USE OF THE

SPECTROPHOTOMETER

Medical Laboratory Practice (MLS 1154)

Prof. Harshini Peiris

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Learning Outcomes

Subject specific knowledge

• Describe the principles of spectrophotometry
• Explain the interaction of radiation with matter
• Define the Beer Lambert law
• Name the components of the spectrophotometer
• Explain the use of the standard curve method and internal standard
method
• Discuss the important characteristics of spectrophotometry
• List the applications of spectrophotometry
Subject specific skills
• Use of spectrophotometer in biochemical analysis
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 Spectrophotometry

• Most important for all the instrumental methods of

analysis in clinical chemistry

• Based on the absorption of electromagnetic radiation in

the
– visible,
– ultraviolet and
– infrared ranges

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Electro-magnetic spectrum

Visible range – 400 nm-700 nm

UV range - 100 nm-400 nm
IR range - 700 nm – 1 mm 4
Interaction of light with matter

• When light is incident on a material, it can either get

reflected, transmitted, absorbed or scattered.
• Typically all these types of interactions are present
in varying proportions

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Absorption Spectrophotometry

Concerned with absorption &

transmission

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• White light is composed of a mixture of colours/
wavelengths of the electromagnetic radiation which is
called the visible region of the electromagnetic
spectrum

Violet: 400 - 420 nm

Indigo: 420 - 440 nm
Blue: 440 - 490 nm
Green: 490 - 570 nm
Yellow: 570 - 585 nm
Orange: 585 - 620 nm
Red: 620 - 780 nm

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• The transmitted colour of an object is said to be
complementary to the color of the wavelength(s)
absorbed.
• The observed colour of the object is determined by
the complementary colour/transmitted portion of
wavelengths.
• This relationship is demonstrated by the color wheel.
• complementary colors are diametrically opposite each
other; the colour that is observed is opposite to the
colour that is absorbed.

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 According to the quantum theory;

• Energy status of an atom or molecule are defined

• Further, changes from one state to another require a

definite amount of energy

• Therefore, when energy is supplied from an external

source, it will be selectively absorbed by different
molecules

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1. The frequency of the Provide information
photons absorbed about the nature of the
material

2. The number of photons Provide information

absorbed about the number of
atoms or molecules of
the material present

Therefore spectrophotometry provide a tool for

qualitative as well as quantitative analysis of
substance
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 Absorbance & Transmission
• A molecule or substance that absorbed light is called a
“Chromophore”
• Chromophores exhibits unique absorption spectra
• When a light at a particular wave length (incident light)
is passed through a solution which contains a
chromophore, some amount of light is absorbed &
therefore the light come out (transmitted light) is
diminished
• The nature of light absorption in a solution is governed
by the “Beer-Lambert law”

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 Beer-Lambert law
• Beer’s law
– the amount of transmitted light decreases exponentially
with an increase in the concentration of the absorbing
molecules

• Lambert’s law
– transmitted light decreases exponentially with the
thickness of the absorbing molecules (thickness of the
medium)

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• By the combination of both laws (Beer-Lambert law)
following mathematical equation has derived

I = I0 10 -εcl

I = Intensity of the transmitted light

I0 = Intensity of the incident light
ε = Molar extinction coefficient (characteristic to the
substance, unit L mol-1 cm-1)
c = concentration of absorbing substrate (unit mol L-1)
l = Thickness of the medium through which light passes
(path length, unit cm)
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 I = I0 10 -εcl or log (I/I0) = -εcl
T= I/I0

A = - log T = - log (I/I0)

Therefore;
A = εcl

T- transmitted light

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Spectrophotometer

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Types of spectrophotometers
1. Visible spectrophotometers
2. UV – Visible spectrophotometers
3. IR - Spectrophotometers 16
 Components of a spectrophotometer

Lamp Lens Monochromater Sample holder

Display Photo tube

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1. Light source
• Tungsten lamp – for visible range
• Higher pressure H2 or D2 lamps – for UV range
2. Lens
• Collect the radiation from the source and directs it
into the slit

3. Monochromator
• Monochromatic light (transmit a mechanically
selected narrow band of wavelengths of light) is
generated by;
1. Filters
2. Movable prism or
3. Diffraction gradients
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• Monochromatic light is projected through the sample
& then measured by a photomultiplier tube

