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E. Paper Chromatography

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Chromatography is used to separate mixtures of substances into their components. All forms of chromatography work on the same principle. They all have a stationary phase (a solid, or a liquid supported on a solid) and a mobile phase (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.

Producing a paper chromatogram

You probably used paper chromatography as one of the first things you ever did in chemistry to separate out mixtures of colored 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 labeled 1, 2 and 3, and the message ink as M.

The paper is suspended in a container with a shallow layer of a suitable solvent or mixture of solvents in it. It is important that the solvent level is below the line with the spots on it. The next diagram doesn't show details of how the paper is suspended because there are too many possible ways of doing it and it clutters the diagram. Sometimes the paper is just coiled into a loose cylinder and fastened with paper clips top and bottom. The cylinder then just stands in the bottom of the container.

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 colored 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 might be the same as the single dye in pen 3.

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 f 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 f value for that component is:

In the example we looked at with the various pens, it wasn't necessary to measure R f 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 color 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 colored compounds with very similar R f values. We'll look at how you can get around that problem further down the page.

What if the substances you are interested in are colorless?

In some cases, it may be possible to make the spots visible by reacting them with something which produces a colored 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 labeled 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. Ninhydrin reacts with amino acids to give colored 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 f values because you can easily compare the spots in the mixture with those of the known amino acids - both from their positions and their colors. In this example, the mixture contains the amino acids labeled 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

Two way paper chromatography gets around the problem of separating out substances which have very similar R f values. I'm going to go back to talking about colored compounds because it is much easier to see what is happening. You can perfectly well do this with colorless 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 labeled 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 f values. They could equally well, of course, both have been the same color - 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 f 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 f 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.

How does paper chromatography work?

Although paper chromatography is simple to do, it is quite difficult to explain compared with thin layer chromatography. The explanation depends to some extent on what sort of solvent you are using, and many sources gloss over the problem completely. If you haven't already done so, it would be helpful if you could read the explanation for how thin layer chromatography works (link below). That will save me a lot of repetition, and I can concentrate on the problems.

The essential structure of paper

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.

Paper chromatography using a non-polar solvent

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 f values.

On the other hand, polar molecules will 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 partition . Paper chromatography using a non-polar solvent is therefore a type of partition chromatography.

Paper chromatography using a water and other polar solvents

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.

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Paper Chromatography

How does paper chromatography work, stationary and mobile phases, paper chromatography experiment, applications.

Paper chromatography is a simple and cost-effective separation technique that separates and identifies different components in a mixture. [1-4]

In paper chromatography, a specialized paper acts as the stationary phase, while a liquid solvent is the mobile phase. The mixture to be analyzed is applied to the paper. As the solvent moves up through capillary action, it carries along the individual components of the mixture at different rates based on their solubility and affinity for the stationary phase.

The principle behind paper chromatography lies in the differential partitioning of compounds between the stationary and mobile phases. The stationary phase typically consists of cellulose fibers embedded in filter paper or thin-layer chromatography plates. These fibers provide an adsorbent surface for compounds to interact with.

Understanding the mechanism behind paper chromatography requires knowledge of several key processes. [1-4]

The first process is capillary action, which refers to the ability of liquids to flow through narrow spaces against gravity. In paper chromatography, capillary action allows the solvent to move up the paper strip due to its attraction to the fibers in the paper. As the solvent moves up, it carries the solutes in the analyzed mixture. This migration of solutes is driven by two main mechanisms: adsorption and partitioning.

Adsorption occurs when solute molecules adhere to the fibers or other surfaces within the paper. It can be influenced by polarity and molecular size, with more polar or larger molecules having stronger interactions with the stationary phase.

Conversely, partitioning involves solute molecules distributing themselves between two immiscible phases – in this case, between the stationary phase (paper) and mobile phase (solvent). The extent of partitioning depends on factors such as solute polarity and affinity for either phase.

As solutes migrate up through capillary action, they may experience different degrees of adsorption and partitioning along their journey. This results in their separation based on their characteristics. By analyzing how far each component migrates on a chromatogram – a visual representation of separated components – scientists can determine properties such as retention factor (R f ) values and identify unknown substances based on known reference compounds.

