BS Chemical Engineering -5TH YEAR Experiment: CARBOHYDRATES

Bonifacio,Rica Joy S. Date Peformed:

Catacutan,Kenneth
Leal,Catalina
Alambra, Karen
Date Submitted:

Experiment: CARBOHYDRATES

OBJECTIVES:

 a. the objectives is to know whether a specific substances contains sugar or not using
Molisch’s test
b. To determine a substance whether it is a non-reducing sugar or a reducing sugar using
Benedict’s test
c. To know whether a substance contains pentose sugar using Bial’s Orcinols test
d. To know whether a substance is ketohexose or aldohexose
e. To know whether a substance is monosaccharide, disaccharide or oligosaccharide
sing Barfoed’s test.
f. To know whether a substance contains an aldehyde groups using Tollen’s test.

DISCUSSION:

CARBOHYDRATES

Theory

Carbohydrates include polyhydroxy aldehydes or ketones and their by-products. They make up
most of our diet and serve as the major source of energy .The play an important part in metabolic
processes by furnishing the carbon chain in for compound synthesis by living organisms.
Carbohydrates can be categorize as monosaccharides, disaccharides and polysaccharides.
Monosaccharides or simple sugars are highly soluble in water,less soluble in ethanol and
insoluble in ether .They cannot further hydrolyzes to simpler units. They are either aldoses and
ketoses depending on the functional group present .They may also be classified into tetroses,
pentoses or hexoses depending on the number of carbon atoms they possess. Free
monosaccharides are all reducing sugars. They also exhibit mutarotation, which means they can
exists in 𝛼- and 𝛽- forms .Disaccharides are formed by two molecules of monosaccharides.
Example of disaccharides are maltose, which are abundant in germinain barley: sucrose, also
known as cane sugar or beet sugar : and lactose or milk sugar,which does not taste very sweet
and is not fermented by yeast. Most polysaccharides found in nature function either as structural
units, (e.g . cellulose) or for storage such as starch, dextrin, glycogen and inulin.
.1. Molisch test

The molisch test is the general test for carbohydrates. The sugars are mixed with 𝛼-napthol. The
test tube is inclined and concentrated 𝐻2 𝑆𝑂4 is added along the side of the tube ,Causing the
formation of a lower layer of acid. The concentrated sulfuric cid will dehydrate the sugar
allowing it to react with the alcohol forming furfural or hydroxymethyl furfural. Formation of a
purple ring at the interface of the two liquids will indicate the presence of a carbohydrate.

2.Bial’s Orcinol test

Bial’s test is used to determine the presence of pentoses and nucleotides that contain pentose
sugars. When pentoses are treated with orcinol, furfural are formed and they will yield a blue
green compound in the presence of ferric ions. The reaction is not specific for pentoses because
other compounds like trioses,uronic acids, and a certainn heptoses will also give blue or green
products. Hydroxymethyl furfural is formed from hexoses to give yellow-brown condensation
products.

3. Benedict’s test

Benedict’s test is a very sensitive test done under mildly alkaline conditions. The reagent
contains Cu𝑆𝑂 4 , 𝑁𝑎2 𝐶𝑂3 and sodium citrate. The formation of a brick red precipitate of 𝐶𝑢2 𝑂 is
considered positive. Most aldehydes have the ability to reduce Benedict’s reagent.Other
compounds like formic acid, hydrazobenzene, phenols, phenylhydrazine, pyrogallol, and uric
acid will also give a positive result in this test.

