An instrument that measures the amount of light that passes through (is transmitted /observed/reflected through) a sample.

1. SPECTROPHOTOMETER
Working principle
and
applications

2.  Spectrophotometer
› An instrument that measures the
amount of light that passes
through (is transmitted
/observed/reflected through) a
sample.

3. ›Uses a type of light to detect
molecules in a solution or
solids
›Light is a type of energy, and
the energy is reported as
wavelengths, in nanometers
(nm).

4.  Ultraviolet (UV)
Spectrophotometers.
Uses ultraviolet light of wave lengths
from 200 nm to 350 nm.
 Visible (VIS) Light Spectrum
Spectrophotometers.
Uses visible light (white light) of wave
lengths from 350 nm to 700 nm.

5.  Shines a beam of light on a sample.
 The molecules in the sample interact
with the light waves in of 3 ways:
› Absorb the energy
› Reflect the energy
› Transmit the energy
between and through the atoms and
molecules of the sample.

6. Blue molecules absorb the other
colors of visible light.
Blue molecules are blue because
they reflect blue light.

7. Consider blue molecules, all the
wavelengths of light are absorbed,
except for the blue ones.
The blue wavelengths are transmitted
or reflected off the molecules. If these
blue wavelengths hit a detector (such
as in the spectrophotometer or the
nerve cells in your eye), they appear
blue.

8. Molecules are whatever color of
light that they do not absorb.
Green molecules appear green
because they absorb most
wavelengths of visible light,
except the green wavelengths.

9. The spectrophotometer measures
the amount of light transmitted
through the sample
(Transmittance).
By using an equation (Beers law),
it converts the transmittance data
to an absorbance value.

10. The concentration of an unknown
sample can be determined by
comparing the absorbance data to
standards of known concentration.
The data generated with the set of
known standards is called a
standard curve.

12. Regions of Electromagnetic Spectrum-the “colour” of light

13. cuvette
source
slit
detector
P0
P

14. Fundamentals of Spectrophotometry
Absorption of Light
Beer’s Law
The relative amount of a certain wavelength of light
absorbed (A) that passes through a sample is dependent
on:
- distance the light must pass through the sample
(cell path length - l)
- amount of absorbing chemicals in the sample
(analyte concentration – c)
- ability of the sample to absorb light (molar
absorptivity - ε)
Absorbance is directly proportional to concentration

15. Fundamentals of Spectrophotometry
Absorption of Light
Beer’s Law
 The relative amount of light making it through the sample
(P/Po) is known as the transmittance (T)
oP
P
T =






×=
oP
P
T% 100Percent transmittance
‘T’ has a range of 0 to 1, %T has a range of 0 to 100%

16. Fundamentals of Spectrophotometry
Absorption of Light
Beer’s Law
 Absorbance is useful since it is directly related to the
analyte concentration, cell pathlength and molar
absorptivity.
 This relationship is known as Beer’s Law
bcA ε=
where: A = absorbance (no units)
ε = molar absorptivity (L/mole-cm)
b = cell pathlength (cm)
c = concentration of analyte (mol/L)Beer’s Law allows
compounds to be
quantified by their ability
to absorb light, Relates
directly to concentration
(c)

17. Fundamentals of Spectrophotometry
Spectrophotometer
Basic Design
 An instrument used to make absorbance or transmittance measurements is
known as a spectrophotometer

18. Working principle
Measures the reflectance of both sample
and reference at each wave length covering all
the visual spectral region from 380 to 720 nm at 10
or 20nm interval.
The light from the source illuminates the
sample and reference material and the reflected
light is passed through monochromator and
detected by photo-detector

19. Important Components of the
instrument
Light source
The light source illuminates the specimen, source used are low
voltage tungston lamp, halogen lamp or Xenon arc.
Monochromator
Which isolates the radiation of desired wave length from the
radiations of incidented or reflected from the object. Uses grating
or prism as light dispersing element.
Photo detectors
Are used to convert the light signal in to electrical signal to transmit
information's to signal processor

20. Fundamentals of Spectrophotometry
Chemical Analysis
Calibration
 To measure the absorbance of a sample, it is necessary to
measure Po and P ratio
- Po – the amount of light passing through the system with
no sample present
- P – the intensity of light when the sample is present
 Po is measured with a blank cuvet
- Cuvet contains all components in the sample solution
except the analyte of interest
 P is measured by placing the sample in the cuvet.
 To accurately measure an unknown concentration, obtain a
calibration curve using a range of known concentrations for
the analyte

21. Measure the spectral reflectance value
Measue the CIE Tristimulus value for desired illuminant
and observer
Used to calculate the chromacity co-ordinates
Calculating CIE L*,a*,b* and L*,U*,v*co-ordinates
To perform colour difference calculations using various
formulae.
Calculating metameric index for different illuminants
Whiteness and yellowness index calculations
Shade sorting
Strength of dye calculation

22. Residual festicide analysis
Residual chlorine analysis
Colour fastness analysis
Dye exhaustion calculation
Other fields
Chemical analysis
Food safety analysis
Blood analysis
DNA/RNA conc. analysis
Soil analysis