1. Spectrophotometry Ability of molecules to absorb and transmit light energy is the basis of one of the most widely used procedures for determining the concentration of substance in a solution

2. Available energy in the universe Electromagnetic radiation gamma rays x-rays UV(visible light = 380 nm to 750 nm) infrared TV and radio waves the smaller the wavelength, the greater the energy

3. Visible light Visible spectrum is approx 380-720nm violet 380 -440 blue-450-495 green-500-575 yellow-580-590 orange-590-620 red 620 -720

4. SPECTROPHOTOMETER Instrument used for spectrophotometry Wavelength control knob 0% transmittance control knob 100% transmittance control knob sample holder absorbance/transmittance scale

5. Ex 3.1 Set wavelength to 620 nm Place a empty tube filled with paper in the sample holder. Look through the sample holder Record wavelength ranges for:red, orange, yellow, green, blue, violet light

6. Ex 3.2 warm up Spec 20 make blank = 1ml water + 5 ml indicator and sample = 1ml protein solution + 5 ml indicator(protein solution = protein + water) wait 5 minutes for color to develop set wavelength at 540 nm set transmittance to 0% using the zero control knob(lid closed and no sample) insert blank, close lid set transmittance to 100% using the 100%T knob record % transmittance and absorbance *check lever of some machines

7. Ex 3.3 Development of a Standard Curve Follow instructions in the book Standard curve is used for determining the unknown conc of protein by plotting absorbance of unknown conc.

8. Results Plot protein concentration in mg/ml vs. absorbance on graph paper measure absorbance of unknown and determine its protein concentration inmg/ml (extrapolate from graph) Graph-X-axis (independent variable) protein concentration in mg/ml and Y-axis (dependent variable) Absorbance