1. SPECTROPHOTOMETRY

2. Spectrophotometry Determines concentration of a substance in solution –Measures light absorbed by solution at a specific wavelength

3. Spectrophotometry One of the simplest and most widely used methods to determine the amount of protein or nucleic acid present in a given solution

4. Spectrophotometry Proteins do not absorb in visible wavelength region unless they have a prosthetic group (e.g., Fe 2+ ), or an unnatural amino acid

5. Spectrophotometry Visible region: low energy electronic transition due to: a. Compounds containing transition metals b. Large aromatic structures & conjugated double bond systems (vitamin A, heme) UV region (200-400 nm): a. Small conjugated ring systems (Phe, Tyr, Trp)

6. Spectrophotometry Lamp Io I A = 0.012  l  Monochromator Cuvette Detector

7. Spectrophotometers Light source (Lamp) Optical filters or prism Tube or cuvette Photocell or photomultiplier tube

8. Light source (Lamp) Visible region = tungsten or tungsten- halogen UV light = deuterium or hydrogen lamp

9. Optical filters/prisms To limit light to a certain wavelength Monochromator can isolate a specific wavelength of white light and allow it to pass through the solution being analyzed

10. Tubes or cuvettes Visible range = glass cuvette UV range = quartz cuvette

11. Photocell To detect transmitted light

12. Spectrophotometry Beer-Lambert’s Law log Io =  cl I Where: Io = intensity of incident light I = intensity of transmitted light  = molar extinction coefficient c = concentration of the absorbing species (mol/L) l = path length of the light-absorbing sample (cm)

13. Beer-Lambert’s Law The fraction of the incident light absorbed by a solution at a given wavelength is related to a. thickness of the absorbing layer (path length) and b. concentration of the absorbing species b. concentration of the absorbing species

14. Visible region wavelength ColorWavelength (nm) Ultraviolet400 and under Violet400 - 450 Blue450 - 500 Green500 - 570 Yellow570 - 590 Orange590 - 620 Red620 - 650 Infrared750 & over

15. Beer-Lambert’s Law Concentration  amount of light absorbed A = abc = log(100/%T) Where A = absorbance a = absorptivity of the compound under standard conditions b = light path of the solution c = concentration of the compound %T = percent transmittance

16. Beer-Lambert’s Law Absorbance A = K x C = Log 10 Io I Where: Io = amount of light absorbed by the solution expressed as absorbance or optical density K = constant C = concentration of the substance

17. Transmittance Defined as the ratio of the intensity of light emerging from the solution (I) to that of incident light entering (Io) T = I Io Io I

18. Transmittance Inversely related to the concentration of the solution and is expressed in % % T = 1 x 100 Io

19. Transmittance 100% transmittance means no light is absorbed by the solution so that incident light is 100% transmitted

20. Absorbance & Transmittance Absorbance  concentration Transmittance 1/  to concentration and absorbance

21. Sample Problem Cytosine has a molar extinction coefficient of 6 x 10 3 mol -1 cm -1 at 270 nm at pH 7. Calculate absorbance of 1 x 10 -3 M cytosine solution in 1mm cell at 270 nm A = Log I 0 =  lc I

22. Sample Problem Solution: 1. A =  lc = (6 x 10 3 )x (0.1) x (1 x 10 -3 ) = 6 x 10 -1 = 0.6 (O.D.)

23. Spectrophotometry Clinical applications: 1. Aromatic amino acids have characteristic strong absorbance of light at a wavelength of 280 nm ex. Tryptophan & tyrosine

24. Calibration Curve Glucose Std. Concn. Absorba nce 60 mg%0.2 120 mg%0.4 U0.5 180 mg%0.6