COLORIMETRY

I. Introduction
1. Color vision and perception
1.1 Color perception
1.2 Color vision
1.3 Factors influencing the color of a stimulus or an object
1.3.1 The color of the stimulus depends on the observation condition
1.3.2 Chromatic Adaptation
1.3.3 Lighting conditions influence the color of an object
1.3.4 Color rendering
2. Colorimetry
II. Principle
1. Beer's Law
2. Lambert's Law
3. Beer's-Lambert's Law
III. Types
1. Visual colorimetry
1.1 Visual Devices
1.1.1 Marten Photometer
1.1.2 Chromaticity-Difference Colorimeter
2. Photo-electric colorimetry
IV. Instruments
1. Colorimeter
1.1 Principle
1.2 Types
1.2.1 Color densitometers
1.2.2 Color photometers
1.3 Parts
1.3.1 Light source
1.3.2 Monochromator
 a. Filters
b. Diffraction gratings
c. Interference filter
d. Prism
1.3.3 Solution holder
a. Test tube
b. Cuvettes
c. Flow-through cells
1.3.4 Photosensitive detector system
a. Barrier layer cell
b. Photo emissive tubes
c. Photomultiplier tubes
1.3.5 Measuring device
2. Spectrophotometer
2.1 Principles
2.2 Types
2.2.1 Single beam spectrophotometer
2.2.2 Double beam spectrophotometer
2.3 Parts
2.3.1 Light source
2.3.2 Wavelength isolator
2.3.3 Cuvettes
2.3.4 Electronic measuring device
V. Application of colorimetry
1. Blood Glucose Analysis
2. Quantitative Determination of Hemoglobin
3. Urinalysis
4. Determination of Potassium
5. Determination of pH
 COLORIMETRY

I. Introduction

We describe the light reaching the eye from an image location by its spectral power
distribution. The spectral power distribution generally specifies the radiant power density at
each wavelength in the visible spectrum. For human vision, the visible spectrum extends
roughly between 400 and 700 nm. Depending on the viewing geometry, measures of radiation
transfer other than radiant power may be used. These measures include radiance, irradiance,
exitance, and intensity.

Color and color perception are limited at the first stage of vision by the spectral properties of
the layer of light-sensitive photoreceptors that cover the rear surface of the eye (upon which
an inverted image of the world is projected by the eye’s optics). These photoreceptors
transduce arriving photons to produce the patterns of electrical signals that eventually lead to
perception. Daytime (photopic) color vision depends mainly upon the three classes of cone
photoreceptor, each with different spectral sensitivity. These are referred to as long-, middle-,
and short-wavelength-sensitive cones (L, M, and S cones), according to the part of the visible
spectrum to which they are most sensitive (see Fig. 6). Night-time (scotopic) vision, by
contrast, depends on a single class of photoreceptor, the rod.

1. Color vision and perception

1.1 Color perception

Color is a visual sensation, a subjective interpretation of some elements of the physical world
we’re living in.
Color originates from the analysis of luminous stimuli by our visual system. Luminous stimuli
are in fact light radiations, and color vision is more particularly related to the spectral
distribution of radiated power within these radiations: light spectrum.

Color sensation is described by words like red, light blue, olive green, dark brown and etc.
These attributes can be classified into color attributes of hue, saturation and lightness.

Following the International Lighting Vocabulary, hue is the attribute of a visual sensation
according to which an area appears to be red, yellow, green, blue, or a combination of two of
them (e.g. orange is a hue since it is a visual combination of red and yellow, meaning that
orange resembles red and yellow and lies somewhere in between).

Lightness is the brightness of an area judged relative to a similarly illuminated white area.
Attributes of lightness are “dark” or “light”.

The accurate definition of saturation is more complex and will not be reproduced here, but
we can imagine that saturation describes the “density” of the hue contained within the color.
Colors for which the hue is well pronounced are called highly saturated (vivid, deep). On the
contrary, as the hue fades away, less saturated colors tend to gray or white: pale colors, such
as pastels.

1.2 Color vision

Color originates from the analysis of light radiation by our visual system. The light stimulus
or radiation is first directed to the retina, through the optical system of the eye. The retina is
covered with sensitive cells which are able of translating light information into electrical
signals. These signals are sent to the brain through the optic nerve.

