1、BRITISH STANDARD BS ISO 8432:1987 Essential Oils Analysis by high performance liquid chromatography General method ICS 71.100.60; 71.040.40 NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBS 8432:1987 This British Standard, having been prepared under the direction of the Consum
2、er Products and services Sector Committee, was published under the authority of the Standards Committee and comes into effect on 15 September 2001 BSI 08-2001 ISBN 0 580 38323 7 National foreword This British Standard reproduces verbatim ISO 8432:1987 and implements it as the UK national standard. T
3、he UK participation in its preparation was entrusted to Technical Committee AW/54, Essential oils, which has the responsibility to: A list of organizations represented on this committee can be obtained on request to its secretary. Cross-references The British Standards which implement international
4、or European publications referred to in this document may be found in the BSI Standards Catalogue under the section entitled “International Standards Correspondence Index”, or by using the “Find” facility of the BSI Standards Electronic Catalogue. A British Standard does not purport to include all t
5、he necessary provisions of a contract. Users of British Standards are responsible for their correct application. Compliance with a British Standard does not of itself confer immunity from legal obligations. aid enquirers to understand the text; present to the responsible international/European commi
6、ttee any enquiries on the interpretation, or proposals for change, and keep the UK interests informed; monitor related international and European developments and promulgate them in the UK. Summary of pages This document comprises a front cover, an inside front cover, the ISO title page, the ISO for
7、eword page, pages 1 to 4, an inside back cover and a back cover. The BSI copyright date displayed in this document indicates when the document was last issued. Amendments issued since publication Amd. No. Date CommentsISO INTERNATIONAL STANDARD 8432 INTERNATIONAL ORGANIZATION FOR STANDARDIZATION ORG
8、ANISATION INTERNATIONALE DE NORMALISATION MEXflYHAPOflHAR OPrAHkl3AMR I-IO CTAH/JAPTM3AMM Essential oils - Analysis by high Performance liquid chromatography - General method Huiles essentielles - Analyse par Chromatographie liquide sous Pression - Mthode g it shall elute as closely as possible to t
9、he compound to be determined and its peak shall not superpose on that of any compound in the essential oil which is detectable by the detection System used. 4.3 Mobile Phase. The composition of the mobile Phase may remain constant dur- ing Operation (isocratic elution) or Change according to a 5 App
10、aratus 5.1 Separation System. 5.1 .l Chromatograph. 5.1.2 Pumping System or any other System, which enables a constant or programmed flow rate to be obtained and maintained. 5.1.3 Solvent supply System. 5.1.4 Solvent degassing System. 5.1.5 Suitable detector System, which enables the quan- tities of
11、 compounds present in the Sample to be determined. 5.2 Recorder, and (optional) integrator, whose perfor- mance is compatible with the rest of the apparatus. 5.3 Column, made of an inert material (for example glass, stainless steel) having mechanical proper-Ges which enable the pressure to be withst
12、ood. The nature and the particle size of the stationary Phase will be specified in each relevant International Standard. The temperature of the column may be stabilized or pro- grammed. ISO 8432: 1987 (El 6 Preparation of the test Sample See ISO 356. If the test Sample to be injected has to undergo
13、special prepara- tion, this will be indicated in the relevant International Stan- dard. 7 Procedure 7.1 Temperature If the temperature is different from ambient, it will be specified in each relevant International Standard. 7.2 Elution solvent flow rate Regulate the flow rate so as to obtain the Sep
14、aration of con- stituents to be determined. 8 Column Performance 8.1 Chemical inertness test Ensure that the products to be identified are not damaged. 8.2 Column efficiency Determine the number of effective plates UV) from the internal Standard peak specified in each relevant International Standard
15、 and using the solvents also specified, at an isothermal temperature, and under isocratic conditions from the equation 8.3 Resolution Calculate the resolution factor, R, of two neighbouring peaks I and II, from the equation R=2 d,(H) - d,(l) bo,5(l) + bo,5(W where d,(l) is the retention distance of
16、peak 1; d,(H) is the retention distance of peak II; b. 5(l) bo5(ll) is the width, in millimetres, at half of peak I height; , is the width, in millimetres, at half of peak II height. (See the figure.) 9 Methods of determination 9.1 General conditions Record the chromatogram as specified in the relev
17、ant lnter- national Standard. For the determination of certain specific constituents, the rele- vant International Standard may specify isocratic Operation or an Operation by elution gradient. In such cases, the flow rate shall be regulated so that the Separation obtained is that specified in the re
18、levant lnter- national Standard. After stabilization of the Operation conditions, inject a suitable quantity of the test Portion. Chromatogram “A” is thus obtained. where d, is the distance, in millimetres, measured on the baseline between the injection Point and the falling edge of the peak being s
19、tudied; 9.2 lnternal Standard method Record the chromatogram of the essential oil and that of the in- ternal Standard (4.2), under the same operating conditions. bo,5 is the width, in millimetres, at half of the peak height. Figure - Calculation of the resolution factor ISO 8432 : 1987 (E) Check on
