BARFOED’S TEST
Comments:

Barfoed’s reagent is similar to Benedict’s reagent except that Barfoed’s is slightly acidic
with a pH of 4.5. At this pH, disaccharides, when heated for 2 minutes will not reduce
Cu2+ to Cu2O whereas monosaccharides will reduce the Cu2+. Longer heating of
disaccharides may lead to some reduction because of the formation of monosaccharides
by hydrolysis so it is important that in this experiment, all sugars be treated in exactly the
same way.
Purpose:
To distinguish between monosaccharides and disaccharides.
Materials:
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
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Hot water bath
Barfoed’s reagent
Solutions to test: Glucose, Galactose, Molasses, Sucrose, Starch, HCl with Sucrose,
fructose, Apple juice, HCl, molasses, lactose, tap water.
Procedure:
1st—In one test tube, add 5 mL of Barfoed’s reagent then add 0.5 mL of a 1%
concentration of the solution to be tested. Do this for all solutions to be tested. Mix well.
2nd—Place the test tubes in a boiling water bath and heat for 2 minutes.
3rd—Record the results and note the time of appearance of the precipitate.
Results:
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A positive test for monosaccharides is the appearance of a red precipitate of cuprous
oxide within 1 or 2 minutes.
Positive: Glucose, Galactose, Molasses, HCl with sucrose, fructose, apple juice.
Negative: Sucrose, starch, lactose, tap water.
Trend/Conclusion:
 Glucose, galactose, fructose, apple juice, and molasses all contain a free carbonyl group
to reduce copper 2+ to cuprous oxide. They are therefore all monosaccharides. Sucrose
with HCl was broken into its monosaccharides glucose and glucose.
 Sucrose is a disaccharide and starch is a polysaccharide, and they will not reduce copper.
BIURET TEST
1. Comments:
 Biuret reagent (a blue-green solution) contains a strong solution of sodium hydroxide
(NaOH) or potassium hydroxide (KOH) and a dilute solution of copper sulfate (CuSO4).
If protein is present, the solution will change to violet because of a reaction with the
peptide bonds that link the individual amino acids into the protein chain.
2. The purpose of this experiment is:
 To test for the presence of protein in a substance.
3. The materials needed for this experiment are:
 7 test tubes
 Biuret reagent
 Solution to test: water, sucrose, albumin, milk, bread, ground peanuts, vegetable oil
4. The procedure:
 1st—Label the test tubes 1-7. Add 2 mL of the following to test tubes 1-4: water, sucrose,
albumin, milk. Test tube 5, add 1 chunk of bread. 6—small amount of ground peanuts.
7—2mL vegetable oil.
 2nd—Add 2 mL of Biuret reagent to each test tube.
 3rd—Replace test tubes in the rack for 2 minutes and record results.
5. The results:
 Color change to purple is a positive test result for protein.
 Positive: Albumin, milk, peanuts
 Negative: Water, sucrose, bread, vegetable oil
6. Conclusions:
 From this experiment, we can conclude that albumin, milk, and peanuts contain protein.
BENEDICT’S TEST
1. Comments:
 All monosaccharides and some disaccharides have the ability to add electrons to (reduce)
other molecules. These sugars are called reducing sugars. Reducing sugars have free
carbonyl groups in close proximity to hydroxyl groups and this is where electrons come
from. When two monosaccharides join to form a disaccharide, the carbonyl group gets
tied up in the linkage so it is not available to release electrons. This is the case with some
disaccharides and all polysaccharides. Benedict’s reagent, which is used to test for
reducing sugars, contains copper ions in alkaline solution. The blue color of the reagent is
characteristic of solutions containing copper ions. When Benedict’s reagent is heated in
the presence of a reducing sugar, the copper ions are reduced to metallic copper. This
forms a precipitate, which colors the contents of the tube green to brick red or brown,
depending on how much reducing sugar is present. Before the development of paper test
strips, this test was used by diabetics to test for sugar in the urine.
2. The purpose of this experiment is:
 To test for the presence of reducing sugars in a substance.
3. The materials needed for this experiment are:
 Test tube rack
 Hot water bath
 9 test tubes
 Benedict’s reagent (Sodium bicarbonate, sodium citrate, copper sulfate)
 Solutions to test: Water, Glucose, Lactose (Milk), Apple juice, starch, molasses (diluted),
sucrose, 10 % HCl, 10% HCL and sucrose
4. The procedure:
 1st—Prepare a hot water bath by setting a beaker about half full of tap water on a
hotplate. Once steam is rising, turn the temperature back to medium.
