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Retention Factor in Chromatography
Chromatography
The separation of a mixture of chemical compounds is extremely important to study the characteristics of each compound accurately. In chemistry, Chromatography is an analytical method that is widely used to separate a mixture of chemical compounds into their individual components, allowing the individual components to be extensively studied. It can also be used to purify the components of a mixture for use in other tests or operations.
Chromatography is a physical separation method in which the components of a mixture are separated by their distribution between two phases; one of these phases is generally immobile, stationary phase, and takes the form of a layer or film, while the other is a fluid, mobile phase, that percolates through or over the stationary phase. The principle of chromatography is based on the idea of separating the constituents, or solutes, of a mixture based on the relative quantities of each solute dispersed between a flowing fluid phase, known as the mobile phase, and a stationary phase
On the basis of the polarity of the mixture, certain components of the mixture remain in the stationary phase for a longer period of time and travel slowly through the chromatographic system, whilst others flow quickly into the mobile phase and leave the system.
Chromatography technique includes these three components :
- Stationary phase: This phase is always made up of a “solid” phase or phases.
- Mobile phase: This phase is always made of “liquid” or a “gaseous component.”
- Separated particles
The primary component effective in separating molecules from one other is the interaction between the stationary phase, mobile phase, and substances contained in the mixture. Partition-based chromatography technologies are particularly successful at separating and identifying small molecules such as amino acids, carbohydrates, and fatty acids.
In chromatography, the compound is separated on the basis of (like-dissolve-like) so the type of mobile phase chosen in separation is critical for example, water is commonly used as a solvent for polar compounds, however, if the chemicals being tested do not dissolve in an aqueous solvent, a non-aqueous solvent (non-polar solvent), such as alcohol, must be employed. The relative speeds at which compounds flow through the column are affected by the polarity of the solvent that is passed through the column. Polar solvents can more effectively compete with polar molecules in a mixture for polar sites on the adsorbent surface and will also solvate the polar components better.
Easy-to-dissolve components come closer to the leading edge of the mobile which is known as the solvent front, as the mobile phase is pulled higher through the stationary phase. Components that do not dissolve in the solvent easily are left behind the solvent front. As a result, the mixture separates into its individual components, which may be observed by an observer. So the solvent front is the point reflecting the furthest distance covered by the developing solvent
In chromatography, a stationary phase is a solid phase or a liquid phase deposited on the surface of a solid phase. The mobile phase flows over the stationary phase. If the mobile phase is liquid, the chromatography technique is liquid chromatography, whereas if it is gas, it is known as gas chromatography (GC) (GC). Gas chromatography is used to analyze volatile liquids and gases. Liquid chromatography is particularly useful for thermally unstable and non-volatile materials.
Thin Layer Chromatography
Also known as TLC, thin layer chromatography is an affinity-based technique for separating chemicals in a mixture. TLC is a flexible separation technology that is commonly used for both qualitative and quantitative sample analysis. Pesticides, steroids, alkaloids, lipids, nucleotides, glycosides, carbohydrates, and fatty acids are just a few of the substances that may be analyzed using TLC.
The stationary phase in TLC is a thin absorbent material layer, commonly silica gel or aluminum oxide, placed onto an inert plate surface, which is frequently glass, plastic, or aluminum. The material is spotted onto one end of the TLC plate and vertically put in a closed container containing an organic solvent (mobile phase). The mobile phase is propelled up the plate by capillary forces, and sample components migrate at variable rates depending on their affinities for the stationary and mobile phases. The plate is taken from the chamber and dried when the components are separated and reach the top of the plate. The divided components show on the plate as spots.
When the molecules in the sample are colorless, the fluorescence, radioactivity, or particular chemical reagent can be utilized to generate a visible colored reaction result. This, then, allows their locations on the chromatograph to be identified. Under room light or UV light, the formation of a visible color can be detected.
