COLORIMETRY &

SPECTROPHOTOMETRY

Dr.Tasnim Ara Jhilky

MD(Part-1)

Department of Biochemistry

Sir Solimullah 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 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 – 700 nm).

Properties of Light

Light is the visible spectrum of electromagnetic radiation,emitted in the form of waves of different wave lengths ranging from380nm to 750nm.

Electromagnetic Spectrum

Colors & Wavelengths

COLOR WAVELENGTH (λ in nm)

Ultraviolet < 380

Violet 380 – 435

Blue 436 – 480

Greenish-blue 481 – 490

Bluish-green 491 – 500

Green 501 – 560

Yellowish-green 561 – 580

Yellow 581 – 595

Orange 596 – 650

Red 651 – 780

Near Infrared > 780

Principle of PhotometrySubstance to be measured by photometry must be

colored to begin with or can be made to produce

color derivatives by using certain reagents and

reactions.

Intensity of colour produced is propotional to the

concentration of the colour producing subs.present

in solution.

Colored subs.absorbs light of a particular wave

length and the extent of light absorption depends on

the conc .of color producing subs.in solution.

Principle (con.)……

A characteristic wavelength of absorption spectrum

is isolated from light passing it through filter

monochromator

Solution with colored subs. is kept in a cuvet&

allowed the subs. to absorb light.

Degree of light absorption by a solute of unknown

conc. Is propotional to degree of light absorption

by same solute in a solution of known conc.

Subs.of unknown conc. is measured by comparing

with same subs .in another solu. Of known conc.

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 :

This law states that,the intensity of

transmitted light decreases exponentially

with the increase in concentration of

colored substance in the solution.

i.e. the amount of light absorbed by a

colored solution is directly proportional to

the conc. Of substance in the solution.

Beer’s law

Beer’s law

Lambert’s law :

This law states that,the intensity of

transmitted light decreases exponentially

with increase in length of light pathway.

(diameter of the cuvette)

i.e. the amount of light absorbed by a colored

solution is directly propotional to the length

of light path.

Transmittance

Transmittance: It is the ratio of intensity of

transmitted light (It) to the intensity of incident

light (lo) across a solution. It is expressed as %

Transmittance (T) = It/ 1o

Transmittance is inversely and logarithmically

proportional to the concentration.i.e Tα log1/C.

Absorbance

• Optical density: it is the amount of light

absorbed by the colored substance. OD

may be defined as the logarithmic ratio

of incident light to that of transmitted

light.

• So A = log ( I/T ) ;= log10 ( 100/T); = 2 -

log10T

Absorbace is directly and linearly

propotional to con.

Relationship between absorbance

and transmittance

2 - log10T

OD %T

OD=

Combined Beer’s- Lambert’s law

Combining the two laws:

A α C x L

A = 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 L

K x CS x L=

AT

AS

CT

CS=

CT =AT

AS

x CS

AS = K x CS x L AT = K x CT x L

CT =AT

AS

xCS

Concentration of TEST sol.

Absorbance of TEST

Absorbance of STANDARDCon. of STANDARDx=

Concentration of TEST/100ml

Absorbance of TEST

Absorbance of STANDARD

Concn of Std X 100x=

X ml

=ODT

ODS

x CS

Standard (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).

Standard Curve / Calibration curve

Preparation of solution for investigation

• In colorimetric estimation it is necessary to

prepare 3 solutions:

BLANK(B)

STANDARD(S)

TEST(T)

10/3/2016 4:36 PM

BLANK.To compensate any non specific color

.To set the instrument 100%T and zero %OD

Water BLANK

Reagent BLANK

STANDARDSolution of known concentration of the substance

Both O.D and concentration are known

So concentration of unknown can be

calculated

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

As per procedure

Complimentary color

• Wavelength between 400nm to 700 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 450 – 479

Red Green 505 – 534

Blue yellow 640 – 689

Green Red 620 - 689

Colorimeter

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

Monochromators/Filters

• This device used for spectral isolation(light of

single wavelength)

• this means of selecting a sufficiently narrow

wave band.

Filter SBW about 50nm (wide band pass

monochromator)

Prism SBW is 5-10 or <5nm (narrow

band pass monochromator)

Diffraction grating SBW is 15-20nm.