Stationary and mobile phases play crucial roles in separating components of a mixture. [1-4]

The stationary phase refers to the absorbent material fixed on the chromatography paper. It can be made of cellulose or other materials with high absorbency. The stationary phase acts as a substrate for the sample mixture to interact with during separation.

On the other hand, the mobile phase is the solvent or liquid that moves through the stationary phase, carrying the sample components. The mobile phase must have good solubility with the components of interest. It should be able to flow easily through the paper.

As the mobile phase moves through the stationary phase, it interacts differently with each mixture component based on their solubility and affinity for both phases. This differential interaction leads to separation as different components travel at different rates along the paper.

Choosing an appropriate combination of stationary and mobile phases is important for effective separation in paper chromatography. Factors such as polarity, viscosity, and compatibility between phases must be considered to achieve optimal results.

Performing a paper chromatography experiment involves several essential steps to ensure accurate results. The process begins with preparing samples for paper chromatography, then spotting the sample on the paper strip, and finally, developing the chromatogram. [1-4]

Preparing the samples is crucial in obtaining reliable data. It involves selecting appropriate substances to analyze and ensuring they are suitable for chromatography. Samples can be liquid or solid and must be dissolved or crushed into a solution before application.

Next, spotting the sample on the paper strip is done carefully to ensure accurate separation. A small spot of the prepared sample is placed near one end of a designated area on the filter paper strip. It is essential to use a capillary tube or micropipette for precise and consistent application.

Once all samples are spotted on the filter paper strip, it is time for the development of the chromatogram. This step involves placing one end of the strip into a solvent traveling up through capillary action. The choice of solvent depends on factors such as solubility and desired separation distance.

As the solvent moves up through the filter paper strip, it carries different components in each sample. These components separate based on their affinity for stationary (filter paper) and mobile (solvent) phases. The separation occurs due to differences in molecular size, polarity, or other physical properties.

Throughout this process, it is important to maintain controlled conditions such as temperature and humidity to ensure reproducibility. Further analysis can be conducted once an optimal separation has been achieved, which can take several minutes or hours depending on various factors, including solvent choice and sample composition.

The diverse applications of paper chromatography across various fields are listed below. [1-4]

• It plays a crucial role in forensic analysis by separating and identifying different components in complex mixtures, such as blood or ink samples.
• Aids in the analysis of crime scene evidence, allowing forensic scientists to determine the presence of specific substances and identify potential suspects based on chromatographic patterns
• Enables the separation of different dyes used in food coloring, helping to ensure compliance with regulatory standards and quality control measures
• Determines the authenticity and safety of food products by identifying and quantifying specific components present in complex food matrices
• Separate and identify active ingredients, impurities, and by-products in pharmaceutical formulations.
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Teach paper chromatography like a maestro

Brighten up your chromatography lessons with these five teacher-tested ideas from teacher fellow Kristy Turner

Source: © GIPhotoStock/Science Photo Library

Use these five top tips to help your students master paper chromatography

Paper chromatography is often one of the first experiments students do in their science careers, sometimes even in primary school. It seems like a simple topic with some nice practical activities, but this simplicity can be misleading. It is a convenient technique with a long history, and it is still used in research today.

In a spiral curriculum, pupils will first meet paper chromatography in 11–14 chemistry, where the focus is carrying out simple experiments to find the numbers of components in mixtures. The focus then develops to building a greater understanding of the role of solubility and the mobile and stationary phases at 14–16, where students are expected to be able to describe and explain key elements of the separation.

1. Keep an easy pace

Don’t rush from introductory concepts into more complex science and, especially, don’t front-load the topic into 11–14. It can be tempting to rush as chromatography seems trivial to experts. Remember to view the curriculum through the novice lens. A broader understanding of concepts like the particle model and solubility supports better understanding of paper chromatography at higher levels. So, try not to bring those difficult concepts down into 11–14, and trust a spiral curriculum to support progression.

2. Add some colour to your lessons

Tailor your lessons to your students’ interests: students can find food fraud investigations or CSI-type activities really engaging. Chromatography of inks or food colourings is a common practical activity, but formulations change frequently so it’s not unusual for reliable experiments to suddenly stop working. Keep an eye on social media and resource sharing sites for new contexts for your experiments. Paper chromatography is very versatile and, with the right conditions, will separate many kinds of mixtures: pigments in leaves, components of universal indicators, amino acids and the coloured coating of hard-shelled sweets.

