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PRELUDE

Most abundant class of organic compounds found in living organism.


n

CO2 + n H2O + energy

CnH2nOn + n O2

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.

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

Fehlings test Benedicts test Barfoeds test

Molischs test
Bials test Seliwanoffs test Iodine test

Fehlings test

Barfoeds test

Molishs test

Dehydrate pentoses

furfural

Bials test

Seliwanoffs test

ketohexoses

5-hydroxymethylfurfural

Benedicts test

Iodine test

QUALITATIVE TESTS
Test solution

Reducing sugars

Non-reducing sugars

Iodine test
monosaccharides Disaccharides Bials test
No change Insulin

positive

Negative
Seliwonoffs test

Blue Starch

Brown glycogen

ketose

Other hexoses

TITRIMETRY METHOD

EDTA has been used in place of fehlings & benedicts 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 PROCEDUREreaction 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= [a]lc [a] = optical activity l= pathlength ,c =589.3nm. The conc of unknown is determined by measuring its angle of of rotation.

Refractive index
n= c/cm

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

Snells law = sin(i)sin(r) = n2/n1


Measurements are made at specific temp (20) & (589.3nm). Quick & simple Determine sugar conc of syrups, honey,molases,tomato products & jams.

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

Benedicts method

1.

This method is value in clinical analysis of glucose in blood & urine. Benedicts sol+ sodium carbonate(conical flask) heat to boil. Std solution is taken in burette.

2.

On titration white bulky ppt is formed (cuprousthiocyanate) Note the vol of sugar solution required.

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

OH OH NO2 NO 2

reduction NO 2

OH

NH2

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 oxidase

H2 O 2 + Gluconic acid

H2 O2

+ O-dianisidine
peroxidase

red colored product

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;
acidmedium hydroxymethyl furfural

Glucose

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


hydrolysed

sugars get remove.


dehydrated

starch

glucose

hydroxymethy furfural anthrone

green color product


Materials : Anthrone sol 80% ethanol 52% perchloric acid Std 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, keiselguhr


THICKNESS

250 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.Gowthami Keerthana Sirisha M.pharm(analysis)