Chapter 2 - Introduction of Optical Instrument

CHAPTER 2:
Introduction of
optical instrument
Prepared by Dr.Kavirajaa, Ph.D., AMRSC

Optical instruments
Based on six phenomena
Absorption
Emission
Fluorescence
Scattering
Phosphorescence
Chemiluminescence

Components of optical
instruments
• Although different configuration; basic components are
similar
• Required properties of components are the same
regardless whether they are applied to different types of
radiation
UV
Visible
IR

Instrumental methods
All of the methods have the same general components:
1.A stable source of radiant energy
2.Wavelength selector – isolate specific region of spectrum
3.A transparent container for holding the sample
4.Radiation detector – converts radiant energy to electrical signal
5.Signal processor and readout – PC, digital meter or recording
device
The actual arrangement of the components will vary based on the

method

Components of various types of optical
Source of thermal energy – spectrometers
flame or plasma
Remarks:
1.Source
2.λ selector
3.Container
4.Radiation
detector
5.Readout
To select emission λ
Sample fixed
at right angle / Fluorescence
To select excitation λ
to avoid
scattering
Sources of Radiation
A source must generate a beam of radiation

• a sufficient radiant power for easy detection and
measurement
• its output power should be stable (reasonable uniform
intensity) for reasonable periods. Typically, the radiant
power varies exponentially with the voltage, hence
 Regulated power source is needed
 Double beam design (ratio of the signal from the sample :
absence of sample measured simultaneously)
TWO TYPES OF SOURCES: Continuum sources and Line sources

Continuum sources
• Emit a broad band of wavelengths, radiation that changes
in intensity only slowly as a function of wavelength
 from which desired wave-length can be selected.
• UV region – D2 lamp
• Intense source (high pressure, gas filled arc lamp) – Ar,
Xe, Hg
• Visible – W lamp
• Infrared – inert solids heated to 1500 – 2000 K (a
temperature where maximum radiant output at 1.5 – 1.9
μm).

Continuum sources
Sources for molecular absorbance and luminescence methods

in UV, visible, and IR regions Prepared by Dr.Kavirajaa, Ph.D., AMRSC
Line sources
 Emit one or more discrete wavelengths with very narrow line
width.
 Most common are hollow cathode lamps, made from element
of interest, used for atomic absorbance and luminescence.
 Also includes LASER = Light Amplification through Stimulated
Emission of Radiation
 LASER – high intensity, narrow bandwidths (related to
frequency), consistent nature of their outputs
 N2 laser (337.1 nm)
 Ar+ laser (488.0 and 514.5 nm)
 He-Ne laser (632.8 nm)
 Nd-YAG laser (1064 nm)
 Dye laser (300 – 700 nm, tunable over 20 – 50 nm)

Spectrum
(a) continuum source

(b) line source
Sources of electromagnetic radiation

Sample Cells and Optics
Must be
 optically transparent in wavelength range of interest
 chemically inert

Sample Cells and Optics

Visible
 Disposable plastic
 Glass
UV
 quartz

Wavelength Selectors
1. Radiation for spectroscopic analyses consists of a limited, narrow,

continuous group of wavelengths known as band
2. A narrow bandwidth
 Increases the sensitivity of measurement
 Provides selectivity
 Obtains a linear relation between output signal and concentration for
quantitative analysis
3. Ideally the output from radiation is single  or  radiation
 We cannot really obtain a single wavelength regardless of the source
 Narrow bandwidth exhibits better performances
 2 types of λ selector: filters or monochromators

Typically, we want to look at a single
wavelength at any given time
Scan a range of λ in sequence
Interested in a single λ (for quantitative
analysis)
GOAL
To allow only specific λ to reach the detector at
any given time

Effective bandwidth

Filter
 The simplest type
 To select a single, narrow range of wavelengths that are
allowed to pass
 If you want to look at several different λ, you would need
many different filters
 Scanning a range of λ would be impossible
Monochromator (Prism)
 Allow selection of a range of wavelength

Filters
1. Two type of filters:
a. Interference filters (Fabry-Perot filter) – Available
for UV, Vis, IR
b. Absorption filters – restricted to visible region only
2. Absorption filters are
a. colored glasses or dye suspended in a gelatin &
sandwiched between glass plates that transmit the
wavelengths of interest
b. wide bandwidth, 30 – 250 nm
c. cheap, but are discontinuous and have fixed
wavelength
 Wavelengths cannot be changed and cannot be scanned.
Monochromators
An alternative to filters
Capable of producing many different wavelengths
Wavelength of interest is selected by proper refraction
angle from a prism or reflection angle from a grating.
Allows wavelengths to be scanned as function of time.
a continuous measurement

