Optical Density Objectives

Analytical methods that measure how light interacts with matter  and Provide information about composition, concentration, and physical properties of samples.

• Optical Techniques

Term for light that includes ultraviolet (UV), visible, infrared radiation and Part of electromagnetic spectrum.

• Optical Radiation

Portion of electromagnetic spectrum detectable by the human eye

• Visible Spectrum

Visible Spectrum Wavelength.

• 380 - 750 nm

Relevant for clinical laboratory specimen  and Used for quantitative analysis of proteins, carbohydrates, lipids, enzymes, nucleic acids, and metabolites 

• UV-Visible Spectrum

UV-Visible Spectrum Wavelength

• 200 - 700 nm

Measures about of light absorbed by a substance

Absorbance

Light passes through a medium with minimal absorption and is key for analyzing transparent materials

• transmittance

What is transmittance used for?

• liquid chromatograph

  • amount of transmitted light indicates concentration

Measures light reflected from a surface or sample 

• reflectance

What is reflectance used for?

• dry chemistry analyzers and point of care testing 

Measures emitted light after excitation  and is Highly sensitive

• fluorescence

What is fluorescence used for?

•  immunoassays, molecular diagnostics, flow cytometry 

Measures light redirected by particles in suspension

• light scatter

What is light scatter used for?

• Provides information on particle size, concentration, and structure 

How is Absorbance measured?

• mathematically relating it to transmittance 

What is the absorbance equation?

• A = log (1/T) = - log T 

  • A = absorbance

  • T = transmittance ( T= %T/100)

• Can be rewritten as:

  • A = log (100/%T) = log100 - log%T = 2 - log%T

What is the relationship between absorbance and transmittance?

• The higher the absorbance, means the lower the transmittance 

  • More light was absorbed in the sample 

Fraction of light transmitted when light is allowed to pass through a solution 

• Transmittance

Transmittance calculation.

• T = I/Io

  • I = transmitted light intensity 

  • Io = original light intensity 

• Percent Transmitance 

  • %T = T x 100 

What is the relationship between %Transmittance and Absorbance

• inverse relationship

measurement of light reflected from the surface of a sample. Light is shined onto a sample, and the light that bounces back is detected. It also allows quantitative measurement of reactions on surfaces such as a dipstick or dry film 

• Reflectance

measure of how much light is reflected by a test sample compared to a standard reflector 

• reflection density

Reflection density calculation.

• Dr = log (Ro/Rtest)

What does a Higher reflection density mean?

• Occur when test sample reflects LESS LIGHT (darker sample/high concentration)

What does a Lower reflection density mean?

• Test sample reflects more light 

 measure the fluorescence emitted by substances when exposed to light 

• fluorometers

What are fluormeters used for?

• Biological research: detecting DNA,RNA, proteins labeled with fluorescent dyes

• Clinical Diagnostics: biomarkers for metabolites in body fluids 

• Environmental Analysis: detecting pollutants in water/air 

Beers Law

• Absorbance is directly proportional to concentration of the absorbing species

• Explains how solute amount affects light absorption 

  • Higher concentration = Higher absorbance 

Lamberts Law

• Absorbance is directly proportional to path length 

• Explains how the diameter of a cuvette affects light absorption 

  • The more material the light passes through, the more it is absorbed 

Beer-Lamberts Law

• Absorbance depends on both path length (b) and concentration (c) 

• Forms the basis of spectrophotometric quantification in clinical labs

Beer Lambert Law Equation

• A = ebc

  • E = molar absorptivity (L/mol * cm) 

What is Beer-Lamberts Law used for?

• spectrophotometric analysis to determine unknown concentrations of analytes by comparing them with a standard solution of known concentrations 

What is a standard solution

• One with known concentration 

• Absorbance is measured using a spectrophotometer under identical conditions as the unknown solution 

Standard Solution formula.

• (A unknown / A standard ) = (C unknown / C standard) 

Supplies electromagnetic radiation for analysis

• Light Source

Light Source Types.

• Deuterium lamp: UV range (200-300 nm)

• Tungsten-halogen lamp: Visible Range (380 - 750 nm)

• Modern instruments have both to cover full UV-visible spectrum

Isolates specific wavelength from the light source and Ensures accurate,specific, and interference free absorbance measurements by using a single, well-defined wavelength 

• Monochromator

Monochromator Parts

• Entrance Slit: controls amount of incoming light 

• Dispersion Elements:

  • Prism: separates wavelengths by refraction 

  • Diffraction grating: most efficient, widely used 

• Exit Slit: allows only selected wavelength to pass through the sample 

Holds the sample for spectrophotometric analysis 

• cuvette

Used for automated/semi-automated system and Allow sample change without moving cuvette 

• flow-through cuvette

Cuvette Materials.

• Quarts: used for UV measurements

• Glass/Plastic: used for visible light 

Converts transmitted or absorbed light into an electrical signal proportional to light intensity

• Detector

Detector: Photomultiplier

• very sensitive

• used for low light levels

Detector: Photodiode

• Common in modern instruments

• wide detection range

Detector: Charge-coupled device

• Highly sensitive, used in advanced spectrophotometers 

Beam splitter

• Splits light into two beams 

  • One for sample

  • One for reference

• Allows simultaneous measurement and correction for light source fluctuations 

• Design ensures great accuracy and reproducibility 

Data Processing and Display System 

• Processes the electrical signal from the detector

• Converts it into an absorbance or transmittance value

• Displays spectrum  results on a digital screen or computer interface 

Measures decrease in transmitted light through a suspension, Detector in line with the light source (0 *).

• Turbidimetry

What is Turbidimetry used for?

• protein assays and  bacterial growth monitoring 

Measures scattered light at an angle and is Sensitive for small particle concentrations

• immunoglobulins, complement proteins, ag-ab complexes 

Cells pass individually through a laser beam and Detects forward scatter (size) and side scatter (granularity)

• flow cytometry

What is flow cytometry used for?

• cell counting, immunophenotyping, leukemia/lymphoma diagnosis