LIBRARY, A & M corrace, CAMPUS. A233-1 131-6,500-L180 TEXAS AGRICULTURAL EXPERIMENT STATION A. B. CONNER, DIRECTOR College Station, Brazos County, Texas BULLETIN NO. 439 NOVEMBER, 1931 DIVISION OF CHEMISTRY Estimation of Nitric and Nitrous Nitrogen in Soils AGRICULTURAL AND MECHANICAL COLLEGE OF TEXAS T. O. WALTON, President STATION STAFF? ADMINISTRATION: A. B. CONNER, M. S., Director R. E. KARPER, M. S., Vice-Director CLARIcE MIxsoN, B. A., Secretary M. P. HOLLEMAN, JR., Chief Clerk J. K. FRANcKLOw, Assistant Chief Clerk CHEsTER HIGGs, Executive Assistant HOWARD BERRY, B. S., Technical Assistant CHEMISTRY: G. S. FRAPS, Ph. D., Chief; State Chemist S. E. AsBURY, M. S., hemist J. F. FUDGE, Ph. D., Chemist E. C. CARLYLE, M. S., Assistant Chemist T. L. OGIER, B. S., Assistant Chemist A. J. STERGEs, M. S., Assistant Chemist RAY TREIGHLER, M. S., Assistant Chemist W WALKER, Assistant Chemist VELMA GRAHAM, Assistant Chemist JEANNE F. DEMOTTIER, Asst. Chemist R. L. SCHWARTZ, B. S., Assistant Chemist C. M. PoUNDERs, B. S., Assistant Chemist HORTICULTURE: S. H. YARNELL, Sc. D., Chief **L. R. HAWTHORN, M. S., Horticulturist H. M. REED, M. S., Horticulturist J. F. WOOD, B. S., Horticulturist L. E. BRoOKs, B. S., Horticulturist RANGE ANIMAL HUSBANDRY: J. M. JoNEs, A. M., Chief B. L.WARwIcK, Ph.D., Breeding Investigations S. P. DAvIs, Wool Grader ENTOMOLOGY: F. L. THOMAs, Ph. D., Chief: State Entomologist _ . J. REINHARD, B. S., Entomologist . K. FLETGHER, Ph. D., Entomologist . L. OWEN, JR., M. S., Entomologist . RONEY, M. S., Entomologist _ . GAINEs, JR., M. S., Entomologist . JoNEs, M. S., Entomologist . BIBBY, B. S., Entomologist . CLARK, B. S., Entomologist . DUNNAM, Ph. D., Entomologist . MORELAND, B. S., Asst. Entomologist . HEARD, B. S., Chief Inspector DDALL, B. S., Foulbrood Inspector . MGGREGOR, B.S., Foulbrood Inspector NOMY: . REYNoLDs, Ph. D., Chief . KARPER, M. S., Agronomist _ . MANGELsDORF, Sc. D., Agronomist . KILLOUGH, M. S., Agronomist REA, B. S., Agronomist . C. LANGLEY, M. S., Agronomist BLICATIONS: . D. JAcKsoN, Chief 213E ma. D102 1m '11 é Fiji/J 8% {'11 pow E “u: 0mm w "U >¢w;Uwwm menww SUBSTATIONS No. l, Beeville, Bee County: R. A. HALL, B. S., Superintendent No. 2, Lindale, Smith County: _ P. R. JOHNSON, M. S., Superintendent _ **B. H. HENDRIcKsON, B. S., Sci. in SoilErosion **R. W. BAIRD, B. S., Assoc. Agr. Engineer No. 3, Angleton, Brazoria County: . . STANsEL, M. S., Superintendent H. M. REED, M. S., Horticulturist No. 4, Beaumont, Jeflerson County: . WYCHE, B. S., Superintendent **H. M. BEAGHELL, B. S., Jr. Agronomist No. 5, Temple, Bell County: _ HENRY DuNLAvY, M. S.. Superintendent C. H. ROGERs‘, Ph. D., Plant Pathologist H. E. REA, B. S., Agronomist S. E. WOLFF, M. S., Botanist . **H. V. GEIB, M. S., Sci. in Soil Erosion **H. O. HILL, B. S., Jr. Civil Engineer No. 6, Denton, Denton County: P. B. DUNKLE, B. S., Superintendent **I. M. ATKINS, B. S., Jr. Agronomist No. 7. Spur, Dickens County: R. E. DIcKsON, B. S., Superintendent B. C. LANGLEY, M. S., Agronomist No. 8. Lubbock, Lubbock County: D. L. JoNEs, Superintendent FRANK GAINEs, Irrig. and Forest Nurs. VETERINARY SCIENCE: *M. FRANcIs, D. V. M., Chief H. SGHMIDT, D. V. M., Veterinarian **F. P. MATHEws, D.V.M., M.S., Veterinarian W. T. HARDY, D. V. M., Veterinarian ———-————i, Veterinarian PLANT PATHOLOGY AND PHYSIOLOGY: J. J. TAUBENHAUs, Ph. D., Chie W. N. EzEKIEL, Ph. D., Plant athologist W. J. BACH, M. S., Plant Pathologist C. H. ROGERS, Ph. D., Plant Pathologist 5 FARM AND RANCH ECONOMICS: g L. P. GABBARD, M. S., Chief 1, W. E. PAuLsON, Ph. D., Marketing ‘ C. A. BONNEN, M. S., Farm Management **W. R. NIsBET, B. S., Ranch Management **A. C. MAGEE, M. S., Farm Management RURAL HOME RESEARCH: JEssIE WHITAcRE, Ph. D., Chief MARY ANNA GRIMEs, M. S., Textiles ELIZABETH D. TERRILL, M. A., Nutrition SOIL SURVEY: **W. T. CARTER, B. S., Chief E. H. TEMPLIN, B. S., Soil Surveyor A. H. BEAN, B. S., Soil Surveyor R. M. MARsHALL, B. S., Soil Surveyor **M. W. BEcK, B. S., Asst. Soil Surveyor BOTANY: V. L. CORY, M. S., Act. Chief S. E. WOLFF, M. S., Botanist SWINE HUSBANDRY: FRED HALE, M. S., Chief DAIRY HUSBANDRY: O. C. COPELAND, M. S., Dairy Husbandman POULTRY HUSBANDRY: R. M. SHERWOOD, M. S., Chief J. R. CoucH, B. S.,Asst. Poultry Husbandman AGRICULTURAL ENGINEERING: . P. SMITH, M. S., Chief MAIN STATION FARM: G. T. McNEss, Superintendent APICULTURE (San Antonio): H. B. PARKs, B. S., Chief A. H. ALEX, B. S., Queen Breeder FEED CONTROL SERVICE: F. D. FULLER, M. S., Chief JAMEs SULLIVAN, Assistant Chief S. D. PEARGE, Secretary J H. ROGERS, Feed Inspector K. L. KIRKLAND, B. S., Feed Inspector IS). REYNoLDs, JR., Feed Inspector ' E. J. . G. MOORE, Feed Inspector WILs0N, B. S., Feed Inspector WIcKEs, B. S., Feed Inspector No. 9, Balmorhea, Reeves County: J. J. BAYLEs, B. S., Superintendent No. l0, College Station, Brazos County: R. SHERWOOD, S., In charge L. J. McCALL, Farm Superintendent No. ll, Nacogdoches, Nacogdoches County: H. F. MoRRIs, M. S., Superintendent **N0. 12, Chillicothe, Hardeman County: J. R. QUINBY, B. S., Superintendent **J. C. STEPHENS, M. A., Assistant Agronomist No. 14, Sonora, Sutton-Edwards Counties: W. H. DAMERON, B. S., Superintendent , Veterinarian W. T. HARDY, D. V. M., Veterinarian O. L. CARPENTER, Shepherd **O. G. BABcOcK, B. S., Asst. Entomologist No. 15, Weslacoul-Iidalgo County: W. I-I. FRIEND, B. S., Superintendent S. W. CLARK, B. S., Entomologist J BAGH, M. S., Plant Pathologist . F. WOOD, B. S., Horticulturist No. l6, Iowa Park, Wichita County: C. H. McDOwELL, B. S., Superintendent L. E. BROOKS, B. S., Horticulturist No. 19, Winterhaven, Dimmit County: E. MORTENsEN, B. S., Superintendent **L. R. HAWTHORN, M. S., Horticulturist Teachers in the School of Agriculture Carrying Cooperative Projects on the Station: G. W. ADRIANcE, Ph. D., Horticulture S. W. BILsING, Ph. D., Entomology V. P. LEE, Ph. D., Marketing and Finance D. ScoATEs, A. E., Agricultural Engineering A. K. MAGKEY, M. S., Animal Husbandry *Dean School of Veterinary Medicine. J. S. IVIOGFORD, M. S., Agronomy F. R. BRIsoN, B. S., Horticulture . R. HORLAGHER, Ph. D., Genetics W i. H. KNOX, M. S., Animal Husbandry L. DARNELL, M. A., Dairy Husbandry TAs of November 1, 1931. **In cooperation with U. S. Department of Agriculture. A study of the changes in the nitrogen of the soil involved in the process of nitrification required study of the method for estimating nitrates in soils and cultures. Considerable discrepancies in the amounts of nitric nitrogen as measured by the colorimetric phenol-disulphonic acid method and by the Tiemann-Schulze and reduction methods, led to considerable study of the methods. The dilferences were found to be due to nitrites in considerable quantities in some of the cultures. Details of the alpha-naphthylamine method for nitrites were modified to render it suitable for estimating nitrites in soils or cultures of soils. The zinc ferrous-sulphate method for nitrates and nitrites was modified for use in testing the methods, the ammonia being finally estimated by titration or by nesslerizing. When allowance was made for nitrites, "the phenol-disulphonic acid method was found to be accurate and best suited to work on soils. Various details of ‘the methods were studied. Detailed descriptions of the methods are given. CONTENTS PAGEAP Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Amount of error due to reading of color in the phenol-disulphonic acid method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 The flocculating reagent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 _; Effect of calcium carbonate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 Number of washings required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 i The phenol-disulphonic acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Quality of phenol . . . . . . . . . . . . . . . . . .’ . . . . . . . . . . . . . . . . . . . . . . . . . . 1O The possibility of interference by organic matter . . . . . . . . . . . . . . . . . 11 Adaptation of the zinc ferrous-sulphate method to soils . . . . . . . . . . . . 12 Connecting bulb tubes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 The zinc ferrous-sulphate titration method . . . . . . . . . . . . . . . . . . . . 12 N esslerizing when quantity of nitrate is small . . . . . . . . . . . . . . . . . 14 " Estimation of nitrates in the presence of nitrites . . . . . . . . . . . . . . . . . 14 Detaileddescription of methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 16 The phenol-disulphonic acid method . . . . . . . . . . . . . . . . . . . . . . . . . . 16 Estimation of nitric nitrogen in soils and cultures of soils . . . . .. 16 The zinc ferrous-sulphate titration method . . . . . . . . . . . . . . . . . . . . 17 Zinc ferrous-sulphate N essler method . . . . . . . . . . . . . . . . . . . . . . . . . 17 Preparation of ammonia-free Water . . . . . . . . . . . . . , . . . . . . . . . .. 17 Preparation of N essler’s reagent . . . . . . . . . . . . . . . . . . . . . . . . . .. 17 Preparation of standard ammonium chloride solution . . . . . . .. 18 Operation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 Nitrite nitrogen in soils. ._ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 Qualitative test for nitrites in soils . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 Quantitative determination of nitrous nitrogen . . . . . . . . . . . . . . . . . . . 2O Summary. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 20 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 21 BULLETIN NO. 439 NOVEMBER, 1931 ESTIMATION OF NITRIC AND NITROUS NITROGEN IN SOILS G. S. FRAPS AND A. J. STERGES In connection with extensive investigations 0n nitrification being conducted at the Texas Agricultural Experiment Station, and on ac- count of irregularities, it was found necessary to study the methods for estimating nitrates in soils. As is well known, the phenol-disul- phonic acid method occasionally gives unreliable results, due to turbid 0r discolored solutions, presence of carbonates, chlorides, organic mat- ter, or other factors. Although the method has received considerable study, more work seemed to be necessary, in order to eliminate diffi- culties as far as possible. - The colorimetric method using phenol-disulphonic acid seemed to give incorrect results on some of the cultures secured in the nitrifica- tion tests. Wide differences were found between some of the results by the colorimetric method and by the Tiemann-Schulze method with some cultures of soils, secured in the- nitrification work. This cast doubt on the reliability of the colorimetric method and required a study of various details and a search for another method to use in checking the results of the colorimetric method, resulting in modifica- tions of the zinc ferrous-sulphate method. The agreement between the colorimetric method and the zinc ferrous-sulphate method was satis- factory on many samples or cultures, but large differences were found between the two methods on other cultures of soils. These differences were subsequently found (3) due chiefly to the presence of nitrites. On account of the need for a method for nitrites in soils, evident after We found large quantities of nitrites in some cultures of soils, the well known colorimetric method (1) for nitrites was modified to make it better adapted for use on soils. When the nitrites were estimated and allowed for, the results of the colorimetric method, which determines nitrates only, were found to check as well as could be expected with the zinc ferrous-sulphate methods, which include both nitrate and nitrite nitrogen. I Some of the results obtained in the course of the investigation out- lined are here presented, together with a detailed description of the methods finally used for the estimation of nitrates and nitrites in soils or cultures of soils secured in nitrification experiments. Studies of the phenol-disulphonic acid method have been made by Lipman and Sharp ('7), Chamot, Pratt and Redfield (2), Harper (5), Van Wijk (10), Whiting, Richmond and Schoonover (11), and others. Comprehensive references to the literature may be found in their papers. 6 BULLETIN NO. 439, TEXAS AGRICULTURAL EXPERIMENT STATION AMOUNT OF ERROR DUE TO READING OF COLOR IN THE PHENOL- DISULPHONIC ACID METHOD T0 ascertain the extent of the variations in the colorimetric method for nitrates, volumes of standard nitrate solution B (1 cc. equals .01 mg. nitrogen) varying from 8 to 25 cc., were measured out by one person and nitrates determined colorimetrically by another. Four series of 12 solutions each were run. The Duboscq colorimeter was used in three series, graduated cylinders in the other. In some cases, there were no differences. The differences found Were as follows (see also Table 1) : Series 1, -—.0 to 3.0, average difference 1.5 cc. (cylinders). Series 2, —— 1.5 to + .2, average 0.5 cc. Series 3, — 6 to 1.8, average difference 0.6 cc. Series 4, I 0.4 to 1.7 cc., average 0.7. Since the amounts taken averaged about 12 cc., the average error with the col- orimeter was 4 to 6 per cent of the total, while it was over 10 per cent for the cylinders. Some error must be, of course, expected in reading the color of the solutions. This error depends, to some extent, on the sensitivity of the eyes of the analyst to thegraduation of the color used. Table 1. Error in colorimetric reading for nitrates—Mg. nitric nitrogen Colorimeter Colorime ter Colorimeter Cylinders Used Found Used Foun Used Foun Used Found Mg. Mg. Mg. Mg. Mg. Mg. Mg. Mg. Average (12) . . . . . . . . . . . .. .125 .129 .121 .116. .124 .133 .119 .128 Average +or——dif............. +.009——.001 +.003—-.0O6 +.0O8 0 +.015——.009 Standard deviation . . . . . . . . . . . . . .008 . . . . . . .007 . . . . . . .009 . . . . . . .017 . . . . . . Another series of experiments was made with additions of the stand- ard solution to quartz sand. Varying quantities of the standard nitrate- solution were measured by one person and nitrates determined by the other. Lime was added, nitrates washed out, and the nitric nitrogen estimated by the colorimetric method described in full on a later page. The maximum difference in Series 1 was 1.0 part per million on 7.7 parts per million, or about 12 per cent; the average difference of the 11 was 0.1 on 7.5 parts per million, or a little over one per cent. In Series 2, the maximum