Human Color vision

Human Eye
Human eye is an organ of human being, which helps to observe any object and helps to detect color.
Human eye can observe objects in the range of electromagnetic wave length of 400-700nm.
It consists of the following parts:

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1. Cornea: The outermost visible part of the eye which is a transparent section of sclerotic
coat. It is the uncovered open portion of sclera. The cornea is kept moist and free from dust
by tear ducts and the blinking of the eyelids. There is a thin transparent layer of elliptical
cells called conjunctiva, which protects cornea.
2. Sclerotic coat: Sclerotic coat is the outer section of the eyeball and known as white of the
eye. It is made of connective tissues and cartilage.
3. Aqueous humor: Aqueous humor is a transparent liquid between the cornea and the lens.
The presence of liquid determines the shape of cornea and normally provides an image
roughly in focus on the retina.
4. Pupil: Pupil is a hole in the center of the iris diaphragm through which light passes into the
eye. The size of the pupil may be increased or reduced by the extension and relaxation of
the muscles or iris.
5. Iris: Iris is a circular diaphragm through the connection of cornea. It controls the
diameter of pupil. The diameter is small under high illumination and expands under low
illumination. It may be as large as 8mm for the dark-adapted eye.
6. Lens: There is a transparent flexible biconvex lens behind the pupil, which remains
hanging by suspensory ligament in between cornea and choroids. Its shape is determined
by radial and sphincter muscles. The muscles adjust the shape of the lens so that an
inverted image of object is formed on the retina.
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7. Retina: Retina is the innermost layer of eye ball. It is the photosensitive part of the
eye made up of a mosaic of rod and cone shaped light sensitive cells. The rods and cones
translate the optical image into a pattern of nerve activity. Rod and cone cell are
responsible for the sensation of color.
8. Vitreous humor: Vitreous humor is the room between lens and retina filled with
viscous transparent fluid. It remains a nearly constant distance between the lens and
retina.
9. Fovial pit: An area of retina that consists entirely of cone cells. This rod free region is
about one square millimeter in area and contains about 5000 cones. It is the highest
color resistive area of the retina.
10. Choroid coat: A brownish black pigment layer behind the retina that absorbs light
transmitted through the retina.
11. Blind spot: The spot where the nerve connections leave the eye to form the optic
nerve, which transmits the signals to the brain. This region contains very few light
sensitive cells and that part of an image falling on this area is invisible.

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 Light sensitive elements of Eye
The retina is the mosaic of light sensitive cells. These light sensitive cells are of two
types namely rods and cones. The distribution of cones in all over retina but is not
uniformly. The distribution is dense in foveal pit and in other sections, they are less
dense. In blind spot, there is no cone cell. In retina, there are 7 million cone cells.

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 Rod and Cone Cells
Rod: Only one type of rod cell is in the eye.
Features of rod cells:
➢ The nerve signals from rods alone unable to provide color vision and provide a
monochromatic view of the world.
➢ Rods are very sensitive to small amount of light and are responsible for our vision in low
level of illumination.
➢ At high level of illumination, rod cells completely loss their sensitivity, because inside
rod presence of a photosensitive pigment which is called RHODOPSIN. It becomes
bleached out under bright light.
➢ When the illumination is low, it takes few minutes for reactivation. This type of vision where
only material is seen but the color is not detected is called SCOTOPIC vision.
➢ Every rod cell is not separately joined with the optic nerve cell. Rather a bundle of rod cell is
connected with the brain via optic nerve. Therefore, the resolution rod cell vision is not so
high.
➢ The illumination response of rods is maximum for light of a wavelength of about 510nm.
➢ At wavelength about 620nm, it has zero visibility. That is rods cannot response at red light.

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Light sensitive elements of Eye (Figure)
➢Rod cells ➢Cone cells

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 Cone Cells
Cone cells are of Three (3) types-
➢ Short(S): Cones with a response peaking at about 440nm
(Blue).
➢ Medium(M): Cones with a response peaking at about 555nm
(Green).
➢ Long(L): Cones with a response peaking at about 585nm
(Red).
Therefore, each type of cell has a different variation of sensitivity with wavelength.
That is to say, cone cells response to primary colors from these three sensitivities.
The combined effect of these three color by three types of cone we can see different
shades. Any defect in these combination results in color blindness.

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 Cone Cells
Features of cone cells:
➢ The combined responses of these types of cone cells provide the ability of
distinguish luminosity and color.
➢ The sensitivity of cones to light is retained up to high level of illumination. So in
normal daylight our vision is totally due to the cone cells. Because in high
illuminations rod cells are completely inactive.
➢ This vision at high level of illumination is called photopic vision.
➢ The peak response of cones is at 555 nm. So green color is healthy for eye. Cones
are less sensitive to blue point of spectrum and more sensitive to red part.
➢ The cone cells are individually connected to the brain with the help of optic nerve.
That is to say each cone cell is separately connected of optic nerve. Because of
this reason the resolution of cone cell is very high.

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 Difference Between Rods and Cones
Rods Cones
1. Shape Rod Shaped Cone Shaped
2. Vision Peripheral vision Center Vision
3. High Density Rod cell are distributed throughout the Cone cell mainly occurs in the fovea.
retina.
4. High density Everywhere except foveal pit At retinal fovea

5. Vision Night vision (Black-white) Colored vision

6. Sensitivity More sensitive to low light Sensitive to bright light
7. Cell Length Comparatively long Comparatively short
8. No. of Cell/Eye 120 million 6 million

9. Vision Scotopic vision Photopic vision

10. Protein Rhodopsin Photopsin
11. Nerve connection Bundle of rod Connected with nerve Separately connected with nerve

12.Integration time Slow response Fast response

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 Types of defective color vision
On the basis of color vision there are four (4) types of people-
1. Trichromacy: Most of the people about 92% are perfect in color vision i.e.
they are trichromates. They can detect all the three primary color perfectly.
2. Dichromacy: About 2-3% people fall under dichromate group. Dichromates
can response to only two primary colors. Again, their capacity to dichromate color
is less than that trichromates.

