Analytical Chemistry

Chapter VIII Molecular Spectrophotometry

Mechanism for molecular absorption Lambert-Beers Law UV-Vis spectrophotometry Fluorescence Instrumentation for Mesuring light absorption and light emission
Chemical Analysis: Chemical Reaction, macro-amount
Titrimetry Gravimetry

Instrumental analysis: Properties of compounds that can be transferred into detectable signals, micro and trace amount
Light
Light absorption-spectrophotometry (UV, IR, etc) Light emission-fluorescence, chemiluminescence (including bio) Light scattering Light infraction-infraction Light polarization-polarization,

Mass-mass spectroscopy Magnetic-NMR (C, H, P, etc) Electric change: charge, voltage, current (Electrochemistry)

Basic Elements for Instrumental Analysis

Properties of compounds that can be transferred into detectable signals Detector that can detect such signals Signal to concentration tranformation

Spectroscopy analytical methods are based on measuring the amount of radiation produced or absorbed by molecular or atomic species of interest.

I0

It
Transmitance Experiment al Design and Applications

Interaction of radiation and Matter

Absorption vs Concentration? Lambert-Beers Law How to measure Absorption -

Now, spectrochemical analysis

Wave nature and particle nature of single frequency electromagnetic radiation

Electric field Y

Z Magnetic field

X
Direction of propagation

E = h = h

Table 24-2, p.714

In light, Mixing Any Two Colors Gives White

Feature 24-3 Why is a read solution red?

Light examples:
Red + Cyan = White Green + Magenta = White Blue + Yellow = White Red + Green + Blue = White Bunsen-Kirchhoff Spectroscope 1859-1860

Then God said, Let there be light, and there was light.
Genesis

Radiation-induced Transitions within a Molecule and Energy Level Accordance with Transition
Electronic transitions
--; Energy associated with the electrons in the various outer orbitals of the molecule

Energy Level Diagram

Regions of UV, visible and infrared spectrum

Region Wavelength Range UV 180-380nm Visible 380-780nm Near-IR 0.78-2.5 um Mid-IR 2.5-50um

Vibrational Transitions
--; Energy level due to the inter-atomic vibrations

Rotational Transitions
--. Energy levels associated with rotation of the molecule about its center of gravity

singlet triplet S2 UV T2

Spectra for Material of Different Types

Atomic Spectraabsorption, emission, fluorescence Line spectra

Molecular Spectra UV, Vis, IR, fluorescence S1 Visible v3 T1 I Band spectra

Ee Infrared S0

v 2 Ev v1

+ r2
r1

r3

Blackbody radiationgenerally produced by heating, light sources

Er

Energy-level diagram

Continuum Spectra

Absorption Band Is Associated with Molecular Structure

Organic Compounds

Aqueous Solutions of Transition Metal Ions Aqueous Charge-transfer Complexes

Fig 24-10, p.724

For different compound, the spectra and and max are different Basis for qualilative analysis For same material, absorbance increases as concentration increase Basis for quantitative analysis Beer-Lambert law (Beers law)

Topic for Today

UV absorption spectrum for organic compounds qualification and others Principle for quantification Instrumentation for spectrum measurement Sensitivity for spectrophotometry

Transition and Absorption for Organic Compounds

Antibonding * Antibonding * Non-bonding n Bonding Bonding

Chromophores
C C
C O C N
C C

HCHO n

C H *
C S C Cl:
C C ,

150nm
, 200 nm

==

n ,

(nm)

*> *> *>n *> *> n *

Transition * n * n * *

max(nm)
~150(<200) ~200 200800 ~200

(Lmol-1 cm-1)
100300 10100 103 ~104

, 200 nm

Isolated double bond <200 nm

C O

C C O

n n

, ,

 Auxochromic Group
Typical auxochromic group and auxochromic ability (bathochromic effect) F<CH3<Cl<Br<OH<OCH3<NH2< NHCH3< N(CH3)2 <NHC6H5<O

Conjugated Double Bonds

Compound Ethene Structure
C C

Butadiene

Hexatriene

Octatetraene

max

185 217 258 1.0104 2.1104 3.5104 Conjugated Aromatic Rings

Molecules Molecular Structure

296 5.2104

B band, max(nm) 256 314 380 480 (yellow) 580 (blue)

