1、 - STD=BSI DD ENV L3070-ENGL I1998 1624bb9 073L4b9 495 DRAFT FOR DEVELOPMENT Fruit and vegetable juices - Determination of the stable carbon isotope ratio ( “Cf2C) in the pulp of fruit juices - Method using isotope ratio mass spectrometry ICs 67.160.20 NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PER
2、MITTED BY COPYRIGHT LAW DD ENV 130701998 STD.BS1 DD ENV 13070-ENGL 1998 m 1624669 0711470 LO7 m DD ENV 130701998 This Draft for Development, hawig been prepared under the direction of the Consumer Protu 613C Carbon 13 (1%) content expressed in parts per thousand (%o) ; 9 Acceleration due to gravity
3、at the surface of the earth (9,81 m/s2). 4 Principle The carbon contained in the pulp is quantitatively combusted into carbon dioxide. The ratio of the two stable carbon isotopes (13C and 12C) is then measured using a isotope ratio mass spectrometer. This value can then be compared with the value ob
4、tained for the sugars (as determined using EM/ 12140). If there is too large a difference between the value in the pulp and the sugars this can indicate the addition of sugars derived from a C, plant such as cane or com. 5 Reagents Use only reagents of recognized analytical grade and only water in a
5、ccordance with at least grade 3 of EN IS0 3696:1995. STD=BSI DD ENV 13070-ENGL 3998 Lb24bb9 0733475 799 m Page 5 ENV 1 3070: 1 998 5.1 Calcium hydroxide 5.2 Sulfuric acid, 95 % to 97 % (mrm) 5.3 Acetone 6 Apparatus Usual laboratory apparatus and, in particular, the following : 6.1 at natural abundan
6、ce with a precision of 0,05 % or better expressed as relative 6 value (see 8) (one measuring cycle with ten integration periods). Isotope ratio mass spectrometer, with the ability to determine the 1% content of CO, gas The mass-spectrometer will generally be fitted with a triple collector to simulta
7、neous register at mass number 44,45 and 46. The mass spectrometer should be fMed either with a dual inlet system, for alternately measuring the unknown sample and a standard, or use an on-line system which combusts the sample in an elemental analyser (6.2) followed by gas chromatographic separation
8、of the combustion products prior to isotopic mass spectrometric determination. The former method offers the highest accuracy for the determination of variations of the isotope content in the range of the natural abundance. However, correct results can also be obtained using the on-line method provid
9、ed a secondary standard is used. 6.2 Combustion apparatus (elemental analyser), which can quantitatively converts all the carbon of the sample into carbon dioxide (CO,), and which is able to convert all nitrous oxides formed, during the oxidation process, into nitrogen gas and to remove all other co
10、mbustion products, including water, from the CO,. 6.3 Centrifuge, capable of producing a centrifugal acceleration of 1 400 g at the base of the centrifuge tube (6.4). NOTE : The rotational frequency required to give correct centrifugal acceleration can be calculated from the following equation : a =
11、 11,18 e r - (n/l OOO)* (1) where : a is the centrifugal acceleration ; r is the radius of the centrifuge in centimetres, measured from the mid point (the centrifuge axis) to the bottom of the centrifuge tube when swung out ; n is the rotational frequency per minute. Page 6 ENV 13070: 1998 6.4 Centr
12、ifuge tubes, of 50 ml capacity. 7 Procedure 7.1 Preparation of the test sample 50 ml of fruit juice (natural or reconstituted) is centrifuged at 1 400 g for 10 min to separate the uspended solids (pulp). To remove any adhering sugars the centrifugate is washed with water. To achieve this the pellet
13、is resuspended in 40 ml of water and stirred for 5 min. This suspension is then recentrifuged and the water discarded. A fresh aliquot of water is added and the process is repeated. A further aliquot of water is used in the third and final wash with water. After this third wash, with water, the pulp
14、 is resuspended in 40 mi of acetone to extract any lipid material. After stimng in acetone the pulp is centrifuged as given above and the pulp separated from the solvent. A fresh aliquot of acetone is added and the process repeated twice more. To obtain meaningful results it is essential that both t
15、he water and acetone washes are carried thoroughly. This ensures that ail the occluded sugars and lipid materials are removed. If the pulp is not properly washed the lipid material will cause a significant negative shift in the 81% content of the pulp. The pulp after washing, as described above, is
16、finally dried under vacuum which gives a white/gray solid which can be easily homogenised. 7.2 Combustion of pulp Combust the samples obtained using the procedures given in 7.1 using the procedure for sugars described in ENV 12140:1996. Care should be taken to ensure that there is a quantitative red
17、uction of all the nitrous oxides formed, from the nitrogen containing compounds in the pulp, during the combustion stage. If this is not achieved nitrous oxide will be present in the carbon dioxide gas and this will contribute to the intensity of the mass 46 ion. This will lead to an incorrect corre
18、ction for oxygen 17 and so give a too low carbon 13 content of the pulp. NOTE : Suitable microcombustion systems are commercially available. 7.3 Determination The 1%/12C isotope ratio in the carbon dioxide obtained from the combustion of the pulp, as described in 7.2, is determined with the aid of a
