Table of Contents
Chromatography Definition
Chromatography is a key biophysical method for separating, identifying, and purifying the components of a mixture for qualitative and quantitative investigation.
- Differentiations in size, binding affinities, charge, and other attributes are used in a variety of chromatographic processes to separate materials.
- It is a potent separation method utilised in many scientific fields, and it is sometimes the only way to separate components from complicated mixtures.
- The ability to separate components of a mixture according to their nature, structure, size, and other characteristics makes chromatography an extremely valuable technology.
- Chromatography, in general, is founded on the idea that parts of a mixture are separated when the mixture added to a mobile phase is passed through a stationary phase, which is typically a solid surface and causes certain parts of the mixture to be attached to it. The mobile phase is passed together with the rest at the same time.
- There are therefore two key elements shared by all chromatography procedures.
What is a stationary phase?
In chromatography, the stationary phase is a solid or liquid particle that is affixed to a glass or metal surface and selectively absorbs the components of the mixture to be separated.
- Since this phase is stationary while the other phase is in motion, it is referred to as stationary.
- The majority of materials used as stationary phases include pores, which enable components to adhere during chromatography.
- Depending on the kind of chromatography being used and the characteristics of the components that need to be separated, a stationary phase must be chosen.
- The stationary phase can be made of gel beads, thin uniform paper, silica, glass, certain gases, or even liquid components, depending on the kind of chromatography being utilised.
What is the mobile phase?
The liquid or gaseous portion of the chromatographic system through which the mixture's components are separated at various rates by adsorbing to the stationary phase is known as the mobile phase.
- The solvent that transports the mixture down the stationary phase is known as the mobile phase.
- When a phase is referred to as mobile, it means that it is moving down the chromatographic system while the other phase is still stationary.
- Depending on the kind of chromatography being employed and the characteristics of the components that need to be separated, materials used as mobile phases are chosen.
- In many chromatographic procedures, the mobile phase is frequently utilised to be alcohol, water, acetic acid, acetone, or certain gases.
Types of Chromatography
1. Affinity chromatography
According to their affinity for the system's stationary phase, the components of a mixture are separated using the separation process known as affinity chromatography.
Principle of Affinity chromatography
- Based on the idea that components of a mixture are separated when an element with an affinity for the stationary phase attaches to the stationary phase, this chromatography method was developed. Other substances, on the other hand, are eluted with the mobile phase.
- The reactive sites for component binding are made visible because the substrate/ligand is attached to the stationary phase.
- The components having substrate binding sites now bond to the substrate on the stationary phase when the mixture is passed through the mobile phase, and the remaining components are rinsed out with the mobile phase.
- The stationary phase's connected components are then released by altering the pH, ionic strength, or other factors.
Steps of Affinity chromatography
- The substrate/ligand with the spacer arm is bonded to the solid support, such as agarose or cellulose, which has been used to construct the column.
- A steady stream of the mixture-containing mobile phase is added to the column.
- Once the procedure is finished, the ligand-molecule complex is separated from the mixture's components and eluted from the stationary phase by altering the conditions that encourage this separation.
Uses of Affinity chromatography
- The standard separation method for enzymes and other proteins is affinity chromatography.
- The in vitro antigen-antibody responses also use this theory.
- This method is employed to separate components from mixtures as well as to purge them of contaminants.
- The identification of mutations and nucleotide polymorphisms in nucleic acids can be done using affinity chromatography.
Examples of Affinity chromatography
- The purification of coli β-galactosidase from a mixture of proteins using the p-aminophenyl-1-thio-β-D-galactopyranosyl agarose as the affinity matrix.
- The removal of excess albumin and α2-macroglobulin from the serum albumin.
2. Anion exchange chromatography
Anion exchange chromatography is the separation technique for negatively charged molecules by their interaction with the positively charged stationary phase in the form of ion-exchange resin.
Principle of Anion exchange chromatography
- This method is based on the idea that positively charged resin and negatively charged analyte would attract to one another. Here, negatively charged molecules are eliminated by the exchange of positively charged ions.
- The components in the mixture with negative charges will bond to the stationary phase, which is first coated with positive charges.
- The negatively charged components are then bound by an anion exchange resin that has a stronger affinity for them, dislodging the positively charged resin.
- The complex made up of the components of the anion exchange resin is then eliminated using various buffers.
