Spectroscopy

1
Spectroscopy

• Making use of the interactions of different kinds
  of light with matter
• Assignment 3

2
1LC1 NMR Solution Structure of Cytochrome c in
30 Acetonitrile
Sivakolundu, S.G. Mabrouk, P.A. J. Am. Chem.
Soc. 2002, in preparation.
3
What is Light?

• Light is electromagnetic radiationIt has a dual
  nature
• wave - explains physical properties of light
  itselfphysicist
• particulate (photon) - explains how light
  interacts with matterbiologist/chemist

4
Wave Nature

• Two waves one electrical and one magnetic
  propagating at 90-degrees with respect to each
  other

y
x
z
5
Particulate

• Photon has a discrete energyE h ? hc /
  ?where h is Plancks constant6.63 x 10-34 J s
• Only discrete energies of light are absorbed by
  matter, i.e.,light is quantized

6
Visible Light
ROYGBIV
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Visible Light and Color
8
Color and Structure

• Fe3 cytochrome c
• Color?
• Fe2cytochrome c (add xs dithionite)
• Color?
• Conclusion UV-vis tells you something about
  electronic structure

9
So, How Does Light Interact With Matter?

• Light can be
• Reflected
• Refracted
• Scattered
• Emitted
• Absorbed

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Absorption
when E hv
11
Emission
when E hv
LUMO
E
hv
HOMO
ExcitedState
GroundState
12
Absorption and Emission

• Absorption - transfer of energy from photon to
  atom or molecule which produces a transition from
  a lower energy to a higher energy level
• Emission - production of photon of energy from
  atom or molecule which is originally in a higher
  energy level and which returns atom or molecule
  to a lower energy level

13
Absorption of Different Types of EM Radiation

• Visible or UV light - produces transition from
  lower to higher energy electronic energy level of
  atom or molecule
• Infrared light - produces vibration of atom or
  molecule
• Radiowaves nuclear transitions for select nuclei

14
Chromophores

• These are functional groups that absorb light.
  Commonly encountered types include
• p-p - aromatics, e.g., benzenevery intense e
  104 - 105 M-1 cm-1appear in ultraviolet region
  usually

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Absorption
when E hv
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Chromophores

• Charge transferintense e 102 - 103 M-1
  cm-1appear in visible region
• MLCT metal (HOMO) to ligand (LUMO) charge
  transfer
• LMCT ligand (HOMO) to metal (LUMO) charge
  transfer
• Ligand field (M to M)very weak e 1 M-1
  cm-1appear in near infrared region

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MLCT (Ru(phen)32)
LUMO (ligand-like)
L
HOMO (metal-like)
M
MLn
18
LMCT
LUMO (metal-like)
M
HOMO (ligand-like)
L
MLn
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3.3 mM KMnO4 (Charge Transfer Transition)
Green light absorbed
20
10 ?M Fe3cytochrome c, pH 7.0 (?-? Transitions)
Blue light absorbed
21
10 ?M Fe3cytochrome c, pH 7.0 (?-? Transitions)
Different ?s absorbed Peaks have different
Intensity
22
Bioanalytical Analytes

• Proteins
• DNA

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Amino Acids
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Molar Absorptivity for Selected Amino Acids
Taken from Franks, F. Protein Biotechnology
Humana Totowa, 1993.
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Nucleic Acids - Bases
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Transmittance
T 100(P/P0)
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Absorbance

• A log(100/T) log (P0/P)

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Relationship between T and A
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Relationship between T and A
Effective Upper Limit
low T high A
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Beer-Lambert Law

• A? (? ? l ) cwhereA ? - absorbance at
  wavelength ? ? ? - Molar absorptivity at ?, M-1
  cm-1c - concentration, M
• Note the larger e, the greater A is
• Significance can measure A for a smaller c

31
Limitations of Beer-Lambert Law

• Light must be monochromatic
• Pathlength must be constant (square cuvette)
• Sample should not
• Fluoresce or phosphoresce
• Scatter light (heterogeneous)
• Change its chemical composition

32
Modes of Spectrometer Operation

• Scan - acquire a spectrum (A vs. l)
• Characterization of new materials
• Time drive - acquire A at one l as a function of
  time
• Quantitation
• Kinetics
• Chromatography
• Wavelength program - acquire A at selected ?
  values)
• Quantitation of Mixtures
• Characterization of Mixtures

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3.3 mM KMnO4 (Charge Transfer Transition)
Green light absorbed
34
Problem

• A 5 ?M solution of ferricytochrome c is put into
  a sample cuvette with a pathlength of 1.0 cm.
  The absorbance at 410 nm is found to be 0.530.
  What is the molar absorptivity of cyt c at that
  wavelength? If a solution of an unknown
  concentration of cyt c exhibits an absorbance of
  0.455 at 410 nm, what is its concentration?
• ANS 106,000 M-1 cm-1 4.3 ?M

35
Homework Problem 1

• One milliliter of a 250 mL stock solution of
  Fe(phen)32 is transferred to a 100 mL volumetric
  flask which is filled to the mark with water. An
  aliquot of the diluted solution is placed in a
  0.1 cm cuvette in a UV-vis and analyzed. The
  absorbance for the diluted solution is 0.560.
  Given that the molar absorptivity for Fe(phen)32
  is 110,000 M-1 cm-1, what is the concentration of
  iron in the stock solution?

