PHOTOMETRY

Presenter: Dr. Anurag Yadav

Moderator: Mr. Arun kumar
 What is light?
 How light interact with matter.

Is form of energy E=hv V=1/λ

Energy is inversely related to wavelength.

Matter react to the light either by

-Absorbing
-Emiting
EMISSION PROCESS

 When electron return to ground state, the energy is

dissipated as radiant energy.

 Used in flame photometry & flurometric methods.

ABSORPTION SPECTROSCOPY

 Ioimpinging on & passing through a

square cell that contain solution of compound that absorb
radiant energy of certain wavelength.
The intensity Is is less than Io.

The transmittance in solution is defined as proportion of

the incident light that is transmitted
T = Is/Io
 As the conc of the compound in solution increases more
the light is absorbed by the solution & less is the light is
transmitted.
EQUATION
T = IE /Io
% T= IE /Io X 100%
A = - log T
A = log 1/T
To convert T to %
A = log 1/T X 100%/100%= log 100%/%T
Rearranging
A = log 100%- log%T

A = 2 – log %T
Laws of light absorption:

BEER’S LAW
• The concentration of a
substance is directly
proportional to the amount of
light absorbed or inversely
proportional to the logarithm
of the transmitted light
 Beer’s law

Beer’s law
LAMBERT’S LAW
• When a ray of
monochromatic light
passes through an
absorbing medium its
intensity decreases
exponentially as the
length of the light path
through light absorbing
material increases
LAMBERT’S LAW
 Bcz of linear relationship btwn absorbance and
concentration, it is possible to relate unknown conc to
single std by a simple proportional equation;
As Cs
Au Cu

Cu Au x Cs
As
 AT
X CS
CT =
AS

Concentration Absorbance of TEST X Concn of STANDARD

of TEST =
solution Absorbance of STANDARD

Concentration Absorbance of TEST X Concn of Std X 100

of TEST /100ml =
Absorbance of STANDARD Xml
Concentration of Absorbance of TEST X Concn of Std X 100
TEST /100ml =
Absorbance of STANDARD Xml

Concentration of O.D of ‘T’- O.D of ‘B’ X Amount of ‘S’ X 100

TEST /100ml =
O.D of ‘S’- O.D of ‘B’ Volume of ‘T’

T-B Amount of ‘S’ X 100

Concentration of X
=
TEST /100ml
S-B Volume of ‘T’
LIMITATIONS:

 Very elevated conc cant be measured.

 Incident radiant energy is not monochromatic.

 Solvent absorption is significant compared to solute

absorbance.

 Radiant energy is transmitted by other mechanism (

stray light).

 The sides of the cell are not parallel.

Note on Stray Light:
- Is radiant energy that reaches the detector at
wavelength other than those indicated by
monochromator setting.

- All radiant energy that reaches detector with/without

having passed through the sample is recorded.

- As the amount of the stray light increases, deviation

from the Beer’s Law also increases.
1. Can be due to Light leaks – excluded by covering cell
compartment
2. Fluorescence- that increases signal to the detector and
causes apparent decrease in A

 Most spectrophotometers are equipped with stray light filters

SPECTROPHOTOMETER:
DEFINITIONS
PHOTOMETER:
If a filter is used as a wavelength selector,
monochromatic light at only discrete wavelength is
avialable & the instrument is called photometer.

SPECTROPHOTOMETER:
If a monochromater is used( prism/grating) as a
wavelength selector, the inst can provide monochromatic
light over a continous range of wavelengths & is called
spectrophotometer.
SPECTROPHOTOMETER:
TYPES:
1. Single beam spectrophotometer
2. Double beam in space spectrophotometer
3. Double beam in time spectrophotometer
4. Multichannel

COMPONENTS:
SOURCE:
1. Tungsten filament lamps – continous spectrum
2. Tungsten iodide lamps – visible & near UV
3. Hydrogen & deuterium discharge lamps – cont UV
4. Mercury vapour lamps – Discontinous/line spectrum
5. Light emitting diode(LED’s) – 2types of semiconductors

