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

  ce

  TEKNIK

  en ci S

  SPEKTROSKOPIK

  of y

• INDRI K.D.,M.Sc.,Apt---

  Teknik analisis fisikokimia yg

mengamati ttg interaksi atom atau

molekul dg radiasi elektromagnetik (REM)

  ce

  Akibat Interasaksi atom/molekul dg REM,

  en

  ada 3 kejadian:

  ci S

  1. hamburan (scattering),

  of

  2. absorpsi (absorption)

  y

  Hamburan REM o/ atom atau molekul 

• Spektofotometri Raman Absorpsi REM o/ atom atau molekul 
• ce spektrofotometri UV-Vis & IR en

  Absorpsi yg disertai emisi REM o/ atom

• ci

  atau molekul  fotoluminesensi

  S

  (flouresensi &fosforesensi)

  of y ce

Spectrofluorimetry Lecture

  en ci S of y

• Images used in this work are distributed under the GNU Free Documentation
• Solution structure of a trans-opened (10S)-dA adduct of +)-(7S,8R,9S,10R)- 7,8-dihydroxy-9,10-epoxy-7,8,9,10-

  y of S ci en ce

  Copyright Statement

  License, Version 1.2 or any later version published by the Free Software Foundation;

  LUMINESCENCE The emission of radiation from a species

• ce after that species has absorbed radiation.

  en ci

  FLUORESCENCE

  S of

  LUMINESCENCE PHOSPHORESCENCE

  y

  SPECTROSCOPY

  LUMINESCENCE

  ce

  Absorption first -

  en ci S of

  Followed by emission

  y

  in all directions , usually

• 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

  y of S ci en ce

  LUMINESCENCE

  ppm levels for absorption

  LUMINESCENCE Collectively, fluorescence and

• ce

  phosphorescence are known as

  en photoluminescence. ci

  A third type of luminescence -

  S

• of

  Chemiluminescence - is based upon

  y

  emission of light from an excited species

  LUMINESCENCE Most chemical species are not naturally

• ce luminescent.

  en

  Derivatisation reactions are often

• ci

  available to form luminescent derivatives

  S of

  of non-luminescent compounds.

  y

  

However, this extra step lessens the

• Fluorimetry is the most commonly used

• The terms fluorimetry and fluorometry are used interchangeably in the chemical

  y of S ci en ce

  LUMINESCENCE

  luminescence method. Phosphorimetry usually requires at liquid nitrogen temperatures (77K).

Energy Level Diagram

  _{SINGLET STATES    } TRIPLET STATES

  VIBRATIONAL

  ce

  s _{2 RELAXATION} T ₂

  en ci

  s ₁ S _INTERSYSTEM T ₁

  of _CROSSING y _{FLUORESCENCE PHOSPHORESCENCE}

Fluorescence and

  Following absorption of radiation, the

• ce

  molecule can lose the absorbed energy by several pathways. The particular

  en ci

  pathway followed is governed by the

  S kinetics of several competing reactions. of

  (Note: in the next slides 1- 10 you need to

  y

  identify each slide with its place with the

Fluorescence and

  One competing process is vibrational

• ce

  relaxation which involves transfer of energy to neighbouring molecules which

  en

• _-13 ci

  is very rapid in solution (10 sec).

  S

  In the gas phase, molecules suffer fewer

• – of

  collisions and it is more common to see the

  y

  emission of a photon equal in energy to that

Fluorescence and

• In solution, the molecule rapidly relaxes to the lowest vibrational energy level of the electronic state to which it is excited (in this case S
₂ ). The kinetically favoured

reaction in solution is then internal

conversion which shifts the molecule

  y of S ci en ce

• 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

  y of S ci en ce

  Fluorescence and

Fluorescence and

  When the molecule has reached the

• ce

  lowest vibrational energy level of the

  en

  lowest singlet electronic energy level

  ci

  then a number of events can take place:

  S of y

Fluorescence and

  the molecule can lose energy by internal

• ce

  conversion without loss of a photon of

  en

  radiation, however, this is the least likely

  ci

  event;

  S of y

Fluorescence and

  the molecule can emit a photon of

• ce

  radiation equal in energy to the difference

  en

  in energy between the singlet electronic

  ci

  level and the ground-state, this is termed

  S

  fluorescence;

  of y

Fluorescence and

  the molecule can undergo intersystem

• ce

  crossing which involves and electron spin

  en

  flip from the singlet state into a triplet

  ci

  state. Following this the molecule decays

  S

  to the lowest vibrational energy level of

  of y

  the triplet state by vibrational relaxation;