Source: Provided by the author

Engage your students and deepen their understanding by getting it wrong! Here, because the solvent started above the origin, some of the sample washed off 

3. Do it wrong (on purpose)

Spotting the errors in diagrams of paper chromatography experiments is a common exam question, but it can be difficult for students to figure this out without first experiencing bad experiments. Engage students with concrete examples by showing them the effects of common errors . Do an experiment where too much solvent is used (washing the sample spots away), or where the origin line is drawn in pen, obscuring the results.

4. Mix it up

Don’t just use water as the solvent or mobile phase. It’s a convenient and safe substance to work with and, of course, it’s a good solvent, but it doesn’t always give the best separations for different mixtures. Just using water can also mean students assume that the solvent will always be water and lead to them not carefully examining the information given in questions. Try switching water for salt solutions when separating inks for better resolution of the different colours. For older students look at how different ratios of ethanol:water in solvent mixtures change how far the components of mixtures travel up the paper and the effect of R f .

More ideas for mastering chromatography

• Check out this collection of useful videos full of paper chromatography experiments. 
• Enhance your approach to practical chromatography  with this CPD guide. 
• Roll out the kitchen roll for this idea for classroom or home activity. 

5. Be ambitious but supportive with terminology

Teach the language of the processes and their explanations explicitly. Chromatography, from ‘chroma’ meaning colour and ‘graph’ meaning to record, stationary phase (not stationery – ‘e’ is for envelopes, after all), mobile phase, origin, solvent front, chromatogram. Practise the use of correct terms using recall items, fill-in-the-blanks exercises and short answers to build student confidence. Where students are really struggling in their explanations and descriptions, ease the cognitive load by providing them with keyword lists to refer to as they work their way through questions.

More from Kristy Turner

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• Chromatography

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Class 11 Chemistry (India)

Course: class 11 chemistry (india)   >   unit 12.

• Simple and fractional distillations

Principles of chromatography

• Basics of chromatography
• Column chromatography
• Thin layer chromatography (TLC)
• Calculating retention factors for TLC
• Gas chromatography
• Take a few leaves and crush them in a mortar.
• Spot a drop of the leaf extract on a strip of chromatographic paper ~ 0.5 cm above the edge of the paper. Chromatographic paper is made of cellulose and is quite polar in nature.
• Place the strip of paper in a jar that contains a small volume of propanone (acetone). There should be just enough propanone that the edge of the paper dips in it comfortably. Place a lid on the jar to avoid any evaporation of the solvent.
• Let the solvent rise up the paper by capillary action. Remove the paper strip from the jar once the solvent has reached the ‘solvent front’ level. 5) What do you think you will notice?
• Higher the adsorption to the stationary phase, the slower the molecule will move through the column.
• Higher the solubility in the mobile phase, the faster the molecule will move through the column.

Different types of chromatography

Thin layer chromatography (tlc): retention factors (r f ‍   ).

• The component that travels the least distance on the TLC plate is the most polar, since it binds to the silica most tightly.
• The component that travels the maximum distance is the least polar; it binds to the silica least tightly and is most soluble in the non-polar solvent (mobile phase), and hence moves up the plate with the solvent.

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Paper Chromatography Experiment

March 17, 2021 By Emma Vanstone Leave a Comment

This simple 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 colour ink particles travel at different speeds through the filter paper allowing you to see the constituent colours of the pen ink.

All types of chromatography have two phases. A mobile phase where the molecules can move and a stationary phase where the molecules can’t move. In the case of paper chromatography, the stationary phase is the filer 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 just the very bottom is touching 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! We call the finished filter paper, a chromatogram.

What happens if you use washable pens?

If the inks are washable, they tend just to contain one type of ink, and so you don’t see any separation of colour.

You can see below that only a couple of the inks have separated out, 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.

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 August 10, 2023 by Emma Vanstone

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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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Separation techniques: Chromatography

Ozlem coskun.