4. Barfoed’s test

Barfoed’s reagent contains cupric acetate in dilute acetic acid and is uded to distinguish between
monosaccharides, disaccharides and oligosaccharides. Barfoed’s reagent oxidizes
monosaccharides but not oligosaccharides .Disaccharides are less easily oxidized but are
oxidized if they undergo prolonged heating, causing hydrolysis of the disaccharides into
monsaccharides which will then give a positive result. The concentration of the sugar solutions
used in this test should be approximately the same, because the use of a more concentrated
disaccharide solution may give a faster reaction than that of a relatively more dilute
monosaccharide solution may give a faster reaction than of that of relatively more dilute
monosaccharide solution. Unlike Benedict’s test, Barfoed’s test is carried out under acidic rather
than basic medium.
5. Seliwanoff’s Test

Ths test is used to differentiate ketohexoses from aldohexoses. Ketosehexoses react faster with
the solution containing hydrochloric acid and resorcinol than aldohexoses. The dehydrated
ketohexoses fom a bright cherry red condensation product, whie the aldohexoses yield only a
pale pink coloration,a negative results. In this test, prolonged heating of samples should be
avoided.

6. Tollen’s test

Sugar with aldehyde groups are capable of reducing Tollen’s reagent (an ammonical solution of
𝐴𝑔+ ) to form a gray to black precipitate. If the rection vessel is clean and rate of deposition is
slow enough, the 𝐴𝑔+ deposit as a silver mirror.

MATERIALS

20 Test tubes Dropper

Test tube rack Glass slide with cover
Test tube holder Microscope
Test tube brush Sucrose solution
Aspirator and pipette 3% solutions of Coke Zero, Wine, Tea, Milk,
Beer, Honey and Wheat
Graduated cylinder Molisch reagent
Spatula Bial’s Orcinol reagent
Cotton balls Seliwanoff’s reagent
Watch glass Benedit’s reagent
Water bath Barfoed’s reagent
Tripod Phenylhydrazine (solid)
Bunsen burner Iodine solution
Wire gauze Concentrated 𝐻2 𝑆𝑂4
Spot plate Concentratred HCl
250 ml beaker

0.5 g of orcinol(3,5 dihydroxy 100 ml water,25 ml distilled

toluene)
250 ml of 30% HCl 60.5 g cupric acetate crystals
10% 𝐹𝑒𝐶𝑙3 distilled water 1 liter.
11.5 g resorcinol in a liter of 6.2 ml of glacial HAc
dilute HCl
73 g sodium citrate 1 drop of NaOH solution to 6
ml of 5% 𝐴𝑔𝑁𝑂3 . Add dilute
 𝑁𝐻4 𝑂𝐻 ( 1ml concentrated
𝑁𝐻4 𝑂𝐻+ 5ml of water )
100 g of anhydrous sodium
carbonate
800 ml of water
17.3 𝐶𝑢𝑆𝑂4

PROCEDURES

1. Molisch’s test -Knowing if the substances wine and soda water contains sugar. Sugar
solution and distilled water as a control.

a) Mix 4 ml of sugar solution and 2 drops of the Molisch reagent in a test tube. This
tube will serve as the control.
b) Place 4 ml of 3% solution of samples in a test tubes. Add 2 drops of the Molisch
reagent and mix the contents by gently shaking the test tube.
c) Incline the test tube and cautiously add about 5 ml of concentration sulfuric
acid,allowing the acid to run down the side of the tube. Sulfuric acid is denser
than water and will form a lower layer. Note the color of the ring formed at the
junction of the two liquids
d) In the same manner of adding acid, add sulfuric acid to the control tube. What do
you observe
e) Repeat the above test with 3% sample solutions on the other samples.
f) Record all results

Substance tested Results

Sugar Solution (control) Positive
Distilled Water (control) Negative
Wine Positive
Soda water Positive
2. Benedict’s test- To know whether a substance contains reducing sugar or not.
Non-reducing: No change in color
Traces of reducing sugar: green/yellow
Moderate: orange red
large amount of reducing sugar: brick red

a) Place 1 ml each of 3% solutions of each samples in separately-labeled test tubes. Add 5

ml of Benedict’s reagent in each test tube.(NOTE : prepare Benedict’s regent by
dissolving 173 g sodium citrate and 100 g of anhydrous sodium carbonate in 800 ml of
water with the aid if heat. Filter if necessary. Dissolve 17.3 𝐶𝑢𝑆𝑂4 in 100 ml water, then
add to the citrate solutionwith constant stirring. Make up to a volume of 1 liter.)
b) Place all the tubes in boiling water bath for 2 to 3 minutes
c) Observe the color the solution and note whether a precipitate was formed. A change in
color of the solution is not considered a positive reaction.
d) Avoid prolonged heating.
e) Record your results.

