Spectrophotometry

Let There Be Light Spectrophotometry: the use of

electromagnetic radiation to measure chemical concentrations

23

2

2hν

3

23

2hν

22

3

hν2

3O2O

OClClOO

OOO

OClOOCl

ClCClFFCCl

ozone OO2

2OO

UV

absorption

The Ozone Hole

Why south pole ?

Properties of light -1

1)

2) Electromagnetic Spectrum

3) Absorption vs Emission of light

ν~hcλ

1hchνE

change of nuclear

configurationγ-ray

change of e distribution X-ray uv.vis

change of configuration

IR

change of orientation microwave

change of spin NMR EPR

Absorption of light -1

1) Spectrophotometer

Absorption of light -2

2) .

b

P o P

When no light is absorbed, P=P0 and A=0

Mmol

cmmolcm

εbcA :Law sBeer'

-logTA Absorbance

P

PT nceTransmitta

0

Absorbance is proportional to the concentration of light-absorbance molecules in the sample.

• A = bc : molar absorptivity (M-1cm-1)

Absorption of light -3

(ex) How effective is sunscreen ? at the peak absorbance near 300 nm ?

A ~ 0.35

T = 10-A

= 10-0.35

= 0.45

= 45%

55%

UV-B is absorbed.

Observed color vs. absorbed color

Practical Matters -1

1) Sample is usually contained in a cell called a cuvet, which has flat, fused-silica faces.A glass made of SiO2 : Vis. UV.

Plastics & ordinary glass: VisNaCl(s) KCl(s) : IR

Practical Matters -22) Good Operating Techniques :

a) Cuvets handle: systematic errors/ random errors

b) Most accurate at A~ 0.3-2Too little light : (high A), P is small & hard to measureToo much light : (low A), it is hard to distinguish P from P0

Practical Matters -3c) Old vs. new curvesd) Greatest sensitivity: λmaxe) Baseline correction

• (a) Proteins at 280 nm: tyr, phe, trp. • (b) A colorimetric reagent to detect phosphate

Using Beer’s Law -1

Using Beer’s Law -2

Ex.1 : Bezene: find molar absorptivity ()

+ hexane

λmax = 256 nmb = 1.000 cmε = ?

250 mL

25.8 mg

Beer’s Law -2Ex.2 : Nitrite in an aquarium

(using a standard curve)

543 nm

Using Beer’s Law -3 (toxic when > 1 ppm) NH3 animals & plant

(toxic when > 1 ppm)

[O]

[O]

NO3-

Using Beer’s Law -4

2) Standard Nitrite

Using Beer’s Law -5

from least square (4.4)

A = 0.1769 [ppm] + 0.0015

Enzyme-based nitrate Analysis - A Green Idea

P.408

NO3- NO2

-

NO3- + NADH + H+ NO2

- + NAD+ + H2O

Cd

Nitrate reductasepH 7

Spectrophotometry :

Instruments & Applications

The Spectrophotometer –1Remote sensing of airborne bacteria:

Optical fiber coated with antibodies to detect spores of a specific bacterium

The Spectrophotometer -21) Spectrophotometera) Single-beam

b) Double-beam

The Spectrophotometer -3

The Spectrophotometer –4

1) Light sourcea. Tungsten lamp:

Vis. near IR (320 nm~2500 nm)

b. Deuterium are lamp: UV (200~400 nm)

c. Electric discharge lamp + Hg(g) or Xenon: Vis & UV

d. Globar (silicon carbide rod): IR (5000~200 cm-1)

e. Laser: intense monochromatic sources.

The Spectrophotometer -5

The Spectrophotometer -6

2) Monochromator disperses light into its component wavelengths and selects a narrow band of wavelengths to pass through the sample

Consists: (1) lenses or mirrors: focus the radiation(2) entrance and exit slits: restrict unwanted

and control the spectral purity of radiation.

(3) dispersing medium: separate the of polychromatic radiation from the source.

(a) prism and (b) diffraction grating

The Spectrophotometer -7

a. entrance slit

b. collimating mirror or lens

c. a prism or grating

d. focal plane

e. exit slit

Monochromator

19.1 The Spectrophotometer -10

Choosing the bandwidth: exit slit width

Resolution

Signal

Monochromator

trade-off

The Spectrophotometer -103) Detector : A detector produces an electric

signal when it is struck by photons (Convert radiant energy (photons) into an electrical signal). Figure 19-8 shows that detector response depends on the wavelength of the incident photons.

• A photomultiplier tube (Figure 19-9) is a very sensitive detector.

