THE NITROGEN FRACTIONAHON OF A CLAY TREATEE SANfiY SGEL “finals gar flu Degree of DH. D. MICEEGEN STETE EKE?ERS§FY Jinan Colo-m 1959 This is to certify that the thesis entitled THE NITROGEN FRACTIONATION OF A CLAY TREATED SANDY SOIL presented by JUAN COLOM has been accepted towards fulfillment of the requirements for _.Eh.D.._degree mSoil Science AR. (om Major professor Date 1 Z 1 0-169 LIBRARY Michigan State University --i.- THE NITROGEN F RACTIONATION OF A CLAY TREATED SANDY SOIL BY JUAN COLOM A THESIS Submitted to the College of Agriculture Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Soil Science 1959 o..- .5‘<- C; AS" ll 9 ”0/9! C. A5729 (we/u v,”_ ."u WWW. 1v Wm , , .— ACKNOWLEDGEMENTS The author wishes to express his deep appreciation to his major professor, Dr. A. R. Wolcott, for his helpful suggestions and as- sistance in this study and in the preparation of this thesis. Grate- ful acknowledgement is also made to Dr. M. M. Mortland and Dr. A. E. Erickson for their suggestions and for supplying the experi- mental soils used in this study. It is a pleasure to acknowledge, with thanks, the assistance of Messers N. Yassoglou and K. Kinra in various phases of the present work. The author wishes to extend his sincere appreciation to the Soil Science and Chemistry Departments of Michigan State University for providing facilities, equipment, and assistance. Special grati- tude is extended to Dr. R. L. Cook, Head, Soil Science Department, under whose direction and help this work was undertaken. Appreciation is extended to Drs. C. L. San Clemente and R. L. Bateman, members of the guidance committee for their help in the counsel and editing of this thesis. Special recognition of gratitude and love is ext ended to the au— thor's wife for her unwearied cooperation and encouragement. $.49.“ ...-———.~ ——--II :-—- «as: T g THE NITROGEN FRACTIONATION OF A CLAY TREATED SANDY SOIL BY J UAN COLOM AN ABSTRACT Submitted to the College of Agriculture Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Soil Science 1959 Approved: Date: JUAN COLOM The distribution of nitrogen in a sandy soil which had received applications of clay three years previously was studied. The addition of Wyoming Bentonite to the sand increased the e- lectr0phoretic mobility of its colloidal fraction. The C:N ratio was found to decrease with increasing application levels of clay, and this was due primarily to increases in total nitrogen. There was no relation between clay treatment and the hydrolyzable ammonia fraction, where inorganic nitrogen would appear. However, the close inverse relationship between cataphoretic mobility and carbon content suggested that organic, rather than inorganic, nitrogen compounds were principal- ly effective in altering the charge on the clay. The increase in nitrogen was due to increases in nitrogenous ma- terials which were hydrolyzed in 6N-HCl and were soluble in the pre- sence of excess Ca(OH)Z. Fractionation of the hydrolyzable nitrogen into basic and non-basic constituents with phosphotungstic acid showed that both constituents increased with clay treatments. However, no relationship between Van Slyke amino nitrogen and clay treatment was found. The acid hydrolyzable materials were then fractionated into func- tional constituents by the process of column separation recommended for protein hydrolysates by Fromageot and Lederer. Basic materials such as diamino acids were retained on a silica column, the acidic or MT— . ""1 we." —~ "W s? magnify? ~ ~~ JUAN COLOM dicarboxylic on an alumina column, the aromatic on a charcoal column, and the final perfusate contained neutral non-aromatic materials. The recovery of basic and non-basic forms of nitrogen by this method agre- ed very closely with the phosphotungstic acid procedure. A considerable proportion of the hydrolyzable nitrogen in these soils was found in the form of neutral non-aromatic constituents. They showed a marked tendency to increase with clay treatments. Aromatic forms of nitrogen were essentially unrelated to clay treatments, while the basic nitrogen constituents increased and the acidic decreased with increasing clay content. Attempts were made to characterize amino acids in the soil sep- arates by paper partition chromatography. Good separation of stand- ard amino acids was achieved. However, no comparable separation was obtained with any fraction or separate from any of the soils. The ninhydrin-reactive compounds in these fractions were not present as free amino acids. Rather, the amino acids were complexed with other organic compounds which appeared to be aromatic in nature. It was not clear whether these complexes had been formed in the soil or whether they had arisen as artifacts during chemical treatment in the laboratory. However, their mobilities in phenol and in butanol- acetic acid on paper chromatograms were clearly influenced by the level of clay application. The possibility that clay could have been present in the soil prep- arations was rejected on evidence from X- ray analyses. Glycerated it! i. ii JUAN COLOM soil hydrolysates proved to be X-ray amorphous. X-ray patterns of unglycerated preparations showed no diffraction angles within the limits of the native soil clay or of Wyoming Bentonite. mw—u TABLE OF CONTENT S Chapter Page I. INTRODUCTION ............... . . . . . 1 II. REVIEW OF THE LITERATURE ...... . . . . . 2 Humus Formation and Chemical Nature of Humus 2 Soil Organic Nitrogen ...... I ......... 10 Evaluation of Some of the Newer Methods Used in 16 Soil Organic Matter Investigations ........ Chromatographic Procedures ......... 16 Electrophoretic Procedures ......... 19 X-Ray Diffraction ............... 21 Infra Red Absorption ............. 22 III. METHODS OF ANALYSIS ............... 27 Total Carbon . ................... 27 Carbon Dioxide Evolution During Incubation . . . 27 Total Nitrogen ................... 27 Nitrifiable Nitrogen ........... . . . . . 27 Primary Hydrolytic Fractionation of Nitrogen . . 27 Van Slyke Nitrogen . . . . ........ . . . . 28 Photometric Ninhydrin Procedure ...... . . ' 28 Desalting ...................... 28 Paper Chromatography .............. 28 Separation of the Phosphotungstic Acid Fractions 30 Column Separation P rocedure ........... 30 ii .- TE“??— J.p__... Chapter Page Electrophoresis .................. 30 X-Rays ...................... 32 Infra Red ..................... 32 Glyc e ration .................... 3 2 IV. CHARACTERIZATION OF THE SOILS ........ 33 Field Description ................. 33 Laboratory Determinations ............ 34 V. NITROGEN FRACTIONATION ............. 53 VI. PAPER CHROMATOGRAPHY ............. 72 VII. SUMMARY ....................... 89 VIII. LITERATURE CITED ................. 91 IX. APPENDIX: GLYCERATION EXPERIMENT ..... 104 INTRODUCTION .................. 105 REVIEW OF THE LITERATURE .. .. . .. .. .. .. . .. . 106 EXPERIMENTAL ................. I 115 DISCUSSION OF RESULTS ............ 116 X-Rays .................... 1 l6 Infra Red ................... 122 CONCLUSIONS .................. l 3 3 iii 13-18. 19-23. LIST OF FIGURES SCHEME FOR PRIMARY FRACTIONATION OF SOIL NITROGEN ................... SCHEME FOR COLUMN SEPARATION OF FRACTIONS A AND B INTO GROUPS BY THE METHOD OF FROMAGEOT ............. X-RAY DIFFRACTION PATTERN OF DIALYZED NATIVE CLAY ISOLATED FROM THE CHECK SOIL ......................... X-RAY DIFFRACTION PATTERNS OF DIALYZED CLAY SUSPENSIONS AND WYOMING BENTONITE AFTER HEATING TO 110°C ............ X-RAY DIFFRACTION PATTERNS OF DIALYZED CLAY SUSPENSIONS AND WYOMING BENTONITE AFTER TREATMENT WITH POTASSIUM CHLORIDE AND HEATING TO 500°C .............. CHROMATOGRAM OF STANDARD AMINO ACIDS CHROMATOGRAPHIC PATTERNS OF THE BASIC- NINHYDRIN COMPONENTS ............. CHROMATOGRAPHIC PATTERNS OF THE ACIDIC NIN HYDRIN COMPONENTS ............. CHROMATOGRAPHIC PATTERNS FOR THE AROMATIC AND THE NEUTRAL NON-AROMATIC NINHYDRIN COMPONENTS ............. iv 29 31 47 49 51 74 76 78 80 10. ll. 12. 13. 14. LIST OF TABLES Carbon and Nitrogen in the Soils of the Experiment . . . Migration Velocities and Calculated Values of 3 Potential for the Clay Suspensions After 15 Minutes . . . . . . . The pH of Soil Suspensions and of 0. 03% Dialyzed Clay Suspensions ............... ........ Carbon and Nitrogen Analyses of the Dialyzed Clay Suspensions ....................... Carbon Mine ralized During Incubation of the Soils of the Experiment ...... . ........ . . . . . . . . X-Ray Diffraction Peaks of the Dialyzed Clay Suspensions and Dialyzed Wyoming Bentonite (in X Units) ...... Recovery of Soil Nitrogen in Hydrolyzable and Non-Hydro- lyzable Forms as Related to Clay Treatment . . . . . . Recovery of Nitrogen in Soil Hydrolysate (Fraction A) as Related to Clay Treatment . . . . . . . . . . . . . . Recovery of Nitrogen in Fraction B as Related to Clay Treatment ................... ..... Van Slyke Nitrogen in Hydrolyzable Fractions from Soils with Varying Clay Treatments . . . . . . . . . . . Distribution of Nitrogen Among Column Separates from Total Hydrolysate (Fraction A), as Related to Clay Treatment O ..... O ...... O O O O O O O O O O 0 Distribution of Nitrogen Among Column Separates from Fraction B, as Related to Clay Treatment . . . . . . . Occurrence of Leucine-Equivalent Ninhydrin-Reactive Compounds in Fraction A and its Column Separates, as Relatedto ClayTreatment. . . . . . . . . . . . . . . . Occurrence of Leucine-Equivalent Ninhydrin-Reactive Compounds in Fraction B and its Column Separates, as Relatedto ClayTreatment. . . . . . . . . . . . . . . . V 35 38 40 41 43 45 54 56 59 61 63 64 68 69 15. Occurrence of Leucine-Equivalent Ninhydrin-Reactive '71 Compounds in Fractions C and D as Related to Clay Treatment......................... 16. X-Ray Diffraction Data for Some of the Soil Fractions 117 Before and After Glyceration . . . . . . . . . . . . . . . 121 17. X-Ray Diffraction Data for Some Amino Acids Before andAfterGlyceration................... V——_-—_—._. -2 ~-- --=-‘—.'_.~‘ LIST OF PLATES I. INF RA RED ABSORPTION SPECTRA OF SOIL NITROGENOUS CONSTITUENTS AND AMINO ACIDS BEFORE AND AFTER GLYCERATION.... ..... .............. 125 Curves l to 8 ...................... 125 Curves 9 to 16 . . . . ................. 126 Curves 17 to 20 . . . ................. . 127 vii . ' ..~. 1‘ _ , -P‘J Id ‘ '. ' 7 fl ; .- 1 I ' l _ ' . i' J)“; Qla/‘I‘n ..."“ i i ri- .«fl »‘ - ii I. :‘f r lr . F ' l-I . ' i _ ’1‘ 'l .- f'l : ' l ”f.‘ b.“ -' _r ' , . _ ' " -.-*=_‘;’.'.‘~A""« ‘-. IN TROD UC TION Practice can never get far ahead of basic principles except acci- dentally (77). A basic understanding of the principles governing the scientific management of soils must take into considerationthe trans- formations undergone by organic matter in the soil. The role of or- ganic matter in fixing and liberating nutrients, as well as its ability to complex metals and react with clay colloids during its decomposi- tion, ,have drawn the attention of many soil scientists. Numerous proximate fractionation procedures have been devised in an effort to isolate constituents of soil organic matter which could be further characterized and related significantly to soil prOperties. The nitrogenous fractions of soil organic matter have been studied in greatest detail. We are still in the initial stages of learning about their importance in humification processes and their role in organic- inorganic soil colloid interactions. The interactions between clay minerals and various organic compounds have been studied in the lab- oratory. Effects of clay on the retention of carbon and nitrogen from organic materials decomposing in soils in the greenhouse have also been studied. The present study was concerned with the effects of clay under field conditions on the retention of carbon and nitrogen and upon the distribution of nitrogen among a number of chemically distinct fractions. an ’ i if??? r . . g *- 6*, F5 'j_.i'3 ;' ' l. _. ‘.l\ . T l .. .‘I. ’ i . Vl—lii REVIEW OF THE LITERATURE For convenience, this review is divided into three general areas: Humus formation and chemical nature of humus, soil organic nitrogen, and evaluation of some of the newer methods used in soil organic mat- ter investigations. Humus Formation and Chemical Nature of Humus From the standpoint of quantity added and residual material re- maining in the soil, residues from higher plants predominate over animal residues and microorganisms as the principal source of soil organic matter. Largely by biochemical processes, organic matter is gradually transformed into a uniform, dark-colored, amorphous mass, designated as humus, in which the products of decomposition can no longer be identified with the original plant and aminal materi.— als from which they came. Waksman (148) has enumerated some properties of humus that distinguish it from the plant and animal ma- terials from which it was formed. Some of the outstanding properties mentioned by Waksman are its insolubility in water, considerable sol- ubility in dilute alkali, ,and its partial solubility in acid. Chemically, it contains about 3 to 6% nitrogen and a C:N ratio close to 10:1. Hu- mus is not a definite organic entity. It is a complex of substances in a state of more or less dynamic equilibrium. Its chemical compo- sition depends, in part, upon the chemical nature of its precursors and, in part, upon the degree of decomposition and the environment- al conditions under which decomposition has occurred. Humus is, therefore, chemically not always the same, but changes constantly as a result of the continuous processes of decomposition brought a- bout mainly by microorganisms (149). The'extent of decomposition of organic matter in soil is found to depend notonly on the nature of the decay pepulation, but also on the nature and amount of available nitrogen (148). Accordingly, for ev- ery unit of carbon decomposed as a source of energy, a certain a- mount of carbon and nitrogen is assimilated into microbial tissues. Nitrogen is present in fresh plant residues largely in the form of proteins. In the process of decomposition, these are hydrolyzed through a series of intermediary complexes with the final liberation of surplus nitrogen as ammonia which, in turn, is oxidized in nor- mally aereated soils to nitrites and then to nitrates (148). Apparently, the rate of decomposition of organic matter in soil is a function of the amount of available energy material added to the soil (31, 117). This viewpoint has been questioned by Allison (3). The latter believes that plant material composition is more important than C:N ratio per se. Maillard (84) claims that microorganisms are .of minor importance in the formation of humus in nature , except for their hydrolytic action in converting proteins to polypeptides and amino acids and complex carbohydrates to sugars. Rubins and Bear (116) are also of the Opinion that it is not the C:N ratio of the material as much as it is the nature of the specific constituent carbon compounds which, ultimately, decide the -rate of decomposition of organic matter 4 in soil. Birch and Friend (17) have observed that it is possible that the so called "priming action" of fresh organic matter on the breakdown of soil humus, generally ascribed to microbial activity is, in fact, a displacement effect caused by an exchange between products of organ- ic matter decomposition and organic compounds previously adsorbed and protected by clay. Although proteins, nucleic acids, and other nitrogenous organic compounds in plant residues are rapidly mineralized when added to soils, not much more than about 1% of the organic nitrogen of the soil is made available during the growing season (24). The low availability of soil organic nitrogen has been the subject of much speculation. Two general types of mechanisms have been proposed to account for the formation of humus of a resistant nature. The first of these includes various organic condensation reactions. The second includes several types of clay-organic interaction. The former theory and the different modalities of it which have been ex- pressed (6, 10, 57, 58, 84) are best represented by the concepts of Waksman 8: Iyer (150, 151, 152, 153) and those of Mattson and Koutler- Andersson (.91). Waksman and Iyer advanced the theory that the avail- ability of soil proteins is reduced through combination with lignins. They advanced evidence in support of this theory from studies on the properties of complexes prepared by mixing and then acidifying alka- line solutions of lignin and protein (150, 151, 152, 153). Such complex- es were found to be highly resistant to biological decomposition. From these results they postulated that "lignoprotein complexes" are form- ed in the soil by a gradual condensation process. As pointed out before, an entirely different explanation of the low availability of soil organic nitrogen has been provided by the pro- ponents of the clay-organic interaction theory. Although the inter- action was originally observed by Demolon and Barbier (42), no theo- retical basis on which the reduced availability of organic nitrogen, in association with clays, could be explained appeared until Ensminger and Gieseking (50, 51, 52, 53) showed that hydrolysis of proteins by proteolytic enzymes was markedly reduced in the presence of certain clay minerals. They also found that the 001 spacing of the clay lat- tice increased with increase in gelat ine adsorption and decrease in pH. Similar interactions have been found for clays and organic mol- ecules other than proteins (19, 29, 48, 68, 80). Lynch and Cotnoir (80) and Birch and Friend (17) observed that organic substrates adsorbed on clay minerals were protected from breakdown by soil microorganisms. Two leguminous crop residues, alfalfa meal and soybean leaf meal, produced, respectively, 29. 0 and 40. 