HALIC UNIVERSITY

DEPARTMENT OF MOLECULAR BIOLOGY AND GENETICS

MBG102 GENERAL BIOLOGY

LABORATORY MANUAL

2024-2025

Course Instructor

Assist. Prof. Deniz KANCA DEMİRCİ

BIOCHEMICAL EXPERIMENTS - Detection of Carbohydrates, Proteins and Fats

Laboratory Assistants

Res. Asst. Hatice KURNAZ

Res. Asst. Şafak ŞENER

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 DETECTION OF CARBOHYDRATES, PROTEINS AND FATS

Part 1: Detection of Carbohydrates

Carbohydrates, or saccharides, are organic compounds containing one or more aldehyde or

ketone groups and multiple hydroxyl groups. Saccharide: Greek for saccharon (sugar)

Polyhydroxy alcohols with active aldehyde or keto groups and organic substances that give
compounds with these properties when hydrolyzed are called carbohydrates.

Some of them also have N, P and S in their structure.

They are generally sweet and are called sugar (-ose):

1. Monosaccharides

2. Disaccharides and oligosaccharides

3. Polysaccharides

General formula: Cn(H2)nOn or Cn(H2O)n or C6H12O6

Classification of Saccharides;

 Monosaccharide: Simple sugars, containing a single polyhydroxy aldehyde or ketone.

Eg. D-glucose (dextrose)

 Oligosaccharide: Short chains in which monosaccharide units or residues are joined by

characteristic linkages called glycosidic bonds.

Eg. sucrose (table sugar) = D-glucose + D-fructose

 Polysaccharide: Consists of hundreds of monosaccharides.

Eg. cellulose, glycogen, starch

Carbon and oxygen (C=O) connected to each other with a double bond are called carbonyl
groups. The carbonyl group forms the framework of functional groups such as aldehyde, ketone,
carboxylic acid, ester, amide, etc.

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Carbohydrates contain either an aldehyde or ketone carbonyl group in their structures; and two
or more hydroxyl groups in both the primary alcohol structure and the secondary alcohol
structure.

Depending on the carbonyl group, compounds with the structure R-CHO carrying one hydrogen
and one radical are called aldehydes, and compounds with the structure R-CO-R carrying two
radicals are called ketones.

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Carbohydrates are resistant to dilute acids, but strong acids break the glycoside bond in
carbohydrates and produce monosaccharides. Monosaccharides are dehydrated in acidic
environment to form furfural and its derivatives.

The colour reactions of carbohydrates are based on the ability of furfural and its derivatives to
give coloured complexes with phenolic substances such as naphthol, resorcin, orsin.

 Monosaccharides can be oxidized by relatively mild oxidizing agents such as Iron and
Copper.

 Glucose and other sugars that can reduce Fe+3 and Cu+ ions are called reducing sugars.

 The end of a chain with a free anomeric carbon (not participating in a glycosidic bond)
is known as the reducing end.

 This property is the basis of the Fehling reaction, a qualitative test for the presence of
a reducing sugar.

Exercise 1. Fehling Test

Fehling test has been used for many years to detect and measure blood and urine glucose levels
in the diagnosis of diabetes. In diabetes and pregnancy, glucose can be observed in the urine
due to the normal glucose threshold being exceeded in the kidney. In order to understand
whether there is sugar in the urine, tests based on the reducing properties of carbohydrates are
applied. This method uses the reducing properties of glucose in a hot and alkaline environment.

However, Fehling test is not specific because it gives positive results with carbohydrates such
as lactose, fructose, galactose, pentose, and other substances with reducing properties such as

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uric acid, creatinine, and glutathione, in addition to glucose. Today, more precise measurements
are made using the glucose oxidase enzyme.

Solutions:
 Fehling I solution
 Fehling II solution
 Glucose, Sucrose solutions (%1)
Method:

1. Equal amounts of Fehling I and Fehling II solution are added to a test tube and shaken.
2. A dark blue coloured solution is formed. This mixture of two solutions is called
‘Fehling's reagent’. It is boiled in open flame. If the colour does not change, the reagent
is clean. It means that there are no reducing substances in it.

Principle of the experiment: The copper (two) hydroxide formed is blue in colour. It
precipitates in alkaline medium. This precipitation is prevented by the tartrate salt in the Fehling
II reagent. Thus, the copper remaining dissolved can be reduced by sugar.