2. ENTRANCE SLIT:
Focuses light on grating/prism, where it can be
dispersed with minimum stray light.
 3. WAVELENGTH SELECTOR:
For isolation of a required wavelength/range of
wavelength.
2types-
a. Filters
b. Monochromators
1. FILTERS:
Consists of only a material that selectively transmits the
desired wavelength & absorbs the rest.
a. Those selective transmission characteristics-
glass & Wratten filter
b. Those based on the principle of inteference.

a. Simple
b. wide wavelength
c. Non adjustable
 2. MONOCHROMATORS:
A grating/prism disperses radiant energy from the
source lamp into a spectrum from which the desired
wavelength is isolated by mechanical slits.
Prism - Nonlinear dispersion
Grating - Linear dispersion

PRISMS:
a. Less linear over lower wavelength over
550nm
b. Give only 1 order of emerging
spectrum thus provide higher optical
efficiency
c. Therefore 3 wavelength checks are
required
b. GRATING:
a. Linear dispersion
b. Therefore only
2wavelength checks
required to certify
accuracy
1000-2000line/mm

4. EXIT SLIT:
Determines the band width of light that will be
selected from the dispersed spectrum.
5. CUVETTES/CELL:
a. Receptacle for sample
b. Optical property
depends on composition.
c. Calibrated to path length
1cm

6. PHOTODETECTORS:
A device that converts light into an electric signal that is
proportional to the number of photons striking its photosensitive
surface.
6. PHOTODETECTORS:
Ideal detector :
a. Photomultiplier tubes high sensitivity,
b. Photodiodes high signal/noise,
constant response for λs,
c. Charged coupled devices and fast response time.

a. Photomultiplier tubes:
An electron tube that is capable of significantly
amplifying a current.
b. PHOTODIODES:
Semiconductors that change their charged voltage upon
being struck by light.
Change is converted to current & measured.

c. Charged coupled devices:

Solid-phase devices that are made of small silicon
cells. Electron released is captured and quantified.

7. READOUT DEVICES:
Electric energy from detector is displayed on a meter or
display system
DOUBLE BEAM SPECTROPHOTOMETER:
Designed to compensate for possible variations in
intensity of light source.
Accomplished by splitting the light beam
DOUBLE BEAM SPECTROPHOTOMETER:

 ADVANTAGES OF A DOUBLE-BEAM OVER A

SINGLE-BEAM INSTRUMENT:
 Compensate for variations in the source intensity.
 Compensate for drift in the detector and amplifier.
 Compensate for variation in intensity as a function of
wavelength
MULTICHANNEL INSTRUMENTS

 Able to “scan” an entire

spectrum in ~ 0.1 sec
 high throughput of radiant
energy due to the minimal
optics
 use a deuterium lamp
source for a spectral range
of 200nm - 820 nm and
have a spectral bandwidth
of 2 nm.
COMPARISION
COLORIMETERS SPECTROPHOTOMETERS

 Light measurement only in  UV, visible, IR

visible region
 Filters  Diffraction gratings, prisms
 Can choose only a  Can choose exact

bandwidth of wavelength wavelength, Colourless

solution can also be
Only coloured solutions
measured
measured
 Absorbance –more accurate
 Absorbance-less accurate
 Kinetic studies and spectrum
can be better studied
QUALITY CONTROL CHECK FOR
SPECTROPHOTOMETER

 Wavelength accuracy: mercury vapor lamp, dueterium

lamp,(strong emmision lines), holmium oxide (strong
absorbtion lines).
 Linearity of detector response: solutions of varying
concentrations of compound(Beer’s law) Eg: oxyhb at
415nm, cobalt ammonium sulphate at 512nm
 Stray radiation : by LiCO3 below 250nm, NaBr below
240nm
 Photometric accuracy: pottasium dichromate soln,
cobalt ammonium sulphate soln
QUALITY CONTROL CHECK FOR
SPECTROPHOTOMETER

 NIST formerly NBS provide the SRM –useful

for calibration/verification of performance of the
instrument
 Eg- SRM 930e –verifies and calibrates T and A –
visible range of spectrophotometer.
 IRMM –provide reference material for
verification of performance of the instrument.
 listed in the IRMM BCR ref material catalogue.
1. WAVELENGTH CALIBRATION
 satisfactoryif its close to λmax of chromogen and if
its reproducible.
 H and Du lamps have built in sources of checking
accuracy
 Prisms(2) and gratings(3) – continuous choice of λ.

