paper chromatography hypothesis

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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

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• Open Library Publishing Platform - DRAFT – Organic and Biochemistry Supplement to Enhanced Introductory College Chemistry - Thin Layer (TLC) And Paper Chromatography (PC)
• Chemistry LibreTexts Library - Paper Chromatography - Separation and Identification of Five Metal Cations
• Oregon State University - College of Engineering - Paper chromatography
• Academia - Paper Chromatography
• Journal of Emerging Technologies and Innovative Research - Paper Chromatography: A Review

paper chromatography , in analytical chemistry , technique for separating dissolved chemical substances by taking advantage of their different rates of migration across sheets of paper. It is an inexpensive but powerful analytical tool that requires very small quantities of material.

The method consists of applying the test solution or sample as a spot near one corner of a sheet of filter paper. The paper is initially impregnated with some suitable solvent to create a stationary liquid phase . An edge of the paper close to the test spot is then immersed in another solvent in which the components of the mixture are soluble in varying degrees. The solvent penetrates the paper by capillary action and, in passing over the sample spot, carries along with it the various components of the sample. The components move with the flowing solvent at velocities that are dependent on their solubilities in the stationary and flowing solvents. Separation of the components is brought about if there are differences in their relative solubilities in the two solvents. Before the flowing solvent reaches the farther edge of the paper, both solvents are evaporated , and the location of the separated components is identified, usually by application of reagents that form coloured compounds with the separated substances. The separated components appear as individual spots on the path of the solvent. If the solvent flowing in one direction is not able to separate all the components satisfactorily, the paper may be turned 90° and the process repeated using another solvent.

Paper chromatography has become standard practice for the separation of complex mixtures of amino acids , peptides , carbohydrates , steroids , purines , and a long list of simple organic compounds . Inorganic ions can also readily be separated on paper. Compare thin-layer chromatography .

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Paper Chromatography Experiment

March 17, 2021 By Emma Vanstone Leave a Comment

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

WHAT IS CHROMATOGRAPHY?

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

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

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

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

The video below shows chromatography in action.

You’ll need:

Filter paper or paper towel

Felt tip pens – not washable or permanent

A container – glass, jar or plate

Instructions

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

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

Our LEGO holder worked well, too!

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

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

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

What happens if you use washable pens?

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

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

Why does chromatography work?

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

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

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

Extension task

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

Try chromatography with sweets .

Steamstational also has a great leaf chromatography investigation.

More separation experiments

Clean up water by making your own filter .

Separate water and sand by evaporation .

Make colourful salt crystals by separating salt and water.

Separate liquid mixtures with a bicycle centrifuge .

Last Updated on May 20, 2024 by Emma Vanstone

Safety Notice

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

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

Candy Chromatography Science Project

Candy chromatography is a type of paper chromatography that is easy, inexpensive, and fun. The basic materials are colored candies, water, and coffee filters. The process separates the pigments in the dyes that color the candies. Here are two sets of candy chromatography instructions. The first targets young children and raises interest in science and exploring how things work. The second set of instructions introduces paper chromatography at the high school or college level.

Candy Chromatography for Kids

Explore color chemistry with this basic candy chromatography project.

• Colored candies
• Paper coffee filters
• Separate the coffee filters and place them onto individual plates.
• Place a single colored candy in the middle of a coffee filter.
• Add a drop of water onto the candy.
• Watch as the dye from the candy spreads outward from the center and separates into its component colors.
• Good candy choices are the ones coated with a shell, like Skittles and M&Ms.
• Green, purple, orange, brown, and black candies are the ones most likely to contains multiple pigment colors. Blue, yellow, and red candies (the primary colors) often only contain one pigment an may not be very exciting for children.
• For more concentrated color, first group candies according to color. Place one or more candies of a single color onto a plate or strip of aluminum foil. Add a few drops of water. Then, drip the resulting colored droplet onto the center of a coffee filter. Repeat with other candy colors. If you like, make custom color mixtures so kids can separate them (e.g., red + yellow = orange; blue + yellow = green; red + blue = purple). This step also reduces the risk of muddying up the colors with chocolate or whatever might be beneath the outer candy shell.

How It Works

The basic principle is that water carries the dissolved pigments into the paper and it’s easier for small pigments to navigate the fibers in the coffee filter than it is for larger pigment molecules . Some food colorings only contain one kind of dye or pigment, so the resulting image (the chromatogram) is just a ring of a single color. Other colorants actually consist of multiple dyes. The chromatogram from these candies shows rings of different colors.

