The development of Fourier transform infrared (FTIR) has had widened its scope of perspective application on different types of substances in terms of technique of material analysis and identification. The tri-step infrared analysis has shown its powerful application in the analysis and interpretation of spectra from pure compound, fraction, raw material, natural product and complex mixture.
- Fourier transform infrared
- pure compound
- raw material
- natural product
- complex mixture
1.1. Types of infrared spectrometer: dispersive and Fourier transform
Dispersive spectrometer has been described as a traditional way in the transformation of Fourier transform infrared (FTIR) spectrometer . The basic function of dispersive spectrometer using diffraction gratings or prisms is to disperse the radiations with wave numbers at several positions. The moving of the gratings is the key that allows the radiations with wave numbers over a short interval of irradiation into the detector. Normally, the spectrum of a sample is created by the ratio between the beam passing through the sample and the reference or background. In comparison, Fourier transform infrared (FTIR) spectrometer is more sensitive and accurate in detecting and determining the higher signal-to-noise ratio within a short period of time. The principle of FTIR is generation of interferogram from the interferometer of the radiation produced by the source. Detecting the signal of radiations with different wave numbers by the Fourier transform to determine frequency domain instead of time domain will enhance the spectrum. In this way, the performance of modern FTIR is advantageous compared with dispersive spectrometer. FTIR also simplifies the complex algorithm into presentable data and is user-friendly.
The development of numerous sampling accessories, such as attenuated total reflection (ATR), sample cell with different window material for liquid sample, 2DIR sample cell etc., widens the utility of FTIR for multi-sample type analysis. Therefore, the origin of the sample material has no barrier for FTIR, albeit the different objectives of the investigation. In fact, FTIR has been recognised as a rapid, direct and non-destructive analytical method. The challenge confronting FTIR is the interpretation of the qualitative or quantitative spectral data from different direction of view.
Factors that influence the frequency vibration mode of a polyatoms molecule included concentration, thermal, time and chemical reaction. This so-called perturbation is an additional input manipulating the vibration mode of the functional group. Typically dominating the motion of the molecules in the normal mode are only one or few groups which vibrate relatively. The establishment of two dimensional correlation spectroscopy via appropriate perturbation on mid-infrared could be used to enhance the detail of infrared spectrum interpretation.
2. Fourier transform infrared spectrum and 2DIR correlation spectroscopy for pure compound analysis
This is the conventional and most widely used spectroscopy in functional group determination. For a known pure compound, each peak of spectrum will identify the main functional group within the specific range of wave number. The matching of peaks in spectrum of unknown and known compound is commonly used as a complimentary analytical method besides LCMS and NMR. Hence, such evidences of compound structure configuration are reliable. Software library incorporated into the system plays a main role in authentication of the compounds. Figure 1 showed the one dimensional Fourier transform infrared spectrum of delphinidin-3-
The 2DIR correlation spectroscopy on pure compound needs to be presented in the form of synchronous and asynchronous spectra . Synchronous spectrum is developed by combination and accumulation of two spectra of the same substance scanned under perturbation. The most commonly used perturbation is the thermal perturbation which supplies the heat at a range of temperature such as 50–120°C, and this supposedly provokes the vibration mode of the relevant bonds. The 2DIR spectra of pure compound, which represents the active sites of different bonding in the molecule, reacts to the heat simultaneously. The region of peak detected on the diagonal positively formed is named as autopeak. It is possible for the autopeak to increase or decrease during the thermal supply phase. The abundance part of 2DIR spectrum is the crosspeaks that are not scattered on the diagonal. They are either positive or negative, depending on the combination of increased or decreased spectral peak from both axes. In term of correlation, the correlation square existing on certain autopeaks and crosspeaks is the best way to interpret the correlation circumstances during the perturbation. The asynchronous spectrum was created on region not related to synchronous spectrum. The Noda’s rules  are abided during the interpretation data process.
