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UV- SPECTROSCOPY PREPARED BY :- MISS ANAMIKA SINGH
S P E C T R O S C O P Y: - S P E C T R O S C O P Y I S T H E B R A N C H O F S C I E N C E D E A L I N G T H E S T U D Y O F I N T E R A C T I O N O F E L E C T R O M A G N E T I C R A D I A T I O N W I T H M A T T E R . O R I T I S T H E M E A S U R E M E N T O F E L E C T R O M A G N E T I C R A D I A T I O N ( E M R ) A B S O R B E D O R E M I T T E D W H E N M O L E C U L E O R I O N S O R A T O M S O F A S A M P L E M O V E F R O M O N E E N E R G Y S T A T E T O A N O T H E R E N E R G Y S T A T E . S P E C T R O S C O P Y I S T H E M O S T P O W E R F U L T O O L A V A I L A B L E F O R T H E S T U D Y O F A T O M I C & M O L E C U L A R S T R U C T U R E A N D I S U S E D I N T H E A N A L Y S I S O F A W I D E R A N G E O F S A M P L E . INTRODUCTION
 T H E P R I N C I P L E I S B A S E D O N T H E M E A S U R M E N T O F S P E C T R U M O F A S A M P L E C O N T A I N I N G A T O M S / M O L E C U L E S S P E C T R U M I S A G R A P H O F I N T E N S I T Y O F A B S O R B E D O R E M I T T E D R A D I A T I O N B Y S A M P L E V E R S E S F R E Q U E N C Y ( V ) O R W A V E L E N G T H ( Λ ) S P E C T R O M E T E R I S A N I N S T R U M E N T D E S I G N T O M E A S U R E T H E S P E C T R U M O F A C O M P O U N D PRINCIPLE OF SPECTROSCOPY
A B S O R P T I O N S P E C T R O S C O P Y : - A N A N A L Y T I C A L T E C H N I Q U E W H I C H C O N C E R N W I T H T H E M E A S U R E M E N T O F A B S O R P T I O N O F E L E C T R O M A G N E T I C R A D I A T I O N E . G . U V ( 1 8 5 - 4 0 0 N M ) / V I S I B L E ( 4 0 0 - 8 0 0 N M ) S P E C T R O S C O P Y , I R S P E C T R O S C O P Y ( 0 . 7 6 – 1 5 U M ) E M I S S I O N S P E C T R O S C O P Y : - A N A N A L Y T I C A L T E C H N I Q U E I N W H I C H E M I S S I O N O F A P A R T I C L E O R R A D I A T I O N I S D I S P E R S E D A C C O R D I N G T O S O M E P R O P E R T Y O F T H E E M I S S I O N & A M O U N T O F D I S P E R S I O N I S M E A S U R E D E . G . M A S S S P E C T R O S C O P Y TYPE OF SPECTROSCOPY
E L E C T R O M A G N E T I C R A D I A T I O N C O N S I S T O F D I S C R E T E P A C K E T S O F E N E R G Y W H I C H A R E C A L L E D P H O T O N S A P H O T O N C O N S I S T O F A N O S C I L L A T I N G E L E C T R I C F I E L D ( E ) & A N O S C I L L A T I N G F I E L D ( M ) W H I C H A R E P E R P E N D I C U L A R T O E A C H O T H E R ELECTROMAGNETIC RADIATION
W A V E L E N G T H : - I T I S T H E D I S T A N C E B E T W E E N T W O S U C C E S S I V E M A X I M U M A N E L E C T R O M A G N E T I C W A V E . U N I T A R E : - M , C M , M M , N M , A N D M I C R O M E T E R F R E Q U E N C Y : - N U M B E R O F W A V E L E N G T H U N I T P A S S T H R O U G H A G I V E N P O I N T I N U N IT T I M E I S C A L L E D A S F R E Q U E N C Y . I T I S D E N O T E D B Y “ V ” A N D U N I T A R E C Y C L E S P E R S E C O N D H E R T Z . W A V E N U M B E R : - A S T H E N U M B E R O F W A V E S P E R C M I N V A C U M M . V E L O C I T Y : - I T I S T H E P R O D U C T O F W A V E L E N G T H A N D F R E Q U E N C Y A N D I S E Q U A L T O T H E V E L O C I T Y O F T H E W A V E I N T H E M E D I U M . V = N × Λ PROPERTIES OF ELECTROMAGNETIC RADIATION
ELECTROMAGNETIC RADIATION
E L E C T R O N I C E N E R G Y L E V E L S : - A T R O O M T E M P E R A T U R E T H E M O L E C U L E A R E I N T H E L O W E S T E N E R G Y L E V E L E 0 . W H E N T H E M O L E C U L E A B S O R B U V - V I S I B L E L I G H T F R O M E M R . O N E O F T H E O U T E R M O S T B O N D / L O N E P A I R E L E C T R O N I S P R O M O T E D T O H I G H E R E N E R G Y S T A T E S U C H A S E 1 E 2 … . E T C I S C A L L E D A S E L E C T R O N I C T R A N S I T I O N A N D T H E D I F F E R E N C E I S A S … V I B R A T I O N A L E N E R G Y L E V E L : - T H E S E A R E L E S S E N E R G Y L E V E L T H A N E L E C T R O N I C E N E R G Y L E V E L S . T H E S P A C I N G B E T W E E N E N E R G Y L E V E L A R E R E L A T I V E L Y S M A L L 0 . 0 1 T O 1 0 K C A L / M O L E . E . G . W H E N I R R A D I A T I O N I S A B S O R B E D M O L E C U L E A R E E X C I T E D F R O M V I B R A T I O N A L T O A N O T H E R O R I T V I B R A T E W I T H H I G H E R A M P L I T U D E . INTERACTION OF EMR WITH MATTER
ROTATIONAL ENERGY LEVELS: - •T H E S E E N E R G Y L E V E L S A RE Q U A NT I ZE D & D I S C R E T E . T H E S PA C I N G B E T WE E N E N E R G Y L E VE L S M A L L E R T H E N V I B R AT I O N A L E N E R G Y L E V E L . INTERACTION OF EMR WITH MATTER
A N EL EC TRO N I N A B O N D I N G S - O R B I TAL I S E XC I TE D TO T H E C O R R E S P O N D I N G A N T I - B O N D I N G O R B I TAL A ND O B S E RV E D W I T H S AT UR AT E D C O M P O U N D S . T H E E NE R G Y R E Q U I R E I S L A R G E . E X : - M E T H A N E ( W I T H H A S O N LY ) C - H B O N D S , A N D CA N O N LY U N DE R G O Σ → Σ * T R A N S I T I O N S H O W A N A B S O R B AN CE M A XI M U M AT 1 2 5 N M . A B S O R P T I O N M A XI M A D U E TO Σ → Σ * T R A N S I T I O N A R E N O T S E E N I N T Y P I C AL U V-V I S S P E CT R A ( 2 0 0 - 7 0 0 N M ) B U T I N U V- R E G I O N ( 1 2 5 - 1 3 5 N M ) ELECTRONIC TRANSITION σ → σ* transition.
N – > Σ * T R A N S I T I O N : - S AT U RATED C O M P O U N D S C O N TA I N I N G ATO M S W I T H L O N E PA I R ( N O N – B O N D I N G E L E CT RO N S ) L I K E O , N , S A N D H A L O G E N A RE C APA B L E O F N – > Σ * T R A N S I T I O N . T H E S E T R AN S I T I O N U S U AL LY N E E D L E SS E NE R G Y T H A N N – > Σ * T R AN S I T I O N . T H E Y CA N B E I N I T I AT E D B Y L I G H T W H O S E WAV E L E N G T H I N T H E R A N G E 1 5 0 - 2 5 0 N M . T H E N U M B E R O F O R G A N I C F U N CT I O N A L G R O U P N – > Σ * PE A K I N T H E U V R E G I O N I S S M A L L ELECTRONIC TRANSITION
Π T O Π * T R A N S I T I O N : - P I E E L E C T R O N I N A B O N D I N G O R B I TA L I S E X C I T E D TO C OR R ES PO N D I N G ANT I - B O N D I N G O R B I TA L PI E S TA R A N D O B S ERV E D I N C O N J U G AT ED CO M P O UN D S . C OM P O U N DS C O N TA I N I NG M U LT I P L E B O N D L I K E A L K E N E S , AL K Y N E S , C AR B ONYL , N I T R I L E S , A R O M AT I C C O M P O UN D S E T C U N D E R G O P I E TO PI E S TA R T R A N S I T I O N . E G : - A L K E N E S G E N E R A L LY A B S O R B I N T H E R E G I O N 1 7 0 TO 2 0 5 N M . ELECTRONIC TRANSITION
N T O Π * T R A N S I T I O N : - A N E L E C T R O N F R O M OR B I TA L I S P R O M O T E D TO A N T I - B O N D I N G P I E S TA R O R B I TA L A N D R E Q U I R E D L O W E R E N E R GY. C O M P O U N DS C O N TA I N I N G D OU B LE B O N D IN V O LV I N G H E T E R O ATO M ( C = O , C N , N = O ) U N D E RG O S UC H T R AN S I T I O N . N TO P I E S TA R T R A NS I T I O N R E Q U I R E M I N I M U M E N E R GY A ND S H O W A B S O R P T I O N L O N G E R WAV E L E N G T H A R O U N D 3 0 0 N M . ELECTRONIC TRANSITION
C H R O M O P H O R E : - T H E PA RT O F A M O L E C U L E R E S P O N S I B L E F O R I M PA RT I N G C O L O R , A R E C A L L E D A S C H R O M O S P H E R S . O R T H E F U N C T I O N A L G R O U P C O N TA I N I N G M U LT I P L E B O N D S C A PA B L E O F A B S O R B I N G R A D I AT I O N A B O V E 2 0 0 N M D U E TO Π TO Π * T R A N S I T I O N TO Π * T R A N S I T I O N E . G . N O 2 , N = O , C = O , C = S E T C . . TERM USED IN UV/ VISIBLE SPECTROSCOPY
A U X O C H R O M E : - T H E F U N C T I O N A L G R O U P A T T A C H E D T O A C H R O M O P H O R E T O A B S O R B L I G H T , A L T E R I N G T H E W A V E L E N G T H O R I N T E N S I T Y O F A B S O R P T I O N . O R T H E F U N C T I O N A L G R O U P W I T H N O N – B O N D IN G E L E C T R O N T H A T D O E S N O T A B S O R B R A D I A T I O N I N N E A R U V R E G I O N W H E N A T T A C H E D T O A C H R O M O P H O R E A L T E R T H E W A V E L E N G T H & I N T E N S I T Y O F A B S O R P T I O N . E G . B E N Z E N E ( Λ M A X ) = 2 5 5 N M , P H E N O L ( Λ M A X ) = 2 7 0 N M , A N I L I N E ( Λ M A X ) = 2 8 0 N M . TERM USED IN UV/ VISIBLE SPECTROSCOPY
