Spectrophotometry

By - Priya TamangSpectrophotometry

CBSE NET JRF EXAM NOTES

Spectrophotometry

Spectrophotometry is a method to measure how much a chemical substance absorbs light by measuring theintensity of lightas a beam of light passes through sample solution. The basic principle is that each compound absorbs or transmits light over a certain range of wavelength. This measurement can also be used to measure the amount of a known chemical substance. Spectrophotometry is one of the most useful methods of quantitative analysis in various fields such as chemistry, physics, biochemistry, material and chemical engineering and clinical applications.Every chemical compound absorbs, transmits, or reflectslight (electromagnetic radiation)over a certain range of wavelength. Spectrophotometry is a measurement of how mucha chemical substanceabsorbs or transmits. Spectrophotometry is widely used for quantitative analysis in various areas (e.g., chemistry, physics, biology, biochemistry, material and chemical engineering, clinical applications, industrial applications, etc). Any application that deals with chemical substances or materials can use this technique. In biochemistry, for example, it is used to determine enzyme-catalyzed reactions. In clinical applications, it is used to examine blood or tissues for clinical diagnosis. There are also several variations of the spectrophotometry such as atomic absorption spectrophotometry and atomic emission spectrophotometry.

A spectrophotometer is an instrumentthat measures the amount of photons (the intensity of light) absorbed after it passes through sample solution. With the spectrophotometer,the amount of a known chemical substance (concentrations) can also bedetermined by measuring the intensity of lightdetected. Depending on the range of wavelength of light source, it can be classified into two different types:UV-visible spectrophotometer:uses lightover the ultraviolet range (185 - 400 nm) and visible range (400 - 700 nm) of electromagnetic radiation spectrum.IR spectrophotometer:uses light over the infrared range (700 - 15000 nm) of electromagnetic radiation spectrum.In visible spectrophotometry, the absorption or the transmission of a certain substance can be determined by the observed color.For instance,a solution sample that absorbs light overall visible ranges (i.e., transmits none of visible wavelengths) appears black in theory. On the other hand, if all visible wavelengths are transmitted (i.e., absorbs nothing), the solution sample appears white. If a solution sample absorbs red light(~700 nm), it appears green because green is the complementary color of red. Visible spectrophotometers, in practice, use a prism to narrow down a certain range of wavelength (to filter out other wavelengths) so that the particular beam of light is passed through a solution sample.

Beer-Lambert Law

Beer-Lambert Law(also known as Beer's Law) states that there is a linear relationship between the absorbance and the concentration of a sample. For this reason, Beer's Law canonlybe applied when there is a linear relationship. Beer's Law is written as:A=lcwhereAAis the measure of absorbance (no units),is the molar extinction coefficient or molar absorptivity (or absorption coefficient),llis the path length, andccis the concentration.The molar extinction coefficient is given as a constant and varies for each molecule. Since absorbance does not carry any units, the units formust cancel out the units of length and concentration. As a result,has the units: Lmol-1cm-1. The path lengthis measured in centimeters.Because a standard spectrometer uses a cuvette that is 1 cm in width,llis always assumed to equal 1 cm. Since absorption,, andpath length are known, we can calculate the concentrationccof the sample.

Devices and mechanism

A spectrophotometer, in general, consists of two devices; a spectrometer anda photometer. A spectrometer isa device that produces, typically disperses and measures light. A photometer indicatesthe photoelectric detector that measures the intensity of light.Spectrometer: Itproduces a desired range of wavelength of light. First a collimator (lens) transmits a straight beam of light (photons) that passes through a monochromator (prism) to split it into several component wavelengths (spectrum). Then a wavelength selector(slit) transmits onlythe desired wavelengths, as shown in Figure 1.Photometer: After thedesired range of wavelength of lightpasses through the solution of a sample in cuvette, the photometer detects the amount of photons that is absorbed and then sends a signal to a galvanometer or a digital display,as illustrated in Figure 1.

Figure 1 illustrates the basic structure of spectrophotometers. It consists of a light source, a collimator, a monochromator, a wavelength selector, a cuvettefor sample solution, a photoelectric detector, and a digital display or a meter. Detailed mechanism is described below.

Figure 2: A single wavelenth spectrophotometer

You need a spectrometer to produce a variety of wavelengths because different compounds absorb best at different wavelengths. For example, p-nitrophenol (acid form) has the maximum absorbance at approximately 320 nm and p-nitrophenolate (basic form) absorb best at 400nm, as shown in Figure 3.

Figure 3: Absorbance of twodifferent compounds

Looking at the graph that measures absorbance and wavelength, an isosbestic point can also be observed. Anisosbestic pointis the wavelength in which the absorbance of two or more species are the same. The appearance of an isosbestic point in a reaction demonstrates that an intermediate is NOT required to form a product from a reactant. Figure4 shows an example of an isosbestic point.

Figure 4: An example of isosbestic point

Referring back to Figure 1 (and Figure 5), the amount of photons that goes through the cuvette and into the detector is dependent on the length of the cuvette and the concentration of the sample. Once you know the intensity of light after it passes through the cuvette, you can relate it to transmittance (T). Transmittance is the fraction of light that passes through the sample. This can be calculated using the equation:Transmittance(T)=It /ItoWhere Itis the light intensity after the beam of light passes through the cuvette and Iois the light intensity before the beam of light passes through the cuvette. Transmittance is related to absorption by the expression:Absorbance(A)=log(T)=log(It/ I0)Where absorbance stands for the amount of photons that is absorbed. With the amount of absorbance known from the above equation, you can determine the unknown concentration of the sample by using Beer-Lambert Law. Figure 5 illustrates transmittance of lightthrough a sample. The lengthllis used for Beer-Lambert Law described below.

