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Introduction to Spectrophotometry: Principles and Applications

Learn about the fundamentals of spectrophotometry, from sources of emission to absorption and fluorescence in samples. Understand the components like monochromators and detectors, data processing techniques, and the analysis of spectra. Gain insights into Beer’s law, curve fitting, and standard procedures for concentration determination.

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Introduction to Spectrophotometry: Principles and Applications

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  1. Lecture 17 Spectrophotometry

  2. Emission source sample Absorption Fluorescence source sample Secondary emission

  3. Monochromator (filter, wavelength selector) Light Source Detector Sample Spectrometer Data Processing

  4. Monochromator (filter, wavelength selector) Light Source Detector Sample Spectrometer Data Processing

  5. Light striking a sample can be 1.reflected2.transmitted3. absorbed 4. scattered

  6. Absorbing plate

  7. I0 IN <1 Transmittance = I0 I1 I2 We almost never use transmittance! I3 I4 I5 T This is a curve! concentration

  8. Absorbing plate N=CVolume Volume= 1  dx N = C  dx dP=  N  P Incident light Emergent light P P0 P1 dP l 0 dx

  9. Absorbing plate absorbance Incident light Emergent light P0 P1 Sensitivity is the same for any power (P) l 0

  10. Bugert, Lambert and Beer Beer’s law A Straight line! concentration

  11. Least-squares curve fitting. The points (1,2) and (6,5) do not fall exactly on the solid line, but they are too close to the line to show their deviations. The Gaussian curve drawn over the point (3,3) is a schematic indication of the fact that each value of y is normally distributed about the straight line. That is, the most probable value of y will fall on the line, but there is a finite probability of measuring y some distance from the line.

  12. y=kx+b straight line equation k = Slope = y / x b - blank! Let us subtract blank: y-b = Y = kx Y1=kx1 Y2=kx2 One standard

  13. Procedure: • Measure blank. • Measure standard. • Measure unknown. • Subtract blank from standard and from unknown. • Calculate concentration of unknown If you have several (N) standards, do it several (N) times

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