II. Spectrophotometry (Chapters 17, 19, 20)

II. Spectrophotometry (Chapters 17, 19, 20) FUNDAMENTALS (Chapter 17) Spectrophotometry: any technique that uses light to measure concentrations (here: U and visible - ~190 800 nm) c = 2.99792 x 10 8 m/s (in vacuum) ν = frequency (s -1 ); λ = wavelength (m) h = Planck s ct. = 6.62608 x 10-34 J/s (this is for 1 photon; for 1 mol of photons: x N) ν is the wavenumber = 1/λ E is inversely related to λ, but directly proportional to ν 1 Additive primary colours: red, green and blue The observed color is called the complementary color. A white object reflects all (visible) light 2

? Airport body scanners Your microwave at home (2.45 GHz) 3 Interaction of molecules with light (EM radiation): Depends on the λ of the radiation: Microwave radiation: rotational transitions of molecule IR radiation: stretching and bending of bonds Irradiation of formaldehyde with the right λ increases the amplitude of the vibrations isible light and U radiation: Energetic enough to cause electronic transitions: electron moves from one MO to higher energy MO (e.g. n π*) Short wavelength U and X-rays: break bonds, ionize molecules All transitions are quantized! selection rules 4

Upon absorption of a photon, the entire molecule is in an excited state, but electronic transitions can usually be assigned to specific functional groups (e.g. C=O, C=C, NO 2, conjugated π system). These chromophores can undergo π π*, n π* and n σ* transitions (inorganic molecules: e.g. d d) With each electronic transition, there are numerous vibrational and rotational transitions broad peaks! E S 1 λ 3 λ 2 ν S 2 0 ν 1 0 (Rotational levels not shown) At room temperature, most transitions are from the lowest vibrational level of the ground state (S 0, ν 0 ). When irradiated with broadband EM radiation, those λ s corresponding with a particular transition can be absorbed. Because of numerous energy levels, numerous λ s are absorbed, giving the impression of a broad absorption peak. In addition, solution-phase spectra are broadened because of interaction of absorbing molecules with solvent molecules. For simple molecules in a dilute gas phase, individual rotational transitions can often be distinguished. There may be several chromophores in one molecule: broad and overlapping peaks 5 Spectra of atoms are line spectra, because they do not have vibrational and rotational transitions. Compare the width of these absorption peaks (notice the different scales) 6

What happens to the absorbed energy? Absorption of radiation increases energy of molecule; two ways to release that energy (besides taking part in a photochemical reaction): Radiationless energy transfer: vibrational relaxation and internal conversion (collisions with other molecules, e.g solvent) Emission of radiation: fluorescence, phosphorescence (= luminescence) 7 Fluorescence always from the lowest vibrational level of the excited state Emission at higher λ (lower frequency, lower E) Fluorescence: much more sensitive analysis technique then absorption spectrophotometry (~no background) 8

Absorption Spectrophotometry (Chapters 19, 20) Schematic of a single beam dispersive spectrophotometer: LIGHT SOURCE - Continuum source Produces a continuous band of λ s in visible and U region of spectrum Deuterium lamp (200-400 nm): electric discharge causes D 2 to dissociate and emit U radiation (~ 110-400 nm) + Tungsten lamp: W filament at 3000 K gives useful radiation in the range of 320-2500 nm (For atomic absorption, line sources are used see further) 9 Typically, the instrument switches automatically from the D 2 lamp to the W lamp when passing through 360 nm so that always the source with the highest intensity is used. MONOCHROMATOR -selects a narrow band of λ s from the continuum band. Historically, prisms were widely used, but they have been surpassed by gratings (optical components with closely spaced lines; different wavelengths of light are reflected at different angles from the grating = diffraction). 10

Polychromatic radiation is collimated The width of the exit slit can be adjusted to allow various bandwidths to reach the detector. A bandwidth of 1.0 nm through the exit slit is quite common, Rotates so that different λs can pass through exit slit A filter is often placed after the exit slit to prevent higher order diffractions of λ 2 /n to reach the detector e.g. 2 nd order of λ 2 /2; third order of λ 2 /3 11 CUETTE- places sample in path of light to be absorbed. P is the irradiance of the beam leaving the cuvette such that P P 0 The pathlength b is the distance the beam has travelled through the sample For U-is spectrophotometry cuvettes are made of quartz LIGHT DETECTOR - converts light to electric current e.g. photomultiplier tube, diode array detector (DAD), or charge coupled device (CCD) Transmittance T: fraction of original light that passes through the sample: P = irradiance (Js -1 m -2 ) % T P/P P 0 P 0 /P A %T = x 100% 100 % 1 1 0 P 0 10% 0.1 10 1 1% 0.01 100 2 Absorbance A: A is dimensionless, but you may see AU or mau as units 12

