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An algorithm is presented that has successfully been utilized in practice for several years. It improves data analysis in chromatography. The program runs in an extremely reliable way and evaluates chromatographic raw data with an acceptable error. The algorithm requires a minimum of preliminaries and integrates even unsmoothed noisy data correctly.
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 μm above the HPTLC plate. Spatial resolutions on the HPTLC plate of better than 160 μm 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.
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 need special pretreatment by the analyst. In HPTLC, however, it is possible to separate ‘dirty’ samples without time-consuming pretreatment, because disposable HPTLC plates are used. A convenient method for quantification of caffeine and quinine in beverages, without sample pretreatment, is presented below. The basic theory of in-situ quantification in HPTLC by use of remitted light is introduced and discussed. Several linearization models are discussed.
A home-made diode-array scanner has been used for quantification; this, for the first time, enables simultaneous measurements at different wavelengths. The new scanner also enables fluorescence evaluation without further equipment. Simultaneous recording at different wavelengths improves the accuracy and reliability of HPTLC analysis. These aspects result in substantial improvement of in-situ quantitative densitometric analysis and enable quantification of compounds in beverages.
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 quantification at reflectance values smaller than 0.8; this can be verified only by use of diode-array scanning. The same theory also recommends quantification by use of peak height data, because the theory predicts best precision only for peak height evaluation. Diode-array scanning with reflectance monitoring enables appropriate validation in TLC and HPTLC analysis. All these aspects result in substantial improvement of in-situ quantitative densitometric analysis, and simultaneous recording at different wavelengths opens the way for chemometric evaluation, e.g. peak purity monitoring, which improves the accuracy and reliability of HPTLC analysis.
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, propyphenazone, tramadol, flupirtine and lidocaine, and second, the detection of the sedative diphenhydramine. In both cases, authentic urine specimens were used. The identifications were carried out by an automatic measurement and computer-based comparison of in situ UV spectra with data from a compiled library of reference spectra using the cross-correlation function. The technique allowed a parallel recording of chromatograms and in situ UV spectra in the range of 197–612 nm. Unlike the conventional densitometry, a dependency of UV spectra by concentration of substance in a range of 250–1000 ng/spot was not observed.
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 measurements, while lately the reflection analysis mode for both types is the most common application. New scanning approaches are designed to aid the analyst who has common demands for TLC-densitometry without using special data, such as scanned images. Two examples that have been developed lately in the laboratories of the authors are described in this paper. These approaches were developed on the basis of current needs for analysts who employ TLC as a tool in research, as well as in routine analysis. One approach is aimed to support analysts in economically disadvantaged areas, where cost intensive apparatus is unsuitable but trace analysis by simple means is required. The other system, allows the spectral determination of chromatographic spots on TLC plates covering the ultraviolet and visible range, thus, revealing highly desired information for the analyst.
A Simple and Reliable HPTLC Method for the Quantification of the Intense Sweetener Sucralose®
(2003)
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
(2003)
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.
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 prove the new theory HPTLC quantification of the analgesic flupirtine was performed over the mass range 300 to 5000 ng per spot. The fluorescence calibration curve was linear over the whole range. The transformation of fluorescence measurements into linear mass-dependent data extends the technique of in-situ fluorescence analysis to the high concentration range. It also extends Kubelka-Munk theory from absorption to fluorescence analysis. The results presented also emphasize the importance of Kubelka-Munk theory for in-situ measurements in scattering media, especially in planar 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 (high-performance 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 EPA PAH 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.
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 absorption of light or by fluorescence detection. The detection limit in absorption mode is approximately 25 ng per spot. In fluorescence mode a detection limit of 15 ng is achievable. A calibration plot for absorption detection is linear in the range 25 to 4000 ng glucosamine. The derivative formed from glucosamine by heating is stable for months, and the relative standard deviation is 1.64% for 600 ng glucosamine. The amounts of glucosamine found in nutritional supplements were in agreement with the label declarations.
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 dense light at 365 nm. The quantification range of urethane is in the lower ng range. By applying 20 µL of sprits, the urethane quantification range is from 320 µg/L to 8.1 mg urethane per litre of spirit. The range of linearity covers nearly two magnitudes. The method is cheap, fast and reliable, and is able to monitor all European legislation limits without time-consuming sample pre-treatments.