Figure 5: Transmittance (illustrated by Heesung Shim)

Ultraviolet and visible spectroscopy

Introduction Many molecules absorb ultraviolet or visible light. The absorbance of a solution increases as attenuation of the beam increases. Absorbance is directly proportional to the path length,b, and the concentration,c, of the absorbing species.Beer's Lawstates that A =ebc, whereeis a constant of proportionality, called theabsorbtivity.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;Transitions involvingp,s, andnelectronsTransitions involving charge-transfer electronsTransitions involvingdandfelectrons (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.

Absorbing species containing, ,, andnelectrons

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.

* TransitionsAn electron in a bondingsorbital is excited to the corresponding antibonding orbital. The energy required is large. For example, methane (which has only C-H bonds, and can only undergos s*transitions) shows an absorbance maximum at 125 nm. Absorption maxima due tos s*transitions are not seen in typical UV-Vis. spectra (200 - 700 nm)n* TransitionsSaturated compounds containing atoms with lone pairs (non-bonding electrons) are capable ofn s*transitions. These transitions usually need less energy thans s*transitions. They can be initiated by light whose wavelength is in the range 150 - 250 nm. The number of organic functional groups withn s*peaks in the UV region is small.n* and * TransitionsMost absorption spectroscopy of organic compounds is based on transitions of n or electrons to the * excited state. This is because the absorption peaks for these transitions fall in an experimentally convenient region of the spectrum (200 - 700 nm). These transitions need an unsaturated group in the molecule to provide thepelectrons.Molar absorbtivities from n* transitions are relatively low, and range from 10 to100 L mol-1cm-1. * transitions normally give molar absorbtivities between 1000 and 10,000 L mol-1cm-1.

The solvent in which the absorbing species is dissolved also has an effect on the spectrum of the species. Peaks resulting from n* transitions are shifted to shorter wavelengths (blue shift) with increasing solvent polarity. This arises from increased solvation of the lone pair, which lowers the energy of thenorbital. Often (butnotalways), the reverse (i.e.red shift) is seen for * transitions. This is caused by attractive polarisation forces between the solvent and the absorber, which lower the energy levels of both the excited and unexcited states. This effect is greater for the excited state, and so the energy difference between the excited and unexcited states is slightly reduced - resulting in a small red shift. This effect also influences n* transitions but is overshadowed by the blue shift resulting from solvation of lone pairs

Charge - Transfer Absorption

Many inorganic species show charge-transfer absorption and are calledcharge-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-1cm-1).

Instrumental components

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 containersDetectorSignal processor and readoutSources of UV radiationIt 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 energyD2*D' + D'' +hvBoth 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 radiationThe 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 beverystable 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 WI2hit 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 slitA collimating lensA dispersing device (usually a prism or a grating)A focusing lensAn exit slitPolychromatic 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

CuvettesThe 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.DetectorsThe photomultiplier tubeis a commonly used detector in UV-Vis spectroscopy. It consists of aphotoemissive cathode(a cathode which emits electrons when struck by photons of radiation), severaldynodes(which emit several electrons for each electron striking them) and ananode.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- 107electrons. 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 arrayis an example of amultichannel 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.

IR Atomic AbsorptionThe term "infra red" covers the range of the electromagnetic spectrum between 0.78 and 1000mm. In the context of infra red spectroscopy, wavelength is measured in "wave numbers", which have the units cm-1.wave number = 1 / wavelength in centimeters It is useful to divide the infra red region into three sections;near,midandfarinfra red;RegionWavelength range(mm)Wave number range(cm-1)Near0.78 - 2.512800 4000Middle2.5 504000 200Far50 -1000200 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 catagories ofstretchingandbending.

Stretching:Change in inter-atomic distance along bond axis

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

Vibrational couplingIn 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 bondsCoupling of bending vibrations occurs when there is a common bond between vibrating groupsCoupling between a stretching vibration and a bending vibration occurs if the stretching bond is one side of an angle varied by bending vibrationCoupling is greatest when the coupled groups have approximately equal energiesNo coupling is seen between groups separated by two or more bonds

Instrumental components

SourcesAn 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 gloweris 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 sourceis 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 5mm) where it's output becomes larger.The incandescent wire sourceis 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.

DetectorsThere are three catagories of detector;ThermalPyroelectricPhotoconducting

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 junctionsPyroelectric detectorsare 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 detectorssuch 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 spectophotometersThese 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, anoptical nullsystem 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 spectrometersAny waveform can be shown in one of two ways; either infrequency domainortime 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 1014Hz). This problem can be solved by using interference to modulate the i.r. signal at a detectable frequency. TheMichelson interferometeris 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 aninterferogram.

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 amovablemirror. 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,fmcan be found by;

wherevis the speed of mirror travel in mm/sBecauseallwavelengths 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 aFast 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 resolutionImproved frequency reproducibility (older dispersive instruments must be recalibrated for each session of use)Higher energy throughputFaster operationComputer 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)