Calabano, Mark Jasper C.

I- BS Medical Technology

SPECTROPHOTOMETER AND BEER'S LAW

What is spectrophotometer and its functions?

Spectrophotometer is an analytical instrument in spectroscopy-the study of the interaction

between matter and electromagnetic radiation, that is used quantitatively to measure the amount of light
absorbed by a substance. The spectrophotometer is used in the process of measuring color and used for
monitoring color accuracy throughout production. It is useful for measuring the absorption spectrum of a
compound, that is, the absorption of light by a solution at each wavelength. This instrument measures the
intensity as a function of light source wavelength. The spectrophotometer measures transmittance (T)
which is the ratio of the transmitted intensity I to the original intensity of light. The main components of
this instrument are the light source, monochromator, a cuvette that will contain the sample and a detector
that is connected to a computer which will then analyze the data. A spectrophotometer is made up of two
instruments: a spectrometer and a photometer. The purpose of spectrometer is to produce light of any
wavelength, while the photometer is to measure the intensity of light. The spectrophotometer is designed
in a way that the liquid or a sample is placed between spectrometer and photometer.  The photometer
measures the amount of light that passes through the sample and delivers a voltage signal to the display. If
the absorbing of light change, the voltage signal also changes. There are two classes of
spectrophotometers: single and dual beam. Single beam specs measure absolute light intensity. A single
beam spectrophotometer uses a reference standard to standardize or blank the instrument before taking
measurements. The dual beam specs measures the ratio of light intensities on two separate light paths-the
reference standard and the sample. It splits the beam of light into two different paths, one of which passes
through the sample while the other passes through a reference standard. Dual beam spectrophotometers
measure the ratio of light intensities and, therefore, are not as sensitive to fluctuations in the light source
or detector. Type so specs include micro-plate spectrophotometers, colorimeters, and florescence micro-
plate readers.
Functions of spectrophotometer are involved in a wide variety of fields. In quantitative analyses,
spectrophotometer is an effective and economical quantitative analysis instrument, especially for
substances that are monotonous and colored that can be analyzed by a single wavelength absorbance
measurement. The quantitative accuracy of a spectrophotometer is typically in ppm level, and in some
situation with well controlled variations, can even in ppb level. In industries, a spectrophotometer is also
useful in quality assurance process, because most of the substance to be tested are known with controlled
conditions. For example, the quality control of the compounds of hexavalent chromium, residual chlorine
in water, nitrates, sulfates and phosphates. In clinical laboratories, spectrophotometers are used for many
applications such as kinetics, spectrum scanning and multiple components DNA/protein testing . In
different areas of science such as microbiology, biochemistry, forensics, physics, and medical health,
spectrophotometers are useful. Spectrophotometers can be used to measure certain ingredients in a drug
to make sure it is effective and safe for consumers. The instrument can also be used in measuring
bacterial growth, or diagnosing a patient based on how much uric acid is present in their urine. Even non-
scientists use spectrophotometers. Wine-makers, for example, use them to determine how much malic
acid (reducing sugars) a particular wine has in it. In biochemistry, for example, it is used to determine
enzyme-catalyzed reactions. Furthermore, some of the major applications of spectrophotometers include
detection of concentration of substances, detection of impurities, structure elucidation of organic
compounds, monitoring dissolved oxygen content in freshwater and marine ecosystem, characterization
of proteins, detection of functional groups, respiratory gas analysis in hospitals, and molecular weight
determination of compounds.

State the Beer-Lambert law?

In spectroscopy, Beer-Lambert law which is also known as Beer's law, states that there is a linear
relationship between the absorbance of light and the concentration of a sample. In simpler terms, a more
concentrated solution absorbs more light than a more dilute solution does. The absorbance of light is
dependent on the number of molecules present in the solution so if the concentration of the solution
increases, then therefore the absorbance of light will also increase. Mathematical statement of Beer's law
is A= εlc, where A is the absorbance of the solution which is expressed in no unit, the epsilon " ε" is a
constant and is the molar absorptivity or the molar extinction coefficient that depends on the nature of the
chemical and the wavelength of the light which is expressed in liter per mol centimeters, l is the distance
that light travels through the solution that is expressed in centimeters, and c wherein it is the
concentration of absorbing species which is expressed in mol per liter. There are three important things
explained in this law. First, when a monochromatic light of initial intensity Io (intensity of incident light
before hitting the sample) passes through a solution in a transparent vessel (cuvette), some of the light is
absorbed so that the intensity of the transmitted light I (intensity of the light after passing through the
sample) is less than Io. As a result of absorption, the intensity of light passing through material bodies as
for example the intensity of transmitted light, decreases. This is due to the law's dependence on a beam of
light being shone through a solution containing solutes which absorb a fraction of the light as it goes
through the solution. As the solution absorbs some of the light (absorbance), the intensity of the light that
will come out of the other side of the solution and then will be decreased in intensity. Second, there is
some loss of light intensity from scattering by particles in the solution and reflection at the interfaces, but
mainly from absorption by the solution. If material bodies are exposed to radiation, part of the incident
radiation is absorbed, a part is scattered and a part is transmitted. And third, the relationship between I
and Io depends on the path length of the absorbing medium, l, and the concentration of the absorbing
solution, c. This means that the transmittance or fraction of incident light absorbed depends on the
thickness of the absorbing medium. The longer the path length, the greater the light absorbance of a
solution. . The relation can be used to determine the concentration of a chemical species in a solution
using a colorimeter or spectrophotometer.

