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Clinical Chemistry 1 > B - CHAPTER V: ANALYTICAL METHODS & INSTRUMENTATION > Flashcards

B - CHAPTER V: ANALYTICAL METHODS & INSTRUMENTATION Flashcards

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1
Q

– radiant energy; photons of energy travelling in a wavelike manner

A

ELECTROMAGNETIC ENERGY

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2
Q
  • The shorter the [?], the higher the [?]
A

wavelength; electromagnetic energy

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3
Q

Types of Electromagnetic energies:

A
  1. Cosmic rays
  2. Gamma rays
  3. X-rays
  4. Visible
  5. Ultra-violet (UV)
  6. Infrared (IR)
  7. Radio, TV, microwave, etc.
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4
Q
  • distance between two peaks as the light travels in a wavelike manner
A

WAVELENGTH

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5
Q

wavelength is expressed in

A

nanometers (nm), angstroms (Å), and millimicron (mµ)

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6
Q
  • 1 nm = [?] = [?]
A

10 Å
1 mµ

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7
Q

Kinds of Wavelength:

A
  1. Visible spectra
  2. Non-visible spectra
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8
Q

= 340 nm – 700 nm

A

Visible spectra

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9
Q

(ultraviolet region)

A

= below 340 nm

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10
Q

(infrared region)

A

= above 700 nm

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11
Q

Kinds of Colorimetry:

A
  1. Visual Colorimetry
  2. Photoelectric Colorimetry
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12
Q

– relies on visual acuity to determine end-point

A

Visual Colorimetry

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13
Q

180 - 220
Short UV

A
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14
Q

220 - 340
Short UV

A
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15
Q

340 - 430
Visible

A

Violet
Yellow green

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16
Q

430 - 475
Visible

A

Blue
Yellow

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17
Q

475 - 495
Visible

A

Green blue
Orange

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18
Q

495 - 505
Visible

A

Blue green
Red

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19
Q

505 - 555
Visible

A

Green
Purple

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20
Q

555 - 575
Visible

A

Yellow green
Violet

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21
Q

575 - 600
Visible

A

Yellow
Blue

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22
Q

600 - 620
Visible

A

Orange
Green blue

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23
Q

620 - 700
Visible

A

Red
Blue green

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24
Q

– measurement of light intensity in a much narrower wavelength

A

SPECTROPHOTOMETRY

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25
Q

 Makes use of prisms and/or diffraction gratings as monochromator to disperse the radiant energy into a continuous spectrum & isolate radiant energy of desired wavelength

A

SPECTROPHOTOMETRY

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26
Q

– measurements of light intensity of multiple wavelength

A

FILTER PHOTOMETRY

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27
Q

 It makes use of filters (interference or transmission) to isolate part of the spectrum

A

FILTER PHOTOMETRY

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28
Q

Light passes through a [?] to provide a selection of the desired region of the spectrum to be used for measurements.

A

monochromator

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29
Q

are used to isolate a narrow beam of light and to improve its chromatic purity.

A

Slits

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30
Q

The light next passes through an [?] where a portion of the [?] is absorbed, depending upon the of the solution.

A

absorption cell
radiant energy
nature and concentration

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31
Q

Any light not absorbed is transmitted to a [?], which converts the light energy to [?] that can be registered on a [?] or a [?].

A

detector
electrical energy
meter/digital read-out

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32
Q

BEER’S LAW: The [?] is directly proportional to the [?] and inversely proportional to the [?]

A

concentration of the solution
amount of light absorbed
logarithm of transmitted light

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33
Q

%T = [?]
Absorbance / Optical Density (O.D.) =

A

ratio of the radiant energy transmitted, divided by the radiant energy incident on the sample
the amount of light absorbed

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34
Q

BOUGUER’S LAW or LAMBERT’S LAW: [?] is directly proportional to the [?] of light path

A

Absorbance
length

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35
Q

? = absorbance

A

A

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36
Q

? = proportionality constant or molar absorptivity or extinction coefficient. Constant for a given compound at a given wavelength under prescribed condition of solvent, temperature, pH, etc.

