Describe the theories behind the science of spectrophotometry.

Differentiate
spectroscopy with spectrophotometry. Identify key applications of this kind of chemical
analysis. Why is this analysis method not as popular as other chemical analysis methods?

Spectrophotometry is a method of determining how much light is reflected by a chemical

substance by measuring the intensity of light as it passes through it. Each substance absorbs or
emits light across a certain wavelength range, according to the fundamental theory. Light emits a
wide range of wavelengths, from extremely short to extremely long. In the context of radiation,
each ray behaves as if it has an electric component and a magnetic component acting at right
angles to each other which is called as electromagnetic radiation.
The working principle of the spectrophotometer is based on Beer-Lambert’s Law which
states that the amount of light absorbed by a color solution is directly proportional to the
concentration of the solution and the length of a light path through the solution. The
spectrophotometer technique is to measure light intensity as a function of wavelength. It does this
by diffracting the light beam into a spectrum of wavelengths, detecting the intensities with a
charge-coupled device, and displaying the results as a graph on the detector and then on the
display device. In the spectrophotometer, a prism (or) grating is used to split the incident beam
into different wavelengths. By suitable mechanisms, waves of specific wavelengths can be
manipulated to fall on the test solution. The range of the wavelengths of the incident light can be
as low as 1 to 2nm. The spectrophotometer is useful for measuring the absorption spectrum of a
compound, that is, the absorption of light by a solution at each wavelength.
Spectroscopy, on the other hand, refers to the dispersion of light into its component hues.
In basic terms, it is a method for determining how much light is absorbed by a chemical material
and how much light flows through it at what intensity. It's a method of examining the absorption
and emission properties of materials when it's subjected to electromagnetic radiation, or light. Its
definition has been broadened to encompass interactions among electrons, protons, and ions.
The type of spectroscopy which deals with the infrared region of the electromagnetic
spectrum is Infrared Spectroscopy. The rays of the infrared region have longer wavelength
whereas having a lower frequency than light. Infrared spectroscopy is based on absorption
spectroscopy. Meanwhile, Raman Spectroscopy is a spectroscopic technique which is used to
analyze vibrational, rotational, and other low-frequency modes in a system. Raman’s
spectroscopy is commonly used in the branch of chemistry to provide a fingerprint by which
molecules can be identified. This phenomenon relies on inelastic scattering of monochromatic
light which is also known as Raman scattering. The energy of the laser photons shifts up & down
due to the interaction of the light with the molecules or phonons of an object. This up down shift
of laser photon forms the vibrational modes of an object or system.
A spectrometer is an aspect of the most responsible spectrophotometer for the calculation
of different objects. A spectrophotometer is a comprehensive device that involves a light source,
a way of collecting the light that has interacted with the objects being measured, and a
measurement spectrometer.
The key applications of spectrophotometry includes the following: (1) detection of
concentration of substances; (2) detection of impurities; (3) structure elucidation of organic
compounds; (4) monitoring of dissolved oxygen content in freshwater and marine ecosystems;
(5) characterization of proteins; (6) detection of functional groups; (7) respiratory gas analysis in
hospitals; (8) molecular weight determination of compounds; and (9) the visible and UV
spectrophotometer may be used to identify classes of compounds in both the pure state and in
biological preparations.

In performing UV-Vis Spectrophotometry, what are the things needed to be considered?

Are there any precautions needed before performing this experiment? What are possible
sources of error for this experiment?

Spectrophotometry nevertheless demands a lot of precautions to avoid errors. It must be

emphasized that handling of cuvettes is very important. Any variation in the cuvettes, such as
stains, scratches, or changes in glass curvature will cause varying results. Thus, it is essential to
follow several rules: (1) Do not handle the lower portion of the cuvette through which the light
passes; (2) Always rinse the cuvette with several portions of the solution to be measured before
taking measurements; (3) Wipe off any liquid drops or smudges with a clean tissue before placing
the cuvette in the spectrophotometer; and (4) When inserting a cuvette, always do so with the
index mark facing the front of the instrument, and after the cuvette is seated, line up the index
marks exactly.
The errors may come from the improper calibration of spectrophotometer modules and
scales, the improper functioning of modules, improper cells, stray light and scattering, as well as
deviations in the chemical reaction, and influences the accuracy of the measurements and the
methods. Other sources of errors include environmental effects on photometer and sample,
temperature, line voltage fluctuations, vibrations, contamination, or heating of the sample by the
photometer. Common sources of error include the use of dirty cuvettes, poorly mixed solutions,
poor pipetting techniques, and incorrect light source or wavelength. Because you have control
over these errors, you must make sure to minimize these problems in your laboratory exercises.
All these factors may impair the measured result, and ways and means are known to test and
eliminate them.

Based on the datasheet from the experiment, how do we determine the wavelength at
maximum absorbance? Can we identify this maximum absorbance at lower
concentrations? What can you say about the color obtained at each wavelength in Table
1? If we were given an unknown solution, and the absorbance obtained is outside that of
the calibration curve, what do we do?

The extent to which a sample absorbs light depends upon the wavelength of light. The
wavelength at which a substance shows the maximum absorbance is called absorption maximum.
The process involves recording the absorbance over the range of 350 nm to 650 nm, usually in
intervals of 25 nm. We can determine the wavelength at maximum absorbance by plotting
absorbance vs. wavelength in graph. The graph allows us to visualize the highest absorbance or
the data pairs can be examined to determine the wavelength. From the data with the highest
absorbance data, we can determine its corresponding wavelength.
Taking into account, the Beer-Lambert’s Law which states that the amount of light
absorbed by a color solution is directly proportional to the concentration of the solution and the
length of a light path through the solution or simple, the absorbance is directly proportional to the
concentration of the solution.
 This means that the higher the molar absorptivity, the higher the absorbance. With higher
molar absorptivity, the lower the concentration of species which still gives a measurable
absorbance value. Therefore, the maximum absorbance can be identified at lower concentrations.
In case of unknown solution and the absorbance obtained is outside of the calibration curve, using
the least square method of curve fitting will determine the unknown concentration.