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:

Should be sensitive,stable

Should have linear response,short response.

• Different detectors used are:

Barrier layer cells (photocells)

– simpliest

Photoconductive cells

(photodiodes) – newest

Read out devices

• The detector response can be measured by any

of the following devices:

a) Galvanometer

b) Ammeter

c) Recorder

d) Digital readout.

The signal may be transmitted to computer or print

out devices.

Criteria for satisfactory colorimetric

estimations

Stability of color

Intensity of color The color of the solution should

be intense. Clarity of the solution Substance under

investigation should be completely soluble.

Specificity Color produced should be specific for the

desired constituent. Validity of Beer’s law The intensity of color

should be proportional to concentration.

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….

Spectrophotometry :Instruments & Applications

37

Principle of Spectrophotometer

– Solutes in a solution show characteristic absorption

spectrum in UV or visible or infrared region of

electromagnetic radiation.

– Characteristic absorption spectrum can be

isolated by passing the electromagnetic

radiation through a prism monochromator.

– Degree of absorption of electromagnetic

radiation depends on the condensation of solute

in solution.

The Spectrophotometer

The Spectrophotometer

Introduction• Spectrophotometer:

a) Single-beam

b) Double-beam

[4]41

Instruments

• Light source: provide a sufficient of light which is

suitable for marking a measurement.

• The light source typically yields a high output of

polychromatic light over a wide range of the

spectrum.[4]

42

• Monochromator : Accepts polychromatic input light from

a lamp and outputs monochromatic light.

• Monochromator consists of these parts:

I. Entrance slit

II.Collimating lens or mirror

III.Dispersion element

IV.Focusing lens or mirror

V.Exit slit [6]

43

Common monochromators:

Filter

Prism

Diffraction grating

Interference filter

Instruments• Dispersion devices: A special plate with hundreds

of parallel grooved lines.

• The grooved lines act to separate the white light into

the visible light spectrum.

44

The more lines

the smaller

the wavelength

resolution.[5]

Instruments

• Focusing devices: Combinations of lenses, slits,

and mirrors.

• relay and focus light through the instrument.[2]

45

Instruments

• Cuvettes: designed to hold samples for spectroscopic

experiments. made of Plastic, glass or optical grade

quartz

• should be as clear as possible, without impurities that

might affect a spectroscopic reading.[2]

46

Instruments

• Detectors: Convert radiant energy (photons) into an

electrical signal.

The photocell and phototube are the simplest

photodetectors, producing current proportional to the

intensity of the light striking Them .[1],[2]

47

Instruments

• Display devices: The data from a detector are

displayed by a readout device, such as an analog meter,

a light beam reflected on a scale, or a digital display , or

LCD .

• The output can also be transmitted to a computer or

printer. [3]

48

Applications

1. Concentration measurement

– Prepare samples

– Make series of standard solutions of known concentrations

[4]

49

Applications

− Set spectrophotometer to the λ of maximum light

absorption

− Measure the absorption of the unknown, and from the

standard plot, read the related concentration[4]

50

Applications

2. Detection of Impurities

• UV absorption spectroscopy is one of the

best methods for determination of impurities in organic

molecules. [7]

51

Additional peaks can be

observed due to impurities

in the sample and it can be

compared with that of

standard raw material.

Applications

3. Structure elucidation of organic compounds.

• From the location of peaks and combination of peaks

UV spectroscopy elucidate structure of organic

molecules:

o the presence or absence of unsaturation,

o the presence of hetero atoms.[7]

52

Advantages:

Ensure higher degree of spectral purity.

Minimum stray light into the exit beam

(wave length outside the desired light is

called stray light).

Greater accuracy.

More sensitivity, specificity and precision.

Source of errors in

spectrophotometer

Stray light

Low resolution of light source

Lacking linearity

Variation in temp.

Low sample volume

Difference between colorimeter and

spectrophotometerTraits Colorimeter Spectrophotometer

Monochromator Filter Prism

Spectral bandwidth Broad band Narrow band

Spectral purity Less More

Spectral isolation Filter has to be changed

Desired wave length can be adjusted

Stray light More Minimum

Accuracy Less More

Sample Larger volume needed Small volume needed

Cost Cheaper More costly

Light source Visible range of light used

Beyond visible range of light used