Transcript Bioanalytical Application of UV/vis Spectroscopy

Bioanalytical Application of
UV/vis Spectroscopy
Week 4 Lab
S117 Fall 2011
Overview
• Post-lab Discussion
– Historical Perspective on Balmer, Rydberg, Bohr
– How science builds
• Pre-lab discussion
– Molecular Spectroscopy
– UV-vis Spectrometer
– Quantitative use of spectroscopy
– Standard curves
Balmer’s Contribution
• Experimental
• Empirical
observation of a
series
• Mathematical
description of
series
n2
Wavelength = constant *
n2 - 22
Rydberg’s Contribution
• Generalization
• Recognizing use of inverse wavelength
Bohr’s Contribution
• Theoretical
• Assume classic interpretation
• BUT…only if angular momentum restricted by
quantum rule
• From classical treatment mixed with quantum
rule:
Why is Bohr Accepted?
• Direct match to experimental results
• Explains Rydberg in terms of more
fundamental constants
• Philosophical question: How right is a “right
theory” and how wrong is a “wrong theory?”
Pre-lab: UV/vis Spectroscopy
• Investigating a Dow
Chemical Resin
• Look at this UV-vis
spectrum
– How is it different than
atomic spectrum?
• Full spectrum and lmax
Molecule for UV/vis
Conjugation leads to molecules absorbing in UV/vis range
463nm, 494 nm
Spectrometer
Source
Lamp
Wavelength Selector
(Monochromator)
Sample
Compartment
Photoelectric
Detector
Beer’s Law
• Absorbance depends on
– Characteristics of molecule
under experimental
conditions (molar
absorptivity)
– Size of cuvette (pathlength)
– Concentration of molecules
A=ebc
Standard Curve
• Determine molar
absorptivity
• Only good for one
wavelength and
one set of
conditions
• Use the data to
determine
concentrations of
unknowns
This Week
• Goal: Determine [protein] using intrinsic
absorbance of protein, then with added dye.
Which is better?
• For intrinsic absorption data
– Determine lmax for protein unknowns and standard
– Use this data to make a standard curve
– Use standard curve to determine concentration of two
unknown protein sample
• For dye test, lmax is given as 595nm
• Need to be able to determine concentrations of
dilutions and convert between concentration
units