Fundamentals of Spectrophotometry

Introduction

1.) Colorimetry  An analytical technique in which the concentration of an analyte is measured by its ability to produce or change the color of a solution

Changes the solution’s ability to absorb light

2.) Spectrophotometry  Any technique that uses light to measure chemical concentrations  A colorimetric method where an instrument is used to determine the amount of analyte in a sample by the sample’s ability or inability to absorb light at a certain wavelength.

Colorimetry Instrumental Methods (spectrophotometry) Non-Instrumental Methods

Fundamentals of Spectrophotometry

Introduction

3.) Illustration  Measurement of Ozone (O 3 ) Above South Pole

O 3 provides protection from ultraviolet radiation Seasonal depletion due to chlorofluorocarbons Chain Reaction Depletion of O 3 O 3 cycle

Spectra analysis of [O 3 ]

Fundamentals of Spectrophotometry

Properties of Light

1.) Particles and Waves  Light waves consist of perpendicular, oscillating electric and magnetic fields  Parameters used to describe light -

amplitude (A): height of wave’s electric vector Wavelength (

l

): distance (nm, cm, m) from peak to peak Frequency (

n

): number of complete oscillations that the waves makes each second



Hertz (Hz): unit of frequency, second -1 (s -1 )



1 megahertz (MHz) = 10 6 s -1 = 10 6 Hz

Fundamentals of Spectrophotometry

Properties of Light

1.) Particles and Waves  Parameters used to describe light -

Energy (E ): the energy of one particle of light (photon) is proportional to its frequency

E



h

n

where: E = photon energy (Joules)

n

= frequency (sec -1 ) h = Planck’s constant (6.626x10

-34 J-s)

As frequency (

n

) increases, energy (E) of light increases

Fundamentals of Spectrophotometry

Properties of Light

1.) Particles and Waves  Relationship between Frequency and Wavelength ln 

c

 n 

c /

l 

where: c = speed of light (3.0x10

8

n

= frequency (sec -1 )

l

= wavelength (m) m/s in vacuum))

Relationship between Energy and Wavelength

E



hc

l 

hc

n

~

where:

n

~

= (1/

l

) = wavenumber

As frequency (

l

) decreases, energy (E) of light increases

Fundamentals of Spectrophotometry

Properties of Light

2.) Types of Light – The Electromagnetic Spectrum  Note again, energy (E) of light increase as frequency ( n ) increases or wavelength ( l ) decreases

Fundamentals of Spectrophotometry

Properties of Light

2.) Types of Light – The Electromagnetic Spectrum

Fundamentals of Spectrophotometry

Absorption of Light

1.) Colors of Visible Light  Many Types of Chemicals Absorb Various Forms of Light  The Color of Light Absorbed and Observed passing through the Compound are

Complimentary

Fundamentals of Spectrophotometry

Absorption of Light

2.) Ground and Excited State  When a chemical absorbs light, it goes from a low energy state ( ground state ) to a higher energy state ( excited state )

Energy required of photon to give this transition:

DE  E 1 - E o   Only photons with energies

exactly

equal to the

energy difference

two electron states will be absorbed between the Since different chemicals have different electron shells which are filled, they will each absorb their own particular type of light

Different electron ground states and excited states

Fundamentals of Spectrophotometry

Absorption of Light

3.) Beer’s Law  The relative amount of a certain wavelength of light absorbed (

A

) that passes through a sample is dependent on: distance the light must pass through the sample (

cell path length - b

) amount of absorbing chemicals in the sample (

analyte concentration – c

) ability of the sample to absorb light (

molar absorptivity -

e )

Increasing [Fe 2+ ]

Absorbance is directly proportional to concentration of Fe +2

Fundamentals of Spectrophotometry

Absorption of Light

3.) Beer’s Law  The relative amount of light making it through the sample (

P/P o

) is known as the

transmittance

(

T

)

T



P P o

Percent transmittance

% T



100

  

P P o

 

T has a range of 0 to 1, %T has a range of 0 to 100%

Fundamentals of Spectrophotometry

Absorption of Light

3.) Beer’s Law  Absorbance (A) is the relative amount of light absorbed by the sample and is related to transmittance (

T

)

Absorbance is sometimes called optical density (OD)

A

 -

log



P P o

  -

log

(

T

)  -

log

(

% T / 100

)

A has a range of 0 to infinity

Fundamentals of Spectrophotometry

Absorption of Light

3.) Beer’s Law  Absorbance is useful since it is directly related to the analyte concentration,  cell pathlength and molar absorptivity.

