Paper Chromatography (using a learning cycle approach)

Welcome: This is the second of a four-lab experiment sequence, covering four important aspects of chemistry, and utilizing a learning cycle. • • • •

Lab 1 Making Experimental Observations: chemical reactions and properties Lab 2 Paper Chromatography: chemical interactions and separation techniques Lab 3 Conservation of Mass Lab 4 Limiting Reagents

Learning cycle: to help you better understand the chemical concepts in each lab experiment, we will employ a three-step learning cycle.

Step 1: We will ask you and a partner to go into the lab, follow a procedure, and make observations.

Step 2: You will then be asked to review material that provides an explanation about what you observed in lab. Step 3: Before going back into the lab, you will be asked to apply your understanding by designing experiments. You and your partner will then go back into the lab to conduct your experiments and gather experimental results. Designing and carrying out your own experiments can be a very challenging part of the lab and requires very careful thought.

Paper Chromatography: to help you better understand the chemical concepts in each lab experiment, we have incorporated descriptive information about paper chromatography into today’s lab activity.

Learning Goals:

 

 

a) To understand the chemical names and symbols for some common metal ions. b) To use the laboratory technique of paper chromatography to separate and identify metal ions in an aqueous (water) solution. c) To learn about paper chromatography d) To design an experiment to determine the metal ions present in an unknown solution.

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Paper chromatography. Chemistry has a wide application in many real-world uses. Have you ever watched a crime-scene investigation TV show that dealt with identification of a written document, such as a ransom note? Aside from their handwriting, the criminal can also be identified by the type of pen they use. Scientists can utilize a method known as chromatography to separate different components of a pen’s ink. Since each model of pen has a different makeup of ink, this separation technique can be used to narrow down the possibilities for the type of pen used. Paper chromatography is a specialized form of chromatography that is relatively fast and inexpensive yet powerful enough to separate a range of different chemicals. This type of chromatography uses a piece of paper and a solvent to separate the components of a mixture. You will use paper chromatography in this experiment to separate and identify metal ions in an aqueous solution. In this experiment, a strip of chromatography paper will be spotted with several known samples of metal ions and developed using a mixture of acetone and hydrochloric acid. After measuring the distance that the solvent moves up the strip of paper (the solvent front distance), the paper is dried and then treated with several solutions that cause the samples to change color. The distance that the sample has moved can be measured and the Rf values (ratio of distance traveled by sample to distance traveled by solvent) for each sample can be determined. The paper strip with the developed sample spots is called a chromatogram. Atoms can readily gain or lose electrons. If electrons are removed or added to a neutral atom, a charged particle called an ion is formed. Because opposites attract, positively and negatively charged ions are attracted to each other and form compounds called ionic compounds. The positively charged ion (called a cation) is often a metal and the negatively charged ion (called an anion) is often a nonmetal. Many of these ionic compounds will dissolve in water to form the individual ions of the compound. For instance, sodium chloride (NaCl) dissolves in water to form sodium ions (Na+) and chloride (Cl-) ions. In fact, natural water sources contain metal ions including sodium (Na+), magnesium (Mg2+), calcium (Ca2+), and iron (Fe3+). They can also contain harmful metal ions such as lead (Pb2+). In this experiment, you will be determining how metal ions move and interact with solvent and paper.  

 

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Pre-lab Assignment In your lab notebook, after reading the lab procedure, please prepare the following information: At the beginning of lab, hand in the copy of your Pre-Lab Assignment and be prepared to discuss it. a) Please write a 2-3 sentence introduction to the lab. b) A table of safety information including the chemicals used in the lab and any safety handling precautions. This information can be obtained using the list of chemicals found below, looking up the appropriate MSDS (material safety data sheets), and understanding how the chemicals will be used in this experiment. The MSDS can be accessed by the link attached to each chemical in the Chemicals & Equipment Table below. c) Please answer the following questions: i. Describe your familiarity with separating things by chromatography. Perhaps you have a water softener in your home or you may have done an experiment using a coffee filter to separate colors in markers. Explain. ii. Imagine a strong versus a weak chemical interaction. On the atomic level, draw what you think might be happening with a strong or weak chemical interaction. Table of Chemicals and Equipment  

Chemicals 0.25 M copper (II) nitrate for the 2+ Cu ion solution 0.25 M nickel (II) nitrate for the 2+ Ni ion solution 0.25 M iron (III) nitrate for the 3+ Fe ion solution 0.25 M cobalt (II) nitrate for the 2+ Co ion solution Unknown consisting of one or more of the above solutions