1 . B A T H O C H R O M I C S H I F T ( R E D S H I F T ) : - W H E N A B S O R P T I O N M A X I M A Λ M A X O F A C O M P O U N D S H I F T S T O L O N G E R W A V E L E N G T H . I T I S K N O W N A S B A T H C H R O M I C S H I F T O R R E D S H I F T . 2 . H Y P S O C H R O M I C S H I F T ( B L U E S H I F T ) : - W H E N A B S O R P T I O N M A X I M A Λ M A X O F A C O M P O U N D S H I F T T O S H O R T E R W A V E L E N G T H , I T I S K N O W N A S H Y P S O C H R O M I C S H I F T O R B L U E S H I F T . 3 . H Y P E R C H R O M I C E F F E C T : - W H E N A B S O R P T I O N I N T E N S I T Y O F A C O M P O U N D I S I N C R E A S E D I T I S K N O W N A S H Y P E R C H R O M I C S H I F T . 4 . H Y P O C H R O M I C E F F E C T : - W H E N A B S O R P T I O N I N T E N S I T Y O F A C O M P O U N D I S D E C R E A S E D I T I S K N O W N A S H Y P O C H R O M I C S H I F T ABSORPTION & INTENSITY SHIFTS
T H E O R Y I N V O L V E D W H E N A B E A M O F L I G H T FA L L S O N A S O L U T I O N O R H O M O G E N O U S M E D I A A P O RT I O N O F L I G H T I S R E F L E C T E D F R O M T H E S U R FA C E O F T H E M E D I A A P O RT I O N I S A B S O R B E D W I T H I N T H E M E D I U M A N D R E M A I N I N G I S T R A N S M I T T E D T H R O U G H T H E M E D I U M .  T H U S I F I 0 I S T H E I N T E N S I T Y O F R A D I AT I O N FA L L I N G O N T H E M E D I A .  I T I S T H E A M O U N T O F R A D I AT I O N S R E F L E C T E D .  I A I S T H E A M O U N T O F R A D I AT I O N A B S O R B E D & .  I T T H E A M O U N T O F R A D I AT I O N T R A N S M I T T E D .  T H E N I 0 = I T + I A + I T UV-VISIBLE SPECTROSCOPY
L AW S I N VO LV E D B E E R ’ S L AW L A M B E RT ’ S L AW B E E R – L A M B E RT ’ S L AW B E E R ’ S L AW : - W H E N A B E A M O F M O N O C H R O M AT I C L I G H T I S PA S S E S T H R O U G H A H O M O G E N O U S A B S O R B I N G M E D I U M . T H E R AT E O F D E C R E A S E O F I N T E N S I T Y O F R A D I AT I O N W I T H I N C R E A S E I N T H E C O N C E N T R AT I O N ( C ) O F A B S O R B I N G S P E C I E S I S D I R E C T LY P R O P O RT I O N A L TO T H E I N T E N S I T Y ( I ) O F T H E I N C I D E N T L I G H T ( R A D I AT I O N ) - d I / d c = kI - d I / I = kd c O N I N T E G R AT I O N O F A B O V E E Q U AT I O N - I n I = k c + b ( b = i n t e g r a t i o n c o n s t a n t ) … … ( 1 ) W H E N C O N C . = 0 , T H E N T H E R E I S N O A B S O R B A N C E . H E R E I = I 0 T H E R E F O R S U B S T I T U TI N G I N E Q U AT I O N ( 1 ) I n I = k  0 + b I n I = b
s u b s t i t u t i n g t h e v a l u e o f b i n t h e e q u a t i o n ( 1 ) I n I = kc - I n I o I n I 0 - I n I o = kc I n I o / I n I = kc ( s i n c e l o g A - l o g B = l o g A / B ) I o / I = e kc ( re mo v i n g n a t u r a l l o g a r i t h m) I o / I = e - kc ( i n v e r s e b o t h s i d e ) I = I o e - kc … … … … … … ……. (2 ) L a mb e r t ’s l a w : - w h e n a b e a m o f mo n o c h ro ma t i c l i g h t i s p a s s e d t h ro u g h a h o mo g e n o u s a b s o r b i n g me d i u m t h e r a t e o f d e c re a s e o f i n t e n s i t y o f r a d i a t i o n w i t h t h i c kn e s s o f a b s o r b i n g me d i u m i s d i re c t l y p ro p o r t i o n a l t o t h e i n t e n s i t y o f t h e i n c i d e n t l i g h t ( r a d i a t i o n ) d I / d t = kI I = i n t e n s i t y o f i n c i d e n t l i g h t o f w a v e l e n g t h Λ & T = T H I C K N E S S O F M E D I U M S i n c e , I = I o e - kc … … … … … …… …(3 ) N o w c o mb i n e t h e e q . ( 2 ) a n d ( 3 ) w e g e t I = I o e - kc t
c o n v e r t i n g n a t u r a l l o g a r i t h m t o b a s e d 1 0 i n v e r s e o n b o t h s i d e I o / I = 1 0 kc t t a k i n g l o g o n b o t h s i d e s l o g I o / I = k c t … … … … … … … … … . . ( 4 ) h e r e t r a n s mi s s i o n ( t ) = I / I o a n d a b s o r b a n c e ( a ) = l o g 1 / T h e n c e A = l o g I 0 / I … … … … … … … … … … … … … … … … … … . ( 5 ) A = k c t I n s t e a d o f K w e c a n u s e ∑ t h e a b o v e e q u a t i o n w i l l b e a s f o l l o w : A = ∑ c t T h i s i s ma t h e ma t i c a l e q u a t i o n f o r B e e r ’s L a mb e r t ’s L a w . A = ∑ c t W h e r e A = A b s o r b a n c e ∑ = M o l e c u l a r e x t i n c t i o n c o e ff i c i e n t C = C o n c e n t r a t i o n o f s a mp l e T = P a t h l e n g t h I=Io e-kct
 S O U R C E O F R A D I AT I O N  C O L L I M AT I N G S Y S T E M  M O N O – C H R O M AT E R S Y S T E M  S A M P L E H O L D E R  D E T E C TO R A M P L I F I E R INSTRUMENTATION
R E Q U I R E M E N T O F A N I D E A L S O U R C E  i t s h o u l d b e s t a b l e a n d s h o u l d n o t a l l o w f l u c t u a t i o n s  i t s h o u l d e mi t l i g h t o f c o n t i n u o u s s p e c t r u m o f h i g h a n d u n i f o r m i n t e n s i t y o v e r t h e e n t i re w a v e l e n g t h re g i o n i n w h i c h i t ’s u s e d . i t s h o u l d p ro v i d e i n c i d e n t l i g h t o f s u f f i c i e n t i n t e n s i t y f o r t h e t r a n s mi t t e d e n e r g y t o b e d e t e c t e d a t t h e e n d o f o p t i c p a t h . i t s h o u l d n o t s h o w f a t i g u e o n c o n t i n u e u s e d . SOURCE OF RADIATION
 I T C O N S T R U C T I O N I S S I M I L A R TO A H O U S E H O L D L A M P.  T H E N B U L B C O N TA I N A F I L A M E N T O F T U N G S T E N F I XE D I N E VA C U AT E D C O N D I T I O N A N D T H E N F I L L E D W I T H I N E RT G A S .  T H E F I L A M E N T C A N B E H E AT E D U P TO 3 0 0 0 K . B E Y O N D T H I S T U N G S T E N S TA RT S S U B L I M AT I N G . I T I S U S E D W H E N P O LY C H R O M AT I C L I G H T I S R E Q U I R E D D E M E R I T  I T E M I T S T H E M A J O R P O RT I O N O F I T S R A D I A N T E N E R G Y I N N E A R I R R E G I O N O F T H E S P E C T R U M TUNGSTEN HALOGEN LAMP
I N H Y D R O G E N D I S C H A R G E L A M P PA I R O F E L E C T R O D E I S E N CL O S E D I N A G L A S S T UB E ( P R O V I D E W I T H S I L I C A O R Q U A RT Z W I N D O W F O R U V RA D I AT I O N TO PA S S T H R O U G H ) F I L L E D W I T H H Y D R O G E N G A S .  W H E N C U R R E NT I S PA S S E D T H R O U G H T H E S E E L E CT R O D E S M A I N TA I NE D AT H I G H VO LTA G E , D I S C H A R G E O F E L E C T R O N O C C U R S W H I C H E XC I T E S H Y D RO G E N M O L E C UL E W H I C H I N T U R N C A U S E E M I S S I O N O F U V R A D I AT I O N I N N E A R U V R E G I O N  T H E Y A R E S TA B L E A N D W I D E LY U S E D . HYDROGEN DISCHARGE LAMP
 I T P O S S E S S E S T W O T U N G S T E N E L E C T R O D E S E PA R AT E D B Y S O M E D I S TA N C E .  T H E S E A R E E N C L O S E D I N A G L A S S T U B E ( W I T H Q U A RT Z O R F U S E D S I L I C A ) A N D XE N O N G A S I S F I L L E D U N D E R P R E S S U R E  A N I N T E N S E A R E I S F O R M E D B E T W E E N E L E C T R O D E B Y A P P LY I N G H I G H VO LTA G E . T H I S I S A G O O D S O U R C E O F C O N T I N U O U S P L U S A D D I T I O N A L I N T E N S E R A D I AT I O N . I T S I N T E N S I T Y I S H I G H E R T H E N T H E H Y D R O G E N D I S C H A R G E L A M P . D E M E R I T : - T H E L A M P S I N C E O P E R AT E S AT H I G H VO LTA G E B E C O M E V E RY H O T D U R I N G O P E R AT I O N A N D H E N C E N E E D S T H E R M A L I N S U L AT I O N . XENON DISCHARGE LAMP
 I N M E R C U RY A R E L A M P , M E R C U RY VA P O R I S S TO R E D U N D E R H I G H P R E S S U R E A N D E XC I TAT I O N O F M E R C U RY ATO M S I S D O N E B Y E L E C T R I C D I S C H A R G E . D E M E R I T : - N O S U I TA B L E F O R C O N T I N U O U S S P E C T R A L S T U D I E S ( B E C A U S E I T D O E S N O T G I V E N C O N T I N U O U S R A D I AT I O N ) MERCURY ARC LAMP
T H E R A D I AT I O N E M I T T E D B Y T H E S O U R C E I S C O L L I M AT E D ( M A D E PA R A L L E L ) B Y L E N S E S M I R R O R A N D S L I T S . L E N S E S  M AT E R I A L U S E D F O R T H E L E N S E S M U S T B E T R A N S PA R E NT TO T H E R A D I AT I O N B E I N G U S E D .  O R D I N A RY S I L I C A G L A S S T R A N S M I T B E T W E E N 3 5 0 TO 3 0 0 0 0 N M A ND I S S U I TA B L E F O R V I S I B L E A N D N E A R I V R E G I O N .  Q U A RT Z O R F U S E D S I L I C A I S U S E D A S A M AT E R I A L F O R L E N S TO W O R K B E L O W 3 0 0 N M . COLLIMATING SYSTEM
M I R R O R S : -  T H E S E A RE U S E D TO R E F L E CT F O C U S O R C O L L I M AT E L I G H T B E A M S I N S P E C T R O P H O TO M E T E R  TO M I N I M I ZE T H E L I G H T L O S S , M I R R O R S A R E A L U M I N I ZE D O N T H E I R F R O N T S U R FA C E . S L I T S : -  S L I T I S A N I M P O RTA N T D E V I CE I N RE S O LV I N G P O LY C H R O M AT I C RA D I AT I O N I NTO M O N O C H R O M AT I C R A D I AT I O N .  TO A C H I E V E T H I S , E N T R A N C E S L I T A N D E XI T S L I T A R E U S E D .  T H E W I D T H O F S L I T P L AY S A N I M P O RTA NT R O L E I N R E S O L U T I O N O F P O LY C H R O M AT I C R A D I AT I O N .
