SPEKTROFLUOROMETRI

M. H. A
 OUTLINE

 3.1. Peristiwa fluoresensi dan fosforisensi, diagram energy fotoluminisensi, dan terbentuknya
pita absorpsi pada panjang gelombang eksitasi dan emisi
3.2. Syarat utama senyawa dapat dideteksi dengan spektrofluorometri
3.3. Selektivitas spektrofluorometri
3.4. Pengertian efisiensi kuantum
3.5. Hubungan antara intensitas total fluoresensi dengan efisiensi kuantum dan konsentrasi
3.6. Faktor-faktor yang mempengaruhi intensitas fluoresensi
3.7. Memahami teknik derivatisasi pada spektrofluorometri
3.8. Aplikasi analisis obat dengan spektrofluorometri
FLUORESENSI DAN FOSFORISENSI
 Singlet State
Triplet State

S2
T2 Internal Conversion
Intersystem
Crossing

S1
T1 Fluorescence
Absorption

Phosphorescence
S0
 Syarat zat yang di analisis dengan fluorometri antara lain
1. Molekul analit dapat menyerap cahaya dengan kuat sehingga analit harus
mengandung gugus kromofor. Seperti senyawa aromatik, heterosiklik dan
sistem konjugasi
2. Struktur molekulnya planar dan kaku, mampu menyerap secara kuat di daerah
200-800 nm pada radiasi elektromagnetik.
3. Transisi energi hingga ke tingkat kondisi eksitasi terendah pasangan elektron
singlet adalah transisi π-π*
4. Molekul yang tereksitasi kembali ke kondisi dasra dengan melepaskan energi
radiatif dengan waktu relaksasi pada fluorometri 10-9 - 10-7 detik dan pada
fosforimetri 10-4 deti
Lots of conjugated electrons
Multiple ring structure, coplanar

Examples:
Fluorescein -- Porphyrins
Anthracene -- Flavins
Rhodamine B -- Cyanines
SENYAWA DAERAH FLUORESENSI ( nm )

Benzena 270 – 310

Toluena 270 – 320

Fluorobenzena 270 – 320

Chlorobenzena 275 – 345

Bromobenzena 290 – 380

Phenol 285 – 365

Anisole 285 – 345

Aniline 310 – 405

Benzoic acid 310 – 390

Benzonitrile 280 - 360

PLANARITY OF THE AROMATIC RING SYSTEM
 EFFECTS OF FUNCTIONAL GROUPS

Electron Withdrawing
Electron Donating
Enhance Quantum Yield Decrease Quantum Yield
_____________ _____________

OH  COOH
NH2  NO2
OCH3
 The absorption of light results in the formation of excited molecules which
can in turn dissipate their energy by decomposition, reaction, or re-emission.
 The efficiency with which these processes take place is called the quantum
efficiency and in the case of photoluminescence can be defined as:

ФE = No. of quanta emitted

No. of quanta absorbed
 and never exceeds unity
INGAT ?????
FLUORESCENCE YIEL/QUANTUM YIELD AND
FACTORS AFFECTING IT
Ø = kf
kf+ki+Kec+kic+kpd+kd
if, kx = ki+Kec+kic+kpd+kd

 Ø = kf
kf+ kx
K is the respective rate constants for the various deactivation processes
Faktor-faktor yang mempengaruhi intensitas fluoresensi
pH
The fluorescent spectra of an ionizable aromatic compound (i.e.,containing
acidic or basic functional groups) are very dependent upon the pH or
hydrogen bonding with the solvent molecules. Intensity and peak
wavelengths can change as a function of pH
SOLVENT
Choice of solvent may influence fluorescent intensity and position of
wavelength maxima
TEMPERATURE
The fluorescence intensity of some compounds will decrease with
increasing temperature (as much as 1 to 2% per degree C).
 Faktor-faktor yang mempengaruhi intensitas fluoresensi
CONCENTRATION
Fluorescence is linear with concentration only under very dilute conditions.
Quenching due to inner filter effects (re-absorption of the emitted light by the
fluorophore, or, the exciting light is absorbed before it can reach the center of
the cuvette) can cause non-linearity
OXYGEN QUENCHING
Fluorescence quenching by molecular oxygen can occur if the solvents have not
been degassed
PHOTODECOMPOSITION
UV radiation may induce photochemical decomposition (bleaching) which can
cause large measurement errors
 Faktor-faktor yang mempengaruhi intensitas fluoresensi

OVERLAPPING BANDS
Overlapping excitation/emission bands can cause problems if
improperly measured. Cross-excitation of neighboring
compounds can be minimized with proper wavelength and slit
selection

CONTAMINATION
Contamination from glassware, pipets, etc. can cause problems
at low fluorescence levels
 DERIVATIV

1.Senyawa di derivative fluoresen

2.Kurva di derivative
Orde ke nol, satu, dua
See materi kemarin
APLIKASI
TERIMA KASIH

Ada pertanyaan??