1.

SPECTROPHOTOMETRY BASIC CONCEPTS, INSTRUMENTATION AND

APPLICATION

2. 2. INTRODUCTION: • Photometry is the measurement of the

amount of luminous light (Luminous Intensity) falling on a surface
from a source. • Spectrophotometry is the measurement of the
intensity of light at selected wavelengths. • The method depends on
the light absorbing property of either the substance or a derivative
of the substance being analyzed • Spectrophotometers use prisms
and gratings for isolating wavelengths while devices that require
filters for this purpose are called Filter Photometers.

3. NATURE OF LIGHT: • Electromagnetic waves are characterized by

their frequency and wavelength. • Light is a spectrum of different
wavelengths which the eye recognizes as “white” but can be
isolated into discrete portions and measured. • Human eye responds
to radiant energy btw 380 and 750nm, but modern instruments can
measure shorter wavelengths (UV) and longer (IR) ones. •
Wavelength describes a position within a spectrum. It is the distance
btw 2peaks as the light travels in a wave-like manner • Light also is
composed of discrete energy packs called photons whose energy is
inversely proportional to the wavelength 3

3. BASIC CONCEPTS: • When light passes through a solution, a

certain fraction is being absorbed. • This fraction is detected,
measured and used to relate the light absorbed or transmitted to
the concentration of the substance. • This enables both qualitative
and quantitative analyses of substances. • The spectrophotometric
technique is used to measure light intensity as a function of
wavelength. It does this by: – Diffracting the light beam into a
spectrum of wavelengths – Direct it to an object – Receiving the light
reflected or returned from the object – Detecting the intensities with
a charge-coupled device – Displaying the results as a graph on the
detector and then the display device

5. • The light absorption is directly related to the concentration of

the compound in the sample. • As Concentration increases, light
Absorption increases linearly and light Transmission decreases,
exponentially 5

6. Transmittance and Absorbance: • When a sample is illuminated, it

absorbs some of the light and transmits the rest. • The transmitted
light (Is) is of lower intensity than the incident light (Io), and the
transmitted light is defined as: T = Is / Io 6

7. • To ensure accuracy (by eliminating effects of reflection by

surface of the cell, absorption by the cell wall and by solvent) an
identical reference cell without the compound of interest is also
used. • Thus, the amount of light absorbed (A) as the incident light
passes through the sample is equivalent to: A = - log Is / IR = - log T
• In practice, the Reference cell is inserted and the instrument
adjusted to an arbitrary scale corresponding to 100% transmittance,
after which the percentage transmittance reading is made on the
sample 7

8. Beer’s Law: • This states that the concentration of a substance if

directly proportional to the amount of light absorbed or inversely
proportional to the logarithm of transmitted light. A = abc Where: A
= Absorbance a = proportionality constant defined as absorptivity b
= light path in centimeters c = concentration in g/L of the absorbing
compound NB: - Absorbance (A) has no units, so the unit for a =
reciprocals those for b and c. • When b is 1cm and c is expressed in
mol/L, ἐ (epsilon) is substituted for the constant, a. • The value of ἐ
is a constant for a given compound at a given wavelength under
prescribed conditions of solvent, temperature, pH, etc., and is called
the Molar absorptivity (ἐ). 8

9. Spectrometry Nomenclature: Application of Beer’s Law: • In

practice, a direct proportionality between absorbance and
concentration must be established for a given instrument under
specific conditions. • A linear relationship exists up to a certain
concentration or absorbance beyond which the solution is said to no
longer obey Beer’s Law • Within this limitation, a calibration
constant (K) may be derived and used to calculate the concentration
of unknown solutions by comparison 9

10. Recall, a = A/bc Thus, A1 / b1c1 = A2 / b2c2 Where 1 and 2

refers to the calibrating (c) and unknown (u) solutions respectively •
But because the Light path b remains constant, b1 = b2 • Then,
A1/c1 = A2 /c2 or Ac/cc = Au/cu • Hence, concentration of the
unknown: cu = Au/Ac x cc And, cu = Au x cc / Ac = Au x K Where, K
= cc / Ac 10

