Visible Spectroscopy Revised 10/07/10 1 VISIBLE SPECTROSCOPY Visible spectroscopy is the study of the interaction of radiation from the

visible part ( = 380 720 nm) of the electromagnetic spectrum with a chemical species. Quantifying the interaction of visible light with a chemical sample allows for the determination of an unknown solution concentration, the monitoring of reaction progress as a function of time, and many other quantitative uses. Understanding visible spectroscopy requires understanding visible light. Light travels in packets of energy called photons. Each photon has a specific energy related to a certain frequency or wavelength (E = h = hc/). Visible light consists of wavelengths ranging from 380 nm (blue violet) to 720 nm (red). When all wavelengths of visible light are present, the light appears "white" to our eyes. If any wavelength is removed (absorbed), we perceive the remaining combination of wavelengths of light as the "complimentary" color (Table 1, Figure 1). Table 1: Absorbed & Perceived Colors Absorbed Wavelength (nm) Absorbed Color Perceived (Transmitted) Color 400 violet green - yellow 450 indigo yellow

480 blue orange 490 blue-green red 530 green purple 570 yellow-green dark blue 600 orange blue 650 red green Figure 1: Color Wheel red orange yellow violet blue green 750 630 590 560 480 430 400 Colored compounds are colored because of the absorption of visible radiation. The color is a result of the compound absorbing a certain color of light, leading to the perception of the compound being the complimentary color. For example, if white light passes through a test tube containing a solution of copper (II) sulfate (CuSO4), the solution will be blue because the Cu 2+

ions strongly absorb orange photons of light (photons of light with ~ 600 nm).Visible Spectroscopy Revised 10/07/10 2 When a photon of colored light is absorbed by a compound an electron transitions from lower energy orbital to higher energy orbital. The energy of absorbed radiation is equal to the energy difference between the highest energy electronic occupied orbital (OO) and the closest unoccupied orbital (UO). Many transition metal complexes and large conjugated organic molecules are brightly colored because this energy difference is equal to an energy within the visible region of the electromagnetic spectrum. Mathematically, this relationship is expressed by equation 1: (1) Elight = hlight = hc/light = E = EUO EOO Before the absorption of a photon, the electrons within the compound are in the lowest energy orbitals possible. Such an electron configuration is called the ground state. When a photon of visible radiation is absorbed by a compound an electron is promoted from an occupied orbital to an unoccupied orbital. The result is a higher energy compound in an excited state. (Figure 2) Occupied Orbital Unoccupied Orbital EOO EUO EOO EUO

hlight EOO EUO Ground State Electron Configuration Excited State Electron Configuration Absorption of a Photon of Visible Light Figure 2. Absorption of light resulting in the excitation of an electron The wavelength (i.e., frequency, energy, or color) of light required to promote an electron from the ground to the excited state is specific to each chemical, just as the energy difference between EOO and EUO is dependent on chemical identity. Most methods of measuring absorbance required that the compound be in the liquid form or dissolved in a liquid solution. Once in solution, the amount of a particular energy of light passing (transmitted) through that solution is quantified as transmittance. Transmittance is Visible Spectroscopy Revised 10/07/10 3 calculated by taking the ratio of the intensity (amount) of light leaving the chemical sample (I) to the intensity (amount) of light entering the chemical sample (Io). (Figure 3) I0 I I0 : Incident Light (light entering sample) I: Transmitted Light

(light leaving sample) colored solution cuvette Figure 3. Transmittance (T = I/I0) of Light by a Sample Absorbance (A) (the amount of light absorbed by the chemical sample) is calculated from the transmittance (2): (2) A = log (1/T) = log Tor A = 2.000 log %T Absorbances are measured by an instrument called a spectrophotometer or spectrometer. A spectrometer contains a light source, focusing lenses, a diffraction grating or prism to split light into different wavelengths, a sample holder or "cell", a photosensitive detector which measures the light passing through the sample, an amplifier, and an output device such as a meter or recorder. The spectrometer used in UCIs General Chemistry Labs is manufactured by Ocean Optics, Inc. The solution to be analyzed is poured into a special vessel called a cuvette. A cuvette is a rectangular box with two opposing clear walls. Typically, two cuvettes are used when measuring a sample. One contains the blank (typically, the solvent only) and the other contains the sample dissolved in the same solvent. To prevent spilling and insure a good measurement, fill a cuvette with solution to about 75% of its total volume. Wipe the outside of a cuvette with a tissue before placing it in the sample holder. Beads of water, fingerprints, or bubbles in the solution interfere with transmission of Visible Spectroscopy Revised 10/07/10 4 light through the sample. Gently insert a cuvette into the sample holder so that the light passes through its opposing clear smooth walls (take care not to spill solutions into the spectrometer). Replace scratched cuvettes. Absorption Spectrum

