Scribd Logo
Upload

Welcome to Scribd!

  • 64 views

    Anthrone Carbohydrate Estn

    Uploaded by

    Dee Shylla
    Notes on Anthrone.

    Copyright:

    © All Rights Reserved
    Download as DOCX, PDF, TXT or read online from Scribd

    Anthrone Carbohydrate Estn

    Uploaded by

    Dee Shylla
    64 views3 pages
    Notes on Anthrone.

    Copyright

    © © All Rights Reserved

    Available Formats

    DOCX, PDF, TXT or read online from Scribd

    Did you find this document useful?

    Is this content inappropriate?

    Notes on Anthrone.

    Copyright:

    © All Rights Reserved
    Download as DOCX, PDF, TXT or read online from Scribd
    Download as docx, pdf, or txt
    64 views3 pages

    Anthrone Carbohydrate Estn

    Uploaded by

    Dee Shylla
    Notes on Anthrone.

    Copyright:

    © All Rights Reserved
    Download as DOCX, PDF, TXT or read online from Scribd
    Download as docx, pdf, or txt
    of 3

    Department of

    Biochemistry, SEC [ Semester ii practical paper ii ]

    Aim: Estimation Of Carbohydrates By Anthrone’s Method

    Principle: This is based on the reaction of carbohydrates with anthrone in the presence of
    concentrated sulphuric acid. Carbohydrate monosaccharides are dehydrated by concentrated
    sulphuric acid to form furfural (from pentoses) or hydroxyl furfural (from hexoses)
    derivatives. The furfural or hydroxyl furfural formed condenses with naphthol from the
    anthrone reagent to form a blue-green complex which shows maximum absorbance at 620
    nm.

    Bluish green complex

    λmax =620nm

    Blank: Light yellow

    Test: Bluish green colour


    with increasing
    intensity as
    concentration
    increases

    Credit: OnePetro.com

    Requirements:

    1. Glucose stock solution (0.1 mg/ml)


    2. Anthrone’s reagent
    3. Glass cuvette
    4. Spectrophotometer
    5. Cyclomixer
    6. Clean and dry glasswares

    Reagent preparation:
    1. Glucose stock is prepared by dissolving 10mg of glucose in 100ml distilled water
    Department of
    Biochemistry, SEC [ Semester ii practical paper ii ]

    2. Anthrone Reagent is prepared by dissolving 2 g of Anthrone, dissolved in 1000 ml of


    concentrated sulphuric acid (handle cautiously)

    Procedure:
    1. 0.1 to 1.0 ml glucose stock solution is pipetted into different labeled test tubes.
    2. The final volume in each test tube is made up to 1 ml by adding appropriate amount of
    distilled water to the test tubes.
    3. Separately, 1 ml of distilled water is taken in a test tube and labeled as blank.
    4. 0.5 ml of unknown solution is taken in two different test tubes. The final volume is
    made to 1 ml with distilled water in each of the unknown test tubes.
    5. 4 ml of Anthrone’s reagent solution is added to each test tube and mixed well.
    6. All the above test tubes are incubated in a boiling water bath for 20 min.
    7. The absorbance of the standard and unknown against the blank is taken at 620 nm.
    8. A standard curve of the absorbance versus concentration is plotted on a graph sheet
    9. The concentration of the unknown is calculated from the curve.

    Tabulation:

    Sl Glucose stock Water Anthrone’ Concentration Absorbance


    No. solution (ml) s Reagent (mg) at 620 nm
    (ml) (ml)
    Blan 0.0 1.0 0 -
    k
    1 0.1 0.9
    2 0.2 0.8
    3 0.3 0.7
    4 0.4 0.6 4
    5 0.5 0.5
    6 0.6 0.4
    7 0.7 0.3
    8 0.8 0.2
    9 0.9 0.1
    10 1.0 0.0

    Sl. Unknown solution Water Reagent Absorbance


    No. (ml) (ml) (ml) at 620 nm
    1

    2 4

    Calculations:

    (I) For standard


    Department of
    Biochemistry, SEC [ Semester ii practical paper ii ]

    Using V1S1=V2S2
    (i) For 0.1 ml
    V1 = 0.1 ml, V2 = 5 ml, S1 = 0.1 mg/ml, S2=?
    S2 = 0.1 ml x 0.1mg/5ml
    S2 = 0.02 mg/ml
    (ii) For 0.2 ml
    V1 = 0.2 ml, V2 = 5ml, S1 = 0.1 mg/ml, S2=?
    S2 = 0.2ml x 0.1 mg/5ml
    S2 = 0.04 mg.
    (iii) For 0.3ml…

    For unknown
    0.5ml of the unknown sample taken gave a reading of x mg from the graph, however the
    sample is diluted (final volume/initial volume= dilution factor) 2 times, therefore, the final
    concentration is x mg *2 = y mg
    Result:

    The concentration of the unknown carbohydrate solution estimated by Anthrone’s method


    is………mg/ml or ……..μg/ml.

    Discussion:

    Anthrone test is used for the detection and quantification of carbohydrates in various samples
    like blood serum, milk, and its variation, etc. This test gives a negative result in samples that
    do not form furfural or hydroxyfurfural in the dehydration step e.g. D-glucose phenylosazone
    and D-glucose phenylosotriaole.

    You might also like