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    Liquid Chromatography Separation
    K-12 Experiments & Background Information
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    Liquid Chromatography Separation Experiments
    This experiment is courtesy of 

    Separation of Unknowns in a Liquid


    Adele P. Hudson
    Horatio B. Hackett School
    Philadelphia, PA

    E. J. Blusiewicz
    Rohm and Haas Company
    Bristol, PA


    4 and 5


    Physical Science


    1. To identify chromatography as a method of separating the components of a liquid.
    2. To develop an understanding that dyes are composed of more than one component in achieving a particular color.
    3. To become familiar with various Chromatography techniques for separating these components.


    Chromatographic separation enables a chemist to separate, isolate, quantify and identify the components of a mixture.

    Chromatography in the broadest sense refers to processes that permit the resolution of a mixture as a consequence of differences in rates at which the individual components of that mixture migrate through a stationary medium under the influence of a mobile phase.

    Paper Chromatography is a method of separation in which a solvent passes through filter paper. The mixture is introduced on the filter. The end of the filter paper is placed in water and the capillary action of the filter causes the solvent to flow. The components that exhibit better solubility characteristics in the solvent move at a faster rate. The result is the formation of moving bands of color.

    The glass column technique involves packing a glass column with adsorbents such as silica, alumina, or calcium carbonate. The sample is introduced at the top of the column. Sufficient solvent is added until each of the adsorbed components has been removed and collected.

    Teacher's Note:

    The principle aim of this week's work in Project Labs was to give the teacher an opportunity to work with an expert in the area of Industrial Chemistry. The result of this experience was the preparation of new lessons that are both motivating and scientifically correct. It is a completely new approach to the teaching of science.

    The result will provide the student with an appreciation for the over-all analytical process. At the same time developing basic laboratory techniques as well as strengthening student's process skills.

    Chromatography at first glance may appear to be too difficult for the young student at the elementary level. It is presented in such a manner that it is viewed as only a part of the whole picture. It is a representative sampling of some of the methods for the completion of an analysis.

    The needs of the student's developmental stage are met and challenged. The progression of such will move the student into the next stages with assurance. The final result will be a student with firm understanding of advanced scientific theory and the skills for the operation of new technological equipment.



    "Modern Liquid Chromatography"; L. R. Snyder & J. J. Kirkland; John Wiley & Sons, NY; 1974.

    "Fundamentals of Analytical Chemistry"; Douglas A. Skoog & Donald M. West; Holt Rinehart & Winston, NY; 1966.

    List of Materials And Where To Buy Them

    Fisher Scientific
    50 Valley Stream Parkway
    Great Valley Corporate Center
    Malvern, PA 19355

    Whatman Filter Paper #1 Diameter 18.5
    Whatman # 1001-185
    Cat. # 09-805H 100/10.65
    Cost $10.65

    Wiretrol Calibrated Micropipets Cap 10 Orange
    Cat # 21-175B 100/12.50
    Cost $12.50

    Short-Length Micro Caps Cap 10
    Cat #21-170F
    Cost $7.50

    Silica Gel Adsorption 25g
    Whatman # 4119-025
    Cat #05-713-368
    Cost $12.50

    Medicine Droppers St. Tip
    Cat #13-700 144/23.00
    Cost $23.00

    Thomas Scientific
    (215) 988-0533
    Micro Extension Clamp
    Cat. #2826-C83
    Cost $13.00

    Sand Hardware Store
    Vegetable Oil Super Market
    Food Dyes Super Market
    Flair Pens Stationers


    Experiment I:

    Index of Experiments Showing Logical Progression

    Paper Chromatography To Demonstrate Chromatography in General

    Experiment II:

    A Comparison through Separation and Isolation Using Paper and Column Chromatography.

    Experiment III:

    A simplistic approach to show partitioning between two liquids. To demonstrate what happens in paper and column chromatography experiments where partitioning occurs between a liquid and a solid.

    Experiment I:

    Determining The Best Paper To Use For Separating Ink Components



    • To determine which filter paper is best for separating the components of black, green, blue and red ink in Flair� Pens.
    • Students will record colors observed in the separation process.
    • Students will measure and graph results.


    Different grades of filter paper or different brands of paper towels
    3 to 4 colors of Flair� Pens
    3 to 4 100-mL jars with lids
    Water bottle filled with tapwater.