 Rare earth glass filters like holmium oxide and

didymium- narrow and wide spectral band widths
 Holmium oxide –
 280-360nm ,

 show sharp absorption

peaks at defined λ
 Another method is by use of solutions.
Disadvantage
absorption peaks are broad and causes spectral
shifts due to
1. contamination
2. aging or
3. preparation errors
2. SPECTRAL BANDWIDTH
 Hg vapor lamp that shows a no of sharp, well
defined emissions lines bet 250 - 580nm .
 Can be calculated from manufactures
specifications.
 Interference filters – 1-2 nm are available and
can be used to check spectral bandwidth of 8nm
or more
3.STRAY RADIATION
 Increases at extreme ends of spectral range
where detector response is lowest.
 Methods to detect

1. filters or solu that’s highly transmitting over a

portion of the spectrum but opaque below an
abrupt cutoff λ
 Egs

1. Li2 CO3 below 250nm

2. NaBr – below 240nm

3. Acetone- below 320nm

 Stray light can also be due to
1. Light leaks – excluded by covering cell
compartment
2. Fluorescence- that increases signal to the
detector and causes apparent decrease in A
 Most spectrophotometers are equipped with stray
light filters
 Blue filters- used with Tungsten lamps for λ
below 400nm

 Red filters- λ range 650 – 800nm

 Eg- spectrophotometer set to 350nm

-stray light - visible range
absorbed by the blue filter
transmits UV portion of spectrum
 If solu / filters –transmitting no radiant energy at measurement
λ
measured T = amount of stray light
T X 100% = % of stray radiation.
If stray light > 1% - instrument malfunction
Liquid cut off filters- UV range where there is more stray light
problem

 UV stray light filters

4. LINEARITY
 Spectrophotometer should exhibit a linear
relationship between radiant energy absorbed and
readout.
 Solid glass may be used for the above

Common method
 use of solu of varying conc of compound
following beer’s law
 DISADV – dilution errors, stability problems,
shifts in Ph,temp effects.
5. PHOTOMETRIC ACCURACY
 Absorbance std – constant stable A with no variation to
spectral band width / light beam.
 NIST –set of 3 neutral density glass filters with known A at 4
λ for each filter.
 They are not always stable- need recalibration by NIST
periodically
Standards for checking accuracy
 - potassium dichromate

 -cobalt amm sulfate

 -nitrate solu
6. MULTIPLE WAVELENGTH
READINGS
 Background interferences – min by including blank or taking
A at 2-3 λ.
 Bichromatic-A is measured at 2 λ

1. corresponds to peak A
2. at a point at the base of the peak serves as baseline.
 Diff in A is related to conc – gives a blank ref point for each
sample.
 Another method to correct background interference- measure
A at 2 λ equidistant from peak and latter is averaged to get a
baseline and that’s subtracted from the peak A –
CORRECTED A
 In cases of spectral overlap –
extinction coeff of each component at
each λ should be known.
 Eg – in blood Hb (red Hb ,oxy Hb
,carboxyHb ,meth Hb , sulfHb)
 ext coeff is known the matrix eq can
be set up to calculate each component
– principle used in COOXIMETERS
APPLICTIONS OF SPECTROPHOTOMETER
 Visible Spectrophotometer Application
- Niacin, Pyridoxine, Vitamin B12, Metal Determination (Fe),
Fat-quality Determination, Enzyme Activity (glucose oxidase)
 UV Spectrophotometer Application
-Protein, Amino Acids (aromatic), Pantothenic Acid, Glucose
Determination, enzyme Activity (Hexokinase)