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SEPARATION OF A STARCH-GLUCOSE MIXTURE USING GEL FILTRATION

Fred Blumenfeld
1993 Woodrow Wilson Biology Institute


INTRODUCTION

For more than thirty years, gel filtration has been used as a biotechnological process for the purification of enzymes, polysaccharides, nucleic acids, proteins and other biological macromolecules. The techniques of gel filtration separate molecules according to differences in their size as they pass through a column packed with a gel media. Gel filtration media are very stable because of their ineptness towards biopolymers. In the experimental laboratory, gel filtration is reliable and simple, little equipment is required, the procedures are straightforward and good separations and yields are usually obtained.

Several years ago, I began experimenting with a number of gels to determine the feasibility of designing high school laboratory experiences using gel filtration. I have successfully completed and utilized a number of these labs in my classroom. One of these is the separation of a starch-glucose mixture using gel packed PD-10 columns.

The gel used, Sephadex G-25, can be purchased pre-packed in polypropylene syringe-like PD-10 columns. The gel is an inert, bead-formed, cross-linked Dextran polymer consisting of many glucose molecules. Sephadex beads are porous. Molecules larger than the largest pores cannot enter the gel and are eluted (pass out) from the column first. Smaller molecules enter the beads and are retarded based on their size. Therefore, molecules are eluted in order of decreasing size. Sephadex G-25 excludes all molecules with a molecular weight greater than 5000, thereby eluting them first.

The following concepts developed are:

  1. Starch is a larger molecule than glucose
  2. An assay based upon selection
  3. The techniques of testing for the presence of specific substances
  4. The basic principles of gel filtration

TARGET AGE/ABILITY GROUP:

A maximum of twenty four students in grades 9-12 of average to high ability can be targeted for each class instruction period.

CLASS TIME:

Forty-five minutes to one hour should be scheduled as operating time, depending on prior preparation.


MATERIALS:

For team of two students

  • Test tube rack to hold 18 small test tubes, 13 x100 mm.
  • Two beakers
  • Wire test tube holder.
  • Masking tape
  • One ring stand with burette clamp.
  • Water bath
  • One PD-10 gel column G-25.
  • Hot plate
  • 0.1% starch and glucose mixture - in dropping bottle
  • 0.9% sodium chloride solution - 30 ml in Barnes dropping bottle
  • Benedict's solution in dropping bottle
  • Lugol's iodine solution in dropping bottle
  • Razor blades, single edge
  • Materials should not exceed twenty dollars. I have used and reused the PD-10 columns for at least 5 years.


    SAFETY PRECAUTIONS:

    Wearing of goggles, use of test tube holders, use of heat resistant gloves, aprons and disposal of end products in evaporating containers placed in hoods.


    TEACHER GUIDE

    Preparation time need not exceed one hour including preparation of solutions which follow.

    1. Starch-glucose mixture.

      Weigh out 1 gm of soluble starch and 1 gm of glucose (dextrose) and place in a two liter beaker. Add one liter of distilled or deionized water and heat to boiling while stirring. Let cool before using.

    2. 0.9% sodium chloride

      Weigh out 9 gms of sodium chloride and add to 991 gms (about a liter) of distilled or deionized water.

    3. Lugol's iodine solution

      This can be purchased from any biological or chemical supply house, or it may be made by dissolving 5.0 grams of iodine plus 10.0 grams of potassium iodide in 100 ml of distilled or deionized water and used as a stock solution. The stock solution should be diluted one to ten parts of water before using.

    4. Benedict's solution

      This is best purchased from a biological or chemical supply house.

    5. PD-10 Columns Prepacked with gel, and swollen in 0.9% salt solution.

      Can be purchased from:
      Pharmacia Fine Chemicals
      800 Centennial Avenue,
      Piscataway, New Jersey 08855-1327
      1-800-526-3593

      PD-10,30/pk. No. 17-0851-01 $112.00


    TEACHERS OUTLINE FOR PRESENTATION OF ACTIVITY

    1. Discuss concepts presented in introduction.

    2. Review safety precautions.

    3. Present flowchart of procedure

    4. Model data collecting technique.

    5. Observe these helpful hints to avoid sources of error:

      a. Step 3 in student procedure pertaining to elution of sodium chloride is to ensure that glucose from previous class usage will be removed from column in case students forgot to completely elute the glucose.

      b. It is important that when filling the column with salt solution for collecting of fractions, there is no overflow into the collecting test tubes.

      c. An alternative method of numbering test tubes is to label the rack openings directly using a strip of masking tape. Be sure all test tubes are thoroughly cleaned.

      d. Make certain that students do not misplace top and spout caps.


    ANSWERS TO QUESTIONS

    1. Student responses.

    2. The first 10 drops contained no starch or glucose, as the remainder of the salt in the column was still eluting through. The starch comes into test tubes 2 and 3, as the starch molecules are much larger than the glucose molecules and cannot penetrate the gel beads. Test tube #4 may indicate a slight presence of both starch and glucose as that fraction may contain the last part of the starch solution, and the initial part of the glucose solution or glucose may be absent showing complete separation. Only test tubes #5-8 should show increasing, then decreasing concentrations of glucose, with no starch being present. Test tube #9 and #10 are negative for both starch and glucose since neither substance is present in the column, and only salt is coming through.

