An introduction to UltravioletVisible Absorption Spectroscopy Lectures 21
An introduction to Ultraviolet/Visible Absorption Spectroscopy Lectures 21 and 22 1
In this chapter: Absorption by molecules, rather than atoms, is considered. v Absorption in the UV and Vis regions: occurs due to electronic transitions from the ground state to excited state. v Broad band spectra are obtained: Because molecules have vibrational and rotational energy levels associated with electronic energy levels. v The signal is either absorbance or percent transmittance of the analyte solution where: 2
v. Absorption measurements based upon ultraviolet and visible radiation: Find widespread application for the quantitative determination of a large variety species. Beer’s Law: A = -log. T = log. P 0/P = bc Where T = P/Po always a fraction) A = absorbance = molar absorptivity [M-1 cm-1] c = concentration [M] P 0 = incident power P = transmitted power (after passing through sample) 3 b= path length in cm
Example: A solution containing 4. 48 ppm KMn. O, had a % transmittance of 30. 9% in a 1. 00 cm cell at 520 nm. Calculate the molar ahsorptivity of KMn. O 4, at 520 nm. A = εbc mmol KMn. O 4/m. L = (4. 48 mg KMn. O 4/1000 m. L) x (mmol KMn. O 4/158 mg KMn. O 4) = 2. 84 x 10 -5 M T = 0. 309 A = - log T = - log 0. 309 = 0. 510 = ε * 1. 00 cm * 2. 84 x 10 -5 mol/L ε = 1. 80 x 104 L mol-1 cm-1 4
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Some losses of radiation power Po Loses about 8. 5 % of incident radiation from reflections 7 P
Measurement of Transmittance and Absorbance: The power of the beam transmitted by the analyte solution is usually compared with the power of the beam transmitted by an identical cell containing only solvent or blank (zero abs). An experimental transmittance and absorbance are then obtained with the equations. = - log T = log 1/T P 0 and P: refers to the power of radiation after it has passed through the solvent and the analyte. Because T is a fraction so generally multiplied by 100 to give %T 8
Beer’s law and mixtures v. Each analyte present in the solution absorbs light! v. The magnitude of the absorption depends on its e A total = A 1+A 2+…+An A total = e 1 bc 1+e 2 bc 2+…+enbcn v. If e 1 = e 2 = en then simultaneous determination is impossible (difficult to separate conc. of substances. seam to be same substance). v. Need to measure A at n ’s (get n 2 e’s) to solve for the concentration of species in the mixture v For 3 substance we need to measure at 9 e 9 Where is max for each substance
b=1 cm Large slope = large e and good sensitivity 10
Limitations to Beer’s Law Deviation from linearity of the A and C relationship (remember no interpolation and extrapolation of the calibration: It is regions of deviation from linearity) Types of deviations: • Real limitations (present in the law itself) • Chemical deviations • Instrumental deviations 11
1. Real Limitations a. Beer’s law is good for dilute analyte solutions only. v High concentrations (> 0. 01 M) will cause a negative error since as the distance between molecules become smaller; the charge distribution will be affected which alter the molecules ability to absorb a specific wavelength. v The same phenomenon is also observed for solutions with high electrolyte concentration, even at low analyte concentration. Both give –ve effect v The molar absorptivity is altered due to electrostatic interactions. 12
b. ( ) depends on refractive index. v The refractive index is a function of concentration. v Therefore, will be concentration dependent. v However, the refractive index changes very slightly for dilute solutions and thus we can practically assume that is constant. c. In rare cases, the molar absorptivity changes widely with concentration, even at dilute solutions. v Therefore, Beer’s law is never a linear relation for such compounds, like methylene blue. 13
2. Chemical Deviations: This factor is an important one which largely affects linearity in Beer’s law. It originates when an analyte 1) dissociates, 2) associates, or 3) reacts in the solvent, or one of matrix constituents. For example: An acid base indicator when dissolved in water will partially dissociate according to its acid dissociation constant: 14
