SPECTROSCOPY. Introduction of Spectrometric Analyses The study how the chemical compound interacts with different wavelenghts in a given region of electromagnetic.

SPECTROSCOPY

Introduction of Spectrometric Analyses

The study how the chemical compound interacts with different wavelenghts in a given region of electromagnetic radiation is called spectroscopy or spectrochemical analysis.

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

Energy Absorption

The mechanism of absorption energy is different in the Ultraviolet, Infrared, and Nuclear magnetic resonance regions. However, the fundamental process is the absorption of certain amount of energy.

The energy required for the transition from a state of lower energy to a state of higher energy is directly related to the frequency of electromagnetic radiation that causes the transition.

 Wave Number (cycles/cm)

X-Ray UV Visible IR Microwave

200nm 400nm 800nm

Wavelength (nm)

Spectral Distribution of Radiant Energy

V = Wave Number (cm-1)

Wave Length

C = Velocity of Radiation (constant) = 3 x 1010 cm/sec.

= Frequency of Radiation (cycles/sec)

The energy of photon:

h (Planck's constant) = 6.62 x 10-27 (Ergsec)

V =C

E = h = hC

C

= C =

Electromagnetic Radiation

Visible

Ultra violet

Radio

Gamma ray

Hz

cmcm-1Kcal/mol eV

Type

Quantum Transition

Type

spectroscopy

Type

Radiation

Frequency

υ

Wavelength

λ

Wave

Number VEnergy

9.4 x 107 4.9 x 106 3.3 x 1010 3 x 10-11 1021

9.4 x 103 4.9 x 102 3.3 x 106 3 x 10-7 1017

9.4 x 101 4.9 x 100 3.3 x 104 3 x 10-5 1015

9.4 x 10-1 4.9 x 10-2 3.3 x 102 3 x 10-3 1013

9.4 x 10-3 4.9 x 10-4 3.3 x 100 3 x 10-1 1011

9.4 x 10-7 4.9 x 10-8 3.3 x 10-4 3 x 103 107

X-ray

Infrared

Micro-wave

Gamma ray emission

X-ray absorption, emission

UV absorption

IR absorption

Microwave absorption

Nuclear magnetic resonance

Nuclear

Electronic (inner shell)

Molecular vibration

Electronic (outer shell)

Molecular rotation

Magnetically induced spin states

Spectral Properties, Application and Interactions of Electromagnetic Radiation

Spectrum of Radiation

Dispersion of Polymagnetic Light with a Prism

Polychromatic Ray

Infrared

RedOrange

Yellow

Green

Blue

Violet

Ultraviolet

monochromatic Ray

SLIT

PRISM

Polychromatic Ray Monochromatic Ray

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

Ultra Violet Spectrometry

The absorption of ultraviolet radiation by molecules is dependent upon the electronic structure of the molecule.

So the ultraviolet spectrum is called electronic spectrum.

Electronic Excitation

The absorption of light energy by organic compounds in the visible and ultraviolet region involves the promotion of electrons in , , 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.

Ene

rgy

*

*

n

*

*

n

*

n

*

Antibonding

Antibonding

Nonbonding

Bonding

Bonding

Electronic Molecular Energy Levels

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

Chromophore is a functional group which absorbs a characteristic ultraviolet or visible region.

UV 210 nm Double Bonds 233 nm Conjugated Diene 268 nm Conjugated Triene 315 nm Conjugated Tetraene

and * orbitals and * orbitals

Spectrophotometer

An instrument which can measure the absorbance of a sample at any wavelength.

Light Lens Slit Monochromator

Sample Detector Quantitative Analysis

Slits

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

Emit fluorescent lightas energy decreases

Ground state

Sample

90C

DetectorUV Light Source

Monochromator Monochromator

Antibonding

Antibonding

Nonbonding

Bonding

BondingEnergy

'

'

'

''

n->n

n->'

Electron's molecular energy levels

Fluorometer

Food Compound

S CH 2 C H 2 CH 3H 3C

Chromophore is a functional group which absorbs a characteristic ultraviolet or visible region.

