UV-Vis Spectroscopy
By: Kimberly Lopez
What is UV-Vis analysis?
 UV-Vis is fast, simple and inexpensive method to
determine the concentration of an analyte in a
solution.
 Used for relatively simple analysis, where the type of
compound to be analyzed is known, to do
quantitative analysis to determine the concentration
of the analytes.
Uses
 Used in a broad range of areas, mainly for routine
measurements
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Hospitals
Petrochemical industry
Food industry
Water quality control labs
Chemical and biological plants
Uses
 In laboratories UV-Vis can be used to monitor the
separations in liquid chromatography
 If a mixture is separated in a column, UV-Vis can be
used to detect the different compounds
 UV-Vis is relatively cheap and an easy detector
compared to Mass spectrometry detectors
How it works?
 In UV-Vis, a beam with a wavelength varying between
180nm-1100nm passes through a solution in a cuvette.
 The sample in the cuvette absorbs this UV or visible
radiation.
I₀ is the radiation coming in
I is the radiation coming
out
How it works?
 The amount of light
that is absorbed by
the solution depends
on:
 The concentration
 The path length of
the light through the
cuvette
 How well the analyte
light absorbs at a
certain wavelength
Violet: 400 - 420 nm
Indigo: 420 - 440 nm
Blue: 440 - 490 nm
Green: 490 - 570 nm
Yellow: 570 - 585 nm
Orange: 585 - 620 nm
Red: 620 - 780 nm
UV-Vis Spectrophotometer
Double Beam UV-Vis
Spectrophotometer
The relation of absorbance to concentration is
given by lambert-Beer’s law (Beer’s Law):
A = ɛlc
Absorbance
Epsilon
Path Length
Molar absorption coefficient of the
analyte for a certain wavelength
Concentration of
analyte
UV-Vis Spectrophotometer
 1st choice in most laboratories concerned with the
identification and measurement of organic and
inorganic compounds
 - in nucleic acids, proteins, foodstuffs, pharmaceuticals
and fertilizers, in mineral oils and in paint.
 In every branch of molecular biology, medicine and the
life sciences, the spectrophotometer is an essential aid
to both research and routine control.
What can be Analyzed?
 Analytes that can be dissolved in solvents like water,
ethanol and hexane
 Analytes that absorb UV or Visible light
 Quantitative measurements with a single analyte in
the solution or more if they do not interfere.
Analyzing Mixtures
 Compounds that absorb UV or visible light have UVVis Spectrum
 Mixtures may be analyzed if the UV-Vis spectra of
compounds differ sufficiently from each other
 In most cases, where there is more than one analyte,
this is where Beer’s Law comes in
What is shown?
 UV-Vis spectrum shows the absorbance of one or more
sample components in the cuvette when scanned through
various wavelengths
Y-axis show the
absorbance
X-axis shows the wavelength
Relevance to Forensics
 Screening methods in drug analysis and some applications in
trace analysis
 Related to forensic pharmacology, color is due to the
absorption of light by two nearby elements of a molecule, in
other substances such as metals, color is due to unique
absorption that is characteristic of that the element(s)
present.
 In Spectrophotometry Cell length is controlled for, the
Coefficient and Concentration for unknowns is not usually
known in forensic analysis, therefore this method is not
utilized often for dealing with unknowns.
Hair samples
 UV-Vis spectrophotometer is used to analyze the
dyes and pigments in both hairs and fibers. This is
done by studying the absorbance and fluorescence
spectra of these microscopic
samples. Microspectrophotometers are also used to
identify the polymers contained in man-made
fibers. This is done by examination of the ultraviolet
absorbance spectra to identify the polymer.
Drug samples
 Since many illegal drugs contain aromatic groups that
produce characteristic UV/Vis spectra. As the
absorption peak depends on the nature of the
aromatic group(s) in the drug compound, additional
spectroscopic information regarding drug derivatives
and their metabolites will be obtained.
Food dyes
 Using a UV-visible spectrometer and a range of food
dyes you will test how the absorbance wavelength
value relates to the color of the solution.
Safety
 Risk of burn. Before replacing the lamp, set the power
switch off and cool down the lamp.
 Room temperature within the range of 15°C to 35°C.
 A position not exposed to direct sunlight.
 A position not subject to strong vibration, or any
continuous (even weak) vibration.
 A position free from strong magnetic or electromagnetic
fields.
 A location free from exposure to corrosive gas, or any
organic or inorganic gas that has an absorption band in the
UV region.
 A location substantially free dirt or dust.
 Before changing a fuse or the inlet voltage, turn off the
power switch and disconnect the power cable.
Advantages & Disadvantages
 Analyze substances quickly and
they are not difficult to use
 UV-Vis spectrometers bring
high-tech spectral analysis to
the food industry, where they
help laboratory technicians
study food products
 These devices are used in fields
as diverse as forensic analysis,
research and medicine
 Dust or grim may coat the mirrors in a
double-beam UV-Vis spectrometer, the
device's performance will degrade
 Repair costs can also double because
you can't replace one mirror if it needs
fixing. Because they work as a pair,
you must replace both mirrors.
 Stray light caused by faulty equipment
design and other factors could
decrease an instrument's linearity
range and reduce the absorbency of
the substance it measures
 Electronic components in the
spectrometer or sample source may
also generate noise that decreases
measurement accuracy and reduces
the device's sensitivity.
Works Cited
 "UV/Vis Spectrometry Basics." - Chromedia. Web. 31 Mar. 2015.
 "Forensic Applications of UV-Vis Spec." Forensic Applications of
UV-Vis Spec. Web. 31 Mar. 2015.
 "Analytical Forensic Pharmacology/UV-VIS Spectroscopy." Wikibooks, Open Books for an Open World. Web. 31 Mar. 2015.
 "CRAIC Technologies Mobile." Fiber and Hair Analysis. Web. 31
Mar. 2015.
 Lee, Kevin. "Advantages & Disadvantages of a UV-VIS
Spectrometer." EHow. Demand Media, 4 May 2010. Web. 31 Mar.
2015.
 "10C: UV/Vis and IR Spectroscopy." - Chemwiki. Web. 31 Mar.
2015.