Department of Biophysics, Canakkale Onsekiz Mart University, Canakkale, Turkey

Chromatography is an important biophysical technique that enables the separation, identification, and purification of the components of a mixture for qualitative and quantitative analysis. Proteins can be purified based on characteristics such as size and shape, total charge, hydrophobic groups present on the surface, and binding capacity with the stationary phase. Four separation techniques based on molecular characteristics and interaction type use mechanisms of ion exchange, surface adsorption, partition, and size exclusion. Other chromatography techniques are based on the stationary bed, including column, thin layer, and paper chromatography. Column chromatography is one of the most common methods of protein purification.

Chromatography is based on the principle where molecules in mixture applied onto the surface or into the solid, and fluid stationary phase (stable phase) is separating from each other while moving with the aid of a mobile phase. The factors effective on this separation process include molecular characteristics related to adsorption (liquid-solid), partition (liquid-solid), and affinity or differences among their molecular weights [ 1 , 2 ]. Because of these differences, some components of the mixture stay longer in the stationary phase, and they move slowly in the chromatography system, while others pass rapidly into mobile phase, and leave the system faster [ 3 ].

Based on this approach three components form the basis of the chromatography technique.

• Stationary phase: This phase is always composed of a “solid” phase or “a layer of a liquid adsorbed on the surface a solid support”.
• Mobile phase: This phase is always composed of “liquid” or a “gaseous component.”
• Separated molecules

The type of interaction between stationary phase, mobile phase, and substances contained in the mixture is the basic component effective on separation of molecules from each other. Chromatography methods based on partition are very effective on separation, and identification of small molecules as amino acids, carbohydrates, and fatty acids. However, affinity chromatographies (ie. ion-exchange chromatography) are more effective in the separation of macromolecules as nucleic acids, and proteins. Paper chromatography is used in the separation of proteins, and in studies related to protein synthesis; gas-liquid chromatography is utilized in the separation of alcohol, esther, lipid, and amino groups, and observation of enzymatic interactions, while molecular-sieve chromatography is employed especially for the determination of molecular weights of proteins. Agarose-gel chromatography is used for the purification of RNA, DNA particles, and viruses [ 4 ].

Stationary phase in chromatography, is a solid phase or a liquid phase coated on the surface of a solid phase. Mobile phase flowing over the stationary phase is a gaseous or liquid phase. If mobile phase is liquid it is termed as liquid chromatography (LC), and if it is gas then it is called gas chromatography (GC). Gas chromatography is applied for gases, and mixtures of volatile liquids, and solid material. Liquid chromatography is used especially for thermal unstable, and non-volatile samples [ 5 ].

The purpose of applying chromatography which is used as a method of quantitative analysis apart from its separation, is to achive a satisfactory separation within a suitable timeinterval. Various chromatography methods have been developed to that end. Some of them include column chromatography, thin-layer chromatography (TLC), paper chromatography, gas chromatography, ion exchange chromatography, gel permeation chromatography, high-pressure liquid chromatography, and affinity chromatography [ 6 ].

Column chromatography

• Ion-exchange chromatography
• Gel-permeation (molecular sieve) chromatography
• Affinity chromatography
• Paper chromatography
• Thin-layer chromatography
• Gas chromatography
• Dye-ligand chromatography
• Hydrophobic interaction chromatography
• Pseudoaffinity chromatography
• High-pressure liquid chromatography (HPLC)

Since proteins have difference characteristic features as size, shape, net charge, stationary phase used, and binding capacity, each one of these characteristic components can be purified using chromatographic methods. Among these methods, most frequently column chromatography is applied. This technique is used for the purification of biomolecules. On a column (stationary phase) firstly the sample to be separated, then wash buffer (mobile phase) are applied ( Figure 1 ). Their flow through inside column material placed on a fiberglass support is ensured. The samples are accumulated at the bottom of the device in a tme-, and volume-dependent manner [ 7 ].

Column chromatography.

Ion- exchange chromatography is based on electrostatic interactions between charged protein groups, and solid support material (matrix). Matrix has an ion load opposite to that of the protein to be separated, and the affinity of the protein to the column is achieved with ionic ties. Proteins are separated from the column either by changing pH, concentration of ion salts or ionic strength of the buffer solution [ 8 ]. Positively charged ion- exchange matrices are called anion-exchange matrices, and adsorb negatively charged proteins. While matrices bound with negatively charged groups are known as cation-exchange matrices, and adsorb positively charged proteins ( Figure 2 ) [ 9 ].

