• Indra gunawan

CONTENTS:-
• Background
• Beer-Lambert law
• The Spectrophotometer
• Basic components
• Types of Spectrophotometers
• Operation
• Maintenance
• Applications of Spectrophotometer
Beer-Lambert law:
Lambert’s Law of Absorption:
• Lambert described how intensity changes with
distance in an absorbing medium.
• The intensity I0 of light beam decreases exponentially
as it passes though a uniform absorbing medium.

I (   )  I ( )   ( ) I ( )
dI ( )
 I ( ) detector   d
I ( )
source I 0

     I ( )  I  I 0 e  
Beer-Lambert law:
Lambert’s Law of Absorption (base 10)
• Typically base 10 is used in photometry

   k
I  I 0e  I 010 k   ln 10
I    k
k is the path length over
 e  10 which the intensity is
I0 attenuated to 1/10.

I
 10  k
I0
Beer-Lambert law:
• Beer’s Law
• Beer found that Lambert’s linear decay constant
k for a solution of an absorbing substance is
linearly related to its concentration c by a
constant.
• the absorptivity ε, a characteristic of the
absorbing substance.
• Restatement: The linear decay constant k is
linear in concentration c with a constant of
proportionality ε.
k c
Beer-Lambert law:
• Photometric Quantities
• In photometry we measure the intensity of light and characterize its
change by substance.
• This change is typically expresses as percent transmittance or
absorbance.

I
T
Transmittance (T) I0
usually given in percent

I 
Absorbance (A) A   log     log T
 I0 
by convention, base 10 logs are used
Absorbance and the extinction coefficient
• Absorbance is useful since it can be summed
for layers of different materials

Atot = A1 + A2 + A
3
+ …
Atot = ε C x + ε C x + ε C x + …
1 1 1 2 2 2 3 3 3

A specialized device to measure the intensity

of light as a function of wavelength is the
spectrophotometer.
 Spectroscopy
• Spectroscopy is the use of the
absorption, emission, or
scattering of electromagnetic
radiation by atoms or molecules
(or atomic or molecular ions) to
qualitatively or quantitatively
study the atoms or molecules.
• The interaction of radiation with
matter can cause redirection of
the radiation and/or transitions
between the energy levels of the
atoms or molecules.
• A transition from a lower level to
a higher level with transfer of
energy from the radiation field to
the atom or molecule is called
absorption. 8
• A transition from a higher level to a lower
level is called emission where energy is
transferred to the radiation field, or
nonradiative decay if no radiation is emitted
• Redirection of light due to its interaction with
matter is called scattering and may or may
not occur with transfer of energy, i.e., the
scattered radiation has a slightly different or
the same wavelength.

9
 SPECTROSCOPY

ABSORPTION EMISSION

SPARC
ATOMIC MOLECULAR FLAME
ABSORPTION EMISSION

ICP

FLUORESCENCE
IR UV VISIBLE

10
 Absorption
• When atoms or molecules absorb light, the incoming energy
excites a quantized structure to a higher energy level.

• The type of excitation depends on the wavelength of the light.

Electrons are promoted to higher orbital by ultraviolet or visible
light, vibrations are excited by infrared light, and microwaves
excite rotations.
• Absorbance is a ratio of the intensity of light that is measured
passing through the sample to the light intensity measured if no
sample was present.

11
 Emission
• When atoms or molecules absorb light, the incoming energy
excites a quantized structure to a higher energy level.

• Atoms or molecules that are excited to high energy levels can

decay to lower levels by emitting radiation (emission or
luminescence).
• For atoms excited by a high-temperature energy source this
light emission is commonly called atomic or optical emission
(atomic emission spectroscopy).
• and for atoms excited with light it is called atomic
fluorescence or molecular fluorescence
• For molecules it is called fluorescence if the transition is
between states of the same spin and phosphorescence if the
transition occurs between states of different spin. 12
 Scattering
• When electromagnetic radiation passes through matter, most of
the radiation continues in its original direction but a small
fraction is scattered in other directions.
• Light that is scattered at the same wavelength as the incoming
light is called Rayleigh scattering.
• Light that is scattered in transparent solids due to vibrations
(phonons) is called. Brillouin scattering is typically shifted by 0.1
to 1 cm-1 from the incident light.
• Light that is scattered due to vibrations in molecules or optical
phonons in solids is called Raman scattering. Raman scattered
light is shifted by as much as 4000 cm-1 from the incident light.

13
 Electromagnetic Spectrum

Radiation Frequency Hz Wavelength Transition

gamma-
1020-1024 <10-12 m Nuclear
rays
x-rays 1017-1020 1 nm-1 pm inner electron

ultraviolet 1015-1017 400 nm-1 nm outer electron

750 nm-400
visible 4-7.5x1014 outer electron
nm
near- 2.5 um-750 outer electron molecular
1x1014-4x1014
infrared nm vibrations
infrared 1013-1014 25 um-2.5 um Molecular vibrations
Molecular rotations, electron
microwaves 3x1011-1013 1 mm-25 um
spin flips*
14
radio waves <3x1011 >1 mm nuclear spin flips*
 Ultraviolet & Visible light Interactions