4. Sample holder
• Cuvette
• A cuvette is a kind of laboratory-ware, usually a
small tube of circular or square cross section, sealed
at one end, made of plastic, glass, or optical grade
quartz (for UV light) and designed to hold samples
for spectroscopic experiments

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Ideal features of a cuvette

• Should be extremely pure

• Thickness should be ideal
everywhere
• Two opposite walls are
transparent
• There’s a mark to indicate
a transparent wall of a
cuvette

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 When using a cuvette;
• about 2/3 of it should be filled with solution

• Before placing it in the spectrophotometer the outsides

must be wiped properly (towards one direction)

• Cuvette should be placed in the spectrophotometer in a

way that the transparent walls face the light beam

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 5. Photomultiplier tube
- Converts the energy of the light photons into
electrons
- The voltage resulting from these electrons is measured
by a meter & converts to an absorbance value
- A blank is used to measure the initial intensity
- Relative difference in the light intensity between blank
& the sample is expressed as the absorbance

6. Display unit
- Displays the readings (The readout system)

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Other accessories of a spectrophotometer

- Chart recorders
- Multiprocessors for data analysis

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Handling of the spectrophotometer
• Spectrophotometer should be placed on a solid
surface which is free from mechanical vibrations

• Power supply should be provided via a stabilizer

• Fuses should be taken to notice

• Manual should be read & understood

• After switching on, it should be allowed for

stabilizing

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• Then a suitable wave length should be selected to
measure the absorbance
• The reagent blank should be inserted in the
instrument first & the absorbance should be made
zero
• After that the absorbance of the sample can be
measured

• Finally the reagent blank should again be returned to

the light path & it should be ensured that the
absorbance is zero
• The concentration of the solution then can be
calculated either using a standard curve or by
internal standard method
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1. Standard curve method

• A set of standard Absorbance

solutions are prepared

• The absorbance of Y = mx
standard solutions is
measured

• A concentration verses
0,0 Concentration
absorbance calibration
curve (standard curve)
is plotted
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• A standard curve should be linear because it follows
the Beer-Lamberts law

• Then the absorbance of the unknown sample should be

measured (using the same blank)

• Concentration of the unknown can be obtained by

interpolating the standard curve

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2. Internal Standard Method

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Important characteristics of
spectrophotometry

• Wide applicability
• High sensitivity
• Moderate to high selectivity
• Good accuracy
• Easy & convenience

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Applications of Spectrophotometry

• Determination of the concentration of substances in

solution

• To identify unknown compounds (using unique

absorption profiles)

• To measure enzyme activity with time

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Classes of spectrophotometers

• Single beam: 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 spectrophotometer 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
the second one is used for reference. This is
beneficial since both reference and sample readings
are taken place at the same time.
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Double Beam Spectrophotometer

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Questions
1. Write the Beer-Lambert law
2. What are the advantages of standard curve method
when compare to that of internal standard method?
3. What is job effect?
4. What are the properties that the solution used
should have?
5. What are the differences between a colorimeter & a
spectrophotometer?
6. What are the advantages & disadvantages of
colorimeter over a spectrophotometer?

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Summary
• Spectrophotometry is a versatile analytical method

• It is based on the absorption of electromagnetic

radiation in the visible, UV & infrared ranges

• According to the Beer Lamberts law; A = εcl

• Light source, monochromator, sample holder,

photomultiplier tube & display unit are the main
components of a spectrophotometer

• The standard curve method or the internal standard

method can be used to determine the concentration of
an analyte 35
• Important characteristics of spectrophotometry
include its wide applicability, high sensitivity, moderate
to high selectivity, good accuracy & convenience
methodology

• A spectrophotometer can be used to determine the

concentration of a sample, identify unknown compounds
& to determine the enzymatic activity

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 References
• Guidelines for Maintenance of Equipment in a Clinical
Chemistry Laboratory (WHO, Ministry of Health and MRI
Publication)
• Estridge, B. H., Reynolds, A. P., and Walters, N. J., 2000. Basic
medical laboratory techniques. 4th ed. Albany, N.Y., Delmar
Publishers.
• Jacobs, D. S., Oxley, D. K., and Demott, W. R., 2001. Jacobs &
DeMott laboratory test handbook: with key word index. 5th
ed. Hudson (Cleveland), OH, Lexi-Comp.
• Sood, R., 2006. Textbook of medical laboratory technology.
New Delhi, Jaypee Brothers.

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