I T I S A D E V I C E U S E D TO I S O L AT E T H E R A D I AT I O N O F T H E D E S I R E D WAV E L E N G T H F R O M WAV E L E N G T H O F T H E C O N T I N U O U S S P E C T R A . T H E E S S E N T I A L E L E M E N TS O F M O N O C H R O M ATO R S A R E  A N E N T R A N C E S L I T  D I S P E R S I N G E L E M E N T  E XI T S L I T T H E E N T R A N C E S L I T S H A R P LY D E F I N E T H E I N C O M I N G B E A M O F H E T R O C H R O M AT I C R A D I AT I O N . T H E D I S P E R S I N G E L E M E N T D I S P E R S E S T H E H E T R O C H R O M AT I C R A D I AT I O N I N TO I T S C O M P O N E N T WAV E L E N G T H . E XI T S L I T A L L O W S T H E N O M I N A L WAV E L E N G T H TO G E T H E R W I T H A B O N D O F WAV E L E N G T H O N E I T H E R S I D E O F I T . MONOCHROMATORS
T W O T Y P E O F F I LT E R A R E U S E D T H E Y A R E A B S O R P T I O N F I LT E R : - W O R K B Y S E L E C T I VE A B S O R P T I O N O F U N WA N T E D R A D I AT I O N A N D T R A N S M I T T H E R A D I AT I O N W H I C H I S R E Q U I R E D . E XA M P L E : - G L A S S A N D G E L AT I N E F I LT E R S E L E C T I O N O F A B S O R P T I O N F I LT E R I S D O N E A C C O R D I N G TO T H E F O L L O W I N G P R O C E D U R E :  D R AW A F I LT E R W H E E L  W R I T E T H E C O L O U R V I B G Y O R I N C L O C K W I S E O R A N T I C L O C K W I S E M A N N E R , O M I T T I N G I N D I G O  I F S O L U T I O N TO B E A N A LY S E D I S B L U E I N C O L O U R A F I LT E R H AV I N G A C O M P L I M E N TA RY C O L O U R O R A N G E I S U S E D I N T H E A N A LY S I S . FILTERS
 A N A B S O R P T I O N G L A S S F I LT E R I S M A D E O F S O L I D S H E E T O F G L A S S T H AT H A S B E E N C O L O U R E D B Y P I G M E N T W H I C H I S D I S S O LV E D O R D I S P E R S E D I N T H E G L A S S . M E R I T S : -  S I M P L E I N C O N S T R U C T I O N  C H E A P E R  S E L E C T I O N O F T H E F I LT E R I S E A S Y D E M E R I T S : -  L E S S A C C U R AT E  B A N D PA S S ( B A N D W I D T H ) I S M O R E ( + 2 0 - 3 0 N M ) I F W E H AV E TO M E A S U R E AT 4 0 0 N M . W E G E T R A D I AT I O N F R O M 3 7 0 - 4 3 0 N M . H E N C E L E S S A C C U R AT E R E S U LT A R E O B TA I N E D .
 W O R K O N T H E I N T E R F E R E N CE P H E N O M E N O N C A U S E R E J E C T I O N O F U N WA N T E D WAV E L E N G T H B Y S E L E C T I V E R E F L E C T I O N .  I T I S C O N S T R U C T E D B Y U S I N G T W O PA R A L L E L G L A S S P L AT E S W H I C H A R E S I LV E R E D I N T E R N A L LY A N D S E PA R AT E D B Y T H I N F I L M O F D I E L E C T R I C M AT E R I A L O F D I F F E R E N T ( C A F 2 , S I O , M G F 2 ) R E F R A C T I V E I N D E X.  T H E S E F I LT E R S H AV E A B A N D PA S S O F 1 0 - 1 5 W I T H P E A K T R A N S M I S S I O N O F 4 0 - 6 0 % INTERFERENCE FILTER
M E R I T S : -  P R O V I D E G R E AT E R T R A N S M I T TA N C E A N D N A R R O W E R B A N D PA S S ( 1 0 - 1 5 N M ) A S C O M PA R E TO A B S O R P T I O N F I LT E R  I N E X P E N S I V E  A D D I T I O N A L F I LT E R C A N B E U S E D TO C U T O F F U N D E S I R E D WAV E L E N G T H .
 P R I S M I S M A D E F R O M G L A S S , Q U A RT Z O R F U S E S S I L I C A .  Q U A RT Z O R F U S E D S I L I C A I S T H E C H O I C E O F M AT E R I A L O F U V S P E C T R U M .  W H E N W H I T E L I G H T I S PA S S E D T H R O U G H G L A S S P R I S M , D I S P E R S I O N O F P O LY C H R O M AT I C L I G H T I N R A I N B O W O C C U R S N O W B Y R O TAT I O N O F T H E P R I S M D I F F E R E N T WAV E L E N G T H O F T H E S P E C T R U M C A N B E M A D E TO PA S S T H R O U G H I N E XI T S L I T O N T H E S A M P L E .  T H E E F F E C T I V E WAV E L E N G T H D E P E N D O N T H E D I S P E R S I V E P O W E R O F P R I S M M AT E R I A L A N D T H E O P T I C A L A N G L E O F T H E P R I S M . PRISM
 T H E S E A RE T W O T Y P E S O F M O U N T I N G I S A N I N S T R U M E NT O N E I S C AL L E D ‘ C O R N T Y PE ’ ( R E F R A CT I VE ) W H I C H H A S O N O P T I C A L AN G L E O F 6 0 0 A ND I T S AD J U ST E D T H AT O N R O TAT I O N T H E E M E R G I N G L I G H T I S A LL O W E D TO FA LL O N E XI T S L I T I N F I G ( A )  T H E O T H E R T Y PE I S C AL L E D “ L I T T R O W T Y PE “ ( R E F L E CT I V E ) , W H I C H H A S O P T I C AL A N G L E 3 0 0 A N D I T S O N E S U R FAC E TO A L U M I N I ZE D W I T H R E F L E CT E D L I G H T B A C K PA S S T H R O U G H P R I S M AN D TO E M E R G E O N T H E S A M E S I D E O F T H E L I G H T S O U R C E . L I G H T D O E S N O T PA S S T H R O U G H T H E P R I S M O N O T H E R S I D E . F I G ( B )
 T H E Y A RE M O S T E F F I C I E N T I N C O N V E RT I NG A P O LY C H R O M AT I C L I G H T . A S A R E S O L U T I O N O F + / - 0 . 1 N M C O U L D B E A C H I E V E D B Y U S I N G G R AT I N G . A S T H E G R AT I N G A R E E XP EN S I VE T H E Y AR E C O M M O N LY U S ED I N S P E C T R O P H O TO M E T E R .  G R AT I N G A R E O F T W O T Y P E : -  D I F F R A C T I O N G R AT I N G  T R A N S M I S S I O N G R AT I N G  D I F F R A C T I O N G R AT I N G : - M O R E R E F I N E D D I S P E R SI O N O F L I G H T I S O B TA I NE D B Y M E AN S O F D I F F R A C T I O N G R AT I N G . T H E S E C O N S I ST A L A R G E N U M B E R O F PA R A L L E L L I N E S ( G R O V E S ) A B O U T 1 5 0 0 0 - 3 0 0 0 0 / - I N C H GRATINGS
I S R U L E D O N H I G H LY P O L I S H E D S U R FA C E O F A L U M I N I U M .  TO M A K E T H E S U R FA C E R E F L E C T I VE A D E P O S I T O F A L U M I N I U M I S M A D E O N T H E S U R FA C E .  D I F F R A C T I O N P R O D U C E R E I N F O R C E M E N T T R A N S M I S S I O N G R AT I N G : - I T I S S I M I L A R TO D I F F R A C T I O N G R AT I N G B U T R E F R A C T I O N TA K E P L A C E I N S T E A D O F R E F L E C T I O N . R E F R A C T I O N P R O D U C E R E I N F O R C E M E N T T H I S O C C U R S R A D I AT I O N T R A N S M I T T E D T H R O U G H G R AT I N G R E I N F O R C E S W I T H T H E PA RT I A L LY R E F R A C T E D R A D I AT I O N . T H E WAV E L E N G T H O F R A D I AT I O N P R O D U C E B Y T R A N S M I S S I O N G R AT I N G C A N B E E XP R E S S E D B Y F O L L O W I N G E Q U AT I O N S .
A D VA N TA G E S : -  G R AT I N G G I V E S H I G H E R A N D L I N E A R D I S P E R S I O N C O M PA R E D TO P R I S M M O N O C H R O M ATO R  C A N B E U S E D O V E R W I D E WAV E L E N G T H R A N G E ,  G R AT I N G C A N B E C O N S T R U C T E D W I T H M AT E R I A L L I K E A L U M I N I U M W H I C H I S R E S I S TA N T TO AT M O S P H E R I C M O I S T U R E .  P R O V I D E L I G H T O F N A R R O W WAV E L E N G T H .  N O L O S S O F E N E R G Y D U E TO A B S O R P T I O N .
 T H E C E L L S O R C U V E T T E S A R E U S E D F O R H A N D L I N G L I Q U I D S A M P L E S .  T H E C E L L M AY E I T H E R B E R E C TA N G U L A R O R C Y L I N D R I C A L I N N AT U R E .  F O R S T U D Y I N U V R E G I O N ; T H E C E L L S A R E P R E PA R E D F R O M Q U A R T Z O R F U S E D S I L I C A W H E R E A S F U S E D G L A S S I S U S E D F O R V I S I B L E R E G I O N .  T H E S U R FA C E O F A B S O R P T I O N C E L L S M U S T B E K E P T C L E A N N O F I N G E R P R I N T S H O U L D B E P R E S E N T O N C E L L S  C L E A N I N G I S C A R R I E D O U T WA S H I N G W I T H D I S T I L L E D WAT E R O R W I T H D I L U T E A L C O H O L , A C E T O N E  T H E C E L L O R C U V E T T E T H AT C O N TA I N S A M P L E S F O R A N A LY S I S S H O U L D F U L F I L C O N D I T I O N S A ) T H E Y M U S T B E U N I F O R M I N C O N S T R U C T I O N T H E T H I C K N E S S M U S T B E C O N S TA N T A N D S U R FA C E FA C I N G T H E I N C I D E N T L I G H T M U S T B E O P T I C A L LY F L AT . B ) T H E M AT E R I A L O F C O N S T R U C T I O N S H O U L D B E I N E R T T O S O LV E N T S C ) T H E Y M U S T T R A N S M I T L I G H T O F T H E WAV E L E N G T H U S E D SAMPLE HOLDER OR CUVETTES
 D E V I C E W H I C H C O N V E RT S L I G H T E N E R G Y I N TO E L E C T R I CA L S I G N A L S T H E Y A R E D I S P L AY E D O N R E A D O U T D E V I C E S .  T H E T R A N S M I T T E D R A D I AT I O N FA L L S O N T H E D E T E C TO R W H I C H D E T E R M I N E S T H E I N T E N S I T Y O F R A D I AT I O N A B S O R B E D B Y S A M P L E  T H E F O L L O W I N G T Y P E O F D E T E C TO R S A R E E M P L O Y E D I N I N S T R U M E N TAT I O N O F A B S O R P T I O N S P E C T R O P H O TO M E T E R 1 ) B A R R I E R L AY E R C E L L 2 ) P H O TO T U B E S 3 ) P H O TO M U LT I P L I E R T U B E DETECTORS
R E Q U I R E M E N T S O F I D E A L C H A R A C T E R  I T S H O U L D G I V E Q U A N T I TAT I V E R E S P O N S E .  I T S H O U L D H AV E H I G H S E N S I T I V I T Y A N D N O I S E L E V E L .  I T S H O U L D H AV E A S H O RT R E S P O N S E T I M E .  I T S H O U L D P R O V I D E S I G N A L S O R Q U A N T I TAT I VE TO W I DE S P E C T R U M O F R A D I AT I O N R E C E I V E D . B A R R I E R L AY E R C E L L : - T H E D E T E CTO R H A S A F I L M M E TA L L I C L AY E R C O AT E D W I T H S I LV E R O R G O L D A N A C T A S E L E C T R O D E . I T AL S O H A S A M E TAL BA SE P LATE W H I C H A CT A S AN O T H E R E L E CT R O D E . T H E Y T W O L AY E R A RE SE PA R AT E D B Y A S E M I C O N D UC TO R L AY E R O F S E L E N I U M . W H E N L I G H T R A D I AT I O N FA L L O N S E L E N I U M L AY E R , E L E C T R O N B E C O M E M O B I L E A ND A R E TA K E N U P B Y T R AN S PA RE N T M E TAL L AY E R . T H I S C R E AT E A P O T E NT I A L D I F F E RE N CE B E T WE E N E L E C T R O D E S C A U SE T H E F L O W O F C U R RE NT .W H E N I T I S C O N N E C TE D TO G A LVA N O M E T E R A F L O W O F C U R R E NT O B SE RVE D W H I C H I S P R O P O RT I O N AL TO T H E I N T E N S I T Y A N D WAVE L E N G T H O F L I G H T FA L L I N G I T .
