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    TLC carotenoids

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    anushka.korpad11
    Its a practical of extracting cartenoids from carrot sample.

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    TLC carotenoids

    Uploaded by

    anushka.korpad11
    2 views2 pages
    Its a practical of extracting cartenoids from carrot sample.

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    © © All Rights Reserved

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    PDF, TXT or read online from Scribd

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    Its a practical of extracting cartenoids from carrot sample.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    2 views2 pages

    TLC carotenoids

    Uploaded by

    anushka.korpad11
    Its a practical of extracting cartenoids from carrot sample.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
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    QUALITATIVE DETERMINATION OF SECONDARY METABOLITE IN THE GIVEN

    SAMPLE

    Aim: To qualitatively determine secondary metabolite (carotenoids) in carrot sample by Thin


    Layer Chromatography
    Principle: TLC is a technique by which non-volatile components in a mixture are separated by a
    fixed stationary phase and a liquid mobile phase by differential affinities between the two phases.
    Thin layer chromatography (TLC) is a widely used separation technique for qualitative analysis.
    It uses a thin layer of stationary phase, usually silica gel, coated on a glass, plastic, or aluminum
    plate. A liquid solvent called the mobile phase carries the sample and separates it as it moves
    across the plate. It offers the advantages of simplicity, sensitivity, and rapid analysis over other
    separation techniques. TLC is used by many industries and fields of research, including
    pharmaceutical production, clinical analysis, industrial chemistry, environmental toxicology,
    food chemistry, water, inorganic, and pesticide analysis, dye purity, cosmetics, plant materials,
    and herbal analysis.
    Retention Factor
    After the separation is complete, individual compounds appear as spots separated vertically.
    Retention factor, or Rf, is a measurement that describes how far a compound travels in a
    chromatographic technique compared to the distance it would have traveled if it moved with the
    solvent. The Rf value is used to compare and identify compounds, and to compare the findings of
    one TLC plate to another. The Rf value ranges from 0 to 1. Rf value of 0 indicates that the
    compound is retained where it was applied as sample spot. Rf value of 1 indicates that the
    compound has migrated with the solvent front.

    Requirements:
    TLC plates, capillary tubes, TLC chamber, hexane, acetone, distilled water, sample (grated
    carrots= 10g), pestle and mortar, muslin cloth, Whatman Filter Paper, funnel, beakers, 500µl
    micropipette and tips, Eppendorf tubes.
    Procedure:
    1. Preparation of extract: Take about 5g of grated carrot sample in dry pestle and mortar.
    Prepare the extract by grinding sample in extraction solvent. Extraction solvent used for
    extraction of carotenoids is hexane : acetone (1:1). Filter the extract through Whatman
    filter paper and take about 1ml extract in Eppendorf tube.
    2. Prepare the mobile phase acetone : distilled water (9:1) about 100ml. Pour mobile phase
    to about 0.5cm ht. in the TLC chamber. Cover and keep for 10-15 mins to saturate the
    TLC chamber.
    3. Mark solvent front on TLC plate (0.5cm from top) and sample application level (1.5cm
    from bottom) with pencil. Apply the spot of extract on the TLC plate three times at the
    same spot with capillary tube. Air dry the applied spot.
    4. Place the TLC plates in chamber taking care that the sample spot should be above the
    level of mobile phase and should not touch the mobile phase directly. Allow the mobile
    phase to run to the solvent front marked. TLC chamber should not be opened during the
    run.
    5. Since carotenoids are coloured pigments, they can be visualized easily as a yellow
    coloured spot without oven baking or reagent. Therefore, no further processing is
    required. Measure the distance travelled by the compound and by the solvent front.
    Calculate the Rf value.

    Observations:

    Results: Rf value of carotenoids from carrot sample =

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