Paper diazotized

Nylon, particularly the positively charged membranes, has largely replaced nitrocellulose or diazotized paper for hybrid selection. DNA, e.g., from linearized plasmid, can be applied in 0.4 N NaOH to denature the DNA and to promote a strong fixation. The nylon membrane (2-cm squares) is soaked for 5 min in water and then 30 min in 0.4 N NaOH. Concurrently, DNA is denatured for 10 min in 0.4 N NaOH. DNA is then applied repeatedly with drying between applications. The membrane is then washed twice with 1 M NH4OAC and twice with 1 X SSG. The membrane is blotted and dried (may contain > 100 p,g/cm ). An alternative method is presented in Section 8.3.1. Disks of 0.5 cm are then punched out with a sterile one-hole paper punch. It is also possible to use Southern blots, or fragments thereof, for hybrid selection. 

Air Monitoring. The atmosphere in work areas is monitored for worker safety. Volatile amines and related compounds can be detected at low concentrations in the air by a number of methods. Suitable methods include chemical, chromatographic, and spectroscopic techniques. For example, the NIOSH Manual of Analytical Methods has methods based on gas chromatography which are suitable for common aromatic and aHphatic amines as well as ethanolamines (67). Aromatic amines which diazotize readily can also be detected photometrically using a treated paper which changes color (68). Other methods based on infrared spectroscopy (69) and mass spectroscopy (70) have also been reported. 

Forty-three grams (0.25 mole) of />-bromoaniline (Note 1) and 20 cc. of water are warmed in a 400-cc. beaker until the bromoaniline melts, and then 50 cc. of concentrated hydrochloric acid (sp. gr. 1.19) is added with mechanical stirring. The mixture is heated and stirred until solution is practically complete (Note 2). The beaker is then set in a dish of ice water and the solution is stirred as it cools in order to precipitate the />-bromoaniline hydrochloride in fine crystals. A few small pieces of ice are added and the cold (about 0-5 °) suspension is diazotized with a solution of 18 g. of sodium nitrite in 36 cc. of water to an end point with starch-iodide paper. 

Testing for excess of nitrous acid at the end of the reaction. For this purpose starch-potassium iodide papers are best used, and these indicate nitrite in acid solution by turning blue instantaneously. With some practice, the nitrite reaction can be clearly distinguished from the coloration caused by certain diazo compounds, such as those bearing nitro substituents. The latter react only after 0.5 to 2 seconds. Often the difference becomes more marked after dilution of the diazo solution with concentrated hydrochloric acid. A properly conducted diazotization should exhibit on completion a very weak nitrite reaction, corresponding to an excess of about 10 4 m. 

The nitrosylsulfuric acid method is particularly suitable for the diazotization of di- and trinitroanilines and aminoanthraquinones. Such amines may be added directly to the nitrosylsulfuric acid, but it is preferable to run the appropriate amount of nitrosylsulfuric acid into a solution of the amine in 96% sulfuric acid. In general, these diazotizations can be carried out at room temperature. The end-point is determined in the usual manner with iodide paper, but only after first diluting a few drops with ice. On completion, the whole is diluted with ice. The test with iodide paper fails in the case of polynitrodiazonium salts. 

Compounds which correspond to 1,2-quinone diazides can also be obtained by diazotization of aromatic and nonaromatic heterocyclic amines with a hydroxy group in the ortho position. Examples include 3,4-quinolinequinone-3-diazide (2.35, Sus et al., 1953 Sus and Moller, 1955) and 3-diazochromane-2,4-dione (2.36, Arndt et al., 1951). Syntheses of more complex heterocyclic quinone diazides have been tabulated by Ershov et al. (1981, p. 105). More recent publications are cited in a paper by Tisler s group (Klotzer et al., 1984). 

In the context of the stability of the nitrosoamine intermediate in the diazotization of heteroaromatic amines relative to that in the case of aromatic amines, the reversibility of diazotization has to be considered. To the best of our knowledge the reverse reaction of a diazotization of an aromatic amine has never been observed in acidic solutions. This fact is the basis of the well-known method for the quantitative analysis of aromatic amines by titration with a calibrated solution of sodium nitrite (see Sec. 3.3). With heteroaromatic amines, however, it has been reported several times that, when using amine and sodium nitrite in the stoichiometric ratio 1 1, after completion of the reaction nitrous acid can still be detected with Kl-starch paper,... 

