3. ANALYTICAL TECHNIQUES Flashcards
(134 cards)
1
Q
Most often used to determine concentration of analytes
in Clinical Chemistry laboratory
A
Spectophotometry and Electrochemistry
2
Q
o Is described as photons of energy traveling in waves
o Can take several forms, the most recognizable being
light and radiant energy
A
Electromagnetic radiation
3
Q
o Is the linear distance between any two equivalent
points on a successive wave
o Unit used in the visible spectrum is nm
A
Wavelength
4
Q
The relationship between wavelength (λ) and energy (E) is
described by
A
Planck’s formula (E = hv)
5
Q
Planck’s constant
A
6.62 X 10 -27 erg sec
6
Q
o The number of oscillations of the waveform in a
second
A
Frequency
7
Q
Changes that may occur over period of time such as movement
Movement of waveform in a second
A
Oscillation
8
Q
The relationship of energy and wavelength is that the frequency is _________________ to wavelength
A
inversely proportional
9
Q
Visible region wavelength
A
400 - 700 nm
10
Q
Ultraviolet region wavelength
A
< 400 nm
11
Q
Infrared region wavelength
A
: > 700 nm
12
Q
states that the concentration of a substance is directly proportional to the amount of light absorbed or inversely proportional to the logarithm of the transmitted light.
A
Beer’s law
13
Q
Is used to measure the light transmitted by a solution to determine the concentration of the light-absorbing substance in the solution.
A
Spectrophotometer
14
Q
Components of a spectrophotometer
A
Light Source
Monochromator
Sample Cell or Cuvet
Photodetector
Meter or read-out device
15
Q
Provides polychromatic light
A
Light Source
16
Q
light source provides light
at several wavelength
A
Polychromatic light
17
Q
light source that provides visible and near-infrared regions continuum type
A
Incandescent tungsten or tungsten-iodide lamp –
18
Q
light source that provides UV
region continuum type
A
Deuterium lamp and mercury arc lamp
19
Q
2 types of Light Source
A
Continuum
Line
20
Q
o Wide applications in the laboratory
o Emits limited number of discrete lines or
bands of radiation
o Examples: Tungsten (visible region) ,
deuterium (UV region), xenon (visible and
UV regions)
A
Continuum
21
Q
o Emits a few discrete lines or bands of
radiation
o Examples:
Mercury and sodium vapor lamps – UV
and visible regions
Hollow cathode lamp - atomic absorption
spectroscopy / spectrophotometry
A
Line
22
Q
Light source that provides visible and
UV regions continuum type
A
xenon
23
Q
Light source that provides UV and visible regions line type
A
Mercury and sodium vapor lamps
24
Q
Line type light source for atomic absorption spectroscopy / spectrophotometry
A
Hollow cathode lamp
25
Isolates individual wavelengths of light
Monochromator
26
Characteristics of monochromators
Nominal wavelength
Spectral bandwidth (or FWHM)
Bandpass
27
Represents nanometers in peak
transmittance
Nominal wavelength
28
Range of wavelengths about ½ peak
transmittance
Spectral bandwidth (or FWHM)
29
Total range of wavelengths, as seen in the chart
Bandpass
30
Types of monochromators
Filters
Prism
Diffraction gratings
31
Simple, inexpensive, and useful monochromator that requires you to determine the analyte of interest and set the wavelength at a specific point
Filters
32
example of filters
Interference and absorption filters
33
Monochromator that can be rotated, allowing only the desired
wavelength to pass through an exit slit
Prism
34
Most commonly used monochromator ; contain parallel grooves
Diffraction gratings
35
may be round or square and must be made of material that is transparent to radiation
used to hold samples; path length is 1 cm (general)
Sample Cell or Cuvet
36
Types of cuvet
Plastic cuvet
Fused silica or quarts
Alumina-silicate glass
37
cuvet for UV region
Fused silica or quarts
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cuvet for 350-2000 nm
Alumina-silicate glass
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cuvet for visible region
Plastic cuvet
40
Converts the transmitted radiant energy into an equivalent amount of electrical energy
Photodetector
41
Types of photodetector
Barrier-layer cell or photocell
Phototube
Photomultiplier tube (PMT)
Photodiode
42
Photodetector
o Least expensive; temperature sensitive
o Composed of selenium on a plate of iron
o Used mainly in filter photometers
Barrier-layer cell or photocell
43
Photodetector
o Contains cathode and anode enclosed in a glass tube
o Has photosensitive material that gives off electrons when light energy strikes it
Phototube
44
Photodetector
o Most common type
o 200 times more sensitive than the phototube
o Highly sensitive to UV and visible radiation
Photomultiplier tube (PMT)
45
Photodetector
o Not as sensitive as PM tube but with
excellent linearity and speed
Excellent linearity: beam of light that
strikes the photodetector reflects the
amount of analyte or concentration of
analyte present in the sample
Speed: concentration is immediately
read out on the read out device or the
meter because it can immediately
transmit the radiant energy into electrical energy
Photodiode
46
Displays output of the detection system
Meter or read-out device
47
Examples of Meter or read-out device
Digital meters, d’Arsonval meters, recorders, light-emitting diodes (LEDs), cathode-ray tubes (CRTs), and liquid crystal displays (LCDs).
