UGANDA STANDARD
 
        US ISO 6632
First Edition
2009-mm-dd
 
Reference numbe r 
     US ISO 6632:1981
 © UNBS 2009
  
Fruits, vegetables and derived products — Determination of 
volatile acidity 
 US ISO 6632: 1981 
 
© 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 
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The Executive Director 
Uganda National Bureau of Standards 
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E-mail: unbs@infocom.co.ug
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      US ISO 6632:1981  
© 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 6632, Fruits, vegetables and derived products — Determination of volatile acidity, 
is identical with and has been reproduced fr om an International Standard, ISO 6632:1981, Fruits, vegetables 
and derived products — Determination of volatile acidity, 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 STANDARDIZATION.MEX,QYHAPO~HAR OPrAHM3ALWl I-IO CTAHC\APTbl3Al@lM*ORGANISATION INTERNATIONALE DE NORMALISATION 
Fruits, vegetables and derived products - Determination 
of volatile acidity 
Fruits, lhgumes et produits d&iv& - Determination de l’acidith vola tile 
First edition - 1981-11-01 
UDC 634/635: 548.813 Ref. No. ISO 66324981 (E) 
Descriptors : agricultural products, fruit and vegetable products, tests, determination, volatility, acidity. 
Price based on 9 pages Foreword 
ISO (the International Organization for Standardization) is a worldwide federation of 
national Standards institutes (ISO member bedies). The work of developing lnter- 
national Standards is carried out through ISO technical committees. Every member 
body interested in a subject for which a technical committee has been set up 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. 
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. 
International Standard ISO 6632 was developed by Technical Committee ISO/TC 34, 
Agricultural food products, and was circulated to the member bodies in May 1980. 
lt has been approved by the member bodies of the following countries : 
Australia 
Austria 
Brazil 
Canada 
Czechoslovakia 
Egypt, Arab Rep. of 
France 
Germany, F. R. 
Hungary 
India 
The member body 
technical grounds 
0 of the foll owing country expressed disapproval of the docu reland 
srael 
<enya 
<orea, Dem. P. Rep. of 
Vlalaysia 
Vetherlands 
Vew Zealand 
Peru 
Philippines 
Poland 
USA Portugal 
Romania 
South Africa, Rep. of 
Spain 
Thailand 
USSR 
Venezuela 
Y ugoslavia 
International Organkation for Standardkation, 1981 
Printed in Switzerland INTERNATIONAL STANDARD ISO 6632-1981 (E) 
Fruits, vegetables and derived products - Determination 
of volatile acidity 
1 Scope and field of application 
This International Standard specifies a method for the deter- 
mination of volatile acidity in fruits, vegetables and derived 
products. 
The method is applicable to all fresh products and to pro- 
ducts preserved without Chemical preservatives, as well as to 
products to which sulphur dioxide has been added with or 
without one of the following preservatives : sorbic acid, ben- 
zoic acid, formic acid. 4.2 Phenolphthalein, IO g/l Solution in 95 % (V/ V) 
ethanol. 
4.3 Tartaric acid, crystallized. 
4.4 Lime water, diluted 1 + 4. 
Dilute one volume of the saturated Calcium hydroxide Solution 
(4.5) with fo ur volumes of water. Allow the mixture to stand 
until Calcium carbonate is precipitated and decant the limpid 
Solution which shall be alkaline to the phenolphthalein sol- 
ution (4.2). 
2 Definition 
This Solution is intended for use in the steam generator (5.2.1). 
2.1 volatile acidity : All lower molecular weight fatty acids, 
such as acetic acid and propionic acid, in free or combined 
form, with the exception of formic acid. 
The volatile acidity, determined by the method specified in this 
International Standard, is expressed either in milliequivalents 
per 100 ml or per 100 g of product, or in grams of acetic acid 
per 100 ml or per 100 g of product. 4.5 Calcium hydroxide, limpid saturated solution. 
