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While a measurement error is the difference between a measured and the true (or best estimate of a true) value, the measurement uncertainty is an estimate of the variation resulting from the measurement of the concentration of the analyte in the sample. In many cases a result maybe meaningless unless accompanied by its uncertainty. ISO 17025, the international standard for general requirements for the competence of testing and calibration laboratories, is used by most accreditation bodies and speciﬁes explicit requirements for laboratories with respect to determining and recording the uncertainty of their measurements. Two approaches are possible: The so-called top down approach uses data such as reproducibility and repeatability from wellconducted collaborative studies as indication of the uncertainty that can be expected of the method. The bottom up approach, as used in the following contribution, applies estimates of the uncertainty associated with each step of the analytical procedure. 1. Principle of the Method Soybeans are milled and then digested with concentrated sulphuric acid and a catalyst. The protein is converted to ammonium sulphate. After digestion alkali is added and the digested sample is distilled with steam, converting the ammonium into ammonia which is absorbed by boric acid in the receiver solution. The distillate is titrated with standard acid solution and the protein is calculated using the consumption of standard acid. 2. General Method Description After grinding, a 0.2g sample is weighed into a digestion tube and digested in a Tecator™ digestion block. The digested sample is inserted into the FOSS Kjeltec™ 2300 Analyzer, where there is an automatic dilution with water, addition of alkali, steam distillation and titration. 3. Identiﬁcation and Analysis of Uncertainty Sources Though of considerable importance sampling, subsampling/sample splitting and sample preparation are not part of this evaluation. On the basis of the ground sample weighed in, the results are calculated using the formula below:
Where: X : Protein content, in % V : Titration volume for the sample, ml V0 : Titration volume for the blank, ml C : Concentration of the titration acid, CHCl mol/l P : Conversion factor for nitrogen to protein (6.25 for soybean) This formula also gives the main components for the calculation of the measurement uncertainty, which can also be shown in a diagram (ﬁgure 1). The sample weight is determined by differential weighing (mgross - mtare). As a calibrated electronic balance is used, certiﬁed values from the supplier can be used for uncertainty calculations. The volume of the titration acid is delivered by a piston burette and is affected by repeatability of the delivered volume, the uncertainty of the calibration of that volume and the uncertainty resulting from the difference in the laboratory and that of the calibration of the piston burette. An addition there is a contribution from the end-point detection (repeatability and bias). The standardization of the titration acid is affected by the weighed mass of anhydrous sodium carbonate, used for the standardization of the hydrochloric acid, the purity of this reagent, the
Grinding Weighing Digestion
Distillation Titration RESULT
2 Uncertainty of the acid concentration Anhydrous sodium carbonate is used to standardize the hydrochloric acid according to Chinese standard GB/T 601-2002 (analogous to AOAC method 936. 1: Cause and effect diagram for the determination of crude protein uncertainty of its molar mass and the volume consumed to neutralize a certain amount of hydrochloric acid. PNa2CO3 in % V : Titration volume for HCl.15) and the concentration of the titration acid is calculated as follows: Fig. Assuming a normal distribution and a conﬁdence interval of 99.1mg.2mg.Vo) = 0.2mg. using a piston burette and a photometric end-point detection. which are usually combined to one contribution for the overall experiment.1 Uncertainty of titrated volumes (instrument error) The titration is carried out automatically by the Kjeltec 2300 instrument.000864 4. CHCl in mol/l m : Mass of Na2CO3. All components include repeatability contributions to the uncertainty. foursquare error ±0.1 /3 = 0. VHCL in ml M : Molar mass of Na2CO3.0333 ur (V – V0) = 0. The uncertainty from this step has been speciﬁed by the manufacturer to 0.u (V . Assuming a normal distribution and a conﬁdence interval of 99.1%. including a term for the overall repeatability contribution: Where: C : Concentration of the titration acid. Quantiﬁcation of the uncertainty of each component 4.7% (k=3). The following information is supplied with the certiﬁcate of the balance: Max error ±0.52% protein and the relative standard uncertainty ur can then be calculated: 4. and for a mean value of 38.52 = 0. mNa2CO3 in g P : Purity of Na2CO3. repeatability error ±0.1 Uncertainty of the weighed mass ur (mNa2CO3) An electronic balance is used to weigh Na2CO3. the uncertainty for the concentration of the titration acid can then be calculated according to: 4. MNa2CO3 in g/mol Including a term for the repeatability. As the components are independent. the standard uncertainty u. the standard uncertainty can be calculated as follows.7% (k=3) the uncertainty for a weighed mass would then be Cont’d on page 26 Con .2.0333 / 38.
