1 Optické metody k stanovení analytu se využívá interakce elektromagnetického záření se zkoumanou látkou (vzorkem). Rotační spektrum mlhoviny v Orionu 2 UV měření koncentrace ozonu (ppb jednotky) Molekulové absorpční spektrum ozonu 3 On-line kontinuální měření koncentrace oxidu uhličitého (IR, 2300 cm-1) a kyslíku (UV, 147 nm) v dechu Absorption Spectra of Hemoglobin Derivatives 4 Vícesložková luminiscenční analýza Vícesložková analýza 5 Polarizované elmg záření el.pole + - , délka vlny čas délka amplituda perioda 6 Duální charakter elmg záření Parametry charakterizující vlnové vlastnosti: ­ vlnová délka, [m, nm] ­ rychlost šíření v určitém prostředí [m/s] (rychlost ve vakuu c = 2,9979.108 m/s) ­ frekvence, [Hz] vztah mezi základními parametry: ­ vlnočet [cm-1] Parametry charakterizující korpuskulární vlastnosti: E = h ­ energie [J] (h = Planckova konstanta, 6,6.10-34 J.s) = 1 =T he distance of one cycle is the wavelength (). The frequency () is the number of cycles passing a fixed point per unit time. = c/ (c = velocity of light, 3 x 1010 cm s-1). The shorter the wavelength, the higher the energy: E = h = hc/ This is why UV radiation from the sun burns you. The distance of one cycle is the wavelength (). The frequency () is the number of cycles passing a fixed point per unit time. = c/ (c = velocity of light, 3 x 1010 cm s-1). The shorter the wavelength, the higher the energy: E = h = hc/ This is why UV radiation from the sun burns you. 7 Visible Fig. 16.2. Electromagnetic spectrum. We see only a very small portion of the electromagnetic spectrum . In spectrochemical methods, we measure the absorption of UV to far IR radiation. UV = 200-380 nm, VIS = 280-780 nm, IR = 0.78 m-300 m We see only a very small portion of the electromagnetic spectrum . In spectrochemical methods, we measure the absorption of UV to far IR radiation. UV = 200-380 nm, VIS = 280-780 nm, IR = 0.78 m-300 m Gary Christian, Analytical Chemistry, 6th Ed. (Wiley) 8 OPTICKÉ METODY při interakci záření/hmota k výměně energie nedochází při interakci záření/hmota k výměně energie dochází REFRAKTOMETRIE POLARIMETRIE NEFELOMETRIE TURBIDIMETRIE SPEKTRÁLNÍ METODY ATOMOVÉ MOLEKULOVÉ Absorpční Emisní Fluorescenční Absorpční Emisní Fluorescenční 2 1 3 1 2 = m3 vzduch vzorek lupa na úhloměrné stupnici 1 2 Refraktometrie n = sin / sin = v1 / v2 9 zdroj záření detektor polarizátor analyzátorkyveta se vzorkem Polarimetrie = [] t L c zdroj záření zdroj záření detektor detektor vzorek vzorek A B Nefelometrie a turbidimetrie (A-turbidimetrie, B-nefelometrie) 10 Příjem energie ­ absorpce Ztráta energie ­ emise (luminiscence) - atomy a molekuly mohou měnit svůj energetický stav přijmutím nebo vyzářením energie, přičemž jak přijatá, tak i vyzářená energie může nabývat pouze určitých diskrétních hodnot; -v atomech přijímají nebo vyzařují energii pouze elektrony, v molekulách jsou elektronové energetické hladiny rozštěpeny na podhladiny vibrační a rotační; pro energetické rozdíly mezi hladinami platí: Erot << Evibr << Eel; E = Erot + Evibr + Eel + Espin + Ecore Franck-Condonův princip Výběrová pravidla 11 Spektrální metody absorpční a emisní ­ společný základ Absorpce záření, X + hv X* L absorbující prostředí 0 Emise záření, X* X + h (excitace absorpcí záření či dodáním tepla) vzorek excitace 0log Lc A == = a.cb Fluorescence, X* X + h' + teplo (excitace absorpcí záření), Excitovaný atom či molekula ztrácí část energie nezářivým způsobem, ' < ; Tok fluorescenčního záření: F = f(c) 2 2 2 2 1 1 1 1 E h= 2 2 E h= 1 1 < E Atomy elektronové hladiny Molekuly E0E0 E E1 E2 vibrační hladiny rotační hladiny elektronovéhladiny nezářivá relaxace E intenzita intenzita čára (linie) čárové spektrum pásové spektrum pás } } A B C D E 12 Fig. 16.29. Energy level diagram showing absorption processes, relaxation processes, and their rates. Absorption of a photon causes electronic transition from the ground state to a higher energy state. The electron relaxes to the lowest energy level