Analytica Chimico Acta. 93 (1977) 349-352 3 Ekevier Scientific Pcblishing Company. Amsterdam - Printed in The Netherlands

Short Communicat~n

COLORMETRIC DETERMPL4TION OF AMINES A?.‘D A!ilIXO ACIDS

JOHN ELLIS’. ASTHONY M. HOLLAND and RHONDA A. HOLLAND

Department o/Chemistry. L’nitvrsity of Wollongong. N.S.W. 2500 (Austmlia)

(Received 30th March 1977)

Nitrous acid reacts rapidly to conve ? the amino group of amino acids to a hydroxyl group: the volume of nitrogen liberated is used in the Van Slyke method [l] to estimate the number of primary amino groups present_ This reaction was extended t.o the determination of primary amino groups in aromatic amines, amides and sulphonamides f 21. Nitrous acid also reacts with secondary amines and N-monosubstituted amides to give N-nitroso derivatives, but without evolution of nitrogen.

By treating the amino compound with a much smaller escess of nitrous acid than that normally used, the unreacted nitrite can be determined calorimetrically and hence the amino compound determined by difference. The two major potential sources of error are loss of nitrogen oxides to the atmosphere, and disproportionation of nitrous acid to nitric oxide and nitric acid with further aerial oxidation of nitric oxide to nitrogen dioxide. Both these sources of error may be eliminated by conducting the reaction in a sealed system under reduced pressure and absorbing the evolved fumes in sodium hydroxide solution: the apparatus involved has been described [3]. Any nitrate ion formed by aerial oxidation is reduced to nitrite ion with copperized cadmium and the total excess of nitrite ion is determined colorimetricaily by reaction with SulphaniIamide and N-(l-naphthyl)-ethylene- diamine [4].

Experimen tat Compounds. Commercially available amines and amino acids were checked

for purity by g.c.-mass spedrometry. All reagents were analytical-reagent grade. Sodium nitrite solution was prepared by dilution of a stock solution prepare&weekly by dissolving ‘i-500 g of sodium nitrite in 100 mi of distilled deionized water. Copperized cadmium was prepared from Merck coarse cadmium powder 1.5 I-

Equipment. Volumetric glassware was all Grade A. The diazotization apparatus consisted of a glass-stoppered (B24) 5 X 2-cm test tube with the extended ends of two 10-ml burettes sealed through the glass stopper; the tips of the burettes were angled away from each other. The vessel is similar to that~described preliiousiy (31 but without the side-arm. A Gary spectra- photometer was used.

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Procedure. Place an aqueous solution of the amino compound (0.5 ml; ca. 0.03 mmol), prepared by successive diluticn of a 2 mg ml-’ stock solution in 1 M HC!, in the reaction vessel. Add hydrochloric acid-sodium acetate buffer (1 ml; pH 3.6), close the burette stopcocks and insert the burette assembly into the reaction vessel. Place sodium nitrite solution (1.0 ml; 6 mg ml-‘) in one burette and create a partial vacuum by cooling t.he reaction vessel in ice. Open the burette tap briefly to admit the nitrite solution. Then place sodium hydroxide solution (1.5 ml; 8 M) in the other burette. Keep the mixture at 0-4°C for 45-60 min, depending on the amino compound used, and then warm to ca. 40°C by immersion in hot water for lo-15 min to complete the reaction. Again cool the vessel in ice and admit small portions of sodium hydroxide solution from the second burette. When all the alkali has been added, sfiake the contents of the vessel to absorb acidic nitrogen oxides. Then wash residual sodium nitrite solution from the fast burette into the reaction vesse! with small portions of dilute sodium hydroxide solution. Transfer the alkaline soIution to a volumetric flask, add cit,ric acid solution (5 %I; 4 ml) to adjust the pH to ‘i- 8, and EDT-4 solution (4 ml; 0.1 XI), and dilute the solution to the mark.

Allow a 50-ml aliquot of this solution to percolate slowly through a column (20 cm X 1 cm) of copperized cadmium, and wash through with 3 X 50-ml portions of distilled water into a 250-ml volumetric flask. Dilute to the mark with distilled water, and analyze a lo-ml aliquot colorimetiically by the sulphanilamide-N-(1-napthyl)&hylenediamine method 141. After suitable dilution, measure the absorbance at 543 nm with a sodium ni’trite blank worked up in exactly the same way as reference.

Resuits and discussion Scope and hitations of method. The method is applicable to primary and

secondary amines and amino acids (Table 1). Primary and secondary amines are not distinguished but the method could be used in conjunction with the direct spectrophotometric determination of secondary amines as their nitroso derivatives [ 61.

