1、BRITISH STANDARD BS 5752-3: 1983 ISO 4052:1983 Methods of test for Coffee and coffee products Part 3: Coffee: determination of caffeine content (reference method) UDC 633.73 + 633.937:620.113BS5752-3:1983 This British Standard, having been prepared under the directionof the Food and Agriculture Stan
2、dards Committee,was published underthe authority of the BoardofBSI and comes into effect on 31 October1983 BSI 10-1999 The following BSI references relate to the work on this standard: Committee reference FAC/15 Draft for comment 76/52236 DC ISBN 0 580 13509 8 Committees responsible for this British
3、 Standard The preparation of this British Standard was entrusted by the Food and Agriculture Standards Committee (FAC/-) to Technical Committee FAC/15 upon which the following bodies were represented: Association of Public Analysts British Soluble Coffee Manufacturers Association Coffee Trade Federa
4、tion Ltd. Department of Industry (Laboratory of the Government Chemist) Food Manufacturers Federation Incorporated Institute of Trading Standards Administration Ministry of Agriculture, Fisheries and Food Overseas Development Administration (Tropical Development and Research Institute) Amendments is
5、sued since publication Amd. No. Date of issue CommentsBS5752-3:1983 BSI 10-1999 i Contents Page Committees responsible Inside front cover National foreword ii 0 Introduction 1 1 Scope and field of application 1 2 References 1 3 Principle 1 4 Reagents 1 5 Apparatus 1 6 Sampling 2 7 Procedure 2 8 Expr
6、ession of results 3 9 Test report 4 Figure 1 Chromatographic columns 5 Figure 2 Example of spectrometric measurement 6 Table Repeatability and reproducibility 4 Publications referred to Inside back coverBS5752-3:1983 ii BSI 10-1999 National foreword This Part of this British Standard has been publis
7、hed under the direction of the Food and Agriculture Standards Committee. It is identical with ISO4052:1983 “Coffee Determination of caffeine content (Reference method)” published by the International Organization for Standardization (ISO). This Part embodies an agreement, in which the UK has taken p
8、art, reached in subcommittee15of Technical Committee34, Agricultural food products, of ISO. BS 5752 consists of Parts corresponding to International Standards as they are published. Subject to UK acceptance of the International Standards the Parts of BS5752 will be identical with the corresponding I
9、SO texts. Terminology 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 f
10、ollowing. The comma has 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 “British Standard”. With reference to ISO
11、1447:1978, there is at present no British Standard equivalent. The International Standard is being revised and, when published, it is expected that it will be published as Part2 of this British Standard. Meanwhile, moisture content of green coffee can be measured using the method given in Part 1 of
12、this standard or by a procedure calibrated against the method given in Part 1. ISO 6670 and ISO 6673 are in course of preparation. It is expected that when they are available they will be published as identical dual-numbered British Standards. With reference to clause6 and footnote 1), until relevan
13、t ISO standards are available, users of this standard should ensure that the sampling procedures employed result in a representative sample. A British Standard does not purport to include all the necessary provisions of a contract. Users of British Standards are responsible for their correct applica
14、tion. Compliance with a British Standard does not of itself confer immunity from legal obligations. Cross-references International Standard Corresponding British Standard BS 5752 Methods of test for coffee and coffee products ISO 3726:1983 Part 6:1983 Instant coffee: determination of loss in mass at
15、70 C under reduced pressure (Identical) ISO 4072:1983 BS 6379 Sampling of coffee and coffee products Part 1:1983 Method of sampling green coffee in bags (Identical) Summary of pages This document comprises a front cover, an inside front cover, pages i and ii, pages1 to 6, an inside back cover and a
16、back cover. This standard has been updated (see copyright date) and may have had amendments incorporated. This will be indicated in the amendment table on the inside front cover.BS5752-3:1983 BSI 10-1999 1 0 Introduction The method described in this International Standard has been chosen from amongs
17、t several methods, a comparative study of which was carried out, because of its general applicability, its reproducibility, its specificity, its ease of application and its rapidity. However, the method is particularly sensitive to variations in its application and it is therefore essential to follo
