DELHI TECHNOLOGICAL UNIVERSITY

Instrumentation Techniques for

Environmental Monitoring
EN303

Practical 7

Submitted to
Prof Anunay Gaur

Submitted by:
Sankalp Purwar (2K20/EN/63)

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OBJECTIVE
Study of UV-VIS Spectrophotometer

Introduction

Ultraviolet-visible (UV-Vis) spectrophotometry is a technique used to measure light absorbance

across the ultraviolet and visible ranges of the electromagnetic spectrum. When incident light
strikes matter, it can either be absorbed, reflected, or transmitted. The absorbance of radiation in
the UV-Vis range causes atomic excitation, which refers to the transition of molecules from a
lowenergy ground state to an excited state.

Before an atom can change excitation states, it must absorb sufficient radiation levels for electrons
to move into higher molecular orbits. Shorter bandgaps typically correlate to the absorption of
shorter wavelengths of light. Therefore, the energy required for molecules to undergo these
transitions is electrochemically specific. A UV-Vis spectrophotometer can use this principle to
quantify the analytes in a sample based on their absorption characteristics.

Working

Whilst there are many variations on the UV-Vis spectrophotometer, to better understand how a
UV-Vis spectrophotometer works, let us consider the main components depicted in Figure.

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Light Source

As a light-based technique, a steady source that can emit light across a wide range of wavelengths
is essential. A single xenon lamp is commonly used as a high-intensity light source for both UV
and visible ranges. However, Xenon lamps are associated with higher costs and are less stable
compared to tungsten and halogen lamps.

For instruments employing two lamps, a tungsten or halogen lamp is commonly used for visible
light,2, whilst a deuterium lamp is the typical UV light source.2 As two different light sources are
needed to scan both the UV and visible wavelengths, the light source in the instrument must
switch during measurement. In practice, this switchover typically occurs during the scan between
300 and 350 nm, where the light emission is similar from both light sources, and the transition
can be made more smoothly.

Wavelength selection

In the next step, specific wavelengths of light suited to the sample type and analyte for detection
must be selected for sample examination from the broad wavelengths emitted by the light source.
Available methods for this include:

Monochromators - A monochromator separates light into a narrow band of wavelengths. It is

often based on diffraction gratings that can be rotated to choose incoming and reflected angles to
select the desired wavelength of light.1,2 The diffraction grating's groove frequency is often
measured as the number of grooves per mm. A higher groove frequency provides a better optical
resolution but a narrower usable wavelength range. A lower groove frequency provides a larger
usable wavelength range but a worse optical resolution. 300 to 2000 grooves per mm is usable for
UV-Vis spectroscopy purposes, but a minimum of 1200 grooves per mm is typical. The quality
of the spectroscopic measurements is sensitive to physical imperfections in the diffraction grating
and in the optical setup. Consequently, ruled diffraction gratings tend to have more defects than
blazed holographic gratings.3 Blazed holographic diffraction gratings tend to provide
significantly better-quality measurements.

Absorption filters - Absorption filters are commonly made of coloured glass or plastic designed
to absorb wavelengths of light.

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Interference filters - Also called dichroic filters, these commonly used filters are made of many
layers of dielectric material where interference occurs between the thin layers of materials. These
filters can eliminate undesirable wavelengths by destructive interference, thus acting as a
wavelength selector.1,2

Cutoff filters - Cutoff filters allow light either below (short pass) or above (longpass) a specific
wavelength to pass through. These are commonly implemented using interference filters.

Bandpass filters -Bandpass filters allow a range of wavelengths to pass through that can be
implemented by combining short pass and long pass filters together.

Monochromators are most used for this process due to their versatility. However, filters are often
used together with monochromators to narrow the wavelengths of light selected further for more
precise measurements and to improve the signal-to-noise ratio.

Sample Analysis

Whichever wavelength selector is used in the spectrophotometer, the light passes through a
sample. For all analyses, measuring a reference sample, often referred to as the "blank sample",
such as a cuvette filled with a similar solvent used to prepare the sample, is imperative. If an
aqueous buffered solution containing the sample is used for measurements, then the aqueous
buffered solution without the substance of interest is used as the reference. When examining
bacterial cultures, the sterile culture media would be used as the reference. The instrument then
uses the reference sample signal automatically to help obtain the true absorbance values of the
analytes.

