Phytochemicals are naturally occurring substances present in plants. Often they are involved in the plants defense mechanism and they can be produced through primary or secondary metabolism. Among the primary plant components there are proteins, amino acids, sugars and chlorophyll while secondary constituents are alkaloids, phenolic compounds, flavonoids, saponin, tannins and many more. [1]
Phytochemicals generally possess a biological activity and have been used and studied since ancient times for their potential health benefits or intoxicating action.
The medicinal power of traditional plant species lies in the phytochemical content producing a specific pharmacological action. [1]
For this reason the extraction, isolation and qualitative and quantitative analysis of the phytochemical content of a plant is necessary in order to reveal the unique plant composition thus the specific phytochemical properties.
The heterogeneity of plant samples and the difficulties linked to the sample preparation can be the hurdles to overcome when analyzing a plant biomass.
The initial extraction of a plant gives generally a crude extract, that is an unrefined mixture of plant components. In order to reveal the plant composition and isolate each compounds from this crude mixture it is possible to employ chromatographic methods including:
- Flash chromatography;
- High-performance liquid chromatography (HPLC);
- Gas chromatography (GC).
The goal of chromatography is the separation of compounds based on polarity, size or chemical functionalization. [2] Once “fractions” of the crude extract are obtained is possible to proceed in their analysis through spectroscopic techniques including:
- Mass spectrometry (MS);
- Nuclear magnetic resonance (NMR);
- Ultraviolet-visible spectroscopy (UV-Vis).
It should be noticed that nowadays hyphenated or hybrid techniques are often used in plant composition analysis. Analytical methods such as chromatographic techniques and spectroscopic or spectrometric methods can be paired in order to analyze samples and exploit the advantages of both methods for qualitative and quantitative sample analysis. [3]
Chromatographic methods
Chromatography can be preparative or analytical. The preparative chromatography is a form of purification in which the crude extract is separated in fractions. This method usually involves large amounts of solvents and it is generally laborious and expensive. Analytical chromatography is generally performed with small amounts of samples and solvents and the purpose is to reveal the presence and the relative proportions of the analytes in a mixture.
Flash chromatography is an example of preparative chromatography while HPLC can be used for both preparative or analytical purposes.
The general principle of this technique involves the separation of analytes from a mixture by dissolving the crude extract in a liquid or gaseous mobile phase that is pumped or flushed through a stationary phase. Depending on the nature of the analytes and the kind of mobile and stationary phases the mixture will separate through the chromatographic column and ideally each component will be eluted in some fractions. In the case of liquid chromatography, before running a column, the behavior of the chemical entities of the mixture is checked through thin layer chromatography (TLC). This permits us to have an idea of the way in which compounds will run in the column and to refine the ratio of the mobile phase solvents in terms of polarity, to elute the desired compounds in an efficient way.
High Performance Liquid Chromatography (HPLC)
This chromatographic method involves the use of small size silica particles (2-50 μm) packed in a column. In case it is used for qualitative and quantitative analysis of botanicals, the process involves first the measurement of a small amount of reference standards of specific analytes of interest. The mobile phase, with specific ratios of solvents, is pumped through the column producing a chromatogram. The fractions or the crude extract is then analyzed and each peak of the chromatogram is compared with the peaks of the reference standards both in terms of elution time for the qualitative analysis and the peak area for the analyte quantification.
Each peak of the chromatogram represents a compound or groups of compounds with a similar characteristic (polarity, size, functionalization etc.) present in the crude extract.
If HPLC is used with the appropriate program\solvents ratio permits to obtain peaks with good resolution meaning that it is possible to discriminate between data of an analyte from another one. The peak resolution is the relative distance between the apexes of two neighboring peaks.
The separation efficiency in the column is measured by the width at half height of the peak: the thinner the peak, the more efficient the column.
Analytical HPLC systems are used in the food safety, manufacturing and pharmaceutical industries and also in the medical research laboratories.
Gas chromatography (GC)
This kind of chromatography can be used to find out how many compounds are present in a mixture. In the case of plant extracts, GC is a suitable and efficient method to quantitatively determine the amount of bioactive components. This analytical method is mainly used to analyze and quantify volatile compounds. In particular the most reliable GC analyses are performed on small molecular weight non-polar volatiles.
In any case GC is generally used on compounds that can be vaporized without decomposition. In fact this method is not reliable on compounds that degrade with the heat.
In order to perform the quantitative analysis of a sample the analyst has to measure the sample size injected, determine the response of the instrument and measure the peak areas of the gas chromatogram.
The target compounds that are heated and vaporized during the injection of the sample are transported to the column by the carrier gas. Commonly used carrier gasses are helium, nitrogen and hydrogen.
In the column the mixture is separated into the various components and the amount of each analyte is measured by the detector.
The detector turns the amount of compounds into electrical signals that can be then sent to a data processing unit and reported into a chromatogram.
The horizontal axis of the chromatogram indicates the time at which the compound reaches the detector while the vertical axis indicates the signal intensity. The retention time is specific for each compound and depends on many factors including:
- type of column;
- analysis conditions;
- contaminations;
- type of compounds analyzed;
- temperature;
- carrier gas;
- column pressure.
Spectroscopic techniques
These techniques are frequently employed to produce highly detailed information regarding the molecular structure and properties of the analyte of interest. In fact, through spectroscopic methods based on the study of the quantized interaction of electromagnetic radiations with matter, it is possible to elucidate bond length and bond angles. [4]
There are various forms of electromagnetic radiation, including light (visible), ultraviolet (UV), infrared (IR), X-rays, microwaves, radio waves, cosmic rays etc.
