photometry i anu

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