SPECTROPHOTOMETERS

Basic definitions:

• Light:
Light is electromagnetic radiations of a wave-length that is visible to human eye (about
400-700 nm).
Light can exhibit properties of both waves and particles. This property is referred to as
wave-particle duality. The study of light is known as optics.

• Waves:
A wave is a disturbance that propagates through space and time, usually with
transference of energy. While a mechanical wave exists in a medium (which on
deformation is capable of producing elastic restoring forces), waves of electromagnetic
radiation (and probably gravitational radiation) can travel through vacuum, that is,
without a medium. Waves travel and transfer energy from one point to another, often
with little or no permanent displacement of the particles of the medium (that is, with little
or no associated mass transport); instead there are oscillations around almost fixed
positions.
Characteristics of waves:
Periodic waves are characterized by crests (highs) and troughs (lows), and may usually be
categorized as either longitudinal or transverse. Transverse waves are those with
vibrations perpendicular to the direction of the propagation of the wave; examples
include waves on a string and electromagnetic waves. Longitudinal waves are those with
vibrations parallel to the direction of the propagation of the wave; examples include most
sound waves.

• Wave-length:
The wavelength (denoted as λ) is the distance between two sequential crests (or
troughs). This generally has the unit of meters; it is also commonly measured in
nanometers for the optical part of the electromagnetic spectrum

• Frequency:
Frequency is a measure of the number of occurrences of a repeating event per unit
time. It is also referred to as temporal frequency. The period is the duration of one cycle
in a repeating event. So the period is the reciprocal of the frequency.
In SI system, the unit of frequency is hertz (Hz), named after the German physicist
Heinrich Hertz. For example, 1 Hz means that an event repeats once per second, 2 Hz is
twice per second, and so on .This unit was originally called a cycle per second (cps),
which is still sometimes used.
Relationship between frequency and wave-length:
Frequency has an inverse relationship to the concept of wavelength, simply, frequency is
inversely proportional to wavelength λ (lambda). The frequency f is equal to the speed v
of the wave divided by the wavelength λ of the wave.
 f=v/λ
Introduction to colorimetry

Physicians need quantitative measurements of various substances in blood, urine, spinal

fluid etc. to make these quantitative measurements, clinical laboratories use various
instruments, but colorimeter is most commonly used. As the name implies colorimetry
involves quantitative estimation of color. This technique is used to measure the
concentration of substances that are colored or that can be converted into colors
compounds by suitable reaction. Furthermore the intensity of color must be dependent
upon concentration.

Types:
Colorimetry is basically of two types:
Visual colorimetry: In this we compare the light intensities transmitted out of the
solutions.
Photometry: In this we compare the absorption of light by solutions.
When absorption measurements are done by photoelectric colorimeters in visible region
of the spectrum (i.e., between 400-760 nm) the device is termed as photoelectric
colorimetry. On the other hand; if these absorption measurements are done in invisible
region i.e., in ultra-violet (<400 nm) and infra-red (>760 nm) regions, the technique is
termed as spectrophotometry, in which we make use of spectrophotometers.

SPECTROPHOTOMETERS

A spectrophotometer is a photometer (a device for measuring light intensity) that can

measure intensity as a function of the color, or more specifically, the wavelength of light.

There are many kinds of spectrophotometers. Among the most important distinctions
used to classify them are the wavelengths they work with, the measurement techniques
they use, how they acquire a spectrum, and the sources of intensity variation they are
designed to measure. Other important features of spectrophotometers include the spectral
bandwidth and linear range.
Perhaps the most common application of spectrophotometers is the measurement of light
absorption, but they can be designed to measure diffuse or specular reflectance.
The use of spectrophotometers is not limited to studies in physics. They are also
commonly used in other scientific fields such as chemistry, biochemistry, and molecular
biology.
Design and working:

The spectrophotometer measures quantitatively the fraction of light that passes through a
given solution. In a spectrophotometer, a light from the lamp is guided through a
monochromator, which picks light of one particular wavelength out of the continuous
spectrum. This light passes through the sample that is being measured. After the sample,
the intensity of the remaining light is measured with a photodiode or other light sensor,
and the transmittance for this wavelength is then calculated.

