Bernd Spangenberg

Offenburg University of Applied Sciences · Environmental Chemistry

We present a video-densitometric quantification method for Sudan red dyes in spices and spice mixtures, separated by TLC. Application was done band-wise in small dots using a 5 μL glass pipette. For separation, the RP-18 plates (20 × 20 cm with fluorescent dye; Merck, Germany, 1.05559) were developed in a vertical developing chamber without vapor s...

We present a planar chromatographic separation method for the phytoestrogenic active compound equol, separated on RP-18 W (Merck, 1.14296) phase. It could be shown that an ethanolic cattle manure extract contains this phytoestrogenic active compound to a larger amount. As solvents for the mobile phase, hexane, ethyl acetate, and acetone (45:15:10,...

trans-Resveratrol is a polyphenolic compound belonging to the group of stilbenes. The compound has gained increasing interest over the last few years. In this study, the authors present a new way to measure trans-resveratrol in fresh and dried fruits like goldenberries (Physalis peruviana L.) using a combination of a “Quick, Easy, Cheap, Effective,...

The main focus of this chapter is the theoretical and instrumental processes that underpin densitometric methods widely used in thin-layer chromatography (TLC). Densitometric methods include UV–vis, luminescence and fluorescence optical measurements as well as infrared and Raman spectroscopic measurements. The chapter is divided in two general part...

In the present study, in vitro toxicity as well as biopersistence and photopersistence of four artificial sweeteners (acesulfame, cyclamate, saccharine, and sucralose) and five antibiotics (levofloxacin, lincomycin, linezolid, marbofloxacin, and sarafloxacin) and of their phototransformation products (PTPs) were investigated. Furthermore, antibioti...

Trans-Resveratrol is a polyphenolic compound belonging to the group of stilbenes. The compound has gained increasing interest over the last years. We now present a new way to measure Trans-resveratrol in red wine. A combination of pure wine application on plate, a TLC separation on TLC silica gel 60 plates (Merck, 1.05554), and chemiluminescence de...

A 2D-separation of 16 polyaromatic hydrocarbons (PAHs) according to the Environmental Protecting Agency (EPA) standard was introduced. Separation took place on a TLC RP-18 plate (Merck, 1.05559). In the first direction, the plate was developed twice using n-pentane at -20°C as the mobile phase. The mixture acetonitrile-methanol-acetone-water (12:8:...

We present a two dimensional (2D) planar chromatographic separation of estrogenic active compounds on RP-18 (Merck, 1.05559) and silica gel (Merck, 1.05721) phase. A mixture of 13 substances was separated using a solvent mix consisting of methanol–acetonitrile–water (2 + 2 + 1, v/v/v) on RP-18 phase in the first direction and cyclohexane–butylaceta...

Data allowing for a complete environmental risk assessment of pharmaceuticals and their photoderatives in the environment are still scarce. In the present study, in vitro toxicity and both bio- and photopersistence of various pharmaceuticals (aciclovir, allopurinol, cetirizine, cimetidine, fluconazole, hydrochlorothiazide, lisinopril, phenytoin, pr...

We present a two dimensional (2D) planar chromatographic separation of ethinyl steroids on cyanopropyl phase. A mixture of nine steroids was separated by using a cyanopropyl-coated silica gel plate (Merck, 1.16464) with the solvent mix of dichloromethane, methanol, cyclohexane (95 + 5 + 60, v/v) in the first direction and with a mixture of water, a...

We present a video-densitometric quantification method for benzocaine in lozenges. The quantification is based on a derivatisation reaction with 4-dimethylaminobenzaldehyde. Measurements were carried out using a 16-bit flatbed scanner. Benzocaine was separated to a distance of 50 mm in a vertical developing chamber without vapour saturation. We pre...

We present a video-densitometric quantification method in combination with diode-array quantification for the methyl-, ethyl-, propyl-, and butylparaben in cosmetics. These parabens were separated on cyanopropyl bonded plates using water-acetonitrile-dioxane-ethanol-NH3 (25%) (8:2:1:1:0.05, v/v) as mobile phase. The quantification is based on UV-me...

