paper chromatography lab hypothesis

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)

Paper Chromatography of Plant Pigments

Learning Objectives

After completing the lab, the student will be able to:

• Extract pigments from plant material.
• Separate pigments by paper chromatography.
• Measure R f (retention factor) values for pigments.

Activity 2: Pre-Assessment

• The leaves of some plants change color in fall. Green foliage appears to turn to hues of yellow and brown. Does the yellow color appear because carotenoids replace the green chlorophylls? Explain your reasoning.
• Examine the molecular structures of photosynthetic pigments in Figure 10.1. Photosynthetic pigments are hydrophobic molecules located in thylakoid membranes. Will these pigments dissolve in water?

Activity 2: Paper Chromatography of Plant Pigments

Paper chromatography is an analytical method that separates compounds based on their solubility in a solvent.

The solvent is used to separate a mixture of molecules that have been applied to filter paper. The paper, made of cellulose, represents the stationary or immobile phase. The separation mixture moves up the paper by capillary action. It is called the mobile phase. The results of chromatography are recorded in a chromatogram. Here, the chromatogram is the piece of filter paper with the separated pigment that you will examine at the end of your experiment (see Figure 10.4).

We separate the compounds based on how quickly they move across the paper. Compounds that are soluble in the solvent mixture will be more concentrated in the mobile phase and move faster up the paper. Polar compounds will bind to the cellulose in the paper and trail behind the solvent front. As a result, the different compounds will separate according to their solubility in the mixture of organic solvents we use for chromatography.

This video demonstrates the principles and examples of chromatography. You will experiment with only paper chromatography in this lab; however, you will see that you are already familiar with some uses of thin layer chromatography.

Safety Precautions

• Work under a hood or in a well-ventilated space and avoid breathing solvents.
• Do not have any open flames when working with flammable solvents.
• Wear aprons and eye protection.
• Do not pour any organic solvent down the drain.
• Dispose of solvents per local regulations.
• Use forceps to handle chromatography paper that has been immersed in solvent and wash your hands after completing this activity.

For this activity, you will need the following:

• Plant material: intact leaves of spinach and Coleus (one leaf of each plant per pair of students)
• Filter or chromatography paper
• Ruler (one per group)
• Colored pencils
• Beakers (400 mL) (Mason jars are an acceptable substitute)
• Aluminum foil
• Petroleum ether: acetone: water in a 3:1:1 proportion
• If no hood or well-ventilated place is available, the mixture can be substituted with 95 percent isopropyl alcohol. Note that, if isopropyl alcohol is used, the pigment bands will smear. You may not be able to separate and identify the chlorophylls or carotene from xanthophyll.

For this activity, you will work in pairs .

Structured Inquiry

Step 1: Hypothesize/Predict: Discuss with your lab partner what color pigments will likely be present in the spinach leaves. Write your predictions in your lab notebook and draw a diagram of how you think the pigments will separate out on the chromatography paper.

Step 2: Student-led Planning: Read step 3 below. Discuss with your lab partner the setup of the experiment. Then agree upon the dimensions of the filter/chromatography paper that you will use. To allow good separation, the paper should not touch the walls of the container. The paper must fit inside the container while being long enough for maximum separation. Write all your calculations in your lab notebook.

Step 3: Follow the steps below to set up your filter paper and perform the chromatography experiment.

• Prepare the chromatogram by cutting a piece of filter paper. Transfer pigments from spinach leaves as in Activity 1. A heavy application line will yield stronger colors when the pigments separate, making it easier to read results. Allow the pigments to dry between applications. Wet extracts diffuse on the paper and yield blurry lines.
• Form a cylinder with the filter paper without overlapping the edges (to avoid edge effects). The sample should face the outside of the cylinder. Secure the top and bottom of the cylinder with staples.
• Pour enough separation mixture to provide a mobile phase while staying below the origin line on the chromatogram. The exact volume is not critical if the origin, the start line where you applied the solvent, is above the solvent. See Figure 10.4.

