1. Introduction to Spectrophotometry

2. Spectroscopy Is the study of the interaction of light & matter
Spectrophotometer – instrument that uses electromagnetic radiation from UV, visible or IR to analyze the absorption or transmission of a sample
We will use visible in our lab

3. Properties of Light Electromagnetic radiation moves in waves
Light (called electromagnetic radiation) moves in waves.
Wavelength = different types of light have different wavelengths. Some are longer than others. For instance, in the visible light spectrum, red light waves are longer than blue light waves.

4. Electromagnetic Spectrum

5. Electromagnetic Spectrum
Wavelengths are commonly given in ????
Lambda λ

6. Visible Light Colors & Wavelengths COLOR WAVELENGTH (λ in nm)
Ultraviolet
< 380
Violet
380 – 435
Blue
436 – 480
Greenish-blue
481 – 490
Bluish-green
491 – 500
Green
501 – 560
Yellowish-green
561 – 580
Yellow
581 – 595
Orange
596 – 650
Red
651 – 780
Near Infrared
> 780
Visible Light

7. What is Colorimetry?
The solutions of many compounds have characteristic colors.
The intensity of such a color is proportional to the concentration of the compound.

8. What are Spectroscopy and Spectrophotometry??
Light can either be transmitted or absorbed by dissolved substances
Presence & concentration of dissolved substances is analyzed by passing light through the sample
Spectroscopes measure electromagnetic emission
Spectrophotometers measure electromagnetic absorption
The presence and concentration of various substances dissolved in a water sample is commonly analyzed by passing different types of light (visible, infrared, or UV) through the sample.
Light can either be transmitted or absorbed by the dissolved substances.

9. Instruments of Measurement
Two most common:
Visible Spectrophotometer
Spect 20, Spect 88
Uses Xe or W lamps as light sources
Glass cuvettes hold the sample
Atomic-Absorption Spectrophotometer

10. Instruments of Measurement
What do visible spectrophotometers measure?
Amount of light absorbed by the dissolved substance
Qualitative – color gives info about the solution composition
Quantitative – provides numerical data for the concentration
The absorption of light indicates the presence of the substance. This is a qualitative measurement.
The amount of light absorbed measures the concentration of the dissolved substance. This is a quantitative measurement.

11. Absorption of Light White light All colors Polychromatic light
When white (polychromatic) light passes through a coloured solution some of the light is absorbed by the substances in the solution, and the rest passes through.
Green solution absorbs light other then green

12. Monochromator-spreads out light into its component wavelength
Absorption of Light
Monochromatic light
Light of one color
Monochromator-spreads out light into its component wavelength
If white light is made to pass through a red filter, all light except red is filtered out and absorbed. Therefore, only red light hits the solution.
Red light is absorbed
by the green solution

13. The Spectrophotometer
The monochromatic light is obtained by allowing the beam of light to pass through a prism or diffraction grating (monochrometer)
The monochromatic light is directed through a cuvette containing the sample, and the light that penetrates hits the photoelectric cell.
The current developed by the photoelectric cell is translated into percent transmission or absorbance through a galvanometer.
Then you can read the absorbance on the galvanometer.
Absorbance is also called extinction and optical density
In review, a spectrophotometer is used to measure the absorption of a certain color of light (specific wavelength) as it passes through a treated sample.
A compound of interest is reacted with reagents creating a colored substance.
It is the blockage or absorption of light by this secondary material (dye) that is actually measured.
The amount of dye created is directly proportional to the amount of the compound of interest present in the sample.
The original material’s concentration can be found by calculations or comparing the sample’s absorption to absorptions of various known strength solutions.

