PRELUDE

Most abundant class of organic compounds found in living organism.

They fill numerous roles in livings,such as storage ,transport of

energy & stuctural components, immune system, fertilisation.

. Classification

monosaccharides ex- glucose,galactose,frutose. simple

disaccharides ex- sucrose,lactose, maltose.

n CO2   +   n H2O   +   energy     CnH2nOn   +   n O2

oligosaccharides ex- raffinose,stachyose.

complex

polysaccharides ex – cellulose,starch,callose,lamarin

General formula – (CH 2 O )n

SIZES–

Trioses C 3 sugars

Tetroses C4sugars

Pentoses C5 sugars

Hexoses C6sugars

stuctures

.

sucrose

starch cellulose

Evaluation methods

Chemical methods

Titrimetrical method

Gravimetrical method

Enzymatic method

Physical methods

Spectrophotometry methods

Chromatography methods

miscellaneous

Chemical methods

Fehling’s test

Benedict’s test

Barfoed’s test

Molisch’s test

Bial’s test

Seliwanoff’s test

Iodine test

Fehling’s test

Barfoed’s test

Molish’s test

Dehydrate pentoses furfural

Bials test

Seliwanoff’s test

ketohexoses 5-hydroxymethylfurfural

Benedict’s test

Iodine test

QUALITATIVE TESTS

Test solution

Reducing sugars Non-reducing sugars

monosaccharides Disaccharides

positive Negative

ketose Other hexoses

Bial’s test

Seliwonoff’s test

Iodine test

BlueStarch

Brownglycogen

No changeInsulin

TITRIMETRY METHOD

EDTA has been used in place of fehling’s & benedict’s reagents to

prevent precipitation of cupric ions in alkaline solution.

Cupric ions form a relatively stable chelation complex with EDTA.

Ferrous ions are detremined by permanganate titrimetry.

Similar approach applied by HAGEDORN-JENSEN

PROCEDURE—reaction of reducing carbohydrates with ferricyanide.

Continution….

Aldehyde function can be deternined by selective oxidation with

hypoiodous acid.

To detremine aldoses– cyanohydrin reaction is done.

To determine polysaccharides– neutralisation of negatively charged

colloidal particles with positively charged ones & relies on

metachromatic dyes.

Limitations.

Results depend on the precise reaction times.temp & reagent

concentration.

It cannot distinguish b/n different types of reducing sugars.

It cannot directly determine the concentration of non reducing

sugars.

It is susceptable to interference from other types of molecules that act as reducing agents.

Gravimetric method

The Menson & walker method –determine the conc of reducing sugars in a sample.

Carbohydrates are oxidised & an excess copper sulfate & alkaline tartarate leads to formation of cuprous oxide ppt

The amount is proportional to conc in the sample.

Merits Accurate

More reproducible.

Enzymatic method

Analytical method based on enzyme rely on their ability to catalyse specific reaction.

Rapid ,highly specific & sensitive to low conc.

Two methods---

1.Allowing the reaction to go to completion & measure concentration of the product.

2. measuring the intial rate of the enzyme catalysed reaction.

Polarimetry

Molecules that contain asymmetric carbon atom have the ability to

rotate the plane polarised light.

The extent of polarisation is related to concentration of the optically

active molecules in sol by equation

a= [a]/c where a=measured angle of rotation [a] = optical activity l= pathlength ,c =589.3nm.

The conc of unknown is determined by measuring its angle of of rotation.

a= [a]lc

Refractive index

Determined by angle of incidence & angle of refraction at a boundary b/n angle & another material of known RI.

Snell’s law = sin(i)sin(r) = n2/n1

Measurements are made at specific temp (20) & λ (589.3nm).

Quick & simple

Determine sugar conc of syrups, honey,molases,tomato products & jams.

n= c/cm

Density

d= m/v The density of aq solution increases as the concentration

increases.

Thus the carbohydrate concentration can be determined

by measuring density.

Routinely used in industry for determination of

carbohydrates concentration of juices & beverages.

Anthrone method

Principle :

carbohydrates + conc H2 SO4

DEHYDRATION

hydroxy methylfurfural+anthrone

CONDENSATION

blue complex (colorimetrically at 620nm)

Procedure

1.Anthrone reagent(0.2 % in conc H 2SO4). 2. std glucose solution (10mg/100ml)

diff volumes of glucose sol

make up to 1ml(water)

4ml of anthrone reagent –mix well

water bath-cool

measure optical density at 620nm

Benedict’s method

This method is value in clinical analysis of glucose in blood & urine.

1. Benedicts sol+ sodium carbonate(conical flask)

heat to boil.

2. Std solution is taken in burette.

On titration white bulky ppt is formed (cuprousthiocyanate)

Note the vol of sugar solution required.

Determination of reducing sugars using 3-5-dinitrosalcylic acid.

Principle This reagent is employed to assay the sugars by using their

reducing properties.

Reagent in alkaline solution is reduced to 3-amino-5-nitrosalicylic acid.

coo- COO-

OH reduction OH

NO2 NO 2 NO 2

NH2

OH

Procedure

Prepare the DNS reagent just before use. 1ml of the reagent to 3ml of sugar solution in a test tube. Prepare blank boil

cool

measure at 510nm

estimate concentration

Determination of glucose by glucose oxidase method Principle

glucose

glucose + oxygen H2 O 2 + Gluconic acid

oxidase

H2 O2 + O-dianisidine red colored productperoxidase

Procedure

Materials; Glucose oxidase peroxidase reagent. Working standard solution. Procedure

0.5ml deprotinised plant extract + 0.5ml dw+1ml reagent

Incubate all tubes at 35c for 40 min.

