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Introduction

Many compounds absorb ultraviolet (UV) or visible (Vis.) light. The diagram below shows a beam of monochromatic radiation of radiant power P0, directed at a sample solution. Absorption takes place and the beam of radiation leaving the sample has radiant power P. The amount of radiation absorbed may be measured in a number of ways: Transmittance, T = P / P0 % Transmittance, %T = 100 T Absorbance, A = log10 P0 / P A = log10 1 / T A = log10 100 / %T A = 2 - log10 %T The last equation, A = 2 - log10 %T , is worth remembering because it allows you to easily calculate absorbance from percentage transmittance data. The relationship between absorbance and transmittance is illustrated in the following diagram:

So, if all the light passes through a solution without any absorption, then absorbance is zero, and percent transmittance is 100%. If all the light is absorbed, then percent transmittance is zero, and absorption is infinite. The Beer-Lambert Law Now let us look at the Beer-Lambert law and explore it's significance. This is important because people who use the law often don't understand it - even though the equation representing the law is so straightforward: A=abc Where A is absorbance (no units, since A = log10 P0 / P ) a is the molar absorbtivity with units of L mol-1 cm-1
b is the path length of the sample - that is, the path length of the cuvette in which the sample is contained. We will express this measurement in

centimetres. c is the concentration of the compound in solution, expressed in mol L-1

The reason why we prefer to express the law with this equation is because absorbance is directly proportional to the other parameters, as long as the law is obeyed. We are not going to deal with deviations from the law. Let's have a look at a few questions... Question : Why do we prefer to express the Beer-Lambert law using absorbance as a measure of the absorption rather than %T ? Answer : To begin, let's think about the equations... A=abc %T = 100 P/P0 = e -abc Now, suppose we have a solution of copper sulphate (which appears blue because it has an absorption maximum at 600 nm). We look at the way in which the intensity of the light (radiant power) changes as it passes through the solution in a 1 cm cuvette. We will look at the reduction every 0.2 cm as shown in the diagram below. The Law says that the fraction of the light absorbed by each layer of solution is the same. For our illustration, we will suppose that this fraction is 0.5 for each 0.2 cm "layer" and calculate the following data: Path length / cm 0 0.2 0.4 0.6 0.8 1.0 %T Absorbance 100 0 50 0.3 25 0.6 12.5 0.9 6.25 1.2 3.125 1.5

A=abc tells us that absorbance depends on the total quantity of the absorbing compound in the light path through the cuvette. If we plot absorbance against concentration, we get a straight line passing through the origin (0,0).

Note that the Law is not obeyed at high concentrations. This deviation from the Law is not dealt with here.

The linear relationship between concentration and absorbance is both simple and straightforward, which is why we prefer to express the Beer-Lambert law using absorbance as a measure of the absorption rather than %T. Question : What is the significance of the molar absorbtivity, a? Answer : To begin we will rearrange the equation A = abc : a = A / bc In words, this relationship can be stated as " ais a measure of the amount of light absorbed per unit concentration". Molar absorbtivity is a constant for a particular substance, so if the concentration of the solution is halved so is the absorbance, which is exactly what you would expect. Question : What is the molar absorbtivity of Cu2+ ions in an aqueous solution of CuSO4 ? It is either 20 or 100,000 L mol-1 cm-1 Answer : I am guessing that you think the higher value is correct, because copper sulphate solutions you have seen are usually a beautiful bright blue colour. However, the actual molar absorbtivity value is 20 L mol-1 cm-1 ! The bright blue colour is seen because the concentration of the solution is very high. -carotene is an organic compound found in vegetables and is responsible for the colour of carrots. It is found at exceedingly low concentrations. You may not be surprised to learn that the molar absorbtivity of -carotene is 100,000 L mol-1 cm-1 !

You should now have a good understanding of the Beer-Lambert Law; the different ways in which we can report absorption, and how they relate to each other. You should also understand the importance of molar absorbtivity.

Theoretical principles

Introduction Different molecules absorb radiation of different wavelengths. An absorption spectrum will show a number of absorption bands corresponding to structural groups within the molecule. For example, the absorption that is observed in the UV region for the carbonyl group in acetone is of the same wavelength as the absorption from the carbonyl group in diethyl ketone.

