THE ACTION OF " I T S ACID UPON A'MTT)lW ETC. 2651 CCCLXI1.-The Action of Nitrous Acid u p Am& and Other G6 Amino "-mlXyund8. By ROBERT HENBY ADERS PLIMXEB. THE action of nitrous acid upon amino-compounds WM apparently k t used by Sachsse and Kormann (LmuZw. Vw&-Stat. 1874, 17 321) for the detection and estimation of these compounds in plant extnrots. H. T. Brown (Trans. Quinm Bes. Lab. 1903) 4 ~ * 2662 PLlMMER THE AmON OF NlTROUS ACID employed the reaction for studying the formation of amino-acids in the brewing process and improved the old type of appmtus. Not until Van Slyke ( J . BbZ. C h . 1911 9 185; 1912 12 275) devised a satisfactory apparatus for the estimation was the method extensively used and as is well known its chief use is for d i s h -guhhing the Merent forms of nitrogen contained in the amino-acids resulting from the hydrolysis of proteins.A special short analpis of proteins oan be made by the method of Van Slyke (J. Bid. Chem. 1911 10 15). The earlier workers tested only a few amino-acids; leucine and alanine gave off the whole of their nitrogen as nitrogen gas asparagine gaxe off only half of its nitrogen as gas. Van Slyke tested a con-siderable number of amino-compounds. All a-amino-acids reacted rapidly; presumably therefore the a-amino-group but not the amide group of asparagine reacted. The nitrogenous groups of guanidine and creatine did not react nor the guanidine group of arginine. The e-amino-group of lysine reacted slowly as also the amino-groups of certain purines. Dunn and Schmidt ( J .Biol. Chem., 1922 53 4-01) and Wright Wilson ( J . Biol. Chem. 1923 56 183) have further studied these slow reactions. Urea was found by Van Slyke to react slowly with sodium nitrite and acetic acid. Werner (J. 1917 111 863) working under different conditions, found no reaction in the presence of acetic acid but obtained an evolution of nitrogen in the presence of mineral acid. He found that the reaction was never complete and considered that the method was of no value for estimating urea. Krall (J. 1915 107 1396) observed an evolution of nitrogen from guanidine in the presence of mineral acid and Wright Wilson (bc. cit.) obtained nitrogen from creatinine using the Van Slyke apparatus. No other reference to the reaction of nitrous acid with " amino "-compound8 has been found on looking through the literature except the statement in the text-book of Organic Chemistry by Meyer and Jacobson (1907, I part 1) that amides give off nitrogen in the presence of strong sulphuric acid.These experiments were therefore undertaken t o find out the con-ditions under which amides guanidine and other amino-compounds react with nitrous acid and to reconcile the various discrepan-cies in the previous results. Some other amino-compounds besides the above-mentioned were also tested. EXPEBIMENTAL. The large form of apparatus described by Van Slyke was used in all the experiments. After the removal of air from the apparatus the acetic acid+mdium nitrite mixfure was always brought to th UPON AMJDES AND OTHEB "AMINO "-COMPOUNDS.2653 same level in the reaction chamber; its volume measured 12 C.C. Generally 5 c.c. sometimes 10 c.c. of the solution of the compound under investigation were delivered from a pipette