In this lecture, you will

learn:
Molecular species that absorb UV/VIS

radiation
Absorption process in UV/VIS region in terms
of its electronic transitions
Important terminologies in UV/VIS
spectroscopy

Organic
compounds

Inorganic
species

MOLECULAR SPECIES THAT

ABSORB UV/VISIBLE
RADIATION

Charge transfer

Definitions:
Organic compound
Chemical compound whose molecule contain carbon.
E.g. C6H6, C3H4

Inorganic species
Chemical compound that does not contain carbon.
E.g. transition metal, lanthanide and actinide elements
Cr, Co, Ni, etc..

Charge transfer
A complex where one species is an electron donor and the

other is an electron acceptor.

E.g. iron(III) thiocyanate complex

NOTE: Transition metals - groups IIIB

through IB

UV-VIS ABSORPTION
In UV/VIS spectroscopy, the transitions which result

in the absorption of EM radiation in this region are

transitions btw electronic energy levels.

- In molecules, not only

have electronic level but
also consist of vibrational
and rotational sub-levels.
- This result in band
spectra.

Type of Transitions
3 types of electronic transitions
, and n electrons
d and f electrons
Charge transfer electrons

What is , and n
electrons?

Sigma ()electron
Electrons involved in single bonds such as those

between carbon and hydrogen in alkanes.

These bonds are called sigma () bonds.
The amount of energy required to excite
electrons in bond is more than UV photons of
wavelength. For this reason, alkanes and other
saturated compounds (compounds with only
single bonds) do not absorb UV radiation and
therefore frequently very useful as transparent
solvents for the study of other molecules. For
example, hexane, C6H14.

Pi () electron
Electrons involved in double and triple bonds

(unsaturated).
These bonds involve a pi () bond.
For example: alkenes, alkynes, conjugated
olefins and aromatic compounds.
Electrons in bonds are excited relatively
easily; these compounds commonly absorb in
the UV or visible region.

Examples of organic molecules containing bonds.

ethylbenzen
e

H3C

CH2CH3

C
C

C
C

benzen
e

propyne

H
H
C
H

H
H

C
H

1,3-butadiene

n electron
Electrons that are not involved in bonding

between atoms are called n electrons.

Organic compounds containing nitrogen,
oxygen, sulfur or halogens frequently contain
electrons that are nonbonding.
Compounds that contain n electrons absorb
UV/VIS radiation.

Examples of organic molecules with non-bonding electrons.

NH2

R
aminobenzen
e

If R = H
aldehyde
ketone

H
C

Br

Carbonyl compound

If R = CnHn

H3C
C

2-bromopropene

Absorption by Organic
Compounds
UV/Vis absorption by organic compounds

requires that the energy absorbed

corresponds to a jump from occupied orbital
unoccupied orbital.
Generally, the most probable transition is

from the highest occupied molecular orbital

(HOMO) to the lowest unoccupied molecular
orbital (LUMO).

Electronic energy levels diagram

Unoccupied levels

Occupied
levels

* transitions

Never observed in normal UV/Vis work.

The absorption maxima are < 150 nm.
The energy required to induce a

*
transition is too great (see the arrow in energy
level diagram)
This type of absorption corresponds to
breaking of C-C, C-H, C-O, C-X, .bonds

vacuum UV region

* transitions

Saturated compounds must contain atoms

with unshared electron pairs.

Compounds containing O, S, N and halogens
can absorb via this type of transition.
Absorptions are typically in the 150 -250 nm
region and are not very intense.
range: 100 3000 Lcm-1mol-1

Some examples of absorption due to n

transitions
Compound

max (nm)

max

H2O

167

1480

CH3OH

184

150

CH3Cl

173

200

CH3I

258

365

(CH3)2O

184

2520

CH3NH2

215

600

* transitions

Unsaturated compounds containing atoms

with unshared electron pairs

These result in some of the most intense
absorption in 200 700 nm region.
range: 10 100 Lcm-1mol-1

* transitions

Unsaturated compounds to provide the

orbitals.
These result in some of the most intense
absorption in 200 700 nm region.
range: 10oo 10,000 Lcm-1mol-1

Some examples of absorption due to n

transitions

* and

CHROMOPHORE
Unsaturated organic functional groups that

absorb in the UV/VIS region are known as

chromophores.

