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Spectrophotometer PowerPoint PPT Presentation


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Spectrophotometer. Two methods to detect molecules:. Indicator – Quickest method. Indicator solutions change colors when a molecule of interest is present. Allows scientist to detect colorless molecules in a solution. Examples: Bradford protein reagent Diphenylamine (DPA) or

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Spectrophotometer

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Spectrophotometer l.jpg

Spectrophotometer


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Two methods to detect molecules:

  • Indicator – Quickest method.

    • Indicator solutions change colors when a molecule of interest is present.

    • Allows scientist to detect colorless molecules in a solution.

    • Examples: Bradford protein reagent

      Diphenylamine (DPA) or

      Biuret (NaOH+CuSO4).

      What kind of data is this?

Qualitative!


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Two methods to detect molecules:

  • Spectrophotometer (Spec)

    • An instrument that measures the amount of light that passes through (is transmitted through) a sample.


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Spectrophotometer cont…

  • Uses a type of light to detect molecules in a solution

  • Light is a type of energy, and the energy is reported as wavelengths, in nanometers (nm).


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Two different types of Spectrophotometer:

  • Ultraviolet (UV) Spectrophotometers.

    • Uses ultraviolet light of wave lengths from 200 nm to 350 nm.

  • Visible (VIS) Light Spectrum Spectrophotometers.

    • Uses visible light (white light) of wave lengths from 350 nm to 700 nm.


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The visible light spectrum

Y

R

O

G

B

I

V


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Spectrophotometer cont…

  • Shines a beam of light on a sample.

  • The molecules in the sample interact with the light waves in of 3 ways:

    • Absorb the energy

    • Reflect the energy

    • Transmit the energy between and through the atoms and molecules of the sample.


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But why is the Box Blue?

Blue molecules are blue because they reflect blue light.

Blue molecules absorb the other

colors of visible light.


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How a spectrophotometer works:

  • Consider blue molecules, all the wavelengths of light are absorbed, except for the blue ones.

  • The blue wavelengths are transmitted or reflected off the molecules. If these blue wavelengths hit a detector (such as in the spectrophotometer or the nerve cells in your eye), they appear blue.


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How a spectrophotometer works:

  • Molecules are whatever color of light that they do not absorb.

  • Green molecules appear green because they absorb most wavelengths of visible light, except the green wavelengths.


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Spectrophotometer cont…

  • The spectrophotometer measures the amount of light transmitted through the sample (Transmittance).

  • By using an equation (Beers law), it converts the transmittance data to an absorbance value.

    What kind of data is this?

Quantitative!


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Spectrophotometer cont…

  • The concentration of an unknown sample can be determined by comparing the absorbance data to standards of known concentration.

  • The data generated with the set of known standards is called a standard curve.


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Parts of a spectrophotometer

  • Inner parts

    • Lamp

    • Prism or grating that direct light of a specific wavelength.


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VIS Spec vs. UV spec

  • Visible spectrophotometer

    • Contains a tungsten lamp that produces whitelight.

  • Ultraviolet spectrophotometer

    • Contains a deuterium lamp that produces light in the UV light part of the spectrum.


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Parts of a Spectrophotometer

  • Outer parts:

WavelengthSelection

Display

Sample Holder

Knobs or buttons used to calibrate the

spec to measure the designated molecule.


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How a spectrophotometer works:

  • Visible Spectrophotometer

    • White light hits the prism or grating, it is split into the colors of the rainbow (Visible Spectrum).

    • The wavelength knob rotates the prism/grating, directing different color of light toward the sample.


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How a spectrophotometer works:

  • The wavelength of light produced by the tungsten lamp range from about 350 nm (Violet light) to 700 nm (red light).

  • The molecules in the sample either absorb or Transmit the light energy of one wavelength or another.


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How a spectrophotometer works:

  • The detector measures the amount of light being transmitted by the sample and reports that value directly (% transmittance) or converts it to the amount of light absorbed in absorbance units (au) using Beers Law.


Beers law aka beers lambert law no you don t need to memorize it yes you need to write it down l.jpg

Beers Law aka Beers Lambert law(No You Don’t Need to Memorize it….YES you need to write it down!)

A = 2 – log10%T


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Absorption Spectrum

  • After collecting data for your concentration an absorption spectrum graph is created.

  • These can be used when attempting to identify unknown substances (e.g. CSI)


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The Absorbance Spectrum

The absorbance spectrum is a

graph of a sample’s absorbance at different wavelengths.


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Example of absorption spectrum of Chlorophyll a & b, and Carotenoids


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The function of a spectrophotometer

The spectrophotometer can

measure the amount of absorbance

or

lack of absorbance of different colored light for a given molecule.


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The relationship of concentration in a solution:

  • The concentration of molecules in a solution affects the solution’s absorbance.

    • Remember [ ] is a ratio – when we change one number – it affects the ratio

  • If there are more molecules in one solution than in another, than there are more molecules to absorb the light.


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    Applications of a spectrophotometer

    • Determines the presence and concentrations of samples.

    • Determines the purity of a sample.

    • Look at the change of samples over time.


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    OVERVIEW OF QUANTITIVE SPECTROPHOTOMETRY

    A. Measure the absorbance of standards containing known concentrations of the analyte

    B. Plot a standard curve with absorbance on the X axis and analyte concentration on the Y axis

    C. Measure the absorbance of the unknown(s)

    D. Determine the concentration of material of interest in the unknowns based on the standard curve


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    Useful Links

    http://web.umr.edu/~gbert/color/AAcolor.html

    http://www.chm.davidson.edu/java/spec/spec.html


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