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Spectrophotometry Techniques and Devices

Paper Type: Free Essay Subject: Chemistry
Wordcount: 3664 words Published: 8th Dec 2017

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Infrared Spectrophotometry is designed to identify or determine the sample by measuring absorption of infrared radiation of wave numbers in a region of 4,000 to 400 cm-1, at various wave numbers, when it passes through the sample. This method uses the property that the infrared absorption spectrum of a substance is Characteristic of its chemical structure. Infrared spectra are shown in charts drawn by plotting the wave numbers on the abscissa and the transmittances or absorbances on the ordinate.

i. Spectrophotometer

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. Important features of spectrophotometers are spectral bandwidth and linear range of absorption measurement.

Perhaps the most common application of spectrophotometers is the measurement of light absorption, but they can be designed to measure diffuse or specular reflectance.

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. [2] They are widely used in many industries including printing and forensic examination.

ii. Design

There are two major classes of devices: single beam and double beam. A double beam spectrophotometer compares the light intensity between two light paths, one path containing a reference sample and the other the test sample. A single beam spectrophotometer measures the relative light intensity of the beam before and after a test sample is inserted. Although comparison measurements from double beam instruments are easier and more stable, single beam instruments can have a larger dynamic range and are optically simpler and more compact.

Historically, spectrophotometers use a monochromator containing a diffraction grating to produce the analytical spectrum. 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 compares the fraction of light that passes through a reference solution and a test solution. Light from the source lamp is passed through a monochromator, which diffracts the light into a “rainbow” of wavelengths and outputs narrow bandwidths of this diffracted spectrum. Discrete frequencies are transmitted through the test sample. Then the intensity of the transmitted light is measured with a photodiode or other light sensor, and the transmittance value for this wavelength is then compared with the transmission through a reference sample.

In short, the sequence of events in a spectrophotometer is as follows:

  1. The light source shines into a monochromator.
  2. A particular output wavelength is selected and beamed at the sample.
  3. The sample absorbs light.

Many spectrophotometers must be calibrated by a procedure known as “zeroing.” The absorbency of a reference 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 initial substance).[2]


i. Ultraviolet spectrophotometry

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.

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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 two 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

ii. IR spectrophotometry

Spectrophotometers 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 photosensors 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 5μm.

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 the cell.


Infrared (IR) radiation is electromagnetic radiation with a wavelength between 700nm and 300µm, which equates to a frequency range between 1THz and 430THz—a span of more than three orders of magnitude.

Its wavelength is longer (and the frequency lower) than that of visible light, but the wavelength is shorter (and the frequency higher) than that of terahertz

radiation microwaves. Bright sunlight provides an irradiance of about 1kilowatt per square meter at sea level. Of this energy, 527 watts is infrared light, 445 watts is visible light, and 32 watts is ultraviolet light.

The infrared part of the electromagnetic spectrum covers the range from roughly 300 GHz (1 mm) to 400 THz (750 nm). It can be divided into three parts:

  1. Far-infrared, from 300 GHz (1 mm) to 30 THz (10 μm). The lower part of this range may also be called microwaves. This radiation is typically absorbed by so-called rotational modes in gas-phase molecules, by molecular motions in liquids, and by phonons in solids. The water in the Earth’s atmosphere absorbs so strongly in this range that it renders the atmosphere effectively opaque. However, there are certain wavelength ranges (“windows”) within the opaque range which allow partial transmission, and can be used for astronomy. The wavelength range from approximately 200 μm up to a few mm is often referred to as “sub-millimeter” in astronomy, reserving far infrared for wavelengths below 200 μm.
  2. Mid-infrared, from 30 to 120 THz (10 to 2.5 μm). Hot objects (black-body radiators) can radiate strongly in this range. It is absorbed by molecular
  3. Vibrations, where the different atoms in a molecule vibrate around their equilibrium positions. This range is sometimes called the fingerprint region since the mid-infrared absorption spectrum of a compound is very specific for that compound.
  4. Near-infrared,

from 120 to 400 THz (2,500 to 750 nm). Physical processes that are relevant for this range are similar to those for visible light.[4]

IV. Infrared spectroscopy

(IR spectroscopy) is the subset of spectroscopy that deals with the infrared region of the electromagnetic spectrum. It covers a range of techniques, the most common being a form of absorption spectroscopy. As with all spectroscopic techniques, it can be used to identify compounds or investigate sample composition. Infrared spectroscopy correlation tables are tabulated in the literature. A common

laboratory instrument that uses this technique is an infrared spectrophotometer.

i. Background and theory

The infrared portion of the electromagnetic spectrum is divided into three regions; the near-, mid- and far- infrared, named for their relation to the visible spectrum. The far-infrared, approximately 400-10cm−1 (1000-30μm), lying adjacent to the microwave region, has low energy and may be used for rotational spectroscopy. The mid-infrared, approximately 4000-400cm−1 (30-2.5μm) may be used to study the fundamental vibrations and associated rotational-vibrational structure. The higher energy near-IR, approximately 14000-4000cm−1 (2.5-0.8μm) can excite overtone or harmonic vibrations. The names and classifications of these subregions are merely conventions. They are neither strict divisions nor based on exact molecular or electromagnetic properties.

