J. Barbara Nebe, Dr. Vice-Chair, Dept. of Cel Biology Address Center of Medical Research, Dept. of Cel Biology Medical Faculty, University of Rostock18057 Rostock, GermanyTel./Fax: +49 (0)381 494 7771/7764 Curriculum Vitae Vice Chair of the Dept. of Cel Biology, Center of Medical Research, Medical Faculty,University of RostockHabilitation, University of Rostock: Integrin receptors and t
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Analysis.ycit.cnIntroduction to Fourier TransformInfrared Spectrometry 2001 Thermo Nicolet CorporationAll rights reserved, worldwide.
What is FT-IR?
FT-IR stands for Fourier Transform InfraRed, the preferred method of infrared spectroscopy. Ininfrared spectroscopy, IR radiation is passed through a sample. Some of the infrared radiation isabsorbed by the sample andsome of it is passed through(transmitted). The resultingspectrum represents the molecularabsorption and transmission,creating a molecular fingerprintof the sample. Like a fingerprintno two unique molecular structures produce the sameinfrared spectrum. This makesinfrared spectroscopy useful forseveral types of analysis.
So, what information can FT-IR provide? • It can identify unknown materials• It can determine the quality or consistency of a sample• It can determine the amount of components in a mixture This booklet is an introduction to the concepts behind FT-IR spectroscopy. It covers both the basic theory of FT-IR and how it works as well as discussing some the practical aspects of FT-IR use.
We hope that it gives you a good understanding of the importance and usefulness of this powerful technique.
Why Infrared Spectroscopy?
Infrared spectroscopy has been a workhorse technique for materials analysis in the laboratory for over
seventy years. An infrared spectrum represents a fingerprint of a sample with absorption peaks which
correspond to the frequencies of vibrations between the bonds of the atoms making up the material.
Because each different material is a unique combination of atoms, no two compounds produce the
exact same infrared spectrum. Therefore, infrared spectroscopy can result in a positive identification
(qualitative analysis) of every different kind of material. In addition, the size of the peaks in the
spectrum is a direct indication of the amount of material present. With modern software algorithms,
infrared is an excellent tool for quantitative analysis.
The original infrared instruments were of the dispersive type. These instruments separated the
individual frequencies of energy emitted from the infrared source. This was accomplished by the use
of a prism or grating. An infrared prism works exactly the same as a visible prism which separates
visible light into its colors (frequencies). A grating is a more modern dispersive element which better
separates the frequencies of infrared energy. The detector measures the amount of energy at each
frequency which has passed through the sample. This results in a spectrum which is a plot of
intensity vs. frequency.
Fourier transform infrared spectroscopy is preferred over dispersive or filter methods of infraredspectral analysis for several reasons: • It is a non-destructive technique• It provides a precise measurement method which requires no external calibration• It can increase speed, collecting a scan every second• It can increase sensitivity – one second scans can be co-added together to ratio out random noise• It has greater optical throughput• It is mechanically simple with only one moving part Why FT-IR?
Fourier Transform Infrared (FT-IR) spectrometry was developed in order to overcome the limitations
encountered with dispersive instruments. The main difficulty was the slow scanning process. A method
for measuring all of the infrared frequencies simultaneously, rather than individually, was needed.
A solution was developed which employed a very simple optical device called an interferometer.
The interferometer produces a unique type of signal which has all of the infrared frequencies “encoded”
into it. The signal can be measured very quickly, usually on the order of one second or so. Thus,
the time element per sample is reduced to a matter of a few seconds rather than several minutes.
Most interferometers employ a beamsplitter which takes the incoming infrared beam and
divides it into two optical beams. One beam reflects off of a flat mirror which is fixed in place. The
other beam reflects off of a flat mirror which is on a
mechanism which allows this mirror to move a very
short distance (typically a few millimeters) away from
the beamsplitter. The two beams reflect off of their
respective mirrors and are recombined when they meet
back at the beamsplitter. Because the path that one beam
travels is a fixed length and the other is constantly
changing as its mirror moves, the signal which exits
the interferometer is the result of these two beams “interfering” with each other. The resulting signal
is called an interferogram which has the unique property that every data point (a function of the
moving mirror position) which makes up the signal has information about every infrared frequency
which comes from the source.