14. Success of spectrophotometry…
Requires sample to absorb light differently to the other chemicals in the solution
How is the correct wavelength selected?
The amount of light absorbed depends on the energy difference between 2 electron energy levels
Optimum wavelength for spectrophotometric analysis is selected by measuring the visible spectrum of the substance
This is done by plotting absorbance (A) versus wavelength (λ)

15. Food Dyes
Only 7 dyes are approved by the FDA for use in foods, drugs & cosmetics
All artificial food colors are mixtures of these 7 dyes
We will be using FD&C Blue in this lab

16. FD&C Blue 1 A solution containing this dye is blue in white light
The colors absorbed by solution are complementary to the transmitted color
Blue solution absorbs yellow, orange, & red light
So expect dye solution to peak at 580 – 650 nm
Optimum wavelength is determined from wavelength of max. absorption λmax = 630 nm for Blue 1
This is given for the blue solution but you will have to claculate this for the red
FD&C Blue 1

17. Wavelength of light absorbed:
Is related to electronic structure of substance
Intensity of light absorbed depends on the concentration of solution
More concentrated, the more intense color & the greater intensity of light absorbed
When light is absorbed, the radiant power (P) of light beam decreases

18. Transmittance (T)
This is the fraction of incident light (P/Po) that passes through the sample
T = P Po
Po = intensity of “blank”
Blank – is solution identical to sample but without solute

19. Definitions & Symbols Intensity (I) Transmittance (T)
It’s also referred to as %T or T x 100
T = P/Po
Where Po is the intensity of the blank
Can also use I = Intensity instead of Power
T = I / Io
I = the rate at which energy in a beam of radiation arrives at a fixed point.
T = the ratio of the intensity in a beam of radiation after it has passed through a sample to the power of the incident beam
T = I/ Io
Where I is the radiation transmitted by the solution and Io is the radiation transmitted by the pure solvent (blank)

20. Graphical Relationship
% transmission and % absorption are not linearly related to concentration
For a graph to be useful, a straight line is needed
ABSORBANCE = log(1/T) = -log(T)
If this is to be done graphically, a graphing problem has to be overcome. Light passage or percent transmittance is not linearly related to the dissolved substances concentration. Percent absorption is also not linearly related to concentration.
For a graph to be useful a straight line is needed (a linear relationship is needed).
A new optical property was derived from percent transmission that is linearly related to concentration called ABSORBANCE.
ABS = log(100/%T)
Absorbance is what is generally recorded from a spectrophotometer.

21. The amount of light absorbed depends upon:
Concentration (c)
Path length of sample cell (b) thru which light passes
Defined by Beer’s Law c P0 P b

22. Beer’s Law %T = Tx100 = P/P0x100% A = - log T A = ε b c
The intensity of a ray of monochromatic light decreases exponentially as the concentration of the absorbing medium increases
More dissolved substance = more absorption and less transmittance
ε = molar absorptivity coefficient and is constant for a substance
%T = Tx100 = P/P0x100%
A = - log T
A = ε b c
Beer’s Law is concerned with light absorption in relation to solution concentration and cell path length.
It states that the intensity of a ray of monochromatic light decreases exponentially as the concentration of the absorbing medium increases.
In other words, the more dissolved substance you have in a solution, the more light that will be absorbed, and the less light that will be transmitted through the solution.
If light is absorbed exponentially with concentration over a reasonable range of concentration, the colored, dissolved material is said to conform to Beer’s Law.
The best way to determine whether a colored compound obeys Beer’s Law is to prepare a series of samples in the desired range of concentration and submit them to a test on the spectrophotometer. If the observations plot on a straight line, the material can be considered to obey Beer’s Law.

23. Spectral Transmission Curve
Optimum wavelength
Before we can create our standardization curve, we must determine the optimum wavelength of the dissolved compound that we’re working with.
The optimum wavelength can be determined at any time by establishing a spectral transmission curve.
The curve is established by making a series of observations of light transmission at several different wavelengths of light.
The results when plotted yield a curve like the one shown above.
The optimum wavelength (characteristic wavelength) is the one with the highest absorbance and will be the wavelength at which the spec. is set for all subsequent absorbance measurements.

24. Standardization Graph
Standards (solutions of known concentration) of the compound of interest are made, treated, and their absorbances (ABS) and concentration values are used to create a Standardization Graph.
Again, specs are calibrated for use by preparing a series of standards and by making observations on light absorbance.
Standards (solutions of known concentration) of the compound of interest are made, treated, and their absorbances (ABS) and concentration values are used to create a Standardization Graph.

25. Standardization Graph
One reads the graph with the sample's ABS (Y axis), then reads across to the line (graphed relationship). The value below this intersection on the Concentration (X) axis is the compound's concentration in the sample.