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 365 nm was used for excitation. The quantification limits of paraquat and diquat in water, using improved solid-phase extraction, was in the low ng L −1 range. The linear range covered more than two orders of magnitude. Recovery was investigated for all the compounds, and was insufficient, ranging from 11 to 92%, but the method is inexpensive, rapid, and works reliably.
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 NH(4)(+)CH(3)COO(-) (17:3, v/v) over a distance of 6 cm. Dequalinium chloride shows an R(F) value of 0.58. Pure dequalinium chloride is measured in the wavelength range from 200 to 500 nm and shows several by-products, contour plot visualized in absorption, fluorescence and using the Kubelka-Munk transformation. Scanning of a single track in absorption and fluorescence measuring 600 spectra in the range from 200 to 1100 nm takes 30s. As a sample pre-treatment of an ointment it is simply dissolved in methanol and can be quantified in absorption from 315 to 340 nm. The same separation can also be quantified using fluorescence spectrometry in the range from 355 to 370 nm. A new staining method for dequalinium chloride, using sodium tetraphenyl borate/HCl in water allows a fluorescence quantification in the range from 445 to 485 nm. The linearity range of absorption and fluorescence measurements is from 10 to 2000 ng. Sugar-containing preparations like liquids or lozenges with a reduced sample pre-treatment can be reliably quantified only in fluorescence. The total for the quantification of an ointment sample (measuring four standards and five samples), including all sample pre-treatment steps takes less than 45 min!
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 fisheri bacteria. Within 10 min a CCD-camera registered the white light of the light-emitting bacteria. Diclofenac and ibuprofen effectively suppressed the bacterial light emission which can be used for quantification within a linear range of 10 to 2000 ng. The detection limit for ibuprofen is 20 ng and the limit of quantification 26 ng per zone. Measurements were carried out using a 16-bit ST-1603ME CCD camera with 1.56 megapixels (from Santa Barbara Instrument Group, Inc., Santa Barbara, USA). The range of linearity covers more than two magnitudes because the extended Kubelka-Munk expression is used for data transformation. The separation method is inexpensive, fast, and reliable.
Limits of quantification of some neonicotinoid insecticides measured by thin-layer chromatography
(2012)
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 neonicotinoid insecticides show light absorptions below 300 nm. The calculated limits of quantification (LOQ) by UV-detection are in the range from 12 ng to 26 ng on plate depending on the different insecticides.Nitenpyram can be stained using fast blue salt B, forming red zones. The observed LOQ is 25 ng on plate. Acetamiprid can be specifically stained using phenylglyoxylic acid forming a yellow/green fluorescent compound. The LOQ is 52 ng per spot.The compounds thiamethoxam, acetamiprid, thiacloprid and clothianidin can be transformed into blue fluorescing zones, using a relatively new staining solution. This consists of tetraphenylborate and HCl. This is the first publication mentioning that neonicotinoids undergo this reaction. The calculated limits of quantification are in the range from 10 ng to 27 ng on plate.A simple pre-treatment procedure using an acetonitrile extraction and a Chromabond SiOH clean up procedure leads to overall LOQs for bee samples of 48 to 108 µg/Kg. The method can be used to measure neonicotinoid contaminations of bees.
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 (1,1-dimethyl-piperidinium), and chloromequat (2-chloroethyltrimethylammonium) were presented by high-performance thin-layer chromatography (HPTLC). The quantification is based on a derivatization reaction, using sodium tetraphenylborate. Measurements were made in the wavelength range from 500 to 535 nm, using a light-emitting diode (LED) for excitation purposes, which emits very dense light at 365 nm. For calculations, a new theory of standard addition method was used, thus leading to a minimal error if exactly the same amount of sample content is added as a standard. The method provides a fast and inexpensive approach to quantification of the five most important quats used for plant protection purposes. The method works reliably because it takes into account losses during pre-treatment procedure. The method meets the European legislation limits for paraquat and diquat in drinking water according to United States Environmental Protection Agency (US EPA) method 549.2 which are 680 ng L−1 for paraquat and 720 ng L−1 for diquat. The method of standard addition in planar chromatography can be beneficially used to reduce systematic errors. Although recovery rates of 33.7% to 65.2% are observed, calculated contents according to the method of standard addition lie between 69% and 127% of the theoretical amounts.