UV-Vis Spectroscopy or Ultraviolet-visible spectroscopy or Ultraviolet-visible

spectrophotometer (UV-Vis) is also called absorption spectroscopy or reflectance spectroscopy

in the ultraviolet-visible spectral region.
Electron transition takes place, so it is also called electron spectroscopy. It is a cost-effective,
simple, versatile, and non-destructive technique that allows the sample to be used again for
further analysis. It is a qualitative, quantitative, and analytical technique that compares a sample
with a blank or reference sample to measure the amount of discrete ultraviolet and visible light
absorbed or transmitted through a particular sample using Beer-Lambert law. It studies under
vacuum conditions.
The wavelength of UV-vis spectroscopy ranges from 190 nm to 800 nm. The UV region ranges
from 190 to 400 nm, and the visible region from 400 to 800 nm. Near UV region is 190 nm to
400 nm, and far UV region is below 200 nm. The shorter the wavelength, the higher will be the
frequency and energy. It occurs in UV region. Similarly, the higher the wavelength, the lower the
frequency and energy in the visible region.

Light Spectrum
Its properties depend on sample composition and concentration. It helps to identify, assess purity,
and quantify the components of the sample by analyzing the pattern of absorption and
transmission of light. It may apply in several sample types, such as monolithic solids, liquids,
glass, powders, and thin films.
Absorbance (A): It, also known as optical density (OD), is the amount of light absorbed by the
object and can be expressed as follows:
Absorbance (A)= -log(T)
Transmittance (T): It is measured by dividing the intensity spectrum of light transmitted through
a sample (I) by the intensity spectrum of light transmitted through the blank (I0).
T= I/Io
UV-Vis Spectroscopy Principle
When a specific wavelength of light hits a molecule, that molecule gets excited. Once the
electron excites, it excites from the ground (lower) energy state to the higher energy state. When
an electron jumps off, it absorbs light energy because electrons in the orbital at a lower energy
state utilize energy to move to a higher energy level.
Energy is neither created nor destroyed but can transform energy from one form to another. On
passing EMR (UV- Vis range 200- 800 nm), only light possessing the precise amount of energy
that can cause transitions from one level to another will absorb because matter’s energy levels
are quantized.
If the energy is utilized, the intensity of light received is lost. At this time, the energy absorbed
by the electrons will equal the energy difference between the two energy levels.
During this stage, electron transition occurs. So, after the interaction of electromagnet radiation,
the spectra received are called absorption spectra. Hence, it is called electron spectroscopy.
Similarly, when electrons in the orbital at a higher energy level move to the ground energy level,
the spectra received are called emissions.
Beer-Lambert Law equation is the principle behind absorbance spectroscopy.
The concentration of the sample can be determined directly from the absorption of spectra
produced by these samples at specific wavelengths using the Beer-Lambert law.
What is Beer-Lambert Law?
When a beam of light allows it to pass through a transparent medium, the rate at which an
intensity decreases with medium thickness is directly proportional to the light beam’s intensity.
According to the Beer-Lambert Law, the absorbance is directly proportional to the concentration
of the substance in the solution. Therefore, a sample’s concentration can also be determined
using UV-visible spectroscopy.
The Beer-Lambert Law can be expressed in the form of the following equation:
A = –log T = –log (I ⁄ Io ) = log(Io ⁄ I)= ecl
A = ecl
Where A = absorbance
l = optical path length of the cell or cuvette or sample holder(cm)
c = concentration of the solution (mol dm-3)
e = molar absorptivity of the compound or molecule in solution, which is constant for a
particular substance at a particular wavelength (dm3 mol-1 cm-1)
Following the Beer-Lambert Law, the plot of absorbance versus concentration should be linear if
the absorbance of a series of sample solutions with known concentrations is measured and
plotted against equivalent concentrations. This graph is known as a calibration graph.
 Instrumentation of UV-Vis Spectroscopy
The main components of UV- Vis spectrophotometer are:
1. Light Source
2. Wavelength selector
3. Sample container
4. Detectors
