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 medium5. 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

Result:
SUMMARY OF RESULTS:

For the summary of results, using distilled water and sugar solution as a control, the wine

and produced a purple ring during the experiment which indicates a positive result.

CONCLUSION:

We conclude that the wine and soda water that is said to be contains zero carbohydrates still

contains sugar. The sugar on the samples get dehydrated by the sulfuric acid to form furfural

products. Those products reacts with sulphonated a-naphthol to give a purple colored complex.

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.

Result:

Substance tested Results

Sugar Solution Non-reducing

Beer Moderate

Energy Drink Large amount

Yakult Moderate

Flour Traces of reducing

sugar

Honey unknown
Before After

SUMMARY OF RESULTS:

For the summary of results, the hue of the solution will identifies reducing sugar. After boiling in

the bath sugar solution color stays unchanged presence of reducing sugar is absent. Yakult and

beer has a complexion yellow-orange that indicates the moderate concentration of reducing sugar

present in the solution. The energy drink has a brick red has a large amount of reducing sugar

while the flour has trace of reducing sugar as it turned into green.

CONCLUSION:

We conclude that Benedict’s reagent starts out blue. As it is heated in the presence of reducing

sugars, it turns yellow to orange. The “hotter” the final color of the reagent, the higher the

concentration of reducing sugar. In general, blue to blue green or yellow green is negative,

yelllowish to bright yellow is a moderate positive and bright orange is very strong positive.
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.

Result:

Substance tested Results

Arabinose (control) Positive

Beer Negative

Energy Drink Negative

Yakult Negative

Flour Negative

Honey Negative

Sucrose Negative
Before :

Arabinose (control) samples

After:
SUMMARY OF RESULTS:

For the summary of results, the yakult,beer,energy drink,flour and honey didn’t produced a

blue-colored product that indicates a negative result while xylitol solution produced a blue-colored

product that indicates a positive result.

CONCLUSION:

We conclude that arabinose contains pentose sugar, a five carbon sugar. Arabinose form

furfural in acidic medium which condense with orcinol in presence of ferric ion to give blue green

colored complex.

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

Before After

SUMMARY OF RESULTS:

For the summary of results, the sucrose, yakult and energy drink change into a red complexion

that indicates the ketose sugar is present in the solution while the flour , honey and beer formed other

color means it is a negative. The flour and honey has a hue of light pink indicates the solution contains

aldoses sugar.
CONCLUSION:

We conclude that Seliwanoff ’s reagent when added to a solution containing ketoses a red color

is formed rapidly indicating a positive test. When added to a solution containing aldoses, a slower forming

light pink is observed instead. This test relies on the principles that, when heated ketoses are more rapidly

dehydrated than aldoses.

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.
Result:

Substance tested Results

Sugar Solution

Beer Disaccharide

Energy Drink Unknown

Yakult Disaccharide

Flour Disaccharide

Honey Monosaccharide
SUMMARY OF RESULTS:

For the summary of results, the beer, yakult and flour has a blue complexion identifies

disaccharides while the honey has a brick red color that indicates the presence of monosaccharides.

CONCLUSION:

We conclude when Barfoed’s reagent ix with the solutions and heated in boiling water

bath, they react and crystal precipitate is formed. Copper acetate which is present in the barfoed

reagent convert to copper oxide and give brick red precipitate when react with monosaccharide.

Disaccharides may also react, but the recation is much lower.

6. Tollen’s test- To know whether a substance contains an aldehyde groups using Tollen’s test.

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.

Result:

Substance tested Results

Sugar Solution Positive

Energy Drink Negative

Yakult Positive

Flour Negative

Honey Positive

Positive- Silver mirror/ Gray to black

Before After
SUMMARY OF RESULTS:

For the summary of results. The sugar solution, honey and flour change into a gray

complexion that indicates the a positive test while the flour, energy drink and beer formed other

color means it is a negative

CONCLUSION:

We conclude that sugar solution , honey and flour turns into gray because it has a presence

of aldehyde, aromatic aldehyde, and alpha-hydroxy ketone functional groups .Tollen’s reagent into

metallic silver which if the test is carried out into a clean glass test tube,forms a mirror on the test

tube. But if not resulted into a gray color.