Acetic anhydrides

Acetic anhydride is the only monocarboxylic acid anhydride that is important in modification reactions. The acetylation of the amino groups of proteins can be made relatively specific if the reaction is done in saturated sodium acetate, since the o-acetyltyrosine derivative is unstable to an excess of acetate ions (Fraenkel-Conrat, 1959). The tyrosine derivative rapidly hydrolyzes in alkaline reaction conditions, even in the absence of added acetate buffer (Uraki et al., 1957 Smyth, 1967). Treatment with hydroxylamine also cleaves any o-acetyltyrosine modifications, forming acetylhydroxamate, which can be followed by its purple complex with Fe3+ at 540 nm 

Dissolve the macromolecule to be modified at a concentration of l-10mg/ml in a buffered solution having a pH between 6.5 and 7.5. Avoid amine-containing buffers such as glycine and Tris. Sodium phosphate buffer at a concentration of 0.1M works well. The addition of an equal volume of a saturated solution of sodium acetate may be done to prevent tyrosine derivatization. 

Cool the solution on ice. With stirring, add an amount of acetic anhydride equal to the mass of macromolecule to be modified. Alternatively, add a 10-fold molar excess of acetic anhydride over the amount of amines present. The addition of the anhydride slowly or in several aliquots over the course of 1 hour will assure good yield of acetylation. 

React with stirring for at least 1 hour while cooling in an ice bath. 

Maleic acid is a linear four carbon molecule with carboxylate groups on both ends and a double bond between the central carbon atoms. The anhydride of maleic acid is a cyclic molecule containing five atoms. Although the reactivity of maleic anhydride is similar to other cyclic anhydrides, the products of maleylation are much more unstable toward hydrolysis, and the site of unsaturation lends itself to additional side reactions. Acylation products of amino groups with maleic anhydride are stable at neutral pH and above, but they readily hydrolyze at acid pH values around pH 3.5 (Butler et al., 1967). Maleylation of sulfhydryls and the phe-nolate of tyrosine are even more sensitive to hydrolysis. Thus, maleic anhydride is an excellent reversible blocker of amino groups to temporarily mask them from reactivity while another 

Acetic anhydride (boiling point 139.5, density 1.0820) may be produced by three different methods. The first procedure involves the in situ production from acetaldehyde of peracetic acid, which in turn reacts with more acetaldehyde to yield the anhydride. 

In the preferred process, acetic acid (or acetone) is pyrolyzed to ketene, which reacts with acetic acid to form acetic anhydride. 

Another process to make acetic anhydride involves carbon monoxide insertion into methyl acetate (Fig. 1). 

FIGURE 1 Acetic anhydride manufacture by carbonylation of methyl acetate 

Approximately 80 percent of acetic anhydride is used as a raw material in the manufacture of cellulose acetate. 

Acetic anhydride [108-24-7], (CH3C0)20, is a mobile, colorless liquid that lias ail acrid odor and is a more piercing lacrimator than acetic acid [64-19-7]. It is the largest commercially produced carboxylic acid anhydride U.S. production capacity is over 900,000 t yeady. Its chief industrial application is for acetylation reactions it is also used in many other applications in oiganic synthesis, and it lias some utility as a solvent in chemical analysis. 

First prepared by C. F. Gerhardt from benzoyl chloride and carefully dried potassium acetate (1), acetic anhydride is a symmetrical intermolecular anhydride of acetic acid die intramolecular anhydride is ketene [463-51 4-]. Benzoic acetic anhydride [2819-08-1] undergoes exchange upon distillation to yield benzoic anhydride [93-97-0] and acetic anhydride. 

No dimerization of acetic anhydride has been observed in either die liquid or solid state. Decomposition, accelerated by heat and catalysts such as mineral acids, leads slowly to acetic acid (2). Acetic anhydride is soluble in many common solvents, including cold water. As much as 10.7 wt % of anhydride will dissolve in water. The unbuffered hydrolysis rate constant k at 20°C is 0.107 min 1 and at 40°C is 0.248 min-1. The corresponding activation energy is about 31.8 kj/inol (7.6 kcal/mol) (3). Aldiougli aqueous solutions are initially neutral to litmus, they show acid properties once hydrolysis appreciably progresses. Acetic anhydride ionizes to acetylium, CH CO+, and acetate, CH - CO, ions in the presence of salts or acids (4). Acetate ions promote anhydride hydrolysis. A summary of acetic anhydride s physical properties is given in Table 1. 