Beer's Law is especially important in the fields of chemistry, physics, and meteorology. Other
uses of Beer's Law is used in chemistry to measure the concentration of chemical solutions, to analyze
oxidation, and to measure polymer degradation. The law also describes the attenuation of radiation
through the Earth's atmosphere. While normally applied to light, the law also helps scientists understand
the attenuation of particle beams, such as neutrons. Beer's law is easy to work with but there still a
limitation to consider. We all know the fact that Beer's law is used to calculate unknown concentration of
a substance in a solution. So in working with Beer's law, there should have standards or the known molar
concentration because these values can help to estimate the value of the unknown concentration. In
measuring the unknown concentration, it's value should fall between the value of known molar
concentration. If not, problems will arise. Problems in Beer's Law is encountered when the value
unknown concentration don't fall between the value of molar concentrations. Beer's Law is only linear or
straight line for short distances, meaning if the unknown concentration do not fall between the standards,
then the result will be inaccurate and cannot be trusted.

What is the principle behind colorimetry?

Colorimetry is the science of quantitative measurement of color and also an analytical technique
for measuring the concentration of a particular compound (solute) in a colored solution (solvent) by the
application of the Beer–Lambert's law, which states that the concentration of a solution is directly
proportional to the absorbance of light. In colorimetry, colored substance absorbs light in relation to their
color intensity or in simpler terms, if the concentration of the solution is greater, more light will be
absorbed. The color intensity is measured photometrically. The measurement is based on the fact that the
component under analysis absorbs or attenuates light of a specific wavelength. This can be identified by
measuring the difference between the amount of light at its origin and that after passing the solution. The
working principle of colorimetry states two things: the amount of light transmitted decreases
exponentially with increase in path length (diameter) of the cuvette or the thickness of colored solution
through which light passes and when a monochromatic light passes through a colored solution, some
specific wavelengths of lights are absorbed which will be related to the color intensity. The amount of
light absorbed or transmitted by a color solution is in accordance to the principle of Beer-Lambert's law.
Inspecting closely to the idea of Beer-Lambert's law, the law is made up of two laws: Beer's law and
Lambert's law. In Beer's law, the concentration of a substance is directly proportional to the amount of
light absorbed and inversely proportional to the logarithm of the transmitted light. So applying the
concept of Beer's law in colorimetry, in relation with the absorbance and concentration, the amount of
light absorbed by a colored solution is directly proportional to the concentration of the substance in the
colored solution. The more concentrated a solution is, the more light will be absorbed. On the other hand,
the Lambert's law states that when a ray of monochromatic light passes through an absorbing medium, its
intensity decreases exponentially as the length of the light path through light absorbing materials
increases. Applying this law also to the concept of colorimetry, in relation with the absorbance and path
length, the amount of light absorbed by a colored solution depends on the path length of the cuvette or
thickness or dept of the colored solution. The longer the path length, the greater the light absorbance of a
solution. Colorimetry is a very quick and efficient way of analyzing colored solutions or any colored
substance. It uses colorimeter as a measuring instrument. Colorimeters helps to detect color and determine
the concentration of the solutions. When a wavelength goes through a sample, some of the light is absorbed
and some passes, this device detects the wavelengths of light that passes through. The colorimeter makes use
of the Beer-Lambert law for the purpose of detecting the wavelength absorbance.

In addition, colorimeters are used to test for water quality, by screening for chemicals such
as chlorine, fluoride, cyanide, dissolved oxygen, iron, molybdenum, zinc, and hydrazine. It also helps to
determine the concentrations of plant nutrients such as phosphorus, nitrate, and ammonia in the soil or
hemoglobin in the blood. Furthermore, it helps in the identification of substandard and counterfeit drugs.
Many food industries use his device. Paints and textile manufacturers use Colorimeter. This device also
checks the quality and consistency of colors in paints and fabrics, to ensure the same quality.