UNIT I COLORIMETER AND SPECTROPHOTOMETERS PART A

Transcription

1 UNIT I COLORIMETER AND SPECTROPHOTOMETERS PART A 1. List any four elements used in spectrophotometers. 1.Radiant source 2.wavelength selector 3.photodetector 4.sample 2. What is meant by flame emission spectrometry? When an analytical sample is introduced into the flame, the flame atomizes the Sample and subsequently they are de-excited with the emission of atomic lines corresponding to different elements present in the sample 3. State Beer s law. The intensity of a beam of monochromatic light decreases exponentially with increase in concentration of the absorbing substance arithmetically. 4. What is monochromators? Monochromators are the optical systems which provide better isolation of spectral energy than the optical filters and are therefore preferred where it is required to isolate narrow bands of radiant energy. 5. What is analytical instrumentation? It is the field of science which deals with the technique by which the sample

2 (chemical species) is analyzed quantitatively. Instruments used for analysis of chemical sample are analytical instrument 6. What is electromagnetic radiation? It is a type of energy that is transmitted through space at a speed of approximately 3x108 m/s. Such radiation doesn t require a medium of propagation and can readily travel through vacuum. It may be considered as discrete packets of energy called photons. 7. Define wave number. It is defined as the number of waves per centimeter. = 1/λ 8. Draw the spectrum of EM regions. Given in the notes. 9. State Lambert s law. When a beam of light is allowed to pass through a transparent medium the rate of decrease of intensity with the thickness of medium is directly proportional to the intensity of light. 10. What is Absorption Spectroscopy? The measurement method based on absorption of radiation of a substance is known as absorption spectroscopy.

3 PART B 1. Explain with necessary diagrams the instrumentation involved in uv visible spectrophotometer? Introduction 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. Sources (UV and visible) 2. Wavelength selector (monochromator) 3. Sample containers 4. Detector 5. Signal processor and readout 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; D 2 + electrical energy D 2 * D' + D'' + hv Both deuterium and hydrogen lamps emit radiation in the range 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.

4 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 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 WI 2. When molecules of WI 2 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

5 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 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

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

7 2. Explain the construction and working of UV-Visible Spectrophotometer Introduction If you pass white light through a coloured substance, some of the light gets absorbed. A solution containing hydrated copper(ii) ions, for example, looks pale blue because the solution absorbs light from the red end of the spectrum. The remaining wavelengths in the light combine in the eye and brain to give the appearance of cyan (pale blue). Some colourless substances also absorb light - but in the ultra-violet region. Since we can't see UV light, we don't notice this absorption. Different substances absorb different wavelengths of light, and this can be used to help to identify the substance - the presence of particular metal ions, for example, or of particular functional groups in organic compounds. The amount of absorption is also dependent on the concentration of the substance if it is in solution. Measurement of the amount of absorption can be used to find concentrations of very dilute solutions. An absorption spectrometer measures the way that the light absorbed by a compound varies across the UV and visible spectrum. A simple double beam spectrometer We'll start with the full diagram, and then explain exactly what is going on at each stage.

8 Working The light source You need a light source which gives the entire visible spectrum plus the near ultraviolet so that you are covering the range from about 200 nm to about 800 nm. (This extends slightly into the near infra-red as well.) You can't get this range of wavelengths from a single lamp, and so a combination of two is used - a deuterium lamp for the UV part of the spectrum, and a tungsten / halogen lamp for the visible part. The combined output of these two bulbs is focussed on to a diffraction grating. The diffraction grating and the slit You are probably familiar with the way that a prism splits light into its component colours. A diffraction grating does the same job, but more efficiently.

