PRELIM LAB 2: INSTRUMENTATION Flashcards
A process by which it MEASURE THE AMOUNT OF LIGHT THAT CAN PASS THROUGH A SOLUTION to determine the concentration of the light-absorbing substance in the solution
SPECTROPHOTOMETRY
“Absorbance is DIRECTLY PROPORTIONAL to the concentration of the solution”
__________ LAW
BEER - LAMBERT LAW
amount of light absorbed as incident light passes through a sample
ABSORBANCE
proportionality constant/a compound that is the measure of the absorption of radiant energy
MOLAR REABSORPTIVITY
SPECTROPHOTOMETRY COMPONENTS:
Emits a broad spectrum of light
LIGHT SOURCE
MOST COMMON LIGHT SOURCE
INCANDESCENT TUNGSTEN/ TUNGSTEN - IODINE LAMP
visible & UV, do not heat up the instrument, reduce warm up time
XENON FLASH
SPECTROPHOTOMETRY COMPONENTS:
Isolation of individual wavelengths of light
MONOCHROMATOR
SPECTROPHOTOMETRY COMPONENTS:
Causes different wavelength of light to be dispersed in different angles
DISPERSION DEVICE
DISPERSION DEVICE TYPES:
- (glass = visible range,3x dispersion than quartz, quartz = visible light & UV)
- Transparent optical elements with flat/curved surfaces that can refract light
- As light passes through it, it is REFRACTED based on the varying refractive indices of different wavelength
PRISM
DISPERSION DEVICE TYPES:
- Absorbs/reflects certain wavelengths & transmitting other wavelengths
FILTER
ABSORB short wavelengths, transmit long wavelengths
ABSORPTION FILTERS
SELECTIVELY TRANSMIT/reflect a certain range of wavelengths; with dielectric films
INTERFERENCE FILTERS
DISPERSION DEVICE TYPES:
(most commonly used)
- Optical components with closely parallel lines/grooves/ruling on its surface (15,000 or 30,000 per inch)
- When the light strikes the gratings, it undergoes DIFFRACTION
- Wavelengths bend as they pass a sharp corner
DIFFRACTION GRATINGS
SPECTROPHOTOMETRY COMPONENTS:
- Small, transparent vessel used to hold a liquid sample to be analyzed
- Scratches may scatter light
- Material:
Glass
Quartz
Plastic
- Shapes:
Rectangular
Cylindrical
SAMPLE CELL (CUVETTE)
SPECTROPHOTOMETRY COMPONENTS:
- Convert the transmitted radiant energy into an equivalent amount of electrical energy, which can be measured & analyzed
- Types:
Barrier layer/photocell
Phototube
Photomultiplier tube (PMT)
PHOTODETECTORS
SPECTROPHOTOMETRY CLASSES:
- Only 1 light path that passes through both the reference and sample cells
- Alternates between measuring the reference (blank) & the sample
SINGLE BEAM
SPECTROPHOTOMETRY CLASSES:
- 2 separate light paths (for sample & reference)
- Measure blank & the sample simultaneously
DOUBLE BEAM
1 photodetector, with chopper/splitter
DOUBLE BEAM IN TIME
2 photodetectors (for sample & reference
DOUBLE BEAM IN SPACE
- Determines the concentration via EXCITATION of the substance via electromagnetic radiation
PRINCIPLE:
- A substance absorbs light energy at a certain wavelength (excitation) and then re-emits light at a longer wavelength (emission)
FLUORESCENCE
FLUORESCENCE COMPONENTS:
- Provides excitation light that is used to illuminate the sample
- Gas discharge lamps (xenon & mercury arc)
LIGHT SOURCE
FLUORESCENCE INTENSITY of fluorophore is reduced (factors: pH, temperature, contamination, UV light changes)
QUENCHING
FLUORESCENCE COMPONENTS:
- CONTROL THE INTENSITY OF EXCITATION LIGHT and optimize the measurement conditions
ATTENUATOR
FLUORESCENCE COMPONENTS:
USED TO SELECT SPECIFIC EXCITATION WAVELENGTH, ensuring that only light of desired wavelength reaches the sample.