difference was 3.3 parts per million on 25 parts per million, or 13 per cent; the average difference of the 10 was 1.5 per million on 28.8, or about 5 per cent, while the standard devia- tion was 2.7. In Series 3, the maximum difference was 3.5 parts per million on 26.5, or about 14 per cent; the average difference of the m, , ._. ~;Q.;>>h>.4b._m ._ U, . . . , ESTIMATION OF NITRIC AND NITROUS NITROGEN IN SOILS 7 l2 was 1.9 parts per million 0n 43, or about 4 per cent, with a standard leviation of 3.0 for the error. In Series 4 the maximum error was 4.3 Jarts per million on 34.0, or about 13 per cent; the average error of zhe 12 was 2.9 parts per millon on 43.1, or about 7 per cent, and the ;tandard deviation for the error 3.2 parts per million. The average error with the colorimetric method is about 5 per cent, while it may run up to 13 per cent occasionally. Thus an error of 20 parts per million may ordinarily occur on a nitrifying culture contain- ing 400 parts per million of nitric nitrogen. THE FLOCCULATING REAGENT As is known, it is sometimes difficult to secure clean filtrates from soils; a good fiocculating reagent is needed to.aid in securing a clear filtrate. The reagents most commonly used for this purpose are potash alum, alumina cream, carbon black, calcium hydroxide, and copper sul- phate. Some of these are satisfactory with some soils and unsatisfac- tory with others. Lipman and Sharp ('7) stated that potash alum had a tendency to give low results, while lime yielded more accurate results. A comparison was made between lime and potash alum, using soils from cultures incubated for 28 days at 35° C. Some of these cultures had received additions of ammonium sulphate. The lime was used in the modified method as described elsewhere in this paper. The alum was added as 40 cc. of a .5 per cent solution of potassium alum to a sample equivalent to 20 grams of dry soil. After being mixed thor- oughly, the liquid was filtered into a ZOO-cc. volumetric flask, the resi- due washed, and an aliquot evaporated to dryness. A few drops of concentrated ammonium hydroxide were added to the filtrate before evaporation in order to neutralize any possible acidity which might cause the volatilization of nitric acid during evaporation. The analysis was completed in the usual way. The results of some of the compari- sons are given in Table 2. Table 2. Effect of flocculating reagent on colorimetric nitric nitrogen, in parts per million of soil. Culture Lime Alum Number flocculent flocculent ____?____________________>____ 9 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287 278 10 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98 107 11 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 430 435 12 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104 107 17 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 10 18 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 4O 19 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 320 312 20 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87 74 21 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 606 606 22 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116 125 23 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 575 606 24 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111 118 Both reagents gave nearly the same results. However, potash alum is an unsatisfactory floceulating reagent because the filtrate comes 8 BULLETIN NO. 439, TEXAS AGRICULTURAL EXPERIMENT STATION through very slowly after 4 or 5 washings, and the filtrates from some soils are very turbid. The lower results man and Sharp-were perhaps due to the potash alum than were used in the experiment here described. Copper sulphate- was tried but did not fiocculate some of the clayey soils in a satisfactory manner. Finely ground lime (calcium oxide) was found to be the best floc- ‘l culent. With some clayey soils, filtration was sloW. The filtrates- but occasionally are slightly COlOTGCl.‘ obtained are usually colorless, a ect the results. Some of the filtrates i The colored solution did not aft obtained with alum by Lip-l use of larger amounts of the? used in the comparison of the phenol-disulphonic acid method and the zinc ferrous-sulphate method, described elsewhere, Were slightly colored but no loss of nitrate could be detected. EFFECT OF CALCIUM CARBONATE According to Chamot et al. (2), as well as Harper (5), a mechanical loss of nitrate may take place upon the addition of the phenol-disul- phonic acid, to a residue containing carbonates, due to the violent eifervescence. When calcium oxide is used as a fiocculent, some cal- cium carbonate remains in the dish after evaporation of the filtrate. The presence of this calcium carbonate might cause loss of nitrates. To test this point, lime was added to measured volumes of standard nitrate solution, the solution was filt estimated in one portion of the fi same filtrate the calcium was removed by ered, and the nitrate Iiitrogen was ltrate. In other portions of the precipitation with ammonium carbonate andsubsequent filtration and nitrates estimated. The excess of ammonium carbonate was volatilized during the subsequent evapora- tion. In order to eliminate individual nitrate solution (1 cc. I 0.01 mg. N) bias, the standard potassium was measured by one person, and the nitrate determination was made by another, who had no knowledge of the amount of nitrates present. The results are in Table 3. Table 3. Effect of presence or absence of carbonates on nitric nitrogen by the colorimetric method Lime present Lime re move d Mg. added Mg. found Mg. added Mg. found 121 . 114 108 . 106 120 . 120 189 . 183 134 . 134 202 . 201 125 .130 228 .234 160 . 166 081 .077 165 . 160 108 . 112 175 . 154 083 .086 243 .248 110 . 110 248 .244 249 .261 104 . 108 138 . 144 O75 .084 225 .222 073 .078 222 234 Average (12) 145 145 162 164 ESTIMATION OF NITRIC AND NITROUS NITROGEN IN SOILS 9 The effect. of lime was also tested in another experiment. One analyst measured certain amounts of standard potassium nitrate solu- tion into evaporating dishes. Another analyst added 10 cc. of lime water to e-ach dish and estimated nitrates. The results of these tests are given in Table 4. Table 4. Effect of addition of lime water in the estimation of nitric nitrogen Dish Number Mg. added Mg. found 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102 107 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110 118 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123 137 4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143 149 5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 058 058 6 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 070 070 7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . O91 091 8 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146 158 9 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 085 090 1O . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 057 065 12 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 089 098 Average (11) . . . . . . . . . . . . . . . . . . . . . . . . . . . .098 . 