Carbohydrates

Carbohydrates also called saccharides (Greek word-Sakchron- sugar). Carbohydrates are

aldehyde or ketone derivatives of higher polyhydroxy alcohols and their anhydrides. General

formula is (CH2O)N. N is no number more then 3.

Classification

Depending upon the monomeric units carbohydrates are classified as

I. Monosaccharides- Monosaccharide are simple sugars Which join in several ways to form

Di, Oligo, poly saccharides. According to the chemical nature of carbonyl group these are

futher subdivided as
 1. Aldoses- A sugar is called aldoses when it has aldehyde group on carbon 1e.g.

Glucose

2. Ketoses- A sugar is called Ketoses when it has ketone group on carbon 2 e.g.

Fructose

According to the number of cabon atoms present monosaccharides are again subdivided

as

1. Trioses- It is smallest unit of monosaccharides e.g. Glyceraldehyde

2. Tetroses-It has four carbonising. E.g. Erythrulose

3. Pentoses- It has five carbonese.g. Ribose, Xylose

4. Hexoses- It has 6 carbon e. g. Glocose

II.Disaccharides- These consist of two similar or dis similar monosaccharides which are

linked by glycosidic bond. eg. Sucrose, maltose. Disaccharides are sub divided as

1. Reducing sugar- These saccharides reduce the Fehling reagent and Tollen’s reagent

2. Nonreducing sugars- These saccharides do not reduce the Fehling reagent and

Tollen’s reagent

Structural features of Reducing sugars

They contain α-Hydroxy aldehyde or α-Hydroxy Ketone functions.

 They contain cyclic hemiacetal or hemi ketal structures in equilibrium with open chainform

having CHO OR C=O group as the case may be. Such sugars, Therefore, reduce Fehling’s and

Tollen’s reagent.

Structural features of Nonreducing sugars

They do not contain free aldehyde or ketone group with OH on the carbon adjacent to the

carbonyl group. They contain acetal and ketal structure which are stable. Their cyclic

structures cannot be open opened in an open -chain form having free carbonyl group.

Thus they are unstable to reduce Fehling and Tollen’s reagent

III.Oligosaccharides- These are the carbohydrates which are build by linking more then two

monosaccharides e.g. maltotriose, Raffinose

IV.Poly saccharides-They are also called glycans, they contain large number of

monosaccharides which are linked together by glycosidic bond.

a) Homo poly saccharides-

b) Heteropolysaccharides-
Properties of carbohydrates-

Carbohydrates are organic compounds of carbon, hydrogen and oxygen. Due to presence of

hydrogen and oxygen they have reducing property. All monosaccharides and some of the

disaccharides participate in oxidation -reduction reaction.

Monosaccharides can be oxidized by mild oxidizing agent such as ferric or cupric ions

1. Oxidation of the carbonyl carbon( aldehyde) of an aldose to carbonyl group produce

aldonic acid such as D- Gluconic acid is formed by Glucose

2. Oxidation of the Carbon 6 forms Uronic acid such as D- glucuronic acid is formed by

glucose.

3. Reduction of Monosaccharides under mild conditions produces cyclic polyhydroxy

alcohols called Alditols eg. Ribitol formed from ribose

Importance of Carbohydrates- Carbohydrate perform numerous functions in living

organisms

1. Poly saccharides serve as the storage form of energy and as structural component.

2. The 5 -carbon monosaccharides in important component of coenzymes. Ribose is back

bone of RNA Deoxyribose is component of DNA

3. Saccharides and their derivatives play important role in the immune system, fertilization,

preventing pathogenesis, blood clotting and development.

4. Carbohydrates (Galactose) is component of milk as lactose. It is found in galactolipids (

in plant membrane) and as glycoprotein ( in many tissues in men and animals).

5. Fructose or fruit sugar, in component of sucrose, It is found in many plants. In human it is

absorbed in intestine and is found in semen.

To estimate the amount of glucose in the whole of the given solution.

Gluucose:
Glucose is a very important monosaccharide in biology. It is one of the major products of

photosynthesis. The living cell uses it as a source of energy and metabolic intermediate. The

name "Gluc" comes from the Greek word "glykys", which means "sweet", plus the suffix "-

ose" which denotes a sugar. Two stereoisomers of the aldohexose sugars are known as

glucose, only one of which (D-glucose) is biologically active. This form (D-glucose) is often

referred to as dextrose monohydrate, or, especially in the food industry, simply dextrose

(from dextrorotatory glucose) .

Fehling's solution:

Fehling's solution is a chemical test used to differentiate between water-soluble aldehyde and

ketone functional groups, and as a test for monosaccharides. The test was developed by

German Chemist Hermann von Fehling in 1849.

Laboratory Preparation:

Fehling's solution is always prepared fresh in the laboratory. It is made initially as two

separate solutions, known as Fehling's A and Fehling's B. Fehling's A is a blue aqueous

solution of copper (II) sulphate pentahydrate crystals, while Fehling's B is a clear solution

of aqueous potassium sodium tartrate (also known as Rochelle salt) and a strong alkali

(commonly sodium hydroxide).

