Anda di halaman 1dari 18

Molisch's Test for arabinose, fructose, glucose, maltose and galactose

(from left to right)


Benedict's and Barfoed's tests are both reduction tests. If carbohydrates have a reducing ability they have, or
are able to form, a free aldehyde or ketone group in solution. In alkaline solution, copper(II) ions are then
reduced to Cu20. All the common monosaccharides are reducing sugars. Some disaccharides and
polysaccharides may initially be nonreducing but will show reducing properties after heating in an acidic
solution because hydrolysis to their monosaccharides has taken place. Although very similar, results from the
two tests will confirm the presence of monosaccharides and distinguish between reducing and nonreducing
disaccharides.
Benedict's Test shows the reduction of Cu2+ to Cu+, forming Cu20, which is a brick-red coloured precipitate.
The colour in a positive Benedict's test may appear as green, yellow, orange, or red depending upon the amount
of Cu20 suspended in the originally dark blue coloured reagent. The amount of Cu20 formed depends on the
concentration of sugar in the solution. Reducing sugars, either mono- or disaccarides, give a positive test. This
is a timed test as continued heating will hydrolyse disaccharides to monosaccharides which will then react with
the Cu2+ to give a positive test.
Barfoed's Test is used to distinguish between reducing mono- and disaccharides. This test is also a copper
reduction reaction but differs from Benedict's in that the reagent is made in an acidic medium [copper(II)
acetate and acetic acid]. A positive reaction within the stated time interval of 5 minutes indicates the presence of
monosaccharides. Because this ion is weak, there is no positive reaction shown by reducing disaccharides
unless they are heated and are hydrolysed by the acid present. A positive reaction after 20 minutes, that is, after
hydrolysis has taken place, confirms the presence of the disaccharide. Characterization information is gained
by recording observations after 5 minutes and again after 20 minutes.

Page 51

Benedict's Test for all nine tested sugars arabinose, fructose, glucose, maltose,
galactose, lactose, starch, sucrose and xylose and distilled water (from left to right )

Barfoed's Test for galactose, glucose, fructose and xylose (from left to right)
Page 52

Iodine reacts with starch, a polysaccaride of glucose, to form a deep blue/purple complex. When an acidified
starch solution is boiled, it hydrolyses to yield oligomers, disaccharides and glucose itself. The iodine test can
be used to follow the course of this hydrolysis. As both starch and sucrose will give a negative Benedicts test,
the iodine test can be used to distinguish sucrose from starch.

Iodine test for starch (left) and sucrose (right)


Bial's Test is used to differentiate between pentoses and hexoses. Pentoses occur in both plants and animals.
The pentoses ribose and deoxyribose are universally found in the nucleic acid portion of nucleoproteins of the
cells. Bial's reagent contains orcinol (5-methylresorcinol) dissolved in concentrated HCl plus a small amount of
FeCl3. When mixed with the reagent, the pentoses are converted to furfural, which reacts with the FeCl3 to yield
a blue coloured compound.

Bial's Test for xylose, arabinose and glucose (from left to right)
Page 53

Seliwanoff's Test distinguishes ketohexoses from aldohexoses and disaccharides. The only common
ketohexose is fructose. The reaction between fructose and the reagent (resorcinol in dilute HCL) occurs within
1-2 minutes in boiling water. A reddish-coloured product is formed; this colour intensifies with further heating.
Other carbohydrates will produce a faint red colour if heating is prolonged. The colour formation follows the
transformation of glucose to fructose (by the catalytic action of HCl) or by the hydrolysis of sucrose.

Seliwanoff's Test for fructose, sucrose, glucose and galactose (from left to right)
SAFETY PRECAUTIONS
- Safety glasses, lab coats and gloves are required at all times.
- Use concentrated acids/bases in the fume hood only.
- Dispose of test solutions in the labelled containers provided.
MATERIALS AND EQUIPMENTS:
Test Tubes (~15 cm in height, 11 test tubes/test)
Test Tube Rack
Graduated Cylinders (10 mL)
Hot Plate
500 mL Beaker
Distilled water
2% solutions of arabinose, fructose, glucose,
maltose, galactose, lactose, starch, sucrose and
xylose
An unknown carbohydrate solution for EACH
group
Tap water for water bath

Iodine solution (1% iodine in 2%


potassium iodide)
Concentrated Sulphuric Acid
Benedicts Reagent
Barfoeds Reagent
Seliwanoffs Reagent
Bials Reagent
Concentrated Hydrochloric Acid
10% Sodium Hydroxide