Acetic anhydride acetylates free hydroxyl groups without a catalyst, but esterification is smoother and more complete in the presence of acids. For example, in the presence of -toluenesulfonic acid [104-15-4], the heat of reaction for ethanol and acetic anhydride is —60.17 kJ/mol (—14.38 kcal/mol) 

ROH + (CH3CO) 20 - CH3COOR + CH3COOH Amines undergo an analogous reaction to yield acetamides, the more basic amines having the greater activity  

Potassium acetate, rubidium acetate, and cesium acetate are very soluble in anhydride in contrast to the only slighdy soluble sodium salt. Barium forms the only soluble alkaline earth acetate. Heavy metal acetates are poorly soluble. 

Acetic anhydride is the largest commercially produced carboxylic acid anhydride, with an annual U.S. production capacity of 2.6 billion lb [40]. There are three principal routes to manufacture acetic anhydride [40,41]. The older process is based on the reaction of ketene with acetic acid  

The ketene could be produced by thermal decomposition of acetone at 700800°C or dehydration at 750°C at reduced pressure over a dehydration agent, such as triethyl phosphate  

Another process is by catalytic oxidation of acetaldehyde. Acetaldehyde is partially oxidized with air in the liquid phase to acetic acid. The acetic acid reacts with the remaining acetaldehyde to form acetaldehyde monoperacetate, which decomposes quantitative to acetic 

A newer process was brought on-stream in the early 1980s by Eastman Chemical Company catalytic carbonylation of methyl acetate produced by methanol acetylation [4145]. The overall reaction of this process is 

The reaction is catalyzed by a rhodium catalysts, with iodide and lithium salt as cocatalysts. This process currently represents about two-third of the U.S. production capacity [41], 

When the acetic acid was replaced by acetic acid anhydride under otherwise unchanged conditions, the reaction became extremely fast. Within a one-minute reaction time the starting material was consumed completely and only a small amount of the wanted para-mononitro compound was found besides a higher nitrated compound as the major product Even if a more favorable selectivity would have been observed, and although a nitration with nitric acid in acetic anhydride can be carried out safely [17] with the micromixer, these conditions have to be avoided because the entire reaction heat is evolved immediately and very probably cannot be controlled on a larger scale. 

In many applications acetic acid is used as the anhydride and the synthesis of the latter is therefore equally important. In the 1970 s Halcon (now Eastman) and Hoechst (now Celanese) developed a process for the conversion of methyl acetate and carbon monoxide to acetic anhydride. The process has been on stream since 1983 and with an annual production of several 100,000 tons, together with some 10-20% acetic acid. The reaction is carried out under similar conditions as the Monsanto process, and also uses methyl iodide as the activator for the methyl group. 

Reaction (9) generates methyl iodide for the oxidative addition, and reaction (10) converts the reductive elimination product acetyl iodide into the product and it regenerates hydrogen iodide. There are, however, a few distinct differences [2,9] between the two processes. The thermodynamics of the acetic anhydride formation are less favourable and the process is operated much closer to equilibrium. (Thus, before studying the catalysis of carbonylations and carboxylations it is always worthwhile to look up the thermodynamic data ) Under standard conditions the AG values are approximately  

Not only does the initiation require hydrogen, but also later in the reaction it may be necessary to reduce rhodium(III) to rhodium(I), since methyl-rhodium(III) is being formed continuously in the reaction  

This protonation may, besides the desired CO insertion, also form inactive trivalent rhodium iodides. In the Monsanto acetic acid process the addition of the reducing agent H2 is not required for two reasons  

Water also causes a change in the reaction medium, which may be advantageous. A drawback of the reducing medium in the Eastman process is that in addition to acetic anhydride, the by-product ethylidene diacetate is formed, CH3CH(AcO)2. This can be thermally decomposed to vinyl acetate and acetic acid, or it can be reduced to ethyl acetate, which in the recycle would lead eventually to propionic acid. 

In mainstream organic synthesis acetylation is usually accomplished using acetic anhydride rather than the more reactive acetyl chloride. However, acetic anhydride has been much less used in carbon-14 synthesis because of the unavoidable loss of 50% of the radioactivity. Therefore, its use is limited to the labeling of substrates that have to be acylated either under very mild conditions or in very small quantities (e.g. proteins ), or to Perkin reactions, where its active methylene group is involved. 