Sensitive cells named cones are responsible for color vision. More densely distributed in the
center of the retina (fovea), they belong to three subclasses: B-cones are more sensible to short
wavelengths, G-cones to the mid ones and R-cones to the longer wavelengths.

Each cone therefore acts as a photoelectric cell, associated with an optical filter. For each “pixel”
of the image, three different signals will reach the brain which therefore will be able to perform
a sort of spectral analysis of the corresponding light stimulus.
 1.3 Factors influencing the color of a stimulus or an object

1.3.1 The color of a stimulus depends on the observation conditions

The color depends on the size of the colored object: if too small on the retina, the image of
this object will not influence the B-cones, since their density drops to zero at the very center
of the fovea. Color vision becomes therefore 2D instead of 3D for very small objects.

The color of an object depends on the dominant color of the previous visual field. This is
illustrated by the experience of phantom images: if we fix a vivid red drawing during one or two
minutes (e.g. a red cross), and then suddenly look at a white paper, the same drawing will
appear as a silhouette on the white paper, but in the complementary color (light cyan) the
color of an object depends on the dominant color of the surrounding visual field. This is
known as simultaneous contrast. For example, the same gray line will appear yellowish against
a blue background and bluish against yellow background.
 1.3.2 Chromatic adaptation

In the phantom images experiment, during the one or two minutes adaptation, the R-cones
strongly excited by the red stimulus, causing some sort of fatigue or under-sensibility
compared with the B and G-cones.

Turning to the white paper, the three cones should deliver the same signal in order to indicate
the white color. However, as the R-cones have lost some sensitivity, the complementary color
(blue-green or cyan) appears for that part of the retina (image) that was strongly exposed to
the red stimulus. After some minutes, the R-cones recover their normal sensitivity, and all the
paper area becomes white again (the silhouette disappears).

1.3.3 Lighting conditions influence the color of an object

Light falling on an object is partly reflected by this object to the observer’s eyes. The reflected
light entering the eye is what we have called the luminous or color stimulus.

In the previous subsections, it has been shown that the color associated with that stimulus
depends on the observation conditions, and in particular on the chromatic adaptation state of
the eye.
Now, it is clear that the color is NOT an intrinsic property of one object, as the light reflected
from it does not only depend on its nature but also on the incident light characteristics.

The color or the light depends on:

 the spectral power distribution of the light source; and
 the spatial distribution of the incident light.

Just have a look at some colored objects illuminated by a low sodium lamp. Under this
monochromatic light, no blue, red or even green colors: everything seems of the same yellow-
orange color, more or less dark more or less light. These are quite unusual lighting conditions,
but it can be shown that even with white illuminants, the color of the same object is more or
less changed by the spectral nature of light source. The fact that spatial distribution of the
incident light influences the color of an object is not so obvious.
Therefore, when lightning conditions influence the color of an object, a tristimulus colorimeter
would also see the effect. This is a major difference with observation conditions’ effects, for which
the stimulus is unchanged, including no influence on the colorimeter.

1.3.4 Color rendering

The color rendering of an illuminant or a light source is its effect on the color appearance of objects
by conscious or subconscious comparison with their color appearance under a reference illuminant
(Lighting Vocabulary).

The color rendering is evaluated by the color rendering index Ra, which equals 100 if there’s no
perceptible effect on color appearance of objects. A common value for interior lighting (offices,
classrooms, working spaces with comfortable color vision) is Ra = 85.

It is interesting to note that the evaluation of this color rendering index takes into account
chromatic adaptation effects induced by the test illuminant. Indeed, in an interior lit by this
illuminant, all objects in the visual field will reflect in some way the “color of the source”,
which would therefore influence the cones adaptation state of the observer such that a white
sheet of paper still appears white, under any illuminant. Not taking this chromatic adaptation effect
into account would lead to a wrong evaluation of color rendering.

2. Colorimetry
Colorimetry, in simple terms, is the measurement of colors and is probably the most widely
used method for determining the concentration of biochemical compounds. This important
laboratory procedure is based on the principle that when white light passes through a colored
solution, some wavelength are absorbed more than others. Many compounds, through not
colored themselves, can be made to absorb light in the visible spectrum by reaction with
suitable reagents. The colored compounds absorbs light at given wavelength at visible
spectrum. The extent to which a solution absorbs depends on the intensity of its color.

