Spot-area method

The spot-area method is the simplest, most rapid, and most inexpensive, though it is less accurate. Usually, a linear relationship between the amount of analyte and the size (or area) of the spot exists. This relationship has been utihzed by several workers for semiquantitative determination of inorganic species [22,25,40,44,45,47-51]. 

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. 

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 very fast method with a low standard deviation is based on the total size of the non-pigmented areas in relation to the total area of the section analyzed (White Spot Area-WSA). 

The spots can be quantitated by removing them from the plate or paper, or they can be measured in situ. Three methods of quantitation on the plate are (1) measuring spot size, (2) densitometering, and (3) radioactive counting. The square root of the spot area is approximately proportional to the log of the weight of the analyte. This is a semiquantitative method, but it is fast. Standards are run along with the unknown so that an estimate of the quantity of analyte can be made visually from the plate with about 25% accuracy. 

It was already mentioned that thin film chromatography is used firstly as a qualitative analysis method. There is no accurate quantitative analysis that is possible by thin film chromatography. However, there is an empirical formula, equation (2.3), which allows the approximate calculation of the weight of the substance (W) from the spot area (A). This method is very inaccurate and can be used only for gross estimation. 

Opium alkaloids are most commonly estimated by separating them on a chromatographic paper (PC) and/or on a thin layer chromatographic plate (TLC). The former needs a longer time for developing spots than on the silica gel layers of TLC. After the separation of different opium alkaloids with different mobile phase systems of PC and TLC, individual alkaloids are quantitatively measured by eluting the spot and using different procedures, like spot area measurement, spectrophotometry, IR spectroscopy, densitometry, etc. Important methods are described below ... 

The systematic development of spot test methods of analysis occupied Fritz Feigl in Vienna and Rio de Janeiro for half a century up to 1970. Although in the past few decades chemical analysis has undergone a formidable process of sophistication with the development of advanced instrumental tools, there has been at the same time a contrary trend toward simplification in selected areas in the form of simple, rapid, and inexpensive spot and screening tests. Commercial companies are selling large numbers of compact spot test systems for the rapid establishment of the presence or absence of particular substances in clinical, food, water, soil, and forensic samples. The tests are essentially qualitative, but often can be semiquantitative if procedures as simple as visual comparison of color intensity are used. 

PoETHKE et al. [125, 173] have worked with belladonna herbs and tinctures. They used silica gel G and chloroform-acetone-diethylamine (50 + 40 + 10) as solvent. Oswald and Flhck [151—153] have chromatographed hyoscyamine and other alkaloids from this material, notably belladonnine, apoatropine, aposcopolamine, scopine and scopoline. Six solvents were used, of which the best was found to be butanone-methanol-7.5% ammonium hydroxide (60 + 30 + 10) the Dragendorff reagent was employed for detection. The method was worked out also for quantitative determination (planimetric evaluation) (limit of error 5.8%). A linear relation was found between spot area and amount of alkaloid, provided that the amount of substance did not fluctuate by more than 20%. In this way it was possible to determine the mixture of hyoscyamine and atropine in the presence of scopolamine (hyoscine) in some of the plant organs of Atropa belladonna. Datura stramonium, Hyoscyamus niger and H. muticus [153]. 

Due to aperture restrictions, selected area methods are only applicable for examining areas of perhaps 0.5 Xm or more in diameter. It is also possible to obtain spot patterns from much smaller areas—down to as little as a nanometer or so in diameter—by using a focused incident electron beam, rather than an aperture, to limit the sample area under examination. Such microdiffraction patterns are somewhat more complex due to the nonparallel illumination, but they may also contain more information. For example, so-called convergent-beam electron diffraction (CBED) patterns provide three-dimensional information about the compounds crystal chemistry and can be used as a sensitive fingerprint for a given atomic structure. ... 

This computer-assisted method for determining the order of elution of spots by comparison of spot area percent and UV spectral data has a distinct advantage over the use of either area percent values or UV spectral data, neither of which is, in isolation, adequate for selection of the optimum mobile phase for separation of complicated samples of unknown composition. Obviously, if spots in a trial run have almost the same spot area and similar UV spectra, complete recognition of spot order may still not be possible. This technique also runs into problems when there is severe spot overlap in the standard run. 

Tracer Type. A discrete quantity of a foreign substance is injected momentarily into the flow stream and the time interval for this substance to reach a detection point, or pass between detection points, is measured. From this time, the average velocity can be computed. Among the tracers that have historically been used are salt, anhydrous ammonia, nitrous oxide, dyes, and radioactive isotopes. The most common appHcation area for tracer methods is in gas pipelines where tracers are used to check existing metered sections and to spot-check unmetered sections. 

Two newer areas of implantation have been receiving attention and development. Focused ion beams have been iavestigated to adow very fine control of implantation dimensions. The beams are focused to spot sizes down to 10 nm, and are used to create single lines of ion-implanted patterns without needing to create or use a mask. Although this method has many attractive features, it is hampered by the fact that the patterning is sequential rather than simultaneous, and only one wafer rather than many can be processed at any one time. This limits the production appHcations of the technique. 

If the drill string becomes differentially stuck, mechanical methods or spotting fluids can be appHed, or the hydrostatic pressure can be reduced (147). In general, penetration of water- or oil-based spotting fluids into the interface between the filter cake and the pipe accompanied by dehydration and cracking results in reduction of differential pressure across the drill string (147,148). Spotting fluids are usually positioned in the open hole to completely cover the problem area. 

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