Analytical Techniques

Chapter Overview

  • Analytic Techniques discussed by Julia C. Drees, Matthew S. Petrie, Alan H.B. Wu

Spectrophotometry and Photometry

Basic Principles

  • Photometric instruments measure light intensity without considering wavelength.

    • Instruments use filters, monochromators, prisms, or gratings to isolate specific wavelengths.

  • Beer's Law (A = abc):

    • Concentration of a substance is directly proportional to light absorbed and inversely proportional to logarithm of transmitted light.

  • Absorbance (A): Amount of light absorbed by a solution, derived from %T ( A = 2 - log(%T)).

Components of a Spectrophotometer

  • Light Source:

    • Types: tungsten (for UV) or deuterium/mercury arc lamps.

  • Monochromators:

    • Function: Isolate wavelengths using filters, prisms, or diffraction gratings.

  • Sample Cell:

    • Material: Typically plastic or quartz (for UV).

    • Care: Scratches or dust can scatter light and affect readings.

  • Photodetectors:

    • Converts radiant energy into electrical energy (includes photocells, phototubes, photomultiplier tubes).

Wavelength Selectors

  • Types include filters, prisms, and diffraction gratings.

    • Quality assessed by nominal wavelength and effective bandwidths.

Signal Processing

Purpose

  • Signal processing is essential for amplifying, rectifying, directing current, altering phase, filtering unwanted components, and performing calculations on signals from transducers.

Quality Assurance in Spectroscopy

Key Parameters

  • Regular monitoring of photometric parameters is required for optimal performance.

Wavelength Accuracy

  • Assessed using special optical filters like didymium and holmium oxide.

  • Wavelength accuracy is crucial to ensure proper monochromator function.

Linearity

  • Ensures the absorbance follows Beer’s Law.

  • Checks include diluting colored solutions to evaluate linearity.

Reflectometry

Clinical Applications

  • Used in urine dipstick analysis and dry chemistry testing.

Reflectometer Functionality

  • Measures quantity of light reflected by samples, such as those on non-polished surfaces (like urine dipstick).

Fluorometry

Basic Instrumentation

  • Filter fluorometers assess concentrations of fluorescing solutions.

Advantages and Disadvantages

  • Advantages: Greater specificity and sensitivity.

  • Disadvantages: Sensitivity to environmental factors that may lead to quenching.

Turbidimetry

  • Measurement of light transmission reduction due to particle formation.

    • Key in microbiology and coagulation studies.

Chemiluminescence

  • Chemical reactions that emit light, requiring no excitation radiation or monochromators.

  • Common in reactions involving luminol, acridinium esters, and dioxetanes.

Electrochemistry

Ion-Selective Electrodes (ISE)

  • Sensitive to individual ions; includes pH electrodes.

Potentiometry

  • Measures potential between two electrodes; uses reference and indicator electrodes.

  • Nernst equation relates cell potential to ion concentration.

Osmometry

Techniques

  • Methods include freezing-point depression and vapor pressure measurement of aqueous solutions (serum, plasma, urine).

Colligative Properties

  • Properties affected by solute concentration include boiling point elevation, vapor pressure lowering, freezing point depression, and osmotic pressure.

  • Primary method used in clinical labs is freezing point depression.

Osmolality vs. Osmolarity

  • Osmolality: Concentration per kg of solvent (mOsmol/kg).

  • Sodium, glucose, and urea determine osmolality.

  • Osmolarity: Concentration per volume of solution.

Osmolal Gap

  • Calculated as measured osmolality minus calculated osmolality; abnormal gaps indicate presence of unmeasured substances.

Freezing Point Measurement

  • Freezing point osmometers supercool samples and initiate freezing, providing readings for solute concentration.