1. Light Source
It is essential for emitting light in a wide range of wavelengths to work in a UV-Vis
spectrometer. Commonly, a high-intensity light source used for both UV and Visible ranges is a
xenon lamp. In contrast to tungsten and halogen lamps, it is less stable and more costly. So, the
two lamps for this instrument are a deuterium lamp for UV light and a halogen or tungsten lamp
for visible light as a source of light. The two lamps provide good intensity. While measuring the
intensity of the light, the spectrometer ought to switch. A smoother transition is possible when
the switchover occurs between 300 and 350 nm because the light emission for both visible and
UV light sources is the same amount of light at that wavelength.
2. Wavelength selector
In order to allow sample examination using the wavelengths that the light source emits,
wavelength selection helps to ascertain which wavelength is appropriate for the type of analyte
and sample. The commonly used wavelength selector in the UV-Vis spectrometer is the
monochromator. It separates light into a narrow band of wavelength.
From the entrance slit, radiation of different wavelengths will enter the monochromator. At a
particular angle, the beam will collide and strike the dispersing element. A monochromator
contains a prism that separates all different wavelengths of light in a single beam. It bends the
monochromatic light and produces non-linear dispersion. Only single radiation or color of a
specific wavelength will allow it to leave the monochromator and pass through its ultimate chain
or exit slit.
3. Sample Container
In a single-beam spectrophotometer, all the radiation coming from the light source passes
through the sample as one beam. Single-beam spectrophotometers can determine color by
comparing the light sources’ intensities before and after a sample is inserted. The wavelength
range measure is 190–750 nm; however, it may go up to 1100 nm.
In a double-beam spectrophotometer, all the radiation coming from the light source splits into
two beams: one passes through the sample, and the other only passes through the reference.
Similarly, Double-beam spectrophotometers offer a wavelength range of 190 to 1100 nm.
Moreover, the double-beam spectrophotometer measures absorbance versus wavelength or
sample and reference beam ratio.
The reference detector is used to adjust lamp brightness fluctuations for each measurement. After
collecting the sample, the sample detector is measured in the sample position and deducted from
the sample spectrum. It contains both a reference chamber and a sample chamber. The sample is
kept in a flat, transparent container called a cuvette or sample chamber. The solvent in which the
sample dissolves is kept in the reference chamber, also known as the blank. The sample cell’s
choice depends on the path length, shape, size, and transmission characteristics at the desired
wavelength and the relative expense.
For each wavelength, the light intensity passes through the beam separator to the reference
chamber (Io) and sample chamber (I). The intensity of light symbolizes as Io measures the
number of photons per second. When the light passes through the blank solution, it does not
absorb light, referred to as (l). If sample I is less than Io, the sample has absorbed some light.
The absorbance (A) of the sample is related to I and Io according to the following equation:
Absorbance (A)= -log(T)= -log (I/Io)
This equation shows the relationships between absorbance and transmittance.
Also, the fraction I divided by Io is called transmittance (T), which expresses how much light has
passed through a sample.
T= I/Io
T= I/Io = e–kbc
Where: – I o is the incident intensity
– I is the transmitted intensity
– e is the base of natural logarithms
– k is a constant
– b is the path length (usually in cms).
The lighter the refracted, the more transmittance occurs. The lower the absorbance, the higher
the transmittance.
In UV and visible regions, fused silica or quartz cuvettes are commonly used.
 4. Detector
Detectors rely on photoelectric coatings or semiconductors. It converts the incoming light from
the sample into an electric signal or current. The higher the current, the greater the intensity. It
has the properties of low noise and high sensitivity, so it gives a linear response. Each detector
has a variety of wavelength ranges and different sensitivity. Finally, The data recorder usually
plots the absorbance against wavelength (nm) in the UV and visible section of the
electromagnetic spectrum.

