MODERN PHARMACEUTICAL ANALYTICAL TECHNIQUES flourimetry instrumentation theory principle quenching applications

1. FLOURIMETRY
PRESENTED BY:
R.SHIVA SAI KIRAN
ROLL.NO.22PH203A09
M.PHARM 1ST YEAR
DEPT. OF PHARMACEUTICS
SUBMITTED TO:
DR. M. Akiful Hoque
ASSOCIATE PROFESSOR
DEPT. OF PHARMACEUTICAL
ANALYSIS

2. CONTENTS
INTRODUCTION
DEFINITION
THEORY
FACTORS AFFECTING FLOURESCENCE
INSTRUMENTATION
APPLICATIONS

3. INTRODUCTION
LUMINESCENCE IS THE EMISSION OF LIGHT BY A SUBSTANCE. IT OCCURS WHEN AN
ELECTRON RETURNS TO THE ELECTRONIC GROUND STATE FROM AN EXCITED STATE
AND LOSES ITS EXCESS ENERGY AS A PHOTON.
IT IS OF 3 TYPES :
FLOURESCENCE
PHOSPHORESCENCE

4. FLOURESCENCE
WHEN A BEAM OF LIGHT IS INCIDENT ON CERTAIN SUBSTANCES THEY
EMIT VISIBLE LIGHT OR RADIATIONS. THIS IS KNOWN AS FLUORESCENCE.
FLUORESCENCE STARTS IMMEDIATELY AFTER THE ABSORPTION OF LIGHT
AND STOPS AS SOON AS THE INCIDENT LIGHT IS CUT OFF.
THE SUBSTANCE SHOWING THIS PHENOMENON ARE KNOWN AS
FLUORESCENT SUBSTANCE .

5. PHOSPHORESCENCE
WHEN LIGHT RADIATION IS INCIDENT ON CERTAIN SUBSTANCES THEY EMIT LIGHT
CONTINUOUSLY EVEN AFTER THE INCIDENT LIGHT IS CUT OFF .
THIS TYPE OF DELAYED FLUORESCENCE IS CALLED PHOSPHORESCENCE.
SUBSTANCES SHOWING PHOSPHORESCENCE ARE PHOSPHORESCENT SUBSTANCES .

6. THEORY
 A MOLECULAR ELECTRONIC STATE IN WHICH ALL OF THE ELECTRONS ARE PAIRED
ARE CALLED SINGLET STATE .
 IN A SINGLET STATE MOLECULES ARE DIAMAGNETIC .
 MOST OF THE MOLECULES IN THEIR GROUND STATE ARE PAIRED .
 WHEN SUCH A MOLECULE ABSORBS UV/VISIBLE RADIATION , ONE OR MORE OF
THE PAIRED ELECTRONS RAISED TO AN EXITED STATE / EXITED TRIPLET STATE .

7. ELECTRONIC STATES
1. SINGLET GROUND STATE : A STATE IN WHICH ALL THE ELECTRONES IN A
MOLECULE ARE PAIRED ↓↑
2. DOUBLET STATE : A STATE IN WHICH AN UNPAIRED ELECTRON IS PRESENT
EX : FREE RADICAL ↑ OR ↓
3. TRIPLET STATE : A STATE IN WHICH UNPAIRED ELECTRONS OF SAME SPIN ARE
PRESENT . ↑ ↑ ( UNPAIRED AND SAME SPIN )
4. SINGLET EXCITED STATE : A STATE IN WHICH UNPAIRED ELECTRONS OF
OPPOSITE SPIN ARE PRESENT .↑ ↓ ( UNPAIRED AND OPPOSITE SPINS )

8. ABSORPTION OF UV/VISIBLE RADIATION CAUSES TRANSITION FROM SINGLET GROUND
STATE TO SINGLET EXCITED STATE . AS THIS EXCITED STATE IS NOT STABLE , IT EMITS THE
EXCESS ENERGY AND RETURNS BACK TO THE GROUND STATE .
TO ACHIEVE THIS TRANSITION THERE ARE 3 POSSIBILITIES :
1. COLLISIONAL DEACTIVATION : IN WHICH THE ENTIRE ENERGY IS LOST DUE TO
COLLISIONAL DEACTIVATION AND NO RADIATION IS EMITTED .
2. FLOURESCENCE : EXCITED SINGLET STATE IS HIGHLY UNSTABLE .RELAXATION OF
ELECTRONS FROM EXCITED SINGLET STATE TO SINGLET STATE WITH EMISSION OF
LIGHT .

