paper chromatography experiment procedure

Chromatography is used to separate mixtures of substances into their components. All forms of chromatography work on the same principle.

They all have a (a solid, or a liquid supported on a solid) and a (a liquid or a gas). The mobile phase flows through the stationary phase and carries the components of the mixture with it. Different components travel at different rates. We'll look at the reasons for this further down the page.

In paper chromatography, the stationary phase is a very uniform absorbent paper. The mobile phase is a suitable liquid solvent or mixture of solvents.

You probably used paper chromatography as one of the first things you ever did in chemistry to separate out mixtures of coloured dyes - for example, the dyes which make up a particular ink. That's an easy example to take, so let's start from there.

Suppose you have three blue pens and you want to find out which one was used to write a message. Samples of each ink are spotted on to a pencil line drawn on a sheet of chromatography paper. Some of the ink from the message is dissolved in the minimum possible amount of a suitable solvent, and that is also spotted onto the same line. In the diagram, the pens are labelled 1, 2 and 3, and the message ink as M.

The reason for covering the container is to make sure that the atmosphere in the beaker is saturated with solvent vapour. Saturating the atmosphere in the beaker with vapour stops the solvent from evaporating as it rises up the paper.

As the solvent slowly travels up the paper, the different components of the ink mixtures travel at different rates and the mixtures are separated into different coloured spots.

The diagram shows what the plate might look like after the solvent has moved almost to the top.

It is fairly easy to see from the final chromatogram that the pen that wrote the message contained the same dyes as pen 2. You can also see that pen 1 contains a mixture of two different blue dyes - one of which be the same as the single dye in pen 3.

values

Some compounds in a mixture travel almost as far as the solvent does; some stay much closer to the base line. The distance travelled relative to the solvent is a constant for a particular compound as long as you keep everything else constant - the type of paper and the exact composition of the solvent, for example.

The distance travelled relative to the solvent is called the R value. For each compound it can be worked out using the formula:

For example, if one component of a mixture travelled 9.6 cm from the base line while the solvent had travelled 12.0 cm, then the R value for that component is:

In the example we looked at with the various pens, it wasn't necessary to measure R values because you are making a direct comparison just by looking at the chromatogram.

You are making the assumption that if you have two spots in the final chromatogram which are the same colour and have travelled the same distance up the paper, they are most likely the same compound. It isn't necessarily true of course - you could have two similarly coloured compounds with very similar R values. We'll look at how you can get around that problem further down the page.

In some cases, it may be possible to make the spots visible by reacting them with something which produces a coloured product. A good example of this is in chromatograms produced from amino acid mixtures.

Suppose you had a mixture of amino acids and wanted to find out which particular amino acids the mixture contained. For simplicity we'll assume that you know the mixture can only possibly contain five of the common amino acids.

A small drop of a solution of the mixture is placed on the base line of the paper, and similar small spots of the known amino acids are placed alongside it. The paper is then stood in a suitable solvent and left to develop as before. In the diagram, the mixture is M, and the known amino acids are labelled 1 to 5.

The position of the solvent front is marked in pencil and the chromatogram is allowed to dry and is then sprayed with a solution of . Ninhydrin reacts with amino acids to give coloured compounds, mainly brown or purple.

The left-hand diagram shows the paper after the solvent front has almost reached the top. The spots are still invisible. The second diagram shows what it might look like after spraying with ninhydrin.

There is no need to measure the R values because you can easily compare the spots in the mixture with those of the known amino acids - both from their positions and their colours.

In this example, the mixture contains the amino acids labelled as 1, 4 and 5.

And what if the mixture contained amino acids other than the ones we have used for comparison? There would be spots in the mixture which didn't match those from the known amino acids. You would have to re-run the experiment using other amino acids for comparison.

Two way paper chromatography gets around the problem of separating out substances which have very similar R values.

I'm going to go back to talking about coloured compounds because it is much easier to see what is happening. You can perfectly well do this with colourless compounds - but you have to use quite a lot of imagination in the explanation of what is going on!

This time a chromatogram is made starting from a single spot of mixture placed towards one end of the base line. It is stood in a solvent as before and left until the solvent front gets close to the top of the paper.