Applications of UV-Vis Spectroscopy

DNA and RNA analysis
It focuses on verifying the concentration and purity of DNA and RNA, which plays a crucial role
in downstream applications like sequencing. It ensures whether the DNA or RNA samples
prepared for sequencing are contaminant or pure.
Since pure DNA has an absorbance ratio of 1.8 and pure RNA has a ratio of 2, the 260 nm/280
nm absorbance ratio is crucial for displaying protein contamination in nucleic acids.
260nm/230nm absorbance ratio varies for RNA and DNA (2.15 to 2.50).
Pharmaceutical analysis
It is essential in drug discovery and development, quantifying impurities in drug ingredients,
dissolution testing of solid oral dosage forms like tablets, and chemical identification and
quantification. It allows overlapping absorbance peaks in the original spectra using mathematical
derivatives to identify pharmaceutical compounds.
Likewise, the Identification of pharmaceutical compounds, Chlortetracycline (antibiotic) and
benzocaine (anesthetic) in veterinary powder formulation, by overlapping the absorbance peaks
in UV spectra using mathematical derivatives.
Food and Beverage Applications
It applies to assessing the sensory attributes, nutritional components of food and its products
such as beer, wine, juices, energy and soft drinks, waters, other thin liquids and thick liquids
(honey, oils), fruits, vegetables, caffeine content, etc., and the chemical composition of
ingredients and detect contaminants or adulterant to ensure the product is safe and healthier.
It can be used in quality control in wine by identifying anthocyanin in blueberries, raspberries,
and cherries. It can evaluate food and food product color, flavor, and aroma.
Bacterial culture
It is essential in the biomass growth curve. It is used in culturing bacteria by estimating cell
concentrations and growth tracking in measuring optical density at 600 nm. 600 nm is best to
preserve the optical properties of culture media where bacteria grow and to avoid cell damage
when there is a need for continuous experimentation.
Other Applications
1. In the cosmetic industry, it is used to evaluate photostability agents and color index, quantify
dyes and antioxidants, and detect adulteration.
2. It is used in material science, like the characterization of small nanoparticles and to determine
battery composition.
3. It is used to examine structural protein changes by tracking changes in peak wavelength
absorbance.
4. In wastewater treatment, it is employed in kinetics and monitoring studies of dyes and dye
byproducts to ensure adequate dye removal by comparing their spectra over time.
5. It is used in cancer research to estimate hemoglobin concentration.
6. It is used to measure color index to monitor transformer oil as a preventive measure to ensure
electric power is delivered safely.
7. It is used in petrochemistry for characterizing crude oil, quality of crude oil gravity,
formulation of indices for aromatic content, and sulfur content.
8. In the biochemistry and genetic fields, it is used to quantify DNA, protein/enzyme, and thermal
denaturation of protein.

Advantages of UV-Vis Spectroscopy

1. It is non-destructive and reusable.
2. It is easy to operate and the fastest method to interpret data because it gives accurate readings.
3. It is an inexpensive technique.
4. It is more convenient.

Disadvantages of UV-Vis Spectroscopy

1. It may take time to prepare using the machine.
2. Spectrometer reading might be affected if it keeps with any electronic noise, outside light, and
other contaminants.
3. The accuracy of the machine’s measurement could be impacted by stray light from defective
equipment design because the linearity range and substance absorbency measuring are likely to
be reduced by stray light.
Frequently Asked Questions

Q1What are the Applications of UV-Visible Spectroscopy?

UV-Visible spectroscopy is widely used in the field of analytical chemistry, especially during the
quantitative analysis of a specific analyte. For example, the quantitative analysis of transition
metal ions can be achieved with the help of UV-Visible spectroscopy. Furthermore, the
quantitative analysis of conjugated organic compounds can also be done with the help of UV-
Visible spectroscopy. It can also be noted that this type of spectroscopy can also be carried out
on solid and gaseous analytes in some conditions.