II. Principle

Colorimetry is the techniques that is frequently used in biochemical investigations. This

involves the quantitative estimation of colors. This means that if you want to measure the
quantity of a substance in a mixture, you could use the technique of colorimetry, by allowing
the substance to bind with color forming chromogens. The difference in color results in the
difference in the absorption of light, which is made use of here in this technique called
colorimetry.

1. Beer’s Law
The proportion of the incident light absorbed by the molecules in a solution is directly
proportional to the number of absorbing molecules in the light path. The ability of the
substance to selectively absorb certain wavelengths of light while transmitting others is
determined by its molecular and atomic structure. Thus, it follows that the concentration of a
substance, i.e., absorbing molecules, is directly proportional to the intensity of the color of the
solution. In Beer’s law, the light path is constant while the concentration varies. As the
concentration of the color substance in the solution increases, absorbance increases and the
amount of light passing through (transmittance) decreases.

t = Ioe-KC

2. Lambert’s Law

When monochromatic light passes through a colored solution, the amount of light absorbed
increases with the increase in the thickness of the layer of the solution through which the light
passes. Thus the concentration of the solution is constant while the light path varies.

It = Ioe-kt

3. Beer-Lambert Law

The basic principles of colorimetry, absorptiometry and spectrophotometry are established,

by combining Beer’s law and Lambert’s law.

The joint law shows that under suitable conditions, the amount of light of absorbed by a
colored solution, when illuminated with the light of suitable wavelength, is directly
proportional to the concentration of the colored solution and the length of the light path
through the solution. Therefore the amount of light decreases exponentially with the increase
in the concentration of the solution and with increase in thickness of the layer of solution
through which light passes.

Therefore, together Beer-Lambert’s law is:

IE/Io = e-KCT
where,
IE = intensity of emerging light
Io = intensity of incident light
e = base of neutral logarithm
K = a constant
C = concentration
 T = thickness of the solution

III. Types

1. Visual colorimetry

Visual colorimetry is one of the oldest form of color measuring techniques which is not used
nowadays, natural or artificial light is used as light source and determination are made with
a colorimetry or color comparator where human eye is used as detector.

1.1 Visual Devices

1.1.1 Martens Photometer

One of the most useful visual devices for determining relative

luminance is the Martens photo meter. This reflectometer is
intended for the measurement of luminous reflectance of opaque
specimens relative to reflecting standards of similar spectral
reflectance. The Priest-Lange instrument is also adaptable to the
measurement of luminous transmittance of transparent plates
relative to transmitting standards similar in spectral
transmittance to the unknown. Finally, the Martens photometer, removed from the mounting,
may be used for the determination of the luminance of an unknown self-luminous surface
relative to a spectrally similar standard of known luminance.

1.1.2 Chromaticity-Difference Colorimeter

The determination of chromaticity coordinates, x,y, by comparison of the unknown specimen

with a working standard of similar spectral reflectance can be carried out visually with high
precision by means of a colorimeter described by Judd [64]. The adjustment of the
chromaticity of the comparison field to match the standard field is by two double wedges, one
of greenish and the other of yellowish glass. Since the light from the comparison field must
pass through both the yellow and the green wedge, some of the radiant energy being
subtracted by each, it is sometimes called a subtractive colorimeter; see figure 11 which gives
a schematic diagram. The standard and comparison fields are brought into juxtaposition by
means of a Lummer-Brodhun cube having a double-trapezoid pattern subtending 9 X 13° at
the observer's eye. The adjustment to near equality of brightness to facilitate detection of
chromaticity differences is by movement of the projection lamp that illuminates both standard
and comparison surfaces.

2. Photoelectric colorimetry

Progress in the development of colorimetric method has

resulted largely due to the application of complicated visual
comparison. In this method human eye is replaced by suitable
photoelectric cell, to afford a direct measure of the light
intensity. Instruments employing photoelectric cell measure
the light absorption and not the color of the substance.

IV. Instruments

1. Colorimeter

A colorimeter is a light-sensitive device used for measuring the

transmittance and absorbance of light passing through a liquid
sample. The device measures the intensity or concentration of the
color that develops upon introducing a specific reagent into a
solution.