5 Apparatus 
Usual laboratory apparatus, and in particular : 
5.1 Mechanical mill. 
3 Principle 
Acidification of a test Portion with tartaric acid, entrainment of 
the volatile acids by steam distillation, and titration of the 
distillate with Standard volumetric sodium hydroxide Solution in 
the presence of phenolphthalein as indicator. If appropriate, 
subtraction of the volatile acidity due to added volatile acid 
antiseptic (preservative) compounds from the volatile acidity 
thus determined. 
4 Reagents 
All the reagents shall be of recognized analytical purity. The 
water used shall be distilled water or water of equivalent purity, 
free from carbon dioxide. 
4.1 Sodium hydroxide, Standard volumetric Solution, 
c( NaOH) = 0,I mol/lI). 
Prepare just before use and check the concentration by titration 
immediately before use. 5.2 Apparatus for extrainment by steam distillation (sec 
the figure), comprising the following elements : 
5.2.1 Steam generator, suitable for the production of steam 
free from carbon dioxide, made of heat-resistant glass or metal, 
and of capacity about 1 500 ml. < $ & .” 
5.2.2 Bubbler, comprising a glass tube, of diameter 30 mm 
and 270 mm long, the lower part of which is sealed and en- 
larged to form a sphere of diameter 60 mm in which the test 
Portion is placed. The bubbler shall be placed on a metal disc 
having an orifice of diameter 40 mm, into which the 
bottom of the bubbler is fitted. 
5.2.3 Fractionating column, comprising a glass tube, of 
diameter 20 mm and 500 mm long, inside which is a spiral- 
shaped stainless steel net, No. 100, with a lead of 15 mm. 
Any other device having the same fractionating efficiency may 
be used (see the note to 5.2.4). 
1) Hitherto expressed as “0,l N Standard volumetric solution”. ISO 6632-1981 (E) 
5.2.4 Condenser (West type), diameter 16 mm and nom- 
inally 400 mm long, placed vertically to ensure condensation of 
the vapour and complete cooling of the distillate. 
NOTE - Apparatus other than that described above may be used, pro- 
vided it camplies with the following requirements : 
a) under normal distillation conditions, at least 99,5 % of a 
known amount of acetic acid added to the Sample shall be found in 
250 ml of distillate. To check this, use 20 ml of acetic acid Solution 
of concentration, cKH$OOH) = 0,l mol/ll); 
b) under the same conditions of distillation, no more than 5 Parts 
per thousand of a known amount of lactic acid added to the Sample 
shall be found in the distillate. To check this, use 20 ml of lactic 
acid Solution of concentration, c(C2H50C00H) = 1 mol/l2); 
c) the steam produced by the steam generator shall be free from 
carbon dioxide. Check this by adding two drops of the 
phenolphthalein Solution (4.2) and 0,l ml of the sodium hydroxide 
solution (4.1) to 250 ml of distillate, and verifying that a pink colour 
(stable for at least IO s) appears. 
5.3 Conical flask, of capacity 500 ml. 
5.4 Graduated pipettes, of capacity 20 ml, complying with 
the requirements of ISO/R 835. 
5.5 Burette, of capacity 25 ml, graduated at 0,I ml intervals, 
complying with the requirements of ISO/R 385, class B. 
5.6 Analytical balance. 
6 Procedure (sec clause 8 for the particular 
products containing vola tile acid preservatives) case of 
61 . Preparation of test Sample 
6.l.l Liquid products and those in which the liquid tan 
be easily separated (juices, syrups, liquid from stewed fruit 
compot, brines, etc. 1 
Thoroughly mix the Sample. If the Sample contains solid par- 
ticles in Suspension, separate the liquid by filtration through a 
fluted filter Paper. If the Sample is in the course of fermenta- 
tion, or if it contains carbon dioxide, transfer, by means of a 
pipette, 50 to 60 ml of the product into a 500 ml flask. Remove 
the carbon dioxide by shaking under reduced pressure for 2 to 
3 min. To avoid formation of foam, a small amount of an- 
tifoaming agent may be added, for example about 0,2 g of tan- 
nie acid, directly to the weighed Sample. 