Q2What kinds of detectors are used in UV-Visible spectroscopy?

A widely used detector in UV-Vis spectroscopy is the Photomultiplier tube. It consists of a

photoemissive cathode (which is a cathode that releases electrons when it is hit by radiation
photons), multiple dynodes (which is a device that emit multiple electrons for each striking
electron), and an anode.

Q3What is UV-Visible spectroscopy?

Ultraviolet and visible (often abbreviated to UV-Vis) absorption spectroscopy is a type of

spectroscopy which involves the calculation of a light beam’s attenuation (strength/intensity
weakening) after it passes through a sample or reflects from a sample surface

Q4What are the limitations of UV Visible Spectroscopy?

The main disadvantage of utilising a UV-VIS spectrometer is the time it requires to prepare to
use one. Setup is crucial when using UV-VIS spectrometers. Any outside light, electrical noise,
or other outside contaminants that could interfere with the spectrometer’s reading must be
removed from the location.

Q5 Why is a glass cuvette not suitable for UV?

Because glass and most plastics absorb ultraviolet light, reusable quartz cuvettes were formerly
required for measurements in the ultraviolet range.
What is UV-Vis spectroscopy?
Ultraviolet (UV) and visible radiation are a small part of the electromagnetic spectrum,
which includes other forms of radiation such as radio, infrared (IR), cosmic, and X rays.

The electromagnetic spectrum, with the visible light section expanded.

Spectroscopy allows the study of how matter interacts with or emits electromagnetic
radiation. There are different types of spectroscopy, depending on the wavelength range
that is being measured. UV-Vis spectroscopy uses the ultraviolet and visible regions of
the electromagnetic spectrum. Infrared spectroscopy uses the lower-energy infrared part
of the spectrum. In UV-Vis spectroscopy, wavelength is usually expressed in nanometers
(1 nm = 10-9 m). The UV range normally extends from 100 to 400 nm, with the visible
range from approximately 400 to 800 nm.

Key principles of UV-Vis spectroscopy—how does it work?

UV-visible spectra
When radiation interacts with matter, several processes can occur, including reflection,
scattering, absorbance, fluorescence/phosphorescence (absorption and re-emission), and
photochemical reactions (absorbance and bond breaking). Typically, when measuring
samples to determine their UV-visible spectrum, absorbance is measured.

Because light is a form of energy, absorption of light by matter causes the energy content
of the molecules (or atoms) in the matter to increase. In some molecules and atoms,
incident photons of UV and visible light have enough energy to cause transitions between
the different electronic energy levels. The wavelength of light absorbed has the energy
required to move an electron from a lower energy level to a higher energy level. The
figure below shows an example of electronic transitions in formaldehyde and the
wavelengths of light that cause them.
Electronic transitions in formaldehyde. UV light at 187 nm causes excitation of an
electron in the C-O bond, and light at 285 nm wavelength causes excitation and transfer
of an electron from the oxygen atom to the C-O bond.

These transitions result in very narrow absorbance bands at wavelengths highly

characteristic of the difference in energy levels of the absorbing species. This is also true
for atoms.
Incident light of a specific wavelength causes excitation of electrons in an atom. The type
of atom or ion and the energy levels the electron is moving between determines the
wavelength of the light that is absorbed. Transitions can be between more than one
energy level, with more energy (i.e., lower wavelengths of light) required to move the
electron further from the nucleus.

However, for molecules, vibrational and rotational energy levels are superimposed on the
electronic energy levels. Because many transitions with different energies can occur, the
bands are broadened. The broadening is even greater in solutions owing to solvent-solute
interactions.
 Electronic transitions and UV-visible spectra in molecules (I is intensity and λ is
wavelength).

