chromatography experiment rf value

Analytical Chemistry
Chromatography

What is R F Value?

The R f (retardation factor) value is the ratio of the solute’s distance travelled to the solvent’s distance travelled.

The word comes from chromatography when it was discovered that a given component will always travel the same distance in a given solvent under the same conditions.

The R f value is a physical constant for organic molecules that can be used to verify a molecule’s identity. Only if the chromatographic settings below are also constant from one trial to the next does the R f for a substance remain constant. Because these variables are challenging to maintain consistency from experiment to experiment, relative R f values are commonly used. “Relative R f ” denotes that the results are provided in comparison to a standard, or that the R f values of compounds run on the same plate at the same time are compared.

R f value explanation.

Why Do We Need the R F Value?

Calculation of R F Value

Factors affecting R f values
Frequently Asked Questions – FAQs

The stationary phase in paper chromatography is water molecules found in the pores of the filter paper, whereas the moving phase is a solvent such as hexane, toluene , acetone, or a mixture of solvents such as methanol-water mixture. As the moving phase passes through the area where the sample has been adsorbed, it dissolves the components more or less quickly, depending on their solubility, and carries them with it as it moves across the spot.

It is possible to determine the characteristic rate of movement of each substance on the chromatography paper as the moving phase moves at a certain temperature and for a specific solvent. This is represented by the R f value, which stands for relative front or retardation factor. Even if the mobile phase (solvent) is the same, various compounds have varying R f values. Furthermore, the R f value of a chemical may vary depending on the solvent. The following expression can be used to calculate R f values:

R f = Distance travelled by the substance from reference line (cm)/Distance travelled by the solvent front from reference line (cm)

Why do we need the R F value?

In chromatography, R f values are the most basic prerequisite of the experiment. These numbers indicate whether the analyte (solute) prefers the stationary or mobile phase. With stationary and mobile phases, R f values are used to determine polarity, relative masses, and relative solubilities, among other things.

When comparing the results of one chromatogram to those of another, retention factors come in handy. The retention factor for a given substance should stay constant if the chromatogram is conducted under the same conditions (same mobile and stationary phases). This enables the comparison of unknown materials to known materials. They are not the same compound if the retention factor of an unknown substance differs from that of a known substance.

Similar retention factors indicate that the two samples may be identical, but this is not proof. The retention variables will vary slightly from sample to sample in reality. The R f value is affected by the interactions of the various components as well as the concentration of the component in the sample.

Depending on the nature of the analytes and the stationary phases, a chromatogram must first be generated with an appropriate solvent (mobile phase). After drying the chromatogram, the locations (migration values) of the analytes and the solvent front are measured.

Using the stated approach and the above experiment, the R f (retardation/retention factor) values can be computed.

On the chromatography paper, a prepared sample solution (A+B) is applied and processed through a mobile phase. Because of their differing affinities with the mobile phase, analytes (A) and (B) separate (solvent). The analytes, the solvent front, and the point where the mixture (A+B) was administered are all measured relative to each other.

Factors Affecting R f Values

1. solvent impacts retention factors.

Because the solvent transports the chemical along the plate, the solvent utilised has a significant impact on the chemical’s retention factor value. A solvent with a stronger interaction with a certain chemical will be able to more easily overcome the chemical’s affinity for the absorbent layer and transfer the chemical further in a given amount of time. Depending on the quantity of each solvent in a mixture, different effects can be achieved.

2. Other factors

In some circumstances, there are a few other elements that may influence the retention factor. The temperature of the solvent and plate may make slight changes, since, the solvent can often better dissolve the chemicals it is transporting at higher temperatures. The retention factor might also be affected by the technician’s method when placing the sample on the plate. Too much sample can cause wide, diffuse bands of chemicals to move up the plate, making it impossible to correctly estimate the distance the chemical has travelled.

Frequently Asked Questions on R f Values

What factors affect chromatography.

Retention factor values in thin layer chromatography are affected by the absorbent, the solvent, the chromatography plate itself, application technique and the temperature of the solvent and plate.

Can R f value be greater than 1?

R f values are always less than 1. An R f value of 1 or too close to it means that the spot and the solvent front travel close together and are therefore unreliable.

How does polarity affect R f value?

The more polar the compound, the more it will adhere to the adsorbent and the smaller the distance it will travel from the baseline, and the lower its R f value.

Why is polarity important for chromatography?

Polarity has a huge effect on how attracted a chemical is to other substances. The larger the charge difference, the more polar a molecule is. You will find that as you increase the polarity of the solvent, all the components of the mixture move faster during your chromatography experiment.

What is the difference between polar and nonpolar molecules?

Polar molecules occur when there is an electronegativity difference between the bonded atoms. Nonpolar molecules occur when electrons are shared equally between atoms of a diatomic molecule or when polar bonds in a larger molecule cancel each other out.

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Chromatography

What are rf values, exploring paper chromatography, using paper chromatography, understanding chromatography and the calculation of rf values.

Chromatography is a widely used technique in chemistry that allows for the separation of components in a mixture. It is used in various fields, including forensics, pharmaceuticals, and environmental sciences. One important aspect of chromatography is the calculation of Rf values. In this article, we will discuss what chromatography is and how Rf values are calculated.

Chromatography is a technique used to separate components in a mixture based on their physical and chemical properties. The sample is first dissolved in a solvent and then placed on a stationary phase, which is often a sheet of paper or a column of beads. The components in the sample then move through the stationary phase at different rates based on their interactions with the solvent and the stationary phase.

Chromatography has two phases: stationary phase and mobile phase.

The Two Phases of Chromatography

Mobile phase and stationary phase are the two phases of chromatography. The mobile phase is the fluid that moves through a column, while stationary phase is the solid material in which molecules are trapped as they pass through it. The mixture of compounds that you want to separate out into their individual components flows down through this column, where each component will be separated based on its affinity for either one or both of these phases.

As they exit at different points along the length of your column (and depending on how long it is), each component will come out separately from all other components in order from most strongly adsorbed at one end to least strongly adsorbed at another end--this process forms an equilibrium between them as well as separating them out by molecular weight and size

Molecules in the Mobile Phase

The mobile phase is a fluid or gas that allows molecules in the stationary phase to move. This is important because it allows us to separate and identify different compounds in a mixture. The most common types of mobile phases are water, organic solvents (such as alcohols), and gases such as helium or nitrogen.

Molecules in the Stationary Phase

The stationary phase is the part of your chromatography system that doesn't move. It can be a solid or thick liquid, which doesn't allow molecules to move through it easily. This allows for separation of different types of molecules in your mixture based on their size and shape.

Distribution Between Phases

Distribution between phases refers to the process of separating a mixture into its components based on their relative solubility in two immiscible liquids. In chromatography, there are two phases: the mobile phase and the stationary phase. The mobile phase consists of a liquid that transports the sample through the chromatography column (such as water), while the stationary phase consists of solid particles that capture substances (such as sand).

The distribution between these two phases can be described by the partition coefficient--this is a ratio describing how a substance will bind to one substance's surface or dissolve in it. If you want to learn more about this concept, check out our article "How do I calculate the partition coefficient?"

Applications of Chromatography

Chromatography is used in many industries, including food and beverage, pharmaceuticals, environmental, and forensic sciences.

In the food industry, it is used to identify the components of a sample or measure the levels of contaminants in food products. The process can also be used to detect pesticides or other harmful substances found in water sources.

Types of Chromatography

Chromatography is a technique that allows you to separate and identify compounds. There are several types of chromatography, including Gas Chromatography (GC), Liquid Chromatography (LC), Thin-Layer Chromatography (TLC), and Affinity Chromatography.

Advantages of Chromatography

Chromatography is an economical, reliable, and sensitive technique that can be used to detect trace amounts of substances. Compared to other analytical methods such as spectroscopy or mass spectrometry, it is relatively easy to use.

Disadvantages of Chromatography

Chromatography is a useful tool for chemists, but it also has some disadvantages. First, chromatography can be time-consuming: you need to wait for the sample to pass through the column to get results. It also requires specialized equipment, and if you are not familiar with it, the results may be difficult to interpret.

Rf values, or the Retention Factor, is a ratio used to describe the relationship between the distance moved by components in a mixture relative to the distance moved by the solvent. It is calculated by dividing the distance moved by the component by the distance moved by the solvent.

Rf = Distance moved by the component / Distance moved by the solvent

The range of Rf values is from 0 to 1, with values closer to 1 indicating that the component has a stronger attraction to the solvent than to the stationary phase.

How to Calculate Rf Values

To calculate Rf values, first measure the distance from the start line to the solvent front. Next, measure each component's distance from the start line to its respective spot. Finally, divide the distance moved by the component by the distance moved by the solvent to determine the Rf value.

It is crucial to note that Rf values can be influenced by various factors, such as the type of stationary phase, solvent, and temperature. Therefore, it is essential to maintain consistent conditions when comparing Rf values.

