COLORIMETRY

BASALINAGAPPA M.SC. MEDICAL BIOCHEMISTRY DEPT. OF BIOCHEMISTRY J S S MEDICAL COLLEGE

Introduction• Photometry is the most common

analytical technique used in the biochemical laboratory. It is designed to measure the intensity of a beam of light.

• Photometric principles are applied to the several kinds of analytical techniques:(a) where absorbed or transmitted light is measured:

• Colorimetry• Spectrophotometry• Atomic absorption, and• Turbidometry

(b) where emitted light is measured:• Flame emission photometry

Introduction (cont.)• The components of most photoelectric

colorimeters are basically the same and the basic method of operation is also similar for all the instruments.

• In analytical chemistry, Colorimetry is a technique “used to determine the concentration of colored compounds (analytes) in sample solution” at visible spectrum of light (400 – 800 nm).

• Colorimeter is instrument which is used in the measurement of the luminious intensity of light.

• Invented by Louis Jules Duboscq in 1870.

Colorimetry

Principle:Colored solutions have the property of absorbing certain wavelength of light when a monochromatic light is passed through them.

• The amount of light absorbed or transmitted by a colored solution is in accordance with two laws: – Beer’s law– Lambert’s law

Beer’s law :• When a monochromatic light passes

through a colored solution, amount of light transmitted decreases exponentially with increase in concentration of colored substance.– i.e. the amount of light absorbed by a colored

solution is directly proportion to the conc. Of substance in the colored solution.

AαC

Beer’s law

Beer’s law

Lambert’s law :• The amount of light transmitted decreases

exponentially with increase in path length (diameter) of the cuvette or thickness of colored solution through which light passes.– i.e. the amount of light absorbed by a colored

solution depends on path length of cuvette or thickness or depth of the colored solution.

AαL

Relationship between absorbance and transmittance

IO IE

OD %T

Transmittance of a solution containing light absorbing substance depends upon1. The nature of light absorbing

substance.

2. Wavelength of light and

3. Amount of light absorbing substance in the light path, which in turn depends on the concentration of light absorbing substance and depth of the solution through which light passes.

Preparation of solution for investigation

• In colorimetric estimation it is necessary to prepare 3 solutions:

BLANK(B)

STANDARD(S)

TEST(T)

05/02/2023 01:01 AM

BLANK

To eliminate the effect of light absorption by the reagent used

Water BLANK

Reagent BLANK

STANDARD

Solution of known concentration of the substance

Both O.D and concentration are known

So concentration of unknown can be

calculated

TEST

Test solution is made by treating a specific volume of the test sample with reagents

As per procedure

Combined Beer’s- Lambert’s law

• Combined Beer’s- Lambert’s law is thus expressed as amount of light transmitted through a colored solution decreases exponentially with increases in conc. Of colored solution & increase in conc. of colored solution & increase in the path length of cuvette or thickness of the colored solution.

• Combining the two laws:A α C x LA = K x C x L

Let AT=absorbance of the test solution CT=concentration of the test solution AS=absorbance of the standard solution CS=concentration of the standard solution

AT

AS

K x CT x LK x CS x L

=

AT

AS

CT

CS=

CT=AT

ASx CS

AS = K x CS x L

AT = K x CT

x L

CT=AT

ASxCS

Concentration of TEST sol.

Absorbance of TESTAbsorbance of STANDARD

Con. of STANDARD

x=

Concentration of TEST/100ml

Absorbance of TESTAbsorbance of STANDARD

Concn of Std X 100x= X ml

=ODT

ODS

xCS

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’

Concentration of TEST /100ml

x=Volume of ‘T’

Amount of ‘S’T - BS - B

x 100

x 100

Standard curve (calibration curve)

• The standard curve is prepared to check whether the method of assaying a particular substance follows Beer’s Law, i.e. whether the absorbance of the substance increases in a linear way with its concentration.

• The standard curve is constructed by plotting a vertical axis (y – axis, ordinate) for optical densities (absorbance) and a horizontal axis (x – axis, abscissa) the concentration of standard solution.

• The concentration of the test/unknown can be measured from the graph (standard curve).

Verification of Beer’s Law• Prepare 1% standard solution of glucose, i.e.