11. • The constant cannot be used once Beer’s Law have been
violated • A non-linear calibration curve can be used if a sufficient
number if calibrators of varying concentration is included to cover
the entire range encountered for reading on the unknowns •
Published constants should be used only if the method is followed in
detail and readings are made on a spectrophotometer capable of
providing light of high spectral purity at a verified wavelength. • Use
of broader band light leads to some decrease in absorbance 11

12. • Beer’s Law is followed only if the following conditions are met:
– Incident radiation on the substance of interest is monochromatic –
Solvent absorption is insignificant compared to the solute
absorbance – Solute concentration is within given limits – Optical
interferant is not present – Chemical reaction does not occur
between the molecule of interest and another solute or solvent
molecule. 12

13. INSTRUMENTATION: The Spectrophotometer 13

14. • The basic components of a spectrophotometer include: a light

source, a means to isolate light of desired wavelength, fiber optics,
cuvets, a photodetector, a readout device, a recorder and a
computer. • Three different types of the device available are: The
Single Beam Spectrophotometer: 14

15. Double-beam-in-space Spectrophotometer: Double-beam-in-time

Spectrophotometer: 15

16. Light Sources: • This provides a sufficient amount of light which

is suitable for making a measurement. • The light source typically
yields a high output of polychromatic light over a wide range of the
spectrum. Electromagnetic spectrum: • Types of light sources used
in spectrophotometers include: Incandescent lamps and lasers.

17. Incandescent Lamps: • Tungsten Filament Lamp: The most

common source of visible and near infrared radiation ( at
wavelength 320 to 2500 nm) • Deuterium lamp: Continuous
spectrum in the ultraviolet region is produced by electrical
excitation of deuterium at low pressure. (160nm- 375nm) •
Hydrogen Gas Lamp and Mercury Lamp, Xenon (wavelengths from
200 to 800 nm): high-pressure mecury and xenon arc lamps are
commonly used in UV absorption measurements as well as visible
light. • Globar (silicon carbide rod): Infra-Red Radiation at
wavelengths: 1200 - 40000 nm • NiChrome wire (750 nm to 20000
nm); ZrO2 (400 nm to 20000 nm): for IR Region 17

18. Laser Sources: • These devices transform light of various

frequencies into an extremely intense, focused, and nearly non-
divergent beam of monochromatic light • Through selection of
different materials, different wavelengths of light emitted by the
laser are obtained. • Used when high intensity line source is
required • Unique properties of laser sources include: – Spatial
coherence: a property that allows beam diameters in the range of
several microns – Production of monochromatic light – Have pulse
widths that vary from microseconds to (flash lamp-pulsed lasers) to
nanoseconds (nitrogen lasers), to picoseconds or less (mode-locked
lasers) 18

19. Spectral Isolation: • A system for isolating radiant energy of a

desired wavelength and excluding that of other wavelength is called
a Monochromator. • Monochromator consists of these parts: –
Entrance slit – Collimating lens or mirror – Dispersion element: A
special plate with hundreds of parallel grooved lines. The grooved
lines act to separate the white light into the visible light spectrum – .
19
20. • Focusing lens or mirror: Combinations of lenses, slits, and
mirrors which relays and focuses light through the instrument • Exit
slit 20

21. • Devices used for spectral isolation include: Filters, Prisms, and
Diffraction gratings. • Variable slits are also used to permit
adjustments in total radiant energy reaching the photocell 21

22. • The spectral purity of a monochromator is usually described as

its spectral bandwidth – width, in nm, of the spectral transmittance
curve at a point equal to half the peak transmittance • Filters: –
Simplest type is a thin layer of coloured glass which is not a true
monochromator because it transmits light over a relatively wide
range of wavelength 22

