UGANDA STANDARD
 
        US ISO 6557-2
First Edition
2009-mm-dd
 
Reference numbe r 
     US ISO 6557-2:1984
 © UNBS 2009
  
Fruits, vegetables and derived products — Determination of 
ascorbic acid content — Part 2: Routine methods 
 US ISO 6557-2: 1984 
 
© 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 
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The Executive Director 
Uganda National Bureau of Standards 
P.O. Box 6329 
Kampala  
Uganda 
Tel: 256 41 505 995 
Fax: 256 41 286 123 
E-mail: unbs@infocom.co.ug
Web: www.unbs.go.ug  
       US ISO 6557-2:1984  
© 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 6557-2, Fruits, vegetables and derived products — Determination
of ascorbic acid co ntent — Part 2: Routine methods,  is identical with and has been reproduced from an
International Standard, ISO 6557-2:1984, Fruits, vegetables and derived products — Determination of ascorbic
acid content — Part 2: Routine methods, 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 
INTERNATIONAL ORGANIZATION FOR STANDARDlZATIONWlEXAYHAPOAHAR OPTAHl43ALU4R fl0 CTAHAAPTM3ALIMM~RGANlSATlON INTERNATIONALE DE NORMALISATION 
Fruits, vegetables and derived products - Determination 
of ascorbic acid content - 
Part 2: Routine methods 
Fruits, lkgumes et produits d&iv& - Ddtermination de la teneur en acide ascorbique - Partie 2: IWthodes pratiques 
First edition - 1984-11-15 
UDC 634.1/635.6 : 543.24 : 547.475.2 Ref. No. IS0 6557/2-1984 (E) 
Descriptors : agricultural products, fruits and vegetable products, tests, determination of content, ascorbic acid. 
Price based on 4 pages Foreword 
IS0 (the International Organization for Standardization) is a worldwide federation of 
national standards bodies (IS0 member bodies). The work of preparing International 
Standards is normally carried out through IS0 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, 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 IS0 Council. They are approved in accordance with IS0 procedures requiring at 
least 75 % approval by the member bodies voting. 
lnternationa I Standard IS0 
Agricultural food products. 655712 was prepared by Technical Commit tee ISO/TC 34, INTERNATIONAL STANDARD IS0 6557/2-1984 (E) 
Fruits, vegetables and derived products - Determination 
of ascorbic acid content - 
Part 2: Routine methods 
1 Scope and field of application 
This part of IS0 6557 specifies two routine methods for the 
determination of the ascorbic acid content’) of fruits, 
vegetables and derived products : 
method A: 2,6-dichlorophenolindophenol titrimetric 
method; 
method B : 2,6-dichlorophenolindophenol spectrometric 
method after extraction with xylene. 
Method A can only be used in the absence of certain in- 
terferences (see 2.6). 
Method B is applicable to derived fruit and vegetable products 
in strongly coloured solutions. 
2 Method A: 2,6-dichlorophenolindophenol 
titrimetric method 
2.1 Principle 
Extraction of the ascorbic acid from a test portion using either 
oxalic acid solution or metaphosphoric acid-acetic acid solu- 
tion. Titration with 2,6-dichlorophenolindophenol dyestuff until 
a salmon pink colour is obtained. 
2.2 Reagents 
All reagents shall be of recognized analytical grade. The water 
used shall be distilled water or water of at least equivalent 
purity. 
2.2.1 Extraction solution. 
Use either a 2 % (m/m) oxalic acid solution or a metaphos- 
phoric acid/acetic acid solution prepared as follows. 
Dissolve 15 g of metaphosphoric acid in 40 ml of glacial acetic 
acid and 200 ml of water in a 500 ml one-mark volumetric flask, 
make up to the mark with water and filter immediately through 
filter paper into a glass bottle. 
This solution can be kept for 7 to 10 days if stored in a 
refrigerator. 2.2.2 2,6-dichlorophenolindophenol, dyestuff solution. 
Dissolve 50 mg of the sodium salt of 2,6-dichloro- 
phenolindophenol in 150 ml of hot (50 to 60 OC) water contain- 
ing 42 mg of sodium hydrogen carbonate in a 200 ml one-mark 
volumetric flask, make up to the mark with water and filter. 
Store the solution in a dark brown bottle in a refrigerator. 
As the dyestuff decomposes with time, fresh solution should 
be prepared periodically. 
2.2.3 Ascorbic acid, 1 g/l standard solution. 
Weigh, to the nearest 0,Ol mg, 50 mg of ascorbic acid which 
has been stored in a desiccator, transfer quantitatively to a 
50 ml one-mark volumetric flask and make up to the mark with 
the extraction solution (2.2.1). 
2.3 Apparatus 
Usual laboratory equipment, and 
2.3.1 Analytical balance. 
2.3.2 Mixer. 
2.3.3 Burette, of capacity 10 to 50 ml. 
2.4 Procedure 
2.4.1 Preparation of the test sample 
If necessary, remove seeds and hard seed-cavity walls and then 
thoroughly mix the sample. Filter, and proceed with the deter- 
mination on the filtrate. 
