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Experiment  726: Paper Chromatography:  A Technique of Separation and Identification  

Section  1:  Purpose and Summary  

One of the problems encountered most frequently in chemistry is that of separating a mixture into the pure substances which compose it. Most natural materials, such as seawater, air, oil, coal, and so on are mixtures. In order to study these materials chemically, we must first treat the mixture in some way to separate it into single, pure substances. (A "pure substance" consists entirely of just one thing; meaning one kind of molecule.) Because it does consist of just one thing, it always behaves in the same way when tested. For example, pure water always boils at 100°C (212°F), provided that the test is done at atmospheric pressure.  

Many different methods have been devised for separating mixtures into their components. In the present experiment, we will use a method called chromatography. It is used quite widely for making small-scale separations and identifications. The method works because of the differences in the ways various components of a dissolved mixture interact with a fixed solid.  The fixed solid can be made of different materials and in different shapes, depending on the version of chromatography that is being done.   One version is called paper chromatography. 

To perform paper chromatography to separate components of a mixture, there are several steps.  First, the mixture that is to be separated is dissolved (if it is not already in liquid form.) Then, a small drop of the solution is applied to a piece of special chromatography paper, which is a porous paper similar to filter paper. This drop makes a tiny spot on the paper. Many such spots (of different materials) may be placed side by side on the same piece of paper. Next, the spotted paper is made to stand in a small amount of some special solvent (liquid), in such a way that only the bottom edge of the paper is submerged in the liquid, not the spots. The paper acts like a wick, drawing the liquid up the paper by capillary action. The solvent then slowly rises up the paper, reaches the spots, and begins to dissolve them and carry the substances up the paper with it.  

The key to this separation is that the different components of the mixture in each spot interact with the paper differently, and so will be found (after a few minutes) to have reached different heights on the paper. This happens because some components are more strongly attracted to the paper (and so move more slowly), while others are more strongly attracted to the solvent. Any particular substance always moves at the same rate, no matter what else it may have been mixed with originally. For that reason, it can always be recognized and identified, because it will always rise to the same height, relative to the heights that the other components rise. 

The movement of any spot on the paper can be quantified by calculating its  R f  ( retention factor ) value after you have stopped the experiment and the paper has dried. 

    R f   =  distance traveled by the solute  

distance traveled by the solvent front 

The distances used in calculating  R f   values are measured as shown in the following figure. To determine the  distance traveled by the solute , measure from the point at which you originally applied the spot to the center or densest part of the spot. The  distance traveled   by the solvent front  is measured from the original point of application of the spot to the limit of movement of the solvent front (which must be marked immediately after the paper is removed from the beaker, because it may be nearly invisible after the solvent evaporates). 

If all conditions could be maintained constant,  R f   values would be constant. However, either variations in temperature or in the composition of the solvent phase or changes in the paper can alter the  R f   value. The  R f   value is useful mainly for expressing the relative mobility of two or more solutes in a particular chromatographic system. The absolute  R f   values may change from day to day, but their values in relation to each other remain nearly constant. 

Figure 1:  Completed paper chromatography containing only 1 dye. 

In this experiment, students will measure the values of several dyes in 3 different solvent systems. Students will also analyze an unknown mixture of dyes in order to identify the dyes present in the mixture.  The three different solvent systems are 1) laboratory water, 2) an aqueous solution of 0.10% sodium chloride (table salt), and 3) rubbing alcohol (70% isopropyl alcohol and 30% water).  By using different solvents, the dyes will travel differently in paper chromatography, and the  R f  values will be different.  The goal is to find a solvent that gives a different value for every dye as this will separate all the dyes from each other (scientists say that the dyes are  resolved  when the solvent system creates distinctly different values for each dye.)  When every dye is separated from all the other dyes, then you can match up  R f   values and identify each dye in the unknown mixture. 

Section 2:  Safety Precautions and Waste Disposal  

Safety Precautions:  

Use of eye protection is recommended for all experimental procedures.   

The rubbing alcohol used in this lab gives off fumes, so keep the beaker containing this solvent covered with plastic wrap when it is not in the hood. 

Waste Disposal:  

The rubbing alcohol should be disposed of in the organic waste container in the hood. The aqueous solvents may be poured down the drain. The chromatography paper can be disposed in the trash. 