Beer-Lambert s law: Absorbance of a sample is linearly related to it s concentration: c = [analyte] (M) b = path length of cuvette (cm) ε = molar absorptivity or extinction coefficient (M -1 cm -1 ) ε is a characteristic of a compound for a particular λ, but can be influenced by intermolecular interactions Ideally as large as possible for good sensitivity Deviations from Beer s law: At high concentrations (>0.01 M), solute molecules are close enough to each other to interact and alter their light absorbing properties (i.e. ε value changes); high conc. of non-absorbing species can have this effect as well Strictly speaking only for truly monochromatic radiation In practice, deviations are not observed with the narrow range of λ s transmitted by spectrophotometers http://www.chem.uoa.gr/applets/appletbeerlaw/appl_beer2.html 13 Analytes that participate in a concentration dependent equilibrium (apparent deviation): e.g. weak acids (HA) are more dissociated at higher dilution. If ε of the undissociated species HA differs from that of the dissociated species A -, a series of dilutions of HA will appear not to obey Beer s law. (Solution: use a buffer to maintain a constant ph constant dissociation) Other apparent deviations: stray light, fluorescence, scattering by particles Application of Beer s law in chemical analysis: Select λ: usually λ of max. intensity (max. ε) to give best sensitivity, and to have the least change in A if drift in monochromator In case of interference with another compound, another λ may be necessary (note: with DAD, the whole spectrum can be recorded instantaneously) Record baseline spectrum of either pure solvent or reagent blank (alternatively, if solvent is used to obtain the baseline spectrum, the value of a reagent blank can be subtracted from the unknown) 14

Measure absorbances of (usually) 3 to 5 standard solutions containing accurately known concentrations of analyte, and the unknown (aim for an absorbance of 0.2 0.9 for best accuracy) Construct a calibration curve: plot A vs. conc. or mass of analyte Use linear regression (least squares) to obtain: A = mc + b where m = slope of curve b = intercept with y-axis c = concentration of amount of analyte With the absorbance of the unknown, and the values of m and b from the calibration curve, the unknown concentration can be found 15 Example 1: The concentration of iron(iii) in an unknown was determined from a calibration curve of absorbance vs concentration of standard solutions. Calculate the concentration of iron (III) in the unknown in mol L 1. A unknown = 0.479; m = 0.2576 ppm -1 ; b = 0.0009 (A - b) unknown 0 479 0 0009 CA = mc = + b.. = = 1. 86 ppm unknown 1 m 0. 2576 ppm 0.479 = 0.2576c + 0.0009 c = 1.86 ppm 1. 86 mg C c = unknown L 3+ 1g 1mol Fe = 3.32 x 10 1000mg 55. 847 g -5 mol Fe Example 2: Gaseous ozone has a molar absorptivity of 2700 M -1 cm -1 at 260 nm. Find the concentration of ozone in air with an absorbance of 0.2335 in a 10.0 cm cell if a blank of air has an absorbance of 0.0011 at this wavelength. A 0.2335-0.0011 c = = -1-1 εb 2700M cm 10.0 cm -6 c = 8.62 x 10 M 3+ L -1 16

Double beam dispersive spectrophotometers: Advantages of double beam over single beam instrument: P 0 reflective surface Reference is left in instrument no switching around Better for following absorbance as a function of time (kinetics experiments) because corrects automatically for drift in both lamp intensity and detector response over time Easier to acquire whole spectrum of analyte by scanning λ-range P 0 17 The photomultiplier tube (PMT) as a sensitive detector: EM radiation hits photosensitive surface (at neg. potential), which emits electrons Emitted electrons strike a dynode (at less neg. potential than photosensitive surface); for each electron that hits the dynode, two or more other electrons are released These electrons hit a second dynode (at less neg. potential then first dynode), which releases even more electrons, which hit a third dynode Signal amplification leads to >10 6 electrons for each photon! (depends on kinetic energy of electrons, thus on potential difference btw photocathode and last dynode) 18

Photodiode array spectrophotometers: Entire spectrum recorded in fraction of a second (signal averaging!) = Type of spectrophotometer used in liquid chromatography Each diode receives a different λ; resolution depends on how closely spaced the diodes are, and the degree of dispersion by the polychromator. Best resolution currently available is ~ 0.5 1.5 nm. Fig. 19-33: Effect of signal averaging on a noisy spectrum 19 A reverse bias is applied to each diode, drawing electrons and holes away from the junction, creating a depletion region. Each photodiode can be thought of as a capacitor, with a fixed charge. When light hits a photodiode, free electrons and holes are created, partially discharging the photodiode. At the end of each measuring cycle, the current needed to recharge the capacitor is proportional to # photons it received (i.e. the irradiance). Charge coupled devices (CCDs) are also used as detectors, and are much more sensitive than photodiodes. They consist of a two-dimensional array of pixels and operate on a slightly different basis than DADs. 20