Examples
max(nm)

CH3Cl
172

CH3Br
204

CH3I
258

Benzene Naphthalene Anthracene

Tetracene/Rubene

Pentacene

Absorption Band of Benzene in iso-Octane

E band(I,E1) K band(II,E2) Fine structural band B(III)

Solvent Effects on Absorption Spectra

In more polar solvent n * transition move toward shorter wavelength, called blue shift or hypsochromic effect Acetone
Solvent hexane 280 chloroform 278 dioxane 277 ethanol 270 H2O 2652

Summary of Absorption bands I II K E2 204

8.0x103

max(nm)

III B B 255
2.0x102

Bands

E E1 180
6.0x104

E1
No solvent effect

E2
With solvent effect

max

E2 > E1 max

In more polar solvent, * transition moves toward longer wavelength, called red shift or bathochromic effect
Solvent Hexane Chloroform Methanol H2O 305 243 More polar

tetrazine

n * 329 * 230
E1

315 238

309 237

max max

No solvent effect

E2

CH3-C-CH=C(CH3)2
Isopropylidene acetone
Skoog Fig 24-14, p.728

With solvent effect

E2 < E1 max

Wavelength Cut-off of Solvents

CHCl3 245 210 210 210 210 210 215 CCl4 DMF 280 265 270 330 303 380

Transmission Ranges for Various Optical Materials

Fused Silica Crystal Quartz 170nm~3.6m *200~600nm, 2m~3.5m KCl 200nm~18m KBr *230nm~25m Glass *360nm~2.5m CsI 230nm~50m NaCl *200nm~15m

235 235 210

210

210250300

The solvents can be used in UV measurement when the wavelength of measurement is greater than the cut-off wavelength. There are also a few more requirements for the solvent: low polarity, analyte is soluble and stable in such solvent.

The Absorption Law: Beer-Lambert Law

(Transmittance
T = I I0

A=lg(I0/It)=kbc
A parallel beam of monochromatic radiation passes through an absorbing solution
For an analyte of a given concentration, the longer the length of the medium through which the light passes (path length of light), the more absorbers are in the path, and the greater the attenuation. For a given path length of the light, the higher the concentration of absorbers, the stronger the attenuation.

Absorbance
A=lg I0/It=lg(1/T) =-lgT = Kbc
I0

T = 10 -Kbc = 10 -A
It

.
For deriving Beers Law, see Skoog book, Feature 24-2, p720-724

1968, IUPAC Regulation: A,T,, b The most used equation: Abc A/bc
Lmol-1cm-1 cm molL-1

Terms Used in Absorption Spectrometry

Akbc A Absorbance (D or O.D ) Optical Density (E) Extinction T Transmission Transmittance b Sample Path Length) in cm generally speaking, b is the thickness of the cell

molar absorptivity
of a species at an absorption maximum is
characteristics of that species, a measure of sensitivity. < 104 low Generally speaking 104~105 middle > 5105 high

Applying Beers Law to Mixtures

Beers Law also applies to solution containing more than one kind of absorbing substance. Provided that there is no interaction among the various species, the total absorbance for a multicomponent system at a single wavelength is the sum of the individual substances.

Limits to Beers law

Real limitations: from interactions between species in the solution
Concentration of analyte should <0.01 mol/L Concentration of other species, esp. electrolytes should not be high

Chemical Deviation
Association, dissociation or reaction with the solvent to give product that absorb differently from the analyte.

Instrumental deviation:
Polychromatic radiation Stray light Mismatched cells

A = A1 + A2 + +An

Instrumental Components
Light source
Fig 24-10, p.724

monochromator

sample cell

Detector

For different compound, the spectra and and max are differentbasis for qualitative

analysis
For same material, absorbance increases as concentration increase basis for quantitative analysis Beer-Lambert law (Beers law)

The 1st Spectrophotometer -Coleman DM Spectrophotometer

Vitamin A showed molecular fingerprint band.