9 The blue arrows show the way the various wavelengths of the light are sent off in different directions. The slit only allows light of a very narrow range of wavelengths through into the rest of the spectrometer. By gradually rotating the diffraction grating, you can allow light from the whole spectrum (a tiny part of the range at a time) through into the rest of the instrument. The rotating discs This is the clever bit! Each disc is made up of a number of different segments. Those in the machine we are describing have three different sections - other designs may have a different number. The light coming from the diffraction grating and slit will hit the rotating disc and one of three things can happen. 1. If it hits the transparent section, it will go straight through and pass through the cell containing the sample. It is then bounced by a mirror onto a second rotating disc. This disc is rotating such that when the light arrives from the first disc, it

10 meets the mirrored section of the second disc. That bounces it onto the detector. It is following the red path in the diagram: 2. If the original beam of light from the slit hits the mirrored section of the first rotating disc, it is bounced down along the green path. After the mirror, it passes through a reference cell (more about that later). Finally the light gets to the second disc which is rotating in such a way that it meets the transparent section. It goes straight through to the detector. 3. If the light meets the first disc at the black section, it is blocked - and for a very short while no light passes through the spectrometer. This just allows the computer to make allowance for any current generated by the detector in the absence of any light.

11 The sample and reference cells These are small rectangular glass or quartz containers. They are often designed so that the light beam travels a distance of 1 cm through the contents. The sample cell contains a solution of the substance you are testing - usually very dilute. The solvent is chosen so that it doesn't absorb any significant amount of light in the wavelength range we are interested in ( nm). The reference cell just contains the pure solvent. The detector and computer The detector converts the incoming light into a current. The higher the current, the greater the intensity of the light. For each wavelength of light passing through the spectrometer, the intensity of the light passing through the reference cell is measured. This is usually referred to as I o - that's I for Intensity. The intensity of the light passing through the sample cell is also measured for that wavelength - given the symbol, I. If I is less than I o, then obviously the sample has absorbed some of the light. A simple bit of maths is then done in the computer to convert this into something called the absorbance of the sample - given the symbol, A. An absorbance of 0 at some wavelength means that no light of that particular wavelength has been absorbed. The intensities of the sample and reference beam are both the same, so the ratio I o /I is 1. Log 10 of 1 is zero.

12 An absorbance of 1 happens when 90% of the light at that wavelength has been absorbed - which means that the intensity is 10% of what it would otherwise be. In that case, I o /I is 100/I0 (=10) and log 10 of 10 is 1. The chart recorder Chart recorders usually plot absorbance against wavelength. The output might look like this: This particular substance has what are known as absorbance peaks at 255 and 395 nm. 3. Explain the construction and working of FTIR? Introduction FTIR spectrometers (Fourier Transform Infrared Spectrometer) are widely used in organic synthesis, polymer science, petrochemical engineering, pharmaceutical industry and food analysis. In addition, since FTIR spectrometers can be hyphenated to chromatography, the mechanism of chemical reactions and the detection of unstable substances can be investigated with such instruments. FTIR spectrometers (Fourier Transform Infrared Spectrometer) are widely used in organic synthesis, polymer science, petrochemical engineering, pharmaceutical industry and food analysis. In addition, since FTIR spectrometers can be

13 hyphenated to chromatography, the mechanism of chemical reactions and the detection of unstable substances can be investigated with such instruments. The Components of FTIR Spectrometers A common FTIR spectrometer consists of a source, interferometer, sample compartment, detector, amplifier, A/D convertor, and a computer. The source generates radiation which passes the sample through the interferometer and reaches the detector. Then the signal is amplified and converted to digital signal by the amplifier and analog-to-digital converter, respectively. Eventually, the signal is transferred to a computer in which Fourier transform is carried out. Figure 2 is a block diagram of an FTIR spectrometer. Figure 2. Block diagram of an FTIR spectrometer Michelson Interferometer The Michelson interferometer, which is the core of FTIR spectrometers, is used to split one beam of light into two so that the paths of the two beams are different. Then the Michelson interferometer recombines the two beams and conducts them into the detector where the difference of the intensity of these two beams are measured as a function of the difference of the paths. Figure 3 is a schematic of the Michelson Interferometer.

