Chapter 4 Analytic Techniques

Analytic Techniques

Chapter Overview

• Introduction to various analytic techniques used in laboratories.

Spectrophotometry

Absorption Spectroscopy

• Definition: Technique measuring absorption of radiation (light) based on frequency or wavelength.

• Instruments: Utilize UV or visible light for analysis.

Beer-Lambert Law (Beer’s Law)

Explanation

• When light passes through a container with an absorbing compound, some light is absorbed while some is transmitted.

• Beer’s Law: Concentration of a substance correlates directly with the light absorbed and inversely with the logarithm of light transmitted.

Transmittance vs Absorbance

Key Equations

• Transmittance (T): T = 10^(-abc)

• Absorbance (A): A = abc

• Variables:

  • a = absorptivity coefficient

  • b = light path length

  • c = concentration (g/L)

Spectrophotometer

Components

• Five components of a spectrophotometer:

  1. Stable source of radiant energy

  2. Device isolating a specific region of the Electromagnetic Spectrum (EMS)

  3. Sample holder (cuvette)

  4. Photo detector

  5. Readout device

Atomic Absorption Spectrophotometry

Purpose

• Measures concentration by detecting absorption of electromagnetic radiation by atoms, not molecules.

• Excellent sensitivity and precision for detecting trace metals.

Fluorometry

Overview

• Uses natural fluorescence to quantify concentrations.

• Process: Electrons absorb electromagnetic radiation, get excited, and emit light with a longer wavelength when returning to a lower energy level.

Chemiluminescence

Principles

• Light emission is a result of a chemical reaction.

• Key Compounds: Acridinium ester and luminol are commonly used.

• Detection: Utilizes a luminometer with photomultiplier tubes (PMTs).

Light Scatter Techniques

Techniques

• For larger particles: Nephlometry and turbidimetry measure light scattered by particles in solution.

• Nephlometry: Measures scattering at angles of 15-90°.

• Turbidimetry: Measures reduction in light transmission due to particle formation.

Osmometry

Measurement

• Measures osmolality in aqueous solutions; increases with concentration of osmotically active particles (e.g., glucose, urea, sodium).

• Principles: Freezing-point depression

  • Freezing-Point Osmometer: Rapidly supercools a sample and initiates freezing, measured to determine osmolality.

Electrochemistry

Types of Cells

1. Galvanic Cells

   • Constructed with two half-cells connected by a salt bridge; demonstrate oxidation and reduction.

2. Electrolytic Cells

   • Both electrodes immersed in the same solution; external EMF drives reactions.

Potentiometry

• Measures voltage between two electrodes.

• Components:

  • Reference electrode (providing constant voltage)

  • Measuring electrode (indicator)

• Calculates ion concentrations using the Nernst equation.

Ion-Selective Electrodes (ISE)

Features

• Membrane-based devices responding to specific ions; generates potential as ions transfer to membrane.

• Utilize ionophores for specific ion measurement.

pH Electrodes

Description

• Measure hydrogen ion activity; typically made of glass.

• Components include pH sensitive glass membrane and chloride-containing buffer solution.

Blood Gas Electrodes

pCO2 Electrode

• Utilizing the Severinghaus electrode to measure PCO2; modified pH electrode with CO2 permeable membrane.

pO2 Electrode

• Clark electrode for measuring oxygen (PO2) using polarographic principles.

Coulometry

Process

• Measures electricity needed to convert an analyte to a different oxidation state; used for chloride ion measurements in the sweat test for cystic fibrosis.

Separation Techniques

Electrophoresis

• Separates charged compounds (e.g., proteins) using electric fields.

• Types include serum protein electrophoresis and hemoglobin electrophoresis.

Chromatography

• Separation based on interactions with mobile and stationary phases.

• Types: Thin-layer, gas, and liquid chromatography (e.g., High-Performance Liquid Chromatography).

Mass Spectrometry

• Identifies compounds, determines concentrations, and studies molecular structures.

• Components: Ion source, mass analyzer, ion detector.

Flow Cytometry

Measurement

• Analyzes multiple cell and particle parameters as they flow past a light source.

• Key features: Cells/particles, light source, fluidics, detector, and computer analysis.

Point of Care (POC)

Microscale Technologies

• Various analytical principles applicable to POC devices include:

  • Reflectance: Urine & blood chemistries (e.g., glucose)

  • Lateral-flow immunoassays: Infectious agents, cardiac markers

  • Electrochemistry: Glucose and other metabolites

  • Light scattering: Coagulation and others

  • Fluorescence: Hemoglobin species and cardiac markers.