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Basic UV/Visible Spectrophotometry
- INTRODUCTION CONTENTS
- THE ELECTROMAGNETIC SPECTRUM
- RADIATION AND THE ATOM
- RADIATION AND THE MOLECULE
- Electronic Transitions
- Vibration and Rotation
- SPECIFIC ABSORPTION
- ABSORPTION AND CONCENTRATION
- INSTRUMENTATION
- Source
- Monochromator
- Filters
- Prisms
- Diffraction Gratings
- Optical geometry
- Single beam optics
- Split (reference) beam optics
- Double beam optics
- Diode array optics
- Sample handling
- Detectors
- Photomultiplier
- Silicon diode
- Diode array
- Measuring Systems
- GOOD OPERATING PRACTICE
- Preferred absorption range
- Absorbance Measurement
- Solvent selection
- SOURCES OF ERROR
- Instrument related sources of error
- Spectral bandwidth and slit width
- Stray Light
- Absorbance Accuracy
- Wavelength Accuracy
- Noise
- Non-instrument sources of error
- EXPERIMENTS
- EXPERIMENT 1 - Calculation of absorbance at λ max, and measurement of natural bandwidth.
- EXPERIMENT 2 - Construction of concentration plots
- EXPERIMENT 3 - Testing for Stray Light
- EXPERIMENT 4 - Demonstration of visible colours and spectral sensitivity of the human eye.
1. Introduction
The spectrophotometer has well been called the workhorse of the modern laboratory. In particular, ultraviolet and visible spectrophotometry is the method of choice in most laboratories concerned with the identification and measurement of organic and inorganic compounds in a wide range of products and processes - in nucleic acids and proteins, foodstuffs, pharmaceuticals and fertilisers, in mineral oils and in paint. In every branch of molecular biology, medicine and the life sciences, the spectrophotometer is an essential aid to both research and routine control.
Modern spectrophotometers are quick, accurate and reliable and make only small demands on the time and skills of the operator. However, the user who wants to optimise the functions of his instrument and to be able to monitor its performance in critical areas will need to understand the elementary physics of the absorption process as well as the basic elements of spectrophotometer design.
This booklet sets out to present that background for both the student and the scientist, primarily neither chemist nor physicist, whose subject makes use of analytical procedures and who may want to know more than the basic outline. The molecular biologist, biochemist, geologist or the pathologist, the pharmacist or the metallurgist - almost any scientist whose discipline involves materials analysis - may feel more secure in his or her mastery of the mechanics of UV/Vis absorption spectrophotometry with this introduction to the underlying principles. He may, for example, prefer to make his own judgements about trade-offs in limiting conditions, and reference to the experiments at the end of the booklet may assist in this.
3. Radiation and the Atom
Although it is convenient to describe electromagnetic radiation in terms of waves, it is necessary to define another model in order to demonstrate clearly the interactions that lead to selective absorption by an atom or molecule. A determining factor is the energy level of the radiation and it is therefore helpful to consider radiation as discrete packages of energy, or quanta. A quantum of light is known as a photon.
The absorption process depends upon an atomic structure in which each of the electrons of an atom has an energy level associated with its position in the atom. Permitted energy levels are finite and well defined, but an electron may be made to change to another level if a quantum of energy is delivered equal to the energy difference between the two levels. The original level is called the ground state and the induced level is known as the excited state. Excited states are generally unstable and the electron will rapidly revert to the ground state, losing the acquired energy in the process.
Whilst the accepted model of atomic and molecular structure has arisen from the wave mechanical treatment of Schroedinger, it is convenient to employ an earlier model (that of Bohr) in order to explain more simply the electronic phenomena of interest in spectrophotometry.
The Bohr model defines an atom as having a number of electron shells, n1 - n2 - n3 etc, in which the increasing values of n represent higher energy levels and greater distance from the nucleus. Electrons rotate about the nucleus in orbits that may be characterised by the space they occupy and are designated s, p, d, etc according to their geometry. An atom may contain several electrons in multiple orbits in each shell (or each n level) but no orbit may contain more than two electrons (Fig. 3).
