Chromatographic properties of mixed cyano/ODS chemically bonded phases for HPLC

Summary A number of different cyano/ODS polymeric modified silica gel phases were prepared by simultaneous bonding of cyanoalkylsilanes and octadecylsilane. Cyano contents between 10–80% were obtained. High surface coverage (above 4 μmole/m²), as well as low hydroxyl content, characterised all phases. The modified silica was suitable for separations in both straight and reversed phase mode. Significantly reduced retention times were obtained in the reversed phase mode for high mole-cular weight polyaromatic compounds (Mw above 300).     

A novel approach to the qualitative and quantitative assay of HPTLC plates: Second and fourth derivative recording of spectrophotodensitograms

One of the limitations due to lack of resolution for a given pair of analytes in TLC or HPTLC is the need to optimize the system. In Practice this requires time, rerunning of the sample in different developing solvents, and a great deal of expertise on the part of the analyst. In our experience, application of first and second derivative recording techniques to HPTLC facilitates and speeds the whole process, permitting qualitative and quantitative assay of most unresolved spots. Consequently, we have now extended our instrumental capabilities to fourth derivative measurements. For this purpose, we have added a homemade electronic unit in series with the one previously used for first and second order derivatives. Thus, we have been able to evaluate the potential advantages of higher order derivatives for HPTLC analysis of unresolved components in various pharmaceutical products. A comparison of second and fourth order derivative measurements of seriously overlapping HPTLC components in a sample of preservatives used in the pharmaceutical industry suggests that the lower order derivatives might be a better choice in view of the higher accuracy and precision of the corresponding data. This is supported by the results of other applications, such as the assay of a commercial colorant, and a syrup formulation. The observed lack of precision of fourth order measurements stems from the fact that although the second and higher order derivatives produce narrower bandwidths, thus contributing to improved resolution, the signal to noise ratio decreases and satellite peak interactions increase, thus rendering correct discrimination of the fine structural detail of overlapping components more difficult.     

Quantitative HPTLC determination of selenium

The present determination of selenium in biological matrices by HPTLC with in situ fluorimetric detection is an accurate alternative method, comparable to other established methods such as photometry, polarography, neutron activation, or X-ray fluorescence analysis, gas chromatography, and atomic absorption spectrometry. The excellent sensitivity of this procedure is proved by the detection limit of 250 fg of selenium per spot (using purified 2,3,1-naphthoselenodiazole). The oxidation of organic matrices, applying a novel digestion procedure, may be carried out with little instrumental expenditure. Sample preparation steps, such as the oxidation of selenium to Se (VI) and subsequent reduction to Se (IV) do not lead to significant random or systematic errors, nor does the digestion step, if an optimized procedure is used. A recovery rate of 103% and nearly parallel calibration curves for digested selenocysteine standards compared with spiked human serum samples demonstrate the accurate quantitative preparation of a biological matrix. Any interfering metal ions can be masked by addition of chelate-forming reagents.     

Quantitative determination of phospholipids in mitochondria using HPTLC and fluorimetric assay in situ

A method is described for quantitative determination of phospholipids of mitochondria following one-dimensional thin-layer chromatography without previous elution. Using high performance thin-layer chromatography (HPTLC) and an in situ fluorescence technique, the time needed for quantitative determination is relatively short. As the method is rather sensitive, small amounts of the extracts can be applied (about 200 ng of total phospholipids per spot). The reproducibility for the phospholipid fractions determined was in the range of 8–17%. The procedure was tested with lipid extracts of rat liver mitochondria. The method allows the determination of cardiolipin, phosphatidyl ethanolamine, phosphatidyl inositol, phosphatidyl choline, and sphingomyelin.     

Quantitative HPTLC analysis of carboxylic acids in wine and juice

The need for quick and specific assay of carboxylic acids in wine and non-fermented beverages is explained. The method developed in this work is based on high performance thin-layer chromatography (HPTLC) followed by densitometric evaluation. Untreated samples, with the exception of red wines which were destained, were directly sprayed onto the cellulose layer. The chromatogram was developed with ethyl acetate-toluene-water-formic acid 60:20:20:15, then stained with xylose-aniline reagent. Densitometric scanning is by absorbance at 546 nm. The method is quick, sensitive, and has a wide dynamic range for the acids analyzed.     

Preparation of reversed-phase high-performance thin-layer plates by in situ reaction with monochlorosilanes

The condition for in situ chemical modification of commercially available thin-layer plates have been varied and the influence thereof upon reversed phase plate properties has been studied. The reaction variables studied were: average silica pore size, silica water content, use of tri- or monochlorosilanes as modifiers, reaction temperature, and addition of base. Coverage and activity of residual silanol groups was studied on modified plates. Desired plate properties are high speed of development in aqueous solvents and low content of active residual silanol groups. Under the conditions tested, the lowest amount of residual silanols was achieved at the expence of the speed of development in aqueous media and vice versa.     

