Data Quality and Post Processing
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1 Data Quality and Post Processing
2 Data Quality and post-processing Quality control Standards e.g. quinine sulphate, raman, solid fluorescence standards Manufacturer supplied QC routines Instrument correction factors Post-processing Inner-filtering Raman or quinine sulphate equivalent units Removal of raman line and scatter
3 Standards A wide range of standards exist: Quinine sulphate solution Raman signal of (deionised) water International Humic Substances Society (IHSS) standards e.g. Suwanee River Fulvic Acid Solid fluorescence standards (e.g. Starna) Each have advantages and disadvantages. e.g. Quinine sulphate units are widely reported. Quantum efficiency known. But dilution series need operator skill and solutions are not stable over time.
4 Em (nm) Em (nm) 600 Sample J - mean corrected EEM 600 Sample M - mean corrected EEM Ex (nm) Ex (nm) Quinine Sulphate SRFA
5 Raman Intensity (peak height, ex=348 nm) Raman Intensity, Varian Cary Eclipse, /01/ /01/ /01/ /01/ /01/ /01/ /01/2009 Date
6 Manufacturer supplied QC routines Most instruments have a wide range of validation / QC procedures. These will check instrument parameters such as excitation and emission wavelength precision, band pass precision and signal to noise. Recommended that these should be run regularly. They will require some accessories (often supplied with instrument)
7 Emission bandpass /01/ /01/ /01/ /01/ /01/ /01/ Excitation wavelength /01/ /01/ /01/ /01/ /01/ /01/2009
8 Instrument correction factors Correction factors can be run to correct excitation and emission spectra for instrument specific characteristics e.g. Variations in lamp emission at different wavelengths. Standards are needed and the procedure isn t trivial. Instrument correction factors might not be available for all wavelengths of interest. Instrument correction allows inter-laboratory comparison, but wouldn t be necessary for single instrument applications.
9 Correction Factor Correction Factor Excitation Correction Emission Correction Excitation Wavelength (nm) Emission Wavelength (nm)
10 Correction Factor Correction Factor Z A x i s Z A x i s Uncorrected Corrected Wavelength (nm) W a v e l e n g t h ( n m ) Wavelength (nm) W a v e l e n g t h ( n m ) Excitation Wavelength (nm) Emission Wavelength (nm)
11 Post Processing Inner Filter Correction is the most common post-processing technique to be needed. This corrects for changes in fluorescence due to strongly coloured / absorbing solutions. A range of techniques are available, but often recommended is an empirical correction based on the absorbance of the solution.
12 Em Lakowicz (1983) CL : F F corr obs [( A em 10 exc A )/ 2] d 2 w 2 Exc w 1 d 1 Parker and Barnes (1957) CP : F corr F obs 2.3 w 1 A exc = 10 d 1A exc (1 10 w 1 A exc ) X 2.3 w 2 A em 10 d 2 A em (1 10 w 2A em )
13 Index (a.u.) IHSS Humic Acid HA no IFE correction Corrected fluorescence Concentration (ppm) Abs. 254 nm (cm -1 ) Data: C. Eliott- Laize, CEH, UK
14 Inner-filter corrections For most samples, fluorescence intensity concentration relationship is linear up to 10 ppm. Empirical corrections simple to implement, but have to be remembered are empirical. Precise absorbance measurements are necessary or errors will be introduced. For many applied uses, a non-linear fluorescence intensity vs concentration relationship might be perfectly acceptable. Other correction techniques are possible, e.g. Dilution, dual pathlength, etc.. No one answer the choice should fit the application.
15 Raman Intensity (peak height, ex=348 nm) Conversion to raman or QS units Fluorescence intensity is measured in arbitary units. These can be converted to QSU if a quinine sulphate standard solution is analysed. Similarly, the raman intensity of water can also be used to standardise the fluorescence intensity /01/ /01/ /01/ /01/ /01/ /01/ /01/2009 Date
16 Removal of scatter lines, raman line, etc. Necessary for some statistical and mathematical treatments (see later presentation). Otherwise, there is no reason to remove these data, and for many applications, both scatter and raman features can be used to calibration / validation.
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