Evaluation Of Carbohydrates

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Evaluation Of Carbohydrates
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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

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• 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

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stuctures

• .

sucrose
starch
cellulose
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Evaluation methods

• Chemical methods
• Titrimetrical method
• Gravimetrical method
• Enzymatic method
• Physical methods
• Spectrophotometry methods
• Chromatography methods
• miscellaneous

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Chemical methods

• Fehlings test
• Benedicts test
• Barfoeds test
• Molischs test
• Bials test
• Seliwanoffs test
• Iodine test

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Fehlings test
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Barfoeds test
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Molishs test
Dehydrate pentoses
furfural
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Bials test
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Seliwanoffs test
ketohexoses
5-hydroxymethylfurfural
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Benedicts test
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Iodine test
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QUALITATIVE TESTS

• Test solution

Reducing sugars
Non-reducing sugars
Iodine test
monosaccharides
Disaccharides
Bials test
Blue Starch
Brown glycogen
No change Insulin
positive
Negative
Seliwonoffs test
ketose
Other hexoses
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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.

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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.

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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.

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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.

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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.

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PHYSICAL METHODS
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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 ameasured 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 alc
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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.

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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.

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Anthrone method

• Principle
• carbohydrates conc H2 SO4
• 
  
  
• DEHYDRATION
• 
  
  
• hydroxy methylfurfuralanthrone
• CONDENSATION
• blue complex
  (colorimetrically at 620nm)

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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

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Benedicts method

• This method is value in clinical analysis of
  glucose in blood urine.
• 1. Benedicts sol sodium carbonate(conical
  flask)
• heat to boil.
• Std solution is taken in burette.
• On titration white bulky ppt is formed
  (cuprousthiocyanate)
• Note the vol of sugar solution required.

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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

OH
NH2
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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

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Determination of glucose by glucose oxidase method

• Principle
• glucose
• glucose oxygen H2 O
  2 Gluconic acid
• oxidase
• H2 O2 O-dianisidine
  red colored product

peroxidase
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Procedure

• Materials
• Glucose oxidase peroxidase reagent.
• Working standard solution.
• Procedure
• 0.5ml deprotinised plant extract 0.5ml
  dw1ml reagent
• Incubate all tubes at 35c for 40
  min.
• Add 2ml of 6N HCL
• Read the color intensity at
  540nm

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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.

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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.

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Estimation of starch by anthrone reagent.

• Sample 80 alcohol sugars get
  remove.
• hydrolysed
  dehydrated
• starch glucose
  hydroxymethy furfural
• 
  anthrone
• 
  green color product
• Materials
• Anthrone sol
• 80 ethanol
• 52 perchloric acid
• Std glucose sol.

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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.

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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.

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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.

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Determination of fructose.

• Principle
• Hydroxymethyl furfural resorcinol
• red color product
• Materials
• Resourcinol reagent
• Dilute HCL
• Standard fructose solution.

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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.

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CHROMATOGRAPHY
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Column chromatography

• Adsorbent materials siliceous earths or
  
• charcoal
• mono disaccharides higher carbohydrates -
  charcoal
• Methyl mannosides cellulose powder
• Mobile solvent butanol pyridine water
  (1033)
• 
  

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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.

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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.

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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.

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Estimation of sugars in ice cream
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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
• 250µ is maintained with controllable
  spreader.

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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.

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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.

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• 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.
• .

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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.

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THANK U
k.Gowthami Keerthana Sirisha M.pharm(analysis)