1.

ABSTRACT
Ultravioletvisible

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 (near-UV and near-infrared [NIR]) 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, molecules undergo electronic transitions.
Ultraviolet-visible spectroscopy or ultraviolet-visible spectrophotometry (UV-VIS)
involves the spectroscopy of photons and spectrophotometry. It uses light in
visible and adjacent near ultraviolet (UV) and near infrared (NIR) ranges. In this
region of energy space molecules undergo electronic transitions.
Electromagnetic radiation in th UV-VIS portion of the spectrum ranges in
wavelength from approximately 200 to 700 nm. The UV range is colorless to the
human eye, while different wavelengths in the visible range each have the
characteristic color, ranging from violet at the short wavelength end of the
spectrum to red at the long wavelength end o the spectrum. The instrument used
in ultraviolet-visible spectroscopy is called Ultraviolet-Visible Spectrophotometer.

2. INTRODUCTION
UV-VIS spectroscopy is one of the oldest methods in molecular spectroscopy.
The definitive formulation of the Bouguer-Lambert Beer law in 1852 created the
basis for the quantitative evaluation of absorption measurements at an early

date. This led firstly to colorimetry, then to photometry and finally to

spectrophotometry. This evolution ran parallel with the development of detectors
for measuring light intensities. With the development of quantum chemistry,
increasing attention was paid to the correlation between light absorption and the
structure of matter with the result that in recent decades a number of excellent
discussions of the theory of electronic spectroscopy (UV-VIS and luminescence
sp,~ctroscopy) have been published.
In the 1930s, vitamin research indicated that several vitamins, particularly vitamin
A, absorb ultraviolet (UV) light. Spurred by the American governments interest in
measuring vitamin content in soldiers rations using ultraviolet and visible (UVVis) light, this research culminated in the commercial launch of UV-Vis
spectrophotometers in

the

early 1940s.

Of

these,

the

Beckman

DU

spectrophotometerfirst sold in 1941 distinguished itself from competing

products by delivering more accurate results and reducing analysis time from
hours, or even weeks, to minutes.
Although modern UV-Vis spectrometers differ greatly from the first DUs, all
operate on the same basic principle.
Whether as standalone instruments or high performance liquid chromatography
(HPLC) detectors, UV-Vis spectrophotometers are indispensible for measuring
analyte concentrationsin scientific research, academic teaching, and QA/QC
laboratories

studying

pharmaceuticals,

proteins,

DNA,

solar

panels,

semiconductors, and coatings.

1930s
In 1935, Arnold O. Beckman founds National Technologies Laboratorieslater
named Beckman Instruments.
1940s
The first commercially available UV-Vis spectrophotometers are
introduced. In 1940, In 1941, Beckman introduces the DU UV-Vis
spectrophotometer, which has higher resolution and lower stray light in the
ultraviolet region than any other commercial instrument.

In 1947, Applied Physics Corporation delivers the first commercially available

recording UV-Vis spectrophotometer, the Cary 11, to the Mellon Institute in
Pittsburgh, PA.
1950s
1950s 1970s Mass production reduces the cost of UV-Vis spectrophotometers.
New photodiode arrays collect all wavelengths simultaneously, reducing the time
required to scan a spectrum from minutes to seconds. In 1950, National
Technologies Laboratories changes its name to Beckman Instruments, Inc.
In 1953, Bausch & Lomb introduces the SPECTRONIC 20 UV-Vis
spectrophotometer, the first massproduced, low-cost UV-Vis spectrophotometer.
In 1954, Applied Physics Corporation launches the Cary 14 spectrophotometer,
the

first

commercially

available

double-beam

spectrophotometer.

The

doublebeam design greatly simplifies and speeds up sample analysis by

simultaneously measuring sample and solvent transmittance over the wide
spectral range of ultraviolet, visible, and near infrared wavelengths.
1960s
In 1969, Cecil Instruments introduces the CE 212, the world's first commercially
available variable wavelength detector for HPLC, allowing users to select
without changing filters or lampsdetection wavelengths on a single detector.
1970s
In 1979, Hewlett-Packard launches the first commercially available diode-array
spectrophotometer, the 8450A. the 8450A utilizes an array of photodiodes to
scan simultaneously the full spectrum of wavelengths in seconds.
1980s
The proliferation of personal computers in the 1980s improves data acquisition
and instrument control. In 1980, Bausch & Lomb introduces the Spectronic 2000
UV-Vis spectrophotometer, the first microprocessor-controlled double-beam UVVis spectrophotometer. Now, instead of measuring sample and solvent
transmittance separately, which the single-beam spectrophotometers required,

the double-beam design greatly simplifies and speeds up sample analysis by

simultaneously measuring sample and solvent transmittance.
In 1987, Pye Unicam Corporation. introduces the PU-8700

