SPECTROPHOTOMETRY Introduction

1
SPECTROPHOTOMETRYIntroduction

• 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

2
Visible Light

• violet 380 nm
• blue
• green
• yellow
• orange
• red 750 nm

3
Parts of the Spec 20

• Wavelength control knob
• 0 transmittance control knob
• 100 transmittance control knob
• sample holder
• absorbance/transmittance scale

4
Visible Portion of the Electromagnetic Spectrum

• Set wavelength to 620 nm
• Record wavelenght ranges for
• red, orange, yellow, green, blue, orange light

5
Using the Spec 20

• warm up Spec 20
• make
• sample 10d water 10d protein 5ml
  indicator
• blank 20d water 5 ml indicator
• wait 5 minutes for color to develop
• set wavelength at 540 nm

6

• set transmittance to 0
• (lid closed and no sample)
• insert blank, close lid
• set transmittance to 100
• record transmittance and absorbance
• check lever of some machines

7
Development of a Standard Curve

• Tube Protein Water Indicator
• 1 20 d 0 5 ml
• 2 10 d 10 d 5 ml
• 3 5 d 15 d 5ml
• 4 2 d 18 d 5 ml
• 5 1d 19 d 5 ml
• 6 0 20 d 5 ml

8
Results

• Plot protein concentration in mg/ml vs.
  absorbance on graph paper
• measure absorbance of unknown and determine its
  protein concentration in mg/ml (extrapolate from
  graph)