 2nd—Number test tubes 1-9 with a wax pencil and place them in a test tube rack.
 3rd—Add 2 mL of each of the following to separate test tubes 1-8: water, glucose,
sucrose, milk, apple juice, starch, molasses, and HCl. Add 2 mL of Benedict’s reagent to
each tube.
 4th—Place in a hot water bath for 2 minutes, remove using a test tube holder, cool and
record the color of each substance.
 5th—In tube 9 combine 2 mL sucrose with 2 mL HCl. Heat in the water bath for 10
minutes. Then remove from heat and add the Benedict’s to tube 9. Reheat, cool, and
record.
5. The results:
 Negative (light blue): Water and HCl (controls); Starch; Sucrose
 Positive (red ppt): Glucose, Milk, Apple juice, Molasses (orange ppt), Sucrose plus HCl
6. Conclusions:
 Glucose, milk, apple juice, and molasses are all reducing sugars because they reacted
with the Benedict’s reagent to form a red precipitate.
 In tube 9, the HCl broke the glycosidic bonds of sucrose to make glucose monomers.
Glucose is a reducing sugar.
STARCH TEST
1. Comments:
 Starch is a polysaccharide consisting of many glucose molecules linked together. Iodine
is added to detect the presence of starch. The iodine molecules get stuck in the spirals of
the starch molecule and cause a blue-black color to appear. A red color is an indication of
the presence of certain dextrins, intermediate products of starch digestion whose spirals
are shorter.
2. The purpose of this experiment is:
 To test for the presence of starch in a solution.
3. The materials needed for this experiment are:
 Iodine (I2-KI)
 Solutions to test: Starch, glucose, water, sucrose, cellulose (cotton), potato, bread, starch
+ saliva
4. The procedure:
 1st—In test tubes 1-7, Add 2 mL of the following to separate test tubes: starch, glucose,
water, sucrose. Place a small piece of the following to separate test tubes: potato, bread,
cotton.
 2nd—Add 5 drops of iodine to each test tube. Observe and record the results.
 3rd—To the 8th test tube, add 1 mL of saliva or bacterial amylase to 1 mL of starch
solution. Let sit for 10-15 minutes, then pour half of the contents into a clean test tube.
 4th—Add iodine to one of the test tubes. Observe and record the reaction.
5. The results:
 Positive test result is indicated by a blue-black color.
 Positive: Starch, Cellulose, Potato, Bread
 Negative: Glucose, Water, Sucrose, Starch + saliva (because amylase breaks down starch
to maltose).
6. Conclusions:
 From this experiment, we can conclude that starch, cellulose, potato, and bread contain
starch.
NINHYDRIN TEST
1. Comments:
 The reaction of ninhydrin with amino acids is of particular importance for the detection
and qualitative estimation of amino acids. Ninhydrin is a powerful oxidizing agent that
removes the amino groups of amino acids. In the process, the reaction liberates ammonia,
carbon dioxide, the corresponding aldehyde, and a reduced form of ninhydrin. The
ammonia then reacts with an additional mole of ninhydrin and the reduced ninhydrin to
form a purple substance. The appearance of the purple color is a positive test for proetein.
2. The purpose of this experiment is:
 To test for the presence of a protein.
3. The materials needed for this experiment are:
 2 test tubes
 Sodium acetate
 Ninhydrin
 Solutions to test: distilled water and egg albumin
4. The procedure:
 1st—Add 3.0 mL of distilled water to one test tube and 3.0 mL of 0.1% egg albumin
solutionto a second tube.
 2nd—Add solid sodium acetate to each test tube (scoop-type spatula loaded to a depth of 1
inch).
 3rd—Add 8 drops of ninhydrin to each test tube.
 4th—Heat for 3 minutes in a boiling water bath and cool.
 5th—Record your observations.
5. The results:
 Positive: Egg albumin
 Negative: Distilled water
6. Conclusions:
 From the results, we can conclude that egg albumin contains protein.
PAULY TEST
1. Comments:
 Only the amino acids tyrosine and/or histidine react in alkaline solution with sulfanilic
acid to give an intense red color.
2. The purpose of this experiment is:
 To test for the presence of the amino acids tyrosine and/or histidine in a solution.
3. The materials needed for this experiment are:
 5 test tubes
 Sulfanilic acid
 5% sodium nitrite NaNO2
 Sodium carbonate NaCO3
 Solutions to test: 10 mg/mL of histidine, tyrosine, glycine. 2 g casein.