104 The results, given in Tables 3 and 4, show clearly that the presence of the carbonate of lime did not affect the accuracy of the nitrate de- termination, as the differences are within the limit of error. NUMBER OF WASHINGS REQUIRED Insufficient washing may leave some of the nitric nitrogen in the soil. It was found, with some samples, after seven washings, that sub- sequent washing removed appreciable amounts of nitrates. Fifteen to twenty washings, with small amounts of water, were found to be sufficient. INTERFERENCE OF CHLORIDES It is known (2, 5) that the presence of chlorides may affect the accuracy of the determination by the phenol-disulphonic acid method. To study this point, extracts from various cultures were prepared, and sodium chloride added to aliquots. The results were compared with those secured on aliquots which had received no additions of sodium chloride. The results are given in Table 5. Amounts of chlorides up to 100 parts per million did not affect the results within the limit of error, but larger amounts reduced the quantity of nitrates found. Soils of the humid regions are not likely to contain enough salt to affect the results. Some soils of arid sections may contain more than 100 parts per million of chlorides, which may be precipitated by silver sulphate, as recommended by Harper (5) if the phenol-disulphonic acid method is to be used, but it would probably be easier to use one of the zinc ferrous-sulphate methods described on a subsequent page. l 10 BULLETIN NO. 439, TEXAS AGRICULTURAL EXPERIMENT STATION Table 5. Nitric nitrogen in parts per million, in cultures, alone and with addition of chlorides Lab. No 50 p.m. 100 p.m. 200 p.m. 500 p.m. 1000 - N0. addition Chlorine Chlorine Chlorine Chlorine ' 29423 Upland blackland . . . . . . . . . . . . . . . . . . . . . .. 500 500 500 . . . . . . . . . . . . . . . . . . . . . . . 29423 Upland blackland . . . . . . . . . . . . . . . . . . . . . . 500 . . . . . . . . . 500 490 . . . . . . . . . . . . . . .. i 29423 Upland blackland . . . . . . . . . . . . . . . . . . . . . . 460 . . . . . . . . . . . . . . . . . . . . . . . . . .. 450. y - 28011 Subsoil (Subsoil to 28010) . . . . . . . . . . . . . .. 80 78 76 . . . . . . . . . . . . . . . . .. ... 28011 subsoil (Subsoil to 28010) . . . . . . . . . . . . . . . 78 . . . . . . . . . 74 74 . . . . . . . . . . . . . . . 31886 Duval fine sandy loam, deep phase. . . . . 45 41 39 . . . . . . . . . . . . . . . . . . . . . . . . .. 31886 Duval fine sandy loam, deep phase 84 . . . . . . . . . 78 76 . . . . . . . . . . . . . . . .. 31323 Amarillo silty clay loa‘m . . . . . . . 152 148 140 . . . . . . . . . . . . . . . . . . . . . . . . .. 31323 Amarillo silty clay loam. . 145 . . . . . . . . . 138 135 . . . . . . . . . 31324 Amarillo silty clay loam. . 31 29 28 . . . . . . . . . . . . . . . . . . . . . . . . .. 31324 Amarillo silty clay loam. . . . . 29 . . . . . . . . . 27 28 . . . . . . . . . . . . . . . . .. 31322 Amarillo silty clay loam . . . . . . . . . . . . . 105 102.5 102.5 . . . . . . . . . . . . . . . . . . . . . . . . . I 31322 Amarillo silty clay loam . . . . . . . . . . . . . . . . . 105 . . . . . . . . . 100 98 . . . . . . . . . . . . . . . . .. ' 31326 Amarillo silty clay loam . . . . . . . . . . . . . . . . . 123 . . . . . . . . . . . . . . . . . . . . . . . . . . . 103 ' 31331 Amarillo fine sandy loam . . . . . . . . . . . . . . . . 82 . . . . . . . . . . . . . . . . . . . . . . . . . . . 64 31880 Duval fine sandy loam . . . . . . . . . . . . . . . . . . 74 . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 31888 Webb fine sandy loam . . . . . . . . . . . . . . . . . . 60 . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 22230 Kirkland clay loam . . . . . . . . . . . . . . . . . . . . . 156 . . . . . . . . . . . . . . . . . . . . . . . . . . . 128 PREFERABLE ALKALI FOR DEVELOPING COLOR Ammonium hydroxide, sodium hydroxide, or potassium hydroxide. can be used for the development of the yellow color in the phenol-l disulphonic acid method. These three reagents were tried and am- monium hydroxide was found to be most satisfactory. It produces a clear yellow solution, and is not disagreeable to handle. Ammonia fumes may be evolved, but this difficulty is overcome, to a large extent, ‘A by using dilute ammonia. Carbonates sometimes present in the sodium or potassium hydroxide, produce a turbidity in the yellow solution, which renders comparison With the standard difficult. They also make glassware very slippery and ‘hard to handle, which is especially unde- sirable when a large number of determinations are being made. THE PHENOL-DISULPHONIC ACID Various proportions of phenol and sulphuric acid are used in the preparation of the phenol-disulphonic acid. A mixture of 15 grams of phenol with 100 cc. of sulphuric acid, heated in boiling water for 6 hours, was used in this work. The reagent was satisfactory and not viscous but easily measured. It was found that 2 cc. of ‘the acid is necessary to saturate the residues; 1 cc., as recommended by some work- ers, is not sufficient for this purpose. QUALITY OF PHENOL A bluish color was occasionally found to occur in some of the prepa- rations of phenol-disulphonic acid. Usually this color disappeared when ammonia was added to develop the yellow color, but with one lot of phenol the color was persistent and so interfered with the yellow color that the phenol had to be discarded. Seven other lots of C. P., or reagent quality phenol obtained from several different manufactur- ESTIMATION OF NITRIC AND NITROUS NITROGEN IN SOILS 11 ers, were tested against one another in 24 estimations of nitric nitro- gen on various samples of soil. Each of these was found to be satis- factory. THE POSSIBILITY OF INTERFERENCE BY ORGANIC MATTER In some instances, it was observed that the yfellow color, which was formed upon the neutralization of the phenol-disulphonic acid residue with the ammonium hydroxide, would fade rapidly. The phenol-disul- phonic acid method gave decidedly lower results than the Tiemann- Schulze method on aliquots taken from the soil extracts of some of these cultures. Chamot et al. (2) state that iron in the filtrate would cause trouble. However, a qualitative test of some of the filtrates showed them prac- tically free of iron, which was to be expected, since any soluble iron salts would have been precipitated by the calcium hydroxide used to. fiocculate the soil particles. It was thought that the low results were perhaps due to soluble or- ganic substances which might be eliminated by the addition of some oxidizing reagent to the filtrate. To test this theory, additions of potassium permanganate (9), of hydrogen peroxide, and of bromine water were made to aliquots of the filtrates from selected cultures of soils. They were evaporated to dryness and the analysis completed in the usual way. Some results are in Table 6. The addition of potassium permanganate or of hydrogen peroxide increased the amounts of nitric nitrogen. The hydrogen peroxide gave higher results than the perman- ganate of potash. Bromine gave lower results. Table 6. Elfect of oxidizing reagents on the colorimetric phcnol-disulphonic acid method Nitric nitrogen Soil Treatment parts per million number No addition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 29208 4 drops potas. permanganate . . . . . . . . . . . . . . . . . . . . . . . . . 