6.1.2 Visco 
concentrated Ius or solid 
juices, dried prod 
fruit 
Remove the seeds and hard seed-cavity Walls if necessary, 
homogenize the Sample in t ,he mechanical mill (5.1). ucts 
and (marmalade, jams, 
vegeta bles, etc. ) 
1) Hitherto expressed 
2) Hitherto expressed 
3) Using 
distillation jellies, 
as “0,l N solution”. 
as “1 N solution”. 
20 ml of liquid test Portion, the volume is not reduced, as the steam is introduced into the flask at the maximum rate at the Start of the 6.1.3 Frozen or deep-frozen products 
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. 
6.2 Test Portion 
6.2.1 Liquid products 
Transfer, by means of a pipette (5.41, 20 ml of the test Sample 
(6.1 .I) to the bubbler (5.2.2). In the case of samples which have 
strong volatile acidity, a smaller amount may be used and the 
total volume tan be made up to 20 ml by addition of the re- 
quired amount of water. 
6.2.2 Viscous or solid or frozen products 
In a beaker, weigh, to the nearest 0,OI g, about IO g of the test 
Sample (6.1.2 or 6.1.3). Transfer the test Portion to the bubbler 
(5.2.2) with the minimum quantity of water necessary for en- 
trainment of the whole test Portion and to render the mixture 
sufficiently fluid. 
6.3 Entrainment sf volatile acids 
Fill the steam generator (5.2.1) to two-thirds of its volume with 
the lime water (4.4). Add 0,5 g of the tartaric acid (4.3) to the 
test Portion (6.2) contained in the bubbler (5.2.2). Connect the 
latter to the steam generator (5.2.1) and to the fractionating 
column (5.2.3) and condenser (5.2.4). Heat the steam 
generator and the bubbler simultaneously on burners. lf the 
initial volume of the contents of the bubbler is more than 20 ml, 
regulate the heating so as to reduce the volume to about 20 ml 
while the steam is slowly introduced.3) Reduce the rate of 
heating of the bubbler to maintain the volume of the contents 
constant (about 20 ml) for the duration of the distillation during 
which the output of steam from the generator (5.2.1) is at its 
maximum. The distillation shall last for IO to 15 min. 
Collect the distillate in the conical flask (5.3), continuing the 
distillation until about 250 ml of distillate has been collected. 
The volume of distillate shall be at least 12 times that of the test 
Portion (6.2). 
6.4 Titration of volatile acids 
Add two drops of the phenolphthalein Solution (4.2) to the 
distillate and titrate with the sodium hydroxide Solution (4.1) 
until a light pink colour, persisting for at least 15 s, appears. 
65 . Number of determinations 
Carry out two determinations on the same test Sample (6.2). 
2 ISO 66324981 (El 
7 Expression of resuks (sec clause 8 for the particular 
case of products containing volatile acid preservatives) 
7.1 Method of calculation and formula 
7.1 .l Liquid products 
The volatile acidity, expressed in milliequivalents per 100 ml of 
product, or in grams of acetic acid per 100 ml of product, is 
given respectively by formulae (1) and (2) : 
IOX v, 
. . . (1) 
VO 
0,6 x V, 
. . . (2) 
VO 
where 
V. is the volume, in millilitres, of the test Portion (6.2.1); 
V, is the volume, in millilitres, of sodium hydroxide solu- 
tion (4.1) required for the titration. 
7.1.2 Viscous or solid or frozen products 
The volatile acidity, expressed in milliequivalents per 100 g of 
product, or in grams of acetic acid per 100 g of product, is 
given respectively by formulae (3) and (4) : 
1ox v, . 
. . . (3) 
m0 
0,6 x V, 
. . . (4) 
m0 
where 
m. is the mass, in grams, of the test Portion (6.2.2); 
VI has the same meaning as in 7.1 .l. 
7.1.3 Resuit 
Take as the result the arithmetic mean of the values obtained in 
the two determinations (6.5), provided that the requirements 
for precision (sec 7.2) are satisfied. 
