———————————————————Fourier-Transform Infrared Radiation Spectroscopy (FTIR) isa technique widely used to obtain an infrared spectrum or absorption of asolid, liquid or gas. The first commercial production of an FTIR Spectrometer wasfirst exhibited in 1969, created by the company Digilab. This was made possibleby the invention of mini computers in 1965 with a greater computing capacity,most notably the PDP-8. This was due to the fact they were needed to performthe intense Fourier transform calculations necessary. This newly inventedproduct had come a long way from the first spectrophotometer with the abilityto record the infrared spectrum created by Perkin-Elmer in 1957.
The FTIR spectrometer consists of an interferometer, this ismost commonly the Michelson Interferometer. Infrared light is sent from a pointsource through a beam splitter which separates the light into two beams, thesetwo separate beams reflect onto two different mirrors, where one is fixed andone is movable. The light reflects and the two beams recombine at the beamsplitter to form one singular beam of light. The interferometer measures theinterference pattern produced for when two beams of light interfere with eachother. When the two waves are in phase they have undergoneconstructive interference and their intensity amplitude is increased.
However,when they are out of phase they undergo destructive interference and theintensity is zero 1. Thedifference in the path travelled by the two waves which have the same frequencyand velocity is known as the path difference. This path difference can beadjusted by moving the mirror which is not fixed to different distances.
This light is then sent through a sample, the chemical bondswithin this sample absorb certain wavelengths of the incident infra-red lightunique to that of the sample. Not all wavelengths are absorbed and thustransmit through the sample reaching the detector. A spectrum is thereforecreated by the light incident on the detector. This spectrum has various peaksand troughs, which are analysed by being compared to a database of knownmeasured elemental spectra which help to determine the samples chemical make-up.In this spectrum there can often be carbon dioxide and water vapour includedwhich can cause a large room for error when analysing the spectrum. If the optical path difference and the light intensity areplotted against one another, an interferogram is produced. This is the foundationof measurement that an FTIR obtains. The interferogram’s, which are producedwhile scanning, are then Fourier Transformed by the FTIR software in order toyield a spectrum onto a computer screen connected to the instrument.
This iswhere the name, FTIR, derives from as the mathematical concept known as theFourier Transform is used in the process to create a spectrum from the raw datareceived 2. This concept is usedto calculate the superposition of sine and cosine waves for a specificfunction. When a sample is placed in the instrument that has an absorbance(absorbing at a wavelength )then the amplitude of that interferogram will decrease 3. When the number of scans is increased, this increases thenumber of times the scanning (movable) mirror is moved forward and backwards. Forexample if there were to be 10 scans then 10 interferograms would be producedand an average of these would be calculated, this is known as signal averaging 4. The instrument experiences background noise when conductingmeasurements which can provide poor results. During the measurement, the signalin the interferogram of the sample is always considered to be positive, howeverthe signal of the noise is arbitrary and fluctuates back and forth between apositive and negative value. If one scan is taken the noise can have a verylarge impact on the resulting spectrum causing poor results, however if 100scans are taken and averaged, as previously mentioned, the noise cancels itselfout.
The interferogram signal however is consistently positive and thereforedoes not cancel itself out. Therefore, the greater the number of scansperformed the less background noise seen in the resulting spectrum. It is foundthat the signal to noise ratio in a spectrum is proportional to the square rootof the number of scans added together 5. The infrared detector has the ability to measure interferogramsas electrical signals, these have different frequencies and are displayed ascosine waves which are presented as the spectrum of the sample.
When aninterferogram is Fourier Transformed, a mathematical function is produced whichcorresponds to it. This function being the spectrum of the infrared beam thatthe detector picks up. As before mentioned, when the light passes through theinterferometer an interference pattern is produced due to the opticaltransformation of the infrared beams spectrum. The interference pattern is thenmeasured by the detector and the Fourier Transform changes it back to thespectrum. So, when a sample is placed in front of the infrared beam it altersthe interference pattern 6. Whenan interferogram is Fourier transformed a single beam spectrum is produced.
Itis noted that FTIR only uses a single beam of IR light as opposed to otherspectrometers which use two beams. A background spectrum is obtained by taking measurementswith the FTIR spectrometer with no sample present. This is a spectrum whichincludes contributions of the surrounding environment (e.g. the contributionsof water vapour and carbon dioxide) and from the instrument. This backgroundspectrum can be used to first determine the quantity of artifacts present, inorder to help analyse the data by subtracting these from the samples spectrum. The data which FTIR collects is high-spectral-resolutiondata which spans over a wide spectral range. This is a clear advantage FTIR has over acommon dispersive spectrometer which has a very narrow range of measurement forintensity of wavelengths at a time.