Understanding Rf Values

Rf values are significant in chromatography as they aid in compound identification. However, it is important to recognize that Rf values may vary depending on the substance and solvent utilized. The choice of solvent impacts Rf values due to polarity discrepancies and interactions with the stationary phase. Hence, using the same solvent is vital for accurate Rf value comparisons.

Differences in Rf values facilitate the identification of substances within a compound. By comparing compound-produced spots to reference values, the contained substances can be determined. This application is valuable in fields like forensics and pharmaceuticals.

During chromatography, a compound separates into distinct spots, each representing a specific substance. In the case of a pure compound, only one spot forms, indicating its purity without additional substances. This underscores the compound's purity.‍

Paper chromatography is a potent technique for separating and identifying chemical compound mixtures. It employs paper as the stationary phase and a liquid as the mobile phase. Through chromatography, mixtures can be separated and compounds identified. This discussion covers paper chromatography basics, phases, and pure substance identification.

Pure substances in paper chromatography produce a single spot on the chromatogram, distinguishing them from mixtures. Comparing the chromatogram's spot count to the known mixture compounds aids in determining compound purity.

Paper chromatography involves two phases: the mobile phase (solvent) and the stationary phase (chromatography paper). The solvent moves through the paper, carrying the mixture and causing compound separation based on chemical properties.

Similar to other chromatography forms, Rf values can be calculated in paper chromatography using the previously mentioned equation. Solvent choice in paper chromatography affects Rf values, as substances move at varying rates based on solubility in the solvent and attraction to the paper. Thus, selecting the appropriate solvent is crucial for effective mixture separation.

One of the most significant advantages of paper chromatography is its ability to identify colourless substances. By adding locating agents to chromatograms with colourless substances, coloured products or ones that glow under ultraviolet light can be formed. An example of a locating agent is iodine vapour, which turns brown when reacted with fats and oils.

Paper chromatography is a method used to separate mixtures and identify compounds. It separates substances based on their different colours, making it useful for analyzing coloured substances like pen ink.

Gather equipment : For this experiment, you will need a solvent, different coloured inks, filter paper, a container, a pencil, a capillary tube, and a ruler.
Draw the origin : Use a ruler and pencil to draw a straight line about 2cm from the bottom of the filter paper. This line serves as the start line for placing the ink droplets to be separated. A graphite pencil is recommended as graphite is insoluble and will not spread in water.
Add ink spots : Using the inks and a capillary tube, place a single spot of each ink on the start line. Using a capillary tube ensures that the ink spots are uniform in size and placement. Make sure to space the spots apart to prevent them from merging.
Place the paper into the solvent : Add a small amount of solvent to the container and then immerse the tip of the filter paper with the ink spots into the solvent. Ensure the paper is upright in the container and that the solvent level is below the ink spots.
Seal the container : Cover the container with a lid to prevent the solvent from evaporating.

Allow the solvent to move up the paper until it reaches near the top. The solvent should not reach the top completely. Once done, remove the paper from the solvent and let it dry. Calculate the Rf values by dividing the distance moved by the substance by the distance moved by the solvent.

What is Chromatography?

Chromatography is a technique used to separate and identify the components of a mixture. It works by using the different physical and chemical properties of the components to separate them.

What is the Rf value in Chromatography?

The Rf value, also known as the retention factor, is a measure of the position of a component in a chromatographic separation. It is calculated by dividing the distance travelled by the component by the distance travelled by the solvent.

Why is the Rf value important in Chromatography?

The Rf value is important in Chromatography because it allows us to identify the components of a mixture. By comparing the Rf value of a component in a mixture to the Rf values of known compounds, we can determine the identity of the component.

What factors can affect the Rf value of a component in Chromatography?

The Rf value of a component in Chromatography can be affected by several factors, including the type of stationary phase, the polarity of the solvent, the temperature, and the concentration of the components in the mixture.

How do you calculate the Rf value in Chromatography?

To calculate the Rf value in Chromatography, you divide the distance travelled by the component by the distance travelled by the solvent. For example, if the component travels 5cm and the solvent travels 10cm, the Rf value is 0.5.

How does Chromatography work?

Chromatography works by using the different physical and chemical properties of the components in a mixture to separate them. The mixture is applied to a stationary phase, such as a piece of paper or a column, and a solvent is added. The components in the mixture move at different speeds through the stationary phase based on their properties, allowing them to be separated.

What are the different types of Chromatography?

There are several different types of Chromatography, including Paper Chromatography, Thin Layer Chromatography (TLC), Column Chromatography, and Gas Chromatography (GC). Each type uses a different stationary phase and a different method for separating the components of a mixture.

How is Chromatography used in real-life applications?

Chromatography is a versatile technique used in various real-world scenarios. It plays a crucial role in analyzing food and beverages, identifying drugs and chemicals, and separating proteins and enzymes in biochemistry. Additionally, chromatography is utilized in environmental testing to detect and quantify pollutants in the air, water, and soil.

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May 13, 2023

Rf value: calculation, significance and affecting factors.

In this article we will learn about Rf value also known as Retention Factor . Here we will discuss calculation of Rf values and its importance in thin layer chromatography .

Key words : Rf value or Retention factor, Thin layer chromatography (TLC), stationary phase, mobile phase.

Definition of Rf value
Thin layer chromatography
Calculation of Rf value
Importance of Rf value
Factors affecting Rf value

1. Definition of Rf value

Rf value or Retention factor is defined as it is ratio of distance travelled by solute to the distance travelled by solvent. Since it is ratio there is no unit for Rf value. (Fig 1)

Figure 1: Calculation of Rf value or Retention factor

2. Thin layer chromatography

It is the chromatographic method to separate the compounds based on Rf value. Here different compounds appears as spots. It is performed on silica gel plate which is also known as stationary phase . Commonly the silica is coated on glass plate or aluminum plate. The sample is deposited on TLC plate and dipped in to a glass chamber which has solvent. This is also known as eluent or mobile phase. Then the sample loaded TLC is placed into the chamber. The solvent moves towards top end due to capillary action. The sample or solute also moves towards top end along with the solvent. Some compounds are having more affinity towards silica whereas some compound has grater affinity towards solvent. Due this difference in affinities compound they moves towards the top end with different speed. Thus, different compounds from a mixture get separated on TLC as spots. (Fig 2)

Figure 2: Thin Layer Chromatography

3. Calculation of Rf value

See the following TLC,

Figure 3: Thin layer chromatography plate

The TLC shows three different spots which corresponds to compounds A, B and C (Fig 3). Here the solvent travelled up to 15cm distance. This is also known as solvent front. The distance is usually calculated from base line. The compound A, B and C are referred as solute. For the calculation of Rf value, we need to consider distance travelled by solute and distance travelled by solvent. The distance should be calculated from base line to the center of the spot.

Therefore; Rf value for spot A can be calculated as

Rf value of A = [Distance travelled by spot A] / [Distance travelled by solvent]

= 5.1 / 15

= 0.34

Hence Rf value of spot A is 0.34 .

Rf value of B = [Distance travelled by spot B] / [Distance travelled by solvent]

= 7.5 / 15

= 0.50

Hence Rf value of spot B is 0.50 .

Rf value of C = [Distance travelled by spot C] / [Distance travelled by solvent]

= 2.0 / 15

= 0.13

Hence Rf value of spot C is 0.13 .

4. Importance of Rf value

Rf value is a physical constant of the compound.
Each organic compound has specific Rf value.
Rf value is used for identification of organic compound.
Rf value is useful in monitoring chemical reactions and purifications.

4.1 Monitoring Chemical Reactions

There are many methods available for identification of organic compounds such as UV spectroscopy, IR spectroscopy, Mass spectrometry and NMR spectroscopy. In addition to this reactions can be monitored by identification of compounds produced in reaction mixture, such as HPLC, LCMS, Gas chromatography etc. These methods are highly advanced and accurate. But it take reasonable time and instruments to perform above analysis.

TLC is very quick method to identify organic compounds. It takes lesser time and efforts to monitor chemical reactions. For example, consider the reaction of benzophenone with sodium borohydride to benzhydrol. (Fig 4)

Figure 4: Reduction of Benzophenone by Sodium borohydride

Figure 5: Monitoring of reaction by TLC

4.2 Monitoring Purification by Column Chromatography

5. factors affecting rf value.

Rf value is a physical property of compound and it get affected by following factors.

5.1 Solvent (Mobile phase)

5.2 solute (sample), 5.3 temperature, 5.4 thickness of silica layer, 6. conclusion.

Rf value or Retention factor is a physical property of compounds.
It is ratio of distance travelled by solute to the distance travelled by solvent.
Rf value denoted by 3 digit number and it does not have unit.
Rf value is useful for monitoring chemical reactions and purification of organic compounds.
There are factors which affects Rf values of compounds. For example, solvent or mobile phase, nature of compounds, temperature and thickness of silica layer.

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What is R F Value?

The Rf value (retardation factor/Retention Factor) is an important parameter in chromatography that represents the distance traveled by a particular compound relative to the distance traveled by the solvent front.