Page 54

Experiment #6 Week 1
PROCEDURE
1. Prepare a boiling water bath by placing approximately 200 ml of tap water in a 500 ml beaker on the hot
plate. Keep enough water in the beaker through this experiment to heat the test tubes of solutions when
required.
2. For each test, set up a test tube rack containing 11 clean test tubes; 9 test tubes for the 2% carbohydrate
solutions; 1 test tube for the unknowns and 1 test tube for the distilled water blank (your negative control).
Check each test for quantities to be measured out.
3. A droppers have been provided for each of the carbohydrate solutions. Please make sure there is no cross
contamination between solutions.
4. When recording results, indicate whether a positive or negative colour change has occurred. For the
conclusion, please be specific. For example, the Molisch test is a test for the presence of a carbohydrate. Your
conclusion should then be either carbohydrates are present or no carbohydrates are present. Refer back to
Figure 1 and your Prelab assignment to help you with your conclusions.
a. Molisch Test
To a set of test tubes (step 2) add 10 drops of each solution.
Add 4 drops of Molisch reagent to each tube and mix well. Be careful not to cross contaminate solutions.
In the Fume Hood, tilt the test tube at an angle of about 45 degrees and very carefully and slowly pour 1mL
of concentrated sulfuric acid from a 10 mL graduated cylinder down the side of the test tube so that the sulfuric
acid forms a layer underneath the solution being tested.
NOTE: It is very important that the lip of the graduated cylinder be touching the inner top of the test
tube containing the carbohydrate and that the acid be poured slowly.
Set the test tubes in the rack and observe for evidence of reaction at the interface of the two liquid layers. A
purple coloured ring at the interface is a positive result. Record your results.
b. Benedict's Test
To a set of test tubes (step 2) add 10 drops of each solution.
Add 1 mL of Benedicts reagent to each test tube and mix well.
Place all tubes in the boiling water bath for a maximum of 3 minutes.
Observe the test tubes and record the results in the data sheets. As soon as a precipitate forms, indicating a
positive result, the test tube can be removed from the water bath and results recorded. Note the appearance and
colour of any precipitate. A colour change with no precipitate is a negative result.
c. Barfoed's Test
To a set of test tubes (step2) add 10 drops of each solution.
Add 1 mL of Barfoeds reagent to each test tube and mix well.
Page 55

Place the tubes in a beaker of boiling water. Note which samples give a precipitate in 5 minutes. Remove tubes
with the precipitate from the water, allow the precipate to settle and record the results. Leave the remaining
tubes (without the precipitate) in the boiling water for a total of 20 minutes. Again, record the results in this
tubes after 20 min and make a conclusions on the data sheets.
d. Iodine Test
For this test, set up five test tubes. To each test tube, add 10 drops of glucose, starch, sucrose, distilled water
and your unknown. Add 10 drops of iodine solution. A colour change to deep purple is considered to be a
positive test.

Experiment #6 Week 2
e. Bials Test
To a set of test tubes (step 2) add 10 drops of each solution. Add 1 mL of Bials reagent to each tube and mix
well. Heat in a boiling water bath just until a blue colour develops. Observe and record the results in the data
sheet.
f. Seliwanoff's Test
To a set of test tubes (step 2) add 10 drops of each solution. Add 1 mL of Seliwanoff's reagent to each tube and
mix well. Heat the tubes in a boiling bath for maximum 3 min until a red color develops. Record the results.
g. Hydrolysis Reactions
Perform the hydrolysis tests detailed below to determine if the test solutions are polymers and, if so, what are
the constituent monomers or monosaccharides.
Set up the following mixtures:
a. 10 drops of starch solution + 5 drops of concentrated HCl
b. 10 drops of sucrose solution + 5 drops of concentrated HCl
c. 10 drops of maltose solution + 5 drops of concentrated HCl
d. 10 drops of your unknown + 5 drops of concentrated HCl
e. 10 drops of distilled water + 5 drops of concentrated HCl
Heat the tubes in a boiling water bath for about 5 minutes. Cool the tubes under the stream of cold tab water.
Neutralize the solutions with 10% NaOH (use red litmus paper).
Withdraw 10 drops of the hydrolyzed starch and test with 10 drops of iodine as described in test (d). Note
differences in colour from that observed in test (d). Record the results on the data sheet. Do not test the other
solutions with iodine.
Repeat the Benedict test using 10 drops of sample of each of the hydrolysed solutions and 1 mL of each test
reagent, as described in procedure (b) above. Record your results on the data sheet. Include results from the first
Benedict test in the Pre-Hydrolysis column.