Dichromates are of three types:

1. Protanopia: Red color vision deficiency.(red blind). Of them 1% male, 1%
female. Most of the dichromate is Protanopia.
2. Deuteranopia: Green color vision deficiency.(green blind). Of them 1% male,
0.01% female.
3. Tritanopia: Blue color vision deficiency.(blue blind). Of them 0.02% male,
0.001% female.
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 Types of defective color vision
3. Monochromacy: Monochromates can detect only one primary color. They are
very insensitive in color vision. This may occur due to severe illness. Rod
monochromate in case of cone absence and cone monochromate in case of rod
absence. Actually, they are very less in number and are very rare. Very less number of
people i.e. 0.003% in case of male and 0.002% incase of female are monochromate.
4. Anomalous trichromacy: The rest 6% people are not perfect in their color
vision. But they do not ignore any primary color. They are actually weak in sensing
one or more primary colors. They are called Anomalous trichromates. They are very
difficult of defect.
Anomalous trichromates are of three types:
1. Protanomaly: weakened sensitivity to long wave length i.e. red primary
weak (red defective). Of them 1% male, 0.03% female.
2. Deuteranomaly: weakened sensitivity to mid wave length i.e. green primary
weak (green defective). Of them 5% male, 0.35% female.
3. Tritanopia: weakened sensitivity to short wave length i.e. blue primary weak
(blue defective). They are very less in number.
15
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 Causes of defective color vision
➢ Genetic or inherited: The inheritance defective color vision
involving red and green cones is due to defect of one of the genes on
one of the X-chromosomes. The probability of color defective vision
is more in male than in female. Inheritance blue cones deficiency is
not sex liked. Blue cone deficiency and monochromacy are
extremely rare case and occurs equally in males and females.
➢ Illness or diseases: Acquired defective color vision may arise from
use of certain drugs. Blood related diseases like diabetes especially
during old age.
➢ Damage or accident: Eye become damaged due to accident and
arises defective vision.
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 Metamerism
➢ Metamerism can only occur when comparing two objects. The color of two objects
match under one set of condition but not under another set of condition.
➢ A small pair can have same tristimulus values at a given light source. That
means, they would look the same, although the reflectance curves are different. If
the illumination changes, a more or less significant color difference can occur
between the two samples. Metamerism happens because of using in different
dyestuffs/pigments. The following example will explain the problem. To quantify,
the so called metameric index is used as measure for metamerism. This index
helps, to choose the best recipe out of a selection.

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 Types of Metamerism
1. Illuminant metamerism: This is the most important type of metamerism which occurs
due to change of illuminating source(light source) under which the matching is done.
2. Observer metamerism: Observer metamerism denotes that a pair of sample is judged
matched by an observer and mismatched by another. An index is used to assess these failures
of color matching among different observers. The mean index for the 20 observers is a
meaningful measure of observer metamerism. According to Strockar-
Observer metamerism index,
3. Geometric metamerism: Geometric metamerism arises when two colored samples
matches under one set of conditions but does not match when the geometries of
illumination and viewing are changed.
4. Field size metamerism: Field size metamerism occurs when the match fails due to change
of the size of visual field or distance of viewing. It is a special case of observer metamerism.
5. Instrumental metamerism: Instrumental metamerism arises when two objects match one
set of instrumental conditions, but fail on another case. This generally happens with different
tristimulus colorimeter and with spectrophotometers with different geometries.

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 Factors of Color
The color perceived by an observer when viewing a sample depends on three factors

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 Color Consistency
➢ Color constancy is a process that allows the brain to recognize a familiar object as being a
consistent color regardless of the amount or wavelengths of light reflecting from it at a given
moment. For example, a red apple will be perceived as red in well or poorly illuminated
surroundings.
➢ Color helps to identify objects. But level of illumination and illuminant color may vary
widely. Color consistency is the variation of an object color under a situation of changing
illuminance. Color consistency phenomena are largely confined to surface or object mode of
color perception. When the illuminant is changed a colorimetric color shift occurs due
to change in spectral radiant power of the illuminant.

Color consistency fails when-

❑ The illuminant is monochromatic.
❑ The illumination varies remarkably across the visual scene.
❑ The objects are viewed against a black background.

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Color & Color Measurement

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 Color
➢ Color is a physical impression of human mind. It is impossible to measure.
➢ Color is an aspect of visual perception dependent on the spectral comparison of observed radiant
energy.
➢ Human eye sense light which is a wave, with color set by its wavelength. When a mixture of
wavelength is detected by the eyes and processed by the brain, a combined color can be seen. Some
colors can be made from a single wavelength (460 nm) (blue) or from a mixture of different wave
length (490-560 nm) (creates green shade)
➢ Modern concept of color was founded in 1704 by Isaac Newton. Newton separated white day light
into a sequence of colored light called ‘spectrum’.

The committee on colorimetry of the optical society of America (1922) defined color:
“Color is the general name for all sensations arising from the activity of the retina of the eye and its
attached nervous mechanism, this activity being in nearly every case in the normal individual, a
specific response to radiant energy of certain wavelength and intensity.”

