Atomic Absorption & Reflectance Photometry

• The material covers Atomic Absorption Spectrometry (AAS) and Reflectance Photometry

Prayer Before Class

• A prayer to the Holy Spirit is offered before class to seek guidance and wisdom
• The prayer requests brilliance to overcome intellectual darkness, a penetrating mind, retentive memory, and ease of learning
• It also asks for guidance in work and successful completion, through Jesus Christ

Atomic Absorption Spectrometry and Reflectance Photometry

• Group 1 members are Burgos, Caspe, and Duran

Atomic Absorption Spectrophotometry

• It is an analytical technique used to determine the concentration of metal atoms/ions in a sample
• Metals can be desirable or contaminants in the sample
• Light at an element's characteristic wavelength shines on a sample
• For instance, a sample containing Cu and Ni, when exposed to light at Cu's wavelength, only Cu will absorb this light
• The amount of light absorbed is directly proportional to the concentration of the absorbing ions or atoms
• This determines to concentration

Advantages

• Low cost per analysis
• Easy to operate
• High sensitivity (up to ppb detection)
• High accuracy
• Mostly free from inter-element interference
• Wide applications across many industries

Limitations

• Cannot detect non-metals
• New equipment is expensive
• More geared towards analysis of liquids
• Sample is destroyed

Parts of the Instrument

• Light Source
• Nebulizers
• Atomizers
• Monochromators
• Detectors
• Readout Devices

AAS Parts: Light Source

• A hollow cathode lamp uses Tungsten to make the anode
• The glass tube is made of quartz
• The Cathode is coated with the sample element
• Inside the glass tube, inactive gasses like Neon or Argon are charged at 1-5 torr atmospheric pressure
• Then it passes through a chopper

AAS Parts: Nebulizers

• Nebulizers create a consistent, fine mist of the liquid sample
• This allows for efficient atomization and absorption of light in the flame

Concentric Nebulizers

• The most common type
• Sample liquid is drawn into a central capillary tube and mixed with a gas stream through a concentric design

Cross-flow Nebulizers

• Sample liquid is sprayed perpendicular to the gas flow
• This provides better sample introduction for viscous liquids

AAS Parts: Atomizers

• Atomizers convert an analyte to a free gaseous atom

Flame Atomizers

• Aqueous sample is drawn into the assembly by passing compressed air past the end of a capillary tube immersed in the sample
• Sample exits the nebulizer and strikes a glass impact bead, which converts it into a fine aerosol mist within the spray chamber
• An aerosol mist is swept through the spray chamber by combustion cases.
• Flame's thermal energy desolvates the aerosol mist to a dry aerosol of small, solid particulates
• The flame's thermal energy then volatilizes the particles, producing a vapor of molecular species, ionic species, and free atoms

Electrothermal Atomizers

• Significant improvement in sensitivity using the resistive heating of a graphite tube in place of flame.
• Typical electrothermal atomizer, graphite furnace, consists of a cylindrical graphite tube approximately 1-3 cm in length and 3–8 mm in diameter.
• Graphite tube is housed in a sealed assembly with an optically transparent window at each end.
• Inert gas is passed through the furnace, which protects graphite tube from oxidation during atomization
• A power supply passes current through the graphite tube for resistive heating

AAS Parts: Monochromator

• It's an optical instrument selectively transmits a narrow range of wavelengths of light/radiation from a broader spectrum

Types of Monochromators

• Prism
• Diffraction Grating

AAS Parts: Detectors

• The process of converting light from a monochromator into a simplified electrical signal can be accomplished through the use of a detector
• The photomultiplier tube serves as the detector in the instrument used for atomic absorption spectrophotometry

AAS Parts: Reading Devices

• Various readout systems are employed in atomic absorption spectroscopy
• Examples of reading devices include meters, chart recorders, and digital display meters
• Hard copies can be produced through the use of chart recorders, printers, or plotters

Operation

• Sample Preparation
  • Dissolving solid samples into an acid to make them into a liquid sample
  • Diluting the analyte into a concentration the machine can detect
  • Preparing standard solution for calibration curve
• Instrument Setup
  • Selecting the light source (HCL)
  • Setting the monochromator
  • Choosing the correct atomizer
  • For Flame AAS, gas flow adjustment is required to avoid too much fuel/oxidant gas used
  • Using the standard solution, calibrate the machine
• Sample Analysis
  • Sample is aspirated into the flame by the nebulizer
  • Atomization occurs
  • Light from the HCL passes through the flame with sample atoms, light at the HCL wavelength is absorbed
  • The light is detected measures the amount of light absorbed
  • After detecting, analyzes sample concentration based on absorbance and calibration curve from standard solution
• Instrument Shutdown and Cleanup
  • Turn off the flame
  • Gas supplies are turned off
  • Instrument cleaning