Chemicals Acetone 6 M Hydrochloric acid (HCl)

Equipment and Supplies Chromatography filter paper, paper towel, pencil, ruler, capillary tube, beaker, 3 pint bottles with corks

8M Ammonia Hydroxide (NH4OH) 1 % dimethylglyoxime (DMG in ethanol) solution Reagent Ethanol

 

Instructions before going into lab: • •

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You should work in pairs for this experiment. If there are an odd number of students, then one group can have three people. Write the name of your partners in your lab notebooks. Each pair of students should discuss and answer questions as they encounter them. These questions are set off in the experiment (i.e. “Q:”). The questions offer guidance during the lab. Answers to these questions do not need to be reproduced in your lab report. Each student is to record notes and observations and all other data in their own lab notebook. Each student is responsible for collecting the information needed to complete the Lab Report and Post Lab Portions of the Experiment. Goggles are required at all times in the lab. There are no exceptions. Gloves and foot covers are available.

 

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Step 1 of our Learning Cycle: the following activities should be completed in the lab: a) Acquire three glass pint bottles with cork stoppers, three 8-inch (20.5 cm) strips of chromatography paper and a solvent mixture of 90 mL of acetone and 10 mL of 6 M HCl. b) Hold the paper vertically. Towards the bottom (about 1.5 cm or 5/8” inch away from the edge), draw a straight line across the paper using a PENCIL. Draw two small X’s spaced evenly on the line to mark the initial spots for your samples. Label these marks "Cu" and "Ni". c) Place the end of a clean capillary tube in the Cu2+ solution, hold the tube perpendicular to the strip, and gently touch the tip of the tube to the previously marked X on the paper. Allow the solution to form a small spot that is about 3 mm in diameter (if the spot is any larger, you will get poor resolution). Allow the spot to dry. Dip the capillary tube in the Cu2+ solution again and dab it on the spot that was just created. Let the spot dry and repeat a third time. The goal is to make a small but concentrated spot. Then clean the capillary tube by rinsing it thoroughly with distilled water in your wash bottle and then repeat the spotting procedure on the other X with the Ni2+ solution. d) When handling these strips, avoid contamination with the solutions and chemicals present in the laboratory. Place them on a clean paper towel and always hold the strips at the sides with your fingertips. e) Repeat the spotting procedure on the second piece of paper, using the Fe3+and Co2+ solutions. f) Repeat the spotting procedure a third time, selecting two known metal ions. This will allow you to gather some duplicate data. You may choose from among any of the known metal ions. g) While the strips are drying, prepare the solvent mixture. Mix 90 mL acetone and 10 mL 6M hydrochloric acid in a beaker. Pour about one third of the mixture into each of the three glass pint bottles. h) Insert one of the prepared chromatography strips into each of the bottles so that the bottom of the strip just touches the liquid. Make sure that the solvent does not touch the ion spot. (You do not want the spots to be submerged, as the ions will dissolve in the solvent and the experiment will be ruined.) The strip should not touch the sides of the bottle and the top part of the paper should extend out of the top of the bottle. Fold the top part of the paper over the edge of the bottle and secure the strip by inserting the cork. Observe your sample as it develops. Repeat this procedure using the other two strips, so they

 

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are all developing at the same time. Q: What is happening to the solvent level on the rectangular piece of paper? Is it moving and if so, in what direction? Q: What is happening to the marked spot you placed on the paper as the solvent moves up the paper? Do you think this is related to anything else going on in the experiment (direction of solvent, etc.)? i) Allow the solvent to move approximately 10 cm up the strip (just to the shoulder of the bottle). Do not allow the solvent front to come near the cork in the top of the bottle. If any colors appear on the strip while it is in the solvent be sure to record the colors you observe. Remove the cork and carefully pull out the paper. Immediately draw a straight line at the highest point of the solvent level (the solvent front) with a PENCIL. Let your sample dry for a few minutes in open air. Mark the leading edge of any spots (the end of the spot closest to the solvent front) that you observe once you take the paper out of the bottle. Some of the spots may not be visible yet.  

 

   

Health Warning: the procedure below is to be done under the hood. Exposure to ammonia at low concentrations may produce rapid skin or eye irritation. Higher concentrations of ammonia may cause severe injury and burns.