14 Figure 3. Schematic of the Michelson interferometer A typical Michelson interferometer consists of two perpendicular mirrors and a beamsplitter. One of the mirror is a stationary mirror and another one is a movable mirror. The beamsplitter is designed to transmit half of the light and reflect half of the light. Subsequently, the transmitted light and the reflected light strike the stationary mirror and the movable mirror, respectively. When reflected back by the mirrors, two beams of light recombine with each other at the beamsplitter. If the distances travelled by two beams are the same which means the distances between two mirrors and the beamsplitter are the same, the situation is defined as zero path difference (ZPD). But imagine if the movable mirror moves away from the beamsplitter, the light beam which strikes the movable mirror will travel a longer distance than the light beam which strikes the stationary mirror. The distance which the movable mirror is away from the ZPD is defined as the mirror displacement and is represented by. It is obvious that the extra distance travelled by the light which strikes the movable mirror is 2. The extra distance is defined as the optical path difference (OPD) and is represented by delta. Therefore,

15 δ=2δ It is well established that when OPD is the multiples of the wavelength, constructive interference occurs because crests overlap with crests, troughs with troughs. As a result, a maximum intensity signal is observed by the detector. This situation can be described by the following equation: δ=nλ (n = 0,1,2,3...) In contrast, when OPD is the half wavelength or half wavelength add multiples of wavelength, destructive interference occurs because crests overlap with troughs. Consequently, a minimum intensity signal is observed by the detector. This situation can be described by the following equation: δ=(n+12)λ (n = 0,1,2,3...) These two situations are two extreme situations. If the OPD is neither n-fold wavelengths nor (n+1/2)-fold wavelengths, the interference should be between constructive and destructive. So the intensity of the signal should be between maximum and minimum. Since the mirror moves back and forth, the intensity of the signal increases and decreases which gives rise to a cosine wave. The plot is defined as an interferogram. When detecting the radiation of a broad band source rather than a single-wavelength source, a peak at ZPD is found in the interferogram. At the other distance scanned, the signal decays quickly since the mirror moves back and forth. Figure 4(a)shows an interferogram of a broad band source. Fourier Transform of Interferogram to Spectrum The interferogram is a function of time and the values outputted by this function of time are said to make up the time domain. The time domain is Fourier transformed to get a frequency domain, which is deconvoluted to product a spectrum. Figure 4 shows the Fast Fourier transform from an interferogram of polychromatic light to its spectrum.

16 (b) (a) Figure 4. (a) Interferogram of a monochromatic light; (b) its spectrum 4. Derive Beer Lambert s law and explain about deviations from Beer s Law? Introduction The Beer-Lambert law (also called the Beer-Lambert-Bouguer law or simply Beer's law) is the linear relationship between absorbance and concentration of an absorber of electromagnetic radiation. The general Beer-Lambert law is usually written as: where A is the measured absorbance, a is a wavelength-dependent absorptivity coefficient, b is the path length, and c is the analyte concentration. When working in concentration units of molarity, the Beer-Lambert law is written as: where is the wavelength-dependent molar absorptivity coefficient with units of M -1 cm -1. The subscript is often dropped with the understanding that a value for is for a specific wavelength. If multiple species that absorb light at a given wavelength are present in a sample, the total absorbance at that wavelength is the sum due to all absorbers:

17 where the subscripts refer to the molar absorptivity and concentration of the different absorbing species that are present. Theory Experimental measurements are usually made in terms of transmittance (T), which is defined as: where P is the power of light after it passes through the sample and P o is the initial light power. The relation between A and T is: The figure shows the case of absorption of light through an optical filter and includes other processes that decreases the transmittance such as surface reflectance and scattering.

