Quantifying the measurement uncertainty for protein determinations in soybeans using the Kjeltec 2300

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 species 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. Identication 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 (gure 1). The sample weight is determined by differential weighing (mgross - mtare). As a calibrated electronic balance is used, certied 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

u (V - Vo) = 0.1 /3 = 0.0333 ur (V V0) = 0.0333 / 38.52 = 0.000864 4.2 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 936.15) and the concentration of the titration acid is calculated as follows:

Fig. 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. All components include repeatability contributions to the uncertainty, which are usually combined to one contribution for the overall experiment. As the components are independent, the standard uncertainty can be calculated as follows, including a term for the overall repeatability contribution:

Where: C : Concentration of the titration acid, CHCl in mol/l m : Mass of Na2CO3, mNa2CO3 in g P : Purity of Na2CO3, PNa2CO3 in % V : Titration volume for HCl, VHCL in ml M : Molar mass of Na2CO3, MNa2CO3 in g/mol Including a term for the repeatability, the uncertainty for the concentration of the titration acid can then be calculated according to:

4. Quantication of the uncertainty of each component 4.1 Uncertainty of titrated volumes (instrument error) The titration is carried out automatically by the Kjeltec 2300 instrument, using a piston burette and a photometric end-point detection. The uncertainty from this step has been specied by the manufacturer to 0.1%. Assuming a normal distribution and a condence interval of 99,7% (k=3), the standard uncertainty u, and for a mean value of 38.52% protein and the relative standard uncertainty ur can then be calculated:

4.2.1 Uncertainty of the weighed mass ur (mNa2CO3) An electronic balance is used to weigh Na2CO3. The following information is supplied with the certicate of the balance: Max error 0.2mg, foursquare error 0.1mg, repeatability error 0.2mg. Assuming a normal distribution and a condence interval of 99,7% (k=3) the uncertainty for a weighed mass would then be

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4.2.2 Uncertainty of the purity ur (PNa2CO3) The purity is (1000.5)%, which is given by the supplier. Assuming a rectangular distribution, k=3, then

4.2.3 Uncertainty of the titrated volume ur (VHCl) 4.2.3.1 Inuence of the calibration error of the burette The calibration error is given in the certicate to 0.05ml. Assuming a triangular distribution, k=6, then

The repeatability term represents a signicant contribution to the uncertainty of the acid concentration. In the calculation of the combined measurement uncertainty this term can be excluded as it is included in the total repeatability term. 4.2.6 The relative uncertainty of the acid concentration is then

4.2.3.2 The i inuence from temperature 4 2 3 2 Th f Suppose the temperature is changing at 5C, swell factor is = 2.1 10-4 / C, titration volume is 40ml. Assuming a rectangular distribution, k= 3, then 4.3 Uncertainty of weighing samples An electronic balance is used for weighing the samples. The certied values are: Max error 0.3mg, foursquare error 0.4mg, and repeatability error 0.2mg. Assuming a normal distribution (k=3) and a sample mass of about 0.2g, then

and the total contribution for the uncertainty of the titrated volume

4.2.4 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[5].
Element Na2 C O3 Calculation 222.9898 112.0107 315.9994 Result 45.9796 12.0107 47.9982 Standard uncertainty 0.0000024 0.00046 0.00051

4.4 Total repeatability term The results of eight repeated protein determinations are shown in table 3, which cover all the contributions from weighing, digestion, distillation and titration.
No. 1 2 3 Results 38.38 38.41 38.41 No. 4 5 6 Results 38.50 38.52 38.58 No. 7 8 Mean Results 38.65 38.68 38.52

Table 1: Atomic weight and uncertainty of each constituent element of Na2CO3

Table 3 Testing results of protein determinations (n=8, %)

4.2.5 Repeatability term ur (rep) The results of eight repeated calibrations of the titration acid are given in table 2.
No. 1 2 3 Results 0.10480 0.10470 0.10483 No. 4 5 6 Results 0.10515 0.10498 0.10463 No. 7 8 Mean Results 0.10428 0.10483 0.10478

Calculation of the combined standard uncertainty

Table 2 Calibration results of standard hydrochloric acid (n=8, mol/l)

6 Expanded uncertainty The expanded uncertainty Uc is obtained by multiplying the combined standard uncertainty by a coverage factor k =2 (95% condence level):
Uc = 2 u(X) = 2 0.073% = 0.15%

Thus the protein content is (38.52 0.15)%.

UX

CHCl

(V-Vo)

m (sample)

rep 0 0,0005 0,001 u(x) (% protein) 0,0015 0,002

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 38.5 % protein. As can be seen, all components are of importance. As the molarity of the titration acid has an important effect, the concentration of the hydrochloric acid used for titration should be assessed to the fourth decimal. Table 4 shows a summary of values and uncertainties for this protein determination. On basis of this information one can now take measures to improve the total uncertainty.
Description rep m (sample) (V-Vo) CHCl UX Repeatability Sample weight titrated volume(s) Acid concentration Protein concentration Value x 1,0 0,2 g 8,4 ml 0,1048 mol/l 38,52% u(x)Standard Rel.standard uncertainty uncertainty 0,00104 % 0,18 mg 7,3 l 0,103 mmol/l 0,073% 0,00104 0,0009 0,000864 0,00098 0,0019

[3] APLAC TC 005 Issue No. 3, 12/06 APLAC Interpretation and Guidance on the Estimation of Uncertainty of Measurement in Testing, http://www.aplac.org/tc_series.html [4] AOAC International, Ofcial Methods of Analysis, Method No 936.15 [5] IUPAC Technical reports, http://www.iupac.org/publications/ by Cheng Shuwei, Gu Jinling, Pang Jing (China Grains & Logistics Corporation Beiliang Co., Ltd., Dalian 116001) Edited by Jrgen Mller, FOSS (jmr@foss.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. This uncertainty is given by a statistical distribution of the results from an interlaboratory test and can therefore, in a top-down approach, also be characterized by the experimental standard deviation. The uncertainty is equal to plus or minus twice the reproducibility standard deviation from a well conducted collaborative study. Example: In the international validation of the global protein standard (AOAC 2001.11, EN ISO 5983-2) fourteen different samples have been analyzed by twelve different laboratories. Amongst the samples soybeans were also represented with a mean crude protein content of 38,8%. The reproducibility standard deviation for this sample was 0,4% protein. The expanded measurement uncertainty (95% condence level, k=2) would then be Ue = 0,8 % protein at a level of 38,8% protein. This can be regarded as a maximum uncertainty that can be expected when applying the method. In a single lab validation using a bottom-up approach and repeatability data better values should be expected.

Table 4: Summary of values and uncertainties for the determination of protein.

References: [1] ISO/IEC 17025:2005, General Requirements for the Competence of Testing and Calibration Laboratories, International Organization for Standardization, Geneva, Switzerland; http://www. iso.org/iso/store.htm [2] Eurachem/Citac Guide (2000), Quantifying Uncertainty in Analitical Measurement, 2nd Ed., S-L-R. Ellison, M. Rosslein, & A. Williams (Eds), http://www.eurachem.org