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Kjeldahl Knowledge Base Decode the Mysteries of the Nitrogen and Protein Determination According to Kjeldahl

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Kjeldahl Knowledge Base Decode the Mysteries of the Nitrogen and Protein Determination According to Kjeldahl Kjeldahl Knowledge Base Decode the mysteries of the nitrogen and protein determination according to Kjeldahl 1. Introduction 2. Procedure 3. Chemicals 4. Regulations Impressum Author Michaela Buffler Reviewer Andrea Mühleis, Melanie Hasler Publisher BÜCHI Labortechnik AG, CH-9230 Flawil, Switzerland Copyright © 2017 by BÜCHI Labortechnik AG, CH-9230 Flawil Order Code 11595478 Comprehensive guide covering all aspects and know-how of the theoretical and practical nitrogen and protein determination according to Kjeldahl. The Kjeldahl Guide helps you to understand the method and enables you to set up experiments correctly. All rights reserved. No part of this book may be reprinted, or reproduced, or utilized in any form or by any electronic or mechanical means, now known or hereafter invented, including photocopying and recording, or in any information storage and retrieval system, without permission in writing from the publisher. Foreword Over 50 years of experience developing Kjeldahl instrumentation and applications are summarized in this Knowledge Base. The aim of it is to share the BUCHI expertise in regards to the nitrogen and protein determination according to the Kjeldahl method. The deeper understanding of the procedure will help laboratory personnel, laboratory supervisors, students and teachers to optimize the individual method and increase the reliability of the results. It is our intention to revive the basic knowledge needed to understand the chemical and physical background associated with nitrogen determinations according to Kjeldahl and provide clear instructions in a wide area of Kjeldahl applications. The mayor theoretical part of the Kjeldahl Knowledge Base contains basic knowledge, consolidation of the Kjeldahl know-how followed by an extensive list of regulations and actual BUCHI Application Notes describing successful nitrogen determinations. With this Kjeldahl Knowledge Base BUCHI would like to live up to its company slogan «Quality in your hands» and support you in your daily work by not only providing high quality instrumentation but also offering comprehensible theoretical background information and showcase applications. Yours sincerely, Michaela Buffler Product Manager Kjeldahl Solutions BÜCHI Labortechnik AG Meierseggstr. 40, Postfach CH-9230 Flawil 1 Switzerland T +41 (0)71 394 63 63 F +41 (0)71 394 64 64 www.buchi.com 1 Introduction 2 1.1 History of the Kjeldahl method 4 1.2 Kjeldahl at BUCHI since 1961 8 1.3 Chemistry of protein 9 1.4 The Kjeldahl method 10 2 Procedure of the kjeldahl nitrogen and protein determination 12 2.1 Sample preparation 15 2.1.1 Amount of sample and sample tube size selection 16 2.1.2 Mincing and homogenization 20 2.1.3 Drying of samples 20 2.2 Digestion 21 2.2.1 Heating technologies 22 2.2.2 IR-digestion vs. Block-digestion 23 2.2.3 Suction manifold and scrubbing 26 2.2.4 Acid mixtures 27 2.2.5 Acid consumption during digestion 27 2.2.6 Catalyst used for digestion 28 2.2.7 Selecting the appropriate catalyst 30 2.2.8 Ideal ratio of salt to sulfuric acid 31 2.2.9 Digestion by hydrogen peroxide 32 2.2.10 Digestion parameters 32 2.2.11 Digestion temperature 34 2.3 Distillation 35 2.3.1 Dilution of the digestion solution 36 2.3.2 Alkalization of the digestion solution 36 2.3.3 Preparation of the receiving vessel for distillate collection 36 2.3.4 Steam distillation 36 2.3.5 Distillation parameters 37 2.4 Titration 39 2.4.1 Integrated vs. External titration 40 2.4.2 Potentiometric vs. Colorimetric titration 40 2.4.3 Boric acid vs. Back titration 42 2.5 Blanks 44 2.6 Calculation of the nitrogen content 46 2.6.1 Calculation for boric acid titration 47 2.6.2 Calculation for back titration 48 2.6.3 Calculation of the protein content 49 2.6.4 Protein factors according to AOAC / ISO / DIN 50 3 Chemicals for Kjeldahl 52 3.1 Kjeldahl Tablets 54 3.2 Boric acid 54 3.3 Indicators for boric acid titration 55 3.3.1 Sher mixed indicator for boric acid titration 56 3.3.2 Bromocresol green / methyl red mixed indicator for boric acid titration 56 4 Regulations 58 About BUCHI 68 Introduction BUCHI offers “Quality in your hands” since 1939 1. Introduction 2. Procedure 3. Chemicals 4. Regulations Kjeldahl Knowledge Base Introduction 1.1 History of the Kjeldahl method For more than 130 years the determination of nitrogen by means of the method developed by the Danish chemist Johan Gustav Christoffer Thorsager Kjeldahl (1849 – 1900) has been an internationally accepted standard. The method was introduced in 1883 at a meeting of the Danish Chemical Society by Johan Kjeldahl as a means to determine nitrogen in barley and yeast1. The method named after its inventor has since found wide-spread application in life science and chemistry and has extended its scope to the determination of nitrogen and proteins in dairy products, meat products, beer, cereals