• UV-VIS spectroscopy is the measurement of the wavelength and intensity of

absorption of near-ultraviolet and visible light by a sample.
• Ultraviolet and visible light are energetic enough to promote outer electrons to higher
energy levels.
• UV-VIS spectroscopy is usually applied to molecules and inorganic ions or
complexes in solution.
• UV-VIS spectra have broad features that are of limited use for sample identification
but are very useful for quantitative measurements. The concentration of an analyte in
solution can be determined by measuring the absorbance at some wavelength and
PCSIR Labs. Karachi Pakistan 15
applying the Beer-Lambert Law
Infrared Interactions

• IR spectroscopy is the measurement of the wavelength and intensity of

the absorption of mid-infrared light by a sample.
• Mid-infrared light (2.5 - 50 µm, 4000 - 200 cm-1) is energetic enough to
excite molecular vibrations to higher energy levels.
• The wavelength of IR absorption bands are characteristic of specific
types of chemical bonds, and IR spectroscopy finds its greatest utility
for identification of organic and organometallic molecules.

16
Background :
 When transitions occurs, the wavelength and
energy decreases, and increases of frequency.
 The Light waves consist of perpendicular,
oscillating electric and magnetic fields ”
Electro- magnetic waves” and described by
1. amplitude(A),
2. wavelength(λ),
3. frequency(F).

 For the light the freq. increases, energy

increases and wavelength decreases through

C C
E = h = h  = C = 
 
Background :
• Visible light is only a
small portion of the
entire electromagnetic
spectrum
• it includes the colors
commonly observed
(red, yellow, green, blue
and violet).
• The visible spectrum
consists of electro-
magnetic radiation
whose wavelengths
range from 380nm to
nearly 760nm.
Background :
 (nm) Region Color Observed
< 380 Ultraviolet Not visible
380-440 Visible Violet
440-500 Visible Blue
500-580 Visible Green
580-600 Visible Yellow
600-620 Visible Orange
620-750 Visible Red
750-2000 Short IR Not visible
Background :
Why do some substances appear colored?
• When light passes through a substance, certain
energies (or colors) of the light are
1. Absorbed
2. Other colors allowed to pass
3. Other are reflected.

• If the substance does not absorb any light, it appears

white (all light is reflected). colorless (all light is
transmitted).
• A solution appears a certain color due to the
absorbance and transmittance of visible light. For
example, a blue solution appears blue because it is
absorbing all of the colors except blue.
 Background :
 At natural state, most of the atoms, molecules and electrons are in
the lowest energy level called ground state.
 To transits from lower energy level to highest the electron need
promotion “as light” and its called energy transition.
 When a chemical absorbs light, it goes from a low energy state
(ground state) to a higher energy state (excited state)

 Only photons with energies exactly equal to the energy difference

between the two electron states will be absorbed
 Since different chemicals have different electron shells which are
filled, they will each absorb their own particular type of light
 The Spectrophotometer
one of the basic medical laboratory
instruments uses to measure light intensity
as a function of wave length(λ).
Measures absorbance as a function of
wavelength
The device important
for determining the
unknown substances
and for calculating
the concentration of
known substances. The Camspec M550 Double Beam Scanning UV/Vis Spectrophotometer
 Spectrophotometer
“Spectrophotometer analyze the concentration of solute in a
solution by measuring the intensity of a particular light beam after it
is directed through and emerges from it.”

• In the Figure below the red part of the spectrum has been almost
completely absorbed by CuSO4 and blue light has been
transmitted. Thus, CuSO4 absorbs little blue light and therefore
appears blue.
• We will get better sensitivity by directing red light through the
solution because CuSO4 absorbs strongest at the red end of the
visible spectrum. But to do this, we have to isolate the red
wavelengths.

PCSIR Labs. Karachi Pakistan 23

 LIGHT IS A TYPE OF
ELECTROMAGNETIC RADIATION

• Imagine electromagnetic radiation like

waves on a pond
– But instead of water, electromagnetic radiation
is energy moving through space
– Distance from one crest to the next is the
wavelength
 WAVELENGTH AND COLOR
• Different wavelengths of light correspond
to different colors
• All colors blended together is called white
light
• The absence of all light is black
• Light of slightly shorter wavelengths is
ultraviolet
– Eyes do not perceive UV light
WAVELENGTH OF VISIBLE
LIGHT AND COLOR
WAVELENGTH COLOR PERCEIVED
380-430 Violet
430-475 Blue
475-495 Greenish Blue
495-505 Bluish Green
505-555 Green
555-575 Yellowish Green
575-600 Yellow
600-650 Orange
650-780 Red
 INTERACTION OF LIGHT WITH
MATERIALS IN SOLUTION
• When light shines on a solution, it
may pass through – be transmitted –
or
• Some or all of the light energy may be
absorbed
 SPECTROPHOTOMETRS MEASURS
INTERACTION OF LIGHT WITH SOLUTIONS
THE ABSORPTION OF LIGHT AND
COLOR OF SOLUTIONS
WAVELENGTH OF COLOR OF LIGHT COLOR OF
LIGHT ABSORBED ABSORBED SOLUTION
380-430 Violet Yellow
430-475 Blue Orange
475-495 Greenish Blue Red-Orange
495-505 Bluish Green Orange-Red
505-555 Green Red
555-575 Yellowish Violet-Red
Green
575-600 Yellow Violet
600-650 Orange Blue
650-780 Red Green
 BIOLOGICAL SOLUTIONS
• Usually appear clear to our eyes – have no color
• DNA, RNA, most proteins do not absorb any
visible light
• But they do absorb UV light, so UV
spectrophotometers are useful to biologists
– Example, can use a detector that measures
absorbance at 280 nm, or 254 nm to detect proteins
 SPECTROPHOTOMETERS
• Are instruments that measure the
interaction of light with materials in
solution
Monochromator Separates Light into Its Component
Wavelengths. Modern Specs Use Diffraction Gratings
 THE BLANK
• Spectrophotometers compare the light
transmitted through a sample to the light
transmitted through a blank.
• The blank is treated just like the sample
• The blank contains everything except the
analyte (the material of interest)
– Contains solvent
– Contains whatever reagents are added to the sample
 WHEN OPERATING SPEC