BARRIER LAYER CELL
 C O N S I S T O F E VA C U AT E D G L A S S T U B E W I T H A P H O TO C AT H O D E A N D A C O L L E C TO R A N O D E .  T H E S U R FA C E O F P H O TO C AT H O D E I S C O AT E D W I T H A L AY E R O F E L E M E N T L I K E C E S I U M , S I LV E R O XI D E O R M I XT U R E O F T H E M .  W H E N R A D I A N T E N E R G Y FA L L O N P H O TO S E N S I T I V E C AT H O D E E L E C T R O N A R E E M I T T E D W H I C H A R E AT T R A C T E D TO A N O D E C A U S I N G C U R R E N T TO F L O W .  M O R E S E N S I T I V E C O M PA R E D TO B A R R I E R L AY E R C E L L A N D T H E R E F O R W I D E LY U S E D . PHOTOUBES
 T H E P R I N CI PA L E M PL O Y E D I N T H I S D E T E C TO R I S T H AT M U LT I PL I CAT I O N O F P H O TO E L E CT R O N S B Y SE C O ND A RY E M I S S I O N O F E L E C T R O N S .  N E AR T H E L A ST D YN O D E I S F I XE D A N O D E I S EL ECT R O N C O L L E C TO R  I N A VA CU UM T UB E A P R I M A RY PH O TO C AT H O D E I S A FI XE D W H I C H R E C E I V E R A D I AT I O N F R O M T H E S A M P L E .  S O M E 8 TO 1 0 D YN O D E S A R E F I XE D E A C H W I T H I N C R E AS I N G P O T E N T I A L O F 7 5 - 1 0 0 V H I G H E R T H E N P R O C E E D I N G O N E .  P H O TO M U LT I P L I E R I S E XT R E M E LY SE N S I T I V I T Y TO L I G H T A N D I S B E ST SU I T E D WH E R E W E AK E R O R L O W R A D I AT I O N I S R E C E I V E D . PHOTOMULTIPLIER TUBE
 T H E S I G N A L F R O M D E T E CTO R A F T E R A M P L I F I C AT I O N A RE F I N A L LY RE CE I VE D B Y R E C O R D I N G S Y S T E M O R R E D O U T D E V I C E .  T H E R E C O R D I N G I S D O N E B Y R E C O R D E R P E N . READ –OUT DEVICE
 L I G H T F R O M T H E S O U R C E I S CA RR I E D T H R O U G H L E N S A N D / O R T H R O U G H A P E RT U R E TO PA S S T H R O U G H A S U I TA B L E F I LT E R .  T H E T Y P E O F F I LTE R TO B E U S E D I S G O V E R NE D B Y T H E C O L O U R O F S O L U T I O N  T H E S A M PL E S O L UT I O N TO B E A N ALY SE D I N P L AC E I N C U V E T T E S .  A F T E R PA S SI N G T H R O U G H T H E S O L UT I O N T H E L I G H T S T R I K E T H E S U R FAC E O F D E T E CTO R ( B A R R I E R L AY E R CE L L O R P H O TO T U B E ) A N D P R O D U C E E L E C T R I CA L C U R R E N T .  T H E O U T P U T O F C U R RE NT I S A M E AS U RE D B Y T HE D E FL E CT I O N O F N E E DL E O F L I G H T S P O T G A LVA N O M E T E R O R M I C R O M E T E R . SINGLE BEAM UV- SPECTROPHOTOMETER
 T H I S M E T E R I S C A L I B R AT E D I N T E R M O F T R A N S M I T TA NC E A S W E L L A S O P T I C A L D E N S I T Y .  T H E RE A D I N G O F S O L U T I O N B O T H S L AN D E R E D A N D UN K N O W N A R E R E C O R D E D I N O P T I C AL DE N S I T Y UN I T A FT E R A DJ U ST I NG I N S T R U M E N T A R E A G E N T B L A N K .
D O U B L E B E A M I N ST RUM E NT I S T H E O N E I N W H I C H T W O B E A M S A R E F O R M E D I N T H E S PA C E B Y A U S H A P E D M I R R O R C A L L E D A S B E A M S P L I T T E R O R B E A M C H O P P E R . C H O P P E R I S A DE V I C E C O N S I S T I NG O F A C I R C UL A R D I S C . O N E T H I R D O F T H E D I SC I S O PA Q U E A N D O N E T H I R D I S T R AN S PA RE N T , RE M A I N I N G I S O N E T H I R D I S M I R R O R E D . I T S P L I T T H E M O N O C H R O M AT I C B E AM O F L I G H T I NTO T W O B E A M S O F E Q U A L I N T E N S I T I E S . DOUBLE BEAM UV- SPECTROPHOTOMETER
S I N G L E B E A M  S I M P L E I N C O N S T R U C T I O N , E A S Y TO U S E E C O N O M I C A L D O U B L E B E A M  I T FA C I L I TAT E S R A P I D S C A N N I N G O V E R W I D E R E G I O N .  F L U C T U AT I O N D U E TO R A D I AT I O N S O U R C E A R E M I N I M I ZE D .  I T D O E S N O T R E Q U I R E A D J U S T M E N T O F T H E T R A N S M I T TA N CE AT 0 % A N D 1 0 0 % AT E A C H WAV E L E N G T H .  I T G I V E S R AT I O O F I N T E N S I T I E S O F S A M P L E & R E F E R E N CE B E A M S I M U LTA N E O U S LY . ADVANTAGE OF SINGLE BEAM & DOUBLE BEAM SPECTROPHOTOMETER
S I N G L E B E A M  A N Y F L U C T U AT I O N I N T H E I N T E N S I T Y O F R A D I AT I O N S O U R C E A F F E C T T H E A B S O R B A N C E .  C O N T I N U O U S S P E C T R U M I S N O T O B TA I N E D . D O U B L E B E A M  C O N S T R U C T I O N I S C O M P L I C AT E D .  I N S T R U M E N T I S E XP E N S I V E . DISADVANTAGE OF SINGLE & DOUBLE BEAM SPECTROPHOTOMETER
COMPARISON
Q U A L I TAT I V E Q U A N T I TAT I VE A N ALY S I S : - I T I S U S E D F O R C H A R A C T E R I ZI N G A R O M AT I C CO M P O U N D S A N D C O N J U G AT E D O L E F I N S . I T C A N B E US E D TO F I ND O U T M O L A R C O N CE NT R AT I O N O F T H E S O L U T E U N D E R S T U D Y . D E T E C T I O N O F I M P U R I T Y : - I T I S O N E O F T H E I M P O RTA N T M E T H O D TO D E T E CT I M P U R I T I E S I N O R G A N I C S O LV E NT S A D D I T I O NA L P E A K C A N B E C O M PA R E D W I T H T H AT O F S TA N D A R D R AW M AT E R I A L . S T RU C T U RE E L U C I D AT I O N O F O R G A N I C C O M P O U N D : - T H E P R E SE N CE O R A B S E N CE O F U N S AT U RAT I O N T H E P R E S E N C E O F H E T E R O ATO M L I K E S , N , O O R H A L O G E N C A N B E D E T E R M I N E D . S T R U C T U R E A N A LY S I S O F O R G A N I C C O M P O U N D : - E F F E CT O F C O N J U G AT I O N : - E XT E N D E D C O N J U G AT I O N S H I F T S T H E TO L O N G E R ( B AT H O C H R O M AT I C SH I F T ) A N D R E DU CT I O N O F T H E C O M P O U N D O R S AT U R AT I O N O F D O U B L E B O N D S L E A D S TO T H E APPLICATION FOR UV- VISIBLE SPECTROSCOPY
O P P O S I T E E F F E C T E X: - H Y P S O C H R O M I C S H I F T E F F E C T O F G E O M E T R I C I S O M E R I S M : - T R A N S I S O M E R A B S O R B AT L O N G E R WAV E L E N G T H T H E N C I S I S O M E R S . C I S TO T R A N S C O N V E R S I O N B AT H O C H R O M I C S H I F T A N D H Y P E R - C H R O M I C E F F E C T . A L K Y L S U B S T I T U T I O N : - S H I F T T H E L A M D A M A X TO L O N G E R WAV E L E N G T H ( B AT H O C H R O M I C S H I F T ) N U M B E R O F R I N G S : - T H E A D D I T I O N O F R I N G C A U S E S B AT H O C H R O M I C S H I F T.
