UGANDA STANDARD 
       US ISO 5518
First Edition
2009-mm-dd
 
Reference numbe r 
    US ISO 5518:2007
 © UNBS 2009
  
Fruits, vegetables and derived products— Determination of 
benzoic acid content— Spectrophotometric method 
 US ISO 5518: 2007 
 
© 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. 
Requests for permission to reproduce this document should be addressed to 
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 5518: 2007  
© 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 5518, Fruits, vegetables and derived products — Determination of benzoic 
acid content — Spectrophotometric method,  is identical with and has been reproduced from an 
International Standard, ISO 5518:2007, Fruits, vegetables and derived products — Determination of benzoic acid content— Spectrophotometric 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
STANDARDISO
5518
Second edition
2007-02-01
Reference number
ISO 5518:2007(E)
© ISO 2007Fruits, vegetables and derived 
products — Determination of benzoic acid content — Spectrophotometric method
Fruits, légumes et produits dérivés — Détermination de la teneur en 
acide benzoïque — Méthode spectrophotométrique
                                    
ISO 5518:2007(E)
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Published in SwitzerlandISO 5518:2007(E)
© ISO 2007 – All rights reserved iiiForeword
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 ISOtechnical committees. Each member body interested in a subject for which a technical committee has beenestablished 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.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.The main task of technical committees is to prepare International Standards. Draft International Standards
adopted by the technical committees are circulated to the member bodies for voting. Publication as an
International Standard requires approval by at least 75 % of the member bodies casting a vote.
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.
ISO 5518 was prepared by Technical Committee ISO/TC 34, 
Food products , Subcommittee SC 3, Fruit and
vegetable products .
This second edition cancels and replaces the first edition (ISO 5518:1978), which has been technically revised.ISO 5518:2007(E)
iv © ISO 2007 – All rights reservedIntroduction
A method for determining the benzoic acid content of fruits, vegetable and derivatives, described in 1959, was
based on the technique of peak emergence. The advantage of this method was that it was specifically intendedfor benzoic acid, with one exception: 
p-chlorobenzoic acid.
However, it was necessary to make a modification, consisting of purifying the ethereal extract by chromic acid
oxidation. This results in the elimination of the effects of colouring substances in certain vegetable productscontaining anthocyanins, and also all the oxybenzoic acids and sorbic acid which may be present if severalantiseptics have been used. Moreover, purification increases the sensitivity of the method. This improvedtechnique was also more rapid.INTERNATIONAL STANDARD ISO 5518:2007(E)
© ISO 2007 – All rights reserved 1Fruits, vegetables and derived products — Determination of 
benzoic acid content — Spectrophotometric method
1S c o p e
This International Standard specifies a method for determining the benzoic acid content of fruits, vegetables
and derived products.
As chlorobenzoic acids are resistant to oxidation, the method cannot be applied in the presence of
p-chlorobenzoic acid, as the absorption spectrum of this acid is close to that of benzoic acid. Neither can it be
used in the presence of cinnamic acid, which is transformed into benzoic acid by chromic acid oxidation.
NOTE The cinnamic acid determined as benzoic acid in this method exists generally only in the form of traces in
vegetables, and therefore has no effect on the result obtained, except in the case of cinnamon bark, which contains higher
quantities.
2 Principle
The test sample is homogenized, followed by dilution and acidification of a test portion. The benzoic acid is
extracted by diethyl ether, then this acid undergoes alkaline re-extraction and purification by oxidation usingacidified potassium dichromate. The purified benzoic acid dissolved in diethyl ether is determined byspectrophotometry.
3R e a g e n t s
Use only reagents of recognized analytical quality and distilled water or water of at least equivalent purity.
3.1 Tartaric acid  [COOH(CHOH)2COOH], crystalline.
3.2 Sodium hydroxide  (NaOH), approximately  solution.
3.3 Potassium dichromate  (K2Cr2O7), solution containing  to .
3.4 Dilute sulfuric acid  (H2SO4), obtained by diluting 2 volumes of concentrated sulfuric acid
( ) with 1 volume of water.
3.5 Diethyl ether  [(CH3CH2)2O], recently distilled.
3.6 Benzoic acid  (C6H5COOH), standard solution in diethyl ether containing .
3.7 Sodium hydrogen carbonate  (NaHCO3), crystalline.
4 Apparatus
Usual laboratory apparatus and, in particular, the following.
4.1 Volumetric flasks , of capacities  and .
4.2 Beakers , of capacities  and .1 mol/l
33 g/l 34 g/l
ρ20=1,84 g/ml
0,100 g/l
50 ml 1 000 ml
50 ml 100 mlISO 5518:2007(E)
2 © ISO 2007 – All rights reserved4.3 Graduated pipettes , of capacities ,  and .