 

 

j) Perform the following procedure under the hood. Move each chromatogram back and forth above an open bottle of Concentrated Ammonia (NH4OH) solution. You will see the vapors of the ammonia gas reacting with the HCl on the paper to form a white gas of ammonium chloride (NH4Cl). If a spot develops, mark its leading edge and record the color. Finally, spray each of your chromatograms with a mist of 1% dimethylglyoxime (DMG in ethanol) solution. Again, mark the leading edge of any new spots observed, and record their colors, if any.

 

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Step 2 of our Learning Cycle: The following activities should be completed out of the lab (e.g. in the break-out room). Chromatography Exercise. Before analyzing the results of the paper  

chromatography experiments you did in Step 1, we’d like you to do the following exercise to help you think about analyzing chromatography strips. (Please work with your lab partner.)  

Consider the following three chromatography strips, representing the same sample at three different times in the developing process: t = 10 min, t = 12 min, and t = 14 min (from left to right in the figure below). Note that in the strips, some of the measurements are indicated to give you an example of the type of data you need to record.

 

Using a ruler, measure the distance, in centimeters, from the starting line to the solvent front on each of the strips and record the data in the table below. [Note: since the figure may print at a different scale, use the distances consistently from your ruler.] Next measure the distance from the starting line to the leading edge of the spot and record the data in the table below. Finally, calculate the ratio of the distance the spot moves to the solvent front; this ratio is called the Rf value.    

 

Time      

Distance Spot Moves      

Distance Solvent Moves (solvent front)      

Ratio: distance spot moves to distance solvent moves [Rf value]      

 

 

 

Q: What characteristic of the spot is independent of the time at which the spot is observed? (Hint: think about the values in your table.) Q: Now consider the two samples 1 and 2 shown at the left. Can you make an educated guess which sample would be the same metal ion used in the exercise above? Explain your reasoning

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Paper Chromatography Reading. Please continue working with your lab partner for the next activity, which involves reading the passages below and answering the questions as a way to understand chromatography. Each student in your group will be asked to fill out a separate worksheet in completing the following activity but you are encouraged to discuss it together. The worksheet should be included in your lab report. Please note that you will be asked to draw a picture, which may be drawn on a separate sheet of paper. This drawing should also be included in your lab report.  

[The following information is based on Chemistry in the Laboratory, 5th Ed, by Postma et al, 2000 and Introductory Chemistry, 2nd Ed Lab Manual by Gloffke & Kimbrough, 2002]: “Chroma-“ means color. Chromatography was first described by a Russian botanist named M.S. Tswett, who used the technique to separate plant pigments. He packed a glass column with an absorbent powder and then poured a solution containing the pigments down the column. The solution separated into different colored bands. Many of today’s techniques don’t produce visible colors, but are still called “chromatography”. What all chromatographic techniques have in common is the separation of components in a mixture by their varying attractions to two materials: the moving phase and the stationary phase. In today’s paper chromatography lab the components of the mixture are the metal ions (Ni+2, Cu+2, Fe+3, and Co+2), the stationary phase is the paper, and the moving phase is the solution of acetone and HCl. As the moving phase travels, the metal ion components spend some of their time on (adsorbed on) the paper, and some of their time in the solution. Metal ions that have a greater attraction for the solution will be carried along quickly. Metal ions that have a greater attraction for the paper will be slowed down. Chromatography takes advantage of differences in properties between the moving phase and the stationary phase. For example, biochemists may need to separate proteins with very different sizes. They often use a column packed with a very porous stationary phase. Small proteins can fit into the pores easily: they travel through the column very slowly as they wind in and out of the pores. Very big proteins can’t fit into the pores at all and move down the column quickly. The properties of the moving and stationary phases that cause the mixture components to separate are consistent, so long as conditions remain the same. So if metal “X” travels up your paper chromatogram faster than metal “Y” today, it will be always be faster. The Rf values will be consistent as well, so long as all the conditions are the same. This is what makes paper chromatography a good identifying technique. Other types of chromatography include thin layer chromatography (TLC), column chromatography, and gas chromatography (GC). In TLC, the stationary phase is a thin plate of plastic or glass covered in fine particles. The mobile phase travels up the plate by capillary action, just as in paper chromatography. TCL is usually much faster than paper chromatography and the resolution of the spots can be easier. However, it can be difficult to get the fine particles on the plates uniform, whereas the paper used in paper chromatography is very uniform. This can lead to more uniform or consistent Rf values with paper chromatography. Column chromatography is what Tswett used. The

 

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stationary phase is a fine solid packed into a vertical column. The mobile phase is a liquid that pours down the column. Gas chromatography is very widely used. In this technique the stationary phase is a liquid packed into a horizontal tube. The mobile phase is a gas that is pushed through the column. Please answer the following questions: Q How did chromatography originate? Q What do all chromatographic techniques have in common? How do they differ? Q Suggest an explanation, at the molecular level, on why the metal ions traveled different distances up the paper. Q: In your lab experiment, what is the evidence that different metal ions interact differently with the chromatography paper?  

