Densitometry, TLC

The benefit of using thin-layer chromatography (TLC) for the identification of unknown vitamins and related compounds by comparing Rf values of the unknown compounds with authentic vitamins is beyond doubt. The quantification of the separated vitamins can be performed by the use of modem densitometry. TLC [or high-performance thin-layer chromatography (HPTLC)], as a powerful separation and analytic tool, is used particularly with pharmaceutical preparations and food products. Because amounts of most hydrophilic vitamins are low, or very low, in tissues or body fluids, bioautography or de-rivatization is used before densitometry. 

The lack of quantitation limits the use of DART in many fields. A new system was proposed, which possesses an option for quantitative analyses [3]. This group demonstrated the analysis of a total extract of Schisandra chinensis fruit on a TLC plate. Three major lignin compounds were identified and quantitated. UV densitometry, TLC-DART-MS, and HPLC-UV were compared as detection methods. For DART analysis, the width of a TLC plate was cut to a narrow slice of 3 mm. The developed... 

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. 

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. 

Another interesting TLC method for the isolation and determination of bromate ion in flour dough and breads has been developed [59]. It involves extraction of BrOj from foodstuff, purification on alumina column, TLC separation on silica gel layer developed with water -1- -butanol + n-propanol (1 1 3), and quantification by densitometry. Bromate ion down to 0.1 pg in bread (1.0 g) was detected with tohdin-FIQ reagent. 

Karuna, R. and Sashidhar, R. B., Use of ion-exchange chromatography coupled with TLC-laser scanning densitometry for the quantitation of fumonisin Bl,... 

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. 

Organotin stabilisers in PVC compositions were determined by means of TLC [518]. The determination of inorganic species (down to ppb range) by TLC has been reviewed on several occasions [519,520]. Instrumental methods successful in the quantitative estimation of inorganic TLC zones directly on the plate are densitometry, fluorimetry, radiometry, planimetry and visual methods. In the framework of recycling studies, in particular as to metal stabilisers in PVC, Braun and Richter... 

Thin-layer chromatography is employed in many areas of QC and routine monitoring of product quality [458]. Fluorescence scanning, densitometry or videodensitometry are used for quantification. Not all polymer additives are amenable to TLC analysis. Some fatty acid amides are virtually insoluble in organic solvents and cannot be isolated by thin-layer or column chromatography. 

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]. 

If no references for the degradation products or impurities are available in the laboratory, the sample should be exposed to stress conditions such as heat (50-80 °C), ultraviolet light (2000 lux), acid and base (0.1-1 M HC1 and NaOH), and oxidant (3% H2O2). After incubation in the allotted time, the purity and identity of the analyte peak/spot should be proved by using DAD or MS detection (for LC), MS (for GC), or in situ UV-Vis measurement using a densitometry or TLC-MALDI MS (for TLC). 

An example of release testing by TLC is shown in Fig. 13.24. In this example, two more polar bands are observed, one that is quantitated at <0.1% and another at 0.3%. Quantitations are based on visual estimation using the standards on the plate. Alternatively, scanning densitometry can be utilized to obtain a more definitive quantitation of impurities. 

A TLC method for determination of quaternary ammonium antiseptics was proposed, using silanized silica plates in combination with triiodide ions and UVV densitometry at 400 nm. The method was applied to cetylpyridinium chloride, cetrimide (197c) and the isomers of benzalkonium chloride (197b)420. 

A number of methods have been described for determination of tetracycline (chlortetracycline, tetracycline, and oxytetracycline) residues in tissues of food-producing animals (53-62), fish (63), eggs (64), and honey (65,66). Most of these methods use reversed-phase HPLC for determination. However, one uses TLC with UV densitometry ( ) and one uses GLC ( ), and one uses a direct mass spectrometric method CAD MIKE spectrometry (collisionally activated decomposition mass-analyzed ion kinetic spectrometry) for oxytetracycline in milk and meat (62). Several use solid-phase extraction in the cleanup procedure using XAD-2 resin (56,58) or Cj g cartridges... 

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