Section 3: Procedure  

Preparing and developing the chromatogram  

Section 4 :    Analysis of the Chromatogra m s  

Section 5:  Identifying the components in the  Unknown  Mixture  

Post Lab Questions:  

Why use a pencil and not a pen to mark your chromatograms? 

2) Why is it important that the chromatography paper not touch the sides of the beaker? 

3) Which solvent system worked the best for you? Explain. 

4) Suppose a student did today’s experiment and obtained the following results: 

4a) Which solvent is best for separating Dyes #1-3? 

4b) Assume that the student receives an Unknown mixture that contains Component Dye #2 and Component Dye #3.  Draw the chromatogram the student would expect to obtain when analyzing Dye #1, Dye #2, Dye #3 and her “Component” mixture when using the best solvent, the same solvent you picked in question 4a.  Include the points of application and the solvent front in your drawing. 

Notes: 

Microbe Notes

Paper Chromatography- Definition, Types, Principle, Steps, Uses

Table of Contents

Interesting Science Videos

What is Paper Chromatography?

Paper chromatography (PC) is a type of planar chromatography whereby chromatography procedures are run on a specialized paper.

PC is considered to be the simplest and most widely used of the chromatographic techniques because of its applicability to isolation, identification, and quantitative determination of organic and inorganic compounds.

It was first introduced by German scientist Christian Friedrich Schonbein (1865).

Types of Paper chromatography

Paper adsorption chromatography.

Paper impregnated with silica or alumina acts as adsorbent (stationary phase) and solvent as mobile phase.

Paper Partition Chromatography

Moisture / Water present in the pores of cellulose fibers present in filter paper acts as stationary phase & another mobile phase is used as solvent In general paper chromatography mostly refers to paper partition chromatography. 

Principle of Paper chromatography

The principle of separation is mainly partition rather than adsorption. Substances are distributed between a stationary phase and a mobile phase. Cellulose layers in filter paper contain moisture which acts as a stationary phase. Organic solvents/buffers are used as mobile phase. The developing solution travels up the stationary phase carrying the sample with it. Components of the sample will separate readily according to how strongly they adsorb onto the stationary phase versus how readily they dissolve in the mobile phase.

Instrumentation of Paper chromatography

• Stationary phase & papers used
• Mobile phase
• Developing Chamber
• Detecting or Visualizing agents

1. STATIONARY PHASE AND PAPERS

• Whatman filter papers of different grades like No.1, No.2, No.3, No.4, No.20, No.40, No.42 etc
• In general the paper contains 98-99% of α-cellulose, 0.3 – 1% β -cellulose.

Other modified papers

• Acid or base washed filter paper
• Glass fiber type paper.
• Hydrophilic Papers – Papers modified with methanol, formamide, glycol, glycerol etc.
• Hydrophobic papers – acetylation of OH groups leads to hydrophobic nature, hence can be used for reverse phase chromatography.
• Impregnation of silica, alumna, or ion exchange resins can also be made.

2. PAPER CHROMATOGRAPHY MOBILE PHASE

• Pure solvents, buffer solutions or mixture of solvents can be used.

Hydrophilic mobile phase

• Isopropanol: ammonia:water 9:1:2
• Methanol : water 4:1
• N-butanol : glacial acetic acid : water 4:1:5

Hydrophobic mobile phases

• dimethyl ether: cyclohexane kerosene : 70% isopropanol
• The commonly employed solvents are the polar solvents, but the choice depends on the nature of the substance to be separated.
• If pure solvents do not give satisfactory separation, a mixture of solvents of suitable polarity may be applied.

3. CHROMATOGRAPHIC CHAMBER

• The chromatographic chambers are made up of many materials like glass, plastic or stainless steel . Glass tanks are preferred most.
• They are available invarious dimensional size depending upon paper length and development type.
• The chamber atmosphere should be saturated with solvent vapor.

Steps in Paper Chromatography

In paper chromatography, the sample mixture is applied to a piece of filter paper, the edge of the paper is immersed in a solvent, and the solvent moves up the paper by capillary action. The basic steps include:

Selection of Solid Support

Fine quality cellulose paper with defined porosity, high resolution, negligible diffusion of the sample, and favoring good rate of movement of solvent.