7.2 Precision 
7.2.1 Repeatability 
The differente between the values obtained in the two deter- 
minations (6.51, carried out simultaneously or in rapid succes- 
sion by the same analyst, shall not exceed 0,2 milliequivalents 
per 100 ml or per 100 g, or 12 mg of acetic acid per 100 ml or 
per 100 g. 
1) Hitherto expressed as “0,Ol N Standard volumetric solution”. 7.2.2 Reproducibility 
The differente between the results obtained in two different 
laboratories, on the same Sample shall not exceed 0,5 milli- 
equivalents per 100 ml or per 100 g, or 30 mg of acetic acid per 
100 ml or per 100 g. 
8 Particular case of products containing 
volatile acid preservatives 
In the case of products containing sulphur dioxide, with or 
without sorbic acid, benzoic acid or formic acid, determine the 
amount of preservative present in the distillate and correct the 
result of the titration accordingly. 
8.1 Determination of sulphur dioxide in the 
distillate (in the absence of other preservatives) 
8.1.1 Reagents 
8.1.1.1 lodine, Standard volumetric Solution, 
ct 1/2 12) = 0,Ol mol/lY 
8.1.1.2 Potassium iodide, crystallized. 
8.1.1.3 Hydrochlorit acid, concentrated. 
8.1.1.4 Sodium borate, saturated Solution (about 60 g of 
sodium borate decahydrate per litre). 
8.1.1.5 Starch indicator : Suspension of 5 g of soluble 
starch in 1 litre of water containing 200 g of sodium chloride as 
preservative. Boil the Solution for 10 min during preparation. 
8.1.2 Titration 
Add 1 drop of the hydrochloric acid (8.1.1.3), 5 ml of the starch 
indicator (8.1.1.5) and about 0,l g of the potassium iodide 
(8.1.1.2) to the distillate after titration of the volatile acids (6.41, 
and titrate the free sulphur dioxide with the iodine Solution 
(8.1.1.1). Record the volume of iodine Solution required for this 
first titration. 
Add 20 ml of the sodium borate Solution (8.1.1.4) to bring the 
pH to between 9 and 9,5 and titrate the combined sulphur diox- 
ide with the iodine Solution (8.1 .l. 1) until a violet-blue colour 
(stable for at least 30 s) is formed. Record the volume of iodine 
Solution required for this second titration. 
8.1.3 Expression of results 
Correct the volume VI of sodium hyd roxide sol ution (4.1) used 
for the titration, for the acidity of the free and combined 
3 ISO 6632-1981 (E) 
sulph ur dioxide, expressed in mil 
soluti on, by mea ns of formu Ia (5) lilitres of 0,l mol/1 alkaline This Solution is unstable and shall be used within 5 h of 
preparation. 
8.2.2 Apparatus 
8.2.2.1 Dish, of porcelain or heat-resistant glass, 
diameter 55 mm. 
8.2.2.2 One-mark volumetric flasks, of capacities 
and 250 ml. 
8.2.2.3 Boiling water bath. h -- 
10 V3 . . . 
20 (5) v; = 5 - 
and of 
where 
Vi is the volume, in m illilitres, of sodium hydroxide solu- 
tion (4. 1) corresponding to the corrected volatile acidity; 10 - 20 
V2 is the volume, in millilitres, of iodine Solution (8.1.1 .l) 
used for the titration of free sulphur dioxide; 
V3 is the volume, in millil itres, of iodine Solution 
used for the titration of CO imbined sulphu r dioxide (8.1.1.1) 8.2.2.4 Spectrophotometer, suitable for use in the vrsrbre 
and ultraviolet regions of the spectrum, fitted with silica cells of 
Optical path length 1 cm. 
The formulae given in 7.1 may be used directly by substituting 
Vi for VI. 8.2.3 Identification and determination 
Collect a known volume V (about 250 ml) of distillate as 
described in 6.3. Confirm the presence of sorbic acid and deter- 
mine the sorbic acid content before determination of the 
volatile acidity. 8.2 Determination of sorbic acid in the distillate 
(with or without the presence of sulphur dioxide) 
8.2.1 Reagents 
8.2.3.1 Identification 
8.2.1 .l Sorbit acid, Standard Solution corresponding to 
20 mg of sorbic acid per litre, or 26,8 mg of potassium sorbate 
per litre. 8.2.3.1.1 Place 10 ml of the distillate in the dish (8.2.2.1). 