Substance tested Results

Sugar Solution Non-reducing
Beer Moderate
Energy Drink Large amount
Yakult Moderate
Flour Traces of reducing sugar
Honey unknown
3. Bial’s Orcinol Test- To know if the substance contains pentose sugar.

Positive: Blue color

Negative: Any color except blue

a. Place 1 ml each of 3% solution of each sample in separately-labeled test tubes.

b. Add 3 m of Bial’s reagent to each test tube.
(NOTE: Bial’s Orcinol reagent dissolce 0.5 g of orcinol(3,5 dihydroxy toluene) in 250 ml
of 30% HCl, to which 10- 15 drop of 10% 𝐹𝑒𝐶𝑙3solution has been added.
c. Carefully heat each over a Bunsen flame until the solution begins to boil. Add 1-2 drops
of 10% 𝐹𝑒𝐶𝑙3 solution.
d. Note the color of the product formed.
e. Record your results in the table below.

Substance tested Results

Sugar Solution Negative
Beer Negative
Energy Drink Negative
Yakult Negative
Flour Negative
Honey Negative
4. Seliwanoff’s test – To distinguish ketose sugars

Ketose: Cherry Red Color (Positive)

a. Place 1 ml each of 3% solution of each sample in separately-labeled test tubes.

b. Add 4 ml of Seliwanoff’s reagent to each test tube.( NOTE: Seliwanoff’s reagent dissolve
11.5 g resorcinol in a liter of dilute HCl( 1 part concentration HCL to 2 parts of water )
c. Place the tube in water filled with boiling water and allow them to stay there for exactly 1
minute.
d. Note the changes and record which test tube gives a positive results in the shortest time.
e. Continue heating and observe the color changes at 1 minute intervals

Record the time required for a positive test for each sample

Substance tested Results

Sugar Solution Positive
Beer Negative
Energy Drink Positive
Yakult Positive
Flour Negative
Honey Negative

5. Barfoed’s test – To distinguish the difference between monosaccharides and

disaccharides
Monossacharide: Brick red precipitate
Disaccharide: Blue precipitate

a. Place 1 ml each of 3% solution of each sample in separately-labeled test tubes.

b. Add 3 ml of Barfoed’s reagent in each test tube.(NOTE: Prepare Barfoed’ reagent by
dissolving 60.5 g cupric acetate crystals in enough distilled water to make 1 liter. Filter
and add 6.2 ml of glacial HAc mixed with 25 ml water.)
c. Prepare a control tube using distilled water instead of sugar solution.
d. Place all the tubes in a boiling water bath for 10 minutes. Record your observations.
Substance tested Results
Sugar Solution
Beer Disaccharide
Energy Drink unknown
Yakult Disaccharide
Flour Disaccharide
Honey Monosaccharide
6. Tollen’s test- determine the presence of aldehyde, aromatic aldehyde, and alpha-
hydroxy ketone functional groups
Positive- Silver mirror/ Gray to black

a) Place 5 drops of 3 % solutions of each sample in separately-labeled test tubes.

b) Add 2 ml of Tollen’s reagent into each tube. (Note: Prepare Tollen’s reagent by adding 1
drop of NaOH solution to 6 ml of 5% 𝐴𝑔𝑁𝑂3 . Add dilute 𝑁𝐻4 𝑂𝐻 (1ml concentrated
𝑁𝐻4 𝑂𝐻+ 5ml of water) until the brown precipitate of silver oxide that form just
dissolves. This reagent must be prepared fresh and not sored since it decomposes when
left standing and yields and explosive decomposition product. Distract all left- over
materials
c) Boil for about 5 minutes. Note and record your observations.

Substance tested Results

Sugar Solution Positive
Beer Negative
Energy Drink Negative
Yakult Positive
Flour Negative
Honey Positive