Fundamentals of UV-Vis

Spectrophotometer
13th July 2009

What is Spectroscopy?
Spectroscopy is the measurement and
interpretation of electromagnetic radiation
absorbed Or emitted when the molecule, or
atom, or ion of a sample moves from one
allowed energy state to another.
Every atom, ion or molecule has a unique and
characteristic relationship with
electromagnetic radiation.
Chemical instrumentation does not create
information; it refines the information already
present in the signal from some transducer.

Electromagnetic radiation
Light as a wave form

Wavelength, frequency and Speed of

light

Electromagnetic radiation

Electromagnetic radiation
Frequency of light and its Energy
(Particle Property of Light)

h = Planks constant = 6.63 X 10 -34 Joule / sec

Electromagnetic radiation
Wave and Particle Property of Light
Energy, E = h c

=h

h = Plancks Constant (erg sec)

c = Speed of light (cm/sec)
= Wavelength (cm)
= Frequency (cycles/sec)

Electromagnetic radiation
The Electromagnetic Spectrum

Electromagnetic radiation
The Electromagnetic Spectrum
Color region

Wavelength
(nm)

violet

380 - 435

blue

435 - 500

cyan

500 - 520

green

520 - 565

yellow

565 - 590

orange

590 - 625

red

625 - 740

Electromagnetic radiation
Complementary colors
Sometimes what you actually see is quite unexpected.
Mixing different wavelengths of light doesn't give you the
same result as mixing paints or other pigments.
If you arrange some colors in a circle, you get a "color
wheel". The diagram shows one possible version of this.

Electromagnetic radiation
Complementary colors
Colours directly opposite each other on the colour wheel
are said to be complementary colours. Blue and yellow
are complementary colours; red and cyan are
complementary; and so are green and magenta.

Electromagnetic radiation
Complementary colors
Mixing together two complementary colours of light will
give you white light.
That is NOT the same as mixing together paint colours.
If you mix yellow and blue paint you don't get white paint.

How and Why absorption?

Anti bonding orbital:
Bonding and anti-bonding orbital in simple Hydrogen
molecule
Two hydrogen atoms -- each has
one electron in the 1s orbital.
These come together to make a
new orbital surrounding both of
the hydrogen nuclei.
The molecule holds together
because both nuclei are strongly
attracted to this same pair of
electrons.

This most simple of bonds is

called a sigma bond - a sigma
bond is one where the electron
pair is most likely to be found on
the line between the two nuclei.

How and Why absorption?

Anti bonding orbital:
Bonding and anti-bonding orbital in simple Hydrogen
molecule
However . . .
Molecular orbital theory demands that if
you start with two atomic orbitals, you
must end up with two molecular
orbitals - and we seem to be only
producing one.
A second molecular orbital is formed,
but in most cases (including the
hydrogen molecule) it is left empty of
electrons. It is described as an antibonding orbital.

How and Why absorption?

Anti bonding orbital:
An anti-bonding orbital is always shown by the use of a star
after its symbol.
When a bonding orbital forms, it is at a lower energy than the
original atoms. Energy is released when the bonding orbital
is formed, and the hydrogen molecule is more energetically
stable than the original atoms.
However, an anti-bonding orbital is less energetically stable
than the original atoms.

How and Why absorption?

Anti bonding orbital:
A bonding orbital is stable because of the attractions
between the nuclei and the electrons. In an anti-bonding
orbital there are no equivalent attractions - instead you get
repulsions. There is very little chance of finding the electrons
between the two nuclei
So in the hydrogen case, both of the electrons go into the
bonding orbital, because that produces the greatest stability more stable than having separate atoms, and a lot more
stable than having the electrons in the anti-bonding orbital.

How and Why absorption?

Why doesn't helium form an He2 molecule?
This time we have a total of 4
electrons in the original atomic
orbitals.
Two atomic orbitals have to form two
molecular orbitals.
That means that this time, we would
have to use both the bonding and
anti-bonding molecular orbitals to
accommodate them.

But any gain in energetic stability due to the

formation of the bonding orbitals would be
countered by the loss of energetic stability
because of the anti-bonding ones. There is
no energetic advantage in He2 forming - and
so it doesn't.

How and Why absorption?

Anti-bonding orbitals in double bonds
The pi bond shown in red is, of
course, a normal bonding orbital. It
was formed by sideways overlap
between a half-filled p-orbital on each
of the two carbon atoms. The two red
shapes shown in the diagram are part
of the same pi bonding orbital.
But if you overlap two atomic orbitals,
you must get two molecular orbitals
according to molecular orbital theory.
The second one is an anti-bonding pi
orbital

The anti-bonding pi orbital is (just like

the anti-bonding sigma one) at a
higher energy than the bonding orbital
- and so isn't used to hold electrons.
Both of the electrons in the pi bond
are found in the pi bonding orbital.

How and Why absorption?