Figure 2 showed the synchronous and asynchronous spectra of delphinidin-3-
2.1. Important usage of FTIR and 2DIR correlation spectroscopy in pure compound
2.1.1. Qualification analysis for special functional group in the compound
Pure compound has the capacity to show the whole corresponded bond taking part in the infrared transmission. The vibration mode of the whole molecule will be determined under the specific assignment. Pure compound normally shows many sharp peaks compared to compound with low purity. This concept is ideal for qualification and the respective peaks are used to identify each component in the molecule itself. In some way, the selective range of particular peak could be used in quantification under the Beer’s Law using the spectrum Quant software .
2.1.2. Identification of the functional groups present in unknown pure compound
In identification of new compound by micro-fingerprinting via spectroscopy, this robust technique can be used to obtain useful information in chemical analysis.
2.1.3. Correlation of main functioning group
Determination of correlation of main functioning group under autopeaks at diagonal line indicates another important interpretation that the bondings in the molecule react positively with mid-infrared. In addition, creating the correlation square among the autopeaks and the crosspeaks for pure compound is advantageous using 2DIR correlation spectroscopy interpretation of inter-molecule.
2.1.4. Quantification study for the sample
The innovative Quant software specific for quantification under mid-infrared is another scope of investigation. The standard compound with different concentration is the conventional method for plotting the standard graph. The new version of Quant software has either the single peak or the range of wave number chosen for the quantification compatible under the standard graph. The single peak is chosen for crude extract spectrum as the way to eliminate the interruption of enormous overlapping vibration mode from uncertain components, while the range of wave number is the best tool for purified compound.
3. Fourier transform infrared spectrum and 2DIR correlation spectroscopy for fractions analysis
Fraction is the intermediate stage of extraction and is situated between crude extract and pure compound. This level eliminates most of the debris, fibre and primary metabolites, and exportation of secondary metabolomics of therapeutic value.
The FTIR spectroscopy analyses with fractions are dependent on the condition of the material. There are two methods on the solid form of fraction, i.e. making a disc with powder KBr, or directly scan with ATR. However, the consideration must be taken on the coverage of wave number using different method. The first method will cover the whole range of wave number, while ATR only scans until 650 cm−1. The KBr method is able to detect more peaks compared to ATR method. Although ATR has its limitations, it is the best choice since in forming the original spectrum, and it is devoid of any problem in transmission percentage.
Liquid fraction is analysed using sample cell with different window material. The exploration of aqueous solution is restricted to any KBr matrix since the O-H bond affects the range around 3600 cm−1. The CaF2 matrix is appropriate for solution containing water, though the transmission is limited to below 900 cm−1. BaF2 has clearer transmission for acidic solution from 800 to 400 cm−1.
The differentiation spectra by another calculation in second derivative has been recognised and proven to be the most proper derivative to correct nonlinear baseline anomalies. The compression of peaks at a single point could be expressed in wider wave number with second derivative. This is crucial as a fraction may consist of more than one compound. The problem of overlapping in 1D FTIR spectrum is caused by similar stretching of vibration mode from different compounds or numerous identical peaks from isomers. It is possible that peak forming in 1D FTIR is due to the combination of closer transmission and clumping together as one. Therefore, the reading on second derivative spectrum is indicative of several aspects initially based on the condition of the fraction.