19、n isotope ratio mass spectrometer (6.1). Determine the ratio for the isotopic species 13CO#*C02 from the corresponding intensities. STD-BSI DD ENV 13070-ENGL 1998 = 1b24bb 0711477 561 m Page 7 ENV 13070: 1998 8 Calculation In addition to the commonly used mass abundance (in %o of atoms), the so-call
20、ed delta value (6) is also used as an alternative system of units for indicating isotope content. Delta values are used exclusively for indicating variations (on the third decimal place) in the natural isotopic abundance. Express the SW values as the relative difference per thousand between the 13C
21、and 12C ratios of a sample in relation to a standard, the Pee Dee Belemnite from South Carolina-USA (the PDB standard). This is a fossil calcium carbonate with an isotope ratio (13C/12C),DB = 0,0112372 for the emitted CO,. This value is the reference point of the common international PDB scale for 6
22、13C values expressed in parts per thousand (%o) which are calculated using the following equation : A suitable secondary standard for routine use in this method is NBS 22 obtained from Intemational Atomic Energy Agency (IAM1), Vienna), which has a 6% value of - 29,80 %o relative to PDB. 9 Precision
23、Details of the interlaboratory test on the precision of the method are summarized in annex B. The values derived from interlaboratory test may not be applicable to analysis of concentration ranges and matrices other than those given in annex 8. 9.1 Repeatability The absolute difference between two s
24、ingle results found on identical test material by one operator using the same apparatus within the shortest feasible time interval will exceed the repeatability limit r in not more than 5 % of the cases. The values are : - orange juice r= 0,32 %o ; - orange juice with added sugar r= 0,31 %o ; - grap
25、efruit juice r= 0,26 %o ; - grapefruit juice with added sugar r= 0,45 960 ; - pineapple juice r = 0,42 %o ; - pineapple juice with added sugar r = 0,46 %o. l) International Atomic Energy Agency P.O. Box 100 A-1400 Wien - Austria. STDDBSI DD ENV 13070-ENGL 1998 1b24bb9 0711478 4T8 M Page 8 EM/ 13070:
26、 1998 9.2 Reproducibility The absolute differences between two single test results on the identical material reported by two laboratories will exceed the reproducibility limit R in not more than 5 % of the cases. The values are : - orange juice R= 1,03%o; - orange juice with added sugar R = 0,92 %o
27、; - grapefruit juice R=0,78%o; - grapefruit juice with added sugar R = 2,35 %o ; - pineapple juice - pineapple juice with added sugar R = 3,OO %o ; R = 2,08 %o. 10 Testreport The test report must contain the following data : - all information necessary for the identification of the sample (kind of s
28、ample, origin of sample, designation) ; a) a reference to this European Standard ; b) the date and type of sampling procedure (if known) ; c) the date of receipt ; d) the date of test ; e) the test results and units in which they have been expressed ; 9 any particular points observed in the course o
29、f the test ; g) any operations not specified in the method or regarded as optional, which might have affected the results. STD-ES1 DD ENV 33070-ENGL 3778 W 3b24bb9 0733479 334 Page 9 ENV 13070: 1998 Annex A (informative) Bibliography H.Craig, Geochim. Cosmochim. Acta, (1 957) 133 G. Hut, Consultants
30、 Group Meeting on Stable Isotope Reference Samples for Geochimical and Hydrological Investigations, IAEA, Vienna, 16-18 September 1985, IAEA Report, April 1987 Determination of carbon-I3 content of sugars of fruit and vegetable juices (Internal Reference Method). Analytica Chimica Acta, (I 997) 340,
31、21-29 - - STD-BSI DD ENV 33070-ENGL 3998 3b24bb9 0733480 O56 Sample Number of laboratories retained after eliminating outliers Number of outliers (laboratories) Mean value (X) (%O) Repeatability standard deviation (s,) (%o) Repeatability relative standard deviation Repeatability limit (i) (%O) Repro
32、ducibility standard deviation (sR) (%O) Reproducibility relative standard deviation Reproducibility limit (R) (%O) . (RSD,) (%) (RSDR) (%) Page 10 EM/ 13070: 1 998 A B C D E F 17 16 16 17 18 17 2 3 3 2 O 2 -25,98 -25,92 -24,92 -24,93 -1 1,47 -1 1,36 0,12 0,ll 0,OQ 0,16 0,15 0,17 0,46 0,42 0,36 0,64
33、1,31 1,50 0,32 0,31 0,26 0,45 0,42 046 0,37 0,33 0,28 0,84 1 ,O7 0,74 1,42 1,27 1 , 12 3,37 9,33 6,51 1,03 0,92 0,78 2,35 3,OO 2,08 Annex B (informative) Statistical results of the interlaboratory test In accordance with IS0 5725:1986, the following parameters have been defined in an interlaboratory
34、 test. (For literature pertaining to the method see annex A). The test was conducted by the Working Group 1 “Isotopes“ of the CENTTC 174. Year of the interlaboratory test 1995 Number of laboratones 19 Number of samples 6 Table B.l NOTE : There was no dependency between r, R and F. Sample types : A o
35、range juice ; B C grapefruit juice ; D E pineapple juice ; F orange juice with added sugar ; grapefruit juice with added sugar ; pineapple juice with added sugar. STD.BSI DD ENV 13070-ENGL 1998 W 1b24bb9 07LL481 T92 D DD ENV 130701998 BSI 389ChiswickHighRoad bndon w4 4AL BSI - British Standards Inst
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