lecture 2 spectroscopy

Spectroscopy radiation by atoms Absorption: Low energy electrons absorb energy to move to higher energy level Emission: Excited electrons return to lower energy states Absorption vs.. Em

PHAM VAN HUNG, PhD

INTRODUCTION

• The study how the chemical compound interacts with different wavelenghts in a given region of

electromagnetic radiationis called spectroscopy

or spectrochemical analysis

• The collection of measurements signals (absorbance) of the compound as a function of electromagnetic radiation is called a spectrum

Spectroscopy

radiation by atoms

Absorption:

Low energy electrons absorb energy to move to higher energy level

Emission:

Excited electrons return to lower energy states

Absorption vs Emission

Ground State

1st 2nd 3rd

Energy is absorbed as electrons jump to higher energy levels

Energy is emitted by electrons returning to lower energy levels Excited

States

Spectroscopic Techniques

• UV-Visible Spectroscopy (UV-Vis).

• Infrared Spectroscopy (IR)

• Atomic Absorption Spectroscopy (AAS).

• Colorimetry.

UV radiation and Electronic Excitations

• The difference in energy between molecular bonding, non-bonding and anti-non-bonding orbitals ranges from 125-650 kJ/mole

• This energy corresponds to electromagnetic radiation in the ultraviolet (UV) region, 100-350 nm, and visible (VIS) regions 350-700 nm of the spectrum

• For comparison, recall the electromagnetic spectrum:

• Using IR we observed vibrational transitions with energies

of 8-40 kJ/mol at wavelengths of 2500-15,000 nm

UV

γ-rays Microwave Radio

Visible

X-ray:

core electron

excitation

UV:

valance

electronic

excitation

IR:

molecular vibrations

Radio waves:

Nuclear spin states (in a magnetic field) Electronic Excitation by UV/Vis Spectroscopy :

3-D structure Anaylysis X-rays

X-ray Crystallography

Elemental Analysis X-rays

X-Ray Spectroscopy

Structure determination Radio waves

FT-NMR

Functional Group Analysis/quant IR/UV

Raman

Functional Group Analysis IR/Microwave

FT-IR

Quantitative analysis Beer’s Law UV-vis region

Atomic Absorption

Quantitative analysis/Beer’s Law UV-vis region

UV-vis Spectroscopic Techniques and Common Uses

Different Spectroscopies

• UV/Vis – electronic states of valence e/d-orbital

transitions for solvated transition metals

• Fluorescence – emission of UV/vis by certain

molecules

• FT-IR – vibrational transitions of molecules

• FT-NMR – nuclear spin transitions

• X-Ray Spectroscopy – electronic transitions of

core electrons

Dispersion of Polymagnetic Light with a Prism

P o ly c h ro m a tic

R a y

I n fra re d

R e d

O ra n g e

G re e n

B lu e

V io le t

U ltr a v io le t

m o n o c h ro m a tic

R a y

S L IT

P R IS M

P o ly c h ro m a tic R a y M o n o c h ro m a tic R a y

• Prism - Spray out the spectrum and choose the certain wavelength (λ) that you want by slit.

• In UV spectroscopy, the sample is irradiated with the broad spectrum of the

UV radiation

• If a particular electronic transition matches the energy of a certain band of

UV, it will be absorbed

Electronic Excitation

The absorption of light energy by organic compounds in the

visible and ultraviolet region involves the promotion of

electrons in σ, π, and n-orbitals from the ground state to higher

energy states This is also called energy transition These higher

energy states are molecular orbitals called antibonding

σ *

π*

n

π

σ

Antibonding

Antibonding

Nonbonding Bonding

Bonding

Electronic Molecular Energy Levels

σ * π*

n π σ

Antibonding Antibonding

Nonbonding Bonding Bonding

• For any bond (pair of electrons) in a molecule, the molecular orbitals are a mixture of the two contributing atomic orbitals; for every bonding orbital “created” from this mixing (s, p), there is a corresponding anti-bonding orbital of symmetrically higher energy (s*, p*).

• The lowest energy bonding orbitals are typically the s; likewise, the corresponding anti-bonding s* orbital is of the highest energy.

• p-orbitals are of somewhat higher energy, and their complementary anti-bonding orbital somewhat lower in energy than s*.

• Unshared pairs lie at the energy of the original atomic orbital, most often this energy is higher than p or s (since no bond is formed, there is no benefit in energy).

• The higher energy transitions (σ →σ*) occur a shorter wavelength and the low energy transitions (π→π*, n →π*) occur at longer wavelength.

Observed electronic transitions

From the molecular orbital diagram, there are several possible electronic

transitions that can occur, each of a different relative energy:

Energy

σ∗

π σ

π∗

n

σ σ π n n

σ ∗

π ∗

π ∗

σ ∗

π ∗

alkanes carbonyls unsaturated cmpds.

O, N, S, halogens carbonyls

Observed electronic transitions

- Routine organic UV spectra are typically collected from 200-700 nm

- This limits the transitions that can be observed:

σ σ π n n

σ ∗

π ∗

π ∗

σ ∗

π ∗

alkanes carbonyls unsaturated cmpds.

