Solution densitometry

Modern TLC is thus an effective tool for separation of complex solutions into their components. Analysis can be qualitative or quantitative. When the composition or the analysed solution is known approximately, identification of the substance contained in a particular spot by its positional coordinates is frequently adequate. HPTLC provides the means not only for flexible screening procedures but also for demanding quantitative determinations. HPTLC features highly sensitive scanning densitometry and video technology for rapid chromatogram evaluation and documentation. 

The greatest strength of 2D planar methods is that they distribute components widely over a 2D space of high peak capacity. Multidimensional TLC development has the advantages of requiring simple equipment is compatible with scanning densitometry for solute identification and quantitation and enables exploitation of the spot reconcentration mechanism. 

Krzek et al. [35] reported the qualitative identification and quantitative analysis of the mixtures of OTC, tiamulin, lincomycin, and spectinomycin in the veterinary preparations by using TLC/densitometry. As stationary phase, they used precoated TLC aluminum sheets, and the mobile phases were mixtures of 10% citric acid solution, hexane, ethanol (80 1 1, v/v), and n-butanol, ethanol, chloroform, 25% ammonia (4 5 2 5, v/v). The other application of TLC or HPTLC for analyzing OTC in the various samples is summarized in Table 2 [36]. 

Wronski [55] described a method to separate and resolve penicillamine from physiological fluids. To urine (100 mL) was added 60 g of (NH4)2S04, the solution was filtered, mixed with 2 g of Na2S03, and 1 mL of 0.1 M EDTA in 20% triethanolamine, and shaken with 5-20 mL of 0.01-0.06 M tributyltin hydroxide in octane for 5 min. A portion (5-8 mL) of the organic phase was shaken with 0.2 mL of HC1 in 20% glycerol solution. A portion of the aqueous phase (5-10 pL per cm of the strip width) was applied to cellulose gel, and electrophoretic separation was performed by the technique described previously. The thiol spots were visualized with o-hydroxymercuri benzoic acid-dithiofluorescein and densitometry with a 588-nm filter. 

Shriadah and Ohzeki [350] determined iron in seawater by densitometry after enrichment as a bathophenanthroline disulfonate complex on a thin layer of anion exchange resin. Seawater samples (50 ml) containing iron (II) and iron (III) were diluted to 150 ml with water, followed by sequential addition of 20% hydrochloric acid (100 xl), 10% hydroxyl-ammonium chloride (2 ml), 5 M ammonium solution (to pH 3.0 for iron (III) reduction), bathophenanthroline disulfonate solution (1.0 ml), and 10% sodium acetate solution (2.0 ml) to give a mixture with a final pH of 4.5. A macroreticular anion exchange resin in the... 

In some instances quantitation, or at least comparison of the proportions of the separated lipids, may be possible by scanning the plate directly by reflectance densitometry, e.g. after charring. Alternatively, the spot may be scraped off the plate and the colour or fluorescence of the eluted solution measured. The eluted compounds can be submitted to further analysis, by GLC for instance, to enable quantitation or identification. For reliable and meaningful results, known standards should always be run and analysed under identical conditions because of variable colour yields with different lipids. Even so, results obtained by different methods will often vary. 

The dye Coomassie Brilliant Blue R250 nonspecifically binds to all the protein. The gel is soaked in the dye for it to seep in and bind to the proteins. The gel is then destained to remove the unbound dye. The dye binds to the protein and not the gel, and hence the protein bands can be visually seen. The binding of the dye to the protein is approximately in stoichiometry, so the relative amounts of protein can be determined by densitometry. For most SDS and native gels, separated proteins can be simultaneously fixed and stained in the same solution. 

Zone electrophoresis is normally carried out horizontally in a suitable medium such as paper, polyacrylamide gel, starch gel or cellulose acetate. The sample components can be completely separated and quantitatively and qualitatively identified in much lower quantities than by the moving-boundary method. The procedure consists of saturating the support material with a buffer solution. The ends of the strip of support are immersed in separate reservoirs of buffer solution to maintain the saturation. The sample is then applied as a narrow band near one end of the support strip. A voltage potential is created down the length of the strip causing the sample components to ionize and then migrate at a rate dependent on their charge, molecular size and interactions with the support medium. When the process is complete, the strip is removed and developed for examination of the separated components. Densitometry is normally used for quantitation of the bands after suitable color development. 

