Nitrophenols, absorption spectra

The initial evidence for the formation of an acyl-enzyme ester intermediate came from studies of the kinetics with which chymotrypsin hydrolyzed analogs of its normal polypeptide substrates. The enzyme turned out to hydrolyze esters as well as peptides and simpler amides. Of particular interest was the reaction with the ester p-nitrophenyl acetate. This substrate is well suited for kinetic studies because one of the products of its hydrolysis, p-nitrophenol, has a yellow color in aqueous solution, whereas p-nitrophenyl acetate itself is colorless. The change in the absorption spectrum makes it easy to follow the progress of the reaction. When rapid-mixing techniques are used to add the substrate to the enzyme, an initial burst of p-nitrophenol is detected within the first few seconds, before the reaction settles down to a constant rate (fig. 8.8). The amount of p-nitrophe-... 

A similar relationship may be found in the adsorption of other atoms or molecules. Many organic substances with peripheric dipoles when adsorbed on salt layers or on the surfaces of metallic oxides show absorption spectra which are shifted appreciably to the red side of the spectrum. Thus p-nitrophenol, having a maximum of light absorption at 316 m. when adsorbed on CaF2, has its absorption spectrum shifted to the red side and is yellow instead of colorless (175), its absorption maximum being at 365 m i. (176). Adsorbed on BaF2 it shows an absorption maxi-... 

The forces responsible for a second or third layer to be adsorbed cannot be very strong. We may, therefore, not expect multimolecular adsorption to occur at moderately low relative pressures on flat and homogeneous surfaces. By means of absorption spectra a bimolecular adsorption of p-nitrophenol could be established on CaF2 or BaF2 layers (250). The absorption spectrum of the first layer was quite different from that of p-nitrophenol itself, the absorption spectrum of the second layer being practically the same as that of pure p-nitrophenol. The first layer is apparently sufficiently polarized by the salt surface, to enable a second layer to adsorb the second layer, however, cannot accumulate a third layer. It may not be entirely excluded, however, that there is no more place in the capillary space between the salt layers to accommodate a third layer of p-nitrophenol. 

The transformation of various substituted phenols has been studied in the presence of nitrite under irradiation dihydroxybenzenes [78,79,113], nitrophenols [109], phenylphenols [114], Obviously, the observed transformation intermediates vary according to the reaction rate of each substrate and intermediate with the various reactive species formed during irradiation and according to the absorption spectrum and direct photolysis quantum yield of each compound. 

The analytical method used for parathion takes advantage of the ease with which the phosphorus-oxygen bond in that material is cleaved by hydroxide. The hydrolysis produces a mole of 4-nitrophenol for each mole of parathion originally present in solution. Because 4-nitrophenol has a well-defined absorption spectrum with a high molar absorptivity at the wavelength of maximum absorption (400 m/x) and because the reaction to form 4-nitrophenol from parathion is rapid and quantitative, the method provides a reliable and sensitive means for analyzing parathion. 

Identification of the Transients. Neutral Solutions. In deaerated 5 X 10"5M p-nitrophenol solution at pH 7, the absorption spectrum with a maximum at 290 n.m. was observed about 20 /xsec. after the end of the radiation pulse (Figure 1-A) when all the hydrated electrons, H and OH radicals have reacted with the p-nitrophenol. The spectra shown in Figure 1-A and 1-B were corrected for the decrease in optical density owing to solute destruction. The G-value for the destruction of p-nitro-phenol was assumed to be equal to the sum of G(e"aq) + G(OH) + G(H) = 6.0 (2, 15, 19). The corrections had a maximum value at 400 n.m. namely, 103% and 130% of the observed optical densities and decreased to 40% and 81% at 290 n.m. for spectrum A and B, respectively. When hydrated electrons are converted to OH radicals by reaction with N20 (II), the absorption at 290 n.m. decreases and the maximum shifts towards 300 n.m. (Figure 1-B). It can be concluded, therefore, that the transient species produced by hydrated electrons as well as OH radicals have an optical absorption in the same wavelength region. 

A portion of the organic by-products was dissolved in carbon tetrachloride or chloroform (spectroscopic grade) for infra-red cuialysis. The absorption peaks corresponded to nitrophenols, nitrocresols, dinitrophenols, dinitrocresols, traces of trinitro conpounds euid nitro-hydroxy carboxylic acids. The presence of individual compounds wais confirmed by comparison with the absorption spectrum of the pure compounds euid by thin-layer chromotography. Quantitative cuialyses of the concentrations of several components of the by-products were made. If a component did not have an absorption bcuid free from interference by euiother constituent of the mixture, queuititative cUialysis could still be achieved by mectsurement of the extinction coefficients of the interfering components at severcLL different frequencies, together with the absorbance of the mixture at these frequencies. Characteristic absorption bands used are shown in Tetble I. 

The plasma level of paracetamol can be determined by gas-liquid chromatography, by its UV absorption spectrum following extraction, and by its colorimetric reaction with nitrous acid to form a yellow coloured nitrophenol (the Glynn and Kendal method). Paracetamol can be detected in urine by a screening test which consists of its hydrolysis to /7-aminophenol, followed by its reaction with o-cresol and ammonia to form a blue in-dophenol. 

In the M. trichosporium OB3b system, a third intermediate, T, with kmax at 325 nm (e = 6000 M-1cm 1) was observed in the presence of the substrate nitrobenzene (70). This species was assigned as the product, 4-nitrophenol, bound to the dinuclear iron site, and its absorption was attributed primarily to the 4-nitrophenol moiety. No analogous intermediate was found with the M. capsulatus (Bath) system in the presence of nitrobenzene. For both systems, addition of methane accelerated the rate of disappearance of the optical spectrum of Q (k > 0.065 s-1) without appreciatively affecting its formation rate constant (51, 70). In the absence of substrate, Q decayed slowly (k 0.065 s-1). This decay may be accompanied by oxidation of a protein side chain. 

Finally,- the alkali salts of phenol itself are more deeply coloured than is phenol. This fact cannot indeed be recognised subjectively, but investigation of the absorption of ultra-violet light demonstrates it. Thus it has been found that the absorption by sodium phenoxide much more nearly approaches the subjectively visible part of the spectrum than does that of the free phenol. The difference is so considerable that it provides also a satisfactory explanation of a subjectively perceptible deepening of colour from colourless to yellow. The colour of the salts of nitrophenols is therefore ascribed to the bathychromic (colour-deepening) efEect of salt-formation. 

Continuous methods do not require a separation step prior to detection. For assays using this method, the substrate and product must differ in some property such that either one may be measured directly in the incubation solution. For example, the activity of an enzyme catalyzes the conversion of 4-nitrophenyl phosphate (4NP), a colorless compound, to 4-nitrophenol, which is yellow and has an absorption maximum at 510 nm. Since the substrate does not absorb in this region of the spectrum, the reaction can be carried out... 

FIGURE 5.49 Expansions of the aromatic-ring-proton multiplets from the 300 MHz spectrum of 2-nitrophenol. The accompanying hydroxyl absorption (OH) is not shown. 

The longest wavelength absorption band of the UV spectrum of 4-nitrophenol is at 263.3 nm (37 980 ) in the gas phase. This band is attributed to a tt tt transition with... 

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