Figure 3 showed the second derivative spectrum of
3.1. Important usage of FTIR and 2DIR correlation spectroscopy in fractions
3.1.1. Investigation of the quality of extraction and isolation
The spectrum of a fraction could be different when compared with the pure compound. A fraction is actually the specific range of peaks chosen from the crude extract chromatogram. Technical experience of the operator will help to determine the nature of the fraction as a single or mixed compound. The pattern of the fraction can be authenticated as macro-fingerprint of the identified material. In fact, each fraction has a specific spectral pattern depending on the quality of extraction. A sample may exhibit different pattern of spectrum when different method of extraction is implemented. For example (Figure 4), the pattern of
3.1.2. Detection of enriched compound
Most of the herbal medicinal products in the market are in the form of fraction. The possibility of the presence of enrich compound added in these products can be detected by FTIR and 2DIR. The concentration of certain active compounds in standard extract is the guide to the formula. Examining this kind of distinctive criteria could be carried out by quantification using spectrum Quant. The plotting of the straight line with various dosage of standard compound is typically prophetic on the targeted compound concentration in the fraction. Normally, the specific spectrum peak has to be determined accordingly because there are procedures for peak selection instead of using the whole spectrum.
3.1.3. Investigation of adulterant in commercial products with similar molecular weight but different structural configuration
The differences could be the pattern of spectrum or the absence or the presence of strangle peak in 1D FTIR. Second derivative is an alternative to detail the differences, but the most powerful is the 2DIR correlation spectroscopy. Tri-step macro-fingerprint infrared method is also able to enhance the discrimination and distinguish the real product.
3.1.4. Determination of halal and non-halal food
Since in the fraction or extract, the main components can be easily mixed up with other ingredients in food, the challenge to separate halal and non-halal food through spectroscopy is choosing the proper range of wave number for comparison. Figure 5 showed the example of determination of halal food with 2DIR.
3.1.5. Elaborate the correlation of the main compounds with 2DIR in the fraction
The fraction spectrum profile of the pure compound usually shows the quality of extraction. The majority of the debris, precipitate and high fibre content has been discarded. The elaboration on the main compound that reacted and correlated in the overall profile of the spectrum could be clearly shown by 2DIR correlation spectroscopy. The exploration of the main compounds in the fraction increases the degree of the correlation and less problematic compared with crude extract.
4. Fourier transform infrared spectrum and 2DIR correlation spectroscopy for raw material analysis
Application of the new method of FTIR and 2DIR on raw material is still raising doubt due to the severely overlapping of peak making interpretation difficult. The detected peak could be due to more than one compound with similar assignment. Methods must be developed to ensure the assignment is interpreted correctly. In real life, raw material can be analysed using FTIR and 2DIR which will indirectly reduce the time for sample preparation. It is convenient in term of preparation and conserves the majority of the raw content.
Traditional Chinese medicines are the primer study model as raw material in the dried form using FTIR and 2DIR . The difficulties, besides the problem of overlapping assignment, also include searching the main compounds which may coincide with the raw material spectrum. This kind of matching is reliable if the sufficient literature background on the raw material exists. Despite interruption of the 1D FTIR spectrum on raw material by mixture of compounds, the spectrum can be confirmed when 60% or more transmission is achieved with KBr. The second derivative is mandatory for the raw material spectrum. Modification on the derivative of spectrum shows that second derivative is the most appropriate for exploring the spectrum since the diagram is easily interpreted. In addition, the 2DIR correlation spectroscopy is another step to enhance the detail of the spectrum for detailed interpretation. Therefore, the tri-step analysis combine method, which involves stepwise progression from the superficial to high level of analysis, is advantageous for raw material using the mid-infrared spectroscopy analysis.
4.1. The advantages of FTIR and 2DIR for raw material
4.1.1. Reduction in sample preparation steps
Raw material from natural product has to be dried completely and pulverised before making disc with KBr. In FTIR and 2DIR analysis, the sample preparation process is simple. Since the presence of moisture in the environment affects the FTIR processing method, it is necessary to use dehumidifier to maintain the dryness of the environment.
By simplifying complicated preparation steps, analysis of raw material is rapid and direct. The analysis of the spectrum with air as background could be performed immediately after the infrared scanning. The pattern of each raw material will possess footage for the identification. Each raw material will be able to show the first image of its content under the scanning. The fingerprint of the original raw material will further confirm the material profile.