A

a

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37
Q

[?] = length of light path in cm

A

b

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38
Q

[?] = molar concentration of absorbing substance

A

c

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39
Q

Internal Parts of the Spectrophotometer

A

source
entrance slit
despersion device
exit slit
sample
detector

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40
Q

A – Light Source
E – Cuvet
B – Entrance slit
F – Detector
C – Monochromator
G – Meter

A
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41
Q

– provides a continuous spectrum of white light which can be separated at different wavelengths

A

LIGHT SOURCE

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42
Q

– isolates a narrow beam of radiant energy; prevents stray light from entering the monochromator

A

ENTRANCE SLIT

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43
Q

– wavelength selector; isolates radiant energy of desired wavelength and excluding that of other wavelengths

A

MONOCHROMATOR

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44
Q

– used to hold the solution whose concentration is to be measures

A

ANALYTICAL / ABSORPTION CELL / CUVETTE

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45
Q

– measure light intensity by converting light signal into electrical signal

A

DETECTORS

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46
Q

– electrical energy from a detector is displayed

A

READ-OUT DEVICES

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47
Q

TYPES OF LIGHT SOURCE

A

A. Tungsten Iodide lamp
B. Quartz Halide lamp
C. Deuterium Discharge lamp
D. Infrared Energy source
E. Mercury Vapor lamp
F. Hollow Cathode lamp

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48
Q

TYPES OF MONOCHROMATOR

A

A. Prism
B. Diffraction Gratings
C. Transmission Filters
D. Interference Filters

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49
Q

TYPES OF ANALYTICAL / ABSORPTION CELL / CUVETTE

A

a. Borosilicate glass
b. Quartz or plastic
c. Alumina silica glass

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50
Q

TYPES OF DETECTORS

A

a. Barrier-Layer cell (Photocell or Photovoltaic cell)
b. Photoemissive tube or Phototube
c. Photoconductive tube or Photoresistive tube
d. Photomultiplier tube

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51
Q

– produces energy wavelength from 340 – 700 nm (visible region); used for moderately diluted solution

A

A. Tungsten Iodide lamp

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52
Q

– contains small amounts of halogen such as iodine to prevent the decomposition of the vaporized tungsten from the very hot filament

A

B. Quartz Halide lamp

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53
Q

– provides energy source with high output in the UV range (down to 165 nm)

A

C. Deuterium Discharge lamp

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54
Q

– used above 800 nm

A

D. Infrared Energy source

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55
Q

– emits narrow bands of energy at well-defined places in the spectrum (UV and visible)

A

E. Mercury Vapor lamp

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56
Q

– consists of a gas-tight chamber containing anode, a cylindrical cathode, and inert gas such as helium or argon

A

F. Hollow Cathode lamp

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57
Q

Infrared Energy source Examples:

A

Merst glower
Globar

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58
Q

– an electrically heated rod of rare earth element oxides

A

Merst glower

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59
Q

– uses silicon carbide

A

Globar

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60
Q

– wedge-shaped pieces of glass, quartz, NaCl, or some other material that allows transmission of light

A

Prism

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61
Q

o Disperses white light into a continuous spectrum of colors by refraction

A

Prism

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62
Q

o Produces a non-linear spectrum.

A

Prism

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63
Q

are for visible region while quartz prisms are for the UV region

A

o Glass prisms

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64
Q

The (?) are close to each other and those of (?) are widely spaced.

A

longer wavelengths
shorter wavelengths

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65
Q

– consist of a thin layer of aluminum-copper alloy on the surface of a flat glass plate that has many small parallel grooves ruled into the metal coating

A

Diffraction Gratings

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66
Q

o Rays of radiant energy bend (refract) around the sharp edges of the grooves

A

Diffraction Gratings

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67
Q

o Extent of refraction varies with the wavelength

A

Diffraction Gratings

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68
Q

– colored glass or colored gelatin sandwiched between two glass plates

A

Transmission Filters

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69
Q

o Light outside the transmission band are absorbed by the colored material

A

Transmission Filters

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70
Q

o Band pass is 35 – 50 nm or more

A

Transmission Filters

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71
Q

– dielectric material (e.g. NaF) sandwiched between two half-silvered pieces of glass

A

Interference Filters

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72
Q

o The thickness of the layer determines the wavelength of energy transmitted.

