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Experiment_726_Paper Chromatography_1_2_1

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    Student Name 


    Laboratory Date: 

    Date Report Submitted: 



    Student ID 


    Experiment Number and Title 

    Experiment 726Paper Chromatography 




    Experiment 726: Paper Chromatography: A Technique of Separation and Identification 


    Section 1:  Purpose and Summary 


    One of the problems encountered most frequently in chemistry is that of separating a mixture into the pure substances which compose it. Most natural materials, such as seawater, air, oil, coal, and so on are mixtures. In order to study these materials chemically, we must first treat the mixture in some way to separate it into single, pure substances. (A "pure substance" consists entirely of just one thing; meaning one kind of molecule.) Because it does consist of just one thing, it always behaves in the same way when tested. For example, pure water always boils at 100°C (212°F), provided that the test is done at atmospheric pressure.  


    Many different methods have been devised for separating mixtures into their components. In the present experiment, we will use a method called chromatography. It is used quite widely for making small-scale separations and identifications. The method works because of the differences in the ways various components of a dissolved mixture interact with a fixed solid.  The fixed solid can be made of different materials and in different shapes, depending on the version of chromatography that is being done.   One version is called paper chromatography. 


    To perform paper chromatography to separate components of a mixture, there are several steps.  First, the mixture that is to be separated is dissolved (if it is not already in liquid form.) Then, a small drop of the solution is applied to a piece of special chromatography paper, which is a porous paper similar to filter paper. This drop makes a tiny spot on the paper. Many such spots (of different materials) may be placed side by side on the same piece of paper. Next, the spotted paper is made to stand in a small amount of some special solvent (liquid), in such a way that only the bottom edge of the paper is submerged in the liquid, not the spots. The paper acts like a wick, drawing the liquid up the paper by capillary action. The solvent then slowly rises up the paper, reaches the spots, and begins to dissolve them and carry the substances up the paper with it.  


    The key to this separation is that the different components of the mixture in each spot interact with the paper differently, and so will be found (after a few minutes) to have reached different heights on the paper. This happens because some components are more strongly attracted to the paper (and so move more slowly), while others are more strongly attracted to the solvent. Any particular substance always moves at the same rate, no matter what else it may have been mixed with originally. For that reason, it can always be recognized and identified, because it will always rise to the same height, relative to the heights that the other components rise. 


    The movement of any spot on the paper can be quantified by calculating its Rf (retention factor) value after you have stopped the experiment and the paper has dried. 


       Rf distance traveled by the solute  

    distance traveled by the solvent front 


    The distances used in calculating Rf values are measured as shown in the following figure. To determine the distance traveled by the solute, measure from the point at which you originally applied the spot to the center or densest part of the spot. The distance traveled by the solvent front is measured from the original point of application of the spot to the limit of movement of the solvent front (which must be marked immediately after the paper is removed from the beaker, because it may be nearly invisible after the solvent evaporates). 


    If all conditions could be maintained constant, Rf values would be constant. However, either variations in temperature or in the composition of the solvent phase or changes in the paper can alter the Rf value. The Rf value is useful mainly for expressing the relative mobility of two or more solutes in a particular chromatographic system. The absolute Rf values may change from day to day, but their values in relation to each other remain nearly constant. 



    Figure 1:  Completed paper chromatography containing only 1 dye. 


    In this experiment, students will measure the values of several dyes in 3 different solvent systems. Students will also analyze an unknown mixture of dyes in order to identify the dyes present in the mixture.  The three different solvent systems are 1) laboratory water, 2) an aqueous solution of 0.10% sodium chloride (table salt), and 3) rubbing alcohol (70% isopropyl alcohol and 30% water).  By using different solvents, the dyes will travel differently in paper chromatography, and the Rf values will be different.  The goal is to find a solvent that gives a different value for every dye as this will separate all the dyes from each other (scientists say that the dyes are resolved when the solvent system creates distinctly different values for each dye.)  When every dye is separated from all the other dyes, then you can match up Rf  values and identify each dye in the unknown mixture. 


    Section 2:  Safety Precautions and Waste Disposal 


    Safety Precautions: 


    Use of eye protection is recommended for all experimental procedures.   

    The rubbing alcohol used in this lab gives off fumes, so keep the beaker containing this solvent covered with plastic wrap when it is not in the hood. 


    Waste Disposal: 

    The rubbing alcohol should be disposed of in the organic waste container in the hood. The aqueous solvents may be poured down the drain. The chromatography paper can be disposed in the trash. 


    Section 3: Procedure 


    Preparing and developing the chromatogram 


    1. Obtain three (3) rectangular pieces of chromatography paper. Touch them only at the edges. (Oils from your hands can affect the separation.) All 3 pieces will be set up the same way: the only difference will be that a different solvent mixture will be used for each. 


    2. Make sure that the chromatography paper will fit into the beaker you plan to use when it is rolled into a cylinder. Make sure that the paper will not touch the sides of the beaker.  


    3. Use a pencil (not a pen) to make a straight line all the way across the long way of the paper, at a distance of about one centimeter above the bottom edge (in Figure 1, this line has an “X” in it). 


    4. You will be testing several dyes as well as your unknown mixture: each chromatography paper will have all the same component dyes including the unknown mixture. For each dye that you test, make a small, light circle in pencil on the line, spacing the circles evenly apart on the paper. Next to each circle put an identifying letter or code so that you will know what the spot contained. (These circles are the “point of application” for each dye: see Figure 1.) 

    Which Unknown Mixture did you use? 