23. – Commonly used glass filters have spectral bandwidth of about

50nm and are refered to as wide-bandpass filters – a cut-off filter
shows a sharp rise in transmittance over a narrow portion of the
spectrum and is used to eliminate light below a given wavelength –
Narrow-bandpass filter is constructed by combining two filters like
shown above, however, the availability of high intensity light
sources now favours the use of narrow bandpass interference filters.
– Alternatively, narrow bandpass filter can be constructed by use of
dielectric material of controlled thickness sandwiched btw two
silvered pieces glass • Prisms and Gratings: – Prisms seperates
white light into a continous spectrum by refraction with shorter
wavelengths being bent or refracted more than longer ones 23

24. – Diffraction gratings are prepared by depositing a thin layer of

aluminuin-copper alloy on the surface of a flat glass plate, then
ruling many small parallel grooves into the metal coating Selection
of a Monochromator: – Narrow spectral bandwidth – for resolving
and identifying sharp absorption peaks that are closely adjacent. –
Lack of agreement with beer’s law will occur when a part of the
spectral energy transmitted by the monochromator is not absorbed
by the analyte as commonly observed with wide-bandpass
instruments – Increase in absorbance and improved linearity with
concentration is usually observed with instruments that operate at
narrower bandwidths of light 24

25. Cuvets: • This is a small vessel used to hold a liquid sample to

be analyzed in the light path of a spectrophotometer. – May be
round, square or rectangular and are constructed from glass, silica
(optical grade quartz) or plastic. – It should be without impunities
that may affect spectrophotometric readings – Reference solution
must be transparent to the radiation which will pass through them. –
Quartz or fused crystalline silica cuvettes for UV spectroscopy. –
Glass cuvettes for Visible Spectrophotometer. – NaCl and KBr
Crystals for IR wavelengths. 25

26. Photodetectors: • These are devices that convert light into an

electric signal that is proportional to the number of photons striking
its photosensitive surface. • The photocell and phototube are the
simplest photodetectors, producing current proportional to the
intensity of the light striking them • The Photomultiplier tube (PMT)
is a commonly used photodetector for measuring light intensity in
the UV and Visible region of the spectrum. They are extremely rapid,
very sensitive and slow to fatigue. 26

27. • The PMT consists of: – A photoemissive cathode (a cathode

which emits electrons when struck by photons) – Several dynodes
(which emit several electrons for each electron striking them) – An
anode – Produces an electric signal proportional to the radiation
intensity – Signal is amplified and made available for direct display –
A sensitivity control amplifies the signal – Examples: Phototube (UV);
Photomultiplier tube (UV-Vis); Thermocouple (IR); Thermister (IR) 27

28. • Other photodetectors include: Barrier layer cells (photovoltaic

cells), Photodiodes, • Photodiodes are made of photosensitive semi-
conductor materials like silicon, gallium, arsenide etc which absorb
light over a characteristic wavelength range e.g 250nm to 1100nm
for silicon. They are capable of measuring light at a multitude of
wavelengths. 28
29. Display or Readout Devices: • Electrical energy from the
detector is displayed on a meter or readout system such as an
analog meter (obsolete), a light beam reflected on a scale, or a
digital display, or LCD • Digital readout devices operate on the
principle of selective illumination of portions of a blank of light
emitting diodes (LEDs), controlled by the voltage signal generated. •
Compared to meters, digital read out devices have faster response
and are easier to read 29

30. Computers: • Using the computer; – output from calibrator is

digitally stored – digital signals from blanks are subtracted from
calibrators and unknowns, and – the concentration of unknowns is
automatically calculated • Data from multiple calibrators often are
used to: – store a complete calibration curve – display or print out
the curve for visible inspection – calculate result of unknowns based
on the curves or some mathematical transformation of the data •
Computers are also used to convert kinetic data into concentration
or enzyme activity. 30

31. Recorders: • Spectrophotometers may be equipped with

recorders in addition to or instead of a digital display • They are
synchronized to provide line traces of transmittance or absorbance
as a function of either time or wavelength. • When a continuous
tracing of absorbance versus wavelength is recorded, the resultant
figure is called an absorption spectrum. If a substance absorbs light,
distinct peaks of absorbance will be observed. • Measuring the
absorption spectra of an unknown sample and comparing them with
spectra from known compounds is very useful for qualitative
purposes. 31

