1、Ref. No. DIN 10780 : 2003-02English price group 10 Sales No. 011010.03DEUTSCHE NORM February 200310780Continued on pages 2 to 12. No part of this translation may be reproduced without the prior permission ofDIN Deutsches Institut fr Normung e.V., Berlin. Beuth Verlag GmbH, 10772 Berlin, Germany,has
2、the exclusive right of sale for German Standards (DIN-Normen).Determination of free and total carbohydrates incoffee extract by high performance anion-exchangechromatographyTranslation by DIN-Sprachendienst.In case of doubt, the German-language original should be consulted as the authoritative text.
3、67.140.20Kaffee-Extrakt Bestimmung der freien und Gesamt-Kohlenhydrate Verfahren mit Hochleistungsanionenaustausch-ChromatographieIn keeping with current practice in standards published by the International Organization for Standardization(ISO), a comma has been used throughout as the decimal marker
4、.ContentsPageForeword . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 Scope . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 Normative references . . . . . . . . . . . . .
5、 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 Concepts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 Des
6、ignation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 Apparatus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7、. . . . . . . . . . . . . . . . . . . . . 38 Sampling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 410 Evaluation . . . .
8、. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 511 Precision . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 512 Test report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9、. . . . . . . . . . . . . . . . . 6Annex A Results of interlaboratory testing . . . . . . . . . . . . . . . . . . . . . . . . . . . 7Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12ForewordThis standard has been prepared by Tec
10、hnical Committee Kaffee of the Normenausschuss Lebensmittel undlandwirtschaftliche Produkte (Foodstuffs and Agricultural Products Standards Committee) and conforms insubstance to International Standard ISO 11292 : 1997. However, it contains new precision data from aninternational interlaboratory tes
11、t performed after publication of the first edition of ISO 11292.1 ScopeThis standard specifies a method of determining the content of free and total carbohydrates, in particularindividual monosaccharides and mannitol, in coffee extract by high performance anion-exchange chroma-tography.Page 2DIN 107
12、80 : 2003-022 Normative referencesThis standard incorporates, by dated or undated reference, provisions from other publications. These normativereferences are cited at the appropriate places in the text, and the titles of the publications are listed below. Fordated references, subsequent amendments
13、to or revisions of any of these publications apply to this standardonly when incorporated in it by amendment or revision. For undated references, the latest edition of the pub-lication referred to applies.DIN 10764-2 Determination of the moisture content of coffee extract by the vacuum cabinet metho
14、dDIN 12680-1 Graduated cylinders for laboratory useDIN EN ISO 1042 Laboratory glassware One-mark volumetric flasks (ISO 1042 : 1998)ISO 3509 : 1989 Coffee and its products VocabularyISO 5725-1 : 1994 Accuracy (trueness and precision) of measurement methods and results Part 1: Generalprinciples and d
15、efinitionsISO 5725-2 : 1994 Accuracy (trueness and precision) of measurement methods and results Part 2: Basicmethod for the determination of repeatability and reproducibility of a standard measure-ment methodISO 6670 : 1983 Instant coffee in cases with liners SamplingISO 11292 : 1997 Instant coffee
16、 Determination of free and total carbohydrate contents Method using high-performance anion-exchange chromatography3 ConceptsIn addition to those given in ISO 3509, the following concepts are used in this standard.3.1 Free carbohydrate contentContent of each individual monosaccharide (arabinose, fruc
17、tose, galactose, glucose, mannose) and of mannitoldetermined by method A as described in this standard (to be reported as a percentage of the dry mass).3.2 Total carbohydrate contentThe content of each individual monosaccharide (arabinose, galactose, glucose, mannose, xylose) determinedby method B a
18、s described in this standard (which involves a hydrolysis step) (to be reported as a percentageof the dry mass).4 Principle4.1 Method AA sample of the extract is dissolved in water and filtered. The carbohydrates in the filtrate are separated by ionchromatography in a high performance anion-exchange