ATOMIC SPECTROSCOPY (Chapter 20) In atomic spectroscopy, samples are decomposed at high temperatures into atoms. Concentrations of atoms in the vapor are measured by absorption or emission of characteristic λs of radiation. μg/g to pg/g levels (sensitive!), with a precision of 1-2% Atomic Absorption Spectrometry (AAS) Nebulizer Liquid sample aspirated into flame Sample atomizes HCL emits light at λ that element can absorb Intensity of light that reaches detector (P) decreases as concentration of analyte increases (Beer s law applies flame = cuvette) 21 Energy level diagram of sodium (1s 2 2s 2 2p 6 3s 1 ): Modified from: http://hyperphysics.phy-astr.gsu.edu/hbase/quantum/sodium.html 22

Hollow Cathode Lamp (HCL) Atomic absorption lines are very narrow (~ 0.01 0.001 nm) Beer s law requires that linewidth of source << linewidth of absorbing sample Cannot use monochromator, instead use HCL with the cathode made from the same element that has to be analyzed P 0 HCL is filled with Ne or Ar (low pressure) applied: gas ionized and cations accelerated towards cathode, where they sputter metal atoms into gas phase. A certain fraction of these are in an excited state, and they fall back to the ground state by emission of photons of specific λs. 23 The λs of the emitted radiation are the same as those that can be absorbed by the analyte (not all λs will be useful) Note that the bandwidth of the emitted radiation (from cool atoms)< that of the absorption bandwidth of the atoms in the hot flame (Doppler linewidth ~ T) A different lamp is needed for each element, but some lamps are made with more than one element in the cathode Monochromator (see slide 8 same type) Selects one specific emission line Rejects as much emission from flame as possible Flame and Nebulizer Sample is aspirated in to the nebulization chamber by the rapid flow of oxidant, where it is turned into a mist (fine droplets) and mixed with fuel and oxidant. The mist flows past baffles which remove large droplets and promotes the formation of finer droplets, resulting in the aerosol, which eventually passes into the flame. (~5% of the solution originally aspirated 95% goes to waste!). 24

MX (solution) nebulization MX (solution aerosol) desolvation MX (solid aerosol) Desolvation: the liquid solvent is evaporated, and the dry sample remains aporization: the solid sample vaporizes to a gas Dissociation: the compounds making up the sample are broken into free atoms. vaporization MX(g) dissociation M 0 X 0 M + X - 25 The flame is long (~10 cm) and narrow; height is controlled by the flow of the fuel mixture The beam of light from the HCL is directed along the long axis and its height has to be optimized for each element because of different temperature regions within the flame (this affects formation of oxides, desolvation, vaporization etc.) Temperature of the flame depends on fuel/oxidant. Acetylene/air is most used (2400 2700 K). Higher temperatures for refractory elements can be obtained with acetylene/oxygen (~3400 K) Detectors: PMT, DAD, CCD Interferences in AAS: Interference = any effect that changes the signal, while analyte conc. stays the same Spectral interference: overlap of spectral lines Chemical interference: e.g. decrease of extent of atomization Ionization interference: ion has different energy levels than atom 26

Flameless methods: Generally better detection limits than flame AAS Graphite Furnace AAS: Sample is deposited (manually or automatically) into a graphite tube which is electrically heated Heating takes place in several steps to remove solvent, destroy organic matter etc. Sample is confined in the optical path for several seconds increases sensitivity (note: transient signal as opposed to continuous signal) Both liquid and solid samples Cold apor AAS: For Hg only (only metallic element with appreciable vapor pressure at ambient temperature) All Hg in a liquid sample is reduced to Hg 0, which is removed by bubbling inert gas through the sample, and introduced into the analysis chamber Hydride generation techniques: For As, Sb, Sn, Se, Bi and Pb: form volatile hydrides after reaction with NaBH 4 (to e.g. AsH 3 ). Most of analyte ends up in the instrument <> flame AAS where 95% of the sample goes to waste. 27 Inductively Coupled Plasma (ICP) Sample is introduced into a plasma (= partially ionized gas free ions and electrons) at a temperature of 6000 10000 K Much of the interferences from AAS are lost (but not all!) At this high temperature, a higher fraction of atoms are in an excited state, and it is the radiation that is emitted by these atoms when they fall back to the ground state that is measured Multielement technique; 70+ elements can be determined at once! 28