into the burette of the apparatus and run into the reaction chamber; the burette was then washed twice with 1 C.C. of water and the washings were run into the apparatus. Reaction was dowed to proceed for p e r i d varying from 1 to 24 hours; during the short periods the appamtns was continuously shaken during the long periods it ww &&en for the last half-hour. If the volume of gas evolved tended to fdl the gas burette the gas was passed into the permanganate vessel for absorption of nitric oxide and not returned to the burette until the reaction time was completed.In the experiments with mineral acid concentrated hydrochloric acid was added 1 C.C. at a time in the same way aa the water used in washing the burette and in its stead. After the addition of 3 C.C. of hydrochloric acid gas evolution was very rapid still more rapid after 4 C.C. In these cases the evolved gas was quickly passed into the permangamte vessel and further quantities were added only when the rate of gas evolution diminished. The rapid gas evolution usually ceased after 5 to 10 minutes and became slow after 1 to 2 hours so that the apparatus could be safely left for the long reaction periods amounting to 24 hours. The evolved nitrogen gas was hally measured and by the use of Van Slyke's table its amount was con-verted into mg. ofmitrogen. Usually 1% solutions of the compound under examination were used.The total amount of nitrogen in these solutions was determined by a Kjeldahl estimation in another aliquot portion. All the figures of the estimations were calculated to g. of nitrogen per 100 C.C. of solution. The results of the experi-ments are in the following tables. D~cussion of R d . Amidm.-The experiments with the above amides show clearly that these do not give off an appreciable quantity of nitrogen with nitrous acid in the presence of acetic acid in 24 hours and are thus sharply distinguished from the simple amino-acids which were shown by Van Slyke to react rapidly and completely in from 5 to 30 minuW. The diITerence in behaviour is clearly shown with asparaghe; only one amino-group reacts in a period of 23 horn under these conditions.On introducing hydrochloric acid to the reaction mixture com-plete reaction of the amide group did not occur until 5 C.C. of con-centrated acid had been added. This amount of acid gave a con-centration of approximately 2N-HC1. In the caae of asparaghe, the whole of its nitrogen was then evolved as gas 2654 PIXMMEB THE AOTION OF NITaOlJS ACID Fomumide. Ezpt. 1. Tdal N per 100 C.C. = 0.2884 9. A m i h . Acetumide. EX@. 1. TOW N per 100 C.C. = 0.1512 9. Nepo*ped %Z N evohed Time @per % o f Time @. Per With (hours). Temp. 1OOkc.). htal N. With @cars). Temp. 1OO.c.c.). N. 2 C.C. H,O 1-5 15' 0.0088 3-1 ,) , 2.5 16 0.0080 2.8 , , 24.5 16 0.0092 3.2 Expt. 2. 2 C.C. IfCl 19 11 0.0191 10.6 4 , , 24 16 0-0923 51.1 6 , , 6 18 0.1609 89.1 6 , ,) 19 14 0.1427 79.0 6 , ,) 22 15 0-1805 100.0 Total N per 100 C.C.= 0.1806 9. Expl. 3. Total N per 100 C.C. = 2 C.C. K&O 26 17 0.0162 2 , HC1 24 16 0.0234 3 , , 24-5 16 0-0228 3 ,) , 24 15 0.0344 4 , ,) 22 14 0.0604 5 , , 22 13 0.1415 6 , , 23 13 0-1732 7 , , 26 12 0-1882 7 , , 24 13 0.2139 0.2240 g. 7.2 10.3 10.2 15-4 27.0 63.2 77.3 84.0 95.5 Propknamide. TO^ N p 50 C.C. = Ezpt. 1 . 