Knowing the materials and conditions used in UV-Vis spectroscopy experiments is essential. For
example, many plastic cuvettes are inappropriate for UV absorption studies because plastic
generally absorbs UV light. Glass can act as a filter, often absorbing the majority of UVC (100-
280 nm)2 and UVB (280-315 nm)2 but allowing some UVA (315‐400 nm)2 to pass through.
Therefore, quartz sample holders are required for UV examination because quartz is transparent
to the majority of UV light. Air may also be thought of as a filter because wavelengths of light
shorter than about 200 nm are absorbed by molecular oxygen in the air. A special and more
expensive setup is required for measurements with wavelengths shorter than 200 nm, usually
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involving an optical system filled with pure argon gas. Cuvette-free systems are also available
that enable the analysis of very small sample volumes, for example, in DNA or RNA analyses.

Detection

After the light has passed through the sample, a detector converts the light into a readable
electronic signal. Generally, detectors are based on photoelectric coatings or semiconductors.

A photoelectric coating ejects negatively charged electrons when exposed to light. An electric
current proportional to the light intensity is generated when electrons are ejected. A
photomultiplier tube (PMT) is one of the more common detectors in UV-Vis spectroscopy. A
PMT is based on the photoelectric effect to initially eject electrons upon exposure to light,
followed by sequential multiplication of the ejected electrons to generate a larger electric
current.4 PMT detectors are especially useful for detecting very low levels of light.

When semiconductors are exposed to light, an electric current proportional to the light intensity
can pass through. More specifically, photodiodes6 and charge‐coupled devices (CCDs)7 are two
of the most common detectors based on semiconductor technology.

After the electric current is generated from whichever detector was used, the signal is recognise
and outputted to a computer or screen. Figures 2 and 3 show simplified example schematic
diagrams of UV-Vis spectrophotometer arrangements.

Principle

UV-Vis spectrophotometer works on the principle of Beer-Lambert’s law. It states that for a
given material sample, the path length and concentration of the sample are directly proportional
to the absorbance of the light.

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Beer–Lambert's law is especially useful for obtaining the concentration of a substance if a linear
relationship exists using a measured set of standard solutions containing the same substance.
Equation 1 shows the mathematical relationships between absorbance, Beer–Lambert's law, the
light intensities measured in the instrument, and transmittance.

UV Vis Spectrophotometer Analysis

The term optical density (OD) is sometimes incorrectly used interchangeably with absorbance.
OD and absorbance measure the amount of light intensity lost in an optical component, but OD
considers loss from light scattering, whereas absorbance does not. If very little light scattering is
present in measurement, then OD may be approximated directly using absorbance and Beer–
Lambert's law may be used.
Knowing the experimental conditions during measurements is important. Cuvettes designed for a
1 cm path length are standard and are the most common. Sometimes, the very little sample is
available for examination and shorter path lengths as small as 1 mm are necessary. Where
quantitation is required, absorbance values should be kept below 1, within the dynamic range of
the instrument. This is because an absorbance of 1 implies that the sample absorbed 90% of the
incoming light, equivalently stating that 10% of the incoming light was transmitted through the
sample. With such little light reaching the detector, some UV‐Vis spectrophotometers are not
sensitive enough to quantify small amounts of light reliably. Two simple possible solutions to
this problem are diluting the sample or decreasing the path length.

As mentioned above, recording a baseline spectrum using a “blank” reference solution is

essential. If the instrument were absolutely perfect in every way, the baseline would have zero
absorbance for every wavelength examined. In a real situation, however, the baseline spectrum
usually has very small positive and negative absorbance values. For best practice, the software
often subtracted these small absorbance values from the sample absorbance values for each
wavelength of light to obtain the true absorbance values.

Depending on the purpose of the analysis, the construction of a calibration curve may be
desirable. Building a calibration curve requires some data analysis and extra work, but it is very
useful to determine the concentration of a particular substance accurately in a sample based on

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absorbance measurements. There are, however, numerous circumstances in which a calibration
curve is not necessary, including OD measurements for bacterial culturing, taking absorbance
ratios at specific wavelengths for assessing the purity of nucleic acids or identifying certain
pharmaceuticals.

In UV-Vis spectroscopy, the wavelength corresponding to the maximum absorbance of the target
substance is chosen for analysis. This choice ensures maximum sensitivity because the largest
response is obtained for a certain analyte concentration.1 An example of a UV Vis absorption
spectrum of Food Green 3 and a corresponding calibration curve using standard solutions are
provided in Figure 5. Note that two maximum absorbance peaks are present in the Food Green 3
dye, a smaller maximum absorbance peak at 435 nm and a more intense maximum absorbance
peak at 619 nm. To gain maximum sensitivity when calculating an unknown concentration of
Food Green 3, the maximum absorbance peak at 619 nm was used for analysis.