HIn (color 1) H+ + In- (color 2) It can be easily appreciated that the amount of HIn present in solution is less than that originally dissolved where: CHIn [HIn] + [In-] Assume an analytical concentration of 2 x 10 -5 M indicator (ka = 1. 42 x 10 -5) was used, we may write: 15
1. 42 x 10 -5 = x 2/(2 x 10 -5 – x) Solving the quadratic equation gives: X = 1. 12 x 10 -5 M which means: [In-] = 1. 12 x 10 -5 M [HIn] = 2 x 10 -5 – 1. 12 x 10 -5 = 0. 88 x 10 -5 M Therefore, the absorbance measured will be the sum of that for HIn and In-. If a 1. 00 cm cell was used and the for both HIn and In- were 7. 12 x 103 and 9. 61 x 102 Lmol-1 cm-1 at 570 nm, respectively, the absorbance of the solution can be calculated: 16
A = AHIn + AIn A = 7. 12 x 103 * 1. 00* 0. 88 x 10 -5 + 9. 61 x 102 * 1. 00 *1. 12 x 10 -5 = 0. 073 However, if no dissociation takes place we may have: A = AHIn (Theoretically) A = 7. 12 x 103 * 1. 00 * 2 x 10 -5 = 0. 142 If the two results are compared we can calculate the % decrease in anticipated signal as: % decrease in signal = {(0. 142 – 0. 073)/0. 142}x 100% = 49% As a result of dissociation 17
However, at 430 nm, the molar absorptivities of HIn and Inare 6. 30*102 and 2. 06*104, respectively. A = AHIn + AIn A = 6. 30*102 * 1. 00* 0. 88 x 10 -5 + 2. 06*104 * 1. 00 *1. 12 x 10 -5 = 0. 236 Again, if no dissociation takes place we may have: A = AHIn (Theoretically) A = 6. 30*102 * 1. 00 * 2 x 10 -5 = 0. 013 If the two results are compared we can calculate the % increase in anticipated signal as: % increase in signal = {(0. 236 – 0. 013)/0. 013}x 100% = V. large Error as a result of dissociation 18
Comparison between results obtained at 570 nm and 430 nm show large dependence on the values of the molar absorptivities of HIn and Inat these wavelength. At 570 nm: A = AHIn + AIn A = 7. 12 x 103 * 1. 00* 0. 88 x 10 -5 + 9. 61 x 102 * 1. 00 *1. 12 x 10 -5 = 0. 073 And at 430 nm: A = 6. 30*102 * 1. 00* 0. 88 x 10 -5 + 2. 06*104 * 1. 00 *1. 12 x 10 -5 = 0. 236 19
Chemical deviations from Beer’s law for unbuffered solutions of the indicator Hln. Note that there are: Ø positive deviations at 430 nm Ønegative deviations at 570 nm. At 430 nm: The absorbance is primarily due to the ionized Inform of the indicator and is proportional to the fraction ionized, which varies nonlinearly with the total indicator concentration. At 570 nm: The absorbance is due principally to the undissociated acid Hln, which increases nonlinearly 20 the total concentration with.
+ve error -ve error 21
Calculated Absorbance Concentrations 22 Data for Various Indicator
An example of association equilibria: Association of chromate in acidic solution to form the dichromate according to the equation below: 2 Cr. O 42 - + 2 H+ D Cr 2 O 72 - + H 2 O The absorbance of the chromate ions will change according to the mentioned equilibrium and will thus be nonlinearly related to concentration. A = Cr. O 4 *b*CCr. O 4 + Cr 2 O 7 *b*CCr 2 O 7 If the two are equals no error 23
3. Instrumental Deviations. a. Beer’s law is good for monochromatic light only since is wavelength dependent. v It is enough to assume a dichromatic beam passing through a sample to appreciate the need for a monochromatic light. v Assume that the radiant power of incident radiation is Po and Po’ while transmitted power is P and P’. v The absorbance ( at two ) of solution can be written as: 24
A = log (Po + Po’)/(P + P’) at the two P = Po 10 - bc, (from the log Eq. ) substituting in the above equation: A = log (Po + Po’)/(Po 10 - bc Po’ 10 - ’bc) for diff. Assume = ’ = A = log (Po + Po’)/(Po + Po’) 10 - bc A = bc However, since ’ # , since is wavelength dependent, then A # bc not a linear relationship between abs. and c Result: Beers law is good for monochromatic radiation. But for different 25 lamps or polych. there is a deviation
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The effect of polychromatic radiation on Beer’s law. v In the spectrum at the left, the molar absorptivity of the analyte is nearly constant over band A. v Note that in Beer’s law plot at the right, using band A gives a linear relationship. v In the spectrum, band B corresponds to a region where the absorptivity shows substantial changes. v In the lower plot, note the dramatic deviation from Beer’s law that results. 27
Sign. change in (diff. abs. ) 28 No sign. change in same abs.