2 C.C. H,O 0.5 13 0-0006 0.5 2 , ) 2 14 0.0017 1.5 2 , , 17.5 14 0.0039 3.4 2 ,) y y 46.5 13 0-0037 3.2 Ex@. 2. 2 C.C. H,O 23 11 0-0014 0.7 2 , HCI 25 11 0.0156 8-0 3 , , 23 11 0.0154 8-0 4 , , 23 12 0.1491 76.6 5 , , 22 12 0.1967 101.0 0.1155 9. Total N per 100 C.C. = 0.1946 g.2c.c. H,O 1 12" 0-0011 0.7 2 , , 4.5 13 0*0011 0.7 2 , ,) 17.5 11 0.0011 0.7 Expt. 2. Total N per 100 C.C. = 2c.c.H,0 23 17 0.0032 2 , HCI 24 17 0-0082 3 ,) , 24 17 0.0360 4 ) , 5 17 0.0644 4 , , 8 16 0.0696 4 , , 12 17 0.0882 4 ,) , 25 17 0-1042 5 , , 6 17 0.0964 5 , ) 17 17 0.1112 5 , , 24 18 0.1185 0.1148 9. 2-8 7.1 31-3 56.1 60.6 76-8 90-1 83-9 96.9 103.2 Aapuragine. Expt. 1. 2 C.C. H,O 0-25 17 0.1039 49.5 2 ,) , 0-5 15 0.1043 49-7 2 , , 1-0 15 0.1060 50.6 2 , , 3-5 16 0.1093 53.0 2 ) , 17-5 13 0.1112 53.0 Expt. 2. 1 C.C. HCl 1 18 0.1061 60.5 2 , , 2.0 18 0.1079 57.4 3 , , 18-0 15 0.1135 54-0 5 , , 7-0 14 0.1615 76.9 7 , , 23-0 16 0.2209 105.2 8 , ) 23 15 0.2142 102.0 Expt. 3. 2 C.C. H,O 23-5 13 0.1699 54.5 2 , HCl 23 13 0.1842 59.1 3 ., , 23 13 0.1917 61.5 4 , , 23 12 0.1881 59.7 5 , , 23-5 13 0.3014 96.7 6 ) , 23.6 15 03264 104.4 TOW N per 100 C.C.= 0.2100 g. Total N per 100 C.C. = 0.2100 g. Total N per 100 C.C. = 0.3115 9 UPOH AMIDES AND OTHER " AMINO "-COMPOUNDS. 2655 EX@. 1. Total N (pet 100 C.C. = 0.2866 9. N evolved Time @.per %of 2c.c.H20 0-5 11" 0.0164 5.7 2 , y 1.0 11 0.0329 11.5 2 ,) ,y 2.5 12 0.0958 33-5 2 , ,y 3.5 12 0.1652 54.3 2 ) , 6.5 12 0.2198 77.0 2 , ,) 8-5 11 0.2315 81.1 2 , , 15.0 11 0.2657 93.0 2 , , 24.5 10 02767 96.9 With (hours.) Temp. 100 c.c.). totel N. Ezpt. 2. 2 C.C. HzO 8-5 20 0.2512 103.7 2 , , 23.0 18 0.2495 103.0 2 , , 47.0 19 0.2456 101.4 Total N v 100 C.C. = 0.2422 9. Expt.1. TOW N p 100 C.C. = Time @Per tdJbl With (hours). Temp. 100c.o.). N. 2 C.C. H,O 1 16O 0-0023 1-3 2 , , 3-5 16 0.0061 3.5 2 y y ) 23-5 8 0.0165 9.4 0-1764 9. N evolved yo of Expt. 2. 2c.c. H,O 23 15 0.0568 30.5 Total N per 100 C.C. = 0.1862 g. 14 0.0929 49.9 1 y HCl 2 , HCI 24 12 0.1137 61.1 3 y ) 23.5 14 0.1309 70-2 4 , , 23 14 0.2143 116.1 Expi. 1. 2 C.O. H,O 0.5 14 04081 12.3 EX@. 1. TOM N 100 C.C. = 2 ,y , 1.5 15 0-0080 12-2 2c.c. H,O 1.0 11 0.0117 4.1 2 ) ,) 17-5 14 0-0132 20.1 2 , , 5.5 11 0-0326 11.3 T ~ k d N v 100 C.C. = 0-0658 9. Biuret. 0.2884 9. 9' '3 16.5 1' 8E :::! E q t . 2. Total N per 100 C.C. = 2 Y 9 40 0.1904 g. EX@. 2. 2 c.c.H,O 6 21 0.0566 2 , ) 17 19 0-0708 2 Y 23 22 0.0763 2 , HE1 24.5 21 0.1035 3 y ,y 23 22 0.1116 4 , yy 24 20 0.1439 5 , , 23 19 0-1411 6 .,) 24 17 0.1448 Total N pt?? 100 C.C. = 0.1551 g. 2 C.C. H,O 0.5 14 0.0161 8.4 2 ) ) 2-6 15 04304 16.0 2 , , 4-5 15 0-0320 16.8 36.4 2 ,) ,) 16.5 14 0.0515 27.0 49-3 2 ,y ) 4-5 15 0.0714 37.5 45.6 ;;:; Ex@. 3. Tdal N p 100 C.C. = 0-1218 9. 92-6 90.8 2 C.C. Hpo 25 16 0.0689 58.6 93.3 2 , HCl 23 22 0.0764 62.7 2 ) , 23.5 17 0.0613 60.3 3 , y 24 18 0.0595 48.8 3 , , 23 19 0.0463 37.2 4 ) , 24 17 0-2037 167-2 5 , , 24 18 0-2817 231.3 6 , , 23 20 0.2859 234. 2666 PIJMMER !FHE AC!CION OF NITaouS ACID Guanidine and Derimfiues. Expt. 1. TofalNper100c.c. = 0.1316g. Expt.l. TotalNperlOOc.c.