The Kjeldahl Method: 140 Years 3031314573, 9783031314575

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Jaime Aguirre

The Kjeldahl Method: 140 Years

The Kjeldahl Method: 140 Years

Jaime Aguirre

The Kjeldahl Method: 140 Years

Jaime Aguirre Guildford, NSW, Australia

ISBN 978-3-031-31457-5 ISBN 978-3-031-31458-2 (eBook) https://doi.org/10.1007/978-3-031-31458-2 © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Switzerland AG The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland

I dedicate this book to my beloved mother, María, for steering me toward the studious life, and to those whom I have endeavored to guide along the same path: Alejandro Julián Melissa I also dedicate this book to the 140th anniversary of the Kjeldahl method.

Preface

My first encounter with the Kjeldahl method occurred in 1976 (that’s 47 years ago!), when I was studying Chemical Technology, in Pereira (Colombia). It was during the Food Analysis course, taught by the late Alcibíades Reyes, a teacher never forgotten. I still remember my earliest Kjeldahl test using a Parnas-Wagner-Pregl apparatus, and although the results obtained were far from correct (understandable on account of my then limited analytical skills), the experience left a significant impression in my mind, and ignited a curiosity that led me to explore the method in depth throughout the subsequent years. Three years later, in 1979, I had the opportunity to manage the Laboratory of Animal Nutrition at the Faculty of Zootechnics and Veterinary Medicine of the University of Antioquia, in Medellín (Colombia). For 10 years I trained undergraduate students on feedstuff analysis techniques, and how to use these methods for testing all kinds of animal feed (fodder, forages, compound feed, and silage) in their thesis/projects. Later, during 1992 and 1993, I was training technical staff in different companies and universities in Colombia, essentially on the setting up and operation of Kjeldahl digestion and distillation systems. While getting ahead with my career I was oriented toward different subjects, but I never lost interest in all kinds of publications on food analysis, and throughout the years I gathered a good deal of information about nitrogen determination. An appraisal of my collection of articles led me to the writing of this review, which is intended as a modest tribute to Johan Kjeldahl on the 140th anniversary of the publication of his method, coming up in March 2023. Guildford, Australia

Jaime Aguirre

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Acknowledgements

I would like to express my deepest appreciation to Mr. Thomas Storgaard, M.A., Historical Consultant and Archivist at Carlsberg Foundation, and to Ms. Merete Yding, Vice President for Publications Activities at Carlsberg Research Laboratory, for their invaluable support with the permission granted by the Carlsberg Foundation and the Carlsberg Research Laboratory to reproduce some excerpts and images from articles published by Johan Kjeldahl in Meddelelser fra Carlsberg laboratoriet (Reports from the Carlsberg Laboratory). I am also very thankful to Mr. Benjamin Rugholm, Head of External Communications and Press Relations at Foss, for his authorization to reproduce the images of Kjeldahl digestion and distillation equipment. Special thanks are extended to Ms. Gabriela Prostko, Associate Editor at Springer Nature, for her assistance throughout the publishing process. In addition, I am grateful to all the institutions that granted permission to reproduce images and/or excerpts from other works in this book, namely: American Chemical Society Bibliothèque nationale de France Carlsberg Archives Carlsberg Foundation Carlsberg Research Laboratory Elsevier B.V. Food and Agriculture Organization of the United Nations Foss Analytical Solutions John Wiley and Sons Royal Society of Chemistry Springer Nature Walter de Gruyter and Company

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Contents

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1 4

2 A Brief History of Organic Elemental Analysis . . . . . . . . . . . . . . . . . . . . 2.1 Carbon and Hydrogen Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Nitrogen Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

7 8 16 29

3 Nitrogen Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 The Kjeldahl Method Applied to Different Nitrogen Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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4 The Kjeldahl Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1 Digestion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.1 Acid Requirements and Temperature . . . . . . . . . . . . . . . . . . . . 4.1.2 Catalysts and Salt Addition . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.3 Hydrogen Peroxide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.4 Sealed Tube Digestion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.5 Microwave Digestion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2 Ammonia Distillation and Determination . . . . . . . . . . . . . . . . . . . . . . 4.2.1 Titration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.2 Spectrophotometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.3 Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.4 Ammonium Ion-Selective Electrode . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

53 53 55 55 60 62 62 63 63 66 71 71 72

5 The Advancement of Kjeldahl Equipment . . . . . . . . . . . . . . . . . . . . . . . . 5.1 Early Devices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2 The Caustic Splash Problem . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3 The Ammonia Distillation Equipment . . . . . . . . . . . . . . . . . . . . . . . . . 5.4 Solving the Problem of Fumes Disposal . . . . . . . . . . . . . . . . . . . . . . . 5.5 The Small Details: Racks and Stands . . . . . . . . . . . . . . . . . . . . . . . . . .

79 79 79 85 88 92

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5.6 The Miniature Equipment: Micro-Kjeldahl Devices . . . . . . . . . . . . . 93 5.7 Kjeldahl Equipment Today . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101 6 Experimental: Evaluation of Titanium Dioxide as a Catalyst in the Determination of Nitrogen by the Kjeldahl Method . . . . . . . . . . 6.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2 Acid-to-Salt Ratio . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3 Catalyst . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4 Experimental . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.5 Production of a Catalytic Mixture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Important Topics Related to the Kjeldahl Method . . . . . . . . . . . . . . . . . 7.1 The Carlsberg Foundation and the Carlsberg Research Laboratory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2 Nitrogen-to-Protein Conversion Factors . . . . . . . . . . . . . . . . . . . . . . . . 7.3 Reference Materials and Primary Standards . . . . . . . . . . . . . . . . . . . . 7.4 Food Standards and the Kjeldahl Method . . . . . . . . . . . . . . . . . . . . . . 7.5 Patents on Kjeldahl Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.6 Reviews of the Kjeldahl Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.7 A Comparison of the Kjeldahl and Dumas Methods . . . . . . . . . . . . . 7.8 Other Methods for Nitrogen Determination Developed in the 1900s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

105 105 106 107 108 111 119 123 123 124 129 131 134 134 138 140 141

About the Author

Jaime Aguirre Education: B.Sc. in Chemical Technology (Technological University of Pereira, Colombia). B.Sc. in Pharmaceutical Chemistry (University of Antioquia, Colombia). M.Sc. in Analytical Chemistry (University of New South Wales, Australia). Employment History: Laboratory of Animal Nutrition, Faculty of Veterinary Medicine and Zootechnics, University of Antioquia (Medellín, Colombia), 10 years. Functional Fluids Laboratory, ICI—Imperial Chemical Industries—(Sydney, Australia), 3 years. Syrup Room, Coca-Cola Amatil (Sydney, Australia), 20 years. Professional Affiliations: Royal Australian Chemical Institute (RACI). American Chemical Society (ACS).

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Chapter 1

Introduction

In 1883, Johan Kjeldahl developed a system for nitrogen determination in organic substances. Soon after its publication, numerous modifications and improvements were made by different chemists. Throughout the years, thousands of different papers have been written on the process, including suggested variations, upgrades, criticisms, and reviews, and many types of devices have been designed for both the digestion and the distillation stages. Since its introduction in 1883, the Kjeldahl method has been an essential analytical tool for nitrogen determination in research, academic, and industrial laboratories. Over the years, the method has been highly commended. Just four years later, Dafert wrote that Kjeldahl’s achievement deserved admiration: “It is and remains one of the most interesting processes in analytical chemistry” [1]. Kebler acknowledged that the method was unique in the sense that it was universally adopted in a short time [2]. Hubert Bradford Vickery stated that Kjeldahl’s contribution was one of the great accomplishments of science [3]. P. L. Kirk wrote: “Many discoveries of far-reaching importance have been fundamentally extremely simple. The discovery that boiling many nitrogen-containing organic compounds in concentrated sulfuric acid liberates the nitrogen in the form of ammonium sulfate is probably one of the most significant analytical discoveries ever made. Attributed to Kjeldahl, who announced the method in 1883, this method has probably been applied in one modification or another to every possible form of nitrogen, and in perhaps more laboratories than almost any other single type of analytical method” [4]. Johan Gustav Christoffer Thorsager Kjeldahl was born on August 16, 1849, in Jægerspris, a Danish town in the northern part of the island of Zealand. He studied applied natural sciences and received a master’s degree from the Polytechnic Institute (officially known today as the Technical University of Denmark) in 1873. In 1876, he was appointed chief of the Chemical Department of the Carlsberg Laboratory, where he had been working as a chemist since 1875. While studying protein metabolism in barley germination, he decided to research a rapid method for the quantitative analysis of proteins. © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 J. Aguirre, The Kjeldahl Method: 140 Years, https://doi.org/10.1007/978-3-031-31458-2_1

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1 Introduction

In Kjeldahl’s words: “This question became particularly urgent for me when some time ago I undertook a special study of the migration and dissolution of proteins during the brewing process, and thus was constantly confronted with an activity that could not be carried out with the existing methods, which is to perform a significant series of nitrogen determinations in a short time. For the time being, I have therefore entirely set aside my primary assignment to devote my attention exclusively to the desired method. I have been fortunate enough to solve this in a very satisfactory manner and have found that I can submit to the chemists a method by which one can determine the amount of nitrogen with sufficient accuracy and with surprising speed” [5]. He shared his method in a lecture to the Danish Chemical Society in Copenhagen on March 7, 1883. He published his findings in March 1883 in Danish (27 pages). A condensed article was published in French (10 pages), a summary (1 page) was published in English in August 1883, and an abbreviated article (17 pages) was published in German in December 1883 [6–8]. Kjeldahl died on July 18, 1900, at the age of fifty. According to Johansen: “He died suddenly while swimming during his stay in Tisvilde beach. His death was due to a brain thrombosis or hemorrhage” [9]. According to Oesper: “…While bathing at Tisvilde, Zealand, he suffered a heart attack and died in the water” [10]. Burns stated that “He died of a brain hemorrhage whilst swimming at Tisvilde …” [11]. On September 15, 1900, the British Medical Journal published a brief note on his death: “We regret to announce the death of Professor Johann Kjeldahl, Director of the Chemical and Physiological Laboratory at Alt-Karlsberg, near Copenhagen, who was seized with cramp while trying to save the life of a drowning child and fell a victim to his courageous philanthropy. Professor Kjeldahl’s name is well known to scientific men from the method for the detection of nitrogen which goes by his name” [12].

More detailed descriptions of the life and achievements of Johan Kjeldahl have been authored by Oesper [10], Burns [11], Jessen-Hansen [13], Veibel [14], Holter and Moller [15], Morries [16], and McKenzie [17] (Figs. 1.1, 1.2).

1 Introduction Fig. 1.1 Johan Kjeldahl (Reproduced with permission of John Wiley and Sons, from Johannsen, W. (1900) Johann Kjeldahl, Berichte der Deutschen Chemischen Gesellschaft, 3:3881–3888. Copyright © 1900 by John Wiley and Sons)

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1 Introduction

Fig. 1.2 Johan Kjeldahl in the Carlsberg Research Laboratory (Reproduced with permission of Carlsberg Foundation/Carlsberg Research Laboratory, from: https://www.carlsberggroup.com/ who-we-are/carlsberg-research-laboratory/. Copyright © 2023 by Carlsberg Foundation/Carlsberg Research Laboratory)

References 1. Dafert, F. W. (1887) Beiträge zur Kenntnis des Kjeldahlschen Stickstoff-BestimmungsVerfahrens (Contributions to the knowledge of the Kjeldahl nitrogen determination method), Die Landwirthschaftlichen Versuchs-Stationen (The Agricultural Experimental Station), 34:311-353. 2. Kebler, Lyman F. (1891) Notes on the estimation of nitrogen in nitrates by Kjeldahl’s method, and an index to the literature on the estimation of nitrogen, The Journal of Analytical and Applied Chemistry, 5:257-278. 3. Vickery, Hubert Bradford (1946) The early years of the Kjeldahl method to determine nitrogen, Yale Journal of Biology and Medicine, 18(6):473-516. 4. Kirk, P. L. (1950) Kjeldahl method for total nitrogen, Analytical Chemistry, 22(2):354–358. Excerpt from page 354 reproduced with permission of American Chemical Society. Copyright © 1950 by ACS. Permission conveyed through Copyright Clearance Center (CCC). 5. Kjeldahl, Johan (1883) En ny Methode til Kvælstofbestemmelse i organiske Stoffer (On a new method for nitrogen determination in organic substances), Meddelelser fra Carlsberg laboratoriet. Udgivne ved Laboratoriets Bestyrelse (Reports from the Carlsberg Laboratory. Published by the Laboratory’s Board of Directors), 2:1–25. Excerpt from page 2 reproduced with permission of Carlsberg Foundation/Carlsberg Research Laboratory. Copyright © 1883 by Carlsberg Foundation/Carlsberg Research Laboratory. 6. Kjeldahl, Johan (1883) Sur une nouvelle méthode de dosage de l’azote dans les substances organiques (On a new method for nitrogen determination in organic substances), Compte-rendu des travaux du laboratoire de Carlsberg (Reports from the Carlsberg Laboratory), 2:3-12. 7. Kjeldahl, Johan (1883) New method for determination of nitrogen, Chemical News, 48:101.

References

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8. Kjeldahl, Johan (1883) Neue Methode zur Bestimmung des Stickstoffs in Organischen Körpern (A new method for nitrogen determination in organic materials), Zeitschrift für Analytische Chemie, 22:366-382. 9. Johannsen, W. (1900) Johann Kjeldahl, Berichte der Deutschen Chemischen Gesellschaft (Reports of the German Chemical Society, now European Journal of Inorganic Chemistry), 3:3881-3888. 10. Oesper, Ralph E. (1934) Kjeldahl and the determination of nitrogen, Journal of Chemical Education, 11:457-462. 11. Burns, D. Thornburn (1984) Kjeldahl, the man, the method and the Carlsberg Laboratory, Analytical Proceedings, 21:210-214 12. The British Medical Journal, 15 September 1900, page 781. 13. Jessen-Hansen, H. (1932) Johan Kjeldahl 1849–1900. In Prominent Danish scientists through the ages, with facsimiles from their works, Meisen, V. (editor), Oxford University Press, Copenhagen, Denmark, pp. 169-172. 14. Veibel, Stig (1949) Johan Kjeldahl (1849–1900), Journal of Chemical Education, 26:459-461. 15. Holter, H. and Moller, K. M. (editors) (1976) The Carlsberg Laboratory 1876–1976, Rhodos International Science and Art Publisher, Copenhagen, Denmark, pp. 50-62. 16. Morries, P. (1983) A century of Kjeldahl (1883–1983), Journal of the Association of Public Analysts, 21(2):53-58. 17. McKenzie, Hugh A. (1994) The Kjeldahl determination of nitrogen: retrospect and prospect, Trends in Analytical Chemistry, 13(4):138-144.

Chapter 2

A Brief History of Organic Elemental Analysis

Organic elemental analysis, also called organic analysis, is the process for determining the contents of carbon, hydrogen, oxygen, nitrogen, sulfur, and halogens in a carbon-based substance. The history of organic elemental analysis began with the first experiments on combustion performed by Lavoisier, who applied a scientific approach to observations made many years before by Jean Rey and John Mayow. Because of the principle involved in the procedure, organic elemental analysis is also known as combustion analysis.

1630

Jean Rey ESSAYS DE JEAN REY

Sur la recherche de la cause pour laquelle l’estain et le plomb augmenten de poids quand on les calcine.

Jean Rey discovered that the weight of lead and tin increases when they are calcined. He explained the larger weight of calcined lead and tin by reasoning that calcination involves the incorporation of air in the metal. In his own words: “To this question I respond…That this additional weight comes from the air which in the vessel has become denser, heavier, and somehow adhesive, by the intense and constant heat of the furnace (aided by the frequent agitation), and attaches itself to its smallest parts, in the same way as water makes sand heavier by moistening it and adhering to the smallest of its grains” [1].

Robert Hooke made observations on the similarity of the action of potassium nitrate (saltpeter) and air in combustion. In his Micrographia Hooke affirmed: “…The dissolution of sulphureous bodies is made by a substance inherent, and mixt with the Air, that is like, if not the very same, with that which is fixt in Salt-peter, which by © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 J. Aguirre, The Kjeldahl Method: 140 Years, https://doi.org/10.1007/978-3-031-31458-2_2

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2 A Brief History of Organic Elemental Analysis multitudes of Experiments that may be made with Salpeter, will, I think, most evidently be demonstrated” [2].

Robert Boyle made observations on the calcination of copper, iron, tin, silver, and lead. Boyle concluded that fire and flame were material things which could be weighed in a balance [3]. Although his observations on calcination agreed closely with those quoted by Jean Rey, “Boyle gave a different explanation of the gain in weight, which he attributed to the absorption of heat instead of to the condensation of air” [4]. Like Hooke, John Mayow [5] also thought that air contains an active principle, similar to saltpeter. He affirmed: “… The air contains certain particles that I have elsewhere called nitro-aerial (oxygen atoms), particles that are absolutely essential for the maintenance of the flame, and which are drawn from the air and absorbed during combustion; so that when this elastic fluid is deprived of these atoms it can no longer support the combustion” [6].

1665

Robert Hooke MICROGRAPHIA

Or some physiological descriptions of minute bodies made by magnifying glasses. With observations and inquiries thereupon.

2.1 Carbon and Hydrogen Analysis Oxygen was first isolated by Carl Wilhelm Scheele in 1771 or 1772. “It is therefore clear that Scheele’s first experiments occurred two or three years before Priestley first produced oxygen because his date is well established” [7]. Scheele obtained oxygen by heating a variety of substances including mercuric oxide, potassium nitrate, silver carbonate, manganese nitrate, and manganese oxide. The discovery was published in his book Chemische Abhandlung von der Luft und dem Feuer (Chemical Treatise on Air and Fire) in 1777. The first methodical experiments on combustion analysis were performed by Antoine Lavoisier between 1772 and 1774. By burning different materials and carefully weighing them before and after combustion, he found that the substances appeared to increase their weight. In August 1774, by heating red mercuric oxide, Joseph Priestley made a gas that caused a glowing splinter to burst into flame and supported life in a mouse in a bottle [8]. He named it dephlogisticated air. A paper outlining the discovery, titled “An account of further discoveries in air”, was published in the Royal Society’s journal Philosophical Transactions. He stated:

2.1 Carbon and Hydrogen Analysis

9

“I have found that the earths of all denominations even the crystalline and the talcky, which are thought to be insoluble in acids, yield a pure dephlogisticated air, when treated in the manner mentioned in my former letters; but that the calcareous earths, and some of the earths of metals, as red lead and the flowers of zinc, yield it in the greatest plenty” [9].

The name oxygen was conceived by Antoine Lavoisier, who was the first to explain combustion as a process of combination with oxygen. He performed several experiments incinerating lead and tin in air enclosed in a glass vessel inverted over water or mercury [10]. He asserted:

1674

John Mayow MEDICO-PHYSICAL WORKS

Being a translation of Tractatus Quinque Medico-Physici. “I thought I could conclude from these experiments that a portion of the air itself, or of any matter contained in the air, and which exists in a state of elasticity, combines with the metals during their calcination, and that this was the cause for the increase in weight of the metallic oxide” [10].

Lavoisier (1775) defined oxygen as “vital air or eminently respirable air.” He found that “…Oxygen has none of the properties of fixed air (carbon dioxide); far from killing animals it seems on the contrary more suited to maintaining their respiration; not only were the candles and burning bodies not extinguished, but the flame spread out in a very remarkable manner; it produced much more light and clarity than in the common air; the charcoal burned with a brightness almost similar to that of phosphorus, and all combustible bodies in general were consumed there with astonishing rapidity. All these circumstances have fully convinced me that this air, far from being fixed air, was in a more breathable, more combustible state, and consequently that it was purer than the very air in which we live.” “It seems proved from this that the principle which combines with metals during their calcination, and which increases their weight, is nothing but the purest portion of the very air which surrounds us, and which we breathe…” [11].

His theory of combustion was accomplished with his article Memoir on combustion in general [12]. The theories and experiments on oxygen and combustion are detailed in the first part of his book Elementary Treatise on Chemistry [13]: Of the formation and decomposition of aeriform fluids; of the combustion of simple bodies, and the formation of acids.

Figure 2.1 shows an apparatus for studying mercury oxidation (left side of image). Experiment described in Traité élémentaire de chimie, Part 1, chapter III, page 35: Analysis of atmospheric air, and its division into two elastic fluids; one fit for respiration, the other incapable of being respired.

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2 A Brief History of Organic Elemental Analysis

Fig. 2.1 DrawingsfromLavoisier’sTraitéélémentaire de Chimie (planche IV). (Reproduced with permission of Bibliotèque nationale de France, from: Lavoiser (1789) Traité élémentaire de Chimie, présenté dans un ordre nouveau et d’après les décuvertes modernes; avec figures. Volume I. Published by Cuche, Paris. Copyright ® 1789 by Bibliotèque nationale de France)

Figure 2.1 shows an apparatus for studying red phosphorus combustion (right side of image). Experiment described in Traité élémentaire de chimie, Part 1, chapter V, pages 58 and 67: Of the decomposition of oxygen gas by sulfur, phosphorus, and charcoal, and of the formation of acids in general.

1775

Joseph Priestley An account of further discoveries in air.

Philosophical Transactions of the Royal Society of London

Figure 2.2 shows an apparatus for studying red phosphorus combustion (left side of image). Experiment described in Traité élémentaire de chimie, Part 1, chapter V, page 61: Of the decomposition of oxygen gas by sulfur, phosphorus, and charcoal, and of the formation of acids in general. Figure 2.2 shows an apparatus to determine the composition of water (right side of image). Experiment described in Traité élémentaire de chimie, Part 1, chapter VIII, page 96: Of the radical principle of water, and of its decomposition by charcoal and iron (experiment fourth).

2.1 Carbon and Hydrogen Analysis

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Fig. 2.2 Drawings from Lavoisier’s Traité élémentaire de Chimie (planche IV). (Reproduced with permission of Bibliotèque nationale de France, from: Lavoiser (1789) Traité élémentaire de Chimie, présenté dans un ordre nouveau et d’après les décuvertes modernes; avec figures. Volume I. Published by Cuche, Paris. Copyright ® 1789 by Bibliotèque nationale de France.)

1775

Antoine-Laurent de Lavoisier

Memoire sur la nature du principe qui se combine avec les metaux pendant leur calcination et qui en augmente le poids. Histoire de l’Académie Royale des Sciences

In 1810 a method of elemental analysis was published in Annales de Chimie, and in Journal de Physique, de Chimie et d’Histoire Naturelle. The procedure was conceived by Joseph Louis Gay-Lussac and Louis Jacques Thénard [14, 15]. The technique was carried out in the apparatus shown in Fig. 2.3. To remove air from the apparatus a blank combustion was conducted before any test. They mixed the sample with potassium chlorate (as an oxidizing agent) and made it into small pellets. A quantity of pellets large enough to represent 0.5–0.6 g of organic matter was dropped into a vertical glass tube heated by an alcohol lamp. The sample was burnt, and the gases given off (carbon dioxide and oxygen) were collected over mercury in a glass vessel, in which they could be measured with precision. The quantity of carbon in the sample was determined from the carbon dioxide absorbed by potassium hydroxide in the mercury vessel. The amount of water produced was calculated from the difference between the oxygen in the gases and that lost by the chlorate. The fraction of the oxygen of the

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2 A Brief History of Organic Elemental Analysis

Fig. 2.3 Gay-Lussac and Thénard apparatus for combustion analysis. (Reproduced with permission of Bibliotèque nationale de France, from Gay-Lussac and Thénard (1811) Sur l’analyse vegetale et animale, Recherches Physico Chimiques, vol 2, pp. 265–350. Copyright ® 1811 by Bibliotèque nationale de France)

potassium chlorate which had disappeared, had formed water with the hydrogen of the substance.

1789

Antoine-Laurent de Lavoisier TRAITÉ ÉLÉMENTAIRE DE CHIMIE Présénté dans un ordre nouveau et d’après les découvertes modernes; avec figures.

The combustion of a substance (glucose as an example) and the collection of carbon dioxide in potassium hydroxide are shown in the following equations:

2.1 Carbon and Hydrogen Analysis

13

2KClO3 → 2KCl + 3O2 C6H12O6 + 6O2 → 6CO2 + 6H2O CO2 + 2KOH → K2CO3 + H2O

Figure 2.3 shows the Gay-Lussac and Thénard apparatus for combustion analysis [16]. The substance was mixed with potassium chlorate, formed into solid pellets, and placed in the stopcock DD' . The rotation of the tap caused the sample to fall into the heated part of the tube AA' . The tube AA' was heated below by a large spirit lamp HH' . The combustion products were led, via the side tube BB' , through mercury into a sealed vessel. The water produced was absorbed by calcium chloride, and the carbon dioxide was absorbed on potassium hydroxide. Both products were then determined by weight. “We thus have all the data necessary to know the proportion of the principles of the vegetable substance: we know how much we have burned of this substance, since we have the weight close to half a milligram; we know how much oxygen was needed to transform it into water and carbonic acid, since the quantity is given by the difference which exists between that contained in the super oxygenated muriate and that contained in the gases; finally, we know how much carbonic acid has been formed, and we calculate how much water must have been formed” [15].

1810

Gay-Lussac et Thénard Extrait d’un mémoire sur l’analyse végétale et animale. Journal de Physique, de Chimie et d’Histoire Naturelle

Gay-Lussac and Thénard used the volumetric approach to the elemental analysis, which is the measuring of the volume of the different gases produced in combustion. Gay-Lussac [17, 18] suggested that copper oxide was preferable as the oxidizing agent. The advantage of this oxide was soon recognized, and the use of potassium chlorate was superseded. Berzelius [19, 20] replaced the volumetric evaluations by gravimetric analysis, improving the method in various ways: he positioned the combustion tube horizontally and added sodium chloride to the potassium perchlorate. The tube was then heated progressively along its length, so that the oxidation was steady. He measured the hydrogen directly by absorbing the water produced on calcium chloride and collected the carbon dioxide on potassium hydroxide. Both products were then determined by weight. Figure 2.4 shows the Berzelius apparatus for combustion analysis [20]. A mixture of organic material and oxidizing agent (potassium chlorate or copper oxide) is

14

2 A Brief History of Organic Elemental Analysis

Fig. 2.4 Berzelius apparatus for combustion analysis. (Reproduced with permission of Bibliotèque nationale de France, from: Berzelius (1831) Traité de chimie, traduit par Me. Esslinger, sur des manuscrits inédits de l’auteur, et sur la dernière édition allemande. Partie 2, Chimie organique, Tome 5. Copyright © 1831 by Bibliothèque nationale de France)

burned progressively inside the combustion tube (bottom left). Water vapor is condensed on the small bulb in the middle, an inclined tube filled with calcium chloride to absorb the water produced in the combustion, and carbon dioxide is absorbed in potassium hydroxide inside the vessel floating on mercury under the bell jar (bottom right). In 1827 William Prout [21] described a method for combustion analysis, essentially based on the volumetric measure of the gases produced. But it did not have much acceptance because the gravimetric method developed by Berzelius gave more accurate results. Additional modifications to the combustion analysis were made by Justus von Liebig [22–24], who used cupric oxide as a source of oxygen, and improved the accuracy of the method by collecting the carbon dioxide in a specially designed glass device: the 5-bulb potassium apparatus (Kaliapparat). The Kaliapparat was a small piece of glass apparatus containing a solution of potassium hydroxide to absorb the carbon dioxide produced when burning an organic substance. Estimating the increase in mass of the Kaliapparat during a combustion analysis, due to carbon dioxide absorption, gave a direct gravimetric measure of the carbon content of the sample. In 1837 Liebig published his Anleitung zur Analyse organischer Körper, in which he described his combustion methods in detail. The book was translated into French: Manuel pour l’analyse des substances organiques [25], and into English: Instructions for the analysis of organic substances [26].

2.1 Carbon and Hydrogen Analysis

15

Figures 2.5, 2.6, and 2.7 show parts of the Liebig apparatus. The five-bulb glass apparatus (Kaliapparat), in which carbon dioxide is absorbed as it bubbles through a concentrated potash solution is shown in Fig. 2.5 Figure 2.6 shows the arrangement of the Liebig apparatus (left side), where: e: Movable brick.

Fig. 2.5 Glass apparatus filled with KOH to collect carbonic acid. (Reproduced with permission of Bibliotèque nationale de France, from: Liebig (1838) Manuel pour l’analyse des substances organiques, translated from German by A. J. L. Jourdan. Copyright © 1838 by Bibliothèque nationale de France)

Fig. 2.6 Arrangement of the Liebig apparatus (left side). (Reproduced with permission of Bibliotèque nationale de France, from: Liebig (1838) Manuel pour l’analyse des substances organiques, translated from German by A. J. L. Jourdan. Copyright © 1838 by Bibliothèque nationale de France)

16

2 A Brief History of Organic Elemental Analysis

Fig. 2.7 Arrangement of the Liebig apparatus (right side). (Reproduced with permission of Bibliotèque nationale de France, from: Liebig (1838) Manuel pour l’analyse des substances organiques, translated from German by A. J. L. Jourdan. Copyright © 1838 by Bibliothèque nationale de France)

f: Wedge intended to elevate the posterior part of the movable brick. h: Tube on top of the horizontal combustion cylinder. A mixture of copper oxide and the sample is burned in the combustion tube. Figure 2.7 shows the arrangement of the Liebig apparatus (right side), where: b: Tube containing calcium chloride to absorb water. c: Glass pieces held together with rubber tubing. m: Kaliapparat.

1786

Claude Louis Berthollet Précis d’observations sur l’analyse animale comparée a l’analyse végétale. Observations et Mémoires sur la Physique, sur l’Histoire Naturelle et sur les Arts

2.2 Nitrogen Analysis In the fifteenth century the alchemist Basil Valentine showed that ammonia could be obtained by the action of alkalis on ammonium chloride.

2.2 Nitrogen Analysis

17

Gaseous ammonia was obtained in pure form from ammonium chloride by Joseph Priestley [8]. He called this gas alkaline air. The composition of ammonia was first determined by Claude Louis Berthollet in 1785 [27]. He proved that ammonia consisted of one part nitrogen to three parts hydrogen. Berthollet made a series of analyses of animal substances with nitric acid, while searching for constituents that differentiate animal from plant substances [28]. He showed that the combustion of animal tissues produced volatile alkali (ammonia), which was a product of the oxidation process. This is clearly the essence of the analytical route followed years later in the exploration of new methods for nitrogen determination. “Organized bodies are mainly composed of two substances, which have very evident distinctive characters; one produces acid when decomposed by the action of fire, and the other gives volatile alkali; one forms ardent spirit by fermentation, the other putrefies immediately, and produces volatile alkali; some leave by calcination a charcoal which burns easily, the others are reduced to a coal whose combustion is difficult; finally, some form the largest part of vegetable substances and the other most of the animal substances, and consequently they are distinguished by these two names” [29].

Antoine de Fourcroy [30, 31] made analogous observations: “M. de Fourcroy concludes from these experiments, that the theory of M. Berthollet on nitrogen in animal substances, and forming one of their characteristics, is founded on solid basis, and is confirmed every day by the experiments made on these materials. He would like the scientists who are concerned with animal structure, to follow the research on this important point, and especially to determine where this principle comes from; how and in what organ it is fixed in animals” [31].

1827

William Prout

On the ultimate composition of simple alimentary substances; with some preliminary remarks on the analysis of organized bodies in general. Philosophical Transactions

Gay-Lussac and Thénard [14–16] analyzed nitrogen in four substances of animal origin, namely fibrin, albumin, gelatin, and casein. The results were not accurate enough to be of significant value. “Having analyzed the main plant substances, naturally we had to try the analysis of animal products; but as they shall contain nitrogen, it was possible that this method of analysis could not be applied straightforwardly: it is in fact what occurred. Whenever animal substances are mixed with excess oxygenated muriate of potash and the mixture is heated, some gaseous nitrous acid is always formed” [16].

18

2 A Brief History of Organic Elemental Analysis

In 1822 Antoine Bussy applied a modification of the Gay-Lussac method to determine nitrogen in morphine [32]. Dumas and Pelletier described the determination of nitrogen in alkaloids using the Gay-Lussac and Thénard combustion apparatus [33]. They analyzed quinine, cinchonine, brucine, strychnine, caffeine, and morphine. Liebig also made determinations by measuring the gaseous nitrogen produced in the combustion [22]. The organic substance is burnt, and the evolved gases (carbon dioxide and nitrogen) are collected in a graduated tube over mercury. Their relative proportions are established by absorbing the carbon dioxide in a solution of potassium hydroxide. A detailed description of his method for nitrogen analysis can be found in Manuel pour l’analyse des substances organiques, pages 67–88 [25], and in Instructions for the analysis of organic bodies, pages 32–41 [26]. “Whenever there is a question of analyzing nitrogenous substances, the quantity of carbon and hydrogen is determined by the means which have previously indicated, and the determination of nitrogen becomes then the object of a distinct experiment in which we have no regard for the other constituent principles” [25].

