Quantitative analysis of some bile acids by differential scanning calorimetry combined with TLC

An analytical method useful for the quantitative determination of some bile acids is proposed. The analysis is carried out in two steps. The first is based on the thin layer chromatographic (TLC) separation of the bile acids on alumina plates. The second is based on the application of differential scanning calorimetry (DSC), which permits the characterization and determination of the amount of compound contained in each spot. The DSC signal is proportional to the amount of sample present in the spot layer, while the various peaks and peak temperatures are used to identify the separated compound.     

Quantitative Densitometry in Situ of Lipids Separated by thin Layer Chromatography

Abstract

Operating parameters are described for a densitometric method to determine in situ eight lipid classes separated by thin layer chromatography. The separated lipids, visualized on the TLC plate by a cupric acetate-phosphoric acid charring method, were quantitatively determined by spectrodensitometry using the Shimadzu CS-910 Dual Wavelength TLC Scanner. Plates were scanned in either a linear scanning mode or in a zigzag scanning mode (flying spot).

Reproducibility of a) sample application (spotting) and b) the lipid separation procedure was determined by scanning. Transmittance measurements yielded response areas that were 2.8 X higher than reflectance measurements. Operating parameters such as scanning direction, wavelength, single and dual-wavelength measurement, scanning speed, and slit geometry were studied. Optimal conditions were established for quantitative densitometry of lipids on thin layer plates.     

Rapid,simple, and highly sensitive analysis of drugs in biological samples using thin-layer chromatography coupled with matrix-assisted laser desorption/ionization mass spectrometry

Rapid and precise identification of toxic substances is necessary for urgent diagnosis and treatment of poisoning cases and for establishing the cause of death in postmortem examinations. However, identification of compounds in biological samples using gas chromatography and liquid chromatography coupled with mass spectrometry entails time-consuming and labor-intensive sample preparations. In this study, we examined a simple preparation and highly sensitive analysis of drugs in biological samples such as urine, plasma, and organs using thin-layer chromatography coupled with matrix-assisted laser desorption/ionization mass spectrometry (TLC/MALDI/MS). When the urine containing 3,4-methylenedioxymethamphetamine (MDMA) without sample dilution was spotted on a thin-layer chromatography (TLC) plate and was analyzed by TLC/MALDI/MS, the detection limit of the MDMA spot was 0.05 ng/spot. The value was the same as that in aqueous solution spotted on a stainless steel plate. All the 11 psychotropic compounds tested (MDMA, 4-hydroxy-3-methoxymethamphetamine, 3,4-methylenedioxyamphetamine, methamphetamine, p-hydroxymethamphetamine, amphetamine, ketamine, caffeine, chlorpromazine, triazolam, and morphine) on a TLC plate were detected at levels of 0.05 − 5 ng, and the type (layer thickness and fluorescence) of TLC plate did not affect detection sensitivity. In addition, when rat liver homogenate obtained after MDMA administration (10 mg/kg) was spotted on a TLC plate, MDMA and its main metabolites were identified using TLC/MALDI/MS, and the spots on a TLC plate were visualized by MALDI/imaging MS. The total analytical time from spotting of intact biological samples to the output of analytical results was within 30 min. TLC/MALDI/MS enabled rapid, simple, and highly sensitive analysis of drugs from intact biological samples and crude extracts. Accordingly, this method could be applied to rapid drug screening and precise identification of toxic substances in poisoning cases and postmortem examinations.     

Post TLC developing technique for tyrosinase inhibitor detection

Post TLC developing technique was developed to detect substances which can inhibit tyrosinase activity. The method involved spraying the TLC plate or chromatographic paper containing sample spot(s) with tyrosinase and l-tyrosine solutions successively. A positive result could be visualized directly as white spot(s) against a brownish-purple background. The method can either be used as a quick screening method for tyrosinase inhibitor detection or a guiding procedure for an isolation of tyrosinase inhibitors from mixtures or natural product extracts. The technique is sensitive enough to give a clear result in the presence of only 6 ng glabridin.     

Systematic and statistical errors in quantitative evaluation in TLC and HPTLC. 3. The determination of the primary statistical errors

The paper describes the measurement and calculation of the primary statistical errors in the evaluation of TLC or HPTLC using in situ reflectance measurements. The primary errors are the errors of the sample volume spotted onto the plate, errors caused by the chromatographic process itself, the positioning error of the plate with respect to the centre of the light beam and the error in the light measurement. By scanning the same spot, the same track or the same plate under different conditions, the various errors can be determined.     