19、 column, pure water being used as eluent. The dissolvedcompounds are determined electrochemically using a pulsed amperometric detector and quantified by compari-son with the peak areas of standard solutions.4.2 Method BA sample of the extract is hydrolysed with aqueous hydrochloric acid and filtered
20、. The carbohydrates in thefiltered hydrolysis solution are analysed as in method A.5 DesignationDesignation of the method of determining the free carbohydrates in coffee extract by high performance anion-exchange chromatography using method A as described in this standard:Method DIN 10780-ADesignati
21、on of the method of determining the total carbohydrates in coffee extract by high performance anion-exchange chromatography using method B as described in this standard:Method DIN 10780-B6 ReagentsOnly analytical grade reagents shall be used and the water used shall be distilled or demineralized.The
22、 following reagents shall be used.Page 3DIN 10780 : 2003-026.1 50 % (m/m) aqueous solution of sodium hydroxide, NaOH, containing a minimum of sodium carbonateand mercury. The solution shall not be shaken or stirred before use.6.2 1 mol/l hydrochloric acid standard solution, HCl.6.3 Demineralized wat
23、er, having a resistivity of 18 MV cm as eluent no. 1, filtered through a membrane filter(as in subclause 7.7) and degassed by gassing with helium for 20 to 30 minutes.6.4 300 mmol/l sodium hydroxide solution, as eluent no. 2, prepared by adding 15,6 ml of sodium hydroxidesolution to 985 ml of degass
24、ed water (as in subclause 6.3) using a pipette.CAUTION. It is crucially important to remove dissolved carbon dioxide from the eluent before use sincecarbonate has a pronounced accelerating effect on the column, with a consequent drastic reduction inresolution and efficiency. The solution shall there
25、fore be prepared one day before the analysis. Eluent no.2 is used in the chromatographic analysis both for column flushing in the cleaning step and as a post-column addition to the analyte (see tables 1 and 2).6.5 Standard carbohydrate solutions containing arabinose, fructose, galactose, glucose, ma
26、nnose, xyloseand mannitol, freshly prepared by weighing about 100 mg of the substance concerned to an accuracy of 0,1 mginto a 100 ml round bottom volumetric flask and making up to the mark with water. These solutions contain1 000 mg/l and shall be kept as stock.Mixed standard solutions may also be
27、prepared from various stock solutions provided the retention time of eachcarbohydrate under the applicable chromatographic conditions is known.The standard solutions shall be diluted until the carbohydrate concentration is the same as in the unhydrolysedcoffee extract test solutions.It is difficult
28、to separate rhamnose and arabinose. If their peaks are not resolved, rhamnose shall not be addedto a mixed standard solution.7 ApparatusIn addition to standard laboratory equipment, the following shall be used.7.1 Analytical balance, capable of being read to an accuracy of 0,1 mg.7.2 Class A volumet
29、ric flask, as in DIN EN ISO 1042, of nominal capacity 100 ml.7.3 Graduated cylinders, tall form, of nominal capacities 1000 ml and 50 ml (e.g. as in DIN 12680-1).7.4 Vacuum filter.7.5 Fluted filter paper, medium-strength, of good quality.7.6 Disposable C18 filter cartridges1), to be used as specifie
30、d by the manufacturer.7.7 Disposable membrane filter, of pore size 0,2 mm.7.8 Water bath, capable of being adjusted to (100 t 5) C.7.9 Metal-free liquid chromatograph1), having a high performance anion-exchange column1) packed witha pellicular polystyrene/divinylbenzene anion-exchange resin, and hav
31、ing a pre-column (guard column)1) anda post-column derivatization system.7.10 Pulsed amperometric detector, with gold electrode1).7.11 Integrator, chromatographic data station1).7.12 Silver-loaded disposable cartridges1), for removing chloride, to be used as specified by the manufac-turer.8 Sampling
32、It is crucial for the laboratory to obtain a representative sample that has not been altered or undergone anydamage in transit or during storage.Sampling is not part of the method described here. A recommended sampling method is specified in ISO 6670.If the coffee extract is not in lined containers,
33、 a well-mixed representative laboratory sample shall be taken fromindividual packages.1) Information on sources of supply is available from the Normenausschuss Lebensmittel und landwirtschaft-liche Produkte, 10772 Berlin, Germany.Page 4DIN 10780 : 2003-029 Procedure9.1 Determination of dry massThe d
34、ry mass shall be determined as specified in DIN 10764-2 using a test portion taken from a laboratorysample.9.2 Preparation of test portion for analysis by method AWeigh about 300 mg of the laboratory sample to an accuracy of 0,1 mg directly into a 100 ml volumetric flask,add 70 ml of water using a g