UV 210 nm Double Bonds 233 nm Conjugated Diene 268 nm Conjugated Triene 315 nm Conjugated Tetraene

and * orbitals and * orbitals

Beer – Lambert Law

Glass cell filled with concentration of solution (C)

IILight

0

As the cell thickness increases, the transmitted intensity of light of I decreases.

R- Transmittance

R = I0 - Original light intensity

I- Transmitted light intensity

% Transmittance = 100 x

Absorbance (A) = Log

= Log = 2 - Log%T

Log is proportional to C (concentration of solution) and is

also proportional to L (length of light path through the solution).

I

I0

I

I0

I0

I

1

T

I

I0

A CL = ECL by definition and it is called the Beer - Lambert Law.

A = ECL

A = ECL

E = Molar Extinction Coefficient ---- Extinction Coefficient of a solution containing 1g molecule of solute per 1 liter of solution

E =Absorbance x Liter

Moles x cm

UNITS

  A = ECL

A = No unit (numerical number only)

E = Liter

Cm x Mole

L = Cm

C = Moles/Liter

A = ECL = (Liter

Cm x Mole) x

Mole

Literx Cm

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 Equation: A = ECL

Wavelength (nm)

Ab

sorb

an

ce

0.0

2.0

200 250 300 350 400 450

Spectrometer Reading

Slope of Standard Curve = AC

1 2 3 4 5

1.0

0.5

Concentration (mg/ml)

A a

t 280

nm

There is some A vs. C where graph is linear.

NEVER extrapolate beyond point known where becomes non-linear.

x

x

x

Spectrometric Analysis Using Standard Curve

1 2 3 4

0.4

0.8

1.2

A a

t 5

40

nm

Concentration (g/l) glucose

Avoid very high or low absorbencies when drawing a standard curve. The best results are obtained with 0.1 < A < 1. Plot the Absorbance vs. Concentration to get a straight line

Sample Cells

UV Spectrophotometer

Quartz (crystalline silica)

 Visible Spectrophotometer

Glass

Light Sources

 UV Spectrophotometer

1. Hydrogen Gas Lamp

2. Mercury Lamp

Visible Spectrophotometer

1. Tungsten Lamp

Chemical Structure & UV Absorption

Chromophoric Group ---- The groupings of the molecules which contain the electronic system which is giving rise to absorption in the ultra-violet region.

Chromophoric Structure

Group Structure nm

Carbonyl > C = O 280

Azo -N = N- 262

Nitro -N=O 270

Thioketone -C =S 330

Nitrite -NO2 230

Conjugated Diene -C=C-C=C- 233

Conjugated Triene -C=C-C=C-C=C- 268

Conjugated Tetraene -C=C-C=C-C=C-C=C- 315

Benzene 261

UV Spectrometer Application

Protein

Amino Acids (aromatic)

Pantothenic Acid

Glucose Determination

Enzyme Activity (Hexokinase)

Flurometric Application

Thiamin (365 nm, 435 nm)

Riboflavin

Vitamin A

Vitamin C

Visible Spectrometer Application

Niacin

Pyridoxine

Vitamin B12

Metal Determination (Fe)

Fat-quality Determination (TBA)

Enzyme Activity (glucose oxidase)

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.

Solution C x 105 M Io I

A 2.23 100 27

B 1.90 100 32

2. The molar extinction coefficient (E) of compound riboflavin is 3 x 103 Liter/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?

3. The concentration of compound Y was 2 x 10-4 moles/liter and 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.

Solution C x 105 M I0 I

A 2.0 100 30

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) Absorbance Transmittance(%) E(L/mole-cm) L(cm)  30 2000 1.00

0.5 2500 1.002.5 x 10-3 0.2 1.004.0 x 10-5 50 50002.0 x 10-4 150  [X](M) = Concentration in Mole/L

3. The molar absorptivity of a pigment (molecular weight 300) 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?