Selection of Mobile Phase

Different combinations of organic and inorganic solvents may be used depending on the analyte.

Example. Butanol: Acetic acid: Water (12:3:5) is a suitable solvent for separating amino acids.

Saturation of Tank

The inner wall of the tank is wrapped with filter paper before the solvent is placed in the tank to achieve better resolution.

Sample Preparation and Loading

If the solid sample is used, it is dissolved in a suitable solvent. Sample (2-20ul) is added on the baseline as a spot using a micropipette and air dried to prevent the diffusion.

Development of the Chromatogram

Different types of development techniques can be used:

ASCENDING DEVELOPMENT

• Like conventional type, the solvent flows against gravity.
• The spots are kept at the bottom portion of paper and kept in a chamber with mobile phase solvent at the bottom.

DESCENDING TYPE

• This is carried out in a special chamber where the solvent holder is at the top.
• The spot is kept at the top and the solvent flows down the paper.
• In this method solvent moves from top to bottom so it is called descending chromatography.

ASCENDING – DESCENDING DEVELOPMENT

• A hybrid of above two techniques is called ascending-descending chromatography.
• Only length of separation increased, first ascending takes place followed by descending.

CIRCULAR / RADIAL DEVELOPMENT

• Spot is kept at the centre of a circular paper.
• The solvent flows through a wick at the centre & spreads in all directions uniformly.

Drying of Chromatogram

After the development, the solvent front is marked and left to dry in a dry cabinet or oven.

Colorless analytes were detected by staining with reagents such as iodine vapor, ninhydrin, etc.

Radiolabeled and fluorescently labeled analytes were detected by measuring radioactivity and fluorescence respectively.

Some compounds in a mixture travel almost as far as the solvent does; some stay much closer to the baseline . The distance traveled relative to the solvent is a constant for a particular compound as long as other parameters such as the type of paper and the exact composition of the solvent are constant. The distance traveled relative to the solvent is called the Rf value.

Thus, in order to obtain a measure of the extent of movement of a component in a paper chromatography experiment, “Rf value” is calculated for each separated component in the developed chromatogram. An Rf value is a number that is defined as the distance traveled by the component from the application point.

Applications of Paper Chromatography

• To check the control of purity of pharmaceuticals,
• For detection of adulterants,
• Detect the contaminants in foods and drinks,
• In the study of ripening and fermentation,
• For the detection of drugs and dopes in animals & humans
• In analysis of cosmetics
• Analysis of the reaction mixtures in biochemical labs.

Advantages of Paper Chromatography

• Paper Chromatography requires very less quantitative material.
• Paper Chromatography is cheaper compared to other chromatography methods.
• Both unknown inorganic as well as organic compounds can be identified by paper chromatography method.
• Paper chromatography does not occupy much space compared to other analytical methods or equipments.
• Excellent resolving power

Limitations of Paper Chromatography

• Large quantity of sample cannot be applied on paper chromatography.
• In quantitative analysis paper chromatography is not effective.
• Complex mixture cannot be separated by paper chromatography.
• Less Accurate compared to HPLC or HPTLC
• http://frndzzz.com/Advantages-and-Disadvantages-of-Paper-Chromatography
• https://www.slideshare.net/shaisejacob/paper-chromatography-pptnew?next_slideshow=1
• https://www.slideshare.net/shaisejacob/paper-chromatography-ppt-new
• https://www.biochemden.com/paper-chromatography/
• http://web.engr.oregonstate.edu/~rochefow/K-12%20Outreach%20Activities/Microfluidics%20&%20Pregnancy%20Test%20Kit%20Lab/paper%20chromatography_Chemguide.pdf
• https://pubs.acs.org/doi/abs/10.1021/ac60051a002

About Author

Sagar Aryal

6 thoughts on “Paper Chromatography- Definition, Types, Principle, Steps, Uses”

Examples of substances that can be effectively separated by paper chromatography is necessary.

I enjoy this write up, but the definition of Rf Values just mention distance traveled by the solute from the point from the point of application of the sample, how about the total distance traveled by the solvent? Some examples of how Rf value can be calculated is necessary.