35、raduated cylinder and shake until the test portion has dissolved. Make up to the markwith water and filter 5 ml to 10 ml of this solution through the filter cartridge, discarding the first few millilitres.9.3 Preparation of test portion for analysis by method BWeigh about 300 mg of the laboratory sa
36、mple to an accuracy of 0,1 mg directly into a 100 ml volumetric flask,add 50 ml of standard hydrochloric acid solution and swirl. Then leave the flask in a boiling water bath for 150minutes, ensuring that the surface of the solution always remains below the surface of the water in the bath andswirli
37、ng the solution by hand every 30 minutes. Then cool the flask to ambient temperature under running water,make it up to the mark with water and filter the solution through a fluted filter. Filter 3 ml of the filtrate througha disposable cartridge, discarding the first few millilitres.9.4 Chromatograp
38、hic analysisSet up the chromatograph, the detector and the integrator and put them into operation. Wait for the chroma-tograph to reach steady-state condition.Filter the standard solutions and the test solutions through 0,2 m membrane filters.Always inject the same volume of the filtered standard so
39、lution and the test solutions into the chromatographand separate the carbohydrates under the conditions specified in tables 1 and 2.After every fourth injection, inject the standard solution once in order to detect and take account of any changesin the retention times or peak integration.Table 1: Co
40、lumn preparationEluentTime, Eluent no. 1, Eluent no. 2,Procedural stepin min in ml in mlIsocratic 0 100 0 Start of data logging50,0 100 0 End of data logging50,1 0 100 Start of clean-up65,0 0 100 End of clean-up65,1 100 0 Start of re-equilibration80,0 100 0 End of re-equilibrationNOTE 1: Retention t
41、imes may vary from column to column. Start the clean-up only when the last monosac-charide (ribose) has been eluted.NOTE 2: To achieve good separation of xylose, it may be necessary to inject the standard solution two to threetimes or to increase the re-equilibration time.Table 2: Analytical conditi
42、onsInjection 20 lFlow rate 1,0 ml/minPost-column addition Eluent no. 2 at a flow rate of 0,6 ml/minTemperature Ambient temperatureDetector Fill the reference cell with eluent no. 2 and use the conditions specifiedas optimum by the manufacturer.Page 5DIN 10780 : 2003-0210 EvaluationCalculate the carb
43、ohydrate content, w, as a percentage by mass, using equation (1):w = G30G30G30G56G6DG41G56G6DG41 100 (1)whereA is the peak area of the relevant carbohydrate in the test solution;A0is the peak area of the relevant carbohydrate in the standard solution;m is the initial sample mass as dry mass in the t
44、est solution (as in subclause 9.2 or 9.3), in g;m0is the mass of the relevant carbohydrate in the standard solution (subclause 6.6), in g;V is the volume of test solution (as in subclause 9.2 or 9.3), in ml;V0is the volume of the standard solution (as in subclause 6.6), in ml.Report the result as th
45、e arithmetic mean from two analyses, either as free carbohydrate (method A) or as totalcarbohydrate (method B), to an accuracy of 0,01 % (m/m) for every relevant carbohydrate.11 Precision11.1 Interlaboratory testDetails of the interlaboratory test performed to determine the precision of the method a
46、re given in Annex A. Thevalues found in the test cannot necessarily be applied to concentration ranges and types of sample other thanthose specified.11.2 Repeatability limit(same operator, same equipment)The absolute difference between two successive results obtained under repeatability conditions w
47、ill not exceedthe repeatability limit, r, given in table 3 in more than 5 % of cases.Table 3Sample A Sample B Sample C Sample D Sample A Sample B Sample C Sample DCarbohydrate_x % r %Free arabinose 0,385 0,728 0,992 0,428 0,097 0,138 0,165 0,073Total arabinose 3,364 3,455 4,080 3,578 0,548 0,848 1,3
48、13 0,890Free galactose 0,418 0,442 0,528 0,486 0,106 0,079 0,109 0,108Total galactose 18,162 17,192 19,117 15,307 2,081 2,597 3,050 1,822Free glucose 0,110 0,075 0,103 0,927 0,038 0,028 0,027 0,180Total glucose 1,126 0,786 1,047 2,492 0,179 0,266 0,292 0,469Free mannose 0,268 0,238 0,224 0,667 0,106
49、 0,064 0,059 0,149Total mannose 9,613 9,187 8,440 9,369 2,524 2,136 2,470 1,873Free mannitol 0,086 0,097 0,106 0,453 0,040 0,034 0,030 0,117Free fructose 0,153 0,091 0,131 1,790 0,097 0,069 0,090 0,412Total xylose 0,522 0,140 0,468 0,596 0,158 0,068 0,155 0,10811.3 Reproducibility limit(different operators, different equipment)The absolute difference between two individual results obtained under reproducibility conditions will not exceedthe reproducibility limit, R, given in table 4 in more than 5 % of cases.Page 6DIN 10780 : 2003-02Table 4Sample A S
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