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									                    Separating Pigments with Liquid Chromatography

RET Program: Summer 2005
RET Teacher: Pamela Maurer

Course(s): Biology Honors, AP Biology, Chemistry Honors, AP Chemistry
Grade level: High school

Sunshine State Standards:

SC.F.1.4.1 Knows that the body processes involve specific biochemical reactions governed by
biochemical principles.

SC.H.1.4.1 Knows that investigations are conducted to explore new phenomena, to check on
previous results, to test how well a theory predicts, and to compare different theories.

SC.H.3.4.2 Knows that technological problems often create a demand for new scientific
knowledge and that new technologies make it possible for scientists to extend their research in a
way that advances science.

SC.H.3.4.6 Knows that scientific knowledge is used by those who engage in design and
technology to solve practical problems, taking human values and limitations into account.

SC.A.2.4.2 Knows the difference between an element, a molecule, and a compound.

S.C.A.1.4.2 Knows that the vast diversity of the properties of materials is primarily due to
variations in the forces that hold molecules together.

Purpose: To observe separation of pigments using a liquid chromatography technique and
understand the chemical and physical properties that allow the separation.

    Students will construct a liquid chromatography column and observe separations.
    Students will understand the chemical properties that allow some substances to pass
       through the column faster than others.
    Students will determine which type of column works best for a particular dye.
    Students will determine what causes the column larger particles separate out first and
       smaller particles separate out last.


        Chromatography is a physical method used to separate one or more substances in a
mixture. There are numerous types of chromatography dependent upon what substances are
being separated.

        Liquid adsorption chromatography is used mainly to separate non-volatile components.
A column is constructed and packed with a hydrophobic adsorbent such as silica gel or acid silica
gel. This adsorbent is called the stationary phase of the column.

        The solvent, which is also known as the liquid phase, will continually wash over the
stationary phase of the column. As the desired mixture washes over the adsorbent bands will
form, each consisting over a different substance from the mixture. Substances in the mixture first
adhere to the adsorbent and then their attraction to the solvent used will cause them to migrate

down the column at different rates. The rate at which each substance in the mixture migrates
down the column is dependent on its attraction to the adsorbent and to the solvent used.

        Separation depends on the type of adsorbent versus the type of solvent used in the
column. Due to the reactivity of the adsorbent, nonpolar compounds will move through the
column faster than polar compounds. Careful consideration needs to be given to the type of
solvent and adsorbent used for the column. Compounds that are highly attracted to the adsorbent
will remain bonded and not pass through the column. Another problem arises if the solvent is too
polar causing the solution to move to fast through the column and not allow separation to occur.


Clear drinking straws                               Wash bottle
Silica gel (70-230 mesh)                            Clamp holder
Ring stand                                          Dropper pipet (disp.)
Extension clamp micro jaws*                         Green food coloring
Steel wool                                          100-mL beakers
Water                                               Microtip dropper*

*=alternative may be used.

Safety requirements: Goggles, gloves, fume hood (if possible)


Part A: Silica Gel Column

    1. Cut off small pieces of steel wool, roll in fingers and plug the bottom of the straw
        (about ½ inch). Be sure not to pack too tightly but tightly enough so that water
        and dye can pass through.
    2. Clamp the straw vertically to the ring stand.
    3. Place a 100-ml beaker under the straw.
    4. In another beaker, add 20ml of water to a small amount of silica gel to form a
        slurry (liquid paste). Use gloves when working with silica gel. If slurry is too
        solid the column will run very slow, if it is too watery the column will run too fast
        and the pigments will not completely separate.
    5. Cut the end of a disposable pipet and draw some slurry into the dropper pipet.
        (See figure 1.1)
    6. Add silica gel slurry to the top of the straw from the dropper pipet. Be careful not
        to create air bubbles within the straw. It is best to add in small amounts and you
        may need to add some DI water first to help it move into column.
    7. Fill the straw with slurry to about five centimeters from the top of the straw.
        Allow the slurry to settle a few minutes, you may need to add more if it is too far
        from the top.
    8. Add water to the top of the straw column using a wash bottle.
    9. Using a microtip dropper, add a 5 l drop of green food color to the top of the
        silica gel in the column. Let the color fall into the gel.
    10. Add water to the top of the column, continuing until the separation is complete.
        Collect the effluent from each band of color in a separate beaker.
    11. Record data in table 1.1

 Figure 1.1
 Separation column

Data & Observations

Table 1.1

Column          # bands         # bands           Colors (list first to last)
                predicted       observed
Silica Gel

Table 1.2

Start time (dye) =_________            End time (dye)=___________

Amount collected= _______ml

Rate of column (ml/min) = _____________________


   1. How did your hypothesis compare to your observed number of bands?

   2. Explain the chemical properties of the first substance to pass through the column

   3. How does the chemical property of the first band compare to the chemical
      property of the last band?

   4. List and explain all complications or errors that occurred.


   1. What is another method of separating substances?

   2. Why was Liquid Chromatography the best method to separate the substances in

   3. How could this lab be extended further?

   4. How is this lab relevant to biological science? Give and example of when it may
      be used.

   5. You performed the procedure one time, what must you do to better support your
      results (data)? Why?

Teacher notes

Preparation time: 20 minutes
Lab time: 90 minutes

Preparation and lab considerations:
   1. Students must wear goggles and gloves when working with 0.1M acetic acid and
       the silica gel throughout this lab.
   2. Separation of the green dye may take up to 45 minutes, be sure preparations are
       done in a timely matter. During this time students can look up the uses of liquid
       chromatography for biology. You can either have them collect each separation in
       different small beakers or you can let them collect the separations into one beaker
       and let them see the original color reappear.
   3. When making the silica gel slurry, add small scoops of silica gel to the water and
       stir after each addition or two. If it becomes too thick add more water back into
       the mixture. USE CAUTION when working with silica gel. Do not breathe it in
       or get it on your hands.
   4. The teacher can assign different groups different columns or each group can do
       both columns at the same time. If each group does both types of columns be sure
       to set them up together since the separation takes at least 45 minutes.
   5. It was determined that green dye worked best but you may want to try other
       alternatives such as vegetable or candy dyes beforehand.

Materials and preparations:

Preparation of silica gel slurry: Depending on the nature of the class, the teacher may
want to make the mixture. Add small increments of silica gel to 20 ml of DI water and
stir. This amount will make at least three columns. If possible use a fume hood to add
silica gel to avoid inhalation. Read MSDS before using silica gel.

Steel wool: Using a steel scrubber will work but some of the silica gel may leak through.
It’s best to use. It is recommended to use the soap free Brillo steel wool balls available
at most grocery stores.

Additional resources:

Slide based web presentation about chromatography is available at:

Accommodation for students with special needs:
   1. Assign lab tasks according to each student’s ability level considering tasks such as
      constructing, designing, calculations, and reading procedures.
   2. You may want to demonstrate how this works with your own model during your
      prelab discussion.


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