Optical Techniques & Spectrophotometry Notes

Photometry

  • Measurement of luminous intensity of light.

  • Types:

    • Filter Photometers

    • Spectrophotometers

Filter Photometers

  • Optical filters isolate narrow wavelength ranges for monochromatic light.

Spectrophotometer

  • Uses a monochromator (prisms or gratings) to isolate specific wavelengths.

  • Measures compound amount in solution by shining light and measuring absorption.

  • Typically uses UV and visible light (290-800nm).

Wavelength, Frequency, and Energy

  • E=h⋅nE = h \cdot n (E = energy, h = Planck's constant, n = frequency)

  • c=l⋅nc = l \cdot n (c = speed of light, l = wavelength)

  • Energy of light is inversely proportional to the wavelength.

Absorption of Electromagnetic Radiation

  • IoI_o: Original intensity

  • IsI_s: Transmitted intensity

  • Use a blank/reference solution to eliminate factors other than the compound of interest.

Derivation of Beer’s Law

  • Transmittance (T) = I<em>s/I</em>oI<em>s/I</em>o

  • %T = I<em>s/I</em>o⋅100I<em>s/I</em>o \cdot 100

  • A=−log(I<em>s/I</em>o)=log(100A = -log(I<em>s/I</em>o) = log(100%) – log %T

  • A=2–logA = 2 – log % T

Beer-Lambert Law (Beer’s Law)

  • Concentration is directly proportional to absorbed light and inversely proportional to the log of transmitted light.

  • A=ebcA = ebc

    • A = absorbance

    • e = molar absorptivity (L/mole-1.cm-1)

    • b = light path (cm)

    • c = concentration (mole/L)

Beer’s Law Application

  • Basis of quantitative analysis using absorption photometry/spectroscopy.

  • Absorbance is unitless.

  • Absorptivity is a proportionality constant.

Review of Beer’s Law

  • Absorbance is directly proportional to concentration; log of transmitted light is inversely proportional to concentration.

  • Increased concentration/cell path increases absorbance and decreases transmittance.

Calculating Unknown Concentrations

  • A<em>s/A</em>u=C<em>s/C</em>uA<em>s / A</em>u = C<em>s /C</em>u

  • C<em>u=(A</em>u⋅C<em>s)/A</em>sC<em>u = (A</em>u \cdot C<em>s) / A</em>s

    • Cu = concentration of the unknown

    • Cs = concentration of the standard

    • Au = absorbance of the unknown

    • As = absorbance of the standard

  • Valid only if Beer’s Law is obeyed and measurements are in the same cell.

  • Data must fall within the standard calibration curve.

Limitations of Beer’s Law

  • Deviations occur at very high concentrations.

  • Non-monochromatic incident light.

  • Significant solvent absorption.

  • Non-parallel cuvette sides.

  • Stray light.

  • Fluorescence.

  • Multiple absorbing chemical species.

Stray Radiation

  • Radiant energy reaching the detector at incorrect wavelengths.

  • Deviations from Beer’s Law increase with stray light.

Design of Spectrometric Methods

  • Analyte absorbs at a unique wavelength.

  • Analyte reacts to produce a product that absorbs at a unique wavelength (chromophore).

Spectrophotometer Components

  • Light Source

  • Wavelength Isolator (filters, prisms, monochromator)

  • Detector

  • Read out Device (meter)

Light Sources

  • Lamps:

    • Tungsten or Tungsten-iodide (visible & near IR)

    • Quartz-halogen (visible)

    • Hydrogen or deuterium (UV, 190-400 nm)

    • Mercury (UV)

  • Lasers: Intense, narrow wavelength, used for fluorescence and specialized testing.

Light Source Factors

  • Range, spectral distribution, radiant production source, radiant energy stability, temperature.

  • Excitor lamp must give intense, cool, constant light.

Light Separation Devices

  • Filters

  • Prisms

  • Gratings

Filters

  • Absorbance Filters (Wratten Absorption Filter): colored glass absorbing unwanted wavelengths.

  • Limited use due to wide band pass (35-50 nm).

  • Simple and inexpensive.

Interference Filters (Fabry-Perot)

  • Semi-transparent silver films on magnesium fluoride.

  • Used for fixed wavelengths; narrow band pass.

  • Monochromatic light via constructive interference.

Prisms

  • Refract light; short wavelengths refract more.

  • Glass (visible); quartz (UV).

  • Nonlinear separation of bands; requires wavelength calibration.

Gratings

  • Transmittance (glass) or Reflection (aluminum).

  • Linear dispersion; holographic gratings minimize stray light.

  • Diffraction grating: reflecting surface with parallel grooves.

  • Resolution depends on groove number.

Other Features: Slits

  • Entrance: focuses light.

  • Exit: isolates narrow wavelength band.

  • Wider exit slit increases intensity but decreases purity.

Bandpass (Bandwidth)

  • Range of transmitted wavelengths.

  • Calculated as width at half maximum transmittance.

  • Example: Bandpass = 10 nm, setting at 455nm transmits 450 nm – 460 nm. Determined by:

    • Light source intensity.

    • System efficiency in isolating wavelengths.

    • Detector sensitivity.

    • Narrow band pass offers greater resolution but decreases intensity.