Molecular Spectrometry (UV and Visible)Part 1: Absorption

InstrumentEvolution

Beckman DU

Replaced prism with grating

Turner Spectrometer

Spec 20 as well

Dual BeamHigh resolution

Cary and Perkin-Elmer

PDAHewlett Packard

Fiber Optic Probes

Ocean Optics

Signal Expressions

Eout = Φλ (Ω/4π) Tm Ts Rλ G

Φλ = source spectral power (W/nm)Tm = monochromator throughput (nm)Ts = Sample transmittanceRλ = Detector Responsivity (A/W)G = Gain of electronics (V/A)

Signal Expressions

Tm = (WH/Aeff) Δλeff Top

W = slit width (cm)

H = slit height (cm)

Aeff = Effective area of source image (cm2)

Δλeff = Effective bandwitdh of the

monochromator (nm)Top = Transmittance of optics

Signal Expressions

Ts = e-εbc = Esam/Eref

Eref = Output voltage for a blank

Esam = Output voltage for sample

Signal Expressions

A = -log (Esam/Eref) = -log (Ts) = ε b c

Notes:1. Since UV-Vis absorbance bandsare much wider than the mono-chromator bandwidth, Beer’s Lawassumes that A does not dependon Δλeff.

2. ε is wavelength dependent, but doesnot depend on any other instrumentalparameter.

A = -log (Esam/Eref) = -log (Ts) = ε b c

A = ε b cDesign Cell to Maximize b

Deviations from Beer’s Law

1. Optical

a) slit widthb) wavelength selectionc) stray radiation

2. Chemical

a) high analyte concentrationb) pH dependencec) mixtures

Deviations from Beer’s Law

Slit Width

Deviations from Beer’s Law

Deviations from Beer’s Law

Wavelength Selection

0

1

2

3

4

5

6

Concentration

Ab

so

rban

ce

Deviations from Beer’s Law

Stray Radiation Effect

No StrayLight

10% Stray Light

0.000

0.100

0.200

0.300

0.400

0.500

0.600

0.700

0.800

0.900

1.000

0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.18 0.2

Concentration (mM)

Tra

ns

mit

tan

ceDeviations from Beer’s Law

High Analyte Concentrations

Deviations fromBeer’s Law pH Dependence

Deviations fromBeer’s Law

Mixtures:Absorbance isAdditive

Deviations fromBeer’s Law

Mixtures:Solve multipleequations!

Deviations fromBeer’s Law

Mixtures: When all else fails,Separate the mixture components

UV Absorbance Detector for HPLC

Final Note on UV-Vis Signals

The Absorbance (Analytical Signal) doesnot depend on the magnitude of the sourcesignal (except for the deviations discussedabove).

The Noise in the measurement often DOESdepend on the source signal level.