4.1.3. No-destructive method and conserve the material information
A raw material conserves most of the natural contents except when contaminated with pollutants. The vulnerable step in the raw material processing is grinding, sufficiently drying and sieving with at least 200 mesh of sieve. However, these are considered less destructive when compared with further extraction on the raw material. In fact, the raw material can be tested under ATR which does not require a disc. The interferon beam has a glimpse of refection on the sample with diamond platform and produce spectrum in the range of 4000–650 cm−1. Comparison of the
4.1.4. Interpretation of poly-nutrient in crude
When the whole profile of the raw material is presented with spectra especially in 2DIR, the correlation square generated under the perturbation is based on the active compounds responding concurrently. Such response is associated with a variety of compounds found in the raw extract. Information derived from such interpretation is vital as it provides a holistic picture of the reaction. Figure 7 showed the correlation square formed between flavonoids peak and carbohydrate peak in
4.1.5. Standardised method for authentication of a raw material derived from natural product
The origin of the raw material in certain commercial medicinal products is difficult to be authenticated especially when they are presented as powder. Such products could be a portion of plant or other types of organism. Screening of the powder using KBr or ATR method will elucidate its chemical profile as the spectrum will confirm the origin as well as the purity. The compendium and monograph of herbal plants should include the spectrum as one of the methods in the authentication of medicinal products.
5. Fourier transform infrared spectrum and 2DIR correlation spectroscopy for natural product analysis
Analysis of natural product with FTIR and 2DIR have to confront with the obstacles coming from the natural texture of the subjects, since their contents consist of water in living cells found leaf, fruit, stem, flower, seed or fruiting body, stalk and sclerotium from medicinal mushroom. The spectroscopy analysis of natural product needs to avoid the interrupting H2O background and preserve the nature of the features. Therefore, the analysis must apply the correct method chosen as well as appropriate spectrum interpretation on natural product.
There are three choices of FTIR spectrum scanning for natural product. Firstly, mixture with KBr if the natural product is completely dried and without the need for further drying process, such as seed, some rhizome or hash portion of fruiting body. Secondly, ATR is the best choice if the sample is sensitive to the environment conditions when they are being removed or still contain water and mucous secretions. Even though there is a slight refection on the sample surface through the diamond platform, the spectrum created is still sufficient to represent the actual profile when using air as a background. In comparison, the juice of the natural product containing water can be analysed using liquid sample cell with calcium fluoride window. The third method is the FTIR imaging attached to a microscope with a spectroscopy system. The combination enriches the scope of analysis and is more powerful and user-friendly.
No doubt that human error is unavoidable when dealing with natural product analysis. The content(s) may have been lost during the sample processing, poor quality of extraction, physical deterioration and extended period of storage. The more the natural content of the subject model is preserved, the more accurate it can be analysed. These kinds of natural product are found in Malay, Chinese  and Ayurvedic medicine such as medicinal mushroom. Many remedies in these communities contain ready to use formulae of the natural products for therapy, e.g. use of turmeric product  for blood clotting. FTIR is one of the methods to study the key chemical contents in such treatment.
5.1. Analysis of natural product and raw material
5.1.1. Appropriate for quantification of chemical marker analysis
In order to determine the exact quantity of the chemical(s) involved in the treatment, the Spectrum Quant is especially effective for this purpose, using different concentrations of standards that will generate the range of spectra for the targeted compound. The other algorithms such as PCR+, PLS and Quant C can also be chosen depending on the respective objectives of the study. The results of analysing an unknown sample can be used to predict its concentration by comparing with the range of standards.
5.1.2. Exposure to various factors
The original natural product is the best type of sample for chemical fingerprint profiling, maturity or storage period determination. Time factors such as oxidation, exposure to air, etc., could affect the whole experiment. The sample of natural product analysed using FTIR and 2DIR is prevented from the detrimental effects of these factors.