Fig. 3 Diagrammatic presentation of simplified electron energy levels in an atom. No two electrons can have identical energies but all can be assigned to groups corresponding to the shells, each of which has a clearly differentiated energy level. The effect of subjecting an atom to appropriate radiation is well demonstrated by considering atoms of sodium vapour.
A sodium atom at ground state (Nao) will absorb a photon at 589 nm to cause a transition of an electron in the outermost shell to a higher energy orbital.
Na (^) o + 589 nm photon Æ Na (^) l
The same ground state atom will also absorb a 330 nm photon to promote a transition to its second excited state.
Na (^) o + 330 nm photon Æ Na (^2)
The diagram illustrates the higher energy required to cause a second level transition, corresponding with the higher energy (shorter wavelength) of the radiation that stimulates the transition (Fig. 4)
Fig. 4 Absorption by gaseous atomic sodium
4. Radiation and the Molecule
Electronic Transitions
Electrons in the atom can be considered as occupying groups of roughly similar energy levels. In the more complicated molecular model, electrons associated with more than one nucleus, the so-called bonding electrons, are particularly susceptible to energy level transitions under the stimulus of appropriate radiation.
The electrons concerned, usually p type electrons in the first or second shell, may be present in one of two conditions: σ (sigma) in localised bonds with a low probability of transitions (and therefore of absorption) or π (pi) where the transition probability is much higher. The presence of a carbon-carbon double bond in the molecule increases the likelihood of π type bonds, especially when conjugated double bonds are involved, i.e. double bonds that alternate with single bonds. The effect is still greater in the presence of nitrogen.
. Chemical bonds are formed by overlapping atomic orbitals that result in molecular orbitals of one of three types: bonding (low energy), antibonding (high energy), or non-bonding. Energy absorption is most typically associated with transitions induced in electrons involved in bonding orbitals, and the atoms involved are, for the most part, those containing s + p electrons. Two types of bond must be mentioned:
(1) σ bond with its related antibonding orbital designated σ* and
(2) π bonds with the corresponding π* antibonding orbital.. The uninvolved n (non-bonding) electrons have no antibonding orbital.
The full series of permitted electronic transitions (by UV/Vis absorption) is:
The figure shows that σ Æ σ* and n Æ σ * transitions require relatively high energy and are therefore associated with shorter wavelength radiation (ultraviolet). Lower energy n Æ π ***** and π Æ π ***** are ultraviolet or visible induced transitions.
The probability that transition (and therefore absorption) will occur is closely related to molecular orbital structure. If the configuration of the molecular orbitals is accurately known, the probability can be calculated with some certainty and an estimate can be made of the energy intensity (relative to other transitions), indicating an approximate value for the molar absorptivity of the species.
Whenever two double bonds are conjugated (i.e. alternate with single bonds), one of the bonding orbitals is raised in energy and the other lowered relative to the energy of an isolated double bond. The same applies to the antibonding orbitals. As a result, transition probability is enhanced, the wavelength of maximum absorption moves to a longer wavelength and the intensity of absorption is frequently increased.
5. Specific Absorption
Because each electron in a molecule has a unique ground state energy, and because the discrete levels to which it may jump are also unique, it follows that there will be a finite and predictable set of transitions possible for the electrons of a given molecule. Each of the transitions, or jumps, requires the absorption of a quantum of energy and if that energy is derived from electromagnetic radiation there will be a direct and permanent relationship between the wavelength of the radiation and the particular transition that it stimulates. That relationship is known as specific absorption and a plot of those points along the wavelength scale at which a given substance shows absorption 'peaks', or maxima, is called an absorption spectrum (Fig. 8).
Fig. 8 Typical absorption spectrum in visible region (Samarium Perchlorate)
The absorption spectrum of a compound is one of its most useful physical characteristics, both as a means of identification (qualitative analysis) and of estimation (quantitative analysis). If there is absorption in the visible and that absorption occurs in the red then the substance will be seen as green/blue since red and green/blue are complementary colours (Fig. 9). [Note: Experiment 4 at the end of this book shows how complementary colours relate to absorption peaks.]
