Lecture_2_-_Spectroscopy

Modern Techniques and Instrumentation [CSU029]

Lecture 02: Single Beam Spectrophotometer

Page 1: Overview

  • Course Name: Modern Techniques and Instrumentation [CSU029]

  • Topic: Single Beam Spectrophotometer

Page 2: Transmittance and Absorbance

Transmittance (T)

  • Formula: T = I / Io

    • Io: Intensity of incident light

    • I: Intensity of transmitted light

  • Transmittance (T) is always less than 1.

  • Measured as a percentage (%T):

    • %T = T * 100 (ranges from 0 to 100%)

Absorbance (A)

  • Defines the amount of light absorbed by the sample.

  • Calculated using:

    • A = - log10(T)

    • A = log10(1/T)

    • A = 2 - log10(%T)

  • Transmittance and absorbance are inversely related.

  • Spectrophotometer displays both transmittance and absorbance.

Page 3: Beer’s Law and Lambert’s Law

Beer’s Law

  • Light intensity decreases exponentially with increasing concentration (C):

    • A α C

    • A: Absorbance

Lambert’s Law

  • Absorbance increases with the thickness of solution (L):

    • A α L

    • L: Length of light path

Combined Equation

  • Combining both laws gives:

    • A α C*L

  • Final form: A = KCL, where K is constant for the colored solution.

Page 4: Beer-Lambert Law

Expression of Beer-Lambert Law

  • Light transmission through a colored solution decreases exponentially with:

    • Increased concentration of colored solution

    • Increased path length of cuvette

Parameters

  • A = ε·b·c

    • A: Absorbance

    • ε: Molar absorptivity (L·mol-1·cm-1)

    • b: Sample path length (typically 1 cm)

    • c: Concentration (mol/L)

Page 5: Deviations from Beer-Lambert Law

Types of Deviations

  1. Real Deviations

    • Occur at high concentrations (>10 mM) leading to changes in analyte behavior due to interactions with solvent/solute.

  2. Chemical Deviations

    • Due to specific attributes of the chemical species, e.g., changes in pH affecting absorption spectra.

  3. Instrument Deviations

    • Occur from non-monochromatic radiation leading to inaccuracies; measurements taken at λmax to minimize this effect.

Page 6: Instrumentation of Single Beam Spectrophotometer

  • A single beam spectrophotometer has a single light path from the source.

  • Polychromatic light is separated into a narrow wavelength band using a wavelength selector and passed through a sample to measure transmitted intensity.

Page 7: Sources of Light and Wavelength Selectors

Light Sources

  • Types: Continuous sources emitting all wavelengths within the spectral region.

    • Deuterium Lamp: 200-400 nm (UV)

    • Tungsten Lamp: 320-2500 nm (Visible and near IR)

Wavelength Selectors

  • Purpose: Select a band of a specific wavelength from the source.

    • Filters and monochromators often used.

    • Performance improves with narrower bandwidths.

Page 8: Monochromators

Functionality

  • Used for spectral scanning across a range of wavelengths.

  • Common mechanical components include slits, mirrors, lenses, gratings, or prisms.

Comparisons

Prism vs. Grating

  • Prism

    • Composed of glass (visible) or quartz (UV).

    • Non-linear dispersion; not sturdy.

  • Grating

    • Provides linear dispersion; better for overlapping issues; more durable.

Page 9: Sample Compartment and Detectors

Sample Compartment (Cuvette)

  • Cuvette used for holding liquid samples in UV/Visible spectroscopy.

    • Suitable materials: Glass (for visible), Quartz (for UV).

Detectors

  • Converts radiant energy into electrical signals.

  • Characteristics: Sensitive, fast response, directly proportional to transmitted intensity.

    • Examples: Phototube and Photomultiplier tube.

Page 10: Acknowledgments

  • Thank you: Jagdish Verma

  • School of Biotechnology, Shoolini University

  • Contact: jagdishverma1@shooliniuniversity.com

  • Phone: +91 8894450102