Fluorescence and

  the molecule can then emit a photon of

• ce

  radiation equal to the energy difference

  en

  between the lowest triplet energy level

  ci

  and the ground-state in a process known

  S as phosphorescence. of y

Fluorescence and

  In fluorescence, the lifetime of the

• ce

  molecule in the excited singlet state is _{-9 -7}

  en 10 to 10 sec. ci S

  In phosphorescence, the lifetime in the

• of
• _-6 y

  excited singlet state is 10 to 10 sec

• 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

  y of S ci en ce

  Quantum Efficiency

CONCENTRATION AND

• The power of fluorescent radiation, F, is

  proportional to the radiant power of the excitation beam absorbed by the species able to undergo fluorescence:

  y of S ci en ce

  F = K'(P - P) where P is the power incident on the sample, P is the power after it traverses a length b of the

CONCENTRATION AND

  Beer's law can be rearranged to give:

• ce
• _-bc

  P/P = 10

  en where A = bc is the absorbance. ci

  Substitution gives:

  S

• _{- bc} of

  F = K'P (1 - 10 )

  y

CONCENTRATION AND

  

This expression can be expanded (Taylor series):

• ce
_{2 3}

   (

2 .

3 bc ) ( 2 . 3 bc )   

  en

   F = K  P 2 . 3 - bc

     

  

  ci

  2! 3 !  

  S ^•

  To a good approximation if bc is small (< 0.05) the

  of

  higher-order terms are nearly zero, we have:

  y

CONCENTRATION AND

  which demonstrates two important points:

  ce en

  that at low concentrations fluorescence

• ci

  intensity is proportional to concentration;

  S of

  that fluorescence is proportional to the

  y

CONCENTRATION AND

  ce

  F

  en ci S of

  For a

  y

  concentration

  _SOURCE

  INSTRUMENTATION _SAMPLE

  ce _EXCITATION en _WAVELENGTH ci _SELECTOR S _EMISSION of _{SELECTOR WAVELENGTH} y

INSTRUMENTATION

  The fluorescence is often viewed at 90°

• ce

  orientation (in order to minimise

  en

  interference from radiation used to excite

  ci the fluorescence). S of y

  The exciting wavelength is provided by

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.

  y of S ci en ce

Types of Fluorescent Molecules

  Experimentally it is found that fluorescence is

• ce

  favoured in rigid molecules, eg., phenolphthalein and fluorescein are structurally

  en ci

  similar as shown below. However, fluorescein

  S

  shows a far greater fluorescence quantum

  of efficiency because of its rigidity. y

Types of Fluorescent Molecules

  It is thought that the extra rigidity

• ce

  imparted by the bridging oxygen group in

  en

  Fluorescein reduces the rate of

  ci

  nonradiative relaxation so that emission

  S

  by fluorescence has sufficient time to

  of y

  occur.

APPLICATIONS

  ce en ci S of y

APPLICATIONS

• ^• Benzo[a]pyrene, is a 5-

  ring polycyclic aromatic hydrocarbon that is

  ce

  mutagenic and highly carcinogenic

  en ci

• ^• It is found in tobacco

  S

  smoke and tar

  of ^•

  The epoxide of this

  y

  molecule intercalates in

APPLICATIONS

  Excitation and fluorescence spectra for benzo(a)pyrene

  ce

  in H SO . In the diagram _{2 4} the solid line is the

  en

  excitation spectrum (the _{Benzo(a)pyrene}

  ci

  fluorescence signal is

  S

  measured at 545 nm as the exciting wavelength is

  of

  varied). The dashed line is

  y

  the fluorescence spectrum

APPLICATIONS

• Many drugs possess high quantum efficiency for fluorescence. For example, quinine can

  y of S ci en ce

  B. Fluorimetric Drug Analysis

• In addition to ethical drugs such as quinine, many drugs of abuse fluoresce directly. For example lysergic acid diethylamide (LSD)

  y of S ci en ce

  APPLICATIONS

APPLICATIONS

• ^• Because LSD is active in minute quantities (as little as 50 g taken orally) an extremely sensitive methods of

  ce

  analysis is required. Fluorimetricaly LSD is usually determined in urine from a sample of about 5mL in

  en

  volume. The sample is made alkaline and the LSD is

  ci

  extracted into an organic phase consisting of n-heptane

  S

  and amyl alcohol. This is a "clean-up" procedure that

  of removes potential interferents and increases sensitivity. y

  The LSD is then back-extracted into an acid solution