SpectrophotometryLecture

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) 350-700 nm Ultraviolet (UV) 200-350 nm

Absorption Spectrophotometer

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 absorbedAbsorbance 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 Lambdamax

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 lambdamax for protein

How are Concentrations Obtained Using a Spec? Most proteins are colorless

Light in visible range will not workLight 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

This project is funded by a grant awarded under the President’s Community Based Job Training Grant as implemented by the U.S. Department of Labor’s Employment and Training Administration (CB-15-162-06-60). NCC is an equal opportunity employer and does not discriminate on the following basis: •against any individual in the United States, on the basis of race, color, religion, sex, national origin, age disability, political affiliation or belief; and•against any beneficiary of programs financially assisted under Title I of the Workforce Investment Act of 1998 (WIA), on the basis of the beneficiary’s citizenship/status as a lawfully admitted immigrant authorized to work in the United States, or his or her participation in any WIA Title I-financially assisted program or activity.

“This workforce solution was funded by a grant awarded under the President’s Community-Based Job Training Grants as implemented by the U.S. Department of Labor’s Employment and Training Administration.  The solution was created by the grantee and does not necessarily reflect the official position of the U.S. Department of Labor.  The Department of Labor makes no guarantees, warranties, or assurances of any kind, express or implied, with respect to such information, including any information on linked sites and including, but not limited to, accuracy of the information or its completeness, timeliness, usefulness, adequacy, continued availability, or ownership.  This solution is copyrighted by the institution that created it.  Internal use by an organization and/or personal use by an individual for non-commercial purposes is permissible.  All other uses require the prior authorization of the copyright owner.”