A

Interference Filters

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73
Q

o Band pass is 10 – 20 nm

A

Interference Filters

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74
Q

– for solutions that do not etch glass

A

a. Borosilicate glass

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75
Q

– does not absorb UV radiation at wavelength below 320 nm

A

b. Quartz or plastic

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76
Q

– good for 340 nm and above (visible region)

A

c. Alumina silica glass

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77
Q
  • Composed of a film of light sensitive material (e.g. Selenium) on an iron plate with a transparent layer of silver
A

a. Barrier-Layer cell (Photocell or Photovoltaic cell)

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78
Q
  • When light passing through the semi-conductive metal layer falls upon the Selenium surface, electrons are released in proportion to the intensity of light and are collected to the silver layer to produce a negative charge
A

a. Barrier-Layer cell (Photocell or Photovoltaic cell)

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79
Q
  • Has photosensitive material that gives off electrons when light energy strikes it
A

Photoemissive tube or Phototube

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80
Q
  • Consists of 2 electrodes (cathode and anode) sealed in an evacuated glass
A

Photomultiplier tube

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81
Q
  • A device whose electrical resistance decreases as the level of incident light is raised
A

Photoconductive tube or Photoresistive tube

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82
Q
  • Cadmium sulfide or cadmium selenide are the light-sensitive materials typically used for the visible region
A

Photoconductive tube or Photoresistive tube

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83
Q
  • Does not require an external power source
A

Photoconductive tube or Photoresistive tube

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84
Q
  • Capable of significantly amplifying a current
A

Photomultiplier tube

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85
Q
  • The cathode is a negative light-sensitive metal that absorbs light and emits electrons in proportion to the radiant energy that strikes the surface
A

Photomultiplier tube

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86
Q
  • Electrons go to the dynodes, where electrons produce 4 – 6 additional electrons
A

Photomultiplier tube

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87
Q
  • The electrons are collected at a final electrode, the positive anode
A

Photomultiplier tube

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88
Q

Photomultiplier tube Advantages:

A
  • rapid response time
  • very sensitive
  • low fatigue
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89
Q

– electrical energy from a detector is displayed

A

READ-OUT DEVICES

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90
Q

– the output of the detector is used to drive a sensitive meter directly without further amplification

A

a. Direct reading system

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91
Q

– the output of the detector is balanced against the output of a reference circuit

A

b. Null Point System

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92
Q

– numerical display of absorbance or converted values of concentrations

A

c. Digital Read-out

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93
Q

K

A
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94
Q

DOUBLE BEAM SPECTROPHOTOMETERS

A
  1. Double Beam-In-Space
  2. Double Beam-In-Time
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95
Q

• All components are duplicated except the light source

A

Double Beam-In-Space

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96
Q

• The beams of light pass through different components but at the same time

A

Double Beam-In-Space

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97
Q

• Uses a light beam chopper (a rotating wheel) – with alternate silvered sections and cut out sections, inserted after the exit slit

A

Double Beam-In-Time

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98
Q

It involves the measurement of emitted light when electrons in an atom become excited by heat energy produced by the flame.

A

FLAME PHOTOMETRY / FLAME EMISSION SPECTROPHOTOMETRY / FILTER PHOTOMETRY

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99
Q

When these electrons return to their ground state, they emit light characteristic of the ions present.