Make alkaline by adding 1 ml of the lime water (4.4) and add 
1 drop of the copper(ll) sulphate Solution (8.2.1.3). (This solu- 
tion catalyses the Oxidation of sulphur dioxide by atmospheric 
Oxygen.) Evaporate to dryness on the boiling water bath 
(8.2.2.3), thus oxidizing the sulphur dioxide, volatilizing the 
ethanol and other alcohols, aldehydes, ketones and aromatic 
esters and fixing the acids as Calcium salts. Dissolve the residue 
in 10 ml of water. 8.2.1.2 Oxidizing agent. 
Dissolve 1 0,5 g of sodium hydrogen carbonate and 0,001 g of 
copper( I 1) sulp Ihate pentahydrate in 1 000 ml of water. 
8.2.1.3 Copper(ll) sulphate, 0,l g/l Solution. 
8.2.3.1.2 Transfer 5 ml of this Solution to a 10 ml volumetric 
flask, and add 2 ml of the chromic-sulphuric acid Solution 
(8.2.1.4). Keep the flask in the boiling water bath for 10 min. 
Add 3 ml of the thiobarbituric acid Solution (8.2.1.5) and keep 
in the boiling water bath for a further 20 min. 8.2.1.4 Chromic-sulphuric acid solution. 
Dissolve 50 mg of potassium dichromate in about 90 ml of 
water in a 200 ml one-mark volumetric flask. Add 10 ml of 
sulphuric acid Solution, c(l/2 H$Od) = 0,3 mol/1 11, and dilute 
to the mark with water. The appearence 
sorbic acid. of a pink colour confirms the presence of 
NOTE - 1 litre of sulphuric acid solution, 
c( 1/2 H$504) = 0,3 mol/1 11, contains 14,7 g of sulphuric acid, or 8 ml 
of sulphuric acid, ~2~ = 1,84 g/ml. 8.2.3.2 Determination 
Carry out the determination of sorbic acid in the 
one of the following methods. distillate 8.2.1.5 Thiobarbituric acid solution. 
Dissolve 0,5 g of thiobarbituric acid in 50 ml of water and add 
IO ml of sodium hydroxide Solution, c(NaOH) = 1 mol/W 
Transfer to a 100 ml one-mark volumetric flask, add 11 ml of 
hydrochloric acid Solution, c(HCI) = 1 mol/l2), and dilute to 
the mark with water. 8.2.3.2.1 Colorimetric method 
After 20 min in the boiling water bath, cool the pink-coloured 
Solution obtained in 8.2.3.1.2 in an ice-water bath. Dilute to 
1) Hitherto expressed as “0,3 N solution”. 
2) Hitherto expressed as “1 N solution”. 
4 ISO 66324981 (E) 
10 ml with water and measure the absorbance of the Solution 
by means of the spectrophotometer (8.2.2.4) at a wavelength 
of 532 nm, using, as the reference liquid, a blank solution 
prepared by the same procedure (8.2.3.1.2) but using 5 ml of 
distilled water in place of the distillate. 
Compare the absorbance with that of suitably diluted stand& 
sorbic acid Solution (8.2.1.11, treated as described in 8.2.3.1.2. 
8.2.3.2.2 Ultraviolet absorption methodl) 
Dilute an aliquot Portion of the distillate 1 + 3 or 1 + 9 or 
1 + 19 with the oxidizing agent (8.2.1.2) in a 10 ml or 20 ml 
volumetric flask (8.2.2.2). (This Solution catalyses the Oxidation 
of sulphur dioxide by atmospheric Oxygen.) Mix and leave ex- 
posed to the air for a few minutes. Fill one of the silica cells (see 
8.2.2.4) with this Solution. 