0% less C02 in the presence of Bentonite over a short period. The inactivation of microbial enzymes by clay minerals, as well as the adsorption of substrates by the clay, were proposed as two hypotheses explaining the observed behavior. Chemical interpretations regarding the manner in which both mech- anisms of humus formation, i. e. the organic condensation and the ‘L", -—- n y. ‘Wu-«$""# _',' ‘P— clay-organic interaction, take place have been proposed by several in- vestigators. Waksman and Iyer (154) suggested that there is a reac- tion between the carbonyl groups of the lignin and the amino groups of the protein to give a Schiff‘s base: Lignin - CO I HZN - R - COOH 5.934311335199-’ Lignin C = N - R - COOH. They considered that this complex is stable and has a high exchange Capacity. Norman (105) suggested that such a complex may be one of physical adsorption, since some of Waksman's data suggest this and that Waksman's proposed re- action would not explain the high resistance to decomposition. Mattson and Koutler-Andersson (91) have postulated a process of autoxidative ammonia fixation by different organic matter fractions. They conclud- ed that the nitrogenous humus complex is a chemical product result- ing from autoxidation and simultaneous ammonia fixation. Alkaline conditions we re found to enhance the process. The following reactions have been suggested by Mattson and Kout- 1er-Andersson(9l) as possible reactions which take place in the oxida- tive fixation of ammonia by humus: Lignins in plant material are considered to be composed of C6-C3 units or C6-C6-C6 units in varying degrees of polymerization. The C6-C3 units are related to degra- dative lignin derivatives of the type: HO-< >- cocnoncn3 (a) in, '1» o~ {"0 W" ‘P-‘g-"J‘u 21".,“ The C6-C6-C6 units are of the type: H CH -CHO (I) c/ 2‘ ”\C HO- C\ CH (b) CHOH- cnfc | 9 0 CH3 H Mattson and Koutler-Andersson found that autoxidation of lignin re- sulted in a loss of phenolic hydroxyls. Oxidation of compounds (3.) and (b) to quinones would account for this. For example, oxidation of (b) would form: (2) . CHOH CH2/ 9 2 CH 3 (1) H 3 Fission of the central six carbon ring in compound (c) at (1) and (2) would give two moles of the aldehyde (d). o =:> = CH-CHz-CHO (d) 8H, The aldehyde (d) would be readily oxidized to an acid: 0 ._- < >= CH-CHz-COOH (e) 21., The formation of organic acids as in (e) would account for the in- creasing cation exchange activity of organic materials which accom- panies chemical autoxidation or normal microbial decomposition. Matt- son and Koutler-Andersson(9l) found an approximately linear relation- ship between nitrogen retained in litter or humus and its acidoid con- tent. They observed that this relationship could be accounted for, if it we re assumed that ammonia fixation occurred simultaneously with the above fission and oxidation reactions to give quinone - imid carbox- ylic acids of the type: HN ._. z CH-CHz-COOH 31., The interactions taking place between organic compounds and clay minerals have been described by several workers (3, 48, 68, 94, 102). As Allison (3) has observed, there is no complete agreement as to the exact nature of the clay-organic combination. Postulated mechanisms _._ fir_ gym vary from mere mechanical mixtures, to close unions that approach the nature of chemical compounds. Several investigators (43, 87, 126) claim to have demonstrated the esterification of the Si - OH groups of clay minerals by reactive organic compounds. Duel and his collabora- tors (43) observed that the charge on the clay was reduced in an amount equivalent to the degree of esterification. Greenland and Russell (68) have accumulated X-ray and chemical data tending to show that the interaction of clays with thionyl and acetyl chloride reported by other workers (87, 126) is to be explained in terms of hydrolysis of the re- agent with adsorbed water, exchange between the cations initially pres- ent and the hydrogen formed by hydrolysis, and adsorption of the or- ganic reagent or organic hydrolysis product. In any event, Glaeser (62) has shown that simple organic compounds adsorbed on the surface of montmorillonite by physical adsorption forces are extremely diffi- cult to remove. The foregoing observations and concepts relating to the origin of humus and its chemical nature, attest the complexity of the organic constituents of soil. Broadbent (29) believes that a sizable portion of soil nitrogen may be in forms which are not closely related to com- pounds produced either by plants or by microorganisms but, instead, are produced in the soil. It is obvious, as Bremner (24) has pointed out, that "pre sent information regarding the chemical nature of the nitrogenous organic complexes of soil is extremely unsatisfactory and that this problem deserves high priority in any scheme of research on 10 soil organic matter". Soil Organic Nitrogen For a long time, it was assumed that organic nitrogen in the soil was largely protein (79, 123, 136, 149, 152). Quite a large volume of recent evidence has accumulated tending to show that soil organic ni- trogen contains considerable amounts of nitrogenous constituents very dissimilar to proteins. The fact that a considerable fraction, as much as 30%, of the soil nitrogen is resistant to acid or alkali hydrolysis has been taken to indicate that much of this nitrogen is of non-protein nature and has led to the suggestion that part of the organic nitrogen of soils is in the form of heterocyclic nitrogen compounds (24). Brem- ner (20) found that as much as 60% of the nitrogen in some humic acid preparations was not dissolved by hydrolysis with strong acids. He has also reported (26) that the amount of ammonia liberated by hydro- lysis of humic acids with 6N - HCl was about maximum after six hours; whereas the amount of ammonia released by hydrolysis of soils increased steadily with time. Anderson (5) has identified purine and pyrimidine bases in hydrolysates of soil humic acid. Dyck and Mc- Kibbin (45) reported that not all organic soil nitrogen is determined by the Kjeldahl method; in every sample tested, the Dumas method (155) gave a considerably higher percentage of nitrogen. "This dis- crepancy in nitrogen values could be attributed to the presence of cer- tain heterocyclic nitrogen compounds or of compounds containing ni- trogen linked directly to nitrogen" (24). r ”*‘V.y :._.. -——-: raw—— ll Mattson and Koutler-Andersson (91) suggest that some of the organ- ic nitrogen of soil that is stable to hydrolysis may be in the form of nitrogenous complexes formed in soil by interaction between oxydized lignin and ammonia. They showed that lignin was able to fix ammonia in a non-exchangeable form under soil conditions. Studies on the fix- ation of ammonia by organic compounds of known constitution led them to the view that fixation of ammonia-nitrogen by lignin takes place at the site of phenolic hydroxyl groups and is preceeded by the oxidation of these groups. Bennett (13) obtained evidence in support of this view. He was able to show that the nitrogen content of a complex prepared by autoxidation of lignin in NH40H was reduced from 7. 22 to 2. 66% by a somewhat drastic, prior methylation. Sohn and Peech (12.8) attributed the capacity of mineral soils to fix ammonia, partly to a neutralization of the exchangeable hydronium and aluminum ions, and partly to the formation of organic nitrogen com- pounds by autofidation of soil organic matter and simultaneous am- monia fixation. They estimated that at least 50% of the ammonia fixed was taken up by the latter mechanism. Bremner and Shaw (27) found that the nitrogen in soil humic acid preparations was mineralized at a rate intermediate between that of nitrogen in the form of lignin-ammonia complexes and that in lignin- protein complexes. They proposed that neither the lignin-protein the- ory advanced by Waksman and Iyer (I 52, 153) nor the lignin-ammonia theory of Mattson and Koutle r-Anders son is adequate, and that the ob- 12 served properties of the humic acid fraction of soil organic matter is more readily explainedby a concept incorporating both of these theo- ries. From hydrolytic fractionation studies it appears that from 20 to 50% of soil organic nitrogen is amino nitrogen, the rest is presumed to be associated with lignin or in heterocyclic combinations (21, 24, 79, 114). The isolation of soil amino compounds is hindered by the interference of other substances present in the hydrolysate and by induced alterations which the analytical procedure imposes by fission and polymerization reactions (20, 25, 91, 108, 120, 156). Bremner (25) briefly reviewed the amino acids which have been isolated as hy- drolysis products of soil organic matter by different investigators. Adding to his list others reported in the literature (10?, 112, 136), the following amino acids have been reported in hydrolysates of soil or soil organic fractions: Leucine, isoleucine, alanine, proline, argi- nine, histidine, lysine, aspartic acid, tyrosine, phenylalanine, valine, glycine, threonine, serine, glutamic acid, dihydroxyphenylalanine, ornithine, J: -aminobutyric acid, methionine sulfoxide, and methionine sulfone. This list is of the order of a first approximation since, in a number of instances, reported amino acids were not rigorously iden- tified (87, 135). Bremner (25), and Stevenson (106), agree that the proteinaceous materials in different soils are similar in their amino acid composi- tion. The evidence in the literature tends to show that the chemical w~%_;‘uu ,pa V5" 7'." W3; ,- o ' ~ " 13 nature of soil organic nitrogen differs considerably from that of plants. In this connection Parker and Sowden (106 ) have compared the amino acid composition in soils and plants and have pointed out striking qual- itative and quantitative differences. Bremner (25) and Stevenson (135) have verified the presence in the soil ofdi , é -diamin0pimelic acid; an amino acid previously identified in bacteria only. Amino acids in soils do not appear to exist as free amino acids (20, 25, 130). Nonetheless, the question arises as to whether failure to show free amino acids may not be due to alterations during the is- olation procedure. Payne (108) was able to show that soil leachates concentrated by freeze ~drying permitted the detection of free ninhydrin reactive compounds, which he designated as free amino acids by com- paring their Rf values in one dimensional paper chromatograms with standard amino acids. He failed to show these spots when concentra- tion was effected in vacuo at 40°C. Putnam and Schmidt (112) also claimed to have identified free amino acids in the soil by column chro- matographic separation. On the other hand, amino acids do not appear to exist in soils as normal proteins (27, 73, 106, 136). Sowden and Parker (130), using the Sanger dinitrofluorobenzene procedure for tagging free amino groups, recovered large quantities of dinitrophenol derivatives of amino acids only from hydrolysates of soil humic acid preparations, none from the whole soil and relatively small amounts from the humic acid preparations themselves. From this, they concluded that soil amino ‘- ash-w l4 acids are essentially associated with humic acid. The same authors have pointed out (1 06) that it seems probable that protein itself is al- tered during humus formation. This concept is consistent with Brem— ner's finding (21) that as much as 60% of the nitrogen in some humic preparations he examined was non-acid-hydrolyzable. Furthermore, soil humates, variously prepared, do not appear to be uniform organic entities. Sowden's work (130) clearly showed that amino acids in their humic acid preparations were distributed among several fractions which showed striking differences in solubility and in their susceptibility to acid hydrolysis. In the water phase of the un- hydrolyzed mate rial, the DNP derivatives of aspartic acid, glycine, é-lysine, valine, and leucine, were identified; the ether phase of the same material contained DNP-glycine, DNP-valine, and DNP,-leucine. Interestingly enough, no DNP derivatives were identified in the water phase of the hydrolyzed mate rial; but DNP-valine and DNP-threonine were found in the ether phase. Stevenson (136), contradicting an opinion expressed by Bremner (23), felt that it was not essential to regard amino acids isolated from soil as structural constituents of proteins. "Evidently, amino acids can be protected from microbial decomposition, either by adsorption on clays or by association with soil organic colloids" (135). Stevenson pointed out that his own data was consistent with the concept that humic acid may fix amino compounds in soils. Amino acid reactions with other organic molecules as well as with ‘- i...l-Ill. i —— 15 clay minerals have been pr0posed by several investigators (17, 29, 30, 91, 102, 103, 136). Beside the amino acids, other nitrogenous organic compounds have been found in the soil. Anderson (5) identified purine and pyrimidine bases from hydrolysates of humic acid from Scottish soils. From his data he concluded that the soil nucleic acid derivatives appear to be of microbial rather than plant or animal origin. Bremner (24) has estim- ated that not more than 10% of the total nitrogen is present as nucleic acid. Adams, Bartholomew and Clark (1) consider that there is very little nucleic acid in soils. Stevenson (133, 134, 137) and Bremner and Shaw (28) have reported amino sugar nitrogen in soils in amounts e- quivalent to 1 to 10% of the total nitrogen. Glucosamine, chondrosa- mine, and their polymers, are some of the amino sugars, which have been identified in soils. Stevenson (134) found that hexosamines had accumulated preponderantly in the B horizon of some profiles studied. It has recently been recognized that clay minerals have high sorp- tive capacity for ammonia (101, 122, 128). Some of the sorbed ammonia is held in non-exchangeable form by entrapment within the clay lattice. Scott, et al. (122) found that drastic treatments with NaOH and heat failed to release considerable quantities of the ammonia present in vermiculite. Stevenson, et al. (138) has presented data which indicates that non-exchangeable, clay-fixed ammonia is included in standard Kjeldahl procedures for determining total nitrogen. This fixed am- monia was found to increase in deeper soil horizons and accounted for 16 the narrowing of soil C:N ratios with depth. Evaluation of Some of the Newer Methods Used in Soil Organic Matter Investigations Chroma togiaphic P roc edur e s According to Cramer (40), chromatography was originally intend- ed for use solely on colored substances, but as a result of numerous improvements and modifications, it has been possible, especially dur- ing the last few years, to adapt the process for employment with color- less materials. In 1941, '42, and '43, Martin, Gordon and Synge (37, 64, 86) developed a process of partition chromatography for the sepa- ration of hydrophilic substances, especially amino acids. In 1944, Consden, Gordon, and Martin (36) developed the paper partition chrom- atographic technique, now extensively used for amino acid analyses. Regarding the experimental conditions desirable for resolving amino acids on'paper, Consden et a1. (36) have found that the most satisfactory solvents are those which are partially miscible with water; solvents completely miscible with water canbe employed provided that the water content is not too high. In this case, presumably, the cel.- lulose of the paper, by a 'salting out' effect, allows the system to func- tion as a partition chromato gram. The main effect of temperature is on the rate of movement of amino acids but is also explained partly in terms of changes in composition of the phases (36). Normally, the paper chromatographic method may be applied to amino acid quantities ranging up to a maximum of 600 micrograms for each amino acid (40). 1? Inorganic salts and ions, if present in fairly high concentration, lead to considerable variance in Rf—values and also to "tail" formation. This is due to the fact that ions act as hydrophilic particle s and attract water, thus upsetting the equilibrium of the water between the solid and liquid phases of the system (40). In rendering visible the amino acids in the chromatogram, the ninhydrin (triketo-hydrindene hydrate ) re- action is recomrnended (18, 37, 40, 144). Chromatographic procedures are finding considerable application in the study of soil organic matter problems. As Bremner (22) has pointed out; "the failure of attempts to achieve useful fractionation of humus by chemical methods, suggests that the time is now ripe for an intensive application of modern chromatographic methods of fraction- ation". Before Bremner (25) first made a qualitative study of the amino acids in soils using the technique of paper chromatography, 0- thers (79, 115, 121, 124, 125) had isolated soil amino acids by chemical means but with limited results as far as yield and number of amino acids identified is concerned. Beside Bremner's (25) report, the fol- lowing workers, among others, have used paper chromatographic tech- niques in identifying and semi-quantitatively evaluating amino acids in soil hydrolysates: Davidson et a1. (41); Sowden & Parker (130); Steven- son (139). Chromatographic analysis of soil preparations has been extended by the use of column separation as in the Moore 8: Stein tech- nique (95, 112, 129, 137). The latter procedure is supposed to elimin- ate interfering substances and separate the amino acids into groups. . . . ’i‘" "" - V ‘ .Wr‘w “Wu-m ‘- .hm w—c—w 18 The procedure is involved and expensive. Furthermore, paper chrom- atography or specific chemical tests have to be used for positive iden- tification purposes. Encouraging results have been obtained by the application of paper chromatographic analyses to soil hydrolysates. More information must be gained regarding the absolute distribution of soil amino nitrogen and its chemical associations in the soil. In this connection, it is inter- esting to recall that Stevenson (135), after observing that basic amino acids appeared to be more resistant to deamination reactions in soils than mono-amino acids, suggested that they are held more firmly by humic colloids and, hence, become less accessible for enzymatic ac- tion. He also suggested that diamino molecules could form points of attachment of humic colloids to clays, hence decreasing their avail- ability. Accordingly, the ability of basic amino acids to resist decom- position in soils may also depend upon the kind and amount of clay min- eral present. Another point of interest is the reported presence (25, 129) of in— terfering materials in the chromatographic fractionation of soil hydro- lysates. Sowden (129) found an interfering material which did not seem likely to be one of the more common amino acids. It reacted readily with the Moore and Stein reagents (96) but very slowly with a ninhydrin- in-butanol spray on paper chromatograms. Full color intensity did not develop for more than 24 hours after spraying and was pink rather than the blue or reddish blue of most amino acids. Because of the 19 interference of this material, they were unable to separate tyrosine and phenylalanine properly. Other studies (5, 17, 20, 94, 106, 108, 112) on soil amino acids and their unknown soil combinations suggest that difficulties encounter- ed in isolating specific amino acids are associated with their origin in the soil. Electrophoretic Procedures Electrophoretic or cataphoretic mobilities, among other electrody- namic properties, have been used for the determination of the sign and the magnitude of the electrokinetic potential on colloidal surfaces. The electtophoretic motion of colloidal particles is due to their electrical charges, i.e. , to the presence of ionized groups (9, 89). Electropho- retic mobilities can be measured either by observing the motion of the particles directly with a microscope, or by observing the motion of the boundaries that the colloidal solution forms when the latter is placed in a U tube (33). The direction of migration indicates the sign of the char- ge on the colloidal particle. The velocity of migration is proportional to the electrokinetic potential (2 potential) existing across the Hemholtz- double layer*. The potential difference in the double layer depends on the number of ions and on the thickness of the atmosphere (89). * "From the principle of the constant ion product, it follows that a Donnan equilibrium must be established between the outer layer sur- rounding the particles, in which the ions of one sign of charge predom- inate over the ions of the opposite charge, and the outside solution which contains an equal number of both kinds of ions" (90). .. ‘i'rV. “'17- —? ‘.w m._._ 20 It has been established (85) that the 2 potential is the mo st import- ant factor governing the stability of dilute 1yophobic solutions. Marshall (85) has summarized average cataphoretic velocities for different pure clays. He has given an average value of 2. 