• Blank is inserted into the

spectrophotometer
• Instrument is set to 100% transmittance
or zero absorbance
 PROPER SELECTION, USE,
AND CARE OF CUVETTES
1. Cuvettes are made from plastic,
glass, or quartz.
a. Use quartz cuvettes for UV
work.
b. Glass, plastic or quartz are
acceptable visible work.
c. There are inexpensive plastic
cuvettes that may be suitable
for some UV work.
PROPER SELECTION, USE,
AND CARE
2. Cuvettes OF CUVETTES
are expensive and fragile
(except for “disposable” plastic ones).
Use them properly and carefully.
a. 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.
 PROPER SELECTION, USE,
AND CARE
3. Disposable cuvettesOF CUVETTES
are often recommended
for colorimetric protein assays, since dyes used
for proteins tend to stain cuvettes and are
difficult to remove.

4. Matched cuvettes are manufactured to absorb

light identically so that one of the pair can be
used for the sample and the other for the blank.
 PROPER SELECTION, USE,
5. AND CARE
Do not touch the OF
baseCUVETTES
of a cuvette or the
sides through which light is directed.

6. Make sure the cuvette is properly aligned

in the spectrophotometer.

7. Be certain to only use clean cuvettes.

OVERVIEW OF QUANTITIVE
SPECTROPHOTOMETRY
A. Measure the absorbance of standards
containing known concentrations of the
analyte
B. Plot a standard curve with absorbance on the
X axis and analyte concentration on the Y axis
C. Measure the absorbance of the unknown(s)
D. Determine the concentration of material of
interest in the unknowns based on the
standard curve
 LINEAR RANGE

• If there is too much or too little analyte,

spectrophotometer cannot read the
absorbance accurately
 COLORIMETRIC ASSAYS
• Quantitative assays of materials that do
not intrinsically absorb visible light
• Combine the sample with reagents that
make the analyte colored
• The amount of color is proportional to the
amount of analyte present
 MORE ABOUT THE CALIBRATION LINE
ON A STANDARD CURVE
• Three things determine the absorbance of
a sample:
– The concentration of analyte in the sample
– The path length through the cuvette
– The intrinsic ability of the analyte to absorb
light at the wavelength of interest
 BEER-LAMBERT LAW
A =  B C
Where:
A = absorbance at a particular wavelength
 = E = absorptivity constant – intrinsic ability
of analyte to absorb light at a particular
wavelength
B = path length through cuvette
C = concentration of analyte
 APPLYING THE EQUATION
• Suppose you have a sample:
– And you know the path length
– And you know the absorptivity constant for the
analyte of interest at a particular wavelength
• Then, measure the sample’s absorbance
at the specified wavelength
• Can calculate the concentration of the
analyte from the Beer-Lambert equation

A =  B C
• But this is a shortcut that may give
inaccurate results!
 EQUATION FOR A LINE
A =  B C
y = m x +0
Y intercept should be zero because of the
blank
– Blank has no analyte (zero concentration) and
is used to set transmittance to 100% =
absorbance to zero
 SLOPE
• Slope relates to the absorptivity constant

A =  B C
y = m x +0
 DETERMINATION OF THE
1.ABSORPTIVITY CONSTANT
Prepare a calibration line based on a
series of standards
Plot concentration on the X axis and
absorbance on the Y axis
2. Calculate the slope of the calibration line:
Y2 – Y1
X2 - X1
3. Determine the path length for the system
(assume 1 cm for a standard sample
holder and cuvette)
 A =  B C
y = m x +0
3. Slope = absorptivity constant X path length
Absorptivity constant = slope
path length