This means that as the interferogram is measured, all frequencies are being measured simultaneously. Thus, the use of the interferometer results in extremely fast measurements.
Because the analyst requires a frequency spectrum (a plot of the intensity at each individual
frequency) in order to make an identification, the measured interferogram signal can not be interpreted
directly. A means of “decoding” the individual frequencies is required. This can be accomplished via a
well-known mathematical technique called the Fourier transformation. This transformation is
performed by the computer which then presents the user with the desired spectral information for analysis.
The Sample Analysis Process
The normal instrumental process is as follows: 1. The Source: Infrared energy is emitted from a glowing black-body source. This beam passes
through an aperture which controls the amount of energy presented to the sample (and, ultimately,to the detector).
2. The Interferometer: The beam enters the interferometer where the “spectral encoding” takes
place. The resulting interferogram signal then exits the interferometer.
3. The Sample: The beam enters the sample compartment where it is transmitted through or reflected
off of the surface of the sample, depending on the type of analysis being accomplished. This iswhere specific frequencies of energy, which are uniquely characteristic of the sample, are absorbed.
4. The Detector: The beam finally passes to the detector for final measurement. The detectors used
are specially designed to measure the special interferogram signal.
5. The Computer: The measured signal is digitized and sent to the computer where the Fourier
transformation takes place. The final infrared spectrum is then presented to the user for interpretation and any further manipulation.
Because there needs to be a relative scale for the absorption intensity, a background spectrum
must also be measured. This is normally a measurement with no sample in the beam. This can be
compared to the measurement with the sample in the beam to determine the “percent transmittance.”
This technique results in a spectrum which has all of the instrumental characteristics removed.
Thus, all spectral features which are present are strictly due to the sample. A single background
measurement can be used for many sample measurements because this spectrum is characteristic of
the instrument itself.
A Simple Spectrometer Layout
Advantages of FT-IR
Some of the major advantages of FT-IR over the dispersive technique include: • Speed: Because all of the frequencies are measured simultaneously, most measurements by FT-IR
are made in a matter of seconds rather than several minutes. This is sometimes referred to as the
• Sensitivity: Sensitivity is dramatically improved with FT-IR for many reasons. The detectors
employed are much more sensitive, the optical throughput is much higher (referred to as the
Jacquinot Advantage) which results in much lower noise levels, and the fast scans enable the
coaddition of several scans in order to reduce the random measurement noise to any desired level
(referred to as signal averaging).
• Mechanical Simplicity: The moving mirror in the interferometer is the only continuously
moving part in the instrument. Thus, there is very little possibility of mechanical breakdown.
• Internally Calibrated: These instruments employ a HeNe laser as an internal wavelength
calibration standard (referred to as the Connes Advantage). These instruments are self-calibrating
and never need to be calibrated by the user.
These advantages, along with several others, make measurements made by FT-IR extremely accurate and reproducible. Thus, it a very reliable technique for positive identification of virtually
any sample. The sensitivity benefits enable identification of even the smallest of contaminants. This
makes FT-IR an invaluable tool for quality control or quality assurance applications whether it be
batch-to-batch comparisons to quality standards or analysis of an unknown contaminant. In addition,
the sensitivity and accuracy of FT-IR detectors, along with a wide variety of software algorithms, have
dramatically increased the practical use of infrared for quantitative analysis. Quantitative methods
can be easily developed and calibrated and can be incorporated into simple procedures for routine analysis.
Thus, the Fourier Transform Infrared (FT-IR) technique has brought significant practical advantages to infrared spectroscopy. It has made possible the development of many new samplingtechniques which were designed to tackle challenging problems which were impossible by older technology. It has made the use of infrared analysis virtually limitless.
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