hypothesis on paper chromatography

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Paper Chromatography Science Projects With a Hypothesis

Chemicals in Dry-Erase Markers

Paper chromatography analyzes mixtures by separating the chemical contents onto paper. For instance, chromatography is used in forensic science to separate chemical substances such as drugs in urine and blood samples. Students can perform paper chromatography projects using ink to understand how scientists are able to determine the presence of different chemicals.

Separate Ink Colors

Form an experiment to separate ink colors using paper chromatography. Hypothesize that regular black ink will show colors on the paper chromatography more noticeably than permanent ink. Set up the experiment using coffee filters and washable and permanent markers. Cut the coffee filters into long strips for each pen. Form a loop by stapling the ends of the strips together. Place a dot of ink on the bottoms of the coffee filter strips. Label each strip using a pencil, specifying the type of pen. Place the strips into a glass, then add water until it touches the bottom of the paper. Observe the strip. Compare your results between permanent marker and washable marker ink. The washable marker colors should spread out onto the paper, while the permanent marker does not because of its permanent ink.

Water vs. Rubbing Alcohol

Create an experiment to separate permanent marker ink colors using paper chromatography in water and rubbing alcohol. Hypothesize that rubbing alcohol will separate the ink colors in permanent markers, while water will not. Set up the experiment using coffee filters and permanent markers. Cut the coffee filters into long strips for each pen. Form a loop by stapling the ends of each strip together. Place a dot of ink on the bottom of the coffee filter strips. Place one strip into a glass of water and place another strip into a glass of rubbing alcohol until the fluid touches the bottom of the paper. Observe the strips. Compare your results between the water and rubbing alcohol solution. The colors should separate on the strip dipped in the rubbing alcohol, but won’t separate when using water.

Different Solvents

Conduct a paper chromatography project to find out if different types of solvents separate ink differently. Set up the experiment using coffee filters and permanent markers. Cut the coffee filters into long strips. Form a loop by stapling the ends of each strip together. Place a dot of ink on the bottom of the coffee filter strips. Place a strip each into a glass of water, rubbing alcohol, vinegar and nail polish remover. Make sure to only add liquid to touch the bottom of the strip. Observe the strips and compare results. Indicate which solvent separated the ink colors the best.

Use a Black Light

Perform an ink paper chromatography test and use a black light to determine if there are any more components visible on the paper than in regular light. Hypothesize that more components will be seen under black light, because some chemicals are invisible under white light. Make sure to look at the paper the same day the paper chromatography test was conducted in order to assure there is no fading on the paper.

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• Science Buddies; Paper Chromatography: Basic Version; Amber Hess; April 2008

About the Author

Based in Huntington Beach, Calif., Dana Schafer has been writing environmental articles and grant proposals since 2006. Schafer has written for Grace Unlimited Corporation and Youth Have Vision. Schafer is in the process of receiving a Master of Science in biology from California State University, Long Beach.

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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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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

Safety Notice

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

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

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Leaf Chromatography 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.

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How to Do Paper Chromatography With Leaves

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You can use paper chromatography to see the different pigments that produce the colors in leaves. Most plants contain several pigment molecules, so experiment with many species of leaves to see the wide range of colors. This is a simple science project that takes about 2 hours.

Key Takeaway: Leaf Paper Chromatography

• Chromatography is a chemical purification method that separates colored substances. In paper chromatography, pigments may be separated based on the different size of the molecules.
• Everyone knows leaves contain chlorophyll, which is green, but plants actually contain a wide range of other pigment molecules.
• For paper chromatography, plant cells are broken open to release their pigment molecules. A solution of plant matter and alcohol is placed at the bottom of a piece of paper. Alcohol moves up the paper, taking pigment molecules with it. It's easier for smaller molecules to move through the fibers in paper, so they travel fastest and move the furthest up the paper. Larger molecules are slower and don't travel as far up the paper.

What You Need

You only need a few simple materials for this project. While you can perform it using only one type of leaf (e.g., chopped spinach), you can experience the greatest range of pigment colors by collecting several types of leaves.

