Ultraviolet absorbance detection

A UV/Vis absorbance detector can also be used if the solute ions absorb ultraviolet or visible radiation. Alternatively, solutions that do not absorb in the UV/Vis range can be detected indirectly if the mobile phase contains a UV/Vis-absorbing species. In this case, when a solute band passes through the detector, a decrease in absorbance is measured at the detector. 

A distinction is normally made between the visible and ultraviolet regions of the spectrum when detecting absorbing substances. Detection in the visible part of the spectrum can be carried out with the eye or with a photomultiplier. 

The side chains of the aromatic amino acids (Phe, Tyr and Trp) are not particularly reactive chemically, but they all absorb ultraviolet (UV) light. Tyr and Trp in particular absorb strongly at 280 nm, allowing detection and quantification of proteins in solution by measuring the absorbance at this wavelength. 

DNA is often present in amounts too small to be detected by direct spectroscopy. In this case, the fluorescent dye EtBr can be used to amplify the absorption. EtBr binds to the DNA molecule by intercalating between adjacent base pairs. It absorbs ultraviolet light at 300 nm and emits light at 590 nm in the red/orange region of the visible spectrum. The method can be used to determine the amount of DNA in a test-tube by comparing the EtBr-mediated fluorescence of the sample with that of standards of known amounts of DNA. 

Most conventional techniques for the determination of biological molecules or other species with similar properties use their ability to absorb ultraviolet or visible light, their fluorescence after excitation with light of the appropriate wavelength, or their electrochemical behaviour. It possible to enhance the detectability of some species by making them react with UV-visible absorbing or fluorescent compounds. Applied to complex matrices, these detection methods are at best only selective, because a wide variety of chromophores will give a response. 

Analytes are detected at the outlet by their ability to absorb ultraviolet radiation from the lamp in Figure 0-4a. The graph of detector response versus time in Figure 0-5 is called a chromatogram. Theobromine and caffeine are the major peaks in the chromatogram. Small peaks arise from other substances extracted from the chocolate. 

Only substances that absorb ultraviolet radiation at a wavelength of 254 nanometers are observed in Figure 0-5. By far, the major components in the aqueous extract are sugars, but they are not detected in this experiment. 

Such solvent systems continued to be used even though the lack of solubility of triacyl-glycerols with carbon numbers greater than 46 in this mobile phase has been noted. The solvent gradients that would be required for optimum separations of complex triacylglycerol mixtures are not compatible with RI detection. Therefore, ultraviolet detectors have also been used, but the range of mobile phases is limited, since TGs absorb only in the far-UV range. 

All compounds with UV or visible (VIS) range absorbance Ultraviolet/visible photometer PAHs (EPA 8310) Nitroaromatics, nitramines (EPA 8330) Nitroglycerine (EPA 8332) —Detection based on a single wavelength is prone to interferences. —Does not have the selectivity for positive compound identification. 

Spectroscopic detection (using ultraviolet/visible (UV/vis) absorbance, refractive index, fluorescence, atomic absorption or atomic emission). Techniques based on postcolumn reactions. 

Detection of tocopherols and tocotrienols after HPLC separation is based on their ability to absorb ultraviolet light and create fluorescence. Tocopherols and tocotrienols show typical UV spectra with maximum absorption at 290-300 nm (Table 1.6). If the samples contain sufficient amounts of analytes, e.g., vegetable oils and supplemented products, a UV detector is sensitive enough. When higher sensitivity and better selectivity is needed, a fluorescence detector is the commonly used detector. With a fluorescence detector, it is possible to analyze tocopherols... 

HPLC = high-performance liquid chromatography LOD = level of detection NPD =nitrogen-phosphorus detector PRA = pararosaniline RSD = relative standard deviation SPE = solid phase extraction PSD = thermionic specific detection UV = ultraviolet absorbance detection... 

Pure polyethylene should not absorb ultraviolet radiation of wavelength above 200 nm since pure paraffins are transparent in that region of the spectrum. However, it is well established [ 20] that even carefully purified polyethylene differs from a simple high molecular weight straight chain paraffin in being to some extent unsaturated. The total unsaturation has been estimated to be about 0.25% C=C by weight [21]. Olefinic unsaturation of different types has been detected by infrared spectroscopy [21, 22] it seems to be mainly of the vinyl type in linear polyethylene, while most unsaturation is of the vinylidene type in branched polyethylene [22]. Attention has also been drawn to the fact that a structure seems to be present in low density polyethylene which leads to a triene on ultraviolet irradiation [23]. 

Quantitation of metabolites is challenging in the absence of radiolabel or authentic standards. The most commonly used methods for metabolite monitoring are detection by ultraviolet (UV) absorbance and by mass spectrometry. When UV absorbance is being used for detection, the peak response will be approximately proportional to the amount of metabolite if the chromophore is not altered, such as hydroxylation of an aliphatic side group. However, if the molecule is cleaved, altering conjugated systems, relative UV peak responses may not accurately represent the relative amounts of parent compound and each metabolite. Mass spectrometry is even less reliable than UV absorbance in the absence of authentic standards, as ionization efficiency for each detected molecule will differ. In practice, a combination of UV and mass spectrometry is most appropriate. However, unlike the substrate depletion approach, the possibility would still exist of missing metabolites if the detection method does not pick up the metabolite of interest. 

Elemental analysis only reveals which atoms are present. Determination of the chemical structure can often be addressed using spectroscopic methods. The moieties in the polymer absorb and emit radiation at frequencies which are characteristic of their chemical structure. Skeletal bond transitions can be detected in the infrared and Raman spectra, electronic transitions typical of unsaturated bonds can be detected at ultraviolet and visible wavelengths, and atomic nuclei with magnetic moments can be detected and their positions found by magnetic resonance experiments. 

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