Analyzing Chemical Observations from Step 1: Now we’d like you to analyze the results from Step 1. Please fill out the table below and answer the questions (you may do this individually and then compare charts or together).   Known Metal Ion

Distance Spot Moves (specify units)

Distance Solvent Moves (solvent front)

Ratio: distance spot

Observations?

moves to distance solvent moves [Rf value]

 

Q: What claims can we make from our laboratory observations (Step 1)? Explain your reasoning.  

 

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Paper Chromatography Worksheet Name: ______________

 

When metal ions are spotted on the paper, placed in a bottle with solvent, what is observed? Please provide an explanation for how this occurs.

Draw a picture of your explanation of what happens during paper chromatography.

Comparing two different metal ions, how would you explain differences in the ratio of the distances the metal ion moves to the distance the solvent moves?

 

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Step 3 of our Learning Cycle. You may start this step in the breakout room before proceeding to lab. Based on what you have learned in the first two steps of the learning cycle, we would like you to design an experiment to identify metals in an unknown solution containing 1-3 unknown metal ions. Write out a procedure to determine your unknown and explain it to your TA.

 

Obtain an unknown, then go back into lab to test your procedure. Good luck!

   

   

 

Answer the following questions once your investigation is completed: Q: Based on your procedure, were you able to successfully identify your unknown solution? This should be part of your claim in your lab report. If you are able to make a claim of identifying the unknown solution, explain your evidence and reasoning. Q: Based on the calculated Rf values, rank the interaction of the metal ions in your unknown solution with the stationary phase (the paper) from least (starting with 1) to greatest. If you have a larger Rf value, does it mean that you have more or less interaction with the stationary phase?

 

Staple your chromatograms into your lab notebook and make a drawing for your lab report. PLACE ALL EXCESS and USED CHEMICALS IN THE PROPERLY MARKED WASTE CONTAINERS.  

 

 

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Designing Experiments Worksheet – Hand in at end of lab You have been asked to design an experiment to identify metals in an unknown solution containing 1-3 unknown metal ions. Please fill out this form and turn it in at the end of lab. Lab Partners: ________________ _______________ Lab performed on date: _________ Signatures:

________________ ________________

Before going into lab:

Designing Experiments: Procedures, Observations, and Results Describe experiments & predictions: (attach extra pages if needed)

Please describe your proposed experiments before performing any experiments Any predictions? We recommend explaining your experiments to your lab instructor before performing them.

While in lab:

Observations and results: (attach extra pages if needed)

Describe data in lab and changes in procedures. Can you support any claims?

While in lab: Any special issues to report?

 

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AFTER LAB: Post-Lab Discussion and Assignments: •

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Before starting the post lab discussion, groups should have already discussed their evidence to identify their assigned unknown, answering the Q: What claims can we make about our experiments? Also, the Q: Were their any issues with our experiments? Next, groups should discuss with the lab instructor and other groups how they designed their investigation and their claims. Particular care should be made to communicate how the experiments were conducted and the reasoning behind the analysis to make their claim. Groups may want to note variations in how experiments were conducted and if some experiments worked out better because of their design. Using your understanding of paper chromatography, describe the chemical interactions that lead to one chemical moving at different rates along the paper.

Laboratory Report Instructions for completing your lab report are posted at: http://umaine.edu/generalchemistry/files/2011/08/Lab_Grading_Guide_Spring_2012_V1.0.pdf Remember to include your paper chromatography worksheet as well as your drawings in your lab report. Please attach a copy of your worksheet to your lab report. Here are some points to include in your lab report: a) An introduction, which includes a description of your experiment to identify your unknown. b) Your claim, underlined, following the introduction. c) The reasoning your group used to make your claim. d) In practical terms, describe how paper chromatography works e) Answering the question: How challenging was it to develop a procedure to identify an unknown? Remember that scientific explanations have three components: claim, evidence and reasoning. More information about claims, evidence, reasoning, and instructions for lab reports can be found at: http://umaine.edu/general-chemistry/files/2011/08/Lab_Grading_Guide_Spring_2012_V1.0.pdf

 

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