• Small Jars with Lids
• Rubbing Alcohol
• Coffee Filters
• Shallow Pan
• Kitchen Utensils

Instructions

• Take 2-3 large leaves (or the equivalent with smaller leaves), tear them into tiny pieces, and place them into small jars with lids.
• Add enough alcohol to just cover the leaves.
• Loosely cover the jars and set them into a shallow pan containing an inch or so of hot tap water.
• Let the jars sit in the hot water for at least a half hour. Replace the hot water as it cools and swirl the jars from time to time.
• The jars are "done" when the alcohol has picked up color from the leaves. The darker the color, the brighter the chromatogram will be.
• Cut or tear a long strip of coffee filter paper for each jar.
• Place one strip of paper into each jar, with one end in the alcohol and the other outside of the jar.
• As the alcohol evaporates, it will pull the pigment up the paper, separating pigments according to size (largest will move the shortest distance).
• After 30-90 minutes (or until the desired separation is obtained), remove the strips of paper and allow them to dry.
• Can you identify which pigments are present? Does the season in which the leaves are picked affect their colors?

Tips for Success

• Try using frozen chopped spinach leaves.
• Experiment with other types of paper.
• You can substitute other alcohols for the rubbing alcohol , such as ethyl alcohol or methyl alcohol.
• If your chromatogram is pale, next time use more leaves and/or smaller pieces to yield more pigment. If you have a blender available, you can use it to finely chop the leaves.

How Leaf Paper Chromatography Works

Pigment molecules, such as chlorophyll and anthocyanins, are contained within plant leaves. Chlorophyll is found in organelles called chloroplasts. The plant cells need to be torn open to expose their pigment molecules.

The macerated leaves are placed in a small amount of alcohol, which acts as a solvent . Hot water helps soften the plant matter, making it easier to extract the pigments into the alcohol.

The end of a piece of paper is placed in the solution of alcohol, water, and pigment. The other end stands straight up. Gravity pulls on the molecules, while alcohol travels up the paper via capillary action, pulling pigment molecules upward with it. The choice of paper is important because if the fiber mesh is too dense (like printer paper), few of the pigment molecules will be small enough to navigate the maze of cellulose fibers to travel upward. If the mesh is too open (like a paper towel), then all of the pigment molecules easily travel up the paper and it's difficult to separate them.

Also, some pigment might be more soluble in water than in alcohol. If a molecule is highly soluble in alcohol, it travels through the paper (the mobile phase). An insoluble molecule might remain in the liquid.

The technique is used to test purity of samples, where a pure solution should only produce a single band. It is also used to purify and isolate fractions. After the chromatogram has developed, the different bands may be cut apart and the pigments recovered.

• Block, Richard J.; Durrum, Emmett L.; Zweig, Gunter (1955). A Manual of Paper Chromatography and Paper Electrophoresis . Elsevier. ISBN 978-1-4832-7680-9.
• Haslam, Edwin (2007). "Vegetable tannins – Lessons of a phytochemical lifetime." Phytochemistry . 68 (22–24): 2713–21. doi: 10.1016/j.phytochem.2007.09.009
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Leaf chromatography

In association with Nuffield Foundation

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

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

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

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

Equipment notes

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

Health, safety and technical notes

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

Source: Royal Society of Chemistry

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

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

Teaching notes

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

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

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

Additional information

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

Practical Chemistry activities accompany  Practical Physics  and  Practical Biology .

© Nuffield Foundation and the Royal Society of Chemistry

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

Specification

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

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Allelochemical root-growth inhibitors in low-molecular-weight cress-seed exudate

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Muhammad Ishfaq Khan, Rifat Ara Begum, Lenka Franková, Stephen C Fry, Allelochemical root-growth inhibitors in low-molecular-weight cress-seed exudate, Annals of Botany , Volume 133, Issue 3, 1 March 2024, Pages 447–458, https://doi.org/10.1093/aob/mcad200

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Cress seeds release allelochemicals that over-stimulate the elongation of hypocotyls of neighbouring (potentially competing) seedlings and inhibit their root growth. The hypocotyl promoter is potassium, but the root inhibitor was unidentified; its nature is investigated here.

Low-molecular-weight cress-seed exudate (LCSE) from imbibed Lepidium sativum seeds was fractionated by phase partitioning, paper chromatography, high-voltage electrophoresis and gel-permeation chromatography (on Bio-Gel P-2). Fractions, compared with pure potassium salts, were bioassayed for effects on Amaranthus caudatus seedling growth in the dark for 4 days.

The LCSE robustly promoted amaranth hypocotyl elongation and inhibited root growth. The hypocotyl inhibitor was non-volatile, hot acid stable, hydrophilic and resistant to incineration, as expected for K + . The root inhibitor(s) had similar properties but were organic (activity lost on incineration). The root inhibitor(s) remained in the aqueous phase (at pH 2.0, 6.5 and 9.0) when partitioned against butan-1-ol or toluene, and were thus hydrophilic. Activity was diminished after electrophoresis, but the remaining root inhibitors were neutral. They became undetectable after paper chromatography; therefore, they probably comprised multiple compounds, which separated from each other, in part, during fractionation. On gel-permeation chromatography, the root inhibitor co-eluted with hexoses.