75 M/sec [volt/cm. for H- bentonite. Mattson (89) has shown that increasing amounts of polyvalent anions associated with clay colloids increase their Z—potential. Thus, polyval- ent anions function as stabilizers of clay suspensions (85). Mattson (89), after studying the behavior of electrodyalized bentonite, concluded that the resulting saturation of the clay with hydrogen leads to a low potential difference between the interior of the atmosphere and the out- side water, as well as a low ionic concentration in the micellar atrnos- phere; thus, to a low 2 potential. As far as the author is aware, only Mattson (88) has studied the ef- fect of polyfunctional ionic molecules, like proteins and humic acid, on the electrophoretic behavior of clay minerals. He observed that the isoelectric point of a protein is lowered by bentonite and explained that change as being due to the formation of a non-ionized compound between bentonite and proteins. He concluded that "there is no doubt that the proteins enter into the make -up of the soil colloidal complex, combin- ing with the basic and acidic constituents according to the pH of the soil solution. But the reactions in the soil are so complex that a discussion of the subject must be postponed. . . " 21 X-RaLDiffraction When an X-ray strikes an object that is made up of repeating units whose distances of separation are commensurate with the wavelength of the X-ray, we obtain diffraction of the X-rays. Diffraction involves the reinforcement of rays whose waves are in phase and the cancella- tion of rays whose waves are out of phase. X-ray diffraction studies of fatty acid crystals (55, 111) have shown three principal lattice spacings. The two small similar spacings are nearly independent of the carbon chain but are dependent on the thickness of two parallel fatty acid molecules, while the long ones do depend on the carbon chain and approximately correspond to two molecular lengths of fatty acids. In a crystal, the carboxyl groups form the main reflect- ing planes and are packed in double layers alternating with double lay- ers of methyl groups; the double layers of methyl groups do not reflect. The individual hydrocarbon chains have parallel orientation. Regarding X- ray diffraction studies with peptides, repeating distan- ces along the fibre axis of 7. 021 have been reported. This is the distan- ce occupied by two amino acid residues. The side chain spacing was 5. 2 X and that of the "back bone" of the peptide of silk fibroin was 4. 6 2(33). Astbury and Street'(7), as cited by Bull (33), were able to show that there is a definite relation bet ween the molecular structure and the extent of stretching of a protein fiber. Thus ordinary hair or wool is reported to exist as an at -Keratin with a repeating distance along the fiber axis of 5. 5 X. When the hair is extended sufficiently, it is 22 changed intofi -Keratin with a repeating distance along the fiber of 3. 32 X, a backbone spacing of 4.6 X, and a side chain spacing of 9.8 X. The structure ofB-Keratin is that of a stretched peptide chain (4, 39). Bernal (14), in an extensive survey, tabulated the results of prelim.- inary studies offifteen amino acids and related compounds. including diketopiperazine, by means of X-ray. No information about X-ray diffraction by glycerated amino acids was found in the literature. The information presented in this work appears to be the first. Regarding X-ray diffraction studies with clay minerals, extensive work has been published, and principal diffraction peaks are well known. Quite reliable techniques for differentiating one clay mine ral from an- other are available (69, 92). Ensminger and Gieseking found that the 001 spacing of bentonite in- creased with the adsorption of proteins and with decrease in pH (52, 53, 61). They inferred that the large protein molecules were adsorbed within the variable portion of the 001 spacing of the crystal lattice. When the amino groups were destroyed by treating the protein with ni- trous acid, the clay with untreated gelatin gave a 001 spacing 8. 0 A higher than that with treated gelatin. No X-ray diffraction patterns have been published of amino acid- clay complexes as such. Infra Red Absorption The infra red region of the electromagnetic spectrum may be div- L a a. w _ 2 A r mun a. ,9 h a, 4 w— 5,. 2 r 23 ided into four sections (71 ); the photographic (visible to 1. 2 microns), overtone (visible to 3 microns), near infra red (2.5 to 25 microns), and far infra red (25 to 300 microns). In the photographic region the principal absorbancies observed are associated with electronic transi- tions. Rotational and vibrational effects are also superimposed. The overtone region is so called because transitions can occur at energy levels which are multiples of the "finger print" section. We find rota- tional and vibrational absorption in this region. Absorption in the near infra red is caused by the vibration of atoms about an equilibrium po- sition in the molecule and the combination of these vibrations with ro- tation of the atoms to produce vibrational spectra. This is also called the "finger print" infra red region. Last, absorption in the very far infra red derived from transitions from radiant to rotational energy only. The region of the spectrum of interest for analysis is from 2. 5 to 25 microns, equivalent to wave numbers of 4, 000 to 400 cm. -1 (71). Infra red absorption has been successfully applied to clay minerals and soil organic fractions (2, 74, 93). Bertramson (15) has observed that with some improvements this method will, probably, be the best single method for clay mineral identification. As far as the infra red spectra of amino acids are concerned, Bellamy (12) has presented a detailed review of the literature on this subject. He points out that no absorption in the usual NH stretching -1 region of 3,500 to 3,300 cm. (2.9 to 3. 0A.) is shown by any amino 24 acid or hydrochloride. This is supported by the work of many inves- tigators cited by Bellamy (12). According to Bellamy, all amino acids capable of possessing the NH: structure and their hydrochlorides, show two characteristic absorptions in the region of 1,600 to 1, 500 cm. -1 (6. 3 to 6. 7,“). In addition, an ionic carbonyl absorption also takes place in this region. The first of these, 1,660 tol,610 cm. '1(6. 0 to 6. 2,“), is often weak while the second, 1, 550 to 1,485 cm. -1 (6. 5 to 6. 7,44,), is usually more intense. The NH vibration for amido acids (NHZ present in an amide instead of an amine) falls in the range 3, 390 to 3,260 cm. '1 (3. o to 3.1/1). No infra red absorption bands characteristic of amides, proteins, polypeptides, and amino acids are reported in the literature (12, 113) for frequencies greater than 4,825 cm. -1 (2.1 ,u.) or less than 880 cm.-1 (11.4fA). There is, nevertheless, information regarding infra red absorption in this region by aromatic compounds, including aromatic amines. Bell (11), studying the absorption spectra of organic derivat- ives of ammonia, particularly anilines, found that the band occurring in the region 2. 8 microns may be regarded as due to amino groups. Alkyl and aryl substitutions on the amino group of aniline caused the band to become shallower and further additions (yielding tertiary am- ines) caused the 2.8 micron band to disappear. A 2. 3 micron (4, 348 cm. -1) band is present in aniline, as well as substituted anilines. Ellis (46) observed bands in toluene, xylene, and mesitylene, in the region -1 2. 3 to 2.4 microns (4, 348 to 4, 167 cm. ) while Glatt and Ellis (63) g. 15' vi v -F [villIIIa'I-Il 25 reported absorption bands in the 4,216, 4, 322, 5,671 cm. -1 (2.4, 2. 3, 1. 8M) and higher frequencies for polythene and Parowax. They also found a 4,291 cm. ‘1 (2. 3 p.) band in nylon, a polyamide. Colthup, as cited by Bellamy (12), found that the band within the range 1,625 to 1,575 cm:1 (6. 2 to 6. 3 IL) was characteristic for most aromatic materials. Again, according to Bellamy, these bands are highly characteristic of the aromatic ring itself and, taken in conjunc- tion with the C-H stretching band near 3, 030 (3. 3,“), they afford a ready means of recognition for this structure. The following remarks are cited from Bellamy (12). "On theoretical grounds, the main 1,600 and l, 500 cm. -1 (6. 3 and 6. 7,14.) bands of the phenyl ring can be expected to occur also in polycyclic materials where there should be expected some broadening of the ranges over which they can occur. ' The 1,600 to 1,500 cm.-1(6. 3 to 6.7g.) absorption bands are notorious for very wide fluctuations in inten- sity. Frequently, the bands are weak in conjugated structures, and are often shown only as shoulders on other bands. 'However, heterocyclic aromatics such as pyridine and pyrimidines also give a similar pair of bands in the 1,600 cm. "1(6.3,u.) region from the c : c and C = N links, although in this case the third skeletal ge— 1m!” 26 vibration is usually at appreciably lower frequencies. In certain cases in which the carbonyl absorption is capable of shifting toward the 1,600 cm. ‘1 (6. 3,0.) region under the'influence of strong hydrogen bonds, this sometimes makes it difficult to differentiate be- tween the two. " Bellamy has presented a detailed discussion of the infra red ab- sorption of aromatic molecules and their derivatives in the region of l, 000 cm. -1 (10. 0,44.) but, as he pointed out, the lower intensity of the- se bands renders them less generally useful than the higher frequency absorptions. "No information is available in the literature on these absorptions, and the correlations depend almost entirely on the unpu- blished work of those workers who have drawn up correlation charts" (12). ME THODS OF ANALYSIS Total Carbon Total carbon was determined by the dry combustion method (110). Carbon Dioxide Evolution Durinflncubation Carbon dioxide evolved during incubation at 35°C was collected in 0. SN sodium hydroxide. One-hundred-gram samples'of soil were placed in two-quart glass fruit jars. Water was added to bring the soil to the moisture equivalent. The alkali for CO; absorption was introduced in small vials, placed on the soil surface, and the jars were sealed. The jars were thoroughly aereated and the vials of alkali replaced at one to four-day intervals. Unused alkali was titrated with standard hydrochloric acid in the presence of phenol- phthalein and an excess of BaClz, and in a COZ-free atmosphere. Total Nitrqgen Total nitrogen in soils and in soil fractions was determined by the mic ro-Kjeldahl method using a 1:1:8 mixture of SeOZ:CuSO4:KZSO4. Ammonia was collected in a 2% boric acid solution and titrated with standard acid in the presence of bromcresol green-methyl red indicator (81). Nitrifiable Nitrogen o Nitrate released during incubation of moistened soils at 35 C was determined by the Iowa incubation procedure (131). Primag Hydrolytic Fractionation of Nitrogen The nitrogen fractionation used in the present work has been des- I _»..9 _ -_. 28 cribed elsewhere (26, 98, 114, 142). The diagram on Figure 1 shows the detailed procedure followed in the fractionation and the determinations which were performed on every fraction: Van Slyke Nitrogen Amino nitrogen was estimated using 5 ml. aliquots and strictly fol- lowing Van Slyke's nitrous acid technique (109, 141, 146, 147). Standard amino acids run prior to the determination of the unknowns demonstrated recoveries of 100 - 0.1% of theoretical amino nitrogen. Photometric Ninhyd rin Proc edur e Alpha amino nitrogen was estimated photometrically with ninhydrin as described by Troll and Cannan (140). De salting Soluble fractions were desalted electrolytically, prior to spotting on paper, with a commercial desalter manufactured by the Research Equipment Corporation, Oakland, California, Model X-lOO (157). Paper Chromatography Two-dimensional ascending paper chromatography was employed, using Whatman No. lpaper, 30 cm. square; 80% phenol was used in the first direction and butanol-acetic acid-water (40:10:50) in the second direction through the fastest grain of the paper (18, 40, 72). The phenol was purified by distilling at reduced pressure and a constant temperature of 84°C, and was never used mo re than once; the butanol solvent was always prepared fresh immediately before using it. A desalted aliquot of 10 mic roliters was always spotted JL Z9 SOIL SAMPLE REFLUX I2 HRS. IN 6 N HCI, FILTER , WASH WITH HOT WATER j FILTRATE INSOLUBLEI RESIOUE l l REPEATEO CONCENTRATION TOTAL N TOTAL C IN VACUO AT 40°C FRACTION A ~ I I I TOTAL N PHOTONETRIC DESALT NINHYDRIN ESTIMATE ELECTROLYTICALLY , FRACTION A'. NAKE ALKALINE WITH CAIOle ~ DISTIL IN VACUO AT 40" C .u..‘_, ... ,., e-F- .4 t a_-l;- gag, '. “WT-Wu_r 30 from a micropipete held at 3 cm. distance from the same corner of the paper while drying with a hair drier and an infra red lamp The papers were dried at 90°C in the oven after each solvent and after spraying with a 0. 2% alcoholic ninhydrin solution. Mixtures of pure amino acids of considerably different Rf values were chromatographed by the above described procedure to pro- vide standard reference chromatograms. Separation of the Phosphotungitic Acid Fractions Basic and non-basic nitrogen compounds in soil hydrolysates were separated using purified phosphotungstic acid (145) and following the procedure outlined by Dunn and Drell (44) for removing the phosphotungstic acid with a 1:1 mixture of amyl alcohol and diethyl ether (143). Column Separation Procedure Hydrolyzable nitrogen compounds were separated into basic, di- carboxylic, aromatic, and neutral non-aromatic fractions by clo- sely following Fromageot and Lederer's chromatographic separa- tion procedure (59). Aliquots of the desalted fraction B1 (see Fi- gure 1) or intact portions of fraction A were used, since the latter could not be directly desalted because of foaming and decomposi- tion shown on trial runs. Figure 2 shows the sequence followed during this part of the work. Electrophoresis Electrophoretic mobilities were observed on 10 ml. aliquots of 31 SOLUBLE FRACTION NEUTRALIZE' WITH Ll OH PERFUSE SILICA COLUMN WASH WATER esnrusns _ SORBATE coucsmans m VACUO AT 40°C ELUTE WITH om HCI l usurssuzs mm u on ELUATE I (ensue u coupouuom PERFUSE ALUMINA cowuu was" | “"7" "23' S‘T’R‘fl" "ATE“ couceursns m VACUO AT 4o°c Pensusns SORBATE TOTAL u PHorousTmc umuvoam ESTIMATE coucsutans m vaggg ELUTE wmt HOT 0.1» 0" " N "2 - AT 40° c H c I TAKE urm sssacsnc amine nno PAPER ACID SATURATED mm (ACIDIC Nlcoupouuom c "”0”""Y a, s 32 . 03% clay suspensions containing particles with diameter less than 0. 7 microns, using the moving boundary technique (33). A potential gradient of 5.25 volt/cm. was used and the medium was prepared by adding 0.1N NaCl to distilled water until the specific conductance equaled that of the clay suspension. X-Rays In the X-ray and infra red analyses, aliquots of the same prepar- ations employed in the paper chromatographic analyses were used. Diffraction patterns of the soil fractions were obtained with a Phillips Norelco X-ray spectrometer (1950 model) equipped with a Copper target. One mililiter of the sample was served over a microscope slide and completely dried under an infra red lamp, except where otherwise indicated. Infra red The infra red absorption studies were conducted with a model-21, double beam, self recording spectrophotometer, using NaCl plates, and Nujol or hexachlorobutadiene as mounting medium whenever the sample was not thick enough to smear directly on the salt plates. Glyceration Glyceration was accomplished by mixing the sample with C. P. glycerine (4:1 sample to glycerine ratio). After glyceration, the samples were dried inside a vaccurn oven at 70°C for 4 days in the case of infra red analysis. “ u. ,,,'-_.‘4 l ”5. .. ' 1"» -,_. q...- I. .......——- ._.. # SI wi ed CHARACTERIZATION OF THE SOILS Field Description“ The soils used in the present work were taken from a field exper- iment on Plainfield sand at the Rose Lake Experiment Station, Michi- gan State University. Various plots in this experiment were treated in 1954 with Wyoming Bentonite at rates of 0, 6.25, 12. 5, 25, and 50 tons per acre. After application, the clay was mixed into the top six inches of the soil, using a rototiller. One ton of limestone per acre and half-a-ton of 10-10—10 fertilizer was also applied. The following cropping and fertilization sequence was followed after treatment (99): Alfalfa was seeded with oats in the Spring of 19 54. Oats were harvest- ed in 1954 and one cutting of alfalta in 1955. Wheat was planted in September 1955 with 640 pounds of a 12-12-12 fertilizer broadcast ahead of planting. Forty pounds of nitrogen per acre was applied as a tOp dressing of ammonium nitrate on wheat in the Spring of 1956. The plots were sampled to a depth of 6 inches in March 1957. The samples were air dried, sieved through a 10 - mesh screen, and com- posited by mixing 300 g. from each of the five replicates. Significant increases in yields of oats, alfalfa, wheat, and corn with the clay treatments in the plots described above have been report- ed (99). Although there were significant increases in the yield of wheat * The field experiment described was established by Dr. M. M. Mortland and Dr. A. E. Erickson, Soil Science Department, Michigan State University. 34 and alfalfa above the check plots for all clay treatments, oat and corn yields showed significant increases for the low and intermediate levels of clay application only. In fact, the yield of oats decreased with the two high clay increments; the yield of the check being higher than the 50 tons per ac re clay treatment, but lower than the rest. Laboratory Determinations Total nitrogen and total carbon were determined in the composited soil samples. It was found that C:N ratio decreased with increasing clay treatment (Table 1). Table 1. Carbon and Nitrogen in the Soils of the Experiment Clay applied tons/acre % Carbon % Nitrogen C:N 0 0. 30 0. 0245 12. 5 6. 25 0.37 0.0295 12.3 12.5 0.36 0.0290 12.4 25 0.34 0.0305 11.3 50 ' 0. 33 0.0310 10. 6 36 These results are in harmony with those of Allison et a1. (3), Bower (l9), and of Ensminger and Gieseking (51). It should be pointed out that only trace amounts of exchangeable ammonia or of nitrate-ni- trogen were found in the soils studied here. Although the carbon con- tent was always higher for the clay-treated soil than for the check, there was, nevertheless, a tendency for the carbon to drop again with the heavier clay treatments. The latter tendency was accompanied by a steady increase in total soil nitrogen. As a result, C:N ratio de- creased with successive clay increments. It is highly interesting to note that these findings were evident three years after the sandy soil was treated with clay. Using artificial sand-colloid preparations Allison showed that certain clay colloids, especially montmo rillonite, protect organic matter against microbial attack (3)—. Because of the field plot arrangements and cropping history of the experimental field, it can be assumed that the whole area was in con- tact with the same type of plant residues. Differences in quantity of residues returned, however, were undoubtedly associated with the re- corded differences in yield of the various crops (99). Nevertheless, it does appear that nitrogenous compounds were preferentially retain- ed by clays during the biochemical transformation of these residues. Ensminger and Gieseking (51) have suggested that soil organic mat- ter occurs in combination with inorganic colloids. Previously, Mattson (88) had suggested that the tendency of proteins to combine with inorg- anic colloids may account for the high nitrogen content of humus and II 37 of the B horizons of soils. He observed that "silicate of gelatin", aluminum "proteinates", and leather ("protein tannate") were very re- sistant to decay. In some respects these substances are comparable to clay-protein or clay-hunus complexes. Mattson found that a steady increase in positive cataphoretic mobility of protein "bentonates" was associated with increasing protein content (88). These observations led us to attempt the characterization of the treated soils in terms of the cataphoretic mobilities of their colloidal fraction. The electrokinetic behavior of 0. 03% clay suspensions extracted* from the composited sandy soil treated with the indicated levels of Wyoming Bentonite in the field for three years, is shown in Table 2. Since it was considered possible that the observed positive migra- tion could have been due to the presence of crystalloidal substances in the suspensions, the clay samples were then dialyzed in collodion bags, against running distilled water for a period of seven days, until first free of chlorides and then calcium, as detected by the silver nitrate and ammonium oxalate tests, respectively. Table 2 contains the migration velocity and the corresponding val- ues of the 2 potential for both the dialyzed and the undialyzed clay suspensions. The 15 minute interval was chosen for comparing the 2. potential of the clay suspensions because this was estimated to be a good average for most treatments. Besides, some determinations had * Particle sizes smaller than 0. 