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1、International Standard INTERNATIONAL ORGANIZATION FOR STANDARDIZATIONWE)KJ1YHAPOtiAfI OPrAHH3ALlHR fl0 CTAHLAPW3AlWWORGANISATION INTERNATIONALE DE NORMALISATION Coffee - Determination of caffeine content (Reference method) Cafbs - Dhtermination de Ia teneur en cafhine - Mhthode de r for the types of
2、 coffee and coffee products have not Yet been elaborated. sampling of instant coffee in cases with liners, see ISO 6670. Methods for other 2) For the determination of the moisture content of green coffee, see ISO 1447, and, for the loss in mass at 105 OC of green coffee, see ISO 6673; for the determ
3、ination of the loss in mass at 70 OC under reduced pressure of instant coffee, see ISO 3726. Methods for other types of coffee and coffee products have not yet been elaborated. 2 Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical S
4、tandards 1/9972545001 Not for Resale, 04/26/2007 22:30:11 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 40524983 (E) 7.4 Determination 7.4.1.1 Column I (alkaline l column) 7.4.1 Filling of columns 7.4.1.1.1 Layer A Mix carefully, by kneading with a flexible spatula bl
5、ade, 3 g of the diatomaceous earth (4.3) and 2 ml of the sodium hydroxide Solution (4.2), until homogeneous (sec the note). A slightly wet powder is obtained. Transfer this powder, in portions of approximately 2 g, into the 21 mm diameter chromatographic column (5.1), the lower part of which is pack
6、ed with a wad of cotton wool or glass wool. Tamp down the mixture after each addition, without excessive forte, using a glass rod one end of which is flattened to the diameter of the column, until a per- fectly homogeneous and compact layer is obtained. A small wad of cotton wool or glass wool may b
7、e placed on the top of layer A. NOTE - Column packing material may be prepared in bulk in advance and stored in closed Containers. A mass of 5,16 g is required for each alkaline column. 7.4.1.1.2 Layer B Transfer the mixture of the diatomaceous earth and the test Portion (7.3) into the column on top
8、 of layer A. Dry the beaker twice with portions of about 1 g 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 wool on the top of this layer 9. 7.4.1.2 Column II (acid column) Place in the 17 mm diamete
9、r chromatographic column (5.1), the lower part of which is packed with a wad of glass wool, 3 g of the diatomaceous earth (4.3) and 3 ml of the sulphuric acid Solution (4.1), carefully mixed and packed into the column as described for layer A of column I in 7.4.1 .l. Place a wad of glass wool on the
10、 top of this layer. NOTE - Column packing material may be prepared in bulk in advance and stored in closed Containers. A mass of 6,36 g is required for each acid column. 7.4.2 Chromatography Mount the columns one above the other so that the effluent from column I tan drip directly into column II. Pa
11、ss 150 ml of diethyl ether (4.5) through the two columns. Adjust the stop- cock of column II so that a quantity of supernatant liquid re- mains above the layer. Remove column 1. Pass 50 ml of diethyl ether (4.5) through column II, using the initial Portion to wash the tip of column I and passing thi
12、s Portion also into column II. Discard the effluent from column II. NOTE - Used 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 no more diethyl ether drips f
13、rom the column and the air flow from the stopcock carries only a faint smell of diethyl ether (see the warning below). Elute column II with 45 to 50 ml of chloroform (4.7). Collect the eluate in a 50 ml one-mark volumetric flask I5.4.3), dilute to the mark with the chloroform (4.7) and mix carefully
14、. The flow rate of the diethyl ether and the chloroform under conditions of natura1 flow should be between 1,5 and 3 ml/min. If this rate is exceeded, channelling should be suspected and the determination recommenced. WARNING - The additions of diethyl ether and chloro- form should be carried out in
15、 a weil-ventilated fume- cupboard to prevent both the possibility of inhalation of solvent vapours and the possibility of an explosion. 7.4.3 Spectrometric measurement (sec figure 2) 7.4.3.1 Measurement of the test Solution Avoiding error from evaporation of chloroform, measure the absorbance of the
16、 Solution of caffeine in chloroform (7.4.2), us- ing the silica cells (5.3), against chloroform (4.7) at the wavelength of maximum absorbance obtained on the spec- trometer used (about 276 nm), and at wavelengths 30 nm above and below this wavelength in Order to verify the purity of the caffeine obt
17、ained. 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 account in the calculation; the appropriate factors of the fo
18、rmulae in 8.1.1, 8.1.2 and 8.1.3 will have to be adjusted accordingly. If the maximum absorbance measured is lower than 0,2, repeat the determination using a test portion of greater mass. 7.4.3.2 Preparation and measurement of reference Solution Prepare a reference Solution of caffeine in the follow