Transmittance and absorbance

When light passes through or is reflected from a sample, the amount of light absorbed is
the difference between the incident radiation (Io) and the transmitted radiation (I). The
amount of light absorbed is expressed as absorbance. Transmittance, or light that passes
through a sample, is usually given in terms of a fraction of 1 or as a percentage. For most
applications, absorbance values are used since the relationship between absorbance and
both concentration and path length is normally linear.

What is a UV-Vis spectrophotometer and how does it work?

Ultraviolet-visible (UV-Vis) spectrophotometers use a light source to illuminate a sample
with light across the UV to the visible wavelength range (typically 190 to 900 nm). The
instruments then measure the light absorbed, transmitted, or reflected by the sample at
each wavelength. Some spectrophotometers have an extended wavelength range, into the
near-infrared (NIR) (800 to 3200 nm).

From the spectrum obtained, it is possible to determine the chemical or physical

properties of the sample. In general, it is possible to:

 Identify molecules in a solid or liquid sample

 Determine the concentration of a particular molecule in solution
 Characterize the absorbance or transmittance through a liquid or solid—over a range of
wavelengths
 Characterize the reflectance properties of a surface or measure the color of a material
 Study chemical reactions or biological processes
A UV absorbance spectrum, showing an absorbance peak at approximately 269 nm.

Various measurements can be performed by combining different accessories and sample

holders with a UV-Vis spectrophotometer. Accessories exist for different measurement
capabilities and sample types (e.g., solids versus liquids) and for different measurement
conditions (see later question on UV-Vis accessories).

The Agilent Cary 3500 UV-Vis spectrophotometer measures up to four temperature

experiments, across eight cuvette positions, simultaneously.

UV-Vis spectrophotometry is a versatile technique and has been used for close to a
century in a wide range of fields. UV-Vis spectrophotometers are in common use in
material testing/research, chemistry/petrochemistry, and biotechnology/pharmaceuticals
laboratories.
What are the main components of a UV-Vis spectrophotometer?
The key components of a UV-Vis spectrophotometer are:

 A light source that generates a broadband of electromagnetic radiation across the UV-
visible spectrum
 A dispersion device that separates the broadband radiation into wavelengths
 A sample area, where the light passes through or reflects off a sample
 One or more detectors to measure the intensity of the reflected or transmitted radiation

Other optical components, such as lenses, mirrors, or fiber optics, relay light through the
instrument.

Schematic of the internal layout of an Agilent Cary 5000 UV-Vis-NIR spectrophotometer,

showing the main components. Note that this is a high-performance instrument. UV-Vis
spectrophotometers for routine measurements have a simpler optical design.

Light sources
See the next question for full details on light sources.
 Monochromators
All the light sources produce a broad-spectrum white light. To narrow the light down to a
selected wavelength band, the light is passed through a monochromator, which consists
of:

 An entrance slit
 A dispersion device, to spread the light into different wavelengths (like a rainbow) and
allow the selection of a nominated band of wavelengths
 An exit slit where the light of the nominated wavelengths passes through and onto the
sample

A single monochromator spectrophotometer is used for general-purpose spectroscopy and

can be integrated into a compact optical system. A double monochromator is typically
found in high-performance instruments.

 For more details on how monochromators work and on holographic gratings, filters, plus
single and double monochromator spectrophotometers, download the Agilent handbook
on the Basics of UV-Vis Spectrophotometry.

Sample compartments
The sample compartment of a UV-Vis spectrophotometer is typically a black-colored box
with a closing lid. The matt black inside the compartment helps to absorb stray light that
may enter the compartment. In the sample compartment, the sample is positioned to
allow the beam from the monochromator to pass through the sample. Glass, plastic, or
quartz cuvettes are used for liquid samples. Solid samples are held in position by a holder
attached to the floor of the sample compartment. The light can also be taken out of the
sample compartment using fiber optics.

Detectors
A detector converts the light from the sample into an electrical signal. Like the light
source, it should give a linear response over a wide wavelength range, with low noise and
high sensitivity.