The preparation of [ C]pacIitaxel 37, by 0-acylation is one of the few examples in which the best results were reportedly achieved with [ CJacetic anhydride. The reaction was carried out with three equivalents of [ C]acetic anhydride in pyridine in the presence of catalytic amounts of 4-dimethylaminopyridine to give the target compound 2S in 89% chemical yield . Acetic anhydride was also preferred in the synthesis of 

Reaction conditions 1. DMAP, pyridine r.t., 20 h 2. benzene reflux, 12-15 h  

Higher alkyl or aryl esters are prepared either by reaction of [ C]acetyl chloride and the respective alcohol in an inert solvent or by alkylation of sodium or triethylammonium salts of acetic acid with primary or secondary alkyl halides, as documented by the procedures for phenyl [ C]acetate 49 , (5)-1,2,2-triphenyl-1,2-ethandiol 2-[ C]acetate 50 and a variety of higher alkyl and aralkyl [ CJacetates 51 °. 

Depending on the respective reaction partner, acetic acid esters can react either as C-H acidic compounds or as acylating agents. Both are illustrated by the self-condensation of ethyl [ 1 acetate in the presence of 0.5 equivalent of sodium ethoxide or triphenymethyl sodium to give ethyl [1,3- C2]acetoacetate (Claisen condensation). In the first case, however, because of the relatively low radiochemical yields (40-45%) obtained by this procedure, it is of minor importance for the preparation of labeled ethyl acetoacetate. The deprotonation of alkyl acetates with LiHMDS followed by acylation with unlabeled or labeled acyl halides to labeled give /3-keto esters is discussed in Section 6.4. Claisen condensation of alkyl [ CJacetates with esters lacking a-hydrogens (i.e. ethyl formate, diethyl oxalate, aromatic/heteroaromatic carboxylic acid esters) proceed unidirectionally and are valuable pathways in the synthesis of ethyl [ C]formyl acetate (521. diethyl [ C]-oxaloacetate (53) and ethyl 3-oxo-3-pyrid-3-yl[2- C]acetate (54). The last example 

of finely-powdered anhydrous sodium acetate to the distillate in order to convert any unchanged acetyl chloride into acetic anhydride, insert a cork carrying a thermometer into the flask, attach a condenser, and distil slowly. Collect the fraction which passes over at 135-140° as acetic anhydride. The yield is 20 g. 

//twv Repeal the three e peiimeuts th serilK d uiidei a((t l ( hloiide. The lesult is the saute in each itise Init is the ai etii anlisdiiile leai Is less readily than acetyl chloride, the luiKline retjuires to hi warmed. 

The simplest method for preparing alkylene oxides involves the use of benzoyl peroxide (Prileschaiev). In absolute ether or, still better, in benzene solution, it is broken by sodium ethoxide into the sodium salt of perbenzoic acid and ethyl benzoate.1 

The unstable per-acid, which like all per-acids is much weaker than the corresponding carboxylic acid, is taken up in chloroform after acidification of the solution of the sodium salt. The chloroform solution serves in the reaction with unsaturated substances which has already been formulated on p. 111. Ethylene itself does not give this reaction. 

For the preparation of acetic anhydride the same apparatus as for acetyl chloride is employed. 

Acetyl chloride (54 g. =075 mole) is allowed to run drop by drop from a tap funnel on to 80 g. of finely powdered anhydrous sodium acetate prepared in the manner described below. When about half of the chloride has been added the experiment is interrupted for a short time in order to stir the pasty mass of material with a bent glass rod, the lower end of which has been flattened. The rest of the acetyl chloride is then run in at such a rate that none passes over unchanged. The anhydride is now distilled from the residual salt by mean of a luminous flame kept constantly in motion. Complete conversion of the last traces of unchanged acetyl chloride to acetic anhydride is attained by adding 3 g. of finely powdered anhydrous sodium acetate to the distillate, which is finally fractionally distilled. Boiling point of acetic anhydride 138°. Yield 55-60 g. Use for acetylation, in Perkin s synthesis (Chap. V. 8, p. 232), preparation of acetophenone (Chap. IX. 3 6, p. 346). 