19、ing man- ner. Weigh, to the nearest 0,l mg, 100 + 20 mg of the pure anhydrous caffeine (4.6). Place in a 1 000 ml one-mark volumetric flask (5.4.3), dissolve in chloroform, and dilute to the mark. Using a pipette (5.4.4), transfer 5,0 ml of this solu- tion into a 50 ml one-mark volumetric flask (5.4
20、.3) and dilute to the mark with chloroform. Measure the absorbance of this Solution as described in 7.4.3.1. The corrected absorbance of the reference Solution (see 8.1 .l and figure 2) should be in the region of 0,4. 7.4.4 Number of determinations Carry out two determinations on separate test porti
21、ons taken from the same test Sample. 7.5 Blank test Carry out a blank test on the reagents, using the procedure described, but omitting the test Portion. Before using repurified reagents (see 4.5 and 4.7), repeat the blank test to verify their purity. 3 Copyright International Organization for Stand
22、ardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/26/2007 22:30:11 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 4052-1983 (El 8 Expression of results 8.1 Method of calculation and formulae 8.1.1 Green
23、coffee and roasted coffee The caffeine content of the Sample, expressed in grams per 100 g of dry matter, is equal to 107 x c x A, A, x in x P where c is the concentration, in grams per millilitre, of caffeine in the reference Solution (7.4.3.2); Al is the corrected absorbance of the purified extrac
24、t (7.4.2) obtained in 7.4.3.1, i.e. (A,lx - (Al)h -XI nm + tAljA + 30nm 2 where the subscript h refers to the wavelength of maximum absorbance (about 276 nm); A2 is the corrected absorbance of the reference Solution of caffeine (7.4.3.21, i.e. M21h - tA2)h -30 nm + lA2)h + 30 nm 2 m is the mass, in
25、grams, of the test Portion; P is the dry matter content, expressed as a percentage by mass, of the Sample (sec 7.2). 8.1.2 Dried coffee extract and liquid coffee extract The caffeine content of the Sample, expressed in grams per 100 g of dry matter, is equal to 25 x 10s x c x A, A2 x m x P where the
26、 Symbols have the same meaning as in 8.1.1. 8.1.3 Decaffeinated green coffee, decaffeinated roasted coffee, dried decaffeinated coffee extract and liquid decaffeinated coffee extract The caffeine content of the Sample, expressed in grams per 100 g of dry matter, is equal to 5 x 105 x c x A, -A, x m
27、x P where the Symbols have the same meaning as in 8.1.1. 8.1.4 Result Take as the result the arithmetic mean of the values obtained, provided that the requirement for repeatability (see 8.2) is satisfied. 8.2 Repeatability The differente between the values obtained in the two deter- minations, carri
28、ed out simultaneously or in rapid succession on the same Sample in one laboratory by the same analyst, shall not exceed the value given in the table. 8.3 Reproducibility The differente between the results of two determinations, car- ried out in two different laboratories on the same Sample, shall no
29、t exceed the value given in the table. 9 Test report The test report shall show the method used and the result obtained. lt shall also mention any operating details not specified in this International Standard, or regarded as optional, together with any circumstances that may have in- fluenced the r
30、esult. The test report shall give all details required for the complete identification of the Sample. Table - Repeatability and reproducibility Sample Amount of caffeine Repeatability” Reproducibility* g/lOO g coffee g caffeine/ g caffeine/ 100 g coffee 100 g coffee green coffee beans about 2 0,12 0
31、,38 about 1 0,08 0,3l decaffeinated 0,l 0,Ol 0,Ol roasted coffee beans about 2 0,lO 0,32 about 1 0,04 0,19 decaffeinated 0,l 0,Ol 0,Ol s”ubecoffee 1 decati;E;i o,3 j g 1 ;z; * The values of repeatability and reproducibility are critical differentes at the 95 % probabili- ty level. 4 Copyright Intern
32、ational Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/26/2007 22:30:11 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 4052-1983 (El 21 mm Cotton wool or glass wool Mixture of di
33、atomaceous earth and test Portion 3 g of diatomaceous earth and 2 ml of NaOH (= 5,16 g) 3g of diatomaceous earth and 3m I of H2S04 ( = 6,36 g) Gotton wool or glass wool Q3 4 - LI * 3 .I Stopcock, preferably PTFE 4 Column I (alkaline) 250 mm long Column II (acid) 250 mm long Figure 1 - Chromatographi
34、e columns 5 Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/26/2007 22:30:11 MDTNo reproduction or networking permitted without license from IHS -,-,- ISO 40524983 (El 12 LO 09 02 w Cell
35、s : silica, of Optical path length 10 mm Absorbance at 276 nm - / Absorbance at 246 nm - Absorbance at 306 nm / 220 240 260 1 w 280 300 320 340 Wavelength, nm Figure 2 - Example of spectrometric measurement Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=NASA Technical Standards 1/9972545001 Not for Resale, 04/26/2007 22:30:11 MDTNo reproduction or networking permitted without license from IHS -,-,-