then 4.00046 0.2g.3 Uncertainty of the titrated volume ur (VHCl) 4. Assuming a normal distribution (k=3) and a sample mass of about 0.41 38. foursquare error ±0.3mg.2.2mg.2. then and the total contribution for the uncertainty of the titrated volume 4. k=√3. 4 5 6 Results 38.4mg. 7 8 Mean Results 0.10480 0. 1 2 3 Results 38. titration volume is 40ml.3. Element Na2 C O3 Calculation 2×22. Assuming a triangular distribution.2. Assuming a rectangular distribution. 4 5 6 Results 0.3 Uncertainty of weighing samples An electronic balance is used for weighing the samples. and repeatability error ±0.2.10470 0.52 38. k= √3.2. 1 2 3 Results 0.10463 No.1 × 10-4 / °C.10483 No.52 Table 1: Atomic weight and uncertainty of each constituent element of Na2CO3 Table 3 Testing results of protein determinations (n=8.10428 0.0000024 0.10515 0. k=√6.38 38. 7 8 Mean Results 38.0107 47.2.41 No.2 The i inﬂuence from temperature 4 2 3 2 Th ﬂ f Suppose the temperature is changing at ±5°C.50 38. which cover all the contributions from weighing.1 Inﬂuence of the calibration error of the burette The calibration error is given in the certiﬁcate to 0.2.4 Total repeatability term The results of eight repeated protein determinations are shown in table 3.68 38. then The repeatability term represents a signiﬁcant contribution to the uncertainty of the acid concentration.Cont’d from page 25 4. In the calculation of the combined measurement uncertainty this term can be excluded as it is included in the total repeatability term.10498 0.9898 1×12.0107 3×15. No.9982 Standard uncertainty 0. The certiﬁed values are: Max error ±0.5)%.9994 Result 45. which is given by the supplier. distillation and titration.9796 12. %) 4.3. No. then 4. digestion.4 Calculation of the molar mass and its uncertainty ur (MNa2CO3) The atomic weights and listed uncertainties for the constituent elements of Na 2CO3 can be found in the latest IUPAC tables.05ml.00051 4. 4.6 The relative uncertainty of the acid concentration is then 4.58 No. swell factor is α= 2.10483 0.10478 5 Calculation of the combined standard uncertainty Table 2 Calibration results of standard hydrochloric acid (n=8. Assuming a rectangular distribution.65 38.5 Repeatability term ur (rep) The results of eight repeated calibrations of the titration acid are given in table 2. mol/l) .2 Uncertainty of the purity ur (PNa2CO3) The purity is (100±0.
Ltd. Rosslein. 12/06 APLAC Interpretation and Guidance on the Estimation of Uncertainty of Measurement in Testing.0005 0.standard uncertainty uncertainty 0. FOSS (jmr@foss. Table 4 shows a summary of values and uncertainties for this protein determination. Method No 936.0019  APLAC TC 005 Issue No. This uncertainty is given by a statistical distribution of the results from an interlaboratory test and can therefore.. Dalian 116001) Edited by Jürgen Möller. As the molarity of the titration acid has an important effect.org . also be characterized by the experimental standard deviation.org/publications/ by Cheng Shuwei. iso. the concentration of the hydrochloric acid used for titration should be assessed to the fourth decimal. As can be seen. Table 4: Summary of values and uncertainties for the determination of protein. 3. http://www.15  IUPAC Technical reports. Pang Jing (China Grains & Logistics Corporation Beiliang Co.0 0. General Requirements for the Competence of Testing and Calibration Laboratories. Gu Jinling.00104 0.5 % protein. Amongst the samples soybeans were also represented with a mean crude protein content of 38. http://www. http://www. in a “top-down” approach.6 Expanded uncertainty The expanded uncertainty Uc is obtained by multiplying the combined standard uncertainty by a coverage factor k =2 (95% conﬁdence level): Uc = 2 × u(X) = 2 × 0.html  AOAC International. In a single lab validation using a “bottom-up” approach and repeatability data better values should be expected.eurachem. References:  ISO/IEC 17025:2005. 2nd Ed.00098 0.. Example: In the international validation of the global protein standard (AOAC 2001. k=2) would then be Ue = ± 0.4% protein.0015 0.00104 % 0.org/iso/store. & A. The reproducibility standard deviation for this sample was 0.073% 0.iupac. International Organization for Standardization. Quantifying Uncertainty in Analitical Measurement.4 ml 0. http://www. S-L-R. UX CHCl (V-Vo) m (sample) rep 0 0.2 g 8. Williams (Eds). Description rep m (sample) (V-Vo) CHCl UX Repeatability Sample weight titrated volume(s) Acid concentration Protein concentration Value x 1. M..073% = 0. Geneva. This can be regarded as a maximum uncertainty that can be expected when applying the method.52% u(x)Standard Rel.000864 0. On basis of this information one can now take measures to improve the total uncertainty. The uncertainty is equal to plus or minus twice the reproducibility standard deviation from a well conducted collaborative study. Ellison.aplac.11. Ofﬁcial Methods of Analysis.3 μl 0.52 ± 0. Switzerland. EN ISO 5983-2) fourteen different samples have been analyzed by twelve different laboratories.0009 0.8%.15)%. all components are of importance.103 mmol/l 0.18 mg 7.15% Thus the protein content is (38. The expanded measurement uncertainty (95% conﬁdence level.001 u(x) (% protein) 0.org/tc_series.002 Figure 2: Contributions to the uncertainty of the protein determination 7 Discussion Figure 2 shows the contributions from the different components to the uncertainty of protein determinations in soybeans at a level of 38.dk) Measurement uncertainty: Top-down approach The expanded measurement uncertainty (Ue) is a parameter representing the distribution of the values that may reasonably be attributed to the result.8% protein.8 % protein at a level of 38.1048 mol/l 38.htm  Eurachem/Citac Guide (2000).
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