In the diagram, the position of the solvent front is marked in pencil before the paper dries out. This is labelled as SF1 - the solvent front for the first solvent. We shall be using two different solvents.

If you look closely, you may be able to see that the large central spot in the chromatogram is partly blue and partly green. Two dyes in the mixture have almost the same R values. They could equally well, of course, both have been the same colour - in which case you couldn't tell whether there was one or more dye present in that spot.

What you do now is to wait for the paper to dry out completely, and then rotate it through 90°, and develop the chromatogram again in a different solvent.

It is very unlikely that the two confusing spots will have the same R values in the second solvent as well as the first, and so the spots will move by a different amount.

The next diagram shows what might happen to the various spots on the original chromatogram. The position of the second solvent front is also marked.

You wouldn't, of course, see these spots in both their original and final positions - they have moved! The final chromatogram would look like this:

Two way chromatography has completely separated out the mixture into four distinct spots.

If you want to identify the spots in the mixture, you obviously can't do it with comparison substances on the same chromatogram as we looked at earlier with the pens or amino acids examples. You would end up with a meaningless mess of spots.

You can, though, work out the R values for each of the spots in both solvents, and then compare these with values that you have measured for known compounds under exactly the same conditions.

Use the BACK button on your browser to return quickly to this page when yhou have read it.

Paper is made of cellulose fibres, and cellulose is a polymer of the simple sugar, glucose.

The key point about cellulose is that the polymer chains have -OH groups sticking out all around them. To that extent, it presents the same sort of surface as silica gel or alumina in thin layer chromatography.

It would be tempting to try to explain paper chromatography in terms of the way that different compounds are adsorbed to different extents on to the paper surface. In other words, it would be nice to be able to use the same explanation for both thin layer and paper chromatography. Unfortunately, it is more complicated than that!

The complication arises because the cellulose fibres attract water vapour from the atmosphere as well as any water that was present when the paper was made. You can therefore think of paper as being cellulose fibres with a very thin layer of water molecules bound to the surface.

It is the interaction with this water which is the most important effect during paper chromatography.

Suppose you use a non-polar solvent such as hexane to develop your chromatogram.

Non-polar molecules in the mixture that you are trying to separate will have little attraction for the water molecules attached to the cellulose, and so will spend most of their time dissolved in the moving solvent. Molecules like this will therefore travel a long way up the paper carried by the solvent. They will have relatively high R values.

On the other hand, polar molecules have a high attraction for the water molecules and much less for the non-polar solvent. They will therefore tend to dissolve in the thin layer of water around the cellulose fibres much more than in the moving solvent.

Because they spend more time dissolved in the stationary phase and less time in the mobile phase, they aren't going to travel very fast up the paper.

The tendency for a compound to divide its time between two immiscible solvents (solvents such as hexane and water which won't mix) is known as . Paper chromatography using a non-polar solvent is therefore a type of .

A moment's thought will tell you that partition can't be the explanation if you are using water as the solvent for your mixture. If you have water as the mobile phase and the water bound on to the cellulose as the stationary phase, there can't be any meaningful difference between the amount of time a substance spends in solution in either of them. All substances should be equally soluble (or equally insoluble) in both.

And yet the first chromatograms that you made were probably of inks using water as your solvent.

If water works as the mobile phase as well being the stationary phase, there has to be some quite different mechanism at work - and that must be equally true for other polar solvents like the alcohols, for example. Partition only happens between solvents which don't mix with each other. Polar solvents like the small alcohols do mix with water.

In researching this topic, I haven't found any easy explanation for what happens in these cases. Most sources ignore the problem altogether and just quote the partition explanation without making any allowance for the type of solvent you are using. Other sources quote mechanisms which have so many strands to them that they are far too complicated for this introductory level. I'm therefore not taking this any further - you shouldn't need to worry about this at UK A level, or its various equivalents.

Where would you like to go now?

To the chromatography menu . . .

To the analysis menu . . .

To Main Menu . . .

