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:
- Starch is a larger molecule than glucose
- An assay based upon selection
- The techniques of testing for the presence of specific substances
- 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
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.
- 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.
- 0.9% sodium chloride
Weigh out 9 gms of sodium chloride and add to
991 gms (about a liter) of distilled or deionized
water.
- 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.
- Benedict's solution
This is best purchased from a biological or
chemical supply house.
- 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
- Discuss concepts presented in introduction.
- Review safety precautions.
- Present flowchart of procedure
- Model data collecting technique.
- Observe these helpful hints to avoid sources of error:
ANSWERS TO QUESTIONS
- Student responses.
- 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.
- 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.
- 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.
- 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.
- 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
State a hypothesis concerning the outcome of this experiment based on background information.
PROCEDURE:
- Mount gel column vertically with burette clamp.
- 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.
- 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.
- When eluant flow has stopped, cap outlet spout.
- 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.
- Remove outlet spout cap, and allow 10 drops to
enter test tube #1S.
- 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.
- 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.
- In test tubes #1G through 10G place one ml or
20 drops of Benedict's solution.
- 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.
- 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
- 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
- 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.
- Was your hypothesis correct? What evidence do
you have to support your hypothesis?
- Explain your results.
- How does dialysis differ from this technique?
How is it similar?
- What role does the sodium chloride solution play
in this experiment?
- 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?
- What other scientific uses can gel filtration
have?
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