of the first excited state. The wavelengths of emitted radiation are independent of the wavelength of excitation. But intensities are not. Absorption of a photon causes electronic transition from the ground state to a higher energy state. The electron relaxes to the lowest energy level of the first excited state. The wavelengths of emitted radiation are independent of the wavelength of excitation. But intensities are not. Gary Christian, Analytical Chemistry, 6th Ed. (Wiley) Fig. 16.30. Excitation and emission spectra of a fluorescing molecule. The excitation spectrum corresponds to the absorption spectrum. In larger molecules, the vibrational spacings of excited states are similar to those in the ground state. So the emission spectrum may be a mirror image of the excitation spectrum. The excitation spectrum corresponds to the absorption spectrum. In larger molecules, the vibrational spacings of excited states are similar to those in the ground state. So the emission spectrum may be a mirror image of the excitation spectrum. Gary Christian, Analytical Chemistry, 6th Ed. (Wiley) 13 ˇ Spektrum ­ uspořádaný soubor absorbovaných či emitovaných vlnových délek počet a hodnoty - kvalitativní údaj intenzita abs./emit. záření ­ kvantitativní údaj. Spektrum ˇ absorpční atomová ˇ emisní molekulová 14 Metody molekulové a atomové spektrometrie ­ společný základ spektra jsou čárováspektra jsou pásová analyticky se využívá absorpce, emise, fluorescence analyticky se využívá absorpce a fluorescence vzorek ve formě oblaku atomůvzorek v kyvetě vyžadují vyšších excitačních energií, VIS, UV, RTG vyžadují malých excitačních energií, UV, VIS, IČ, -vlny, RF využití k důkazu a stanovenívyužití k identifikaci a stanovení informace o přítomnosti atomůinformace o přítomnosti molekul, vazeb, funkčních skupin Atomová spektrometrieMolekulová spektrometrie Transmission and Color The human eye sees the complementary color to that which is absorbed 15 Absorbance and Complementary Colors The complement of the absorbed light gets transmitted. The color of an object we see is due to the wavelengths transmitted or reflected. Other wavelengths are absorbed. The more absorbed, the darker the color (the more concentrated the solution). In spectrochemical methods, we measure the absorbed radiation. The complement of the absorbed light gets transmitted. The color of an object we see is due to the wavelengths transmitted or reflected. Other wavelengths are absorbed. The more absorbed, the darker the color (the more concentrated the solution). In spectrochemical methods, we measure the absorbed radiation. Gary Christian, Analytical Chemistry, 6th Ed. (Wiley) 16 17 18 Fig. 16.3. Energy level diagram illustrating energy changes associated with absorption of electromagnetic radiation. E0 is electronic ground state and E1 is first electronic excited state. A - pure rotational changes (far IR). B - rotational-vibrational changes (near IR). C - rotational-vibrational-electronic changes (visible and UV). These transitions are all quantized. A - pure rotational changes (far IR). B - rotational-vibrational changes (near IR). C - rotational-vibrational-electronic changes (visible and UV). These transitions are all quantized. Gary Christian, Analytical Chemistry, 6th Ed. (Wiley) 19 Fig. 16.4. Typical infrared spectra. The peaks are associated with vibrational modes within the molecule. (More in Fig. 16.8 on types of bonds that give peaks.) The peaks are associated with vibrational modes within the molecule. (More in Fig. 16.8 on types of bonds that give peaks.) Gary Christian, Analytical Chemistry, 6th Ed. (Wiley) 20 Fig. 16.5. Typical visible absorption spectrum. 