Ion- exchange chromatography.

The basic principle of this method is to use dextran containing materials to separate macromolecules based on their differences in molecular sizes. This procedure is basically used to determine molecular weights of proteins, and to decrease salt concentrations of protein solutions [ 10 ]. In a gel- permeation column stationary phase consists of inert molecules with small pores. The solution containing molecules of different dimensions are passed continuously with a constant flow rate through the column. Molecules larger than pores can not permeate into gel particles, and they are retained between particles within a restricted area. Larger molecules pass through spaces between porous particles, and move rapidly through inside the column. Molecules smaller than the pores are diffused into pores, and as molecules get smaller, they leave the column with proportionally longer retention times ( Figure 3 ) [ 11 ]. Sephadeks G type is the most frequently used column material. Besides, dextran, agorose, polyacrylamide are also used as column materials [ 12 ].

Gel-permeation (molecular sieve) chromatography.

This chromatography technique is used for the purification of enzymes, hormones, antibodies, nucleic acids, and specific proteins [ 13 ]. A ligand which can make a complex with specific protein (dextran, polyacrylamide, cellulose etc) binds the filling material of the column. The specific protein which makes a complex with the ligand is attached to the solid support (matrix), and retained in the column, while free proteins leave the column. Then the bound protein leaves the column by means of changing its ionic strength through alteration of pH or addition of a salt solution ( Figure 4 ) [ 14 ].

Affinity chromatography.

In paper chromatography support material consists of a layer of cellulose highly saturated with water. In this method a thick filter paper comprised the support, and water drops settled in its pores made up the stationary “liquid phase.” Mobile phase consists of an appropriate fluid placed in a developing tank. Paper chromatography is a “liquid-liquid” chromatography [ 15 ].

Thin-layer chromatography is a “solid-liquid adsorption” chromatography. In this method stationary phase is a solid adsorbent substance coated on glass plates. As adsorbent material all solid substances used. in column chromatography (alumina, silica gel, cellulose) can be utilized. In this method, the mobile phase travels upward through the stationary phase The solvent travels up the thin plate soaked with the solvent by means of capillary action. During this procedure, it also drives the mixture priorly dropped on the lower parts of the plate with a pipette upwards with different flow rates. Thus the separation of analytes is achieved. This upward travelling rate depends on the polarity of the material, solid phase, and of the solvent [ 16 ].

In cases where molecules of the sample are colorless, florescence, radioactivity or a specific chemical substance can be used to produce a visible coloured reactive product so as to identify their positions on the chromatogram. Formation of a visible colour can be observed under room light or UV light. The position of each molecule in the mixture can be measured by calculating the ratio between the the distances travelled by the molecule and the solvent. This measurement value is called relative mobility, and expressed with a symbol R f . R f . value is used for qualitative description of the molecules [ 17 ].

In this method stationary phase is a column which is placed in the device, and contains a liquid stationary phase which is adsorbed onto the surface of an inert solid. Gas chromatography is a “gas-liquid” chromatography. Its carrier phase consists of gases as He or N 2 . Mobile phase which is an inert gas is passed through a column under high pressure. The sample to be analyzed is vaporized, and enters into a gaseous mobile phase phase. The components contained in the sample are dispersed between mobile phase, and stationary phase on the solid support. Gas chromatography is a simple, multifaceted, highly sensitive, and rapidly applied technique for the extremely excellent separation of very minute molecules. It is used in the separation of very little amounts of analytes [ 18 ].

Development of this technique was based on the demonstration of the ability of many enzymes to bind purine nucleotides for Cibacron Blue F3GA dye [ 19 ]. The planar ring structure with negatively charged groups is analogous to the structure of NAD. This analogy has been evidenced by demonstration of the binding of Cibacron Blue F3GA dye to adenine, ribose binding sites of NAD. The dye behaves as an analogue of ADP-ribose. The binding capacity of this type adsorbents is 10–20-fold stronger rhat that of the affinity of other adsorbents. Under appropriate pH conditions, elution with high-ionic strength solutions, and using ion-exchange property of adsorbent, the adsorbed proteins are separated from the column [ 20 , 21 ].