Each detector has a different sensitivity and wavelength range. For systems with multiple
detectors, the system will switch to the detector corresponding to the required wavelength
range for the measurement. UV-Vis spectrophotometer detectors include photomultiplier
tubes (PMT) and silicon diodes (Si). Indium gallium arsenide (InGaAs) photodiodes and
lead sulfide (PbS) detectors are found on high-performance UV-Vis-NIR systems to
improve wavelength coverage or sensitivity.
What light sources do UV-Vis spectrophotometers use?
The ideal light source would yield a constant intensity over all wavelengths with low
noise and long-term stability of the output. Unfortunately, such a source does not exist.
Two different light sources have historically been used in UV-Vis spectrophotometers:

 The deuterium arc lamp was used to provide a good intensity continuum in the UV region
and useful intensity in the visible region.
 The tungsten-halogen lamp yielded good intensity over the entire visible range and part
of the UV spectrum.

More recently, a single xenon flash lamp has been used more widely. The use of a xenon
flash lamp as a single source has significant advantages over the use of the two
conventional lamps.

Deuterium (D2) arc lamp

The deuterium arc lamp uses arc discharge from deuterium gas and yields a good
intensity continuum in the UV region, and useful intensity in the visible region, 185 to
400 nm. Although modern deuterium arc lamps have low signal noise, noise from the
lamp is often the limiting factor in overall instrument noise performance. Such a lamp
typically has a half-life (the time required for the intensity to fall to half of its initial
value) of approximately 1,000 hours. This short half-life means the D2 lamp needs to be
replaced relatively frequently.

Tungsten-halogen lamp
The tungsten-halogen lamp uses a filament. When a current is passed through the
filament, it becomes heated and emits light. The lamp yields good intensity over part of
the UV spectrum and over the entire visible and NIR range (350 to 3000 nm). This type
of lamp has very low noise and low drift and typically has a functional life of 10,000 h.

In UV-Vis spectrophotometers using both a D2 and a tungsten-halogen lamp, either a

source selector is used to switch between the lamps as appropriate, or the light from the
two sources is mixed to yield a single broadband source.

Xenon flash lamp

A xenon flash lamp emits light for an extremely short time, in flashes. Since it emits only
for a short time and only during sample measurement, it has a long life. The sample is
 only irradiated with light at the time of measurement. This short illumination time makes
the xenon flash lamp suitable for measuring samples that may be sensitive to
photobleaching. Photobleaching can be observed on sensitive samples when exposed to a
constant long exposure by a continuous light source.

The xenon flash lamp emits high intensity light from 185 to 2,500 nm, which means no
secondary light source is required. The xenon flash lamp may be used for many years
before requiring replacement and it does not require warmup time, making it a popular
choice. Xenon flash lamps are used in Agilent Cary 60 and Cary 3500 UV-Vis
spectrophotometers.

How do you use a UV-Vis spectrophotometer?

In the sample compartment, the sample is positioned to allow the beam from the
monochromator to pass through the sample. For the measurement of absorbance, liquid
samples would typically be held in a cuvette with a known, fixed pathlength. A cuvette is
a rectangular liquid holder as shown below. They are made from glass, quartz, plastic, or
another material that transmits UV or visible light. Standard cuvettes have a 10 mm
pathlength and are made from quartz, to ensure good transmittance of UV wavelengths.
Cheaper plastic cuvettes can also be used, but generally do not transmit UV light so are
only useful if measurements in the visible wavelength region are required. A multitude of
cuvettes for special applications are available—from cuvettes that hold tiny volumes of
liquids through to those that have much longer pathlengths, for use with very dilute
samples.

Cuvettes for measuring liquid samples. From left to right: A standard 10 mm pathlength,
3 mL cuvette, an ultramicro cell for measuring very low volumes, and a long pathlength
cuvette for dilute solutions.