Allow frozen or deep frozen products to thaw in a closed vessel 
and add the liquid formed during this process to the product 
before mixing. 
2.4.2 Test portion 
Weigh, to the nearest 0,l mg, 10 to 100 g of the sample. 
1) The ascorbic acid is determined as dehydroascorbic acid. IS0 6557/2-1984 (El 
2.4.3 Determination *1 is the mass, in milligrams, of ascorbic acid equivalent 
to 1,O ml of the dyestuff solution (see 24.32); 
2.4.3.1 Extraction 
VO is the volume, 
the titration; in millilitres, of dyestuff solution used for 
Mix the test portion with the extraction solution (2.2.1) so that 
the volume, in millilitres, of the latter is numerically between 1 
to 5 times the mass, in grams, of the test portion. v, is the volume, 
the blank test. in millilitres, of dyestuff solution used in 
Filter the solution, discarding the first few millilitres of filtrate. Take as the result the arithmetic 
in the three determinations. means of the values obtained 
The ascorb 
between 0, ic acid concent 
and 1 mg/ml ration in this test solution should be 
2.6 Notes on procedure 
2.4.3.2 Standardization of the dyestuff solution A number of interferences exist, in particular iron, copper, tin, 
reducing agents, hydrosulfides, sulfites and sulfur dioxide. 
Reducing agents, in particular, are present in products which 
have been overheated or stored for too long a period. Dilute a 5 ml aliquot portion of the standard ascorbic acid soiu- 
tion (2.2.3) with 5 ml of the extraction solution (2.2.1) and 
titrate rapidly with the dyestuff solution (2.2.2) until a salmon 
pink coloration, persisting for at least 5 s, is obtained. Repeat 
this procedure twice more, and record the volume of dyestuff 
solution used each time to the nearest 0,l ml. If the presence of 
teed as follows: any of these interferences is suspected, pro- 
Add 2 drops of 0,05 % methylene blue solution to 10 ml of a 
solution containing equal volumes of the test solution and the 
extraction solution. Mix. The disappearance of the coloration 
within 5 to 10 s indicates the presence of interferences. Proceed in the same way to establish the blank test, replacing 
the 5 ml of standard ascorbic acid solution with 5 ml of extrac- 
tion solution. 
Subtract the result of the blank test from the volumes of 
dyestuff solution used for the three standardization titrations 
and express the concentration of the dyestuff solution as the 
mass, in milligrams, of ascorbic acid equivalent to 1,0 ml of 
solution. NOTE - Tin cannot be 
cedure should be used. detected in this way, and the following pro- 
Add 5 drops of 0,05 % carmine indigo to 10 ml of the test solution to 
which 10 ml of (1 + 3) hydrochloric acid has been added. Mix. The 
disappearance of the coloration within 5 to 10 s indicates the presence 
of tin or other interferences. 
2.4.3.3 Titration 
Take three aliquot portions of the filtrate obtained in 2.4.3.1 so 
that each contains about 2 mg of ascorbic acid, and titrate 
rapidly with the dyestuff solution until a salmon pink coloration, 
persisting for at least 5 s, is obtained. Take for the calculation 
(see 2.5) the arithmetic mean volume of dyestuff solution used. 3 Method B : 2,6-dichlorophenolindophenol 
spectrometric method, after extraction with 
xylene 
3.1 Principle 
2.4.4 Blank test 
Extraction of the ascorbic acid from a test portion using either 
oxalic acid solution or metaphosphoric acid-acetic acid solu- 
tion. Quantitative reduction of 2,6-dichlorophenolindophenol 
dyestuff by the ascorbic acid, extraction of the excess dyestuff 
using xylene, and determination of the excess by spectro- 
metric measurement at a wavelength of 500 nm. Carry out a blank test, proceeding as specified in 2.4.3, using 
the same volume of extraction solution as in 2.4.3.1, but omit- 
ting the test portion. 
2.4.5 Number of determinations 
Carry out three determinations on test 
same test sample. portions taken from the 3.2 Reagents 
All reagents shall be of recognized analytical grade. The water 
used shall be distilled water or water of at least equivalent 
purity. 2.5 Expression of results 
The ascorbic acid content, 
product, is equal to expressed in mil ligrams per 100 g of 
3.2.1 Extraction solution. 
(Vo - v,, x ml 
x loo See 2.2.1. 
where 3.2.2 Sodium acetate/acetic acid, buffer solution, pH 4,0. 
Add 300 g of anhydrous sodium acetate to 700 ml of water and 
1000 ml of g lacial acetic acid. m0 is the mass, in grams, 
tion taken for the titration ; of test portion in the aliquot por- 
2 IS0 6557/2-1984 E) 
3.2.3 2,6-dichlorophenolindophenol, dyestuff solution. 3.4.3.2 Standardization of the dyestuff solution. 
Proceed as specified in 2.4.3.2. See 2.2.2. 
