INSTITUT TEKNOLOGI BANDUNG - 2011

Spectrofotometry UV-
Visibel
FA-3112
[07-09: 9012]

Elin Julianti, Ph.D

School of Pharmacy
 SEKOLAH FARMASI
INSTITUT TEKNOLOGI BANDUNG

Fundamental of Spectrophotometry

Properties of Light
• Light waves consist of perpendicular, oscillating electric and magnetic
fields.

• Wavelength () is the crest-to-crest distance between waves.

• Frequency () is the number of complete oscillations that the wave makes
each second. The unit of frequency is 1/second. One oscillation per second
is called one hertz (Hz).
Relation between frequency and wavelength:

c = .  where c is the speed of light (2.998 x 108 m/s in vacuum)

With regard to energy, it is more convenient to think of light as particles

called photons. Each photon carries the energy, E, which is given by

Relation between energy and frequency

E = . h
where h is Planck’s constant ( 6.626 x 1034 J s).

~ energy is proportional to frequency.

 is called wavenumber

Energy is inversely proportional to wavelength and directly proportional to

wavenumber. Red light, with a longer wavelength than blue light, is
less energetic than blue light. The most common unit of wavenumber in the
literature is cm1, read “reciprocal centimeters” or “wavenumbers

.
Spectrum electromagnetic
UV region : 200nm- 400nm.

Visible region: 400nm-800nm.

 Absorption of Light
• When a molecule absorbs a • The lowest energy state of a
photon, the energy of the molecule is called the
molecule increases  the ground state.
molecule is promoted to an
excited state

• If a molecule emits a
photon, the energy of the
molecule is lowered
• When light is absorbed by a sample, the
irradiance of the beam of light is decreased.
• Irradiance, P, is the energy per second per
unit area of the light beam
• Irradiance (P) = intensity (I)
 Schematic diagram of a single-beam
spectrophotometric experiment

• Light is passed through a monochromator (a prism, a grating, or even a

filter) to select one wavelength.
• Light with a very narrow range of wavelength is said to be monochromatic
(“one color.”)
• The monochromatic light, with irradiance P0, strikes a sample of length b.
• The irradiance of the beam emerging from the other side of the sample is
P. Some of the light may be absorbed by the sample, so P ≤ P0
• Transmittance, T, is defined as the fraction of the original light that passes
through the sample.

Transmittance:

• Therefore, T has the range 0 to 1. The percent transmittance is simply

100T and ranges between 0 and 100%.

• Absorbance is defined as

• Relation between transmittance and absorbance:

• Absorbance is sometimes called optical density

• Absorbance is so important because it is directly proportional to the
concentration, c,

• Beer-Lambert law (Beer’s Law) :

A = Absorbance (dimensionless)
c = concentration (M)
b = pathlength (cm)
 = molar absorptivity (M1cm1)

Molar absorptivity is the characteristic of a substance that tells how much

light is absorbed at a particular wavelength. The greater the molar
absorptivity, the greater the absorbance. The part of a molecule responsible
for light absorption is called a chromophore
• The substance
absorbs certain wavelengths of the white light, and our eyes detect the
wavelengths that are not absorbed
• Beer’s law states that absorbance is proportional to the concentration of
the absorbing species.

• It applies to monochromatic radiation, and it works very well for dilute

solutions ( ≤ 0.01M) of most substances.

• In concentrated solutions, solute molecules influence one another as a

result of their proximity.

• When solute molecules get close to one another, their properties

(including molar absorptivity) change somewhat.
 Measuring Absorbance

• Light from a continuous source is passed through a monochromator,

which selects a narrow band of wavelengths from the incident beam. This
“monochromatic” light travels through a sample of pathlength b, and the
irradiance of the emergent light is measured.
• For visible and ultraviolet spectroscopy, a liquid sample is usually
contained in a cell called a cuvet.
• Glass is suitable for visible, but not ultraviolet spectroscopy, because it
absorbs ultraviolet radiation
• The most common cuvets have a 1.000-cm pathlength and are sold in
matched sets for sample and reference.
Spectrum UV
 Single-beam instrument
• Has only one beam of light.

• The irradiance of light passing through a reference cuvet containing pure

solvent (or a reagent blank) is defined as P0. This cuvet is then removed
and replaced by an identical one containing sample.

• The irradiance of light striking the detector after passing through the
sample is the quantity P.

• Knowing both P and P0 allows T or A to be determined.