UV-Vis

spectrophotometer, the first mouse-driven, graphical interface UV-Visible

spectrophotometer.
In 1989, Dr. Arnold O. Beckman, now 88 years old, receives the National Medal
of Science for his leadership in analytical instrumentation development.
1990s
1990s, External software now provides PC control, onscreen spectra display, and
spectra reprocessing and storage. Fiber optics reduce instrument size, and fiber
optic sampling accessories allow sample measurement outside the UV-Vis
spectrophotometers sample compartment, eliminating the need to fill sample
cells and cuvettes.
In 1995, Hewlett-Packard launches the 8453A, the first small-footprint and fullfeatured diode-array spectrophotometer.
In 1997, Beckman Instruments, Inc. acquires Coulter Corporation, the leading
manufacturer of systems for blood and cell analysis. In 1998, the company is
renamed Beckman Coulter, Inc.
In 1999, Hewlett-Packard announces a strategic realignment to create an
independent measurement company, Agilent Technologies.
2000s
2000s, Significant progress is made in the ability to measure micro volume liquid
samples (< 1 L) in biotechnology and pharmaceutical applications. UV-Vis
spectroscopy is applied to alternative energy R&D such as solar energy.
Instrument manufacturers start to miniaturize instruments and develop dedicated
instruments

for

specific

applications,

such

as

biological

applications.

In 2000, Thermo Scientific introduces the GENESYS 10 instruments with out-ofplane optics that minimize stray light and reduce noise caused by instrument
optics.
In 2002, Varian Inc. releases the 6000i UV-Vis-NIR spectrophotometer. The Cary
6000i uses an InGaAs detector that improves signal-tonoise ratio over

conventional lead sulfide detectors. Its operating range of 175 nm to 1800 nm is

applicable to materials science research.
In 2003, Thermo Scientific introduces

the

Evolution

300

spectrophotometer, the first double-beam xenon lamp-based

spectrophotometer. The double-beam design simplifies and speeds up sample
analysis. Xenon flash lamps provide a high-energy light source with a shorter
warm up time and longer lamp life than traditional tungsten and
deuterium lamps.
In 2004, Shimadzu introduces the SolidSpec-3700/3700DUV
series of UV-Vis-NIR spectrophotometers, the first UV-Vis-NIR
spectrophotometer with three detectorsa photomultiplier for the UVVis region,
and an InGaAs detector and a cooled PbS detector for the NIR region.
In 2005, the NanoDrop ND-1000 UV-Vis spectrophotometer (from NanoDrop
Technologies) for micro-quantitation of only 1 l of sample enters the market. The
sample is directly pipetted onto a fiber optic measurement surface where it is
held in place by surface tension, eliminating the need for cuvettes or capillaries.
In 2006, JASCO manufactures a new range of UV-Vis-NIR spectrophotometers
with compatible accessories for life sciences, materials analysis, and
semiconductor R&D.
In 2008, Shimadzu launches the UV- 1800 compact UV-Vis spectrophotometer. It
occupies a 15% smaller footprint than the model it replaces, the UV-1700.
Also in 2008, Perkin Elmer releases the LAMBDA Bio UV-Vis spectrophotometer
pre-configured with standard methods for biological applications including protein
assays, cell density measurements, as well as DNA, RNA, and oligonucleotides
concentration and purity.
2010s
In 2010, Agilent

Technologies

acquires

Varian

Inc.

and

continues to offer the Cary spectrophotometer series under the Cary name.
Also in 2010, Thermo Scientific introduces the Evolution 200 Series
spectrophotometer.

Its

application-focused

beam

geometry

tailors

the

instrument's optical system to specific applications for microcells, solid sampling,

and fiber optics.

Also in 2010, JASCO offers the SAH-769 One Drop accessory to measure micro
volume samples of proteins and nucleic acids with UVVis spectrophotometers.
In 2011, Agilent Technologies releases the Cary 60 UV-Vis spectrophotometer
with low cost of ownershipthe xenon lap typically lasts 10 yearsand remote
sampling options that minimize sample handling.
Future of UV-Vis Spectrophotometers
Future improvements in UV-Vis spectrophotometers will focus
on ease-of-use, portability, and application-specific instruments.
UV-Vis analysis of solid samples and materials continues to grow in areas such
as solar cell research, semiconductor products, and coating materials. Advances
in

light

sources

will

provide

new

developments

in

conventional

spectrophotometers and handheld UV-Vis instruments. Further development in

remote sensors will enable more types of samples to be measured outside the
laboratory.