Pls how is charging a disadvantage of filter paper in chromatography

Hi! I enjoyed reading it. Hmm just wanna know somepoints.

Why it is best to use the farthest distance traveled by a sugar if and when the solvent went over the paper and what is the purpose of heating the chromatographic paper after running the procedure?

This links are so much useful and it’s very helpful also….

Thanks for sharing important details like this. I enjoyed reading your site, and love to know the latest updates.

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Paper Chromatography – Principle, procedure, Applications

What is Paper Chromatography?

Paper chromatography definition explains that is an inexpensive and powerful analytical technique, which requires a piece of paper or strips serving as an adsorbent in the stationary phase across which a particular solution is allowed to pass.

For the separation of dissolved chemical substances and lipid samples (in particular), paper chromatography is found to be very trustable. This analytical tool employs very few quantities of material.

Principle of Paper Chromatography

Paper chromatography is a form of liquid chromatography where the basic principle involved can be either partition chromatography or adsorption chromatography.

In paper chromatography separation of component is distributed between phases of liquid. Here, one phase of liquid is water that is held amidst the pores of filter paper and the other liquid is the mobile phase that travels along with the filter paper. Separation of the mixture is the result that is obtained from the differences in the affinities towards the water and mobile phase when travelling under capillary action between the pores of the filter paper.

Though in a majority of paper chromatography applications, the principle is based on partition chromatography but sometimes, adsorption chromatography can take place where the stationary phase is the solid surface of the paper and the mobile phase is the liquid phase.

Paper Chromatography procedure

• Selection of the ideal type of development: Based on factors such as the complexity of the solvent, mixture, paper, etc. the development type is chosen. Mostly either Radial or Ascending type of paper chromatography is employed because of the easiness they offer while handling and performing which ultimately leads to obtaining the chromatogram faster within a shorter duration of time.
• Selection of Filter paper: As per the pores’ size and the sample quality.
• Sample preparation: This involves the dissolution of the sample in an ideal solvent that is being utilized in developing the mobile phase.
• Sample loading or spotting on the paper: With the help of a capillary tube, micropipette, the sample is spotted on the paper at an accurate position. This promotes the interpretation of the chromatogram more quickly and easily.
• Chromatogram development: This is carried by the paper immersion in the mobile phase. The mobile phase crosses over the sample on the paper because of the capillary action of the paper.
• Drying of paper and detection of the compound: With the aid of air drier, the paper is dried as soon as the chromatogram is developed. On the chromatogram developed paper, the detecting solution is sprayed and dried thoroughly for the identification of the sample chromatogram spots.

Types of Paper Chromatography

Ascending Paper Chromatography As per the name, the developing solvent is found to be moving in an upward direction. Here, a sufficient quantity of mobile phase is poured into the development chamber. Sample and reference are spotted on the line drawn a few centimetres from the bottom edge of the paper suspended from a hook or clip at the top.

Descending Paper Chromatography Here, the solvent front travels down the length of paper suspended from the top inside the developing chamber. The mobile phase is kept in a trough in the upper chamber. The paper with spotting on the line drawn a few centimetres from the top is clamped to the top. Before elution, the jar is covered and equilibrated with the mobile phase vapour.

Ascending – Descending Chromatography It is a mixed type of chromatography where the solvent first travels upwards on the paper that is folded over a rod and after crossing the rod it moves downwards. 

Horizontal or Circular Paper Chromatography This allows the separation of sample components in the form of concentric circular zones through the radial movement of the liquid phase. 

Two-Dimensional Chromatography This helps in resolving substances that have similar Rf values.

