Wavelength Methods

An assumption inherent in the one-wavelength method is that the distribution of lipofuscin is uniform throughout the area of the retina being analyzed. Certainly if lipofuscin concentration... 

Trieschmann, M. et al. (2006). Macular pigment optical density measurement in autofluorescence imaging Comparison of one- and two-wavelength methods. Graefe s Archive for Clinical and Experimental Ophthalmology 244 1565-1574. 

Mendelsohn ML. Absorption cytophotometry comparative methodology for heterogeneous objects, and the two-wavelength method, in Introduction to Quantitative Cytochemistry (Wied GL, ed.), Academic Press, New York, 1966, pp. 201-214. 

Galassi L, Della Vecchia B. Selection of optimal transmittances with the cytopho-tometric two-wavelength method. Basic Appl Histochem 1988 32 279-291. 

COLOR LOSS BY INDIVIDUAL WAVELENGTH METHOD (% OF STARTING MATERIAL)... 

Figure 2. Relationship between color loss by the integration method as compared to that by the individual wavelength method selected values of several lignin types 350 nm (0) and 500 nm (A).

There is reasonably good agreement between the measurements made by spectral reconstruction and the linear wavelength method. This supports the validity of this method which takes considerably less instrument time (see above). 

Comparisons can also be made by wavelength methods. These have the advantage that two lasers of quite different frequency can be compared but are currently limited in accuracy to about the to 10 - - - range. 

Gangwal and Sharma [16] described a simultaneous spectrophotometric method for the determination of mefenamic acid and paracetamol in their combined dosage forms based on the native UV absorbance maxima of mefenamic acid and paracetamol in 0.02 M NaOH. Mefenamic acid exhibits two absorption maxima at 285 nm and 333 nm, while paracetamol has one absorbance maxima at 257 nm. In a separate study, the same group [17] also reported a spectrophotometric procedure for mefenamic acid and paracetamol in two component tablet formulations. The method is based on the two-wavelength method of calculation. The difference in absorbances at 217 nm and 285 nm was used for determination of mefenamic acid, and the difference in absorbances at 257 nm and 308.8 nm was used for the determination of paracetamol. Beer s law is obeyed by both the drugs within the concentration ranges employed for analysis. The method has been statistically validated, and was found to be satisfactory. 

Any calculation involving Eqs. (9) and (10) presupposes a two-component system. It must be emphasized that this condition is usually fulfilled only as a good approximation. This obviously limits the accuracy of every two-wavelength method. 

In a series of 30 blood samples taken during heart catheterization the So, was determined using Van Slyke s manometric technique and the two-wavelength method using X = 560 and 522 nm. The results are given in Table 14. The mean difference between the spectrophotometric and manometric methods was -t-0.57% saturation with a standard deviation of 2.8% saturation. 

In this section, 2 two-wavelength methods making use of an isobestic point, which have proven to be quite suitable in our laboratories for many years, are described. The general requirements dealt with in Section 4 are also valid for these techniques. 

Hemiglobin can be estimated in the presence of HbOa, using a two-wavelength method (Section 4). A suitable isobestic point is situated at X = 523 nm, while a large difference between eni and Hbo, is found at X = 558 nm. Other possibilities are referred to by Henry (HI). 

Two Wavelengths Method The spectral overlapping of the components of a mixture is one of the most important limitations of the spectrophotometric methods when one component has to be determined in the UV-visible range. When two analytes have to be determined in the same solution, two different wavelengths have to be chosen in such a way that one analyte does not interfere with the other. The general relation between the two concentrations to be determined and the two measured absorbance values is ... 

RESULTS ILLUSTRATING THE SCOPE OF THE METHOD VARIABLE SINGLE WAVELENGTH AND MULTI-WAVELENGTH METHODS... 

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