1. B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000 H. Albus Basic Course Experiments to Demonstrate Validation 1 Basic Course Experiments to Demonstrate Validation (1) Fundamental concepts (2) Experimental arrangements (3) Results

2. B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000 H. Albus Basic Course Experiments to Demonstrate Validation 2 Objective of the Lesson Elaboration of the fundamental meaning of validation: Validation is the process of making sure that an analytical method is fit for the intended purpose. or in other words: Make sure that the obtained results are as good as you need them! (But first define properly what is „good enough“!)

3. B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000 H. Albus Basic Course Experiments to Demonstrate Validation 3 Fit for Purpose Question: How can one achieve good results? Answer: by using a method with the appropriate performance characteristics  Task of method validation Investigation and evaluation of the performance characteristics of an analytical method

4. B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000 H. Albus Basic Course Experiments to Demonstrate Validation 4 Basic Performance Characteristics of Analytical Methods Precision or: how good is the closeness of agreement between the obtained measurement results? Note: Precision is only influenced by random errors. This type of error shows no systematics and occurs randomly with statistical variability. measured values true value measured values true value good precision poor precision

5. B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000 H. Albus Basic Course Experiments to Demonstrate Validation 5 Basic Performance Characteristics of Analytical Methods Trueness or:how good is the closeness of agreement between the average value calculated from a series of test results and the (assumed) true value of the analyte (= substance under investigation)? Note: Trueness is only influenced by systematic errors. This type of error modifies the result only in one direction (too low or too high results).

6. B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000 H. Albus Basic Course Experiments to Demonstrate Validation 6 Basic Performance Characteristics of Analytical Methods Accuracy or: how good is the closeness of agreement between the result of a single measurement and the true value of the analyte? Note: Accuracy is a measure which combines precision and trueness (i.e. the effects of random and systematic errors respectively). If the obtained results are not affected by systematic errors, their accuracy becomes equivalent to their precision.

7. B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000 H. Albus Basic Course Experiments to Demonstrate Validation 7 Key Question of Analytical Chemistry How can one achieve accurate results? Answer: (1) by performing measurements using appropriate samples and a well described analytical method (  estimation of precision) (2) by performing measurements using an independent, validated comparison method (  estimation of trueness) Note: “Independent“ in this connection means that two methods are based on different physical and/or chemical principles.

8. B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000 H. Albus Basic Course Experiments to Demonstrate Validation 8 Strategy Method A is suitable / not suitable for the intended purpose YES  NO  ?? Sample (homogeneous) Subsamples Measurement Method A (under investigation) Method B (validated) independent Result agreement

9. B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000 H. Albus Basic Course Experiments to Demonstrate Validation 9 Example 1 Validation of the gravimetric determination of Fe 3+, Al 3+, PO 4 3- and SO 4 2- by application of a one-channel FIA-system. Background: Gravimetric determinations are difficult for beginners!  The flexible constructed One-channel FIA-system allows reliable control measurements which can be performed by the students very fast and without great expenditure.

10. B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000 H. Albus Basic Course Experiments to Demonstrate Validation 10 One-Channel FIA-System detector recorder ion-exchange column Injection - valve peristaltic pump sample route Pos. B reagent (carrier-) stream chemical reactor sample route Pos. A waste

11. B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000 H. Albus Basic Course Experiments to Demonstrate Validation 11 Working parameters Detector: photometer with flow cell Flow rate: 1,8 mL/min Injection volume: 40 µL Wavelength: 470 nm (Fe 3+ ); 532 nm (Al 3+ ); 390 nm (PO 4 3- ); 232 nm (SO 4 2- ) Reagent solutions Fe 3+ : Sulfosalicylic acid solution (1.0 % (m/m)); pH = 1.8 Al 3+ : Xylenol orange (0.1 % (m/m)) in buffer solution; pH = 4.4 PO 4 3- : Ammonium heptamolybdate (c = 0.005 mol/L) in nitric acid (c = 0.4 mol/L) SO 4 2- : dist. water (carrier)

12. B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000 H. Albus Basic Course Experiments to Demonstrate Validation 12 Working Procedure Determination of Fe 3+, Al 3+ and PO 4 3- Sample route Position A  direct injection of sample into reagent stream via injection valve Linear measurement range Fe 3+ 100 - 700 mg / L Al 3+ 10 - 70 mg / L PO 4 3- 100 - 700 mg / L

13. B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000 H. Albus Basic Course Experiments to Demonstrate Validation 13 Working Procedure Determination of SO 4 2- Sample route Position B pumping of sample solution through ion-exchange column (nitrate-form); quantitative replacement of sulphate: R(NR 3 + ) n (NO 3 - ) n + n/2 SO 4 2-  R(NR 3 + ) n (SO 4 2- ) n/2 + n NO 3 -  Direct UV-detection of NO 3 - at 232 nm Calculation factor: 0.775 1.29 mg NO 3 -  1 mg SO 4 2 - Linear measurement range 100 - 800 mg / L 

14. B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000 H. Albus Basic Course Experiments to Demonstrate Validation 14 Results Typical analyte concentrations  500 mg /L ( Fe 3+, PO 4 3- and SO 4 2- )  50 mg /L (Al 3+ ) Typical performance parameters

15. B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000 H. Albus Basic Course Experiments to Demonstrate Validation 15 Conclusion Gravimetry, performed with typical practical course equipment, and FIA show comparable accuracy!  One-channel FIA is an appropriate independent comparison method for the validation of gravimetric determination of Fe 3+, Al 3+, PO 4 3- and SO 4 2-.

16. B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000 H. Albus Basic Course Experiments to Demonstrate Validation 16 Example 2 Validation of the electrogravimetric determination of Cu 2+ by application of a didactically designed photometer. Background: Modern analytical instruments are „black box“ for beginners!  The use of an „open“ instrument enhances the learning effect to a great extent.

17. B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000 H. Albus Basic Course Experiments to Demonstrate Validation 17 Basic Concept of Photometer cheap (unit price 500 DM) no complex optical system robust and easy to handle modular construction; one electronic and one measurement case, connected with cables All important parts (lamp; slit; filter; detector) can directly be seen in full view. Display shows transmission; corresponding absorbance has to be calculated by the students.

18. B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000 H. Albus Basic Course Experiments to Demonstrate Validation 18 Diagram of Course of Beam within the Measurement Case tungsten lampslit colour filter cuvettephotodiode

19. B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000 H. Albus Basic Course Experiments to Demonstrate Validation 19 Electronic Case

20. B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000 H. Albus Basic Course Experiments to Demonstrate Validation 20 Measurement Case

21. B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000 H. Albus Basic Course Experiments to Demonstrate Validation 21 Procedure Cu 2+ - solution + conc. ammonia (25 % (m/m))  formation of [Cu(NH 3 ) 4 (H 2 O) 2 ] 2+ ( max = 580 nm)  Transfer in 1cm cuvette and threefold measurement (every time with new solution!)  calculation of concentration Linear measurement range: 100-700 mg / L

22. B. Neidhart, W. Wegscheider (Eds.): Quality in Chemical Measurements © Springer-Verlag Berlin Heidelberg 2000 H. Albus Basic Course Experiments to Demonstrate Validation 22 Results Typical analyte concentration:  500 mg Cu 2+ / L Typical performance parameters:  Photometry is easy to implement into first practical courses und of great usefulness for validation purposes in this context! average deviation mean value nominal value average rel. standard deviation (n = 3) El.-GravimetryPhotometryEl.-GravimetryPhotometry 1.2 %0.8 %1.5 %1.0 %