Bernd Spangenberg

Quantitative thin-layer chromatography : a practical survey

History of Planar Chromatography.- The Theoretical Basis of Thin-Layer Chromatography (TLC).- The Stationary Phase in Thin Layer Chromatography (TLC).- The Mobile Phase in Adsorption and Partition Chromatography.- Preparing and Applying Samples.- Basis for TLC Development Techniques.- Specific Staining Reactions.- Bioeffective-linked Analysis in Modern HPTLC.- Planar Chromatography Detectors.- Diffuse Reflectance from TLC Layers.- Fluorescence in TLC Layers.- Chemometrics in HPTLC.- Reciprocal Model.- Statistics for Quantitative TLC.- Planning an Analysis and Validation in TLC.

Fibre optical scanning with high resolution in thin-layer chromatography.

In this paper a high-performance thin-layer chromatography (HPTLC) scanner is presented in which a special fibre arrangement is used as HPTLC plate scanning interface. Measurements are taken with a set of 50 fibres at a distance of 400 to 500 microm above the HPTLC plate. Spatial resolutions on the HPTLC plate of better than 160 microm are possible. It takes less than 2 min to scan 450 spectra simultaneously in a range of 198 to 610 nm. The basic improvement of the item is the use of highly transparent glass fibres which provide excellent transmission at 200 nm and the use of a special fibre arrangement for plate illumination and detection.

New evaluation algorithm in diode-array thin-layer chromatography

In-situ densitometry for qualitative or quantitative purposes is a key step in thin-layer chromatography (TLC). It is a simple means of quantification by measurement of the optical density of the separated spots directly on the plate. A new scanner has been developed which is capable of measuring TLC or HPTLC (highperformance thin-layer chromatography) plates simultaneously at different wavelengths without damaging the plate surface. Fiber optics and special fiber interfaces are used in combination with a diode-array detector. With this new scanner sophisticated plate evaluation is now possible, which enables use of chemometric methods in HPTLC. Different regression models have been introduced which enable appropriate evaluation of all analytical questions. Fluorescent measurements are possible without filters or special lamps and signal-to-noise ratios can be improved by wavelength bundling. Because of the richly structured spectra obtained from PAH, diode-array HPTLC enables quantification of all 16 EPAPAH on one track. Although the separation is incomplete all 16 compounds can be quantified by use of suitable wavelengths. All these aspects are enable substantial improvement of in-situ quantitative densitometric analysis.

Does the Kubelka-Munk theory describe TLC evaluations correctly?

In thin-layer chromatography the development step distributes the sample throughout the layer, a process which strongly affects the reflection signals. The essential requirement for quantitative thinlayer chromatography is not a constant sample concentration but constant sample distribution in each sample spot. This makes evaporation of the mobile phase extremely important, because all tracks of a TLC plate must be dried uniformly. This paper shows that quantitative TLC is possible even if the concentration of the sample is not constant throughout the layer or if the distribution of the sample is not known. With uniform sample distribution, classical Kubelka-Munk theory is valid for isotropic scattering only. In the absence of this constraint classical Kubelka-Munk theory must be extended to situations where scattering is asymmetric. This can be achieved by modification of the original Kubelka-Munk equation. Extended theory is presented which is not only capable of describing asymmetrical scattering in TLC layers but also includes a formula for absorption and fluorescence in diode-array TLC. With this new theory all different formulas for diode-array thin-layer chromatographic evaluation are combined in one expression.

Metal π complexes of benzene derivatives

Abstract Bis(cyclobuta-η 6 -benzene)metal complexes of chromium(0) ( 4 ), molybdenum(0) ( 14 ) and tungsten(0) ( 15 ) as well as bis([1,2:4,5]-dicyclobuta-η 6 -benzene)chromium(0) ( 5 ) have been prepared by metal atom ligand vapor cocondensation techniques. The 1 H NMR coordination shift &δ for the endo - and exo -protons of the cyclobutene ring are discussed in the context of anisotropic shielding in the periphery of bis(η 6 -arene)chromium. The ESR spectra of the chromium centered radical cations 4 + and 5 + are also reported. In conjunction with hyperfine coupling data for (η 12 -[2.2]paracyclophane)chromium + ( 16 + ), which also possesses monitor protons at defined positions, and for bis(η 6 -paraxylene)chromium + ( 17 + ), containing freely rotating methyl groups, the contributions of conformation dependent, and conformation independent pathways of metal ligand electron-spin transfer are assessed.