1.1 Principles

The colorimeter is based on Beer-Lambert's law, according to which the absorption of light
transmitted through the medium is directly proportional to the medium concentration.
In a colorimeter, a beam of light with a specific wavelength is passed through a solution via a
series of lenses, which navigate the colored light to the measuring device. This analyzes the
color compared to an existing standard. A microprocessor then calculates the absorbance or
percent transmittance. If the concentration of the solution is greater, more light will be
absorbed, which can be identified by measuring the difference between the amount of light at
its origin and that after passing the solution.

1.2 Types

1.2.1 Color densitometers

A densitometer is a device that

measures the degree of darkness (the
optical density) of a photographic or
semitransparent material or of a
reflecting surface. The densitometer is
basically a light source aimed at a
photoelectric cell. It determines the
density of a sample placed between the
light source and the photoelectric cell
from differences in the readings.
Modern densitometers have the same components, but also have electronic integrated
circuitry for better reading. Densitometer, device that measures the density, or the degree of
darkening, of a photographic film or plate by recording photometrically its transparency
(fraction of incident light transmitted). In visual methods, two beams of equal intensity are
used. One is directed through the plate, while the intensity of the other is adjusted by an
optical wedge, by an iris diaphragm, or by moving the source, until the two beams have equal
intensity, judged by the eye or by a photoelectric cell. With proper calibration, the density can
be read directly. Other methods employ photoelectric cells to measure the intensity of the
same beam with and without film or plate inserted in the path, the difference in intensity being
a measure of density.

The same techniques can be used to measure the density of semitransparent materials other
than photographic plates—for example, sunglasses.
 1.2.2 Color photometers

A photometer is an instrument that measures the strength of

electromagnetic radiation in the range from ultraviolet to
infrared and including the visible spectrum. Most photometers
convert light into an electric current using a photo resistor,
photodiode, or photomultiplier. Color photometers measure
the amount of light falling on an object along with the intensity
of color coming from it. Such photometers are used to balance
out the saturation of color and are particularly useful when it comes to photography.

1.3 Parts

1.3.1 Light source

The type of light source will depend upon the region of the spectrum required. For visible
light, the most common source is a tungsten-filament lamp, or the higher powered tungsten-
halogen (quartz-iodine) lamp. For the ultraviolet region, hydrogen or deuterium lamps are
used.

1.3.2 Monochromator

To select the wavelength, filters or monochromators are used to split the light from the light
source. The monochromator can be a diffraction grating or prism and is usually used in most
expensive colorimeters.

A. Filters – The simpler instruments such as colorimeters, use filters as means of

selecting a band wavelength. The simplest filters are either colored glass or
suitably dyed gelatin sandwiched in a glass. They have limited transmission
band and are usually complimentary to the color of the solution to be measured.
The simple filters have a range of 400-800 nm. Table 1.2 shows the filter used
for colored solutions.
 B. Interference filter – This is more sophisticated type of filter. It consist of two
partially transmitting films of metal separated by a transparent spacer of low
refractive index, or of a piece of glass or silicon coated with materials of various
refractive indices. The wavelength of the interference filter is from 330-1200 nm.

C. Prism – Prisms are composed of glass, for visible wavelength, and of quartz or
silica for the ultraviolet region. They are designed to be turned to allow the
required light to pass through the focusing slit on the solution. They are
however much more expensive and require more advanced precision
instruments.

D. Diffraction gratings – Most modern spectrophotometers use diffraction

grating method for wavelength selection. The grating is made of a series of
finely etched parallel grooves on a shining reflecting surface. The grating is
placed at an angle to a beam of light. It disperses the white light into a
continuous spectrum, each groove acting as a vey narrow prism.

1.3.3 Solution holder

These are used to hold colored solutions and must be scrupulously clean.

A. Test tubes – They are used usually in the simpler instruments. They must be
matched for transmission; sometimes a mark is etched on them, and this mark
 is lined up against a mark on the instrument to ensure consistent optical
pathway.

B. Cuvettes – These are rectangular cells with one pair of opposite sides optically
clear, while the other parallel sides are opaque and should not be placed in the
light path. They are glass for use at visible wavelengths, and of silica or quartz
for use in the ultraviolet region.

C. Flow-through cells – These are used in absorptiometry to speed up analytical

procedures. The cells or cuvettes are drained without being removed from the
instruments. In this way, readings are taken more quickly. These cells ate used
in automated instruments.

