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Absorption and Fluorescence

Lecture

Dr Mark Selby

E413D (GP)

m.selby@qut.edu.au

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Spectrochemical Analysis

• In spectrochemical analysis, the electromagnetic

spectrum of radiation is used to identify and/or quantify

chemical species.

• A "spectrum" is a plot of some measurable property of

the radiation, as a function of the frequency f(ν), or

wavelength, f(λ) , of the radiation .

For instance, the Near-

infrared absorbance

f(λ), spectrum of

chloroform over the

wavelength (λ) range

from 1100 nm to about

1700 nm shown

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Spectrochemical Analysis

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The Star Trek Tricorder

• The perfect biochemical scanner!

We don’t have a

tricorder – BUT

we do have UV-

vis absorption

and Fluorescence

Spectrometry!

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Spectrochemical Analysis• For a photon of electromagnetic radiation, the frequency

(ν) is related to the energy by the Planck equation:

E = h ν

where E is the energy of the photon, ν is its frequency and h is the Planck constant (6.624 × 10-34 J s).

• Since ν λ = c (where c is the speed of light in vaccuumand λ is the wavelength then:

hchcc

hE 1

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Absorption Spectrophotometry

• If a beam of radiation is sent into a chemical sample, it is possible that the sample will absorb some portion of that radiation, as shown

• The incident radiant power of the beam being Po and that transmitted being P.

b

Thickness

Chemical Sample

Concentration, c

Po P

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Fundamental Laws For Absorption

of Radiation

• The transmission of electromagnetic radiation

through a sample depends upon the number of

encounters between photons and species

capable of absorbing them. This is turn

depends upon:

(i) the power of the radiation;

(ii) the concentration of the sample species and

(iii) the thickness of the sample container.

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Fundamental Laws For Absorption

of Radiation

• The relationship between radiant power, concentration and rate of absorption is known as the Beer-Lambert law, or often, simply as Beer's Law:

A = log(I0/I) = εbc

b

Thickness

Chemical Sample

Concentration, c

Po P

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Fundamental Laws For Absorption

of Radiation

• where I0 is the power of the incident radiation, I is the power of the

transmitted radiation, A is the absorbance, b is the thickness of the cell, c is

the concentration (in mol L-1) of the sample and is the molar absorptivity

constant (in units of mol-1 L cm-1).

• If the concentration, c, of the sample is expressed in g L-1, then Beer's Law

can be written as:

A = log(I0/I) = abc

• where A is the absorbance (as before) and a is the absorptivity in

g-1 L cm-1.

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Fundamental Laws For Absorption

of Radiation

• The ratio I/I0 is called the transmittance,

T, whereas 100T is the percent

transmittance (%T).

• Instruments for absorption spectro-

photometry are generally calibrated in

terms of both transmittance and

absorbance:

• A = log(I0/I) = log(1/T) = log(100/%T).

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Absorption and Transmittance

• Absorption (NOT absorbance) and

transmittance are complementary:

absorption = 1 – T

This is usually expressed as a percentage:

% absorption = 100 - %T

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Analytical Working Curves

• It is seldom safe to assume adherence to

Beer's law. In general, a number of

calibration standards should be prepared

and measured in turn. The concentration

of an unknown sample is then determined

from an analytical working curve (also

known as a calibration curve).

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Analytical Working Curves

• Example: The determination of formaldehyde by the addition of

chromatropic acid and conc. sulfuric acid recording the absorbance

on a spectrophotometer at 570 nm.

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Deviations from Beer's Law

• Generally, the data over a wide range of concentrations will deviate from Beer's law, similarly to the plot above. This indicates that Beer's law is only applicable up to a concentration of c1.

A

Conc. of absorbing species

A

Conc. of absorbing species

c1

Beer’s Law ObeyedDeviations from

Beer’s Law

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Deviations from Beer's Law

• Nevertheless, it is still possible to determine the concentration of the absorbing substance from such a curve.

• The most common reason for departures from Beer's law is the use of non-monochromatic light. Beer's law is rigorously applicable only for absorption of radiation at a single frequency.

• In practice, therefore, some deviation from Beer's law will generally be found in instrumental systems

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UV-vis Spectroscopy -

Dr Mark Selby

EFFECT OF POLYCHROMATIC

RADIATION

• In the diagram below, the Beer’s Law

linear relationship is maintained for Band

A but not for Band B

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UV-vis Spectroscopy - Dr Mark Selby

Single-beam Spectrophotometer

• Instruments with a continuous source have a dispersing element and aperture or slit to select a single wavelength before the light passes through the sample.