The product should be tested for chlorine by boiling a sample with water and adding dilute nitric acid and a few drops of silver nitrate. 

On the guinea pig skin, the liquid in concentrations in excess of 80% produced severe burns concentrations of 50-80% produced moderate to severe burns solutions below 50% caused relatively mild injury no injury was produced by 5-10% solutions.  

Although ingestion is unlikely to occur in industrial use, as little as 1.0 ml of glacial acetic acid has resulted in perforation of the esophagus.  

Acetic acid was not clastogenic in Chinese hamster ovary (CHO) cells in vitro when the pH of the culture medium was neutralized. The 2003 ACGIH threshold limit value-time-weighted average (TLV-TWA) for acetic acid is 10 ppm (25mg/m ) with a short-term exposure limit of 15 ppm (37mg/m ). 

AIHA Hygienic Guide Series Acetic Acid. Akron, OH, American Industrial Hygiene Association, 1978 

Rajan KG, Davies BH Reversible airways obstruction and interstitial pneumonitis due to acetic acid. BrJ Ind Med 46 67-68, 1989 

Colorless liquid with strong acrid odor bp, 139°C.1 

Flammable. Flash point, 52°C explosive limits, 3-10% ignition temperature, 332°C. Extinguish fire with water spray, dry chemical foam, or carbon dioxide.2 

Reacts slowly with water to form acetic acid.1 

Boric Acid. Heating a mixture to 60°C may result in explosion.3,4 

Chromic Acid. Strongly exothermic reaction occurs when the anhydride is added to an aqueous solution of chromium trioxide.5,6 

Vinylacetate. Based on methyl acetate, Halcon has also developed a route to vinylacetate, which is technologically ready for commercialization [32] (F,quation (16)). 

The ethylidene diacetate formed can be converted to vinylacetate and acetic acid in high yield by passage over a metal oxide catalyst at high temperatures. The net reaction is given in Equation (17). 

The carbonylation of methyl acetate is the critical step. For instance, it can he carried out with Co2(CO)B/NBu3/MeJ in acetic acid [35], with RhClj/picohn/. MeJ [36] or Ni-/Co-compounds [37]. 

For synthesis of ethylidene diacetate, other startmg materials have also been proposed. Hydrogenation of acetic anhydride by a palladium catalyst [3gj or caibonylafion of CH3CH(OCIl3)2 with RhCl3/Ph3P/MeJ (39] are two alternative routes. 

Dissolve the protein or other amine-containing macromolecule at a concentration of 1—10 mg/ml in 0.1 M sodium phosphate, 0.15 M NaCl, pH 7.5. 

Add a 25 molar excess of sulfo-NHS acetate over the amount of amines present in the sample. If the precise amount of amines is not known, adding an equal mass of reagent to the mass of protein will provide a large excess of reactivity to completely block all amines. 

FiyurG 99 Sulfo-NHS acetate may be used to block amine groups, forming permanent amide bond derivatives. 

At physiological pH values, acetylation of amine groups proceeds rapidly, requiring less than an hour to go to completion (Fig. 100). 

Physical Data bp 138-140°C mp —73 °C d 1.082gcm . Solubility sol most organic solvents. Reacts with water rapidly and alcohol solvents slowly. 

Form Supplied in commercially available in 98% and 99+% purities. Acetic anhydride-rfe is also commercially available. Analysis of Reagent Purity IR, NMR.  

Preparative Methods acetic anhydride is prepared industrially by the acylation oi Acetic Acid with Jfetene. A laboratory preparation of acetic anhydride involves the reaction of sodium acetate and Acetyl Chloride followed by fractional distillation. Purification adequate purification is readily achieved by fractional distillation. Acetic acid, if present, can be removed by refluxing with CaC2 or with coarse magnesium filings at 80-90 °C for 5 days. Drying and acid removal can be achieved by azeotropic distillation with toluene.  

Handling, Storage, and Precautions acetic anhydride is corrosive and a lachrymator and should be handled in a fume hood. 

Recently, Vedejs found that a mixture of Tri-n-butylphosphine and acetic anhydride acylates alcohols faster than acetic anhydride with DM AP. However, the combination of acetic anhydride with DMAP and Triethylamine proved superior. It is believed that the EtsN prevents HOAc from destroying the DMAP catalyst. 