Steps of Anion exchange chromatography
- The stationary phase is a column filled with resin that is positively charged.
- The combination of charged particles is then sent down the column where the positively and negatively charged resins attach to one another.
- The negatively charged molecules now bond to the anion exchange resin, displace the positively charged resin, and the anion exchange resin is then transported through the column.
- In order to separate the complex of anion exchange resins and the charged molecules, a suitable buffer is now added to the column.
Uses of Anion exchange chromatography
- Proteins and amino acids are separated from their mixes using anion exchange chromatography.
- It is possible to isolate negatively charged nucleic acids, which aids in subsequent nucleic acid analysis.
- This process may also be applied to the hydroxyl ion exchange in water treatment.
- Metal separation can be accomplished using anion exchange resins because these materials frequently include complexes that are negatively charged and bind to the anion exchangers.
Examples of Anion exchange chromatography
- the process of separating nucleic acids from a mixture that is produced following cell death.
- the process of separating proteins from a blood serum-derived crude mixture.
3. Cation exchange chromatography
Anion exchange chromatography is the separation technique for positively charged molecules by their interaction with negatively charged stationary phase in the form of ion-exchange resin.
Principle of Cation exchange chromatography
- This method is based on the idea that negatively charged resin and positively charged analyte would attract one another. Here, positively charged molecules are eliminated by the exchange of negatively charged ions.
- The components of the combination with positive charges are initially covered with negative charges on the stationary phase, where they will bond.
- The positively charged components are then bound by a cation exchange resin that has a stronger affinity for them, dislodging the negatively charged resin.
- The complex made up of the components of the cation exchange resin is subsequently eliminated using various buffers.
Steps of Cation exchange chromatography
- The stationary phase is a column filled with resin that is negatively charged.
- After passing the combination of charged particles down the column, positively charged molecules attach to the negatively charged resins.
- The positively charged molecules now bond to the cation exchange resin, displace the negatively charged resin, and the cation exchange resin is then transported through the column.
- In order to separate the complex of cation exchange resins and the charged molecules, the proper buffer is now added to the column.
Uses of Cation exchange chromatography
- Analysing the byproducts of the breakdown of nucleic acids is done using cation exchange chromatography.
- In order to eliminate the negatively charged complexes, the metal ions themselves might bond to the negatively charged resins while separating metals.
- By exchanging the positively charged ion for hydrogen ions, cation exchange chromatography aids in the purification of water.
- Inorganic molecules and rocks are both analysed using it.
Examples of Cation exchange chromatography
- the process of separating positively charged lanthanoid ions from the crust of the earth.
- the analysis of the presence of calcium ions to determine the total amount of salts dissolved in natural waterways.
4. Column chromatography
- In column chromatography, the components in a mixture are separated based on how differently they adhere to the stationary phase, which causes them to move at various rates when passed through a column.
- It uses a solid-liquid chromatography method where the mobile phase is a liquid or gas and the stationary phase is a solid.
Principle of Column chromatography
- This method is founded on the concept of differential adsorption, in which various molecules in a mixture have varying affinities for the absorbent present on the stationary phase.
- Higher affinity molecules stay adsorbent for a longer period of time, slowing down their passage down the column.
- The molecules may be divided into distinct fractions because the molecules with lesser affinity move more quickly.
- Here, the mobile phase is a liquid that enables molecules to flow along the column smoothly whereas the stationary phase, also known as the absorbent in column chromatography, is a solid (usually silica) in this case.
Steps of Column chromatography
- A glass tube that has been dried and covered with a thin, homogeneous coating of stationary phase (cellulose, silica) is used to create the column.
- The mixture is then poured into the mobile phase to create the sample. The sample enters the column from the top and is let to flow through the column while being affected by gravity.
- By using an elution technique, the molecules coupled to the column are separated, either by using a solution with the same polarity (isocratic approach) or by using multiple samples with various polarities (gradient technique).
- The separated molecules can then be examined further for a variety of reasons.
Uses of Column chromatography
- Column chromatography is frequently used to separate contaminants from diverse biological mixtures and purify them.
- Additionally, active compounds and metabolites from diverse substances may be isolated using this method.
- More and more labs are using column chromatography to find medicines in raw extracts.
Examples of Column chromatography
- extraction of pesticides from animal-sourced solid food samples that comprise lipids, waxes, and colours.