We will present the first example of a two-dimensional scanned TLC-plate, measured by use of a diode-array scanner. A spatial resolution of 250 mu m was achieved on plate. The system provides real 2D fluorescence and absorption spectra in the wavelength-range from 190 to 1000 nm with a spectral resolution of greater than 1 nm. A mixture of 12 sulph...

A simple method to quantify the neonicotinoid insecticides nitenpyram, thiamethoxam, acetamiprid, imidacloprid, thiacloprid, and clothianidin directly on an HPTLC-plate is presented. As stationary phase silica gel 60 RP-18WF254 s plates were used and a mixture of methyl-t-butyl ether, 2-butanone, NH3 (25%) (5 + 2+0.1, v/v) was used as solvent. All...

Improved separation of highly toxic contact herbicides paraquat (1,1'-dimethyl-4-4'-bipyridinium), diquat (6,7-dihydrodipyridol[1,2-a:2', 1'-c]pyrazine-5,8-di-ium), difenzoquat (1,2-dimethyl-3,5- diphenyl-1H-pyrazolium-methyl sulfate), mepiquat (1,1dimethyl- piperidinium), and chloromequat (2-chloroethyltrimethyl-ammonium) were presented by high-pe...

We present a video-densitometric quantification method for the pain killer known as diclofenac and ibuprofen. These non-steroidal anti-inflammatory drugs were separated on cyanopropyl bonded plates using CH2Cl2, methanol, cyclohexane (95 + 5 + 40, v/v) as mobile phase. The quantification is based on a bio-effective-linked analysis using Vibrio fish...

Thin-layer chromatography (TLC) is widely used particularly for pharmaceutical and food analysis. While there are a number of books on the qualitative identification of chemical substances by TLC, the unique focus here is on quantitative analysis. The authors describe all steps of the analytical procedure, beginning with the basics and equipment fo...

In thin-layer chromatography (TLC), there are only three degrees of freedom in which the chromatographic results can be seriously influenced. They are (1) the polarity of the stationary phase in partition chromatography or its activity in adsorption chromatography; (2) the selection of the mobile phase; and (3) the composition of the vapour phase i...

Generally speaking, spectroscopy and spectrometric methods such as infra-red (IR) spectroscopy and ultra-violet (UV) spectrophotometry are incapable of carrying out complete analysis of complicated mixtures. As a consequence, the individual substances must be separated from one another before spectrophotometric determination can take place.

In column chromatography a defined sample amount is injected into a flowing mobile phase. The mix of sample and mobile phase then migrates through the column. If the separation conditions are arranged such that the migration rate of the sample components is different then a separation is obtained. Often a target compound (analyte) has to be separat...

The purpose of bioeffective-linked analysis is to bridge the gap between cause and effect, that is, primarily not to identify a given analyte, but to identify a class of compounds with a defined bioactivity. This is the basis of screening tests for substances with special properties (e.g. fungicides) and for screening tests on new compounds with a...

Sample application decisively influences the separation quality and fidelity of quantitative information. The important fact is that they both can be affected by the choice of experimental conditions Sample application, however, only indirectly influences the selectivity of a separation system. A poor application merely hinders the achievement of t...

From the beginning of the 1950s, paper chromatography was used to quantify samples. The paper was made transparent by liquids and transmitted light was measured according to the Lambert-Beer law. Silver-impregnated media were used as detectors, adapted from paper photography.

The measurement of fluorescence in planar chromatography is a sensitive method for the determination of fluorescing substances [1–9]. Fluorescence emission provides more selectivity and increased sensitivity compared to UV–visible absorption. Molecular fluorescence can be described as the immediate emission of energy from a molecule after irradiati...

An undoubted advantage of TLC is the simplicity of qualifying a separation. Coloured compounds can be seen by eye. Compounds absorbing only below 400 nm can be visualized by incorporating a fluorescent dye in the layer. Illuminating the plate with light of 256 nm will induce fluorescence. A sample zone covers the dye and inhibits fluorescence, so t...