Drinking water analysis (EPA certified method) GFAA for clinical use determination of trace elements in blood serum. Trace elements in food Trace element analysis of hair (forensics) For accurate quantitative analysis, multiply DL by 10 For analysis of elements at DL afforded by GFAAS and ICP-MS, a clean room with filtered air supply is necessary! 29 MATRIX EFFECTS (Chapter 5) Matrix: All non-analyte components of a sample Matrix effects: Influence of the matrix on the response of the instrument to the analyte Why are matrix effects a problem in analysis of real samples? If the response of the analyte in the unknown solution is different from the one in the standard solutions (because of matrix effects), then large errors can be introduced in the analysis. Methods used to minimize matrix effects: Separation of analyte from the matrix - Must be quantitative (e.g. chromatography, precipitation and filtration of interfering compound) Prepare standard solutions in the same matrix as the samples (e.g. standards in river water has to be free of analytes that are analyzed!) Standard addition: Allows the standard and analyte to be measured in same or nearly identical matrix. 30

Overcoming Matrix Effects using Standard Addition M. Bader, Journal of Chemical Education 57 (1980) 703 Known quantities of analyte are added to the unknown (small amounts of a concentrated standard are added, so that the matrix composition does not change a lot) From increase in signal, original amount or concentration is deduced Requires linear increase in response = conc. of analyte X in sample (volume 0 ); gives a signal intensity of I x I x = k A known conc. of a standard S (which is the same analyte as X) is added (conc. [S] i ; volume s ). The intensity of the signal is now I S+X I S+X = k ([X] f + [S] f ) [X] f = [X] 0 i [S] f = [S] s i ( = 0 + s ) 31 Dividing first equation by second equation: I X I S+X = [X] f + [S] f I X and I S+X are measured [X] f and [S] f are calculated can be determined There are a number of different ways in which standard addition can be performed. We will only discuss three cases: CASE 1: Same flask volume changes Accurately measure a certain volume of sample acquire signal Accurately add a known amount of standard into the sample solution acquire signal (e.g. ion selective electrode) EXAMPLE: A 50.0 ml sample of orange juice gave a signal of 1.78 μa using an electrochemical method. A standard addition of 0.400 ml of 0.279 M ascorbic acid increased the signal to 3.35 μa. Find the concentration of ascorbic acid in the orange juice. 32

I x = 1.78 μa I S+X = 3.35 μa 0 = 50.0 ml ; = 50.0 ml + 0.400 ml = 50.4 ml [X] f = 0 [S] f = [S] i = 50.0 ml 50.4 ml s = 0.279 M 0.400 ml 50.4 ml = 0.992 1 = 2.21 4 x 10-3 M 1.78 μa 3.35 μa = 0.992 1 + 2.21 4 x 10-3 M = 2.49 x 10-3 M CASE 2: Sample in one flask Sample and standard in 2 nd flask both diluted to same final volume Usually done when reagents have to be added (e.g. colorimetric reaction) and/or when the measurement itself consumes the sample 33 Same volume of sample is added to both volumetric flasks; small volume of standard solution added to one of the flasks Reagents added to both volumetric flasks, which are then diluted to volume Signals for both solutions are acquired In the formula on slide 32, the numerator corresponds to the measurement of the undiluted sample. However, here the sample is diluted to its final volume when we measure it, so the formula we have to use changes to: I X I S+X [X] = f Standard addition at constant volume [X] f + [S] f EXAMPLE: Two 10.0 ml samples of run-off water are added to separate 25.00 ml flasks. A 0.200 ml aliquot of 500.0 ppm iron(iii) is added to the second flask. Both flasks have reagents added to give a colored product and are then diluted to volume (25.00 ml). The absorbance of the two solutions in a 1.00 cm cell at 490 nm were 0.426 and 0.753 respectively. Calculate the concentration of iron (III) in the run-off water. 34

I x = 0.426 I S+X = 0.753 0 = 10.00 ml ; = 25.00 ml [X] f = 0 = 10.00 ml 25.00 ml = 0.4000 [S] f = [S] i s = 500.0 ppm 0.200 ml = 4.00 ppm 25.00 ml 0.426 0.753 = 0.4000 0.4000 + 4.00 ppm = 13.0 ppm Case 3: Multiple Standard Addition at Constant Total olume Calibration Curves A series of solutions are prepared in volumetric flasks, each containing the same volume of unknown ( 0 ), and different volumes of standard ( s ) 35 (from slide 27) I S+X = k 0 + k [S] i s and add reagents, if any y = b + m x Plot I S+X vs. s from the equation of this curve, determine b and m: b = k m = k [S] i 0 b m = 0 [S] i = b m [S] i 0 36

I S+X -10-5 0 5 10 15 20 s Alternatively, one can plot I S+X vs. [S] i s i.e. I S+X vs. conc. of standard in the flasks We now find: b = k m = k 0 b m = 0 = b m 0 Note that the factor / 0 takes into account the dilution of the sample in the volumetric flask. If we plot I S+X vs. [S] i s, the concentration of the 37 unknown in the volumetric flask is the neg. intercept with the x-axis: I S+X -2-1 0 1 2 3 4 conc. (mm) - conc. of X in volumetric flask; has to be corrected with dilution factor to find 38