4.4 Flasks , of capacity , having ground-glass stoppers and made of borosilicate glass.
4.5 Separating funnels , of capacities  and .
4.6 Evaporating dish , of diameter about .
4.7 Water bath , capable of being controlled at a temperature of  to .
4.8 Homogenizer  or mortar , as appropriate.
4.9 Spectrophotometer  for determination in the ultraviolet range, equipped with a monochromator allowing
measurement to the nearest , with silica cells of optical path length  or  (preferably so as to increase sensitivity), equipped with ground glass covers.
4.10 Analytical balance .
5 Sampling
A representative sample should have been sent to the laboratory. It should not have been damaged or changed
during transport or storage.
6 Procedure
6.1 Preparation of test sample
6.1.1 Liquid products  (e.g. juices, pulpy fluid products, syrups)
Thoroughly mix the laboratory sample.
6.1.2 Thick products  (e.g. marmalades, jams)
Homogenize the laboratory sample after having carefully mixed it.
6.1.3 Solid products  (e.g. fruits, vegetables)
Cut a part of the laboratory sample into small pieces and remove seeds, stalks and carpellary cells, if
necessary. Carefully homogenize approximately  of the sample.
6.1.4 Frozen or deep-frozen products
After thawing the sample in a closed container and removing, if necessary, seeds, stalks and carpellary cells,
mix the product with the liquid formed during the thawing process and proceed as described in 6.2.1, 6.2.2 or6.2.3 as appropriate.
6.2 Preparation of test portion
6.2.1 Liquid products
Using a pipette (4.3), take  of the test sample (6.1), free from substances in suspension, dilute it with
approximately  of water and transfer it to a  separating funnel (4.5) (separating funnel A).10 ml 20 ml 50 ml
250 ml
100 ml 500 ml
10 cm
70◦C 80◦C
0,5 nm 10 mm 20 mm 20 mm
40 g
20 ml
50 ml 500 mlISO 5518:2007(E)
© ISO 2007 – All rights reserved 3The test portion may also be taken by mass by weighing, to the nearest , approximately  of the test
sample.
6.2.2 Pulpy and fluid products
Take  of the test sample (6.1). Place in a mortar (4.8) and dilute with  of water. After decanting, filter
the liquid.
Twice successively, take up the residue in  of water and filter after decanting.
Collect all the filtrates directly in a  separating funnel (4.5) (separating funnel A).The test portion may also be taken by mass by weighing, to the nearest , approximately  of the test
sample.
6.2.3 Thick or solid products
Weigh, to the nearest , approximately  of the test sample (6.1) and, using  to  of water,
transfer it to a  flask (4.4).
Add approximately  of sodium hydrogen carbonate (3.7) (see Note). Shake, then place the flask on the
water bath (4.7), controlled at  to , and leave for  to . Filter the contents of the flask andrinse twice using  to  of water each time.
Collect all the filtrates in a  separating funnel (4.5) (separating funnel A). Allow to cool.
NOTE The addition of sodium hydrogen carbonate is intended to neutralize the benzoic acid, traces of which could be lost
by volatilization.
6.3 Extraction of the benzoic acid
WARNING — Attention is drawn to the hazard derived from the use of diethyl ether, which is a highly
flammable, explosive and harmful substance.
6.3.1 Introduce  of the tartaric acid (3.1) into the separating funnel A (4.5) containing the diluted test portion
(6.2), add  of the diethyl ether (3.5) and shake carefully.
Allow to separate, then collect the ethereal layer in a second  separating funnel (4.5) (separating
funnel B).
Wash the aqueous phase in the first separating funnel (A) with  of the diethyl ether.
Allow to separate, then collect the ethereal layer in the separating funnel (B) containing the first layer collected.
Proceed similarly with a third extraction with  of the diethyl ether and combine the ethereal layer with the
first two in the separating funnel (B).
6.3.2 Extract the benzoic acid from the ethereal solution by adding successively  and then  of the
sodium hydroxide solution (3.2), and then twice  of water. After each addition, shake, then allow toseparate and collect the aqueous phase.