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Separation of Color

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 Color Model
A color model is a system that uses three primary colors to create a larger range of colors.
There are different kinds of color models used for different purposes, and each has a slightly
different range of colors they can produce. The whole range of colors that a specific type of
color model produces is called a color space. All color results from how our eye processes
light waves, but depending on the type of media, creating that color comes from different
methods.

WHAT’S THE DIFFERENCE BETWEEN A COLOR MODEL AND A COLOR SPACE?

A color model is a system used to describe a color. For example with Red, Green and Blue
(RGB) elements or Cyan, Magenta, Yellow and Black (CMYK).
A color space is a way of mapping real colors to the color model's particular values. For
example, sRGB and Adobe RGB are color spaces that both use RGB as a color model,
however the way they are displayed and represented will be slightly different.

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 The Munsell color order system
➢ The Munsell system is a subjective color ordering system developed by and
American Albert Munsek in 1915. Using water colors he painted many hues on
small squares of white papers and arranged those in the order that became known
as Munshell color order system. Samples in the Munsell set differ from adjacent
samples in equal visual steps.
➢ Munsell color space can be described by the attributes hue, chroma and value.
➢ Munsell hue is that quality of color by which we distinguish blue from yellow,
red from green etc. i.e. the common name of color to perceive them. The 10
principal hues in Munsell system are yellow, yellow-red, red, red-purple, purple,
purple-blue, blue, blue-green, green, green-yellow. Each hue as 10 subdivisions
and the principal hue is designated as ‘5’.

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The Munsell color order system

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The Munsell color order system

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 The Munsell color order system
➢ Value is the lightness on darkness of color along a white to black scale calibrated from 1 to 10 ;
where 1 is perfect black and 10 is perfect white.
➢ Chroma is used to describe the intensity of color. Chroma has no upper limit theoretically. It
is the vividness or dullness of color.
➢ The Munsell color system can be viewed as a book of 100 pages having its pages spreaded out to
make a cylinder (100 equal steps) with the binding at the center of the cylinder. The pages of the
book would represent the various hues. The centre binding would represent the value scale and the
distance from the centre binding towards the edge of the page would represent the chroma of a
color.
➢ Each sample in the Munsell system has a designation. For example, a sample is represented as 5Y
2/12. Here 5Y indicates hue of 5 yellow, 2 indicates the value and 12 indicates the chroma.
➢ The greatest advantage of Munsell system is that the colors can be viewed and measured and it is
easy to supply a set of a Munsell color samples to the trading organizations. The disadvantages are
lack of permanency of color, discoloration of colors due to handling the samples and the fact that the
construction of the color space is subjective.
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 Light
Light is an electro radiation with particular wave length.
Light is that aspect of radiant energy of which a human observer is aware, through visual
sensations arising from simulation to the retina by the radiant energy.
CIE: In 1931, CIE (Commission Internationale de I’Eclairage) was established at Paris in
France.
The aim of CIE system is to tell us how a color might be produced (by a mixture of three primary
sources) rather than describe.
Trichromatic Theory of color: Color that aspect of visual perception dependent on the spectral
comparison of observed radiant energy. Trichromatic or three-color theory was proposed by Thomas
Young in 1801 but remained unnoticed until Helmholtz rediscovered it, about 50 years later in
1852 and experimented by Maxwell in 1860.
1. Thomas Young (1801): Proposed that the eye perceive color interms of three colour stimuli,
these are Red, Green and Blue.
2. Helmholtz theory (1852): First recognize the difference between the additive and subtractive
color mixing.
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 Light
Wavelength

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 Additive or Light theory
In light theory, the primary colors are red, blue and green. The mixing of these three colors
in equal quantity produces white light

The addition of these three primaries gives the following colors-

❑ Red + Green=Yellow
❑ Green+ Blue =Cyan
❑ Blue+ Red=Magenta

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 Subtractive or Pigment theory
In pigment/subtractive theory, the primary colors are red, yellow and blue. The mixing
of these three colors in equal quantity produces black shade.

The addition of these three primaries gives the following colors-

❑ Red + yellow=Orange
❑ Yellow + Blue=Green
❑ Blue + Red=Violet

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 XYZ Theory
In 1931, CIE (Commission Internationale de I’Eclairage) was established. They
formulated XYZ-system of color specification. In this system, the response of the eye
was defined in terms of three reforms (X), (Y) and (Z) derived mathematically from
(R), (G) and (B).
• X- Represents a red stimuli more saturated than any
spectral Red.
• Y- Represents a Green stimuli more saturated than any
spectral Green.
• Z- Represents a Blue stimuli more saturated than any
spectral Blue.
• X+Y+Z= White and R+Y+B= White

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 XYZ Theory
The unit amount of (X), (Y) and (Z) were adjusted so that equal quantities are required
to match as equal energy spectrum white.

The chromaticity diagram was derived depending on XYZ-system of

color specification.
1.0X 2.3646(R)-0.5151(G)+0.0052(B)
1.0Y -0.8965(R)+1.4264(G)-0.0144(B)
1.0Z -0.4681(R)+0.0887(G)+1.0092(B)
1.0X +1.0(Y)+1.0(Z) 1.0000(R)+1.0000(G)+1.0000(B)
• The chromaticity diagram was developed depending on XYZ
system of color specifications.
• The chromaticity co-ordinates X and Y are defined in an analogous
way to r and g.