Monochromators
Consists of
1.Entrance slit - to restrict incident of unwanted
radiation
2.Collimating lens or mirrors - to focus radiation
3.Prism or grating - to ‘separate’ the wavelengths of the
polychromatic radiation from the source
4.Focusing element – reforms the image at the entrance
slit and focus it on a planar surface called a focal plane
5.exit slit - to help control spectral purity of the
radiation emitted from the monochromator
> in addition most monochromators have
entrance and exit windows designed to protect
the components from dust and corrosive media.
Bunsen prism monochromator Angular dispersion of the λ
results from diffraction,
which occurs at the
reflective surface
Czerny-Turner grating monochromator -
Refraction at
the two faces
results in
angular
dispersion of
the radiation
Cheaper fabrication, better λ Most of the modern

separation, disperse radiation monochromators are based on
linearly along the focal plane the gratings instead of prism
Dispersing medium

Performance Characteristics of
Monochromators
 Linear Dispersion: change in distance with wavelength at focal plane
at Figure 7-18 (A – B) dy f dr
D 
d d
 Resolving Power: minimum wavelength difference that can be
separated (better resolution) 
R  nN

 Light gathering power of monochromator: efficiency of light
gathering, depends on diameter (d) of limiting aperture, want low F-
number
f f:focal length; d: diameter; F: speed (f-number)
F
d
 Stray Light Rejection: eliminate light not at desired wavelength
Wavelength Selector

Detectors of EMR
We need a method of detecting any

radiation that has made it through the
system
To convert the responses into measurable
signals
Based upon the type of radiation

Radiation transducers
Transducers convert power (P) of

radiation to a proportional electrical
signal (S)
SP

Properties of ideal transducers
S = current/voltage output of transducer Prepared by Dr.Kavirajaa, Ph.D., AMRSC
k = calibration sensitivity
Real transducers
 Exhibits a small, constant response in the absence
of radiation
 Dark current (kd)
S = k P + kd
S = current/voltage output of transducer
k = calibration sensitivity
kd = dark current which is usually constant over
short measurement periods.
Equips with a compensating circuit
Reduces kd to zero
S=kP
Types of transducers
1. Two types:
a.Photon transducers (photoelectric or quantum
detectors
i. Responds to photons
b.Thermal
ii. Responds to heat
Note: The distinction between photon and heat transducers is important because shot
(Skoog, Pg: 112) noise often limits the behavior of photon transducer and thermal noise
often limits thermal transducers.

Photon Transducers
1. Also known as photoelectric detectors or
quantum detectors
2. Have an active surface that absorbs radiation
3. Sometimes, absorbed energy causes emission of
electrons and production of photocurrent.
or….
4. The radiation promotes electrons into conduction
bands
 Thus detection is based on enhanced conductivity
(photoconduction)

Photon Transducers
For measurement of
 UV possess enough energy
to cause photoemission
 Visible
to occur
 Near IR

Types of Photon Transducers
Photovoltaic cell
 Radiant energy generates a current at the interface of a semiconductor layer and a metal
Phototube (PT)
 Radiation causes emission of electrons from a photosensitive solid surface
Photomultiplier tube (PMT)

 Contains photoemissive surface as well as several additional surfaces that emit a cascade of
electrons when struck by electrons from the photosensitive area
Photoconductivity
 Absorption of radiation by a semiconductor produces electrons and holes, thus leading to
enhanced conductivity
Silicon photodiode/Photodiode arrays (PDA)
 Photons can cause the formation of electron hole pairs and a current across a reverse-biased
pn junction
Charge-transfer device (CTD)
 Charges developed in a silicon crystal as a result of absorption of photons are collected and
measured

Phototube (PT)
1. Consists of semicylindrical
cathode: Supports a layer of
photoemissive material that tend to
emit electron when it is radiated.
Variety of photoemissive materials:
highly sensitive, red sensitive, UV
sensitive, flat response
2. Wire anode:
Sealed in an evacuated transparent
envelope

More info on Pg 193
Phototube
 Works via a photoelectric effect

 A photon hits the cathode which is covered with a
photoemissive surface
 Emits electrons when it is irradiated
 When a voltage is applied across the electrodes, the emitted
electrons flow to the wire anode generating
 Generating a photocurrent
 The current is small
 Amplification is easily accomplished because PT has a high
electrical resistance Prepared by Dr.Kavirajaa, Ph.D., AMRSC
Phototube
1. The number of electrons ejected from the photoemissive surface is directly

proportional to the radiant power of the beam that strikes that surface.
2. Current directly proportional to the intensity of the photon.
3. PT frequently produce a small dark current that results from thermally
induced electron emission and natural radioactivity from 40K in the glass
housing of the tube.
4. Use UV transparent window to be UV sensitive.