• Either type of single-beam instrument, the instrument is

calibrated with a reference cell containing only solvent to

determine the I0 value.

The simplest

instruments use a

single-wavelength

light source, such

as a light-emitting

diode (LED), a

sample container,

and a photodiode

detector.

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UV-vis Spectroscopy - Dr Mark Selby

Double-beam Spectrophotometer

•The double-beam design greatly simplifies this process by

simultaneously measuring I and I0 of the sample and reference

cells, respectively. Most spectrometers use a mirrored rotating

chopper wheel to alternately direct the light beam through the

sample and reference cells. The detection electronics or software

program can then manipulate the I and I0 values as the

wavelength scans to produce the spectrum of absorbance or

transmittance as a function of wavelength.

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LUMINESCENCE

SPECTROSCOPY

Absorption first -

Followed by emission

in all directions , usually

at a lower frequency

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LUMINESCENCE

SPECTROSCOPY

• Collectively, fluorescence and

phosphorescence are known as

photoluminescence.

• A third type of luminescence -

Chemiluminescence - is based upon

emission of light from an excited species

formed as a result of a chemical reaction.

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Jablonski Diagram(energy levels)

s2

SINGLET STATES TRIPLET STATES

Ground

State

s1T

T

1

2

INTERSYSTEMCROSSING

VIBRATIONALRELAXATION

FLUORESCENCE PHOSPHORESCENCE

INTERNALCONVERSION CONVERSION

INTERNAL

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Fluorescence and

Phosphorescence - 1

• Following absorption of radiation, the molecule can lose the absorbed energy by several pathways. The particular pathway followed is governed by the kinetics of several competing reactions.

(Note: in the next slides 1- 10 you need to identify each slide with its place with the energy level diagram from the previous slide)

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Fluorescence and

Phosphorescence - 2

• One competing process is vibrational relaxation which involves transfer of energy to neighbouring molecules which is very rapid in solution (10-13 sec).

– In the gas phase, molecules suffer fewer collisions and it is more common to see the emission of a photon equal in energy to that absorbed in a process known as resonance fluorescence.

(Energy level diagram)

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Fluorescence and

Phosphorescence - 3

• In solution, the molecule rapidly relaxes

to the lowest vibrational energy level of

the electronic state to which it is excited

(in this case S2). The kinetically favoured

reaction in solution is then internal

conversion which shifts the molecule

from S2 to an excited vibrational energy

level in S1.

(Energy level diagram)

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Fluorescence and

Phosphorescence - 4

• Following internal conversion, the molecule loses further energy by vibrational relaxation. Because of internal conversion and vibrational relaxation, most molecules in solution will decay to the lowest vibrational energy level of the lowest singlet electronic state before any radiation is emitted.

(Energy level diagram)

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Fluorescence and

Phosphorescence - 5

• When the molecule has reached the

lowest vibrational energy level of the

lowest singlet electronic energy level then

a number of events can take place:

(Energy level diagram)

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Fluorescence and

Phosphorescence - 6

• the molecule can lose energy by internal

conversion without loss of a photon of

radiation, however, this is the least likely

event;

(Energy level diagram)

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Fluorescence and

Phosphorescence - 7

• the molecule can emit a photon of

radiation equal in energy to the difference

in energy between the singlet electronic

level and the ground-state, this is termed

fluorescence;

(Energy level diagram)

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Fluorescence and

Phosphorescence - 8

• the molecule can undergo intersystem

crossing which involves and electron spin

flip from the singlet state into a triplet

state. Following this the molecule decays

to the lowest vibrational energy level of

the triplet state by vibrational relaxation;

(Energy level diagram)

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Fluorescence and

Phosphorescence - 9

• the molecule can then emit a photon of

radiation equal to the energy difference

between the lowest triplet energy level

and the ground-state in a process known

as phosphorescence.

(Energy level diagram)

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Fluorescence and

Phosphorescence - 10

• In fluorescence, the lifetime of the

molecule in the excited singlet state is

10-9 to 10-7 sec.

• In phosphorescence, the lifetime in the

excited singlet state is 10-6 to 10 sec

(because a transition from T1 to the

ground state is spin forbidden).

