Absorbance, measurement wavelength

Separate sample blanking requires an additional analytical channel, and is therefore wasteflil of both reagents and hardware. An alternative approach that is used on several automated systems, eg, Du Pont ACA, BM-Hitachi 704, Technicon RA-1000, is that of bichromatic analysis (5) where absorbance measurements are taken at two, rather than one, wavelength. When the spectral curves for the interference material and the chromogen of the species measured differ sufficiently, this can be an effective technique for reducing blank contributions to assay error. Bichromatic analysis is effective for blanks of both the first and second type. 

Atomic Absorption An analytical method in which the sample is converted into a vapor by passing it through a flame or other energy source and the absorbance at a particular wavelength is measured and compared with that of a reference substance. The absorbance measured is proportional to the concentration ot that substance in the sample. 

The three normal means of presenting the spectrophotometric data are described below by far the most common procedure is to plot absorbance against wavelength (measured in nanometres). The wavelength corresponding to the absorbance maximum (or minimum transmission) is read from the plot and is used for the preparation of the calibration curve. This point is chosen... 

If the wavelength of maximum absorption of the analyte (Xmax) is known, it can be monitored and the detector may be considered to be selective for that analyte(s). Since UV absorptions are, however, generally broad, this form of detection is rarely sufficiently selective. If a diode-array instrument is available, more than one wavelength may be monitored and the ratio of absorbances measured. Agreement of the ratio measured from the unknown with that measured in a reference sample provides greater confidence that the analyte of interest is being measured, although it still does not provide absolute certainty. 

UV-absorbance measurement at a given wavelength, either on a photometer, which is known to be sufficiently selective, or on a HPLC... 

Rosseinsky and Nicol have investigated in detail the reaction in highly acidic perchlorate media which leads to the production of Mn ". Using [Mn(II)] in the range 2x 10 to 1 x 10 M with [MnO ] 10 M the rate law, found from absorbance measurements at a wavelength of 525 rn.fi, is... 

All other approaches try and relate the child spectra to the parent spectra. In the patented method of Shenk and Westerhaus [41 Sh], in its simplest form, one first applies a wavelength correction and then a correction for the absorbance. Each wavelength channel i of the parent instrument is linked to a nearby wavelength channel j(i) in the child instrument, namely the one to which it is maximally correlated. Then, for each pair of wavelengths, i for the parent and j i) for the child, a simple linear regression is carried out, linking the pair of measured absorbances... 

Step j Wavelength jc, CI2 X2 Br Absorbance Measured ) Estimated ) Innovation... 

Step j Wavelength Ch 2 Brj Absorbance Measured Estimated Innovation... 

Aa is the absorbance in the tilted geometry, Az the absorbance measured in normal incidence geometry with light polarized along the Z-axis, and n the refractive index of the sample. The isotropic refractive index in the visible range is often used as a first approximation, but quantitative measurements require knowledge of the anisotropic refractive indices at the wavelength of interest [29]. 

Fig. 17.4. Absorbance versus wavelength (nM) spectroscopic measurement for acetazolamide compound. The solubility equilibrium is obtained at 72 hours after dissolution.

Figure 10. Absorbance response of an indicator to pH change. (The calculations were made for the absorbance measured at a wavelength where the absorptivity of the base form of the indicator is larger than that of the acid form so that Ab>Aa. Ab is the absorbance at a high pH when all the indicator is in its base form and Aa is the absorbance at a low pH when all the indicator is in its acid form).

A spectrum is a plot of some measure of the electromagnetic radiation absorbed by a sample versus the wavelength or energy of the electromagnetic radiation. For example, it is common practice to plot the absorbance versus wavelength for spectra in the ultraviolet and visible spectral regions as shown below (Fig. 5.1). 

In the laboratory, solutions of analytes that fluoresce are tested by measuring the intensity of the light emitted. The instrument for measuring fluorescence intensity is called a fluorometer. Inexpensive instruments used for routine work utilize absorption filters similar to what was described previously for absorption spectrophotometers (see Figure 8.2 and accompanying discussion) and are called filter fluorometers. Two such filters are needed—one to isolate the wavelength from the source to be absorbed, the wavelength... 

Spectral interferences are due to substances in the flame that absorb the same wavelength as the analyte, causing the absorbance measurement to be high. The interfering substance is rarely an element, however, because it is rare for another element to have a spectral line at exactly the same wavelength, or near the same wavelength, as the primary line of the analyte. However, if such an interference is suspected, the analyst can tune the monochromator to a secondary line of the analyte to solve the problem. 

A plot of absorbance versus wavelength may be used to identify a component of a solution or to determine the wavelength of maximum absorbance (maximum molar absorptivity = a). A more common plot is one of absorbance versus concentration. For this type of plot the instrument is set at the wavelength of maximum molar absorptivity and the absorbances of solutions of various known concentrations (c) are measured. This plot should be a straight line. This linear relationship is called Beer s law and has the form of A = abc. The concentration of an unknown solution may be determined by measuring its absorbance and using the plot to find its concentration. 

Imperfections in monochromators result in the presence of a small proportion of unwanted wavelengths in the incident radiation. Such stray light results in a deviation from a Beer-Lambert relationship (Figure 2.14) and the effect is that absorbance measurements are lower than they should be. 

Molecule Absorbed laser wavelength [Mm] Kind of measurement References... 

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