Tan, Denise Mariano, Andrea

Perez, Daennielle Valenzuela, Nyx

Ramos, Carl Joshua 2Biology1
UV-VIS

Ultraviolet-visible spectrophotometer

From ultra-high-performance UV, Vis and NIR systems to the smallest

spectrophotometers, our analytical solutions offer reliable data and the highest available
performance specifications using superior optical features. If you are working in a
regulated industry, need a wide range of sampling options, or need to process a large
number of samples, you'll find our UV-Vis systems are easy to operate, and deliver
results you can trust with a minimum of operator training. And for the most advanced
applications, the newest additions to our molecular spectrophotometry portfolio redefine
your range of capabilities by providing a previously impossible level of sensitivity,
resolution, and scanning speed in the near infrared range.

The Electromagnetic Spectrum

A continuum of all electromagnetic waves arranged according to frequency and

wavelength. The sun, earth, and other bodies radiate electromagnetic energy of varying
wavelengths. Electromagnetic energy passes through space at the speed of light in the
form of sinusoidal waves.

Electromagnetic radiation can be expressed in terms of energy, wavelength, or

frequency. Frequency is measured in cycles per second, or Hertz. Wavelength is
measured in meters. Energy is measured in electron volts. Each of these three
quantities for describing EM radiation are related to each other in a precise
mathematical way.

The laws of quantum mechanics

Scientists interpret quantum mechanics to mean that a tiny piece of material like a
photon or electron is both a particle and a wave. It can be either, depending on how one
looks at it or what kind of an experiment one is doing. In fact, it might be more accurate
to say that photons and electrons are neither a particle or a wave -- they're undefined up
until the very moment someone looks at them or performs an experiment, thus forcing
them to be either a particle or a wave. This comes with other side effects: namely that a
number of qualities for particles aren't well-defined. For example, there is a theory by
Werner Heisenberg called the Uncertainty Principle. It states that if a researcher wants
to measure the speed and position of a particle, he can't do both very accurately. If he
measures the speed carefully, then he can't measure the position nearly as well. This
doesn't just mean he doesn't have good enough measurement tools -- it's more
fundamental than that. If the speed is well-established then there simply does not exist
a well-established position (the electron is smeared out like a wave) and vice versa.

Albert Einstein disliked this idea. When confronted with the notion that the laws of
physics left room for such vagueness he announced: "God does not play dice with the
universe." Nevertheless, most physicists today accept the laws of quantum mechanics
as an accurate description of the subatomic world. And certainly it was a thorough
understanding of these new laws which helped Bardeen, Brattain, and Shockley invent
the transistor.

Frequency is how often an event repeats itself over a set amount of time.
In physics, the frequency of a wave is the number of wave crests that pass a point in
one second (A wave crest is the peak of the wave).
Hertz (symbol Hz) is the unit of frequency.
The relationship between Frequency and wavelength is expressed by the formula: f = v /
λ

f=c/λ

All electromagnetic waves travel at the speed of light in a vacuum but they travel
at slower speeds when they travel through a medium that is not a vacuum. Other
waves, such as sound waves, travel at much much lower speeds and cannot travel
through a vacuum.

Examples of electromagnetic waves are; light waves, radio waves, infrared

radiation, microwaves, and gamma waves.

The speed of light in vacuum, commonly denoted c, is a universal physical

constant important in many areas of physics. Its exact value is 299792458 metres per
second (approximately 3.00×108 m/s, approximately 186,282 mi/s); it is exact because
the unit of length, the metre, is defined from this constant and the international standard
for time.

E=hxv

This equation is used to calculate the energy of electromagnetic radiation, and is

known as the Planck-Einstein relation. It involves the energy of a particle of light (E),
called a photon, is proportional to its frequency (v), by a constant factor (h). This means
that photons with low frequencies, like radio waves, have lower energies than photons
with high frequencies, like x-rays. Plancks constant (h) is a physical constant of
proportionality connecting the energy of a photon to the frequency of the photon with an
approximate value of 6.626 x 10-34 J.s.

𝒉𝒄
E= 𝝀

The photon energy is the energy carried by a single photon with a

certain electromagnetic wavelength and frequency. The higher the photon's frequency,
the higher its energy. Equally, the longer the photon's wavelength, the lower its energy.

Photon energy is solely a function of the photon's wavelength. Other factors, such as
the intensity of the radiation, do not affect photon energy. In other words, two photons
of light with the same color (and, therefore, same wavelength) will have the same
photon energy, even if one was emitted from a wax candle and the other from the Sun.

The photon energy can be represented by any unit of energy. Among the units
commonly used to denote photon energy are the electronvolt (eV) and the joule (as well
as its multiples, such as the microjoule). As one joule equals 6.24 × 10 18 eV, the larger
units may be more useful in denoting the energy of photons with higher frequency and
higher energy, such as gamma rays, as opposed to lower energy photons, such as
those in the radiofrequency region of the electromagnetic spectrum.

