The influence of the reference method on the results of the secondary method via calibration

Transcription

1 The influence of the reference method on the results of the secondary method via calibration Heinz-Dieter Isengard 1, Georg Merkh 1 and Clément Dubois 2 1 University of Hohenheim, Institute of Food Science and Biotechnology, Stuttgart, Germany 2 École Supérieure d Agriculture, Angers, France 5 th Conference on Water in Food 6 to 8 April 2008 Nürtingen, Germany

2 Direct methods measure the property of the sample or the analyte as such. Direct methods are therefore also called primary methods and often serve as reference methods.

3 Indirect methods measure something that depends on the extent of the property or the concentration or amount of the analyte. They are therefore also called secondary methods.

4 Indirect methods need therefore relation to a direct method (to which they are referred ) in a way that the value measured allows a conclusion to the property of the sample or the concentration or the amount of the analyte.

5 Samples with a known property (analysed with a reference method) are measured with an indirect method and the value found is related to the true value.

6 Such data pairs are plotted versus each other and a regression curve (mostly a straight line) is laid through these data points. This is the calibration line.

7 When chemometric methods are applied, the analytical data points are calculated from many measurements each. These values are often called predicted values and are plotted against the true values obtained by the reference method.

8 So, where is the problem?

9 The whole approach depends on the correctness of the reference method, because it is always possible to draw a regression line through data points.

10 It is therefore essential that the reference method be correct.

11 Example: Water determination The Karl Fischer titration (KFT) detects water selectively, whereas drying techniques determine mass loss under the applied conditions (other volatiles are also included and strongly bound water is not detected completely).

12 Near infrared (NIR) spectra depend on water content. NIR calibrations should therefore be based on a reference method that really determines water and nothing but water (like the Karl Fischer titration).

13 Calibration can, however, also be carried out on the basis of a drying technique. Chemometrics will always find a calibration line (even when random numbers are used!).

14 NIR calibration line for water content of wheat semolina, based on Karl Fischer titration Water content in g/100 g predicted by NIR spectroscopy g g /100 in h alt g e e r s a W g ter a s g e e r V orh B Kalibriergerade "KFT - A2 und A3" Alle Spektren Property Outlier Spectra Loading Outlier Spectra Residual Outlier Spectra Validation Spectra f(x)=0.9918x r= Calibration Spectra f(x)=0.9967x r= Gemessener Wassergehalt in g/100 g Water content in g/100g measured by KFT ( true value ) ga n 3 I 2 2 : 0 : ,./ , UṆIR E N A 3 A 2+ K FT ß G RIE C al: N I R

15 Water content in g/100 g predicted by NIR spectroscopy g g /100 in h alt g e e r s a W g ter a s g e e r V orh B NIR calibration line for water content of wheat semolina, based on oven drying Kalibriergerade "Trockenschrank - A2 und A3" Alle Spektren Leverage Outlier Spectra Property Outlier Spectra Residual Outlier Spectra Validation Spectra f(x)=0.9899x r= Calibration Spectra f(x)=0.9970x r= Gemessener Wassergehalt in g/100 g Water content in g/100g measured by oven drying ( true value ) ga n 9 I 1 8 : 1 1 : ,./ , uṇi r e n u e n n k ra h n sc e c k ro A 3T A 2+ C al: N I R

16 Curves are nearly identical.

17 Results follow the respective calibration curve (KFT slightly better than OD) and are as correct (or not) as the reference method. In this case the differences are only small.

18 Another example: Lactoserum containing much lactose

19 Calibration against gravimetric mass lost by drying at 145 C of Lactoserum (Euvoserum from Nestlé)

20 The calibration is acceptable if the mass loss on drying is really the property to be analysed.

21 The calibration is acceptable if the mass loss on drying is really the property to be analysed. It is, however, useless if water content is to be analysed.

22 Calibration against gravimetric mass lost by drying at 145 C of Lactoserum (Euvoserum from Nestlé)

23 Lactoserum Euvoserum 16,0 14,0 12,0 10,0 8,0 6,0 4,0 2,0 0, Drying time in minutes Water content [%] of the original sample determined by Karl Fischer titration Gravimetric mass loss [%] of the sample in a drying oven at 145 C Residual water content [%] by Karl Fischer titration of the sample in a drying oven at 145 C

24 Calibration against water content determined by Karl Fischer titration of Lactoserum (Euvoserum from Nestlé)

25 A further example: Lasana, a baby food

26 9 Mass loss and water content in % of Lasana samples by oven drying at 140 C 8 0,478 7 Water Content %Mass Lost 6 % 5 4 0,8303 0,3844 0,398 7, KF titr. = 4,0698 % 3 2,1228 6, , , , , , Time in min

27 Calibration of mass loss against mass loss Mass loss in % predicted Mass loss in % measured by oven drying at 140 C

28 Calibration water content against water content (of dried product!) Water content in % predicted Water content in % measured after drying at 140 C

29 Calibration true water content minus mass loss against true water content minus mass loss True water content minus mass loss in % predicted True water content minus mass loss in % measured

30 Absorbance Units Wavenumber cm D:\Clement\Lasana original.0 Lasana original sample form D:\Clement\Lasana oven drying 140 1h.0 Lasana oven drying 140 1h sample form D:\Clement\Lasana oven drying 140 2h.0 Lasana oven drying 140 2h sample form D:\Clement\Lasana oven drying 140 3h.0 Lasana oven drying 140 3h sample form D:\Clement\Lasana oven drying 140 5min.0 Lasana oven drying 140 5min sample form D:\Clement\Lasana oven drying min.0 Lasana oven drying min sample form D:\Clement\Lasana oven drying min.0 Lasana oven drying min sample form D:\Clement\Lasana oven drying min.0 Lasana oven drying min sample form Seite 1 von

31 Mass loss and water content in % of Lasana samples by vacuum-oven drying at 48 C with P 2 O 5 4,5 4 0,6617 0,446 KF titr. = 4,0698 % 3,5 1,4794 1, ,4185 % 2,5 2 3,4201 2,9698 3, , Water Content %Mass Lost 1,5 2, , , ,5 1, , min 40min 1h30 3h 4h40 24h 48h Time

32 Calibration of mass loss against mass loss Mass loss in % predicted Mass loss in % measured by vacuum-oven drying at 48 C using P 2 O 5

33 Calibration water content against water content (of dried product!) Water content in % predicted Water content in % measured after vacuum-oven drying at 48 C using P 2 O 5

34 Calibration true water content minus mass loss against true water content minus mass loss True water content minus mass loss in % predicted True water content minus mass loss in % measured

35 Absorbance Units Wavenumber cm D:\Clement\Lasana original.0 Lasana original sample form D:\Clement\Lasana vacuum Jo 1h30.0 Lasana vacuum Jo 1h30 sample form D:\Clement\Lasana vacuum Jo 3h.0 Lasana vacuum Jo 3h sample form D:\Clement\Lasana vacuum Jo 4h40.0 Lasana vacuum Jo 4h40 sample form D:\Clement\Lasana vacuum Jo 15min.0 Lasana vacuum Jo 15min sample form D:\Clement\Lasana vacuum Jo 24H.0 Lasana vacuum Jo 24H sample form D:\Clement\Lasana vacuum Jo 40min.0 Lasana vacuum Jo 40min sample form D:\Clement\Lasana vacuum Jo 48H.0 Lasana vacuum Jo 48H sample form Seite 1 von

36 Summary:

37 Summary: Be sure to have good references!