In this method the adsorbents prepared as column material for the ligand binding in affinity chromatography are used. HIC technique is based on hydrophobic interactions between side chains bound to chromatography matrix [ 22 , 23 ].

Some compounds as anthraquinone dyes, and azo-dyes can be used as ligands because of their affinity especially for dehydrogenases, kinases, transferases, and reductases The mostly known type of this kind of chromatography is immobilized metal affinity chromatography (IMAC) [ 24 ].

Using this chromatography technique it is possible to perform structural, and functional analysis, and purification of many molecules within a short time, This technique yields perfect results in the separation, and identification of amino acids, carbohydrates, lipids, nucleic acids, proteins, steroids, and other biologically active molecules, In HPLC, mobile phase passes throuıgh columns under 10–400 atmospheric pressure, and with a high (0.1–5 cm//sec) flow rate. In this technique, use of small particles, and application of high presure on the rate of solvent flow increases separation power, of HPLC and the analysis is completed within a short time.

Essential components of a HPLC device are solvent depot, high- pressure pump, commercially prepared column, detector, and recorder. Duration of separation is controlled with the aid of a computerized system, and material is accrued [ 25 ].

Chromatography technique is a valuable tool for biochemists, besides it can be applied easily during studies performed in clinical laboratories For instance, paper chromatography is used to determine some types of sugar, and amino acids in bodily fluids which are associated with hereditary metabolic disorders. Gas chromatography is used in laboratories to measure steroids, barbiturates, and lipids. Chromatographic technique is also used in the separation of vitamins, and proteins.

Initially chromatographic techniques were used to separate substances based on their color as was the case with herbal pigments. With time its application area was extended considerably. Nowadays, chromatography is accepted as an extremely sensitive, and effective separation method. Column chromatography is one of the useful separation, and determination methods. Column chromatography is a protein purification method realized especially based on one of the characteristic features of proteins. Besides, these methods are used to control purity of a protein. HPLC technique which has many superior features including especially its higher sensitivity, rapid turnover rate, its use as a quantitative method, can purify amino acids, proteins, nucleic acids, hydrocarbons, carbohydrates, drugs, antibiotics, and steroids.

Conflict of Interest: None declared.

Financial Disclosure: The authors declared that this study has received no financial support.

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New treatment could reverse hair loss caused by an autoimmune skin disease

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Researchers at MIT, Brigham and Women’s Hospital, and Harvard Medical School have developed a potential new treatment for alopecia areata, an autoimmune disorder that causes hair loss and affects people of all ages, including children.

For most patients with this type of hair loss, there is no effective treatment. The team developed a microneedle patch that can be painlessly applied to the scalp and releases drugs that help to rebalance the immune response at the site, halting the autoimmune attack.

In a study of mice, the researchers found that this treatment allowed hair to regrow and dramatically reduced inflammation at the treatment site, while avoiding systemic immune effects elsewhere in the body. This strategy could also be adapted to treat other autoimmune skin diseases such as vitiligo, atopic dermatitis, and psoriasis, the researchers say.

“This innovative approach marks a paradigm shift. Rather than suppressing the immune system, we’re now focusing on regulating it precisely at the site of antigen encounter to generate immune tolerance,” says Natalie Artzi, a principal research scientist in MIT’s Institute for Medical Engineering and Science, an associate professor of medicine at Harvard Medical School and Brigham and Women’s Hospital, and an associate faculty member at the Wyss Institute of Harvard University.

Artzi and Jamil R. Azzi, an associate professor of medicine at Harvard Medical School and Brigham and Women’s Hospital, are the senior authors of the new study , which appears in the journal Advanced Materials . Nour Younis, a Brigham and Women’s postdoc, and Nuria Puigmal, a Brigham and Women’s postdoc and former MIT research affiliate, are the lead authors of the paper.

The researchers are now working on launching a company to further develop the technology, led by Puigmal, who was recently awarded a Harvard Business School Blavatnik Fellowship.