36
Quantitation using a Linear Calibration Curve

• Idea
• Prepare a series of solutions containing the
  analyte in the concentration range expected
• Record the absorbances for each solution
• Plot the data (c vs. A)
• Measure absorbance of unknown
• Assumption know sample matrix all chemical
  species and concentrations

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Linear Regression

• Purpose examine direct (linear) relationship
  between two variables x and y
• y m x bwhere x is independent variabley
  is dependent variablem is slope (dy/dx)b is
  y-intercept

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Linear Regression

• Method minimize square of the deviations (why?)
  from each datum to the best fit line (hence least
  squares)
• Goodness of fit indicated by r, correlation
  coefficient(mm)0.5 r gt 0.999 implies
  linearitym is slope for the line x m x
  b

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Example Determination of Fe in Drinking Water

• Consider relationship between Absorbance and
  concentration of Fe(phen)32
• What type of compound is this?
• What type of transition?
• Where will this compound absorb in the UV-vis
  spectral range (nm)?

40
Linear Regression

• Note correlation coefficient lt 0.99
• What test can we use to remove 1 bad datum?
• How can we use that test here?

41
Analytical Figures of Merit

• Accuracy
• Precision
• Detection limit
• Sensitivity (dy/dx) what is this for UV-vis?
• Dynamic range (linearity)
• Practical time, cost, sample prep required?

42
Question

• Could we use a calibration curve in the following
  problems? Why/why not?
• Determination of hemoglobin in blood
• Determination of drugs in urine of athletes
• Determination of pesticides in drinking water
• Can you make any generalizations?

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Method of Standard Addition

• Purpose make standard using complex matrix of
  sample of unknown composition
• Make two measurements
• sample
• spike sample with concentrated analyte standard
  (spike add small amount of concentrated
  solution)

44
Standard Addition

• l is a constant

45
Problem

• The concentration of zinc in seawater was
  determined polarographically by the method of
  standard addition. The diffusion current
  measured in a 25 mL sample of seawater was 0.14
  ?A. After the addition of 1 mL of 0.2 mM zinc
  standard, the measured diffusion current was 0.32
  ?A. Given there is a linear relationship between
  Zn and diffusion current, calculate the Zn in
  the original seawater sample.
• Zn 5.8 ?M

46
Homework Problem 2

• The concentration of phosphate in urine was
  determined spectrophotometrically based on the
  reaction of phosphate with molybdenum blue.
  Excess molybdenum blue is added to a 1.0 mL urine
  sample. The sample is diluted to 5 mL and
  analyzed by UV-vis. The absorbance of the
  resulting sample measured at 710 nm is 0.139. 1
  mL of a 5 ppm phosphate solution is added to a
  second 1.0 mL aliquot of the same urine sample,
  excess molybdenum blue is added, the sample is
  diluted to 5.0 mL and the absorbance is again
  measured. If the absorbance of this solution
  measured at 710 nm is found to be 0.836, what is
  the concentration, in ppm, of phosphate in the
  urine sample?

47
Problem

• Ay Dot Student prepared a standardized solution
  of potassium permanganate (FW 158.0) as follows.
  Ay transferred a spatula of potassium
  permanganate to a 2 L volumetric flask and then
  filled the flask to the mark with distilled
  water. Ay quantitatively transferred 3 mL of
  this solution to a 500 mL volumetric flask and
  filled this volumetric flask to the mark with
  distilled water. Next Ay transferred some of
  this solution to a 2 mm pathlength cuvette and
  found the T at 550 nm. The T was 72.5 .
  Given that the molar absorptivity of potassium
  permanganate at 550 nm is 3,000 M-1 cm-1, how
  many grams of potassium permanganate were there
  in the spatula of potassium permanganate that Ay
  used to prepare the original 2 L solution?