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. 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. Absorption of ultraviolet and visible radiation in organic molecules is restricted to certain functional groups (chromophores) that contain valence electrons of low excitation energy. The spectrum of a molecule containing these chromophores is complex. This is because the superposition of rotational and vibrational transitions on the electronic transitions gives a combination of overlapping lines. This appears as a continuous absorption band. Charge - Transfer Absorption Many inorganic species show charge-transfer absorption and are called charge-transfer 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. Absorption of radiation then involves the transfer of an electron from the donor to an orbital associated with the acceptor. Molar absorbtivities from charge-transfer absorption are large (greater that 10,000 L mol-1 cm-1).

You should now be aware of why molecules absorb radiation in the UV and visible light regions, and why absorption spectra look the way they do.

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), several dynodes (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 diodecapacitor 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. Review your learning You should now have an understanding of the separate components which make up a spectrophotometer, and how they fit together. Have a look at this schematic of the Hitachi 100-60 manual double-beam spectrophotometer;

Do you understand what each component does?

Theoretical Principles

Introduction The term "infra red" covers the range of the electromagnetic spectrum between 0.78 and 1000 m. In the context of infra red spectroscopy, wavelength is measured in "wavenumbers", which have the units cm-1. wavenumber = 1 / wavelength in centimeters It is useful to divide the infra red region into three sections; near, mid and far infra red; Region Wavelength range ( m) Wavenumber range (cm-1) Near Middle 0.78 - 2.5 2.5 - 50 12800 - 4000 4000 - 200

Far

50 -1000

200 - 10

The most useful I.R. region lies between 4000 - 670cm-1. Theory of infra red absorption IR radiation does not have enough energy to induce electronic transitions as seen with UV. Absorption of IR is restricted to compounds with small energy differences in the possible vibrational and rotational states. For a molecule to absorb IR, the vibrations or rotations within a molecule must cause a net change in the dipole moment of the molecule. The alternating electrical field of the radiation (remember that electromagnetic radation consists of an oscillating electrical field and an oscillating magnetic field, perpendicular to each other) interacts with fluctuations in the dipole moment of the molecule. If the frequency of the radiation matches the vibrational frequency of the molecule then radiation will be absorbed, causing a change in the amplitude of molecular vibration. Molecular rotations Rotational transitions are of little use to the spectroscopist. Rotational levels are quantized, and absorption of IR by gases yields line spectra. However, in liquids or solids, these lines broaden into a continuum due to molecular collisions and other interactions. Molecular vibrations The positions of atoms in a molecules are not fixed; they are subject to a number of different vibrations. Vibrations fall into the two main categories of stretching and bending. Stretching: Change in inter-atomic distance along bond axis

Bending: Change in angle between two bonds. There are four types of bend:

Rocking Scissoring Wagging Twisting

Vibrational coupling In addition to the vibrations mentioned above, interaction between vibrations can occur (coupling) if the vibrating bonds are joined to a single, central atom. Vibrational coupling is influenced by a number of factors;

Strong coupling of stretching vibrations occurs when there is a common atom between the two vibrating bonds Coupling of bending vibrations occurs when there is a common bond between vibrating groups Coupling between a stretching vibration and a bending vibration occurs if the stretching bond is one side of an angle varied by bending vibration Coupling is greatest when the coupled groups have approximately equal energies No coupling is seen between groups separated by two or more bonds

You should now understand how certain molecules absorb infra red radiation, and the effects that this absorption has. You should be familiar with the ways in which molecules can vibrate, and factors which influence how these vibrations interact with each other.

Instrumentation

Introduction In this look at instrumentation for IR spectroscopy, we will be limiting our attention to instrumentation concerned with spectroscopy in the middle region (4000 - 200cm-1). It is absorption in this region which gives structural information about a compound. Instrumental components Sources An inert solid is electrically heated to a temperature in the range 1500-2000 K. The heated material will then emit infra red radiation. The Nernst glower is a cylinder (1-2 mm diameter, approximately 20 mm long) of rare earth oxides. Platinum wires are sealed to the ends, and a current passed through the cylinder. The Nernst glower can reach temperatures of 2200 K. The Globar source is a silicon carbide rod (5mm diameter, 50mm long) which is electrically heated to about 1500 K. Water cooling of the electrical contacts is needed to prevent arcing. The spectral output is comparable with the Nernst glower, execept at short wavelengths (less than 5 m) where it's output becomes larger.