1, Sample; 2, blank. Electronic transitions (at higher energy ­ shorter wavelengths) are superimposed on rotational and vibrational trasitions. These many discrete transitions result in a broad band of unresolved fine structure. (double or triple bonds) and n (outer shell) electrons are responsible for most UV and Vis electronic transitions. Electronic transitions (at higher energy ­ shorter wavelengths) are superimposed on rotational and vibrational trasitions. These many discrete transitions result in a broad band of unresolved fine structure. (double or triple bonds) and n (outer shell) electrons are responsible for most UV and Vis electronic transitions. Gary Christian, Analytical Chemistry, 6th Ed. (Wiley) Fig. 16.6. Typical ultraviolet spectrum. These are similar in structure to visible spectra.These are similar in structure to visible spectra. Gary Christian, Analytical Chemistry, 6th Ed. (Wiley) 21 These groups absorb in the UV or visible regions.These groups absorb in the UV or visible regions. Gary Christian, Analytical Chemistry, 6th Ed. (Wiley) Fig. 16.7. Ultraviolet spectrum of benzene. Aromatic compounds are good absorbers of UV radiation.Aromatic compounds are good absorbers of UV radiation. Gary Christian, Analytical Chemistry, 6th Ed. (Wiley) 22 Fig. 16.8. Simple correlation of group vibrations to regions of infrared absorption. Absorption in the 6- to 15-m region is very dependent on the molecular environment. This is called the fingerprint region. Absorption in the 6- to 15-m region is very dependent on the molecular environment. This is called the fingerprint region. Gary Christian, Analytical Chemistry, 6th Ed. (Wiley) Organic substances measured in the UV must usually be dissolved in organic solvents. The solvent may affect the spectrum due to solute-solvent interactions. A polar solvent may cause loss of fine structure. Organic substances measured in the UV must usually be dissolved in organic solvents. The solvent may affect the spectrum due to solute-solvent interactions. A polar solvent may cause loss of fine structure. Gary Christian, Analytical Chemistry, 6th Ed. (Wiley) 23 0 0 0 0 ln( ) log( ) ( ) ln(10) P b P dP Pc dx dP c dx P dP c dx P P cb P P cb P = - - = - = = = 24 Absorption of radiation. P0 = power of incident radiation, P = power of transmitted radiation, c = concentration, b = pathlength. Transmittance = P/P0 = 10-abc a = proportionality constant = absorptivity -log T = abc = A = absorbance Transmittance = P/P0 = 10-abc a = proportionality constant = absorptivity -log T = abc = A = absorbance Transmittance and Concentration The Bouguer-Lambert Law 0/ Const Pathlength T I I e- = = 25 Transmittance and Path Length Beer's Law 0/ Const Concentration T I I e- = = Concentration The Beer-Bouguer-Lambert Law ( ) ( )0 0log log / log /A T I I I I b c= - = - = = 26 Stanovení nanomolárních koncentrací dusičnanů a dusitanů pomocí ,,Long Path Length Absorbance" spektroskopie Use the newer recommended nomenclature.Use the newer recommended nomenclature. Gary Christian, Analytical Chemistry, 6th Ed. (Wiley) 27 Fig. 16.11. Baseline method for quantitative determination in infrared region of spectrum. This empirical ratio method is used because of deviations from Beer's law, scattered radiation, etc. Log P0/P is plotted against C. This empirical ratio method is used because of deviations from Beer's law, scattered radiation, etc. Log P0/P is plotted against C. Gary Christian, Analytical Chemistry, 6th Ed. (Wiley) Fig. 16.21. Distribution of wavelengths leaving the slit of monochromator. The nominal wavelength is that set on the instrument. The slit passes a band of wavelengths. The bandwidth varies with wavelength with a prism, but is constant with a grating. The nominal wavelength is that set on the instrument. The slit passes a band of wavelengths. The bandwidth varies with wavelength with a prism, but is constant with a grating. Gary Christian, Analytical Chemistry, 6th Ed. (Wiley) 28 UV/VIS spektroskopie Gaussovský pás IR, NMR spektroskopie Lorenzovský pás Ideální případ !!! Většinou kombinace obou funkcí Šířka čáry Ďopplerovo rozšíření ­ detektor vs. zdroj ˇ,,lifetime" rozšíření - princip neurčitosti Derivative Spectra of a Gaussian Absorbance Band 1st Derivative: 2nd Derivative: Absorbance: )(' f d dA = )(fA = )('' 2 2 f d Ad = 29 Resolution Enhancement ˇ Overlay of 2 Gaussian bands with a NBW of 40 nm separated by 30 nm ˇ Separated by 4th derivative Discrimination of Broad Bands ˇ Derivatives can eliminate background absorption ˇ Derivatives discriminate against broad absorbance bands 30 Akumulace signálu 31 32 Vícesložková analýza 33 Instrumentace Základní části spektrálních přístrojů 1. zdroj záření 2. selektor vlnových délek 3. vzorek 4. detektor záření 5. vyhodnocovací zařízení a displej 34 1 zdroj záření vlnový selektor2 3 vzorek detektor záření4 zpracování a zobrazení signálu 5 A Základní uspořádání pro absorpční spektrometrii: Základní uspořádání pro emisní spektrometrii: vlnový selektor2 3 vzorek detektor záření4 zpracování a zobrazení signálu 5 1 zdroj záření B 35 Základní uspořádání pro fluorescenční spektrometrii: 1 zdroj záření vlnový selektor2 3 vzorek detektor záření4 zpracování a zobrazení signálu 5 C Jednopaprskové diferenční uspořádání spektrometru: zdroj záření monochro- mátor kyveta se vzorkem zpracování a zobrazení signálu referentní kyveta clona detektor A 36 Diode-Array Spectrophotometer Schematic of a diode-array spectrophotometer Diode-Array Spectrophotometer Optical diagram of the HP 8453 diode-array spectrophotometer 37 Dvoupaprskové uspořádání spektrometru: zdroj záření monochro- mátor kyveta se vzorkem zpracování a zobrazení signálu referentní kyveta detektory diferenciální zesilovač dělič paprsku Conventional Spectrophotometer Optical system of a double-beam spectrophotometer 38 Diode-Array Spectrophotometer Optical system of the HP 8450A diode-array spectrophotometer Conventional Spectrophotometer Optical system of a split-beam spectrophotometer 39 d) v emisních spektrálních metodách je zdrojem záření excitovaný vzorek RTG spektrometrieRTG lampa c) zdroj spojitého i čárového záření atom. abs. spektr., UV, VIS; atom. fluoresc. spektr. Hg výbojka atom. abs. spektr., UV, VIS; atom. fluoresc. spektr. výbojka s dutou katodou molek. abs. spektr., UV, VIS, IČ; molek. fluoresc. spektr. laser b) zdroje čárového spektra molek. abs. spektr., IČglobar (SiC, 1500oC) molek. abs. spektr., UV, VIS, IČW, W-X žárovka molek. abs. spektr., UVH2, D2 výbojka molek. fluoresc. spektr.xenonová lampa a) zdroje spojitého záření použití1. zdroj 40 Záření černého tělesa M = T4 astronomie Intensity Spectrum of the Deuterium Arc Lamp ˇ Good intensity in UV range ˇ Useful intensity in visible range ˇ Low noise ˇ Intensity decreases over lifetime 41 Intensity Spectrum of the Tungsten-Halogen Lamp ˇ Weak intensity in UV range ˇ Good intensity in visible range ˇ Very low noise ˇ Low drift 42 Intensity Spectrum of the Xenon Lamp ˇ High intensity in UV range ˇ High intensity in visible range ˇ Medium noise Lasery 43 44 Vlastnosti laserů ˇ monochromatické záření ˇ extrémně úzké ­ vysoký výkon při jedné ˇ kolimované ˇ polarizované ˇ koherentní Nevýhody ˇ drahé ˇ náročné údržba ˇ omezený počet l pro použití 45 Lasers are intense monochromatic sources, good as fluorescence sources, since F Intensity. Lasers are intense monochromatic sources, good as fluorescence sources, since F Intensity. Gary Christian, Analytical Chemistry, 6th Ed. (Wiley) 2. Vlnové selektory Filtry ˇabsorpční ˇinterferenční Monochromátory ˇ hranolové ˇ mřížkové 3. Vzorek Molekulové spektrální. metody ­ ˇ kyvety, (hranaté, válcové) z vhodného materiálu; Atomové spektrální metody ˇ oblak atomů. 