The theoretical basis of thin-layer densitometry by transmission is the same as that for absorption in solution. The medium provides the main differences between the two processes. For example, in solution the effects of reflected and scattered light are at a minimum, whereas such effects are significant with samples adsorbed on layers. Also, the distribution of a compound in solution is homogeneous, while that of a spot on an adsorbent is not, neither vertically nor horizontally in the layer. Shibata [42] described the phenomenon of light striking a translucent material as... 

In addition to its obvious routine importance, UV spectrophotometry has been used as an assay method the berbamine content of crude ethanolic extracts of 22 Berberis species was determined, with standard deviation 3.83%, by measuring the absorption at 282 nm (171). Thalicarpine was similarly assayed at 280 nm and also by TLC densitometry and titrimetry or potentiometry (308). Tetrandrine was determined in drug preparations by UV spectrophotometry of a dilute HC1 solution at 280 nm (standard deviation 0.40%) (309). 

In 1980, Klug reported a method to detect morphine in head hair of drug abusers by TLC. He dissolved the hair in sodium hydroxide and hydrolyzed the solution with HCL. He extracted the solution with amyl alcohol and separated the components by TLC. Detection and quantitation were made by fluorimetry. The findings were between traces and 4 ng/mg. A HPTLC method was used to determine morphine in human hair. From 20 to 200 mg of hair were washed four times with water to remove surface contamination. Then the hair was incubated in sodium hydroxide and subsequently in HCL at 80°C in both cases. After SPE (type "Extrelut 3"/Merck) the solution was dansylated, analyzed by HPTLC (methanol (MeOH)/ammonia 99 1), and quantitated by densitometry. 

The relative response factor of DneiK and RCMLA to Coomassle Blue R staining was determined by SDS-PAGE analysis of samples of known concentrations of DnaK and RCMLA. Aliquots of RCMLA and DnaK stock solutions of identical concentrations and Tris-HCl buffer (20 mM 19 mM NaCl. pH 7.3) were mixed to afford different [DnaK] [RCMLA] molar ratios (2 1 1 1 1 2 1 3) the concentration of DnaK was kept constant at 4.4 pM. These solutions were treated with SDS sample buffer and analyzed by densitometry as described above. The relative... 

K-edge densitometry Hybrid XRF K-edge Amount of U, Pu, Th, Np in solutions... 

The following solution is used for staining 0.5% acetic acid, 0.1% pyronine Y and 1 mM citric acid. The gels are stained overnight and destained by washing in 10% acetic acid. Formamide does not interfere with densitometry in the ultraviolet at 280 nm. 

Reflection spectra of the spots of lac color standard at Rf values of 0.60 and 0.29 and cochineal color standard at Rf value of 0.52 on the TLC plate were taken under the conditions described above. The obtained spectra showed good agreement with the spectra obtained from methanol solutions. Therefore, we considered that TLC/scanning densitometry is effective for the identification of these natural colors. 

Although TLC-MS (mass spectrometry) has been shown to be technically feasible and applicable to a variety of problems, thin-layer chromatography is generally coupled with spectrophotometric methods for quantitative analysis of enantiomers. Optical quantitation can be achieved by in situ densitometry by measurement of UV-vis absorption, fluorescence or fluorescence quenching, or after exctraction of solutes from the scraped layer. The evaluation of detection limits for separated enantiomers is essential because precise determinations of trace levels of a d- or L-en-antiomer in an excess of the other become more and more important. Detection limits as low as 0.1% of an enantiomer in the other have been obtained. 

Ten pi serum sample is applied to the thin-film element. After a 5 minutes incubation at 37 C, 10 pi of hydrogen peroxide solution is applied. The rate of formation of the dye, monitored at 670 nm by reflectance densitometry, is measured during a second 5 minutes incubation at 37 C. 

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