Dumas remarked that ammonia is produced when treating animal tissue with potassium hydroxide. “In fact, when animal substances are treated with potash, ammonia is released; but all chemists know that this liberation is not so instantaneous as if an ammoniacal salt were treated with this base. Quite the contrary, if we operate on a rather large quantity of animal matter, several hours of sustained boiling are necessary to drive out the ammonia, even when care is taken to use a large excess of concentrated potash” [34].

1830

Anselme Payen Notice sur les moyens d’utiliser toutes les parties des animaux morts dans les campagnes.

Mémoire couronné par la Société royale et centrale d’Agriculture dans sa séance publique du 18 avril 1830.

He also observed that ammonium sulfate is produced when treating urea with potassium hydroxide or concentrated sulfuric acid: “I submitted urea to the action of boiling concentrated sulfuric acid. Pure carbonic acid is released, and ammonium sulfate remains with an excess of acid” [35]. “It suffices in effect to establish that under the influence of potash all nitrogen turns into ammonia. It does not form other nitrogen products. In the residue I have found only potash carbonate and no trace of cyanide. Like sulfuric acid, potash therefore transforms the elements of urea into ammonia and carbonic acid” [35].

It was Anselme Payen who first mentioned a method for nitrogen determination based on collecting the ammonia produced by dry distillation of the sample [36].

2.2 Nitrogen Analysis

19

Payen published a procedure for assessing the quality of organic fertilizer obtained from animal substances. “The sample is decomposed at the red temperature in a retort made of cast iron or clay, and the volatile products of the distillation are received in a flask cooled by cold water and closed by a tube immersed in water. The condensed vapors produce the crystallization of carbonate of ammonia; the amount of this salt is relative to that of the animal substance… A still more rigorous appreciation could be obtained of the quality of these fertilizers, as well as of those resulting from bones, horns, etc., by collecting the gaseous products in an excess sulfuric acid, then determining the quantity of the acid consumed, which would be easy to calculate by measuring the unbound acid with a known alkaline solution” [36].

His approach will be improved years later by Varrentrapp and Will, and further developments will lead to the Kjeldahl method. In 1831 Jean-Baptiste Dumas described a method for nitrogen determination [37] (which he had already used in 1823 [33]), based on the Gay-Lussac and Thénard combustion apparatus. The sample is mixed with copper oxide and burned in a tube containing copper turnings. The tube has been previously purged with carbon dioxide. All nitrogen oxides are reduced to elemental nitrogen, which is collected in a vertical graduated cylinder. Carbon dioxide is absorbed in caustic potash in the same cylinder.

1831

Jean-Baptiste Dumas Lettre de M. Dumas à M. Gay-Lussac, sur les prócedés de l’analyse organique. Annales de Chimie et de Physique

The skeleton equations are: Nsample + CuO → N2 + Noxides + Cu Noxides + Cu → N2 + CuO Dumas sent a letter to Guy-Lussac describing this method, and criticizing the method developed by Liebig. Curiously the letter was published in Annales de Chimie on the pages following Liebig’s article on his own method. “…Nitrogenous matters present more difficulty. M. Liebig’s memoir will be noticed in this respect, for it clearly posed the question; but a close examination of his method will show, perhaps, that he has solved it in a way which still leaves much to be desired. We will see what are the procedures that I use myself, and with the help of which this analysis can be done with certainty” [37].

20

2 A Brief History of Organic Elemental Analysis

Figures 2.8 and 2.9 show Dumas and Stas apparatuses for graphite combustion and organic analysis [39, 40]. Figures 2.10 and 2.11 show Dumas apparatuses for hydrogen, carbon, and nitrogen determination. It is worth mentioning that the publication date of the book containing the image of the nitrogen apparatus is 1828 [38]. Dumas described his method in 1831, but he had already analyzed nitrogen in alkaloids in 1823, together with Pelletier. The method is explained in great detail in the Journal de Pharmacie et des Sciences Accesoires [41]. The Dumas method underwent many improvements through the years, and it is still in use today. A critical development was made by Hugo Schiff, who invented the azotometer to measure the total volume of nitrogen generated [42]. Due to the difficulties posed by the combustion method of determining nitrogen, chemists endeavored to find a wet method. It was by then well known that nitrogencontaining compounds release ammonia upon treatment with strong alkaline substances.

Fig. 2.8 Dumas and Stas apparatus for graphite combustion. (Reproduced with permission of Bibliotèque nationale de France, from: Dumas and Stas (1841) Sur lé veritable poids atomique du carbone, Annales de Chimie et de physique, volume 38 (3rd series, 1st volume), pp. 5–59. Copyright © 1841 by Bibliothèque nationale de France)

Fig. 2.9 Dumas and Stas apparatus for organic analysis. (Reproduced with permission of Bibliotèque nationale de France, From: Dumas and Stas (1841) Sur lé veritable poids atomique du carbone, Annales de Chimie et de physique, volume 38 (3rd series, 1st volume), pp. 5–59. Copyright © 1841 by Bibliothèque nationale de France)

2.2 Nitrogen Analysis

21

Gay-Lussac highlighted the universal presence of nitrogenous matter in seeds, which he demonstrated by the formation of ammonia during their distillation. “To be convinced of this truth, it suffices to submit to distillation any seed in its natural state, or better, stripped of its woody envelope” [43].

Figure 2.8. shows the arrangement of the Dumas apparatus for graphite combustion, where: 1. CuO and KClO3 2. CuO 3. Sample 4. CuO 5. CaCl2 (for water absorption)

6. H2 SO4 (for water absorption) 7. Liebig’s 5-bulb apparatus 8. Liebig’s 5-bulb apparatus 9. Solid KOH 10. Aspirator with potash

Figure 2.9 shows the arrangement of the Dumas apparatus for organic analysis, where: 1. CuO and KClO3 2. CuO 3. Sample mixed with CuO 4. CuO

5. CaCl2 plus H2 SO4 6. Liebig’s 5-bulb apparatus 7. Solid KOH 8. Aspirator with potash

Figure 2.10 shows the arrangement of the Dumas apparatus for hydrogen and carbon determination. Figure 2.11 depicts the Dumas apparatus for nitrogen determination. A detailed explanation of the parts and the procedure can be found in Traité de chimie appliquée aux arts. Volume 1, partie organique, azote, pages 17–19 [44]. The whole system was previously purged with pure carbon dioxide. After the combustion the liberated nitrogen is swept from the tube with carbon dioxide and collected over potassium hydroxide. The volume of nitrogen is read at atmospheric pressure and its weight is calculated from the volume, pressure, and temperature.

Fig. 2.10 Dumas apparatus for hydrogen and carbon determination. (Reproduced with permission of Bibliotèque nationale de France, from: Dumas (1828) Traité de chimie appliquée aux arts. Atlas, Béchet Jeune, Libraire de l’Académie royale de médecine, Paris. Copyright © 1828 by Bibliothèque nationale de France)

22

2 A Brief History of Organic Elemental Analysis

Fig. 2.11 Dumas apparatus for nitrogen determination. (Reproduced with permission of Bibliotèque nationale de France, From: Dumas (1828) Traité de chimie appliquée aux arts. Atlas, Béchet Jeune, Libraire de l’Académie royale de médecine, Paris. Copyright © 1828 by Bibliothèque nationale de France)

1841

F. Varrentrapp and H. Will Neue Methode zur Bestimmung des Stickstoffs in organischen Verbindungen. Annalen der Chemie und Pharmacie

In 1841 Varrentrapp and Will [45–47] published a new method for nitrogen determination: the sample is blended with a mixture of potassium hydroxide (hydrate of potash) and calcium hydroxide (caustic lime), in a specially designed apparatus, and then heated. The ammonia produced was collected over hydrochloric acid and gravimetrically determined as hexachloroplatinate (Fig. 2.12). Table 2.1 shows some results of samples analyzed by Varrentrapp and Will. “MM. Varrentrapp and Will have applied their process to the determination of nitrogen in numerous organic substances, some rich and others poor in this element, which had been previously analyzed by the best chemists (melamine, urea, uric acid, oxamide, caffeine,

2.2 Nitrogen Analysis

23

Fig. 2.12 Varrentrapp and Will apparatus for nitrogen determination. (Reproduced with permission of John Wiley & Sons, from: Varrentrapp and Will (1841) Neue Methode zur Bestimmung des Stickstoffs in organischen Verbindungen, Annalen der Chemie und Pharmacie, 39(3):257–296. Copyright © 1841 by John Wiley and sons) Table 2.1 Substances analyzed by Varrentrapp and Will

Percent nitrogen Found

Calculated

Theoretical

Urea

46.79

46.76

46.64

Uric acid

33.18

33.37

33.32

Taurine

11.00

11.27

11.19

Oxamide

31.70

31.80

31.81

Caffeine

28.90

28.83

28.85

Asparagine

21.27

21.27

21.20

Melamine

66.22

66.56

66.63

Hippuric acid

7.78

7.82

7.82

Amygdalin

3.12, 2.96

3.06

Narcotine

3.77, 3.72

3.39

Piperine

4.61, 4.31

4.91

Brucine

6.60, 6.69, 7.24

7.10

24

2 A Brief History of Organic Elemental Analysis asparagine, taurine, hippuric acid, brucine, etc.). The results which they have obtained possess a strong resemblance with those of their predecessors and prove in an incontestable manner the advantages of their process” [47].

The Varrentrapp-Will method was rapidly accepted, and was widely known as the soda-lime method. But it was not the ideal method. Mulder remarked various circumstances as a consequence of which the method gave erroneous results [48]. Van der Burg remarked: “Only after I had carried out the above analyzes did I consult the original paper by Varrentrapp and Will, and learned to my astonishment that of the bases we examined only brucine and narcotine, which had also given us good results, were analyzed by them. Nowhere did we find any mention of the difficulties encountered in following Varrentrapp and Will’s method, particularly in the case of the cinchona alkaloids, except in a treatise by Mulder” [49]. Atwater wrote extensively on the procedure [50–52]. “Leaving out of account the apparent gain of nitrogen which may come from impure soda-lime, or from obvious errors in manipulation, to be mentioned beyond, the principal sources of error involve loss of nitrogen. Among the sources of loss are: 1. Incomplete ammonification of the nitrogen. 2. Dissociation or oxidation of the ammonia formed. 3. Failure of the ammonia to be completely caught and retained by the acid solution” [52]. (Atwater, 1888, page 197).

1847

Eugène Péligot

Sur un procedé propre à déterminer, d’une manière rapide, la quantité d’azote contenue dans les substances organiques. Compte Rendu des Séances de l’Académie des Sciences

From its publication in 1841 to the appearance of Kjeldahl’s method in 1883 (42 years), Varrentrapp-Will’s technique was the prevailing procedure for nitrogen analysis. In 1843 Jean Louis Lassaigne presented a procedure for detecting the presence of nitrogen in organic compounds by heating them with potassium [53]. Wilhelm Heinrich Heintz used sulfuric acid digestion in the analysis of urea. “I decided to study more closely the action of sulfuric acid on urea. Dumas has already proven that urea is decomposed into carbon dioxide and ammonia by heating with concentrated sulfuric acid” [54]. A variant was presented by Eugène Péligot, in which ammonia was determined by titration with sulfuric acid:

2.2 Nitrogen Analysis

25

“The process which I propose to adopt is a very simple modification brought to the method of MM. Will and Varrentrapp…The combustion of nitrogenous matter is carried out by means of mixing the sample with lime and soda. The ammonia, which comes from this decomposition, is condensed, and received into a known quantity of sulfuric acid. Now, like ammonia which combines with this acid lowers the titer, it becomes easy to determine the composition of this liquor and compare it to that which it presented before, to know the quantity of ammonia which it has condensed, and, consequently, the quantity of nitrogen provided by the material which has been subjected to analysis” [55].

In 1867 Wanklyn, Chapman and Smith published a method to determine nitrogen in sewage water: “The peculiar feature in our method of dealing with the organic matters present in waters, is to estimate the amount of nitrogenous organic matter by the amount of ammonia which is actually formed during distillation with carbonate of soda, caustic potash, and permanganate of potash..” “Direct experiments in which a known quantity of urea, gelatine, and albumin were taken, have shown that all the nitrogen present in these substances is obtainable in the form of ammonia when they are subjected to the treatment about to be described, and has disclosed the very singular fact that boiling with a caustic alkali liberates one-third of the nitrogen, both of albumin and of gelatine, in the form of ammonia, and that, a subsequent boiling with permanganate of potash liberates the other two-thirds. This property may be taken advantage of as a test of albuminoid matter” [56].

A review of the method was done by Wanklyn [57]. He also studied the action of potassium permanganate in excess caustic solution on different organic compounds.

1867

A. Wanklyn, E. Chapman, and M. Smith

Water analysis: determination of the nitrogenous organic matter. Journal of the Chemical Society

His conclusion was: “On referring to the results given by different substances, as described in this paper, it will be seen that, putting nitro compounds on one side, organic nitrogenous substances in general evolve ammonia on being heated to 100 °C, with strongly alkaline solution of permanganates. This reaction is very general, as an inspection of the very varied list of substances contained in this paper is sufficient to show. The compound ammonias of all kinds, the amides of the acids, such substances as pipeline, hippuric acid, creatine, the natural alkaloids, albumin, gelatin, and uric acid, evolve ammonia when treated in this way. Even so tough a substance as picoline, which, as is well known, is one of the most stubborn of organic compounds, yields ammonia when subjected to this treatment” [58]. In 1868 Frankland and Armstrong developed a combustion process for direct measurement of organic carbon and nitrogen in water: “Since we began to use this process for the estimation of organic carbon and nitrogen in waters, Wanklyn, Chapman, and Smith have proposed a new method for the determination of the latter

26

2 A Brief History of Organic Elemental Analysis

element in potable waters. Their process is founded upon a highly remarkable change which albumin and some other organic substances undergo during prolonged ebullition with an alkaline solution of potassic permanganate, by which their nitrogen is converted into ammonia. Unfortunately, however, this conversion is never complete; neither is there any guarantee that all the different forms of nitrogenous organic substances in water will thus yield up their nitrogen in the form of ammonia” [59]. In 1877 Wanklyn and Cooper presented a method to determine the amount of protein in vegetable substances, based on the amount of ammonia generated when the sample is subjected to the action of a boiling solution of potassium permanganate, a special adaptation of the ammonia process of water analysis [60]. “Between 1868 and 1877 the relative merits of Frankland’s combustion and Wanklyn’s ‘ammonia’ processes dominated discussions of water analysis in Britain” [61].

In 1878 Grete recommended the treatment of samples with concentrated sulfuric acid, before testing for nitrogen [62]. In 1883 Edmond Dreyfus used concentrated sulfuric acid to determine nitrogen in fertilizers: “One gram of sample is introduced in a small glass capsule; concentrated sulfuric acid is added to cover the fertilizer, and then heated on a Bunsen burner. Sulfuric acid displaces nitric nitrogen and dissolves organic matter”. “With this operation the fertilizer is freed of any traces of nitrate nitrogen, and in the sulfuric solution remain the total amount of organic nitrogen, and the ammonia nitrogen combined as ammonium sulfate” [63].

1883

Johan Kjeldahl

En ny Methode til Kvælstofbestemmelse i organiske Stoffer. Meddelelser fra Carlsberg laboratoriet

Johan Kjeldahl published his method for nitrogen determination in 1883 [64–66]. Since the Varrentrapp and Will method was too intricate to be used in consecutive determinations, Kjeldahl attempted a modification to Wanklyn’s method by using dilute sulfuric acid and potassium permanganate to boil the sample, concluding that ammonia would be easily formed in the presence of an acid, and should be liberated by alkalinizing the final solution. “Due to the great importance of the matter, especially for this laboratory, I have for several years performed a number of experiments according to Wanklyn’s method, to a certain extent with the extremely small amounts of substance used by the English author, and determination of the ammonia colorimetrically… In all cases, however, I found that ammonia formation was quite incomplete and, what was worse, the results were rather inconsistent” [64].

He then made a series of tests adding dilute sulfuric acid to albumin, and an excess of potassium permanganate. In this way he obtained in some experiments about 70% of the nitrogen present in the sample.

2.2 Nitrogen Analysis

27

His next step was to attempt the digestion with concentrated sulfuric acid. “The situation is quite different, however, when the samples are first subjected to a strong heating with concentrated sulfuric acid. The solution thus obtained is oxidized with an excess of dry powdered permanganate. Under these circumstances the organic nitrogen is transformed, almost without exception, into ammonium sulfate. The solution is then supersaturated with soda, distilled off and ammonia determined according to the usual methods” [64].

After having analyzed a lot of different types of nitrogenous compounds he concluded that the method was not effective with all substances: “…The formation of ammonia is even much less affected by the action of sulfuric acid when we switch to substances in which the nitrogen must be assumed to be bound in a non-amide like manner. This has been assumed for many alkaloids, in which there is a probability that the nitrogen is included as part of the benzene nucleus itself. In accordance with this, the ammonia formation by heating with sulfuric acid is also found to be extremely incomplete here. Thus, to give an example, equal amounts of ordinary albumin, morphine and quinine were treated for the same length of time with the same amount of sulfuric acid; 92% of the nitrogen in albumin was obtained as ammonia, whereas only 40% of the nitrogen in morphine, and only 25% of the nitrogen in quinine were obtained as ammonia. Of all the compounds I have examined quinine was the most difficult to decompose in the said process” [64].

Kjeldahl analyzed several substances by his method as well as by the VarrentrappWill method. “I have sought to provide the proof of the reliability of the method partly by the analysis of various pure substances with known content of nitrogen, and partly by the study of a large quantity of substances of animal or vegetable origin, with which I have at the same time carried out control provisions according to Will and Varrentrapp’s method” [64].

Table 2.2 shows the results obtained by Kjeldahl using both his method and the Varrentrapp-Will’s. The last column shows the calculated (theoretical) values. “A comparison of the two series of numbers shows that there is good conformity in the results, for both pure substances and mixtures, with the new method and Will-Varrentrapp’s method. Only with a few alkaloids the conversion to ammonia is incomplete” [64].

In 1884 Heffter, Hollrung, and Morgen compared the Kjeldahl procedure with the Varrentrapp-Will method [67]. They examined a total of 65 substances, mainly artificial fertilizers and animal feed. In each of these the nitrogen content was determined twice by the Kjeldahl’s method and three times by the Varrentrapp-Will’s method. They found that the Kjeldahl technique provided more accurate results. The fundamental steps of the final Kjeldahl method, as published in 1883, are as follows: • The sample is heated in concentrated sulfuric acid for one or two hours. • At the end of this period powdered potassium permanganate is added to complete the reaction. • After cooling and diluting the digest, a solution of concentrated sodium hydroxide is added, and distillation of the ammonia is performed.

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2 A Brief History of Organic Elemental Analysis

Table 2.2 Kjeldahl nitrogen results compared to the Varrentrapp-Will method Substance

Per cent nitrogen Kjeldahl

Varrentrapp-Will

Calculated

Triethylamine.HCl

10.16

10.18

Asparagine

18.7

18.67

Uric acid

33.1

33.3

Urea

46.6

46.7

Aniline.HCl

10.65

10.82

Indigotine

10.60

10.68

Hippuric acid

7.75

7.82

Morphine.HCl

4.21

4.36

Quinine.HCl

7.47

7.77

Caffeine

28.6

Casein

15.6

15.6

Egg albumin

15.3

15.6

28.86

Conglutin

17.5

17.6

Amygdalin

3.01

3.03

White beans

3.20

3.21

Squarehead wheat

1.94

1.96

Rye

1.46

1.47

Barley

1.33, 1.72, 1.53

1.33, 1.71, 1.55

Wort extract

0.81

0.83

Beer extract

1.10

1.12

Dried yeast

10.4

10.6

Ox meat

12.49

12.43

Witte’s peptone

13.2

13.2

• The ammonia is recovered in a solution of a standard acid. • A mixture of potassium iodide and potassium iodate is then added to the receiving flask in order to determine the free acid by titrating the generated iodine with sodium thiosulfate. The publication of this technique was followed by numerous works including improvements and modifications, amongst them: • The development of different apparatus for the method: Petri and Lehman [68], Heffter et al. [67], Reitmair and Stutzer [69]. • The introduction of mercury and copper as catalysts by Wilfarth in 1885. • The addition of potassium sulfate by J. W. Gunning in 1889, in order to increase the boiling point of the digesting solution. • The direct titration of ammonia after absorption in boric acid, introduced by Winkler in 1913.

References

29

• The use of hydrogen peroxide, first reported by Victor C. Myers in 1920. • The search for new catalysts, the most significant being selenium, introduced by Lauro in 1931, and titanium dioxide, introduced by Williams in 1973.

References 1. Rey, Jean (1630) Essays de Jean Rey, docteur en médecine. Sur la recherche de la cause pour laquelle l’estain et le plomb augmentent de poids quand on les calcine. Guillaume Millanges, imprimeur ordinaire du roy, Paris. Excerpt from pages 96–97 reproduced with permission of Bibliothèque nationale de France. Copyright © 1630 by Bibliothèque nationale de France. 2. Hooke, Robert (1665) Micrographia: or some physiological descriptions of minute bodies made by magnifying glasses. With observations and inquiries thereupon. Observation xvi. Of charcoal, or burnt vegetables. Printed by Jo. Martyn and Ja. Allestry, printers to the Royal Society, London. Excerpt from page103 reproduced with permission of Bibliothèque nationale de France. Copyright © 1665 by Bibliothèque nationale de France. 3. Boyle, Robert (1673) New experiments to make the parts of fire and flame stable and ponderable, included in The philosophical works of the honourable Robert Boyle. By Peter Shaw, M.D. Vol. II, Fire and flame weighed in a balance. Printed by W.G. for M. Pitt, 1725, London. 4. Lowry, T. M. (1915) Historical Introduction to Chemistry, MacMillan and Co., London. 5. Mayow, John (1674) Medico-physical Works. Being a translation of Tractatus Quinque Medicophysici. Published by The Alembic Club, 1904, London. 6. Ledru, L. and Gaubert, H. C. (1840) Traduction des ouvres chimiques et physiologiques de Jean Mayow, Paris. Excerpt from page 41 reproduced with permission of Bibliothèque nationale de France. Copyright © 1840 by Bibliothèque nationale de France. 7. West, John B. (2014) Carl Wilhelm Scheele, the discoverer of oxygen, and a very productive chemist, American Journal of Physiology: Lung Cellular and Molecular Physiology, pp. L811L816. 8. Priestley, Joseph (1775) Experiments and observations on different kinds of air, 2nd edition, Printed for J. Johnson, No. 72, in St. Paul’s Church Yard, London. 9. Priestley, Joseph (1775) An account of further discoveries in air, Philosophical Transactions of the Royal Society, 65:384–394. Excerpt from page 392 reproduced with permission of Royal Society of Chemistry (through PLSclear). Copyright © 1775 by Royal Society of Chemistry. 10. Lavoisier, Antoine Laurent (1774) Mémoire sur la calcination de l’étain dans les vaisseaux fermés et sur la cause de l’augmentation du poids qu’acquiert ce métal pendant cette operation (Memoir on the calcination of tin in closed vessels and on the cause of the gain in weight which this metal acquires in the operation). Histoire de l’Académie Royale des Sciences, pp. 351– 367. Excerpt from page 351 reproduced with permission of Bibliothèque nationale de France. Copyright © 1774 by Bibliothèque nationale de France. 11. Lavoisier, Antoine Laurent (1775) Mémoire sur la nature du principe qui se combine avec les métaux pendant leur calcination et qui en augmente le poids (Memoir on the nature of the principle which combines with metals during calcination and increases their weight). Histoire de l’Académie Royale des Sciences, pp. 520–526. Excerpt from page 525 reproduced with permission of Bibliothèque nationale de France. Copyright © 1775 by Bibliothèque nationale de France. 12. Lavoisier, Antoine Laurent (1775) Mémoire sur la combustion en general (Memoir on combustion in general). Histoire de l’Académie Royale des Sciences (published in 1777), pp. 592–600. 13. Lavoisier, Antoine Laurent (1789) Traité élémentaire de chimie, présénte dans un ordre nouveau et d’après les découvertes modernes; avec figures. Published by Cuchet, Paris.

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2 A Brief History of Organic Elemental Analysis

14. Gay-Lussac, J. L., and Thénard, L. J. (1810) Extrait d’un mémoire sur l’analyse végétale et animale (Extract from a dissertation on plant and animal analysis), Annales de Chimie, 74:47-64. 15. Gay-Lussac, J. L., and Thénard, L. J. (1810) Extrait d’un mémoire sur l’analyse vegétale et animale (Extract from a dissertation on plant and animal analysis), Journal de Physique, de Chimie, d’Histoire Naturelle et des Arts, 70:257–266. Excerpt from page 260 reproduced with permission of Bibliothèque nationale de France. Copyright © 1811 by Bibliothèque nationale de France. 16. Gay-Lussac, J. L., and Thénard, L. J. (1811) Sur l’Analyse végétale et animale, “Quatrième partie: Méthode pour déterminer la proportion des principes qui constituent les substances végétales et animales, et application de cette méthode d’analyse d’un grand nombre de ces substances. Présenté à l’Institut le 15 janvier 1810”, Recherches Physico-Chimiques, Volume 2, pp. 265–350. Excerpt from pages 322–323 reproduced with permission of Bibliothèque nationale de France. Copyright © 1811 by Bibliothèque nationale de France. 17. Gay-Lussac, J. L. (1815) Observation sur l’acide urique. Annales de Chimie et de Physique, 96:53–54. 18. Gay-Lussac, J. L. (1815) Recherches sur l’acide prussique. Annales de Chimie et de Physique, 95:136–231. 19. Berzelius, J. J. (1814) Experiments to determine the definite proportions in which the elements of organic nature are combined. Annals of Philosophy, 4:323–331, and 4:401–409. 20. Berzelius, J. J. (1831) Traité de chimie, traduit par Me. Esslinger, sur des manuscrits inédits de l’auteur, et sur la dernière édition allemande. Partie 2, Chimie organique, Tome 5. 21. Prout, William (1827) On the ultimate composition of simple alimentary substances; with some preliminary remarks on the analysis of organized bodies in general, Philosophical Transactions, 117:355–388. 22. Liebig, Justus (1830) Ueber die Analyse organischen Substanzen (On the analysis of organic substances), Annalen der Physik und Chemie, 94(3):357–367. 23. Liebig Justus (1831) Sur un nouvel appareil pour l’analyse des substances organiques; et sur la composition de quelques-unes de ces substances (On a new apparatus for the analysis of organic substances; and on the composition of some of these substances), Annales de Chimie et de Physique, 47(2):147–197. 24. Liebig, Justus (1831) Ueber einen neuen Apparat zur Analyse organischer Körper, und über die Zusammensetzung einiger organischen Substanzen (On a new apparatus for the analysis of organic bodies, and on the composition of some organic substances), Annalen der Physik und Chemie, 97(1):1–43. 25. Liebig, Justus (1838) Manuel pour l’analyse des substances organiques, translated from German by A. J. L. Jourdan. Excerpt from page 67 reproduced with permission of Bibliothèque nationale de France. Copyright © 1838 by Bibliothèque nationale de France. 26. Liebig, Justus (1839) Instructions for the chemical analysis of organic bodies, translated from German by William Gregory. 27. Berthollet, C. L. (1785) Analyse de l’alkali volatile (Analysis of volatile alkali), Histoire de l’Académie Royale des Sciences, pp. 316–326. 28. Berthollet, C. L. (1780) Recherches sur la nature des substances ani-males, et sur leurs rapports avec les substances végétales (Research on the nature of animal substances, and on their relationship with vegetable substances), Histoire de l’Académie Royale des Sciences, pp. 120–125. 29. Berthollet, C. L. (1786) Précis d’observations sur l’analyse animale comparée a l’analyse végétale (Detailed observations on animal analysis compared to plant analysis), –Read at the public session of the Faculty of Medicine on 29 December 1785–, Observations sur la Physique, sur l’Histoire Naturelle et sur les Arts, 28:272–275. Excerpt from page 272 reproduced with permission of Bibliothèque nationale de France. Copyright © 1786 by Bibliothèque nationale de France. 30. Fourcroy, Antoine François de (1789) Mémoire sur l’existence de la matiere albumineuse dans les végétaux (Memoir on the existence of albuminous matter in plants), Annales de Chimie, pp. 252–262.

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31. Fourcroy, Antoine François de (1789) Extrait d’un memoire ayant pour titrer Recherches pour server a l’histoire du gaz azote ou de la mofette, come principe des matières animales (Extract from a memoir entitled Research to serve the history of nitrogen gas or mofette, as the principle of animal matters), Annales de Chimie, pp. 40–46. Excerpt from page 46 reproduced with permission of Bibliothèque nationale de France. Copyright © 1789 by Bibliothèque nationale de France. 32. Bussy, Antoine (1822) Sur l’analyse des substances végétales ou animales (On the analysis of plant or animal substances), Journal de Pharmacie et des Sciences Accessoires, 8:580-590. 33. Dumas, J. B. and Pelletier, P. J. (1823) Recherches sur la composition élémentaire et quelques propriétés charactéristiques des bases salifiables organiques, Annales de Chimie et de Physique, 24:163-191. 34. Dumas, J. B. (1830) Sur l’oxamide, matière qui se rapproche de quelques substances animales (On oxamide, a material similar to some animal substances), Annales de Chimie et de Physique, 44:129–143. Excerpt from page 129 reproduced with permission of Bibliothèque nationale de France. Copyright © 1830 by Bibliothèque nationale de France. 35. Dumas, J. B. (1830) Sur la composition de l’urée (On the composition of urea), Annales de Chimie et de Physique, 44:273–278. Excerpts from pages 274–275 reproduced with permission of Bibliothèque nationale de France. Copyright © 1830 by Bibliothèque nationale de France. 36. Payen, Anselme (1830) Notice sur les moyens d’utiliser toutes les parties des animaux morts dans les campagnes (Notification on how to use all parts of dead animals in the countryside), Mémoire couronné par la Société royale et centrale d’Agriculture dans sa séance publique du 18 avril 1830. Printed by M. Huzard, Paris. Excerpts from pages 103–104 reproduced with permission of Bibliothèque nationale de France. Copyright © 1811 by Bibliothèque nationale de France. 37. Dumas, J. B. (1831) Lettre de M. Dumas à M. Gay-Lussac, sur les prócedés de l’analyse organique (Letter of Mr Dumas to Mr Gay-Lussac about the procedures for organic analysis), Annales de Chimie et de Physique, 47:198–213. Excerpt from page 204 reproduced with permission of Bibliothèque nationale de France. Copyright © 1831 by Bibliothèque nationale de France. 38. Dumas, J. B. (1828) Traité de chimie appliquée aux arts. Atlas, Béchet Jeune, Libraire de l’Académie royale de médecine, Paris. 39. Dumas, J. B. and Stas, J. S. (1841) Sur lé veritable poids atomique du carbone (On the true atomic weight of carbon), read to the Academy of Sciences on 21 December 1840, Annales de Chimie et de Physique, volume 38 (3rd series, 1st volume), pp. 5–59. 40. Dumas, J. B. and Stas, J. S. (1841) Untersuchungenn über das wahre Atomgewicht des Kohlenstoffs (Investigations on the true atomic weight of carbon), Annalen der Chemie und Pharmacie, 38:141-195. 41. Dumas, J. B. (1834) De l’analyse elementaire des substances organiques, Journal de Pharmacie et des Sciences Accessoires, 20:129–156. 42. Schiff, Hugo Josef (1868) Zur Azotometrie (On azotometry), Zeitschrift für Analytische Chemie, 7(1):430–432. 43. Gay-Lussac L. J. (1833) Sur la présence de l’azote dans toutes les semences (On the presence of nitrogen in all seeds), Annales de Chimie et de Physique, 53:110–111. Excerpt from page 110 reproduced with permission of Bibliothèque nationale de France. Copyright © 1833 by Bibliothèque nationale de France. 44. Dumas J. B. (1847) Traité de chimie appliquée aux arts. Volume I - Partie organique (Analyse élémentaire des matières organiques. Détermination de l’azote), pages 17–19, Félix Oudart, éditeur, Liége. 45. Varrentrapp, Franz, and Will, Heinrich (1841) Neue Methode zur Bestimmung des Stickstoffs in organischen Verbindungen (A new method for nitrogen determination in organic substances), Annalen der Chemie und Pharmacie, 39(3):257-296. 46. Varrentrapp, Franz, and Will, Heinrich (1841) Neue Methode zur Bestimmung des Stickstoffes in organischen Verbindungen (A new method for nitro-gen determination in organic substances), Journal für Praktische Chemie, 24(1):303-327.

