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) (ISO 8968-4:2016)

ISO 8968-4|IDF 20-4:2016 specifies a method for the direct and indirect determination of the protein nitrogen content of liquid, whole or skimmed milk.

Milch und Milcherzeugnisse - Bestimmung des Stickstoffgehaltes - Teil 4: Bestimmung des Proteinstickstoff-Gehaltes und des Nichtproteinstickstoff-Gehaltes und Berechnung des tatsächlichen Proteingehaltes (Referenzverfahren) (ISO 8968-4:2016)

Dieser Teil von ISO 8968|IDF 20 legt ein Verfahren zur direkten und indirekten Bestimmung des Proteinstickstoff-Gehaltes von flüssiger, unbehandelter oder entrahmter Milch fest.

Lait et produits laitiers - Détermination de la teneur en azote - Partie 4: Détermination de la teneur en azote protéique et non protéique et calcul de la teneur en protéines vraies (Méthode de référence) (ISO 8968-4:2016)

ISO 8968-4|FIL 20-4:2016 spécifie une méthode pour la détermination directe et indirecte de la teneur en azote protéique du lait liquide, entier ou écrémé.

Mleko in mlečni proizvodi - Določevanje vsebnosti dušika - 4. del: Določevanje vsebnosti beljakovinskega in nebeljakovinskega dušika ter izračun dejanske vsebnosti beljakovin (referenčna metoda) (ISO 8968-4:2016)

Ta del standarda ISO 8968|IDF 20 določa metodo za neposredno in posredno določevanje beljakovinskega dušika v tekočem, polnomastnem in posnetem mleku.

General Information

Status
Withdrawn
Publication Date
30-Jul-2016
Withdrawal Date
30-Oct-2016
Current Stage
6060 - Definitive text made available (DAV) - Publishing
Start Date
20-Apr-2016
Completion Date
20-Apr-2016

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SLOVENSKI STANDARD
SIST EN ISO 8968-4:2016
01-julij-2016
1DGRPHãþD
SIST EN ISO 8968-4:2002
SIST EN ISO 8968-4:2002/AC:2012
SIST EN ISO 8968-5:2002
0OHNRLQPOHþQLSURL]YRGL'RORþHYDQMHYVHEQRVWLGXãLNDGHO'RORþHYDQMH
YVHEQRVWLEHOMDNRYLQVNHJDLQQHEHOMDNRYLQVNHJDGXãLNDWHUL]UDþXQGHMDQVNH
YVHEQRVWLEHOMDNRYLQ UHIHUHQþQDPHWRGD  ,62

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) (ISO 8968-4:2016)

Milch und Milcherzeugnisse - Bestimmung des Stickstoffgehaltes - Teil 4: Bestimmung

des Proteinstickstoff-Gehaltes und des Nichtproteinstickstoff-Gehaltes und Berechnung

des tatsächlichen Proteingehaltes (Referenzverfahren) (ISO 8968-4:2016)

Lait et produits laitiers - Détermination de la teneur en azote - Partie 4: Détermination de

la teneur en azote protéique et non protéique et calcul de la teneur en protéines vraies

(Méthode de référence) (ISO 8968-4:2016)
Ta slovenski standard je istoveten z: EN ISO 8968-4:2016
ICS:
67.100.10 0OHNRLQSUHGHODQLPOHþQL Milk and processed milk
SURL]YRGL products
SIST EN ISO 8968-4:2016 en

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

---------------------- Page: 1 ----------------------
SIST EN ISO 8968-4:2016
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SIST EN ISO 8968-4:2016
EN ISO 8968-4
EUROPEAN STANDARD
NORME EUROPÉENNE
April 2016
EUROPÄISCHE NORM
ICS 67.100.10 Supersedes EN ISO 8968-4:2001, EN ISO 8968-5:2001
English Version
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) (ISO 8968-4:2016)

Lait et produits laitiers - Détermination de la teneur en Milch und Milcherzeugnisse - Bestimmung des

azote - Partie 4: Détermination de la teneur en azote Stickstoffgehaltes - Teil 4: Bestimmung des

protéique et non protéique et calcul de la teneur en Proteinstickstoff-Gehaltes und des

protéines vraies (Méthode de référence) (ISO 8968- Nichtproteinstickstoff-Gehaltes und Berechnung des

4:2016) tatsächlichen Proteingehaltes (Referenzverfahren)
(ISO 8968-4:2016)
This European Standard was approved by CEN on 19 March 2016.

CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this

European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references

concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN

member.

This European Standard exists in three official versions (English, French, German). A version in any other language made by

translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management

Centre has the same status as the official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,

Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,

Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and

United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels

© 2016 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 8968-4:2016 E

worldwide for CEN national Members.
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SIST EN ISO 8968-4:2016
EN ISO 8968-4:2016 (E)
Contents Page

European foreword ....................................................................................................................................................... 3

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SIST EN ISO 8968-4:2016
EN ISO 8968-4:2016 (E)
European foreword

This document (EN ISO 8968-4:2016) has been prepared by Technical Committee ISO/TC 34 "Food

products" in collaboration with Technical Committee CEN/TC 278 “Milk and milk products - Methods of

sampling and analysis” the secretariat of which is held by NEN.

This European Standard shall be given the status of a national standard, either by publication of an

identical text or by endorsement, at the latest by October 2016, and conflicting national standards shall

be withdrawn at the latest by October 2016.

Attention is drawn to the possibility that some of the elements of this document may be the subject of

patent rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent

rights.
This document supersedes EN ISO 8968-4:2001 and EN ISO 8968-5:2001.

According to the CEN-CENELEC Internal Regulations, the national standards organizations of the

following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria,

Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia,

France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta,

Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland,

Turkey and the United Kingdom.
Endorsement notice

The text of ISO 8968-4:2016 has been approved by CEN as EN ISO 8968-4:2016 without any

modification.
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SIST EN ISO 8968-4:2016
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SIST EN ISO 8968-4:2016
INTERNATIONAL ISO
STANDARD 8968-4
IDF
20-4
Second edition
2016-04-01
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)
Lait et produits laitiers — Détermination de la teneur en azote —
Partie 4: Détermination de la teneur en azote protéique et non
protéique et calcul de la teneur en protéines vraies (Méthode de
référence)
Reference numbers
ISO 8968-4:2016(E)
IDF 20-4:2016(E)
ISO and IDF 2016
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SIST EN ISO 8968-4:2016
ISO 8968-4:2016(E)
IDF 20-4:2016(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO and IDF 2016, Published in Switzerland

All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form

or by any means, electronic or mechanical, including photocopying, or posting on the internet or an intranet, without prior

written permission. Permission can be requested from either ISO at the address below or ISO’s member body in the country of

the requester.
ISO copyright office International Dairy Federation

Ch. de Blandonnet 8 • CP 401 Silver Building • Bd Auguste Reyers 70/B • B-1030 Brussels

CH-1214 Vernier, Geneva, Switzerland
Tel. +41 22 749 01 11 Tel. + 32 2 325 67 40
Fax +41 22 749 09 47 Fax + 32 2 325 67 41
copyright@iso.org info@fil-idf.org
www.iso.org www.fil-idf.org
ii © ISO and IDF 2016 – All rights reserved
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SIST EN ISO 8968-4:2016
ISO 8968-4:2016(E)
IDF 20-4:2016(E)
Contents Page

Forewords .......................................................................................................................................................................................................................................v

1 Scope ................................................................................................................................................................................................................................. 1

2 Normative references ...................................................................................................................................................................................... 1

3 Terms and definitions ..................................................................................................................................................................................... 1

4 Principle ........................................................................................................................................................................................................................ 1

4.1 Indirect protein nitrogen ............................................................................................................................................................... 1

4.2 Direct protein nitrogen .................................................................................................................................................................... 2

5 Reagents ........................................................................................................................................................................................................................ 2

5.1 General ........................................................................................................................................................................................................... 2

5.2 Trichloroacetic acid (CCl COOH) solution ...................................................................................................................... 2

5.3 Hydrochloric acid standard volumetric solution ....................................................................................................... 2

6 Apparatus ..................................................................................................................................................................................................................... 2

7 Sampling ........................................................................................................................................................................................................................ 3

8 Preparation of test sample ......................................................................................................................................................................... 3

9 Procedure — Direct protein nitrogen approach ................................................................................................................. 3

9.1 Test portion ................................................................................................................................................................................................ 3

9.2 Determination ......................................................................................................................................................................................... 3

9.2.1 Precipitation and filtration ..................................................................................................................................... 3

9.2.2 Preparation of the precipitate .............................................................................................................................. 4

9.2.3 Digestion and distillation ......................................................................................................................................... 4

