UVVis Spectrophotometry

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UV/Vis Spectrophotometry

• Sec. 3.1 -3.3 Robyt and White

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UV/Vis

• Light interacts with molecules
• Microwave Causes rotations
• IR causes vibrations
• UV/Vis excites electrons in bonds
• Vacuum UV excites them so much that bonds break
• X-rays and Gamma rays excite electrons in atoms.
  (some to the point of escaping the atom)

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UV/Vis

• ROYGBIV
• Molecules absorb opposite there color
• Red molecules absorb Blue Green
• Blue molecules absorb Yellow Red

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UV/Vis

• Why Stay below 1 AU
• More important for cheaper Spectrophotometers
• -log(I/Io) A
• 1AU10 transmittance 90 of light absorbed

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limitations on assays

• BSA
• incubation times
• Lowry
• Store base (alkaline) in plastic
• Incubation times
• Bradford
• Making the stuff
• BCA

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Sample Size

• Cuvette size
• Sample limits
• make up volumes
• Dilution solutions

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Reagent solutions

• Ease of prep
• Prep at least 1 more assay than needed

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Things to consider

• Sample prep the ½ solution
• how would you make a guess for an assay
• Standards
• Minimums for good lines
• 3x5
• 2x7
• you can pitch 2 points

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Data analysis and blanks

• Blank with water
• results in non zero zero
• Graphs and analyzes just fine
• Blank with blank
• Which one of the three?
• lines typically dont hit zero anyway
• sometimes improves measurements of solutions with
  high blanks.

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Miscellaneous stuff

• Start thinking in µL below 1mL
• You can multiply/divide the prefixes on things to
  get values
• 1µl x 1mg/mL µg
• 1mg/mL 1µg/µL