PRINCIPLES

OF
PHOTOMETR
Y
Seminar By- Dr Manisha Guliya
JR Lab Medicine
Moderator- Dr Sudip (Assoc.Prof Lab med)
Dr Kundan (SR Lab med)
OVERVIEW
• Definition
• EM Spectrum
• Beer’s Lambert Law
• Transmittance and Absorbance
• Concept of Non Linearity
• Lambda max
• Standard curve
• Limitation of law
• Application of law
• Types of Photometry
• Colorimetry and Spectrophotometry
• Few terminologies
• Turbidimetry and Nephelometry
 PHOTOMETRY

• Photometry- word is composed of the Greek word Photo “light” & Metry
“measurement”
• It is the measurement of intensity of light
• It is the most commonly used analytical technique in clinical biochemistry
• Term photometric measurement originally measure light intensity independent
of wavelength
• Modern instruments (Spectrophotometers), however, isolate a narrow
wavelength range of the spectrum or measurements
Beer’s Lambert Law
Transmittance(T)
• It is the ratio of the intensity of light
emerging from the solution (It) to that of
incident light entering (Io)
• It is defined as proportion of the incident
light that get transmitted

T= I t

Io
% T= T
• Lambert derived a quantitative relationship between decrease in
intensity of a monochromatic light due to passage through a
homogenous medium of thickness x and the intensity of light I
• 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
• The path distance that travels in the
sample is proportional to the
absorbance
A b
 Beer’s Lambert Law
Beer’s Law-
• Lambert law was extended by Beer who showed that
the fraction of incident light absorbed is not only
dependent on the intensity of light but also on the
concentration of the solution
• It states that is amount of light absorbed A by a
substance in solution is directly proportional to the
concentration C of a substance & inversely
proportional to the logarithm of the transmitted light

A C
or
A log
 Absorbance
• It is the amount of light absorbed by a sample
• It is calculated from T or % T using the following equation
• A = - log T or A = - log10 (1/T)
• Equation- T = It/Io
%T = It/Io × 100
A = - log T
A = log 1/T
To convert T to %
A = log 1/T × 100 % , on rearranging
A = log (100%) – log(%) T
A = 2 – log %T
 Beer’s Law Lambert’s Law
AC Ab

A bC
A = bC

• Here is molar absorptivity

• A is unitless
• a has units M-1cm-1
Lambda max
• It is a characteristic wavelength for a UV
active compound
• It is also known as absorption
maxima/maximum absorbance
• It is a particular wavelength at which
substance absorbs maximum light in the
form of photon
• It is independent of concentration
 Standard curve
• RULE- The unknown must be within
range of the measuring device you are
using
LIMITATION OF LAW-

• Law is valid for low concentration analyte

• Only applicable to monochromatic light
• Temperature constant
• The solution should be homogenous and diluted
• Non linearity arises at high concentration
• Scattering of light due to particulates in sample
• Non monochromatic radiation
• Changes in refractive index
Application of law
The law finds application in various fields-
• Analytical chemistry-
 To determine the concentration of unknown absorbing substance
 Measurement of substance concentration such as protein,DNA or RNA,
growth or bacterial cells and enzymatic reaction
 This analysis mainly concentrates on the separation, quantification and
identification of matter by spectrophotometry without the need for
extensive pre-processing of the sample

• In Atmosphere-
 Solar or stellar radiation can be described using the law
Types of Photometry
• Colorimetry & Spectrophotometry
• Flame Emission Spectrophotometry
It measures light emitted by excited atoms, was widely used to determine
concentrations of Na, K, Li.
• Atomic absorption Spectrophotometry
It measures concentration by detecting the absorption of EM radiation by
atoms rather than molecules
• UV Visible Spectrophotometry
• Fluorometry
• Nephelometry and Turbidimetry
Principle of photometry is applied in
the following:
Measurement of light Equipment used (some ex)