Ideal detector : high sensitivity, high signal/noise, constant response for λs, and fast response time.

The Spectrophotometer -11

Detector response depends on the λ of the incident photons.

3) Detector

The Spectrophotometer -12Photomultiplier tube: very sensitive detector

Analysis of a mixture -11) Absorbance of a mixture :

Analysis of a mixture -22) Isosbestic points : for rxn: X Y, every spectrum

recorded during chemical reaction will cross at the same point. Good evidence for only two principle species in rxn.

Ex: HIn In- + H+

Analysis of a mixture -3Why isosbestic point?

HInIn bε

In bεHIn bεA

:mixture aFor

εεεInHIn when

In εA

HIn εA

465

465

In

465HIn

465

465465

In

465HIn

465

In

465

465HIn

465

Spectrophotometric Titrations -1

Ferric nitrilotriacetate

[used to avoid Fe(OH)3 ]

Spectrophotometric Titrations-2

125 μL ferric nitrilotriancetate

2 mL apotransferrin A = 0.260

A corrected = ?

Spectrophotometric Titrations-3

ex.at p.425

Corrected A for the effect of dilution

Corrected A = (Vt / Vi) (observed A) (Beer’s law)

What happens when a molecule absorbs light ?

1) Absorbing species :

M + hν M* (lifetime : 10-8 ~ 10-9 sec)

Relaxation processes :

a) M* M + heat (most common)

b) M* new species (photochemical reaction)

c) M* M + h (fluorescence, phosphorescence)

Geometry of formaldehyde

Electronic States of FormaldehydeElectronic States of Formaldehydeexcited state are shown in Figure:

MO of CH2O

Molecular orbitals describe the distribution of electrons in a molecule, just as atomic orbitals describe the distribution of electrons in an atom.

In Figure, four low-lying orbitals of formaldehyde, labeled σ1 through σ4, are each occupied by a pair of electrons with opposite spin (spin quantum numbers= +1/2 and -1/2 represented by ↑and↓).

2) Types of absorbing electrons

Consider formaldehyde: three types of molecular orbitals

n

H

HC O

In a electronic transition, an electron moves from one orbital to another.

Four types of electronic transitions:

σ*

π*

σ

π

nE

< 125 nm

150~250 nm

200~700 nm

Two possible electronic states arising:

n * transition

singlet state: The state in which the spins are opposed.

triplet state: spins are parallel

E: T1 < S1

4) Electronic transition of formaldehyde

n * (T1), absorption of light at λ = 397 nm

green-yellow

n * (S1), absorption of light at λ = 355 nm

colorless (more probable)

Vibration and Rotational States of FormaldehydeVibration and Rotational States of Formaldehyde

• The six modes of vibration of formaldehyde.

Combined Electronic, Vibrational, and Rotational Combined Electronic, Vibrational, and Rotational

TransitionsTransitions

• Electronic absorption bands are usually very broad (~100 nm) because many different vibrational and rotational levels are excited at slightly different energies.

5) Vibrational & Rotational states of CH3CO

(IR and microwave radiation)

6) What happens to absorbed energy

7) Luminescence procedures : emission spectrum of M* provides information for qualitative or quantitative analysis.

① Photoluminescence :

a) Fluorescence : S1 S0, no change in electron spin. (< 10-5 s)

b) Phosphorescence : T1 S0, with a change in electron spin. (10-4~102 s)

② Chemiluminescence : Chemical reaction (not initiated by light) release

energy in the form of light. ex : firefly.

a molecule absorbs light

7) In which your class really shines ?

emission spectrum

a molecule absorbs light

Hg + E1 Hg* Hg + h185 nm

Sb3+ + Mn2+ + h185 nm M*

M* M + h

a molecule absorbs light8) Absorption & Emission Spectra

Luminescence in analytical chemistry

1) Instrument

① .hνout (photon)

② heat

③ breaking a chemical bond

hνin

Luminescence

2) I = kPoC

incident radiation

sensitivity by P0 or C

3) more sensitive than Absorption

4) Fluorimetric Assay of Selenium in Brazil Nuts– Se is a trace element essential to life: destruct

ROOH (free radical)

– Derivatized:

– Self-absorption: quench

Luminescence

Luminescence

5) Immunoassarys employ anitbody to detect analyte.

Ex: ELISA

Luminescence

a. pregnancy test. sensitive to < 1 ng of analyteb. Enviromental Analysis. (ppm) or (ppt)

pesticides, industrial chemicals, & microbialtoxins.