Cress-seed allelochemicals inhibiting root growth are different from the agent (K + ) that over-stimulates hypocotyl elongation and the former probably comprise a mixture of small, non-volatile, hydrophilic, organic substances. Abundant components identified chromatographically and by electrophoresis in cress-seed exudate fitting this description include glucose, fructose, sucrose and galacturonic acid. However, none of these sugars co-chromatographed and co-electrophoresed with the root-inhibitory principle of LCSE, and none of them (in pure form at naturally occurring concentrations) inhibited root growth. We conclude that the root-inhibiting allelochemicals of cress-seed exudate remain unidentified.

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Separation of Plant Pigments by Paper Chromatography

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

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

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

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

Content: Separation of Plant Pigments by Paper Chromatography

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

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

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

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

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

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

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

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

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

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

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

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

You can separate all the above pigments using paper chromatography.

Video: Separation of Plant Pigments

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

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Experiment_729_Qualitative Testing of Amino Acids and Proteins 1_2

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Experiment 72 9 :  Qualitative Analysis of  Amino Acids and Proteins  

Section 1:  Purpose and Summary:    

Develop an understanding of the structure and bonding in amino acids and proteins. 

Identify different amino acids and an unknown by paper chromatography. 

Observe how different amino acids and proteins react in different chemical tests. 

What are  proteins ?  

Proteins  are polymers of amino acids linked together by peptide bonds.  Proteins are important biological molecules with many functions. There are transport proteins (hemoglobin is an example), storage proteins, structural proteins, proteins for muscular contraction, and others. Proteins that catalyze biological reactions are called  enzymes . All proteins are very large molecules with high molecular weights that range from 6500 to 205,000 or more grams per mole.  

An  amino acid  is a molecule that contains an amino group and a carboxyl group in the same molecule. Amino acids found in proteins are α-amino acids.  This means the amino group (NH 2 - or NH 3 + -) is attached to the alpha carbon--this is the carbon next to the carboxyl group. There are 20 amino acids that differ from each other only in the identity of the side chain attached to the alpha-carbon. The amino acid side chains can be classified based on whether they are nonpolar, polar, acidic, or basic. The general structure of an amino acid is shown below. In the following structure, R represents the side chain. 

Since proteins are biological molecules, they are usually found in a neutral solution that is buffered between pH 7.0 and 7.4. In this situation, the acidic and basic groups on the amino acids will be ionized. Most amino acids exist as “ zwitterions ” (dipolar ions) at pH 7. The structure of a zwitterion is shown below. Because of the charges, amino acids are water-soluble. 

Amino acids can be linked together – the amino group on one amino acid can react with the carboxyl group on another amino acid, forming an amide. Therefore, the bonds linking together the amino acid residues are amide bonds, but in proteins they are called  peptide bonds . Shown on the next page is the structure of a  dipeptide  (the prefix "di" means two--there are two amino acids in a dipeptide).  A typical protein molecule contains around 100 amino acids joined by peptide bonds. 

alanylglycine, a dipeptide 

There are several aspects to describe structure of a protein. The  primary   structure  of a protein is the sequence of amino acids. This sequence determines the overall shape of the protein.  The  secondary   structure  consists of regular and repeating structures held together by hydrogen bonds between the C=O and the N-H groups along the backbone of the molecule. The alpha helix and beta sheet are examples of secondary structure. The  tertiary   structure  refers to the overall folding of the entire polypeptide chain and is determined by interactions between the side chains off the amino acids. These interactions include hydrogen bonding, hydrophobic interactions, salt bridges, and disulfide bridges.  Quaternary   structure  involves the same types of interactions as tertiary structure, but the interactions occur between the side chains on different polypeptide chains. These interactions hold together different subunits of a protein.   

If the overall folding of a protein is changed, the protein is called  denatured . A denatured protein is no longer biological active. Some common denaturing agents include heat, organic solvents, acids, bases, agitation, detergents, and heavy metal ions. It is important to note that denaturation affects the secondary, tertiary, and quaternary structure of a protein, but does not affect the primary structure. Sometimes the denaturation is  reversible,  and the protein can be renatured (which reestablishes its biological activity). 

Paper Chromatography of Amino Acids   

Chromatography is a technique of separation and identification. There are many types of chromatography, including but not limited to paper chromatography, thin layer chromatography, gas chromatography, liquid chromatography, and ion-exchange chromatography. In this experiment, paper chromatography will be used to identify amino acids.    

