1、BRITISH STANDARD BS 6248-11: 1987 ISO 8195:1987 Caseins and caseinates Part 11: Method for determination of nitrate and nitrite contents ISO title: Caseins and caseinates Determination of nitrate and nitrite contents Method by cadmium reduction and spectrometry NOTEThis Part should be read in conjun
2、ction with Part 1 “General introduction, including preparation of laboratory samples”, published separately. UDC 637.142.2.04:546.173/.175:546.48 + 543.42:620.168.2BS6248-11:1987 This British Standard, having been prepared under the directionof the Dairying Standards Committee, was published under t
3、he authority ofthe Board of BSI and comes intoeffect on 31 December 1987 BSI 12-1999 The following BSI references relate to the work on this standard: Committee reference DAC/3 Draft for comment 85/54934 DC ISBN 0 580 16339 3 Amendments issued since publication Amd. No. Date of issue CommentsBS6248-
4、11:1987 BSI 12-1999 i Contents Page National foreword ii 1 Scope and field of application 1 2 Reference 1 3 Definition 1 4 Principle 1 5 Reagents 1 6 Apparatus 2 7 Sampling 2 8 Procedure 2 9 Expression of results 4 10 Test report 5 Figure Apparatus for nitrate reduction 6 Publications referred to In
5、side back coverBS6248-11:1987 ii BSI 12-1999 National foreword This Part of BS 6248, which has been prepared under the direction of the Dairying Standards Committee, is identical with ISO 8195:1987 “Caseins and caseinates Determination of nitrate and nitrite contents Method by cadmium reduction and
6、spectrometry”, prepared by ISO/TC 34, Agricultural food products, of the International Organization for Standardization (ISO). The method described in ISO 8195 was developed jointly with the International Dairy Federation (IDF) and the Association of Official Analytical Chemists (AOAC). Terminology
7、and conventions. The text of the International Standard has been approved as suitable for publication as a British Standard without deviation. Some terminology and certain conventions are not identical with those used in British Standards; attention is drawn especially to the following. The comma ha
8、s been used as a decimal marker. In British Standards it is current practice to use a full point on the baseline as the decimal marker. Where the words “International Standard” appear, referring to this standard, they should be read as “Part of this British Standard”. The symbol “l” has been used to
9、 denote litre (and in its sub-multiples). In British Standards it is current practice to use the symbol “L”. In British Standards it is current practice to use the spelling “sulphur”, etc. instead of “sulfur”, etc. Additional information. With reference to clause 5, water complying with grade 3 of B
10、S 3978:1987 “Specification for water for laboratory use” may be used. With reference to 6.6, class A graduated pipettes complying with BS700“Graduated pipettes” Part 1:1982 “Specification for general requirements” or Part2:1982 “Specification for pipettes for which no waiting time is specified” may
11、be used in place of those complying with ISO 8351, and class A one-mark pipettes complying with BS 1583:1986 “Specification for one-mark pipettes” may be used in place of those complying with ISO 648, class A. With reference to 6.12, a wire cloth test sieve of diameter 200 mm and aperture size 500 4
12、m, complying with BS 410:1986 “Specification for test sieves”, is suitable. It is recommended that analysts should periodically undertake duplicate determinations to verify that results complying with the repeatability requirements of 9.1.2 or 9.2.2, as appropriate, are being obtained. When a determ
13、ination is being undertaken for reference purposes, or in cases of dispute, it is strongly recommended that duplicate determinations be undertaken. Provided the duplicate values, calculated as described in 9.1.1 or9.1.2, as appropriate, comply with the relevant repeatability requirement (9.1.2 or 9.