Photomultiplier tube (PMT)
1. PMT usually used for low radiant power measurements.
2. Photocathode surface is similar to PT.
3. The incoming photon will cause the electron to be ejected by the initial
cathode.
4. Signals are amplified using a series of electrodes known as dynodes
(additional nine electrodes).
Working principle of PMT:

1. Each photon energy that strikes the dynodes causes emission of several
additional electons.
2. The process repeated at each dynodes (9 times ~ 106 – 107) and finally
cascade electrons collected at the anode and resulting current is then
converted to voltage and measured.

More info on Pg 194-195
PMT amplification (106–
108) gives higher
sensitivity!
Silicon Photodiodes
 are semiconductor light sensors that
generate a current or voltage when p-n
junction in the semiconductor is
illuminated by light
 Current is proportional to the amount of
radiation
 more sensitive than PT
 less sensitive than PMT
 Spectral ranges from 190 to 1100 nm

Si Photodiodes Arrays (PDA)
 Information about the entire spectrum is accumulated
simultaneously in discrete elements rather than
continuously
 Equivalent to that recorded during scanning
 Cannot match the performance of PMT with respect to:
 Sensitivity
 Dynamic range
 Signal-to-noise ratio
 Thus, they have been used most often for applications in
which high sensitivity and large linear range are not
required
 HPLC coupled to UV-vis absorption spectrometry
Array detectors allow simultaneous
detection of multiple wavelengths and
analytes! Prepared by Dr.Kavirajaa, Ph.D., AMRSC
PDA
 Si photodiode arrays consist of multiple elements

of the same size, formed in a linear or matrix
arrangement at an equal spacing in one package.
 Can measure several wavelengths at once

Thermal transducers
• photon energies in the IR region of the

spectrum are insufficient to cause
photoemission of electrons
• IR radiation is detected by
– Thermal transducers
– Photo-conductive transducers

Thermal transducers
1. Radiation impinges (restricts) on and is absorbed by
a small blackbody (an object that absorb all
electromagnetic radiation that falls on it) and the
resultant temperature rise is measured.
2. The radiant power from IR beam is
extremely small (10-7 – 10-9W) so that,
 The heat capacity of the absorbing element must
be as small as possible
 To detect a temperature change

Thermal transducers
Thermocouples
Bolometers
Pyroelectric transducers

Thermocouples
1.In it’s simplest form, a thermocouple

consists of a pair of junctions formed when
two dissimilar metals are welded together.
2.A voltage develops between the two

junctions with the different in their
Temperatures.

Bolometers
Resistance thermometer constructed of

strips of metals or semiconductors
Semiconductor bolometers
Known as thermistors
A large change in resistance as a
function of temperature

Signal processor and read-outs
All instruments are expected to have
Signal processor
Proper amplification to produce measurable
signal
Signal processing to remove, average data, drive
a readout, AC/DC conversion
A readout – chart, plot ….
Digital meters, recorders, LCD panels
Numerical processing capability
Most spectrometers use a PC
Performance characteristics of
instruments
Precision
Bias
Sensitivity
Detection limit
Dynamic range
Selectivity

Other characteristics
Speed
Ease and convenience
Skill required by operators
Cost
Availability of equipment
Per sample cost

Numerical Criteria for selecting
Analytical Methods

Figures of merits for precision
of analytical methods

Key Points of Comparison of
Instrumental Methods
 Selectivity – What does the instrument respond to? Universality vs.
specificity
 “Sensitivity” – What is the detection limit (LOD)?
 Speed of analysis (throughput)
 Cost per analysis
 Precision (%RSD) – How similar are the results for repeated analysis
of the same sample?
 Accuracy – How close is your result to the ‘true’ answer?
 Reproducibility – If you analyze a sample today, and the same
sample one year from now, how close will the results be?
 Compatibility – Given the chemistry of the molecule, does it make
sense to use method X to analyze analyte Y?