© Jim Clark 2007 (modified July 2016)

What is Paper Chromatography? Principle, Procedure, Types, and Applications

Paper chromatography has proved to be very successful in the analysis of chemical compounds and lipid samples in particular.

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.

It is the simplest and commonest form of liquid-liquid chromatography.

Whatman filter paper or commercially prepared cellulose plates are used for chromatographic separation.

The basic principle of this procedure was described for the first time by  Consden ,  Gordon , and  Martin (1944).

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Components of the mixture are carried along with the solvent up the paper to varying degrees, depending on the compound’s preference to be adsorbed onto the paper versus being carried along with the solvent.

The paper is  composed of cellulose to which polar water molecules  are adsorbed, while the solvent is less polar, usually comprising a mixture of water and an organic liquid.

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The paper is called the stationary phase while the solvent is referred to as the mobile phase.

To get a measure of the extent of movement of a component in a paper chromatography experiment, we can calculate an “ Rf value ” 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 an application point.

Table of Contents

Nature of Paper

The paper commonly used consists of highly purified cellulose. Cellulose, is a homopolysaccharide of glucose. It contains several thousand anhydrous-glucose units linked through oxygen atoms. The paper exhibits weak ion exchange and absorptive properties.

Modified forms of paper have been produced in which the paper has been impregnated with alumina, silica gel, and ion-exchange resin , etc.

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The chemical composition of Whatman filter paper no: 1 is: a-cellulose (98 to 99%), b-cellulose (0.3 to 1%), Pentosans (0.4 to 0.8%), Ash (0.07 to 0.1%) & ether soluble matter (0.015 to 0.1%).

The apparatus required for paper chromatography are

• Support for paper
• Solvent trough
• Airtight chamber
• Whatman filter paper number 1
• Capillary tubes
• Samples–Amino acids (or) Pigments
• Platinum loop

Paper Development

There are two main techniques, which may be employed for the development of paper Chromatograms.

1. Ascending techniques

The filter paper is then dried and equilibrated by putting it into an airtight cylindrical jar, which contains an aqueous solution of a solvent.

The most widely applicable solvent mixture is n- B utanol: A cetic acid: W ater (4:1:5), which is abbreviated as BAW .

The sheet of paper is supported on a frame with the button edge in contact with a trough with a solvent.

The arrangement is contained in an airtight tank lined with paper saturated with the solvent to prove a constant atmosphere and separations are carried out in a constant temperature room.

Thus, the solvent will ascend into the paper this process is, therefore, termed “ Ascending Chromatography ”.

2. Descending techniques

The end of the filter paper may be put into the solvent mixture contained in a narrow trough mounted near the top of the container.

In this chromatography, the solvent will descend into the paper and this process is then termed “Descending Chromatography”.

This method is convenient for compounds, which have similar Rf values since the solvent drips off the bottom of the paper, thus giving a wider separation.

3. Two-dimensional chromatography (2D)

The mixture is separated than the first solvent, which should be volatile: then after drying, the paper is turned through 90 0 and separation is carried out in the second solvent.

After locating the migrated unknown sample along with a standard known sample, a map is obtained and comparing their position with a map of known compounds can identify compounds

Locating the compounds:

The strip is removed when the solvent has migrated over most of the available space.

The distance to which the solvent has run is marked. In most cases, the completed Chromatogram is colorless with no indication of the presence of any compounds.

Such a chromatogram is said as “Undeveloped” for locating the various compounds.

The filter paper strip is first dried, then sprayed with 0.5% Ninhydrin in acetone and at least heated for a few minutes at 80 to 100 0 C.

The reaction occurs and the colored spots appear at the sites of the amino acids, such as Chromatogram is now called “ Developed ”.

In paper chromatography, the stationary cellulose phase is more polar than the mobile organic phase.

Identifying the compounds:

The ratio of the distance traveled by a component (i.e. amino acid) to that traveled by the solvent front, both measured from the marked point of the application of the mixture, is called the “Resolution front (Rf)” value for that component.

   Distance from origin run by the compound

Rf = —————————————————————————–

Distance from origin run by the solvent

The filter paper strip may be sprayed with ninhydrin and heated so that the colored spots showing the location of amino acids may develop. The color densities of these spots may be measured with a recording(or) reflectance photometer device.

