Many molecules absorb ultraviolet or visible light.

Absorbance is directly proportional to the path length, b, and the concentration, c, of the absorbing species. Beer's Law states that A = bc, where is a constant of proportionality, called the absorbtivity. Different molecules absorb radiation of different wavelengths..

The absortion of UV Vis radiation by an atom or molecular two step process 1. Excitation M + hv M* ( 10 -8 10 -9 2. M * M + M + energy

Absorbing spesies containing , , and n elektron for organic molekul ion of inorganic anion

 Electronic transitions The absorption of UV or visible radiation corresponds to the excitation of outer electrons. There are three types of electronic transition which can be considered; Transitions involving , , and n electrons Transitions involving charge-transfer electrons Transitions involving d and f electrons (not covered in this Unit) When an atom or molecule absorbs energy, electrons are promoted from their ground state to an excited state. In a molecule, the atoms can rotate and vibrate with respect to each other. These vibrations and rotations also have discrete energy levels, which can be considered as being packed on top of each electronic level.

organic dan anorganic

 * Transitions An electron in a bonding orbital is excited to the corresponding anti bonding orbital. For example, methane (which has only CH bonds, and can only undergo * transitions) shows an absorbance maximum at 125 nm.

 n * Transitions Saturated compounds containing atoms with lone pairs (non-bonding electrons) are capable of n * transitions. These transitions usually need less energy than * transitions. They can be initiated by light whose wavelength is in the range 150 - 250 nm. The number of organic functional groups with n * peaks in the UV region is small.

 n * and * Transitions Most absorption spectroscopy of organic compounds is based on transitions of n or electrons to the * excited state. absorption peaks for these transitions spectrum (200 - 700 nm). These transitions need an unsaturated group in the molecule to provide the p electrons. Molar absorbtivities from n * transitions are relatively low, and range from 10 to100 L mol-1 cm-1 * transitions normally give molar absorbtivities between 1000 and 10,000 L mol-1 cm-1 .

2. Transitions involving d and f electrons

3. Transitions charge-transfer electrons Many inorganic species show chargetransfer absorption and are called chargetransfer complexes. For a complex to demonstrate charge-transfer behaviour, one of its components must have electron donating properties and another component must be able to accept electrons.

Schematic of a single beam uv-vis spectrophotometer

schematic diagram of a doublebeam UV-Vis. spectrophotometer;

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

 Sources of UV radiation 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/Wolfram filament lamp is commonly employed as a source of visible light. wavelength range of 350 - 2500 nm. Tungsten/halogen lamps contain a small amount of iodine in a quartz The iodine reacts with gaseous tungsten, formed by sublimation, producing the volatile compound WI2. W + I2 WI2 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), several dynodes (which emit several electrons for each electron striking them) and an anode Photomultipliers are very sensitive to UV and visible radiation. They have fast response times.

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 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.

Scematic of the hitachi 100-60 manual double- beam spectrophotometer

Chromophore

Example

Excitation

max, nm

Solvent

C=C

Ethene

__

> *

171

15,000

hexane

CC

1-Hexyne

__

> *

180

10,000

hexane

C=O

Ethanal

__

> * __> *

290 180

15 10, 000

hexane hex ane

N=O

Nitromethane

__

> * __> *

275 200

17 5,0 00

ethanol eth ano l

C-X X=Br X=I

Methyl bromide Methyl Iodide

__

> * n __> *

205 255

200 360

hexane hex ane

 The Importance of Conjugation A comparison of the absorption spectrum of 1pentene, max = 178 nm, with that of isoprene (above) clearly demonstrates the importance of chromophore conjugation. Further evidence of this effect is shown below. The spectrum on the left illustrates that conjugation of double and triple bonds also shifts the absorption maximum to longer wavelengths. From the polyene spectra displayed in the center diagram, it is clear that each additional double bond in the conjugated pi-electron system shifts the absorption maximum about 30 nm in the same direction. Also, the molar absorptivity () roughly doubles with each new conjugated double bond.

Larger picture of the Spectronic 20D with labels.

Pictures of single beam uv-vis spectrophotometers (Spectronic 20 and 20D

 Luminescence is the emission of light by a substance. It occurs when an electron returns to the electronic ground state from an excited state and loses it's excess energy as a photon.