Summarizing the relative energies of various kinds of orbitals
"n" (for non-bonding -- non-bonding
orbitals contain lone pairs of
electrons
The non-bonding orbitals we are
interested in contain a lone pair of
electrons - a perfectly stable, filled
orbital.
Anti-bonding orbitals are normally
empty and lower the stability of a
compound if they contain electrons.

How and Why absorption?

When light passes through a compound,
some of the energy in the light kicks an
electron from one of the bonding or nonbonding orbitals into one of the anti-bonding
ones.

How and Why absorption?

In each possible case, an electron is
excited from a full orbital into an empty
anti-bonding orbital. Each jump takes
energy from the light, and a big jump
obviously needs more energy than a
small one.
The energy gaps between these levels
determine the frequency (or wavelength)
of the light absorbed, and those gaps will
be different in different compounds

How and Why absorption?

Each wavelength of light has a particular energy
associated with it. If that particular amount of
energy is just right for making one of these
energy jumps, then that wavelength will be
absorbed - its energy will have been used in
promoting an electron.
Groups in molecule which absorb light are
known as Chromophores

Absorption of Simple isolated

Chromophores
Class
R - OH
R-O-R
R - NH2
R - SH
R2C = CR2
R-C C-R
R-C N
R-N=N-R

Transition
n
n
n
n

n
n

max (nm)
180
180
190
210
175
170
160
340

Class

Transition

max (nm)

R - NO2
R - CHO

n

n

271
190
290

R2CO

n
n
n
n

180
280
205
205
210

RCOOH
RCOOR
RCONH2

Absorption of Common
Chromophores
Chromophore

Example

Solvent

max
(nm)

Type of
transition

Alkene

C6H13CH=CH 2

n-Heptane

177

Alkyne

C5H 11C C-CH 3

n-Heptane

O
||
CH 3CCH3
O
||
CH3CH
O
||
CH 3COH

n-Heptane

178
196
225
186
280

n
n

n-Heptane

180
293

n
n

Ethanol

204

Carboxyl

Absorption of Common
Chromophores
Chromophore

Example

Solvent

max (nm)

Type of
transition

Amido

O
||
CH3CNH2

Water

214

Azo

CH3N=NCH3

Ethanol

339

Nitro

CH3NO2

Isooctane

280

Nitroso

C4H9NO

Ethyl ether

300
665

---n

Nitrate

C2H5ONO2

Dioxane

270

Absorption of Multichromophores
Compound

Type

max (nm)

CH3(CH2)2CH=CH2

Olefin

184

CH2=CH(CH2)2CH=CH2

Diolefin (unconjugated)

185

H2C-CHCH-CH2

Diolefin (conjugated)

217

H2C=CHCH=CHCH=CH2

Triolefin (conjugated)

250

Absorption of Multichromophores
Compound

Type

max (nm)

O
||
CH3(CH2)3CCH3

Ketone

278

O
||
CH2=CH(CH2)2CCH3

Unsaturated ketone
(unconjugated)

324

O
||
CH2-CHCCH3

- Unsaturated
ketone (conjugated)

219

When Light Strikes a Molecule

Low energy causes
Vibrational transitions
Rotational transitions

High energy causes

Electronic transitions

Electronic Transition
Energy level

Excited state E1
Energy difference
E

E = E1 - E0 = h

Radiation

Ground state E0

Absorption of
Radiationv

Energy

v1
v0
Rotational
transition R

5
4
3
2
1
0

v2

E1
Electronic
transition

v1

Vibrational transition

v0

E0

E = h in which h is Plancks constant (6.626 * 10 -34 Joules

and is the frequency of the radiation

What causes Absorption in

UV / VIS Region?

Some single bonds

Many double bonds
Conjugated double bonds
Some metal and inorganic
compounds
N, O, S, halogen

Calibration and Beers Law

Cuvette containing analyte

Transmitted light
To
I1
detector

Incident light
I0
From light source

Optical path length

I0
Transmittance T =
I1

Lambert Beers
Law

1
Absorbance A = log
T

= C l

where
T
: Transmittance

: Molar extinction coefficient

l
: Path length
C
: Analyte concentration

Absorbance

CALIBRATION
AbsGRAPH
= m*Concentration + K

Slope m = l

Analyte concentration (ppm)

Real Limitations of Beers

Law
Increase in analyte concentration

Chemical deviation due to physico-chemical

interaction
Stray light
Fluorescence by the absorbing species
Change in refractive index of analyte
Non-parallel light beam
Instrumental limitation due to noise

Effect of analyte concentration

Applicable to diluted solution
Deviation from linearity occurs at high concentration
(> 0.01 M)
Dilute the sample to a lower concentration
The obtained absorbance value is multiply by the
dilution factor
High concentration
more molecules are
packed in one volume

Effect of chemical interaction

Deviation from linearity may caused by
Dissociation of analytes
Association of analytes
Chemical reactions with solvent to produce a
product having different absorption spectrum from
the analyte