In physics, spectrophotometry is the quantifiable study of electromagnetic spectra. It is more specific than the general term electromagnetic spectroscopy in that spectrophotometry deals with visible light, near-ultraviolet, and near-infrared. Also, the term does not cover time-resolved spectroscopic techniques.
Spectrophotometry involves the use of a spectrophotometer. A spectrophotometer is a photometer (a device for measuring light intensity) that can measure intensity as a function of the color, or more specifically, the wavelength of light. There are many kinds of spectrophotometers. Among the most important distinctions used to classify them are the wavelengths they work with, the measurement techniques they use, how they acquire a spectrum, and the sources of intensity variation they are designed to measure. Other important features of spectrophotometers include the spectral bandwidth and linear range.
Perhaps the most common application of spectrophotometers is the measurement of light absorption, but they can be designed to measure diffuse or specular reflectance. Strictly, even the emission half of a luminescence instrument is a kind of spectrophotometer.
The use of spectrophotometers is not limited to studies in physics. They are also commonly used in other scientific fields such as chemistry, biochemistry, and molecular biology. They are widely used in many industries including printing and forensic examination.
There are two major classes of spectrophotometers; single beam and double beam. A double beam spectrophotometer measures the ratio of the light intensity on two different light paths, and a single beam spectrophotometer measures the absolute light intensity. Although ratio measurements are easier, and generally more stable, single beam instruments have advantages; for instance, they can have a larger dynamic range, and they can be more compact.
Historically, spectrophotometers use a monochromator to analyze the spectrum, but there are also spectrophotometers that use arrays of photosensors. Especially for infrared spectrophotometers, there are spectrophotometers that use a Fourier transform technique to acquire the spectral information quicker in a technique called Fourier Transform InfraRed...
The spectrophotometer quantitatively measures the fraction of light that passes through a given solution. In a spectrophotometer, a light from the lamp is guided through a monochromator, which picks light of one particular wavelength out of the continuous spectrum. This light passes through the sample that is being measured. After the sample, the intensity of the remaining light is measured with a photodiode or other light sensor, and the transmittance for this wavelength is then calculated.
In short, the sequence of events in a spectrophotometer is as follows:
The light source shines through the sample.
The sample absorbs light.
The detector detects how much light the sample has absorbed.
The detector then converts how much light the sample absorbed into a number.
The numbers are either plotted straight away, or are transmitted to a computer to be further manipulated (e.g. curve smoothing, baseline correction)
Many spectrophotometers must be calibrated by
a procedure known as "zeroing." The absorbency of some standard
substance is set as a baseline value, so the absorbencies of all other substances
are recorded relative to the initial "zeroed" substance. The spectrophotometer
then displays % absorbency (the amount of light absorbed relative to the
UV and IR spectrophotometers
The most common spectrophotometers are used in the UV and visible regions
of the spectrum, and some of these instruments also operate into the near-infrared
region as well.
Visible region 400-700nm spectrophotometry is used extensively in colorimetry
science. Ink manufacturers, printing companies, textiles vendors, and many
more, need the data provided through colorimetry. They take readings in
the region of every 10- 20 nanometers along the visible region, and produce
a spectral reflectance curve or a data stream for alternative presentations.
These curves can be used to test a new batch of colorant to check if it
makes a match to specifications e.g., iso printing standards.
Traditional visual region spectrophotometers cannot detect if a colorant
or the base material has fluorescence. This can make it difficult to manage
color issues if for example one or more of the printing inks is fluorescent.
Where a colorant contains fluorescence, a bi-spectral fluorescent spectrophotometer
is used. There are two major setups for visual spectrum spectrophotometers,
d/8 (spherical) and 0/45. The names are due to the geometry of the light
source, observer and interior of the measurement chamber. Scientists use
this machine to measure the amount of compounds in a sample. If the compound
is more concentrated more light will be absorbed by the sample; within small
ranges, the Beer-Lambert law holds and the absorbance between samples vary
with concentration linearly. In the case of printing measurements 2 alternative
settings are commonly used- without/with uv filter to control better the
effect of uv brighteners within the paper stock.
Samples are usually prepared in cuvettes; depending on the region of
interest, they may be constructed of glass, plastic, or quartz.
designed for the main infrared region are quite different because of the
technical requirements of measurement in that region. One major factor is
the type of photo-sensors that are available for different spectral regions,
but infrared measurement is also challenging because virtually everything
emits IR light as thermal radiation, especially at wavelengths beyond about
Another complication is that quite a few materials such as glass and
plastic absorb infrared light, making it incompatible as an optical medium.
Ideal optical materials are salts, which do not absorb strongly. Samples
for IR spectrophotometry may be smeared between two discs of potassium bromide
or ground with potassium bromide and pressed into a pellet. Where aqueous
solutions are to be measured, insoluble silver chloride is used to construct
Spectroradiometers, which operate almost like the visible region spectrophotometers,
are designed to measure the spectral density of illuminants in order to
evaluate and categorize lighting for sales by the manufacturer, or for the
customers to confirm the lamp they decided to purchase is within their specifications.
The light source shines onto or through the sample.
The sample transmits or reflects light.
The detector detects how much light was reflected
from or transmitted through the sample.
The detector then converts how much light the sample
transmitted or reflected into a number.
Atomic Absorption Spectrophotometry : Atomic absorption spectroscopy (AAS)
and atomic emission spectroscopy (AES) is a spectroanalytical procedure for
the quantitative determination of chemical elements using the absorption of
optical radiation (light) by free atoms in the gaseous state. Atomic
absorption spectroscopy is based on absorption of light by free metallic
Spectroradiometry : Radiometry is a set of techniques for measuring
electromagnetic radiation, including visible light. Radiometric techniques
in optics characterize the distribution of the radiation's power in space,
as opposed to photometric techniques, which characterize the light's
interaction with the human eye. Radiometry is distinct from quantum
techniques such as photon counting.
References ^ a b Rendina, George. Experimental Methods
in Modern Biochemistry W. B. Saunders Company: Philadelphia, PA. 1976. pp.
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