CHARACTERISTICS OF AN ANALYTICAL METHODS
Accuracy: The degree to which an experimental result

approaches the true or accepted answer.
Ways to Describe Accuracy:
Error: An experimental measure of accuracy. The difference between

the result obtained by a method and the true or accepted value.
Absolute Error = (X – m)
(X  m)
Relative Error (%) =  100
m
where: X = The experimental result
m = The true result
All Methods, except counting, contain errors – do not know “true” value
Two types of error: random or systematic

Random Error: results in a scatter of results centered on the true

value for repeated measurements on a single sample.
Systematic Error: results in all measurements exhibiting a definite

difference from the true value
Random Error Systematic Error
Plot of the number of occurrences or population

Prepared of each
by Dr.Kavirajaa, Ph.D., AMRSC
measurement (Gaussian curve)

Precision
 Degree of mutual agreement among data obtained

in the same way
 A measure of random or indeterminate error of an
analysis

Precision: The reproducibility of results. The degree to which an
experimental result varies from one determination to
the next.
Precision is related to random error and Accuracy is related to

systematic error.
Illustrating the difference between “accuracy” and “precision”
Low accuracy, low precision Low accuracy, high precision
High accuracy, low precision High accuracy, high precision

Ways to Describe Precision:
Range: the high to low values measured in a repeat series of

experiments.
Standard Deviation: describes the distribution of the measured results about

the mean or average value.
Absolute
n Standard Deviation (SD):
SD   ( X
i 1
i  X ) 2
/(n  1)
Relative Standard Deviation (RSD) or

Coefficient of Variation (CV):
RSD(%)  ( SD / X )100
where: n = total number of measurements

Xi = measurement made for the ith trial
X Prepared by Dr.Kavirajaa, Ph.D., AMRSC
= mean result for the data sample

Response: The way in which the result or signal of a method
varies with the amount of compound or property being
measured.
Ways to Describe Response:
Calibration Curve: A plot of the result or signal vs. the known amount of a
known compound or property (standard) being measured.
s ulfate calibration curve

y = 14427x - 12024
R2 = 0.999
1400000
1200000
1000000
peak area
800000
600000
400000
200000
0
0 10 20 30 40 50 60 70 80 90
conce ntration (ppm )
by area Linear (by area)

Sensitivity
 A measure of its ability to discriminate between

small differences in analyte concentration
 is related to the slope of a calibration curve.
 A steeper calibration curve
 More sensitive
Analytical sensitivity = γ = slope/std. dev.

Parameters used to Describe a Calibration Curve: S = mc + Sbl

S – measured signal
c – analyte concentration
Sbl – instrument signal for blank
Sensitivity: calibration sensitivity = slope (m) of calibration curve.

analytical sensitivity () = slope (m)/standard deviation (Ss)
Ability to discriminate between small 70
differences in analyte concentration.

60
Slope and reproducibility of the
calibration curve. 50
Method A
40
30
Method B
20
10
0
0 2 4 6 8 10 12
Concentration (mM)
Selectivity: degree to which the method is free from interference by other
species in the sample
70
No method is totally free from
60 interference from other species.
50
Selectivity coefficient (k):
40
Species A
kB,A = mB/mA
30
20
Species B Relative slopes of calibration
10
curves indicate selectivity:
0
0 2 4 6 8 10 12
S = mA(cA + kB,Acb) + Sbl
Concentration (mM)
Interested in detecting species A, but signal will be abycombination

Prepared Dr.Kavirajaa, Ph.D., of signal
AMRSC
from the presence of species A and species B.

Limits of Detection (cm ): (minimum analyte signal (Sm) - mean blank signal(Sbl ))/slope(m)
minimum/maximum concentration or mass of analyte that can be

detected at a known confidence level.
Signal-to-noise Ratio (S/N):

Noise: random variation in signal or background
Signal: net response recorded by a method for a sample
(Note: a value of S/N = 2 or better is considered to be the minimum ratio

needed for the reliable detection of a true signal from a sample.)
Estimate S/N:
signal
1) Multiple determination of
blank samples.
noise 2) Estimation of best-fit to
calibration curves

Detection limit
 The minimum concentration or mass of analyte that can be detected at a known

confidence level
(cm ) = (minimum analyte signal (Sm) - mean blank signal( ))/slope(m)
Sbl

Dynamic range
Extends from
 the lowest concentration at which quantitative
measurements can be made (LOQ)
to
 The concentration at which the calibration departs
from linearity by a specified amount (LOL)
 Upper limit
 a deviation of 5% from linearity

Dynamic Range: linear region of calibration curve where the lower limit is ten times
the standard deviation of the blank.
LOQ - limit of quantitation
LOL - limit of linearity

Example 1: The data in the table below were obtained during a colorimetric
determination of glucose in blood serum.
Glucose Concentration, Absorbance, A
mM
0.0 0.002
2.0 0.150
4.0 0.294
6.0 0.434
8.0 0.570
10.0 0.704
A serum sample gave an absorbance of 0.350. Find the glucose concentration and its
standard deviation, calibration sensitivity, detection limit and dynamic range.