Inorganic and organic mercury analysis by HPTLC and in situ densitometric detection. Application to real samples

Inorganic mercury and some organo-mercury species have been separated as dithizonates by HPTLC and determined in situ at the subnanogram level by densitometric automatic scanning of the plate. The extraction efficiency of the dithizonates was tested at pH 1, 7, and 10. The R_f values for HgD, CH₃HgD, C₂H₅HgD, and C₆H₅HgD are 0.15, 0.32, 0.35, and 0.28 respectively. The recovery of the tested mercury species in simulated and real samples has been investigated. Conditioning of the plates with NH₃ prior to elution eliminates interference by many other metal ions. Some examples of real sample analysis (tap water, sea water, human urine) are reported.     

High performance thin-layer chromatography of some sixteen-membered ring macrolide antibiotics

The use of HPTLC in the analysis of some sixteen-membered ring macrolide antibiotices was examined. In the case of Spiramycins, instrumentalized HPTLC proved to be very efficient for the separation and determination of these antibiotics. With the use of an internal standard together with the datapair technique in sampling and evaluation of the HPTLC plates, a coefficient of variation less than 1.5% could be achieved when determining the different Spiramycins. Other sixteen-membered macrolides, such as Tylosins, Turimycins and 9-Propionylmaridomycins can be separated with sufficient resolution for quantitative work, in spite of their extremely simular structures and large molecular weights. Detection is always at wavelengths which agree with the intrinsic absorption maximum of the chromophors of the components (e. g. 282 nm for Tylosins, 232 nm for Spiramycins and Turimycins, 195 nm for 9-Propionylmaridomycins).     

The application of gradient saturation in unidimensional planar chromatography of polar lipids. Part 1: Presentation of the HPTLC system

A thermostated HPTLC system was assembled to test the influence of gradient chamber saturation upon resolution of polar lipids chromatographed on boric acid-impregnated silica gel plates. Inexpensive mixtures of nonacidic (14 component peaks) and acidic (8 component peaks) lipids were prepared from soya lecithin by a simplified procedure involving DEAE-Sephadex liquid column chromatography to monitor performance of the system using a readily available reference material. The system requires ca. 5 min chamber presaturation after which the polar lipids are chromatographed in less than 30 min. Retention times (t_R) as well as peak area % values of all component peaks and measured by scanning photodensitometry to demonstrate the influence of different modes of chamber saturation upon the resolution of polar lopids.     

HPTLC analysis of organotin compounds in preservative solutions and preservative-treated wood

A high-performance thin-layer chromatographic (HPTLC) method is described for the determination of tributyltin compounds (bis(tri-n-butyltin) oxide, TBTO, and tri-n-butyltin naphthenate, TBTN) and their degradation products (dibutyltin and monobutyltin compounds). The organotin compounds are extracted from wood with ethanol containing 0.5% (v/v) of hydrochloric acid and the separation of the defferent kinds of organotin compounds is achieved by thin-layer chromatography. The sample spots are measured using a scanning densitometer after decomposing the organotin compounds to inorganic tin by ultraviolet irradiation and visualization of the spots with pyrocatechol violet. Applications of the method to detection and quantification of organotin compounds in preservative solutions, in recently impregnated wood, and in wood samples from five-year-old window frames are described.     

Influence of solvent strength in possible optimization of adsorption thin-layer chromatography

Considerations of TLC process optimization have been based on the thermodynamic theory of adsorption from multicomponent solvents using experimental and theoretical R_{M1, 2} = f (Φ₁) relationships. It was found that a relationship exists between the A_z parameter (log k where k is the partition coefficient of the substance chromatographed) of the above theory and pK_a values of substances as well as the solubility parameter δ of the mobile phase components. Analysis of the A_z values of substances shows that a slight variation therein is associated with lower selectivity of chromatographic separation.     

Separation,quantification, spectral properties and stability of photosynthetic pigments on CN-coated HPTLC plates

Summary Chlorophylls, phaeophytins a and b, -carotene, lutein, violaxanthin and neoxanthin can be separated in 15 min on HPTLC, CN-coated sheets using chloroform-hexane-methanol (25-70-05 v/v). The algebraic characteristics of calibration curves and of the absorbance decay with time have been determined for each component. Solid phase spectra have been established from 370 to 700 nm and their variations have been examined with respect to the amounts of pigments spotted on the plates and to the storage time of chromatograms in the dark at 4°C.Pigments extracted with chloroform from barley leaves were analysed using the described method. A 5% accuracy is normally to be expected, and the sensitivity ranges from 0.5 pmol (-carotene) or 1 pmol (chlorophylls a and b) to 12 pmols (lutein) in quantitative determinations at 425 nm.