Photons being massless, the notion of "photon energy" is not related to mass through
the equivalence E = mc2.
 Ultraviolet–visible spectroscopy or ultraviolet-visible spectrophotometry (UV-
Vis or UV/Vis) refers to absorption spectroscopy or reflectance spectroscopy in
the ultraviolet-visible spectral region. This means it uses light in the visible and adjacent
ranges. The absorption or reflectance in the visible range directly affects the
perceived color of the chemicals involved. In this region of the electromagnetic
spectrum, atoms and molecules undergo electronic transitions. Absorption spectroscopy
is complementary to fluorescence spectroscopy, in that fluorescence deals with
transitions from the excited state to the ground state, while absorption measures
transitions from the ground state to the excited state.

Principle of ultraviolet-visible absorption

Molecules containing π-electrons or non-bonding electrons (n-electrons) can

absorb the energy in the form of ultraviolet or visible light to excite these electrons to
higher anti-bonding molecular orbitals.[2] The more easily excited the electrons (i.e.
lower energy gap between the HOMO and the LUMO), the longer the wavelength of
light it can absorb. Based on the fact of four type of transition- π-π*, n-π*, σ-σ*, and n-
σ*. The energy required for various transitions obey the following order σ-σ*>n-σ*>π-
π*>n-π*.

Instruments for measuring the absorption of U.V. or visible radiation are made up of
the following components;

1. Sources (UV and visible)

2. Wavelength selector (monochromator)
3. Sample containers
4. Detector
5. Signal processor and readout

Sources of UV radiation

It is important that the power of the radiation source does not change abruptly over its
wavelength range.
Wavelength selector (monochromator)

All monochromators contain the following component parts;

 An entrance slit
 A collimating lens
 A dispersing device (usually a prism or a grating)
 A focusing lens
 An exit slit

Cuvettes

The containers for the sample and reference solution must be transparent to the
radiation which will pass through them. Quartz or fused silica cuvettes are required for
spectroscopy in the UV region. These cells are also transparent in the visible region.
Silicate glasses can be used for the manufacture of cuvettes for use between 350 and
2000 nm.

Detectors

The photomultiplier tube is a commonly used detector in UV-Vis spectroscopy. It

consists of a photoemissive cathode (a cathode which emits electrons when struck by
photons of radiation), several dynodes (which emit several electrons for each electron
striking them) and an anode.

The linear photodiode array is an example of a multichannel photon detector. These

detectors are capable of measuring all elements of a beam of dispersed radiation
simultaneously.

Charge-Coupled Devices (CCDs) are similar to diode array detectors, but instead of
diodes, they consist of an array of photocapacitors.

*If the sample compound does not absorb light of of a given wavelength, I = I0.
However, if the sample compound absorbs light then I is less than I0, and this difference
may be plotted on a graph versus wavelength, as shown on the right. Absorption may
be presented as transmittance (T = I/I0) or absorbance (A= log I0/I). If no absorption has
occurred, T = 1.0 and A= 0. Most spectrometers display absorbance on the vertical axis,
and the commonly observed range is from 0 (100% transmittance) to 2 (1%
transmittance). The wavelength of maximum absorbance is a characteristic value,
designated as λma
 I. Lambert’s Law

The amount of monochromatic light absorbed by a sample is determined by

comparing the intensities of the incident light (Io) and transmitted light (I). The
ratio of the intensity of the transmitted light (I) to the intensity if the incident light
𝐼
(Io) is called transmittance (T) . Transmittance: (T) = 𝐼𝑜

II. Beer’s Law

For each wavelength of light passing through the spectrometer, the intensity of the
light passing through the reference cell is measured. This is usually referred to as Io
- that's I for Intensity. The intensity of the light passing through the sample cell is
also measured for that wavelength - given the symbol, I. If I is less than Io, then
obviously the sample has absorbed some of the light. A simple bit of maths is then
done in the computer to convert this into something called the absorbance of the
sample - given the symbol, A. For reasons to do with the form of the Beer-Lambert
Law (below), the relationship between A (the absorbance) and the two intensities is
given by:
𝐼𝑜
Absorbance (A) = log 𝐼

100
A = log 𝑇

Transmittance, T = P / P0
% Transmittance, %T = 100 T

Absorbance,

A = log10 P0 / P
A = log10 1 / T
A = log10 100 / %T
A = 2 - log10 %T

The last equation, A = 2 - log10 %T , is worth remembering because it allows you to

easily calculate absorbance from percentage transmittance data.

The relationship between absorbance and transmittance is illustrated in the following

diagram:
So, if all the light passes through a solution without any absorption, then absorbance is
zero, and percent transmittance is 100%. If all the light is absorbed, then percent
transmittance is zero, and absorption is infinite.

A = 𝜀𝑐𝑏

Where A is absorbance (no units, since A = log10 P0 / P)

e is the molar absorbtivity with units of L mol cm-1
-1

b is the path length of the sample - that is, the path length of the cuvette in which the
sample is contained. We will express this measurement in centimetres.
c is the concentration of the compound in solution, expressed in mol L-1

Example of an Experiment using UV-VIS

Determination of Iron in Water