(Observe that the constant has units that depend on

how concentration was expressed in the standards)
 UV SPECTROPHOTOMETRY
• DNA, RNA and proteins are commonly
analyzed with UV spectrophotometry
because these molecules absorb UV light
 UV METHODS
• Used to evaluate the quality of DNA or
RNA in a sample
• Used to estimate the quantity of DNA or
RNA in a sample
• Procedure: Take single wavelength
readings of samples at 260 and 280 nm
 CONCENTRATION
• The absorbance at 260 nm is related to the
concentration of DNA or RNA in the sample
– Pure, double-stranded DNA has an absorbance of
about 1 at 260 nm when it is at a concentration of
about 50 micrograms/mL
– Pure, single-stranded DNA has an absorbance of
about 1 at 260 nm when it is at a concentration of
about 33 micrograms/mL
– Values for proteins vary considerably from protein to
protein
 PURITY
• The ratio of the absorbance at 260 and
280 nm is related to the purity of the
sample
– An A260/A280 ratio of 2.0 is characteristic of
pure RNA
– An A260/A280 ratio of 1.8 is characteristic of
pure DNA
– An A260/A280 ratio of 0.6 is characteristic of
pure protein
 UV METHODS
• These UV methods for estimating
concentration and purity of DNA, RNA,
and proteins are very commonly used, are
very quick, and easy to perform
• However, they values obtained are not
very accurate – they are rough estimates
 CALIBRATION OF A
• SPECTROPHOTOMETER
Brings the readings of the
spectrophotometer into accordance with
nationally accepted values
• Part of routine quality control/maintenance
 CALIBRATION
Two parts:
1. Wavelength accuracy, the agreement
between the wavelength selected by the
operator and the actual wavelength of light
that shines on sample
2. Photometric accuracy, or absorbance scale
accuracy, the extent to which a measured
absorbance or transmittance value agrees with
an accepted reference value
• Wavelength accuracy is determined using
certified standard reference materials (SRMs)
available from NIST or traceable to NIST
– An absorbance spectrum for the reference material is
prepared
– The absorbance peaks for reference standards are
known, so the wavelengths of the peaks generated by
the instrument can be checked
• Manufacturers specify the wavelength
accuracy of a given instrument
– For example, a high performance instrument
may be specified to have a wavelength
accuracy with a tolerance of + 0.5 nm
– A less expensive instrument may be specified
to have a wavelength accuracy of + 3 nm
 PHOTOMETRIC ACCURACY
• Assures that:
– If the absorbance of a given sample is
measured in two spectrophotometers at the
same wavelength and under identical
conditions
• then the readings will be the same
• and the readings will correspond to nationally
accepted values
• Photometric accuracy is difficult to achieve
due to different instrument designs and
optics
• Usually photometric accuracy is not critical
if the same instrument is used consistently
and if its readings are linear and
reproducible
• Photometric accuracy is required where
values from different labs and instruments
are compared
• Required if rely on published absorptivity
constants
• Likely required in a GMP-compliant facility
 PROBLEM
• Assume that a spectrophotometer is able
to read accurately in the range from 0.1 to
1.8 AU. The molar absorptivity constant
for NADH is 15,000 L/mole-cm at 260 nm.
Using Beer's Law, calculate the
concentration range of NADH that can be
accurately quantitated at 260 nm based on
the limits of the spectrophotometer.
 ANSWER
• This involves calculation of the molar
concentrations which will produce absorbances
of 0.1 and 1.8. From Beer's Law:

C = A
ε b

Substituting 0.1 and 1.8 into the equation:

C = 0.1 = 6.7 X 10-6 mole/L
(15,000 L)(1 cm)
Mole-cm

C = 1.8 = 120.0 X 10-6 mole/L

(15,000 L)(1 cm)
mole-cm
Thus, the range of NADH concentrations
that can be detected at this wavelength
with this spectrophotometer is from 6.7 X
10-6 mole/L to 120.0 X 10-6 mole/L. These
are very dilute solutions of NADH.
78
PCSIR Labs. Karachi Pakistan 79
 Spectrophotometer Types
• 1- Single Beam

• 2- Double Beam
Single Beam Spectrophotometer
Double Beam Spectrophotometer
Spectrophotometer Block Diagram
 Double-Beam
Spectrophotometer
 Introduction
• Principle

• What is the Spectrophotometry?

• Purpose of the instrument.

• Types of Spectrophotometer.
 Device Description
• Mirror.
• Light Source.
• Monochromator.
• Entrance and exit slit.
• Rotating Discs.
 Device Description
• Sample cell & Reference cell in cuvettes.
• Photo Detector (PMT).
• Computer.
• Optical Wedge.
 Applications
• Determination of Alanine
Aminotransferase (ALT/GPT) in clinical
samples.
• Determination of cholesterol in clinical
samples.
• Determination of glucose in clinical
samples.
• Determination of urea in clinical samples.
• Determination of phosphate in clinical
samples.
• Io is the incident light and represents 100% of
the light striking the cuvette.
• I is the transmitted light. This is the light, which
has not been absorbed by the solution in the
cuvette and will strike the phototube.
• The photons of light, which do strike the
phototube, will be converted into electrical
energy. This current, which has been produced,
is very small and must be amplified before it can
be efficiently detected by the galvanometer.
• The deflection of the needle on the
galvanometer is proportional to the amount of
light, which originally struck the phototube and is
thus an accurate measurement of the amount of
light which has passed through (been
transmitted by) the sample.

PCSIR Labs. Karachi Pakistan 91

 BLANK
• In order to effectively use a spectrophotometer we must first zero the
machine, we do this using "the blank."
• The blank contains everything except the compound of interest, which
absorbs light. Thus, by zeroing the machine using "the blank," any
measured absorbance is due to the presence of the solute of interest.

ABSORPTION SPECTRUM
• Different compounds having dissimilar atomic and molecular interactions
have characteristic absorption phenomena and absorption spectra, which
differ.
• The point (wavelength) at which any given solute exhibits maximum
absorption of light (the peaks on the curves on the figure below) is defined
as that compounds particular max.