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 L*a*b* theory
In 1976, CIE recommended the CIE L*a*b* system.
Properties of CIE LAB (L*a*b*) Color space:
The system is intended to be a uniform color space with the following properties-
• A rectangular, three dimensional color space (L*a*b*) in which all surface colors
can be represented.
• The distance between the points representing the colors of two samples is
proportional to the visual color difference between them.
• The axes are scaled so that a just perceptible color difference is represented by unit
distance.
• The L*a*b* values can be easily interpreted in terms of the hue, lightness and
depth of shade.

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 L*a*b* theory
• L* is the vertical axis and represents lightness. 0 being a perfect
black and 100 a perfect white.
• a* an axis in the plane normal to L which represents the
redness/greenness quality of the color, positive values denote redness
and negative values denote greenness.
• b* an axis normal to both L and a which represents the
yellowness/blueness quality of the color, positive values denote
yellowness and negative value denote blueness.

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L*a*b* theory

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 L*a*b* theory
The co-ordinates of the points representing the color of a sample can also be expressed
in terms of the cylindrical co-ordinates lightness (L*), chroma (c*), and hue (h0).

When,
• h0 = 0, red • h0 = 270, blue
• h0 = 45, orange • h0 = 315, Magenta
• h0 = 90, yellow • h0 = 360, red
• h0 = 135, yellow • h0 = 180, green
• h0 = 225, cyan

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 Recent CIE LCH (L*C*h0)

• While CIE LAB uses Cartesian co-ordinates to calculate a color in a

color space, CIE LCH (L*C*h0) uses polar co-ordinates. This color
expression can be derived form CIE LAB. The L* defines lightness,
C* specifies chroma and h0 denotes hue angle.
• The (L*C*h0) expression offers an advantage over CIE LAB
because it can be easily related to the earlier based on physical
samples like the Munsell color scale. Different hue angle indicated
different hue.

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Recent CIE LCH (L*C*h0)

When,
• h0 = 0, red • h0 = 270, blue
• h0 = 45, orange • h0 = 315, Magenta
• h0 = 90, yellow • h0 = 360, red
• h0 = 135, yellow • h0 = 180, green
• h0 = 225, cyan

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 Attributes of Color
Each color has its own distinct appearance based on three elements: Hue, Value, and
Chroma. By describing a color using these three attributes, we can accurately identify
a particular color and distinguish it from any other.
• Hue (dH): When asked to identify the color of an object, we must likely speak first its
hue. Hue is we perceive an objects color. Red, Orange, Green, Blue etc.
• In CIE color space, hue can be expressed by hue angle. That is to say in CIE LCH
(L*C*h0) system h0 denotes hue angle as CIE LCH uses polar co-ordinates. By
different hue angles i.e. different values h0 we can express different hues as below-
When,
• h0=0, red • h0=270, blue
• h0=45, orange • h0=315 , Magenta
• h0=90, yellow • h0=360, red
• h0=135 yellow • h0=180, green
• h0=225, cyan
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 Attributes of Color
• Value(dL): The second characteristic of colour describes its luminous intensity
that is its degree of lightness. Colour can be classified as light or dark color when
comparing the value. The scale of value ranges from zero (0) for pure black to 100
for pure white. In CIE L*a*b* system, L represents value. L= 0 means perfect black
(total absorption) and L=100 means perfect white (total reflection).
For example, when placing a tomato and a radish side by side by the red of tomato, in
contrast the radish has a dark red value.
• Chroma(dC): The vividness or dullness of color describing its chroma. Again
comparing the tomato and radish the red of the tomato is much more vivid, the
radish appears duller.

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Attributes of Color

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 Color difference (ΔE)
In CIE L*,a*, b* space, the color difference between a standard and a trial sample is
expressed numerically by ΔE.

• Where, the subscripts T and S denote trial and

standard samples respectively.
• The color difference can also be specified in terms
of co-ordinates L*,C*and H* where C* represents
Chroma and H* represents hue angle.
• C* = the distance between the sample and L-axis.
• H* = the angle made by the chroma line and a*
axis.
• The total color difference (in CIE LCH system) is:

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 Acceptable limit of Color difference (Δ E)

• Practically zero color difference ( E=0) is impossible. Even in a

same place of a colored sample, if we test twice the instrument
will show color difference. But there is an acceptable range of
color difference.
• Δ E>1, colors do not match
• Δ E ≤1, colors match.
• But it depends on visual assessment, if color matches visually
(i.e. visually looks good), the value of E is ignored.

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Acceptable limit of Color difference (Δ E)

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 Spectrophotometer
Spectrophotometer is a photometric device that measures spectral
transmittance, spectral reflectance relative spectral emittance. It compares
light leaving form the object with that incident on it at each wavelength. The
data are primarily related to the color of the object and are presented as
curves (curves of reflectance% against wavelength). The shape of the
spectrophotometric curves can give some indications of perceived color by
comparison with those of known hues.
Color is measured by spectrophotometer by reflectance value.
Reflectance can be defined as:

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Spectrophotometer

• The material absorbs some light. So reflectance

value ranges from 0-1.
• For perfect black, reflectance = 0
• For perfect white, reflectance = 1
• Reflectance is often expressed as percentage (%).

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 Types of Spectrophotometer
There are present different types of Spectrophotometer. They are classified based
on their application/uses.

1. Visible light spectrophotometer: use visible light from a tungsten lamp; mainly
used for routine laboratory practice, particularly the portable and bench-top
spectrophotometer models.
2. UV vis spectrophotometer: As compared to visible light spectrophotometer, this
type of microscope contains a second lamp. UV/Visible spectrophotometer can
measure up to 1100 wavelengths. It has different characteristics such as scanning
function, user interface, integral printer, and multiple cell setting.