For paper chromatography, the samples to be tested are spotted on one edge of a rectangular piece of filter paper. The paper is placed in a container that has a solvent that wets the bottom of the paper. The paper acts as a wick for the solvent.  The solvent passed through the samples as it wicks up the paper, carrying the samples. The samples move at different rates: those that are more attracted to the solvent will travel faster, and those that are more attracted to the paper will move slower. After the solvent has moved about 3/4 of the way up the paper, the chromatography is complete.  The paper is removed from the container and dried. The amino acid solutions are colorless, so a developer needs to be used to make the amino acids visible.  For amino acids, it is sprayed with a developer called ninhydrin. This will react with amino acids and produce a purple color.  Ninhydrin will also react with proteins. 

A photo of the chromatography setup is below: 

<<  PHOTO WILL BE IN NEXT UPDATE  >> 

After the paper is developed, the distance that the solvent traveled and the distance that each amino acid has traveled are measured. This is called the retention factor, R f , and has a different value for each amino acid is then calculated. By comparing R f  values of unknown amino acids to those of known values, the unknowns can be identified.  

A sample calculation of R f  values is shown below. 

\(R_{f}=\frac{\text { distance traveled by amino acid }}{\text { distance traveled by solvent }}\)

Spot A:  \(R_{f}=\frac{2.3 \mathrm{~cm}}{5.1 \mathrm{~cm}}=0.45\)      Spot B:   \(R_{f}=\frac{4.1 \mathrm{~cm}}{5.1 \mathrm{~cm}}=0.80\)

          Spot C:  \(R_{f}=\frac{3.1 \mathrm{~cm}}{5.1 \mathrm{~cm}}=0.61\)

In this experiment, students will test several known amino acid solutions and one unknown. The results for the unknown should match one of the known amino acids. 

Reactions of Amino Acids and Proteins  

Hydrolysis  

Proteins can be hydrolyzed either partially or completely in the presence of acids, bases, or digestive enzymes. When a protein is hydrolyzed, some or all of the peptide bonds are broken. The products obtained depend on how long the hydrolysis is allowed to take place. The products of partial hydrolysis are peptides and the products of complete hydrolysis are amino acids. 

Biuret Test  

Compounds that contain two or more peptide bonds will react with Cu 2 +  in a basic solution to form a violet-pink complex. The original Cu 2 +  solution is blue, so if the solution remains blue, the compound being tested could be an amino acid or a dipeptide or neither. 

Xanthoproteic Test  

The aromatic rings on tyrosine and tryptophan react with nitric acid. In this reaction, the aromatic rings become nitrated. When nitric acid is added to a sample and the mixture is heated, a yellow solution will result if the sample contains tyrosine or tryptophan. When this yellow solution is treated with a strong base (such as NaOH), it turns orange. Since most proteins contain one or both amino acids, most proteins will show a positive reaction in this test. 

Ninhydrin Test  

Free amino groups will react with the ninhydrin reagent to yield a purple solution. Almost all amino acids contain a free amino group (except proline and hydroxyproline). Some proteins also give a positive test with ninhydrin. 

Sulfur Test  

Sulfur-containing amino acids include cysteine and methionine. If a sample that contains one or both of these amino acids is acidified, the gas H 2 S is produced, which smells like rotten eggs. When a piece of moistened lead (II) acetate paper is held over the solution as the H 2 S is being produced, the H 2 S reacts with the lead ion, forming a black or gray coating of PbS (lead (II) sulfide). Appearance of this black color is taken as a positive test for a sulfur-containing amino acid. 

Denaturation  

There are many ways to denature a protein. Recall that denaturation refers to a disruption of the secondary, tertiary, and quaternary structures – denaturation destroys the normal folding of the protein, making it inactive. When a protein is denatured, it often coagulates, forming a visible solid. Some denaturing agents include heat, organic solvents, agitation, acid or base, and heavy metal ions. 

Section 2:  Safety Precautions and Waste Disposal  

Safety Precautions:  

Wear your safety goggles. 

Nitric acid is very corrosive – avoid skin contact with it. If skin contact occurs, flood the area with plenty of running water for 10 minutes.  

Heavy metal ions such as Pb 2 +  are poisonous. Avoid skin contact with solutions containing these ions and with the lead acetate paper. Wash your hands thoroughly before leaving the lab. 

Ninhydrin spray causes stains. 

Waste Disposal:  

At the end of the experiment, all liquid wastes go into the inorganic waste container.  Solid wastes go into the solid waste container. 