5.1.3. Determination of different geographical origin of natural product using principal component analysis or SIMCA
Natural product especially the medicinal plants and mushroom of different geographical origin would have a variety of chemical contents with respect to the quality and the quantity. Mean spectral data from randomly chosen plants from a plantation and those from individual plants can be compared. This kind of statistical value could be plotted using a software such as Assured ID which will provide overall comparison of the similar type of natural product collected from different geographical locations. The differences are due to factors such as different types of soil, water, weather, pH of the soil, water, etc. These factors will influence the distance and the percentage of rejection of SIMCA created by three principle components. Figure 8 showed the example of SIMCA result of
6. Fourier transform infrared spectrum and 2DIR correlation spectroscopy for complex mixture analysis
Today, use of combo medicinal contents has been reported to be more efficient compared to single compound when treating viral diseases. Research on the effects of combination of different substances has been reported
6.1. Difficulty in interpretation
Analysis of the complex mixture of samples required pre-knowledge of the main compounds involved. The addition of different types of standards is needed to determine the assignment of bond in 1D FTIR. There could be no actual matching of standards peak with the complex mixture because of the overlapping and interruption of the bonds from each other. Consequently, more standards or fractions need to be used for different scope of comparison. The additional library in the software could be helpful in this aspect. When the peaks have been confirmed, then the next step will be carried out as usual. The 2DIR with synchronous spectrum is the best option to investigate the interaction of mixture under the appropriate perturbation.
6.2. Level of sensitivity
It is often difficult to address the interaction of a mixture as it may contain totally dissimilar types of compounds. However, they can react concurrently when perturbation is applied. The degree of sensitivity, either from the user or the system is very much relied on the experience and practice, e.g. two possible interpretations based on the similar spectrum. Therefore, the analysis on the mixture with FTIR and 2DIR must be accurate and detailed initially to prevent subsequent misinterpretation.
6.3. Strengthening of data
It is not advisable to use the asynchronous spectrum in 2DIR for the mixture due to the tremendous overlapping of assignment which is confusing. Other types of data generated from HPTLC, HPLC, GCMS, LCMS and NMR could be additional evidences for mixture complex analysis.
7. Limitations of FTIR and 2DIR
FTIR and 2DIR technique used in analysis has their limitations that could reduce the degree of efficiency. Fractions in solution form are not eligible for 2DIR analysis except using liquid sample cell, since special sample cell is needed to create high temperature changes and the rate of evaporation of the natural product solution is unknown. However, liquid sample cell is expensive. Besides, there is no appropriate FTIR equipment to capture the changes of spectrum within a few seconds, such as oxidation from the natural product. The thermal perturbation is commonly utilised for analysis of different substances and difficult to interpret compound with more than one isomers structure. However, no standardise method exist to determine the degree of peak differences. Some researchers agree that the range 2–3 cm−1 is for detecting the pure compound and the fraction, while the range 5–8 cm−1 is for the raw material and other materials. However, the acceptance of these ranges is still debatable. Lastly, there will be confusion on spectra for comparison when the quality of extraction is not the same especially the fractions.
In conclusion, much research on FTIR and 2DIR is still ongoing in order to overcome the various limitations confronting their widespread use. With accumulated experience and sophisticated innovation such as imaging microscope connect to FTIR, the techniques will be improved and enhanced in the near future.
We would like to thank the Director General of Health of Malaysia for his permission to publish this article and the Director of Institute for Medical Research (IMR), Kuala Lumpur for the support of this project. This work was financially supported by the NKEA AGRICULTURE (EPP#1), NKEA Research Grant Scheme (NRGS) (Grant No: NH1014D060). The author also thanks Dr.Aswir (IMR), Dr. Hussin (IMR) and Dr. Lee Han Lim (IMR) for critically reviewing this paper and technical support from Perkin Elmer Sdn Bhd. (Malaysia). The previous support of Islam Development Bank (IDB) for training in FTIR and 2DIR under the Merit Fellowship is gratefully acknowledged.