92 29208 6 drops hydrogen peroxide . . . . . . . . . . . . . . . . . . . . . . . . . . . 126 29208 6 dropsbromine water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 29208 No addition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . '. . . . . . . 2 29209 4 drops potas. permanganate . . . . . . . . . . . . . . . . . . . . . . . . . 7 29209 6 drops hydrogen peroxide . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 29209 6 dropsbromine water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0 29209 N0 addition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169 29423 4 drops potas. permanganate . . . . . . . . . . . . . . . . . . . . . . . . . 210 29423 6 drops hydrogen peroxide . . . . . . . . . . . . . . . . . . . . . . . . . . . 425 29423 6 dropsbromine water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128 29423 No addition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235 29424 4 drops potas. permanganate . . . . . . . . . . . . . . . . . . . . . . . . . 235 29424 6 drops hydrogen peroxide . . . . . . . . . . . . . . . . . . . . . . . . . . . 450 29424 6 drops bromine water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1'75 29424 Considerable study was given to methods based upon the oxidation of the soil extract with hydrogen peroxide, often with quite satisfactory results, but sometimes the results were not satisfactory. The differ- ences were afterwards found to be partly caused by the presence of 12 BULLETIN NO. 439, TEXAS AGRICULTURAL EXPERIMENT STATION nitrites, which were oxidized to nitrates by hydrogen peroxide, and 3 i partly tothe production of an interfering color by the hydrogen per- ‘ oxide. After a large number of tests, the use of hydrogen peroxide 1 was abandoned. ADAPTATION OF THE ZINC FERROUS-SULPHATE METHOD TO SOILS a The uncertainty with respect to the accuracy of the phenol-disul- phonic acid method rendered it necessary to find another method with which to compare the results. The Tiemann-Schulze method Was used » at first, but required much time and was not altogether satisfactory. The A. O. A. C. zinc ferrous-sulphate method (1) was tested, and after considerable preliminary work, methods of manipulation were de- vised which gave satisfactory results. Two procedures were used on the soil extract. If the amount of ammonia expected was large, it was distilled into standard acid and titrated. If the amount of ammonia expected was small, it was estimated by the Nessler method. Colmefling bulb tllbfis- Preliminary blank tests showed the impor- tance of selecting effective Kjeldahl bulb tubes for use in the distilla- tion. Tests showed the ordinary Kjeldahl bulb tube to be unsatisfac- tory. The ordinary Kjeldahl bulb tubes were then compared with the Clark bulb and the McHargue bulb, with the results given in Table '7. These include the blank on the reagents as well as the effect of the bulb tubes. Use of the ordinary bulb tube may cause erratic results, when the quantity of ammonia distilled is small. Since 1 cc. of the 0.2N acid on 50 grams of soil is equal to 56 parts per million of nitric nitrogen, it is seen from the table that the use of the ordinary bulb may introduce an error of as much as 56 parts per million, while the error with the Clark bulb may be only 11 parts per million. The McHargue bulb gave much better results than the ordinary bulb tube, but the Clark bulb tube gave the lowest blank and the least variation and was adopted for use in our work. Table 7. Comparison of blanks on Kieldahl distillation bulb tubes, in cubic centimeters of 0.2 N acid Ordinary Clark McHargue tube tube tube cc. | cc. cc. I Average, 3 tests, Set 1 . . . . . . . . . . . . . . . . . . . . . . . . .. .97 .17 .35 Average, 3 tests, Set 2 . . . . . . . . . . . . . . . . . . . . . . . . . . .63 .22 .39 Average, 3 tests, Set 3 . . . . . . . . . . . . . . . . . . . . . . . . .. .82 .26 .41 The zinc ferrous-sulphate titration method. Preliminary work seemed to show that it would be advisable to use hydrogen peroxide to destroy the organic matter in the soil extract used in the zinc ferrous-sulphate method, but further work showed that its use gave erratic and unsatis- factory results. Sodium peroxide, which was recommended by Whiting ESTIMATION OF NITRIC AND NITROUS NITROGEN IN SOILS 13 i V al. (11), was also found to be unsatisfactory, as results were much ‘ 10w in many cases. The use of both reagents was abandoned, after A siderable work, which will not be detailed. The use of 2 cc. of strong caustic soda in the preliminary distillation r remove the ammonia was insufficient, giving high results in the bsequent reduction. This was probably due to the incomplete decom- vsition of organic compounds which yield ammonia. The use of 10 to p’! cc. of strong caustic soda (1.43 sp. gr.), followed by distillation to move the ammonia, then by reduction of the nitrates, and distilla- on of the ammonia produced from the nitrates, proved quite satisfac- iry. This method is based upon the Jones method (6) for nitrates in i; fertilizers. The larger amount of the strong caustic soda not ffi y removes ammonia, but also decomposes organic nitrogenous sub- nces which might interfere with the reduction of nitrates 0r yield fmonia in the subsequent distillation. able 8. The nitrates by the colorimetric method compared with nitrates by the modified zinc ‘1 ferrous-sulphate titration method when nitrites are absent. Nitric nitrogen 1n parts per million of cultures >- Colorimetric Zinc ferrous- method sulphate method 432 429 179 176 112 115 144 139 250 261 166 163 416 406 163 155 344 361 262 267 280 274 176 174 A 392 386 67 69 368 346 160 160 456 439 82 82 272 271 44 45 384 392 208 204 408 400 7O 68 64 _66 480 470 131 133 157 156 368 378 54 50 368 370 54 59 510 492 104 106 344 343 132 143 172 177 80 84 490 492 384 399 230 231 48 50 114 121 140 145 14 BULLETIN NO. 439, TEXAS AGRICULTURAL EXPERIMENT STATION Solutions obtained from cultures of soils which received additio“ ammonium sulphate, and which contained large amounts of nit were used in one se-ries of tests. Some of the results of these , are given in Table 8 and show that there is a close agreement be ~_ the two methods, and that the results agree Within the limit of erro Nesslerizing when the quantity of nitrates was small. The very ,’ amounts of nitrate in some soils could not be-accurately measured ~ the zinc ferrous-sulphate method of titration, since 1 cc. of 0.2 ' equals 56 parts per million of nitric nitrogen. The Nessler met was adapted to estimating the small amounts of nitric nitrogen n" usually occur in field soils, as described elsewhere. '- Table 9. Nitric nitrogen in parts per million by the colorimetric method and by the 2'1 ferrous-sulphate Nessler method, mtrites absent 4 Colorimetric Zinc ferrous- method sulphate Nessler ‘method 12.5 13.1 27.5 25.4 1.9 3.0 3.5 6.5 11.3 12.3 4.9 6_7 5.5 6.9 2.0 2.1 7.8 7.8 9.3 8.0 8.0 8.0 3.6 5.1 10.3 13.0 25.5 28.1 20.5 22.6 9.8 12.1 10.5 12.7 17.5 17.6 30.6 30.1 15.0 16.8 20.0 19.0 14.5 16.5 20.0 21.8 18.0 18.8 18.0 19.0 23.0 19.8 The 62.5 gm. of soil mixed with