Spectroscopy: the science that deals with “interactions of matter

with electromagnetic radiation or other forms energy”

Spectrophotometry: a more restrictive term,

- any procedure that uses light to measure chemical concentrations.

- the quantitative measurement of the intensity of electromagnetic

radiation at one or more wavelengths with photoelectric detector.

acoustic waves, beams of particles such as ions and electrons

Chapter 17:

Fundamentals of Spectrophotometry

17-1. Properties of Light

Electromagnetic radiation ; EM wave ; radiation ; radient ray ; ray ; light

One linearly (or plane) polarized and consists of a single frequency, that is, is monochromatic.

17-1. Properties of Light

Electromagnetic radiation ; EM wave ; radiation ; radient ray ; ray ; light

One linearly (or plane) polarized and consists of a single frequency, that is, is monochromatic.

Dual properties:

1) wave: perpendicular, oscillating electric and magnetic fields

explains diffraction, reflection

= C/ (: frequency, C; speed of light, ; wavelength)

2) particle (photon): absorption, emission

E = h• =h • C/ =hc

where E is the energy in joules (J)

h; plank constant ( 6x10-34J •s)

; wavenumber (1/ , cm-1 reciprocal centimeter ; Kayser)

17-2 Absorption of light

Absorption of light: increases the energy of molecule

(the molecule is promoted to an excited state)

Emission of light: decreases the energy of molecule

Ground state: lowest energy state of a molecule

M + h• υ M*

(life time: 10-6 ~10-9 S)

M* M + light (fluorescence, phosphorescence)

or M* M + heat

Excitation

Relaxation

Absorption

When light is absorbed by a sample

the radiant power of the beam of light is decreased

Radiant power (P): the energy per second per unit area of the light beam

Transmittance (T): T = P/Po (T = 0 ~ 1)

Absorbance (A), or optical density: A = log (Po/P) = -log T

(if 90% light is absorbed, 10% transmitted: T = 0.1Po/Po = 0.1, A= - log T=1)

Absorption spectrum: absorbance vs wavelength

Absorption: Beer’s Law

The part of molecule responsible for light absorption: chromophore

Absorbance is directly proportional to the concentration

Beer-Lambert law: A = εbc

ε : molar absorptivity (extinction coefficient)

characteristic of a substance that tells how much light is absorbed

at a particular wavelength

b: path length

c: concentration

Beer’s law works for monochromatic radiation passing through a dilute solution< 10 mM

Colorimetry: a procedure based on absorption of visible light

17-3 Measuring Absorbance

For visible and UV spectroscopy,

a liquid sample is contained in a cell called cuvet:

- fused silica: UV and VIS

- glass; suitable for VIS but not for UV spectroscopy (it absorbs UV)

For IR spectroscopy:

- cells of NaCl or KBr

17-4 Beer’s Law in Chemical Analysis

Determination of serum iron (Fe3+, from transferrin)

Fe2+ + 3 ferrozine2- (ferrozine)3Fe4-

purple complex (max = 562 nm)

Colorimetry: based on visible radiation

17-6 What happens when a molecule absorbs light ?

Ground state (planar)

Excited state (pyramidal)

electronic transition ( n π* (S1))e movement from one MO to another MO

Infra Red (IR)

Absorption of IR radiation: vibration

Absorption of microwave radiation: rotation

Nonlinear molecule with n atom: 3N-6

Linear molecule with n atom: 3N-5

(Formaldehyde: 3N-6 = 3x4 -6 = 6)

Electronic transitions involve simultaneous

vibrational and rotational transition

IR spectroscopy: good for structural information

T1: triplet excited state

S0: singlet ground state

S1: singlet excited state

IC: raditionless transition between states with the same quantum state (S2 S1, S1 S0)

ISC: raditionless transition between states with different quantum state (S1 T1)

What happens to absorbed energy?

Life time: 10-8-10-4 s Life time: 10-4-102 s

What happens to absorbed energy?

R: vibrational relaxation

Fluorescence: S1 S0

Phosphorescence: T1 S0 (very rare)

ISC (T1 S0)can occur before phosphorescence: cooling required

The relative rate of internal conversion, intersystem crossing, fluorescence, and phosphorescence depend on the molecule, the solvent, and conditions such as temperature and pressure.

Fluorescence vs Phosphorescence

Luminescence: 1) photoluminescence (fluorescence, phosphorescence)

2) chemiluminescence

3) bioluminescence

17-7. Luminescence

0 (abs) < 0 (emission)

The absorption and emission spectra will have an approximate mirror image if the spacings between vibrational levels are roughly equal and if the transition probabilities are similar

The o does not exactly overlap:

In the emission spectrum, o comes at slightly lower energy than

in the absorption spectrum

Relationship between absorption and emission spectra

Frank-Condon Principle:

Electronic transition are so fast, relative to nuclear motion

o (absorption) < o (emission)

Fluorescence Spectrometer

Luminescence is observed at 90o to the incident light

Emission spectrum: constant ex and variable em

Excitation spectrum: constant em and variable ex

Excitation and emission spectra

Emission spectrum: constant ex and variable em

Excitation spectrum: constant em and variable ex

An excitation spectrum looks very much like an absorption spectrum

Luminescence in Analytical Chemistry

Relation of emission intensity to concentration:

I = kPoC

I: emission intensity

Po: radiant power of incident light

C: concentration of emitting species

In FL: Higher radiation power higher intensity better detection

In Absorbance: Higher radiation power no change in absorbance

(Laser-induced fluorescence; LIF) good for the detection of trace amount

Emission intensity is not proportional to analyte concentration

at high concentration, or in the presence of significant amount of absorbing species

Self-absorption

Luminescence in Analytical Chemistry

Self-absorption

Luminescence in Analytical Chemistry

N

S

CH3CH3

OOCl

N

S

CH3CH3

OON H

CR

COOH

H

H

C

COOH

RNH2+

Dansyl Chloride Amino AcidDansyl Amino Acid

Fluorophore

ChromophoreNo FL, no absorbance

Most compounds are not fluorescent:

Fluorescent moiety (fluorophore) should be coupled to the compound.

Derivatization: the chemical alteration of analyte so that it can be detected easily or separated easily from other species

NH

NH

NH2 O

O

O-

O-

NH2 O

O

NH

NH

O

O

3-APA + ligh

N

OH-, H2O2, catalyst

(CH2)4

C2H2

3-aminophtalate*(3-APA*)

(a)

H2N

(b)

aminobutylethylisoluminol (ABEI)

*

Catalysts: Heme proteins (HRP, hemoglobin..)transition metal ions (Co2+, Cu2+, Fe2+, ...)

Application:- H2O2 detection at submicromolar concentration:

H2O2 producing substrates using oxidase enzymes

- Immunoassay using HRP, isoluminol, or ABEI as labels

- Amino acid detection using isoluminol or ABEI as labels

- Transition metal detection

- BOD sensor (HRP used)

- Forensic science (blood trace)

(425 nm blue emission)

Luminol Chemiluminescence

Luminol Chemiluminescence

Catalysts: Heme proteins (HRP, hemoglobin..)transition metal ions (Co2+, Cu2+, Fe2+, ...)

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