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    Ajay Krishnanv
    The document describes a colorimetric method using ninhydrin to assay amino acids. Ninhydrin reacts with amino acids to produce a purple color. The intensity of this color can then be quantified using a spectrophotometer. The method involves reacting the amino acid sample with ninhydrin, then measuring absorbance at 570nm. While fast and sensitive, the method is not specific to individual amino acids and the color intensity depends on the amino acid. The document also provides details on the reagents, equipment, and procedures needed to perform the assay.

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    Experiment

    Uploaded by

    Ajay Krishnanv
    63 views5 pages
    The document describes a colorimetric method using ninhydrin to assay amino acids. Ninhydrin reacts with amino acids to produce a purple color. The intensity of this color can then be quantified using a spectrophotometer. The method involves reacting the amino acid sample with ninhydrin, then measuring absorbance at 570nm. While fast and sensitive, the method is not specific to individual amino acids and the color intensity depends on the amino acid. The document also provides details on the reagents, equipment, and procedures needed to perform the assay.

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    The document describes a colorimetric method using ninhydrin to assay amino acids. Ninhydrin reacts with amino acids to produce a purple color. The intensity of this color can then be quantified using a spectrophotometer. The method involves reacting the amino acid sample with ninhydrin, then measuring absorbance at 570nm. While fast and sensitive, the method is not specific to individual amino acids and the color intensity depends on the amino acid. The document also provides details on the reagents, equipment, and procedures needed to perform the assay.

    Copyright:

    © All Rights Reserved
    Download as DOCX, PDF, TXT or read online from Scribd
    Download as docx, pdf, or txt
    63 views5 pages

    Experiment

    Uploaded by

    Ajay Krishnanv
    The document describes a colorimetric method using ninhydrin to assay amino acids. Ninhydrin reacts with amino acids to produce a purple color. The intensity of this color can then be quantified using a spectrophotometer. The method involves reacting the amino acid sample with ninhydrin, then measuring absorbance at 570nm. While fast and sensitive, the method is not specific to individual amino acids and the color intensity depends on the amino acid. The document also provides details on the reagents, equipment, and procedures needed to perform the assay.

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    © All Rights Reserved
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    AMINO ACID ASSAY

    BY NINHYDRIN COLORIMETRIC METHOD

    Method
    The reaction between alpha-amino acid and ninhydrin involved in the development of color are
    described by the following five mechanistic steps:

    alpha-amino acid + ninhydrin ---> reduced ninhydrin + alpha-amino acid + H2O

    alpha-amino acid + H2O ---> alpha-keto acid +NH3

    alpha-keto acid + NH3 ---> aldehyde + CO2


    Step (1) is an oxidative deamination reaction that removes two hydrogen from the alpha-amino
    acid to yield an alpha-imino acid. Simultaneously, the original ninhydrin is reduced and loses an
    oxygen atom with the formation of a water molecule. In Step (2), the NH group in the alpha-
    imino acid is rapidly hydrolyzed to form an alpha-keto acid with the production of an ammonia
    molecule. This alpha-keto acid further undergoes decarboxylation reaction of Step (3) under a
    heated condition to form an aldehyde that has one less carbon atom than the original amino acid.
    A carbon dioxide molecule is produced here. These first three steps produce the reduced
    ninhydrin and ammonia that are required for the production of color in the last two Steps (4) and
    (5). The overall reaction for the above reactions is simply (slightly inaccurately) expressed in
    Reaction (6) as follows:

    alpha-amino acid + 2 ninhydrin ---> CO2 + aldehyde + final complex(BlUE) + 3H2O


    In summary, ninhydrin, which is originally yellow, reacts with amino acid and turns deep purple.
    It is this purple color that is detected in this method.

    Ninhydrin will react with a free alpha-amino group, NH2-C-COOH. This group is contained in
    all amino acids, peptides, or proteins. Whereas, the decarboxylation reaction will proceed for a
    free amino acid, it will not happen for peptides and proteins. Thus, theoretically only amino acids
    will lead to the color development. However, one should always check out the possible
    interference from peptides and proteins by performing blank tests especially when such solutions
    are readily available. For example, one can simply add the ninhydrin reagent to a solution of only
    proteins and see if there is any color development. There is no excuse for failing to perform such
    a vital test when the sample mixture contains both proteins and amino acids. There are also
    reports that chemical compounds other than amino acids also yield positive results.

    This test can be used routinely for the detection of glycine in the absence of other interfering
    species. Although this is a fast and sensitive test for the presence of alpha-amino acids, because
    of the nonselectivity, it cannot be used to analyze the relative individual contents of a mixture of
    different amino acids. Furthermore, the color intensity developed is dependent on the type of
    amino acid. Finally, it does not react with tertiary or aromatic amines.

    Note that since ninhydrin is a strong oxidizing agent, proper caution should be exercised in
    handling this compound. It is especially potent at the elevated temperature under which the
    reaction is carried out. The ninhydrin reagent will stain the skin blue and cannot be immediately
    washed off completely if it comes in contact with the skin. However, as in any other stain on the
    skin, the color will gradually rub off after about a day.

