Two-wavelength methods

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. 

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 ... 

The probes are assumed to be of contact type but are otherwise quite arbitrary. To model the probe the traction beneath it is prescribed and the resulting boundary value problem is first solved exactly by way of a double Fourier transform. To get managable expressions a far field approximation is then performed using the stationary phase method. As to not be too restrictive the probe is if necessary divided into elements which are each treated separately. Keeping the elements small enough the far field restriction becomes very week so that it is in fact enough if the separation between the probe and defect is one or two wavelengths. As each element can be controlled separately it is possible to have phased arrays and also point or line focussed probes. 

Two additional methods for determining the composition of a mixture deserve mention. In multiwavelength linear regression analysis (MLRA) the absorbance of a mixture is compared with that of standard solutions at several wavelengths. If Asx and Asy are the absorbances of standard solutions of components X and Y at any wavelength, then... 

See the end of Section 5.3. Various investigators have used differential absorptiometry at two wavelengths that enclose, but do not locate, an absorption edge. This procedure resembles simple absorptiometry at more than one wavelength, and it also resembles the absorption-edge method. 

Kulkarni [68] described an ultraviolet absorption method for the measurement of the organic carbon level in seawater which compares the intensities at two wavelengths, one less than 350 nm and the other greater than 400 nm. 

Since the slope of the calibration curve is sb, we need to construct four calibration curves, two at the first wavelength for compounds a and b and two at the second wavelength for compounds a and b. Once the four slopes are determined along with the absorbance of the unknown at the two wavelengths, we have two equations in two unknowns that can be solved algebraically or with simple matrix methods. 

Three steady-state methods can be used to determine the energy transfer efficiency. In the following description of these methods, the fluorescence intensity is indicated with two wavelengths in parentheses the first one is the excitation wavelength, and the second is the observation wavelength. Because the characteristics of the donor and/or acceptor are measured in the presence and in the absence of transfer, the concentrations of donor and acceptor and their microenvironments must be the same under both these conditions. 

It is recommended, whenever possible, to carry out ratiometric measurements, i.e. to determine the ratio of the fluorescence intensities measured at two wavelengths according to the following methods ... 

In the ratiometric method, the fluorescence intensity of the liposomes containing pyranine (F) and in the presence of the quencher DPX was determined at 520 nm upon excitation at two wavelengths 460 nm (of the charged unprotonated pyranine) and 415 nm (of the pH-independent isosbestic wavelength that describe the total pyranine concentration). The ratio of is described as F. The ratiometric measurement is used to determine the intraliposome aqueous phase pH (18,22). Then nigericin (or nonactine) at final concentration of 5pM was added to disrupt the pH and/or ammonium ion gradient that induce complete gradient collapse and the measurement at the above two excitations was repeated, and indeed it demonstrated a shift of the intraliposome aqueous pH to be identical to the extraliposome medium pH (10). 

The precise configuration can be chosen according to the laboratory workload. Table 2.2 clearly illustrates the capacities of the various models. These instruments will support a wide range of analytical methods including end-point assays at one or two wavelengths and with one or two steps as well as rate analyses. 

Giacovazzo, C. and SUiqi, D. (2001). The method of joint probability distribution functions apphed to MAD techniques. The two-wavelength case for acentric crystals. Acta Crystallogr. A 57, 700-707. 

Figure 5.4 The difference in the observed response when two wavelengths are tested, 2 and 3, compared to the actual effect of changing from the method value to either 2 or 3...

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