Section 3: Procedure  

Part I. Paper Chromatography   

Identification of Unknown  

Part 2.  Chemical Tests for Amino Acids   

2.  Biuret  Test  

In this part, you will test solutions of egg albumin, gelatin, casein, glycine, and proline. Put 2 mL of each solution to be tested in its own test tube. Add 3 mL of 10% NaOH to each tube and mix well. (Remember, if you are using a stirring rod, rinse it and wipe it off between solutions so that you don’t contaminate the solutions.) Add 2 drops of 2% copper sulfate (CuSO 4 ) solution (which is blue) to each tube and mix well. Record your observations on the data table below. 

3.  Xanthoproteic  Test  

You will test solutions of egg albumin and tyrosine. Put about 1 mL of each solution to be tested in its own test tube. In the hood, carefully add 5 drops of concentrated nitric acid (HNO 3 ). Caution: nitric acid must be kept under the hood at all times. Avoid all skin contact with nitric acid. Mix each test tube carefully and place the tubes in a boiling water bath for two minutes. Observe and record the color of the solutions. Remove them from the water bath and let them cool. When they are cool, add 10% NaOH to each tube dropwise until the solutions are basic. (You can do this by adding one drop of NaOH, mixing well with a stirring rod, and touching the stirring rod to a piece of red litmus paper. If the litmus paper does not turn blue, add another drop of NaOH to the solution, mix with the stirring rod, and again touch it to a piece of red litmus paper. Repeat this sequence until the litmus paper turns blue – then your solution is basic. You can use the same piece of litmus paper, if you touch the stirring rod to a different dry section each time.) Observe and record any color changes. 

4 .   Ninhydrin  Test  

You will test solutions of glycine, proline, and egg albumin. Put 2 mL of each solution to be tested in separate test tubes. Add 1 mL of 1 % ninhydrin solution to each tube. Heat the tubes for several minutes in a boiling water bath. Record your observations.   

5. Sulfur  Test  

Test solid egg albumin and solid cysteine. Weigh out about 0.2 g of each of these solids and place the samples in separate test tubes. Add 10 mL of 3 M NaOH to each tube and place the tubes in a boiling water bath for 15 minutes. If the solutions start to foam, remove them from the water bath briefly. After 15 minutes of heating, remove the tubes from the water bath and let them cool to room temperature. Add about 10 mL of 3 M HCl to each tube to acidify the contents. Moisten two pieces of lead acetate paper with deionized water and place a piece over the top of each tube. Put the tubes in the boiling water bath again. Do not let the lead acetate paper fall into the solutions. Record your observations. Wash your hands after handling the lead acetate paper, as lead compounds are toxic. 

6. Denaturation Tests  

a. Put 3 mL of egg albumin solution in a test tube and place the test tube in a boiling water bath. Observe the appearance of the solution before and after heating.  

b. Put 3 mL of egg albumin solution in a test tube and add 7 mL of 95 % ethanol. Mix well and record your observations.  

c. Put 3 mL of egg albumin solution in a test tube and add 5-8 drops of FeCl 3  solution. Mix well and record your observations.  

d. Put 3 mL of egg albumin solution in a test tube and add 5-8 drops of 0.2 M ZnCl 2  solution. Mix well and record your observations. Put the waste in the inorganic waste container in the hood. 

Observations  

Denaturation Tests  

Post Lab Questions :  

1. According to the results of the paper chromatography, which amino acids were most attracted to the solvent? Which amino acids were most attracted to the paper? Explain.  

2. According to your answer to question #1, which do you think is more polar, the chromatography solvent or the paper? Explain.  

3. If you had completely hydrolyzed the egg albumin before doing the Biuret test on that sample, what results would you expect for the Biuret test? Explain why. Include what color you would expect to see, whether it is a positive or negative test, and what that means.  

4. Suggest a reason why alcohol or other disinfectants are often applied to a person’s skin before an injection is given (based on something you learned in this lab).  

5. Draw the structure of phenylalanine in its regular form and in its zwitterion form.  

6. What substances react in the Biuret test?  

7. What substances react in the Xanthoproteic test?  

8. Suggest a reason why milk is used as an antidote for lead poisoning. Hint: milk contains a lot of protein.  

9. Predict the results of each of the following reactions. Include the observed colors and whether it corresponds to a positive or a negative test.   

The Biuret test on proline   

The Biuret test on egg albumin   

The ninhydrin test on lysine   

The xanthoproteic test on egg albumin  

The sulfur test on cysteine