48
uses 2 photodetectors 2 sample cuvets and 2 photodetectors
Double beam in space
49
– uses 1 photodetector; chopper is used to pass the monochromatic
radiation through the sample cuvet and then to the reference cuvet
Double-beam in time
50
a device that rotates or breaks up
radiation beams
Chopper
51
Wavelength or photometric accuracy
Implies that a photometer is measuring at the wavelength that it is set to
Can be checked by Special glass-type optical filters like
– didymium glass (600 nm); holmium oxide (360 nm)
52
Closeness of a measured value to its true or target value
Accuracy
53
Using glass filters or solutions that have known absorbance values for a specific wavelength
Absorbance check
54
The ability of a photometric system to yield a linear relationship between the radiant power incident upon its detector and the concentration
Linearity
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Any light that impinges upon the detector that does not originate from a polychromatic light source
Stray light
56
Measures concentration by detecting the absorption of electromagnetic radiation by atoms rather than by
molecule
Atomic Absorption Spectrophotometer
57
Atomic Absorption Spectrophotometer light source
hollow-cathode lamp; electrodeless
discharge lamp
58
In AAS the use of chopper comes in the form of?
Flame
59
AAS Photodetector
PM tube
60
Application of AAS
to measure concentrations of trace metals [lead, mercury, cadmium]
61
o Uses an electric furnace to break chemical bonds (electrothermal atomization)
o It does not require burner to produce flame but requires electric current
o Sample is atomized still but does not require burner
Flameless AAS
62
o Measures light emitted by excited atoms
o No longer routinely used
FLAME PHOTOMETRY
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Light source of flame photometer
Flame
64
Internal standard of flame photometer
Lithium
Cesium
65
Where is flame photometer used
To measure concentrations of sodium, potassium, and lithium
66
measure the concentrations of
solutions that contain fluorescing molecules
Filter fluorometers
67
Light source of filter fluorometers
mercury (filter fluorometers)
68
Light source of spectrofluorometers
xenon arc
69
Disadvantages of fluorometry
Sensitive to environmental changes
70
Is different from fluorescence in that no excitation radiation is required and no monochromators are needed
CHEMILUMINESCENCE
71
Chemiluminescence reactions are oxidation reactions of
___________________ characterized
by a rapid increase in intensity of emitted light followed by a gradual breakdown/decay
luminol, acridinium esters, and dioxetanes
72
Advantages of chemiluminescence
Subpicomolar detection limits, speed, ease of use, and simple instrumentation
73
Disadvantage of chemiluminescence
Impurities can degrade sensitivity and specificity
74
is the production of electromagnetic
radiation when a chemical reaction yields an excited products/excited immediate product, in short, it is the
emission of light as a result of chemical reaction
Chemiluminiscence
75
TURBIDITY AND NEPHELOMETRY Applications:
measurement of antigen–antibody reactions,
prealbumin, and other serum proteins
76
is the measurement of the light scattered by a particulate solution
Nephelometry
77
o 3 types of light scatters:
Rayleigh theory
Mie theory
Rayleigh-Debye theory
78
If the wavelength (λ) of light > the
diameter (d) of the particle (d < 0.1λ), the light scatter is symmetrical around the particle. Minimum light scatter occurs at 90 degrees to the incident beam.
Rayleigh theory
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- If the wavelength of light < the particle
diameter (d > 0.1λ), then the light scatters forward.
Mie theory
80
If the wavelength of light is approximately the same as the particle size, more light scatters in the forward direction than in other directions.
Rayleigh–Debye theory
81
Nephelometer Components
Light Source
Collimator / lens
Monochromator
Sample cuvet
Photodetector
82
Measurements are made with a spectrophotometer to determine the concentration of particulate matter in a
sample
o Determines the amount of light blocked by a suspension of particles
Turbidimetry
83
Applications of turbidimetry
microbiology analyzers,
coagulation analyzers
quantify protein concentration
in biologic fluids such as urine and CSF.