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2 A Brief History of Organic Elemental Analysis

47. Varrentrapp, Franz, and Will, Heinrich (1843) New method to determine the amount of nitrogen in organic compounds, American Journal of Pharmacy, New Series, 8:141-151. 48. Mulder, E. (1860) Ueber Vermeidung von Fehlern bei der Stickstoffbestim mung mittels Natronkalk (On avoiding errors in nitrogen determination using soda lime), Chemisches Zentral Blatt, 6(1):44-45. 49. van der Burg, E. A. (1865) Chemische Mittheilungen in Betreff der China-Alkaloide und der Stickstoffbestimmung mittelst Natronkalks (Chemical communications regarding the cinchona alkaloids and the determination of nitrogen by means of soda lime), Zeitschrift für Analytische Chemie, 4(1):273-330. 50. Atwater, W. O. and Woods, C. D. (1887) IV - Notes on the soda-lime method for determining nitrogen, American Chemical Journal, 9(5):311-324. 51. Atwater, W. O. and Ball, E. M. (1888) VII - On certain sources of loss in the determination of nitrogen by soda-lime, American Chemical Journal, 10(2):113-119. 52. Atwater, W. O. (1888) VIII - On sources of errors in determinations of nitrogen by soda-lime, and means of avoiding them, American Chemical Journal, 10(3):197–209, 10(4):262–282. 53. Lassaigne, Jean Louis (1843) Mémoire sur un procédé simple pour constater la presence de l’azote dans des quantités minimes de mati˙ere organique (Dissertation on a simple process for determining the presence of nitrogen in minimal amounts of organic matter), Compte Rendu des Séances de l’Académie des Sciences, 16(4):387-391. 54. Heintz, W. (1845) Ueber die quantitative Bestimmung des Harnstoffs, des Kalis und Ammoniaks im Harn, und über die Zusammensetzung des salpetersauren Harnstoffs (On the quantitative determination of urea, potash and ammonia in urine, and on the composition of the nitric acid urea), Annalen der Physik, 142(9):114-160. 55. Péligot, Eugene (1847) Sur un procédé propre à déterminer, d’une manière rapide, la quantité d’azote contenue dans les substances organiques (On a specific process to quickly determine the amount of nitrogen contained in organic substances), Compte Rendu des Séances de l’Académie des Sciences, Vol. 24, n. 1, pp. 550–553. Excerpt from page 551 reproduced with permission of Bibliothèque nationale de France. Copyright © 1847 by Bibliothèque nationale de France. 56. Wanklyn, J. A., Chapman, E. T., and Smith, M. H. (1867) Water analysis: determination of the nitrogenous organic matter, Journal of the Chemical Society, 20:445–454. Excerpt from page 448 reproduced with permission of Royal Society of Chemistry (through PLSclear). Copyright © 1867 by Royal Society of Chemistry. 57. Wanklyn, J. A. (1867) Verification of Wanklyn, Chapman and Smith’s water analysis on a series of artificial waters, Journal of the Chemical Society, 20:591-595. 58. Wanklyn, J. A. (1868) On the action of oxidizing agents on organic compounds in presence of excess of alkali, Journal of the Chemical Society, 21:161–172. Excerpt from page 170 reproduced with permission of Royal Society of Chemistry (through PLSclear). Copyright © 1868 by Royal Society of Chemistry. 59. Frankland, E. and Armstrong, H. E. (1868) On the analysis of potable waters, Journal of the Chemical Society, 21:77–108. Excerpt from page 97 reproduced with permission of Royal Society of Chemistry (through PLSclear). Copyright © 1868 by Royal Society of Chemistry. 60. Wanklyn, J. Alfred, and Cooper, W. J. (1877) On a method of determining the amount of protein compounds in vegetable substances, The London, Edinburgh and Dublin Philosophical Magazine and Journal of Science, Volume III, fifth series, 3(19):282-285. 61. Hamlin, Christopher (1990) A science of impurity: water analysis in nineteenth century Britain. University of California Press, Berkeley. 62. Grete, E. A. (1878) Ueber die Bestimmung stickstoff haltiger organischer Substanzen (On the analysis of organic substances containing nitrogen), Berichte der Deutschen Chemischen Gesellschaft, 11:1558. 63. Dreyfus, Edmond (1883) Détermination de l’azote total dans les engrais (Determination of total nitrogen in fertilizers), Bulletin de la Société Chimique de Paris, 40:267–271. Excerpt from page 269 reproduced with permission of Bibliothèque nationale de France. Copyright © 1883 by Bibliothèque nationale de France.

References

33

64. Kjeldahl, J. (1883) En ny Methode til Kvælstofbestemmelse i organiske Stoffer (A new method for nitrogen determination in organic substances), Meddelelser fra Carlsberg laboratoriet (Reports from the Carlsberg Laboratory), 2:1–25. Excerpts from pages 4, 8, 14, 23, and table on page 25 reproduced with permission of Carlsberg Foundation/Carlsberg Research Laboratory. Copyright © 1883 by Carlsberg Foundation/Carlsberg Research Laboratory. 65. Kjeldahl, J. (1883) A new method for nitrogen determination in organic materials, Chemical News, 48:101-102. 66. Kjeldahl, J. (1883) Neue Methode zur Bestimmung des Stickstoffs in organischen Körpern (A new method for nitrogen determination in organic materials), Zeitschrift für Analytische Chemie, 22:366-382. 67. Heffter, Hollrung and Morgen (1884) Ein Beitrag zu der Methode der Stick-stoffbestimmung nach Kjeldal (A contribution to the Kjeldahl method for nitrogen determination), Chemiker Zeitung, 8(25):432-435. 68. Petri and Lehman, Th. (1884) Die Bestimmung des Gesamtstickstoffs im Harn (The determination of total nitrogen in urine), Zeitschrift für Physiologische Chemie, 8:200-213. 69. Reitmair, O. and Stutzer, A. (1885) Bestimmung des Stickstoffs in Stoffen vegetabilischen oder animalischen Ursprungs (Determination of nitrogen in substances of vegetable or animal origin), Chemiker-Zeitung, 9:1357-1358.

Chapter 3

Nitrogen Compounds

Nitrogen is the first member in Group 15 of the periodic table; it is one of the most important elements in the chemistry of life. All organisms are built from the same six essential elemental ingredients: carbon, hydrogen, nitrogen, oxygen, phosphorus, and sulfur. Nitrogen chemistry is complex and extensive. Both organic and inorganic nitrogen-containing compounds present an immense structural diversity. The most significant use of nitrogen in industrial chemistry is the production of ammonia, which is used to make other compounds, such as ammonium sulfate, ammonium nitrate, urea, and nitric acid. Nitrogen forms a large number of organic compounds. The amines, amino acids, and amides are derived from or closely related to ammonia. Nitrogen heterocyclic compounds are of extraordinary significance in organic chemistry. They are widely distributed in Nature, playing a vital role in the metabolism of all living cells. Many N-heterocyclic compounds possess physiological and pharmacological properties, and are constituents of many biologically active molecules, including many vitamins, nucleic acids, pharmaceuticals, antibiotics, amongst many others. A nitrogen atom has seven electrons arranged in the electron configuration 1s2 2s2 2p3 . There are five valence electrons in the 2s and 2p orbitals. The 2s electrons form a lone pair, whereas the 3p electrons are unpaired (Fig. 3.1). Nitrogen can have oxidation states that range from −3 to +5. Some examples are described in Table 3.1. List of some nitrogen compounds: 1. Inorganic compounds 1. 2. 3. 4. 5.

Ammonia Cyanides (sodium cyanide) Hydrazine Hydrazoic acid (hydrogen azide) Hydroxylamine

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 J. Aguirre, The Kjeldahl Method: 140 Years, https://doi.org/10.1007/978-3-031-31458-2_3

35

36

3 Nitrogen Compounds

Fig. 3.1 Structural formula of a nitrogen compound showing three covalent bonds and a lone pair

Table 3.1 Different oxidation states of nitrogen Oxidation number Electronic configuration

Chemical formula

Compound

NH3

Ammonia

– 3

N3−

1s2 2s2 2p6

−2

N2−

1s2 2s2 2p5

H2 N–NH2

Hydrazine

−1

N1−

1s2 2s2 2p4

NH2 OH

Hydroxylamine

0

N0

1s2 2s2 2p3

N2

Nitrogen (g)

+1

N1+

1s2 2s2 2p2

N2 O

Nitrous oxide

+2

N2+

1s2 2s2 2p1

NO

Nitric oxide

+3

N3+

1s2 2s2 2p0

N2 O3

Dinitrogen trioxide

+4

N4+

1s2 2s1 2p0

N2 O4

Dinitrogen tetroxide

+5

N5+

1s2 2s0 2p0

N2 O5

Dinitrogen pentoxide

6. Nitrogen halides and oxohalides (nitrogen trichloride, nitrosyl chloride, nytril chloride) 7. Nitrogen oxides (nitric oxide, nitrogen dioxide) 8. Nitrogen oxoacids and their salts (nitric acid, sodium nitrate) 2. Organic compounds 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

Aliphatic amines (dimethylamine) Amides (acetamide) Amino acids (glycine, L-lysine, tryptophan) Anilides Aromatic amines (aniline) Azides Azo compounds Azoxy compounds Bicyclic compounds (tropane) Diazo compounds Heterocyclic aromatic amines (imidazole, pyrazole, pyrazolone, pyridazine, pyridine, pyrimidine, pyrrole, triazine, triazole,)

3 Nitrogen Compounds

37

12. Heterocyclic non-aromatic amines (azepane, azepine, azetidine, aziridine, piperazine, piperidine, pyrrolidine) 13. Hydrazides (acyl hydrazide, sulfonyl hydrazide) 14. Hydrazones 15. Imides 16. Imines 17. Iso cyanates 18. Isonitriles 19. Nitriles 20. Nitro amines 21. Nitro compounds 22. Nitrosamines 23. Opioids (morphine, codeine, papaverine, thebaine) 24. Oximes (aldoximes, ketoximes) 25. Polynuclear aromatic amines (acridine, benzimidazole, cinnoline, indole, isoquinoline, phenazine, phtalazine, pteridine, purine, quinazoline, quinoline, quinoxaline) 26. Purine bases (adenine, guanine) 27. Pyrimidine bases (cytosine, thymin, uracyl) 28. Quaternary ammonium compounds 29. Semicarbazones 30. Vitamins containing nitrogen (biotin, cobalamin, folic acid, niacin, pantothenic acid, pyridoxal phosphate, riboflavin, tiamine) Examples of some structural formulas of nitrogen compounds: Ammonia Ammonia is an inorganic compound of nitrogen and hydrogen with the chemical formula NH3 .

Hydrazine Hydrazine, N2 H4 , is a molecule in which one hydrogen atom in NH3 is replaced by an –NH2 group.

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3 Nitrogen Compounds

Hydroxylamine Hydroxylamine, NH2 OH, is a molecule in which one hydrogen atom in NH3 is replaced by a –OH group.

Potassium nitrate Potassium nitrate is an inorganic compound with the chemical formula KNO3 .

Sodium cyanide Sodium cyanide is an inorganic compound with the chemical formula NaCN.

Dimethylamine Dimethylamine is an aliphatic amine with the chemical formula (CH3 )2 NH. It is a secondary amine.

Aniline Aniline is an aromatic amine with the chemical formula C6 H5 NH2 . It is the simplest aromatic amine.

3 Nitrogen Compounds

39

Pyrrole Pyrrole is a heterocyclic aromatic amine with the chemical formula C4 H4 NH. It is a five-membered ring.

Pyridine Pyridine is a heterocyclic aromatic amine with the chemical formula C5 H5 N. It is a six-membered ring. Pyridine alkaloids are a class of alkaloids that contain a pyridine ring. Typical examples are nicotine and ricinine. The leaves of Nicotiana tabacum (tobacco) contain nicotine as a major constituent and several other pyridine alkaloids as minor constituents.

Pyridine

Nicotine

Pyrimidine Pyrimidine is a heterocyclic aromatic amine with the chemical formula C4 H4 N2 . It is a six-membered ring.

Purine Purine is a heterocyclic polynuclear aromatic amine that consists of two rings (pyrimidine and imidazole) fused together. Caffeine and theobromine, methylated derivatives of xanthine, are generally the main purine alkaloids.

40

Purine

3 Nitrogen Compounds

Caffeine

Quinoline Quinoline is a heterocyclic polynuclear aromatic amine with the chemical formula C9 H7 N. Cinchona alkaloids have so far been one of the most researched natural products. Quinine is a quinidine alkaloid isolated from the bark of the cinchona tree. In quinine the hydrogen at the 6-position of the quinoline ring is substituted by a methoxy group.

Quinoline

Quinine

Isoquinoline Isoquinoline is a heterocyclic polynuclear aromatic amine. It is a structural isomer of quinoline. Isoquinoline and quinoline are benzopyridines, which are composed of a benzene ring fused to a pyridine ring. Morphine is a benzylisoquinoline alkaloid with two additional ring closures.

Isoquinoline

Morphine

Pyrrolidine Pyrrolidine, also known as tetrahydropyrrole, is a heterocyclic non-aromatic amine with the chemical formula (CH2 )4 NH.

3 Nitrogen Compounds

41

Piperidine Piperidine is a heterocyclic non-aromatic amine with chemical formula (CH2 )5 NH. It consists of a six-membered ring containing five methylene bridges (–CH2 –) and one amine bridge (–NH–).

Piperazine Piperazine is a heterocyclic non-aromatic amine with chemical formula C4 H10 N2 . It consists of a six-membered ring containing two nitrogen atoms at opposite positions in the ring.

Tropane Tropane is a nitrogenous bicyclic organic compound. Other alkaloids are derived from it, such as atropine, cocaine, hyoscyamine, and scopolamine.

Tropane

Cocaine

Acetamide Acetamide is the amide of acetic acid with the formula CH3 CONH2 .

42

3 Nitrogen Compounds

Nitriles Nitriles are compounds with the functional group –C≡N.

Azo compounds Azo compounds are organic compounds with the functional group diazenyl (R– N=N–R’, in which R and R’ can be either aryl or alkyl groups).

Nitro compounds Nitro compounds are organic compounds with one or more nitro functional groups (−NO2 ).

Nitrosamines Nitrosamines are organic compounds with the chemical structure R2 N–N=O, where R is usually an alkyl group.

Iso cyanates Iso cyanates are organic compounds with the chemical structure −N=C=O.

Quaternary ammonium compounds Quaternary ammonium compounds are salts of quaternary ammonium cations.

3 Nitrogen Compounds

43

Amino acids Amino acids are organic compounds that contain both amino and carboxylic acid functional groups.

Tryptophan

Glycine

Nucleobases Nucleobases are nitrogen-containing biological compounds that form nucleosides, which, in turn, are components of nucleotides. Purine bases

Guanine

Adenine

Pyrimidine bases

Cytosine

Thymine

Uracil

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3 Nitrogen Compounds

Vitamins containing nitrogen

Biotin

Folic acid

Niacin

Pantothenic acid

3.1 The Kjeldahl Method Applied to Different Nitrogen Compounds Soon after the publication of the method different laboratories began trials applying it to an assortment of nitrogen compounds. Petri and Lehmann found the method excellently suited for the determination of the total nitrogen in urine. “Absolute accuracy, handiness and little time expenditure. We have obtained excellent results for food, feces, and other substances to be considered here… The numerical evidence supplied therefore proves the applicability of the method in the clearest possible way” [1]. Some results are shown in Table 3.2. Heffter, Hollrung and Morgen carried out analyses on a diverse number of substances [2]. A total of 65 samples were examined. In each of them the nitrogen content was determined by both Kjeldahl’s and Varrentrapp-Will’s methods, obtaining very favorable conclusions for the Kjeldahl method in every respect. Some results are shown in Table 3.3. Bosshard, at the Zurich Agricultural Chemistry Laboratory of the Polytechnic, applied Kjeldahl’s method to determine nitrogen in allantoin, aminovaleric acid, Table 3.2 List of some compounds analyzed by Petri and Lehmann

Substance

Per cent nitrogen Found

Calculated

Theoretical

Ammonium sulfate

21.26

21.17

21.20

Urea

46.68

46.72

46.64

Uric acid

33.36

33.33

33.32

7.83

7.82

7.82

Hippuric acid

3.1 The Kjeldahl Method Applied to Different Nitrogen Compounds Table 3.3 List of some compounds analyzed by Heffter, Hollrung, and Morgen

Substance

45

Percent nitrogen Method Varrentrapp-Will

Kjeldahl

Cottonseed meal

6.71, 6.85, 6.90

6.77, 6.99, 6.90

Coconut meal

3.32

3.32

Pea meal

2.57

2.58

Peanut meal

7.16

7.29

Barley flour

2.06, 2.06, 1.69

2.14, 2.16, 1.71

Blood meal

14.41

14.48

Ammonium sulfate

20.51, 21.21

20.51, 21.21

asparagine, aspartic acid, glutamic acid, glutamine, leucine, and tyrosine [3]. His results were very close to the theoretical values, and “confirmed the favorable judgment of Kjeldahl’s method made by others” (Table 3.4). The Kjeldahl method has been subjected to numerous modifications in order to be applied to a wide range of different nitrogen compounds. Complete recovery of nitrogen cannot be obtained from certain organic compounds such as nitrogen present in azo, diazo, nitro, nitroso, oxime, hydrazine forms and heterocyclic rings because it is not converted quantitatively to ammonia. Kjeldahl found lower than expected results with a couple of alkaloids: “Only with a few alkaloids the full yield of ammonia is not achieved; with morphine there is thus an error of 0.15%, with quinine the deviation is 0.30%, even after a very energetic effect of sulfuric acid and phosphoric anhydride…” [4]. He observed that van der Burg (1865) obtained very variable results for nitrogen in quinine with the Varrentrapp-Will method, depending on the experiment conditions [5] (Table 3.5). Nevertheless Meusel, using the same method, although with different conditions, found a value of 8.46%, which is closer to the theoretical 8.64% [6]. Kjeldahl affirmed that the most frequent reason for errors in the Varrentrapp-Will’s method is an incomplete ammonia formation. Table 3.4 List of compounds analyzed by Bosshard

Substance

Per cent nitrogen Found

Calculated

Theoretical

Asparagin

18.65, 18.53

18.67

21.20

Aspartic acid

10.38, 10.53

10.53

10.52

Leucine

10.61, 10.77

10.69

10.68

Glutamic acid

9.37, 9.63

9.52

9.52

Tyrosine

7.60, 7.70

7.73

7.73

Aminovaleric acid

11.65, 11.89

11.96

11.96

Allantoin

35.52, 35.33

35.44

35.43

46

3 Nitrogen Compounds

Table 3.5 van der Burg results for N in quinine by the Varrentrapp-Will method, with different experimental conditions Test

Sample weight

Conditions

Per cent N

(a)

0.527

Burned in the usual way with soda lime

0.59

(b)

0.435

Burned in the usual way with soda lime

2.35

(c)

0.432

Heated in a porcelain tube

0.93

(d)

0.371

Tube surrounded with copper sheet and heated in a modified furnace

5.04

(c)

0.409

Heated in the same way for 1.5 h (was strongly incandescent)

3.48

In 1885 Stebbins analyzed nitrogen in some aromatic compounds, obtaining low results, so he decided to add sucrose as a reducing agent. “This great discrepancy led to the conclusion that the small yield of nitrogen was simply due to the fact that a part of the nitrogen had been driven off by the sulfuric acid as NO. It was therefore suggested to me to mix with the sulfuric acid a substance, which, upon heating, would evolve a large volume of sulfuric acid, and thus act as a reducing agent upon the NO. The substance best suited for this purpose is pure cane sugar” [7]. See Table 3.7. Asboth recommended adding two parts of sucrose to one part of sample, in organic substances which contain nitrogen as oxide or in the cyano group, before adding the sulfuric acid for digestion [8]. In the case of nitrates, the sugar is replaced by benzoic acid (Table 3.6). From his results Asboth concludes: 1. The nitrobenzene, azobenzene and trinitrophenol mixed with sugar produce good results, with low errors. 2. The same happens with the cyanide compounds, namely the potassium cyanide, the potassium ferrocyanide and ferricyanide, and sodium nitroprusside. 3. A better result is obtained with morphine when using Wilfarth’s modification. 4. The addition of permanganate is superfluous. 5. The potassium nitrate results are very variable.

Table 3.6 List of compounds analyzed by Asboth

Ammonium rhodanilate

Potassium cyanide

Azobenzene

Potassium ferricyanide

Cyanuric acid

Potassium ferrocyanide

Morphine

Prussic acid (Hydrogen cyanide)

Nitrobenzene

Potassium nitrate

Picric acid (2,4,2-trinitrophenol)

Sodium nitroprusside

3.1 The Kjeldahl Method Applied to Different Nitrogen Compounds

47

Table 3.7 List of compounds analyzed by Stebbins Substance

Per cent nitrogen Found

Theoretical Adding sucrose

p-Toluidine

10.69, 11.44

Diphenylamine

7.78

Azobenzene

14.00

1,2-Dinitrobenzene

7.84

13.07 8.28 15.37 16.12

16.66

2-Nitrophenol

9.74

10.07

3-Nitroaniline

20.04

20.28

Jodlbauer used phenol, which is readily nitrated, to analyze nitrogen in potassium nitrate [9]. He obtained an average of 13.79% nitrogen in twenty two determinations, very close to the theoretical value of 13.85%. In 1887 Dafert performed an extensive study in order to investigate the applicability of Kjeldahl’s method to a wide variety of nitrogen substances [10]. He divided them into two groups (Table 3.8). (a) Compounds which need no previous treatment, such as amides, amines, ammonium compounds, pyridine and quinoline derivatives, alkaloids, proteins, and related substances. (b) Compounds requiring previous treatment, such as nitro, nitroso, azo, diazo, aminoazo, hydrazines, and others. Arnold [11, 12] repeated Asboth’s modifications in a wider range of samples and concluded that “Nitro compounds are very easily and completely converted into ammonia; azo compounds give only partially good results, and diazo compounds give no ammonia at all, the nitrogen escapes as such. Sodium nitroprusside gives off some of the nitrogen as nitrogen oxide gas, as does all inorganic nitrites.” (see Table 3.9). Table 3.8 List of compounds analyzed by Dafert Acridine picrate

p-Nitroso-N,N-dimethylaniline

Aniline

p-Nitrosophenol

Carbazole

p-Nitrotoluene

Cyanuric acid

Potassium ferrocyanide

Diazoaminobenzene

Propionitrile

Nitric acid

p-Toluidine

Nitroaniline

Pyridine

Nitrobenzene

Trimethylphenylammonium chloride

Phenylhydrazine

Xylidine

Phenylhydrazine hydrochloride

48

3 Nitrogen Compounds

Table 3.9 List of compounds analyzed by Arnold

Ammonium nitrate

Orange G

Crocein scarlet (azo compound)

Potassium ferricyanide

Cyanuric acid

Potassium nitrate

Diazo salicylic acid

Prussic acid (Hydrogen cyanide)

Nitromethyltoluidine

Sodium nitroprusside

Orange II

Strychnine nitrate

Orange IV

Scovell used salicylic acid instead of phenol. He obtained an average of 13.80% nitrogen in eight determinations on potassium nitrate [13]. In 1894 Devarda proposed an alloy made of 45 parts aluminium, 50 parts copper, and 5 parts zinc (Devarda’s alloy), for reducing nitrates to ammonia [14]. This blend has been commonly used since then [18, 31]. The reduction process is shown by the equation −  − 3NO− 3 + 8Al + 5OH + 18H2 O → 3NH3 + 8 Al(OH)4 In 1895 Dyer applied the Kjeldahl method to a number of representative organic compounds and concluded that it has many advantages over the soda-lime (Varrentrapp-Will) process [15]. Dakin and Dudley (1914) determined nitrogen by the Kjeldahl method in different derivatives of pyrrole, pyridine, piperidine, quinoline, and pyrazole. They found good results for the pyrrole derivatives. The method was unsatisfactory for the nitrogen analysis of pyridine, quinoline and pyrazole derivatives. Accurate results for piperidine could only be obtained after very prolonged heating [16]. Cope (1916) found that the modification of the Kjeldahl method with salicylic acid yields good results in picric acid and the tri-nitrotoluenes [17]. Davisson and Parsons (1919) improved a method for nitrogen determination, including nitrates, in soil biology studies, including reduction of nitric nitrogen in an alkaline solution with Devarda’s alloy [18]. Phelps (1920) investigated the Kjeldahl method in different refractory compounds, using mercuric oxide as a catalyst. He found that a complete recovery of nitrogen was achieved with the following compounds: glucosamine hydrochloride, isatin, atropine, cocaine, nicotine, nicotinic acid, eucaine hydrochloride, hydroxyquinoline, cinchonidine, strychnine, brucine, papaverine, narcotine (noscapine), morphine, hydrastinine, caffeine, and some imidazole derivatives [19]. Nitrogen was not completely recovered from azo compounds, hydrazine sulphate nor semicarbazide hydrochloride. Oesper and Ballard (1925) successfully applied the Kjeldahl method to determine the nitrogen content in twenty-eight salts of hydroxylamine with organic acids by the Kjeldahl method [20].

3.1 The Kjeldahl Method Applied to Different Nitrogen Compounds

49

Kaye and Weiner (1945) analyzed several nitrogen heterocyclic compounds and nitro compounds, using mercuric oxide as catalyst. Excellent results were obtained in all but one case [21]. Shirley and Becker (1945) tested the effectiveness of various digestion catalysts on caffeine, quinoline, and nicotine. They found that mercury alone or mercury plus selenium oxychloride are satisfactory catalysts for the Kjeldahl determination of compounds containing a refractory ring type nitrogen [22]. Ogg et al. (1948) used a catalytic mixture of mercuric oxide and selenium to determine nitrogen in a series of compounds containing heterocyclic ring nitrogen. His method proved successful, requiring a digestion time of four hours. With acetanilide (compound not containing ring nitrogen) the digestion time required was one hour [23]. Vanetten and Wiele (1951) obtained very good results when nitrile-type nitrogen was determined by the Kjeldahl procedure without preliminary reducing treatment [24]. Fish (1952) determined nitrogen in azines, hydrazones, oximes, and semicarbazones, applying a special reduction treatment previous to the Kjeldahl digestion. The purpose of the reductive pretreatment was to rupture the N–N and N–O linkages to convert the nitrogen to either ammonia or amino compounds. He obtained good results [25]. Bradstreet (1954) used a mixture of equal parts of 1-naphthol and pyrogallol to convert nitro groups into a more easily reducible form [26]. Steyermark et al. (1958) applied a zinc-iron reduction method before the Kjeldahl digestion. They found excellent values with a variety of compounds, including those containing N–N, N=N, NO, and NO2 groups, where the nitrogen to nitrogen linkage is not part of the ring [27]. Ashraf et al. (1961) described a method for the determination of nitrogen in nitro, nitroso and azo compounds by a Kjeldahl method without distillation. The compound is reduced with glucose or zinc, and the resultant ammonium sulfate is titrated by hypochlorite-arsenite [28]. Raney nickel catalyst (developed in 1926 by Murray Raney for the hydrogenation of vegetable oils) has also been used as reducing agent in the Kjeldahl determination of nitrogen in nitrates [29, 30]. Liao (1981) described a modified semimicro-Kjeldahl procedure for total nitrogen determination to include nitrates and nitrites. The method involves use of Devarda’s alloy as a reducing agent [31]. Yoshikuni (1992) applied an acidic lithium sulfate reduction method before the Kjeldahl digestion [32]. He found good results for pyrazine, pyrimidine, purine, quinoxaline, quinazoline and phtalazine. Shirai and Kawashima (1993) formulated a comprehensive Kjeldahl digestion method based on the oxidation numbers of nitrogen [33]. Table 3.10 below shows the basic digestion procedure to be followed depending on the functional group to be analyzed.

50

3 Nitrogen Compounds

Table 3.10 Digestion process for some nitrogen compounds Type of bond H–N

C–N

Straight digestion

O–N

N–N

Pre-treatment required

Ammonia

Amides

Nitrates

Azo compounds

Ammonium compounds

Amines

Nitrites

Azoxy compounds

Amino acids

Diazo compounds

Anilines

Hydrazides

Azides

Hydrazines

Aziridines

Nitroamines

Imines

Nitrosamines

Isonitriles

Nitrosohydroxylamines

Nitriles

Pyrazoles and indazoles

Proteins

Pyridazines and cinnolines

Pyridines

Triazines and derivatives

Pyrroles

Triazoles and derivatives

Quinoline

References 1. Petri, and Lehmann, Th. (1884) Die Bestimmung des Gesamtstickstoffs im Harn (The determination of total nitrogen in urine), Zeitschrift für Physiologische Chemie, 8:200-213. 2. Heffter, Hollrung and Morgen (1884) Ein Beitrag zu der Methode der Stickstoffbestimmung nach Kjeldal (A contribution to the Kjeldahl method for nitrogen determination), Chemiker Zeitung, 8(25):432-435. 3. Bosshard, E. (1885) Zur Stickstoffbestimmung nach Kjeldahl (On nitrogen determination according to Kjeldahl), Zeitschrift für Analytische Chemie, 24(1):199-201. 4. Kjeldahl, J. (1883) En ny Methode til Kvælstofbestemmelse i organiske Stoffer (A new method for nitrogen determination in organic substances), Meddelelser fra Carlsberg laboratoriet (Reports from the Carlsberg Laboratory), 2:1–25. Excerpts from page 23 reproduced with permission of Carlsberg Foundation/Carlsberg Research Laboratory. Copyright © 1883 by Carlsberg Foundation/Carlsberg Research Laboratory. 5. van der Burg, E. A. (1865) Chemische Mittheilungen in Betreff der China-Alkaloide und der Stickstoffbestimmung mittelst Natronkalks (Chemical communications regarding the cinchona alkaloids and the determination of nitrogen by means of soda lime), Zeitschrift für Analytische Chemie, 4(1):273-330. 6. Meusel, E. (1866) Ueber die für Chinabasen in Frage gestellte Anwendbarkeit der Stickstoffbestimmung nach Varrentrapp und Will (On the question about the applicability of the Varrentrapp and Will nitrogen determination to cinchona alkaloids), Zeitschrift für Analytische Chemie, 5(1)197-200. 7. Stebbins, James H. (1885) On the estimation of nitrogen in compounds of the auromatic series by the Kjeldahl method, Journal of the American Chemical Society, 7(4):108-112. 8. Asboth, A. von (1886) Über allgemeinere Anwendung der Kjeldahl’schen Methode der Stickstoffbestimmung (On a more general application of the Kjeldahl’s method for nitrogen determination), Chemisches Zentral-Blatt, 17(9):161-165.