9.2.4 Titration................................................................................................................................................................................... 4

9.3 Blank test ..................................................................................................................................................................................................... 4

9.4 Calculation and expression of results.................................................................................................................................. 4

9.4.1 Calculation of protein nitrogen content ....................................................................................................... 4

9.4.2 Expression of results .................................................................................................................................................... 5

9.4.3 Calculation of true protein content .................................................................................................................. 5

9.4.4 Expression of the true protein content results ...................................................................................... 5

10 Procedure — Indirect protein nitrogen approach ............................................................................................................ 6

10.1 Nitrogen determination .................................................................................................................................................................. 6

10.2 Non-protein nitrogen determination ......... .......................................................................................................................... 6

10.2.1 Test portion .......................................................................................................................................................................... 6

10.2.2 Precipitation and filtration ..................................................................................................................................... 6

10.2.3 Preparation of the filtrate......................................................................................................................................... 6

10.2.4 Digestion and distillation ......................................................................................................................................... 6

10.2.5 Titration................................................................................................................................................................................... 6

10.3 Blank test ..................................................................................................................................................................................................... 6

10.4 Calculation and expression of results.................................................................................................................................. 7

10.4.1 Calculation of non-protein nitrogen content ........................................................................................... 7

10.4.2 Calculation of protein nitrogen content ....................................................................................................... 7

10.4.3 Expression of results .................................................................................................................................................... 8

10.4.4 Calculation of true protein content .................................................................................................................. 8

10.4.5 Expression of the true protein content results ...................................................................................... 8

11 Precision ....................................................................................................................................................................................................................... 8

11.1 Interlaboratory test............................................................................................................................................................................. 8

11.2 Repeatability ............................................................................................................................................................................................. 8

11.3 Reproducibility ....................................................................................................................................................................................... 8

12 Test report ................................................................................................................................................................................................................... 9

Annex A (informative) Results of a collaborative study — Estimation of precision data of

direct and indirect methods for the true protein determination ...................................................................10

© ISO and IDF 2016 – All rights reserved iii
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SIST EN ISO 8968-4:2016
ISO 8968-4:2016(E)
IDF 20-4:2016(E)

Bibliography .............................................................................................................................................................................................................................11

iv © ISO and IDF 2016 – All rights reserved
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SIST EN ISO 8968-4:2016
ISO 8968-4:2016(E)
IDF 20-4:2016(E)
Forewords

ISO (the International Organization for Standardization) is a worldwide federation of national standards

bodies (ISO member bodies). The work of preparing International Standards is normally carried out

through ISO technical committees. Each member body interested in a subject for which a technical

committee has been established has the right to be represented on that committee. International

organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.

ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of

electrotechnical standardization.

The procedures used to develop this document and those intended for its further maintenance are

described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the

different types of ISO documents should be noted. This document was drafted in accordance with the

editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).

Attention is drawn to the possibility that some of the elements of this document may be the subject of

patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of

any patent rights identified during the development of the document will be in the Introduction and/or

on the ISO list of patent declarations received (see www.iso.org/patents).

Any trade name used in this document is information given for the convenience of users and does not

constitute an endorsement.

For an explanation on the meaning of ISO specific terms and expressions related to conformity

assessment, as well as information about ISO’s adherence to the WTO principles in the Technical

Barriers to Trade (TBT) see the following URL: Foreword - Supplementary information

The committee responsible for this document is ISO/TC 34, Food products, Subcommittee SC 5, Milk and

milk products and the International Dairy Federation (IDF). It is being published jointly by ISO and IDF.

This second edition of ISO 8968-4|IDF 20-4 cancels and replaces ISO 8968-4|IDF 20-4:2001 and

ISO 8968-5|IDF 20-5:2001, which have been technically revised.

ISO 8968|IDF 20 consists of the following parts, under the general title Milk and milk products —

Determination of nitrogen content:
— Part 1: Kjeldahl principle and crude protein calculation
— Part 3: Block-digestion method (Semi-micro rapid routine method)

— Part 4: Determination of protein and non-protein nitrogen content and true protein content calculation

(Reference method)

1) It is intended that upon revision, the main element of the title of ISO 8968-3|IDF 20-3 (i.e. “Milk”) will be aligned

with the main element of the titles of ISO 8968-1|IDF 20-1 and ISO 8968-4|IDF 20-4.

