UGANDA STANDARD
 
        US ISO 6557-1
First Edition
2009-mm-dd
 
Reference numbe r 
     US ISO 6557-1:1986
 © UNBS 2009
  
Fruits, vegetables and derived products — Determination of 
ascorbic acid — Part 1: Reference method 
 US ISO 6557-1: 1986 
 
© UNBS  2009 – All rights reserved ii Compliance with this standard does not, of itself confer immunity from legal obligations 
A Uganda Standard does not purport to include all necessary provisions of a contract. Users are 
responsible for its correct application  
© UNBS 2009 
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilised in any 
form or by any means, electronic or mechanical, in cluding photocopying and microfilm, without prior written 
permission from UNBS. 
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The Executive Director 
Uganda National Bureau of Standards 
P.O. Box 6329 
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Uganda 
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E-mail: unbs@infocom.co.ug
Web: www.unbs.go.ug  
       US ISO 6557-1:1986  
© UNBS  2009 - All rights rese rved                iii National foreword 
Uganda National Bureau of Standards (UNBS) is a parastatal under the Ministry  of Tourism, Trade and 
Industry established under Cap 327, of the Laws of U ganda. UNBS is mandated to co-ordinate the elaboration 
of standards and is  
(a) a member of International Organisation for Standardisation (ISO) and  
(b) a contact point for the WHO/FAO Codex A limentarius Commission on Food Standards, and  
(c) the National Enquiry Point on TBT/SPS Agr eements of the World Trade Organisation (WTO). 
The work of preparing Uganda Standards is carried out through Technical Committees. A Technical 
Committee is established to deliberate on standards in a gi ven field or area and consists of representatives of 
consumers, traders, academician s, manufacturers, governme nt and other stakeholders. 
Draft Uganda Standards adopted by the Technical Committ ee are widely circulated to stakeholders and the 
general public for comments. The committee reviews the comments before recommending the draft standards 
for approval and declaration as Uganda Standards by the National Standards Council. 
This Uganda Standard, US ISO 6557-1, Fruits, vegetables and derived products — Determination  
of ascorbic acid — Part 1: Reference method,  is identical with and has been reproduced from an
International Standard, ISO 6557-1:1986, Fruits, vegetables and derived products — Determination of 
ascorbic acid — Part 1: Reference method, and adopted as a Uganda Standard.  
This standard was developed under the subcommittee on fruits and vegetables by the Food and agriculture 
Standards Technical Committee (UNBS/TC 2). 
Wherever the words, "International Standard" a ppear, they should be replaced by "Uganda Standard." 
 International Standard 655711 
INTERNATIONAL ORGANIZATION FOR STANDARDIZATIONWblEHtAYHAPOAHAR OPfAHM3At&lR fl0 CTAH~APTbl3ALWWORGANISATlON INTERNATIONALE DE NORMALISATION 
Fruits, vegetables and derived products - Determination 
of ascorbic acid - 
Part 1: Reference method 
Fruits, ldgumes et produits d4rivhs - LXtermination de la teneur en acide ascorbique - Partie 7 : Methode de rhf&rence 
First edition - 1986-10-01 
w - 
8 Q, UDC 635.1/.6 : 543.426 : 547.475.2 Ref. No. ISO 6557/1-1986 (E) 
Descriptors : agricultural products, fruits, vegetables, fruit and vegetable products, Chemical analysis, determination of content, ascorbic acid, 
F 
5l spectrochemical analysis. 
5: Price based on 3 pages Foreword 
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. Esch 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, govern- 
mental and non-governmental, in liaison with ISO, also take part in the work. 
Draft International Standards adopted by the technical committees are circulated to 
the member bodies for approval before their acceptance as International Standards by 
the ISO Council. They are approved in accordance with ISO procedures requiring at 
least 75 % approval by the member bodies voting. 
International Standard ISO 6557/1 was prepared by Technical Committee ISO/TC 34, 
Agricultural food products. 
Users should note that all International Standards undergo revision from time to time 
and that any reference made herein to any other International Standard implies its 
Iatest edition, unless otherwise stated. 