lime was extracted with water and the filtrate made up to 250 cc. One portion Was used for nitric nitro- gen by the phenol-disulphonic acid method. Another portion of 200 cc. was placed in a Kjeldahl flask with 10 cc. of caustic soda and about 300 cc. water. It was distilled as usual, testing the distillate for am- g monia with Nesslefs reagents at intervals. After all the ammonia was -: driven 01f, the gas was turned off; then, 2.5 grams of zinc dust, 1 gram of ferrous sulphate, and a small piece of paraflin, With ammonia- free-water, were added and the distillation performed as usual. The 7 distillate was collected in 250 cc. volumetric flasks to a little below the mark. It was then made to the mark with ammonia-free water. Of this, 50 cc. was transferred to a 100-cc. flask and diluted with ESTIMATION OF NITRIC AND NITROUS NITROGEN IN SOILS 15 rcammonia-free water; 2 cc. of Nessler’s reagent was added and made to iithe mark. AThe solution was then compared with the standard in the olorimeter. Some of the results of the comparison with the phenol- isulphonic acid method are in Table 9, showing reasonably close agree- ment. These estimations were made on cultures of soils. While some Tlj of the results do not agree as well as could be desire-d, most of them _ agree within the limit of error." ESTIMATION OF NITRATES IN THE PRESENCE OF NITRITES -_ As already stated, the phenol-disulphonic acid method gave low re- isults with some cultures of soil. This loss at first was thought to _e due to the presence of soluble organic matter in the soil solution, , ‘ut later it was found that nitrites were present, and that the nitrous nitrogen was not estimated by the phenol-disulphonic acid method, while it was included in the results secured by the Tiemann- v Schulze or the reduction method. Table 10_. Nitric and nitrcus nitrogen by the colorimetric methods compared with the nitrogen by the zinc ferrous-sulphate titration method, 1n parts per million of soil cultures '9 Laboratory Colorimetric Zinc-ferrous , Number of S0llS Colorimetric Colorimetric Nitric and sulphate g; sed for cultures Nitric N. Nitrous N. Nitrous method 5958 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 368 56 424 407 9225 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66 20 86 98 7346 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 22 41 39 7350 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140 ~ 13 153 159 7350 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98 1 8 1 16 1 15 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 141 194 190 23549 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128 39 167 157 5935 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66 1 6 82 95 5937 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 22 67 67 5958 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 200 248 232 5935 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106 54 160 154 5958 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 344 14 358 347 20720 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160 75 235 216 5958 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147 148 295 268 20720 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109 3 1 13 109 20721 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 7 67 68 33135 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 26 91 87 9277 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229 23 252 249 33134 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219 O 219 226 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6O 78 138 141 23321 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155 82 237 233 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70 1 1 1 181 178 25785 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219 30 249 253 I" The alpha-naphthylamine colorimetric method for nitrites was mod- lfied to adapt it to soils and is described in full on another page. ~ The methods were compared on extracts of cultures of soils contain- idling nitrites to which sulphate of ammonia had been added before incu- ‘bation. Of the 250 cc. soil extract, 10 cc. was taken for nitrite deter- mination with the alpha-naphthylamine method, 10 cc. for nitrate de- termination with the phenol-disulphonic acid method, and 200 cc. for total nitric and nitrous nitrogen with the zinc ferrous-sulphate titra- tion method- Some of the results are given in Table 10. The agree- cement indicates the accuracy of the methods. As pointed out in another 16 BULLETIN NO. 439, TEXAS AGRICULTURAL EXPERIMENT STATION part of this paper, the average error in reading the color in the co orimetric method for nitrates in solution is about 5 per cent, while p may run t0 13 per cent. ‘ DETAILED DESCRIPTION OF METHODS On account of the importance of apparently small details, which ma nevertheless save time and increase accuracy, it is considered desirabl to give in detail the methods as they were finally used. The Phenol-Disulphonic Acid Method Preparation. of the standard nitrate solution. Dissolve 7,25 grams C. P. potassium nitrate in water, and make up to one liter. Deter? mine nitrogen in 25 cc. with the zinc ferrous-sulphate method. Adjust so that the solution (A) contains 1.0 milligram of nitric nitrogen per one cubic centimeter.' Dilute 10 cc. of A to eiie liter. This SOlUtiOII (B) contains .01 milligram of nitrogen per cubic centimeter. Evaporate 5 cc. of solution B to dryness, and saturate the residue, with 2 cc. phenol-disulphonic acid. Develop the color with ammonia p and make up to volume in a 100-cc. volumetric flask. This standard I yellow solution contains .0005 milligram of nitrogen per cubic cen- ; timeter. Estimation of Nitric Nitrogen in Soils and Cultures of Soils I To 20 grams of cultures of soils, or 40 grams of field soil or the equivalent if wet, in a beaker, add 40 cc. of distilled water, and about 2 grams of finely ground calcium oxide and stir thoroughly. Allow the soil to settle for 10 to 15 minutes. Filter the supernatant liquid into - a 200-cc. volumetric flask provided with a wire between the neck of the flask and the funnel, to allow the displaced air to escape from the flask. Transfer the soil to the filter with as little water as possible, and wash about 15 to 20 times with small portions of Water. Make up to the mark and mix thoroughly by placing the thumb on the mouth of the flask and inverting it several times. Rinse a 10-cc. pipette thor- oughly twice with the filtrate; then transfer 10 cc. to an evaporating dish and evaporate to dryness on a steam bath. Use 10 cc. of filtrate obtained from cultures of soils or 25 cc. obtained from field soils. Add 2 cc. of phenol-disulphonic acid to the cool dry residue. Rotate the acid about the dish several times in order to secure thorough con- tact with all the residue. Afte-r 10 minutes, wash the sides of the dish with a fine stream of water and allow the acid solution to cool for about 30 minutes. Transfer the solution carefully to a 100-cc. volumetric flask, add 25 cc. of dilute ammonium hydroxide (1 part of the concen- trated ammonia to 4 parts of distilled water), and mix thoroughly Make to volume with water and again mix thoroughly. ESTIMATION OF NITRIC AND