AUXOCHROME
Groups such as OH, -NH2 & halogens that

attached to the doubley bonded atoms cause the

normal chromophoric absorption to occur at
longer (red shift). These groups are called
auxochrome.

Effect of Multichromophores on
Absorption

More chromophores in the same molecule

cause bathochromic effect ( shift to longer )

and hyperchromic effect(increase in intensity)

In the conjugated chromophores * electrons

are delocalized over larger number of atoms

causing a decrease in the energy of *
transitions and an increase in due to an
increase in probability for transition.

Other Factor that Influenced

Absorption
Factors that influenced the :

i) Solvent effects (shift to shorter : blue shift)

ii) Structural details of the molecules

Important terminologies

hypsochromic shift (blue shift)

- Absorption maximum shifted to shorter

bathochromic shift (red shift)

- Absorption maximum shifted to longer

Terminology for Absorption

Shifts
Nature of Shift

Descriptive Term

To Longer Wavelength

Bathochromic

To Shorter Wavelength

Hypsochromic

To Greater Absorbance

Hyperchromic

To Lower Absorbance

Hypochromic

Absorption by Inorganic
Species
Involving d and f electrons absorption

3d & 4d electrons
- 1st and 2nd transition metal series
e.g. Cr, Co, Ni & Cu
- Absorb broad bands of VIS radiation
- Absorption involved transitions btw filled and
unfilled d-orbitals with energies that depend on
the ligands, such as Cl-, H2O, NH3 or CN- which
are bonded to the metal ions.

4f & 5f electrons
- Ions of lanthanide and actinide elements
- Their spectra consists of narrow, welldefined characteristic absorption peaks

Charge Transfer Absorption

Absorption involved transfer of electron from

the donor to an orbital that is largely

associated with the acceptor.
an electron occupying in a or orbital
(electron donor) in the ligand is transferred to
an unfilled orbital of the metal (electron
acceptor) and vice-versa.
e.g. red colour of the iron(III) thiocyanate
complex

Important components in a UV-Vis

spectrophotometer
1

Source
lamp

Sample
holder

selector

Detecto
r

UV region:
- Deuterium
lamp;

Quartz/fused
silica

Prism/monochroma
tor

Glass/quartz

Prism/monochromat
or

H2 discharge
tube

Phototube,
PM tube,
diode array

Visible region:
- Tungsten lamp

Phototube,
PM tube,
diode array

Signal
processor
& readout

UV-VIS INSTRUMENT
Single beam
Double beam

Single beam instrument

One radiation source
Filter/monochromator ( selector)
Cells
Detector
Readout device

Single beam instrument

Disadvantages:

Two separate readings has to be made on

the light. This results in some error
because the fluctuations in the intensity
of the light do occur in the line voltage,
the power source and in the light bulb
btw measurements.
Changing of wavelength is accompanied
by a change in light intensity. Thus
spectral scanning is not possible.

Double-beam instrument with beams separated in

space

Double beam instrument

Advantages:

1. Compensate for all but most short-term

fluctuations in the radiant output of the source as
well as for drift in the transducer and amplifier
2. Compensate for wide variations in source
intensity with
3. Continuous recording of transmittance or
absorbance spectra

Quantitative Analysis
The fundamental law on which absorption

methods are based on Beers law (BeerLambert law).

Measuring absorbance
You must always attempt to work at the

wavelength of maximum absorbance (max)

This is the point of maximum response, so

better sensitivity and lower detection limits.

You will also have reduced error in your
measurement.

Quantitative Analysis

Calibration curve method

Standard addition method

Calibration curve method

A general method for determining the

concentration of a substance in an unknown

sample by comparing the unknown to a set of
std sample of known concentration

Standard Calibration Curve

Concentration,
ppm

How to measure the concentration of unknown?

Practically, you have measure the absorbance of your unknown.

Once you know the absorbance value, you can just read the
corresponding concentration from the graph .