Where, Retardation factor (Rf) = The distance travelled by the solute/ distance travelled by the solvent front

Applications of paper chromatography 

In the analysis of different classes of compounds namely:

• Amino acids and organic acids
• Polysaccharides
• Proteins and peptides
• Natural and artificial pigments
• Inorganic cations
• Plant extracts

Applications of paper chromatography in different key areas

Paper chromatography uses are not confined to any particular field. A number of the necessary areas include:

• Reaction monitoring – The progress of the reaction can be estimated by developing the chromatogram over different time intervals by spotting the reactants.
• Isolation & Purification – This technique is useful in the purification and isolation of components of mixtures. Here, the separated components on the paper are cut, dissolved in suitable solvents and using spectroscopic methods, their absorption is characterised at specific wavelengths.
• Foods – Analysis of food colours in synthetic drinks and beverages, ice creams, sweets, etc. Only edible colours are permitted for use, this is why identification and quantification are of utmost importance.
• Forensics – Provides a basis for identification and comparison against reference standards for drugs and their metabolites. Paper chromatography offers a vital role in the viable analysis of samples that are available in milligrams or microlitre quantities. 
• Pharmaceuticals – Provides information related to the development of new drugs molecules, reaction completion and progress of manufacturing processes. This process is cost-effective and hence used as an alternative method in monitoring the active ingredients present in the drug forms. Paper chromatography is also applicable in colour identifications of pharmaceutical formulations.

The technique of Paper Chromatography is being extensively used for the last several years and still have preserved their ground associated with the separation of different classes of compounds.

• https://byjus.com/chemistry/paper-chromatography/
• https://biochemden.com/paper-chromatography/
• https://laboratoryinfo.com/paper-chromatography/

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Paper Chromatography

• First Online: 25 July 2023

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• V. K. Ahluwalia 2  

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Paper chromatography is the simplest most widely used technique for the separation and identification of the constituents of a mixture. The stationary phase in paper chromatography is a liquid, i.e. , water (adsorbed on cellulose of the Whatman filter paper) and the moving phase (known as irrigant) is also a liquid, which may be a solvent or mixture of solvents. Paper chromatography is a type of partition chromatography (partition between two liquid phases). The components of the mixture to be separated travel at different rates and appear as spots on different points of the Whatman filter paper.

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Ahluwalia, V.K. (2023). Paper Chromatography. In: Instrumental Methods of Chemical Analysis. Springer, Cham. https://doi.org/10.1007/978-3-031-38355-7_3

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• Analytical Chemistry

Paper Chromatography

What Is Paper Chromatography?

Chromatography technique that uses paper sheets or strips as the adsorbent being the stationary phase through which a solution is made to pass is called paper chromatography. It is an inexpensive method of separating dissolved chemical substances by their different migration rates across the sheets of paper. It is a powerful analytical tool that uses very small quantities of material. Paper chromatography was discovered by Synge and Martin in the year 1943.

Table of Contents

Paper chromatography principle, paper chromatography diagram, paper chromatography procedure, paper chromatography applications.

• Types of Paper Chromatography
• Frequently Asked Questions – FAQs

The principle involved can be partition chromatography or adsorption chromatography. Partition chromatography because the substances are partitioned or distributed between liquid phases. The two phases are water held in pores of the filter paper and the other phase is a mobile phase which passes through the paper. When the mobile phase moves, the separation of the mixture takes place. The compounds in the mixture separate themselves based on the differences in their affinity towards stationary and mobile phase solvents under the capillary action of pores in the paper. Adsorption chromatography between solid and liquid phases, wherein the solid surface of the paper is the stationary phase and the liquid phase is the mobile phase.

Below we have explained the procedure to conduct Paper Chromatography Experiment for easy understanding of students.

• Selecting a suitable type of development: It is decided based on the complexity of the solvent, paper, mixture, etc. Usually ascending type or radial paper chromatography is used as they are easy to perform. Also, it is easy to handle, the chromatogram obtained is faster and the process is less time-consuming.
• Selecting a suitable filter paper : Selection of filter paper is done based on the size of the pores and the sample quality.
• Prepare the sample: Sample preparation includes the dissolution of the sample in a suitable solvent (inert with the sample under analysis) used in making the mobile phase.
• Spot the sample on the paper: Samples should be spotted at a proper position on the paper by using a capillary tube.
• Chromatogram development: Chromatogram development is spotted by immersing the paper in the mobile phase. Due to the capillary action of paper, the mobile phase moves over the sample on the paper.
• Paper drying and compound detection : Once the chromatogram is developed, the paper is dried using an air drier. Also, detecting solution can be sprayed on the chromatogram developed paper and dried to identify the sample chromatogram spots.

