Basic Course Experiments to Demonstrate Validation

1. H. Albus Basic Course Experiments to Demonstrate Validation Basic Course Experiments to Demonstrate Validation (1) Fundamental concepts (2) Experimental arrangements (3) Results

2. H. Albus Basic Course Experiments to Demonstrate Validation 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. H. Albus Basic Course Experiments to Demonstrate Validation 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. H. Albus Basic Course Experiments to Demonstrate Validation measured values true value good precision measured values true value poor precision 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.

5. H. Albus Basic Course Experiments to Demonstrate Validation 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. H. Albus Basic Course Experiments to Demonstrate Validation 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. H. Albus Basic Course Experiments to Demonstrate Validation 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. H. Albus Basic Course Experiments to Demonstrate Validation Strategy Method A is suitable / not suitable for the intended purpose Sample (homogeneous) Subsamples Measurement Method A Method B (under investigation) (validated) independent Result Result agreement  ? NO  YES 

9. H. Albus Basic Course Experiments to Demonstrate Validation Example 1 • Validation of the gravimetric determination of • Fe3+ , Al3+ , PO43-and SO42- • 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. H. Albus Basic Course Experiments to Demonstrate Validation sample route recorder Pos. A ion-exchange column sample route Pos. B detector reagent (carrier-) stream Injection - valve chemical peristaltic reactor pump waste One-Channel FIA-System

11. H. Albus Basic Course Experiments to Demonstrate Validation • Working parameters • Detector: photometer with flow cell • Flow rate: 1,8 mL/min • Injection volume: 40 µL • Wavelength:             470 nm (Fe3+); 532 nm (Al3+); •                                   390 nm (PO43-); 232 nm (SO42-) • Reagent solutions • Fe3+: • Sulfosalicylic acid solution (1.0 % (m/m)); pH = 1.8 • Al3+: • Xylenol orange (0.1 % (m/m)) in buffer solution; pH = 4.4 • PO43- : • Ammonium heptamolybdate (c = 0.005 mol/L) • in nitric acid (c = 0.4 mol/L) • SO42- : • dist. water (carrier)

12. H. Albus Basic Course Experiments to Demonstrate Validation Working Procedure • Determination of Fe3+, Al3+and PO43- • Sample route Position A •  direct injection of sample into reagent stream • via injection valve • Linear measurement range • Fe3+ 100 - 700 mg / L • Al3+ 10 - 70 mg / L • PO43- 100 - 700 mg / L

13. H. Albus Basic Course Experiments to Demonstrate Validation Working Procedure • Determination of SO42- • Sample route Position B • pumping of sample solution through • ion-exchange column (nitrate-form); • quantitative replacement of sulphate: • R(NR3+)n(NO3-)n + n/2SO42- R(NR3+)n(SO42-)n/2 + n NO3- •  • Direct UV-detection of NO3- at 232 nm • Calculation factor: 0.775 • 1.29 mg NO3- 1 mg SO42- • Linear measurement range • 100 - 800 mg / L 

14. H. Albus Basic Course Experiments to Demonstrate Validation Results • Typical analyte concentrations •  500 mg /L ( Fe3+, PO43- and SO42-) •  50 mg /L (Al3+) • Typical performance parameters

15. H. Albus Basic Course Experiments to Demonstrate Validation 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 Fe3+, Al3+, PO43- and SO42-.

16. H. Albus Basic Course Experiments to Demonstrate Validation Example 2 • Validation of the electrogravimetric determination of Cu2+ 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. H. Albus Basic Course Experiments to Demonstrate Validation 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. H. Albus Basic Course Experiments to Demonstrate Validation colour filter tungsten lamp slit cuvette photodiode Diagram of Course of Beam within the Measurement Case

19. H. Albus Basic Course Experiments to Demonstrate Validation Electronic Case

20. H. Albus Basic Course Experiments to Demonstrate Validation Measurement Case

21. H. Albus Basic Course Experiments to Demonstrate Validation Procedure • Cu2+- solution + • conc. ammonia (25 % (m/m)) •  formation of [Cu(NH3)4(H2O)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. H. Albus Basic Course Experiments to Demonstrate Validation average deviation average rel. standard mean value nominal value deviation (n = 3) El.-Gravimetry Photometry El.-Gravimetry Photometry 1.2 % 0.8 % 1.5 % 1.0 % Results • Typical analyte concentration: •  500 mg Cu2+/ L • Typical performance parameters: •  • Photometry is easy to implement into first practical courses und of great usefulness for validation purposes in this context!