Add 2ml of 6N HCL

Read the color intensity at 540nm

Phenol sulphuric acid method for total carbohydrate determination.

Principle; Glucose acidmedium hydroxymethyl furfural

phenol

green color product.

Materials ;

1.Phenol 5%

2.Sulphuric acid 96% reagent grade.

3.Standard glucose.

Procedure

Pipette out 0.2 ,0.4, 0.6,0. 8 & 1ml of working std.

pipette out 0.1 & 0.2ml of sample sol . Makeup to 1mlH2O

set a blank with 1ml water

Add 1ml of phenol sol to each tube.

Add 5ml of sulphuric acid to each tube & shake well.

After 10 min shake & place in water bath for 25-30c. Read the color at 490nm.

Estimation of starch by anthrone reagent. Sample + 80% alcohol sugars get remove. hydrolysed dehydrated starch glucose hydroxymethy furfural anthrone

green color productMaterials :Anthrone sol 80% ethanol52% perchloric acidStd glucose sol.

Procedure

Homogenise 0.1 to 0.5g of sample in 80% alcohol.

Centrifuge & retain residue, wash & dry.

Add 5ml of water & 6.5ml of 52% perchloric acid.

Extract at 0c for 20min. Centrifuge & save supernatant.

Repeat the extraction using fresh perchloric acid,centifuge , makeup

Pipette out 0.1 or 0.2 ml of supernatant & make up to 1ml.

Prepare the std & add 4ml of anthrone reagent.heat & cool.

Determination of amylose

Principle

The iodine is adsorbed within the helical coils of amylose to produce a

blue colored complex.

Materials

Distilled ethanol 1N NAOH

0.1% phenolphthalein

Iodine reagent

Standard solution.

Procedure

Weigh 100mg ofsample,&add1ml ofethanol,add10ml of 1n NAOH.

Make up to vol to 100ml.

2.5 extract ,add 20ml dw & 3 drops of phenolphthalein.

Add 0.1n HCLdrop by drop until pink color just disappears.

Add 1ml of iodine reagent & make up to 50ml.

Take 0.2,0.4,0.6, &1ml of std amylose & develop color.

Calculate the amt of amylose.

Determination of fructose.

Principle Hydroxymethyl furfural + resorcinol

red color product

Materials Resourcinol reagent

Dilute HCL

Standard fructose solution.

Procedure

2ml of solution ,add1ml of resourcinol reageant

Add 7ml of dil HCL

Pipette out 0.2, 0.4, 0.6, 0.8 & 1ml of std & makeup to 2ml with H2O

Add 1ml of resourcinol & 7ml of dilute HCL

heat all tubes in water bath , cool

Read the color at520nm within 3omin.

Column chromatography

Adsorbent materials – siliceous earths or

charcoal mono & disaccharides & higher carbohydrates - charcoal Methyl mannosides – cellulose powder

Mobile solvent – butanol: pyridine: water (10:3:3)

Estimation by HPLC

Problems with detection system for carbohydrates have limited the application of HPLC to carbohydrates.

The ploar bonded phases – lichrosorb – NH2

Mobile phases– acetonitrile – water mixture.

This system is used for determination of sugar content of soyabeans,dairy roducts ,molases.

The determination of glycoproteins levels & of protein/carbohydrate ratio in glycoprotein is very imp in diagnosis of cancer patients.

Ion exchange chromatography

Derivitization ----bisulphite addtion product of a carbonyl compound & the production of carbohydrates –substituted boric acid.

Separation of larger quantities of sugar - anion exchange columns.

Mixtures of sugars – on the bases of the relative stability of their borate complexes.

Paper chromatography

Developing solvent – mixture of butanol ,acetic acid& water.

Reagents– aniline, diphenyl amine, phosphoric acid.

Ascending paper chromatography – separation of monosaccharides.

Chromatogram sprayed with p-anisidine, eluted with aq.stannous chloride.

Benzidine with stannous chloride ---aldose analysis.

Triphenyltetrazolium,benzidine – direct photometric examination of paper chromatography spots.

Estimation of sugars in ice cream

Thin layer chromatography

Principle.

Thin even layer of adsorbent is coated on glass plate.

spots are applied

Development takes place & spots are identified

BINDING MATERIALS.

silicis acid, alumina, keiselguhrTHICKNESS

250µ is maintained with controllable spreader.

Procedure

Prepare silica gel plate – activates at 105c for 30 min.

Solvent preparation & std sugar solution preparation.

Spot sugars & unknown

Development

Spray coating reagent

Calculate Rf .

Rf = distance travelled by compound

distance moved by glucose.

Miscellaneous methods

Gasometry is applicable to carbohydrate analysis.

Hagedorn-jenson reaction--- reaction of sugars with

ferricyanide. Best range10 – 120mcg of reducing sugar

Biochemical procedures also prove useful for

identification of carbohydrates.

Electrochemical procedures have been adopted to

carbohydrate analysis.

Reduction of carbonyl function with a sodium borohydride

followed by titration of residual borohydride with acid to

produce hydrogen gas.

.

Significances

Generally available as immediate energy source.

Cellulose, polysaccharides is an important structural component of plant cells.

Glucose is essential for cell function.

Significance of carbohydrates for gastrointestinal function.

Majore role in working process of immune system, fertilisation,pathogenesis,blood clotting.

k.GowthamiKeerthanaSirishaM.pharm(analysis)