and other food materials. Kjeldahl was a member of the innovative laboratory team at the Carlsberg brewery in Copenhagen, also famous in microbiology for isolating the famous beer yeast Saccharomyces Carlsbergensis which is still used today. As the head of the chemistry department at the Carlsberg brewery he was involved in a very modern problem: quality management and optimization of productivity. Kjeldahl intended to determine the protein content of grain in order to find out how the protein content influences quality and quantity of the brewed beer. Johan Gustav Christoffer Thorsager Kjeldahl in his laboratory ∙ 1849: born in Jagerpris in Denmark ∙ 1867: graduated from university ∙ 1876: Scientist and head of lab at Carlsberg Breweries ∙ Fermentation studies ∙ Research on carbohydrate in barley and malt ∙ Developing of method for exact determination of nitrogen in animal and vegetable material ∙ 1883: published the “Kjeldahl method” for protein determination ∙ 1900: died in Tisvilde 1 J. Kjeldahl, Sur une nouvelle méthode de dosage de l’azote dans les substances organiques, Résumé du compte-rendu des travaux du Laboratoire de Carlsberg, Copenhague, en commission chez H. Hagerup, Imprimerie de Thiele 1983 4 Citation from Kjeldahl’s original publication of 1883 Original citation Translation «Le principe de la nouvelle méthode consiste donc «The principle of the new method is to heat the à chauffer pendant quelque temps la matière test material for some time with a large quantity of à analyser avec une forte proportion d’acide concentrated sulfuric acid at a temperature close sulfurique concentré jusqu’à une température to the acid’s boiling point and to oxidize the solution voisine du point d’ébullition de l’acide, et à thus obtained with an excess of dry potassium oxyder la dissolution ainsi obtenue avec un excès permanganate powder. Under these conditions d’hyperpermanganate de potasse sec en poudre. the nitrogen of the organic substances, … , is Dans ces conditions, l’azote des substances completely transformed into ammonium sulfate organiques, … , se transforme complètement en which, once the oxidation is completed and after sulfate d’ammoniaque, qui, l’oxydation une fois saturation with caustic soda can be distilled and terminée et après saturation avec la soude, peut determined by ordinary methods.» être distillé et dosé par les méthodes ordinaires.» Although individual chemicals used in the Kjeldahl method have changed over the years it is possible to give a concise general definition: The Kjeldahl Method consists in a procedure of catalytically supported mineralization of organic material in a boiling mixture of sulfuric acid and sulfate salts at boiling temperatures between 340 and 370 °C. In the digestion process the organically bonded nitrogen is converted into ammonium sulfate. Alkalizing the solution liberates ammonia which is quantitatively steam-distilled and determined by titration. Original Kjeldahl apparatus from 1883 5 Introduction BUCHI Kjeldahl evolution 1960 1970 1980 Steam B-322 + generator Vapodest Büchi 325 + 345 B-342 / B-343 B-321 Nitrogen Nitrogen determination determination apperatus apperatus Distillation B-320 B-315 IH 4.2 / IH 2.1 Büchi 425 / 430 Ventilator Digestion Scrubber B-412 Scrubbing 1990 2000 2010 AutoKjeldahl System KjelMaster K-375 B-339 K-370 / K-371 KjelSampler K-376 / K-377 B-323 B-324 KjelFlex K-360 Distillation Unit B-316 K-314 K-350 / K-355 Rapid Digester Digest System K-437 KjelDigester Büchi 420 Digest Automat K-438 K-446 / K-449 B-424 / B-435 + B-436 Digest System K-431 SpeedDigester K-424 / K-435 + B-436 Digest Automat K-432 K-425 / K-436 / K-439 Scrubber Scrubber B-414 K-415 Kjeldahl Knowledge Base Introduction 1.2 Kjeldahl at BUCHI since 1961 At the time when Johan Kjeldahl published his method for the determination of nitrogen in 1883 the electric lamp was just patented and the technical age in its childhood. Seldom in human history has an invention remained basically unchanged for such a long time as Kjeldahl’s method for nitrogen determination. As in 1883 the Kjeldahl nitrogen determination starts with sample preparation, proceeds to the digestion followed by separation using steam distillation and subsequent volumetric determination of the amount of ammonia formed in the process. Kjeldahl’s visionary idea of providing a simple method for nitrogen and protein determinations, which can also be carried out by non-academic lab personnel, has been put into practice by BUCHI’s Kjeldahl systems since 1961. With this guide BUCHI wants to support you in your daily work not only by providing high quality instrumentation but also offering theoretical background information for your daily routine. 1961: BUCHI Market Innovation For more efficient distillations Launch of the first automatic nitrogen distillation instrument with integrated steam generator. 2012: BUCHI Market Innovation For highest productivity Launch of the first nitrogen distillation instrument with integrated titration for potentiometric and colorimetric titration and sample changer for a fully automated process.
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