 Luminescence spectroscopy is a collective name given to three related spectroscopic techniques. They are;
Molecular fluorescence spectroscopy Molecular phosphorescence spectroscopy Chemiluminescence spectroscopy

Fluorescence and phosphorescence (photoluminescence)

The electronic states of most organic molecules can be divided into singlet states and triplet states;

Singlet state: All electrons in the molecule are spin-paired Triplet state: One set of electron spins is unpaired

Fluorescence
Absorption of UV radiation by a molecule excites it from a vibrational level in the electronic ground state to one of the many vibrational levels in the electronic excited state. This excited state is usually the first excited singlet state. A molecule in a high vibrational level of the excited state will quickly fall to the lowest vibrational level of this state by losing energy to other molecules through collision. The molecule will also partition the excess energy to other possible modes of vibration and rotation. Fluorescence occurs when the molecule returns to the electronic ground state, from the excited singlet state, by emission of a photon. If a molecule which absorbs UV radiation does not fluoresce it means that it must have lost its energy some other way. These processes are called radiationless transfer of energy.

Intra-molecular redistribution of energy between possible electronic and vibrational states

The molecule returns to the electronic ground state.The excess energy is converted to vibrational energy (internal conversion), and so the molecule is placed in an extremely high vibrational level of the electronic ground state. This excess vibrational energy is lost by collision with other molecules (vibrational relaxation). The conversion of electronic energy to vibrational energy is helped if the molecule is "loose and floppy", because it can reorient itself in ways which aid the internal transfer of energy.

A combination of intra- and intermolecular energy redistribution

The spin of an excited electron can be reversed, leaving the molecule in an excited triplet state; this is called intersystem crossing. The triplet state is of a lower electronic energy than the excited singlet state. The probability of this happening is increased if the vibrational levels of these two states overlap. For example, the lowest singlet vibrational level can overlap one of the higher vibrational levels of the triplet state. A molecule in a high vibrational level of the excited triplet state can lose energy in collision with solvent molecules, leaving it at the lowest vibrational level of the triplet state. It can then undergo a second intersystem crossing to a high vibrational level of the electronic ground state. Finally, the molecule returns to the lowest vibrational level of the electronic ground state by vibrational relaxation.

Phosphorescence
A molecule in the excited triplet state may not always use intersystem crossing to return to the ground state. It could lose energy by emission of a photon. A triplet/singlet transition is much less probable than a singlet/singlet transition. The lifetime of the excited triplet state can be up to 10 seconds, in comparison with 10-5 s to 10-8 s average lifetime of an excited singlet state. Emission from triplet/singlet transitions can continue after initial irradiation. Internal conversion and other radiationless transfers of energy compete so successfully with phosphorescence that it is usually seen only at low temperatures or in highly viscous media.

Chemiluminescence
Chemiluminescence occurs when a chemical reaction produces an electronically excited species which emits a photon in order to reach the ground state. These sort of reactions can be encountered in biological systems; the effect is then known as bioluminescence. The number of chemical reactions which produce chemiluminescence is small. However, some of the compounds which do react to produce this phenomenon are environmentally significant. A good example of chemiluminescence is the determination of nitric oxide: NO + O3 NO2* + O2 NO2* NO2 + hv (l = 600 - 2800 nm)

The following graph shows the spectral distribution of radiation emitted by the above reaction:

schematic diagram of a fluorometer / spectrofluorometer

 Often, fluorescence spectrometers use double-beam optics to compensate for power fluctuations in the source. The fluorescent emission is measured at right angles to the incident beam. Emitted radiation passes through a second filter or monochromator to isolate the fluorescent peak for measurement. The reference beam passes through an attenuator to reduce its power to that of the fluorescent radiation. Fluorometers use filters to restrict excitation and emission beam wavelengths. Spectrofluorometers have two monochromators; one allowing choice of excitation wavelength and the other allowing fluorescence emission spectra to be scanned.

 Sources Generally, the source must be more intense than that required for UV-Vis. absorption spectroscopy; magnitude of the emitted radiation is directly proportional to the power of the source. Filter fluorometers often employ a low-pressure mercury vapour lamp. This source produces intense lines at certain wavelengths. One of these lines will usually be suitable for excitation of a fluorescent sample. Spectrofluorometers, which need a continuous radiation source, are often equipped with a 75-450 W highpressure xenon arc lamp. Lasers are sometimes used as excitation sources. A tunable dye laser, using a pulsed nitrogen laser as the primary source can produce monochromatic radiation between 360 and 650 nm. Since the radiation produced is monochromatic, there is no need for an excitation monochromator.

 Filters and monochromators Fluorometers use either interference or absorption filters. Spectrofluorometers are usually fitted with grating monochromators.

 Detectors Fluorescence signals are usually of low intensity. Photomultiplier tubes are in common use as detectors. Diode-array detectors are sometimes used.

 Phosphorimeters Instruments for measuring phosphorescence are very similar to those used for fluorescence. However, two additional components are needed: A mechanism or electronic circuit is required which allows the sample to be irradiated, and then after a time delay, allows measurement of phosphorescent intensity. Since phosphorescence is aided by low temperatures and a viscous medium, the analyte is present as a solute in a solid solvent "glass" in liquid nitrogen. A Dewar flask with quartz windows is often used.