Optimization wavelengths

Fig. 41.6. Multicomponent analysis (see Fig. 41.5) with an optimized wavelength sequence.

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. 

Sulfo-SAND, SANPAH, and Sulfo-SANPAH all contain a nitrated phenyl azide photoreactive group. The presence of the nitro group shifts the optimal wavelength for photoactivation... 

Scenario A student determined that the optimal wavelength for the absorbance of FeSCN2+ experiment was 445 nm. Then the student prepared samples of known concentrations of FeSCN2+ ranging from 4.0 x 10 5 M to 1.4 x 10 4 M. The samples were then examined by means of a spectrophotometer and their transmittances recorded. From the transmittance, the absorbance was calculated and graphed. Next, he mixed 5.0 mL of 2.0 x 10 3 M Fe(N03)3 with 5.0 mL of 2.0 x 10 3 M KSCN. This solution was then analyzed in the spectrophotometer and through extrapolation, he was able to determine that the concentration of FeSCN2+ at equilibrium was 1.3 x 10" M. 

Before the concentration could be determined through colorimetry, the student needed to know the wavelength of light that was most absorbed by the complex ion in order to set the spectrophotometer properly. The student calibrated the spectrophotometer by setting the transmittance to 100% with the FeCl3-KCl-HCl solution as a reference. The optimal wavelength was found to be 525 nm (see Figure 1). 

In some cases, a less optimal wavelength is used. The absorption observed will be sufficient to stay in the dynamic range of the detector. This is documented by the example of the nitrate ion that still absorbs at 230 nm, but almost not at all at 254 nm. When the analysis is run at 254nm instead of 233 nm, it results in an overall higher signal (Eigure 5). 

The difference between the two forms is greatest at 595 nm therefore, this is the optimal wavelength to measure the blue color from the Coomassie dye-protein complex. If desired, the blue color can be read at any wavelength between 575 and 615 nm. At the two extremes (575 and 615 nm) there is a loss of -10% in the measured amount of color (absorbance) compared to the value obtained at 595 nm. 

Ultraviolet intensity is easily controlled through the selection of the lamp. Low-pressure mercury cathode lamps emit most of their energy at the optimal wavelength of 253.7 nm. The electrical input ranges from 15 to 30%. The advantage of medium-pressure, mercury-vapor lamps is their high electrical rating from 0.1 to 20 kW. However, medium-pressure mercury lamps have... 

The function of the monochromator in AES is to isolate the determinant spectral wavelength of interest from the emission from all concomitant matrix emitting elemental or molecular species. This frequently means that a narrow spectral bandpass must be selected. It is however generally slightly easier to make sure in AES than in AAS that the optimal wavelength is being employed since emission spectra often may be scanned directly. 

For separations in the ng/mL-to-pg/mL range and solutes that absorb in the UV/visible portion of the spectrum, optimize the detector wavelength. The optimal wavelength is usually in the low-UV portion of the spectrum. Using LC, the UV cutoff of the mobile phase often prevents such low wavelengths from being employed. Limits of detection usually approach 10"6 M without heroic measures. The downside of low-UV detection is a loss of selectivity, since more solutes will absorb there. This is countered in part by the high peak capacity of CE. In some cases, appropriate sample preparation may be required for selectivity. 

The reactants and products were separated on an MOS Hypersil column (4.6 millimeters x 200 mm, 5 /urn). The mobile phase was composed of a 90 10 mixture of solvent A, consisting of 0.1 M sodium acetate, 0.02 M citric acid, 0.93 mM sodium octanesulfonate, and 0.12 mM disodium EDTA (pH 4.6), and solvent B, methanol UV detection was used, with the optimal wavelength being 258 nm for the adenoxyl derivatives and 279 nm for adrenaline and noradrenaline. Quantitation was normally based on the S-adenosyl-L-homocysteine formed. 

Figure 8-13. Optimal wavelength selection for API and related impurities.

Many modern scanners have a computer-controlled motor-driven monochromator that allovre automatic recording of in situ absorption and fluorescence excitation spectra. These spectra can aid compound identification by comparison with stored standard spectra, test for identity by superimposition of spectra from different zones on a plate, and check zone purity by superimposition of spectra from different areas of a single zone. The spectral maximum determined from the in situ spectrum is usually the optimal wavelength for scanning standard and sample areas for quantitative analysis. 

Figure 4 shows theoretical losses of (1) silver hollow-optical fiber, (2) dielectric-coated silver hollow-fiber with coating thickness of 0.39 pm, and (3) d = 0.66 pm. The thickness of 0.39 pm is the optimized value for 2 = 3 pm and 0.66 pm is the one for 2 = 5 pm. Parameters used in the calculation are rii = 1.53, z = 1 m, 2T = 1 mm, and cr = 0 and complex refractive index of silver is taken from literature [11]. In the calculation, a Gaussian beam with the divergence angle of 6° in full-width-half-maximum is assumed as an input beam. As seen from the calculated spectra, one can obtain a low loss region around the optimized wavelength. 

Wang and Kerker" and Chew and Wang" " have presented a theoretical treatment, based on the electrodynamic approaches used for the SERS problem, and provide a full description of the extinction of the dye-coated spheroids. They also calculated the luminescence enhancement, and find it to be up to 10" on silver for optimal wavelengths and particle shapes. 

The library was linked to the solid support via the photo-labile linker (see Fig. 8.6) which was cleaved at the 337 nm of the nitrogen laser used in MALDI-TOF mass spectrometers (optimal wavelength for cleavage of linker is 365 nm).The linker facilitated the... 

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