Fig.9 Complementary colours.
The chemical group most strongly influencing molecular absorption characteristics is called a chromophore. Chromophores which can be detected by UV/Vis spectrophotometers always involve a multiple bond (such as C=C, C=O or C≡N) and may be conjugated with other groups to form complex chromophores. A typical example is the benzene ring which has an absorption peak at 254 nm.
Increasingly complex chromophores move the associated absorption peak towards longer wavelengths and generally increase the absorption at the maxima.
Although the emphasis on the value of UV/Vis spectrophotometry is naturally towards organic compounds, there is a wide range of inorganic substances that lend themselves to similar methods of analysis. Species with a non-metal atom double bonded to oxygen absorb in the ultraviolet region, and there are several inorganic double-bond chromophores that show characteristic absorption peaks. In some instances, measurement of inorganic materials may demand a secondary process, such as complexation with a colour-forming reagent or oxidation - e.g. manganese (II) oxidised to manganese (VII) and measured as the MnO 4 -^ ion (permanganate).
If a solution has this colour….
….it will absorb this colour….
6. Absorption and Concentration
For analytical purposes, two main propositions define the laws of light absorption.
Note : In this section ideal conditions are assumed throughout. The potential errors arising from scatter, reflection, fluorescence etc. are discussed in section 9.
A. Lambert's Law. The proportion of incident light absorbed by a transparent medium is independent of the intensity of the light (provided that there is no other physical or chemical change to the medium). Therefore successive layers of equal thickness will transmit an equal proportion of the incident energy (Fig. 10).
Fig. 10 Diagrammatic representation of relationship between Transmission and Absorption.
Lambert's law is expressed by I = T I (^) o
where I is the intensity of the transmitted light, Io is the intensity of the incident light, and T is the Transmittance.
It is customary to express transmittance as a percentage:
%T = I x I (^) o
It can at once be seen that a plot of absorbance against concentration will be linear (Fig. 12 ). It is therefore more convenient to express results in absorbance rather than transmission when measuring unknown concentrations, since linear calibration plots will be available.
Fig. 12 Absorbance plotted against concentration.
An alternative to plotting calibration curves is to make use of the relationship:
C = k A
where C is the concentration of the unknown, A is the measured absorbance of the unknown, and k is a factor derived from the reference or standard solution.
To determine the factor, measure the absorbance of a standard solution of known concentration and divide the concentration by the absorbance.
k = concentration (standard) absorbance (standard)
The factor k may be applied to a series of absorbance measurements on similar solutions measured in the same conditions to give results directly in concentration.
In many of today's spectrophotometers, the output electronics provide the means of entering the concentration value of the standard or the factor to the calculation so that instrument readings are directly in concentration units.
7. Instrumentation
The minimum requirements of an instrument to study absorption spectra (a spectrophotometer) are shown below (Fig 13):
- a source of radiation of appropriate wavelengths.
- a means of isolating light of a single wavelength and getting it to the sample compartment - monochromator and optical geometry.
- a means of introducing the test sample into the light beam - sample handling.
- a means of detecting and measuring the light intensity.
Fig. 13 Basic construction of a spectrophotometer.
Source
The requirements are that the source should be stable during the measurement period, i.e. that the intensity of emitted radiation should not fluctuate, and that there should be adequate intensity over as large a wavelength region as possible.
Ultraviolet light is generally derived from a deuterium arc that provides emission of high intensity and adequate continuity in the 190 - 380 nm range. A quartz or silica envelope is necessary not only because of the heat generated but also to transmit the shorter wavelengths of the ultraviolet radiation. The limiting factor is normally the lower limit of atmospheric transmission at about 190 nm (Fig. 14).
