ANALYTICAL TECHNIQUES AND - Flame Emission Spectrophotometry

o Measures
INSTRUMENTATION o Internal standard?
Basic Disciplines: o Components
1. Measurements of Radiant Energy o Examples
a. Absorption o pI
b. Emission - Atomic Absorption Spectrophotometry
c. Polarization o Measures
d. Scattering techniques o Sensitivity
2. Luminescence o pI
3. Electroanalytical Method o components
4. Chromatography o kind of method
o correction
- Photometry o Spectral VS Non-Spectral interferences
- Spectrophotometry o Elements measured
- Types of Photometers - Fluorometry
- Wavelength o Location of photodetector
- Photons o Only what can pass through the detector?
- Photometric instruments o Absorbance and emission
o What does it not consider? o Definition of fluorescence
- Photometer o Advantage
o Similar to a monochromator o Disadvantage
- Electromagnetic Radiation o Decrease in fluorescence
o Planck’s formula (shows what relationship) o Application
o Indirect and direct proportional o Fluorophore
o Planck’s constant o Types of Fluorometers and
o Wavelength, amplitude, trough and crest Spectrofluorometers (4)
- UV and IR radiation  Measures what
o Numerical values - Phosphorescence
- 2 kinds of lights emitted by spectrophotometry o Difference from fluorescence
- Single Beam Spectrophotometer - Types of Luminescence
1. Light Source o Chemiluminescence
o Important Factors  Chemical reaction
2. Entrance Slit  Oxidant
o Stray Light o Bioluminescence
3. Monochromator  Biological catalyst
4. Exit Slit o Electrochemiluminescence
5. Cuvette
 Label
6. Photodetector
7. Read Out
 Application
- Light Scattering Techniques
- Colorimetry (law used) o Factors influencing light scatter
- Beer-Lambert’s law - Turbidimetry
o Absorbance o Application
o Transmittance - Nephelometry
- Absorbance formula [3] o Application
- Transmittance vs Absorbance Graph o Instruments used
o Easier to work with? o Limitations
- Three Methods for Calculation of Concentration - Turbidimetry VS Nephelometry
of an Unknown - Electrophoresis
o Direct (formula) o Definition
o Standard Curve (how to remedy out of o pI
range) o CHON (acidic and basic)
o Use of Molar absorptivity value o Migration of small molecules
o Use porous supporting medium (example)
- Reflectance Photometry o Electropherogram
o Measures? o Quantitative profiling
o Formulas that convert data into linear o Factors that affect migration rate
format (2) o Components
o Components  Support media
o Used in
 Buffer
  Stains  emzymes
o History  fluorescent
- Types of Electrophoresis  luminescent
o Zone Electrophoresis - RIA
 Classification
o Slab gel electrophoresis - EIA
 Separation - IFA
o Disc electrophoresis - Types of ELISA
 Zones yielded - Immunofluorescence
o Isoelectric focusing electrophoresis o Direct VS Indirect
 Separation  Commonly used
o Two-dimensional electrophoresis  Advantage
 Dimension composition  Disadvantage
 Used in o Dyes
o Blotting techniques o Quantitation
o Capillary electrophoresis o Principle
o Microchip Electrophoresis o Stokes Shift (Fluorescein VS Phycocythrin)
 Alternative to - Ultracentrifugation
 Separation - Scintillation Counters
- Common problems o Solid, Liquid
- Basic Concepts of Automation
- Description of Conventional Electrophoretic
o Intelligent Automation
Procedure
- New Areas in Automation
- Steps in Gel electrophoresis - Automation of Analytical Process
o What does it separate o Specimen Delivery
o Temperature of  Courier Service
o Machine used by PCR  Pneumatic Tube System
o Which travels further  Electric Track Vehicles
o Purposes  Mobile Robots
o Pros and Cons o Specimen Preparation
- Potentiometry o Specimen loading and aspiration
o Measures what o Reagent Handling and Storage
o Another term o Reagent Delivery
o Substances measured - Chemical reaction Phase
o (ISE) ion selective electrode o Mixing Reactants
o Basic components of pH meter - Measurement Approaches
o 3 types of ion selective electrode - Types of Automation
 membrane electrode types o Random access analyzers
- Coulometry o Continuous flow analyzers (example) (air
o Titration is used to determine bubbles)
o Definition o Centrifugal (another term) (instrument
- Chromatography used)
o pI o Dry Slide Analyzer (technique used)
o Components (composition)
o Planar (example: stationary and mobile - Advantages of the different Types of Automation
phase) - POCT
o Column o Example of tests
o Environment done
- Separation Mechanisms
o Advantages
o Steric Exclusion Chromatography
- Implementation and Management
o Partition Chromatography
Considerations
o Ion exchange chromatography
o QC, QA and Audit
o Adsorption chromatography
o Maintenance and Inventory Control
 Requirement
 Can be used for
- Miscellaneous techniques/instruments
o Immunoassays
 Binding is related to what?
 labelling (purpose, with what,
improves)
 radioactive