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1、INTERNATIONAL STANDARD ISO 5518 Second edition 2007-02-01 Reference number ISO 5518:2007(E) ISO 2007 Fruits, vegetables and derived products Determination of benzoic acid content Spectrophotometric method Fruits, lgumes et produits drivs Dtermination de la teneur en acide benzoque Mthode spectrophot
2、omtrique Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=Defense Supply Ctr/9960866100 Not for Resale, 04/24/2007 11:01:44 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 5518:2007(E) ii ISO 2007 All rights reserv
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6、 2007 All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below or ISOs member body in the
7、country of the requester. ISO copyright office Case postale 56 CH-1211 Geneva 20 Tel. + 41 22 749 01 11 Fax + 41 22 749 09 47 E-mail copyrightiso.org Web www.iso.org Published in Switzerland Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=Defe
8、nse Supply Ctr/9960866100 Not for Resale, 04/24/2007 11:01:44 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 5518:2007(E) ISO 2007 All rights reserved iii Foreword ISO (the International Organization for Standardization) is a worldwide federation of national standards
9、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 organizat
10、ions, 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 th
11、e 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
12、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 produc
13、ts, Subcommittee SC 3, Fruit and vegetable products. This second edition cancels and replaces the first edition (ISO 5518:1978), which has been technically revised. Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=Defense Supply Ctr/9960866100
14、Not for Resale, 04/24/2007 11:01:44 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 5518:2007(E) iv ISO 2007 All rights reserved Introduction A method for determining the benzoic acid content of fruits, vegetable and derivatives, described in 1959, was based on the tech
15、nique of peak emergence. The advantage of this method was that it was specifically intended for 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 elimin
16、ation of the effects of colouring substances in certain vegetable products containing anthocyanins, and also all the oxybenzoic acids and sorbic acid which may be present if several antiseptics have been used. Moreover, purification increases the sensitivity of the method. This improved technique wa
17、s also more rapid. Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=Defense Supply Ctr/9960866100 Not for Resale, 04/24/2007 11:01:44 MDTNo reproduction or networking permitted without license from IHS -,-,- INTERNATIONAL STANDARDISO 5518:2007(
18、E) ISO 2007 All rights reserved 1 Fruits, vegetables and derived products Determination of benzoic acid content Spectrophotometric method 1Scope This International Standard specifies a method for determining the benzoic acid content of fruits, vegetables and derived products. As chlorobenzoic acids
19、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. NOTET
20、he 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. 2Principle The test sample is homogenized, followed by dilu
21、tion 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 using acidified potassium dichromate. The purified benzoic acid dissolved in diethyl ether is determined by spectrophotometry. 3Reag
22、ents Use only reagents of recognized analytical quality and distilled water or water of at least equivalent purity. 3.1Tartaric acid COOH(CHOH)2COOH, crystalline. 3.2Sodium hydroxide (NaOH), approximately solution. 3.3Potassium dichromate (K2Cr2O7), solution containing to . 3.4Dilute sulfuric acid (
23、H2SO4), obtained by diluting 2 volumes of concentrated sulfuric acid () with 1 volume of water. 3.5Diethyl ether (CH3CH2)2O, recently distilled. 3.6Benzoic acid (C6H5COOH), standard solution in diethyl ether containing . 3.7Sodium hydrogen carbonate (NaHCO3), crystalline. 4Apparatus Usual laboratory
24、 apparatus and, in particular, the following. 4.1Volumetric flasks, of capacities and . 4.2Beakers, of capacities and . 1 mol/l 33 g/l34 g/l 20=1,84 g/ml 0,100 g/l 50 ml1 000 ml 50 ml100 ml Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=Defen
25、se Supply Ctr/9960866100 Not for Resale, 04/24/2007 11:01:44 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 5518:2007(E) 2 ISO 2007 All rights reserved 4.3Graduated pipettes, of capacities , and . 4.4Flasks, of capacity , having ground-glass stoppers and made of borosi