• Label the beaker with a piece of tape with your initials and your partner’s initials.
• Lower the paper into the container with the band from the extraction in the lower section. The paper must touch the solvent, but not reach the band of pigment you applied. Why must the band be above the solvent line? Write your answer in your notebook.
• Cover the container tightly with a piece of aluminum foil.
• Track the rising of the solvent front. Can you see a separation of colors on the paper?
• When the solvent front is within 1 cm of the upper edge of the paper, remove the cylinder from the beaker using forceps. Trace the solvent front with a pencil before it evaporates and disappears! Draw the colored bands seen on your chromatography paper in your lab notebook immediately. The colors will fade upon drying. If no colored pencils are available, record the colors of the lines.
• Let the paper dry in a well-ventilated area before making measurements because the wet paper is fragile and may break when handled. This is also a precaution to avoid breathing fumes from the chromatogram.
• Discard solvent mixture per your instructor’s directions. Do not pour down the drain.

Step 4: Critical Analysis: Open the dried cylinder by removing the staples. Measure the distance from the first pencil line to the solvent front, as shown in Figure 10.5. This is the distance traveled by the solvent front. Measure the distance from the pencil line to the middle point of each color band and the original pencil line. Record your results in your notebook in a table modeled after Table 10.1. The retention factor (R f ) is the ratio of the distance traveled by a colored band to the distance traveled by the solvent front. Calculate R f values for each pigment using the following equation:

R f=Distance traveled by colored band/Distance traveled by solvent front

Step 5: After determining the color of the band, tentatively identify each band. Did your results support your hypothesis about the color of each band? Discuss which aspects of the experiments may have yielded inconclusive results. How could you improve the experiment?

Guided Inquiry

Step 1: Hypothesize/Predict: What type of pigments are present in Coleus leaves and where are the different colors located? Can you make a hypothesis based on the coloration of the variegated leaves? Write your hypothesis down in your lab notebook. Would there be a difference if you performed chromatography on pigment composition from different colored regions of the leaves?

Step 2: Student-led Planning: Cut the chromatography/filter paper to the dimensions needed. Apply pigments from different parts of the Coleus leaves following the procedure described under Activity 1, keeping in mind that a darker line will yield stronger colors when the pigments are separated, which will make it easier to read the results. Allow the pigments to dry between applications. Wet extracts diffuse on the paper and yield blurry lines.

Step 3: When the solvent front reaches 1 cm from the top of the filter paper, stop the procedure. Draw the pigment bands you see on the filter paper in your lab notebook. Clearly indicate the color you observed for each band.

Step 4: Let the cylinder dry and measure the distance the front traveled from the origin and the distances traveled by each of the pigments. If the bands broadened during separation, take measurements to the middle of each band.

Step 5: Critical Analysis: Calculate R f for each of the bands and record them in a table in your notebook. Compare the R f you obtained with those of other groups. Are the R f values similar? What may have altered R f values?

Assessments

• Carotenoids and chlorophylls are hydrophobic molecules that dissolve in organic solvents. Where would you find these molecules in the cell? What would happen if you ran the chromatography in this lab with water as the solvent?
• All chlorophyll molecules contain a complexed magnesium ion. Your houseplant is developing yellow leaves. What may cause this, and how can you restore your plant’s health?
• Seeds that grow under dim light are said to be etiolated, which describes their pale and spindly appearance. They soon waste away after exhausting their food reserves. Can you explain this observation?

Lab Manual for Biology Part I Copyright © 2022 by LOUIS: The Louisiana Library Network is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License , except where otherwise noted.

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Leaf Chromatography Experiment – Easy Paper Chromatography

Leaf chromatography is paper chromatography using leaves. Paper chromatography is a separation technique. When applied to leaves, it separates the pigment molecules mostly according to their size. The main pigment molecule in green leaves is chlorophyll, which performs photosynthesis in the plant. Other pigments also occur, such as carotenoids and anthocyanins. When leaves change color in the fall , the amount and type of pigment molecules changes. Leaf chromatography is a fun science project that lets you see these different pigments.