 Double-beam instrument
• Splits the light to pass alternately between sample and reference cuvets
• For spectrophotometric analysis, normally choose the wavelength of
maximum absorbance for two reasons:
(1) The sensitivity of the analysis is greatest at maximum
absorbance; that is, we get the maximum response for a given
concentration of analyte.
(2) The curve is relatively flat at the maximum, so there is little
variation in absorbance if the monochromator drifts a little or if
the width of the transmitted band changes slightly.

• Modern spectrophotometers are most precise (reproducible) at

intermediate levels of absorbance (A~ 0.3 to 2).

• If too little light gets through the sample (high absorbance), intensity is
hard to measure.
• If too much light gets through (low absorbance), it is hard to distinguish
transmittance of the sample from that of the reference. It is desirable to
adjust sample concentration so that absorbance falls in this intermediate
range.

• Compartments must be tightly closed to avoid stray light, which leads to

false readings
 Drill
0.10 mM KMnO4 has an absorbance maximum
of 0.26 near 525 nm in a 1.000-cm cell. Find the
molar absorptivity and the concentration of a
solution whose absorbance is 0.52 at 525 nm in
the same cell
Need =  (M1cm1)?
Know = c KMnO4 = 0.10 mM, A = 0.26, b = 1 cm
How = A = . b. c
Solve =
0.26 = . 1. 0.1. 10-3
 = 0.26/10-4
 = 2600 M1cm1

Need = c (M)?
Know = A = 0.52, b = 1 cm,  = 2600 M1cm1
How = A = . b. c
Solve =
0.52 = 2600. 1. c
c= 0.52/2600 = 0.0002 M = 0.02 mM
 Simultaneous equation method
• If sample contains two absorbing compounds (X and Y) of
which absorb at the max of the other (1 and 2), it may be
possible to determine both the compounds by the
simultaneous equation method
The absorbance of the mixture is the sum of the individual
absorbances of X and Y.

The information required :

The absorptivities molar of X at λ1 and λ2 ( X1 and X2)
The absorptivities molar of Y at λ1 and λ2 ( Y1 and Y2)
The absorbances of the diluted sample at λ1 and λ2 (A1 and A2)
A = xbcx + ybcy
(1) at λ1 A1 = x1cx + y1cy
(2) at λ2 A2 = x2cx + y2cy

Multiply the equation (1) with x2 and (2) with x1, as a result obtained the
following equation:
Cy = (A1 x2 - A2 x1 ) / (y1 x2 - y2 x1 )
Cx = (A2 y1 – A1 y2) / (y1 x2 - y2 x1 )

by solving these simultaneous equations we can determine the concentration

of X and Y in the sample
These criteria are satisfied only when:
• the λmax of the two components are
reasonably dissimilar.
• that the two components must not interact
chemically, thereby negating the initial
assumption that the total absorbance is the
sum of individual absorbance.
 Derivative Spectroscopy
• for analytical situations in which mixture contribute
interfering absorption, a method of manipulating the spectral
data is called derivative spectroscopy.

• Derivative spectrophotometry involves the conversions of a

normal spectrum to it’s first, second or higher derivative
spectrum.

• In the context of derivative spectrophotometry, the normal

absorption spectrum is referred to as the fundamental, zero
order spectrum.
• The first derivative spectrum of an absorption
band is characterized by a maximum, a
minimum & a cross-over point at the λmax of
the absorption band
• The second derivative spectrum is
characterized by a minimum corresponds to
λmax of the fundamental band (Maximum
negative curvature).
Zero (a) , 1st (b), & 2nd (c) derivative spectra
• The maximum absorption wavelength on a compound will be the
wavelength zero-crossing on the first derivative spectrogram, the
wavelength has no absorption or dA / dλ = 0.

• The zero-crossing method separates the binary mixture from the

derivative spectrum on wavelength at the first component there is no
signal. Measurements on each zero-crossing components in the mixture
are a single function of concentration from the other
Example of Zero (a) , 1st (b) & (c) derivative
spectra of binary mixture
• (a) The absorption spectra of substances A
and B
• (b) the first derivative spectrum showing the
zero crossing of the two substances
• (c) the first derivative spectrum of mixtures A
and B, at the zero crossing the signal depends
only on one analyte
 Isosbestic Points
• Often one absorbing species, X, is converted into another absorbing
species, Y, in the course of a chemical reaction.
• If the spectra of pure X and pure Y cross each other at any wavelength,
then every spectrum recorded during this chemical reaction will cross at
that same point, called an isosbestic point