 Absorbed or transmitted light  Colorimeter, Spectrophotometer,

Semiautoanalyser and Autoanalyser

 Emitted light  Turbidimeter, Flame photometer

 Reflected/Scatter light  Nephelometer

 Filters
• Filters are made up of colored glasses.
Filters are used for selecting light of
narrow wavelength
• Filters will absorb light of unwanted
wavelength and allow only
monochromatic light to pass through
• Example- A green filter absorbs all color,
except green light which is allowed to
pass through a green filter (500-560nm)
• Filter used is always complimentary in
color to the color of solution
In colorimetric estimation, it is necessary to prepare a Test,
Blank, Standard
• TEST- This solution is prepared by treating a specific volume of
specimen (Blood, urine, CSF, etc)
• STANDARD- Prepared by treating a solution of the pure substance
of concentrations
• BLANK- Is prepared to rule out color produced by reagents alone
a) Distilled water as blank
b) Reagent blank (reagent used in the estimation is taken as blank)
Standard, Control, Calibration & Blank
Beer’s Law-
A C
 Standard
• It is a solution based on which we
can compare colors and give correct
concentration
• It should be of known value and
stable
• Needs to be of similar matrix
 Matrix
• Consider a normal plasma test for
glucose
• In plasma, all the components apart
from glucose makes up the “matrix of
the sample”
Usually all the biochemical kits (especially the kits for semiautoanalyser) come with
standards.

• PRIMARY STANDARD:
Weigh actual quantity of standard and dissolve it in accurate amount of matrix- all
measurement need to be by the most perfect system of measurement

• SECONDARY STANDARD:
Made by comparing standard to primary standard. This is what commonly being
used by all the kits
 CONTROL
• We do a test by comparing our
results with a standard.
• Whatever test we do we will get a
result
• But the most important question-
is my result correct ?
 Levels of control
Control can have different levels-
• Normal level control- Here the control has the analyte in the normal
range and we test if we get correct result or not

• High level control- When I want to know the system gives good results
when testing parameters at high range i.e. (for ex when creatine is
6.3mg/dl)

• Low level control- While doing platelet count I want to know if my

system will give me correct reading if the platelet count is 7500/cumm
• Control are also called Level 1, Level 2
or Level 3 controls
• Controls are also sometimes refered to
as normal and pathological etc
Calibration and Calibrator

• Standard that come with a kit are usually used to caliberate one
parameter
• The level of purity and accuracy is not very high
• In big labs where the quality and accuracy is very imp,we use
calibrators
• They are high quality standards.Multiple parameters can be
calibradted with one calibrator
• Irrespective of the reagents we use or the analyser we use, when
we use a good calibrator, we will get accurate results
• STANDARD- A solution having a known concentration of an analyte
which is used for comparing absorbance.
• CALIBRATOR- A Very high quality of standard.Usually multiple
parameters can be calibrated with one calibrator
• CONTROL- A solution used to test if our system is functioning
correctly or not

NOTE- A control can not and should not be used for calibrating an
analyser. It should be used only to test if an analyser is giving correct
results or not.
BLANK
• The objective of blank analysis resuts assessment is to determine the
existence and magnitude of contamination resulting from laboratory
activities
• A lot of blank are required for the analysis according to the methods
• A blank is prepared to represent the matrix as closely as possible
• Various blanks are- Reagent Blank
Sample Blank
Method Blank
Calibrator Blank
Spectrophotometer: Types
• Single beam spectrophotometer
• Double beam spectrophotometer
• Double beam in time spectrophotometer
• Multichannel
Nephelometry and Turbidimetry

• Turbidimetric measurements are made with a spectrophotometer to determine

the concentration of particulate matter in a sample. The amount of light blocked
by a suspension of particles depends not only on concentration but also on size.
Because particles tend to aggregate and settle out of suspension, sample
handling becomes critical. Instrument operation is the same as for any
spectrophotometer.
• Nephelometry is similar, except that light scattered by the small particles is
measured at an angle to the beam incident on the cuvette.