- To treat type 1 and type 2 diabetics, Pramlintide, a peptide hormone analogue of Amylin, was created.
- Purification of bioactive glycolipids with HSV-1 (the herpes virus) antiviral activity.
5. Flash chromatography
A separation method called flash chromatography uses smaller gel particle sizes as the stationary phase and pressurised gas to move the solvent through the column.
Principle of Flash chromatography
- Similar to column chromatography, which separates the components based on their differential adsorption on the stationary phase, flash chromatography works on a similar premise.
- A pressurised gas is used to pass the applied sample, speeding up and improving the procedure.
- According to their affinity, molecules attach to the stationary phase, and the remaining solvent is rinsed out by injecting pressurised gas to speed up the process.
- Here, the mobile phase, the elution solution, and an extra pressurised gas are all liquids while the stationary phase is solid.
Steps of Flash chromatography
- A glass tube that has been dried and covered with a thin, homogeneous coating of stationary phase (cellulose, silica) is used to create the column. To keep the gel from escaping, cotton wool is stuffed into the bottom and top of the column.
- The mixture is then poured into the mobile phase to create the sample. A pumped sample is used to pass the sample at a steady pace after being injected into the column from the top.
- Either a solution with the same polarity is used (isocratic approach) or several samples with various polarities are utilised (gradient technique) to elute the bound molecules from the column.
- A constant minimum pressure is used to apply the elution solvent in order to transport the solute down the column.
- The separated molecules can then be examined further for a variety of reasons.
Uses of Flash chromatography
- As a quicker and more effective way of separating the constituents of various mixes, flash chromatography is employed.
- It is used to clean out contaminants from unprocessed natural and manufactured mixed extracts.
6. Gas chromatography
With the use of the separation technique known as gas chromatography, molecules are separated according to their retention times, which are determined by their affinity for the stationary phase.
The sample, which is vaporised at the injection site, is either a liquid or a gas.
Principle of Gas chromatography
- The basic principle of gas chromatography is the idea that substances with a higher affinity for the stationary phase have a longer retention period because they take longer to leave the column.
- However, when they move with the mobile phase, the components with a higher affinity for the stationary phase have a shorter retention duration.
- A gas, often helium, known as the mobile phase is what moves the sample through the column.
- Once the sample has been injected, it is converted to the vapour stage, and the retention time is then measured by passing the sample through a detector.
- As the components exit the stationary phase at various periods, they are collected individually.
Steps of Gas chromatography
- After being injected into the column, the sample is vaporised and transformed into a gas. The component that has been vaporised then combines with the mobile phase to move through the remainder of the column.
- The stationary phase is placed in the column, and the molecules are separated according to their affinities to the stationary phase.
- Due to variations in how long each component is maintained in the column, the mixture's components arrive to the detector at different times.
Uses of Gas chromatography
- Calculating the concentration of various compounds in various samples is done using this method.
- Oil spills, air pollution, and other samples are all analysed using this.
- The identification and measurement of numerous biological substances discovered at the crime scene may also be done using gas chromatography in forensic science.
Examples of Gas chromatography
- the detection of performance-enhancing substance in an athlete's urine.
- the identification and measurement of a solid medicine in water and soil samples.
7. Gel filtration chromatography/ Gel permeation chromatography/ Size exclusion chromatography/ Molecular sieve chromatography
A type of partition chromatography used to separate molecules with various molecular sizes is gel-filtration chromatography.
Numerous other names, such as gel-permeation, gel-exclusion, size-exclusion, and molecular-sieve chromatography, have also been commonly used to refer to this method.
Principle
- According to their respective sizes, molecules are divided between a mobile phase and a stationary phase.
- A porous polymer matrix with pores of a certain size makes up the stationary phase.
- The mobile phase fills the pores of the stationary phase when it is injected into the sample.
- If the molecules are the right size to fit within the pores, they partially or completely stay there.
- However, bigger molecules are prevented from entering the pores and are instead transported out of the column by the mobile phase.
- Gel filtration chromatography is the name of the procedure when the mobile phase is utilised in an aqueous solution.
- It is known as gel permeation chromatography if an organic solvent is employed as the mobile phase.
Steps
- Semi-permeable, porous polymer gel beads with a well specified range of pore diameters are used to fill the column.