Uncoated Silica gel is the dominant stationary phase used in thin-layer chromatography (TLC). This constitutes an important difference to HPLC where most separations are carried out on chemically bonded phases by partition chromatography in the reversed-phase mode. Normal-phase separations by HPLC on inorganic oxide sorbents are uncommon due in par...

Selecting the sorbent and solvent and minimizing the application zone widths are important factors on the way to obtaining optimum separating conditions. In this respect, TLC is very flexible with many possible methods. Thus, it provides additional opportunities not available to other chromatic processes, for example, using the vapour phase to infl...

In general, the aim of analytical measurements is the quantification of the analyte. The conversion of the observed signal into absolute sample amounts is usually achieved through calibration. For this purpose, response measurements are always indicated as the dependent variable (y-data), whereas the independent variable (x-data) is some function o...

From the beginning of the 1950s, paper chromatography was used to quantify samples. The paper was made transparent by liquids and transmitted light was measured according to the Lambert-Beer law. Silver-impregnated media were used as detectors, adapted from paper photography.

In planar chromatography light is used for detecting separated sample zones. To achieve this the stationary phase is commonly illuminated from the top by light of known intensity. Light of reduced intensity reflected from the top surface of the layer is used for quantification.

This short overview section is dedicated to the planning stage of an analysis by TLC to ensure that the methods used are fit for their intended purpose. This process of evaluation is called validation. It consists of checking the quality of the whole analytical process and making improvements wherever necessary. Validation also encompasses document...

Thin-layer chromatography is a rapid and reliable working method for quantification of mycotoxins which is suitable for checking EC legislation aflatoxin limits for dried figs without an RP-18 pre-column cleaning step. We describe normal-phase chromatography on silica gel plates with 2.4:0.05:0.1:0.05 (v/v) methyl t-butyl ether water methanol cyclo...

We present a video-densitometric quantification method for the triazine herbicides atraton, terbumeton, simazine, atrazine, and terbutylazine. Triazine herbicides were separated on silica gel using methyl-t-butyl ether, cyclohexane (1 + 1, v/v) as mobile phase. The quantification is based on a derivation reaction using chlorine and starch-iodine wh...

An interlaboratory comparison was carried out to evaluate the effectiveness of a method based on HPTLC in which reagent-free derivatization is followed by UV/fluorescence detection. The method was tested for the determination of sucralose (C12H19C13O8; (2R,3R,4R,5S,6R)-2-[(2R,3S,4S,5S)-2,5-bis(chloromethyl)-3,4-dihydroxyoxolan-2-yl]oxy-5-chloro-6-h...

An interlaboratory comparison was carried out to evaluate the effectiveness of a method based on HPTLC in which reagent-free derivatization is followed by UV/fluorescence detection. The method was tested for the determination of sucralose (C12H19Cl3O8; (2R,3R,4R,5S,6R)-2- [(2R,3S,4S,5S)-2,5-bis(chloromethyl)-3,4-dihydroxyoxolan- 2-yl]oxy-5-chloro-6...

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...

A diode array HPTLC method for dequalinium chloride in pharmaceutical preparations is presented. For separation a Nano TLC silica gel plate (Merck) is used with the mobile phase methanol—7.8% aqueous NH4+CH3COO− (17:3, v/v) over a distance of 6 cm. Dequalinium chloride shows an RF value of 0.58. Pure dequalinium chloride is measured in the waveleng...

We report improved separation of the highly toxic contact herbicides paraquat, diquat, difenzoquat, mepiquat, and chloromequat by HPTLC. Quantification was based on a new derivatization reaction using sodium tetraphenylborate. Measurements were in the wavelength range from 440 to 480 nm or from 440 to 590 nm. An LED emitting very intense light at 3...

A new quantification method for melamine and cyanuric acid in milk and milk products was developed. For quantification 2 mL milk was diluted with 2 mL THF and 1 g NaCl. The upper phase was applied band-wise (7 mm) on a TLC K60 silica gel 60 foil (No. 1.05554.0001 from Merck, Darmstadt, Germany) in the amount of 10 µL. The plate was developed in a v...