A

FLAME PHOTOMETRY / FLAME EMISSION SPECTROPHOTOMETRY / FILTER PHOTOMETRY

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100
Q

It is used primarily to determine concentration of sodium, potassium or lithium since these alkali metals are easy to excite

A

FLAME PHOTOMETRY / FLAME EMISSION SPECTROPHOTOMETRY / FILTER PHOTOMETRY

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101
Q

Sodium =

A

yellow

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102
Q

Rubidium =

A

red

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103
Q

Potassium =

A

violet

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104
Q

Magnesium =

A

blue

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105
Q

Lithium =

A

red

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106
Q

COMPONENTS of the FLAME PHOTOMETER

A
  1. GASES
  2. BURNER ASSEMBLY
  3. INTERFERENCE FILTERS as MONOCHROMATOR
  4. DETECTOR
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107
Q

Types of Burner:

A

A. Total Consumption Burner
B. Premix Burner

108
Q

mixture of hydrogen and oxygen gas

A
  • acetylene
  • propane
  • natural gas
109
Q

BURNER ASSEMBLY

A

a. Aspirator
b. Atomizer (Nebulizer )
c. Flame

110
Q

– draws sample into the flame

A

a. Aspirator

111
Q

– creates a fine spray of sample solution to be fed into the flame of the burner

A

b. Atomizer (Nebulizer )

112
Q

– provides heat energy for excitation

A

c. Flame

113
Q

– aspirate sample directly into the flame, the gases are passed at high velocity over the end of the capillary suspended in the solution

A

A. Total Consumption Burner

114
Q

– involves the gravitational feeding of solution through a restricting capillary into an area of high velocity gas flow where small droplets are produced and passed into the flame

A

B. Premix Burner

115
Q
  • transmit yellow light (589 nm)
A

Na filter

116
Q
  • transmit violet light (767 nm)
A

K filter

117
Q
  • transmit red light (761 nm)
A

Lithium

118
Q

DETECTOR – uses photocell as detector

A

FLAME PHOTOMETRY

119
Q

The Internal Standard in Flame Photometry: Uses (?)

A

Lithium or Cesium

120
Q

In AAS, the element is not excited in the flame but merely dissociated from its (?) and placed in an unexcited state. The atom, at a (?), absorbs light. The (?) emits radiant energy to be absorbed by the .

A

chemical bond
lower energy level
light source; element

121
Q
  • Measures the amount of light absorbed by ground state atom
A

ATOMIC ABSORPTION SPECTROPHOTOMETRY (AAS)

122
Q

COMPONENTS OF ATOMIC ABSORPTION SPECTROPHOTOMETRY (AAS)

A
  1. LIGHT SOURCE
  2. MECHANICAL ROTATING CHOPPER
  3. BURNER
  4. MONOCHROMATOR
  5. DETECTOR
  6. METER or READ-OUT DEVICE
123
Q

– hollow cathode lamp, which produces a wavelength of light specific for the kind of metal in the cathode

A
  1. LIGHT SOURCE
124
Q

– modulates light beam coming from the hollow cathode lamp

A
  1. MECHANICAL ROTATING CHOPPER
125
Q

– uses flame to dissociate the chemical bonds and form free, unexcited atoms

A
  1. BURNER
126
Q

Two types of Burner:

A

a. Total Consumption burner
b. Pre-mix burner

127
Q

– flame is more concentrated and can be made hotter, thus lessening chemical interferences.

A

a. Total Consumption burner

128
Q

– gases are mixed and the sample is atomized before entering the flame and the large droplets go to waste and not in the flame.

A

b. Pre-mix burner

129
Q

It has less noisy signals with longer pathlength and greater absorption and sensitivity.

A

b. Pre-mix burner

130
Q

– selects the desired wavelength from a spectrum of wavelength which could either be a prism or a diffraction grating.

A
  1. MONOCHROMATOR
131
Q

– uses photomultiplier tubes to measure the intensity of the light signal.

A

DETECTOR

132
Q

– energy emission that occurs when certain compounds absorb electromagnetic radiation, become excited and then return to an energy level that is usually slightly higher than their original level.

