8.5
Analysis of Phytochemicals
Purification of the active compound is the key step in analysing the phytochemicals
(Obouayeba et al. 2015; Thakor et al. 2016). Column chromatography and thin layer
chromatography are the most widely used purification technique. Chromatography is
used to separate the active components based on polarity of stationary phases like
silica, alumina, cellulose, and polyamide (Coskun 2016). In both chromatography
techniques, the analytes are separated based on the amount of affinity between two
phases. The mobile phase carries the solution to be purified. After purifying the
bioactive compounds, it is subjected to structural clarification using spectroscopic
techniques viz., mass spectroscopy, infrared spectroscopy, UV visible spectroscopy,
and nuclear magnetic resonance (Dias et al. 2016).
8.5.1 IR Spectroscopy
Infrared light is an electromagnetic radiation which has a wavelength longer than
visible light (0.7–1000 μm). Usually tungsten bulbs were used to produce infrared
light and analytical applications are confined to the middle infrared range (3–8 μm),
as the absorption of organic molecules is high in this region (Schulz and Baranska
2007; Baker et al. 2014). The light source is focused using a concave mirror to the
sample area and the amount of light which pass through the sample is detected using
thermocouple, pyroelectric, or photo-conducting detectors. Frequencies of infrared
light absorbed are detected by the detector and plotted on a chart (frequency on Xaxis and intensity of absorption on Y-axis).
8.5.2 UV Visible Spectroscopy
UV visible spectrophotometer measures the absorbance based on the transmittance
by the light source (UV range: 185–400 nm, visible range: 400–800 nm) when it
passed through a sample. It is calculated using Beer Lambert’s law which states that
absorbance is equal to the multiplication of absorbance constant, path length, and
sample concentration (Priya et al. 2012). It uses deuterium arc lamp (190–420 nm),
tungsten lamp (350–2500 nm), or xenon lamp (180–900 nm) as light source and it is
focused to a monochromator using mirror or lens. The monochromatic light passes
through the sample in the absorption cell (quartz, fused silica, or glass) and gets
detected using photodiode or photo multiplier tube detector (Banu and Cathrine
2015; Altemimi et al. 2017).
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L. Manickam et al.
Analysis of Phytochemicals
Purification of the active compound is the key step in analysing the phytochemicals
(Obouayeba et al. 2015; Thakor et al. 2016). Column chromatography and thin layer
chromatography are the most widely used purification technique. Chromatography is
used to separate the active components based on polarity of stationary phases like
silica, alumina, cellulose, and polyamide (Coskun 2016). In both chromatography
techniques, the analytes are separated based on the amount of affinity between two
phases. The mobile phase carries the solution to be purified. After purifying the
bioactive compounds, it is subjected to structural clarification using spectroscopic
techniques viz., mass spectroscopy, infrared spectroscopy, UV visible spectroscopy,
and nuclear magnetic resonance (Dias et al. 2016).
8.5.1 IR Spectroscopy
Infrared light is an electromagnetic radiation which has a wavelength longer than
visible light (0.7–1000 μm). Usually tungsten bulbs were used to produce infrared
light and analytical applications are confined to the middle infrared range (3–8 μm),
as the absorption of organic molecules is high in this region (Schulz and Baranska
2007; Baker et al. 2014). The light source is focused using a concave mirror to the
sample area and the amount of light which pass through the sample is detected using
thermocouple, pyroelectric, or photo-conducting detectors. Frequencies of infrared
light absorbed are detected by the detector and plotted on a chart (frequency on Xaxis and intensity of absorption on Y-axis).
8.5.2 UV Visible Spectroscopy
UV visible spectrophotometer measures the absorbance based on the transmittance
by the light source (UV range: 185–400 nm, visible range: 400–800 nm) when it
passed through a sample. It is calculated using Beer Lambert’s law which states that
absorbance is equal to the multiplication of absorbance constant, path length, and
sample concentration (Priya et al. 2012). It uses deuterium arc lamp (190–420 nm),
tungsten lamp (350–2500 nm), or xenon lamp (180–900 nm) as light source and it is
focused to a monochromator using mirror or lens. The monochromatic light passes
through the sample in the absorption cell (quartz, fused silica, or glass) and gets
detected using photodiode or photo multiplier tube detector (Banu and Cathrine
2015; Altemimi et al. 2017).
178
L. Manickam et al.