Direct delivery

Alopecia areata, which affects more than 6 million Americans, occurs when the body’s own T cells attack hair follicles, leading the hair to fall out. The only treatment available to most patients — injections of immunosuppressant steroids into the scalp — is painful and patients often can’t tolerate it.

Some patients with alopecia areata and other autoimmune skin diseases can also be treated with immunosuppressant drugs that are given orally, but these drugs lead to widespread suppression of the immune system, which can have adverse side effects.

“This approach silences the entire immune system, offering relief from inflammation symptoms but leading to frequent recurrences. Moreover, it increases susceptibility to infections, cardiovascular diseases, and cancer,” Artzi says.

A few years ago, at a working group meeting in Washington, Artzi happened to be seated next to Azzi (the seating was alphabetical), an immunologist and transplant physican who was seeking new ways to deliver drugs directly to the skin to treat skin-related diseases.

Their conversation led to a new collaboration, and the two labs joined forces to work on a microneedle patch to deliver drugs to the skin. In 2021, they reported that such a patch can be used to prevent rejection following skin transplant. In the new study, they began applying this approach to autoimmune skin disorders.

“The skin is the only organ in our body that we can see and touch, and yet when it comes to drug delivery to the skin, we revert to systemic administration. We saw great potential in utilizing the microneedle patch to reprogram the immune system locally,” Azzi says.

The microneedle patches used in this study are made from hyaluronic acid crosslinked with polyethylene glycol (PEG), both of which are biocompatible and commonly used in medical applications. With this delivery method, drugs can pass through the tough outer layer of the epidermis, which can’t be penetrated by creams applied to the skin.

“This polymer formulation allows us to create highly durable needles capable of effectively penetrating the skin. Additionally, it gives us the flexibility to incorporate any desired drug,” Artzi says. For this study, the researchers loaded the patches with a combination of the cytokines IL-2 and CCL-22. Together, these immune molecules help to recruit regulatory T cells, which proliferate and help to tamp down inflammation. These cells also help the immune system learn to recognize that hair follicles are not foreign antigens, so that it will stop attacking them.

Hair regrowth

The researchers found that mice treated with this patch every other day for three weeks had many more regulatory T cells present at the site, along with a reduction in inflammation. Hair was able to regrow at those sites, and this growth was maintained for several weeks after the treatment ended. In these mice, there were no changes in the levels of regulatory T cells in the spleen or lymph nodes, suggesting that the treatment affected only the site where the patch was applied.

In another set of experiments, the researchers grafted human skin onto mice with a humanized immune system. In these mice, the microneedle treatment also induced proliferation of regulatory T cells and a reduction in inflammation.

The researchers designed the microneedle patches so that after releasing their drug payload, they can also collect samples that could be used to monitor the progress of the treatment. Hyaluronic acid causes the needles to swell about tenfold after entering the skin, which allows them to absorb interstitial fluid containing biomolecules and immune cells from the skin.

Following patch removal, researchers can analyze samples to measure levels of regulatory T cells and inflammation markers. This could prove valuable for monitoring future patients who may undergo this treatment.

The researchers now plan to further develop this approach for treating alopecia, and to expand into other autoimmune skin diseases.

The research was funded by the Ignite Fund and Shark Tank Fund awards from the Department of Medicine at Brigham and Women’s Hospital.

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MIT researchers have developed microneedle patches that are capable of restoring hair growth in alopecia areata patients, reports Ernie Mundell for HealthDay . The team’s approach includes a, “patch containing myriad microneedles that is applied to the scalp,” writes Mundell. “It releases drugs to reset the immune system so it stops attacking follicles.” 

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The Macroeconomic Impact of Climate Change: Global vs. Local Temperature

This paper estimates that the macroeconomic damages from climate change are six times larger than previously thought. We exploit natural variability in global temperature and rely on time-series variation. A 1°C increase in global temperature leads to a 12% decline in world GDP. Global temperature shocks correlate much more strongly with extreme climatic events than the country-level temperature shocks commonly used in the panel literature, explaining why our estimate is substantially larger. We use our reduced-form evidence to estimate structural damage functions in a standard neoclassical growth model. Our results imply a Social Cost of Carbon of $1,056 per ton of carbon dioxide. A business-as-usual warming scenario leads to a present value welfare loss of 31%. Both are multiple orders of magnitude above previous estimates and imply that unilateral decarbonization policy is cost-effective for large countries such as the United States.

