Measurement of Spot Areas

Methods of area measurement, independent of judgment by the eye, more objective and without outlay of apparatus, have been studied intensively. 

If the spots are outlined relatively distinctly, the areas can be measured in several ways comparison with suitable standard sizes [459] evaluation with a planimeter [493, 494] copying on to writing paper and 

Spot area Deviation with a Deviation with five 

Should be spot edges be indistinct against the backgroimd, their more objective location may be attempted by copying on hard photographic paper [624]. 

The spot area however increases with increase in substance amount more rapidly than the theory predicts. The linear relation between the square root of the surface area and the logarithm of the applied substance amount, ascertained by Purdy and Truter [547, 548, 549, 720] is generally valid over a wider range of concentration (cf. Fig. 73 c). With the help of a standard solution, the analysis solution and a diluted analysis solution, a quantitative determination can be carried out in accordance with the formula  

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Caffeine was extracted from ficw varieties of roasted coffee beans and was determined in parallel by (1) measurement of spot area after thin layer chromatography on silica gel GF plates (development with chloroform/ cyclohexane/glacial acetic acid, 8 2 1, visualization in UV light), and (2) Kjeldahl N determination. Caffeine contents by (1) and (2), respectively, in the five varieties analyzed were (percent in DM) Santos lave 0, 1.10, and 1.12 Java Robusta 3, 1.19, and 1.22 Camerun Robusta 2, 1.16, and 1.19 Mocca 2, 1.21, and 1.26 Guatemala 0, 1.18, and 1.20. (1) is considered slightly less accurate than (2) but rather easier and more rapid.21... 

Measurement of Spot-areas This method is solely based on a mathematical relationship existing between the prevailing spot area and the amount of component present. It is not quite accurate due to high random errors. 

Quantitative TLC by the measurement of spot areas or by computerized scanning reflectance densitometry (Topic E8) is possible, but, unless HPTLC plates are used, the relative precision attainable is generally only 5-10%. The principal source of error is in applying sample spots to the plate, although automated systems can reduce this. 

Quantitative analysis may be performed directly on the thin-layer plate in a variety of ways by visual comparison by measurement of spot areas by the transmittance of spots that are colored, are charred, or that absorb ultraviolet light by reflectance or by fluorescence. Alternatively, the zone of absorbent containing the constituent can be scraped from the plate and extracted. The extract is then analyzed by... 

If the height fluctuations for a specified laser beam area a are uncorrelated and characterized by a standard deviation Aha, then the standard error on N measurements of spot height is simply / mln = Ah, j /N. To express this in terms of the total scanned area A of a protein layer, it becomes... 

For these reasons, Purdy and Truter(20 ) and several other workers have stressed methods by which measurement of the area of the developed colored spot on TLC plates can be used directly for quantitative analysis. Methods for measuring the spot area may be classified as follows ... 

A Q-switched, frequency-quadrupled Nd—YAG laser (X, = 266 nm) and its accompanying optical components produce and focus the laser pulse onto the sample surface. The typical laser spot size in this instrument is approximately 2 pm. A He-Ne pilot laser, coaxial with the UV laser, enables the desired area to be located. A calibrated photodiode for the measurement of laser energy levels is also present... 

Filter measured volume of air through lead-acetate-impregenated filter paper tape compare optical density with unexposed impregnated spot of similar area. Lead-acetate-impregnated filter paper tape No data NR EPA 1978... 

Thin-layer chromatography does not provide quantitative information of the highest precision and accuracy. Linear relationships between the mass of a substance and the logarithm or square-root of the spot area can sometimes be established under very closely controlled conditions. The optical absorbance of a spot determined by reflectance measurements can be similarly related to mass, or the substances can be scraped from the plate and dissolved in a suitable solvent for a spectrometric determination. The main difficulties with area and density measurements lie in defining the boundaries of spots and controlling chromogenic reactions in a reproducible manner. Relative precision can be as good as 1-2% but is more usually 5-10%. 

In the manufacture of SAN and ABS and polymer dispersions (and also of chemical intermediates) under normal conditions, spot measurements of 5 ppm [11 mg/m ] were found during 1963-74, and it was assumed that higher levels occurred under some conditions. In 1975-77, monthly readings averaged 1.5 ppm [3.3 mg/m ] (Thiess Fleig, 1978). In ABS factories in France, short-tenn (< 2 h) area measurements averaged... 

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