Standard solutions across a range of known concentrations were prepared by diluting a stock
solution, taking absorbance measurements and then plotting these on a graph of absorbance
versus concentration to build a numerical relation between concentration and absorbance. A
calibration curve was created using the least squares linear regression equation. The closer the
data points are to a straight line, the better the fit. The y-intercept in the straight-line equation was
set to zero to indicate no absorbance when no dye was present. The equation shown in Figure 5
calculates the concentration of Food Green 3 (variable x) in an unknown sample based on the
measured absorbance (variable y).

For data analysis, the graph of absorbance versus concentration can indicate how sensitive the
system is when building a calibration curve. When a linear least squares regression equation is
used, the slope from the line of best fit indicates sensitivity. If the slope is steeper, the sensitivity
is higher. Sensitivity is the ability to differentiate between small differences in the sample
concentration. From Beer–Lambert's Law, the sensitivity can be partially indicated by the molar
absorptivity ε. Knowing the ε values beforehand, if available, can help determine the required
samples' concentrations, particularly where samples are limited or expensive.

UV spectroscopy experiments and readings should be repeated for reliability and best practice.
When repeating the examination of a sample, in general, a minimum of three replicate trials is
common, but many more replicates are required.

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in certain fields of work. A calculated quantity, such as the concentration of an unknown sample,
is usually reported as an average with a standard deviation. Reproducible results are essential to
ensure precise, high-quality measurements. Standard deviation, relative standard deviation, or the
coefficient of variation help to determine how precise the system and measurements are. A low
deviation or variation indicates a higher level of precision and reliability.

Applications of UV-Vis Spectrophotometer

DNA and RNA analysis

Quickly verifying the purity and concentration of RNA and DNA is one particularly widespread
application. A summary of the wavelengths used in their analysis and what they indicate is given
in Table 1. When preparing DNA or RNA samples, for example, for downstream applications
such as sequencing, it is often important to verify that there is no contamination of one with the
other or with protein or chemicals carried over from the isolation process.
The 260 nm/280 nm absorbance (260/280) ratio is useful for revealing possible contamination in
nucleic acid samples, summarized in Table 2. Pure DNA typically has a 260/280 ratio of 1.8,
while the ratio for pure RNA is usually 2.0. Pure DNA has a lower 260/280 ratio than RNA
because thymine, replaced by uracil in RNA, has a lower 260/280 ratio than uracil. Samples
contaminated with proteins will lower the 260/280 ratio due to higher absorbance at 280 nm.

Pharmaceutical analysis
One of the most common uses of UV-Vis spectroscopy is in the pharmaceutical industry. In
particular, processing UV-Vis spectra using mathematical derivatives allows overlapping
absorbance peaks in the original spectra to be resolved to identify individual pharmaceutical
compounds. For example, benzocaine, a local anaesthetic, and chlortetracycline, an antibiotic,
can be identified simultaneously in commercial veterinary powder formulations by applying the
first mathematical derivative to the absorbance spectra. Simultaneous quantification of both
substances was possible on a microgram per millilitre concentration range by building a
calibration function for each compound.

Bacterial Culture

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UV-Vis spectroscopy is often used in bacterial culturing. OD measurements are routinely and
quickly taken using a wavelength of 600 nm to estimate the cell concentration and to track
growth.18 600 nm is commonly used and preferred due to the optical properties of bacterial
culture media in which they are grown and to avoid damaging the cells in cases where they are
required for continued experimentation.

Beverages Analysis
The identification of particular compounds in drinks is another common application of UV-Vis
spectroscopy. Caffeine content must be within certain legal limits, for which UV light can
facilitate quantification. Certain classes of coloured substances, such as anthocyanin found in
blueberries, raspberries, blackberries, and cherries, are easily identified by matching their known
peak absorbance wavelengths in wine for quality control using UV-Vis absorbance.

References

1. Harris DC. Quantitative Chemical Analysis. 7th ed, 3rd printing. W. H. Freeman; 2007.

2. Diffey BL. Sources and measurement of ultraviolet radiation. Methods. 2002;28(1):4-13.

doi:10.1016/S1046-2023(02)00204-9

3. Namioka T. Diffraction Gratings. In: Vacuum Ultraviolet Spectroscopy. Vol 1. Experimental

Methods in Physical Sciences. Elsevier; 2000:347-377. doi:10.1016/B978-012617560-8/50018-9

4. Mortimer Abramowitz and Michael W. Davidson. Photomultiplier Tubes. Molecular

Expressions. Accessed April 25, 2021.
https://micro.magnet.fsu.edu/primer/digitalimaging/concepts/photomultip liers.html