Collect the aqueous phases in an evaporating dish (4.6). Place the dish on the water bath (4.7), controlled at
 to , and leave until the volume of the alkaline solution is reduced by approximately half, to remove
the residual dissolved diethyl ether.0,01 g 20 g
20 ml 20 ml
20 ml
500 ml
0,01 g 20 g
0,01 g 10 g 30 ml 40 ml
250 ml
50 mg
70◦C80◦C 15 min 30 min
15 ml 20 ml
500 ml
1g
60 ml
500 ml
60 ml
30 ml
10 ml 5m l
10 ml
70◦C 80◦CISO 5518:2007(E)
4 © ISO 2007 – All rights reserved6.4 Purification of the benzoic acid
After cooling, pour the contents of the dish into a  flask (4.4) containing a mixture of  of the dilute
sulfuric acid (3.4) and  of the potassium dichromate solution (3.3). Stopper the flask and leave for at least
.
Other preservatives derived from benzoic acid may be present. In this case, leave the flask for at least  to
oxidize completely the three hydroxybenzoic acids and prevent any interference in the determination. The
extension of the reaction time creates no problem as the benzoic acid resists this oxidizing mixture.
When the initial product also contains sorbic acid, it is necessary to prolong oxidation for  so as to ensure
the complete destruction of this acid.
6.5 Extraction of the purified benzoic acid
Extract the benzoic acid by treating the above solution (6.4) twice with  to  of the diethyl ether (3.5),
collecting the ethereal solutions. Wash the ethereal solutions twice with several millilitres of water. Afterdecanting very carefully, filter through a dry filter paper and collect the filtrate in a  volumetric flask (4.1).Then wash the filter with several millilitres of the diethyl ether, adding sufficient washing solvent to dilute to themark.
6.6 Determination
Using the spectrophotometer (4.9), measure the absorbance of the ethereal solution (6.5) in relation to theabsorbance of the pure diethyl ether at ,  and  (see Note).
The absorbance due to benzoic acid is given by the formula for the differential measure of emergence at
:
where
is the absorbance at ;is the absorbance at ;is the absorbance at .
NOTE Examination of the absorption spectrum of the ethereal solution of purified benzoic acid allows characterization of
this product by the presence of two peaks at  and .
The benzoic acid extracted by the diethyl ether is determined by the measurement of the relative height of the peak at
 with respect to the straight line which joins the points on the abscissa between  and .
6.7 Number of determinations
Carry out two determinations on the same test sample (6.1).
6.8 Plotting the calibration curve
Into a series of six  volumetric flasks (4.1), introduce respectively , , , ,  and
 of the standard benzoic acid solution (3.6). Dilute to the mark with the diethyl ether (3.5).
The solutions obtained contain respectively , , , ,  and  of benzoic acid per
litre.250 ml 20 ml
20 ml
1h
3h
24 h
20 ml 25 ml
50 ml
267,5 nm 272 nm 276,5 nm
272 nm
A2−A1+A3
2
A1 267,5 nm
A2 272 nm
A3 276,5 nm
272 nm 279 nm
272 nm 267,5 nm 276,5 nm
50 ml 5m l 7,5 ml 10 ml 12,5 ml 15 ml
20 ml
10 mg 15 mg 20 mg 25 mg 30 mg 40 mgISO 5518:2007(E)
© ISO 2007 – All rights reserved 5Plot the curve showing the different measurements according to the content of benzoic acid, in milligrams per
litre, indicated above.
7 Calculation and expression of results
7.1 Test portion taken by pipetting
The benzoic acid content, expressed in milligrams per litre of product, is given by
where  is the mass, in milligrams, of benzoic acid read on the calibration curve (6.8).
7.2 Test portion taken by weighing
The benzoic acid content, expressed in milligrams per kilogram of product, is given by
where
is the mass, in grams, of the test portion (6.2);
is the mass, in milligrams of benzoic acid, read on the calibration curve (6.8).
8 Repeatability
The absolute difference between two independent single test results, obtained using the same method on
identical test material in the same laboratory by the same operator using the same equipment within a shortinterval of time, will in not more than  of cases exceed  of benzoic acid per litre or per kilogram,
depending on the individual circumstances.
NOTE The method allows determination of the quantity of benzoic acid to the nearest  when the product contains less
than  per litre or per kilogram.
9 Test report
The test report shall specify:
a) all information necessary for the complete identification of the sample;b) the sampling method used, if known;
c) the test method used, with reference to this International Standard;
d) all operating details not specified in this International Standard, or regarded as optional, together with
details of any incidents which may have influenced the test result(s);
e) the test result(s) obtained or, if the repeatability has been checked, the final quoted result obtained.m2×50
20=2,5m2
m2
m2×50
m1
m1
m2
5% 10 mg
2m g
50 mgISO 5518:2007(E)
ICS 67.080.01
Price based on 5 pages
© ISO 2007 – All rights reserved