Sensitivity and Selectivity in Optical Spectroscopy and Imaging: A Molecular Approach
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1 Sensitivity and Selectivity in Optical Spectroscopy and Imaging: A Molecular Approach Process Analysis & Technology Prof. Dr. R. W. Kessler STZ Technology Process Control and Data Analysis Reutlingen, Germany Process Analysis and Technology PA&T Reutlingen University, Germany Prof. Dr. Rudolf Kessler Prof. Dipl. Phys. W. Kessler STZ Technology Transfer Center Process Control and Data Analysis Reutlingen, Germany
2 Sensitivity and Selectivity in Optical Spectroscopy and Imaging: A Reminder Process Analysis & Technology Prof. Dr. R. W. Kessler STZ Technology Process Control and Data Analysis Reutlingen, Germany Process Analysis and Technology PA&T Reutlingen University, Germany Prof. Dr. Rudolf Kessler Prof. Dipl. Phys. W. Kessler STZ Technology Transfer Center Process Control and Data Analysis Reutlingen, Germany
3 Further Reading R. W. Kessler, Perspectives in process analysis. J. Chemometrics, 2013, 27: doi: /cem.2549 B. Boldrini, W. Kessler, K. Rebner and R. W. Kessler Hyperspectral imaging: a review of best practice, performance and pitfalls for inline and online applications, Journal of Near Infrared Spectroscopy 2012, 20,
4 Agenda Molecular Optical Spectroscopy Sensitivity cross sections of absorption and scatter information depth sensitivity detectors Examples Selectivity resolution chemometrics 2D-fluorescence derivatives Robustness S/N, drift etc. inline illumination and specular refelctance flutter signal enhancement Summary
5 Molecular optical Spectroscopy: Absorption E π* π s 2 v n v 2 v 1 v 0 S 1 s 1 π* See Energie Difference π s 0 v 2 v 1 v 0 s 0 IR/NIR VIS UV v 0 v 1
6 Molecular Spectroscopy: Fluorescence Emission E s 2 s 1 v 1 s 0 v 0 IR/NIR VIS UV
7 Molecular Spectroscopy: Raman Scatter E s 2 s 1 v 1 s 0 v 0 IR/NIR VIS UV
8 Toolbox Optical Spectroscopy wide spectral range I UV/VIS N NIR N MIR/Raman N Fluorescence N Reflection Fluorescence UV NIR IR Raman Different experimental setup Transmission y x spatial scan (x,y) Hyperspectral imaging
9 Measurements for Knowledge Based Production and Causality First Principle & Variable Material Input Adaptive Process Knowledge based Production Consistent Output Mechanistic Model Causality Correlative and Descriptive Model Fixed Process Variable Output Process AND Functionality Design
10 Measurements Data Knowledge The Japanese eat very little fat and suffer fewer heart attacks than the British or the Americans. The French eat a lot of fat and also suffer fewer heart attacks than the British or the Americans. The Japanese drink very little red wine and suffer fewer heart attacks than the British or the Americans. The Italians drink a lot of red wine and also suffer fewer heart attacks than the British or the Americans. Conclusion: Eat and drink whatever you like. It's speaking English that kills you. What is wrong? To draw conclusions from random or spurious correlations
11 Important: Spectroscopy detects ALL chemical information measuring the absorption of light ALL morphological information (e.g. texture of the particle, colloids, etc.) measuring the scattering of light PROBLEM: Superposition of Manifold Informationen Use Multivariate Data Analysis Develop Smart Sensors and Integrate First Principles into Spectroscopy (Causality!!!) Increase Photon Flux and Sensitivity!
12 Technology Review (see Kessler (ed.)) UV/VIS/ s-nir NIR MIR Fluorescence Raman Selectivity Sensitivity (+) (+) ++(+) Sampling Working in aqueous media Applicability Process analytical tool Light guide glass (+) Signal Absorption Absorption Absorption Emission Scattering Samling online/inline Techniques s, l, g s, l s, l, g s, l (g) s, l, (g) Transmission Reflectance ATR Transmission Reflectance ATR ATR (Transmission) Reflectance Transmission Reflectance Relative costs
13 Sensitivity: absorption, scatter ANALYTICAL Sensitivity represents the smallest amount of a substance that can accurately be measured by a given technology From a Quantum Mechnical Point of View: σ a [cm 2 ] the absorption cross section σ is given usually in cm 2 /molecule and depend on the individual molecular structure of the compound and the quantum mechanical selection rules. The term cross section is used in physics to quantify the probability of a certain interaction, e.g. scattering, electromagnetic absorption, etc. Absorption: Technology log σ a [cm 2 /molecule] Fluorescence Extinction ε [Mol -1 cm -1 ] UV-Vis app Mid-Infrared app Near-Infrared app Raman app Rayleigh scatter per molecule Mie, scatter per particle Scattering intensity [a.u.] Unpolarized light at 532 nm, polystyrene sphere 20 orders of magnitude!!!! I s ~ d 6 Rayleigh RGD 4 90 d λ Mie Diameter [µm] RGD= Rayleigh-Gans-Debye I s ~ d 2 Fraunhofer
14 Example Darkfield Glioblastoma Vis Backscattering Light RGB: TP53
15 Example: New MCR Marker Free Karyotyping of a Chromosome Pushbroom Imaging Pushbroom Slit Rel. Transmission RGB-Image of Chromosome - Chromosome - Border resolution in x-axis: 64.5nm per pixel Wavelength [nm] MCR Component 1 MCR Component 2 MCR Component 3 false colour karyotype compare FISH
16 UV-Vis Absorption: e.g. Woodward Fieser Rules calculating λ max : λ max = M + n ( n) 16.5 R endo 10 R exo calculating ε max : ε max = (1.74 x 10 4 ) n λ max is the wavelength of maximum absorption in nm ε max is the maximum absorptivity in [cm -1 mole -1 ] M: is the number of alkyl substituents / ring residues in the conjugated system n: is the number of conjugated double bonds R endo : is the number of rings with endocyclic double bonds in the conjugated system R exo : is the number of rings with exocyclic double bonds in the conjugated system. Predicted: λ max : 453 nm ε max : x 10 4 Measured: λ max : 452 nm ε max : x 10 4
17 Example Tablet: Absorption and Scattering measured Real Life ASA tablet calculated R Kubelka Munk T Penetration Depth!!!!