Estimation of Ethyl Cellulose Layer Thickness of Coated Polyethylene Oxide Particles Using Differential Scanning Calorimetry

Particles of poly(ethylene oxide) (PEO) were coated using ethyl cellulose (EC). Equations and a method were proposed to estimate the EC layer thickness by using differential scanning calorimetry (DSC) based on melting or crystallization heat of phase-change materials. The result shows that EC layer thickness of polyethylene oxide particles determined using DSC is consistent with the result using an optical microscope.     

Quantitative evaluation of thin-layer chromatograms. 3. The calculation of fluorescence using multilayer models

Changes of fluorescence intensity at the far side (transmission) and near side (remission) in cases where a spot of a fluorogen lies in different sublayers are calculated using a multilayer model. Examples shown are: the relative intensity of fluorescence in the layer as a function of the absorption coefficient of a sorbent at the excitation wavelength; the relative intensity of fluorescence at the far and near sides of a TLC plate; the influence of using too broad a monochromatic filter at the emission side on the results of fluorescence measurements. Advantages of near-side scanning of fluorescence compared to far-side scanning are discussed. The most important source of errors in fluorodensitometric measurements is the effect of secondary chromatography.     

药物分析中薄层色谱的方法认证

在药物分析中,针对所要求的性能参数,对一个薄层色谱程序的各个环节必须进行的认证方法和认可标准进行了讨论。建议当提出结果报告时,应附上关于对方法的认证参数和认证方法的说明。     

Thermal behaviour of diclofenac sodium: decomposition and melting characteristics

The thermal behaviour and melting characteristics of diclofenac sodium were investigated using various instrumental techniques--differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), Fourier-transform infrared (FT-IR) spectroscopy and thin layer chromatography (TLC). DSC analysis of diclofenac sodium performed under dynamic flow of either synthetic air or helium or nitrogen did not produce any sharp endothermic peak characteristic of melting peak of a pure substance. Both the rate of scanning of the sample and the environmental atmospheric condition significantly affected the thermographic profile of diclofenac sodium. An exothermic peak prior to an endothermic peak corresponding to melting of the substance appeared when heated under dynamic flow of synthetic air suggesting oxidation (decomposition) of diclofenac sodium before reaching its melting point. In fact, at a scanning rate of 1 degree C/min only the exothermic peak appeared in the thermogram, suggesting complete decomposition prior to melting under the dynamic flow of synthetic air. DSC, FT-IR and TLC data obtained from samples heated under the dynamic flow of either helium or nitrogen revealed formation of a related compound, 1-(2,6-dichlorophenyl)-indolin-2-one, an indol-cyclic amide, as a result of an intramolecular cyclization reaction during the heating process. TGA data demonstrated a loss of 11.4-20.2% of the mass of diclofenac sodium when heated under various environmental conditions, and also supported the oxidative nature of degraded product(s) when the thermal process occurred slowly under a dynamic flow of synthetic air.     

Thin-layer chromatographic scanner, spectrophotometric and high-performance liquid chromatographic methods for the determination of colchicine

One high-performance liquid chromatographic (HPLC) and two thin-layer chromatographic (TLC) methods are proposed for the determination of colchicine in crude drugs and pharmaceutical preparations. The TLC scanner method is based on measurement of the absorbance of the separated colchicine spot; alternatively, after scraping the spot from the plate and elution the absorbance can be measured spectrophotometrically. The HPLC assay was carried out isocratically on a reversed-phase column using MeOH-H2O (60 + 40). The recoveries were 99.2 +/- 1.23, 99.1 +/- 1.12 and 99.1 +/- 2.01% for the TLC scanner, spectrophotometric and HPLC methods, respectively. The methods were shown to be sensitive and specific and can be used as an alternative to the pharmacopoeial methods having been applied to the determination of colchicine in corms of Merendera persica and in three pharmaceutical preparations.     

Thin-layer chromatography/liquid secondary ion mass spectrometry in the determination of major bile salts in dog bile

Positive and negative ion liquid-state secondary-ion mass spectrometry (LSIMS) was applied to several bile acids and bile salts and their spectra were measured directly from the surface of silica gel thin-layer chromatograms. Such spectra were identical to the LSIMS spectra of the pure compound at the same concentration. Three-dimensional ion images were obtained of a model mixture of cholic, chenodeoxycholic and lithocholic acids in both the positive and negative ion modes. A sample of dog bile was prepared, and the bile acids extracted from it were separated by high-performance TLC; TLC/LSIMS spectra were obtained for sodium taurocholate and sodium taurochenodeoxycholate/taurodeoxycholate, the predominant bile salts present. Quantitative estimates of the analytes were obtained by monitoring the ion intensity for the sample spot and a standard spot that had been developed in parallel on the same TLC plate. The concentration of sodium taurocholate in the bile of this particular dog was found to be 38 mg/ml.     