• See if children can predict the colors of the pigments in a candy.
• For chromatograms with multiple rings, see if they can identify which ring represents the smallest pigment (the color that travels the farthest) and the largest pigment (the one that travels the least distance).
• If you like, introduce more complex concepts. Chromatography separates molecules according to multiple factors (not just size). Cellulose in paper is polar , so some pigments bind to it or are attracted by it. So, whether a pigment is polar or nonpolar or whether it carries an electrical charge also determines its movement through the paper.

Candy Chromatography for More Advanced Students

Although candy chromatography is simple, it actually introduces most of the basic terms and concepts of chromatography. Slightly changing the design makes it possible to directly compare the pigments in different candies or to compare candies against a standard mixture of dyes.

• Coffee filters or filter paper
• Plate or foil
• First, cut the coffee filter or filter paper into rectangular strips. Each strip will form one chromatogram.
• Using a pencil, draw a line 1 cm or 1/2″ from the end of each strip. Place pencil dots for each candy color in the test. Label the dots.
• Place colored candies on a place or piece of foil. Separate the candies according to color and leave space between them so they don’t touch. Drip water onto each candy so you get a spot of dyed liquid around each one.
• Using a toothpick, pick up a droplet of color and place it onto the labeled dot on the paper. Try keeping each dot as small as possible. It helps applying a tiny dot, letting it dry, and then applying more color. Repeat the process using other colors, using a clean toothpick for each color.
• Prepare a 1% salt solution . Mix 1/8 teaspoon of salt with three cups of water (1 milliliter or cm 3 of salt and 1 liter of water). Shake or stir the solution until the salt dissolves.
• Pour the salt solution into the bottom of a glass so the liquid level is 1/4″ or 0.5 cm. Basically, make certain the liquid level is below the pencil and sample line on the paper.
• Stand the filter paper in the glass so the pencil line is above the liquid level.
• Remove the paper when the liquid level is 1/4″ or 0.5 cm from the end of the paper. Mark this location with a pencil so you will know how far the solvent progressed through the paper. Set the paper aside so it dries. This is your chromatogram.

After the paper dries, compare the results for different candy colors. Do any candies contain the same dyes? You can tell because these bands are the same color and distance along the paper. Which candies contain multiple dyes? A candy that contains multiple pigments has bands or lines that are different distances from the pencil line.

How Candy Chromatography Works

In this project, the paper is the stationary phase. It does not move, but it separates the components of the mixture. The paper is cellulose, which is a polar molecule. So, the pigments move at different rates through the paper based not only on size and shape, but also by polarity and electric charge. The salt water is the mobile phase. It carries the sample through the stationary phase in a definite direction. The liquid phase moves through the stationary phase via capillary action , which depends on surface tension, adhesion, and cohesion .

One way of analyzing a chromatograph is according to R f values. An R f value is the distance traveled by the sample component divided by the distance traveled by the solvent. The Rf value makes it easier comparing different components of a sample and also has some use when comparing the results of chromatograms made at different times.

Further Investigation

• Compare the effect of the composition of the fluid phase. For example, compare what happens if you use water or ethanol instead of salt water.
• Consider sample solubility. What if you repeat the project using organic dyes instead of water soluble colorants? What solvent should you use?
• Repeat the project using food coloring, marker ink, or other colorants.
• See what happens if you change the solid phase. What are the results replacing the coffee filter with a paper towel or strip of cotton?

Chromatography Terms and Definitions

• Chromatography : Chromatography is a physical separation method. Components separate into the stationary phase and mobile phase.
• Chromatogram : A chromatogram is a physical representation that measures the movement of solvent and sample over time.
• Chromatograph : A chromatograph is the apparatus that performs chromatography. When used as a verb, to chromatograph a sample is to separate it using chromatography.
• Stationary Phase : The stationary phase is one of the two phases in the chromatography system. For example, in candy chromatography, the stationary phase is the coffee filter paper.
• Mobile Phase : The mobile phase is the fluid that moves in a definite direction. For example, in candy chromatography, the water or salt water is the mobile phase.
• Sample : The sample is the mixture that the chromatograph separates into components. For example, the sample is the candy dye in this project.
• Solute : The solute is another name for the sample.
• Solvent : The solvent is another name for the liquid phase.
• Standard : A standard is a mixture of known composition. Comparing the sample against a standard helps identify components of the mixture.
• Ettre, L.S.; Zlatkis, A., eds. (2011). 75 Years of Chromatography: A Historical Dialogue . Elsevier.. ISBN 978-0-08-085817-3.
• Ettre, L.S. (1993). “Nomenclature for Chromatography (IUPAC Recommendations 1993)”. Pure and Applied Chemistry . 65 (4): 819–872. doi: 10.1351/pac199365040819
• 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.
• Ninfa, A.J. (2009). Fundamental Laboratory Approaches for Biochemistry and Biotechnology . ISBN 978-0-470-47131-9.

