Wavelength selection, quantitative absorption

In order to plot the absorption spectrum of a compound or complex ion, we must be able to carefully control the wavelengths from the broad spectrum of wavelengths emitted by the source so that we can measure the absorbance at each wavelength. Additionally, in order to perform quantitative analysis by Beer s law, we need to be able to carefully select the wavelength of maximum absorption, also from this broad spectrum of wavelengths, in order to plot the proper absorbance at each concentration. These facts dictate that we must be able to filter out the unwanted wavelengths and allow only the wavelength of interest to pass. 

The basic principle of quantitative absorption spectroscopy lies in comparing the extent of absorption of a sample solution with that of a set of standards under radiation at a selected wavelength. 

To increase the precision of quantitative analysis, the plasticizer sample is diluted with carbon disulfide, its infrared absorption measured, and compared with absorptions standard of standard samples prepared also in CS2 to cover the range of concentrations from 0.5 to 3 mg/ml. For each suspected (identified) plasticizer, a series of standards has to be prepared and measured. It is also important to select a suitable wavelength for quantitative analysis. For dioctyl phthalate bands at 1725 and 1121 cm" are usually used. For tricresyl phosphate band at 1191 cm is used. Similar to gravimetric method, the results are subject to various interferences when mixtirres of plasticizers or mixture of plasticizers with other additives ate used. 

Interference filter photometers (dispersive Fig. 5) provide a low-cost, rugged alternative to grating or interferometer-based instrumentation. These instruments typically contain from 5 to 40 interference filters that select the proper wavelengths for quantitative analysis based on previous work with scanning instruments or based on theoretical positions for absorption. 

Absorption spectra and, within certain restrictions, also fluorescence excitation spectra can be measured. The stored spectral data are used postrun for various purposes the operator selects i) spectra are useful to determine at which wavelength(s) quantitative scanning shall be performed ii) spectra of individual fractions can be displayed for identification by comparison with spectra of authentic standards stored in a spectrum library iii) superimposed spectra of all equidistantly migrated fractions of a chromatogram, e.g. for identity check or iv) superimposed spectra from different positions within a spot, for checking purity. 

When considering the optical resonances of the sample to aid in the selection of laser wavelength, the self-absorption of the sample also needs to be considered. When exciting organic materials or aqueous solutions in the NIR, this can mean absorption due to overtones and combinations of vibrational fundamentals. The self-absorption will affect relative intensities, which can severely affect the ability for quantitation. 

When the DAD is used, a selection of wavelengths may be available for individual monitoring as well as the selection of the best wavelength for quantitation in consideration of background noise that may be largely chemical noise or interferences from the matrix. In the case of PNAs, the full absorption spectrum for benzo [a] pyrene is given in Figure 2. 

Atomic emission is used for the analysis of the same types of samples that may be analyzed by atomic absorption. The development of a quantitative atomic emission method requires several considerations, including choosing a source for atomization and excitation, selecting a wavelength and slit width, preparing the sample for analysis, minimizing spectral and chemical interferences, and selecting a method of standardization. 

Quantitative accuracy and precision (see Section 2.5 below) often depend upon the selectivity of the detector because of the presence of background and/or co-eluted materials. The most widely used detector for HPLC, the UV detector, does not have such selectivity as it normally gives rise to relatively broad signals, and if more than one component is present, these overlap and deconvolution is difficult. The related technique of fluorescence has more selectivity, since both absorption and emission wavelengths are utilized, but is only applicable to a limited number of analytes, even when derivatization procedures are used. 

IR spectroscopy is a powerful and readily available orientation characterization technique. It offers a high chemical selectivity since most functional groups absorb at distinct wavelengths (typically in the 2.5-25 pm range (4,000 00 cm-1 range)), which often depend on their local environment. IR spectroscopy thus provides qualitative and quantitative information about the chemical nature of a sample, its structure, interactions, etc. The potential of IR spectroscopy for orientation characterization stems from the fact that absorption only occurs if the electric field vector of the incident radiation, E, has a component parallel to the transition dipole moment, M, of the absorbing entity. The absorbance, A, is given... 

The difficulties in the use of fluorescence for quantitative measurement of hydrocarbons are much like those for the ultraviolet absorption methods. Each compound has its own excitation and emission maxima, with the fluorescence quantum yields varying sometimes by an order of magnitude. Thus the amount of hydrocarbon reported by an analysis will depend upon the emission and excitation wavelengths chosen, and upon the compound selected as the standard. 

The use of visible and UV spectrometry for quantitative analysis by comparing the absorbance of standards and samples at a selected wavelength is one of the most widespread of all analytical techniques. It is also one of the most sensitive. The analysis of mixtures of two or more components is facilitated by the additivity of absorbances. This has been discussed earlier (p. 356). Other applications include measurement of the absorption of complexes as a function of solution conditions or time to establish their composition, and to determine thermodynamic and kinetic stability for analytical purposes or for more fundamental studies. 

The quantitation of known compounds is carried out at a selected wavelength, usually that where the compound exhibits its strongest absorption. Calibration graphs are constructed, and corrections for the reagent and sample background are made when possible. 

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