Characterization of the morphology of co-extruded, thermoplastic/rubber multi-layer tapes

Tapes with alternating semi-crystalline thermoplastic/rubber layers with thicknesses varying from 100 nm up to several μm were prepared by multi-layer co-extrusion. The variation in layer thickness was obtained by varying the thermoplastic/rubber feed ratio. A systematic study on the use of various microscopy techniques to visualize the morphology of the layered systems is presented. The relatively large length scales and the sample preparation make optical microscopy (OM) unsuitable to study the morphology of the multi-layer tapes. Although excellent contrast between the thermoplastic and rubber layers can be obtained, the usually applied, relatively large magnifications limit the use of transmission electron microscopy (TEM) and atomic force microscopy (AFM) to small sample areas. The large range of applicable magnifications makes scanning electron microscopy (SEM) the most suitable technique to study the morphology of the multi-layer tapes. The sample preparation for SEM with a secondary electron (SE) detector is often based on the removal of one of the components, which may induce changes in the morphology. SEM with a back-scattered electron (BSE) detector is a very convenient method to study the morphology over a wide range of length scales, where the contrast between the different layers can be enhanced by chemical staining. Finally, the nucleation behavior (homogeneous versus heterogeneous) of the semi-crystalline layers, as probed by differential scanning calorimetry (DSC), provides valuable information on the layered morphology. The use of relatively straightforward DSC measurements shows a clear advantage with respect to the discussed microscopy techniques, since no sample preparation is required and relatively large samples can be studied, which are more representative for the bulk.     

Application of thin-layer chromatography with fluorescence scanning densitometry for analysing saturates in heavy liquids derived from Co-pyrolysis of biomass and plastics

Summary Two alternative methods, based on Thin-Layer Chromatography (TLC) with Fluorescence Scanning Densitometry have been developed for characterization of heavy liquids from copyrolysis of different kinds of biomass and plastics in autoclaves under inert atmosphere. A conventional TLC system, which includes a vertical developing tank, and a High Performance TLC (HPTLC) system, with a horizontal developing chamber and the use of HPTLC plates, have been used. The analytical method involves in both cases the measurement of two chromatograms per sample: the first, on a silica gel berberine-impregnated plate, for detection of saturates using the phenomenon of berberine-induced fluorescence; and the second, on a silica gel plate, for detection of aromatic-polars and polars, by native fluorescence. Although the HPTLC system is more sensitive and faster, both techniques represent an improvement with regard to current methods for analyzing these kinds of products. However their application depends on the particular solubility of each sample and on its slope of the fluorescent response-sample load regression.     

Steuergerät zur automatischen spektralphotometrischen Direktauswertung von Dünnschicht-Chromatogrammen

An instrument has been constructed for the automatic control of a TLC spectrophotometer. In addition to the automatic scanning of all tracks of a thin-layer chromatogram the positions and values of diffuse reflectance minima are recognized and printed. A second mode allows the manual scanning and measurement of a spot at any position on the TLC plate. A digital output is provided for the evaluation of the results with a computer. The errors of reproducibility and of concentration are much smaller using the automatic scanning of bands than using the manual positioning of circular spots as the examples show. The linearity of several analytical functions is analysed within two decades of concentration. The Kubelka-Munk function was found to be superior in linearity to all other functions studied. Calculations of costs show, that the total costs of an analysis in a routine laboratory can be lowered to an extend of about 70% by the use of an application instrument and a controlling instrument for automatic scanning inspite of the higher costs of investments.     

Molecular Ionization-Desorption Analysis Source (MIDAS) for Mass Spectrometry: Thin-Layer Chromatography

Molecular ionization-desorption analysis source (MIDAS), which is a desorption atmospheric pressure chemical ionization (DAPCI) type source, for mass spectrometry has been developed as a multi-functional platform for the direct sampling of surfaces. In this article, its utility for the analysis of thin-layer chromatography (TLC) plates is highlighted. Amino acids, which are difficult to visualize without staining reagents or charring, were detected and identified directly from a TLC plate. To demonstrate the full potential of MIDAS, all active ingredients from an analgesic tablet, separated on a TLC plate, were successfully detected using both positive and negative ion modes. The identity of each of the compounds was confirmed from their mass spectra and compared against standards. Post separation, the chemical signal (blue permanent marker) as reference marks placed at the origin and solvent front were used to calculate retention factor (R_f) values from the resulting ion chromatogram. The quantitative capabilities of the device were exhibited by scanning caffeine spots on a TLC plate of increasing sample amount. A linear curve based on peak are, R² = 0.994, was generated for seven spots ranging from 50 to 1000 ng of caffeine per spot.

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Graphical Abstract ?