48
Instrumentation

• A Look at How UV-vis measurements are made

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Spectrometer
hv
sample
hv
hv
lightsource
monochromator
detector
50
Basic Components of Spectrometer

• light source - W or D2
• monochromator - turns polychromatic light into
  monochromatic light
• Sample contained in cuvette
• detector - phototube, photomultiplier, or PDA

51
UV-vis Light Sources

• W halogen lamptungsten wire heated to
  incandescence at 2900 Kalmost continuous ?
  coverage from 320 - 2500 nm
• D arc lamp ? coverage 180 - 375 continuous

52
Monochromator

• used to produce monochromatic (single ?)
  lightcommonly use two types of dispersive
  elements
• prisms - quartz or glass cut at an angle
  (Refraction)
• gratings - finely ruled highly reflective surface
  (Diffraction)

53
Cuvette

• commonly a rectangular container made of
  nonabsorbing (light) material used to contain
  sample for analysisMaterials
• Pyrex - 340 - 2500 nm
• Suprasil - 200 - 2500 nm
• Infrasil - 225 - 3600 nm
• Polystyrene (visible) or methacrylate(UV) -
  disposable

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3 Common Detectors

• Phototube
• Photomultiplier (PMT)
• Photodiode array (PDA)

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Phototube

• Light strikes photocathode (-)
• Photocathode emits photoelectrons
• Photoelectrons accelerate toward anode ()
• flow of electrons current
• current proportional to photons incident on
  photocathode

-

-
hv
-
e-
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Photomultiplier

• Light strikes photocathode (-)
• Photocathode emits photoelectrons
• Photoelectrons accelerate toward series of
  increasingly positive anodes () at which
  photoelectrons and secondary electrons are
  emitted (dynodes)
• Electrons accelerated toward collection anode

57
Analysis of Mixtures

• Principle of Additivity Absorbance of mixture at
  ?1 should be the sum of the absorbance of the
  components at ?1
• A(mixture) ?1 A(1) ?1 A(2) ?1

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Analysis of Mixture Containing 2 Components
Absorbance
?1
?2
?, nm
59
Analysis of Mixture Containing 2 Components
(contd)

• Amixture(?1) Acomponent1(?1) Acomponent2(?1)
  ?comp1 (?1)ccomp1l ?comp2 (?1)ccomp2l
• same relationship holds at ?2 Amixture(?1)
  Acomponent1(?1) Acomponent2(?1) ?comp1
  (?1)ccomp1l ?comp2 (?1)ccomp2l

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Problem

• A 1.0 mM solution of a dye A shows an absorbance
  of 0.20 at 450 nm and an absorbance of 0.05 at
  620 nm. An 0.1 mM solution of a dye B shows 0.00
  absorbance at 450 nm and an absorbance of 0.42 at
  620 nm. All measurements were made in a 2 mm
  pathlength cuvette.

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Analysis of Mixture Containing 2 Components
BGreen ABlue
Absorbance
?1
?2
?, nm
62
Problem (continued)

• Fill in the table below

63
Problem (continued)

• Fill in the table below

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Problem (continued)

• Calculate the concentration of each dye present
  in a solution that exhibits an absorbance of 0.38
  and 0.71 at 450 nm and 620 nm, respectively. A
  1.0 cm pathlength cuvette is used for both
  measurements.
• ANS cA 3.7 x 10-5 M cB3.4 x 10-5 M

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Standards for Instrument Performance

• Wavelength accuracy and precision
• Photometric accuracy and precision
• Stray Light - linearity
• Resolution
• Noise
• Baseline flatness
• Stability

66
Primary Applications of Analytical Spectroscopy

• Structural identification
• Quantitation
• Determining concentration of analyte
• Determining change in concentration of analyte

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Where Used?

• Process Analysis
• Analytical Separations

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Hypsochromism of DNA

• A(DNA) A(G) A(T) A(C) A(A)
• hyposchromism - reduced ?Abs(DNA) attributed to
  interaction of ?-electrons of nearby bases in
  helical DNA
• hyperchromism - increased Abs(DNA) attributed to
  unfolding/denaturation of DNA often at elevated
  temperatures

69
Quantitation of Protein

• Absorbance at 280 nm
• Lowry Method - sensitive but many interferents
• Bradford Method -sensitive, specific, few
  interferents (detergent)
• Biuret Method - least sensitive, specific, few
  interferents, and easy to do

70
Commonly Used Protein Assays
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Bradford (Coomassie Brilliant Blue)

• Red complex protein ? Blue complex465 nm
  ?max 595 nm ?max
• 5 ?g protein A595 0.1 (blue complex)
• Rxn conditions RT mix and measure
• Interferents detergents and color pH sensitive
• 2 -3 x more sensitive than Lowry

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Quantitation of DNA

• A(260)/A(280) - DNA purityprotein most common
  impurityA(260)/A(280) ? 1.7 - 2.0 pure DNA
• Warburg Christian Assay
• Melting point, Tm

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Frequently Used Types of Spectroscopy

• UV-vis - quantitation
• Fluorescence - quantitation
• Fourier-Transform Infrared (FTIR) - both
• NMR - structural identification
• Raman - structural identification

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Journals

• Analytical Chemistry (ACS)
• Analytical Biochemistry (ACS)
• Applied Spectroscopy (SAS)
• Clinical Chemistry (AACC)
• Trends in Analytical Chemistry
• Analytica Chimica Acta
• The Analyst (RSC)
• also specialty journals
• Journal of Raman Spectroscopy
• BioSpectroscopy