7 microns were obtained by the pipette method, following dispersion in distilled water. Table 2. Migration Velocities and Calculated Values of 2 Potential for the Clay Suspensions After 15 Minutes Clay treatment Migration velocity 3 potential tons lac re mic rons lsec . millivolts Undialyzed o 9.4 22. 34 6. 25 14. 7 34. 70 12. 5 1 3. 1 31. 02 25 15.6 36. 80 50 18. 0 42. 58 Dialyzed 0 0 --* 6. 25 10. 0 23. 66 12. 5 5. 6 13. 14 25 11. 1 26. 29 50 12. 22 28. 91 * Precipitation after closing the circuit for running the trial invalidat- ed this determination. 39 to be discontinued after 15 minutes because of lack of uniformity in the ascending boundary. It should be noted that figures on Table 2 are relative rather than absolute. The 3 potential has never been measured directly, but al- ways calculated from observed movements which, in turn, are depend- ent on factors such as viscocity and dielectric constant of the medium, particle size and shape, and the electrical potential imposed. These figures show the comparative electrophoretic behavior of the colloidal fractions of the different soils. There was, in fact, a reduction in Z potential when the suspensions were dialyzed, indicating that adsorbed ions had contributed to the high mobility of the undialyzed clays. The native soil clay in the check was completely precipitated at pH 6.1 (Table 3). There was a general trend for increasing 2 potential with increasing level of clay treatment. At first glance, it might appear that such behavior could be attributed so- lely to dilution by bentonite of the native soil clay. As will be shown later, the native clay was largely chlorite, a slow moving clay. However, the re was an inversion of mobilities in the dialyzed sus- pensions betvfien the 6. 25 and 12. 5 ton per acre levels of clay treat- ment. The low 2 potential for the dialyzed clay from the 12. 5 ton treatment was associated with a relatively high carbon content of the dialyzed suspension (Table 4). A generally inverse relationship be- tween 2 potential and carbon content existed for the whole group of soils, which suggests that organic materials associated with the clay 40 Table 3. The pH of Soil Suspensions and of 0. 03% Dialyzed Clay Suspensions pH Clay treatment 0. 03% Dialyzed tons / ac re 1:1 Soil Suspension clay suspension 0 5. 3 6. 1 6. 25 5. 3 6. 0 12. 5 5. 5 6. 0 25 6. 0 6. 1 ‘Var’ Table 4. 41 Carbon and Nitrogen Analyses"I of the Dialyzed Clay Suspensions Clay treatment tons / ac re % Carbon % Nitrogen C:N 0 0- 32 not detectable CD 6.25 0.20 0.84 0.24 12.5 0.34 0.84 0.40 25 0. 15 0. 92 0. 16 50 0. 18 l. 00 0. 18 * The assumption is made that the clay suspensions were . 03% WIv and the percentage figures are based on air dry weight of the initially added clay. .—-—.—-.w_ ‘rv— _ ._4...‘ .. |\-. ‘33—‘3- 42 influenced its electrophoretic behavior. Associated carbon appears to have been more significant than nitrogen, since nitrogen content in- creased rather consistently with clay treatment and did not reflect any corresponding deviation for the erratic 12. 5 ton treatment. The deviating behavior of the soil from the 12. 5 ton treatment does not appear to be due to sampling error, since the clay content was in line with the level of treatment. Moisture equivalents also showed the expected trend: 3. 1% for the check soil and 3.1, 3. 6, 4.1, and 10. 3% for successive clay applications. The high carbon content of this soil appears to be related to the fact that maximum crop yields have been the highest with this treatment, from which it may be inferred that larger quantities of residues have also been returned to the soil with this treatment. The low C:N ratios for the clay suspensions (Table 4) indicate that only a portion of the nitrogen in the dialyzed clay could have been or- ganic. Since it was not removed by dialysis, much of it may have been ammonia entrapped within the clay lattice. The results of carbon dioxide evolution during a. thirty-day incuba- tion study are shown on Table 5. In contrast with the observed decom- position of the organic matter after two weeks, the initial decomposi- tion of the organic matter in these soils was independent of the clay or nitrogen content. Recalling that the C:N ratio for the whole soil at the three lower clay application levels was rather similar (Table 1), the observation is made that the increased evolution of C02 with increased Table 5. Carbon Mineralized During Incubation of the Soils of the Experiment Milligrams of carbon evolved as CO; per 100 grams of soil - Clay applied 2 3 5 7 ll 16 30 tons lacre days days days dgays day_s days days 0' 4.0 6.8 10.2 12.8 17.6 21.9 27.5 6.25 4.3 7.3 11.1 12.9 18.2 24.3 31.7 12.5 4.5 7.4 11.0 13.6 18.9 24.0 34.2 25 4.6 7.2 11.9 14.3 20.5 25.5 36.8 50 4.9 7.5 11.1 13.2 19.3 26.1 37.3 '1' . fl ‘ fl- . ‘x... "~._ -. 1.. tfi-‘g'rfl t- . ' 1 . v. . a . . - l' g B§Wf£fl' E JV 5 "2 i "‘ ' 6 ‘ ‘ ‘ ‘ ' g i’ .' t, ‘ ‘ - .— — .—. v — - — _ -‘ "‘7 Tw-I-vz: 44 clay treatment after two weeks appears to be associated with the che- mical nature of the organic constituent rather than with its C:N ratio or its nitrogen content. These results are further supported by the fact that the NO3-nitrogen accumulation for two weeks' incubation was 15 parts per million for all soils except the check. The latter nitrified 6 parts per million NO3-nitrogen during the same period. The protec- tion of soil organic matter by montmorillonite has been demonstrated by several investigators (3, 51, 88): The results reported here indi- cate that nitrogen is stabilized more effectively by this protective ac- tion of clay than is carbon. It would also appear that residual carbon retained during previous decomposition in the presence of clay is more readily available to microbial attack as the prOportion of clay is in- creased. The X-ray diffraction data for the clay suspensions is shown in Table 6 and Figures 3, 4, and 5. Chlorite appears to be the principal native clay mineral (Figure 3): This is supported by its non-expanding behavior demonstrated by its non-pcollapsing lattice when heated to 500°C after KCl treatment. It shows high intensity of its second and third order reflections. Figure 4 shows that all clay mineral prepara- tions, except thelcheck, show an expanded lattice above the limits of pure Bentonite. Interstratification of the Wyoming Bentonite was evi- dent from the behavior of the clay mineral suspensions (other than the check) after heating to 500°C (Figure 5). Bands, rather than peaks, having a very low intensity were observed; diffraction hardly occurred, 13.? ‘ 45 o A: ”BOA m .NTm .w .o .3 m .31.... .w .o .vm m .NTm .w .m .vH o .3 Uooom 3 powwow“ u "UM m A: umod o .vH o .«A m :3 o .3 Uoo: 0» won—do: : HUM N .v 930: o o .3 m 6H .w .mH N .vH .w .2 o .3 .o .mu m :3 .o :3 .m .o 6 $192 haw Doc: 3 68.603 m .3 undo: vN ham v.3 m s: .w .wu .m .3 .w .2 N .vfi .m .5; m JH .m .wH o .3 neueofinmop E UUEQ «.3 9:: .~.3 vaH .~.$ m4; 3...: ml“: 5.2 o.: 0:02 euflcofinom om mm m .3 mN 6 o «confides-ohm 9:50.»? hdunx o.” u< you much. 5 «nogumouh >20 A325 .4. a: 339.com maggot»? wouzmwfl can snowmcomudm >30 woubdwfl 05 mo 330nm fioflodumflfl tnwmuun o .o 34MB 46 FIGURE 3. X-RAY DIFFRACTION PATTERN OF DIALYZED NATIVE CLAY ISOLATED FROM THE CHECK SOIL J L. .g-< «H- . v-‘-‘> a a! 47 4 no 4w.m. <06; $3 335 225$,qu m w x. m o o. : .2 mi 3 n. 2 t m. m. om _~ - _ p _ _ _ _ . p . . . r _ _ _ T _ u <~é FIGURE 4. X-RAY DIFFRACTION PATTERNS OF DIALYZED CLAY SUSPENSIONS AND WYOMING BENTONITE AFTER HEATING Pattern A Pattern B Pattern C Pattern D TO 110°C Corresponds to the check soil Corresponds to the 6. 25 tons of clay/ac re treatment Corresponds to the 50 tons of clay/ac re treatment Corre sponds to Wyoming Bentonite 48 i..— -27. .- H.011 W S DuFFRncnou RNQLE (2 e) 49 50 FIGURE 5 . X-RAY DIFFRACTION PATTERNS OF DIALYZED CLAY SUSPENSIONS AND WYOMING BENTONITE AFTER TREATMENT WITH POTASSIUM CHLORIDE AND HEATING TO 500°C Pattern A - Corresponds to Wyoming Bentonite Pattern B - Corresponds to the check soil Pattern C - Corresponds to the 6.25, 12. 5, 25, and 50 tons of clay/acre treatments / 14.“ JA 3\K DIFFRRC'HON RNC‘LE (as) ‘Er'p M . :“V' .‘7‘ 1;."- '..‘v-“. ' -w ‘ xiii??? if ‘~ "1*:-"V -,i, _ ._.-s._—— 52 suggesting disorientation of the crystal lattice, probably due to an in- complete burning of entrapped organic molecules between the clay lat- tice. The high nitrogen content of the clay suspensions, along with the results of X-ray and electrophoresis studies, made us suspect that some nitrogenous organic constituents were interacting with the Wyoming Bentonite applied to the sandy soil. The low C:N ratios of the colloidal suspensions indicated that not all of the nitrogen associated with the clays was organic. However, as will be shown in the next chapter, there was no relation between clay treatment and the hydrolyzable ammonia fraction, where inorganic nitrogen would appear. The close inverse relationship between cataphoretic mobility and carbon content suggested that organic, rather than inorganic, nitrogen compounds were principal- 1y effective in altering the charge of the clay. , v.1 ; ..._ - . - . ~ 1 - ni'f.~-r_mww:sr~m 9.x" ;.v11r:11--._ , .--... NITROGEN FRACTIONATION The results of fractionating the total soil nitrogen of the different soil treatments into acid and non-acid hydrolyzable forms are present- ed in Table 7. As pointed out before, there was a definite trend for total soil nitrogen to increase with level of clay application. There was, essentially, no difference in the non-acid hydrolyzable nitrogen, especially in the case of the clay-treated soils; the check soil showed a somewhat lower absolute amount of acid resistant nitrogen, but this represented a larger proportion of the total soil nitrogen than in the soils treated with clay. The proportions of nitrogen liberated during hydrolysis are in agreement with those reported in the literature (26, 79, 97, 106). The data indicates that there was a distinct trend for the acid-hydrolyzable nitrogen to increase absolutely and percentagewise with increasing clay treatments. It appears that the clay-associated organic nitrogen may have been protected from enzymatic transformation in the soil (100), but yet, it was susceptible to release by strong acid hydrolysis. On the other hand, the non-acid hydrolyzable nitrogen appears to have been inde- pendent of the amount of clay and of the acid hydrolyzable or total ni- trogen. This suggests that this acid resistant soil nitrogenous fraction was associated with the original soil organic matter or humus, rather than with any nitrogenous organic fractions formed during the three- year period since clay treatment. Mattson and Koutler-Andersson (91) regard this acid-resistant fraction to be the product of chemical fixation Table 7. ”212; 3....-- 54 Recovery of Soil Nitrogen in Hydrolyzable and Non-Hydrolyzable Forms as Related to Clay Treatment Clay treatment N fraction None 6. 2.5T 12. 5T 25T SOT N, - mg. per 100 g. soil Total soil 24. 5 29. 5 29. 0 30. 5 31. 0 Non-hydrolyzable 6. 8 7. 2 7. 2 7. 4 7. 4 Hydrolyzable 17.4 21.4 21.8 22. 6 24. 3 (Fraction A) Not accounted for -0. 3 -O. 9 0 -0. 5 l0. 7 N, - percent of total soil N Non-hydrolyzable 27. 8 24. 4 24. 8 24. 3 23. 9 Hydrolyzable 71. 0 72. 5 75. 5 74. 1 78. 4 (Fraction A) Not accounted for -1. 2 -3. 0 0 -1. 6 {2. 3 55 of ammonia which occurs simultaneously with the autoxidation of lignin or other aromatic constituents or decomposition products of plant ma- terials. Stevenson (137) has suggested that amino acids or peptides may be similarly complexed by oxidized humic materials. Broadbent (29) has expressed the opinion that a sizable part of the organic nitrogen in soils may be in forms not closely related to compounds produced by plants or microorganisms, but may, instead, be produced in the soil. The data in Table 7 suggest that the non-hydrolyzable nitrogen fraction may have been more directly related to the degree of oxidation of such soil-formed nitrogen complexes than to the quantity or quality of plant residues returned or to the quantity of clay associated with their de- composition during the three-year period since treatment. A further fractionation of the acid-hydrolyzable nitrogen was a- chieved by making it alkaline with calcium hydroxide and subjecting it to distillation in vacuo at 40°C. This treatment results in the release of hydrolyzable ammonia and the precipitation of an alkali insoluble fraction, called humin. There is left an acid-and alkali-soluble super~ natant (Fraction B). Table 8 shows the results of this fractionation. The proportion of humin nitrogen decreased about half as the result of clay treatment, but there was no apparent significance in the differences between the several clay treatments. It should be noted that the abso- lute amount of nitrogen as well as the percent of the total nitrogen pre- sent in the soil hydrolysates after removal of ammonia and humin in- creased with successive clay treatments. This trend was less evident 1*:" .. .. , 1 ”HEX. if _ ~ fit?n y”: 1: 'VI 0- 56 Table 8. Recovery of Nitrogen in Soil Hydrolysate (Fraction A) as Related to Clay Treatment Clay treatment N fraction None 6. 25T 12. 5T 25T SOT N, - mg. per 100 g. soil FractionA 17.4 21.4 21.8 22.6 24.3 NH3-N 1. 8* 4. 5 3. 8 4. z 3. 5 "Hmnin" N 3.7 2.2 2.6 2. 5 1.9 Fraction B 11.9 14.2 14.2 16.2 19. 5 Not accounted for -- -0.5 -1.2 [0. 3 -0.4 N, - percent of total soil N Fraction A 71. 0 72. 5 75. 5 74. l 78. 4 NH3-N 11.4 15.2 13.1 13.8 11.3 "Humin" N 15. 1 7.4 9. O 8. 2 6.1 Fraction B 48. 6 48. 1 49 . 0 53.1 62. 9 Not accounted for -- -1.7 -4.1 {1.0 -1.3 N, - percent of total N in Fraction A NH3-N 16.1 21.0 17.4 18.6 14.4 "Humin"N 21.3 10.3 11.9 11.1 7.8 Fraction B 68.3 66.3 65.1 71.7 80.2 Not accounted for -- -2. 3 -5. 5 l4. 4 -l. 6 *Calculated by difference rather than analyzed in this one sample only. - . ,.’... if ’1‘ 1.7,. 57 when the nitrogen in Fraction B was compared with the total nitrogen in the acid hydrolyzable fraction (Fraction A). It is possible that this lack of consistent trend in the proportion of hydrolyzable nitrogen re- covered in Fraction B may have been related to the trends in hydroly- zable nitrogen not accounted for. It is known that Kjeldahl procedures do not give as complete recovery of nitrogen from organic materials as can be achieved, with the Dumas catalytic ignition procedure (23, 29). The origin of the hydrolyzable ammonia has been regarded as spe- culative (136). It may come from decomposition during hydrolysis of acid amides (124) or amino sugars (132) or organic complexes (91). Without doubt, exchangeable ammonia is included, as well as some of the ammonia fixed irreversibly by clay minerals (122). According to Shorey (124), secondary products formed by polyme- rization and condensation during the laboratory procedures of some of the primary splitting products can account for the presence of humin nitrogen. Stevenson (132) considers that the acid hydrolyzable nitrogen left after removing the ammonia and humin is essentially amino-acid nitro- gen. By this criterion the values of amino acid nitrogen reported here were observed to be higher than those reported by some workers (26, 79), but similar to those of others (53, 132). The results of the nitrogen fractionation so far presented showed that the preferential accumulation of nitrogen over carbon with inc reas- ing clay treatments observed in Table l was principally due to an in- ""13”,. r. W, ._‘Q’Jt’. . --—‘. — -_. . - 58 crease in acid hydrolyzable nitrogen. Since the increase was largely in the acid-and alkali soluble Fraction B, it was inferred that clay treatment had resulted in an accumulation of amino nitrogen. To find out how this amino nitrogen was distributed in the soil with respect to the clay treatments was the next objective in the present work. The first step in this direction was to fractionate the acid and alkali soluble nitrogen (Fraction B) into its basic (Fraction D) and non-basic (Fraction C) constituents. This was achieved by precipitating the basic nitrogen compounds with phosphotungstic acid, as the Hausmann protein frac- tionating procedure (65). The results of this fractionation are present- ed in Table 9. The values in Table 9 are quantitatively similar to those reported by Kojima (79). The results of this step revealed that both basic and non-basic forms of hydrolyzable nitrogen increased following applica- tions of Wyoming Bentonite to the soil. In the case of the non-basic Fraction C, there was no consistently direct relationship with the am- ounts of clay which had been applied. However, the nitrogen in the basic Fraction D increased quite consistently with clay application up to 25 tons per ac re. Although there was no additional increase where 50 tons of clay was applied, the basic nitrogen recovered was still three times that found in the check or the soil which received the 6. 26 tons per ac re application. This finding appears to be in agreement with Stevenson's proposal (135) that the ability of basic amino acids to resist decomposition in soils may depend upon the kind and amount of Recovery of Nitrogen in Fraction B as Related to Clay Treatment Table 9. 59 N fraction Clay treatment Fraction B Fraction C (non-basic) Fraction D (basic) Not accounted for Fraction B Fraction C (non-basic) Fraction D (basic) Not accounted for Fraction C (non-basic) Fraction D (basic) Not accounted for None 6.25T 12. 5T 25T SOT N, - mg. per 100 g. soil 11.9 14.2 14.2 16.2 19. 5 9.3 13.6 12.7 11.8 16. 5 1.3 1.3 2.1 4.4 4. 0 -1.3 {0.7 {0.6 0 l1 0 N, - percent of total soil N 48. 6 48.1 49. 0 53.1 62. 9 38. 0 46 .8 43.8 38.7 53.2 5.3 4.4 7.2 14.4 12.9 -5. 3 {2.4 {2. 1 0 {3.2 N, - percent of total N in Fraction B 78.1 95.8 89.4 72.8 84.6 10.9 9.2 14.8 27.2 20.5 .10.9 {4.9 {4.2 o {5.1 60 clay mine ral present. Other workers (3, 53) have, previously, sug— gested a similar interaction. In order to determine how much of the nitrogen in the basic and non- bas ic fractions could be accounted by the Van Slyke nitrous acid proce- dure , these two separates were subjected to this treatment following the recommendations of Peters and Van Slyke (109). The elemental ni- tro gen evolved was measured manometrically. A reaction time of three minutes was allowed. Alpha-amino nitrogen, if present, is deaminated by this procedure. However, Kojima (79) and Bremner (20) have ob- served that Van Slyke's nitrous acid method for“ -amino nitrogen de- termination of soil hydrolysates yields results higher than expected. The results of this step are shown on Table 10. There appeared to be no relation between the Van Slyke-nitrogen and clay treatment in eithe :- fraction. We were, therefore, lead to attempt a more detailed frac tionation of the acid hydrolyzable nitrogen left after removal of am- monia and humin. The acid hydrolyzable supernatant was then fractionated into func- tional constituents by the process of column separation recommended for protein hydrolysates by Fromageot (59). Here, basic materials, such as diamino acids, are retained in a silica column, the acidic or diearboxylicin an alumina column, the aromatic in a charcoal column, and the final perfusate contains neutral non-aromatic materials. The Procedure was tested using pure amino acid mixtures belonging to the ba- sic, acidic, neutral non-aromatic, and aromatic groups. These ~1 $11W1'4‘c‘u it: . if”! ' 77::31f'ificrz‘2 rev» '5 ‘4': M“; Table 1 0. Van Slyke Nitrogen in Hydrolyzable Fractions from Soils with Varying Clay Treatments 61 N fraction Clay treatment None 6.25T 12.5T 25T 50T C (non-basic) D (basic) C l D C (non-basic) D (basic) C I D C (non-basic) D (basic) C J D C (non-basic) D (basic) C I D 6. 