18 Derivation of the Beer-Lambert law The Beer-Lambert law can be derived from an approximation for the absorption coefficient for a molecule by approximating the molecule by an opaque disk whose cross-sectional area,, represents the effective area seen by a photon of frequency w. If the frequency of the light is far from resonance, the area is approximately 0, and if w is close to resonance the area is a maximum. Taking an infinitesimal slab, dz, of sample: I o is the intensity entering the sample at z=0, I z is the intensity entering the infinitesimal slab at z, di is the intensity absorbed in the slab, and I is the intensity of light leaving the sample. Then, the total opaque area on the slab due to the absorbers is * N * A * dz. Then, the fraction of photons absorbed will be * N * A * dz / A so, Integrating this equation from z = 0 to z = b gives:

19 or Since N (molecules/cm 3 ) * (1 mole / 6.023x10 23 molecules) * 1000 cm 3 / liter = c (moles/liter) and * log(x) = ln(x), then or where = * (6.023x10 20 / 2.303) = * 2.61x10 20 Limitations of the Beer-Lambert law The linearity of the Beer-Lambert law is limited by chemical and instrumental factors. Causes of nonlinearity include: deviations in absorptivity coefficients at high concentrations (>0.01M) due to electrostatic interactions between molecules in close proximity scattering of light due to particulates in the sample fluoresecence or phosphorescence of the sample changes in refractive index at high analyte concentration shifts in chemical equilibria as a function of concentration non-monochromatic radiation, deviations can be minimized by using a relatively flat part of the absorption spectrum such as the maximum of an absorption band stray light

20 5. Explain with neat diagram the construction and working of atomic absorption spectrophotometer? A diagram of a Flame Atomic Absorption Spectrometer is shown in figure 11. The light source is a cold cathode lamp that produces (almost exclusively) the light that would be naturally emitted by the element to be measured at a high temperature. A large range of such lamps are available that includes the vast majority of the elements of general analytical interest. Consequently, the light will contain specifically those wavelengths that the element in the flame will selectively absorb. The light passes through the flame, which is usually rectangular in shape so as to provide an adequate path length of flame for the light to be absorbed, and then into the optical system of the spectrometer. The flame is fed with a combustible gas, customarily air/acetylene, nitrous oxide/acetylene or air/propane or butane. The sample, dissolved in a suitable solvent, is nebulized and fed into the gas stream at the base of the burner. The light, having passed through the flame, can be focused directly onto a photo-cell or onto a Diffraction Grating by means of a spherical mirror. The Diffraction Grating can be made movable, and so it can be set to monitor a particular wavelength that is characteristic of the element being measured, or it can be scanned to produce a complete absorption Spectrum of the sample. After leaving the grating, light of a selected wavelength, or range of wavelengths, is focused onto the photocell. The position of the Diffraction Grating determines the wavelength of the light that is to be monitored.

21 Figure 1. The Basic System of a Flame Atomic Absorption Spectrometer The flame absorption spectrometer is fairly sensitive, and can be readily used as a tandem instrument combined with a chromatograph, providing that an appropriate interface is employed. This instrument is normally fitted with 4 different cold cathode lamps and, thus, can examine 4 different elements, automatically, from the same analysis; 8 lamp units are also available. A diagram of a hollow cathode lamp is shown in figure 13. The cylindrical hollow cathode of the lamp contains one or more of the elements of interest in the analysis. The cylindrical cathode is screened from the anode connections by means of a ceramic cylinder The anode is situated above the cathode and is made of tungsten or nickel and the whole electrode system is enclosed in a glass envelope. The glass envelope is filled with neon or argon to a pressure of 1kPa and on applying a potential of 100 to 200 volts across the electrodes a glow discharge is formed.