Leaf Chromatography Materials

You only need a few simple materials for the leaf chromatography project:

• Rubbing alcohol (isopropyl alcohol)
• Coffee filters or thick paper towels
• Small clear jars or glasses with lids (or plastic wrap to cover the jars)
• Shallow pan
• Kitchen utensils

You can use any leaves for this project. A single plant leaf contains several pigment molecules, but for the most colors, use a variety of leaves. Or, collect several of each kind of leaf and compare them to each other. Good choices are colorful autumn leaves or chopped spinach.

Perform Paper Chromatography on Leaves

The key steps are breaking open the cells in leaves and extracting the pigment molecule and then separating the pigment using the alcohol and paper.

• Finely chop 2-3 leaves or several small leaves. If available, use a blender to break open the plant cells. The pigment molecules are in the chloroplasts of the cells, which are organelles encased within the plant cell walls. The more you break up the leave, the more pigment you’ll collect.
• Add enough alcohol to just cover the leaves.
• If you have more samples of leaves, repeat this process.
• Cover the container of leaves and alcohol and set it in a shallow pan filled with enough hot tap water to surround and heat the container. You don’t want water getting into your container of leaves.
• Replace the hot water with fresh water as it cools. Swirl the container of leaves around from time to time to aid the pigment extraction into the alcohol. The extraction is ready when the alcohol is deeply colored. The darker its color, the brighter the resulting chromatogram.
• Cut a long strip of coffee filter or sturdy paper towel for each chromatography jar. Paper with an open mesh (like a paper towel) works quickly, but paper with a denser mesh (like a coffee filter) is slower but gives a better pigment separation.
• Place a strip of paper into jar, with one end in the leaf and alcohol mixture and the other end extending upward and out of the jar.
• The alcohol moves via capillary action and evaporation, pulling the pigment molecules along with it. Ultimately, you get bands of color, each containing different pigments. After 30 to 90 minutes (or whenever you achieve pigment separation), remove the paper strips and let them dry.

How Leaf Chromatography Works

Paper chromatography separates pigments in leaf cells on the basis of three criteria:

• Molecule size

Solubility is a measure of how well a pigment molecule dissolves in the sol vent. In this project, the solvent is alcohol . Crushing the leaves breaks open cells so pigments interact with alcohol. Only molecules that are soluble in alcohol migrate with it up the paper.

Assuming a pigment is soluble, the biggest factor in how far it travels up the paper is particle size. Smaller molecules travel further up the paper than larger molecules. Small molecules fit between fibers in the paper more easily than big ones. So, they take a more direct path through the paper and get further in less time. Large molecules slowly work their way through the paper. In the beginning, not much space separates large and small molecules. The paper needs to be long enough that the different-sized molecules have enough time to separate enough to tell them apart.

Paper consists of cellulose, a polysaccharide found in wood, cotton, and other plants. Cellulose is a polar molecule . Polar molecules stick to cellulose and don’t travel very far in paper chromatography. Nonpolar molecules aren’t attracted to cellulose, so they travel further.

Of course, none of this matters if the solvent doesn’t move through the paper. Alcohol moves through paper via capillary action . The adhesive force between the liquid and the paper is greater than the cohesive force of the solvent molecules. So, the alcohol moves, carrying more alcohol and the pigment molecules along with it.

Interpreting the Chromatogram

• The smallest pigment molecules are the ones that traveled the greatest distance. The largest molecules are the ones that traveled the least distance.
• If you compare chromatograms from different jars, you can identify common pigments in their leaves. All things being equal, the lines made by the pigments should be the same distance from the origin as each other. But, usually conditions are not exactly the same, so you compare colors of lines and whether they traveled a short or long distance.
• Try identifying the pigments responsible for the colors.