=0*2590g. N evolved N evolved % of Time Time @.per total 2 C.C. H,O 1.0 15" 0.0029 2-2 2 C.C.H,O 0-5 12" 0.0177 6.8 2 , ,) 3.5 15 0-0016 1.2 2 , ,) 1.5 11 04465 17.9 2 , , 5.5 15 0.0029 2-2 2 , y y 2.5 14 0.0510 19.7 2 , ) 15-5 11 0-0060 4.6 2 ,) , 17.5 11 0.0608 23.5 2 ,) yy 40.5 14 0.0104 7.9 2 , )) 24-5 15 0-0617 23.8 E:E:E:E:E:E:E:E:E:E:E:~,~. TotulNper lOOc.c.=O-3185 g. 9' " 2575 l6 0.0659 25*4 2 )) , 48-5 11 0-0709 27.4 2 C.C. H,O 25 13 0.0319 10.0 2 ,) ) 50 15 0.0777 30.0 2 , HCI 24 11 0.0639 20-1 Guanidine Carbonate. Aminogwznidinc Acetde. With (hours). Temp. 106 ' per c.c.). t~&lN. ' Of with @oars). Temp. 100c.c.). N. ; : i ii t:ig; ;;: Expt. 2. Total N per 100 C.C. = 0.3402 9. 6 , , 24 10 0-2553 80.1 7 ,) , 24 13 0.2784 87.4 2c.c. H,O 24 13 0.0731 21.5 2 HC1 24 13 0.1052 30.9 Ezpt.3. TotalNper 1OOc.c. = 0.2968g. 3 : ) 24 12 0.1067 31.4 2 C.C.HzO 24 10 0.0104 3-5 4 , , 24 12 0-1855 54.5 2 , HCI 23.5 9 0.0368 12.4 5 , , 24 12 0.2750 80.8 3 , y9 23.5 10 04437 14.7 6 ,) , 25 14 0.3112 91.5 4 1 23.5 11 0.0826 27.8 7 , , 24 16 0.3168 93.3 5 19 9) 25 11 0.2132 71.8 8 , 24 16 0.3154 92.7 6 , ) 24 12 0.2420 81.5 7 y y , 23.6 14 0-2622 88.4 8 ,) ) 23 14 0.2688 90-5 Arginine Carbonate. Expt. 1. Total N per 100 C.C. = 0.1078 g. 2 C.C. H,O 0.5 16 0-0267 24.8 2 )) ) 1 16 0-0294 27.3 2 ., , 1 15 0-0280 26.0 2 )) , 1 16 0.0267 24-8 2 , ,) 2 14 0-0280 26.0 2 ) , 4 14 0.0304 28-2 2 Y 21-5 17 0.0504 46.7 2 , HG 4 19 0.0402 37-3 2 )) ) 26 16 0.0698 6 4 7 2 , , 30 16 0.0680 63-1 Expt. 1. Totul N per 100 C.C. = 0.07 g. ~ c . c . H,O 1 15 0.0018 2.6 2 ,) , 3.5 15 0-0011 1.6 2 , , 4.0 15 0-0023 3-3 2 , , 18-5 15 0.0045 6-4 Creatinc.,2. Totd N per 100 C.C. = 0-1456 g. H,O 23.5 14 0.0231 15.9 HCI 25 14 0-0394 27.0 ,) 24 14 0-0494 33.9 , 24 16 0.0483 33.2 , 24 13 0.0574 39.4 ) 24 14 0.0967 66-4 , 24 15 0.1081 74-2 , 24 14 0.1008 69.2 Arginine carbonate. Total AT per 100 C.C. = Expt. 2. 2 C.C. H,O 0.5 20 0-0212 17.5 2 , ,) 1.0 19 0.0266 22-0 2 9 Y 24 13 0-0566 46-7 2 ,) He1 24 14 0.1044 86-2 3 ) 24-5 13 0.1006 83.1 4 ) ) 24.5 14 0.1118 92.3 6 ) , 24 15 0.1173 96.9 7 ) , 25 19 0.1167 964 8 , , 24 17 0.1237 102-1 0.1211 9. Creatinine. Expt. 1. Total N per 1OOc.c. =0*2898 g. 2 C.C. H,O 1 15 0%766 22.9 2 , ) 4 17 0.1027 35-4 2 , , 15.5 15 0.1075 37-1 2 .. .. 23 17 0.1164 40.1 19 17 13 14 16 14 17 15 14 0.1211 0.1208 0.1221 0.0886 0.0540 0-0646 0.0478 0-0506 0-0455 41.8 41.7 42-1 -30.6 18-6 22.3 16.5 17.5 15.7 Amnumiurn acetate.Hydrazine mdphtc. T0ta2 N per 100 C.C. = 0-3304 9. 2 C.C. H,O 0.5 13 0.1061 32.1 2 C.C. H,O 0.5 13 0.0641 111.7 2 , , 2-5 16 0.3385 102.4 2 , , 4.5 13 0.1424 258.5 2 , ) 18.5 14 0.3408 103.1 2 ) ) 17.5 14 0.1573 274-0 Total N per 100 C.C. = 0.0574 9 UPON ABIDES AND OTHES " AMINO "-COMFOUNDS. 2667 The formation of nitrogen in the presence of hydrochloric acid is not due to hydrolysis of the amide. Experiments were made fo fest this possibility by alloffing 20 C.C. of the amide solution to stand for 24 hours with 6 C.C. of concentrated hydrochloric acid. The solution was then rendered alkaline with sodium carbonate and any ammonia produced estimated by the eration method of Polin.Acetamide gave 26.2 propionamide 26-3 aspamgine 4.9%. Formamide was apprently completely hydrolysed with the forma-ation of 96.2% of ammonia. If the reaction of amino-acids with nitrous acid ih acetic acid solution is taken as an indication of the presence of a primary amino-group the difference in the behaviour of amides should be represented by giving amidea the alternative formula R*C(OH):N€€ which may be regarded as being converted in the presence of mineral acid into the more usual formula R*CO*m which shows the presence of an -NK2 group. This alternative formula is supported by the form-ation of unstable salts of amides which are decomposed by water. Urea and Derimtives.