    3. This technique differs from dialysis in that the gel process can be considered in two ways. If you view the entire column as the membrane, then only the glucose should pass through, starch should be left behind- which obviously is not the case. If you view each gel bead as the membrane, there is no difference.

    4. Sodium chloride elutes the solutes through the gel column as well as cleansing the gel column at the conclusion of the lab. Sodium chloride also keeps the concentration of the salt the same both inside and outside the beads.

    5. Ten drops are collected first, as the bottom of the column contains only salt. Salt comes through the gel column with the starch. This could be proven by adding lead nitrate to any fraction collected, and obtaining a white precipitate.

    6. Purification of biological macromolecules e.g. proteins, nucleic acids, photosynthetic pigments, inorganic mixtures.

    OTHER

    Another biological experiment that uses the principle of gel filtration is the separation of pigments from Coleus leaves. The gel, LH-20, is required. The organic solvent, 95% Ethanol separates the extract into distinct bands of chlorophyll, xanthophyll and anthocyanin.


    SOURCES OF MATERIALS AND REFERENCES

    Carolina Biological Supply Co.
    Burlington, North Carolina
    Kemtec Co. Cincinnati, Ohio

    Pharmacia Fine Chemicals
    Pharmacia LKB Biotechnology Manual
    "Principles and Methods of Gel Filtration"

    James Gardner, Consultant,
    Fairleigh Dickinson University,
    C.E. Div. Madison, NJ


    SEPARATION OF STARCH FROM GLUCOSE USING GEL FILTRATION

    PURPOSE:

    To separate starch from glucose in a mixture.

    MATERIALS:

    For team of two students

  • Test tube rack to hold 18 small test tubes, 13 x100 mm
  • Two beakers
  • Wire test tube holder
  • Masking tape
  • One ring stand with burette clamp
  • Water bath
  • One PD-10 gel column G-25
  • Hot plate
  • 0.1% starch and glucose mixture - in dropping bottle
  • 0.9% sodium chloride solution 30 ml in Barnes dropping bottle
  • Benedict's solution in dropping bottle
  • Lugol's iodine solution in dropping bottle
  • Razor blades, single edge

  • State a hypothesis concerning the outcome of this experiment based on background information.

    PROCEDURE:

    1. Mount gel column vertically with burette clamp.

    2. Number test tubes 1S through 8S and 1G through 10G ("S" is for Starch and "G" is for Glucose) and place in test tube rack.

    3. Allow eluant (a substance used to remove any material already present in the gel column) to drip out into beaker until flow stops. Add 1.0 ml or 20 drops of 0.1% glucose and starch mixture to the top of the gel column, continuing to collect eluant in the beaker. Discard all eluant washing.

    4. When eluant flow has stopped, cap outlet spout.

    5. Add 0.9% salt solution to fill top of column, and lower the gel column so that the spout is about 2.0 cm above test tube #1S.

    6. Remove outlet spout cap, and allow 10 drops to enter test tube #1S.

    7. Moving the test tube rack, collect an additional 25 drops in each test tube numbered 2S through 8S. Collect an additional 10 drops in test tube 9G and 10G. The column will have to be refilled at least twice with 0.9% sodium chloride during this part of the lab.

    8. Cap the outlet spout after test tube #10G has been collected. Also make certain that the space above the gel column is only about 1/2 filled with 0.9% sodium chloride solution before replacing the top cap.

    9. In test tubes #1G through 10G place one ml or 20 drops of Benedict's solution.

    10. Pour all but a few drops of eluant collected in test tube #1S into test tube #1G containing the Benedict's solution. Similarly, repeat pouring eluants from test tube #2S into test tube #2G, test tube #3S into test tube #3G, . . . through test tube #8S into test tube #8G. This effectively divides the eluant of each test tube numbered 1S through 8S into two parts. The largest portion, in test tubes numbered 1G through 8G will be used to test for glucose. In addition test tube #9G and 10G will be tested for glucose. The other portion, containing only a few drops, in test tubes numbered 1S through 8S will be used for starch identification.

    11. Place one drop of iodine solution into each test tube numbered 1S through 8S containing a few drops of eluant. Observe results for starch identification (blue-black color) by holding against a white background and note observation in data chart as follows: (++) for intense color change (+) for slight color change (-) for a negative test

    12. Gently heat contents of test tubes numbered 1G through 10G in water bath for a few minutes or until any color change occurs. Observe color changes, if any, in each test tube for glucose identification, and note observations in data chart as follows: (++) for a dark green to brick red (+) for a yellow to light green (-) for a blue color

    13. Clean all glassware thoroughly.


    TEST TUBES1S  2S  3S  4S  5S  6S  7S  8S  
    Starch Id.     *   *   *   *   *   *   *   *
    

    TEST TUBES1G  2G  3G  4G  5G  6G  7G  8G  9G  10G
    Glucose Id.   *    *   *     *    *    *   *    *     *     *
    

    CONCLUSIONS:

    Answer the following questions.

    1. Was your hypothesis correct? What evidence do you have to support your hypothesis?

    2. Explain your results.

    3. How does dialysis differ from this technique?
      How is it similar?

    4. What role does the sodium chloride solution play in this experiment?

    5. In sample 1S, why were only 10 drops collected? What eluant came through the gel column with the starch, and with the glucose? How could you prove this?

    6. What other scientific uses can gel filtration have?


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