© ISO and IDF 2016 – All rights reserved v
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SIST EN ISO 8968-4:2016
ISO 8968-4:2016(E)
IDF 20-4:2016(E)

IDF (the International Dairy Federation) is a non-profit private sector organization representing the

interests of various stakeholders in dairying at the global level. IDF members are organized in National

Committees, which are national associations composed of representatives of dairy-related national

interest groups including dairy farmers, dairy processing industry, dairy suppliers, academics and

governments/food control authorities.

ISO and IDF collaborate closely on all matters of standardization relating to methods of analysis

and sampling for milk and milk products. Since 2001, ISO and IDF jointly publish their International

Standards using the logos and reference numbers of both organizations.

Attention is drawn to the possibility that some of the elements of this document may be the subject of

patent rights. IDF shall not be held responsible for identifying any or all such patent rights. Details of

any patent rights identified during the development of the document will be in the Introduction and/or

on the ISO list of patent declarations received (see www.iso.org/patents).

Any trade name used in this document is information given for the convenience of users and does not

constitute and endorsement

ISO 8968-4|IDF 20-4 was prepared by the IDF Standing Committee on Analytical methods for composition

and ISO Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products.

The work was carried out by the IDF/ISO Project Group (C13) of the Standing Committee on Analytical

methods for composition under the aegis of its project leaders, D. Barbano (US) and P. Trossat (FR).

This ISO|IDF International Standard cancels and replaces ISO 8968-4|IDF 20-4:2001 and

ISO 8968-5|IDF 20-5:2001, which have been technically revised.

ISO 8968|IDF 20 consists of the following parts, under the general title Milk and milk products —

Determination of nitrogen content:
— Part 1: Kjeldahl principle and crude protein calculation
— Part 3: Block-digestion method (Semi-micro rapid routine method)

— Part 4: Determination of protein and non-protein nitrogen content and true protein content calculation

(Reference method)
vi © ISO and IDF 2016 – All rights reserved
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SIST EN ISO 8968-4:2016
ISO 8968-4:2016(E)
INTERNATIONAL STANDARD
IDF 20-4:2016(E)
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)

WARNING — The use of this part of ISO 8968|IDF 20 can involve the use of hazardous materials,

operations and equipment. This part of ISO 8968|IDF 20 does not purport to address all the

safety risks associated with its use. It is the responsibility of the user of this part of ISO 8968|IDF

20 to establish appropriate safety and healthy practices and determine the applicability of local

regulatory limitations prior to use.
1 Scope

This part of ISO 8968|IDF 20 specifies a method for the direct and indirect determination of the protein

nitrogen content of liquid, whole or skimmed milk.
2 Normative references

The following documents, in whole or in part, are normatively referenced in this document and are

indispensable for its application. For dated references, only the edition cited applies. For undated

references, the latest edition of the referenced document (including any amendments) applies.

ISO 8968-1|IDF 20-1, Milk and milk products — Determination of nitrogen content — Part 1: Kjeldahl

principle and crude protein calculation
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
non-protein nitrogen content
NPN
mass fraction of substances determined by the specified procedure

Note 1 to entry: The non-protein nitrogen content is expressed as a percentage by mass.

3.2
protein nitrogen content

mass fraction of substances determined by the specified procedure, directly or, alternatively, indirectly

Note 1 to entry: The protein nitrogen content is expressed as a percentage by mass.

4 Principle
4.1 Indirect protein nitrogen

Precipitation of protein from a test portion by addition of trichloroacetic acid solution such that the final

concentration of trichloroacetic acid in the mixture is approximately 12 %. Removal of the precipitated

© ISO and IDF 2016 – All rights reserved 1
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SIST EN ISO 8968-4:2016
ISO 8968-4:2016(E)
IDF 20-4:2016(E)

milk protein by filtration, with the remaining filtrate containing the non-protein nitrogen components.

Determination of the nitrogen content of the filtrate by the procedure described in ISO 8968-1|IDF 20-1.

Where the total nitrogen content of the milk sample has previously been determined, the true protein

nitrogen content may be calculated as the difference between the total nitrogen content and the non-

protein nitrogen content.
4.2 Direct protein nitrogen

Precipitation of protein from a test portion by addition of trichloroacetic acid solution such that the final

concentration of trichloroacetic acid in the mixture is approximately 12 %. Separation of the protein

precipitate by filtration. (The precipitate contains the protein nitrogen of the sample.) Determination of

the nitrogen content of the precipitate by the procedure described in ISO 8968-1|IDF 20-1.

