ANALYTICAL METHODS Flashcards
1
Q
Distance between two successive peaks and expressed in terms of nanometer (nm)
A
Wavelength
2
Q
Infrared region
A
> 700 nm
3
Q
Visible spectrum
A
400-700 nm
4
Q
Ultraviolet region
A
<400 nm
5
Q
Wavelength is ________________ to Frequency and Energy
A
INVERSELY related
6
Q
Measurement of light intensity in a narrower wavelength
A
Spectrophotometric measurement
7
Q
Measurement of light intensity
A
Photometric measurement
8
Q
Involves measurement of light transmitted by a solution to determine the concentration of the light-absorbing substances in the solution
A
Spectrophotometry
9
Q
Simplest type of absorption spectrometer
Designed to make on measurement at a time at one specified wavelength
A
Single beam spectrophotometer
10
Q
Instrument that splits the monochromatic light into two components:
1 beam passes through the sample, 1 beam passes through a reference solution or blank
A
Double beam spectrophotometer
11
Q
Function of additional beam
A
Corrects for variation in light source intensity
12
Q
Provides polychromatic light and must generate sufficient radiant energy or power to measure the analyte of interest
A
Light or Radiant Source
13
Q
Emits radiation that changes in intensity;
Widely used in the labroatory
Type of Light source
A
Continuum source
14
Q
Commonly used light source in the visibule and near infrared region
Example of Continuum source
A
Tungsten light bulb
15
Q
Routinely used to provide UV radiation
Example of Continuum source
A
Deuterium lamp
16
Q
Produces a continous source of radiation, which covers both the UV & Visible range
Example of Continuum source
A
Xenon discharge lamp
17
Q
Emits limited radiation and wavelength
Type of Light Source
A
Line Source
18
Q
Also used as light sources for spectrophotometry
A
LASER
Light Amplification by Stimulated Emission of Radiation
19
Q
Tungsten Light Bulb
Mercury Arc
A
Visible Region
20
Q
Deuterium lamp
Mercury lamp
Xenon lamp
Hydrogen lamp
A
Ultraviolet Region
21
Q
Merst glower
Globar (Silicone carbide)
A
Infrared Region
22
Q
Minimizes unwanted or stray light and prevents the entrance of scattered light into the monochromator system
A
Entrance Slit
23
Q
Any wavelengths outside the band transmitted by the monochromator
Does not originate from the polychromatic light source
Causes Absorbance error
A
Stray light
Most common cause of loss of linearity at high-analyte concentration
24
Q
Any wavelengths outside the band transmitted by the monochromator
Does not originate from the polychromatic light source
Causes Absorbance error
A
Stray light
Most common cause of loss of linearity at high-analyte concentration
25
Any wavelengths outside the band transmitted by the monochromator
Does not originate from the polychromatic light source
Causes Absorbance error
Stray light
## Footnote
Most common cause of loss of linearity at high-analyte concentration
26
Isolates specific or individual wavelength of light
Monochromator
27
Most commonly used Monochromator
Made by cutting grooves or slits into an aluminized surface of a flat piece of crown glass
Diffraction gratings
28
Controls the width of light beam
Allows only a narrow fraction of the spectrum to reach the sample cuvette
Exit Slit
29
Holds the solution whose concentration is to be measured
Cuvette
| Aka Absorption Cell/ Analytical Cell/ Sample Cell
30
Holds the solution whose concentration is to be measured
Cuvette
| Aka Absorption Cell/ Analytical Cell/ Sample Cell
31
Most commonly used cuvette
| Can be used in 350-2000 nm
Alumina silica glass
32
Used for measurement of solution requiring visible and ultraviolet spectra
| Kinds of Cuvets
Quartz/ Plastic
33
Used for measurement of solution requiring visible and ultraviolet spectra
| Kinds of Cuvets
Quartz/ Plastic
34
Detects and converts ***transmitted light into photoelectric energy*** Detects the amount of light that passes through the sample in cuvet
Photodetector
35
Simplest detector; Least expensive; Temperature sensitive
Used for detecting and measuring radiation in the visible region
| Kinds of Detector
Barrier layer cell/ Photocell/ Photovoltaic cell
36
Contains Cathode and Anode enclosed in a glass case
| Kinds of Detector
Phototube
37
Most commonly used detector
Measures visible & UV regions
| Kinds of Detector
## Footnote
Should never be exposed to room light because it will burn out