The Rf value is a dimensionless number that ranges from 0 to 1. It is calculated by dividing the distance traveled by the compound by the distance traveled by the solvent front. The resulting Rf value represents the relative mobility of the compound in the chromatogram.

Calculation of RF Value

The Rf value can be calculated using the following formula:

Rf = distance traveled by the compound / distance traveled by the solvent front

The distance traveled by the compound is measured from the origin to the center of the spot, while the distance traveled by the solvent front is measured from the origin to the solvent front.

Why Do We Need the RF Value?

The Rf value is an essential parameter in chromatography because it allows for the identification and analysis of individual components in a mixture. By comparing the Rf values of an unknown sample to those of known compounds, scientists can determine the identity of the unknown compound.

The Rf value also provides information on the polarity of the compound. Polar compounds tend to have a lower Rf value, while nonpolar compounds tend to have a higher Rf value. This information can be useful in determining the chemical properties of a compound.

Factors Affecting Rf Values

The Rf value is influenced by various factors, including:

The nature of the stationary phase: Different stationary phases have different affinities for compounds, which can affect their mobility in the chromatogram.
The composition of the mobile phase: The composition of the mobile phase can affect the solubility and polarity of the compounds, which can in turn affect their mobility.
The temperature and pressure of the system: Changes in temperature and pressure can affect the viscosity and density of the solvent, which can affect the mobility of the compounds.
The size and shape of the spot: The size and shape of the spot containing the compound can affect its Rf value.

Further Insights on RF Value

The Rf value is dependent on the type of chromatography technique being used, such as thin-layer chromatography (TLC), high-performance liquid chromatography ( HPLC ), or gas chromatography (GC).
The Rf value can be affected by the pH of the solvent system. Compounds with ionizable groups may exhibit different Rf values at different pH values.
The Rf value can be used to determine the purity of a compound. A pure compound will have a single spot with a consistent Rf value, while impure compounds will have multiple spots with varying Rf values.
The Rf value can also be used to estimate the molecular weight of a compound. Larger molecules tend to have a lower Rf value, while smaller molecules tend to have a higher Rf value.
In some cases, the Rf value may not be the best parameter to use for compound identification. Other parameters such as retention time, peak area, and peak height may be more appropriate for certain types of chromatography.

FAQs Related to RF value

What is a good Rf value?

A good Rf value is one that is consistent with the known Rf values of the compound under the same experimental conditions. However, there is no universal “good” Rf value, as it can vary depending on the system and the compound being analyzed.

Can two compounds have the same Rf value?

Yes, two compounds can have the same Rf value if they have similar chemical properties and interact similarly with the stationary and mobile phases in the chromatography system.

Can the Rf value be greater than 1?

No, the Rf value cannot be greater than 1. If the compound travels farther than the solvent front, it may have been over-spotted or over-applied, or the solvent system may not be appropriate for the compound.

Principle of LCMS

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How to Find the Rf Value in Chromatography

by Taha Cheema

As an Chemistry student , knowing how to find Rf value in chromatography is crucial. If you have been practicing O Level or IGCSE Chemistry past papers, you would have noticed that Rf value questions are common in Paper 1 (MCQs) and chromatography experiments are also tested in the ATP . So let’s dive in and understand this calculation better!

As an O Level and IGCSE Chemistry student, knowing how to find Rf value in chromatography is crucial.

What is the Rf value in Chromatography?

Before we dive into what an Rf value is, let's first understand what is chromatography. Chromatography is a technique used to separate and identify the components of a mixture. It works by using the different physical and chemical properties of the components to separate them. It is used in various ways, including in the analysis of food and drinks, the identification of drugs and chemicals and the separation of proteins and enzymes in biochemistry.

The Rf value is used to identify a chemical compound . It is also known as the retention factor as it is a measure of the position of a component in a chromatographic. For a given compound, you can determine the Rf value in chromatography using this formula:

Rf value = Distance moved by the substance / Distance moved by the solvent front

In the example above, the Rf value of the green substance can be calculated by dividing the distance b by a. For example, if the component travels 6cm and the solvent travels 10cm, the Rf value is 0.6.

What is the Purpose of the Rf value in Chromatography?

Different compounds have different Rf values depending on how much they travel with respect to the solvent front. Hence we can identify substances or components using their unique Rf values.

Different compounds end up having different Rf values depending on how much they travel with respect to the solvent front. Hence we can identify substances using their unique Rf values.

Differences Between Rf Values

Rf values can differ. It is important to understand that Rf values are not the same for every substance and every solvent. It will change depending on the solvent used.

Rf values identify compounds. Due to the differing Rf values, substances can be identified by chromatography. This allows us to see the substances within one compound by comparing them to reference values.

Moreover, when chromatography is carried out on a compound, these substances separate to create a series of spots. Each spot will represent one specific substance.

*Tip: Pure compounds produce a single spot. When we carry out chromatography on a pure compound, one single spot will be formed. This indicates that there are no other substances present, reinforcing that the compound is pure.

What are the Factors Affecting Rf value in Paper Chromatography?

Rf values can vary if you don’t standardize the following factors in your experiment:

The type of stationary phase

The polarity of the solvent

The concentration of compounds in the mixture

The temperature

For example, if you use a different solvent across two experiments, the Rf value for the same chemical compound will be different!

Conclusion

If you were confused about Rf value chromatography concepts, we hope this guide helped solidify your understanding. Feel free to check out our O Level/IGCSE Chemistry crash course where we have included the most common past paper questions at the end of each chapter’s content!

Here's a sneak peek into how Out-Class video lessons look like:

Q. What is Chromatography?

Chromatography is a technique used to separate and identify the components of a mixture. It works by using the different physical and chemical properties of the components to separate them.

Q. What is an Rf value in Chromatography?

The Rf value is used to identify a chemical compound . It is also known as the retention factor as it is a measure of the position of a component in a chromatographic.

Q. How do you calculate the Rf value in Chromatography?

To calculate the Rf value, you divide the distance travelled by component by the distance travelled by solvent.

Q. What is the purpose of the Rf value in chromatography?

Q. how is chromatography used in real-life applications.

Chromatography is used in various ways, including in the analysis of food and drinks, the identification of drugs and chemicals and the separation of proteins and enzymes in biochemistry.

Q. What are the Factors Affecting Rf value in Paper Chromatography?

The Rf values can be affected by several factors:

If you want to dive right in, let's start with selecting the course you want

What course are you interested in, choose from the list.

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Thin layer chromatography (TLC) is a widely-used analytical technique for separating complex mixtures of organic compounds. By using a stationary phase – the adsorbent, and a mobile phase – the solvent, compounds in the mixture are separated based on their different adhesion rates. One of the key parameters in interpreting TLC results is the retention factor, or Rf value. In this article, we will discuss how to calculate Rf values for TLC analysis.

What is an Rf Value?

The Rf value, short for “retention factor,” is a dimensionless ratio that represents the relative distance traveled by a compound on the TLC plate with respect to the solvent front’s distance. By comparing the Rf values of several compounds to known standards, it is possible to identify or approximate those components that are present in a mixture.

Steps to Calculate Rf Values for TLC

1. Run your TLC experiment: Apply a small amount of your mixture onto the base of a clean TLC plate alongside known reference samples. Carefully place your prepared TLC plate in a development chamber containing an appropriate solvent system as the mobile phase, making sure that the initial spots are above the solvent level. Let the solvent rise up by capillary action until it reaches around three-quarters of the height of your plate.

2. Remove your plate from the chamber: Once the solvent reaches the desired position on the TLC plate, carefully remove it from the chamber and immediately mark both your sample and reference spots’ locations and also mark where the solvent front has traveled.

3. Measure distances: Measure two distances using a ruler: (a) The distance each separated compound has traveled from start position (baseline), which we will label as “distance traveled by solute” (D_solute), and (b) The overall distance traveled by solvent front from start position, which we will label as “distance traveled by solvent” (D_solvent).

4. Calculate Rf values : Divide the distance traveled by solute (D_solute) for each compound by the distance traveled by solvent (D_solvent). Rf = D_solute / D_solvent.

Example Calculation:

Imagine an example where the solvent front has traveled a total of 9 centimeters (cm), and one compound has traveled 3 cm from the baseline. You will calculate its Rf value as follows:

Rf = D_solute / D_solvent = 3 cm / 9 cm = 0.33

Important Notes

– Be precise in all measurements while marking spots and measuring distances.

– Always compare the Rf values of unknown compounds to known reference standards or literature data.

– The same compound might exhibit different Rf values if different solvent systems or adsorbents are used.

– Ideally, compare compounds that have similar structures to ensure better identification.

Rf values for thin-layer chromatography play an essential role in interpreting results and identifying compounds within complex mixtures. By following the steps outlined in this article, you can easily calculate Rf values for your TLC experiment with accuracy and reliability.

How to Calculate RF Value

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AQA GCSE Chemistry Revision Notes

Chromatography and Rf Values (GCSE Chemistry)

Chromatography.