0 International Organkation for Standardkation, 1986 0 
Printed in Switzerland INTERNATIONAL STANDARD ISO 6557/1-1986 (E) 
Fruits, vegetables and derived products - Determination 
of ascorbic acid - 
Part 1: Reference method 
1 Scope and field of application 
This part of ISO 6557 specifies the reference method, using 
molecular fluorescence spectrometry, for the determination of 
the combined ascorbic and dehydroascorbic acid content of 
fruits, vegetables and derived products. 
2 Principle 
Transformation of ascorbic acid into dehydroascorbic acid 
using activated charcoal. 
Reaction of the dehydroascorbic acid obtained with 
o-phenylenediamine (OPDA) to give a fluorescent compound 
according to the reaction 
/ a N”z I + \ N”z ’ 
0 
(OPDA) (Dehydroascorbic acid) 
t 
0 
( 1 -Oxo-2,4 H-3-t 1,2-d’h d I y roxyethyl) furo 13,4-bl quinoxaline) 
In a control test in the presence sf boric acid, formation of the 
complex HsB03 - dehydroascorbic acid to prevent reaction 
with OPDA. Any fluorescent interference tan thus be taken 
into account in the calculation. 
NOTE - The initial dehydroascorbic acid content alone tan be deter- 
mined by omitting the step using activated charcoal. lt is then possible, 
by subtraction, to determine the initial content of ascorbic acid alone. 3 Reagents and materials 
All reagents shall be of recognized analytical grade and the 
water used shall be distilled or water of equivalent purity. 
3.1 o-Phenylenediamine dihydrochloride 
(C6H8N2 9 2HCI), 0,2 g/l Solution. 
Prepare this Solution just before use. 
3.2 Sodium acetate trihydrate (CH,COONa~3H20}, 
500 gll Solution. 
3.3 Boric acidkodium acetate Solution. 
Dissolve 3 g of boric acid (H3B03) in 100 ml of the sodium 
acetate Solution (3.2). 
Prepare this Solution just before use. 
3.4 Ascorbic acid, 1 g/l Standard solution. 
Weigh, to the nearest 0,Ol mg, 50 mg of ascorbic acid, 
previously dehydrated in a desiccator protected from iight. 
Transfer quantitativeiy to a 50 ml one-mark volumetric fiask and 
make up to the mark with the extraction Solution (3.5), just 
before use. 
3.5 Extraction soiution. 
3.5.1 Metaphosphoric acidlacetic acid. 
Transfer 30 g of metaphosphoric acid (HPO,) to a 1 000 m$ 
beaker or conical fiask containing 80 mi of giaciai acetic acid 
(CH$OOH) and about 500 mi sf water. Warm and stir gently 
until complete dissolution. 
Aliow the soiution to cool. Transfer it quantitativeiy to a 
1 000 mi one-mark voiumetric fiask and make up to the mark 
with water. 
or 
3.52 Metaphosphoric acid/methanol. 
Mix 3 voiumes of a 4 % (mlm) metaphosphoric acid solution 
with 1 volume of methanoi. 
NOTE - Metaphosphoric acid is commercialiy availabie with an HPO, 
content of 40 to 44 %. ISO 6557/1-1986 (E) 
3.6 Activated charcoaI.1) 
Weigh 200 g of activated charcoal and add 1 litre of 10 % (V/ v) 
hydrochloric acid. 
Heat to boiling, then filter through a sintered glass filter of 
porosity P 40 (16 to 40 Pm). Transfer the “cake” of carbon into 
a beaker. Add 1 litre of water, Shake and filter through a 
sintered glass filter. Repeat three times the Operation of 
washing with water and filtering. 
Place the residue in a drying oven maintained at 115 rt 5 OC and 
leave for 12 h (for example overnight). 
4 Apparatus 
Usual laboratory equipment and in particular 
4.1 Mechanical grinder. 
4.2 Centrifuge. 
4.3 Stirrer, for conical flasks and test tubes. 
4.4 Molecular fluorescence spectrometer. The Optimum 
excitation and emission wavelengths for the test should be 
determined beforehand and depend on the instrument used. It 
shall be fitted with a lamp giving a continuous spectrum. 
4.5 Conical flasks, of appropriate capacities. 
4.6 Volumetric flasks, of capacity 100 ml. 
4.7 Test tubes, of diameter 10 mm. 
4.8 Pipettes, of appropriate capacities. 
4.9 Filter paper-. 
5 Procedure 
5.1 Preparation of the test Sample 
Mix the laboratory Sample weil. If necessary, first remove seeds 
and hard seed-cavity Walls and pass the laboratory Sample 
through a mechanical grinder (4.1). Allow frozen or deep- 
frozen products to thaw in a closed vessel and add the liquid 
formed during this process to the laboratory Sample before mix- 
ing. 
