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.
monosaccharides ex- glucose,galactose,frutose.
disaccharides ex- sucrose,lactose, maltose.
oligosaccharides ex- raffinose,stachyose.
polysaccharides ex – cellulose,starch,callose,lamarin
General formula – (CH 2 O )n
Trioses C 3 sugars
Pentoses C5 sugars
Dehydrate pentoses furfural
Reducing sugars Non-reducing sugars
ketose Other hexoses
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.
Aldehyde function can be deternined by selective oxidation with
To detremine aldoses– cyanohydrin reaction is done.
To determine polysaccharides– neutralisation of negatively charged
colloidal particles with positively charged ones & relies on
Results depend on the precise reaction times.temp & reagent
It cannot distinguish b/n different types of reducing sugars.
It cannot directly determine the concentration of non reducing
It is susceptable to interference from other types of molecules that
act as reducing agents.
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.
Analytical method based on enzyme rely on their ability to catalyse
Rapid ,highly specific & sensitive to low conc.
1.Allowing the reaction to go to completion & measure concentration
of the product.
2. measuring the intial rate of the enzyme catalysed reaction.
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
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 &
The density of aq solution increases as the concentration
Thus the carbohydrate concentration can be determined
by measuring density.
Routinely used in industry for determination of
carbohydrates concentration of juices & beverages.
carbohydrates + conc H2 SO4
blue complex (colorimetrically at 620nm)
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
measure optical density at 620nm
This method is value in clinical analysis of glucose in blood &
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-
This reagent is employed to assay the sugars by using their
Reagent in alkaline solution is reduced to 3-amino-5-nitrosalicylic
OH reduction OH
NO2 NO 2 NO 2
Prepare the DNS reagent just before use.
1ml of the reagent to 3ml of sugar solution in a test tube.
measure at 510nm
Determination of glucose by glucose
glucose + oxygen H2 O 2 + Gluconic acid
H2 O2 + O-dianisidine red colored product
Glucose oxidase peroxidase reagent.
Working standard solution.
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
Glucose acidmedium hydroxymethyl furfural
green color product.
2.Sulphuric acid 96% reagent grade.
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.
starch glucose hydroxymethy furfural
green color product
52% perchloric acid
Std glucose sol.
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
The iodine is adsorbed within the helical coils of amylose to produce a
blue colored complex.
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.
Hydroxymethyl furfural + resorcinol
red color product
Standard fructose solution.
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.
Adsorbent materials – siliceous earths or
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
Mixtures of sugars – on the bases of the relative stability
of their borate complexes.
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
Benzidine with stannous chloride ---aldose analysis.
Triphenyltetrazolium,benzidine – direct photometric examination of
paper chromatography spots.
Estimation of sugars in ice cream
Thin layer chromatography
Thin even layer of adsorbent is coated on glass plate.
spots are applied
Development takes place & spots are identified
silicis acid, alumina, keiselguhr
250µ is maintained with controllable spreader.
Prepare silica gel plate – activates at 105c for 30 min.
Solvent preparation & std sugar solution preparation.
Spot sugars & unknown
Spray coating reagent
Calculate Rf .
Rf = distance travelled by compound
distance moved by glucose.
Gasometry is applicable to carbohydrate analysis.
Hagedorn-jenson reaction--- reaction of sugars with
Best range10 – 120mcg of reducing sugar
Biochemical procedures also prove useful for
identification of carbohydrates.
Electrochemical procedures have been adopted to
Reduction of carbonyl function with a sodium borohydride
followed by titration of residual borohydride with acid to
produce hydrogen gas.
Generally available as immediate energy source.
Cellulose, polysaccharides is an important structural component of
Glucose is essential for cell function.
Significance of carbohydrates for gastrointestinal function.
Majore role in working process of immune system,