4. The procedure:
 1st—Pipet 2.0 mL of the following into separate test tubes: histidine, tyrosine, glycine,
distilled water. Add 2 g casein to another test tube.
 2nd—Add 1.0 mL of sulfanilic acid reagent and 1 mL of 5% sodium nitrite to each tube.
Mix and let stand for 30 minutes.
 3rd—Add 3.0 mL of 20% sodium carbonate to each tube and mix.
 4th—Record observations.
5. The results:
 Positive: tyrosine, histidine, casein (turned red)
 Negative: glycine, distilled water
6. Conclusions:
 Because tyrosine, histidine, and casein tested positive, they contain either one or both the
amino acids tyrosine and histidine.
TEST FOR ORGANIC AND INORGANIC COMPOUNDS
1. Comments:
 A standard definition of an organic compound is one whose molecules contain carbon in
the form of chains or rings.
2. The purpose of this experiment is:
 To test whether a compound is organic or not, meaning it contains carbon and hydrogen
molecules.
3. The materials needed for this experiment are:
 6 test tubes
 Bunsen burner
 Compounds to test: Bread, potato, sucrose, Meat, Salt (NaCl), Chalk (CaCO3)
4. The procedure:
 1st—Place a small amount of each substance into separate test tubes.
 2nd—Hold the test tube over an open flame until all reactions inside the tubes seem to
stop.
 3rd—Record observations.
5. The results:
 Positive test is indicated by a black residue left by the substance.
 Positive: Bread, potato, sucrose, meat
 Negative: NaCl, CaCO3
6. Conclusions:
 From the results, we can see that bread, potato, sucrose, and meat are all organic
compounds.
SUDAN STAIN FOR LIPIDS
1. Comments:
 Lipids are generally insoluble in water but are soluble in organic solvents, such as ether,
acetone, carbon tetrachloride, and chloroform.
2. The purpose of this experiment is:
 To test for the presence of a lipid.
3. The materials needed for this experiment are:
 1 test tube
 Liquid detergent
 Vegetable oil
 Red Sudan stain
4. The procedure:
 1st—Add 3 drops of vegetable oil to a test tube half-filled with tap water.
 2nd—Shake thoroughly and observe how the oil is dispersed only temporarily. This is an
emulsion—a mixture of two liquids, each insoluble in the other.
 3rd—Add a small amount of red Sudan stain and mix again.
 4th—Add several droppersful of a liquid detergent to the tube and shake again. Allow the
tube to stand and note that the two phases (oil and water) are no longer distinctly
separated. Detergent is often termed as an emulsifier. Its molecules are water-soluble on
one end and lipid-soluble on the other. These surround small oil droplets, water soluble
end out, and allow the droplets to stay suspended in the water.
5. The results:
 Vegetable oil separates from water in step 2.
 In step 3, the Sudan red stains the vegetable oil and is concentrated at the surface of the
water.
 In step 4, the red Sudan stain is dispersed throughout the water.
6. Conclusions:
 Oil and water do not mix because oil is nonpolar and insoluble in the polar water
molecules. The liquid detergent acts as an emulsifier and separates the oil droplets from
congregating together.
SAKAGUCHI TEST
1. Comments:
 Alkaline solutions of proteins that contain the amino acid arginine react with a-napthol
and sodium hypobromite to produce an intense red color. The color will disappear rapidly
if it is not stabilized by the addition of urea. It is useful in detecting arginine.
2. The Purpose of this experiment is:
 To test for the amino acid arginine.
3. The materials needed for this experiment are:
 3 test tubes
 Pipette
 1.0ml of 10 N sodium hydroxide
 1.0ml of a 0.02% a-napthol
 Sodium hypobromite
 Compounds to test: distilled water, 1% albumin, 0.1% arginine
 40% Urea solution
4. The Procedure:
 1st – Pipet 3.0ml of distilled water into Test Tube 1, 3.0 ml of a 1 % albumin solution into
Test Tube 2, and a 3.0ml of a 0.1% arginine solution into Test Tube 3.
 2nd- Add 1.0ml of 10 N sodium hydroxide to each tube, followed by 1.0ml of a .02% anapthol solution.
 3rd – Add two drops of sodium hypobromite to each tube, followed immediately (within
10 seconds) by 1.0ml of a 40% urea solution.
 Record your observations.
5. The Results:
 Positive Test is indicated by an intense red color.
 Positive: 0.1% arginine
 Negative: Distilled water and 1% albumin
6. Conclusion:
 From the results, we can see that arginine contains the amino acid arginine.