Analytical Methods Flashcards
1
Q
Energy are electromagnetic waves that are characterized by ___________ and ___________.
A
Frequency and Wavelength
2
Q
Wavelength
A
HORIZONTAL (width) distance from each wave
3
Q
Amplitude
A
VERTICAL (height) distance from each wave
4
Q
Frequency
A
Number of waves/vibrations
5
Q
Wavelength is ___________ proportional to Frequency and Energy
A
Inversely proportional
6
Q
Frequency is _________ proportional to Energy
A
Directly Proportional
7
Q
Wavelengths are expressed in _________.
A
Nanometer
8
Q
Increased Color Reaction: ________________
A
Increased analyte concentration
9
Q
Colorimetric Methods
A
Spectrophotometry
Flame Emission Photometry
Atomic absorption Spectrophotometry
10
Q
Principle in Spectrophotometry
A
Measurement of the LIGHT TRANSMITTED in the solution to determine the concentration of the LIGHT ABSORBED in the solution
11
Q
______________ is used to verify WAVELENGTH ACCURACY and for instruments with narrower bandpass.
A
Mercury Vapor Lamp
12
Q
Light Source
A
a.k.a Radiant energy source/Lamps
Provide limited radiation and polychromatic light
13
Q
Most common light source for VISIBLE-INFRARED light
A
Tungesten
14
Q
Routinely used for UV light
A
Deuterium
15
Q
Enumerate: Light Source (UV Light)
Mnemonics: MXHD
A
Mercury Arc
Xenon
Hydrogen Lamp
Deuterium
16
Q
Enumerate: Light Source (Infrared Light)
Mnemonics: MG
A
Merst Glower
Globar
17
Q
Number of Photodetectors in Double Beam in SPACE
A
2 photodetectors
18
Q
Number of Photodetectors in Double Beam in TIME
A
1 photodetector
19
Q
SIMPLEST type of absorption spectrophotometry
A
Single Beam spectrophotometry
20
Q
Single Beam Spectrophotometry
A
One measurement at a time
21
Q
Double Beam Spectrophotometry
A
Splits monochromator light into 2 components
22
Q
Emits radiation that CHANGES INTENSITY
A
Continuum Source
23
Q
Emits limited radiation in discrete lines
A
Line Source
24
Q
Monochromator
A
“Wavelength Selector” - Isolate specific or individual light
25
Most common and Best utilized monochromator
Diffraction Gratings
26
Synonyms of "Cuvette"
Sample Holder
Absorption Cell
Analytical Cell
Sample Cell
27
Cuvette
Holds the solution which concentration is to be measured
28
Most Common sample holder
Aluminum Silica Glass
29
Sample holder BEST utilized for UV
Quartz
30
Photodetector
Detect and Convert transmitted ight into PHOTOELECTRIC ENERGY
31
Most common Photodetector
Photomultiplier Tube (PMT)
32
Excellent in LINEARITY
Photodiode
33
Routine path length of of Cuvettes
1 cm
34
Range of Aluminum Silica Glass cuvettes to transmit light effectively
> 220 nm
35
What to do when results are NON-LINEAR?
1. Blank Technique
2. Reagent Blank
3. Sample Blank
36
Most common cause of loss of Linearity
Stray Lights
37
Principle in Flame Emission Spectrophotometry (FEP)
Light Emitted from EXCITATION of electrons from lower to higher energy source.
38
Flame Emission Photometry measures light emitted by a ___________ burned in a ________.
Single atom
Flame
39
FEP.
Light Source: ___________
Photodetector: _______
Light Source: Flame
Photodetector: Photocell
40
Indirect Internal standard Method (Recalls: March, 2019)
Sodium: ________
Potassium: _________
Calcium: __________
Lithium: __________
Sodium: Yellow
Potassium: Purple
Calcium: Orange
Lithium: Red
41
Quality Assurance in FEP. Flickering Lights indicates changes in the ____________ of the instrument.
Fuel reading
42
Preferred Internal Standard Method (FEP)
Lithium
Cesium
43
Principle: Atomic Absorption Spectrophotometry
Elements are not excited by merely dissociating from its chemical bond and are placed in an unexcited, unionized, ground state.
44
Atomic Absorption Spectrophotometry (AAS)
Light Source: ________
Photodetector: _________
Light Source: Hallow Cathode Lamp
Photodetector: PMT
45
Reference method for CALCIUM, MAGNESIUM, TRACE METALS.
Atomic Absorption Spectrophotometry
46
Principle: Volumetry/Titrimetry
Unknown sample is made to react with Known sample in the presence of INDICATOR
47
Methods using Volumetry
Schales and Schales: Chloride
EDTA Titration Method: Calcium
48
Principle: Turbidimetry
Measures the amount of light BLOCKED in a turbid solution.
