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?

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.
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

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.
 Light sources

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 photo bleaching. Photo bleaching 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 warm up time, making it a popular choice..
     
 Monochromator
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.
      
     
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 is the difference between a single beam and double beam spectrophotometer?
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.