14、2.2), the result should be reported as the mean value and in such cases the test report (see clause 10) should include a reference to the fact that such a result has been reported. Cross-references International Standard Corresponding British Standard ISO 707:1985 BS 809:1985 Methods for sampling of
15、 milk and milk products (Identical) ISO 1042:1983 BS 1792:1982 Specification for one-mark volumetric flasks (Identical)BS6248-11:1987 BSI 12-1999 iii A British Standard does not purport to include all the necessary provisions of a contract. Users of British Standards are responsible for their correc
16、t application. 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, pagesi toiv, pages1 to6, an inside back cover and a back cover. This standard has been updated (see copyright da
17、te) and may have had amendments incorporated. This will be indicated in the amendment table on the inside front cover.iv blankBS6248-11:1987 BSI 12-1999 1 1 Scope and field of application This International Standard specifies a method by cadmium reduction and spectrometry for the determination of th
18、e nitrate and nitrite contents of caseins and caseinates. 2 Reference ISO 707, Milk and milk products Methods of sampling. 3 Definition nitrate and nitrite contents of caseins and caseinates the contents of substances determined by the procedure specified in this International Standard and expressed
19、 respectively as milligrams of nitrate ion (NO) and of nitrite ion (NO) per kilogram 4 Principle Dispersion of the casein or caseinate in warm water, precipitation of the fat and proteins, and filtration. Reduction of the nitrate to nitrite in a portion of the filtrate by means of copperized cadmium
20、. Development of a red colour, in portions of both unreduced filtrate and of the reduced solution, by addition of sulfanilamide and N-1-naphthyl ethylenediamine dihydrochloride, and spectrometric measurement at a wavelength of 538 nm. Calculation of the nitrite content of the sample and of the total
21、 nitrite content after reduction of nitrate, by comparing the measured absorbances with those of a set of sodium nitrite calibration solutions; calculation of the nitrate content from the difference between these two contents. 5 Reagents All reagents shall be of recognized analytical grade. The wate
22、r used shall be distilled or deionized water, free from nitrate and nitrite. NOTEIn order to avoid possible inclusion of small gas bubbles in the copperized cadmium column (6.10), the distilled or deionized water used for the preparation of the column (8.1), for checking the reducing capacity of the
23、 column (8.2), and for regeneration of the column (8.3) should preferably be freshly boiled and afterwards cooled to room temperature. 5.1 Cadmium, granules, diameter 0,3 to 0,8 mm. If cadmium granules are not available commercially, they may be prepared as follows. Place a suitable number of zinc r
24、ods in a beaker and cover with a 40 g/l solution of cadmium sulfate octahydrate (CdSO 4 8H 2 O). From time to time scrape the cadmium sponge from the rods over a period of 24 h. Remove the zinc rods and decant the liquid until only sufficient remains to cover the cadmium. Wash the sponge two or thre
25、e times with water. Transfer the cadmium to a laboratory blender together with 400 ml of hydrochloric acid solution, c(HCl) . 0,1 mol/l, and blend for a few seconds to obtain granules of the required size. Return the contents of the blender to the beaker and leave to stand for several hours, occasio
26、nally stirring to remove bubbles. Decant most of the liquid and immediately copperize as described in 8.1.1 to8.1.5. 5.2 Copper(II) sulfate solution. Dissolve 20 g of copper(II) sulfate pentahydrate (CuSO 4 5H 2 O) in water and dilute to 1 000 ml. 5.3 Hydrochloric acid solution, c(HCl) . 2 mol/l. Di
27、lute 160 ml of concentrated hydrochloric acid ( 201,19 g/ml) to 1 000 ml with water. 5.4 Hydrochloric acid solution, c(HCl) . 0,1 mol/l. Dilute 50 ml of the hydrochloric acid (5.3) to1000ml with water. 5.5 Solutions for precipitation of proteins and fat 5.5.1 Zinc sulfate solution. Dissolve 53,5 g o
28、f zinc sulfate heptahydrate (ZnSO 4 7H 2 O) in water and dilute to 100 ml. 5.5.2 Potassium hexacyanoferrate(II) solution. Dissolve 17,2 g of potassium hexacyanoferrate(II) trihydrate (K 4 Fe(CN) 6 3H 2 O) in water and dilute to 100 ml. 5.6 EDTA solution. Dissolve 33,5 g of disodium ethylenediaminete
29、traacetate dihydrate (Na 2 C 10 H 14 N 2 O 8 2H 2 O) in water and dilute to1000ml. 5.7 Solutions for colour development 5.7.1 Solution I Dilute 450 ml of concentrated hydrochloric acid ( 201,19 g/ml) to 1 000 ml with water. 5.7.2 Solution II Dissolve, by heating on a water-bath, 0,5 g of sulfanilami
30、de (NH 2 C 6 H 4 SO 2 NH 2 ) in a mixture of75ml of water and 5 ml of concentrated hydrochloric acid ( 201,19 g/ml). Cool to room temperature and dilute to 100 ml with water. Filter if necessary. 3 2BS6248-11:1987 2 BSI 12-1999 5.7.3 Solution III Dissolve 0,1 g of N-1-naphthyl ethylenediamine dihydr
31、ochloride (C 10 H 7 NHCH 2 CH 2 NH 2 2HCl) in water. Dilute to 100 ml. Filter if necessary. The solution may be stored for up to 1 week in a well-stoppered brown bottle in a refrigerator. 5.8 Sodium nitrite, standard solution corresponding to 0,001 g of NO per litre. 5.8.1 Stock solution Dry a few g
32、rams of sodium nitrite (NaNO 2 ) at 110 to120 C to constant mass, i.e. until the difference between two successive weighings does not exceed1mg. Dissolve 0,150 g of the sodium nitrite in water in a 1 000 ml one-mark volumetric flask. Dilute to the mark with water and mix. Prepare this solution on th