PCSIR Labs. Karachi Pakistan 92

 Cuvettes

A cuvette is a kind of cell usually a small square tube,

sealed at one end, made of Plastic, glass or optical
grade quartz and designed to hold samples for
spectroscopic experiments. Cuvette should be as clear
as possible, without impurities that might affect a
spectroscopic reading. Like a test-tube, a cuvette may
be open to the atmosphere on top or have a glass or
Teflon cap to seal it shut.

Quartz Cells
170-2700 nm wavelength range
Disposable Cuvettes
UV-Cuvettes for the range between 220-900 nm
VIS-Cuvettes for the range 350-900 nm

93
 Filters
Filters separate different parts of the electromagnetic spectrum by
absorbing or reflecting certain wavelengths and transmitting other
wavelengths.
• Color Filters
Color filters are glass substrates containing absorbing species that absorb
certain wavelengths. A typical example is a cut-on color filter, which blocks
short wavelength light such as an excitation source, and transmits longer
wavelength light such as fluorescence that reaches a detector.
• Interference Filters
Interference filters are made of multiple dielectric thin films on a substrate.
They use interference to selectively transmit or reflect a certain range of
wavelengths. A typical example is a bandpass interference filter that
transmits a narrow range of wavelengths, and can isolate a single emission
line from a discharge lamp. Prisms

94
 Calibration Filters
Didymium filters
This glass filter is designed for checking the wavelength
calibration of spectrophotometers in both the visible and
near infrared regions of the spectrum. The usable range
is 430nm to 890nm, instruments of SBW of less than
10nm.

Holmium filters
This filter is intended exclusively for checking the
wavelength of moderate to high resolution
spectrophotometers.They are custom made to order to
required size and supplied either un-mounted or in
anodised holders.
95
 Design of a Spectrophotometer

• Light Source
• Monochromator assembly
• Sample holder assembly
• Detector

96
 Light Source
Lamps convert electrical energy into radiation. Different designs
and materials are needed to produce light in different parts of the
EMS

Blackbody Sources
• A hot material, such as an electrically-heated filament in a light
bulb, emits a continuum spectrum of light. The spectrum is
approximated by Planck's radiation law for blackbody radiators:
• The most common incandescent lamps and their wavelength
ranges are:
tungsten filament lamps : 350 nm - 2.5 mm
glowbar : 1 - 40 mm
Nernst glower : 400 nm - 20 mm
• Tungsten lamps are used in visible and Near-infrared (NIR)
absorption spectroscopy and the glowbar and Nernst glower are
used for infrared spectroscopy.
97
 Discharge Lamps
Discharge lamps, such as neon signs, pass an electric
current through a rare gas or metal vapor to produce light.
The electrons collide with gas atoms, exciting them to
higher energy levels which then decay to lower levels by
emitting light. Low-pressure lamps have sharp line
emission characteristic of the atoms in the lamp, and high-
pressure lamps have broadened lines superimposed on a
continuum.
• Common discharge lamps and their wavelength ranges
are:
hydrogen or deuterium: 160 - 360 nm
mercury : 253.7 nm, and weaker lines in the near-UV and
visible
Ne, Ar, Kr, Xe discharge lamps : many sharp lines
throughout the near-uv to near-IR
xenon arc : 300 - 1300 nm
• Deuterium lamps are the UV source in UV-VIS absorption
spectrophotometers. The sharp lines of the mercury and
rare gas discharge lamps are useful for wavelength 98
calibration of optical instrumentation. Mercury and xenon
 Monochromator

A monochromator is a spectrometer capable of measuring a single

wavelength which can be scanned through a wide wavelength
range. A common form of monochromator is the Czerny-Turner
design, consisting of fixed entrance and exit slits, fixed focussing
mirrors and a rotatable diffraction grating. As the grating rotates a
different wavelength is focused onto the exit slit.

99
The wavelength range of a monochromator varies with the choice of grating, but
commonly they can scan from 160 nm to 500 nm or ever wider ranges. The spectral
resolution depends on the widths of the slits, the choice of grating and focal length,
but commonly can be less than 10 pm for high resolution OES.
A key to the performance of monochromators is the design of the grating movement:
the grating is place on a large drive wheel with motor control, allowing fine and
precise positioning of the grating

100
 Monochromator (Optical Spectrometers)

Monochromator parameters
• Bandpass
The wavelength range that the monochromator transmits.
• Dispersion
The wavelength dispersing power, usually given as spectral range / slit width
(nm/mm). Dispersion depends on the focal length, grating resolving power,
and the grating order.
• Resolution
The minimum bandpass of the spectrometer, usually determined by the
aberrations of the optical system.
• Acceptance angle (f/#)
A measure of light collecting ability, focal length / mirror diameter
• Blaze wavelength
The wavelength of maximum intensity in first order.

101
 Detector
Silicon PIN Photodiodes Photoconductive sensors
Blue enhanced for a spectral range from 350nm to 1100nm;
designed for low-capacitance, high speed, wide bandwidth
applications. Active areas vary from .17 mm² to 100 mm².
Applications include: chemical and analytical measurement,
laser detection, bar code, smoke detector, appliances,
industrial controls, instrumentation,

Silicon PIN Photodiodes Photovoltaic V-Series

Blue enhanced for spectral range from 350nm to 1100nm; designed for low-
noise, D.C. to medium bandwidth applications. Active areas range from
.31mm² to 100mm². Applications include: low light level measurements,
particle counting, chemical and analytical measurement and detection.

