Quantitation densitometric method

The spectrodensitometric analysis allows the separation and quantitation of zaleplon from its degradate on silica gel plates using chloroform-acetone-ammonia solution (9 1 0.2, v/v/v) as a mobile phase. This method depends on quantitative densitometric evaluation of thin-layer chromatogram of zaleplon at 338 nm over a concentration range of 0.2-1 yg/band, with mean percentage recovery 99.73 1.35.7%. 

Fatty acids released by lipases can be determined quantitatively by TLC, GC and HPLC. TLC methods are used in conjunction with densitometric methods or autoradiographic methods using radiolabelled TAG. Many GC and HPLC methods that have been outlined earlier are widely used to isolate and quantify FFAs in lipolytic assays. Additionally a method using p-nitrophenyllaurate as a substrate was described by Maurich et al. (1991) who quantified activity by the release of p-nitrophenol. Veeraragavan (1990) used a RP-HPLC method with triolein as the substrate. Triolein was emulsified in buffer with the aid of a surface active agent and the lipase added under controlled conditions. Lipolytic activity was measured by the release of oleic acid and quantified by absorbance at 208 nm. 

Graham, R. J. T., Bark, L. S., Tinsley, D. A. Quantitative thin-layer chromatography on liquid anion exchangers, Part II. A comparison of a spot removal method with a in situ direct densitometric method. J. Chromatogr. 39, 218 (1969)... 

A thin layer chromatographic (TLC) system suitable for determination of clofazimine in plasma has been developed (Lanyi and Dubois, 1982). The plasma samples were acidified using acetate buffer pH 5 and extracted with toluene, evaporated to dryness under nitrogen, reconstituted in toluene and applied to the TLC plate. The adsorbent used was HPTLC silica gel 60. The plates were developed in toluene - acetic acid - water (50 50 4), allowed to stand for 30 min at room temperature, the Rf value of clofazimine was 0.36. Detection and quantitation is carried out using a densitometric method. The limit of detection reported for this method was 5ng/g. 

Densitometric methods. In situ densitometry is an often-used technique for lipid quantitation and has been extensively reviewed by Prosek and Pukl (1996). Lipids are generally sprayed with reagent and their absorption or fluorescence can be measured under UV or visible light by means of a densitometer. The method needs to be standardized and suitable calibration curves need to be constructed to avoid errors. There are several models of densitometer available and some of them are highly automated and coupled to computer systems. Apart from these the use of CCD (charge-coupled device) cameras and colour printers have further improved the densitometric capabilities for accurate quantitations (Prosek and Pukl, 1996). A recent review by Ebel (1996) compares quantitative analysis in TLC with that in HPTLC, including factors that can effect quantitation, the need for careful calibration and errors in quantitative HPTLC analyses. Ebel is of the opinion that as both HPTLC and HPLC are based on the same absorption and fluorescence phenomena they should obtain similar results with respect to quantitation. 

Some non-densitometric methods that can be used for TLC quantification are described only briefly in this entry. The majority of the coverage is given to densitometry, which is the most widely used, highly sensitive [typical detection limits are in the medium picogram (fluorescence) to low nanogram (absorbance) range of analyte per zone], versatile, convenient, and reliable quantitative TLC method. 

Struck [165] and Jacobsohn [92, 93] have worked on the quantitative determination of oestrogens in TLC, using spectrophotometric and photogram-densitometric methods respectively. 

Detection and quantitative analysis of separated enantiomers on these plates were carried out by using densitometric methods. 

In conjunction with densitometric detection, it can be used as a quantitative technique for compounds which are difficult to analyse by other chromatographic methods because of the absence of a chromophore. 

Jamshidi and Nateghi reported a two-step isocratic HPTLC method on silica gel 60F254 plates that used densitometric quantitation at a wavelength of 280 nm for the separation of atorvastatin from plasma... 

Recently, the availability of capillary electrophoresis provides a sensitive method for enumerating the serum protein fractions without the use of staining and densitometric scanning because the electrophoretically separated fractions are quantitated by the measurement of UV absorbance. 

Two methods for DNA quantitation are described spectrophotometric and densitometric. We have used both methods for analyzing hundreds of samples. The first method is simpler and more precise however, more DNA is required. The second method requires much less DNA (several nanograms) and is the only option when little DNA is available. A method for rapidly extracting DNA from many samples of milligram (and larger) amounts of plant,2 3 algal,34 and fungal4 tissues has been described. We do... 

Methods of quantitation other than the listed beta scanner and the use of Biomax film can be used (e.g., Kodak X-OMAT AR film, AMBIS beta scanner, phosphorimager, densitometric scans on films, and so on). However, the authors have found that these items are, by far, the most sensitive and efficient tools for quantitation of HBV DNA in this assay system. 

Two approaches have been proposed to improve the electrophoretic separation between bone and liver ALPs. Both methods exploit differences in the carbohydrate portions of the two forms of ALP. In one, electrophoresis is carried out in the presence of wheat germ lectin, which retards bone ALP migration more than the liver enzyme migration. In the other, serum is treated briefly (i.e.> for 15 min at 37 °C), with neuraminidase to remove part of the terminal sialic acid residues. As the sialic acid residues of bone ALP are more readily attacked than those of liver ALP, the electrophoretic mobility of the bone form is reduced more than that of liver ALP. The improved separation allows quantitative estimates to be made by densitometric scanning (Figure 21-5). ... 

SDS-PAGE provides molecular weight-based separation, as migration in the gel depends on size, the gel being stained for detection purposes. The method can be used either quahtatively or quantitatively (with densitometric evaluation). Immuno-blotting is a frequently used add-on immunological characterization in which the electrophoreticaUy separated bands are subjected to reaction with a corresponding antibody on a nitrocellulose membrane. 

TLC is useful both as an analytical and a preparative technique, and substances tentatively identified by TLC may be further characterized by various analytical techniques such as nuclear magnetic resonance spectrometry, mass spectrometry, or gas liquid chromatography. Moreover, many specific chemical detection tests are available to help identify substances separated by TLC. TLC is a microanalytical procedure and provides for separations and at least tentative identification of substances in the milligram (mg), microgram (/ig), and nanogram (ng) range. TLC can provide the biochemist with a method of eluting separated substances from plates for quantitative analyses. Recent studies indicate that elution techniques may not be the best alternative for quantitative analyses of many substances separated by TLC and that the preferred method may involve quantitative in situ densitometric analysis [1,2]. 

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