    1. Cut strips of paper so that strips do not touch sides of jar when inserted. The strips must be long enough to overlap the rim of the jar.
    2. Fill 100-mL jars 1/8 full with tap water. Use a water bottle to prevent sides of jar from getting wet.
    3. Place 3 dots of each colored ink 1.5 cm from bottom of testing strip. Use a separate strip for each color.
      Caution, the dots must be the smallest that you can make them.
    4. Carefully immerse only the lower part of strip in water.
    5. Cap the jar being careful not to create turbulence in the water.
    6. Allow to develop until the water front moves past the original ink dots by about 10 cm.
    7. Record the color changes.
    8. Carefully remove strips and allow to dry. Mount on sheet of paper.
    9. Measure and record lengths of color bands


    Color Bands

    Paper #1

    Paper #2

    Paper #3

    Paper #4





    Experiment I:

    Lengths of Color Bands


    Green _____________________________________

    Blue ______________________________________

    Red _______________________________________

    Construct a Bargraph of the colors of each ink.



    Which paper had the least streaming of the color band?


    Which paper had the most streaming of the color bands?


    Which paper showed the best color separations?


    Which paper would you use to do other tests of this kind?


    1. Repeat the experiment but substitute different pens.
    2. Record results in the same manner.
    3. Finally, repeat the experiment by selecting two of the same color but different manufacturers. Make a comparison and record results.

    Experiment IIa

    A Comparison of Paper Chromatography and Column Chromatography


    Paper strips (towels or filter paper) cut to fit jar not touching sides and overlapping rim of jar.
    100-mL glass jars with lids
    Blue, green, yellow and red food dye
    Micro caps


    1. Fill 4 (100-mL) jars 1/8 full with tap water. Use a water bottle to prevent sides of jar from getting wet.
    2. Place three dots of dye 1.5 cm from bottom of paper strip. Use a microliter cap to make dots. Use a separate strip for each color.
    3. Carefully immerse only the lower part of strip in the water. Do not allow the water to touch the dots.
    4. Cap the jar being careful not to create turbulence in the water.
    5. Allow to develop until the water front moves past the original ink dots by about 10 cm.
    6. Record the color changes
    7. Allow to dry and mount on sheet of paper.


    Food Dye Color Bands


    Green _____________________________________

    Blue ______________________________________

    Red _______________________________________

    Tape Dye Paper Samples









    Experiment IIa

    A Comparison of Paper Chromatography and Column Chromatography


    To separate food dye into its component parts by using a liquid chromatography column and to compare this with results from paper chromatography experiment.


    8cm rubber bulb pipettes

    Washed sand

    Glass wool

    Blue and green food dye

    Silica gel


    1. Remove the rubber bulb from the glass pipette.
    2. Pack a small amount of glass wool in the narrow end of the pipette. Add a layer of silica gel, approximately 3 cm. Add a 3mm layer of washed sand to the top of the silica gel. Carefully drop 10 microliters of blue dye to the top of the sand. Add 2 drops of water to start the dye through the sand. After the water has passed through add 2 more drops. Wait until this has passed through the sand. At this point fill the tube with water.
    3. Collect the liquid from the separated layers.
    4. Continue to add water in small amounts until all of the bands of color have been washed out.
    5. Repeat procedure with green dye.
    6. Save the collected material in separate bottles.


    While the two methods, paper chromatography and column chromatography give similar information, there are differences. What are the differences and when would you use each method or possibly both methods?



    Paper chromatography is used primarily to determine if a material is pure or a mixture and in some cases can be used to identify the presence of a material by comparing the distance an unknown travels as related to how far a known travels. Column chromatography is usually used to separate and isolate a quantity of a material from a mixture, to purify a material, i.e., separate it from impurities, and to separate and identify components of a mixture. Both methods are used when paper chromatography is used to determine if a separation is possible and what solvents are best to achieve that separation.

    Experiment III

    Partitioning in Water and Oil


    To demonstrate partitioning of materials in an aqueous or oil base.


    Light colored olive oil

    Fresh fruit dye


    Kerosene soluble dye

    Commercially prepared food dyes



    1. Use 2 small jars with lids. Select 2 colors of food dye. Fill each jar 1/4 full with water. Pour an equal amount of oil to the water. Add 2 drops of food dye. Put the lid on the jar and shake. Observe and record the results.
    2. Repeat step 1 but for the sample add 6 drops of colored kerosene. Observe and record results.
    3. Repeat step 1 but for the sample add 2 drops of fruit dye. Observe and record results.
    4. Fill small jar with lid half full with water. Add the pollen of a lily. Cap the bottle and shake. Observe and record results. Repeat the process using oil.


    Food Dye

    Kerosene Dye



    Water soluble

    Water insoluble

    Water insoluble

    Water insoluble

    Oil insoluble

    Oil insoluble

    Oil insoluble

    Oil insoluble



    Pollen displayed a solubility in both water and oil. The color red was extracted in the water. The color orange was extracted in the oil.

    The food dye was soluble only in water. The kerosene dye was soluble only in oil.

    We wanted to demonstrate that materials soluble in both phases would position between the two phases. We were not able to find a "Safe" dye for the purpose.

    This experiment is courtesy of 

    My Dog Kelly

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    Last updated: June 2013
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