This relationship is known as Beer’s Law

A

 e

bc

where: A = absorbance (no units)

e

= molar absorptivity (L/mole-cm) b = cell pathlength (cm) c = concentration of analyte (mol/L) Beer’s Law allows compounds to be quantified by their ability to absorb light, Relates directly to concentration (c)

Fundamentals of Spectrophotometry

Absorption of Light

4.) Absorption Spectrum  Different chemicals have different energy levels

different ground vs. excited electron states

 -

will have different abilities to absorb light at any given wavelength

Absorption Spectrum – plot of absorbance (or e ) vs. wavelength for a compound  The greater the absorbance of a compound at a given wavelength (high e ), the easier it will be to detect at low concentrations

Fundamentals of Spectrophotometry

Absorption of Light

4.) Absorption Spectrum  By choosing different wavelengths of light ( l A can be measured vs. l B ) different compounds l

A

l

B

Fundamentals of Spectrophotometry

Spectrophotometer

1.) Basic Design  An instrument used to make absorbance or transmittance measurements is known as a spectrophotometer

Fundamentals of Spectrophotometry

Spectrophotometer

1.) Basic Design 

Light Source

: provides the light to be passed through the sample -

Tungsten Lamp: visible light (320-2500 nm)

Low pressure (vacuum) Tungsten Filament - based on black body radiation: heat solid filament to glowing, light emitted will be characteristic of temperature more than nature of solid filament

-

Deuterium Lamp: ultraviolet Light (160-375 nm)

40V Electric Arc Filament Electrode

In presence of arc, some of the electrical energy is absorbed by D 2

Sealed Quartz Tube

(or H 2 ) which results in the disassociation of the gas and release of light

D 2 + E elect  D * 2  Excited state D ’ + D ’’ + h n (light produced)

Fundamentals of Spectrophotometry

Spectrophotometer

1.) Basic Design 

Wavelength Selector (monochromator)

: used to select a given wavelength of light from the light source

Prism:

-

Filter:

Fundamentals of Spectrophotometry

Spectrophotometer

1.) Basic Design 

Wavelength Selector (monochromator)

: used to select a given wavelength of light from the light source

Reflection or Diffraction Grating:

Fundamentals of Spectrophotometry

Spectrophotometer

1.) Basic Design  -

Sample Cell

: sample container of fixed length (b).

Usually round or square cuvet Made of material that does not absorb light in the wavelength range of interest 1.

2.

3.

Glass – visible region Quartz – ultraviolet NaCl, KBr – Infrared region

Fundamentals of Spectrophotometry

Spectrophotometer

1.) Basic Design 

Light Detector:

measures the amount of light passing through the sample.

Usually works by converting light signal into electrical signal

Photomultiplier tube Process: a) light hits photoemissive cathode and

e -

b) an emitted

e -

is emitted.

is attracted to electrode #1 c) (dynode 1), which is 90V more positive.

Causes several more

e -

to be emitted.

these e are attracted to dynode 2, which is d) 90V more positive then dynode 1, emitting more e .

process continues until e are collected at e) anode after amplification at 9 dynodes.

overall voltage between anode and cathode f) is 900V.

one photon produces 10 6 – 10 7 electrons.

g) current is amplified and measured

Fundamentals of Spectrophotometry

Spectrophotometer

2.) Types of Spectrophotometers 

Single-Beam Instrument:

sample and blank are alternatively measured in same sample chamber.