 T H E C H O I C E O F T H E S O LV E NT TO B E U SE D I N ULT R AVI O L E T S P E C T R O S C O P Y I S Q U I T E I M P O RTA N T .  A S E C O N D C R I T E R I O N F O R A G O O D S O LV E NT I S I T S E FF E CT O N T H E F I N E S T R U C T U RE O F A N A B S O R P T I O N B A N D .  I N A P O L A R S O LV E NT T H E H Y D R O G E N B O N D I N G F R O M A S O L U T E - S O LVE NT S C O M P L E X A ND T H E F I N E S T RU C TU RE M AY D I S A P P E A R .  A N O N – P O L A R S O LV E N T D O E S N O T H Y D R O G E N B O N D W I T H T H E S O L U T E A N D T H E S P E C T R U M O F T H E S O L U T E CL O S E LY A P P R O XI M AT E S T H E S PE CT R U M T H AT W O U L D B E P R O D U CE D I N T H E G A S E O U S S TAT E , I N W H I C H F I N E S T R U CT UR E I S O F T E N O B S E RV E D .  T H E F I R ST C R I T E R I O N F O R A G O O D S O LV E NT I S T H AT I T S H O U L D N O T AB S O R B U LT RAV I O L E T R A DI AT I O N I N T H E S A M E R E G I O N A S T H E S UB STA NC E W H O S E S P E CT R U M I S B E I N G D E T E R M I N E D . CHOICE OF SOLVENT
 U V- V I S S P E C ( E L E C T R O N I C S P E C T R O P H O T O M E T E R )  Q U A L I T A T I V E A N D Q U A N T I T A T I V E A N A L Y S I S  D E T E C T I O N O F I M P U R I T Y F R O M O R G A N I C M I X T U R E  E L U C I D A T I O N O F S T R U C T U R E  F O R E N S I C T O X I C O L O G Y  M O L E C U L A R W E I G H T D E T E R M I N A T I O N  F O R Q U A L I T Y D E T E R M I N A T I O N  D E T E R M I N A T I O N O F M E T A L C O N T A M I N A N T S .  Q U A L I T A T I V E A N A L Y S I S  C H E M I C A L K I N E T I C APPLICATION
UV Visible spectroscopy

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UV Visible spectroscopy

  • 1. UV- SPECTROSCOPY PREPARED BY :- MISS ANAMIKA SINGH
  • 2. S P E C T R O S C O P Y: - S P E C T R O S C O P Y I S T H E B R A N C H O F S C I E N C E D E A L I N G T H E S T U D Y O F I N T E R A C T I O N O F E L E C T R O M A G N E T I C R A D I A T I O N W I T H M A T T E R . O R I T I S T H E M E A S U R E M E N T O F E L E C T R O M A G N E T I C R A D I A T I O N ( E M R ) A B S O R B E D O R E M I T T E D W H E N M O L E C U L E O R I O N S O R A T O M S O F A S A M P L E M O V E F R O M O N E E N E R G Y S T A T E T O A N O T H E R E N E R G Y S T A T E . S P E C T R O S C O P Y I S T H E M O S T P O W E R F U L T O O L A V A I L A B L E F O R T H E S T U D Y O F A T O M I C & M O L E C U L A R S T R U C T U R E A N D I S U S E D I N T H E A N A L Y S I S O F A W I D E R A N G E O F S A M P L E . INTRODUCTION
  • 3.  T H E P R I N C I P L E I S B A S E D O N T H E M E A S U R M E N T O F S P E C T R U M O F A S A M P L E C O N T A I N I N G A T O M S / M O L E C U L E S S P E C T R U M I S A G R A P H O F I N T E N S I T Y O F A B S O R B E D O R E M I T T E D R A D I A T I O N B Y S A M P L E V E R S E S F R E Q U E N C Y ( V ) O R W A V E L E N G T H ( Λ ) S P E C T R O M E T E R I S A N I N S T R U M E N T D E S I G N T O M E A S U R E T H E S P E C T R U M O F A C O M P O U N D PRINCIPLE OF SPECTROSCOPY
  • 4. A B S O R P T I O N S P E C T R O S C O P Y : - A N A N A L Y T I C A L T E C H N I Q U E W H I C H C O N C E R N W I T H T H E M E A S U R E M E N T O F A B S O R P T I O N O F E L E C T R O M A G N E T I C R A D I A T I O N E . G . U V ( 1 8 5 - 4 0 0 N M ) / V I S I B L E ( 4 0 0 - 8 0 0 N M ) S P E C T R O S C O P Y , I R S P E C T R O S C O P Y ( 0 . 7 6 – 1 5 U M ) E M I S S I O N S P E C T R O S C O P Y : - A N A N A L Y T I C A L T E C H N I Q U E I N W H I C H E M I S S I O N O F A P A R T I C L E O R R A D I A T I O N I S D I S P E R S E D A C C O R D I N G T O S O M E P R O P E R T Y O F T H E E M I S S I O N & A M O U N T O F D I S P E R S I O N I S M E A S U R E D E . G . M A S S S P E C T R O S C O P Y TYPE OF SPECTROSCOPY
  • 5. E L E C T R O M A G N E T I C R A D I A T I O N C O N S I S T O F D I S C R E T E P A C K E T S O F E N E R G Y W H I C H A R E C A L L E D P H O T O N S A P H O T O N C O N S I S T O F A N O S C I L L A T I N G E L E C T R I C F I E L D ( E ) & A N O S C I L L A T I N G F I E L D ( M ) W H I C H A R E P E R P E N D I C U L A R T O E A C H O T H E R ELECTROMAGNETIC RADIATION
  • 6. W A V E L E N G T H : - I T I S T H E D I S T A N C E B E T W E E N T W O S U C C E S S I V E M A X I M U M A N E L E C T R O M A G N E T I C W A V E . U N I T A R E : - M , C M , M M , N M , A N D M I C R O M E T E R F R E Q U E N C Y : - N U M B E R O F W A V E L E N G T H U N I T P A S S T H R O U G H A G I V E N P O I N T I N U N IT T I M E I S C A L L E D A S F R E Q U E N C Y . I T I S D E N O T E D B Y “ V ” A N D U N I T A R E C Y C L E S P E R S E C O N D H E R T Z . W A V E N U M B E R : - A S T H E N U M B E R O F W A V E S P E R C M I N V A C U M M . V E L O C I T Y : - I T I S T H E P R O D U C T O F W A V E L E N G T H A N D F R E Q U E N C Y A N D I S E Q U A L T O T H E V E L O C I T Y O F T H E W A V E I N T H E M E D I U M . V = N × Λ PROPERTIES OF ELECTROMAGNETIC RADIATION
  • 8. E L E C T R O N I C E N E R G Y L E V E L S : - A T R O O M T E M P E R A T U R E T H E M O L E C U L E A R E I N T H E L O W E S T E N E R G Y L E V E L E 0 . W H E N T H E M O L E C U L E A B S O R B U V - V I S I B L E L I G H T F R O M E M R . O N E O F T H E O U T E R M O S T B O N D / L O N E P A I R E L E C T R O N I S P R O M O T E D T O H I G H E R E N E R G Y S T A T E S U C H A S E 1 E 2 … . E T C I S C A L L E D A S E L E C T R O N I C T R A N S I T I O N A N D T H E D I F F E R E N C E I S A S … V I B R A T I O N A L E N E R G Y L E V E L : - T H E S E A R E L E S S E N E R G Y L E V E L T H A N E L E C T R O N I C E N E R G Y L E V E L S . T H E S P A C I N G B E T W E E N E N E R G Y L E V E L A R E R E L A T I V E L Y S M A L L 0 . 0 1 T O 1 0 K C A L / M O L E . E . G . W H E N I R R A D I A T I O N I S A B S O R B E D M O L E C U L E A R E E X C I T E D F R O M V I B R A T I O N A L T O A N O T H E R O R I T V I B R A T E W I T H H I G H E R A M P L I T U D E . INTERACTION OF EMR WITH MATTER
  • 9. ROTATIONAL ENERGY LEVELS: - •T H E S E E N E R G Y L E V E L S A RE Q U A NT I ZE D & D I S C R E T E . T H E S PA C I N G B E T WE E N E N E R G Y L E VE L S M A L L E R T H E N V I B R AT I O N A L E N E R G Y L E V E L . INTERACTION OF EMR WITH MATTER
  • 10. A N EL EC TRO N I N A B O N D I N G S - O R B I TAL I S E XC I TE D TO T H E C O R R E S P O N D I N G A N T I - B O N D I N G O R B I TAL A ND O B S E RV E D W I T H S AT UR AT E D C O M P O U N D S . T H E E NE R G Y R E Q U I R E I S L A R G E . E X : - M E T H A N E ( W I T H H A S O N LY ) C - H B O N D S , A N D CA N O N LY U N DE R G O Σ → Σ * T R A N S I T I O N S H O W A N A B S O R B AN CE M A XI M U M AT 1 2 5 N M . A B S O R P T I O N M A XI M A D U E TO Σ → Σ * T R A N S I T I O N A R E N O T S E E N I N T Y P I C AL U V-V I S S P E CT R A ( 2 0 0 - 7 0 0 N M ) B U T I N U V- R E G I O N ( 1 2 5 - 1 3 5 N M ) ELECTRONIC TRANSITION σ → σ* transition.
  • 11. N – > Σ * T R A N S I T I O N : - S AT U RATED C O M P O U N D S C O N TA I N I N G ATO M S W I T H L O N E PA I R ( N O N – B O N D I N G E L E CT RO N S ) L I K E O , N , S A N D H A L O G E N A RE C APA B L E O F N – > Σ * T R A N S I T I O N . T H E S E T R AN S I T I O N U S U AL LY N E E D L E SS E NE R G Y T H A N N – > Σ * T R AN S I T I O N . T H E Y CA N B E I N I T I AT E D B Y L I G H T W H O S E WAV E L E N G T H I N T H E R A N G E 1 5 0 - 2 5 0 N M . T H E N U M B E R O F O R G A N I C F U N CT I O N A L G R O U P N – > Σ * PE A K I N T H E U V R E G I O N I S S M A L L ELECTRONIC TRANSITION
  • 12. Π T O Π * T R A N S I T I O N : - P I E E L E C T R O N I N A B O N D I N G O R B I TA L I S E X C I T E D TO C OR R ES PO N D I N G ANT I - B O N D I N G O R B I TA L PI E S TA R A N D O B S ERV E D I N C O N J U G AT ED CO M P O UN D S . C OM P O U N DS C O N TA I N I NG M U LT I P L E B O N D L I K E A L K E N E S , AL K Y N E S , C AR B ONYL , N I T R I L E S , A R O M AT I C C O M P O UN D S E T C U N D E R G O P I E TO PI E S TA R T R A N S I T I O N . E G : - A L K E N E S G E N E R A L LY A B S O R B I N T H E R E G I O N 1 7 0 TO 2 0 5 N M . ELECTRONIC TRANSITION
  • 13. N T O Π * T R A N S I T I O N : - A N E L E C T R O N F R O M OR B I TA L I S P R O M O T E D TO A N T I - B O N D I N G P I E S TA R O R B I TA L A N D R E Q U I R E D L O W E R E N E R GY. C O M P O U N DS C O N TA I N I N G D OU B LE B O N D IN V O LV I N G H E T E R O ATO M ( C = O , C N , N = O ) U N D E RG O S UC H T R AN S I T I O N . N TO P I E S TA R T R A NS I T I O N R E Q U I R E M I N I M U M E N E R GY A ND S H O W A B S O R P T I O N L O N G E R WAV E L E N G T H A R O U N D 3 0 0 N M . ELECTRONIC TRANSITION
  • 14. C H R O M O P H O R E : - T H E PA RT O F A M O L E C U L E R E S P O N S I B L E F O R I M PA RT I N G C O L O R , A R E C A L L E D A S C H R O M O S P H E R S . O R T H E F U N C T I O N A L G R O U P C O N TA I N I N G M U LT I P L E B O N D S C A PA B L E O F A B S O R B I N G R A D I AT I O N A B O V E 2 0 0 N M D U E TO Π TO Π * T R A N S I T I O N TO Π * T R A N S I T I O N E . G . N O 2 , N = O , C = O , C = S E T C . . TERM USED IN UV/ VISIBLE SPECTROSCOPY