10 0.77 6.87 65. 59 59.23 64.81 35. 06 4. 42 39.48 24. 89 3.41 28. 04 * Amino N, - mg. per 100 g. soil 7.46 9. 02 8.50 5.46 0.75 0.81 3. 09 o. 10 8.21 9. 83 11.59 5.56 Amino N, - percent of N in Fraction B 54. 85 71. 02 72. 03 33. 09 57.69 38.57 70.23 2.50 55.10 66. 42 71.54 27.12 Amino N, - percent of hydrolyzable N (Fraction A) 34.86 41.38 37.61 22.46 3.50 3. 72 13.72 0.41 38. 36 45. 09 51.28 22.88 Amino N, percent of total soil N 25. 28 31.10 27.87 17.61 2.54 2.79 10.13 0.32 27.83 33.90 38. 00 17.94 m * Nitrous acid releases other -NHZ groups besides OC -amino. 62 were simultaneously fractionated by Fromageot's procedure singly, in mixtures, and in mixtures added to soil fractions. Micro Kjeldahl an- alysis for total N showed reliable recoveries and the procedure was, therefore, adopted. The results of applying Fromageot and Lederer fractionation pro- cedure to the soils are shown in Tables 11 and 12. They are consider- ed highly informative and suggest that this procedure can be utilized in studying soil nitrogenous fractions. It is significant to note in Table 12 that the results of this fractionation of the acid hydrolyzable nitro- gen (ammonia and humin free) served to check our previous findings and revealed further details about active functional groups. The basic and non-basic nitrogen recovered by this new method, expressed as percentage of the total soil nitrogen or as percentage of total nitrogen in Fraction B, was found to be very close to the recovery of the same nitrogenous groups obtained by phosphotungstic acid separation as shown in Table 9. Functional group separation by columns is a more detail - ed separation and, therefore, more informative. Moreover, it is quicker and less laborious. No similar agreement for these two me- thods of fractionation was detected for Fraction A (Table 11). This is taken to imply that humin present in Fraction A interfered in the fur- ther separation of the soil hydrolyzable nitrogen into functional group constituents by this method. . A considerable proportion of the hydrolyzable nitrogen in these soils was in the form of neutralaion-aromatic constituents (Tables 11 and 12). AR' Distribution of Nitrogen Among Column Separates from Total Hydrolysate Table 1 1 . (Fraction A), as Related to Clay Treatment 63 Column separate Clay treatment None 6.25T 12. 5T 25T SOT Basic Acidic Aromatic Neutral, non-aromatic Total of separates Fraction A Basic Acidic Aromatic Neutral, non-aromatic Total of separates Not accounted for Total N, - as percent of total soil N 1.80 7.26 5.80 45.06 59.92 71.02 9. 05 9. 05 14.03 41. 39 73. 52 72. 54 1.72 14.51 3.66 59.86 79.76 75.52 2.43 2.62 0 60. 72 65. 77 74. 09 5. 81 6. 00 0 41.87 53.67 78. 39 Total N, - as percent of total N in Fraction A 2. 53 10. 22 8. 16 63.45 84. 36 -15. 64 12.48 12.48 19. 35 57. 06 101. 36 {1. 36- 2.29 19.31 4.86 79.63 106. 10 {6. 10 3.27 3.54 0.00 81.95 91. 16 -8. 84 7.41 7.65 0.00 53.41 68.48 -31. 52 Table l 2. Distribution of Nitrogen Among Column Separates from Fraction B, as Related to Clay Treatment 64 Clay treatment Column separate None 6. 25T 12. 5T 25T 50T Total N, - as percent of total soil N Basic 4. 69 4-75 7. 14 14. 1 0 13. 23 Acidic 13.42 10. 51 10. 86 9. 38 6. 39 Aromatic 5. l4 4. 75 4.90 6. 23 6. 84 Neutral, non-aromatic 23. 88 24. 14 25. 86 26. 23 36. 38 Total of separates 47.15 44. 15 48. 76 55-94 62. 84 Fraction B 48.50 48. 13 48.90 53. 11 62.90 Total N, - as percent of total N in Fraction B Basic 9. 66 9. 86 14. 57 26. 54 21. 02 Acidic 27.65 21.83 22.18 17.65 10.15 Aromatic 1 0. 59 9. 86 10. 00 1 1. 73 1 0. 87 Neutral, nonaaromatic 49. 16 50. 14 52. 82 49. 38 57. 84 Total of separates 97. O6 91. 69 99. 56 105. 31 99. 90 Not accounted for -2. 94 -8. 31 -0. 44 {5. 31 -0. 10 65 These increased with clay treatments up to one and a half times the check soil, for the highest level. An outstanding feature of the results of this fractionation is that while the basic constituents increased with clay treatments, the acidic decreased. Their range of variation (Table 12) was of a similar magnitude, since they ‘gave rise to not very divergent maxima and minima. The maximum for the basic amino separates was about 13 to 14 mg. of nitrogen per 100 g. of soil for the higher clay treatments and a minimum of 5 mg. for the check. These changes cor- responded to a maximum of about 13 mg. of nitrogen in the acidic se- parate from the check soil and about 6 mg. minimum for the highest clay treatment. The latter results point toward some state of inverse equilibrium between the basic and the acidic nitrogenous constituents in their rela- tionship to increasing clay treatment. It is recalled that Bremner (23) is of the Opinion that basic nitrogenous compounds are associated with humic acid. Stevenson (136) has suggested that diamino molecules could form points of attachment of humic colloids to clays. Our results sug- gest that acidic and basic nitrogenous constituents are dependently in- terrelated in the associative interactions which occur in soils between organic materials and clay minerals. It thus appears that equilibrium states achieved in the humification process reflect some sort of chemical balance between clay minerals and the acidic and basic constituents of humus. The total charge of the mineral-organic colloidal complexes in soil apparently modifies the 66 course of decomposition of fresh plant residues in such a way as to maintain the electrical stability of the system. The negatively charged clay minerals were found here to promote a preferential retention of hydrolyzable basic nitrogen compounds. It is conceivable that these were held by cation exchange forces, as was inferred by Birch and Friend (17) when they suggested that the so called "priming" action of fresh organic matter on the breakdown of soil humus is a displacement reaction caused by exchange between products of decomposition of the fresh organic matter and similar organic compounds previously protect.- ed by the clay. The observed reduction in hydrolyzable acidic nitrogen compounds (Table 12) might reflect an enhanced microbial dissipation of these ma- terials. However, the increase in neutral non-aromatic nitrogen was of the order of 3 times greater than the decrease in acidic nitrogen. This would indicate that, under the influence of clay, an increasing quantity of both basic and acidic nitrogen compounds were incorporated into neutral organic combinations chemically very resistant in nature. As will be shown later, the chromatographic behavior of the neutral non-aromatic fractions on paper was such as to conclude that they were indeed incompletely hydrolyzed complexes, rather than mixtures of ul- timate splitting products. Their chemically resistant nature may be inferred from their apparent resistance to hydrolysis. However, the possibility must yet be entertained that the observed complexes may have arisen as artifacts by oxidative condensation and polymerization 67 following the neutralization of the hydrolysates with LiOH (Figure 2). With the purpose of obtaining some idea as to the distribution of the ninhydrin reacting compounds in the different soil nitrogenous fractions, the modified photometric ninhydrin procedure of Troll and Cannan (140) was applied. Several workers (41, 66, 95, 127) have expressed the re- sults of this procedure in terms of the leucine equivalent nitrogen. This is done by comparing color development by unknown solutions after re- action with ninhydrin with that for a series of leucine standards. The 570 mnfilter is used. The procedure was originally elaborated by Harding (70). This is just an empirical estimation of amino groups since the different amino acids give not only differences in the shade of color, but differences in the depth of color per unit of the amino radi- cal (65). Gortner and Gortner (65) have regarded the ninhydrin reac- tion as the most delicate reagent for detecting the presence of an 0C - amino acid. The reaction is not Specific. Tables 13 and 14 contain the results of the photometric ninhydrin reaction applied to Fractions A and B and their column separates. The leucine equivalent nitrogen of the total hydrolysate (Fraction A) was less than the total for the column separates (Table 13). This was not expected, since Fraction A contains ammonia and it is known that am- monia gives the purple color with ninhydrin. Apparently, passing the whole acid hydrolyzable fraction through the columns promoted some kind of liberation of the amino and carboxylic groups required to de- ve10p the. purple color (65). This is an indication that the ammonia in ‘iL‘Q..F"v-—- ces—'V—VW 1 1 Table 1 3. Occurrence of Leucine-Equivalent Ninhydrin-Reactive Compounds in Fraction A and its Column Separates, as Related to Clay Treatment 68 Clay treatment Column separate None 6. 25T 12. 5T 25T 50T Leucine-equivalent amino - N, as total soil N. Basic 2. 00 2. 54 1. 00 1.15 Acidic 4. 24 1.12 2. 62 1. 84 Aromatic 3. 59 3. 05 2. 66 2. 95 Neutral, non-aromatic 3. 06 2. 58 3. 34 2. 88 Total of separates 12. 90 9. 28 9. 62 8. 82 Fraction A 2. 41 3. 80 4. 34 3. 34 Total N in Fraction A 71 . 02 72. 54 75. 52 74. l 0 Leucine-equivalent amino - N, as total N in Fraction A Basic 2.82 3. 50 1.33 l. 55 Acidic 5.98 1. 54 3.49 2.48 Aromatic 5. 06 4. 20 3. 60 3. 98 Neutral, non-aromatic 4. 31 3. 55 4. 45 3. 89 Total of separates 18.16 12.80 12.80 11.90 Fraction A 3. 39 5. 23 5. 78 4, 51 pe rc ent of 2.84 1. 55 2.61 2. 03 9. 03 3. 35 78.40 percent of 3.62 1.90 3.33 2.59 11.52 4.28 RC: ‘9‘...- w Occurrence of Leucine-Equivalent Ninhydrin-Reactive Compounds Table 14. 69 in Fraction B and its Column Separates, as Related to Clay Treatment Column separate Clay treatment None 6.25T 12. 5T 25T 50T Basic Acidic Aromatic Neutral, non-aromatic Total of separates Fraction B Total N in Fraction B Basic Acidic Aromatic Neutral, non-aromatic Total of separates Fraction B Leucine-equivalent amino - N, as percent of total soil N 1.84 4.20 3.26 5.95 15.26 2.45 48.60 Leucine-equivalent amino - N, as percent of total N in Fraction B 3. 78 8.66 6. 72 12. 26 31.42 5. 04 0.71 2.61 1.97 2.74 8.00 8.61 48.10 1.41 5.42 4. 08 5. 70 16.62 17.88 0.75 2.48 2.76 1. 38 7. 38 4.14 49. 00 1. 55 5. 07 5.62 2.82 15. 07 8.45 1.11 2.10 1.97 1.61 6.79 6.56 53.10 2. 09 3.95 3.70 3. 02 12.77 12.34 3.16 1.94 3.61 1.45 10.16 6.90 62.90 5. 02 3. 08 5. 74 2. 30 16.15 10.97 70 Fraction A is not present as such but rather as an interfering compound or complex. The recovery of ninhydrin reactive compounds from the acid hydro— lyzable fractions after removing ammonia and humin (Fraction B, Table 14) was similar to the summation of the corresponding column separat- es. This emphasizes the necessity for getting rid of ammonia and humin during the process of soil amino nitrogen fractionation. With the reservations already pointed out above, the quantities of ninhydrin reactive nitrogen are considered to be low when compared with the total nitrogen in the fractions in Tables 11 and 12. Leucine-equivalent nitrogen found in the phosphotungstic acid sepa- rates in Fraction B is shown in Table 15. It is interesting to note that the values for basic leucine-equivalent nitrogen presented here are roughly of the same order as those presented in Table 10 for the Van Slyke -nitrogen of the same basic separate. The nitrogen content for the non-basic phosphotungstic acid separa- te obtained by the Van Slyke method is much higher than by the colori- metric ninhydrin procedure. The lack of uniformity in the behavior of these two fractions makes it impossible to draw any definite conclu- sions as to their amino nitrogen content. It does appear that the Van Slyke nitrogen of the basic phosphotungstic acid separate (Fraction D) is of a similar chemical nature to the leucine-equivalent nitrogen of the same separate. The same cannot be said for these forms of nitro- gen in the non-basic separate (Fraction C). *9 ~~‘— -- 7 . ._.... _=' mama mew-‘1'”? W“- 7%!‘Ffl'tfirr ._. _ _ ‘1" F - 71 Table 1 5. Occurrence of Leucine-Equivalent Ninhydrin-Reactive Compounds in Fractions C and D as Related to Clay Treatment N fraction C (non-basic) D (basic) C l D C (non-basic) D (basic) C { D Clay treatment None 6. 25T 12. 5T 25T SOT Leucine-equivalent amino - N, as percent of total soil N 9. 42 12. 92 4. 38 12. O6 10. 35 1.71 1.76 2.62 8.79 4.42 11.15 114.68 9.00 20.85 14.77 Leucine-equivalent amino - N, as percent of total N in fraction 24.84 28. 02 10-00 31.19 19.45 32. 31 40. 00 34.28 60.91 34.25 25.75 29.06 15.07 41.73 22.34 _ w. .1. .. -.. , . PAPER CHROMATOGRAPHY It was hoped that column separates would be adequately purified for identification of constituent amino acids by paper chromatography. Ac- cordingly, two dimensional paper chromatograms were run for each fraction and column separate of each of the five soils. The technique utilized has been described under Methods of Analyses. The results are presented in Figures 6 to 23. In these figures we have shown gen- eralized chromatographic patterns for the different column separates. The spots represent material which gave a positive test when the pa- pers were sprayed with ninhydrin. These figures were pantographed from the original papers. A good separation of standard amino acids was achieved with this procedure (Figure 6). However, no comparable separation was obtain- ed with any fraction or separate from any of the soils. There were dif- ferences in the paper patterns for different clay treatments. but these were not interpretable. Nevertheless, the results are considered val.- id, in that they were reproducible and certain patterns of migration were associated with given fractions or separates. Definite trends in mobility of ninhydrin reactive materials we re also associated with in- creasing clay treatment. It is considered worth mentioning that the basic group of ninhydrin reactive compounds separated with the phosphotungstic acid were an- alyzed before and after desalting. The intensity and the purple area on the paper was considerably greater before desalting. Moreover, FIGURE 6. CHROMATOGRAM OF STANDARD AMINO ACIDS 73 B; in PhQNOl . 74 g _. m. _ Prelim. q; ~ 0 , Phcn lulamnc Methionineo / V k 1— Q . ' ~ ‘ Velma. Leucme Hnst'dnne ‘9 ' C? Threonine Tyrosme so. 9 Argmmc Aian‘ng ‘1' )- 0 Lgsine oszrine . a! —- O glycme 06,1111qu acid 9! r- Aquvlloc. acid '7 - OCys‘leme o _ e 1 1 1 L 1 1 4 1 1 4 .o .1 .2. .3 4 .5 .6 .7 .8 .9 Rf 1n Bu: Anti-110 Fig.6 'fl—r ‘— v“ FIGURES 7 TO 12. CHROMATOGRAPHIC PATTERNS OF THE BASIC-NINHYDRIN COMPONENTS Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Phosphotungstic acid-precipitated fraction of the check soil. Phosphotungstic acid-precipitated fraction of the 6. 25 tons of clay/ac re treatment. Phosphotungstic acid-precipitated fraction of the 12. 5 tons of clay/acre treatment. Pho sphotungstic acid-precipitated fraction of the 50 tons of clay/ac re treatment. Generalized pattern for the components of all the soils retained by the silica columns before removing ammonia and humin from the hydrolysate. Generalized pattern for the components of all the soils retained by the silica columns after removing ammonia and humin from the hydrolysate. 75 R5 in P he no! .4 .6 .8 1.0 .2 .0 Rf'mphcnol .2 .4 .5 .3 .0 1.0 m Phenol .4- .e .6 .2R F 76 9 .8 2 .1. is. 3 F119. 7 (Ba—12:90 1 1 J_ 1 1 .o 2 .4 .6 5 1119.9 0 C) O O .o .2 4 .6 .8 Rf an'suzAcznzo Fig. 11 $ 0 Z .4 6 8 F '19. 8 G 1 1 1 1 4 o 2 .4 6 8 F 19.10 9 J L L o .0 ,z .4 .6 .8 Rf m BquctHzo 1719.12 77 FIGURES 13 TO 18. CHROMATOGRAPHIC PATTERNS OF THE ACIDIC NINHYDRIN COMPONENTS Figure Figure Figure Figure Figure Figure 13 14 15 16 17 18 Generalized pattern of the phosphotungstic acid supernatant of all soils. Components of the check soil retained in the alumina columns before removing ammonia and humin from the hydrolysate. Components of the 6. 25 tons of clay/ac re treatment retained in the alumina column before removing ammonia and humin from the hydrolysate. Components of the 2.5. 5 tons of clay/ac re treatment retained in the alumina column before removing ammonia and humin from the hydrolysate. Components of the check soil retained in the alumina column after removing ammonia and humin from the hydrolysate. Generalized pattern of the components of all soils, except the check, retained in the alumina columns after removing ammonia and humin from the hydrolysate. 78 LO L s- 3"- “9‘ C sou)L 4" \Qr' a. E i" Q'" L.- CC Q'— N- O ._ Q 0+ as 0:11.. 1 1 1 1 1 A l l 1 J 1 l O 2. 4 -6 8 1.0 0 .2 4 .6 8 IO 0 F19 13 F“; 14 “P Q @- a 09. 1: °W+ .f.‘ \o- o. .E 2M4 .2 4f O 2. .4 6 8 I'.O o z 4 6 8 FLC) 15 PH) \6 O. -F- o... .30. c . d 0.. g . (1% .s “‘h 4'- E" 0 er J ~b o ‘ <2.- 9 0CD 9L 0 f .‘z .1; is .3 Lo .5 .£ .4 is .33 Rf in BUIAcZHZO - Rf in BuiAcin Fig.1? Fi9- ‘8 "'V. '3'. v .- 79 FIGURES 19 TO 23. CHROMATOGRAPHIC PATTERNS FOR THE AROMATIC AND THE NEUTRAL NON-AROMATIC NINHYDRIN COMPONENTS Figure 19 Figure 20 Figure 21 Figure 22 Figure 23 Generalized pattern for the aromatic components of all the soils retained in the charcoal columns before removing ammonia and humin from the hydrolysate. Generalized pattern for the aromatic components of the check soil and the 6. 25 and 12. 5 tons of clay/acre treatment retained in the charcbal columns after removing ammonia and humin from the hydrolysate. Generalized pattern for the aromatic components of the 24. 4 and 50 tons of clay /ac re treatment retained in the char- coal columns after removing ammonia and humin from the hydrolysate. Generalized pattern for the neutral non- aromatic components of all the soils be- fore removing ammonia and humin from the hydrolysate. Generalized pattern for the neutral non- aromatic components of all the soils after removing ammonia and humin from the hydrolysate. mu 8 R; ‘m P hqnol .4 .6 -2 .4 -6 '8 \.o R{ m P homo! .z .8 l .6 I R? \n P henol .4 .Z ‘ +- Fi9.Zo I l l L J .o z 4 .6 .a .Rfin Bu=AdH20 Fig.22 I-O l-O .0 .2 .4- a6 . Rf In ENACTHzO Fig. 23 .m' w- m. ‘w .1..— .1‘ ‘._"_t 81 the ninhydrin photometric procedure (140) applied to this group before and after desalting definitely showed a loss of ninhydrin reactivity with desalting. Before de salting, the pho sphotungstic acid-precipitated fractions showed considerable "trailing", especially toward the phenol front. It was, therefore, considered important to desalt the samples following recommendations of Zweig and Hood (157) and also to find out by X-ray if there was clay present in the fractions. The results of the X-ray studies are presented in the Appendix. The chromatograms of the desalted basic amino fraction precipitat- ed with phosphotungstic acid are presented in Figures 7 to 10. All soils showed components with low mobility in butanol-acetic acid (Rf 8 0. 1 - 0. 2), but with variable mobility in 80% phenol. Maximum mobility in phenol (Rf = O. 5 - 0. 7) occurred with the soils treated with 12. 