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MOL. BIO LAB > PRELIM LAB 2: INSTRUMENTATION > Flashcards

PRELIM LAB 2: INSTRUMENTATION Flashcards

1
Q

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

A

SPECTROPHOTOMETRY

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2
Q

“Absorbance is DIRECTLY PROPORTIONAL to the concentration of the solution”

__________ LAW

A

BEER - LAMBERT LAW

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3
Q

amount of light absorbed as incident light passes through a sample

A

ABSORBANCE

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4
Q

proportionality constant/a compound that is the measure of the absorption of radiant energy

A

MOLAR REABSORPTIVITY

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5
Q

SPECTROPHOTOMETRY COMPONENTS:
Emits a broad spectrum of light

A

LIGHT SOURCE

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6
Q

MOST COMMON LIGHT SOURCE

A

INCANDESCENT TUNGSTEN/ TUNGSTEN - IODINE LAMP

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7
Q

visible & UV, do not heat up the instrument, reduce warm up time

A

XENON FLASH

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8
Q

SPECTROPHOTOMETRY COMPONENTS:
Isolation of individual wavelengths of light

A

MONOCHROMATOR

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9
Q

SPECTROPHOTOMETRY COMPONENTS:
Causes different wavelength of light to be dispersed in different angles

A

DISPERSION DEVICE

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10
Q

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

A

PRISM

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11
Q

DISPERSION DEVICE TYPES:
- Absorbs/reflects certain wavelengths & transmitting other wavelengths

A

FILTER

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12
Q

ABSORB short wavelengths, transmit long wavelengths

A

ABSORPTION FILTERS

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13
Q

SELECTIVELY TRANSMIT/reflect a certain range of wavelengths; with dielectric films

A

INTERFERENCE FILTERS

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14
Q

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

A

DIFFRACTION GRATINGS

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15
Q

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

A

SAMPLE CELL (CUVETTE)

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16
Q

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)

A

PHOTODETECTORS

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17
Q

SPECTROPHOTOMETRY CLASSES:
- Only 1 light path that passes through both the reference and sample cells
- Alternates between measuring the reference (blank) & the sample

A

SINGLE BEAM

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18
Q

SPECTROPHOTOMETRY CLASSES:
- 2 separate light paths (for sample & reference)
- Measure blank & the sample simultaneously

A

DOUBLE BEAM

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19
Q

1 photodetector, with chopper/splitter

A

DOUBLE BEAM IN TIME

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20
Q

2 photodetectors (for sample & reference

A

DOUBLE BEAM IN SPACE

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21
Q
  • 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)

A

FLUORESCENCE

22
Q

FLUORESCENCE COMPONENTS:
- Provides excitation light that is used to illuminate the sample
- Gas discharge lamps (xenon & mercury arc)

A

LIGHT SOURCE

23
Q

FLUORESCENCE INTENSITY of fluorophore is reduced (factors: pH, temperature, contamination, UV light changes)

24
Q

FLUORESCENCE COMPONENTS:
- CONTROL THE INTENSITY OF EXCITATION LIGHT and optimize the measurement conditions

A

ATTENUATOR

25
FLUORESCENCE COMPONENTS: USED TO SELECT SPECIFIC EXCITATION WAVELENGTH, ensuring that only light of desired wavelength reaches the sample.
PRIMARY FILTER (EXCITATION MONOCHROMATIC/FILTER)
26
FLUORESCENCE COMPONENTS: Holds the sample being analyzed (cuvette)
SAMPLE HOLDER
27
FLUORESCENCE COMPONENTS: Used to select a specific wavelength
SECONDARY FILTER (EMISSION MONOCHROMATIC/FILTER)
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FLUORESCENCE COMPONENTS: - Capture and measures the intensity of the emitted fluorescence
DETECTOR
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- 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
30
Emission of light resulting from chemical reaction wherein chemical energy is directly converted into light energy
CHEMILUMINESCENCE
31
Chemiluminescence found in biological (organ) systems (certain fungi, bacteria, jellyfish, fireflies)
BIOLUMINESCENCE
32
Luminescence resulting from electrochemical reaction
ELECTROCHEMILUMINESCENCE
33
Most commonly used electrochemiluminescence label
Ruthenium (Ru2+) & tris (bipyridyl) chelate:
34
are ULTRASENSITIVE with WIDE DYNAMICS RANGES, & is utilized in molecular biology laboratory
CHEMILUMINESCENCE ASSAYS
35
DEPENDS ON WAVELENGTH & particle size & measurements have been applied to immunoassays of specific proteins & haptens
LIGHT SCATTERING DEVICES
36
- 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
37
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
38
detector is positioned IN LINE with the incident light beam
Turbidimetry
39
detector is placed PERPENDICULAR to the incident light beam
Nephelometry
40
- 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
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- 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
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SODIUM
589 nm, yellow
43
potassium
66nm, violet
44
lithium
670 nm , red
45
- 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
46
- 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
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ELECTROCHEMICAL DEVICES: - Measures the CONCENTRATION OF SPECIFIC IONS in a solution by responding to that particular ion
ION SELECTIVE ELECTRODES
48
ELECTROCHEMICAL DEVICES: - Measures the acidity/alkalinity (pH) of a solution
pH ELECTRODES
49
ELECTROCHEMICAL DEVICES: - Detects the presence and concentration of specific gasses in a given environment
GAS SENSING ELECTRODES
50
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
51
ELECTROCHEMICAL DEVICES: - Measure the concentration of solute particles in a solution (osmotic concentration)
OSMOMETRY

MOL. BIO LAB (9 decks)

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