Teknik analisis fisikokimia yg mengamati ttg interaksi atom atau molekul dg radiasi elektromagnetik (REM)

Fa

cu

lt

y

of

S

ci

en

ce

TEKNIK

SPEKTROSKOPIK

Fa

cu

lt

y

of

S

ci

en

ce

_{Teknik analisis fisikokimia yg}

mengamati ttg interaksi atom atau molekul dg radiasi elektromagnetik (REM)

Akibat Interasaksi atom/molekul dg REM, ada 3 kejadian:

Fa

cu

lt

y

of

S

ci

en

ce

• _{Hamburan REM o/ atom atau molekul }

Spektofotometri Raman

• _{Absorpsi REM o/ atom atau molekul }

spektrofotometri UV-Vis & IR

• _{Absorpsi yg disertai emisi REM o/ atom}

Fa

cu

lt

y

of

S

ci

en

ce

Fa

cu

lt

y

of

S

ci

en

ce

Copyright Statement

• _{Images used in this work are distributed}

under the GNU Free Documentation

License, Version 1.2 or any later

version published by the Free Software Foundation;

• _{Solution structure of a trans-opened}

(10S)-dA adduct of +)-(7S,8R,9S,10R)-

7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene in a DNA

duplex is by Richard Wheeler (Zephyris

Fa

cu

lt

y

of

S

ci

en

ce

LUMINESCENCE

SPECTROSCOPY

• _{The emission of radiation from a species}

after that species has absorbed radiation.

LUMINESCENCE

FLUORESCENCE

PHOSPHORESCENCE

SPECTROSCOPY

Fa

cu

lt

y

of

S

ci

en

ce

LUMINESCENCE

SPECTROSCOPY

Absorption first

Fa

cu

lt

y

of

S

ci

en

ce

LUMINESCENCE

SPECTROSCOPY

• _{In favorable cases, luminescence methods}

are amongst some of the most sensitive

and selective of analytical methods available.

• _{Detection Limits are as a general rule at}

ppm levels for absorption

Fa

cu

lt

y

of

S

ci

en

ce

LUMINESCENCE

SPECTROSCOPY

• _{Collectively, fluorescence and}

phosphorescence are known as

photoluminescence.

• _{A third type of luminescence -}

Chemiluminescence - is based upon

Fa

cu

lt

y

of

S

ci

en

ce

LUMINESCENCE

SPECTROSCOPY

• _{Most chemical species are not naturally}

luminescent.

• _{Derivatisation reactions are often}

available to form luminescent derivatives of non-luminescent compounds.

• _{However, this extra step lessens the}

Fa

cu

lt

y

of

S

ci

en

ce

LUMINESCENCE

SPECTROSCOPY

• Fluorimetry is the most commonly used

luminescence method. Phosphorimetry

usually requires at liquid nitrogen temperatures (77K).

• _{The terms fluorimetry and fluorometry are}

used interchangeably in the chemical literature.

• _{Chemiluminescence won’t be further}

Fa

cu

lt

y

of

S

ci

en

ce

Energy Level Diagram

s₂

SINGLET STATES TRIPLET STATES

Ground State

s₁

T T

1 2

INTERSYSTEM CROSSING VIBRATIONAL

RELAXATION

FLUORESCENCE PHOSPHORESCENCE

INTERNAL

CONVERSION CONVERSIONINTERNAL

Fa

cu

lt

y

of

S

ci

en

ce

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.

Fa

cu

lt

y

of

S

ci

en

ce

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

Fa

cu

lt

y

of

S

ci

en

ce

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

Fa

cu

lt

y

of

S

ci

en

ce

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

Fa

cu

lt

y

of

S

ci

en

ce

Fluorescence and

Phosphorescence - 5

• _{When the molecule has reached the}

Fa

cu

lt

y

of

S

ci

en

ce

Fluorescence and

Phosphorescence - 6

• _{the molecule can lose energy by internal}

Fa

cu

lt

y

of

S

ci

en

ce

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

Fa

cu

lt

y

of

S

ci

en

ce

Fluorescence and

Phosphorescence - 8

• _{the molecule can undergo intersystem}

crossing which involves and electron spin flip from the singlet state into a triplet

Fa

cu

lt

y

of

S

ci

en

ce

Fluorescence and

Phosphorescence - 9

• _{the molecule can then emit a photon of}

Fa

cu

lt

y

of

S

ci

en

ce

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}

Fa

cu

lt

y

of

S

ci

en

ce

Quantum Efficiency

• _{Fluorescence, phosphorescence and}

internal conversion are competing

Fa

cu

lt

y

of

S

ci

en

ce

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'(P₀ - P)

where P₀ is the power incident on the sample, P

Fa

cu

lt

y

of

S

ci

en

ce

CONCENTRATION AND

FLUORESCENCE INTENSITY

• Beer's law can be rearranged to give:

P/P₀ = 10-bc

where A = bc is the absorbance.

Substitution gives:

F = K'P₀(1 - 10- bc)

• This is the fluorescence law

• _{Unlike Beer’s Law fluorescence isn’t in}

CONCENTRATION AND

FLUORESCENCE INTENSITY

• _{This expression can be expanded (Taylor series):}

• _{To a good approximation if bc is small (< 0.05) the}

higher-order terms are nearly zero, we have:

Fa

cu

lt

y

of

S

ci

en

ce

CONCENTRATION AND

FLUORESCENCE INTENSITY

which demonstrates two important points:

• _{that at low concentrations fluorescence}

intensity is proportional to concentration;

• _{that fluorescence is proportional to the}

Fa

cu

lt

y

of

S

ci

en

ce

CONCENTRATION AND

FLUORESCENCE INTENSITY

F

Conc. of fuorescing species c₁

For a

concentration

above c₁ the

Fa

cu

lt

y

of

S

ci

en

ce

INSTRUMENTATION

SOURCE

EXCITATION WAVELENGTH

SELECTOR

EMISSION

WAVELENGTH SELECTOR

DETECTOR

Fa

cu

lt

y

of

S

ci

en

ce

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}

Fa

cu

lt

y

of

S

ci

en

ce

INSTRUMENTATION

• _{Because An intense monochromatic light source}

is required ...

• _{Lasers are an almost ideal light source for}

fluorimetry (laser-induced fluorescence) but are too expensive and/or impractical for most

routine applications.

• _{Two wavelength selectors are required filters}

Fa

cu

lt

y

of

S

ci

en

ce

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.

•

Fa

cu

lt

y

of

S

ci

en

ce

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.

Fa

cu

lt

y

of

S

ci

en

ce

APPLICATIONS

A. Determination of polyaromatic hydrocarbons

– _{Benzo[a]pyrene is a product of incomplete}

Fa

cu

lt

y

of

S

ci

en

ce

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

Fa

cu

lt

y

of

S

ci

en

ce

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).

Fa

cu

lt

y

of

S

ci

en

ce

APPLICATIONS

B. Fluorimetric Drug Analysis

• _{Many drugs possess}

high quantum efficiency for

fluorescence. For

example, quinine can be detected at levels

Fa

cu

lt

y

of

S

ci

en

ce

APPLICATIONS

• _{In addition to ethical}

drugs such as

quinine, many drugs of abuse fluoresce directly. For

Fa

cu

lt

y

of

S

ci

en

ce

APPLICATIONS

• _{Because LSD is active in minute quantities (as little as}