There are various applications of paper chromatography . Some of the uses of Paper Chromatography in different fields are discussed below:

• To study the process of fermentation and ripening.
• To check the purity of pharmaceuticals.
• To inspect cosmetics.
• To detect the adulterants.
• To detect the contaminants in drinks and foods.
• To examine the reaction mixtures in biochemical laboratories.
• To determine dopes and drugs in humans and animals.

Types of paper chromatography:

• Ascending Paper Chromatography – The techniques goes with its name as the solvent moves in an upward direction.
• Descending Paper Chromatography – The movement of the flow of solvent due to gravitational pull and capillary action is downwards, hence the name descending paper chromatography.
• Ascending – Descending Paper Chromatography – In this version of paper chromatography, movement of solvent occurs in two directions after a particular point. Initially, the solvent travels upwards on the paper which is folded over a rod and after crossing the rod it continues with its travel in the downward direction.
• Radial or Circular Paper Chromatography – The sample is deposited at the centre of the circular filter paper. Once the spot is dried, the filter paper is tied horizontally on a Petri dish which contains the solvent.
• Two Dimensional Paper Chromatography – Substances which have the same r f values can be resolved with the help of two-dimensional paper chromatography.

Frequently Asked Questions – FAQs

What are the advantages of paper chromatography.

Paper Chromatography Has Many Benefits Simple and rapid Paper chromatography necessitates a minimal amount of quantitative material. Paper chromatography is less expensive than other chromatography methods. The paper chromatography method can identify both unknown inorganic and organic compounds. Paper chromatography takes up little space when compared to other analytical methods or equipment. Outstanding resolving power

Why water is not used in paper chromatography?

It is preferable to use a less polar solvent, such as ethanol, so that the non-polar compounds will travel up the paper while the polar compounds will stick to the paper, separating them.

What are the limitations of Paper Chromatography?

Limitations of Paper Chromatography are as follows- Paper chromatography cannot handle large amounts of sample. Paper chromatography is ineffective in quantitative analysis. Paper chromatography cannot separate complex mixtures. Less Accurate than HPLC or HPTLC

What is the importance of paper chromatography?

Paper chromatography has traditionally been used to analyse food colours in ice creams, sweets, drinks and beverages, jams and jellies. Only edible colours are permitted for use to ensure that no non-permitted colouring agents are added to the foods. This is where quantification and identification come into play.

Is paper chromatography partition or adsorption?

A type of partition chromatography is paper chromatography.

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It is so easy to understand by students Explained with applications also.

Paper Chromatography Experiment

March 17, 2021 By Emma Vanstone Leave a Comment

This simple felt tip pen paper chromatography experiment is a great way to learn about this particular method of separating mixtures .

WHAT IS CHROMATOGRAPHY?

Chromatography   is a technique used to separate mixtures. Information from a chromatography investigation can also be used to identify different substances.

In chromatography, the mixture is passed through another substance, in this case, filter paper. The different-coloured ink particles travel at different speeds through the filter paper, allowing the constituent colours of the pen ink to be seen.

All types of chromatography have two phases: a mobile phase where the molecules can move and a stationary phase where they can’t move. In the case of paper chromatography, the stationary phase is the filter paper, and the mobile phase is the solvent ( water ).

The more soluble the ink molecules, the further they are carried up the paper.

The video below shows chromatography in action.

You’ll need:

Filter paper or paper towel

Felt tip pens – not washable or permanent

A container – glass, jar or plate

Instructions

Pour a small amount of water onto a plate or into the bottom of a jar.

Find a way to suspend the filter paper over the water so that just the very bottom touches the water. If you do the experiment in a jar, the easiest way to do this is to wrap the top of the filter paper around a pencil, clip it in place, and suspend it over the top of the jar.

Our LEGO holder worked well, too!

Use the felt tip pens to draw a small circle about 1cm from the bottom of the filter paper with each colour pen you want to test.

Suspend the filter paper in the water and watch as the ink moves up the filter paper.

You should end up with something like this! The end result is called a chromatogram.

What happens if you use washable pens?

If the inks are washable, they tend to contain just one type of ink, so there is no separation of colour.

Below, only a couple of the inks have separated compared to the non-washable pens above.

Why does chromatography work?