References

51

9. Jodlbauer, M. (1886) Die Bestimmung des Stickstoffes in Nitraten nach der Kjeldahl’schen Methode (Nitrogen determination of nitrates by the Kjeldahl method), Chemisches ZentralBlatt, 17(24):433-434. 10. Dafert, F. W. (1887) Beiträge zur Kenntnis des Kjeldahlschen Stickstoff-BestimmungsVerfahrens (Contributions to the knowledge of the Kjeldahl nitrogen determination method), Die Landwirthschaftlichen Versuchs-Stationen, 34:311-353. 11. Arnold, C. (1886) Die allgemeinere Anwendbarkeit der Kjeldahl’schen Stickstoff bestimmungsmethode (The more general applicability of Kjeldahl’s nitrogen determination method), Archiv der Pharmazie, 224(18):785-794. 12. Arnold, C. (1887) Ueber die allgemeinere Anwendbarkeit der Kjeldahl’schen Stickstoffbestimmungsmethode (On the more general applicability of the Kjeldahl nitrogen determination method), Zeitschrift für Analytische Chemie, 26(1):525-533. 13. Scovell, M. A. (1887) The Kjeldahl method applicable to determination of nitrogen in nitrates, U. S. Department of Agriculture, Division of Chemistry, Bulletin No. 16, pp. 51–54. 14. Devarda, A. (1894) Eine neue Methode zur Bestimmung des Stickstoffs im Chilisalpeter (A new method for determining nitrogen in sodium nitrate –Chile saltpetre–), Zeitschrift für Analytische Chemie, 33(1):113-114. 15. Dyer, B. (1895) Kjeldahl’s method for the determination of nitrogen, Journal of the Chemical Society, Transactions, 67:812-817. 16. Dakin, H. D. and H.W. Dudley, H. W. (1914) Some limitations of the Kjeldahl method, Journal of Biological Chemistry, 17(2):275–280. 17. Cope, W. C. (1916) Kjeldahl modification for determination of nitrogen in nitro substitution compounds, Industrial and Engineering Chemistry, 8(7):592-593. 18. Davisson, B. S. and Parsons, J. T. (1919) The determination of total nitrogen including nitric nitrogen, Industrial and Engineering Chemistry, 11(4):306-311. 19. Phelps, I. K. and Daudt, H. W. (1920) Investigations of the Kjeldahl method for determining nitrogen, Journal of Association of Official Agricultural Chemists, 3(3):306-315. 20. Oesper, R. E. and Ballard, M. P. (1925) Hydroxylamine salts of organic acids, J Am Chem Soc, 47 (9):2424-2427 21. Kaye, I. A. and Weiner, N. (1945) Semimicro-Kjeldahl nitrogen determination, Industrial & Engineering Chemistry Analytical Edition, 17 (6):397-398. 22. Shirley, R. and Becker, W.W. (1945) Determination of nitrogen in pyridine ring-type compounds by Kjeldahl method, Industrial & Engineering Chemistry Analytical Edition, 17(7): 437-438. 23. Ogg, C. L. et al (1948) Micro and semimicro determination of nitrogen in heterocyclic nitrogen ring compounds by a Kjeldahl method, Journal of Association of Official Agricultural Chemists, 31(3): 663–669. 24. Vanetten, C. H. and Wiele, M. B. (1951) Determination of nitrile-type nitrogen with ordinary Kjeldahl digestion, Analytical Chemistry, 23(9):1338-1339. 25. Fish, V. B. (1952) Hydrazones, semicarbazones, and other nitrogenous substances requiring a reductive pretreatment, Analytical Chemistry, 24(4):760-762. 26. Bradstreet, R. B. (1954) Determination of nitro nitrogen by Kjeldahl method, Analytical Chemistry, 26(1):235-236. 27. Steyermark, A. L. et al (1958) Micro-Kjeldahl method for nitrogen in certain organic compounds containing nitrogen-nitrogen and nitrogen-oxygen linkages, Analytical Chemistry, 30(9):1561-1563. 28. Ashraf, M. et al (1961) Elimination of distillation in the Kjeldahl method for the micro- and semimicro-determination of nitrogen in nitro, nitroso and azo compounds, Analytica Chimica Acta, 25:448-452. 29. Brabson, J. A. and Burch, W. G. Jr. (1964) Reduction of nitrates in acid medium with Raney catalyst powders, Journal of the Association of Official Analytical Chemists, 47(6):1035-1040. 30. Brabson, J. A. and Woodis Jr, T. C. (1969) A new approach to the Raney catalyst powder method for total nitrogen in fertilizers. Journal of the Association of Official Analytical Chemists, 52(1):23-30.

52

3 Nitrogen Compounds

31. Liao, C. F. H. (1981) Devarda’s alloy method for total nitrogen determination, Soil Science Society of America Journal, 45(5):852-855. 32. Yoshikuni, N. (1992) Rapid decomposition of heterocyclic ring compounds with molten acidic lithium sulphate flux containing catalysts and Kjeldahl determination of nitrogen, Talanta, 39(7):805-808. 33. Shirai, M. and Kawashima, T. (1993) Kjeldahl digestion method formulated by a criterion related to the oxidation number of nitrogen, Bulletin of the Chemical Society of Japan, 66(9):2541-2546.

Chapter 4

The Kjeldahl Method

The basis of the Kjeldahl method is the conversion of organic nitrogen into ammonium sulfate, and subsequent separation and determination of the ammonium ion. Although initially developed by Johan Kjeldahl to determine nitrogen content in proteins, many further modifications were made to the method by different researchers with the purpose of applying it to other types of nitrogen compounds as well. Three basic steps are required to perform the Kjeldahl analysis: • Conversion of nitrogen in the sample to ammonium sulfate. • Separation of the ammonium ion. • Quantitative determination of the ammonium ion and subsequent stoichiometric conversion to nitrogen.

4.1 Digestion The conversion of organic nitrogen to ammonium sulfate is achieved by digesting the sample with concentrated sulfuric acid. It is basically here where Kjeldahl succeeded over the Varrentrapp–Will method: while the recovery of nitrogen in an alkaline media was incomplete for many types of samples, the acidic media showed a much higher yield, closer to the actual content in the original sample, and the use of concentrated sulfuric acid permitted a complete nitrogen recovery in numerous products. Under strong digestion conditions, sulfuric acid breaks down all organic material into carbon dioxide, water, and ammonium bisulfate. The digestion process is illustrated in the following equation, with glycine as an example: NH2 -CH2 -COOH + 4H2 SO4 → 2CO2 + 3SO2 + 4H2 O + (NH4 )HSO4

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 J. Aguirre, The Kjeldahl Method: 140 Years, https://doi.org/10.1007/978-3-031-31458-2_4

53

54

4 The Kjeldahl Method

The digestion process has been thoroughly studied, although the reaction mechanism has not been sufficiently explored. The first studies known on the kinetics of digestion were performed by Bredig [1]. Milbauer investigated the kinetics of the digestion with selenium as a catalyst [2]. Further studies of the reaction mechanism have been made by Schwab and Schwab-Agallidis [3, 4], Schwab and Caramanos [5], Schwab and Neuwirth [6], Morita [7], and Suard et al. [8]. Carbon and hydrogen atoms in the sample are oxidized to carbon dioxide and water, and nitrogen atoms are converted into ammonium sulfate. A considerable amount of work has been done since the inception of the method to find the optimum digestion conditions. The fundamental issues studied are acid requirements, digestion temperature, catalysts, salt addition, and hydrogen peroxide addition.

Fig. 4.1 Johan Kjeldahl in the Carlsberg Research Laboratory (Reproduced with permission of Carlsberg Foundation/Carlsberg Research Laboratory, from Holter, H., and Moller, K. M. (editors) 1976, The Carlsberg Laboratory 1876–1976, Rhodos International Science and Art Publisher, Denmark, pp. 50–62. Copyright © 1976 by Carlsberg Foundation/Carlsberg Research Laboratory)

4.1 Digestion

55

4.1.1 Acid Requirements and Temperature In 1912, Self found that digestion temperature determines the efficiency of the method. In one of the initial studies on acid requirements Self indicated: “So far as it is permissible to generalize from the results of so few experiments, these appear to justify the conclusion that when 25 ml of sulfuric acid and 12 gm of potassium sulphate are taken, at least 15 gm of acid should remain at the end of the experiment” [9]. This was written more than a hundred years ago. Today, with the large variety of automated digestion systems available, each company has established the optimum amount of acid to be used in their procedure. Extensive studies have been done on acid requirements and temperature by Kirk [10], Bradstreet [11], and many more.

1885

H. Wilfarth Eine Modifikation der Kjeldahl’schen Stickstoffbestimmungs Methode. Chemisches Zentral-Blatt

4.1.2 Catalysts and Salt Addition The use of catalysts was first investigated by Wilfarth in 1885. He examined several metal oxides (iron, mercury, manganese, bismuth, tin, lead, and copper), and found mercuric oxide to be the most significant [12]. In 1886, Arnold considered a mixture of copper sulfate and mercury to be more efficient than either metal alone: “In the course of the investigation I found that the presence of two metals accelerates the oxidation…The most suitable metals being mercury and copper” [13]. Arnold and Wedemeyer (1892) published an article describing the use of a mixture of copper sulfate, mercuric oxide, and potassium sulfate to digest the sample [14]. In 1931, Lauro used selenium and selenium oxychloride as single catalysts, comparing the results with those obtained with mercuric oxide and copper sulfate used separately. He obtained very good results [15]. Wieninger suggested a mixture of copper sulfate and selenium to be used as a catalyst in the Kjeldahl method, with very reliable outcomes [16, 17]. Sreenivasan and Sadasivan (1939) studied the mechanism of the selenium catalytic reactions, showing that selenium acts as an efficient carrier of oxygen when heated

56

4 The Kjeldahl Method

with sulfuric acid [18]. Patel and Sreenivasan (1948) found that selenium causes loss of nitrogen during prolonged heating, and recommended mixing it with mercuric oxide to obtain a better recovery [19]. Osborn and Krasnitz made different systematic investigations of mercuric oxide, and comparisons of the catalytic effects of mercuric oxide, selenium, and copper sulfate [20–22]. Osborn and Wilkie made a comparison study of the catalytic effects of thirty-nine metals [23]. They found that a combination of selenium with mercuric oxide or with copper sulfate has a great advantage over any of them used alone. From the results of the shorter digestion times, the catalysts were arranged in decreasing order of their efficiency (best and safest), as follows: mercury, tellurium, titanium, iron, and copper.

1886

Carl Arnold Die allgemeinere Anwendbarkeit der Kjeldahl’schen Stickstoff bestimmungsmethode. Archiv der Pharmazie

Bradstreet (1938) used a mixture of equal parts of ferrous sulfate and selenium, a combination never used before [24]. He obtained results comparable to those with a mixture of copper sulfate and selenium. Bradstreet (1940) studied the effect of selenium, and concluded that the quantity of selenium should not exceed 0.25 g, as an increase of selenium appears to cause a loss of nitrogen [25]. In 1949, Bradstreet investigated tellurium as a catalyst, given the fact that it is analog to selenium. He concluded that tellurium is not suitable as a catalyst for the Kjeldahl digestion [26]. In 1961, Baker investigated the performance of twenty-one different catalysts (fifteen single catalysts and six mixtures). His conclusion was: “The results show clearly that none of the catalysts investigated is demonstrably superior to mercury, used alone…” [27]. See Table 4.1. During the decades of 1960 and 1970, worldwide awareness of mercury toxicity was on the rise, as a result of the Minamata Bay disaster, the first large-scale incident of methylmercury poisoning. Many industrial and academic laboratories introduced programs to reduce the use of mercury. In 1972, Rexroad recommended the use of cupric sulfate instead of mercuric oxide, due to the high toxicity of mercury and its compounds [28]. In 1973, Williams compared four different catalysts for nitrogen determination in cereal grains [29]. See Table 4.2.

4.1 Digestion

57

The results obtained with cupric sulfate–titanium dioxide were equivalent to those obtained with cupric sulfate–mercuric oxide (routinely used), and in consequence the CuSO4 /TiO2 catalytic mixture was approved by the laboratories of the Canadian Grain Commission for protein determination in wheat and other grains. In 1975, Stirrup and Hartley used the CuSO4 /TiO2 catalytic mixture in the protein analysis of different feeding stuff ingredients and found satisfactory results [30]. Klopper (1976) used titanium dioxide for nitrogen determination in barley, malt, and beer with excellent results [31]. Rexroad and Cathey (1976) developed a successful Kjeldahl process using only cupric sulfate as the catalyst (the Missouri copper method) [32]. Table 4.1 List of catalysts examined by Baker

Single catalysts

Mercury

HgO

Selenium

Se

Copper

CuSO4

Titanium

TiO2

Iron

FeCl3

Platinum

H2 PtCl6

Molybdenum

MoO3

Vanadium

V2 O5

Chromium

CrO3

Tellurium

Te

H2 SeO3

H2 SeO4

FeSO4

Fe2 (SO4 )3

VOSO4

Mixed catalysts

Table 4.2 Catalysts examined by Williams

Mercury and selenium

HgO + Se

Mercury and copper

HgO + CuSO4

Selenium and copper

Se + CuSO4

Mercury, selenium, and copper

HgO + Se + CuSO4

Vanadium and selenium

V2 O5 + Se

Mercury and tellurium

HgO + Te

Single catalysts

Copper

CuSO4

Titanium

TiO2

Mixed catalysts

Copper and titanium

CuSO4 + TiO2

Mercury and copper

HgO + CuSO4

58

4 The Kjeldahl Method

In 1984, a collaborative study was carried out by Kane. Fifty-four samples were sent to different laboratories. They consisted of twenty-six blind duplicates plus two standard materials [33]. The samples tested were dehydrated alfalfa meal, soy protein concentrate, pullet grower, meat and bone meal, custom mix cattle feed,

1889

J. W. Gunning Ueber eine Modification der Kjeldahl-Methode. Zeitschrift für Analystische Chemie

swine base mix, dry dog food, hydrolyzed poultry feathers, cottonseed meal, blood meal, swine developer, milk replacer, soybean meal, ammonium dihydrogen phosphate, and lysine hydrochloride. The sample materials were to be analyzed once each by the official method (using HgO as catalyst), and by the alternative method (using CuSO4 as a catalyst). After the statistical analysis of the results, the recommendation was that the copper sulfate catalyst method be adopted. In 1986, Kane did a preliminary study on titanium dioxide and cupric sulfate mixture as a catalyst and found the results worth of a collaborative study [34]. A collaborative study was carried out by Kane in 1987. Thirty-eight samples were sent to different laboratories. They consisted of eighteen blind duplicates plus two standard materials [35]. The samples examined were cattle concentrate, dehydrated alfalfa meal, broiler finisher, soy protein concentrate, soybean meal, blood meal, dry milk powder, feather meal, meat meal, ammonium sulfate, and lysine hydrochloride. The sample materials were to be analyzed once each by the official method (using HgO as catalyst), and by the alternative method (using CuSO4 /TiO2 as a catalytic mixture). After the statistical analysis of the results, the recommendation was that the copper sulfate/titanium dioxide catalyst method be adopted. In 1889, Gunning introduced the use of potassium sulfate to increase the digestion temperature [36, 37]. He mixed one part of K2 SO4 with two parts of H2 SO4 . When heating the mixture of sulfuric acid potassium sulfate with organic matter, the acidity increases and the boiling point rises. Gunning reported results for different samples with this modification. See Table 4.3. Latshaw (1916) concluded that sodium sulfate can be used as well as potassium sulfate [38]. Other authors have investigated sodium sulfate (Dowell and Friedeman [39], Jarrell [40]). In 1912, a modification of the method was designated as the Kjeldahl–Gunning– Arnold method by the U.S. Department of Agriculture. This was comprehensively

4.1 Digestion Table 4.3 Substances analyzed by Gunning with added K2 SO4

59

Percent nitrogen

Substance

Found

Flax-seed food

4.69

Bread

1.22

Milk

0.449

Beer

0.0896

Calculated

Peptone (commercial)

10.2

Uric acid

33.3

33.33

Acetanilidea

10.34

10.37

(NH4 )2 SO4

21.01b

21.21

NH4 Cl

26.17

26.17

4.60

Morphine

10.0

Aniline oxalate

4.62

10.1

a In

the French summary wrongly translated as acetamide thought that the low value was due to water in the sample b Gunnings

discussed by Vickery (1946), who suggested that the proper designation should include Wilfarth: “It seems proper to suggest that the procedure that is currently known as the Kjeldahl-Gunning-Arnold method should in the future be referred as the Kjeldahl-Wilfarth-Gunning method; this is the only designation that places the credit for this brilliant analytical achievement where it really belongs” [41] (Table 4.4). There were then three official methods for nitrogen determination approved by the USDA: • The Kjeldahl method • The Gunning method • The Kjeldahl–Gunning–Arnold method. Self performed a number of experiments to investigate the optimal conditions to avoid ammonia loss during the Kjeldahl digestion [9]. He found that the loss occurs when the solution becomes more concentrated during heating, and calculated the theoretical quantity of H2 SO4 to be used in order to avoid the drawback:

Table 4.4 Reagents in the methods approved by the USDA in 1946

Kjeldahl

Gunning

Kjeldahl–Gunning–Arnold

H2 SO4

H2 SO4

H2 SO4

HgO

K2 SO4

K2 SO4

KMnO4

CuSO4

HgO

CuSO4

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4 The Kjeldahl Method

Grams (approx.) of H2 SO4 required per 1 g of constituent

7.3

Carbohydrates

9.0

Proteins

17.8

Lipids

In 1940, Bradstreet reviewed all the variations of the method, including duration of digestion, loss of ammonia, sample size, use of sodium sulfate, and other variables [42]. In 1950, Kirk reviewed the most important generalizations that could be made on the Kjeldahl digestion, based on the large number of empirical studies available then [10]. Middleton and Stuckey (1951) thoroughly studied the digestion process [43]. McKenzie and Wallace (1954) made experimental examinations of different features of the Kjeldahl method, such as the effect of temperature, the effect of catalyst, the use of hydrogen peroxide as an oxidizing agent, and equipment and conditions for the distillation and titration [44]. Bradstreet (1957) studied the acid requirements and the acid-to-salt ratio of the Kjeldahl digestion [11]. The total amount of acid required varied depending on the sample size, the rate of heating, and other factors. Baker (1961) made a systematic investigation of the effects of varying salt concentrations and catalysts [27]. The boiling points of solutions of varying concentrations of added salt were also determined.

1920

Victor C. Myers Chemical changes in the blood in disease. I. Nonprotein and urea nitrogen. Journal of Laboratory and Clinical Medicine

4.1.3 Hydrogen Peroxide The use of hydrogen peroxide to complete the oxidation in the Kjeldahl digestion process was first reported by Victor C. Myers in 1920. When analyzing nitrogen in blood by the Kjeldahl method, he observed that “The final oxidation may be greatly facilitated by the addition of 1 or 2 drops of hydrogen peroxide” [45]. Kleemann (1921) added H2 O2 to the digestion, given that the action of the oxygen peroxide on concentrated sulfuric acid produces oxysulfuric acid (peroxymonosulfuric acid, H2 SO5 ), which has a strong oxidizing effect [46]:

4.1 Digestion

61

H2 O2 + H2 SO4  H2 SO5 + H2 O Koch and McMeekin (1924) used a 30% hydrogen peroxide solution and concentrated sulfuric acid to analyze samples of urine, blood, milk, and some pure substances, namely acetanilide, tryptophan, histidine, caffeine, strychnine, and uric acid [47]. The results obtained were within acceptable limits. Myers (1931) published a note confirming that he described the use of hydrogen peroxide in the Kjeldahl method as early as 1920 [48]. Miller and Miller (1948) carried out extensive research on the use of hydrogen peroxide in nitrogen tests with amino acids, pyrimidines, purines, yeast nucleic acid, creatine, vitamins, and some other substances, such as purified proteins [49]. As a result of the study, specific procedures were developed to meet different requirements, depending on the substance to be determined and the precision required. Florence and Milner (1979) analyzed a variety of samples, such as barley, hay, grass meal, whole milk, and others, in order to verify the efficiency of the digestion using hydrogen peroxide [50]. Their results were satisfactory. Srikar and Chandru (1983) applied the digestion method with hydrogen peroxide to different samples of fish, egg albumin, and casein [51]. The procedure was rapid, reliable, and accurate. Singh et al. (1984) made comparisons on the digestion of plant materials with different catalysts, namely mercury and selenium, and

1921

Dr. Kleemann Über die Wirkung des Wasserstoffsuperoxydes bei der Aufschließung pflanzlicher und tierischer Stoffe. Zeitschrift für Angewandte Chemie

hydrogen peroxide [52]. They found that the hydrogen peroxide digestion technique can be adapted for total nitrogen determination in plant tissues. Hach et al. (1985) used peroxymonosulfuric acid (resulting from hydrogen peroxide mixed with sulfuric acid) to digest a range of samples including reagent grade organic compounds (amines, amino acids, porphyrins, and heterocyclic compounds), grains, feeds, and cereals [53]. This method has several advantages over the conventional catalyst digestion methods. The technique was improved by the use of a Vigreux fractionating reflux head [54]. A comparison of two conventional methods (Kjeltec and Kjeldahl) with the Hach hydrogen peroxide method was performed by Watkins et al. in 1987 [55]. Results indicated that the Hach procedure was more sensitive than the other two methods. Guebel et al. (1991) analyzed nitrogen in bovine serum albumin and other substances, using a mixture of sodium selenite and hydrogen peroxide as catalyst

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4 The Kjeldahl Method

[56]. They recommended the technique for the assessment of nitrogen content in small samples from complex fermentation media. Feinberg et al. (1993) studied the use of an open vessel microwave digestion system, decomposing the sample with sulfuric acid and further oxidation with hydrogen peroxide. Their conclusion was “…The use of hydrogen peroxide greatly simplifies this method, whereas official methods require the addition of catalyst and salts” [57]. Buondonno et al. (1995) investigated hydrogen peroxide digestion in the analysis of soils [58]. They found a very good consistency between the traditional catalytic digestion and the hydrogen peroxide–sulfuric acid treatment.

4.1.4 Sealed Tube Digestion The sealed tube digestion procedure was conceived by White and Long in 1951 [59]. This method of microanalysis nitrogen determination is carried out in heavy-walled, sealed glass tubes at 470 °C, with concentrated sulfuric acid and mercuric oxide catalyst. Further studies by Grunbaum, Schaffer, and Kirk showed that the catalyst was unnecessary [60]. An investigation of conditions for the sealed tube Kjeldahl digestion demonstrated that ammonia losses can occur due to the oxidation of ammonia to nitrogen gas when the temperature is higher than 500 °C [61].

4.1.5 Microwave Digestion Abu-Samra et al. (1975) adapted a modified commercial microwave oven into a system suitable for wet ashing of some biological samples, in what appears to be the first account of this procedure [62]. Alvarado et al. (1988) applied microwave heating to the Kjeldahl digestion, and found that in most cases the precision and accuracy of the results obtained were comparable to those obtained by the conventional method [63]. Feinberg et al. (1993) combined the use of microwave heating and hydrogen peroxide for the Kjeldahl digestion [57]. They concluded that open-vessel microwave digestion allows an appreciable decrease in sample preparation time compared to other methods. Collins et al. (1996) developed a microwave digestion method using the Prolabo Microdigest 401® . “Atmospheric pressure microwave sample preparation can be as effective a method as closed vessel microwave sample preparation methods, if not better…” [64]. Chemat et al. (1998) studied various parameters (microwave power, concentrations, and time) of microwave digestion in order to define the optimal digestion conditions [65].

4.2 Ammonia Distillation and Determination

63

Lo et al. (2005) developed a closed-vessel microwave digestion process for the determination of total Kjeldahl nitrogen (TKN) in sewage and wastewater [66]. The method has advantages over the conventional digestion method; it significantly reduces the digestion temperature and length.

1913

L. W. Winkler Beitrag zur titrimetrischen Bestimmung des Ammoniaks. Zeitschrift für Angewandte Chemie

4.2 Ammonia Distillation and Determination Once the digestion process is finished, an excess sodium hydroxide is added to convert the ammonium sulfate into ammonia. A quantitative method is then applied to determine the ammonium ion, with subsequent mathematical conversion to nitrogen. Numerous techniques have been employed in the analysis of ammonia after the digestion process, namely acid–base titration, amperometric titration, coulometric titration, chromatography, ion-selective electrode, spectrophotometry, and more. For an extensive review of all alternate methods, see Sáez-Plaza et al. [67] and Zhou et al. [68].

4.2.1 Titration In his original method, Kjeldahl distilled the ammonia into a known volume of standard acid, added potassium iodade and iodate, and titrated the liberated iodine with standard sodium thiosulfate (iodometric method). In 1888, Kjeldahl published an article with recommendations on the appropriate preparation and storage of the thiosulfate solution [69]. The iodometric titration was superseded over the subsequent years: the ammonia was collected in a known amount of standard sulfuric acid, and the excess acid was titrated with a standard sodium hydroxide solution. The boric acid modification introduced by Winkler in 1913 is generally used today [70]. In this adaptation, the ammonia is distilled into a 3% boric acid solution, and then titrated with a standard sulfuric acid solution. It has the benefit that only one standard solution is needed. Scales and Harrison (1920) verified Winkler’s modification by distilling known quantities of ammonia (obtained by the action of sodium hydroxide on ammonium

64

4 The Kjeldahl Method

sulfate) into both standard sulfuric acid and 4% boric acid solutions [71]. They also analyzed different samples of soil, corn fodder, wheat grain, and sweet clover. Their results showed that both variations were accurate. Markley and Hann (1925) did extensive tests on the boric acid modification and obtained good results [72]. Meeker and Wagner (1933) achieved 99.9% recovery of ammonia in different substances, using the boric acid titration variant [73]. A significant investigation of the boric acid modification was made by Yuen and Pollard (1953). They studied several variables, such as the true ammonia-fixing capacity of the boric acid, cooling of the condensate, effect of carbon dioxide, and best indicators [74]. Siemer (1986) used solid crystalline boric acid as an ammonia absorbent by inserting an ammonia trap assembly into the neck of the distillation flask [75]. The boric acid was subsequently dissolved in water and the ammonium ion was measured by either acidimetry, conductometry, or colorimetry. A variety of single and mixed indicators have been used for the titration in the boric acid variant. The most used are shown in Table 4.5. Table 4.5 Different indicators used in the boric acid variant

Indicator

References

Single indicators

Bromophenol blue

1920 Scales and Harrison [71]

1925 Markley and Hann [72]

Congo red

1913 Winkler [70]

1920 Scales and Harrison

Methyl orange

1913 Winkler

1920 Scales and Harrison

Methyl red

1933 Meeker and Wagner [73]

1940 Wagner [77]

Mixed indicators

Sodium alizarine sulfonate + Guinea green

1930 Johnson and Green [78]

Sodium alizarine sulfonate + Indigo carmine

1930 Johnson and Green

Methylene blue + Methyl red

1930 Johnson and Green

1933 Mecker and Wagner

1937 Sobel et al. [79]

1953 Yuen and Pollard [74]

Tetrabromophenol blue + Methyl red

1931 Stover and Sandin [80]

Bromocresol green + Methyl red

1942 Ma and Zuasaga [81]

1944 Sobel et al. [82]

Bromocresol green + New coccine (Ponceau 4R) + p-Nitrophenol

1955 Sher [76]

4.2 Ammonia Distillation and Determination

65

Sher’s mixed indicator (1955) has been largely employed in many laboratories [76]. Detailed studies of the titration by the boric acid modification have been made by Michalowski et al. [83] and Cruz [84]. Chemical reactions in the boric acid variant Alkalinization

(NH4 )HSO4 + 2NaOH → NH3 + Na2 SO4 + 2H2 O

Distillation − NH3 + H3 BO3 → NH+ 4 + H2 BO3

Titration − NH+ 4 + H2 BO3 + H2 SO4 → (NH4 )HSO4 + H3 BO3

Other titration methods have been applied directly to the final digestion solution, without distillation. Based on the principle that ammonia reacts with formaldehyde to form hexamethylenetetramine, Shaw (1924) tested the accuracy of the method, and especially its application to the estimation of nitrogen in ammonium sulfate (the form in which it is present after the Kjeldahl digestion). The results only varied very slightly from the theoretical [85]. The reactions are 6HCHO + 4NH3 → (CH2 )6 N4 + 6H2 O + 6HCHO + 4NH+ 4 → (CH2 )6 N4 + 6H2 O + 4H

The acid formed is titrated with standard alkali. Marcalli and Rieman (1946) proved the formaldehyde method by analyzing nitrogen in eleven organic compounds, obtaining high accuracy [86]. Willard and Cake [87], and Harvey [88] described a method in which nitrogen is estimated without distillation. After the digestion stage, the ammonia in the solution is treated with an excess of sodium hypobromite: 3NaBrO + 2NH3 → N2 + 3NaBr + 3H2 O The remaining hypobromite is determined by titrating the iodine liberated (after the addition of potassium iodide and hydrochloric acid) with standard sodium thiosulfate. They analyzed several organic substances by this method, with very good results. Kolthoff and Stenger (1935) obtained good results using calcium hypochlorite instead of hypobromite as a standard solution [89]. Belcher (1950) used tartrazine as a reversible indicator for this titration [90]. Belcher and Bhatty (1956) also replaced hypobromite with hypochlorite [91]. They analyzed various organic compounds, namely phenacetin, hippuric acid,

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4 The Kjeldahl Method

S-benzylthiouronium chloride, phenyl thiourea, and 8-hydroxyquinoline. They obtained satisfactory results. Hashmi and Umar (1962) developed a simple and accurate method for the Kjeldahl determination of nitrogen without distillation, by using the sodium hypobromite method [92]. They analyzed many nitrogenous compounds. The results indicated that nitrogen could be determined accurately by this technique.

4.2.2 Spectrophotometry Different spectrophotometric methods have been applied to the determination of nitrogen in the Kjeldahl distillate. Nessler reaction The Nessler reaction was first described by Julius Nessler (1827–1905) in his doctoral dissertation “A new reaction to ammonia”, published in 1856. The reagent has a high sensitivity for the detection of ammonia and is used for its quantitative determination in water analysis. Nessler’s reagent is a solution consisting of mercury(II) iodide and potassium iodide in a highly alkaline solution. The chemical name is potassium tetraiodomercurate(II). It will react to form a yellow color in proportion to total ammonium nitrogen. Extremely small quantities of ammonia can be detected. Its reaction with ammonia produces a yellow color with small amounts, an orange with large amounts, and a brown precipitate when very large amounts are found. The reaction is 2K2 HgI4 + NH3 + 3KOH → HgO.Hg(NH2 )I + 7KI + 2H2 O Folin and Farmer (1912) developed a new method for nitrogen analysis in urine using the Nessler reaction for ammonia determination [93]. Folin and Denis (1916) performed an extensive investigation on nitrogen determination in urine by direct nesslerization [94]. Koch and McMeekin (1924) described a modification of the Nessler–Folin reagent [47]. They added 30% hydrogen peroxide to a solution of organic matter in concentrated sulfuric acid. This causes very rapid oxidation with complete retention of nitrogen as ammonia. Chiles (1928) applied the nesslerization method to the analysis of blood, urine, milk, grain feeds, tankage, and cottonseed meal [95]. Nichols and Willitt (1934) studied the interaction of ammonia and the various components of Nessler’s solution [96]. Miller and Miller (1948) examined the Koch and McMeekin method in a wider range of substances [49]. The Nessler method is widely used in clinical and water analysis, where small amounts of ammonia are to be determined.

4.2 Ammonia Distillation and Determination

67

Berthelot reaction The Berthelot reaction (also called the indophenol reaction) is based on the development of a deep blue color when ammonia reacts with phenol and alkaline hypochlorite. Ammonia is converted to monochloramine at pH 9.7–11.5, which reacts with phenol to form indophenol. The reaction was discovered by Marcellin Berthelot in 1859 [97]. The reaction (unbalanced) is

Phenol

Indophenol

The salicylate method is a modification of the phenol method in which sodium salicylate is substituted for phenol, as it is less toxic. Catalysts for this reaction (acetone, manganous ions, and sodium nitroprusside) have been developed to facilitate color development to proceed at room temperature. Weatherburn (1967) studied the reaction in detail and recommended a simple but reliable technique [98]. Harwood and Huyser (1970) studied the effects of changes in phenol, hypochlorite, and catalyst concentrations on the indophenol reaction, using acetone and sodium nitroprusside [99]. Sodium nitroprusside was generally found to be far superior to acetone. Gehrke et al. (1971) developed an automated spectrophotometry method [100]. A continuous digestor is coupled with an automated colorimetric system, based on the ammonia–phenate–hypochlorite reaction. Smith (1980) studied the Berthelot method applied to plant tissues [101]. A thorough review of the Berthelot method was made by Searle in 1984 [102]. The use of the Berthelot reaction in the Kjeldahl method has been considerably studied. Some examples are as follows. Reardon et al. (1966) found that a mixture of sodium salicylate and sodium dichloroisocyanurate was suitable for determining ammonia nitrogen derived from urea in clinical analyses [103]. Solórzano (1969) analyzed samples of different concentrations of ammonia in freshwater and filtered seawater by the phenol–hypochlorite method, with good results [104]. Scheiner (1976) proposed a modification to the phenol–hypochlorite method by introducing a buffering system that allows reliable and reproducible determination of ammonia and Kjeldahl nitrogen in fresh and wastewater [105].