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Homogeneous catalysts. With a homogeneous catalyst, the reaction proceeds entirely in the vapor or liquid phase. The catalyst may modify the reaction mechanism by participation in the reaction but is regenerated in a subsequent step. The catalyst is then free to promote further reaction. An example of such a homogeneous catalytic reaction is the production of acetic anhydride. In the first stage of the process, acetic acid is pyrolyzed to ketene in the gas phase at TOO C ... 

Figure 14.8a shows a simplified flowsheet for the manufacture of acetic anhydride as presented by Jeffries. Acetone feed is cracked in a furnace to ketene and the byproduct methane. The methane is used as furnace fuel. A second reactor forms acetic anhydride by the reaction between ketene from the first reaction and acetic acid. 

Figure 14.8 Simplified fiowsheet for the acetic anhydride process. The composite curves show both distillation columns to be across the pinch. (From Smith and Linnhoff, Trans. IChemE, ChERD, 66 195, 1988 reproduxxd by permission of the Institution of Chemical Engineers.)...

Heating with a mixture of acetic anhydride and acetic acid. 

One disadvantage of using acetic anhydride is that with primary amines, traces of the diacctyl compound, RN(COCH3)2, niay be formed the chances of this secondary acetylation are, however, usually remote, and recrystallisation from an aqueous solvent will generally hydrolyse the diacetyl derivative rapidly back to the mono-acetyl compound. 

For complete acetylation of polyhydric compounds, such as glucose (p. 141) and mannitol (p. I42), even undiluted acetic anhydride is insufficient, and a catalyst must also be employed. In such cases, the addition of zinc chloride or anhydrous sodium acetate to the acetic anhydride usually induces complete acetylation. ... 

Some amines, e.g., aniline, can on the other hand be readily acetylated by dissolving them in cold dil. acetic acid and adding acetic anhydride the method is not however general. 

Add 20 ml. of a mixture of equal volumes of acetic anhydride and glacial acetic acid to 10 ml. (10 3 g.) of aniline contained in a 150 ml. conical flask. Fit a reflux water-condenser to the flask, and boil the mixture gently for 10 minutes. Then pour the hot liquid into 200 ml. of cold water, stirring the latter well... 

Although the acetylation of alcohols and amines by acetic anhydride is almost invariably carried out under anhydrous conditions owing to the ready hydrolysis of the anhydride, it has been shown by Chattaway (1931) that phenols, when dissolved in aqueous sodium hydroxide solution and shaken with acetic anhydride, undergo rapid and almost quantitative acetylation if ice is present to keep the temperature low throughout the reaction. The success of this method is due primarily to the acidic nature of the phenols, which enables them to form soluble sodium derivatives, capable of reacting with the acetic... 

Naphthyl Acetate. CHgCOOCi H,. Dissolve 1 g. of pure 2-naphtnol in 5 ml. (r8 mols.) of 10% sodium hydroxide solution as before, add 10 g. of crushed ice, and i-i ml. (1-14 g., 1 5 mols.) of acetic anhydride. Shake the mixture vigorously for about 10-15 minutes the 2-naphthyl acetate separates as colourless crystals. Filter at the pump, wash with water, drain, and dry thoroughly. Yield of crude material, 1-4 g. (theoretical). Recrystallise from petroleum (b.p. 60-80 ), from which, on cooling and scratching, the 2-naphthyl acetate separates as colourless crystals, m.p, 71 yield, 10 g. 

It should be emphasised that salicylic acid can be readily acetylated by Method 1, and that the above preparation of acetylsalicyclic acid is given solely as an illustration of Method 2. To employ Method 1, add 10 g. of salicylic acid to 20 ml. of a mixture of equal volumes of acetic anhydride and acetic acid, and boil gently under reflux for 30 minutes. Then pour into about 200 ml. of cold water in order to precipitate the acetylsalicylic acid (11 g.) and finally recrystallise as above. Method 2, however, gives the purer product. 

It should be emphasised that whereas the interaction of a sodium salt and an acid chloride is a convenient general laboratory method for preparing all classes of anhydrides, acetic anhydride is prepared on a large scale by other and cheaper methods. Industrial processes are based on reactions indicated by the equations ... 

CH,CO O OCCH, + H NH, CH,CONH, + CH,COOH Acetic anhydride Acetamide... 