Visible light is normally supplied by a tungsten lamp or, in modern systems, by a tungsten-halogen (also described as quartz-iodine) lamp which has higher relative output in the cross-over region (320 - 380 nm). The long wavelength limit is usually the cut-off of the glass or quartz envelope, normally well beyond the useful visible limit at 900 nm (Fig. 14).
In most modern spectrophotometers the power supply arrangements, including any necessary start-up sequences for arc lamps, as well as the cross-over between sources at the appropriate wavelength, are automatic mechanical sequences. Lamps are usually supplied on pre-set focus mounts or incorporate simple adjustment mechanisms for easy replacement.
Recently, xenon lamp sources have been introduced, and these cover the UV and visible range. There is a trade off, however, because the instrumental stray light is higher and there is less energy at the far visible end. For general measurements, however, they are ideal and have the benefit of a long lifetime as they are only consumed during the actual measurement cycle (“press to read”). In addition, no cross-over wavelength is necessary.
Fig 14 UV/Vis light sources
Diffraction Gratings
Gratings provide an alternative means of producing monochromatic light. A diffraction grating consists of a series of parallel grooves (lines) on a reflecting surface that is produced by taking a replica from a master carefully prepared using a machine or, increasingly, from one which is holographically generated. The grooves can be considered as separate mirrors from which the reflected light interacts with light reflected from neighbouring grooves to produce interference, and so to select preferentially the wavelength that is reflected when the angle of the grating to the incident beam is changed.
Among the advantages that gratings offer (compared to prisms) are better resolution, linear dispersion and therefore constant bandwidth and simpler mechanical design for wavelength selection ( Fig. 16).
Fig. 16 Diffraction grating monochromator.
When parallel radiation illuminates a reflecting diffraction grating, the multiple reflections from the mirror grooves will overlap and interfere with each other. If the reflected waves are in phase interference is said to be constructive and the reflected light is not affected. If the reflected waves are out of phase there is destructive interference and light of the wavelength at which such interference occurs will not be propagated.
The relationship that determines the wavelength of the reflected light is expressed by:
n λ = 2d sin θ
where n is the order (see below), d is the separation of the reflecting surfaces (or lines) and θ is the angle of incidence of the radiation. Rotating the grating in the light beam changes θ and so selects the wavelength reflected (Figs. 17, 18 and 19)
Fig. 1 7 Operating principles of a reflecting diffraction grating. Fig. 18 Spectral distribution after diffraction.
Two additional characteristics of gratings may be noted:
- If wavelength λ is reflected for a given angle θ, then λ/2, λ/3 and so on are also reflected at that angle. These overlapping spectra, known as second and third orders etc, can be removed with filters or with a pre-monochromator. Careful selection of the blaze angle (the angle at which the groove is cut) will peak the energy at the wavelength of the blaze, typically 250 nm for instruments of the kind under discussion.
- Both the energy and the resolution of a grating are directly proportional to the number of lines. For maximum efficiency the line separation should be as close as possible to one wavelength and for UV/Vis gratings the line density is typically 1200 per mm.
Gratings have the following advantages over prisms:
(a) better resolution and energy transfer. Fig 19 Schematic relationship of energy (b) linear dispersion and therefore constant bandwidth. distribution and grating line density (c) less complicated wavelength drive mechanism is required. (d) stray light is limited to imperfections at the grating surface.
Optical geometry
As all absorption measurements are ratio dependent (I/Io ), it is necessary to record a reference solution before bringing the sample under test into the light path. These measurements are done using a cuvette (matched, if possible, to that containing the test sample) in the light path filled with the appropriate solvent. The reference intensity (Io) varies with wavelength in a complicated multi-function way (due mainly to source energy, monochromator transmission, slit width and detector response), so it is essential, when measuring absorption, to re-measure the reference for each discrete wavelength at which measurement is to be made. All modern instruments are microprocessor based, and have the facility to store a baseline, that is 100 %T or 0 A set at each wavelength in the range, overcoming this requirement. This has allowed single beam spectrophotometers to compete on performance with the more expensive double beam instruments.