When the filter paper containing the ink spots is placed in the solvent ( in this case, water ), the dyes travel through the paper.

Different dyes in ink travel through the chromatography filter paper at different speeds. The most soluble colours dissolve and travel further and faster than less soluble dyes, which stick to the paper more.

I’ve created a free instruction sheet and chromatography experiment write up to make the activity even easier.

Extension task

Experiment with different types and colours of pens. Depending on the type of ink used, some will work better than others.

Try chromatography with sweets .

Steamstational also has a great leaf chromatography investigation.

More separation experiments

Clean up water by making your own filter .

Separate water and sand by evaporation .

Make colourful salt crystals by separating salt and water.

Separate liquid mixtures with a bicycle centrifuge .

Last Updated on May 20, 2024 by Emma Vanstone

Safety Notice

Science Sparks ( Wild Sparks Enterprises Ltd ) are not liable for the actions of activity of any person who uses the information in this resource or in any of the suggested further resources. Science Sparks assume no liability with regard to injuries or damage to property that may occur as a result of using the information and carrying out the practical activities contained in this resource or in any of the suggested further resources.

These activities are designed to be carried out by children working with a parent, guardian or other appropriate adult. The adult involved is fully responsible for ensuring that the activities are carried out safely.

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Leaf chromatography

In association with Nuffield Foundation

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Try this class practical using paper chromatography to separate and investigate the pigments in a leaf

Most leaves are green due to chlorophyll. This substance is important in photosynthesis (the process by which plants make their food). In this experiment, students investigate the different pigments present in a leaf, from chlorophyll to carotenes, using paper chromatography.

The experiment takes about 30 minutes and can be carried out in groups of two or three students.

• Eye protection
• Pestle and mortar
• Chromatography paper
• Beaker, 100 cm 3
• Small capillary tube (see note 1)
• Cut-up leaves, or leaves and scissors (see note 2)
• Propanone (HIGHLY FLAMMABLE, IRRITANT), supplied in a small bottle fitted with a teat pipette (see note 3)

Equipment notes

• The capillary tubing can be ‘home-made’ from lengths of ordinary glass tubing (diameter: 3–4 mm) using a Bunsen burner fitted with a flame-spreading (‘fish-tail’) jet.
• A variety of leaves can be used. Best results are obtained from trees or bushes with dark green leaves, eg holly.
• Preferably use teat pipettes that do not allow squirting, eg those fitted to dropper bottles of universal indicator.

Health, safety and technical notes

• Read our standard health and safety guidance.
• Wear eye protection throughout.
• Propanone, CH 3 COCH 3 (l), (HIGHLY FLAMMABLE, IRRITANT) – see CLEAPSS Hazcard HC085A .  The vapour of propanone is HIGHLY FLAMMABLE. Do not have any source of ignition nearby.
• Finely cut up some leaves and fill a mortar to about 2 cm depth.
• Add a pinch of sand and about six drops of propanone from the teat pipette.
• Grind the mixture with a pestle for at least three minutes.
• On a strip of chromatography paper, draw a pencil line 3 cm from the bottom.
• Use a fine glass tube to put liquid from the leaf extract onto the centre of the line. Keep the spot as small as possible.
• Allow the spot to dry, then add another spot on top. Add five more drops of solution, letting each one dry before putting on the next. The idea is to build up a very concentrated small spot on the paper.
• Attach the paper to the pencil using sellotape so that when placed in the beaker, the paper is just clear of its base.
• Place no more than about 10 cm 3 of propanone in the beaker and hang the paper so it dips in the propanone. Ensure the propanone level is below the spot.

Source: Royal Society of Chemistry

The equipment required for using paper chromatography to separate the different pigments in leaves

• Avoid moving the beaker in any way once the chromatography has started.
• Leave the experiment until the propanone has soaked near to the top, and then remove the paper from the beaker.
• Mark how high the propanone gets on the paper with a pencil and let the chromatogram dry.

Teaching notes

This experiment works very well providing care is taken over preparing the spot on the chromatography paper. It should be as small and as concentrated as possible. Encourage students to be patient and to wait until each application is dry before adding the next.

At least three spots should be obtained, and one of these should be yellow due to carotenes.