v Therefore, the linearity between absorbance and concentration breaks down if incident radiation was polychromatic. v In most cases with UV-Vis spectroscopy, the effect is small especially at the wavelength maximum. v The small changes in signal is insignificant since differs only slightly. 29
UV-Vis Absorption Spectroscopy Lecture 23 30
b. Stray Radiation ﺃﺸﻌﺔ ﺿﺎﻟﺔ Stray radiation resulting from scattering or various reflections in the instrument will reach the detector without passing through the sample. v. The problem can be severe in cases of: 1 - High absorbance. 2 - When the wavelengths of stray radiation is in such a range where the detector is highly sensitive as well as at wavelengths extremes of an instrument. v The absorbance recorded can be represented by the relation: A = log (Po + Ps)/(P + Ps) Solvent sample Error due to Ps: Ps is added not multiplied Where; Ps is the radiant power of stray radiation. 31
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Instrumental Noise as a Function in Transmittance The uncertainty in concentration as a function of the uncertainty in transmittance can be statistically represented as: sc 2 = (dc/d. T)2 s. T is uncertainty of transmitance A = -log T = bc = -0. 434 ln T c = -(1/ b)*0. 434 ln T dc/d. T = - 0. 434/ b. T (1) sc 2 = (-0. 434/ b. T)2 s. T 2 (2) 33
Dividing equation 2 by the square of equation 1 (sc /c)2 = (-0. 434/ b. T)2 s. T 2/{(-0. 434 ln T)2/( b)2} sc/c = (s. T / T ln T) v Therefore, it is clear that the uncertainty in concentration of a sample is nonlinearly related to the magnitude of the transmittance. v Substitution for different values of transmittance and assuming s. T is constant, we get: 34
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Very lower unc. in con at : Abs: 0. 2 -0. 7 36
v Therefore, an absorbance between 0. 2 -0. 7 may be advantageous in terms of a lower uncertainty in concentration measurements. v At higher or lower absorbances, an increase in uncertainty is encountered. v It is therefore advised that the test solution be in the concentration range which gives an absorbance value in the range from 0. 2 -0. 7 for best precision. v However, it should also be remembered that we ended up with this conclusion provided that s. T is constant. v Unfortunately, s. T is not always constant which complicates the conclusions above. 37
EFFECT OF bandwidth which is related to slit width Effect of bandwidth on spectral detail for a sample of benzene vapor. Note that as the spectral bandwidth increases, the fine structure in the spectrum is lost. At a bandwidth of 10 nm, only a broad absorption band is 38 observed.
Effect of slit width (spectral bandwidth) on peak heights. Here, the sample was a solution of praseodymium chloride. Note that as the spectral bandwidth decreases by decreasing the slit width from 1. 0 mm to 0. 1 mm, the peak heights increase. 39 ﻟﺘﺤﺴﻴﻦ ﺍﻻﺷﺎﺭﺓ ﻳﺘﻢ ﺍﻟﺘﻀﻴﻴﻖ ﺗﺪﺭﻳﺠﻴﺎ ﻟﻠﺤﺼﻮﻝ ﻋﻠﻰ ﺃﺤﺴﻦ ﺍﻣﺘﺼﺎﺹ ﺍﻱ ﺛﺒﺎﺕ ﻟﻼﺷﺎﺭﺓ ﻗﺒﻞ ﺍﻟﻨﺰﻭﻝ
Effect of Scattered Radiation at Wavelength Extremes of an Instrument Wavelength extremes of an instrument are dependent on: v Type of source v Type of detector v Type of optical components used in the manufacture of the instrument. Outside the working range of the instrument: It is not possible to use it for accurate determinations (at extremes unc. increases). However, the extremes of the instrument are very close to the region of invalid instrumental performance and would thus be not very accurate. 40
An example: A visible photometer which, in principle, can be used in the range from 340 -780 nm. It may be obvious that glass windows, cells and prism will start to absorb significantly below 380 nm and thus a decrease in the incident radiant power is significant. 41
What defines the instrumental wavelength extremes? Three main Factors: 1. Source 2. Detector 3. Optical components (lenses, windows, etc) Measurements at wavelength extremes should be avoided since errors are very possible due to: 1. Source limitations 2. Detector limitations 3. Sample cell limitations 4. Scattered radiation 42
B: UV-VIS spectrophotometer A: VIS spectrophotometer EFFECT OF SCATTERED RADIATION Spectrum of cerium (IV) obtained with a spectrophotometer having glass optics (A) and quartz optics (B). The false peak in A arises from transmission of stray radiation of longer wavelengths. 43
v. The output from the source at the low wavelength range is minimal. v Also, the detector has best sensitivities around 550 nm which means that away up and down this value, the sensitivity significantly decrease. v However, scattered radiation, and stray radiation in general, will reach the detector without passing through these surfaces as well as these radiation are constituted from wavelengths for which the detector is highly sensitive. v In some cases, stray and scattered radiation reaching the detector can be far more intense than the monochromatic beam from the source. v. False peaks may appear in such cases and one should be aware of this cause of such peaks. 44