102
UV Enhanced Silicon Photodiodes
Spectral enhanced from UV (190nm) response out to Near IR (900nm).
Processed for high shunt resistances, low noise and medium electrical
bandwidth, these silicon diodes are designed for photovoltaic, low-signal
applications. Active areas vary from 0.073mm² to 100mm². Package
options include T0-46, T0-18, T0-5, T0-8, Jumbo T0-8, and ceramic
packages with quartz and UV transmitting windows. Applications include:
pollution monitors, UV exposure meters, water purification, fluorescence,
and other spectroscopic applications.

Silicon Carbide (SiC) UV Photodiodes

Standard and custom UV sensors are available with spectral ranges from
200nm to 400nm (optically non-sensitive from 400nm to 1200nm). Active
areas include 0.09mm² and special orders for 1.0mm² and larger. Package
configurations include: isolated hermetic T0-46 with UV windows. Detector-
amplifier hybrid configurations are also available. Applications include:
combustion, flame and arc detection, solar blind UV sensing, solar radiation,
spectroscopy, sterilization, UV curing detection, and phototherapy control.

103
Detector-Filter Combination Photodiodes
Standard and custom silicon photodiodes, with integrated optical long-pass
(IR), short-pass (VIS), ultra-violet (UV) bandpass, narrow "notch" filters,
low-cost plastic long-pass (IR) filters, are offered. Standard configurations
include: visible light detectors (500nm), Near-IR (>800nm), UV-A (360nm),
UV-B (320nm), UV filter detectors (254 & 310nm), CIE (human eye)
response detectors, and neutral-density detector-filter combinations. Active
area sizes include 1.55mm² to 100mm². Packages include two-leaded
ceramic, T0-46, T0-5, T0-8, Jumbo T0-8, and BNC. Applications include
analytical instrumentation, photometry/radiometry, medical instrumentation,
and other spectra-radiometry applications.

Gallium Nitride (GaN) UV Detectors

This family of Gallium Nitride (GaN) UV Detectors are Schottky
processed fully passivated U.V. photodiodes. Spectral range from 200
nm to 365 nm and is ideal for UVA or UVB sensing applications and is
packaged with a quartz window.
.

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Charge-Coupled Devices (CCD)
A CCD is an integrated-circuit chip that contains an array of
capacitors that store charge when light creates e-hole pairs. The
charge accumulates and is read in a fixed time interval. CCDs are
used in similar applications to other detectors, although the CCD is
much more sensitive for measurement of low light levels.

105
Photomultiplier Tubes
Photomultiplier Tubes (PMTS) are light detectors that are useful in
low intensity applications and due to high internal gain, PMTs are
very sensitive detectors.
Design
PMTs are similar to phototubes. They consist of a photocathode
and a series of dynodes in an evacuated glass enclosure. Photons
that strikes the photoemissive cathode emits electrons due to the
photoelectric effect. Instead of collecting these few electrons (there
should not be a lot, since the primarily use for PMT is for verly low
signal) at an anode like in the phototubes, the electrons are
accelerated towards a series of additional electrodes called
dynodes.

PCSIR Labs. Karachi Pakistan 106

 These electrodes are each maintained at a more positive potential.
Additional electrons are generated at each dynode. This cascading
effect creates 105 to 107 electrons for each photon hitting the first
cathode depending on the number of dynodes and the accelerating
voltage. This amplified signal is finally collected at the anode where
it can be measured.
Typical specifications

• Wavelength range: 110-1100 nm

(wavelength sensitivity dependent on wavelength, uv-sensitive
PMTs must have uv-transmitting windows, see optical materials)
• Quantum efficiency (Q.E., number of electrons ejected by the
photocathode / number of incident photons): 1-10%
• Response time: 1-15 ns

107
 UV/Visible Spectrophotometer
• It is the measurement of the wavelength and intensity of absorption of
near-ultraviolet and visible light by a sample.
• Ultraviolet and visible light are energetic enough to promote outer
electrons to higher energy levels.
• UV-VIS spectroscopy is usually applied to molecules and inorganic
ions or complexes in solution.
• The UV-VIS spectra have broad features that are of limited use for
sample identification but are very useful for quantitative measurements.
• The concentration of an analyte in solution can be determined by
measuring the absorbance at some wavelength and applying the Beer-
Lambert Law.

108
• The light source is usually a hydrogen or deuterium lamp for UV measurements
and a tungsten lamp for visible measurements.
• The wavelengths of these continuous light sources are selected with a
wavelength separator such as a prism or grating monochromator.
• Spectra are obtained by scanning the wavelength separator and quantitative
measurements can be made from a spectrum or at a single wavelength.

109
 Optics of UV/Visible Spectrophotometer
The UV-Visible spectrophotometer uses two light sources, a deuterium
(D2) lamp for ultraviolet light and a tungsten (W) lamp for visible light. After
bouncing off a mirror (mirror 1), the light beam passes through a slit and
hits a diffraction grating. The grating can be rotated allowing for a specific
wavelength to be selected. At any specific orientation of the grating, only
monochromatic (single wavelength) successfully passes through a slit.