1gm/dl 1000mg/100ml.• Make different dilutions of standard solution

using the general formula given as following for obtaining different concentrations of a solution by dilution with diluent (DW):Tube

no.Conc. (mg%)

Amount of mL needed, (V1)C1 x V1 = C2 x V2

DW (mL)

Total vol. V2 (ml)

1 50 1000 x V1 = 50 x 2, V1= 0.1

1.9 2

2 100 1000 x V1 = 100 x 2, V1= 0.2

1.8 2

3 150 1000 x V1 = 150 x 2, V1= 0.3

1.7 2

4 200 1000 x V1 = 200 x 2, V1= 0.4

1.6 2

5 250 1000 x V1 = 250 x 2, V1= 0.5

1.5 2

6 300 1000 x V1 = 300 x 2, V1= 0.6

1.4 2

7 350 1000 x V1 = 350 x 2, V1= 0.7

1.3 2

8 400 1000 x V1 = 400 x 2, V1= 0.8

1.2 2

0.06 0.12 0.18 0.24 0.3 0.36 0.42 0.480

50

100

150

200

250

300

350

400

450

OD of solutionCO

NC.

OF

GLU

COSE

(m

g/dL

)

Standard Curve / Calibration curve

TYPES OF COLORS

Cannot be mixed

Mixing primary color

Mixing a primary and secondary color

Light spectrum and their wavelengths

Complimentary color• Wavelength between 400nm to 760 nm form

the visible spectrum of light• Light passed through a solution which

selectivity absorbs radiation at fixed wave lengths,then the color of the transmitted light is complementary to that of the absored light.

Colors and complimentary colors of visible spectrum

Color of the solution/ solution color transmitted

Filter used/ color absorbed

Wavelength (nm)

Yellow blue Violet 380 – 430Yellow Blue 430 – 475Orange Green blue 475 – 495Red Blue green 495 – 505Purple Green 505 – 555Violet Yellow green 555 – 575Blue yellow 575 – 600Green blue Orange 600 – 650Blue green Red 650 - 750

Colorimeter

Components of Colorimetry

Components of Colorimetry

1. Light source:The light source is usually a tungten lamp, for wavelength in the visible range (320 –

700nm) and a deutarium or hydrogen lamps for ultraviolet light (below 350nm).

a) Tungsten lamp Visible range

b) Deutarium/hydrogen lamp (preferred) UV Rays

c) Black body radiators (Nerst glower) Infrared radiations

Light source

1. Tungsten lamp:filament mode of tungsten sealed in a glass

envelopeFiled with inert gas.Life time is limited due to gaseous tungsten

formed by sublimation.

Light source

Carbon arc lamp• If sufficient intensity of light is not

obtained from tungsten lamp then carbon arc lamp can be use as a source for color measurement.

Monochromators/Filters

• This is a means of selecting a sufficiently narrow wave band

• Filter will absorb light of unwanted wavelength and allow only monochromatic light to pass through.– E.g.: a green filter absorbs all color, except

green light which is allowed to pass through.Filter

Absorption filter Interference filter Ex: Glass filter,Gelatin filter

Monochromators

• Early colorimeters used Absorption filters (i.e. glass filter, Gelatin filter) that transmitted a wide segment of spectrum (50nm or more).

• Newer instrument use Interface filters that consist of thin layer of magnesium fluoride crystals with a semitransparent coating of silver on each side.

• Monochromator consists of:– Entrance slit– Absorption/ interface filter and– Prisms or diffraction grating for wavelength

selection– Exit slit

Sample Holder/ Cuvette• Cuvettes are rectangular cell , square cell or

circular one.• Made up of optical glass for visible wavelength

(quartz or fused silica for UV).• Common one is square, rectangular to avoid

refraction artifacts.• Optical path (length) of cuvette is always1cm.• Capacity may be 3ml/2ml/1ml depending upon

the thickness of the wall of the cuvette.• For accurate and precise reading cuvette must be

transparent, clean, devoid of any scratches and there should be no bubble adhering to the inner surface of the filled cuvette.

Photosensitive detectors

• Detectors are the transducers, which convert light energy to electrical enagery. A detector should be possess follwing characteristics:1.Should be sensitive 2.Should have linear response 3.Its noise level Should be low 4.Should have short response time5.Should stable.

Photosensitive detectors• Different detectors used are:

1. Barrier layer cells (photocells) – simplest2. Photoemmisive cells3. Photomultiplier tube (for low intensity lights)4. Photoconductive cells (photodiodes) –

newest.