1.3.4 Photosensitive detector system

When light falls on these elements electric current is generated which deflects a galvanometer
needle. The meter reading is proportional to the light intensity. These photosensitive detectors
are also referred to as photoelectric cells.

A. Barrier layer cell – This is made up of a metal disc on which the selenium is
thinly layered, and this is also covered by a thin transparent layer of metal with
a thick end to which one of the terminals of a galvanometer is attached. A
strong incident light is required for the barrier cell layer since the current
produced cannot be easily amplified.

B. Photoemissive tubes – These consist of an evacuated glass tube or a tube

containing inert glass at low pressure. The inside of the tubes are thinly layered
with cesium or potassium oxide and silver oxide to act as cathode; while the
anode is a metal ring inserted close to the center of the valve.

C. Photomultiplier tube – This is the most sensitive detector which is an

improvements of the photoemissive tubes. It has enhanced sensitivity because
the elements within the tube are connected in series. Light falling on the first
element releases secondary electrons in large number resulting in an increase
 in current flow from the cell. It is used in the most sophisticated instruments
where it is capable of measuring intensities of light 100 times weaker than those
measured by the photoemissive tube.

1.3.5 Measuring Device

The current from the detector is fed to a sensitive suitable measuring device, usually a
galvanometer. The read-out can be means of a scale or digital display. The scale may show
both the absorbance and percent transmission. The absorbance scale ranges from zero to
infinite, while the percent transmission scale ranges from 0-100.

2. Spectrophotometer

This is an instrument used to measure absorbance at various

wavelength. It is similar to the absorptiometer except that it uses
diffraction gratings or glass prism to produce monochromatic light.
 2.1 Principle

Spectrophotometers analyze the light reflected or transmitted by a sample at each wavelength

in the visible spectrum, compared to that from a reference sample. They are fitted with a
device, typically a diffracting element, which breaks the incident light into individual
wavelengths. The resulting data are referred to as spectral data. Spectral data are an
independent, relative measurement of an object that serves as a fingerprint of the color. This
data never changes for the object, and can be used in several different ways.

2.2 Types

2.2.1 Single beam spectrophotometer

It operates between 325-1000 nm wavelength; using a single source of light, e.g., tungsten
filament lamp. It has two photocells. The light travels along only one pathway. The test
solutions and blank are read on the same position.

2.2.2 Double beam spectrophotometer

It operates between wavelength range 185-1000 nm. It has two light sources and two
photocells. This instrument splits the light from the monochrometer into two beams. One
beam is used for reference and the other for sample reading. It eliminates errors due to
fluctuations in the light output, and sensitivity of the detector.
 2.3 Parts

2.3.1 Light source

Each spectrophotometer must have a light source. This can be a light bulb constructed to give
the optimum amount of light. The light source must be steady and constant; therefore, use of
a voltage regulator or an electronic power supply is recommended. The most common light
source for work in the visible or near-infrared region is the incandescent tungsten or tungsten-
iodide lamp.

2.3.2 Wavelength isolator

Before the light from the light source reaches the sample of solution to be measured, the
interfering wavelengths must be removed. A system of isolating a desired wavelength and
excluding others is called a monochromator; the light is actually being reduced to a particular
wavelength. Light of a desired wavelength can also be provided by other means. One common
instrument employ a diffraction grating with a special plate and slit to reduce the spectrum to
the desired wavelengths.

2.3.3 Cuvettes

Any light (of the wavelength selected) coming from a filter or diffraction grating will pass on
to the solution in the cuvette. Glass cuvette are relatively inexpensive and satisfactory,
provided they are matched or calibrated. Calibrated cuvettes are tubes that have been optically
matched so that the same solution in each will give the same reading on the photometer.

2.3.4 Electronic measuring device

In the more common spectrophotometers, the electronic measuring device consist of a

photoelectric cell and a galvanometer. The amount of light transmitted by the solution in the
cuvette is measured by a photoelectric cell, a sensitive instrument producing electrons in
proportion to the amount of light hitting it. The electrons ate passed on to a galvanometer,
where they are measured.