TLC-analysis in forensic sciences using a diode-array detector

SummaryHPTLC (High Performance Thin Layer Chromatography) is a well known and versatile separation method which shows many advantages when compared to other separation techniques. The method is fast and inexpensive and does not need time-consuming pretreatments. For visualisation of the sample distribution on a HPTLC-plate we developed a new and sturdy HPTLC-scanner. The scanner allows simultaneous registrations of spectra in a range from 198 nm to 612 nm with a spectral resolution of better than 0.8 nm. The on-plate spatial resolution is better than 160 μm. The measurement of 450 spectra in one separation track does not need more than two minutes. The new diode-array scanner offers a fast survey over a TLC-separation and makes various chemometric applications possible. For compound identification a cross-correlation function is described to compare UV sample spectra with appropriate library data. The cross-correlation function herein described can also be used for purity testing. Unresolved peaks can be virtually separated by use of a least squares fit algorithm. In summary, the diode arry system delivers much more information than the commonly used TLC-scanner.

A new method for quantification of melamine in milk by absorption diode-array thin-layer chromatography

Melamine (1,3,5-triazine-2,4,6-triamine or cyanuramide, C 3 H 6 N 6 ) is a trimer of cyanamide, with a 1,3,5-triazine skeleton (Figure 1). The molecule contains 66% nitrogen by mass and, if mixed with resins, has fire-retardant properties because it releases nitrogen gas when burned or charred. The word melamine (from German) is a combination of the word melam (which is a distillation derivative of ammonium thiocyanate) and amine [1]. Melamine is also a metabolite of cyromazine, an insecticide in which the proton of an NH 2 group is substituted by a cyclopropyl group.

Determination of the Mycotoxin, Sterigmatocystin, by Thin-Layer Chromatography and "Reagent-free" Derivatisation.

Abstract This paper describes a thin‐layer chromatographic (TLC) method, which allows the determination of sterigmatocystin at a level of 2 µg/kg in various cereal grains of interest. Sterigmatocystin is extracted from the food matrix and further purified by phenyl‐bond solid‐phase extraction (SPE). The separation and identification is performed on an amino‐derivatised high‐performance TLC (HP‐TLC) plate. The derivatisation for densitometric measurement and visual inspection is achieved reagent free by heating the plate. Sterigmatocystin results in highly fluorescent spots. The method has shown good recovery values for the various cereals analysed (e.g., around 80% for wheat). The method was applied for monitoring the sterigmatocystin content in 85 cereal samples, purchased from the local (Italian) market in 2002. However, none of the samples was found to be positive, indicating that this mycotoxin was not a problematic contaminant in products of this particular region and in this specific year.

A Simple and Reliable HPTLC Method for the Quantification of the Intense Sweetener Sucralose

Abstract This paper describes a simple and fast thin layer chromatography (TLC) method for the monitoring of the relatively new intense sweetener Sucralose® in various food matrices. The method requires little or no sample preparation to isolate or concentrate the analyte. The Sucralose® extract is separated on amino‐TLC‐plates, and the analyte is derivatized “reagent‐free” by heating the developed plate for 20 min at 190°C. Spots can be measured either in the absorption or fluorescence mode. The method allows the determination of Sucralose® at the levels of interest regarding foreseen European legislation (>50 mg/kg) with excellent repeatability (RSD = 3.4%) and recovery data (95%).

Fluorescence Enhancement of Pyrene Measured by Thin-Layer Chromatography with Diode-Array Detection

In-situ densitometry for qualitative or quantitative purposes is a key step in thin-layer chromatography. It offers a simple way of quantifying by measuring the optical density of the separated spots directly on the plate. A new TLC scanner has been developed which is able to measure TLC plates or HPTLC plates, at different wavelengths simultaneously, without destroying the plate surface. The system enables absorbance and fluorescence measurements in one run. Fluorescence measurements are possible without filters or other adjustments. The measurement of fluorescence from a TLC plate is a versatile means of making TLC analysis more sensitive. Fluorescence measurements with the new scanner are possible without filters or special lamps. Improvement of the signal-to-noise ratio is achieved by wavelength bundling. During plate scanning the scattered light and the fluorescence are both emitted from the surface of the TLC plate and this emitted light provides the desired spectral information from substances on the TLC plate. The measurement of fluorescence spectra and absorbance spectra directly from a TLC plate is based on differential measurement of light emerging from sample-free and sample-containing zones. The literature recommends dipping TLC plates in viscous liquids to enhance fluorescence. Measurement of the fluorescence and absorbance spectra of pyrene spots reveals the mechanism of enhancement of plate dipping in viscous liquids—blocked contact of the fluorescent molecules with the stationary phase or other sample molecules is responsible for the enhanced fluorescence at lower concentrations. In conclusion, dipping in TLC analysis is no miracle. It is based on similar mechanisms observable in liquids. The measured TLC spectra are also very similar to liquid spectra and this makes TLC spec-troscopy an important tool in separation analysis.