B) [i-PENTACETYLGLUCOSE. Required Anhydrous sodium acetate, 2 5 g. acetic anhydride, 25 ml. glucose, 5 g. 

Carry out this preparation precisely as described for the a-compound, but instead of zinc chloride add 2 5 g. of anhydrous powdered sodium acetate (preparation, p. 116) to the acetic anhydride. When this mixture has been heated on the water-bath for 5 minutes, and the greater part of the acetate has dissolved, add the 5 g. of powdered glucose. After heating for I hour, pour into cold water as before. The viscous oil crystallises more readily than that obtained in the preparation of the a-compound. Filter the solid material at the pump, breaking up any lumps as before, wash thoroughly with water and drain. (Yield of crude product, io o-io 5 g.). Recrystallise from rectified spirit until the pure -pentacetylglucose is obtained as colourless crystals, m.p- 130-131° again two recrystallisations are usually sufficient for this purpose. 

Place a mixture of 5 g. of camphor, 6 g. of powdered selenium dioxide and 5 ml. of acetic anhydride in flask fitted with a reflux water-condenser. Heat the flask in an oil-bath for 3 hours at 140-150 so that gentle boiling occurs shake the mixture from time to time. 

Cinnamic acid is usually prepared by Perkin s reaction, benzaldehyde being heated with sodium acetate in the presence of acetic anhydride. It is probable that the benzaldehyde and the acetic anhydride combine under the catalytic action of the sodium acetate, and the product then readily loses water to give mono-benzylidene acetic anhydride (. ). The latter, when subsequently... 

Required Benzaldehyde, 20 ml. acetic anhydride, 30 ml. anhydrous sodium acetate, 10 g. 

When cinnamaldehyde, succinic acid and acetic anhydride are heated in the presence of litharge (PbO), the aldehyde and the succinic acid condense to give the dicarboxylic acid (I), which undergoes decarboxylation to give the pale yellow crystalline 1,8-diphenyloctatetrene (II), Kuhn has shown that as the... 

The mixed bases are boiled with an acetic acid-acetic anhydride mixture to convert the aniline into acetanilide. The product is poured into water, when the acetanilide crystallises out while the quinoline remains in solution as quinoline acetate. The acetanilide is filtered off, and the filtrate made alkaline and steam-distilled. 

When the reaction has subsided, boil the reaction-mixture under reflux for 2 hours then make it alkaline with sodium hydroxide solution, and distil it in steam until oily drops no longer come over in the aqueous distillate (1 2 litres). Extract the distillate thoroughly with ether ca. 150 ml.), and dry the ethereal extract over powdered sodium hydroxide. Filter the dry extract through a fluted filter-paper moistened with ether into a 200 ml. flask. Fit the flask with a distillation-head, or a knee-tube , and distil off the ether. Now replace the distillation-head by a reflux water-condenser, add 10 ml. of acetic anhydride, and boil the mixture under reflux for 10 15 minutes. 

Coumarin is usually prepared by heating salicylaldehyde with acetic anhydride and sodium acetate (i.e., the Perkin cinnamic acid synthesis, p. 23 6), whereby the 0" hydroxy-cinnamic acid (I) undergoes cyclisation to coumarin. Coumarins having substituents in the benzene ring can often be similarly prepared. 

Acetylation. Boil i g. of salicylic acid with 4 ml. of an acetic anhydride-acetic acid mixture (equal volumes) under reflux for 10 minutes. Pour into water. Filter off the aspirin (p. 111), wash with water and recrystallise from aqueous acetic acid (1 1) m.p. l36 ... 

B) ACID ANHYDRIDES. Acetic anhydride, succinic anhydride phthalic anhydride (and substituted derivatives). 

Physical properties. Acetic anhydride, (CH3C0).20, is a colourless liquid with a sharp pungent odour, decomposed slowly by water, in which it is only slightly soluble. 

Z>) To 1 ml. of aniline in a small conical flask add about 2 ml. of acetic anhydride and heat on a boiling water-bath for 5 minutes. Now add just sufficient water to dissolve the product on boiling. On cooling, crystals of acetanilide separate out. 

Hydroxamic acid formation. To 0 1 g. of acetic anhydride, add 0 1 g. of hydroxylamine hydrochloride and 5 ml. of 10% NaOH solution. Boil the mixture for i minute, cool and acidify with dilute... 

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