Chromatography can separate mixtures . By using chromatography , we can separate mixtures. This will help us to identify the substances within the mixture.
Chromatography has two phases. There are two phases in chromatography, called the mobile phase and the stationary phase. These phases allow the mixture to separate.
The two phases reach an equilibrium. As compounds move through the two phases of chromatography, an equilibrium is formed.
Molecules can move in the mobile phase . The mobile phase can be a liquid or a gas , allowing molecules to move.
Molecules cannot move in the stationary phase. The stationary phase can be a solid or thick liquid , which doesn’t allow the molecules to move. In this phase, molecules can separate out of the mixture.
Distribution between phases determines separation. Depending on the distribution of the compound between the mobile and stationary phases, molecules will separate.

Substances start at the origin . The starting point of the compound is called the origin . This is where we measure the distance moved by the substance.
Substances move different distances. Each substance will move a different distance . We can measure the distance travelled by substance by going from the centre of the spot to the origin.
Rf values are ratios. An Rf value is the ratio of the distance moved by the compound as compared to the distance moved by the solvent. If the solvent only moves a short distance, then the Rf value will be small.

Table of Contents

Differences Between Rf Values

Rf values can differ. Rf values are not the same for every substance and every solvent. The Rf value will change depending on the solvent used.
Rf values identify compounds. Due to the differing Rf values, substances can be identified by chromatography. This allows us to see the substances contained within one compound by comparing them to reference values.
Substances create a series of spots. When chromatography is carried out on a compound, the substances separate out to create a series of spots . Each spot will represent one specific substance .
Pure compounds produce a single spot. When we carry out chromatography on a pure compound, one single spot will be formed. This indicates that there are no other substances present, reinforcing that the compound is pure.

Paper Chromatography

Paper chromatography separates mixtures. Paper chromatography is a form of chromatography. By using chromatography, we can separate mixtures and identify compounds.
Paper chromatography identifies pure substances. As previously mentioned, we can identify pure substances from chromatograms, as they produce one single spot on the chromatogram.
Paper chromatography has two phases. Paper chromatography has a mobile phase and a stationary phase. The mobile phase is the solvent , whilst the stationary phase is the chromatography paper .
Paper chromatography gives Rf values . Like any other form of chromatography, we can calculate Rf values from paper chromatography using the equation we saw previously.
Rf values are affected by the solvent. In paper chromatography, the Rf values are affected by the solvent used. The substances will move at different rates depending on how soluble they are in the solvent, and how attracted they are to the paper.
Paper chromatography can identify colourless substances . By adding locating agents to chromatograms with colourless substances, coloured products or ones which glow under ultraviolet light can be formed. An example is iodine vapour. It turns brown when reacted with fats and oils.

Using Paper Chromatography

Paper chromatography separates mixtures. As we’ve seen, paper chromatography is a form of chromatography. By using chromatography, we can separate mixtures and identify compounds .
Paper chromatography separates coloured substances . Paper chromatography can be used to separate the compounds within coloured substances such as pen ink.
Gather equipment . For this experiment, we will need a solvent, some substances (in this case, different coloured inks), filter paper, a container, a pencil, a capillary tube , and a ruler.
Draw the origin . Using the ruler and a pencil, draw a straight line about 2cm from the bottom of the filter paper. This is the start line , where the ink droplets to be separated will be placed. A graphite pencil is used as graphite is insoluble and will not spread in water.
Add ink spots . Using the inks and a capillary tube, place a single spot of each ink on the start line. Using a capillary tube will make sure that the spots of ink are all a similar size and on the start line. Make sure the spots are far enough apart so they will not spread into each other.
Place the paper into the solvent . Put a small amount of solvent into the container, then place the tip of the filter paper with the ink spots into the solvent. Make sure to place the paper upright into the container and ensure that the solvent is below the ink spots.
Place a lid onto the container . Make sure to place a lid onto the container so that the solvent does not evaporate .

GCSE Chemistry - Chromatography and Rf Values

Wait for the solvent to travel up the paper. Now, we must wait for the solvent to travel most of the way up the paper. The solvent should not completely reach the top of the paper. When the paper is removed, the final line of the solvent is called the ‘ solvent front . ’
Allow chromatogram to dry. When the chromatogram is ready, remove the paper from the solvent and allow the chromatogram to dry .
Calculate Rf values. Using the chromatogram, Rf values can be calculated with the equation distance moved by substance / distance moved by solvent . As seen earlier, the distance moved by substance is the distance from the start line to the centre of the spot.

Worked example: Look at the chromatogram in the diagram above.

Draw three conclusions about the three substances analysed by the chromatogram.

Answer: Any three from:

Substance A has four different substances, as there are four spots.

Substance B has three different substances, as there are three spots.

Substance C has three different substances, as there are three spots.

Substance A and B both have two of the same substances (red and pink spots).

Substances A and C both have two of the same substances (blue and yellow spots).

Substances B and C both have one of the same substances (green spot).

Worked example: Look at the chromatogram given here. Calculate the Rf value of the red spot.

Answer: Rf value = distance travelled by the spot/ distance travelled by the solvent = ⅗ = 0.6

Chromatography is a technique used to separate and identify the components of a mixture. It works by using the different physical and chemical properties of the components to separate them.

Chromatography is used in a variety of real-life applications, including the analysis of food and drink, the identification of drugs and chemicals, and the separation of proteins and enzymes in biochemistry. It is also used in environmental testing to detect and measure pollutants in air, water, and soil.

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Science in School

Colour, chlorophyll and chromatography teach article.

Author(s): Josep Tarragó-Celada, Josep M Fernández Novell

Use thin-layer chromatography to discover the variety of pigments that play a role in photosynthesis and give leaves their colour.

Looking out over a lush green valley or forest, it is fascinating to see the array of different shades. Leaves range from light to dark and even speckled. The colours are determined by the presence of different pigments, many of which are responsible for one of the most interesting and important metabolic reactions in living organisms: photosynthesis.

Photosynthetic pigments are located in the chloroplasts of the leaf. They capture energy from the visible light spectrum, which they use to synthesise carbohydrates from inorganic matter. There are many types of photosynthetic pigments, but the two main groups are chlorophylls and carotenoids (which are further split into two classes: carotenes and xanthophylls). Each type absorbs a different wavelength, so that together they capture more light.

Chlorophylls are the pigments primarily responsible for photosynthesis. They absorb red and blue light, and reflect green light, which is what gives leaves their green colour. Carotenoids, on the other hand, reflect yellow, orange and red – the colour of leaves during autumn. During this time of year, chlorophyll breaks down so the carotenoid pigments become visible.

Carotenoids assist with photosynthesis by absorbing wavelengths of light that chlorophylls cannot absorb. They transfer energy to chlorophyll molecules and also help to protect the leaf from excess light – they absorb surplus light energy and dissipate it as heat to prevent it from damaging the leaf.

Other non-photosynthetic pigments, such as anthocyanins or other flavonoids, determine the colour of flowers, so their absorption spectra vary. The function of these pigments is to attract insects or birds for pollination.

Absorption spectrum for photosynthetic pigments

Separating leaf pigments using thin-layer chromatography

This article presents a simple laboratory experiment to understand leaf pigments. Students use thin-layer chromatography to separate the various pigments that are present in two different leaf extracts. They identify each pigment and determine whether the two extracts have any pigments in common. The experiment is suitable for students aged 11–16 and takes 1–2 hours to complete.

Note that we used leaves from Epipremnum aureum (commonly known as devil’s ivy) and Ficus benjamina (commonly known as weeping fig) , but any species could be used for the leaf extracts. You might also like to carry out the experiment using a brightly coloured flower, such as those in the Petunia genus, and also a yellow or orange leaf.

For the thin-layer chromatography, we use a combined mobile phase of hexane, acetone and trichloromethane (3:1:1) as it provides the best separation result. However, it requires part of the activity to be carried out inside a fume hood by the teacher. This mobile phase separates the pigments most clearly, but you could adapt the activity to use mobile phases of hexane or ethanol alone, which the students can carry out themselves. Both hexane and ethanol successfully separate the pigments, but the distinction between each pigment is not as clear as when the combined solvent is used.

Leaf samples (e.g. E. aureum and F. benjamina ), cut into pieces measuring approximately 2 cm x 2 cm
Thin-layer chromatography plates (10 cm x 5 cm) pre-coated with silica gel
3 parts hexane, C 6 H 14
1 part acetone, (CH 3 ) 2 CO
1 part trichloromethane, CHCl 3
A beaker and watch glass (or chromatography chamber)
Spotting tile
Mortar and pestle
1 ml Pasteur pipettes (one for each leaf sample)

Safety note

A lab coat, gloves and eye protection should be worn. The solvents used in this experiment are flammable, so they must not be used near flames. The combined solvent (hexane, acetone and trichloromethane) must only be used inside a fume hood due to the volatility, smell and health risks associated with it.

