UV SPECTROPHOTOMETER
Introduction
Ultraviolet-visible (UV-Vis) spectroscopy is one of the most popular
analytical techniques because it is very versatile and able to detect nearly every molecule.
With UV-Vis spectroscopy, the UV-Vis light is passed through a sample and the
transmittance of light by a sample is measured. From the transmittance (T), the absorbance
can be calculated as A=-log (T). An absorbance spectrum is obtained that shows the
absorbance of a compound at different wavelengths. The amount of absorbance at any
wavelength is due to the chemical structure of the molecule.
UV-Vis can be used in a qualitative manner, to identify functional groups or
confirm the identity of a compound by matching the absorbance spectrum. It can also be
used in a quantitative manner, as concentration of the analyte is related to the absorbance
using Beer'sLaw. UV-Vis spectroscopy is used to quantify the amount of DNA or protein in
a sample, for water analysis, and as a detector for many types of chromatography. Kinetics
of chemical reactions is also measured with UV-Vis spectroscopy by taking repeated UVVis measurements over time. UVVis measurements are generally taken with a
spectrophotometer. UV-Vis is also a very popular detector for other analytical techniques,
such as chromatography, because it can detect many compounds.
Principle
UV-Vis is often called a general technique because most molecules will absorb in
the UV-Vis wavelength range. The UV extends from 100–400 nm and the visible spectrum
from 400–700 nm. The 100–200 nm range is called the deep UV. Light sources are more
difficult to find for this range, so it is not routinely used for UV-Vis measurements. Typical
UV-Vis spectrometers use a deuterium lamp for the UV that produces light from 170–375
nm and a tungsten filament lamp for visible, which produces light from 350–2,500 nm.
When a photon hits a molecule and is absorbed, the molecule is promoted into a
more excited energetic state. UV-visible light has enough energy to promote electrons to a
higher electronic state, from the highest occupied molecular orbital (HOMO) to the lowest
unoccupied molecular orbital (LUMO). The energy difference between the HOMO and the
LUMO is called the band gap. Typically, these orbitals are called bonding and anti-bonding.
The energy of the photon must exactly match the band gap for the photon to be absorbed.
Thus, molecules with different chemical structures have different energy band gaps and
different absorption spectra.
The most common transitions that fall in the UV-Vis range are π-π* and n- π*. Pi orbitals
arise due to double bonds, and n- orbitals are for non-bonding electrons. Pi star are antibonding pi orbitals. Thus, the best UV-Vis absorption is by molecules that contain double
bonds. Pi orbitals adjacent to each other that are connected, called conjugation, typically
increases absorption.
Sigma-σ* transitions, associated with single bonds, are higher energy and fall in the deep
UV, so they are less useful for routine use. The appearance of broad bands or shoulders on
the UV-Vis structure is due to the numerous vibrational and rotational states of a molecule,
which lead to separate energy band gaps of slightly different energies.
For molecules with absorption in the visible region, the compounds will often appear
colored.
However, a common misconception is that the wavelength of peak absorption (λmax) for a
compound is the color it appears. A compound that appears red does not have much
absorption in the red region of the spectrum. Instead, the λmax for a compound that looks red
is green. The color of a compound arises because those wavelengths of light are selectively
transmitted through the sample, and thus they are not absorbed. A color wheel is helpful in
determining what color a compound will absorb and what range the λmax will be, as the color
directly across the wheel from the observed color is the color that is most absorbed.
Absorption follows Beer's Law, A= εbC where ε is the molar attenuation coefficient, b is
path length, and C is concentration. The molar attenuation coefficient is the characteristic of
an individual compound to absorb at a given wavelength and this property is due to
functional groups, conjugation, etc. If a compound does not have a high attenuation coefficient, it could be tagged with an appropriate group to increase its absorbance. Path
length is generally related to the size of the cuvette and is 1 cm in standard
spectrophotometers.
UV-Vis is performed on a variety of instruments, from traditional spectrophotometers
to more modern-day plate readers. The absorbance wavelength must be chosen, either using
a filter or a monochromator. A monochromator is a device that separates the wavelengths of
light spatially and then places an exit slit where the desired wavelength of light is.
Monochromators can be scanned to provide a whole absorbance spectrum. Alternatively, a
diode-array instrument allows all colors of light to be transmitted through the sample, and
then the light is separated into different wavelengths spatially and detected using
photodiodes. Diode-array instruments collect full spectra faster, but are more complicated
and more expensive.
Working Procedure
1. Calibrate the Spectrometer
• Turn on the UV-Vis spectrometer and allow the lamps to warm up for an
appropriate period of time (around 20 min) to stabilize them.
• Fill a cuvette with the solvent for the sample and make sure the outside is clean.
This will serve as a blank and help account for light losses due to scattering or
absorption by the solvent.
• Place the cuvette in the spectrometer. Make sure to align the cuvette properly, as
often the cuvette has two sides, which are meant for handling (may be grooved)
and are not meant to shine light through.
• Take a reading for the blank. The absorbance should be minimal, but any
absorbance should be subtracted out from future samples. Some instruments might
store the blank data and perform the subtraction automatically.
2. Perform an Absorbance Spectrum
• Fill the cuvette with the sample. To make sure the transfer is quantitative, rinse
the cuvette twice with the sample and then fill it about ¾ full. Make sure the
outside is clean of any fingerprints, etc.
• Place the cuvette in the spectrometer in the correct direction.
• Cover the cuvette to prevent any ambient light.
• Collect an absorbance spectrum by allowing the instrument to scan through
different wavelengths and collect the absorbance. The wavelength range can be
set with information about the specific sample, but a range of 200–800 nm is
standard. A diodearray instrument is able to collect an entire absorbance spectrum
in one run.
• From the collected absorbance spectrum, determine the absorbance maximum
(λmax). Repeat the collection of spectra to get an estimate of error in λmax.
• To make a calibration curve, collect the UV-Vis spectrum of a variety of different
concentration samples. Spectrometers are often limited in linear range and will not
be able to measure an absorbance value greater than 1.5. If the absorbance values
for the sample are outside the instrument's linear range, dilute the sample to get
the values within the linear range.
3. Kinetics Experiments with UV-Vis Spectroscopy
• UV-Vis can be used for kinetics experiments by examining the change in
absorbance over time. For a kinetics experiment, take an initial reading of the
sample
• Quickly add the reagent to start the chemical reaction
• Stir it well to mix with the sample. If a small amount is added, this could be done
in a cuvette. Alternatively, mix the reagent with sample and quickly pour some in
a cuvette for a measurement
• Measure the absorbance at the λmax for the analyte of interest over time. If using
up the reagent being measuring (i.e. absorbance is going up because there is less
reagent to absorb), then the decay will indicate the order of the reaction
• Using a calibration curve, make a plot of analyte concentration vs time, converting
the absorbance value into concentration. From there, this graph can be fit with
appropriate equations to determine the reaction rate constants
Applications
1. UV-Vis is used in many chemical analyses. It is used to quantitate the amount of
protein in a solution, as most proteins absorb strongly at 280 nm
2. UV-Vis is also used as a standard technique to quantify the amount of DNA in a
sample, as all the bases absorb strongly at 260 nm
3. RNA and proteins also absorb at 260 nm, so absorbance at other wavelengths can be
measured to check for interferences. Specifically, proteins absorb strongly at 280 nm, so
the ratio of absorbance at 280/260 can give a measure of the ratio of protein to DNA in a
sample
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