Fundamentals of Spectrophotometry

Spectrophotometer

2.) Types of Spectrophotometers  -

Double-Beam Instrument Continuously compares sample and blank Automatically corrects for changes in electronic signal or light intensity of source

Fundamentals of Spectrophotometry

Chemical Analysis

1.) Calibration  To measure the absorbance of a sample, it is necessary to measure P o

P o

and P ratio

– the amount of light passing through the system with no sample present P – the intensity of light when the sample is present

  P o is measured with a blank cuvet

Cuvet contains all components in the sample solution except the analyte of interest

P is measured by placing the sample in the cuvet.

 To accurately measure an unknown concentration, obtain a calibration curve using a range of known concentrations for the analyte

Fundamentals of Spectrophotometry

Chemical Analysis

2.) Limitations in Beer’s Law 

Results in non-linear calibration curve

 At high concentrations, solute molecules influence one another because of their proximity

Molar absorptivity changes Affect on equilibrium, (HA and A difference absorption) have

  Analyte properties change in different solvents Errors in reproducible positioning of cuvet

Also problems with dirt & fingerprints

 Instrument electrical noise

Keep A in range of 0.1 – 1.5 absorbance units (80 -3%T)

Fundamentals of Spectrophotometry

Chemical Analysis

3.) Precautions in Quantitative Absorbance Measurements  -

Choice of Wavelength



Choose a wavelength at an absorption maximum Minimizes deviations from Beer’s law, which assumes

e

is constant Pick peak in absorption spectrum where analyte is only compound absorbing light Or choose a wavelength where the analyte has the largest difference in its absorbance relative to other sample components

Bad choice for either Best choice compound (b) compound (a) or (b)

Fundamentals of Spectrophotometry

Chemical Analysis

4.) Example:

A 3.96x10-4 M solution of compound A exhibited an absorbance of 0.624 at 238 nm in a 1.000 cm cuvet. A blank had an absorbance of 0.029. The absorbance of an unknown solution of compound A was 0.375. Find the concentration of A in the unknown.

Fundamentals of Spectrophotometry

What Happens When a Molecule Absorbs Light?

1.) Molecule Promoted to a More Energetic

Excited State

 Absorption of UV-vis light results in an electron promoted to a higher energy molecular orbital  s  s

*

transition in vacuum UV 

n

 s

*

saturated compounds with non-bonding electrons 

n

 p

*,

p  p

*

requires unsaturated functional groups (eq. double bonds) most commonly used, energy good range for UV/Vis

Fundamentals of Spectrophotometry

What Happens When a Molecule Absorbs Light?

1.) Molecule Promoted to a More Energetic

Excited State

 Geometrical Structure of the Excited State will Differ from the Ground State

Ground State

Excitation of an electron to the pi

antibonding

orbital ( p *) in formaldehyde produces repulsion instead of attraction between the carbon and oxygen atom

Excited State

Fundamentals of Spectrophotometry

What Happens When a Molecule Absorbs Light?

1.) Molecule Promoted to a More Energetic

Excited State

  Two Possible Transitions in Excited State

Single state – electron spins opposed Triplet state – electron spins are parallel

In general, triplet state has lower energy than singlet state   Singlet to Triplet transition has a very low probability Singlet to Singlet Transition are more probable

Fundamentals of Spectrophotometry

What Happens When a Molecule Absorbs Light?

2.) Infrared and Microwave Radiation  Not energetic enough to induce electronic transition  Change vibrational, translational and rotational motion of the molecule

The entire molecule and each atom can move along the x, y, z-axis When correct wavelength is absorbed,



Oscillations of the atom vibration is increased in amplitude



Molecule rotates or moves (translation) faster

Vibrational States of Formaldehyde Energy: Electronic >> Vibrational > Rotational

symmetric asymmetric In-plane scissoring Out-of-plane twisting In-plane rocking Out-of-plane wagging

Fundamentals of Spectrophotometry

What Happens When a Molecule Absorbs Light?