The range of infrared isapproximately 700nm ~ 1mm long 7. FTIRis the third generation of Infrared spectroscopy, there are several reasons whyit has replaced the previous 2nd generation technique firstintroduced in the 1960’s 8. Someof the advantages include; high accuracy of wavenumber to an uncertainty of±0.01,a wide scan range (1000 ~ 10),extremely high resolution (0.
1 ~ 0.005)and the scan time of all frequencies is short. The FTIR has a major advantage over other infraredspectrometers and that is due to the fact they can measure spectra with highsignal-to-noise ratios (SNR).
The SNR is the measurement of the quality ofspectra, the more light that hits the detector the greater the amount of signalin a spectrum. Many other instruments force light to pass through gratings andprisms which reduces the amount of light hitting the detector and thereforeproduce a weaker signal within a spectrum. However, in a FTIR spectrometer, thebeam of light does not pass through any such thing and thus has a greateramount of light hitting the detector, giving it the ability to measure spectrawith high SNR 9. It is also found that the SNR is directly proportional tothe number of scans taken added together to create a spectrum in FTIRspectroscopy, therefore the greater number of scans taken, the greater the SNR.This is known as the multiplex advantage. A high SNR is very advantageous as itimproves the instruments sensitivity making it easier to detect smaller peaks.
Another reason a high SNR is beneficial is quantitative accuracy, this is howaccurately peak areas and heights can be measured, as the absorbance spectra isproportional to the concentration. These SNR advantages of FTIR spectroscopy have provided anabundance of applications that were never available using other types ofspectrometers. Different techniques of sample preparation, most notablyAttenuated Total Reflection (ATR), are now performing constructive analysis ofvarious samples with speed and ease. In a more medical frame, the spectra ofcancerous cells in the human body is now currently possible, due to these SNRadvantages which are being used to save lives by targeting cancer at an earlystage 10. ATR is an accessory of FTIR spectrometry, it is used toperform measurements of surface properties of thin or solid film samples directlywithout further preparation, as opposed to their bulk properties. This processtends to have a 1 – 2 micrometre penetration depth which can vary depending onthe conditions of the sample.
ATR produces an evanescent wave using propertiesof total internal reflection. IR light passes through a crystal and reflectsonce, at the very least, off of the internal surface which is in contact withthe sample. An evanescent wave is produced in the process which penetrates thesample. The value to which it penetrates is dependent on; the angle ofincidence, the wavelength of the light and the refraction of the ATR crystalbeing used. Changing the angle of incidence can alter the amount of reflectionsproduced. Once the beam exits the crystal it is collected by a detector.
Evert instrument has imperfections and the FTIR Spectrometer’slie in the disadvantages of ‘artifacts’. These are features which are not fromthe sample in question but are detected as part of the samples measurements.Two very common artifacts are water vapour and carbon dioxide peaks.
When usingFTIR spectroscopy the sample and background spectra must be measured atdifferent points in time, this means that if anything is to change within thespectrometer between the two spectra measured, such as a change in H2O orCarbon Dioxide concentration, then the peaks produced by this will contaminatethe spectrum of the sample 11. It thus causes the need for one to use great caution inavoiding making the mistake of interpreting atmospheric gas peaks as being fromthe sample. The spectrum of the atmosphere needs to be well understood to avoidthis problem. There also must be great care when preparing the sample, allscientists new to working in this field require time to perfect the art of thepreparation of a sample making sure no air bubbles are present to avoid spectracontamination. Albeit this is a very common way to make mistakes and is adisadvantage of FTIR spectroscopy, as a whole the advantages that have beendiscussed far outweigh this inconvenience. FTIR spectroscopy, as before mentioned, can produce informationabout different samples in multiple states. This can help to identify anunknown composition of elements within a material which can provide veryhelpful in many areas.
One of the most common applications of FTIR is in thepharmaceutical industry. The strong regulatory nature of this industry and itsmass production scale require quick and effective analysis of the drugs or samplesin use, making FTIR spectroscopy an obvious choice and very beneficial. The applicationsof the technique in this field ranges from drug formula characterisations toclarification of kinetic processes in drug delivery.
12 Apaper written by H. Masmoudi titled “The evaluation of cosmetic andpharmaceutical emulsions aging process using classical techniques and a newmethod: FTIR” found that FTIR was the only technique found to be able tocharacterise the evolution and chemical functions of emulsions during theprocess of thermal aging. This paper also stated that whilst FTIR wasbeneficial for the chemical modifications of emulsions, conductivitymeasurements were necessary for the physical aspect of the emulsions and theirstability measurements. It was concluded the two techniques were complimentaryand should be used together to obtain the most accurate data 13.
Another field where FTIR plays an important role is in Forensics. Drugenforcement agencies around the world must be able to effectively identifydrugs or illegal substances which are being brought into the country. Policedepartments require the same speed of analysis when working with crime sceneevidence. One important application of FTIR within this field is in the fightagainst endangered animal hunting and poaching for the reward of highly soughtafter hair or fur.
