Spectrophotometry

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Spectrophotometry

• Lab One

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Electromagnetic Spectrum
3 properties of light How do we see colors?
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Concentration

• The solute is dissolved or suspended in a solvent
• Molarity refers to the number of moles of solute
  in the solvent
• mole - The amount of a substance that contains as
  many atoms, molecules, ions, or other elementary
  units as the number of atoms in 0.012 kilogram of
  carbon 12. The number is 6.0225 1023,
  orAvagadro's number. Also called gram molecule.
  (from Dictionary.com)
• Todays unit mM (milli Molarity)

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Examples of Concentration

• Water Quality Assessment
• Phosphorus
• Nitrogen
• DO
• Ammonia
• CHLORINE

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Serial Dilution
1. Concentrations? 2. What will happen to the
color of the solutions ?
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Pipetting

• Reading a pipette
• Drawing liquid to a particular volume
• Meniscus
• Blow out

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Spectrophotometer

• -Cuvette Well
• Wavelength
• Calibration knobs
• Readings - T and A
• page 6

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Calibration write out page 7

• Warm up
• Set wavelength (This experiment 540nm)
• Solvent Blank (what is the solvent?)
• Nothing in 0T (Left Left knob)
• Blank in 100T (Right Right knob)
• Take out, back to 0
• KIMWIPES!!

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Taking a Reading

• Wipe with Kimwipe
• Place into spec 20
• Close lid and Read!
• Note T and A
• Remove cuvette

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Determining the Concentration of Chlorine in Tap
Water

• First Add DPD Free Chlorine Reagent to 5ml of
  stock solution
• The more chlorine present, the more the solution
  will turn pink
• Perform a serial dilution and determine T and
  Abs. of each known concentration
• Record on page 9
• Add DPD Free Chlorine Reagent to 5ml of tap water
  and determine T and Abs.
• Record on page 9

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Collect Data!!!
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Graphing page 10

• Make graphs 1-3 as described on page 10
• Make sure to include the following
• Titles
• Y vs. X axis (Dependent vs. Independent
  variables)
• Units
• Hand drawn and on computer versions

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Linear vs. Logarithmic

• Logarithmic scales
• Not like a ruler increments increase as scale
  increases
• Illustrates proportions (ratios)
• Pertains to T only
• T is a log function
• Based on multiplication

• Linear
• Like a ruler all increments equal
• Will show trends of plotted points
• Based on addition

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Application

• Standard Curve of known concentrations
• T on a linear graph
• Use this graph to determine the concentration of
  Chlorine in Tap Water

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T Reading of unknown 55 What is the unknown
solutions concentration? (unknown aka tap water)
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Now.making graphs on Excel - see Appendix in
lab manual