The 1st Commercialized Spectrophotometer -Beckman DU by Arnold Beckman

Single-beam Spectrophotometer

Henry Howard Cary

Source
Dual-beam Spectrophotometer

Blackbody Radiation Curves for Various Light Sources

Xenon arc lamp

H2 lamp

Tungsten lamp

Bunsen Design Prism monochromator

Monochromator
Focal plane
1

Radiation Filters
Interference filter Absorption filter
Nominal wavelength

collimating Entrance slit lens

Prism Focusing lens

2

Exit slit

Czerny-Turner Design Grating monochromator

(phototube)

Picture of a Photo Multiplier Tube

Photomultiplier Tube

Sample Containers-cells or Cuvettes

1106107

160-700 nm

Glass for visible Quartz for UV

A Single Beam Spectrophotometer

Design of Dual-beam Spectrophotometer

hv

Designs of Dual-Beam Spectrophotometer

Double-beam-in-space

Multiple Channel Diode Array Spectrophotometer

Double-beam-in-time

DAD-Diode Array Detector

Sensitivity of Spectrophotometry
Molar absorption =A/bc (L mol-1cm-1) of a species at an absorption maximum is characteristics of that species, a measure of sensitivity. Generally speaking < 104 low 104~105 middle > 5105 high

Sandell sensitivity S
1cm2 0.001 Sgcm-2
A=bc, hence bc(cmmol/1000cm3)0.001/

Homework for Today

101415171

S=

bc 0.001 103 M M 106 = 3 10

S=

The smaller S is, the more sensitive the method is; in another word, the higher is, the more sensitive the method is.

(g cm2 )

Outline for Today

Instrumental uncertainties Spectrophotometric application Introduction to Molecular Fluorescence and Phosphorescence Presentations of Today Answer to problems in the second half of the semester Summary

The Effect of Instrumental Uncertainties

A spectrophotometric absorbance measurement entails three steps: 0% T adjustment 100% T adjustment Measurement of %T

c
c

0.434 T log T T

Instrumental Indeterminate Errors in UV-Vis Spectrophotometry

Uncertainties in spectrophotometric concentration measurements depend on the magnitude of the transmittance (absorbance) in a complex way. The uncertainties can be independent of T, proportional to T2+T, or proportional to T.

Quantitative Applications of UV-Vis Spectroscopy Reaction with chromophores Reaction conditions Eliminating interference by separation and masking

Organic Reagents
OH

A Good Spectrophotometric Method

Reasonable sensitivity>104 Reasonably good selectivity Reagent reacts selectively with analyte Reagent does not absorb light at the wavelength where the absorbance is taken, max>60 nm Complex formed has a determinate stoichiometry and is stable during determination Procedure is easy to manipulate and repeatable

OO type: Sulfosalicylic Acid NN type:

dimethylglyoxime

COOH

Fe3+complex: 520 nm Fe2+complex: colorless

SO3H H3C C C HO N N OH CH3

Ni2+

ON type: PAR
N N N OH

Cu2+ Pb2+

S type: dithiozone

HO
H N NH

S
N N

10

Wavelength Selection
K2Cr2O7 A (380nm) A(350nm)

Examples of Selecting Reaction Conditions

0.8

0.8

0.4

0.4

c(R)

c(R)

c(R)

0.0

/nm

350 380

0.0 c

Reaction time, temperature, pH, etc.

Molecular Fluorescence Spectroscopy

Mechanism of Fluorescence and Phosphorescence
Jablonski Diagram Fluorescence and molecular structure

Time Frame for Development Fluorescence

157(6)5N. Monardes (Spanish) 1852Sir George Stokes interpreted the mechanism of fluorescence and created the term fluorescence 1867application in analytical chemistry 1929Jette & West-proposed 1st fluorimeter with electrophotodetector 19521st commercialized fluorimeter

Instrumentation
Radiation sources sample geometry Cells for fluorescence Instrumentation for phosphorescence

Quantitative Fluorimetry
Calibration graphs The inner filter effect Light gives color and brilliance to all works of nature and of art; it multiplies the universe by painting it in the eyes of all that breathe.

Mechanism (Jablonski Diagram) Diagrams on the Electronic Configurations

* n

Ground singlet state

Excited singlet state

Excited singlet state

Excited triplet state

According to Hunds Rule, the triplet state has a lower energy than that of the singlet state of the same configuration.

