1. 8. Internal standards Adv. Chromatography

2. Instrument variations problems caused by instrument or sample introduction: standard addition or matrix-matched standards will not help often intermittent examples injection volumes for GC & HPLC flame/plasma temperature for flame photometry and ICP nebuliser uptake rates for flame photometry and ICP need a checking mechanism in solution that indicates when variations occur known as internal standard

3. Example 8.1 A sample with an internal standard included is measured for both species twice If these were two different samples, and the internal standard wasn’t used, you would conclude that #2 was 10% lower in concentration than #1 IS used in calcs to correct for variations AnalyteIS Run 110050 Run 29045

4. Choosing a suitable internal standard internal standard is NOT the analyte, but a totally different species must be chosen carefully 1.The chosen compound cannot be present in the sample. 2.The chosen compound should have similar physical and chemical properties to the analytes. 3.The chosen compound should be readily available. Exercise 8.1: Why are these important? 1.It would add to the IS response, but not be due to instrument variation 2.The two species need to behave similarly with instrument variation 3.Convenience and practicality

5. what constitutes similar properties depends on the technique: emission spectro: chemical => same periodic table group chromatography: physical => similar retention times (more in Ch 6) Exercise 8.2 How would you determine whether the chosen IS was present in the sample? analyse the sample for the proposed IS

6. Adding the IS 1.The IS is added to all solutions – standards, samples and recovery checks. 2.The same concentration of IS is added to all solutions. 3.The response of the IS should be similar to that of the main analyte. Exercise 8.3: Why are these important? 1.Instrument variation can occur at any time in any solution 2.A change in the IS response is supposed to indicate instrument variation, not differences in concentration 3.The instrument response at very different intensities could be different anyway

7. In calculations

8. Example 8.2 SolutionA resp.IS resp.Ratio 1% std12575125 ÷ 75 = 1.67 5% std648798.20 10% std12387316.96 Sample7035712.33

9. Exercise 8.4 Answer from graph: 0.478 %w/v Original conc: x 5 = 2.390% in 100 mL = 0.239g %w/w = 1.94 0.05%w/v std15759 2.66 0.2% std75372 10.46 1 % std352666 53.42 Sample170667 25.46