3.) Combined Electronic, Vibrational and Rotational Transitions  Absorption of photon with sufficient energy to excite an electron will also   cause vibrational and rotational transitions There are multiple vibrational and rotational energy levels associated with each electronic state

Excited vibrational and rotational states are lower energy than electronic state

Therefore, transition between electronic states can occur between different vibrational and rotational states

Vibrational and rotational states associated with an electronic state

Fundamentals of Spectrophotometry

What Happens When a Molecule Absorbs Light?

4.) Relaxation Processes from Excited State  There are multiple possible relaxation pathways   Vibrational, Rotational relaxation occurs through collision with solvent or other molecules

energy is converted to heat (radiationless transition)

Electronic relaxation occurs through the release of a photon (light)

Fundamentals of Spectrophotometry

What Happens When a Molecule Absorbs Light?

4.) Relaxation Processes from Excited State 

Internal conversion

– transition between

singlet

overlapping vibrational states electronic states through 

Intersystem crossing

– transition between a singlet electronic state to a triplet electronic state by overlapping vibrational states

Fundamentals of Spectrophotometry

What Happens When a Molecule Absorbs Light?

4.) Relaxation Processes from Excited State 

Fluorescence

– emitting a photon by relaxing from an excited

singlet

electronic states to a ground singlet state

S 1



S o



Phosphorescence

– emitting a photon by relaxing from an excited

triplet

electronic states to a ground singlet state

T 1



S o

Fundamentals of Spectrophotometry

What Happens When a Molecule Absorbs Light?

5.) Fluorescence and Phosphorescence  Relative rates of relaxation depends on the molecule, the solvent,    temperature, pressure, etc.

Energy of Phosphorescence is less than the energy of fluorescence Phosphorescence occurs at a longer wavelengths than fluorescence

Lifetime

of Fluorescence (10 -8 to 10 -4 s) is very short compared to phosphorescence (10 -4 to 10 2 s) Fluorescence and phosphorescence are relatively rare

Fundamentals of Spectrophotometry

What Happens When a Molecule Absorbs Light?

5.) Fluorescence and Phosphorescence  Fluorescence and phosphorescence come at lower energy than absorbance  Emission spectrum is roughly mirror image of absorption spectrum

Color Change Due to Fluorescence at Higher Wavelength

Fundamentals of Spectrophotometry

What Happens When a Molecule Absorbs Light?

5.) Fluorescence and Phosphorescence  Emission spectrum are of lower energy or higher wavelength because of the efficiency of vibrational relaxation

Absorption to an excited vibrational state will relax quickly to a ground vibrational state before the electronic relaxation

Fundamentals of Spectrophotometry

What Happens When a Molecule Absorbs Light?

5.) Fluorescence and Phosphorescence  Also, differences in stability of excited and ground state structure contribute to energy difference

Fundamentals of Spectrophotometry

Chemical Analysis

1.) Excitation and Emission Spectra

Excitation Spectra – measure fluorescence or phosphorescence at a fixed wavelength while varying the excitation wavelength.

Emission Spectra – measure fluorescence or phosphorescence over a range of wavelengths using a fixed varying excitation wavelength.

Fundamentals of Spectrophotometry

Chemical Analysis

2.)  Fluorescence and Phosphorescence Intensity

At low concentration, emission intensity is proportional to analyte

-

concentration

Related to Beer’s law

I



kP o c

where: k = constant P o = light intensity c = concentration of analyte (mol/L)

 At high concentrations, deviation from linearity occurs

Emission decreases because absorption increases more rapidly Emission is

quenched



absorption of excitation or emission energy by analyte molecules in solution

Fundamentals of Spectrophotometry

Chemical Analysis

3.) Example

In formaldehyde, the transition n

 p

*(T 1 ) occurs at 397 nm, and the n

 p

*(S 1 ) transition comes at 355 nm. What is the difference in energy (kJ/mol) between the S 1 and T 1 states?