There are three broad classes of plant pigments: porphyrins, carotenoids, and flavonoids. The main porphyrins are chlorophyll molecules. There are actually multiple forms of chlorophyll, but you can recognize them because they are green. Carotenoids include carotene (yellow or orange), lycopene (orange or red), and xanthophyll (yellow). Flavonoids include flavone and flavonol (both yellow) and anthocyanin (red, purple, or even blue).

Experiment Ideas

• Collect leaves from a single tree or species of tree as they change color in the fall. Compare chromatograms from different colors of leaves. Are the same pigments always present in the leaves? Some plants produce the same pigments, just in differing amounts. Other plants start producing different pigments as the seasons change.
• Compare the pigments in leaves of different kinds of trees.
• Separate leaves according to color and perform leaf chromatography on the different sets. See if you can tell the color of leaves just by looking at the relative amount of different pigments.
• The solvent you use affects the pigments you see. Repeat the experiment using acetone (nail polish remover) instead of alcohol.
• Block, Richard J.; Durrum, Emmett L.; Zweig, Gunter (1955).  A Manual of Paper Chromatography and Paper Electrophoresis . Elsevier. ISBN 978-1-4832-7680-9.
• Ettre, L.S.; Zlatkis, A. (eds.) (2011). 75 Years of Chromatography: A Historical Dialogue . Elsevier. ISBN 978-0-08-085817-3.
• Gross, J. (1991). Pigments in Vegetables: Chlorophylls and Carotenoids . Van Nostrand Reinhold. ISBN 978-0442006570.
• Haslam, Edwin (2007). “Vegetable tannins – Lessons of a phytochemical lifetime.”  Phytochemistry . 68 (22–24): 2713–21. doi: 10.1016/j.phytochem.2007.09.009
• McMurry, J. (2011). Organic chemistry With Biological Applications (2nd ed.). Belmont, CA: Brooks/Cole. ISBN 9780495391470.

Separation of Plant Pigments by Paper Chromatography

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

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

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

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

Content: Separation of Plant Pigments by Paper Chromatography

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

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

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

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

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

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

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

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

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

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

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

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

You can separate all the above pigments using paper chromatography.

Video: Separation of Plant Pigments

Preparation of Concentrated Leaf Extract

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

Load the Leaf Extract onto Absorbent Paper

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

Setup the Chromatography Chamber

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

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

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

Observation Table

1. Light green spot indicates chlorophyll-b pigment.

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

2. Dark green spot represents chlorophyll-a pigment.

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

3. The yellow band represents xanthophyll pigment.

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

4. The yellow-orange band indicates carotene pigment.

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

Factors affecting the Rf values of a particular analyte are:

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

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

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

Related Topics:

• Difference Between Budding and Grafting
• Phototropism in Plants
• Potometer Experiment

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

Nice experiment and understanding.

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Jove Lab Bio

Lab 7: photosynthesis — procedure.