The absorption spectrum is a plot of the absorbance of a sample as a function of wavelength. This plot can be used to help identify an unknown sample since some compounds have characteristic absorption spectra. Figure 6 shows the absorbance spectrum in the visible region for a complex metal ion. Maximum absorption for this ion occurs at a wavelength of approx. 560 nm. Figure 6: A Plot of Absorbance vs. Wavelength for a Metal Ion Complex Beer's Law Plot For dilute solutions, the amount of light absorbed at a specific wavelength is directly proportional to the concentration of the solution. This relationship is called Beer's Law (3). (3) A = C l A = absorbance (no units) = molar absorptivity coefficient (units = L/mol-cm) C = concentration of absorbing species (units = mol/L) l = path length (units = cm)Visible Spectroscopy Revised 10/07/10 5 A Beers Law Plot is a calibration curve of absorption plotted as a function of concentration. An absorption spectrum must be acquired first to determine the wavelength of maximum absorbance, max, for the compound being studied. All absorbances are acquired at this wavelength setting because the signal is the strongest and least likely to be obscured by instrument fluctuations. To create the plot, the absorbances of at least three solutions of known concentration are measured. A graph of absorbance versus concentration is constructed and a best fit straight line is drawn through the data points. Then the absorbance of a solution of unknown concentration is measured and its concentration is determined by comparison to the Beer's Law Plot. The Plot must be used in two ways to determine the unknowns concentration. The first way is visual: a horizontal line is drawn from the value of the experimentally found absorbance on the y-axis to the calibration curve; a vertical line is then

drawn to the x-axis to determine the value of the independent variable (the unknown solution concentration). The second way is mathematical, utilizing the line equation for the calibration curve. This equation is easily generated by a graphing program (See Graphing Techniques for Excel instructions). Plug in the experimentally found value for y and solve for x to find the solution concentration. Both methods must be utilized. Example: Absorbance readings are taken for seven standard cobalt (II) chloride solutions and a Beers Law Plot is created with the equation: y = 4.8571x + 0.0038. A cobalt (II) chloride solution of unknown concentration is found to have an absorbance of 0.28. The unknown concentration can be calculated by setting y = 0.28 and solving for x. Also, by a visual comparison to the plot (Figure 7), the concentration of the unknown is 0.057 mol/L. Figure 7: A Beer's Law Plot for a CoCl2(aq) at 560 nm. Visible Spectroscopy Revised 10/07/10 6 Notice that the best-fit line in the plot appears to pass through the origin; that is, absorbance equals zero when concentration equals zero, as expected. (As shown, the y-intercept is very close to zero, 0.0038. What experimental error might have resulted from the deviation?) However, Beer's Law is linear only for very dilute solutions and may deviate from linearity at higher concentrations. The range of dilution for a particular compound must be determined by experiment. (For some compounds the range may be 0.01-0.1 M, for others it may be closer to 10 -5 to 10 -6 M.) Interferences Because absorption is dependent on concentration, identity of the absorbing species, and path

length, experiments need to be conducted carefully so that there is no error caused by changes in the path length of light during the experiment or interferences caused by other absorbing species in the solution. The path length of light can be held constant if the same cuvette or identical cuvettes are used for each measurement. The cuvettes provided to you will have standard, reproducible diameters and volumes. The solvent, impurities in the solvent, and/or the cuvette glass can absorb light in the selected region. These interferences can be corrected or eliminated by calibrating the spectrometer with the blank in the sample compartment. In effect, this act instructs the instrument to ignore any absorbance from materials in the glass or solvent and to detect only the absorbance from the particular species to be measured in the sample. (A similar analogy is the taring of a balance.) Review Questions: Define: absorbance, transmittance, blank, path length, Beer's Law, cuvette. If a solution is blue-colored, what wavelength of light (in nm) is being absorbed? What is absorbance when percent transmittance is equal to 58%? When absorbance is infinite, what is %T?Visible Spectroscopy Revised 10/07/10 7 If fingerprints are left on the cuvette, how does it affect A and %T? A student rinses a cuvette with DI water and then pours his solution into the wet cuvette. How does this affect A and %T? How does a student correct for an absorbing impurity present in the solvent? How does the student correct for any stray light that might enter the sample holder? What is the purpose of the mark on the edge of the sample holder? What is being measured in an "absorption spectra"? What is being measured in a Beer's Law Plot? What two factors are held constant when

constructing the plot? Why is the wavelength of light set at maximum absorbance when making a Beer's Law Plot? Does the wavelength change while doing the experiment? Which solution is expected to deviate from the straight line in a Beer's Law plot: the more concentrated or more dilute solution? Is the straight line of a Beer's Law plot expected to pass through the origin of the graph or at some point above the origin?