9. 3. phosphorescence : at favorable conditions like low temperature and absence
of oxygen there is a transition from excited singlet state to triplet state which is
called as inner system crossing . the emission of radiation when electrons
undergo transition from triplet state to singlet ground state called as
phosphorescence .
FACTORS EFFECTING FLOURESCENCE INTENSITY :
1. conjugation
2. nature of substituent groups
3. rigidity of structures
4. effect of temperature
5. viscosity
6. oxygen
7. effect of pH
8. photochemical decomposition

10. QUENCHING
QUENCHING IS THE DECREASE IN FLUORESCENCE INTENSITY DUE TO SPECIFIC EFFECTS
OF CONSTITUENTS OF THE SOLUTION.
THESE EFFECTS MAY BE DUE TO THE FACTORS LIKE CONCENTRATION , PH , PRESENCE
OF SPECIFIC CHEMICAL SUBSTANCES , TEMPERATURE , VISCOSITY , ETC.
VARIOUS TYPES OF QUENCHING ARE :
1. SELF QUENCHING OR CONCENTRATION QUENCHING .
2. CHEMICAL QUENCHING .
3. STATIC QUENCHING .
4. COLLISIONAL QUENCHING .

11. SELF QUENCHING OR CONCENTRATION QUENCHING
AT LOW CONCENTRATIONS [ΜG OR NG/ML] , LINEARITY IS OBSERVED . AT HIGH
CONCENTRATIONS [MG/ML] OF THE SAME SUBSTANCE , PROPORTIONATE INCREASE IN
FLUORESCENCE INTENSITY DOES NOT OCCUR . THIS PHENOMENON IS CALLED AS SELF
QUENCHING OR CONCENTRATION QUENCHING .
CHEMICAL QUENCHING : IN THIS TYPE , QUENCHING IS DUE TO FACTORS LIKE CHANGE IN
PH , PRESENCE OF OXYGEN , HALIDES OR HEAVY METALS .

12. CHEMICAL QUENCHING
a. PH : ANILINE AT PH 5 TO 13 GIVES BLUE FLUORESCENCE WHEN EXCITED AT
290NM. BUT AT PH < 5 AND >13 IT DOES NOT EXHIBIT FLUORESCENCE .
b. OXYGEN : PRESENCE OF OXYGEN LEADS TO QUENCHING BECAUSE OF ITS
PARAMAGNETIC PROPERTY .
c. HALIDES AND ELECTRONIC WITHDRAWING GROUPS : HALIDES LIKE CHLORIDE,
BROMIDE, IODIDE AND ELECTRONIC WITHDRAWING GROUPS LIKE NITRO AND
CARBOXYLIC GROUP LEADS TO QUENCHING .
d. HEAVY METALS : PRESENCE OF HEAVY METALS ALSO LEADS TO QUENCHING
BECAUSE OF COLLISIONS AND TRIPLET GROUND STATE .

13. STATIC QUENCHING
THIS OCCURS BECAUSE OF COMPLEX FORMATION .
EX ; CAFFIENE REDUCES THE FLUORESCENT INTENSITY OF RIBOFLAVIN , BY COMPLEX
FORMATION.
COLLISION QUENCHING : IT REDUCES THE FLUORESCENCE BY COLLISION .
NUMBER OF COLLISIONS INCREASED HENCE QUENCHING TAKES PLACE .

14. INSTRUMENTATION
 SOURCE OF LIGHT
 FILTERS AND MONOCHROMATORS
 SAMPLE CELLS
 DETECTORS

15. SOURSE OF LIGHT
MERCURY ARC LAMP : MERCURY VAPOUR AT HIGH PRESSURE GIVE INTENCE
LINES ON CONTINUOUS BACKGROUND ABOVE 350NM. LOW PRESSURE
MERCURY VAPOUR GIVES AN ADDITIONAL LINE AT 254NM. IT IS USED IN
FILTER FLUORIMETER .