22 Figure 2. The Hollow Cathode Element Lamp. A simple explanation of the process is as follows, Accelerated electrons from the cathode collide with the gas atoms and produce ions. The ions are accelerated to the cathode by the electric field and when they strike the cathode surface the elements of interest are ejected from the surface and are excited to provide radiation in the discharge environment. These lamps can be combined in groups so that a given instrument can determine a number of different elements by merely switching the lamps. A single lamp can be made to generate characteristic radiation for up to two or three elements without interference problems. Modern atomic adsorption spectrometers are deigned to provide extremely simple operation, easy maintenance and, when required, fast and inexpensive servicing. Many ancillary and alternative devices are available to suit specific applications. A number of different sampling devices are available including very sophisticated automatic samplers that allow instruments to be operated 24 hours a day.

23 6. With a neat sketch explain flame emission spectroscopy The analytical apparatus The functions of an analytical flame spectrometer in general are: (a) transformation of the solution to be analyzed into a vapour containing free atoms or molecular compounds of the analyte in the flame; (b) Selection and detection of the optical signal (arising from the analyte vapour) which carries information on the kind and concentration of the analyte; (c) Amplification and read-out of the electrical signal. Transformation of sample into vapour With a pneumatic nebulizer operated by a compressed gas, the solution is aspirated from the sample container and nebulized into a mist or aerosol of fine droplets. By desolvation, i.e., evaporation of the solvent from the droplets, this mist is converted into a dry aerosol which is volatilized in the flame. The atomization, i.e., the conversion ofvolatilized analyte into free atoms is performed by the flame or other atomizer. The total consumption time is the time required to consume the sample entirely. The minimum consumption time is the time for which nebulization must be carried out to perform an analysis with a given precision. Nebulizers can be described as follows: According to the source of energy used for nebulization as, for example,

24 pneumatic or ultrasonic nebulizers. According to the way the liquid is taken up, e.g., suction, gravity-fed, controlled flow, and reflux-nebulizers. According to the relative position of the capillaries for the nebulizing gas and the aspirated liquid, e.g., angular and concentric nebulizers. In the chamber-type nebulizer, the nebulizing gas-jet stream emerges from the sprayer into a spray chamber. Special devices are the nebulizer with heated spray chamber, the twin nebulizer, and the drop generator. Construction and working: Flames are produced by means of a burner to which fuel and oxidant are supplied in thenform of gases. With the premix burner, fuel and oxidant are thoroughly mixed inside the burner housing before they leave the burner ports and enter the primary combustion or inner zone of the flame. This type of burner usually produces an approximately laminar flame, and is commonly combined with a separate unit for nebulizing the sample. In contrast, a direct-injection burner combines the function of nebulizer and burner. Here oxidant and fuel emerge from separate ports and are mixed above the burner orifice to produce a turbulent flame. Most commonly, the oxidant is also used for aspirating and nebulizing the sample. However, when the fuel is used for this purpose, the term reversed direct-injection burner is applied. In each case, the mist droplets enter the flame directly, without passing through a spray chamber. The term total-consumption burner, which is often used, is not recommended. Premix burners are distinguished as Bunsen-, Meker-, or slot-burners according to whether they have one large hole, a number of small holes, or a slot as outlet for the gas mixture, respectively. When several parallel slots are present, they are identified as multislot burners (e.g., a three-slot burner). The small diameter of the holes in the Meker burner or the narrowness of the slot in the slot-burner prevents the unwanted flash-back of the flame into the burner housing. At the edge of the flame where the hot gas comes into contact with the surrounding air, secondary combustion occurs and the secondary combustion or outer zone is formed.

25 The region of the flame confined by the inner and outer zones, where in many instances the conditions for flame analysis are optimum, is called the interzonal region, or, when the combustion zones have the form of a cone, the interconal zone. Sometimes provision is made to screen the observed portion of the flame gases from direct contact with the surrounding air. This may be done either mechanically, by placing a tube on the top of the burner around the flame, which produces a zonal separation (separated flame), or aerodynamically, by surrounding the flame with a sheath of inert gas that emerges from openings at the rim of the burner top (shielded flame). Observations can thus be made without disturbances from the secondary-combustion zone.