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4 The Kjeldahl Method

Verdouw et al. (1978) established the optimal reaction conditions for the sodium salicylate variation of the Berthelot method [106]. They found this modification to have very good reproducibility when applied to seawater analysis. Krom (1980) suggested a modification in which sodium salicylate, sodium dichloroisocyanurate, and complex cyanides are the principal reagents [107]. He presented experimental results that were consistent with the reaction scheme proposed. Bower and Holm-Hansen (1980) determined total ammonia nitrogen in artificial seawater by the salicylate–hypochlorite method [108]. The recoveries of ammonia showed high accuracy and low standard deviations. Fukumoto and Chang (1982) made a comparison between the conventional microKjeldahl method involving distillation and titration, and the salicylate–hypochlorite method, using urine and fecal samples [109]. Conformity between the two methods was excellent; a correlation coefficient of 0.9992 and a coefficient of variation of the estimate of 2.1% were obtained. Wang and Øien (1986) studied the Berthelot method applied to soil extracts [110]. Baethgen and Alley (1989) analyzed soil and plant tissue samples by the salicylate–hypochlorite method, obtaining recoveries of 99% or higher, compared to the distillation–titration technique [111]. Giner-Sánz et al. (2020) used the salicylate method for ammonia quantification in nitrogen electroreduction experiments [112]. Zhou and Boyd (2016) compared Nessler, phenate, salicylate, and ion-selective electrode procedures for the determination of total ammonia nitrogen in aquaculture [113]. The results of this study confirmed that the standard salicylate method is appropriate for application in aquaculture water. According to most researchers, the phenol–hypochlorite reaction for the detection of ammonia is more sensitive than Nessler’s solution. Ninhydrin reaction Ninhydrin is a chemical used to detect ammonia or primary and secondary amines. When reacting with these free amines, a deep blue or purple color known as Ruhemann’s purple is produced. The reaction was discovered by Siegfried Ruhemann in 1910 [114]. Ninhydrin is also known as indanetrione hydrate. The reaction below is just an overview. For the actual reactions and mechanism, see Bottom et al. [115].

Ninhydrin

Glycine

Ruhemann’s purple

4.2 Ammonia Distillation and Determination

69

An extensive review of the chemistry and applications of ninhydrin reactions with a variety of substrates was made by Friedman [116]. Starcher (2001) developed a rapid and sensitive microtiter plate ninhydrin method to quantitate total protein based on the total amino acid content of protein hydrolysates [117]. Abernathy et al. (2009) presented a microwell ninhydrin assay suitable for determining protein as well as total usable nitrogen in beer or grape juice/wine [118]. The assay is inexpensive, rapid, accurate, and applicable to large numbers of samples. Jacobs applied this method to the determination of nitrogen in serum, protein hydrolysates, and heterocyclic compounds (nicotinic acid, methionine, 8-hydroxyquinoline, and streptomycin) on the micro scale [119, 120]. Hantzsch reaction The reaction of ammonia with a β-keto ester and an aldehyde is known to form 1,4dihydropyridine derivative. The Hantzsch reaction allows the preparation of dihydropyridine derivatives by condensation of an aldehyde with two equivalents of a β-ketoester in the presence of ammonia. This reaction was reported by Arthur Rudolf Hantzsch in 1881 [121]. The reaction below is just an overview. For the actual reactions and mechanism, see Vanden Eynde and Mayence [122].

Devani et al. (1989) developed a spectrophotometric method for the determination of nitrogen in the Kjeldahl digest, based on the above reaction [123]. The method is simple, rapid, and precise. They analyzed a variety of organic compounds including amines, amino acids, anilide, sulfonamides, sulfonylurea, piperidine, pyrimidine, and so forth. The results were comparable with those obtained by the AOAC method 47.021.

70

4 The Kjeldahl Method

Summary of the main colorimetric methods Nessler reaction

HgO·Hg(NH2 )I

Potassium tetraiodomercuriate (II)

Mercury(II) amido-iodide (Iodide of Millon’s base)

Main reagent

Final product

Ammonia reacts with [HgI4 ]2− to form a yellow coloration, or a brown precipitate, according to the amount of ammonium ions present.

Berthelot reaction

Phenol + sodium hypochlorite

Indophenol

Main reagent

Final product

Ammonia reacts with hypochlorite to form monochloramine, which in turn reacts with phenol to form an indophenol.

Ninhydrin reaction

Ninhydrin

Ruhemann’s purple

Main reagent

Final product

Ammonia reacts with ninhydrin. The development of a deep blue color indicates the presence of ammonia, primary/secondary amines, or amino acids.

4.2 Ammonia Distillation and Determination

71

Hantzsch reaction

Acetylacetone + Formaldehyde

3,5-Diacetyl-1,4-dihydrolutidine

Main reagent

Final product

Ammonia reacts with acetylacetone and formaldehyde. The initial reaction product is a dihydropyridine, which can be oxidized to a pyridine in a subsequent step.

4.2.3 Chromatography The use of both gas and ion chromatography to determine total nitrogen as ammonium ion after sample digestion has been a common technique. The methods have shown reasonable agreement with the results obtained by the conventional titration practice. Moldoveanu (1988) proposed a gas chromatographic determination: the ammonia is collected in acetic acid after adding a strong base to the digest [124]. The ammonium acetate solution is then injected into a chromatographic column and identified with a thermal conductivity detector. The procedure is based on the fact that, by injecting a solution of ammonium acetate into the gas chromatograph, it will behave like a mixture of ammonia and acetic acid. Jackson et al. (1991) applied ion chromatography to determine total nitrogen as an ammonium ion after sample digestion [125]. The method showed reasonable agreement with the results obtained by the conventional Kjeldahl procedure. Wang et al. (2016) reported a simple and rapid method for the determination of nitrogen in biological samples [126]. The Kjeldahl digestion is followed by ion chromatography analysis of the digest after dilution. The method provides accurate and precise results in the range of 0.01 to 1.0 mg/mL of ammonium.

4.2.4 Ammonium Ion-Selective Electrode An ion-selective electrode (ISE)—often called a specific ion electrode—is used to sense the strength of a particular ion in a solution. The concentration is measured as an electric potential difference. During the early 1970s, there was an active interest in the application of ionselective electrodes to quantitative analytical techniques. Various authors applied the ammonium ion-selective electrode to the Kjeldahl method.

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4 The Kjeldahl Method

Bremner and Tabatabai (1972) used an Orion® ammonia electrode for nitrogen analysis of soils [127]. Rogers and Pool (1973) described a method for the determination of ammonia and urea in raw sewage samples using an Orion® ammonia gas-sensing electrode [128]. Buckee (1974) described a procedure using an ammonia probe for estimating the total nitrogen content of barley, malt, wort, and beer [129]. Byrne and Power (1974) used an ammonia electrode to determine ammonium nitrogen in animal slurries [130]. Eastin (1976) used an Orion® ammonia electrode for nitrogen analysis in plants [131]. Byrne and McCormack (1978) determined ammonium nitrogen in silage samples with an ammonia electrode [132]. Roy et al. (1980) developed an automated method incorporating an on-line ionselective electrode ammonia probe in conjunction with sparging for the routine analysis of total Kjeldahl nitrogen in feed samples [133]. Powers et al. (1981) used an ammonia electrode in the analysis of nitrogen in micro-Kjeldahl digests of forest vegetation [134]. In all reports, the ammonia electrode compares favorably with the standard titration procedure, and it is faster and simpler.

References 1. Bredig, G. and Brown, J. W. (1903) Katalytische Oxydationen organischer Substanzen mit konzentrierter Schwefelsaure. I Beitrage zur chemischen Kinetik der Kjeldahlanalyse und Naphtalinoxydation (Catalytic oxidation of organic substances with concentrated sulfuric acid. I Contributions to the chemical kinetics of Kjeldahl analysis and naphthalene oxidation), Zeitschrift für Physikalische Chemie, 46(1):502–520. 2. Milbauer, Jaroslav (1941) Reaktionen im Schwefelsäuremedium. XXX Noch ein Wort über den Selenkatalysator (Reactions in sulfuric acid medium. XXX One more word about the selenium catalyst, Chemisches Zentralblatt, 18:2172. 3. Schwab, Georg-Maria and Schwab-Agallidis, Elly (1951) Kinetics of the Kjeldahl reaction, Journal of the American Chemical Society, 73:803-809. 4. Schwab, Georg-Maria and Schwab-Agallidis, Elly (1953) Die Kieldahlisierung des Harnstoffs Kinetik der Kieldahl-Reaktion, II. Mitteilung (The Kjeldahlization of urea kinetics of the Kieldahl reaction, II Notice), Angewandte Chemie, 65(16):418–421. 5. Schwab, GM. and Caramanos, S. (1955) Zur Kenntnis der Kjeldahl-Reaktion III (About the Kjeldahl reaction III), Monatshefte für Chemie, 86:341–347. 6. Schwab, GM. and Neuwirth, O. (1957) Zur Kenntnis der Kjeldahl-Reaktion IV (About the Kjeldahl reaction IV), Monatshefte für Chemie 88:288–291. 7. Morita, Yazaemon (1968) A theoretical consideration on chemical reactions in the Kjeldahl digestion, Bulletin of the Chemical Society of Japan, 41:2029-2032. 8. Suard, Carolle. L. et al. (1997) Mechanisms involved in Kjeldahl microwave digestion of amino acids, Journal of Agricultural and Food Chemistry, 45:1202-1208. 9. Self, P. A. W. (1912) An unrecognized source of error in the Kjeldahl-Gunning method for the determination of nitrogen, The Pharmaceutical Journal and Pharmacist, 34:384-385. 10. Kirk, P. L. (1950) Kjeldahl method for total nitrogen, Analytical Chemistry, 22:354-358.

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11. Bradstreet, R.B. (1957) Acid requirements of the Kjeldahl digestion, Analytical Chemistry, 29(6):944-947. 12. Wilfarth, H. (1885) Eine Modifikation der Kjeldahl’schen Stickstoffbestimmungs Methode (A modification of the Kjeldahl method for nitrogen determination), Chemisches Zentral-Blatt, 16:17–19 and 113–115. 13. Arnold, Carl. (1886) Die allgemeinere Anwendbarkeit der Kjeldahl’schen Stickstoff bestimmungsmethode (The more general applicability of Kjeldahl’s nitrogen determination method), Archiv der Pharmazie, 224(18):785-794. 14. Arnold, C., and Wedemeyer, Konrad (1892) Beitrige zur Stichstoffbestimug nach Kjeldahl. (Contribution to Kjeldahl’s nitrogen determination), Zeitschrift für Analytische Chemie, 31:525-533. 15. Lauro, M. F. (1931) Use of selenium as catalyst in determination of nitrogen by Kjeldahl method, Industrial and Engineering Chemistry, Analytical Edition, 3(4):401-402. 16. Wieninger, F. M. (1933) Neue Schnellstickstoffbestimmungs-methode mit Selen als Beschleuniger (A new rapid method of estimating nitrogen with selenium as catalyst), Wochenzeitschrift für Brauerei, 50:124. 17. Wieninger, F. M. (1936) Nachtrag zur Schnellstickstoffbestimmungs-methode mit Selen als Beschleuniger (Addendum to the rapid method of estimating nitrogen with selenium as catalyst). Wochenzeitschrift für Brauerei, 53:251-252 18. Sreenivasan, A. and Sadasivan, V. (1939) Estimation of nitrogen by the Kjeldahl method. Nature of the action of selenium, Industrial and Engineering Chemistry, Analytical Edition, 11(6):314-315. 19. Patel, S. M. and Sreenivasan, Arunachala (1948) Selenium as Catalyst in Kjeldahl Digestions, Analytical Chemistry, 20(1):63-65. 20. Osborn, R. A. and Krasnitz, A. (1933) A study of the Kjeldahl method. I. Mercuric oxide as a catalyst when block tin condensers are used, Journal of the Association of Official Agricultural Chemists, 16(1):107–110. 21. Osborn, R. A. and Krasnitz, A. (1933) A study of the Kjeldahl method. II. Comparison of selenium with mercury and with copper catalysts, Journal of the Association of Official Agricultural Chemists, 16(1):110–113. 22. Osborn, R. A. and Krasnitz, A. (1934) A study of the Kjeldahl method. III. Further comparisons of selenium with mercury and with copper catalysts, Journal of the Association of Official Agricultural Chemists, 17:339-342. 23. Osborn, R. A. and Wilkie, J. B. (1935) A study of the Kjeldahl method. IV. Metallic catalysts and metallic interferences, Journal of the Association of Official Agricultural Chemists, 18(4):604–609. 24. Bradstreet, R. B. (1938) A new catalyst for the determination of nitrogen by the Kjeldahl method, Industrial and Engineering Chemistry, Analytical Edition, 10 (12):696. 25. Bradstreet, R. B. (1940) Effect of selenium on the Kjeldahl digestion, Industrial and Engineering Chemistry, Analytical Edition, 1940, 12:657. 26. Bradstreet, R.B. (1949) Comparison of tellurium and selenium as catalysts for the Kjeldahl digestion. Analytical Chemistry, 21:1012-1013. 27. Baker, P. R. W. (1961) The micro-Kjeldahl determination of nitrogen. An investigation of the effects of added salt and catalysts, Talanta, 8:57-71. 28. Rexroad, Paul R. (1972) Total Nitrogen in Fertilizers, Journal of the Association of Official Analytical Chemists, 55 (4): 707–708. 29. Williams, Philip C. (1973) The use of titanium dioxide as a catalyst for large-scale Kjeldahl determination of the total nitrogen content of cereal grains. Journal of the Science of Food and Agriculture, 24:343-348. 30. Stirrup, J. E., and Hartley, A. W. (1975) The use of titanium dioxide and copper sulfate as a catalyst mixture for the Kjeldahl determination of nitrogen in feeding stuffs, Journal of the Association of Public Analysts, 13:72-75. 31. Klopper, W. J. (1976) Use of titanium dioxide as a catalyst in the Kjeldahl determination of the total nitrogen content of barley, malt and beer, Journal of the Institute of Brewing, 82(6):353-354.

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32. Rexroad, Paul R., and Cathey, Robert D. (1976) Pollution-reduced Kjeldahl method for crude protein, Journal of the Association of Official Analytical Chemists, 59(6): 1213–1217. 33. Kane, Peter F. (1984) Comparison of HgO and CuSO4 as digestion catalysts in manual Kjeldahl determination of crude protein in animal feeds: collaborative study, Journal of the Association of Official Analytical Chemists, 67:869-877. 34. Kane, Peter F. (1986) CuSO4-TiO2 as Kjeldahl digestion catalyst in manual determination of crude protein in animal feeds, Journal of the Association of Official Analytical Chemists, 69(4):664-666. 35. Kane, Peter F. (1987) Comparison of HgO and CuSO4 /TiO2 as catalysts in manual Kjeldahl digestion for determination of crude protein in animal feed: Collaborative study, Journal of the Association of Official Analytical Chemists, 70:907-911. 36. Gunning, J.W. (1889) Ueber eine Modification der Kjeldahl-Methode (On a modification of the Kjeldahl method), Zeitschrift für Analystische Chemie, 28:188. 37. Gunning, J.W. (1889) Amélioration de la méthode Kjeldahl pour le dosage de l’azote, Recueil des Travaux Chimiques des Pays-Bas, 8(8):254-256. 38. Latshaw, W. L. (1916) Sodium sulfate as a substitute for potassium sulfate in the Gunning modifications for determining nitrogen, Industrial and Engineering Chemistry, 8(7):586-587. 39. Dowell, C. T. and Friedeman, W. G. (1918) The use of sodium sulfate in the Kjeldahl-Gunning method, Industrial and Engineering Chemistry, 10(8):599–600. 40. Jarrell, T. D. (1920) Substitution of sodium sulphate for potassium sulphate in the KjeldahlGunning-Arnold method for the determination of ammonia in fertilizers, Journal of Association of Official Agricultural Chemists, 3(3):304-306. 41. Vickery, Hubert Bradford (1946) The position of Arnold in relationship to the Kjeldahl method. Journal of the Association of Official Analytical Chemists, 29(4):358-370. 42. Bradstreet, R. B. (1940) A review of the Kjeldahl determination of organic nitrogen, Chemical Reviews, 27:331-350 43. Middleton, G. and Stuckey, R. E. (1951) The standardization of the digestion process in the Kjeldahl determination of nitrogen, Journal of Pharmacy and Pharmacology, 3(1):829-842. 44. McKenzie, H. A. and Wallace, H. S. (1954) The Kjeldahl determination of nitrogen: A critical study of digestion conditions–temperature, catalyst, and oxidizing agent–, Australian Journal of Chemistry, 7(1):55-70. 45. Myers, Victor C. (1920) Chemical changes in the blood in disease. I. Nonprotein and urea nitrogen, Journal of Laboratory and Clinical Medicine, 5(7):418–428. 46. Kleemann (1921) Über die Wirkung des Wasserstoffsuperoxydes bei der Aufschließung pflanzlicher und tierischer Stoffe (The effect of hydrogen peroxide upon the decomposition of plant and animal substances), Zeitschrift für Angewandte Chemie, 34:625. 47. Koch, F. C. and McMeekin, T. L. (1924) A new direct nesslerization micro-Kjeldahl method and a modification of the Nessler-Folin reagent for ammonia, Journal of the American Chemical Society, 46(9):2066-2069. 48. Myers, Victor C. (1931) The use of hydrogen peroxide in the micro Kjeldahl nitrogen method, Journal of Laboratory and Clinical Medicine, 17(3):272-273. 49. Miller, Gail Lorenz and Miller, Elizabeth Eshelman (1948) Determination of nitrogen in biological materials. Improved Kjeldahl-Nessler method, Analytical Chemistry, 20(5):481488. 50. Florence, Eric, and Milner, Douglas Frank (1979) Routine determination of nitrogen by Kjeldahl digestion without use of catalyst, Analyst, 104:378-381. 51. Srikar, L. N., and Chandru, R. (1983) Technical note: determination of organic nitrogen by Kjeldahl digestion using hydrogen peroxide, Journal of Food Technology, 18:129-133. 52. Singh, Umaid et al. (1984) The use of hydrogen peroxide for the digestion and determination of total nitrogen in chickpea (Cicer arietinum L.) and pigeon pea (Cajanus cajan L.), Journal of the Science of Food and Agriculture, 35:640-646. 53. Hach, Clifford C. et al. (1985) A powerful Kjeldahl nitrogen method using peroxymonosulfuric acid. Journal of Agricultural and Food Chemistry, 33:1117-1123.

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54. Hach, Clifford C. et al. (1987) More powerful peroxide Kjeldahl digestion method. Journal of the Association of Official Analytical Chemists, 70:783-787. 55. Watkins, Kevin L., Veum, Trygve L., and Krause, Gary F. (1987) Total nitrogen determination of various sample types: A comparison of the Hach, Kjeltec, and Kjeldahl methods. Journal of the Association of Official Analytical Chemists, 70(3):410-412. 56. Guebel, D. V., Nudel, B. C., and Giulietti, A. M. (1991) A simple and rapid micro-Kjeldahl method for total nitrogen analysis, Biotechnology Techniques, 5(6):427-430. 57. Feinberg, M. H., J. Ireland-Ripert, J., and Mourel, R. M. (1993) Optimization procedure of open vessel microwave digestion for Kjeldahl nitrogen determination in foods, Analytica Chimica Acta, 272:83-90. 58. Buondonno, A., Rashad, A. A., and Coppola, E. (1995) Comparing tests for soil fertility. II. The hydrogen peroxide/sulfuric acid treatment as an alternative to the copper/selenium catalyzed digestion process for routine determination of soil nitrogen- Kjeldahl. Communications in Soil Science and Plant Analysis, 26 (9 & 10):1607–1619. 59. White, L. M. and Long, M. C. (1951) Kjeldahl microdigestions in sealed tubes at 470°C, Analytical Chemistry, 23(2):363-365. 60. Grunbaum, B. W., Schaffer, F. L. and Kirk, P. L. (1952) Kjeldahl Determination of Nitrogen with Sealed Tube Digestion, Analytical Chemistry, 24(9):1487-1490. 61. Grunbaum, et al (1955) Kjeldahl method with sealed tube digestion, Analytical Chemistry, 27(3):384–388. 62. Abu-Samra, A., Morris, J. S., and Koirtyohann, S. R. (1975) Wet ashing of some biological samples in a microwave oven, Analytical Chemistry, 47(8):1475-1477. 63. Alvarado, José, Márquez, Manuel, and León, Luis E. (1988) Determination of organic nitrogen by the Kjeldahl method using microwave acid digestion, Analytical Letters, 21(3):357-365. 64. Collins, Leo W., Chalk, Stuart J., and Kingston, H. M. (1996) Atmospheric pressure microwave sample preparation procedure for the combined analysis of total phosphorus and Kjeldahl nitrogen, Analytical Chemistry, 68(15):2610-2614. 65. Chemat, Z. et al (1998) Application of atmospheric pressure microwave digestion to total Kjeldahl nitrogen determination in pharmaceutical, agricultural and food products, Analusis, 26:205-209. 66. Lo, K. V. , Wong, W. T. and Liao, P. H. (2005) Rapid determination of total Kjeldahl nitrogen using microwave digestion, Journal of Environmental Science and Health, Part A: Toxic/ Hazardous Substances and Environmental Engineering, 40(3):609–615. 67. Sáez-Plaza, Purificación, et al. (2013) An overview of the Kjeldahl method of nitrogen determination. Part II. Sample preparation, working scale, instrumental finish, and quality control, Critical Reviews in Analytical Chemistry, 43:224-272. 68. Zhu, Yong, et al. (2019) Development of analytical methods for ammonium determination in seawater over the last two decades, Trends in Analytical Chemistry, 119:1-14. 69. Kjeldahl, J. (1888) Nogle Bemærkninger om den jodometriske Syretitrering (Some remarks on the iodometric acid titration), Meddelelser fra Carlsberg laboratoriet (Reports from the Carlsberg Laboratory), 2:323-329 70. Winkler, L. W. (1913) Beitrag zur titrimetrischen Bestimmung des Ammoniaks (Contribution to the volumetric determination of the ammonia), Zeitschrift für Angewandte Chemie, 26:231232. 71. Scales, F. M., and Harrison, A. P. (1920) Boric acid modification of the Kjeldahl method for crop and soil analysis, The Journal of Industrial and Engineering Chemistry, 12(4):350-352. 72. Markley, K. S., and Hann, Raymond M. (1925) A comparative study of the Gunning-Arnold and Winkler boric acid modifications of the Kjeldahl method for the determination of nitrogen, Journal of the Association of Official Agricultural Chemists, 8(4):455-467. 73. Meeker, E. W. and Wagner, E. C. (1933) Titration of ammonia in the presence of boric acid, Industrial and Engineering Chemistry, Analytical Edition, 5:396. 74. Yuen, S. H., and Pollard, A. G. (1953) Determination of nitrogen in soil and plant materials: Use of boric acid in the micro-Kjeldahl method, Journal of the Science of Food and Agriculture, 4:490-496.

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75. Siemer, Darryl D. (1986) Use of solid boric acid as an ammonia absorbent in the determination of nitrogen, The Analyst, 111:1013-1015. 76. Sher, Irving H. (1955) Two-step mixed indicator for Kjeldahl nitrogen titration, Analytical Chemistry, 831–832. 77. Wagner, E. C. (1940) Titration of ammonia in the presence of boric acid, Industrial and Engineering Chemistry, Analytical Edition, 12(12):771-772. 78. Johnson, Arnold H., and Green, Jesse R (1930) Modified methyl red and sodium alizarin sulfonate indicators, Industrial and Engineering Chemistry, Analytical Edition, 2(1):240-242. 79. Sobel, Albert E. et al. (1937) A convenient method of determining small amounts of ammonia and other bases by the use of boric acid, Journal of Biological Chemistry, 118(2):443-446. 80. Stover, Norman M., and Sandin, R. B. (1931) Use of boric acid in micro-Kjeldahl determination of nitrogen, Industrial and Engineering Chemistry, Analytical Edition, 3(3):240-242. 81. Ma, T. S., and Zuazaga, G. (1942) Micro-Kjeldahl determination of nitrogen. A new indicator and an improved rapid method, Industrial and Engineering Chemistry, Analytical Edition, 14(3):280-282. 82. Sobel, Albert E. et al. (1944) A convenient titrimetric ultra-micro method for the estimation of urea, Journal of Biological Chemistry, 156(1):355-363. 83. Michalowski, Tadeusz, et al. (2013) The titration in the Kjeldahl method of nitrogen determination: base or acid as titrant?, Journal of Chemical Education, 90(2):191-197. 84. Cruz, Gregorio (2013) Boric acid in Kjeldahl analysis, Journal of Chemical Education, 90(12):1645-1648. 85. Shaw, W. S. (1924) Application of “formol titration” to the Kjeldahl method of estimating nitrogen, Analyst, 49:558-565. 86. Marcalli, Kalman, and Rieman, William (1946) Kjeldahl determination of nitrogen. Elimination of the distillation, Industrial and Engineering Chemistry, Analytical Edition, 18(11):709710. 87. Willard, H. H., and Cake, W. E. (1920) The iodometric determination of amino nitrogen in organic substances, Journal of the American Chemical Society, 42(12):2646-2650. 88. Harvey, H. W. (1951) Micro-determination of nitrogen in organic matter without distillation, Analyst, 76:657-660. 89. Kolthoff, I. M. and Stenger, V. A. (1935) Calcium hypochlorite as a volumetric oxidizing agent: stability and standardization of the solution. Determination of ammonia, Industrial and Engineering Chemistry, Analytical Edition, 7(2):79-81. 90. Belcher, R. (1950) A reversible indicator for use in oxidimetric titrations with standard sodium hypochlorite, Analytica Chimica Acta, 4:468-471. 91. Belcher, R., and Bhatty, M. K. (1956) The elimination of the distillation procedure in the Kjeldahl method, Mikrochimica Acta, 44(7-8):1183-1186. 92. Hashmi, M. H., Ali, Ehsan, and Umar, Muhammad (1962) Kjeldahl determination of nitrogen without distillation, Analytical Chemistry, 34(8):988-990. 93. Folin, Otto and Farmer, Chester J. (1912) A new method for the determination of total nitrogen in urine, Journal of Biological Chemistry, 11:493-501. 94. Folin, Otto and Denis, W. (1916) Nitrogen determinations by direct nesslerization, Journal of Biological Chemistry, 26:473-489. 95. Chiles, H. M. (1928) Direct Nesslerization of Kjeldahl digestions, Journal of the American Chemical Society, 50:217–221. 96. Nichols, M. L. and Willitt, C. O. (1934) Reactions of Nessler’s solution, Journal of the American Chemical Society, 56(4):769–774. 97. Berthelot, M. (1859) Violet d’aniline (Aniline violet), Répertoire de Chimie Appliquée : Compte rendu des applications de la chimie en France et a l’étranger, p. 284. 98. Weatherburn, M. W. (1967) Phenol-hypochlorite reaction for determination of ammonia, Analytical Chemistry, 39(8):971-974. 99. Harwood, J. E. and Huyser, D. J. (1970) Some aspects of the phenol-hypochlorite reaction as applied to ammonia analysis, Water Research, 4(7):501-515.

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100. Gehrke, Charles W. et al. (1971) Missouri automated method for total nitrogen in all fertilizers, Journal of the Association of Official Analytical Chemists, 5(54):651-657 101. Smith, V. R. (1980) A phenol-hypochlorite manual determination of ammonium-nitrogen in Kjeldahl digests of plant tissue, Communications in Soil Science and Plant Analysis, 11(7):709-722. 102. Searle, Philip L. (1984) The Berthelot or indophenol reaction and its use in the analytical chemistry of nitrogen, Analyst, 109:549-566. 103. Reardon J. et al. (1966) New reactants for the colorimetric determination of ammonia, Clinica Chimica Acta, 14(3):203-205. 104. Solórzano, Lucía (1969) Determination of ammonia in natural waters by the phenol hypochlorite method, Limnology and Oceanography, 14(5):799-801. 105. Scheiner, Dora (1976) Determination of ammonia and Kjeldahl nitrogen by indophenol method, Water Research, 10(1):31-36. 106. Verdouw, H. et al. (1978) Ammonia determination based on indophenol formation with sodium salicylate, Water Research, 12(6):399-402. 107. Krom, Michael David (1980) Spectrophotometric determination of ammonia: A study of a modified Berthelot reaction using salicylate and dichloroisocyanurate, The Analyst, 105:1249108. Bower, Carol E. and Holm-Hansen, Thomas (1980) A salicylate–hypochlorite method for determining ammonia in seawater, Canadian Journal of Fisheries and Aquatic Sciences, 37(5):794-798. 109. Fukumoto, Helen E. and Chang, George W. (1982) Manual salicylate-hypochlorite procedure for determination of ammonia in Kjeldahl digests, Journal of the Association of Official Analytical Chemists, 65(5):1076-1079. 110. Wang, L. and Øien, A. (1986) Determination of Kjeldahl nitrogen and exchangeable ammonium in soil by the indophenol method, Acta Agriculturae Scandinavica, 36(1):60-70. 111. Baethgen, W. E. and Alley, M. M. (1989) A manual colorimetric procedure for measuring ammonium nitrogen in soil and plant Kjeldahl digests, Communications in Soil Science and Plant Analysis, 20(9 & 10):961-969. 112. Giner-Sanz, Juan José et al. (2020) Salicylate method for ammonia quantification in nitrogen electroreduction experiments: the correction of iron III interference, Journal of The Electrochemical Society, 167 134519. 113. Zhou, Li and Boyd, Claude E. (2016) Comparison of Nessler, phenate, salicylate and ion selective electrode procedures for determination of total ammonia nitrogen in aquaculture, Aquaculture, 450:187-193. 114. Ruhemann, Siegfried (1910) CXXXII Cyclic di- and tri-ketones, Journal of the Chemical Society, Transactions, 97:1438-1449. 115. Bottom, Carey B. et al. (1978) Mechanism of the ninhydrin reaction, Biochemical Education, 6(1):4-5. 116. Friedman, Mendel (2004) Applications of the ninhydrin reaction for analysis of amino acids, peptides, and proteins to agricultural and biomedical sciences, Journal of Agriculture and Food Chemistry, 52:385-406. 117. Starcher, Barry (2001) A ninhydrin-based assay to quantitate the total protein content of tissue samples, Analytical Biochemistry, 292(1):125-129. 118. Abernathy, Daniel G., et al. (2009) Analysis of protein and total usable nitrogen in beer and wine using a microwell ninhydrin assay, Journal of the Institute of Brewing, 115(2):122-127. 119. Jacobs, S. (1959) Determination of nitrogen in proteins by means of indanetrione hydrate, Nature, 183:262. 120. Jacobs, S. (1960) The determination of nitrogen in organic compounds by the indanetrione hydrate method, Analyst, 85:257-264. 121. Hantzsch, Arthur (1881) Condensationsprodukte aus Aldehydammoniak und ketonartigen Verbindungen (Condensation products from aldehyde ammonia and ketone-like compounds), Chemische Berichte, 14(2):1637-1638. 122. Vanden Eynde, J. J. and Mayence, Annie (2003) Synthesis and aromatization of Hantzsch 1,4-dihydropyridines under microwave irradiation. An overview, Molecules, 8(4):381-391.

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123. Devani, Muljibhai B., et al. (1989) Spectrophotometric method for micro determination of nitrogen in Kjeldahl digest, Journal of the Association of Official Analytical Chemists, 72(6):953-956. 124. Moldoveanu, Serban (1988) Chromatographic determination of total nitrogen following the Kjeldahl oxidation, Journal of Chromatographic Science, 26:12-14. 125. Jackson, Peter E. et al. (1991) Determination of total nitrogen in food, environmental and other samples by ion chromatography after Kjeldahl digestion, Journal of Chromatography, 546:405-410. 126. Wang, Hsiaoling et al. (2016) Protein nitrogen determination by Kjeldahl digestion and ion chromatography, Journal of Pharmaceutical Sciences, 105:1851-1857. 127. Bremner, J. M., and Tabatabai, M. A. (1972) Use of an ammonia electrode for determination of ammonium in Kjeldahl analysis of soils, Communications in Soil Science and Plant Analysis, 3(2):159-165. 128. Rogers, Darius S. and Pool, Karl H. (1973) Analysis of urea nitrogen in the presence of ammonia using an ammonia gas sensitive electrode, Analytical Letters, 6(9):801-808. 129. Buckee, G. K. (1974) Estimation of nitrogen with an ammonia probe, Journal of the Institute of Brewing, 80(3):291-294. 130. Byrne, E. and Power, T. (1974) Determination of ammonium nitrogen in animal slurries by an ammonia electrode, Communications in Soil Science and Plant Analysis, 5(1):55-65. 131. Eastin, E. F. (1976) Use of ammonia electrode for total nitrogen determination in plants, Communications in Soil Science and Plant Analysis, 7(5):477-481. 132. Byrne, E. and McCormack, S. (1978) Determination of ammonium nitrogen in silage samples by an ammonia electrode, Communications in Soil Science and Plant Analysis, 9(8):667-684. 133. Roy, Ram B. et al. (1980) Application of sparging to the automated ion selective electrode determination of Kjeldahl nitrogen, Journal of the Association of Official Analytical Chemists, 63(4):931-936. 134. Powers, Robert F. et al. (1981) Ammonia electrode analysis of nitrogen in Microkjeldahl digests of forest vegetation, Communications in Soil Science and Plant Analysis, 12(1):19-30.

