Date : ......................

COLORIMETRY

Introduction :

A colorimeter is an instrument used to measure the intensity of colour in a given solution,with

reference to a similarly colored standard solution. In this way, if the concentration of the colour- producing
substance in the standard solution is known, its concentration in the unknown solution can be readily
ascertained.
Principle of colorimetry :
Colored solutions have the property of absorbing certain wavelength of light and transmitting others.
This property is based on Beer Lamberts law.
Beer's law :
When monochromatic light passes through a colored solution, the amount of light transmitted
decreases exponentially with the increase in concentration of the colored substance. The intensity of the
color is directly proportional to the concentration of the colored particle in solution.
Lambert’s law :
The amount of light transmitted decreases exponentially with the increase in the thickness of the layer
of the colored solution. The amount of light absorbed by the colored solution depends on the column or
depth of the liquid through light passes.
Beer's Lamberts law is expressed as Ie
= e-kct
Io

Io= Intensity of light

Ie= Intensity of incident light
K= constant
c = concentration of colored substance
t = thickness or length of the layer through which light passes
Io/Ie is called transmission and the absorbance is expressed as –log T
Parts of Colorimeter :
1. Source of light –Filament lamp
2. Monochromatic filters
3. Cuvette - sample holder
4. Photo cell - Detector
5. Display - Galvanometer

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The photoelectric colorimeter :
This instrument measures the intensity of colour of a solution by registering the quantity of light
absorbed by the given solution. Hence, a photoelectric colorimeter is better known as absorptiometer.

1. Light source : Filament Lamp

2. Filter :
It will absorb light of unwanted wavelength and allow only monochromatic light to pass through. the
monochromatic light after passing the filter is allowed to fall on the colored solution kept in the cuvette.
The solution absorbs part of the light and the remaining light is allowed to fall on photo cells which
convert light into electrical signals. The electrical signal generated is directly proportional to the intensity
of light falling on the detector. The signals are measured by galvanometer and read as absorbance or
optical density.
3. Cuvettes :
These are made of glass, which is inert. The solution, the OD of which is to be determined, It is poured
into a cuvette and placed in the colorimeter, for measurements to be made. Cuvettes usually have a
constant internal diameter of 1cm.
4. Photosensitive element :
In most absorptiometers, "barrier layer" cells are used for the photosensitive element. These cells are
furnished with a layer of selenium. When light falls on the selenium layer, it is activated and emits
electrons. The emission is proportionate to the light falling on it. If a galvanometer is attached, electron
flow can be measured, which in turn will indicate the amount of light falling on the cells.
5. A highly sensitive galvanometer :
This measures the output of the photosensitive element.
6. Significance of blanks solutions :
In all colorimetric analyses, preparation of a 'blank' solution is necessary. This is because of the fact
that some color in the colored solution (of interest) is, inevitably, due to the color of the reagents
themselves. Hence, if a blank solution is prepared using all the reagents, except the substance which is
being measured, and its OD value is subtracted from the OD of the unknown ands standard, the resulting
value represents the true optical density.

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PROCEDURE :
In all colorimeter analysis it is necessary to prepare 3 solution namely a blank (distilled water), test
specified volume of the blood filtrate or other specimens) and a standard. The standard solution is Prepared
by from a solution of the pure substance of known concentration. A standard graph with different
concentration should be prepared.The appropriate coloured filter is chosen and the cuvette to about three
fourth with distilled water placed in the cuvette slot. Instrument is switched on and allow to warm up for 4-
5 minutes. Zero optic density should be adjusted first. Note the distilled water should be removed and the
solution in the test tube labeled 'blank' should be taken in the cuvette and O.D is read and noted. Similarly
O.D for test and standard solution are read and noted.
Calculation :
Concentration of the sample = ( O.D of Test (T) – O.D of Blank (B) ) X Conc of Std

( OD of Std (S) – O.D. of Blank (B) ) X Vol of Test

= ( T-B ) X Conc. of Std x

100 ( S-B )

CALCULATION :

Concentration of test solution= ( OD Test –OD Blank )

X Conc of Std

( OD Test –OD Std

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 Date : ......................
ESTIMATION OF PLASMA GLUCOSE
AIM :

To determine the amount of GLUCOSE present in the given sample.

METHOD : Glucoseoxidase / peroxidase method.

PRINCIPLE :
Glucose oxidase (GOD) acts on glucose to produce gluconic acid and hydrogen peroxide. Breakdown
of hydrogen peroxide by peroxidase (POD) is coupled with oxidation of phenol. The oxidized phenol
complexes with 4-amino antipyrine to give a red colour which is measured at 530nm,using a colorimeter.
GOD

D. Glucose + H2O-------------- Gluconic acid+ H2O2.

POD

H2O2 + Phenol+ 4-amino antipyrine--------------- quinoneimine+H2O

( Pinkcolour)
PROCEDURE :
Take three tubes marked B.S.T. Pipette 10μl of distilled water into B, 10μl of standard into test tube S,
10μl Test solution in the test tube labeled T. To all the three test tubes add 1000μl of glucozyme reagent
and mix well. Keep the tubes at room temperature for 15 minutes. Cool and read the optical density at 530
nm.

CONTENTS Blank(μl) Standard (μl) Test (μl)

Distilled water 10 - -
Glucose Standard solution - 10 -
Test solution - - 10
Glucozyme reagent 1000 1000 1000
Mix well and keep at RT for 15 minutes read
at 530 nm within 60 minutes
Optical density at 530nm

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CALCULATION :
Conc of glucose = OD Test - OD Blank x Conc. of standard
OD Std - OD Blank

Conc of std = 100mg / dl

RESULT :
The amount of glucose in 100 ml of given sample =----------mg/dl.

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CLINICAL SIGNIFICANCE :
Normal Range : 70-110 mg/dl. (Fasting )

up to 140 mg /dL( Post prandial )

An increase in blood glucose level (more than the normal) is known as hyperglycemia and decreased
level is known as hypoglycemia.
1. Hyperglycemia- elevated blood sugar ( >120 mg/dl) occur in
o Diabetes mellitus
o Hyperpituitarism
o Hyper thyroidism
o Cushing syndrome
o Insulin resistance
o Stress
o Pancreatitis
o Chronic liver damage
o Brain trauma and damage.
2. Hypoglycemia-lower blood glucose ( <50 mg/dl) occur in
o Overdose of insulin in the treatment of diabetes.
o Carcinoma of pancreas
o Addison's disease
o Hypothyroidism
o Glycogen storage diseases
o Severe exercise.
o Starvation
OTHER METHODS :
o Hexokinase method (enzymatic)
o Folin Wu method.
o O-Toluidine method.
o Nelson-Somogyi method

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