4. Detektory ˇfotonky, fotonásobiče ˇpolovodičové detektory ďetektory s diodovým polem ťermočlánky 46 Fig. 16.25. Schematic of interferometer for FTIR spectroscopy. Modern IR spectrometers are Fourier transform spectrometers, rather than dispersive. The beam is spit into two paths. When reflected, they are out of phase due to the moving mirror. They recombine to give an interference pattern of all wavelengths (pattern changes with time). A time-domain spectrum is recorded (interferogram ­ see next slide). Modern IR spectrometers are Fourier transform spectrometers, rather than dispersive. The beam is spit into two paths. When reflected, they are out of phase due to the moving mirror. They recombine to give an interference pattern of all wavelengths (pattern changes with time). A time-domain spectrum is recorded (interferogram ­ see next slide). Gary Christian, Analytical Chemistry, 6th Ed. (Wiley) 47 48 49 Kalibrace přístroje 50 Dispersion Devices ˇ Non-linear dispersion ˇ Temperature sensitive ˇ Linear Dispersion ˇ Different orders Fig. 16.14. Dispersion of polychromatic light by prism. Dispersion by prisms is good at short wavelengths, poor at long wavelengths (IR). Dispersion by prisms is good at short wavelengths, poor at long wavelengths (IR). Gary Christian, Analytical Chemistry, 6th Ed. (Wiley) 51 52 Fig. 16.15. Diffraction radiation from grating. Dispersion by a grating is independent of wavelength, but intensity varies with wavelength. Gratings are blazed for certain wavelength regions. Higher order radiation is produced (multiples of the primary, 1st order, radiation). Radiation at wavelengths shorter than the spectral region must be filtered out to prevent its 2nd order radiation from overlapping the spectral region. Dispersion by a grating is independent of wavelength, but intensity varies with wavelength. Gratings are blazed for certain wavelength regions. Higher order radiation is produced (multiples of the primary, 1st order, radiation). Radiation at wavelengths shorter than the spectral region must be filtered out to prevent its 2nd order radiation from overlapping the spectral region. Gary Christian, Analytical Chemistry, 6th Ed. (Wiley) 53 Fig. 16.16 Some typical UV and visible absorption cells. The standard cell is 1 x 1 cm. Quartz is used for UV and visible. Glass and clear plastic are used for visible. The standard cell is 1 x 1 cm. Quartz is used for UV and visible. Glass and clear plastic are used for visible. Gary Christian, Analytical Chemistry, 6th Ed. (Wiley) Transmission Characteristics of Cell Materials Note that all materials exhibit at least approximately 10% loss in transmittance at all wavelengths 54 Open-topped rectangular standard cell (a) and apertured cell (b) for limited sample volume Cell Types I Cell Types II Micro cell (a) for very small volumes and flow-through cell (b) for automated applications 55 Salt crystals are used as cell material for the IR region. NaCl must be protected from moisture, and is polished to remove "fogging". AgCl can be used for wet samples. Salt crystals are used as cell material for the IR region. NaCl must be protected from moisture, and is polished to remove "fogging". AgCl can be used for wet samples. Gary Christian, Analytical Chemistry, 6th Ed. (Wiley) Fig. 16.17. Typical infrared cells. (a) Fixed-path cell. (b) Variable length cell. A short path cell is used with pure substances for qualitative measurements (e.g., 0.01-0.05 mm). High concentration solutions are usually used since most solvent absorb some in the IR (ca. 0.1 mm pathlength). A short path cell is used with pure substances for qualitative measurements (e.g., 0.01-0.05 mm). High concentration solutions are usually used since most solvent absorb some in the IR (ca. 0.1 mm pathlength). Gary Christian, Analytical Chemistry, 6th Ed. (Wiley) 56 h e - +- katoda anoda A h +- + + + + + + + - - - - - - - polovodič p polovodič n C h fotokatoda dynody anoda B +- A. Fotonka B. Fotonásobič C. Polovodičový detektor 57 Photomultiplier Tube Detector Anode ˇ High sensitivity at low light levels ˇ Cathode material determines spectral sensitivity ˇ Good signal/noise ˇ Shock sensitive Fig. 16.18. Some spectral responses of photomultipliers. S-5 = RCA 1P28; S-4 = RCA 1P21; S-1 = RCA 7102. PM tubes are sensitive, but different photoemissive surfaces are responsive to different wavelengths. Einstein received the 1921 Nobel Prize in Physics for explaining the photoelectric effect in 1905. PM tubes are sensitive, but different photoemissive