3. THEORY
Molecules containing -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 longer the wavelength of light it can absorb.

The Beer-Lambert law states that the absorbance of a solution is directly

proportional to the concentration of the absorbing species in the solution and the
path length.[3] Thus, for a fixed path length, UV/Vis spectroscopy can be used to
determine the concentration of the absorber in a solution. It is necessary to know
how quickly the absorbance changes with concentration. This can be taken from
references (tables of molar extinction coefficients), or more accurately,
determined from a calibration curve.

AC
Lamberts Law is defined as the absorbance (A) is directly proportional to
thickness of solution (b) when beam of monochromatic light is passed through a
solution of constant concentration.

Ab

Combining Beers and Lamberts expression, we have :

A bC

Thus,

= bc,

where = molar
.
absorptivity
The wavelengths of
absorption peaks can be correlated with the types of bonds
in a given molecule and are valuable in determining the functional groups within
a molecule. The nature of the solvent, the pH of the solution, temperature, high
electrolyte concentrations, and the presence of interfering substances can
influence the absorption spectrum. Experimental variations such as the slit width
(effective bandwidth) of the spectrophotometer will also alter the spectrum. To
apply UV/Vis spectroscopy to analysis, these variables must be controlled or
accounted for in order to identify the substances present. [4]

Schematic of UV- visible spectrophotometer.

The instrument used

in

ultraviolet-visible

spectroscopy

is

called

UV/Vis spectrophotometer. It measures the intensity of light passing through a

sample ( ), and compares it to the intensity of light before it passes through the
sample (

). The ratio

is called the transmittance, and is usually expressed

as a percentage (%T). The absorbance,

, is based on the transmittance:

A spectrophotometer can be either single beam or double beam. In a single

beam instrument, all of the light passes through the sample cell.

must be

measured by removing the sample. This was the earliest design and is still in
common use in both teaching and industrial labs.
In a double-beam instrument, the light is split into two beams before it reaches
the sample. One beam is used as the reference; the other beam passes through
the sample. The reference beam intensity is taken as 100% Transmission (or 0
Absorbance), and the measurement displayed is the ratio of the two beam
intensities. Some double-beam instruments have two detectors (photodiodes),
and the sample and reference beam are measured at the same time.

Furthermore, the energy of a compound can be ascertained from this technology

by using the equation E = hc/ (where E = energy, h = Plancks constant, c =
speed of light, and = wavelength). Since photons travel at the speed of light,
and h and c are constants, one can find the energy

4. SAMPLE PREPARATION
Samples for UV/Vis spectrophotometry are most often liquids, although the
absorbance of gases and even of solids can also be measured. Samples are
typically placed in a transparent cell, known as a cuvette. Cuvettes are typically
rectangular in shape, commonly with an internal width of 1 cm. (This width
becomes the path length, , in the Beer-Lambert law.) Test tubes can also be
used as cuvettes in some instruments. The type of sample container used must
allow radiation to pass over the spectral region of interest. The most widely
applicable cuvettes are made of high quality fused silica or quartz glass because
these are transparent throughout the UV, visible and near infrared regions. Glass

and plastic cuvettes are also common, although glass and most plastics absorb
in the UV, which limits their usefulness to visible wavelengths. [9]

5. Mmm

6. Ddd

7. LIMITATIONS
1. mixtures of molecules can be a problem due to overlap (hence, routinely
requires significant sample preparation)
2. spectra are not highly specific for particular molecules
3. absorption can be dependent on solution conditions; hence, often optimal to
combine with a HPLC in order to standardize solution conditions
4. result only show a peak which determine a compound but do not show the
structure
5. the solvent must be dilute enough if not high peak will be obtsined

http://chemwiki.ucdavis.edu/Physical_Chemistry/Kinetics/Reaction_Rates/Experimental
_Determination_of_Kinetcs/Spectrophotometry
https://www2.chemistry.msu.edu/faculty/reusch/virttxtjml/Spectrpy/UV-Vis/uvspec.htm
http://www.labmanager.com/lab-product/2011/07/evolution-of-uv-visspectrophotometers?fw1pk=2#.VROAa_mUeEw
https://medicine.yale.edu/labmed/Images/spectroscopic%20techniques
%20lecture_tcm45-9318.pdf

https://www.ucmo.edu/chemphys/about/documents/cary_300_bio_uv.pdf
http://sphinxsai.com/sphinxsaiVol_2No.1/ChemTech_Vol_2No.1/ChemTech_Vol_2No.1
PDF/CT=108%20%28695-699%29.pdf