  • 15. A U X O C H R O M E : - T H E F U N C T I O N A L G R O U P A T T A C H E D T O A C H R O M O P H O R E T O A B S O R B L I G H T , A L T E R I N G T H E W A V E L E N G T H O R I N T E N S I T Y O F A B S O R P T I O N . O R T H E F U N C T I O N A L G R O U P W I T H N O N – B O N D IN G E L E C T R O N T H A T D O E S N O T A B S O R B R A D I A T I O N I N N E A R U V R E G I O N W H E N A T T A C H E D T O A C H R O M O P H O R E A L T E R T H E W A V E L E N G T H & I N T E N S I T Y O F A B S O R P T I O N . E G . B E N Z E N E ( Λ M A X ) = 2 5 5 N M , P H E N O L ( Λ M A X ) = 2 7 0 N M , A N I L I N E ( Λ M A X ) = 2 8 0 N M . TERM USED IN UV/ VISIBLE SPECTROSCOPY
  • 16. 1 . B A T H O C H R O M I C S H I F T ( R E D S H I F T ) : - W H E N A B S O R P T I O N M A X I M A Λ M A X O F A C O M P O U N D S H I F T S T O L O N G E R W A V E L E N G T H . I T I S K N O W N A S B A T H C H R O M I C S H I F T O R R E D S H I F T . 2 . H Y P S O C H R O M I C S H I F T ( B L U E S H I F T ) : - W H E N A B S O R P T I O N M A X I M A Λ M A X O F A C O M P O U N D S H I F T T O S H O R T E R W A V E L E N G T H , I T I S K N O W N A S H Y P S O C H R O M I C S H I F T O R B L U E S H I F T . 3 . H Y P E R C H R O M I C E F F E C T : - W H E N A B S O R P T I O N I N T E N S I T Y O F A C O M P O U N D I S I N C R E A S E D I T I S K N O W N A S H Y P E R C H R O M I C S H I F T . 4 . H Y P O C H R O M I C E F F E C T : - W H E N A B S O R P T I O N I N T E N S I T Y O F A C O M P O U N D I S D E C R E A S E D I T I S K N O W N A S H Y P O C H R O M I C S H I F T ABSORPTION & INTENSITY SHIFTS
  • 17. T H E O R Y I N V O L V E D W H E N A B E A M O F L I G H T FA L L S O N A S O L U T I O N O R H O M O G E N O U S M E D I A A P O RT I O N O F L I G H T I S R E F L E C T E D F R O M T H E S U R FA C E O F T H E M E D I A A P O RT I O N I S A B S O R B E D W I T H I N T H E M E D I U M A N D R E M A I N I N G I S T R A N S M I T T E D T H R O U G H T H E M E D I U M .  T H U S I F I 0 I S T H E I N T E N S I T Y O F R A D I AT I O N FA L L I N G O N T H E M E D I A .  I T I S T H E A M O U N T O F R A D I AT I O N S R E F L E C T E D .  I A I S T H E A M O U N T O F R A D I AT I O N A B S O R B E D & .  I T T H E A M O U N T O F R A D I AT I O N T R A N S M I T T E D .  T H E N I 0 = I T + I A + I T UV-VISIBLE SPECTROSCOPY
  • 18. L AW S I N VO LV E D B E E R ’ S L AW L A M B E RT ’ S L AW B E E R – L A M B E RT ’ S L AW B E E R ’ S L AW : - W H E N A B E A M O F M O N O C H R O M AT I C L I G H T I S PA S S E S T H R O U G H A H O M O G E N O U S A B S O R B I N G M E D I U M . T H E R AT E O F D E C R E A S E O F I N T E N S I T Y O F R A D I AT I O N W I T H I N C R E A S E I N T H E C O N C E N T R AT I O N ( C ) O F A B S O R B I N G S P E C I E S I S D I R E C T LY P R O P O RT I O N A L TO T H E I N T E N S I T Y ( I ) O F T H E I N C I D E N T L I G H T ( R A D I AT I O N ) - d I / d c = kI - d I / I = kd c O N I N T E G R AT I O N O F A B O V E E Q U AT I O N - I n I = k c + b ( b = i n t e g r a t i o n c o n s t a n t ) … … ( 1 ) W H E N C O N C . = 0 , T H E N T H E R E I S N O A B S O R B A N C E . H E R E I = I 0 T H E R E F O R S U B S T I T U TI N G I N E Q U AT I O N ( 1 ) I n I = k  0 + b I n I = b
  • 19. s u b s t i t u t i n g t h e v a l u e o f b i n t h e e q u a t i o n ( 1 ) I n I = kc - I n I o I n I 0 - I n I o = kc I n I o / I n I = kc ( s i n c e l o g A - l o g B = l o g A / B ) I o / I = e kc ( re mo v i n g n a t u r a l l o g a r i t h m) I o / I = e - kc ( i n v e r s e b o t h s i d e ) I = I o e - kc … … … … … … ……. (2 ) L a mb e r t ’s l a w : - w h e n a b e a m o f mo n o c h ro ma t i c l i g h t i s p a s s e d t h ro u g h a h o mo g e n o u s a b s o r b i n g me d i u m t h e r a t e o f d e c re a s e o f i n t e n s i t y o f r a d i a t i o n w i t h t h i c kn e s s o f a b s o r b i n g me d i u m i s d i re c t l y p ro p o r t i o n a l t o t h e i n t e n s i t y o f t h e i n c i d e n t l i g h t ( r a d i a t i o n ) d I / d t = kI I = i n t e n s i t y o f i n c i d e n t l i g h t o f w a v e l e n g t h Λ & T = T H I C K N E S S O F M E D I U M S i n c e , I = I o e - kc … … … … … …… …(3 ) N o w c o mb i n e t h e e q . ( 2 ) a n d ( 3 ) w e g e t I = I o e - kc t
  • 20. c o n v e r t i n g n a t u r a l l o g a r i t h m t o b a s e d 1 0 i n v e r s e o n b o t h s i d e I o / I = 1 0 kc t t a k i n g l o g o n b o t h s i d e s l o g I o / I = k c t … … … … … … … … … . . ( 4 ) h e r e t r a n s mi s s i o n ( t ) = I / I o a n d a b s o r b a n c e ( a ) = l o g 1 / T h e n c e A = l o g I 0 / I … … … … … … … … … … … … … … … … … … . ( 5 ) A = k c t I n s t e a d o f K w e c a n u s e ∑ t h e a b o v e e q u a t i o n w i l l b e a s f o l l o w : A = ∑ c t T h i s i s ma t h e ma t i c a l e q u a t i o n f o r B e e r ’s L a mb e r t ’s L a w . A = ∑ c t W h e r e A = A b s o r b a n c e ∑ = M o l e c u l a r e x t i n c t i o n c o e ff i c i e n t C = C o n c e n t r a t i o n o f s a mp l e T = P a t h l e n g t h I=Io e-kct
  • 21.  S O U R C E O F R A D I AT I O N  C O L L I M AT I N G S Y S T E M  M O N O – C H R O M AT E R S Y S T E M  S A M P L E H O L D E R  D E T E C TO R A M P L I F I E R INSTRUMENTATION
  • 22. R E Q U I R E M E N T O F A N I D E A L S O U R C E  i t s h o u l d b e s t a b l e a n d s h o u l d n o t a l l o w f l u c t u a t i o n s  i t s h o u l d e mi t l i g h t o f c o n t i n u o u s s p e c t r u m o f h i g h a n d u n i f o r m i n t e n s i t y o v e r t h e e n t i re w a v e l e n g t h re g i o n i n w h i c h i t ’s u s e d . i t s h o u l d p ro v i d e i n c i d e n t l i g h t o f s u f f i c i e n t i n t e n s i t y f o r t h e t r a n s mi t t e d e n e r g y t o b e d e t e c t e d a t t h e e n d o f o p t i c p a t h . i t s h o u l d n o t s h o w f a t i g u e o n c o n t i n u e u s e d . SOURCE OF RADIATION
  • 23.  I T C O N S T R U C T I O N I S S I M I L A R TO A H O U S E H O L D L A M P.  T H E N B U L B C O N TA I N A F I L A M E N T O F T U N G S T E N F I XE D I N E VA C U AT E D C O N D I T I O N A N D T H E N F I L L E D W I T H I N E RT G A S .  T H E F I L A M E N T C A N B E H E AT E D U P TO 3 0 0 0 K . B E Y O N D T H I S T U N G S T E N S TA RT S S U B L I M AT I N G . I T I S U S E D W H E N P O LY C H R O M AT I C L I G H T I S R E Q U I R E D D E M E R I T  I T E M I T S T H E M A J O R P O RT I O N O F I T S R A D I A N T E N E R G Y I N N E A R I R R E G I O N O F T H E S P E C T R U M TUNGSTEN HALOGEN LAMP
  • 24. I N H Y D R O G E N D I S C H A R G E L A M P PA I R O F E L E C T R O D E I S E N CL O S E D I N A G L A S S T UB E ( P R O V I D E W I T H S I L I C A O R Q U A RT Z W I N D O W F O R U V RA D I AT I O N TO PA S S T H R O U G H ) F I L L E D W I T H H Y D R O G E N G A S .  W H E N C U R R E NT I S PA S S E D T H R O U G H T H E S E E L E CT R O D E S M A I N TA I NE D AT H I G H VO LTA G E , D I S C H A R G E O F E L E C T R O N O C C U R S W H I C H E XC I T E S H Y D RO G E N M O L E C UL E W H I C H I N T U R N C A U S E E M I S S I O N O F U V R A D I AT I O N I N N E A R U V R E G I O N  T H E Y A R E S TA B L E A N D W I D E LY U S E D . HYDROGEN DISCHARGE LAMP
  • 25.  I T P O S S E S S E S T W O T U N G S T E N E L E C T R O D E S E PA R AT E D B Y S O M E D I S TA N C E .  T H E S E A R E E N C L O S E D I N A G L A S S T U B E ( W I T H Q U A RT Z O R F U S E D S I L I C A ) A N D XE N O N G A S I S F I L L E D U N D E R P R E S S U R E  A N I N T E N S E A R E I S F O R M E D B E T W E E N E L E C T R O D E B Y A P P LY I N G H I G H VO LTA G E . T H I S I S A G O O D S O U R C E O F C O N T I N U O U S P L U S A D D I T I O N A L I N T E N S E R A D I AT I O N . I T S I N T E N S I T Y I S H I G H E R T H E N T H E H Y D R O G E N D I S C H A R G E L A M P . D E M E R I T : - T H E L A M P S I N C E O P E R AT E S AT H I G H VO LTA G E B E C O M E V E RY H O T D U R I N G O P E R AT I O N A N D H E N C E N E E D S T H E R M A L I N S U L AT I O N . XENON DISCHARGE LAMP
  • 26.  I N M E R C U RY A R E L A M P , M E R C U RY VA P O R I S S TO R E D U N D E R H I G H P R E S S U R E A N D E XC I TAT I O N O F M E R C U RY ATO M S I S D O N E B Y E L E C T R I C D I S C H A R G E . D E M E R I T : - N O S U I TA B L E F O R C O N T I N U O U S S P E C T R A L S T U D I E S ( B E C A U S E I T D O E S N O T G I V E N C O N T I N U O U S R A D I AT I O N ) MERCURY ARC LAMP
  • 27. T H E R A D I AT I O N E M I T T E D B Y T H E S O U R C E I S C O L L I M AT E D ( M A D E PA R A L L E L ) B Y L E N S E S M I R R O R A N D S L I T S . L E N S E S  M AT E R I A L U S E D F O R T H E L E N S E S M U S T B E T R A N S PA R E NT TO T H E R A D I AT I O N B E I N G U S E D .  O R D I N A RY S I L I C A G L A S S T R A N S M I T B E T W E E N 3 5 0 TO 3 0 0 0 0 N M A ND I S S U I TA B L E F O R V I S I B L E A N D N E A R I V R E G I O N .  Q U A RT Z O R F U S E D S I L I C A I S U S E D A S A M AT E R I A L F O R L E N S TO W O R K B E L O W 3 0 0 N M . COLLIMATING SYSTEM
  • 28. M I R R O R S : -  T H E S E A RE U S E D TO R E F L E CT F O C U S O R C O L L I M AT E L I G H T B E A M S I N S P E C T R O P H O TO M E T E R  TO M I N I M I ZE T H E L I G H T L O S S , M I R R O R S A R E A L U M I N I ZE D O N T H E I R F R O N T S U R FA C E . S L I T S : -  S L I T I S A N I M P O RTA N T D E V I CE I N RE S O LV I N G P O LY C H R O M AT I C RA D I AT I O N I NTO M O N O C H R O M AT I C R A D I AT I O N .  TO A C H I E V E T H I S , E N T R A N C E S L I T A N D E XI T S L I T A R E U S E D .  T H E W I D T H O F S L I T P L AY S A N I M P O RTA NT R O L E I N R E S O L U T I O N O F P O LY C H R O M AT I C R A D I AT I O N .