surfaces are responsive to different wavelengths. Einstein received the 1921 Nobel Prize in Physics for explaining the photoelectric effect in 1905. Gary Christian, Analytical Chemistry, 6th Ed. (Wiley) 58 The Photodiode Detector ˇ Wide dynamic range ˇ Very good signal/noise at high light levels ˇ Solid-state device Schematic Diagram of a Photodiode Array ˇ Same characteristics as photodiodes ˇ Solid-state device ˇ Fast read-out cycles 59 Fig. 16.19. Photo of 1024-element diode arrays. These detectors allow recording of an entire spectrum at once.These detectors allow recording of an entire spectrum at once. Gary Christian, Analytical Chemistry, 6th Ed. (Wiley) Fig. 16.20. Typical spectral response of diode array. Diode arrays are useful for UV to IR radiation.Diode arrays are useful for UV to IR radiation. Gary Christian, Analytical Chemistry, 6th Ed. (Wiley) 60 Charged Coupled Device 61 62 63 Fig. 16.32. Fiber-optic structure. The cladding has a higher refractive index than the core. The buffer layer is a protective layer. Light entering at no greater angle than a will be internally reflected and transmitted. The cladding has a higher refractive index than the core. The buffer layer is a protective layer. Light entering at no greater angle than a will be internally reflected and transmitted. Gary Christian, Analytical Chemistry, 6th Ed. (Wiley) 64 System Pb(II)-XO 65 66 Fig. 16.34. Miniature fiber spectrometer. Box is the spectrometer. Light source is to right, and fiber-optic cable guides light to cuvet. Second cable takes transmitted light to spectrometer. The detector is a 2048-element charge-couple device (CCD). The light from the fiber optic cable is dispersed across the array via a fixed grating. The detector is a 2048-element charge-couple device (CCD). The light from the fiber optic cable is dispersed across the array via a fixed grating. Gary Christian, Analytical Chemistry, 6th Ed. (Wiley) 67 Fig. 16.27. relative concentration error as function of transmittance for 1% uncertainy in %T. It is difficult to precisely measure either very small or very large decreases in absorbance. For thermal-noise limited detectors (as used in IR), the error is minimum for A = 0.434; working range 0.1-1 A. For shot-noise limited phototube and photomultiplier detectors, the error is minimum at A = 0.87; working range is 0.1-1.5 A. It is difficult to precisely measure either very small or very large decreases in absorbance. For thermal-noise limited detectors (as used in IR), the error is minimum for A = 0.434; working range 0.1-1 A. For shot-noise limited phototube and photomultiplier detectors, the error is minimum at A = 0.87; working range is 0.1-1.5 A. Gary Christian, Analytical Chemistry, 6th Ed. (Wiley) 68 69 Definition of Resolution Spectral resolution is a measure of the ability of an instrument to differentiate between two adjacent wavelengths Instrumental Spectral Bandwidth The SBW is defined as the width, at half the maximum intensity, of the band of light leaving the monochromator 70 Natural Spectral Bandwidth The NBW is the width of the sample absorption band at half the absorption maximum Effect of SBW on Band Shape The SBW/NBW ratio should be 0.1 or better to yield an absorbance measurement with an accuracy of 99.5% or better 71 Effect of Digital Sampling The sampling interval used to digitize the spectrum for computer evaluation and storage also effects resolution Wavelength Resettability Influence of wavelength resettability on measurements at the maximum and slope of an absorption band 72 Effect of Stray Light Effect of various levels of stray light on measured absorbance compared with actual absorbance Theoretical Absorbance Error The total error at any absorbance is the sum of the errors due to stray light and noise (photon noise and electronic noise) 73 Effect of Drift Drift is a potential cause of photometric error and results from variations between the measurement of I0 and I Effect of