Additional advantages of microprocessor handling of the detector output are the ability to introduce component factors (e.g. concentration or molar absorption data) and to present results in alternative formats without additional manual calculation. An important consideration in some laboratories is the ability to interface with personal computers (Fig. 24), so that results can be incorporated into a laboratory information management system (LIMS) or transferred to disk for archiving or data manipulation purposes.
Single beam optics
The development of the microprocessor has made it possible to achieve excellent results using a single beam configuration when compared to a double beam configuration; this results in greater optical and mechanical simplicity, as shown in Fig, 16. The process of comparison between reference and sample cells can be achieved with single beam instrumentation by feeding the post detector signal to a microprocessor which stores the reference data for subtraction from the sample signal prior to printing or displaying the reference corrected result (the baseline). Signal levels can be compared between different samples at one wavelength, at a series of predetermined wavelengths or, if wavelength drive is provided, a complete absorption spectrum can be obtained.
Split (reference) beam optics
With the introduction of xenon flash lamps into spectrophotometers, the split beam configuration has become necessary; this is because the high intensity flashes from the xenon pulse lamp are not always of equal magnitude. Thus approx. 70 %, f the energy from the monochromator is passed through the sample, with the rest going to a separate feedback detector, enabling a means of taking into account drops/gains in energy via a feedback gain loop in the detector electronics. This stabilises the system, and there are no large extra cost elements involved.
Sample handling
In practice, by far the greater part of all measurements will be made on samples in solution. Vapours and solids can be accommodated, but most instruments are designed with a standard cell (or cuvette) as the normal sample container. The design, construction and material of the cuvette are all important to accurate measurements as are operator practice and sample preparation.
Cuvettes are typically made of glass or UV grade silica (according to the wavelength range of interest), are fused rather than cemented (to resist the action of some solvents), and have the following characteristics:
- optical windows (the sides through which the beam passes) are highly polished, parallel and flat
- entrance and exit surfaces are exactly parallel and orthogonal
- light path (distance between inner surfaces of windows) is tightly controlled.
The holder that locates the cuvette in the light beam must ensure precise and reproducible location with respect to the beam.
The most commonly used cuvette has a light pathlength of 10 mm, but longer or shorter pathlengths are useful if concentration or absorbance fall outside normal ranges without further processing - e.g. solvent extraction or dilution. Microcells are particularly useful where sample volumes are restricted: gas cells, flow cells and disposable cells are all available to extend the usefulness of the technique. A range of sample cuvettes is shown in Fig. 21.
Fig. 21 Selection of sample cuvettes.
Disposable plastic cuvettes are used to avoid tedious washing procedures, particularly in higher throughput applications such as QC. More recently, new plastic materials that transmit down into UV have become available.
Absorption spectrophotometry is a powerful aid, both to the identification of the components of an unknown (qualitative analysis), and to the measurement of the amounts of individual components present (quantitative analysis). In qualitative work it is usually necessary to measure absorption over a range of wavelengths. Some form of wavelength scanning mechanism and a means of comparing the unknown solution with a reference are desirable features of spectrophotometers to be used for spectrum plotting. However, in many laboratories the requirement is to measure large numbers of similar samples at one or a few wavelengths only - e.g. blood samples in a busy pathology laboratory. The need to plot a complete absorption spectrum is rare. In most practical situations, therefore, single beam instrumentation is preferred for speed, convenience and simplicity of operation, and consequently reduced likelihood of operator error. Instrument requirements are usually less complex and therefore less expensive.
The technique may be assisted by a means of manually introducing a standard or reference solution into the light path at set intervals to check 0 %T and 100 %T settings. In such cases accuracy will be enhanced if the reference and sample solutions are contained in cuvettes with closely matching transmission characteristics at the wavelength of interest.
Detectors
Of the four principal types of detectors found in spectrophotometers one, the photoconductive cell (typically a light sensitive layer, e.g. selenium, on a metal substrate) is so severely restricted in both wavelength response and sensitivity, that it is almost never found in instruments of the class under consideration. The most commonly encountered detectors are the photomultiplier, the silicon diode and the diode array.