Retention Factor in Chromatography
The location of each molecule in the mixture may be calculated by dividing the distance traveled by the molecule by the distance traveled by the solvent. This measurement value is known as the retention factor and is denoted by the sign Rf. The Rf value is used to provide a qualitative description of the compounds.
The longer the retention and the greater the retention factor, the stronger the interactions of the analyte with the surface.
This calculation enables unknown materials to be compared to known materials. They are not the same compound if the retention factor of an unknown substance does not match that of a known material. Similar retention factors imply that the two samples are perhaps the same, but this is not evidence. In practice, the retention variables will differ somewhat from one sample to the next. The Rf value is affected by the interactions of the different components with one another as well as the concentration of the component in the sample. The retention factor is important in comparing the findings of one TLC plate to the results of another.
Retention Factor Formula
Frequently Asked Questions
Retention factor is a very useful chromatographic descriptor since it is dimensionless and independent of the flow rate and column dimensions of mobile phases.
The formula for retention time is given as
According to the retention factor, inert tracer which are not absorbed is zero.
According to the retention factor the non absorbed inert tracer is zero. The retention factor has various significance as the most significant quantity that can be derived directly from the chromatogram. It is a partition without dimension or the distribution ratio given by.
What is the Rf value formula?
To calculate the retention factor, divide the distance traveled by the chemical of interest by the distance from the baseline to the solvent front.
How do you interpret Rf values?
Rf values range from 0 to 1, with 0 representing extremely low solvent polarity and 1 representing extremely high solvent polarity. When carrying out the experiment, keep in mind that the separated components have unique polarities. If the result is 0, the polarity of the solvent should be increased since the sample is stationary and not moving. If the result is 1, the solvent’s polarity must be lowered since the chemical was unable to separate and is now at the solvent’s front point.
How do you calculate retention value in chromatography?
Step 1: Determine the distance between the spotting line and the compound of interest’s distance traveled as determined by the spot.
Step 2: Measure the space between the spotting line and the solvent front.
Step 3: To calculate the retention factor, divide the distance traveled by the chemical of interest by the distance from the baseline to the solvent front.
What does a high RF value mean?
The high value of the retention factor means the interaction between the compound of interest and the surface is strong. It also means that the compound of interest has a high solubility in the mobile phase.
How to Calculate Retention Factors in Thin-Layer Chromatography
Step 1: Find or identify the distance from the baseline to the solvent front.
Step 2: Find or identify the distance from the baseline to the point of interest.
Step 3: Calculate the retention factor by dividing the distance from the baseline to the solvent front by the distance from baseline to the point of interest.
Calculating Retention Factors in Thin-Layer Chromatography Vocabulary and Formulas
Thin-Layer Chromatography: Abbreviated as TLC, a technique used to identify and determine the purity of some samples. TLC involves spotting a very small amount of a sample, called the solute, close to the bottom of a silica plate. When the plate is placed into a small amount of solvent, the solvent climbs up the plate, bringing the solvent with it. Sometimes a single spot on the baseline will separate into multiple spots on the plate.
Baseline: The starting point where the solvent starts carrying the solute. This is where the sample is spotted.
Point of Interest: A spot that moves up the TLC plate.
Solvent Front: The line where the solvent stops moving. The solvent does not stop moving on its own and the plate must manually be removed and the solvent front marked before the solvent reaches the top of the plate.
Retention Factor: Abbreviated as Rf, the ratio of the distance a spot travelled to the distance the solvent travelled.
Completed TLC Plate:
Next, we will look at two examples of TLC plates and we will calculate the Rf for certain points of interest. The first example will cover a point of interest that is close to the baseline and the second will cover a point of interest that is close to the solvent front.
Calculating Retention Factors in Thin-Layer Chromatography Example: TLC with Point of Interest Close to Baseline
What is the retention factor of the point of interest in this solvent?
Step 1: Find or identify the distance from the baseline to the solvent front.