Refractive Index Changes in the refractive index of reference and sample measurement can cause wrong absorbance measurements 74 Non-planar Sample Geometry Some sample can act as an active optical component in the system and deviate or defocus the light beam Averaging of data points reduces noise by the square root of the number of points averaged Effect of Integration Time 75 ˇ Wavelength averaging reduces also the noise (square root of data points) ˇ Amplitude of the signal is affected Effect of Wavelength Averaging Selection of a wavelength in the slope of a absorption band can increase the dynamic range and avoid sample preparation like dilution Increasing Dynamic Range 76 Scattering causes an apparent absorbance because less light reaches the detector Scattering Scatter Spectra ˇ Rayleigh scattering: Particles small relative to wavelength ˇ Tyndall scattering: Particles large relative to wavelength 77 Absorbance at the reference wavelength must be equivalent to the interference at the analytical wavelength Isoabsorbance Corrections Background modeling can be done if the interference is due to a physical process Background Modeling 78 Corrects for constant background absorbance over a range Internal Referencing Three-Point Correction ˇ Uses two reference wavelengths ˇ Corrects for sloped linear background absorbance 79 Scatter Correction by Derivative Spectroscopy Scatter is discriminated like a broad-band absorbance band Effect of Fluorescence The emitted light of a fluorescing sample causes an error in the absorbance measurement 80 Acceptance Angles and Magnitude of Fluorescence Error ˇ Forward optics: Absorbance at the excitation wavelengths are too low ˇ Reversed optics: Absorbance at the emission wavelengths are too low Inadequate Calibration ˇ Theoretically only one standard is required to calibrate ˇ In practice, deviations from Beer's law can cause wrong results 81 Calibration Data Sets ˇ Forward optics: Absorbance at the excitation wavelengths are too low ˇ Reversed optics: Absorbance at the emission wavelengths are too low Wavelength(s) for Best Linearity ˇ A linear calibration curve is calculated at each wavelength ˇ The correlation coefficient gives an estimate on the linearity 82 Wavelength(s) for Best Accuracy ˇ The quantification results are calculated at each wavelength ˇ The calculated concentration are giving an estimate of the accuracy Precision of an Analysis Precision of a method is the degree of agreement among individual test results when the procedure is applied repeatedly to multiple samplings 83 Wavelength(s) for Best Sensitivity ˇ Calculation of relative standard deviation of the measured values at each wavelength ˇ The wavelength with lowest %RSD likely will yield the best sensitivity Wavelength(s) for Best Selectivity Selectivity is the ability of a method to quantify accurately and specifically the analyte or analytes in the presence of other compounds 84 Ideal Absorbance and Wavelength Standards ˇ An ideal absorbance standard would have a constant absorbance at all wavelengths ˇ An ideal wavelength standard would have very narrow, well-defined peaks Ideal Stray Light Filter An ideal stray light filter would transmit all wavelengths except the wavelength used to measure the stray light 85 Holmium Perchlorate Solution The most common wavelength accuracy standard is a holmium perchlorate solution Potassium Dichromate Solution The photometric accuracy standard required by several pharmacopoeias is a potassium dichromate solution 86 Stray Light Standard Solutions The most common stray light standard and the respectively used wavelengths Toluene in Hexane (0.02% v/v) The resolution is estimated by taking the ratio of the absorbance of the maximum near 269 nm and minimum near 266 nm 87 Confirmation Analysis In confirmation analysis, the absorbance at one or more additional wavelengths are used to quantify a sample Spectral Similarity Comparative plots of similar and dissimilar spectra