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CH 150: Introduction to Biochemistry
Carbohydrate
Characterization Objective: to learn four tests to characterize carbohydrate structure and then characterize the characteristics of the sugars found in corn meal, corn syrup, honey, and saw dust extract. Introduction:
The structures and chemical reactivities of carbohydrates are widely
varied. A sugar may be composed of simple monosaccharide subunits, or
may be a more complex polysaccharide. A sugar may or may not have a reducing
end, and its subunits may have 3,4,5,6 or more carbon units!
The presence of different structural features of a carbohydrate can
determined by specific chemical tests. Benedict's Test assays for the presence of reducing sugars. The positive results is the formation of a precipitate within 5 minutes, with the color of the solid ranging from green to yellow to orange to brick red. The acetal or ketal group on the reducing end of a carbohydrate is isomerized to an aldehyde under alkaline conditions. This aldehyde may be oxidized by certain metal cations, thus reducing the metal ion forming a colored precipitate. Most commonly, copper (I) ion is used. Barfoed's
Test is a test for monosaccharides. A
positive result is indicated by the formation of brick red precipitate within 5
minutes. Disaccharides usually do not form a precipitate even after ten minutes.
The precipitate that does form may adhere to wall of the test tube in which the
assay is performed. Seilwanoff's
Test is a test for ketoses. In this assay, concentrated HCl is used.
Ketoses usually dehydrate at a faster rate than do aldoses, allowing for
the discrimination of the two. The limit of detection is 20 mg/mL. A
positive test is the formation of orange to red color within 5 minutes without
the formation of a precipitate. Sucrose also tests positive with this assay. An
apricot color is not a positive test.
It is interesting to note that glucose gives no color even after 10
minutes Bial's Test
is a test for pentoses. In the
presence of concentrated HCl, the furfural that is produced (see experiment 3)
generates a blue-green color is the presence of orcinol and ferric ions. Thus a
positive test is the formation of a blue to green color within 5 minutes, again
without the formation of a precipitate. PreLab Exercise: Draw the structures of the following carbohydrates: glucose, fructose, sucrose, lactose, maltose, ribose, sorbose, and xylose. Identify each as a mono- vs. disaccharide; reducing vs. nonreducing; pentose vs. hexose; aldose vs. ketose. Fill in the table below with the information.
Procedure: A. Known Carbohydrate
Analysis In a test tube containing 20 drops of the
respective reagent, add 5 drops of the sugar.
Mix well and place in a boiling water bath for 5-10 minutes.
Record whether a precipitate is formed or not and how long it take to
form.
B. Carbohydrates in
Natural Samples Prepare acid hydolysates of cornmeal (0.1 g)
and saw dust (0.2 g) by heating in 2 mL of 1M HCl in a boiling water bath for 1
hour. Dilute each sample with 3 mL
water and decant the liquid to a clean test tube. Remove 2 mL of the decantate
to a clean test tube and add 4-8 drops of 10 % NaOH to neutralize the solutions
(Check with pH paper). Perform each of the tests above on each sample, using the
neutralized solution for Benedict's test and the acidic sample for Seliwanoff's,
Bial's, and Barfoed's tests. Perform
the same tests on the sample of corn syrup (2% in water) and honey.
Record your observations.
Results and Discussion: 1.
For what feature of carbohydrate structure does each assay test? Describe
positive and negative results for each test in terms of color, formation of a
precipitate, etc.
2.
Based on the structures of the 8 known sugars you tested, predict how
each would react in each test. Do
your experimental results support your hypotheses?
3.
Describe the sugars in each of your natural samples.
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