Photomultiplier
Photomultipliers have an internal amplification that gives them great sensitivity and a wide spectral range. Light causes emission of electrons from a photocathode which accelerate past a series of dynodes maintained at progressively increasing potentials. Electrons striking the first dynode release a secondary emission that is stronger than the original beam and so on through the dynode chain to produce a cascade effect. The electron density released by the final dynode to the anode can be many orders of magnitude greater than that from the cathode, but it remains proportional to the intensity of the incident radiation.
Two types of photomultiplier geometry may be noted: side window and end window. In most cases the end window permits the exposure to the beam of a larger cathode area and is the preferred form for higher specification instruments (Fig. 22). A single photomultiplier will provide adequate sensitivity over the whole UV/Vis region, but at some considerable cost both of the component itself and the necessary power supply and control circuitry.
Fig. 22 End window photomultiplier.
Silicon diode
Silicon diode detectors have performance characteristics which (when the device is integrated with an operational amplifier) compare with those of a photomultiplier, but having a wider wavelength range but less sensitivity. They are mechanically robust (being solid-state devices), and electronic benefits include reduced power supply and control circuit requirements (Fig. 23).
Fig. 23 Spectral response of silicon diode detectors
Diode array
A diode array is an assembly of individual detector elements in linear or matrix form which, in a spectrophotometer, can be mounted so that the complete spectrum is focused on to an array of appropriate size. No wavelength change mechanism is required and output presentation is virtually instantaneous. Resolution, however, is limited by the physical size of individual detector elements (typically > 2 nm) and wavelength range is usually limited to about 1 octave (200- 400nm, 300-600nm, 400-800nm). This is because in a diode array instrument, it is difficult to introduce filters to mitigate the effects of stray light from the second order spectrum.
8. Good operating practice
The good housekeeping routines normal to any analytical laboratory are equally essential in the preparation of samples for spectrophotometric assays. The cleanliness of all materials and equipment, especially the sample cuvettes, is mandatory, as are weighing and volumetric accuracy. Temperature variations can be a source of significant error too.
It has already been pointed out that ideally the sample and reference cuvettes should be optically identical and their orientation in the light path should be carefully controlled; this is particularly the case when using a double beam spectrophotometer. Most equipment manufacturers will supply (to special order) cells with transmission characteristics matched to 1% or better over a defined wavelength range. In summary, it should be noted that the surface finish and parallelism of cuvette windows are more likely to introduce errors than pathlength variations.
Preferred absorption range
The ability of a spectrophotometer to measure accurately throughout its absorption range is impaired by a number of factors, chief of which is the inherent noise in the detector. It can be shown that instruments equipped with photo- emissive detectors (phototube or photomultiplier) have minimum relative error when absorbance = 0.86 (13.5%T); for photoconductive detectors the equivalent value is 0.43.
In practice the error will not be significant for any absorbance between 0.8 and 1.5A, except where light levels are low - e.g. at the extreme limits of any of the components of the system. If it is required to adjust the sample to bring absorbance within the preferred range, it is normally better to change the sample pathlength rather than the concentration, e.g. by diluting.
Note that spectrophotometers equipped with silicon diode detectors do not suffer from this limitation. In such instruments performance limits are usually dependent on stray light (see below) and a quality system will measure absorbance up to 3A with accuracy and reliability.
