1、| | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | BRITISH STANDARD BS EN 1379 : 1997 The Eur
2、opean Standard EN 1379 : 1996 has the status of a British Standard ICS 67.180.10 NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAW Foodstuffs Determination of cyclamate and saccharin in liquid table top sweetener preparations Method by high performance liquid chromatographyBS EN
3、 1379 : 1997 This British Standard, having been prepared under the direction of the Consumer Products and Services Sector Board, was published under the authority of the Standards Board and comes into effect on 15 May 1997 BSI 1997 The following BSI references relate to the work on this standard: Co
4、mmittee reference AW/-/3 Draft for comment 94/501927 DC ISBN 0 580 27125 0 Amendments issued since publication Amd. No. Date Text affected Committees responsible for this British Standard The preparation of this British Standard was entrusted to Technical Panel AW/-/3, Food analysis Horizontal metho
5、ds, upon which the following bodies were represented: Association of Public Analysts Food and Drink Federation Institute of Food Science and Technology Laboratory of the Government Chemist Ministry of Agriculture, Fisheries and Food Royal Society of ChemistryBS EN 1379 : 1997 BSI 1997 i Contents Pag
6、e Committees responsible Inside front cover National foreword ii Foreword 2 Text of EN 1379 3ii BSI 1997 BS EN 1379 : 1997 National foreword This British Standard has been prepared by Technical Panel AW/-/3 and is the English language version of EN 1379 : 1996 Foodstuffs Determination of cyclamate a
7、nd saccharin in liquid table top sweetener preparations Method by high performance liquid chromatography, published by the European Committee for Standardization (CEN). EN 1379 was produced as a result of international discussions in which the United Kingdom took an active part. Cross-references Pub
8、lication referred to Corresponding British Standard EN ISO 3696 : 1995 BS EN ISO 3696 : 1995 Water for analytical laboratory use. Specification and test methods ISO 5725 : 1986, to which informative reference is made in the text, has been superseded by ISO 5725-1 : 1994, ISO 5725-2 : 1994, ISO 5725-
9、3 : 1994, ISO 5725-4 : 1994 and ISO 5725-6 : 1994 which are identical with BS ISO 5725 Accuracy (trueness and precision) of measurement methods and results, BS ISO 5725-1 : 1994 General principles and definitions, BS ISO 5725-2 : 1994 Basic method for the determination of repeatability and reproduci
10、bility of a standard measurement method, BS ISO 5725-3 : 1994 Intermediate measures of the precision of a standard measurement method, BS ISO 5725-4 : 1994 Basic method for the determination of the trueness of a standard measurement method, and BS ISO 5725-6 : 1994 Use in practice of accuracy values
11、. Compliance with a British Standard does not of itself confer immunity from legal obligations. Summary of pages This document comprises a front cover, an inside front cover, pages i and ii, the EN title page, pages 2 to 10, an inside back cover and a back cover.CEN European Committee for Standardiz
12、ation Comite Europe en de Normalisation Europa isches Komitee fu r Normung Central Secretariat: rue de Stassart 36, B-1050 Brussels 1996 Copyright reserved to CEN members Ref. No. EN 1379 : 1996 E EUROPEAN STANDARD EN 1379 NORME EUROPE ENNE EUROPA ISCHE NORM September 1996 ICS 67.180.10 Descriptors:
13、 Food products, intense sweeteners, chemical analysis, determination of content, high performance liquid chromatography English version Foodstuffs Determination of cyclamate and saccharin in liquid table top sweetener preparations Method by high performance liquid chromatography Produits alimentaire
14、s Dosage du cyclamate et de la saccharine dans les e dulcorants de table liquides Me thode par chromatographie liquide a haute performance Lebensmittel Bestimmung von Cyclamat und Saccharin in Flu ssigtafelsu en Hochleistungs- Flu ssigkeitschromatographisches Verfahren This European Standard was app
15、roved by CEN on 1996-06-08. CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national st
16、andards may be obtained on application to the Central Secretariat or to any CEN member. This European Standard exists in three official versions (English, French, German). A version in any other language made by translation under the responsibility of a CEN member into its own language and notified
17、to the Central Secretariat has the same status as the official versions. CEN members are the national standards bodies of Austria, Belgium, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and United Kingdom.Pag
18、e 2 EN 1379 : 1996 BSI 1997 Foreword This European Standard has been prepared by the Technical Committee CEN/TC 275, Food analysis Horizontal methods, the Secretariat of which is held by DIN. This European Standard shall be given the status of a national standard, either by publication of an identic