Instrumentation

Introduction Have a look at this schematic diagram of a double-beam UV-Vis. spectrophotometer;

Instruments for measuring the absorption of U.V. or visible radiation are made up of the following components; 1. 2. 3. 4. 5. Sources (UV and visible) Wavelength selector (monochromator) Sample containers Detector Signal processor and readout

Each of these components will be considered in turn.

Instrumental components Sources of UV radiation It is important that the power of the radiation source does not change abruptly over it's wavelength range. The electrical excitation of deuterium or hydrogen at low pressure produces a continuous UV spectrum. The mechanism for this involves formation of an excited molecular species, which breaks up to give two atomic species and an ultraviolet photon. This can be shown as; D2 + electrical energy D2* D' + D'' + hv Both deuterium and hydrogen lamps emit radiation in the range 160 - 375 nm. Quartz windows must be used in these lamps, and quartz cuvettes must be used, because glass absorbs radiation of wavelengths less than 350 nm. Sources of visible radiation

The tungsten filament lamp is commonly employed as a source of visible light. This type of lamp is used in the wavelength range of 350 - 2500 nm. The energy emitted by a tungsten filament lamp is proportional to the fourth power of the operating voltage. This means that for the energy output to be stable, the voltage to the lamp must be very stable indeed. Electronic voltage regulators or constant-voltage transformers are used to ensure this stability. Tungsten/halogen lamps contain a small amount of iodine in a quartz "envelope" which also contains the tungsten filament. The iodine reacts with gaseous tungsten, formed by sublimation, producing the volatile compound WI2. When molecules of WI2 hit the filament they decompose, redepositing tungsten back on the filament. The lifetime of a tungsten/halogen lamp is approximately double that of an ordinary tungsten filament lamp. Tungsten/halogen lamps are very efficient, and their output extends well into the ultra-violet. They are used in many modern spectrophotometers. Wavelength selector (monochromator) All monochromators contain the following component parts;

An entrance slit A collimating lens A dispersing device (usually a prism or a grating) A focusing lens An exit slit

Polychromatic radiation (radiation of more than one wavelength) enters the monochromator through the entrance slit. The beam is collimated, and then strikes the dispersing element at an angle. The beam is split into its component wavelengths by the grating or prism. By moving the dispersing element or the exit slit, radiation of only a particular wavelength leaves the monochromator through the exit slit. Czerney-Turner grating monochromator

Cuvettes

The containers for the sample and reference solution must be transparent to the radiation which will pass through them. Quartz or fused silica cuvettes are required for spectroscopy in the UV region. These cells are also transparent in the visible region. Silicate glasses can be used for the manufacture of cuvettes for use between 350 and 2000 nm. Detectors The photomultiplier tube is a commonly used detector in UV-Vis spectroscopy. It consists of a photoemissive cathode (a cathode which emits electrons when struck by photons of radiation), severaldynodes (which emit several electrons for each electron striking them) and an anode. A photon of radiation entering the tube strikes the cathode, causing the emission of several electrons. These electrons are accelerated towards the first dynode (which is 90V more positive than the cathode). The electrons strike the first dynode, causing the emission of several electrons for each incident electron. These electrons are then accelerated towards the second dynode, to produce more electrons which are accelerated towards dynode three and so on. Eventually, the electrons are collected at the anode. By this time, each original photon has produced 106 - 107 electrons. The resulting current is amplified and measured. Photomultipliers are very sensitive to UV and visible radiation. They have fast response times. Intense light damages photomultipliers; they are limited to measuring low power radiation. Cross section of a photomultiplier tube

The linear photodiode array is an example of a multichannel photon detector. These detectors are capable of measuring all elements of a beam of dispersed radiation simultaneously. A linear photodiode array comprises many small silicon photodiodes formed on a single silicon chip. There can be between 64 to 4096 sensor elements on a chip, the most common being 1024 photodiodes. For each diode, there is also a storage capacitor and a switch. The individual diode-capacitor circuits can be sequentially scanned. In use, the photodiode array is positioned at the focal plane of the monochromator (after the dispersing element) such that the spectrum falls on the diode array. They are useful for recording UV-Vis. absorption spectra of samples that are rapidly passing through a sample flow cell, such as in an HPLC detector. Charge-Coupled Devices (CCDs) are similar to diode array detectors, but instead of diodes, they consist of an array of photocapacitors.