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UV-VIS Spectroscopy - Chemical Analysis Chemical Analysis Solutions Unit Sonia S i R R. S Sousa PhD Marketing Manager - Spectroscopy 21 January 2009 Page 1 Group/Presentation Title Agilent Restricted Month ##, 200X Topics • Basic UV-VIS Theory • UV-VIS UV VIS history and product offerings • key Instrumental parameters • Method development • Sample handling and measurements • Questions and answers Page 2 Group/Presentation Title Agilent Restricted Month ##, 200X THEORY Page 3 Group/Presentation Title Agilent Restricted Month ##, 200X Theory – UV-VIS The wavelength and amount of light that a compound absorbs depends on its molecular structure and the concentration of the compound used. Concentration dependence follows Beer’s Law A = -log T A=εbc A εbc A = is absorbance (no units, since A = log(P0 / P ) ε = the molar absorbtivity with units of L mol-1 cm-1 b = the p path length g of the sample p (typically ( yp y in cm)) c = the concentration of the compound in solution mol L-1 Io = incident light intensity I = Intensity of light transmitted through solution T= transmittance Group/Presentation Title Agilent Restricted Electronic Excitation by UV-VIS Spectroscopy Energy Scale in UV-VIS Spectroscopy Ref Fundamental of Molecular Spectroscopy (C Ref. (C. N N. Banwell) Page 6 Group/Presentation Title Agilent Restricted Month ##, 200X Table of Chromophore Page 7 N Name Ch Chromophore h W Wavelength l th [nm] [ ] acetylide -C=C 175-180 Aldehyde -CHO 210 amine -NH2 195 azo -N=N- 285-400 bromide -Br Br 208 carboxyl -COOH 200-210 ester -COOR 205 ether -OO 18 185 ketone >C=O 195 nitrate -ONO2 270 nitrile -C=N 160 nitrite -ONO 220 - 230 nitro -NO2 210 Group/Presentation Title Agilent Restricted Month ##, 200X UV-VIS Naphthalene Anthracene Pyrene Conjugated Carbonyl Compounds UV-VIS HISTORY AND PRODUCT OFFERINGS Page 11 Group/Presentation Title Agilent Restricted Month ##, 200X Agilent UV-VIS Spectroscopy History Page 12 Group/Presentation Title Agilent Restricted Month ##, 200X Product Offerings and Solutions • Hardware and Software • Accessories • Fully test systems • Compliance services for IQ/OQ/PV • Supplies G1120A Multicelltransport 89090A Tempco Page 13 Group/Presentation Title Agilent Restricted Month ##, 200X Agilent UV-VIS 8453 Spectrometer • Specifications • Wavelength range: 190 – 1100 nm • Slit width: 1 nm UV-VIS 8453 G1103A • Full spectrum scan: 0.1 sec • Low stray light • High wavelength reproducibility and accuracy • Fully EP and USP compliant • GLP • Advanced mode: 21 CFR 11 compliant Page 14 Group/Presentation Title Agilent Restricted Month ##, 200X Spectrometer Instrument Design Dual Beam Spectrometer p Diode Array Spectrometer Conventional single beam spectrometer Page 15 Group/Presentation Title Agilent Restricted Month ##, 200X Diode Array Advantages • Simple mechanical and optical design • Open sample area • Fixed arrangement of grating and detectors • Fast acquisition of complete spectra • Statistics Page 16 Group/Presentation Title Agilent Restricted Month ##, 200X Open Sample Area Reverse Optics • Room light cannot reach detector • Narrow “acceptance angle” after sample • Geometry of spectrograph • Easy and convenient access • Better productivity • Less error prone • Easy installation of acessories Page 17 Group/Presentation Title Agilent Restricted Month ##, 200X ChemStation Software Mode Structure Page 18 Group/Presentation Title Agilent Restricted Month ##, 200X Common Features to all Applications • Universal data structure • Common basic features for • Instrument control • Accessory configuration and handling • Method handling • Raw data storage, retrieval and export • Different Diff t view i modes d for f raw data d t and d results lt • Graphical window • Table window • New: Access to Agilent OpenLab Enterprise Content Management (ECM) Page 19 Group/Presentation Title Agilent Restricted Month ##, 200X Solution for General Purpose Page 20 Group/Presentation Title Agilent Restricted Month ##, 200X Solution for General Purpose Page 21 Group/Presentation Title Agilent Restricted Month ##, 200X General Purpose SW - Key Features • Graphical User Interface (GUI) with integrated status display • Includes all common application tasks • Single dialog box for method setup • Easy automation • One page report • Verification and diagnostics • Execute advances method capability p y Page 22 Group/Presentation Title Agilent Restricted Month ##, 200X Solution for Research and Development Page 23 Group/Presentation Title Agilent Restricted Month ##, 200X Solution for Research and Development Page 24 Group/Presentation Title Agilent Restricted Month ##, 200X Advanced SW – Key Features • Flexible method setup • Time based measurements • Multiple data analysis- confirmation analysis • Single components analysis SCA and multi components analysis MCA • Automation • Sophisticated interactive mathematical functions • Sophisticated results statics • Customization – macro programming • Customer report generator Page 25 Group/Presentation Title Agilent Restricted Month ##, 200X KEY INSTRUMENTAL PARAMETERS Page 26 Group/Presentation Title Agilent Restricted Month ##, 200X Key Instrumental Parameters • Spectral Resolution • Wavelength accuracy and precision • Photometric accuracy and precision • Stray light • Noise • Drift Page 27 Group/Presentation Title Agilent Restricted Month ##, 200X Key Instrumental Parameters • Spectral Resolution – is the ability of an instrument to differentiate between two adjacent j wavelengths g Two wavelength are considered resolved if the minimum between the two peaks of the detector output signal is lower than 80% of maximum Page 28 Group/Presentation Title Agilent Restricted Month ##, 200X Key Instrumental Parameters • Wavelength accuracy and precision – important for the comparison p of measurements made on different instruments Page 29 Group/Presentation Title Agilent Restricted Month ##, 200X Key Instrumental Parameters • Photometric accuracy and precision • Stray light – is defined as detected light of any wavelength that lies outside t id th the b bandwidth d idth off the th selected l t d wavelength l th • Stray light causes a negative bias in instrument response and eventually is the limiting factor for the absorbance, and thereby concentration, that can be measured Page 30 Group/Presentation Title Agilent Restricted Month ##, 200X Key Instrumental Parameters • Photometric accuracy and precision • Noise – affects the precision of the measurement, for a single measurement, t may introduced i t d d error iin accuracy as well. ll The total error at any absorbance is the Sum of the errors due to stray light and noise ( h t noise (photon i and d electronic l t i noise) i ) Page 31 Averaging of data points reduce noise Group/Presentation Title Agilent Restricted Month ##, 200X Key Instrumental Parameters • Photometric accuracy and precision • Drift – normally results from variations in lamp intensity between the measurements. Change in the instrument electronics also can cause drift. Page 32 Group/Presentation Title Agilent Restricted Month ##, 200X METHOD DEVELOPMENT Page 33 Group/Presentation Title Agilent Restricted Month ##, 200X Method Development • Linearity • Accuracy • Precision • Sensitivity • Range • Selectivity Page 34 Group/Presentation Title Agilent Restricted Month ##, 200X Method Development • Linearity – is the ability of the method to produce test results that are proportional, either directly or by a well-defined mathematically transformation transformation, to the concentration of analyte in sample within a given range. Page 35 Group/Presentation Title Agilent Restricted Month ##, 200X Method Development • Accuracy – is the degree of agreement between an individual test result g generated by y the method and the true value Page 36 Group/Presentation Title Agilent Restricted Month ##, 200X Method Development • Precision – is the agreement among individual test results when the p procedure is applied pp repeatelly p y to multiple p sampling p g Page 37 Group/Presentation Title Agilent Restricted Month ##, 200X Method Development • Sensitivity – response obtained by a given analyte Page 38 Group/Presentation Title Agilent Restricted Month ##, 200X Method Development • Selectivity – the ability of a method to quantify accurately and specifically p y the analyte y or analytes y in the p presence of other compounds Page 39 Group/Presentation Title Agilent Restricted Month ##, 200X SAMPLE HANDLING AND MEASUREMENTS Page 40 Group/Presentation Title Agilent Restricted Month ##, 200X Sample Handling and Measurement • Cell • Material, Material type, type and care • Choice of solvent • Effect of solvent solvent, concentration concentration, pH pH, and temperature • Sample • Type, Type strong or week absorbance absorbance, interference Page 41 Group/Presentation Title Agilent Restricted Month ##, 200X Cell Material Page 42 Group/Presentation Title Agilent Restricted Month ##, 200X Summary • Advantage of diode array spectrometer • Fast spectral acquisition • The full spectra can be used for error correction • Background modeling • Derivative spectroscopy • Simultaneous wavelength measurement • Sensitivity • Reliability Page 43 Group/Presentation Title Agilent Restricted Month ##, 200X Acknowledgement • Thomas Klink – Agilent Technologies • Tony Owens – Agilent Technologies Fundamental of UV-VIS spectroscopy – Primer and Workbook Agilent technologies Page 44 Group/Presentation Title Agilent Restricted Month ##, 200X Page 45 Group/Presentation Title Agilent Restricted Month ##, 200X