(Abs. Inst. )Instrumentation • Light source • - selector • Sample container • Detector • Signal processing Light Sources (commercial instruments) – D 2 lamp (UV: 160 – 375 nm) – W lamp (vis: 350 – 2500 nm) 45
Sources (UV) 1) Deuterium and hydrogen lamps (160 – 375 nm) D 2 + Ee → D 2* → D’ + D’’ + h 46
Deuterium lamp UV region (a) A deuterium lamp of the type used in spectrophotometers and (b) its spectrum. The plot is of irradiance Eλ (proportional to radiant power) versus wavelength. Note that the maximum intensity occurs at ~225 m. Typically: Instruments switch from deuterium to tungsten at ~350 nm. 47
2) Tungsten lamp: Visible and near-IR region (a) A tungsten lamp of the type used in spectroscopy and its spectrum. (b) Intensity of the tungsten source is usually quite low at wavelengths shorter than about 350 nm. Note that the intensity reaches a maximum in the near-IR region of the spectrum. 48
v The tungsten lamp is by far the most common source in the visible and near IR region with a continuum output wavelength in the range from 350 -2500 nm. v The lamp is formed from a tungsten filament heated to about 3000 o. C housed in a glass envelope. v The output of the lamp approaches a black body radiation where it is observed that the energy of a tungsten lamp varies as the fourth power of the operating voltage. 49
v. Tungsten halogen lamps are currently more popular than just tungsten lamps since they have longer lifetime. v. Tungsten halogen lamps contain small quantities of iodine in a quartz envelope. v. The quartz envelope is necessary due to the higher temperature of the tungsten halogen lamps (3500 o. C). v The longer lifetime of tungsten halogen lamps stems from the fact that sublimed tungsten forms volatile WI 2 which redeposits on the filament thus increasing its lifetime. v The output of tungsten halogen lamps are more efficient and extend well into the UV. 50
Tungsten lamps (350 -2500 nm) Why add I 2 in the lamps? W + I 2 → WI 2 Low limit: 330 nm 1)Low intensity 2)Glass or quartz envelope 51
3. Xenon Arc Lamps: Passage of current through an atmosphere of high pressured xenon excites xenon and produces a continuum in the range from 200 -1000 nm with maximum output at about 500 nm. v Although the output of the xenon arc lamp covers the whole UV and visible regions, it is seldom used as a conventional source in the UVVis. v The radiant power of the lamp is very high as to preclude the use of the lamp in UV-Vis instruments (very low amount absorbed not detected). v However, an important application of this source will be discussed in luminescence spectroscopy which will be discussed later. 52
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UV-Vis Absorption Spectroscopy Lecture 24 54
Instrumental Components • Source • - selector (monochromators) • Sample holders • Cuvettes (b = 1 cm, typically) 1. Glass (Vis) 2. Fused silica (UV+Vis) • Detectors – Photodiodes – PMTs 55
Sample Containers v. Sample containers are called cells or cuvettes and are made of either glass or quartz depending on the region of the electromagnetic spectrum. v. The path length of the cell varies between 0. 1 and 10 cm but the most common path length is 1. 0 cm. v Rectangular cells or cylindrical cells are routinely used. v In addition, disposable polypropylene cells are used in the visible region. v The quality of the absorbance signal is dependent on the quality of the cells used in terms of matching, cleaning as well as freedom from scratches. 56
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Types of Instruments Instrumental designs for UV-visible photometers or spectrophotometers. In (a), a single-beam instrument is shown. Radiation from the filter or monochromator passes through either the reference cell or the sample cell before striking the photodetector. 60
1. Single beam – Place cuvette with blank in place in the instrument and take a reading 100% T – Replace blank with sample and take reading % T for analyte (from which absorbance is calc’d) 61
Most common spectrophotometer Spectronic 20 1. On/Off switch and zero transmission adjustment knob 2. Wavelength selector/Readout 3. Sample chamber 4. Blank adjustment knob 5. Absorbance/Transmitt ance scale 62
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End view of the exit slit of the Spectronic 20 spectrophotometer pictured earlier 64
• Single-Beam Instruments for the Ultraviolet/Visible Region 65
• Single-Beam Computerized Spectrophotometers 66 Inside of a singlebeam spectropho tometer connected to a computer.