The incandescent wire source is a tightly wound coil of nichrome wire, electrically heated to 1100 K. It produces a lower intensity of radiation than the Nernst or Globar sources, but has a longer working life. Detectors There are three catagories of detector;

Thermal Pyroelectric Photoconducting

Thermocouples consist of a pair of junctions of different metals; for example, two pieces of bismuth fused to either end of a piece of antimony. The potential difference (voltage) between the junctions changes according to the difference in temperature between the junctions Pyroelectric detectors are made from a single crystalline wafer of a pyroelectric material, such as triglycerine sulphate. The properties of a pyroelectric material are such that when an electric field is applied across it, electric polarisation occurs (this happens in any dielectric material). In a pyroelectric material, when the field is removed, the polarisation persists. The degree of polarisation is temperature dependant. So, by sandwiching the pyroelectric material between two electrodes, a temperature dependant capacitor is made. The heating effect of incident IR radiation causes a change in the capacitance of the material. Pyroelectric detectors have a fast response time. They are used in most Fourier transform IR instruments. Photoelectric detectors such as the mercury cadmium telluride detector comprise a film of semiconducting material deposited on a glass surface, sealed in an evacuated envelope. Absorption of IR promotes nonconducting valence electrons to a higher, conducting, state. The electrical resistance of the semiconductor decreases. These detectors have better response characteristics than pyroelectric detectors and are used in FT-IR instruments - particularly in GC - FT-IR. Types of instrument Dispersive infra red spectophotometers These are often double-beam recording instruments, employing diffraction gratings for dispersion of radiation. Radiation from the source is flicked between the reference and sample paths. Often, an optical null system is used. This is when the detector only responds if the intensity of the two beams is unequal. If the intensities are unequal, a light attenuator restores equality by moving in or out of the reference beam. The recording pen is attached to this attenuator. Fourier-transform spectrometers Any waveform can be shown in one of two ways; either in frequency domain or time domain.

Dispersive IR instruments operate in the frequency domain. There are, however, advantages to be gained from measurement in the time domain followed by computer transformation into the frequency domain. If we wished to record a trace in the time domain, it could be possible to do so by allowing radiation to fall on a detector and recording its response over time. In practice, no detector can respond

quickly enough (the radiation has a frequency greater than 1014 Hz). This problem can be solved by using interference to modulate the i.r. signal at a detectable frequency. The Michelson interferometer is used to produce a new signal of a much lower frequency which contains the same information as the original IR signal. The output from the interferometer is an interferogram. The Michelson interferometer

Radiation leaves the source and is split. Half is reflected to a stationary mirror and then back to the splitter. This radiation has travelled a fixed distance. The other half of the radiation from the source passes through the splitter and is reflected back by a movable mirror. Therefore, the path length of this beam is variable. The two reflected beams recombine at the splitter, and they interfere (e.g. for any one wavelength, interference will be constructive if the difference in path lengths is an exact multiple of the wavelength. If the difference in path lengths is half the wavelength then destructive interference will result). If the movable mirror moves away from the beam splitter at a constant speed, radiation reaching the detector goes through a steady sequence of maxima and minima as the interference alternates between constructive and destructive phases. If monochromatic IR radiation of frequency, f ( ir ) enters the interferometer, then the output frequency, fm can be found by;

where v is the speed of mirror travel in mm/s Because all wavelengths emitted by the source are present, the interferogram is extremely complicated. The moving mirror must travel smoothly; a frictionless bearing is used with electromagnetic drive. The position of the mirror is measured by a laser shining on a corner of the mirror. A simple sine wave interference paatern is produced. Each peak indicates mirror travel of one half the wavelength of the laser. The accuracy of this measurement system means that the IR frequency scale is accurate and precise. In the FT-IR instrument, the sample is placed between the output of the interferometer and the detector. The sample absorbs radiation of particular wavelengths. Therefore, the interferogram contains the spectrum of the source minus the spectrum of the sample. An interferogram of a reference (sample cell and solvent) is needed to obtain the spectrum of the sample. After an interferogram has been collected, a computer performs a Fast Fourier Transform, which results in a frequency domain trace (i.e intensity vs. wavenumber) that we all know and love. The detector used in an FT-IR instrument must respond quickly because intensity changes are rapid (the moving mirror moves quickly). Pyroelectric detectors or liquid nitrogen cooled photon detectors must be used. Thermal detectors are too slow. To acheive a good signal to noise ratio, many interferograms are obtained and then averaged. This can be done in less time than it would take a dipersive instrument to record one scan.

Advantages of Fourier transform IR over dispersive IR;

Improved frequency resolution Improved frequency reproducibility (older dispersive instruments must be recalibrated for each session of use) Higher energy throughput Faster operation Computer based (allowing storage of spectra and facilities for processing spectra) Easily adapted for remote use (such as diverting the beam to pass through an external cell and detector, as in GC - FT-IR)