Measure the absorbance of the Solution by means of the spec- 
trophotometer (8.2.2.4) at a wavelength of 256 nm, using, as 
the reference liquid, a blank Solution prepared by the same pro- 
cedure but using water in place of the distillate. The measured 
absorbance shall be less than 0,7; this determines the Order of 
dilution to adopt. 
Compare the absorbance with that of the Standard sorbic acid 
Solution (8.2.1 .l) diluted 1 + 3 with the oxidizing agent 
(8.2.1.2). 
8.2.4 Expression sf results 
The sorbic acid content of the distillate, expressed in milligrams 
per litre, is given by formula (6) : 
A 
20 x2 
4 
where . . . (6) 
A, is the absorbance of the diluted distillate, at 532 nm 
(see 8.2.3.2.1) or at 256 nm (see 8.2.3.2.2); 
A, is the absorbance of the reference Solution (8.2.1.1), 
diluted in the same manner, at 532 nm (see 8.2.3.2.1) or at 
256 nm (see 8.2.3.2.2). 
8.2.5 Determination of distillate acidity 
The acidity of the sorbic acid, expressed in millilitres of 
0,l mol/1 alkaline Solution, is given by formula (7) : 
20 A V’ -xxx- 
11,2 A, 1000 
where l . . (7) 
V’ is the remaining volume, in millilitres, of distillate, 
slightly less than V (the volume of distillate collected - see 
8.2.3); 
1) A, and A, have the same meanings as in 8.2.4; 
11,2 is the mass, in milligrams, of sorbic acid 
1 ml of 0,l mol/1 sodium hydroxide Solution. equivalent to 
8.2.6 Method of calculation and formulae 
Correct the volume V4 of sodium hydroxide Solution (4.1) 
used for the titration for the acidity of the sorbic acid as 
follows. 
8.2.6.1 Liquid products 
The volatile acidity of the Sample, expressed in milliequivalents 
per 100 ml, or in grams of acetic acid per 100 ml, is given 
respectively by formulae (8) and (9) : 
1 V 
F- . . . (8) 
(9) 
V4 is the corrected volume, in millilitres, 
hydroxide Solution (4.1 used for the titration; of sodium 
A, and A, have the same meanings as in 8.2.4; 
V and V’ have the same meanings as in 8.2.5; 
VO has the same meaning as in 7.1.1. 
8.2.6.2 Viscous or solid or frozen products 
The volatile acidity of the Sample, expressed in milliequivalents 
per 100 g, or in grams of acetic acid per 100 g, is given 
respectively by formulae (10) and (11) : 
. . . 
. . . 
where 
V4 has the same meaning as in 8.2.6.1; 
A, and A, 
V and V’ have the same meanings as in 8.2.4; 
have the same meanings as in 8.2.5; 
m0 has the same meaning as in 7.1.2. (10) 
(11) 
For products containing volatile oils and volatile aromatic compounds (such as the aurantiaceae), only the colorimetric method may be used. 
5 ISO 6632-1981 (EI 
8.3 Determination of benzoic acid in the distillate 
(with or without the presence of sulphur dioxide) Compare the absorbance with that of the Standard benzoic acid 
Solution (8.3.1 .l) diluted 1 + 1 with water. 
8.3.1 Reagent 8.3.3 Expression of results 
The benzoic acid content of the distillate, expressed 
milligrams per litre, is given by formula (6’) : 8.3.1 .l Benzoic ac ‘id, Standard so lution containing 20 mg of 
benzoic acid, or 23,6 mg of sodium benzoate, per litre. 