-Van Slyke has stated that urea reacted slowly with nitrous acid in the presence of acetic acid.These results show that at low temperatures (from 10" to 12") the reaction is not complete but that complete decomposition occurs at 18" In 20". Werner under different experimental conditions did not obseme complete reaction and attributed the incompleteness to the formation of ammonium salts. As seen from the experiment with ammonium acetate it is completely decomposed in 24 hours. The difference in the results seems to be due to the length of time of the reaction. As urea was decomposed in the presence of acetic acid no experiments were made in the presence of hydrochloric acid. On comparing the results with those of amides it appears that urea possesses the alternative formula HN=C<? which changes mzl in presence of acids to HN=CLNH, /OH aa proposed by Werner.The substance with the latter form& showing an -NH p u p would be attacked by nitrous acid; imyanic acid which is eady hydrolysed to ammonium carbonate would also yield nitrogen. This alternative formula for urea is supported by the behaviour of semicarbazide and urethane. One-third of the nitrogen of semi-carbazide was obtained as gas in the presence of acetic acid rather more in the presence of 2 or 3 C.C. of hydrochloric acid. One of the three nitrogen atoms would thus be present as an - group. In presence of more hydrochloric acid large volumes of gas were evolved suggesting that hydrazine was formed by decomposition. Hydrazine in another experiment was observed to produce larg 2658 PLTMMEB TEE A W O N OF NITROUS ACID volumes of gas probably resulting from reduction of nitrous acid by hydrazine.Urethane behaved like the simple amides no evolution of nitrogen in presence of acetic acid but complete reaction in presence of hydrochloric acid. It would thus appear to have the alternative formula OEt.C(OH):NH which changa into OEtCO-NH in presence of mineral acid. The behaviour of biuret with nitrous mid is most easily explained by Werner's formula NH:C(OH)*NH*C(OH):NH. In presence of acetic acid this would change to NH:C(OH)mNEl-CO*NH with liberation of one-third of its nitrogen as gas as found by experi-ment ; in the presence of 2 to 3 C.C. of hydrochloric acid the formula would become N€&-CO-NH*CO*NH ; two-thirds of the nitrogen was given off.In presence of 4 to 6 C.C. of hydrochloric acid the whole of the nitrogen was evolved indicating that the molecule was completely broken down. Ghunidine and Derivatives.