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 Types of Spectrophotometer
The UV/Visible spectrophotometer is divided into three classes such as:
1. Single beam: In a single beam spectrophotometer, a reference standard is used to
measure light intensity before and after the sample is loaded. These are typically less
expensive and have higher sensitivity.
2. Double beam: In a double beam spectrophotometer the beam is split with one beam
passing through the standard and the other through the sample to compare intensities.
The double beam spectrophotometers tend to be more stable and easier to use.
3. Split beam: In this type of spectrophotometer, the light emitted by the same
monochromator is split into two beams, one of which reaches the detector directly
and the other passes through the sample and reaches the other detector. The
advantage of this instrument is that it monitors errors in the light source, but does
not eliminate the effects of the reference.
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 Types of Spectrophotometer
1. Near-infrared spectrophotometer: This type of microscope is used to measure the
response of a sample when exposed to infrared light. It gives a non-invasive analysis
and a quantitative finding with only the least specimen preparation. Those solid
samples have a high absorbance, they are mainly monitored by using this Near-
infrared spectrophotometer.
2. Nuclear Magnetic Resonance spectroscopy: It is used to identify the structure of
organic compounds. It gives structural detail of the entire molecule as well as
dynamic information of organic reactions.
3. Mercury spectrophotometer/analyzer: Used to measure the amount of mercury in
water.

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 Types of Spectrophotometer
4. Fluorometers: It is used to measure the fluorescence discharge once the given
sample is exposed to a single wavelength of light.
5. Atomic absorption spectrophotometer: A flame fumes the water from the
specimen causing it to separate into ions. The dissociation leads to changes in the
intensity of light as seen by the detector. Hence, help in finding out the concentration
of the sample. Atomic absorption spectrophotometer’s high precision analysis is
useful in toxicology, environmental testing, and quality control laboratories.

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Types of Spectrophotometer

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Types of Spectrophotometer

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 Components of spectrophotometer
A spectrophotometer contain these following components;
1. Light Source: The light source is required to generate lights within the
spectrophotometer. The material is used in light sources; it should be excited to high
energy states by a high voltage electric discharge (or) by electrical heating to serve as
excellent radiant energy sources.
2. Collimator (lens): This lens points the light to a monochromator or prism from the
light source.
3. A monochromator (Prisms or Grating): It is used to separate the polychromatic
radiation into component wavelength (or) bands of wavelengths. A monochromator
determines polychromatic radiation into its individual wavelengths and isolates these
wavelengths into very narrow bands.

Md. Golam Mostufa, Lecturer, STEC 56

 Components of spectrophotometer
4. Wavelength selector (slit): It selects a particular wavelength of light from the
splited wavelengths and passes it through the cuvette.
5. Transport vessels (cuvettes): The cuvette is used to hold the sample to be
studied. It is made up of Quartz.
6. A Photosensitive detector and an associated readout system: Most of the
detectors depend on the photoelectric effect. The current is then proportional to the
light intensity and therefore a measure of it. Radiation detectors help to produce
electronic signals which are equivalent to the transmitter light. These signals require
to be turned into a form that is simple to interpret. This is achieved by using
amplifiers, Ammeters, Potentiometers and Potentiometric recorders.

Md. Golam Mostufa, Lecturer, STEC 57

Components of spectrophotometer

Md. Golam Mostufa, Lecturer, STEC 58

 Working principle of Spectrophotometer
• The specimen to be studied is placed within the spectrophotometer.
• After that, a light source is shone to generate the light which is then passed through a
monochromator where the light splits into each color/individual wavelength.
• These split individual wavelengths hit the subject that is held within a tiny container
known as a cuvette. Gently handle the cuvette because the slightest fingerprint can
alter the result.
• The light that crosses through the sample is read and interpreted as seen on the output
screen.
• The resulting light strikes the photodetector device which compares the intensity of
the beam.
• Electronic circuits convert the relative currents into linear transmission percentages
and/or absorbance/concentration values.
Md. Golam Mostufa, Lecturer, STEC 59
 Spectrophotometer uses/Application
• Used for qualitative analysis of a sample.
• Used for quantitative analysis of biochemistry practical for example determine the concentration of an
unknown given sample.
• It also used for Enzyme assay.
• To determine the molecular weight of a particular sample, for example amine picrates, ketone
compounds, aldehyde, and sugar.
• Used to Identify the impurities.
• To detect the concentration of supplied speciemen.
• To elucidate the structure of Organic compounds.
• To determine the characteristics of a protein.
• To determine the amount of dissolved oxygen in a body of water.
• To analyze the respiratory gas in hospitals.
• Used to detect the Functional group.
• To identify the molecular weight in a particular compound.
• Used to determine the classes of compounds.
• Used in Bial’s test for concentration of pentoses.
• To Estimate the dissolved organic carbon concentration.
• Specific ultraviolet absorbance for metric of aromaticity.
Md. Golam Mostufa, Lecturer, STEC 60
 CIE standard Illuminant
Illuminants are standard light sources defined by specific spectral power
distribution. The reliability and reproducibility of any color measurement is highly
dependent on the particular illuminant employed on the color. Standard Illuminants
are introduced to provide standard conditions of illumination characterized by their
standard chromaticity co-ordinates x, y and z. So CIE in 1931 defined 4 standard
sources of illuminants. These are-
• Illuminant A
• Illuminant B
• Illuminant C
• Illuminant D65

