Spectrophotometry Lecture

Interaction of Radiation and Matter

Absorption and Fluorescence

Terms

Interaction of Light with Matter

Molecules Absorption Wavelengths

Spectrophotometry Although a number of different types of spectrophotometers exist all have one thing in common.  Utilize light energy to detect molecules in a solution  Light energy is reported to the user as wavelengths in nanometers (nm) Different spec’s utilize wavelengths that fall into different ranges.  Visible (VIS) nm  Ultraviolet (UV) nm

Absorption Spectrophotometer

Spectrophotometer

spectrophotometer measures intensity of a light beam after it is directed through and emerges from a solution  Ex: solution of copper sulfate (CuSO4) absorbs light  The red part of spectrum has been almost complete absorbed by CuSO4 and blue light has been transmitted  Gain greater sensitivity by directing red light through the solution because CuSO4 absorbs strongest at the red end of the visible spectrum  But to do this, we have to isolate the red wavelengths

Spectrum of visible light How do you isolate red wavelengths of light?  In a spectrophotometer, a light source gives off white light which strikes a prism, separating light into its component wavelengths:  Red wavelengths pass through CuSO4 solution and measure amount of red light absorbed Colored compounds absorb light differently depending on the l of incident light l = Wavelength, nm=nanometers

Design of Spectrophotometer THE BLANK  In order to effectively use a spectrophotometer we must first zero the machine  Blank contains everything except compound of interest which absorbs light. By zeroing machine using "the blank," any measured absorbance is due to the presence of solute of interest ABSORPTION SPECTRUM  Different compounds having dissimilar atomic and molecular interactions have characteristic absorption phenomena and absorption spectra which differ  The point (wavelength) at which any given solute exhibits maximum absorption of light (the peaks on the curves on the figure below) is defined as that compounds particular lmax

Design of Spectrophotometer

Electromagnetic Spectrum

How is Does a Spectrophotometer Work? Amount of a particular molecule of interest is measured according to amount of light that is absorbed  Absorbance data is compared to a standard of a known concentration to determine the concentration of the unknown.

How is Does a Spectrophotometer Work? All spec’s share following common features:  Lamp i.e. tungsten or deuterium  Prism or grating  Sample holder  Display

Absorption Spectrophotometer

Absorption Curve

Background, B

Detection Limit DL or LOD

Dynamic or linear range

Sensitivity

Calibration Curve

Calibration Curve Procedures

Calibration Curve Plot

Example Nitrite Analysis

Results

Plot of Results

Best Fit Line

How are Concentrations Obtained Using a Spec? More molec…more to absorb light  Note peaks of absorption curves Lambda max  Wavelength at which a molecule absorbs the most light Proteins, like other molecules, interact with certain wavelengths of light  Proteins absorption spectrum can be determined by measuring proteins light absorbance at different wavelengths.  Determine the lambda max for protein

How are Concentrations Obtained Using a Spec? Most proteins are colorless  Light in visible range will not work  Light in UV range will work for a colorless solution ~280 nm Does NOT distinguish between different protein types in a solution

Using Bradford Reagent Way to colorize proteins and use white light spectroscopy  Solution changes from brown to blue when proteins present. Degree of “blueness” of Bradford-protein mixture can be used to determine concentration of protein in a solution

How are Concentrations Obtained Using a Spec? Calculating protein concentration in an unknown sample  Known standards are mixed with Bradford reagent and their absorbance values are determined Standard curve generated.  known absorbance values can be plotted and concentrations determined

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