PCSIR Labs. Karachi Pakistan 110

A filter is used to remove unwanted higher orders of diffraction.The light
beam hits a second mirror before it gets split by a half mirror (half of the
light is reflected, the other half passes through). One of the beams is
allowed to pass through a reference cuvette (which contains the solvent
only), the other passes through the sample cuvette. The intensities of the
light beams are then measured at the end.

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Dual-Beam UV-VIS Spectrophotometer

In single-beam UV-VIS Absorption Spectroscopy,

obtaining a spectrum requires manually measuring the
transmittance of the sample and solvent at each
wavelength. The double-beam design greatly simplifies
this process by measuring the transmittance of the
sample and solvent simultaneously. The detection
electronics can then manipulate the measurements to
give the absorbance.

PCSIR Labs. Karachi Pakistan 112

 Optical Materials
Mirrors
• X-rays Ultraviolet aluminium
• Visible aluminium
• Near infrared gold
• infrared copper, gold
Lenses
• X-rays Ultraviolet fused silica
(synthetic quartz), sapphire
• Visible glass, sapphire
• Near infrared glass, sapphire
• Infrared CaF2, ZnSe

113
 Aberrations
Lenses (and curved mirrors) do not focus light perfectly. Chromatic
and spherical aberations occur on-axis and coma and astigmitism
occur off-axis.
• Chromatic aberration
Chromatic aberration occurs due to the variation of refractive index
with wavelength for a lens material (there is no chromatic aberation
in curved mirrors). This wavelength dependence results in slightly
different focal lengths for different wavelengths of light. Compound
lenses, called achromats, can reduce or eliminate chromatic
aberation because the components are chosen such that the
variation in refractive index as a function of wavelength cancels out.

• Spherical aberration
Spherical aberation results because the actual focal point of a light
ray depends on its distance from the optic axis.

114
• Coma
Coma is caused by the distortion of a wave front as it encounters an
optic asymmetrically. The result for collimated incoming light is a
circle instead of a point image. The light rays farther from the optic
axis have more severe aberation and the resulting image looks like
a comet-shaped series of circles.
• Astigmatism
The projection of an optic off-axis looks squashed in one direction.
The squashed direction focuses light to a greater extent than the
normal dimension. The result is two line images.

Minimizing aberrations
• work on or near the optic axis
• use compound lenses (achromats, doublets, triplets) which
can be designed to reduce chromatic aberation, spherical
aberation, and coma
• use computer optimized aspheric lenses
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 The Spectrophotometer
Components: light source, monochromator,
sample cell, detector & optical system.
monochromator
sample cell
slit detector
diffraction grating

optical system
light source
 Basic components:
1- Light Source: provides the light to be passed
through the sample.
 a source must generate a beam of radiation
that is sufficiently powerful for easy
detection and measurement.
- Hollow Cathode Lamps (HCL)
- tungsten Lamp: visible light .
- Hydrogen discharge: ultraviolet Light.

its output power should be stable for reasonable periods.

 Basic components:
2- monochromator: used to select a given wavelength of
the light from the light source.
mono single.
chroma color.
ator denoting an agent

There exists many techniques for that

• Diffraction gratings
• Prisms
• Collimation
• Stray light
• Wavelength range
• Double monochromator
Basic components:
2- monochromator:
• Diffraction gratings

To obtain of specific wavelength:

1. entrance slit.
2. concave mirror or lens.
3. a prism or grating.
4. focal plane.
5. exit slit.
Basic components:
2- monochromator:
• Prisms spray out the spectrum and choose the certain
wavelength that you want by slit.
 Basic components:
3- Sample Cell:
• A container that contains a sample is usually called
"cell"
• has fixed length & volume.
• usually round or square cuvette.
• made of material that does not absorb light in the
wavelength range.
• two types are available:-
- Glass – visible region.
- Quartz – ultraviolet.
Basic components:
4- Detector:
• to convert the radiant energy to a
measurable signal; and to a readout device
• “Detector” is a device that indicates the
existence of some physical phenomenon.
• The term transducer is used to indicate the
type of detector that converts quantities,
such as light intensity, into such electrical
signals that can be subsequently amplified,
manipulated, and finally converted into
numbers.
Basic components:
Ideal detector : high sensitivity.
high signal/noise.
fast response time.
constant response for λs.
responds to low levels of energy.
 Types of Spectrophotometers:
Single-Beam Instrument: -
 sample and blank are alternatively measured in same sample
chamber.
 use a single-wavelength light source, such as a light
 emitting diode (LED), a sample container, and a photodiode
detector.
 can utilize a fixed wavelength light source or a continuous source.
 and offers a small and inexpensive device configuration
 Types of Spectrophotometers:
2-Double-Beam Instrument:
 A double beam system has two dedicated
positions for the sample and the blank
 Continuously compares sample and blank.
 The change of the light source can be
corrected, and it is possible to measure the
system with stability.
Operation
Calibration Method
 Calibration Steps
1. Turn on your instrument and allow it to warm up for 20
minutes.
2. Select the %Transmittance operating mode by pressing the
MODE key.
3. Set the wavelength to 519nm.
4. Insert the cuvette filled with distilled water in the well of the
cuvette holder.
5. Pressing the 100%T/0-Abs key until display read 100.0%T
or 0.000A, then remove the cuvette with water.
Troubleshooting
Power Supply Circuit for
Spectrophotometer
Detector Circuit
 Maintenance:
Periodically maintenance:
• Change the light source.
• Clean lenses, mirrors and light paths
periodically
• Keep lenses and mirror away from touch
and dust. “Affect the readings”
• Be careful when dealing with mirrors and
lenses and clean them with soft material
 Maintenance:
Basic faults:
• Damage of light source, detector.
• Illogical measurements-dusty of
mirrors and lenses
• IC’s damaged –check each IC’s
individually
 Maintenance:
Safety notes:
• Keep U-light away from human
eyes
• There exist high voltage – be
aware when removing the cover
• Remove the power supply when
change device kits
Trouble Shooting

