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
Io impinging 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.
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
EQUATION
A = 2 – log %T
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
AαC
Laws of light absorption:
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
AαL
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
=
=
CT =
AT
AS
X CS
Concentration of TEST solution
Absorbance of TEST
Absorbance of STANDARD
Concn of STANDARDX=
Concentration of TEST /100ml
Absorbance of TEST
Absorbance of STANDARD
Concn of Std X 100X=
Xml
Concentration of TEST /100ml
Absorbance of TEST
Absorbance of STANDARD
X
=
Xml
Concn of Std X 100
Concentration of TEST /100ml
O.D of ‘T’- O.D of ‘B’
O.D of ‘S’- O.D of ‘B’
X
=
Volume of ‘T’
Amount of ‘S’ X 100
Concentration of TEST /100ml
T - B
S - B
X=
Volume of ‘T’
Amount of ‘S’ X 100
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 spectrum2. Tungsten iodide lamps – visible & near UV3. Hydrogen & deuterium discharge lamps – cont UV4. Mercury vapour lamps – Discontinous/line
spectrum5. 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 filterb. Those based on the principle of inteference.
a. Simpleb. wide wavelengthc. 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 dispersionPRISMS:
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
4. EXIT SLIT:
Determines the band width of light that will be selected from the dispersed spectrum.
1000-2000line/mm
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:
a. Photomultiplier tubes
b. Photodiodes
c. Charged coupled devices
a. Photomultiplier tubes: An electron tube that is capable of significantly amplifying a current.
Ideal detector : high sensitivity, high signal/noise, constant response for λs, and fast response time.
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
Light measurement only in visible region
Filters Can choose only a
bandwidth of wavelength Only coloured solutions measured
Absorbance-less accurate
SPECTROPHOTOMETERS
UV, visible, IR
Diffraction gratings, prisms Can choose exact
wavelength, Colourless solution can also be measured
Absorbance –more accurate Kinetic studies and spectrum
can be better studied
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.
QUALITY CONTROL CHECK FOR SPECTROPHOTOMETER
satisfactory if 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
1. WAVELENGTH CALIBRATION
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
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
2. SPECTRAL BANDWIDTH
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
3.STRAY RADIATION
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
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.
4. LINEARITY
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
5. PHOTOMETRIC ACCURACY
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
6. MULTIPLE WAVELENGTH READINGS
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
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)
APPLICTIONS OF SPECTROPHOTOMETER
Thank You