A

FLUORESCENCE

133
Q

COMPONENTS OF FLUORESCENCE SPECTROPHOTOMETRY

A
  1. LIGHT SOURCE
  2. MONOCHROMATORS:
    a. Primary filter / Excitation filter
    b. Secondary filter / Emission filter
  3. PHOTOMULTIPLIER
  4. READ-OUT DEVICE
134
Q

– hydrogen discharge lamp or xenon lamp

A
  1. LIGHT SOURCE
135
Q
  1. MONOCHROMATORS:
A

a. Primary filter / Excitation filter
b. Secondary filter / Emission filter

136
Q

– isolates the ultraviolet light

A

a. Primary filter / Excitation filter

137
Q

– isolates secondary emission (filter, prism or diffraction grating)

A

b. Secondary filter / Emission filter

138
Q

– separation of a substance in a pure form and then determining its dry weight

A

GRAVIMETRIC METHOD

139
Q

GRAVIMETRIC METHOD Example:

A

Total Lipid determination

140
Q

– the unknown sample is made to react with a known solution (titrating agent) in the presence of an indicator

A

VOLUMETRIC / TITRIMETRIC METHOD

141
Q

VOLUMETRIC / TITRIMETRIC METHOD Example:

A

Chloride determination (Schales & Schales)

142
Q

– measurement of the amount of light blocked by a particulate matter suspended in solution (180° to the incident beam)

A

TURBIDIMETRY

143
Q

Factors affecting turbidimetry:

A

o Size and number of particles
o The depth of the tube
o Cross-sectional area of each particle

144
Q

– detection of light energy scattered or reflected toward a detector that is not in the direct path of the transmitted light (90° to the incident beam)

A

NEPHELOMETRY

145
Q

– detection of light energy scattered or reflected toward a detector that is not in the direct path of the transmitted light (90° to the incident beam)

A

NEPHELOMETRY

146
Q

• The factors affecting turbidimetric measurements are the same factors affecting (?) measurements

A

nephelometric

147
Q

• It is more specific than turbidimetry

A

NEPHELOMETRY

148
Q

– it is used to measure the disintegration of a radioisotope per minute

A

SCINTILLATION COUNTER

149
Q

Types of Radiation:

A
  1. Alpha
  2. Beta
  3. Gamma
150
Q

– positively charged particles; resemble the nucleus of helium atom with a mass of 4 o Have very little energy

A
  1. alpha
151
Q

– resembles an electron with both negative (β-) and positive (β+) charges but essentially no mass o Exists in two forms: soft and hard beta

A
  1. Beta
152
Q

– a form of electromagnetic energy with no mass, only energy o Exists in two forms: soft and hard gamma

A
  1. Gamma
153
Q

Types of Scintillation Counters:

A
  1. Solid Scintillation Counter
  2. Liquid Scintillation Counter
154
Q

– measures gamma radiation using thallium activated NaI crystal as scintillator and PM tube as detector with preamplifier circuit

A
  1. Solid Scintillation Counter
155
Q

– measures beta radiation using liquid flour as scintillator

A
  1. Liquid Scintillation Counter
156
Q

– an immunologic procedure involving the use of radioisotope

A

RADIOIMMUNOASSAY

157
Q

Substances involved in RIA:

A
  1. Unlabelled antigen (Ag)
  2. Radiolabelled antigen (Ag)
  3. Antibody
158
Q

– substance being analyzed

A
  1. Unlabelled antigen (Ag)
159
Q

– acts as label 3.

A
  1. Radiolabelled antigen (Ag)
160
Q

– provide binding site for the two antigens

A
  1. Antibody
161
Q

Types of RIA:

A
  1. Solid RIA
  2. Liquid RIA
162
Q

– measurement of differences in voltage at a constant current

A

POTENTIOMETRY

163
Q

o The unknown voltage introduced into the potentiometer circuit opposes a known reference voltage

A

POTENTIOMETRY

164
Q

o The voltage of the unknown is measured by comparison to determine the voltage required to exactly oppose the flow of current in the test circuit

A

POTENTIOMETRY

165
Q

o The relationship between the measured voltage and the sought-for concentration

A

POTENTIOMETRY

166
Q

The relationship between the measured voltage and the sought-for concentration is shown by the Nernst Equation

A

POTENTIOMETRY

167
Q

– measurement of differences in current at a constant voltage

A

POLAROGRAPHY

168
Q

o Used to measure trace metals, oxygen, Vit. C, and amino acid concentration

A

POLAROGRAPHY

169
Q

o The relationship between the differences in current and voltage is shown by the Ilkovic Equation