In short, the sequence of events in a spectrophotometer is as follows:

1. The light source shines through the sample.

2. The sample absorbs light.
3. The detector detects how much light the sample has absorbed.
4. The detector then converts how much light the sample absorbed into a number.
5. The numbers are either plotted straight away, or are transmitted to a computer to
be further manipulated (e.g. curve smoothing, baseline correction)

Many spectrophotometers must be calibrated by a procedure known as "zeroing." The

absorbency of some standard substance is set as a baseline value, so the absorbencies of
all other substances are recorded relative to the initial "zeroed" substance. The
spectrophotometer then displays % absorbency (the amount of light absorbed relative to
the initial substance).

Principle:

When white light passes through a colored solution, the coloring substance
(chromogen) absorbs a portion of light and the rest is transmitted. The extent to which the
different components of white light are absorbed by a solution depends upon the nature of
chromogen. Thus if a chromogen forms a red solution, it means that the absorption of the
red component is the minimum whereas the other components are absorbed to a larger
extent. The complementary color is absorbed to the greater extent. Even in the
complementary color, the light waves of a particular wave-length are absorbed
maximally. In colorimetry, it is preferable to use monochromatic light of a specific wave-
length which is absorbed maximally by the chromogen being measured. The extent of
actual absorption of light depends upon the number of chromogen particles in the path
traversed by light. This in turn depends upon the concentration of the chromogen and the
thickness of the solution traversed by light.

The relationship between the absorption of the light and the concentration of the
chromogen was described by Beer, and that between the absorption of the light and the
thickness of the solution was described by Lambert.
Beer’s law
This states that the log of the ratio of intensities of incident light and the emergent
light is directly proportional to the concentration of the solution through which the
light passes

log Io α C
I
or log Io = K1C
I
Where
Io = intensity of the incident light
I = intensity of the emergent light
C = concentration of the chromogen.
K1= constant depending upon the wave-length of the light, the nature of the chromogen
and the thickness of the solution.

Lambert’s law:
This states that the log of the ratio of intensities of incident light and the emergent
light is directly proportional to the thickness of the solution through which the light
passes provided that the concentration of the chromogen is constant.

log Io α t
I
or log Io = K2t
I

Where
Io = intensity of the incident light
I = intensity of the emergent light
t = thickness of the solution traversed by light
K2 = constant depending upon the wave-length of the light, the nature of the chromogen
and the thickness of the solution.
Beer-Lambert’s law:
It is the combination of Beer‘s law and Lambert’s law and is:

log Io α C t
I

log Io = K C t
I
Where
K is a constant depending upon the log of wave-length of the incident light and the
nature of the chromogen.
Thus the Beer –Lambert’s law states that the ratio of the intensities of incident
light and emergent light is directly proportional to the concentration of the
chromogen and the thickness of the solution through which the light passes. The
ratio of the intensities of the emergent light and the incident light (I /Io) is known as the
transmittance (T). This is a measure of the ability of a solution to transmit light.

log 1 = K C t
T
log 1/T is known as the optical density(O.D) or the absorbance (A). This is a
measure of the ability of a solution to absorb light. So,

A= KCt and
A α Ct
In other words, whereas transmittance (T) has a logarithmic relationship with c and t,
absorbance (A) has a direct relationship with C and t. therefore, absorbance is used in
calculation in all colorimetric determinations.
 PROCEDURE
Colometry is used based upon the comparison of two solutions of the substance that is
being measured. The concentration of one solution (standard solution) is known, and that
of other (unknown solution) is to be determined. The intensities of colors (absorbance) of
the unknown and the standard solution is measured while the path traversed by the light
(thickness of the solution) is kept equal.

If
Au = absorbance of unknown solution
Cu = concentration of the substance being measured in the unknown solution
As = absorbance of standard solution
Cs = concentration of the substance being measured in the standard solution
K = constant (common to both solutions),
t = the thickness of both the solution,

Then Au = KCut or Au = Kt
Cu

And As = KCst or As = Kt
Cs

So, Au = As and Cu = Au x Cs
Cu Cs As
Generally, the concentration of the substance being measured is expressed in mg/dl of
blood or other physiological fluids. Suppose x ml of sample was present in the final
volume of the unknown solution.

The substance in xml of the sample = Cu = Au x Cs

As

The substance in 100 ml of sample = Cu =( Au x Cs x 100) mg

As x
The concentration of the substance

(Au x Cs x 100) mg /100 ml (or mg/dl) of the sample

As x