Clinical Chemistry 9th Edition Chapter 4

Chapter Outline

1. Spectrophotometry

   • The technique for measuring how much light a chemical substance absorbs by determining the intensity of light transmitted through the sample. It is crucial for quantifying the concentration of solutes in various solutions, especially in clinical laboratories.

2. Beer’s Law

   • A fundamental principle in spectrophotometry stating that absorbance (A) is directly proportional to concentration (c) of an absorbing species in a solution, path length (b), and molar absorptivity (ε). The formula is written as A = ε × b × c, and it indicates that as the concentration of a solution increases, the absorbance will also increase, which can be plotted on a calibration curve to determine unknown concentrations.

3. Spectrophotometric Instruments

   • These instruments consist of various essential components:

     • Light Source: (e.g., tungsten for visible light, deuterium lamps for UV).

     • Monochromator: Used to isolate specific wavelengths of light needed for measurement.

     • Sample Cell (Cuvette): The container that holds the solution, typically made from materials like quartz or glass that do not absorb the wavelengths being measured.

     • Detector: Converts light into an electrical signal for quantification.

4. Spectrophotometer Quality Assurance

   • Regular calibration and maintenance of spectrophotometers are crucial to ensure accuracy in the measurements. Quality assurance protocols include checks on wavelength accuracy, absorbance accuracy, and performance validation against known standards.

5. Atomic Absorption Spectrophotometry

   • A technique utilized for measuring the concentration of trace metals in samples by detecting the light absorbed by free atoms in a vaporized state, typically in a flame or graphite furnace. It is highly accurate and is often used for analyzing environmental samples and biological fluids.

6. Fluorometry

   • This technique measures the intensity of fluorescence emitted from a sample when it is excited by specific wavelengths of light. Its high sensitivity and specificity make it ideal for clinical applications such as drug testing, biomarker detection, and environmental monitoring.

   • Instrumentation: Comprises light sources, interference filters, and photomultiplier tubes or CCD detectors.

7. Fluorescence Polarization

   • A method used to study molecular interactions based on the rotational mobility of fluorophore-labeled molecules. It is employed in various applications including immunoassays.

8. Advantages and Disadvantages of Fluorometry

   • Advantages: High sensitivity, specificity, and capability for multiplexing.

   • Disadvantages: Sensitivity to environmental factors such as temperature and pH; may require extensive calibration.

9. Chemiluminescence

   • A detection method where light is emitted as a result of a chemical reaction without the need for external light sources. It is a valuable technique in various immunoassays and is worth exploring for its application in diagnostics.

10. Turbidimetry and Nephelometry

    • Techniques used to measure the concentration of suspended particles in a solution by analyzing the scattering of light (nephelometry) or the reduction of transmitted light (turbidimetry). They are useful in assessing sample clarity and particle levels in industrial and medical applications.

11. Laser Applications

    • Lasers are used in many analytical techniques due to their ability to provide highly focused light and increased sensitivity in measurements.

12. Electrochemistry

    • This field studies the connection between chemical reactions and electrical energy. Key components include galvanic cells (which produce electrical energy from spontaneous reactions) and electrolytic cells (which require energy input to drive chemical reactions). Applications include blood glucose monitoring and blood gas analysis.

13. Electrophoresis

    • A technique for separating charged particles in a solution using an electric field. It is heavily employed for protein analysis, nucleic acids separation, and various diagnostics.

    • Procedure: Involves preparing the gel, loading samples, applying voltage, and visualizing results, typically through staining.

14. Chromatography

    • A collection of techniques that separate chemical mixtures based on their interactions with stationary and mobile phases. High-Performance Liquid Chromatography (HPLC) and Gas Chromatography (GC) are widely used for analyzing compounds in biological samples.

15. Mass Spectrometry

    • A powerful analytical method used for identifying the amount and type of chemicals present in a sample by measuring the mass-to-charge ratio of ions. It is crucial in toxicology, proteomics, and microbial identification, and often combines with chromatography techniques for enhanced analysis.

Key Terms

• Atomic Absorption Spectrophotometry: Quantitative analysis technique for detecting metals in various samples.

• Chemiluminescence: Emission of light as a result of a chemical reaction.

• Chromatography: Method for separating components based on differential partitioning behavior.

• Electrochemistry: Study of chemistry related to electrical potentials and currents.

• Electrophoresis: Technique that separates charged molecules in a gel by using an electric field.

• Fluorometry: Measurement of fluorescence from a sample under specific excitation conditions.

• Gas Chromatography: Technique used to separate and analyze compounds that can be vaporized without decomposition.

• High-Performance Liquid Chromatography: Advanced form of liquid chromatography for separating mixtures.

• Ion-Selective Electrodes: Electrodes that respond selectively to particular ions in a sample.

• Mass Spectrometry: Technique for identifying compounds based on mass-to-charge ratios.

• Osmometry: Measurement of osmotic pressure in solutions.

• Spectrophotometry: Technique measuring light absorption to assess solution concentrations.

Chapter Objectives

• Understand and explain analytic methods utilized in clinical laboratories.

• Assess operational principles and limitations associated with different techniques.

• Identify clinical applications derived from these diverse analytic techniques.