33、e day of use. 5.8.2 Preparation of standard solution Transfer, by means of pipettes, 10 ml of the stock solution (5.8.1) and 20 ml of the buffer solution(5.10) to a 1 000 ml one-mark volumetric flask. Dilute to the mark with water and mix. 1 ml of this standard solution contains 1,00 4g of NO. 5.9 P
34、otassium nitrate, standard solution corresponding to 0,004 5 g of NO per litre. 5.9.1 Stock solution Dry a few grams of potassium nitrate (KNO 3 ) at 110 to 120 C to constant mass, i.e. until the difference between two successive weighings does not exceed1mg. Dissolve 1,468 g of the potassium nitrat
35、e in water in a 1 000 ml one-mark volumetric flask. Dilute to the mark with water and mix. 5.9.2 Preparation of standard solution Transfer, by means of pipettes, 5 ml of the stock solution (5.9.1) and 20 ml of the buffer solution(5.10) to a 1 000 ml one-mark volumetric flask. Dilute to the mark with
36、 water and mix. Prepare this solution on the day of use. 1 ml of this standard solution contains 4,50 4g of NO. 5.10 Buffer solution, pH 9,6 to 9,7. Dilute 50 ml of concentrated hydrochloric acid 20 1,19 g/ml; about 38 % (m/m) HCl solution with600 ml of water. After mixing, add 135 ml of ammonia sol
37、ution 200,91 g/ml; about 25 % (m/m) NH 3solution. Dilute to 1 000 ml with water and mix. Adjust the pH to 9,6 to 9,7 if necessary. NOTEIf ammonia solution of this concentration is not available, an equivalent amount of a more concentrated solution may be used, for example 100 ml of 35 % (m/m) soluti
38、on ( 200,88 g/ml). 6 Apparatus NOTE ON THE PREPARATION OF GLASSWARE All glassware shall be thoroughly cleaned and rinsed with distilled water to ensure that it is free from nitrate and nitrite. Usual laboratory equipment and in particular 6.1 Analytical balance 6.2 Sample container, provided with an
39、 airtight lid. 6.3 Magnetic stirrer 6.4 Conical flasks, of capacities 250 and 500 ml. 6.5 One-mark volumetric flasks, of capacities 100; 500; and 1 000 ml, complying with the requirements of ISO 1042, class B. 6.6 Pipettes, to deliver 2; 4; 5; 6; 8; 10; 12; 20; and25ml, complying with the requiremen
40、ts of ISO648, class A, or ISO 835-1. NOTEWhere appropriate, burettes may be used instead of pipettes. 6.7 Measuring cylinders, of capacities 5; 10; 25; 100; 250; 500; and 1 000 ml. 6.8 Glass funnels, of diameter 7 cm, with short stem. 6.9 Filter paper, medium grade, of diameter about15 cm, free from
41、 nitrate and nitrite. 6.10 Reduction column (for example as shown in the Figure). 6.11 Spectrometer, suitable for measuring absorbance at a wavelength of 538 nm, with cells of optical path length 1 to 2 cm. 6.12 Test sieve, of woven wire cloth, diameter200mm, nominal size of openings 500 4m, with re
42、ceiver, complying with the requirements of ISO 565. 6.13 Grinding device, for grinding the laboratory sample if necessary. This device should be such that no undue heat will be developed and no loss of moisture occurs. (A hammer mill shall not be used.) 7 Sampling See ISO 707. 8 Procedure 8.1 Prepar
43、ation of the copperized cadmium column 8.1.1 Transfer the cadmium granules (5.1) (approximately 40 to 60 g for each column) into a250 ml conical flask (6.4). 8.1.2 Add sufficient of the hydrochloric acid solution(5.4) to cover the cadmium. Swirl for a few minutes. 2 2 3 3BS6248-11:1987 BSI 12-1999 3
44、 8.1.3 Decant the solution and wash the cadmium in the flask with water, until it is free from chloride (i.e. until reaction with silver nitrate is negative). 8.1.4 Copperize the cadmium granules by adding the copper(II) sulfate solution (5.2) (about 2,5 ml per gram of cadmium) and swirling for 1 mi
45、n. 8.1.5 Decant the solution and wash the copperized cadmium immediately with water, taking care that the cadmium is continuously covered with water. Terminate the washing when the wash water is free from precipitated copper. 8.1.6 Fit a glass wool plug to the bottom of the glass column intended to
46、contain the copperized cadmium (see the Figure). Fill the glass column with water. 8.1.7 Transfer the copperized cadmium into the glass column with minimum exposure to air. Theheight of the copperized cadmium shall be 15 to20 cm. NOTE 1Avoid trapping air bubbles between the copperized cadmium granul
47、es. NOTE 2Take care that the level of the liquid does not fall below the top of the copperized cadmium. 8.1.8 Condition the newly prepared column by running through it a mixture of 750 ml of water,225ml of the potassium nitrate solution(5.9), 20 ml of the buffer solution (5.10) and20 ml of the EDTA
48、solution (5.6), at a flow rate not exceeding 6 ml/min, then wash the column with50 ml of water. 8.2 Checking the reducing capacity of the column Carry out this check at least twice a day, at the beginning and at the end of a series of determinations. 8.2.1 Pipette 20 ml of the standard potassium nit
49、rate solution (5.9) into the reservoir on top of the column. Immediately add 5 ml of the buffer solution(5.10) to the contents of the reservoir. Collect the eluate in a 100 ml one-mark volumetric flask. The flow rate shall not exceed 6 ml/min. 8.2.2 When the reservoir has nearly run empty, wash the walls of the reservoir with about 15 ml of water and, when this has run off, repeat the same treatment with another 15 ml portion of water. After this second portion of water has run into the column as well, completely fill the rese