We present an improved quantification method for urethane found in spirits. The quantification is based on a derivatization reaction using cinnamaldehyde in combination with phosphoric acid. Measurements were carried out in the wavelength range from 445 to 460 nm using a diode-TLC device. An LED was used for illumination purposes. It emits very den...

HPTLC on amino plates, with simple heating of the plates for derivatization, has been used for quantification of glucosamine in nutritional supplements. On heating the plate glucosamine reacts to form a compound which strongly absorbs light between 305 and 330 nm, with weak fluorescence. The reaction product can be detected sensitively either by ab...

The identification and quantification of compounds in the gas phase becomes of increasing interest in the context of environmental protection, as well as in the analytical field. In this respect, the high extinction coefficients of vapours and gases in the ultraviolet wavelength region allow a very sensitive measurement system. In addition, the inc...

Previous studies of the hyphenation of gas chromatographic separation and spectrophotometric detection in the ultraviolet wavelength range between 168 and 330 nm showed a high potential for applications where the analysis of complex samples is required. Within this paper the development of a state-of-the-art detection system for compounds in the va...

In thin-layer chromatography, fiber-bundle arrays have been introduced for spectral absorption measurements in the UV-region. Using all-silica fiber bundles, the exciting light will be detected after re-emission on the plate with a fiberoptic spectrometer. In addition, fluorescence light can be detected which will be masked by the re-emitted light....

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 thin-layer chromatography is not a constant sample concentration but constant sample distribution in each sample spot. This makes evaporation of the mobile...

Diode-array planar chromatography is a versatile tool for identification of pharmaceutical substances In this paper thirty-three compounds with benzodiazepine properties were investigated and the separating conditions for silica gel HPTLC plates and three mobile phases were optimized. Diode-array HPTLC makes it possible to identify all the compound...

A new formula is presented for transforming fluorescence measurements in accordance with Kubelka-Munk theory. The fluorescence signals, the absorption signals, and data from a selected reference are combined in one expression. Only diode-array techniques can measure all the required data simultaneously to linearize fluorescence data correctly. To p...

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 simultaneous...

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...

The use of a TLC scanner can be regarded as a key step in high performance thin layer chromatography (HPTLC). Densitometric measurements transform the substance distribution on a TLC plate into digital computer data. Systems that allow quantitative measurements have been available for many years for either fluorescence or ultraviolet absorption mea...

HPTLC (High Performance Thin Layer Chromatography) is a well known and versatile separation method which shows a lot of advantages and options in comparison to other separation techniques. The method is fast and inexpensive and does not need time-consuming pretreatments. Using fiber-optic elements for controlled light-guiding, the TLC-method was si...

A systematic toxicological analysis procedure using high-performance thin layer chromatography in combination with fibre optical scanning densitometry for identification of drugs in biological samples is presented. Two examples illustrate the practicability of the technique. First, the identification of a multiple intake of analgesics: codeine, pro...

A new diode-array scanner in combination with a computer-controlled application system meets all the demands of modern HPTLC measurement. Automatic application, simultaneous measurements at different wavelengths, and different linearization models enable appropriate evaluation of all analytical questions. The theory of error propagation recommends...

High performance thin layer chromatography (HPTLC) is a frequently used separation technique which works well for quantification of caffeine and quinine in beverages. Competing separation techniques, e.g. high-performance liquid chromatography (HPLC) or gas chromatography (GC), are not suitable for sugar-containing samples, because these methods ne...

The sample analysis with Thin Layer Chromatography is an off-line method, in contrast to other separation methods. In addition to the sample preparation, the key-step is the in- situ densitometry for qualitative or quantitative purposes by measuring the optical density of the separated spots directly on the plate. With a newly developed HPTLC-syste...

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 (high-performance thin-layer chromatogr...

HPTLC (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 HP...

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...

Mainly due to the unexpected progress in manufacturing of solarization-reduced all-silica fibers, new fiber-optic applications in the UV-region are feasible. However, the other components like the UV-sources and the detector- systems have to be improved, too. Especially, the miniaturization is very important fitting to the small-sized fiber-optic a...