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Infrared spectroscopy exploits the fact that molecules have specific frequencies at which they rotate or vibrate corresponding to discrete energy levels (vibrational modes). These resonant frequencies are determined by the shape of the molecular potential energy surfaces, the masses of the atoms and, by the associated vibronic coupling. In order for a vibrational mode in a molecule to be IR active, it must be associated with changes in the permanent dipole. In particular, in the Born-Oppenheimer and harmonic approximations, i.e. when the molecular Hamiltonian corresponding to the electronic ground state can be approximated by a harmonic oscillator in the neighborhood of the equilibrium molecular geometry, the resonant frequencies are determined by the normal modes corresponding to the molecular electronic ground state potential energy surface. Nevertheless, the resonant frequencies can be in a first approach related to the strength of the bond, and the mass of the atoms at either end of it. Thus, the frequency of the vibrations can be associated with a particular bond type. Simple diatomic molecules have only one bond, which may stretch. More

complex molecules have many bonds, and vibrations can be conjugated, leading

to infrared absorptions at characteristic frequencies that may be related to chemical groups. For example, the atoms in a CH2 group, commonly found in organic compounds can vibrate in six different ways: symmetrical and antisymmetrical stretching, scissoring, rocking, wagging and twisting:

The infrared spectrum of a sample is collected by passing a beam of infrared light through the sample. Examination of the transmitted light reveals how much energy was absorbed at each wavelength. This can be done with a monochromatic beam, which changes in wavelength over time, or by using a Fourier transform instrument to measure all wavelengths at once. From this, a transmittance or absorbance spectrum can be produced, showing at which IR wavelengths the sample absorbs. Analysis of these absorption characteristics reveals details about the molecular structure of the sample. When the frequency of the IR is the same as the vibrational frequency of a bond, absorption occurs.

This technique works almost exclusively on samples with covalent bonds. Simple spectra are obtained from samples with few IR active bonds and high levels of purity. More complex molecular structures lead to more absorption bands and more complex spectra. The technique has been used for the characterization of very complex mixtures.

ii. Adjustment and Instrument

Use a dispersive infrared spectrophotometer or a Fourier-transform infrared spectrophotometer. Before using the infrared spectrophotometer, adjust it as specified in the operating manual. The linearity of the absorbance between 20% and 80% of transmittance (%) should be within 1%. The reproducibility of the transmittance should be within 0.5% in two consecutive measurements. The reproducibility of wave number should be within 5 cm-1 at about 3,000 cm-1 and within 1 cm-1 at About 1,000 cm-1. In addition, adjust the instrument so that a spectrum exhibits absorptions at the wave numbers as indicated in the following figure when measurement is made on a polystyrene film (about 0.03 mm thick).[5]

iii. Preparation of Sample

According to an appropriate one of the methods below,Prepare the sample so that the transmittance of the most intense absorption bands should be within a range of 20 to 80%. For the optic plate, use sodium chloride, potassium bromide, or thallium iodide bromide.

  1. Potassium Bromide Disk Method Place 1 to 2 mg of a solid sample and 100 to 200 mg of dried potassium bromide for infrared spectrophotometry into an Agate mortar, quickly reduce to fine particles protecting from moisture, mix Completely, and transfer into a die. Press the surface of the disk at 500 to 1,000 N/cm2 under reduced pressure of not more than 0.7 kPa for 5 to 8 minutes, and use this disk for the measurement.
  2. Solution Method Prepare a solution of the solid or liquid sample in the Specified solvent, inject the solution into a fixed cell for liquid, and use this cell for the measurement. Place the similar cell containing the same solvent for the Compensation beam. The thickness of the fixed cell is generally 0.1 mm or 0.5 mm.
  3. Paste Method Crush finely a solid sample and knead well with liquid Paraffin in the mortar. Hold the paste between two optic plates without any air gap, and measure.
  4. Liquid Film Method Hold 1 to 2 drops of liquid sample as a capillary film Held between two optic plates, and measure the liquid layer between the plates. If it is necessary to thicken the liquid layer, place rings of aluminum foil or a similar material between the two optic plates so that the liquid sample lies between the plates.
  5. Thin Film Method Dissolve the sample in the specified solvent, and apply it to one optic plate. Evaporate the solvent by drying with hot air, and measure the thin film adhered on the plate. If the sample is a film with a thickness of not more than 0.02 mm, measure the film just as it is.
  6. Gas Sample Measurement Put the sample gas in a gas cell with a light Path of 5 to 10 cm in length, previously evacuated, under pressure specified in the individual monograph, and measure. A long cell with the light path of not shorter than 1 m is also used if necessary.

iv. Conventional method

A beam of infrared light is produced and split into two separate beams. One is passed through the sample, the other passed through a reference which is often the substance the sample is dissolved in. The beams are both reflected back towards a detector, however first they pass through a splitter which quickly alternates which of the two beams enters the detector. The two signals are then compared and a printout is obtained.