How to produce
standard
calibration curve?
Prepare a series of

standard solution
with known
concentration.
Measure the
absorbance of the
standard solutions.
Plot the graph A vs
concentration of std.
Find the best
straight line by using
least-squares
method.

Finding the Least-Squares Line

Concentrati
on
xi

Absorban
ce
yi

0.201

10

0.420

15

0.654

20

0.862

25

1.084

xi

yi

x2i

xi2

N=5
N is the number of points
used

y2i

yi2

xiyi

xiyi

The calculation of the slope and intercept is

simplified by defining three quantities Sxx, Syy and

Sxy.

Sxx =

(xi x)2 = xi2 ( xi)2

N

(1)

Syy

= (yi y)2 = yi2 ( yi)2 (2)

Sxy

= (xi x) (yi y)2 = xiyi xi

yi (3)

The slope of the line, m:

m =

Sxy
Sxx

The intercept, b:
b = y - mx

Thus, the equation for the least-squares line is

y = mx + b

Concentration
x

y = mx + b

5
10
15
20
25

From the least-squares line equation, you can

calculate the new y values by substituting the x

value.
Then plot the graph.

Most linear regression implementations have an option

to force the line through the origin, which means

forcing the intercept of the line through the point
(0,0). This might seem reasonable, since a sample
with no detectable concentration should produce no
response in a detector, but must be used with care.
HOWEVER, forcing the plot through (0,0) is not always

recommended, since most curves are run well above

the instrumental limit of detection (LOD). Randomly,
adding a point (0,0) can skew the curve because the
instruments response near the LOD is not predictable
and is rarely linear. As illustrated next page, forcing a
curve through the origin can, under some
circumstances, bias results.

Standard addition method

used to overcome matrix effect
involves adding one or more increments of a

standard solution to sample aliquots of the

same size.
each solution is diluted to a fixed volume before
measuring its absorbance

Absorbanc
e

Concentration,
ppm

How to produce standard addition

curve?

1. Add same quantity of unknown sample to a series of flasks

2. Add varying amounts of standard (made in solvent) to each

flask, e.g. 0,5,10,15mL)

3. Fill each flask to line, mix and measure

Single-point standard
addition method

Multiple additions
method

Standard addition
- if Beers law is obeyed,

A = bVstdCstd

bVxCx

Vt
= kVstdCstd

Vt
+

kVxCx

k is a constant equal to
b
Vt

Standard Addition
- Plot a graph: A vs Vstd
A = mVstd + b

where the slope m and intercept b are:

m = kCstd

b = kVxCx

Cx can be obtained from the ratio of

these two quantities: m and b
b

= kVxCx

kCstd

Cx

= bCstd
mVx

Standard Addition
For single-point standard addition
Absorbance
of diluted
sample

A1 =
bVxCx

Eq. 1

Vt
Absorbance
of diluted
sample +
std

A2 = bVxCx
+
Vt

bVsCs
Vt

Eq. 2

Standard Addition
For single-point standard addition
Dividing the 2nd equation by the first & then
rearrange it will give:
Cx

A 1 C s Vs
(A2 A1 ) Vx

Example Standard Addition (single point

addition)
Example 14-2 (page 376)
A 2.00-mL urine specimen was treated with
reagent to generate a color with phosphate,
following which the sample was diluted to 100
mL. To a second 2.00mL sample was added
exactly 5.00mL of a phosphate solution
containing 0.03 mg phosphate /mL, which was
treated in the same way as the original sample.
The absorbance of the first solution was 0.428,
while the second one was 0.538. Calculate the
concentration of phosphate in milligrams per
millimeter of the specimen.

Solution:
Cx = (0.428) (0.03 mg PO43-/mL) (5.00mL)
(0.538 0.428)(2.00mL)
= 0.292 mg PO43- / mL sample

Exercise
The concentration of an unknown chromium solution was
determined by pipetting 10.0mL of the unknown into each of
five 50.0 mL volumetric flasks. Various volumes of a standard
containing 12.2 ppm chromium were added to the flasks and
then the solutions were diluted to the mark.
Standard, mL
0.0

Absorbance
0.201

10.0

0.292

20.0

0.378

30.0

0.467

40.0

0.554

Determine the concentration of chromium (in ppm) in the unknown.