SPY Lab

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SPY Lab

• Michael Levine
• mnlevine_at_wisc.edu

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The Electromagnetic Spectrum
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Spectrophotometer
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Lambert-Beer Law

• A bec log I0/I - log T
• b is pathlength (usually 1 cm)
• e is the molar absorptivity (M-1cm-1)
• c is the concentration (M)
• T stands for transmittance
• If absorbance has multiple components
• A b ?ei8ci

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Hemoglobin
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Hemoglobin

• Hemoglobin is a tetrameric protein
• 4 subunits a2ß2
• One heme group per subunit
• 4 moles heme per mole hemoglobin
• Three redox states are known in vivo
• met- , deoxy- and oxy-hemoglobin

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Hemoglobin, cont.

• Deoxy-hemoglobin oxy-hemoglobin are the
  functional forms
• Met-hemoglobin is inactive
• Apo-hemoglobin can also exist in vitro
• Ascorbate and Sodium Dithionite will be used to
  reduce hemoglobin samples

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Hemoglobin Binding Curve
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Pulse Oximeter
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Pulse Oximeter

• Used in hospitals.
• Uses ratio of red light to infrared light to
  measure oxy to total hemoglobin ratio (oxygen
  saturation)
• Measures changing spectra with each pulse as
  arteries expand
• Works even if patient is wearing red nail polish
• Uses 2 wavelengths of light (660, 905 nm)

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Pulse Oximeter

• Used to see if a patient needs supplemental
  oxygen
• Limitations
• Does not measure total oxygen
• Cant tell if patient is anemic
• Reads 85 if there is met heme present
• Falsely high for carbon dioxide or cyanide
  poisoning
• There are expensive pulse oximeters that measure
  carboxy hemoglobin and met hemoglobin that use 7
  wavelengths

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Experimental Overview

• Perform experiment in order 1, 2, 5, 4, 3
• 1 Turn on spectrophotometer and allow it to warm
  up
• 2 Absorbance spectrum of hemoglobin from 670-230
  nm (do background correction first with buffer
  blank in back).
• Absorbance at 3 wavelengths (415, 420, 430 nm)
• Do NOT rezero at this point
• Use Quartz Cuvette

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Overview

• 5 Dithionite Reduced Hemoglobin
• Change Scan to 670-320 nm
• Use plastic cuvettes from this point on
• Dont shake sample

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Overview

• 5 (Cont.) Measure absorbance at 415, 420, and
  430 nm
• Dont re-zero and move on to step 4
• 4 Changes in spectrum in the presence of
  ascorbate
• Two scans and then fixed wavelength scan
• Go on to step 3

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Overview

• 3 Molar Absorptivity of hemoglobin in the soret
  region
• Use plastic cuvettes
• Be careful while making standard solutions
• Label your tubes

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Common Errors

• Lamp not warmed up
• Wrong Wavelength
• Improper blanking
• Dilution Mistake
• Dirty or mismatched cuvette
• Improper seating of cuvette
• Operation Errors

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Calculations

• Show calculations in lab report
• One of each kind of calculation
• Calculated concentrations
• 4 moles heme groups per mole hemoglobin
• Divide concentration by 4 to get hemoglobin
  concentration

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Calculations

• Try to put a file on the web site for matrix
  calculations
• Use cautiously because it doesnt always work
• Email me if you need help

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Considerations

• Read Manual
• Review spec instructions
• Warm up
• Zero
• Be patient
• Perform experiment steps 1, 2, 5, 4, 3
• Obtain dithionite (Step 5) directly from TA
• Be careful with quartz cuvettes
• Write all data in manual (get signed by TA before
  leaving)

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Report

• Due Dates
• Group A Feb 15
• Group B Feb 22
• Reports should include the following
• Cover Page
• Optical spectra, data sheets and other primary
  results
• Clearly labeled graphs
• Example calculations
• Watch units and significant figures
• Answers to questions
• Clear and Concise