49
Turbidimetry measures abundant _________ like proteins and bacteria.
Large Particles
50
Principle: Nephelometry
Measures the amount of Light SCATTERED in a turbid solution.
51
Analytical method used to measure antigen-antibody complex
Nephelometry
52
Measure angle from scattered light in cuvet
15 - 90 degrees
53
Principle: Fluorometry
Measures the amount of light emitted by a MOLECULE after EXCITATION through ELECTROMAGNETIC RADIATION.
54
Only method that uses 2 Monochromators
Fluorometry
55
Fluorometry measures light intensity in a _______ background.
Zero background
56
Purpose of 2 Monochromators in Fluorometry
1st - Primary/Excitation Filter: selects WL that is best absorbed
2nd - Secondary/Emission Filter: prevent incident light
57
Fluorometry
Light Source: ________
Photodetector: ________
Light Source: Mercury, Xenon, UV light
Photodetector: PMT/Phototube
58
What is Quenching?
Reduction or limitation of a particle in an excited state through;
pH changes
Temperatures
UV
Chemical changes
59
Recalls: September 2019
Increase Light: ______
Increase Temperature: _______
Increase QUENCHING: ________
Increased Absorbing Analyte: _______
Increase Light: Increase Fluorescence
Increase Temperature: Decrease Fluorescence
Increase QUENCHING: Decrease Fluorescence
Increased Absorbing Analyte: Decrease Fluorescence
60
Scoring of FITC: Apple green Fluorescence
2+
61
Difference of Maximum WL of excitation and Emitted Fluorescence or Max. WL of excitation - Emitted Fluorescence
Stokes Shift
62
Auramine Rhodamine: _________
Calcofluor White: ________
Acridine Orange: __________
Auramine Rhodamine: Mycobacterium
Calcofluor White: Fungi and Yeasts
Acridine Orange: DNA - nucleus, nucleoid, plasmids.
63
Principle: Chemiluminescence
Chemical/Electrochemical Reaction that yields an electronically EXCITED COMPOUND that emits light as it returns to its ground state.
64
Chemiluminescence ________ use Light source and Monochromator
DO NOT use light Source and Monochromator
65
Which is not part of Chemiluminescence?
1. Light Source
2. Photodetector
3. Monochromator
4. Cuvette
Light Source and Monochromator
Note: If there is BOTH Light Source and Monochromator are present in the choices, then Both is the answer. If BOTH is none from the choices, answer Monochromator only.
66
Main Components of Luminometer
Cuvette
Photodetector
Read-out device.
67
Principle: Osmometry
Measure of changes in Colligative Properties due to variation in solute/particle concentration.
68
Colligative Properties
Osmotic Pressure
Boiling Point
Freezing Point
Vapor Pressure
69
Increased Osmolarity (Recalls. September 2019, January 2021)
Increased - ________, _________
Decreased - __________, ________
Increased: Osmotic Pressure, Boiling Point
Decreased: Freezing Point, Vapor Pressure
70
Principle: Denisitometry
Measures the concentration of ABSORBED stain from PROTEIN FRACTIONS
71
Unit of Densitometry
Optical Density (Absorption)
72
OD 450
Cut-off detection limit - BILIRUBIN
73
OD 650
Cut-off detection limit - LAMELLAR BODIES
74
Principle: Chromatograhy
SEPARATION of soluble materials in a solution by their CHEMICAL and PHYSICAL differences.
75
Factors affecting Chromatraphy
Rate of Diffusion
Solubility
Size
Ionic Charges
76
Planar Form Chromatography: __________
Column Form Chromatography: __________
Planar Form Chromatography: diffusion of molecules in 2D PLANE SYSTEM
Column Form Chromatography: diffusion of molecules in 3D MULTIDIRECTIONAL SYSTEM
77
Paper Chromatography
Fractionation of _________ and _________.
Sorbent: ____________
Fractions: Sugar and Amino Acid
Sorbent: Whattman paper
78
Thin Layer Chromatography
Use: _____________
Sorbent: _____________
Use: TDM and SEMI-QUANTITATIVE DRUG SCREENING TEST
Sorbent: Thin Plastic plate (Impregnated with a layer of Silica gel/Alumina)
79
Used for VOLATILE compounds
Gas Chromatography
80
Gas Chromatography separates ____________, __________, ___________, __________.
Mnemonics: LABS
Lipids
Alcohol
Barbiturates
Steroids
81
Absorption of gases at SOLID phase surfaces
Gas Solid Chromatography
82
Gas liquid chromatography is the partition between gaseous _________ phase, and liquid ________ phase.
Gaseous Mobile Phase and Liquid Stationary Phase
83
Liquid Chromatography
Distribution of solute between liquid mobile phase and liquid stationary phase.
