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    COLORIMETER

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    ahteshammirji575
    The document discusses the principles and components of colorimetry, which is a technique used to measure the concentration of compounds in a solution by measuring the intensity of color. It describes how colorimetry works based on Beer's law and Lambert's law, relating absorbance to concentration and path length. The key components of a colorimeter are described along with the process used to measure absorbance and calculate concentration of an unknown sample.

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    COLORIMETER

    Uploaded by

    ahteshammirji575
    24 views5 pages
    The document discusses the principles and components of colorimetry, which is a technique used to measure the concentration of compounds in a solution by measuring the intensity of color. It describes how colorimetry works based on Beer's law and Lambert's law, relating absorbance to concentration and path length. The key components of a colorimeter are described along with the process used to measure absorbance and calculate concentration of an unknown sample.

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    The document discusses the principles and components of colorimetry, which is a technique used to measure the concentration of compounds in a solution by measuring the intensity of color. It describes how colorimetry works based on Beer's law and Lambert's law, relating absorbance to concentration and path length. The key components of a colorimeter are described along with the process used to measure absorbance and calculate concentration of an unknown sample.

    Copyright:

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

    COLORIMETER

    Uploaded by

    ahteshammirji575
    The document discusses the principles and components of colorimetry, which is a technique used to measure the concentration of compounds in a solution by measuring the intensity of color. It describes how colorimetry works based on Beer's law and Lambert's law, relating absorbance to concentration and path length. The key components of a colorimeter are described along with the process used to measure absorbance and calculate concentration of an unknown sample.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    of 5

    COLORIMETRY

    AIM:
    To study the principles of colorimetry and verify the Lambert’s and Beer’s law.
    Many biochemical experiments involve the measurement of compound
    present in a complex mixture. The most widely used method for determining the
    concentration of biochemical compounds is colorimetry.
    PHOTOMETRY:
    Photometry means measurement of light. The color of light is a function of its wavelength.
    As the wavelength is changed within the visible range, an alteration in color is detected.
    PRINCIPLE:
    When a beam of monochromatic light is passes through a colored solution, some light is
    absorbed and some light is transmitted. The absorbed light is measured as optical
    density(OD). This absorbed light is made to fall on photocell, which converts light energy
    into electrical energy which is measured by a galvanometer. The intensity of the coloured
    solution is proportional to the concentration of the substance.
    COLORIMETRY:
    This refers to the measurement of intensity of colour in a solution. thus, the concentration
    of colourless biochemical compounds and metabolites can be estimated if they are
    converted to coloured compounds using chemical reactions under defined conditions, by
    measuring the intensity of colour which is proportional to the concentration of the
    compound. this is the basic principle of all colorimetric measurement.
    COMPONENTS OF THE COLORIMETER:
    1.source of light
    2.adjustable slit
    3.condensing lens
    4.filter
    5.cuvette (sample holder)
    6.A photocell/detector
    7.recorder/galvanometer

    1.SOURCE OF LIGHT:
    A lamp provides light in visible region of the spectrum. Usually tungsten lamp is the
    source of light.
    2.ADJUSTABLE SLIT:
    The light emerging from tungsten lamp is allowed to pass through a narrow adjustable slit.
    3.CONDENSING LENS:
    Provides parallel beam of light.
    4.FILTER:
    It provides the monochromatic light (of single wavelength) by filtering other wavelengths.
    The colour of the filter is complementary to the colour of the solution. This allows only
    appropriate wavelength of light to pass through the coloured solution.
    5.CUVETTE (SAMPLE HOLDER):
    Cuvette is a special glass tube with some absorptive capacity. It holds the solution to be
    analyzed in a colorimeter. Cuvette should have uniform thickness, inner diameter and
    refractive index. Cuvettes usually have 1 cm light path.
    6.A PHOTOCELL/DETECTOR:
    It is a photo sensitive element usually made of selenium. It is activated when light falls on
    it. It emits electrons proportional to the amount of light falling on it. It converts the light
    energy into electrical energy.
    7.GALVANOMETER:
    To measure the output electrical energy.
    SELECTION OF FILTERS:
    COLOUR OF SOLUTION FILTER COLOUR WAVELENGTH(nm)
    Green-Yellow Violet 400-435
    Yellow Blue 435-500
    Red Green 500-570
    Blue Yellow 570-600
    Green-Blue Orange 600-630
    Green Red 630-700
    LAWS OF PHOTOMETRY:
    The measurement of colour by photometry is governed by Beer’s law and Lambert’s law.
    BEER’s LAW:
    The amount of light absorbed by a coloured solution is proportional to the concentration of
    the solution.
    If A is the light absorbed (absorbance) and C is the concentration of the colour in the
    solution then, AαC or A=KC.
    LAMBERT’s LAW:
    The amount of light absorbed by a coloured solution is proportional to the depth through
    which the light passes in the solution.
    If L is the depth through which the light passes in the solution then, AαL or A=KL.

    Combining the two law Beer’s -Lambert’s law stats that,


    The amount of light absorbed by a coloured solution is proportional to distance travelled
    in solution and concentration of the coloured substance in the solution.
    AαCαL or A=KCL
    K= the constant of coloured solution
    AT =Absorbance of the test solution
    CT =Concentration of test solution
    AS =Absorbance of the standard solution
    CS = Concentration of standard solution
    Since in the colorimetric measurements, optically similar cuvetts having the same length
    of light path are used for blank, test and standard. Hence,
    OPTICAL DENSITY (OD) OR ABSORBANCE:
    It is defined as the log of the ratio of incident light (I) to the transmitted light (T).
    When OD is plotted against concentration a straight line passing through origin should be
    absorbance as directly proportional to concentration of absorbing substance.
    Absorbance has no unit-all photometric instrument convert transmittance (T) into
    absorbance is also called optical density (OD) which is in fact the more commonly used
    term .
    CONCENTRATION OF TEST SOLUTION=
    absorbance of test
    x concentration of standard
    absorbance of standard
    SIGNIFICANCE OF TEST:
    In colorimetric assays some colour developed, due to reagent themselves. Hence a blank
    (B) is prepared using all reagents except the substance being measure and its OD
    value(OD of Blank) is substracted from the OD of test & OD of standard to get true
    optical density of both test and standard solution.
    CONCENTRATION OF TEST SOLUTION=
    OD of Test−OD of Blank
    x concentration of standard
    OD of Standard−OD od Blank

    CONCENTRATION IN 100ML OF TEST SAMPLE


    OD of Test−OD of Blank Concentration of standard
    = X X 100
    OD of Standard−OD od Blank effective volume of sample

    STEPS IN OPERATION OFPHOTOELECTRIC COLORIMETER:


    Switch on the instrument and allow it to warm up 4-5 mins.
    Select proper filter by rotating the turret.
    Fill the cuvette to about 3/4th with the distilled water and place it in the cuvette slot.
    Pressing the button “coarse” and “fine” knobs to give zero optical activity in the
    galvanometer. Release the button.
    Replace the cuvette with distilled water in sample holder with cuvette containing blank
    solution. Let the OD be ‘B’.
    Transfer the blank solution to the original test tube.
    Take “standard” solution in the same cuvette and record the OD. Let it be ‘S’.
    Next take “Test” solution in the same cuvette and read the OD. Let it be ‘T’.
    Transfer the test solution back to the original test tube and wash the cuvette.
    NOTE:
    Take the sample from lower to higher concentration to avoid error in reading. Rinse the
    cuvette with distilled water every time before going to next sample.

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