• Ninhydrin test or Ninhydrin reagent

Ninhydrin Test:

• Amines (including α-amino acids) react with ninhydrin to give a colored product.
• It can be used qualitatively ( e.g.  for chromatographic visualization) or quantitatively ( e.g. for pep tide sequencing).
• The α-amino acids typically give a  blue-purple product .
• Proline, a secondary amine, gives a  yellow-orange product .
• The test is sensitive enough that ninhydrin can be used for the visualization of fingerprints.

Applications of Paper Chromatography

By using this technique

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

Reference: Read for more details

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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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Practical videos | 14–16 years

• 1 Access free videos to support your teaching
• 2 Paper chromatography
• 3 Rates of reaction
• 4 Simple distillation
• 5 Enthalpy change of combustion
• 6 Conservation of mass
• 7 Electrolysis of aqueous solutions
• 8 Halogen displacement reactions
• 9 Identifying ions
• 10 Preparing a soluble salt
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• 12 Simple titration
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• 14 Potable water

Paper chromatography

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Investigate the separation of inks using paper chromatography

The value of experiencing live practical work cannot be overstated. Numerous studies provide evidence of its value in terms of learner engagement, understanding, results and the likelihood of continuing to study chemistry or work in a related field.

Use this video to complement live practical work, or to help learners understand the methods, equipment and skills when they cannot access the lab.

Source: © Royal Society of Chemistry

Investigate the separation of inks using this paper chromatography video, including a step-by-step method, animation and calculation support for learners

Chapter titles: 00:10 Introduction to chromatography; 00:44 Carrying out the experiment; 03:59 Results; 04:43 Animation; 05:11 Alternative phases; 06:05 Calculating R f values.

• Teacher notes

Full teacher notes are available in the  supporting resources booklet  (also available in  MS Word ), including ideas for  how to use this video  and the accompanying activites and answers to use as part of your teaching. 

Get the resources

Supporting resources

Detailed teacher notes, learner activities and answers

Learner slides

Integrated instructions, Frayer models and Johnstone's triangle

• Technician notes

Equipment, chemicals, hazards and disposal information

Notes on running the practical experiments

This experiment is designed to cover most of the questions asked in a variety of syllabuses. You or your school’s technician will have to do some trial and error to get the ink composition correct to get the desired results. The suggestion is that one ink is not soluble in the water (solvent) and so doesn’t move, one colour moves to the top of the paper (learners can identify this as the most soluble) and there is a colour in common between two of the samples. Although the video and worksheets refer to particular colours seen in the video, the actual colours don’t matter and it is their characteristics that are key to the success of the practical.

Use the video’s animation and  Johnstone’s triangle , available in the PowerPoint slides , to help learners link their observations to what’s going on at the submicroscopic level.

Health, safety and technical notes

You may want to demonstrate the experiment using a different solvent, such as ethanol ( CLEAPSS student safety sheet 60 ). Use a lid and wear eye protection (safety glasses to EN166 F) when using the alcohol. To show a different stationary phase, use a TLC plate.

Read our standard health and safety guidance and carry out a risk assessment before running any live practical. Refer to SSERC/CLEAPSS Hazcards, recipe books and student safety sheets. Hazard classification may vary depending on supplier. Download the technician notes for the full equipment list, safety notes and tips.

• Cut a piece of filter paper to fit inside a beaker (size of the beaker is not relevant) so that it does not curve and lies flat, not touching the edges of the beaker.
• Using a pencil, draw an origin line on the paper and label 1, 2 and 3 at equal distances below the line.
• Using a separate capillary tube for each ink, transfer a small spot onto the correct labelled position on the origin line.
• Add a small amount of the solvent to just cover the bottom of the beaker.
• Check the paper is the right length by lining it up on the outside of the beaker so that the water is below the origin line. Roll the paper round a splint and hold it with a paper clip.
• Place the paper inside the beaker. Make sure it just touches the water and it is vertical.
• Remove the filter paper from the beaker when the solvent has nearly reached the top of the filter paper.
• Leave the filter paper to dry or use a hairdryer.
• Measure the distances from the origin line to the middle of the colours and from the origin line to the solvent front to find the R f values.

