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    Determination of Absorption Spectra

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    say.ellese
    The document discusses spectrophotometry principles and experiments involving the absorption spectra of bromphenol blue (BPB) and methyl orange (MO). It defines spectrophotometry and the underlying principles of absorbance, transmittance, and Beer's Law. The experiment measured absorbance of BPB and MO solutions at various wavelengths and plotted the absorption spectra. Results showed maximum absorbance of 505nm for MO and 600nm for BPB, matching theoretical wavelengths. Clinical applications of spectrophotometry in measuring bilirubin levels for hemolytic disease of newborns are also mentioned.

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    Determination of Absorption Spectra

    Uploaded by

    say.ellese
    23 views30 pages
    The document discusses spectrophotometry principles and experiments involving the absorption spectra of bromphenol blue (BPB) and methyl orange (MO). It defines spectrophotometry and the underlying principles of absorbance, transmittance, and Beer's Law. The experiment measured absorbance of BPB and MO solutions at various wavelengths and plotted the absorption spectra. Results showed maximum absorbance of 505nm for MO and 600nm for BPB, matching theoretical wavelengths. Clinical applications of spectrophotometry in measuring bilirubin levels for hemolytic disease of newborns are also mentioned.

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    The document discusses spectrophotometry principles and experiments involving the absorption spectra of bromphenol blue (BPB) and methyl orange (MO). It defines spectrophotometry and the underlying principles of absorbance, transmittance, and Beer's Law. The experiment measured absorbance of BPB and MO solutions at various wavelengths and plotted the absorption spectra. Results showed maximum absorbance of 505nm for MO and 600nm for BPB, matching theoretical wavelengths. Clinical applications of spectrophotometry in measuring bilirubin levels for hemolytic disease of newborns are also mentioned.

    Copyright:

    © All Rights Reserved
    Download as PPTX, PDF, TXT or read online from Scribd
    Download as pptx, pdf, or txt
    23 views30 pages

    Determination of Absorption Spectra

    Uploaded by

    say.ellese
    The document discusses spectrophotometry principles and experiments involving the absorption spectra of bromphenol blue (BPB) and methyl orange (MO). It defines spectrophotometry and the underlying principles of absorbance, transmittance, and Beer's Law. The experiment measured absorbance of BPB and MO solutions at various wavelengths and plotted the absorption spectra. Results showed maximum absorbance of 505nm for MO and 600nm for BPB, matching theoretical wavelengths. Clinical applications of spectrophotometry in measuring bilirubin levels for hemolytic disease of newborns are also mentioned.

    Copyright:

    © All Rights Reserved
    Download as PPTX, PDF, TXT or read online from Scribd
    Download as pptx, pdf, or txt
    of 30

    Determination of Absorption Spectra

    Group 6
    Salvador
    San Andres
    Sanchez
    Santos
    Say
    Learning Objectives:

    1. Demonstrate the principles of spectrophotometry

    2. Plot the absorption spectra of bromphenol blue (BPB) and methyl


    orange (MO)

    3. Identify the point of maximal absorption of BPB and MO


    Definition and Principles
    Spectrophotometry – a study
    of how much a chemical substance
    absorbs light by measuring the
    intensity of light as a beam passes
    through sample solution.

    Spectrophotometer
    Light
    Light is a form of electromagnetic
    radiation,
    transmitted via wavelengths,
    measured in nanometers (nm)

    a. Light can be reflected by the substance


    b. It can be absorbed by the substance
    c. It can be absorbed at certain wavelengths
    and the remainder will be transmitted or
    reflected
    Electromagnetic Spectrum
    the plot of a sample’s absorbance of light at various wavelengths
    Underlying Principles
    Transmittance: ratio of the amount of light transmitted to the
    amount of light that initially fell on the surface;

    %T= I0 x 100

    Absorbance: the negative logarithm of the transmittance

    A = 2 – log%T
    REMEMBER!!!

    Absorbance and transmittance


    bear an

    Inverse relationship
    Absorbance & Transmittance

    Computation
    ▪ First, the intensity of light (I0) passing through a blank is measured.
    The blank is a solution that is identical to the sample solution
    except that the blank does not contain the solute that absorbs
    light. This measurement is necessary, because the cell itself scatters
    some of the light.

    %T= I0 x 100 and A = 2 – log%T


    Absorbance & Transmittance
    Computation
    ▪ Second, the intensity of light (I) passing through the sample
    solution is measured.
    ▪ Third, the experimental data is used to calculate two
    quantities: the transmittance (T) and the absorbance (A).

