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

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

Visible light Visible spectrum is approx nm violet blue green yellow orange red

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

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

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

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.

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