A

POLAROGRAPHY

170
Q

– the measurement of the amount of electricity (in coulombs) at a fixed potential

A

COULOMETRY

171
Q

is equal to a current flow of 1 ampere per second

A

coulomb

172
Q

o The (?) consumed can be related directly to the concentration of the unknown

A

number of coulombs

173
Q

o The relationship is expressed by the Faraday’s Law

A

COULOMETRY

174
Q

– measurement of the amount of current that flows when a constant voltage is applied to the measuring electrode

A

AMPEROMETRY

175
Q

– measurement of the current flow between two non-polarizable electrodes between which a known electrical potential is established

A

CONDUCTOMETRY

176
Q

• Separation is based on solubility

A

PRECIPITATION

177
Q

The precipitate is studied by:

A

a. Turbidimetric method
b. Chemical reaction (after being dissolved)
c. Gravimetric method

178
Q
  • separates dissolved materials
A

ULTRAFILTRATION

179
Q
  • removes particulate matter
A

ULTRAFILTRATION

180
Q
  • separation of dissolved molecules
A

DIALYSIS

181
Q
  • movement through a semi-permeable membrane driven by a force or pulled through a vacuum
A

ULTRAFILTRATION

182
Q
  • movement through a semi-permeable membrane more freely
A

DIALYSIS

183
Q

Cellulose esters
Cellulose acetate Polyamide
Polyvinyl chloride
Sheets, disks or hair thin fibers, conical

A

ULTRAFILTRATION

184
Q

Cellophane (regenerated cellulose) in sheets or tubing

A

DIALYSIS

185
Q
  • For desalting, fractionation of protein solutions and the preparation of pff
A

ULTRAFILTRATION

186
Q
  • Employed in continuous flow automated systems
A

DIALYSIS

187
Q
  • used to purify or concentrate samples
A

DIALYSIS

188
Q

CHROMATOGRAPHY Requires two (2) phases:

A

(1) Solid support
(2) Mobile phase

189
Q

– coated or uncoated

A

(1) Solid support

190
Q

– flowing gas or liquid

A

(2) Mobile phase

191
Q

TWO GENERAL TYPES of CHROMATOGRAPHY:

A

A. Adsorption Chromatography
B. Partition Chromatography

192
Q

– molecules separated are adsorbed at the surface of a solid support or flow with the mobile phase

A

A. Adsorption Chromatography

193
Q

– solid support is coated with a film of water or non-volatile organic liquid

A

B. Partition Chromatography

194
Q

Partition Chromatography Examples:

A

TLC, GLC

195
Q
  1. Paper Chromatography
A

a. Solid or immobile phase
b. Mobile phase

196
Q

– paper is composed of cellulose; the matrix of cellulose is bound to water

A

a. Solid or immobile phase

197
Q

– organic solvent

A

b. Mobile phase

198
Q

o involves partition between water and organic solvent

A

Mobile phase

199
Q

o if the molecules are more soluble in the flowing solvent, the faster it will move along the paper

A

Mobile phase

200
Q

o if the molecules are more soluble in water, they do not move very fast

A

Mobile phase

201
Q

Rf = ratio of the distance of movement by a (?) to the distance of the (?) front

A

compound
solvent

202
Q

Rf = a/B

Where:
a = distance travelled by (?) from origin to front of spot
b = distance travelled by (?)

A

compound
solvent

203
Q

o Uses a flat sheet of chromatographic material

A

Thin Layer Chromatography

204
Q

Thin Layer Chromatography Advantages over paper chromatography

A

• Solid Support
• Mobile Phase

205
Q

Thin Layer Chromatography

• Solid Support: water bound to

A

a. Silica or silicic acid
b. Alumina – aluminum oxide + aluminum hydroxide

206
Q

Thin Layer Chromatography

• Mobile Phase:

A

organic solvent

207
Q

Thin Layer Chromatography

Qualitative analysis:

A

based on colors and positions

208
Q

Thin Layer Chromatography

Quantitative analysis:

A

remove spots and extract

209
Q

o Separation is based on electrical charge

A

Ion-Exchange Chromatography

210
Q

– capture anions

A

o Anion exchangers

211
Q

– capture cations

A

o Cation exchangers

212
Q

Ion-Exchange Chromatography

• Stationary / Immobile / Solid support

A

a. Aluminum silicate
b. Polysaccharide
c. Synthetic resins – polystyrene beads

213
Q

Ion-Exchange Chromatography

• Mobile Phase:

A

water

214
Q

o Separation is based on differences in molecular size

A

Gel Filtration / Molecular Sieve / Gel Permeation / Size Exclusion / Molecular Exclusion

215
Q

Gel Filtration / Molecular Sieve / Gel Permeation / Size Exclusion / Molecular Exclusion

• Stationary phase

A

a. Polyacrylamide (plastic)
b. Sephadex (cross-linked polysaccharide)
c. Porous beads

216
Q

Gel Filtration / Molecular Sieve / Gel Permeation / Size Exclusion / Molecular Exclusion

• Mobile phase:

A

flowing water

217
Q

Gas Chromatography
Two general types:

A

a. Gas – Liquid Chromatography (GLC)
b. Gas – Solid Chromatography (GSL)

218
Q

Gas Chromatography
• Stationary Phase –

A

diatomaceous earth (silica) coated with a non-volatile organic liquid = silicone polymer or alcoholic wax

219
Q

Gas Chromatography
– inert carrier gas (Helium or Nitrogen)

A

• Mobile Phase

220
Q

a. Gas – Liquid Chromatography (GLC) –
b. Gas – Solid Chromatography (GSL) –

A

based on partition
based on adsorption

221
Q

❖ Involves the migration of charged solutes or particles in a supporting medium under the influence of an electric field

A

ELECTROPHORESIS

222
Q

– migration of small ions or molecules

A

• Iontophoresis

223
Q

– migration of charged macromolecules in a porous medium such as cellulose acetate, paper or agarose

A

• Zone electrophoresis

224
Q

➢ it generates an ELECTROPHORETOGRAM – a display of protein zones

A

• Zone electrophoresis

225
Q

– a display of protein zones

A

ELECTROPHORETOGRAM

226
Q

ELECTROPHORESIS

Principle:
• An ampholyte carries either a positive (+) or negative (-) charge; a (?)
• In an acid solution, an ampholyte receives protons and thereby carries a net (+) charge and migrates towards the (?)
• In an alkaline solution, an ampholyte gives up protons and thereby carries a net (-) charge and migrates towards the (?)

A

zwitterion
CATHODE
ANODE

227
Q

Migration depends on:

A
  1. Net electrical charge of molecule
  2. Size and shape of the molecule
  3. Strength of the electrical field
  4. Properties of the support medium
  5. Temperature of operation
228
Q

STAINS:

A

Protein Stains
Isoenzymes: Nitrotetrazoleum Blue
Lipoprotein

229
Q

Protein Stains

A

o Amido Black
o Bromphenol Blue
o Coomasie Brilliant Blue
o Nigrosin
o Ponceau S

230
Q

Lipoprotein

A

o Fat Red 7B (Sudan Red)
o Oil Red O
o Sudan Black B

231
Q

TYPES of ELECTROPHORESIS:

A
  1. Paper Electrophoresis (PE)
  2. Agarose Gel Electrophoresis (AGE)
  3. Cellulose Acetate Electrophoresis (CAE)
  4. Polyacrylamide Gel Electrophoresis (PAGE)
  5. Starch Gel Electrophoresis 6. Isoelectric Focusing
232
Q

ISOELECTRIC FOCUSING  An electrophoretic method in w/c proteins are separated on the basis of their pI (isoelectric pH)

A
233
Q

 An electrophoretic method in w/c proteins are separated on the basis of their pI (isoelectric pH)

A

ISOELECTRIC FOCUSING

234
Q

 Makes use of the property of proteins that their net charges are determined by the pH of their local environment

A

ISOELECTRIC FOCUSING

235
Q

 Proteins show considerable variation in pI, but pI values fall in the range pH 3–12 (many having pIs between pH 4–7)