Chapter 5

The Advancement of Kjeldahl Equipment

5.1 Early Devices The designs of Kjeldahl digestion and distillation equipment have evolved notably throughout the years. In 1884, Petri and Lehman [1] developed one of the very first Kjeldahl distillation apparatuses, in which steam was used to distill the ammonia (Fig. 5.1). Also, in 1884 Heffter, Hollrung, and Morgen designed two types of equipment, one for digestion, and one for distillation (Fig. 5.2): “The six burners, each of which can be regulated by a gas tap, can each be fed jointly by an ordinary gas tap. We have four heating devices set up in this way, in which 24 determinations can be prepared at the same time. Each apparatus can be filled three times a day without any effort, i.e., 72 determinations can be prepared daily” [2].

In 1885, Kreusler designed a gas furnace as a heating device for the Kjeldahl analysis [3]. See Fig. 5.3.

5.2 The Caustic Splash Problem One of the problems encountered with the original method was the splashing of caustic soda into the distillate. During the boiling of the alkaline solution, minute drops of the liquid are formed by bubble bursting, and are carried away into the condenser. Kjeldahl himself acknowledged this: “Shortly after my announcement on nitrogen determination, I became aware of two small sources of error in the ammonia distillation, namely the effect on the glass of the cooling tube of the water vapor, whereby a little alkali is given off, and the splashing caused by the evolution of hydrogen from zinc. Particles of the alkaline content of the distillation flask could be transferred to the condenser… Several places where the method was introduced became aware of © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 J. Aguirre, The Kjeldahl Method: 140 Years, https://doi.org/10.1007/978-3-031-31458-2_5

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Fig. 5.1 Petri and Lehman distillation apparatus (Reproduced with permission of Walter de Gruyter and Company, from Petri and Lehmann [1]. Copyright © 1884 by Walter de Gruyter and Company. Permission conveyed through Copyright Clearance Center)

the same disadvantages, and a significant number of distillation apparatus have been described to solve this limitation” [4]. Some of the first Kjeldahl equipment focused on the solution to this problem by including traps to stop the droplets and allow only vapors to enter the distillation column. Throughout the years, many chemists would try to solve this problem in a different manner. Kjeldahl distillation units usually include a glass connecting bulb to attach the flask to the condensing apparatus. The connecting bulb holds back any spray from the boiling liquid and breaks up any foaming which may occur. In 1885, Arnold introduced a distillation apparatus, with further revisions in 1886, solving the caustic splash problem by using a Péligot tube [5, 6]. See Fig. 5.4. In 1885, Pfeiffer and Lehmann [7] designed a “safety tube” (Fig. 5.5). The lower part of A is drawn out into a narrow tube covered by a small platinum cone c. Above this is a layer of glass beads. This simple device is connected to the ordinary distillation tube B by a second rubber stopper. Rindell and Hannin found that the tube was not sufficient to hold back all the caustic soda [8]. They rectified the problem by inserting the bead tube into another jacket tube. They found that adequate safety is gained if the pearls are stacked at least 8 cm high (Fig. 5.6).

5.2 The Caustic Splash Problem

81

Fig. 5.2 Heffter, Hollrung, and Morgen digestion apparatus (Reproduced with permission of John Wiley and Sons, from Heffter et al. [2]. Copyright © 1884 by John Wiley and Sons. Permission conveyed through Copyright Clearance Center)

Fig. 5.3 Kreusler digestion apparatus (Reproduced with permission of Springer Nature, from Kreusler [3]. Copyright © 1885 by Springer Nature. Permission conveyed through Copyright Clearance Center)

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Fig. 5.4 Arnold’s distillation apparatus (Reproduced with permission of John Wiley and Sons, from Arnold [6]. Copyright © 1886 by John Wiley & Sons. Permission conveyed through Copyright Clearance Center)

In 1888, Kjeldahl designed a distillation system that included a built-in trap (Fig. 5.7). “I therefore now use the washing apparatus shown in the drawing: through the side tube a, which is kept closed with a hose and a glass stopper during the distillation, a few drops of water are introduced before. The water that collects in the wash retains no ammonia; after distillation, it is sucked back into the flask as soon as the burner is turned off” [4]. In 1896, Hopkins introduced a new device to solve the caustic splash problem. “In doing a large amount of work with fodders and fertilizers involving several hundred determinations of nitrogen (by the Kjeldahl method), I have observed that the distillation tube (generally known as ‘Kjeldahl’s connecting bulb tube’, but doubtless more properly as Reitmeir’s distillation tube) is frequently a source of error, due to the

5.2 The Caustic Splash Problem Fig. 5.5 Pfeiffer and Lehmann caustic trap (Reproduced with permission of Springer Nature, from Pfeiffer and Lehmann [7]. Copyright © 1885 by Springer Nature)

Fig. 5.6 Rindell and Hannin caustic trap (Reproduced with permission of Springer Nature, from Rindell and Hannin [8]. Copyright © 1886 by Springer Nature)

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Fig. 5.7 Kjeldahl distillation apparatus (Reproduced with permission of Carlsberg Foundation/Carlsberg Research Laboratory, from Kjeldahl [4]. Copyright © 1888 by Carlsberg Foundation/Carlsberg Research Laboratory)

fact that it allows fixed alkali to be carried over mechanically” [9]. The modification was criticized by Davisson, who affirmed that “the Hopkins bulb, which has been long in use, does not prevent alkali from passing into the receiving acid” [10]. Davisson found that the entrained alkali can be satisfactorily removed when the vapors are scrubbed through water prior to condensation. He constructed a scrubber of Pyrex glass, shown in Fig. 5.8. The small bulb on the inlet tube has three openings in the same horizontal plane. The large bulb has a capacity of 200 ml, which gives it a satisfactory condensing surface. The small bulb on the inlet tube has three openings in the same horizontal plane. West used a distillation trap in which vapors pass up the inner tube, through the side holes at the end, and are deflected down through the wet narrow annular space between the inner tube and cap [11]. See Fig. 5.9. The vapor then passes out between the wet walls of the cap and bulb. Condensed liquid returns to the boiling flask through a hole at the bottom of the inner tube near its seal to the outer tube. Figure 5.10 shows a modified Kjeldahl distillation apparatus which was successfully used by Weakley in 1936 [12]. It has the peculiarity that metal heads take the place of the usual glass connecting bulbs. The block-tin condenser tube which comes with this particular apparatus is the right length to be soldered into the head and should extend to within 2 or 3 mm of the top (soldered cap). The wire gauze and glass beads completely break up and return to the flask any spray or foam which may occur.

5.3 The Ammonia Distillation Equipment

85

Fig. 5.8 Davisson’s scrubber for ammonia distillation (Reproduced with permission of American Chemical Society, from Davisson [10]. Copyright © 1919 by American Chemical Society. Permission conveyed through Copyright Clearance Center)

Although not very common, ammonia traps were used at the receiver end of the distillation apparatus. In 1946, Potts described a glass device to seal the receiver (Erlenmeyer flask) collecting the ammonia distillate (Fig. 5.11). It traps any ammonia gas that is driven through the condenser and retains it in solution. “When the distillation is complete and the heat is turned off, the lower pressure inside the system will cause the solution in the trap to siphon into the receiving flask” [13].

5.3 The Ammonia Distillation Equipment In 1889, Stein and Schwarz [14] published an article on the separation of ammonia by distillation. They presented an apparatus which solved the problem of caustic soda droplets splashing into the distillate (Fig. 5.12). Weston described an apparatus used for both Wanklyn and Kjeldahl methods [15]. The first set of stills of this design was built for the laboratory of the Cincinnati Water Commission in 1898. The apparatus is shown in Fig. 5.13 (front and side views). Kober described an apparatus for the quantitative distillation of ammonia [16]. As shown in Fig. 5.14, cylinder A contains the standard sulfuric acid, Kjeldahl flask

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Fig. 5.9 West’s improved distillation trap (Reproduced with permission of American Chemical Society, from West [11]. Copyright © 1932 by American Chemical Society. Permission conveyed through Copyright Clearance Center)

B contains the solution after digestion, and cylinder C contains the caustic solution to alkalinize the digest. This solution is transferred to the Kjeldahl flask by applying suction on the tube at the left, while at the same time shaking the Kjeldahl flask. Green described a steam still for nitrogen determination, with accuracy equal to a still heated by gas or electricity [17]. The steam passes throughout the needle valves to flow freely into the liquid in the distillation flasks, J. The steam rises through the liquid carrying the ammonia up through the traps and down through the condenser as distillate into the receiving flasks. Figure 5.15 shows the front and side views of the apparatus.

5.3 The Ammonia Distillation Equipment Fig. 5.10 Weakley’s special distillation head (Reproduced with permission of American Chemical Society, from Weakley [12]. Copyright © 1936 by American Chemical Society. Permission conveyed through Copyright Clearance Center)

Fig. 5.11 Potts ammonia trap (Reproduced with permission of American Chemical Society, from Potts [13]. Copyright © 1946 by American Chemical Society. Permission conveyed through Copyright Clearance Center)

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Fig. 5.12 Stein and Schwarz distillation apparatus (Reproduced with permission of Springer Nature, from Stein and Schwarz [14]. Copyright © 1889 by Springer Nature. Permission conveyed through Copyright Clearance Center)

5.4 Solving the Problem of Fumes Disposal Sy [18] used an arrangement specially designed to dispose of the large amount of fumes that evolved during the Kjeldahl digestion. See Fig. 5.16. “Three years ago, the writer began using an arrangement similar in principle to the one here described and shown. The whole is mounted on a portable iron stand, occupies but a little space and is easily moved. In the apparatus here shown, four ordinary long-necked 500 cc. Kjeldahl flasks suitable for digestion and distillation, are used” [18]. The bulb tubes on top of the Kjeldahl flasks are connected with a large glass suction pump having four branches. To operate, the pump is coupled with the water supply. Hastings et al. described an apparatus for extracting fumes from the Kjeldahl flasks during digestion [19]. It consists of a large lead tube, with lead side arm tubes, and carrying movable lead stoppers. These lead stoppers move freely up and down the side arm tubes and thus accommodate Kjeldahl flasks with varying sizes of necks.

5.4 Solving the Problem of Fumes Disposal

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Fig. 5.13 Weston distillation apparatus (Reproduced with permission of American Chemical Society, from Weston [15]. Copyright © 1900 by American Chemical Society. Permission conveyed through Copyright Clearance Center) Fig. 5.14 Kober’s ammonia distillation apparatus (Reproduced with permission of American Chemical Society, from Kober [16]. Copyright © 1908 by American Chemical Society. Permission conveyed through Copyright Clearance Center)

The digestion tube is connected by a coupling to a water injector. This consists of a small lead pipe passing into the larger fume tube. The end of this small pipe is closed except for the small holes as shown in the figure. When the water is turned on, it escapes through these holes and passes down through the fume tube, creating a suction. Figure 5.17 shows a cross section of the water injector, and one branch of the Kjeldahl digestion apparatus.

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Fig. 5.15 Green’s distillation apparatus (Reproduced with permission of American Chemical Society, from Green [17]. Copyright © 1927 by American Chemical Society. Permission conveyed through Copyright Clearance Center)

Fig. 5.16 Sy’s fume extractor (Reproduced with permission of American Chemical Society, from Sy [18]. Copyright © 1912 by American Chemical Society. Permission conveyed through Copyright Clearance Center)

5.4 Solving the Problem of Fumes Disposal

91

Fig. 5.17 Hastings, Fred, and Peterson Kjeldahl apparatus (Reproduced with permission of American Chemical Society, from Hastings et al. [19]. Copyright © 1927 by American Chemical Society. Permission conveyed through Copyright Clearance Center)

In 1937, Clark described a digestion apparatus with a fume extractor [20]. See Fig. 5.18. It consists of the following parts: A: Horizontal water reservoir pipe B: Couplings C: Valves D: Short pipe fittings E: Glass suction pumps

F: Two bulb stoppers G: Half section of acid-proofed pipe H: Pieces of glass tubing I: Ring supports for the flasks J: Burners.

Tyner (1948) described a manifold that achieves fume disposal through their disolution in water [21]. The junction of the Kjeldahl flask with the manifold is not tight or rigid (Fig. 5.19).

92

5 The Advancement of Kjeldahl Equipment

Fig. 5.18 Clark’s Kjeldahl digestion apparatus (Reproduced with permission from American Chemical Society, from Clark [20]. Copyright © 1937 by American Chemical Society. Permission conveyed through Copyright Clearance Center)

5.5 The Small Details: Racks and Stands Throughout the years, innovations were presented for all parts of both digestion and distillation equipment, namely fume removal tubes, racks and supports for Kjeldahl flasks, and so on. Rice described an adjustable rack for Kjeldahl digestion flasks [22]. Figure 5.20 shows side and front elevations.

5.6 The Miniature Equipment: Micro-Kjeldahl Devices

93

Fig. 5.19 Tyner’s manifold for disposal of fumes (Reproduced with permission of American Chemical Society, from Tyner [21]. Copyright © 1948 by American Chemical Society. Permission conveyed through Copyright Clearance Center)

Front described a support which holds the flasks firmly and vertically, and provides easy access to each flask [23]. See Fig. 5.21.

5.6 The Miniature Equipment: Micro-Kjeldahl Devices Quantitative organic microanalysis was conceived by Fritz Pregl in the early 1900s. He was awarded the Chemistry Nobel Prize in 1923 for his invention of the method of microanalysis of organic substances. Pregl designed an apparatus for micro-Kjeldahl nitrogen determination, in which distillation can be accelerated and bumping prevented by passing steam through the boiling solution [24]. Parnas and Wagner designed a micro apparatus for carrying out the distillation [25]. It was basically a modification of Pregl’s design (Fig. 5.22). Parnas conceived a macro apparatus, being basically a scale-up of the micro type [26].

94

5 The Advancement of Kjeldahl Equipment

Fig. 5.20 Rice’s adjustable rack for Kjeldahl digestion flasks (Reproduced with permission of American Chemical Society, from Rice [22]. Copyright © 1918 by American Chemical Society. Permission conveyed through Copyright Clearance Center)

Kemmerer and Hallett described an improved apparatus for ammonia distillation [27]. See Fig. 5.23. A is the steam generator, with openings at L and K to allow the steam to pass through B, E, F, and M to the distilling flask, N. Ammonia-free water is added to A through funnel D. The digested sample and an excess of potassium hydroxide are added to N through the funnel P. The steam generated in A passes to N, helps to liberate and carry the ammonia and steam through tube H, and on passing through T is condensed and collected in a 50 ml graduated flask containing 25 ml of ammonia-free water. The condenser tube is placed below the surface of the liquid and 25 ml of distillate is collected.

5.6 The Miniature Equipment: Micro-Kjeldahl Devices

95

Fig. 5.21 Front’s stand for Kjeldahl flasks (Reproduced with permission of American Chemical Society, from Front [23]. Copyright © 1944 by American Chemical Society. Permission conveyed through Copyright Clearance Center)

Fig. 5.22 Parnas and Wagner micro-Kjeldahl apparatus (Reproduced with permission of Springer Nature, from Parnas and Wagner [25]. Copyright © 1921 by Springer Nature)

96

5 The Advancement of Kjeldahl Equipment

Fig. 5.23 Kemmerer and Hallett micro-Kjeldahl apparatus (Reproduced with permission of American Chemical Society, from Kemmerer and Hallett [27]. Copyright © 1927 by American Chemical Society. Permission conveyed through Copyright Clearance Center)

Scott and West presented a micro-Kjeldahl apparatus [28]. It consisted of a 100 ml digestion and distillation flask (Kjeldahl flask) attached by a ground-glass joint to a distilling head composed of a West distilling trap and a West condenser (Fig. 5.24). The device was significant because of its relative simplicity, compactness, strength, and accuracy. Kirk designed a micro-Kjeldahl apparatus in which all rubber connections were eliminated. It showed a number of points of superiority over the usual type of equipment (Fig. 5.25). “It is made as a single piece of glass, with a steam generator surrounding the distillation flask, and an internal cold finger condenser, which gives a rugged and compact unit, that is nearly automatic in its operation” [29, 30]. In 1951, the Committee for the Standardization of Microchemical Apparatus, Division of Analytical Chemistry of the American Chemical Society, published a paper including recommendations for the apparatus used in the Kjeldahl nitrogen determination [31]. See Figs. 5.26 and 5.27. Steyermark published a comprehensive treatise on quantitative organic microanalysis [32]. The micro-Kjeldahl method is thoroughly described. See Fig. 5.28.

5.6 The Miniature Equipment: Micro-Kjeldahl Devices Fig. 5.24 Scott and West micro-Kjeldahl apparatus (Reproduced with permission from American Chemical Society, from Scott and West [28]. Copyright © 1937 by American Chemical Society. Permission conveyed through Copyright Clearance Center)

Fig. 5.25 Kirk’s micro-Kjeldahl apparatus (Reproduced with permission of American Chemical Society, from Kirk [30]. Copyright © 1950 by American Chemical Society. Permission conveyed through Copyright Clearance Center)

97

98 Fig. 5.26 Micro-Kjeldahl distillation apparatus—one-piece model (Reproduced with permission of American Chemical Society, from Steyermark et al. [31]. Copyright © 1951 by American Chemical Society. Permission conveyed through Copyright Clearance Center)

Fig. 5.27 Micro-Kjeldahl distillation apparatus—Pregl type (Reproduced with permission of American Chemical Society, from Steyermark et al. [31]. Copyright © 1951 by American Chemical Society. Permission conveyed through Copyright Clearance Center)

5 The Advancement of Kjeldahl Equipment

5.7 Kjeldahl Equipment Today

99

Fig. 5.28 Circular and straight type micro-Kjeldahl digestion racks (Reproduced with permission of Elsevier B. V., from Steyermark [32]. Copyright © 1961 by Elsevier B. V. Permission conveyed through PLSclear)

5.7 Kjeldahl Equipment Today Numerous designs for both digestion and distillation devices have been made in Europe and the United States since the publication of Kjeldahl’s method. Today’s Kjeldahl equipment is semi-automated and considerably computerized, offering high efficiency, energy saving, and safety for the user and the environment. See Figs. 5.29 and 5.30.

100 Fig. 5.29 Foss Digestor™ 2520 (Reproduced with permission of Foss Analytics. Copyright © 2023 by Foss Analytics, Denmark)

5 The Advancement of Kjeldahl Equipment

References

101

Fig. 5.30 Foss Kjeltec™ 8400 (Reproduced with permission of Foss Analytics. Copyright © 2023 by Foss Analytics, Denmark)

References 1. Petri, and Lehmann, Th. (1884) Die Bestimmung des Gesamtstickstoffs im Harn (The determination of total nitrogen in urine), Zeitschrift für Physiologische Chemie, 8:200–213. 2. Heffter, Hollrung and Morgen (1884) Ein Beitrag zu der Methode der Stickstoffbestimmung nach Kjeldal (A contribution to the Kjeldahl method for nitrogen determination), Chemiker Zeitung, 8(25):432–435. 3. Kreusler, U. (1885) Digestionsofen zur Stickstoffbestimmung nach Kjeldahl (Digestion furnace for nitrogen determination according to Kjeldahl), Zeitschrift für Analytische Chemie, 24:393– 394. 4. Kjeldahl, J. (1888) Et Destillationsapparat til Brug ved Kvælstofbestemmelse (A distillation apparatus for use in nitrogen determination), Meddelelser fra Carlsberg laboratoriet (Reports from the Carlsberg Laboratory), 2:330–331. Excerpts from pages 330–331 reproduced with permission of Carlsberg Foundation/Carlsberg Research Laboratory, from: Kjeldahl, Johan. 1888, Et Destillationsapparat til Brug ved Kvælstofbestemmelse, Meddelelser fra Carlsberg laboratoriet, 2:330–331. Copyright © 1888 by Carlsberg Foundation/Carlsberg Research Laboratory. 5. Arnold, Carl (1885) Die Kjeldahl’che Methode der Stickstoffbestimmung. (The Kjeldahl method of nitrogen determination), Archiv der Pharmazie, 223(5):177–185.

102

5 The Advancement of Kjeldahl Equipment

6. Arnold, Carl (1886) Die allgemeinere Anwendbarkeit der Kjeldahl’schen Stickstoff bestimmungsmethode (The more general applicability of Kjeldahl’s nitrogen determination method), Archiv der Pharmazie, 224(18):785–794. 7. Pfeiffer, T. and Lehmann, F. (1885) Notiz zur Kjeldahl’schen Stickstoff-BestimmungsMethode, Zeitschrift für Analytische Chemie, 24:388–393. 8. Rindell, A. and Hannin, F. (1886) Zur Stickstoffbestimmung nach Kjeldahl’s Methode, Zeitschrift für Analytische Chemie, 25:155–156. 9. Hopkins, Cyril G. (1896) A new safety distillation tube for rapid work in nitrogen determinations, Journal of the American Chemical Society, 18(3):227–228. 10. Davisson, B. S. (1919) A scrubber for ammonia distillation, Journal of Industrial & Engineering Chemistry, (5):465–466. 11. West, Edward S. (1932) Improved distillation trap, Industrial and Engineering Chemistry, Analytical Edition, 4(4):445. 12. Weakley, Chas. E. (1936) Special head for Kjeldahl distillation apparatus, Industrial and Engineering Chemistry, Analytical Edition, 8(5):367. 13. Potts, T. J. (1946) Apparatus for trapping ammonia in the Kjeldahl method for nitrogen, Industrial & Engineering Chemistry Analytical Edition, 18(1):78. 14. Stein, Walter M. and Schwarz, Paul W. (1889) Beitrag zur Bestimmung von Ammoniak durch Destillation (Contribution to the determination of ammonia by distillation), Zeitschrift für Analytische Chemie, 28:428–431. 15. Weston, R. S. (1900) Apparatus for the determination of ammonia in water, by the Wanklyn method, and total nitrogen by the Kjeldahl method, Journal of the American Chemical Society, 22 (8), 468–473. 16. Kober, Philip Adolph (1908) A new apparatus for the quantitative distillation of ammonia, Journal of the American Chemical Society 30(7):1131–1135 17. Green, Jesse (1931) Use of steam for Kjeldahl distillation of nitrogen, Industrial & Engineering Chemistry Analytical Edition, 3(2): 160–161. 18. Sy, A. P. (1912) Apparatus for fumeless Kjeldahl nitrogen digestion, Industrial and Engineering Chemistry, 4(9):680–681. 19. Hastings, E. G. et al (1927) A simple and inexpensive Kjeldahl digestion apparatus, Industrial & Engineering Chemistry, 19(3):397. 20. Clark, Wesley M. A (1937) Kjeldahl digestion apparatus, Journal of Industrial & Engineering Chemistry 9(7):338–339. 21. Tyner, E. H. (1948) Manifold for disposal of fumes given off during macro-Kjeldahl digestive process, Analytical Chemistry, 20(3):273. 22. Rice, Frank E. (1918) A Simple and entirely adjustable rack for Kjeldahl digestion flasks, Journal of Industrial & Engineering Chemistry, 10(8):631–632. 23. Front, Jacqueline (1944) Support for Kjeldahl flasks, Industrial & Engineering Chemistry, Analytical Edition, 16(5):324. 24. Pregl, Fritz. (1899) Ueber die Verwendung eines einfachen Apparates bei der StickstoffBestimmung nach Kjeldahl (On the use of a simple apparatus for the determination of nitrogen according to Kjeldahl), Zeitschrift für Analytische Chemie, 38:166–167. 25. Parnas, J. K. and R. Wagner, R. (1921) Über die Ausführung von Bestimmungen leiner Stickstoffmengen nach Kjeldahl (On the execution of determinations of small amounts of nitrogen according to Kjeldahl), Biochemische Zeitschrift, 125:253–256 26. Parnas, J. K. (1938) Über die Ausführung der stickstoffbestimmung nach Kjeldahl in der Modifikation von Parnas und Wagner (About the execution of the nitrogen determination according to Kjeldahl in the modification of Parnas and Wagner), Zeitschrift für Analytische Chemie, 114:261–275. 27. Kemmerer, George and Hallett, L. T. (1927) Improved micro-Kjeldahl ammonia distillation apparatus, Industrial & Engineering Chemistry, 19(11):1295–1296. 28. Scott, Joseph E. and West, Edward S. (1937) A simplified micro-Kjeldahl apparatus, Industrial & Engineering Chemistry Analytical Edition, 9(1):50.

References

103

29. Kirk, P. L. (1936) A one-piece glass micro-Kjeldahl distillation apparatus, Industrial and Engineering Chemistry, Analytical Edition, 8(3):223–224. 30. Kirk, P. L. (1950) Kjeldahl method for total nitrogen, Analytical Chemistry, 22(2):354–358. 31. Steyermark, Al et al. (1951) Recommended specifications for microchemical apparatus. MicroKjeldahl nitrogen, Analytical Chemistry, 23(3):523–528. 32. Steyermark, Al (1961) Quantitative Organic Microanalysis, Academic Press, New York and London.

Chapter 6

Experimental: Evaluation of Titanium Dioxide as a Catalyst in the Determination of Nitrogen by the Kjeldahl Method

The use of different catalysts in the Kjeldahl method has been a subject of investigation since the inception of the technique. Four catalysts have proved successful throughout the years, namely HgO, Se, CuSO4 , and TiO2 . Toxicity of both mercuric oxide and selenium is widely known, leaving cupric sulfate and titanium dioxide as the most appropriate options. In this research, selected samples of pure amino acids and feed ingredients were used to evaluate the catalytic action of the mixture TiO2 –CuSO4 against HgO and Se–CuSO4. Comparison of these catalysts resulted in statistically reliable outcomes. The substitution of other catalysts by a mixture of titanium and copper sulfate is recommended, in order to eliminate the risks of environmental contamination and toxicity. In the second part of this work, the catalytic mixture of potassium sulfate, copper sulfate, and titanium dioxide was produced by recrystallization of commercial-grade chemicals. This catalytic mixture showed to be as reliable as the one made of reagentgrade chemicals.1

6.1 Introduction Since its publication in 1883 by Johan Kjeldahl, this method to determine nitrogen has reached wide diffusion, being within analytical chemistry one of the most used quantitative procedures.

1

Jaime Aguirre (1988) Laboratory of Animal Nutrition, Faculty of Veterinary Medicine and Zootechnics, University of Antioquia, Colombia.

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 J. Aguirre, The Kjeldahl Method: 140 Years, https://doi.org/10.1007/978-3-031-31458-2_6

105

106

6 Experimental: Evaluation of Titanium Dioxide as a Catalyst …

Essentially the method is performed in two stages: 1. Digestion of organic matter with sulfuric acid. 2. Separation and determination of the ammonium ion generated in the digestion. This last step has had a few difficulties to carry out, and there are various methods of great reproducibility applicable, according to the convenience of each laboratory. See, for example, Fukumoto and Chang [1], Novamsky et al. [2], Searle [3], and Siemer [4]. The organic matter digestion process involves several variables, which make it the critical point of the method. In the initial experiments, Kjeldahl accelerated the oxidation by adding potassium permanganate. Wilfarth introduced HgO as a catalyst [5]. Gunning used potassium sulfate to increase the boiling point with the consequent increase in the reaction rate [6]. Kirk [7] studied the first stage considering the following aspects: Amount of sulfuric acid. Amount of added salt. Catalyst. Oxidizing agents. Reducing agents. Digestion time and conditions. His conclusions highlighted the importance of controlling the proportion of sulfuric acid to potassium sulfate, the differences in the effectiveness of different catalysts, and the good prospects offered by hydrogen peroxide as an oxidizing agent. The use of the latter has currently led to the elimination of the catalyst and the design of more efficient equipment and methods. See Florence and Millner [8], Hach [9], and Singh et al. [10].

6.2 Acid-to-Salt Ratio Very little has been investigated on the reaction mechanism of sulfuric acid digestion. Fundamentally, two reactions occur: oxidation of carbon to carbon dioxide, and conversion of organic nitrogen to ammonium bisulfate. The reaction rate is favored by an increase in temperature, and this is achieved by adding potassium sulfate. The boiling point of concentrated sulfuric acid is 338 °C (at a pressure of one atmosphere), according to Kunzler [11]. The addition of salt makes it possible to obtain a boiling range from 370 °C to 410 °C. Lake et al. [12] found that when digesting pyridine ring structures (in this structure, the nitrogen is particularly refractory to Kjeldahl digestion), at a temperature lower than 370 °C either requires too long a digestion time or fails to give good results, while a temperature higher than 410 °C may result in loss of nitrogen. Control of this range can be effected by maintaining an appropriate acid-to-salt ratio. This aspect of the analysis was studied by Bradstreet [13].

6.3 Catalyst

107

Rexroad and Cathey analyzed nitrogen content in a variety of samples with CuSO4 as catalyst and a salt-to-acid ratio of 15 g K2 SO4 to 20 ml H2 SO4 (this is equivalent to a 1.3 acid-to-salt ratio). The precision and accuracy of the results were quite acceptable [14].

6.3 Catalyst The sulfuric acid digestion is enhanced by some catalysts. Mercuric oxide has proved to be an excellent choice, but its high toxicity has always been a drawback [15]. Consequently, investigations on this method were focused on the search for an optimal catalyst that is risk-free to the analyst and to the environment. Lauro used selenium to replace mercury [16]. The effectiveness of selenium was questioned by Patel and Sreenivasan [17] and Kirk [7]; they found low nitrogen values in the analysis performed using selenium. Selenium toxicity has also been observed [18]. Despite this, the use of mercury and selenium as catalysts has not been completely discontinued [19, 20]. Osborn and Wilkie evaluated 39 different metals, from which they selected the ten best ones in decreasing order of effectiveness: Hg, Se, Te, Ti, Mo, Fe, Cu, V, W, and Ag [21]. Bradstreet studied tellurium obtaining negative results [22]. Liao used tellurium mixed with cupric sulfate and its effectiveness was increased [23]. Other compounds have been evaluated as catalysts, but their use did not progress, namely zirconium dioxide [24] and antimony [25]. Odland obtained good accuracy in the analysis carried out with cupric sulfate as a catalyst [26]. Rexroad [27] and Rexroad and Cathey [14], in the laboratories of the Experimental Station of the University of Missouri, proposed a procedure based on an acid-to-salt ratio of 1.3, 0.04 gm of cupric sulfate as catalyst, and 90 min of heating, using lysine hydrochloride and tryptophan as standards (these amino acids are refractory to digestion and require stringent conditions for full nitrogen recovery). Kane applied the Missouri method to animal feeds with good results [28]. In addition to studying the toxicity risks posed by the catalysts, the investigations were oriented toward the reduction of the digestion time. Williams incorporated titanium dioxide into the mixture of potassium sulfate and cupric sulfate, managing to considerably reduce the digestion time [29]. Several works on TiO2 were carried out later [30]. Kane performed a collaborative study obtaining results comparable to those achieved with mercury [31]. He proposed that the titanium method be officially adopted by the AOAC, with the use of lysine hydrochloride in routine tests as a standard for full nitrogen recovery by any method. This catalytic mixture meets the following requirements: Able to maintain a reaction rate comparable to mercury. Total nitrogen recovery. Nontoxic.

108

6 Experimental: Evaluation of Titanium Dioxide as a Catalyst …

Cost-effective. In Colombia, Laredo and Torres (1979) analyzed the protein composition of various raw materials used in the manufacture of animal feed, comparing mercury oxide, selenium, and titanium dioxide [32]. Their research showed that titanium dioxide produces reliable results, and they recommended its use. The present work seeks to evaluate the application of titanium dioxide in samples of different nature, in order to contribute to the diminution of the risk of ambient pollution with toxic catalysts.