The following steps should be carried out by the students:

Place your first leaf sample in the mortar. Pipette 1 ml of acetone into the mortar and use the pestle to grind the sample until the leaf is broken down.
Transfer the mixture to a well of the spotting tile using the pipette.
Wash the mortar and pestle, and repeat steps 1–2 using the second leaf sample. Use a new pipette to add 1 ml of acetone and use this pipette to transfer the mixture to a new well of the spotting tile.
Take the chromatography plate and draw a horizontal line 1.5 cm from the bottom using a pencil. Take care not to touch the plate with your fingers.
Using your first pipette (take care not to mix up which pipettes were used for each leaf sample), draw up some of your first leaf sample. Apply a single, small drop to the pencil line on the left hand side of the chromatography plate. Make sure to leave enough space to fit the second sample on the right hand side.
Wait a few seconds until it dries, and apply a second drop on the same spot. Continue until you have added around 10 drops.
Using your second pipette, repeat steps 5 and 6 for the second leaf sample by adding it to the right hand side of the plate.
Allow the plate to dry completely.

The following steps must be carried out by the teacher:

Inside the fume hood, combine the solvents in the following proportions: hexane, acetone and trichloromethane, 3:1:1.
Add the combined solvent to the beaker. You should add only a shallow layer of solvent, so that the pencil line on the chromatography plate will not be submerged.
Place the chromatography plate vertically into the beaker, with the pencil line at the bottom, and cover the beaker with a watch glass. Students can watch as the solvent moves up the plate and the pigments separate.
Wait until the solvent has travelled roughly 6 cm from the starting point (this will take approximately 15–30 minutes) before removing the plate from the beaker, leaving it inside the fume hood.
Use a pencil to quickly mark the furthest point reached by the solvent. Allow the plate to dry completely before removing it from the fume hood.
Photograph the chromatogram as soon as it is dry. The colours will fade within a few hours. Print out a copy of the photograph for your notes.
Using the chromatogram photo, try to work out how many pigments are present in each leaf extract.
Now look at the chemical structures of different pigments (see figure 1). Can you determine which pigment is which (see the explanation section for more guidance)? Write down your answers.
Measure the distances travelled by the solvent and the pigments, and calculate the retardation factor (Rf) using the following equation: Rf = (distance travelled by pigment) / (distance travelled by solvent)

Record your results in a table. Compare these to the values in table 1: were your answers correct?

Chemical structures of photosynthetic pigments

Explanation

The different pigments in a leaf extract are separated based on their affinities for the stationary phase (the silica on the thin-layer chromatography plate – a polar substance) and the mobile phase (the solvent – a nonpolar substance). Compounds with a high affinity for the solvent (i.e. nonpolar compounds) will move much further than compounds with a high affinity for silica (i.e. polar compounds).

In our example (see figure 2), both leaf extracts contained four pigments. Pigment 4 moved a shorter distance than pigment 1, indicating that pigment 4 is more polar and pigment 1 is less polar. By looking at the chemical structures of different pigments and the polar and nonpolar groups, students can try to identify the pigments in each of the leaf extracts.

They will need to know that, of the functional groups present in the pigments in figure 1, alcohol groups are the most polar, ester and ether groups the least polar, and aldehyde and ketone groups are in between. From this, we can deduce that carotenes are the least polar pigments (no polar groups), and xanthophylls are the most polar (two alcohol groups, one at each end of the molecule). Therefore, pigments 1 and 2 are likely to be carotenes, and pigment 4 is likely to be a xanthophyll. Pigment 3 is likely to be chlorophyll, since it is more polar than carotenes but less polar than xanthophylls. You can observe the characteristic green colour from chlorophyll on the chromatogram.

Chromatograms and corresponding Rf values for two leaf samples

Now look at the Rf values, which range between 0 and 1, with 0 being a pigment that does not move at all, and 1 indicating a pigment that moves the same distance as the solvent. The Rf value varies depending on the solvent used, but the general order of the pigments (from the highest to the lowest Rf value) usually remains the same, because the nonpolar compounds move further than the polar compounds. Rf values for various pigments (using hexane, acetone and trichloromethane (3:1:1) for the solvent) are shown in table 1.

).

β-carotene	0.98
Chlorophyll a	0.59
Chlorophyll b	0.42
Anthocyanins	0.32-0.62
Xanthophylls	0.15-0.35

After the experiment, you can ask your students some of the following questions to gauge their understanding of plant pigments and thin-layer chromatography.

Look at absorption spectra for various plant pigments. Which pigments absorb the most light from the red end of the spectrum? What colour are they?
If chlorophyll is the most important photosynthetic pigment, which colours of the visible spectrum are most useful to a plant for photosynthesis?
Seaweeds are often yellow-brown in colour. Do you think light from the red end or the blue end of the spectrum penetrates water best?
What species of plants have non-green leaves? How could you find out what pigments they contained?
Where are photosynthetic pigments located within a leaf?
Why is it useful for plants to contain several different photosynthetic pigments?
Why is it important to use a nonpolar solvent (such as hexane, acetone and trichloromethane) and not a polar solvent (such as water) to investigate plant pigments using thin-layer chromatography?
Why should you avoid touching the thin-layer chromatography plate?
Why should the plate be completely dry before putting it into the beaker?
Why do some pigments have a larger Rf value than others?
Reiss C (1994) Experiments in Plant Physiology . Englewood Cliffs, NJ, USA: Prentice Hall. ISBN: 0137012853
For an infographic explaining the chemicals behind the colour of leaves, visit the Compound Interest website .
Read more about the chemical structure of different plant pigments by visiting the Harvard Forest website from Harvard University.

Josep Tarragó-Celada is a PhD student in biochemistry at the faculty of biology in the Universitat de Barcelona, Spain. His work focuses on the metabolic reprogramming of cancer metastasis.

Josep M Fernández Novell is a professor in the department of biochemistry and molecular biomedicine at the Universitat de Barcelona.

Together, they presented this activity at the 2018 Hands-on Science conference in Barcelona, and they frequently organise and participate in educational activities to help bridge the gap between university and secondary school students.

Combining the outdoor element of nature with the identification of different chemical structures produces a perfect applied science lesson. The analysis of the different pigments in leaves has a clear visual outcome that can then be related to the chemical structures of the different photosynthetic pigments.

This practical activity affords students the opportunity to move beyond basic paper chromatography to the more complex technique of thin-layer chromatography. This cross-curricular task will engage students who enjoy biology-based topics such as photosynthesis as well as students who enjoy the problem-solving aspect of analytical techniques in chemistry.

The activity is most suitable for students aged 14–16 as part of a science club or extension activity. In addition to the main method, the authors provide suggestions for using different solvents to enable students to carry out the experiment entirely independently. With further detail, the activity could also be useful for students aged 16–19.

Many new terms are introduced, so the article provides an excellent chance to challenge students to understand concepts such as mobile and stationary phases, polarity of molecules and how biology is fundamentally based on chemical building blocks.

Caroline Evans, head of chemistry, Wellington College, UK

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Chromatography

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Chromatography is used to separate colours from mixtures of coloured substances such as inks, dyes and food colourings. A simple chromatography experiment can be carried out using chromatography paper or filter paper. The solvent travels up through the paper and the ink components dissolve into it and are carried up through the paper by the solvent.

The distance travelled by each of the components depends upon their solubility in that solvent. Some components are more soluble so will travel further with the solvent, whereas some are less soluble and will remain on the paper and not travel as far.

As the different components travel at different rates up the paper, this allows them to be separated. The pattern produced from the spreading out of the components is known as a chromatogram and the number of spots seen on the chromatogram indicates the composition of the original mixture.

In figure 1, a spot of the original ink mixture was placed at position x and, as the solvent soaked up the paper, was separated into three different spots – A, B and C. This tells us that the original ink was a mixture of three different chemicals. Spot C has barely moved up the paper suggesting it is not very soluble in the solvent. However, spot A has moved as far as the solvent; this suggests it must be very soluble in the solvent. Spot B has moved up the paper a good distance and sits between C and A so its solubility in the solvent must be somewhere in the middle of the other two.

Producing a paper chromatogram

Paper chromatography is used in forensic sciences to match signatures on documents to the pen belonging to a suspect. The method used in paper chromatography is simple but must be followed carefully to achieve accurate results. For example, imagine that a ransom letter has been written and there are three suspects with three different black pens. One of the suspects has written the letter but in order to decide which one, scientists must perform chromatography using the ink from the letter and comparing it to all three pens.

The ink from the pen which was used to write the letter can then be identified as the one which produces a pattern which matches to the pattern produced by the letter ink on the chromatogram.

A line is drawn in pencil approximately 2cm up from the bottom of the paper. Ink from the message is then dissolved in the smallest amount of solvent and a spot of this solution is placed onto the pencil line and labelled M. Spots of the inks from the suspects’ pens are also placed on the same line and labelled 1, 2 and 3, as shown below:

The chromatography paper is then placed into a container containing a shallow depth of solvent. The depth of the solvent must always be lower than the pencil line so that the spots of ink do not wash off the paper into the solvent and are therefore able to travel up the paper with the solvent instead.