Selectivity
 The degree to which the method is free from

interference by other species contained in the
sample matrix
 No method is free from interference
 Minimize the effects of interference

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Chapter 2 - Introduction of Optical Instrument

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CHAPTER 2:

Prepared by Dr.Kavirajaa, Ph.D., AMRSC

Prepared by Dr.Kavirajaa, Ph.D., AMRSC

Prepared by Dr.Kavirajaa, Ph.D., AMRSC

The actual arrangement of the components will vary based on the

Prepared by Dr.Kavirajaa, Ph.D., AMRSC

A source must generate a beam of radiation

TWO TYPES OF SOURCES: Continuum sources and Line sources

Prepared by Dr.Kavirajaa, Ph.D., AMRSC

Sources for molecular absorbance and luminescence methods

Prepared by Dr.Kavirajaa, Ph.D., AMRSC

(a) continuum source

Prepared by Dr.Kavirajaa, Ph.D., AMRSC

Prepared by Dr.Kavirajaa, Ph.D., AMRSC

Prepared by Dr.Kavirajaa, Ph.D., AMRSC

Prepared by Dr.Kavirajaa, Ph.D., AMRSC

1. Radiation for spectroscopic analyses consists of a limited, narrow,

Prepared by Dr.Kavirajaa, Ph.D., AMRSC

Prepared by Dr.Kavirajaa, Ph.D., AMRSC

Prepared by Dr.Kavirajaa, Ph.D., AMRSC

Prepared by Dr.Kavirajaa, Ph.D., AMRSC

Prepared by Dr.Kavirajaa, Ph.D., AMRSC

Czerny-Turner grating monochromator -

Cheaper fabrication, better λ Most of the modern

Prepared by Dr.Kavirajaa, Ph.D., AMRSC

Prepared by Dr.Kavirajaa, Ph.D., AMRSC

We need a method of detecting any

Prepared by Dr.Kavirajaa, Ph.D., AMRSC

Transducers convert power (P) of

Prepared by Dr.Kavirajaa, Ph.D., AMRSC

S = current/voltage output of transducer Prepared by Dr.Kavirajaa, Ph.D., AMRSC

Prepared by Dr.Kavirajaa, Ph.D., AMRSC

Prepared by Dr.Kavirajaa, Ph.D., AMRSC

Prepared by Dr.Kavirajaa, Ph.D., AMRSC

Photomultiplier tube (PMT)

Prepared by Dr.Kavirajaa, Ph.D., AMRSC

Prepared by Dr.Kavirajaa, Ph.D., AMRSC

 Works via a photoelectric effect

1. The number of electrons ejected from the photoemissive surface is directly

Prepared by Dr.Kavirajaa, Ph.D., AMRSC

Working principle of PMT:

Prepared by Dr.Kavirajaa, Ph.D., AMRSC

Prepared by Dr.Kavirajaa, Ph.D., AMRSC

 Si photodiode arrays consist of multiple elements

Prepared by Dr.Kavirajaa, Ph.D., AMRSC

• photon energies in the IR region of the

Prepared by Dr.Kavirajaa, Ph.D., AMRSC

Prepared by Dr.Kavirajaa, Ph.D., AMRSC

Prepared by Dr.Kavirajaa, Ph.D., AMRSC

1.In it’s simplest form, a thermocouple

2.A voltage develops between the two

Prepared by Dr.Kavirajaa, Ph.D., AMRSC

Resistance thermometer constructed of

Prepared by Dr.Kavirajaa, Ph.D., AMRSC

Prepared by Dr.Kavirajaa, Ph.D., AMRSC

Prepared by Dr.Kavirajaa, Ph.D., AMRSC

Prepared by Dr.Kavirajaa, Ph.D., AMRSC

Prepared by Dr.Kavirajaa, Ph.D., AMRSC

Prepared by Dr.Kavirajaa, Ph.D., AMRSC

Accuracy: The degree to which an experimental result

Ways to Describe Accuracy:

Error: An experimental measure of accuracy. The difference between

Two types of error: random or systematic

Random Error: results in a scatter of results centered on the true