O, N, S, halogens carbonyls

150 nm

170 nm

180 nm √ - if conjugated!

190 nm

300 nm √

UV

Observed electronic transitions

- Remember the electrons present in organic molecules are involved

in covalent bonds or lone pairs of electrons on atoms such as O or N

- A functional group capable of having characteristic electronic

transitions is called a chromophore (color loving).

- Chromophore is a functional group which absorbs a characteristic

ultraviolet or visible region.

C C

C C

C O

C O H

λ A

180 nm

279 nm

C O

Spectrum

Spectrum

Glass cell filled with concentration of solution (C)

I I

Light

0

Transmittance is defined as the ratio of the electromagnetic radiation’s power

exiting the sample, I, to that incident on the sample from the source, I 0,

I

I0

T =

An alternative method for expressing the attenuation of electromagnetic

radiation is absorbance, A, which is defined as

A = - Log T = - Log = Log I0

I I

I0

Transmittance and Absorbance

Beer – Lambert Law

• There is a logarithmic dependence between the transmission, T, of light

through a substance and the product of the absorption coefficient of the

substance, α, and the distance the light travels through the material, ℓ

• The absorption coefficient can, in turn, be written as a product of either a

molar absorptivity (extinction coefficient) of the absorber, ε, and the molar

concentrationc of absorbing species in the material.

• The molar absorptivity give, in effect, the probability that the analyte will absorb

a photon of given energy As a result, value for ε depend on the wavelength of

electromagnetic radiation Compound x has a unique e at different wavelengths.

• Unit of ε: L*cm -1 *M -1

Steps in Developing a Spectrometric Analytical Method

1 Run the sample for spectrum

2 Obtain a monochromatic wavelength for the maximum absorption wavelength

3 Calculate the concentration of your sample using Beer Lambert

0.0 2.0

200 250 300 350 400 450

Spectrophotometer

An instrument which can measure the absorbance of a

sample at any wavelength

Slits

Instrument to measures the intensity of fluorescent light emitted by a sample exposed to UV light under specific conditions.

Emit fluorescent light

as energy decreases

Ground state

Sample 90°C

Detector

UV Light Source

Monochromator Monochromator

Antibonding Antibonding Nonbonding Bonding Bonding Energy

σ π

σ π

σ −>σ

π −>π '

' '

' n->

n

σ n->π'

Electron's molecular energy levels

Fluorometer

The optics of the light source in UV-visible spectroscopy

allow either visible [approx 400nm (blue end) to 750nm

(red end) ] or ultraviolet (below 400nm) to be directed at

the sample under analysis (common range: 200 – 800 nm)

UV/Vis Spectrophotometer

UV Spectrophotometer

Quartz (crystalline silica)

Visible Spectrophotometer

Glass, Plastic

Light Sources

UV Spectrophotometer

Visible Spectrophotometer

UV Spectrometer Application

Protein

Amino Acids (aromatic)

Pantothenic Acid

Glucose Determination

Enzyme Activity (Hexokinase)

Visible Spectrometer Application

Niacin Pyridoxine Vitamin B12 Metal Determination (Fe) Fat-quality Determination (TBA) Enzyme Activity (glucose oxidase)

Flurometric Application

Thiamin (365 nm, 435 nm)

Riboflavin

Vitamin A

Vitamin C

Standard Practice

• Prepare standards of known concentration

• Measure absorbance at λmax of solution

at different concentration

• Plot A vs concentration

• Obtain slope

• Use slope (and intercept) to determine the concentration of the analyte in the

unknown

Typical Beer’s Law Plot

y = 0.02x

0

0.2

0.4

0.6

0.8

1

1.2

concentration (uM)

R 2 = 0.995

Characteristics of Beer’s Law Plots

• One wavelength

• Good plots have a range of absorbances from 0.010 to 1.000

• Absorbances over 1.000 are not that valid and should be avoided

Chromophoric Structure

Practice Examples

1 Calculate the Molar Extinction Coefficient E at 351 nm for aquocobalamin in 0.1 M phosphate buffer pH = 7.0 from the following data which were obtained in 1 Cm cell

2 The molar extinction coefficient (E) of compound riboflavinis 3 x 103Liter/Cm x Mole If the absorbance reading (A) at 350 nm is 0.9 using a cell of 1 Cm, what is the concentration of compound riboflavin in sample?

the absorption of the solution at 300 nm using 1 Cm quartz cell

was 0.4 What is the molar extinction coefficient of compound

Y?

4 Calculate the molar extinction coefficient E at 351 nm for

aquocobalamin in 0.1 M phosphate buffer pH =7.0 from the

following data which were obtained in 1 Cm cell

Spectroscopy Homework

1 A substance absorbs at 600 nm and 4000 nm What type of energy transition most likely accounts for each of these absorption processes?

2 Complete the following table

[X](M) = Concentration in Mole/L

is 30,000 at 550 nm What is the absorptivity in L/g-cm.

4 The iron complex of o-phenanthroline (Molecular weight

236) has molar absorptivity of 10,000 at 525 nm If the

absorbance of 0.01 is the lowest detectable signal, what

concentration in part per million can be detected in a 1-cm

cell?