Md. Golam Mostufa, Lecturer, STEC 61

 CIE standard Illuminant
1. Illuminant A: Illuminant A is defined as an illuminant having the same SPD (Spectral
Power Distribution) as that of Planckian radiator at a color temperature of 2856K. It
represents interior illumination by a gas filled called tungsten filament lamp. The illuminant
is incandescent, yellow-orange in color with a wavelength range of 380-700nm.
2. Illuminant B: It represents illumination by direct sunlight. It is obtained by combining
illuminant A and liquid filters containing different chemical solutions. It has lack in Ultra-
Violet light which exists in real day light.
3. Illuminant C: It also represents illumination by average daylight coming from clear
northern sky (average 5500Lux). It is a tungsten illumination, bluish in color with a
correlated color of 6774K. It also obtained by combing illuminant A with chemical solution
filters and also has lack in UV lights.
4. Illuminant D65: Artificial daylight, correlated color temperature 6500K within the tolerance
descried in BS 905 PART I, illuminate for color matching appraisal for most applications
where there is need to maintain color consistency and quality conforming highly to the C IE
specifications for accurate color matching and CRI value 98.
Md. Golam Mostufa, Lecturer, STEC 62
 Specific Viewing Geometry
CIE has recommended four different instrument geometries for
the measurement of color :

1. 45/0 (45/normal)
2. 0/45 (normal/45)
3. D/O (diffuse/normal)
4. O/D (normal/diffuse)

Md. Golam Mostufa, Lecturer, STEC 63

 Specific Viewing Geometry
1. 45/0 (45/normal): Illumination of the sample by one or more beams at an angle
of 450 to its surface and direction of the reflected light at close to right angle.
2. 0/45 (normal/45): The specimen is illuminated by one or more beams whose
effective axis is normal to the specimen and direction of reflected light at 450 to
the specimen surface.

Md. Golam Mostufa, Lecturer, STEC 64

 Specific Viewing Geometry
3. D/O (diffuse/normal): The specimen is illuminated diffusely by an integrating
sphere. The angle between the normal the specimen and the axis of the viewing
beam should not exceed 100. The direction of the reflected light at close to
perpendicular to the sample. The typical integrating sphere is a hollow sphere
coated inside with a spectrally non-selective white material like BaSO4 (reflectance
98.5%, previously MgO was used).The sphere may be of any diameter (most
commonly 150mm) provided that the total area of the pores does not exceed 10% of
total internal reflecting area.
4. O/d (normal/diffuse): The light source is placed at perpendicular to the specimen
and the detector receives the reflected light which comes diffusely from the
integrating sphere. That is to say, the sample is illuminated vertically and the
diffusely reflected light is collected in the integrating sphere and measured.

Md. Golam Mostufa, Lecturer, STEC 65

Specific Viewing Geometry

Md. Golam Mostufa, Lecturer, STEC 66

 Recipe Calculation
1. Reference measurement: First of all, the reference is measured and the reflectance
values are stored in the system so they can be called off at a later stage. It is very
often the case, that the reference is too small which makes a reliable measurement
impossible. The smallest aperture of the different measuring instruments is around
5-10mm.
2. Dyestuff selection: The dyestuff used for a color matching is depending on the
different requirement such as fastness, dyeing conditions, dye machines etc. But
further also the costs, availability and finally the color match are important. The
knowledge of the experienced colorist is very useful by the selection of the dyestuff
groups. In general, the computer is allowed to calculate with 6-8 different dyestuff
(more are possible ). All the possible solutions are shown in a table and the colorist
can choose the best recipe and print that out. More than one recipe can be chosen
of course.

Md. Golam Mostufa, Lecturer, STEC 67

 Recipe Calculation
3. Recipe calculation: With the different dyestuffs, all the possible combinations for match
are calculated. First of all, three dyestuff combinations are calculated. This is because the
color of the reference is clearly defined with the three values XYZ. To improve the
metamerism, sometimes four dyestuffs in a combination are necessary. With two dyestuffs it
is normally difficult to get a good color matching. If it is possible to match with two or even
one dyestuffs, the concentration of the other dyestuff components are set to zero.
The basics of recipe calculation :
• First of all, a rough calculation of the three dyestuff concentrations is made.
• From that, the K/S, the reflectance and the XYZ values are evaluated and compared with
the values of the reference.
• A significant deviation appears. To improve that, an iteration procedure is maintained.
• The computer checks, how a slight change in the concentration affects the XYZ values
and then calculates a new better formula.
• Normally more then one iteration is necessary until the values of reference and recipe
are comparable.
Md. Golam Mostufa, Lecturer, STEC 68
 Recipe Calculation
4. Results: The evaluated recipes are shown on the monitor in a table. In the table, the recipes are in order
either according to price or to quality (metamerism). Out of that table, the best recipe can be chosen. A
metamerism index < 1 is tolerable although the deviation can be quite significant. If the requirement is
high, Metameric index < 0.5 is recommended. Index > 2 should be avoided.
• The colorist is choosing the recipe with the best compromise according to metamerism, price and
fastness etc.
5. Corrections: First of all, the first trial is measured. The computer needs fooling data:
I. R%-values of the reference.
II. R%-values of the first trial
III. First recipe
IV. R%-values of the substrate.
• The first trial deviates from the reference because of different reasons. The system would start from a
wrong basis. The concentration can not be reduce incase the dyeing is too dark. The modern
programmes allow to adjust tolerances for example in lightness to make it possible to adjust at least
the shade to the reference, even it is a little bit darker. The dyeing concentration is depending on the
local conditions in the production and has to be found out with tests.