NITROUS NITROGEN IN SOILS 17 Rinse the comparison tube of the colorimeter twice with small por- tions of this yellow solution. Match a third portion against freshly prepared yellow standard solution in the colorimeter. If the color of the solution to be tested is too strong for compari- son, take 10, 20, or 25 cc. With a pipette and make up to 50 cc., 100 cc., or 200 cc. in a graduated flask. Then make the comparison as usual. The Zinc Ferrous-Sulphate Titration Method To 50 grams of dry soil, or its equivalent if wet, in a beaker, add 50-60 cc. of distilled water and 2 to 3 grams of finely ground cal- cium oxide and stir thoroughly. Allow to settle for 10 minutes and then transfer the supernatant liquid first and then the soil to a filter. Wash the soil 2O to 25 times, collecting the filtrate in a Kjeldahl flask. Add 20 cc. of caustic soda (about 1.4 sp. gr.) and enough dis- tilled water to make the volume of the filtrate about 500 cc., and con- nect to the condenser. Distill off 150 to 200 cc. and then begin to test distillate- for ammonia with Nessler’s reagent. When no more ammonia can be detected, disconnect and allow to cool. Then add a small piece of paraffin to prevent foaming, 5 grams of zinc dust, and 2 grams of ferrous sulphate. Connect to the condenser and distill into 12 cc. of .2 N acid. Titrate with .1 N sodium hydrox- ide; 1 cc. of 0.2 acid equals 56 parts per million of nitric nitrogen, on 50 grams of soil. Run 2 blanks with each set and subtract the average amount of nitro- gen found in the blanks. Nitric and nitrous nitrogen combined are determined by this method. If nitrites are present, they should be determined separately and the nitrite nitrogen subtracted from the total nitrogen. Ferrous Sulphate-Nessler Method Preparation 0f ammonia-free water. Prgparev Negglefg reagent 3g de- scribed below (about 250 cc.) with distilled water if ammonia-free water is not available, and allow to settle over night. In the mean- time, prepare distilling apparatus with glass condenser and see that the inner tube is not in direct contact with any rubber tubing. The distilling apparatus should be set up in a room free from ammonia. Place in a 6-liter flask 10-15 grams of sodium carbonate, a few pieces of ignited pumice stone, and fill it with condensed Water. Con- nect with the distilling apparatus and distill off about one-fourth of the water in the flask. Then collect distillate in 50 cc. portions at a time and test it with 2 cc. of Nessler’s reagent. When no more am- monia can be detected, begin to collect the distillate in a flask protected with a U tube, and save as ammonia-free Water. ' Preparation 0f Nesslefs reagent: Dissolve 61.75 grams of potassium iodide in 250 cc. of ammonia-free water, and add a cold solution of 18 BULLETIN NO. 439, TEXAS AGRICULTURAL EXPERIMENT STATION mercuric chloride which has been saturated by boiling with an exc, of the salt. About 25 grams of mercuric chloride dissolved in 425 of ammonium-free water will be sufficient for this purpose. Pour I; mercury solution cautiously into the potassium iodide solution until precipitate begins to form. Then with a pipette, drop by drop, ad enough solution to make the color a permanent bright red. Dissolv the red precipitate by adding exactly .75 gram of potassium iodide Stir well,‘ and transfer to a one-liter flask. Then add 150 grams 0 potassium hydroxide dissolved i11 250 cc. of ammonia-free water Shake well, and make up to the mark with ammonia-free water. thoroughly, transfer to -a clean flask, and allow to settle over night; Pour off the supernatant liquid and keep it in a place free from am-i moma. Preparation of standard ammonium chloride solution: To 715 (j(3_ of; .1 N ammonium hydroxide solution, add 3.58 cc. of .2 N hydrochlorief acid. Transfer to a liter flask and make up to the mark with am-f monia-free water. Mix thoroughly and then transfer to a bottle. 1 - cc. of this solution I 0.00001 gram of nitrogen. Operation! Weigh out 50 grams of soil (or 25 grams of soil obtained from cultures containing larger amounts of nitrates) into a beaker. Add about 50 cc. of distilled water, 2 to 3 grams of finely ground calcium = oxide g stir, and after 10 minutes, transfer the supernatant liquid and soil . to a filter, collecting the filtrate i11 a Kjeldahl flask, and wash "thoroughly . about 20 times. Add 10 cc. of caustic soda, enough distilled water to make the volume about 500 cc., and connect to the condenser. Distill off about 200 cc. and then begin to test the distillate with N essler’s re- agent. Continue to test until no more ammonia can be detected. Then turn off the heat and put a 250-00. volumetric flask at the delivery tube to collect the distillate. Disconnect the Kjeldahl flask; add a small piece of paraflin to prevent foaming, 2.5 grams of zinc dust, and 1 gram of ferrous sulphate. Add also a little ammonia-free water if necessary. Connect with the condenser and distill off nearly 250 cc. Make distillate up to 250 cc. with ammonia-free water. To 50 cc. in a 100-cc. volumetric flask, add about 20 to 30 cc. of ammonia-free water, shake and then add 2 cc. of N essler’s reagent. Make up to the mark, shake, and then allow to stand 10 minutes. Compare a portion of this with the standard in the colorimeter. For the preparation of the standardsolution, dilute 10 cc. of the standard ammonium chloride in a 100-cc. volumetric flask, with 60 to '70 cc. of ammonia-free water, add 2 cc. of N essler’s reagent, and make up to the mark. Shake thoroughly and allow to stand 10 minutes. Then use for comparison in the colorimeter. If Nessler’s reagent is added directly to the standard ammonia solution before dilution, a pre- cipitate is formed, which renders the solution useless. ESTIMATION OF NITRIC AND NITROUS NITROGEN IN SOILS 19 NITRITE NITROGEN IN SOILS Sulphani1i¢ acid- Dissolve 1.6 grams of sulphanilic acid in 200 cc. Ilof dilute acetic acid (29 per cent or 1.04 specific gravity‘). Heat gently iand stir thoroughly in order to dissolve the sulphanilic acid. Cool and ipreserve in a glass-stoppered bottle. Naphthylamille flcetflte- Dissolve .5 gram of alpha-naphthylamine in i100 cc. of dilute acetic acid (29 per cent or 1.04 specific gravity). Heat gently and stir thoroughly to dissolve the naphthylamine. Cool and place in a brown glass-stoppered bottle. This reagent is good for 2 to 3 days. After this period of time, a. new supply should be prepared. Silver nitrite- Dissolve 10 grams of silver nitrate in 20 cc. of hot distilled water (8). Dissolve 10 grams of sodium nitrite in 15 cc. of distilled water and heat. Mix both solutions While hot and stir thor- oughly, then allow to cool. Filter and Wash about 10 times With ice- cold water. Place the washed precipitate with the filter paper between white blotting paper and press it gently to get the moisture out. Then Wrap it well with another filter paper, and dry it in a desiccator in a _dark place for a Week or longer. Standard sodium nitrite solution: Solution A, Dissolve 0,55 gram of silver nitrite in 50 cc. of distilled water in a beaker by heating. In another beaker dissolve 0.3 gram of sodium chloride in 25 cc. of water. Add the sodium chloride solution to the silver