6.4 Experimental Equipment. Digestion unit, Büchi 430. Distillation unit, Büchi 321. Electronic burette, Metrohm E 415. pH Meter, Metrohm. Reagents. (NH4)2 SO4 ≥ 99.5, Merck 101217. H2 SO4 97%, Merck 731. NaOH (pellets), Merck 6498. H2 SO4 0.1 N titrisol, Carlo Erba 410731. K2 SO4 Merck 5153. HgO Carlo Erba 461303. TiO2 Baker 1–4162. CuSO4 Carlo Erba 476245. H3 BO3 Carlo Erba 402767. Lysine.HCl Merck 5700. Tryptophane Merck 8375. Glycine Merck 4201. Tris(hydroxymethyl)aminomethane Merck 8382. Mixed indicator according to Sher, Büchi. Catalytic tablets according to Wieninger, Merck 10958. Catalytic tablets free of mercury and selenium, Merck 15348. Distillation. The effectiveness of the distillation was verified with ammonium sulfate. The distilled ammonia was received in a 3% boric acid solution, and was titrated with 0.1 N H2 SO4 , using the mixed indicator according to Sher and a pH meter (pH at the end point is 4.65). The normality of H2 SO4 was verified with the primary standard tris(hydroxymethyl)aminomethane. The results obtained are shown in Table 6.1. Theoretical nitrogen: 21.20% Recovery: 99.95%

6.4 Experimental Table 6.1 Percentage of nitrogen recovered by distillation of ammonium sulfate

109

(NH4 )2 SO4 (g)

H2 SO4 ( ml)

Percent N

0.0762

11.53

21.19

0.0937

14.21

21.23

0.0878

13.29

21.19

0.1054

15.92

21.15

0.0910

13.82

21.26

0.0655

9.89

21.14

Mean

21.19

Acid-to-salt ratio. Initially, nitrogen determinations of lysine hydrochloride with selenium and titanium dioxide were performed separately, in order to find the best acid-to-salt ratio for each catalyst. Catalyst A: 1 tablet (5 g) Wieninger catalyst: Na2 SO4 + CuSO4 + Se. Catalyst B: 4.85 g K2 SO4 + 0.05 g CuSO4 + 0.10 g TiO2 . Catalyst C: 7.35 g K2 SO4 + 0.15 g HgO. Catalyst D: 1 tablet (5 g) free of Hg and Se: K2 SO4 + Na2 SO4 + CuSO4 + TiO2 Sample: 0.1 gm lysine.HCl (0.2 g of starch is added to increase the bulk). Digestion time: 60 min. Analyses were carried out with mixtures A and B keeping the amount of sample and the digestion time constant, which produces different acid/salt ratios. Each result is the arithmetic mean of three determinations. The theoretical content of nitrogen in lysine.HCl is 15.34%. Catalyst C was not included in the first part of the work. Analyses carried out with catalyst D were inconsistent, therefore they are not included in this work. The results for catalytic mixture A are shown in Table 6.2 and Fig. 6.1. The results for catalytic mixture B are shown in Table 6.3 and Fig. 6.2. If nitrogen content is plotted against the ratio of acid (volume) to salt (weight), the curve shown in Fig. 6.1 is obtained.

Table 6.2 Percentage of nitrogen in lysine.HCl with catalyst A and different acid/ salt ratios

H2 SO4 97% (ml) Acid/salt ratio Percent N (ml/g) 6

1.2

Mean

14.44, 14.38, 14.40 14.41

9

1.8

14.45, 14.39, 14.26 14.37

12

2.4

14.73, 14.65, 14.83 14.74

15

3.0

15.09, 14.99, 15.03 15.04

20

4.0

15.38, 15.42, 15.27 15.36

110

6 Experimental: Evaluation of Titanium Dioxide as a Catalyst …

Fig. 6.1 Acid-to-salt ratio versus nitrogen content with catalyst A

Table 6.3 Percentage of nitrogen in lysine.HCl with catalyst B and different acid/ salt ratios

H2 SO4 97% (ml) Acid/salt ratio Percent N (ml/g)

Mean

6

1.2

15.27, 15.28, 15.26 15.27

7

1.4

14.71, 14.62, 14.73 14.69

8

1.6

14.24, 14.21, 14.13 14.19

9

1.8

13.60, 13.75, 13.27 13.54

10

2.0

13.39, 13.11, 12.98 13.16

11

2.2

12.89, 12.74, 13.18 12.94

12

2.4

12.73, 12.76, 12.59 12.69

The optimal acid-to-salt ratio for catalyst A (Na2 SO4 + CuSO4 + Se) is 4.0 If nitrogen content is plotted against the ratio of acid (volume) to salt (weight), the curve shown in Fig. 6.2 is obtained. The optimal acid-to-salt ratio for catalyst B (K2 SO4 + CuSO4 + TiO2 ) is 1.2. Determination of nitrogen in amino acids and feeding stuffs. The following samples were analyzed: Lysine.HCl, tryptophan, glycine, pulverized gelatin, blood meal, soybean meal, dried Leucaena leucocephala (a tree species of the genus Leucaena of the leguminous or Fabaceae family; cultivated for use as fodder), and pulverized whey. The following catalysts were used: Catalytic mixture A: 1 Tablet (5 g) Wieninger Catalyst: Na2 SO4 + CuSO4 + Se Sample amount: from 0.3 to 0.5 g.

6.5 Production of a Catalytic Mixture

111

Fig. 6.2 Acid-to-salt ratio versus nitrogen content with catalyst B

Sulfuric acid: 18 ml (acid/salt ratio = 3.6). Digestion time: 60 min. Catalytic mixture B: 7.35 g K2 SO4 + 0.05 g CuSO4 + 0.10 g TiO2 Sample amount: from 0.3 to 0.5 g. Sulfuric acid: 9 ml (acid/salt ratio = 1.2). Digestion time: 60 min. Catalytic mixture C: 7.35 g K2 SO4 + 0.15 g HgO Sample amount: from 0.3 to 0.5 g. Sulfuric acid: 15 ml (acid/salt ratio = 2.0). Digestion time: 60 min (after cooling, 0.2 g of Na2 S2 O3 is added to precipitate the mercury). Note: Results with catalytic mixture C (mercuric oxide catalyst) are used as reference or true values for the feeding stuff samples (Tables 6.4, 6.5, 6.6, 6.7, 6.8, and 6.9). The results are essentially the same for both catalytic mixtures, meaning that a TiO2 -based catalyst is an excellent replacement for a selenium-based catalyst.

6.5 Production of a Catalytic Mixture In the 1980s, at the Laboratory of Animal Nutrition, Faculty of Veterinary Medicine and Zootechnics (University of Antioquia, Colombia), a large number of products used for animal feed was required to be analyzed for total Kjeldahl nitrogen. These

112

6 Experimental: Evaluation of Titanium Dioxide as a Catalyst …

Table 6.4 Nitrogen content in amino acids using catalytic mixtures A and B L-Lysine.HCl

L-Tryptophan

Glycine

(N = 15.34%)

(N = 13.72%)

(N = 18.66%)

Catalytic mixture A

B

15.38

15.27

A

B

A

B

13.63

18.51

18.55

15.27

15.36

13.68

18.55

18.61

15.34

15.28

13.65

18.53

18.58

15.27

15.31

13.63

18.59

18.55

15.30

15.29

13.64

18.47

18.48

15.19

15.26

13.64

18.62

18.59

15.26

15.28

13.62

18.55

18.60

15.24

15.30

13.67

18.61

18.60

15.27

15.27

13.66

18.59

18.58

15.23

15.28

13.64

18.54

18.62

15.18

15.26

13.64

18.58

18.61

15.30

15.29

13.64

18.51

18.56

15.27a

15.29a

13.65a

18.55a

18.58a

a

Mean Note: Results with catalytic mixture A for L-Tryptophan were inconsistent, and therefore not considered Table 6.5 Nitrogen content in gelatin powder, blood meal, and soybean meal using catalyst mixtures A and B Blood meal

Gelatin powder

Soybean meal

Catalytic mixture A

B

A

B

A

B

14.85

14.95

12.16

12.30

7.49

7.59

14.85

14.96

12.14

12.32

7.50

7.60

14.89

14.96

12.13

12.30

7.54

7.63

14.86

14.95

12.15

12.30

7.49

7.58

14.93

14.92

12.18

12.27

7.55

7.58

14.81

14.97

12.11

12.26

7.54

7.58

14.90

14.98

12.18

12.27

7.51

7.57

14.87

14.94

12.17

12.27

7.51

7.60

14.82

14.99

12.16

12.27

7.48

7.56

14.82

14.98

12.14

12.30

7.52

7.63

14.84

14.99

12.11

12.28

7.50

7.62

14.88

15.00

12.11

12.25

7.53

7.56

14.86a

14.97a

12.15a

12.28a

7.51a

7.59a

a

Mean

6.5 Production of a Catalytic Mixture

113

Table 6.6 Nitrogen content in Leucaena leucocephala and whey powder using catalyst mixtures A and B Whey powder

Leucaena leucocephala Catalytic mixture A

B

A

B

4.07

4.14

1.93

1.97

4.06

4.17

1.93

1.96

4.07

4.15

1.92

1.96

4.07

4.16

1.91

1.96

4.06

4.12

1.93

1.96

4.07

4.12

1.92

1.96

4.06

4.17

1.92

1.96

4.06

4.11

1.91

1.93

4.05

4.15

1.90

1.96

4.07

4.12

1.91

1.95

4.08

4.17

1.91

1.95

4.03

4.14

1.92

1.95

4.06a

4.14a

1.92a

1.96a

a

Mean

Table 6.7 Nitrogen content in feeds using catalyst mixture C Gelatin powder

Blood meal

Soybean meal

Leucaena leucocephala

Whey powder

% Nitrogen 14.94

12.26

7.60

4.03

1.96

14.91

12.27

7.61

4.08

1.95

14.93a

12.27a

7.61a

4.06a

1.96a

a

Mean

Table 6.8 Statistical analysis catalyst A Lysine.HCl Trp

Gly

Gel. pwdr. Blood meal Soy. meal L. l .

Whey pwdr.

n

12

12

12

12

TVa

12

12

12

12

15.34

13.72 18.66 14.93

12.27

7.61

4.06

1.96

Mean 15.27

18.55 14.86

12.15

7.51

4.06

1.92

CVb

0.004

0.002 0.002

0.002

0.003

0.003 0.005

SDc

0.057

0.045 0.036

0.026

0.023

0.013 0.010

SEd

0.018

0.014 0.011

0.008

0.007

0.004 0.003

a True Value (theoretical nitrogen content for amino acids, and results with HgO for feed ingredients) b

Coefficient of Variation Standard Deviation d Standard Error c

114

6 Experimental: Evaluation of Titanium Dioxide as a Catalyst …

Table 6.9 Statistical analysis catalyst B Lysine.HCl Trp

Gly

Gel. pwdr. Blood meal Soy. meal L. l .

Whey pwdr.

n

12

12

12

12

TVa

12

12

12

12

15.34

13.72 18.66 14.93

12.27

7.61

4.06

1.96

Mean 15.29

13.65 18.58 14.97

12.28

7.59

4.14

1.96

CVb

0.002

0.001 0.002 0.002

0.002

0.003

0.005 0.005

SDc

0.027

0.017 0.039 0.024

0.021

0.025

0.022 0.010

SEd

0.009

0.005 0.012 0.007

0.007

0.008

0.007 0.003

a True Value (theoretical nitrogen content for amino acids, and results with HgO for feed ingredients) b

Coefficient of Variation Standard Deviation d Standard Error c

included processed products from fodder crops, feedstocks, silages, and manufactured compound feeds, consisting of various mixed feeds of vegetal and animal origin. Graduate theses involving feed nutritional evaluation usually required proximate analysis to be performed on numerous samples. Chemical reagents, imported from European and American companies, were high-priced. Given the fact that some of the reagents used in the method were just inorganic unsophisticated products (potassium sulfate, boric acid, titanium dioxide), it was then considered to investigate a way of producing some of these chemicals in the laboratory, not by a complex synthetic process but by way of recrystallization. Many chemicals are available inexpensively in impure forms, such as technical and laboratory grades. These grades are inadequately pure for analytical purposes, but some of them can be purified to the equivalent reagent grade, by means of crystallization and further recrystallization. In the particular case of the Kjeldahl method, the main requirement is to get a very low nitrogen content, even if other elements remain present at a bit higher level. The manufacturing process involved the purification of technical-grade chemicals [33]. The purity of these chemicals is usually over 98% (see Table 6.10), and they can be further purified by crystallization and recrystallization, reaching a higher purity. The main chemical grades used in analytical laboratories are 1. Reagent grade: it is generally equal to ACS grade (≥95%) and is acceptable for food, drug, or medicinal use and is suitable for use in many laboratory and analytical applications. Table 6.10 Purity of some commercial-grade chemicals

Product

% Purity

Potassium sulfate

98.0

Cupric sulfate pentahydrate

99.0

Boric acid

99.5

Titanium dioxide

99.0

6.5 Production of a Catalytic Mixture

115

2. Laboratory grade: it is the most popular grade for use in educational applications, but its exact levels of impurities are unknown. 3. Technical grade: it is used for commercial and industrial purposes; however, like many others, it is not pure enough to be offered for food, drug, or medicinal use of any kind. On some occasions, the in-house manufacture of some chemical reagents was implemented as an alternative to reduce expenses. The reagents produced were Potassium sulfate. Cupric sulfate. Titanium dioxide. Boric acid. After recrystallization of technical K2 SO4 , CuSO4 , H3 BO3 , and leaching of technical TiO2 , these chemicals will have an extremely low nitrogen content. Nevertheless, a procedural blank should always be run on the final catalytic mixture [34]. The catalytic mixture was produced by mixing three components as indicated by Williams [29]. The process of purification for each chemical is as follows: Potassium sulfate.

Molecular formula: K2SO4 Molecular weight: 174.26 g/mol Solubility in water: 0° C: 7.35 g/100 g water 100° C: 24.1 g/100 g water

Technical (fertilizer) grade potassium sulfate usually has between 75 and 98 percent K2 SO4 . As an example, a commercial brand picked at random has the following composition:

116

6 Experimental: Evaluation of Titanium Dioxide as a Catalyst …

Potassium sulfate – Technical grade Potassium oxide (equivalent):

K2O 53% min.

Sulfur:

S 18% min.

Chlorine:

Cl 0.05% max.

Insoluble matter:

0.1% max.

The amount of potassium is reported in terms of the K2 O equivalent. To convert to K2 SO4 , multiply by 1.85: K2 SO4 /K2 O = 174.259/94.196 = 1.85. % K2 SO4 = % K2 O × 1.85 = 53 × 1.85 = 98.05. Technical (fertilizer)-grade potassium sulfate is purified by crystallization and recrystallization. Dry crystals at 110° C. Test final K2 SO4 for nitrogen according to the ACS method [35] (Committee on Analytical Reagents 1968). The solubility of potassium sulfate in water is detailed in the IUPAC-NIST solubility data series [36]. For properties of potassium sulfate aqueous solution and crystals, see Ishii and Fujita [37]. Mullin et al. [38] studied the potassium sulfate precipitation from aqueous solution by salting out with acetone. Taboada et al. [39] studied the crystallization of potassium sulfate by cooling and salting out using 1-propanol. Li et al. [40] studied the purification process of agricultural K2 SO4 . Purification was achieved by dissolving agricultural potassium sulfate at a high temperature, filtering to remove the insoluble content, and then crystallizing at a low temperature. The purity of K2 SO4 was nearly 100%, with a yield of 36.28%. Cupric sulfate pentahydrate.

Molecular formula: CuSO4.5H2O Molecular weight: 249.685 g/mol Solubility in water: 0° C: 14.3 g/100 g water 100° C: 75.4 g/100 g water

6.5 Production of a Catalytic Mixture

117

Commercial- and technical-grade copper sulfate are used in fungicide and dyes industries, and also in the cattle and poultry feed, agricultural, electroplating, and fertilizer industries. As an example, a commercial brand picked at random has the following composition:

Copper sulfate pentahydrate – Technical grade Assay:

96% min.

Nitrate:

10 ppm max.

Technical (fertilizer)-grade cupric sulfate is purified by crystallization and recrystallization. CuSO4 .5H2 O can be precipitated by adding a small amount of ethanol [41]. An alternative process is the dissolution of high-purity copper (such as copper wires obtained from scrap cable) in H2 SO4 /H2 O2 . Different techniques for CuSO4 recrystallization from water are described by Armarego [42] and Geballe and Giauque [43]. Test final CuSO4 for nitrogen according to the ACS method [35] (Committee on Analytical Reagents 1968). Anhydrous copper(II) sulfate can be produced by dehydration of pentahydrate copper sulfate in a microwave oven. The anhydrous salt is produced by heating the hydrate to 150 °C. CuSO4 .5H2 O/CuSO4 = 249.685/159.60 = 1.56. Boric acid.

Molecular formula: H3BO3 Molecular weight: 61.83 g/mol Solubility in water: 0° C: 2.66 g/100 g water 100° C: 40.25 g/100 g water

Technical-grade boric acid is used as a pesticide, fungicide, herbicide, and antibacterial product. It can have up to 99.9% purity. Technical-grade boric acid is purified by crystallization and recrystallization. Recrystallization from water is described by Armarego [42].

118

6 Experimental: Evaluation of Titanium Dioxide as a Catalyst …

The solubility curve for boric acid was determined by Blasdale and Slansky [44]. Titanium dioxide. Molecular formula: TiO2 Molecular weight: 79.866 g/mol Solubility in water: Insoluble

Titanium dioxide is used as a white pigment in a variety of products such as sunscreens, cosmetics, paints, and foods. As a food additive (E171), it is often used to give a natural whiteness and opacity to foods. Technical-grade titanium dioxide was purified by leaching impurities with dilute sulfuric acid and further drying at 110° C. Procedure for Nitrogen Determination Nitrogen determinations were performed on different amino acids (reagent grade) and animal feeds. Catalytic mixture according to Williams (made in the laboratory from recrystallized CuSO4 and K2 SO4 , and purified TiO2 ): CuSO4 : 0.10 g. TiO2 : 0.10 g. K2 SO4 : 7.35 g. Titrations were performed with 3% boric acid solution, prepared from recrystallized commercial grade boric acid. Determination of Nitrogen in Amino Acids and Feeding Stuffs The following samples were analyzed: Lysine.HCl, tryptophan (Trp), glycine (Gly), gelatin powder (Gel.), blood meal, soybean meal (Soy. meal), dried Leucaena leucocephala (L. leuco.), and whey powder (Whey). Sample amount: from 0.3 to 0.5 g. Digestion time: 60 min. Reagents: Catalytic mixture: 7.35 g K2 SO4 + 0.16 g CuSO4 .5H2 O + 0.10 g TiO2 . (Factor to convert anhydrous cupric sulfate to pentahydrate cupric sulfate: CuSO4 .5H2 O/CuSO4 = 249.685/159.60 = 1.56). 98% sulfuric acid: 9 ml. Boric acid: 3% solution. Results are shown in Table 6.11, and statistical analysis in Table 6.12. The results obtained were excellent, with higher than 99% accuracy in both amino acids and animal feeds.

References

119

Table 6.11 Nitrogen content in amino acids and feed ingredients using catalyst mixture made in the laboratory Lysine.HCl Trp

Gly

Gelatin powder Blood meal Soy. meal L. Whey powder leuco.

15.30

13.62 18.66 14.93

12.21

7.61

4.06

1.96

15.27

13.59 18.60 14.89

12.13

7.48

4.00

2.00

15.32

13.58 18.59 14.83

12.20

7.50

3.95

1.85

15.26

13.65 18.56 14.88

12.19

7.55

3.96

1.79

15.32

13.62 18.62 14.92

12.18

7.49

4.00

1.82

15.24

13.67 18.62 14.90

12.07

7.51

3.95

1.85

15.28

13.64 18.58 14.88

12.11

7.40

3.85

1.90

15.31

13.56 18.42 14.80

12.18

7.74

3.86

1.80

15.20

13.68 18.66 14.92

12.17

7.50

3.78

1.95

15.25

13.65 18.59 14.85

12.14

7.52

4.08

1.96

15.28a

13.63a

12.16a

7.53a

3.95a

1.89a

a

18.59a

14.88a

Mean

Table 6.12 Statistical analysis Lysine.HCl

Trp

Gly

Gel.

Blood meal

Soy. meal

L. leuco.

Whey

n

10

10

10

10

10

10

10

10

TVa

15.34

13.72

18.66

14.93

12.27

7.61

4.06

1.96

Mean

15.28

13.63

18.59

14.88

12.16

7.53

3.95

1.89

CVb

0.003

0.003

0.004

0.003

0.004

0.012

0.024

0.040

SDc

0.039

0.039

0.068

0.042

0.044

0.091

0.095

0.076

SEd

0.012

0.012

0.021

0.013

0.014

0.029

0.030

0.024

a True Value (theoretical nitrogen content for amino acids, and results with HgO for feed ingredients) b

Coefficient of Variation Standard Deviation d Standard Error c

References 1. Fukumoto, H. E. and Chang, G.W. (1982) Manual salicylate-hypochlorite procedure for determination of ammonia in Kjeldahl digests, Journal of AOAC International, 65(5):1076–1079. 2. Novamsky, I. et al. (1974) Total nitrogen determination in plant material by means of the indophenol-blue method, Netherlands Journal of Agricultural Science, 22(1):3–5. 3. Searle, P.L. (1984) The Berthelot or Indophenol reaction and its use in the analytical chemistry of nitrogen. A Review, Analyst, 109(5):549-568. 4. Siemer, D.D. (1986) Use of solid boric acid as an ammonia absorbent in the determination of nitrogen, Analyst, 111(9):1013-1015. 5. Wilfarth, H. (1885) Eine Modifikation der Kjeldahl’schen Stickstoffbestimmungs Methode (A modification of the Kjeldahl method for nitrogen determination), Chemisches Zentral-Blatt, 16:17–19 and 113–115.

120

6 Experimental: Evaluation of Titanium Dioxide as a Catalyst …

6. Gunning, J.W. (1889) Ueber eine modification der Kjeldahl-Methode, Fresenius’ Zeitschrift für analytische Chemie, 28(1):188–191. 7. Kirk, P.L. (1950) Kjeldahl method for total nitrogen, Analytical Chemistry, 22(2):354–358. 8. Florence, E. and Milner, D.F. (1979) Routine determination of nitrogen by Kjeldahl digestion without use of catalyst, Analyst, 104:378-381. 9. Hach, C.C., Brayton, S.V. and Kopelove, A.B. (1985) A powerful Kjeldahl nitrogen method using peroxymonosulfuric acid, Journal of Agricultural and Food Chemistry, 33(6):1117–1123. 10. Singh, U. et al (1984) The use of hydrogen peroxide for the digestion and determination of total nitrogen in Chickpea (Cicer arietinum L.) and pigeonpea (Cajanus cajan L.) Journal of the Science of Food and Agriculture, 35(6):640–646. 11. Kunzler, J. E. (1953) Absolute sulfuric acid, highly accurate primary standard: constant boiling sulfuric acid and other reference standards, Analytical Chemistry, 25(1):93–103. 12. Lake, K. L. et al (1951) Effects of digestion temperature on Kjeldahl analyses, Analytical Chemistry, 23(11):1634–1638. 13. Bradstreet, R. B. (1957) Acid requirements of Kjeldahl digestion, Analytical Chemistry, 29(6):944–947. 14. Rexroad, P. R. and Cathey, R. D. (1976) Pollution-reduced Kjeldahl method for crude protein, J Assoc Off Anal Chem 59(6):1213–1217. 15. Wren, C. D. (1986) A review of metal accumulation and toxicity in wild mammals: I. Mercury, Environmental Research, 40(1):210–244, 16. Lauro, M. F. (1931) Use of selenium as catalyst in determination of nitrogen by Kjeldahl method, Industrial and Engineering Chemistry, Analytical Edition, 3(4):401-402. 17. Patel, S. M. and Sreenivasan, A. (1948) Selenium as catalyst in Kjeldahl digestions, Analytical Chemistry, 20(1):63–65. 18. Olson, O. E. (1986) Selenium toxicity in animals with emphasis on man, J of the American College of Toxicology, 5(1):45–70. 19. Kollonitsch, V. and Kline, C. H (1963) Catalytic activity of selenium, Industrial & Engineering Chemistry, 55 (12):18–26. 20. Lennox, L. J. and Flanagan, M. J. (1982) An automated procedure for the determination of total Kjeldahl nitrogen, Water Research, 16(7):1127–1133. 21. Osborn, R. A. and Wilkie, J. B. (1935) A study of the Kjeldahl method. IV. Metallic catalysts and metallic interferences, Journal of the Association of Official Agricultural Chemists, 18(4):604– 609. 22. Bradstreet, R. B. (1949) Comparison of tellurium and selenium as catalysts for Kjeldahl digestion, Analytical Chemistry 21(8):1012–1013. 23. Liao, C. F. H. (1982) Tellurium as catalyst in semimicro Kjeldahl method for total nitrogen determination, J Assoc Off Anal Chem, 65(4):786–790. 24. Glowa, W. (1974) Zirconium dioxide, a new catalyst in the Kjeldahl method for total nitrogen determination, J Assoc Off Anal Chem, 57(5):1228–1230. 25. Bjarnø, Ole-Christian (1980) Kjel-Foss automatic analysis using an antimony-based catalyst: collaborative study, J Assoc Off Anal Chem, 63(3):657–663. 26. Odland, R. (1972) Revised Kjeldahl total nitrogen method for feeds and premixes, J Assoc Off Anal Chem 55(5):984–985. 27. Rexroad, P. R. (1973) Nitrogen in feed and fertilizer, J Assoc Off Anal Chem 56(4):862–863. 28. Kane P. F. (1984) Comparison of HgO and CuSO4 as digestion catalysts in manual Kjeldahl determination of crude protein in animal feeds: collaborative study, J Assoc Off Anal Chem 67(5):869–77. 29. Williams, P. C. (1973) The use of titanium dioxide as a catalyst for large-scale Kjeldahl determination of the total nitrogen content of cereal grains, Journal of the Science of Food and Agriculture, 24(3):343–348. 30. Klopper, W. J. (1976) Use of titanium dioxide as a catalyst in the Kjeldahl determination of the total nitrogen content of barley, malt and beer, J Inst Brew, 82:353–354. 31. Kane P. F. (1987) Comparison of HgO and CuS04/Ti02 as catalysts in manual Kjeldahl digestion for determination of crude protein in animal feed: collaborative study, J Assoc Off Anal Chem 70(5):907–911.

References

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32. Laredo, M. A. and Torres, S. H. (1979) Efecto del catalizador en la determinación de nitrógeno en material vegetal, Revista ICA, 14(4):215–220. 33. Schieving, A. (2017) Understanding Chemical Reagents, Lab Manager, 12(10):22-23. 34. Eurachem. (2019) Blanks in Method Validation. Supplement to Eurachem guide. The Fitness for purpose of analytical methods. 35. Committee on Analytical Reagents (Editors) (1968) Reagent Chemicals. American Chemical Society Specifications, American Chemical Society, Washington 36. Eysseltováa, J. and Bouaziz, R. (2012) IUPAC-NIST solubility data series. 93. Potassium sulfate in water, Journal of Physical and Chemical Reference Data, 41(1):1–48. 37. Ishii, T. and Fujita, S. (1978) Properties of potassium sulfate aqueous solution and crystals, Journal of Chemical and Engineering Data, 23(1):19-23. 38. Mullin, J. W. et al (1989) Potassium sulfate precipitation from aqueous solution by salting-out with acetone, Chemical Engineering and Processing: Process Intensification, 26(2):93-99. 39. Taboada, M. E. et al (2003) Crystallization of potassium sulfate by cooling and salting-out using 1-propanol in a calorimetric reactor, Crystal Research and Technology, 38(1):21-29. 40. Li, Shoujiang et al (2019) Purification and rapid dissolution of potassium sulfate in aqueous solutions, RSC Advances, 9:2156-2161. 41. Ayala, J. and Fernández, B. (2014) Synthesis of commercial products from copper wire-drawing waste, The journal of the Minerals, Metals and Materials Society, 66(6):1099-1105. 42. Armarego, W. L. F. (2017) Purification of Laboratory Chemicals, 8th edition, ButterworthHeinemann, Amsterdam. 43. Geballe, T. H. and Giauque, W. F. (1952) The heat capacity and magnetic properties of single crystal copper sulfate pentahydrate from 0.25 to 4°K, Journal of the American Chemical Society, 74(14):3513–3519. 44. Blasdale, Walter C. and Slansky, Cyril M. (1939) The solubility curves of boric acid and the borates of sodium, Journal of the American Chemical Society, 61(4):917-920.

Chapter 7

Important Topics Related to the Kjeldahl Method

7.1 The Carlsberg Foundation and the Carlsberg Research Laboratory The Carlsberg Research Laboratory was founded by the brewer J. C. Jacobsen in 1875. “The principal task of The Carlsberg Laboratory shall be to develop as complete a scientific basis as possible for malting, brewing and fermenting operations.” (J.C. Jacobsen).1 Johan Kjeldahl was appointed head of the Chemical Department in 1876. He held this position until his death in 1900. Søren Peter Lauritz Sørensen was Director of the Carlsberg Laboratory’s Department of Chemistry from 1901 to 1938. Sørensen established research in proteins and proteolytic enzymes as the main activities of the laboratory. In 1909, he developed the pH scale and demonstrated the significance of pH for biochemical reactions, including those involved in brewing [1]: “With the invention of the pH scale, Carlsberg could ensure high quality of every beer. The applications of the pH scale have since been countless throughout all fields”.2

It is worth mentioning that Sørensen (1905) published two articles about the Kjeldahl method in the journal Comptes Rendus des Travaux du Laboratoire Carlsberg (Reports of the Works of the Carlsberg Laboratory): – On the Kjeldahl method for the determination of nitrogen [2]. – Can the nitrogen content of lysine and similar compounds be determined by the Kjeldahl method? [3] “In 1883, Emil Christian Hansen, head of the Physiology Department of the Carlsberg Research Laboratory, made a ground-breaking discovery that would revolutionize the brewing industry.”

1 2

https://www.carlsberggroup.com/who-we-are/carlsberg-research-laboratory/. https://www.carlsberggroup.com/pursuit-of-better/scientific-discoveries/ph-scale/.

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 J. Aguirre, The Kjeldahl Method: 140 Years, https://doi.org/10.1007/978-3-031-31458-2_7

123

124

7 Important Topics Related to the Kjeldahl Method

“Hansen’s method on how to purify yeast made it possible to make quality beer from every brew and the new Saccharomyces Carlsbergensis was used for the first time, and with great success, on a production scale in November 1883”.3

The Carlsberg Foundation was established in 1876 to support science. One of his main objectives is to support basic scientific research within the natural sciences, social sciences, and humanities.

7.2 Nitrogen-to-Protein Conversion Factors The protein content in foods is estimated by multiplying the nitrogen content by a nitrogen-to-protein conversion factor (NPCF). According to Salo-Väänänen and Koivistoinen [4], it was Wilhelm Henneberg who in 1865 asserted that the protein content of a natural product in general could be calculated by multiplying the total nitrogen content by a conversion factor of 6.25. Henneberg was the director of the former Agricultural Experimental Station Goettingen, in Weende (now the Institute for Animal Nutrition and Animal Physiology in Goettingen). At the Weende Agricultural Station, together with Friedrich Stohmann, he developed a proximate system for routine analysis of animal feed that is now referred to as the Weende analysis (proximate analysis). In the 12th Annual Report of the Storrs Agricultural Experiment Station (1899), it is stated: “It has for a considerable time been the general custom to assume an average of 16 per cent of nitrogen in protein, including both proteids and non-proteids. In other words, the total nitrogen has been multiplied by the factor 6.25 to obtain the amount of protein in the given material. The proteids of muscular tissue, such as are found in ordinary meats, appear to contain, in general, about 16 per cent and the non-proteids of such tissue a somewhat larger proportion of nitrogen. However, neither the amount of these non-proteids nor their proportion of nitrogen seems to be sufficient to cause any large error in the use of the factor 6.25 for estimating the total protein” [5].

Jones (1941) presented some new conversion factors: “The new protein conversion factors presented in this circular are based upon the most reliable information available regarding the nature and composition of the proteins in the materials concerned. Although it is realized that their use will not give values which will express the quantity of protein in the different food materials with absolute accuracy, it is believed that they will give values representing the real protein content more closely than those obtained by the indiscriminate application of the factor 6.25, now in general use. How these factors are to be applied must be left to the discretion of those who wish to use them in their own particular fields” [6]. Jones included a list of different proteins and the percentages of nitrogen (see Table 7.1). 3

https://www.carlsberggroup.com/pursuit-of-better/scientific-discoveries/purifying-yeast/.