11

Time Course for Radiation and Radiationless De-activation

Time for transitions between electronic states 10-15 s Time period for molecular vibration 10-14 s Time period for molecular rotation 10-11 s Average time between collisions for a molecule in the liquid phase at room temperature 10-12 s Lifetime for fluorescence:10-8 s Life time for phosphorescence:10-410 s

Use the Data Above to Calculate

The number of vibrations The number of rotations The number of collisions
1,000,000 1,000 10,000

Which occur with an electronically excited molecule before it returns to the ground state with the emission of fluorescence

Aleksander Jablonski (1898-1980)

Born in Voskresenovka, Ukraine. Started studying physics at Kharkov University and continued (after the 1st World War at Warsaw under S. Pienkowski) . He received his PhD in 1930. In 1935, he suggested the famous diagram, commonly known under his name, which makes it possible to explain both the kinetics and spectra of fluorescence, phosphorescence and delayed fluorescence.

Fluorescence Spectra for Anthracene

Absorption Fluorescence

Excitation Spectrum Emission Spectrum The world of fluorescence is a world of beautiful color. In the darkness all the ordinary colors of our daylight world disappear. Only the intensely glowing hues of fluorescent substances touched by the ultraviolet beam shine out with striking clarity. Sterling Gleason, 1960

Characteristics of Fluorescence Spectra

Stokes shiftThe emitted wavelength is always longer (if
single photons are absorbed) or equal to the incident wavelength, due to energy conservation

1852, George Stokes, professor of mathematics and physics at Cambridge, interpreted the light-emitting phenomenon and formulated the law (the Stokes Law or the Stokes Shift) that the fluorescent light is of longer wavelength than the exciting light. 1853, Stokes coined the term "fluorescence" from the term "internal dispersion." 1864, Stokes lectured "On the application of the optical properties to detection and discrimination of organic substances" before the Chemical Society and the Royal Institution.

Emission spectrum is irrelevant to excitation wavelength Mirror-image relationship

12

Fluorescence and Molecular Structure

Fluorescence Efficiency() is described by the quantum yield of fluorescence ()f.
I no. of photons emitted f = = f no. of photons absorbed I a also f = kf kf + knr

Fluorescence and Molecular Structure

-O O C O

COO-

kf the 1st order rate constant for fluorescence relaxation knr the rate constant for radiationless relaxation

f falls in the range 0 to 1.

A high value of f is generally associated with molecules possessing an extensive delocalized system of conjugated double bonds which results in a relatively rigid structure. ,

Fluorescence and Molecular Structure

Temperature and Solvent Effects

f decreases with increasing temperature because the increased frequency of collision at elevated temperatures increases the probability of collisional relaxation A decrease in solvent viscosity( ) leads to the same results
Rigid molecules or complexes tend to fluorescence Examples

Chemical Reaction De-excitation Emission of Photons Radiationless Transfer External Quenching Collision Resonance Transfer

Fluorescence Phosphorescence Internal Conversion Vibrational Relaxation Inter-system Crossing

Quantitative Fluorimetry-IF versus c

In very diluted solution (2.303bc0.05) If2.3bcI02.3AI0
Molar absorptivity
blight path I0Radiation intensity

Internal Transfer

To a given b and I0 value, IfKc

13

Inner filter effect happens at high concentration, which makes the calibration curve show curvature.
F

Factors Affecting Linearity of If vs. c

Concentrationdiluted solutionFc (bc0.05) Sources for fluorescenceI0 pH Solvent Temperaturet

Diagram of Fluorimeter
( 200-800 nm)

F
F

Skoog Fig 27-8b, p.830

Light Source and Cells for Fluorescence

Light source Xenon arc lamp Solid state sources
Light emitting diodes (LEDs) Laser diodes emit monochromatic radiation Both can be pulsed or modulated for TR-Fluorescence

Sample Geometry for Fluorescence Measurement

Sample cell fused quartz to avoid natural fluorescence in the impurity of natural quartz with all four faces polished

Sample Geometry for UV-vis Absorption Measurement

14

Sample Cells for Phosphorescence

Room temperature Phosphorescence for Solid sample Low temperature Phosphorescence for liquid sample

Detectors for Fluorescence

Photomultiplier tube (PMT) Charge coupled devices (CCDs)

Io

Sample cell for low temperature phosphorescence

15