Find the integrated instructions for this experiment in the PowerPoint slides . 

Real-world contexts

• Highlight real-world uses of chromatography with the article Poisoned by milk .
• Complete this project on the chemistry of food in a sequence of timetabled chemistry lessons, STEM clubs or during an activity day to provide context to analytical techniques.
• Engage younger learners with this investigation to solve who stole a famous painting.
• Watch this  video to see how senior science manager Paul uses liquid chromatography in his job at British Sugar.

Learners will need to have a clear understanding of the following scientific terminology:

• Chromatography – a technique for separating mixtures of soluble substances.
• Chromatogram – the results of separating mixtures by chromatography.
• Mixture – two or more different substances, not chemically joined together.
• Solvent – the substance a solute dissolves in to form a solution.
• Solute – a substance that will dissolve in a solvent.
• Dissolve – when a solute mixes completely with a solvent to produce a solution.
• Solution – a mixture formed by a solute and a solvent.
• Soluble – a substance that will dissolve.
• Insoluble – a substance that will not dissolve.
• Pure – a substance made of only one element or compound.
• Impure – a substance made of more than one element or compound.
• R f value – the ratio of the solute’s distance travelled to the solvent’s distance travelled.
• Stationary phase – the substance that does not move, eg paper.
• Mobile phase – the solvent that moves up the stationary phase, eg water.
• Origin line – the mark you add the samples to at the start of chromatography.
• Solvent front – the distance travelled by the solvent.
• Volatile – evaporates easily.

You will find a template, example Frayer model and suggested answers for the terms: ‘chromatography’, ‘mobile phase’, ‘stationary phase’, ‘origin line’ and ‘solvent front’ in the PowerPoint slides . Find more examples and tips on  how to use Frayer models  in your teaching. 

Cross-curriculum links and skills

Learners will use key maths skills in the practical including:

• Measuring distances accurately.
• Giving answers to a specified decimal place.

Common misconceptions

Using pen to draw the origin line. Bring attention to using pencil and encourage learners to think why pens might interfere with the separation.

Putting too much solvent so learners submerge the origin line. Ensure the solvent is below the origin line and learners understand why.

Allowing the solvent to travel too far up the paper. The solvent front will be lost meaning the R f value cannot be calculated.

All mixtures are separated using only one separating technique . Practise a range of techniques with learners and highlight that chromatography is a method of separation for analysing a mixture but does not achieve separation of the entire test mixture. Carry out further methods once purity has been determined and/or the impurities identified.

The solvent/mobile phase can only be water . Use different solvents, such as sodium chloride solution and ethanol.

The stationary phase in chromatography can only be paper . Introduce alternatives, such as thin layer chromatography using silica on a plastic backing or aluminium oxide coated plates.

Dyes have a preference or ‘like’ to be in the stationary or the mobile phase . Avoid anthropomorphising as analogies like this can be a barrier to deep learning. Use ‘is attracted to’ or ‘hydrophobic/philic’ instead.

More resources

• Do this chromatography practical  using dye from different coloured sweets with your 11–16 year-old learners and read the accompanying article to find out more about baking with sprinkles.
• Make cross-curricular links to biology and investigate the pigments in a leaf .
• Practical planning: spot the mistakes  includes an exam-style answer on chromatography and is part of the  Teaching science skills series.

Paper chromatography supporting resources

Paper chromatography technician notes, paper chromatography slides, additional information.

The original video script, supporting resources and slides were written by Karen Marshall. The technician notes were adapted by Sandrine Bouchelkia.