- The sample and mobile phase are then combined before being injected into the column from its top.
- Either a solution with the same polarity is used (isocratic approach) or several samples with various polarities are utilised (gradient technique) to elute the bound molecules from the column.
- You can choose elution conditions (pH, necessary ions, cofactors, protease inhibitors, etc.) that will complement the needs of the target molecule.
Uses
- The ability to accomplish separation in conditions carefully created to preserve the stability and activity of the target molecule without sacrificing resolution is one of the key benefits of gel-filtration chromatography.
- Since there is no molecule-matrix binding stage, delicate molecules are not unnecessarily harmed, making gel-filtration separations often result in high activity recoveries.
- Gel-filtration chromatography has been used successfully to separate proteins and peptides from a variety of sources because of its distinctive mechanism of separation.
- Different nucleic acid species, including DNA, RNA, and tRNA, as well as their component bases, adenine, guanine, thymine, cytosine, and uracil, have been separated using gel-filtration chromatography.
Examples
- high yield urea-gradient size-exclusion chromatography for the separation of recombinant human granulocyte colony-stimulating factor (rhG-CSF) from inclusion bodies.
- employing gel columns made of both acrylamide and dextran to separate the lysozyme from hen eggs.
8. High-performance liquid chromatography (HPLC)
In a modified version of column chromatography known as high-performance liquid chromatography, the components of a mixture are separated according to their affinity for the stationary phase.
Principle of HPLC
- This method is based on the idea of differential adsorption, in which various molecules in a mixture interact with the absorbent on the stationary phase to varied degrees.
- Higher affinity molecules stay adsorbent for a longer period of time, slowing down their passage down the column.
- The molecules may be divided into distinct fractions because the molecules with lesser affinity move more quickly.
- This procedure differs slightly from column chromatography in that the solvent is pressed under strong pressures of up to 400 atmospheres in this instance rather than being allowed to drop down naturally.
Steps of HPLC
- A glass tube that has been dried and covered with a thin, homogeneous coating of stationary phase (cellulose, silica) is used to create the column.
- The mixture is then poured into the mobile phase to create the sample. A high-pressure pump is utilised to pass the sample at a steady pace once it is inserted into the column from the top.
- The mobile phase then descends to a detector that looks for molecules at a certain wavelength of absorbance.
- The separated molecules can then be examined further for a variety of reasons.
Uses of HPLC
- Pollutants found in environmental samples are analysed using high-performance liquid chromatography.
- It is done to keep industrial productions' diverse industrial productions' quality and product purity under control.
- Additionally, various biological substances including proteins and nucleic acids may be separated using this method.
- The procedure is quicker and more efficient because to the rapid pace of this technology.
Example of HPLC
A technique called high-performance liquid chromatography has been used to evaluate how well certain antibodies protect against illnesses like Ebola.
9. Hydrophobic interaction chromatography
Hydrophobic interaction chromatography is the separation technique that separates molecules on the basis of their degree of hydrophobicity.
Principle of Hydrophobic interaction chromatography
- The principle of hydrophobic interaction chromatography is based on the interaction between two molecules with hydrophobic groups.
- The stationary phase in this instance is a solid support that has hydrophobic and hydrophilic groups added to it.
- The hydrophobic groups interact with the solvent molecules with hydrophobic regions, isolating them from the molecules with hydrophilic groups.
- By administering an elution solution with a decreasing salt gradient, the contact is then reversed, resulting in the separation of the hydrophobic molecules from the stationary phase.
Steps of Hydrophobic interaction chromatography
- The column is made by covering a glass tube with a solid support, such as silica gel, then attaching hydrophobic groups, such as phenyl and octyl butyl, to it.
- The mixture is added to the mobile phase to create the sample.
- After that, the sample is injected into the column from the top.
- The molecules with hydrophobic groups form an interaction with the hydrophobic groups of the stationary phase. The molecules lacking these groups, on the other hand, migrate out of the column with the mobile phase.
- The attached molecules are subsequently extracted from the stationary phase using a specific elution solution that has a decreasing salt gradient.
Uses of Hydrophobic interaction chromatography
- Hydrophobic interaction chromatography is extremely important for the separation of proteins with hydrophobic groups.
- As it causes the least amount of denaturation activities, this technique is more suited than other ways.