Page 56

Iodine test for starch before and after the hydrolysis (from left to right)

Benedict test for starch after and before the hydrolysis (from left to right)
Page 57

Experiment #6 Table 1. Test Results


Solution

Molisch

Conclusion

Benedicts

arabinose

fructose

glucose

maltose

galactose

lactose

starch

sucrose

xylose

control
(distilled
water)
unknown
#---------Page 58

Conclusion

Experiment #6 Table 2. Test Results


Solution

Barfoeds
(5 min.)

Barfoeds
(20 min.)

Conclusion

arabinose

Iodine

N/A

fructose

N/A

glucose

maltose

N/A

galactose

N/A

lactose

N/A

starch

sucrose

xylose

N/A

control
(distilled
water)
unknown
#---------Page 59

Conclusion

Experiment #6 Table 3. Test Results


Solution

Bials

Conclusion

Seliwanoff

arabinose

fructose

glucose

maltose

galactose

lactose

starch

sucrose

xylose

control
(distilled
water)
unknown
# ______
Page 60

Conclusion

Experiment #6 Table 4. Test Results


Solution

Iodine

Conclusion

Pre-Hydrolysis
Benedict
(from Table 1)

starch

sucrose

maltose

unknown

N/A

N/A

N/A

N/A

N/A

N/A

N/A

N/A

# ______
control
(distilled
water)

Page 61

Conclusion

Post-Hydrolysis
Benedict

Conclusion

POST-LAB QUESTIONS
1. You are given the following carbohydrates: arabinose, glucose, fructose, maltose, sucrose, and starch.
Explain the results described below.
(a) A carbohydrate solution (from the list above) gave a positive Molisch test and negative
Benedict's, Barfoed's, Bial's, and Seliwanoff's tests. When treated with hydrochloric acid and boiled
for several minutes, the solution showed positive Benedict's, Barfoed's, and Seliwanoffs tests and a
negative Bial's test. What carbohydrate was in the original solution?
(b) A solution (from the list above) containing only one carbohydrate gave a Cu2O precipitate with
Benedict's reagent. Which possible carbohydrates are present in the solution?
(c) Another sample of the solution (from Q1b) failed to give a Cu2O precipitate with Barfoed's
reagent. Which carbohydrate is present in the solution?
2. Describe how a solution of lactose would react toward these reagents.
(a) Benedict's
(b) Barfoed's
(c) Seliwanoff s
4. What functional groups(s) are present in the reducing carbohydrates?
5. Which of the carbohydrates shown on the picture below is s reducing sugar and why?
5. Why is sucrose a non-reducing sugar?
6. How can you tell when the hydrolyses of starch is complete? Why does the test work this way? What is
the monosaccharide that results at the end?

Page 62

Experiment #7
Action of Enzymes
BACKGROUND
Enzymes are a special group of proteins that act as catalysts for specific chemical reactions that take place in
cells of living organisms. There are over 1,000 different enzymes known. Most enzyme names end in ase.
Without enzymes, chemical reactions in cells would occur slowly and would require the input of
considerable heat or other forms of energy. By using the enzymes, cells are able to perform reactions at
great speed and at regular body temperatures. During the reaction, the enzyme is not changed.
Enzymes are very specific they act to speed up one particular chemical reaction by changing the alignment
of the reactants making it easier for them to come together. The chemical, or chemicals, that the enzyme acts
upon is known as its substrate. On the surface of the enzyme is an active site that the substrate fits into,
much like a key fits into a lock. The specificity of the enzymes is due to the geometrical relationship of the
substrate to the enzyme. The enzyme will only accept a molecule that has a complementary fit. There is
usually only one active site in each enzyme molecule.
When the substrate has combined with the enzyme, the enzyme molecule becomes deformed, placing a strain
on the geometry of the substrate molecule, making it easier for H+, OH- or specific functional groups of the
enzyme (remember they are proteins so they contain amino acids) to attack the substrate. The substrate is
converted to its products which then diffuse away from the active site. The enzyme returns to its normal
shape, combines with another substrate molecule and repeats the cycle. The substrate is held at the active
site by weak bonds for the split second required for the catalytic action to take place. In one minute, a single
enzyme molecule can carry out as many as several million catalytic cycles.
In this experiment, four different chemical reactions and the effect of their enzymes on the rate of reaction
will be studied. A brief description of each of these reactions is listed below.
Pepsin: Pepsin is an enzyme commonly found in gastric juices, that is, in the stomach. Protease or pepsin
catalyzes one of the chemical reactions that hydrolyse the peptide bonds in proteins. Enzyme produced in the
mucosal lining of the stomach that acts to degrade protein. Pepsin is one of three principal protein-degrading,
or proteolytic, enzymes found in the digestive system, the other two being chymotrypsin and trypsin. The
three enzymes were among the first to be isolated in crystalline form. During the process of digestion, these
enzymes, each of which is particularly effective in severing links between particular types of amino acids,
collaborate to break down dietary proteins to their components, i.e., peptides and amino acids, which can be
readily absorbed by the intestinal lining. In laboratory studies, pepsin is most efficient in cleaving bonds
involving the aromatic amino acids, phenylalanine, tryptophan, and tyrosine. Pepsin is synthesized in an
inactive form by the stomach lining; hydrochloric acid, also produced by the gastric mucosa, is necessary to
convert the inactive enzyme and to maintain the optimum acidity (pH 1-3) for pepsin function.
Amylase: Amylase is the enzyme that is found in saliva and it catalyzes the hydrolysis of complex
carbohydrates or polysaccharides to simpler sugars. Amylase hydrolyzes starch, glycogen, and dextrin to
form in all three cases glucose and maltose. Salivary amylase is also known as ptyalin; although humans
have this enzyme in their saliva, some mammals, such as horses, dogs, and cats, do not. Ptyalin begins
polysaccharide digestion in the mouth; the process is completed in the small intestine by the pancreatic
amylase, sometimes called amylopsin. The amylase of malt digests barley starch to the disaccharides that are
attacked by yeast in the fermentation process.
Antioxidant Enzymes-Catalase and Polyphenoloxydase (PPO): Catalase is an example of a
coenzyme.Fe2+ /Fe 3+ must be present for it to work. Catalase catalyzes the following reaction:
Page 63