A reference is used for two reasons:

  • This prevents fluctuations in the output of the source affecting the data
  • This allows the effects of the solvent to be cancelled out (the reference is usually a pure form of the solvent the sample is in)

v. Fourier transform infrared spectroscopy

Fourier transform infrared (FTIR) spectroscopy is a measurement technique for collecting infrared spectra. Instead of recording the amount of energy absorbed when the frequency of the infra-red light is varied (monochromator), the IR light is guided through an interferometer. After passing through the sample, the measured signal is the interferogram. Performing a Fourier transform on this signal data results in a spectrum identical to that from conventional (dispersive) infrared spectroscopy.

FTIR spectrometers are cheaper than conventional spectrometers because building an interferometer is easier than the fabrication of a monochromator. In addition, measurement of a single spectrum is faster for the FTIR technique because the information at all frequencies is collected simultaneously. This allows multiple samples to be collected and averaged together resulting in an improvement in sensitivity. Virtually all modern infrared spectrometers are FTIR instruments.

Summary of absorptions of bonds in organic molecules

vi. Uses and applications

Infrared spectroscopy is widely used in both research and industry as a simple and reliable technique for measurement, quality control and dynamic measurement. It is of especial use in forensic analysis in both criminal and civil cases, enabling identification of polymer degradation for example. It is perhaps the most widely used method of applied spectroscopy.[citation needed]

The instruments are now small, and can be transported, even for use in field trials. With increasing technology in computer filtering and manipulation of the results, samples in solution can now be measured accurately (water produces a broad absorbance across the range of interest, and thus renders the spectra unreadable without this computer treatment). Some instruments will also automatically tell you what substance is being measured from a store of thousands of reference spectra held in storage.

By measuring at a specific frequency over time, changes in the character or quantity of a particular bond can be measured. This is especially useful in measuring the degree of polymerization in polymer manufacture. Modern research instruments can take infrared measurements across the whole range of interest as frequently as 32 times a second. This can be done whilst simultaneous measurements are made using other techniques. This makes the observations of chemical reactions and processes quicker and more accurate.

Techniques have been developed to assess the quality of tea-leaves using infrared spectroscopy. This will mean that highly trained experts (also called ‘noses’) can be used more sparingly, at a significant cost saving.

Infrared spectroscopy has been highly successful for applications in both organic and inorganic chemistry. Infrared spectroscopy has also been successfully utilized in the field of semiconductor microelectronics[8]: for example, infrared spectroscopy can be applied to semiconductors like silicon, gallium arsenide, gallium nitride, zinc selenide, amorphous silicon, silicon nitride, etc.


A technique to identify materials including organic polymers. An infrared spectrometer directs infrared radiation through a sample and records the relative amount of energy absorbed by the sample as a function of the wavelength or frequency of the infrared radiation. The method is applicable particularly to organic materials, because the vibrational frequencies of the constituent groups within the molecules coincide with the electromagnetic frequencies of the infrared radiation. Therefore, the infrared radiation is selectively absorbed by the material to produce an absorption spectrum. The spectrum produced is compared with correlation spectra from known substances.


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. Components:

  1. The light source shines onto or through the sample.
  2. The sample transmits or reflects light.
  3. The detector detects how much light was reflected from or transmitted through the sample.
  4. The detector then converts how much light the sample transmitted or reflected into a number.


In this topic which is infrared spectrophotometry I have introduced what is spectrophotometry. And it is used in a device called spectrophotometer which is explained in the above thesis. Followed on single beam spectrophotometer is also explained with its design & working.

Spectrophotometry is generally of two types UV & IR spectrophotometry, UV spectrophotometry is explained in short but IR spectrophotometry is explained briefly. The word INFRARED is explained i.e. what it means, infrared region is explained in EM radiation. In EM spectrum there comes a topic infrared spectroscopy which is explained briefly with its background & theory. Its preparation of sample followed by conventional method of it. There is other phenomenon called FITR (Fourier transform infrared spectroscopy) is a measurement technique for collecting infrared spectra. FTIR spectrometers are cheaper than conventional spectrometers. Uses & application is also explained in the above thesis. At last but not the least its use in organic is explained. The idea of Spectroradiometers is also given, which operate almost like the visible region spectrophotometers. This is end of the conclusion of my thesis infrared spectrophotometry.


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