A lid must be placed on top of the container to provide an atmosphere which is saturated with solvent vapour. The presence of a saturated atmosphere means that the solvent is less likely to evaporate as it travels up the paper.

The solvent will be absorbed by and travel up through the paper, carrying the different components of the ink mixtures with it. Each component travels at a different rate and is therefore separated into different coloured spots.

From the chromatogram produced, we can see that the pattern produced by the letter ink can be matched exactly to the pattern produced from suspect number 2’s pen. We can also see that pen 1 contains a combination of two different black dyes due to the presence of two individual spots.

While some components travel as far up as the solvent, some stay close to the base line. The components of a mixture can be identified by comparing the distance travelled by the substance to the distance travelled by the solvent. This comparison is known as the retention factor (Rf) value, and is compared to a database of known values to confirm the identity of the component.

The Rf value of a compound always remains the same for any component providing that the chromatography has been carried out in the same way. The equation used to calculate the Rf value is:

$\text{Rf} = \frac{\text{distance moved by the compound}}{\text{distance travelled by the substance}}}$

For example, a component of a mixture travels 8.4cm from the base line and the solvent travels 13.5cm. The Rf value for this component is calculated as:

$\text{Rf} = \frac{8.4}{13.5} = 0.62$

There are no units for the Rf value.

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How To Calculate

How to calculate the rf value., what is rf value.

In order to calculate the Rf value, we will need to understand the meaning of rf values.

Rf is short for retention or retardation factor. We can define it as the ratio of the distance the spot moved above the origin to the distance the solvent front moved above the origin.

Retention factors range between zero and one due to the fact that the solvent front is always larger from the distance traveled by the solute.

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, the retention factor for a given material should remain constant. This allows unknowns to be compared to the known materials.

Factors affecting Rf values.

The rf values of a particular substance should be constant irregardless of the concentration of the analyte. However, sometimes the rf value of a substance may change due to the following factors.;

Concentration of stationary phase
Mobile phase
Concentration of mobile phase
Temperature
Stationary phase

The rf values are applied in chromatography in order to make the technique more scientific rather than a mere interpretation by sight.

Formula to Calculate the Rf Value.

The Rf value of a compound is equal to the distance traveled by the compound divided by the distance traveled by the solvent front.

A solvent front traveled for 0.7cm on a thin-layer chromatography paper (TLC) while a compound traveled for 0.5 cm. Calculate the Rf value.

Therefore, the rf value is 0.7.

Calculate the Rf value if a compound travels 2.5 cm and the solvent front travels 6.0 cm.

Therefore, the rf value is 0.42.

How to Calculate Molecular Formula.

How to Calculate pH from Molarity.

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Separation of Plant Pigments by Paper Chromatography

The separation of plant pigments by paper chromatography is an analysis of pigment molecules of the given plant. Chromatography refers to colour writing . This method separates molecules based on size, density and absorption capacity.

Chromatography depends upon absorption and capillarity . The absorbent paper holds the substance by absorption. Capillarity pulls the substance up the absorbent medium at different rates.

Separated pigments show up as coloured streaks . In paper chromatography, the coloured bands separate on the absorbent paper. Chlorophylls, anthocyanins, carotenoids, and betalains are the four plant pigments.

This post discusses the steps of separating plant pigments through paper chromatography. Also, you will get to know the observation table and the calculation of the Rf value.

Content: Separation of Plant Pigments by Paper Chromatography

Paper chromatography, plant pigments, steps of plant pigment separation, observation, calculation.

It is the simplest chromatography method given by Christian Friedrich Schonbein in 1865. Paper chromatography uses filter paper with uniform porosity and high resolution.

The mixtures in compounds have different solubilities . For this reason, they get separated distinctly between the stationary and running phase.

The mobile phase is a combination of non-polar organic solvents. The solvent runs up the stationary phase via capillary movement.
The stationary phase is polar inorganic solvent, i.e. water. Here, the absorbent paper supports the stationary phase, i.e. water.

Plant pigments are coloured organic substances derived from plants. Pigments absorb visible radiation between 380 nm (violet) and 760 nm (red).

They give colour to stems, leaves, flowers, and fruits. Also, they regulate processes like photosynthesis, growth, and development.

Plants produce various forms of pigments. Based on origin, function and water solubility, plant pigments are grouped into:

Chlorophylls (green)
Carotenoids (yellow, orange-red)
Anthocyanins (red to blue, depending on pH)
Betalains (red or yellow)

Chlorophyll : It is a green photosynthetic pigment. Chlorophyll a and b are present within the chloroplasts of plants. Because of the phytol side chain, they are water-repelling . Their structure resembles haemoglobin. But, they contain magnesium as a central metal instead of iron.

Carotenoids : These are yellow to yellow-orange coloured pigments. Also, they are very long water-repelling pigments. Carotenoids are present within the plastids or chromoplasts of plants.

Anthocyanins : These appear as red coloured pigments in vacuoles of plant cells. Anthocyanins are water-soluble pigments. They give pink-red colour to the petals, fruits and leaves.

Betalains : These are tyrosine derived water-soluble pigments in plants. Betacyanins (red-violet) and betaxanthins (yellow-orange) are the two pigments coming in this category. They are present in vacuoles of plant cells.

You can separate all the above pigments using paper chromatography.

Video: Separation of Plant Pigments

Separation of Plant Pigments by Paper Chromatography

Preparation of Concentrated Leaf Extract

requirements to prepare concentrated leaf extract

Wash spinach leaves in distilled water.
Then take out the spinach leaves and allow the moisture to dry out.
After that, take a scissor and cut the leaves into the mortar.
Take a little volume of acetone into the mortar. Note : Acetone is used instead of water to mash the leaves because it is less polar than the water. This allows a high resolution of the molecules in the sample between the absorbent paper.
Then, grind spinach leaves using a pestle until liquid paste forms. Note : The liquid in the crushed leaf paste is the pigment extract.
After that, take out the mixture into the watch glass or Petri dish.

Load the Leaf Extract onto Absorbent Paper

Take Whatman filter paper and draw a line above 2 cm from the bottom margin. You can use a pencil and scale to draw a fainted line. Note : A pencil is used because pencil marks are insoluble in the solvent.
Then, cut the filter paper to make a conical edge from the line drawn towards the margin end. You can use a scissor to cut the Whatman filter paper. Note : The conical end at the bottom of the filter paper results in better separation.
Put a drop of leaf extract on the centre of a line drawn on the absorbent paper.
Then, at the same time dry the absorbent paper.
Repeat the above two steps many times so that the spot becomes concentrated enough.

Setup the Chromatography Chamber

requirements to setup chromatography chamber

Take a clean measuring cylinder and add rising solvent (ether acetone) up to 4 ml.
Bend the strip of paper from the top. Then, using a pushpin attach the paper to the bottom of the cork.
Adjust the length of the paper. The absorbent paper should not touch the surface of the measuring cylinder.
After that, allow the solvent to move up the absorbent paper.
When the solvent front has stopped moving, remove the paper.
Allow it to dry for a while until the colours completely elute from the paper.
At last, mark the front edge travelled by each pigment.

Over the dried paper strip, you will see four different bands. Different colour streaks form because of different affinities with the mobile phase (solvent).

The carotene pigment appears at the top as a yellow-orange band.
A yellowish band appears below the carotene, which indicates xanthophyll pigment.
Then a dark green band represents the chlorophyll-a pigment.
The chlorophyll-b pigment appears at the bottom as a light green band.

Observation Table

Band Colour	Plant Pigment	Distance from sample spot (cm)	Solvent front (cm)	Rf Value
Light green	Chlorophyll-b	2 cm	10 cm	0.2
Dark green	Chlorophyll-a	3.7 cm	10 cm	0.37
Yellow	Xanthophyll	5.6 cm	10 cm	0.56
Yellow-orange	Carotene	9 cm	10 cm	0.9

1. Light green spot indicates chlorophyll-b pigment.

Rf value= Distance chlorophyll-b travelled / Distance solvent travelled = 2/10 = 0.2

2. Dark green spot represents chlorophyll-a pigment.

Rf value= Distance chlorophyll-a travelled / Distance solvent travelled = 3.7/10 = 0.37

3. The yellow band represents xanthophyll pigment.

Rf value= Distance xanthophyll travelled / Distance solvent travelled = 5.6/10 = 0.56

4. The yellow-orange band indicates carotene pigment.

Rf value= Distance carotene travelled / Distance solvent travelled = 9/10 = 0.9

Factors affecting the Rf values of a particular analyte are:

Stationary phase
The concentration of the stationary phase
Mobile phase
The concentration of the mobile phase
Temperature

The Rf value of compounds in the mixture differs by any changes in the concentration of stationary and mobile phases.

Temperature affects the solvent capillary movement and the analyte’s solubility in the solvent. Rf value is independent of the sample concentration. Its value is always positive .

1 thought on “Separation of Plant Pigments by Paper Chromatography”

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Practical chromatography

By Dorothy Warren and Kay Stephenson 2017-03-09T08:10:00+00:00

No comments

Ideas for enhancing chromatography practical work

Why study chromatography?