16. • XENON ARC LAMP : IT GIVE MORE INTENSE RADIATION THAN MERCURY VAPOUR LAMP .
IT IS USED IN SPECTRO FLOURIMETER .
• TUNGSTEN LAMP : IF EXCITATION HAS TO BE DONE IN VISIBLE REGION THIS CAN BE
USED IN LOW COST INSTRUMENTS .

17. FILTERS AND MONOCHROMATORS
• FILTERS : THESE ARE NOTHING BUT OPTICAL FILTERS WORKS ON THE
PRINCIPLE OF ABSORPTION OF UNWANTED LIGHT AND TRANSMITTING THE
REQUIRED WAVELENGTH OF LIGHT . IN INEXPENSIVE INSTRUMENTS
FLUORIMETER PRIMARY FILTER AND SECONDARY FILTER ARE PRESENT .
1. PRIMARY FILTER : ABSORBS VISIBLE RADIATION AND TRANSMITS UV
RADIATION .
2. SECONDARY FILTER : ABSORBS UV RADIATION AND TRANSMITS VISIBLE
RADIATION .

19. MONOCHROMATORS
THEY CONVERT POLYCHROMATIC LIGHT INTO MONOCHROMATIC LIGHT THEN ISOLATE A
SPECIFIC RANGE OF WAVELENGTH OR A PARTICULAR WAVELENGTH OF RADIATIONS FROM A
SOURCE .
• EXCITATION MONOCHROMATORS : PROVIDES SUITABLE RADIATION FOR EXCITATION OF
MOLECULE .
• EMISSION MONOCHROMATORS : ISOLATE ONLY THE RADIATION EMITTED BY THE
FLUORESCENT MOLECULES .

21. SAMPLE CELLS
THESE ARE MENT FOR HOLDING LIQUID SAMPLES . THESE ARE MADE UP
OF QUARTZ AND CAN HAVE VARIOUS SHAPES .
EX : CYLINDRICAL OR RECTANGULAR ETC.

22. DETECTORS
PHOTOMETRIC DETECTORS ARE USED THEY ARE :
 BARRIER LAYER / PHOTOVOLTAIC CELL .
PHOTO TUBE .
PHOTOMULTIPLIER CELLS .

23. PHOTOMULTIPLIER TUBES
• THE PRINCIPLE INVOLVED IN THIS DETECTOR IS THAT , MULTIPLICATION OF
PHOTOELECTRONS BY SECONDARY EMISSION OF ELECTRONS .
• THIS IS ACHIEVED BY USING A PHOTO CATHODE AND A SERIES OF ANODES
(DYNODES) . UP TO 10 DYNODES , EACH DYNODE IS MAINTAINED AT 75 –
100 V HIGHER THAN THE PRECEDING ONE .
• AT EACH STAGE , THE ELECTRON EMISSION IS MULTIPLIED BY A FACTOR OF 4
TO 5 DUE TO SECONDARY EMISSION OF ELECTRONS AND HENCE AN
OVERALL FACTOR OF 106 IS ACHIEVED.
• PHOTOMULTIPLIER TUBE CAN DETECT VERY WEAK SIGNALS , EVEN 200
TIMES WEAKER THEN THAT COULD BE DONE USING PHOTOVOLTAIC CELL.
HENCE IT IS USEFUL IN FLOURESCENCE MEASUREMENTS .

24. APPLICATIONS :
1. DETERMINATION OF SUBSTANCES LIKE AL , LI , ZN .
2. DETERMINATION OF THIAMIN HCL .
3. DETERMINATION OF PHENYTOIN .
4. DETERMINATION OF INDOLES , PHENOLS & PHENOTHIAZINES .
5. DETERMINATION OF NAPHTHOLS , PROTIENS , PLANT PIGMENTS AND STEROIDS.
6. NOW A DAYS FLOURIMETRY CAN ALSO BE USED IN DETECTION OF IMPURITIES IN
NANOGRAM LEVEL BETTER THAN ABSORBENCE SPECTROPHOTOMETER.