18 Mid-IR- Absorption (separate three regions) IR: Changes in Dipol Moment Raman: Changes in Polarisation fingerprint
19 Be Aware: QM Absorption Cross Sections NIR 1.2 Absorption Oberton aller Arten an -CH von nm 1. Oberton -OH bei nm Ethanol 1. Oberton aller Arten an -CH von nm Propionsäure Kombination aller Arten an -CH von nm Kombination -OH bei 2100 nm Xylol ν near IR v 0 v 1 v 2 v v Wellenlänge / nm
20 Absorption Absorption Example: MIR and NIR Spectra of Water ,45 0,4 0,35 0,3 0,25 0,2 0,15 0,1 0,05 MIR (ATR: app. 5µm pathlength) µm pathlength 0.5 mm Wavenumber[cm -1 ] mm µm pathlength mm 10 mm 5 mm 0.1 mm 50 mm 2 mm wavelength [nm]
21 Penetration depth/cm Scale of Scrutiny: Information Depth Si-based CCD 281 Mpa mixed??? 156 Mpa 0.2 InGas-based detectors rd overtone in NIR wavelength/nm Optical penetration depth of Theophyllin tablets with different API concentrations, calculated from S and K many small measurement spots are better than one large spot in spectroscopy!!!
22 Sensitivity: Technology courtesy of Bruker Technology log σ a [cm 2 /molecule] Extinction ε [Mol -1 cm -1 ] Fluorescence UV-Vis app Mid-Infrared app Near-Infrared app Raman app Rayleigh scatter per molecule Mie, scatter per particle
23 Selection Criteria: Sensitivity (incl. technology and robustness) UV/VIS/ s-nir NIR MIR Fluorescence Raman Selectivity Sensitivity (+) (+) ++(+) Sampling Working in aqueous media Applicability Process analytical tool Light guide glass (+) Signal Absorption Absorption Absorption Emission Scattering Samling online/inline Techniques s, l, g s, l s, l, g s, l (g) s, l, (g) Transmission Reflectance ATR Transmission Reflectance ATR ATR (Transmission) Reflectance Transmission Reflectance Relative costs
24 Selectivity: Definition describes the capability of the technology to deliver signals that are free from interferences and give true results. This implies, that if the signal of interferent and analyte can be separated, the sensitivity increases. (ICHQ2) distinct Rayleigh resolution 1 cm -1 2 cm -1 MIR Polypropylene ATR selectivity/ noise Sparrow FWHM of PSF 4 cm -1 8 cm cm -1 A Δ A = cm -1 robustness 64 cm cm-1 wavenumber cm -1
25 e.g. PP/PES in Non Wovens: enhanced Selectivity using Chemometrics 1.2 PC /100 50/50 70/30 75/25 100/0 predicted PLS / PP/PES PC measured E 0.4 PES PP 0.2 ~210 C [nm] NIR spectra
26 2D- Fluoreszenz: e.g. E Coli Fermentation Cytosin Guanin Adenin Samples/Scores Plot of data Scores on Comp 1 (32.00%) Scores on Comp 2 (29.33%) Scores on Comp 3 (37.63%) Thymin? UV/Vis-Transmission log (T) Wellenlänge /nm Sample
27 Derivative Spectroscopy: UV-Vis, NIR.. concentration A + 2B concentration A + B original concentration A 1 st derivative be aware: E = h c/λ 200nm = cm nm = cm -1 Δ = 50nm, Δ = cm -1 2 nd derivative Δ Raman, MIR app cm -1!!!!
28 Robustness ICHQ2 guideline: robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage.
29 Specrograph Specrograph Specrograph Robustness: Inline Illumination Detection Set Up CCD Array CCD Array CCD Array light source light source diffuse light source sample moving direction sample moving direction sample moving direction 45R45 45R0 dr0
30 Robustness: Specular and Diffuse Reflectance e.g. of a Cavity on a Surface parallell high concentration? Model System: cellulose/ dyed cellulose high lateral resolution!! Specular Reflection I 0 Δ n low lateral resolution due to photon diffusion!! crossed
31 Inline Illumination pushbroom imager light source diffusors
32 On-Line Control of Wood Chips : Flutter Diffuse Reflectance Probe at the Conveyer Belt Measures and Controls Wood Chips On-Line.
33 Summary: Technology Review UV/VIS/ s-nir NIR MIR Fluorescence Raman Selectivity Sensitivity (+) (+) ++(+) Sampling Working in aqueous media Applicability Process analytical tool Light guide glass (+) Signal Absorption Absorption Absorption Emission Scattering Samling online/inline Techniques s, l, g s, l s, l, g s, l (g) s, l, (g) Transmission Reflectance ATR Transmission Reflectance ATR ATR (Transmission) Reflectance Transmission Reflectance Relative costs
34 LIVE LONG AND PROSPER THANK YOU FOR YOUR ATTENTION
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