    %T= I0 x 100 and A = 2 – log%T


    Discussion on Experiment on Beer-
    Lambert’s Law
    Learning Objectives:

    1. To discuss the Beer-Lambert’s Law

    2. To demonstrate the Beer-Lambert’s Law using the experimental set-


    up of bromphenol blue and methyl orange
    Beer-Lambert Law

    deals with the relationship between


    absorbance and concentration, and between
    absorbance and path length

    ▪ The concentration of a
    substance is directly
    proportional to the amount of
    radiant energy absorbed
    Beer-Lambert Law
    Absorbance ∝ path length (l) • concentration
    A= a•b•c = 2-log%T
    Where:
    a or ε = proportionality constant, the molar extinction
    coefficient or molar absorptivity
    b = path length of light traveled (cm)
    c = concentration of absorbing molecules in mol/L
    The relationship between absorbance and transmittance is illustrated in
    the following diagram:

    If all the light passes through a solution without any absorption,


    then absorbance is zero, and percent transmittance is 100%.
    If all the light is absorbed, then percent transmittance alpha is
    zero, and absorption is infinite.
    Flow of Experiment
    Flow Chart of the Experiment
    Determination of Absorption Spectrum
    (Methyl Orange, Bromphenol Blue)

    Take Readings of Optical Density of Diluted Sample

    Set Spectrometer at 100 Transmission

    Read test tube at different wavelength

    Plot wavelength versus optical density

    Draw a smooth curve through the points

    Indicate a point of max absorption


    Results
    Results of the
    Absorbance of Methyl Orange at Different Wavelengths

    Absorbance of Methyl Orange at Different Wavelengths

    Wavelength: Group 2: Group 4: Group Group Group


    6: 8: 10:

    340 0.899 0.908 0.905 0.906 0.896

    405 1.798 1.885 1.888 1.879 1.876

    450 1.633 1.630 1.632 1.631 1.635

    505 2.492 2.495 2.485 2.492 2.472

    550 1.474 1.463 1.465 1.466 1.461

    600 0.012 0.015 0.019 0.025 0.021


    Result of the
    Absorbance of Bromphenol Blue at Different Wavelengths
    Absorbance of Bromphenol Blue at Different Wavelengths

    Wavelengt Group 1: Group 3: Group Group


    h: 5: 7: Group
    9:

    340 1.402 1.355 1.295 1.119 1.218

    405 2.056 2.048 2.040 2.046 2.038

    450 1.662 1.647 1.637 1.628 1.538

    505 2.060 2.017 2.020 2.038 2.011

    550 2.476 2.209 2.418 2.420 2.416

    600 2.916 2.921 2.925 2.939 2.941


    Theoretical Results vs Actual Results

    Analyte Theoretical Result Actual Result

    Methyl Orange 450nm 505nm

    Bromphenol Blue 580nm 600nm


    Absorbance of MO 460 nm

    Test tube: Dilution Absorbance


    (MO:Water)

    1 1:4 1.612

    2 2:3 1.540
    Absorbance

    3 3:2 1.631

    4 4:1 1.649

    5 Pure MO 1.675

    Test tube and Dilution


    Absorbance of BPB at 580 nm

    Test tube: Dilution Absorbance


    (BPB:Water)

    1 1:4 3.021

    2 2:3 3.090
    Absorbance

    3 3:2 3.050

    4 4:1 2.992

    5 Pure BPB 2.925

    Test Tube and Dilution


    Common Errors in Spectrometry

    1. Error found in any dilution procedure is in the thoroughness of mixing the


    solution
    a. it is extremely important to mix the solutions thoroughly
    before taking aliquots.
    Common Errors in Spectrometry

    2. Error involving the reading of the photometric values.

    a. may be observed by glancing at the scale of optical density


    that certain parts of the scale re-read with greater ease than
    other. (Calibration)
    Common Errors in Spectrometry

    3. Improper fit of tubings

    a. may produce “wiggling”, successive readings in the tube may


    not be alike.
    b. Scattering of light produced by the tubes may not be
    perfectly clean.
    Clinical Application
    Clinical Application and Other Applications
    HDN (Hemolytic Disease of Newborn)

    RBC RBC ↑Unconjugated Jaundice ↑kernicterus


    lysis bilirubin

    Treatment / management

    Measurement: Reading:
    Spectrophotometry Zone 1 - “mildly affected”
    Maximum absorbance Zone 2 - “moderately affected”
    at 540nm Zone 3 - “ severely affected
    SUMMARY

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