Applications of Spectrophotometry

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Applications of Spectrophotometry

• Ch. 19

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Analysis of a Mixture

• The absorbance of a solution at any wavelength is
  the sum of the absorbances of all species in the
  solution
• A exbX eybY ezbZ.
• We can use simultaneous equations at different
  wavelengths if there is little overlap at these
  wavelengths in order to solve for two species,
  but for more it is best to use excel

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• When we have species whose spectra overlap, we
  can use beers law, to calculate the molar
  absorptivities of a standard for the species
• ex Axs / bXs ey Ays / bYs
• However, we often do not know the concentrations.
  We can solve for this using excel
• Using the equation Acalc exbXguess
  eybYguess

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• The terms Xguess and Yguess are inserted
  into the spreadsheet and the sum of the squares
  (Acalc Am)2 set equal to them. Using the solver
  function to vary Xguess and Yguess to
  minimize the sum of the squares, we can compute
  the most likely concentrations.

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• When spectra are well resolved, we use a matrix
  determination of the functions
• A exbX eybY
• A exbX eybY
• in order to compute the concentrations of X and
  Y.
• Often one absorbing species, X, will convert to
  another absorbing species, Y, which will result
  in a very distinct pattern in which the graphs
  cross at a point known as the Isosbestic point

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Measuring an Equilibrium Constant The Scatchard
Plot

• Equilibrium constants are measured by activities.
  For example, for the equilibrium P X PX,
• K PX/PX
• If there were a series of solutions in which
  increments of X are added to a constnant amount
  of P, so that P0 is the total concentration of P,
• P P0 -PX

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• Substituting this into the equilibrium
  expression, and rearranging terms, we get
• PX/P KP K(P0 - PX)
• Using spetrophotometric absorbance to measure PX,
  we get
• A epxPX epP
• Substituting P P0 -PX again, we get
• A epxPX epP0 -epPX

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• Since epP0 is A0,
• A PX (epx -ep) A0 ? PX?A/?e
• So that
• ?A/X K?eP0 - K?A

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Flow injection analysis

• In flow injection analysis, a sample is injected
  into a moving liquid stream to which various
  reagents can be added. After a suitable time, the
  reacted sample reaches a detector, which is
  usually a spectrophotometric cell. Flow injection
  is widely used in medical and pharmaceutical
  analysis, water analysis, and industrial process
  control
• A key feature of Flow injection analysis is
  rapid, repetitive analysis.

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Immunoassays and Aptamers

• Immunoassays, an important application of
  fluorescence employs antibodies to detect
  analytes. The analyte will bind to an antibody,
  and then a second antibody, which has an enzyme
  that results in either a colored or fluorescent
  product attached to it, binds to the analyte.

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Time Resolved Immunoassays

• The sensitivity of fluorescence immunoassays can
  be enhanced with time-resolved meausrements of
  luminescence from the lanthanide ion Eu3.
• A chelatin group binds the Eu3 to the antibody
  after completing all required steps, the pH is
  lowered and the Eu3 is released as an ion in the
  presence of a soluble chelator, which attracts
  metal to the solution. Strong luminescence from
  the metal is easily detected

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Aptamers

• Aptamers are 15-40 base pieces of DNA or RNA
  that strongly and selectively bind to a specific
  molecule. An aptamer for a desired target
  molecule is chosen from a pool of random DNA or
  RNA sequences by successive cyclings of binding
  to the target, removing unbound material, and
  replicating the bound nucleic acid. . This
  aptamer then behaves as a custom made, synthetic
  antibody.

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Sensors based on Luminescent Quenching

• Luminescence intensity is proportional to the
  concentration of the emitting species if
  concentrations are low enough. We can measure
  some analytes, such as O2, by their ability to
  quench the luminescence of another compound.
• In a molecular beacon, fluorescent and
  quenching groups are built onto one molecule of
  DNA or RNA. When the beacon binds to
  complementary molecules of DNA or RNA, the
  fluorescent group separates from the quencher and
  the molecule becomes strongly fluorescent