2. Double beam (most commercial instruments) – Light is split and directed towards both reference cell (blank) and sample cell – One or two detectors; electronics measure ratio (i. e. , measure/calculate absorbance) – Advantages: Ø Compensates for fluctuations in source intensity and drift in detector Ø Better design for continuous recording of spectra Ø Faster 67
General Instrument Designs Double Beam: In – Space (not populated) Use two detectors Control first the a two ref. by the knob Needs two detectors 68
General Instrument Designs Double Beam: In – Time Use only one detector To give zero difference between the two Ref cells Half the radiation pass and the other reflected not (needed) Chopper transmit half the beam (sample) and reflect the other (Ref ) 69
A prefer design practically : few components, UV-Vis regions Two sources: We can choose any of the two sources Self collimation 70
The two choppers opened and closed alternatively with the same motor 71
Merits of Double Beam Instruments 1. Compensate for all but the most short term fluctuation in radiant output of the source. 2. Compensate amplifier. drift in transducer and 3. Compensate for wide variations in source intensity with wavelength. 4. Much faster than single beam. 72
3. Dual Beam Instruments (single beam) Measure the ratio of the two beams at the two detectors 73
4. Multichannel Instruments v Photodiode array detectors used (multichannel detector, can measure all wavelengths dispersed by grating simultaneously). v Advantage: Ø scan spectrum very quickly “snapshot” < 1 sec. Ø Powerful tool for studies of transient intermediates in moderately fast reactions. Ø Useful for kinetic studies. Ø Useful for qualitative and quantitative determination of the components exiting from a liquid chromatographic column. 74
Sample located before monochromator as the sample exposed to radiation at very short time A multichannel diode-array spectrophotometer 75
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Location of Sample cell Ø In all photometers and scanning spectrophotpmeters described above, the cell has been positioned after the monochromators. Ø This is important to decrease the possibility of sample photodecomposition due to prolonged exposure to all frequencies coming from the source. Ø However, the sample is positioned before the monochromator in multichannel instruments like a photodiode array spectrophotometer. Ø This can be done without fear of photodecomposition since the sample exposure time is usually less than 1 s. Ø Therefore, it is now clear that in UV-Vis: v Where photodecomposition of samples can take place, the sample is placed after the monochromators in scanning instruments. v. While positioning of the sample before the 77 monochromators is advised in multichannel instruments.
5. Probe Type Instruments v These are the same as conventional single beam instruments but the beam from the monochromators is guided through a bifurcated optical fiber to the sample container where absorption takes place. v The attenuation in reflected beam at the specified wavelength is thus measured and related to concentration of analyte in the sample. 78
A fiber optic cable: Ø Can be referred to as a light pipe where light can be transmitted by the fiber without loss in intensity (when light hits the internal surface of the fiber at an angle larger than a critical angle). Ø Therefore, fiber optics can be used to transmit light for very long distances without losses. Ø A group of fibers can be combined together to form a fiber optic cable or bundle. Ø A bifurcated fiber optic cable: has three terminals where fibers from two separate cables are combined at one end to form the 79 new configuration.
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Fiber optic probe instrument 83
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6. Double Dispersing Instruments v. The instrument in this case has two gratings where the light beam leaving the first monochromators at a specified wavelength is directed to the second grating. v This procedure results in better spectral resolution as well as decreased scattered radiation. v However, double dispersing instruments are expensive and seem to offer limited advantages as compared to cost; especially in the UV-Vis region where exact wavelength may not be crucial. 85
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ØOptical diagram of the Varian Cary 300 double-dispersing spectrophotometer. ØA second monochromator is added immediately after the source. ØUsed for Qualitative analysis. Note: not needed for quant. Analysis; as we measure at max 87
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