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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.
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
Enzymatic method
Physical methods Spectrophotometry methods Chromatography methods miscellaneous
Chemical methods
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.
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.
It cannot distinguish b/n different types of reducing sugars. It cannot directly determine the concentration of non reducing sugars.
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.
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
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.
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
Principle
glucose
H2 O 2 + Gluconic acid
H2 O2
+ O-dianisidine
peroxidase
Procedure
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.
starch
glucose
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
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.
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.
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.
Principle.
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.
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.