84
Most widely used Liquid Chromatography
High Performance Liquid Chromatography (HPLC)
85
Separate non-volatile substances
Liquid Chromatography - Mass Spectroscopy (LC-MS)
86
Principle of Mass Spectrophotometry
Fragmentation and Ionization of molecules
87
Gold Standard for Drug Testing
GC-MS
88
Gold Standard for New Born Screening
MS-MS
89
Migration based on ELECTRICAL CHARGE
Electrophoresis
90
Migration based on PHYSICAL and CHEMICAL properties
Chromatography
91
Migration based on pH GRADIENT
Isoelectric focusing
92
Migration based on ELECTRO-OSMOTIC FLOW (EOF)
Capillary Electrophoresis
93
Isoelectric Point
pH at which substances have a ZERO NET CHARGE (positively charged groups are equal to negatively charged groups)
94
Electro-osmotic flow (EOF)
A bulk flow resulting from excess positive ions at the inner capillary surface moving towards the cathode
95
Principle: Potentiometry
Measures electrical potential due to its activity of free ions
96
Change in Voltage: _______________
Change in Voltage: Analyte Activity
97
Potentiometry follows __________ equation
NERNST Equation
98
Potentiometry measures _______ and _______.
pH and pCO2
99
Two Types of ISE
Direct ISE - Undiluted Sample
Indirect ISE - Diluted Sample
100
ISE Membranes
Glass Aluminum Silicate: _________
Valinomycin: ____________
Organic Liquid: __________
Glass Membrane: _________
Glass Aluminum Silicate: Sodium
Valinomycin: Potassium
Organic Liquid: Calcium, Lithium
Glass Membrane: pH
101
ISE Electrodes
Clarke:
Severinghaus:
Glass Aluminum Silicate:
Valinomycin Gel:
Glass Membrane:
Clarke: pO2
Severinghaus: pCO2
Glass Aluminum Silicate: Na
Valinomycin Gel: K
Glass Membrane: pH
102
Principle: Coulometry
Measures electricity at FIXED POTENTIAL
103
The endpoint in Coulometry is detected by __________.
Amperometry
104
Measured by Coulometry
Chloride Tests - CSF, Sweat, Serum
105
Coulometry follows _________ Law
Faraday's Law
106
Measured by Amperometry
Mnemonics: PoGI C Papi
pO2
Glucose
Chloride
Peroxidase
107
Measurement of differences in current at a constant voltage
Polarography
108
Polarography uses __________ equation
Ilkovic Equation
109
Nernst equation: ___________
Faraday's Law: ___________
Ilkovic Equation: __________
Nernst equation: Potentiometry
Faraday's Law: Coulometry
Ilkovic Equation: Polarography`
110
Principle: Voltametry
Measurement of CURRENT AFTER A POTENTIAL IS APPLIED to an ELECTROCHEMICAL CELL.
111
Anodic Striping Voltametry is utilized for _______, and ________ studies.
Lead and Iron
112
Gene Mutation in Cystic Fibrosis
Chromosome 7
113
Cystic Fibrosis (MUCOVISCIDOSIS)
Defect:
Sweat Inducer:
Diagnostic Test:
Reference Method:
Defect: Trans-epithelial Chloride Transport
Sweat Inducer: Pilocarpine
Diagnostic Test: Sweat Test Coulometry
Reference Method: Gibson and Cooke Pilocarpine Iontophoresisi
114
Most Common
UV Lamp:
Visible - Infrared Lamp:
Cuvet:
Monochromator:
Photodetector:
Most Common
UV Lamp: Deuterium
Visible - Infrared Lamp: Tungsten
Cuvette: Aluminum Silica Glass
Monochromator: Diffraction Gratings
Photodetector: PMT
115
Most sensitive and specific method in Colorimetry
AAS
116
Quality assurance of Spectrophotometry
Wavelength Accuracy
Stray Light
Linearity
117
PMT is _____ times more sensitive than phototube
200x
118
Photocell vs. Phototube
Photocell - no external voltage
Phototube - require external voltage
119
Stray Light
Causes:
Major Effect:
Detected:
: light outside the band by the monochromator
Causes: Scratches, Dust
Major Effect: absorbance error
Detected: Cutoff Filters
120
Reference Method for Ca and Mg
AAS
121
Modulate the light source in AAS
Mechanical Rotating Chopper
122
Degree of Sensitivity of Analytical Method
Chemiluminescence > Fluorometry > Spectrophotometry
123
Advantages of Chemiluminescence
Subpicomolar detection limits
Speed (Flash type rxns. & Light measured within 10 secs.)
Ease of use
Simple Instrumentation
124
This technique contains the serum but without the reagent
Blanking Technique
125
Interferes mainly in blanking technique, thus increasing light scatter (turbidity)
Lipids
126
Verify Absorbance accuracy on Linearity
Neutral Density
Dichromate Soln