The optimum pH for the diazotization of amines has been studied [T] ; the reaction rate declines markedly below pH 3. At the low pH necessary to achieve rapid diazotization of amides (6 M sulphuric acid IS]), amines are fully protonated and therefore unreactive. Hence, by appropriate adjustment of the pH used for the diazotization step, the procedure can be used to determine amines in the presence of amides and vice versa.

The reaction is not seriously affected by steric hindrance and gives better results than the Van Slyke procedure for “anomalous” amino acids; for e-=mple, glycine gives a quantitative result whereas the Van Siyke method leads to the formation of nitrous oxide and nitrogen 19, lo]. The production of nitrous oxide has been attributed ti the formation of :I nitrolic acid inter- mediate 1111, especially at high temperature and high nitrite concentration. The present method uses a low sample conczntzation and orJy a moderate

353

TABLE 1

Analysis of amines and amino acids

Amino compound’

n-Butylamine i-Butybmine t-Butylamine

Reaction &mple mass Recoveryb time (mm) (mtz!) (%‘c)

40 1.8 96.7-101.6 60 3.7 98.3-100.5 60 1.7 97.7- 99.4

Piperidine 60 2.1 97.7-106.5 Dicyclopentylamine 60 3.2 98.1-100.9

Glycine 40 3.0 99.8-101.0 1.0 99.7-103.0

.Blycylglycine 50 4.0 98.8-102.0 Phenylalanine 50 2.0 100.5-103.5 Lmucine 50 4.0 96.3-102.0 Tyrrosine 15 4.0 99.5-101.0

t-Butylamine 60 1.7 98.8- 99.4 (t 4 mg isobutymmide) Isobutymmide 15 1.0 96.0-103.0 (+ l-7 mg t-butylamine)

‘Amines and amino acids diazotized at pH 3.5-4, and amide groups in 6 11 sulphuric acid. bThree determinations of each amino compound.

excess of nitrous acid; the probability of reaction of the intermediate with water is therefore enhanced relative to its reaction with nitrous acid.

The principal classes of interfering substances are as for the Van Sly& procedure [12]. Positive interference may be expected from isonitroso and active methylene groups and from monohalogenocarboxylic acids, phenols, sulphur compounds oxidizable by nitrous acid, indoles and oxindoles, and s lactams. Negative errors will arise for amines which ate difficult to d;?otize or dissolve. The Van Slyke procedure has two additional sources of negative error: amines which give relatively stable diazonium salts and side-reactions of the intermediate diazonium salt. Bromine-acetic acid has been used to block the interference of active methylene compounds, phenols and sulphur compounds [121. Treatment of a tyrosine solution with a few drops of bromine water before the diazotization gives quantitative results (Table 1).

Inorganic ions which can reduce nitrite interfere, but minor concentrations of osidizing inorganic ions do not: any nitrate produced is reduced to nitrite by the cadmium column_ High concentrations of metal ions could interfere with the function of the EDT-A, which complexes the Cd*’ rekased by oxidation of the cladmium [Sl .

Sensitivity and precision_ The precision was studied by performing five replicate determinations at each of two different concentration levels with phmy&he and t-butylamine (Table 2). When all the manipulations of

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TABLE 2

Effect of sar$ple mass on precision

Phenylalanine (mg) 2 0.2 t-Butylamine (mg) 2 0.2 +(%,n=5! 2.1 2.7 s,(%, n = 5) 1.1 5.0

the procedure are taken into account, solutions containing as little as 0.017 mg of amino nitrogen can be determined with a relative standard deviation of 2.7%. Bemuse the method is bzsed on comparison with tie absorption of a blank subjected to the entire procedure, errors associated with nitrate impurities in zhe nitrite reknent and uptake of nitrogen oxides from the atmosphere are minimized. The sensitivity depends on the absorptivity of the azo dye used to measure nitrite concentration. With lo-cm cells, nitrite concentrations as low as 14 pg N 1-l have been measured [5] with a relative standard deviation of 4%.

Conclusions. This procedure affords a sensitive and accurate method for determiningprimary and secondary amines and amino acids. The apparatus is simpler to construct and use than the Van Slyke apparatus. It avoids the chromophoric substituent effects associated with direct spectrophotometric determination of the derivatives formed by 2,4-dinitrofluorobenzer.e [ 131 or 2,4.6-trinitrobenzenesulphonic acid 1141 and givesa stable colour in contrast to that given by ninhydrin [151_

One of us (A.M.H.) acknowledges the support of a Commonwealth Postgraduate Award.

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