5 and 25 tons of clay per acre. With these two treatments rather distinct spots were obtained which correspond to lysine, arginine, and histidine in Figure 6. Both of these treatments showed a very similar chroma- tographic pattern. Figure 9 represents the pattern for both soils. In these two soils only, there appeared ninhydrin-reactive components immobile in phenol but with varying mobility in butanol-acetic acid. These correspond to none of the standard amino acids. If it could be assumed that these phenol immobile components were present in organ- ic complexes with basic amino acids in the other soils, it would account for the reduced mobility and indistinct separation of components with 82 phenol affinity in the soils of lower clay treatment (Figures 7 and 8). The effect of intermediate clay treatments in this case would appear to be due to absorption of basic amino acids by clays, preventing their reaction with other organic molecules to form strictly organic complex- es. The disappearance of the phenol immobile constituent at the high- est level of clay treatment (Figure 10) may have been related to the de— pressed yield of crops and reduced level of residue return, or to pro- gressively greater breakdown of these constituents during de salting. Gas evolution was more vigorous with desalting at higher clay contents. The latter explanation appears more likely, considering the results depicted in Figure 11. The generalized chromatogram in Figure 11 re- presents basic constituents retained on the silica column in the From- ageot procedure. They were eluted with 0. l N hydrochloric acid and concentrated in vacuo at 40°C. However, the eluate was not desalted prior to spotting on paper. The distribution of ninhydrin-reactive ma- terials was essentially the same for all soils. An intense purple spot in the check soil with Rf = 0. 28 in phenol and 0. 27 in butanol-acetic acid-water was found to become progressively smaller and less intense with increasing clay treatment. Coincident with the disappearance of this spot, three other spots were observed to increase in intensity. These three spots, all exhibited greater mobility in butanol-acetic acid- water than the first spot. Two exhibited greater mobility in phenol, the other a lower mobility than the first. None of these spots corres- ponded to any of the standard amino acids shown on Figure 6 or with ‘3 ~' 6”.” ‘fi—v 83 phosphotungstate -precipitated basic constituents which were fairly well resolved in Figure 9. The three well defined spots in Figure 11 may be amino acids with substituted organic groups which have altered their mobility in the solvents used. While phosphotungstic precipitation and careful desalting may, more effectively, separate constituent basic ninhydrin-reactive compounds, the less drastic separation achieved by silica absorption may serve to isolate multimolecular groupings or complexes which represent significant units in the larger humic complex. The relation to clay treatment which was observed among the silica ab- sorbed constituents suggest that these materials were formed in the soil rather than as artifacts arising during hydrolysis. Attention is called to the distribution of the ninhydrin-reactive com- pounds in Figure 12. These also represent basic constituents retained in the silica column, but are free of ammonia and humin. The elimi- nation of ammonia and humin did not help to separate ninhydrin- react- ive constituents for identificational purposes. In fact, the distinct spots observed in Figure 11 were lost completely. There was an increase in mate rials ,with a low mobility in both solvents which resulted in the closing of the "L" shaped pattern at its vertex. This behavior appears to have resulted from the complexing of compounds with differential mobility in phenol and in butanol-acetic acid. This complex formation must have occurred during the distillation following addition of C3a(OH)2 to remove ammonia and humin. When paper chromatograms of the phosphotungstate-precipitated 84 basic constituents (Figures 7 to 10) are compared with the corresponding fractions separated from the silica column by Fromageot's procedure (Figures 11 and 12), it is noted that by the latter procedure ninhydrin- reactive constituents were separated which had a greater range of mo- bility in butanol-acetic acid than those obtained by the precipitation pro- cedure. The elimination of ammonia and humin (Figure 12) brought about a smaller resolution of the ninhydrin-reactive components in this basic fraction. The foregoing suggests that Fromageot's procedure permits a bet- ter separation of the ninhydrin-reactive components of the soil hydro— lysates. This-is further suggested when Figure 13, showing the non- basic constituents from the phosphotungstate supernatant, is compared with the acidic constituents retained in the alumina column (Figures 14 to 16). The former (Figure 13) showed practically no migration of the ninhydrin-reactive components. There appeared a single very in- tense pink spot. Figures 14 to 16 show a greater resolution with in- creasing clay treatments. There was a distinct difference in the degree of resolution between the acidic column fractions before and after removing the ammonia and humin. As was observed in Figures 11 and 12, there was better resol- ution of ninhydrin-reactive constituents in the column separates from clay-treated soils before removal of ammonia and humin (Figures 15, 16 and 18). On the other hand, removal of ammonia and humin improv- ed the resolution of acidic ninhydrin-reactive constituents in the check 85 soil (Figures 14 and 17). This interaction bet ween clay content of the sample and the alkaline distillation employed to remove ammonia and humin from the hydrolysate is difficult to interpret. It is apparent, however, that clay treatment in the field markedly altered the propor- tions of complexable constituents in the hydrolysate. The general chromatograms of the aromatic fractions are shown in Figures 19 to 21. These showed a distinct tendency for the "L" shaped migration patterns found in the case of the basic fractions after removal (Figure 12), and of the acidic fractions before removal, of ammonia and humin (Figures 15 and 16). The chromatographic behavior of the neutral non-aromatic fractions is shown in Figures 22 and 23. These did not show much migration in either solvent. It is recalled that these fractions were quickly desalt- ed. The neutral non-aromatic fraction before removing humin and am- monia is shown in Figure 22. This showed a rather larger spot contain- ing an amalgam of color which varied from a deep purple, grading through pink into a yellow border at the farmost butanol front. The col- ors appeared uniformly distributed along the phenol front. Figure 23 represents this same fraction after removing ammonia and humin. There appears to be not much difference in resolution in the neutral non-aromatic fractions before and after removing ammonia and humin. The total area and intensity of the spots for the neutral non-arom- atic fractions clearly increased with increasing clay treatments, ex- cept for the 12. 5 tons of clay per acre treatment, which showed the J&. v ~'~1VMWv«u-mw1=~w_vvra;4“ 86 least area and color intensity. This is consistent with the fact that total nitrogen in this separate increased with clay treatment (see Table 12). The low mobility of these. materials in both solvents suggest that they were relatively complex, large molecules. From their behavior during paper chromatography it is clear that the various hydrolytic fractions and separates studied did not contain any significant quantities of free amino acids but, rather, amino acids complexed with other organic compounds. It would appear that the mix- ture of colors observed in the neutral non-aromatic fractions represent undissociated amino acids complexed to an organo-phyllic counterpor- tion. The resulting complexes exhibited a neutral, non-migratory be- havior, due to internal electrochemical compensation of their function- al groups. Most of these complexes are not definite compounds. If they were, they would give distinct spots instead of trailing across the paper or producing spots with a mixture of colors. The fact that they were neutral to silica and alumina columns suggests that both the amino and carboxylic groups were tied up. The organic compounds which are tied up with the amino acids ap- pear to be aromatic in nature. This is suggested by the fact that the "L" shaped pattern of the aromatic group is to be observed also in the basic group after, and in the acidic group before, alkaline distillation to remove ammonia and humin. This is compatible with the concept that amino acids in soils are held in complexes with humic acids. It was found that the ninhydrin color reaction did not reach max- d 87 imum intensity for several days after the papers were sprayed. This, too, would suggest that amino acid groups were tied up in some way so that they could react only slowly with ninhydrin. Most workers (25, 29, 48, 73, 106, 130, 136) infer from the high amount of amino derivatives obtained from soil humate hydrolysates, and the high amount of non- acid hydrolyzable nitrogen in humus, that the soil amino acids are es- sentially associated with hurnic acid rather than being present as free amino acids or as normal proteins. Sowden and Parker (130) and Bremner (23, 27) have shown that soil amino acids are very strongly associated with humus. Their work also showed that humus is not uniform since the dinitrophenyl amino acid derivatives of humic acid preparations showed striking differences in susceptibility to hydrolysis and solubility. Sowden has reported (129) the presence of an unknown interfering material in his chromatographic elution columns. According to his own description, "it did not seem likely that it would be one of the more common amino acids". It react- ed readily with the Moore & Stein reagentit but very slowly with a nin- hydrin-in-butanol spray. Full color intensity did not develop for more than 24 hours after spraying and was pink rather than blue or reddish blue as is true for most amino acids. Because of the interference of this material they were unable to properly separate tyrosine and phe- nylalanine, two aromatic amino acids. * Sequence of pyridine, 80% phenol, and ninhydrin. 88 The possibility that clay could have been present in the soil pre- parations was rejected on the basis of X-ray analyses, since it was found that glycerated soil hydrolysates proved to be X-ray amorphous. Diffraction patterns of unglycerated preparations showed no maxima which could be attributed to the native soil clay or Wyoming Bentonite. The details of this part of the work is fully discussed in the Appendix where a mechanism is tentatively suggested to explain the results. {"m- ”Ya-and; as: M‘. ’2?" (J M {IM‘ 'O‘ SUMMARY Chemical and physical studies with a clay-amended Plainfield sand three years after field applications of Wyoming Bentonite gave the fol- lowing results: 1.. Increased rate of treatment with Wyoming Bentonite resulted in an increased positive electrophoretic mobility at pH 6. 0 to 6. 1 of the col- loidal fraction of the soil. Calculated Z potentials were inversely re- lated to the carbon content of the colloidal fraction. 2. The C:N ratio of the soil decreased with increasing levels of 'clay application. This was due, principally, to an increase in total nitrogen and more specifically to an increase in nitrogenous materials which were solubilized by hydrolysis in 6N HCl. 3. Fractionation of the hydrolyzable nitrogen showed an increase with increasing level of clay application in basic and non-basic components soluble in the presence of excess Ca(OI—I)2, while ammonia nitrogen was essentially unaltered and humin nitrogen decreased. 4. The recovery of basic and non-basic nitrogen by phosphotungstate precipitation was in close agreement with the recovery by column sepa- ration. (The results with column separation revealed that the observed tendency for non-basic forms of nitrogen to increase with clay treat- ment was very largely due to an increase in neutral non-aromatic cons- tituents. 5. Aromatic forms of nitrogen were essentially unrelated to treatment, whereas nitrogen in acidic constituents decreased and basic forms of .— u s... "“~"’T' 4' «rawmmmhi'h‘W zvv—finr'wv- ”n7... .. 90 nitrogen increased with increasing amounts of clay. 6. Paper chromatograms showed that the ninhydrin-reactive nitrogen in the various hydrolyzable fractions and separates was not present in the form of free amino acids. The amino acids appeared to be complex- ed with other organic compounds. These complexing compounds appear- ed to be aromatic in nature. 7. It was not clear whether the amino nitrogen complexes observed had been formed in the soil or whether they had arisen as artifacts during chemical treatment. However, their distribution and their mobilities in phenol and in butanol-acetic acid-water on paper chromatograms were clearly influenced by the level of clay application. 8. The release of C02 and nitrate during incubation indicated that the added clay had a greater stabilizing influence on residual soil nitrogen than carbon. .p. LITERATURE CITED Adams, A. P. , Bartholomew, W. V. , and Clark, F. E. Measu- rement of nucleic acid components in soil. Soil Sci. Soc. Amer. Proc. 18: 40—46. 1954. Adler, H. H., Bray, E. E. , Stevens, N. P., Hunt, J. M., Keller, W. D. , Pickett, E. E. , and Kerr, P. F. Infrared spectra of re- ference clay minerals. Prelim. Rept. No. 8, Amer. Petroleum Institute Res. Proj. 49. Columbia Univ. , New York. 1950. Allison, F. E.. Sherman, M. S., and Pinck, L. A. Maintenance of soil organic matter: 1. Inorganic soil colloid as a factor in retention of carbon during formation of humus. Soil Sci. 68: 463-478. 1949. Ambrose, E. J. , Elliot, A. , and Temple, R. B. The use of pol- arized infra red radiation in the study of crystal structure. Proc. Roy. Soc. London, Ser. A. 206: 192-219. 1951. Anderson, G. Identification of derivatives of desoxyribonucleic acid in humic acid. Soil Sci. 86: 169-174. 1958. André, G. Sur la constitution des matiéres hurniques. Compt. Rend. Sci. 128: 513-516. 1899. Astbury, W. T. , and Street, A. X-ray studies of the structure of hair, wool, and related fibers: I. General. Trans. Roy. Soc. London, Ser. A. 230: 75-101. 1931. Bamférd, C. H. , ‘Hanby, W. E. , and Happey, F. The structure of synthetic polypeptides: III. Roy. Soc. London, Ser. A. 206: 407-424. 1951. Baver, A. H. , and Marshall, C. E. The cataphoresis of clay min— erals and factors affecting their separation. Soil Sci. Soc. Amer. Proc. 15: 142-145. 1950. Beckley, V. A. The formation of humus. Jour. Agric. Sci. 11: 69-77. 1921. Bell, K. F. The infra red absorption spectra of organic deriva- tives of ammonia: I. Aniline and some mono-and dialkyl an- ilines. J. Am. Chem. Soc. 47: 2192-2207. 1925. Bellamy, L. J. The Infra- red Spectra of Complex Molecules. John Wiley & Sons. , Inc. , New York. 1958. - ~.- _ 3" ‘1- JMLQW'W .,._.—._ .‘J» Yr”!- w. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 92 Bennett, E. Fixation of ammonia by lignin. Soil Sci. 68: 399- 400. 1949. Bernal, J. D. Kurzere originalmitteilungen und notizen. Z. Krist 78: 363-369. 1931. Bertramson, B. R. Soil Chemistry Notes. Students Book Corpo- ration, Washington. 1955. Berry, H. K., Sutton, H. E. , Cain, L. , and Berry, S. J. Devel- opment of paper chromatography for use in the study of meta- bolic patterns. Univ. of Texas Pub. No. 5109: 22-55. 1951. Birch, H. F. , and Friend, M. J. Humus decomposition in East African soils. Nature 178: 500-501. 1956. Block, R. J. , Durrum, E. ,L. , and Zweig, G. A Manual of Paper Chromatcggaphy and Paper Electrophoresis. Academic Press Inc. , New York. 1955. Bower, C. A. Separation and identification of phytin and its der- ivatives from soils. Soil Sci. 59: 277-285. 1945. Bremner, J. M. Studies on soil humic acids. J. Agric. Sci. 48: 352-360. 1956. . Studies on soil humic acids: I. The chemical nature of humin nitrogen. J. Agric. Sci. 46: 247-256. 1955. . A review of recent work on soil organic matter: II. J. Soil Sci. 5: 214-232. 1954. . The nature of soil nitrogen complexes. J. Sci. Food Agr. 3: 497-500. 1952. . A review on recent work on soil organic matter: I. J. Soil Sci. 2: 67-82. 1951. . The amino acid composition of the protein mater- ial in soil. Bioch. J. 47: 538-547. 1951. . Studies on soil organic matter: I. The chemical nature of soil organic nitrogen. J. Agric. Sci. 39: 183-193. 1949. , and Shaw, K. The mineralization of some nitro- genous materials in soils. J. Sci. Food Agric. 8: 341-347. 1957. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 93 , and . Studies on the estimation and decomposition of amino sugars in soil. J. Agric. Sci. 44: 152-159. 1944. Broadbent, F. E. Basic problems in organic matter transform- ations. Soil Sci. 79: 107-114. 1955. . Modification in chemical properties of straw during decomposition. Soil Sci. Soc. Amer. Proc. 18: 165- 169. 1954. , and Norman, A. G. Some factors affecting the availability of the organic nitrogen in soil. Soil Sci. Soc. Amer. Brown, C. J., Coleman, D., and Farthing, A. C. Further stud- ies in synthetic polypeptides. Nature 163: 834-835. 1949. Bull, H. B. Physical Biochemistry. John Wiley & Sonc, Inc. , New York. 1951. Colthup, N. B. Spectra-structure correlations in the infra-red region. J. Opt. Soc. Amer. 40: 397-400. 1940. Conant, J. B. , and Blatt, A. H, The Chemistrl of Organic Com- pounds. The MacMillan Co. , New York. 1947. Consden, R. , Gordon, A. H. , and Martin, A. J. P. Partition chromatography with paper. Bioch. J. 38: 224-232, 1944. , , . Gramicidin S: The sequence of the amino acid residues. Bioch. J. 41: 596- 602. 1947. Corey, R. B. The crystal structure of diketOpiperazine. J. Am. Chem. Soc. 60: 1598-1604. 1939. , and Donohue, J. Interatomic distances and bond angles in the polypeptide chain of proteins. J. Amer. Chem. Soc. 72: 2899-2900. 1950. Cramer, F. Paper Chromatggraphy. McMillan Co. Ltd. , London. 1955. / ,J 1 ‘11 ‘1. L. 1' 'F ‘ 1. ' 1 I 94 41. Davidson, D. 1., Sowden, F. J., and Atkinson, H. J. Application of paper chromatography to identification and quantitative es- timation of amino acids in soil organic matter fractions. Soil Sci. 71: 347-352. 1951. 42. Demolon, A. , and Barbier, G. Conditions de formation et constitution du complexe argilo-hurnique des sols. Compt. Rend. Acad. Sci. 188: 654-656. 1929. 43. Duel, H. , and Huber, G. Organische derivate von tonmineralien alkoxy-alkyl-und arylderivate des montmorillonite. Helv. Chim. Acta 34: 1697-1701. 1951. 44. Dunn, M. S., and Drell, W. Experiments in Biochemistry. McGraw Hill Book Co. , Inc. , New York. 1951. 45. Dyck, A. W. J., and McKibbin, R. R. The non-protein nature of a fraction of soil organic nitrogen. Can. J. Res. 13: 264- 268. 1935. 46. Ellis, J. W. Band series in infra-red absorption spectra of or- ganic compounds: 1. Phys. Rev. 27: 298-313. 1926. 47. , and Bath, J. Infra-red and Fermi resonance as- sociated with CH2 groups in crystals. Phys. Rev. 55: 1098- 1099. 1939. 48. Emmerson, W. W. Comparison between the mode of action of organic matter and synthetic polymers in stabilizing soil crumbs. J. Agric. Sci. 47: 350-353. 1956. 49. English, J. , and Cassidy, H. G. Principles of Organic Chemis- EEK McGraw Hill Book Co. , Inc. , New York. 1949. 50. Ensminger, L. E. Factors affecting the interaction between or- ganic matter and montmorillonite. Soil Sci. 54: 191-198. 1942. 51. , and Gieseking, J. Resistance of clay adsorbed proteins to proteolytic hydrolysis. Soil Sci. 53: 205-209. 1942. 52. , and . Adsorption of proteins by montmorillonite clays and its effect on base exchange capacity. Soil Sci. 51: 125-132. 1941. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 95 , and . Adsorption of pro- teins by montmorillonite clays. Soil Sci. 48: 467-473. 1939. Fieser, L. F., and Fieser, M. Organic Chemistry: Reinhold Pub. Co., New York. 1950. Francis, F. , Piper, S. H. , and Malkin, T. The n-fatty acids. Proc. Roy. Soc. London, Ser. A. 128: 214-252. 1930. Frankel, M., and Katchalski, E. Poly-condensation ofOC - amino acid esters: I. Poly-condensation of glycine esters. J. Am. Chem. Soc. 64: 2264-2271. 1942. Fremy, E. Recherches chimiques sur les combustibles mineraux. Compt. Rend. Acad. Sci. 88: 1048-1055. 1879. . Recherches chimiques sur les combustibles mineraux. Compt. Rend. Acad. Sci. 52: 114-118. 1861. Fromageot, M. J. , and Lederer, E. Separations chromatogra- phiques d'acides aminés et de peptides: IV. Séparation en qua- tre groupes. Biochim. Biophys. Acta 2: 487-498. 