Measurement wavelength, optimal

The excitation maximum for Br-BODIPY 493/503 is 515nm and its emission occurs at 525 nm when dissolved in methanol. Upon coupling to a sulfhydryl compound, however, the excitation wavelength of the adduct decreases to 493 nm and its emission drops to 503 nm. The very small lOnm Stoke s shift may be a problem, particularly in avoiding interference due to of excitation-light scattering in critical emission measurements. Sub-optimal excitation wavelengths... 

The use of UV-absorbance measurements to optimize the base extraction was demonstrated. UV measurements over a wide wavelength range were demonstrated to be necessary because many humic materials being removed may not have the same maximum absorbance wavelength. 

Improved separation of natural oil TGs using short columns packed with 3-//m alkyl bonded-phase particles was reported by Dong and DiCesare (88). The HPLC columns used were HS-3 high-speed columns packed with 3-/um C18 bonded-phase particle (100 X 4.6-mm ID) with a column void volume of ca. 0.8 ml and efficiencies in the range of 13,000-15,000 theoretical plates (measured under optimized conditions) and HS5 C,8 columns (125 X 4.6-mm ID packed with 5-yttm particles). Two detectors were used a modified refractive index detector having an 8-/rl flow cell and 0.007-in. ID inlet tubing and a variable-wavelength UV/visible detector. 

Table 6-4. Optimal UV measuring wavelengths for some selected inorganic anions.

Direct UV detection gained great significance in the determination of nitrite and nitrate [18,19] as well as bromide and iodide in the presence of high chloride concentrations. The optimal measuring wavelength for the determination of those anions is listed in Table 6-4. Fig. 6-11 illustrates the superiority of direct UV detection over conductometric detection with a nitrite determination in the presence of a 100-fold chloride excess. Determinations of this kind may be performed in all saline samples such as body... 

With older equipment which cannot be used to obtain spectra, the optimal measurement wavelengths are determined as follows If it is known, for example, that the maximum is at a wavelength less than 300 nm, the chromatogram lane containing the substance to be determined is scanned several times using a scheme such as the following ... 

Fluorescence spectra were measured at wavelength scanning of tunable dye-laser. In spite of the monochromic excitation the fluorescence spectmm has quite complex composition. Such variety of wavelengths allows to optimize fluorescence excitation and registration for any technological conditions. 

Ratzlaff, K. L., and bin Darus, H., Optimization of Precision in Dual Wavelength Spectrophotometric Measurement, Anal. Chem. 51, 1979, 256-261. 

The emission spectra of BODIPY derivatives normally display narrow bandwidths, providing intensely fluorescent labels for biomolecules. Unfortunately, they also have very small Stoke s shifts, typically on the order of only 10-20 nm. Excitation at the optimal wavelength may cause some interference in measurements at the emission wavelength due to light scattering or cross-over from the wide bandwidth of the excitation source. The dyes usually require excitation at sub-optimal wavelengths to prevent this problem. 

The excitation maximum for BODIPY 493/503 C3 hydrazide occurs at 498 nm and its emission at 506 nm. Since this is an extremely small Stoke s shift, it may be difficult to avoid completely problems of excitation-light scattering interference in critical emission measurements unless sub-optimal excitation wavelengths are used. The molecule has an extinction coefficient in methanol of about 92,000M-1cm 1 at 493 nm. 

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