7.2 Nitrogen-to-Protein Conversion Factors Table 7.1 Nitrogen content of various proteins [6]

125

Source

Protein

Percent nitrogen

Almond

Amandin

19.3

Barley

Hordein

17.2

Corn

Cottonseed Eggs

Milk (cow’s) Oat

Pea

Peanut Rye

Soybean

Globulin

18.1

Alpha Glutelin

16.2

Albumin

16.6

Zein

16.1

Glutelin

16.1

Globulin

18.0

Alpha Globulin

18.2

Beta Globulin

17.8

Ovoalbumin

15.5

Conalbumin

16.1

Vitellin

16.3

Livetin

15.1

Casein

15.9

Lactalbumin

15.4

Prolamin

16.4

Glutelin

17.5

Globulin

17.9

Legumin

18.0

Legumelin

16.3

Vicilin

17.4

Arachin

18.3

Conarachin

18.2

Gliadin

17.7

Glutelin

16.7

Albumin

16.7

Glycinin

17.5

Legumelin

16.1

Sorghum

Prolamin

14.3

Wheat endosperm

Gliadin

17.6

Wheat germ

Glutenin

17.5

Alpha glutelin

17.1

Beta glutelin

16.1

Leucosin

16.8

Globulin

18.3

Proteose

17.0

126

7 Important Topics Related to the Kjeldahl Method

Jones presented the nitrogen-to-protein conversion factors for “some food substances concerning the proteins of which, it is believed, there is sufficient knowledge to justify the use of special factors for converting the percentages of nitrogen into that of protein.” (see Table 7.2). With improved analytical technologies, it has been found that some of the factors published by Jones and others were not accurate. Feed matrices contain other nitrogenous compounds that are not proteins. These non-protein nitrogenous compounds include nucleic acids, amines, urea, biuret, ammonia, nitrates, nitrites, vitamins, alkaloids, phospholipids, and nitrogenous glycosides. Some of these compounds can be present in a relatively significant amount. Nitrogen-to-protein conversion factors based on amino acid composition are recommended whenever a more accurate approximation of the protein content of individual food is required. Tkachuk [7] calculated the conversion factors on cereals and oilseed meals from quantitative amino acid data. He obtained values varying from 5.4 to 5.8, which indicated that the common factors 5.7 and 6.25 can overestimate the protein content of these foodstuffs. Heidelbaugh et al. [8] compared three methods for calculating the protein content of 68 foods: 1. Multiplication of Kjeldahl nitrogen by 6.25. 2. Multiplication of Kjeldahl nitrogen by factors varying from 5.30 to 6.38 depending on food type. 3. Summation of amino acid content as determined by chemical analyses. They calculated new conversion factors based on amino acid analyses. Substantial differences were found in protein content for many foods depending on the calculation. Sosulski and Holt [9] studied the amino acid composition of a wide range of grain legumes and used the data to calculate nitrogen-to-protein factors. They concluded that the factor of 6.25 commonly used for legumes is not a realistic factor for estimating protein and should be reduced. If a universal factor for all plant proteins is preferred, then 5.7 could be the best choice. Morr [10] determined the variations among nitrogen conversion factors of different soy protein products, namely soy flour/meal and laboratory and commercial soy protein isolates. Computed nitrogen conversion factors were 5.64–6.08 (Kjeldahl method) and 6.70–6.84 (factor method-based on amino acid composition). He endorsed the factor method for providing the most accurate conversion. In 1982, he recalculated the factors for a variety of soy protein isolate products [11]. Boisen et al. [12] determined total nitrogen, amino acid nitrogen, and true protein in seven common feedstuffs: skim milk powder, barley, grass meal, soybean meal, fish meal, peas, and meat and bone meal. They found that non-protein nitrogen could make up as much as 20% of total nitrogen, and therefore the calculation of protein from total nitrogen by conversion factors overestimates the protein content in diets.

7.2 Nitrogen-to-Protein Conversion Factors Table 7.2 Factors suggested by Jones [6]

Substance

127

Factor suggested

Cereal grains Wheat, endosperm

5.70

Wheat, embryo

5.80

Wheat, bran

6.31

Wheat, whole kernel

5.83

Rye

5.83

Barley

5.83

Oats

5.83

Rice

5.95

Corn (maize)

6.25

Oilseeds and nuts Hempseed

5.30

Cottonseed

5.30

Sunflower seed

5.30

Flaxseed

5.30

Squash seed

5.30

Pumpkin seed

5.30

Sesame seed

5.30

Cantaloupe seed

5.30

Almonds

5.18

Coconut

5.30

Brazil nut

5.46

Hazelnut

5.30

Walnut

5.30

Peanut

5.46

Soybean

5.71

Butternut

5.30

Castor bean

5.30

Substances of animal origin Milk

6.38

Eggs

6.25

Meats

6.25

Gelatine

5.55

Leguminous seeds Navy bean

6.25

Lima bean

6.25

Mung bean

6.25 (continued)

128 Table 7.2 (continued)

7 Important Topics Related to the Kjeldahl Method

Substance

Factor suggested

Velvet bean

6.25

Aduski bean

6.25

Jack bean

6.25

Mossé [13] showed that, in the absence of perfectly accurate values of the conversion factor, it is still possible to correctly determine its upper and lower limits, which are often close to each other. Sosulski and Imafidon [14] recommended a factor of 5.70 to be used in all mixed or blended foods or diets where accurate protein values are required for nutritional applications. Salo-Väänänen and Koivistoinen [4] 1. Determined the true protein values as sums of amino acid residues and compared them with crude protein values. 2. Determined the nitrogen-to-true protein conversion factors by means of linear regression analyses between the true protein values and nitrogen content. They concluded that the conversion factor of 6.25 overestimates the true protein content of foods to the extent that its acceptability as a method for determining protein content is questionable. The International Dairy Federation [15] published a comprehensive review of scientific literature on nitrogen-to-protein conversion factors. It comprises three tables, which provide an extensive listing of literature values of nitrogen-to-protein conversion factors for three different groups: 1. Vegetable and nut protein sources, with values ranging from 5.38 to 5.8. 2. Dairy protein sources, showing a prevalence of 6.38 for milk and most milk and dairy products. 3. Dairy protein components, with values for the different milk proteins ranging from 6.29 to 6.38. Mariotti et al. [16] recommended that a set of data (a summary of the average factors for the main classes of dietary protein foodstuffs) “should be used when the aim is to specifically express nitrogen in terms of protein.” A summary of the average factors for the main classes of dietary protein foodstuffs is given. An extensive review of the literature on dairy proteins and soy proteins in infant foods NPCF was performed by Maubois and Lorient [17]. They concluded that “considering the values of the conversion factors for milk proteins (6.38) and for soy proteins (5.71), it is scientifically justified that this difference is kept.” Krul [18] reviewed the NPCF with a focus on soy protein. She suggested setting up amino acid analyses in foods intended as single sources in vulnerable populations “where more accurate measurement and reducing any risk of adulteration with nonprotein nitrogen are essential.”

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129

A systematic review on nitrogen-to-protein conversion factors for dairy and soy protein-based foods has been published by the Joint Expert Meetings on Nutrition of the World Health Organization and the Food and Agriculture Organization of the United Nations [19, 20]: “Nitrogen to protein conversion factors (NPCFs) allow for the estimation of protein content in food samples from the amount of nitrogen in the sample, based on two assumptions: that most of the nitrogen is associated with amino acids and that most of the amino acids in foods are associated with protein. The accuracy of the estimation depends on the value of the conversion factor. A value of 6.25 is applied for measuring protein content in most foods and food ingredients, again based on two assumptions: that proteins contain about 16% nitrogen by weight (i.e. of the total mass of a protein, 16% is nitrogen), and that all nitrogen in food is derived from protein. However, using the same conversion factor for all protein sources can introduce errors that lead to significant overestimation or underestimation of the actual protein content of most foods. Hence, a default value of 6.25 may not be an appropriate conversion factor for all protein sources; instead, specific values should be considered for different foods and food ingredients.” “Across Codex standards, there is no single universally agreed NPCF for dairy and soy. The Codex Committee on Nutrition and Foods for Special Dietary Uses (CCNFSDU) has recently raised the question of the appropriate NPCFs to be used for milk and soy protein in infant formula and follow-up formula. To this end, CCNFSDU sought advice from the Joint Food and Agriculture Organization of the United Nations (FAO)/WHO Expert Meetings on Nutrition (JEMNU)1—and requested that JEMNU be convened to review the evidence and develop evidence-informed guidance regarding NPCFs.” “This review was commissioned by FAO and WHO to serve as a background document for JEMNU. The objective was to assess known conversion factors for dairy-based and soy-based ingredients through a systematic review of the literature, including methods and approaches used for the determination of NPCFs, with the aim of supporting the selection of appropriate conversion factors for dairy-based and soy-based ingredients used in infant formula and follow-up formula [20].”

7.3 Reference Materials and Primary Standards Method Validation “Validation is the confirmation by examination and the provision of objective evidence that the particular requirements for a specific intended use are fulfilled” [21, 22]. The most common validation method for an analytical procedure is the collaborative study. In this method, an unknown material is analyzed by several laboratories or analysts [23]. Fisher and Gurnsey (1987) devised an alternative validation scheme for semiautomated determination of total nitrogen in plant material [24]. They used five methodologies: 1. 2. 3. 4.

Tests of robustness. Determination of fortified internal reference materials. Measurements of precision. Comparison with alternative assay procedures.

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5. Assay of analogous NBS reference materials. The results obtained by applying this scheme allow the analyst to obtain a performance profile for the assay of nitrogen by the Kjeldahl method in biological samples. Nelsen and Wehling (2008) examined in detail the use of collaborative studies for quantitative chemical analytical methods [25]. Reference materials are used for method validation and calibration. Reference Material ISO Guide 30 defines a reference material (RM) as a “material, sufficiently homogeneous and stable with respect to one or more specified properties, which has been established to be fit for its intended use in a measurement process” [26]. The Springer Handbook of Metrology defines a RM as a “material, sufficiently homogeneous and stable with regards to specified properties, which has been established to be fit for its intended use in measurement or in examination of nominal properties” [27]. The European co-operation for Accreditation (EA) published an informative guide on reference materials [28]. Certified Reference Material ISO Guide 30 defines a certified reference material (CRM) as a “reference material characterized by a metrologically valid procedure for one or more specified properties, accompanied by a reference material certificate that provides the value of the specified property, its associated uncertainty, and a statement of metrological traceability” [26]. The Springer Handbook of Metrology defines a CRM as “a reference material, accompanied by documentation issued by an authoritative body and providing one or more specified property values with associated uncertainties and traceabilities, using a valid procedure” [27]. The National Institute of Standards and Technology (NIST) provides a wide variety of standard reference materials (SRMs) for validating and calibrating analytical methods. Some examples are shown in Table 7.5. Primary Standard In analytical chemistry, a primary standard is “a highly purified compound that serves as a reference material in titrations and in other analytical methods. The accuracy of a method critically depends on the properties of the primary standard” [29]. A primary standard should satisfy the following conditions [30, 31]: 1. It must be a stable substance of definite composition. 2. It should have a high equivalent weight so that weighing errors may be insignificant. 3. It must be a substance that can be dried at 105°–110°. 4. It should be unaltered in air during weighing; this condition implies that it should not be hygroscopic, oxidized by air, or affected by carbon dioxide. It should neither be deliquescent nor efflorescent. 5. The substance should be readily soluble under the conditions in which it is employed. It should react stoichiometrically.

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131

The principles for manufacturing primary standards are summarized by Farr et al. [32]. Tris-(hydroxymethyl)-aminomethane (also known as Tris or THAM) was first recommended by Fossum et al. as an alternative general acidimetric primary standard [33]. Whitehead showed a Tris potentiometric titration curve to illustrate the pH range of the single stoichiometric point and compared it with the two stoichiometric points of sodium carbonate [34]. Rodkey (1964) suggested that Tris can be used to standardize the acid (HCl or H2 SO4 ) by direct titration, as well as a reference material for the Kjeldahl digestion [35]. He showed experimental data to demonstrate that a solution of Tris may be used as a standard to control the entire Kjeldahl procedure. It is worth to mention an article in which the authors (Koch et al. 1975) caution analysts to exercise “wariness if not downright skepticism toward statements on the labels of bottles of this material… Although the THAM ground and sifted through a 100-mesh sieve, and found to be 99.914% pure, would be satisfactory for standardization in most routine work, it could hardly be considered a primary standard” [36]. Baker and Chesterfield (1977) showed that THAM is very suitable as a complete standard for Kjeldahl analysis. It is interesting to note that they remark “…Its use as a nitrogen standard for the entire method including the digestion phase has not been described previously” [37]. Tellefson (1980) discussed the use of some potential nitrogen standards, such as ammonium oxalate, ammonium sulfate, ferrous ammonium sulfate, and ferrous ammonium sulfate hexahydrate, the latter being preferred because of its much higher equivalent weight, more freely flowing crystals, and better stability [38]. Chen et al. (1988) published an article describing the properties of three nitrogen compounds as primary standards for the Kjeldahl method, namely ammonium p-toluenesulfonic acid, glycine p-toluenesulfonic acid, and nicotinic acid p-toluenesulfonic acid [39]. These chemicals were developed by Hach®. The USP Council of Experts and the USP Reference Standards Committee issued an article dealing with the complexity of monographs and reference materials with a focus on qualifying one reference material relative to another [40] (Tables 7.3 and 7.4).

7.4 Food Standards and the Kjeldahl Method A “standard of identity” is an agreed upon legal definition for what food actually is. The United States Food and Drug Administration (FDA) began establishing Standards of Identity (SOI) in 1939. Standards of identity were introduced as a means of consumer protection. A food standards agency is a branch of government dedicated to ensuring the safety and minimum nutritional standards of food supplied within the country. The Kjeldahl

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Table 7.3 Some NIST Standard Reference Materials (SRM) NIST SRM

Description

Percent Protein Certified Value

Reference

1546a

Meat homogenate

15.68 ± 0.18

[41]

1549a

Whole Milk Powder

25.64 ± 0.31

[42]

1566b

Oyster Tissue

42.6 ± 1.3

[43]

1845a

Whole Egg Powder

43.32 ± 0.47

[44]

1849a

Infant/Adult Nutritional Formula I (milk-based)

13.225 ± 0.056

[45]

1869

Infant/Adult Nutritional Formula II (milk/whey/soy-based)

14.498 ± 0.83

[46]

2384

Baking Chocolate

13.18 ± 0.46

[47]

2387

Peanut Butter

22.2 ± 0.5

[48]

3233

Fortified Breakfast Cereal

7.25 ± 0.18

[49]

3234

Soy Flour

53.37 ± 0.36

[50]

3252

Protein Drink Mix

66.92 ± 0.61

[51]

method is included in the most acknowledged food standards. Some examples of those standards including the Kjeldahl method are ISO—International Organization for Standardization ISO 3188:1978

Starches and derived products—Determination of nitrogen content by the Kjeldahl method—Titrimetric method

ISO 5378:1978

Starches and derived products—Determination of nitrogen content by the Kjeldahl method—Spectrophotometric method

ISO 5663:1984

Water quality—Determination of Kjeldahl nitrogen—Method after mineralization with selenium

ISO 5397:1984

Leather—Determination of nitrogen content and “hide substance”—Titrimetric method

ISO 11261:1995

Soil quality—Determination of total nitrogen—Modified Kjeldahl method

ISO 6851:2001

Photography—Processing waste—Determination of total amino nitrogen (microdiffusion Kjeldahl method)

ISO 5983–1:2005

Animal feeding stuffs—Determination of nitrogen content and calculation of crude protein content—Part 1: Kjeldahl method

ISO 5983–2:2009

Animal feeding stuffs—Determination of nitrogen content and calculation of crude protein content—Part 2: Block digestion and steam distillation method

ISO 8988:2006

Plastics—Phenolic resins—Determination of hexamethylenetetramine content—Kjeldahl method, perchloric acid method, and hydrochloric acid method

ISO 1871:2009

Food and feed products—General guidelines for the determination of nitrogen by the Kjeldahl method (continued)

7.4 Food Standards and the Kjeldahl Method

133

(continued) ISO 20483:2013

Cereals and pulses—Determination of the nitrogen content and calculation of the crude protein content—Kjeldahl method

ISO 8968–1:2014

Milk and milk products—Determination of nitrogen content—Part 1: Kjeldahl principle and crude protein calculation

ISO 14244:2014

Oilseed meals—Determination of soluble proteins in potassium hydroxide solution

ISO 8968–4:2016

Milk and milk products—Determination of nitrogen content—Part 4: Determination of protein and non-protein nitrogen content and true protein content calculation (Reference method)

AOCS—American Oils Chemists’ Society AOCS Official Method Aa 5–91. Revised 2017 Nitrogen and Protein in Cottonseed and Cottonseed Meals, Modified Kjeldahl Method AOCS Official Method Ab 4–91. Reapproved 2017

Nitrogen and Protein in Peanuts, Modified Kjeldahl Method

AOCS Official Method Ac 4–91. Reapproved 2017

Nitrogen and Protein in Soybeans, Modified Kjeldahl Method

AOCS Official Method Ai 4–91. Revised 2017 Nitrogen and Protein in Sunflower Seed, Modified Kjeldahl Method AOCS Official Method Ba 4d-90. Revised 2022

Nitrogen and Protein Modified Kjeldahl Method

AOAC—Association of Official Analytical Chemists AOAC 928.08–1974

Nitrogen in meat—Kjeldahl method

AOAC 920.53–1978

Protein in beer—Kjeldahl method

AOAC 960.52–1961(2010)

Microchemical determination of nitrogen

AOAC 991.23–1994(1996)

Protein nitrogen content of milk

AOAC 988.05–1990(1996)

Protein (Crude) in animal feed and pet food—CuSO4 /TiO2 mixed catalyst Kjeldahl method

AOAC 984.13–1994(1996)

Protein (Crude) in animal feed and pet food—Copper catalyst Kjeldahl method

AOAC 954.01–1954(1996)

Protein (Crude) in animal feed and pet food—Kjeldahl method

AOAC 991.20–1994

Nitrogen (Total) in Milk—Kjeldahl Method

AOAC 981.10–1983

Crude protein in meat—Block digestion method

AOAC 955.04–1955(1997)

Nitrogen (Total) in fertilizers—Kjeldahl method

AOAC 959.04–1964

Nitrogen in tobacco—Kjeldahl method for products containing nitrates (continued)

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(continued) AOAC 978.04–1978

Nitrogen (Total) (Crude protein) in plants—Kjeldahl methods

AACC—American Association of Cereal Chemists 46–11.02

Crude Protein—Improved Kjeldahl Method, Copper Catalyst Modification

46–12.01

Crude Protein—Kjeldahl Method, Boric Acid Modification

46–13.01

Crude Protein—Micro-Kjeldahl Method

46–16.01

Crude Protein—Improved Kjeldahl Method, Copper–Titanium Dioxide Catalyst Modification

EPA—Environmental Protection Agency Method 351.1

Nitrogen, Kjeldahl, Total (Colorimetric, Automated Phenate) by Autoanalyzer

Method 351.2, Revision 2.0

Determination of Total Kjeldahl Nitrogen by Semi-Automated Colorimetry

7.5 Patents on Kjeldahl Equipment Hundreds of patents have been filed worldwide regarding the Kjeldahl method apparatus and/or variants. Some examples taken from the World Intellectual Property Organization (WIPO) are shown in Table 7.5.

7.6 Reviews of the Kjeldahl Method Numerous reviews of the Kjeldahl method have been written since its introduction. In 1891, Kebler published an article including an index to the literature on modifications of the procedure, and an index to publications on nitrogen determination by other methods. He stated: “In the history of analytical chemistry, no method has been so universally adopted, in so short a time, as the Kjeldahl Method for the estimation of nitrogen” [52].

7.6 Reviews of the Kjeldahl Method

135

Table 7.4 Nitrogen primary standards Standard

Molecular weight

(NH4 )2 Fe(SO4 )2 .6H2 O Ferrous ammonium sulfate hexahydrate

392.14

Percent nitrogen 7.14

165.19

8.48

284.05

9.86

121.136

11.56

115.025

12.18

204.229

13.72

182.65

15.34

(NH4 )2 SO4 Ammonium sulfate

132.14

21.20

(NH4 )2 C2 O4 Ammonium oxalate

124.096

22.57

L-Phenyl alanine (NH4 )2 Fe(SO4 )2 Ferrous ammonium sulfate

Tris-(hydroxymethyl)-Aminomethane (NH4 )(H2 PO4 ) Ammonium dihydrogen phosphate

L-Tryptophan

L-Lysine hydrochloride

In 1908, Hepburn [53] examined the most noteworthy articles written during the first twenty-five years of the Kjeldahl method (1883 to 1908). A broad experimental investigation taking into consideration various details of the method was carried out by Paul and Berry (1921). They studied the effect of the use of different types of apparatus, the proper method of applying heat during the digestion, and the details for the successful decomposition of cottonseed meal and

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Table 7.5 Some Patent Documents on Kjeldahl Equipment/Devices Patent

Priority date

Automatic sample injector of Kjeldahl apparatus

2022

Kjeldahl determination instrument for food protein content detection

2022

Full-automatic Kjeldahl determination device

2021

Novel automatic Kjeldahl type azotometer

2020

Sodium hydroxide reagent bottle provided with funnel and used for Kjeldahl apparatus

2020

Kjeldahl type nitrogen determination distillation device

2020

Novel automatic Kjeldahl apparatus

2020

Sample injection device for automatic Kjeldahl apparatus

2020

Kjeldahl nitrogen determination digestion device

2020

Full-automatic Kjeldahl apparatus for food detection

2020

Automatic Kjeldahl apparatus

2019

Kjeldahl nitrogen determination distillation reaction chamber device

2019

Kjeldahl apparatus

2018

Automatic sampler, sample injection method, and Kjeldahl apparatus detecting system

2017

Kjeldahl digests processing apparatus

2016

Kjeldahl electric heating jacket

2016

Steam generator device for Kjeldahl apparatus

2015

Kjeldahl flask rack

2014

Kjeldahl nitrogen determination and distillation device

2014

Semimicro-Kjeldahl determination device for protein determination

2014

Kjeldahl device for protein determination

2012

Novel Kjeldahl nitrogen determination flask

2011

Kjeldahl titrator

2010

Tube-form distiller for Kjeldahl-Gunning nitrogen determination apparatus

1990

Kjeldahl nitrogen determination device

1987

Microwave-based apparatus for the Kjeldahl method

1986

Analytical apparatus for serial determination of nitrogen in samples by the Kjeldahl method

1973

Apparatus for use in determination of nitrogen by the Kjeldahl method

1972

Apparatus for distillation with steam according to Kjeldahl

1969

other common nitrogenous substances, such as wheat flour, powdered milk, gelatin, and egg white [54]. Friedrich (1933) published a general review on the 50th anniversary of the Kjeldahl method: “This method, originally developed for the analytic work of the brewery chemist, has now been accepted in all branches of scientific and technical analysis, and thus became the most widespread determination all analytical methods” [55].

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137

Oesper (1934) also wrote a review to commemorate the first 50 years of the method [56]. Schuette and Oppen (1935) wrote a general review [57]. In 1940, Bradstreet published his first review of the method, analyzing the different inputs on every step: digestion, oxidizing agents, catalysts, distillation and determination of ammonia, and application of the method to refractory nitrogen compounds [58]. Vickery (1946) reviewed the modifications of the method, its range of applicability, and the development of apparatus during the early years [59]. Exceptionally informative is his description of the progress of research in both the method and the equipment in the United States, pursuits which started soon after the creation of the Association of Official Agricultural Chemists in 1884. Examinations of all the features of the method were done by Kirk in 1947 (a chapter in the book Advances in Protein Chemistry [60]), and 1950 (an article in Analytical Chemistry [61]). In 1954, Bradstreet published his second review of the method, covering the different papers published between 1939 and 1954 [62]. McKenzie and Wallace (1954) studied several variables of the method, including the effects of temperature, catalyst, and oxidizing agent on the digestion speed [63]. A survey of the application and history of the method was done by Neill [64]. Charlett (1965) did a brief review [65]. Jacobs (1965) briefly reviewed the determination of nitrogen in biological materials [66]. Bradstreet (1965) published his book The Kjeldahl Method for Organic Nitrogen, the most comprehensive work on the subject available today. Even after 58 years, most of the facts included in the book are still relevant and meaningful [67]. Jacobs (1978) examined the different methods applied to the determination of nitrogen in biological materials, fundamentally the Dumas and the Kjeldahl techniques [68]. On the centenary of the Kjeldahl method, Morries (1983) presented a review [69]. Two brief but significant reviews were published by D. Thornburn Burns [70], and W. I. Stephen [71]. Midkiff (1984) briefly examined the history of feed analysis, as chronicled in the development of AOAC official methods between 1884 and 1994, including an outstanding review of the Kjeldahl method [72]. A book on the Kjeldahl method was published by Jones in 1991 [73]. McKenzie (1994) published a second review of the method, emphasizing the new dimension given to the analysis by the development of microwave digestion [74]. On the 130th anniversary of the Kjeldahl method, a comprehensive review was presented by Sáez-Plaza et al. [75, 76]. Their work is the most complete survey of the method since that of Bradstreet in 1965. A brief review of the relevance of the Kjeldahl method in clinical laboratories was made by Chromýa et al. [77].

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7 Important Topics Related to the Kjeldahl Method

7.7 A Comparison of the Kjeldahl and Dumas Methods The Dumas method comprises three steps: 1. All forms of N are converted into gaseous nitrogen oxides NOx by combustion. 2. A heated reduction tube, filled with copper, is used to convert nitrogen oxides NOx to N2 . 3. N2 is measured by a thermal conductivity cell. Several studies have compared the effectiveness of the Dumas method with that of the Kjeldahl method for various food and feed products. The LECO FP-228 was compared with the AOAC copper catalyst Kjeldahl method for the determination of crude protein in feed materials [78]. A wide variety of feed materials of various nitrogen levels were analyzed. Results were precise, accurate, and rapid. The combined average recovery of nitrogen from tryptophan, lysine-HCl, and EDTA determined by the LECO combustion method was 99.94% compared to 99.88% determined by the AOAC Kjeldahl method. Nine laboratories participated in a collaborative study on the determination of crude protein in animal feeds to compare a combustion method based on the Dumas procedure with the AOAC mercury catalyst Kjeldahl method [79]. The generic combustion method was approved interim official first action. A collaborative study was done by the Analysis Committee of the Institute of Brewing to compare the Kjeldahl and Dumas method in barley, malt, and beer [80]. They found no significant difference between the precision data obtained for barley and malt by the Dumas method and the Kjeldahl procedure. The Committee approved the inclusion of the Dumas method for determining total nitrogen in barley and malt in the Recommended Methods of Analysis. Daun and DeClerq (1994) compared the two methods in oilseeds (canola seed, flaxseed, sunflower seed, mustard seed, and soybeans) at the Grain Research Laboratory, in Winnipeg (Canada). They found better results with the Dumas method, and, as a result, the method was adopted as official [81]. Simonne et al. (1997) analyzed a large number of samples (dairy products, oilseeds, chemicals, animal feed, infant formulas and baby foods, cereal, meats and meat products, vegetables and vegetable products, fish, and fruit by both the Kjeldahl and the Dumas methods [82]. They found that the Dumas method may replace the Kjeldahl method for the determination of nitrogen and crude protein in selected group foods. A comparison of nitrogen values obtained utilizing the Kjeldahl nitrogen and Dumas combustion methodologies on samples typical of an animal nutrition analytical laboratory (feed, excreta, carcass, egg yolk, milk, and urine) was carried out by Etheridge et al. (1998). They concluded that the Dumas combustion procedure for the measurement of nitrogen in the samples analyzed is reasonably accurate and precise [83]. Accordingly, the Dumas procedure is capable of replacing the Kjeldahl procedure for routine animal nutrition laboratory nitrogen analyses. Wiles et al. (1998) compared the Kjeldahl and Dumas methods in eleven laboratories analyzing eight dairy products and two pure reference compounds [84]. They

7.7 A Comparison of the Kjeldahl and Dumas Methods

139

found no evidence for a significant generic difference between the two methods. A summary of previous comparisons of the two methods is included in the article. The determination of the total soluble nitrogen content of malt and beer by the Dumas combustion method was collaboratively tested by the Analysis Committee of the Institute of Brewing and the European Brewery Convention (Johnson and Johansson 1999). They found the repeatability and reproducibility precision values to be acceptable, and approved the method to be included in Analytica EBC and the IOB Recommended Methods of Analysis [85]. Rhee (2001) affirmed that because the new instruments for the Dumas method can handle one gram quantities of dry or wet samples, it is rapidly becoming favored. The major benefits of this method are the very rapid turnaround time, and the elimination of contaminants associated with the use of catalysts [86]. A comparison of the Kjeldahl and Dumas methods for the determination of protein in foods was done by Thompson et al. (2002), using data accumulated over several years (taken from a proficiency testing scheme) on samples of milk, meat, fish, and cereal [87]. On average, the Dumas method provided results that were relatively higher than the Kjeldahl method (by about 1.4%). The difference between the methods depended on the type of foodstuff. Marcó et al. (2002) compared the features of the Dumas and Kjeldahl methods as they apply to total nitrogen determination in animal feed [88]. They found that both methods achieved similar repeatability and similar intra-laboratory reproducibility. A comparison of Kjeldahl and Dumas methods for determining protein contents of soybean products was done by Jung et al. (2003), using nine soybean products having protein contents ranging from 0.5 to 90%. The Kjeldahl method gave slightly, although significantly lower values than the Dumas method [89]. In 2015, a comparison of the Kjeldahl, Dumas, and NIR methods for total nitrogen determination in meat and meat products was done by Mihaljev et al. [90]. The precision rate for the Dumas method in all the analyzed samples was higher than for the Kjeldahl method. The Dumas method was at least as precise as the Kjeldahl method, but significantly faster. They concluded that the Dumas procedure can be replaced with the advantage of the Kjeldahl procedure in animal nutrition laboratory analysis. In 2022, a comparative study between four different total protein determination methods (Kjeldahl, Dumas, bicinchoninic acid assay, and Bradford) was carried out in milk from cows, goats, and sheep and their ultrafiltration products by Hueso et al. [91]. They found the Bradford assay to be the most suitable since it allows the obtention of fast and accurate results compared to the Kjeldahl reference method. The Dumas method also provided accurate results. It can be concluded from the above studies that the Dumas method has some advantages over the Kjeldahl method. But this was not always the case. Although the Dumas method is over fifty years older than Kjeldahl’s, it proved extremely difficult to come by with the proper apparatus for the Dumas method, whereas a lot of research on equipment began almost immediately after Kjeldahl published his procedure. Progress in equipment design for the Dumas method advanced rapidly after the invention of the combustion analysis crucible [92] and the combustion apparatus

140

7 Important Topics Related to the Kjeldahl Method

for analytical instruments [93]. After modern devices were developed, numerous laboratories evaluated the Dumas technique, and more research papers were published. Currently, some international food standards have the Dumas method as an official method for nitrogen analysis. ISO 16634–1:2008

Food products—Determination of the total nitrogen content by combustion according to the Dumas principle and calculation of the crude protein content—Part 1: Oilseeds and animal feeding stuff

ISO/TS 16634–2:2009

Food products—Determination of the total nitrogen content by combustion according to the Dumas principle and calculation of the crude protein content—Part 2: Cereals, pulses, and milled cereal products

ISO 14891:2002 IDF 185:2002

Milk and milk products—Determination of nitrogen content—Routine method using combustion according to the Dumas principle

AACC 46–30.01

Crude Protein—Combustion Method Nitrogen, freed by pyrolysis and subsequent combustion at high temperature in pure oxygen, is quantified by thermal conductivity detection. This generic combustion method is applicable to all flours, cereal grains, oilseeds, and animal feeds. Different conversion factors are used for various cereal grains and oilseeds

ICC 167

Determination of crude protein in grain and grain products for food and feed by the Dumas Combustion Principle

AOAC 990.03

Protein (crude) in animal feed, combustion method

AOAC 992.23–1992(1998)

Crude protein in cereal grains and oilseeds. Generic combustion method

AOAC 997.09–2008

Nitrogen in beer, wort, and brewing grains protein (total) by calculation—Combustion method

AOCS Ba 4e-93

Generic Combustion Method for Crude Protein

AOCS Ba 4f-00

Combustion Method for Crude Protein in Soybean Meal

7.8 Other Methods for Nitrogen Determination Developed in the 1900s The 1800s saw the creation of three important methods for nitrogen determination, namely Dumas (1831), Varrentrapp and Will (1841), and Kjeldahl (1883) methods. During the twentieth century, many modifications and improvements were made to these methods, mainly to the Dumas and the Kjeldahl techniques. But some other approaches to nitrogen determination are worth mentioning, which were developed in the 1900s. Meulen (1924) described a procedure in which the organic compound is heated at 350 °C in a current of hydrogen, and passes the resulting gases over a nickel catalyst [94]. Under these conditions, organic nitrogen is reduced to ammonia, which is

References

141

subsequently titrated. The method has been studied by different authors, namely Smith and West [95], Russell and Marks [96], and Hollowchak et al. [97]. Another method was developed by Ohashi in 1995: when an organic compound containing nitrogen is heated with iodic acid in strong phosphoric acid, carbon and hydrogen are oxidized, and nitrogen is reduced to its elemental form. The gases are led to an azotometer, which is filled with potassium hydroxide solution; the nitrogen can then be determined volumetrically. “This method is, so to speak, the wet Dumas method” [98]. But the classical Dumas and Kjeldahl methods are still the forerunners, bettered by the advancement in electronic technology and automated systems.

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