Solid samples can be held in place for simple transmission measurements. They can also
be measured at various angles of incidence. For more complex measurements, like
 diffuse reflectance or transmission, other accessories may be installed into the sample
compartment.

The light can also be taken out of the sample compartment using fiber optics. Fiber optics
are useful when measuring very large, hot, cold, radioactive, or other dangerous samples.
As shown below, fiber optics can take the light from the spectrophotometer through a
fiber-optic probe, to measure solutions outside of the sample compartment. Alternatively,
a fiber-optic device that allows the measurement of light reflectance, fluorescence, or
transmission through a solid sample can be used.

What is a double beam spectrophotometer?

A double beam spectrophotometer has two separate light paths, often referred to as the
sample beam and the reference beam. By comparing the sample and reference beams, a
double beam spectrophotometer provides more accurate and reliable results, since
fluctuations in absorbance over time are instantaneously corrected, which is not the case
on single beam spectrophotometers. Some UV-Vis spectrophotometers provide access to
both the sample and the reference beam, like the Cary 4000 UV-Vis and the Cary 3500
Flexible UV-Vis. The Cary 60 UV-Vis gives users access to the sample beam and
the Cary 3500 Multicell UV-Vis provides analysts with one reference beam and up to
seven sample beams.

Single beam spectrophotometer

The simplest UV-Vis spectrophotometer has a single beam optical system, where the
light from the monochromator passes through the sample and then to the detector. This
simple design means that fewer optical components are used, reducing the size and cost
of the instrument.

However, before a sample can be measured, a baseline or blank sample must be

measured. For liquid measurements, the baseline reading is taken to allow for any
absorbance of the cuvette and solvent used. With a single beam system, the baseline
needs to be measured separately from the sample. The separate readings mean that if
there is any variation of light intensity, or system optical performance, between the
baseline and sample being read, the measurement may be less accurate. This inaccuracy
is a concern for sample measurements that take a long time, or where the blank may vary
over time. In practice, this means that a baseline/blank measurement should be run
frequently and regularly during a measurement session if using a single beam system.

Double beam spectrophotometer

Many UV-Vis instruments use a double beam optical setup, where the light emitted from
the monochromator is split into two beams: a reference beam and a sample beam. The
light is usually split with an optical component such as a rotating wheel that has a
mirrored segment, or a half-silvered mirror called a beam splitter. Each beam enters the
sample chamber through separate optical paths. Since two beams of the same
wavelengths are available, the reference/blank and sample can be measured at the same
time. This means that the sample measurement can be corrected for any instrument
 fluctuations in real time. This real-time correction delivers a highly accurate
measurement.

Schematic diagram of double-beam optical system, with dual detectors.

Dual beam spectrophotometer

Another, more recent, spectrophotometer design uses a dual beam optical layout with a
sample and reference detector. The reference detector is used to correct lamp brightness
fluctuations for each measurement, while the solvent or blank (in the case of a solid
sample) is measured in the sample position and then subtracted from the sample spectrum
after collection. With improvements in electronics and software, this design keeps the
measurement process simple and reduces the chance of user error due to mismatched
cuvettes or incorrect sample placement. Dual beam design has the same performance as a
routine double beam instrument, while double beam design is now typically reserved for
research-grade instruments.

What does a UV-Vis spectrophotometer measure?

UV-Vis and UV-Vis-NIR instruments measure the light absorbed, transmitted, or
reflected by the sample across a certain wavelength range. Absorbance (A or Abs) is
frequently measured in UV-Vis spectroscopy due to the linear relationship between
concentration and absorbance as described by the Beer-Lambert law. For other
applications, the percentage of light transmitted or absorbed may be more meaningful.
When comparing the optical properties of a material, for example, it may be more useful
to compare the percent transmission or absorbance difference.