20、the chromatogram that the products to be deter- mined are separated from the constituents of the essential Oil, and that the internal Standard does not interfere with any con- stituent of the essential Oil. 9.2.1 Determination of response factor If, for quantitative determinations, the response fact
21、or of a constituent relative to the internal Standard has to be deter- mined, weigh appropriate quantities of the internal Standard (4.2) and that of the reference substance (4.1) such that the corresponding peak areas will be approximately equal. The solvent to be used for di vant International Sta
22、ndard. lutions will be specif ied in the rele- After stabilization of the whole System, inject a suitable quan- tity of this mixture and carry out the analysis under conditions specified in 9.1. Chromatogram “B” is thus obtained. Calculate the response factor, K, by means of the equation mRAE KZ- mE
23、AR where AR is the peak area, in integrator units, corresponding to the reference substance the response factor of which is to be calculated ; AE is the peak area, the internal Standard ; mR mE in integrator units, corresponding to is the mass, in milligrams, of the reference substance; is the mass,
24、 in milligrams, of the internal Standard. 9.2.2 Determination If the relevant International Standard specifies the use of an internal Standard, prepare a mixture by weighing, to the nearest 0,l mg, appropriate quantities of the essential oil and of the internal Standard. Choose the amount of interna
25、l Standard so that the peak area of the compound to be determined and that of the internal Standard will be approximately equal. After stabilization of the whole System, inject a suitable quan- tity of the mixture and carry out the analysis under the con- ditions specified in 9.1. Chromatogram “C” i
26、s thus obtained. 9.3 Addition method If it is not possible to use the internal Standard method for a particular determination, use the addition method. For this, inject a suitable quantity of the essential oil in which X is the compound to be determined, and Y is a compound giving a neighbouring pea
27、k on the chromatogram “D” obtained. Then prepare, by weighing to the nearest 0,l mg, a mixture of m g of the test Sample and mx g of the reference substance (4.1) corresponding to the compound X to be determined. Inject an appropriate quantity of this mixture. Chromato- gram “E” is thus obtained. 9.
28、4 External Standard method If it is not possible to use the methods specified in 9.2 and 9.3, use the external Standard method. This method is only applicable if the apparatus has an injection System which enables identical volumes to be injected (e.g. loop valve). Prepare solutions of increasing co
29、ncentrations of the reference substance. lnject successively the same quantity of each of these different solutions. Plot a graph of peak areas against concentration of reference substance in the different solutions. A plot which gives peak area of reference substance according to the concentration
30、in the different solutions is thus obtained. Inject the same quantity of the essential oil where “Z“ is the compound to be determined. Measure the area of the peak given by “Z“. From the area given by “Z“, read off the “Z“ concentration from the graph. 10 Expression of results 10.1 lnternal Standard
31、 Calculate the content, cx, expressed as a percentage by mass, of the compound to be determined, by means of the formula AxVIE X K x 100 AEmX where A, is the peak area, in integrator units, corresponding to the compound to be determined; AE is the peak area, in integrator units, corresponding to the
32、 internal Standard ; mx is the mass, in milligrams, of the essential Oil; mE is the mass, in milligrams, of the internal Standard; K is the response factor for the compound to be deter- mined relative to the internal Standard. 10.2 Addition method Calculate the content, cx, expressed as a percentage
33、 by mass, of the compound to be determined, by means of the formula mR r - x - x 100 (r r) m f-r where mR is the mass, in grams, of the reference substance (4.1); m is the mass, in grams, of the essential Oil; AX r - = A, ISO 8432: 1987 (EI in which means by more than a certain percentage. This perc
34、entage and the number of determinations will be specified in the relevant International Standards. A, is the peak area corresponding to the compound X on chromatogram “D”; A, is the peak area, corresponding to the compound Y close to X on chromatogram “D”; 11 Test report AX rf = - A, The test report
35、 shall include the following information: in which A, is the peak area corresponding to the compound X on chromatogram “E”; AY is the peak area corresponding to the compound Y close to X on chromatogram “EI. a) the apparatus System; b) reference to this International Standard; c) the characteristics
36、 of the column (material, dimensions, packing, stationary Phase) ; d) the characteristics of the detector (optional) and the operating conditions; 10.3 Results and repeatabilty Take as the result for the response factor K and the content cx of the compound to be determined the mean values of several
37、 (at least three) determinations carried out on the same test Sample. These different values should not differ from their e) the nature) ; characteristics of the mobile Phase (flow rate and f) identification of the test Sample (quantity injected and final dilution) ; g) results obtained. BS ISO 8432
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