Catalase is present in nearly all animal cells and organs and in aerobic microorganisms. It is found in high
concentrations in a compartment in cells called the peroxisome. Catalase catalyses conversion of hydrogen
peroxide, a powerful and potentially harmful oxidizing agent, to water and molecular oxygen. By preventing
excessive hydrogen peroxide build up, catalase allows important cellular processes which produce hydrogen
peroxide as a byproduct to take place safely. Catalase also uses hydrogen peroxide to oxidise toxins
including phenols, formic acid, formaldehyde and alcohols.
Polyphenoloxydase (PPO) is enzymes that were first discovered in mushrooms and are widely distributed in
nature. They appear to reside in the plastids and chloroplasts of plants, although freely existing in the
cytoplasm of senescing or ripening plants. In the presence of oxygen from air, the enzyme catalyzes the first
steps in the biochemical conversion of phenolics to produce quinones, which undergo further polymerization
to yield dark, insoluble polymers referred to as melanins. This process is known as a plant enzymatic
browning. These melanins form barriers and have antimicrobial properties which prevent the spread of
infection or bruising in plant tissues. Polyphenoloxidase also occurs in animals and is thought to increase
disease resistance in insects and crustaceans. Since it is also antioxidant, PPO will be used in this experiment
to illustrate the action of catalase.
Rennin: Rennin, also known as chymosin, is a proteolytic enzyme that is synthesized by cells in the
stomach and is consequently present in gastric or stomach juices. It helps break apart the protein casein in
milk and triggers the formation of another compound that bonds to the calcium in the milk, forming curds.
Its role in digestion of curdling or coagulating milk in the stomach is a process of considerable importance,
particularly in the young. If milk were not coagulated, it would rapidly flow through the stomach and its
proteins could not be digested in the stomach. Chymosin secretion is maximal during the first few days after
birth, and declines thereafter, replaced in effect by secretion of pepsin as the major gastric protease.
Preservatives: Preservatives are static agents used to inhibit the growth of microorganisms, most often in
foods. If eaten they should be nontoxic. Preservatives can be grouped into three general types: antimicrobials
that block growth of bacteria, molds or yeasts; antioxidants that slow oxidation of fats and lipids that leads to
rancidity, and a third type that fights enzymes that promote the natural ripening that occurs after fruits or
vegetables are picked. This experiment will focus on the third type of the preservatives. Examples are
calcium propionate, sodium benzoate, formaldehyde, nitrate, sulfur dioxide.
Antiseptics: Antiseptics are agents that kill microorganisms. They may denature microbial enzymes and
other proteins, usually by disrupting the hydrogen and disulfide bonds that give the enzyme its threedimensional functional shape. This blocks metabolism. They are harmless enough to be applied to the skin
and mucous membrane; should not be taken internally (not to be used in foods). Examples are mercurial,
silver nitrate, iodine solution, alcohols, detergents.
MATERIALS AND EQUIPMENTS:
Beaker 250mL
Starch solution
Saliva (provided by students)