nitrite solution. Stir thoroughly. Allow to cool and then filter into a. 500-cc. volumetric flask. Wash thoroughly about 15 times. Then add 1 cc. of chloro- form, make it up to the mark, and shake thoroughly. Transfer the solution to a brown bottle and stopper Well. Keep in a dark place. Determine total nitrogen in 50 cc. by the zinc ferrous-sulphate method, and nitric nitrogen in 10 cc. by the phenol-disulphonic acid method, and adjust if necessary. One cc. should contain 0.1 mg. of nitrous nitrogen. Solution B. Dilute 10 cc. of solution A to one liter. This solution contains .001 mg. of nitrogen in 1 cc. Keep in a brown bottle. Standard colflred SOIHtiOII- Transfer 10 cc. of solution B to a 100- cc. flask, add about '75 cc. of water and 2 cc. each of the sulfanilic acid reagent and the alpha-naphthylamine acetate. Shake, make up to the mark, and shake again thoroughly. Set aside for 15 or 20 minutes and then compare with the unknown in the colorimeter. One cc. of this pink solution (C) contains 0.0001 mg. of nitrous nitrogen. Qualitative Test for Nitrites in Soils Prepare soil solution as described under the colorimetric nitrate method, using 20 grams of dry soil (or equivalent of a wet soil), and 2 to 3 grams of calcium oxide. Dilute after filtration to 200 cc. in a 20 BULLETIN NO. 439, TEXAS AGRICULTURAL EXPERIMENT STATION graduated flask. Transfer 2 cc. of this solution to a test tube mark at 1O cc. Make up slightly below the mark with distilled water. A 5 drops each of the sulp-hanilic acid and of the alpha-naphthylam" acetate. Shake thoroughly. Allow color to form 15 to 20 minu Then compare with 10 cc. of the standard (C) in another test tut The standard is equal to 5 parts per million of nitrous nitrogen, in t soil taken. " Quantitative Determination of Nitrous Nitrogen First observe the unknown in the qualitative test. If a heavy pr cipitate has been formed, make up 10 cc. of the soil solution to l? cc. Transfer 10 cc. of this dilution to a 100-cc. volumetric flask, dilu a to '75 cc. and add 2 cc. of each reagent. Make it to the mark and shak it as usual. Allow color to form 15 to 20 minutes. Then compar with the standard in the colorimeter. If the color of the unknown still too deep, dilute 25 cc. of this to 5O cc. or to 100 cc. and compare as usual. y: If only a slight precipitate is formed in the qualitative test, make up 10 cc. of the soil extract to 100 cc., transfer 10 cc. to a 100-cc.‘ flask, and develop the color as previously described. 1; If no precipitate is present in the qualitative test, transfer 10 cc. I to a 100-cc. volumetric flask, and dilute and develop the color. If the color is too deep, make up 1O cc. of the soil solution to 100 cc.; place 25 cc. of this dilution to a 100-cc. flask, and develop the color. 1 The pink color should always be developed in a 100-cc. volumetric flask. 5‘ In case of field soils, or soils low in nitrites (lower than 5 parts '_ per million), determine the nitrites directly in the filtrate from the soil, without dilution. SUMMARY Differences in the quantities of nitrogen found by the phenol-sul- t phonic acid method and by the Tiemann-Schulze method, and by re- duction methods were found to be chiefly due to the presence of nitrites, in large quantities, in some cultures, which were not included in the results by the phenol-disulphonic acid method, but were included with the other methods. For the purpose of checking the colorimetric method, the zinc fer- rous-sulphate method for combined nitrate and nitrite nitrogen was adapted for use with soils. When moderate amounts of nitrogen were present, the ammonia was collected in acid and the acid titrated with alkali. For small amounts of ammonia, the ammonia in the distillate ' was estimated by the Nessler method. Both methods are described in detail. ‘_ Details of the alpha-naphthylamine method for nitrites was modified ’ to adapt it to estimation of nitrites in soils. " When nitrites were allowed for, the results by the phenol-disulphonic P ESTIMATION OF NITRIC AND NITROUS NITROGEN IN SOILS 21 acid method checked very well against the zinc ferrous-sulphate methods. The Clark bulb was superior to the ordinary Kjeldahl distillation bulb. The error due to matching the color with the standard in the phenol- disulphonic acid method for nitric nitrogen was found to average about 5 per cent of the amount of nitrogen present, while it was sometimes found to be as much as 13 per cent. Lime was a good fiocculating agent for soils in nitrate work. Potash alum may be used, but was less satisfactory. I The presence of calcium carbonate did not appear to affect the results by the colorimetric phenol-disulphonic acid method. Washing the soil residue 15 to 2O times is recommended. Ammonium hydroxide for developing the yellow color is preferable to sodium or potassium hydroxides. Additions of oxidizing agents such as hydrogen peroxide gave higher results for nitrates on some of the cultures, when the colorimetric method was used, but their use was found to be unsatisfactory. Ordinarily the colorimetric methods for nitrates and for nitrites should be used for soils or cultures secured in nitrification experiments. The zinc ferrous-sulphate methods may be used for testing the method or for special purposes. On account of the importance of apparently minor points to secure accurate results or to save the time of the analyst, the methods for nitrates and nitrites as finally used are described in detail. REFERENCES 1. Association of Oflicial Agricultural Chemists, 1925. Official and Tentative Methods of Analysis. 2. Chamot, E. M., Pratt, D. S., and Redfield, H. W., 1911. A study of the phenol-disulphonic acid method for the determination of nitrates in Water. J our. Am. Chem. Society, 33:336. 3. Fraps, G. S., and Sterges, A. J ., 1930. Occurrence of nitrites in soils. Texas Agr. Exp. Sta. Bull. 4112. 4. Greaves, J. E., and Hirst, C. T., 1917. Some factors influenc- ing the quantitative determination of nitric nitrogen in the soil. Soil Science, 4:179. 5. Harper, H. J ., 1924. The accurate determination of nitrates in soils, phenol-disulphonic acid method. Ind. and Eng. Chem. 16:180. 6. Jones, C. H., 1927. Determination of mineral nitrogen in fer- ‘ tilizers. Ind. and Eng. Chem., 19:269. '7. Lipman, C. 13., and Sharp, L. T., 1912. Studies of the phenol- disulphonic acid method for determining nitrates in soils. Univ. Calif. Publications in Agr. Sciences, 1 :21. 8. Morse, H. N, 1905. Exercises in Quantitative Chemistry. Ginn & Company, Boston. 22 BULLETIN N O. 439, TEXAS AGRICULTURAL EXPERIMENT STATION 9. Syme, W. A., 1909. The colorimetric estimation of nitrates r" soil solutions containing organic matter. Jour. Ind. and Eng. Chem g 1 Z188. ' 10. Van Wiijk, D. J. H., 1924. The quantitative determination nitrates in soils. Soil Science, 17 :163. g 11. Whiting, A. L., Richmond, T. C., and Schoonover, W. R, 1920. The determination of nitrates in soil. Jour. Ind. and Eng. Chem., 12:982. 12. Wiley, H. W., 1908. Principles and Practice of Agricultural Analysis. Vol. 2, p. 403, Chemical Publishing Co. a 13. Yoe, J. H., 1928. Photometric Chemical Analysis. Chap. V, g Errors in Colorimetry. John Wiley & Sons, New York. ‘