Spectrophotometry – Fundamentals

1. Electromagnetic radiation – properties

Relationships

a. Frequency/Wavelength - νλ = c where c = the speed of light in vacuum (2.998 x 108 m/s) b. Energy/Frequency – E = hν where h = Planck’s constant (6.626 x 10-34 J s-1), ν (Hz)

E = λhc = hcν

where ν =

ν1 and called the wavenumber

Electromagnetic radiation

Photons

E

M Propagation direction

2. Electromagnetic Spectrum

Why is a red solution red?

Wavelength (nm)

e.g. FeSCN2+

Color Complementary color

400-435 Violet Yellow-green 435-480 Blue Yellow 480-490 Green-blue Orange 490-500 Blue-green Red 500-560 Green Purple

3. Absorption of Light

Molar extinction coefficient

A/bc Molar absorptivity ε

Extinction coefficient k

A/bc Absorptivity a

l,d ----- Path length of radiation b

Transmission T P/ P0 Transmittance T

Optical density D Log P0/P

Absorbance A

Radiation intensity I, I0

Energy of radiationRadiant power P, P0

Term & Symbol* Definition Alt. Name & Symbol

* Recommended by American Chemical Society

a. Beer’s Law A = abc(g L

-1) or A = εbc(mol L

-1)

Ex: A 7.50 x 10-5 M solution of KMnO4 has T of 36.4% when measured in a 1.05-cm cell at 525nm. (1) Calculate the absorbance of this solution: (2) Calculate the molar absorptivity of KMnO4:

P0

b

P

Absorbing solution (concentration c)

Transmittance T = P/ P0

Absorbance A = log P0/P = -logT = abc

%T A 100 0.00

90

80

70

60

50

40

30

20

10

0.05

0.10 0.15

0.20 0.25 0.30 0.35 0.40 0.45 0.50

0.60 0.70 0.80 1.00 1.50

Beer’s Law

b. Limitations to Beer’s Law (1). Real Limitations – high concentrated solutions, concentrated electrolyte solutions (proximity alters molecular absorption).

(2) Chemical Limitations – absorbing species participate in association or dissociation reactions, e.g. weak acids in concentrated solutions, complexation.

−++ →← 2NO O3H O2H2HNO

(3) Instrumental Deviations – polychromatic radiation used to measure absorbance, stray light.

- Use of filters, diffraction gratings - Molar absorptivities must be equal

0 2 4 6 8 100.0

0.2

0.4

0.6

0.8

1.0 1 - 10 mg l -1 NO 3-N

r = 0.8791

[NO 3-N] (mg l -1)

Stray radiation

A = log PoP

A = log sP PsPoP

+

+

where Ps = incident power of stray radiation 4. Absorption Spectra

- Plotting of spectral data - λ maximum, nm

- Solutions of different concentrations

400 450 500 550 600 650 7000.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6

523 nm 0.5 A.U. 1.0 A.U. 1.5 A.U. 2.0 A.U.

Wavelength (nm)

5. Theory of Molecular Absorption -every molecular species has a unique set of energy states; the lowest termed the ground state. Excited State – energy of photon transferred to molecule:

*MM →+ νh (excited state)

heatM*M +→ (relaxation)

a. Types of Molecular Transitions

(1) Electronic transition – electron between two orbitals (UV-vis).

hν = Energy Difference of Orbitals

(2) Vibrational transitions – vibrational states associated with bonds holding molecule. Involve simultaneous:

(3) Rotational transitions – changes in rotational states,

about its center of gravity.

Matter Absorption

Energy transition

Alkenes, carbonyl compounds, alkynes, azo compounds

Oxygen, nitrogen, sulfur and halogen compounds

Types of electronic transitions

σ to σ* Alkanes

σ to π* Carbonyl compounds

π to π*

η to σ *

η to π* Carbonyl compounds

σ*

π*

π to π* η

π σ σ to σ*

η to σ* η to π*

Empty levels

Occupied levels

Highest occupied molecular orbital (HOMO)

Lowest unoccupied molecular orbital (LUMO)

E = h ν = h v/ λ = E (LUMO) – E (HOMO)

Therefore - Overall change in Energy with an orbital of a molecule is:

E = Eelectronic + Evibrational + Erotational

E electronic ≈ 10 Evibrational

≈ 100 Erotational

Emission Processes b. Luminescence – emission of light from any excited state of a molecule. - more sensitive than absorption measurements

(1) Fluorescence – emission of photon during transition between states with same spin quantum #’s (e.g.

01 SS → )

(2) Phosphorescence - emission of photon during

transition between states with different spin quantum #’s (e.g. 01 ST → )

6. Instrumentation (a) Basic components

(1) Radiation source – Visible = tungsten filament lamp; UV = deuterium and hydrogen lamps

(2) Monochromators/filters – Restricts wavelength to offer narrow band of radiation

(3) Sample containers (cuvettes or cells) – glass, plastic or quartz (1 cm)

(4) Phototubes/photomultiplier tubes – emit photoelectrons after being irradiated, current is then amplified and measured

- Single Beam Instruments - Double Beam Instruments

- Array Detector Instruments

Monochromator Detector

Radiation source Sample cell

Signal processor

Sample cuvette

Grating

Dispersed spectral radiation Focusing lens

Source Photodiode array

7. Scope - Application to absorbing species

Chromophore Example Solvent λmax Alkenes C6H13CH=CH2 n-Heptane 177 nm

Alkynes CH2=CHCH=CH2 n-Heptane 196 nm

Nitro CH3NO2 Isooctane 280 nm

Nitrate C2H5ONO2 Dioxane 270 nm

Absorption by inorganic ions (η to σ *) Carbonate, 217 nm; nitrite, 313 nm; azido, 230 nm Absorption due to σ to σ* Methane, 125 nm; ethane, 135 nm Absorption of aromatic compounds Compound Formula λmax Benzene C6H6 256

Toluene C6H5CH3 261

Phenol C6H5OH 270

Styrene C6H5CH=CH2 282

8. Optimizing Experimental Conditions

a. Wavelength Selection

b. pH of Solution

Absorbance

350 400 450 500 550 600 650 700 -0.2

-0.1

0.0

0.1

0.2

0.3

0.4

0.5

Wavelength (nm)

λmax.= 620

550 nm

510 nm

0 0.02 0.04 0.06

0.08

0.10 0.12

0 2 4 6 8PO4-P / mg l-1

c. Reagent Concentration

d. Temperature e. Interfering Substances 9. Applications a. Health Sciences – 95% of all analyses are performed by spectrophotometry. b. Biological Sciences c. Chemical & Environmental Sciences Organic, inorganic systems and biochemical systems

0.06

0

0.01

0.02

0.03

0.04

0.05

0 50 100 150Time / s

30 g l-1

15 g l-1

10. Analysis of Mixtures

MixtureAbs = ......][][ ++ YbYxbx εε

where ε = molar absorptivity of each species at wavelength

specified b = cell pathlength

-measure A at more wavelengths then there are components in mixture Ex. Two-component mixture, at least three wavelengths. More is better – increase in accuracy.

11. Flow Injection Analysis

300 350 400 450 500 550 600 650 700 750 8000

0.1

0.2

0.3

0.4

0.5

WAVELENGTH / nm

ABSORBANCE

IRON

CHROMIUM

COPPER

COBALTNICKEL

0 50 100

150 200 250

300

-0.002 0.000 0.002 0.004 0.006 0.008 0.010 0.012 0.014 0.016 0.018 0.020

Time (sec)

Absorbance