V. Application of colorimetry

1. Blood Glucose Analysis

The "Gold standard" for testing blood glucose in Colorimeters is the measurement of glucose
in a plasma sample obtained from a vein. This method involves a chemical reaction activated
by an enzyme called Glucose Oxidase. Most of the Colorimeters used for the Glucose
measurement in Clinical laboratories perform well in the 100 – 400 mg/dl range of blood
glucose. In Self Monitoring Blood Glucometers (SMBG), a drop of blood is placed on a small
window in a test strip. Blood glucose acts as a reagent in a chemical reaction that produces a
color change. The color change is detected by a reflectance-meter and reported as a glucose
value. They too perform well in the range of 60 – 400 mg/dl range of Blood Glucose.
Moreover, requirement of special purpose Dry slides is a big problem for the users of
Glucometers. This paper aims to sandwich the technologies of Colorimeter and SMBG and
interface the Clinical instrument with the Computer for a better clinical diagnostic result. The
existing Colorimeter is upgraded with RGB Color sensor and Microcontroller and interfaced
with the Computer to make Clinical measurement easier. Experimental results show that this
modified Colorimeter can perform better than the existing colorimeter.
 2. Quantitative Determination of Hemoglobin

Principle

Hemoglobin is oxidized by potassium ferricyanide into methaemoglobin, which is converted

into cyanomethaemoglobin, by potassium cyanide. The intensity of the color formed is
proportional to the Hemoglobin concentration in the sample.

Clinical manifestations

The Hemoglobin is a protein that contains iron and that the red color to the blood. The
Hemoglobin is in red globules and it is the one in charge of oxygen transport by the blood
from the lungs to weaves. When the level of Hemoglobin appears underneath the normal
levels is describing an anemia that can be of different origins: primary anemia, cancer,
pregnancy, renal diseases, and hemorrhages.

If the Hemoglobin levels appear high it can be due to cardiopathies dehydration and stays in
places of much altitude Clinical diagnosis should not be made on a single test result; it should
integrate clinical and other laboratory data.

3. Urinalysis

All sites used the Atlas, the CT-200+, and visual

evaluation by at least two readers. Some sites also carried
out additional chemical analyses, e.g., glucose by a
hexokinase procedure, pH testing with a pH meter, and
specific gravity by a Total Solids (T.S.) meter (2).

The Atlas (Fig. 1, 2) is a fully automated analyzer and has

a capacity of 490 reagent strips attached to a continuous
plastic carrier. The instrument pipettes urine onto the
reagent strip, and optical reading is made after the
appropriate time interval for each test. In daily use, only operator calibration and loading into
a specimen wheel is required. The Atlas can assay 40 urines plus calibrators and controls in
an unattended mode.

4. Determination of Potassium

This spectrophotometric method for the direct determination of potassium in serum or plasma
is based on the selective complexing of potassium by a specific macrocyclic polyether, with
the subsequent formation of an ion-pair with a colored anion. The colored anion is extracted
into an organic solvent, clarified by centrifugation, and then measured at 415 nm. The
absorbance of the chromogen varies linearly with [K+] to at least 15 mmol/L. Results of this
colorimetric method (y) correlate well with the results obtained by a flame-photometric
method (y = 1.04x - 0.22, r = 0.97, n = 81), with CVs ranging from 2 to 4%. We observed no
interferences from lipemia, added bilirubin, or various electrolytes. We also evaluated the use
of this reagent in a new automated blood analyzer developed by Abbott, a two-dimensional
centrifugal system (Clin Chem 31:1457-1463, 1985). Potassium determined with this system
(y) correlated well with results by flame photometry: y = 1.02x + 0.02 (r = 0.94, n = 168).
With this system one can use whole-blood specimens in measuring potassium.

5. Determination of pH

A colorimetric method of pH determination is depicted to measure the pH of a liquid/

aqueous solution with the 0-01 pH unit accuracy. It is emphasized that the obvious pH
measured by sulphonephthalein indicators is not in the same amount as it gets
electrometrically. The progress of glass electrodes, involved in the pH meter, has certainly
made the electrometric method the most common way of pH measurement.

Since it can be applied in a highly diluted solution, colloids, and colored solution, where the
indicators are inconvenient, in addition to these particular cases, the indicators provide some
benefits, and it appears to us a mistake to regard that the electrometric method is a standard,
and always the most precise method. The simple Comparator is used, about 0-01 pH unit of
reproducibility readily gives by an indicator.
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