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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


   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

                                                                        Iodine test
monosaccharides         Disaccharides

                         Bial’s test
                                                                                      No change
                                                             Blue        Brown
                                                                                       Insulin
                                                            Starch      glycogen
             positive                  Negative

                                   Seliwonoff’s test


                             ketose               Other hexoses
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= [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.

   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.
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
    OH

          NO2              NO 2                 NO 2
                                                                          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                        H2 O 2 + Gluconic acid
                      oxidase



H2 O2    + O-dianisidine                   red colored product
                           peroxidase
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 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
     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.Gowthami
Keerthana
Sirisha
M.pharm(analysis)

				
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