18、w the instructions in every detail. 1 Scope and field of application This International Standard specifies the reference method for the determination of the caffeine content of coffee. The method is applicable to green coffee, decaffeinated green coffee, roasted coffee, decaffeinated roasted coffee,
19、 extracts of coffee, both dried and liquid, and decaffeinated extracts, both dried and liquid. The lower limit of detection is0,02% caffeine on the dry basis. 2 References ISO 1447, Green coffee Determination of moisture content (Routine method). ISO 3726, Instant coffee Determination of loss in mas
20、s at70 C under reduced pressure. ISO 4072, Green coffee in bags Sampling. ISO 6670, Instant coffee in cases with liners Sampling 1) . ISO 6673, Green coffee Determination of loss in mass at 105 C 1) . 3 Principle Extraction of the caffeine from a test portion, in an ammoniacal medium. Successive pur
21、ification, with diethyl ether, on two chromatographic columns, the first in an alkaline medium, the second in an acid medium, followed by elution of the caffeine by chloroform. Spectrometric measurement of the eluate at the wavelength of maximum absorbance (in the ultraviolet region). 4 Reagents All
22、 reagents shall be of recognized analytical quality. The water used shall be distilled water or water of at least equivalent purity. 4.1 Sulphuric acid, 200 g/l solution c(H 2 SO 4 ). 2mol/l. 4.2 Sodium hydroxide, 80 g/l solution c(NaOH) . 2 mol/l. 4.3 Diatomaceous earth The product used shall ensur
23、e at least98% recovery of caffeine from the test portion. NOTECelite 545 has been found to be suitable. 4.4 Ammonia, 70 g/l solution (1 volume of concentrated ammonia solution, 20. 0,9g/ml,+2 volumes of water). 4.5 Diethyl ether, pure, or repurified (see7.5) by chromatography as follows, and saturat
24、ed with water. Pass 800 ml of diethyl ether through a column containing100g of basic aluminium oxide of activity grade 1. The diethyl ether, thus repurified, shall be kept in dark bottles until used. (Alternatively, diethyl ether, recently distilled and free of peroxides, can be used instead of diet
25、hyl ether repurified by chromatography.) 4.6 Caffeine 1,3,7-trimethyl-2,6-dioxopurine (C 8 H 10 N 4 O 2 ), pure, anhydrous. 4.7 Chloroform, pure, or repurified (see7.5) by chromatography as described in4.5, and saturated with water. 5 Apparatus 5.1 Chromatographic columns (seeFigure 1), 250mm long,
26、21mm in internal diameter (column I) and17mm in internal diameter (column II), with stopcocks preferably of PTFE. 5.2 Ultraviolet spectrometer, accurate to within0,004absorbance unit within the range used. 5.3 Silica cells, of optical path length 10 mm. 5.4 Usual laboratory equipment, including 5.4.
27、1 Beakers, of capacity 100 ml. 5.4.2 Boiling water bath 5.4.3 One-mark volumetric flasks, of capacities50, 100 and1000 ml. 5.4.4 One-mark pipettes, of capacities2 and5 ml. 5.4.5 Analytical balance 5.5 Coffee-mill, suitable for roasted coffee beans. 5.6 Toothed-disk mill, with cooling jacket, or anal
28、ytical mill, with sparecutter and cooling jacket, or similar mill suitable for green coffee beans. 5.7 Sieve, of woven metal wire cloth, nominal aperture size6004m or 6304m, complying with the requirements of ISO3310-1. 1) At present at the stage of draft.BS5752-3:1983 2 BSI 10-1999 6 Sampling Sampl
29、e in accordance with the method specified in the relevant International Standard. 2) 7 Procedure 7.1 Preparation of test sample If necessary, mill the sample, using the apparatus specified in 5.5 or 5.6, as appropriate, until it passes the sieve (5.7). 7.2 Determination of dry matter content Calcula
30、te the dry matter content after determining the moisture content on part of the test sample (7.1) in accordance with the method specified in the relevant International Standard. 3) 7.3 Test portion 7.3.1 Green coffee and roasted coffee Weigh, to the nearest0,1mg, about1g of the test sample (7.1). Tr
31、ansfer it to a100ml beaker (5.4.1), add5ml of the ammonia solution (4.4) and warm for2min on the boiling water bath (5.4.2). Allow to cool, then transfer to a100ml volumetric flask(5.4.3), dilute to the mark with water and mix. Allow this turbid solution to settle, and then, using a pipette (5.4.4),
32、 transfer5,0ml of the solution into a100ml beaker (5.4.1), add6g of the diatomaceous earth (4.3) and mix carefully. 7.3.2 Dried coffee extract Proceed in accordance with 7.3.1, but using a test portion of0,5g, and an aliquot portion of the turbid solution of2ml, and3g of the diatomaceous earth. 7.3.