When both reagents are mixed, the following reaction occurs:

1. After this check, the sample to be analysed (e.g. urine) is added drop by drop and the
boiling is continued.

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 2. In the presence of excess sugar, yellow turbidity or orange-red precipitate is observed.
3. If the sugar is in small amounts, add an amount of sample equal to the volume of
Fehling's reagent.
4. If a yellow or red precipitate forms, sugar is present.
5. If no precipitate is formed, it is certain that there is no sugar in the analysed sample.

Exercise 2: Molisch Testi

It is a general test for carbohydrates. It can be used for all carbohydrates except amino sugars,
sugar alcohols and carboxylic acids.

Solutions:

 Molisch reagent (α-naphthol reagent): A 5% solution of alpha-naphthol in alcohol

should be freshly prepared.
 Glucose, lactose, glycine solutions (1%), concentrated H2SO4

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Principle of the experiment: Furfural and its derivatives formed in acidic medium form a
coloured complex with α-naphthol.

Method:

1. Add 1-2 drops of Molisch reagent (alternative: 1ml α-naphthol reagent to 3ml sugar
solution) to 2 mL of fruit juice and mix. Add this solution to 2 mL of concentrated
H2SO4 in a tube (inclined at 30°) and wait without stirring.
2. In the presence of carbohydrate, a colour change (red-violet ring) is observed at the
junction of the two solutions.

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Exercise 3: Benedict Test

The Benedict test is used to determine whether the solution known to be carbohydrate is
reducing or not. With this experiment, the presence of sugar in urine can be determined and its
concentration can be determined semiquantitatively.

Solutions:

 Qualitative Benedict reagent: 17.3 g sodium citrate, 10 g anhydrous sodium carbonate

(Na2CO3) and 1.73 g copper sulphate (CuSO4.5H2O) dissolved in 100 mL distilled
water.

Principle of the experiment: Carbohydrates carrying free aldehyde or ketone group (carboxyl
group) reduce copper ions from +2 to +l in alkaline medium with the effect of heat and a colour
change is observed. Since all monosaccharides and some disaccharides have free carbonyl
group, they give positive results in this experiment. When reducing sugars are heated with
Benedict reagent, they give different colour reactions depending on the type and amount of
sugar.

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Method:

1. Grate the onion and potato separately and squeeze the water into tubes.
2. The steps given in the table below are applied respectively.

Tube 1 Tube 2 Tube 3 Tube 4 Tube 5 Tube 6

0,5 ml water 0,5 ml onion 0,5 ml potato 0,5 ml glucose 0,5 ml sucrose 0,5 ml starch
water water
1,5 ml 1,5 ml 1,5 ml 1,5 ml 1,5 ml 1,5 ml
Benedict Benedict Benedict Benedict Benedict Benedict
Boil for 5 min. and the colour changes are interpreted

Exercise 4: Methylene Blue Test

Solutions:

 Methylene Blue (%0,1)

 2 N NaOH
 Glucose solutions (%2)

Principle of the experiment: When monosaccharides with free aldehyde or ketone group and
carbohydrates with semi-acetal or semi-ketal group are heated in basic medium, they are
oxidised to form carboxylic acids and reduce the methylene blue in the medium, causing the

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blue colour to disappear. The disappearance of the blue colour indicates that the carbohydrate
in the solution is oxidised.

Method:

1. 5 mL of water, 1 drop of methylene blue (0.1%) and 2 drops of 2 N NaOH are added to
the test tube.
2. After heating the solution in a water bath, 4-5 drops of glucose solution (2%) is added.
The blue colour of the solution disappears at the end of heating. However, if the tube is
shaken, the blue colour reappears.
3. The same experiment is repeated with tea sugar.

Exercise 5: Moore Test

Under the influence of intense alkalis, sugars polymerise and also form reductants. If NaOH
solution is added to the glucose solution and heated, the colour of the mixture changes first to
yellow and then to brown as a result of the effects mentioned above and a caramel smell can be
smelled.

Materials:

1. Bunzen beaker
2. Glucose solution
3. NaOH solution
4. Tong
5. Test tube
6. Pipette

Method:
1. Take a test tube, add 2 ml of glucose solution and 1 ml of NaOH.
2. Keeping the test tube tilted and stirring, it is heated in the flame of a flame burner.
3. If the colour of the mixture changes and a butterscotch smell appears, the experiment
has given a positive result.

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