- Similar to the separation of other organic molecules having hydrophobic groups, this technique can be used.
- This makes it possible to separate biological molecules that are hydrophilic from those that are hydrophobic.
Example of Hydrophobic interaction chromatography
The separation of plant proteins from the crude extracts.
10. Ion exchange chromatography
In ion exchange chromatography, charged molecules are separated from one another by interacting with an oppositely charged stationary phase, such as ion-exchange resin.
Principle of Ion exchange chromatography
- This method is based on the idea that charged resin will attract an analyte that is negatively charged. In order to remove the charged molecules, here an interchange of negatively and positively charged ions occurs.
- The components of the mixture with opposite charges will bond when the stationary phase is initially covered with specific charges.
- The charged components are then bound by a cation or anion exchange resin that has a stronger affinity for them, replacing the oppositely charged resin.
- The complex of the cation or anion exchange resin is subsequently eliminated using various buffers.
Steps of Ion exchange chromatography
- The stationary phase is a column filled with charged resin that may be positively or negatively charged.
- The charged molecules then bond to the resins that have opposing charges when the mixture containing the charged particles is moved down the column.
- The positively charged molecules now bind to the cation exchange resin, replacing the negatively charged resin, if a cation exchange resin is utilised.
- Similar to this, when using an anion exchange resin, the positively charged resin is replaced with negatively charged molecules that bond to the anion exchange resin.
- In order to separate the complex of charged exchange resins and the charged molecules, the proper buffer is now added to the column.
Uses of Ion exchange chromatography
- In the process of purifying water, ion exchange chromatography replaces positively charged ions with hydrogen ions and negatively charged ions with hydroxyl ions.
- This technique also functions well for the analysis of the byproducts of nucleic acid hydrolysis.
- Ion-exchange chromatography also makes it easier to separate metals and other inorganic substances.
Examples of Ion exchange chromatography
- the process of separating positively charged lanthanoid ions from the crust of the earth.
- the process of separating proteins from a blood serum-derived crude mixture.
11. Liquid chromatography
Liquid chromatography is a method of separation where the mobile phase is a liquid and the separation can occur on a simple surface or in a column.
Principle of Liquid chromatography
- The concept underlying the molecules' affinity to the mobile phase serves as the foundation for liquid chromatography.
- The molecules travel with the mobile phase and exit the column more quickly if the components to be separated have a higher affinity for it.
- The molecules travel slowly and exit the column later, however, if the components interact with the mobile phase to a lesser extent.
- The two molecules will thus travel through the stationary phase at different rates if two molecules in a mixture have different polarities and the mobile phase is of a different polarity.
Steps of Liquid chromatography
- The stationary phase (cellulose or silica) is put to the solid support to create the column or paper.
- The liquid mobile phase, which includes the sample in it, is then injected into the chromatographic apparatus.
- Before exiting the column or the edge of the paper, the mobile phase passes through the stationary phase.
- To remove the molecules from the stationary phase, the system is exposed to an elution solution.
Uses of Liquid chromatography
- A coloured solution may be separated using liquid chromatography, and the separated coloured solution forms two distinct bands.
- Due to its ease of use and lower cost, this approach might also be preferred to others.
- It may be used to separate solid molecules from liquid ones that cannot dissolve in water.
Examples of Liquid chromatography
In the study of biological molecules, a modified version of liquid chromatography known as high-performance liquid chromatography is employed.
12. Paper chromatography
Paper chromatography is a method of separation in which the separation is carried out on a particular type of paper.
Principle of Paper chromatography
- Two distinct forms of paper chromatography are based on two distinct ideas.
- The first type of chromatography uses paper adsorption and is based on the different levels of contact that exist between the molecules and the stationary phase.
- Higher affinity molecules stay adsorbent for a longer period of time, slowing down their passage down the column.
- The molecules may be divided into distinct fractions because the molecules with lesser affinity move more quickly.
- Paper partition chromatography is the second kind of paper chromatography. It is based on the idea that the molecules travelling with the mobile phase use the moisture on the cellulose paper as a stationary phase to stop.
- Thus, the degree to which the molecules adsorb to the stationary phase determines how the molecules are separated.
- When separating molecules using paper chromatography, a further notion of "retention factor" is used.
- The ratio of the molecule's travel distance to the mobile phase's travel distance yields the retention value for a given molecule.
