchi samples were analyzed for the food additives, with sweeteners detected in more than half of these samples. The most commonly detected sweetener was saccharin, and six of the kimchi samples contained a combination of sweeteners; dulcin, sucralose, acesulfame K, preservatives, and synthetic dyes were not detected. Based on these results, the developed method can be used for the rapid quality control of food additives in kimchi.     

Chromatographic behavior of selected dyes on silica and cellulose micro-TLC plates: Potential application as target substances for extraction,chromatographic, and/or microfluidic systems

ABSTRACT

The main goal of this research communication focuses on screening of chromatographic behavior of 18 colorants including amaranth, black PN, bromophenol blue, bromocresol green, bromocresol purple, bromothymol blue, carmine, dimethyl yellow, erythrosine, fluorescein, methyl red, naphthalene black, patent blue, phenol red, Sudan II, Sudan III, Sudan IV, and p-xylenol blue. They are commonly used as food and industrial colorants or sensing molecules for analytical and biomedical applications. We studied retention (R_F), peak base width (W_b), and peak asymmetry factor (A_S) data collected under thermostatic conditions (303?K) using silica and cellulose-coated microplates (5?×?5?cm) and simple as possible mono/binary eluents. Presented retention database may help to select the proper conditions for, for example, chromatographic separation, extraction to solid phase (e.g. bar-type extraction devices) or application of such chemicals as the internal standard substances for planar/column chromatography and/or microfluidic devices (e.g., microfluidic paper-based analytical devices—μPADs). Furthermore, the microplates peak capacity (maximum number of resolvable spots, n) was calculated and discussed. As the second goal, we presented results of literature concerning dyes quantification in complex samples. We compared latest methodologies involving simple spectrophotometric analysis and more sophisticated protocols like high-throughput separation/detection procedures based on high-performance planar/liquid chromatography, capillary electrophoresis and microfluidic devices.     

Gas Chromatographic Determination of Trimethylsilyl Derivatives of Free Amino Acids from a Botanical Source

Abstract

The application of gas chromatography for the separation of TMS-amino acids from a botanical source was demonstrated. The trimethyl-silyl derivatives of the extracts from germ free tobacco tissue cultures were prepared by reacting amino acid extracts with bis(trimethylsilyl)-trifluoroacetamide (BSTFA) using acetonitrile as a reaction solvent following preliminary separation of the free acids by ion exchange chromatography. Gas chromatographic separation was accomplished with a 10% OV-11 glass column and temperature programming. The findings compare favorably with other chromatographic methods. Structures of the TMS-amino acids were confirmed by gas chromatography-mass spectrometry combination. Mass spectral data for each derivative is presented for the principal protein amino acids observed as well as γ-aminobutyric acid, asparagine, α-aminobutyric acid and β-alanine.     

HPLC Separation of Sugar Anomers in a Very Low Temperature Region

Abstract

High-performance liquid chromatographic (HPLC) separation of sugar anomers was carried out on -NH₂ columns in acidic eluents in a very low temperature region. Chromatogram patterns were found to show remarkable changes with decreasing column temperatures. This was attributed to the fact that interconversion between sugar anomers (mutarotation) occurred during the chromatographic process. Since mutarotation was suppressed in a very low temperature region, pyranose anomers of each mono- and di-saccharide were separated completely. Furanose anomers were also separated from an equilibrium mixture, and the necessary conditions for complete separation of furanose anomers were discussed.     

Structure–retardation factor relationship of natural amino acids in two different mobile phases of RP-TLC

Abstract

Separation of amino acids (AAs) and their simple and inexpensive determination/identification is an interesting topic in biological and protein science, different food industries, and drug factories. Also, the presentation of the chromatographic behavior of compounds in a predictive model can be effective to estimate the structural/chemical properties of analyte and mobile phases. In this work for the first time, retardation factor (R_F) of 42 AAs in reversed-phase thin layer chromatography (RP-TLC) was modeled. Acetonitrile-sodium azide solution and 1,2 dioxane-sodium azide solution were two mobile phases which have been studied in this work. Results showed that the values of R_F are correlated with the structural properties of AAs and these properties had some similarities and differences in two noted mobile phases. For the TLC data in two mobile phases, five parametric linear models were suggested (R²_train = 0.93 and 0.97; R²_test = 0.93 and 0.99). The models were also evaluated with different statistical approaches. It was shown that increasing the sum of geometrical distances between N and O in AAs causes decreasing their R_F in RP-TLC using both mobile phases. Other structural effects of AAs on their separation in the desired RP-TLC system were also discussed.     

Separation of Hydrocarbon Mixtures in the Microgram Range by Inclusion in a Urea-packed Mini Column

Abstract

The use of classical urea inclusion techniques for the separation of straight chain hydrocarbons from branched and cyclic compounds is satisfactory when applied to mixtures in the milligram to gram ranges, but leads to low separation efficiencies when quantities in the microgram to milligram range are involved. In this study, a modified inclusion technique using an urea-packed milli-bore column and a catalytic eluent is described. Examples of its use for the separation of mixtures of linear and cyclic hydrocarbons from 30μg up to a few milligrams are given. The versatility of this technique for the analyses of low amounts of environmental samples is described, and an application to the hydrocarbon fraction of surface sediment from lake Leman (Switzerland) is presented.     