Using modern applications and contexts is a great way to hook your students and capture their interest. Look at figure 1 and think about what the images have in common.

Source: Shutterstock

Figure 1: A crime scene, Egyptian mummy, hamburger and mobile phone

Chromatography is the link: Chromatography Today is a great place to find interesting stories about chromatography and its applications to share with your students.

Chromatography is a key analytical technique students will meet several times during their secondary education (see the chromatography curriculum information sheet – a summary of the programme of study and subject content at key stages 3, 4 and 5). It also a required practical activity in most GCSE courses (table 1).

Table 1: Practical work required by GCSE specifications
Specification	Practical
AQA GCSE Chemistry (8462)	Investigate how paper chromatography can be used to separate and tell the difference between coloured substances. Students should calculate RF values.
Edexcel GCSE (9-1) Chemistry	Investigate the composition of inks using simple distillation and paper chromatography.
OCR GCSE (9-1) Gateway Chemistry A	Using chromatography to identify mixtures of dyes in an unknown ink.
OCR GCSE (9-1) Twenty First Century Science Chemistry B	Using chromatography to identify mixtures of dyes in a sample of an unknown composition
WJEC Eduqas GCSE (9-1) Chemistry	Separation of liquids by distillation and by paper chromatography

How can we ensure there is a clear progression in the knowledge students develop? How can we maintain their interest and help them develop practical skills? How can we make chromatography in the classroom more engaging?

Underpinning chemistry and progression

The chemistry behind chromatography involves some quite basic concepts (figure 2) that are first introduced in primary schools and then revisited during key stage 3. These important ideas lay the foundations for understanding chromatography at key stage 4, when they can be further developed and applied to this technique.

Figure 2: Basic chemical concepts underpinning chromatography

It is worth taking a moment to check your student’s understanding of the basic concepts (see table 2 for suitable resources) before launching into chromatography at key stage 4.

Resource	Comment
	Assessment for Learning is an effective way of actively involving students in their learning. This lesson plan comes with student worksheets and suggestions of how to organise activities
	These activities provide strategies for dealing with some misconceptions students may have, in the form of ready-to-use classroom resources
	Use this book chapter to enhance your own knowledge of different types of chromatography
	Further background information, including how to develop chromatograms so colourless spots become visible, and how to get better separation by using two-way chromatography

Developing practical skills and progression

There is a danger students will simply repeat the same chromatography experiment throughout their education. Popular activities with key stage 2 children include separating the mixtures found in felt-tip pens and sweets. At key stage 3 students separate the mixtures in inks and food colourings. Some exam boards specify the chromatography of inks and dyes as GCSE practical activities (table 1).

One of the challenges faced by teachers is how to develop students’ practical skills and maintain their interest if they are repeating experiments. There are several possible tactics: using different contexts; setting investigative or problem-solving activites; and taking a synoptic approach.

Another challenge with practical work is for students to understand what they are doing and why they are doing it. Getting this correct during lessons will help students answer practical-based exam questions. One tactic is to provide plenty of opportunities for students to do more than just follow a given procedure.

Source: Flo~commonswiki

Synoptic approach

Example: the chromatography of pigments in leaves

Some possible questions to get your students started:

What is the green pigment in leaves?
Where have you come across it before?
Where have you extracted chlorophyll before?
How did you extract the chlorophyll?
What techniques did you use?
How could you carry out the chromatography experiment?

Planning activity

Explain how you would carry out each stage. Give details of the equipment needed and experimental procedures.

Practical problems and suggested solutions

Figure 3: Typical paper chromatograms of water soluble inks or food dyes obtained by students (a) and as shown by textbooks or exam papers (b)

Chromatography is often seen as a quick and easy activity. However, the chromatograms students see in their textbooks and on exam papers often look nothing at all like the chromatograms from their own experiments. Typical examples are shown in figure 3.

So, faced with these results:

How do we convince students they have carried out a similar process?
Where is the centre of the spot?
What value should the student use to calculate the R F value?
How can you compare R F values with such big error bars?

Calculating R F

R F = Distance moved by compound / Distance moved by solvent

For the purple spot in figure 3b (the textbook diagram), the R F is 0.8. For the same spot in figure 3a (typical student results), what is the distance moved by the spot? Where would you draw the line? There will be a very large error in any value calculated.

Improving the results

Comparison of paper and thin layer chromatography plates

Figure 4: The effect of changing only the stationary phase: (a) paper (b) thin layer (silica on a plastic backing)

Paper chromatography doesn’t give very good separation in classroom experiments. You would need a long piece of paper and lengthy elution time to get good separation. However, there are several things you can do to make improvements.

Changing the stationary phase by using thin layer chromatography (TLC) can lead to better separation. In figure 4, TLC gives clear bands with more easily measurable RF values: the solvent in both cases was a dilute solution of sodium chloride and the same black ink is used in both. Note the difference in the order in which the component dyes move up the stationary phase.

TLC is not covered in all GCSE specifications (see the curriculum information sheet, below) but it gives great results and the principles and underlying chemistry are the same as paper chromatography. The only real difference is the stationary phase, which may be silica on a polyester backing or aluminium oxide coated plates. The mobile phase remains the same but the way the two phases interact with the sample component is different. 1

Figure 5 shows how changing the mobile phase, ie using different solvents, can also give better separation.

Figure 5: TLC chromatogram of ink showing the effect of changing only the mobile phase (the eluting solvent): a) H 2 O, b) NaCl (aq) (~2% w/v) c) NaHCO 3 (aq) (~2% w/v) d) Ethanol (aq) (~2% v/v)

Changing the phases is the basis of a good investigation. The activity could be a problem solving exercise or competition, where students have to work out how to get good separation and then go on to produce the best chromatogram.

Hopefully your students will be able to accept that the chromatograms seen in figures 4b and 5 are a little closer to what they might meet in the exam (figure 3b).

Chromatography equipment

Figure 6: Chromatography equipment

Although TLC plates are expensive, a classroom set of small plates can be cut from one standard plate. (Tip: If using right-handed scissors, try cutting with the backing side of the sheet uppermost. This seems to minimise flaking of the fragile coating. Alternatively, use a scalpel, ruler and cutting board.)

An expensive chromatography tank is not necessary to get good chromatograms. There are many methods that give good results. A one-holed boiling tube and crocodile clip (figure 6a) is useful for large classes and when volatile solvents (eg ethanol or propanone) are needed. Only small quantities of solvents are needed and fumes can be kept to a minimum.

A small vial and thin layer plate (figure 6b) can also give good separation over a very short distance. This small-scale approach is described by several sources , including these activities on plant pigments .

TLC plates with a fluorescing agent in the coating can be obtained (figure 6c). These are useful for locating colourless components – many colourless compounds absorb UV light and appear as darker spots.

Learn Chemistry resources

There are a number of chromatography resources on Learn Chemistry for students of all levels. Some provide further background information, such as how to link chromatography with particle theory and how chromatography is used in the workplace. Others are experiments ready to use with your students, for example the chromatography of leaves or sweets.

Download a table of learn chemistry resources, with ideas on how to include them in your teaching, below.

Kay Stephenson and Dorothy Warren are independent science education consultants. Figures 3, 4, 5, and 6 courtesy of Kay Stephenson

Resources on Learn Chemistry

Curriculum information.

B Faust, Modern chemical techniques , p119. Royal Society of Chemistry, 1997 ( rsc.li/2kmh457 )
Chromatography
Developing teaching practice
Practical skills and safety
Professional development

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RF Value Calculator

Introduction.

The RF (Retention Factor) Value is a crucial parameter in chromatography, a technique widely used in chemistry to separate and analyze components of a mixture. It represents the ratio of the distance traveled by a solute to the distance traveled by the solvent in a chromatographic process. Calculating the RF Value is essential for identifying and characterizing substances within a mixture. The RF Value Calculator simplifies this process by allowing you to quickly determine the RF Value based on the distances traveled by the solute and solvent. In this article, we will introduce the concept of the RF Value Calculator, provide the formula for calculating RF Value, explain how to use it effectively, offer an example scenario, address common questions in the FAQs section, and conclude with the significance of RF Values in chromatography.

The formula for calculating the RF Value is straightforward:

RF Value = Distance Traveled by Solute (DSU) / Distance Traveled by Solvent (DSV)

Distance Traveled by Solute (DSU) represents the distance the solute travels from its origin on the chromatographic medium.
Distance Traveled by Solvent (DSV) signifies the distance the solvent travels from the same origin.

By using this formula, you can quickly determine the RF Value, which is used to identify compounds and assess the quality of a chromatographic separation.

To use the RF Value Calculator effectively, follow these steps:

Set Up Your Chromatography Experiment : Perform your chromatography experiment, ensuring that you have accurate measurements of DSU (Distance Traveled by Solute) and DSV (Distance Traveled by Solvent).
Input Data : Enter the values of DSU and DSV into the respective fields of the calculator.
Click Calculate : Press the “Calculate” button to initiate the calculation.
Get the Result : The calculator will compute the RF Value and display it in the designated field.
Interpret the Result : Examine the RF Value to identify and characterize the components of your mixture.