Data Analysis and Interpretation

• A calibration graph of iron is performed by atomic absorption

Reflectance Photometry

• Reflectance photometry measures the amount of light reflected by a surface
• It analyzes color, composition, or physical properties
• Quantitative analysis of body fluids

Reflectance Photometer Principles

• Operation is based on the incidence of a light beam on the surface to be examined
• Reflected light is detected and analyzed by the photometer
• Different wavelengths of the visible spectrum are used to obtain information about the material

Components of a Reflectance Photometer

• Light Source
• Test Pad Surface
• Photodetector
• Analog-to-Digital Converter
• Microprocessor

Light Source

• A key component emits a beam of light directed at a measured surface that determines the amount of light reflected

Light Source List

• LEDs are commonly used, emits small, energy-saving lights that are long-lasting and precise
• Cold Lamps are bright and accurate. High sensitivity and reliable sources of light
• Halogen Lamps are stable, good for older models, and use filters

Test Pad Surface

• Made from plastic or paper with chemical reagents
• Pads change color upon contact with urine, can be compared against color chart for interpretation

Common Parameters Tested

• Leukocytes
• Nitrites
• Urobilinogen
• Proteins
• pH
• Specific Gravity
• Ketones
• Bilirubin
• Glucose

Photodetector

• Measures light intensity/color changes on test strips
• Used to detect substances like glucose, proteins, or pH levels
• The most commonly used photodetector in urine analyzers is the reflective photometer

Digital Cameras With Flow Cell Imaging

• Captures high-resolution images of urine sediment flowing through a cell for particle identification

Analog-to-Digital Converter

• The sensors detect color changes on reagent pads caused by chemical reactions with the urine

Microprocessor

• Control Operations: Automate testing processes, like moving strips and timing reactions
• Data Processing: Analyze sensor signals measure things like pH and glucose.
• Image Analysis: Process and classify images of urine particles
• Error Correction: Adjust for measurement errors to ensure accuracy
• Data Storage: Store results send data lab systems print reports

How the Instrument Works:

• Printer Cover is lifted to insert paper
• Strip Loading Plate moves the test strip to the measuring position
• LCD Display shows working process

Operation

• Place the plate in the middle of the instrument
• Connect the power cord to the back of the instrument, and turn it on
• System Check:
• The instrument will do a system check automatically
• The plate will move to the middle of the reader

Operation

• Place Test Strips
• Choose a mode: General, One by One, or Quick
• General Mode: Put the first strip in the groove near the center. Add more strips (up to 10) and press the End key after the last one
• One by One Mode: Put the first strip, press Start. Add each strip one by one, pressing Start after each. Press End after the last strip
• Quick Mode: Put all strips in and press End
• Wait until the color on the first strip changes completely
• The instrument will start measuring after the first strip is ready
• Once the measurement is done, the instrument will wait
• Read results and throw away the used strips

How Does It Work?

• The strip is illuminated by white light, and the reflected light from the strip is detected by the Sensor
• The RGB signals are digitized, interpreted by the processor
• The intelligent image analyzer SW locates the strip and the pads, color data determines parameter values
• Results including the date, time, sequence number, and ID are stored and printed out by the internal print

Reference Values

• Are as follows:
  • Color - Yellow (light/pale to dark/deep amber)
  • Clarity/turbidity – Clear or cloudy
  • pH – 4.5-8
  • Specific gravity – 1.005-1.025
  • Glucose - ≤130 mg/d
  • Ketones - None
  • Nitrites - Negative
  • Leukocyte esterase – Negative
  • Bilirubin - Negative
  • Urobilirubin – Small amount (0.5-1 mg/dL)
  • Blood - ≤3 RBCs
  • Protein - ≤150 mg/d
  • RBCs - ≤2 RBCs/hpf
  • WBCs - ≤2-5 WBCs/hpf
  • Squamous epithelial cells - ≤15-20 squamous epithelial cells/hpf
  • Casts – 0-5 hyaline casts/lpf
  • Crystals - Occasionally
  • Bacteria - None
  • Yeast – None

Data Analysis and Interpretation

• The graphs illustrate the correlation between urinary protein concentrations and reflectance values using reflectance photometry in urine analysis