1948. Fuson, N. , Josien, M. L. , and Powell, R. L. Infrared spectros- copy of compounds having biological interest: 11. A comprehen- sive study of mercapturic acids and related compounds. J. Am. Chem. Soc. 74: 1-5. 1952. Gieseking, J. Mechanism of cation exchange in montmorillonite, beidellite, and montronite type of clay minerals. Soil Sci. 47: 1-13. 1939. Glaeser, R. Retention of organic molecules on montmorillonite. Compt. Rend. 232: 1496-1498. 1951. Glatt, L. , and Ellis, J. W. Infrared dichroism in aligned poly- thene and "Parowax". J. Chem. Phys. 15: 884. 1947. Gordon, A. H. , Martin, A. J. P., and Synge, R. L. M. Parti- tion chromatography in the study of protein constituents. Bioch. J. 37: 79-86. 1943. Gortner, R. A., Gortner, Jr., R. A., and Gortner, W. A. Outlines of Biochemistry- John Wiley 8: Sons, Inc. , New York. 1953. a 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78. 96 Green, M., and Stahmann, M. R. Preparation and enzymatic hydrolysis of poly-T -ethylglutamic bovine plasma albumin. J. Biol. Chem. 213: 259-269. 1955. Greenland, D. J. The adsorption of sugars by montmorillonite. J. Soil Sci. 7: 319-334. 1956. , and Russell, E. W. Organo-clay derivatives and the origin of the negative charge on clay particles. Trans. Faraday Soc. 51: 1300-1307. 1955. ' Grim, R. E. Clay Mineralogy. McGraw-Hill Book Company, Inc. , New York. 1953. Harding, V. J. , and McLean, R. M. A colorimetric method for the estimation of amino-acidd‘ -nitrogen: H. Application to the hydrolysis of proteins by pancreatic enzymes. J. Biol. Chem. 24: 503-507. 1916. Harley, J. H., and Wiberley, S. E. Instrumental Analysi . John Wiley & Sons, Inc. , New York. 1954. Jirgensons, B. The influence of solvent composition, tempera- ture, and some other factors in the Rf values of amino acids in paper chromatography. Univ. of Texas Pub. No. 5109: 56- 70. 1951. Johnston, H. H. Studies on the chemical, physical, and biological properties of soil organic matter. Michigan State University. Ph. D. Dissertation. 1958. . Soil organic matter: I. Electrophoresis separa- tion of acid-resistant components. Agronomy Abstr. 15: 1958. Katchalski, E. Poly-0C -amino acids. Advances in Protein Chem- istry VI: 123-185. Acad. Press Inc., New York. 1951. Kellner, L. Infrared spectrum and molecular structure of diketo- piperazine andtetramethyl -diketopiperazine. Nature (Lond.) 140: 193. 1937. Kellog, C. E. We seek; we learn. U. S.D.A. Yearbook Agric. 1957. 1-11. 1957. Klarman, E. Recent advances in the determination of the struc- ture of proteins. Chem. Rev. 4: 51-107. 1927. 97 Nature of the organic Some studies I. 79. Kojima, Ruth T. Soil organic nitrogen: nitrogen from a muck soil from Geneva, N. Y. II. in amino acids of protein material in 'soil. Soil Sci. 64: 157- 165, 245-252. 1947. The influence of clay minerals Soil Sci. Soc. 80. Lynch, D. L., and Cotnoir, L. J. on the breakdown of certain organic substances. Amer. Proc. 20: 367-370. 1956. Linstead, R. P., Elvidge, J. A. , and Whalley, M. A Course in Modern Techniques of Organic Chemistry. Butterworths Scient— 1955. 81. ific Publications, London. J. Am. Chem. 82. Magee, M. Z. , and Hofmann, K. The application of the Curtius reaction to the polymerization of triglycine. 1949. Soc. 71: 1515-1516. Maillard, L. C. Synthesis of polypeptides by the action of glyce- 519-578. 1914. 83. rol upon glycocoll. Ann. Chem. 1 Formation d'humus et de combustibles mineraux sans intervention de l'oxygene atmospherique, des microorgan- Compt. 84. izmes, de hautes temperatures, ou de fortes pressions. 155: 1554-1556. 1911. Rend. Acad. Sci. Marshall, C. E. The Colloid Chemistry of the Silicate Minerals. 1949. 85. Academic Press Inc. , New York. Martin, J. P. , and Synge, R. L. M. Separation of the higher monoamino-acids by counter-current liquid-liquid extraction: Bioch. J. 35: 91-121. 86. The amino acid composition of wool. 1941. Martin-Vivaldi, J. L. , and Hendrick, S. B. Reactividad de los iones-H de las arcillas en disolventes no polares: I. Accién An. Edaf. Fisiol. Veg. 11: 601-629. 1952. Mattson, S. The laws of colloidal behavior: VII. Soil Sci. 33: 41-72. 87. Proteins and del diazometano. 1932. 88. proteinated complexes. The laws of colloidal behavior: 1. Soil Sci. 28: 89. 179-220. 1929. The relation between the electrokinetic behavior and J. Am. .Soc. Agron. 90. o the base exchange capacity of soil colloids. 1926. 18: 459-510. 91. 92. 93. 94. 95. 96. 97. 98. 99. 1 00. 101. 102. 103. 98 , and Koutler-Andersson, E. The acid-base condi- tion in vegetation, litter, and humus: VI. Ammonia fixation and humus nitrogen. Lantbruks Hogskol Ann. 11: 107-134. 194 3. MacEwan, D. M. C. Identification of the montmorillonite groups of minerals by X-rays. Nature (Lond.) 154: 577-578. 1944. McCreery, R. A. Mineralogy of Palouse and related series. Ph.D. Thesis. Library, State College of Washington. 1954. McLaren, A. D. The adsorption and reactions of enzymes and proteins on kaolinite. J. Phys. Chem. 58: 129-137. 1954. Moore, S. , and Stein, H. W. Chromatography of amino acids on sulfonated polystyrene resins. J. Biol. Chem. 192: 663- 681. 1951. , and . Photometric ninhydrin method for use in the chromatography of amino acids. J. Biol. Chem. 176: 367-388. 1948. Morrow, C. A. , and Gortner, R. A. The organic matter of the soil: V. A study of the nitrogen distribution in different soil types. Soil Sci. 3: 297-331. 1917. , and Sandstrom, W. M. Biochemical Laboratory Methods. John Wiley & Sons, Inc. , New York. 1935. Mortland, M. M. , Erickson, A. E. , and Davis, J. F. Clay am- mendments on sand and organic soils. Michigan Agric. Exp. Sta., Quarterly Bul. 40: 23-30. 1957. , and Gieseking, J. Influence of clay on the en- zymatic hydrolysis of organic phosphorus compounds. Soil Sci. Soc. Amer. Proc. 16: 10-13. 1952. , Lawton, K. , and Uehara, G. Alteration of bio- tite to vermiculite by plant growth. Soil Sci. 82: 477-481. 1956. Myers, H. E. Physicochemical reactions between organic and inorganic soil colloids as related to aggregate formation. Soil Nehéz, R. Physicochemical properties of soil organic matter. ASIOkém. Talajt 4: 19-30. 1955. 104. 105. 106. 107. 108. 109. 110. 111. 112. 113. 114. 115. 116. 117. 99 Noller, C. R. The Chemistry of Organic Compounds. W. B. Sanders Co. , Philadelphia. 1951. Norman. A. G. Problems in the chemistry of soil organic mat- ter. Soil Sci. Soc. Amer. Proc. 7: 7-15. 1942. Parker, D. I. , Sowden, F. J. , and Atkinson, H. J. The nitro- gen distribution and amino acid content of certain soil organic matter fractions. Sci. Agric. 32: 163-169, 127-133. 1952. Pavel, L. , Kolousek, J. , and Smatlak, V. Humic substances: II. Soils and Fertilizers, Commonwealth Bur. Soil Sci. 17: 412. 1954. Payne, T. M. B., Rouatt, J. W., and Katznelson, H. Detec- tion of free amino acids in soils. Soil Sci. 82: 521-524. 1956. Peters, J. P. , and Van Slyke, D. D. Quantitative Clinical Chem- istry, Methods (vol. 2). The Williams and Wilkins Co. , Balt- imore. 1932. Piper, C. S. Soil and Plant Analysis. Interscience Publishers, Inc. , New York. 1944. Piper, S. H. Some examples of information obtainable from the long spacings of fatty acids. Trans. Faraday Soc. 25: 348- 351. 1929. Putnam, H. D., and Schmidt, E. L. Free amino acids in soils. Bact. Proc. 56: 24. 1956. Randall, H. M., Fowler, R. G., Fuson, N. , and Dangl, J. R. Infra-red Determination of Organic Structures. D. Van Nos- trand Co. , Inc. , New York. 1949. Reding, V. V. Fractionation of soil nitrogen and factors affect- ing distribution. Soil Sci. 71: 253-267. 1951. Robinson, C. S. Two compounds isolated from peat soils. J. Am. Chem. Soc. 33: 564-568. 1911. Rubins, E. J. , and Bear, F. E. Carbon-nitrogen ratios in or- ganic fertilizer mate rials in relation to the availability of their nitrogen. Soil Sci. 54: 411-423. 1942. Russell, E. J. Soil Conditions and Plant Growth. Longmans, Green and Company, London. 1953. " -. we. "r -. .1 .w _ '=‘. , I ”a," R: "2‘... w-ww‘.w 118. 119. 120. 121. 122. 123. 12.4. 125. 126. 127. 128. 129. 130. 131. 100 Sanborn, I. H. Glycine anhydride. Phys. Chem. 36: 1799-1830. 1932. Scheffer, F., Welte, E., and Ziechmann, W. Synthetic humic acids. 2. Pflanzeneriiahr. Dung. Bodenk. 69: 58-65. 1955. Schwartz, H. M. , and Lea, C. H. The reaction between proteins and reducing sugars in the dry state. Relative reactivity of the 0C - and (- -amino groups of insulin. Bioch. J. 50: 713-717. 1952. Schreiner, O. . and Shorey, E. C. The presence of arginine and histidine in soils. J. Biol. Chem. 8: 381-384. 1910. Scott, A. D. , Hanway, J. J. , and Stanford, G. Thermal studies on ammonium fixation and release in certain clay minerals. Amer. Miner. 41: 701-721. 1956. Servici de Rondini, M. A. Estudio analftico del complejo coloi- dal organico en suelos Argentinos. Rev. Invest. Agric. Buenos Aires 8: 225-247. 1954. Shorey, E. C. Some methods for detecting differences in soils. U. S. Dept. Agric. Tech. Bul. 211. 1930. . Some organic soil constituents. U. S. Dept. Agric. Bur. Chem. Bul. 88. 1913. Slabaugh, W. H. The synthesis of organo-bentonite anhydrides. J. Phys. Chem. 56: 748-751. 1952. Smith, E. , and Stockell, A. Amino acid composition of crystal- line carboxypeptidase. J. Biol. Chem. 207: 501-514. 1954. Sohn, J. B. , and Peech. M. Retention and fixation of ammonia by soils. Soil Sci. 85: 1-9. 1958. Sowden, F. J. Determination of amino-acids in soil hydrolysates by the Moore and Stein method. Soil Sci. 80: 181-188. 1955. , and Parker, D. 1. Amino nitrogen of soils and certain fractions isolated from them. Soil Sci. 76: 201-208. 1953. Stanford, G. , and Hanway, J. A simplified technique for det- ermining relative nitrate production in soils. Soil Sci. Soc. Amer. Proc. 19: 69-73. 1955. 132. 133. 134. 135. 136. 137. 138. 139. 140. 141. 142. 101 Distribution of the forms of nitrogen in some 283-287. 1957. Stevenson, F. J. soil profiles. Soil Sci. Soc. Amer. Proc. 21: . Investigations of amino polysacharides in soils: I. Colorimetric determination of hexosamines in soil hydroly- sates. Soil Sci. 83: 113-122. 1957. . Investigations of amino polysacharides in soils: 11. Distribution of hexosamines in some soil profiles. Soil Sci. . Isolation and identification of some amino com- pounds in soils. Soil Sci. Soc. Amer. Proc. 20: 201-204. 1956. . Effect of some long-time rotations on the ami- no acid composition of the soil. Soil Sci. Soc. Amer. Proc. 20: 204-208. 1956. . Ion exchange chromatography of the amino acids in soil hydrolysates. Soil Sci. Soc. Amer. Proc. 18: 373-376. 1954. , Dhariwal, A. P. S. , and Choudhri, M. B. Further evidence for naturally occurring fixed ammonium in soils. Soil Sci. 85: 42-46. 1958. , Marks, J. D. , Varner, J. E. , and Martin, W. P. Electrophoretic and chromatographic investigations of clay- adsorbed organic colloids. 1. Preliminary investigation. Soil Sci. Soc. Amer. Proc. 16: 69-73. 1952. Troll, W. , and Cannan, R. K. A modified photometric ninhydrin method for the analysis of amino and imino acids. J. Biol. Chem. 200: 806-810. 1953. Van Slyke, D. D. Manometric determination of primary amino nitrogen and its applications to blood analysis. J. Biol. Chem. 83: 425-461. 1929. . The analysis of proteins by determination of the chemical groups characteristic of the different amino-acids. J. Biol. Chem. 10: 15-55. 1911. 143. 144. 145. 146. 147. 148. 149. 150. 151. 152. 153. 102 . Improvements in the method for analysis of proteins by the determination of the chemical groups charac- teristic of the different amino acids. J. Biol. Chem. 22: 281- 285. 1915. , MacFadden, D. A. , and Hamilton, P. Deter- mination of free amino acids by titration of the carbon dioxide formed in the reaction with ninhydrin. J. Biol. Chem. 141: 671-680. 1941. , Hiller, A. , and Dillon, R. T. Solubilities and compositions of the phospho-lZ-tungstates of the diamino acids and of proline, glycine, and tryptophane. J. Biol. Chem. 146: 137-157. 1942. , and . Gaseometric I determination of carboxyl groups in free amino acids. J. Biol. Chem. 141: 627-669. 1941. , and Folch, J. Manometric carbon determina- tion. J. Biol. Chem. 136: 509-541. 1940. Waksman, S. A. Soil Microbiology. John Wiley 8: Sons, Inc. , New York. 1952. . Humus. The Williams and Wilkins Company, Baltimore. 1938. , and Iyer, K. R. N. Contribution to our know- ledge of the chemical nature and origin of humus: III. The base exchange capacity of "synthesized humus" (ligno-protein and "natural humus" complexes). Soil Sci. 36: 57—67. 1933. , and . Contribution to our know- ledge of the chemical nature and origin of humus: IV. Fixa- tion of proteins by lignins and formation of complexes resistant to microbial decomposition. Soil Sci. 36: 69-82. 1933. , and . Contribution to our know- ledge of the chemical nature and origin of humus: I. On the synthesis of the "humus nucleus". Soil Sci. 34: 43-69. 1932. , and . Contribution to our know- ledge of the chemical nature and origin of humus: II. The influence of "synthesized" humus compounds and of "natural humus" upon soil microbiological processes. Soil Sci. 34: 71- 79. 1932. 154. 155. 156. 157. 103 , and . Synthesis of the humus-nu- cleus, an important constituent of humus in soils. peats, and compost. J. Wash. Acad. Sci. 22: 41-50. 1932. Willard, H. H. , and Diehl, H. Advance Quantitative Analysis. D. Van Nostrand Co. , Inc. , New York. 1943. Winsor, G. W., and Pollard, A. G. Carbon-nitrogen relation- ships in soil. J. Sci. Food Agric. 7: 134-149. 1956. Zweig, G. , and Hood, S. L. Microdesalter for qualitative paper chromatography of amino acids. Anal. Chem. 29: 438-441. 1957. APPENDIX: GLYCERATION EXPERIMENT INTRODUCTION X-rays were used in an effort to detect clay in the hydrolyzable fractions used in paper chromatography. The so called 'grauhumin- saure',, which are high molecular weight substances from which amino acids are isolated, have been reported (48, 119) to form complexes with clay minerals. Preliminary results with X-rays showed evidence that clay was not present in the hydrolyzable fractions used in paper chro- matography. Nevertheless, glyceration was adopted in order to achieve greater discrimination by expanding the lattice of the clay mineral. Glyceration caused the disappearance of the X-ray patterns. As far as the author is aware, this is the first time such a reaction has been reported in the literature. Its significance in identifying amino acid derivatives is not understood. Moreover, it is suspected that a simi- lar mechanism could mask soil amino acids. In fact, Scheffer et al. (119), were able to prepare synthetic humic acids which proved to be X- ray amorphous. REVIEW OF THE LITERATURE Glycerol is a triatomic alcohol having the formula CHzOH - CHOH - CHZOH. It shows the characteristic reactions of a primary and a se- condary alcohol together in a single molecule. Oxidation of a primary alcohol yields an acid, while a secondary alcohol yields a ketone. Al- cohols react with many acids to form esters. They can also undergo dehydration reactions to olefins in the presence of strong acids and other strongly electrophilic reagents. Glycerol is a polyfunctional mo- lecule, as are amino acids. The interaction of polyfunctional molecul- es may lead to condensation reactions giving rise to polymers of mul- tiple inter-and intra-molecular linkages, as in vulcanized rubber (35). Thus, a dibasic acid, for instance, and glycerol, give esters of high molecular weight in which there are cross links joining the chain to- gether (35). Alpha andS -amino acids tend, like the corresponding hydroxy acids, to form cyclic derivatives on heating. 1 Alpha and 5 -hydroxy acids form cyclic inner esters which are called lactones. These heterocyclic ring systems are most easily formed when the ring contains five or six atoms in all (35, 49, 54). The im- ides are similar compounds where the substituting group is not an OH group like in the hydroxy acids, but an NHz. These cyclic imides are called lactams, and their stability is also dependent upon configura- tions involving five or six atom members (54, 104). When heated, OC-amino acids and their esters form a type of cyclic 107 amide known as diketopiperazine, according to a reaction of the fol- lowing type: 0 £5 11% CH2 COOH =. / —’ ' HzC \C HZ I ZHzO *— \ NHZ N— C H Glycine H O Diketopiperazine As they are readily formed whenOC -amino acids or esters are sub- jected to high temperature, they are found among the products of pro- tein hydrolysis which involves long heating (49, 65, 75). Glycerol and amino-dicarboxylic acid additions, leading to the formation of polyamides, are known (35). When glycerol is heated in a solution of Oc-amino acids, water is lost and diketopiperazine cyclic amides are formed (75, 104). In 1900, Balbiano and Trasciatti, as cited by Katchalski (75) described the preparation of polyglycine by heating glycine in glycerol. The polymer formed is insoluble in water and the usual organic solvents. Its properties are described as re- sembling those of a horny protein and yield glycine methyl ester and diglycine methyl ester, respectively. When Balbiano‘s polymerization reaction was studied by Maillard (75, 83) he was able to isolate, in ad- dition to the horny, water-insoluble polymeric product, the water-so- luble intermediate triglycyl-glycine and glycine anhydride. The ratio between the final horny product and the stable six-membered diketo- piperazine varied depending on the conditions used. 108 Because diket0pipe razines appear to be present as an artifact in protein hydrolysates and because it was suspected to be present in the present work, it is considered appropriate to discuss some of the li- te rature describing its behavior. The following remarks are cited from Katchalski (75): "No data on the mechanism of polymerization of glycine in glycerol are available. Carothers suggests that the condensation reaction, probably, involves the formation of glycerol esters as transitory intermediates. Maillard made the curious observation that an acqueous solution of triglycyl-glycine and diketOpiperazine deposits an insolu- ble material on standing. Analytical evidence indicated that the insoluble material is a hexapeptide. Except for the solubility effect in acqueous solution, the re is no ob- vious reason why this coupling of diketOpiperazine with the polypeptide should stop at the hexapeptide stage. The glycine anhydride may, therefore, play its part as an in- termediate in the polymerization process. Esters of amino acids and peptides, generally, undergo condensa- tion more readily than the free amino acids and free peptides. The linear product of polycondensation of es- ters of amino acids are often accompanied by diketOpi- perazines in varying quantities. Frequently, a diketo- piperazine is the only compound which can be isolated 109 from the condensation mixture". Diketopiperazines have been reported to react readily with aniline, ammonia, and primary and secondary amines (75). They may, also, undergo polymerization in the solid or liquid state, as well as in sol- ution (75). Water acts as a catalyst in this polymerization reaction. The following factors were considered (56) to favor the polymeriza- tion reaction: a) elevated temperature b) use of solvents c) the passage of gases through solutions of the esters, -CO2 was found to promote condensation d) reduced pressure The polycondensation of peptide derivatives, as studied by Magee and Hofmann (82), deserves our attention in connection with Bremner's (20) finding that some constituent of humic acid preparations interfered with the estimation of free amino groups by the Van Slyke manometric technique (109). The former observed that the rate of interaction of nitrous acid with the hydrazide group of diglycyl-glycine hydrazide greatly exceeded its rate of interaction with the free amino group of the peptide. The hydrazide can be formed when the NaNOz is added to the peptide. DiketOpipe razine has been studied extensively by itself and in its connection with protein hydrolysates (4, 38, 47, 76, 78, 118). Klarman (78) found that glycine-alanine-tyrosine piperazine did not give a pos- l ,1 of» 37. .