Evaluation of novel dendrimer chiral stationary phases using HPLC

Summary The reversed phase chromatographic properties of the [G1]-L-glutamic and ethyl ester-AC-silica (1), [G2]-L-glutamic acid ethyl ester-AC-silica (2) and the [G1]-L-glutamic acidt-butyl ester-AC-silica (3) dendrimer stationary phases were evaluated. Initial studies involved the comparison between these phases with a classic reversed phase (i.e. ODS1) by the separation of a standard reversed phase test mixture composed of dimethylphthalate, nitrobenzene, anisole, diphenylamine and fluorene. Separations were achieved with comparable performance to those obtained with the conventional reversed phase (ODS1). However, it was apparent that the chromatographic selectivity exhibited by the dendrimer stationary phases was different from that of the ODS1 phase. On a per mole basis, the dendrimers exhibited similar (and sometimes greater) affinity for these analytes compared with the ODS1 ligand. Subsequent chromatographic experiments were conducted upon the dendrimer chiral stationary phases using chiral analytes under reversed phase and normal phase conditions. Chiral resolution was not observed.     

Preparative separation of phytosterol analogues from green alga Chlorella vulgaris using recycling counter‐current chromatography

The unicellular alga Chlorella vulgaris is a well‐known health food. It has been proven that the minor phytosterols, ergosterol and its analogue, are an important class of bioactive substances in C. vulgaris . In this work, a recycling counter‐current chromatographic approach was proposed for preparative separation of two analogue sterols from crude extract of C. vulgaris . The separation unit was set up with a type‐J instrument coupled with a column switching valve. A two‐phase solvent system composed of n‐hexane/dichloromethane/acetonitrile (10:3:7, v/v/v) was selected and optimized. After five cycles of separation, two analogue sterols were baseline separated, producing 11.7 mg 26‐nor‐25‐isopropyl‐5,7,22‐trien‐3β‐ol and 20.3 mg ergosterol from 300 mg of C. vulgaris extract. Their purities were both above 95%. The structures of two sterols were identified by using NMR spectroscopy.     

Analysis of the Sulfonylurea Herbicide Metsulfuron-Methyl and Its Metabolites in the Soil of Cereal Crops. Comparative Analytical Chemistry of the Sulfonylureas

Abstract

For the analysis of metsulfuron-methyl in the crop soils with a sensitivity limit of 0.3 μg kg^?1 dry soil, in the soil extract metsulfuron-methyl was separated from its soil metabolites and the soil impurities by repeated thin-layer chromatographies (TLC). In the cleaned soil extract, diazomethane transformed metsulfuron-methyl 1 into N,N′ -dimethyl metsulfuron-methyl 2 (methyl 2-[[[[(4-methoxy-6-methyl-1,3,5-triazin-2-yl)methylamino]carbonyl]methylamino]sulfonyl]benzoate). In the gas-liquid chromatograph with detection by electron capture (GC-EC) and in the combined gas chromatograph-mass spectrometer (GC-MS), 2 was transformed into 1-dioxy-2-N-methyl-3-keto-1,2-benzisothiazole 3 which was measured by GC-EC with confirmation by GC-MS. The metsulfuron-methyl soil metabolites 2-sulfonamido-methylbenzoate 6, 1-dioxy-3-keto-1,2-benzisothiazole (saccharin) 7 and 2-sulfonamidobenzoic acid 8 were analyzed in the soil of winter wheat crops by a procedure similar to the one for metsulfuron-methyl. After their separation and purification in the soil extracts by TLC, 7 and 8 were methylated, and analyzed as 3 in the GC-EC and GC-MS apparatus where the generated 6 was quantitatively transformed into 3; 6 was analyzed as such with the GC and GC-MS apparatus wherein it was transformed into 3. The sensitivity limit for each metabolite was 0.3 μg of equivalents of metsulfuron-methyl kg^?1dry soil. The syntheses of the analysis standards of the metsulfuron-methyl derivatives 2 and 3, and of the metsulfuron-methyl metabolites 6, 7 and 8 are described. The transformation pathways of metsulfuron-methyl and of its derivatives are different from those of the pyridine-pyrimidine sulfonylurea herbicides flupyrsulfuron-methyl and rimsulfuron. The soil analysis of a sulfonylurea -by means of one of its transformation product- needs a previous study of the chemical reactivity of the sulfonylurea. This leads to the analysis procedures for the main soil metabolites of the sulfonylurea.     

Thin-Layer Chromatographic Separation of the Chloroplast Pigments

Abstract

The separation of the chloroplast pigments is achieved by two-way chromatography on cellulose sheets. The chromatographic system employed is superior to any other thin-layer or paper system yet found.     

MLC Determination of Preservatives in Cranberry Foodstuffs

Optimized conditions for the determination of 14 food preservatives were selected by using interpretative optimization strategy and Derringer’s desirability function for combining three main chromatographic goals: resolution, separation time and ruggedness. The best mobile phase for separation by micellar liquid chromatography contains 0.045 M SDS and 1.0% (v/v) 1-pentanol at pH 2.5 adjusted with trichloroacetic acid. The accuracy of the method was confirmed by analysis of spiked samples and is useful for routine analysis of food preservatives in drinks due to simple sample preparation, safety and low cost of micellar mobile phases. Presented in part by A.P. Boichenko at the 4th Black Sea Basin Conference on Analytical Chemistry, 19–23 September, 2007, Sunny Beach, Bulgaria.