Depending on the intended purpose, the wavelength region used and the type of the material, different spectroscopic techniques exist.
Mass spectrometry (MS)
The mass-to-charge ratio (m/z) of charged molecules can be measured using this analytical technique. The main principle of MS consists in generating ions or molecular fragments through electron bombardment (electronic ionization) and to measure the quantity of the matter related to the electric charge of a given particle.
In order to produce ionized particles various methods can be used including:
- Electronic ionization (EI);
- Chemical ionization (CI);
- Electrospray ionization (ESI);
- Atmospheric pressure chemical ionization (APCI);
- Atmospheric solid analysis probe ionization (ASAP);
- Atmospheric pressure photoionization (APPI);
- Matrix-assisted laser desorption/ionization (MALDI).
Among the specific applications of MS technique there are: drug testing, drug discovery, pesticide residue analysis, protein identification, food contamination detection, isotope ratio determination, etc.
With the combination of MS, computer technology and software, the range of MS applications is always more extensive.
Nuclear magnetic resonance (NMR)
In the case of NMR radio frequency waves are used to promote transitions between different nuclear energy levels (resonance). [5]
This spectroscopic technique has been employed on solids, gas, liquids to perform kinetic and structural studies.
The molecule or the mixture can be analyzed by placing it in a strong magnetic field. This allows the nuclear spins to be visible and measurable, elucidating details regarding the molecular structure and conformation.
In NMR spectroscopy the number of signals describes the different environment in which the hydrogens atoms are in. In order to reveal the number of atoms in each molecular environment it is possible to do the peaks area ratio.
Ultraviolet-visible spectroscopy (UV-Vis)
The amount of absorbed or transmitted discrete UV-wavelengths of visible light can be measured by a UV-Vis spectrophotometer. The measurement involves the sample comparison to a reference also called “blank” that is usually the solvent made to prepare the sample. The amount of absorbed or transmitted light is dependent on the sample composition and can give a hint about the presence or not of analytes in the sample. Moreover the UV-Vis measurement can also give an indication of the sample concentration.
The info is collected in an absorbance graph as a function of the wavelength.
This spectroscopic method finds many uses including: DNA and RNA analysis, pharmaceutical analysis, bacterial culture, food and beverage analysis, etc. [6]
Combined techniques
Many instruments combine two or more techniques in order to provide more detailed and reliable analysis results. Among the most widely used analytical hybrid techniques there are:
- gas chromatography-mass spectrometry (GC-MS);
- liquid chromatography-mass spectrometry (LC-MS);
- liquid chromatography-nuclear magnetic resonance spectroscopy (LC-NMR);
- gas chromatography-infrared spectroscopy (GC-IR).
The separation capability of chromatographic methods is usually combined with Mass Spectrometry (MS).
The main advantage of HPLC combined with MS (HPLC-MS) and GC-MS is that the mixture can be separated and then the individual components can be identified using MS.
When compounds reach the MS sensor they are fragmented into ions by electron impact ionization. Each fragment is reported in the mass spectrum. The peculiar fragment formation helps define the structure of the molecule of interest.
If the chromatographic techniques are combined with NMR, the components are first separated into single compounds or less complex mixtures and then analyzed exploiting their magnetic properties.
Depending on the chemical environment each atom will vibrate differently giving microscopic chemical and physical information. As the signals are unique or highly characteristic to individual compounds, NMR is considered a definitive technique to identify organic compounds.
Using IR techniques when performing chromatographic analysis permits the visualization of results almost in real time. The principle is the different frequencies absorbed by molecules in order to deduce their structure. The different IR signals permit the characterization of functional groups and help elucidate the molecular structure of the analyte.
These hybrid techniques are widely used in research laboratories, medical, environmental and pharmaceutical industries.
References:
[1] Agidew, M.G. Phytochemical analysis of some selected traditional medicinal plants in Ethiopia. Bull Natl Res Cent 46, 87 (2022). https://doi.org/10.1186/s42269-022-00770-8
[2] Albuquerque, Ulysses Paulino; de Lucena, Reinaldo Farias Paiva; Cruz da Cunha, Luiz Vital Fernandes; Alves, Rômulo Romeu Nóbrega (2019). [Springer Protocols Handbooks] Methods and Techniques in Ethnobiology and Ethnoecology || Methods in the Extraction and Chemical Analysis of Medicinal Plants. , 10.1007/978-1-4939-8919-5(Chapter 17), 257–283.doi:10.1007/978-1-4939-8919-5_17
[3] Patel KN, Patel JK, Patel MP, Rajput GC, Patel HA. Introduction to hyphenated techniques and their applications in pharmacy. Pharm Methods. 2010 Oct;1(1):2-13. doi: 10.4103/2229-4708.72222. PMID: 23781411; PMCID: PMC3658024.
[4] Douglas J. Henderson, Charles T. Rettner, inEncyclopedia of Physical Science and Technology (Third Edition), 2003
[5]https://chem.libretexts.org/Bookshelves/Physical_and_Theoretical_Chemistry_Textbook_Maps/Supplemental_Modules_(Physical_and_Theoretical_Chemistry)/Spectroscopy/Magnetic_Resonance_Spectroscopies/Nuclear_Magnetic_Resonance/Nuclear_Magnetic_Resonance_II
[6] https://www.technologynetworks.com/analysis/articles/uv-vis-spectroscopy-principle-strengths-and-limitations-and-applications-349865