3.2.4 Ascorbic acid, 1 g/l standard solution. 3.4.3.3 
See 2.2.3. . Transfer, by means of a pipette, 1 to 5 ml of the test solution to 
a centrifuge tube (3.3.4) and add an equal volume of the buffer 
solution (3.2.2). Immediately add an excess of the dyestuff 
solution I3.2.3), mix, and add 10 ml of the xylene (3.2.5). Stop- 
per the tube and shake vigorously for 6 to 10 s. Centrifuge to 
separate the layers. Carefully remove the upper xylene layer 
and fill a spectrometric cell. Reduction 
3.2.5 Xylene. 
WARNING - In view of the narcotic properties of xylene 
in high concentrations, all operations involving its use 
should be carried out under a fume hood. 
Check the purity of the xylene as follows: 3.4.3.4 Spectrometric measurement 
Add ascorbic acid to a small quantity of the dyestuff solution 
(3.2.3) until the solution is decolorized, and shake with 10 ml of 
the xylene. Leave for 10 min. If there is any trace of colour in 
the xylene layer, the xylene shall be distilled. Measure the absorbance of the xylene layer at 500 nm. 
3.4.4 Blank test 
Measure the absorbance of the xylene (3.2.5) at 500 nm. The xylene used in the determination may be recovered by 
shaking with 20 % (m/m) sodium hydroxide solution to 
neutralize the acetic acid, followed by redistillation. 3.4.5 Preparation of the calibration graph 
Transfer to each of four centrifuge tubes (3.3.4) the same 
volume of extraction solution as used for the determination 
(3.4.3.3). Add to each an equal volume of the buffer solution 
(3.2.2) and then add, respectively, 0,2; 0,4; 0,6; and 0,8 ml of 
the dyestuff solution (3.2.3). 3.3 Apparatus 
Usual laboratory equipment, and 
3.3.1 Analytical balance. 
Proceed as specified in 3.4.3.3. 
3.3.2 Mixer. Plot a g raph of absorbance 
dyestuff solution added. as a function of the volume of 
3.3.3 Microburettes, of capacities 2, 5 and 10 ml. 
3.4.6 Number of determinations 
3.3.4 
pers. Centrifuge tubes, of capacity 25 ml, with glass stop- 
Carry out two determinations on the same test sample. 
3.3.5 Centrifuge. 
3.5 Expression of results 
3.3.6 Spectrometer, suitable for making measurements at a 
wavelength of 500 nm. The ascorbic acid content, 
product, is equal to expressed in milligrams per 100 g of 
3.4 Procedure (V() - vl, x ml 
x 100 
m0 3.4.1 Preparation of the test sample 
Proceed as specified in 2.4.1. where 
m0 is the mass, i n grams, of test 
tion taken for the determination ; portion in the aliquot por- 
3.4.2 Test portion 
Proceed as specified in 2.4.2. 
ml is the mass, in milligrams, of 
to 1, 0 ml of the d yestuff solution ; asco rbic acid equi valent 
3.4.3 Determination 
V. is the volume, in millilitres, of dyestuff solution added 
in 3.4.3.3; 3.4.3.1 Extraction 
Proceed as specified in 2.4.3.1 in order to obtain a test solution 
containing between O,O5 and 0,5 mg of ascorbic acid per 
millilitre. V, is the volume, in millilitres, of the excess of dyestuff 
corresponding to the absorbance measured in 3.4.3.4, read 
from the calibration graph. IS0 6557/Z-1984 W 
3.6 Repeatability 
The difference between the results of the two determinations 
(3.4.61, carried out simultaneously or in rapid succession by the 
same analyst on the same test sample, shall not exceed 3 % of 
the mean. 
3.7 Notes on procedure 
3.7.1 If the product contains xylene-extractable pig 
carry out a hydroquinone blank correction as follows. ments, 
After measuring the absorbance of the xylene layer (see 
3.4.3.4), add 2 drops of a semi-saturated solution of hydro- 
quinone (prepared by adding twice the volume of acetone re- 
quired to obtain a saturated solution of hydroquinone), mix, 
leave for 30 s and measure the absorbance again. Deduct this 
absorbance from the initial absorbance of the xylene layer. 
3.7.2 If the product has been overheated or stored for too 
long a period, or, in some cases, in the case of natural products 
(for example blackcurrant juice), a treatment with for- 
maldehyde may be used to correct for the presence of reducing substances not related to ascorbic acid. For this purpose, carry 
out a check test (control) in parallel with the determination, 
proceeding as specified in 3.4 up to the addition of the 
dyestuff. Before this addition, add to the test solution 1 ml of 
water and to the control solution 1 ml of 40 % formaldehyde 
solution. Leave for 10 min and then proceed with the deter- 
mination. . 
From the calibration graph, determine the volume of dyestuff 
solution decolorized by the interferences and correct the result 
accordingly. 
4 Test report 
The test report shall show the method used and the result ob- 
tained. It 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 rep0 rt shall include all the information 
the complete identification o f the sa mple. necessary for This page intentionally left blank This page intentionally left blank