In lab, measurements are usually taken manually by ruler. Here, the distance from the baseline to the solvent front is shown to be 5.2 cm.
Step 2: Find or identify the distance from the baseline to the point of interest.
The distance from the baseline to the point of interest is shown to be 0.34 cm.
Step 3: Calculate the retention factor by dividing the distance from the baseline to the solvent front by the distance from baseline to the point of interest.
Because units are the same in both the numerator and denominator, units cancel and Rf is a unitless ratio.
If we round our answer to two decimal places, the Rf for this point of interest is 0.06.
Calculating Retention Factors in Thin-Layer Chromatography Example: TLC with Point of Interest Close to Solvent Front
What is the retention factor of the point of interest in this solvent?
Step 1: Find or identify the distance from the baseline to the solvent front.
The distance from the baseline to the solvent front is shown to be 4.3 cm.
Step 2: Find or identify the distance from the baseline to the point of interest.
The distance from the baseline to the point of interest is shown to be 3.6 cm.
Step 3: Calculate the retention factor by dividing the distance from the baseline to the solvent front by the distance from baseline to the point of interest.
If we round our answer to two decimal places, the Rf for this point of interest is 0.84.
Sample Problems
Question 1: Calculate the retention factor if the solute and solvent moved from 5cm and 10cm respectively from the baseline which is at 0cm.
Solution:
Given,
Distance moved by solute from base line = 5cm
Distance moved by solvent from base line = 10cm
= 5/10
= 1/2
= 0.5
Retention Factor for the given solution is 0.5
Question 2: Calculate the retention factor if the solute and solvent moved from 7cm and 20cm respectively from the baseline which is at 0cm.
Solution:
Given,
Distance moved by solute from base line = 7cm
Distance moved by solvent from base line = 20cm
= 7/20
= 0.35
Retention Factor for the given solution is 0.35
Question 3: Calculate the retention factors for solute A and B if they moved from 2cm and 5cm from base level and solvent moved to 10cm up from the baseline which is at 0cm.
Solution:
Given,
Distance moved by solute A from base line = 2cm
Distance moved by solute B from base line = 5cm
Distance moved by solvent from base line = 10cm
= 5/10
= 0.5
Retention Factor of solute A, B are 0.2, 0.5
Question 4: Calculate the retention factors for solute A and B if they moved from 10cm and 20cm from base level and solvent moved to 60cm up from the baseline which is at 0cm.
Solution:
Given,
Distance moved by solute A from base line = 10cm
Distance moved by solute B from base line = 20cm
Distance moved by solvent from base line = 60cm
= 20/60
= 0.33
Retention Factor of solute A, B are 0.16, 0.33
Question 5: Calculate the retention factor if the solute is moved to 23% above the base level and solvent moved up to 50%.
Solution:
Given,
Distance moved by solute from base line = 23% = 23/100 = 0.23 units
Distance moved by solvent from base line = 50% = 50/100 = 0.5 units
= 0.23/0.5
= 0.46
Retention Factor for the given solution is 0.46
Retention Factors
The amount that each component of a mixture travels can be quantified using retention factors (Rf). The retention factor of a particular material is the ratio of the distance the spot moved above the origin to the distance the solvent front moved above the origin. It can be calculated using the formula:
The Rf values for each of the components in the previous example can be calculated:
Notice that (1) the bigger the Rf, the further the spot moved and (2) that the Rf should be the same for a component regardless of how far the solvent moves.
Retention factors are useful in comparing the results of one chromatogram to the results of another. If the conditions in which the chromatogram are run are unchanged (same mobile and stationary phases), the retention factor for a given material should remain constant. This allows unknowns to be compared to known materials. If the retention factor of an unknown does not match that of a known material, they are not the same compound. Similar retention factors suggest that the two samples could be the same, but is not proof. In reality, the retention factors will vary slightly from sample to sample. Interactions of the individual components with each other and the concentration of the component in the sample will both affect the Rf value.
Retention Factor Formula Problems