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BIOLOGY JUNCTION

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

Paper Chromatography Report

Introduction The purpose of this experiment is to observe how chromatography can be used to separate mixtures of chemical substances. Chromatography serves mainly as a tool for the examination and separation of mixtures of chemical substances. Chromatography is using a flow of solvent or gas to cause the components of a mixture to migrate differently from a narrow starting point in a specific medium, in the case of this experiment, filter paper. It is used for the purification and isolation of various substances. A chromatographically pure substance is the result of the separation. Because purification of substances is required to determine their properties, chromatography is an indispensable tool in the sciences concerned with chemical substances and their reactions.

Chromatography is also used to compare and describe chemical substances. The chromatographic sequence of sorbed substances is related to their atomic and molecular structures. A change in a chemical substance produced by a chemical or biological reaction often alters the solubility and migration rate. With this knowledge, alterations or changes can be detected in the substance.

In all chromatographic separations, there is an important relationship between the solvent, the chromatography paper, and the mixture. For a particular mixture, the solvent and the paper must be chosen so the solubility is reversible and be selective for the components of the mixture. The main requirement, though, of the solvent is to dissolve the mixture needing to be separated. The porous paper used  must also absorb the components of the mixtures selectively and reversibly. For the separation of a mixture, the substances making up the mixture must be evenly dispersed in a solution, a vapor, or a gas. Once all of the above criteria have been met, chromatography can be a simple tool for separating and comparing chemical mixtures.

Hypothesis Paper can be used to separate mixed chemicals.

Materials The materials used for this lab are paper, pencil, eraser, filter paper, test tube, rubber stopper, paper clip, metric ruler, black felt-tip pen, and a computer.

Methods The first step of the method is to bend a paper clip so that it is straight with a hook at one end. Push the straight end of the paper clip into the bottom of the rubber stopper. Next, you hang a thin strip of filter paper on the hooked end of the paper clip. Insert the paper strip into the test tube. The paper should not touch the sides of the test tube and should almost touch the bottom of the test tube. Now you will remove the paper strip from the test tube. Draw a solid 5-mm-wide band about 25 mm from the bottom of the paper, using the black felt-tip pen. Use a pencil to draw a line across the paper strip 10 cm above the black band.

Pour about 2 mL of water into the test tube. The water will act as a solvent. Put the filter paper back into the test tube with the bottom of the paper in the water and the black band above the water. Observe what happens as the liquid travels up the paper. Record the changes you see. When the solvent has reached the pencil line, remove the paper from the test tube. Measure how far the solvent traveled before the strip dries. Finally, let the strip dry on the desk. With the metric ruler, measure the distance from the starting point to the top edge of each color. Record this data in a data table. Calculate a ratio for each color by dividing the distance the color traveled by the distance the solvent traveled.

Results The results of the experiment are shown in a chart and a graph.

1. How many colors separated from the black ink? Five colors separated from the black ink: yellow, pink, red, purple, and blue.

2. What served as the solvent for the ink? Water served as the solvent for the ink. As the solvent traveled up the paper, which color of ink appeared first? The color orange first appeared as the solvent traveled up the paper.

3. List the colors in order, from top to bottom, which separated from the black ink. The colors separated in this order, from top to bottom: blue, purple, red, pink, and then yellow.

4. In millimeters, how far did the solvent travel? The solvent traveled 111 mm.

5. From your results, what can you conclude is true about black ink? Black ink is a mixture of several different colors.

6 . Why did the inks separate? The inks separated because the black ink was a mixture of different pigments with different molecular characteristics. These differences allow for different rates of absorption by the filter paper.

7. Why did some inks move a greater distance? The ink least readily absorbed by the paper would then travel the farthest from the starting mark. You can conclude from this information that the different pigments were absorbed at different rates.

Error Analysis Possible errors could include inaccurate measurements of the distances traveled by the inks and mistakes when calculating the ratio traveled by the water and colors. If a longer test tube was used, a longer strip of filter paper could have been used. This may have changed the ratios. Another color may have been present, but not detected because of the filter paper length.

Conclusion The proposed hypothesis was correct. The paper chromatography did show that black ink could be separated into various colors. The black ink gets its color from a mixture of various colored inks blended together. The first color of ink to appear on the filter paper was yellow followed by pink, red, purple then blue. The colors separated the way they did because of the differences in their molecular characteristics, specifically, their solubility in water and their rate of absorption by the paper. The most soluble and readily absorbed ink color was the yellow. The least soluble and least absorbable ink color was the blue.

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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.

More resources

Add context and inspire your learners with our short career videos showing how chemistry is making a difference .

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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