- The retention value of different molecules can be used to differentiate those molecules.
Steps of Paper chromatography
- Exceptionally high-quality cellulosic paper is chosen as the stationary phase.
- Different mixtures of organic and inorganic solvents are employed as the mobile phase.
- At the baseline of the paper, 2-200 l of the sample solution are injected, and the paper is then left to air dry.
- The sample laden paper is then slowly lowered to a height of no more than one centimetre in the mobile phase.
- The paper is removed when the mobile phase is close to the edge of the paper.
- The separated components are recognised using various methods, and the retention factor is determined.
Uses of Paper chromatography
- To determine the purity of various medicinal goods, paper chromatography is used.
- Additionally, it may be used to identify contamination in a variety of samples, including food and drinks.
- The removal of contaminants from a variety of industrial items may also be done using this technique.
- Paper chromatography is another method used in chemistry labs to analyse reaction mixtures.
Examples of Paper chromatography
The separation of ink mixes or other coloured liquids using paper chromatography.
13. Reverse-phase chromatography
Reverse-phase chromatography is a type of liquid chromatography in which the liquid mobile phase and stationary phase interact hydrophobically to separate molecules.
Principle of Reverse-phase chromatography
- The interaction between two molecules with hydrophobic groups is the foundation of the reverse phase chromatography method.
- The stationary phase in this instance is a solid support that has hydrophobic and hydrophilic groups added to it.
- The hydrophobic groups interact with the solvent molecules with hydrophobic regions, isolating them from the molecules with hydrophilic groups.
- By administering an elution solution with a decreasing salt gradient, the contact is then reversed, resulting in the separation of the hydrophobic molecules from the stationary phase.
Steps of Reverse-phase chromatography
- The column is made by covering a glass tube with a solid support, such as silica gel, then attaching hydrophobic groups, such as phenyl and octyl butyl, to it.
- The combination is added to the mobile phase of organic and inorganic solvents to prepare the sample.
- After that, the sample is injected into the column from the top.
- The hydrophobic groups of the stationary phase's hydrophobic molecules interact with the molecules having hydrophobic groups. The molecules lacking these groups, on the other hand, migrate out of the column with the mobile phase.
- The attached molecules are subsequently extracted from the stationary phase using a specific elution solution that has a decreasing salt gradient.
Uses of Reverse-phase chromatography
- For the separation of biomolecules, reverse chromatography is increasingly employed in conjunction with high-performance liquid chromatography.
- This is also used to the research of metabolite and active molecule analysis.
- It may also be used to clean up different environmental samples that include contaminants.
Examples of Reverse-phase chromatography
Reverse phase chromatography is an example where this method is used to separate proteins from their mixtures, as in the case of hydrophobic interaction chromatography.
14. Thin-layer chromatography (TLC)
The separation method known as thin-layer chromatography uses a liquid mobile phase and a stationary phase that is deposited as a thin layer on a solid support plate.
Principle of Thin-layer chromatography (TLC)
- This method of chromatography is based on the idea that when a component with an affinity for the stationary phase attaches to the stationary phase, the mixture's components are separated. Other substances, on the other hand, are eluted with the mobile phase.
- The reactive sites for component binding are made visible because the substrate/ligand is attached to the stationary phase.
- The components having substrate binding sites now bond to the substrate on the stationary phase when the mixture is passed through the mobile phase, and the remaining components are rinsed out with the mobile phase.
- Following separation, the molecules appear as dots throughout the stationary phase, each in a separate place.
- Many different strategies are used to find molecules.
Steps of Thin-layer chromatography (TLC)
- On a solid support (glass, a thin plate, or aluminium foil), the stationary phase is evenly placed and cured.
- About a centimetre above the plate's edge, the sample is injected as spots on the stationary phase.
- After that, the sample-loaded plate is gently dipped into the mobile phase to a maximum depth of 1 cm.
- The plate is removed when the mobile phase is close to the plate's edge.
- The retention factor is measured similarly to paper chromatography, and several methods are used to identify the separated components.
Uses of Thin-layer chromatography (TLC)
- To distinguish between the many compounds contained in a combination, thin-layer chromatography is frequently used in labs.
- This method aids in the forensic investigation of fibres.
- The assay of numerous medicinal goods is also possible using TLC.
- It assists in identifying therapeutic plants and their makeup.
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