    List of Reagents and Instruments

    A. Equipment

     Test tubes
     Pipets
     Spectrophotometer

    B. Reagents

     Ninhydrin Reagent Solution


    o Ninhydrin: 0.35 g
    o Add ethanol to: 100 ml
    Procedures

    1. Add 1 ml of the ninhydrin solution to 5 ml of sample. Cover the test tube with a piece of
    paraffin film to avoid the loss of solvent due to evaporation. A capped test tube can also
    be used instead.
    2. With gentle stirring, react at 80-100ºC for 4-7 minutes. (How would one find out the
    amount of time needed to ensure a complete reaction?) If a large heated water bath is
    used for the entire class and if there is no good provision for holding the test tube in the
    hot water bath, the test tube may be held with a piece of wire and hang on the side of the
    water container. A clamp usually does not work too well.
    3. After cooling to room temperature in a cold water bath, record the absorbance with a
    spectrophotometer. (How would you find out the best wavelength to use for this purpose?
    Hint: the final purple colored complex of ninhydrin absorbs the most amount of light at
    the wavelength of 570 nm.)
    4. 1ml O.D.

    starch soln -->----+ +-------> at 570nm


    ||| |||
    |-+-| |-+-|
    | | heat | |
    | | --------> | |
    | | | |
    | | | |
    +---+ +---+

    5ml sample soln


    Notes

    1. Iso-propanol or a 1:1 mixture of acetone/butanol may be used in lieu of ethanol in


    preparing the ninhydrin reagent.

    Exp. 2Analysis of Leaf Pigments

    The most abundant plant pigments are chlorophyll a and b, which occur in a ratio approx. 3:1.
    The second group of plant pigment, the carotenoids can be divided into two different types; the
    carotenes and the xanthophylls. Chlorophyll extracted in 80% acetone from a green leaf appears
    green. The chlorophyll extract can vary in the depth of green or tint of green depending on the
    plant material from which it was extracted. In Angiosperms (most land plants) there are typically
    two types of Chlorophyll (Chl) molecules, namely, chlorophyll a (Chl a) and chlorophyll b (Chl
    b ). Both of these pigments absorb photons of light in the blue and red spectral regions, but the
    specific wavelengths of light they absorb are different. The absorbance of photons at 663 nm and
    645 nm, specific for Chl a and Chl b, respectively, will be determined. Because the absorption
    spectra (an integrated picture of the wavelengths of light absorbed) of these two Chl molecules
    overlap, you will use a simultaneous equation to solve for the amount of both pigments. The
    equation for this has been worked out and is known as Arnon's equation. Arnon's equation will
    provide you with quanitative information about the Chl a and Chl b. In contrast to the
    chlorophylls, which absorb light in two regions of the visible spectrum, the carotenoids exhibit
    intense absorption in just one, 350-500nm. These equations were published by D.I. Arnon (1949)
    Plant Physiol. 24: 1. You will be given different types of leaves from which you will determine
    the amounts of chlorophyll a, b and carotenoids.

    Spectrophotometric Determination of Chlorophyll Procedure:

    For the different types of leaves provided follow the procedure given below:
    1. Cut the leaves into small pieces. Discard major veins and any tough, fibrous tissue. Weigh the
    pieces: you should keep about 0.10 g (100 mg) of material for grinding (record total fresh weight
    of each sample).

    2. Put the tissue into a mortar and add 10 ml of 80% acetone (acetone:water 80:20 v:v). Grind the
    tissue with a pestle. You want to pulverize the tissue completely, thus a few grains of sand may
    help. This is your leaf homogenate.

    3. Filter the leaf homogenate through the filter paper. The retentate is removed by the filter paper
    (and discarded) and the extract (or filtrate) is collected in a test-tube.

    Determination of chlorophyll concentration

    1. Obtain a clean cuvette for the spectrophotometer/colorimeter and fill two-thirds full with 80%
    acetone; this is the blank. Wipe the cuvette with a tissue and put it into the spectrophotometer,
    then set the wavelength to 663 nm. Cover the cuvette chamber and set the spectrophotometer to 0
    absorbance with the blank in place. Remove the blank and save for the next measurement.

    2. Gently swirl your first extract in the test-tube and fill a second cuvette two-thirds full. Wipe it
    clean, insert into the spectrophotometer, and close the hatch. The readout should give you the
    absorbance at 663 nm, the A663. Record this number, and repeat step 2 with the other extracts.

    3. Change the wavelength to 645 nm. Reinsert the blank cuvette, and re-zero the
    spectrophotometer at the new wavelength. Remove the blank and insert a cuvette containing your
    first extract. Read and record A645. Repeat for the other extracts.

    Calculations:

    Use Arnon's equation (below) to convert absorbance measurements to mg Chl g-1 leaf tissue Chl
    a (mg g-1) = [(12.7 × A663) - (2.6 × A645)] × ml acetone / mg leaf tissue Chl b (mg g-1) =
    [(22.9 × A645) - (4.68 × A663)] × ml acetone / mg leaf tissue Total Chl = Chl a + Chl b. C x+c =
    1000 A470 – 1.90Chla – 63.14 Chlb/214, (x = xanthophylls and carotenes)

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