The reactions of arenediazonium ions with 7V-alkyl- or 7V-arylhydroxylamines were investigated by Bamberger (1920b, and earlier papers). Mitsuhashi et al. (1965) showed that the l,3-diaryl-3-hydroxytriazenes are tautomeric with 1,3-diaryltriazene-3-oxides (Scheme 6-16). Oxidation of 1,3-diaryltriazenes with peroxybenzoic acid in ether yields the same product as that from diazonium salts and TV-arylhydroxyl-amine. The infrared spectrum of the product obtained by coupling diazotized relabeled aniline with A/-phenylhydroxylamine indicates that the diaryltriazene-oxide is the preponderant tautomer. 

Before discussing Beckwith s data on that basis, a brief mention must be made of investigations by Lown s group (Naghipur et al., 1989, 1990, and other papers cited there) who claim to have observed the formation of benzoxathiete (10.63) and its valence isomer monothio-l,2-benzoquinone (10.64) in the aprotic diazotization of 2-[(2,-acetoxyethyl)sulfinyl]aniline (10.62). Scheme 10-82 is an abbreviated form of the mechanism proposed by the authors. A more detailed experimental study is clearly required. 

The new method of Yoneda s group132 is also a one-pot diazotization-fluoro-de-diazoniation in a liquid-liquid two-phase mixture of pyridine and hydrogen fluoride. Yields for 25 aromatic amines and diamines are 50-100%, except for 2-and 3-fluorobenzoic acid, the three nitroanilines, 3- and 4-diaminobenzene and 4,4 -diaminodiphenyl-oxide (10-50%). In their 1994 paper the authors demonstrate that, in the same system, the photochemical decomposition gives in many cases significantly higher yields than the thermal reaction. The most spectacular increase in yield was found for the fluorination of 2-fluoroaniline where o-difluorobenzene was obtained photochemically in 80.2% yield, but thermally only in 0.6% ... 

Several amino compounds are being used extensively in industrial processes. Most of these compounds are manufactured, except hydrazine. Azo dyes are produced by diazotization of aromatic amines and currently there are at least 3000 azo dyes in use. These dyes are used widely in textiles, leather, printing, paper making, drug and food industries. In the past three decades many food, drug and cosmetic colours have been banned from commercial use as food colourants. This section gives a brief account of adverse affects caused by the use of various amino compounds. 

The nucleotide 5-astato-2-deoxyuridine has been obtained from 2-deoxyuridine in a manner similar to that described for preparing 5-astatouridine with a radiochemical yield of 85% (170). Attempts to synthesize 5-astato-2-deoxyuridine from the diazonium salt of 5-amino-2-deoxyuridine led only to a 2-3% yield of desired product, whereas 20-25% of the bound astatine was found in the form of 5-astatouracil (99,123). This was apparently due to hydrolysis of the 7V-glycosyl bond in the course of the diazotization reaction. The final product, 5-astato-2-deoxyuridine, was identified by TLC, paper electrophoresis (170), and HPLC (99,122,123). 

Diazotization is an important reaction because of the practical uses of diazo compds. One use of diazo compds is in the prepn of light-sensitive printing papers, since most diazo compds are stable in the dark but decomp in light. The largest most varied use of diazotization and diazo compds is for coupling with amino or hydroxy aromatic compds to produce azo dyes. The coupling occurs accdg to the equation ... 

TNT in air by collecting the sample in iso-ftroDanol hv means of a midvet imninger. reducing the TNT to triaminotoluene with TiCl, and estimating the triamino compd colorimetric ally after diazotization and coupling as described in the paper) 10)F. 

To 450 cc. of concentrated hydrochloric acid (sp. gr. 1.19) and 500 cc. of water in a 4-I. (i-gal.) earthenware crock equipped with an efficient stirrer is added 143 g. (1 mole) of /3-naphthyla-mine. The suspension of the amine hydrochloride is cooled by the addition of 500 g. of cracked ice. When the temperature reaches 5° solid sodium nitrite (about 69 g.) is added until starch-iodide paper shows an excess. During the diazotization about 600 g. of cracked ice is introduced at such a rate as to keep the temperature at 50. The cold solution of the diazonium salt is filtered to remove a small amount of precipitate and returned to the crock. A solution of 271 g. (1 mole) of mercuric chloride in 300 cc. of concentrated hydrochloric acid is mixed with 300 g. of ice and added slowly to the rapidly stirred solution. A heavy yellow solid separates. Stirring is continued for one-half hour to secure complete reaction. The yellow addition compound of /3-naphthalene diazonium chloride and mercuric chloride is collected on a 20-cm. Buchner funnel, sucked as dry as possible, and then washed with two 400-cc. portions of water and two 150-cc. portions of acetone (Note 1). The... 

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