Most UV-Vis measurements are reported against wavelengths measured in nanometers (1

× 10−9 m). In some older literature, the reciprocal length or wavenumber (cm-1) is used.
For UV-Vis spectroscopy, wavelength is generally preferred as a convenient way to
visualize the displayed spectrum over a spectral range. Most UV-Vis spectrophotometer
systems will enable you to collect a spectrum in either wavelength or wavenumber.
How do I select optimum parameters for UV-Vis measurements?
Selecting the most suitable sample holder, solvent, and instrument parameters is critical
for the success of your measurement. Factors to consider includes:

1. Optical cell selection—this is based on desired pathlength, sample volume, and the
optical properties of the material used in the cuvette windows.
2. Thermostatting your samples—there are some circumstances that require samples to be
heated or cooled.
3. Stirring your sample—this ensures that both solution and temperature homogeneity are
always maintained.
4. Measurements at low temperatures—these may cause condensation to form on the
outside of cuvettes, which can interfere with the measurement.
5. Solvent transparency for liquid samples or dissolving of solid samples—solvents are
selected based on sample solubility, stability, pH requirements, and the UV-visible cut-
off wavelength.
6. Optimum spectral bandwidth—when measuring a sample, consideration should be given
to the measurement resolution required.
7. Stray light—the percentage of radiation reaching the detector whose wavelengths are
outside the selected spectral band. Most systems are provided with instrument
performance checks that identify stray light issues.
8. The linear range of a UV-Vis instrument—understanding the limits of your system allows
you to avoid measuring samples or performing calibrations that are outside the
capabilities of your instrument.
 Difference between single beam and double beam spectrophotometer?

Single-beam and double-beam spectrophotometers are both analytical instruments used to

measure the absorption or transmission of light by a sample in order to determine its
concentration or other properties. However, they differ in their design and operation, leading to
variations in performance and application. Here's a breakdown of the differences between the
two:

1. Design:
 Single-Beam Spectrophotometer: In a single-beam spectrophotometer, only one beam
of light passes through the sample. The sample is placed in the path of this beam, and the
intensity of the light transmitted through or absorbed by the sample is measured.
 Double-Beam Spectrophotometer: In a double-beam spectrophotometer, the light beam
is split into two paths - one passes through the sample, while the other serves as a
reference and typically passes through a blank solvent or reference material. The two
beams are then recombined, and the difference in intensity between them is measured.
2. Baseline Stability:
 Single-Beam Spectrophotometer: Since there's no separate reference beam, any
fluctuations in the light source or detector can affect the accuracy of the measurement.
Baseline stability can be an issue in single-beam instruments.
 Double-Beam Spectrophotometer: Because it utilizes a separate reference beam,
double-beam spectrophotometers offer better baseline stability. Any fluctuations in the
light source or detector are accounted for by the reference beam, resulting in more
accurate measurements.
3. Accuracy and Precision:
  Single-Beam Spectrophotometer: Single-beam instruments may suffer from
inaccuracies due to fluctuations in the light source or detector. However, they can still
provide accurate measurements for many applications with careful calibration and control
of experimental conditions.
 Double-Beam Spectrophotometer: Double-beam spectrophotometers generally offer
higher accuracy and precision compared to single-beam instruments, particularly for
quantitative analysis. The ability to compensate for fluctuations in the light source or
detector enhances the reliability of measurements.
4. Applications:
 Single-Beam Spectrophotometer: Single-beam instruments are often suitable for
qualitative analysis, routine measurements, and educational purposes where high
precision is not critical.
 Double-Beam Spectrophotometer: Double-beam spectrophotometers are preferred for
quantitative analysis, especially in research laboratories and industries where precise and
accurate measurements are essential.

In summary, while both single-beam and double-beam spectrophotometers serve the same
fundamental purpose of measuring light absorption or transmission by samples, double-beam
instruments offer enhanced accuracy, precision, and baseline stability due to their design with
separate reference beams. Therefore, they are often preferred for demanding analytical
applications.