26、licate glass. 4.5Separating funnels, of capacities and . 4.6Evaporating dish, of diameter about . 4.7Water bath, capable of being controlled at a temperature of to . 4.8Homogenizer or mortar, as appropriate. 4.9Spectrophotometer for determination in the ultraviolet range, equipped with a monochromat
27、or allowing measurement to the nearest , with silica cells of optical path length or (preferably so as to increase sensitivity), equipped with ground glass covers. 4.10Analytical balance. 5Sampling A representative sample should have been sent to the laboratory. It should not have been damaged or ch
28、anged during transport or storage. 6Procedure 6.1Preparation of test sample 6.1.1Liquid products (e.g. juices, pulpy fluid products, syrups) Thoroughly mix the laboratory sample. 6.1.2Thick products (e.g. marmalades, jams) Homogenize the laboratory sample after having carefully mixed it. 6.1.3Solid
29、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. 6.1.4Frozen or deep-frozen products After thawing the sample in a closed container and removing, if
30、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 or 6.2.3 as appropriate. 6.2Preparation of test portion 6.2.1Liquid products Using a pipette (4.3), take of the test sample (6.1), free from subst
31、ances in suspension, dilute it with approximately of water and transfer it to a separating funnel (4.5) (separating funnel A). 10 ml 20 ml50 ml 250 ml 100 ml500 ml 10 cm 70 C 80 C 0,5 nm10 mm20 mm20 mm 40 g 20 ml 50 ml500 ml Copyright International Organization for Standardization Provided by IHS un
32、der license with ISO Licensee=Defense Supply Ctr/9960866100 Not for Resale, 04/24/2007 11:01:44 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 5518:2007(E) ISO 2007 All rights reserved 3 The test portion may also be taken by mass by weighing, to the nearest , approxima
33、tely of the test sample. 6.2.2Pulpy 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 separati
34、ng 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. 6.2.3Thick 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 appr
35、oximately 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 and rinse twice using to of water each time. Collect all the filtrates in a separating funnel (4.5) (separating funnel
36、A). Allow to cool. NOTEThe addition of sodium hydrogen carbonate is intended to neutralize the benzoic acid, traces of which could be lost by volatilization. 6.3Extraction of the benzoic acid WARNING Attention is drawn to the hazard derived from the use of diethyl ether, which is a highly flammable,
37、 explosive and harmful substance. 6.3.1Introduce 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) (separa
38、ting 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 t
39、he ethereal layer with the first two in the separating funnel (B). 6.3.2Extract 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 to separate and collect the aqueous phas
40、e. 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 g20 g 20 ml20 ml 20 ml 500 ml 0,01 g20 g 0
41、,01 g10 g30 ml40 ml 250 ml 50 mg 70 C 80 C 15 min30 min 15 ml20 ml 500 ml 1 g 60 ml 500 ml 60 ml 30 ml 10 ml5 ml 10 ml 70 C 80 C Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=Defense Supply Ctr/9960866100 Not for Resale, 04/24/2007 11:01:44
42、MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 5518:2007(E) 4 ISO 2007 All rights reserved 6.4Purification 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
43、 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
44、 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. 6.5Extraction of the purified benzoic acid Extract the benzoic aci
45、d 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. After decanting very carefully, filter through a dry filter paper and collect the filtrate in a volumetric flask (4
46、.1). Then wash the filter with several millilitres of the diethyl ether, adding sufficient washing solvent to dilute to the mark. 6.6Determination Using the spectrophotometer (4.9), measure the absorbance of the ethereal solution (6.5) in relation to the absorbance of the pure diethyl ether at , and
47、 (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 . NOTEExamination of the absorption spectrum of the ethereal solution of purified benzoic acid allows charac
48、terization 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 . 6.7Number of determinations Carry out two determinations on the same test sample (6.1). 6.8Plotting 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).