More Sandrine Bouchelkia

Enthalpy change of combustion of ethanol | practical videos | 14–16 years

Simple distillation | practical videos | 14–16 years

Rates of reaction | practical videos | 14–16 years

Access free videos to support your teaching

Rates of reaction

Simple distillation

Enthalpy change of combustion

Conservation of mass

Electrolysis of aqueous solutions

Halogen displacement reactions

Identifying ions

Preparing a soluble salt

Reactivity series of metals

Simple titration

Temperature change (neutralisation)

Potable water

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• Chromatography

Specification

• the distance travelled by the substance divided by the distance travelled by the solvent
• the Rf value should be the same if it is the same substance (under the same conditions)

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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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One of the first-ever practical experiments you probably carried out in chemistry at school is a simple example of paper chromatography : separating a coloured ink. You draw a pencil line across the bottom of a sheet of paper, place a dot of ink on the line, and place the paper upright in a beaker of solvent. The solvent travels up the paper, carrying the ink with it, and the ink separates out into all of its different coloured components. 

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What is the mobile phase in paper chromatography?

What is the stationary phase in paper chromatography?

In paper chromatography, the starting solvent level should be _____.

In paper chromatography, components that travel faster up the paper have a greater affinity to the ______.

In paper chromatography, more soluble components have a _____ affinity to the mobile phase.

What are the units for Rf values?

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This is a simple but effective way of separating mixtures. You'll learn exactly how it works here.

Paper chromatography is an analytical technique used to separate and analyse mixtures of soluble substances. It is a type of chromatography.

• This article is all about the type of chromatography known as paper chromatography.
• We will start by looking at the principles of chromatography before applying them specifically to paper chromatography.
• We'll then run through the method, and you'll be able to calculate Rf values using an example to guide you through the process.
• After that, we'll explore how you read chromatograms and interpret the results.
• To finish, we'll look at the advantages and uses of paper chromatography.

Principles of paper chromatography

All types of chromatography follow the same basic principles.

• We use a solvent, called the mobile phase, to dissolve a sample of a soluble mixture. The solvent carries the mixture up a solid called the stationary phase .
• Some components of the mixture are carried up the solid by the solvent more quickly than others. We say that components that travel faster have a greater affinity to the mobile phase. This separates the mixture out into its component parts and produces a chromatogram .
• We then use the distances travelled by the components to work out Rf values. These help us identify the component.

If you haven't already done so, we'd recommend looking at Chromatography for a more detailed explanation of these ideas.

Let's now look at some of those ideas in terms of paper chromatography.

Stationary phase

The stationary phase is a static solid, liquid, or gel. In chromatography, the solvent carries the soluble mixture through the stationary phase.

In paper chromatography, the stationary phase is - as the name suggests - paper. However, it's a bit more complicated than that. Paper is made of cellulose, a polymer of glucose. Cellulose fibres bind to water vapour in the air, alongside any water that was around when the paper was made. You can actually think of the stationary phase as being a complex matrix made of water and paper, not just the paper itself.

Mobile phase

The mobile phase is the solvent used to carry the mixture analysed through the stationary phase.

The stationary phase - the paper - is placed in a solvent. This is our mobile phase. In paper chromatography, we typically use a nonpolar solvent. The solvent travels up the paper, carrying the different components within the mixture with it.

Retention factors

The components of the sample mixture travel up the stationary phase at different speeds. This means that in a given time period, the different components will all travel different distances. We'll look at why this is in just a second.

You'll see these components as spots on the chromatogram , which is the name for your paper once the experiment has finished. The ratio between the distance travelled by each component and the total distance travelled by the solvent gives us Rf values .

To calculate an Rf value, divide the distance travelled by the component - in other words, the distance from the starting pencil line to the coloured spot -by the distance travelled by the solvent.

Rf values are important because each component has a fixed Rf value under a specific set of conditions. If you repeated the experiment again, keeping the mobile phase, the stationary phase, and the temperature exactly the same, you would get the same Rf value for the same component. We can then compare Rf values to ones in a database to identify the components in the mixture.

Relative affinity

What determines how quickly a substance travels up the paper? This is all to do with relative affinity .

In chromatography, relative affinity describes how well a component is attracted to either the stationary or mobile phase. It determines how quickly the component moves through the stationary phase.

Components with a greater affinity to the mobile phase will move faster up the plate than those with a greater affinity to the stationary phase. They are more soluble in the solvent and travel a greater distance in a given time period.

Let's look more closely at the mobile and stationary phases in paper chromatography to work out why some components have a greater affinity to one or the other.