33、3 Liquid coffee extract Proceed in accordance with 7.3.1, but using a test portion between 1 and2,5g corresponding to approximately0,5g of coffee solids, and an aliquot portion of the turbid solution of2ml, and3g of the diatomaceous earth. 7.3.4 Decaffeinated green coffee and decaffeinated roasted c
34、offee Weigh, to the nearest0,1mg, about1g of the test sample (7.1). Transfer it to a beaker (5.4.1), add5ml of the ammonia solution (4.4) and warm for2min on the boiling water bath (5.4.2). Add6g of diatomaceous earth (4.3) and mix carefully. 7.3.5 Dried decaffeinated coffee extract Proceed in accor
35、dance with 7.3.4, but using a test portion of0,5g. 7.3.6 Liquid decaffeinated coffee extract Proceed in accordance with 7.3.4, but using a test portion between1and2,5g corresponding to approximately0,5g of coffee solids, and7to8g of the diatomaceous earth. 7.4 Determination 7.4.1 Filling of columns
36、7.4.1.1 Column I (alkaline column) 7.4.1.1.1 Layer A Mix carefully, by kneading with a flexible spatula blade, 3g of the diatomaceous earth (4.3) and2ml of the sodium hydroxide solution (4.2), until homogeneous (see the note). A slightly wet powder is obtained. Transfer this powder, in portions of a
37、pproximately2g, into the21mm diameter chromatographic column (5.1), the lower part of which is packed with a wad of cotton wool or glass wool. Tamp down the mixture after each addition, without excessive force, using a glass rod one end of which is flattened to the diameter of the column, until a pe
38、rfectly homogeneous and compact layer is obtained. A small wad of cotton wool or glass wool may be placed on the top of layer A. NOTEColumn packing material may be prepared in bulk in advance and stored in closed containers. A mass of5,16g is required for each alkaline column. 7.4.1.1.2 Layer B Tran
39、sfer the mixture of the diatomaceous earth and the test portion (7.3) into the column on top of layerA. Dry the beaker twice with portions of about1g of the diatomaceous earth (4.3), transferring this into the column. Tamp down to obtain a homogeneous layer and place a wad of cotton wool or glass wo
40、ol on the top of this layer B. 7.4.1.2 Column II (acid column) Place in the17mm diameter chromatographic column (5.1), the lower part of which is packed with a wad of glass wool, 3g of the diatomaceous earth(4.3) and3ml of the sulphuric acid solution(4.1), carefully mixed and packed into the column
41、as described for layer A of column I in7.4.1.1. Place a wad of glass wool on the top of this layer. NOTEColumn packing material may be prepared in bulk in advance and stored in closed containers. A mass of6,36g is required for each acid column. 2) For the sampling of green coffee in bags, see ISO407
42、2; for the sampling of instant coffee in cases with liners, see ISO6670. Methods for other types of coffee and coffee products have not yet been elaborated. 3) For the determination of the moisture content of green coffee, seeISO1447, and, for the loss in mass at105 C of green coffee, seeISO6673; fo
43、r the determination of the loss in mass at70 C under reduced pressure of instant coffee, see ISO3726. Methods for other types of coffee and coffee products have not yet been elaborated.BS5752-3:1983 BSI 10-1999 3 7.4.2 Chromatography Mount the columns one above the other so that the effluent from co
44、lumn I can drip directly into columnII. Pass150ml of diethyl ether (4.5) through the two columns. Adjust the stopcock of column II so that a quantity of supernatant liquid remains above the layer. Remove column I. Pass50ml of diethyl ether(4.5) through column II, using the initial portion to wash th
45、e tip of column I and passing this portion also into column II. Discard the effluent from column II. NOTEUsed diethyl ether may be recovered by shaking it with iron(II) sulphate. Pass a stream of air from the top to the lower part of column II (for example by using an inflated rubber blower), until
46、no more diethyl ether drips from the column and the air flow from the stopcock carries only a faint smell of diethyl ether (see the warning below). Elute column II with45to50ml of chloroform (4.7). Collect the eluate in a50ml one-mark volumetric flask (5.4.3), dilute to the mark with the chloroform
47、(4.7) and mix carefully. The flow rate of the diethyl ether and the chloroform under conditions of natural flow should be between1,5and3ml/min. If this rate is exceeded, channelling should be suspected and the determination recommenced. WARNING The additions of diethyl ether and chloroform should be
48、 carried out in a well-ventilated fume-cupboard to prevent both the possibility of inhalation of solvent vapours and the possibility of an explosion. 7.4.3 Spectrometric measurement (see Figure 2) 7.4.3.1 Measurement of the test solution Avoiding error from evaporation of chloroform, measure the abs
49、orbance of the solution of caffeine in chloroform (7.4.2), using the silica cells (5.3), against chloroform (4.7) at the wavelength of maximum absorbance obtained on the spectrometer used (about276nm), and at wavelengths30nm above and below this wavelength in order to verify the purity of the caffeine obtained. If the maximum absorbance exceeds the limit of accurate measurement of the instrument used, repeat the measurement on a diluted aliquot portion of the solution of caffeine in chloroform (7.4.2). In this case, take the dilution into acco
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