Adrien Bilal gratefully acknowledges support from the Chae Family Economics Research Fund at Harvard University. The views expressed herein are those of the authors and do not necessarily reflect the views of the National Bureau of Economic Research.

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Department of Agricultural, Food, and Resource Economics Innovation Lab for Food Security Policy, Research, Capacity and Influence

Crop Production Diversity and the Well-being of Smallholder Farm Households: Evidence from Nigeria

May 14, 2024 - Ibukun James Olaoye, Sarah Edore Edewor, Tarana Chauhan and David Stifel

This paper explores the implications of farmers choosing to diversify their crop production rather than to specialize in one crop on household welfare. Specifically, we estimate the association between household crop production diversity (CD) and household welfare outcomes. To better understand the farmers’ production decisions, we also explore the influence that market access and rainfall shocks may have on CD practices. Using fixed-effects models applied to nationally representative panel data for 2010, 2012 and 2015 from the Nigerian Living Standard Measurement Survey, we find that CD is positive and significantly associated with improved household welfare outcomes for households situated father away from markets but the association is not significant for children anthropometric well-being. While a positive association between CD and farm income exist, we find that smallholder households uptake CD due to limited market access, and the exposure to positive and negative rainfall shocks. Our findings contribute to understanding farm household production and consumption behavior and are relevant for policy responses towards reinforcing smallholders’ capacity to cope with and adapt to shocks. It can also serve as a guide in prioritizing development efforts to stimulate relevant and well-informed policy and interventions.

Crop diversification, climate shocks, market access, household well-being, panel regression, Nigeria.

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Creating a green composite material from Japanese washi paper

Washi: the traditional Japanese paper, known for its beauty and strength, has been used in bookbinding, art, furniture, and architecture for hundreds of years. But more recently, washi's usage is on the decline, as people opt for more western style housing designs. In a bid to revive interest in this traditional craft, a group of Tohoku University researchers has developed an environmentally friendly material from washi that boasts improved strength and biodegradability.

Details of the research were published in the journal Composites Part A: Applied Science and Manufacturing on May, 9, 2024.

Bio-based and biodegradable materials are increasingly sought after as the world seeks to move away from fossil based-plastic materials and build a more sustainable society. Green composites combine plastics with natural fibers, producing materials with higher strength, improved biodegradability, and a lower environmental footprint.

"We created a green composite from washi, which itself stems from plant fibers, improving its properties further whilst still maintaining its classical beauty," points out Hiroki Kurita, co-author of the paper and an associate professor at Tohoku University's Graduate School of Environmental Studies.

To produce the material, Kurita and his colleagues layered and hot pressed sheets of Washi with polybutylene succinate (PBS). To source the Washi, they worked with an artisan from a Miyagi-based washi-workshop. The material's ultimate tensile strength, i.e., the amount of stress the paper could withstand, stood at 59.85 MPa, representing an improvement of over 60%.

Washi has a lot of space between its entangled fibers. When combined with PBS, the plastic filled these spaces, thereby locking the fibers in place and preventing the fibers from moving.

PBS is also notable for its biodegradability, and the resultant composite material degraded much faster than pure plastic. After 35 days, it had biodegraded by 82%.The biodegradation was calculated by measuring the amount of CO2 released from the material when it was buried in compost. At the same time, researchers measured weight loss and loss of strength during degradation.

Not only was the team successful in producing a new material, but Kurita believes they were able to raise the standard of biodegradation testing and provide blueprints for future research into biodegradable composite materials. "We utilized both standardized and non-standardized methods for measuring biodegradability. The differing methods used will help researchers compare biodegradability between different materials moving forward."

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• Lovisa Rova, Alia Gallet-Pandellé, Zhenjin Wang, Hiroki Kurita, Fumio Narita. Japanese washi-paper-based green composites: Fabrication, mechanical characterization, and evaluation of biodegradability . Composites Part A: Applied Science and Manufacturing , 2024; 108261 DOI: 10.1016/j.compositesa.2024.108261

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