Remember how the stationary phase is a matrix of cellulose and water? This means that it is polar and can experience permanent dipole-dipole forces . The water molecules can also form hydrogen bonds with suitable substances. In contrast, the mobile phase in paper chromatography is typically nonpolar . It can only form weak van der Waals forces. From this we can deduce the following:

• If any of the components within the sample mixture are polar, or contain chemical groups that can form hydrogen bonds, they will bond more strongly to the polar cellulose-water structure than to the nonpolar solvent. This means that they have a greater affinity to the stationary phase and will travel more slowly up the paper. These components give lower Rf values.
• However, nonpolar components will bond more strongly to the nonpolar solvent than to the polar paper. They are more soluble. They, therefore, have a greater affinity to the mobile phase and will travel more quickly up the paper. These components give higher Rf values.

Check out Intermolecular Forces for more on permanent dipole-dipole forces, hydrogen bonds, and van der Waals forces.

Method for paper chromatography

That's enough of the technical details - how do you actually carry out paper chromatography?

• Draw a pencil line along the bottom of a sheet of chromatography paper.
• Place a spot of the mixture you want to analyse on the middle of the line.
• Place the sheet of paper in a beaker filled with a shallow layer of your solvent of choice. Make sure the level of the solvent is below the pencil line.
• Place a lid on the beaker and leave the solvent to travel up the paper, carrying the components of the mixture with it.
• When the solvent level almost reaches the top of the paper, remove the paper from the beaker and mark the position of the solvent with another pencil mark. Your chromatogram is now ready to be analysed.

What's the importance of, say, drawing the line in pencil? Here are some of the reasons behind particular steps in the method.

• We draw the line in pencil because pencil is insoluble. This prevents it being carried up the paper with the mobile phase. If we were to use ink, for example, the ink would also dissolve in the solvent and travel up the paper, producing confusing results.
• The solvent level must be below the spot of your mixture to prevent the spot fully dissolving in the solvent and being washed away.
• You should also make sure that you handle the paper by its edges, to avoid getting fingerprints on it. This could dirty the paper and give misleading results.
• We use a lid to keep the environment saturated with solvent and to prevent the solvent from evaporating. You could also line the sides of the beaker with filter paper soaked in the solvent to saturate it even more.

At the end of the experiment, the setup should look a little something like this:

The dot of ink has travelled up the paper and separated into several spots. Each spot represents a different component found in the original mixture. Each component moves up the paper at a different speed, depending on its relative affinity to the stationary phase and its relative affinity to the mobile phase.

We can now use these results to calculate Rf values for each spot.

Calculating Rf values

Earlier in the article, we mentioned Rf values. These are values that show the ratio between the distance travelled by each component and the total distance travelled by the solvent.

Let's look at calculating Rf values for the chromatogram we showed above.

• Measure the distance between the base pencil line and one of the coloured spots on the chromatogram. This is the distance travelled by the component that produced that spot.
• Measure the distance between the base pencil line and the pencil line you used to mark the solvent front. This is the distance travelled by the solvent.
• Divide the distance travelled by the component by the distance travelled by the solvent. This gives you your Rf value.
• Repeat for all of the coloured spots.

Rf value = distance travelled by solute distance travelled by solvent

Let's calculate the Rf value for the green spot. The green spot has travelled 3.0 cm whilst the solvent front has travelled 9.8 cm. Divide 3.0 by 9.8 to get your answer:

3 . 0 ÷ 9 . 8 = 0 . 306

We tend to round Rf values to two decimal places. this gives us an overall answer of 0.31

Remember that the distance travelled by a substance all depends on its relative affinities to each of the stages. A substance with a greater affinity to the stationary phase will travel more slowly up the paper and will travel less far in a given time period. This means that it will have a lower Rf value. In contrast, a substance with a greater affinity to the mobile phase will travel more quickly up the paper and will have a higher Rf value.

Analysing chromatograms

Chromatograms show us two things.

• The number of different components in our starting mixture.
• The identity of each component in our starting mixture.

Number of different components

Remember, each spot represents a different component found in the original solute mixture. In our example above, we have three different spots on our chromatogram. We, therefore, know that we have three different substances present.