Md. Golam Mostufa, Lecturer, STEC 69

 Recipe Calculation
Possibilities and limits of match predictions:
Advantages:
• Better recipe
• Reduction of metamerism
• Selection of an optimal recipe.
Limits:
• Different material
• Different pre treatment
• Different dyeing conditions
• Incomplete exhausion of the dyestuffs
• Interactions between dyestuffs
• Sample properties
• Measurement accuracy
• Deviation from the theories.
Md. Golam Mostufa, Lecturer, STEC 70
Light Wave and Color

Md. Golam Mostufa, Lecturer, STEC 71

 LIGHT
Light, or Visible Light, commonly refers to electromagnetic radiation that can be
detected by the human eye. The entire electromagnetic spectrum is extremely broad,
ranging from low energy radio waves with wavelengths that are measured in meters, to
high energy gamma rays with wavelengths that are less than 1x10-11 meters.
Electromagnetic radiation, as the name suggests, describes fluctuations of electric and
magnetic fields, transporting energy at the Speed of Light (which is ~300,000 km/sec
through a vacuum). Light can also be described in terms of a stream of photons,
massless packets of energy, each travelling with wavelike properties at the speed of
light. A photon is the smallest quantity (quantum) of energy which can be transported,
and it was the realization that light travelled in discrete quanta that was the origins of
Quantum Theory.

Md. Golam Mostufa, Lecturer, STEC 72

 Characteristics Of Light

Five characteristics of light are:

1. Light consists of packets of energy called photons.
2. Light is the relatively narrow frequency band of
electromagnetic waves.
3. Light travels at such a high speed, 3×108 m/sec.
4. Light behaves both as a wave and particle.
5. Light travels fast in a vacuum.

Md. Golam Mostufa, Lecturer, STEC 73

 Light Sources
Light is electromagnetic radiation. A particular frequency of this
radiation (around 390-700 nm) is visible to the human eye. Everything
that we see around us is because of light. Light is a form of energy and
like all energies, it is produced from a source. In Physics, these are called
light sources.

Types of Light Sources:

There are countless sources of light, but they can all be categorized
under either of the two following categories-
• Natural sources
• Artificial sources
Md. Golam Mostufa, Lecturer, STEC 74
 Natural Light Sources
➢ The universe is filled with objects that emit light. Some light from these sources
reaches the earth. The following things in nature have the ability to emit light:
➢ The Sun is the major source of light for the earth. The sun is a massive ball of fire, at the
center of which nuclear fusion produces massive energy. This energy comes out as heat
and light. The light from the sun is one of the major factors behind the sustainability of
life on earth.
➢ Every other star produces light too, but only a small or no amount of it reaches the
earth because of the huge distance.
➢ The moon provides light as well but it cannot produce light on its own. The light that we
get from the moon is the light reflected by it from the sun.
➢ Some living organisms have the ability to produce light too. It is called
bioluminescence. It is the effect of certain chemical reactions within the organism.
Fireflies, jellyfish, glow-worm, certain deep-sea plants, and microorganisms can be
cited as examples.
➢ Certain other natural phenomena such as lightning and volcanic eruptions also emit
light. Md. Golam Mostufa, Lecturer, STEC 75
 Artificial Light Sources
Apart from natural sources, light can be produced artificially too. The different light
sources produced artificially can be put under three broad categories-
Incandescent Sources:
• When certain objects are heated to a high temperature, they begin to emit light. Both
infrared and visible light is produced in the process.
• Example- Candle, incandescent lamp etc.
Luminescent Sources:
• Light can be produced by accelerating charges in a luminescent material. One common way
of doing it is bypassing current through the material.
• Example- Fluorescent tube light, electric bulb etc.
Gas Discharge Sources:
• Passing electricity through certain gases at very low pressure can produce light too.
• Example – Neon lamp, Sodium lamp.

Md. Golam Mostufa, Lecturer, STEC 76

 How does light interact with matter?
Matter is composed of atoms, ions or molecules and it is through their interactions with
light which gives rise to the various phenomena which can help us understand the
nature of matter. The atoms, ions or molecules have defined energy levels, usually
associated with energy levels that electrons in the matter can hold. Light sometimes be
generated by the matter, or more commonly, a photon of light can interact with the energy
levels in a number of ways.

Figure 2 – Jablonski Diagram example, illustrating transitions between the various energy states of molecules
following interaction with a photon.
Md. Golam Mostufa, Lecturer, STEC 77
 How does light interact with matter?

We can represent the energy levels of matter in a scheme known as a Jablonski diagram,
represented in Figure . An atom or molecule in the lowest energy state possible, known as
the ground state, can absorb a photon which will allow the atom or molecule to be raised
to a higher energy level state, known as an excited state. Hence the matter can absorb light
of characteristic wavelengths. The atom or molecule typically stays in in an excited state
only for a very short time and it relaxes back to the ground state by a number of
mechanisms. In the example shown, the excited atom or molecule initially loses energy,
not by emitting a photon, but instead it relaxes to the lower energy intermediate state by
internal processes which typically heat up the matter. The intermediate energy level then
relaxes to the ground state by the emission of a photon of lower energy (longer
wavelength) than the photon that was initially absorbed.

Md. Golam Mostufa, Lecturer, STEC 78

 What is light absorption?
Light absorption is the process in which light is absorbed by matter and converted into
energy. In an atom, electrons vibrate at a specific frequency – this is called the natural
frequency. If a wave of light hits a material in which the electrons are vibrating at the
same frequency as the wave of light, the electrons will absorb the energy and convert it
into vibrational motion. This is why objects have different colours – different materials’
electrons will vibrate at different rates, and therefore absorb different frequencies of light.

