Fundamentals of UV-Vis Spectrophotometer

8/18/2019 Fundamentals of UV-Vis Spectrophotometer

1/36

Fundamentals of UV-Vis

Spectrophotometer

13th July 2009


2/36

What is Spectroscopy?

Spectroscopy is the measurement andinterpretation of electromagnetic radiationabsorbed Or emitted when the molecule, or

atom, or ion of a sample moves from oneallowed energy state to another.

Every atom, ion or molecule has a unique andcharacteristic relationship with

electromagnetic radiation. Chemical instrumentation does not createinformation; it refines the information alreadypresent in the signal from some transducer.


3/36

!lectroma"netic radiation

Light as a wave form

avelength, frequency and Speed of

light


4/36



5/36


!requency of light and its Energy

"#article #roperty of Light$

h # $lan%s& constant # '('3 ) 10 -3* Joule + sec


6/36


ave and #article #roperty of Light

h = Planck’s Constant (erg sec)

c = Speed of light (cm/sec)

λ = Wavelength (cm) ν = Frequenc (ccles/sec)

!nerg" ! = =h cλ

h ν


7/36


%he Electromagnetic Spectrum


8/36


%he Electromagnetic Spectrum

Color regionWavelength

(nm)

violet #$% & '#

blue '# & %%

cyan %% & %

green % & *

yellow * & +%

orange +% & *

red * & ,'%


9/36


Complementary colors Sometimes ,hat you actually see is uite une.pected(

/i.in" different ,aelen"ths of li"ht doesn&t "ie you the

same result as mi.in" paints or other pi"ments(

f you arran"e some colors in a circle you "et a color

,heel( 4he dia"ram sho,s one possi5le ersion of this(


10/36


Complementary colors 6olours directly opposite each other on the colour ,heel

are said to 5e complementary colours( 7lue and yello,

are complementary colours8 red and cyan are

complementary8 and so are "reen and ma"enta(


11/36


Complementary colors /i.in" to"ether t,o complementary colours of li"ht ,ill

"ie you ,hite li"ht(

4hat is :4 the same as mi.in" to"ether paint colours(

f you mi. yello, and 5lue paint you don;t "et ,hite paint(


12/36


13/36


14/36


15/36


16/36


17/36

ust li%e

the anti-5ondin" si"ma one@ at a

hi"her ener"y than the 5ondin" or5ital

- and so isn;t used to hold electrons(

7oth of the electrons in the pi 5ond

are found in the pi 5ondin" or5ital(


18/36

for non-5ondin" -- non-5ondin"

or5itals contain lone pairs of

electrons

4he non-5ondin" or5itals ,e areinterested in contain a lone pair of

electrons - a perfectly sta5le filled

or5ital(

=nti-5ondin" or5itals are normally

empty and lo,er the sta5ility of acompound if they contain electrons(


19/36


20/36


21/36


22/36

=5sorption of Simple isolated

6hromophores

Class -ransition λma. (nm)

& 01

& 0 & & 21 & S1 C = C & C C & & C 2

& 2 = 2 &

σ∗n σ∗n σ∗n σ∗n π∗π

π∗π

σ∗n

σ∗n

3$%

3$%3+%3%3,3,%3*%

#'%

Class -ransition λma. (nm)

& 20

& C10

C0

C001

C00’C021

π∗n π∗π π∗n

π∗π

π∗n π∗n

π∗n π∗n

,3

3+%+%

3$%$%%

%3%


23/36

=5sorption of 6ommon

6hromophoresChromophore Example Solvent

max

(nm)

Type of

transition

4lkene C*13#C1=C1) n&1eptane 3,, π −> π∗

4lkne C(133C C&C1# n&1eptane 3,$

3+*))(

π −> π∗

−−−

−−−

0

55

C1#CC1# 0

55

C1#C1

n&1eptane

n&1eptane

3$*

)$%

3$%

)+#

n −> σ∗

n −> π∗

n −> σ∗

n−> π∗

Car6o.l 0

55

C1#C01

!thanol )%' n −> π∗

≡7


24/36

=5sorption of 6ommon

6hromophores

Chromophore Example Solvent max (nm) Type of

transition

4mido 0

55

C1#C21)

Water )3' n −> π∗

48o C1# 2=2C1# !thanol ##+ n −> π∗

2itro C1# 20) 9sooctane )$% n −> π∗

2itroso C'1+ 20 !thl ether #%%**(

&&&&

n −> π∗

2itrate C)1(020) :io.ane ),% n −> π∗


25/36

=5sorption of /ultichromophores

Compound Type max (nm)

C1#(C1)C1=C1 0lefin 3$'

C1=C1(C1)C1=C1 :iolefin (uncon;ugated) 3$

1C&C1C1&C1 :iolefin (con;ugated) 3,

1C=C1C1=C1C1=C1 -riolefin (con;ugated) %


26/36

=5sorption of /ultichromophores

Compound Type max (nm)

0

55

C1#(C1)#CC1#


27/36

When Bi"ht Stri%es a /olecule

Low energy causes C 4ibrational transitions

C 5otational transitions

+igh energy causes C Electronic transitions


28/36

!lectronic 4ransition

>round state !%

!.cited state !3

∆! = !3 & !% = h ν

!nerg level

Radiation

e!nerg difference

∆!


29/36

=5sorption of

Dadiation!nerg

ibrational transition

Rotationaltransition R

v!

v"

v#

$%&"

!#

Electronictransition

E!

E#

v#

v!

v"

E ' h in ?hich h is Planck’s constant (*7** @ 3%' Aoulesand ν is the frequenc of the radiation


30/36

What causes =5sorption in

UV + VS De"ion?

Some sin"le 5onds

/any dou5le 5onds 6onu"ated dou5le 5onds

Some metal and inor"anic

compounds

: S halo"en


31/36

6ali5ration and 7eer&s Ba,

Cuvette containing analte

9ncident light

9%

-ransmitted light

93

0ptical path length

From light source-odetector

-ransmittance - =9%

93


32/36

Bam5ert C 7eer&s

Ba,46sor6ance 4 = log = ε C l

3

-

?here- B -ransmittance

ε B olar e.tinction coefficient

l B Path lengthC B 4nalte concentration


33/36

6=B7D=4:

ED=$<

4nalte concentration (ppm)

4 6 s o r 6 a

n c e

Slope m = ε l

46s = m@Concentration D


34/36

Deal Bimitations of 7eers

Ba, ncrease in analyte concentration 6hemical deiation due to physico-chemical

interaction

Stray li"ht

Fluorescence 5y the a5sor5in" species 6han"e in refractie inde. of analyte

on-parallel li"ht 5eam

nstrumental limitation due to noise


35/36

!ffect of analyte concentration

=pplica5le to diluted solution eiation from linearity occurs at hi"h concentration

>G 0(01 /@

ilute the sample to a lo,er concentration

C 4he o5tained a5sor5ance alue is multiply 5y thedilution factor

1igh concentrationmore molecules are packed in one volume


36/36

!ffect of chemical interaction

eiation from linearity may caused 5y

C issociation of analytes

C =ssociation of analytes

C 6hemical reactions ,ith solent to produce a

product hain" different a5sorption spectrum from

the analyte

Documents

Fundamentals of UV-Vis Spectrophotometer