PRIMARY FILTER (EXCITATION MONOCHROMATIC/FILTER)
FLUORESCENCE COMPONENTS:
Holds the sample being analyzed (cuvette)
SAMPLE HOLDER
FLUORESCENCE COMPONENTS:
Used to select a specific wavelength
SECONDARY FILTER (EMISSION MONOCHROMATIC/FILTER)
FLUORESCENCE COMPONENTS:
- Capture and measures the intensity of the emitted fluorescence
DETECTOR
- Excitation of the substance in the sample is caused by a CHEMICAL, BIOCHEMICAL or , ELECTROCHEMICAL reaction
- Emission is measured by LUMINOMETERS
-No monochromator or light source
LUMINESCENCE
Emission of light resulting from chemical reaction wherein chemical energy is directly converted into light energy
CHEMILUMINESCENCE
Chemiluminescence found in biological (organ) systems (certain fungi, bacteria, jellyfish, fireflies)
BIOLUMINESCENCE
Luminescence resulting from electrochemical reaction
ELECTROCHEMILUMINESCENCE
Most commonly used electrochemiluminescence label
Ruthenium (Ru2+) & tris (bipyridyl) chelate:
are ULTRASENSITIVE with WIDE DYNAMICS RANGES, & is utilized in molecular biology laboratory
CHEMILUMINESCENCE ASSAYS
DEPENDS ON WAVELENGTH & particle size & measurements have been applied to immunoassays of specific proteins & haptens
LIGHT SCATTERING DEVICES
- Determines the AMOUNT OF LIGHT BLOCKED (light reduction) by a particulate matter in a turbid solution
- When particles are present in the sample, they scatter & absorb light, reducing the amount of light that reaches the detector
- Applications: protein measurements (CSF & urine); bacterial growth detection in broths, AST (antimicrobial susceptibility) in broths, detection of clots
TURBIDIMETRY
Determines the AMOUNT OF SCATTERED LIGHT (at an angle 90 degrees from the indecent light) by a particular matter suspend in a turbid solution
Physical phenomenon that results from light particle interactions in a solution
Application: antigen-antibody complexes (proteins)
NEPHELOMETRY
detector is positioned IN LINE with the incident light beam
Turbidimetry
detector is placed PERPENDICULAR to the incident light beam
Nephelometry
- Used to identify presence of relatively small number of metal ions in a compound
- Heat of the flame converts the metal ions into atoms which become excited and emit visible light
FLAME TESTS
- Measures the light emitted by a single atom burned in a flame
- Principle: excitation of electrons from lower to higher energy state
- Cuvette & light source: flame
For the determination of sodium, potassium, lithium, & other metal/ions
FLAME EMISSION PHOTOMETERS
SODIUM
589 nm, yellow
potassium
66nm, violet
lithium
670 nm , red
- Based on INTERACTION OF RADIANT ENERGY with suitably excited atoms/molecules
- For determination of structure, ID of samples, quantification, diagnosis
- Employed in hematology as flow cytometers for the differential analysis of WBCs
Side scatter: complexity/granularity of cell
Forward scatter: size of cell
LASER SPECTROPHOTOMETRY
- Measures the light absorbed by atoms dissociated by heat
Principle: an element is not excited by merely dissociated from its chemical bond & placed in unionized, unexcited ground state
Light source: hollow - cathode lamp
Cuvette: flame
Very precise & for detection of metals and heavy metals
ATOMIC ABSORPTION SPECTROPHOTOMETRY
ELECTROCHEMICAL DEVICES:
- Measures the CONCENTRATION OF SPECIFIC IONS in a solution by responding to that particular ion
ION SELECTIVE ELECTRODES
ELECTROCHEMICAL DEVICES:
- Measures the acidity/alkalinity (pH) of a solution
pH ELECTRODES
ELECTROCHEMICAL DEVICES:
- Detects the presence and concentration of specific gasses in a given environment
GAS SENSING ELECTRODES
ELECTROCHEMICAL DEVICES:
- Migration of charged solutes/particles in an electrical field (macromolecules of interest are proteins, nucleic acids)
- Rate of movement is influenced by the size and charge of the particles, leading to their separation based on these characteristics
ELECTROPHORESIS
ELECTROCHEMICAL DEVICES:
- Measure the concentration of solute particles in a solution (osmotic concentration)
OSMOMETRY