Photomultiplier Tube
38
Not as sensitive as PMT but with excellent linearity
Most useful as a simultaneous multichannel detector
| Kinds of Detector
Photodiode
39
Displays output of the detection system
Meter or Read-out device
40
Mathematically establishes the relationship between concentration and absorbance
Beer's Law
41
Concentration is ____ to Absorbed light
DIRECTLY
42
Concentration is ____ to Transmitted light
INVERSELY
43
Amount of light absorbed
Proportional to the INVERSE log of transmittance
Absorbance
44
Formula of Absorbance
A = abc
A = 2-log%T
45
350 - 430 nm
A: Violet
O: Yellow Blue
| A for Absorbed, O for Observed
46
431 - 475 nm
A: Blue
O: Yellow
47
476 - 495 nm
A: Green-blue
O: Orange
48
496 - 505 nm
Blue-green
Red
49
506 - 555 nm
Green
Purple
50
556 - 575 nm
Yellow-Green
Violet
51
576 - 600 nm
Yellow
Blue
52
601 - 650 nm
Orange
Green-Blue
53
651 - 700 nm
Red
Blue-green
54
Blank contains serum but without the reagent to complete the assay
Blanking technique
55
Corrects absorbance caused by the color of the reagent
Reagent blank
56
Measures absorbance of the sample and reagent in the absence of the endproduct
Sample Blank
57
Measures the light emitted by a single atom burned in a flame
Excitation of elevtrons from LOWER to HIGHER energy state
Flame Emission Photometry
| Measurement of excited ions: Na & K
58
Measures the light absorbed by atoms dissociated by heat
More sensitive than FEP
Atomic Absorption Spectrophotometry (AAS)
| Measurement of unexcited trace metals: Ca & Mg
59
Unknown sample is made to react with a known solution in presence of an indicator
Volumetric or Titrimetric
60
Schales and Schales method
| Example of Volumetric
Chloride test
61
EDTA Titration method
| Example of Volumetric
Calcium test
62
Measuring large particles and bacterial suspensions
Determines amount of light BLOCKED by a particulate matter
Turbidimetry
| Depends on specimen concentration and particle size
63
Measuring amount of antigen-antibody complexes
Determines amount of SCATTERED light by a particulate matter
Nephelometry
| Depens on wavelength and particle size
64
Measuring amount of antigen-antibody complexes
Determines amount of SCATTERED light by a particulate matter
Nephelometry
| Depens on wavelength and particle size
65
Migration of charged particles in an electric field
Separates protein on the basis of their electric charge densities
Electrophoresis
| Protein are negatively vharged (anions) and move towards the anode
66
Has a net charge that can be either positive or negative depending on pH conditions
Amphoteric
67
Migration of small charged ions
Iontophoresis
68
Migration of charged MACROmolecules
Zone Electrophoresis
69
Factors affecting rate of Migration
1. Net electric charge
2. Size and Shape
3. Electric field strength
4. Nature of supporting medium
5. Temperature
70
Separates by molecular size
| type of supporting media
Cellulose Acetate
71
Separates by electrical charge
Does not bind protein
| type of supporting media
Agarose gel
72
Separates on basis of charge and molecular size
Separates porteins into 20 fracrions
Used to study Isoenzymes
| type of supporting media
Polyacrylamide gel
73
Stains for Visualization of Fractions
1. Amido black
2. Ponceau S
3. Oil Red O
4. Sudan black
5. Fat Red 7B
6. Coomassie Blue
7. Gold/ Silver stain
74
Measures the absorbance of stain - concentration of the dye and protein fraction
Densitometer
75
Electrophoretic mobility is ____ to Net charge
Directly proportional
76
Electrophoretic mobility is ------- to molecular size and viscosity of supporting medium
Inversely Proportional
77
Separates molecules by migration through a pH gradient
Ideal for separating proteins of identical sizes but with different net charges
Isoelectric Focusing
| pH = pI
78
Advantages of Isoelectric focusing
1. Ability to resolve mixture of proteins
2. Detect isoenzymes of ACP, CK and ALP
3. Identify genetic variants of proteins such as alpha-1-antitrypsin
4. Detect CSF oligoclonal banding
79
Sample molecules are separated by electro-osmotic flow
(+) charged emerge early at the capillary outlet
(-) charged move towards the capillary outlet but at slower rate
Capillary Electrophoresis
80
Southern blot
DNA
81
Northern blot
RNA
82
Western blot
Proteins
83