Equal volumes of the two mixtures are mixed together to get the final Fehling's solution,

which is a deep blue colour. In this final mixture, aqueous tartrate ions from the dissolved

Rochelle salt chelate to Cu2+ (aq) ions from the dissolved copper sulphate crystals, as

bidentate ligands giving the bistartratocuprate(II) complex as shown below.

Uses of Fehling's solution:

Fehling's solution can be used to determine whether a carbonyl-containing compound is an

aldehyde or a ketone. The bistartratocuprate (II) complex in Fehling's solution is an oxidizing

agent and the active reagent in the test. The compound to be tested is added to the Fehling's
solution and the mixture is heated. Aldehydes are oxidized, giving a positive result, but

ketones do not react, unless they are alpha-hydroxy-ketones. The bistartratocuprate (II)

complex oxidizes the aldehyde to a carboxylate anion, and in this process the copper (II) ions

of the complex are reduced to copper (I) ions. Red copper (I) oxide then precipitates out of

the reaction mixture, which indicates a positive result. That is redox reaction takes place. A

negative result is the absence of the red precipitate; it is important to note that Fehling's will

not work with aromatic aldehydes; in this case Tollens' reagent should be used.

Fehling's test can be used as a generic test for monosaccharides. It will give a positive result

for aldose monosaccharides (due to the oxidisable aldehyde group) but also for ketose

monosaccharides, as they are converted to aldose by the base in the reagent, and then give a

positive result. For this reason, Fehling's reagent is sometimes referred to as a general test for

monosaccharides.

Fehling's can be used to screen for glucose in urine, thus detecting diabetes. Another use is in

conversion / breakdown of starch to glucose syrup and maltodextrins, to measure the amount

of reducing sugars and calculating the dextrose equivalent (DE) of the starch sugar.

Methylene Blue:

Methylene blue is a heterocyclic aromatic chemical compound with the molecular formula

C16H18N3SCl. It has many uses in different fields, such as biology and chemistry. At room

temperature it appears as a dark green odourless solid powder, which yields a blue solution

when dissolved in water.The International Nonproprietary Name (INN) of methylene blue

is methylthioninium chloride.

Methylene Blue as Redox indicator:

Methylene blue is widely used as redox indicator in analytical chemistry. In an oxidizing

environment the solutions of this substance are blue, if exposed to a reducing agent it will

turn colourless. The redox properties can be seen in a classical demonstration of chemical
kinetics in general chemistry, the "blue bottle" experiment. Typically, a solution is made of

dextrose, methylene blue, and sodium hydroxide. Upon shaking the bottle, oxygen oxidizes

methylene blue, and the solution turns blue. The dextrose will gradually reduce the methylene

blue to its colourless, reduced form. Hence, when the dissolved oxygen is entirely consumed,

the solution will turn colourless.

Theory of Estimation of Glucose:

A freshly prepared Fehling’s solution is first standardized by titration against a standard

solution of pure glucose A.R. The standardized Fehling’s solution is then used to determine

the amount of glucose in an unknown sample or solution by direct titration.

The Fehling’s solution being a solution of cupric ions is blue in colour and at the end point

changes to red colour precipitate of cuprous oxide. As the supernatant liquid is blue and the

precipitate is red in colour, there may be some difficulty in determination of end point

accurately. Hence sometimes a methylene-blue indicator is employed for accurate

determination of the end point.

C6H12O6 + 2CuO → C6H11O5.COOH + Cu2O

Glucose Cupric Oxide Gluconic Acid Cuprous oxide
(Fehling’s solution)

Standardisation of Fehling’s Solution:

Prepare a solution (known standard solution) of glucose AR by weighing accurately 1.25gm

and dissolving it in 250 mL standard flask in water. Make up the volume to the mark.

Pipette out 20 mL each of Fehling’s A & B in a dry conical flask and shake thoroughly.

Pipette out 20 mL of this freshly mixed Fehling’s solution in a clean conical flask and dilute

it with 20 mL water. Heat the solution up to 70° over wire gauze. Take the standard solution

of glucose prepared in a burette and run this solution slowly into the boiling Fehling’s

solution until the blue colour has completely disappeared. Take care to maintain this

temperature for every addition of glucose solution. Repeat the above titration by running the
glucose solution steadily into the boiling Fehling’s solution until the end point is approached

and then cautiously add glucose solution drop-by-drop till the end point is reached.

Alternatively, to detect the end point more accurately, 5-6 drops of methylene- blue indicator

may be added to the Fehling’s solution and then glucose solution added drop by drop.

However, if methylene-blue is used as indicator the Fehling’s solution should not boil for

more than 2-3 minutes at a stretch. The end-point here also is marked by the disappearance of

the blue colour.

Simulator Procedure:

1. Choose the titrant.

2. Choose the titrate.
3. Select the normality of the titrate.
4. Select the volume of the titrate.
5. Start titration.
6. When the blue colour just fades Select the indicator.
7. Continue the titration.
8. End point is noted at the colour change of the solution.
9. From the final reading the normality of titrant can be calculated by the equation:

1. After finding the normality, the amount of substance in the whole of the given
solution can be calculated by the equation:

▪ Note: 10 times dilute the stoke solution and that is used as titrant.
▪ Atomic weight of Glucose=180.1559 g/mol.