‘.’ . ”I’ "‘3” ~'2~.- 2'! 4.21m . . r. w -ana -w- up ‘w --. tux", ‘4,’7.‘... .- 110 itive Millon's test despite the fact that it contained tyrosine. He con- cluded that the piperazine nucleus interferes with this test. Katchalsky (75) has emphasized that the presence of functional groups in addition to the nonnalOC-amino and oC-carboxyl groups may tend to produce unexpected complications in these condensation reactions of amino acids leading to diketopiperazines formation. Klarman (78) has described the color reactions of diketopiperazines. Aromatic dinitro compounds (carbonyl reagents) were found to give a characte ristic reaction with diketopiperazines when heated for a short time. Precipitated moist copper oxide which reacts readily with amino acids in proteins, and in ninhydrin and biuret tests, does not give any reaction with diketopiper- azines. Ambrose and Ellis (4, 47, 76) have described the infra red proper- ties of diketopiperazine. Kellner (76) assigned a C = O vibration of 2. 9M3,448 cm.-1‘) for the first ove rtone of diketopiperazine and 3. Op- (3, 333 cm. -1) for the fundamental valence vibration of N - H groups for diketopiperazine. The CH3 vibration was found at 3. 4 to 3. 5 [6 (2,941 to 2,857 cm. 4). No information about X-ray studies with glycerated amino acids was found in the literature. The information presented in this work appears to be the first. Katchalsky (75) has presented and discussed some of the work which has been done in X-ray of polyamino acids polymerized in ethyl acetate and benzene. Brown et al. (32), has re- 0 ported interplanar spacings of 5. 2 and 11. 7 A as the two principal dif- 111 fraction rings observed from the X-ray powder diagrams of (1:1 mole) copolymers of DL-fl -phenylalanine and L-leucine, and DL-phenyl- alanine and 0C -amino isobutyric acid. Shifting of the 11. 7 X diffraction peak was observed according to the X-ray technique employed. Bernal (14) has prepared an extensive report on a preliminary X-ray study of fifteen amino acids and related compounds, including diketopiperazine. 7 As far as the infra red spectra of amino acids is concerned, Bellamy (12) has presented a detailed review of the literature on this subject. He points out that no absorption in the usual NH stretching region of 3, 500 to 3, 300 cm. -1 (2. 9 to 3. 0)“) is shown by any amino acid or hy- drochloride. This is supported by the work of many investigators cited (12). According to Bellamy, all amino acids capable of possessing the l NH3 structure, and their hydrochloride, show two characteristic ab- sorption bands at l, 600 to 1, 500 cm. -1 (6. 3 to 6. 7,“), in addition to an ionic carbonyl absorption which also takes place in this region. The first of these, at l, 660 to 1,610 cm. -1(6. 0 to 6.2,u, ), is often weak, while the second, at 1, 550 to 1,485 cm. -1 (6. 5 to 6. 7“), is usually more intense. The NH vibration for amido acids (NHZ present in an amide, instead of an amine) falls in the range 3, 390 to 3,260 cm. -1 (3. 0 to 3. 1 [1,). The location of the amide I absorption peak (carbon-carbon and C-H links) was found by Randall et al. (113), to fall between 1,600 to 1,620 -1 cm. (6. 3 to 6.2,“) in twelve out of fifteen cases. Other investigators have observed the same specific absorption (12, 60)for the amide II e\2-:’ .‘nrxr 112 (carbon-nitrogen and nitrogen-hydrogen) links in 25 compounds studied. No infra red absorption bands characteristic of amides, proteins, polypeptides, and amino acids are reported in the literature (12) for frequencies greater than 4,825 cm. -1 (2. 111.) or less than 880 cm. -1 (11.411). There is, nevertheless, information on infra red absorption by aromatic compounds, including aromatic amines, in the region a- bout 4, 000 cm. -1 (2. 5M). Bell (11), for instance, studying the absorp- tion spectra of organic derivatives of ammonia, particularly anilines, found that the band occurring in the region 2. 8 [M3, 571 cm. -1) may be regarded as due to amino groups. Alkyl and aryl introductions on the amino group of aniline caused the band to become shallower and further additions (yielding tertiary amines) caused the 2. 8/1. (3, 571 cm. -1) band to disappear. A 2. 3 #44, 348 cm. -1) band is present in aniline, as well as in substituted anilines. Ellis (46) observed a band in toluene, xyle- ne, and mesitylene in the region 2. 3 to 2.4,“.(4, 348 to 4, 167 cm. -1) while Glatt and Ellis (63) reported absorption bands at 4,216, 4, 322, -1 5,671 cm. (2.4, 2. 3, 1.8 M) and higher frequencies for polythene and Parowax. They also found a 4,291 cm. -1 (2. 3 [1.) band in nylon, a polyamide. Colthup (34) reported that the band within the range I, 625 to l, 575 cm. -1 (6. 2 to 6. 3,“) was characteristic for most aromatic materials. Again, according to Bellamy (12), these bands are highly characteris- tic of the aromatic ring itself and taken in conjunction with the C-H stretching band near 3, 030 cm. (3. Bit), they afford a ready means 113 of recognition for this structure. The following remarks are cited from Bellamy (12): "On theoretical grounds the main 1,600 and l , 500 cm. -1 (6. 3 and 6. 7,1) bands of the phenyl ring can be expected to occur also in polycyclic materials where there should be expected some broadening of the over all ranges in which they can occur. " The 1,600 to l, 500 cm. -1 (6. 3 to 6. 7p.) aromatic absorption bands are notorious for the very wide fluctuations in intensity which are en- countered in this region. Frequently, the bands are weak in conjugated structures, and are often shown only as shoulders on other bands (12). However, heterocyclic aromatics such as pyridine and pyrimidines also give a similar pair of bands in the 1,600 cm. -1 (6. 3/U.) region from the C'-'C and C:N links, although in this case the third skeletal vibra- tion is usually at appreciably lower frequencies (12). In certain cases, where the carbonyl absorption is capable of shifting toward the 1, 600 cm. - (6. 3 M) region under the influence of strong hydrogen bonds, it is difficult to differentiate between the two (12). Bellamy has presented a detailed discussion of the infra red absorp- tion of aromatic molecules and their derivatives in the region about 1, 000 cm. -1 (10,“). But, as he pointed out (12), the lower intensity of these bands renders them less generally useful than the higher-frequen- cy absorptions already described. "No information is available in the literature on these absorptions, and the correlations depend, almost 114 entirely, on the unpublished work of those workers who have drawn up correlation charts (12)". EXPERIMENTAL The samples used in the X- ray and infra red analyses were the sa- me ones used to spot the paper chromatograms. Diffraction patterns of the soil fractions were obtained with a Philips Norelco X-ray spec- trometer (1950 model) equipped with a copper target. One mililiter of the sample was served over a microscope slide and completely dried under an infra red lamp, except when otherwise indicated. The infra red absorption analyses were done with a model-21, dou- ble beam, self recording spectrophotometer, using NaCl plates, and Nujol or hexachlorobutadiene as mounting medium whenever the sample was not thick enough to smear it directly on the salt plates. Glyceration was done by mixing the sample with C. P. glycerine (4:1 sample to glycerine ratio). For the infra red studies, the samples were dried inside a vaccum oven at 70°C for four days after glycera- tion. For both analyses pure amino acid systems were prepared in a- queous solution and half of them were glyce rated as indicated above. DISCUSSION OF RESULTS X-Rays The X-ray diffraction peaks for the dicarboxylic fraction retained by the alumina column from the Ca(OH)Z treated hydrolysate of the check soil are shown in Table 16. Three outstanding diffraction peaks are readily recognized: 11. 3 X, 5.6 X, and 3.8 X. The last peak is about 1. 5 times stronger than the other two. The 11. 3 X and 3. 8 X diffraction peaks are considered complex in the sense that they showed a compact zig-zagging at their tips. The 11. 3 A peak has an accom- panying 11. 8 2. peak close to its base. Several other small peaks are also present. The similarity of those peaks with those reported by Brown et a1. (32) for their amino acid c0polymers is noticeable. The glycerated counter portion of the above mentioned fraction showed no diffraction peaks at all. Heating the glycerated samples was then considered worthwhile. Heating for 4 hours at 200°C did not alter the results. But when the sample was heated for 2 hours at 500°C, the mate rial over the plate turned, from a dark brown, to a whitish color and two small clear peaks appeared at 4. 22 X and 3. 50 X and a strong peak at 3. 35 X. These peaks could be interpreted as being due to salts which may have been able to show up when the rest of the material was "burned". Salt metal chelation by organic matter is, therefore, indi- cated. It should be pointed out that these samples were all desalted by the procedure described in the paper chromatography section of this work, so that salt protection by chelation seems to be plausible during Table 16. X-Ray Diffraction Data for Some of the Soil Fractions Before and After Glyceration Soil fraction Alumina column sorbate of the check soil after removing ammonia and humin Alumina column sorbate of the 50 tons of clay/ac re before removing ammonia and humin Alumina column sorbate of the 6. 25 tons of clay/acre after removing ammonia and humin Alumina column sorbate of the 50 tons of clay/acre after removing ammonia and humin Silica column sorbate of the 50 tons of clay/acre before removing ammonia and humin Treatment Unglycerated Glycerated Unglyc e rated Glyce rated Unglyce rated Glycerated Unglyce rated Glycerated Unglyc e rated Glyce rated Most prominent peaks observed 0 A 11.3, 5.6 and 3.8 No diffraction 7. 3. l , 4.3, 3.8 2.5, and \OI-IO‘ No diffraction No diffraction No diffraction o 13. A No diffraction 7.6, 4.3, 3.8, and 3.0 No diffraction Remarks Small peaks also present at 11.8, 3. 6, 3. 5, 3. 3, and 3. 2 Band around 17. 4 and small peaks at 8.4, 5.9, and 2. 9 One single sharp peak Band around 17. 4 and small peaks at 8.4, 5.9, and 2. 5 118 amino acid isolation from soils. Table 16 also shows the diffraction pattern of the unglycerated por- tion of the separate retained by the alumina column from the soil hydro- lysate (Fraction A) for the treatment which received 50 tons of clay per acre. Sharp peaks are observed at 7.6 R, 4. 3 X, 3. 78 X, 3.1 X, 2. 5 X, and 1.9 X. Small, but distinct, peaks are present also at 17.4, 8.4, 5. 9, and 2. 9 X. Except for the 3. 8 X peak, which was very prominent here again, the rest do not correspond to those for the dicarboxylic acid separate from the soil hydrolysate after ammonia and humin se- paration (Fraction B). It might be inferred that the removal of the hu- min precipitate or of ammonia caused the elimination of peaks for or- ganic compounds capable of reacting with glycerol. Since all the peaks disappeared after glyceration of this fraction, too, the possibility must also be entertained that crystal orientation of organic molecules over the plate was altered by the presence of glycerol. The behavior of glycerated soil amino fractions is emphasized he re for the reason that similar reactions with other organic soil constitu- ents may, seriously, interfere with the isolation of amino acids from soils. Furthermore, the possibility that such reactions may go on in the soil itself should not be overlooked. That mechanisms similar to the one just described may take place without glyceration, is suggested by the results observed in the dicar- boxylic acid fraction of the soil hydrolysate after removing the humin precipitate and ammonia from the soil hydrolysate of the 6. 25 tons of 119 clay per ac re treatment. No reflections we re shown by this sample even before glyceration. Still, this Sample showed a positive ninhydrin reaction. It is appropriate to point out that a careful visual inspection of the ninhydrin color test run on other of the glycerated dicarboxylic acid fractions after they were concentrated in vacuo to a gummy syrup, demonstrated that they had lost their sensitivity to the ninhydrin reac- tion. These samples showed a gradual blue-violet coloration when heated, not with ninhydrin itself, but with the phenol-pyridine reagent of Troll and Cannan (140) so that, when ninhydrin was actually added to the sample, there was little increase in purple coloration. It should be recalled that Sowden (129) recently reported that some peculiar com- pound eluted from the Moore and Stein column between phenylalanine and tyrosine showed an abnormal behavior toward ninhydrin. "It react- ed readily with Moore and Stein reagent but very slowly with ninhydrin in butanol spray. Full color intensity did not develop in the paper chro- matograms for more than 24 hours after spraying. " Attention is brought to the fact that a similar retardation in color over the paper was also observed, in many instances, in the present work (see paper chroma- tographic section). A color change similar to that described above when the glycerated dicarboxylic acid concentrates were treated with the phenol-pyridine reagent, has also been observed for diketOpipera- zines by Klarman (56, 78). The former observed that aromatic nitro compounds (carbonyl reagents) were found to give characteristic color reactions with diketOpiperazines. It was observed, in the present work, 120 that most of the gmnmy concentrates, prepared as indicated above, were capable of decolorizing an alcoholic solution (0. 1%) of p-nitrOphe- nol with the consequent precipitation of a white substance which is be- lieved to be of the same salty nature as the one described above when one of the dicarboxylic fractions used in X-ray analysis was ignited at 500°C. This belief is partially supported by the fact that a similar white precipitate separated out of the brown gummy concentrate after boiling in 95% alcohol, but without the apparent disappearance of the brown gummy compounds. The X- ray pattern for the unglycerated dicarboxylic acid fraction of the soil hydrolysate after humin and ammonia separation for the 50 tons per acre clay treatment showed only one peak at 13. 0 X, of very high intensity. Here again, glyceration caused the peak to disappear. The behavior toward glyceration of other samples was similar to that described above. The basic amino fraction of the hydrolysate from the 50 tons per acre clay treatment showed very intense peaks at 7. 56, 4. 29, 3.8, and 3. 5 X, before glyceration. Glyceration caused the dis- appearance of those peaks. Other basic fractions showed no peaks be- fore or after glyceration. As a matter of comparison, some pure amino acids were run (see Table 17). In the particular cases of lysine and glutamic acid, glyce- ration caused the disappearance of peaks characteristic for the pure amino acids. It is recalled that Scheffer et al. (119) found that synthetic humic Table 1 7. 121 X-Ray Diffraction Data for Some Amino Acids Before and After Glyceration Most prominent peaks observed Amino acids Treatment Remarks Glutamic acid -- 8. 7, 4. 9, 4.4, Smaller peaks were 2. 7, 2. 5 present incluging one at 2. 33 A Glutamic acid Glycerated 4.4, 2. 7, 2. 3 Lysine -- 19.6, 10. 3, 3.4 Bands rather than peaks were found at about 5. 0 6° | 4.6 and 3. 9 A Lysine Glycerated No peaks No peaks Cystine -- 4.8 and 3. 13 Other small peaks at 9.4, 4.2, 4. 0, 3. 3, .2.8, 2.7 X Histidine -- 7. 7, 6. 2, 4. 3, Other smaller 3.9, 3.6, 3.5, peaks at 8.7, 4.4, 3.4, 3.1, 2.6 3.3, 3.2, 2.9,o 2.8, 2. 7, 2.4 A Arginine -- 5. 0, 4. 4, 3. 5, Small peaks at 3.4, 2.7, 2.4, 10.2,4.5b4.3, 2 3 3.1, 2. 5 A Phenylalanine -- 16. 4, 5. 2, 5. 0, Other peaks at 4.9. 4.8. 4.3, 3.6,03.l, and 9, 3. 4 2. 6 A 122 acids, produced on the basis of multivalent phenol, were X-ray amor- phous and formed difficultly insoluble humates with polyvalent cations. The fact that glycerol is capable of "erasing" the X-ray diffraction pat- tern of soil amino hydrolysates and two pure amino acids tested, is considered very interesting. The results of X-ray analyses, together with the heating procedure, demonstrated that clay was not present in the soil nitrogenous fractions. Infra Red The infra red spectra of some of the fractions discussed above are shown on Plate 1. The broken line portion of these Spectrograms re- present the portions which can be accounted for, either by glycerol or by the solvent used in mounting the preparation. Except for curves 1?, 18, 19, and 20 on Plate 1, the rest show in successive pairs the infra red spectrograms of glycerated soil fractions or pure amino acids and their unglycerated counterparts. Curves 18, 19, and 20 show the infra red spectra of the solvents employed for smearing the samples on the salt plates. The glyce ration behavior of the acidic separate from Fraction B for the 12. 5 tons of clay per acre treatment is shown in Curves 1 and 2. Glyceration suppressed absorption at 3,840 cm. -1 (2. 6ft), enhanced the absorption which appears as a shoulder at 3,257 cm. -1 (3. 1w), and left intact the shoulder at 2,941 cm. -1 (3.4 ,(L). The glyce ration of this fraction brought about a pronnounced absorption band at 1, 198 cm. - -1 (8.4M) and broadened the band at 1,053 cm. (9. 5f»). 123 PLATE 1 INFRA RED ABSORPTION SPECTRA OF SOIL NITROGENOUS CONSTITUENTS AND AMINO ACIDS BEFORE AND AFTER Curve 1 Curve 2 - Curve 3 - Curve 4 - Curve 5 Curve 6 - Curve 7 - Curve 8 - Curve 9 - Curve 1 0- GLY CE RATION Unglycerated constituents of Fraction B retained in the al- umina colurrm from the 12. 5 tons of clay per acre treat- ment. Nujol was used as mounting medium. Glycerated constituents of Fraction B retained in the alum- ina column from the 12. 5 tons of clay per acre treatment. Glycerol served as mounting medium. Unglycerated constituents of Fraction A retained in the si- lica column from the 6. 25 tons of clay per acre treatment. Nujol was used as mounting medium. Glycerated constituents of Fraction A retained in the silica column from the 6. 25 tons of clay per acre treatment. Glycerol served as mounting medium. Unglyce rated neutral non-aromatic, final perfusate of Fraction B from the check soil. Nujol was used as mount- ing medium. Glycerated neutral non-aromatic, final pe rfusate of Fraction B from the check soil. Nujol was used as mounting medium. Unglycerated constituents of Fraction A retained in the al- umina column from the 12. 5 tons of clay per acre treat- ment. Nujol was used as mounting medium. GlyCerated constituents of Fraction A retained in the alu- mina column from the 12. 5 tons of clay per acre treatment. Glycerol served as mounting medium. Unglycerated constituents of Fraction B retained in the alu- mina column from the 25 tons of clay per acre treatment. Nujol was used as mounting medium. Glycerated constituents of Fraction B retained in the alu- mina column from the 25 tons of clay per acre treatment. Nujol was used as mounting medium. Curve 11 Curve 12 Curve 1 3 Curve 14 Curve 15 Curve 16 Curve 1 7 Curve 18 Curve 19 Curve 20 124 Unglycerated asparagine. Nujol was used as mounting medium. Glycerated asparagine. Glycerol served as mounting me- dium. Unglycerated glutamic acid. Nujol was used as mounting medium. Glycerated glutamic acid. Glycerol served as mounting medium. Unglycerated lysine. Nujol was used as mounting medium. Glycerated lysine. Glycerol served as mounting medium. Glycerated constituents of Fraction B retained in the alu- mina column from the check soil. Hexachlorobutadiene was used as mounting medium. Glycerol. Nuj ol. Hexachlo robutadiene. 125 Curve 5'“ Curve 1 :muz