Identity of each component

There are two ways of identifying substances in a chromatogram. Firstly, when setting up the experiment, you could also place a small dot of a known substance, such as a particular amino acid or organic molecule, on the pencil line to the side of your solute dot. This known substance acts as a reference molecule. It will also be carried up the plate by the solvent, producing a visible spot. If any of the spots from your mixture match the known substance's spot, you know that substance is present in your mixture.

Sound a little confusing? Here's what it looks like in practice.

The red spot on the left is from a known substance. One of the spots produced by our mixture matches it exactly. We can therefore deduce that the mixture contains this particular substance.

But there is another way of identifying components. We also mentioned earlier that, provided you keep the conditions the same, a particular component will always produce the same Rf value. Let's say that a particular component has an Rf value of 0.4. If we look in a database, we should be able to find a substance that also produces an Rf value of 0.4 under the same conditions - the same mobile phase, stationary phase, and temperature. These two substances are one and the same.

Two-way paper chromatography uses two different solvents, one after the other, on the same sample. It is useful for separating out components with similar Rf values.

To carry out this technique, place a small spot of your mixture at one edge of the base pencil line. Place the paper in a beaker with your first solvent, removing it when the solvent front has almost reached the top of the paper. Mark the position of this first solvent front.

Your paper should look a little something like the diagram below.

You'll notice that two components produce one merged spot - they haven't clearly separated. This is because they have similar relative affinities to the stationary and mobile phases and so have travelled at similar speeds up the paper.

Now, rotate your paper by 90° so that the separated spots now lie along the bottom of the paper. Choose a different solvent and repeat the experiment again. It is very unlikely that the two substances that produced the merged spot will also have similar affinities to the stationary and mobile phases in this solvent. Therefore, they will travel at different speeds up the paper and separate out into clear, distinct spots.

Advantages of paper chromatography

Paper chromatography is a relatively simple technique. However, it does have its advantages.

• It is cheap and easy to run, with a simpler setup than other types of chromatography.
• It only uses small amounts of the sample mixture.
• It can analyse organic and inorganic compounds.

However, compared to other chromatography techniques such as gas chromatography and thin-layer chromatography , paper chromatography is less accurate. This is one of its main disadvantages.

Uses of paper chromatography

Paper chromatography is more than just a way of making pretty coloured patterns. It has a variety of different uses, many of which it shares with other chromatography techniques. These include:

• Separating mixtures. For example, in the early 20th century, paper chromatography was widely used to separate plant extracts.
• Obtaining pure compounds and removing impurities.
• Analysing drugs.
• Testing wastewater.

Paper Chromatography - Key takeaways

Paper chromatography is an analytical technique used to separate and analyse mixtures of soluble substances.

• In paper chromatography, the stationary phase is a sheet of paper and the mobile phase is a solvent.
• You can identify components in paper chromatography by calculating their Rf values and comparing them to those in a database.
• Two-way chromatography is a variation of paper chromatography that uses two different solvents to separate out components with similar Rf values.
• Paper chromatography is cheap, simple, and uses small sample sizes.

Flashcards in Paper Chromatography 6

Chromatography paper.

Below the pencil line containing the spot of mixture.

Mobile phase.

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Frequently Asked Questions about Paper Chromatography

What is paper chromatography?      

How does paper chromatography work?

In paper chromatography, a sheet of paper known as the stationary phase is placed in a solvent, known as the mobile phase. A small spot of a soluble mixture is placed on the paper. The solvent carries the mixture up the paper. Different components of the mixture have different relative affinities to the stationary and mobile phases and so travel up the paper at different speeds. This separates the components out.

What is paper chromatography used for? 

Paper chromatography is used for separating mixtures, obtaining pure compounds, and analysing drugs.

What is the principle of paper chromatography? 

Different components within a mixture have different affinities to the stationary phase - the paper, and the mobile phase - the solvent. This means that they travel up the paper at different speeds. Some will travel much further than others in a given time period. This separates the components.

What is an example of paper chromatography? 

An example of paper chromatography is separating the different dyes within an ink.

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