(Energy level diagram)

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Quantum Efficiency

• Fluorescence, phosphorescence and

internal conversion are competing

processes. The fluorescence quantum

efficiency () and the phosphorescence

quantum efficiency are defined as the

fraction of molecules which undergo

fluorescence and phosphorescence

respectively.

(Energy level diagram)

,

. ,

.

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CONCENTRATION AND

FLUORESCENCE INTENSITY

• The power of fluorescent radiation, F, is proportional to the radiant power of the excitation beam absorbed by the species able to undergo fluorescence:

F = k(I0 - I)

where I0 is the power incident on the sample, Iis the power after it traverses a length b of the solution and k is a constant which depends upon experimental factors and the quantum efficiency of fluorescence.

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CONCENTRATION AND

FLUORESCENCE INTENSITY

• Beer's law can be rearranged to give:

I/I0 = 10-bc

where A = bc is the absorbance.

Substitution gives:

F = kI0(1 - 10- bc)

• This is the fluorescence law

• Unlike Beer’s Law fluorescence isn’t in general linear with concentration.

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CONCENTRATION AND

FLUORESCENCE INTENSITY

For low concentration this simplifies to:

F = kI0 bc

which demonstrates two important points:

– that at low concentrations fluorescence

intensity is proportional to concentration;

– that fluorescence is proportional to the

incident power in the incident radiation at the

absorption frequency.

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CONCENTRATION AND

FLUORESCENCE INTENSITY

F

Conc. of fluorescing species

c1

For a

concentration

above c1 the

calibration

curve is no

longer linear.

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INSTRUMENTATION

Schematic Diagram of Fluorescence Spectrometer. M1 =

excitation monochromator, M2 emission monochromator,

L light source. s = sample cell, PM photo multiplier

detector.

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INSTRUMENTATION

• The fluorescence is often viewed at 90°

orientation (in order to minimise interference

from radiation used to excite the fluorescence).

• The exciting wavelength is provided by an

intense source such as a xenon arc lamp

(remember F I0).

• Two wavelength selectors are required - filters

(in fluorimeters) or monochromators (in

spectrofluorometers).

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Types of Fluorescent Molecules

• Experimentally it is found that fluorescence is

favoured in rigid molecules, eg.,

phenolphthalein and fluorescein are structurally

similar as shown below. However, fluorescein

shows a far greater fluorescence quantum

efficiency because of its rigidity.

•

phenolphthalein

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Types of Fluorescent Molecules

• It is thought that the extra rigidity

imparted by the bridging oxygen group in

Fluorescein reduces the rate of

nonradiative relaxation so that emission

by fluorescence has sufficient time to

occur.

Fluorescein

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APPLICATIONS

A. Determination of polyaromatic hydrocarbons

– Benzo[a]pyrene is a product of incomplete

combustion and found in coal tar.

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APPLICATIONS

• Benzo[a]pyrene, is a 5-ring polycyclic aromatic hydrocarbon that is mutagenic and highly carcinogenic

• It is found in tobacco smoke and tar

• The epoxide of this molecule intercalates in DNA, covalently bonding to the guanine base nucleotide

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APPLICATIONS

Excitation and fluorescence spectra for benzo(a)pyrene in H2SO4. In the diagram the solid line is the excitation spectrum (the fluorescence signal is measured at 545 nm as the exciting wavelength is varied). The dashed line is the fluorescence spectrum (the exciting wavelength is fixed at 520 nm while the wavelength of collected fluorescence is varied).

Benzo(a)pyrene

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APPLICATIONS

B. Fluorimetric Drug

Analysis

– Many drugs possess

high quantum

efficiency for

fluorescence. For

example, quinine can

be detected at levels

below 1 ppb.Quinine

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APPLICATIONS

• In addition to ethical

drugs such as

quinine, many drugs

of abuse fluoresce

directly. For

example lysergic

acid diethylamide

(LSD) whose

structure is:

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APPLICATIONS

Because LSD is active in minute quantities (as little as 50 g taken orally) an extremely sensitive methods of analysis is required. Fluorimetrically LSD is usually determined in urine from a sample of about 5mL in volume. The sample is made alkaline and the LSD is extracted into an organic phase consisting of n-heptane and amyl alcohol. This is a "clean-up" procedure that removes potential interferentsand increases sensitivity. The LSD is then back-extracted into an acid solution and measured directly using and excitation wavelength of 335 nm and a fluorescence wavelength of 435 nm. The limit of detection is approximately 1 ppb: An old method – but still a

goodie in certain circumstances!