Densitometry in situ

In situ densitometry is rapidly becoming the method of choice for the quantitative analyses of substances separated by TLC [2]. This method of analysis involves the use of a commercially available densitometer for scanning TLC plates. Biochemists interested in using this procedure should contact commercial suppliers of densitometers such as Kontes or Schoeffel. 

Selected examples of TLC separations of different compounds of interest to the biochemist 

Amino acids (dansyl) Amino acids (PTH) Anilines Antibiotics 

Aromatic acids Aromatic alcohol 3,5-dinitrobenzoates Aromatic aldehydes 

Carotene and Canthaxanthin Carotenoid aldehydes Ceramides Ceramide acetates 

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Measurement of optical density directly on the layer (i.e., in situ densitometry)... 

In situ densitometry has been the most preferred method for quantitative analysis of substances. The important applications of densitometry in inorganic PLC include the determination of boron in water and soil samples [38], N03 and FefCNfg in molasses [56], Se in food and biological samples [28,30], rare earths in lanthanum, glass, and monazite sand [22], Mg in aluminum alloys [57], metallic complexes in ground water and electroplating waste water [58], and the bromate ion in bread [59]. TLC in combination with in situ fluorometry has been used for the isolation and determination of zirconium in bauxite and almnimun alloys [34]. The chromatographic system was silica gel as the stationary phase and butanol + methanol + HCl -H water -n HF (30 15 30 10 7) as the mobile phase. 

Very High Density Amorphous Ice (VHDA). By annealing HDA to T > 160 K at pressures > 0.8 GPa, a state structurally distinct from HDA can be produced, which is called VHDA ice [152]. The structural change of HDA to a distinct state by pressure annealing was first noticed in 2001 [152]. Even though VHDA was produced in experiments prior to 2001 [170], the structural difference and the density difference of about 10% at 77 K, and 1 bar in comparison with HDA remained unnoticed. Powder X-ray diffraction, flotation, Raman spectroscopy, [152] neutron diffraction [171], and in situ densitometry [172, 173] were employed to show that VHDA is a structural state distinct from HDA. Alternatively, VHDA can be prepared by pressurization of LDA to P > 1.1 GPa at 125 K [173, 174] or by pressure-induced amorphization of hexagonal ice at temperatures 130 K < T < 150 K [170]. The density of this amorphous state at 77 K and 1 bar is 1.26 g/cm3 [152]. 

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. 

Phenols occurring in water have been quantified by in situ densitometry after coupling with diazotized p-... 

Quantitation based on visual assessment of spot size compared to standards can be accurate within 10-30%, which may be adequate for many applications of TLC and PC. Quantitative results with accuracy and precision rivaling those in gas chromatography and high performance column liquid chromatography can be obtained by zone elution and microanalysis or by in situ densitometry. 

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. 

The newest area of TLC involves quantitative in situ densitometry of compounds (Chapter 10). The area has been well explored in lipid TLC, and reference is made to the works of Privett et al. (1973), Downing (1979), and Fried (1991). For in situ densitometric quantification, the chromatographer will need a suitable densitometer and recorder, integrator, or computer. We have described one quantitative experiment (Experiment 8) using cholesterol as the lipid fraction quantified. The experiment described utilizes a Kontes densitometer. 

This experiment is based on the use of a Kontes fiber optics densitometer (K-49500) with baseline corrector and strip-chart recorder. The description given in the second paragraph of Experiment 8, Section 8 is based on the use of that equipment. Readers will have to modify their approaches to in situ densitometry based on the available instrumentation in their laboratories. In our laboratory, we no longer use the K-49500 model and do most of our lipid in situ densitometry with either a Kontes Model 800 densitometer equipped with a Hewlett-Packard Model 3992A integrator/recorder (Morris et al., 1987) or a Shimadzu CS-930 computerized TLC densitometer operated in the single-beam, reflectance mode. [See Morris et al. (1987), Park et al. (1991), and Masterson et al. (1993) for further information on the use of these instruments in lipid quantification by TLC-densitometry.]... 

Lang et al. (1992) stated that although TLC has value as an inexpensive qualitative and semiquantitative assay for vitamin E, HPLC or GC is more suitable for quantitative analysis. However, given proper sample preparation, TLC is suitable for all sample types and for separating tocopherol and tocotrienol homologs. Although one-dimensional systems are usually satisfactory, additional resolution can be achieved by developing in a second direction. Quantification can be done by in situ densitometry. TLC is also useful for sample cleanup prior to other quantitative procedures such as GC or HPLC. 

Otsuka et al. (1986) used TLC to isolate and partially characterize degradation products of 2,3-diketo-L-gulonic acid (intermediates in the biosynthesis of ascorbic acid) the products were characterized by different spectrometric methods. Mandrou et al. (1988) devised a TLC procedure to determine ascorbic and dehydroascorbic acids in fruit juices and wine the sample was reacted with 2,4-dinitrophenylhydrazine to form the osazones the osazones were spotted on the TLC plate and quantified by. in situ densitometry at 494 nm. 

The three main approaches related to quantitative TLC are (a) visual estimation and spot size measurement, (b) zone-elution and spectrophotometry, and (c) in situ densitometry. 

By way of an example of in situ densitometry of lipids, the following study is detailed from Morris et al. (87) on the quantification of cholesterol in hen s egg yolk. Because of the availability of hen s egg yolk, this experiment can easily be replicated by chromatographers who seek an introduction to simple in situ lipid densitometric procedures. 

As mentioned previously, an extensive body of literature exists on the quantification of lipids. Table 10 provides selective examples from the 1979-1995 literature on the quantification of both neutral and complex lipids mainly by in situ densitometry. 

Quantification of lipids mainly by in situ densitometry has been described, and a detailed description has been provided of this procedure from the author s work on the quantification of cholesterol in hen s egg yolk (87). In situ quantification techniques continue to become more automated and will be used more frequently in the future for lipid analyses in clinical, industrial, and research labs. 

In situ densitometry quantifying the chromatogram directly on the plate by measurement of optical density, native or induced fluorescence, etc., of the separated spots. 

Table 3 Advantageous and Disadvantageous Properties on Spot Elution Technique and In Situ Densitometry...

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