19、al text or by endorsement, at the latest by March 1997, and conflicting national standards shall be withdrawn at the latest by March 1997. According to the CEN/CENELEC Internal Regulations, the following countries are bound to implement this European Standard: Austria, Belgium, Denmark, Finland, Fra
20、nce, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and the United Kingdom. Contents Page Foreword 2 1 Scope 3 2 Normative references 3 3 Principle 3 4 Reagents 3 5 Apparatus and equipment 4 6 Procedure 4 7 Expression of results 5 8 Pr
21、ecision 5 9 Test report 6 Annexes A (informative) Figures 7 B (informative) Precision data 10 C (informative) Bibliography 10Page 3 EN 1379 : 1996 BSI 1997 1) c is the substance concentration. 2) r is the mass concentration. 3) conversion factor from sodium cyclamate to cyclohexane sulfamic acid = 0
22、,8906. 4) conversion factor from sodium saccharin to saccharin as free imide = 0,7593. 1 Scope This European Standard specifies a high performance liquid chromatography (HPLC) method for the determination of sodium cyclamate and saccharin in liquid table top sweetener preparations. It also allows th
23、e determination of sorbic acid in liquid table top sweetener preparations. An inter-laboratory test has been carried out with liquid table top sweetener preparation 1. 2 Normative references This European Standard incorporates by dated or undated reference, provisions from other publications. These
24、normative references are cited at the appropriate places in the text and the publications are listed hereafter. For dated references, subsequent amendments to or revisions of any of these publications apply to this European Standard only when incorporated in it by amendment or revision. For undated
25、references the latest edition of the publication referred to applies. EN ISO 3696 Water for analytical laboratory use Specification and test methods 3 Principle Determination of sodium cyclamate, saccharin and sorbic acid in an appropriate dilution of a liquid table top sweetener preparation in wate
26、r by HPLC and subsequent photometric detection in the ultraviolet (UV) range. Identification on the basis of the retention times, and quantitative determination by the external standard method using peak areas or peak heights. 4 Reagents During the analysis, unless otherwise stated, use only reagent
27、s of recognized analytical grade and water of at least grade 1 as defined in EN ISO 3696. 4.1 Potassium dihydrogen orthophosphate solution, c (KH 2 PO 4 ) = 0,0125 mol/l. 1) 4.2 Methanol, suitable for HPLC analysis. 4.3 Phosphoric acid. 4.4 HPLC mobile phase. Mix 70 parts by volume of the potassium
28、dihydrogen orthophosphate solution (4.1) and 30 parts by volume of methanol (4.2) and adjust the pH to 4,5 with phosphoric acid (4.3). Remove particles by membrane filtration (see 5.2). In order to avoid corrosion problems due to prolonged contact with phosphate containing eluents and as a precautio
29、n against blockages occurring due to precipitation of phosphate, water should be pumped through the HPLC equipment after carrying out this method. 4.5 Standard substances 4.5.1 Sodium cyclamate standard substance, with a known content of at least 98 % in dry matter (105 C 2 C, to constant mass). The
30、 loss in mass on drying shall not exceed 1 %. NOTE. For further information on identification and purity, see 2. 4.5.2 Sodium saccharin standard substance, with a known content of at least 98 % in dry matter (105 C 2 C, to constant mass). The loss in mass on drying shall not exceed 15 %. NOTE. For f
31、urther information on identification and purity, see 2. 4.5.3 Sorbic acid standard substance, with a known content of at least 99 % after being stored for 4 h in a vacuum of about 33 hPa over concentrated sulfuric acid at room temperature. 4.6 Stock solutions 4.6.1 Sodium cyclamate stock solution, r
32、 (C 6 H 11 NHSO 3 Na) 4 mg/ml 2) Finely grind at least about 1,7 g of the sodium cyclamate standard substance (4.5.1), then, without delay, dissolve about 400 mg of the undried sodium cyclamate standard substance (equivalent to about 356 mg of cyclamate as free acid) 3) , weighed to the nearest 0,1
33、mg, in the mobile phase (4.4)i n a 100 ml volumetric flask and dilute to the mark with the mobile phase (4.4). Reserve the remaining finely ground sodium cyclamate standard substance for the determination of loss in mass on drying (see 6.1.1). The determination of loss in mass on drying is carried o
34、ut immediately. 4.6.2 Saccharin stock solution, r (C 7 H 5 NO 3 S) 2 mg/ml. Finely grind at least about 1,5 g of the undried sodium saccharin standard substance (4.5.2) then, without delay, dissolve about 263 mg of the undried sodium saccharin standard substance (equivalent to about 200 mg of saccha