Let’s consider an example to illustrate how to use the RF Value Calculator:

Distance Traveled by Solute (DSU): 5.2 cm
Distance Traveled by Solvent (DSV): 10.4 cm

Using the formula, you can calculate the RF Value as follows:

RF Value = 5.2 cm / 10.4 cm = 0.5

In this scenario, the RF Value for the chromatographic separation is 0.5 .

Why is the RF Value important in chromatography?

The RF Value is crucial for identifying and characterizing substances within a mixture. It provides a reference point for comparing the migration distances of different compounds in chromatography.

What does an RF Value of 1 signify?

An RF Value of 1 indicates that the solute and solvent traveled the same distance, meaning the solute did not interact with the chromatographic medium. It can suggest that the solute is highly soluble in the solvent.

Can the RF Value be used for quantitative analysis?

While the RF Value is primarily used for qualitative analysis, it can provide relative quantification when comparing the RF Values of different compounds in the same chromatographic system.

The RF Value Calculator simplifies the determination of RF Values in chromatography, a technique widely used in chemistry for component separation and analysis. By using the formula and following the steps outlined in this article, you can quickly calculate the RF Value and use it to identify and characterize substances within mixtures. RF Values are essential tools for chemists and researchers, providing valuable insights into the behavior of compounds in chromatographic systems and aiding in the interpretation of experimental results. Understanding and calculating RF Values are fundamental skills for anyone working in the field of chromatography and chemical analysis.

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COMMENTS

RF Value
RF Value - RF value (in chromatography) The distance travelled by a given component divided by the distance travelled by the solvent front. ... Because these variables are challenging to maintain consistency from experiment to experiment, relative R f values are commonly used. "Relative R f " denotes that the results are provided in ...
Understanding Chromatography and the Calculation of Rf Values
Gather equipment: For this experiment, you will need a solvent, different coloured inks, filter paper, a container, a pencil, ... To calculate the Rf value in Chromatography, you divide the distance travelled by the component by the distance travelled by the solvent. For example, if the component travels 5cm and the solvent travels 10cm, the Rf ...
Paper Chromatography Full Experiment Including Calculating Rf Values
Demonstration to show hot to carry out paper chromatography to identify components in an unknown mixture. Includes explanations and how to calculate Rf value...
Rf Value: Calculation, Significance and Affecting Factors
1. Definition of Rf value. Rf value or Retention factor is defined as it is ratio of distance travelled by solute to the distance travelled by solvent. Since it is ratio there is no unit for Rf value. (Fig 1) Figure 1: Calculation of Rf value or Retention factor. The Rf value is reported in Paper chromatography or Thin layer chromatography (TLC ...
PDF CHEM 2219: Exp. #1 Thin Layer Chromatography (TLC)
Microsoft Word - 2219_EXP1_TLC_procedure_FS22.docx. Objective: In this experiment you will learn to separate the components of a solution using thin layer chromatography; and, to determine the solvent polarity effects. Retention factors will be calculated to determine the identity of the unknown compounds and theoretical plates will be ...
RF Value
The Rf value (retardation factor/Retention Factor) is an important parameter in chromatography that represents the distance traveled by a particular compound relative to the distance traveled by the solvent front. The Rf value is a dimensionless number that ranges from 0 to 1. It is calculated by dividing the distance traveled by the compound ...
How to Find the Rf Value in Chromatography
How to Find the Rf Value in Chromatography. by Taha Cheema. As an Chemistry student, knowing how to find Rf value in chromatography is crucial. If you have been practicing O Level or IGCSE Chemistry past papers, you would have noticed that Rf value questions are common in Paper 1 (MCQs) and chromatography experiments are also tested in the ATP.So let's dive in and understand this calculation ...
How to Calculate Rf Value for Thin Layer Chromatography (TLC)
You will calculate its Rf value as follows: Rf = D_solute / D_solvent = 3 cm / 9 cm = 0.33. Important Notes. - Be precise in all measurements while marking spots and measuring distances. - Always compare the Rf values of unknown compounds to known reference standards or literature data. - The same compound might exhibit different Rf ...
Chromatography and Rf Values (GCSE Chemistry)
Substances B and C both have one of the same substances (green spot). Worked example: Look at the chromatogram given here. Calculate the Rf value of the red spot. GCSE Chemistry - Chromatography and Rf Values. Answer: Rf value = distance travelled by the spot/ distance travelled by the solvent = ⅗ = 0.6.
Colour, chlorophyll and chromatography
The Rf value varies depending on the solvent used, but the general order of the pigments (from the highest to the lowest Rf value) usually remains the same, because the nonpolar compounds move further than the polar compounds. Rf values for various pigments (using hexane, acetone and trichloromethane (3:1:1) for the solvent) are shown in table 1.
Chromatography
Rf values. Chromatography is used to separate colours from mixtures of coloured substances such as inks, dyes and food colourings. A simple chromatography experiment can be carried out using chromatography paper or filter paper. The solvent travels up through the paper and the ink components dissolve into it and are carried up through the paper ...
PDF Thin layer chromatography
Rf values always lie between 0 and 1 (0 being a pigment that doesn't move at all and 1 being a pigment that is so soluble, it moves the same distance as the solvent). Because for a given pigment, the Rf value will vary according to the solvent (or mixture of solvents) used, Rf values are often written with the
How to Calculate the Rf Value
Formula to Calculate the Rf Value. The Rf value of a compound is equal to the distance traveled by the compound divided by the distance traveled by the solvent front. Example 1: A solvent front traveled for 0.7cm on a thin-layer chromatography paper (TLC) while a compound traveled for 0.5 cm. Calculate the Rf value. Therefore, the rf value is 0.7.
Separation of Plant Pigments by Paper Chromatography
1 Comment / Botany / By Supriya N. The separation of plant pigments by paper chromatography is an analysis of pigment molecules of the given plant. Chromatography refers to colour writing. This method separates molecules based on size, density and absorption capacity. Chromatography depends upon absorption and capillarity.
Practical chromatography
Students should calculate RF values. Edexcel GCSE (9-1) Chemistry: 2.11 Core practical Investigate the composition of inks using simple distillation and paper chromatography. ... There is a danger students will simply repeat the same chromatography experiment throughout their education. Popular activities with key stage 2 children include ...
PDF Plant Pigment Paper Chromatography
Separation of Pigments: Place the test tube in the test tube rack. Using the 6mL syringe, dispense 5 mL of chromatography solvent in the test tube. Carefully lower the paper strip into the test tube and secure the cork in the top. The solvent must touch the pointed end of the paper but should not touch the green line.
RF Value Calculator
The RF Value Calculator simplifies the determination of RF Values in chromatography, a technique widely used in chemistry for component separation and analysis. By using the formula and following the steps outlined in this article, you can quickly calculate the RF Value and use it to identify and characterize substances within mixtures.
Lab 11 (pdf)
Pre Lab-Questions 1. In a chromatography experiment measuring the polarity of amino acid functional groups, one amino acid has an Rf value of 0.32 and another had an Rf of 0.68 - which one traveled furthest on the chromatography paper? a. The fastest moving spot had the highest Rf value, so it traveled far in the chromatography paper. 2.

What is R F Value?

Table of Contents

Calculation of R F Value

Why do we need the R F value?

Factors Affecting R f Values

2. Other factors

Frequently Asked Questions on R f Values

Can R f value be greater than 1?

How does polarity affect R f value?

Why is polarity important for chromatography?

What is the difference between polar and nonpolar molecules?

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Chromatography

The Two Phases of Chromatography

Molecules in the Mobile Phase

Molecules in the Stationary Phase

Distribution Between Phases

Applications of Chromatography

Types of Chromatography

Advantages of Chromatography

Disadvantages of Chromatography

How to Calculate Rf Values

Understanding Rf Values

What is Chromatography?

What is the Rf value in Chromatography?

Why is the Rf value important in Chromatography?

What factors can affect the Rf value of a component in Chromatography?

How do you calculate the Rf value in Chromatography?

How does Chromatography work?

What are the different types of Chromatography?

How is Chromatography used in real-life applications?

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Table of Contents

1. Definition of Rf value

2. Thin layer chromatography

3. Calculation of Rf value

4. Importance of Rf value

4.1 Monitoring Chemical Reactions

4.2 Monitoring Purification by Column Chromatography

5.1 Solvent (Mobile phase)

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How to Find the Rf Value in Chromatography

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Differences Between Rf Values

What are the Factors Affecting Rf value in Paper Chromatography?

Conclusion

Q. What is Chromatography?

Q. What is an Rf value in Chromatography?

Q. How do you calculate the Rf value in Chromatography?

Q. What is the purpose of the Rf value in chromatography?

Q. What are the Factors Affecting Rf value in Paper Chromatography?

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Separating leaf pigments using thin-layer chromatography