PRINCIPLE OF COLORIMETRY

ESTIMATION O F UREA, G LU CO S E
& PROTEIN
COLORIMETRY
Colorimetry means
measurement of colour. This
technique is used in Biochemistry
to measure the concentration of
substances that are coloured or
that can be converted to
coloured compounds by suitable
reactions. This technique is very
sensitive and requires small
quantity of sample.
 •When ordinary light passes through a coloured solution, a portion of
the light is absorbed by the coloured substances (chromogen) and the
rest is transmitted.

INCIDENT TRANSMITTED
LIGHT LIGHT

l
OPTICAL DENSITY: Ability of a material to transmit light throught it . It measures
the
speedof light passing through a substance and is affected by the wavelenght of
light.

TRANSMITTENCE : is the quantity of light that passes through a solution or defined

as the ratio of the transmitted intensity, I, over the incident intensity, I0
The amount of light absorbed or transmitted by a colour solution is in accordance
C O L O R I M E T E R -P R I N C I P L E
• BEER’S LAW
When a parallel beam of monochromatic light is passed throuh a
solution the absorbance (A) of the solution is directly proportional
to the concentration of the substance to the solution

A ∝ C
• LAMBERT’S LAW
According to this law the absorbance is directly
proportional to the pathlength which is the thickness of the
solution

A ∝ l
B E E R - L A M B E RT L AW
A∝C WHERE :
A∝l C IS

A ∝Cl CONCENTRATION L
IS PATH LENGTH
A=
K IS
KC l PROPORTIONALITY
As A has a direct relationship with C and L, A is used in
CONSTANT
calculations in all colorimetric estimations.
Calculations are based on comparison of two solutions, one
standard whose concentration is known and the other
unknown whose concentration is to be determined.
COMPONENT O F COLOMETER
SPRECTOPHOTOMETER
A sprectophotometer has all the basic component of
photoelectric colorimeter with more sophistication .

A DVA N TAG E O F SPRACTOPHOTOMETER OV E R THE COLORIMETER

Spractophotometer is thousend time more sensitive
then colorimeter. Therefor even minute quantites of substance or
over dilute solution can be assesd in sprectophotometer.
it cam measure the concentration of colourless solution too.
QUANTATIVE ESTIMATION OF BLOOD GLUCOSE (BY GOD-POD
METHODE)
PRINCIPLE
:□ Glucose + H₂O + O₂ GOD Gluconic acid +
□ H₂O₂ POD
H₂O +
□ [O]
4 Aminoantipyrine + Phenol + [O] Quinonimine (Pink colour comp.)
□ Intensity is determined at on 505 nm filter.

PROCEDURE :
Mix & keep it for incubation at 37 Cͦ for 15 min or at room temperature for
30 min. Measure the intensity of colour at 505 nm filter (Green filter)

CALCULATION
: O.D. o f Test - O.D. o f Blank
Concentration of Substance = O.D. of Std.- O.D. of Blank X Concern. of Std.
I N T E R P R E TAT I O N : [NORMAL RANG : 70 TO 100
Hyperglycemia
•mg/dL] • Hypoglycemia:

: in • It is found in following
• It is found following conditions:
• I. Physiological:
•conditions
I. Physiological: –During starvation
• 1. Alimentary : Afte hig –After Severe Exercise
carbohydrate r h • II. Pathological:

•diet
2. Emotional: Stress, anger, –Prolonged fasting
–Due to excess of insulin e.g.
anxiety etc. • Excessive dose of insulin
• II. Pathological:
insuli
• No food intake after n
administration
• 1. Diabetes mellitus • Tumours of pancreas (insulinoma)
• 2. Hyperadrenalism –Glycogen storage disease
• 3. Hyperpituitarism –Hypoactivity of adrenal and
pituitary gland
QUANTATIVE ESTIMATION OF TOTAL PROTEIN (BY BIURET
METHODE)
When serum is treated with biuret reagent, the peptide bonds of proteins react with cupric
ions in alkaline medium to form a violet coloured copper co-ordinate complex.
The absorbance of this complex is measured colorimetrically at 540 nm using green filter. A
standard protein solution is also treated similarly and the colour intensities are compared.

PROCEDURE :
Reagents
1.Biuret reagent: Sodium - potassium tartarate. Copper
sulphate, Potassium iodide and NaOH.
NORMAL RANGE : 6.0 T0 8.0 mg/dl

CA LCULATION :

O.D. o f Test - O.D. o f Blank

Concentration of Substance = O.D. of Std.- O.D. of Blank X Concern. of Std.
 INTERPRETATION

A low total protein A high total protein

level level
liver disorder chronic inflammation
kidney
viral hepatitis or HIV
disorder
bone marrow
malnutrition
disorders multiple
malabsorption
celiac disease / myeloma.
inflammatory bowel disease
(IBD).
 QUANTATIVE ESTIMATION OF BLOOD UREA
:Urea is Hydrolyzed by water and Urease into Ammonia and Carbon
Dioxide. Urease
UREA+H2O 2NH3 +
CO₂
➢The Ammonia produced is further acted with Hypochlorite and Salicylate to
form Green Complex.
➢ The intensity of Color is proportional to the Urea Concentration in the Sample

PROCEDURE :
In this test (Urea test) Wavelength used is 578 nm. Sample used is Serum.Mix, incubate for 5 min at RT or for 3 min at
37C

NORMAL RANGE : 15 TO 20 mg/dl

CA LCULATION :
Absorbance of test
Concentration of Substance = X Concern. of Std.
Absorbance of standard
 INTERPRETATION
Increased blood urea or Low levels of urea in
BUN (azotemia) may be due the blood suggest:
to:
1 High protein diet.
1 Malnutrition.

2 GIT bleeding. 2 Low protein diet.

3Increased protein catabolism 3 Liver disease.

(trauma, surgery,, extreme starvation.
4 Pregnancy.
4 Impaired renal function.

5 Dehydration, volume depletion.

6 Cardiac failure.

7 Urinary tract obstruction

THANK YOU
VERY
MUCH!