84
Involves measurement of the current or voltage generated by the activity of specific ions
ELECTROCHEMISTRY
85
Measurement of potential (voltage) between two electrodes in a solution
POTENTIOMETRY
86
Tow electrodes in POTENTIOMETRY
Reference electrode
Indicator electrode
87
– electrode with a constant voltage
o Calomel and Silver/silver chloride
Reference electrode
88
– measuring electrode
Indicator electrode –
89
o Is very sensitive and selective for the ion it measures
o Consists of a membrane separating a reference solution and a reference electrode from the solution to be analyzed
Ion-Selective Electrode (ISE)
90
ISE Membrane for sodium
Glass aluminum silicate
91
ISE Membrane for potassium
Valinomycin gel
92
ISE Membrane for calcium and
lithium
Organic liquid ion exchangers
93
ISE Membrane for carbon dioxide and ammonia
Gas electrodes
94
ISE Membrane for urease and glucose oxidase
Enzyme electrodes
95
2 types of ISE
Direct: it does not require sample dilution
Indirect: the sample requires dilution before the analysis phase
96
o Used to measure hydrogen ion activity
o Buffer: has known hydrogen ion concentration
pH electrode
97
Internal reference electrode of pH electrodes
silver/silver chloride
98
External reference electrode of pH electrodes
Calomel electrode
99
o A pH electrode within a plastic jacket (has sodium bicarbonate buffer and gas-permeable membrane)
o When whole blood contains CO2 contact with gas permeable membrane, it mixes with the buffer
pCO2
100
Measures the quantity of electricity (in coulombs) needed to convert an analyte to a different oxidation state
COULOMETRY
101
COULOMETRY follows what equation
Faraday’s equation
102
COULOMETRY Application:
to measure chloride ion in serum, plasma, CSF, and sweat samples
103
Is the measurement of the current flow produced by an oxidation–reduction reaction
AMPELOMETRY
104
AMPELOMETRY application
to measure chloride ion in serum, plasma, CSF, and sweat samples; pO2 electrode in blood gas analyzers
105
Is a method in which a potential is applied to an electrochemical cell and the resulting current is measured
VOLTAMMETRY
106
used to measure heavy metals such as lead
Anodic stripping voltammetry
107
is the measurement of the osmolality of an aqueous solution such as serum, plasma, or urine
OSMOMETRY
108
Osmotically active particles
glucose, urea nitrogen,
sodium
109
is the separation of charged
compounds based on their electrical charge
Electrophoresis
110
2 TYPES OF ELECTROPHORESIS
Iontophoresis
Zone Electrophoresis
111
Migration of small ions
Very much associated with Cystic fibrosis
Sweat test
Iontophoresis
112
o Migration of charged macromolecules in a porous support medium
o DNA proteins
o Lipoproteins
Zone Electrophoresis
113
o A substance that can have a negative, zero or a positive charge depending on conditions
o If the pH is basic, the sample could have a positive or negative charge
o There are also case that if the pH is acidic, the sample can be transformed from being ampholyte (can either
be a positive, negative or zero charge)
Amphotheric
114
Negatively charge ion
Anion
115
Postively charged ion
Cation
116
Both positively and negatively charge at a same time
Zwitterion
117
FACTORS AFFECTING MOBILITY OF PARTICLES
Net charge of the particle
Size and shape of the particle
Strength of the electric field
Chemical and physical properties of the medium
Electrophoretic temperature
COMPONENT
118
COMPONENTS of ELECTROPHORESIS
POWER SUPPLY
BUFFER
SUPPORT MEDIUM
SAMPLE
DETECTING SYSTEM
119
Supplies constant current or voltage in the system.
This drives the molecules through the support medium
Driving force
POWER SUPPLY
120
Used to provide ions that carry a current and to maintain the pH at a relatively constant value
BUFFER
121
A network of interacting fibres or a polymer that is solid but traps large amount of solvent in pores or channel inside
SUPPORT MEDIUM
122
SUPPORT MEDIUM types
Cellulose Acetate
Agarose Gel
Polyacrilamide Gel
123
Separates serum proteins into 5 bands
Isoelectric focusing
Very much famous and very much useful in performing electrophoresis for protein and is usually support medium of choice would be
cellulose acetate
124
Used as a purified fraction of agar
From red algae
It is neutral and, thus, does not produce
electroendosmosis
Separates proteins into 10 – 15 bands
Agarose Gel
125
Must not interact with the analyte: what you want is for the analyte to just patch through the support media and you do not want your support media to be affecting your analyte which is called
electroendosmosis
126
Separation of protein base on charge and molecular
size
Separates serum proteins into 20 or more fractions
Isoenzyme determination
Polyacrilamide Gel
127
Is a physical technique that separates mixtures into individual components
Used to separate complex mixtures on the basis of different physical interactions between the individual compounds and the stationary phase of the system
CHROMATOGRAPHY
128
2 CATEGORIES
Planar Chromatography
Column Chromatography
129
Fractionation of sugar and amino acid
Paper chromatography
130
Paper chromatography Sorbent
Whatman paper
131
Uses pressure for fast separation of
thermolabile substances
HPLC – High – Performance Liquid
Chromatography
132
Fragmentation and ionization of molecules using a suitable
source of energy
MASS SPECTROPHOTOMETER
133
MASS SPECTROPHOTOMETER Has two distinct portion
fragmentation and ionization of
molecules
134
Is an analytical technique in which chemical compounds are
ionized into charged molecules and ratio of their mass to
charged is measured (m/z)
o Quadropole Mass Analyszers
o Ion Trap Analyzers
o Time of Flight Analyzers
