Definition of RF Value
Retardation or factor of retention on a chromatogram, the (RF) value is the ratio of the distance travelled by the analyte to the distance travelled by the solvent front. The movement of analytes with mobile solvents differs between chromatographic techniques in which analytes are added to stationary phases (phases). This difference is due to analyte’s affinities for stationary and mobile solvents. The greater an analyte's relative affinity with a stationary phase, the longer it will stay in place and the lower the RF value, and vice versa.
The characteristic identification value for analytes at given temperatures is RF value. It means that compounds can be identified and analysed based on their RF values. When a new compound is discovered, however, this is not the case.
How to Find RF Value?
To begin, a chromatogram must be created using an appropriate solvent, depending on the nature of the analytes and stationary phases (mobile phase). The locations (migration values) of the analytes and the solvent front are measured after drying the chromatogram.
The RF (retardation/retention factor) values can be calculated using the above approach and experiment. A prepared sample solution (A+B) is applied to the chromatography paper and processed through a mobile phase. Analytes (A) and (B) separate due to their different affinities with the mobile phase (solvent). The analytes, the solvent front, and the location of administration of the mixture (A+B) are all measured in relation to one another.
RF Value Explanation
In paper chromatography, the stationary phase is water molecules found in the pores of the filter paper, while the moving phase is a solvent such as hexane, toluene, acetone, or a solvent mixture such as methanol-water mixture. The moving phase dissolves the components more or less quickly, depending on their solubility, as it passes through the area where the sample RF has been adsorbed, and carries them with it as it moves across the spot.
As the moving phase moves at a specific temperature and solvent, the characteristic rate of movement of each substance on the chromatography paper can be determined. The value, which stands for relative front or retardation factor, represents this. Even when the mobile phase (solvent) is the same, the RF values of different compounds vary. Furthermore, a chemical's RF value can vary depending on the solvent. RF values can be calculated using the following expression:
RF = Distance of the substance from the reference line (cm)/Distance of the solvent front from the reference line (cm)
Factors Affecting RF Values
1. Solvent Impacts Retention Factors
As the solvent transports the chemical along the plate, the solvent used has a significant impact on the retention factor value of the chemical. A solvent with a stronger interaction with a specific chemical will be able to overcome the chemical's affinity for the absorbent layer more easily and transfer the chemical further in a given amount of time. Different effects can be obtained depending on the amount of each solvent in a mixture.
2. Other Factors
There are a few other factors that may influence the retention factor in some cases. Since the solvent can often better dissolve the chemicals it transports at higher temperatures, the temperature of the solvent and plate may change slightly. The technician's method of placing the sample on the plate may also have an effect on the retention factor. Too many samples can cause wide, diffuse bands of chemicals to move up the plate, making it impossible to accurately estimate the distance travelled by the chemical. RF
Important Questions
1. What is chromatography?
Chromatography is a technique for separating the components of a mixture, or solutes, based on the relative amounts of each solute distributed between a moving fluid stream, known as the mobile phase, and a contiguous stationary phase. The mobile phase can be a liquid or a gas, whereas the stationary phase can be solid or liquid. The term "chromatography" is derived from Greek, chroma meaning "colour" and graphein meaning "to write," and was directly inherited from the invention of the first technique used to separate pigments.
2. What are the solvents used in chromatography?
In most cases, chromatography employs a liquid solvent. Water, methanol, isopropanol, acetonitrile, and formic acid are common liquid solvents used in fast protein liquid chromatography (FPLC), high-performance liquid chromatography (HPLC), and liquid chromatography-mass spectrometry (LC-MS). These chromatography solvents extract, dissolve, and move samples without permanently altering their chemical structure, making them an important component of standard separation techniques. Solvents are frequently used in water or in combination with another solvent. Some solvents, on the other hand, are not miscible and must be used as a pure reagent. Water and other polar solvents will only dissolve in other polar solvents.
Key Features
Retardation or factor of retention on a chromatogram, the RF value is the ratio of the distance travelled by the analyte to the distance travelled by the solvent front.
In paper chromatography, the stationary phase is water molecules found in the pores of the filter paper, while the moving phase is a solvent such as hexane, toluene, acetone, or a solvent mixture such as methanol-water mixture.
A chromatogram must first be generated with an appropriate solvent, depending on the nature of the analytes and stationary phases (mobile phase).
Multiple Choice Questions
1.Which of the following is used as a spraying reagent in paper chromatography?
(a) Concentrated hydrochloric acid solution
(b) Sodium chloride solution
(c) Ninhydrin solution
(d) Copper sulphate solution
Answer: (c)
2.Which of the following types of chromatography involves the process where the mobile phase moves through the stationary phase by the influence of gravity or capillary action?
(a) Column Chromatography
(b) High-Pressure Liquid Chromatography
(c) Gas Chromatography
(d) Paper Chromatography
Answer: (a)
FAQs on RF Value
1.What are forces involved in chromatography?
The London dispersion, dipole-dipole, hydrogen bonding, and ion-dipole forces are the four forces involved in chromatography.
London Dispersion Force – This is a temporary attractive force that occurs when electrons in two adjacent atoms are displaced to form temporary dipoles.
Dipole-Dipole Force – This is the attractive force that exists between one polar molecule's negative end and the positive end of another polar molecule, or between two dipoles.
Hydrogen Bonding Force – This is a type of dipole-dipole attraction that results in the formation of hydrogen bonds between molecules.
Ion-Dipole Force – A partially charged dipole and a fully charged ion form an ion-dipole force. The strength of the ion-dipole force is proportional to the ion charge. This force is most commonly encountered in solutions.
2.What is Thin Layer Chromatography analysis?
TLC (Thin Layer Chromatography) is a method of separating chemicals from mixtures. A thin coating of adsorbent material, typically silica gel, aluminium oxide, or cellulose, is coated on a sheet of glass, plastic, or aluminium foil in this method. The adsorbent layer is also called the stationary phase. After the sample is placed on the plate, capillary action draws a solvent or solvent combination (known as the mobile phase) up the plate. Different analytes ascend the TLC plate at different rates, resulting in separation.
3. What is the application of chromatography?
Chromatography is primarily used in bio analytical chemistry for the separation, isolation, and purification of proteins from complex sample matrices. Proteins, for example, coexist with numerous other compounds in cells, such as lipids and nucleic acids. These proteins must be separated from the rest of the cell in order to be studied. The proteins of interest may then need to be separated from other proteins and purified further.
Chromatography is a critical component of almost every protein purification strategy. Protein purification and analysis are carried out using a variety of chromatographic techniques. They are classified based on the physical principle at work in the separation process. Reversed phase chromatography, ion exchange chromatography, affinity chromatography, and size exclusion chromatography are a few examples.