Absorbance Measurement
In practice, the unwanted effects of spectral bandwidth on peak absorbance measurement can be eliminated by constructing a set of calibration curves at known concentrations. Providing Beer's law is obeyed, a plot of concentration against absorbance at a given wavelength will yield a straight line from which unknown concentrations can be accurately determined if absorbance is measured under similar instrument conditions. If the measuring system of the spectrophotometer is microprocessor-based, relevant data or programmes can be entered or kept in memory. Direct readout in concentration is made easy. [Note: Experiment 2 at the end of this book shows how to construct concentration curves.] The importance of measuring absorbance precisely at the wavelength of an absorption peak, i.e. at λ max, is demonstrated in Fig 25. In the hypothetical case illustrated, any wavelength setting within the narrow band indicated would have no significant effect on absorbance measured at the peak. However, the same band of wavelengths displaced to shorter wavelength would introduce the possibility of major error, depending on the precise point within the band at which the measurement was taken. Errors due to either wavelength setting or instrument calibration will be at a minimum when measurements are made at the wavelength of maximum absorption. [Note: Experiment 1 at the end of this book shows how to measure both absorbance at λ max and natural bandwidth.] Fig. 25 Importance of measurement at λ max
Solvent selection
A common characteristic of the wide range of solvents available is the rapid fall of transmission towards the short wavelength limits. Care must be taken when working below (say) 250 nm that solvent absorption is not so high that the incremental absorption due to the sample is small compared to total absorption. The problem is exacerbated in the presence of stray light, but it may be noted here that the effects are reduced at lower absorbance levels.
9. Sources of error
There has been an increase in laboratory requirements to conform with Good Laboratory Practice (GLP) techniques. These require that results obtained can be traced back to an instrument and that the instrument can be proved to be working correctly, for validation purposes. Instrument performance criteria for spectrophotometers have been defined by the Pharmacopoeia as being spectral bandwidth, stray light, absorbance accuracy and wavelength accuracy, and it is therefore important to check these potential sources of error periodically. Modern instruments in the mid to upper price bracket often have GLP self diagnostic tests as part of the calibration procedure, accompanied by a print out of these results.
Instrument related sources of error
Spectral bandwidth and slit width
The resolution of a spectrophotometer - the minimum separation between narrow absorption bands that can be observed
- is usually limited by the spectral purity and intensity of the monochromator light output and the detector sensitivity at that wavelength.
In some (older) instruments the control of the energy level reaching the detector is achieved by adjusting the aperture of the slit at the monochromator exit. As the exit slit defines the spectral bandwidth (the range of wavelength, Fig. 26) at the detector, it is important to realise that both photometric accuracy and wavelength accuracy may be affected. In general, narrower slit widths will reduce error providing the overall energy level remains adequate and electronic noise levels are not significant. Most instruments using diffraction gratings take advantage of the linear dispersion and provide fixed slit widths to give known and controlled bandwidth at the exit slit of the monochromator. More than one slit width may be available to give the user a means of trading energy against spectral sensitivity. Fig.26 Spectral bandwidth.
The total energy at the exit slit of a monochromator at wavelength λ may be assumed to have a triangular function as shown in Fig. 26. The spectral bandwidth for a given slit width is the band of wavelength that corresponds to the half- peak intensity. A well defined absorption peak for a specific compound has a similar triangular function and can be assigned a natural bandwidth (assuming infinite resolution), at half-peak height, similar to the definition of spectral bandwidth.
The ratio of spectral bandwidth to natural bandwidth is a determining factor in absorption measurement. As the ratio of the spectral bandwidth to natural bandwidth increases, the deviation of observed absorbance from true absorbance will be greater. The natural bandwidth of most commonly encountered compounds in UV/Vis work, particularly bio- molecules in the Life Sciences, lie within the range 5 - 50 nm. Thus a spectrophotometer with a fixed bandwidth of 2 - 6 nm is ideal for bio-molecule measurement, since there is no spectral fine detail. A narrower bandwidth is required for measurements involving rare earth and transition metal complexes and conjugated organic species, where critical fine detail may be present.
A diagrammatic representation of an absorbing species measured at progressively increasing spectral bandwidths is shown in Fig. 27. As bandwidth increases beyond a certain value separation of the two bands is less well defined, the apparent absorbance at the maxima decreases and the observed bandwidth of the peaks increases.
Fig. 27 Typical absorption peaks plotted at varying spectral bandwidths.
Fig. 28 Effect of slit width on observed absorbance at λ max.
