Introduction to Spectrophotometry

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

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.

Electromagnetic Spectrum

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

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

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.

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.

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

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.

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

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

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.

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)

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

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

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

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

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)

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.

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

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.

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.

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.

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.