2. Spectrophotometry Measuring Concentrations of Substances in Body Fluids

3. Spectrophotometer Measures light absorbance by solutions at specific wavelengths (colors) of light Different chemicals absorb light at different wavelengths Used to determine concentrations solutes in solutions

4. Beer’s Law Version #1… –“The concentration of a substance in solution is proportional to the amount of light absorbed” –[X] α A [X] = concentration of substance X A = amount of light absorbed (absorbance)

5. Beer’s Law

6. Version #2 –“The concentration of a substance in solution is inversely proportional to the logarithm of the amount of light transmitted through the solution” –[X] α 1/log %T %T = % transmittance (% light not absorbed while passing through the solution)

7. Beer’s Law

8. How Do We Use Beer’s Law? A = 0.250 A = 0.500

9. How Do We Use Beer’s Law? We know that [X] α A So for two solutions containing the same solute (standard and sample): [X STD ] α A STD and [X unk ] α A unk Therefore [X unk ] / [X STD ] = A unk /A STD Rearrange equation to solve for [X unk ]

10. Determining the Unknown [X unk ] = 50 mg/dL × 0.250 0.500 = 25 mg/dL [X unk ] = [X STD ] × A unk A std

11. Directions 1.Obtain 4 clean test tubes 2.Add 5.0 ml glucose reagent to each tube 3.Add the following to the tubes: 1.0.1 ml distilled water (will act as blank) 2.0.1 ml dog blood serum 3.0.1 ml unknown #1 4.0.1 ml unknown #2 4.Immerse each tube in 100  C water bath for 10 min 5.Remove tubes and immediately immerse in ice bath for 3 min 6.Use tube 1 as blank – record absorbance values for remaining three tubes

12. Using the Spectrophotometer 1.Turn on the machine w/ front left knob 2.Set wavelength with top right knob 3.With nothing in the holder and the holder lid closed, set transmittance to 0% with front left knob 4.Place the blank in the cuvette holder, close lid. Use right front knob to set the transmittance to 100%. Push mode button to set absorbance to.000 5.Remove blank 6.Insert tube to be tested, close lid, and read absorbance

13. Experimental Error Experiments are NEVER perfect –May mix reagents differently –Temperature may change –Instrument may vary High levels of error can lead to erroneous results Is a single reading representative?

14. Standard Curves Measure absorbance of multiple standards of different known concentrations [Solute] (g/dL)Absorbance 0.10.050 0.20.090 0.40.210 0.60.290 0.80.400

15. Standard Curves Plot Absorbance values vs. [Solute]

16. Standard Curves “Best Fit” Line

17. Standard Curves Extrapolation (e.g., Unknown A = 0.200)