136
Sensitivity problems
• Check the lamp usage hours if it is expired then
change it with the new one.(be careful about the
warm-up time).
• Check the sample holder and windows, they should
be dust free.
• The Cuvettes should be used on the proper side
and there should be no scratches on the light
exposer side.
• Check the light chopper, the direct exposer also
reduces the sensitivity.
• Check the grounding and shielding caps and of
detector
• Check the photo cell and its preamplifier circuitry.137
Displacement of peaks (shift in the
wavelength)
It is caused due to the miss alignment and over traveling of monochromator
arm, knobs, scale or the reference mark, can be removed by calibrating the
hardware assembly with the help of calibration filters (didymium, holmium) or
reference samples.

Calibration
Advance Spectrophotometers have their own calibration
routines and the user have to just run it, the controller then
selects the desired filter and optimized the hardware. In
case if there is no auto calibration function one have to use
reference sample and calibration filters for adjusting the
wavelength scale and intensity readings.
138
 Monochromator Assembly problems
• In advanced spectrophotometers the Monochromators are derived
through stepper motors. To identify the initial position (home
position) a reference hole, mark or notch is usually given. A position
sensor (encoder, micro switch, transmitter receiver pair) is installed
in the assembly so as to monitor the movement and selection of
wavelength. On startup the controller rotates the stepper motor so
that to reach the home position or reference mark.
• The common problems are the failure of position sensor,
malfunctioning of the stepper motor, and improper homing on
startup.
• For proper homing the reference mark and position sensor should
be properly aligned and free of dust.
• The working of position sensor can be easily checked by pressing
(micro switch), inserting a paper (Optical sensor) and using
oscilloscope(encoder).
• The stepper motors used are normally from 5 to 12volt DC(1~5A)
and can be checked by applying DC pulses on its winding.
139
 Detectors and Preamplifier section

• The common problems in detectors are the damaging and fading of

the active areas.
• It is difficult to directly check the response of the detectors, but after
some working on the preamplifier circuit one can easily identify the
first amplifier IC and by using an oscilloscope the pulsating response
can be observed.
• The pulsating response on the output of the amplifier IC is due to the
chopping of light from the lamp, and this frequency varies from 50 to
1000Hz.
• Normally one or two variable resistors are given in the Preamplifier
section, they are for the adjustment of gain, and offset of the
preamplifier.

140
 Light Chopper assembly
• To minimize the effect of stray light a Chopping Technique
is normally used in which a fan choppes the light coming
from the lamp with frequency corresponding to the
number of wings and RPM. This chopping frequency is
used as synchronizing pulls for the chopping stabilized
opamps.
• For simplicity and minimum component requirement,
usually AC fans are used, which gives chopping frequency
of 50Hz. Some manufacturer use DC fans with stabilized
supply voltages and PWM speed control technique.
• The common problems are the faulty fans, displacement
of fan and lamp, or the fan not properly choppes the light
beam. This chopping can be observed on the output of
first amplifier or synchronizing input. The failure of
chopper circuitry misguides the chopper stabilized
opamps, which in turn produce the erratic behavior.
141
 Lamp power supply

• To stabilized the intensity of lamp through out the

analysis the voltage and current regulation is made which
uses Regulator IC’s, MOSFET’s or Transistors.
• The common problems are the burnouts of the lamps,
shortening of the Transistors, or Regulator IC’s.
• Tungsten lamps can be checked by using its filament
resistance(1~200ohms), where as the discharge lamps
such as deuterium lamps shows open on its terminal and
can only be checked by its rated power supply.

142
Data Acquisition System
• The Acquisition system uses 12bit, 16bit or 18bit Analog
to Digital Converters depending on the resolution and
accuracy requirements.
• The common problems are the variation or noise in the
reference voltage of ADC, malfunctioning of the ADC,
RAM or Processor.
• The main program in the EPROM / EEPROM may
sometimes be deleted or corrupted due to the frequent
power failure or variation.
• To get a rough idea of working and functioning of
processor and peripherals, start with the checking of
pulses on ALE, Address and Data bus, they should
have relation with each other.
• The reset circuitry of the Processor are sometimes
triggered by the Power-on, watch-dog and lamp-supply
monitoring circuitries, malfunction of these sections lead
143
the processor to hang.
 _

Detector

PCSIR Labs. Karachi Pakistan 144

Applications of Spectrophotometer
• Spectroscopy
• Chemical Analysis: concentration ,trace
analysis, pH and remote monitoring
• Geology.
• Astronomy.
• Particle size.
• Thin film characterization
• Color matching
• Optics