A 
20 xx . . . (6’) 
4 8.3.2 Identification and determination 
Collect a known volume V (about 250 ml) of distillate as 
described in 6.3. Confirm the presence of benzoic acid and 
determine the benzoic acid content before the determination of 
volatile acidity. A, is the absorbance of the diluted distillate (see 8.3.2.2); 
‘A, is the absorbance of the reference Solution (8.3.1.11, 
diluted in the same manner (see 8.3.2.2). 8.3.2.1 Identificationl) 
Place 2 ml of distillate in a silica cell (see 8.2.2.41, and record 
the absorption spectrum between 220 and 300 nm. If the 
absorbance exceeds 0,7, carry out the same examination on the 
distillate diluted 1 + 1, 1 + 3 or 1 + 9, with water. 8.3.4 Determination of distillate acidity 
The acidity of benzoic acid, expressed in millilitres of 0,l mol/1 
alkaline Solution, is given by formula (12) : Benzoic acid is characterized in this solvent by two maximum 
absorption bands, one between 225 and 230 nm, and the other 
at 268 nm. 20 A V” XX- 
12,2 ’ A, . . . (12) 1000 
8.3.2.2 Determination 
where 
8.3.2.2.1 In the absence of volatile oils, aromatic compounds 
and sulphur dioxide, measure the maximum absorbance, bet- 
ween 225 and 230 nm, of the distillate, suitably diluted, in a 
silica cell (see 8.2.2.41, using water as the reference liquid. V” is the 
slightly less 
8.3.2); remaining volume, in millilitres, of distillate, 
than V (the volume of d istillate collected - see 
A, and A, have the same meanings as in 8.3.3; 
Compare the absorbance with that of the Standard 
Solution (8.3. 1.1) diluted 1 + 1 with water. benzoic 
12,2 is the mass, in milligrams, of benzoic acid 
to 1 ml of 0, 1 mol/1 sodium hyd roxide solu tion. equivalent 
8.3.2.2.2 If the product contains sulphur dioxide, dilute the 
distillate 1 + 1, 1 + 3 or 1 + 9 with the oxidizing agent 
(8.2.1.21, Shake and leave exposed to the air for a few minutes. 8.3.5 Method of calculation and formulae 
Correct the volume Vk of sodium hydroxide Solution ( 4.1) used 
for the titration for the acidity of the benzoic acid as follows. Measure the maximum absorbance, between 225 and 230 nm, 
using, as the reference liquid, a blank Solution prepared by 
diluting the oxidizing agent (8.2.1.2) with yvater, to replace the 
distillate. 8.3.5.1 Liquid products 
8.3.2.2.3 If the product contains volatile oils or volatile 
aromatic compounds (such as the aurantiaceae), with or 
Githout sulphur dioxide, place 10 ml of the distillate in the dish 
(8.2.2.1). Make alkaline by adding 1 ml of the lime water (4.4), 
add 1 drop of the copper(ll) sulphate Solution (8.2.1.3) and 
evaporate to dryness on the boiling water bath (8.2.2.3). 
Dissolve the residue in 10 ml of water. 20 A V” V xx- - 
il . . . 1 ooo V" (13) 
Measure the maximum absorbance of the Solution at about 
230 nm using, as the reference liquid, a blank Solution prepared 
by the same procedure, but using 10 ml of water in place of the 
distillate. 20 A, VI, V --X-- 
12,2 x A, 11 . . . 1 ooo V” (14) 
compounds (such as the aurantiaceae), the distillate should be treated as indicated in 1) For products CO ntaining volatile oils and volatile aromatic 
2.2.3 before exa mination of the ultraviolet spectrum. 8.3. ISO 6632-1981 (EI 
where 
55 is the corrected volume, In millilitres, 
hYd roxide Solution (4.1 ) used for the titration; 
A, and A, of sodium 
have the same meanings as in 8.3.3; 
V and V” have the same meanings as in 8.3.4; 
8.3.5.2 has the same meaning as in 7.1.1. 
Viscous or solid or frozen products 
The volatile acidity of the Sample, expressed in milliequivalents 
per 100 g, or in grams of acetic acid per 100 g, is given respect- 
ively by formulae (15) and (16) : 
. . (15) 
. . (16) 
where 
V5 has the same meaning as in 8.3.5; 
A, and A,. have the same meanings as in 8.3.3; 
V and V” have the same meanings as in 8.3.4; 
mg has the same meaning as in 7.1.2. 
8.4 Determination of volatile acidity in the 
presence of formic acid (with or without the presence of 
sulphur dioxide) 
8.4.1 Reagents 
8.4.1.1 Sodium acetate, crystallized. 
8.4.1.2 MercuryW chloride Solution. 
Dissolve 1’5 g of mercury(II) chloride in 100 ml of water. 