1) A suitabie product, available commercially, is Norite. 
not constitute an endorsement of this product by ISO. 5.2 Test Portion 
Transfer a quantity, weighed to the nearest 0,l mg, of the test 
Sample (5.1) to a conical flask (4.51, so that after dilution with 
the extraction Solution the ascorbic acid and dehydroascorbic 
acid concentration may be expected to be between 0 and 
50 mg/l. 
5.3 Preparation of the test solution 
5.3.1 Add a known quantity of the extraction solution (3.5) to 
the test Portion so that the ascorbic acid and dehydroascorbic 
acid content is between 0 and 50 mg/l. Agitate for 30 min, and 
centrifuge. Adjust the pH to 1,2 with a measured volume of the 
extraction Solution (3.5). 
Take 100 ml of this solution and add 1 g of activated charcoal 
(3.6). Mix weil, then filter it through filter Paper (4.9), discard- 
ing the first few millilitres of the filtrate. 
5.3.2 Introduce, by means of a pipette (4.81, 5 ml of the 
sodium acetate Solution (3.2) and 5 ml of the filtrate (5.3.1) into 
a 100 ml one-mark volumetric flask (4.6). Mix and make up to 
the mark with water. 
5.4 Matthing test 
Introduce, by means of a pipette, 5 ml of boric acid/sodium 
acetate Solution (3.3) and 5 ml of the filtrate (5.3.1) into a 
100 ml one-mark volumetric flask. Allow to stand for 15 min, 
mixing occasionally, then make up to the mark with water. 
5.5 Determination 
Transfer 2 ml of the test Solution (5.3.2) into a test tube (4.7) 
and 2 ml of the matthing test Solution (5.4) into another test 
tube. 
Add 5 ml of the o-phenylenediamine dihydrochloride Solution 
(3.1) to the contents of each tube, protected from light. Mix 
well using the stirrer (4.31, then allow the reaction to proceed 
for 30 min in the dark. 
Carry out measurements on both tubes by means of the 
previously adjusted molecular fluorescence spectrometer (4.4) 
using minimum lamp power. Subtract the matthing test sol- 
ution reading from that of the test solution. 
5.6 Calibration graph 
5.6.1 Transfer, by means of a pipette, 2 and 5 ml of the stan- 
dard Solution (3.4) to two 100 ml one-mark volumetric flasks. 
Make up each to the mark with the extraction Solution (3.5). 
These solutions contain 20 and 50 mg of ascorbic acid per litre. 
Add 1 g of activated charcoal (3.6) to each of these solutions. 
Mix weil, then filter through filter Paper (4.9), discarding the 
first few millilitres of each filtrate. 
This information is given for the convenience of the user of this part of ISO 6557 and does 
2 ISO 6557/1-1986 (E) 
5.6.2 Repeat the operations 5.3.2, 5.4 and 5.5 with both 
calibration solutions (5.6.11, replacing the 5 ml of filtrate with 
5 ml of each calibration Solution. 
Establish the calibration graph by plotting the spectrometer 
readings against concentration, expressed in milligrams per 
litre, of both calibration solutions. 
Draw the calibration 
two Points obtained. graph passing through the origin and the 
5.7 Number of determinations 
Carry out two determinations on the same test Sample (5.1). 
Expression of results 
The ascorbic acid and dehydroascorbic acid content, expressed 
in milligrams per 100 g of product, is given by the formula 
CV 
lOm, m0 is the mass, in grams, of the test Portion; 
V is 
added the volume, in millilitres, of the extraction Solution 
c is the concentration, expressed in milligrams per litre, of 
ascorbic acid and dehydroascorbic acid in the test Solution 
read from the calibration graph and corrected for the 
matthing test Solution. 
7 Test report 
The test report shall show the method used and the results ob- 
tained. lt shall also mention any operating details not specified 
in this International Standard, or regarded as optional, together 
with details of any incidents likely to have influenced the 
results. 
The test report shall include all the information 
the complete identifi cation of the Sample. necessary for This page intentionally left blank This page intentionally left blank This page intentionally left blank