Você está na página 1de 45

# PHOTOMETRY

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

-Absorbing
-Emiting
EMISSION PROCESS

##  Used in flame photometry & flurometric methods.

ABSORPTION SPECTROSCOPY

##  Ioimpinging on & passing through a

square cell that contain solution of compound that absorb
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:

absorbance.

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
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.

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
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.
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
 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

##  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
 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
 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)