Raman Spectroscopy

Fourier transform Raman spectroscopy has been described as a successful non-destructive technique employed in analytical investigations of nat-ural products (Edwards et al., 2005a,b). Raman spectroscopy is a very promising tool in analytical chemistry as many samples can be examined

non-destructively in a short time with no sample preparation. Moreover, Raman spectra exhibit well-resolved bands of fundamental vibrational transitions, thus providing a high content of molecular structural in-formation (Muik et al., 2003b; Table 7.4). In combination with chemo-metric data evaluation, Raman spectrometry is a powerful tool capable of extracting quantitative chemical information from complex matrix (Himmelsbach et al., 2001; Schulz et al., 2002). Recent advances in in-strumentation technology have contributed decisively to a rapid increase in the industrial applications of Raman spectroscopy (Chalmers and Dent, 1997; Cooper, 1999).

Raman spectra of raw materials and solvent extracts of Cacao seeds were recorded using the macroscopic mode with a specimen footprint of about 100 mm using a Bruker IFS 66 instrument with an FRA 106 Raman module attachment and an Nd³⁺/YAG laser operating at 1064 nm in the near infrared and an InGaAs detector cooled with liquid nitrogen. The spectrum consisted of several C-H stretching vibrations at 3000–2850 cm^–1 and some sharp bands that could be considered as key features of the kernel and fat matrix, including C=O stretching bands at 1743 and 1730 cm^–1 (shoulder), (C=C) + (C=O) stretching at 1659 cm^–1 and a broad unre-solved aromatic C-CH quadrant stretching mode feature centred at 1609 cm^–1. The identification of biomarker bands for theobromine, a member of the caffeine group of alkaloids, was done successfully.

Raman spectroscopy was used to investigate polyacetylenes in American ginseng roots (Baranska et al., 2006). The Raman spectra taken in situ from fresh ginseng root revealed a characteristic polyacetylene band at 2237 nm^–1, whereas in the spectrum obtained from the dried root, this band was shifted to about 2258 nm^–1. The data obtained had a good agreement with the spectra obtained using isolated standards (falcarinol 2258 nm^–1 and panaxydol 2260 nm^–1). Further, application of the Raman mapping technique to ginseng roots showed that the content of both main polyacet-ylenes decreased with increased root size. This finding was confirmed by HPLC analysis.

Raman spectroscopic studies of Guarana, an important product of the Amazon rainforest, was investigated using Raman spectroscopy (Edwards et al., 2005a). The therapeutic properties of guarana and its extracts have been attributed to guaranine, which could be a complex of caffeine and tannins, or to a xanthine natural product. Comparison of the Raman spectra of pure and anhydrous caffeine and of guarana methanolic extracts showed that the extracts of guarana contained anhydrous caf-feine, confirmed by the presence of the doublet at 1698 and 1654 cm^–1. Also, the composition of guarana was very similar for the whole seed and for the outer and inner portions of the dissected seed. The above findings indicated that Fourier transform Raman spectroscopy could be used to monitor quality control of guarana products in the phytopharmaceutical industry.

Raman spectrometry has been applied sucessfully in determining the total unsaturation in oils, the classification of oil and fat and for the

detection of adulterants in virgin olive oil (Sadeghi-Jorabchi et al., 1990;

Baeten and Meurens, 1996; Marigheto et al., 1998; Barthus and Poppi, 2001). Fourier transform Raman spectroscopy in combination with PLS regression was used for the direct, reagent-free determination of free fatty acid content in olive oil and olives (Muik et al., 2003a). Oils were inves-tigated directly in a simple flow cell. Milled olives were measured in a dedicated sample cup, which was rotated eccentrically to the horizontal laser beam during spectrum acquisition to compensate sample heterogen-eity. The samples were illuminated by a Nd:YAG laser line at 1064 nm with a power of 500 mW using a focused laser beam. Both external and internal (leave-one-out) validation was used to assess the ability of PLS calibration models to predict the free fatty acid (FFA) content (in terms of oleic acid) in oil and olives in the range 0.24–6.14 and 0.15–3.79%, re-spectively. The root mean square error of prediction (RMSEP) was 0.29%

for oil and 0.26% for olives. Ninety per cent of the oil samples and 80% of the olives were classified correctly.

7.4 Biosensors

Biosensors have also found application in phytochemical analysis.

Biosensors consist of a biological element that has a direct contact with a transducer. Biological components are immobilized on the surface of the biological recognition element, where they react with the target com-pounds. The transducers convert a particular biochemical phenomenon such as electrochemical, mass, optical or temperature change into elec-trical signals (Angelova et al., 2008). A superoxide dismutase biosensor has been developed to evaluate the antioxidant capacity of ginseng tea (Campanella et al., 2004). An optical biosensor based on optical fibres and laser technology has been developed to distinguish ginseng from sawdust (Yap et al., 2005). Spectroscopic analysis was performed directly on pow-dered root samples. It was found that a PCA of the fingerprint region be-tween 2000 and 600 cm^–1 could be applied to distinguish between ginseng and sawdust, as well as between ginsengs.

7.5 Conclusion

NIRS belongs to indirect or secondary methods. To obtain information, sample spectra have to be compared to the primary or reference methods.

Mathematical means are applied to spectra to establish an NIR predictive model to predict the composition of the investigated samples.

NIRS technology is developing and improving continuously. Its wider application for the quantitative and qualitative analysis of natural products will certainly improve their quality control and safety in clinical use. The further development of NIRS will serve to strengthen quality monitoring and control of natural products and regulate the market. Both solid and liquid

samples in different types of packaging can be tested because of the better penetrability of the fibre optics used in NIRS (Xing and Zhang, 2010). NIRS is a full-spectrum method in constrast to chromatographic or electrophoretic analytical methods, which focus only on the separation and detection of cer-tain analytes. Further, the NIRS method not only allows offline but also online or even in-line analysis. Even though the costs of NIRS equipment are high and calibration needs a lot of time, NIRS has the great advantage of reducing time and costs, especially in combination with the fast reference method.

Since, in principle, NIRS measurements can also be performed on fresh plant material, it is possible to use this method to predict the op-timal time to harvest or for the selection of individual genotypes directly in field. Continuing developments in diode array instruments are ex-pected to extend the potential of NIR for on-site determination to various plant constituents.

Non-destructive techniques could be utilized as a control procedure or as an alternative rapid and effective quantification method. These ana-lytical tools perform as well as established conventional methods, while reducing the cost of analysis in terms of chemical reagent use, which is more environmental friendly. The following points are worthy to mention for the proper application of NIRS:

1. Prior to NIRS analysis, reference methods such as GC, HPLC, etc., have to be used. HPLC has to be used in order to build a reference data set, as the NIRS method was developed using a statistical comparison of the re-sults obtained from the reference data.

2. Samples are divided randomly into two groups: calibration set