  • 29. I T I S A D E V I C E U S E D TO I S O L AT E T H E R A D I AT I O N O F T H E D E S I R E D WAV E L E N G T H F R O M WAV E L E N G T H O F T H E C O N T I N U O U S S P E C T R A . T H E E S S E N T I A L E L E M E N TS O F M O N O C H R O M ATO R S A R E  A N E N T R A N C E S L I T  D I S P E R S I N G E L E M E N T  E XI T S L I T T H E E N T R A N C E S L I T S H A R P LY D E F I N E T H E I N C O M I N G B E A M O F H E T R O C H R O M AT I C R A D I AT I O N . T H E D I S P E R S I N G E L E M E N T D I S P E R S E S T H E H E T R O C H R O M AT I C R A D I AT I O N I N TO I T S C O M P O N E N T WAV E L E N G T H . E XI T S L I T A L L O W S T H E N O M I N A L WAV E L E N G T H TO G E T H E R W I T H A B O N D O F WAV E L E N G T H O N E I T H E R S I D E O F I T . MONOCHROMATORS
  • 30. T W O T Y P E O F F I LT E R A R E U S E D T H E Y A R E A B S O R P T I O N F I LT E R : - W O R K B Y S E L E C T I VE A B S O R P T I O N O F U N WA N T E D R A D I AT I O N A N D T R A N S M I T T H E R A D I AT I O N W H I C H I S R E Q U I R E D . E XA M P L E : - G L A S S A N D G E L AT I N E F I LT E R S E L E C T I O N O F A B S O R P T I O N F I LT E R I S D O N E A C C O R D I N G TO T H E F O L L O W I N G P R O C E D U R E :  D R AW A F I LT E R W H E E L  W R I T E T H E C O L O U R V I B G Y O R I N C L O C K W I S E O R A N T I C L O C K W I S E M A N N E R , O M I T T I N G I N D I G O  I F S O L U T I O N TO B E A N A LY S E D I S B L U E I N C O L O U R A F I LT E R H AV I N G A C O M P L I M E N TA RY C O L O U R O R A N G E I S U S E D I N T H E A N A LY S I S . FILTERS
  • 31.  A N A B S O R P T I O N G L A S S F I LT E R I S M A D E O F S O L I D S H E E T O F G L A S S T H AT H A S B E E N C O L O U R E D B Y P I G M E N T W H I C H I S D I S S O LV E D O R D I S P E R S E D I N T H E G L A S S . M E R I T S : -  S I M P L E I N C O N S T R U C T I O N  C H E A P E R  S E L E C T I O N O F T H E F I LT E R I S E A S Y D E M E R I T S : -  L E S S A C C U R AT E  B A N D PA S S ( B A N D W I D T H ) I S M O R E ( + 2 0 - 3 0 N M ) I F W E H AV E TO M E A S U R E AT 4 0 0 N M . W E G E T R A D I AT I O N F R O M 3 7 0 - 4 3 0 N M . H E N C E L E S S A C C U R AT E R E S U LT A R E O B TA I N E D .
  • 32.  W O R K O N T H E I N T E R F E R E N CE P H E N O M E N O N C A U S E R E J E C T I O N O F U N WA N T E D WAV E L E N G T H B Y S E L E C T I V E R E F L E C T I O N .  I T I S C O N S T R U C T E D B Y U S I N G T W O PA R A L L E L G L A S S P L AT E S W H I C H A R E S I LV E R E D I N T E R N A L LY A N D S E PA R AT E D B Y T H I N F I L M O F D I E L E C T R I C M AT E R I A L O F D I F F E R E N T ( C A F 2 , S I O , M G F 2 ) R E F R A C T I V E I N D E X.  T H E S E F I LT E R S H AV E A B A N D PA S S O F 1 0 - 1 5 W I T H P E A K T R A N S M I S S I O N O F 4 0 - 6 0 % INTERFERENCE FILTER
  • 33. M E R I T S : -  P R O V I D E G R E AT E R T R A N S M I T TA N C E A N D N A R R O W E R B A N D PA S S ( 1 0 - 1 5 N M ) A S C O M PA R E TO A B S O R P T I O N F I LT E R  I N E X P E N S I V E  A D D I T I O N A L F I LT E R C A N B E U S E D TO C U T O F F U N D E S I R E D WAV E L E N G T H .
  • 34.  P R I S M I S M A D E F R O M G L A S S , Q U A RT Z O R F U S E S S I L I C A .  Q U A RT Z O R F U S E D S I L I C A I S T H E C H O I C E O F M AT E R I A L O F U V S P E C T R U M .  W H E N W H I T E L I G H T I S PA S S E D T H R O U G H G L A S S P R I S M , D I S P E R S I O N O F P O LY C H R O M AT I C L I G H T I N R A I N B O W O C C U R S N O W B Y R O TAT I O N O F T H E P R I S M D I F F E R E N T WAV E L E N G T H O F T H E S P E C T R U M C A N B E M A D E TO PA S S T H R O U G H I N E XI T S L I T O N T H E S A M P L E .  T H E E F F E C T I V E WAV E L E N G T H D E P E N D O N T H E D I S P E R S I V E P O W E R O F P R I S M M AT E R I A L A N D T H E O P T I C A L A N G L E O F T H E P R I S M . PRISM
  • 35.  T H E S E A RE T W O T Y P E S O F M O U N T I N G I S A N I N S T R U M E NT O N E I S C AL L E D ‘ C O R N T Y PE ’ ( R E F R A CT I VE ) W H I C H H A S O N O P T I C A L AN G L E O F 6 0 0 A ND I T S AD J U ST E D T H AT O N R O TAT I O N T H E E M E R G I N G L I G H T I S A LL O W E D TO FA LL O N E XI T S L I T I N F I G ( A )  T H E O T H E R T Y PE I S C AL L E D “ L I T T R O W T Y PE “ ( R E F L E CT I V E ) , W H I C H H A S O P T I C AL A N G L E 3 0 0 A N D I T S O N E S U R FAC E TO A L U M I N I ZE D W I T H R E F L E CT E D L I G H T B A C K PA S S T H R O U G H P R I S M AN D TO E M E R G E O N T H E S A M E S I D E O F T H E L I G H T S O U R C E . L I G H T D O E S N O T PA S S T H R O U G H T H E P R I S M O N O T H E R S I D E . F I G ( B )
  • 36.  T H E Y A RE M O S T E F F I C I E N T I N C O N V E RT I NG A P O LY C H R O M AT I C L I G H T . A S A R E S O L U T I O N O F + / - 0 . 1 N M C O U L D B E A C H I E V E D B Y U S I N G G R AT I N G . A S T H E G R AT I N G A R E E XP EN S I VE T H E Y AR E C O M M O N LY U S ED I N S P E C T R O P H O TO M E T E R .  G R AT I N G A R E O F T W O T Y P E : -  D I F F R A C T I O N G R AT I N G  T R A N S M I S S I O N G R AT I N G  D I F F R A C T I O N G R AT I N G : - M O R E R E F I N E D D I S P E R SI O N O F L I G H T I S O B TA I NE D B Y M E AN S O F D I F F R A C T I O N G R AT I N G . T H E S E C O N S I ST A L A R G E N U M B E R O F PA R A L L E L L I N E S ( G R O V E S ) A B O U T 1 5 0 0 0 - 3 0 0 0 0 / - I N C H GRATINGS
  • 37. I S R U L E D O N H I G H LY P O L I S H E D S U R FA C E O F A L U M I N I U M .  TO M A K E T H E S U R FA C E R E F L E C T I VE A D E P O S I T O F A L U M I N I U M I S M A D E O N T H E S U R FA C E .  D I F F R A C T I O N P R O D U C E R E I N F O R C E M E N T T R A N S M I S S I O N G R AT I N G : - I T I S S I M I L A R TO D I F F R A C T I O N G R AT I N G B U T R E F R A C T I O N TA K E P L A C E I N S T E A D O F R E F L E C T I O N . R E F R A C T I O N P R O D U C E R E I N F O R C E M E N T T H I S O C C U R S R A D I AT I O N T R A N S M I T T E D T H R O U G H G R AT I N G R E I N F O R C E S W I T H T H E PA RT I A L LY R E F R A C T E D R A D I AT I O N . T H E WAV E L E N G T H O F R A D I AT I O N P R O D U C E B Y T R A N S M I S S I O N G R AT I N G C A N B E E XP R E S S E D B Y F O L L O W I N G E Q U AT I O N S .
  • 38. A D VA N TA G E S : -  G R AT I N G G I V E S H I G H E R A N D L I N E A R D I S P E R S I O N C O M PA R E D TO P R I S M M O N O C H R O M ATO R  C A N B E U S E D O V E R W I D E WAV E L E N G T H R A N G E ,  G R AT I N G C A N B E C O N S T R U C T E D W I T H M AT E R I A L L I K E A L U M I N I U M W H I C H I S R E S I S TA N T TO AT M O S P H E R I C M O I S T U R E .  P R O V I D E L I G H T O F N A R R O W WAV E L E N G T H .  N O L O S S O F E N E R G Y D U E TO A B S O R P T I O N .
  • 39.  T H E C E L L S O R C U V E T T E S A R E U S E D F O R H A N D L I N G L I Q U I D S A M P L E S .  T H E C E L L M AY E I T H E R B E R E C TA N G U L A R O R C Y L I N D R I C A L I N N AT U R E .  F O R S T U D Y I N U V R E G I O N ; T H E C E L L S A R E P R E PA R E D F R O M Q U A R T Z O R F U S E D S I L I C A W H E R E A S F U S E D G L A S S I S U S E D F O R V I S I B L E R E G I O N .  T H E S U R FA C E O F A B S O R P T I O N C E L L S M U S T B E K E P T C L E A N N O F I N G E R P R I N T S H O U L D B E P R E S E N T O N C E L L S  C L E A N I N G I S C A R R I E D O U T WA S H I N G W I T H D I S T I L L E D WAT E R O R W I T H D I L U T E A L C O H O L , A C E T O N E  T H E C E L L O R C U V E T T E T H AT C O N TA I N S A M P L E S F O R A N A LY S I S S H O U L D F U L F I L C O N D I T I O N S A ) T H E Y M U S T B E U N I F O R M I N C O N S T R U C T I O N T H E T H I C K N E S S M U S T B E C O N S TA N T A N D S U R FA C E FA C I N G T H E I N C I D E N T L I G H T M U S T B E O P T I C A L LY F L AT . B ) T H E M AT E R I A L O F C O N S T R U C T I O N S H O U L D B E I N E R T T O S O LV E N T S C ) T H E Y M U S T T R A N S M I T L I G H T O F T H E WAV E L E N G T H U S E D SAMPLE HOLDER OR CUVETTES
  • 40.  D E V I C E W H I C H C O N V E RT S L I G H T E N E R G Y I N TO E L E C T R I CA L S I G N A L S T H E Y A R E D I S P L AY E D O N R E A D O U T D E V I C E S .  T H E T R A N S M I T T E D R A D I AT I O N FA L L S O N T H E D E T E C TO R W H I C H D E T E R M I N E S T H E I N T E N S I T Y O F R A D I AT I O N A B S O R B E D B Y S A M P L E  T H E F O L L O W I N G T Y P E O F D E T E C TO R S A R E E M P L O Y E D I N I N S T R U M E N TAT I O N O F A B S O R P T I O N S P E C T R O P H O TO M E T E R 1 ) B A R R I E R L AY E R C E L L 2 ) P H O TO T U B E S 3 ) P H O TO M U LT I P L I E R T U B E DETECTORS
  • 41. R E Q U I R E M E N T S O F I D E A L C H A R A C T E R  I T S H O U L D G I V E Q U A N T I TAT I V E R E S P O N S E .  I T S H O U L D H AV E H I G H S E N S I T I V I T Y A N D N O I S E L E V E L .  I T S H O U L D H AV E A S H O RT R E S P O N S E T I M E .  I T S H O U L D P R O V I D E S I G N A L S O R Q U A N T I TAT I VE TO W I DE S P E C T R U M O F R A D I AT I O N R E C E I V E D . B A R R I E R L AY E R C E L L : - T H E D E T E CTO R H A S A F I L M M E TA L L I C L AY E R C O AT E D W I T H S I LV E R O R G O L D A N A C T A S E L E C T R O D E . I T AL S O H A S A M E TAL BA SE P LATE W H I C H A CT A S AN O T H E R E L E CT R O D E . T H E Y T W O L AY E R A RE SE PA R AT E D B Y A S E M I C O N D UC TO R L AY E R O F S E L E N I U M . W H E N L I G H T R A D I AT I O N FA L L O N S E L E N I U M L AY E R , E L E C T R O N B E C O M E M O B I L E A ND A R E TA K E N U P B Y T R AN S PA RE N T M E TAL L AY E R . T H I S C R E AT E A P O T E NT I A L D I F F E RE N CE B E T WE E N E L E C T R O D E S C A U SE T H E F L O W O F C U R RE NT .W H E N I T I S C O N N E C TE D TO G A LVA N O M E T E R A F L O W O F C U R R E NT O B SE RVE D W H I C H I S P R O P O RT I O N AL TO T H E I N T E N S I T Y A N D WAVE L E N G T H O F L I G H T FA L L I N G I T .
