**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

*2.1.2. Identification of the functional groups present in unknown pure compound*

nique can be used to obtain useful information in chemical analysis.

the range of wave number is the best tool for purified compound.

*2.1.3. Correlation of main functioning group*

178 Fourier Transforms - High-tech Application and Current Trends

*2.1.4. Quantification study for the sample*

pretation of inter-molecule.

autopeak spectrum of the 2DIR.

In identification of new compound by micro-fingerprinting via spectroscopy, this robust tech-

**Figure 2.** The 2DIR spectra of delphinidin-3-O-sambubioside in the range of 1700–700 cm−1. (a) Synchronous spectrum. The correlation square is created from negative crosspeak at (1177, 1650), autopeak 1650 cm−1, negative crosspeak (1650, 1177) and autopeak 1177 cm−1. There is another bigger correlation square which is created from negative crosspeak (1019, 1650), autopeak 1649 cm−1, negative crosspeak (1650, 1019) and autopeak 1019 cm−1. A smaller correlation square is created from four red areas. They are positive crosspeak (1019, 1177), autopeak 1177 cm−1, positive crosspeak (1177, 1019) and autopeak 1019 cm−1. (b) Asynchronous spectrum. The positive crosspeak at (1177, 1650) determines the sequence action against perturbation of the area at 1650 cm−1 first reacted than area at 1177 cm−1. The similar scenario for the bigger correlation square where the peak area at 1650 cm−1 reacted first than area at 1019 cm−1. When compared with the area at 1019 cm−1 and 1177 cm−1, 1019 cm−1 is reacted first than 1177 cm−1. The sequence of decrease reacted to the thermal perturbation in The series is 1650 cm-1, 1019 cm-1, 1177 cm-1. (c) The

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 inter-

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

**Figure 3.** Second derivative spectra of CPC delphinidin fraction of *H. sabdariffa* ethanol extract in the range of 1850–900 cm−1. There are 10 base peaks which are compatible when matching the peaks from fraction and the pure compound. The correlation between the fraction and the compound is 0.52. The base peak of the fraction within 1807 and 1643 cm−1 does not appear in the spectrum of delphinidin-3-*O*-sambubioside, since these could be the natural substances of *H. sabdariffa*.

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 *Hibiscus sabdariffa* L ethanol fraction purified by HPLC preparative in the range of 1850–900 cm−1.

### **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 *H. sabdariffa* raw material spectrum showed 20% dissimilarity with *H. sabdariffa* ethanol crude extract when both spectral relatively correlated with spectrum of residue. The fraction of *H. sabdariffa* spectrum showed content of anthocyanins at the peak 1071 cm−1 and the pattern is completely different from the anthocyanin pure compound. Hence, the spectrum of material can be used to estimate the quality of extraction and purification. Assignment of peak in spectrum is an alternative method for identification of the main compounds in the fraction isolated from HPLC preparative. The spectrum of fraction has the higher percentage of similarity with pure compound compared with extract. The quality of extraction from the raw material and compound can be clearly discerned monitored by spectroscopy.

### *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.

**Figure 4.** FTIR spectra of *H. sabdariffa* sample material in the range of 1850–400 cm−1 from different level of extraction (a) *H. sabdariffa* raw material. The whole spectrum is divided into three areas and represented most of the primary metabolism. The first three peaks started from peak 1790 cm−1 and is unique for this plant. Peak 1741 cm−1, assigned for C-O bonding, normally refers to ester components, while peak 1630 cm−1 refers to amide I. There is no amide II bonding, since the range from 1500 to 400 cm−1 consisted of the bonding for fatty acid and carbohydrate. (b) *H. sabdariffa* ethanol extract. The first three peaks are still maintained except peaks in the range of 1500–1000 cm−1 are replaced by few new peaks. Intensity of peak 1632 cm−1 is reduced and peak 956 cm−1 and 863 cm−1 appear and are not found in raw material spectrum. (c) *H. sabdariffa* Centrifugal Partition Chromatography (CPC) delphinidin fraction. The curve between the peak 1214 cm−1 and 1098 cm−1 is getting wider. The intensity of peak 1640 cm-1 and 1037 cm−1 is getting lower compared to the raw. But the peak 1073 cm−1 and 1037 cm−1 which matched with delphenidin-3-*O*-sambubioside are sharp in appearance. (d) Delphenidin-3-O-sambubioside pure compound. There are only three peaks that matched with *H. sabdariffa* fraction and two with *H. sabdariffa* ethanol extract. (e) The *H. sabdariffa* residue. The peak 1640 cm−1 is lost and 89% of the spectrum correlated with raw, 69% correlated with extract, and 47% correlated with fraction. Only 11% of the content is extracted from the raw material.

### *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*

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

**Figure 3** showed the second derivative spectrum of *Hibiscus sabdariffa* L ethanol fraction puri-

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 *H. sabdariffa* raw material spectrum showed 20% dissimilarity with *H. sabdariffa* ethanol crude extract when both spectral relatively correlated with spectrum of residue. The fraction of *H. sabdariffa* spectrum showed content of anthocyanins at the peak 1071 cm−1 and the pattern is completely different from the anthocyanin pure compound. Hence, the spectrum of material can be used to estimate the quality of extraction and purification. Assignment of peak in spectrum is an alternative method for identification of the main compounds in the fraction isolated from HPLC preparative. The spectrum of fraction has the higher percentage of similarity with pure compound compared with extract. The quality of extraction from the raw material and compound can be clearly discerned monitored

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

**3.1. Important usage of FTIR and 2DIR correlation spectroscopy in fractions**

based on the condition of the fraction.

180 Fourier Transforms - High-tech Application and Current Trends

by spectroscopy.

spectrum.

*3.1.2. Detection of enriched compound*

fied by HPLC preparative in the range of 1850–900 cm−1.

*3.1.1. Investigation of the quality of extraction and isolation*

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 f or comparison. **Figure 5** showed the example of determination of halal food with 2DIR.

**Figure 5.** Comparison of bovine gelatin and halal gelatin using 2DIR. (a) Synchronous spectrum of bovine gelatin in the range of 1800–600 cm−1. The higher intensity in the area of 1100–1000 cm−1 compared to spectrum of halal gelatin. (b) Synchronous spectrum of halal gelatin in the range of 1800–600 cm−1. (c) Asynchronous spectrum of bovine gelatin in the range 1800–1400 cm−1. The differences with halal spectrum are the intensity of the crosspeaks at (1480, 1625), (1572, 1733) and (1620, 1733) is more intense. (d) Asynchronous spectrum of halal gelatin in the range of 1800–1400 cm−1. (e) The autopeak spectrum of bovine gelatin in the range of 1800–600 cm−1. (f) The autopeak spectrum of halal gelatin in the range of 1800–600 cm−1.

### *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.