Md. Golam Mostufa, Lecturer, STEC 79

 Light absorption and matter
• Electrons can only exist in discrete energy levels (these can also be called electron
shells) – they can’t exist halfway between. The lowest energy level that an electron
can be in is called the ground state. For an electron to move from a lower energy level
to a higher energy level, it must absorb a set amount of energy because energy levels
are quantised. This means that the energy absorbed by the electron must be exactly
the same as the energy difference between the two levels.
• When an electron absorbs energy, is it promoted to a higher energy level further away
from the nucleus of the atom and is described as being ‘excited’.

Md. Golam Mostufa, Lecturer, STEC 80

What happens after an electron becomes excited?

Electrons don’t like being in an excited state. This means that after
becoming excited and moving to a higher energy level, they soon fall
back to their original energy level. However, to do this, they have to
release a packet of energy – this is called a photon. The size of the
photon released is exactly equal to the size of the jump the electron had
to make in the first place.

Md. Golam Mostufa, Lecturer, STEC 81

 Absorption Spectroscopy
• Absorption spectroscopy is a technique used to measure the absorption of energy. The
absorption spectrum of a certain material is shown by a continuous band of colour with black
lines between them. The coloured parts represent the total light that is focused on the material.
The black lines show an absence of this light – these are the parts of the spectrum where the
electrons have absorbed the light photons. There are two types of absorption spectroscopy:
atomic and molecular.
• Atomic absorption spectroscopy is the method of producing a spectrum when free atoms absorb
different wavelengths of light–this is usually used for gases.
• Molecular absorption spectroscopy is the method of producing a spectrum when whole
molecules absorb different wavelengths of light (usually ultraviolet or visible).
• Absorption spectrums are the exact opposite of emission spectrums.

Md. Golam Mostufa, Lecturer, STEC 82

 Emission Spectroscopy
• Emission spectroscopy is used to measure the photons released when an electron
falls to a lower energy level after becoming excited. The emission spectrum of a
certain material is shown by a black band with separated coloured lines. These colours
lines are the parts of the spectrum where photons have been released from the
electrons when they fall to a lower energy level.
• There are two types of emission spectroscopy: line and continuous. When the spectrum
is shown as lots of lines separated by black spaces, it is a line emission spectrum.
When the spectrum is shown as lots of colours in one particular wavelength, it is a
continuous emission spectrum. Emission spectroscopy is used to identify a substance
because the energy released when the electrons fall back to their ground state is
different for every substance.
• Emission spectrums are the exact opposite of absorption
spectrums.

Md. Golam Mostufa, Lecturer, STEC 83

 What is Scattering of light?
• When sunlight enters the atmosphereof the earth,the atoms and molecules of different gasses present in
the air absorb the light. Then these atoms re-emit light in all directions. This process is known as
Scattering of light.
• Theatomsorparticlesthatscatterlight arecalledscatterers.
• Light can be examined entirely from its source. When light passes from one medium
to any other medium say air, a glass of water then a part of the light is absorbed by
particles of the medium preceded by its subsequent radiation in a particular direction.
This phenomenon is termed as a scattering of light. The intensity of scattered light
depends on the size of the particles and the wavelength of the light.
• Shorter wavelength and high frequency scatter more due to the waviness of the line
and its intersection with a particle. The wavier the line, the more are the chances of it
intersecting with a particle. On the other hand, longer wavelengths have low
frequency, and they are straighter and chances of colliding with the particle are less
so the chances are less.
Md. Golam Mostufa, Lecturer, STEC 84
 What is Scattering of light?
• The bending of multicoloured light can be seen in the afternoon due to the
refraction and total internal reflection of light. The wavelength of the sunlight forms
different colours in different directions. Rayleigh Scattering Theory is reasoned for
the red colour of the sun in the morning and blue colour of the sky.
• Let p be considered as the probability of scattering and λ is the wavelength of
radiation, then it is given as:
P⋉1λ4P⋉1λ4
• The probability for scattering will give a high rise for shorter wavelength and it is
inversely proportional to the fourth power of the wavelength of radiation.

Md. Golam Mostufa, Lecturer, STEC 85

 Law of Scattering

The intensity of scattered light is

inversely proportional with the
fourth power of the wavelength of
the incident light when the particle
(Scatterer) is of smaller diameter
than the wavelength of light.

Md. Golam Mostufa, Lecturer, STEC 86

 Why is colour of clear sky blue? And why are
the clouds white?
• Molecules with a larger size than the wavelength of light, experience the scattering
effect differently, the phenomenon is known as Mie effect. Due to the largeness of
particles, the light appears white. That is why the clouds, which are made of droplets
of water are white. The blue colour is present in the major percentage among the
lower wavelengths. With the wavelength of the light, the scattering efficiency of the
small molecules in the atmosphere decreases. Sun radiates its light and its rays fall
into the earth’s envelope thus, sunlight gets scattered in the atmosphere.
• There are some examples that also show scattering, particles like dust, and smoke can
also scatter radiation. In the same manner, we can explain the red colour appearance of
the sun. For red light, the wavelength is more, and it is easy to go through the
atmosphere as the scattering is less for the red light. When the light is on any other
object, it gets scattered depending on its properties as different light has different
intensity and each particle has different characteristics.
Md. Golam Mostufa, Lecturer, STEC 87
Red sky and sun on the time of Sunset or Sunrise

At the time of sunrise or sunset, the position of the sun is very far
away from the earth. The sunlight travels a longer distance
through the atmosphere. Scattering blue light is more than red
light. As a result, more red lights reach our eyes than the blue
light. Hence sunrise and sunset appear reddish.

Md. Golam Mostufa, Lecturer, STEC 88

Md. Golam Mostufa, Lecturer, STEC 89