Spectroscopy in pharmacognosy is an essential process that helps identify and quantify the active compounds in an extract.
The basis of spectroscopy is the interaction of matter with radiation. This interaction causes emittance or absorbance of energy discretely in the form of quanta. The matter could redirect the radiation or cause a transition of energy levels. This transmission can be emission, absorption, or scattering.
Isolated and purified plant compounds can be identified based on their spectroscopic characteristics. Different spectroscopic methods are used for qualitative and qualitative study of atoms, molecules, or physical properties of a compound.
Types of Spectroscopy in Pharmacognosy
Although different types of spectroscopic methods exist, only four are used for phytopharmaceutical analysis. They are,
- Ultraviolet (UV) and Visible Absorption Spectroscopy
- Infrared (IR) Spectroscopy
- NMR or Nuclear Magnetic Resonance Spectroscopy
- Mass Spectrometry (MS)
1. Ultraviolet-Visible Absorption Spectroscopy
Many organic molecules have specific functional groups or chromophores that have low-energy valence electrons that can absorb UV-visible radiation at various wavelengths. The absorption spectrum of such molecules will reveal absorption bands, which refer to those specific functional groups.
Solvents are used to solubilize the compounds to make the process easier. 95% ethanol is the most commonly used solvent. Other solvents include methanol, water, hexane, petroleum, ether, etc.
A detector such as a Photo Diode Array or PDA is used to study the absorption and identify the functional group. When the PDA is connected to the HPLC (High-Performance Liquid Chromatography) system, it helps determine the purity of the compound having different chromatophores. It is the detection wavelength that determines the nature of the chromatophore.
Compounds that have chromatophores have a wavelength between 200-700 nm on the visible spectrum while those lack chromatophores or such functional groups are detected at 200-400 nm. The value of these spectra is related to the relative complexity of the spectrum and the position of wavelength maxima.
2. Infrared Spectroscopy or IR Spectroscopy
Infrared spectroscopy uses the ability of a compound to absorb infrared radiation from the sunlight. This ability is restricted to those compounds that have a slight difference in their energy levels in their rotational and vibrational states.
The molecule can absorb IR radiation when these rotations and vibrations cause a dipole net change. This happens when the alternating electric field in the radiation matches the fluctuations of the dipole moment. The IR absorption causes an amplitude change in the vibrations. This change is detected by the spectrometer.
Each functional group has a specific vibration frequency by which it is identified. The plant compound is used in its liquid or solid state. They are prepared using chloroform and potassium bromide respectively. The absorption is measured in wavenumbers having a unit of cm-1.
3. NMR Spectroscopy
Nuclear Magnetic Resonance Spectroscopy is a powerful analytical tool that helps get valuable information about the structure of the atom in a molecule, identify the content, and also find its purity in the sample used. Proton NMR Spectroscopy is the most widely used tool.
NMR Spectroscopy works on the principle that every nuclei is electrically charged and many of them have a spin on its axis. When a strong magnetic field is applied on these nuclei, there could be a transfer of energy from the base to a higher energy level. This transfer of energy when coincides with the frequency applied, that reading is detected to determine the value.
At the same time, the energy is also emitted at a specific frequency when it comes back to the base level. It is the electrons, protons, and neutrons that are spinning which can be imaged.
Molecules of H-1 and C-13 have spinning in their nuclei. When such a sample is placed in an inert solvent between strong magnetic poles, the atoms absorb radiation to move from the base energy level to a higher level.
At different frequency levels, the absorption of energy will also be different. The NMR spectrum is taken by plotting the energy absorbed versus the frequency of radiation applied.
According to the atom targetted, NMR can be H NMR or C-13 NMR. Modern NMR techniques include Correlated Spectroscopy (COSY), HMBC – Heteronucelar Multiple Bond Correlation, HSQC – Heteronuclear Single Quantum Coherence, NOESY – Nuclear Overhaucer Enhancement Spectroscopy, TOCSY – Total Correlated Spectroscopy, etc.
4. Mass Spectroscopy
Mass spectroscopy helps determine the molecular weight of compounds in a sample in any physical form. Here, the sample will be subjected to ionization, ion detection, mass analysis, etc. The common ionization methods used are,
- Chemical ionization
- Electron Impact Ionization
- Desorption Ionization
Samples in liquid or solid states are volatilized before or during ionization. Charged molecules in a gaseous state are generated by FAB- Fast Atom Bombardment, PD – Plasma Desorption, thermospray, and particle beam methods.
Ions are accelerated inside and are separated. They are detected as per their mass per charge. This data will help generate the chemical structure possible.
Gas Chromatography-Mass Spectroscopy, Liquid Chromatography-Mass Spectroscopy, capillary electrophoresis mass spectroscopy, etc., help identify the molecule.
References
- A Review on Spectroscopic Analysis of Phytopharmaceuticals
- Shah, Biren N, Avinash Seth. (2010). Textbook of Pharmacognosy and Phytochemistry. Elsevier.
Additional Reading
- Classification of Crude Drugs in Pharmacognosy
- Types of Chromatographic Techniques & Applications