The extent to which any particular component moves up the paper is dependent not only on its solubility in propanone but also on its attraction for the cellulose in the chromatography paper. The yellow carotene spot (with a higher RF value) tends to move up the paper the furthest.

Additional information

This is a resource from the  Practical Chemistry project , developed by the Nuffield Foundation and the Royal Society of Chemistry.

Practical Chemistry activities accompany  Practical Physics  and  Practical Biology .

© Nuffield Foundation and the Royal Society of Chemistry

• 11-14 years
• 14-16 years
• Practical experiments
• Chromatography

Specification

• 2. Develop and use models to describe the nature of matter; demonstrate how they provide a simple way to to account for the conservation of mass, changes of state, physical change, chemical change, mixtures, and their separation.
• Chromatography as a separation technique in which a mobile phase carrying a mixture is caused to move in contact with a selectively absorbent stationary phase.
• 6 Investigate how paper chromatography can be used to separate and tell the difference between coloured substances. Students should calculate Rf values.
• Chromatography involves a stationary phase and a mobile phase. Separation depends on the distribution of substances between the phases.
• The ratio of the distance moved by a compound (centre of spot from origin) to the distance moved by the solvent can be expressed as its Rf value: Rf = (distance moved by substance / distance moved by solvent)
• Mixtures can be separated by physical processes such as filtration, crystallisation, simple distillation, fractional distillation and chromatography. These physical processes do not involve chemical reactions and no new substances are made.
• Recall that chromatography involves a stationary and a mobile phase and that separation depends on the distribution between the phases.
• Interpret chromatograms, including measuring Rf values.
• Suggest chromatographic methods for distinguishing pure from impure substances.
• 12 Investigate how paper chromatography can be used to separate and tell the difference between coloured substances. Students should calculate Rf values.
• 2.11 Investigate the composition of inks using simple distillation and paper chromatography
• 2.9 Describe paper chromatography as the separation of mixtures of soluble substances by running a solvent (mobile phase) through the mixture on the paper (the paper contains the stationary phase), which causes the substances to move at different rates…
• C2.1g describe the techniques of paper and thin layer chromatography
• 2.9 Describe paper chromatography as the separation of mixtures of soluble substances by running a solvent (mobile phase) through the mixture on the paper (the paper contains the stationary phase), which causes the substances to move at different rates o…
• C5.1.4 recall that chromatography involves a stationary and a mobile phase and that separation depends on the distribution between the phases
• 3 Using chromatography to identify mixtures of dyes in a sample of an unknown composition
• C3 Using chromatography to identify mixtures of dyes in a sample of an unknown composition
• 1.9.5 investigate practically how mixtures can be separated using filtration, crystallisation, paper chromatography, simple distillation or fractional distillation (including using fractional distillation in the laboratory to separate miscible liquids…
• 1.9.7 interpret a paper chromatogram including calculating Rf values;
• carry out paper and thin-layer chromatography and measure the Rf values of the components and interpret the chromatograms;

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• Published: 19 June 1954

Preparative Circular Paper Chromatography

• K. V. GIRI 1  

Nature volume  173 ,  pages 1194–1195 ( 1954 ) Cite this article

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FROM the preparative aspect, the conventional paper chromatographic technique is rather limited, in that only a few micrograms of material can be spotted on a single strip or sheet, and the labour involved in the separation of even milligram quantities in this way is enormous. From this point of view the circular paper chromatographic technique 1 offers great possibilities. By a suitable modification of this technique, it has been found possible to obtain from a single chromatogram material of individual components from a mixture of sugars sufficient for the determination of physical constants and preparation of characteristic derivatives.

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Giri, K. V., and Rao, N. A. N., Nature , 169 , 923 (1952); J. Ind. Inst. Sci. , 34 , 95 (1952); Nature , 171 , 1159 (1953).

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Giri, K. V., and Nigam, V. N., Naturwissenschaften , 40 , 343 (1953); J. Ind. Inst. Sci. , 36 , 49 (1954).

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GIRI, K. Preparative Circular Paper Chromatography. Nature 173 , 1194–1195 (1954). https://doi.org/10.1038/1731194b0

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Issue Date : 19 June 1954

DOI : https://doi.org/10.1038/1731194b0

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