  • 43.  C O N S I S T O F E VA C U AT E D G L A S S T U B E W I T H A P H O TO C AT H O D E A N D A C O L L E C TO R A N O D E .  T H E S U R FA C E O F P H O TO C AT H O D E I S C O AT E D W I T H A L AY E R O F E L E M E N T L I K E C E S I U M , S I LV E R O XI D E O R M I XT U R E O F T H E M .  W H E N R A D I A N T E N E R G Y FA L L O N P H O TO S E N S I T I V E C AT H O D E E L E C T R O N A R E E M I T T E D W H I C H A R E AT T R A C T E D TO A N O D E C A U S I N G C U R R E N T TO F L O W .  M O R E S E N S I T I V E C O M PA R E D TO B A R R I E R L AY E R C E L L A N D T H E R E F O R W I D E LY U S E D . PHOTOUBES
  • 44.  T H E P R I N CI PA L E M PL O Y E D I N T H I S D E T E C TO R I S T H AT M U LT I PL I CAT I O N O F P H O TO E L E CT R O N S B Y SE C O ND A RY E M I S S I O N O F E L E C T R O N S .  N E AR T H E L A ST D YN O D E I S F I XE D A N O D E I S EL ECT R O N C O L L E C TO R  I N A VA CU UM T UB E A P R I M A RY PH O TO C AT H O D E I S A FI XE D W H I C H R E C E I V E R A D I AT I O N F R O M T H E S A M P L E .  S O M E 8 TO 1 0 D YN O D E S A R E F I XE D E A C H W I T H I N C R E AS I N G P O T E N T I A L O F 7 5 - 1 0 0 V H I G H E R T H E N P R O C E E D I N G O N E .  P H O TO M U LT I P L I E R I S E XT R E M E LY SE N S I T I V I T Y TO L I G H T A N D I S B E ST SU I T E D WH E R E W E AK E R O R L O W R A D I AT I O N I S R E C E I V E D . PHOTOMULTIPLIER TUBE
  • 45.  T H E S I G N A L F R O M D E T E CTO R A F T E R A M P L I F I C AT I O N A RE F I N A L LY RE CE I VE D B Y R E C O R D I N G S Y S T E M O R R E D O U T D E V I C E .  T H E R E C O R D I N G I S D O N E B Y R E C O R D E R P E N . READ –OUT DEVICE
  • 46.  L I G H T F R O M T H E S O U R C E I S CA RR I E D T H R O U G H L E N S A N D / O R T H R O U G H A P E RT U R E TO PA S S T H R O U G H A S U I TA B L E F I LT E R .  T H E T Y P E O F F I LTE R TO B E U S E D I S G O V E R NE D B Y T H E C O L O U R O F S O L U T I O N  T H E S A M PL E S O L UT I O N TO B E A N ALY SE D I N P L AC E I N C U V E T T E S .  A F T E R PA S SI N G T H R O U G H T H E S O L UT I O N T H E L I G H T S T R I K E T H E S U R FAC E O F D E T E CTO R ( B A R R I E R L AY E R CE L L O R P H O TO T U B E ) A N D P R O D U C E E L E C T R I CA L C U R R E N T .  T H E O U T P U T O F C U R RE NT I S A M E AS U RE D B Y T HE D E FL E CT I O N O F N E E DL E O F L I G H T S P O T G A LVA N O M E T E R O R M I C R O M E T E R . SINGLE BEAM UV- SPECTROPHOTOMETER
  • 47.  T H I S M E T E R I S C A L I B R AT E D I N T E R M O F T R A N S M I T TA NC E A S W E L L A S O P T I C A L D E N S I T Y .  T H E RE A D I N G O F S O L U T I O N B O T H S L AN D E R E D A N D UN K N O W N A R E R E C O R D E D I N O P T I C AL DE N S I T Y UN I T A FT E R A DJ U ST I NG I N S T R U M E N T A R E A G E N T B L A N K .
  • 48. D O U B L E B E A M I N ST RUM E NT I S T H E O N E I N W H I C H T W O B E A M S A R E F O R M E D I N T H E S PA C E B Y A U S H A P E D M I R R O R C A L L E D A S B E A M S P L I T T E R O R B E A M C H O P P E R . C H O P P E R I S A DE V I C E C O N S I S T I NG O F A C I R C UL A R D I S C . O N E T H I R D O F T H E D I SC I S O PA Q U E A N D O N E T H I R D I S T R AN S PA RE N T , RE M A I N I N G I S O N E T H I R D I S M I R R O R E D . I T S P L I T T H E M O N O C H R O M AT I C B E AM O F L I G H T I NTO T W O B E A M S O F E Q U A L I N T E N S I T I E S . DOUBLE BEAM UV- SPECTROPHOTOMETER
  • 49. S I N G L E B E A M  S I M P L E I N C O N S T R U C T I O N , E A S Y TO U S E E C O N O M I C A L D O U B L E B E A M  I T FA C I L I TAT E S R A P I D S C A N N I N G O V E R W I D E R E G I O N .  F L U C T U AT I O N D U E TO R A D I AT I O N S O U R C E A R E M I N I M I ZE D .  I T D O E S N O T R E Q U I R E A D J U S T M E N T O F T H E T R A N S M I T TA N CE AT 0 % A N D 1 0 0 % AT E A C H WAV E L E N G T H .  I T G I V E S R AT I O O F I N T E N S I T I E S O F S A M P L E & R E F E R E N CE B E A M S I M U LTA N E O U S LY . ADVANTAGE OF SINGLE BEAM & DOUBLE BEAM SPECTROPHOTOMETER
  • 50. S I N G L E B E A M  A N Y F L U C T U AT I O N I N T H E I N T E N S I T Y O F R A D I AT I O N S O U R C E A F F E C T T H E A B S O R B A N C E .  C O N T I N U O U S S P E C T R U M I S N O T O B TA I N E D . D O U B L E B E A M  C O N S T R U C T I O N I S C O M P L I C AT E D .  I N S T R U M E N T I S E XP E N S I V E . DISADVANTAGE OF SINGLE & DOUBLE BEAM SPECTROPHOTOMETER
  • 52. Q U A L I TAT I V E Q U A N T I TAT I VE A N ALY S I S : - I T I S U S E D F O R C H A R A C T E R I ZI N G A R O M AT I C CO M P O U N D S A N D C O N J U G AT E D O L E F I N S . I T C A N B E US E D TO F I ND O U T M O L A R C O N CE NT R AT I O N O F T H E S O L U T E U N D E R S T U D Y . D E T E C T I O N O F I M P U R I T Y : - I T I S O N E O F T H E I M P O RTA N T M E T H O D TO D E T E CT I M P U R I T I E S I N O R G A N I C S O LV E NT S A D D I T I O NA L P E A K C A N B E C O M PA R E D W I T H T H AT O F S TA N D A R D R AW M AT E R I A L . S T RU C T U RE E L U C I D AT I O N O F O R G A N I C C O M P O U N D : - T H E P R E SE N CE O R A B S E N CE O F U N S AT U RAT I O N T H E P R E S E N C E O F H E T E R O ATO M L I K E S , N , O O R H A L O G E N C A N B E D E T E R M I N E D . S T R U C T U R E A N A LY S I S O F O R G A N I C C O M P O U N D : - E F F E CT O F C O N J U G AT I O N : - E XT E N D E D C O N J U G AT I O N S H I F T S T H E TO L O N G E R ( B AT H O C H R O M AT I C SH I F T ) A N D R E DU CT I O N O F T H E C O M P O U N D O R S AT U R AT I O N O F D O U B L E B O N D S L E A D S TO T H E APPLICATION FOR UV- VISIBLE SPECTROSCOPY
  • 53. O P P O S I T E E F F E C T E X: - H Y P S O C H R O M I C S H I F T E F F E C T O F G E O M E T R I C I S O M E R I S M : - T R A N S I S O M E R A B S O R B AT L O N G E R WAV E L E N G T H T H E N C I S I S O M E R S . C I S TO T R A N S C O N V E R S I O N B AT H O C H R O M I C S H I F T A N D H Y P E R - C H R O M I C E F F E C T . A L K Y L S U B S T I T U T I O N : - S H I F T T H E L A M D A M A X TO L O N G E R WAV E L E N G T H ( B AT H O C H R O M I C S H I F T ) N U M B E R O F R I N G S : - T H E A D D I T I O N O F R I N G C A U S E S B AT H O C H R O M I C S H I F T.
  • 54.  T H E C H O I C E O F T H E S O LV E NT TO B E U SE D I N ULT R AVI O L E T S P E C T R O S C O P Y I S Q U I T E I M P O RTA N T .  A S E C O N D C R I T E R I O N F O R A G O O D S O LV E NT I S I T S E FF E CT O N T H E F I N E S T R U C T U RE O F A N A B S O R P T I O N B A N D .  I N A P O L A R S O LV E NT T H E H Y D R O G E N B O N D I N G F R O M A S O L U T E - S O LVE NT S C O M P L E X A ND T H E F I N E S T RU C TU RE M AY D I S A P P E A R .  A N O N – P O L A R S O LV E N T D O E S N O T H Y D R O G E N B O N D W I T H T H E S O L U T E A N D T H E S P E C T R U M O F T H E S O L U T E CL O S E LY A P P R O XI M AT E S T H E S PE CT R U M T H AT W O U L D B E P R O D U CE D I N T H E G A S E O U S S TAT E , I N W H I C H F I N E S T R U CT UR E I S O F T E N O B S E RV E D .  T H E F I R ST C R I T E R I O N F O R A G O O D S O LV E NT I S T H AT I T S H O U L D N O T AB S O R B U LT RAV I O L E T R A DI AT I O N I N T H E S A M E R E G I O N A S T H E S UB STA NC E W H O S E S P E CT R U M I S B E I N G D E T E R M I N E D . CHOICE OF SOLVENT
  • 55.  U V- V I S S P E C ( E L E C T R O N I C S P E C T R O P H O T O M E T E R )  Q U A L I T A T I V E A N D Q U A N T I T A T I V E A N A L Y S I S  D E T E C T I O N O F I M P U R I T Y F R O M O R G A N I C M I X T U R E  E L U C I D A T I O N O F S T R U C T U R E  F O R E N S I C T O X I C O L O G Y  M O L E C U L A R W E I G H T D E T E R M I N A T I O N  F O R Q U A L I T Y D E T E R M I N A T I O N  D E T E R M I N A T I O N O F M E T A L C O N T A M I N A N T S .  Q U A L I T A T I V E A N A L Y S I S  C H E M I C A L K I N E T I C APPLICATION