A

ISOELECTRIC FOCUSING

236
Q
  1. Establishing pH gradients
    Accomplished w/ the use of:
A

a. Carrier Ampholytes (Amphoteric electrolytes)
b. Acrylamide buffers
2. Gel for Isoelectric Focusing

237
Q
  • Mixtures of molecules containing multiple aliphatic amino & carboxylate groups (buffer molecules)
A

Carrier Ampholytes (Amphoteric electrolytes)

238
Q
  • Included directly in IEF gels
A

Carrier Ampholytes (Amphoteric electrolytes)

239
Q
  • Derivatives of Acrylamide containing both reactive double bonds & buffering groups
A

Acrylamide buffers

240
Q
  • Covalently incorporated in PAG at the time of casting
A

Acrylamide buffers

241
Q

Gel for Isoelectric Focusing

A

❖ Polyacrylamide Gel

242
Q

– large-pore convective matrices - Polymerized with an initiator system including Riboflavin for photo-polymerization

A

❖ Polyacrylamide Gel

243
Q

– introduction of the 1st automated analyzer by Technicon
- Continuous-flow

A

1957

244
Q

: sequential batch analyzer capable of providing single test result on approx. 40 samples / hour

A
  • Continuous-flow
245
Q

– Technicon instruments w/c were next developed

A

 Simultaneous Multiple Analyzer (SMA)

246
Q
  • With multiple channels (for diff. tests)
A

 Simultaneous Multiple Analyzer (SMA)

247
Q
  • 6-12 test results simultaneously at the rate of 360
A

 Simultaneous Multiple Analyzer (SMA)

248
Q

– 720 tests per hour

A

 Simultaneous Multiple Analyzer (SMA)

249
Q

– specialty area w/ rapidly developing arsenal of analyzers

A

 IMMUNOCHEMISTRY

250
Q
  • Immunological techniques for assaying drugs, specific proteins, tumor markers & hormones
A

 IMMUNOCHEMISTRY

251
Q
  • Fluorescence Polarization Immunoassay
  • Nephelometry
  • Chemiluminescent Detection
A

 IMMUNOCHEMISTRY

252
Q

BASIC APPROACHES OF AUTOMATED ANALYZERS

A

I. CONTINUOUS - FLOW
II. CENTRIFUGAL ANALYSIS
III. DISCRETE ANALYSIS

253
Q
  • Liquids (reagents, diluents & samples) are pumped through a system of continuous tubing
A

CONTINUOUS - FLOW

254
Q
  • Samples are introduced in a sequential manner, following each other through the same network
A

CONTINUOUS - FLOW

255
Q
  • Batch analysis can be used (e.g. large # of specimen in one run)
A

CONTINUOUS - FLOW

256
Q

 More sophisticated continuous flow anayzers Use parallel single channels to run multiple tests on each sample (e.g. SMA & SMAC)

A

CONTINUOUS - FLOW

257
Q

 Major drawbacks: significant carry-over problems & wasteful use of continuously flowing reagents

A

CONTINUOUS - FLOW

258
Q
  • Uses the force generated by centrifugation to transfer & then contain liquids in separate cuvets for measurement
A

CENTRIFUGAL ANALYSIS

259
Q
  • Capable of running multiple samples, one test at a time, in a batch MAJOR ADV.: batch analysis (e.g. COBAS - Bio by Roche Diagnostics)
A

CENTRIFUGAL ANALYSIS

260
Q

– Bio by Roche Diagnostics)

A

CENTRIFUGAL ANALYSIS

261
Q
  • Most popular & versatile
A

DISCRETE ANALYSIS

262
Q
  • Separation of each sample & accompanying reagents in a separate container
A

DISCRETE ANALYSIS

263
Q
  • Capability of running multiple tests one sample at a time OR multiple samples one test at a time
A

DISCRETE ANALYSIS

264
Q
  • Random access, stat capabilities
A

DISCRETE ANALYSIS

265
Q

Use parallel single channels to run multiple
tests on each sample (e.g. SMA & SMAC)

A

CONTINUOUS - FLOW

Clinical Chemistry 1 (10 decks)

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