Read out devices

• The detector response can be measured by any of the following devices:a) Galvanometerb) Ammeterc) Recorderd) Digital readout.The signal may be transmitted to computer or print out devices.

Criteria for satisfactory colorimetric estimations

• Stability of colorColor may be fade of air oxidation,

photochemical decomposition, temperature.

• Intensity of colorThe color of the solution should be

intense in order to detect small amount of constituents and for making accurate result in low concentration.

Criteria for satisfactory colorimetric estimations

• Clarity of the solutionSubstance under investigation should

be completely soluble in the solvent, since turbid solution, suspension or colloidal solution absorb as well as scatter light.

• ReproducibilityThe intensity of the colored solution

must be reproducible. The effect of order of adding reagent, pH and other variable should be clearly studied

Criteria for satisfactory colorimetric estimations

• Specificity Color produced should be specific for the

desired constituent. If other constituents interfere with color reaction they be removed or prevented from or prevented from functioning through appropriate treatment like use of other coloring agent, altering the oxidation state.

• Validity of Beer’s lawThe intensity of color should be proportional

to concentration. It can be easily assessed by plotting absorbance Vs concentration, where a straight line passing through origin should be obtained.

Steps In OperationGlass/gel filter is placed in the filter

slot

3/4th of cuvette is filled with distilled water and placed in the

cuvette slot

Instrument is switched ‘on’ and allowed to warm-up for 4-5 minutes

Button is adjusted using ‘coarse’ and ‘fine’ knobs to give zero optical

activity in the galvanometer

Blank solution is placed in an identical cuvette and the OD is read (‘B’)

Blank solution is transferred to the original test tube

Test solution is taken in the same cuvette and O.D. is read (‘T’)

Test solution is transferred back to the original test tube

Standard solution is taken in same cuvette and O.D. is read (‘S’)

Standard solution is transferred back to the test tube

Cuvette is washed

Calculation

X X 100

Cs Conc. Of standardV Volume of test sample

NOTE :- Satisfactory results are obtained only when the O.D. values are in the range 0.1 – 0.7

Applications Of Colorimeter• Estimation of biochemical compounds in

blood, plasma, serum, CSF, urine, etc.:– Glucose– Urea– Creatinine– Uric Acid– Bilirubin– Lipids– Total Proteins– Enzymes [e.g. ALT, AST, ALP]– Minerals [Calcium, Phosphorus etc.]

etc….

• It is widely used in hospital & laboratory for estimation of biochemical samples , like plasma, serum, cerebrospinal fluid ( csf ) , urine.

• It is also used to quantitative estimation of serum components as well as glucose, proteins and other various biochemical compound.

• They are used by the food industry and by manufacturers of paints and textiles

• They are used to test for water quality, by screening for chemicals such as chlorine, fluoride, cyanide, dissolved oxygen, iron, molybdenum, zinc and hydrazine.

• They are also used to determine the concentrations of plant nutrients (such as phosphorus, nitrate and ammonia) in the soil or hemoglobin in the blood and to identify substandard and counterfeit drugs. 

Advantage It is inexpensive .

Very well applicable for quantitative analysis of colored compounds.

Easily carriable and transportable.

Disadvantage

Cannot be used for colorless compounds.

It does not work in UV and IR regions.

We cannot set specific wavelength, as we have to set a range as a parameter.

Similar colors from interfering substances can produce errors in results .

Use, care and preventive maintenance of a

Colorimeter:• Read the user manual carefully.• Use the correct type of cuvette in the

colorimeter as recommended by the manufacturer.

• Make sure that the cuvette is clean and it’s optical surfaces are dry and free from finger marks and scratches.

• Bring filter in to place before switching on the colorimeter.

• Before reading the absorbance of a solution, check that it is clear, there are no air bubbles in it.

• Remove the cuvettes from the instrument when not in use

• Clean the outside of the cuvette with tissue paper to remove any marks from the optical surfaces.

• To prolong the life of the lamp, switch off the colorimeter after use.

• At the end of the day, disconnect It from the main switch and cover the colorimeter with its protective cover.

• At regular intervals check the mains power adapter and cable for wear and tear and replace if damaged.

• Keep in cool place away from corrosive chemicals or fumes.

References• Clinical chemistry –Michael bishop

• A book of medical science- J.ochei

• Practical biochemistry- Keith Wilson & john walker

• Clinical chemistry &molecular diagnostics-Tietz