This can be beneficial by determining a specific animal’shair, which has been used for indigenous products and accessories to aid in theattempt to save these endangered species and punish those responsible for doingso. A paper from 2008 titled ‘Forensic identification of elephant and giraffe hairartifacts using HATR FTIR spectroscopy and discriminant analysis’ found successby using horizontal-attenuatedtotal-reflection Fourier transform infrared spectroscopy to differentiatebetween Elephant and Giraffe hair. This could prove very beneficial by usingFTIR to aid in uncovering illegal wildlife trade 14.FTIR spectroscopyhas also proven very useful and is in fact a front running method which iswidely used in biophysics. This is a very broad field with seemingly limitlessapplications for FTIR, however one of the most common is in the detection andstudy of biological tissue, mainly proteins. Proteins are a verycommon sample used in FTIR. Whilst FTIR Spectroscopy may not provide thesame level of detail of a proteins structure as other techniques such as NMR orX-Ray crystallography, it can be used readily to help our understanding of how thesebiological components function.
15This is due to the fact the data whichit collects has a high-spectral-resolution which spans over a wide spectralrange 16. The spectrum produced when exposing a sample to infraredlight in a FTIR spectrometer can give a large amount of information about theprotein. When exposed to Infrared Radiation, sample molecules absorb radiationof certain wavelengths, this then changes the dipole moment of the molecule 17.The absorption peak intensity is notably related to the change of dipole momentand the possibility of the transition of energy levels. The number ofvibrational freedoms of the molecule is directly related to the number ofabsorption peaks recorded. Furthermore, the frequency of the absorption peaksis determined by the vibrational energy gap 18.
As can be seen, theanalysis of the infrared spectrum using FTIR spectroscopy can therefore providean abundance of structural information of the protein in question, which willin turn provide information which can be applied to the enhancement of modernday challenges in the medicinal industry such as drug binding. A paper from 2007 titled “Fourier Transform Infrared SpectroscopicAnalysis of Protein Secondary Structures” written by J. Kong studied theprocess of protein analysis by Infrared Spectroscopy and found that FTIRspectroscopy is far more convenient to use in the process than othertechniques. This is because it can obtain IR spectra in a wide range ofenvironments with little sample volume. They also state the potential errorsthat could arise from using the background subtraction procedure which is verycommon when working with FTIR Spectroscopy. They concluded that it wasbeneficial to use both CD and FTIR Spectroscopy together for more accurateanalysis 19.
Written by J. Zhang, “Solid-film sampling method for thedetermination of protein secondary structure by Fourier transform infraredspectroscopy” was published in 2017 and studies the ‘solid film method’ ofsample preparation which is used before the application of FTIR spectroscopy.They found that this method renders the FTIR technique suitable for determiningthe secondary structure of proteins in aqueous solutions at lowerconcentrations, less than 0.5mg/mL, which is much lower than the traditionalmethod 20. In 2016, A. C. S.
Talari produced a paper named “Advances in Fouriertransform infrared (FTIR) spectroscopy of biological tissues”. In thispaper, there is a wide set of data collected of spectral peaks that are presentin FTIR Spectroscopy of biological tissues which were recorded and tabled toaid in the study of spectral analysis to reduce time spent by scientists. Thedata incorporates all varieties of biological tissue 21. “MultivariateAnalysis for Fourier Transform Infrared Spectra of Complex Biological Systemsand Processes” which waswritten by D.
Ami and published in 2013 by InTechOpen confirmed FTIRmicro-spectroscopy to be a very useful tool as it is a quick and effective wayto provide a ‘chemical fingerprint’ of biological samples. However, the studydid highlight the problems that the method encounters such as overlapping ofmain biomolecules which shows that multivariate analysis is needed if the datais going to be successfully interpreted 22. In conclusion FTIRspectroscopy consistently remains as the main choice of spectroscopy for manyscientists when determining structural properties or elemental make up of asolid in a gas, water or vapour state. There are many advantages to using thistechnique when compared to other forms of IR spectroscopy, although there arestill issues with artifacts throughout the process, this can be disregarded as enoughof a reason to avoid using the technique once the large amount of advantageshave been taken into account, such as the high spectral resolution and theability attained to measure spectra with high SNR. We have also discussedvarious applications of FTIR, however this only scratches the surface of fieldsthat it is currently being used for, not to mention the possible applicationsof the future. We have already seen the advancement in recent years of FTIR, thatbeing the new ability to produce high resolution imaging which is imperativefor characterising tissue structures and cell types leading to accurate diseasediagnosis through ATR-FTIR. The future aim for FTIR would certainly be toovercome the contamination of sample results by removing artifacts completelyfrom the instruments measurements.
A modification to the instrument couldperhaps provide an elimination process for artifacts by running more intenseand thorough background checks before conducting the measurements. The futurelooks bright for the application and development of FTIR regardless, it willcontinue to progress and provide important information to make improvements inmany different fields.