8.4.1.3 Potassium dichromate, 33’79 g/l solution. 
8.4.1.4 Sulphuric acid, diluted 2 + 1. 
8.4.1.5 IronW) sulphate. 
8.4.2 Apparatus 
8.4.2.1 Microscope. 8.4.2.2 Boiling water bath. 
8.4.2.3 Heat-resistant dish, of capacity 500 ml. 
8.4.3 Identification of formic acid and determination of 
volatile acidity 
8.4.3.1 Identification of formic acid 
Collect a known volume V (about 250 ml) of distillate as 
described in 6.3. Confirm the presence of formic acid before 
the determination of volatile acidity. 
Transfer 2 ml of distillatel) to a test tube. Add 0’25 g of the 
sodium acetate (8.4.1.1) and 1 ml of the mercury(II) chloride 
Solution (8.4.1.2). Heat on the boiling water bath (8.4.2.2) for 
10 min. After cooling, examine the crystalline precipitate of 
mercury(lI) chloride under the microscope for Cross- or 
X-shaped crystals which characterize the presence of formic 
acid. 
Under the conditions indicated, the presence of formic acid is 
detected in the initial product (6.2) at a minimum level of detec- 
tion of 0’5 g/l, which is much less than the effective antiseptic 
dose. However, smaller traces of formic acid which may occur 
naturally in vegetable products cannot be detected in this way. 
8.4.3.2 Oxidation of formic acid and of any sulphur dioxide 
Titrate the volatile acidity of the distillate with the sodium 
hydroxide Solution (4.1) using the phenolphthalein Solution 
(4.2) as indicator. Add 10 ml of the sodium hydroxide Solution 
to make the distillate strongly alkaline, and concentrate the 
volume in the dish (8.4.2.3) on the boiling water bath (8.4.2.2) 
to obtain 2 to 5 ml of liquid residue. Alcohols, aldehydes, 
ketones, esters, volatile oils and aromatic compounds are thus 
removed. Put the residue in the bubbler (5.2.2) of the steam 
distillation apparatus (5.21, rinse the dish with 10 ml of the 
potassium dichromate Solution (8.4.1.3)’ then with 10 ml of the 
sulphuric acid Solution (8.4.1.41, collecting these liquids suc- 
cessively in the bubbler. Shake and leave for at least 15 h. 
Under these conditions, the formic acid and sulphur dioxide are 
totally oxidized while the acetic acid and other higher 
homologous compounds which constitute the volatile acidity 
remain unaffected. (This procedure does not allow specific 
determination of the formic acid contained in the distillate.) 
Reduce the pressure in the bubbler, and Shake to remove the 
carbon dioxide formed. Reduce the excess of potassium 
dichromate by adding 2 to 3 g of the iron(ll) sulphate (8.4.1.5)’ 
collect the distillate and titrate the volatile acids by the pro- 
cedure described in 6.3 and 6.4. 
8.4.4 Expression of results 
See 7.1. 
1) Taking 2 ml of distillate for the identification of the formic acid (8.4.3.1) introduces a negligible error in the result of the determination (8.4.3.2). 
7 ISO 6632-1981 (El 
9 Test report International Standard or regarded as optional, as well as any 
incidents likely to have affected the results. 
The test report shall show the method used and the result ob- 
tained, indicating clearly the method of calculation used. lt 
shall also mention all operating details not specified in this The test report shall give all the information necessary for the 
complete identification of the Sample. ISO 66324981 (EI 
Steam generator (5.2.1) 
Lime water (4.4) Fractionating 
column (5.2.3) 
Bubbler (5.2.2)--- 
-2 -- - r / 
x 
> -0 
% 
x 
k 
Y 4 
\ Condenser (5.2.4)--- 
Test Portion (6.2) 
f + tartaric acid (4.3) 7 
i Conical flask (5.3) 
Distillate 
Figure Steam distillation apparatus (5.2) This page intentionally left blank