Observations and Calculations:

S. Volume of Burette reading Volume of titrant

no titrate used
Initial Final
1
2

Normality of Titrate used, N1=.............N.

Volume of Titrate used, V1 =..............mL.

Volume of Titrant Used, V2 =..............mL.

Therefore, the Normality of Titrant N2= =................N.

The amount of substance in the whole of the given solution =

=.......................g.

Result:

• The amount of substance in the whole of the given solution =..............g.

Points to Remember while Performing the Experiment in a Real Laboratory:

1. Always wear lab coat and gloves when you are in the lab. When you enter the lab,

switch on the exhaust fan and make sure that all the chemicals and reagents required

for the experiment are available. If it is not available, prepare the reagents using the

components for reagent preparation.

2. Properly adjust the flame of the Bunsen burner. The proper flame is a small blue cone;

it is not a large plume, nor is it orange.

3. Make sure to clean all your working apparatus with chromic acid and distilled water

and ensure that all the apparatus are free from water droplets while performing the

experiment.

4. Make sure to calibrate the electronic weigh balance before taking the measurements.

5. Clean all glass wares with soap and distilled water. Once the experiment completed

recap the reagent bottles. Switch off the light, exhaust fan and Gas cylinder before

leaving the lab.

6. Discard the used gloves in a waste Normal values of blood glucose:

Normal fasting level = 60-90mg/100ml of blood

Glucose level half an hour (after meal) (post prandial) = 120-150mg/100ml of blood
In normal healthy individuals the peak glucose level (at any time of the day) = 60-110

mg/100ml of blood is considered normal.

Clinical significance of blood sugar level:

Blood glucose level increases in diabetes mellitus, acute stress, hyperthyroidism and chronic

liver disease.

Blood glucose level decreases in Addison’s disease, hypothyroidism and cancer of the

pancreas.

The increase in the blood glucose level is called hyperglycemia and decrease in blood glucose

level as hypoglycemia.

People suffering from diabetes mellitus need to get their blood glucose tested frequently.

Methods used to measure blood glucose level

Although a number of methods are used for glucose determination, commonly used two

methods are discussed here.These can be grouped into two categories- chemical and

enzymatic.

▪ Chemical method

▪ Folin-Wu method

▪ Ortho-Toluidine method

▪ Enzymatic method

▪ GOD-POD method. (Glucose oxidase method)

Chemical method to estimate blood glucose:

Folin-Wu method:

• It is based on the principle that glucose when heated with an alkaline copper solution,

reduces cupric ions to cuprous ions.

 • The cuprous ions are then measured photometrically (colorimetrically) by adding

phosphomolybdic acid which gets reduced to molybdenum blue.

• In this method, whole blood is used and the blood glucose value is determined by the

intensity of blue color.

Ortho-Toluidine method:

• This is an ideal manual method used for its rapidity, sensitivity, accuracy, and relative

simplicity.

• It is based on the principle that the aldose sugar i.e. glucose on condensation with

ortho-toluidine in glacial acetic acid gives a green colour that can be measured

spectrophotometrically.

Procedure: O-toluidine method is performed on plasma or serum.

• To a trichloroacetic acid filtrate of blood add O-toluidine dissolved in glacial acetic

acid.

• The mixture is heated to 100oC for about 10 minutes.

• The mixture gives a stable green color.

• Measure the density photometrically.

Enzymatic method- GOD-POD method to estimate blood glucose:

Enzymatic methods provide maximum degree of glucose specificity, hence are very good in

estimating true blood glucose.For this method, only blood plasma or serum is used. The

glucose remains stable for 24 hours at 2-8oC if serum or plasma is prepared within 30 minutes

after collection. The enzyme peroxidase catalyzes the following reaction. The hydrogen

peroxide formed reacts with phenol and 4 amino-phenazone to a red-violet dye as indicator.

The intensity of the color formed is measured colorimetrically (or spectrophotometrically)

which is directly proportional to the blood glucose level.

 • Glucose + O2 + H2O ——(glucose oxiadase)———–> Gluconic acid + H2O2

• H2O2 + phenol + 4 aminophenazone ——(Hydrogen peroxidase)—–> Quinoneimine

+ 4H2O

This test is not influenced by the pressure of uric acid, ascorbic acid, anticoagulants or

bilirubin in blood.

Enzyme reagent:

It is ready for use reagent that consists of-glucose oxidase, peroxidase, phenol, 4-amino

phenazone phosphate buffer and stabilize.

Standard:

It is also ready, for use and consists of glucose conc. 100mg/100ml.

Procedure to estimate blood glucose:

• Take 3 test-tubes and mark them as blank, sample and standard. Add the following

contents:

• Blank – 2ml of water + 2ml of enzyme reagent

• Test or sample – 2ml of sample + 2ml of enzyme reagent

• Standard- 2ml of standard + 2ml of enzyme reagent

Mix well and incubate all test-tubes for 10 minutes at 20-25oC. Measure the absorbance of

the standard and the sample against the reagent blank at 500-546onm colorimetrically.