35、rin as free imide) 4) , weighed to the nearest 0,1 mg, in the mobile phase (4.4)i n a 100 ml volumetric flask and dilute to the mark with the mobile phase (4.4). Reserve the remaining finely ground sodium saccharin standard substance for the determination of loss in mass on drying (see 6.1.2). The d
36、etermination of loss in mass on drying is carried out immediately.Page 4 EN 1379 : 1996 BSI 1997 4.6.3 Calculation of the exact concentrations of sodium cyclamate and saccharin stock solutions Calculate the exact concentration of anhydrous sodium cyclamate and anhydrous saccharin, r, in milligrams p
37、er litre of the stock solutions, using the following equation: (1) r = m 1 3 (1002 L D )3 10 100 where: m 1 is the mass of standard substances of sodium cyclamate or saccharin, in V 2 (see equation (2), in milligrams; L D is the loss in mass on drying, in %. 4.6.4 Sorbic acid stock solution, r (CH 3
38、 (CH) 4 COOH) 0,1 mg/ml. Dissolve about 100 mg of sorbic acid standard substance (4.5.3), weighed to the nearest 0,1 mg, in the mobile phase (4.4) in a 100 ml volumetric flask and dilute to the mark. Pipette 10 ml of this solution in a 100 ml volumetric flask and dilute to the mark with the mobile p
39、hase (4.4). 4.7 Standard test solutions NOTE. Additional solutions with concentrations within the linear range may be prepared for the calibration graph. 4.7.1 Standard test solution I. Pipette 50 ml of the sodium cyclamate stock solution (4.6.1), 10 ml of the saccharin stock solution (4.6.2) and 10
40、 ml of the sorbic acid stock solution (4.6.4) into a 100 ml volumetric flask and dilute to the mark with the mobile phase (4.4). 1 l of this solution contains about 2000 mg of sodium cyclamate, 200 mg of saccharin and 10 mg of sorbic acid. 4.7.2 Standard test solution II. Pipette 25 ml of the sodium
41、 cyclamate stock solution (4.6.1), 5 ml of the saccharin stock solution (4.6.2) and 5 ml of the sorbic acid stock solution (4.6.4) into a 100 ml volumetric flask and dilute to the mark with the mobile phase (4.4). 1 l of this solution contains about 1000 mg of sodium cyclamate, 100 mg of saccharin a
42、nd 5 mg of sorbic acid. 4.7.3 Standard test solution III. Pipette 10 ml of the sodium cyclamate stock solution (4.6.1), 2 ml of the saccharin stock solution (4.6.2) and 2 ml of the sorbic acid stock solution (4.6.4) into a 100 ml volumetric flask and dilute to the mark with the mobile phase (4.4). 1
43、 l of this solution contains about 400 mg of sodium cyclamate, 40 mg of saccharin and 2 mg of sorbic acid. 5 Apparatus and equipment Usual laboratory apparatus and, in particular, the following. 5.1 Filtration unit, e.g. glass vacuum filtration unit consisting of a glass sinter disk (diameter 50 mm)
44、 a 250 ml top section and a 1 l conical flask, all with ground glass joints. 5.2 Membrane filter, suitable for mobile phase (4.4), pore size# 5mm. 5.3 High performance liquid chromatograph consisting of a pump, a sample applicator, a UV detector with variable wavelength setting and an evaluation sys
45、tem, e.g. a chart recorder or integrator. 5.4 Analytical reversed phase separating column, e.g. C 18 reversed phase, particle size 10mm, diameter 4,6 mm, length 250 mm. Other particle sizes than specified in this European Standard may be used. Separation parameters have to be adapted to such materia
46、ls to guarantee equivalent results. NOTE. Minimal theoretical plates at the retention volume of the resolved analyte should preferably be not less than 7700 for saccharin and not less than 4600 for sodium cyclamate under the conditions of chromatography employed. 6 Procedure 6.1 Determination of los
47、s in mass on drying of standard substances 6.1.1 Determination of loss in mass on drying of sodium cyclamate standard substance Weigh, to the nearest 0,1 mg, about 1,0 g of the reserved finely ground sodium cyclamate standard substance (4.5.1). Dry this portion to constant mass at (105 2) C and dete
48、rmine the loss in mass on drying (LD) in per cent by weighing. The loss in mass on drying shall not exceed 1 %. 6.1.2 Determination of loss in mass on drying of sodium saccharin standard substance Weigh, to the nearest 0,1 mg, about 1,0 g of the reserved finely ground sodium saccharin standard subst
49、ance (4.5.2). Dry this portion to constant mass at (105 2) C and determine the loss in mass on drying (LD) in per cent by weighing. The loss in mass on drying shall not exceed 15 %. 6.2 Preparation of the sample test solution Dilute 10 ml of the liquid table top sweetener preparation to 100 ml (V 1 ) with the mobile phase (4.4) and filter through a membrane filter (5.2). Dilute 10 ml of this solution to 100 ml.Page 5 EN 1379 : 1996 BSI 1997 6.3 Identification by HPLC Identify the sweeteners to be determ