Involves the separation of soluble components by specific differences in physical-chemical characteristics of the different constituents
Chromatography
84
Used for fractionation of sugar and amino acid
Paper Chromatography
85
Sorbent or Stationary phase in Paper chromatography
Whatman paper
86
Semiquantitative drug screening test
Sample components are identified by comparison with standards on the same plate
Thin Layer Chromatography
87
Sorbent in TLC
Thin plastic plates impregnated with a layer of silica gel or alumina
88
Rf value formula
Rf = distance leading edge of component moves/ total distance
89
Separation of steroid, barbiturates, blood, alcohol and lipids
Useful in compounds that are naturally volatile or can be easily converted into a volatile form
Gas Chromatography
90
Based on the fragmentation and ionization of molecules using a suitable source of energy
Detects structural information and determination of molecular weight
Mass Spectroscopy
91
Gold Standard for drug testing
Also used for xenobiotics, anabolic steroids and pesticides
Gas Chromatography-Mass Spectroscopy (GC-MS)
| Every drug has its own fingerprint pattern
92
Can detect 20 inborn errors of metabolism from a single blood spot
Tandem Mass Spectroscopy (MS/MS)
93
Most widely used liquid chromatography
Fractionation of drugs, hormones, lipids, carbohydrates and proteins
| Uses pressure for fast separations, controlled temp, in-line detectors
High Performace Liquid Chromatography (HPLC)
94
Detecting nonvolatile substances in body fluids
Confirm positive results from screening of illicited drugs
Used in TDM, Toxicology & studies of drug metabolites
Liquid Chromatography - Mass Spectroscopy (LC/MS)
95
Gel filtration
Separation of enzymes, antibodies and proteins
| Dextran & Agarose
Hydrophilic gel
96
Gel permeation
Separation of triglyceride & fatty acids
| Sephadex
Hydrophobic gel
97
Exchange of sample ions and mobile phase ions with the charged group of the stationary phase
Ion Exchange Chromatography
| Separation of AA, CHON & NA
98
Separation of compounds based on their partition between a liquid mobile phase and a liquid stationaty phase
| Separation of TDM and their metabolite
Partition Chromatography
99
Uses the so-called lock-and-key binding
| Separation of lipoproteins, CHO, and glycated hemoglobins & Antibodies
Affinity Chromatography
100
Based on differences between the adsorption and desroption of solutes at the surface of a solid particel
Adsorption Chromatography
101
Measures the amount of light intensity present over a zero background
Determines the amount of light emitted by a molecule after excitation by electromagnetic radiation
| 1000x more sensitive than Spectrometry
Fluorometry/
Molecular Luminescence Spectrophotometry
102
Major disadvantage of Fluorometry
pH and temperature changes, chemical contaminants and UVL changes
Quenching
103
Differs from fluorescence and phosphorescence in that the emission of light is created from a chemical or electrochemical reaction and not from absorption of electromagnetic energy
| More sensitive than fluorescence
Chemiluminescence
104
Measurement of osmolality of an aqueous solution
Based on measuring changes in colligative properties
Osmometry
105
Osmolality is ___ to Osmotic pressure and boiling point
DIRECTLY Proportional
106
Osmolality is ____ to Freezing point and Vapor Pressure
INVERSELY Proportional
| More commonly used: Freezing point
107
Most commonly used method for measuring the changes in colligative properties of a solution
Addition of solute molecules LOWERS the temperature at which a solution freezes
Freezing-point depression osmometry
108
Measurement of electrical potential due to the activity of free ions
Measurement of differences in voltage at a constant current
Follows the Nernst Equation
Potentiometry
| Use pH and pCO2
109
Reference Electrodes in potentiometry
Calomel &Silver-Silver Chloride
110
Electrochemical transducer capable of responding to one given ion
Very sensitive and selective for the ion it measures
Ion Selective Electrode
111
Measurement of amount of electricity at a fixed potential
Follows the Faraday's law
Use: Chloride test
Coulometry
112
Measurement of the current flow produced by an oxidation-reaction
Amperometry
| Use: pO2, Glucose, Chloride, Peroxidase
113
Measurement of current after which a potential is applied to an electrochemical cell
Voltammetry