-Guanidine reacted only slightly in presence of acetic acid two-thirds of its nitrogen was given off in presence of 5 C.C. of hydrochloric acid and the reaction was nearly complete in presence of 8 C.C. of hydrochloric acid in 23 hours. The alternative formula HN=C<yG proposed by Gall is indicated for guanidine. This changes to the usually adopted formula NH:C(NH,), in presence of mineral acid which explains the liberation of two-thirds of its nitrogen in presence of hydro-chloric acid. Arginine behaved in a similar way to guanidine.Only the a-amino-group reacts with nitrous acid in presence of acetic acid. This reaction is used in its analysis. An excew of nitrogen over one-third was found by Plimmer (Bimhem. J. 1924 18 105) if the reaction were prolonged. The whole of its nitrogen is given off as gas in the presence of hydrochloric acid. Aminoguanidine gave off one-quarter of its nitrogen &s gas in presence of acetic acid but larger quantities in presence of hydro-chloric acid. Corresponding with guanidine the whole of the nitrogen was not evolved as gas. Creatine reacted in a similar way to guanidine and appeam to have an alternative formula such as C02H*C€&*NMe*C<xH3, which changes to the usual formula CO,H~CH,=NMe*C(:NEl)*~, in presence of hydrochloric acid. A volume of nitrogen was evolved corresponding to two nitrogen atoms.The third nitrogen atom to which the methyl group is attached would not be expected to yield nitrogen. Creatinine which was also found by Wright Wilson to N UPON ~ E S AND cmnm “ ~ O ” - C O M P O U N D S . 2659 react with nitrous acid in presence of acetic acid showed an un-expected behaviour indicating the presence of an amino-group. It would thus appear to have an alternative formula such as (I) insw of (II). N NH /\ m:(( yo (11.1 A-(1.) Nlq ((0 M e L q Me3T-W The effect of mineral acid in diminishing the volume of nitrogen evolved may be due to a change of the new alternative formula to the commonly adopted one. The formation of the smaller amounts of nitrogen in the experiments may be due to the method of adding the hydrochloric acid I C.C.at a time; a certain volume would be liberated before the whole of the 6 or 8 C.C. could be introduced. Summary. 1. Amides and urethane do not react with nitrous acid in presence of acetic acid. 2. Both react quantitatively in presence of approximately 2N-hydrochloric acid. 3. Urea reacts quantitatively with nitrous acid in presence of acetic acid. 4. Biuret reacts with one nitrogen atom in presence of acetic acid, with two nihgen atoms in presence of small amounts of hydrochloric acid with three nitrogen atoms in presence of 2N-hydrochloric acid. 5. Guanidine and creatine do not react with nitrow acid in presence of acetic acid but give off nitrogen in presence of hydro-chloric acid. Arginine excepting its primary a-amino-group behaves in a similar way. 6. Creatinine gives off nitrogen corresponding to one nitrogen atom with nitrous acid in presence of metic acid; the volume of nitrogen evolved is dimirriclhed in presence of hydrochloric acid. 7. If nitrous acid in presence of acetic acid is a reagent for the presence of an -NH group amides and the other compounds investigated wiU possess alternative formulae which in presence of hydrochloric acid change to the usually accepted formulae for these compounds. The author d e s h to express his thanks to the Government Grant Committee of the Royal Society for a grant out of which the expenses of this remarch have been defrayed. ST. THOU’S HOSPITAL MEDICAL SCHOOL, LONDON. [Rmeiued Aecgzcat 5& 1925.