Milk
1% rennin
Small potato or potato extract (see
Procedure)
3% hydrogen peroxide
Test tubes (~15 cm, x 12)
Test tube rack
Water bath at 38 oC

Benedicts Solution
Iodine solution
1% pepsin in 0.2% HCl
Jello (already prepared)
Page 64

PROCEDURE
A. Action of Pepsin
1. Place 1 mL of 2% pepsin (dissolved in 0.2% HCl) in each of two test tubes.
2. Heat one tube to boiling and continue to boil for 1 minute. Cool to room temperature.
3. Placed just a pinch of Jello in a each of second set of two test tubes
4. Add the unboiled 2% pepsin to the surface of the Jello in one test tube and the boiled 2% pepsin to
the other tube.
5. Observe the changes in the Jello in each test tube carefully for the next 2 minutes.
6. Observe the changes after 15 minutes.
Action of boiled and unboiled pepsin on jello
after 2min of reaction (from left to right)

B. Action of Amylase
1. Place 2 mL of 1% starch solution in each of two test tubes.
2. Heat one tube to boiling, add 1 mL of saliva (approximate), and continue boiling for 1 minute and
cool.
3. Add 1 mL of saliva to the unboiled tube.
4. Place both tubes in a water bath set at 38o C for 30 minutes.
5. Transfer the half of each solution in another test tube.
6. Test one half of each solution with 1 ml of Benedicts solution. Boil the tubes (see Exp#5)
7. Test the other half of each solution with 5 drops of Iodine solution (see Exp#5)

Benedict's test after action of boiled and unboiled


amylase on starch (from left to right)

Iodine test after action of boiled and unboiled


amylase on starch(from left to right)

Page 65

C. Action of Catalase (Note: Steps 1 and 2 are already done for you)
1. Pare a small potato, cut it into approximately 3 cm chunks and add to the blender. Add 100 mL of
distilled water and blend for 30 seconds on high. Let stand for 15 minutes.
2. Filter through a plug of glass wool in a powder funnel. Keep this P.P.O. (poly-phenol-oxidase)
enzyme solution in an ice bath until ready to use.
3. Place 1 mL of the filtrate in each of two test tubes.
4. Heat one tube to boiling and allow it to boil for 1 minute. Cool.
5. Add to each test tube 5 drops of 3% hydrogen peroxide. Note what happens
D. Effect of Preservatives and Antiseptics
1. Place 1 mL of 2% WARM milk in six test tubes
2. First test tube is considered as a positive controller.
3. Add 5 drops of the following solutions in remaining five test tubes
a. toluene
b. pectin
c. chloroform (CHCl3)
d. 5% phenol in water
e. Lab germicide
4. Add 1mL of 2% rennin solution to each of six test tubes
5. Shake gently and observe formation of clotting
6. If clotting does not happen in 5min place the tubes in the water bath at 38 oC for 1-2 min.
7. If clotting does not happen in 15min terminate the experiment
Experiment #7 Table 1. Action of Pepsin
Test Tube
After 0-2min
After 15 min

Conclusions

Boiled

Not Boiled

Experiment #7 Table 2. Action of Amylase


Test Tube
Appearance Benedicts
Before water Solution
bath
Boiled

Iodine
Solution

Not Boiled

Page 66

Conclusion

Experiment #7 Table 3. Action of Catalase


Test Tube Initial Appearance
With 3% Hydrogen
Peroxide
Boiled

Conclusion

Not
Boiled

Experiment #7 Table 4. Effect of Preservatives and Antiseptics


Solution
Appearance
Conclusion
Milk
Toluene
Pectin
Chloroform
(CHCl3)
5% phenol in
water
Lab Germicide

Page 67

POST-LAB QUESTIONS
1. At what pH dose pepsin work best?
2. Describe the changes to the Jello, comparing them to the expected changes.
3. What does starch give on hydrolysis by salivary amylase? What does starch give on complete
hydrolysis?
4. What were the results of the Iodine and Benedicts test? What is the reason for the difference?

5. The experiment with Amylase simulates the environment in the mouth. How would the absence of
the amylase in the mouth will affect the taste of pasta or breathe when they are chew in the mouth?
6. What is catalase do?

7. How do you explain the change in the tube with the active catalase?
8. Where is rennin found?
9. What reaction does rennin catalyze?
10. The enzyme of bacterium is optimal at 40oC. Would this enzyme be more active or less active at
normal body temperature or when a person has a fever?

11. Why does an enzyme lose activity at the high temperature?


12. Can an enzyme function at any pH? Why?
13. What is the difference between antiseptics and preservatives?

Page 68