    Unknown Mixture #________ 

    (Use this space for drawing a sketch of your chromatography paper. Just sketch 1 chromatography paper because all 3 should be exactly the same. In the same below also recording the identifying letters in the same order they appear on the rectangular piece of chromatography paper. Include enough information here so you know what each identifying letter stands for.) 














    List your identifying letters in the same order they appear on the rectangular piece of chromatography paper: 


    5. Apply tiny drops of each liquid to be tested to the appropriate spot on the pencil line. This can be done by repeatedly dipping a toothpick into the liquid and then touching it to the paper. The darker and smaller your spots are, the better results you will obtain. You may wish to practice one spot first on a scrap of paper towel before spotting your chromatography paper. The chromatography paper is expensive, so please do not waste it. 


    6. Roll the spotted paper into a cylinder with the spots on the outside and staple the ends together. Use two staples, placing them about one-third of the way from the upper and lower edges. Staple the paper so that the ends of the paper do not touch each other, because if they do, it will affect the flow of the solvent at that point. 


    7. Obtain 3 clean, dry beakers. A 250-mL beaker is a good size for this experiment, but any size can work if it has enough room for the rolled up chromatography paper without touching the sides of the beaker. Label each beaker with a different solvent system.  The 3 different solvent systems are 1) laboratory water, 2) an aqueous solution of 0.10% sodium chloride (table salt), and 3) rubbing alcohol (70% isopropyl alcohol and 30% water). 


    8. To the empty beaker, add a few milliliters of the appropriate solvent to each beaker.  Use just enough solvent so that it will wet the bottom edge of the cylinder of paper but not deep enough to touch the spots. 


    9.  Write the name of the solvent you are using at the top of the chromatography paper, and place it in each beaker.  Carefully stand the paper cylinder in the beaker that has the solvent in it. Make sure that the paper does not touch the sides of the beaker. Remember that the spots must not be immersed in the solvent. Cover the beaker with plastic wrap or aluminum foil. 


    10. Let the beaker stand undisturbed. Do not pick it up or slide it around because that will cause sloshing. Watch the solvent rise up the paper (it looks wet where solvent is), and watch the original spots spreading out into different colored components. 


    11. The chromatogram is finished when the separations are complete. However, since separations are not always perfect, you should stop yours when the solvent has risen about three-fourths of the way toward the top of the paper, even if the results are not absolutely perfect. (Sometimes it is necessary, in actual scientific research, to try a different solvent or use a larger piece of paper.) 


    12. Stop the chromatography by lifting the paper out of the beaker. Immediately mark (with a pencil) the height reached by the solvent. (This is called the solvent front and is useful as a reference point.) Unless otherwise specified by local requirements, all solutions used in this experiment may be disposed of down the drain.   


    13. Let the paper dry. You may use a hair dryer if you wish to speed up the process.  


    Section 4 Analysis of the Chromatograms 


    For each spot, measure the distance (in centimeters) traveled by the spot (use the center of the spot) and the distance traveled by the solvent front. Record the color of each spot, then calculate Rf  values for each of the spots. 

    Unknown Mixtures may contain 2 or 3 or 4 different component dyes mixed together. Measure every component dye in your unknown mixture. 


    Chromatograph using laboratory water                  Distance the solvent front moved = __________ cm  

    Name of dye 

    Color of dye 

    Distance dye moved 

    Rf values 





























    Unknown Mixture: 

    Color of dye 

    Distance dye moved 

    Rf value 

    Component dye #1 




    Component dye #2 




    Component dye #3 




    Component dye #4 






    Chromatograph using 0.10% salt water               Distance the solvent front moved = __________ cm  

    Name of dye 

    Color of dye 

    Distance dye moved 

    Rf values 





























    Unknown Mixture: 

    Color of dye 

    Distance dye moved 

    Rf value 

    Component dye #1 




    Component dye #2 




    Component dye #3 




    Component dye #4 










    Chromatograph using rubbing alcohol                Distance the solvent front moved = __________ cm  

    Name of dye 

    Color of dye 

    Distance dye moved 

    Rf values 





























    Unknown Mixture: 

    Color of dye 

    Distance dye moved 

    Rf value 

    Component dye #1 




    Component dye #2 




    Component dye #3 




    Component dye #4 






    Section 5: Identifying the components in the Unknown Mixture 


    Use the colors and the Rf  values of the known dyes to identify the dyes found in your Unknown mixture. 


    Unknown Mixture # _______ 

    Component dye #1 =  


    Component dye #2 = 


    Component dye #3 = 


    Component dye #4 = 





    Post Lab Questions: 


    1. Why use a pencil and not a pen to mark your chromatograms? 






    2) Why is it important that the chromatography paper not touch the sides of the beaker? 







    3) Which solvent system worked the best for you? Explain. 







    4) Suppose a student did today’s experiment and obtained the following results: 


    Solvent system 

    Dye #1 

    Dye #2 

    Dye #3 

    Laboratory water 

    Rf = 0.64 

    Rf = 0.38 

    Rf = 0.55 

    0.10% salt water 

    Rf = 0.92 

    Rf = 0.27 

    Rf = 0.59 

    Rubbing alcohol 

    Rf = 0.79 

    Rf = 0.43 

    Rf = 0.45 


    4a) Which solvent is best for separating Dyes #1-3? 



    4b) Assume that the student receives an Unknown mixture that contains Component Dye #2 and Component Dye #3.  Draw the chromatogram the student would expect to obtain when analyzing Dye #1, Dye #2, Dye #3 and her “Component” mixture when using the best solvent, the same solvent you picked in question 4a.  Include the points of application and the solvent front in your drawing. 















    Experiment_726_Paper Chromatography_1_2_1 is shared under a CC BY license and was authored, remixed, and/or curated by LibreTexts.

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