• NOTE: In this experiment you will separate pigments from spinach leaves using chromatography paper. Individual pigments travel along the paper at different rates and may have different colors. By calculating the relative distance the pigments travel, their resolution factor, and comparing them with literature values, you can identify different pigments. HYPOTHESES: In this exercise the experimental hypothesis is that there will be multiple pigments within the spinach leaves that absorb different wavelengths of sunlight. The null hypothesis is that there is only one type of pigment within the spinach leaf.
• Use a pencil to make a line two centimeters from one end of the chromatography paper.
• Then, lay a pipe cleaner horizontally across the top of a clean 400 mL beaker.
• Place the pencil-marked side of the chromatography strip at the bottom of the beaker.
• Next, wrap the paper around the pipe cleaner so that the bottom edge is barely touching the bottom of the beaker and then secure it with a paperclip.
• When the paper is secured around the pipe cleaner, remove it from the beaker, and then place a patted-dry spinach leaf over the marked line on the chromatography paper.
• Roll a coin over the spinach leaf along the pencil line going back and forth multiple times and applying steady pressure. When the leaf is removed, a green line should be clearly present.
• Next, place 8 mL of chromatography solvent in the beaker.
• Lower the chromatography strip into the beaker so that the edge of the paper touches the solvent but the green line does not. Adjust the pipe cleaner if needed.
• Without disturbing the beaker, observe the solvent as it moves up the paper and the individual pigments separate.
• When the solvent has traveled half way up the chromatography paper, which will take approximately 10 minutes, and the pigments have separated into well-defined bands, remove the paper from the beaker.
• Mark how far the solvent traveled with a pencil and then allow the paper to dry. NOTE: The solvent evaporates quickly.
• Next, record the number of visible bands and describe their color and relative size.
• Measure how far the solvent and pigments traveled, and record this information for each pigment in Table 1. Click Here to download Table 1
• Dispose of the chromatography solvent in a waste container under a fume hood. Throw the chromatography strips into the regular trash, and then clean the beakers with soap and water.
• NOTE: In this experiment you will indirectly observe photosynthesis and cellular respiration using a floating leaf disc in a solution. During photosynthesis, air bubbles will cause the leaves to float, and during respiration, the discs will sink. HYPOTHESES: In this exercise, the experimental hypothesis is that the leaf discs will have a greater rate of photosynthesis in the bicarbonate solution, because bicarbonate provides added CO 2 to fuel photosynthesis, causing more leaf discs to float. Additionally, all of the discs will sink in dark conditions as they perform cellular respiration. The null hypothesis is that there will be no difference in the rate of photosynthesis, and therefore the number of floating discs, between the bicarbonate and water, or light and dark treatments.
• To place leaf discs under vacuum, first remove the plungers from two 20 mL syringes, and then place 10 leaf discs inside each syringe tube. Label one syringe “bicarbonate”, and label the other syringe “water”.
• Replace the plungers and push the plunger until only a small amount of air remains in the syringe. Take care not to damage the leaf discs.
• Pull 5 mL of the bicarbonate solution into one of the syringes. Invert and swirl the syringe to suspend the leaf discs in solution.
• Push as much air out as possible without expelling the solution or damaging the leaf discs.
• Then pull 5 mL of the water solution into the other syringe and swirl it as previously described (step 3).
• To create a vacuum, hold one finger over the tip of the syringe while pulling back on the plunger. Hold this for 10 seconds while swirling the syringe to keep the leaf discs in suspension.
• Then, release the vacuum. NOTE: The discs should have absorbed the solution into the air spaces in their tissues and you should see them sink. If the discs don't sink, you can repeat the vacuum creation up to three times.
• Next, add 50 mL of bicarbonate solution to a plastic cup or a glass beaker, and then gently add the discs from the bicarbonate vacuum syringe.
• For the control, add the same amount of water to an identical cup, and then add the leaf discs from the water vacuum syringe. Label the containers appropriately.
• Place both cups under a light source.
• Every five minutes record the number of discs floating on the surface of the cup in Table 3 until 20 minutes have passed. Click Here to download Table 3
• Next, remove the cups from the light source and then swirl them so that the discs at the surface intermix with any gases also at the surface.
• Move the cups to a dark place. Every five minutes record the number of leaf discs floating at the surface until 20 minutes have passed. Swirl the cup each time before placing it back in the dark.
• To clean up, dispose of the leaf discs in the trash, and pour the bicarbonate solution down the drain. Wash the syringes and cups thoroughly.
• NOTE: In the first experiment, you observed how far pigments from spinach leaves traveled on chromatography paper. Different pigments absorb light at different wavelengths.
• Using colored pens or pencils, draw the positions of the pigment bands and the solvent on Figure 3.
• Calculate the retention factor, or Rf values for the pigments, which is done by dividing the distance the pigment in question moved up the paper from the line by the distance the solvent moved up the paper from the line.
• Compare your calculated Rf values to those in Table 2 to determine the identity of the pigment. Click Here to download Table 2
• Record these data in Table 1. NOTE: In the second experiment, you observed floating and sinking leaf discs as an indirect measurement of photosynthesis and respiration.
• Graph the results with time and minutes on the x-axis and number of floating discs on the y-axis. Use two different lines to represent the water control and the bicarbonate treatment.
• Add a line to the graph to indicate the point where the discs were removed from the light condition and placed into the dark.
• Next, starting with the bicarbonate condition, use the graph to determine the point at which 50% of the leaf discs were floating. This is referred to as the effective time, or ET50. NOTE: You will notice that the discs likely hit the 50% floating mark once in the light condition and then again in the dark condition.
• Your water samples may or may not have reached the ET50 mark. If they did, add the line for this sample also.
• Finally, compare your ET50 values and graphs with the rest of the class.