5 Reagents
5.1 General

Use only reagents of recognized analytical grade, unless otherwise specified, and distilled or

demineralized water or water of equivalent purity.

The reagents specified for the determination of total nitrogen by the method described in

ISO 8968-1 | IDF 20-1, together with the following, are required.
5.2 Trichloroacetic acid (CCl COOH) solution

Dissolve 15,0 g of trichloroacetic acid in water in a 100 ml one-mark volumetric flask. Dilute to the

mark with water. Do not use concentrations of trichloroacetic acid and volumes of solutions other than

those specified.

NOTE The performance of the method with respect to mean value and between-laboratory performance

characteristics will be different, if using other than specified concentrations of trichloroacetic acid and volumes

of solutions.
5.3 Hydrochloric acid standard volumetric solution

For the direct approach, the 0,1 mol/l hydrochloric acid solution is as described in ISO 8968-1| IDF 20-1.

For the indirect protein nitrogen approach, the following hydrochloric acid solution is required

c(HCl) = (0,01 ± 0,000 1) mol/l in addition to the 0,1 mol/l hydrochloric acid solution required as

described in ISO 8968-1|IDF 20-1.
6 Apparatus

Usual laboratory apparatus and that specified for the determination of total nitrogen described in

ISO 8968-1|IDF 20-1 and, in particular, the following.
6.1 Water bath, capable of maintaining a temperature of between 38 °C and 40 °C.
6.2 Conical flasks, of capacity 125 ml (indirect approach only).
6.3 Pipettes, of capacities 5 ml, 10 ml and 20 ml.
6.4 Filter funnel, made of glass, of diameter 75 mm.
2 © ISO and IDF 2016 – All rights reserved
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SIST EN ISO 8968-4:2016
ISO 8968-4:2016(E)
IDF 20-4:2016(E)
6.5 Filter paper, nitrogen free, of diameter 15 cm.
6.6 Automatic pipette, piston pump, capable of delivering 10 ml.
6.7 Beakers, of capacity 50 ml (indirect approach only).
7 Sampling

Sampling is not part of the method specified in this part of ISO 8968|IDF 20. A recommended sampling

method is given in ISO 707|IDF 50.

A representative sample should have been sent to the laboratory. It should not have been damaged or

changed during transport or storage.
8 Preparation of test sample

Warm the test sample to between 38 °C to 40 °C in the water bath (6.1). Cool the sample to room

temperature while gently mixing the test sample immediately prior to weighing the test portion (9.1 or

10.2.1).
9 Procedure — Direct protein nitrogen approach
9.1 Test portion

Pipette 5,0 ml ± 0,1 ml of the prepared test sample (see Clause 8) either into a dry and clean Kjeldahl

flask or digestion tube, pre-weighed to the nearest 0,1 mg. Weigh the test sample to the nearest 0,1 mg.

Immediately add 5,0 ml ± 0,1 ml of water to the flask or tube, rinsing any test sample on its neck into

its bottom.

NOTE The use of either a Kjeldahl flask or a digestion tube is dependent on the laboratory’s choice of

digestion apparatus.
9.2 Determination
9.2.1 Precipitation and filtration

Add 40 ml ± 0,5 ml of trichloroacetic acid solution (5.2) to the Kjeldahl flask or digestion tube containing

the test portion (9.1) and swirl to mix the contents. Let the flask or tube stand for approximately 5 min

to allow the precipitate to settle. Pour the contents of the flask or tube through a filter paper (6.5)

placed in a filter funnel (6.4). Collect the filtrate in a clean conical flask. Some of the precipitate will

remain in the Kjeldahl flask or digestion tube and some will be collected on the filter paper. It is not

necessary to remove all of the precipitate from the flask or tube.

Immediately after pouring the mixture and so as not to allow any precipitate to dry on the neck of the

flask or tube, add by means of an automatic pipette (6.6), 10 ml of the trichloroacetic acid solution (5.2).

Use the solution to rinse any precipitate from the neck of the flask or tube down into the bottom. Swirl

to mix the contents. Pour the thus-obtained contents of the flask or tube through the same filter paper.

Add the filtrate to that previously collected in the conical flask. Again, immediately rinse the neck of the

flask or tube with a further 10 ml of trichloroacetic acid solution and swirl to mix the contents. Pour the

contents of the flask or tube for the third time through the same filter paper, adding the f

...

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