Spectrophotometry involves using a spectrophotometer to measure how much light is absorbed or transmitted by a sample at different wavelengths. It is commonly used to determine the concentration of known substances in solution. A spectrophotometer works by shining light through a sample and measuring the intensity of the transmitted light, then using Beer's Law to calculate absorbance from transmittance. Absorbance measurements at different concentrations can be used to generate a standard curve to determine the concentration of unknown samples. Spectrophotometers are versatile tools that are widely used in laboratory analysis across various industries.

1. Spectrophotometry at
a Glance
Presented by:
Nasir Nazeer

2. Introduction

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Spectrophotometry is the quantitative measurement of the reflection or
transmission properties of a material as a function of wavelength. It is more
specific than the general term electromagnetic spectroscopy in that
spectrophotometry deals with visible light, near-ultraviolet, and nearinfrared, but does not cover time-resolved spectroscopic techniques.
Spectrophotometry involves the use of a spectrophotometer. A
spectrophotometer is a photometer that can measure intensity as a function
of the light source wavelength. Important features of spectrophotometers
are spectral bandwidth and linear range of absorption or reflectance
measurement.
The spectrophotometer has well been called the workhorse of the modern
laboratory. In particular, ultraviolet and visible spectrophotometry is the
method of choice in most laboratories concerned with the identification and
measurement of wide range of products and processes.
Modern spectrophotometers are quick, accurate and reliable and make only
small demands on the time and skills of the operator.

3. The Spectrophotometer

4. Spectrophotometric Analysis

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Spectrophotometric techniques are used to
measure the concentration of solutes in solution by
measuring the amount of light that is absorbed by
the solution in a cuvette placed in the
spectrophotometer.
The spectrophotometer can measure the amount
of light or electromagnetic radiation (of certain
frequency) transmitted or absorbed by the solution.
If there is too much or too little analyte,
spectrophotometer cannot read the absorbance
accurately.

5. Properties of Light
Electromagnetic radiation moves in waves

6. Regions of Electromagnetic Spectrumthe “colour” of light

7. Colors & Wavelengths
COLOR
WAVELENGTH (λ in nm)
< 380
Violet
380 – 435
Blue
436 – 480
Greenish-blue
481 – 490
Bluish-green
491 – 500
Green
501 – 560
Yellowish-green
561 – 580
Yellow
581 – 595
Orange
i si V
Ultraviolet
596 – 650
Red
651 – 780
Near Infrared
> 780

8. R
O
Y
G
B IV

9. The absorption process
How does matter absorb radiation

When polychromatic light (white light), which contains the whole spectrum of
wavelengths in visible region, is passed through an object will absorb certain
of the wavelengths, leaving the unabsorbed wavelengths to be transmitted.
These residual transmitted wavelengths will be seen as a color. This color is
complementary to the absorbed colors.

10. Classes of
Spectrophotometers
Single beam and double beam are the two major
classes of spectrophotometer.
 Single Beam: In this type, all the light passes through
the sample .To measure the intensity of the incident
light the sample must be removed so that all the light
can pass through. This type of spectrometer is usually
less expensive and less complicated.
 Double Beam: In this type, before reaches the sample
the light source is split into two separate beams. From
these one passes through the sample and second one
is used for reference. This gives the advantageous
because at the same time the reference reading and
sample reading can take place.

11. Single Beam
Spectrophotometer

12. Double Beam
Spectrophotometer

13. Different types of
Spectrophotometers

Visible Light:
Visible spectrophotometers use incandescent, halogen, LED, or a
combination of these sources and these spectrophotometers vary in
accuracy. Plastic and glass cuvettes can be used for visible light
spectroscopy.

Ultraviolet Light:
UV spectroscopy is used for fluids, and even for solids. Cuvettes, only
made of quartz, are used for placing the samples.

Infrared Light:
IR spectroscopy, which helps to study different structures of
molecules and its vibrations. Different chemical structures vibrate in different
ways due to variation of energy associated with each wave length. For
example, mid-range and near infrared (higher energy) infrared tends to
cause rotational vibrations and harmonic vibrations respectively.

14. Different types of Lamps used
in Spectrophotometer

Visible spectrophotometer
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Contains a tungsten lamp that produces white light. Tungsten
ght
lamp consists of a Tungsten filament, enclosed in a glass
envelop with the wavelength range of 330 to 900nm, are used for
visible region. They are generally useful for measuring
moderately dilute solutions in which change in color intensity
varies significantly with the change in solute. It has long life about
1200hr
Ultraviolet spectrophotometer

Contains a Deuterium/ Hydrogen lamp that produces light in
the UV light part of the spectrum. It ranges about 200 to 450nm
in wavelength. This lamp is generally more stable.

15. How a Spectrophotometer
works?
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Shines a beam of light on a sample.
The molecules in the sample interact with the light waves in 3 ways:
 Absorb the energy
 Reflect the energy
 Transmit the energy between and through the atoms and
molecules of the sample.
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.

16. Cuvettes are made from plastic, glass, or quartz.
a.
Use quartz cuvettes for UV work.
b.
Glass, plastic or quartz are acceptable in visible work.
c.
There are inexpensive plastic cuvettes that may be suitable for
some UV work.
2. Cuvettes are expensive and fragile (except for “disposable”
plastic ones). Use them properly and carefully.
1.
Do not scratch cuvettes; do not store them in wire
racks or clean with brushes or abrasives.
b. Do not allow samples to sit in a cuvette for a long
period of time.
c. Wash cuvettes immediately after use.
a.

18. Beer’s Law
 The
intensity of a ray of monochromatic light
decreases exponentially as the concentration
of the absorbing medium increases.
 More
dissolved substance = more absorption
and less transmittance

19. Beer’s Law
source
slit
cuvette
detector

20. How absorbance is
calculated?
Lambert's law is expressed by
 I/Io = T
 where I is the intensity of the transmitted light, Io is the intensity
of the incident light, and T is the Transmittance. It is customary to
express transmittance as a percentage:
%T = I/Io x100
 A combination of the two laws (known jointly as the BeerLambert Law) defines the relationship between absorbance
 (A) and transmittance (T).
 A = log Io/I = log 100/T = ε c b
ε : is molar absorptivity ( L.mol-1. cm-1)
b : is path length (cm)
c : concentration (M)
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21. 
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After collecting data for your
concentration
an
absorption
spectrum graph is created.
These can be used when attempting
to identify unknown substances
The absorbance spectrum is a graph
of a sample’s absorbance at different
wavelengths.
Measure
the
absorbance
of
standards
containing
known
concentrations of the analyte
Plot
a
standard
curve
with
absorbance on the X axis and
analyte concentration on the Y axis
Measure the absorbance of the
unknown(s).
Determine the concentration of
material of interest in the unknowns
based on the standard curve.

22. Standardization graph

23. Difference between
Spectrophotometer and Colorimeter

A colorimeter measures the
absorbance
of
a
particular
wavelength by a solution. It is
usually used to determine the
concentration of a known solute in
a known solvent through the
application of the Beer-Lambert
law.

A spectrophotometer is employed
to measure the amount of light
that a sample absorbs. The
instrument operates by passing a
beam of light through a sample
and measuring the intensity of
light reaching a detector.
(1) Wavelength selection, (2) Printer button,
(3) Concentration factor adjustment, (4) UV
mode selector (Deuterium lamp), (5)
Readout, (6) Sample compartment, (7) Zero
control (100% T), (8) Sensitivity switch,
(9)ON/OFF switch