BIOLOGY JUNCTION

Test And Quizzes for Biology, Pre-AP, Or AP Biology For Teachers And Students

The Unique Burial of a Child of Early Scythian Time at the Cemetery of Saryg-Bulun (Tuva)

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Pages:  379-406

In 1988, the Tuvan Archaeological Expedition (led by M. E. Kilunovskaya and V. A. Semenov) discovered a unique burial of the early Iron Age at Saryg-Bulun in Central Tuva. There are two burial mounds of the Aldy-Bel culture dated by 7th century BC. Within the barrows, which adjoined one another, forming a figure-of-eight, there were discovered 7 burials, from which a representative collection of artifacts was recovered. Burial 5 was the most unique, it was found in a coffin made of a larch trunk, with a tightly closed lid. Due to the preservative properties of larch and lack of air access, the coffin contained a well-preserved mummy of a child with an accompanying set of grave goods. The interred individual retained the skin on his face and had a leather headdress painted with red pigment and a coat, sewn from jerboa fur. The coat was belted with a leather belt with bronze ornaments and buckles. Besides that, a leather quiver with arrows with the shafts decorated with painted ornaments, fully preserved battle pick and a bow were buried in the coffin. Unexpectedly, the full-genomic analysis, showed that the individual was female. This fact opens a new aspect in the study of the social history of the Scythian society and perhaps brings us back to the myth of the Amazons, discussed by Herodotus. Of course, this discovery is unique in its preservation for the Scythian culture of Tuva and requires careful study and conservation.

Keywords: Tuva, Early Iron Age, early Scythian period, Aldy-Bel culture, barrow, burial in the coffin, mummy, full genome sequencing, aDNA

Information about authors: Marina Kilunovskaya (Saint Petersburg, Russian Federation). Candidate of Historical Sciences. Institute for the History of Material Culture of the Russian Academy of Sciences. Dvortsovaya Emb., 18, Saint Petersburg, 191186, Russian Federation E-mail: [email protected] Vladimir Semenov (Saint Petersburg, Russian Federation). Candidate of Historical Sciences. Institute for the History of Material Culture of the Russian Academy of Sciences. Dvortsovaya Emb., 18, Saint Petersburg, 191186, Russian Federation E-mail: [email protected] Varvara Busova  (Moscow, Russian Federation).  (Saint Petersburg, Russian Federation). Institute for the History of Material Culture of the Russian Academy of Sciences.  Dvortsovaya Emb., 18, Saint Petersburg, 191186, Russian Federation E-mail:  [email protected] Kharis Mustafin  (Moscow, Russian Federation). Candidate of Technical Sciences. Moscow Institute of Physics and Technology.  Institutsky Lane, 9, Dolgoprudny, 141701, Moscow Oblast, Russian Federation E-mail:  [email protected] Irina Alborova  (Moscow, Russian Federation). Candidate of Biological Sciences. Moscow Institute of Physics and Technology.  Institutsky Lane, 9, Dolgoprudny, 141701, Moscow Oblast, Russian Federation E-mail:  [email protected] Alina Matzvai  (Moscow, Russian Federation). Moscow Institute of Physics and Technology.  Institutsky Lane, 9, Dolgoprudny, 141701, Moscow Oblast, Russian Federation E-mail:  [email protected]

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