32. PERFORMANCE PARAMETERS: Parameters tested to verify that

the spectrophotometer is performing satisfactorily include: •
Wavelength Accuracy: – Holmium oxide in dilute perchloric acid is
used for calibration of Narrow-spectral-bandwidth instruments –
sharp absorbance peaks are obtained at defined wavelengths which
can be compared with established values. – Didymuin filter is used
in broader-bandpass instruments • Spectral Bandwidth: • Width of
and emission band at half-peak height – Maybe calculated from the
manufacturer’s specifications – Interference filters maybe used to
check the instrument32
33. • Stray Light: • radiation of wavelengths outside the narrow
band nominally transmitted by the monochromator that hits the
detector – Defined as a ratio or percent of the total detected light –
Causes an absorbance error and can be minimized by an inbuilt
stray light filter – Cutoff filters are used for the detection of stray
light which they are capable of absorbing with 0% transmission. In
the UV range, liquid cutoff filters are very effective – Emanates from
scattering and diffraction inside the monochromator, light leaks, and
fluorescence of the sample • Photometric Accuracy: • Neutral
density filters are used to check an instrument’s photometric
accuracy. – Solutions of potassium dichromate may be used for
overall checks on photometric accuracy which is compared with
literature values. 33

34. APPLICATIONS: 1. Measurement of Concentration: – Prepare

samples – Make series of standard solutions of known
concentrations – Set spectrophotometer to the λ of maximum light
absorption – Measure the absorption of the unknown, and from the
standard plot, read the related concentration 34

35. 2. Detection of impurities: – UV absorption spectroscopy is one

of the best methods for determination of impurities in organic
molecules – Additional peaks can be observed due to impurities in
the sample and it can be compared with that of standard raw
material 35

36. 3. Elucidation of the structure of Organic Compounds: • From the

location of peaks and combination of peaks UV spectroscopy
elucidate structure of organic molecules: – the presence or absence
of unsaturation, – the presence of hetero atoms 4. Chemical
Kinetics: – Kinetics of reaction can also be studied using UV
spectroscopy. The UV radiation is passed through the reaction cell
and the absorbance changes can be observed 36

37. 5. Detection of Functional Groups: – Absence of a band at

particular wavelength regarded as an evidence for absence of
particular group 6. Molecular weight determination: – Molecular
weights of compounds can be measured spectrophotometrically by
preparing the suitable derivatives of these compounds. – For
example, if we want to determine the molecular weight of amine
then it is converted in to amine picrate 37

38. REFLECTANCE PHOTOMETRY • In Reflectance photometry,

diffused light illuminates a reaction mixture in a carrier and the
reflected light is measured. – Alternatively, the carrier is illuminated
and the reaction mixture generates a diffuse reflected light which is
measured. • The intensity of the reflected light from the reagent
carrier is compared with the intensity of light reflected from a
reference surface. • The intensity of reflected light is non-linear in
relation to the concentration of analyte, – The data is first converted
to linear format using the Kubelka- Munk equation or the Clapper-
Williams transformation. • Reflectance photometry is used as the
measurement method with dry-film chemistry systems. • The
Electro-optical components used in reflectance photometry are
essentially the same as that used in absorbance photometry. 38

39. FLAME EMISSION SPECTROPHOTOMETRY • This is based on the

characteristic emission of light by atoms of many metallic elements
when given sufficient energy, such as that supplied by a hot flame •
The wavelength to be used in the measurement of an element
depends on the selection of a line of sufficient intensity to provide
adequate sensitivity and freedom from other interfering lines at or
near the selected wavelength (e.g lithium – red, sodium – yellow,
potassium – violet, rubidium – red, magnesium – blue) • These
colours are characteristic of the metal of the metal atoms present as
cations in solution. • Under constant and controlled conditions, the
intensity of the characteristic wavelength produced by each of the
atoms is directly proportional to the number of atoms that are
emitting energy, which in turn is directly proportional to the
concentration of the substance of interest in the sample.