     

Biennial review of planar chromatography: 2011–2013

The most important advances in planar chromatography published between November 1, 2011 and November 1, 2013 are reviewed in this paper. Included are an introduction to the current status of the field; student experiments, books, and reviews; theory and fundamental studies; apparatus and techniques for sample preparation and TLC separations (sample application and plate development with the mobile phase); detection and identification of separated zones (chemical and biological detection, TLC/mass spectrometry, and TLC coupled with other spectrometric methods); techniques and instruments for quantitative analysis; preparative layer chromatography; and thin layer radiochromatography. Numerous applications to a great number of compound types and sample matrices are presented in all sections of the review.     

Biennial review of planar chromatography: 2009-2011

The most important advances in the planar chromatography published between November 1, 2009 and November 1, 2011 are reviewed in this paper. Included are an introduction to the current status of the field; history, student experiments, books, and reviews; theory and fundamental studies; apparatus and techniques for sample preparation and TLC separations (sample application and plate development with the mobile phase); detection and identification of separated zones (chemical and biological detection, TLC/MS, and TLC coupled with other methods); techniques and instruments for quantitative analysis; preparative layer chromatography; and thin layer radiochromatography. Selected applications are given in the various sections of the review, especially for modern HPTLC-densitometry.     

Computational Quantification of Spot Tests by Image Scanning-A New Analytical Technique for Micro Samples

The benefits of spectrophotometric quantitative analysis and the small sample requirements of a spot test have been merged in this new methodology for metal analysis at the sub-micro level. A 1 µL sample is transferred to a suitable medium (TLC plate) with subsequent application of reagent(s) at the same spot; the color density of the resulting spot is then measured using a flatbed color scanner connected to a PC running specially developed software. Calibration data was collected for As, Hg, and Pb in 1–5 ppb range with a correlation co-efficient better than 0.9962. The method is especially suitable for precipitation systems where absorption spectroscopy fails.     

Identification of Polyethylene by Differential Scanning Calorimetry: Application to forensic science

A forensic sample consisting of melt-recrystallized polymers that was recovered from the scene of a fire in a factory was identified by differential scanning calorimetry. The factory commonly used two kinds of film sheets, A and B, made by different manufacturers. It was necessary to decide whether the forensic sample related to material A or B. The forensic sample and reference samples of materials A and B were subjected to infrared spectroscopy and pyrolysis gas chromatograph mass spectrometry measurements, which revealed their polyethylene nature. The thermal behaviour of the samples was examined by differential scanning calorimetry (DSC) and they were found to be blends of two kinds of polyethylenes, low-density polyethylene and linear low-density polyethylene. The samples could be identified and distinguished from each other via the DSC measurements.This revised version was published online in November 2005 with corrections to the Cover Date.     

Quantitative thin-layer chromatographic method of analysis of azithromycin in pure and capsule forms

A validated stability-indicating thin-layer chromatographic (TLC) method of the analysis of azithromycin (AZT) in bulk and capsule forms is developed. Both AZT potential impurity and degradation products can be selectively and accurately estimated in both raw material and product onto one precoated silica-gel TLC plate 60F254. The development system used is n-hexane-ethyl acetate-diethylamine (75:25:10, v/v/v). The separated bands are detected as brown to brownish red spots after spraying with modified Dragendorff's solution. The Rf values of AZT, azaerythromycin A, and the three degradation products are 0.54, 0.35, 0.40, 0.20, and 0.12, respectively. The optical densities of the separated spots are found to be linear in proportion to the amount used. The stress testing of AZT shows that azaerythromycin A is the major impurity and degradation product, accompanied by three other unknown degradation products. The stability of AZT is studied under accelerated conditions in order to provide a rapid indication of differences that might result from a change in the manufacturing process or source of the sample. The forced degradation conditions include the effect of heat, moisture, light, acid-base hydrolysis, sonication, and oxidation. The compatibility of AZT with the excipients used is also studied in the presence and absence of moisture. The amounts of AZT and azaerythromycin A are calculated from the corresponding linear calibration curve; however, the amounts of any other generated or detected unknown impurities are calculated as if it were AZT. This method shows enough selectivity, sensitivity, accuracy, precision, linearity-range, and robustness to satisfy Federal Drug Administration/International Conference of Harmonization regulatory requirements. The method developed can also be used for the purity testing of AZT raw material and capsules, content uniformity testing, dissolution testing, and stability testing of AZT capsules. The potential impurity profiles of both active AZT material and capsule forms are found comparable. The linear range of AZT is between 5 and 30 mcg/spot with a limit of quantitation of 2 mcg/spot. The intraassay relative standard deviation percentage is not more than 0.54%, and the day-to-day variation is not more than 0.86%, calculated on the amounts of AZT RS recovered using different TLC plates.