3Spectrophotometry

3Spectrophotometry - make blank = 1ml water + 5 ml...

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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
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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
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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
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SPECTROPHOTOMETER Instrument used for spectrophotometry Wavelength control knob • 0% transmittance control knob • 100% transmittance control knob • sample holder • absorbance/transmittance scale
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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
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Ex 3.2 • warm up Spec 20
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Unformatted text preview: 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...
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3Spectrophotometry - make blank = 1ml water + 5 ml...

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